Ptbp1 knockdown induces conversion of rat spinal cord astrocytes into neuron like cells

Polypyrimidine tract-binding protein 1 (Ptbp1) is crucial in regulating neuronal differentiation and maturation through alternative splicing. Direct neuronal reprogramming via Ptbp1 knockdown has shown therapeutic effects in various neurodegenerative disease models. However, these findings are not reproducible when stringent lineage-tracing methods are used, and few studies have strictly examined the process of Ptbp1-mediated conversion in in vitro models. In this study, we knocked down Ptbp1 in rat spinal cord astrocytes using two different methods, i.e., short hairpin RNAs (shRNAs) and small interfering RNAs (siRNAs), without introducing small-molecule reprogramming agents or neural transcription factors. Over time, we observed astrocyte-to-neuron-like cell conversion, accompanied by neuron-like morphological and neuronal-specific immunofluorescent alterations in these cells. Our results also suggest that the initial state of the astrocytes primarily determines conversion efficiency, offering a reasonable explanation for the existing controversy. Reactive spinal cord astrocytes induced by lipopolysaccharide (LPS) showed lower conversion efficiency, but dexamethasone (DEX) partially reversed their reactive state, thereby restoring their capacity for neuronal conversion. Although the converted neuron-like cells gradually adopted the neuronal phenotype over time, microelectrode array (MEA) recordings indicated that they still exhibited immature neuronal characteristics and lacked full electrophysiological functionality. Treatment with smoothened agonist (SAG) significantly enhanced their maturation and electrophysiological properties. These findings suggest that Ptbp1 knockdown, combined with SAG, could provide a promising strategy for spinal cord injury repair in the future.