Reducing polypyrimidine tract­binding protein 1 fails to promote neuronal transdifferentiation on HT22 and mouse astrocyte cells under physiological conditions.

ABSTRACT

In contrast to prior findings that have illustrated the conversion of non-neuronal cells into functional neurons through the specific targeting of polypyrimidine tract-binding protein 1 (PTBP1), accumulated evidence suggests the impracticality of inducing neuronal transdifferentiation through suppressing PTBP1 expression in pathological circumstances. Therefore, the present study explored the effect of knocking down PTBP1 under physiological conditions on the transdifferentiation of mouse hippocampal neuron HT22 cells and mouse astrocyte (MA) cells. A total of 20 µM negative control small interfering (si)RNA and siRNA targeting PTBP1 were transfected into HT22 and MA cells using Lipo8000™ for 3 and 5 days, respectively. The expression of early neuronal marker ßIII-Tubulin and mature neuronal markers NeuN and microtubule-associated protein 2 (MAP2) were detected using western blotting. In addition, ßIII-tubulin, NeuN and MAP2 were labeled with immunofluorescence staining to evaluate neuronal cell differentiation in response to PTBP1 downregulation. Under physiological conditions, no significant changes in the expression of ßIII-Tubulin, NeuN and MAP2 were found after 3 and 5 days of knockdown of PTBP1 protein in both HT22 and MA cells. In addition, the immunofluorescence staining results showed no apparent transdifferentiation in maker levels and morphology. The results suggested that the knockdown of PTBP1 failed to induce neuronal differentiation under physiological conditions.