Project description:Single cell RNA sequencing analysis was performed on microglia isolated from brain slices (timepoints: 1 day, 1 week or 3 weeks in culture), 2D in vitro cultured microglia, and acutely isolated adult microglia. Differences in gene expression were examined to determine relative similarity to acutely isolated adult microglia.
Project description:Organotypic brain slice culture microglia exhibit molecular similarity to acutely-isolated adult microglia and provide a platform to study neuroinflammation
Project description:Organotypic slice cultures from prostate cancer patients were generated and treated with or without 17b-estradiol or DHT to study estrogen and androgen signalling pathways.
Project description:The numerous neurological syndromes associated with COVID-19 implicate an effect of viral pathogenesis on neuronal function, yet reports of direct SARS-CoV-2 infection in the brain are conflicting. We used a well-established organotypic brain slice culture to determine the permissivity of hamster brain tissues to SARS-CoV-2 infection. We found levels of live virus waned after inoculation and observed no evidence of cell-to-cell spread, indicating that SARS-CoV-2 infection was non-productive. Nonetheless, we identified a small number of infected cells with glial phenotypes; yet no evidence of viral infection or replication was observed in neurons. Our data corroborates several clinical studies that have assessed patients with COVID-19 and their association with neurological involvement.
Project description:Gene expression changes induced by alpha-secretase cleaved amyloid precursor protein (sAPPalpha) in organotypic hippocampal slice cultures of male, postnatal day 15 mice (C57B6/SJL). Hippocampal slice cultures were treated with phosphate buffered saline (GSM26700, GSM26701, GSM26702) or 1 nM sAPPalpha (GSM26703, GSM26704, GSM26705) for 24 h. Each sample consists of total RNA isolated from 8-12 slices from 4 mice. Data were analyzed with MAS 5.0 and scaled to 2500. sAPPalpha induces the amyloid sequestration protein transthyretin, insulin-like growth factor 2, insulin-like growth factor binding protein 2, and other genes involved in protective pathways such as apoptosis inhibition, detoxification, and retinol transport. Keywords = Alzheimer's disease Keywords = neuroprotection Keywords = sAPPalpha
Project description:We generated single-cell RNAseq profiles of microglia and brain myeloid cells (1922 total; 1816 cells passed quality control) from different developmental stages (E14.5, P7 and P60) to study their heterogeneity. Cells were isolated from either the whole developing brain (for the E14.5 stage) or six separate regions (for P7 and P60 stages): Cortex (CTX), Cerebellum (CB), Hippocampus (HIP), Striatum (STR), Olfactory bulb (OB), Choroid plexus (CP). Single cells were FACS index sorted followed by Smart-seq2 library preparation and Illumina Nextseq (sequence depth > 1 million per cell). All 1816 cells were grouped into 15 clusters using Seurat package (Macosko, Basu, Satija et al. Cell. 2015), and manually annotated based on gene expression signatures and meta data. We found that the majority of adult microglia expressing homeostatic genes are remarkably similar in transcriptomes, regardless of brain region. By contrast, postnatal microglia represent a more heterogeneous population. We discovered a proliferative region-associated microglia (PAM) subset, mainly found in developing white matter, that share a characteristic gene signature with degenerative disease-associated microglia (DAM). Such PAM have amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.
Project description:DNA double strand breaks (DSBs) are linked to neurodegeneration and senescence. However, it is not clear how DSB-bearing neurons influence neuroinflammation associated with neurodegeneration. Here, we characterize DSB-bearing neurons from the CK-p25 mouse model of neurodegeneration using single-nucleus, bulk, and spatial transcriptomic techniques. DSB-bearing neurons enter a late-stage DNA damage response marked by NFκB-activated senescent and antiviral immune pathways. In humans, Alzheimer’s disease pathology is significantly associated with immune activation in excitatory neurons. Spatial transcriptomics reveal that regions of CK-p25 brain tissue dense with DSB-bearing neurons harbor signatures of inflammatory microglia, which is ameliorated by NFκB knock-down in neurons. Inhibition of NFκB in DSB-bearing neurons also reduces microglia activation in organotypic mouse brain slice culture. In conclusion, DSBs activate immune pathways in neurons, which in turn adopt a senescence-associated secretory phenotype to elicit microglia activation. These findings highlight a novel role for neurons in the mechanism of disease-associated neuroinflammation.
Project description:We assessed the roles of repopulating microglia in brain repair using mouse models. In this project, we show that removal of microglia from the mouse brain has little impact on the outcome of TBI but inducing the turnover of these cells through either pharmacologic or genetic approaches can yield a neuroprotective microglial phenotype that profoundly aids recovery. As a part of the experimental approaches, we perform bulk RNA sequencing experiments to unbiasedly profile the transcriptome of repopulating microglia. We identified unique gene signatures from repopulating microglia cells and infer how these cells modulate the microenvironment after TBI.