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Astrocyte Transcriptomics in a Three-Dimensional Tissue-Engineered Rostral Migratory Stream

ABSTRACT: The glial tube is a longitudinal structure predominantly composed of densely bundled, aligned astrocytes that projects from subventricular zone (SVZ) to olfactory bulb. Neural precursor cells (NPCs) generated in the SVZ migrate through this glial tube – referred to as the rostral migratory stream (RMS) – to replace olfactory bulb interneurons in the mammalian brain. RMS astrocytes have distinct morphological and functional characteristics. These characteristics facilitate the unique purpose of the RMS as an endogenous living scaffold directing NPC migration and maturation. However, the transcriptomic factors underlying these unique structure-function attributes versus standard stellate astrocytes have not been examined. We previously developed biofabrication techniques to create the first tissue-engineered rostral migratory stream (TE-RMS) that replicates key features of the glial tube in vivo. We have shown that TE-RMS astrocytes exhibit elongated nuclei, longitudinally aligned intermediate filaments, and enrichment of key functional proteins – cytoarchitectural and surface features characteristic of native RMS astrocytes. In the current study, we performed RNAseq on TE-RMS astrocytes in comparison to planar astrocyte cultures to identify gene expression patterns that may underlie their profound morphological and functional differences. Remarkably, we found 4008 differentially expressed genes in TE-RMS astrocytes, with 2076 downregulated (e.g. LOC690251, ccn5) and 1932 upregulated (e.g. lrrc45, cntn1) compared to planar astrocytes. Moreover, there were 256 downregulated and 91 upregulated genes with >3-fold change. We also conducted analyses of gene sets related to cytoskeleton and nuclear structure, revealing greatest enrichment of actin-related components. Overall, the TE-RMS offers a platform to study interplay between transcriptomic and cytoarchitectural dynamics in a unique astrocyte population.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE309109 | GEO | 2025/12/04

REPOSITORIES: GEO

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Project description:The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 157 intraventricularly injected barcoded AAV variants profiled by transcriptome sequencing. Transcriptome analysis identified two synthetic capsids, AAV9_A2 and AAV1_P5 transducing active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of promoter and dose of injected viral genomes enabled targeting of 57% of the NSC compartment. Importantly, transduced NSC readily produced neurons. The present study identifies AAV variants with a high specificity and transduction efficiency of adult NSCs thereby paving the way for preclinical testing of regenerative gene therapy. We identified a synthetic Adeno-associated virus (AAV) capsid, AAV1_P5, that transduces active and quiescent neural stem cells (NSCs). In order to fully characterize the identity of AAV1_P5-transduced cells, as well as potential changes arising by the AAV-transduction itself, we profiled these cells and untransduced ones from the same mouse by single cell RNA sequencing. To this end, three months-old eYFP-reporter mice were injected with 109 vg/mouse AAV1_P5 harboring the CMV_Cre construct. Transduction induces expression of eYFP. 37 days post injection, we isolated cells from the v-SVZ and other brain regions. More precisely, we isolated labeled cells of the ventricular-subventricular zone and the striatum, rostral migratory stream (RMS) and olfactory bulb, here referred to as rest of the brain (RoB). To capture the remaining unlabeled cells of the NSC-lineage in the v-SVZ, we also isolated GLAST+ v-SVZ cells. Two samples of two pooled mice each were subjected to single cell RNA sequencing.

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Astrocyte Transcriptomics in a Three-Dimensional Tissue-Engineered Rostral Migratory Stream

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