P2X7R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes.

The purinoceptor P2X7R is a promising therapeutic target for tauopathies, including Alzheimer's disease (AD). Pharmacological inhibition or genetic knockdown of P2X7R ameliorates cognitive deficits and reduces pathological tau burden in mice that model aspects of tauopathy, including mice expressing mutant human frontotemporal dementia (FTD)-causing forms of tau. However, disagreements remain over which glial cell types express P2X7R and therefore the mechanism of action is unresolved. Here, we show that P2X7R protein levels increase in human AD post-mortem brain, in agreement with an upregulation of P2RX7 mRNA observed in transcriptome profiles from the AMP-AD consortium. P2X7R protein increases mirror advancing Braak stage and coincide with synapse loss. Using RNAScope we detect P2RX7 mRNA in microglia and astrocytes in human AD brain, including in the vicinity of senile plaques. In cultured microglia, P2X7R activation modulates the NLRP3 inflammasome pathway by promoting the formation of active complexes and release of IL-1ß. In astrocytes, P2X7R activates NFκB signalling and increases production of the cytokines CCL2, CXCL1 and IL-6 together with the acute phase protein Lcn2. To further explore the role of P2X7R in a disease-relevant context, we expressed wild-type or FTD-causing mutant forms of tau in mouse organotypic brain slice cultures. Inhibition of P2X7R reduces insoluble tau levels without altering soluble tau phosphorylation or synaptic localisation, suggesting a non-cell autonomous role of glial P2X7R on pathological tau aggregation. These findings support further investigations into the cell-type specific effects of P2X7R-targeting therapies in tauopathies.

Astrocytic C-X-C motif chemokine ligand-1 mediates ß-amyloid-induced synaptotoxicity.

BACKGROUND: Pathological interactions between ß-amyloid (Aß) and tau drive synapse loss and cognitive decline in Alzheimer's disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to Aß-induced synaptotoxicity in AD is not well understood. METHODS: We stimulated mouse and human astrocytes with conditioned medium containing concentrations and species of human Aß that mimic those in human AD brain. Medium from stimulated astrocytes was collected and immunodepleted of Aß before being added to naïve rodent or human neuron cultures. A cytokine, identified in unbiased screens of stimulated astrocyte media and in postmortem human AD brain lysates was also applied to neurons, including those pre-treated with a chemokine receptor antagonist. Tau mislocalisation, synaptic markers and dendritic spine numbers were measured in cultured neurons and organotypic brain slice cultures. RESULTS: We found that conditioned medium from stimulated astrocytes induces exaggerated synaptotoxicity that is recapitulated following spiking of neuron culture medium with recombinant C-X-C motif chemokine ligand-1 (CXCL1), a chemokine upregulated in AD brain. Antagonism of neuronal C-X-C motif chemokine receptor 2 (CXCR2) prevented synaptotoxicity in response to CXCL1 and Aß-stimulated astrocyte secretions. CONCLUSIONS: Our data indicate that astrocytes exacerbate the synaptotoxic effects of Aß via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokine-receptor pair as a novel target for therapeutic intervention in AD.

Distinct cytokine profiles in human brains resilient to Alzheimer's pathology.

