Joëlle L Nader

Cinzia Rapino

Benjamin Gennequin

François Chavant

Maureen Francheteau

Alexandros Makriyannis

Andrea Duranti

Mauro Maccarrone
email: m.maccarrone@unicampus.it

Marcello Solinas

Nathalie Thiriet
email: nathalie.thiriet@univ-poitiers.fr

Francois Chavant

Prior stimulation of the endocannabinoid system prevents methamphetamine-induced dopaminergic neurotoxicity in the striatum through activation of CB2 receptors

Keywords: Endocannabinoids, Methamphetamine, Neurotoxicity, Neuroprotection, THC, Neuroinflammation

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Introduction

The endocannabinoid system (ECS) is an endogenous neuromodulatory system comprised of: i) two main endogenous ligands of type-1 (CB 1 ) and type-2 (CB 2 ) cannabinoid receptors [START_REF] Howlett | CB(1) cannabinoid receptors and their associated proteins[END_REF], namely N-arachidonoylethanolamine (anandamide, AEA) [START_REF] Devane | A novel probe for the cannabinoid receptor[END_REF] and 2-arachidonoyl glycerol (2-AG) [START_REF] Mechoulam | Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors[END_REF]; ii) a putative membrane transport system [START_REF] Fowler | Transport of endocannabinoids across the plasma membrane and within the cell[END_REF]; iii) intracellular storage organelles [START_REF] Oddi | Molecular identification of albumin and Hsp70 as cytosolic anandamidebinding proteins[END_REF] and cytosolic carriers [START_REF] Maccarrone | Intracellular trafficking of anandamide: new concepts for signaling[END_REF]; and iv) enzymes responsible for the synthesis and degradation of these compounds. Fatty acid amide hydrolase [START_REF] Mckinney | Structure and function of fatty acid amide hydrolase[END_REF][START_REF] Fezza | Fatty acid amide hydrolase: a gate-keeper of the endocannabinoid system[END_REF], and monoacylglycerol lipase [START_REF] Dinh | Brain monoglyceride lipase participating in endocannabinoid inactivation[END_REF] are the most important degradative enzymes for AEA and 2-AG, respectively. The ECS has been shown to participate in a broad range of functions including anxiety, depression, neurogenesis, reward, cognition, learning, and memory [START_REF] Mechoulam | The endocannabinoid system and the brain[END_REF]. Converging evidence suggests that endogenous cannabinoids also play a neuroprotective role in pathological situations [START_REF] Van Der Stelt | Cannabinoid receptors and their role in neuroprotection[END_REF]. Endocannabinoids bind mainly (but not exclusively) to CB 1 [START_REF] Matsuda | Structure of a cannabinoid receptor and functional expression of the cloned cDNA[END_REF] and CB 2 [START_REF] Munro | Molecular characterization of a peripheral receptor for cannabinoids[END_REF] receptors that are G-protein coupled receptors. CB 1 receptor appears to be largely expressed in the brain, and particularly in the striatum, the hippocampus, the amygdaloid nucleus and the substantia nigra, and it is believed to play a major role in the ECS activity on movement control, learning, memory, reward and emotions [START_REF] Freund | Role of endogenous cannabinoids in synaptic signaling[END_REF][START_REF] Solinas | The endocannabinoid system in brain reward processes[END_REF][START_REF] Viscomi | Selective CB2 receptor agonism protects central neurons from remote axotomy-induced apoptosis through the PI3K/Akt pathway[END_REF]. For more than 10 years, it was believed that CB 2 receptors were absent from the brain, and that they were involved only in peripheral effects of endocannabinoids [START_REF] Raitio | Targeting the cannabinoid CB2 receptor: mutations, modeling and development of CB2 selective ligands[END_REF]. However, later on the presence of these receptors was clearly demonstrated also in the brain, and since then their functional role in pathological conditions has been suggested by several studies [START_REF] Van Sickle | Identification and functional characterization of brainstem cannabinoid CB2 receptors[END_REF][START_REF] Viscomi | Selective CB2 receptor agonism protects central neurons from remote axotomy-induced apoptosis through the PI3K/Akt pathway[END_REF][START_REF] Onaivi | CNS effects of CB2 cannabinoid receptors: beyond neuroimmuno-cannabinoid activity[END_REF]. Recently, these receptors were also proposed to play a role in addiction and neuropsychiatric diseases [START_REF] Morales | Getting to the core of addiction: Hooking CB2 receptor into drug abuse?[END_REF][START_REF] Onaivi | CNS effects of CB2 cannabinoid receptors: beyond neuroimmuno-cannabinoid activity[END_REF].

Methamphetamine is one of the most abused psychostimulants around the world, due to its inexpensive production and long lasting action [START_REF] Krasnova | Methamphetamine toxicity and messengers of death[END_REF]. A plethora of Internet websites exists and are easily accessible to explain how to synthesize methamphetamine. Therefore, methamphetamine is often homemade clandestinely resulting in important variations in the concentration of different batches, which increase the risks of methamphetamine overdosing. Chronic use of methamphetamine is often associated with cognitive impairments such as deficits in attention, working memory and decision-making [START_REF] Simon | Cognitive impairment in individuals currently using methamphetamine[END_REF][START_REF] Volkow | Association of dopamine transporter reduction with psychomotor impairment in methamphetamine abusers[END_REF]. Post-mortem and brain imaging studies have shown that long-term use of methamphetamine induces specific damages for the dopaminergic neurotransmitter system, such as a reduction of dopamine levels, as well as a decreased level of dopamine transporter and tyrosine hydroxylase, the key player in dopamine synthesis [START_REF] Wilson | Striatal dopamine nerve terminal markers in human, chronic methamphetamine users[END_REF][START_REF] Volkow | Association of dopamine transporter reduction with psychomotor impairment in methamphetamine abusers[END_REF]. In animals, several protocols of administration have been developed to investigate neurotoxic effects of methamphetamine. These protocols mostly consist in repeated methamphetamine injections (4 × 10 mg/kg at 2 h intervals) or to single injections of 30 or 40 mg/kg of methamphetamine [START_REF] Davidson | Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse and treatment[END_REF][START_REF] Zhu | Disparity in the temporal appearance of methamphetamine-induced apoptosis and depletion of dopamine terminal markers in the striatum of mice[END_REF][START_REF] Zhu | Methamphetamine-induced striatal apoptosis in the mouse brain: comparison of a binge to an acute bolus drug administration[END_REF] which can be considered models of methamphetamine overdose. Importantly, these protocols produce similar neurotoxicity for the dopamine neurotransmission [START_REF] Davidson | Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse and treatment[END_REF][START_REF] Zhu | Disparity in the temporal appearance of methamphetamine-induced apoptosis and depletion of dopamine terminal markers in the striatum of mice[END_REF][START_REF] Zhu | Methamphetamine-induced striatal apoptosis in the mouse brain: comparison of a binge to an acute bolus drug administration[END_REF]. As a matter of fact, these treatments reproduce the reductions in tyrosine hydroxylase activity and level, DAT activity and vesicular monoamine transporter-2 protein level in striatum and cortex observed in humans, and, thus these effects are often considered as hallmarks of the dopamine terminal loss induced by the drug [START_REF] Wilson | Striatal dopamine nerve terminal markers in human, chronic methamphetamine users[END_REF][START_REF] Cadet | Speed kills: cellular and molecular bases of methamphetamine-induced nerve terminal degeneration and neuronal apoptosis[END_REF][START_REF] Krasnova | Methamphetamine toxicity and messengers of death[END_REF]. In addition to the loss of monoaminergic terminals, methamphetamine treatment causes death of neuronal cell bodies in the striatum [START_REF] Cadet | Speed kills: cellular and molecular bases of methamphetamine-induced nerve terminal degeneration and neuronal apoptosis[END_REF][START_REF] Thiriet | Neuropeptide Y protects against methamphetamine-induced neuronal apoptosis in the mouse striatum[END_REF]. All these damages have been linked to the massive dopamine release induced by high doses of methamphetamine through an increased reverse transport of this monoamine through the DAT [START_REF] Sulzer | Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport[END_REF] and to the subsequent production of free radicals triggered by dopamine oxidation [START_REF] Yamamoto | The effects of methamphetamine on the production of free radicals and oxidative stress[END_REF]. The understanding of the mechanisms involved in methamphetamine neurotoxicity could lead to the discovery of new strategies to prevent or counter neurotoxic and neurodegenerative processes.

The aim of this study was to determine whether stimulation of the ECS could reduce methamphetamine neurotoxicity for dopa-mine terminals. We first evaluated whether a toxic regimen of methamphetamine modulates levels of endocannabinoids in the striatum. We then investigated whether THC, the main psychoactive ingredient of cannabis extracts, or synthetic compounds like URB597, a selective inhibitor of the fatty acid amide hydrolase that increases the in vivo levels of AEA [START_REF] Kathuria | Modulation of anxiety through blockade of anandamide hydrolysis[END_REF], and JZL184, a selective inhibitor of the monoacylglycerol lipase that increases the in vivo levels of 2-AG [START_REF] Long | Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects[END_REF], could reduce the toxicity of methamphetamine on dopamine terminals. In addition, we sought to ascertain which cannabinoid receptor sub-type was involved in these effects of AEA and 2-AG. Finally, because the effects of endocannabinoids appeared to depend on CB 2 rather than CB 1 receptors, we investigated whether their effects were associated with neuroinflammatory mechanisms.

Materials and methods

Animals

Adult male mice C57Bl/6J were housed in a temperature-controlled environment on a 12 h light / 12 h dark cycle (light from 7 am till 7 pm). They were bred on-site and randomly housed in groups of four, directly after weaning (3 weeks of age). Mice were given free access to food and water. All experiments were conducted during the light period. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) for the care of laboratory animals.

