669 research outputs found

    The role of AD protective variant PLCγ2P522R in modulating microglia mediated clearance and synaptic pruning

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    PLCG2-P522R, a rare coding variant in the Phospholipase C gamma-2 (PLCG2) gene, has been found to be protective against late onset Alzheimer's disease (AD). Within the central nervous system, PLCγ2 is most abundantly expressed in microglia, and microglial mediated neuroinflammatory system has emerged as a major contributor to the molecular and phenotypic changes observed in the AD brain. However, the mechanism by which the P522R variant of PLCγ2 reduces AD pathology is still unknown. BV2 (mouse microglia) cells and human induced pluripotent stem-cells (hiPSC) derived microglia were used in this thesis work to evaluate the role of PLCγ2 in modifying various disease-relevant microglia functions. PLCγ2WT and PLCγ2P522R expression constructs were transfected into BV2 cells to examine the effects of PLCγ2 overexpression on various microglia functions including amyloid beta (Aβ) clearance and synaptic targeting, and various transcriptional changes linked to AD. hiPSCs were genome edited using CRISPR/Cas9 to generate both heterozygous and homozygous forms of the PLCG2_P522R variant in healthy controls. These hiPSC derived microglia were used to explore the effects of the PLCγ2P522R basal level on disease-relevant processes, such as microglial capacity to uptake Aβ and synapses. Microglia transcriptional changes were examined using targeted qPCR analysis to investigate changes in expression of key microglial genes. Mitochondrial function and calcium level changes were also investigated in these microglia cells to determine their metabolic fitness. In addition, the microglia were subjected to acute and chronic treatment of oligomeric Aβ to examine the impact of PLCγ2P522R on microglia's ability to respond to acute and chronic stress. As a result, the effects of Aβ oligomers on lysosomal biogenesis and phagocytic capacities of these microglia were examined further. As a result of PLCγ2 overexpression, Aβ uptake and other immune- provoking cargoes like zymosan were significantly increased. In contrast, the uptake of synaptosomes in BV2 cells overexpressing PLCγ2 was considerably reduced. Similarly, microglia generated from hiPSCs also showed enhanced clearance of Aβ and preservation of synapses by PLCγ2P522R variants. In the PLCγ2P522R microglia variants, the expression of multiple genes, including IL-10 and CX3CR1, as well as mitochondrial function, cytoplasmic calcium flux, and cellular motility were all increased. It was found that the protective effect of PLCγ2P522R was vitally dependent on 'allelic-dose', as homozygous cells displayed a lower preservation of synapse and a distinct gene expression profile compared to heterozygous cells. Similarly, microglia with the protective mutation PLCγ2P522R displayed higher inflammatory cytokine IL-1β level, and better response to acute treatment with Aβ oligomers. PLCγ2P522R appeared to resist the quiescence that was seen in WT microglia variants, by increasing cytokine production and lysosomal biogenesis. My findings suggest that the P522R variant in PLCγ2 increases microglia capacity to clear toxic aggregates such as Aβ while preserving synapses. Furthermore, my findings suggests that PLCγ2P522R contributes to greater microglial surveillance, as well as microglia priming towards a pro-inflammatory state, along with an increased capacity to adapt to growing energy demands. This, however, also shows the delicate balance of this system, as increasing the 'dosage' of PLCγ2P522R may result in diminished favourable benefits

    Investigating neural differentiation capacity in Alzheimer’s disease iPSC-derived neural stem cells