Our group has previously studied the brains of some unique individuals who are able to tolerate robust amounts of Alzheimer's pathological lesions (amyloid plaques and neurofibrillary tangles) without experiencing dementia while alive. These rare resilient cases do not demonstrate the patterns of neuronal/synaptic loss that are normally found in the brains of typical demented Alzheimer's patients. Moreover, they exhibit decreased astrocyte and microglial activation markers GFAP and CD68, suggesting that a suppressed neuroinflammatory response may be implicated in human brain resilience to Alzheimer's pathology. In the present work, we used a multiplexed immunoassay to profile a panel of 27 cytokines in the brains of controls, typical demented Alzheimer's cases, and two groups of resilient cases, which possessed pathology consistent with either high probability (HP, Braak stage V-VI and CERAD 2-3) or intermediate probability (IP, Braak state III-IV and CERAD 1-3) of Alzheimer's disease in the absence of dementia. We used a multivariate partial least squares regression approach to study differences in cytokine expression between resilient cases and both Alzheimer's and control cases. Our analysis identified distinct profiles of cytokines in the entorhinal cortex (one of the earliest and most severely affected brain regions in Alzheimer's disease) that are up-regulated in both HP and IP resilient cases relative to Alzheimer's and control cases. These cytokines, including IL-1ß, IL-6, IL-13, and IL-4 in HP resilient cases and IL-6, IL-10, and IP-10 in IP resilient cases, delineate differential inflammatory activity in brains resilient to Alzheimer's pathology compared to Alzheimer's cases. Of note, these cytokines all have been associated with pathogen clearance and/or the resolution of inflammation. Moreover, our analysis in the superior temporal sulcus (a multimodal association cortex that consistently accumulates Alzheimer's pathology at later stages of the disease along with overt symptoms of dementia) revealed increased expression of neurotrophic factors, such as PDGF-bb and basic FGF in resilient compared to AD cases. The same region also had reduced expression of chemokines associated with microglial recruitment, including MCP-1 in HP resilient cases and MIP-1α in IP resilient cases compared to AD. Altogether, our data suggest that different patterns of cytokine expression exist in the brains of resilient and Alzheimer's cases, link these differences to reduced glial activation, increased neuronal survival and preserved cognition in resilient cases, and reveal specific cytokine targets that may prove relevant to the identification of novel mechanisms of brain resiliency to Alzheimer's pathology.

Stress Increases the Negative Effects of Chronic Pain on Hippocampal Neurogenesis.

BACKGROUND: Patients with chronic pain often suffer from affective disorders and cognitive decline, which significantly impairs their quality of life. In addition, many of these patients also experience stress unrelated to their illness, which can aggravate their symptoms. These nociceptive inputs are received by the hippocampus, in which maladaptive neuroplastic changes may occur in the conditions of chronic pain. The hippocampus is a structure involved in emotionality, learning, and memory, and the proliferating cells in the granular layer of the hippocampal dentate gyrus respond to chronic pain by slowing their turnover. However, whether the maturation, survival, and integration of newborn cells in the hippocampus are affected by chronic pain remains unclear. In addition, it is unknown whether an added stress may increase this effect. METHODS: We have evaluated the proliferation, differentiation, and survival of newborn hippocampal cells in a rat model of neuropathic pain (chronic constriction injury), with or without stress (chronic immobilization), by assessing the incorporation of bromodeoxyuridine into proliferating cells and immunostaining. RESULTS: The data obtained indicated that there was a decrease in the number of proliferating cells 8 days after nerve injury in animals subjected to neuropathic pain, an effect that was exacerbated by stress. Moreover, 4 weeks after nerve injury, neuropathic pain was associated with a loss of neuroblasts and the reduced survival of new mature neurons in the hippocampal granular layer, phenomena that also were increased by stress. By contrast, the rate of differentiation was not affected in this paradigm. CONCLUSIONS: Neuropathic pain negatively influences hippocampal neurogenesis (proliferation and survival), and this effect is exacerbated by stress. These neuroplastic changes may account for the affective and cognitive impairment seen in patients with chronic pain.

Dissecting phenotypic traits linked to human resilience to Alzheimer's pathology.

