Marcello D'amelio

Virve Cavallucci

Silvia Middei

Cristina Marchetti

Simone Pacioni

Alberto Ferri

Adamo Diamantini

Daniela De Zio

Paolo Carrara

Luca Battistini

Sandra Moreno

Alberto Bacci

Martine Ammassari-Teule

Hélène Marie

Francesco Cecconi
email: francesco.cecconi@uniroma2.it

Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer's Disease

Keywords: AMPA receptor, GluR1, Tg2576 mouse model, APPswe, calcineurin, cell death, synaptic degeneration, synaptic plasticity, memory function

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mice expressing mutated forms of the amyloid precursor protein (APP), associated with familial Alzheimer's Disease, show age-dependent reductions in spine density, prior to plaque deposition [START_REF] Lanz | Dendritic spine loss in the hippocampus of young PDAPP and Tg2576 mice and its prevention by the ApoE2 genotype[END_REF] .

Nevertheless, the molecular link between APP mutations triggering Alzheimer's Disease, and the occurrence of early spine loss remains elusive. Interestingly, a localized caspase-3 activity, causing synaptic failure, has been observed in vitro [START_REF] Mattson | Evidence for synaptic apoptosis[END_REF] , but the molecular mechanism linking caspase-3 activity to synaptic loss is far from being elucidated.

Here, we analyzed caspase-3 activity in hippocampal synapses of the Tg2576 transgenic mouse model, harboring the human APPswe mutant allele linked to familial Alzheimer's Disease [START_REF] Hsiao | Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice[END_REF] . These mice develop early synaptic deficits [START_REF] Jacobsen | Early-onset behavioral and synaptic deficits in a mouse model of Alzheimer's disease[END_REF] and several neuropathological features at older age, including amyloid plaques and dystrophic neurites [START_REF] Kawarabayashi | Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease[END_REF] . Although Tg2576 mice lack neurofibrillary tangles and significant neuronal loss [START_REF] Irizarry | APPSw transgenic mice develop age-related A beta deposits and neuropil abnormalities, but no neuronal loss in CA1[END_REF] , there is strong evidence that accumulation of the amyloid-β (Aβ) peptide, derived via APP proteolysis, is responsible for age-related memory decline in this model 15- 17 .

By means of several experimental approaches, we demonstrate an increase of caspase-3 activity in Tg2576 hippocampal synapses at 3 months of age, much earlier than amyloid plaque deposition is detectable [START_REF] Hsiao | Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice[END_REF] . We report that this enhanced local caspase-3 activity leads to a permanent activation of calcineurin which causes, in turn, AMPA receptor (AMPAR) GluR1 subunit dephosphorylation and its removal from the post-synaptic sites. Furthermore, we show that this caspase-3 activity increase is matched by i) a deficit in hippocampal-dependent contextual fear conditioning (CFC), ii) a reduction of spine density in CA1 pyramidal neurons, iii) an alteration in basic glutamatergic synaptic transmission and enhanced LTD in these same neurons. Importantly, we demonstrate that pharmacological inhibition of caspase-3 activity in Tg2576 mice restores post-synaptic densities (PSD) composition, glutamatergic synaptic transmission and spine size, and provides a partial rescue of memory during CFC. Collectively, our data indicate that caspase-3 activity plays a crucial role in the early synaptic dysfunction reported during Alzheimer pathology in Tg2576 mice.

Results

Onset of memory decline in Tg2576 mice

We evaluated hippocampal-dependent memory in Tg2576 mice and their wild-type controls by means of the CFC task [START_REF] Phillips | Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning[END_REF][START_REF] Lee | Differential contributions of dorsal hippocampal subregions to memory acquisition and retrieval in contextual fear-conditioning[END_REF] . We observed that, although wild-type and Tg2576 mice exhibit similar baseline activity during the training trials before the shock delivery (Supplementary Fig. 1), memory performance, as assessed by freezing behavior upon re-exposure of mice to the context where they were previously shocked, was reduced in Tg2576 mice compared to wild-type mice (Fig. 1a). Notably, this effect reached statistical significance at 3 but not at 2 months of age, suggesting the 3-month age-point as the onset of hippocampal-dependent memory impairment.

To assess whether early cognitive decline in Tg2576 mice was associated with dendritic spine alterations, we measured spine density and spine size in Golgi-stained CA1 pyramidal neurons of the dorsal hippocampus in 2-and 3-month-old wild-type and Tg2576 mice (Fig. 1b). Number of spines along basal dendrites laying in the stratum oriens is unaltered in Tg2576 mice at both 2 and 3 months of age. We detected on apical dendrites laying in the stratum radiatum a significant reduction in spine density in 3-month-old Tg2576 mice compared to both wild-type littermate and 2-month-old Tg2576 mice (Fig 1c). Along the same apical dendrites, Tg2576 mice exhibited a higher proportion of spines with smaller head diameters (Fig. 1d) and the total dendrite length was also significantly reduced (Fig. 1e) in 3-month-old Tg2576 mice (1429.10±97.10 µm) compared to age-matched wild-type mice (2057.99±195.30 µm). Altogether, these alterations result in a reduced synaptic input in the hippocampus of Tg2576 mice that likely contributes to their cognitive impairment.

Detection of early synaptic deficits in Tg2576 mice

Structural modifications in the PSD precede morphological alterations of the dendritic spine [START_REF] Lajtha | Handbook of Neurochemistry and Molecular Neurobiology[END_REF] . In order to investigate the molecular basis of dendritic spine loss in Tg2576 mice we thus evaluated the PSD composition in wild-type and Tg2576 mice at 3 months of age. Isolated PSD preparation extracts from hippocampi of Tg2576 and age-matched wild-type mice were blotted and probed with antibodies against the main PSD components (Fig. 2a and Supplementary Fig. 2a,b). Whereas the relative amounts of most post-synaptic components remained essentially unchanged in PSD from both genotypes, the levels of the AMPAR subunit GluR1 and one of its phosphorylated form (GluR1pSer845) were significantly reduced in the PSD obtained from Tg2576 mice compared to wild-type (Fig. 2a). However, in total hippocampal homogenates, the GluR1 levels were unaffected while GluR1pSer845 levels were reduced in Tg2576 hippocampus (Fig. 2b). Furthermore, the subcellular distribution of GluR1 was also markedly changed in Tg2576 mice, with a significant decrease in the PSD-enriched fraction (TxP) and a significant increase in the microsome-enriched fraction (P3) (Fig. 2c and Supplementary Fig. 2c). Altogether, these results argue against a global degradation of GluR1 in the whole hippocampus of Tg2576 mice, but suggest a synaptic redistribution of GluR1 and specific dephosphorylation of its Ser845.

Since it has been shown that Aβ impairs synaptic plasticity [START_REF] Li | Soluble oligomers of amyloid Beta protein facilitate hippocampal long-term depression by disrupting neuronal glutamate uptake[END_REF] , we asked whether modulating Aβ load in Tg2576 mice could affect GluR1 distribution. We injected intra-hippocampally the γsecretase inhibitor (DAPT) in 3-month-old Tg2576 mice and we found that there was a marked translocation of GluR1 to the PSD-enriched fraction in the DAPT-injected ipsilateral side, compared to the vehicle-injected controlateral hippocampus already 15 hrs after injection (Supplementary Fig. 3a). We also found that the head diameter of dendritic spines was enlarged in the DAPT-injected ipsilateral side, compared to the vehicle-injected controlateral hippocampus (Supplementary Fig. 3b). Finally, bilateral injection of DAPT in the hippocampus of Tg2576 mice 15hrs before training significantly rescued memory deficits without affecting motor activity (Supplementary Fig. 3c,d). Together with previous in vitro studies [START_REF] Almeida | Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses[END_REF] , these in vivo results indicate that Aβ contributes to synaptic modifications, since alterations in GluR1 distribution and dendritic spine alterations were blocked by γ-secretase-dependent APP processing inhibition.

Alterations of the PSD composition of hippocampal neurons in 3-month-old Tg2576 mice may lead to changes in glutamatergic synaptic transmission. We first evaluated basic synaptic transmission by measuring input-output relationships of field excitatory post-synaptic potentials (fEPSPs) of CA1 pyramidal neurons evoked by stimulation of the Schaffer collateral pathway from acute hippocampal slices obtained from wild-type and Tg2576 mice. No difference was detected between Tg2576 and wild-type mice using this analysis (Supplementary Fig. 4). To detect more subtle alterations in synaptic transmission we next performed whole-cell patch-clamp recordings of CA1 pyramidal neurons. As the main alteration in the PSD of 3-month-old Tg2576 mice was a downregulation of GluR1 levels whereas the levels of NMDA receptor (NMDAR) subunits remained relatively stable, we first assessed the relative contribution of AMPARs and NMDARs to evoked excitatory post-synaptic currents (EPSCs), measured as an AMPA/NMDA ratio. We found that this ratio was significantly decreased in Tg2576 mice (wild-type 39.34±6.76, Tg2576 20.24±4.64; Fig. 3a). We evaluated pre-synaptic release short-term plasticity by measuring the paired-pulse ratios (PPR) of synaptic responses. PPR did not differ in Tg2576 (1.42±0. 19) compared to wild-type (1.40±0.07) mice, arguing against any alterations in pre-synaptic release properties at this age (Fig. 3b). We also examined AMPAR transmission by measuring spontaneous miniature excitatory postsynaptic currents (mEPSCs), which represent AMPAR-mediated post-synaptic responses to spontaneous release of individual glutamate vesicles (Fig. 3c,d). We did not observe a statistically significant difference in mEPSC amplitude in Tg2576 (12.77±0.66) with respect to wild-type (14.12±0.68) mice, although a trend towards a decrease was apparent (Fig. 3d). We however observed that mEPSC frequency was significantly decreased in Tg2576 mice (wild-type 1.15±0.14, Tg2576 0.80±0.09; Fig. 3d). A lack of PPR alterations together with a reduction of mEPSC frequency is consistent with a decrease in the number of AMPAR-containing synapses. These results are in line with the reduction of GluR1 levels in the PSD and a loss of spines observed in 3month-old Tg2576 mice, although we cannot exclude additional NMDAR alterations.

The role of GluR1 phosphorylation in synaptic plasticity paradigms, such as long term potentiation (LTP) and long term depression (LTD), has been extensively studied [START_REF] Malinow | AMPA receptor trafficking and synaptic plasticity[END_REF] . We therefore tested whether the specific reduction of GluR1pSer845 in the PSD affected LTP and LTD expression. Similar levels of synaptic potentiation were observed in wild-type and Tg2576 mice (Fig. 3e). This result suggests that the mechanisms of GluR1 trafficking to the PSD during LTP are still intact in Tg2576 mice at this age, as previously reported [START_REF] Chapman | Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice[END_REF] .

By contrast, we found that the magnitude of LTD observed in Tg2576 mice was slightly, but significantly, enhanced compared to wild-type mice (Fig. 3f). These data suggest that the mechanisms driving synaptic depression, including specific dephosphorylation of GluR1Ser845, are more efficient in Tg2576 than in wild-type mice [START_REF] Lee | NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus[END_REF] .

Active caspase-3 increases in Tg2576 hippocampal synapses

By means of in vitro studies, a localized caspase-3-dependent apoptosis has been proposed to contribute to early synaptic loss in Alzheimer and other neurodegenerative diseases [START_REF] Mattson | Evidence for synaptic apoptosis[END_REF] ; the mechanism(s) underlying this process, however, remains elusive.

We therefore tested whether caspase-3 activity was responsible for the observed PSD alteration and spine loss in the Tg2576 hippocampus. We compared caspase-3-like activity in whole cell extracts and synaptosomal fraction prepared from hippocampi of 2-and 3-month-old wild-type and Tg2576 mice. A significant increase in caspase-3 activity, in both total hippocampal homogenates and synaptosomes, was observed in Tg2576 mice at 3 but not at 2 months of age (Fig. 4a).

