ESTABLISHING A NOVEL PROTOCOL FOR THE TRANSCRIPTOMIC PROFILING OF NEURONS ENCAPSULATED BY A PERINEURONAL NET IN THE HUMAN BRAIN

Perineuronal nets (PNN) are specialized extracellular matrix structures that predominantly enwrap parvalbumin-expressing interneurons in neocortex, stabilize their synapses, and contribute to the closure of critical periods of heightened brain plasticity during development. Our group previously reported PNN dysregulation in the ventromedial prefrontal cortex of individuals with a history of child abuse (CA). The molecular mechanisms underlying PNN regulation in humans remain largely unexplored, however. Here, we developed a protocol to characterize the transcriptome of cells enwrapped by PNNs in post-mortem human brain tissue, focusing on a cohort of individuals with a history of CA. We combined laser-capture microdissection (LCM) to isolate single neurons surrounded by PNNs with next-generation sequencing to profile their transcriptomes. Following targeted LCM, RNA was isolated from cells encapsulated by PNN and converted to cDNA using a non-commercial protocol optimized for the low RNA yields typical of LCM from post-mortem tissue. We then generated sequencing libraries and sequenced a pilot set on the Illumina NovaSeq platform. Finally, we implemented a bioinformatics pipeline to assess biological signals and technical quality metrics for each library. To our knowledge, this is the first protocol enabling transcriptomic profiling specifically of PNN+ neurons in post-mortem human brain, providing a framework to identify PNN-related components expressed by these cells and study how differences in genetic expression can relate to network dysfunctions associated with CA.