Clinico-pathological correlation studies and positron emission tomography amyloid imaging studies have shown that some individuals can tolerate substantial amounts of Alzheimer's pathology in their brains without experiencing dementia. Few details are known about the neuropathological phenotype of these unique cases that might prove relevant to understanding human resilience to Alzheimer's pathology. We conducted detailed quantitative histopathological and biochemical assessments on brains from non-demented individuals before death whose brains were free of substantial Alzheimer's pathology, non-demented individuals before death but whose post-mortem examination demonstrated significant amounts of Alzheimer's changes ('mismatches'), and demented Alzheimer's cases. Quantification of amyloid-ß plaque burden, stereologically-based counts of neurofibrillary tangles, neurons and reactive glia, and morphological analyses of axons were performed in the multimodal association cortex lining the superior temporal sulcus. Levels of synaptic integrity markers, and soluble monomeric and multimeric amyloid-ß and tau species were measured. Our results indicate that some individuals can accumulate equivalent loads of amyloid-ß plaques and tangles to those found in demented Alzheimer's cases without experiencing dementia. Analyses revealed four main phenotypic differences among these two groups: (i) mismatches had striking preservation of neuron numbers, synaptic markers and axonal geometry compared to demented cases; (ii) demented cases had significantly higher burdens of fibrillar thioflavin-S-positive plaques and of oligomeric amyloid-ß deposits reactive to conformer-specific antibody NAB61 than mismatches; (iii) strong and selective accumulation of hyperphosphorylated soluble tau multimers into the synaptic compartment was noted in demented cases compared with controls but not in mismatches; and (iv) the robust glial activation accompanying amyloid-ß and tau pathologies in demented cases was remarkably reduced in mismatches. Further biochemical measurements of soluble amyloid-ß species-monomers, dimers and higher molecular weight oligomers-in total brain homogenates and synaptoneurosomal preparations failed to demonstrate significant differences between mismatches and demented cases. Together, these data suggest that amyloid-ß plaques and tangles do not inevitably result in neural system derangement and dementia in all individuals. We identified distinct phenotypic characteristics in the profile of brain fibrillar and soluble amyloid-ß and tau accrual and in the glial response that discriminated demented and non-demented individuals with high loads of Alzheimer's pathology. Amyloid-ß deposition in the form of fibrillar plaques and intimately related oligomeric amyloid-ß assemblies, hyperphosphorylated soluble tau species localized in synapses, and glial activation emerged in this series as likely mediators of neurotoxicity and altered cognition, providing further insight into factors and pathways potentially involved in human susceptibility or resilience to Alzheimer's pathological changes.

Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection.

Molecular chaperones are protective in neurodegenerative diseases by preventing protein misfolding and aggregation, such as extracellular amyloid plaques and intracellular tau neurofibrillary tangles in Alzheimer's disease (AD). In addition, AD is characterized by an increase in astrocyte reactivity. The chaperone HSPB1 has been proposed as a marker for reactive astrocytes; however, its astrocytic functions in neurodegeneration remain to be elucidated. Here, we identify that HSPB1 is secreted from astrocytes to exert non-cell-autonomous protective functions. We show that in human AD brain, HSPB1 levels increase in astrocytes that cluster around amyloid plaques, as well as in the adjacent extracellular space. Moreover, in conditions that mimic an inflammatory reactive response, astrocytes increase HSPB1 secretion. Concomitantly, astrocytes and neurons can uptake astrocyte-secreted HSPB1, which is accompanied by an attenuation of the inflammatory response in reactive astrocytes and reduced pathological tau inclusions. Our findings highlight a protective mechanism in disease conditions that encompasses the secretion of a chaperone typically regarded as intracellular.

Astrocyte adaptation in Alzheimer's disease: a focus on astrocytic P2X7R.

Astrocytes are key homeostatic and defensive cells of the central nervous system (CNS). They undertake numerous functions during development and in adulthood to support and protect the brain through finely regulated communication with other cellular elements of the nervous tissue. In Alzheimer's disease (AD), astrocytes undergo heterogeneous morphological, molecular and functional alterations represented by reactive remodelling, asthenia and loss of function. Reactive astrocytes closely associate with amyloid ß (Aß) plaques and neurofibrillary tangles in advanced AD. The specific contribution of astrocytes to AD could potentially evolve along the disease process and includes alterations in their signalling, interactions with pathological protein aggregates, metabolic and synaptic impairments. In this review, we focus on the purinergic receptor, P2X7R, and discuss the evidence that P2X7R activation contributes to altered astrocyte functions in AD. Expression of P2X7R is increased in AD brain relative to non-demented controls, and animal studies have shown that P2X7R antagonism improves cognitive and synaptic impairments in models of amyloidosis and tauopathy. While P2X7R activation can induce inflammatory signalling pathways, particularly in microglia, we focus here specifically on the contributions of astrocytic P2X7R to synaptic changes and protein aggregate clearance in AD, highlighting cell-specific roles of this purinoceptor activation that could be targeted to slow disease progression.