A significant increase in caspase-3 activity was also detected in Tg2576-derived synaptosomes when compared to those from age-matched wild-type mice at 6 and 18 months after birth (Supplementary Fig. 5).

The cleaved forms of caspase-3 were detected in synaptosomes showing a stronger immunoreactivity in Tg2576 compared to wild-type mice at 3 months of age (Fig. 4b). Intriguingly, we found that procaspase-3 is also more abundant in the synaptosomal fraction, when compared to the total homogenate. These findings suggest that caspase-3 is preferentially localized and selectively activated within the synaptic compartment, whereas the other effector caspases (caspase-7 and -6) remain uncleaved (Supplementary Fig. 6).

To provide additional evidence of the synaptic localization of active caspase-3 in the hippocampus, we performed pre-embedding immunoelectron microscopy, using an antibody specific to cleaved caspase-3. Immunostained spines were significantly more abundant in Tg2576 neurons than in their wild-type counterparts (Fig. 4c and Supplementary Fig. 7). Within the spine, the immunoprecipitates were specifically concentrated within the PSD. No immunoreactivity was found in cell soma, nuclei, pre-synaptic terminals, or surrounding glial projections.

The exposure of phosphatidylserine (PS) residue on the plasma membrane is usually considered as a downstream event of caspase-3 activation and a hallmark of apoptosis [START_REF] Van Engeland | Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure[END_REF] . In order to examine apoptosis in hippocampal synapses, we analyzed the exposure of PS in the synaptosomal fraction where active caspase-3 accumulates. By labeling with annexin V and a viability marker (calcein AM) prior to flow cytometry, local synaptic death was measured in intact synaptosomes prepared from the hippocampus of 3-month-old wild-type and Tg2576 mice. We found that PS exposure is increased in Tg2576-derived synaptosomes when compared to wild-type (Fig. 4d). The absence of genotype-dependent difference in propidium iodide (PI) labeling of hippocampal cell nuclei, isolated from the same hippocampi used for synaptosome preparation, rules out the possibility of wide-spread neuronal apoptosis (Fig. 4e). The reported ~5% of nuclei incorporating PI is merely due to the experimental procedure (background). Unstained controls are shown in Supplementary Fig. 8a,b. In line with these results, no cell death was detected by TUNEL assay in wild-type nor in Tg2576 hippocampi (Supplementary Fig. 8c).

An increase in caspase-3 activity in the absence of neuronal death suggests a sub-lethal mitochondrial stress. This is in line with the finding that, in the hippocampus of 3-month-old Tg2576 mice, protein oxidation level is significantly higher than in those from age-matched controls (Supplementary Fig. 9).

Comprehensively, these data demonstrate that oxidative stress is initiated in Tg2576 mice already at 3 months of age, synchronous with the occurrence of some apoptotic biochemical features which can occur, locally, at the synapses.

The canonical activation of caspase-3 occurs in a wide range of cellular and in vivo systems by means of two main pathways [START_REF] Green | The pathophysiology of mitochondrial cell death[END_REF][START_REF] Schulze-Osthoff | Apoptosis signaling by death receptors[END_REF] : the extrinsic pathway of cell death, mediated by the death receptor family; and the mitochondrial (intrinsic) pathway of cell death, mediated by the Apaf1centered apoptosome. Since the main route of neuronal death is along the mitochondrion/apoptosome pathway, we expected caspase-3 to be activated via the intrinsic pathway. To confirm the involvement of the Apaf1-centered apoptosome in this context, we used Tg2576-derived primary hippocampal cultured neurons lacking Apaf1 (Tg + /Apaf1 -/-double mutants). In both total lysates and synaptosomes from Tg + /Apaf1 -/-neurons, we did not observe caspase-3 cleavage (Supplementary Fig. 10a,b). In addition, in Tg + /Apaf1 +/+ and Tg + /Apaf1 -/- neurons, caspase-7 and -6 were not activated (Supplementary Fig. 10c). These results suggest that, in Tg2576 neurons, caspase-3 activation is apoptosome-dependent.

Caspase-3 mediates GluR1 removal from post-synaptic sites

As caspase-3 activity was increased in the post-synaptic compartments and synaptic GluR1 trafficking was altered in Tg2576 hippocampal neurons, we tested whether these two molecules interacted epistatically. We performed direct fluorimetric measurement of caspase-3-like activity in acute hippocampal slices from 3-month-old Tg2576 mice after 2 hrs of incubation with the caspase-3 inhibitor z-DEVD-fmk and we observed that z-DEVD-fmk inhibits caspase-3-like activity in a concentration-dependent manner (Fig. 5a). Notably, cleaved PARP (a nuclear substrate of caspase-3) was undetectable in all experimental conditions, ruling out the possibility that z-DEVD-fmk protects neurons from cell death induced by slicing and incubation conditions (Supplementary Fig. 11).

Immunoblot analysis of PSD from Tg2576 slices treated with z-DEVD-fmk showed an increase of GluR1 and GluR1pSer845, compared to the vehicle-treated controls. By contrast, caspase-3 inhibition had no effect on NMDAR subunits and PSD-95 levels (Fig. 5b). A linear regression analysis showed that the GluR1 levels in the PSD are inversely correlated to caspase-3-like activity (Fig. 5c). Conversely, analysis of PSD from wild-type slices treated with z-DEVD-fmk showed that caspase-3 inhibition causes an inverse effect on GluR1 surface expression (Supplementary Fig.

12).

We also analyzed the content of GluR1 in total homogenate of Tg2576 hippocampal slices treated with z-DEVD-fmk compared to untreated slices. Caspase-3 inhibition caused an increase of GluR1pSer845 but no difference was observed in total GluR1 levels (Fig. 5d).

The treatment of Tg2576 hippocampal slices with the caspase negative control inhibiting other proteases, z-FA-fmk, caused an expected reduction of cathepsin B activity but it did not affect the phosphorylation of GluR1 (Supplementary Fig. 13a,b). Comprehensively, these results indicate that caspase-3, under these experimental conditions, is specifically involved in GluR1 phosphorylation and mobility, but not degradation.

To confirm the involvement of caspase-3 in the regulation of synaptic transmission, we measured AMPA/NMDA ratio of CA1 neurons in acute Tg2576 hippocampal slices after 2 hrs of incubation with caspase-3 inhibitor or vehicle. We found that the AMPA/NMDA ratio in z-DEVD-fmk-treated slices was significantly increased with respect to vehicle-incubated slices restoring the ratio to wildtype value (vehicle control 19.46±2.77, z-DEVD-fmk 46.43±8.48, wild-type 39.34±6.76; Fig. 5e).

PPR of synaptic responses did not differ in z-DEVD-fmk-treated compared to vehicle-treated slices (vehicle control 1.34±0.05, z-DEVD-fmk 1.32 ±0.05, wild-type 1.40±0.07; Fig. 5f), indicating that caspase-3 inhibition does not affect pre-synaptic release properties. These data suggest that blocking caspase-3 activity in Tg2576 neurons is sufficient to specifically restore post-synaptic glutamate transmission to wild-type levels.

As calcineurin is required for Aβ-mediated synaptic removal of AMPAR, leading to loss of dendritic spines [START_REF] Hsieh | AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss[END_REF] , and calcineurin A (CN-A) is known to be proteolytically activated by caspase-3 [START_REF] Mukerjee | Caspase-mediated proteolytic activation of calcineurin in thapsigargin-mediated apoptosis in SH-SY5Y neuroblastoma cells[END_REF] , we hypothesized that hippocampal CN-A activity would increase upon caspase-3 cleavage thus inducing GluR1 removal from the PSD. To test whether CN-A is a hippocampal caspase-3 substrate, we performed an immunoblot analysis of the total homogenate from Tg2576 slices treated with z-DEVD-fmk or the vehicle control by using an antibody specific to the N-terminal of CN-A. We found that CN-A processing is reduced in Tg2576 slices treated with z-DEVD-fmk (Fig. 6a). Given that the 45 kDa CN-A fragment produced by caspase-3 cleavage contains the whole catalytic domain but lacks the autoinhibitory one, we assayed CN-A activity in response to caspase-3 inhibition. We measured CN-A activity in a calcium-free medium, to exclude a calcium-dependent regulation of CN-A activation. In addition, the specificity of the activity assay was confirmed by using the calcineurin inhibitor FK506 (Fig. 6b). We observed a significant reduction of calciumindependent CN-A activity in z-DEVD-fmk-treated slices, as compared to vehicle-treated slices, indicating that caspase-3-dependent cleavage of CN-A causes its activation.

To confirm the effect of calcineurin activity on GluR1 phosporylation we also analyzed Tg2576 hippocampal slices treated with FK506 compared to untreated slices. Incubation with calcineurin inhibitor for 2 hrs caused an increase of GluR1pSer845 but no differences were observed in total GluR1 from Tg2576 slices compared to vehicle-treated controls (Supplementary Fig. 14).

In addition, caspase-3 activity increase in 3-month-old Tg2576 hippocampus was associated in vivo with an increase of both activity and processing of calcineurin when compared to wild-type (Supplementary Fig. 15a,b). The levels of active calpain-1 were similar in young wild-type and Tg2576 mice, even though an increase of calpain-1 cleavage was observed in older mice (Supplementary Fig. 15c). This result excludes the involvement of calpain-1 in CN-A processing at this very early stage of disease in Tg2576 mouse model. Taken together, these results highlight a role for caspase-3 in GluR1 synaptic removal via CN-A cleavage.

Caspase-3 inhibition in vivo rescues Tg2576 phenotype

We next addressed the possibility of rescuing GluR1 PSD levels, spine size and memory deficits by in vivo caspase-3 inhibition. As z-DEVD-fmk cannot cross the blood brain barrier, and to minimize the pharmacological effect on other brain regions, we injected z-DEVD-fmk directly in one hippocampus and the vehicle in the controlateral hippocampus as control. Fifteen hours after injection, we observed a reduction of both caspase-3 and calcineurin activity together with a reduction of calcineurin processing in the ipsilateral z-DEVD-fmk-injected side, compared to the controlateral vehicle-injected hippocampus (Fig. 7a,b). Then, we analyzed the synaptic GluR1 content, and we observed a marked translocation of GluR1 from the microsomal fraction to the PSD (Fig. 7c) together with an increase of GluR1pSer845 in the PSD (Supplementary Fig. 16). To confirm the effect of caspase-3 inhibition on GluR1 synaptic distribution we used the polyglutamic acid (PGA)-peptoid QM56 to inhibit the activity of the apoptosome [START_REF] Vicent | Poly-L-glutamic acid (PGA) aided inhibitors of apoptotic protease activating factor 1 (Apaf-1): an antiapoptotic polymeric nanomedicine[END_REF] , a macromolecular complex activating caspase-3 and the mitochondrial-dependent apoptosis pathway [START_REF] Santamaría | A nanoconjugate Apaf-1 inhibitor protects mesothelial cells from cytokine-induced injury[END_REF] .

Forty-eight hours after QM56 intra-hippocampal injection, we analyzed caspase-3 activity and GluR1 PSD levels. We observed a reduction of caspase-3 activity in the ipsilateral QM56-injected side, compared to the controlateral vehicle-injected hippocampus (Supplementary Fig. 17a) and, as reported by using z-DEVD-fmk, we observed a marked translocation of GluR1 from the microsomal fraction to the PSD, with an increase of GluR1pSer845 in the PSD (Supplementary Fig. 17b,c).

Since we previously showed that the DAPT injection in the hippocampus affected GluR1 synaptic distribution (Supplementary Fig. 3), we also assessed whether this effect was mediated by caspase-3 by analyzing caspase-3 activity in Tg2576 hippocampus 15 hrs after DAPT injection. We observed a reduction of caspase-3 activity in the ipsilateral DAPT-injected side, compared to the controlateral vehicle-injected hippocampus (Supplementary Fig. 18).

