Keywords: Neuroinflammation, endothelial cells, NMDA receptor, tPA, non-conventional. ABSTRAIT Neuroinflammation, cellules endothéliales, Récepteur NMDA, TPA, non-conventionnel, Métabotropique

NMDA receptors of the Blood Brai Barrier: Mecha is of actio a d i teractio with tPA

Neuroinflammation is a common denominator of several central nervous system disorders. Inflammatory reactions are often mediated by several signaling pathways which lead to the opening of the blood brain barrier. Tissue plasminogen activator (tPA) is a serine protease induces opening of the blood brain barrier. In recent years, it has also been shown that NMDA receptors located in endothelial cells can play a crucial role in propagation of inflammatory reaction. My doctoral study focused on the finding the underlying mechanisms of action(s) by which NMDA receptor mediates tPA induced opening of the blood brain barrier. In our first study we show that endothelial NMDA receptors are potential therapeutic targets to prevent EAE mediated immune cell infiltration and inflammation. We show that NMDA receptor specific mouse monoclonal antibody Glunomab could prevent the brain spinal cord barrier from inflammatory damage. We also show that NMDA receptors are expressed in close association of tight junction proteins in cerebral endothelial cells. In our second study, we show for the first time that, neuroendothelial NMDA receptors can exhibit metabotropic mode of action during inflammation. We also highlight that these receptors are indeed GluN3A expressing non-conventional NMDA receptors. In addition, we report that tPA accelerates the opening of blood brain barrier in presence of an uncommon agonist glycine by RhoA activation dependent mechanism. My project results provide a nouvelle insight for the role of metabotropic NMDA receptors in cerebral endothelial cells. In addition it also provides more precise details of blood brain barrier opening mediated by tissue plasminogen activator.

Acknowledgment

First of all, I would like to express my gratitude to Pr. Denis Vivien, who I am bound to have a great deal of gratitude to Dr. Fabian Docagne. Thank you for being an encouraging team leader and motivator throughout this process. You have immense patience for which I will always be grateful. Your words of encouragement, every now and then, were a big thrust to my confidence. Your criticism has thrived me to do better. Your understanding and compassion towards your pupil is valuable.

You have taught me of what all qualities a good boss must have.

I would like to thank Dr. Sandrine Bourdoulous for accepting my request to be the member of this jury as the rapporteur for my thesis evaluation. Thank you for devoting time out of your schedule for this purpose.

I would like to mention that your commitment is highly valued.

I would like to thank Pr. Vincent Berezowski for devoting time from his schedule to evaluation of my thesis as the rapporteur and be member of the jury for my thesis defense. I would like to mention that your commitment is highly respected.

I would like to thank Dr. Elga de Vries for taking our time, agreeing to be member of my thesis defense. I am grateful to you for hosting me at your lab in Amsterdam where I spent two months of secondment as a part of my project. I am thankful to you and your lab for equipping me with all the resources required by the project for that duration.

I owe a big vote of gratitude is Pr. Carine Ali. You have always been compassionate and concerned. Your support towards me, through the rough patches will always be of very high regards. I am glad you are one of the friends I found here in Caen. Your criticism is valuable and knows no filter. Your skeptical view towards this project has helped me throughout. Thank you for kindly accepting my request to be the member of my thesis committee.

Marina Rubio, you are sweet spoken and sympathetic. Your friendly gestures were always been uplifting and motivating. Thank you for being there. A special vote of thanks to Sara and Max, your helpfulness was always appreciated.

A special vote of gratitude for my friends in our office, C054, Pauline Helie, Anna Rose and Helene Party. You are understanding, funny and witty. All the good times with you all are worth cherishing. Your support through my thesis is highly appreciated. Thank you for all the small French lessons I was bestowed with.

Thank you Ben and Yannick for your help and all the weird jokes. Thank you Richard for your kind words.

Thank you Isa and Eric for your suggestions and for teaching me new things. Thanks to all my former mates of the unit U919 and present mates of unit U1237, your every little gesture has been valuable. Last but not the least, a heartfelt vote of gratitude to my grandmother, my parents and my brother, who have supported me all through this far. A special regard for my best friends Ruma and Govind, whom with I have shared my bests and worsts. Obermeier, Daneman, and Ransohoff, 2013...................... Figure 2. Blood Brain-Barrie . Adapted from Engelhardt and Ransohoff, 2012................................... Figure 3. Tight Junction Proteins and Associated Intracellular Components of the Endothelial and Epithelial Junctions. Adapted from Aktories and Barbieri, 2005 ........................................................ Figure 4. Dual localization of Zoluna Occludens Nucleic Acid Binding (ZONAB) protein. Adapted from Tsapara et al. 2006. ............................................................................................................................. Figure 5. Transmembrane Occludin Tight Junction Protein Associated with ZO-1 and Cytoskeletal Protein F-actin. Adapted from Cummins et al. 2012 ............................................................................ Figure 6. Diversity Tsukita et al. 2001 ............................................................... Figure 7. VE cadherins. Adapted from P. A. Vincent et al. 2004............................................................ Figure 8. MAP kinase signaling cascade. Adapted from Hommes et al. 2017..................................... Figure 9. RhoA Signaling Cascade . Adapted from González-Mariscal, Tapia, and Chamorro 2008.... Figure 10. Myosin Light Chain. Adapted from González-Mariscal, Tapia, and Chamorro 2008.......... Figure 11. Ribbon Diagram of the CREB bZIP.DNA Complex. Adapted from Schumacher, Goodman, and Brennan 2000 ................................................................................................................................ Figure 12. Functional Structure of Tissue-type Plasminogen Activator. Adapted from Vivien et al. 2011 ...................................................................................................................................................... Figure 13. tPA Mediated Neuroinflammatory Channels in Brain Endothelial Cells andAstrocyte . Adapted from Mehra et al. 2016 .......................................................................................................... Figure 14. Cytokine like Effects of tPA on Microglia. Adapted from Mehra et al. 2016 ...................... Figure 15. Characterization of NMDA Receptor Subtypes and their Differential Expression in Region of Brain. Adapted from Pierre Paoletti, Bellone, andZhou 2013 ............................................................. Figure 16. Transmembrane Topology andModular Structure of the GluN3A Subuni . Adapted from Pérez-Otaño, Larsen, andWesseling 2016 .......................................................................................... Figure 17. The hypothesis of this study ................................................................................................ Figure 18. Neuroendothelial NMDA receptors are composed of GluN2B andGluN3A subunits. ...... Figure 19. Neuroendothelial NMDA receptors are expressed in closely associated with transmembrane tight junction proteins . ............................................................................................ Figure 20. Inflammation like conditions promote upregulation of NMDA receptor levels in cerebral endothelial cells . ................................................................................................................................. Figure 21. NMDA and tPA induce CREB activation. ............................................................................ Figure 22. NMDA and tPA induce activation RhoA signaling ............................................................. Figure 23. NMDA and tPA induce blood brain barrier opening. ......................................................... Figure 24. tPA prolongs glycine induced CREB activation . ................................................................. Figure 25. tPA prolongs glycine induced RhoA activation . ................................................................. Figure 26. tPA accelerates glycine induced increase in endothelial barrier permeability ..................

I would like to acknowledge Marie Skłodowska-Curie

of structure between the tight junction proteins occludin, claudin and junction adhesion molecule (JAM). Adapted from

INTRODUCTION Preface

Inflammation is a common denominator of many neurological disorders of the central nervous system. The inflammatory reactions can be part of a long lines cascade of events. And inflammation can play the instrumental role in the pathological conditions at the different time points of the disease. In many cases inflammation can be the last of the symptoms, on the other hand it can be seen on the onset of the disease. The central nervous system is guarded by endothelial barrier, which forms a physical barrier between the circulation and brain parenchyma. These endothelial barriers are also further categorized as blood brain barrier and blood spinal cord barrier. Cerebral endothelial cells play crucial role in the phenomena of inflammation. The endothelial ells pro ide the interfa e for the rolling of the ir ulating ells hi h is follo ed y the a ti ation of cells. Under the inflammatory conditions provided by high levels of inflammatory cytokines and factors, endothelial cells (under stress) respond via upregulating the expression of the surface proteins and factors. This upregulation of proteins thereby aggravates the speed of interaction of endothelial cells with the different external factors (Antigen presenting cells, cytokines, chemokines, etc.). The interaction of these factors with receptors, then triggers several signaling cascades in endothelial cells. These signaling pathways hence articulate the endothelial barriers which causes endothelial barrier dysfunction. This dysfunction may lead leakage of fluids and infiltration of cells from the lumen of the blood vessels to brain parenchyma. Although this should be noted that the opening of the blood brain barrier could be an eventual well-planned, non-invasive mechanism, regulated by the membrane and channel proteins, which would allow a programmed passage of molecules and cells without damaging the blood brain barrier.

The blood brain barrier and blood spinal cord barrier are two complex entities comprised of several cellular and molecular components, which are responsible for various signaling cascades. These signaling pathways of the blood brain and blood spinal cord barriers play a vital role in development and regulation of the barrier function.

Another aspect of blood brain and blood spinal cord barrier dysfunction is the factors and the pathological conditions that lead on the CNS barrier malfunctioning. These are the biophysical and molecular factors, which bring about the barrier dysfunction via signal transduction. Tissue plasminogen activator is one of these factors, which plays a potential role in promoting the barrier dysfunction. These adverse effects though have been shown to interrupt the barrier function by damaging the tight junction proteins, but the signaling pathway responsible for these consequences has not been described clearly. It is noticeable that tissue plasminogen activator interacts with N-Methyl D-Aspartate receptors by its proteolytic and non-proteolytic activity to promote barrier dysfunction.

In the following review of literature, aforementioned aspects have been discussed in details with respect to inflammation and its consequences. To begin with, we discuss the CNS-barriers and their molecular components.

Following these, we will review the key signaling pathways which are associated with the maintenance and dysregulation of the CNS-barriers. In special regard to this project, a detailed focus has been made on RhoA/ROCK signaling cascade and the cAMP responsive element binding (CREB) activation.

Following this, Tissue plasminogen activator has been proven malicious for its deleterious effects on the endothelial integrity. The focus of this study is the determination of the underlying role of neuroendothelial NMDA receptors in neuroinflammation mediated by tissue plasminogen activator which promotes the endothelial barrier dysfunction.

The N-Methyl D-Aspartate (NMDA) receptors are calcium permeable glutamate ionotropic receptors, which play crucial role in synaptic functions. These receptors are suggested to be expressed almost universally in all the regions of the brain and spinal cord. Apart from the classical pattern of these receptors in neurons, expression of N-Methyl D-Aspartate receptors has also been reported in astrocytes, oligodendrocytes and the cerebral endothelial cells.

The neuroendothelial N-Methyl D-Aspartate receptors are recently reported to be a potential therapeutic targets to evade the inflammatory outcomes [START_REF] Macrez R 1 | Chevilley A 1[END_REF]).

Though the importance of NMDA receptors in neuroinflammation has always been an interesting matter of interest, the underlying mechanism by which neuroendothelial cells orchestrate these outcomes is still unknown.

Along with this, the possible and probable NMDA receptor subunits expressed in endothelial cells is still unknown. This lack of information brings along the unawareness about the behavior these receptors in endothelial cells. At this point it is important to note because NMDA receptors, as versatile as they can be in their expression in different parts of the brain, their behavior also changes in accordance with their respective subunit subtypes. Just to introduce the terms here, NMDA receptors are categorized in classical ionotropic NMDA receptors and a special kind, often regarded as non-classical NMDA receptors whose activity is completely independent of calcium ions influx (a characteristic feature of classical NMDA receptor).

In the following study we will explore these differential behaviors of neuroendothelial NMDA receptors during neuroinflammation.

Neuroinflammation

Central nervous system (CNS) is an excellent example of a well-synchronized machinery which is maintained by the regular supply of nutrients, exchange of metabolites and prevention from toxins and pathogens. Cerebral endothelial cells together form the blood brain barrier. When the tightly attached cell to cell integrity is lost, we term it as BBB breakdown (shown in figure 1), leading to a dysfunctional barrier. In this unfortunate event, the lood rai arrier s fu tio s are o pro ised hence the barrier is dysfunctional which leads to neuroinflammation (the inflammation in nervous tissue). Neuroinflammation can leave a short term, long lasting or a permanent damage to the CNS. Neuroinflammation is defined in many ways in accordance with different studies. The opening of the blood brain barrier and a blood brain barrier dysfunction, both can be regarded as the BBB breakdown. Although, these both processes are not referred rupture of the cells at the blood brain barrier.

BBB breakdown is a synchronized procedure taken care by several ligands, receptors, antibodies, inflammatory cytokines and factors, messenger molecules and enzymes, which regulate several signaling pathways. These where the allowance of the passage for molecules varies gradually and the tight junction proteins may not be necessarily involves in a low grade opening of the blood brain barrier. Another important point to note is that in many cases the BBB breakdown does not necessarily involves the tight junction protein activity. For instance, studies have reported that leukocytes migration across the endothelial cell barrier may employ the tight junction proteins or a large pore or a vacuole in the endothelial cells. It is also interesting to note that apart from the paracellular passage, leukocytes can also cross the endothelial barrier by transcytosis (Godessart and Kunkel 2001;Filous and Silver 2016;Biber et al. 2002). Hence, the activity of the tight junctions is not a necessity for the transcellular passage.

Despite of the variety of NDs, neuroinflammation is one the most common pathophysiological outcome amongst the , hi h also i reases the se erit of the diseases, su h as Alzhei er s disease AD , Multiple S lerosis MS , Parki so s disease PS a d rai i fe tio s. There are several signaling cascades and molecules that are associated with many neurological disorders, which regulate the blood, brain barrier integrity. For example, during

Alzhei er s disease (AD), several in vitro studies suggests that the high cytokines activity and caspases activation regulate W t/β-Catenin signaling which can influence the blood brain barrier (Machhi et al. 2016; Zolezzi and Inestrosa 2017). The depletion of Brain Derived Neurotrophic Factor (BDNF) reduces the neurodegenerative progression during AD. Another study suggests that the depletion in BDNF levels reduces the plaque size and numbers, causing reduced neuropathology of the disease an in vivo BDNF knockout mice model (Braun, Kalinin, and Feinstein 2017). In another in vitro primary astrocyte culture study it has been shown that TNF-α i du es

PKCσ phosphor latio a d a late phase NFκB sig ali g. These i fla ator respo ses hea il sti ulate neuroinflammation during AD (C. Lee et al. 2017). An in vitro BV2 microglial cells study shows that the inflammatory responses during AD are intensified by the activation of RhoA/ROCK pathways. RhoA/ROCK activation is induced with inducible nitric oxide synthase (iNOS) activation [START_REF] Chen | a. I hi itio of AGEs/RAGE/Rho/ROCK Path a Supp esses No -Specific Neuroinflammation by Regulating BV2 Microglial M1/M2 Polarization through the NF-κB Path a[END_REF]). Inflammation has also been shown to be regulated by TGF-β sig aling via multiple factors, such as, through chemokines CCL5 and CCL2 as shown in an in vitro study with BV2 microglial cells (Huang et al. 2010), and TGF-β apoptoti fa tor as

shown in an in vivo study (M.-H. Lee et al. 2010).

Another examples of a neurological disorder effected by inflammation is Parki so s disease (PD) which also shows association with signaling pathways. An in vivo & in vitro (in peripheral blood mononuclear cells (PBMCs))

study suggests that LPS induced UDP secretion promotes ERK1/2 pathway activation, which in turn accelerates neuroinflammation during PD. Mutually, UDP/P2Y6R (PD biomarker) signaling aggravates the inflammatory reactions (Yang et al. 2017). High mobility group box 1 (HMGB1) which is a pro-inflammatory cytokine induces TLR /NFκB sig ali g hi h i du es the i fla ator rea tio duri g PD, as shown in an in vitro BV2 microglial cells study (Li et al. 2017). The resident and non-resident cells of CNS also play a vital role in many neurological disorders. In an interesting in vitro study (with immortalized embryonic mouse microglia N9 cell line) it has been shown that the PD symptoms worsen in the effect of microglia induced NFκB sig ali g. This i roglial a ti atio induced inflammatory response is associated with high ROS and nitric oxide levels, which can be prevented by silver nanoparticles (Gonzalez-Carter et al. 2017). Neuroinflammation is a very complex phenomenon and signaling cascades mentioned above are a very few examples to quote. These signaling pathways may do not entirely lead deleterious effect on CNS barriers but they mediate their effect by activating inflammatory factors and promoting opening of the blood brain barrier. All these aspects have been discussed in detail ahead.

The blood brain barrier and blood spinal cord barrier are regulated by several signaling pathways which function in synchrony to maintain the barrier function. These signaling molecules can interact with the internal cellular structures such as cytoskeleton and tight junction proteins which leads to the barrier dysfunction. The tight junctions and adherens junctions proteins associated pathways regulate the function, dysfunction, assembly, disassembly, fluidity, and permeability of the CNS-barriers.

Blood Brain Barrier/ Blood Spinal Cord Barrier

Cerebral microvessels are composed of highly specialized endothelial cells (which form the core component of the blood brain barrier). These endothelial cells have several distinguished features which enable CNS homeostasis for the highly controlled influx of solutes as well as facilitated efflux of metabolites. These endothelial cells express a unique pattern of transmembrane tight junction proteins, have low pinocytic activity and lack fenestrations (Kniesel and Wolburg 2000;Gloor et al. 2001). Cerebral endothelial cells have negative charge on the luminal surface which repels highly negative charges compounds (de Boer and Gaillard 2006). No fenestrations and presence of tight junction proteins enables them to stay tightly conjoint with each other, thereby reducing the rate of paracellular and transcellular transport across the monolayer layer (Abbott, Rönnbäck, and Hansson 2006).

The central nervous system possesses a dense network of blood vessels, as of arteries and veins, arterioles and venules, and dense capillaries that go deeper into the tissues (Daneman 2012). These capillaries play a crucial role in delivering the nutrients to the neurons residing in proximity as well as regulating the cellular traffic in parenchymal space (Aird 2007). This makes the endothelium of the blood brain barrier a highly specialized machinery. In addition, to maintain this functional variability, endothelial cells of the blood brain barrier are supported by neighboring astrocyte, pericytes, extracellular matrix (ECM) and the basement membrane. The astrocytes ensheath 99% of the microvessels in brain though their end-feet [START_REF] Hawkins | The Blood-Brain Barrier/Neurovascular Unit in Health and Disease[END_REF]. Astrocytes In addition, the blood brain barrier is also equipped with transporter molecules. Transporter molecules exist in two states of brain to blood (efflux) and blood to brain transporter. These molecules regulate the brain uptake by regulating the retention of the substrates. The transporter molecules facilitate the uptake of the water soluble polar molecules such as glucose and amino acids across the blood brain barrier. These transporter molecules mediate the uptake of large molecules such as regulatory proteins which are not taken in by transendothelial diffusion [START_REF] Pan | Interactions of Cytokines with the Blood-Brain Barrier: Implications for Feeding[END_REF]. The carrier mediated transporter molecules mediate the transport of molecules in both blood to brain and brain to blood directions whereas the efflux transporters are unidirectional i.e. from brain to blood. A category of transporters also form the ion channels which facilitate the passage of ions. These include sodium pump which mediates the passage of sodium and potassium ions mediated by Na + K + ATPase enzyme. The chloride-bicarbonate exchanger, Na + -K + -2Cl -cotransporter and sodium hydrogen exchanger is also ion transporters of the blood brain barrier (Redzic 2011).

It is conclusive that the blood brain barrier is a one highly complex machinery which has numerous components responsible for its regulated functioning. The tight junction proteins are one crucial component of the blood brain barrier which defines its functions.

Tight Junction Proteins and the Adherens Junctions

The endothelial tight junction proteins are located in the apical intercellular space. In the extracellular space, these transmembrane proteins interact via their extracellular domains that are utilized for barrier strength and for 1999;Kirk et al. 2003;Vorbrodt and Dobrogowska 2003). The ZO-1, primary member of this family, links the membranal tight junctions and adherens junctions with the cytoplasmic actin cytoskeleton (Fanning et al. 1998;M. Itoh, Morita, and Tsukita 1999). ZO-1 organizes the tight junctions, both in endothelial and epithelial cells, and also maintains the VE-cadherin dependent adherens junctions (Tornavaca et al. 2015).

Zonula occludens (ZO)

Several in vitro studies have shown that Zonula Occludens (ZOs) are regulatory phosphoproteins and they are phosphorylated at their serine residue (Anderson et al. 1988;Avila-Flores et al. 2001). ZO-2 proteins are generally observed more phosphorylated in serine residues in the absence of calcium in culture media. Similarly, when the tight junction assembly is enhanced, the phosphorylation level of ZO-1 decreases along. In addition, translocation of the ZOs proteins into the nucleus is also an interesting characteristic.

Key points: Zonula occludens are membrane associated scaffold proteins. They are closely associated with the cytoskeletal elements such as actin. ZO-1 undergoes dephosphorylation during tight junction assembly

Nuclear shuttling of ZOs proteins

The nuclear shuttling of ZOs proteins is one the significant and unique characteristic of these tight junction proteins. ZOs proteins remarkably show nuclear localization (NLS) and nuclear export signals (NES) (Islas et al. 2002;Jaramillo et al. 2004;Traweger et al. 2003). An in vitro study suggests that the localization of the ZO-1 protein in nucleus was found to be inversely related to the extent of maturity of cell-to-cell contact. Before the maturation of tight junctions, ZO-1 is accumulated in the nucleus. Nuclear accumulation of ZO-1 proteins is also a result of wounding in cultured epithelial cells (Gottardi et al. 1996). Another study also supported nuclear localization of this protein as nuclear localization of ZO-2 proteins is determined by the state of the cell-to-cell contacts of epithelial monolayer cells (in epithelial MDCK, Madin Darby canine kidney cultures) (Islas et al. 2002).

Interestingly, it has been shown that, nuclear localization of ZO-1 gradually decreases as ZO-1 eventually exits the nucleus as the monolayer reaches to confluency. Interestingly, it was demonstrated that nuclear localization of ZO-2 decreases gradually as ZO-1 eventually exits the nucleus under the effect of leptomycin-sensitivity as the monolayer reaches to confluency. An in vivo study suggests that, the localization of ZOs proteins is dependent on the condition of cell-to-cell contacts in epithelial cells (Traweger et al. 2003). It is also very interesting to know that the localization of ZOs proteins in the nuclear environment can also be a consequence of chemical or physical shock (figure 4). For instance, nuclear accumulation of ZO-2 increased in epithelial and endothelial cells when the cells were subjected to heat shock and chemical insult (Traweger et al. 2003). Similar pattern of nuclear localization is observed upon disruption of the cell-to-cell contact by mechanical injury (Islas et al. 2002). These studies are evident enough to confirm that ZOs proteins are potentially mobile and highly sensitive proteins, which exhibit nuclear localization during barrier development, immature barrier Key points: It can be concluded that in the event of physical/heat shock and chemical insult ZO-1 proteins attain mobility and translocate. In addition, eventual immature junctions and damaged cell junctions result in accumulation of ZO-1 in nucleus. These high ZO-1 nuclear levels are further reduced when cells attain healthy tight junctions and recover from shock.

integrity, chemical stress, and mechanical shock.

Occludin

Occludin was the first tight junction transmembrane molecule discovered which is highly expressed in brain microvessels (M Furuse et al. 1993;T Hirase et al. 1997). Later, it was shown that occludin is a type II transmembrane protein and share a MARVEL domain, which belongs to the protein family of tricellulin and MarcelD3 (D. R. [START_REF] Raleigh | Tight Ju tio -Associated MARVEL Proteins MarvelD3, Tricellulin, and Occludin Ha e Disti t ut O e lappi g Fu tio s[END_REF]). This protein links its transmembrane domain with the actin cytoskeleton protein via membrane associated guanylyl kinase-like domain (MAGUK) scaffold proteins (such as ZO-1, ZO-2 and ZO-3), as shown in figure 5 [START_REF] Muller | The Tight Ju tio P otei O ludi a d the Adhe e s Ju tio P otei -Catenin Share a Common Interaction Mechanism with ZO[END_REF]Masahiko Itoh et al. 1999;Nasdala 2002).

Occludin regulates the ion transport across the blood brain barrier, as is shown in an in vivo study with occludin deficient mice [START_REF] Schulzke | Epithelial Transport and Barrier Function in Occludin-Defi ie t Mi e[END_REF]). This tight junction protein is important for the formation of the junction during the development of the blood brain barrier but not essentially. It has been shown in an occludin deficient embryonic stem cells study, that occludin deficient mice develop chronic inflammation and hyperplasia of gastric strain and shear stress can also monitor the blood brain barrier integrity as they modulate the expression and phosphorylation (by activation of tyrosine phosphatase) of occludin and ZO-1 tight junction protein in vascular endothelial cells [START_REF] Collins | C li St ai -Mediated Regulation of Vascular Endothelial Occludin and ZO-. A te ios le osis, Th o osis, a d Vas ula Biolog[END_REF]Pang, Antonetti, and Tarbell 2005). Interestingly, in an in vitro study with porcine brain capillary endothelial cells shows that, inhibition of tyrosine phosphatase activity can influence the barrier activity by decreasing the permeability across the barrier through an increase in TEER (Trans-Endothelial Electrical Resistance) (Lohmann et al. 2004).

Tyrosine phosphorylation is also one of the key downstream events leading in opening of the blood brain barrier in neurological disorders. For instance, in cerebral ischemia, blood brain dysfunction is observed with a decrease in occludin and ZO-1 levels along with tyrosine phosphorylation of the occludin tight junction protein (Kago et al. 2006).

Claudins

Claudins 3 is expressed in choroid plexus epithelial cells and is localized at tight junctions (Hartwig Wolburg et al. 2003;Kratzer et al. 2012;Kooij et al. 2014). On the other hand, Claudin-5 is specifically expressed in endothelial cells at the tight junctions (K. Morita et al. 1999) and exhibits significant heterogeneity in the endothelial cells of microvessels (Paul et al. 2013). This claudin is highly expressed in the brain endothelial cells of rodents, non-human primates and humans (Nitta et al. 2003;Hoshi et al. 2013). It is interesting to note that Claudin-5 assembly is crucial for the prevention of BBB leakage. An in vivo study suggests that the loss of function of claudin-5 leads to the blood brain barrier dysfunction (Nitta et al. 2003).

An in vitro study in MDCK II cells via stable expression of Claudin-5 protein suggests that second extracellular loop of the claudin-5 plays a crucial role in the tightening of the blood brain barrier that regulates the paracellular movement of ions, small and large molecules (Piehl et al. 2010). Claudin-11 induces tight junction parallel strands in oligodendrocytes myelin sheaths (Gow et al. 1999) and is localized in the epithelial tight junctions of BCSFB as parallel running particles strands (Hartwig Wolburg and Paulus 2010) whereas Claudin-12 is expressed in tight junction of brain endothelial cells and but its functions are still unknown.

As well as occludin regulates the tight junction via phosphorylation and dephosphorylation, the specific but different amino acid residues of claudins also undergo phosphorylation by the activity of several protein kinases.

High levels of claudin-5 expression have been reported in endothelial cells (Kazumasa Morita et al. 1999). As the expression of claudin-5 in brain endothelium comes into attention, the regulatory role of claudin-5 in the blood brain barrier has been proved too (Nitta et al. 2003). For instance, cAMP promoted the barrier functions of the brain endothelial cells, which is exhibited as an increase in claudin-5 gene expression, is independent of the PKA signaling. On the other hand, an in vitro study in porcine cerebral endothelial cells suggests that PKA activation in brain endothelia induces phosphorylation of claudin-5 which is independent of the cAMP, though both of these processes are involved in promotion of the barrier function in brain endothelial cells (Ishizaki et al. 2003). Claudin

proteins undergo phosphorylation at threonine, tyrosine and serine residues. The residue that undergoes phosphorylation differs from one type of claudin protein to another. For instance, an in vitro study in epithelial cells of carcinomas, phosphorylation of the threonine 192 residue by the cAMP dependent protein kinase A (PKA) activation regulates the arrier fu tio s D Souza, Agar al, a d Mori . Follo i g the sa e patter , mitogen-activated protein kinase (MAPK) induces phosphorylation of claudin-1 at the threonine 203 residue in rat lung endothelial cells, promoting the barrier function of tight junctions (Fujibe et al. 2004).

