Exposure to 6-PPD Quinone at Environmentally Relevant Concentrations Causes Abnormal Locomotion Behaviors and Neurodegeneration in Caenorhabditis elegans.

6-PPD quinone (6-PPDQ) can be transformed from 6-PPD through ozonation. Nevertheless, the potential neurotoxicity of 6-PPDQ after long-term exposure and the underlying mechanism are largely unclear. In Caenorhabditis elegans, we here observed that 0.1-10 µg/L of 6-PPDQ caused several forms of abnormal locomotion behaviors. Meanwhile, the neurodegeneration of D-type motor neurons was observed in 10 µg/L of 6-PPDQ-exposed nematodes. The observed neurodegeneration was associated with the activation of the Ca2+ channel DEG-3-mediated signaling cascade. In this signaling cascade, expressions of deg-3, unc-68, itr-1, crt-1, clp-1, and tra-3 were increased by 10 µg/L of 6-PPDQ. Moreover, among genes encoding neuronal signals required for the control of stress response, expressions of jnk-1 and dbl-1 were decreased by 0.1-10 µg/L of 6-PPDQ, and expressions of daf-7 and glb-10 were decreased by 10 µg/L of 6-PPDQ. RNAi of jnk-1, dbl-1, daf-7, and glb-10 resulted in the susceptibility to 6-PPDQ toxicity in decreasing locomotory ability and in inducing neurodegeneration, suggesting that JNK-1, DBL-1, DAF-7, and GLB-10 were also required for the induction of 6-PPDQ neurotoxicity. Molecular docking analysis further demonstrated the binding potential of 6-PPDQ to DEG-3, JNK-1, DBL-1, DAF-7, and GLB-10. Together, our data suggested the exposure risk of 6-PPDQ at environmentally relevant concentrations in causing neurotoxicity in organisms.

Notoginsenoside R1 alleviates spinal cord injury by inhibiting oxidative stress, neuronal apoptosis, and inflammation via activating the nuclear factor erythroid 2 related factor 2/heme oxygenase-1 signaling pathway.

The secondary injury plays a vital role in the development of spinal cord injury (SCI), which is characterized by the occurrence of oxidative stress, neuronal apoptosis, and inflammatory response. Notoginsenoside R1 (NGR1) has been involved in the modulation of antioxidative stress and anti-inflammatory response. However, its roles in SCI-induced injury are still unknown. We explored the therapeutic effect of NGR1 and its underlying mechanism after SCI by using behavioral, biochemical, and immunohistochemical techniques. The administration of NGR1 after SCI enhanced the neurological function, and mitigated tissue damage and motor neuron loss than those in SCI + vehicle group. Meanwhile, significantly increased expression of Nrf2 protein and HO-1 protein was found in the SCI + NGR1 group compared with those in the SCI + vehicle group. In addition, the inhibitory effects of oxidative stress, apoptotic neuron ratio, and neuronal inflammation in the SCI + NGR1 group can be partially reversed when the Nrf2/HO-1 signaling pathway was inhibited by ML385. Our results indicate that the administration of NGR1 can attenuate oxidative stress, neuronal apoptosis, and inflammation by activating the Nrf2/HO-1 signaling pathway after SCI, thereby improving neurological function.

VEGF is an essential retrograde trophic factor for motoneurons.

VEGF was initially discovered due to its angiogenic activity and therefore named "vascular endothelial growth factor." However, its more recently discovered neurotrophic activity may be evolutionarily more ancient. Our previous work showed that all the changes produced by axotomy on the firing activity and synaptic inputs of abducens motoneurons were completely restored after VEGF administration. Therefore, we hypothesized that the lack of VEGF delivered by retrograde transport from the periphery should also affect the physiology of otherwise intact abducens motoneurons. For VEGF retrograde blockade, we chronically applied a neutralizing VEGF antibody to the lateral rectus muscle. Recordings of extracellular single-unit activity and eye movements were made in alert cats before and after the application of the neutralizing antibody. Our data revealed that intact, noninjured abducens motoneurons retrogradely deprived of VEGF exhibited noticeable changes in their firing pattern. There is a general decrease in firing rate and a significant reduction in eye position and eye velocity sensitivity (i.e., a decrease in the tonic and phasic components of their discharge, respectively). Moreover, by means of confocal immunocytochemistry, motoneurons under VEGF blockade showed a marked reduction in the density of afferent synaptic terminals contacting with their cell bodies. Altogether, the present findings demonstrate that the lack of retrogradely delivered VEGF renders abducens motoneurons into an axotomy-like state. This indicates that VEGF is an essential retrograde factor for motoneuronal synaptic drive and discharge activity.

