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Although nestin has served as a marker of neural stem/progenitor cells for close to twenty years, its function is still poorly understood. During development, this intermediate filament protein is expressed in many different progenitors including those of the central nervous system, heart, skeletal muscle and kidney. The adult expression of nestin is mainly restricted to the subependymal zone and dentate gyrus of the brain, the neuromuscular junction and renal podocytes. I have used two approaches of gain of function and loss of function to elucidate the role of nestin in vivo. Although I was able to generate transgenic lines in which the transgene was ubiquitously expressed at the RNA level, over-expression of nestin at the protein level was not achieved possibly due to post transcriptional regulation of this gene. My data from loss of function approach indicates that nestin-deficient mice have impaired coordination. Balance and muscle strength are not affected and there are no apparent anatomical defects. I found that nestin deficiency is compatible with normal development of the central nervous system but results in abnormal clustering of acetylcholine receptors in the neuromuscular junctions, similar to the phenotype described for deficiency of cyclin-dependent kinase 5 (Cdk5) a candidate downstream effector of nestin. In renal podocytes, where both nestin and Cdk5 are normally expressed, we found reduced branching and abnormally contoured podocyte processes. To further connect the phenotype of nestin deficiency to Cdk5, I demonstrated that nestin deficiency can rescue maintenance of acetylcholine receptor clusters in the absence of agrin, similar to Cdk5/agrin double knockouts, indicating that the observed nestin deficiency phenotypes are the consequence of aberrant Cdk5 activity.

Although nestin has served as a marker of neural stem/progenitor cells for close to twenty years, its function is still poorly understood. During development, this intermediate filament protein is expressed in many different progenitors including those of the central nervous system, heart, skeletal muscle and kidney. The adult expression of nestin is mainly restricted to the subependymal zone and dentate gyrus of the brain, the neuromuscular junction and renal podocytes. I have used two approaches of gain of function and loss of function to elucidate the role of nestin in vivo. Although I was able to generate transgenic lines in which the transgene was ubiquitously expressed at the RNA level, over-expression of nestin at the protein level was not achieved possibly due to post transcriptional regulation of this gene. My data from loss of function approach indicates that nestin-deficient mice have impaired coordination. Balance and muscle strength are not affected and there are no apparent anatomical defects. I found that nestin deficiency is compatible with normal development of the central nervous system but results in abnormal clustering of acetylcholine receptors in the neuromuscular junctions, similar to the phenotype described for deficiency of cyclin-dependent kinase 5 (Cdk5) a candidate downstream effector of nestin. In renal podocytes, where both nestin and Cdk5 are normally expressed, we found reduced branching and abnormally contoured podocyte processes. To further connect the phenotype of nestin deficiency to Cdk5, I demonstrated that nestin deficiency can rescue maintenance of acetylcholine receptor clusters in the absence of agrin, similar to Cdk5/agrin double knockouts, indicating that the observed nestin deficiency phenotypes are the consequence of aberrant Cdk5 activity.

Hydrogen peroxide (H2O2), a membrane-permeable reactive oxygen species, is continuously produced by mitochondrial respiration, the membrane-associated NADPH oxidase complex, xathine oxidase catalyzed reaction. Although the cytotoxic effect of H2O2 is well documented, the role of H2O2 in synapse formation if still in its infancy. Here we test the role of H2O2 on the frequency of spontaneous synaptic currents (SSCs) at developing Xenopus neuromuscular synapse by using whole-cell patch clamp recording. Bath application of H2O2 dose-dependently enhances the frequency of spontaneous synaptic currents (SSC frequency). Treatment of the culture with membrane-permeable antioxidants N-acetylcysteine and sodium pyruvate significantly decreased SSC frequency, indicating endogenous reactive oxygen species play important roles in the regulation of spontaneous ACh release. Bath application of membrane non-permeable catalase, which breaks down H2O2 specifically, has no significant effect on SSC frequency, suggesting H2O2 is not an intercellular signaling molecule being produced and released from postsynaptic myocyte and affects the neurotransmitter release of presynaptic motoneuron. Much to our surprise is that the SSC frequency was significantly decreased while catalase was vii loaded into the myocyte through recording pipette. Furthermore, the SSC frequency facilitation induced by exogenously applied H2O2 was completely hampered while catalase was loaded into the myocyte. These results indicate although endogenous H2O2 in myocyte plays a crucial role on SSC frequency facilitation, this facilitation on the neurotransmitter release of presynaptic motoneuron is achieved through a retrograde factor other than H2O2 itself. Treatment of the culture with inhibitor of either NADPH oxidase does not have significant effect on SSC frequency. Bath application of mitochondria complex I, II and xanthine oxidase inhibitor significantly decreased SSC frequency, suggesting H2O2 derived from xanthine oxidase and mitochondria is responsible for the regulation of SSC frequency. Bath application of translation blocker anisomycin and cycloheximide could not attenuate the facilitation of H2O2. Addition of IGF-1 receptor inhibitor JB-1 to the culture significantly attenuated SSC frequency. Overall, our current results suggest that xanthine oxidase activity-derived H2O2 in myocyte induce the release of IGF-1 which retrogradely enhance the spontaneous neurotransmitter release from presynaptic motoneuron. Since synaptic activity is crucial in synaptogenesis and synapse maturation, results form viii our studies have shed some light on the molecular mechanism of the formation of developing neuromuscular synapse.