An overexpression of this protein WNK4 (serine-threonine protein kinase) induces high paracellular permeability through an increase in chloride shunt across MDCKII cells observed along with phosphorylation of claudin-4 and claudin-1 (Yamauchi et al. 2004;Ohta et al. 2006). It has also been reported in an in vitro study that phosphorylation of the carboxyl terminus of claudin-4 at tyrosine 208 residue in epithelial tissue is observed along with paracellular permeability. An increase in paracellular permeability is mediated by disassembly of the claudin-4 with ZO-1 upon phosphorylation (Tanaka, Kamata, and Sakai 2005). These studies put forward the clear idea that claudins are dynamic in their roles as regulators of the barrier function.

Along with the regulatory role of phosphorylation, expression levels of claudins and their tight integrated conformation are also major factors that alter the endothelial barrier functions. For instance, arsenic μM produces substantial damage to the tight junctions as it decreases the expression of claudin-1 and claudin- . 2015). An in vitro study in bEnd.3 endothelial cells suggests that claudin-5 may disrupt during an early or a late phase. This biphasic modulation of paracellular claudin-5 alters the blood brain barrier through PI3K/AKT signaling [START_REF] Camire | Biphasi Modulatio of Pa a ellula Claudi -5 Expression in Mouse Brain Endothelial Cells Is Mediated through the Phosphoinositide-3-Ki ase/AKT Path a[END_REF]). These studies confirm that claudins (via its phosphorylation on different amino acid residues, or an alteration in levels of expression and an eventful degradation) can monitor the endothelial and epithelial monolayer tight junction integrity.

There are several other proteins of the tight junctions which are crucial for the assembly and maintenance of the endothelial barriers. Key points: These studies project a clear idea that claudins are highly versatile tight junction proteins. These molecules undergo phosphorylation at many sites. In claudins, different threonine, tyrosine and serine residues undergo phosphorylation which thereby regulate the claudin protein conformation at the tight junctions. For example, phosphorylation at threonine residues promotes a high TEER, whereas tyrosine phosphorylation observed when there is high paracellular permeability. These studies confirm that claudins, via its phosphorylation on different amino acid residues or by an alteration in levels of expression and an eventual degradation can monitor the endothelial and epithelial monolayer tight junction integrity.

Junction Adhesion Molecules

On the other hand, junctional adhesion molecules are members of the immunoglobulin subfamily, expressed in endothelial and epithelial cells in which they localize at cell-to-cell contacts at tight junctions. [START_REF] Ludwig | Ju tio al Adhesio Mole ule JAM -B Supports Lymphocyte Rolling and Adhesio th ough I te a tio ith α β I teg i[END_REF]).

E Cadherins, N Cadherins and VE Cadherins, Proteins of Adherens Junctions

Transmembrane adherens junctions are another component of the BBB and BCSFB, which have functions different from tight junctions. Adherens junctions maintain the tensile forces and the plasticity of the cells. They initiate the cell-to-cell contact and promote the tight junction development, which fastens the barrier (figure 7). Ecadherin is a member of the cadherin family, which is a membrane-integrated protein expressed in epithelial cells.

Key points: It is conclusive that JAMs play a vital role in cell trafficking and opening of the blood brain barrier. α-Catenin binds directly or indirectly to the actin cytoskeleton and maintains the adherens junctions (Vestweber 2015).

In addition, some of the other components of the blood brain barrier are worth a mention. Though they are not one of the key regulators of the blood brain barrier system. For instance, Platelet endothelial cells adhesion molecule, PECAM-1 is an integral membrane protein highly expressed at the endothelial cell-to-cell contacts but outside the adherens or tight junctions (Graesser et al. 2002;Lyck et al. 2009). Upon activation it undergoes phosphorylation at the serine and tyrosine residues in its long cytoplasmic terminal (Privratsky and Newman 2014 Afadin links them with cytoplasmic actin cytoskeleton proteins (Indra et al. 2013). Nectin is observed in choroid plexus and its afadin interaction promotes adherens junction and tight junction formation [START_REF] Lagaraine | Tight Ju tio P otei s Va i the Cho oid Ple us of E es a o di g to Photope iod[END_REF]Ikeda et al. 1999).

Tight Junctions and Signaling Cascades

Tight junctions of the blood brain barrier are heterogeneous multiprotein complexes, which associate themselves with an array of cytoplasmic and intermediate cytoskeletal proteins. As tight junctions maintain the endothelial barrier integrity, the cytoplasmic and intermediate cytoskeletal proteins maintain the bridge between the tight junction proteins with actin cytoskeleton. All these components are regulated by several signaling pathways.

These signaling pathways maintain the endothelial monolayer integrity through phosphorylation and dephosphorylation of different molecules at different check points which alter the tight junction and adherens junction proteins. Some of these signaling pathways have been discussed here.

Protein Kinase C (PKC) Signaling

PKC (includes a family of serine-threonine specific kinases) signaling pathway has been shown to influence the endothelial barrier. factor Epac in cultures neurons, plays a critical role in inflammatory pain (Hucho, Dina, and Levine 2005). In another study it has been shown that, 12-O-tetradecanoylphorbol-13-acetate (TPA) induces edema and leucocyte influx, which is regulated by PKC signaling during inflammatory responses in skin diseases (Passos et al. 2013).

TNF-α (10ng/ml) induced PKC isoenzyme activation triggers P115RhoGEF phosphorylation, which leads to the RhoA activation. RhoA activation promotes opening of the brain microvascular endothelial barrier (Peng et al.

2011).

Protein Kinase A (PKA) Signaling

PKA signaling is closely associated with regulation of the tight junctions proteins of the blood brain barrier. PKA activity in high when permeable cAMP levels are elevated in the endothelial cells. It has been shown in many studies that cAMP levels stabilize the tight junction proteins and decrease the permeability through an increase in the complexity of junctional strands (Raub 1996;Ishizaki et al. 2003;H Wolburg et al. 1994). Studies suggest that high levels of cAMP, acts as a stimulus and triggers activation of PKA signaling which alters the endothelial barrier integrity. In cell culture in vitro models, the brain endothelial TEER is as high as 100-Ω 2 , but it is increased when the cells are grown with astrocytes or in astrocytes conditioned media. Culture with astrocytes or in astrocytes conditioned media i reases as high as Ω 2 through an increase in the level of intracellular cAMP (Krause et al. 1991;Raub 1996). The tight junction proteins undergo damage by the oxidative stress and removal of extracellular calcium, although these deleterious effects are reversed by active PKA signaling. (M. Nilsson, Fagman, and Ericson 1996;Pérez et al. 2006). An in vitro stud i MDCK epithelial ells suggests that s during the development of tight junction proteins, Rab 13 (a GTPase), binds to PKA, which blocks PKA activity. This results in a delayed arrival of Claudin-1 and ZO-1 at the tight junctions (Köhler, Louvard, and Zahraoui 2004). PKA signaling also regulates the endothelial barrier functions by modulating the levels of pro-inflammatory molecules.

For instance, in peripheral blood mononuclear cell, high cAMP levels induce PKA signaling attenuates inflammation by inhibit the production of pro-inflammatory molecules (IL-2 and IFN-gamma) in the effect of μg/ l a d μg/ l of lipoi a id (Salinthone et al. 2010).

Exhibiting its protective role against inflammation, an in vitro study (in P3 primary rat microglia and immortalized murine microglia cells (BV2) suggests that cAMP/PKA signaling decreases LPS (100 ng/ml)-induced microglial activation, which is also observed with decrease in the mRNA expression of inflammatory chemokines and cytokines (Park et al. 2016). It is understood that many signaling pathways function with a co-activation with PKA signaling. For example, PKA signaling in coaction with CREB activation suppresses LPS-induced neuroinflammation in BV2 microglial cells (Jung et al. 2017). Although this should be noted that, the near nature of two or more coactivated signaling pathways and their effect on the blood brain barrier is debatable.

Protein phosphatases PP1, PP2A, and PP2B

These are group of serine-threonine specific phosphatases, which can cause change in phosphorylation states of some of the tight junction proteins thereby alter the endothelial barrier permeability. For instance, it has been shown in in vitro studies with Caco-2 and MDCK epithelial cells that the dephosphorylation of ZO-1, occludin and claudin-1 increases the epithelial paracellular permeability, which is mediated by serine-threonine protein phosphatase 2A (PP2A) and PKC signaling (Seth et al. 2007;[START_REF] Nunbhakdi-Craig | P otei Phosphatase A Asso iates ith a d Regulates At pi al PKC a d the Epithelial Tight Ju tio Co ple[END_REF]. It has been shown that, PP2A and PP2B interact with one of the PKC isoforms which is found close to tight junctions. This interaction leads in an alteration of the tight junction proteins. An in vitro study in bovine pulmonary artery endothelial cells suggests that PP2B activity increases under the effect of 100nM thrombin, which causes tight junction proteins dysfunction by the phosphorylation of myosin light chain proteins [START_REF] Verin | Role of Ca +/ al oduli -Dependent Phosphatase 2B in Thrombin-Induced E dothelial Cell Co t a tile Respo ses[END_REF]).

MAPK Signaling

Mitogen-activated protein (MAP) kinases are a group of serine-threonine protein kinases. The MAP kinase signaling is biphasic as it has a membranous and a cytoplasmic phases. In the primary phase, a small GTP binding proteins mediate the activation of the small membranous proteins. This activity is followed by sequential activation of kinases (MAPKs) in the cytoplasm (figure 8). This signaling cascade occurs in a systematic activation Key points: The functions of PP1, PP2A and PP2B in neuroinflammation are not entirely clear, though they play a regulatory role in maintenance of the paracellular permeability.

Key points: Hence, it is conclusive from these studies that cAMP/PKA signaling has a rescuing effect on endothelial barrier against neuroinflammation. This signaling pathway is also influences the blood brain barrier when it functions singly or in action with other signaling pathways.

of MAP3K followed by MAP2K, which finally activates MAPK by phosphorylation at serine and threonine residues.

MAPKs play an important role of activating a large population of cytoplasmic proteins. In addition, MAPKs also activate proteins of the nucleus and various transcription factors, which results in modulation of protein expression in the endothelial cells. There are several categories of MAPKs which functional in regulation of the endothelial barrier. It is well understood that that reactive oxygen species have a disruptive effect on the endothelial barrier integrity.

Oxidative stress in bovine lung microvascular endothelial cells (BLMVECs) can lead to an extended damage to the endothelial barrier functions which is prevented by thiols (Usatyuk, Parinandi, and Natarajan 2006). Oxidative stress can cause disruption of cadherin-beta-catenin complex. This damage is observed under ERK1/2 activation in cultured murine brain endothelial cells [START_REF] Krizbai | Effe t of O idati e St ess o the Ju tio al P otei s of Cultu ed Ce e al E dothelial Cells[END_REF]. In an in vitro study with brain-derived microvascular endothelial cells (BMEC) it is shown that hydrogen peroxide inflicts a similar kind of damage with activation of p38/ ERK1/2 MAP kinases activation under the oxidative stress. The activation of p38/ ERK1/2 signaling results in an increase in the paracellular permeability (FISCHER et al. 2005). In addition, another in vitro study in Human

RhoA/ROCK/MLC Signaling

RhoA/ROCK Activation

Ras homolog gene family, member A (RhoA) is a small GTPase protein of Rho family. RhoA is a small GTP binding protein that exists in GTP bound active or GDP bound inactive form, which is regulated by GTPase activation The Rho kinase is a serine-threonine kinase which has two members namely Rho-ki ase α/ROCK /ROK a d Rhoki ase β/ROCK /ROK . Rho-kinase activation influences the cellular contraction, motility, and paracellular permeability (L. Yao et al. 2010). An in vitro study in primary bovine pulmonary artery endothelial cells suggests that ROCK activation controls mechano-chemical processes in endothelial cells by exercising myosin light chain phosphorylation (Bhadriraju et al. 2007). The inhibition of ROCK activity reduces the inflammatory outcomes. In mouse microglial BV2 cells the suppression of ROCK activity by fasudil inhibits the reactive oxygen species (ROS) production and downregulates inducible nitric oxide synthase (iNOS). This inhibitory activity by fasudil therefore prevents neuroinflammatory outcomes (J. [START_REF] Chen | a. I hi itio of AGEs/RAGE/Rho/ROCK Path a Supp esses No -Specific Neuroinflammation by Regulating BV2 Microglial M1/M2 Polarization through the NF-κB Path a[END_REF]). An in vivo study suggests that RhoA/ROCK signaling induces vascular leakage under the effect of histamine, which results in anaphylactic shock (Mikelis et al. 2015). RhoA/ROCK signaling inhibitors such as fasudil have been shown to be effective as in a therapeutic approach for preventing inflammatory reactions. For instance, Y27632 is one well-known ROCK inhibitor.

Inhibition of ROCK activity downregulates the MLC phosphatase activation thereby regulates the cell morphology.

For instance, a recent study suggests that ROCK inhibition by L-F001 leads substantial depletion in neuroinflammation in in vitro and in vivo conditions (J. Chen, Yin, et al. 2017). Inhibition of ROCK and NADPH oxidase (NOX2) can be used and a potential treatment for neuroinflammation in in vivo conditions (Alokam et al.

2015).

It is worth noting that destabilization of the tight junction proteins can also be a result of internalization of proteins in form of vacuoles. It has been shown in an in vitro stud that IFNγ i du es for atio of large a ti oated vacuoles (VAC) originating from the membrane apices. These VAC internalize the tight junction proteins namely occludin, JAM-A and claudin-1, which induces endothelial hyperpermeability (Bogatcheva et al. 2006). GEF-H1 is the linking factor between RhoA activation and microtubule destabilization (Krendel, Zenke, and Bokoch 2002).

Thrombin or nocodazol mediated microtubule depolymerization induces GEF-H1 activation which results in RhoA activation which results in the downstream consequences of RhoA signaling pathway (A. A. Birukova et al. 2005;Krendel, Zenke, and Bokoch 2002). It has been shown that RhoA activation can be inhibited by the interaction of GEF-H1 with cingulin protein (cytosolic protein localized at tight junctions, interact with microtubules) (Aijaz et al. 2005;Benais-Pont et al. 2003).

Key points:

The common idea that can be taken from all these studies is that RhoA/ROCK signaling pathway plays a crucial role in endothelial barrier opening and an inhibitory control on this pathway evades the system from inflammatory outcomes. RhoA activation is the very first milestone of signaling, which triggers activation of many other signaling molecules which play an active role in modulation of the endothelial barrier.

Myosin Light Chain (MLC)

The Reorganization of actin proteins and alteration of tight junction proteins result in increase in paracellular permeability. It is a result of the overexpression of MLCK catalytic domain upon RhoA activation [START_REF] Hecht | E p essio of the Catal ti Do ai of M osi Light Chai Ki ase I creases Pa a ellula Pe ea ilit . The A e i a[END_REF]Shen et al. 2006). An increased expression of the catalytic domain of MLCK increases epithelial paracellular permeability [START_REF] Turner | Ph siologi al Regulatio of Epithelial Tight Ju tio s Is Asso iated ith M osi Light-Chai Phospho latio . The A e i a[END_REF]Zolotarevsky et al. 2002).

The activity of MLC2 proteins are driven by the association of these proteins with cytoskeletal elements. For instance, an in vitro study in T84 intestinal epithelial cells suggest that during the assembly of the apical tight junction protein complex, the involvement of polymerized actin and phosphorylation of MLC2 protein are two important requirements (A. I. Ivanov et al. 2005). In the same study, it has been shown that tight junction proteins are colocalized with N-WASP (neuronal Wiskott-Aldrich syndrome protein) myosin II and double phosphorylated MLC2 at threonine 18 and serine 19. It is observed that calcium promotes the arrival (assembly) of the tight junction proteins on the cell borders. Evidently, this assembly is blocked by several inhibitors such as wiskostatin and blebbistatin. Hence, we can conclude that phosphorylation of serine 19 residue of MLC2 is crucial as this leads to the contraction of actomyosin complex.

PKC signaling is also actively involved in MLCK activity. In an in vitro study with Caco-2 cells, it is suggested that PKC activation induces a progressive decay in MLC2 phosphorylation, which eventually restores the barrier integrity by increase in TER (Trans epithelial resistance) (Turner et al. 1999) influences NF-κB sig ali g resulti g i TNF-induced inflammation (X. Wu et al. 2009). The activation of RhoA and MLC phosphorylation leads to endothelial hyperpermeability induced by IL-β, hi h manipulates the f-actin protein morphology leading to an increase in permeability (L. Wu et al. 2016). AGEs/RAGE/RhoA/ROCK pathway inhibition by fasudil and FPS-ZM-1 evades the BV2 cells from non-specific neuroinflammation. This results in a decrease in M1 and increase in M2 microglial phenotypes (J. [START_REF] Chen | a. I hi itio of AGEs/RAGE/Rho/ROCK Path a Supp esses No -Specific Neuroinflammation by Regulating BV2 Microglial M1/M2 Polarization through the NF-κB Path a[END_REF]).

Cyclic AMP Response Element Binding Protein (CREB)

CREB is the first transcription factor, which was shown to be regulated by phosphorylation along with other members of the same family, such as activating transcription factor 1 (ATF1) and cAMP response element CREB family members also contain a basic leucine zipper dimerization domain located at its carboxyl terminal, which supports the dimer conformation of the protein during the transcription and provides stability. Followed by the leucine zipper, the basic residue domain interacts with the CRE DNA strand. The activation of CREB is a phosphorylation activity, which is a response to an increased cAMP signals. cAMP is an intracellular secondary messenger. This increase in cAMP level is an indicator of guanine nucleotide binding G protein coupled receptor activation. The increase in cAMP levels is then followed by increase in cAMP dependent protein kinase A (PKA) activity. Upon activation, PKA kinase passively diffuses from cytoplasm into the nucleus and phosphorylates CREB at its serine residue 133, as shown in an in vitro study in PC12 and F9 teratocarcinoma cells (Gonzalez and Montminy 1989). The active cAMP responsive genes mediate the activation of CREB, which allows the binding of the CREB on -TGACGTCA-, cAMP responsive element (CRE) [START_REF] Montminy | Ide tifi atio of a C li -AMP-Responsive Element within the Rat So atostati Ge e. P o eedi gs of the Natio al A ade of S ie es[END_REF]Comb et al. 1986). After the binding of CREB with CRE, the transcription of genes is seen at its peak in 30 minutes after stimulation with cAMP. In the time lapse of two to four hours CREB activity goes down. It is led by dephosphorylation of CREB by PP-1 and PP-2A protein phosphatase [START_REF] Wadzinski | Nu lea P otei Phosphatase A Dephospho lates P otei Ki ase A-Phosphorylated CREB and Regulates CREB Transcriptio al Sti ulatio[END_REF]Hagiwara et al. 1992).

In mechanism, CREB forms a dimer while binding to a palindromic sequence CRE with a nanomolar affinity (Kwok The CREB sig ali g a d protei ki ase C epsilo PKCε sig ali g path a s are also i ol ed i i fla ator a d neuropathic pain reactions. PKCε a ti atio upregulates the NF-κB, COX-1, and CREB levels. For instance, in pain and inflammatory in vivo models, 30mg/kg euphol (an anti-inflammatory tetracyclic triterpene) decreases the PKCε sig ali g and CREB levels, which inhibit the pro-inflammatory factors, and prevented the putative effect of prostaglandin E2 (PGE2) induced acute and persistent mechanical hypersensitivity in mice and rats (Dutra et al. 2015). Another in vivo study suggests that PKC zeta enhances lung inflammation in the effect of cigarette smoke/aldehyde and LPS. This effect is exhibited as PKC zeta activation followed by its translocation in nucleus and binding with CREB binding protein which leads to the phosphorylation and acetylation on promoters of proinflammatory genes which promote the lung inflammation (H. Yao et al. 2010).

TNF-α is a ell-studied pro-inflammatory factor known to have its mechanism of action via activation of NF-κB signaling in inflammation and its outcomes. An in vitro study in adenocarcinomic human alveolar basal epithelial cells (A549) and Human bronchial epithelial cells (BEAS-2B) suggests that TNF-α i reases the phosphorylation of CREB protein, which is inhibited by PKC signaling [START_REF] Catley | I hi ito s of P otei Ki ase C PKC P e e t A ti ated T a s iptio : ROLE OF EVENTS DOWNSTREAM OF NF-B DNA BINDING[END_REF]). In some studies, Ca

CREB Rho Signaling

In

Tissue Plasminogen Activator System

Tissue plasminogen activator system is comprised of group of active serine proteases proteins which are responsible for the fibrinolysis of the blood clots, and thereby regularization of the blood flow. Another important role of this system, which is seen most commonly, is in maintenance of the extra cellular matrix (ECM), which enables the cell migration and proliferation by allowing the cell processes to extend and grow. Two well-known plasminogen molecules have fibrinolytic activity. They are tissue Plasminogen Activator (tPA) and urokinase

Plasminogen Activator (uPA). Plasminogen Activator Inhibitor 1 (PAI 1) and neuroserpin (NS) inhibit the catalytic activity of these molecules.

Commonly known, as Alteplase (referred for commercial use, a form of recombinant tissue type plasminogen activator) is a premier therapeutic compound used in the treatment of ischemic stroke and hemorrhagic stroke.

tPA provides the proteolytic activity that is required for conversion of plasminogen into plasmin. domain exhibits very high affinity for fibrin, interaction with PAI-1 and neuroserpin, which act as tPA antagonists.

The fi ger do ai s high affi it to fi ri is e essar for the effi ie t fi ri ol ti a ti it . This do ai is also associated with microglial activation. The next domain composed of the amino acid residues 44 to 92, is called epidermal growth factor domain (EGF domain). Hepatic clearance is one of the important functions of this domain along with leading stabilization of catalytic site and binding to the EGF receptor. Liberatore et al. 2003). The finger domain and K2 kringle domain together form the interaction site for fibrin. This domain also interacts with tPA antagonists PAI-1, neuroserpin and Platelet-derived growth factor-CC as shown in in vitro studies (Sillaber et al. 1999;Yepes et al. 2000;Fredriksson et al. 2004). An in vitro study in HEK239 cells suggests that tPA consists a lysine-binding site, which plays a key role in the tPA promoted excitotoxicity (Parcq et al. 2013). The last domain is termed as B chain, which is composed of last 230 residues. The active sites of this domain are histidine 322, asparagine 371 and serine 478.

Sources of tPA in CNS

Adult human brain likewise an adult mouse brain has many sources of tPA. The mRNA expression levels show that there are many regions in brain, which are continuous source of tPA, such as cortex, cerebellum, amygdala, hippocampus, olfactory bulb, and thalamus (Cops et al. 2013;Sappino et al. 1993;Teesalu et al. 2004). Though studied in detail, expression of tPA in mouse hippocampus is associated with mossy fiber pathway, in which mossy fiber tPA activity showed a biphasic activity of increase in tPA activity at 8 hours followed by decrease in activity at 24 hours after the injury. This study exhibits a highly regulated mechanism of tPA release and activity (Sallés and Strickland 2002). Apart from the classical cells of CNS, tPA is also expressed by vascular endothelial cells throughout the brain parenchyma (Levin, Santell, and Osborn 1997;Teesalu et al. 2004).

It is also shown that neurons also express tPA, as seen localized on the growth cone during neuritogenesis, which facilitates the progression of cone (Shin, Kundel, and Wells 2004; Yamada, Spooner, and Wessells 1970). During the event of neuronal depolarization, tPA is released from axon terminals via calcium dependent pathway [START_REF] Parmer | Tissue Plas i oge A ti ato T-PA) Is Targeted to the Regulated Secretory Pathway. Catecholamine Storage Vesicles as a Reservoir for the Rapid Release of T-PA[END_REF]Gualandris et al. 1996). Moreover, there are many cells of CNS, which have shown tPA (either one or both of) expression and activity such as microglia (Rogove et al. 1999;Joo et al. 2010), oligodendrocytes and astrocytes (V. [START_REF] Vincent | Role of Ast o te-Derived Tissue-Type Plasminogen Activator in the Regulation of Endotoxin-Sti ulated Nit i O ide P odu tio Mi oglial Cells[END_REF]Hultman et al. 2008;Xin et al. 2011). Perivascular mast cells (Sillaber et al. 1999;Kose et al. 2007), pericytes (Moonen, Grau-Wagemans, and Selak 1982), and interestingly blood are also a reported source of tPA in CNS (Krystosek and Seeds 1981).

Key points: tPA is a structurally dynamic molecule which contains 5 structurally and functionally different domains. tPA interacts with the GluN1 subunit of NMDA receptor via its kringle 2 domain.

tPA in Neuroinflammation

There are many studies, which have suggested that tPA is a potential agent to mediate blood brain barrier opening.

tPA influences the blood brain barrier by the regulation of many molecular factors which play crucial role during inflammatory reactions such as cytokines, tight junction proteins and matrix metalloproteinases (MMPs). For instance, an interesting in vivo study with tPA -/-, Plg -/-, uPA -/-and MMP9 -/-mice (after 6 hours of MCAO) suggests that tPA (0.14-. μM) induces an increase in vascular permeability. In the same study it is shown that opening of blood brain barrier is mediated by the LDL (Low-density lipoprotein) receptor-related protein (Yepes et al. 2003).

Another study suggested in their in vivo and in vitro (co-culture of bovine brain endothelial cells and glial cells) models study that tPA crosses the blood brain barrier. However, this study also suggests that tPA (20µg/ml) alone tPA is a potent molecule to mediate the disassembly or disorganization of the neurovascular unit by inducing detachment of astrocytic end-feet projection. This phenomenon commences as the shedding of the astrocytic LRP re eptor i du es NFκB/AKT sig ali g and (X. Zhang et al. 2007;An et al. 2008), which leads to increase in MMP-9 levels destabilizes the cellular anchorage. These events together result in detachment of astrocytic end-feet (S.

does
Wang et al. 2006;Polavarapu et al. 2007). Interestingly, the conversion of PDGF-CC to PDGF-C is abolished in LRP1 -/-animals (Su et al. 2008), which indicates that PDGFR-α a ti ation is mediated by interaction of tPA with LRP1 on astrocytes.

In neuroendothelial cells, it has also been shown that tPA induces an upregulation of adhesion molecules From the studies mentioned here, we can conclude that tPA is a potential factor, which can lead BBB dysfunction.

tPA and Microglial Activation

tPA (independent of its proteolytic activity) also exhibits cytokine like activities, one of which is microglial activation mediated by tPA binding with membrane protein annexin II (figure 14) (Siao and Tsirka 2002). An in vitro study in glial cells (isolated form cerebral cortices of C57BL/6, tPA -/-, or Gal1 -/-mice) suggests that tPA ( μg/ L) induces glial activation which is mediated by ERK1/2 and Jnk pathways. This tPA activity is independent of its catalytic activity. This study also suggests that Annexin-2 and Galectin-1 play crucial role in tPA induced

tPA and Interaction with NMDA Receptor

The NMDA receptors are expressed on all regions of brain. The interaction of tPA with NMDA receptors is a crucial event in tPA related outcomes and neuroinflammation. tPA is long studied for its role in regulation of the extracellular matrix (ECM) clearance during neuronal growth. tPA activity via degradation of ECM provides the motility which is required for the extension and expansion of the growth cone in parenchyma. (Krystosek and Seeds 1981). It has been also confirmed that ECM degradation which is an essential phenomenon for cell proliferation and neuronal growth, is dependent on plasminogen activation system (Krystosek and Seeds 1981).

Along with this as function, tPA (20 µg/ml) proteolytically cleaves the GluN1 subunit of the NMDA receptor upon The mode and mechanism of action of tPA on NMDA receptors is debatable since tPA has been reported to exhibit the neuroprotective role in some studies whereas other studies have shown the delete (Matys and Strickland 2003;Vivien et al. 2003). For instance, in an in vivo study (tPA -/-and Plg -/-mice) it has been shown that tPA induced ischemic preconditioning (IPC) requires the proteolytic activity of tPA, and triggers plasmin induced NDMA receptor mediated Akt phosphorylation and exhibits delayed neuroprotection (Echeverry et al. 2010). The same study also suggests that in case of acute injury, the tPA activity is rapidly enhanced in the extracellular space.

