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, 2009. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2009" Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 76-85).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 137-161).

Spinal Muscular Atrophy (SMA) is an autosomal, recessive form of childhood motor neuron disease and the most common genetic cause of infant mortality in the western world. SMA displays the characteristic hallmarks of a motor neuron disease, including loss of motor neurons in the spinal cord and atrophy of skeletal muscles. However, mounting evidence suggests that multiple tissues and body systems, beyond the neuromuscular system, are affected in SMA. Previous studies have highlighted alterations in the vascular system in both SMA patients and in a variety of mouse models of the disease, reporting alterations in vessel structure and perfusion abnormalities in peripheral tissues. In this project a detailed morphological investigation of the capillary beds of skeletal muscle and the spinal cord, two of the key pathological tissues in SMA was undertaken. This work was conducted in the Smn-/-;SMN2, Smn-/-;SMN2tg/+ and Smn-/-;SMN2;Δ7 mouse models of SMA. Significant alterations in the form and extent of the skeletal muscle and spinal cord capillary bed in SMA mice were identified, the most striking of which being a reduction in capillary density in SMA tissue when compared to control littermate tissue. In skeletal muscle, this reduction in capillary density was found to be a postnatal phenomenon, which occurred independently of denervation, in a variety of phenotypically distinct muscles and in all three SMA mouse models investigated. In the spinal cord, the capillary defect was seen to develop in a similar postnatal pattern to that observed in skeletal muscle. Importantly, a reduction in capillary density was observed in the ventral horn of the spinal cord, which houses motor neuron cell bodies, a known pathological target in SMA. These motor neurons were seen to be surrounded by fewer capillaries than their control counterparts. Using an injectable marker of hypoxia, it was determined that the cells of the ventral horn of SMA spinal cords are hypoxic. This suggests that the capillary defect identified has a functional impact on the tissues it is observed in. Having established the presence of capillary defect in SMA tissue, the effect of potential SMA therapeutics on the capillary defect was then investigated. The effect of HDAC inhibitors, which have been successfully shown to increase the levels of the disease causing Smn protein, was investigated. Treatment with the HDAC inhibitor SAHA was found to ameliorate the capillary defect, significantly improving capillary density in SMA skeletal muscle. This implies that the capillary defect is related to Smn levels in tissue and is amenable to therapeutics which increase Smn levels. Having characterised the capillary defect in SMA tissues in detail, a selection of tools were then used to investigate the underlying mechanisms resulting in the defect. First, using primary cell cultures, the growth and morphology of the key cellular component of capillaries, the endothelial cell, was examined. While displaying reduced levels of the Smn protein, endothelial cells isolated from SMA tissues showed no difference in growth rate, morphology or endothelial cell marker expression when compared to endothelial cells isolated from control tissue. This suggests that the defects seen in SMA capillary beds are not the result of defects in the structure and growth of endothelial cells. Second, retinas from SMA mice were found to exhibit similar capillary defects to those observed in SMA skeletal muscle and spinal cord. Given the entirely postnatal development of the retinal capillary network, the retina was identified as a useful experimental preparation for the further investigation of the mechanisms underlying the capillary defect in SMA. In summary, this work highlights the incidence and importance of capillary defects in mouse models of spinal muscular atrophy.

The childhood neuromuscular disease spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN) protein. Historically, SMA has been characterised as a disease primarily affecting lower motor neurons. However, recent breakthroughs have revealed defects in other non-neuronal cells and tissues. In vivo analysis of peripheral nerve showed defects in Schwann cells, manifesting as abnormal myelination and delayed maturation of axo-glia interactions. The experiments in this thesis were designed to build on these observations and examine whether Schwann cell defects are intrinsic and occur as a primary result of low levels of SMN in that cell type, or rather represent a secondary consequence of pathology in neighbouring motor neurons. I initially developed a protocol to allow isolation of high-yields of purified, myelination-competent Schwann cells from ‘Taiwanese’ SMA mice. SMA-derived Schwann cells had significantly reduced SMN levels and failed to respond normally to differentiation cues. Increasing SMN levels restored myelin protein expression in Schwann cells from SMA mice. Perturbations in expression of key myelin proteins were likely due to failure of protein translation and/or stability rather than transcriptional defects. Co-cultures of healthy neurons with SMA Schwann cells revealed a significant reduction in myelination compared to cultures where wild-type Schwann cells were used. The presence of SMA Schwann cells also disrupted neurite stability. Perturbations in the expression of key extracellular matrix proteins, such as laminin α2, in SMA-derived Schwann cells suggests that Schwann cells were influencing neurite stability by modulating the composition of the extracellular matrix. Previous studies have demonstrated that low levels of SMN lead to disruption of ubiquitin homeostasis and decreased expression of ubiquitin-like modifier activating enzyme (UBA1) in the neuromuscular system, driving neuromuscular pathology via a beta-catenin dependent pathway. Label-free proteomics analysis of SMA and control Schwann cells identified 195 proteins with modified expression profiles. Bioinformatic analysis of these proteins using Ingenuity Pathway Analysis (IPA) software confirmed that major disruption of protein ubiquitination pathways was also present in Schwann cells from SMA mice. Immunolabeling and proteomics data both revealed that UBA1 levels were significantly reduced in SMA-derived Schwann cells. However, loss of UBA1 in Schwann cells did not lead to downstream modifications in beta-catenin pathways. Pharmacological inhibition of UBA1 in healthy Schwann cells was sufficient to induce defects in myelin protein expression, suggesting that UBA1 defects contribute directly to Schwann cell disruption in SMA. I conclude that low levels of SMN induce intrinsic defects in Schwann cells, mediated at least in part through disruption to ubiquitination pathways.

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.

No description available.