Methylmercury (MeHg) is a ubiquitous environmental toxin. Exposure to MeHg in humans occurs primarily through the consumption of contaminated seafood. MeHg has been shown to act most strongly during neural development. Epidemiological data on the effect MeHg exposure through seafood has on children and fetuses is conflicted, with large cohort studies showing both presence and absence of MeHg-induced deficits in achieving developmental milestones. Because of this uncertainty in the literature it is important that we come to understand the mechanisms of MeHg toxicity so that we might advise the public more accurately on the risks of MeHg exposure. Research into the mechanisms of MeHg toxicity has found a number of cellular and molecular effects including disruptions of microtubule formation, Ca2+ homeostasis, and glutamate signaling. However, none of these effects of MeHg fully explains its neurodevelopmental specificity. Previous work in Drosophila neural-derived cell lines has shown that MeHg causes upregulation of the canonical Notch response gene E(spl)m . The Notch pathway is crucial to neural development and perturbation of a Notch target may explain the developmental specificity of MeHg. In this dissertation I describe experiments I performed to test the hypothesis that the observed upregulation of E(spl)m plays an important role in MeHg toxicity in Drosophila. I first describe experimental evidence that E(spl)m is upregulated by MeHg treatment in vivo in Drosophila embryos in addition to cells, as has previously been shown. By contrasting the effects of the toxic inorganic mercurial HgCl2 with MeHg I show that the E(spl)m expression response to MeHg is not simply a stress response and is a likely specific activity of MeHg. I also show that the effect of MeHg on E(spl)m expression is not simply due to a developmental delay induced by the toxin. I also identify two neural phenotypes of MeHg toxicity in Drosophila embryos, in the outgrowth of the intersegmental and segmental motor nerves. Genetic manipulation causing overactivity of the Notch pathway in neurons can mimic these phenotypes. However, induced expression of E(spl)m in neurons does not cause a failure of motor nerve outgrowth. Upon further examination I demonstrate that endogenous expression of E(spl)m occurs in the muscle. Induced E(spl)m expression in the muscle causes a segmental nerve phenotype similar to MeHg treatment, indicating a role for E(spl)m in MeHg toxicity in this system. MeHg treatment and E(spl)m overexpression in the muscle causes a failure of normal muscle development. Yet, this gross developmental abnormality only partially explains the observed motor nerve phenotype. E(spl)m is unique among the E(spl) genes in its ability to cause these muscle and motor nerve phenotypes as shown by contrasting genetic manipulation of the closely related E(spl)m . Overall my findings support the hypothesis that MeHg toxicity in Drosophila is mediated in part by E(spl)m . They also suggest that E(spl)m plays an important role in the formation of the muscle during embryonic development, contributing to the literature describing disparate functions for E(spl) genes despite structural similarities. Finally, my findings suggest that MeHg may be able to impact neural development through toxicity in supporting tissues rather than neurons themselves. This final finding has implications for the study of MeHg toxicity in humans, and is supported by previous findings that describe a role of glia in modulating MeHg neurotoxicity.

<p>The purpose of this study was to investigate the difference in amplitude between M-response from m. Abductor pollicis brevis/m. Pronator quadratus and m. Abductor pollicis brevis/m. Abductor digiti minimi on patients with carpal tunnel syndrome, compared with control subjects. We wanted to see if m. Pronator quadratus is a better alternative than m. Abductor digiti minimi as comparison with m. Abductor pollicis brevis on patients with carpal tunnel syndrome.</p><p>Nerve conduction studies were performed on 20 patients with carpal tunnel syndrome and on 31 healthy subjects.</p><p>The test-retest result shows that this method was reproducible. The amplitude difference of m. Abductor pollicis brevis-m. Abductor digiti minimi, for the patients, was 1,5mV lower and the amplitude for m. Abductor pollicis brevis-m. Pronator quadratus was 2mV lower than for healthy subjects. Two of the patients were outside the 2SD for the m. Abductor pollicis brevis-m. Pronator quadratus difference but not on the m. Abductor pollicis brevis-m. Abductor digiti minimi. This may indicate that m. Pronator quadratus was better than m. Abductor digiti minimi in the comparison with the m. Abductor pollicis brevis amplitude.</p>

The purpose of this study was to investigate the difference in amplitude between M-response from m. Abductor pollicis brevis/m. Pronator quadratus and m. Abductor pollicis brevis/m. Abductor digiti minimi on patients with carpal tunnel syndrome, compared with control subjects. We wanted to see if m. Pronator quadratus is a better alternative than m. Abductor digiti minimi as comparison with m. Abductor pollicis brevis on patients with carpal tunnel syndrome. Nerve conduction studies were performed on 20 patients with carpal tunnel syndrome and on 31 healthy subjects. The test-retest result shows that this method was reproducible. The amplitude difference of m. Abductor pollicis brevis-m. Abductor digiti minimi, for the patients, was 1,5mV lower and the amplitude for m. Abductor pollicis brevis-m. Pronator quadratus was 2mV lower than for healthy subjects. Two of the patients were outside the 2SD for the m. Abductor pollicis brevis-m. Pronator quadratus difference but not on the m. Abductor pollicis brevis-m. Abductor digiti minimi. This may indicate that m. Pronator quadratus was better than m. Abductor digiti minimi in the comparison with the m. Abductor pollicis brevis amplitude.