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    Neurodegeneration in Alzheimer’s disease (AD) may be exacerbated by dysregulated hippocampal neurogenesis. Neural stem cells (NSC) maintain adult neurogenesis and depletion of the NSC niche has been associated with age-related cognitive decline and dementia. We hypothesise that familial AD (FAD) mutations bias NSC toward premature neural specification, reducing the stem cell niche over time and accelerating disease progression. Somatic cells derived from patients with FAD (PSEN1 A246E and PSEN1 M146L heterozygous mutations) and healthy controls were reprogrammed to generate induced pluripotent stem cells (iPSC). Pluripotency for patient and control iPSC lines was confirmed, then cells were amplified and cryopreserved as stores. iPSC were subjected to neural specification to rosette-forming SOX2+/nestin+ NSCs for comparative evaluations between FAD and age-matched controls. FAD patient and control NSC were passaged under defined steady state culture conditions to assess stem cell maintenance using quantitative molecular markers (SOX2, nestin, NeuN, MAP2 and βIII-tubulin). We observed trends towards downregulated expression of the nestin coding gene NES (p=0.051) and upregulated expression of MAP2 (p=0.16) in PSEN1 NSC compared with control NSC, indicative of a premature differentiation phenotype induced by presence of the PSEN1 mutation. Cell cycle analysis of PSEN1 NSC showed that compared with controls, a greater number of PSEN1 NSC were retained in G0/G1 phase of the cell cycle (p=0.39), fewer progressed to S-phase (p=0.11) and fewer still reached G2 phase (p=0.23), suggesting cell cycle progression may be impaired in PSEN1 NSC. Nuclear DNA fragmentation was increased (p=0.10) in FAD NSC compared with controls, indicative of elevated cell death/apoptosis. Flow cytometry-based analysis of live, nestin+ NSC and NPC indicated increased apoptosis (p=0.14) in FAD NSC compared with controls, as well as increasing levels of apoptosis (p=0.33) in FAD NSC as they specified to neural progenitor cells. Global RNA sequencing was used to identify transcriptomic changes occurring during both disease and control neural specification. GO analysis of DEGs between PSEN1 and control NSC at P3 revealed significant upregulation (FDR<0.0000259) of 5 biological processes related to transcription and gene expression as well as significant upregulation (FDR<0.000000725) of 12 molecular functions related to DNA binding and transcription factor activity. These data suggest significant changes in gene expression were occurring in PSEN1 NSC at P3 compared with control NSC at the same stage in neural specification. The number of DEGs (p<0.05) between PSEN1 and control NSC at P3 was 9.92-fold higher than the number of DEGs between PSEN1 and control NSC at P2, suggesting transcriptomic differences between PSEN1 and control NSC become more pronounced as cells specify further down the neural lineage. Gene ontology (GO) analysis of differentially expressed genes (DEGs) specific to AD neural differentiation revealed significant dysregulation (FDR p<0.05) of genes related to neurogenesis, apoptosis, cell cycle, transcriptional control, and cell growth/maintenance as PSEN1 NSC matured from P2 to P3. The number of DEGs (p<0.05) in PSEN1 neural differentiation was 4.7-fold higher than the number of DEGs seen in control neural differentiation, indicating more transcriptional changes occurred in PSEN1 NSC than in controls at the same time point in neural specification. Dysregulation of Notch signalling was specific to PSEN1 neural differentiation and Notch related DEGs significantly upregulated (p<0.05) in PSEN1 NSC at P3 compared with P2 included NCOR2, JAG2, CHAC1 and RFNG. qPCR based validation displayed significant upregulation of RFNG (p=0.04) in PSEN1 NSC at P3 compared with PSEN1 NSC at P2, and indicated a trend towards upregulation of JAG2 expression, correlating with RNA sequencing data. Data generated in this study indicate that presence of the PSEN1 mutation significantly increases the number of transcriptional changes occurring during neural differentiation. It is plausible that transcriptional changes to Notch signalling cause dysregulated neural specification and increased apoptosis in PSEN1 NSC, ultimately resulting in depletion of the NSC niche

    The Genomics of Autism-Related Genes IL1RAPL1 and IL1RAPL2: Insights into Their Cortical Distribution, Cell-Type Specificity, and Developmental Trajectories

