Conditional deletion of KCC2 impairs synaptic plasticity and both spatial and nonspatial memory.

The postsynaptic inhibition through GABAA receptors (GABAAR) relies on two mechanisms, a shunting effect due to an increase in the postsynaptic membrane conductance and, in mature neurons, a hyperpolarization effect due to an entry of chloride into postsynaptic neurons. The second effect requires the action of the K+-Cl- cotransporter KCC2 which extrudes Cl- from the cell and maintains its cytosolic concentration very low. Neuronal chloride equilibrium seems to be dysregulated in several neurological and psychiatric conditions such as epilepsy, anxiety, schizophrenia, Down syndrome, or Alzheimer's disease. In the present study, we used the KCC2 Cre-lox knockdown system to investigate the role of KCC2 in synaptic plasticity and memory formation in adult mice. Tamoxifen-induced conditional deletion of KCC2 in glutamatergic neurons of the forebrain was performed at 3 months of age and resulted in spatial and nonspatial learning impairment. On brain slices, the stimulation of Schaffer collaterals by a theta burst induced long-term potentiation (LTP). The lack of KCC2 did not affect potentiation of field excitatory postsynaptic potentials (fEPSP) measured in the stratum radiatum (dendrites) but increased population spike (PS) amplitudes measured in the CA1 somatic layer, suggesting a reinforcement of the EPSP-PS potentiation, i.e., an increased ability of EPSPs to generate action potentials. At the cellular level, KCC2 deletion induced a positive shift in the reversal potential of GABAAR-driven Cl- currents (EGABA), suggesting an intracellular accumulation of chloride subsequent to the downregulation of KCC2. After treatment with bumetanide, an antagonist of the Na+-K+-Cl- cotransporter NKCC1, spatial memory impairment, chloride accumulation, and EPSP-PS potentiation were rescued in mice lacking KCC2. The presented results emphasize the importance of chloride equilibrium and GABA-inhibiting ability in synaptic plasticity and memory formation.

Overexpression of wild-type human amyloid precursor protein alters GABAergic transmission.

The function of the amyloid precursor protein (APP) is not fully understood, but its cleavage product amyloid beta (Aß) together with neurofibrillary tangles constitute the hallmarks of Alzheimer's disease (AD). Yet, imbalance of excitatory and inhibitory neurotransmission accompanied by loss of synaptic functions, has been reported much earlier and independent of any detectable pathological markers. Recently, soluble APP fragments have been shown to bind to presynaptic GABAB receptors (GABABRs), subsequently decreasing the probability of neurotransmitter release. In this body of work, we were able to show that overexpression of wild-type human APP in mice (hAPPwt) causes early cognitive impairment, neuronal loss, and electrophysiological abnormalities in the absence of amyloid plaques and at very low levels of Aß. hAPPwt mice exhibited neuronal overexcitation that was evident in EEG and increased long-term potentiation (LTP). Overexpression of hAPPwt did not alter GABAergic/glutamatergic receptor components or GABA production ability. Nonetheless, we detected a decrease of GABA but not glutamate that could be linked to soluble APP fragments, acting on presynaptic GABABRs and subsequently reducing GABA release. By using a specific presynaptic GABABR antagonist, we were able to rescue hyperexcitation in hAPPwt animals. Our results provide evidence that APP plays a crucial role in regulating inhibitory neurotransmission.

Regulation of PPARα by APP in Alzheimer disease affects the pharmacological modulation of synaptic activity.

Among genetic susceptibility loci associated with late-onset Alzheimer disease (LOAD), genetic polymorphisms identified in genes encoding lipid carriers led to the hypothesis that a disruption of lipid metabolism could promote disease progression. We previously reported that amyloid precursor protein (APP) involved in Alzheimer disease (AD) physiopathology impairs lipid synthesis needed for cortical networks' activity and that activation of peroxisome proliferator-activated receptor α (PPARα), a metabolic regulator involved in lipid metabolism, improves synaptic plasticity in an AD mouse model. These observations led us to investigate a possible correlation between PPARα function and full-length APP expression. Here, we report that PPARα expression and activation were inversely related to APP expression both in LOAD brains and in early-onset AD cases with a duplication of the APP gene, but not in control human brains. Moreover, human APP expression decreased PPARA expression and its related target genes in transgenic mice and in cultured cortical cells, while opposite results were observed in APP-silenced cortical networks. In cultured neurons, APP-mediated decrease or increase in synaptic activity was corrected by a PPARα-specific agonist and antagonist, respectively. APP-mediated control of synaptic activity was abolished following PPARα deficiency, indicating a key function of PPARα in this process.