Drugs and treatment

Adult male mice (about 4 months of age) received a single intraperitoneal (i.p.) injection of physiological saline or of a high dose of methamphetamine (Research Triangle Institute) (30 mg/kg), which aims to mimic an overdose of the drug. The fatty acid amide hydrolase inhibitor URB597 (synthesized at the University of Urbino Carlo Bo as previously reported) [START_REF] Mor | Cyclohexylcarbamic acid 3'-or 4'-substituted biphenyl-3-yl esters as fatty acid amide hydrolase inhibitors: synthesis, quantitative structure-activity relationships, and molecular modeling studies[END_REF], the CB1 antagonist rimonabant (donated by the Research Triangle Institute, USA) and the CB2 antagonist AM630 (synthesized at Northeastern University) (1 mg/kg) were dissolved in 5% DMSO (Sigma, France), 5% Tween-80 (Sigma, France) and 90% sterile saline. The monoacylglycerol lipase inhibitor JZL184 (Interchim, France) (16 mg/kg) was dissolved in 20% DMSO (Sigma, France), 5% Tween-80 and 75% sterile saline. Δ9-Tetrahydrocannabinol (THC) (3 mg/kg) was dissolved in a solution of 5% ethanol, 5% Tween-80 and 90% physiological saline. Doses of each compound were chosen based on previously published papers (respectively: for URB597 [START_REF] Kathuria | Modulation of anxiety through blockade of anandamide hydrolysis[END_REF][START_REF] Moreira | Reduced anxiety-like behaviour induced by genetic and pharmacological inhibition of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) is mediated by CB1 receptors[END_REF]; JZL184 [START_REF] Kinsey | Inhibition of endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the marble burying assay[END_REF][START_REF] Sumislawski | Reversible gating of endocannabinoid plasticity in the amygdala by chronic stress: a potential role for monoacylglycerol lipase inhibition in the prevention of stress-induced behavioral adaptation[END_REF]; for AM630 and THC [START_REF] Tourino | THC prevents MDMA neurotoxicity in mice[END_REF]). Whereas some papers have used doses of rimonabant as high as 3 mg/kg in mice, this dose produces behavioral effects even in CB1 knock-out mice [START_REF] Haller | The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety[END_REF][START_REF] Haller | CB1 cannabinoid receptors mediate anxiolytic effects: convergent genetic and pharmacological evidence with CB1-specific agents[END_REF], suggesting that at such doses rimonabant produces non-specific effects likely related to its reported inverse agonist activity [START_REF] Bergman | Some effects of CB1 antagonists with inverse agonist and neutral biochemical properties[END_REF]. Therefore, we decided to use a dose of 1 mg/kg that is high enough to be effective in blocking the effects of exogenous cannabinoid [START_REF] Solinas | The cannabinoid CB1 antagonist Npiperidinyl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl) -4-methylpyrazole-3-carboxamide (SR-141716A) differentially alters the reinforcing effects of heroin under continuous reinforcement, fixed ratio, and progressive ratio schedules of drug self-administration in rats[END_REF] while limiting the non-specific effects [START_REF] Haller | The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety[END_REF][START_REF] Haller | CB1 cannabinoid receptors mediate anxiolytic effects: convergent genetic and pharmacological evidence with CB1-specific agents[END_REF].

Measurement of endocannabinoids levels

For the detection of endocannabinoids, mice were treated with methamphetamine (30 mg/kg, i.p.) or physiological saline, and decapitated 1 h, 6 h, 12 h or 24 h after the treatment. Brains were removed and the striata were dissected on ice and frozen on dry ice, then tissues were subjected to lipid extraction with chloroform/ methanol (2:1, v/v), in the presence of d 8 -AEA and d 8 -2-AG as internal standards [START_REF] Pucci | Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid receptor[END_REF]. The organic phase was dried and then analyzed by liquid chromatography-electrospray ionization mass spectrometry (LCeESI-MS), using a single quadrupole API-150EX mass spectrometer (Applied Biosystem, CA, USA) in conjunction with a PerkinElmer LC system (PerkinElmer, MA, USA). Quantitative analysis was performed by selected ion recording over the respective sodiated molecular ions, as reported [START_REF] Pucci | Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid receptor[END_REF].

Western blot analysis

To evaluate dopamine terminal loss, we measured tyrosine hydroxylase levels in the striatum 7 days after the drug treatment by Western Blot. Mice were treated with selective antagonists of CB 1 or CB 2 receptors (or their vehicle as a control) 1 h prior to methamphetamine injection. Inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase (or their vehicle as a control) were administered 40 min prior to methamphetamine injection. THC (or its vehicle as a control) was administered 30 min prior to methamphetamine injection. Animals were sacrificed 7 days after drug treatment by decapitation. Brains were removed and striatum was dissected on ice, frozen on dry ice and stored at 80 °C until Western blotting was performed [START_REF] Thiriet | Environmental enrichment does not reduce the rewarding and neurotoxic effects of methamphetamine[END_REF]. Tissues were homogenized (350 ml/25 mg) by ultra-sonication in a 0.2 M TriseHCl (pH 6.9) buffer solution containing 8% SDS and 35% glycerol. Samples were then heated to 90 °C for 3 min and centrifuged at 4 °C for 10 min (13000 g). Supernatants were collected and analyzed using a DC protein assay kit (BioRad, France). Fifty mg of proteins per sample were loaded on an SDS-page gel, and then gels were transferred onto nitrocellulose membranes that were blocked for 1 h at room temperature in Tris-Buffered Saline (TBS) solution containing 3% Bovine Serum Albumin (BSA) and 0.1% Tween-20. Membranes were incubated with primary antibodies: mouse anti-tyrosine hydroxylase antibody (1:5000) (Immunostar, France) and rabbit anti-Actin antibody (1:10,000) (Sigma, France), diluted in the same buffer for 2 h at room temperature. Several rinses were made (3 × 10 min) with TBS containing 0.1% Tween-20 before incubation for 1 h with secondary antibodies: goat anti-rabbit coupled to Alexa Fluor 488, and goat anti-mouse coupled to Alexa Fluor 568 (1:1000, Invitrogen, France), diluted in the same buffer. Membranes were rinsed as described before and analyzed using Typhoon Imaging system (GE Healthcare). Image J software was used for quantification. Actin signal was used to normalize tyrosine hydroxylase signal, in order to avoid loading artifacts.

Enzyme-linked immunosorbent assay (ELISA)

For the ELISA experiments, mice were killed 24 h after methamphetamine injections. Striata were dissected and homogenized in 25 mM Tris buffer (pH 7.4), containing 150 mM NaCl and 1 mM EDTA. Commercially available ELISA kits were used in order to assess TNF alpha levels (sensitivity: 4 pg/mL), according to the manufacturer's instructions (BioLegend, Ozyme, St Quentin Yvelines, France). The range of analysis was between 4 and 500 pg/ml. Lysates were diluted (1:2) with the assay diluents and all steps were performed at room temperature, except for plate coating and samples incubation (4 °C). The enzymatic reaction was performed by incubating for 15 min with the tetramethylbenzidine substrate, and then was stopped by adding 2N H 2 SO 4 . The optical density (OD) was read at 450 nm within 30 min, using the Mithras LB940™ (Berthold) multimode microplate reader. The TNF alpha levels were then calculated by plotting OD values of each sample against the standard curve. For convenience all results were expressed as pg/mg protein.

Statistical analysis

All results are presented as group means (±SEM). Differences between groups were assessed by ANOVA. For the effects of methamphetamine on endogenous cannabinoid levels a one-way ANOVA was used with time after methamphetamine as a factor. For the effects of THC on tyrosine hydroxylase protein levels a two-way ANOVA with pretreatment (Veh or THC) and treatment (Sal or METH) as factors. For the effects of URB597 or JZL184 on methamphetamine-induced decreases in TH levels and increases in TNF-alpha levels, we used a one-way ANOVA with treatment as a factor. Results showing significant differences were subjected to Fischer LSD post-hoc test except for the data of the endocannabinoids levels that were subjected to a Dunnet's post-hoc test. The level of significance was always set at p < 0.05.

Results

Methamphetamine treatment modulates endocannabinoids levels in the striatum

In order to evaluate the impact of methamphetamine treatment on the levels of endocannabinoids in the striatum of mice, we measured AEA and 2-AG levels at different time points after an acute toxic methamphetamine treatment (30 mg/kg). We found that striatal AEA levels show two peaks one at 1 h and one at 12 h after methamphetamine injection (p < 0.05) (Fig. 1A), two time points that appear critical for the toxic effects of methamphetamine (see Discussion). Then, AEA levels returned to basal levels after 24 h. Instead, 2-AG levels slowly decreased after methamphetamine administration, and this decrease reached significance 12 h after drug injection (p < 0.05), returning to basal levels after 24 h (Fig. 1B).

THC prevents methamphetamine-induced reduction of tyrosine hydroxylase levels in the striatum

To evaluate the protective role of endocannabinoids, mice were treated with the natural agonist of CB 1 /CB 2 receptors THC (3 mg/kg), 30 min prior to methamphetamine (30 mg/kg) injection. After seven days, tyrosine hydroxylase levels were measured in the striatum by Western blotting in order to evaluate the long-term impact of methamphetamine treatment on dopamine neurotrans-mission (Fig. 2). Tyrosine hydroxylase levels of methamphetamine-treated mice were significantly reduced (by 40%) when compared to vehicle-treated animals (p < 0.001), a result that extends the known toxicity of methamphetamine for dopamine neurotrans-mission within the striatum [START_REF] Bowyer | Neurotoxic-related changes in tyrosine hydroxylase, microglia, myelin, and the blood-brain barrier in the caudate-putamen from acute methamphetamine exposure[END_REF]. When mice were treated with THC prior to methamphetamine injection, their striatal tyrosine hydroxylase levels remained similar to those of control mice, and were significantly higher than those of mice that received methamphetamine alone (p < 0.01). Administration of THC alone had no effect on striatal tyrosine hydroxylase levels. These results indicate that THC pretreatment prevents methamphetamine-induced toxicity in the striatum.