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    Neuropsychiatric disorders have a significant impact on modern society. These disorders affect a large percentage of the population: schizophrenia has a world-wide prevalence of 1% and autism spectrum disorders (ASD) affects 1 in 59 school-aged children in the US. There is substantial evidence that most neuropsychiatric disorders have a genetic component. Thus, with the advent of high throughput sequencing much effort has gone into identifying genetic variants associated with these disorders. The emerging picture from these studies is a complex one where hundreds of genes with small effects interact with a varied landscape of common variants to result in disease. Despite this complexity, individual disease-associated genes have been identified but studies of the functional role of each of these genes in brain development and function have only just begun. In addition to efforts designed to identify disease-relevant genetic variants, large consortia have been formed to generate other genomic datasets (e.g., bulk, or single cell RNA expression) to uncover both cell-type specific and tissue-specific transcriptional networks where disease-associated genes are involved. This study integrated several types of bulk tissue datasets with single-cell datasets to investigate the cortical distribution, cell-type specificity, and developmental trajectories of two ASD-linked genes: IL1RAPL1 and its paralog IL1RAPL2. Genetics studies linked IL1RAPL1 and IL1RAPL2 with ASD and intellectual disabilities (ID) and both are strong ASD risk gene candidates with a SFARI score of two. Even though IL1RAPL1 has been shown to have a role in synaptic development and synaptic strength, little is known about IL1RAPL2. Therefore, whether or how IL1RAPL2 functions in synapse development is a significant gap in knowledge and given the role of cortical excitatory neurons in ASD and intellectual ability, IL1RAPL2 is highly likely to serve a critical role. Recently, IL1RAPL2 has been identified as a hub gene in an ASD module associated with memory oscillations and it is strongly co-expressed with other ASD-risk genes. These data further confirm the potential role of IL1RAPL2 in neuronal etiology and ASD. To further gain insights into the role of these genes in the human brain and ASD, we analyzed the transcriptomic landscapes of IL1RAPL1 and IL1RAPL2 in cortical region and cell types of the human brain. We hypothesized that IL1RAPL1 and IL1RAPL2 gene expression significantly differs within cortical regions involved in higher order cognitive function, both at cell-type level and during development. This project took advantage of publicly available genomic data from PsychENCODE, Allen Institute, and Lister Lab. We analyzed bulk RNA-seq from neurotypical and ASD patients (N = 103) identifying gene expression and co-expression differences between IL1RAPL2 and IL1RAPL1 in 11 cortical regions. We next used a time series bulk RNA-seq data from neurotypical subjects (N = 39) to infer the developmental trajectories of IL1RAPL2 and IL1RAPL1 in 11 cortical regions. Finally, by using single cell RNA-seq data, we uncovered the cell-type gene expression distribution differences between IL1RAPL2 and IL1RAPL1 in multiple regions and cortical-layers in human and mice samples, and to find differences in expression across development. In summary, this comprehensive genomic project highlighted significant differences between IL1RAPL1 and IL1RAPL2 in specific brain regions, cell-types, and developmental trajectories across the human lifespan. The identified differences provide preliminary evidence that IL1RAPL1 and IL1RAPL2 might play a different role in human cortical development and cell-type

    Activation of beta2-adrenergic receptors alleviates neuropathic pain hypersensitivity in mice: focus on spinal glial cells

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    Chronic pain affects roughly one-fifth of the world’s population, and many patients do not respond to current therapies or conventional analgesics. Thus, studying the molecular mechanisms underlying neuropathic pain is crucial in identifying novel molecular targets that can be used to develop effective pain relief therapies. Previous studies have thus far focused on α2-adrenergic receptors (α2-ARs) and neuronal excitability, among others. However, recent research suggests that astrocytes and microglia, which express adrenergic receptors, contribute significantly to neuropathic pain. In particular, microglia have been found to express elevated levels of Gs-coupled β2-AR and they are responsive to norepinephrine application. Additionally, systemic administration of β2-AR agonists, such as Formoterol, has anti-inflammatory and anti-nociceptive properties in neuropathic pain, but the underlying processes are poorly understood. Therefore, this thesis work focuses on investigating glial noradrenergic signaling via β2-AR, specifically on microglia and its contribution to the modulation of neuropathic pain in mice. In the present study, activation of the β2-ARs through Formoterol induced a decrease of anti-inflammatory cytokine levels in primary isolated microglia and reversed nerve injury-induced morphological alterations in spinal dorsal horn microglia. Systemic administration of Formoterol inhibited evoked behaviors as well as aversive components related to neuropathic pain and reduced chronically-established neuropathic pain. The analgesic effects of Formoterol were mainly mediated by microglia, as demonstrated by employing the conditional knock-out mouse line lacking the β2-AR specifically in microglia. Remarkably, the effect of Formoterol on neuropathic pain-related behavior and microgliosis was lost in mice with the microglia-specific deletion of β2-ARs. In addition, microglia phenotype showed a sex-dependency in the late phase of neuropathic pain, which was not observed in response to β2-AR stimulation. Notably, Formoterol also reduced astrogliosis in the late stage of neuropathic pain independently of β2-AR signaling in microglia. Collectively, this work highlights the impact of microglial β2-AR stimulation in mediating the inhibition of pro-inflammatory signaling in the spinal cord during the initial phase of neuropathic pain. These results emphasize the importance of exploring microglial β2-AR agonists in alleviating neuropathic pain and elucidating the underlying mechanisms
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