A la fin du XVIIIe siècle, la population de la région stéphanoise est caractérisée par sa maîtrise des processus industriels, une culture spécifique du corps, influencée par les rituels carnavalesques, le mouvement convulsionnaire janséniste, puis le magnétisme animal. L'industrie dépend alors des corps des ouvriers, des Sublimes. Les critiques des élites face à un système industriel passent des discours à une lente immixtion dans la chair des ouvriers qu'il faut enfermer, contrôler et rationaliser. La disqualification des corps ouvriers commence par un déclassement esthétique, qui débouche sur une racialisation, confortée par les théories de la dégénérescence. La rationalisation, la morale hygiéniste et l'éducation physique participent de cette prise de contrôle de la chair. Les nombreuses résistances des ouvriers face à une industrialisation rationalisée et mécanisée aliénante se caractérisent par une contre-culture ouvrière tenace, le maintien de la petite industrie qui maintient des espaces de liberté, et des violences, qui passe par l'anarchisme violent la grève, fracassée par une répression d'Etat. / At the end of the eighteenth century, the population of the Stephan region was characterized by its mastery of industrial processes, a specific culture of the body, influenced by carnival rituals, the Jansenist convulsion movement, and then animal magnetism. lndustry then depends on the bodies of the workers, the Sublimes. The criticisms of the elites in the face of an industrial system pass !rom speeches to a slow interference in the workers' flesh, which must be locked up, controlled and rationalized. The disqualification of the workers' bodies begins with an aesthetic downgrading, which leads to racialization, reinforced by theories of degeneration. Rationalization, hygienic morality and physical education are part of this takeover of the flesh. The many resistance of the workers to an alienating mechanized and rationalized industrialization are characterized by a tenacious working-class counter-culture, the maintenance of small industry which maintains areas of freedom and violence, which passes through anarchism violate the strike, Shattered by a state repression.

Patients with giant axonal neuropathy (GAN) exhibit loss of motor and sensory function and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin, a ubiquitously-expressed cytoplasmic protein whose cellular roles are poorly understood. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. Disorganization of the neuronal intermediate filament (nIF) network is a feature of several neurodegenerative disorders, including amyotrophic lateral sclerosis, Parkinson's disease and axonal Charcot-Marie-Tooth disease. In GAN such changes are often striking: peripheral nerve biopsies show enlarged axons with accumulations of neurofilaments; so called "giant axons." Interestingly, IFs also accumulate in other cell types in patients. These include desmin in muscle fibers, GFAP (glial fibrillary acidic protein) in astrocytes, and vimentin in multiple cell types including primary cultures of biopsied fibroblasts. These findings suggest that gigaxonin may be a master regulator of IFs, and understanding its function(s) could shed light on GAN as well as the numerous other diseases in which IFs accumulate. However, an interaction between gigaxonin and IFs has not been detected and how IF accumulation is triggered in the absence of functional gigaxonin has not been determined. To address these questions I undertook a proteomic screen to identify the normal binding partners of gigaxonin. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. Strikingly, human motor neurons (MNs) differentiated from GAN iPSCs recapitulate this key phenotype. Accumulation of nIFs can be rescued by reintroduction of gigaxonin, by viral delivery or genetic correction. GAN iPS-MNs do not display survival vulnerability in the presence of trophic factors, but do display increased cell death in the presence of oxidative stress. Preliminary experiments suggest that in iPS-MNs nIFs are degraded by contributions from both the proteasome and lysosome. Gigaxonin interacts with the autophagy protein p62 which has been implicated in the clearance of ubiquitin aggregates by the lysosome, and this interaction is greatly enhanced in conditions of oxidative stress. My data provide the first direct link between gigaxonin loss and IF aggregation, and suggest that gigaxonin may be a substrate adaptor for the degradation of IFs by autophagy, pointing to future approaches for reversing the phenotype in human patients.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the degeneration of cortical and spinal motor neurons. Animal models of ALS based on known ALS-causing mutations are instrumental in advancing our understanding of the pathophysiology of motor neuron degeneration. Recent identification of mutations in the genes encoding RNA-binding proteins TDP-43 and FUS has suggested that aberrant RNA processing may underlie common mechanisms of neurodegeneration in ALS and focused attention on the normal activities of TDP-43 and FUS. However, the role of the normal functions of RNA-binding proteins in ALS pathogenesis has not yet been established. In this thesis I present my work on novel FUS-based mouse lines aimed at clarifying the relationships between ALS-causing FUS mutations, normal FUS function and motor neuron degeneration. Experiments in mutant FUS knock-in mice show evidence of both loss- and gain-of-function effects as well as misfolding of mutant FUS protein. Characterization of mice expressing ALS-mutant human FUS cDNA in the nervous system reveals selective, early onset and slowly progressive motor neuron degeneration that is mutation dependent, involves both cell autonomous and non-cell autonomous mechanisms and models key aspects of ALS-FUS. Using a novel conditional FUS knockout mutant mouse, I also demonstrate that postnatal elimination of FUS selectively in motor neurons or more broadly in the nervous system has no effect on long-term motor neuron survival. Collectively, our findings suggest that a novel toxic function of mutant FUS, and not the loss of normal FUS function, is the primary mechanism of motor neuron degeneration in ALS-FUS.