Taken together, these results clearly prove i) the specificity of caspase-3 inhibition on synaptic function; ii) the main route of caspase-3 activation is mitochondria-dependent.

We also found that the head diameter of dendritic spines was enlarged in the ipsilateral z-DEVDfmk-injected side compared to the controlateral vehicle-injected hippocampus restoring spine size to wild-type levels (Figs. 7d and1c). Finally, bilateral injection of z-DEVD-fmk in the hippocampus of Tg2576 mice 15 hrs before training in CFC significantly rescued the memory deficits (Fig. 7e) without affecting motor activity (Supplementary Fig. 19). Interestingly, intra-hippocampal injection of z-DEVD-fmk caused an inverse effect on wild-type mice, confirming a physiological role of caspase-3 in hippocampal function. Specifically, synaptic fractionation of z-DEVD-fmktreated wild-type hippocampi demonstrated a reduction of GluR1 at PSD compared to vehicle controls (Supplementary Fig. 20). Moreover, z-DEVD-fmk-treated wild-type mice worsened memory in the CFC task (Fig. 7e).

Discussion

Caspase-3 activation is considered the terminal step in the biochemical cascade leading to apoptotic cell death [START_REF] Salvesen | Caspases: intracellular signaling by proteolysis[END_REF] . However, the opinion that caspase-3 is more than just a 'killer' involved in neuronal programmed cell death is supported by studies implicating caspase-3 in regulation of synaptic plasticity [START_REF] Bravarenko | Caspase-like activity is essential for long-term synaptic plasticity in the terrestrial snail Helix[END_REF][START_REF] Huesmann | Dynamic role of postsynaptic caspase-3 and BIRC4 in zebra finch song-response habituation[END_REF][START_REF] Li | Caspase-3 activation via mitochondria is required for long-term depression and AMPA receptor internalization[END_REF] .

Evidence for a role of caspase-3 in Alzheimer's Disease includes caspase-3 activation in Aβ-treated neurons [START_REF] Marín | Beta-amyloid-induced activation caspase-3 in primary cultures of rat neurons[END_REF] , immunodetection of activated caspase-3 in patient brain [START_REF] Gervais | Involvement of caspases in proteolytic cleavage of Alzheimer's amyloidbeta precursor protein and amyloidogenic A beta peptide formation[END_REF][START_REF] Stadelmann | Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer's disease. Evidence for apoptotic cell death[END_REF] , involvement of caspase-3 in APP proteolysis and Aβ peptide formation [START_REF] Gervais | Involvement of caspases in proteolytic cleavage of Alzheimer's amyloidbeta precursor protein and amyloidogenic A beta peptide formation[END_REF] , increase of caspase-3 in the PSD fractions of Alzheimer patients [START_REF] Louneva | Caspase-3 is enriched in postsynaptic densities and increased in Alzheimer's disease[END_REF] .

Despite this exciting array of data indicating caspase-3 as an etiologic factor in Alzheimer's Disease, the molecular mechanism(s) linking Alzheimer-related synaptic dysfunction to caspase-3 activity remains elusive.

We now identify local up-regulation of caspase-3 in hippocampal CA1 dendritic spines as a key process triggering functional and structural hippocampal synaptic alterations correlating with the onset of memory deficits in the APPswe Tg2576 mouse model. Our combined ex-vivo and in vivo analyses provide strong evidence that Tg2576 mice display enhanced caspase-3 activity in dendritic spines, resulting in a down-regulation of the surface expression of GluR1-containing AMPAR via proteolytic activation of calcineurin. The alterations of PSD composition are accompanied by synaptic transmission deficits, enhanced LTD and reduced spine size and density. These data are supported by a study reporting that synaptic AMPAR removal is both necessary and sufficient for Aβ-induced spine loss [START_REF] Vicent | Poly-L-glutamic acid (PGA) aided inhibitors of apoptotic protease activating factor 1 (Apaf-1): an antiapoptotic polymeric nanomedicine[END_REF] . The implication of this novel non-apoptotic caspase-3 dependent mechanism at the onset of Alzheimer neurodegeneration is proved by ex-vivo and in vivo rescue experiments using the caspase-3 inhibitor z-DEVD-fmk. Indeed, we demonstrate that caspase-3 inhibition restores glutamatergic synaptic transmission in Tg2576 slices to wild-type levels and that, in vivo, a single injection of caspase-3 inhibitor is sufficient to down-regulate caspase-3 and calcineurin activity thus restoring GluR1 levels at PSD, spine size and memory performance. The pharmacological approach used in this work affords the unprecedented possibility of acutely reducing caspase-3 activity at the onset of Alzheimer-related cognitive decline in the Tg2576 mouse model. Indeed, this approach avoids any interference with developmental parameters, postnatal differentiation and neuron physiology which could be perturbed by using genetic approaches, as highlighted by abnormal brain development in knock-out mice with an altered caspase-3 activation [START_REF] Kuida | Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice[END_REF][START_REF] Hakem | Differential requirement for caspase 9 in apoptotic pathways in vivo[END_REF][START_REF] Kuida | Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9[END_REF][START_REF] Cecconi | Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development[END_REF] .

Our data strongly support the possibility of targeting caspase-3 activity to reverse hippocampal function deficits at the onset of Alzheimer pathology. Nevertheless, the opposite effects produced by in vivo injection of the caspase-3 inhibitor in wild-type mice suggest that therapeutic intervention should take into account the critical physiological role of caspase-3-dependent proteolysis in synaptic plasticity and memory, independent of its role in apoptosis.

Our proposed model explaining the role of caspase-3 in dendritic spine degeneration is illustrated in Supplementary Fig. 21. Intraneuronal accumulation of Aβ within distal processes and synaptic compartment [START_REF] Takahashi | Oligomerization of Alzheimer's beta-amyloid within processes and synapses of cultured neurons and brain[END_REF] leads to the generation of free radicals [START_REF] Manczak | Mitochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression[END_REF] . These reactive species, in turn, disrupt the electron transport chain thus leading to mitochondrial dysfunction and consequent cytochrome c release from the outer membrane. The release of cytochrome c into the cytosol induces the formation of the apoptosome which, in turn, recruits and activates caspase-3. Proteolytic cleavage of calcineurin induces a loss of its calcium-calmodulin sensitivity [START_REF] Santamaría | A nanoconjugate Apaf-1 inhibitor protects mesothelial cells from cytokine-induced injury[END_REF] . This causes an increase in its phosphatase activity resulting in an increased dephosphorylation of GluR1 at Ser845, followed by AMPAR removal from PSD and progressive spine loss. However, the cleavage of other synaptic substrates by caspase-3 cannot be excluded.

Consistent with our model, recent evidence identified the mitochondria as a key organelles in Aβinduced dendritic spine loss and neuronal damage [START_REF] Cho | S-nytrosylation of Drp1 mediates beta-amyloid-related mitochondrial fission and neuronal injury[END_REF] . Here, we propose a mechanism downstream of mitochondrial dysfunction, which orchestrates dendritic spine shrinkage and loss during early Alzheimer-related neurodegeneration.

In conclusion, the above identified mechanism points to caspase-3 as a possible early biomarker and should encourage further studies on its regulation to develop rationale "synaptoprotective" therapies for preventing or slowing down Alzheimer's Disease progression.

Methods

Mice

Heterozygous male Tg2576 mice 11 and wild-type littermates were used for all studies. All experiments were performed on independent groups of mice. Experiments were carried out in accordance with the European Community's Council Directive 86/609/EEC. Formal approval of the described experiments was obtained by the Italian Ministry of Health (D.L.vo 116/92).

Contextual fear conditioning (CFC)

After four days of handling each mouse was placed in the conditioning chamber (transparent Plexiglass cage, 28x28x10 cm) for 120 s of free exploration period, followed by a series of 5 non signaled footshocks (0.7 mA, 2 s duration, separated by 60 s intervals) delivered through the grid floor. CFC was assessed 24 hrs later by returning mice for 5 min to the conditioning chamber where no footshock was delivered. Fear memory was estimated by recording the time spent in freezing behavior, defined as the total absence of all but respiratory movements associated with a crouching posture. Mice behavior was videotaped by means of a video camera connected to a computer equipped with a dedicated software (Noldus Ethovision System, The Netherlands). Motor activity during the free exploration period was automatically scored by means of Noldus Ethovision system.

Golgi Cox staining and dendritic spines measurements

Naïve male mice were intracardially perfused with 0.9% saline solution. The brains were impregnated by immersion in a standard Golgi-Cox solution (1% potassium dichromate, 1% mercuric chloride, 0.8% potassium chromate) for 6 days, transferred to a 30% sucrose solution for 2 days, and then sectioned coronally (100 µm). Sections were mounted on gelatinized slides and stained according to the Gibb and Kolb method. Fully impregnated pyramidal neurons laying in the CA1 region of the dorsal hippocampus were identified under low magnification (20x/0.5NA). In each brain, 11 neurons displaying dendritic tree without obvious truncations and balanced per hemisphere were analyzed under higher magnification (63x/0.75NA). Measurements were carried out using a microscope (DMLB, Leica) equipped with a camera (resolution 2600x2600, Axiocam, Zeiss), and the KS300 3.0 system (Zeiss). A computer-based neuron tracing system (Neurolucida, Microbrightfield) was used to trace single neurons. Spine density was assessed by counting the number of spines in five 20 μm dendrite segments per neuron chosen in branch orders 2-4 of apical and basal dendrites. All protrusions, with or without bulbous expansion but no longer than 2 µm, were counted as spines if they were in continuity with the dendritic shaft. The average spine density (number of spines per 1 μm length segment) was averaged for a neuron mean. To determine dendritic spine morphology, the spines on five 20 μm apical dendrite segments per neuron were acquired under higher magnification (100x/1.25NA) using a digital camera (Axiocam, Zeiss).

Morphology was estimated by measuring spine head diameter by means of the public domain

ImageJ software (HYPERLINK "http://.info.nih.gov/ij/"). Values were averaged for neuron and cumulative frequencies compared among groups. Total dendrite length was automatically calculated by the Neurolucida software after each neuron was traced.

In vitro slice preparation and electrophysiology

Hippocampal slices were prepared as described previously [START_REF] Marie | Generation of silent synapses by acute in vivo expression of CaMKIV and CREB[END_REF] . Slices were recorded at 32 ± 1ºC in oxygenated artificial cerebrospinal fluid (aCSF) containing 126 mM NaCl, 26 mM NaHCO 3 , 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 2 mM MgSO 4 , 2 mM CaCl 2 , 10 mM glucose, gassed with 95% O 2 / 5% CO 2 , pH 7.4, in the continuous presence of 100 µM picrotoxin. Field excitatory post-synaptic potentials (fEPSPs) were recorded in the stratum radiatum of the CA1 and stimuli delivered by a monopolar electrode. Input/output relationships were generated by calculating the fEPSP initial slope (to avoid population spike contamination) corresponding to a given fiber volley amplitude (100 to 200 mV in increments of 50 mV). For LTP experiments, fEPSPs were stimulated at 0.1 Hz and LTP was induced by 1 train of 100 stimuli at 100 Hz. For LTD, slices were superfused with aCSF containing [Ca 2+ ]=4 mM and LTD was induced with a low frequency stimulation (LFS) protocol of 900 pulses at 1 Hz.