This leads to LDL receptor mediated cell signaling which results in neuroprotection.

NMDA receptor itself serves interacting pocket for the activity of tPA. tPA interacts with GluN1 subunit of NMDA receptor via its kringle 2 domain because peptides of the kringle 2 domain of tPA prevents the tPA induced GluN1

cleavage. The same study suggests that tPA exacerbates NMDA induced calcium influx and neurotoxicity as shown in an in vitro study with neuronal cortical cell cultures (Lopez-Atalaya et al. 2008). An in vivo and in vitro study suggests that OGD (oxygen and glucose deprivation) conditions aggravate the passage of tPA across the blood brain barrier (López-Atalaya et al. 2007). Furthermore, it is notable from an in vitro study in primary cortical neuronal cultures suggests that NMDA receptor activity in neurons requires an activity of a co-receptor, which in turn enhances the calcium influx mediated by NMDA receptor [START_REF] Samson | Tissue-Type Plasminogen Activator Requires a Co-Receptor to Enhance NMDA Re epto Fu tio[END_REF]. From the previous studies, we can conclude that NMDA receptors play essential role in tPA mediated loss of function of BBB.

N-Methyl D-Aspartate (NMDA) Receptor

Excitatory neurotransmission is one of the crucial and vital mechanism carried out in brain and spinal cord by the amino acid glutamate [START_REF] Traynelis | Gluta ate Re epto Io Cha els: St u tu e, Regulatio , a d Fu tio[END_REF]). The calcium permeable ion channel receptors that have high regulatory role in excitatory neurotransmission via glutamate are ategorized as α-amino-3-hydroxy-5-methyl-4- The GluN1 subunit is one functionally crucial subunit expressed with certain specific differences as GluN1 isoforms. GluN1-a and GluN1-b isoforms are expressed in abundance in various regions of brain. As, GluN1-a is expressed in all hippocampus principle cells while GluN1-b is specifically expressed in CA3 layer (Laurie and Seeburg 1994).

The NMDA receptor heterogeneity is highly dependent on the expression of four GluN2 subunits in different regions of brain and the expression of these subunits varies chronologically. mRNA studies suggest that GluN1, GluN2A, GluN2B, and GluN2D are expressed in postnatal day 0, while GluN2C levels are detected at postnatal day 14 (Akazawa et al. 1994). These results are supported by the immunoprecipitation protein studies (Sheng et al. 1994), thus distinctive expression of subtypes of receptor contributes to its functional properties (Monyer et al. 1994). GluN2A expression is observed starts after birth and increases readily which is evident in every area of CNS in adult brain. The expression of GluN2B also increases at the same pace following birth, following the first postnatal weeks, after which the expression of this subunit is mostly restricted to the forebrain. On the other hand, expression of GluN2D drops steadily after birth and in expressed brain is expressed in very low levels in diencephalon and mesencephalon. Expression of GluN2C is confined to the regions of cerebellum and the olfactory bulb.

On the other hand, GluN3 subunits exhibit a slight different expression profiles. High GluN3A expression is observed in early postnatal life and steadily declines. GluN3B is expressed at very high levels in motor neurons and other regions. Studies suggest that GluN3A containing NMDA receptors exhibit regional and temporal expression. GluN3A dysfunction may contribute in neurological disorders since its expression specifically in the early stage of brain development shows its role in synaptogenesis and synapse maturation (Henson et al. 2010;Henson et al. 2010).

All the NMDA receptor subtypes are a combination of two obligatory GluN1 and a pair of GluN2 and/or GluN3 subunits. In fact, two forms of GluN1 (GluN1-a and GluN1-b) have also been reported (Sheng et al. 1994). This

NMDA Receptor Subunit Structure

The NMDA receptors have four different modules/domains structurally (shown in figure 12). They possess an extracellular region which expresses a large globular clamshell like domain. This domain exposes the N-terminal domain (NTD) which plays crucial role in allosteric regulation and assembly of subunits. The extracellular NTD is followed by antigen binding domain (ABD), which is joined by a linker strand. ABD is constitute of two discontinuous segments termed as S1 and S2. This domain serves as the binding domain for different agonists such as glycine (or D-serine) for GluN1 and GluN3A and glutamate for Glun2B subunits. This domain is then followed by a transmembrane domain termed as TMD, which has an ion selective filter contributed by three transmembrane helices and a pore loop (M2). The internal carboxyl terminal makes the carboxyl terminal domain CTD, which plays crucial role in the receptor trafficking, anchorage and transduction of the signaling pathways.

Information regarding the structure of NMDA receptor has been studied from the crystal structures of NTDs and ABDs which detail the binding and interaction domains for various agonists, ions and antagonists (Karakas, Simorowski, and Furukawa 2011;Furukawa et al. 2005). When the subunits of the receptors undergo assembly to form a tetramer, the NTDs and ABDs assemble as dimers, hence the receptor functions as dimers of dimers (Salussolia et al. 2011;Riou et al. 2012).

NMDA

Gating Ability and Ion Influx of NMDA Receptors

The basic functioning of the classical (conventional) ionotropic NMDA receptors relies on the influx of ions and re eptor s hara teristi se siti it to ards differe t io s. The su u it o positio highl i flue es the a ilit of Mg 2+ blockage and Ca 2+ permeability of the receptor. GluN2C and GluN2D subunits expressing heterodimeric subtypes have very low conductance, low sensitivity for Mg 2+ and low permeability for calcium ions.

On the other hand, a very high conductance, calcium permeability and high sensitivity to magnesium blockage is GluN2C enables signaling along with incomplete Mg 2+ blockade [START_REF] Schwartz | NMDA Re epto s ith I o plete Mg + Blo k E a le Lo -Frequency Transmission through the Cere ella Co te[END_REF]). Apart from GluN2, NMDA receptor subtypes expressing GluN3A subunits exhibit much stronger Mg 2+ ion blockade and least Ca 2+ permeability, and this effect, surprisingly, is prevalent in GluN1-GluN2B-GluN3A NMDA receptor subtype (Henson et al. 2010;Pachernegg, Strutz-Seebohm, and Hollmann 2012). The gating properties, agonist sensitivity and deactivation kinetics of receptor are the function of GluN2 subunits. GluN1-GluN2C and GluN1-GluN2D NMDA receptor subtypes show low probability of an open channel whereas GluN1-GluN2B shows relatively higher probability, which is even lower than GluN1-GluN2A. Among these subtypes, GluN1 and GluN2A expressing receptors have least sensitivity to the NMDA receptor agonists glycine and glutamate.

Dissociation kinetics of glutamate, which can also be termed as glutamate deactivation kinetics, determines excitatory postsynaptic current decay (EPSC) which is a key parameter for the regulation of integration of synapse and exercises gating. Hence, GluN2 subunits play crucial role in its regulation (Vicini 1998). The EPSC varies amongst different NMDA receptor subunits depending on the affinity of individual subunit for the agonist glutamate. For example, while GluN1-GluN2D has the slowest decay, it is fastest in GluN1-Glun2A subtype of NMDA receptors. In GluN2 subunits, most of extracellular domains (N-terminal domain and the agonist-binding domain) of the receptor are determinants of its probability of gating (Gielen et [START_REF] Ludwig | Ju tio al Adhesio Mole ule JAM -B Supports Lymphocyte Rolling and Adhesio th ough I te a tio ith α β I teg i[END_REF]H. Yuan et al. 2009). This NMDA receptor structurally possesses many small molecules ligand interaction sites that act as the negative or positive allosteric modulators, which result in functional variability of these receptors. There are many factors or molecules which naturally act as effective modulators of NMDA receptors. These can be protons, polyamine and Zn 2+ (Pierre Paoletti 2011). NMDA receptors are highly sensitive to cations as they interact with them via NTDs of the specific subunits where sensitivity varies from constitution of the subunits. For instance, Zn 2+ is highly specific for its interaction with Glun1-GluN2A receptors, as zinc ion acts as an antagonist. NMDA receptor subtypes expressing GluN1-GluN2A and GluN1-GluN2B both are highly responsive to the Zn 2+ (Rachline et al. 2005).

Interestingly, GluN2A is more sensitive to Zn 2+ as the ion concentrations as low as 1µM are sufficient for receptor to exhibit its effect (P Paoletti, Ascher, and Neyton 1997; Nozaki 2011). Along with zinc ions, the protons also influence NMDA receptors. Protons exhibit an inhibitory effect on GluN2B and GluN2D bearing receptor subtypes (Banke, Dravid, and Traynelis 2005). There are several pharmacological factors which can be used to identify a particular subunit expressed in NMDA receptor subtype. For instance, GluN1-Glun2B N terminal domain is effectively inhibited by a subunits specific drug ifenprodil (Karakas, Simorowski, and Furukawa 2011). Hence, a varied sensitivity of GluN1-GluN2A and GluN1-GluN2B receptor subtypes towards Zn 2+ and ifenprodil, is a classical way of discriminating between GluN2B and GluN2A expressing receptors. In case of heterotrimer GluN1-GluN2B-GluN2A, this receptor exhibits strong affinity for the interaction with Zn 2+ and ifenprodil, but the inhibitory effects of the antagonists are not as strong as observed in individual heterodimers of GluN1 with GluN2A and Glun2B

Involvement of NMDA Receptor in Disorders of CNS

Being an important entity to regulate the proper functioning of neurons, NMDA receptors are equally crucial in their roles in the neurological disorders of the central nervous system. NMDA receptors malfunctions lead to synaptic defects and other dysfunctions that lead to neurological disorders leading upon inflammatory reactions.

It is well known that high extracellular glutamate results in neuronal death (Rothman and Olney 1995). In the chronic cases of focal contusions and ischemic stroke, release of excitatory amino acids like glutamate can exacerbate the neuronal damage and glutamate antagonists can be potential molecules for treatment (Bullock et al. 1998). Most of the CNS disorders share a common pathophysiological condition i.e. inflammation and more often NMDA receptors have been reported to play crucial roles on progression of inflammatory reactions.

NMDA Receptors in Cerebral Ischemia

There are many studies which have established the role of NMDA receptors in cerebral ischemia. For instance to start with, NMDA receptor activation mediated neuronal death can be prevented via NMDA blockers in in vitro and in vivo models of ischemic stroke (Y. Liu et al. 2007; M. [START_REF] Chang | GEF-H1 Couples Nocodazole-Induced Microtubule Disassembly to Cell Co t a tilit ia RhoA[END_REF]). It is also evident that NMDA receptor activation mediated neuronal toxicity is highly dependent on NMDA receptor subunit, as GluN2B blocking inhibits Key points: Their specific agonists and antagonists are characteristic of NMDA receptor subtypes. It is also interesting to note that GluN3 expressing NMDA receptor subtypes show very low calcium permeability and low magnesium sensitivity. On the contrary, GluN2 subtype expressing exhibit high sensitivity to magnesium ion and high calcium permeability. In addition, NMDA receptors possess mobility, as studies have suggested that through the activation of certain signaling pathways NMDA receptors can accumulate at certain regions of the cells.

Key points: Neuroendothelial NMDA receptors may serve as a potential therapeutic targets. The inhibition NMDA receptor interaction site on its GluN1 subunit potentially prevents the inflammatory outcomes the neuronal damage whereas outcomes are not similar in case of GluN2A blocking (M. [START_REF] Chang | GEF-H1 Couples Nocodazole-Induced Microtubule Disassembly to Cell Co t a tilit ia RhoA[END_REF]Y. Liu et al. 2007). The attempts for the trials with NMDA subunits antagonists have not been successful as one of the reasons behind it has been the unavoidable side effects of these antagonists. Another constraint of a very small therapeutic window of these antagonists results in an excessive increase in the extracellular glutamate levels, which is a result of the reverse activity of NMDA receptors (Lai, Shyu, and Wang 2011). However, in animal ischemia models, NMDA receptor antagonists have shown a protective effect against neuronal damage. However, these protective effects are more effective when antagonists were administered prior to the onset of the stroke (M. [START_REF] Chang | GEF-H1 Couples Nocodazole-Induced Microtubule Disassembly to Cell Co t a tilit ia RhoA[END_REF]). Interestingly, these evasive effects are not seen in 30 mins or 3 hours after the onset of stroke as shown in the transient focal cerebral ischemia in normotensive rats (Margaill et al. 1996). NMDA receptors of capillaries regulate the blood brain barrier function and they can mediate the barrier opening following a cryogenic injury, as shown in an in vivo study (Koenig et al. 1992).

It has also been observed that disrupting the interaction between the NMDA receptors scaffold proteins domains and the associated signaling molecules can effectively reduce the neuronal cell death in stroke models. Interaction of GluN2B NMDA receptors with phosphatase, PSD-95 (postsynaptic density-95) and death-associated protein kinase 1 (DAPK-1) can increase the outcomes of NMDA receptor activation mediated neuronal cell death. When these protein interaction are blocked using synthetic peptides, the ischemic stroke driven brain damage is reduced (Aarts et al. 2002;Tu et al. 2010;Cook, Teves, and Tymianski 2012). Interestingly, similar results are observed when PSD-95 inhibitor NA-1(Tat-NR2B9c) reduces ischemic driven damage in human brain (Hill et al. 2012).

Although in the conditions of the traumatic brain injuries, the protective role of NMDA GluN2B antagonists is debatable (Beauchamp et al. 2008). A study suggests that hyperactivation of NMDA receptors which is likely because of the levels of extracellular glutamate, is short lived and is followed by a long lasting loss of function. On the contrary, an in vivo study in model of traumatic brain injury suggests that NMDA induced activation of receptor activation after 24 and 48 hours post-injury resulted in decrease in neurological deficits (Biegon et al. 2004).

To understand the role of NMDA receptor in neurological disorders, in an indirect way, the influence of the NMDA receptor agonists is also an important aspect during the progression of diseases and finally leading to inflammation. This fact has been established by several studies. Notably, an in vitro study in immortalized human brain endothelial cells (HBEC) suggests that upon NMDA receptor activation glutamate causes considerable decrease in cerebral endothelial cell barrier integrity which is exhibited as decrease in trans-endothelial electrical resistance (TEER) (Sharp et al. 2003). It has been established that blood brain barrier permeability increases in effect of topical N-methyl-D-aspartate (NMDA, NMDA receptor specific agonist that mimics the action of glutamate). The BBB transfer coefficient (ki) determined by 14C-alpha-aminoisobutyric acid shows that NMDA promotes blood brain barrier s i rease i per ea ilit , as shown on in vivo model of chronic hypertensive rats (Chi, Chang, and Weiss 1997). Interestingly, the effect and role of thrombolytic agents, and their interaction with NMDA receptor is also an efficient approach for prevention and evasion from the stroke and its outcomes. Tissue plasminogen activator (tPA), which has been discussed before and is employed in stroke treatment. Interestingly, in cortical neuronal cultures, tPA induces NMDA receptor signaling via its proteolytic activity (Nicole et al. 2001).

In addition, it has also been reported that in neurons, arginine 260 amino acid residue at the amino terminal domain of GluN1 subunit is crucial for tPA induced ATD cleavage and potentiation of NMDA receptor mediated signaling (Fernández-Monreal et al. 2004). Hence, a therapeutic strategy involving prevention of the interaction of tPA with NMDA receptors. In an in vivo study with model of intracerebral hemorrhage in rats, it has been shown that the αATD-GluN1 (anti-amino terminal domain of GluN1) antibody reduces the brain edema and neuronal death. Also with decrease in microglial activation, the possible outcomes of intracerebral hemorrhage are considerably reduced (Gaberel et al. 2013). These studies provide us enough evidence that NMDA receptors are actively involved in the pathological outcomes of stroke. In addition, it is also a potential therapeutic target for the treatment of stroke.

NMDA Receptors in Multiple Sclerosis

Similarly Multiple sclerosis, which exhibits inflammation as one of its pathological outcomes, is a mechanism driven by inflammatory cytokines and signaling cascades associated along. Interestingly, pharmacological inhibition of the NMDA receptors by classical antagonists amantadine and memantine results in substantial decrease in levels of pro-inflammatory cytokines, thereby decreasing the neurological symptoms of disease in EAE rats (Sulkowski et al. 2013). In a study ahead, it has been found that monoclonal antibody against the NMDA receptor can inhibit the interaction of the receptor with tissue plasminogen activator. This inhibition of interaction results in evasion from the pathological outcomes of EAE and preservation of the blood-spinal cord barrier and reduced leucocyte infiltration [START_REF] Macrez R 1 | Chevilley A 1[END_REF]).

These studies prove the crucial role of NMDA receptors in the simulated pathogenesis of multiple sclerosis (in EAE). The inhibitory action on the NMDA receptor activity can be an effective tool to prevent the neuroinflammatory outcomes of this neurological disorder.

NMDA Receptors in other Neurological Diseases

Widel studied Alzhei er s disease AD is so asso iated ith a d i flue ed NMDA re eptors. The produ tio NMDA receptors are universal in their expression and hence they possess a high probability to influence and mediate several neurological disorders. This unique property not only makes them one of the receptors of interest, but also lets us to explore them as potential therapeutic targets.

Non-conventional NMDA Receptors Metabotropic NMDA Receptors

The ionotropic mechanism of action in NMDA receptors is well known and under understood. However, eventually there have been many studies, which suggest that NMDA receptors may not behave as the ionotropic receptors.

For instance, it has been shown that upon glutamate binding, NMDA receptors signaling can be driven in the Ca 2+

influx independent mechanism and this mechanism is mediated by the conformational changes in GluN2 subunits of the NMDA receptors. This metabotropic mode of action is mediated by activation of p38 MAPK activation via NMDA receptors [START_REF] Nabavi | Meta ot opi NMDA Re epto Fu tio Is Re ui ed fo NMDA Re epto -Dependent Long-Te Dep essio[END_REF]. There are several studies, which have suggested the metabotropic NMDA receptor signaling which is coupled with Src family kinases. Src kinase activation equips pannexin-1 to form a signaling complex, which is accompanied by phosphorylation of tyrosine 308 residue in pannexin-1. Pannexin-1 is Key points: NMDA receptors on the cerebral endothelial cells play a crucial role during the blood brain barrier opening. Yet the exact mechanism by which these inflammatory outcomes are mediated by NMDA receptors is still unknown.

a member of proteins expressed in vertebrates and have homology with innexins, which are expressed in invertebrates. Pannexin-1 and pannexin-2 play critical role in cerebral ischemic damage, as it is evident from the study, which suggests that pannexin-1 and panenxin-2 knockout mice exhibited better functional outcome and smaller infarct when compared to the wild type mice upon ischemic stroke [START_REF] Bargiotas | Pa e i s i Is he ia-I du ed Neu odege e atio[END_REF]). These studies clearly reflect upon the non-classical metabotropic role of NMDA receptors, which lets us focus on further collection of more details and studies of these receptors.

GluN3 Subunit Expressing Subtype of NMDA Receptor

The NMDA receptor subtypes which exhibit the rare metabotropic mechanism of action are often categorized as non-conventional (non-classical) NMDA receptors. Expression of GluN3 subunits with GluN1 and GluN2 subunits alters the NMDA receptor activity. The non-conventional NMDA receptor subtypes express the GluN3 subunits as dimers of dimers (GluN1-GluN3) or heterotrimers (GluN1-GluN2-GluN3), and these receptors exhibit different biophysical and trafficking properties (figure 16). In addition to these characteristics, these receptors are associated with signaling pathways, which are independent of ion gradient.

GluN3 subunits are expressed by GRIN3A and GRIN3B located on human chromosome 9 (M Eriksson 2002) and chromosome 19 (Andersson et al. 2001) respectively. A protein functional expression studies in Xenopus oocytes, it has been found that GluN3A protein amino acid sequence shares sequence homology with different NMDA receptor subunits at different magnitudes. For instance, the GluN3A shares 57% of maximum homology with GluN3B. On the other hand, this shared homology is significantly less with GluN1 and GluN2 subunits as 27% and in the range of 24-29% respectively [START_REF] Ciabarra | Clo i g a d Cha a te izatio of Chi-1: A Developmentally Regulated Member of a No el Class of the Io ot opi Gluta ate Re epto Fa il . The Jou al of eu os ie e : the offi[END_REF].

The protein structure of the GluN3 subunits are very much similar to GluN1, and hold the interacting site for glycine or D-serine, and have nearly no affinity for glutamate (a characteristic agonist of GluN2 subunits). The differential affinity of GluN3A expressing receptors has been discussed ahead. GluN3 subunits of NMDA receptors share a discrete profile in terms of the receptor assembly and their biophysical properties. NMDA receptors expressing GluN3 subunits is rare and so are their functional characteristics. It has been found that GluN3B NMDA Key points: Metabotropic NMDA receptors are non-conventional receptors, which can induce activation of metabotropic signaling pathways.

receptor subtype is abundant in motor neurons. GluN3B subtypes expression has restricted expression in somatic motor neurons of the brain stem and spinal cord (Nishi et al. 2001).

On the other hand, GluN3A expressing NMDA receptors are widely distributed. A co-expression (GluN3A or GluN3B with GluN1) study in Xenopus oocytes, strongly suggests that these receptors are unresponsive for the classical NMDA receptor agonists glutamate or NMDA, while exhibit high response towards glycine. Contributing to the fact that GluN3 expressing receptors do not exhibit the characteristics of classical NMDA receptors, these receptors form Ca 2+ impermeable cation channels, which are also unresponsive to Mg 2+ , MK-801, memantine and competitive antagonists [START_REF] Chatterton | E itato Gl i e Re epto s Co tai i g the NR Fa il of NMDA Re epto Su u its[END_REF]).

GluN3 expressing NMDA receptors exhibit specific characteristics of assembly and receptor biology. It is known that in hippocampal neurons, assembly of GluN3B with GluN1 and GluN2A can reduce the Ca 2+ permeability of NMDA currents. GluN1 and GluN3B single transportation to the cell surface is not possible, but the co-expression of these subunits mutually support the NMDA receptor transport to the surface (Matsuda et al. 2003). A similar pattern of assembly and expression has been observed in GluN3A expressing NMDA receptors.

In a study by means membrane expression and trafficking it has been suggested that GluN3A co-assembles with GluN1-a and GluN2A to form a receptor complex and these receptors show relatively low calcium permeability.

GluN1 facilitate the GluN3A NMDA receptor subtype expression of the surface. This characteristic is also confirmed by the fact that Glun3A-Glu2A homodimers and GluN3A homomers were not detected on the surface and rather found accumulated in endoplasmic reticulum (Perez-Otano 2001).

GluN3A subunits influence the NMDA receptor function, specifically in the presence of this subunit largely influences NMDA currents. The single channel recordings from Xenopus oocytes reveal that GluN3A expressing NMDA receptors shows very small unitary conductance. In fact, GluN3A knockout mice had an enhanced NMDA response and increased dendritic spines in early postnatal cerebrocortical neurons, which suggests that GluN3A expression regulates the NMDA receptor activity and development of synapse [START_REF] Lipton | I eased NMDA Cu e t a d Spi e De sit i Mi e La ki g the NMDA Re epto[END_REF].

It is also interesting to note that GluN3A subunits also alter the Mg 2+ sensitivity, response to NMDA/glycine and NMDA receptor mediated excitatory postsynaptic currents (EPSCs). For instance, cultured GluN3A transgenic neurons exhibited two different NMDA receptor populations, in which Glun3A subunit constituted the NMDA receptors (composed of GluN1, GluN2 and GluN3), and these receptors exhibited a decrease in magnesium sensitivity, reduced calcium permeability when treated with glutamate/NMDA/glycine (Tong et al. 2007). There have been many studies, which have suggested the native NMDA receptors, expressing GluN1-GluN2A/B-GluN3A assemblies (Al-Hallaq et al. 2002;A. Nilsson et al. 2007;Burzomato et al. 2010). These features of GluN3A expressing NMDA receptors (decreased NMDA receptor unitary conductance, lower open probability and reduced magnesium sensitivity) are also considered to be characteristics of GluN3A containing receptors, and used for their characterization, comparison and differentiation (Sasaki et al. 2002).

In terms of expression, GluN3A subunit is often seen at its peak in early postnatal phase that declines in adulthood.

GluN3A is widely expressed in many different regions of brain. For instance, high GluN3A mRNA levels are observed in occipital and entorhinal cortices, thalamus, olfactory bulb and cerebellum (L. Sun et al. 1998;Al-Hallaq et al. 2002). Cultured hippocampal neurons, rodent cortical neurons and trigeminal motor neurons also express GluN3A positive NMDA receptors (Sasaki et al. 2002;Hirai and Yoneda 2004;Ishihama et al. 2005). HuGluN3A has also been detected in human fetal spinal cord and forebrain (Maria Eriksson et al. 2002). GluN3A expression in regions of brain decreases in the adult brain. Since the expression of GluN3A is classified to the time of brain development, there are many studies, which have suggested on the probable role of this subunit. The levels of

GluN3A proteins are at its peak, which suggests that GluN3A might play a role in neuronal differentiation, synapse formation and migration [START_REF] Ciabarra | Clo i g a d Cha a te izatio of Chi-1: A Developmentally Regulated Member of a No el Class of the Io ot opi Gluta ate Re epto Fa il . The Jou al of eu os ie e : the offi[END_REF]N J Sucher et al. 1995;H.-K. Wong et al. 2002). An internal pyramidal layer of large neurons form a V layer in neocortex. GluN3A is expressed in the V layer of the neocortex (Nikolaus J Sucher et al. 2003). It has been suggested that GluN3A might be involved in neuronal migration during cortical development and neurogenesis. In addition, (Nikolaus J Sucher et al. 2003) suggests that GluN3A (attributed to its ability reduce calcium influx via classical NMDA receptors) may have a neuroprotective role for the neurons of the V layer in primitive postnatal week. Expression of GluN3A has also been reported in cerebellar Purkinje cells and granule cells (Wee et al. 2008;Andersson et al. 2001). Expression of GluN3A and GluN3B is also observed in neurites, neuronal somata and on the postsynaptic synapse [START_REF] Lipton | I eased NMDA Cu e t a d Spi e De sit i Mi e La ki g the NMDA Re epto[END_REF]H.-K. Wong et al. 2002).

GluN3A expression is also reported in astrocytes in fetal and adult telencephalon, where GluN3A is believed to modulate the calcium waves, which facilitates glia signaling, extracellular glutamate release from astrocytes and reduce NMDA receptor mediated calcium influx in astrocytes (M.-C. Lee et al. 2010;Palygin, Lalo, and Pankratov 2011). Oligodendrocytes of the cerebellum and microglia in the cortex have also been observed to express GluN3A which may alter the magnesium block and other classical NMDA receptor functions (Karadottir et al. 2005).

GluN3A subunit shares much common features with GluN1 subunit when compared with GluN2 subunits. Glycine is one common agonist for GluN1 and GluN3A whereas GluN2 subunit bund with glutamate. Interestingly, studies with rodent ligand binding domain show that GluN3A has a very high affinity for glycine with Kd of 40nM, which is 650 times less that Kd for GluN1. Glutamate binds to GluN3A with a very low affinity (dissociation constant Kd = 9.6 mM) (Y. Yao and Mayer 2006). The receptors expressing GluN1-GluN3A exhibit much higher affinity for glycine binding than GluN1-GluN2 receptor subtypes [START_REF] Chatterton | E itato Gl i e Re epto s Co tai i g the NR Fa il of NMDA Re epto Su u its[END_REF]).

In addition, human GluN3A exhibits dissociation constant of 535 nM (A. Nilsson et al. 2007), which is comparably lesser than rodent Glun3A subtypes. These differences could be attributed to the difference of the full protein length varying among the species (Y. Yao and Mayer 2006). GluN1-GluN3A subtype of NMDA receptor exhibit glycine evoked currents. GluN1-GluN3A and GluN1-GluN3B co expressed receptors display small and a quick desensitization of glycine inducible currents, which are efficiently reduced by 6-cyano-2, 3-dihydroxy-7-nitroquinoxaline (CNQX) which prevents glycine binding with GluN3A subunit. In contrast, GluN1 specific antagonist MDL-29951 (MDL) which inhibits glycine binding with GluN1, does not inhibit glycine mediated evoked currents in GluN1-GluN3A subtype receptors (Madry et al. 2007).

