Fibrillar Aβ (beta) triggers microglial proteome alterations and dysfunction in Alzheimer mouse models

Microglial dysfunction is a key pathological feature of Alzheimer's disease (AD), but little is known about proteome-wide changes in microglia during the course of AD and their functional consequences. Here, we performed an in-depth and time-resolved proteomic characterization of microglia in two mouse models of amyloid beta (A beta) pathology, the overexpression APPPS1 and the knock-in APP-NL-G-F (APP-KI) model. We identified a large panel of Microglial A beta Response Proteins (MARPs) that reflect heterogeneity of microglial alterations during early, middle and advanced stages of A beta deposition and occur earlier in the APPPS1 mice. Strikingly, the kinetic differences in proteomic profiles correlated with the presence of fibrillar A beta, rather than dystrophic neurites, suggesting that fibrillar A beta may trigger the AD-associated microglial phenotype and the observed functional decline. The identified microglial proteomic fingerprints of AD provide a valuable resource for functional studies of novel molecular targets and potential biomarkers for monitoring AD progression or therapeutic efficacy

Microbiota-derived short chain fatty acids modulate microglia and promote Aβ plaque deposition.

Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer's disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aβ deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aβ plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aβ plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aβ plaques upon SCFA supplementation, microglia contained less intracellular Aβ. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aβ deposition likely via modulation of the microglial phenotype

Streptozotocin-induced beta-cell damage, high fat diet, and metformin administration regulate Hes3 expression in the adult mouse brain

Diabetes mellitus is a group of disorders characterized by prolonged high levels of circulating blood glucose. Type 1 diabetes is caused by decreased insulin production in the pancreas whereas type 2 diabetes may develop due to obesity and lack of exercise;it begins with insulin resistance whereby cells fail to respond properly to insulin and it may also progress to decreased insulin levels. The brain is an important target for insulin, and there is great interest in understanding how diabetes affects the brain. In addition to the direct effects of insulin on the brain, diabetes may also impact the brain through modulation of the inflammatory system. Here we investigate how perturbation of circulating insulin levels affects the expression of Hes3, a transcription factor expressed in neural stem and progenitor cells that is involved in tissue regeneration. Our data show that streptozotocin-induced beta-cell damage, high fat diet, as well as metformin, a common type 2 diabetes medication, regulate Hes3 levels in the brain. This work suggests that Hes3 is a valuable biomarker helping to monitor the state of endogenous neural stem and progenitor cells in the context of diabetes mellitus

The COP9 signalosome reduces neuroinflammation and attenuates ischemic neuronal stress in organotypic brain slice culture model

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deNEDDylase controlling ubiquitination activity of cullin-RING-E3 ligases (CRLs) and thus the levels of key cellular proteins. While the CSN and its catalytic subunit CSN5 have been extensively studied in cancer, its role in inflammatory and neurological diseases is less understood. Following verification that CSN5 is expressed in mouse and human brain, here we studied the role of the CSN in neuroinflammation and ischemic neuronal damage employing models of relevant brain-resident cell types, an ex vivo organotypic brain slice culture model, and the CRL NEDDylation state-modifying drugs MLN4924 and CSN5i-3, which mimic and inhibit, respectively, CSN5 deNEDDylase activity. Untargeted mass spectrometry-based proteomics revealed that MLN4924 and CSN5i-3 substantially alter the microglial proteome, including inflammation-related proteins. Applying these drugs and mimicking microglial and endothelial inflammation as well as ischemic neuronal stress by TNF and oxygen-glucose-deprivation/reoxygenation (OGD/RO) treatment, respectively, we could link CSN5/CSN-mediated cullin deNEDDylation to reduction of microglial inflammation, attenuated cerebral endothelial inflammation, improved barrier integrity, as well as protection from ischemic stress-induced neuronal cell death. Specifically, MLN4924 reduced phagocytic activity, motility, and inflammatory cytokine expression of microglial cells, and this was linked to inhibition of inflammation-induced NF-& kappa;B and Akt signaling. Inversely, Csn5 knockdown and CSN5i-3 increased NF-& kappa;B signaling. Moreover, MLN4924 abrogated TNF-induced NF-& kappa;B signaling in cerebral microvascular endothelial cells (hCMECs) and rescued hCMEC monolayers from OGD/RO-triggered barrier leakage, while CSN5i-3 exacerbated permeability. In an ex vivo organotypic brain slice model of ischemia/reperfusion stress, MLN4924 protected from neuronal death, while CSN5i-3 impaired neuronal survival. Neuronal damage was attributable to microglial activation and inflammatory cytokines, as indicated by microglial shape tracking and TNF-blocking experiments. Our results indicate a protective role of the CSN in neuroinflammation via brain-resident cell types involved in ischemic brain disease and implicate CSN activity-mimicking deNEDDylating drugs as potential therapeutics

Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia.

Niemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in NPC1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent pathological feature, direct consequences of NPC1 loss on microglial function remain not fully characterized. We discovered pathological proteomic signatures and phenotypes in NPC1-deficient murine models and demonstrate a cell autonomous function of NPC1 in microglia. Loss of NPC1 triggers enhanced phagocytic uptake and impaired myelin turnover in microglia that precede neuronal death. Npc1-/- microglia feature a striking accumulation of multivesicular bodies and impaired trafficking of lipids to lysosomes while lysosomal degradation function remains preserved. Molecular and functional defects were also detected in blood-derived macrophages of NPC patients that provide a potential tool for monitoring disease. Our study underscores an essential cell autonomous role for NPC1 in immune cells and implies microglial therapeutic potential

Human stem cell-derived monocytes and microglia-like cells reveal impaired amyloid plaque clearance upon heterozygous or homozygous loss of TREM2

INTRODUCTION: Murine microglia expressing the Alzheimer's disease-linked TREM2R47H mutation display variable decrease in phagocytosis, while impaired phagocytosis is reported following loss of TREM2. However, no data exist on TREM2+/R47H human microglia. Therefore, we created human pluripotent stem cell (hPSC) monocytes and transdifferentiated microglia-like cells (tMGs) to examine the effect of the TREM2+/R47H mutation and loss of TREM2 on phagocytosis. METHODS: We generated isogenic TREM2+/R47H, TREM2+/-, and TREM2-/- hPSCs using CRISPR/Cas9. Following differentiation to monocytes and tMGs, we studied the uptake of Escherichia coli fragments and analyzed amyloid plaque clearance from cryosections of APP/PS1+/- mouse brains. RESULTS: We demonstrated that tMGs resemble cultured human microglia. TREM2+/- and TREM2-/- hPSC monocytes and tMGs phagocytosed significantly less E. coli fragments and cleared less amyloid plaques than wild-type hPSC progeny, with no difference for TREM2+/R47H progeny. DISCUSSION: In vitro phagocytosis of hPSC monocytes and tMGs was not affected by the TREM2+/R47H mutation but was significantly impaired in TREM2+/- and TREM2-/- progeny.status: publishe

Glitter in the Darkness? Non-fibrillar β-amyloid Plaque Components Significantly Impact the β-amyloid PET Signal in Mouse Models of Alzheimer's Disease.

