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.
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.
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.
It has been shown previously in a number of systems that after an extended block of activity, synaptic strength is increased. We found that an extended block of synaptic activity at the mouse neuromuscular junction, using a tetrodotoxin cuff in vivo, increased synaptic strength by prolonging the evoked endplate current (EPC) decay. Prolongation of EPC decay was accompanied by only modest prolongation of spontaneous miniature EPC (MEPC) decay. Prolongation of EPC decay was reversed when quantal content was lowered by reducing extracellular calcium. These findings suggested that the cause of EPC prolongation was presynaptic in origin. However, when we acutely inhibited fetal-type acetylcholine receptors (AChRs) using a novel peptide toxin (αA-conotoxin OIVA[K15N]), prolongation of both EPC and MEPC decay were reversed. We also blocked synaptic activity in a mutant strain of mice in which persistent muscle activity prevents upregulation of fetal-type AChRs. In these mice, there was no prolongation of EPC decay. We conclude that upregulation of fetal-type AChRs after blocking synaptic activity causes modest prolongation of MEPC decay that is accompanied by much greater prolongation of EPC decay. This might occur if acetylcholine escapes from endplates and binds to extrajunctional fetal-type AChRs only during large, evoked EPCs. Our study is the first to demonstrate a functional role for upregulation of extrajunctional AChRs.
Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate, N-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (1H), with excellent in vitro UT inhibitory activity at the submicromolar level. The half maximal inhibitory concentrations of 1H against UT-B in mouse, rat, and human erythrocyte were 1.60, 0.64, and 0.13 μmol/L, respectively. Further investigation suggested that 8 μmol/L 1H more powerfully inhibited UT-A1 at a rate of 86.8% than UT-B at a rate of 73.9% in MDCK cell models. Most interestingly, we found for the first time that oral administration of 1H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats. Additionally, 1H did not exhibit apparent toxicity in vivo and in vitro, and possessed favorable pharmacokinetic characteristics. 1H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects.
by
Megan F. Duffy;
Timothy J. Collier;
Joseph R. Patterson;
Christopher J. Kemp;
Kelvin C. Luk;
Malu Tansey;
Katrina L. Paumier;
Nicholas M. Kanaan;
Luke D. Fischer;
Nicole K. Polinski;
Olivia L. Barth;
Jacob W. Howe;
Nishant N. Vaikath;
Nour K. Majbour;
Omar M. A. El-Agnaf;
Caryl E. Sortwell
Background: Converging evidence suggests a role for microglia-mediated neuroinflammation in Parkinson's disease (PD). Animal models of PD can serve as a platform to investigate the role of neuroinflammation in degeneration in PD. However, due to features of the previously available PD models, interpretations of the role of neuroinflammation as a contributor to or a consequence of neurodegeneration have remained elusive. In the present study, we investigated the temporal relationship of neuroinflammation in a model of synucleinopathy following intrastriatal injection of pre-formed alpha-synuclein fibrils (α-syn PFFS). Methods: Male Fischer 344 rats (N = 114) received unilateral intrastriatal injections of α-syn PFFs, PBS, or rat serum albumin with cohorts euthanized at monthly intervals up to 6 months. Quantification of dopamine neurons, total neurons, phosphorylated α-syn (pS129) aggregates, major histocompatibility complex-II (MHC-II) antigen-presenting microglia, and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactive microglial soma size was performed in the substantia nigra. In addition, the cortex and striatum were also examined for the presence of pS129 aggregates and MHC-II antigen-presenting microglia to compare the temporal patterns of pSyn accumulation and reactive microgliosis. Results: Intrastriatal injection of α-syn PFFs to rats resulted in widespread accumulation of phosphorylated α-syn inclusions in several areas that innervate the striatum followed by significant loss (~35%) of substantia nigra pars compacta dopamine neurons within 5-6 months. The peak magnitudes of α-syn inclusion formation, MHC-II expression, and reactive microglial morphology were all observed in the SN 2 months following injection and 3 months prior to nigral dopamine neuron loss. Surprisingly, MHC-II immunoreactivity in α-syn PFF injected rats was relatively limited during the later interval of degeneration. Moreover, we observed a significant correlation between substantia nigra pSyn inclusion load and number of microglia expressing MHC-II. In addition, we observed a similar relationship between α-syn inclusion load and number of microglia expressing MHC-II in cortical regions, but not in the striatum. Conclusions: Our results demonstrate that increases in microglia displaying a reactive morphology and MHC-II expression occur in the substantia nigra in close association with peak numbers of pSyn inclusions, months prior to nigral dopamine neuron degeneration, and suggest that reactive microglia may contribute to vulnerability of SNc neurons to degeneration. The rat α-syn PFF model provides an opportunity to examine the innate immune response to accumulation of pathological α-syn in the context of normal levels of endogenous α-syn and provides insight into the earliest neuroinflammatory events in PD.
Psychological stress can have devastating and lasting effects on a variety of behaviors, especially those associated with mental illnesses such as anxiety and depression. Animal models of chronic stress are frequently used to elucidate the mechanisms underlying the relationship between stress and mental health disorders and to develop improved treatment options. The current study expands upon a novel chronic stress paradigm for mice: predatory stress. The predatory stress model incorporates the natural predator-prey relationship that exists among rats and mice and allows for greater interaction between the animals, in turn increasing the extent of the stressful experience. In this study, we evaluated the behavioral effects of exposure to 15 days of predatory stress on an array of behavioral indices. Up to 2 weeks after the end of stress, adult male mice showed an increase of anxiety-like behaviors as measured by the open field and social interaction tests. Animals also expressed an increase in depressive-like behavior in the sucrose preference test. Notably, performance on the novel object recognition task, a memory test, improved after predatory stress. Taken as a whole, our results indicate that 15 exposures to this innovative predatory stress paradigm are sufficient to elicit robust anxiety-like behaviors with evidence of co-morbid depressive-like behavior, as well as changes in cognitive behavior in male mice.
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.
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.
Urea transporters are a family of urea-selective channel proteins expressed in multiple tissues that play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of urea transporter (UT)-B, UT-A1/A3, or all UTs leads to urea-selective diuresis, indicating that urea transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UTA1–knockout mouse model. Phenotypically, daily urine output in UT-A1–knockout mice was nearly 3-fold that of WT mice and 82% of all-UT–knockout mice, and the UT-A1–knockout mice had significantly lower urine osmolality than WT mice. After 24-h water restriction, acute urea loading, or high-protein (40%) intake, UT-A1–knockout mice were unable to increase urine-concentrating ability. Compared with all-UT–knockout mice, the UT-A1–knockout mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in urea-dependent urine-concentrating mechanisms, suggesting that UTA1 represents a promising diuretic target.