Whole cell patch clamp recordings were obtained from CA1 pyramidal cells as described previously [START_REF] Marie | Generation of silent synapses by acute in vivo expression of CaMKIV and CREB[END_REF] . Recordings were made using intracellular solution (117.5 mM CsMeSO 4 , 15.5 mM, CsCl, 10 mM TEACl, 8 mM NaCl, 1.25 mM NaH 2 PO 4 , 10 mM HEPES, 0.25 mM EGTA, 4 mM MgATP, 0.3 mM NaGTP, pH=7.3, osmolarity ~290 mOsm). Series (10-20 MΩ) and input (100-200 MΩ) resistances were monitored online throughout each experiment. For the AMPAR/NMDAR ratio, cells were held at -65 mV to record AMPAR mediated currents and at +20 mV, to record AMPAR+NMDAR mediated currents. The AMPAR excitatory post-synaptic current (EPSC) amplitude was calculated at the peak (averaged over a 1 ms window) and the NMDA EPSC amplitude was calculated by measuring the current (2 ms window) at 60 ms after stimulation artifact. For paired pulse ratios (PPR) measurements, two pulses were delivered 100 ms apart at -65 mV. Spontaneous AMPAR-mediated miniature EPSCs (mEPSCs) were recorded at -65 mV holding potential in [Ca 2+ ]out=4mM and [K + ]out=7mM, in the presence of 0,5 μM tetrodoxin (Latoxan) and 100 μM picrotoxin (Sigma-Aldrich). Spontaneous events were analysed using using the Clampfit software (Molecular Devices) through a template search with variable amplitude.

For experiments in Fig. 5, slices were incubated for 2 hrs with 5 µM z-DEVD-fmk at RT, and then transferred to the recording chamber. Only cells successfully recorded within 1 hr of the end of incubation were included for analysis.

Surgical procedures

Mice were anesthetized with chloral hydrate (400 mg/kg) and mounted on stereotaxic apparatus. Bilateral injecting cannulae were inserted in the hippocampus (coordinates relative to bregma: anteroposterior -2 mm, lateral ±1.7 mm, ventral 1.9 mm). Injections were performed using a 10 μl Hamilton syringe connected to a gauge injector.

One group of mice was injected in one hemi-hippocampus with 1 μl of 174 μM z-DEVD-fmk-DMSO solution (Imgenex) diluted in aCSF. As control, 1 μl of aCSF-DMSO solution was injected in the controlateral hemi-hippocampus. A separate group of mice was injected with 1 μl of 3.5 mM γ-secretase inhibitor IX (DAPT, Calbiochem) in one hemi-hippocampus and 1 μl of DMSO in the controlateral hemi-hippocampus. Another group of mice was injected with 1 μl of 0.

Post-synaptic density (PSD) preparation

The brain was rapidly dissected and the hippocampus was homogenized in homogenization buffer (320 mM sucrose, 1 mM HEPES pH 7.4, 1 mM MgCl 2 , 1 mM EDTA, 1 mM NaHCO 3 , 1 mM PMSF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 5mM NaF, with protease inhibitors -Sigma) with 10 strokes of a tight-fitting glass Dounce tissue grinder (7ml; Wheaton). The homogenate was centrifuged at 1,000 × g for 10 min and the resulting supernatant was centrifuged at 3,000 × g for 15 min. The pellet was resuspended in 1 mM HEPES pH 7.4 containing protease inhibitors and centrifuged at 100,000 × g for 1 hr. The resulting pellet was resuspended in a buffer containing 75 mM KCl, 1% Triton X-100 and proteases inhibitors and centrifuged at 100,000 × g for 1 hr. The supernatant was referred as Triton X-100 Soluble Fraction (TSF). The pellet containing the PSD fraction (Triton X-100 Insoluble Fraction, TIF) was resuspended in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% Triton X-100, 0.25% Na-deoxycholate, 150 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 0,1% SDS, protease inhibitors), sonicated and incubated on ice for 20 min. The samples were centrifuged at 11,500 × g for 10 min and the protein concentration of resulting supernatant was determined.

Sub-synaptic fractionation

Hippocampus was homogenized in homogenization buffer (320 mM sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM NaHCO 3 , 1 mM PMSF, 1 mM Na 3 VO 4 , 5mM NaF, with protease inhibitors) with 10 strokes of a tight-fitting glass Dounce tissue grinder. The homogenate was centrifuged at 1,000 × g for 10 min and the resulting supernatant was centrifuged at 10,000 × g for 15 min. Both the pellet (P2) and the supernatant (S2) were stored. The P2 was resuspended in homogenization buffer, incubated 40 min on ice and centrifuged at 32,000 × g for 20 min. The resulting pellet (TxP) containing PSD was processed for protein extraction as described for "PSD preparation". The S2 was centrifuged at 100,000 × g for 1 hr and the microsomal-enriched proteins were extracted from resulting pellet (P3).

Synaptosome preparation

Hippocampus was homogenized in homogenization buffer (320 mM sucrose, 4 mM Hepes pH 7.4, 1 mM EGTA, 1 mM PMSF, with protease inhibitors) with 10 strokes of a tight-fitting glass Dounce tissue grinder. The homogenate was centrifuged at 1,000 × g for 10 min and the resulting supernatant was centrifuged at 12,000 × g for 15 min. The pellet was resuspended in homogenization buffer and centrifuged at 13,000 × g for 15 min to obtain the final pellet containing synaptosome-enriched fraction.

Nuclei isolation

Nuclei enriched fraction was prepared by the same hippocampal homogenate used for synaptosomes preparation. The homogenate was centrifuged at 800 × g for 5 min, the pellet was washed twice with cold PBS, resuspended in hypotonic lysis buffer (10 mM Tris-HCl pH 7.4, 0.1 M sucrose, 10 mM NaCl, 1 mM DTT, 1 mM PMSF, with protease inhibitors), vortexed for 15 s and incubated on ice for 15 min. Then, 1.8 M sucrose was added, samples were mixed and loaded on top of a 1.1 M sucrose cushion and centrifuged at 14,700 × g for 25 min in a SW60Ti rotor. The pellet was resuspended in PBS and centrifuged at 1000 × g for 5 min to obtain the pellet containing nuclei.

Total homogenate preparation from hippocampal tissue

Hippocampal tissue was homogenized in lysis buffer (320 mM sucrose, 50 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.5 mM Na-orthovanadate, 5 mM β-glycerophosphate, with proteases inhibitors), incubated on ice for 30 min and centrifuged at 13,000 × g for 10 min. The total protein content of resulting supernatant was determined.

Fluorimetric assay of caspase-3 activity

Synaptosomes-containing pellet, total hippocampal tissue and hippocampal slices were homogenized in lysis assay buffer (100 mM Hepes pH 7.4, 0.1% Chaps, 1 mM EDTA, 10 mM DTT, 1 mM PMSF) and lysed by freezing in liquid N 2 and thawing at 37°C three times. After centrifugation at 11,500 × g for 5 min the protein concentration of resulting supernatant was determined and the same amount of proteins was incubated at 37°C in lysis assay buffer containing 50 µM Ac-DEVD-AMC (Calbiochem). The fluorescence was measured with 380 nm excitation wavelength and 460 nm emission wavelength.

Immunoelectron microscopy

Animals were intracardially perfused with a calcium-free Ringer's variant, followed by fixative solution (4% depolymerized paraformaldehyde in 0.12 M sodium phosphate buffer, pH 7.2). Brains were sagittally cut to obtain 80 µm thick sections. The free-floating sections were incubated with an anti-cleaved caspase-3 antibody (1:100; Cell Signaling). Then, sections were post-fixed with 1% OsO 4 , dehydrated, infiltrated in epoxy resin and flat-embedded in the same medium. Hippocampal CA1 fields were re-embedded on epon blanks and further cut by a Reichert Ultracut S ultramicrotome (Leica Microsystems), to obtain ultrathin (70-80 nm) sections. These were collected on nickel grids, lightly contrasted with uranyl acetate and lead citrate and finally observed under CM120 electron microscope (Philips), equipped with a videocamera. Digitized images were electronically captured and composed in Adobe Photoshop CS3. To quantitatively evaluate the occurrence of caspase-3-containing spines in the different genetic conditions, we analyzed DAB stained ultrathin sections.

Flow Cytometry

Synaptosome flow cytometry was performed as previously described [START_REF] Gylys | Rapid annexin-V labeling in synaptosomes[END_REF] . Synaptosomes-containing pellet was resuspended in binding buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM KCl, 5 mM MgCl 2 , 1.8 mM CaCl 2 ), 100 µl aliquots were incubated 10 min at RT with dye solution and then diluted in PBS (final volume 500 µl) for immediate flow cytometry analysis. Final dye concentration was 100 nM for calcein AM (Molecular Probes) and 1 mg/ml for PE-annexin V (BD Pharmingen). The samples were acquired on a FACS Canto flow cytometer. Polystyrene beads (0.75, 1.4 and 4.5 µm) were run on the instrument to standardize forward scatter measurements.

Nuclei-containing pellet was resuspended in PBS, and propidium iodide, PI (Sigma), solution was added to give a final concentration of 1 mg/ml. The samples were immediately analyzed with appropriate software (FACS Canto and FACS Diva; Becton Dickson). Scatter profiles for fluorescence intensities FL-2 (PI) were plotted. The percentage of cells in each quadrant was determined with Flowjo version 8.6.1.

Hippocampal slices treatment

Coronal brain slices [START_REF] Marie | Generation of silent synapses by acute in vivo expression of CaMKIV and CREB[END_REF] were cut along a sagittal plane into 2 hemisections. The homolateral sections were incubated for 2 hrs at RT in aCSF (gassed with 95% O 2 / 5% CO 2 , pH 7.4) containing 2.5 or 5

Proteins were applied to SDS-PAGE and electroblotted on a PVDF membrane. Immunoblotting analysis was performed using a chemiluminescence detection kit. The relative levels of immunoreactivity were determined by densitometry using the software ImageQuant 5.0. Antibodies to caspase-3, cleaved caspase-3, cleaved PARP, caspase-6 and caspase-7 were purchased from Cell Signaling, Apaf1 from Alexis Biochemicals, GluR1, GluR1pSer831 and NMDAε2 from Upstate, GluR1pSer845, GluR2/3, PSD-95 and calcineurin from Chemicon, NMDAε1 and NMDAζ1 from Santa Cruz, NSF from Synaptic Systems, αCaMKII from Zymed Laboratories, mGluR5 and synaptophysin from Abcam, Rab11 from BD Biosciences, calpain-1 from Calbiochem, actin and βtubulin from Sigma.

Coomassie staining

PSD proteins were resolved by SDS-PAGE. After electrophoresis the gels were fixed for 30 min in cold fixing solution (50% MetOH, 10% acetic acid, 40% ddH 2 O), stained with Coomassie solution (0.2% Coomassie blue, 20% MetOH, 10% acetic acid) for 30 min and washed abundantly with cold destaining solution (45% MetOH, 10% acetic acid, 45% ddH 2 O).

TUNEL assay

TUNEL assay was performed on 10 µm paraffin-embedded brain sections by using the ApopTag Fluorescein In Situ Apoptosis Detection Kit purchased from Chemicon. The assay was carried out according to manufacturer's instructions.

Measurement of carbonylated proteins

Carbonylated proteins were detected using the Oxyblot kit (Chemicon) according to manufacturer's instructions. Briefly, 5 µg of hippocampal proteins were reacted with dinitrophenylhydrazine (DNP) solution for 15 min at 25°C. The DNP-reacted proteins were resolved on 10% SDS-polyacrylamide gel and were identified by immunoblotting using an anti-DNP antibody.

Primary neuronal cell culture: preparation and analysis

The primary neuronal cell cultures were prepared from cortical and hippocampal tissue of E15 embryos. Females Apaf1 +/-44 were crossed with males Tg2576/Apaf1 +/-to obtain transgenic and transgenic lacking of Apaf1 protein neurons.