Endogenous cannabinoid system regulates intestinal barrier function in vivo through cannabinoid type 1 receptor activation.

The deleterious effects of stress on the gastrointestinal tract seem to be mainly mediated by the induction of intestinal barrier dysfunction and subsequent subtle mucosal inflammation. Cannabinoid 1 receptor (CB1R) is expressed in the mammalian gut under physiological circumstances. The aim of this investigation is to study the possible role of CB1R in the maintenance of mucosal homeostasis after stress exposure. CB1R knockout mice (CB1R(-/-)) and their wild-type (WT) counterparts were exposed to immobilization and acoustic (IA) stress for 2 h per day during 4 consecutive days. Colonic protein expression of the inducible forms of the nitric oxide synthase and cyclooxygenase (NOS2 and COX2), IgA production, permeability to (51)Cr-EDTA, and bacterial translocation to mesenteric lymph nodes were evaluated. Stress exposure induced greater expression of proinflammatory enzymes NOS2 and COX2 in colonic mucosa of CB1R(-/-) mice when compared with WT animals. These changes were related with a greater degree of colonic barrier dysfunction in CB1R(-/-) animals determined by 1) a significantly lower IgA secretion, 2) higher paracellular permeability to (51)Cr-EDTA, and 3) higher bacterial translocation, both under basal conditions and after IA stress exposure. Pharmacological antagonism with rimonabant reproduced stress-induced increase of proinflammatory enzymes in the colon described in CB1R(-/-) mice. In conclusion, CB1R exerts a protective role in the colon in vivo through the regulation of intestinal secretion of IgA and paracellular permeability. Pharmacological modulation of cannabinoid system within the gastrointestinal tract might be therapeutically useful in conditions on which intestinal inflammation and barrier dysfunction takes place after exposure to stress.

Restraint stress increases neuroinflammation independently of amyloid ß levels in amyloid precursor protein/PS1 transgenic mice.

Both hypercortisolemia and hippocampal damage are features found in patients diagnosed of Alzheimer's disease (AD) and epidemiological evidence supports a role for stress as a risk factor for AD. It is known that immobilization stress is followed by accumulation of oxidative/nitrosative mediators in brain after the release of proinflammatory cytokines, nuclear factor kappa B activation, nitric oxide synthase-2 and cyclooxygenase-2 expression. Long-term exposure to elevated corticosteroid levels is known to affect the hippocampus which plays a central role in the regulation of the hypothalamic-pituitary-adrenal axis. We therefore studied the effect of chronic immobilization stress on amyloid precursor protein/PS1 mice. Stress exposure increased AD-induced neuroinflammation characterized by astrogliosis, increased inflammatory gene transcription and lipid peroxidation. Importantly, immobilization stress did not increase the soluble or insoluble amyloid ß levels suggesting that increased cortisol levels lower the threshold for a neuroinflammatory response, independently from amyloid ß. Since inflammation may act as a factor that contributes disease progression, the stress-inflammation relation described here may be relevant to understand the initial mechanisms in underlying the risk enhancing action of stress on AD.

Cannabinoid/agonist WIN 55,212-2 reduces cardiac ischaemia­reperfusion injury in Zucker diabetic fatty rats: role of CB2 receptors and iNOS/eNOS.