Neurodegenerative disorders constitute a class of diseases that express characteristic misfolded proteins that aggregate and induce neuronal toxicity and death. Huntington’s disease is one such fatal protein misfolding disease. Currently no therapeutic avenue can delay or stop the progression of the disease. In this context, there is a need to identify therapeutic pathways and drug targets that can prevent or delay pathogenesis in neurodegenerative diseases involving protein misfolding. This dissertation describes how our search for new drug targets have led us to identify protein disulfide isomerase and three unique small molecules that modulate its activity as a means to protect neuronal cells from neurodegenerative protein misfolding diseases, such as Huntington’s disease. Protein disulfide isomerase is a thiol-oxidoreductase in the endoplasmic reticulum that has garnered increased attention because of its implicated role in numerous human diseases, including cancer, human immunodeficiency virus pathogenesis, and thrombosis. Validating protein disulfide isomerase as target for neurodegenerative disorders may open up new therapeutic strategies to understand and treat these diseases. First, I describe the identification and validation of protein disulfide isomerase as a target of the neuroprotective small molecule, 16F16. I show that 16F16 is an irreversible inhibitor of protein disulfide isomerase that binds covalently to both cysteines in the active site. This inhibition is protective in cell and brain-slice models of Huntington’s disease, as well as in the brain-slice model of Alzheimer’s disease. Next, I describe the neuroprotective small molecule IBS141 that was originally incorrectly annotated with a chemical structure. I elucidate the correct structure of the active compound using analytical chemistry, revealing it to be the natural product securinine. Furthermore, I identify the binding site of securinine to protein disulfide isomerase and show that the inhibition of the protein is protective in cell and brain-slice models of neurodegenerative diseases. In addition to finding this unexpected activity of securinine, I provide a systematic roadmap to those who encounter compounds with incorrect structural annotation in the course of screening campaigns. Last, I describe the discovery of LOC14, a nanomolar, reversible, modulator of protein disulfide isomerase that protects cells and medium spiny neurons from the toxic mutant huntingtin protein. I find that this protection results from LOC14 binding adjacent to the active site and inducing protein disulfide isomerase to adopt an oxidized conformation. LOC14, has dramatically improved potency for protein disulfide isomerase over previously identified inhibitors and displays favorable pharmaceutical properties, making it an idea compound to evaluate the therapeutic potential of modulating protein disulfide isomerase in in vivo models of neurodegenerative diseases.

