Prion diseases are fatal, transmissible neurodegenerative disorders characterised by accumulation throughout the brain of PrPSc, an abnormally folded isoform of the normal cellular prion protein, PrP. PrPSc is associated with infectivity but is not directly neurotoxic and targeting it is of limited efficacy in prion therapeutics. However, PrP-null mice are resistant to prion infection and neurotoxicity. Transgene-c mediated depletion of neuronal PrP in mice with established prion infection reverses early spongiosis, neuronal loss and cognitive deficits, and prevents clinical disease progression. Thus, reducing PrPC expression in the brain through extrinsic means is likely to be an effective therapy for prion diseases. RNA interference can be exploited to mediate gene silencing and can be stably achieved in non-dividing cells such as neurons by incorporation of the small interfering RNAs into replication-deficient lentiviruses. The work described in this thesis strongly validates the use of lentiviral-mediated RNA interference as a therapeutic approach in prion disease. Reducing PrPc expression with siRNA duplexes enabled clearance of PrPSc and infectivity from prion-infected cells in vitro. Lentiviruses constructed to express the interfering sequences demonstrated effective reduction of PrPc expression in vitro. Stable expression of the interfering RNA molecules in vivo through lentiviral transduction of the hippocampus reduced local pathology and significantly prolonged survival in a mouse model of prion disease. This represents an important and novel advance in the treatment of established prion disease with relevance for all prion strains.

Many neurodegenerative disorders, including Alzheimer's (AD), Parkinson's (PD) and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. During prion disease, an increase in misfolded prion protein (PrP) generated by prion replication leads to sustained overactivation of the branch of the unfolded protein response (UPR) that controls the initiation of protein synthesis. The UPR is a protective cellular mechanism that is induced during periods of cellular and endoplasmic reticulum stress. UPR activation aims to restore protein homeostasis, by reducing protein translation, and up-regulating chaperone proteins that assist with proper protein folding. However, sustained activation of this pathway results in persistent repression of translation, resulting in the loss of critical proteins that leads to synaptic failure and neuronal death. Inhibiting the UPR by genetic means has recently been shown to be neuroprotective in prion disease (Moreno et al., 2012). A drug screen was performed in the model organism C. elegans to search for inhibitors of the UPR. 34 compounds were identified, of which five were selected for further analysis in C. elegans before being tested as a potential treatment in prion diseased mice. Two compounds, dibenzoylmethane and trazodone hydrochloride displayed efficacy against prion disease, and represent novel therapeutic targets. GSK2606414, a specific inhibitor of PERK (protein kinase RNA-like endoplasmic reticulum kinase), a key mediator of the UPR induced translational repression was also tested in prion diseased mice. It restored protein synthesis and prevented the development of clinical prion disease. These data validate the UPR as a viable target in prion disease, and uncover promising potential therapeutics.

Alterations in glutamate uptake and in expression of glutamate transporters have been identified in ALS patients; however studies on human specimens are done using post mortem samples that are only representative of the very late stage of the disease, characterized by massive motor neuron degeneration and severe tissue compromise. Mutant wobbler mice and transgenic SOD1G93A mice are the most used animal models for human ALS, and are considered a useful tool for studying mechanisms of motor neuron death potentially relevant to human disease. In this study a specific method for isolation of highly enriched synaptosomal fraction was set up to allow the evaluation of glutamate uptake characteristics in the presynaptic compartment. Results indicated that presynaptic terminals were able to clear glutamate through a high-affinity mechanism likely mediated by GLT1, in the absence of glial contamination.

The aim of this thesis was to develop and apply Diffusion Tensor Imaging (DTI) and functional Magnetic Resonance Imaging (fMRI) in patients with demyelinating diseases in order to provide further insights into the mechanisms of damage and recovery. We used DTI to investigate pathological changes in the normal-appearing (NA) brain of patients with multiple sclerosis (MS), using regions of interest. Our results showed that diffusion changes were associated with disability and progression in MS, and that the NA white matter damage related to focal lesions. We then extended the investigation to the whole white matter tracts, using the novel technique of diffusion-based tractography, in three separate studies. In the frst study, we assessed the reproducibility of fast marching tractography in quantifying the major white-matter pathways. In the second, we created probabilistic group maps of these tracts in healthy volunteers, in order to estimate their normal inter-subjects variability. In the third study, we employed these group mapping techniques to investigate pathologic changes in the optic radiations of patients affected by optic neuritis, and found that patients had a lower voxel-scale connectivity in the optic radiations when compared with controls. To complement these studies, we developed a motor paradigm that allows investigation of the brain functional response to active and passive movements of the foot. We then used this paradigm to determine the role of cortical reorganisation in patients with primary progressive MS, and concluded that brain functional changes, in our cohort of patients, represented both adaptive and non-adaptive responses to central nervous system damage. Finally, we combined the data obtained from tractography with those obtained by fMRI, in patients with optic neuritis, and demonstrated the potential of this methodology to understand the relationship between structural and functional mechanisms.

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