BACKGROUND: Diabetes increases cardiac damage after myocardial ischaemia. Cannabinoids can protect against myocardial ischaemia/reperfusion injury. The aim of this study was to examine the cardioprotective effect of the cannabinoid agonist WIN 55,212-2 (WIN) against ischaemia/reperfusion injury in an experimental model of type 2 diabetes. We performed these experiments in the Zucker diabetic fatty rat, and focused on the role of cannabinoid receptors in modulation of cardiac inducible nitric oxide synthase (iNOS)/endothelial-type nitric oxide synthase (eNOS) expression. METHODS: Male 20-week-old Zucker diabetic fatty rats were treated with vehicle, WIN, the selective CB1 or CB2 receptor antagonists AM251 and AM630, respectively, AM251 + WIN or AM630 + WIN. Hearts were isolated from these rats, and the cardiac functional response to ischaemia/reperfusion injury was evaluated. In addition, cardiac iNOS and eNOS expression were determined by western blot. RESULTS: WIN significantly improved cardiac recovery after ischaemia/ reperfusion in the hearts from Zucker diabetic fatty rats by restoring coronary perfusion pressure and heart rate to preischaemic levels. Additionally, WIN decreased cardiac iNOS expression and increased eNOS expression after ischaemia/reperfusion in diabetic hearts. WIN-induced cardiac functional recovery was completely blocked by the CB2 antagonist AM630. However, changes in NOS isoenzyme expression were not affected by the CB antagonists. CONCLUSIONS: This study shows a cardioprotective effect of a cannabinoid agonist on ischaemia/reperfusion injury in an experimental model of a metabolic disorder. The activation mainly of CB2 receptors and the restoration of iNOS/eNOS cardiac equilibrium are mechanisms involved in this protective effect. These initial studies have provided the basis for future research in this field.

Isoform-selective decrease of glycogen synthase kinase-3-beta (GSK-3ß) reduces synaptic tau phosphorylation, transcellular spreading, and aggregation.

It has been suggested that aberrant activation of glycogen synthase kinase-3-beta (GSK-3ß) can trigger abnormal tau hyperphosphorylation and aggregation, which ultimately leads to neuronal/synaptic damage and impaired cognition in Alzheimer disease (AD). We examined if isoform-selective partial reduction of GSK-3ß can decrease pathological tau changes, including hyperphosphorylation, aggregation, and spreading, in mice with localized human wild-type tau (hTau) expression in the brain. We used adeno-associated viruses (AAVs) to express hTau locally in the entorhinal cortex of wild-type and GSK-3ß hemi-knockout (GSK-3ß-HK) mice. GSK-3ß-HK mice had significantly less accumulation of hyperphosphorylated tau in synapses and showed a significant decrease of tau protein spread between neurons. In primary neuronal cultures from GSK-3ß-HK mice, the aggregation of exogenous FTD-mutant tau was also significantly reduced. These results show that a partial decrease of GSK-3ß significantly represses tau-initiated neurodegenerative changes in the brain, and therefore is a promising therapeutic target for AD and other tauopathies.

Chronic immobilisation stress ameliorates clinical score and neuroinflammation in a MOG-induced EAE in Dark Agouti rats: mechanisms implicated.

BACKGROUND: Multiple sclerosis (MS) is the endpoint of a complex and still poorly understood process which results in inflammation, demyelination and axonal and neuronal degeneration. Since the first description of MS, psychological stress has been suggested to be one of the trigger factors in the onset and/or relapse of symptoms. However, data from animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) are inconsistent and the effect of stress on EAE onset and severity depends on duration and time of application of the stress protocol and the underlying mechanisms. METHODS: Dark Agouti rats were inoculated with MOG/CFA to induce EAE, and an immobilisation stress protocol with two different durations (12 and 21 days, starting at the moment of MOG-inoculation) was applied in order to analyse the effect of stress on disease onset and neuroinflammation. RESULTS: Twelve days of stress exposure increased EAE clinical score in Dark Agouti rats. In addition, these animals presented higher levels of MMP-9 and proinflammatory PGE2 in spinal cord. In contrast, animals chronically exposed to stress (21 days) showed a significantly lower incidence of EAE clinical signs and reduced myelin loss, leukocyte infiltration and accumulation of inflammatory/oxidative mediators in spinal cord. Interestingly, chronically stressed animals showed a parallel increase in levels of the anti-inflammatory prostaglandin 15d-PGJ2, the main endogenous agonist of PPARγ. CONCLUSIONS: Our results demonstrate that, depending on duration, stress exposure elicits opposite effects on PGE2/15d-PGJ2 ratios in spinal cord of EAE-induced Dark Agouti rats. Further studies are needed to elucidate if these changes in prostaglandin balance are sufficient to mediate the differences in clinical score and inflammation here reported, and to establish the potential utility of pharmacological intervention in MS directed toward anti-inflammatory pathways.