Modified Industrial Three-Dimensional Polylactic Acid Scaffold Cell Chip Promotes the Proliferation and Differentiation of Human Neural Stem Cells.

In this study, we fabricated a three-dimensional (3D) scaffold using industrial polylactic acid (PLA), which promoted the proliferation and differentiation of human neural stem cells. An industrial PLA 3D scaffold (IPTS) cell chip with a square-shaped pattern was fabricated via computer-aided design and printed using a fused deposition modeling technique. To improve cell adhesion and cell differentiation, we coated the IPTS cell chip with gold nanoparticles (Au-NPs), nerve growth factor (NGF) protein, an NGF peptide fragment, and sonic hedgehog (SHH) protein. The proliferation of F3.Olig2 neural stem cells was increased in the IPTS cell chips coated with Au-NPs and NGF peptide fragments when compared with that of the cells cultured on non-coated IPTS cell chips. Cells cultured on the IPTS-SHH cell chip also showed high expression of motor neuron cell-specific markers, such as HB9 and TUJ-1. Therefore, we suggest that the newly engineered industrial PLA scaffold is an innovative tool for cell proliferation and motor neuron differentiation.

Collagen VI Regulates Motor Circuit Plasticity and Motor Performance by Cannabinoid Modulation.

Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystonia-related collagen α3 VI (COL6A3) C-terminal domain (CTD). These Col6a3CTT mice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner. Col6a3CTT mice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid (eCB) signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance in Col6a3CTT mice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control.SIGNIFICANCE STATEMENT Dystonia is a movement disorder characterized by involuntary movements. We previously identified genetic variants affecting a specific domain of the COL6A3 protein as a cause of dystonia. Here, we created mice lacking the affected domain and observed an analogous movement disorder. Using a protein interaction screen, we found that the affected COL6A3 domain mediates an interaction with the cannabinoid receptor 1 (CB1R). Concordantly, our COL6A3-deficient mice showed a deficit in synaptic plasticity linked to a deficit in cannabinoid signaling. Pharmacological cannabinoid augmentation rescued the motor impairment of the mice. Thus, cannabinoid augmentation could be a promising avenue for treating dystonia, and we have identified a possible molecular mechanism mediating this.

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

Nonsteroidal anti-inflammatory drug (ketoprofen) delivery differentially impacts phrenic long-term facilitation in rats with motor neuron death induced by intrapleural CTB-SAP injections.

Intrapleural injections of cholera toxin B conjugated to saporin (CTB-SAP) selectively eliminates respiratory (e.g., phrenic) motor neurons, and mimics motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Additionally, microglial density increases in the phrenic motor nucleus following CTB-SAP. This CTB-SAP rodent model allows us to study the impact of motor neuron death on the output of surviving phrenic motor neurons, and the underlying mechanisms that contribute to enhancing or constraining their output at 7 days (d) or 28d post-CTB-SAP injection. 7d CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) through the Gs-pathway (inflammation-resistant in naïve rats), while pLTF is elicited though the Gq-pathway (inflammation-sensitive in naïve rats) in control and 28d CTB-SAP rats. In 7d and 28d male CTB-SAP rats and controls, we evaluated the effect of cyclooxygenase-1/2 enzymes on pLTF by delivery of the nonsteroidal anti-inflammatory drug, ketoprofen (IP), and we hypothesized that pLTF would be unaffected by ketoprofen in 7d CTB-SAP rats, but pLTF would be enhanced in 28d CTB-SAP rats. In anesthetized, paralyzed and ventilated rats, pLTF was surprisingly attenuated in 7d CTB-SAP rats and enhanced in 28d CTB-SAP rats (both p < 0.05) following ketoprofen delivery. Additionally in CTB-SAP rats: 1) microglia were more amoeboid in the phrenic motor nucleus; and 2) cervical spinal inflammatory-associated factor expression (TNF-α, BDNF, and IL-10) was increased vs. controls in the absence of ketoprofen (p < 0.05). Following ketoprofen delivery, TNF-α and IL-10 expression was decreased back to control levels, while BDNF expression was differentially affected over the course of motor neuron death in CTB-SAP rats. This study furthers our understanding of factors (e.g., cyclooxygenase-1/2-induced inflammation) that contribute to enhancing or constraining pLTF and its implications for breathing following respiratory motor neuron death.

