Variants identified in genome‐wide association studies have implicated immune pathways in the development of Alzheimer’s disease (AD). Here, we investigated the mechanistic basis for protection from AD associated with PLCγ2 R522, a rare coding variant of the PLCG2 gene. We studied the variant's role in macrophages and microglia of newly generated PLCG2‐R522‐expressing human induced pluripotent cell lines (hiPSC) and knockin mice, which exhibit normal endogenous PLCG2 expression. In all models, cells expressing the R522 mutation show a consistent non‐redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This manifests as enhanced release of cellular calcium ion stores in response to physiologically relevant stimuli like Fc‐receptor ligation or exposure to Aβ oligomers. Expression of the PLCγ2‐R522 variant resulted in increased stimulus‐dependent PIP2 depletion and reduced basal PIP2 levels in vivo. Furthermore, it was associated with impaired phagocytosis and enhanced endocytosis. PLCγ2 acts downstream of other AD‐related factors, such as TREM2 and CSF1R, and alterations in its activity directly impact cell function. The inherent druggability of enzymes such as PLCγ2 raises the prospect of PLCγ2 manipulation as a future therapeutic approach in AD.
Ca 2þ signaling is vital for various cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, gene transcription, and cellular proliferation. The endoplasmic reticulum (ER) is the largest intracellular Ca 2þ store, and dysregulation of ER Ca 2þ signaling and homeostasis contributes to the pathogenesis of various complex disorders and Mendelian disease traits. We describe four unrelated individuals with a complex multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay. Through whole-exome sequencing and family-based genomics, we identified bi-allelic variants in CCDC47 that encodes the Ca 2þ-binding ER transmembrane protein CCDC47. CCDC47, also known as calumin, has been shown to bind Ca 2þ with low affinity and high capacity. In mice, loss of Ccdc47 leads to embryonic lethality, suggesting that Ccdc47 is essential for early development. Characterization of cells from individuals with predicted likely damaging alleles showed decreased CCDC47 mRNA expression and protein levels. In vitro cellular experiments showed decreased total ER Ca 2þ storage, impaired Ca 2þ signaling mediated by the IP 3 R Ca 2þ release channel, and reduced ER Ca 2þ refilling via store-operated Ca 2þ entry. These results, together with the previously described role of CCDC47 in Ca 2þ signaling and development, suggest that bi-allelic loss-offunction variants in CCDC47 underlie the pathogenesis of this multisystem disorder. Ca 2þ signaling is a multipurpose intracellular signaling system that regulates a number of cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, transcription, and cellular proliferation. 1 The endoplasmic reticulum (ER), or the sarcoplasmic reticulum (SR) in muscle cells, is the largest store of intracellular Ca 2þ. 2 ER Ca 2þ depletion is also observed in a number of genetic disorders due to variants in Ca 2þ channels and sensors. For example, Brody myopathy (MIM: 601003) is caused by recessive variants in ATP2A1 (MIM: 611974), which encodes the fast-twitch skeletal muscle sarcoplasmic reticulum Ca 2þ ATPase (SERCA1), 3 while Darier disease (MIM: 124200) occurs due to variants in ATP2A2 (MIM: 108740), which encodes another sarcoplasmic reticulum Ca 2þ ATPase, SERCA2. 4 Minicore myopathy (MIM: 255320) and central core disease (MIM: 117000) result from variants in RYR1 (MIM: 180901), which encodes a major Ca 2þ release channel, 5 and autosomal centronuclear myopathy (MIM: 160150) is associated with variants in MTMR14 (MIM: 611089), which encodes a muscle-specific inositol phosphatase. 6 Stormorken syndrome (MIM: 185070), tubular aggregate myopathy 1 (MIM: 160565), and immunodeficiency 10 (MIM: 612783) are caused by variants in STIM1 (MIM: 605921), 7-9 which encodes a Ca 2þ sensor. Tubular aggregate myopathy 2 (MIM: 615883) and immunodeficiency 9 (MIM: 612782) are caused by variants in ORAI1 (MIM: 610277), 10,11 which encodes a Ca 2þ channel that...
Microglia are increasingly recognized as vital players in the pathology of a variety of neurodegenerative conditions including Alzheimer’s (AD) and Parkinson’s (PD) disease. While microglia have a protective role in the brain, their dysfunction can lead to neuroinflammation and contributes to disease progression. Also, a growing body of literature highlights the seven phosphoinositides, or PIPs, as key players in the regulation of microglial-mediated neuroinflammation. These small signaling lipids are phosphorylated derivates of phosphatidylinositol, are enriched in the brain, and have well-established roles in both homeostasis and disease.Disrupted PIP levels and signaling has been detected in a variety of dementias. Moreover, many known AD disease modifiers identified via genetic studies are expressed in microglia and are involved in phospholipid metabolism. One of these, the enzyme PLCγ2 that hydrolyzes the PIP species PI(4,5)P2, displays altered expression in AD and PD and is currently being investigated as a potential therapeutic target.Perhaps unsurprisingly, neurodegenerative conditions exhibiting PIP dyshomeostasis also tend to show alterations in aspects of microglial function regulated by these lipids. In particular, phosphoinositides regulate the activities of proteins and enzymes required for endocytosis, toll-like receptor signaling, purinergic signaling, chemotaxis, and migration, all of which are affected in a variety of neurodegenerative conditions. These functions are crucial to allow microglia to adequately survey the brain and respond appropriately to invading pathogens and other abnormalities, including misfolded proteins. AD and PD therapies are being developed to target many of the above pathways, and although not yet investigated, simultaneous PIP manipulation might enhance the beneficial effects observed. Currently, only limited therapeutics are available for dementia, and although these show some benefits for symptom severity and progression, they are far from curative. Given the importance of microglia and PIPs in dementia development, this review summarizes current research and asks whether we can exploit this information to design more targeted, or perhaps combined, dementia therapeutics. More work is needed to fully characterize the pathways discussed in this review, but given the strength of the current literature, insights in this area could be invaluable for the future of neurodegenerative disease research.