D-serine is another glycine site agonist to NMDA receptors, in fact D-serine exhibits high affinity and strong binding kinetics with human and rat GluN3A (Y. Yao and Mayer 2006;A. Nilsson et al. 2007). On the contrary some studies have suggested that D-serine behaves as functional antagonist of GluN1-GluN3A receptors [START_REF] Chatterton | E itato Gl i e Re epto s Co tai i g the NR Fa il of NMDA Re epto Su u its[END_REF]). This functional discrepancy can be attributed to a fact that GluN3A subunit assembly with other subunits brings about the allosteric interactions that modulates ligand binding properties of receptor (Laurie and Seeburg 1994).

There are a few pharmacologically active antagonists against GluN3 subunits. GluN1-GluN3A expressing NMDA receptor subtypes exhibit nominal electrophysiological inhibition by AP5. MK-801 and memantine, which are classical NMDA receptor blockers [START_REF] Chatterton | E itato Gl i e Re epto s Co tai i g the NR Fa il of NMDA Re epto Su u its[END_REF]).

GluN1-GluN2-GluN3A heterotrimers can be distinguished from GluN1-GluN3A homodimers by AP5, as it suggested that AP5 blocks the small conductance by GluN1-GluN2-GluN3A (Sasaki et al. 2002). The heterotrimers are also blocked by GluN2 targeting antagonists, which also makes them detectable against GluN1-GluN2 NMDA receptor subtypes (Smothers and Woodward 2003). Ifenprodil, a GluN2B specific NMDA receptor antagonist, exhibits its blocking effect on the number of GluN2B subunits expressed in NMDA receptor complex (Hatton and Paoletti 2005).

Glun3A expressing NMDA receptors has possible association with protein phosphatase, cytoskeletal proteins and signaling pathways regulating receptor trafficking. GluN3A expressing NMDA receptor subtypes have been shown

to be associated with the several signaling pathways. GluN3A subunits contain a C terminal binding domain by which it interacts with protein phosphatase 2 (PP2). This interaction regulates synaptic activity, which involves dephosphorylation of GluN1 subunit at residue serine 897, which attenuates NMDA receptor single channel currents and decreases calcium permeability (Chan and Sucher 2001). GluN3A also modulates synapse maturation, GluN3A binds with a neuron specific intracellular adaptor protein PACSIN/synapdin1 via C terminus of the NMDA receptor subunit. This interaction recruits endocytic proteins dynamin and clathrin which promotes endocytosis of GluN3A containing NMDA receptors. PASCIN-1 interaction with GluN3A regulates the NMDA receptor replacement that which is followed by synapse maturation (Pérez-Otaño et al. 2006).

Interestingly, GluN3A also interacts with the cytoskeletal proteins that are associated with cell cycle and regulation of apoptosis. GluN3A binds with scaffolding protein plectin along PKC activation. In addition, it interacts with GPS2/AMF1, a regulatory factor in G protein signaling and MAPK pathways. The apoptosis regulatory proteins also interact with CARP-1 (cell cycle and apoptosis regulatory protein-1) (Maria Eriksson et al. 2007). Though the interaction of the cytoskeletal proteins with GluN3A is not entirely clear, but these interaction explain the cellular dynamic during synapse development and receptor replacement. The signaling factors and the signaling cascades associated with GluN3A may be involved with the role of GluN3A expressing NMDA receptor subtypes in various diseases.

GluN3A in brain injury

We can recollect from the previous studies that GluN1-GluN3A homodimeric and GluN1-GluN2-GluN3A heterotrimeric NMDA receptor subtypes are expressed in various cells of various regions and of the CNS such as neurons, astrocytes, and oligodendrocytes. These receptors exhibit minimal calcium ion permeability, lesser magnesium sensitivity. They respond positively via glycine evoked currents and glutamate activation. These properties are attributed to the pharmacological role of GluN3 expressing NMDA receptors. Though expression of GluN3A subunits with NMDA receptor is highly critical mechanism since the overexpression of GluN3A may lead to dangerous decrease in NMDA receptor currents [START_REF] Lipton | I eased NMDA Cu e t a d Spi e De sit i Mi e La ki g the NMDA Re epto[END_REF]Lipton 2006). A study using the GluN3A knockout and transgenic mice has revealed the neuroprotective role of GluN3A expression in retinal cells against apoptotic damage to cells (Nakanishi et al. 2009).

Key points: Though the exact role of GluN3 expressing NMDA receptors is not clear but their protective role has been shown promising against neurological disorders. Inclusive of these facts, the role of GluN3A NMDA receptors in inflammation is yet to be explored, although their association with cytoskeletal proteins may imply on their probable role in inflammation.

To summarize this review of literature we can pin down following points:

• A healthy blood brain barrier is a function of integrated tight junction proteins and signaling pathways, which is maintained via support of the other components of the blood brain barrier.

• • In the event of mechanical and chemical shock and stress, the tight junction protein ZO-1 seeks accumulation in nuclei.

• CREB-RhoA signaling pathway is an arguable signaling pair, since CREB pathways alter the gene expression on transcription level. This mechanism often supports the evasion from the RhoA mediated cell-to-cell damage. Other the other hand, in some studies the opposite effect of CREB signaling is questionable.

• In addition, tissue plasminogen activator is a potential factor that can induce and promote the blood brain barrier damage driven inflammation. tPA can aggravate inflammatory outcomes by prompting cellular infiltration and microglial activation.

• NMDA receptors, glutamate ionotropic receptors of the CNS, may serve as the connecting link for the tPA driven inflammatory outcomes.

• More interestingly, a special category of metabotropic endothelial NMDA receptors may be assumed to be involved in inflammatory reactions.

The hypothesis from the beginning of this study was inspired by the previous studies, which supported the probable role of neuroendothelial NMDA receptors in EAE mediated inflammation. A study suggests that the antibodies which prevent the interaction of tissue plasminogen activator and NMDA receptor reduce the damages on stroke (Macrez et al. 2011). In our first study we show that a therapeutic mouse monoclonal antibody Glunomab prevents the inflammatory outcomes by blocking activation of NMDA receptors [START_REF] Macrez R 1 | Chevilley A 1[END_REF]). Hence, the inhibition of this interaction shall be able to inhibit the progressive damage of neuroinflammation.

As discussed in the introduction, tPA is a potential molecule that (despite of being a therapeutic agent for stroke treatments) causes adverse effects by inflicting hemorrhages, leakage following the pathophysiological effects of neuroinflammation (Vivien et al. 2011)(Benchenane et al. 2005). From the previous studies it is evident that tPA influences the BBB via its possible interaction with NMDA receptor (Nicole et al. 2001). According to our hypothesis, endothelial cells express active NMDA receptors, which act as the interacting and activating pocket in presence of TPA. These interactions thereby may trigger some signaling cascade inside the cells figure 17.

Figure 17. The hypothesis of this study. According to our hypothesis, neuroendothelial NMDA receptors can trigger one or several signaling cascades, which may or may not depend on the calcium ion concentration for activation. These signaling pathways may affect the endothelial tight junctions and cause increase in endothelial barrier permeability. This increase in permeability can be a result of alteration of the tight junction proteins and adherens junction proteins.

In this study, whether this activation of receptor and eventual signaling cascade is calcium ion influx dependent or independent will be another interesting fact to know. This fact would let us understand the real nature of neuroendothelial NMDA receptors. Interestingly, NMDA receptors may behave as conventional ionotropic or non-conventional metabotropic NMDA receptors. Another, aspect of our hypothesis is the signaling cascade may play as the key modulator of the integrity of blood brain barrier by influencing the tight junction proteins that maintain the barrier junctions intact. On the basis of the evidence put forward to us by previous studies (Reijerkerk et al. 2008)(MacRez et al. 2011) , we decided the following objectives.

Study 1. : Neuroendothelial NMDA receptors as therapeutic targets in experimental autoimmune encephalomyelitis

 To understand the interaction of Glunomab with NMDA receptor.

An ex vivo study to determine whether or not cerebral endothelial cells express NMDA receptors

We checked the NMDA receptor expression via Glunomab, a monoclonal antibody developed against NMDA receptor subunit GluN1.

 To determine the effect of Glunomab antibody on EAE mediated cell infiltration in blood spinal cord-barrier.

EAE is often characterized by the infiltration of immune cells in the parenchyma leading edema and hypoxia induced cell damage. Hence, since the glunomab antibody was directed towards the NMDA receptors, we decided to check the counter effects of glunomab binding with NMDA receptor.

Study 2. : Neuroendothelial NMDA Receptors as Modulators of Blood Brain Barrier: Mechanism of Action and Interaction with tPA.

 Characterization of neuroendothelial NMDA receptor subunit composition.

The subunits expressed in specific NMDA receptor subtype determine the basic function and nature of the receptor. Expression of the functional subunit GluN1 was reported in study 1.

 Characterization between the proximity of NMDA receptor expression with tight junction and adherens junction proteins.

Previously mentioned in (Macrez et al. 2016), the colocalization studies shall reveal, to which protein of endothelial junction the NMDA receptors are most closely associated with. This also will provide as tentative target of alteration upon NMDA receptor activation in endothelial cells.

 Effect of inflammatory like conditions on neuroendothelial NMDA receptor expression.

Ideally, an increase in surface expressed adhesion molecules aggravates the inflammatory outcomes. This will provide us an additional information about the nature of expression of neuroendothelial NMDA receptors.

 Impact of a common NMDA receptor agonist on blood brain-barrier permeability.

GluN2 subunits are most commonly expressed NMDA receptor subtypes, which are largely responsive to glutamate and NMDA (an NMDA receptor agonist that mimic the pharmacological activity of glutamate).

 Impact of an uncommon NMDA receptor agonist on blood brain-barrier permeability.

GluN3 expressing NMDA receptors are regarded as non-conventional non-ionotropic NMDA receptors. Since their role in inflammation is unknown, it will be interesting to explore the probable role of GluN3 expressing NMDA receptors in opening of the blood brain barrier.

CONTEXT OF THE STUDY

In recent years, tPA and its effects on the blood brain-barrier have been in highlight because of tPA being It has been observed that tPA controls the neurotoxicity via binding and cleaving the GluN1 subunit of NMDA receptor (Liu et al. 2004;Benchenane et al. 2007). The cleaving of the ATD domain of the GluN1 subunit of NMDA receptor by tPA leads to the NMDA induced neurotoxicity [START_REF] Lopez-Atalaya | To a d Safe Th o ol ti Age ts i St oke: Mole ula Re ui e e ts for NMDA Receptor-Mediated Neu oto i it[END_REF]).

In addition, interestingly, the severe outcomes of the hemorrhagic and ischemic stroke could be highly reduced by the antibodies that could prevent the interaction of tPA and NMDA receptor (Macrez et al.

2011).

HYPOTHESIS

The previous studies have lead us to hypothesize that disease progression in the EAE could be effectively prevented by the immuno-intervention. In this study, we tested a strategy of immuno-intervention aimed at blocking the tPA-induced potentiation of endothelial NMDA receptor function. Interestingly, this strategy led to prevention of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). We then show that this therapeutic effect occurs via a direct action on endothelial cells, preventing the alteration of the blood-spinal cord barrier, resulting in a dramatic reduction of immune cell entry into the nervous system and a subsequent preservation of myelin.

MATERIALS AND METHODS * C57BL6/J, male, 8 weeks old mice were used for tissue sampling. These tissues were chiefly used for immunohistochemistry studies. The anti-GluN1 antibody (Glunomab, 1:500) staining was observed in these tissues with the positive control of GluN1 staining in the neurons. In another sets, the sections were incubated for overnight at ˚C ith goat pol lo al a ti-GluN1 antibody, rabbit monoclonal anti-claudin-5 antibody, rabbit monoclonal anti-occludin antibody, rabbit monoclonal ZO-1 antibody, goat polyclonal anti-collagen IV antibody, rat monoclonal anti-podocalyxin antibody and rat monoclonal anti-CD31

antibody. The tissues were later incubated with respective fluorescent secondary antibodies. The slides were mounted with DAPI containing protective medium to prevent fading.

The images were captured with Leica SP5 confocal microscope at immersion mode with 100X objective.

All the images were captured in 1024 X 1024-pixel resolution with the z-step of . μ . The digital data have been processed and analyzed in image processing software ImageJ and Imaris.

KEY RESULTS

 Glunomab®, a monoclonal antibody directed against NMDA receptor, binds to the receptor in the vicinity of tight junction proteins in endothelial cells of the blood spinal-and blood brain-barrier.

 The profile of binding of this antibody with NMDA receptor also confirms that NMDA receptors are expressed on the luminal side of the blood vessel.

 The monoclonal antibody Glunomab prevents the migration of leucocytes across the BBB.

 The progression of the pathological symptoms in EAE model is ceased when the monoclonal antibody Glunomab is administered against the NMDA receptors.

* The following experiments were conducted as the contribution to this study.

Context of study

The blood brain-barrier (BBB) is equipped with highly specialized endothelial cells that form the continuous barrier between the CNS parenchyma and the circulating system. The BBB is composed of endothelial cells which possess well-constructed tight junction proteins and lack fenestrations. The BBB maintains the CNS homeostasis by the regulation of metabolites and the gases. Not only it forms a strong impermeable barrier for the pathogens but also it makes it impregnable for the therapeutic drugs. The latter feature implies harm to the CNS and most importantly in the therapies of neurological disorders.

Still with regard to all these characteristics, the blood brain-barrier is primary and a soft target to the progression of the pathophysiological symptoms in many neurological disorders.

The tissue plasminogen activator has been long studied for its role in the manipulation of blood brain- NMDA receptors expressed in brain endothelial cells play an instrumental role in blood brain barrier

opening. An in vitro study in endothelial cells suggests that GluN1 regulates tPA induced signaling pathways and controls the endothelial monolayer permeability for monocytes (Reijerkerk et al. 2010).

In our previous study Macrez et al. 2016, we have shown the critical role of neuroendothelial NMDA receptors in neuroinflammation during multiple sclerosis. We also show the therapeutic potential of the strategies targeting the protease regulated site of NMDA receptor. However, the mechanism by which NMDA receptor mediates the opening of the blood brain-barrier, remains unclear.

In this study, we show the mechanism which explains the speculated role of neuroendothelial NMDA receptors in neuroinflammation. In addition, this study will also put forward the influence of NMDA receptor agonists on opening of the blood brain-barrier. This study will also bring attention to a nonconventional role of NMDA receptors in brain endothelial cells.

Materials and Methods

Cell Culture

The hCMEC/D3 cell line was kind present from Dr. P. O. Couraud from the Institute Cochin, Université René Descartes, Paris, France. The hCMEC/D3 cells were cultured from passage 29-34 as per the guidelines provided previously (Weksler, Romero, and Couraud 2013). The cell culture dishes and tanks were coated with 15 μg/ml Cultrex® Rat Collagen I, overnight under standard cell culture parameters. Post-seeding the cell cultures were maintained in proprietary Endothelial Growth Medium-2 (EGM-2) provided by Lonza ((Walkersville, MD). The media was supplemented with 2.5% Fetal Bovine Serum, 0.5 ml human recombinant Epidermal Growth Factor (hEGF), 2.0 ml human Fibroblast Growth Factor -Basic with heparin (hFEF-B), 0.5 ml Vascular Endothelial Growth Factor (VEGF), 0.5 ml Ascorbic acid, 0.2 ml Hydrocortisone, 0.5 ml Insulin like Growth Factor, 0.5 ml Gentamycin and Amphotericin. The cells were g o u de ˚C a d % CO2. Fresh media was added on the 3 rd day of culture and cells were passaged and used for experiments at approximately 90%-100% confluence.

Animals

The animals used for the histology were all male C57BL6/J WT, 8 weeks old, weighing 20-30 gm, housed in temperature controlled environment on a 12 hour dark/light cycle with access to food and water ad libitum facilitated by the local conventional animal facility (CURB, University of Caen). The guidelines respective omissions of primary or secondary antibodies in singular-immunostaining were used to detect unspecific labelling.

Immunocytochemistry

HCMEC/D3 cells cultured on chamber slides were incubated with ice-cold 2% paraformaldehyde for 10 minutes. The fixative was carefully removed and cells were rinsed with PBS buffer (pH 7.4) for three times at room temperature. The cells were then permeabilized with PBS buffer with 0.1% triton (100X) for 10 minutes. Followed by three rinses with PBS the cells were blocked with PBS buffer with 1-2% BSA for 30 i utes at oo te pe atu e. The ells e e i u ated o e ight at ˚C ith p i a a ti odies rabbit polyclonal anti-VE Cadherin antibody ab33168, iFluor 488 reagent Phalloidin cytopainter ab176753, moue monoclonal anti-GluN1 Glunomab [START_REF] Macrez R 1 | Chevilley A 1[END_REF]), rabbit polyclonal anti-GluN2B sc9057 antibody and rabbit polyclonal anti-GluN3A 07-356 antibody) diluted in PBS. After three rinses with PBS, the cells were incubated for 90 mins at room temperature with secondary antibodies (1:1000). Post-incubation the cells were rinsed thrice with PBS, cells were mounted with DAPI containing protective medium under the coverslip and slides were stored in dark. The cells devoid of any primary antibodies were used as negative controls.

Imaging and analysis

BBB Permeability assay

To the understand the effects of glycine and NMDA in presence of tPA on endothelium, the permeability of hCMEC/D3 monolayer was assessed under the treatments w.r. was assessed by collecting samples of SFM from the lower compartment of the transwell at the different time points (5, 10, 15, 30, 45, 60, 90, 180 minutes). The fluorescence intensity was captured using BIOTEK FLx800 microplate reader, excitation 485 nm and emission 520 nm.

Statistical analysis

Colocalization statistics have ee des i ed i Pea so s oeffi ie t , hi h is a esti ate of the goodness of an approximate value of relationship between the intensities of two images. The value ranges from -1 to 1, exhibiting total negative correlation and positive correlation respectively, and zero for no

Results

NMDA receptors expressed in endothelial cells of CNS are composed of two different subunits with GluN1.

In the beginning, we analyzed the mouse spinal cord tissue for the expression of the NMDA receptor subunits. Figure 18 shows the expression of the subunits in blood vessels of the CNS. We observed positive immunostaining with Glunomab with in the microvessels of the spinal cord tissue. We show that these

GluN1 positive blood vessels also exhibit positive staining for GluN2B (fig. 18A) and GluN3A (fig. 18C). The 3D reconstruction with IMARIS software and the correlation studies with ImageJ software we show that GluN2B (fig. 18B) and GluN3A (fig. 18D) subunits are expressed in complete co-localization with GluN1 subunit. In addition, in fig. 18F, Table 1 we sho the deg ee of olo alizatio i te s of the Pea so s

Monolayer transwell permeability

In this part of our study, we checked the permeability of 70kDa FITC dextran across 0.4 microns pore size filter seized transwell, on which the endothelial cells were allowed to reach confluency for 7-10 days (Poller et al. 2008). Thereafter the stimulated cells were treated with 25 μM NMDA and, with 2 μg/ml tPA. In our data (in fig. 23A), we show that under the effect of NMDA-tPA treatment, the relative permeability of the monolayer for 70kDa FITC dextran is increased by 5 folds (n=3, *p < 0.05, **p < 0.005), w.r.t control. In fact, we observed minimal increase is permeability under the effect of tPA and NMDA individually. This information coincides with our previous data on MLC activation, as phosphorylated MLC levels are intact and considerably high even in third hour of treatment.

Morphological changes in cellular structure

MLC activation is often attributed to the change in cell morphology, which is mediated by the retraction of the cytoskeletal components. This leads to the contraction of cells and subsequent loosening of cell-tocell contacts. Hence, we analyzed the f-actin cytoskeletal protein in endothelial cells after NMDA-tPA treatment. We show that NMDA-tPA co-treatment induces an increase in the paracellular permeability.

We performed immunocytochemistry for f-actin, on the hCMEC/D3 monolayer (grown to confluency).

These cells were stimulated and further treated with 25 μM NMDA with 2 μg/ml tPA for 3 hours (since, maximum permeability across the endothelial monolayer was observed at 3 hours point in our transwell experiments). In this part of our study, we observed the f-actin staining by phalloidin tagged with FITC.

For better interpretation of data, we performed the 3D reconstruction of the f-actin staining with IMARIS (shown in fig. 23B). We processed the control and NMDA-tPA treated cells images on the same threshold of signal intensity. Interestingly, we found that endothelial cells (which form the monolayer) become slender or thin in appearance, loose down cell-to-cell contacts, which is also visible with increasing distance between the cell borders.

Previous part of this study provided many prerequisites for the following study.

CREB Activation

In this part of the study we studied the role of glycine as the GluN3A agonist on endothelial NMDA receptors for which we checked the CREB activation at 1 and 3 hour after glycine and glycine-tPA

Monolayer transwell permeability

Moving forward, we checked the effect of glycine and glycine-tPA on endothelium permeability for which we conducted a transwell permeability study. The cells were treated with 25 μM glycine and, 25 μM glycine with 2 μg/ml tPA until 3 hours. The media samples were collected form the lower chamber at the finite time points (5, 10, 15, 30, 45, 60, 90, and 180 minutes). In our data (fig. 26A) we show that tPA in presence of glycine increases the relative monolayer permeability by four folds, whereas there is nearly two folds increase is permeability in effect of only glycine(n=3, *p < 0.05, **p < 0.005).

Morphological changes in cellular structure

With evident RhoA activation and substantial increase in endothelial monolayer permeability, we checked the change in morphology of endothelial cells upon glycine-tPA treatment. We performed immunocytochemistry for f-actin on the hCMEC/D3 monolayer treated with 25 μM glycine with 2 μg/ml tPA for 3 hours (since, maximum permeability across the endothelial monolayer was observed at 3 hours point in our transwell experiments). In our data (fig. 26B), we observed the f-actin staining and the 3D reconstruct of images shows that post glycine-tPA treatment the endothelial cells lose cell-to-cell contact.

This increases the relative distance between the cells and which results in an increase the monolayer permeability.

Discussion

Our current study reports that neuroendothelial NMDA receptors show the characteristic expression of non-conventional non-ionotropic NMDA receptors. The non-conventional NMDA receptors are a heterodimer (GluN1-GluN3) or a heterotrimer (GluN1-GluN2-GluN3) NMDA receptor subtypes. In accordance with our data, we confirm that cerebral endothelial cells express GluN1, GluN2B and GluN3A possessing NMDA receptors. Our correlation study suggests a strong colocalization of GluN2B and GluN3A with GluN1 subunits in brain microvessels. Our findings will provide more details to the studies which have shown the expression of GluN1 subunit on brain endothelial and suggested the role of NMDA receptor in monocyte transmigration and immune cell infiltration across the blood brain barrier (Reijerkerk et al. 2010;[START_REF] Macrez R 1 | Chevilley A 1[END_REF]). Though the technical constrains have held us from finding out if these receptors in fact are GluN1-GluN2B-GluN3A expressing heterotrimer or the GluN1-GluN2B and GluN1-GluN3A expressing homodimers. Yet we can confirm that these receptors do express the three different subunits.

In this study we also confirm that NMDA receptors of endothelial cells are expressed in a very close vicinity of transmembrane tight junction protein occludin and claudin-5, which are expressed at the apical end toward the lumen of the microvessels. The ag itudes of the Pea so s oeffi ie ts suggest that positive correlation of NMDA receptor with ZO-1 and VE-cadherin is comparatively less than that of occludin and claudin-5 with the receptor. This new finding shall be supportive of studies which have suggested the regulatory role of NMDA receptors in blood brain barrier via modulation of the tight junction proteins. [START_REF] Beard | No -Muscle Mlck Is Required for -Catenin-and FoxO1-Dependent Downregulation of Cldn5 in IL-1 -Mediated Ba ie D sfu tio i B ai E dothelial Cells[END_REF]Schwartz 2004). After the analysis of f-actin staining in endothelial monolayers, we confirm that NMDA-tPA and glycine-tPA treatments induce morphological changes in endothelial cells along with loosening of the cell-to-cell contacts.

In some studies it has been shown that RhoA signaling can induce CREB phosphorylation and thereby regulate the transcription levels (Kimura et al. 1998;Sordella et al. 2002). It has also been suggested that CREB and RhoA signaling activation leads to the alteration of microvascular integrity (Zhong et al. 2012b).

However, the mutual dependence of either of the CREB abd RhoA signaling pathways is not clear yet.

In this study we also suggest that NMDA-tPA and glycine-tPA co-treatments induce opening of the blood brain-barrier. Our permeability assay confirms that the endothelial monolayer is compromised in presence of NMDA receptor agonist with tPA. NMDA and tPA co-treatment trigger the opening of the blood brain barrier. Interestingly, glycine alone induces the increased permeability across endothelial unilayer. However, tPA accelerates the glycine induced opening of the blood brain barrier since the unilayer permeability increases at a faster rate. It is also known that tPA mediated inflammatory outcomes can be prevented by the therapeutic approach of blocking the NMDA receptor. Adding more to detail, this study will provide information to understand the role of NMDA receptor agonists driven opening of the blood brain barrier and will establish more detail for neuroendothelial NMDA receptor activation and action (Reijerkerk et al. 2010;Macrez, Vivien, and Docagne 2015;Macrez et al. 2011;[START_REF] Macrez R 1 | Chevilley A 1[END_REF] With the confirmation of expression of GluN1, GluN2B and GluN3A subunits in brain endothelial cells, we also confirm that these receptors are nonconventional NMDA receptors and show metabotropic activity.

Firstly, AP5, a classical competitive antagonist of NMDA did not block the NMDA-tPA and glycine-tPA induced activation of CREB in endothelial cells. This brings our attention to an interesting study which suggests that non-conventional NMDA receptors can exhibit mechanoreceptor like response and these receptors do not respond even to very high doses of AP-5 [START_REF] Cahusac | A e U o e tio al NMDA Re epto s I ol ed i Slo l Adapti g T pe I Me ha o e epto Respo ses?[END_REF]. When GluN3 subunits are co-expressed with GluN2 NMDA receptor subtypes, GluN3 can suppress the classical receptor activity (Henson et al. 2010). Hence, the subsequent inability of AP-5 to inhibit the CREB and RhoA activation can be attributed to the change of activity of NMDA receptors. Secondly, glycine-tPA induced activity of CREB and RhoA was inhibited by the glycine antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Non-R E S U L T S conventional GluN3A expressing NMDA receptors show high affinity for glycine. Although it is also known that GluN1 subunit also exhibits affinity for glycine but it is far less than GluN3A (Madry et [START_REF] López-Atalaya | Re o i a t Des odus Rotu dus Salivary Plasminogen Activator Crosses the Blood-Brain Barrier through a Low-Density Lipoprotein Receptor-Related Protein-Dependent Me ha is ithout E e ti g Neu oto i Effe ts[END_REF]Madry et al. 2008;Balasuriya et al. 2014). Thirdly, we show that neither NMDA nor NMDA-tPA treatment triggered any calcium influx in endothelial cells. Similarly, neither glycine nor glycine-tPA induced any calcium influx. In addition, MK801, which is a NMDA receptor ion channel blocker did not inhibit neither CREB nor RhoA activation in endothelial cells. This indicated that NMDA-tPA and glycine-tPA driven CREB and RhoA activation in endothelial cells is independent of calcium influx. studies suggest that nonconventional NMDA receptors exhibit decrease in unitary conductance, very low calcium permeability and low magnesium sensitivity (Sasaki et al. 2002;[START_REF] Chatterton | E itato Gl i e Re epto s Co tai i g the NR Fa il of NMDA Re epto Su u its[END_REF]Kvist et al. 2013). These all characteristics confirm that neuroendothelial NMDA receptors are non-conventional NMDA receptors.

Conclusion

From

Endothelial cells express NMDA receptors in close vicinity of tight junction proteins

As we observed expression of GluN1 subunit in small vessels of brain, we confirm that NMDA receptors

Neuroendothelial cells express GluN1, GluN2B and GluN3A expressing subtypes of nonclassical metabotropic NMDA receptors

In our current study we have been successful to show that NMDA receptors expressed in cerebral endothelial cells are a GluN1, GluN2B and GluN3A expressing subtypes. These finding were consistent with the immunohistochemistry and protein studies. Moreover it also interesting to notice that GluN3A and GluN2B are expressed highly colocalized with GluN1 subunit.