CSF1R inhibition rescues tau pathology and neurodegeneration in an A/T/N model with combined AD pathologies, while preserving plaque associated microglia.

Alzheimer's disease (AD) is characterized by a sequential progression of amyloid plaques (A), neurofibrillary tangles (T) and neurodegeneration (N), constituting ATN pathology. While microglia are considered key contributors to AD pathogenesis, their contribution in the combined presence of ATN pathologies remains incompletely understood. As sensors of the brain microenvironment, microglial phenotypes and contributions are importantly defined by the pathologies in the brain, indicating the need for their analysis in preclinical models that recapitulate combined ATN pathologies, besides their role in A and T models only. Here, we report a new tau-seed model in which amyloid pathology facilitates bilateral tau propagation associated with brain atrophy, thereby recapitulating robust ATN pathology. Single-cell RNA sequencing revealed that ATN pathology exacerbated microglial activation towards disease-associated microglia states, with a significant upregulation of Apoe as compared to amyloid-only models (A). Importantly, Colony-Stimulating Factor 1 Receptor inhibition preferentially eliminated non-plaque-associated versus plaque associated microglia. The preferential depletion of non-plaque-associated microglia significantly attenuated tau pathology and neuronal atrophy, indicating their detrimental role during ATN progression. Together, our data reveal the intricacies of microglial activation and their contributions to pathology in a model that recapitulates the combined ATN pathologies of AD. Our data may provide a basis for microglia-targeting therapies selectively targeting detrimental microglial populations, while conserving protective populations.

Alzheimer's Disease, a Lipid Story: Involvement of Peroxisome Proliferator-Activated Receptor α.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Mutations in genes encoding proteins involved in amyloid-ß peptide (Aß) production are responsible for inherited AD cases. The amyloid cascade hypothesis was proposed to explain the pathogeny. Despite the fact that Aß is considered as the main culprit of the pathology, most clinical trials focusing on Aß failed and suggested that earlier interventions are needed to influence the course of AD. Therefore, identifying risk factors that predispose to AD is crucial. Among them, the epsilon 4 allele of the apolipoprotein E gene that encodes the major brain lipid carrier and metabolic disorders such as obesity and type 2 diabetes were identified as AD risk factors, suggesting that abnormal lipid metabolism could influence the progression of the disease. Among lipids, fatty acids (FAs) play a fundamental role in proper brain function, including memory. Peroxisome proliferator-activated receptor α (PPARα) is a master metabolic regulator that regulates the catabolism of FA. Several studies report an essential role of PPARα in neuronal function governing synaptic plasticity and cognition. In this review, we explore the implication of lipid metabolism in AD, with a special focus on PPARα and its potential role in AD therapy.

Amyloid Precursor Protein (APP) Controls the Expression of the Transcriptional Activator Neuronal PAS Domain Protein 4 (NPAS4) and Synaptic GABA Release.

The amyloid precursor protein (APP) has been extensively studied as the precursor of the ß-amyloid (Aß) peptide, the major component of the senile plaques found in the brain of Alzheimer's disease (AD) patients. However, the function of APP per se in neuronal physiology remains to be fully elucidated. APP is expressed at high levels in the brain. It resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell-cell interaction and/or activation of intracellular signaling pathways. In this respect, the APP intracellular domain (AICD) was reported to act as a transcriptional regulator. Here, we used a transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and on maturation of primary cortical neurons. Surprisingly, the overall transcriptional changes were subtle, but a more detailed analysis pointed to genes clustered in neuronal-activity dependent pathways. In particular, we observed a decreased transcription of neuronal PAS domain protein 4 (NPAS4) in APP-/- neurons. NPAS4 is an inducible transcription factor (ITF) regulated by neuronal depolarization. The downregulation of NPAS4 co-occurs with an increased production of the inhibitory neurotransmitter GABA and a reduced expression of the GABAA receptors α1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABAA receptors activity, but long-term potentiation (LTP) measurement supported an increased GABA component in synaptic transmission of APP-/- mice. Together, NPAS4 appears to be a downstream target involved in APP-dependent regulation of inhibitory synaptic transmission.