Blockade of endocannabinoids degradation prevents methamphetamine-induced reduction of tyrosine hydroxylase levels in the striatum

To activate the ECS, we then used two different synthetic compounds: URB597, an inhibitor of the fatty acid amide hydrolase activity that increases brain levels of AEA [START_REF] Kathuria | Modulation of anxiety through blockade of anandamide hydrolysis[END_REF], and JZL184, an inhibitor of the monoacylglycerol lipase activity that increases brain levels of 2-AG [START_REF] Long | Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects[END_REF]. Again, striatal tyrosine hydroxylase levels were significantly reduced by methamphetamine injection, compared to controls (Figs. 3 and4). Pretreatment with URB597 at the dose of 1 mg/kg completely prevented methamphetamine-induced decreases in tyrosine hydroxylase striatal levels, suggesting that fatty acid amide hydrolase inhibition blocked methamphetamine-induced striatal dopaminergic terminal loss (p < 0.001) (Fig. 3). Importantly, URB597 produced similar protective effects on methamphetamine-induced reduction in striatal dopamine levels in the striatum (Supplementary Fig. 1) and these effects were independent from effects on methamphetamine-induced hyperthermia (Supplementary Fig. 2). Administration of JZL184 at a dose of 16 mg/kg 40 min prior to methamphetamine treatment also prevented methamphetamine-induced decrease in striatal tyrosine hydroxylase levels (p < 0.001) (Fig. 4). Incidentally, URB597 or JZL184 did not modify striatal tyrosine hydroxylase levels when used alone (Figs. 3 and4, respectively).

CB 2 , but not CB 1 , receptors mediate the protective effects of URB597 and JZL184 against methamphetamine-induced toxicity

To further dissect the mechanisms by which endocannabinoids can provide neuroprotection against methamphetamine-induced dopamine terminal loss, we used selective antagonists of CB 1 and CB 2 receptors, namely rimonabant and AM630, which were injected 1 h prior to methamphetamine injection, and 20 min prior to URB597 or JZL184 injection. Blockade of CB 1 receptors by rimonabant (1 mg/kg) did not modify the protection against methamphetamine-induced decreases in tyrosine hydroxylase levels exerted by URB597 (Fig. 3) or JZL184 (Fig. 4) pre-administration, suggesting that this receptor subtype was not engaged in the neuroprotective effects of endocannabinoids. Conversely, when the CB 2 antagonist AM630 (1 mg/kg) was pre-administered, the beneficial effects of both URB597 (p < 0.05) (Fig. 3) and JZL184 (p < 0.01) (Fig. 4) on methamphetamine-induced decreases in striatal tyrosine hydroxylase levels were blunted, suggesting that the neuroprotective actions of endocannabinoids engaged mainly the CB 2 receptor subtype. Neither CB 1 nor CB 2 receptor antagonists affected methamphetamine-induced decreases in striatal tyrosine hydroxylase levels, when used alone (data not shown).

The protective effects of endocannabinoids are associated with a reduced striatal accumulation of TNF alpha after methamphetamine administration

Because CB 2 receptors are known to play a role in regulating neuroinflammatory reactions [START_REF] Cabral | CB2 receptors in the brain: role in central immune function[END_REF], which are involved in methamphetamine-induced neurotoxicity [START_REF] Kita | Dopamine-induced behavioral changes and oxidative stress in methamphetamine-induced neurotoxicity[END_REF][START_REF] Goncalves | Methamphetamine-induced neuroinflammation and neuronal dysfunction in the mice hippocampus: preventive effect of indomethacin[END_REF], we measured striatal levels of the cytokine TNF alpha as a marker of neuroinflammation 24 h after methamphetamine injection and we evaluated whether ECS stimulation could modulate these levels (Fig. 5). As previously reported [START_REF] Kita | Dopamine-induced behavioral changes and oxidative stress in methamphetamine-induced neurotoxicity[END_REF], we found that methamphetamine administration increased the level of TNF alpha (+100%, p < 0.001) in the striatum. This increase was blunted by pre-administration of THC, URB597 or JZL184 (p < 0.01), though TNF alpha levels remained slightly higher than those of controls (p < 0.01).

Discussion

In this study, we found that stimulation of the ECS prior to methamphetamine administration has protective effects against methamphetamine-induced neurotoxicity to dopamine neurons. Indeed, administration of a toxic dose of methamphetamine alters striatal levels of endocannabinoids, the endogenous ligands of CB 1 and CB 2 receptors, and inhibition of the hydrolysis of these compounds prevents methamphetamine-induced reduction of tyrosine hydroxylase levels, a hallmark of dopamine terminal loss in the striatum [START_REF] Cadet | Speed kills: cellular and molecular bases of methamphetamine-induced nerve terminal degeneration and neuronal apoptosis[END_REF]. In particular, it appears that the beneficial effects of endocannabinoids are mediated by stimulation of CB 2 , rather than CB 1 receptors, and involve a reduction of neuroinflammatory processes triggered by methamphetamine.

Methamphetamine alters the ECS

In this study, we found that administration of a toxic dose (30 mg/kg) of methamphetamine altered striatal levels of endogenous cannabinoids in a time-dependent manner. First of all, we observed two peak increases in AEA striatal levels at 1 and 12 h after injection of methamphetamine, and these levels returned to basal values after 24 h. As discussed bellow, these time points have already been demonstrated to be crucial for methamphetamineinduced toxicity [START_REF] Pereira | Acute changes in dopamine release and turnover in rat caudate nucleus following a single dose of methamphetamine[END_REF][START_REF] Bowyer | Neurotoxic-related changes in tyrosine hydroxylase, microglia, myelin, and the blood-brain barrier in the caudate-putamen from acute methamphetamine exposure[END_REF][START_REF] Krasnova | Methamphetamine toxicity and messengers of death[END_REF]. Indeed, within the synapse, methamphetamine stimulates a massive release of dopamine as soon as 30 min after its injection, and this release is maximal after about 90 min [START_REF] Pereira | Acute changes in dopamine release and turnover in rat caudate nucleus following a single dose of methamphetamine[END_REF][START_REF] Lan | Enhancing effects of morphine on methamphetamine-induced reinforcing behavior and its association with dopamine release and metabolism in mice[END_REF]. Since endocannabinoids have been described to play a neuromodulatory role within the brain [START_REF] Marsicano | Neuromodulatory functions of the endocannabinoid system[END_REF], it could be speculated that the methamphetamine-induced dopamine release leads to the synthesis and release of AEA [START_REF] Giuffrida | Dopamine activation of endogenous cannabinoid signaling in dorsal striatum[END_REF], which would act retrogradely [START_REF] Wilson | Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses[END_REF] to produce a negative feedback on dopamine release [START_REF] Melis | Endocannabinoids mediate presynaptic inhibition of glutamatergic transmission in rat ventral tegmental area dopamine neurons through activation of CB1 receptors[END_REF]. As a result, AEA would limit the damage triggered by dopamine oxidation, as well as by generation of reactive oxygen species [START_REF] Cadet | Speed kills: cellular and molecular bases of methamphetamine-induced nerve terminal degeneration and neuronal apoptosis[END_REF]. Twelve hours after injection, methamphetamine toxicity reaches a peak in terms of striatal tyrosine hydroxylase immunoreactivity, and glial reactions become apparent [START_REF] Bowyer | Neurotoxic-related changes in tyrosine hydroxylase, microglia, myelin, and the blood-brain barrier in the caudate-putamen from acute methamphetamine exposure[END_REF]; therefore, increases in AEA levels at this later time point may represent a second wave of reactions to help the system to recover from the toxic effects of methamphetamine.

Interestingly, at variance with what we found with AEA, the administration of methamphetamine did not produce an increase but rather a decrease in striatal 2-AG levels that became significant 12 h after methamphetamine administration. Because 2-AG has been suggested to play a house-keeping role that is necessary for basal functioning and survival of the cell [START_REF] Piomelli | The molecular logic of endocannabinoid signalling[END_REF], it could be speculated that the decreases in 2-AG levels represent a sign of cell damage that takes place 12 h after the injection of methamphetamine [START_REF] Bowyer | Neurotoxic-related changes in tyrosine hydroxylase, microglia, myelin, and the blood-brain barrier in the caudate-putamen from acute methamphetamine exposure[END_REF][START_REF] Krasnova | Methamphetamine toxicity and messengers of death[END_REF]. Therefore, the brain would respond to toxic insults induced by methamphetamine by releasing AEA and, if this reaction is not sufficient to avoid brain damage, subsequent toxicity and temporary malfunctioning would be associated with low levels of 2-AG.

Endocannabinoids provide neuroprotection against methamphetamine-induced dopamine terminal loss

To evaluate the potential protective effect of endocannabinoids against methamphetamineinduced degeneration of dopamine terminals, we stimulated the ECS by using a direct agonist of CB 1 and CB 2 receptors, or inhibitors of the degradation of their endogenous ligands AEA and 2-AG, previously used to increase endocannabinoids levels in the brain [START_REF] Kathuria | Modulation of anxiety through blockade of anandamide hydrolysis[END_REF][START_REF] Long | Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects[END_REF] and we measured striatal levels of tyrosine hydroxylase. In fact, in models of methamphetamine toxicity, tyrosine hydroxylase levels are often used as an index of loss of dopamine terminals and their reduction closely parallels depletion in dopa-mine levels measured by HPLC [START_REF] Hayashi | Delta opioid peptide [D-Ala2, D-Leu5]enkephalin causes a near complete blockade of the neuronal damage caused by a single high dose of methamphetamine: examining the role of p53[END_REF][START_REF] Thiriet | Neuropeptide Y protects against methamphetamine-induced neuronal apoptosis in the mouse striatum[END_REF], and Supplementary Fig. 1). Administration of the CB 1 /CB 2 receptors agonist THC significantly reduced methamphetamine-induced decreases in tyrosine hydroxylase levels. This finding is in agreement with previous cell culture studies that showed that THC could reduce the neurotoxicity induced by the neurotoxin 1-methyl-4-phenyl pyridinium (MPP+), which shares mechanisms of action with methamphetamine [START_REF] Moldzio | Effects of cannabinoids Delta(9)-tetrahydrocannabinol, Delta(9)-tetrahydrocannabinolic acid and cannabidiol in MPP+ affected murine mesencephalic cultures[END_REF]. In line with this, in an in vivo mouse model of Parkinson's disease, dopamine neurons are also protected against MPP+ neurotoxicity by the injection of WIN55,212-2, another nonspecific CB 1 /CB 2 receptor agonist [START_REF] Price | WIN55,212-2, a cannabinoid receptor agonist, protects against nigrostriatal cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease[END_REF]. Similarly, the administration of exogenous plant-derived cannabinoids (i.e., THC or cannabidiol) also reduces the dopaminergic toxicity induced by 6-OHDA lesion in rats, another model of Parkinson's disease [START_REF] Lastres-Becker | Cannabinoids provide neuroprotection against 6-hydroxydopamine toxicity in vivo and in vitro: relevance to Parkinson's disease[END_REF]. Altogether, our results and literature data concur to suggest that THC injection could have neuroprotective properties, thus preventing damage to dopamine neurons.

