The discovery of TREM2 as a myeloid-specific Alzheimer's disease (AD) risk gene has accelerated research into the role of microglia in AD. While TREM2 mouse models have provided critical insight, the ...normal and disease-associated functions of TREM2 in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we find that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1α/CXCR4-mediated chemotaxis, culminating in an impaired response to beta-amyloid plaques in vivo. Single-cell sequencing of xenotransplanted human microglia further highlights a loss of disease-associated microglial (DAM) responses in human TREM2 knockout microglia that we validate by flow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles in the development and progression of AD.
The P522R variant of PLCG2, expressed by microglia, is associated with reduced risk of Alzheimer's disease (AD). Yet, the impact of this protective mutation on microglial responses to AD pathology ...remains unknown. Chimeric AD and wild‐type mice were generated by transplanting PLCG2‐P522R or isogenic wild‐type human induced pluripotent stem cell microglia. At 7 months of age, single‐cell and bulk RNA sequencing, and histological analyses were performed. The PLCG2‐P522R variant induced a significant increase in microglial human leukocyte antigen (HLA) expression and the induction of antigen presentation, chemokine signaling, and T cell proliferation pathways. Examination of immune‐intact AD mice further demonstrated that the PLCG2‐P522R variant promotes the recruitment of CD8+ T cells to the brain. These data provide the first evidence that the PLCG2‐P522R variant increases the capacity of microglia to recruit T cells and present antigens, promoting a microglial transcriptional state that has recently been shown to be reduced in AD patient brains.
Disease-associated microglia (DAMs), that surround beta-amyloid plaques, represent a transcriptionally-distinct microglial profile in Alzheimer's disease (AD). Activation of DAMs is dependent on ...triggering receptor expressed on myeloid cells 2 (TREM2) in mouse models and the AD TREM2-R47H risk variant reduces microglial activation and plaque association in human carriers. Interestingly, TREM2 has also been identified as a microglial lipid-sensor, and recent data indicates lipid droplet accumulation in aged microglia, that is in turn associated with a dysfunctional proinflammatory phenotype. However, whether lipid droplets (LDs) are present in human microglia in AD and how the R47H mutation affects this remains unknown.
To determine the impact of the TREM2 R47H mutation on human microglial function in vivo, we transplanted wild-type and isogenic TREM2-R47H iPSC-derived microglial progenitors into our recently developed chimeric Alzheimer mouse model. At 7 months of age scRNA-seq and histological analyses were performed.
Here we report that the transcriptome of human wild-type TREM2 and isogenic TREM2-R47H DAM xenografted microglia (xMGs), isolated from chimeric AD mice, closely resembles that of human atherosclerotic foam cells. In addition, much like foam cells, plaque-bound xMGs are highly enriched in lipid droplets. Somewhat surprisingly and in contrast to a recent in vitro study, TREM2-R47H mutant xMGs exhibit an overall reduction in the accumulation of lipid droplets in vivo. Notably, TREM2-R47H xMGs also show overall reduced reactivity to plaques, including diminished plaque-proximity, reduced CD9 expression, and lower secretion of plaque-associated APOE.
Altogether, these results indicate lipid droplet accumulation occurs in human DAM xMGs in AD, but is reduced in TREM2-R47H DAM xMGs, as it occurs secondary to TREM2-mediated changes in plaque proximity and reactivity.
Microglia are strongly implicated in the development and progression of Alzheimer’s disease (AD), yet their impact on pathology and lifespan remains unclear. Here we utilize a CSF1R hypomorphic mouse ...to generate a model of AD that genetically lacks microglia. The resulting microglial-deficient mice exhibit a profound shift from parenchymal amyloid plaques to cerebral amyloid angiopathy (CAA), which is accompanied by numerous transcriptional changes, greatly increased brain calcification and hemorrhages, and premature lethality. Remarkably, a single injection of wild-type microglia into adult mice repopulates the microglial niche and prevents each of these pathological changes. Taken together, these results indicate the protective functions of microglia in reducing CAA, blood-brain barrier dysfunction, and brain calcification. To further understand the clinical implications of these findings, human AD tissue and iPSC-microglia were examined, providing evidence that microglia phagocytose calcium crystals, and this process is impaired by loss of the AD risk gene, TREM2.
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•Absence of microglia in Alzheimer’s disease mice promotes cerebral amyloid angiopathy•Increased vascular amyloid is accompanied by hemorrhages, calcification, and lethality•Adult transplantation of microglia prevents these diverse pathological changes•snRNA-seq reveals the impact of microglial absence and replacement on other cell types
Kiani Shabestari et al. show that genetic absence of microglia in Alzheimer’s disease mice causes a shift from parenchymal amyloid plaques to cerebral amyloid angiopathy, brain calcification and hemorrhages, and premature lethality. Adult microglial transplantation rescues these pathological changes, demonstrating that microglia protect the brain against detrimental Alzheimer’s disease co-pathologies.