Sex-regulated gene dosage effect of PPARα on synaptic plasticity.

Mechanisms driving cognitive improvements following nuclear receptor activation are poorly understood. The peroxisome proliferator-activated nuclear receptor alpha (PPARα) forms heterodimers with the nuclear retinoid X receptor (RXR). We report that PPARα mediates the improvement of hippocampal synaptic plasticity upon RXR activation in a transgenic mouse model with cognitive deficits. This improvement results from an increase in GluA1 subunit expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, eliciting an AMPA response at the excitatory synapses. Associated with a two times higher PPARα expression in males than in females, we show that male, but not female, PPARα null mutants display impaired hippocampal long-term potentiation. Moreover, PPARα knockdown in the hippocampus of cognition-impaired mice compromises the beneficial effects of RXR activation on synaptic plasticity only in males. Furthermore, selective PPARα activation with pemafibrate improves synaptic plasticity in male cognition-impaired mice, but not in females. We conclude that striking sex differences in hippocampal synaptic plasticity are observed in mice, related to differences in PPARα expression levels.

Aggregated Tau activates NLRP3-ASC inflammasome exacerbating exogenously seeded and non-exogenously seeded Tau pathology in vivo.

Brains of Alzheimer's disease patients are characterized by the presence of amyloid plaques and neurofibrillary tangles, both invariably associated with neuroinflammation. A crucial role for NLRP3-ASC inflammasome [NACHT, LRR and PYD domains-containing protein 3 (NLRP3)-Apoptosis-associated speck-like protein containing a CARD (ASC)] in amyloid-beta (Aß)-induced microgliosis and Aß pathology has been unequivocally identified. Aß aggregates activate NLRP3-ASC inflammasome (Halle et al. in Nat Immunol 9:857-865, 2008) and conversely NLRP3-ASC inflammasome activation exacerbates amyloid pathology in vivo (Heneka et al. in Nature 493:674-678, 2013), including by prion-like ASC-speck cross-seeding (Venegas et al. in Nature 552:355-361, 2017). However, the link between inflammasome activation, as crucial sensor of innate immunity, and Tau remains unexplored. Here, we analyzed whether Tau aggregates acting as prion-like Tau seeds can activate NLRP3-ASC inflammasome. We demonstrate that Tau seeds activate NLRP3-ASC-dependent inflammasome in primary microglia, following microglial uptake and lysosomal sorting of Tau seeds. Next, we analyzed the role of inflammasome activation in prion-like or templated seeding of Tau pathology and found significant inhibition of exogenously seeded Tau pathology by ASC deficiency in Tau transgenic mice. We furthermore demonstrate that chronic intracerebral administration of the NLRP3 inhibitor, MCC950, inhibits exogenously seeded Tau pathology. Finally, ASC deficiency also decreased non-exogenously seeded Tau pathology in Tau transgenic mice. Overall our findings demonstrate that Tau-seeding competent, aggregated Tau activates the ASC inflammasome through the NLRP3-ASC axis, and we demonstrate an exacerbating role of the NLRP3-ASC axis on exogenously and non-exogenously seeded Tau pathology in Tau mice in vivo. The NLRP3-ASC inflammasome, which is an important sensor of innate immunity and intensively explored for its role in health and disease, hence presents as an interesting therapeutic approach to target three crucial pathogenetic processes in AD, including prion-like seeding of Tau pathology, Aß pathology and neuroinflammation.