The medical use of THC or other direct CB 1 /CB 2 receptor agonists is complicated by their reinforcing effects and their well-known abuse liability. The discovery of the ECS has led to the development of new pharmacological tools that allow indirect stimulation of CB 1 /CB 2 receptors by blocking metabolic degradation of AEA and 2-AG [START_REF] Kathuria | Modulation of anxiety through blockade of anandamide hydrolysis[END_REF][START_REF] Piomelli | The molecular logic of endocannabinoid signalling[END_REF][START_REF] Marzo | The endocannabinoid system and its therapeutic exploitation[END_REF][START_REF] Long | Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects[END_REF][START_REF] Bisogno | Latest advances in the discovery of fatty acid amide hydrolase inhibitors[END_REF]. Although these compounds have not yet been tested in humans, several studies in animals suggest their lack of abuse liability [START_REF] Gobbi | Antidepressant-like activity and modulation of brain monoaminergic transmission by blockade of anandamide hydrolysis[END_REF][START_REF] Solinas | Anandamide administration alone and after inhibition of fatty acid amide hydrolase (FAAH) increases dopa-mine levels in the nucleus accumbens shell in rats[END_REF][START_REF] Justinova | Fatty acid amide hydrolase inhibition heightens anandamide signaling without producing reinforcing effects in primates[END_REF]. Interestingly, the administration of the fatty acid amide hydrolase inhibitor URB597 or of the monoacylglycerol lipase inhibitor JZL184 blocked the effects of the neurotoxic regimen of methamphetamine on striatal tyrosine hydroxylase levels. For URB597, this protection was also confirmed for methamphetamine-induced reductions in striatal dopamine levels (Supplementary Fig. 1). In addition, URB97 did not alter methamphetamine-induced hyperthermia suggesting that the effects of stimulation of the endogenous cannabinoid system are not simply secondary to non-specific effects on temperature (Supplementary Fig. 2). The protection by the endocannabinoids is consistent with previous studies showing that N-acylethanolamines, to which AEA belongs [START_REF] Degn | Changes in brain levels of N-acylethanolamines and 2-arachidonoylglycerol in focal cerebral ischemia in mice[END_REF], and 2-AG [START_REF] Melis | Protective activation of the endocannabinoid system during ischemia in dopamine neurons[END_REF] can protect neurons against oxidative stress due to ischemia, both in vitro and in vivo. These substances have also beneficial effects against excitotoxicity [START_REF] Marsicano | CB1 cannabinoid receptors and on-demand defense against excitotoxicity[END_REF], a process that contributes with oxidative stress to the neurotoxicity of methamphetamine [START_REF] Cadet | Speed kills: cellular and molecular bases of methamphetamine-induced nerve terminal degeneration and neuronal apoptosis[END_REF][START_REF] Krasnova | Methamphetamine toxicity and messengers of death[END_REF]. Therefore, drugs that enhance the levels of endogenous CB 1 /CB 2 receptor agonists may be more useful than THC for therapeutic use in humans, because they are devoid of risks of abuse [START_REF] Gobbi | Antidepressant-like activity and modulation of brain monoaminergic transmission by blockade of anandamide hydrolysis[END_REF][START_REF] Solinas | Anandamide administration alone and after inhibition of fatty acid amide hydrolase (FAAH) increases dopa-mine levels in the nucleus accumbens shell in rats[END_REF][START_REF] Justinova | Fatty acid amide hydrolase inhibition heightens anandamide signaling without producing reinforcing effects in primates[END_REF].

Role of CB 2 receptors in the effects of endocannabinoids

In this study, the protective effects of the ECS against methamphetamine-induced toxicity appear to be mediated by CB 2 receptors. THC, AEA and 2-AG all activate both CB 1 and CB 2 receptors, although AEA and 2-AG have a slightly higher affinity for CB 1 receptors [START_REF] Pertwee | International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB(1) and CB(2)[END_REF]. Our results are in agreement with previous literature data that document a neuroprotective function for CB 2 receptor within the brain [START_REF] Fernandez-Ruiz | Role of CB2 receptors in neuroprotective effects of cannabinoids[END_REF][START_REF] Viscomi | Selective CB2 receptor agonism protects central neurons from remote axotomy-induced apoptosis through the PI3K/Akt pathway[END_REF][START_REF] Onaivi | CNS effects of CB2 cannabinoid receptors: beyond neuroimmuno-cannabinoid activity[END_REF]. For example, a recent study has shown that protection provided by WIN52,212-2 against MPP+ toxicity is mediated by the stimulation of CB 2 receptors, since ablation of these receptors exacerbated the effects of the neurotoxin [START_REF] Price | WIN55,212-2, a cannabinoid receptor agonist, protects against nigrostriatal cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease[END_REF]. In addition, other studies have shown that CB 2 receptors stimulation reduce the neurotoxicity of MDMA [START_REF] Torres | Evidence that MDMA ('ecstasy') increases cannabinoid CB2 receptor expression in microglial cells: role in the neuroinflammatory response in rat brain[END_REF][START_REF] Tourino | THC prevents MDMA neurotoxicity in mice[END_REF][START_REF] Torres | Changes in interleukin-1 signal modulators induced by 3,4-methylenedioxymethamphetamine (MDMA): regulation by CB2 receptors and implications for neurotoxicity[END_REF], a substituted amphetamine that produces toxicity in the brain with mechanisms that are largely overlapping on those induced by methamphetamine [START_REF] Quinton | Causes and consequences of methamphetamine and MDMA toxicity[END_REF]. These results suggest that CB 2 agonists may be beneficial in preventing the toxic consequences of overdose induced by methamphetamine or other amphetamine derivates.

Neuroinflammation, endocannabinoids and methamphetamine

The prominent role of CB 2 receptors in the neuroprotective effects of endocannabinoids against methamphetamine toxicity suggests that the effects of endocannabinoids may depend on the reduction of methamphetamine-induced neuroinflammation. Indeed, endocannabinoids modulate inflammatory responses by regulating microglia function via receptor dependent and independent mechanisms, and consequently they control the generation of cytotoxic factors such as TNF alpha [START_REF] Walter | Cannabinoids and neuroinflammation[END_REF]. In this study, we found that pretreatment with a CB 1 /CB 2 receptor agonist, or with inhibitors of the degradation of their endogenous ligands (AEA and 2-AG), both reduce the accumulation of TNF alpha in the striatum 24 h after methamphetamine injection. In agreement with this finding, several studies have demonstrated the involvement of CB 2 receptors in the progression/arrest of brain damage, by influencing events such as microglial cell proliferation, differentiation and migration at neuroinflammatory lesion sites [START_REF] Walter | Nonpsychotropic cannabinoid receptors regulate microglial cell migration[END_REF][START_REF] Fernandez-Ruiz | Role of CB2 receptors in neuroprotective effects of cannabinoids[END_REF], 2011), for example in models of neurodegenerative diseases like Parkinson's and Huntington's diseases [START_REF] Price | WIN55,212-2, a cannabinoid receptor agonist, protects against nigrostriatal cell loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease[END_REF][START_REF] Fernandez-Ruiz | Prospects for cannabinoid therapies in basal ganglia disorders[END_REF]. THC also reduces MDMA-induced microglial activation by acting on CB 2 receptors [START_REF] Torres | Evidence that MDMA ('ecstasy') increases cannabinoid CB2 receptor expression in microglial cells: role in the neuroinflammatory response in rat brain[END_REF][START_REF] Tourino | THC prevents MDMA neurotoxicity in mice[END_REF]. It is also noteworthy that CB 2 receptor expression increases in micro-glial cells in case of noxious conditions associated with inflamma-tory events [START_REF] Fernandez-Ruiz | Cannabinoid CB2 receptor: a new target for controlling neural cell survival?[END_REF]. Interestingly, such inflammatory processes appear to be part of methamphetamine-induced toxicity, because this drug stimulates microglial activation in dopaminergic regions such as the striatum [START_REF] Sekine | Methamphetamine causes microglial activation in the brains of human abusers[END_REF], and increases TNF alpha levels [START_REF] Nakajima | Role of tumor necrosis factor-alpha in methamphetamineinduced drug dependence and neurotoxicity[END_REF][START_REF] Kita | Dopamine-induced behavioral changes and oxidative stress in methamphetamine-induced neurotoxicity[END_REF]. Altogether, these observations suggest that activation of CB 2 receptors may modulate neuroinflammatory responses induced by methamphetamine, may involved a reduction of TNF alpha production triggered by methamphetamine [START_REF] Fernandez-Ruiz | Role of CB2 receptors in neuroprotective effects of cannabinoids[END_REF] and, consequently, limiting methamphetamine-induced neurotoxicity.