Selective neuronal loss in response to loss or dysfunction of a ubiquitously expressed protein is a hallmark of neurodegenerative disease. Proximal spinal muscular atrophy (SMA) is caused by homozygous loss of the ubiquitously expressed survival motor neuron 1 (SMN1) gene, resulting in progressive neuromuscular weakness that eventually leads to flaccid paralysis and death from respiratory failure by two years of age in the most severely affected patients. Despite widespread motor neuron loss, certain motor pools are clinically spared. Type I SMA patients exhibit intercostal recession in conjunction with diaphragmatic sparing that produces a characteristic “bell-shaped chest.” Additionally, patients retain extraocular and external sphincter function, even in late disease stages. In order to fully define this differential vulnerability, I performed an extensive characterization of neuromuscular autopsies from Type I SMA patients and age-matched control patients. I found highly divergent degrees of motor unit degeneration, even within individual cranial nerves or a select anatomical region such as the neck. Remarkably, the diaphragm in a Type I SMA patient kept alive on life support for 17 years was still relatively preserved, despite virtually complete fibro-fatty infiltration in other muscles. Extraocular functions were also normal in this patient. These findings suggest that the molecular determinants of SMA-resistance provide indefinite protection against low SMN protein. Thus, identification and modulation of these genes and pathways represents a promising potential therapeutic strategy. Remarkably, this exquisite pattern of selectivity was preserved in the SMNΔ7 mouse, a widely used SMA mouse model. This suggests that the molecular determinants of differential vulnerability are conserved between mouse and human. Given the high degree of diversity between motor pools, I performed a comparative transcriptional microarray between multiple SMA-vulnerable and –resistant motor pools in healthy mice. This analysis revealed a small number of candidate therapeutic genes that segregate closely with vulnerability. I present a series of preliminary studies evaluating these targets in the SMNΔ7 mouse. Ongoing and future studies combine pharmacological, viral, and genetic approaches to modulate these candidate targets in the SMNΔ7 mouse and assess for improvements in neuromuscular pathology. Given the remarkable preservation of select motor pools in SMA patients, changing expression levels of the candidate targets I have identified may provide substantial clinical benefit.

Neuronal circuit perturbations are emerging as important determinants in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, and spinal muscular atrophy (SMA). SMA is a motor neuron disease caused by deficiency in the ubiquitously expressed survival motor neuron (SMN) protein. The hallmarks of SMA include loss of motor neurons, muscle atrophy, and abnormal postural reflexes. Although cell-autonomous mechanisms of motor neuron death have received much attention, recent studies in animal models of SMA revealed that motor circuit deficits resulting from early impairment of synaptic function and sensory-motor connectivity precede motor neuron death. It remains to be established whether motor circuit dysfunction is a consequence of SMN-deficiency in the motor neuron or SMN-dependent alterations in the activity of premotor neurons. Here I sought to address these outstanding issues through the development and characterization of a simplified in vitro model of the motor circuit based on the use of embryonic stem cell-derived motor neurons and interneurons. I found that SMN deficiency caused death of motor neurons in co-culture with other neurons as well as in isolation, demonstrating the cell autonomous origin of this defect. SMN requirement for motor neuron function was investigated using intracellular patch clamp recordings to measure both passive and active membrane properties. Remarkably, SMN deficiency induced hyperexcitability of motor neurons only when they are cultured in the presence of interneurons but not in isolation, providing initial evidence that SMN deficiency induces motor neuron hyperexcitability in a non-cell autonomous manner and that dysfunction and death of motor neurons are uncoupled. To determine the role of SMN-dependent interneuron dysfunction on motor neuron hyperexcitability, the effect of selective SMN depletion in either motor neurons or interneurons was investigated. Importantly, I found that SMN-deficient motor neurons cultured in the presence of wild type interneurons are not hyperexcitable, while the presence of SMN-deficient interneurons is necessary and sufficient to elicit hyperexcitability of wild type motor neurons. Therefore, in the context of SMN deficiency, increased excitability of motor neurons is a homeostatic response to interneuron dysfunction. Although the exact mechanism is currently unknown, reduced glutamatergic drive appears to play a role since glutamatergic receptor blockers phenocopied SMN deficiency in inducing motor neuron hyperexcitability but not neuronal death. Moreover, SMN deficiency caused reduction of excitatory VGluT2 synapses on motor neurons. In addition to changes in intrinsic membrane properties, SMN deficiency caused severe reduction in the spontaneous activity and firing pattern of motor neurons. However, in contrast to death and hyperexcitability, SMN-dependent deficits in both motor neurons and interneurons appear to underlie this complex phenotype. The findings presented in this study validate the use of in vitro models to study SMA disease mechanisms and shed new light on the cellular basis of motor circuit dysfunction induced by SMN deficiency that can have predictive value in vivo.