Is there a role for the nuclear receptor PPARγ in neuropsychiatric diseases?

The aetiology of psychiatric diseases such as depression or schizophrenia remains largely unknown, even though multiple theories have been proposed. Although monoamine theory is the cornerstone of available pharmacological therapies, relapses, incomplete control of symptoms or failure in treatment occur frequently. From an inflammatory/immune point of view, both entities share several common hallmarks in their pathophysiology, e.g. neuroendocrine/immune alterations, structural/functional abnormalities in particular brain areas, and cognitive deficits, suggesting a dysregulated inflammatory-related component of these diseases that better explains the myriad of symptoms presented by affected individuals. In this review we aimed to explore the role and relevance of inflammatory related lipids (prostanoids) derived from arachidonic acid metabolism by identification of new inflammatory markers and possible pharmacological/dietary modulation of these compounds, with the aim of improving some of the symptoms developed by individuals affected with psychiatric diseases (a critical review of basic and clinical studies about inflammatory-related arachidonic acid metabolism on neuropsychiatric diseases is included). As a specific candidate, one of these immunoregulatory lipids, the anti-inflammatory prostaglandin 15d-PGJ2 and its nuclear receptor peroxisome proliferator-activated nuclear receptor (PPARγ) could be used as a biological marker for psychiatric diseases. In addition, its pharmacological activation can be considered as a multi-faceted therapeutic target due to its anti-inflammatory/antioxidant/anti-excitotoxic/pro-energetic profile, reported in some inflammatory-related scenarios (neurological and stress-related diseases). PPARs are activated by a great variety of compounds, the most relevant being the currently prescribed group of anti-diabetic drugs thiazolidinediones, and some cannabinoids (both endocannabinoids, phytocannabinoids or synthetic), as possible novel therapeutical strategy.

Bridging Integrator-1 protein loss in Alzheimer's disease promotes synaptic tau accumulation and disrupts tau release.

Polymorphisms associated with BIN1 confer the second greatest risk for developing late onset Alzheimer's disease. The biological consequences of this genetic variation are not fully understood, however BIN1 is a binding partner for tau. Tau is normally a highly soluble cytoplasmic protein, but in Alzheimer's disease tau is abnormally phosphorylated and accumulates at synapses to exert synaptotoxicity. The purpose of this study was to determine if alterations to BIN1 and tau in Alzheimer's disease promote the damaging redistribution of tau to synapses, as a mechanism by which BIN1 polymorphisms may increase risk of developing Alzheimer's disease. We show that BIN1 is lost from the cytoplasmic fraction of Alzheimer's disease cortex, and this is accompanied by the progressive mislocalization of phosphorylated tau to synapses. We confirmed proline 216 in tau as critical for tau interaction with the BIN1-SH3 domain and show that phosphorylation of tau disrupts this binding, suggesting that tau phosphorylation in Alzheimer's disease disrupts tau-BIN1 associations. Moreover, we show that BIN1 knockdown in rat primary neurons to mimic BIN1 loss in Alzheimer's disease brain, causes the damaging accumulation of phosphorylated tau at synapses and alterations in dendritic spine morphology. We also observed reduced release of tau from neurons upon BIN1 silencing, suggesting that BIN1 loss disrupts the function of extracellular tau. Together, these data indicate that polymorphisms associated with BIN1 that reduce BIN1 protein levels in the brain likely act synergistically with increased tau phosphorylation to increase risk of Alzheimer's disease by disrupting cytoplasmic tau-BIN1 interactions, promoting the damaging mis-sorting of phosphorylated tau to synapses to alter synapse structure, and by reducing the release of physiological forms of tau to disrupt tau function.

The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by "tethering proteins" that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson's disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson's disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson's disease and FTD/ALS.

Stress mediators regulate brain prostaglandin synthesis and peroxisome proliferator-activated receptor-gamma activation after stress in rats.

