In several animal models of motor neuron disease, degeneration begins in the periphery. Clarifying the possible role of Schwann cells remains a priority. We recently showed that terminal Schwann cells (TSCs) exhibit abnormalities in postnatal mice that express mutations of the SOD1 enzyme found in inherited human motor neuron disease. TSC abnormalities appeared before disease-related denervation commenced and the extent of TSC abnormality at P30 correlated with the extent of subsequent denervation. Denervated neuromuscular junctions (NMJs) were also observed that lacked any labeling for TSCs. This suggested that SOD1 TSCs may respond differently than wildtype TSCs to denervation which remain at denervated NMJs for several months. In the present study, the response of SOD1 TSCs to experimental denervation was examined. At P30 and P60, SC-specific S100 labeling was quickly lost from SOD1 NMJs and from preterminal regions. Evidence indicates that this loss eventually becomes complete at most SOD1 NMJs before reinnervation occurs. The loss of labeling was not specific for S100 and did not depend on loss of activity. Although some post-denervation labeling loss occurred at wildtype NMJs, this loss was never complete. Soon after denervation, large cells appeared near SOD1 NMJ bands which colabeled for SC markers as well as for activated caspase-suggesting that distal SOD1 SCs may experience cell death following denervation. Denervated SOD1 NMJs viewed 7 days after denervation with the electron microscope confirmed the absence of TSCs overlying endplates. These observations demonstrate that SOD1 TSCs and distal SCs respond abnormally to denervation. This behavior can be expected to hinder reinnervation and raises further questions concerning the ability of SOD1 TSCs to support normal functioning of motor terminals.
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
Homeostatic regulation is essential for the maintenance of synaptic strength within the physiological range. The current study is the first to demonstrate that both induction and reversal of homeostatic upregulation of synaptic vesicle release can occur within seconds of blocking or unblocking acetylcholine receptors at the mouse neuromuscular junction. Our data suggest that the homeostatic upregula-tion of release is due to Ca2+-dependent increase in the size of the readily releasable pool (RRP). Blocking vesicle refilling prevented upregulation of quantal content (QC), while leaving baseline release relatively unaffected. This suggested that the upregulation of QC was due to mobilization of a distinct pool of vesicles that were rapidly recycled and thus were dependent on continued vesicle refilling. We term this pool the “homeostatic reserve pool.” A detailed analysis of the time course of vesicle release triggered by a presynaptic action potential suggests that the homeostatic reserve pool of vesicles is normally released more slowly than other vesicles, but the rate of their release becomes similar to that of the major pool during homeostatic upregulation of QC. Remarkably, instead of finding a generalized increase in the recruitment of vesicles into RRP, we identified a distinct homeostatic reserve pool of vesicles that appear to only participate in synchronized release following homeostatic upregulation of QC. Once this small pool of vesicles is depleted by the block of vesicle refilling, homeostatic upregulation of QC is no longer observed. This is the first identification of the population of vesicles responsible for the blockade-induced upregulation of release previously described.
In mice that express SOD1 mutations found in human motor neuron disease, degeneration begins in the periphery for reasons that remain unknown. At the neuromuscular junction (NMJ), terminal Schwann cells (TSCs) have an intimate relationship with motor terminals and are believed to help maintain the integrity of the motor terminal. Recent evidence indicates that TSCs in some SOD1 mice exhibit abnormal functional properties, but other aspects of possible TSC involvement remain unknown. In this study, an analysis of TSC morphology and number was performed in relation to NMJ innervation status in mice which express the G93A SOD1 mutation. At P30, all NMJs of the fast medial gastrocnemius (MG) muscle were fully innervated by a single motor axon but 50% of NMJs lacked TSC cell bodies and were instead covered by the processes of Schwann cells with cell bodies located on the preterminal axons. NMJs in P30 slow soleus muscles were also fully innervated by single motor axons and only 5% of NMJs lacked a TSC cell body. At P60, about 25% of MG NMJs were denervated and lacked labeling for TSCs while about 60% of innervated NMJs lacked TSC cell bodies. In contrast, 96% of P60 soleus NMJs were innervated while 9% of innervated NMJs lacked TSC cell bodies. The pattern of TSC abnormalities found at P30 thus correlates with the pattern of denervation found at P60. Evidence from mice that express the G85R SOD1 mutation indicate that TSC abnormalities are not unique for mice that express G93A SOD1 mutations. These results add to an emerging understanding that TSCs may play a role in motor terminal degeneration and denervation in animal models of motor neuron disease.
Patients with Charcot–Marie–Tooth Type 2D (CMT2D), caused by dominant mutations in Glycl tRNA synthetase (GARS), present with progressive weakness, consistently in the hands, but often in the feet also. Electromyography shows denervation, and patients often report that early symptoms include cramps brought on by cold or exertion. Based on reported clinical observations, and studies of mouse models of CMT2D, we sought to determine whether weakened synaptic transmission at the neuromuscular junction (NMJ) is an aspect of CMT2D. Quantal analysis of NMJs in two different mouse models of CMT2D (GarsP278KY, GarsC201R), found synaptic deficits that correlated with disease severity and progressed with age. Results of voltage-clamp studies revealed presynaptic defects characterized by: (1) decreased frequency of spontaneous release without any change in quantal amplitude (miniature endplate current), (2) reduced amplitude of evoked release (endplate current) and quantal content, (3) age-dependent changes in the extent of depression in response to repetitive stimulation, and (4) release failures at some NMJs with high-frequency, long-duration stimulation. Drugs that modify synaptic efficacy were tested to see whether neuromuscular performance improved. The presynaptic action of 3,4 diaminopyridine was not beneficial, whereas postsynaptic-acting physostigmine did improve performance. Smaller mutant NMJs with correspondingly fewer vesicles and partial denervation that eliminates some release sites also contribute to the reduction of release at a proportion of mutant NMJs. Together, these voltage-clamp data suggest that a number of release processes, while essentially intact, likely operate suboptimally at most NMJs of CMT2D mice.
Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.