Influence of the familial Alzheimer's disease-associated T43I mutation on the transmembrane structure and γ-secretase processing of the C99 peptide.

Extracellular deposition of ß-amyloid (Aß) peptides in the brain is a hallmark of Alzheimer's disease (AD). Upon ß-secretase-mediated cleavage of the ß C-terminal fragment (ß-CTF) from the Aß precursor protein, the γ-secretase complex produces the Aß peptides associated with AD. The familial T43I mutation within the transmembrane domain of the ß-CTF (also referred to as C99) increases the ratio between the Aß42 and Aß40 peptides largely due to a decrease in Aß40 formation. Aß42 is the principal component of amyloid deposits within the brain parenchyma, and an increase in the Aß42/Aß40 ratio is correlated with early-onset AD. Using NMR and FTIR spectroscopy, here we addressed how the T43I substitution influences the structure of C55, the minimal sequence containing the entire extracellular and transmembrane (TM) domains of C99 needed for γ-secretase processing. 13C NMR chemical shifts indicated that the T43I substitution increases helical structure within the TM domain of C55. These structural changes were associated with a shift of the C55 dimer to the monomer and an increase in the tilt of the TM helix relative to the membrane normal in the T43I mutant compared with that of WT C55. The A21G (Flemish) mutation was previously found to increase secreted Aß40 levels; here, we combined this mutation in the extracellular domain of C99 with T43I and observed that the T43I/A21G double mutant decreases Aß40 formation. We discuss how the observed structural changes in the T43I mutant may decrease Aß40 formation and increase the Aß42/Aß40 ratio.

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.

A Role for GDNF and Soluble APP as Biomarkers of Amyotrophic Lateral Sclerosis Pathophysiology.

The current inability of clinical criteria to accurately identify the "at-risk group" for Amyotrophic Lateral Sclerosis (ALS) development as well as its unknown etiology are fueling the interest in biomarkers aimed at completing clinical approaches for the diagnosis. The Glial cell line-derived neurotrophic factor (GDNF) is a diffusible peptide critically involved in neuronal differentiation and survival. GDNF is largely studied in various neurological and neuromuscular diseases, with a great interest in the peripheral nervous system (PNS). The recent discovery of Amyloid Precursor Protein (APP)-dependent GDNF regulation driving neuro-muscular junctions' formation in APP null transgenic mice, prompts to study whether neurodegeneration relies on loss or gain of APP function and suggests that it could affect peripheral processes. Here, we explored a brand-new aspect of the loss of trophic support in ALS by measuring GDNF, APP, soluble APP fragments and Aß peptides levels in SOD1WT or SOD1G93A transgenic mouse models of ALS and in human biological fluids [i.e. serum and cerebrospinal fluid (CSF)] from ALS patients and control subjects. Our results show that both GDNF and soluble APP fragments levels are altered at the onset of motor deficits in mice and that their levels are also modified in patient samples. This study indicates that both GDNF and soluble APPα represent possible biomarkers for ALS.

ß-Sheet Structure within the Extracellular Domain of C99 Regulates Amyloidogenic Processing.

Familial mutations in C99 can increase the total level of the soluble Aß peptides produced by proteolysis, as well as the Aß42/Aß40 ratio, both of which are linked to the progression of Alzheimer's disease. We show that the extracellular sequence of C99 forms ß-sheet structure upon interaction with membrane bilayers. Mutations that disrupt this structure result in a significant increase in Aß production and, in specific cases, result in an increase in the amount of Aß42 relative to Aß40. Fourier transform infrared and solid-state NMR spectroscopic studies reveal a central ß-hairpin within the extracellular sequence comprising Y10-E11-V12 and L17-V18-F19 connected by a loop involving H13-H14-Q15. These results suggest how familial mutations in the extracellular sequence influence C99 processing and provide a structural basis for the development of small molecule modulators that would reduce Aß production.

Presenilin 2-Dependent Maintenance of Mitochondrial Oxidative Capacity and Morphology.