Conclusions

Altogether, the present results demonstrate that stimulation of the ECS prior to the administration of a neurotoxic dose of meth-amphetamine considerably reduced the toxicity of this drug for dopamine terminals in the striatum, mainly by stimulating CB 2 receptors. These results highlight the importance of endocannabinoids as neuroprotective modulators against neurotoxic processes observed within the brain during some neurodegenerative diseases. Further investigation will be needed to determine whether similar neuroprotective effects could be found if the ECS is stimulated after the administration of methamphetamine. AEA (A) and 2-AG (B) levels in the striatum at 1 h, 6 h, 12 h and 24 h after methamphetamine treatment (30 mg/kg). Columns represent the means, and vertical lines the standard error of the mean (SEM) (n = 8-9 mice per group). Administration of methamphetamine induced an increase in AEA levels 1 h and 12 h after drug injection (A), and a decrease in 2-AG levels 12 h after drug injection (B). One-way ANOVA followed by post-hoc Dunnet's test. *p < 0.05 compared values from the methamphetamine-treated mice to vehicle-treated mice. Effect of pre-treatment with URB597 and cannabinoid receptors antagonists on the striatal levels of tyrosine hydroxylase protein of mice treated with methamphetamine. Graphical representation of the striatal tyrosine hydroxylase protein levels (data are expressed as means + SEM, and n = 6-9 mice per group). Administration of methamphetamine (METH; 30 mg/kg) induced a decrease of tyrosine hydroxylase expression, and pre-administration of URB597 (1 mg/kg) 40 min before blunted this decrease. The prior administration (1 h before methamphetamine) of the CB 2 antagonist AM630 (1 mg/kg), but not of the CB 1 antagonist rimonabant (1 mg/kg), abolished the beneficial effect of URB597. One-way ANOVA followed by post-hoc Fischer LSD's test. *p < 0.05 and ***p < 0.001 compared to vehicletreated group. $$$ p < 0.001 compared with methamphetamine-treated group. ¤ p < 0.05 compared to Vehicle-URB-methamphetamine-treated animals. Effect of pre-treatment with JZL184 and cannabinoid receptors antagonists on the striatal levels of tyrosine hydroxylase protein of mice treated with methamphetamine. Graphical representation of the striatal tyrosine hydroxylase protein levels (data are expressed as means + SEM, and n = 6-7 mice per group). Administration of methamphetamine (METH; 30 mg/kg) induced a decrease of tyrosine hydroxylase expression, and pre-administration of JZL184 (16 mg/kg) 40 min before blunted this decrease. The prior administration (1 h before methamphetamine) of the CB 2 antagonist AM630 (1 mg/kg), but not of the CB 1 antagonist rimonabant (1 mg/kg), abolished the beneficial effect of JZL184. One-way ANOVA followed by post-hoc Fischer LSD's test. **p < 0.01 and ***p < 0.001 compared to vehicle-treated group. $$$ p < 0.001 compared with methamphetamine-treated group. ¤¤ p < 0.01 compared to Vehicle-JZL-methamphetamine-treated animals.

Fig

  Fig. 1.

Fig. 2 .

 2 Fig. 2.Effect of THC pre-treatment on the striatal levels of tyrosine hydroxylase protein in mice treated with methamphetamine. (A) Representative immunoblots of levels of tyrosine hydroxylase (TH) in the striatum of mice treated with vehicle (NaCl 0.9%), THC (3 mg/kg), methamphetamine-HCl (METH; 30 mg/kg), or with THC (3 mg/kg) 30 min prior to methamphetamine injection, and killed 7 days later. (B) Graphical representation of tyrosine hydroxylase levels (data are expressed as means + SEM, and n = 6-7 mice per group). Administration of methamphetamine induced a decrease in the striatal tyrosine hydroxylase expression of about 40%, and THC pre-administration blunted this decrease. Two-way ANOVA followed by post-hoc Fischer LSD's test. ***p < 0.001 compared with vehicletreated group. ¤¤ p < 0.01 compared with methamphetamine-treated group.

Fig

  Fig. 3.

Fig

  Fig. 4.

Fig. 5 .

 5 Fig. 5. Changes in TNF alpha striatal levels in response to methamphetamine, alone or in combination with cannabinoid receptors agonists Graphical representation of the TNF alpha levels (data are expressed as means + SEM, and n = 7-8 mice per group). Administration of methamphetamine (METH; 30 mg/kg) increased TNF alpha tissue concentration and pre-administration of THC (3 mg/kg) 30 min before, and of URB597 (1 mg/kg) or JZL184 (16 mg/kg) 40 min before, reduced this increase. Oneway ANOVA followed by post-hoc Fischer LSD's test. **p < 0.01 and ***p < 0.001 compared to vehicle-treated group. $$ p < 0.01 compared to methamphetamine-treated group.

Neuropharmacology. Author manuscript; available in PMC 2016 July 11.

Acknowledgments

This work was supported by the Institut National pour la Santé et la Recherche Médicale, le Centre National pour la Recherche Scientifique, University of Poitiers, the Contrat de Projet Etat Region (CPER) #5, and by the Italian Ministero dell'Istruzione, dell'Università e della Ricerca (grant PRIN 2010-2011 to MM). Joëlle Nader was a recipient of PhD fellowship by the French Minister of Research. We thank NIDA IRP for providing methamphetamine-HCL. We thank Arnaud François for help with the ELISA experiments.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Longlong Wang

Shihua Shi

Adeline Unterreiner

Ronan Kapetanovic

Sucheta Ghosh

Jacint Sanchez

Selma Aslani

Yuan Xiong

Chi-Lin Hsu

Katherine A Donovan

Christopher J Farady

Eric S Fischer

Frédéric Bornancin

Patrick Matthias

HDAC6/aggresome processing pathway importance for inflammasome formation is context dependent

Keywords: Inflammasome, HDAC6, degrader, inhibitors, DARPin, interleukin

Introduction

The inflammasome is a critical protein complex involved in the body's immune response [START_REF] Mariathasan | Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf[END_REF]. When activated, the inflammasome releases the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 , which can result in tissue damage and cell death through pyroptosis [START_REF] Broz | Inflammasomes: of assembly, regulation and signalling[END_REF]. Several sensor proteins can nucleate inflammasome assembly, including NLRP and NLRC subfamilies, AIM2, and pyrin. Among these, the NLRP3 inflammasome, which is key for sterile inflammation has been extensively studied (3). Activation of the NLRP3 inflammasome often requires a transcriptional priming step to upregulate the inflammasome components such as NLRP3 and IL-1β.

Priming agents, e.g., bacterial lipopolysaccharide (LPS), tumor necrosis factor (TNF) or phorbol 12-myristate 13-acetate (PMA), also trigger critical post-translational modifications that sensitize the inflammasome components to subsequent activation.

Various stimuli, including the pore-forming toxin nigericin, adenosine-triphosphate (ATP) (4) and monosodium urate crystals (MSU) [START_REF] Martinon | Goutassociated uric acid crystals activate the NALP3 inflammasome[END_REF] can stimulate NLRP3 inflammasome assembly, leading to pro-caspase 1 activation, followed by cleavage and release of IL-1β (3). Aberrant inflammasome activity has been linked to many diseases (3), and a detailed understanding of the inflammasome's activation process is crucial for the development of potential treatments for diseases related to inflammation and immune responses.

The aggresome processing pathway (APP) is a stress-response pathway that is activated when misfolded proteins accumulate; it promotes the formation of the aggresome, a deposit of misfolded and ubiquitinated proteins near the microtubuleorganizing center (MTOC), which is subsequently eliminated by autophagy [START_REF] Johnston | Aggresomes: A Cellular Response to Misfolded Proteins The[END_REF]. An essential component of the APP is the lysine deacetylase HDAC6 [START_REF] Kawaguchi | The Deacetylase HDAC6 Regulates Aggresome Formation and Cell Viability in Response to Misfolded[END_REF], an atypical enzyme with two catalytic domains and a zinc finger domain (ZnF-UbP, hereafter ZnF) binding the small protein ubiquitin (Ub). HDAC6 has been implicated in diverse normal or pathological cellular processes, such as stress response (e.g. APP and stress granules formation) [START_REF] Saito | Acetylation of intrinsically disordered regions regulates phase separation[END_REF], cancer [START_REF] Rodriguez-Gonzalez | Role of the aggresome pathway in cancer: targeting histone deacetylase 6-dependent protein degradation[END_REF], inflammation and viral infection [START_REF] Indranil Banerjee | Influenza A virus uses the aggresome processing machinery for host cell entry[END_REF]. Depending on the situation, the catalytic domains (regulating acetylation levels of the relevant substrate) and/or the ZnF domain (recruiting Ub) are important for optimal HDAC6 function. In its role in the APP, HDAC6 acts as an adapter protein that promotes J o u r n a l P r e -p r o o f transport of ubiquitinated misfolded proteins by dynein motor proteins along the MTs towards the MTOC. For this, both the ZnF and the catalytic domains are critical.

It has recently been proposed that formation of the NLRP3 and pyrin inflammasomes takes place near the MTOC and uses components of the APP machinery and that consequently inflammasome formation is impaired in cells lacking HDAC6 or expressing HDAC6 with a mutated ZnF domain [START_REF] Magupalli | HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation[END_REF]. Furthermore, the lysosomal Ragulator complex was recently reported to activate the NLRP3 inflammasome in vivo via HDAC6 [START_REF] Tsujimoto | The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6 The[END_REF]. Given the apparent similarity between inflammasome formation and APPin both cases assembly of a complex near the MTOCit is plausible that HDAC6 could be involved in both pathways, but to define the absolute requirement of HDAC6 in inflammasome activation, a robust profiling of orthogonal methods needs to be tested in different settings. Here we have examined the importance of HDAC6 for inflammasome activation in different cellular systems, including primary mouse cells (BMDMs), peripheral blood mononuclear cells (PMBCs) and human cell lines and manipulated HDAC6 levels through different approaches. Under the conditions tested, we found that the activation of NLRP3 was only partially dependent on HDAC6.

