Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative condition which is almost universally fatal. Some sufferers have an identifiable genetic mutation (<2%), but the majority of cases are sporadic (SALS). There is a body of evidence suggesting involvement of oxidative stress and mitochondrial abnormalities in the pathogenesis of ALS. Mitochondrial function was assessed in remote tissues (muscle, cultured myoblasts, cultured fibroblasts) from patients with ALS and controls. Muscle tissue was examined for histological features consistent with a mitochondrial disorder. Mitochondrial respiratory chain function was measured in mitochondrial homogenates. Mitochondrial protein expression was determined using immunofluorescent antibodies to a mitochondrial DNA (mtDNA)-encoded respiratory chain enzyme subunit with image analysis. MtDNA quantity and quality was assessed using Southern blot and long-range PCR. Analysis of inheritance patterns was performed using a database of familial ALS cases. No mitochondrial abnormalities were identified using these techniques. There was no evidence of anticipation or a sex effect on inheritance. In order to look for a difference in susceptibility to oxidative stress, cell cultures were examined for markers of oxidative damage and apoptosis, following growth with a free radical generating agent (paraquat). These studies demonstrated an increased susceptibility to oxidative damage in patient myoblasts and fibroblasts. This effect was not seen in cybrid studies combining mtDNA from patient platelets with experimental cells devoid of mtDNA (p cells), where no difference in oxidative damage was seen, confirming the absence of a systemic mtDNA abnormality. These results suggest that patients with SALS have an increased systemic sensitivity to oxidative stress which becomes pathological in tissues, such as motor neurons, which are considered to be vulnerable to such stress due to their metabolic activity and structure. This altered sensitivity is due to a factor other than a mitochondrial DNA abnormality, such as a nuclear DNA mutation or an unidentified environmental factor.

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Motor neurone disease (MND) is a fatal neuromuscular illness defined by progressive muscle weakness and wastage. Death typically occurs within 2-3 years from symptom onset and is most often attributable to respiratory complications. Weakness in the respiratory muscles increases the risk of mortality and is also a significant predictor of quality of life in MND, which makes management of respiratory aspects of the condition a vital component of care. The first signs of respiratory insufficiency are typically related to the onset of nocturnal hypoventilation, which can disturb sleep and lead to waking headaches, somnolence, fatigue, impaired concentration, cognitive impairment, reduced appetite, and low mood. The primary means of managing these symptoms is through the use of non-invasive ventilation (NIV). There is a vast quantitative research base charting the impact of NIV. It is known to decrease the number of arousals from sleep, reduce somnolence/fatigue, eliminate waking-headaches, improve cognitive functioning, and may also exert a positive impact on emotional/social functioning in some patients. NIV can also benefit daytime respiratory performance, improving self-rated dyspnoea and slowing the rate of respiratory decline as the illness progresses. Crucially, NIV also affords selected MND patients a significant survival advantage. Existing research offers limited insight into the lived experience of respiratory impairment and NIV use; it is unknown how patients themselves feel about using a ventilator or how they receive the positive clinical outcomes associated with the treatment. This thesis presents a body of qualitative research using interpretative phenomenological analysis (IPA) to explore MND patients' experiences of respiratory impairment and NIV use. The first study, a small cross- sectional enquiry (11=5), reports the experiences of patients already established on the ventilator. This study found complex emotional •and psychological responses to NIV, including reluctance to initiate, fear of dependence and threats to control. Respiratory masks were also found to have a significant negative impact on identity and self-esteem. However, patients felt that the positive physical effects of NIV made this experience acceptable. This cross-sectional study was a preparative step for a subsequent longitudinal study, recruiting patients (11=26) and carers (n=26) prior to ventilation and interviewing them over time as they started using NIV. Two data sets are reported: 'pre-ventilation' and 'post-ventilation' analysis. Pre-ventilation analysis also explored the lived experience of respiratory impairment and treatment decision-making. This longitudinal study found that referral into a respiratory service was an emotionally stressful event for patients; most had been unaware of the prospect of respiratory impairment and were vulnerable to anxiety. Patients seemed to lack insight into their own respiratory status, which appeared to be attributable to both a lack of understanding of respiratory aspects of the condition and difficulties in making sense of respiratory changes within the wider physical context of the illness. Most patients responded negatively to the idea of ventilation, seeing it both as a 'defeat' and as an ominous sign of illness progression, yet they were also comforted by the idea that there was 'help' available to prevent suffering. Patients typically elected not to think about NIV ahead of time, which appeared to be part of a wider 'one day at a time' approach to MND. Patients who went on to trial NIV (11= 12) reported markedly different responses to initiation and variable degrees of involvement in treatment decision-making. Many patients did not feel that intervention was needed. Positive physiological! clinical outcomes did not necessary lead to positive psychological and emotional responses to the treatment and did not ensure that the experience of using a ventilator was a positive one. There were a number of practical and psychological challenges that determined tolerance and compliance with the treatment; however, the same challenges were often perceived differently by different patients. It was not possible to predict 'success' on NIV based on clinical or demographic variables alone. Patients' experiences of NIV were best understood in the individual context of each patient and in light of their personal illness experiences. These findings make a significant contribution to the established research base, providing an alternative perspective to substantiate the quantitative evidence. It is intended that the research presented in this thesis will be of direct practical utility, helping clinicians who are supporting MND patients to ensure that patients' experiences of respiratory care and NIV use are as positive as they can be.

