The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates.

BACKGROUND: Effective neurorestorative therapies of neurodegenerative diseases must be developed. There is increasing interest in using human platelet lysates, rich in neurotrophic factors, as novel disease-modifying strategy of neurodegeneration. To ensure virus safety, pathogen reduction treatments should be incorporated in the preparation process of the platelet concentrates used as source material. We therefore investigated whether platelet concentrates (PC) pathogen-inactivated using a licensed photo-inactivation treatment combining photosensitive psoralen (amotosalen) and UVA irradiation (Intercept) can serve as source material to prepare platelet lysates with preserved neuroprotective activity in Parkinson's disease models. METHODS: Intercept treated-PCs were centrifuged, when reaching expiry day (7 days after collection), to remove plasma and platelet additive solution. The platelet pellet was re-suspended and concentrated in phosphate buffer saline, subjected to 3 freeze-thaw cycles (- 80 °C/37 °C) then centrifuged to remove cell debris. The supernatant was recovered and further purified, or not, by heat-treatment as in our previous investigations. The content in proteins and neurotrophic factors was determined and the toxicity and neuroprotective activity of the platelet lysates towards LUHMES cells or primary cortical/hippocampal neurons were assessed using ELISA, flow cytometry, cell viability and cytotoxicity assays and proteins analysis by Western blot. RESULTS: Platelet lysates contained the expected level of total proteins (ca. 7-14 mg/mL) and neurotrophic factors. Virally inactivated and heat-treated platelet lysates did not exert detectable toxic effects on neither Lund human mesencephalic dopaminergic LUHMES cell line nor primary neurons. When used at doses of 5 and 0.5%, they enhanced the expression of tyrosine hydroxylase and neuron-specific enolase in LUHMES cells and did not significantly impact synaptic protein expression in primary neurons, respectively. Furthermore, virally-inactivated platelet lysates tested were found to exert very strong neuroprotection effects on both LUHMES and primary neurons exposed to erastin, an inducer of ferroptosis cell death. CONCLUSION: Outdated Intercept pathogen-reduced platelet concentrates can be used to prepare safe and highly neuroprotective human heat-treated platelet pellet lysates. These data open reassuring perspectives in the possibility to develop an effective biotherapy using virally-inactivated platelet lysates rich in functional neurotrophins for neuroregenerative medicine, and for further bio-industrial development. However, the data should be confirmed in animal models.

New piperazine multi-effect drugs prevent neurofibrillary degeneration and amyloid deposition, and preserve memory in animal models of Alzheimer's disease.

Alzheimer's Disease is a devastating dementing disease involving amyloid deposits, neurofibrillary tangles, progressive and irreversible cognitive impairment. Today, only symptomatic drugs are available and therapeutic treatments, possibly acting at a multiscale level, are thus urgently needed. To that purpose, we designed multi-effects compounds by synthesizing drug candidates derived by substituting a novel N,N'-disubstituted piperazine anti-amyloid scaffold and adding acetylcholinesterase inhibition property. Two compounds were synthesized and evaluated. The most promising hybrid molecule reduces both the amyloid pathology and the Tau pathology as well as the memory impairments in a preclinical model of Alzheimer's disease. In vitro also, the compound reduces the phosphorylation of Tau and inhibits the release of Aß peptides while preserving the processing of other metabolites of the amyloid precursor protein. We synthetized and tested the first drug capable of ameliorating both the amyloid and Tau pathology in animal models of AD as well as preventing the major brain lesions and associated memory impairments. This work paves the way for future compound medicines against both Alzheimer's-related brain lesions development and the associated cognitive impairments.

Neuronal AMP-activated protein kinase hyper-activation induces synaptic loss by an autophagy-mediated process.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by synaptic loss that leads to the development of cognitive deficits. Synapses are neuronal structures that play a crucial role in memory formation and are known to consume most of the energy used in the brain. Interestingly, AMP-activated protein kinase (AMPK), the main intracellular energy sensor, is hyper-activated in degenerating neurons in several neurodegenerative diseases, including AD. In this context, we asked whether AMPK hyper-activation could influence synapses' integrity and function. AMPK hyper-activation in differentiated primary neurons led to a time-dependent decrease in pre- and post-synaptic markers, which was accompanied by a reduction in synapses number and a loss of neuronal networks functionality. The loss of post-synaptic proteins was mediated by an AMPK-regulated autophagy-dependent pathway. Finally, this process was also observed in vivo, where AMPK hyper-activation primed synaptic loss. Overall, our data demonstrate that during energetic stress condition, AMPK might play a fundamental role in the maintenance of synaptic integrity, at least in part through the regulation of autophagy. Thus, AMPK might represent a potential link between energetic failure and synaptic integrity in neurodegenerative conditions such as AD.