Mitochondrial dysfunction plays a pivotal role in the progression of Alzheimer's disease (AD), and yet the mechanisms underlying the impairment of mitochondrial function in AD remain elusive. Recent evidence suggested a role for Presenilins (PS1 or PS2) in mitochondrial function. Mutations of PSs, the catalytic subunits of the γ-secretase complex, are responsible for the majority of inherited AD cases (FAD). PSs were shown to be present in mitochondria and particularly enriched in mitochondria-associated membranes (MAM), where PS2 is involved in the calcium shuttling between mitochondria and the endoplasmic reticulum (ER). We investigated the precise contribution of PS1 and PS2 to the bioenergetics of the cell and to mitochondrial morphology in cell lines derived from wild type (PS+/+), PS1/2 double knock-out (PSdKO), PS2KO and PS1KO embryos. Our results showed a significant impairment in the respiratory capacity of PSdKO and PS2KO cells with reduction of basal oxygen consumption, oxygen utilization dedicated to ATP production and spare respiratory capacity. In line with these functional defects, we found a decrease in the expression of subunits responsible for mitochondrial oxidative phosphorylation (OXPHOS) associated with an altered morphology of the mitochondrial cristae. This OXPHOS disruption was accompanied by a reduction of the NAD+/NADH ratio. Still, neither ADP/ATP ratio nor mitochondrial membrane potential (ΔΨ) were affected, suggesting the existence of a compensatory mechanism for energetic balance. We observed indeed an increase in glycolytic flux in PSdKO and PS2KO cells. All these effects were truly dependent on PS2 since its stable re-expression in a PS2KO background led to a complete restoration of the parameters impaired in the absence of PS2. Our data clearly demonstrate here the crucial role of PS2 in mitochondrial function and cellular bioenergetics, pointing toward its peculiar role in the formation and integrity of the electron transport chain.

Amyloid precursor protein reduction enhances the formation of neurofibrillary tangles in a mutant tau transgenic mouse model.

Alzheimer's disease is characterized by the presence of 2 neuropathological lesions: neurofibrillary tangles, composed of tau proteins which are highly phosphorylated and phosphorylated on uncommon sites, and amyloid plaques, containing the Aß peptides generated from the amyloid precursor protein (APP). Reduction of some APP proteolytic derivatives in Alzheimer's disease such as sAPPα fragment has been reported and sAPPα has been shown to affect tau phosphorylation. To investigate in vivo the effect of absence of APP protein and its fragments on tau phosphorylation and the formation of neurofibrillary tangles, we have generated mice deleted for APP gene and overexpressing a human mutant tau protein and developing neurofibrillary tangles (APPKOTg30 mice). These APPKOTg30 mice showed more severe motor and cognitive deficits, increased tau phosphorylation, increased load of neurofibrillary tangles, and increased p25/35 ratio in the brain, compared with Tg30 mice. These data suggest that APP and/or its proteolytic derivatives interfere with the formation of neurofibrillary tangles in a transgenic mouse model that will be useful for investigating the relationship between APP and tau.

Cortical cells reveal APP as a new player in the regulation of GABAergic neurotransmission.

The amyloid precursor protein (APP) modulates synaptic activity, resulting from the fine tuning of excitatory and inhibitory neurotransmission. GABAergic inhibitory neurotransmission is affected by modifications in intracellular chloride concentrations regulated by Na+-K+-2Cl- cotransporter 1 (NKCC1) and neuronal K+-Cl- cotransporter 2 (KCC2), allowing entrance and efflux of chloride, respectively. Modifications in NKCC1 and KCC2 expression during maturation of cortical cells induce a shift in GABAergic signaling. Here, we demonstrated that APP affects this GABA shift. Expression of APP in cortical cells decreased the expression of KCC2, without modifying NKCC1, eliciting a less inhibitory GABA response. Downregulation of KCC2 expression by APP was independent of the APP intracellular domain, but correlated with decreased expression of upstream stimulating factor 1 (USF1), a potent regulator of Slc12a5 gene expression (encoding KCC2). KCC2 was also downregulated in vivo following APP expression in neonatal mouse brain. These results argue for a key role of APP in the regulation of GABAergic neurotransmission.

