Abstract:
Ischemic strokes disrupt brain networks, leading to remote effects in key regions like the thalamus, a critical hub for brain functions. However, non-invasive methods to quantify these remote consequences still need to be explored. This study aimed to demonstrate that MRI-derived R2* changes can capture iron accumulation linked with inflammation secondary to stroke-induced disconnection. In order to link remote R2* changes to stroke-induced disconnection, we first conducted a secondary analysis of 156 prospectively included stroke patients who underwent MRI at baseline and 1-year follow-up. We mapped fibers disconnected by baseline infarcts to compare the R2* changes over 1 year according to the disconnectivity status in specific thalamic nuclei groups. We also identified the variables associated with elevated R2* at 1 year in a multivariate context through linear regressions. In parallel, to understand the biological underpinning of the remote R2* changes, we set up a translational mouse model through photothrombotic induction of focal cortical infarcts or sham procedures in 110 C57BL/6J mice. We explored the mice through combinations of in vivo MRI at 72h, 2-, 4-, and 8-weeks, histology, qPCR for gene expression, mass spectrometry for iron concentration quantification, and additional ex vivo high-resolution diffusion tensor imaging. In stroke patients, we found a significant increase of R2* within severely disconnected medial and lateral thalamic nuclei groups from baseline to 1 year. At the same time, no change occurred if these structures were not disconnected. We also showed that the disconnectivity status at baseline was significantly associated with R2* at follow-up, independently from confounders, establishing a direct and independent relationship between baseline disconnection and the subsequent R2* increase within the associated locations. In mice, we recapitulated the patients' conditions by observing increased R2* in the stroke groups, specifically within the disconnected thalamic nuclei. Such remote and focal R2* changes peaked at 2 weeks, preceding and correlating with longer-term atrophy at 8 weeks. We established that the remote R2* increase was spatially and temporally correlated with a significant increase of chemically determined iron load bound to ferritin within activated microglial cells. This study provides critical evidence that R2* is a sensitive marker of inflammation secondary to network disconnection, potentially informing future neuroprotective strategies targeting remote brain regions after stroke.

Abstract:
Memory impairment is one of the disabling manifestations of multiple sclerosis (MS) possibly present from the early stages of the disease and for which there is no specific treatment. Hippocampal synaptic dysfunction and dendritic loss, associated with microglial activation, can underlie memory deficits, yet the molecular mechanisms driving such hippocampal neurodegeneration need to be elucidated. In early-stage experimental autoimmune encephalomyelitis (EAE) female mice, we assessed the expression level of molecules involved in microglia-neuron interactions within the dentate gyrus and found overexpression of genes of the complement pathway. Compared to sham immunized mice, the central element of the complement cascade, C3, showed the strongest and 10-fold upregulation, while there was no increase of downstream factors such as the terminal component C5. The combination of in situ hybridization with immunofluorescence showed that C3 transcripts were essentially produced by activated microglia. Pharmacological inhibition of C3 activity, by daily administration of rosmarinic acid, was sufficient to prevent early dendritic loss, microglia-mediated phagocytosis of synapses in the dentate gyrus, and memory impairment in EAE mice, while morphological markers of microglial activation were still observed. In line, when EAE was induced in C3 deficient mice (C3KO), dendrites and spines of the dentate gyrus as well as memory abilities were preserved. Altogether, these data highlight the central role of microglial C3 in early hippocampal neurodegeneration and memory impairment in EAE and, therefore, pave the way toward new neuroprotective strategies in MS to prevent cognitive deficit using complement inhibitors.

Abstract:
OBJECTIVE: To assess the ability of a combination of synaptic CSF biomarkers to separate AD and non-AD disorders and to help in the differential diagnosis between neurocognitive diseases. METHODS: Retrospective cross-sectional monocentric study. All participants explored with CSF assessments for neurocognitive decline were invited to participate. After complete clinical and imaging evaluations, 243 patients were included. CSF synaptic (GAP-43, neurogranin, SNAP-25 total, SNAP-25 aa40, synaptotagmin-1) and AD biomarkers were blindly quantified using ELISA or mass spectrometry. Statistical analysis compared CSF levels between various groups AD dementias n=81, MCI-AD n=30, other MCI n=49, other dementias (OD) n=49, neurological controls n=35) as well as their discriminatory powers. RESULTS: All synaptic biomarkers were significantly increased in MCI-AD and AD -dementias patients compared to other groups. All synaptic biomarkers could efficiently discriminate AD dementias from OD (AUC >/=0.80). All but synaptotagmin were also able to discriminate MCI-AD from controls (AUC >/=0.85) and AD dementias from controls (AUC >/=0.80). Overall, CSF SNAP 25aa40 had the highest discriminative power (AUC=0.93) between AD dementias and controls or OD, and AUC=0.90 between MCI-AD and controls. Higher levels were associated with two alleles of apolipoprotein E (APOE) epsilon4. CONCLUSION: All synaptic biomarkers tested had a good discriminatory power to distinguish patients with AD abnormal CSF from non-AD disorders. SNAP25aa40 demonstrated the highest power to discriminate AD CSF positive patients from non-AD patients and neurological controls in this cohort. CLASSIFICATION OF EVIDENCE: This retrospective study provides Class II evidence that CSF synaptic biomarkers discriminate patients with AD from non-AD patients.