Objective: β-amyloid PET (Aβ-PET) is an important tool for quantification of amyloidosis in the brain of suspected Alzheimer's disease (AD) patients and transgenic AD mouse models. Despite the excellent correlation of Aβ-PET with gold standard immunohistochemical assessments, the relative contributions of fibrillar and non-fibrillar Aβ components to the in vivo Aβ-PET signal remain unclear. Thus, we obtained two murine cerebral amyloidosis models that present with distinct Aβ plaque compositions and performed regression analysis between immunohistochemistry and Aβ PET to determine the biochemical contributions to Aβ-PET signal in vivo. Methods: We investigated groups of AppNL-G-F and APPPS1 mice at three, six and 12 months of age by longitudinal 18F-florbetaben Aβ-PET and with immunohistochemical analysis of the fibrillar and total Aβ burdens. We then applied group level inter-modality regression models using age and genotype matched sets of fibrillar/ non-fibrillar Aβ data (predictors) and Aβ-PET results (outcome) for both transgenic models. An independent group of double-hit APPPS1 mice with dysfunctional microglia due to knock-out of triggering receptor expression on myeloid cells 2 (Trem2-/-) served for validation and evaluation of translational impact. Results: Neither fibrillar nor non-fibrillar Aβ content alone sufficed to explain the Aβ-PET findings in either transgenic AD model. However, a regression model compiling fibrillar and non-fibrillar Aβ together with the estimate of individual heterogeneity and age at scanning could explain a 93% of variance of the Aβ-PET signal (P<0.001). Fibrillar Aβ burden had a 16-fold higher contribution to the Aβ-PET signal when compared to non-fibrillar Aβ. However, given the relatively greater abundance of non-fibrillar Aβ, we estimate that non-fibrillar Aβ produced 79±25% of the net in vivo Aβ-PET signal in AppNL-G-F mice, and 25±12% in the APPPS1 mice. Corresponding results in separate groups of APPPS1/Trem2-/- and APPPS1/Trem2+/+ mice validated the calculated regression factors and revealed that the altered fibrillarity due to Trem2 knockout impacts the Aβ-PET signal. Conclusion: Taken together, the in vivo Aβ-PET signal derives from the composite of fibrillar and non-fibrillar Aβ plaque components. While fibrillar Aβ has inherently higher PET tracer binding, the greater abundance of non-fibrillar Aβ plaque in AD model mice contributes importantly to the PET signal

Transcriptome sequencing during mouse brain development identifies long non-coding RNAs functionally involved in neurogenic commitment

Transcriptome analysis of somatic stem cells and their progeny is fundamental to identify new factors controlling proliferation versus differentiation during tissue formation. Here, we generated a combinatorial, fluorescent reporter mouse line to isolate proliferating neural stem cells, differentiating progenitors and newborn neurons that coexist as intermingled cell populations during brain development. Transcriptome sequencing revealed numerous novel long non-coding (lnc)RNAs and uncharacterized protein-coding transcripts identifying the signature of neurogenic commitment. Importantly, most lncRNAs overlapped neurogenic genes and shared with them a nearly identical expression pattern suggesting that lncRNAs control corticogenesis by tuning the expression of nearby cell fate determinants. We assessed the power of our approach by manipulating lncRNAs and protein-coding transcripts with no function in corticogenesis reported to date. This led to several evident phenotypes in neurogenic commitment and neuronal survival, indicating that our study provides a remarkably high number of uncharacterized transcripts with hitherto unsuspected roles in brain development. Finally, we focussed on one lncRNA, Miat, whose manipulation was found to trigger pleiotropic effects on brain development and aberrant splicing of Wnt7b. Hence, our study suggests that lncRNA-mediated alternative splicing of cell fate determinants controls stem-cell commitment during neurogenesis. © 2013 European Molecular Biology Organization

Microglia contribute to the propagation of A beta into unaffected brain tissue

Microglia appear activated in the vicinity of amyloid beta (A beta) plaques, but whether microglia contribute to A beta propagation into unaffected brain regions remains unknown. Using transplantation of wild-type (WT) neurons, we show that A beta enters WT grafts, and that this is accompanied by microglia infiltration. Manipulation of microglia function reduced A beta deposition within grafts. Furthermore, in vivo imaging identified microglia as carriers of A beta pathology in previously unaffected tissue. Our data thus argue for a hitherto unexplored mechanism of A beta propagation. This study shows that A beta from transgenic host tissue is able to enter and deposit within wild-type grafts via microglia, thus identifying microglia as carriers of A beta deposition into previously unaffected brain tissue

Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE

Coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with late-onset Alzheimer's disease (AD). We demonstrate that amyloid plaque seeding is increased in the absence of functional Trem2. Increased seeding is accompanied by decreased microglial clustering around newly seeded plaques and reduced plaque-associated apolipoprotein E (ApoE). Reduced ApoE deposition in plaques is also observed in brains of AD patients carrying TREM2 coding variants. Proteomic analyses and microglia depletion experiments revealed microglia as one origin of plaque-associated ApoE. Longitudinal amyloid small animal positron emission tomography demonstrates accelerated amyloidogenesis in Trem2 loss-of-function mutants at early stages, which progressed at a lower rate with aging. These findings suggest that in the absence of functional Trem2, early amyloidogenesis is accelerated due to reduced phagocytic clearance of amyloid seeds despite reduced plaque-associated ApoE