Alzheimer’s disease (AD) is a neurodegenerative disease which affects over 500,000 people in the UK. Worldwide 44 million people are affected by AD and other dementias. Most cases occur over the age of 65 and is characterised by gradual and increasing loss of cognitive function and behavioural abnormalities. The main causes are a build-up of the toxic protein amyloid-β (Aβ) and hyperphosphorylation of the microtubule stabilising protein: tau, leading to neurofibrillary tangles (NFT). These two hallmarks of disease result in neuronal damage and cell death causing associated symptoms and eventually death. Orexins (OX) are neuropeptides which function to regulate the sleep-wake cycle and feeding behaviour. They are produced from a prepro-orexin (PPO) molecule and cleaved into two isoforms: orexin-A (OXA) and orexin-B (OXB). OXA and OXB are the ligands for two G-protein coupled receptors (GPCR): orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). 50-80,000 OX producing neurons project to many areas of the brain including the lateral hypothalamus (LHA), locus coeruleus (LC), tuberomammillary nucleus (TMN), paraventricular nucleus (PVN) and raphe nuclei and from these areas regulate feeding and appetite and the sleep wake cycle through their receptors. QRFP is a newly discovered neuropeptide which exerts similar orexigenic activity including the control of feeding behaviour. It is the ligand for the GPCR GPR103, both of which are widely expressed in the brain and also in the retina, testes, thyroid, pituitary and prostate. GPR103 also shares 48 and 47% protein sequence homology with OX1R and OX2R respectively. It is in these tissues where it can exert other physiological functions including regulation of feeding, control of the gonadotropic axis and bone formation. The exact expression and signalling characteristics and physiological actions of QRFP/GPR103 are still poorly understood. It is through the physiological functions of the orexigenic system and the clinical symptoms observed in AD which suggests a possible link between the two. For example, in AD one of the main reasons for institutionalisation is the severely dysregulated sleep pattern that is experienced by sufferers. They experience increased nocturnal activity and early awakenings as well as hypersomnia and excessive daytime sleepiness; all of which is beyond what someone of the same age experiences. As well as this AD patients suffer from significant weight loss and a significant negative correlation has been identified between progression of disease and appetite. All of this points towards an involvement of the orexigenic system in AD. AD patients have been found to have a 40% loss of immunoreactive OX neurons and have severe reductions in circulating OXA. This led us to believe that the OX system is of vital importance in AD and could be targeted to ameliorate symptoms. Studies have implicated OX and OXR in memory processes, appetite regulation, and severe disturbances of the sleep-wake cycle all of which are phenotypes of AD. Given that they play a key role in energy homeostasis and physiological behaviour, we hypothesise that OXs and their receptors are implicated in the pathophysiology of AD. Therefore, in this study we will investigate the detailed expression and signalling characteristics of OXR and GPR103 in vitro and in clinical samples In this study we neuronally differentiated two human neuroblastoma cell lines: IMR32 and SH-SY5Y. Neuronally acquired phenotype was confirmed through increased neurite length, increased expression of key neuronal proteins and increases in microtubule-associated protein tau (MAPT), neurogenin1 (NG1) and neuron-specific enolase (NSE) as well as a reduction in the neuronal marker of immaturity; nestin (NES). OXR and GPR103 were confirmed in both cell lines after differentiation at mRNA and protein level and were shown to be fully functional through phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). We also identified possible cross talk of GPR103 with the OXR though addition of selective OXR antagonists, which blocked QRFP induced ERK1/2 phosphorylation. We show for the first time that addition of Aβ42 and zinc sulphate to mimic AD in vitro, results in a significant reduction of OX1R and GPR103 in the cell lines SH-SY5Y and we have performed the first comprehensive study in clinical AD patients which demonstrate a loss of OX1R, OX2R and GPR103 at mRNA and protein level compared to age matched controls in the hippocampus. We performed microarray analysis which identified many genes and pathways regulated by the OXA, OXB and QRFP; including corticotropin-releasing hormone receptor (CRHR1), regulated in development and DNA damage responses 1 (REDD1), erythropoietin (EPO), Bcl-2-like protein 1 (BCL2L11), myb proto-oncogene protein (c-myb), vasoactive intestinal peptide (VIP), endothelin 1 (EDN1) as well as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) and hypoxia-inducible factor-1α (HIF-1α) pathways. These genes are all implicated in neuroprotection, particularly in AD. This represents the first comprehensive gene expression data in a neuroblastoma cell line for these orexigenic proteins. Collectively these data suggest a potential role of the orexigenic system in neuroprotection and a functional loss of the receptors in AD patients which could confer a loss of neuroprotection through the orexigenic system. Pharmacological intervention directed at the orexigenic system may prove to be an attractive avenue towards the discovery of novel therapeutics for diseases such as AD and improving neuroprotective signalling pathways.