J o u r n a l P r e -p r o o f

Results

NLRP3 inflammasome is activated efficiently in HDAC6 KO and ZnF m BMDMs

Previous work identified the role for HDAC6 in inflammasome activation in immortalized bone marrow-derived macrophages (iBMDMs) [START_REF] Magupalli | HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation[END_REF]. To avoid alterations in transcriptome and other cellular processes associated with establishment of an immortalized cell line, we first used primary BMDMs from wild-type (WT) mice as well as two mouse lines in which HDAC6 is either ablated (KO) [START_REF] Zhang | Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally[END_REF] or contains a W1116A point mutation in the ZnF domain (ZnF m , manuscript in preparation), which abolishes ubiquitin binding [START_REF] Indranil Banerjee | Influenza A virus uses the aggresome processing machinery for host cell entry[END_REF]. BMDMs of the different genotypes were first primed by LPS and then challenged by nigericin, which activates the NLRP3 inflammasome by raising intracellular potassium (K + ) efflux [START_REF] Muñoz-Planillo | K⁺ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter[END_REF] or MSU, which leads to generation of reactive oxygen species (ROS) through activation of NADPH oxidases [START_REF] Martinon | Goutassociated uric acid crystals activate the NALP3 inflammasome[END_REF]. Inflammasome activation was monitored by examining the cleaved Caspase-1 p20 fragment by immunoblotting and by measuring release of the cytokine IL-1β in the culture supernatant (Fig. 1A). As shown in Figure 1B and 1C, generation of Caspase-1 p20 and production of IL-1β were similar in WT, KO and ZnF m cells. The inflammasome dependence of the IL-1β release was confirmed by using the specific NLRP3 inhibitor MCC950 [START_REF] Natsume | Degrons for Controlling Protein Expression at the Protein Level[END_REF]. Because strong inflammasome activation eventually leads to cell membrane damage and release of cellular contents, we examined cellular toxicity by measuring the level of lactate dehydrogenase (LDH) release from cells. Also in this case, the differences between the genotypes were minimal and did not reach statistical significance (Fig. 1D); thus, deletion or mutation of HDAC6 in primary BMDMs does not appear to protect cells from cell death. We next examined the kinetic of IL-1β release in BMDMs of the different genotypes. Interestingly, the IL-1β amount in HDAC6 KO and ZnF m BMDMs reached a plateau rapidly in only 15 mins after nigericin challenge, while WT BMDMs responded to stimulation more slowly (Fig. S1). At later time points (between 30 and 90 mins), the IL-1β difference between the three BMDMs types were less significant, although the ZnF m BMDMs showed a slightly reduced level.

Collectively, our results in primary BMDMs did not demonstrate a clear dependency on HDAC6 or its ZnF domain for NLRP3 inflammasome activation, but indicate that HDAC6 may participate in the early timepoint response.

J o u r n a l P r e -p r o o f A ZnF-specific DARPin moderately inhibits NLRP3 inflammasome activation in THP-1 cells

To investigate more broadly the role of the HDAC6 ZnF in NLRP3 inflammasome activation, we switched from primary BMDMs to the leukemic THP-1 monocyte/macrophage cell line, a widely used surrogate for human monocytes or macrophages. We recently reported a synthetic protein based on a designed ankyrin repeat protein (DARPin) scaffold, DARPin F10, which can selectively bind to the HDAC6 ZnF Ub binding pocket in cells and blocks Ub recruitment (15) (Fig. 2A). We previously showed that DARPin F10 is highly specific for the HDAC6 ZnF [START_REF] Wang | Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways[END_REF]. We had made a degradable version of DARPin F10 by fusing it to the FKBP F36V degron tag [START_REF] Natsume | Degrons for Controlling Protein Expression at the Protein Level[END_REF] and showed that it is efficiently degraded in A549 cells upon addition of the chemical dTAG-13 (hereafter dTAG). We used this system to generate THP-1 cells expressing DARPin F10 (F10-FKBP F36V THP-1 cells) and found that the protein was well-expressed and could be efficiently degraded within 6 hours of dTAG addition (Fig. 2B); furthermore, expression of DARPin F10 does not alter the level of endogenous HDAC6, or of the major class I HDACs, HDAC1 and 2 (Fig. S2A). Nigericin-induced NLRP3 activation was comparable in dTAG treated parental (WT) THP-1 cells (Fig. 2C) and in control cells expressing only the FKBP F36V moiety (FKBP F36V THP-1 cells, Fig. S2B), thus excluding a possible inhibitory effect of FKBP F36V expression or dTAG on IL-1β release. When parental WT THP-1 cells were treated with MCC950 [START_REF] Coll | A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases[END_REF], or CGP084892, a Caspase-1 inhibitor (18), IL-1β release was strongly reduced, as expected. A modest reduction of IL-1 secretion was observed in F10-FKBP F36V THP-1 cells, when compared to the WT THP-1 cells. Degradation of DARPin F10 by dTAG treatment led to a partial, but significant, recovery of IL-1β release (Fig. 2C). We next examined early timepoints -0.5 and 1.5 hr post-nigericin activation -to study the impact of DARPin F10 on the kinetic of IL-1β release. In agreement with Figure 2C, we observed again a slight increase of IL-1β at 3.5 hr in dTAG-treated cells (Fig. 2D).

However, a larger and significant difference was seen at 1.5 hr post activation. These data indicate that blockade of the HDAC6 ZnF domain moderately inhibits the NLRP3 inflammasome, particularly during the early phase of activation by nigericin in THP-1 cells.

HDAC6 degradation moderately inhibits NLRP3 inflammasome in PBMCs

J o u r n a l P r e -p r o o f

Blocking specifically the ZnF domain in THP-1 cells by DARPin F10 showed a partial inhibition of NLRP3 inflammasome activation, confirming that HDAC6 contributes to this pathway. To extend these observations in other cell settings, we next made use of a novel HDAC6 proteolysis targeting chimera (PROTAC) degrader, XY-07-35 [START_REF] Xiong | Chemo-proteomics exploration of HDAC degradability by small molecule degraders[END_REF].

Treatment of cells with XY-07-35 leads to recruitment of the E3 ligase cereblon (CRBN) to HDAC6, ubiquitination and subsequent proteasomal degradation of HDAC6 (Fig. 3A). This degrader is based on the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) fused by a linker to pomalidomide as a CRBN-binding moiety; the negative control compound XY-07-191 contains a methyl group on the pomalidomide moiety which disrupts CRBN recruitment (Fig. 3B). In vitro enzymatic assays with purified HDACs showed that XY-07-35 inhibited HDAC6 with a low IC50=0.0485 μM, which is 10 times lower than for HDAC8 (0.621 μM) or other HDACs (1.04-5.75 μM; Fig. 3C); in addition, the degradation profile of XY-07-35 is highly specific for HDAC6 [START_REF] Xiong | Chemo-proteomics exploration of HDAC degradability by small molecule degraders[END_REF]. This compound does not show cellular toxicity when tested over a broad concentration range in either HEK293T, Jurkat or U937 cells (Fig. S3A).

We first tested the efficacy of XY-07-35 in immune cell lines. Unexpectedly, XY-07-35 treatment did not lead to HDAC6 degradation in both non-differentiated and PMAdifferentiated THP-1 cells (Fig. S3B). Therefore, to assess its effect on NLRP3 inflammasome activation in human cells we used peripheral blood mononuclear cells (PBMCs). HDAC6 was efficiently degraded in PBMCs at 1 μM XY-07-35 concentration; by contrast, the negative control compound XY-07-191 or the IκKβ inhibitor AFN700 (18) had only minimal or no effect on HDAC6 levels (Fig. 3D). To test the functional consequence of HDAC6 degraders, PBMCs were first treated with the HDAC6 degrader or the negative control compound for 24 hrs, followed by LPS priming and subsequent stimulation with MSU, nigericin, or ATP to activate the NLRP3 inflammasome (Fig. 3E). The HDAC6 degrader XY-07-35 clearly reduced the IL-1β release under all stimulatory conditions as compared to DMSO, though partial inhibition was also seen with the negative control XY-07-191, suggesting some nonspecific effects of the scaffold. HDAC6 degradation had the most profound inhibition of MSU-triggered IL-1β release, achieving ca. 60% of IL-1β reduction as compared to DMSO, while under nigericin and ATP stimulation, the reduction was 40%. A statistically significant reduction of IL-1 between XY-07-35 and the control compound XY-07-191, was only seen in the MSU conditions. Inhibitors of other nodes of the J o u r n a l P r e -p r o o f pathway, the NLRP3 inflammasome inhibitor NP3-948 and AFN700 both led to an almost complete (>90%) inhibition of IL-1β release. The negative control compound XY-07-191 also minimally inhibited IL-1β release, but less effectively than XY-07-35, suggesting a beneficial effect of HDAC6 degradation. We also measured cell viability by using resazurin fluorescence in PBMCs, but since monocytes constitute only a minor fraction of the cell population, the pyroptosis could not be tracked efficiently.

Overall, these results show partial reduction in NLRP3 inflammasome activation following degradation of HDAC6, with the magnitude of the effect depending on the stimulus.

HDAC6 degradation does not affect pyrin inflammasome activation

According to a previous report [START_REF] Magupalli | HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation[END_REF], activation of both NLRP3 and pyrin inflammasomes depends on HDAC6. To evaluate the importance of HDAC6 for the pyrin inflammasome, we used a previously established U937 pyrin-overexpressing cell line [START_REF] Alimov | Bile acid analogues are activators of pyrin inflammasome[END_REF]. In these cells, treatment with the bile acid derivative BAA473 activates the pyrin inflammasome, leading to the release of cytokines (e.g., IL-1β, IL-18) and pyroptosis; IL-18 expression is induced without the need for prior priming and was used for monitoring inflammasome activation. HDAC6 was efficiently degraded in the U937-pyrin expressing cells by XY-07-35 at concentrations ranging from 0.1 μM to 10 μM (Fig. 4A). However, treatment with XY-07-35 did not result in any change in IL-18 secretion compared to either DMSO control or the XY-07-191 control (Fig. 4B).