Stress exposure leads to oxidative/nitrosative and neuroinflammatory changes that have been shown to be regulated by antiinflammatory pathways in the brain. In particular, acute restraint stress is followed by cyclooxygenase (COX)-2 up-regulation and subsequent proinflammatory prostaglandin (PG) E2 release in rat brain cortex. Concomitantly, the synthesis of the antiinflammatory prostaglandin 15d-PGJ(2) and the activation of its nuclear target the peroxisome proliferator-activated receptor (PPAR)-gamma are also produced. This study aimed to determine the possible role of the main stress mediators: catecholamines, glucocorticoids, and excitatory amino acids (glutamate) in the above-mentioned stress-related effects. By using specific pharmacological tools, our results show that the main mediators of the stress response are implicated in the regulation of prostaglandin synthesis and PPARgamma activation in rat brain cortex described after acute restraint stress exposure. Pharmacological inhibition (predominantly through beta-adrenergic receptor) of the stress-released catecholamines in the central nervous system regulates 15d-PGJ(2) and PGE(2) synthesis, by reducing COX-2 overexpression, and reduces PPARgamma activation. Stress-produced glucocorticoids carry out their effects on prostaglandin synthesis through their interaction with mineralocorticoid and glucocorticoid receptors to a very similar degree. However, in the case of PPARgamma regulation, only the actions through the glucocorticoid receptor seem to be relevant. Finally, the selective blockade of the N-methyl-d-aspartate type of glutamate receptor after stress also negatively regulates 15d-PGJ(2) and PGE(2) production by COX-2 down-regulation and decrease in PPARgamma transcriptional activity and expression. In conclusion, we show here that the main stress mediators, catecholamines, GCs, and glutamate, concomitantly regulate the activation of proinflammatory and antiinflammatory pathways in a possible coregulatory mechanism of the inflammatory process induced in rat brain cortex by acute restraint stress exposure.

Expression of nitrotyrosine and oxidative consequences in the trabecular meshwork of patients with primary open-angle glaucoma.

PURPOSE: To evaluate the possible correlation between the visual field defects in patients with primary open-angle glaucoma (POAG) and the expression and enzymatic activity of nitric oxide synthase (NOS) isoenzymes and nitrotyrosine in trabecular meshwork (TM) samples. METHODS: TM specimens were collected from 146 patients with POAG by using standard filtration surgery. Visual field defects were evaluated by perimetry. Expression of endothelial (e)NOS and inducible (i)NOS were evaluated by quantitative RT-PCR. Constitutive (Ca2+-dependent) and iNOS (Ca2+-independent) activities were measured by the conversion of L-[14C]-arginine to L-[14C]-citrulline. In four TM specimens from POAG-affected eyes and in three human donor control eyes, 3-nitrotyrosine was localized by immunohistochemistry. The marker of lipid peroxidation malondialdehyde (MDA) was measured by the thiobarbituric acid test in samples of aqueous humor (AH) from 48 patients with either POAG or cataracts. RESULTS: The results showed an upregulation of iNOS and a downregulation of calcium-dependent NOS correlated with visual field defects. Expression and activity of iNOS increased in parallel with visual field defects. However, constitutive activity decreased as the visual field defect increased. Nitrotyrosine was observed only in the cells of the TM specimens from eyes with severe POAG. CONCLUSIONS: The increased expression and activity of iNOS in the TM of patients with POAG are proportional to the visual field defect and could lead to the increased of nitrotyrosine levels which may serve as marker of oxidative stress in the progression of cell death of the TM in POAG.

Deciphering the Astrocyte Reaction in Alzheimer's Disease.

Reactive astrocytes were identified as a component of senile amyloid plaques in the cortex of Alzheimer's disease (AD) patients several decades ago. However, their role in AD pathophysiology has remained elusive ever since, in part owing to the extrapolation of the literature from primary astrocyte cultures and acute brain injury models to a chronic neurodegenerative scenario. Recent accumulating evidence supports the idea that reactive astrocytes in AD acquire neurotoxic properties, likely due to both a gain of toxic function and a loss of their neurotrophic effects. However, the diversity and complexity of this glial cell is only beginning to be unveiled, anticipating that astrocyte reaction might be heterogeneous as well. Herein we review the evidence from mouse models of AD and human neuropathological studies and attempt to decipher the main conundrums that astrocytes pose to our understanding of AD development and progression. We discuss the morphological features that characterize astrocyte reaction in the AD brain, the consequences of astrocyte reaction for both astrocyte biology and AD pathological hallmarks, and the molecular pathways that have been implicated in this reaction.