Contribution of the Endosomal-Lysosomal and Proteasomal Systems in Amyloid-ß Precursor Protein Derived Fragments Processing.

Aß peptides, the major components of Alzheimer's disease (AD) amyloid deposits, are released following sequential cleavages by secretases of its precursor named the amyloid precursor protein (APP). In addition to secretases, degradation pathways, in particular the endosomal/lysosomal and proteasomal systems have been reported to contribute to APP processing. However, the respective role of each of these pathways toward APP metabolism remains to be established. To address this, we used HEK 293 cells and primary neurons expressing full-length wild type APP or the ß-secretase-derived C99 fragment (ß-CTF) in which degradation pathways were selectively blocked using pharmacological drugs. APP metabolites, including carboxy-terminal fragments (CTFs), soluble APP (sAPP) and Aß peptides were studied. In this report, we show that APP-CTFs produced from endogenous or overexpressed full-length APP are mainly processed by γ-secretase and the endosomal/lysosomal pathway, while in sharp contrast, overexpressed C99 is mainly degraded by the proteasome and to a lesser extent by γ-secretase.

AMP-activated Protein Kinase Controls Immediate Early Genes Expression Following Synaptic Activation Through the PKA/CREB Pathway.

Long-term memory formation depends on the expression of immediate early genes (IEGs). Their expression, which is induced by synaptic activation, is mainly regulated by the 3',5'-cyclic AMP (cAMP)-dependent protein kinase/cAMP response element binding protein (cAMP-dependent protein kinase (PKA)/ cAMP response element binding (CREB)) signaling pathway. Synaptic activation being highly energy demanding, neurons must maintain their energetic homeostasis in order to successfully induce long-term memory formation. In this context, we previously demonstrated that the expression of IEGs required the activation of AMP-activated protein kinase (AMPK) to sustain the energetic requirements linked to synaptic transmission. Here, we sought to determine the molecular mechanisms by which AMPK regulates the expression of IEGs. To this end, we assessed the involvement of AMPK in the regulation of pathways involved in the expression of IEGs upon synaptic activation in differentiated primary neurons. Our data demonstrated that AMPK regulated IEGs transcription via the PKA/CREB pathway, which relied on the activity of the soluble adenylyl cyclase. Our data highlight the interplay between AMPK and PKA/CREB signaling pathways that allows synaptic activation to be transduced into the expression of IEGs, thus exemplifying how learning and memory mechanisms are under metabolic control.

AMP-Activated Protein Kinase Is Essential for the Maintenance of Energy Levels during Synaptic Activation.

Although the brain accounts for only 2% of the total body mass, it consumes the most energy. Neuronal metabolism is tightly controlled, but it remains poorly understood how neurons meet their energy demands to sustain synaptic transmission. Here we provide evidence that AMP-activated protein kinase (AMPK) is pivotal to sustain neuronal energy levels upon synaptic activation by adapting the rate of glycolysis and mitochondrial respiration. Furthermore, this metabolic plasticity is required for the expression of immediate-early genes, synaptic plasticity, and memory formation. Important in this context, in neurodegenerative disorders such as Alzheimer disease, dysregulation of AMPK impairs the metabolic response to synaptic activation and processes that are central to neuronal plasticity. Altogether, our data provide proof of concept that AMPK is an essential player in the regulation of neuroenergetic metabolic plasticity induced in response to synaptic activation and that its deregulation might lead to cognitive impairments.

A phenotypic approach to the discovery of compounds that promote non-amyloidogenic processing of the amyloid precursor protein: Toward a new profile of indirect ß-secretase inhibitors.

