Neurons transfected with the noneffective shRNA vector were used as control. to adulthood. Among the adult tissues we examined, the expression of Nes-S PE859 is restricted to the sensory and motor PE859 neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons. Keywords: Alternative Splicing, Cytoskeleton, Intermediate Filaments, Neurons, Protein Aggregation, Dorsal Root Ganglia, Nestin Isoform, Sensory and Motor Neurons Introduction Nestin is a type VI intermediate filament (IF)2 protein (1). It is also known as IFAP-70/280kD for hamster nestin and transitin for avian nestin (2C5). It is widely accepted that the IF cytoskeleton maintains the mechanical stability of most animal cells (6C8). This function is further enhanced by its cross-linking to actin filaments and microtubules, as well as to junctional complexes via intermediate filament-associated proteins (IFAPs), such as plectin/IFAP300 and the plakin family proteins (9C14). All of the IF proteins contain a central rod domain, which mediates coiled-coil dimer formation and leads to the assembly of the IFs. The rod domain is flanked at both ends with nonhelical sequences, the so-called N-terminal head and C-terminal tail domains, which are different in length and properties among IF proteins (15C18). Based on gene structure as well as PE859 nucleotide sequence homology of the region coding for the rod domain, the IF proteins are classified into six subtypes (5, 16, 19). Nestin and its homologue synemin (5, 20) comprise the type VI IF protein family, which is characterized by a distinct intron-exon pattern and a long C-terminal tail that often contains tandem repeat motifs (1, 5, 21). The rat nestin protein is 1893 amino acids (aa) in length, comprising a short 7-aa N-terminal head, a 307-aa rod domain, and a long, 1579-aa C-terminal tail that contains a 536-aa tandem repeat region (aa 641C1176) (1, 4, 22). The rat nestin gene consists of four exons and three introns, with the first two introns interrupting its rod domain in a manner similar to the three type IV neurofilament proteins. Thus, it has been proposed that nestin should be classified as a type IV IF protein (19). However, the presence of the distinct third intron within the C-terminal domain, as well as the low sequence homology between the rod domain of nestin and neurofilament proteins, favors its classification, together with synemin, as a distinct type VI IF protein family (1, 3, 5, 21, 22). It is well known that nestin is mainly Rabbit Polyclonal to CADM2 expressed in cells with high proliferating capacity, predominately in myogenic and neurogenic progenitor cells (for review, see Refs. 23 and 24). Enhancer elements that specifically regulate nestin expression in myogenic and neurogenic progenitor cells are located, respectively, in the first and second intron (25C27). In primarily cultivated rat aortic smooth muscle cells, nestin expression is induced by epidermal growth factor (EGF), thrombin, and platelet-derived growth factor (PDGF) (28, 29). During development of the nervous system, nestin is predominately expressed in neuronogenic and gliogenic progenitors. In central nervous system (CNS), the expression of nestin ceases in most mature neurons and astrocytes (1, 30, 31). In adult CNS, nestin can only be observed in neural progenitors at regions undergoing active neurono- and gliogenesis, such as hippocampus and olfactory bulb, as well as a subset of neurons located in the basal forebrain (23, 30, 32). In rat peripheral nervous system, nestin expression ceases in differentiating neurons as soon as they express neurofilament proteins during early neurogenesis, but it is still expressed in satellite and Schwann cells of adult rats (23, 30). Notably, it is well known that nestin is re-expressed in the proliferating reactive astrocytes (33C35). However, the expression of nestin is rarely reported in adult neurons under both normal.