Cortical and hippocampal tissues of embryos were dissociated in 0.025% Trypsin (7 min at 37°C), dissociation was blocked with FBS and samples were centrifuged for 3 min at 220 × g. The tissue was resuspended in MEM#1 medium (25 mM glucose, 1% FBS, 2 mM L-glutamine, 0.1 mg/ml gentamicin, MEM medium) and centrifuged 10 min at 220 × g. The resulting cells pellet was resuspended in MEM#2 medium (25 mM glucose, 5% FBS, 5% HS, 2 mM L-glutamine, 0.1 mg/ml gentamicin, MEM medium). Cells were plated in MEM#2 medium and incubated in a humidified 37°C, 5% CO 2 incubator; after 1 hr all medium were replaced with Neurobasal medium (2% B-27 minus AO, 2 mM L-glutamine, 0.1 mg/ml gentamicin, Neurobasal medium) and primary neurons were cultured for 7 days.

To obtain total lysates for immunoblotting analysis, neurons were scraped in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% Triton X-100, 0.25% Na-deoxycholate, 150 mM NaCl, 5 mM MgCl 2 , 1 mM EDTA, 0.1% SDS, protease inhibitors), sonicated, incubated 20 min on ice and centrifuged at 11500 × g for 10 min. The protein content of resulting supernatant was determined.

Data analysis and statistics

Behavioral, total dendrite length, immunoblotting, immunohistochemistry, electrophysiology, flow cytometry and enzyme activity data analysis were performed with a two tailed Student's t test.

Spine density data were analyzed by two-way ANOVA with age and genotype as independent variables, Behavioral data from mice treated with z-DEVD-fmk were analyzed by two-way ANOVA with treatment and genotype as independent variables.

Figure 1 .

 1 Figure 1. Contextual Fear Conditioning (CFC) performance and morphology of CA1 neuron

Figure 2 .

 2 Figure 2. Three-month-old Tg2576 mice show altered hippocampal GluR1 distribution.

Figure 3 .

 3 Figure 3. Three-month-old Tg2576 mice show altered basic glutamatergic synaptic

Figure 4 .

 4 Figure 4. Tg2576 hippocampal dendritic spines accumulate active caspase-3 in the post-

Figure 5 .

 5 Figure 5. Inhibition of caspase-3 activity reduces GluR1 dephosphorylation, its removal from

Figure 6 .

 6 Figure 6. Inhibition of caspase-3 activity reduces calcineurin cleavage and activity.

Figure 7 .

 7 Figure 7. Caspase-3 inhibition in vivo influences GluR1 distribution and rescues spine head

  7 mM apoptosome inhibitor (QM56, kindly provided by Dr. Pérez-Payá) diluted in PBS. As control, 1 μl of PBS was injected in the controlateral hemi-hippocampus. Injections of inhibitor or vehicle were balanced for hemispheres. Mice were sacrificed 15 hrs (z-DEVD-fmk-and DAPT-injected mice) and 48 hrs (QM56-injected mice) after the injections for brains collection. Two independent groups of naïve mice were injected bilaterally in the hippocampus with either z-DEVD-fmk or DAPT 15 hrs before training in CFC task. Relative controls were injected respectively with vehicle solutions.

Acknowledgements

We thank the Animal Facility of the IRCCS Fondazione Santa Lucia/EBRI/CNR for the mouse work, Taconic for the Tg2576 mice (NY), M. Acuña-Villa and M. W. Bennett for editorial and secretarial work, R. Nardacci, F. Fanelli, and M. Nencini for research assistance and help with image processing. A. Roberto and A. Pignataro for help with Neurolucida measurements. We are grateful to Dr. E. Pérez-Payá for providing us with apoptosome inhibitor QM56. This work was supported in part by grants from the Telethon Foundation, Ricerca Corrente and Ricerca Finalizzata from the Italian Ministry of Health, the Italian Ministry of University and Research, and Compagnia di San Paolo.

The Kolmogorov-Smirnov test was used to compare cumulative frequencies of spines heads diameters. In all cases P<0.05 was considered statistically significant.

Author contributions

M.DA. and V.C. designed and carried out all molecular biology experiments and caspase-3 analysis. All authors discussed the results and commented on the manuscript. µM z-DEVD-fmk (Imgenex), 5 µM z-FA-fmk (Imgenex) or 10 µM FK506 (Sigma), and the controlateral sections in aCSF containing DMSO.

Cathepsin B activity assay

Hippocampal slices were homogenized in lysis buffer (20 mM Hepes pH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 0.5% NP40, 1 mM DTT, 1 mM PMSF), sonicated for 10 s and centrifuged at 15,000 × g for 30 min. The protein concentration of resulting supernatant was determined, the same amount of proteins was incubated at 37°C in assay buffer (50 mM Na-acetate pH 6.0, 4 mM EDTA, 8 mM DTT) containing 50 µM z-RR-AMC (Biomol International) and the fluorescence was measured with 380 nm excitation wavelength and 460 nm emission wavelength.

Calcineurin activity assay

CaN activity was assayed by monitoring 32 P release from radiolabeled purified RII substrate peptide [START_REF] Ferri | Calcineurin activity is regulated both by redox compounds and by mutant familial amyotrophic lateral sclerosis-superoxide dismutase[END_REF] . The hippocampal tissues were homogenized in buffer A (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM CaCl 2 , 1 mM PMSF, 1 mM DTT), subjected to repeated freezing in liquid N 2 and thawing at 37°C, and centrifuged at 17,000 × g for 10 min. 10 µg of total proteins from the clear supernatant was diluted with an equal volume of buffer B (100 mM Tris-HCl pH 7.4, 1 mM MnCl 2 , 0.1 mM CaCl 2 , 1 mM DTT) and immediately processed for enzyme assay. Addition of 1 mM NiSO 4 in buffer B was used to obtain maximal activation of the enzyme together with 200 nM okadaic acid at a final concentration known to inhibit phosphatase activities other than calcineurin.

Addition of MgCl 2 was avoided to prevent activation of protein phosphatase 2C. 'Ca-free' conditions of calcineurin assay were obtained by omission of calcium in both A an B buffers.

Addition of calcineurin inhibitor (10 µM FK506) to clear supernatant was used as control.

Immunoblotting analysis and antibodies


Professor Rose A Cooper
email: rcooper@uwic.ac.uk

BSc Leighton Jenkins

PhD Ana F M Henriques

R S Duggan

PhD Neil F Burton

BSc Sean Duggan

Absence of bacterial resistance to medical-grade manuka honey

Keywords: manuka honey, resistance, wounds

Clinical use of honey in the topical treatment of wounds has increased in Europe and North America since licensed wound care products became available in 2004 and 2007 respectively. Honeyresistant bacteria have not been isolated from wounds, but there is a need to investigate whether honey has the potential to select for honey resistance. Two cultures of bacteria from reference collections (Staphylococcus aureus NCTC 10017 and Pseudomonas aeruginosa ATCC 27853) and four cultures isolated from wounds (Escherichia coli, MRSA, Pseudomonas aeruginosa and Staphylococcus epidermidis) were exposed to sub-lethal concentrations of manuka honey in continuous and stepwise training experiments to determine whether susceptibility to honey diminished. Reduced susceptibilities to manuka honey in the test organisms during long-term stepwise resistance training were found, but these changes were not permanent and honey-resistant mutants were not detected. The risk of bacteria acquiring resistance to honey will be low if high concentrations are maintained clinically.

Response to Reviewers: Dear Editor, Thank you for the reviewers' comments on our paper entitled "Absence of bacterial resistance to medical-grade manuka honey". We were pleased that they liked the design and execution of our study and we understood the reservations that reviewer had about our conclusions. We have made the following changes in response to each specific observation,

also used the EUCAST definition of clinical resistance to show that honey susceptibility which had increased by a factor of 1.4 is unlikely to lead to therapeutic failure when wound care products normally contain at least 80% manuka honey and normally 95%(w/v) (lines 258 to 260). We have removed the statement "gradually increased towards pre-treatment levels". We have made clear the proportional changes in MICs (lines 195 to 197 and 208 and 251). We have commented on the need to maintain high concentrations of manuka honey in wounds to avoid the selection of resistant strains (lines 278 to 284). We have modified the Abstract (lines 42 to 45) to explain that reduced susceptibility was found during long-term training and we have commented on the need to maintain high concentrations of manuka honey during clinical use (lines 45 to 46). In the concluding paragraph we also note that prolonged exposure to antimicrobial agents should be avoided. We have deleted statements about the development of antibiotic resistance from the Introduction and the Discussion. We have inserted "preceding days' culture" into line 169, and "day" into line 194. We have removed the reference to high sugar content, low water content and acidity. We have changed the captions to Figure 1 and2. Reviewer 2. We were encouraged that our conclusions were considered to be robust. We have changed MIC against manuka honey to "MICs to manuka honey" throughout the paper. We have clarified which honey was used in which experiments by changing "preliminary" to "shortterm"(line 91) and explained the activity associated with each honey (lines 100 to 103). We have inserted "preceding days' culture" in line 133. In order to clarify the number of MIC tests performed and the experimental design we have inserted a brief explanation in lines 210 to 217. We hope that you will now be able to accept our manuscript for publication,

Yours sincerely, Rose Cooper

Absence of bacterial resistance to medical-grade manuka honey training were found, but these changes were not permanent and honey-44 resistant mutants were not detected. The risk of bacteria acquiring resistance 45 to honey will be low if high concentrations are maintained clinically.

Introduction 47

For thousands of years wounds were treated topically with relatively poorly 48 characterised preparations of antimicrobial agents derived from animal, 49 vegetable and mineral sources [START_REF] Forrest | Early history of wound treatment[END_REF]. Apart from honey, many of those 50 substances were too toxic for internal use [START_REF] Moellering | Past, present, and future of antimicrobial agents[END_REF]. During the nineteenth century, 51 the chemical industry yielded a variety of antiseptic compounds that were 52 utilised for preventing and treating wound infections [START_REF] Hugo | A brief history of heat and chemical preservative and 293 disinfection[END_REF], but in recent times 53 cytotoxicity has limited their acceptance [START_REF] Brennan | The effect of antiseptics on the healing 295 wound: a study using the rabbit ear chamber[END_REF]. A systematic strategy of discovering and evaluating promising chemotherapeutic antimicrobial agents 55 was initiated by Paul Ehrlich at the beginning of the twentieth century; it paved 56 the way for the advent of antibiotics, which revolutionised the treatment of 57 infection. These antimicrobial agents with their specific mechanisms of 58 inhibition exhibited selective toxicity against microbial species, rather than 59 patients and were initially effective in controlling infections. Although not 60 universally susceptible to all antibiotics, most microbial species were 61 susceptible to at least one type of antibiotic. To date seventeen classes of 62 antibiotic have been introduced, but the rate of discovery of new antibiotics 63 has slowed within the past twenty years. The therapeutic value of many 64 antibiotics has been curtailed by the acquisition of resistance in species that 65 were once susceptible, as well as the emergence of species with resistance to 66 multiple antibiotics. This is a real concern because species resistant to every 67 known antibiotic have emerged [START_REF] Payne | Drugs for bad 297 bugs: confronting the challenges of antibacterial discovery[END_REF], antiseptic resistant strains have been 68 isolated and organisms with both antibiotic and antiseptic resistance 69 determinants have been discovered [START_REF] Russell | Introduction of biocides into clinical practice and the 300 impact on antibiotic-resistant bacteria[END_REF]. The speed of emergence of 70 antimicrobial resistance is unpredictable [START_REF] Davies | Bacteria on the rampage[END_REF], but whenever a new inhibitory 71 agent is introduced, resistant organisms emerge in due course. With fewer 72 effective antimicrobial agents to manage wound infections, impacts such as 73 increased morbidity, treatment costs and mortalities have ensued [START_REF] Filius | Impact of increasing antimicrobial 303 resistance on wound management[END_REF]. Novel 74 antimicrobial remedies are needed, but there is always the possibility that 75 organisms with resistance to these agents will arise. 76 77 Since medical-grade honey was first licensed for treating wounds in Australia 78 in 1999, the clinical use of wound care products containing honey has America. As with antibiotics, extensive use of honey may well provide a 81 selective pressure for the emergence of honey resistant strains of wound 82 pathogens. This can be tested in vitro by either repeated cultivation of 83 bacteria in a sub-lethal concentration of the agent, or by incubation in 84 stepwise increasing concentrations. The aim of this study, therefore, was to 85 determine whether bacteria continuously exposed to low concentrations of 86 manuka honey changed their susceptibility to manuka honey and whether 87 resistant strains were selected. 88 the well of the previous microtitre plate containing the highest concentration of 154 honey with viable cells (i.e. one well below the actual MIC). Also, the purity of 155 each inoculum was checked by dropping 20 µl onto nutrient agar plates and 156 streaking out to achieve isolated colonies after incubation at 37 0 C for 24 157 hours. The experiment was continued for 28 successive days with each test 158 organism using an appropriate range of honey concentrations above and 159 below the last recorded MIC value. Cultures obtained after 28 days 160 continuous exposure to a sub-lethal concentration of honey were frozen and 161 stored at -80 0 C for future reference. 162

