Quantitative Analysis of Novel Chemical and shRNA Based Methods to Increase Survival of Motor Neuron Protein Levels
Authors
Evans, Matthew C.Faculty Advisor
Elliot J. Androphy, M.D.Academic Program
Interdisciplinary Graduate ProgramUMass Chan Affiliations
MedicineDocument Type
Doctoral DissertationPublication Date
2011-06-20Keywords
Muscular AtrophySpinal
Survival of Motor Neuron 2 Protein
Histone Deacetylases
Amino Acids, Peptides, and Proteins
Enzymes and Coenzymes
Nervous System Diseases
Neuroscience and Neurobiology
Nucleic Acids, Nucleotides, and Nucleosides
Pathological Conditions, Signs and Symptoms
Metadata
Show full item recordAbstract
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder that is the leading genetic cause of infantile death. SMA is caused by homozygous deletion or mutation of the survival of motor neuron 1 gene (SMN1). The SMN2 gene is nearly identical to SMN1, however is alternatively spliced. The close relationship to SMN1 results in SMN2 being a very power genetic modifier of SMA disease severity and a target for therapies. In this study we attempt to characterize novel chemical compounds identified as potential activators of the SMN2 gene. Additionally, we sought to determine the regulatory role individual HDAC proteins use to control expression of full length protein from the SMN2 gene. We used quantitative PCR to determine the effects of novel compounds and shRNA silencing of individual HDACs on the steady state levels of a SMN2-luciferase reporter transcripts. We determined that the compounds identified in multiple reporter high throughput screens increased SMN protein levels via transcriptional activation of the SMN2 gene. Other compounds identified in the same screen functioned post-transcriptionally, possibly stabilizing the SMN protein itself by decreasing degradation. Furthermore, we determined that reduction of individual HDAC proteins was sufficient to increase SMN protein levels in a transgenic reporter system. Knockdown of class I HDAC proteins preferentially activated the reporter by increased promoter transcription. Silencing of class II HDAC proteins maintained transcriptional activity; however silencing of HDAC 5 and 6 also appeared to enhance inclusion of an alternatively spliced exon. This collective work defines a quantitative RNA based protocol to determine mechanism of SMN reporter increase in response to any chosen treatment method. Additionally, this work highlights HDAC proteins 2 and 6 as excellent investigative targets. These data are important to the basic understanding of SMN expression regulation and the refinements of current therapeutic compounds as well as the development of novel SMA therapeutics.DOI
10.13028/r8sr-5t09Permanent Link to this Item
http://hdl.handle.net/20.500.14038/31908Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/r8sr-5t09