Background: Despite its identification as a key checkpoint regulator of microglial activation in Alzheimer’s disease, the overarching role of CX3CR1 signaling in modulating mechanisms of Aβ driven neurodegeneration, including accumulation of hyperphosphorylated tau is not well understood. Methodology: Accumulation of soluble and insoluble Aβ species, microglial activation, synaptic dysregulation, and neurodegeneration is investigated in 4- and 6-month old 5xFAD;Cx3cr1^+/+ and 5xFAD;Cx3cr1^−/− mice using immunohistochemistry, western blotting, transcriptomic and quantitative real time PCR analyses of purified microglia. Flow cytometry based, in-vivo Aβ uptake assays are used for characterization of the effects of CX3CR1-signaling on microglial phagocytosis and lysosomal acidification as indicators of clearance of methoxy-X-04⁺ fibrillar Aβ. Lastly, we use Y-maze testing to analyze the effects of Cx3cr1 deficiency on working memory. Results: Disease progression in 5xFAD;Cx3cr1^−/− mice is characterized by increased deposition of filamentous plaques that display defective microglial plaque engagement. Microglial Aβ phagocytosis and lysosomal acidification in 5xFAD;Cx3cr1^−/− mice is impaired in-vivo. Interestingly, Cx3cr1 deficiency results in heighted accumulation of neurotoxic, oligomeric Aβ, along with severe neuritic dystrophy, preferential loss of post-synaptic densities, exacerbated tau pathology, neuronal loss and cognitive impairment. Transcriptomic analyses using cortical RNA, coupled with qRT-PCR using purified microglia from 6 month-old mice indicate dysregulated TGFβ-signaling and heightened ROS metabolism in 5xFAD;Cx3cr1^−/− mice. Lastly, microglia in 6 month-old 5xFAD;Cx3cr1^−/− mice express a ‘degenerative’ phenotype characterized by increased levels of Ccl2, Ccl5, Il-1β, Pten and Cybb along with reduced Tnf, Il-6 and Tgfβ1 mRNA. Conclusions: Cx3cr1 deficiency impairs microglial uptake and degradation of fibrillar Aβ, thereby triggering increased accumulation of neurotoxic Aβ species. Furthermore, loss of Cx3cr1 results in microglial dysfunction typified by dampened TGFβ-signaling, increased oxidative stress responses and dysregulated pro-inflammatory activation. Our results indicate that Aβ-driven microglial dysfunction in Cx3cr1^−/− mice aggravates tau hyperphosphorylation, neurodegeneration, synaptic dysregulation and impairs working memory.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, robust microgliosis, neuroinflammation, and neuronal loss. Genome-wide association studies recently highlighted a prominent role for microglia in late-onset AD (LOAD). Specifically, inositol polyphosphate-5-phosphatase (INPP5D), also known as SHIP1, is selectively expressed in brain microglia and has been reported to be associated with LOAD. Although INPP5D is likely a crucial player in AD pathophysiology, its role in disease onset and progression remains unclear. We performed differential gene expression analysis to investigate INPP5D expression in AD and its association with plaque density and microglial markers using transcriptomic (RNA-Seq) data from the Accelerating Medicines Partnership for Alzheimer's Disease (AMP-AD) cohort. We also performed quantitative real-time PCR, immunoblotting, and immunofluorescence assays to assess INPP5D expression in the 5xFAD amyloid mouse model. Differential gene expression analysis found that INPP5D expression was upregulated in LOAD and positively correlated with amyloid plaque density. In addition, in 5xFAD mice, Inpp5d expression increased as the disease progressed, and selectively in plaque-associated microglia. Increased Inpp5d expression levels in 5xFAD mice were abolished entirely by depleting microglia with the colony-stimulating factor receptor-1 antagonist PLX5622. Our findings show that INPP5D expression increases as AD progresses, predominantly in plaque-associated microglia. Importantly, we provide the first evidence that increased INPP5D expression might be a risk factor in AD, highlighting INPP5D as a potential therapeutic target. Moreover, we have shown that the 5xFAD mouse model is appropriate for studying INPP5D in AD.

