ABSTRACT Direct lineage reprogramming represents a promising strategy to convert somatic cells into neurons, offering regenerative potential. While transcription factor‐based approaches have been extensively studied, the role of post‐transcriptional regulation, particularly alternative splicing (AS), in neuronal fate acquisition remains poorly defined. Here, we demonstrate that the concurrent knockdown of the splicing regulator PTBP2 and the barrier protein p53 enhances the neuronal conversion of human retinal pigment epithelial (hRPE‐19) cells when combined with ASCL1 and miR‐9/9*‐124 (AMnp). Transcriptomic and splicing analyzes reveal that PTBP2 depletion induces widespread AS changes, most notably promoting near‐complete inclusion of exon 36 in the ANK2 gene, which encodes a key regulator of axon initial segment assembly. Functional and rescue assays confirm that loss of exon 36 significantly impairs neuronal induction, whereas re‐expression restores neuronal conversion efficiency, establishing ANK2 isoform switching as a mechanistic requirement for reprogramming. Moreover, photoreceptor markers expression in AMnp‐reprogrammed neurons suggests partial photoreceptor‐like features potentially reflecting residual epigenetic memory, with chromatin remodeling potentially cooperating with splicing to influence subtype specification. These findings identify the PTBP2 ‐ ANK2 splicing axis as an isoform‐specific molecular switch for RPE‐to‐neuron conversion, offering a strategy to enhance the precision and efficiency of neuronal reprogramming. image
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