Alzheimer disease (AD) is characterized by the accumulation of beta-amyloid (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau, which together lead to neurodegeneration and ...cognitive decline. Current therapeutic approaches have primarily aimed to reduce pathological aggregates of either Aβ or tau, yet phase 3 clinical trials of these approaches have thus far failed to delay disease progression in humans. Strong preclinical evidence indicates that these two abnormally aggregated proteins interact synergistically to drive downstream neurodegeneration. Therefore, combinatorial therapies that concurrently target both Aβ and tau might be needed for effective disease modification.
A combinatorial vaccination approach was designed to concurrently target both Aβ and tau pathologies. Tau22/5xFAD (T5x) bigenic mice that develop both pathological Aβ and tau aggregates were injected intramuscularly with a mixture of two MultiTEP epitope vaccines: AV-1959R and AV-1980R, targeting Aβ and tau, respectively, and formulated in Advax
, a potent polysaccharide adjuvant. Antibody responses of vaccinated animals were measured by ELISA, and neuropathological changes were determined in brain homogenates of vaccinated and control mice using ELISA and Meso Scale Discovery (MSD) multiplex assays.
T5x mice immunized with a mixture of Aβ- and tau-targeting vaccines generated high Aβ- and tau-specific antibody titers that recognized senile plaques and neurofibrillary tangles/neuropil threads in human AD brain sections. Production of these antibodies in turn led to significant reductions in the levels of soluble and insoluble total tau, and hyperphosphorylated tau as well as insoluble Aβ
, within the brains of bigenic T5x mice.
AV-1959R and AV-1980R formulated with Advax
adjuvant are immunogenic and therapeutically potent vaccines that in combination can effectively reduce both of the hallmark pathologies of AD in bigenic mice. Taken together, these findings warrant further development of this vaccine technology for ultimate testing in human AD.
Lysosome dysfunction is a shared feature of rare lysosomal storage diseases and common age-related neurodegenerative diseases. Microglia, the brain-resident macrophages, are particularly vulnerable ...to lysosome dysfunction because of the phagocytic stress of clearing dying neurons, myelin, and debris. CD22 is a negative regulator of microglial homeostasis in the aging mouse brain, and soluble CD22 (sCD22) is increased in the cerebrospinal fluid of patients with Niemann-Pick type C disease (NPC). However, the role of CD22 in the human brain remains unknown. In contrast to previous findings in mice, here, we show that CD22 is expressed by oligodendrocytes in the human brain and binds to sialic acid–dependent ligands on microglia. Using unbiased genetic and proteomic screens, we identify insulin-like growth factor 2 receptor (IGF2R) as the binding partner of sCD22 on human myeloid cells. Targeted truncation of IGF2R revealed that sCD22 docks near critical mannose 6-phosphate–binding domains, where it disrupts lysosomal protein trafficking. Interfering with the sCD22-IGF2R interaction using CD22 blocking antibodies ameliorated lysosome dysfunction in human
mutant induced pluripotent stem cell–derived microglia-like cells without harming oligodendrocytes in vitro. These findings reinforce the differences between mouse and human microglia and provide a candidate microglia-directed immunotherapeutic to treat NPC.
Amyloid-beta impairs TOM1-mediated IL-1R1 signaling Martini, Alessandra Cadete; Gomez-Arboledas, Angela; Forner, Stefania ...
Proceedings of the National Academy of Sciences - PNAS,
10/2019, Letnik:
116, Številka:
42
Journal Article
Recenzirano
Odprti dostop
Defects in interleukin-1β (IL-1β)–mediated cellular responses contribute to Alzheimer’s disease (AD). To decipher the mechanism associated with its pathogenesis, we investigated the molecular events ...associated with the termination of IL-1β inflammatory responses by focusing on the role played by the target of Myb1 (TOM1), a negative regulator of the interleukin-1β receptor-1 (IL-1R1). We first show that TOM1 steady-state levels are reduced in human AD hippocampi and in the brain of an AD mouse model versus respective controls. Experimentally reducing TOM1 affected microglia activity, substantially increased amyloid-beta levels, and impaired cognition, whereas enhancing its levels was therapeutic. These data show that reparation of the TOM1-signaling pathway represents a therapeutic target for brain inflammatory disorders such as AD. A better understanding of the age-related changes in the immune system will allow us to craft therapies to limit detrimental aspects of inflammation, with the broader purpose of sharply reducing the number of people afflicted by AD.