Recovery of cultures of clinical isolates following long-term exposure to 163

honey. 164

Each 'honey-trained' culture obtained after 28 days continuous exposure to 165 manuka honey was inoculated into 20 ml isosensitest broth (ISB) contained 166 within a 100 ml conical flask and incubated in a shaking water bath at 37 o C for 167 24 hours. On each successive day, one of four fresh flasks of ISB were 168 inoculated with 100 µl of each culture transferred from the preceding days' 169 culture, respectively until 28 days had elapsed. At each daily transfer MIC 170 against manuka honey was determined. Cultures obtained after the 28 day 171 recovery period were frozen and stored at -80ºC. 172

Cultural characteristics. 173

Identity of the test organisms before the start of the long-term training regime, 174 collected after the 28 day training period and after the 28 day recovery period 175 was determined by Crystal kits (Becton Dickinson, Basingstoke, UK) and by When S. aureus NCTC 10017 was cultured in a sub-lethal concentration of 180 manuka honey for 10 consecutive days, susceptibility to manuka honey did 181 not decrease significantly (Table 1). Conversely the MIC for P. aeruginosa 182 ATCC 27853 increased significantly from 15.7 to 19.7%(w/v), indicating that 183 the culture had become less susceptible to manuka honey. However, after 6 184 subcultures in honey free NB, there was no significant difference in MIC 185 compared to the starting value. 186 When these two test organisms were successively cultured in stepwise 187 increasing concentrations of honey, viable bacteria were not recovered above 188 aeruginosa respectively, demonstrating that honey-resistant strains of these 199 bacteria had not been selected (Fig. 2). Neither MRSA nor E. coli returned to 200 the pre-training MIC values within the 28 day recovery period. Several months 201 later the cultures frozen at the end of the recovery period were thawed and 202

MICs were determined in duplicate on three separate occasions (Table 2). 203

MBCs were also determined by streaking out samples from microtitre plate 204 wells showing no growth onto nutrient agar. It was seen that the susceptibility 205 of three of the clinical isolates was similar to those values obtained before 206 resistance training (Fig. 1), but the MIC of manuka honey in E.coli remained 207 1. [START_REF] Brennan | The effect of antiseptics on the healing 295 wound: a study using the rabbit ear chamber[END_REF] The excessive use and misuse of antibiotics is known to favour the selection 222 of resistant strains and to inhibit sensitive strains. In Norway, for example, 223 where mupirocin was not available, mupirocin-resistant strains of S. aureus 224 were not detected [START_REF] Afset | Susceptibility of skin and soft-tissue isolates 307 of Staphylococcus aureus and Streptococcus pyogenes to topical antibiotics[END_REF]. Restricting mupirocin use in an American medical 225 centre caused an immediate reduction in the occurrence of mupirocin-226 resistant MRSA [START_REF] Walker | A 310 decline in mupirocin resistance in methicillin resistance in methicillin-resistant 311 Staphylococcus aureus accompanied administrative control of prescriptions[END_REF] and an association between exposure to fusidic acid and 227 fusidic acid-resistance in S.aureus has been established [START_REF] Mason | Fusidic acid resistance in community 314 isolates of methicillin susceptible Staphylococcus aureus and the use of 315 topical fusidic acid: a retrospective case-controlled study[END_REF]. Resistance to 228 biocides used for skin and wound care has also been demonstrated [START_REF] Russell | Introduction of biocides into clinical practice and the 300 impact on antibiotic-resistant bacteria[END_REF].In the

  In short-term resistance training experiments Staphylococcus aureus NCTC 91 10017 and Pseudomonas aeruginosa ATCC 27853 were utilised with a 92 sample of sterile manuka honey provided by the New Zealand Natural Food 93 Company. For the rest of the study clinical isolates of MRSA, Staphylococcus 94 epidermidis, Escherichia coli and Pseudomonas aeruginosa were tested with 95 a sample of manuka honey that was provided by Comvita UK (Manukacare 96 18+). This product is available as Medihoney in Canada and the United 97 States. The clinical isolates were cultures of bacteria that had been isolated 98 from patients with chronic wounds attending a local out-patient clinic at 99 University Hospital of Wales, Cardiff. Using a bioassay [9], the antibacterial 100 activity of New Zealand Natural Food company honey sample was found to be 101 equivalent to 19%(w/v) phenol and Manukacare 18+ was equivalent to 102 18%(w/v) phenol. 103 Determination of Minimum Inhibitory Concentrations (MICs) MICs were determined in 96 well, flat bottomed microtitre plates (Nunc, 105 Roskilde, Denmark) using manuka honey dissolved in either nutrient broth 106 (NB; Oxoid, Basingstoke, UK) or isosensitest broth (ISB; Oxoid, Basingstoke, 107 UK). Honey concentrations varying by 1%(w/v) were used in successive wells 108 and all wells contained 200 µl. Wells were inoculated with 1µl undiluted 109 overnight broth cultures of test organisms (approximately 1 x 10 6 cfu/ml) and 110 incubated at 37ºC for 24 h. Positive (broth and inoculum) and negative 111 controls (broth and honey) were included. MICs were determined visually and 112 confirmed by measuring absorbance at 400 nm in a microtitre plate reader 113 (Anthos Labtec Instruments). 114 Short-term resistance training by exposure to a single sub-lethal concentration 115 of manuka honey. 116 To one 100 ml conical flask with 20 ml NB containing 2.5%(w/v) manuka 117 honey, 40 µl overnight broth culture of S. aureus was inoculated and to 118 another 100 ml conical flask with 20 ml NB containing 8%(w/v) manuka 119 honey, 40 µl P. aeruginosa was inoculated. Both flasks were incubated at 120 37ºC in a shaking water bath (100 cycles per min) for 24 h. On 10 successive 121 days similar, freshly prepared flasks were inoculated with 40 µl from each 122 preceding days' culture, respectively. MICs for each test organism were 123 determined on days 0 and 10 and compared by the Mann-Whitney test using 124 Minitab version 14. 125 Short-term resistance training by exposure to stepwise increasing 126 concentrations of manuka honey. 127 Cultures were set up as above using an initial concentration of 1%(w/v) 128 manuka honey in NB for S. aureus and 5%(w/v) manuka honey in NB for P. aeruginosa and incubated at 37ºC with shaking for 24 h. At 24 h intervals on 130 10 consecutive days, freshly prepared broths with honey concentrations 131 increased by 0.5%(w/v) for S. aureus and 1%(w/v) for P. aeruginosa were 132 inoculated with 40 µl from the preceding days' culture. Throughout the 133 training period the identity of each culture was confirmed daily by BBL Crystal 134 kits (Becton Dickinson, Basingstoke, UK). For both test organisms MICs of 135 manuka honey were determined before and after the training period. 136 Stepwise resistance training of clinical isolates by long-term exposure to sub-137 inhibitory concentrations of manuka honey. 138 MICs of manuka honey for each of the clinical isolates were determined 139 before continuous exposure to honey. In a 96 well, flat bottomed microtitre 140 plate for each of the 4 test organisms a range of 15 concentrations of manuka 141 honey (Comvita manukacare 18+) in ISB that varied by 1%(w/v) intervals and 142 included concentrations above and below the initial MIC of each test organism 143 was prepared. Each well contained 200 µL ISB and honey and was inoculated 144 on day 0 with 5 µl overnight broth culture, then incubated at 37ºC for 24 h. 145 MIC was determined visually as the lowest concentration of honey to prevent 146 growth. A fresh microtitre plate was prepared every 24 h with a new range of 147 15 honey concentrations contained in ISB that increased by 1%(w/v) 148 increments and included the last MIC in the middle of the range. With time, 149 increasingly higher concentrations of honey in ISB were required, but the 150 range always included honey concentrations above and below the expected 151 MIC. On each successive day of the experiment, wells in the new microtitre 152 plate were inoculated with 5 µl of each respective test organism taken from 153

  their starting MIC values. Hence honey resistance was not observed in either 189 S. aureus or P.aeruginosa. 190 191 Resistance training of clinical isolates by long-term exposure to honey. 192 The susceptibility of the 4 clinical isolates tested in this study for manuka 193 honey decreased during the 28 day resistance training period (Fig. 1), with the 194 greatest proportional change observed for E. coli (2.1) and the least in P. 195 aeruginosa (1.4). MICs of MRSA and S. epidermidis increased by factors of 196 1.6 and 1.7, respectively . However during the recovery period, MIC values 197 returned to pre-training values within 9 and 14 days for S. epidermidis and P. 198

Fig. 1 :Fig. 2 :

 12 Fig.1: Sensitivity of four clinical isolates to manuka honey during exposure to 358

  Clinical use of honey in the topical treatment of wounds has increased in 32Europe and North America since licensed wound care products became 33 available in 2004 and 2007 respectively. Honey-resistant bacteria have not 34 been isolated from wounds, but there is a need to investigate whether honey 35 has the potential to select for honey resistance. Two cultures of bacteria from 36 reference collections (Staphylococcus aureus NCTC 10017 and 37Pseudomonas aeruginosa ATCC 27853) and four cultures isolated from 38 wounds (Escherichia coli, MRSA, Pseudomonas aeruginosa and 39

	1
	2 3	*RA Cooper, L Jenkins, AFM Henriques † , RS Duggan, NF Burton
	4
	5	University of Wales Institute Cardiff.
	6
	7
	8
	9
	10	*Corresponding author:
	11	Professor RA Cooper,
	12	Centre for Biomedical Sciences,
	13	Department of Applied Sciences,
	14	Cardiff School of Health Sciences,
	40	Staphylococcus epidermidis) were exposed to sub-lethal concentrations of
	15	University of Wales Institute Cardiff,
	41	manuka honey in continuous and stepwise training experiments to determine
	16	Western Avenue,
	42	whether susceptibility to honey diminished. Reduced susceptibilities to
	17	Cardiff CF5 2YB
	43	manuka honey in the test organisms during long-term stepwise resistance
	18	UK.

  times higher. MBC values were close to MICs indicating that manuka 208 honey was bactericidal for the recovered cultures. 209 It is perhaps worth noting that by exposing each test organism to a range of 210 honey concentrations simultaneously during the long-term training 211 experiment, MIC values were obtained daily at each transfer step. This meant 212 that only one determination was obtained at each time point. MICs obtained 213 later on stored cultures were done in replicate (Table 2). Unlike antibiotic 214 testing where serial doubling dilutions series are normally utilised, here honey 215 concentrations differing by 1%(w/v) intervals were used to get a more precise 216

	217	estimate of MIC.
	218	Purity plates and biochemical characteristics confirmed that culture identities
	219	were maintained throughout the training and recovery periods; RAPD profiles
	220	remained constant.
	221	Discussion

Table 1 :

 1 Sensitivity of two reference cultures to manuka honey before and 354 after culture in sub-lethal concentrations of honey for 10 successive days. 355

			Mean MIC %(w/v) ± sd (n)
			P.aeruginosa ATCC 27853	S.aureus NCTC 10017
		Before training	15.7 ± 2.0 (8)	2.8 ± 0.4 (5)
		After training	19.7 ± 1.4 (7)	3.8 ± 0.08 (5)
		p	0.03	>0.05
	356
	357	sd = standard deviation; n = number of assays

Table 2 :

 2 Sensitivity of clinical isolates to manuka honey following long-term 371 exposure, recovery in honey free media and storage at -80⁰C. 372

	Test organism	MIC*	MBC*
	E. coli	16.17 ± 1.47	18.0 ± 1.67
	MRSA	5.83 ± 1.6	8.5 ± 2.5
	P. aeruginosa	15.33 ± 2.25	15.67 ± 3.2
	S. epidermidis	5.67 ± 1.63	8.33 ± 2.56

*Mean manuka honey concentration as %(w/v) ± standard deviation (tests 373 were done in duplicate on three separate occasions) 374

Short-term resistance training with reference cultures.

