Evaluation of antioxidant and neuroprotective therapies in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by dysfunction and degeneration of motor neurons innervating skeletal muscle. ALS patients experience progressive muscle weakness and atrophy, leading to paralysis and death within 3-5 years of diagnosis. The mechanisms underlying neurodegeneration in ALS are unknown; studies of patient tissue and of transgenic mouse models of ALS have implicated oxidative stress, neuroinflammation, aberrant RNA metabolism, excitotoxicity, protein misfolding, autophagy and proteasome dysfunction, and intracellular transport deficits in disease processes. Current ALS therapeutics can only extend lifespan by a matter of months, so it is vital that novel therapeutic targets and therapeutic molecules are identified. The many putative triggers‚ÄövÑvp of ALS are predicted to converge upon common mechanisms of degeneration, with oxidative stress being identified as one of the major pathological hallmarks of ALS. Therapeutics capable of modulating oxidative stress and preventing neuronal death may be of value in treating human ALS. In this thesis, the temporal correlations between microglial activation, development of pathological alterations in the spinal cord, and functional decline, were explored in the transgenic SOD1 mouse model of ALS (carrying the ALS-linked mutant human Cu,Zn-superoxide dismutase gene SOD1G93A), with non-transgenic (WT) mice used as controls. The ability of three putative therapeutic compounds for ALS ‚Äö- Gemals, metallothionein-2 protein, and Emtin peptides ‚Äö- to increase survival time in SOD1 mice was also examined. Pathological alterations in motor neurons preceded an increase in microglial numbers, suggesting microglial activation occurs as a reactive response to neuronal degeneration or dysfunction. Microglial activation occurred concurrently with disease onset at 14 weeks of age, but preceded the development of overt functional deficits around 18 weeks of age. Interestingly, microglial activation was associated with an increase in the number of microglia expressing the M2-like, putative neuroprotective, marker arginase1 (Arg1), and to a lesser extent with an increase in the number of microglia expressing the M1-like, putative neurotoxic, marker inducible nitric oxide synthase (iNOS). These data suggest the concurrent presence of ongoing neuroprotective and neuroinflammatory processes in the spinal cord of SOD1 mice; microglial activation may not be a primary cause of neurodegeneration, but may drive disease progression after onset. Additionally, the expression of the antioxidant protein metallothionein-1/2 (MT-1/2) increased from 18 weeks of age, possibly in response to oxidative stress or neuronal degeneration. Gemals, an antioxidant and anti-inflammatory combination therapy, has been previously shown to extend the lifespan of an ALS rat model, but has not been tested in ALS mice. Here, Gemals was administered subcutaneously to SOD1 mice from the age of symptom onset through to disease endpoint. No significant changes in survival time were identified in Gemals-treated SOD1 mice compared to controls, indicating that Gemals treatment may be less effective when administered after symptom onset. MT-1/2 protein has previously shown both antioxidant and neuroprotective properties, and its ablation in SOD1 mice has been shown to accelerate disease progression. In this study, SOD1 mice were treated with MT2 injections, and/or with treadmill running exercise to upregulate endogenous MT-1/2. MT2 treatment slightly but significantly delayed disease onset, and tended to increase survival time, in SOD1 mice, whereas treadmill running exercise had little effect. However, the mechanism of action for MT2 is as yet unknown ‚Äö- preliminary data suggest that MT2 treatment did not substantially prevent spinal cord motor neuron degeneration or muscle endplate denervation. Peptide derivatives of MT-1/2, termed Emtins, have previously displayed similar neuroprotective properties to their parent MT-1/2 protein in vitro and in vivo, and additionally can readily cross the blood-brain barrier. Emtins were administered subcutaneously to SOD1 mice from the onset of disease symptoms, resulting in increased survival time compared to control mice, although this result was not significant due to a smaller number of animals used during this trial. These data indicate that both MT2 and Emtins have pro-survival effects in the SOD1 mice. Emtin peptides are thought to have limited metal-binding and antioxidant properties; however, both MT-1/2 and Emtins are known to interact with low-density lipoprotein receptor-related proteins (LRPs) and activate the Akt pathway, leading to increased cellular survival. It is possible that the pro-survival effects of MT2 and Emtins seen in these studies were mediated through LRP binding and activation of downstream pathways. MT2 and Emtins show potential as therapeutic molecules for ALS, but more work is required to elucidate the mechanism of action.