Abstract:
Brain lesions in Alzheimer's disease (AD) include amyloid plaques made of Abeta peptides and neurofibrillary tangles composed of hyperphosphorylated tau protein with synaptic and neuronal loss and neuroinflammation. Abeta oligomers can trigger tau phosphorylation and neuronal alterations through activation of neuronal kinases leading to progressive cognitive decline. PKR is a ubiquitous pro-apoptotic serine/threonine kinase, and levels of activated PKR are increased in AD brains and AD CSF. In addition, PKR regulates negatively memory formation in mice. To assess the role of PKR in an AD in vivo model, we crossed 5xFAD transgenic mice with PKR knockout (PKRKO) mice and we explored the contribution of PKR on cognition and brain lesions in the 5xFAD mouse model of AD as well as in neuron-microglia co-cultures exposed to the innate immunity activator lipopolysaccharide (LPS). Nine-month-old double-mutant mice revealed significantly improved memory consolidation with the new object location test, starmaze test, and elevated plus maze test as compared to 5xFAD mice. Brain amyloid accumulation and BACE1 levels were statistically decreased in double-mutant mice. Apoptosis, neurodegeneration markers, and synaptic alterations were significantly reduced in double-mutant mice as well as neuroinflammation markers such as microglial load and brain cytokine levels. Using cocultures, we found that PKR in neurons was essential for LPS microglia-induced neuronal death. Our results demonstrate the clear involvement of PKR in abnormal spatial memory and brain lesions in the 5xFAD model and underline its interest as a target for neuroprotection in AD.

Abstract:
Cerebral autosomal dominant arteriolopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is the most common form of hereditary small vessel disease (SVD) of the brain. Neuronal apoptosis has been demonstrated in the cortex of patients. Whether it is associated with an activation of the pro-apoptotic protein PKR pathway is unknown. Similarly, activation of autophagy in CADASIL has never been explored. Immunostaining of four CADASIL brains previously analyzed for cortical neuronal apoptosis and five control brains for PKR (phosphoPKR) and autophagy (ATG5, LC3II) activation markers. Significant nuclear pPKR staining was observed in CADASIL neurons comparatively to controls (p = 0.001). No difference was observed between patients and controls with autophagy markers. We demonstrated the activation of PKR pathway in CADASIL. This was not associated with a detectable modulation of autophagy. These results open a new field to explore in order to better understand the mechanisms underlying cortical neurons apoptosis.

Abstract:
Pyk2 is a Ca(2+)-activated non-receptor tyrosine kinase enriched in forebrain neurons and involved in synaptic regulation. Human genetic studies associated PTK2B, the gene coding Pyk2, with risk for Alzheimer's disease (AD). We previously showed that Pyk2 is important for hippocampal function, plasticity, and spine structure. However, its potential role in AD is unknown. To address this question we used human brain samples and 5XFAD mice, an amyloid mouse model of AD expressing mutated human amyloid precursor protein and presenilin1. In the hippocampus of 5XFAD mice and in human AD patients' cortex and hippocampus, Pyk2 total levels were normal. However, Pyk2 Tyr-402 phosphorylation levels, reflecting its autophosphorylation-dependent activity, were reduced in 5XFAD mice at 8months of age but not 3months. We crossed these mice with Pyk2(-/-) mice to generate 5XFAD animals devoid of Pyk2. At 8months the phenotype of 5XFAD x Pyk2(-/-) double mutant mice was not different from that of 5XFAD. In contrast, overexpression of Pyk2 in the hippocampus of 5XFAD mice, using adeno-associated virus, rescued autophosphorylated Pyk2 levels and improved synaptic markers and performance in several behavioral tasks. Both Pyk2(-/-) and 5XFAD mice showed an increase of potentially neurotoxic Src cleavage product, which was rescued by Pyk2 overexpression. Manipulating Pyk2 levels had only minor effects on Abeta plaques, which were slightly decreased in hippocampus CA3 region of double mutant mice and increased following overexpression. Our results show that Pyk2 is not essential for the pathogenic effects of human amyloidogenic mutations in the 5XFAD mouse model. However, the slight decrease in plaque number observed in these mice in the absence of Pyk2 and their increase following Pyk2 overexpression suggest a contribution of this kinase in plaque formation. Importantly, a decreased function of Pyk2 was observed in 5XFAD mice, indicated by its decreased autophosphorylation and associated Src alterations. Overcoming this deficit by Pyk2 overexpression improved the behavioral and molecular phenotype of 5XFAD mice. Thus, our results in a mouse model of AD suggest that Pyk2 impairment may play a role in the symptoms of the disease.

