Intercellular Transfer of PTBP1 Drives Human Neural Stem Cell Fate

During fetal brain development, temporally defined alternative splicing (AS) programs control human neural stem cell (hNSC) self-renewal and differentiation, thereby regulating neurogenesis and gliogenesis. Polypyrimidine tract-binding protein 1 (PTBP1) is a master regulator of AS during neurogenesis; however, its functional role and dynamics in hNSCs remain largely unexplored. Here, we investigate the cellular and molecular functions, nucleocytoplasmic distribution, and intercellular trafficking of PTBP1 in primary hNSCs. We found that PTBP1 knockdown (KD) alters self-renewal capacity, mitochondrial dynamics and membrane potential, lipid droplet abundance, and PTBP2 expression. RNA sequencing analysis revealed that PTBP1 depletion affects the expression profiling of hundreds of coding and non-coding genes, collectively orchestrating a neuronal differentiation program. Super-resolution {tau}-STED microscopy and live-cell imaging demonstrated that PTBP1 localizes not only to the nucleus but also to the cytoplasm, tunneling nanotubes (TNTs), migrasomes, and extracellular vesicles (EVs). Co-culture experiments and EV uptake assays showed that cytosolic PTBP1 can be transferred between hNSCs and delivered to the nuclei of recipient cells via TNTs and EVs. Moreover, EVs were found to contain specific and previously uncharacterized PTBP1 isoforms and were efficiently transferred to PTBP1-KD cells, rescuing their proliferative capacity. Analysis of the mouse brain reveals the presence of PTBP1 in the V-SVZ and within TNT-like structures connecting NSCs, suggesting a role for TNT-mediated PTBP1 trafficking in vivo. Together, these findings uncover previously unrecognized roles for PTBP1 in hNSCs and provide the first evidence that PTBP1 can be transferred between hNSCs via TNTs and EVs, revealing a novel mechanism by which hNSCs may regulate fetal neurogenesis. Graphical AbstractA: PTBP1 regulates hNSC fate by controlling cell proliferation, lipid droplet dynamics, mitochondrial function, and post-transcriptional programs involved in neuronal commitment. B: Cytosolic PTBP1 is transferred between hNSCs via tunneling nanotubes (TNTs) and extracellular vesicles (EVs). Abbreviations: LV, lateral ventricle; aRG, apical radial glial cells; bRG, basal radial glial cells; V-SVZ, ventricular-subventricular zone; EVs, extracellular vesicles; TNTs, tunneling nanotubes.