Brain-derived neurotrophic factor (BDNF) is a critical growth factor involved in the maturation of the CNS, including neuronal morphology and synapse refinement. Herein, we demonstrate astrocytes express high levels of BDNF’s receptor, TrkB (in the top 20 of protein-coding transcripts), with nearly exclusive expression of the truncated isoform, TrkB.T1, which peaks in expression during astrocyte morphological maturation. Using a novel culture paradigm, we show that astrocyte morphological complexity is increased in the presence of BDNF and is dependent upon BDNF/TrkB.T1 signaling. Deletion of TrkB.T1, globally and astrocyte-specifically, in mice revealed morphologically immature astrocytes with significantly reduced volume, as well as dysregulated expression of perisynaptic genes associated with mature astrocyte function. Indicating a role for functional astrocyte maturation via BDNF/TrkB.T1 signaling, TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function. These data suggest a significant role for BDNF/TrkB.T1 signaling in astrocyte morphological maturation, a critical process for CNS development.

BackgroundRett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. Much of our understanding of MeCP2 function is derived from transcriptomic studies with the general assumption that alterations in the transcriptome correlate with proteomic changes. Advances in mass spectrometry-based proteomics have facilitated recent interest in the examination of global protein expression to better understand the biology between transcriptional and translational regulation.MethodsWe therefore performed the first comprehensive transcriptome-proteome comparison in a RTT mouse model to elucidate RTT pathophysiology, identify potential therapeutic targets, and further our understanding of MeCP2 function. The whole cortex of wild-type and symptomatic RTT male littermates (n = 4 per genotype) were analyzed using RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Ingenuity® Pathway Analysis was used to identify significantly affected pathways in the transcriptomic and proteomic data sets.ResultsOur results indicate these two “omics” data sets supplement one another. In addition to confirming previous works regarding mRNA expression in Mecp2-deficient animals, the current study identified hundreds of novel protein targets. Several selected protein targets were validated by Western blot analysis. These data indicate RNA metabolism, proteostasis, monoamine metabolism, and cholesterol synthesis are disrupted in the RTT proteome. Hits common to both data sets indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding, and cytoskeletal cell structure. Finally, in addition to confirming disrupted pathways and identifying novel hits in neuronal structure and synaptic transmission, our data indicate aberrant myelination, inflammation, and vascular disruption. Intriguingly, there is no evidence of reactive gliosis, but instead, gene, protein, and pathway analysis suggest astrocytic maturation and morphological deficits.ConclusionsThis comparative omics analysis supports previous works indicating widespread CNS dysfunction and may serve as a valuable resource for those interested in cellular dysfunction in RTT.Electronic supplementary materialThe online version of this article (10.1186/s13229-017-0174-4) contains supplementary material, which is available to authorized users.

The isolation and study of cell-specific populations in the central nervous system (CNS) has gained significant interest in the neuroscience community. The ability to examine cell-specific gene and protein expression patterns in healthy and pathological tissue is critical for our understanding of CNS function. Several techniques currently exist to isolate cell-specific populations, each having their own inherent advantages and shortcomings. Isolation of distinct cell populations using magnetic sorting is a technique which has been available for nearly 3 decades, although rarely used in adult whole CNS tissue homogenate. In the current study we demonstrate that distinct cell populations can be isolated in rodents from early postnatal development through adulthood. We found this technique to be amendable to customization using commercially available membrane-targeted antibodies, allowing for cell-specific isolation across development and animal species. This technique yields RNA which can be utilized for downstream applications—including quantitative PCR and RNA sequencing—at relatively low cost and without the need for specialized equipment or fluorescently labeled cells. Adding to its utility, we demonstrate that cells can be isolated largely intact, retaining their processes, enabling analysis of extrasomatic proteins. We propose that magnetic cell sorting will prove to be a highly useful technique for the examination of cell specific CNS populations.