Interestingly, GluN3A expressing NMDA receptors are categorized as non-classical NMDA receptors which exhibit their characteristic features a very low calcium permeability, low magnesium ion sensitivity and high binding affinity to glycine (Sasaki et al. 2002;Nikolaus J Sucher et al. 2003). Interestingly, it is also reported that GluN3A co-expression with GluN1 and GluN2 subunits considerably decreases the classical GluN2 subunit driven calcium influx. We confirm that neither the treatments with glycine nor NMDA could trigger a calcium ion influx on cerebral endothelial cells. This confirms that the NMDA receptors expressed in cerebral endothelial cells are non-ionotropic.

Notably, this finding falls in line with the study which suggests that the conformational changes in NMDA receptor contributes to its metabotropic mode of action without involving the influx of ions [START_REF] Nabavi | Meta ot opi NMDA Re epto Fu tio Is Re ui ed fo NMDA Re epto -Dependent Long-Te Dep essio[END_REF]. In another study it has also been shown that the inability of NMDA receptor for the ion influx could be a result of conformational changes, which promotes Aβ ediated sy apti depressio (Kessels, Nabavi, and Malinow 2013) which can be seen as the metabotropic effect these receptors. In addition, previous studies have also suggested that GluN3A expression in NMDA receptors promotes the inhibition of calcium influx (Al-Hallaq et al. 2002), which again favors the metabotropic mechanism of action by these receptors.

In addition, as we show in our study, NMDA receptor agonists induce CREB and RhoA signaling in brain Pro-inflammatory cytokine TNF-α upregulates the levels of GluN1 and GluN3A subunits in brain endothelial cells.

Furthermore, in our study we confirm that pro-inflammatory conditions promote the upregulation of GluN1 and GluN3A subunits in cerebral endothelial cells. Pro inflammatory conditions are implied by proinflammatory cytokines like tumor necrosis factor (TNF-α hi h a elerate and promote the blood brain barrier damage. An increase in expression of protein of interest is one of the strategy by which proinflammatory cytokines aggravate inflammatory outcomes. Although, the expression of GluN2B under the pro-inflammatory conditions remains unchanged.

An increase in ligand specific receptor expression promises an accelerated response upon ligand receptor activation. For example, TNF enhances ICAM-1 and VCAM-1 which enhances leucocytes adhesiveness to endothelial cells [START_REF] Min | TNF-Related Activation-Induced Cytokine Enhances Leukocyte Adhesiveness: Induction of ICAM-1 and VCAM-1 via TNF Receptor-Associated Factor and Protein Kinase C-Dependent NF-κB A ti atio i E dothelial Cells[END_REF]). Similarly, coinciding with our finding, expression of GluN1 enhanced in hippocampal neurons under the effect of pro-inflammatory cytokine TNF-α (Wheeler et [START_REF] Ludwig | Ju tio al Adhesio Mole ule JAM -B Supports Lymphocyte Rolling and Adhesio th ough I te a tio ith α β I teg i[END_REF]Shen et al. 2011).

Until here in our studies, we confirm that neuroendothelial cells express metabotropic type nonionotropic NMDA receptors. In addition, we show that these receptors are in a close vicinity of tight junction transmembrane occludin and claudin-5 proteins. We also confirmed that these NMDA receptors show no calcium influx activity and they express GluN1, GluN2B and GluN3A subunits. The in vitro data also confirms that pro-inflammatory conditions lead an increase in expression level of GluN1 and GluN3A subunits in hCMEC/D3 cells. From here we confirm that neuroendothelial NMDA receptors exhibit an increase in expression under proinflammatory conditions.

Neuroendothelial NMDA receptors mediate CREB activation

To further investigate the role of these GluN1, GluN2B and GluN3A expressing NMDA receptor subtypes in cerebral endothelial cells, we checked the effect of N-methyl D-aspartate (an NMDA receptor agonist that mimics the interaction of glutamate to GluN2B subunit of the NMDA receptor). Since NMDA receptors are known to trigger cAMP element-binding protein (CREB) signaling and associated signaling pathways (Sala, Rudolph-Correia, and Sheng 2000;Dudman et al. 2003). CREB activation facilitates NMDA receptor channel activity via phosphorylation of CREB protein at serine 133 residue. Hence, coinciding with previous studies, we also confirm that NMDA receptor activation in presence of NMDA and tissue plasminogen activator (tPA) triggers substantial CREB activation in cerebral endothelial cells. In the time span of three hours, we observed excessive levels of phosphorylated CREB (phospho serine 133) levels.

In addition, the activation exhibits a pattern of gradual increase in CREB phosphorylation with an increase in NMDA agonist levels in presence of tPA. We also show that NMDA causes NMDA receptor activation mediated CREB activation only in presence of tissue plasminogen activator. Since there was no apparent increase in phospho CREB levels by NMDA and tPA treatments separately, this shows that tPA is pharmacologically important for NMDA receptor driven CREB activation in endothelial cells.

Similarly, when we observed the effect of glycine (a strong GluN3A agonist, binds with very low affinity with GluN1) on phosphorylated CREB levels, we conclude that glycine alone causes an increase in phosphorylated CREB levels and this increase in CREB activation does not last until late 3 hours. Although.

tPA prolongs the CREB activation until 3 hours in the presence of glycine. and in vivo study it is suggested that tPA activates NMDA receptor via plasminogen independent mechanism that prevent neurons from the damaging excitotoxic effect. This protective role of tPA is D I S C U S S I O N observed via ERK1/2 activation mediated CREB activation in neurons (Wu et al. 2013). Unfortunately, since the effects of tPA during inflammation are more damaging than protective, hence, the role of NMDA-tPA and glycine-tPA induced CREB activation and its total role in inflammation is arguable.

We also show that, NMDA-tPA, glycine and glycine-tPA mediated CREB activation is uninhibited in presence of the calcium channel blocker, MK801. This information also supports our previous finding in calcium influx studies, which suggests that upon NMDA and glycine administration (with or without tPA)

there is no influx of calcium ions in cerebral endothelial cells.

As we suggest that these NMDA receptors exhibit no characteristics of classical ionotropic NMDA receptors. It is conclusive that the NMDA-tPA and glycine-tPA driven CREB activation is indeed a metabotropic signaling activity by GluN1, GluN2B, and GluN3A expressing NMDA receptors is independent of the calcium influx (Balasuriya et al. 2014;Pachernegg, Strutz-Seebohm, and Hollmann 2012). Another interesting thing to mention is, CNQX (6-cyano-2, 3-dihydroxy-7-nitro-quinoxaline, a selective antagonist against GluN3A binding to glycine) completely inhibits the CREB activation induced by glycine and glycine-tPA, which confirms that CREB activation is driven by interaction of glycine with glycine binding site on GluN3A. Noticeably, CNQX did not inhibit the CREB phosphorylation by NMDA-tPA.

This indirectly concludes two important points in regard of the NMDA receptor activity in endothelial cells.

Firstly, the inability of a glycine inhibitor to prevent the CREB activation clearly states that the CREB activation upon NMDA-tPA treatment is induced by NMDA agonist interaction with GluN2B subunit of NMDA receptor hence a glycine antagonist shows no effect. Secondly, since in some studies it has been suggested that CNQX is also an AMPA inhibitor (Morrell et al. 2008;Isa et al. 1996). Following from our data we can confirm that NMDA-tPA, glycine and glycine-tPA mediated CREB activation in not mediated by AMPA receptors. We can confirm so because in case it is driven by AMPA receptors, hence CNQX should inhibit the CREB activation irrespective of presence of the GluN2B agonist NMDA, GluN3A agonist glycine, and tPA in the system, which does not happen.

In addition, it is also interesting that AP5, an NMDA antagonist does not inhibit glycine and glycine-tPA mediated CREB activation, which is coherent with our conclusions that a GluN2B antagonist does not inhibit a GluN3A subunit driven mode of action. However, we find that AP5 does not inhibit the NMDA-tPA mediated CREB activation either. Surprisingly, the inability of AP5 to prevent the interaction of NMDA with GluN2B is disputable. In support of this phenomena, to the best of our knowledge, we bring attention to a very interesting study which suggests that non-conventional NMDA receptors can exhibit mechanoreceptor like response and these receptors do not respond even to very high doses of AP-5 [START_REF] Cahusac | A e U o e tio al NMDA Re epto s I ol ed i Slo l Adapti g T pe I Me ha o e epto Respo ses?[END_REF]). As we mention that the neuroendothelial NMDA receptors are indeed metabotropic receptors, hence they may possess a characteristic mechanoreceptor like features.

Activation of CREB signaling pathway with no exhibit of calcium influx explains the metabotropic mode of action of neuroendothelial NMDA receptors. Since, CREB activation and its role in blood brain barrier is an arguable matter, since we are unaware of the downstream targets of activated CREB.

Neuroendothelial NMDA receptors mediate RhoA/MLC activation

RhoA/ROCK/MLC signaling cascades are associated with the regulation of cell morphology, cell-to-cell contacts, and cytoskeletal arrangements. RhoA signaling activation can lead to opening of the blood brain barrier and blood spinal cord barrier (van Nieuw Amerongen et [START_REF] Van Nieuw Amerongen | A ti atio of RhoA Th o i i E dothelial H pe pe ea ilit : Role of Rho Ki ase a d P otei T osi e Ki ases[END_REF]Stamatovic et al. 2006). In our study we confirm that NMDA mediates an increase in levels of GTP bound RhoA protein in presence of tPA. Similarly, glycine and glycine-tPA induce RhoA activation, observed with an increase in levels of GTP bound RhoA protein. Interestingly, tPA accelerates the blood brain barrier opening induced by glycine.

This can be explained since tPA can show cytokine like effect and promote the opening of the blood brain barrier (J. Wang et al. 2014). Moreover, tPA compromises the blood brain barrier integrity even to promote cellular permeability across the endothelial barrier (Reijerkerk et al. 2008;Yepes et al. 2003).

Although an exact mechanism by which tPA mediates its damage to blood brain barrier remains unclear.

As we confirm that tPA promotes RhoA activation in presence of NMDA receptor agonists. This finding coincides with studies, which suggest that tPA activates Rho kinases activity and signaling. For instance, tPA alters the astrocytic cytoskeletal protein f-actin and increases myosin phosphorylation, which thereby increases the blood brain barrier permeability. This effect is driven by RhoA/ROCK pathway, which is inhibited by the ROCK inhibitor Y27632 (Niego et al. 2012). Also, statin induced Rho signaling inhibition prevents from deleterious effects of tPA stimulated MMP-9 (S. Wang et al. 2006).

In our study we also confirm that RhoA activation by NMDA-tPA, glycine and glycine-tPA, lead the activation of MLC (myosin light chain) proteins. In these treatments, we confirm that the level of phosphorylated MLC protein is increased. Phosphorylation of MLC proteins brings out the conformational changes in cellular structure because the cytoskeleton undergoes rearrangement, which leads to changes in cell-to-cell contact. MLC activation causes the actin-myosin fibers to retract which causes cell contraction (Goldberg et al. 2002;Mckenzie and Ridley 2007).

Furthermore, we suggest that upon RhoA/MLC activation by NMDA-tPA and glycine-tPA, the cerebral endothelial cells undergo conformational changes, a 3D reconstruction of f-actin fiber arrangement in endothelial monolayer clearly shows that endothelial cells lose more cell-to-cell contact and attain a slender conformation. In addition, we confirm that the NMDA-tPA and glycine-tPA treatments compromise the blood brain barrier integrity. This effect is evident from an increased endothelial unilayer permeability. These findings are supported as, RhoA signaling and its components regulate the endothelial

Conclusion

From our current study, the metabotropic role of GluN3A expressing NMDA receptors has been brought in light. Neuroendothelial NMDA receptors mediated CREB and RhoA activation. GluN2B agonist NMDA and tissue plasminogen activator (tPA) induce opening of the endothelial unilayer by compromising the cell-to-cell contacts. Similar pattern of damage is observed under the effects of GluN3A agonist glycine.

tPA accelerates the glycine-induced opening of the endothelial barrier. In addition, the close association of endothelial NMDA receptors expression in close vicinity of the transmembrane occludin and claudin-5

proteins also leaves a large opportunity of discussion for their associated role in blood brain barrier and blood spinal cord barrier regulation.

In our previous study, we confirmed that neuroendothelial NMDA receptors are a potential therapeutic target to evade from EAE mediated blood brain barrier and blood spinal cord barrier damage. This study also put up query on the unknown mechanism by which NMDA receptors could mediate this blood brain barrier and blood spinal cord barrier damage. Henceforth, we also confirm the metabotropic mode of action by NMDA receptors, which include CREB and RhoA signaling cascade.

In this study we have confirmed that NMDA receptors expressed by cerebral endothelial cells. These receptors are expressed in close association with the tight junction proteins. As previous studies have shown that NMDA receptors on endothelial cells are potential therapeutic targets against the inflammatory outcomes of EAE. The close association of NMDA receptors with tight junction proteins and involvement of RhoA signaling pathway seem to cross roads somewhere, which still needs to be explored.

Another interesting aspect of this study is CREB signaling pathway. However, so far, the influence or the role of CREB activation in increased permeability of endothelial unilayer is still not clear. We believe that it will be another intriguing exploration since some studies have suggested that during inflammation CREB can have a dual course of action. The first is an early short-term effect, which propagates inflammatory reactions. While another a late phase long-term effect mediated by increase in protein expression and recovery of cells from the inflammatory damage.

Another interesting prospect of this study will be nuclear accumulation of ZO-1 proteins. It is known that when endothelial cells are under a chemical stress, a heat shock or a mechanical injury, the membranal ZO-1 protein Trans-locates to cytoplasm and is accumulated in the nuclei. Similar behavior is observed when cells have immature tight junctions or damaged tight junctions due to environmental stress.

In addition, it is now very important to see these effects of co-treatment of NMDA and glycine with tPA in an extended time of frame of 24 hours. This aspect of this study shall be able to provide us the entire profile of the signaling proteins in the course of time. Secondly, it may also provide us the information for the role of CREB signaling pathway.

This study has opened up a new window of opportunity to be explored in the field inflammation in different neurological disorders and related impact of neuroendothelial NMDA receptors. As we highlight the association of the tight junction proteins with NDMA receptors, it will now be interesting to study more in this context.

It is conclusive that NMDA receptors of the cerebral endothelial cells are an important entity in the phenomena of the neuroinflammation. This study hence provides us the clear picture of the impact of NMDA receptors in the blood brain barrier. Siegle Ret hless, B, W Gao, a d J W Joh so .

.

  chose to provide me the opportunity to work in his esteemed INSERM Unit U1237. You have directed my work with great enthusiasm and support. I thank you for guiding me throughout my project with a welcoming gesture of new ideas. I thank you for your patience through the troughs of the project. I will always cherish what I have learned from you.

  Actions-European Commission for providing the financial support through fellowship for my research, stay and mobility throughout these years of my doctoral studies. I show my gratitude to University of Caen and Cyceron for hosting my research and maintained the continuum of resources.

Figure 1 .

 1 Figure1. BBB Breakdown. Adapted from Obermeier, Daneman, and Ransohoff, 2013 ......................

Figure 1 .

 1 Figure 1. BBB Breakdown. The factors that support BBB dysfunction and damage followed by the chief characteristics of BBB damage and their outcomes. Figure adapted from (Obermeier, Daneman, and Ransohoff 2013).

  pathways can result in BBB dysfunction in various mechanisms such as excess in transporter or receptor mediated transport, loss of specificity for transport or exacerbated transcytosis at the BBB which may involve the I N T R O D U C T I O N degradation or disassembly of the tight junction proteins. The BBB breakdown shall be considered as a gradient

  are source of several factors which regulate the BBB permeability and the formation of the tight junctions (Alvarez, Cayrol, and Prat 2011). In addition, pericytes are present in blood brain barrier at the level of capillaries and the post capillary venules and cover 22-23% of capillaries in brain (Dore-Duffy 2008). Pericytes regulate vascular stability, angiogenesis and clearance of the toxic metabolites (Winkler, Bell, and Zlokovic 2011). Pericytes and endothelial cells together construct the basement membrane, which supports the development and maintenance of the blood brain barrier, shown in figure 2. At the functional level, these components engage with microglial ells a d euro s to for a euro as ular u it NVU (Abbott, Rönnbäck, and Hansson 2006).

Figure 2 .

 2 Figure 2. Blood brain barrier. An overview of arrangement of the cellular components of the blood brain barrier across capillary and post-capillary venule. The endothelial monolayer is surrounded by the endothelial and parenchymal basement membranes. Peri tes a d astro te s feet e ds further support this arrangement.Figure adapted from (Engelhardt and Ransohoff 2012).

  communications across the cells. The tight junction proteins also include Zonula Occludens (ZOs) and other intracellular proteins, which are associated with the cytoskeleton. The tight junction and adherens junction proteins are a set of characteristic proteins expressed on cell-to-cell junction points of endothelial and epithelial cells. These proteins play a crucial role of developing a tightly integrated junction between endothelial cells which are employed to regulate transcellular, paracellular traffic of cells, drugs and metabolites. The most important entities of tight junctions are ZOs (zonula occludens), claudins, occludin, and JAMs (Junction Adhesion Molecules), shown in figure 3.

First

  observed via immunofluorescence and immunoelectron microscopy, a 65-kDa protei o ludi is transmembranal tight junction protein expressed in endothelial and epithelial cells (M Furuse et al. 1993). Zonula occludens are scaffolding proteins of the MAGUK family, which have a membrane associated domain with guanylyl kinase-like domain (Christina M. Van Itallie and Anderson 2014). This family includes ZO-1, ZO-2, and ZO-3 scaffolding proteins. ZO-1 is localized at the blood brain barrier junctions in mouse and human brain (Nico et al.

Figure 4 .

 4 Figure 4. Dual localization of Zoluna Occludens Nucleic Acid Binding (ZONAB) protein. Epifluorescence images of ZONAB staining (A) before and (B) after heat shock. Adapted from Tsapara et al. 2006.

  epithelium. They also developed severe calcification in brain and the parenchyma around the vessels (Saitou et al. 1998). Occludin proteins play a vital role in the maintenance of the endothelial barrier permeability. It has been reported in an in vitro study with MDCK I (Madin Darby Canine Kidney Cell line), that the cytoplasmic domain of occludin is highly phosphorylated when localized in tight junctions (Sakakibara et al. 1997). An in vitro study in human umbilical vein endothelial cell line (EVC304) shows that phosphorylation of occludin regulates junction permeability in a G protein dependent or independent mechanism (Tetsuaki Hirase et al. 2001).

Figure 5 .

 5 Figure 5. Transmembrane Occludin Tight Junction Protein Associated with ZO-1 and Cytoskeletal Protein F-actin. The figure shows amino acid chain of occludin running across the plasma membrane. The carboxyl terminal of this chain interacts with the ZO-1 GUK domain. Notably, the ZO-1 PDZ domain interacts with claudins. Occludin exhibits two extracellular loops (EL1 and EL2) along with four transmembrane domains and an intermediate loop. The carboxyl domain amino acid chain (257 aa) is longer than the amino terminal domain (66 aa).Figure adapted from (Cummins 2012).

  on the other hand, are a family of tetra-spanning tight junction transmembrane proteins, which fulfil the task of barrier function (figure6). They share four transmembrane domains with occludin, with no sequence homology (M Furuse etal. 1998). It has been shown in Xenopus kidney epithelial cells that these proteins are considered as the effectors of the restricted permeability across the barrier (V. Wong and Gumbiner 1997). The electron microscopy and molecular modeling data reveals the hemophilic and heterophilic interactions in their cis and trans conformations of their extracellular loops to form an integrated tight junction(Piontek et al. 2007).Key points: Hence, from these studies we can easily conclude that, occludin protein is a vital component of the tight junctions, which regulates the blood brain barrier integrity via the status of the protein phosphorylation, degradation, and expression. Occludin is regulated by many signaling cascades, such as PKC and RhoA/ROCK activation.

Figure 6 .

 6 Figure 6. Diversity of structure between the tight junction proteins occludin, claudin and junction adhesion molecule (JAM). (a) Occludin and (b) claudin have four transmembrane domains with two extracellular loops. Occludin consists of a longer cytoplasmic domain. (c) JAM has a single transmembrane domain, and its extracellular portion bears two immunoglobulin-like loops that are formed by disulphide bonds. Adapted from Tsukita et al. 2001.

Figure 7 .

 7 Figure 7. VE cadherins. VE cadherins engage with several different intracellular proteins of cytoskeleton, which regulate cell orpholog . β-catenin and plakoglobin associate with the cytoplasmic domain of cadherin protein. These structures establish the interaction between the VE cadherin with intracellular actin strands and intermediate filaments. Also refer to figure 1. Figure adapted from (P. A.Vincent et al. 2004)

Figure 8 .

 8 Figure 8. MAP kinase signaling cascade. Members of this family activate each other by adding phosphate groups to serine/threonine amino acids. MAPK, MAP kinase; MAPKK, MAP kinase kinase; MAPKKK, MAP kinase kinase kinase. Adapted from Hommes et al. 2017.

  umbilical vein endothelial cells (HUVECs) suggests that activation of ERK1/2 signaling pathway results in the delocalization of occludin and its phosphorylation at serine residues(Kevil et al. 2000). In an in vivo study it is suggested that ERK activation along with p38 MAPK activation under the effect of LPS (100 ng/ml), in state of chronic alcohol consumption can cause tight junction proteins phosphorylation. This results in transient disruption of the blood brain barrier and barrier abnormalities with a decrease in tight junction proteins mRNA expression levels(Singh et al. 2007). Proinflammatory molecules also alter the blood brain barrier through MAPKs activation. An in vitro study MDCK cells suggests that TNF-α a d IFN-γ proi fla ator toki es i du e ERK / a d p a ti atio , which results in delocalization of the tight junction proteins and cytoskeletal protein reorganization. These deleterious effects are observed as alterations in occludin, claudin-1 and claudin-2 protein levels, which result in modulated endothelial barrier functions (Patrick et al. 2006). ERK1/2 activation also alters the ZO-1 protein levels in brain endothelial cells. Under the oxidative stress induced by HIV-1 Tat protein, ERK1/2 activation results in decrease in Key points: Hence, it is understood that MAPK signaling pathways are potential modulators of the e dothelial a d epithelial arrier's fu tio s, hi h is go er ed y o-activation of several other signaling cascades. The effect of MAPK activity in endothelial and epithelial cells gives a mixed idea of a deleterious effect sometimes while protective effect in some other cases. levels of ZO-1, which is prevented by N-acetylcysteine (NAC, a potent antioxidant) Pu et al. 2005). In addition, Matrix metalloproteinases (MMPs) also induce damage to the tight junction proteins. It is also suggested that MMP-7 EGFR mediates the MEK-ERK signaling. MEK-ERK signaling induces disruption of the tight junction protei s structures (Tan et al. 2005). An in vivo study shows that inhibition of the ERK and related MAPK signaling pathways, reduces tight junction protein ZO-1 degradation and significantly reduces the tissue edema. This indicates a crucial role of MAPK signaling in barrier function (Mori et al. 2002). It is not clear yet whether the MAPKs activity only has a deleterious effect on the blood brain barrier. Some studies suggest that steroids are potent molecules to positively influence the endothelial barrier integrity via MAPKs activity. For instance, a study in HUVECs suggests that 10nM concentrations of estradiol (E(2)) and dihydrotesterone (DHT) enhance the expression levels of the tight junction protein occludin. Increased occludin helps in maintenance of the endothelial barrier integrity (Wasana K. Sumanasekera et al. 2006).

  proteins and nucleotide exchange factors (shown in figure9). Rho GTPases family includes RhoA, Rac and Cdc2 proteins. In addition, RhoA, Rho-GEFs (Rho guanine nucleotide exchange factors), Rho associated protein kinase (ROCK) , myosin light chain kinase (MLCK) and myosin light chain protein (MLC) are key complexes involved in RhoA signaling. These all proteins regulate the actin cytoskeleton protein arrangement in cells(Kaibuchi, Kuroda, and Amano 1999; Jaffe and Hall 2005). Rac and Cdc2 regulate the protrusive activities of the cell whereas RhoA regulates cell contraction (Hall 2005). Rho GTPases signaling has several downstream targets, which are actively involved in maintenance of cytoskeleton rearrangement. These Rho effectors include a group of molecules such as myosin phosphatase-targeting subunit 1 (MYPT1). These molecules possess an active motif for the interaction with myosin light chain (MLC) phosphatase, Rhotekin and Rhophilin (Mutsuki Amano, Fukata, and Kaibuchi 2000; Narumiya, Tanji, and Ishizaki 2009). RhoA/ROCK/MLC signaling maintains cell-to-cell contacts by the modulation of tight junction proteins. RhoA/ROCK/MLC signaling influences the endothelial and epithelial barriers with the help of these modulations. For instance, in an in vitro study (in MDCK cells) during depletion of the ATP, Rho GTPase signaling protects the tight junctions from disruption (Gopalakrishnan et al. 1998). Moreover, in intestinal (T84) cells when RhoA activity is inhibited with C3 transferase, tight junction are dysfunctional and there is compromised (Walsh et al. 2001; Zhou and Zheng 2013). In human TM cells (GTM3) the Rho-Rho kinase pathway regulates the cell contractility and attachment with the extracellular matrix through the alteration of MLC proteins (Ramachandran et al. 2011). RhoA signaling is an integral part of the cell cycle. As shown in an in vitro study with human mesenchymal stem cells (hMSCs), RhoA signaling regulates the actin-myosin generated tension, which regulates the cell shape and morphology (McBeath et al. 2004). Rho family of small GTPases regulates the cytoskeletal dynamics by coordinating the cytoskeleton rearrangement (Spiering and Hodgson 2011). A study in MDA-MB-231 mesenchymal breast cancer cells suggests that RhoA, Rac1, and PAK family kinases exist in a bi-stable (active and inactive) switch like responses to maintain cell morphology and migrations (Byrne et al. 2016). It has been shown in an in vitro study in HEK293 cells that RhoA is closely associated with microtubules, which regulates the anchorage of endothelial cells with basement membrane (Nakaya et al. 2008). signaling influences the actomyosin contractility and microtubule stability, which regulate the migratory polarity during cell proliferation (Takesono et al. 2010). In fact, in neuronal cells, RhoA specific guanine nucleotide exchange factors (such as p190RhoGEF) interact with the microtubules (van Horck et al. 2001). These factors induce microtubule disassembly and cell contractility (Chang et al. 2008). It is interesting to note that different RhoA signaling pathway influences the tight junction proteins through ROCK activity. If ROCK activity is blocked during tight junction assembly, then tight junction protein distribution on cellular junctions is disrupted in intestinal T84 cells (Walsh et al. 2001), indicating that ROCK is important for the regulation of tight junction assembly. However, direct inhibition of ROCK by Y27632 (ROCK inhibitor) can disorganize the apical ring of actin, but has no effect on tight junction protein localization.

Figure 9 .

