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Transcriptomic approaches in the brain at cell type resolution: Analysis of neuron-glia interaction in Plp1 and Cnp1 null-mutant mice

ABSTRACT: Global gene expression profiling is a powerful tool to obtain deep insights into physiological and pathological cellular mechanisms. The enormous cellular complexity of the mammalian brain, however, is a major obstacle for gene expression profiling. Physiologically relevant changes of transcription that occur in specific cell populations are likely to remain undetected in cellularly complex samples. The purification of single populations of neural cell types eliminates these difficulties. We have approached this problem in transgenic mice by labelling genetically-defined neural cell types that express variants of GFP. When combined with isolation techniques such as fluorescence activated cell sorting (FACS) or laser capture microdissection (LCM), the isolation of intact RNA from unfixed cells for global transcriptome analysis is possible. In this study we applied FACS and LCM to isolate neurons and glia to gain insight into cell type specific gene expression. We profiled glial precursor cells, microglia and oligodendrocytes using FACS and generated a transcriptome database for glial cell types. LCM was used to study single isolated callosal projection neurons from the cortex of mouse models, which develop adult onset axonal degeneration a hallmark of many neurodegenerative diseases such as parkinson disease, Alzheimer disease and Multiple Sclerosis (MS). The identification of molecular mechanisms underlying axonal degeneration is critical to design rational therapies for neurodegenerative diseases. Therefore we profiled wild type controls, Cnp1 and Plp1 null mutant mice (myelin specific genes) at 4 different time points during adulthood. Cnp1 and Plp1 null mutants show axonal swellings and with age severe neurodegeneration, although CNS myelin seems ultra structural not affected. In summary, we have shown the feasibility to snapshot gene expression profiles of genetically defined neuronal subtypes in vivo and to compare morphologically similar neurons at a given time in pathological conditions. We followed gene expression changes starting from early disease states until stages of severe pathological signs, focusing on cells known to be susceptible to a genetic predisposition. Our analysis revealed several known and novel candidate genes and mechanisms that likely play a role in the early adaptive responses of cortical projection neurons to cope with axonal stress.

ORGANISM(S): Mus musculus

PROVIDER: GSE185092 | GEO | 2021/10/01

REPOSITORIES: GEO

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Project description:Expression profiling of distinct central nervous system (CNS) cell populations has been employed to facilitate disease classification and to provide insights into the molecular basis of brain pathology. One important cell type implicated in a wide variety of CNS disease states is the resident brain macrophage (microglia). In these studies, microglia are often isolated from dissociated brain tissue by flow sorting procedures [fluorescence-activated cell sorting (FACS)] or from postnatal glial cultures by mechanic isolation. Given the highly dynamic and state-dependent functions of these cells, the use of FACS or short-term culture methods may not accurately capture the biology of brain microglia. In the current study, we performed RNA-sequencing using Cx3cr1+/GFP labeled microglia isolated from the brainstem of 6-week-old mice to compare the transcriptomes of FACS-sorted versus laser capture microdissection (LCM). While both isolation techniques resulted in a large number of shared (common) transcripts, we identified transcripts unique to FACS-isolated and LCM-captured microglia. In particular, M-bM-^HM-<50% of these LCM-isolated microglial transcripts represented genes typically associated with neurons and glia. While these transcripts clearly localized to microglia using complementary methods, they were not translated into protein. Following the induction of murine experimental autoimmune encephalomyelitis, increased oligodendrocyte and neuronal transcripts were detected in microglia, while only the myelin basic protein oligodendrocyte transcript was increased in microglia after traumatic brain injury. Collectively, these findings have implications for the design and interpretation of microglia transcriptome-based investigations. Wildtype and GFP expressing microglia from mouse brainstems were flow sorted or captured by laser microdissection. Differences between the two isolation methods were verified and further examined in neurodegenerative disease models.

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Transcriptomic approaches in the brain at cell type resolution: Analysis of neuron-glia interaction in Plp1 and Cnp1 null-mutant mice

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