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Prion diseases are a group of fatal neurodegenerative diseases, including CJD and scrapie, which are thought to be caused by a protein termed a prion (PrP). As manganese has previously been suggested to be involved in prion disease we have investigated manganese binding to PrP and its role in the toxicity of the protein. We have shown that manganese bound PrP (MnPrP) has several of the characteristics of the disease form of PrP, including protease resistance and toxicity that is dependent on cellular PrP expression. Further investigation into the mechanism of toxicity revealed that MnPrP is significantly more toxic to neuronal cells than nonmanganese bound PrP and that toxicity requires the presence of known metal binding residues within the protein. We have demonstrated that treatment of neuronal cells with MnPrP causes caspase 3 activation and apoptosis, as demonstrated by DNA laddering, and we hypothesise that caspase 3 is activated by a p38 pathway. Treatment of neurones with MnPrP also caused a significant increase in cellular ROS production, although this did not appear to be a major cause of cell death as antioxidants were unable to save cells from cell death. We also investigated mechanisms by which cells can survive scrapie infection and MnPrP toxicity. We have shown that cells infected with scrapie have increased ERK activation which was important for their survival. Cells that survived MnPrP treatment were also found to have increased ERK activation. This suggests that ERK may have a protective role in prion diseases and may be a potential therapeutic target.

Le cerveau de drosophile est constitué entre autres des mushroom body, siège de la mémoire et de l’apprentissage. Cette structure est composée de différents types de neurones, parmi lesquels les neurones /. Ces neurones se présentent sous une forme orthogonale, avec l’axone qui se divise en une branche dorsale : la branche et une branche médiale : la branche . Le but de cette étude est de comprendre les mécanismes et voies de signalisation mis en jeu lors du développement de ces neurones.Chez la drosophile, la protéine APPL (Amyloïd Precursor Protein-Like) est l’homologue de la protéine APP humaine, connue pour son implication dans la maladie d’Alzheimer chez l’homme. Cette pathologie est caractérisée par une dégénérescence neuronale entraînant des défauts cognitifs et mnésiques. Malgré les nombreuses études focalisées sur la fonction pathologique d’APP durant les dernières décennies, peu de choses sont actuellement connues sur les fonctions physiologiques de cette protéine et notamment pendant le développement. C’est dans cette optique que nous avons étudié la fonction d’APPL et son interaction avec différentes protéines lors du développement des mushroom body. La protéine APPL a été identifiée comme étant un co-récepteur de la voie PCP (Planar Cell Polarity), permettant la régulation de la croissance axonale. Lors du développement, APPL permet le recrutement et l’activation de la protéine ABL (Abelson Tyrosine Kinase), qui phosphoryle DSH (dishevelled) et ainsi active la voie de signalisation permettant la croissance axonale.Le premier volet de cette thèse porte sur la régulation de l’activité ABL lors du développement des neurones /. S’il est établi qu’APPL permet une régulation positive de l’activité kinase d’ABL, je montre ici que la protéine HTT (huntingtine) permet de réguler négativement cette activité. Cette protéine HTT est impliquée dans la maladie de Huntington chez l’homme, une autre pathologie neurodégénérative. Ces travaux démontrent qu’HTT régule le niveau de phosphorylation d’ABL et par conséquent son activité. Le deuxième volet de cette thèse porte sur l’interaction d’APPL avec la protéine ARM (armadillo), homologue de la -caténine, lors du développement des neurones /. Je démontre que cette interaction est indépendante du rôle d’APPL dans la voie PCP. Je démontre aussi que cette interaction entre APPL et ARM est dépendante uniquement de la fonction d’ARM dans la dynamique du cytosquelette d’actine.Enfin le troisième volet de cette thèse porte sur la création de nouveaux allèles mutants pour Appl grâce à la technique du CRISPR-CAS9. La production de ces allèles permet d’avancer d’une part un possible rôle du gène voisin vnd (ventral nervous system defective) dans le développement des mushroom body, et d’autre part une interaction génétique entre Appl et vnd. / In the drosophila brain, mushroom bodies are involved in olfactory memory and learning. This structure is composed of different types of / neurons. These neurons form an orthogonal structure, with the branch projecting dorsally and the branch projecting medially. The aim of this study is to understand mechanisms and pathways involved during the development of these neurons.The drosophila APPL protein (Amyloïd Precursor Protein-Like) is the homologue of the human APP, known to be involved in Alzheimer’s disease. This pathology is characterized by neuronal degeneration inducing cognitive and memory defects. In spite of the numerous studies focused on the pathological function of APP during the last decades, few things are known on the physiological functions of this protein and more particularly during the development. This is from this perspective that we studied the APPL function and its interaction with proteins during the mushroom bodies development.The APPL protein was identified as a co-receptor of the PCP pathway (Planar Cell Polarity), involved in the axonal growth regulation. During the development, APPL allows the recruitment and the activation of the ABL protein (Abelson Tyrosine Kinase), which phosphorylates DSH (dishevelled) and so activates the axonal growth pathway.The first part investigates the regulation of ABL activity during the / neuron development. If it’s already established that APPL regulates positively the kinase activity of ABL, I show here that the HTT protein (Huntingtin) allows a negative regulation of ABL activity. In human, HTT is involved in the Huntington’s disease, another neurodegenerative disorder. This thesis work shows that HTT regulates the phosphorylation level of ABL, and therefore its activity.The second part investigates the interaction between APPL and ARM (armadillo), the homologue of the human -catenin, during the development of the / neurons. I show that this interaction is independent of the APPL function in the PCP pathway. Moreover, this interaction between APPL and ARM involves the actin cytoskeleton dynamic function of ARM, and not its Wnt pathway function.The third and last part presents new mutant alleles of APPL obtained with the CRISPR-CAS9 technique. The creation and analysis of these new alleles lead us to propose that vnd (ventral nervous system defective), neighbor gene of Appl, is also involved in / neurons development, and can interact genetically with Appl.

