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.

Duchenne muscular dystrophy (DMD) is characterized by necrosis and progressive loss of muscle fibers. DMD patients have a mutation in the gene encoding dystrophin, a large membrane-associated cytoskeletal protein on the cytoplasmic side of the sarcolemma. Gene therapy using fully deleted adenoviral vectors shows great potential for the eventual treatment of DMD and other genetic diseases. These vectors are less immunogenic than their predecessors and have the capacity to carry large DNA inserts such as the full-length dystrophin (12 kb). However, the lack of viral genes results in a weakened and subsiding (short) transgene expression in muscle. Findings in the lung and liver have shown the adenoviral E4 region, in particular E4 open reading frame 3 (ORF3) to contribute to the maintenance of transgene expression. We constructed an adenovirus in which E4 ORF3 was reintroduced into a fully-deleted adenovirus along with full-length dystrophin (AdCBDysORF3). Dystrophin levels produced by AdCBDysORF3 were found to be not sustained in mdx mice, dropping significantly by day 90. However, expression levels did increase when AdCBDysORF3 was complemented with other viral proteins such as EIB. Likewise, increasing the expression of the primary adenovirus receptor (CAR) in muscle also resulted in a higher initial dystrophin expression in myofibers.

No description available.

Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy 1A (MDC1A) are the two most common forms of muscular dystrophy in humans, caused by mutations in dystrophin and laminin α2 genes respectively. Both are severe forms of the disease that lead to premature death due and are both now known to have a significant effect on the central nervous system. This project investigated the role of both proteins involved in each of these diseases in cerebellar Purkinje cells of two murine models of disease: the mdx mouse a dystrophin-deficient model of DMD and the dy2J a laminin α2-deficient murine model of MDC1A. In the case of dystrophin further studies were undertaken in order to determine if increasing age had any effects on cerebellar function. It was found that there is no difference in electrophysiological characteristics (RMP, IR, eEPSP) of the cells when compared to appropriate control groups, nor was there any difference when young and aged dystrophin-deficient mdx groups were compared. Evoked IPSP characteristics were examined in young mdx cerebellar Purkinje cells and again no difference was found when compared to wildtype. There was a significant difference in response to the GABAA antagonist bicuculline, with wildtype increasing eEPSP amplitude by almost double that found in mdx. There was no difference in short term plasticity as measured by paired pulse facilitation in any of these groups. There was no difference in paired pulse depression at the inhibitory interneuron- Purkinje cell synapse of young wildtype and mdx cerebellar Purkinje cells. There a significant blunting of long term depression (LTD, (a form of long term synaptic plasticity) between young wildtype and mdx. When young wildtype animals were compared to aged wildtype animals LTD was found to be similar, when young mdx was compared to aged mdx, there was a recovery of LTD seen in the aged population. There was also significant differences in LTD found when littermate controls were compared to dy2J (laminin α2 mutants). A third of the phenotypic animals (dy2J) potentiated. Finally when rebound potentiation (a GABA-ergic form of long term synaptic plasticity in the cerebellum) was compared in young wildtype and mdx mice, mdx mice displayed depression, rather than the expected potentiation in contrast to potentiation (or no change) as seen in all wildtype cells.

Duchenne muscular dystrophy (DMD) is an X-linked lethal condition associated with high morbidity and mortality. There is currently no cure for this disease. Several gene-based therapeutic approaches for treating DMD are currently under development but all are dependent on the knowledge of the causative dystrophin gene mutation. A combined mutation detection approach consisting of a quantitative PCR based analysis and DNA sequencing of the dystrophin gene resulted in a mutation etection rate of 96% in the New South Wales (NSW) DMD cohort. The proportion of exon duplication mutations was twice that generally reported for similar patient opulations. The clinical utility of the combined mutation protocol for DMD carrier testing clarified the carrier status of an additional one-third (33%) of female relatives compared to a conventional approach of biochemical, pedigree and linkage studies. The generally accepted view that two-thirds of mothers of isolated cases of DMD are themselves mutation carriers is challenged. Although this assumption is valid for duplication and DNA sequence mutations, it is not valid for deletion mutations in the NSW cohort. The incidence of new cases of DMD in the New South Wales population was educed from approximately 1 in 3594 live male births to 1 in 6022 live male births over a 25 year period, indicative of a significant effect of the combination of genetic counselling and improved methods of carrier detection over that period. In a study of a cohort of boys with DMD, who had both psychological and mutational analysis, it was shown that mutations affecting the shorter, C-terminal isoforms of dystrophin are associated with decreased mean intellectual function. A hypothesis is presented that mutations within the long 5' untranslated region of the Dp140 isoform are unlikely to significantly affect expression of this brain-expressed isoform. During the course of studying the NSW DMD cohort a family was identified which exhibited X-linkage and a unique clinical presentation involving episodes of severe and prolonged muscle weakness. A novel variant in the pyruvate dehydrogenase E1 alpha subunit (PDHA 1) was identified. The phenotypic effect of this variant is not proven but a body of evidence implicates this as likely to be causative of the observed phenotype.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

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