Background
Many ongoing studies for AD immunotherapy are focused on the immunodominant N‐terminus of Aβ. This region is often truncated and post‐translationally modified in amyloid. The pyroglutamate ...modified variant of Aβ40/42 (pE3‐Aβ) is more neurotoxic and aggregation‐prone and is thought to play an important role in disease onset. We have previously demonstrated that our proprietary vaccine platform MultiTEP is extremely immunogenic in various mammals, including disease models of AD and aged monkeys, and can be used to generate a robust immune response towards various targets. Based on these we developed a MutiTEP‐based vaccine targeting pE3‐Aβ.
Method
A genetically engineered variant of MultiTEP was generated to enable chemoselective bioconjugation of synthetic peptides under mild aqueous conditions. The peptide‐protein stoichiometry was selected at 3:1 based on previous data. C57Bl6 and 5xFAD mice were monthly immunized with pE3‐Aβ‐MultiTEP adjuvanted vaccine, AV‐1986R. Antibody titers were tested via ELISA on plates coated with pE3‐Aβ42 and unmodified Aβ42 as control. The reduction of brain pathology in 5xFAD was assessed via biochemical assessment (MSD ELISA) as well as IHC and confocal microscopy using various commercial antibodies. The binding of immune sera to brain sections from non‐AD, MCI, and AD cases was examined.
Result
The AV‐1986R generates a robust immune response in transgenic and WT mice after two immunizations. The generated antibodies exhibit three orders of magnitude higher affinity (one to a million avg endpoint titer) to the pE3‐Aβ compared to Aβ42 and recognized Aβ plaques in brain tissue slices from both transgenic animals and AD cases. Importantly, vaccination significantly reduced AD‐like pathology in 5xFAD mice.
Conclusion
AV‐1986R is immunogenic and effective. A recent report from Elly‐Lilly about the pE3‐Aβ specific monoclonal antibody donanemab shows that treatment slows down the cognitive decline in humans. While this is promising and re‐affirms the selection of pE3‐Aβ as a therapeutic target, the regular injections of monoclonal antibodies are extremely expensive and have low patient compliance due to the frequent and invasive nature of the treatment. We suggest using active vaccine as a feasible alternative, allowing the maintenance of a constant level of therapeutic antibodies with minimal costs and infrequent vaccine injections.
Background
Amyloid‐beta (Aβ) plaque formation is a well‐established hallmark for Alzheimer’s disease (AD). However, the processes behind plaque formation are not understood. Previous work from our ...group identified rare coding variants for PLD3, with one variant in particular, p.A442A, disrupting a splicing enhancer binding site within the mRNA transcript. In this study, we present findings that PLD3 regulates Aβ clearance in the brain through both cell autonomous and non‐autonomous means.
Method
To replicate the splicing effect observed in patient brain samples in vitro, PLD3 p.A442A expressing induced pluripotent stem cells (iPSCs) were differentiated into neurons or microglia and evaluated for extracellular Aβ and Aβ uptake, respectively. In an APP/PSEN1xPld3KO mouse model, Aβ dynamics were assessed with in vivo microdialysis and plaque pathology was assessed with immunohistochemistry. Frozen cortical tissue from these mice was analyzed through bulk RNAseq to identify transcriptomic and molecular changes that are attributed to Pld3 deficiency. PLD3 expression was assessed in an independent single‐cell RNAseq (scRNAseq) dataset from iPSC‐derived microglia to assess a potential cell‐autonomous role for PLD3 in microglia.
Result
Transcriptomics from human samples reveal a significant decrease of PLD3 expression in AD brains versus controls, particularly in the brain regions most susceptible to amyloid pathology. Similar to PLD3‐variant brains, iPSC‐derived neurons carrying PLD3 p.A442A exhibited alternative splicing and displayed elevated Aβ levels in the conditioned media. Loss of Pld3 in APP/PSEN1 mice led to increased interstitial levels of Aβ and reduced Aβ clearance. Immunohistochemistry revealed an increase in diffuse, nonfibrillar plaque area and reduced microglial recruitment in APP/PSEN1xPld3KO. Transcriptomics data from brain tissue in these mice revealed altered expression of genes that define the disease associated microglia signatures. To begin to understand the role of PLD3 in microglia, we analyzed scRNAseq from CRISPRi screen in iPSC‐derived microglia. We found that PLD3 expression is enriched in interferon‐associated microglia. Finally, we demonstrate that iPSC‐derived microglia carrying PLD3 p.A442A exhibit reduced internalization of Aβ and myelin, suggesting a defect in phagocytic capacity.
Conclusion
Together, we show that PLD3 plays a critical role in the brain by facilitating the microglial response to Aβ plaque pathology, modulating plaque composition.
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