Blockade of 5-HT2 receptors suppresses rate of torque development and motor unit discharge rate during rapid contractions.

Serotonin (5-HT) is a neuromodulator that is critical for regulating the excitability of spinal motoneurons and the generation of muscle torque. However, the role of 5-HT in modulating human motor unit activity during rapid contractions has yet to be assessed. Nine healthy participants (23.7 ± 2.2 yr) ingested 8 mg of the competitive 5-HT2 antagonist cyproheptadine in a double-blinded, placebo-controlled, repeated-measures experiment. Rapid dorsiflexion contractions were performed at 30%, 50%, and 70% of maximal voluntary contraction (MVC), where motor unit activity was assessed by high-density surface electromyographic decomposition. A second protocol was performed where a sustained, fatigue-inducing dorsiflexion contraction was completed before undertaking the same 30%, 50%, and 70% MVC rapid contractions and motor unit analysis. Motor unit discharge rate (P < 0.001) and rate of torque development (RTD; P = 0.019) for the unfatigued muscle were both significantly lower for the cyproheptadine condition. Following the fatigue inducing contraction, cyproheptadine reduced motor unit discharge rate (P < 0.001) and RTD (P = 0.024), whereas the effects of cyproheptadine on motor unit discharge rate and RTD increased with increasing contraction intensity. Overall, these results support the viewpoint that serotonergic effects in the central nervous system occur fast enough to regulate motor unit discharge rate during rapid powerful contractions.NEW & NOTEWORTHY We have shown that serotonin activity in the central nervous system plays a role in regulating human motor unit discharge rate during rapid contractions. Our findings support the viewpoint that serotonergic effects in the central nervous system are fast and are most prominent during contractions that are characterized by high motor unit discharge rates and large amounts of torque development.

Evaluation of a 5-HT2B receptor agonist in a murine model of amyotrophic lateral sclerosis.

Degeneration of brainstem serotonin neurons has been demonstrated in ALS patients and mouse models and was found responsible for the development of spasticity. Consistent with involvement of central serotonin pathways, 5-HT2B receptor (5-HT2BR) was upregulated in microglia of ALS mice. Its deletion worsened disease outcome in the Sod1G86R mouse model and led to microglial degeneration. In ALS patients, a polymorphism in HTR2B gene leading to higher receptor expression in CNS, was associated with increased survival in patients as well as prevention of microglial degeneration. Thus, the aim of our study was to determine the effect of a 5-HT2BR agonist : BW723C86 (BW), in the Sod1G86R mouse model. Despite good pharmacokinetic and pharmacological profiles, BW did not ameliorate disease outcome or motor neuron degeneration in a fast progressing mouse model of ALS despite evidence of modulation of microglial gene expression.

Contingent intramuscular boosting of P2XR7 axis improves motor function in transgenic ALS mice.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder that leads to progressive degeneration of motor neurons and severe muscle atrophy without effective treatment. Most research on the disease has been focused on studying motor neurons and supporting cells of the central nervous system. Strikingly, the recent observations have suggested that morpho-functional alterations in skeletal muscle precede motor neuron degeneration, bolstering the interest in studying muscle tissue as a potential target for the delivery of therapies. We previously showed that the systemic administration of the P2XR7 agonist, 2'(3')-O-(4-benzoylbenzoyl) adenosine 5-triphosphate (BzATP), enhanced the metabolism and promoted the myogenesis of new fibres in the skeletal muscles of SOD1G93A mice. Here we further corroborated this evidence showing that intramuscular administration of BzATP improved the motor performance of ALS mice by enhancing satellite cells and the muscle pro-regenerative activity of infiltrating macrophages. The preservation of the skeletal muscle retrogradely propagated along with the motor unit, suggesting that backward signalling from the muscle could impinge on motor neuron death. In addition to providing the basis for a suitable adjunct multisystem therapeutic approach in ALS, these data point out that the muscle should be at the centre of ALS research as a target tissue to address novel therapies in combination with those oriented to the CNS.

Differences of Transport Activity of Arginine and Regulation on Neuronal Nitric Oxide Synthase and Oxidative Stress in Amyotrophic Lateral Sclerosis Model Cell Lines.