Dysregulation of the Amyloid Precursor Protein (APP) processing leading to toxic species of amyloid ß peptides (Aß) is central to Alzheimer's disease (AD) etiology. Aß peptides are produced by sequential cleavage of APP by ß-secretase (BACE-1) and γ-secretase. Lysosomotropic agent, chloroquine (CQ), has been reported to inhibit Aß peptide production. However, this effect is accompanied by an inhibition of lysosome-mediated degradation pathways. Following on from the promising activity of two series of APP metabolism modulators derived from CQ, we sought to develop new series of compounds that would retain the inhibitory effects on Aß production without altering lysosome functions. Herein, we applied a ligand-based pharmacophore modeling approach coupled with de novo design that led to the discovery of a series of biaryl compounds. Structure-activity relationship studies revealed that minor modifications like replacing a piperidine moiety of compound 30 by a cyclohexyl (compound 31) allowed for the identification of compounds with the desired profile. Further studies have demonstrated that compounds 30 and 31 act through an indirect mechanism to inhibit ß-secretase activity. This work shows that it is possible to dissociate the inhibitory effect on Aß peptide secretion of CQ-derived compounds from the lysosome-mediated degradation effect, providing a new profile of indirect ß-secretase inhibitors.

Study of AMPK-Regulated Metabolic Fluxes in Neurons Using the Seahorse XFe Analyzer.

AMP-activated protein kinase (AMPK) is the intracellular master energy sensor and metabolic regulator. AMPK is involved in cell energy homeostasis through the regulation of glycolytic flux and mitochondrial biogenesis. Interestingly, metabolic dysfunctions and AMPK deregulations are observed in many neurodegenerative diseases, including Alzheimer's. While these deregulations could play a key role in the development of these diseases, the study of metabolic fluxes has remained quite challenging and time-consuming. In this chapter, we describe the Seahorse XFe respirometry assay as a fundamental experimental tool to investigate the role of AMPK in controlling and modulating cell metabolic fluxes in living and intact differentiated primary neurons. The Seahorse XFe respirometry assay allows the real-time monitoring of glycolytic flux and mitochondrial respiration from different kind of cells, tissues, and isolated mitochondria. Here, we specify a protocol optimized for primary neuronal cells using several energy substrates such as glucose, pyruvate, lactate, glutamine, and ketone bodies. Nevertheless, this protocol can easily be adapted to monitor metabolic fluxes from other types of cells, tissues, or isolated mitochondria by taking into account the notes proposed for each key step of this assay.

A modification-specific peptide-based immunization approach using CRM197 carrier protein: Development of a selective vaccine against pyroglutamate Aß peptides.

Strategies aimed at reducing cerebral accumulation of the amyloid-ß (Aß) peptides have therapeutic potential in Alzheimer's disease (AD). Aß immunization has proven to be effective at promoting Aß clearance in animal models but adverse effects have hampered its clinical evaluation. The first anti-Aß immunization clinical trial, which assessed a full-length Aß1-42 vaccine, increased the risk of encephalitis most likely because of autoimmune pro-inflammatory T helper 1 (Th1) response against all forms of Aß. Immunization against less abundant but potentially more pathologically relevant Aß products, such as N-terminally-truncated pyroglutamate-3 Aß (AßpE3), could provide efficacy and improve tolerability in Aß immunotherapy. Here, we describe a selective vaccine against AßpE3, which uses the diphtheria toxin mutant CRM197 as carrier protein for epitope presentation. CRM197 is currently used in licensed vaccines and has demonstrated excellent immunogenicity and safety in humans. In mice, our AßpE3:CRM197 vaccine triggered the production of specific anti-AßpE3 antibodies that did not cross-react with Aß1-42, non-cyclized AßE3, or N-terminally-truncated pyroglutamate-11 Aß (AßpE11). AßpE3:CRM197 antiserum strongly labeled AßpE3 in insoluble protein extracts and decorated cortical amyloid plaques in human AD brains. Anti-AßpE3 antibodies were almost exclusively of the IgG1 isotype, suggesting an anti-inflammatory Th2 response bias to the AßpE3:CRM197 vaccine. To the best of our knowledge, this study shows for the first time that CRM197 has potential as a safe and suitable vaccine carrier for active and selective immunization against specific protein sequence modifications or conformations, such as AßpE3.

AMPK in Neurodegenerative Diseases.

Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis are neurodegenerative disorders that are characterized by a progressive degeneration of nerve cells eventually leading to dementia. While these diseases affect different neuronal populations and present distinct clinical features, they share in common several features and signaling pathways. In particular, energy metabolism defects, oxidative stress, and excitotoxicity are commonly described and might be correlated with AMP-activated protein kinase (AMPK) deregulation. AMPK is a master energy sensor which was reported to be overactivated in the brain of patients affected by these neurodegenerative disorders. While the exact role played by AMPK in these diseases remains to be clearly established, several studies reported the implication of AMPK in various signaling pathways that are involved in these diseases' progression. In this chapter, we review the current literature regarding the involvement of AMPK in the development of these diseases and discuss the common pathways involved.

AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo.

Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer's disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology.

CALHM1 deficiency impairs cerebral neuron activity and memory flexibility in mice.

CALHM1 is a cell surface calcium channel expressed in cerebral neurons. CALHM1 function in the brain remains unknown, but recent results showed that neuronal CALHM1 controls intracellular calcium signaling and cell excitability, two mechanisms required for synaptic function. Here, we describe the generation of Calhm1 knockout (Calhm1(-/-)) mice and investigate CALHM1 role in neuronal and cognitive functions. Structural analysis revealed that Calhm1(-/-) brains had normal regional and cellular architecture, and showed no evidence of neuronal or synaptic loss, indicating that CALHM1 deficiency does not affect brain development or brain integrity in adulthood. However, Calhm1(-/-) mice showed a severe impairment in memory flexibility, assessed in the Morris water maze, and a significant disruption of long-term potentiation without alteration of long-term depression, measured in ex vivo hippocampal slices. Importantly, in primary neurons and hippocampal slices, CALHM1 activation facilitated the phosphorylation of NMDA and AMPA receptors by protein kinase A. Furthermore, neuronal CALHM1 activation potentiated the effect of glutamate on the expression of c-Fos and C/EBPß, two immediate-early gene markers of neuronal activity. Thus, CALHM1 controls synaptic activity in cerebral neurons and is required for the flexible processing of memory in mice. These results shed light on CALHM1 physiology in the mammalian brain.

AMPK in Neurodegenerative Diseases: Implications and Therapeutic Perspectives.

Maintaining proper energy levels in brain neurons is crucial for many cerebral functions such as synaptic transmission, vesicle recycling and axonal transport. AMP-activated protein kinase (AMPK) is the main energy sensor of all living cells. Beside its role as a crucial whole-body energy sensor in hypothalamic neurons, AMPK is also expressed in neurons throughout the brain where it might play additional fundamental roles. For instance, AMPK might be involved in brain development, neuronal polarization and neuronal activity. In addition, recent evidences suggest that AMPK deregulation might participate in neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis and ischemic stroke. Therefore, AMPK is emerging as a potential therapeutic target for these neurodegenerative diseases. Here, we will review the recent literature regarding the physiological and pathological role of AMPK in the brain and discuss the resulting potential therapeutic implications.

Postnatal neurodevelopmental expression and glutamate-dependent regulation of the ZNF804A rodent homologue.

The zinc finger protein ZNF804A rs1344706 variant is a replicated genome-wide significant risk variant for schizophrenia and bipolar disorder. While its association with altered brain structure and cognition in patients and healthy risk allele carriers is well documented, the characteristics and function of the gene in the brain remains poorly understood. Here, we used in situ hybridization to determine mRNA expression levels of the ZNF804A rodent homologue, Zfp804a, across multiple postnatal neurodevelopmental time points in the rat brain. We found changes in Zfp804a expression in the rat hippocampus, frontal cortex, and thalamus across postnatal neurodevelopment. Zfp804a mRNA peaked at postnatal day (P) 21 in hippocampal CA1 and DG regions and was highest in the lower cortical layers of frontal cortex at P1, possibly highlighting a role in developmental migration. Using immunofluorescence, we found that Zfp804a mRNA and ZFP804A co-localized with neurons and not astrocytes. In primary cultured cortical neurons, we found that Zfp804a expression was significantly increased when neurons were exposed to glutamate [20µM], but this increase was blocked by the N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801. Expression of Comt, Pde4b, and Drd2, genes previously shown to be regulated by ZNF804A overexpression, was also significantly changed in an NMDA-dependent manner. Our results describe, for the first time, the unique postnatal neurodevelopmental expression of Zfp804a in the rodent brain and demonstrate that glutamate potentially plays an important role in the regulation of this psychiatric susceptibility gene. These are critical steps toward understanding the biological function of ZNF804A in the mammalian brain.

CALHM1 ion channel elicits amyloid-ß clearance by insulin-degrading enzyme in cell lines and in vivo in the mouse brain.

