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.
Overall design
For this study 1, 3, 6 and 12 month old male Cnp1-/-, Plp1-/Y and age-matched control mice (n = 3/genotype and timepoint) were used.
Less...
More...