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This dissertation, Production and Characterization of Recombinant Mouse ProGDNF by Mingxi, Wang, 王明席, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of Thesis Entitled Production and Characterization of Recombinant Mouse ProGDNF Submitted by WANG Mingxi for the Degree of Doctor of Philosophy at The University of Hong Kong in December 2006 The mechanisms of selective spinal motor neuronal loss in spinal and bulbar muscular atrophy (SBMA) patients, caused by mutated expanded polyglutamine (poly Q) in androgen receptor (AR), are still not clearly understood. SBMA patients often have elevated plasma creatine phosphokinase led us to hypothesize that the subclinical pathologic changes involving skeletal muscles contribute to such selectivity. Especially, SBMA AR might impair the capacity of skeletal muscles in providing neurotrophic factors to support the viability of motor neurons innervating the skeletal muscles. Based on this hypothesis, an in vivo SBMA muscle model was previously established by direct electrotransfer of SBMA AR constructs into biceps of neonatal mice, and a 52 kDa putative dimeric proGDNF was found to be induced while mature GDNF was drastically reduced. In the current study, this inductive effect on proGDNF of SBMA AR was further confirmed in both in vitro and improved in vivo SBMA model. To explore the functional/pathogenic roles proGDNF on motor neurons, cleavage-resistant proGDNF constructs were generated for expressing proGDNF. Two expression systems, mammalian cell and baculovirus insect cell production, were adopted. With carefully refinement of these systems, cleavage-resistant proGDNF were expressed, and purified through serial procedures including Immobilize Metal Affinity chromatography, Heparin Affinity Chromatography, Concanavalin A Affinity Chromatography etc. In vitro study showed that proGDNF retained some common properties as mature GDNF, such as dimerization. On the other hand, proGDNF exhibited different functional profiles from mature GDNF, including being less potent in inducing neurite growth. For better characterization of the neurite-promoting potency of proGDNF, a more motor neuron-like cell line NSC34-N was derived from NSC-34 and shown to be more prone to sprout neurites, more prone to be infected by AAV1 virus and expressed all the receptors responsible for known GDNF signaling pathways. Transient and long lasting in vivo proGDNF expression in skeletal muscles were also successfully generated by electrotransferring proGDNF expressing constructs into biceps muscles or by infecting them with AAV1 vector respectively. Characterizations of GDNF precursor expression in vitro driven by wild type GDNF constructs in transiently transduced mammalian cells and in vivo driven by AAV1 virus provided evidences to suggest that the pro domain of proGDNF might render proGDNF cleavage less efficient. ProGDNF expressed in these ways also behaved differently from mature GDNF in not being transported to the neuromuscular junctions (NMJs). These proGDNF properties, including less potence in inducing neurite growth, plausible inability to bind glial cell line-derived neurotrophic factor receptor alpha-1 (GFRα1), failure to be transported to NMJs, and less efficiency in being processed into GDNF, suggested that the skeletal muscle-derived proGDNF could be less effective than mature GDNF to protect against progressive motor neuron apoptosis in SBMA patients. Taken together with other currently known cellular mechanisms exerted by poly Q's toxici

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