Effects of peroxisome proliferator-activated receptor gamma agonists on brain glucose and glutamate transporters after stress in rats.

Repeated stress causes an energy-compromised status in the brain, with a decrease in glucose utilization by the brain cells, which might account for excitotoxicity processes seen in this condition. In fact, brain glucose metabolism mechanisms are impaired in some neurodegenerative disorders, including stress-related neuropsychopathologies. More recently, it has been demonstrated that some synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) agonists increase glucose utilization in rat cortical slices and astrocytes, as well as inhibit brain oxidative damage after repeated stress, which add support for considering these drugs as potential neuroprotective agents. To assess if stress causes glucose utilization impairment in the brain and to study the mechanisms by which this effect is achieved, young-adult male Wistar rats (control and immobilized for 6 h during 7 or 14 consecutive days, S7, S14) were i.p. injected with the natural ligand 15-deoxy-Delta-12,14-prostaglandin J2 (PGJ2, 120 microg/kg) or the high-affinity ligand rosiglitazone (RG, 3 mg/kg) at the onset of stress. Repeated immobilization during 1 or 2 weeks produces a decrease in brain cortical synaptosomal glucose uptake, and this effect was prevented by treatment with both natural and synthetic PPARgamma ligands by restoring protein expression of the neuronal glucose transporter, GLUT-3 in membrane fractions. On the other hand, treatment with PPARgamma ligands prevents stress-induced ATP loss in rat brain. Finally, repeated immobilization stress also produces a decrease in brain cortical synaptosomal glutamate uptake, and this effect was prevented by treatment with PPARgamma ligands by restoring synaptosomal protein expression of the glial glutamate transporter, EAAT2. In summary, our results demonstrate that 15d-PGJ2 and the thiazolidinedione rosiglitazone increase neuronal glucose metabolism, restore brain ATP levels and prevent the impairment in glutamate uptake mechanisms induced by exposure to stress, suggesting that this class of drugs may be therapeutically useful in conditions in which brain glucose levels or availability are limited after exposure to stress.

Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer's disease brain.

Alterations in calcium homeostasis are widely reported to contribute to synaptic degeneration and neuronal loss in Alzheimer's disease. Elevated cytosolic calcium concentrations lead to activation of the calcium-sensitive cysteine protease, calpain, which has a number of substrates known to be abnormally regulated in disease. Analysis of human brain has shown that calpain activity is elevated in AD compared to controls, and that calpain-mediated proteolysis regulates the activity of important disease-associated proteins including the tau kinases cyclin-dependent kinase 5 and glycogen kinase synthase-3. Here, we sought to investigate the likely temporal association between these changes during the development of sporadic AD using Braak staged post-mortem brain. Quantification of protein amounts in these tissues showed increased activity of calpain-1 from Braak stage III onwards in comparison to controls, extending previous findings that calpain-1 is upregulated at end-stage disease, and suggesting that activation of calcium-sensitive signalling pathways are sustained from early stages of disease development. Increases in calpain-1 activity were associated with elevated activity of the endogenous calpain inhibitor, calpastatin, itself a known calpain substrate. Activation of the tau kinases, glycogen-kinase synthase-3 and cyclin-dependent kinase 5 were also found to occur in Braak stage II-III brain, and these preceded global elevations in tau phosphorylation and the loss of post-synaptic markers. In addition, we identified transient increases in total amyloid precursor protein and pre-synaptic markers in Braak stage II-III brain, that were lost by end stage Alzheimer's disease, that may be indicative of endogenous compensatory responses to the initial stages of neurodegeneration. These findings provide insight into the molecular events that underpin the progression of Alzheimer's disease, and further highlight the rationale for investigating novel treatment strategies that are based on preventing abnormal calcium homeostasis or blocking increases in the activity of calpain or important calpain substrates.