Tau interactome mapping based identification of Otub1 as Tau deubiquitinase involved in accumulation of pathological Tau forms in vitro and in vivo.

Dysregulated proteostasis is a key feature of a variety of neurodegenerative disorders. In Alzheimer's disease (AD), progression of symptoms closely correlates with spatiotemporal progression of Tau aggregation, with "early" oligomeric Tau forms rather than mature neurofibrillary tangles (NFTs) considered to be pathogenetic culprits. The ubiquitin-proteasome system (UPS) controls degradation of soluble normal and abnormally folded cytosolic proteins. The UPS is affected in AD and is identified by genomewide association study (GWAS) as a risk pathway for AD. The UPS is determined by balanced regulation of ubiquitination and deubiquitination. In this work, we performed isobaric tags for relative and absolute quantitation (iTRAQ)-based Tau interactome mapping to gain unbiased insight into Tau pathophysiology and to identify novel Tau-directed therapeutic targets. Focusing on Tau deubiquitination, we here identify Otub1 as a Tau-deubiquitinating enzyme. Otub1 directly affected Lys48-linked Tau deubiquitination, impairing Tau degradation, dependent on its catalytically active cysteine, but independent of its noncanonical pathway modulated by its N-terminal domain in primary neurons. Otub1 strongly increased AT8-positive Tau and oligomeric Tau forms and increased Tau-seeded Tau aggregation in primary neurons. Finally, we demonstrated that expression of Otub1 but not its catalytically inactive form induced pathological Tau forms after 2 months in Tau transgenic mice in vivo, including AT8-positive Tau and oligomeric Tau forms. Taken together, we here identified Otub1 as a Tau deubiquitinase in vitro and in vivo, involved in formation of pathological Tau forms, including small soluble oligomeric forms. Otub1 and particularly Otub1 inhibitors, currently under development for cancer therapies, may therefore yield interesting novel therapeutic avenues for Tauopathies and AD.

Glycines from the APP GXXXG/GXXXA Transmembrane Motifs Promote Formation of Pathogenic Aß Oligomers in Cells.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline leading to dementia. The amyloid precursor protein (APP) is a ubiquitous type I transmembrane (TM) protein sequentially processed to generate the ß-amyloid peptide (Aß), the major constituent of senile plaques that are typical AD lesions. There is a growing body of evidence that soluble Aß oligomers correlate with clinical symptoms associated with the disease. The Aß sequence begins in the extracellular juxtamembrane region of APP and includes roughly half of the TM domain. This region contains GXXXG and GXXXA motifs, which are critical for both TM protein interactions and fibrillogenic properties of peptides derived from TM α-helices. Glycine-to-leucine mutations of these motifs were previously shown to affect APP processing and Aß production in cells. However, the detailed contribution of these motifs to APP dimerization, their relation to processing, and the conformational changes they can induce within Aß species remains undefined. Here, we describe highly resistant Aß42 oligomers that are produced in cellular membrane compartments. They are formed in cells by processing of the APP amyloidogenic C-terminal fragment (C99), or by direct expression of a peptide corresponding to Aß42, but not to Aß40. By a point-mutation approach, we demonstrate that glycine-to-leucine mutations in the G(29)XXXG(33) and G(38)XXXA(42) motifs dramatically affect the Aß oligomerization process. G33 and G38 in these motifs are specifically involved in Aß oligomerization; the G33L mutation strongly promotes oligomerization, while G38L blocks it with a dominant effect on G33 residue modification. Finally, we report that the secreted Aß42 oligomers display pathological properties consistent with their suggested role in AD, but do not induce toxicity in survival assays with neuronal cells. Exposure of neurons to these Aß42 oligomers dramatically affects neuronal differentiation and, consequently, neuronal network maturation.

Activation of phagocytic activity in astrocytes by reduced expression of the inflammasome component ASC and its implication in a mouse model of Alzheimer disease.

