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- Professor
- Vice Chair, Faculty Development & Engagement
1020 Locust Street
416 Jefferson Alumni Hall
Philadelphia, PA 19107
How does expansion of the polyglutamine tract in the androgen receptor cause neuromuscular dysfunction, motor neuron death and muscle atrophy?
The research in my lab centers on areas of investigation to understand the molecular pathogenesis of the neurodegenerative disease spinal and bulbar muscular atrophy, which is caused by polyglutamine expansion within the androgen receptor (AR). 1) To understand the molecular basis for DHT-dependent AR misfolding, aggregation and toxicity; and 2) To develop an understanding of the molecular pathways by which the neuromuscular system becomes dysfunctional in response to expression of the mutant protein. These studies are generally designed to understand how spinal motor neurons and muscle fibers respond to the accumulation of misfolded proteins.
Research Projects
The overarching research goals of the Merry Laboratory are to understand the molecular mechanisms of spinal and bulbar muscular atrophy (SBMA), an X-linked neurodegenerative disease caused by an expansion of a polyglutamine (polyQ) tract within the androgen receptor (AR), and to identify potential therapeutic targets. Our laboratory uses a combination of cell and mouse models, and a variety of experimental approaches including biochemistry, cell biology, and molecular biology approaches. The pathology of SBMA is characterized by muscle atrophy and is associated with loss of motor neurons from the spinal cord and brainstem. The disease is a proteinopathy, whereby misfolding and aggregation of the mutant AR protein lead to disease symptoms. Although the molecular mechanisms underlying mutant AR-induced disease pathology remain poorly understood, we and others have begun to develop an understanding of the molecular and biochemical features of the mutant AR that are required for its misfolding, aggregation, and toxicity. These features include a requirement for hormone binding, the translocation of the mutant AR to the nucleus, and several post-translational modifications, such as acetylation of lysines 630, 632 and 633, methylation of arginines at the consensus site RXRXXS, and phosphorylation of serine 94. Moreover, a conformational change that involves physical interaction between the N- and C-termini of the AR protein (N/C interaction) is also required for disease (see Project 2). Finally, polyQ length-dependent, toxicity occurs prior to the formation of frank inclusion bodies, suggesting that toxicity results from the presence of soluble, full-length, mutant AR species.
The Role of the AR Interactome in SBMA
Spinal and bulbar muscular atrophy (SBMA) is an X-linked neurodegenerative disease caused by an expansion of a polyglutamine (polyQ) tract within the androgen receptor (AR). The pathology of the disease is characterized by muscle atrophy and is associated with loss of motor neurons from the spinal cord and brainstem. Surviving motor neurons and myofiber nuclei display intranuclear inclusions made up of insoluble, aggregated, and predominantly truncated AR protein. Although the molecular mechanisms underlying mutant AR-induced disease pathology remain poorly understood, we and others have begun to develop an understanding of the molecular and biochemical features of the mutant AR that are required for its misfolding, aggregation, and toxicity. These features include a requirement for hormone binding, the translocation of the mutant AR to the nucleus, and several post-translational modifications, such as acetylation of lysines 630, 632 and 633, methylation of arginines at the consensus site RXRXXS, and phosphorylation of serine 94. Moreover, a conformational change that involves physical interaction between the N- and C-termini of the AR protein (N/C interaction) is also required for disease (see Project 2). Finally, polyQ length-dependent, toxicity occurs prior to the formation of frank inclusion bodies, suggesting that toxicity results from the presence of soluble, full-length, mutant AR species.