Alzheimer's disease is characterized by amyloid-ß (Aß) peptide accumulation in the brain. CALHM1, a cell-surface Ca(2+) channel expressed in brain neurons, has anti-amyloidogenic properties in cell cultures. Here, we show that CALHM1 controls Aß levels in vivo in the mouse brain through a previously unrecognized mechanism of regulation of Aß clearance. Using pharmacological and genetic approaches in cell lines, we found that CALHM1 ion permeability and extracellular Ca(2+) were required for the Aß-lowering effect of CALHM1. Aß level reduction by CALHM1 could be explained by an increase in extracellular Aß degradation by insulin-degrading enzyme (IDE), extracellular secretion of which was strongly potentiated by CALHM1 activation. Importantly, Calhm1 knockout in mice reduced IDE enzymatic activity in the brain, and increased endogenous Aß concentrations by up to ∼50% in both the whole brain and primary neurons. Thus, CALHM1 controls Aß levels in cell lines and in vivo by facilitating neuronal and Ca(2+)-dependent degradation of extracellular Aß by IDE. This work identifies CALHM1 ion channel as a potential target for promoting amyloid clearance in Alzheimer's disease.

Chloroquine and chloroquinoline derivatives as models for the design of modulators of amyloid Peptide precursor metabolism.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD). Preventing deregulated APP processing by inhibiting amyloidogenic processing of carboxy-terminal fragments (APP-CTFs), and reducing the toxic effect of amyloid beta (Aß) peptides remain an effective therapeutic strategy. We report the design of piperazine-containing compounds derived from chloroquine structure and evaluation of their effects on APP metabolism and ability to modulate the processing of APP-CTF and the production of Aß peptide. Compounds which retained alkaline properties and high affinity for acidic cell compartments were the most effective. The present study demonstrates that (1) the amino side chain of chloroquine can be efficiently substituted by a bis(alkylamino)piperazine chain, (2) the quinoline nucleus can be replaced by a benzyl or a benzimidazole moiety, and (3) pharmacomodulation of the chemical structure allows the redirection of APP metabolism toward a decrease of Aß peptide release, and increased stability of APP-CTFs and amyloid intracellular fragment. Moreover, the benzimidazole compound 29 increases APP-CTFs in vivo and shows promising activity by the oral route. Together, this family of compounds retains a lysosomotropic activity which inhibits lysosome-related Aß production, and is likely to be beneficial for therapeutic applications in AD.

Effect of the CALHM1 G330D and R154H human variants on the control of cytosolic Ca2+ and Aß levels.

CALHM1 is a plasma membrane voltage-gated Ca2+-permeable ion channel that controls amyloid-ß (Aß) metabolism and is potentially involved in the onset of Alzheimer's disease (AD). Recently, Rubio-Moscardo et al. (PLoS One (2013) 8: e74203) reported the identification of two CALHM1 variants, G330D and R154H, in early-onset AD (EOAD) patients. The authors provided evidence that these two human variants were rare and resulted in a complete loss of CALHM1 function. Recent publicly available large-scale exome sequencing data confirmed that R154H is a rare CALHM1 variant (minor allele frequency (MAF)  = 0.015%), but that G330D is not (MAF  = 3.5% in an African American cohort). Here, we show that both CALHM1 variants exhibited gating and permeation properties indistinguishable from wild-type CALHM1 when expressed in Xenopus oocytes. While there was also no effect of the G330D mutation on Ca2+ uptake by CALHM1 in transfected mammalian cells, the R154H mutation was associated with defects in the control by CALHM1 of both Ca2+ uptake and Aß levels in this cell system. Together, our data show that the frequent CALHM1 G330D variant has no obvious functional consequences and is therefore unlikely to contribute to EOAD. Our data also demonstrate that the rare R154H variant interferes with CALHM1 control of cytosolic Ca2+ and Aß accumulation. While these results strengthen the notion that CALHM1 influences Aß metabolism, further investigation will be required to determine whether CALHM1 R154H, or other natural variants in CALHM1, is/are associated with EOAD.

Inhibition of AMP-activated protein kinase signaling alleviates impairments in hippocampal synaptic plasticity induced by amyloid ß.