BACKGROUND: The proinflammatory cytokine interleukin-1ß (IL-1ß) is overexpressed in Alzheimer disease (AD) as a key regulator of neuroinflammation. Amyloid-ß (Aß) peptide triggers activation of inflammasomes, protein complexes responsible for IL-1ß maturation in microglial cells. Downregulation of NALP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome has been shown to decrease amyloid load and rescue cognitive deficits in a mouse model of AD. Whereas activation of inflammasome in microglial cells has been described in AD, no data are currently available concerning activation of inflammasome in astrocytes, although they are involved in inflammatory response and phagocytosis. Here, by targeting the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD domain), we investigated the influence of activation of the inflammasome on the phagocytic activity of astrocytes. METHODS: We used an ASC knockout mouse model, as ASC is a central protein in the inflammasome, acting as an adaptor and stabilizer of the complex and thus critical for its activation. Lipopolysaccharide (LPS)-primed primary cultures of astrocytes from newborn mice were utilized to evaluate Aß-induced inflammasome activation by measuring IL-1ß release by ECLIA (electro-chemiluminescence immunoassay). Phagocytosis efficiency was measured by incorporation of bioparticles, and the release of the chemokine CCL3 (C-C motif ligand 3) was measured by ECLIA. ASC mice were crossbred with 5xFAD (familial Alzheimer disease) mice and tested for spatial reference memory using the Morris water maze (MWM) at 7-8 months of age. Amyloid load and CCL3 were quantified by thioflavine S staining and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RESULTS: Cultured astrocytes primed with LPS and treated with Aß showed an ASC-dependent production of IL-1ß resulting from inflammasome activation mediated by Aß phagocytosis and cathepsin B enzymatic activity. ASC+/- astrocytes displayed a higher phagocytic activity as compared to ASC+/+ and ASC -/- cells, resulting from a higher release of the chemokine CCL3. A significant decrease in amyloid load was measured in the brain of 7-8-month-old 5xFAD mice carrying the ASC +/- genotype, correlated with an increase in CCL3 gene expression. In addition, the ASC +/- genotype rescued spatial reference memory deficits observed in 5xFAD mice. CONCLUSIONS: Our results demonstrate that Aß is able to activate astrocytic inflammasome. Downregulation of inflammasome activity increases phagocytosis in astrocytes due to the release of CCL3. This could explain why downregulation of inflammasome activity decreases amyloid load and rescues memory deficits in a mouse model of AD.

Heterotypic seeding of Tau fibrillization by pre-aggregated Abeta provides potent seeds for prion-like seeding and propagation of Tau-pathology in vivo.

Genetic, clinical, histopathological and biomarker data strongly support Beta-amyloid (Aß) induced spreading of Tau-pathology beyond entorhinal cortex (EC), as a crucial process in conversion from preclinical cognitively normal to Alzheimer's Disease (AD), while the underlying mechanism remains unclear. In vivo preclinical models have reproducibly recapitulated Aß-induced Tau-pathology. Tau pathology was thereby also induced by aggregated Aß, in functionally connected brain areas, reminiscent of a prion-like seeding process. In this work we demonstrate, that pre-aggregated Aß can directly induce Tau fibrillization by cross-seeding, in a cell-free assay, comparable to that demonstrated before for alpha-synuclein and Tau. We furthermore demonstrate, in a well-characterized cellular Tau-aggregation assay that Aß-seeds cross-seeded Tau-pathology and strongly catalyzed pre-existing Tau-aggregation, reminiscent of the pathogenetic process in AD. Finally, we demonstrate that heterotypic seeded Tau by pre-aggregated Aß provides efficient seeds for induction and propagation of Tau-pathology in vivo. Prion-like, heterotypic seeding of Tau fibrillization by Aß, providing potent seeds for propagating Tau pathology in vivo, as demonstrated here, provides a compelling molecular mechanism for Aß-induced propagation of Tau-pathology, beyond regions with pre-existing Tau-pathology (entorhinal cortex/locus coeruleus). Cross-seeding along functional connections could thereby resolve the initial spatial dissociation between amyloid- and Tau-pathology, and preferential propagation of Tau-pathology in regions with pre-existing 'silent' Tau-pathology, by conversion of a 'silent' Tau pathology to a 'spreading' Tau-pathology, observed in AD.