Amyotrophic lateral sclerosis (ALS) is a devastating degenerative disease, affecting both upper and lower motor neurons in the eNS. Around 10% of ALS cases are classed as familial, and around 20% of these are due to a mutation in the gene superoxide dismutase-t (SOot). The SOOtG ,,. mouse is one of the transgenic rodent models of this disease. Both in humans and animal models of ALS, neuroinflammation is an important theme that has attracted much research attention over the past few decades. An area that has attracted much less attention, though, is the neurovascular aspect of pathology. In both these fields , the literature is biased towards the spinal cord, with little information available about SOOt pathology in other eNS reg ions. In this context, we sought to investigate pathological components of ALS pathology throughout the eNS, using a multi-disciplinary approach. First, I explore the use of MRI as in in vivo biomarker of brainstem pathology, showing that ALS pathology can be tracked using short sequences, paving the way for MRI as a high-throughput biomarker for clinical trials. Secondly, I use in vivo MRI and immunohistochemistry to investigate vascular and inflammatory components of the pathology. I show that there is very little cellular adhesion molecule expression in the eNS of SOOtG'JA mice, despite postsymptomatic expression of cytokines and chemokines, and no disruption to the BBB at any point in the disease. Finally, I investigate whether steroids have therapeutic value in SOOtG'3A mice, when del ivered specifically into the eNS using targeted liposomes, and show that centrally-targeted methylprednisolone has a significant impact of brainstem pathology, but no effect on spinal pathology in this model. In summary, inflammation is a key aspect of SOOtG'3A pathology, although changes in the penmeabilily of the BBB may be more contentious than the previous literature would suggest. Targeting inflammatory processes using more targeted approaches may offer hope for development of therapies for ALS in the future.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease targeting preferentially motor neurons. Cyclophilin A (CypA) was identified as a hallmark of disease in mutant SODl (mSOD1) animal models of familial ALS (fAlS) at a presymptomatic stage, and in sporadic (sALS) patients (Massignan 2007; Nardo 2011). Moreover, CypA was enriched in the spinal cord aggregates of mSODl mice and sALS patients (Basso 2009). CypA is an ubiquitous protein with multiple functions relevant to the CNS, where it is abundantly expressed. Insights into CypA function in ALS were provided via a proteomic analysis of its interacting proteins, that functionally associated CypA with different proteins networks. In particular, it extensively binds proteins regulating RNA metabolism, including several hnRNPs and TDP-43, a major disease protein in AlS. TDP-43 and CypA interact in the nucleus, in an RNA-dependent way. CypA has a key role in the stabilization of TDP- 43/hnRNP A2/Bl interaction, and TDP-43-mediated DAC6 expression regulation, properties impaired in TDP-43 ALS-mutants, possibly because of a loss-of-interaction with CypA. CypA interacts also with mSOD1, suggesting a gain-of-interaction specifically linked to fAlS. Mice expressing mSODl and lacking CypA show increased levels of insoluble mSODl and hyperphosphorylated TDP-43 in the spinal cord at the onset. This thesis work shows that CypA has a protective role in AlS: as a chaperone (for mSOD1) and in maintenance of multi-protein (TDP-43/hnRNPs) complex stability. Regardless the cause of the disease, mSODl or alterations in TDP-43, the interaction with CypA is impaired and it is co-sequestered in proteinaceous aggregates, altering its protective activities. The net effect is the formation of pathological inclusions that may lead to a compromised RNA metabolism. CypA being a key interacting partner of both mSODl and TDP-43 can represent the "missing link" of these two patho-mechanisms in ALS and an interesting ta rget for therapeutic interventions.