Acknowledgements 286

This study was supported in part by Derma Sciences Inc. New Jersey, USA.


Prof Manuel Luis

M.D.; Fernández Guerrero

Jiménez Fundación

Díaz

M.D.; Julio José Gonzalez Lopez

M.D.. Miguel Górgolas

M.D. ; Manuel L Fernández Guerrero

M.D Julio González López

Infectious Endocarditis in Patients with Cirrhosis of the Liver: A Model of Infection in the Frail

Keywords: infectious endocarditis, beta-hemolytic streptococci, cirrhosis of the liver Infectious endocarditis, liver cirrhosis, nosocomial endocarditis

To know whether cirrhosis is a predisposing cause of IE and to determine the microbiology, prognosis and the role of cardiac surgery on mortality. Review of cases of infectious endocarditis in a university-affiliated hospital during 10 years. Thirty-one(9.8%) patients among 316 cases of infectious endocarditis had hepatic cirrhosis. Valve disorders were present in 62.2% of cirrhotic patients and infection occurred on aortic(48%) and mitral valves(45%).Endocarditis was hospital-acquired in 14(45%) and 11(17.7%) of cirrhotic and controls respectively(OR 3.82;95%CI:1.46-9.99;p 0.005).Staphylococcus aureus was the most common causative microorganism but βeta-hemolytic streptococci were most frequently isolated in cirrhotic patients(OR 8.75; 95% CI: 1.7-45.2;p 0.001).Renal failure was more frequent in patients with cirrhosis(OR 8.23;95%CI: 3.06-22.2; p 0.001).Cirrhotic patients had a higher mortality(51% vs 17.7%; OR 4.95;95%CI:1.89-12.91;p 0.001) associated with the severity of liver disease. Valve replacement was performed less frequently in cirrhotic patients(56.2% vs 92%) and the operative mortality was extremely high in patients with stages B and C. Hepatic cirrhosis is a frequent comorbid condition in patients with endocarditis. Due to the presence severe hepatic dysfunction, cardiac surgery is not undertaken even when indicated and mortality is high in stages B and C. Endocarditis is a serious hazard for hospitalized cirrhotic patients.

Response to Reviewers: Dear Dr. Van Belkum: Thank you very much

Abstract

To know whether cirrhosis is a predisposing cause of IE and to determine the microbiology, prognosis and the role of cardiac surgery on mortality.

Review of cases of infectious endocarditis in a universityaffiliated hospital during 10 years.

Thirty-one(9.8%) patients among 316 cases of infectious endocarditis had hepatic cirrhosis. Valve disorders were present in 62.2% of cirrhotic patients and infection occurred on aortic(48%) and mitral valves(45%).Endocarditis was hospital-acquired in 14(45%) and 11(17.7%) of cirrhotic and controls respectively(OR 3.82;95%CI:1.46-9.99;p 0.005).Staphylococcus aureus was the most common causative microorganism but βeta-hemolytic streptococci were most frequently isolated in cirrhotic patients(OR 8.75; 95% CI: 1.7-45.2;p 0.001).Renal failure was more frequent in patients with cirrhosis(OR 8.23;95%CI: 3.06-22.2; p 0.001).Cirrhotic patients had a higher mortality(51% vs 17.7%; OR 4.95;95%CI:1.89-12.91;p 0.001) associated with the severity of liver disease. Valve replacement was performed less frequently in cirrhotic patients(56.2% vs 92%) and the operative mortality was extremely high in patients with stages B and C. Hepatic cirrhosis is a frequent comorbid condition in patients with endocarditis. Due to the presence severe hepatic dysfunction, cardiac surgery is not undertaken even when indicated and mortality is high in stages B and C. Endocarditis is a serious hazard for hospitalized cirrhotic patients.

INTRODUCTION.

Patients with cirrhosis of the liver are prone to develop severe bacterial infections due to the presence of various facilitating mechanisms including changes in the reticuloendothelial system, decreased opsonic activity, leukocyte dysfunction and iatrogenic factors among others [START_REF] Christou | Bacterial infection-related morbidity and mortality in cirrhosis[END_REF]. Infections such as primary peritonitis, pneumonia, or tuberculosis are acknowledged causes of morbidity and mortality in patients with cirrhosis of the liver; yet, apart from these particular diseases, other infection issues have not been the subject of in-dept investigation [START_REF] Christou | Bacterial infection-related morbidity and mortality in cirrhosis[END_REF].

Infectious endocarditis occurring in patients with hepatic cirrhosis is rarely reported [START_REF] Mccashland | Bacterial endocarditis in patients with chronic liver disease[END_REF]. Anecdotal case reports of endocarditis associated with invasive procedures -TIPS, upper endoscopy, sclerotherapy of esophageal varices or liver biopsy -, and caused by unusual pathogens comprise the majority of the articles [START_REF] Finkielman | Endocarditis as a complication of a transjugular intrahepatic portosystemic stent-shunt[END_REF][START_REF] Schalaeffer | Serious bacterial infections after endoscopic procedures[END_REF][START_REF] Tripodi | Streptococcus bovis endocarditis and its association with chronic liver disease: an underestimated risk factor[END_REF][START_REF] Fayad | Pasteurella multocida aortic valve endocarditis: case report and literature review[END_REF][START_REF] Guerrero | Hospital-acquired infectious endocarditis not associated with cardiac surgery: an emerging problem[END_REF]. However, to the best of our knowledge, studies have not analyzed the impact of liver cirrhosis, a common morbid condition, on the outcome of patients with endocarditis and thus, some important clinical and prognostic factors of endocarditis in patients with cirrhosis are still poorly known.

The aim of this study were: (i) to know whether cirrhosis is in fact a predisposing cause of IE; (ii) to determine the microbial etiology of IE in this population and other clinically relevant features; and (iii) to assess prognostic factors and how frequently cardiac surgery is needed and performed.

PATIENTS AND METHODS.

Retrospective review of consecutive cases of infectious endocarditis in a tertiary, university affiliated hospital that provides service for a population of 350.000 people in downtown Madrid, during a period of 10 years (1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009). A control group composed of 2 patients per case (the previous and the consecutive to the index case) was used for comparison purposes. IE was diagnosed by the usual modifed Duke's criteria [START_REF] Li | Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis[END_REF].

Only patients with a definite diagnosis of endocarditis were included. Hospital-acquired infectious endocarditis was defined as endocarditis developing ≥ 72 hours after admission in association with a hospital-based procedure or during another hospitalization within the preceding 8 weeks [START_REF] Guerrero | Hospital-acquired infectious endocarditis not associated with cardiac surgery: an emerging problem[END_REF].

Renal failure was defined as an increase in serum creatinin levels ≥ 1.6 mg/dl persisting ≥ 24 hours despite of the administration of fluids. Central nervous system complications included embolic events and brain hemorrhage and were evaluated by CT-scan and MR-imaging.

Mortality per episode was calculated. All death that occurred during hospitalization as a direct consequence of infection, including operative and perioperative mortality and any death associated with bleeding or acute liver failure occurring during the active phase of infection were considered related with endocarditis.

Statistics: SPSS v 11.0 (SPSS Inc., Chicago, IL) was used for the statistical analysis of the data. Continuous variables were expressed as mean (range). Discrete variables were expressed as percentages.

Associations were tested by chi-square test or the Fisher exact test when samples were small. The strength of associations was measured by odds ratio (OR) or the Haldane estimator for small sample 2 x 2 tables, and their 95% confidence interval (CI).

Differences in continuous variables were measured by Student t test for independent variables. Differences between groups were considered significant at p value < 0.05.

RESULTS.

Thirty-one (9.8%) patients among 316 cases of infectious endocarditis had cirrhosis of the liver. The mean age of cirrhotic patients was 59.6 years, lower than that in the control group. Most patients (71%) were males. The etiology of hepatic disease was alcoholic cirrhosis in 16 patients (51.6%), posthepatitis cirrhosis caused by hepatitis C or B viruses in 11 patients (35.4%) or cryptogenic cirrhosis in 4 (13%) and were classified as stages A (12), B (8), C [START_REF] Li | Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis[END_REF] of Child-Turcotte-Pugh.

Table 1 shows some relevant epidemiologic and clinical features of endocarditis in patients with cirrhosis of the liver. An underlying predisposing valve disease was present in 62.2% of cirrhotic patients and infection occurred mainly on aortic (48%) and mitral valves (45%). Infectious endocarditis was hospital-acquired in 14 (45%) and 11 (17.7%) of cirrhotic and control patients respectively (OR 3.82;95% CI: 1.46-9.99 ; p 0.005). Central venous catheters (4 cases), urinary tract instrumentation (3 cases), colonoscopy (1 case), total parenteral nutrition (1 case), liver biopsy (1 case), sclerotherapy of esophageal varices (1) and Sengstaken-Blakemore balloon (1 case) were the recognized sources of bacteremia.

No differences were observed among cirrhotic and control patients in the relative frequency of complications such as cardiac failure or central nervous system events. However, renal failure was significantly more frequent in patients with cirrhosis of the liver (OR 8.23; 95% CI: 3.06-22.2; p 0.001).

Table 2 shows the microbial etiology of infectious endocarditis in patients with cirrhosis of the liver. None of these patients were on chronic, low-dose antimicrobial therapy with fluoroquinolones or other drugs for prevention of primary bacterial peritonitis.

Staphylococcus aureus was the most common causative microorganism in both cirrhotic and control patients. Beta-hemolytic streptococci, including S. agalactiae and S. pyogenes, were most frequently isolated in patients with cirrhosis of the liver than in controls (OR 8.75; 95% CI: 1.7-45.2; p 0.001). On the other hand, viridans streptococci were uncommonly found in cirrhotic patients (OR 0.1; 95% CI: 0.01-0.83; p 0.002). Staphylococcus aureus [START_REF] Tripodi | Streptococcus bovis endocarditis and its association with chronic liver disease: an underestimated risk factor[END_REF], enterococci (4), coagulasenegative staphylococci (2), Escherichia coli (1), Candida albicans (1) and P aeruginosa (1) were the microorganisms isolated in cirrhotic patients with nosocomial endocarditis.

Sixteen patients (51%) with cirrhosis of the liver and 11 risk of these last patients ranged from 57% to 80% at 30 days and from 69% to 81% at 1-year. In the rest of the cases, valve replacement was declined due to the presence of end-stage liver failure (3 in stage B, 4 in stage C). The postoperative mortality risk in this group of patients was retrospectively calculated and ranged from 33% to 85% at 30 days and from 54% to 91% at 1-year. On the other hand, valve replacement was indicated in 24 control patients and was performed in 22 (91.6%) of whom 18 (81.8%) survived the infection.