Lower motor neuron diseases (LMNDs) include a large spectrum of clinically and genetically heterogeneous disorders, characterized by progressive anterior horn cell degeneration. The aims of this thesis were on the one hand to refine the phenotypic description and the clinical classification of hereditary LMNDs, and on the other hand to improve our knowledge of the genetic bases of these disorders. This work was performed in collaboration with the Centre of Human Genetics of the Necker-Enfants-Malades Hospital in Paris. We focused our researches on autosomal recessive variants of LMNDs. First, we selected patients with Spinal Muscular Atrophy with Respiratory Distress (SMARD or d-HMN VI). This severe variant of autosomal recessive LMND is characterized by neurogenic muscular atrophy associated with early life-threatening respiratory failure due to diaphragmatic dysfunction. SMARD type 1 has been ascribed to mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2) gene on chromosome 11q13-q21. We reported the identification of 9 novel IGHMBP2 mutations in five SMARD1 patients, Seven of them occurred at highly conserved residues of the putative DNA helicase domain, suggesting that this particular domain plays a major role in the SMARD1 disease causing mechanism (Hum Mutat. 2004; 23(5):525-6). Then, we collected families and sporadic patients affected by chronic distal spinal muscular atrophy (d-HMN III/IV), an autosomal recessive variant of LMND characterized by a progressive motor weakness and muscular atrophy, predominating in the distal parts of the limbs. A form of chronic dSMA gene had been mapped to a 10.3 cM interval on chromosome 11q13. By linkage analysis in 12 European chronic dSMA families, we reduced the genetic interval to a 2.6cM region on chromosome 11q13.3 and showed partial linkage disequilibrium between 3 rare alleles and the mutant chromosome in European patients, suggesting that most chronic dSMA chromosomes are derived from a single ancestor (Eur J Hum Genet. 2004;12(6):483-8). Additional experiments are now in progress at Necker-Enfants-Malades Hospital, in order to identify the disease-causing gene. At last, we described the clinical features of a novel variant of autosomal recessive LMND, characterized by childhood onset, generalized muscle involvement, and severe outcome. Studying a large inbred African family, we mapped the disease gene to a 3.9-cM interval on chromosome 1p36 (Neurology.2006;67(1):120-4). We identified a homozygous missense mutation of the PLEKHG5 gene and performed in vitro experiments to clarify the pathogenic function of this mutation. In transiently transfected HEK293 and MCF10A cell lines, we found that wild-type PLEKHG5 activated the NFkB signaling pathway and that both the stability and the intracellular location of mutant PLEKHG5 protein were altered, severely impairing the NFkB transduction pathway. Moreover, we observed aggregates in transiently transfected NSC34 murine motor neurons overexpressing the mutant PLEKHG5 protein. In conclusion, we showed that both loss of PLEKHG5 function and aggregate formation might contribute to neurotoxicity in this novel form of LMND (Am J Hum Genet. 2007;81(1):67-76). Further experiments should now be planned, in particular to understand the role of aggregates in neurodegeneration, and to precise the links between the PLEKHG5 gene and the other LMNDs-causing genes. In conclusion, we hope that this work, contributing to a better understanding of the molecular mechanisms involved in motor neuron degeneration, will open the way to new therapeutic strategies. / Les amyotrophies spinales (SMA) se caractérisent par une dégénérescence des motoneurones des cornes antérieures de la moelle épinière ou des noyaux du tronc cérébral. Elles comprennent de nombreuses entités, très hétérogènes tant sur le plan clinique que sur le plan génétique. Cette