Increased dietary intake of niacin has been correlated with reduced risk of Alzheimer’s disease (AD). Niacin serves as a high-affinity ligand for the receptor HCAR2 (GPR109A). In the brain, HCAR2 is expressed selectively by microglia and is robustly induced by amyloid pathology in AD. The genetic inactivation of Hcar2 in 5xFAD mice, a model of AD, results in impairment of the microglial response to amyloid deposition, including deficits in gene expression, proliferation, envelopment of amyloid plaques, and uptake of amyloid-β (Aβ), ultimately leading to exacerbation of amyloid burden, neuronal loss, and cognitive deficits. In contrast, activation of HCAR2 with an FDA-approved formulation of niacin (Niaspan) in 5xFAD mice leads to reduced plaque burden and neuronal dystrophy, attenuation of neuronal loss, and rescue of working memory deficits. These data provide direct evidence that HCAR2 is required for an efficient and neuroprotective response of microglia to amyloid pathology. Administration of Niaspan potentiates the HCAR2-mediated microglial protective response and consequently attenuates amyloid-induced pathology, suggesting that its use may be a promising therapeutic approach to AD that specifically targets the neuroimmune response.

Background: Alzheimer’s disease (AD) is characterized by robust microgliosis and phenotypic changes that accompany disease pathogenesis. Accumulating evidence from genetic studies suggests the importance of phospholipase C γ 2 (PLCG2) in late-onset AD (LOAD) pathophysiology. However, the role ofPLCG2in AD is still poorly understood.Methods: Using bulk RNA-Seq (N=1249) data from the Accelerating Medicines Partnership-Alzheimer’s Disease Consortium (AMP-AD), we investigated whetherPLCG2expression increased in the brains of LOAD patients. We also evaluated the relationship betweenPLCG2expression levels, amyloid plaque density, and expression levels of microglia specific markers (AIF1 and TMEM119). Finally, we investigated the longitudinal changes ofPLCG2expression in the 5xFAD mouse model of AD. To further understand the role ofPLCG2in different signaling pathways, differential gene expression and co-expression network analyses were performed using bulk RNA-Seq and microglial single-cell RNA-Seq data. To substantiate the human analyses, we performed differential gene expression analysis on wild-type (WT) and inactivatedPlcg2mice and used immunostaining to determine if the differentially expressed genes/pathways were altered by microglial cell coverage or morphology.Results: We observed significant upregulation ofPLCG2expression in three brain regions of LOAD patients and significant positive correlation ofPLCG2expression with amyloid plaque density. These findings in the human brain were validated in the 5xFAD amyloid mouse model, which showed disease progression-dependent increases inPlcg2expression associated with amyloid pathology. Of note, increasedPlcg2expression levels in 5xFAD mice were abolished by reducing microglia. Furthermore, using bulk RNA-Seq data, we performed differential expression analysis by comparing cognitively normal older adults (CN) with 75th percentile (high) and 25th percentile (low)PLCG2gene expression levels to identify pathways related to inflammation and the inflammatory response. The findings in the human brain were validated by differential expression analyses between WT andplcg2inactivated mice.PLCG2co-expression network analysis of microglial single-cell RNA-Seq data identified pathways related to the inflammatory response including regulation of I-kappaB/NF-kappa B signaling and response to lipopolysaccharide.Conclusions: Our results provide further evidence thatPLCG2plays an important role in AD pathophysiology and may be a potential target for microglia-targeted AD therapies.

A cytoskeleton-associated protein regulates microglial function and affects Alzheimer’s disease phenotypes.

High-fat diet (HFD) has been shown to accelerate Alzheimer’s disease (AD) pathology, but the exact molecular and cellular mechanisms remain incompletely understood. Moreover, it is unknown whether AD mice are more susceptible to HFD-induced metabolic dysfunctions. To address these questions, we used 5xFAD mice as an Alzheimer’s disease model to study the physiological and molecular underpinning between HFD-induced metabolic defects and AD pathology. We systematically profiled the metabolic parameters, the gut microbiome composition, and hippocampal gene expression in 5xFAD and wild type (WT) mice fed normal chow diet and HFD. HFD feeding impaired energy metabolism in male 5xFAD mice, leading to increased locomotor activity, energy expenditure, and food intake. 5xFAD mice on HFD had elevated circulating lipids and worsened glucose intolerance. HFD caused profound changes in gut microbiome compositions, though no difference between genotype was detected. We measured hippocampal mRNAs related to AD neuropathology and neuroinflammation and showed that HFD elevated the expression of apoptotic, microglial, and amyloidogenic genes in 5xFAD mice. Pathway analysis revealed that differentially regulated genes were involved in insulin signaling, cytokine signaling, cellular stress, and neurotransmission. Collectively, our results showed that 5xFAD mice were more susceptible to HFD-induced metabolic dysregulation and suggest that targeting metabolic dysfunctions can ameliorate AD symptoms via effects on insulin signaling and neuroinflammation in the hippocampus.