Inherited retinal dystrophies encompass a broad group of genetic disorders affecting visual functions in as high as 1 in 3,000 individuals around the world. Common symptoms include loss of central, periphery, or night visions, and in severe cases progression to complete blindness. Syndromic forms also exist involving abnormalities in other parts of the body. Currently, more than 250 genes representing a wide variety of functional roles have been shown to be responsible for the disease phenotypes. Moreover, mutations in the same gene sometimes cause different phenotypes while mutations in multiple genes can give rise to the same clinical subtype, further demonstrating the level of complexity in these disorders. Such genetic heterogeneity has substantially complicated the process of pinpointing precise genetic causes underlying these conditions. The goal of my thesis research is to clarify the genetic causes underlying retinal dystrophies, with a primary focus on phenotypes resembling ABCA4-associated diseases and retinitis pigmentosa in both syndromic and non-syndromic forms. Recent advances in the next-generation sequencing (NGS), the high-throughput, ‘deep’ sequencing technology, have enabled several novel genes to be identified, or found new mutations in known genes. Nevertheless, a substantial fraction of unsolved cases still remain. The primary work in this thesis involves utilizing NGS, particularly whole-exome sequencing, to identify disease-causal mutations in families where at least one parent and affected or unaffected siblings are available. Determining all genetic variation underlying retinal diseases is necessary for precise molecular genetic diagnosis and improved prognosis of these conditions. The first part of my thesis highlights the complexity in genetic inheritance of diseases caused by mutations in the ABCA4 gene. In a substantial fraction of Stargardt Disease cases with only one mutation in the ABCA4 coding region, deep sequencing of the entire locus identified the second mutation in the intronic region of the gene in 10% of cases. The genetic heterogeneity of ABCA4 was further demonstrated by the identification of 4 different pathogenic ABCA4 mutations and 4 phenotypes in a single family. These findings epitomized the extremely complex mutational spectrum underlying the ABCA4-associated diseases and suggested thorough sequencing of variations in the entire genomic locus, including copy number variant analysis. In the second part of my thesis, exome-sequencing has led to findings of phenotypic expansions in known disease gene, and in one case the precise molecular diagnosis resulted in an immediate treatment. A family with 2 affected siblings presented novel phenotype of a macular dystrophy caused by mutations in CRB1. In another family where 9 members were affected with late-onset BEM, a mutation was found in CRX given incomplete penetrance. In one family with an affected adult, two well-documented mutations in MMACHC - a gene causal for a potentially debilitating disorder of cobalamin deficiency, were found to segregate with bull’s eye maculopathy (BEM) and minimal systemic features in the proband. Early diagnosis in this patient resulted in hydroxycobalamin treatment for her condition, and possibly an improvement of her systemic prognosis. Together, these findings revealed that clinical phenotype can be very divergent from those described, and only genetic testing can unequivocally determine the cause of a disease. The third part of my thesis work highlights first-time discovery, and co-discovery of new genes associated with retinal diseases. A new form of syndromic RP was investigated in a family presenting a previously undescribed constellation of phenotypic features. Exome sequencing analysis of 3 affected siblings and their unaffected parents revealed deleterious mutations in the RDH11 gene. In another family where 2 affected siblings presented with a remarkably similar phenotype, no mutations in RDH11 were detected. However, analysis of absence of heterozygosity revealed causal mutations in the CWC27 gene. In the search for novel genes in cone-rod dystrophy cases negative of ABCA4 mutations, WES identified new rare, deleterious mutations in RAB28 in two families of Spanish descent. These findings revealed novel genetic causes underlying hereditary retinal diseases, and demonstrated the effectiveness of WES analysis in rare disease gene discovery. In summary, this work represents a comprehensive mutational analysis of inherited retinal dystrophies with complex genotype and phenotype correlations, utilizing next-generation DNA sequencing in large study cohorts. The power of whole-exome sequencing for gene discovery was well demonstrated by unequivocally solving close to 50% of all patients examined in this study. Establishing precise correlations between genotype and clinical phenotype is important for facilitating patient care, counseling, and therapeutic intervention for inherited diseases.

This thesis describes the development of an systems biology method to study transcriptional programs that are activated during early and late phases of cell-fusion mediated reprogramming, as well as an implementation of systems-level analysis using reverse-engineered regulatory networks to study CNS disorders like Alcohol Addiction, and neurodegenerative disorders like Alzheimer's Disease (AD), and Parkinson's Disease (PD). The results will show an unprecedented view into the mechanisms underlying complex processes and diseases, and will demonstrate the predictive power of these methodologies that extended far beyond their original contexts.