L-Arginine, a semi-essential amino acid, was shown to delay dysfunction of motor neurons and to prolong the lifespan, upon analysis of transgenic mouse models of amyotrophic lateral sclerosis (ALS). We investigated the transport function of arginine and neuronal nitric oxide synthase (nNOS) expression after pretreatment with L-arginine in NSC-34 hSOD1WT (wild-type, WT) and hSOD1G93A (mutant-type, MT) cell lines. [3H]L-Arginine uptake was concentration-dependent, voltage-sensitive, and sodium-independent in both cell lines. Among the cationic amino acid transporters family, including system y+, b0,+, B0,+, and y+L, system y+ is mainly involved in [3H]L-arginine transport in ALS cell lines. System b0,+ accounted for 23% of the transport in both cell lines. System B0,+ was found only in MT, and whereas, system y+L was found only in WT. Lysine competitively inhibited [3H]L-arginine uptake in both cell lines. The nNOS mRNA expression was significantly lower in MT than in WT. Pretreatment with arginine elevated nNOS mRNA levels in MT. Oxidizing stressor, H2O2, significantly decreased their uptake; however, pretreatment with arginine restored the transport activity in both cell lines. In conclusion, arginine transport is associated with system y+, and neuroprotection by L-arginine may provide an edge as a possible therapeutic target in the treatment of ALS.

cAMP controls a trafficking mechanism that maintains the neuron specificity and subcellular placement of electrical synapses.

Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here, we show that a developmental program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. We use live-cell imaging to visualize electrical synapses in vivo and an optogenetic assay to confirm that they are functional. In ventral A class (VA) motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments establish that cAMP regulates gap junction trafficking for the biogenesis of functional electrical synapses.

Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice.

Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na+ and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na+-permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca2+ release and turned off by Ca2+ reuptake through the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output.

Tocotrienol-Rich Vitamin E (Tocovid) Improved Nerve Conduction Velocity in Type 2 Diabetes Mellitus Patients in a Phase II Double-Blind, Randomized Controlled Clinical Trial.

Diabetic peripheral neuropathy (DPN) is the most common microvascular complication of diabetes that affects approximately half of the diabetic population. Up to 53% of DPN patients experience neuropathic pain, which leads to a reduction in the quality of life and work productivity. Tocotrienols have been shown to possess antioxidant, anti-inflammatory, and neuroprotective properties in preclinical and clinical studies. This study aimed to investigate the effects of tocotrienol-rich vitamin E (Tocovid SuprabioTM) on nerve conduction parameters and serum biomarkers among patients with type 2 diabetes mellitus (T2DM). A total of 88 patients were randomized to receive 200 mg of Tocovid twice daily, or a matching placebo for 12 months. Fasting blood samples were collected for measurements of HbA1c, renal profile, lipid profile, and biomarkers. A nerve conduction study (NCS) was performed on all patients at baseline and subsequently at 2, 6, 12 months. Patients were reassessed after 6 months of washout. After 12 months of supplementation, patients in the Tocovid group exhibited highly significant improvements in conduction velocity (CV) of both median and sural sensory nerves as compared to those in the placebo group. The between-intervention-group differences (treatment effects) in CV were 1.60 m/s (95% CI: 0.70, 2.40) for the median nerve and 2.10 m/s (95% CI: 1.50, 2.90) for the sural nerve. A significant difference in peak velocity (PV) was also observed in the sural nerve (2.10 m/s; 95% CI: 1.00, 3.20) after 12 months. Significant improvements in CV were only observed up to 6 months in the tibial motor nerve, 1.30 m/s (95% CI: 0.60, 2.20). There were no significant changes in serum biomarkers, transforming growth factor beta-1 (TGFß-1), or vascular endothelial growth factor A (VEGF-A). After 6 months of washout, there were no significant differences from baseline between groups in nerve conduction parameters of all three nerves. Tocovid at 400 mg/day significantly improve tibial motor nerve CV up to 6 months, but median and sural sensory nerve CV in up to 12 months of supplementation. All improvements diminished after 6 months of washout.