Amyotrophic Lateral Sclerosis (ALS) is characterised by motor neuronal degeneration leading to muscular wasting and eventually death. 5% of ALS cases are familial (F ALS) and 20% of these are due to mutations in the gene encoding for copperlzinc superoxide dismutase-l (SOD1). Mice overexpressing human mutant SODl develop a neurodegenerative condition closely resembling ALS. Beyond increasing age, male gender and family history of ALS, there are no other validated risk factors. However, recently, association between intense physical activity and ALS has been proposed by epidemiological studies. In the first part of this thesis, the response of motor neurones to the stress caused by the presence of the mutant human SOD 1 in the mouse model of F ALS was investigated. In the second part, the genes and pathways involved in the physiological response to the stress caused by voluntary exercise in motor neurones were identified. The final section of this thesis investigated whether single nucleotide polymorphisms (SNPs) in key genes involved in the response to exercise were associated with the pathophysiology of ALS. Microarray analysis of motor neurones isolated using laser capture microdissection from the lumbar spinal cord of G93A-SOD 1 mice and their littermates at different stages of disease has identified several potential targets for drug therapy, especially at the presymptomatic stage. Applying the same techniques, the transcription profile of motor neurones isolated from the lumbar spinal cord of exercised and sedentary mice was analysed as well as gastrocnemius muscles. Motor neurones showed transcriptional changes involved in neurotrophic factor signalling, electrophysiological changes and synaptic reorganization, while gastrocnemius muscle revealed mcreases in transcripts responsible for neovascularisation and myogenesis. The comparison between the two studies reveals that, although the pathways activated in motor neurones in response to the two stresses are different, the physiological response to exercise requires production of neurotrophic factors and activation of pathways that have been shown to be implicated in the pathology of ALS. In particular, VEGF receptor 2 (KDR) was upregulated in response to exercise. In order to investigate the hypothesis that genetic susceptibility could be implicated in a failure to mount the physiological response to exercise, non-synonymous SNPs in KDR were screened in sporadic ALS cases and controls. Although the screening did not reveal any positive associations, the present study provides additional candidate genes that might confer a genetic susceptibility to ALS in response to intense physical activity.

Motor neurone disease (MND) is the third most common adult-onset neurodegenerative disorder. It is relentlessly progressive and universally fatal, usually as a result of respiratory failure. In 3-5% of MND cases overt dementia is present, however, subtle impairment to cognitive function is present in up to 50% of sufferers. In approximately 10% of MND cases there is a clear pattern of inheritance, however, genetic causes are believed to make a substantial contribution to apparently 'sporadic' disease. Mutations in ANG were first identified in a large cohort of MND cases from several diverse geographical regions, four of the patients suffered from familial disease, whereas the remaining 11 had no family history of MND. A mutation in CHMP2B was originally identified in a Danish pedigree with autosomal dominant FTD, which was subsequently followed by the identification of missense mutations in two, unrelated, patients with familial MND, one of whom also showed features ofFTD. The initial aim of the present study was to determine whether mutations in ANG and CHMP2B contribute to MND pathogenesis by mutation screening a large cohort of MND patients for whom serial clinical details were available. Neuropathological tissue was available for a proportion of these cases. Sequencing of ANG revealed a mutation in one case diagnosed with an early-onset, classical amyotrophic lateral sclerosis (ALS) phenotype, who showed rapid deterioration and characteristic ALS neuropathology. Four cases carrying 3 missense mutations in CHlvfP2B, including one novel mutation, p. Thrl04Asn, \vere identified. Only 1 case had a family history of MND, the remaining 3 were affected by apparently sporadic disease. In all 4 cases analysis of clinical and neuropathological data was consistent \vith a diagnosis of the progressive muscular atrophy (PMA) variant of MND. To analyse the affect of mutant CHMP2B on the transcriptional response, gene expression profiling was performed on RNA extracted from motor neurones (MNs) from CHMP2B cases and controls. Significant changes in the expression of genes from multiple pathways were identified, including: axon guidance; actin cytoskeleton regulation and SNARE interactions in vesicular transport; cell cycle; apoptosis; mTOR signalling and autophagy regulation; MAPK signalling; calcium signalling and Wnt signalling. The alterations to these pathways were predicted to result in: disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of A TP; downregulation of the classical and p38 MAPK signalling pathways; reduction in autophagy initiation and a global repression of translation. Finally, to determine the effect of CHMP2B mutations on cellular phenotypes, HEK- 293 cells were transiently-transfected. This demonstrated that mutant CHMP2B expression resulted in the formation of large cytoplasmic vacuoles and aberrant lysosomal localisation.