Substantial evidence supports a mechanism involving protein toxicity, rather than loss of AR function. We hypothesize that, since expanded polyQ segments modify the overall conformation of the protein, such structural changes likely result in a variety of aberrant (gained and lost) protein interactions. Identification of this mutant interactome will provide insights into pathogenic mechanisms that link polyQ-expanded AR to neuromuscular dysfunction and degeneration and will offer new targets for therapeutic development. In order to identify the mutant AR interactome, we utilized a quantitative proteomics approach involving stable isotopic labeling with amino acids in cell culture (SILAC) in a robust cell model that reproduces the metabolism of mutant AR, including its nuclear aggregation and proteolysis, features that are observed in diseased SBMA patient tissue. This strategy has identified approximately 30 novel protein candidates whose interactions with AR are significantly altered by the polyQ expansion. We began with the analysis of one of these interactors, USP7, and not only validated its differential interaction with mutant AR but, importantly, identified a role for USP7 in SBMA. We are continuing our mechanistic studies of the role of USP7 in SBMA, 2) carrying out additional interactome screens using biotin ligase-based proximity labeling, and 3) investigating the roles of the other differentially interacting proteins identified in our screens.
The AR N/C Interaction in SBMA – Mechanistic Role & Therapeutic Potential
My group discovered that a normal structural rearrangement of the AR, the “N/C interaction,” which involves the interaction between the amino-terminal FxxLF motif and the hormone-bound, carboxyl-terminal AF-2 domain, is required for disease features in SBMA, both in vitro and in vivo. While it is clear that preventing the AR N/C interaction is protective, how this manipulation reduces toxicity is unknown. It may be that it reduces dimerization of the AR, thereby preventing the formation of higher order oligomers that can seed aggregation and drive toxicity. Such a mechanism would predict that other mutations that prevent AR dimerization would have similar effects on AR aggregation and toxicity. Answering this question is one of the major goals of this project. In addition, we will take advantage of our expertise in quantitative proteomics to identify proteins that differentially interact with AR N/C-intact and N/C-inhibited states. In our studies of the AR N/C interaction, we observed, both in vitro and in vivo, that its inhibition leads to Serine16 (S16) hyperphosphorylation; moreover, mutation of Ser-16 to prevent its phosphorylation eliminated the neuroprotection afforded by N/C inhibition. Understanding the mechanistic basis for the role of S16 phosphorylation, including identification of the responsible kinase, in the neuroprotection afforded by N/C inhibition is a second major focus of this project.
Determining the role of AR transcriptional function in SBMA
Studies in recent years, from our group and others, have revealed that disease symptoms and pathology in SBMA require the presence of both androgens and the nuclear localization of polyglutamine (Q)-expanded AR (polyQ-AR). Moreover, our studies revealed that an inter-domain (N/C) interaction of the AR, which occurs upon hormone binding, described above, is required for polyQ-AR aggregation and toxicity in vitro and in vivo. These results predict that a structural or functional state of polyQ-AR that occurs within the nucleus and is regulated by the AR N/C interaction mediates its toxicity. One such state, a functional AR state, is represented by the transcriptional activity of polyQ-AR. The AR is a hormone-activated transcriptional regulator that binds to DNA at canonical androgen response elements (AREs), thereby regulating the transcription of its target genes. The focus of our studies in project 3 is the investigation of the role of this specific feature of normal AR function in the aggregation and toxicity of the mutant AR. Understanding the role of AR transcriptional activity in the molecular pathogenesis of SBMA is a critical requirement for the development of ideal therapeutic strategies, given the important functional role played by the AR in both motor neuron and muscle physiology; removal of the positive, trophic effects of AR upon androgen depletion or AR knockout/knockdown may counter the protection afforded by functional AR reduction against the neurotoxic effects of the mutant AR. Understanding whether therapeutic strategies designed to prevent mutant AR misfolding and aggregation should prevent or promote AR transcriptional activity is a central question in SBMA research.
A primary focus of this project is to utilize a novel mouse model and genetically modified cell lines (both iPSC lines from SBMA patients and controls and other cell models), modified to prevent AR DNA-binding, to identify pathways that mediate disease using transcriptomic and proteomic studies. These combined studies represent a robust approach to elucidate the role of AR transcriptional activity in SBMA.