 9 Figure 9. RhoA Signaling Cascade. A crosstalk between PKC, Rho, and MLCK signaling pathways. The figure shows different downstream targets of RhoA signaling pathway. The activation process, which starts with GTP binding to RhoA protein, following ROCK activation. Activation of ROCK on one end mediates phosphorylation of the tight junctions resulting in tight junction disassembly. It also regulates activation of MLC proteins, which regulate rearrangement of cytoskeletal components. Figure adapted from (González-Mariscal, Tapia, and Chamorro 2008) Notably, the damaging effects of RhoA signaling pathway are more significant since they aggravate the severity of the inflammatory outcomes. As shown in figure 9, the downstream molecules of RhoA signaling influence the tight junction proteins configuration. For instance, in an in vitro T84 epithelial cells RhoA signaling pathways induce the endothelial barrier by altering the tight junction proteins under the effect of proinflammatory cytokines like IFNgamma (Utech et al. 2005). Increase in ROCK expression during RhoA activation causes loosening of the tight junction proteins induced by pro-inflammatory cytokines in intestinal T-84 cells (Utech et al. 2005). Tight junction proteins are disrupted by an increased MLC phosphorylation and MLC kinase activity during RhoA signaling pathway.In vitro studies suggest that RhoA activity destabilizes the microtubules in the human pulmonary endothelial cells(Bogatcheva et al. 2006; A. A. Birukova et al. 2005). RhoA activation leads to endothelial hyperpermeability in a calcium ion dependent mechanism in an in vitro study in HUVECs (van Nieuw Amerongen etal. 2000). RhoA activation derived tight junction protei s disasse l also promotes migration of small lung

  roles of myosin light chain (MLC) phosphorylation and MLC kinase (MLCK) are other important factors that are involved in Rho signaling cascade. MLC protein (as depicted in figure 10) is composed of two 200 kDa heavy chains and four 20 kDa small chains, termed as essential and regulatory light chains (MLC1 and MLC2) respectively. These heavy and light chains are coiled together forming an amino terminal globular head, which interacts with actin. MLCK is a serine-threonine specific protein kinase, which regulates the MLC phosphorylation. MLC phosphorylation regulates cell motility, cytokinesis, cell contractility, paracellular permeability, and cell morphology (M Amano et al. 1996; S. Y. Yuan 2002; Komarova and Malik 2010; Mehta and Malik 2006). An in vitro study in Caco-2 suggests that the monolayer barrier functions are restored when TNF-α a d IFN-gamma induced MLCK activity is blocked by membrane permeant peptide (Zolotarevsky et al. 2002). MLC phosphorylation is a crucial phenomenon which regulates the cell shape and morphology. Hence, any change in cell shape alters its contact with the other cells which results in opening of the blood brain barrier. For instance, MLC phosphorylation induced by active p38 MAPK in calf pulmonary artery endothelial cells (CCL 209), results in increased paracellular permeability of the endothelial barrier in in vitro conditions (Goldberg et al. 2002). As shown in an in vitro study with porcine aortic endothelial cells (PAECs) it is interesting to note that monophosphorylation of MLC protein at either T18 or S19 is functionally sufficient for endothelial barrier opening (Hirano and Hirano 2016). This should be noted that, the contraction of the acto-myosin belt is derived by the phosphorylation of the MLC2 chain at serine 19 position. When the extracellular concentration of calcium is reduced to minimal micromolar of concentrations, the disassembly of the tight junctions and adherens junctions is triggered. It is followed by retraction of actin and myosin fibers close to the junctions (T. Y. Ma et al. 2000; Andrei I Ivanov, Parkos, and Nusrat 2010).

Figure 10 .

 10 Figure 10. Myosin Light Chain. The heavy and light chain with the amino terminal head of MLC protein. Figure adapted from (González-Mariscal, Tapia, and Chamorro 2008).

  modulator (CREM). Functions of the CREB activity is understood as a response to the extracellular stimuli, which results in the altered gene expression in target cells. The CREB pathway has critical roles in the cell proliferation and differentiation. It binds to the short DNA sequence called cyclic AMP response element (CRE) which are highly conserved nucleotide sequence. CRE sites are typically found upstream of genes, within the promoter or enhancer regions. Upon stimulation, an increase in cAMP levels and/or Ca 2+ levels triggers the CREB protein activation, which i ds to the CRE ele e t at the e d ge e to u dergo tra s riptio .The CREB family includes CREB, CREM and ATF-1 molecules. Structurally, CREB family of activators contain a 60 amino acid long kinase inducible domain (KID) which exhibits PKA phosphorylation site and other phosphorylation sites for kinase I and II(de Groot et al. 1993; Brindle, Linke, and Montminy 1993). An In vitro study suggests that KID domain is attached with glutamine rich domains Q1 and Q2(Brindle, Linke, and Montminy 1993; Quinn 1993).

Figure 11 .

 11 Figure 11. Ribbon Diagram of the CREB bZIP.DNA Complex. The leucine zipper residues are seen in purple from residue 308 to 336 and residue 337 to 339. The basic region residues are seen in green from 285 to residue 307 which interact with the CRE DNA element. Figure adapted from (Schumacher, Goodman, and Brennan 2000).

  1996). The CREB zipper domain possesses both conserved and variable sequences relative to the members of the same protein family(Schumacher, Goodman, and Brennan 2000). The palindromic DNA base sequence recognition is carried out by the basic residues which line the inner face of the helix of the CREB DNA binding domain. These residues on the inner face of the helix possess the basic residues namely Asn293 and Arg301.When bound to the CRE DNA strand the Lys304 residue interacts with a hexahydrated magnesium ion as seen in the figure 11 as yellow entity with hydration in red. It is believed that magnesium selectively stabilizes binding of CREB with DNA strand (Craig 2001). CREB signaling, upon activation works in coaction with many other signaling pathways. These signaling pathways play a critical role in endothelial homeostasis, inflammation and its outcomes. Classically, CREB activation is phosphorylation at its serine 133 residue (Mayr and Montminy 2001). It is carried out along with involvement of the CREM proteins of CREB with cAMP dependent protein kinase A (PKA) (Brindle, Linke, and Montminy 1993) , protein kinase C (PKC) and calmodulin kinases (CaMK-IV, signaling pathway responsive to calcium influxes from extracellular environment) (Enslen, Tokumitsu, and Soderling 1995). The CREB signaling and its role in neuroinflammation is debatable, since some studies have shown the protective role of CREB activity against inflammation whereas in others CREB signaling can have a negative effect, promoting the inflammatory outcomes. Hence, it is difficult to define its effect individually and in action with other signaling pathways. Heam oxygenase-1 (HO-1) is an inducible form of heam oxygenase, which increases the endothelial resistance against inflammation and apoptosis. An in vitro study in human umbilical vein ECs (HUVECs) suggests that, PKC activity with CREB and Nrf2 signaling pathways regulate the HO-1 levels, which helps in maintenance of endothelial homeostasis (Mylroie et al. 2015). Interestingly, in BV2 microglial cells, Morin (a flavonoid that possess various medicinal properties) increases PKA/CREB signaling. PKA/CREB signaling promotes Nrf2/HO-1 activation which suppress the LPS induced neuroinflammation and its outcomes (Jung et al. 2017). In addition, the neuro-inflammatory effects of LPS by Akt/NF-κB signaling and MAPKs activation in BV-2 microglial cells are inhibited by fucoxanthin by PKA/CREB pathway in (Zhao et al. 2017). In an in vivo study, the learning and memory deficits in mice are resumed by an increase in levels of cAMP/CREB/PKA proteins, which also decreases in inflammatory factors such as NF-κB, iNOS and TNF-α Guo et al. . Similar deleterious inflammatory effects by 100ng/mL LPS were prevented by 10nM N-Docosahexaenoylethanolamine induced cAMP/PKA CREB signal activation in P3 primary rat microglia and immortalized BV2 murine microglia cells (Park et al. 2016). In an in vivo study, 70 and 100 mg/kg doses of topiramate decrease in levels of CREB, BDNF, and PKA proteins. This results in a reduction in inflammatory outcomes induced by 10mg/kg methylphenidate (MPH) in rats (Motaghinejad et al. 2016).

  It has been shown before that PKC signaling plays regulatory role in inflammatory reactions. Though apart from PKC, NF-κB and CREB signaling alter endothelial and epithelial barriers through other ways also. For instance, an in vitro study in A549 and HEK293 cells suggests that the upregulation of pro-inflammatory NF-κB target genes along with CREBBP (CREB binding protein) mediates RelA acetylation, which promotes inflammatory reaction. This effect is reversed under the a ti atio of WNT/β-catenin signaling which reinforces the barrier functions (B. Ma, Fey, and Hottiger 2015). In an in vivo study it has been shown that the inflammatory outcomes induced by (20mg/kg/body) nickel sulfate are prevented when the TLR4/p38/CREB signaling pathways are effectively inhibited (C.-M. Liu et al. 2016). It has been shown in an in vivo study that the changes in neurotransmitters levels and brain inflammatory cytokines in type-2 diabetic rats can be prevented by inhibition of NF-κB phosphor latio . This e asio fro the i fla ator outcomes is also observed in line with decrease in IL-6 and TNF-α, ith an increase in CREB phosphorylation (Datusalia and Sharma 2016). In addition, an in vivo/in vitro study shows that LPS (100ng/ml in vitro) induced PI3K/AKT and CYP2E/Nrf2/ROS signaling promotes inflammation and apoptosis and this damage is reduced by curcumin (W. Zhong et al. 2016). NF-κB sig ali g deri ed TNF-α pla s sig ifi a t role i regulatio of inflammation. Moreover, followed endothelial barrier dysfunction and CREB activation can play an additional regulatory role in inflammatory outcomes.

  many studies, CREB activation with RhoA signaling has been shown to be associated with regulation of inflammation, cell proliferation, and embryogenesis. Though the dependency of the either of the signaling pathway on each other is still not clear, but these signaling pathways orchestrate the endothelial barrier functioning by altering the cell morphology. For instance, an in vivo study (with mice lacking p190-B RhoGAP protein) suggests that Rho GTPases modulates CREB activity to regulate cell size and animal size during embryogenesis(Sordella et al. 2002). In an in vitro study with human chondrocyte cell line (C28/I2) suggests that RhoA activation in coaction with increased CREB phosphorylation prevents hypoxia-induced apoptosis in chondrocytes (decrease in ROS) and mitochondrial dysfunction (K. Zhang and Jiang 2017). CREB RhoA signaling also regulates the cytoskeletal remodeling for the maintenance and formation of dendritic spines. The brain derived neurotrophic factor (BDNF) induces an increase in dendritic spine formation via CREB regulation in hippocampal neuronal cultured cells. CREB regulates the RhoA inhibitors namely Par6C and Rnd3 (RhoE) that promote synaptogenesis upon RhoA inhibition (Lesiak et al. 2013). On the other hand, an in vivo study suggests Key points: Hence, it can be concluded that CREB signaling pathway does not have any deleterious effect on the endothelial cells, entirely. Activated by high level of cAMP levels, this signaling pathway functions in synchrony with the other signaling pathways. Although the effects of CREB activity in coaction with other signaling pathways on the blood brain barrier is debatable based on the fact that it can induce considerable damage to the endothelial barrier when activated with several other signaling pathways. that netrin-1 promotes cell invasiveness and tumor cell sprouting which is a result of the RhoA and CREB activation. These pathways enhance the capillary endothelial cells sprouting and endothelial cells infiltration (Shimizu et al. 2013). It has been suggested in an in vitro study that RhoA GTPases signaling along with p38 pathway induces Cry61/CCN1 (angiogenic factor, which has a regulatory role in cell adhesion, proliferation, and differentiation) through phosphorylation of CREB protein in primary cultures of smooth muscle cells (Han et al. 2003). Interestingly another in vitro study in human pulmonary arterial endothelial cells (HPAE) suggests that endothelial permeability is controlled by CREB activation, which is a result of regulation of GTPase-activating protein p190RhoGAP-A protein (a RhoA GTPase inhibitor). This regulatory role of CREB signaling pathway on RhoA activity suggests a pivotal role of CREB in regulation of endothelial barrier function (Chava et al. 2012). The epithelial barrier integrity is enhanced under the effect of adenosine. Adenosine increases the TER, which is mediated by dephosphorylation of myosin light chain protein (MLC). The phosphorylation of MLC protein is a downstream event of an activated RhoA signaling along with active ROCK proteins. Interestingly, an in vitro study suggests the same effect of adenosine in cultured bovine corneal endothelial cells (BCECs). This study shows that adenosine increases MLC dephosphorylation and increases TER. These outcomes are observed with an elevation in cAMP levels, which triggers CREB phosphorylation in BCECs (Srinivas et al. 2004). On the contrary, CREB signaling pathway also influences the tight junctions at the endothelial barrier as it induces RhoA signaling through upregulation of VEGF like growth factors. For instance, an in vitro study in granulosa-lutein cells (from patients) suggests that VEGF and IL-8 increase the endothelial permeability through VEGFR-2 mediated RhoA/ROCK activation, which results in cytoskeletal reorganization. These signaling pathways also mediate VE-cadherin and occludin phosphorylation which result in dysfunction of adherens and tight junctions (S.-U. Chen et al. 2010). The CREB RhoA signaling pathway also modulates the endothelial permeability (Human cerebral microvascular endothelial cells model [HCMEC/D3]) by regulating the levels of ZO-1 tight junction protein. It has been suggested that human immunodeficiency virus (HIV) specific trans-activator of transcription (Tat) mediates alterations in integrity of endothelial monolayers via RhoA signaling. While CREB activation induces upregulation of ZO-1 upon CREB binding to the proximal region of the ZO-1 promoter in in vitro conditions (Y. Zhong et al. 2012). Key points: These studies confirm that CREB-RhoA signaling pathway alter the normal function of the blood brain barrier through the modulation of tight junction and the adherens junction proteins.

Figure 12 .

 12 Figure 12. Functional Structure of Tissue-type Plasminogen Activator. Figure adapted from (Vivien et al. 2011).

  not alter the blood brain barrier structure or permeability(Benchenane et al. 2005). Interestingly, an in vitro study has shown that only tPA (20μg/ l) increases the permeability of EBA E a s lue al u i duri g or o ia which is enhanced under the hypoxia/reoxygenation conditions(Hiu et al. 2008). Another in vitro study in rat brain microvascular endothelial cell line (GP8/3.9) suggests that tPA and mediates diapedesis of monocytes across the barrier. It also suggested from the same study that tPA triggers a ROS dependent blood brain barrier dysfunction mediated through the damage to occludin tight junction protein (Reijerkerk etal. 2008).There are many signaling pathways, which are associated with deleterious effects of tPA in blood brain barrier shown in figure 13. There are many factors, receptors and pathways which have been shown to play crucial role in tPA regulated blood brain barrier function. For instance, an in vivo study in tPA-defi ie t tPA-/-i e suggests that tPA ( μM) triggers cleavage of the substrate binding ectodomain of surface expressed astrocytic LRP receptor. This process of clea i g is ter ed as sheddi g (Polavarapu etal. 2007). It also suggested that tPA stimulates endothelial LRP induced NFκB sig ali g hi h ediates upregulatio of MMP-3 and MMP-9 matrix metalloproteases. This results in matrix degradation and BBB dysfunction (X.Wang et al. 2003; Suzuki et al. 2009).

Figure

  Figure 13. tPA Mediated Neuroinflammatory Channels in Brain Endothelial Cells and Astrocytes. tPA acts on LRP receptors on endothelial cells and astrocytes. In both cases, this effect is mediated through its proteolytic a tio sheddi g of LRP re eptors , leadi g to the re ruit e t of NFκB a d the produ tio of MMP9 by target cells. MMP9 then plays its action of BBB, enhancing its permeability and leukocytes diapedesis. In endothelial cells, NMDA receptors may also in part mediate tPA effects. Alternatively, the proteolytic activation of PDGF-CC in active PDGF-C by tPA can also contribute to astrocyte endfeet detachment by activating astrocytic PDGFR-α. The binding of tPA to LRP receptors on astrocytes facilitates the activation of PDGF-CC.Figure adapted from (Mehra et al. 2016)

  expression. Intercellular adhesion molecule 1 (ICAM-1) is one such adhesion molecule which is expressed on endothelial surface. In an in vivo study (in EAE mouse model) and an in vitro study (in bEnd.3 endothelial cells) it has been shown that tPA (in in vivo μg/kg a d in vitro 2.5-μg/ l) induces an upregulated expression of ICAM-1 (J. Wang et al. 2014). This should be noted that adhesion molecules are crucial mediators of cell contact a d apture, ra l a d ross phe o e o E gelhardt a d Ra sohoff .

  inflammatory response in microglial cells(Pineda et al. 2012). tPA interacts with Galectin -1 and this phenomenon triggers the inflammatory outcomes with microglial activation(Pineda et al. 2012; Roda et al. 2009; Nagaoka, Kouyoumdjian, and Borges 2003). An in vivo study in neuroserpin knockout mice (Ns -/-) suggests that in absence of neuroserpin (a tPA inhibitor) the level of tPA is increased which promotes microglial activation with an upregulation of TNF-α. I rease i proi fla ator fa tors and high fibrinolytic activity results in an increased inflammatory outcome as increased infarct size and elevated microglial activation (Gelderblom et al. 2013). Under high proteolytic activity of tPA, the level of chemokine (C-C) ligand -3 (CCL3) is also enhanced which facilitates the microglial recruitment (Lenglet et al. 2014). Interestingly, an in vivo study in tPA -/-mice suggests that tPA released Key points: tPA potentially influences the blood brain barrier by various mechanism of action. tPA can mediate damage to the tight junction proteins like occludin. tPA also promotes paracellular transport across the endothelial barrier. It can also engage many signaling pathways such NF-κB sig ali g, that ay ha e deleterious effect on blood brain barrier by inducing inflammation.from neurons triggers microglial activation, which induces inflammation and neuronal toxicity(Siao, Fernandez, and Tsirka 2003). In addition with tPA, microglial activation and recruitment is also associated with proteolytic activity of plasminogen. An in vitro study in primary microglial cells suggests that plasminogen ( μg/ l) induces the expression of IL-β a d TNF-α which is induced under the effect of reactive oxygen species (ROS). Together these phenomena mediate microglial activation(Min, Jou, and Joe 2003).

Figure 14 .

 14 Figure 14. Cytokine like Effects of tPA on Microglia. tPA induces non-proteol ti , toki e-like effe ts on microglia, mediated by Annexin II, possibly via a co-operation with Galectin 1. The interaction of tPA with these membrane targets leads to the activation of microglia via Akt, Erk1/2 and Jnk pathways, leading to enhanced production of proinflammatory cytokines. Figure adapted from (Mehra et al. 2016)

  interaction. tPA also promotes NMDA induced neuronal death and enhances the NMDA receptor dependent calcium ion influx, as shown in an in vitro study with mixed cortical cultures containing both neuronal and glial cells(Nicole et al. 2001). Generally, interaction of tPA with NMDA receptor is studied in context of excitotoxicity (i.e. toxicity by excessive stimulation of neurotransmitters which results in neuronal death and damage)(Parathath, Parathath, and Tsirka 2006; Chevilley et al. 2015; Jullienne et al. 2011) and long-term potentiation(Centonze et al. 2002; Qian et al. 1993). Interestingly, tPA has been exhaustively studied now for its crucial role in neuroinflammation(Passos et al. 2013; East et al. 2005). Although in very recent studies, role NMDA receptors has also been discussed and explored. In an in vivo study passive immunization technique it has been suggested that immunotherapy blocking of NMDA receptor by mouse pol lo al a ti odies αATD-GluN1, anti-amino terminal domain (ATD) antibody), inhibits the tPA induced NMDA receptor activation by which improves the hemorrhagic stroke outcome(Gaberel et al. 2013). Another in vivo and in vitro study (in tPA knockout and primary cultures of cortical neurons, respectively) suggests that anti-ATD GluN1 antibodies prevent the NMDA receptor mediated excitotoxicity and improve provide brain tissue from stroke. In addition, these antibodies also prevent tPA mediated alteration of the neurovascular unit(Macrez et al. 2011).

  isoxazolepropionic acid (AMPA) receptors, Kainate receptors and N-methyl D-aspartate (NMDA) receptor. The NMDA receptors are glutamate-gated ion channels, which have been studied for a very long time. These receptors Key points: The interaction of tPA with NMDA receptor brings about the NMDA receptor activation like activity like long term potentiation. However, in cerebral endothelial cells, this interaction leads in increase in endothelial monolayer permeability. tPA can interact with NMDA receptors both in its proteolytic and non-proteolytic mode of action. An inhibitory control by specific antibodies against the interaction of tPA and NMDA can prevent the inflammatory outcomes in stroke. This inhibition technique prevents the tPA and NMDA receptor interaction which preserves the parenchyma from leucocytes transmigration.are mediators of brain plasticity and lead the change in neuronal activity patterns into long-term changes in the structure and function of synapse[START_REF] Traynelis | Gluta ate Re epto Io Cha els: St u tu e, Regulatio , a d Fu tio[END_REF]). Role of NMDA receptor has been studied in various neurological and psychiatric neurological disorders, such as schizophrenia, neurodegenerative disease, stroke, pathological pain and more recently in neuroinflammation. NMDA receptors are almost universal w.r.t. their expression in brain regions. Yet the subunit composition of the NMDA receptor varies across the CNS.NMDA Receptor Subtypes and their Variance of ExpressionNMDA receptors are one of the most dynamic receptor, figure 15. Their functional versatility is attributed to the huge variance of expression receptor, which comes from different subunits of NMDA receptors and their combination of expression. The binding studies on neurons have revealed that NMDA receptor has multiple subtypes, which have different properties understood by the response of different subtypes to different but specific agonists and definitive antagonists[START_REF] Traynelis | Gluta ate Re epto Io Cha els: St u tu e, Regulatio , a d Fu tio[END_REF] Pierre Paoletti 2011). NMDA receptors are heteromers with multiple combinations of multiple subunits. Till date there are seven different subunits of NMDA receptors that have been reported across different regions of brain in different subtypes of receptor (Cull-Candy and Leszkiewicz 2004). This set of subunits (encoding number of amino acids ranging from 900 to 1480) include GluN1, four GluN2 subunits which are categorized as GluN2A, GluN2B, GluN2C and GluN2D which are encoded by 4 different genes, and GluN3 which includes 2 members GluN3A and GluN3B which are encoded by two different genes. The varied lengths of the different subunits are attributed to their intracellular carboxyl terminal domain (CTD) which plays critical role in transmission of signal transduction[START_REF] Traynelis | Gluta ate Re epto Io Cha els: St u tu e, Regulatio , a d Fu tio[END_REF]). NMDA receptors are heteromers which have several subtypes in expression of a pair of GluN1 with pair of similar GluN2 or a different GluN2 subunit, similar GluN3 or different GluN3 subunits[START_REF] Traynelis | Gluta ate Re epto Io Cha els: St u tu e, Regulatio , a d Fu tio[END_REF]) (Pierre Paoletti 2011).

Figure 15 .

 15 Figure 15. Characterization of NMDA Receptor Subtypes and their Differential Expression in Region of Brain. In panel a. subunit heterogeneity is showed with the details of the alternative splicing. Further, b. describes a generalized scheme of NMDA receptor subunits depicting its N terminal domain (NTD), linker followed by Agonist binding domain (ABD), transmembrane domain and finally carboxyl CTD. In c. the X-ray crystal structure shows different motifs and sites on interaction for different agonists (glutamate, glycine) and antagonists (ifenprodil, Zn 2+ , MK801). Panel d. and e. exhibit the variance of expression of NMDA receptor subtypes in different regions of brain and their prevalence in different stages of development. Figure adapted from (Pierre Paoletti, Bellone, and Zhou 2013).

  variety in expression has attributed a differentiation of a different category for NMDA receptors, which is conventional (or classical) and non-conventional (or non-classical) NMDA receptors. Conventional receptors are the dimers of dimers specifically of GluN1-GluN2 subunits(Salussolia et al. 2011; Riou et al. 2012). Other the other hand non-conventional NMDA receptors are a set of heterotrimer of GluN1-Glun2-GluN3 and heterodimer of GluN1-GluN2 subunits(Piña-Crespo et al. 2010; Burzomato et al. 2010; Pachernegg, Strutz-Seebohm, and Hollmann 2012; Henson et al. 2010).Traditionally, NMDA receptors have been considered as the classical receptors of neurons, but in recent years they have been reported in other cells of CNS such as oligodendrocytes(Burzomato et al. 2010; Káradóttir et al. 2005), astrocytes(Jimenez-Blasco et al. 2015; M.-C. Lee et al. 2010; Verkhratsky and Kirchhoff 2007) and cerebral endothelial cells(Reijerkerk et al. 2010;[START_REF] Macrez R 1 | Chevilley A 1[END_REF]). Heterodimer GluN1-GluN2B and GluN1-GluN2A subtypes of NMDA receptors form an important fraction of young and adult brain. Heterotrimer GluN1-GluN2A-GluN2B are expressed in abundance in many regions in adult brain specifically in hippocampus and cortex. All together GluN1-GluN2A-GluN2B receptor subtype includes 15 to less than 50% of the total receptor population in brain(Al-Hallaq et al. 2007; Rauner and Kohr 2011; Gray et al. 2011). GluN1-GluN2A-GluN2C subtypes and GluN1-GluN2B-GluN2D have also been reported (Cull-Candy and Leszkiewicz 2004; Pierre Paoletti 2011).Expression and functional diversities associated with heterodimer GluN1-GluN3 and heterotrimer GluN1-GluN2-GluN3 subtypes of NMDA receptors have been discussed in detail ahead. The expression of NMDA receptors is also specific to the neuronal types. For example, hippocampal and cortical interneurons exhibit high levels of GRIN2C and GRIN2D, which is generally not the case in principal cells(Monyer et al. 1994). Many NMDA receptor subtypes can be co-expressed in the same cell type. These co-existing NMDA receptor subunits also segregate from each other in specific manner (Cull-Candy and Leszkiewicz 2004). The expression of Glun2B expressing subtype is more prevalent in CA3-CA1 synapses, interestingly the content of these GluN2B receptors differ from right and left hemispheres (Shinohara et al. 2008). The aggregation of NMDA receptors can also change in accordance with the function. For instance, in ganglion retinal cells, at ON, synapses are rich in Glun2B subtypes whereas OFF synapses are enriched with GluN2A NMDA receptor subtypes (J. Zhang and Diamond 2009). GluN2A and GluN2B expressing subtypes of NMDA receptors exhibit differential expression which attributes to synaptic timing and properties (Kumar and Huguenard 2003; Lei and McBain 2002). Additionally, it has been shown that peri-synaptic and extra-synaptic sites have very high expression GluN2B expressing NMDA receptor subtype. GluN1-GluN2A and heterotrimer of GluN1-GluN2A-GluN2B are expressed on post synaptic site and their input is definitive of their input (Hardingham and Bading 2010; Gladding and Raymond 2011). It has also been reported that GluN2A subtype receptors can accumulate on the synaptic site whereas there is separate segregation outside the synapse, which is decided, by the stimulus and the response (Lopez de Armentia and Sah 2003; A. Z. Harris and Pettit 2007).

  receptors express the antigen-binding domain sandwiched between the N-terminal domain and carboxyl terminal domain. The receptor employs an agonist-induced closure of antigen binding domain the gating operations(Mayer 2011; Pierre Paoletti 2011). The structural alignment of the amino terminal domain NTD (relatively loosed conformation) is such that structural rearrangements happening on the distal amino end is sensed by the downstream gating machinery at the transmembrane domain of the receptor(Karakas, Simorowski, and Furukawa 2011; Gielen 2008; Gielen et al. 2009). The structural variability of the NMDA receptor in alignment with its structural flexibility contributes to the dynamic nature to recognize the host ligands and respond with an allosteric modulation leading to the functional and pharmacological outcomes of the receptor. The receptor properties are also determined by the specific subunit, which are co-expressed with GluN1. NMDA receptors are highly permeable to Ca 2+ ions, and respond to voltage dependent block by Mg2+. NMDA receptors activation is glutamate (to which the receptor exhibits a slow kinetics in the event of slow glutamate dissociation) agonist mediated in the presence co-agonists glycine or D-serine. These receptors are responsive to their microenvironment with the help of an array of modulatory sites in their structure.

  observed in GluN2A and GluN2B expressing NMDA receptor subtypes. The relative responses to agonists and change in sensitivities of NMDA receptor subtypes are controlled by the specific GluN2 subunit (Siegler Retchless, Gao, and Johnson 2012). For example, during high polarized potentials, even at very low frequency stimulation Key points: NMDA receptors are extremely versatile proteins. Their functional diversity is attributed to the different subunits (GluN2A, GluN2B, GluN2C, GluN2D, GluN3A, and GluN3B) expressed in combinations with GluN1 subunits. Based on expression of GluN2 and GluN3 subunits, the NMDA receptors are categorized as conventional (GluN1-GluN2 homodimers and heterodimers) and non-conventional (GluN1-GluN3 homodimer and GluN1-GluN2-GluN3 heterodimer) NMDA receptors. They are expresses in the most neuronal cells and a few non-neuronal cells. The GluN1 subunit expression has been confirmed in the cerebral endothelial cells.

  characteristic of the receptor is also determined by the GluN1 isoforms, as GluN1-a have lower EPSC than GluN1b (Rumbaugh et[START_REF] Van Nieuw Amerongen | A ti atio of RhoA Th o i i E dothelial H pe pe ea ilit : Role of Rho Ki ase a d P otei T osi e Ki ases[END_REF] Vance, Hansen, and Traynelis 2012). The synaptic responses are vital to trigger longterm potentiation (LTP). Under low frequency stimulation GluN1-GluN2B subtype receptors induce a large LTD whereas under a strong high frequency stimulation GluN1-GluN2A subtypes exhibit strong long-term potentiation(Erreger et al. 2005). These studies clearly provide us the evidence about the versatile characteristics of the NMDA receptors. Aforementioned characteristics, hence make them unique in their functions w.r.t. their subtypes and the region of the CNS they are expressed.