Bisperoxovanadium promotes motor neuron survival and neuromuscular innervation in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease, with no present cure. The progressive loss of MNs is the hallmark of ALS. We have previously shown the therapeutic effects of the phosphatase and tensin homolog (PTEN) inhibitor, potassium bisperoxo (picolinato) vanadium (bpV[pic]), in models of neurological injury and demonstrated significant neuroprotective effects on MN survival. However, accumulating evidence suggests PTEN is detrimental for MN survival in ALS. Therefore, we hypothesized that treating the mutant superoxide dismutase 1 G93A (mSOD1G93A) mouse model of ALS during motor neuron degeneration and an in vitro model of mSOD1G93A motor neuron injury with bpV(pic) would prevent motor neuron loss. To test our hypothesis, we treated mSOD1G93A mice intraperitoneally daily with 400 µg/kg bpV(pic) from 70 to 90 days of age. Immunolabeled MNs and microglial reactivity were analyzed in lumbar spinal cord tissue, and bpV(pic) treatment significantly ameliorated ventral horn motor neuron loss in mSOD1G93A mice (p = 0.003) while not significantly altering microglial reactivity (p = 0.701). Treatment with bpV(pic) also significantly increased neuromuscular innervation (p = 0.018) but did not affect muscle atrophy. We also cultured motor neuron-like NSC-34 cells transfected with a plasmid to overexpress mutant SOD1G93A and starved them in serum-free medium for 24 h with and without bpV(pic) and downstream inhibitor of Akt signaling, LY294002. In vitro, bpV(pic) improved neuronal viability, and Akt inhibition reversed this protective effect (p < 0.05). In conclusion, our study indicates systemic bpV(pic) treatment could be a valuable neuroprotective therapy for ALS.

Biodistribution of onasemnogene abeparvovec DNA, mRNA and SMN protein in human tissue.

Spinal muscular atrophy type 1 (SMA1) is a debilitating neurodegenerative disease resulting from survival motor neuron 1 gene (SMN1) deletion/mutation. Onasemnogene abeparvovec (formerly AVXS-101) is a gene therapy that restores SMN production via one-time systemic administration. The present study demonstrates widespread biodistribution of vector genomes and transgenes throughout the central nervous system (CNS) and peripheral organs, after intravenous administration of an AAV9-mediated gene therapy. Two symptomatic infants with SMA1 enrolled in phase III studies received onasemnogene abeparvovec. Both patients died of respiratory complications unrelated to onasemnogene abeparvovec. One patient had improved motor function and the other died shortly after administration before appreciable clinical benefit could be observed. In both patients, onasemnogene abeparvovec DNA and messenger RNA distribution were widespread among peripheral organs and in the CNS. The greatest concentration of vector genomes was detected in the liver, with an increase over that detected in CNS tissues of 300-1,000-fold. SMN protein, which was low in an untreated SMA1 control, was clearly detectable in motor neurons, brain, skeletal muscle and multiple peripheral organs in treated patients. These data support the fact that onasemnogene abeparvovec has effective distribution, transduction and expression throughout the CNS after intravenous administration and restores SMN expression in humans.

Confocal Endomicroscopy of Neuromuscular Junctions Stained with Physiologically Inert Protein Fragments of Tetanus Toxin.

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066-1315 (emGFP-1066:TetC), 1093-1315 (emGFP-1093:TetC) and 1109-1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

A microfluidic approach to rescue ALS motor neuron degeneration using rapamycin.

TAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

The Ryanodine receptor stabilizer S107 fails to support motor neuronal neuritogenesis in vitro.

Calcium homeostasis is essential for neuronal cell survival/differentiation. Imbalance of the Ca2+ homeostasis due to excessive Ca2+ overload is essential for spinal cord injury (SCI). The overload resulted from Ca2+ flux across the plasma membrane and from internal Ca2+ store release (mitochondria, endoplasmic reticulum, ER). Inositol trisphosphate receptors (IP3R) and ryanodine receptors (RyR) are involved in releasing Ca2+ from ER contributing to axonal degeneration following SCI. In turn, block of both receptors is axoprotective. The calstabin RyR subunit, stabilizing the channel in a state of reduced activity, prevents pathological Ca2+ release too. We investigated whether S107, a RyR-stabilizing compound (Rycal), is beneficial for survival and neuritogenesis of spinal cord motor neurons in vitro. We used a spinal cord slice model and the motor neuron-like NSC-34 cell line. Effects of S107 were tested by propidium iodide/fluorescein diacetate vital staining, mitotic index determination via BrdU-incorporation, and neurite sprouting parameters. Results showed that S107 (i) had no effect on gliosis resulting from slices preparation; (ii) had no effect on motor neuronal survival and proliferation; and (iii) impaired neurite sprouting, no matter whether it was a differentiation (NSC-34 cells) or regeneration (spinal cord slices) process. The results underline the need for a flexible Ca2+homeostasis provided by the ER for re-initiation of neuritogenesis.