PTBP2 attenuation facilitates fibroblast to neuron conversion by promoting alternative splicing of neuronal genes

Area of Science:

• Neuroscience
• Molecular Biology
• Cell Biology

Background:

• Direct conversion of somatic cells to neurons offers a promising avenue for regenerative medicine.
• However, the efficiency and underlying mechanisms of this transdifferentiation process remain incompletely understood.

Purpose of the Study:

• To elucidate the molecular mechanisms governing the direct conversion of human skin fibroblasts into neurons.
• To identify key regulators, particularly RNA splicing factors, that enhance transdifferentiation efficiency.

Main Methods:

• Utilized knockdown and overexpression strategies for PTBP2 and RBFOX3 (NeuN) in human skin fibroblasts.
• Employed a cocktail of transcription factors (ASCL1, MIR9/9*-124, p53 shRNA) to induce neuronal transdifferentiation.
• Conducted longitudinal RNA sequencing to analyze gene expression and alternative splicing events.

Main Results:

• Knockdown of PTBP2 significantly enhanced the efficiency of fibroblast-to-neuron transdifferentiation, yielding predominantly GABAergic neurons.
• RBFOX3 (NeuN) was identified as a critical factor; its knockdown abrogated transdifferentiation, while its overexpression enhanced it.
• PTBP2 attenuation promoted neuron-specific alternative splicing in genes crucial for neuronal function, an effect modulated by RBFOX3 levels.

Conclusions:

• PTBP2 plays a crucial role in regulating alternative splicing during neuronal differentiation.
• RBFOX3 acts in concert with PTBP2 to facilitate neuron-specific alternative splicing, thereby enhancing direct fibroblast-to-neuron conversion.
• This study reveals a novel mechanism involving PTBP2 and RBFOX3 in optimizing direct neuronal reprogramming.