Spatial analysis of in vivo neuronal reprogramming

Astrocyte-to-neuron lineage conversion, or direct neuronal reprogramming, is a potential therapeutic strategy for neurodegenerative disease. While neuronal reprogramming has been shown definitively in vitro, whether it occurs in vivo is under debate, partly due to the cis-effects of neurogenic transcription factor coding sequences on the glial fibrillary acid protein (GFAP) promoter, which switches downstream reporter expression from endogenous astrocytes to neurons. Here, we used spatial transcriptomics as a reporter agnostic method to assess the effects of expressing Ascl1, a basic-helix-loop-helix (bHLH) transcription factor, and Ascl1SA6, a mutated bHLH gene with enhanced neurogenic capacity, in cortical astrocytes in vivo. Adeno-associated viruses expressing iCre alone, Ascl1-t2a-iCre or Ascl1SA6-t2a-iCre from the GFAP promoter were injected into the motor cortex of hSOD1G93A mice, an amyotrophic lateral sclerosis (ALS) model that exhibits deep-layer neurodegeneration. Control iCre-transduced cells, visualized with a Rosa-zsGreen reporter, formed a distinct cluster associated with a pro-inflammatory transcriptional signature, including reactive astrocyte, microglia, macrophage and complement system genes. Conversely, Ascl1 and Ascl1SA6 activated gene regulatory networks comprised of neurogenic genes, including Zbtb18, Id2 and Id3 transcriptional regulators also identified as Ascl1 transcriptional targets in prior in vitro neuronal reprogramming studies. Strikingly, in silico knock-out of Zbtb18 alters expression of 27% of the genes within the human ASCL1 and ASCL1SA5 gene regulatory networks associated with neuronal reprogramming in vitro. Our spatially resolved transcriptomic data thus supports the notion that bHLH transcription factors promote neuronal lineage conversion when expressed in brain astrocytes in vivo, and identifies Zbtb18 as a new candidate neuronal conversion factor.