The AMP-activated protein kinase (AMPK) is a Ser/Thr kinase that is activated in response to low-energy states to coordinate multiple signaling pathways to maintain cellular energy homeostasis. Dysregulation of AMPK signaling has been observed in Alzheimer's disease (AD), which is associated with abnormal neuronal energy metabolism. In the current study we tested the hypothesis that aberrant AMPK signaling underlies AD-associated synaptic plasticity impairments by using pharmacological and genetic approaches. We found that amyloid ß (Aß)-induced inhibition of long-term potentiation (LTP) and enhancement of long-term depression were corrected by the AMPK inhibitor compound C (CC). Similarly, LTP impairments in APP/PS1 transgenic mice that model AD were improved by CC treatment. In addition, Aß-induced LTP failure was prevented in mice with genetic deletion of the AMPK α2-subunit, the predominant AMPK catalytic subunit in the brain. Furthermore, we found that eukaryotic elongation factor 2 (eEF2) and its kinase eEF2K are key downstream effectors that mediate the detrimental effects of hyperactive AMPK in AD pathophysiology. Our findings describe a previously unrecognized role of aberrant AMPK signaling in AD-related synaptic pathophysiology and reveal a potential therapeutic target for AD.

Recovery of brain biomarkers following peroxisome proliferator-activated receptor agonist neuroprotective treatment before ischemic stroke.

BACKGROUND: Lipid lowering agent such as agonists of peroxisome proliferator-activated receptors (PPAR) are suggested as neuroprotective agents and may protect from the sequelae of brain ischemic stroke. Although the demonstration is not clearly established in human, the underlying molecular mechanism may be of interest for future therapeutic purposes. To this end, we have used our well established rodent model of ischemia-reperfusion pre-treated or not with fenofibrate or atorvastatin and performed a differential proteomics analyses of the brain and analysed the protein markers which levels returned to "normal" following pre-treatments with PPARα agonists. RESULTS: In order to identify potential therapeutic targets positively modulated by pre-treatment with the PPARα agonists, two-dimensional gel electrophoresis proteome profiles between control, ischemia-reperfusion and pre-treated or not, were compared. The polypeptide which expression was altered following ischemia - reperfusion but whose levels remain unchanged after pre-treatment were characterized by mass spectrometry and further investigated by Western-blotting and immunohistochemistry. A series of 28 polypeptides were characterized among which the protein disulfide isomerase reduction - a protein instrumental to the unfolded protein response system - was shown to be reduced following PPARα agonists treatment while it was strongly increased in ischemia-reperfusion. CONCLUSIONS: Pre-treatment with PPARα agonist or atorvastatin show potential neuroprotective effects by inhibiting the PDI overexpression in conjunction with the preservation of other neuronal markers, several of which are associated with the regulation of protein homeostasis, signal transduction and maintenance of synaptic plasticity. This proteomic study therefore suggests that neuroprotective effect of PPARα agonists supposes the preservation of the expression of several proteins essential for the maintenance of protein homeostasis not necessarily directly linked to PPARα known-regulated targets.

CB2 receptor deficiency increases amyloid pathology and alters tau processing in a transgenic mouse model of Alzheimer's disease.

The endocannabinoid CB2 receptor system has been implicated in the neuropathology of Alzheimer's disease (AD). In order to investigate the impact of the CB2 receptor system on AD pathology, a colony of mice with a deleted CB2 receptor gene, CNR2, was established on a transgenic human mutant APP background for pathological comparison with CB2 receptor-sufficient transgenic mice. J20 APP (PDGFB-APPSwInd) mice were bred over two generations with CNR2(-/-) (Cnr2(tm1Dgen)/J) mice to produce a colony of J20 CNR2(+/+) and J20 CNR2(-/-) mice. Seventeen J20 CNR2(+/+) mice (12 females, 5 males) and 16 J20 CNR2(-/-) mice (11 females, 5 males) were killed at 12 months, and their brains were interrogated for AD-related pathology with both biochemistry and immunocytochemistry (ICC). In addition to amyloid-dependent endpoints such as soluble Aß production and plaque deposition quantified with 6E10 staining, the effect of CB2 receptor deletion on total soluble mouse tau production was assayed by using a recently developed high-sensitivity assay. Results revealed that soluble Aß42 and plaque deposition were significantly increased in J20 CNR2(-/-) mice relative to CNR2(+/+) mice. Microgliosis, quantified with ionized calcium-binding adapter molecule 1 (Iba-1) staining, did not differ between groups, whereas plaque associated microglia was more abundant in J20 CNR2(-/-) mice. Total tau was significantly suppressed in J20 CNR2(-/-) mice relative to J20 CNR2(+/+) mice. The results confirm the constitutive role of the CB2 receptor system both in reducing amyloid plaque pathology in AD and also support tehpotential of cannabinoid therapies targeting CB2 to reduce Aß; however, the results suggest that interventions may have a divergent effect on tau pathology.