Additionally, we measured cell viability and found that HDAC6 degradation by XY-07-35 did not protect cells from pyroptosis (Fig. S4), confirming that in this setting, HDAC6 degradation has no effect on pyrin inflammasome activation.

J o u r n a l P r e -p r o o f

Discussion

In this study, we utilized a range of cellular systems and several orthogonal approaches to modulate HDAC6 activity and probe its role in NLRP3 and pyrin inflammasome activation. HDAC6 protein levels were modulated by both knock-out and chemically induced degradation, while function was probed by a knock-in unable to bind to ubiquitin and a DARPin inhibitor blocking ubiquitin recruitment. In contrast to a previous study reporting on the HDAC6-ubiquitin interaction as a crucial regulator of inflammasome activation [START_REF] Magupalli | HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation[END_REF], we did not observe a significant difference in NLRP3 inflammasome activation between WT and HDAC6 knock-out or HDAC6 ZnF m (W1116A) BMDM cells. To investigate this further, we examined the effect of a small protein (DARPin F10) binding to the HDAC6 ZnF domain and preventing ubiquitin recruitment. This protein was shown to have a strong inhibitory impact on processes involving the ZnF-Ub interaction, such as the formation of aggresomes or stress granules, or infection by influenza or Zika virus [START_REF] Wang | Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways[END_REF]. But under conditions of inflammasome induction, we observed only partial downregulation of IL-1β release in THP-1 cells expressing DARPin F10 when stimulated with nigericin. In primary PBMCs and U937 cells, the selective HDAC6 degrader XY-07-35 efficiently degraded HDAC6, but HDAC6 had only modest, stimulation-dependent effects on NLRP3 inflammasome activation. Furthermore, HDAC6 degradation had no effect on IL-18 release and pyroptosis mediated by pyrin. With respect to NLRP3 inflammasome activation, HDAC6 degradation had the strongest effect on MSU-stimulated IL-1 release, which is a milder, non-pyroptotic form of NLRP3 activation (Fig. 3E). Similarly, in THP1 cells, HDAC6 inhibition seemed to have its most pronounced effect at early timepoints. It is possible that HDAC6 plays a more pronounced role in response to crystalline inflammasome activators (in agreement with the results in Magupali et al, 2022). It has also been noted that other NLRP3 regulatory mechanisms such as PKD phosphorylation play an important role at early time points in cell culture experiments, but compensatory pathways override these effects at later timepoints [START_REF] Heiser | Evaluation of protein kinase D auto-phosphorylation as biomarker for NLRP3 inflammasome activation[END_REF].

Based on our results it seems that the importance of HDAC6 for NLRP3 inflammasome activation is not absolute, but condition and context dependent. On one hand, we noted that blocking the HDAC6 ZnF-Ub interaction with DARPin F10 in THP-1 cells and degrading HDAC6 in PBMCs both resulted in an inhibitory effect, thus J o u r n a l P r e -p r o o f supporting a role for HDAC6 in this pathway. When degrading DARPin F10 with dTAG we only observed a partial rescue of IL-1β release; this can possibly be explained by incomplete degradation of DARPin F10, leading to some residual F10 (even in tiny amounts) that could interfere with ubiquitin recruitment. Similarly, incomplete HDAC6 clearance by the proteasome system might be responsible for the moderate downregulation of IL-1β release or cell death protection in XY-07-35 treated PBMCs. Furthermore, we found that the negative control compound XY-07-191 also exhibited slight inhibition of NLRP3 inflammasome activation, which we attribute to its possible catalytic inhibition of HDAC6. Indeed, both compounds are derived from SAHA (Fig. 3B), which is known to inhibit all HDACs including HDAC6, and previous studies have demonstrated that HDAC inhibition can downregulate IL-1β release [START_REF] Magupalli | HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation[END_REF][START_REF] Xu | Tubastatin A Improves Post-Resuscitation Myocardial Dysfunction by Inhibiting NLRP3-Mediated Pyroptosis Through Enhancing Transcription Factor EB Signaling[END_REF]. Context dependence for HDAC6 inflammasome activation has also been observed in HDAC6 inhibitor studies. For instance, a recent study in THP-1 cells [START_REF] Bockstiegel | Pharmacological inhibition of HDAC6 suppresses NLRP3 inflammasomemediated IL-1β release[END_REF] showed that inhibiting HDAC6 by Tubastatin A leads to a reduced IL-1b release, but in another study in BMDMs (24), Tubastatinn A did not impact the mIL-1b level. We notice these differences and remark that current HDAC inhibitors, including HDAC6-selective inhibitors, may not be as specific as previously reported [START_REF] Lechner | Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target[END_REF].

On the other hand, we found that the activation of the NLRP3 inflammasome in BMDMs from HDAC6 KO and ZnF m (W1116A) mice took place normally; likewise, activation of the pyrin inflammasome in U937-pyrin cells was not affected by HDAC6 depletion. In a previous study using immortalized BMDMs [START_REF] Magupalli | HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation[END_REF], researchers described a HDAC6-mediated aggresome-like pathway for NLRP3 inflammasome formation, and reported that deletion of HDAC6 or mutations in the ZnF domain completely blocked inflammasome activation, in contrast to our results. Likewise, another recent study reported that the lysosomal Ragulator complex is important for NLRP3 inflammasome activation in vivo via interaction with HDAC6 (12).

We hypothesize that the partial discrepancy may result from the differences in the cell types and specific experimental conditions used. Immortalized cells are known to carry chromosomal abnormalities that allow them to proliferate indefinitely, leading to changes in their transcriptome, proteome and reliance on specific pathways compared to primary cells. As a result, it is reasonable to speculate that established iBMDMs may behave differently from primary BMDMs. As for the pyrin inflammasome, we used U937 cells instead of iBMDMs, and bile acid derivatives instead of Clostridioides J o u r n a l P r e -p r o o f difficile toxin B (TcdB) to activate the pathway, which may also contribute to the observed differences in results obtained. In summary, we conclude that HDAC6 can participate in NLRP3 inflammasome activation, but its importance appears to be context dependent.

Experimental procedures

Cell Culture THP-1, U937, PBMC and BMDM cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium-stable Glutamine (Bioconcept#1-41F03-1) supplemented with 10% fetal bovine serum (FBS). For BMDMs the medium also contained M-CSF 1 (100 ng/mL). All cells were cultured in 5% CO2 at 37°C.

BMDMs isolation and culture

BMDMs were isolated as previously described [START_REF] Afroz | Quantifying Regulated Mitochondrial Fission in Macrophages[END_REF]. In short, mice (8~14 weeks old) were sacrificed and both legs were sterilized in 70% ethanol. Attached tissues were removed by dissection with scissors and the isolated bones were flushed or crushed twice in a mortar with 5 mL RPMI 1640 medium. The suspension from crushed bones was filtered through 100 μm cell strainer (Falcon#352360) to remove debris. Filtered cells were centrifuged at 500 x g, 15 mins at room temperature. Supernatant was removed and cell pellets were resuspended in RPMI 1640 (+M-CSF 1, 100 ng/mL).

The cell suspension (from 1 leg) was divided into four Nunc Square 25-cm dishes (Thermo Scientific#166508), with each plate containing 10 mL cell suspension. On Day 3 post-seeding, 5 mL fresh RPMI 1640 (+M-CSF 1) medium was added to each plate. On Day 6, BMDMs were ready for collection or re-seeding for new experiments.

All mice handling was done according to Swiss federal guidelines for animal experimentation and approved by the FMI Animal committee and the local veterinary authorities (Kantonales Veterinäramt of Kanton Basel-Stadt).

Immunoblotting

Treated BMDM cells were lysed with RIPA buffer (supplied with cOmplete™ Protease Inhibitor Cocktail, Merck#11697498001), and protein concentration was determined by Bradford assay. Same protein amounts were loaded onto 4-12% NuPAGE Gels J o u r n a l P r e -p r o o f and protein was transferred to PVDF membranes by using Trans-Blot® Turbo™ Transfer System (Biorad#17001917). Membrane was blocked with 5% milk-TBS, and then incubated with primary antibodies overnight at 4°C. Signal was developed with HRP-conjugated secondary antibodies and Amersham ECL Western Blotting Detection Reagent (Cytiva#RPN2106). Antibody information: mouse Caspase-1 (p20)

(AdipoGen#AG-20B-0042-C100), mHDAC6 (homemade), human HDAC6
(CellSignaling#7558), α-tubulin (Sigma-Aldrich #T9026), HA (CellSignaling#3724), pan Actin (Sigma-Aldrich#SAB4502632).

ELISA (for IL-1β and IL-18)

Supernatant from treated cells were collected and frozen at -80°C before determining cytokine concentrations. mIL-1β was determined by ELISA MAX™ Standard Set Mouse IL-1β kit (BioLegend# 432601). Human IL-1β and IL-18 were determined with Cisbio HTRF Cisbio Human IL1β (#62HIL1BPET) and Human IL18 (#62HIL18PEG) protocols. All the data were from 3 or 4 independent biological replicates.

LDH measurement

Culture supernatants from the different BMDMs samples were analyzed with the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega# G1780). Total cell lysate of non-treated (i.e., DMSO only -no inhibitor-, but induced for inflammasome formation) BMDM cells was defined as 100% cell death or cytotoxicity. Values from other conditions were calculated as follows: (ODtreated cells-ODno-inhibitor control)÷(ODtotal lysate-ODno-inhibitor control) * 100.

Single cell clonal THP-1 F10 and FKBP cells establishment

Lentiviral vectors for HA-DARPin F10-FKBP F36V and HA-FKBP F36V was from previous work [START_REF] Wang | Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways[END_REF]. Lentivirus was generated with 3 rd generation lentivirus packaging systems.

