From the Department of Cell and Molecular Biology Karolinska Institutet, Stockholm, Sweden SPINAL AND BULBAR MUSCULAR ATROPHY: NEW INSIGHTS INTO THE DISEASE MECHANISM AND PROSPECTS FOR PHARMACOLOGICAL THERAPY

Expansion of a polyglutamine-encoding trinucleotide CAG repeat in the androgen receptor (AR) gene causes spinal and bulbar muscular atrophy (SBMA, or Kennedy’s disease). SBMA is an adult-onset disease characterized by progressive muscle weakness and atrophy due to the degeneration of lower motor neurons in the brainstem and spinal cord. At present, effective disease-modifying treatment is not available for this disorder. Neuronal dysfunction in SBMA is at least in part due to a toxic gain of function of the mutant AR, however, the underlying mechanism in the pathogenesis is not known. Work in this thesis identified new disease features of SBMA (Study I), investigated the effect of the polyglutamine expansion in the context of normal AR function (Study II), and explored pharmacological strategies for reducing mutant AR as potential treatments (Studies III and IV). In Study I, we describe a 29-year old SBMA patient with a 68 CAG repeat, the largest reported to date. The patient had an unusually early onset and novel clinical features, including developmental defects and autonomic dysfunction. In Study II, we examined the effect of the polyglutamine expansion in the AR on androgen-induced differentiation of neuronal cells. We show that mutant AR expression in this model leads to aberrant neurite outgrowth and reduced cell cycle arrest. The expanded polyglutamine tract in the AR interferes with the activity of the ubiquitin ligase APC/C-Cdh1, a critical regulator of cell cycle exit and neuronal architecture. These findings suggest that cellular abnormalities due to the stabilization of APC/C-Cdh1-dependent substrates may contribute to the pathogenic mechanism in SBMA. Augmentation of insulin-like growth factor (IGF)-1/Akt signaling was previously shown to promote the degradation of polyglutamine-expanded AR. In Study III, we tested the efficacy of exogenous IGF-1 administration in a transgenic mouse model of SBMA. We report that systemic delivery of IGF-1 reduces mutant AR accumulation and ameliorates disease manifestations in SBMA mice. We also tested a novel curcumin analog in cell and animal models of SBMA and investigated its mechanism of action. We show in Study IV that this compound enhances the clearance of mutant AR and mitigates the SBMA phenotype in Drosophila melanogaster and mice. The protective effect of the compound on mutant ARinduced degeneration in Drosophila is mediated through the Nrf1/Nrf2-dependent antioxidant response. Our results establish IGF-1 and curcumin analogs as candidates for therapeutic intervention in SBMA. In summary, our findings extend the known phenotype of SBMA. We also provide evidence that the mutant AR alters ubiquitin-dependent degradation pathways that are necessary for neuronal differentiation and function. Lastly, our results demonstrate the preclinical efficacy of IGF-1 and curcumin analogs in SBMA and warrant further investigation of these compounds in clinical studies. LIST OF PUBLICATIONS This thesis is based on the following articles and manuscripts: I Grunseich C, Kats IR, Bott LC, Rinaldi C, Kokkinis A, Fox D, Chen KL, Schindler AB, Mankodi AK, Shrader JA, Schwartz DP, Lehky TJ, Liu CY, and Fischbeck KH (2014). Early onset and novel features in a spinal and bulbar muscular atrophy patient with a 68 CAG repeat. Neuromuscular Disorders, 24, 978-981 II Bott LC, Salomons FA, Maric D, Fischbeck KH, and Dantuma NP. APC/C-Cdh1 dysregulation by the polyglutamine-expanded androgen receptor causes cell cycle reentry in spinal and bulbar muscular atrophy. Manuscript in preparation III Rinaldi C*, Bott LC*, Chen KL, Harmison GG, Katsuno M, Sobue G, Pennuto M, and Fischbeck KH (2012). Insulinlike growth factor (IGF)-1 administration ameliorates disease manifestations in a mouse model of spinal and bulbar muscular atrophy. Molecular Medicine, 18, 1261-1268 IV Bott LC, Badders N, Chen KL, Harmison GG, Bautista E, Shih CCY, Taylor JP, Dantuma NP, Fischbeck KH, and Rinaldi C. A small-molecule activator of Nrf1 and Nrf2 mitigates polyglutamine toxicity in spinal and bulbar muscular atrophy models. Manuscript in preparation * These authors contributed equally to the work LIST OF PUBLICATIONS (CONTINUED) Related publications not included in the thesis: Beskow A, Grimberg KB, Bott LC, Salomons FA, Dantuma NP, and Young P (2009). A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation. Journal of Molecular Biology, 394, 732-746 Tresse E, Salomons FA, Vesa J, Bott LC, Kimonis V, Yao TP, Dantuma NP, and Taylor JP (2010). VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD. Autophagy, 6, 217-227 Svensson C, Ceder J, Iglesias-Gato D, Chuan YC, Pang ST, Bjartell A, Martinez RM, Bott L, Helczynski L, Ulmert D, Wang Y, Niu Y, Collins C, and Flores-Morales A (2014). REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer. Nucleic Acids Research, 42, 999-1015 Grunseich C, Zukosky K, Kats IR, Ghosh L, Harmison GG, Bott LC, Rinaldi C, Chen KL, Chen G, Boehm M, and Fischbeck KH (2014). Stem cell-derived motor neurons from spinal and bulbar muscular atrophy patients. Neurobiology of Disease, 70, 12-20 Dantuma NP, and Bott LC (2014). The ubiquitin-proteasome system in neurodegenerative diseases: Precipitating factor, yet part of the solution. Frontiers in Molecular Neuroscience, 7:70 Rinaldi C*, Bott LC*, and Fischbeck KH (2014). Muscle matters in Kennedy’s disease. Neuron, 82, 251-253 * These authors contributed equally to the work