背根神經元巢蛋白異構物Nes-S：辨識、鑑定與細胞保護功能

Abstract

Nestin is a type VI intermediate filament (IF) protein predominantly expressed in neurogenic and myogenic stem cells. Nestin has hardly been discovered in mature neurons, and its isoforms have never been reported. In this study, the first nestin isoform, Nes-S, was identified by RT-PCR on RNA obtained from single neurons of dorsal root ganglia (DRG) of adult rats. In this isoform, the majority of the nestin C-terminal domain, including the whole repeat region, is removed. This splicing also changes the open reading frame, and results in a novel 32-aa tail sequence and a premature stop codon. The Nes-S protein is 408-amino acid (aa) in length, containing a complete 7-aa N-terminal domain, a complete 307-aa rod domain, and a 94-aa C-terminal domain, with a predicted molecular weight of 45.9 kD. Nes-S cannot form filaments by itself in cytoplasmic IF-free SW13 cells. Instead, it co-assembles into filaments with vimentin in vimentin+ SW13 cells, and with peripherin and neurofilament heavy chain in both N2a neuroblastoma cells and primarily cultivated DRG neurons. To study its distribution pattern, an isoform specific antibody was generated against a peptide derived from the novel tail sequence of Nes-S. Immunoblotting assay with this antibody showed that its expression first appears in DRG at postnatal day 5 and persist to adulthood. In addition, immunoblotting and immunofluorescence microscopy with the same antibody showed that, among the adult neural tissues we examined, expression of Nes-S was only observed in neurons whose processes extend towards the limbs or the periphery of body, including neurons of DRG, trigeminal ganglia, superior cervical ganglia, as well as motor and sympathetic neurons of the thoracic spinal cord. Finally, we showed by MTT assay that Nes-S exerts a cytoprotective function when transfected into N2a neuroblastoma cells, and knockdown of endogenous Nes-S impaired the survival of primary DRG neurons during prolonged cultures. For the last decade, emerging evidences have revealed that alternative splicing serves as an additional way to regulate IF functions, and the IF isoforms may be involved in cellular stress responses as well as the pathogenesis of neural degenerative diseases. Nes-S is a new neuronal IF protein that exerts a cytoprotective function in mature neurons, and future studies on this protein may shed new lights on the research of neural degenerative diseases.