The neuromuscular junction (NMJ), the synapse formed between lower motor neuron and skeletal muscle fibre, is known to be a target in a number of neurodegenerative conditions, including motor neuron disease (MND). Located in an accessible part of the peripheral nervous system, the NMJ can be used as a ‘model synapse’ in the context of ‘connectomics’ – the study of synaptic connectivity throughout the nervous system as a whole. Although the NMJ has been studied in a number of species, relatively little is known about its structure in humans, complicating the translation of animal models of disease to the human condition. Described here is the first detailed cellular and molecular characterization of the human NMJ. A standardized methodology for comparative morphometric analysis of NMJs was developed and validated (‘NMJ-morph’). NMJ-morph was used to generate baseline data for 2160 NMJs from a single litter of wild type mice, representing 9 distinct muscles across 3 body regions. Principal components analysis (PCA) revealed synaptic size and fragmentation to be the key determinants of synaptic variability. Correlation data revealed the pre-synaptic cell (motor neuron) to be a stronger predictor of synaptic morphology than the post-synaptic cell (muscle fibre). Other factors influencing synaptic variability were in a clear hierarchy: muscle identity accounted for more variation in synaptic form than animal identity, with side having no effect. Human tissue was obtained from 20 patients (aged 34 to 92 years) undergoing lower limb amputation, primarily for the complications of peripheral vascular disease (PVD). Muscle samples were harvested from non-pathological regions of the surgical discard tissue. 2860 human NMJs were analyzed from 4 distinct muscles (extensor digitorum longus, soleus, peroneus longus and peroneus brevis), and compared with equivalent NMJs from wild type mice. Human NMJs displayed unique morphological characteristics, including small size, thin axons, rudimentary nerve terminals and distinctive ‘nummular’ endplates, all of which distinguished them from equivalent mouse NMJs. The previous notion of partial occupancy in human NMJs was disproved. As in mice, the pre-synaptic cell was shown to correlate more strongly with NMJ morphology; in contrast to mice, the human NMJ was found to be relatively stable throughout its 90+ year lifespan. In support of the tissue harvesting procedure, patient co-morbidities (diabetes mellitus and vascular disease) did not significantly impact NMJ morphology. Super-resolution imaging of the NMJ revealed significant differences in the functional architecture of human and mouse active zones. Despite the smaller synaptic size in humans, the total quantity of active zone material was conserved between the species, suggesting a homeostatic mechanism to preserve effective neurotransmission. Parallel proteomic profiling demonstrated further species-specific differences in the broader molecular composition of the NMJ. The cellular and molecular anatomy of the human NMJ is fundamentally different to that of other mammalian species. These differences must be taken into account when translating animal models of disease to the human condition.

Background: Language impairment associated with Motor Neurone Disease (MND) has been documented since the late 19th century, yet little is understood about the pervasiveness or nature of these deficits. The common clinical view among healthcare professionals is that communication difficulties can be attributed solely to the motor speech disorder dysarthria. Recent literature raises the possibility of more central processing deficits. Impairments in naming ability and comprehension of complex grammatical constructs have been frequently reported in some patients with MND. However, there is now growing evidence of spelling impairment, which could suggest the contribution of a more phonologically based deficit. In addition, the close relationship between MND and frontotemporal dementia (FTD) raises questions about the connection between the language impairments seen in MND patients and those documented in patients with the primary progressive aphasia (PPA) syndromes associated with FTD. Aims: This thesis examines the nature of speech and language deficits in people with MND and the extent to which expressive communication impairment can occur above and beyond dysarthria. In particular, the study explores: i) to what extent these language impairments can be attributed to deficits in working memory, executive functioning and/or disease severity; ii) what spelling errors can reveal about the integrity of lexical, phonological and orthographic processing; iii) whether similar patterns of impairment can be seen in PPA syndromes; iv) the relationship between language impairment and bulbar onset; and v) the impact these findings have on clinical management of MND patients. Methods: MND patients from across Scotland with changes in speech and/or language were tested using a neuropsychological battery of experimental and standardised tests of naming, spelling, syntactic comprehension, prosody and phonological and orthographical awareness. Patients were also screened for levels of dysarthria, executive functioning and working memory deficits, and results compared to those of matched controls. Findings: As a group, MND participants performed significantly worse than matched controls on measures of naming, spelling, orthographical awareness, grammatical comprehension, affective prosody and verbal fluency, but not working memory. However, based on patterns of individual impairment, of which spelling impairment formed a distinctive marker, the patient group divided into dichotomous subgroups, with 44% of participants categorised as ‘linguistically impaired’, while the remainder displayed little to no impairment. Those participants identified as linguistically impaired did not differ significantly from other MND participants on measures of disease severity, disease duration or dysarthria severity, although significantly more bulbar onset than limb onset participants were linguistically impaired. Spelling error patterns were suggestive of deficits at both a lexical and sublexical level, and were comparable to those reported in PPA literature. These findings suggest that dysarthria may be masking linguistic deficits in almost half of dysarthric MND patients, and highlight the importance of multidimensional assessment of language for effective clinical management.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.