DISCUSSION.

Cirrhosis of the liver was a common underlying condition in patients with IE observed in almost 10% of cases. Cirrhotic patients, particularly those with advanced disease exhibit leukocyte dysfunction and phagocytic defects that make them prone to develop focal and systemic bacterial infections. The presence of portosystemic shunts and the common occurrence of cutaneous abrasions in oedematous legs are potential sources of bacteremia from gastrointestinal tract and skin. In addition, patients with hepatic cirrhosis are frequently admitted to hospital where they are the subject of invasive diagnostic and therapeutic procedures with the potential of producing bacteremia and endocarditis.

In this series, endocarditis was a nosocomial infection in 45% of cases, a proportion significantly higher than that found in the control group and much higher than that observed in previous reports on hospital-acquired infectious endocarditis [START_REF] Guerrero | Hospital-acquired infectious endocarditis not associated with cardiac surgery: an emerging problem[END_REF][START_REF] Benito | Health care-associated native valve endocarditis: importance of non-nosocomial acquisition[END_REF]. This observation suggests that infectious endocarditis is a serious hazard for hospitalized patients with cirrhosis of the liver. As shown in previous studies, venous catheters and urinary tract instrumentation were the main sources for bacteremia leading to endocarditis in cirrhotic patients [START_REF] Guerrero | Nosocomial enterococcal endocarditis: a serious hazard for hospitalized patients with enterococcal bacteremia[END_REF]. However it is interesting to note that other procedures that are commonly undertaken in these patients such as liver biopsy or urgent measures for the control of esophageal bleeding such as sclerotherapy of varices and Sengstaken-Blakemore balloon tamponade may be associated with bacteremia and endocarditis.

While S aureus and enterococci were the organisms most frequently found in nosocomial cases, β-hemolytic streptococci were common causes of community-acquired endocarditis in patients with cirrhosis. In previous studies, endocarditis caused by β-hemolytic streptococci has been associated with chronic underlying diseases such as alcoholism and cirrhosis of the liver [START_REF] Lefort | the Beta-hemolytic streptococci infective endocarditis study group. Comparison between adult endocarditis due to beta-hemolytic streptococci (serogroups A, B, C, and G) and Streptococcus milleri: a multicenter study in France[END_REF]. Specifically S agalactiae, that in pre-and early antibiotic era was almost exclusively an infection seen in postpartum and postabortal women, is now a microrganism causing severe infections, including bacteremia and endocarditis, in chronically ill patients with cirrhosis of the liver, malignancies and some other conditions [START_REF] Lefort | the Beta-hemolytic streptococci infective endocarditis study group. Comparison between adult endocarditis due to beta-hemolytic streptococci (serogroups A, B, C, and G) and Streptococcus milleri: a multicenter study in France[END_REF][START_REF] Sambola | Streptococcus agalactiae infective endocarditis: analysis of 30 cases and review of the literature, 1962-1998[END_REF]. As noted before, endocarditis caused by S agalactiae followed a complicated course characterized by large vegetations, rapid destruction of cardiac valves and heart failure [START_REF] Sambola | Streptococcus agalactiae infective endocarditis: analysis of 30 cases and review of the literature, 1962-1998[END_REF][START_REF] Rollan | Clinical profile of Streptococcus agalactiae native valve endocarditis[END_REF]. In addition, chronic liver disease has been recognized as a predisposing factor for S bovis bacteremia which may explain the increased frequency of endocarditis caused by these microorganisms in our series [START_REF] Gonzalez Quintela | Prevalence of liver disease in patients with Streptococcus bovis bacteremia[END_REF].

Liver cirrhosis has a significant impact on the mortality of patients with bacteremic infections and high production of proinflammatory cytokines seems to play a role in the worsening of liver function and the development of organ-system failures such as hepato-renal syndrome or hepatic encephalopathy [START_REF] Gustot | Severe sepsis in cirrhosis[END_REF][START_REF] Chen | Impact of liver cirrhosis on mortality in patients with community-acquired bacteremia[END_REF].

Remarkably, renal failure occurred in most of our cases and seemed to contribute to the poor outcome of patients. Besides heart failure, acute renal failure was a major determinant of mortality.

Mortality of cardiac surgery of patients with advanced liver cirrhosis is extremely high [START_REF] Takahashi | Succesful aortic valve replacement for infective endocarditis in a patient with severe liver cirrhosis[END_REF] and so, the decision to operate on these patients is difficult. In this series, although indicated in most patients with cardiac failure, valve replacement was precluded in many due to the presence of end-stage liver disease and when performed, only patients in good functional state survived. MELD score, which has been recently validated to quantify the risk of mortality postoperatively in patients with cirrhosis, may be a useful tool in the task of deciding whether or not these patients must been sent to the operating room for valve replacement [START_REF] Teh | Risk factors for mortality after surgery in patients with cirrhosis[END_REF].

In summary, we show herein data that suggest that infectious endocarditis in patients with cirrhosis of the liver is a severe disease with unique community-acquired etiologic agents. Besides, infectious endocarditis is frequently acquired during hospitalization as a consequence of invasive diagnostic or therapeutic procedures. ------------------------------------------------------ ------------------------------------------------------ ---------------------------------------------------------- In doing the revision, I have taken into account all the suggestions of the reviewer.

Firstly, let me tell you why we chose to perform a case-control study instead of a case-cohort comparison. A case-control study is a simple and very useful investigative tool to identify factors that may contribute to the understanding of many medical conditions. In this case, we compared patients with cirrhosis of the liver and endocarditis with subjects with endocarditis but without such underlying disease. In short, we looked back to see if there were characteristics of these patients that differ from those who did not have cirrhosis. The main reasons of our choice were:

1. Simplicity. 2. Accuracy. Because patients were seen over a relatively long period -10 years-we tried to bring nearer cases and controls to avoid biases associated with changes in diagnostic techniques (use of TEE, blood cultures), antibiotic usage or surgical team over the time. For this reason we selected two controls for each case: the previous and the consecutive to the index case.

Reviewer:

1. Our patients with cirrhosis of the liver were not on any type of chronic antimicrobial therapy with fluoroquinolones or other agents such as those that are administered for the prevention of primary bacterial peritonitis. We have included a short comment on this in the last paragraph of page 7. This is an interesting comment because such intervention may eventually favour enterococcal infections. Our cases, enterococcal endocarditis mainly occurred as a result of urinary tract instrumentation. Patients with endocarditis caused by beta-hemolytic endocarditis developed the disease in the community and was not associated with a previous use of antibiotics. 2. As suggested, we have included a short summary of our results and a recommendation on the management of cirrhotic patients with endocarditis in the last paragraph of page 11.

In addition, one new quotation (no.13) has been included.

  Hepatic cirrhosis was diagnosed by clinical and laboratory criteria and in some patients by means of liver biopsy. The severity of liver dysfunction was categorized according to the modified Child-Turcotte-Pugh classification with a scoring system of 5 to 15: scores of 5 and 6 being class A consistent with compensated cirrhosis; scores of 7 to 9 indicating class B characterized by mild ascitis and minimal encephalopathy; and scores of 10 to 15 class C with poorly controlled ascitis and severe hepatic encephalopathy [8]. The risk of postoperative mortality was assessed by the Model for End-stage Liver Damage (MELD) [9] with an on-line tool that used age, type of hepatic disease, the American Society of Anesthesiolologists Physical Status Classification (ASA score) and patient's laboratory values for serum bilirubin, serum creatinin and the international normalized ratio for prothrombin time (INR) [10].

( 17 .

 17 7%) in the control group died during hospitalization for causes associated with endocarditis (OR 4.95;95% CI: 1.89-12.91; p 0.001). Mortality was associated with the severity of liver disease and so, while only 2 of 12 (16.6%) patients in stage A died, 6 out of 8 (75%) in stage B and 8 of 11 (72.7%) in stage C died. Cardiac failure, hepato-renal syndrome with acute renal failure and brain bleeding were the most common causes of death. Valve replacement was considered in 16 patients with cirrhosis of the liver who developed cardiac failure but was performed in only 9 (56.2%) cases in whom it seemed feasible and indicated. Five of theses patients survived (4 in stage A, and 1 in stage B) and 4 died (3 in stage B and 1 in stage C). The calculated postoperative mortality

  When indicated, valve replacement can be successfully performed in patients with minor or mild hepatic dysfunction. However, operative mortality of patients with Child stages B and C is extremely high and probably should be undertaken only in selected individuals with cardiac failure without renal failure and other systemic complications. Finally, because most of the factors facilitating the development of bacterial infections in cirrhotic patients are unchangeable and many cases occur during hospitalization, prevention should focus on measures to avoid bacteremia and other nosocomial infections. New studies are required to better determine the role of cardiac surgery in the management of infectious endocarditis in cirrhotic patients.

  Ref. EJCMID-D-10-00196 "Infectious Endocarditis in Patients with Cirrosis of the Liver:A Model of Infection in the Frail Patient". Dear Dr. Van Belkum: Thank you very much for the opportunity of revising the above mentioned manuscript.

Table 1 .

 1 Epidemiologic, Clinical Features and Mortality of Infectious Endocarditis in Patients with Cirrhosis of the Liver. Patients no (%)

Table 2 .

 2 -Microbial Etiology of Infectious Endocarditis In Patients withCirrhosis of the Liver.

	Mean age	59.6 (32-71) 68.9 (45-86)
	Gender: Male	22 (70.9)	41 (66.1)
	Female	9 (29.19)	21 (33.8)
	Underlying valve disease			ns
	Rheumatic disease	5 (16.1)	9 (14.5)
	Degenerative	6 (19.3)	16 (25.8)
	Prosthetic valve	4 (12.9)	10 (16.1)
	Mi valve prolapse	3 (9.6)	3 (4.8)
	Other	1 (3.2)	7 (11.2)
	Undetermined	12 (38.7)	17 (27.4)
	Valve involvement
	Mitral valve	14 (45.1)	25 (40.3)
	Aortic valve	15 (48.3)	23 (37)
	Mi & Ao valves	2	3
	Tricuspid valve	-	9	0.09:0.01-1.59	.03
	Other*	-	2
	Acquisition
	Hospital-acquired	14(45.1)	11(17.7)	3.82;1.46-9.99 .005
	Community-acquired	17 (54.8)	51 (82.2)
	Complications
	Cardiac failure	16 (51.6)	25 (40.3)
	Brain emboli	5 (16)	10 (16.1)
	Renal failure	19 (61.2)	10 (16.1) 8.23;3.06-22.2	.001
	Mortality	16 (51.6)	11 (17.7) 4.95;1.89-12.91 .001

Table

  -

	Staphylococcus aureus	8 (25.8)	17 (27.4)	ns
	Coagulase-staphylococci	2 (6.4)	5 (8.6)	ns
	Enterococci	6 (19.3)	6 (9.6)	ns
	β-hemolytic streptococci:	7 (22.5)	2 (3.2) 8.75;1.7-45.2 .001
	S agalactiae	4	1
	S pyogenes	3	-
	Group C streptococci	-	1
	Streptococcus bovis	3 (9.6)	3 (4.8)
	Streptococcus pneumoniae	1	-
	Streptococcus viridans	1 (3.2)	15 (24.1) 0.1; 0.01-0.83 .002
	Listeria monocytogenes	1	1
	Gram-negative bacteria*	1 (3.2)	6 (9.6)
	Fungi	1	1
	Coxiella burnetii	-	3
	Unknown	-	3
	* HACEK group, Enterobacteriaceae, Pseudomonas.

Authors' Response to Reviewers' Comments Click here to download Authors' Response to Reviewers' Comments: Letter to the editor.doc

  Thank you once more for this opportunity and do not hesitate if further questions are raised.Looking forward to hearing from you.

	Yours sincerely
	Manuel L. Fernández Guerrero