thèse vise à préciser les caractéristiques phénotypiques et génétiques des différentes formes d’amyotrophie spinale, en particulier celles des variants de transmission autosomique récessive. En premier lieu, nous avons sélectionné une cohorte de patients dont le tableau clinique était compatible avec la variante SMARD d’amyotrophie spinale (pour Spinal Muscular Atrophy with Respiratory Distress). Il s’agit d’une forme très sévère d’amyotrophie spinale autosomique récessive, qui associe une faiblesse musculaire à prédominance distale et une détresse respiratoire précoce secondaire à une paralysie diaphragmatique. Nous avons décrit 9 nouvelles mutations au sein du gène IGHMBP2 (pour immunoglobulin µ-binding protein 2) chez 5 patients atteints et confirmé ainsi le rôle pathogène de ce gène. Sept des 9 mutations décrites concernent des acides aminés conservés dans les espèces et localisés dans le domaine hélicase. Ce domaine pourrait donc jouer un rôle essentiel dans la physiopathologie de la maladie (Hum Mutat. 2004; 23(5):525-6). Deuxièmement, nous avons rassemblé des patients atteints d’amyotrophie spinale chronique distale (d-HMN III/IV), de transmission autosomique récessive. Cette affection se définit par une amyotrophie et une faiblesse musculaire progressive qui prédomine au niveau des pieds et des mains. Grâce à des analyses de liaison réalisées dans 12 familles européennes, nous avons restreint la localisation génétique sur le chromosome 11 (en 11q13.3) à un intervalle de 2.6 cM. Nous avons également mis en évidence un déséquilibre de liaison entre 3 allèles rares et le locus génétique, ce qui suggère un phénomène d’effet fondateur dans la population caucasienne (Eur J Hum Genet. 2004;12(6):483-8). Des études complémentaires sont actuellement en cours à l’Hôpital Necker-Enfants-malades à Paris pour identifier le gène responsable de cette forme chronique d’amyotrophie spinale distale. Troisièment, nous avons décrit un nouveau variant d’amyotrophie spinale de transmission autosomique récessive. L’amyotrophie et la faiblesse musculaire débutent vers l’âge de 3 ans et concernent rapidement l’ensemble de la musculature. Le pronostic est sévère, avec perte de la marche durant l’enfance et altération de l’autonomie respiratoire à l’adolescence. L’étude d’une grande famille Malienne consanguine nous a permis de localiser le gène responsable de la maladie dans un intervalle de 3.9 cM sur le chromosome 1, en 1p36 (Neurology. 2006;67(1):120-4). Nous avons ensuite mis en évidence une mutation faux-sens à l’état homozygote dans le gène PLEKHG5 chez les patients atteints et avons prouvé le caractère pathogène de cette mutation grâce à une série d’études fonctionnelles. Nous avons montré que la protéine PLEKHG5 sauvage a une distribution cytoplasmique homogène dans des cellules rénales (HEK293) et mammaires (MCF10A) humaines transfectées et qu’elle y active la voie de signalisation NF-kappaB. La protéine PLEKHG5 mutée est quant à elle instable, ce qui entraîne une perte de sa fonction activatrice sur NF-kappaB. De plus, grâce à des études de transfection transitoire de motoneurones murins (cellules NSC34), nous avons montré que la protéine PLEKHG5 mutée entraîne la formation d’importants agrégats cytoplasmiques. Dans cette nouvelle forme d’amyotrophie spinale, la perte de la fonction activatrice de la voie de signalisation NF-kappaB et la formation d’agrégats pourraient toutes deux contribuer à la neurotoxicité de la protéine PLEKHG5 mutée et conduire ainsi à la dégénérescence des motoneurones (Am J Hum Genet. 2007;81(1):67-76). En conclusion, nous espérons que ces résultats, qui contribuent à améliorer la connaissance des mécanismes physiopathologiques responsables de la dégénérescence des motoneurones, ouvriront à l’avenir la voie vers de nouvelles perspectives thérapeutiques.

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