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.

No description available.

by Wu Kwok Hang. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 213-229.

Dissertation (MSc)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

The androgen receptor (AR) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. Mutations in the androgen receptor are associated with androgen insensitivity syndrome (AIS), and a neurodegenerative disease, spinal bulbar muscular atrophy (SBMA). Most of the mutations causing AIS are loss-of-function missense mutations whereas SBMA is caused by a gain-of-function polyglutamine expansion in the N-terminal domain of the protein. Characterization of AR mutations has led to a better understanding of structure-function relationships of the AR and serves as a prototype for steroid receptors mechanisms of action. / In the first paper, we examine the role of an AR mutation in causing mild androgen insensitivity syndrome. We found that this mutation conferred reduced transactivation by AR through impaired interactions with the AR coactivator, TIF2, and impaired homodimerization. / In the second paper, we investigate the role of the AR polyGln expansion mutation in SBMA pathogenesis. Recent evidence has implicated proteolytic degradation of polyGln-expanded proteins and their subsequent intracellular aggregation in polyGn-expanded disease pathogenesis. We examined the role and composition of aggregates using fluorescently-tagged AR and found that proteolysis need not be a prerequisite for aggregation and that aggregation is not necessary for poly-Gln-induced cellular toxicity. / Finally, we characterize the novel heterodimerization of AR and ERalpha. We determined that this direct interaction has functional implications for the transactivational properties of both receptors.

The disruption of the survival motor neuron (SMN1) gene leads to the children’s genetic disease spinal muscular atrophy (SMA). SMA is characterized by the degeneration of α-motor neurons and skeletal muscle atrophy. Although SMA is primarily considered a motor neuron disease, the involvement of muscle in its pathophysiology has not been ruled out. To gain a better understanding of the involvement of skeletal muscle pathophysiology in SMA, we have developed three aims: to identify cell-specific Smn-interacting proteins, to characterize postnatal skeletal muscle development in mouse models of SMA, and to assess the functional capacity of muscles from SMA model mice. We have used tandem affinity purification to discover Smn interacting partners in disease relevant cell types. We have identified novel cell-specific Smn interacting proteins of which we have validated myosin regulatory light chain as a muscle-specific Smn associated protein in vivo. We have taken advantage of two different mouse models of SMA, the severe Smn-/-;SMN2 mouse and the less severe Smn2B/- mouse, to study the postnatal development of skeletal muscle. Primary myoblasts from Smn2B/- mice demonstrate delayed myotube fusion and aberrant expression of the myogenic program. In addition, the expression of myogenic proteins was delayed in muscles from severe Smn-/-;SMN2 and less severe Smn2B/- SMA model mice. Muscle denervation and degeneration, however, are not the cause of the aberrant myogenic program. At the functional level, we demonstrate a significant decrease in force production in pre-symptomatic Smn-/-;SMN2 and Smn2B/- mice indicating that muscle weakness is an early event in these mice. Immunoblot analyses from hindlimb skeletal muscle samples revealed aberrant levels of developmentally regulated proteins important for muscle function, which may impact muscle force production in skeletal muscle of SMA model mice. The present study demonstrates early and profound intrinsic muscle weakness and aberrant expression of muscle proteins in mouse models of SMA, thus demonstrating how muscle defects can contribute to the disease phenotype independently of and in addition to that caused by motor neuron pathology.

Spinal muscular atrophy (SMA) is the most commonly inherited neurodegenerative disease that leads to infant mortality worldwide. There are no known cures for SMA, but small increase in protein levels of SMN can be beneficial. We have developed adenoviral (Ad) vectors that express a human transgene of SMN and have tested their safety in vitro. We have demonstrated that these viruses can effectively express the transgene following cell entry and that the levels are relative to the virus dose. The viruses do not appear to impact the health and function of the cells, and are capable of increasing the number of Gems. We also attempted to change the tropism of the viruses through fiber protein modifications in order to target muscles and motor neurons. Our results suggest that a therapy based on an Ad-SMN fiber-modified vector may ultimately be successful in treating patients of SMA.

The autosomal recessive disorder spinal muscular atrophy (SMA) causes motor neuron degeneration and muscle wasting, progressing to paralysis and death in severe cases. The disease is caused by deficiency of survival motor neuron protein (SMN) due to deletion or mutation of the SMN1 gene. We seek to develop a protein-based therapy for SMA using an adenoviral vector which encodes a secretable form of SMN fused to a protein transduction domain (PTD) derived from the trans-acting activator of transcription (TAT) from HIV. We generated secretable GFP proteins using transient transfection in mammalian cells and determined that the secretory peptide was inefficient when paired with the native PTD. We generated TAT-GFP proteins in bacteria and observed that the variant TAT3 most reliably tranduced cells in vitro. We did not observe uptake of the therapeutic protein following infection with an adenoviral vector and subsequent secretion of the protein from infected cells.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "May 1997." Typescript. Vita. Includes bibliographical references (leaves 81-91). Also available on the Internet.

Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on January 20, 2009. Major professor: Scott Reaves, Ph.D. "Presented to the faculty of California Polytechnic State University." "In partial fulfillment of the requirement of the degree [of] Master of Science in Agriculture with a specialization in Food Science and Nutrition." "December 2008." Includes bibliographical references (p. 81-90). Also available on microfiche.