The effects of Hg2+, methyl mercury, and flufenamic acid, all of which inhibit mitochondria, were examined at frog motor nerve terminals. Unbiased estimates of m (no. of transmitter quanta released), n (no. of functional release sites), p (probability of release), and vars p (spatial variance in p) were obtained using K+-induced asynchronous neurosecretion (m, n and p not having the same definitions as with nerve-evoked release). Transient but significant increases in m, n, p and vars p were found with all three agents. These findings indicate that mitochondrial inhibition and release of sequestered Ca2+ can be detected as a real-time increase in vars p. The results also suggest that changes in vars p might be used to differentiate between cellular (membrane) and subcellular (organellar) actions of drugs at the nerve terminal.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by widespread loss of lower motor neurons from the spinal cord. Lower motor neuron degeneration leads to a progressive decline in motor development, manifesting as muscle atrophy and weakness. It is now well characterised that ubiquitin homeostasis is altered in SMA and that reduction of the ubiquitin-like modifier-activating enzyme 1 (UBA1) is central to this disruption. UBA1 is responsible for activating ubiquitin as the first step in the ubiquitin conjugation process, marking unwanted proteins for degradation by the proteasome. While it is known that therapies targeting UBA1 rescue neuromuscular phenotypes in SMA models, the mechanism by which UBA1 mediates neurodegeneration is unclear. In fact, very little is known about the function of UBA1 beyond its canonical role in the ubiquitin proteasome system. To better understand the role of UBA1 in motor neuron degeneration, a robust set of antibodies for both in vivo and in vitro work to study UBA1 have been identified. This enabled a novel characterisation of UBA1 distribution throughout disease progression in SMA spinal motor neurons to be performed, revealing that UBA1 reduction is an important pre-symptomatic molecular feature of SMA. To identify downstream targets of UBA1 critical for UBA1-mediated degeneration in SMA, label-free proteomics was performed on HEK293 cells after overexpression or knockdown of UBA1. The proteomics data was analysed across multiple platforms, including Biolayout, IPA and DAVID to identify UBA1-dependent pathways and demonstrated that modulation of UBA1 levels lead to disruption of key cellular pathways including translation elongation, nuclear transport, and tRNA synthetases. Validation of target proteins from these UBA1-dependent pathways identified that the tRNA synthetease GARS behaves in a UBA1-dependent manner across a range of model systems in vitro and in vivo. It was then identified that GARS expression is significantly dysregulated across a range of neuronal tissues in a mouse model of SMA. Interestingly, mutations in GARS cause Charcot-Marie-Tooth disease type 2D (CMT2D), an axonal neuropathy, in which a disruption to sensory neuron fate in dorsal root ganglia has recently been identified. In a mouse model of SMA we identified a phenotype consistent with that in the CMT2D mouse model and showed that disruption to sensory neuron fate is reversible and dependent on changes in UBA1 and GARS expression in SMA. In conclusion, modulation of UBA1 levels leads to disruption of key cellular pathways, with dysregulation of tRNA synthetases a prominent feature that is likely to play a role in the pathogenesis of SMA.

The mammalian spinal cord contains the neuronal circuitry necessary to generate rhythmic locomotor activity in the absence of inputs from the higher brain centre or sensory system. This circuitry is regulated by local neuromodulatory inputs, which can adjust the strength and timing of locomotor output. The free radical gas nitric oxide has been shown to act as an important neuromodulator of spinal circuits, which control locomotion in other vertebrate models such as the tadpole and lamprey. Despite this, the involvement of the NO-mediated soluble guanylate cyclase/cyclic guanosine monophosphate secondary messenger-signalling pathway (NO/sGC/cGMP) in mammalian locomotion has largely been under-investigated. The NADPH diaphorase histochemical reaction was used to identify sources of NO in the lumbar spinal cord. The largest population NADPH diaphorase reactive neurons were located in the dorsal horn, followed by the laminae of the ventral horn, particularly around the central canal (lamina X) and lamina VII. NADPH diaphorase reactive neurons were found along a rostrocaudal gradient between lumbar segments L1 to L5. These results show that that discrete neuronal sources of NO are present in the developing mouse spinal cord, and that these cells increase in number during the developmental period postnatal day P1 – P12. NADPH diaphorase was subsequently used to identify NADPH diaphorase reactive neurons at P12 in the mouse model of ALS using the SODG93A transgenic mouse. Physiological recordings of ventral root output were made to assess the contribution of NO to the regulation induced rhythmic fictive locomotion in the in vitro isolated spinal cord preparation. Exogenous NO inhibits central pattern generator (CPG) output while facilitating and inhibiting motor neuron output at low and high concentrations respectively. Removal of endogenous NO increases CPG output while decreasing motor neuron output and these effects are mediated by cGMP. These data suggest that an endogenous tone of NO is involved in the regulation of fictive locomotion and that this involves the NO/sGC/cGMP pathway. Intracellular recordings from presumed motor neurons and a heterogeneous, unidentified sample of interneurons shows that NO modulates the intrinsic properties of spinal neurons. These data suggest that the net effect of NO appears to be a reduction in motor neuron excitability.