Genetic Ptbp1 depletion does not induce neuronal RNA splicing patterns in mature astrocytes (Bulk RNA-seq)

Neuronal reprogramming of astrocytes poses a promising therapeutic approach to replace degenerating cells with newly converted neurons. Recent studies reported depletion of polyprimidine tract binding protein PTBP1, a repressor of neuronal alternative splicing, could induce astrocyte to neuron conversion and rescue functional deficits in models of neurodegenerative disease, while others disputed the astrocytic origin of converted neurons. Mechanistic understanding of the conversion, or the lack thereof, requires investigating the transcriptomic effects of Ptbp1 loss of function in mature astrocytes on RNA splicing, in conjunction with lineage tracing, which have not yet been examined. Here, we used genetic depletion of Ptbp1 in adult Aldh1l1-Cre/ERT2 reporter mice to determine whether lineage traced Ptbp1 knockout astrocytes exhibited RNA splicing alterations underlying potential neuronal transdifferentiation. Our analysis employed an alternative genetic Ptbp1 knockout mouse model and an extended 12-week window of Ptbp1 knockout to match previous reports of positive conversion. We found no widespread induction of neuronal identity, despite detecting a minuscule fraction of Cre-positive cells with neuronal transcriptomic features following Ptbp1 deletion. Importantly, PTBP1 depletion in mature astrocytes induces splicing alternations unlike neuronal features of alternative splicing. These findings highlight the challenge of targeting astrocytes for neuronal conversion via Ptbp1 knockout and underscore the importance of cellular gene expression profiling and genetic lineage tracing for in vivo studies of cell type conversion.