In detail, DARPin F10 vector and lentivirus packaging plasmids (Tat, Rev, Gag/pol, Vsv-g) were co-transfected in 293T cells in 6 well-plates. After 3 days culturing, supernatants were harvested and concentrated with Lenti-X™ Concentrator (Takara# 631232). Virus pellet was resuspended in 1~4 mL RPMI1640 medium (with 10% FBS).

Next, THP-1 cells were seeded in 6 well-plates (each well with 1 x10 6 cells), and then lentivirus was added to the plate in the presence of polybrene (8 μg/mL), followed by J o u r n a l P r e -p r o o f centrifugation at 1500 rpm / 453 x g, for 45 mins at room T°. After transduction, THP-1 cells were cultured at 37°C for 7~14 days. The cell number and density were monitored carefully; when a concentration of > 2 x10 6 cells per mL was reached, 2 μg/mL puromycin was added for selection for 3~6 days. Surviving cells were single cell sorted by FACS into 96 well plates (in RPMI1640 without puromycin). After about 1 month in culture, single cell clones were analyzed by immunoblotting to test for DARPin F10 expression. Cell clones with a good expression level were stored in cryosolution (90% FBS + 10% DMSO) in liquid nitrogen.

Inflammasome activation

For BMDM cells, 1.5 x10 6 cells per well were seeded in 6 well-plates. After priming with 1 μg/ml LPS (Invivogen #tlrl-b5lps), cells were challenged with 200 μg/ml MSU crystals (Invivogen #tlrl-msu-25) for 6 hours or 20 μM nigericin (Sigma-Aldrich #N7143-5MG) for 30 mins. For THP-1 cells, 5x10 5 cells/mL were primed with 0.5 μM PMA (Invivogen #tlrl-pma) in T75 Ultra-low binding flask (CORNING #3814) for 3 hours, and then cells were collected by centrifugation at 500 x g, for 5 mins. The supernatant was removed, and fresh medium was added to have cells at a density of around 5x10 5 cells/mL. 100 μL cells were seeded in 96 well plate and incubated for 1~2 days. During the culturing, dTAG was added when needed to degrade the DARPin. After 1~2 days culturing, inflammasome was activated by adding 15 μM nigericin for up to 4 hours. For PBMCs, 3x10 4 cells per were seeded, and primed with 0.2 ng/mL LPS for 4 hours and then incubated with MSU 200 μg/mL (5 hours), nigericin 2 μM (2 hours) or ATP 3 mM (0.5 hours). For pyrin inflammasome, U937-pyrin cells were seeded in 96 well after 0.5 μM PMA treatment for 3 hours and then challenged by BAA473 (200 μM) for 4 hrs at 37°C. For control experiments inhibitors were used at the following final concentration: MCC950 at 10 μM, CGP084892 at 20 μM. NP3-948 is a potent NLRP3 inhibitor developed at Novartis that competes with MCC950 for binding to the NLRP3 protein [START_REF] Dekker | Crystal Structure of NLRP3 NACHT Domain With an Inhibitor Defines Mechanism of Inflammasome Inhibition[END_REF].

Cell viability Assay

Cell viability of PBMCs and U937 cells was checked with PrestoBlue™ HS Cell Viability Reagent (Invitrogen # P50200) according to the manufacturer's instructions. J o u r n a l P r e -p r o o f

J o u r

  n a l P r e -p r o o f research funding from Deerfield, Novartis, Ajax, Interline and Astellas. K.A.D is a consultant to Kronos Bio and Neomorph Inc.

Figure 1 :

 1 Figure 1: The NLRP3 inflammasome is activated normally in HDAC6 KO or ZnF m primary mouse BMDMs

Figure 2 :

 2 Figure 2: A DARPin blocking the HDAC6-Ub interaction has a modest effect on IL-1β release from THP-1 cells (A) Structure of the DARPin F10 -HDAC6 ZnF domain complex (PDB: 7ZYU) showing blockade of Ub recruitment. The surface representation shows HDAC6 ZnF (yellow) and the C-terminal LRGG of Ub (light pink) (overlayed with PDB:3GV4), the ribbon representation depicts DARPIN F10 (cyan).

Figure 3 :

 3 Figure 3: Effect of XY-07-35 mediated HDAC6 degradation on NLRP3 inflammasome activation in PBMCs (A) Schematic of the mode of action of an HDAC6 degrader. The PROTAC engages both HDAC6 and the E3 ligase cereblon (CRBN), which then leads to ubiquitination and degradation of HDAC6.

Figure 4 :

 4 Figure 4: Effect of HDAC6 degradation on pyrin inflammasome activation in U937 cells(A) XY-07-35 mediated HDAC6 degradation in pyrin-expressing U937 cells. Cells were treated with the indicated compounds and HDAC6 was detected by immunoblotting with an anti-HDAC6 antibody. Detection of actin was used as a control for protein loading.

Figure S3 (

 S3 Figure S3 (related to Fig. 3):

Figure S4 (

 S4 Figure S4 (related to Fig. 4): Cell viability was measured by resazurin assay, and fluorescence intensity was a representative of the viability. No significant difference was found among DMSO, XY-07-35 and XY-07-191 groups. Maximum survival was obtained when cells were treated with the inflammasome inhibitor CGP. J o u r n a l P r e -p r o o f

Acknowledgements and funding

We thank Christelle Arnold (NIBR) for her help in setting up the collaboration with FMI.

We also thank Chun Cao and Gabriele Matthias for maintaining and providing HDAC6 KO and ZnF m mice. Financial support through the SystemsX.ch MRD project VirX J o u r n a l P r e -p r o o f

Funding acquisition.

J o u r n a l P r e -p r o o f

Declaration of interests

Part of the results presented herein have been used in the patent application EP-A-20213494.6. E.S.F is a founder, scientific advisory board (SAB) member, and equity holder of Civetta Therapeutics, Lighthorse Therapeutics, Proximity Therapeutics, and Neomorph, Inc. (also board of directors). He is an equity holder and SAB member for Avilar Therapeutics and Photys Therapeutics and a consultant to Novartis, Sanofi, EcoR1 Capital, Ajax Therapeutics, Odyssey Therapeutics and Deerfield. The Fischer lab receives or has received research funding from Deerfield, Novartis, Ajax, Interline and Astellas. K.A.D is a consultant to Kronos Bio and Neomorph Inc. J o u r n a l P r e -p r o o f

Data availability

Mass spectrometry data related to HDAC6 degrader XY-07-35 and XY-07-191 are deposited to PRIDE under archive number PXD023652. The data that supports the findings described in this work are available from the corresponding author upon request.

Abstract

The inflammasome is a large multiprotein complex that assembles in the cell cytoplasm in response to stress or pathogenic infection. Its primary function is to defend the cell and promote the secretion of pro-inflammatory cytokines, including IL-1β and IL-18. Previous research has shown that in immortalized bone marrow-derived macrophages (iBMDMs) inflammasome assembly is dependent on the deacetylase HDAC6 and the aggresome processing pathway (APP), a cellular pathway involved in the disposal of misfolded proteins. Here we used primary BMDMs from mice in which HDAC6 is ablated or impaired and found that inflammasome activation was largely normal. We also used human peripheral blood mononuclear cells and monocytes cell lines expressing a synthetic protein blocking the HDAC6-ubiquitin interaction and impairing the APP and found that inflammasome activation was moderately affected.

Finally, we used a novel HDAC6 degrader and showed that inflammasome activation was partially impaired in human macrophage cell lines with depleted HDAC6. Our results therefore show that HDAC6 importance in inflammasome activation is context dependent.

J o u r n a l P r e -p r o o f

After incubation for 1 to 2 hrs, fluorescence was determined with a PheraStar plate reader under Ex/Em=540/590 nm.

Cell viability with CTG assay U937, Jurkat, and HEK293T cells were plated with a density of 10 5 cells/mL, with 50 µL/ well in a white 384 well plate (Corning #3570). Two hours after plating, XY-07-035 was dispensed to each well using HPd300e and incubated at 37℃ for 70 hours.

CellTiter-Glo® (CTG) Luminescent Cell Viability Assay (Promega #G7572) was then used to measure cell viability. Aliquoted frozen CTG reagent and cell plates were warmed to room temperature prior to loading. After the plates were cooled to room temperature, the CTG reagent was loaded 15 µL/ well with an electronic multichannel pipette. To ensure the reagent had fully reacted with cells, plates were centrifuged at 500 g, 3 min followed by a 30 min incubation in a dark box at room temperature. The luminescence signal was read over 10 cycles for each data point on a PHERAstar FS microplate reader (BMG Labtech). The Relative abundance was calculated as the sum of the averages of duplicate individual 12-point dose-response data points.

Statistical analysis

All the statistical analysis were done with One-way or Two-way ANOVA test, corrected for multiple comparisons using post-hoc significance Tukey test. A significance level of p < 0.05 was used.


domain: spi spi

text:
Nuno Gonçalo

Martins

Maria Guerrero Sanchez

Angela Daniela Carboni
email: angela.carboni@hotmail.com

Fernanda Fonseca
email: fernanda.fonseca@inrae.fr

Sonia Campoy
email: sonia.campoy@kerry.com

Andrea Gómez-Zavaglia

Paula Cristina Castilho

Keywords: galacto-oligosaccharides, legumes, lactic acid bacteria, stabilization

HAL is

  Stabilization of Lactiplantibacillus plantarum WCFS1 during freezing, freeze-drying, and storage using legume wastewater extracts Gonçalo N. Martins, Maria Guerrero Sanchez, Angela D. Carboni, Fernanda Fonseca, Sonia Campoy, Andrea Gómez-Zavaglia, Paula C. Castilho To cite this version: Gonçalo N. Martins, Maria Guerrero Sanchez, Angela D. Carboni, Fernanda Fonseca, Sonia Campoy, et al.. Stabilization of Lactiplantibacillus plantarum WCFS1 during freezing, freeze-drying, and storage using legume wastewater extracts. 3rd Food Chemistry Conference, Oct 2023, Dresden, Germany. hal-04395559