Regulation of the Intermediate Filament Protein Nestin at Rodent Neuromuscular Junctions by Innervation and Activity

Abstract

The intermediate filament nestin is localized postsynaptically at rodent neuromuscular junctions. The protein forms a filamentous network beneath and between the synaptic gutters, surrounds myofiber nuclei, and is associated with Z-discs adjacent to the junction. In situ hybridization shows that nestin mRNA is synthesized selectively by synaptic myonuclei. Although weak immunoreactivity is present in myelinating Schwann cells that wrap the preterminal axon, nestin is not detected in the terminal Schwann cells (tSCs) that cover the nerve terminal branches. However, after denervation of muscle, nestin is upregulated in tSCs and in SCs within the nerve distal to the lesion site. In contrast, immunoreactivity is strongly downregulated in the muscle fiber. Transgenic mice in which the nestin neural enhancer drives expression of a green fluorescent protein (GFP) reporter show that the regulation in SCs is transcriptional. However, the postsynaptic expression occurs through enhancer elements distinct from those responsible for regulation in SCs. Application of botulinum toxin shows that the upregulation in tSCs and the loss of immunoreactivity in muscle fibers occurs with blockade of transmitter release. Extrinsic stimulation of denervated muscle maintains the postsynaptic expression of nestin but does not affect the upregulation in SCs. Thus, a nestin-containing cytoskeleton is promoted in the postsynaptic muscle fiber by nerve-evoked muscle activity but suppressed in tSCs by transmitter release. Nestin antibodies and GFP driven by nestin promoter elements serve as excellent markers for the reactive state of SCs. Vital imaging of GFP shows that SCs grow a dynamic set of processes after denervation.

Identity, developmental restriction and reactivity of extralaminar cells capping mammalian neuromuscular junctions

“…Next, we partially denervated TS muscles in adult mice, then immunostained them 1-6 days later with nestin antibody, a marker Journal of Cell Science 121 (23) of reactive terminal Schwann cells (Hayworth et al, 2006;Kang et al, 2007). As expected, nestin was expressed only in muscle fibres at innervated neuromuscular junctions; terminal Schwann cells remained nestin-negative for at least 2 days after denervation (Fig.…”

Biology and pathology of nonmyelinating Schwann cells

“…There were limitations in the stains that were available to distinguish among these cells at the NMJ and, as argued above for Remak fibers, an inability to resolve them in light microscopy. Now from electron micrographs (Birks et al, 1960a;Heuser and Reese, 1973;Slater, 1968, 1970;Peper et al, 1974;Robertson, 1956), the filling of individual cells with fluorescent dyes using microelectrodes (McMahan et al, 1972), and most recently from immunostaining (Astrow et al, 1994(Astrow et al, , 1998Reynolds and Woolf, 1992;Young et al, 2005) and use of GFP transgenes (Kang et al, 2000(Kang et al, , 2007Zuo et al, 2004) (see Fig. 2), it is apparent that NMSCs cover the synapse.…”

“…These processes grow steadily with time and are quite dynamic (Kang et al, 2007); some of these processes can reach lengths of up to 800 lm. This process growth occurs in concert with the expression by the cells of the protein GAP-43 (Mehta et al, 1993).…”

Nestin Is Not Essential for Development of the CNS But Required for Dispersion of Acetylcholine Receptor Clusters at the Area of Neuromuscular Junctions

“…؊/؊ mice Both nestin and Cdk5 are expressed at the NMJ (Vaittinen et al, 1999;Fu et al, 2001;Kang et al, 2007), and the latter has recently been shown to play a major role in dispersion of AChR clusters during NMJ development (Delalle et al, 1997;Fu et al, 2005). In vitro studies have also shown that nestin serves as a scaffold for Cdk5 and through this interaction can regulate Cdk5/p35 signaling (Sahlgren et al, 2006).…”

“…In addition, nestin has been detected in the neuromuscular junction (NMJ) (Vaittinen et al, 1999;Kang et al, 2007) and renal podocytes (Chen et al, 2006).…”