Polyglutamine (polyQ) diseases describe a group of late-onset progressive neurodegenerative disorders which are caused by the CAG triplet repeat expansion in the coding region of disease genes. Such expansions result in expanded polyQ tracts in the disease proteins which confer neurotoxicity. To date, nine such diseases are reported including Huntington's disease and several types of spinocerebellar ataxias. Misfolding of polyQ proteins and formation of intracellular SDS-insoluble protein aggregates are closely associated with the toxicity of these diseases. In particular, impairment of the ubiquitin-proteasome system (UPS) which is responsible for protein degradation has been observed in polyQ diseases. Recently, ubiquitin ligases, which govern substrate specificity of the UPS, have gained huge attention in polyQ disease pathogenesis studies. In humans, cullin (Cul) proteins, including Cul1, 2, 3, 4 & 5, are integral components of a group of ubiquitin ligases called cullin- RING ubiquitin ligases (CRLs). Each CRL displays distinct substrate specificity through specific substrate receptors. Cullin proteins are evolutionarily conserved and Cul orthologues are found in the Drosophila genome. In the present study, it was found that individual Culs displayed distinct effects on polyQ pathogenesis in Drosophila polyQ models. Particularly, it was found that Cul1 modulated polyQ-induced toxic phenotype. This modification was accompanied with an alteration in the ubiquitination level and SDS-solubility properties of expanded polyQ protein. Through genetic interaction studies and biochemical analyses, it is suggested that Cul1-based CRL specifically targets SDS-insoluble species of expanded polyQ protein for ubiquitination via selective recognition by CG2010 substrate receptor. On the other hand, it was found that expanded polyQ protein induced accumulation of CRL substrates in cells. Current data support a hypothesis that polyQ protein would impair the ubiquitin ligase activity of CRLs upon expansion of the polyQ domain, through interfering with neddylation of cullin and other uncharacterized mechanisms. Taken together, the present study identifies Cul1-CRL as a novel E3 ligase that modifies polyQ toxicity through modulating ubiquitination of expanded polyQ protein, and demonstrates a pathological mechanism by expanded polyQ protein through impairing CRL activity. These findings would lead to a better understanding of polyQ pathogenesis and give insights on developing treatments against polyQ diseases. / Wong, Kam Yan. / Adviser: Ho-Yin Edwin Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 260-273). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Polyglutamine disease is a class of neurodegenerative diseases, which is manifested by the atrophy of nervous system that results in dementia and/or motor dysfunction. The major pathological characteristics include progressive loss of neuronal cells as well as the appearance of insoluble nuclear inclusions in degenerating neuronal cells. Polyglutamine disease is caused by CAG triplet expansion in the genome. When translated, such expansion leads to the formation of expanded polyglutamine domain within the respective disease proteins and promotes abnormal protein conformational changes. Owing to their misfolded nature, the expanded polyglutamine proteins form insoluble nuclear inclusions. These insoluble nuclear inclusions are heterogeneous in nature, in which polyglutamine protein and molecular chaperones are the recruited components. All eukaryotic cells express molecular chaperones which function to mediate the proper folding of proteins. The recruitment of molecular chaperones into nuclear inclusions that contain misfolded triplet-expanded proteins strongly suggests the involvement of molecular chaperones in polyglutamine disease progression. It has been shown that over-expression of molecular chaperones in polyglutamine disease models can lead to a suppression of polyglutamine toxicity and a concomitant increase in the solubility of disease proteins, i.e. the solubility of polyglutamine disease protein is related to its toxicity. Intrigued by these observations, I aimed at elucidating the mechanism of polyglutamine disease pathogenesis by first studying the cellular regulation of endogenous chaperone expression in neurodegeneration in a transgenic Drosophila model of polyglutamine disease. A biphasic regulation of Hsp70 expression was observed, which the regulation was at the transcription level. Moreover, over-expression of Hsp70 could alter the endogenous Hsp70 protein and mRNA level of polyglutamine disease fly model. The study may help the understanding of how the chaperone expression is regulated under the effects of polyglutamine expression and thus to find out the mechanism of pathogenesis. In addition, cellular proteins that change in solubility other than disease protein will also be identified. Small heat shock proteins, glutathione S transferase and alpha 4 proteasome subunit, etc., showed change in solubility or expression by 2D gel electrophoresis analysis. Identifying the proteins that change in solubility or expression may help the finding of the interplay of proteins and thus the pathways involve in the mechanism of polyglutamine disease pathogenesis. Understanding pathogenic pathways can give ideas on how polyglutamine lead to the disease, up- or down-regulation of those protein interplays may provide direction and therapeutic candidates to suppress polyglutamine disease. / Huen Ngar Yee. / "September 2006." / Advisers: Ho Yin Chan; Siu Kai Kong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1465. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 134-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.