(

  Hatton and Paoletti 2005). NMDA receptors employ many signaling pathways upon activation, which are responsible for the traverse and translation (resulting into an activated signaling pathway) of the stimulus for the receptor. In this part of NMDA receptor functioning, carboxyl terminal domain (CTD) plays a crucial role. Primarily, because they form the connecting bridge between the NTDs and ABDs with the cytoplasmic messengers which carry out the stimulus specific response. Secondly, the CTDs are the domains of the NMDA receptor subunits, which have least conserved regions, which attributes to the variability in functions and responses of different subunits (Sprengel 1998; Martel 2012; Sanz-Clemente, Nicoll, and Roche 2012). These signaling cascades play important role in the receptor localization, receptor trafficking and signaling. For instance, a PDZ domain containing scaffold protein (MAGUKs) which are membrane associated guanylate kinases, play crucial role in retention of GluN2B expressing receptors at the synapse where as there is accumulation of GluN2A receptors on postsynaptic sites independent of guanylate kinase binding (Prybylowski 2005). NMDA receptors exhibit mobility on the synapses. Phosphorylationof the PDZ domain guanylate kinases binding motif of GluN2B by casein kinase 2 (CK2) could be one the reasons to explain that how GluN2B receptors can move in and out of the synapses while this activity is not observed in GluN2A receptor subtypes contributing to the fact that they do not interact with PDZ domain kinases (Chung etal. 2004; Sanz-Clemente et al. 2010). There are several other signaling molecules which have been shown to i tera t ith GluN su u its a d o tri ute i re eptor s su u it spe ifi traffi ki g a d sig ali g. These molecules include protein kinase A (PKA), protein kinase C (PKC), cyclin dependent kinase 5 (CDK5) and SRC tyrosine kinases. These molecules can interact with GluN2 subunits and can be actively involved in receptor trafficking(Sanz-Clemente et al. 2010; M. W. Salter and Kalia 2004). GluN2B CTD also interacts with Calmodulin dependent protein kinase II (calcium dependent, CaMKII) (Barria and Malinow 2005; Lisman, Yasuda, and Raghavachari 2012). GluN2B CaMKII interaction play important role in synaptic plasticity. NMDA receptor expression, regulation, mobility, trafficking, and functions make it complex entity of the CNS.

  , NMDA receptors have been shown to be involved in pathogenesis of EAE. They can mediate the characteristic neuroinflammatory outcomes governed by the pathological condition. Hyperactivity of the NMDA receptors on pre-synapse site results in rise in glutamate release by a voltage gates sodium channel dependent mechanism. In addition, pharmacological blockade of the NMDA receptors in the in vivo study shows an improvement in clinical disease course of the EAE mice (Grasselli et al. 2013). Interestingly, a GluN2B blocker RO25-6981 effectively decreases the extent of inflammation, neuro-axonal damage and myelin degradation. Selective inhibition of GluN2B containing NMDA receptors hence a control of neurotoxicity can modulate Key points: NMDA receptor plays critical role in the progression of cerebral ischemia. Activation of NMDA receptor signaling via its proteolytic activity. Ischemia induced brain edema and neuronal death is reduced when NMDA receptor activity is inhibited by an anti-amino terminal domain antibody αATD-GLuN1 antibody. Hence, we can conclude that NMDA receptors play a key role in inflammatory reactions during stroke. pathological changes in EAE mice (Farjam et al. 2014). An in vivo study in EAE model of optic neuritis suggests that blocking of NMDA receptors also show a neuroprotective role via inhibition of NMDA receptor mediated glutamate toxicity by memantine or MK801, during the induction phase of experimental autoimmune optic neuritis (Sühs et al. 2014). Similarly, Matrine (MAT, a quinolizidine alkaloid) can effectively reduce the glutamate excitotoxicity by reducing the level of NMDA receptor in EAE rats, thereby decreasing the extent of neuronal death (Kan et al. 2014). It is also suggested that pharmacological inhibition of NMDA receptors by amantadine and memantine reduces the neurological symptoms in EAE rats, and the elevated mRNAs expression levels of MBP and glycoproteins MAG and MOG supports the europrote ti e role of NMDA re eptor a tago ists Dą ro ska-Bouta et al. 2015).

  a d depositio of β-a loid peptide Aβ is the dri i g fa tor for the progressio of this disease. The fu tio al Key points: NMDA receptor plays critical role in the progression of multiple sclerosis. NMDA activation blocked by a monoclonal antibody Glunomab effectively resulted in evasion from the pathological outcomes of EAE and preservation of the blood-spinal cord barrier and reduced leucocyte infiltration. This study again shows that NMDA receptors play a crucial role in neuroinflammatory outcomes. However, the underlying mechanism for this outcome is still unknown. abilities of NMDA receptor are questionable due to their involvement in the advancement of the disease. It has ee sho that solu le for of Aβ peptide a distur the s apti fu tio a d plasti it , hi h are the ajor functional abilities of NMDA receptors (Sheng, Sa ati i, a d Südhof . The deposits of Aβ peptide a also inhibit the long-term potentiation (LTP) and prolong the long-term depression (LTD) of glutamatergic transmission along with disrupting the synaptic plasticity (Ondrejcak et al. 2010). It is also e ide t that Aβ peptide i du es activation of GluN2B-GluN1 NMDA receptor subtypes, which results in the alteration in the synaptic functions i hi it LTP a d e ha e LTD a d plasti it . These ad erse effe ts of Aβ peptide are i hi ited upo GluN B blockade by ifenprodil in primary neuronal cell culture (Rönicke et al. 2011). High magnitude of NMDA receptor ediated e itoto i it also a elerates euro al death i se ere eurodege erati e disease su h as Hu ti gto s disease(HD), also that CREB activation can act as a linking pathways which can HD pathogenesis (Milnerwood and Raymond 2010; Milnerwood et al. 2010).

Figure 15 .

 15 Figure 15. Transmembrane Topology and Modular Structure of the GluN3A Subunit. Panel a. suggests the extracellular amino terminal domain along with the S1 and S2 domains of subunit which together form the cleft for glycine binding, four transmembrane and along cytoplasmic domain which provides the interaction sites for signaling factors for cell cycle, receptor trafficking, endocytosis and phosphatase signaling. Panel b. describes the functional differences between the GluN1-GluN2 NMDA receptor subtypes and GluN3 expressing NMDA receptor subtypes (the homodimer and heterotrimer). GluN3A expression at synaptic sites in adult rat hippocampus, revealed by post embedding immunogold electron microscopy. Adapted from (Pérez-Otaño, Larsen, and Wesseling 2016)

  GluN3A expressing glial NMDA receptors in have been suggested to crucial role in white matter injury. Recently, it has been shown that GluN1-GluN2-GluN3 expressing heterodimer in expressed in oligodendrocyte exhibit NMDA evoked currents and very low magnesium sensitivity. NMDA receptors partly mediate an inward current developed in oligodendrocytes during ischemia(Karadottir et al. 2005). Another study suggests that during Key points: GluN3 expressing NMDA receptor subtypes exhibit very low magnesium sensitivity; also, they have very low calcium permeability. These subunits when expressed with GluN2 subunits of NMDA receptors, may alter the behavior of these classical NMDA subunits. In addition, GluN3A expressing NMDA receptor subtypes show a very high affinity towards glycine and D-serine and very low affinity for glutamate. Glycine binding NMDA receptor antagonist CNQX inhibits the glycine binding to GluN3A NMDA ischemia axon cylinders and myelin undergo an ultrastructural damage and this damage is greatly reduced by NMDA receptor antagonists. The immunohistochemistry studies exhibit expression of GluN1, GluN2 and GluN3 subunits in myelin and also suggested the involvement of NMDA receptors activation in mechanism of axomyelinic signaling. These roles make GluN3A NMDA receptors subtypes a potential therapeutic target in disorders which exhibit demyelination during multiple sclerosis, neurotrauma, and infections(Micu et al. 2005). GluN3A expressing NMDA receptor activation results in quick calcium dependent detachment and disintegration of oligodendrocytes processes in the white matter (M.G. Salter and Fern 2005). Although with a very limited number of studies, it is conclusive that while GluN3A expressing NMDA receptor exhibit lower calcium permeability, this insufficient calcium buffering may promote oligodendrocytes to cell death. While these damaging effects can be evaded by NMDA receptor antagonists.NMDA receptors are often involved and responsible for excitotoxicity mediated cell damage, which worsens many pathoph siologi al o ditio s like Hu ti gto s disease, Parki so s disease, Alzhei er s disease, stroke a d traumatic injury. It has been understood that excessive NMDA receptor activation mediates excitotoxicity because of depolarization dependent calcium influx promotes cell death (Rothman and Olney 1995). These neurotoxic damages are effectively attenuated by antagonists. The activity of NMDA receptor (excitotoxic damages) can be manipulated via GluN3A subunits because of their ability to reduce calcium permeability of classical NMDA receptors.

•

  Tight junction proteins claudins, occludin and ZO-1 are vital components of blood brain barrier which regulate barrier integrity by undergoing physicochemical changes such as change in phosphorylation, degradation, change in level of expression, accumulation in nucleus and endocytosis. RhoA/ROCK/MLC signaling pathways are a potential mechanism for inducing the change in morphology and alteration in cell-to-cell contact. This signaling mechanism is a multistep signaling cascade which leads to an alteration in cell-to-cell contact and tight junctions.

a

  potent molecule leading to the opening of blood brain-barrier. An in vivo study suggests that tPA can cross the blood brain-barrier and infiltrate in the parenchyma without alteration of BBB with a LRP dependent signaling pathway (Benchenane et al. 2005). On the contrary, tPA exhibits the characteristics of cytokines by upregulating the expression of ICAM-1 like adhesion molecules in the endothelial cells (Wang et al. 2014). tPA can induce the BBB breakdown independent of the role conventional MMPs (Copin et al. 2011). In addition, tPA can act as an active regulator of diapedesis of the monocytes across the BBB (Reijerkerk et al. 2008).The NMDA receptors are shown to be expressed in other cell types of the CNS, like the microglia (Kaindl etal. 2012). The presence of NMDA receptors in non-neuronal cells of CNS has been proved by glutamatergic signaling(Verkhratsky and Kirchhoff 2007; Boldyrev, Carpenter, and Johnson 2005). In fact, it has been also shown that NMDA receptors are expressed in cerebral endothelial cells. These endothelial NMDA receptors play an active role in alteration of the blood brain-barrier (András et[START_REF] López-Atalaya | Re o i a t Des odus Rotu dus Salivary Plasminogen Activator Crosses the Blood-Brain Barrier through a Low-Density Lipoprotein Receptor-Related Protein-Dependent Me ha is ithout E e ti g Neu oto i Effe ts[END_REF] Reijerkerk, Kooij, van der Pol, et al. 2010).

STUDY 2 :

 2 NEUROENDOTHELIAL NMDA RECEPTORS AS MODULATORS OF BLOOD BRAIN-BARRIER: MECHANISM OF ACTION AND INTERACTION WITH tPA(Article in preparation)

  barrier. under in vivo conditions tPA induces the opening of the blood brain-barrier by enhancing the vascular permeability of the capillaries via LDL receptor-related protein(LRP)(Yepes et al. 2003). Several in vivo studies confirm that tPA arbitrates the passage of tPA across the blood brain-barrier by transcytosis via LRP receptor (Benchenane etal. 2005;[START_REF] López-Atalaya | Re o i a t Des odus Rotu dus Salivary Plasminogen Activator Crosses the Blood-Brain Barrier through a Low-Density Lipoprotein Receptor-Related Protein-Dependent Me ha is ithout E e ti g Neu oto i Effe ts[END_REF]). tPA also inflicts a cytokine like effect on the brain endothelial cells inducing an upregulation of surface expression of the adhesion molecules(Wang et al. 2014). Under its deleterious effects on blood brain barrier, tPA also inflicts damage to the tight junction proteins such as occludin and ZO-1 (Reijerkerk etal. 2008; Hiu et al. 2008). It is also known that tPA mediates microglial activation and inflammatory outcomes(Pineda et al. 2012).N-Methyl D-Aspartate (NMDA) receptor are excitatory receptors which are involved in synaptic function of neurotransmission. tPA interacts with NMDA receptors and activates its receptor mediated signaling(Nicole et al. 2001). An in vitro study in neurons showed that tissue plasminogen activator specifically interacts with amino acid residue Arginine 260, where the plasminogen activator binds and cleaves the amino terminal domain (ATD) of GluN1 and induce NMDA receptor signaling pathways(Fernández- Monreal et al. 2004). It is also known that ATD of GluN1, on interaction with tPA, is responsible for initiation of calcium influx and associated neurotoxicity[START_REF] Lopez-Atalaya | To a d Safe Th o ol ti Age ts i St oke: Mole ula Re ui e e ts for NMDA Receptor-Mediated Neu oto i it[END_REF]).

  Images were digitally captured by Coolsnap camera (ORCA Flash 4-LT; Hamamtsu) coupled with Leica DM6000B microscope at 100X oil-immersion mode. Additionally, Leica SP5 confocal microscope by Lecia microsystems was used at 100X oil-immersion mode. Confocal images were captured in 1024 X 1024 high quality resolution with a z-step of 0.45 μ . Fo i age isualizatio , Meta o ph . soft a e ole ula devices) was used and while data analysis and processing were done ImageJ1.5h software (NIH). Confocal images were further analyzed and reconstructed into a 3D project by IMARIS software (version 5.5, Bitplane, Zurich, Switzerland). Treatments Upon reaching the desired confluency, the monocultures were maintained overnight (18 hours) in 1ng TNF-α i se u f ee edia ith o added g o th fa to s . Post-stimulation the cells underwent t eat e ts at ˚C a d % CO2 with different concentrations of Glycine (control {the EGM-2 SFM contains μM gl i e le els} 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM, vehicle SFM) for 1 and 3 hour. E e tuall , si ila o e t atio s e e late ad i iste ed ith μg/ l of tPA i uffe fo the sa e R E S U L T S time durations and at same conditions. In other sets of experiments after stimulation the cells were treated with different concentrations of NMDA(control, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM) and tPA (2 μg/ml and 4 μg/ml, vehicle control is buffer). In addition, similar concentrations of NMDA were administered with 2 μg/ml of tPA (in buffer) for 1 and 3 hour. The concentrations of glycine and NMDA were first decided to be gradients, so with the progression of study the appropriate EC50 concentration could be determined. Post treatments, the cells remain intact exhibiting no cell damage. .Western blotting Post-treatment, HCMEC/D3 cells were lysed in ice-cold TNT buffer containing protease inhibitor and phosphatase i hi ito a d e t ifuged at p fo i utes at ˚. The p otei o e t atio s were determined with Thermo S ie tifi ™ Pie e™ BCA P otei Assa based on bicinchoninic acid (BCA) for the colorimetric quantification of protein in samples. Denatured samples containing proteins (12-15 μg ea h e e loaded i SDS-PAGE (4-15%) and proteins were transferred by electro-blotting onto the PVDF membranes. Membranes were blocked for 1 hour in 2-3% BSA with TTBS buffer (0.1% Tween-20 in TBS buffer, pH 7.4). After three consecutive rinses (10 minutes each) with TTBS, membranes were i u ated o e ight at ˚C ith p i a a tibody diluted in TTBS. After three times rinses with TTBS (10 minutes each) the membranes were incubated with the HRP-conjugated secondary antibodies at the room temperature. Membranes were subsequently stripped for the detection of other proteins. GAPDH levels have been used as the loading controls. Protein bands were enhanced for analysis and detection by chemiluminescence agent Thermo Scientific Pierce ECL Plus substrate and captured digitally by ImageQuant LAS 4000 (GE Healthcare Life Sciences). The data analysis was done in ImageJ software (NIH).

  t. controls. The hCMEC/D3 cells were seeded onto collagen-coated Corning® Costar® Transwell® cell culture TC treated 12 inserts 24 well plate and grown in EGM-2 supplemented media and grown for 7-10 days. Post stimulation with TNF-α fo hours, the cells were treated with Glycine and NMDA in effect of tPA, along with required controls. The trans-monolayer permeability of FITC-Dextran (70 kDa, 1mg/ml in SFM, administered in the upper well)

  correlation. Another statistics e tio ed fo the stud of olo alizatio is Ma de s oeffi ie ts M a d M2 are the determinants of summed up intensities of pixels of one channel for which the intensities of the other channel is above zero, and vice versa. All the statistics demonstrate the ±SEM. For in vitro, the n value corresponds to the number of independent experiments of different passages. For determining the significance, Mann-Whitney U test was used.

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  Ma de s oeffi ie ts et ee the GluN B a d GluN A su u its ith GluN NMDA e epto su u it in the blood vessels. The immunostainings for other subunits of NMDA receptor (GluN2A, GluN2C, GluN2D and GluN3B) as shown in fig. 18E, show negative expression but positive GluN1 staining in vessels. Tissue plasminogen activator potentiates NMDA driven CREB activation and RhoA signaling activation, independently of AP5 blocking. CREB activation Our next step was to understand the mechanism of NMDA receptor driven opening of the blood brain barrier. Since we observed that cerebral endothelial cells show positive expression of GluN2B, hence we decided to check the effect of NMDA receptor agonist NMDA (which mimics the activity of glutamate). The NMDA receptor activation promotes CREB activation in neurons (Dudman et al. 2003; Sala, Rudolph-R E S U L T S Correia, and Sheng 2000). Hence, here in our study, we checked the CREB activation in cerebral endothelial cells. In fig. 21C, we show that tPA 20 μg/ l with NMDA (control, 5 μM, 10 μM, 25 μM, 50 μM, 750 and 100 μM) induces CREB activation after 1 and 3 hours of treatment in endothelial cells (n=3, *p < 0.05). Interestingly, individual treatments of tPA (buffer vs & μg/ l a d NMDA o t ol, μM, e ts did ot sho a CREB a ti it (shown in fig. 21A & 21B).It is also noticeable that NMDA-tPA induced CREB activity remains high until 1 hour and slightly decreases by 3 hours of treatment. We also analyzed the effect of classical NMDA antagonists on CREB activation, to reverse this effect. We show that the CREB activation by 25 μM of NMDA in presence of 2 μg/ml of tPA remains completely uninhibited (shown in fig.21D) by classical NMDA receptor antagonists 1 μM MK801 (NMDA receptor calcium channel blocker), 1 μM CNQX (glycine inhibitor) and 20 μM AP5 (competitive NMDA antagonist) at 1 and 3 hour (n=3, *p < 0.05). These results were highly intriguing, since these a tago ists effi ie tl lo k lassi al NMDA e epto s a ti it . Furthermore, we checked the calcium influx in hCMEC/D3 cells for which we performed calcium imaging. Adding more speculation to our study, we show that neither NMDA (fig.21E) alone nor in co-treatment with tPA (fig.21F) induced any calcium ion influx.RhoA ActivationSubsequently, we checked the RhoA-GTP levels in reference to the study referring to an increased endothelial permeability in the effect of RhoA signaling pathway along with CREB activation (Zhong etal. 2012). Here we show that that NMDA-tPA co-treatment induces a dose dependent increase in RhoA-GTP activation, which is shown fig.22A(n=4, *p < 0.05). Interestingly, we also observed that RhoA-GTP protein levels slightly decrease after 3 hours of treatment in endothelial cells. Further, we analyzed the MLC activation, which is mediated by RhoA associated kinase, ROCK. We show in fig.22B(n=4, **p < 0.005) that NMDA-tPA co-treatment increase in levels of phosphorylated MLC protein. Interestingly, ROCK inhibitor Y27632, completely blocks the MLC phosphorylation (fig. 22C, (n=4, *p < 0.05)). This data also falls in line with subsequent increase in GTP RhoA levels at the same concentrations. RhoA signaling pathway is one well known mechanism causing alteration of endothelial monolayer integrity. This signaling pathway can manipulate the cell morphology by rearrangement of cytoskeletal components and loosening of cell-to-cell contacts, change in cell motility and cell structure (Takaishi et al. 1993). R E S U L T S Tissue plasminogen activator with NMDA potentiates the damage to the endothelial monolayer integrity.

  treatments. GAPDH was used as loading control, and 12-μg p otei as used pe ell. We observed that tPA treat e t ith μg/ l a d μg/ l of tPA did not change levels of PCREB, as shown in fig.24A.Interestingly, we observed an increase in CREB activity after just 1 hour treatment with glycine (n=3, *p < 0.05). Although this effect was not persistent until the 3 hours of glycine treatment (fig.24B). However, we observed a prolonged CREB activation by glycine until 3 hours in presence of tPA (fig.24C, (n=3, *p < 0.05)). We also checked whether the NMDA receptor antagonists inhibited glycine-tPA driven CREB activation. Interestingly, as shown in fig.24D, we observed that CNQX (a glycine binding site NMDA receptor antagonist) completely reversed the effect of glycine-tPA to trigger CREB activation in endothelial cells ((n=3, *p < 0.05)).R E S U L T SAs a counter check, we again checked if this activity of CREB signaling was dependent on calcium influx.

Fig .

 . Fig. 24E and 24F show that neither glycine alone nor glycine-tPA could induce any calcium influx. This again falls in line with the fact that Glun3A receptor subtypes have very low calcium permeability.

  NMDA receptors have been shown to regulate the adherens and tight junctions in brain microvasculature(Beard, Reynolds, and Bearden 2011). It has been shown that glutamate stimulated NMDA receptors are involved in occludin expression and phosphorylation in brain endothelial cells(András et al. 2007). This close association of the NMDA receptors with transmembrane tight junction proteins claudin-5 and occludin may allow the receptor driven alteration of the blood brain barrier.Furthermore, we also suggest that the NMDA receptor expression in cerebral endothelial cells is upregulated under the proinflammatory conditions. TNF-α upregulates the expression of the GluN1 and R E S U L T S subsequent activation of myosin light chain protein (MLC), which is completely blocked by ROCK (Rho associated kinase) inhibitor Y27632. It is suggested that MLC activation leads to change in cytoskeleton arrangement there by altering cell morphology, motility and association of adherens and tight junctions with cytoskeleton (Ramachandran et al. 2011; R. S.

  are localized in the luminal surface of the brain vascular endothelium. Our finding falls in line with a previous in vivo study which suggests the NMDA receptor expression by brain endothelial cells(Reijerkerk et al. 2010). Furthermore, we also confirm that NMDA receptors are expressed in a close association with the tight junction proteins occludin, claudin-5, zonula occludens -1 (ZO-1) and VE cadherins. Alongside, we also show that NMDA receptors are much closely associated with the transmembrane occludin and claudin-5 proteins, while distant from the intracellular ZO-1 and adherens junction protein VE cadherins.NMDA receptors in brain endothelial cells are long discussed and investigated for their role in the blood brain barrier opening. The close association of the endothelial NMDA receptors with the tight junction proteins is highly interesting, adding to the fact that NMDA receptor activation potentially leads to the blood brain barrier and blood spinal cord barrier opening via alteration of the tight junction protein occludin distribution and subsequent expression (András etal. 2007). In addition, we also show that the specific endothelial cells of the CNS, which are deprived of tight junctions, and other characteristic features of blood brain barrier, also lack NMDA receptor expression.The knowledge of close association of NMDA receptor expression with the tight junction proteins allows the exploration of underlying mechanism by which NMDA receptor modulate the endothelial barrier. Our findings of close association of NMDA receptors supports the study that NMDA receptor activation via MEK1/2-ERK1/2 signaling, suppresses the transendothelial electrical resistance and occludin levels in mouse cerebrovascular endothelial cells[START_REF] Chen | Roles of NMDARs i Maintenance of the Mouse Cerebrovascular Endothelial Cell-Co st u ted Tight Ju tio Ba ie[END_REF]).The luminal marking of NMDA receptor subunit by the mouse monoclonal antibody Glunomab suggests that the NMDA receptors are expressed towards the lumen of the vessel. Eventual blockage of blood brain barrier and blood spinal cord barrier alteration and prevention of immune cell infiltration by Glunomab implies the potential role of luminal NMDA receptor expression in endothelial cells of microvessels[START_REF] Macrez R 1 | Chevilley A 1[END_REF]).

  endothelial cells. Activation of metabotropic signaling pathways is one of the characteristics of nonionotropic NMDA receptors. For instance, metabotropic NMDA receptors induce Src kinases signaling during excitotoxicity, while engaging the pannexin-1 Src complex formation (Weilinger etal. 2016). The independency of metabotropic receptors from ion flux and other synaptic functions makes them feasibly functional for endothelial cells, which are neither excitable nor ionotropic. The expression of GluN3A expressing NMDA receptors in endothelial cells ensures their probable role in blood brain barrier dysfunction independently of its ionotropic effects of classical NMDA receptors.

  There are several studies, which show how CREB activation can influence the cell fate. For instance, NMDA mediated toxicity is evaded by tat-NR2B peptide, which enhances calcium dependent phosphorylation of CREB protein and CaMKIV phosphorylation. This results in reduction in infarct size during ischemic injury(Bell et al. 2013). Similarly, phosphorylation of CREB and activation of CaMKII signaling cascade prevents the damaging effects of cerebral ischemia reperfusion injury(Zhang et al. 2017). CREB can modulate the expression of tight junction proteins. CREB upregulates the ZO-1 levels in nucleus via binding to the ZO-1 promoter, probably which leads to the recovery of the endothelial barrier junctional properties (Zhong etal. 2012). Although, in our current study, the effect of CREB activation on the endothelial barrier is still unknown.The specific role of tissue plasminogen activator (tPA) in CREB activation in the presence of NMDA and glycine is also interesting. As we confirm that tPA in presence of NMDA triggers CREB activation, on the other hand, tPA prolongs the CREB activation which is induced by glycine. This role of tPA brings many studies in light, which have shown the role of tPA in CREB signaling pathway. Interestingly, in an in vitro

  permeability. For instance, chemoattractant CCL2 induces endothelial hyperpermeability via RhoA/PKC signaling pathways (Stamatovic et al. 2006). Another study suggests that TNF-α i du ed MLC phosphorylation increases endothelial permeability which results in inflammation (Mong and Wang 2009). VEGF induces venular hyperpermeability stimulated by MLC phosphorylation (Sun et al. 2006).RhoA signaling activation mediates Rho-kinase (ROCK) activation. ROCK activation promotes MLC activation by stimulating MLC phosphatase. To confirm this event, we checked the inhibitory effect of Y-27632 (a ROCK inhibitor). In theory when ROCK activation is inhibited by Y-27632, it halts the MLC phosphorylation. This inhibitory effect prevents from RhoA signaling induced blood brain barrier damage(Liao, Seto, and Noma 2007; Sun et al. 2006). In our study, we confirm that NMDA-tPA treatment induced MLC phosphorylation is inhibited by Y-27632. We know that MLC activation is one of the high ends where RhoA signaling cascades finally imposes the alterations on endothelial cells morphology by compromising the cell-to-cell contacts. Hence the activation RhoA signaling cascade is a promising mechanism employed in metabotropic role of endothelial NMDA receptors.

Akaza a ,

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Figure 3. Tight Junction Proteins and Associated Intracellular Components of the Endothelial and Epithelial Junctions.

	The
	endothelial and epithelial junctions are formed
	by the transmembrane proteins such as
	occludin, claudins, JAMs in association with
	intramembranous linking proteins ZOs (ZO-1,
	ZO-2 and ZO-3) which form connection with
	cytoskeletal actin strands. Figure adapted from
	(Aktories and Barbieri 2005).

  The potent excitatory neurotransmitter glutamate dramatically alters the integrity of the blood brain barrier when it is present in very high levels under pathophysiological conditions such as cerebral ischemia. It has been shown in an in vitro study with cultured brain microvascular endothelial cells (BMECs) that a high level of

glutamate (1mM) increases tyrosine phosphorylation and decreases threonine phosphorylation of occludin

(András et al. 2007

).