Purpose To describe the baseline participant characteristics in the ASPREE-AMD study, investigating the effect of aspirin on AMD incidence and progression. Methods Australian participants from the ASPirin in Reducing Events in the Elderly (ASPREE) trial, randomized to 100 mg aspirin daily or placebo, had non-mydriatic, digital color fundus images graded according to the Beckman AMD classification. Associations with AMD were determined for baseline characteristics and genetic risk variants. Results ASPREE-AMD sub-study enrolled 4993 participants with gradable macular images. Median age was 73.4 years (IQR, 71.5, 76.6), 52% were female, 10% had diabetes mellitus, 73% had hypertension, and 44% were former/current smokers. Early, intermediate and late AMD (detected in 20.6%, 16.1%, 1.1%, respectively), significantly associated with age, were also associated with increasing HDL levels: OR = 1.52 (95%CI, 1.26, 1.84), OR = 1.43 (1.17, 1.77) and OR = 1.96 (1.02, 3.76), respectively. Female sex was associated with early [OR = 1.37 (1.16, 1.62)], and intermediate [OR = 1.35 (1.12, 1.63)] AMD, as was previous regular use of aspirin, with OR = 1.46 (1.11, 1.92) and OR = 1.37 (1.01, 1.85), respectively. Current smoking had increased odds for late AMD, OR = 4.02 (1.42, 11.36). Genetic risk variant rs3750846 ( ARMS2/HTRA1 ) was associated with each AMD stage ( p < 0.001), risk variants rs570618 and rs10922109 ( CFH) with intermediate and late AMD ( p < 0.001), and rare variant rs147859257 ( C3 ) with late AMD ( p < 0.001). The randomized groups were well balanced for all analyzed AMD risk factors. Conclusions Observed associations are typical of AMD. The ASPREE-AMD clinical trial provides a unique opportunity to determine the risks and benefits of low-dose aspirin for AMD incidence and progression in elderly population. Trial registration Australian New Zealand Clinical Trial Registry: ACTRN 12613000755730.
Recent genome-wide association studies of Alzheimer's disease (AD) have identified variants implicating immune pathways in disease development. A rare coding variant of PLCG2, which encodes PLCγ2, shows a significant protective effect for AD (rs72824905, P522R, P=5.38x10 -10 , Odds Ratio = 0.68). Molecular dynamic modelling of the PLCγ2-R522 variant, situated within the auto-inhibitory domain of PLCγ2, suggests a structural change to the protein. Through CRISPR-engineering we have generated novel PLCG2-R522 harbouring human induced pluripotent cell lines (hiPSC) and a mouse knockin model, neither of which exhibits alterations in endogenous PLCG2 expression. Mouse microglia and macrophages and hiPSC-derived microglia-like cells with the R522 mutation, all demonstrate a consistent non-redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This signalling alteration manifests as enhanced cellular Ca 2+ store release (~20-40% increase) in response to physiologically-relevant stimuli (e.g. Fc receptor ligation and Aβ oligomers). This hyperfunctionality resulted in increased PIP2 depletion in the cells with the PLCγ2-R522 variant after exposure to stimuli and reduced basal detection of PIP2 levels in vivo. These PLCγ2-R522 associated abnormalities resulted in impairments to phagocytosis (fungal and bacterial particles) and enhanced endocytosis (Aβ oligomers and dextran). PLCγ2 sits downstream of disease relevant pathways, such as TREM2 and CSF1R and alterations in its activity, direct impacts cell function, which in the context of the inherent drugability of enzymes such as PLCγ2, raise the prospect of manipulation of PLCγ2 as a therapeutic target in Alzheimer's Disease.
Microglia, the main immune modulators of the central nervous system, have key roles in both the developing and adult brain. These functions include shaping healthy neuronal networks, carrying out immune surveillance, mediating inflammatory responses, and disposing of unwanted material. A wide variety of pathological conditions present with microglia dysregulation, highlighting the importance of these cells in both normal brain function and disease. Studies into microglial function in the context of both health and disease thus have the potential to provide tremendous insight across a broad range of research areas. In vitro culture of microglia, using primary cells, cell lines, or induced pluripotent stem cell derived microglia, allows researchers to generate reproducible, robust, and quantifiable data regarding microglia function. A broad range of assays have been successfully developed and optimised for characterizing microglial morphology, mediation of inflammation, endocytosis, phagocytosis, chemotaxis and random motility, and mediation of immunometabolism. This review describes the main functions of microglia, compares existing protocols for measuring these functions in vitro, and highlights common pitfalls and future areas for development. We aim to provide a comprehensive methodological guide for researchers planning to characterise microglial functions within a range of contexts and in vitro models.
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