Abstract:
Metabolic disorders including obesity and type 2 diabetes are known to be associated with chronic inflammation and are obvious risk factors for Alzheimer's disease. Recent evidences concerning obesity and diabetes suggest that the metabolic inflammasome ('metaflammasome') mediates chronic inflammation. The double-stranded RNA-dependent protein kinase (PKR) is a central component of the metaflammasome. In wild type (WT) and PKR-/- mice, blood glucose, insulin and lipid levels and the brain expression of the phosphorylated components of the metaflammasome-PKR, JNK, IRS1 and IKKbeta-were studied after the induction of obesity by a high fat diet (HFD). The results showed significant increased levels of activated brain metaflammasome proteins in exposed WT mice but the changes were not significant in PKR-/- mice. In addition, gain weight was observed in WT mice and also in PKR-/- mice exposed to HFD. Increased blood insulin level was more accentuated in PKR -/- mice. The modulation of PKR activity could be an appropriate therapeutic approach, aimed at reducing abnormal brain metabolism and inflammation linked to metabolic disorders in order to reduce the risk of neurodegeneration.

Abstract:
Alzheimer's disease (AD) is characterized by accumulations of amyloid-beta (Abeta42) and hyperphosphorylated tau proteins, associated with neuroinflammation, synaptic loss, and neuronal death. Several studies indicate that c-Jun N-terminal kinase (JNK) is implicated in the pathological features of AD. We have investigated in 5XFAD mice, the therapeutic effects of Brimapitide, a JNK-specific inhibitory peptide previously tested with higher concentrations in another AD model (TgCRND8). Three-month-old 5XFAD and wild-type littermate mice were treated by intravenous injections of low doses (10 mg/kg) of Brimapitide every 3 weeks, for 3 or 6 months (n = 6-9 per group). Cognitive deficits and brain lesions were assessed using Y-maze, fear-conditioning test, and histological and biochemical methods. Chronic treatment of Brimapitide for 3 months resulted in a reduction of Abeta plaque burden in the cortex of 5XFAD treated mice. After 6 months of treatment, cognitive deficits were reduced but also a significant reduction of cell death markers and the pro-inflammatory IL-1beta cytokine in treated mice were detected. The Abeta plaque burden was not anymore modified by the 6 months of treatment. In addition to modulating cognition and amyloid plaque accumulation, depending on the treatment duration, Brimapitide seems experimentally to reduce neuronal stress in 5XFAD mice.

Abstract:
PKR is a cellular kinase involved in the regulation of the integrative stress response (ISR) and pro-inflammatory pathways. Two N-terminal dsRNA Binding Domains (DRBD) are required for activation of PKR, by interaction with either dsRNA or PACT, another cellular DRBD-containing protein. A role for PKR and PACT in inflammatory processes linked to neurodegenerative diseases has been proposed and raised interest for pharmacological PKR inhibitors. However, the role of PKR in inflammation is subject to controversy. We identified the flavonoid luteolin as an inhibitor of the PKR/PACT interaction at the level of their DRBDs using high-throughput screening of chemical libraries by homogeneous time-resolved fluorescence. This was further validated using NanoLuc-Based Protein Complementation Assay. Luteolin inhibits PKR phosphorylation, the ISR and the induction of pro-inflammatory cytokines in human THP1 macrophages submitted to oxidative stress and toll-like receptor (TLR) agonist. Similarly, luteolin inhibits induction of pro-inflammatory cytokines in murine microglial macrophages. In contrast, luteolin increased activation of the inflammasome, in a PKR-independent manner. Collectively, these data delineate the importance of PKR in the inflammation process to the ISR and induction of pro-inflammatory cytokines. Pharmacological inhibitors of PKR should be used in combination with drugs targeting directly the inflammasome.

Abstract:
Alzheimer's disease (AD) is a neurodegenerative disorder, marked by senile plaques composed of amyloid-beta (Abeta) peptide, neurofibrillary tangles, neuronal loss and neuroinflammation. Previous works have suggested that systemic inflammation could contribute to neuroinflammation and enhanced Abeta cerebral concentrations. The molecular pathways leading to these events are not fully understood. PKR is a pro-apoptotic kinase that can trigger inflammation and accumulates in the brain and cerebrospinal fluid of AD patients. The goal of the present study was to assess if LPS-induced neuroinflammation and Abeta production could be altered by genetic PKR down regulation. The results show that, in the hippocampus of LPS-injected wild type mice, neuroinflammation, cytokine release and Abeta production are significantly increased and not in LPS-treated PKR knock-out mice. In addition BACE1 and activated STAT3 levels, a putative transcriptional regulator of BACE1, were not found increased in the brain of PKR knock-out mice as observed in wild type mice. Using PET imaging, the decrease of hippocampal metabolism induced by systemic LPS was not observed in LPS-treated PKR knock-out mice. Altogether, these findings demonstrate that PKR plays a major role in brain changes induced by LPS and could be a valid target to modulate neuroinflammation and Abeta production.