Key points: It is conclusive from these studies that cadherins regulate the endothelial barrier integrity and stabilization. These proteins are associated with intracellular intermediate filaments and cytoskeletal proteins. Wnt-β ate i sig ali g plays a crucial role in the regulation of cadherins.

	2014). Such characteristics are found with adherens junction at the BBB (Liebner, Gerhardt, and Wolburg 2000)
	and BCSFB (Lippoldt et al. 2000).
	β-Catenin is also localized in the endothelial nuclei for the transcription control during brain angiogenesis
	regulatio a d BBB differe tiatio	i du i g W t/β-Catenin signaling pathway (Engelhardt and Liebner 2014).
	Using the immunogold cytochemistry applied to electron microscopy, it has been shown that P120-Catenin is
	detected at ultrastructural levels in adherens junctions in human brain microvessels (Dobrogowska and Vorbrodt
	2004) where they bind to the juxtamembrane region of the VE-and E-cadherin. p120-Catenin maintains actin
	cytoskeleton dynamics by regulating the Rho family GTPases, and stabilizes the adherens junctions by preventing
	e do tosis of adheri s Vest e er	. γ-Catenin, another member of the family, binds with VE-cadherin
	cytoplasmic domain and links the cadherins to the intermediate filaments. α-Catenin lastly, are localized at the
	ultrastructural level, observed in mouse and human endothelial cells at the interendothelial junction in blood
	vessels (Vorbrodt and Dobrogowska 2003) and in adherens junction in BCSFB epithelial cells (Lippoldt et al. 2000).
		. These homophilic
	adhesion molecules control the stabilization of blood vessel. Moreover, VE cadherin prevents the disassembly of
	blood vessels (Carmeliet et al. 1999; Crosby 2005). E-cadherin is highly expressed in the epithelial cells of the
	choroid plexus (Marrs 1993; Figarella-Branger et al. 1995), while VE-cadherin is localized in the blood vessels of
	the CNS (Vorbrodt and Dobrogowska 2003). Another in vitro and in vivo in brain endothelial cells suggests that N-
	cadherin is a transmembrane protein from the cadherin family, which mediates pericyte endothelial interactions
	during angiogenesis (Liebner, Gerhardt, and Wolburg 2000). An in vivo study suggests that VE-PTP (phospho
	tyrosine phosphate), controls the phosphorylation of VE-cadherin, thereby influencing the vascular integrity and
	leucocyte diapedesis (Küppers et al. 2014).
	Catenins
	Catenin proteins are another family of intracellular scaffolding proteins associated with the adherens junctions. It
	has been shown in Drosophila embryo model for adherens junction assembly that β-Catenin and p120-Catenin
	are armadillo repeat proteins (T. J. C. Harris 2012) which are associated with the cytoplasmic domain of the VE-
	cadherin and E-cadherin. An in vivo study suggests that β-Catenin binds to the distal regions of the cytoplasmic
	domains of the cadherins, and hence they form a link between f-actin proteins of cytoskeleton (Buckley et al.

Key points: Catenin all together regulate BBB differentiation by Wnt-β ate i sig ali g. They ai tai cytoskeleton dynamics and stabilize adherens junctions. Key points: These studies provide put forward a clear idea that tight junctions and adherens junctions are essential components of the blood brain barrier. Tight junctions and adherens junctions are strictly regulated by many signaling pathways. These signaling pathways regulate the protein expression, degradation, assembly, disassembly, translocation, and accumulation of these junction proteins which thereby regulate the endothelial barrier integrity. The signaling pathways are do not only have a regulatory role in tight junction proteins necessarily. Many signaling cascades have been reported which mediate the opening of the blood brain barrier. barrier

  ). PECAM-1 promotes a steady-state barrier function of endothelial cells and restoration of the blood brain integrity (Graesser etal. 2002; Privratsky and Newman 2014). Moreover, Nectin is another adherens junction molecule, a member of the Ig superfamily. They exercise their interaction with adaptor protein afadin.

Key points: A direct effect of PKC signaling on endothelial tight junction and adherens junctions is not clear because this pathway is much more involved in maintenance of cell polarity, but PKC signaling can show a coaction with other signaling pathways and influence the endothelial barrier.

	These proteins have two different (calcium dependent and calcium independent) modes of
	mechanism upon activation, which depends on exactly which of the one isoform is activated. Conventional
	isoforms (cPKC) are calcium dependent and novel isoforms (nPKC) are calcium independent. PKC is involved in the
	disassembly of the tight junction proteins. Under the oxidative stress, PKC mediates alteration in actin
	cytoskeleton and tight junction proteins (Pérez et al. 2006). In many studies it has been shown that PKC signaling
	can induce inflammatory outcomes. For example, PKC signaling triggered by cAMP-activated guanine exchange

We can conclude that RhoA/ROCK/MLCK/MLC signaling is one of the crucial signaling cascades in neuroinflammation, which individually or in coaction of other signaling pathways can adversely affect endothelial integrity by inducing changes in cell morphology, contractility, and cell-to-cell contacts. This cascade can be blocked by several inhibitors, which in the end prevents the MLC activation. Rhosin, Fasudil, Y27632 are a few very common inhibitors which halt the RhoA/ROCK/MLCK/MLC signaling pathway at different checkpoints.

. Although PKC and MLC activation can have debatable influences, since PKC activation and excessive increase in MLCK phosphorylation can be concurrent activities. Despite of the discrepancies, PKC activation does show a protective mode of action leading the junction assembly at cell borders against RhoA signaling. RhoA and MLCK pathways can induce caspase activation and production of inflammatory cytokines, which promotes neuroinflammation. An in vitro study suggests that RhoA/ROCK can act a potential target for the inflammatory mediators leading to MLC activation and results in endothelial contraction. The same study suggests that cyclic AMP can block bacterial LPS induced MLC phosphorylation in HUVECs (Essler et al. 2000). MLC activation Key points:

  2+ /calmodulin-dependent protein kinase II (CaMKII)/CREB signaling pathway has been suggested to modulate the endothelial barrier function. For instance, in an in vivo study a neuroprotective drug (paeoniflorin) regulates the Ca2+/CaMKII/CREB signaling pathway, which acts as a regulating factor to attenuate the damaging effects of cerebral ischemic injury decreasing the neurological deficit scores and infarct volume in vivo (Y.Zhang et al. 2017). An in vitro study in HUVECs suggest that endothelial dysfunction and inflammation under high glucose levels were prevented by the inhibition of CaMKII/CREB phosphorylation and suppression of PP2A expression by Propofol (a widely used anesthetic drug) (Q.Wu et al. 2017). An in vivo study in the valproic acid (VPA) rat model of autism shows that an increase in levels of Ca 2+ /CaMKII and CREB under the neuroprotective agent, results in a weakens the social deficits, learning and memory impairment, neuronal loss, and inflammation (H.Wu et al. 2017). These studies put forward a clear indication that CaMKII signaling in coaction with activated CREB can regulate the inflammatory outcomes, but the entire idea, whether it is a positive regulation or a negative regulation, is not clear yet.

serine proteases, which catalyzes the fibrinolysis of the blood clots, and thereby regularization of the blood flow during stroke. tPA also has deleterious effects on the BBB. There have been various mechanisms by which it has been shown that tPA can induce apoptosis, blood brain barrier breakdown, and neuroinflammation. This involves its effect on microvessels, pericytes, astrocytes, microglia, and oligodendrocytes.

  Synthesized as single polypeptide molecule with 34 disulfide bonds, it is released as single chain tPA (sctPA). The other form of tPA is two chain tPA (tctPA). It is formed when this long polypeptide is cleaved at arginine 275 and isoleucine 276, creating a heavy chain A and a light chain B. The heavy chain A has four distinct regions which are functionally different. First domain is a finger domain which is represented by amino acid

Though not fibrinolytic by itself, tPA catalyzes fibrin-bound plasminogen into plasmin. While its interaction with tPA, plas i oge ha ges its o for atio fro lose to ope to fa ilitate the lea age tPA Fle ig a d Melzig 2012). However, the therapeutic effects of tPA are much celebrated but in recent years, many studies have reported the deleterious damaging effects of tPA. There are various mechanisms by which it has been shown that tPA can induce apoptosis, blood brain barrier breakdown, and neuroinflammation. This involves its effect on microvessels, pericytes, astrocytes, microglia and oligodendrocytes. tPA and its Structure tPA is a 69-kDa glycoprotein, which is composed of 527 or 530 amino acids containing single polypeptide chain (figure 12). residues 6 to 43. It is termed as the finger (F) domain and is homologous to the first domain of fibronectin. This Key points: tPA, a

  our current study, we have achieved a new insight into the role of neuroendothelial NMDA receptors in the opening of the blood brain barrier. The NMDA receptors of the brain endothelial cells are non-Bibliography Alkabie, Samir et al. 2016. SPARC E p essio Ce e al Mi o as ula E dothelial Cells i Vit o a d Its Influence on Blood-Brain-barrier P ope ties. Journal of neuroinflammation 13(1): 225. Asha i, Gho a gol et al.. NMDA Re eptor Adjusted Co-Administration of Ecstasy and Cannabinoid Receptor-1 Agonist in the Amygdala via Stimulation of BDNF/Trk-B/CREB Path a i Adult Male Rats. Jassa , Sa ah A et al. . TNF-α E ha e e t of CD E Mediates Adhesio of No -Small Cell Lung Cancer Cells to Brain Endothelium via CD15 in Lung-B ai Metastasis. Neu o-oncology 18(5): 679-90. Kahles, Ti o et al. . Tissue Plas i oge A ti ato Mediated Blood-Brain-barrier Damage in Transient Focal Cerebral Ischemia in Rats: Relevance of Interactions between Thrombotic Material and Th o ol ti Age t. Vas ula pha a olog : -59. Kaindl, Angela M. et al. 2012. A ti atio of Mi oglial N-Methyl-D-Aspartate Receptors Triggers Inflammation and Neuronal Cell Death in the Developing and Matu e B ai . A als of Neu olog : 536-49. Kása, A ita et al. . P otei Phosphatase A A ti it Is Re ui ed fo Fu tio al Adhe e t Ju tio s i E dothelial Cells. Mi o as ula Resea h : -94. Ki u a, K. et al. . Regulatio of the Asso iation of Adducin with Actin Filaments by Rho-Associated Kinase (Rho-Ki ase a d M osi Phosphatase. Jou al of Biologi al Che ist : -48. K ist, T i e et al. . St u tu e-Based Discovery of Antagonists for GluN3-Containing N-Methyl-D-Aspa tate Re epto s.Lester, R A et al. 1989. I te a tio of -Cyano-7-Nitroquinoxaline-2,3-Dione with the N-Methyl-D-Aspartate Receptor-Asso iated Gl i e Bi di g Site. Mole ula pha a olog : -70. Lopes Pinheiro, Melissa A. et al. 2016. I u e Cell T affi ki g a oss the Ba ie s of the Ce t al Ne ous S ste i Multiple S le osis a d St oke. Bio hi i a et Bioph si a A ta BBA -Molecular Basis of Disease 1862(3): 461-71. Lopez-Atalaya, Jose P et al. 2008. To a d Safe Th o ol ti Agents in Stroke: Molecular Requirements for NMDA Receptor-Mediated Neu oto i it . Jou al of Ce e al Blood Flo & Meta olis : -21. López-Atalaya, José P et al. 2007. Re o i a t Des odus Rotu dus Sali a Plas i oge A ti ato Crosses the Blood-Brain-barrier through a Low-Density Lipoprotein Receptor-Related Protein-Dependent Characterization of a Novel NMDA Receptor Subunit NR3B: A Dominant Subunit That Reduces Calcium Pe ea ilit . B ai esea h. Mole ula ai esea h -2): 43-52.

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conventional NMDA receptors expressing GluN1, GluN2B and GluN3A subunits. These receptors exhibit metabotropic activity upon activation by NMDA receptor agonists NMDA, and glycine with tPA. NMDA-tPA and glycine-tPA induce RhoA/MLC activation and CREB activation. Although the link between these two pathways is not clear yet. NMDA receptor agonists, in presence of tPA induce an increase in permeability across the unilayer and change in morphology of endothelial cells. Recent data that we have presented here provides us an enough table to understand NMDA receptor activation mediated neuroinflammation.

Matsuda, Keiko, Yoshi o i Ka i a, Shi ji Matsuda, a d Mi hisuke Yuzaki.

. Clo i g a d

Discussion

  The objective of our studies[START_REF] Macrez R 1 | Chevilley A 1[END_REF]) a d Pote tial role of eta otropi NMDA re eptors i tPA i du ed CREB a d RhoA a ti atio ediated lood rai arrier dysfu tio arti le i preparatio has been focused on the role of neuroendothelial NMDA receptors during inflammation. These studies so far have been conclusive of the following.

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  A , Jie et al.. Tissue-Type Plasminogen Activator and the Low-Density Lipoprotein Receptor-Related P otei I du e Akt Phospho latio i the Is he i B ai . Blood : -94.Anderson, J M etal. 1988. Cha a te izatio of ZO-1, a Protein Component of the Tight Junction from Mouse Liver and Madin-Da Ca i e Kid e Cells. The Jou al of Cell Biology 106(4): 1141-49. A de sso , O, A Ste ist, A Atte sa d, a d G o Eule . . Nu leotide Se ue e, Genomic Organization, and Chromosomal Localization of Genes Encoding the Human NMDA Receptor Subunits NR A a d NR B. Genomics 78: 178-84. András, Ibolya E et al. 2007. The NMDA a d AMPA/KA Re epto s A e I ol ed i Gluta ate-Induced Alterations of Occludi E p essio a d Phospho latio i B ai E dothelial Cells. Jou al of Ce e al Blood Flow & Metabolism 27(8): 1431-43. Antonetti, D A et al. 1999. Vas ula E dothelial G o th Fa to I du es Rapid Phospho latio of Tight Junction Proteins Occludin and Zonula Occluden 1. A Potential Mechanism for Vascular Permeability in Dia eti Reti opath a d Tu o s. The Jou al of Biological Chemistry 274(33): 23463-67. Antonetti, David A. et al. 2002. H d o o tiso e De eases Reti al E dothelial Cell Wate a d Solute Flux Coi ide t ith I eased Co te t a d De eased Phospho latio of O ludi . Jou al of Neurochemistry 80(4): 667-77. Arrate, M. P. et al. 2001. Clo i g of Hu a Ju tio al Adhesio Mole ule JAM a d Its Ide tifi atio as the JAM2 Counter-Re epto . Jou al of Biologi al Che ist : -32. Asha i, Gho a gol et al. . NMDA Re epto Adjusted Co-Administration of Ecstasy and Cannabinoid Receptor-1 Agonist in the Amygdala via Stimulation of BDNF/Trk-B/CREB Pathway in Adult Male Rats. Brain Research Bulletin 130: 221-30. Aurrand-Lio s, M. . Hete oge eit of E dothelial Ju tio s Is Refle ted Diffe e tial E p essio a d Spe ifi Su ellula Lo alizatio of the Th ee JAM Fa il Me e s. Blood : -3707. Avila-Flores, A et al. 2001. Tight-Junction Protein Zonula Occludens 2 Is a Target of Phosphorylation by P otei Ki ase C. The Biochemical Journal 360(2): 295-304. Balasuriya, Dilshan, Hirohide Takahashi, Shyam Srivats, and J. Michael Edwardson. 2014. Activation-Induced Structural Change in the GluN1/GluN3A Excitatory Glycine Receptor. Biochemical and Biophysical Research Communications 450(4): 1452-1457.

  Copin, Jean Christophe et al. 2011. Re o i a t Tissue Plas i oge A ti ato I du es Blood-Brain Barrier Breakdown by a Matrix Metalloproteinase-9-Independent Pathway after Transient Focal Cerebral Is he ia i Mouse. Eu opea Jou al of Neu os ie e : -92. Cops, Elisa J. et al. . Tissue-Type Plasminogen Activator Is an Extracellular Mediator of Purkinje Cell Da age a d Alte ed Gait. E pe i e tal Neu olog : -19. C aig, J C. . Co se sus a d Va ia t AMP-regulated Enhancers Have Distinct CREB Binding P ope ties. Journal of Biological Chemistry 276: 11719-28. C os , C. V. . VE-Cadherin Is Not Required for the Formation of Nascent Blood Vessels but Acts to P e e t Thei Disasse l . Blood : -76. Cuadrado, Elo et al. . Tissue Plas i oge A ti ato T-PA) Promotes Neutrophil Degranulation and MMP-Release. Jou al of Leukocyte Biology 84(1): 207-14. Cull-Ca d , S. G., a d D. N. Leszkie i z. . Role of Disti t NMDA Re epto Su t pes at Ce t al S apses. S ie e Sig ali g : e -re16.

  A Si gle GluN Su u it Residue Co t ols NMDA Receptor Channel Properties via Intersubunit I te a tio . Natu e Neu os ience 15: 406-13. Silla e , C et al. . The Mast Cell as Site of Tissue-Type Plasminogen Activator Expression and Fi i ol sis. Jou al of Immunology Balti o e, Md. : : -41. Simonovic, Ivana, Jonathan Rose e g, Atha asia Koutsou is, a d Gail He ht. . E te opathoge i Es he i hia Coli Dephospho lates a d Disso iates O ludi f o I testi al Epithelial Tight Ju tio s. Singh, A. K, Y. Jiang, S. Gupta, and E. Benlha i . . Effe ts of Ch o i Etha ol D i ki g o the Blood Brain Barrier and Ensuing Neuronal Toxicity in Alcohol-Preferring Rats Subjected to Intraperitoneal LPS I je tio . Al ohol a d Al oholis : -99. Smothers, C. Thetford, and John J. Wood a d. . Pha a ologi al Cha a te izatio of Gl i e-Activated Currents in HEK 293 Cells Expressing N-Methyl-D-Aspa tate NR a d NR Su u its. The Jou al of Pharmacology and Experimental Therapeutics 322(2): 739-48. Smothers, C.Thetford, and John J. Wood a d. . Effe t of the NR Su u it o Etha ol I hi itio of Re o i a t NMDA Re epto s. B ai Resea h : -21. Sordella, Raffaella, Marie Classon, Kang-Quan Hu, Stephen F. Matheson, Madeleine R. Brouns, Barry Fine, Le Zhang, et al. 2002. Modulatio of CREB A ti it the Rho GTPase Regulates Cell a d O ga is Size du i g Mouse E o i De elop e t. De elop e tal Cell : -65. Spie i g, Dési ée, a d Louis Hodgso . . D a i s of the Rho-Family Small GTPases in Actin Regulatio a d Motilit . Cell Adhesio a d Mig atio : -80. Sp e gel, R. . I po ta e of the I t a ellula Do ai of NR Su u its fo NMDA Re epto Fu tio i Vi o. Cell 92: 279-89. Sreekanthreddy, Peddagangannagari et al. 2015. A Th ee-Dimensional Model of the Human Blood-Brain Ba ie to A al se the T a spo t of Na opa ti les a d Ast o te/e dothelial I te a tio s. F Resea h 4: 1279. Srinivas, S.P. et al. 2004. Ade osi e I du es Dephospho latio of M osi II Regulato Light Chai i Cultu ed Bo i e Co eal E dothelial Cells. E pe i e tal E e Resea h : -51. Sta ato i , S etla a M, Oli e B Di it ije i , Ri ha d F Keep, a d A uska V A djelko i . . P otei Kinase Calpha-RhoA Cross-Talk in CCL2-Induced Alterations in Brain Endothelial Pe ea ilit . The Jou al of Biological Chemistry 281(13): 8379-88. St e , A ke, A ette Ja i g, Holge Ba th, a d Volke Ge ke. . E dothelial Rho Sig ali g Is Re ui ed fo Mo o te T a se dothelial Mig atio . FEBS Lette s -3): 261-66.

	Cellular Microbiology 2(4): 305-15.

DISCUSSION

R E S U L T S provided by the institutional ethics committee (Co ite´ No a d d'e´thi ue e

atie` e d'expe´ i e tatio a i ale, CeNo ExA) were strictly followed.

Immunohistochemistry

C57BL6/J mice were deeply anesthetized and perfused transcardially only with cold saline with heparin.

The ai a d spi al o d tissue e e e ised a d i sed o e i sali e. The tissues e e sto ed at ˚C i increasing different gradient concentrations of sucrose (w/v) in phosphate buffer saline at pH 7.4 until the tissue sank down in each incubation. Later, the tissues were secured at -˚C i tissue-tek (miles Scientific). The tissue sections as thick as 1 μm from cryomicrotome were collected at on poly-lysine coated slides, mounted with Superfrost® Plus slide and stored at -˚C.

Staining protocol

The poly-lysine slides were taken out from -˚C to ea h at oo te pe atu e, afte hi h l of % paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) was added on the top of the tissues and allowed to rest for 15 minutes. The paraformaldehyde solution was carefully discarded and slides were rinsed with veronal buffer for five minutes, followed by incubation of slides in veronal buffer with 0.2% BSA for 15 minutes. After blocking, the slides were rinsed with veronal buffer for fourfive times for 10 minutes each. Hence, the tissue samples were incubated in primary antibody (with goat polyclonal anti-GluN1 sc1467 antibody, goat polyclonal anti-GluN2A sc1468 antibody, rabbit polyclonal anti-GluN2B sc9057 antibody, mouse monoclonal anti-GluN2B sc365597 antibody, goat polyclonal anti-GluN2C sc1470 antibody, goat polyclonal anti-GluN2D sc31551 antibody, rabbit polyclonal anti-GluN3A 07-356 antibody and rabbit polyclonal anti-GluN3B sc-50474 antibody, mouse specific goat polyclonal anti-podocalyxin antibody (R&D systems, goat anti-Collagen IV antibody (southern Biotech), mouse monoclonal Glunomab, rabbit monoclonal anti-occludin ab167171 antibody, rabbit monoclonal anti-claudin-5 ab131259

antibody, rabbit polyclonal anti-ZO-1 ab59720 antibody, rabbit polyclonal anti-VE cadherin ab33168 antibody antibody) dilutions prepared in veronal buffer with 0.25% of triton (100X) at 4°C. Following a rinsing with veronal buffer for three times, tissues samples were incubated with secondary antibodies diluted (1:600) in veronal buffer with 0.25% of triton (100X) for 90 minutes at room temperature. Again, followed by three times rinsing with veronal buffer the tissues were mounted with DAPI containing protective medium (to prevent evanescing) under the coverslip. Each experiment of immunolabeling was accompanied with required controls. Absence of relevant signals in multi-immunolabeling was detected with only the cocktail of secondary antibody and no primary antibody incubation with the tissue. Similarly, R E S U L T S

NMDA receptors of the brain endothelial cells are expressed in close vicinity of claudin-5 and occludin tight junction proteins but far from ZO-1

We further analyzed the association of endothelial NMDA receptors with tight junction proteins through immunohistochemistry. In fig. 19, we show that the NMDA receptors (positive GluN1 staining in microvessels) are very closely expressed to the tight junction proteins claudin-5 and occludin whereas they are farther from ZO-1 and the adherens junction protein VE cadherin. In fig. 19A, we show we show the brain microvessels exhibiting claudin-5, occludin, ZO-1 and VE-cadherin expressed in close vicinity of NMDA receptor. Through 3D reconstructs of microvessels in fig. 19B, we show that upon analysis, at comparatively similar thresholds, we find high quantity of colocalized channel (seen in yellow pixels)

shared by GluN1 with occludin and claudin-5. In addition we observe that the intensity of colocalization is decreased in vessels stained of ZO-1 and VE cadherin with GluN1. Interestingly, in line with the observations above, we also found that occludin (r = 0.684) and claudin-5 (r = 0.806) demonstrate high positi e Pea so s o elatio (r) with GluN1 as shown in fig. 19C. Moreover, ZO-1 (r = 0.404) and VE cadherin (r = 0.350) co-immunostaining with GluN1, colocalization correlation is observed lesser than the other tight junction proteins.

TNF-α sti ulated hu a e e al e dothelial ells exp ess elevated levels of NMDA e epto su u its.

We also analyzed the expression levels of GluN1, GluN2B and GluN3A in hCMEC/D3. Firstly, via immunocytochemistry (in fig. 20A) we show that hCMEC/D3 cells exhibit positive staining for GluN1, GluN2B, and GluN3A subunits. We also show that TNF-α i du es a i ease i GluN a d GluN A expression in endothelial cells (in representative pictures, magenta (GluN1, GluN2B, and GluN3A) with nuclear staining in blue). Secondly, we analyzed the corrected total cell fluorescence (CTCF, method used to calculate rectified total cell fluorescence by background subtraction) in TNF-α sti ulated ells . .t.

control (fig. 20B).

We observed that the GluN1 CTCF levels increased by two folds (n=5, ****p < 0.0001) in TNF-α stimulated cells. Similarly, GluN3A C levels increased by three folds (n=5, ****p < 0.0001) after TNF-α sti ulatio . Although we did not observe any significant difference in GluN2B CTCF levels after TNF-α treatment. Thirdly, we checked the protein levels of these subunits in HCMEC/D3 cells after TNF-α treatment. In fig. 20C we show that the level of GluN1 expression in increased by 50% (n=3, *p < 0.05)

after TNF-α sti ulatio i e dothelial ells. I te estingly, TNF-α t eat e t doubles the GluN3A levels in these cells (n=3, *p < 0.05) while GluN2B levels remain the same. We have analyzed glyceraldehyde 3phosphate dehydrogenase (GAPDH) levels as loading controls.

R E S U L T S

RhoA Activation

We checked the RhoA activation in endothelial cells under the effect of glycine and glycine-tPA Hence, we decided to check the levels of GTP bound RhoA after 25 μM glycine-μg/ l tPA t eatment in endothelial cells for 1 and 3 hours. In Fig. 25A, we show that the increase in RhoA-GTP levels under the glycine is small. Although these levels are increased under the effect of tPA after 1 and 3 hour of treatment (n=3, p < 0.05). To check the effect of NMDA receptor antagonists to reverse RhoA activation, we administered the 250 µM glycine with 20µg/ml tPA along with 10 µM MK801, 200 µM AP5 and 10 µM CNQX. In fig. 25B, we show that CNQX completely inhibits the RhoA activation, which is observed as a steep decrease in GTP bound RhoA levels (n=3, p < 0.05).

R E S U L T S

GluN3A subunits of the NMDA receptors in brain endothelial cells. This result also coincides with an increase in GluN1 surface expression in hippocampal neurons under proinflammatory cytokine effect of TNF-α (Wheeler et al. 2009;Shen et al. 2011;Olmos and Lladó 2014). Upregulation of the surface expressed receptors on the endothelial surface is one of a crucial and potentiating activity on the onset of neuroinflammation.

The objective of this study is to determine the mechanism by which NMDA receptor mediates the opening of blood brain-barrier. For this purpose a clear establishment of facts regarding the expression of NMDA receptor subtype in brain endothelial cells and their association with tight junction was essential and was accomplished in the first part of the study.

In this study we confirm that NMDA receptor agonists NMDA (a GluN2B agonist) and glycine (a GluN3A agonist) have a huge impact on the blood brain barrier under the influence of tissue plasminogen activator. We suggest that NMDA with tPA triggers CREB activation on brain endothelial cells. In a slight dissimilar pattern, we observed that glycine, which is a GluN3A specific agonist induces CREB activation too. Although, this effect does not persist for long. However, in presence of tPA, the effect of glycine induced CREB activation is prolonged and is noticeable until 3 hours. Interestingly, these data have provided us a new insight into the catalytic and a pharmacologically important role tPA for NMDA receptor activation in endothelial cells. High cytoplasmic cAMP levels serves as a stimulus for CREB activation. Upon activation, CREB regulates the transcription of target genes and hence alters the protein levels. Our data falls in line with studies which suggest that NMDA receptor regulates the cAMP element-binding protein (CREB) signaling and associated signaling pathways (Sala, Rudolph-Correia, and Sheng 2000;Dudman et al. 2003;Perkinton et al. 2002).

Furthermore, we confirm that NMDA receptor agonists NMDA and glycine under the effect of tPA induce RhoA signaling pathway in brain endothelial cells. RhoA signaling includes activation of many downstream proteins, which alter the cell morphology via cytoskeletal organization