Scott Wilson, PhD - US grants

DESCRIPTION (provided by applicant): The ubiquitin-proteasome system (UPS) is a central pathway common to all eukaryotic cells for regulating protein turnover. There are numerous regulatory pathways that rely on the timely removal of critical proteins. These pathways include the cell cycle, DNA repair, receptor-mediated endocytosis and the induction of long-term memory. The inability to remove unwanted proteins from cells has been linked to several chronic neurological diseases including Parkinson's disease, Alzheimer's disease, and the Spinocerebellar ataxias. While it is clear that these diseases are associated with polyubiquitinated protein aggregates, it is not clear how these aggregates contribute to neuronal dysfunction. In contrast to the polyubiquitination signal that targets proteins for proteasomal degradation, a monoubiquintin tag can signal receptor internalization and sorting of intracellular vesicles. This modification by monoubiquitin is reversible and, akin to phosphorylation, can regulate protein localization and activity. We have recently demonstrated that Uspl4, a deubiquitinating enzyme (DUB) that specifically removes ubiquitin from proteins, is mutated in the neurological mouse mutant ataxia (ax/j). The axJ mice do not show protein aggregation defects or neuronal loss. Instead, these mice exhibit defects in synaptic transmission, indicating that neurological disease may be rooted in synaptic dysfunction. Our working hypothesis is that loss of Uspl4 disrupts the ubiquitinated state of specific components of the neurotransmitter release machinery, thereby resulting in synaptic defects. This proposal is therefore directed at addressing the role of Uspl4 in regulating synaptic function. The first Aim will determine if Usp 14 associates with the 26S proteasome in neurons and if it has a role in ubiquitin-dependent proteolysis. In the second Aim, we will identify components and pathways that are regulated by Usp14 in order to better understand the regulation of ubiquitin modification in normal physiology and disease. The third Specific Aim will determine which neuronal circuits are disrupted by the loss of Uspl4 and examine how these circuits contribute to the tremor, ataxia and muscle wasting phenotypes of the ax J mice. Completion of these Specific Aims will enable us to uncover new processes that rely on ubiquitin-signaling and to determine how alterations in these pathways can lead to neurological disease.

DESCRIPTION (provided by applicant): Neuroscience is one of the most important areas of modern biomedical research. Despite the declaration of the 1990's as the "Decade of the Brain" and significant scientific advances over the last 15 years, effective treatments for neurological and psychiatric diseases remains the largest and fastest growing unmet medical need in the United States. To meet this need, a multi-disciplinary and integrative approach to neuroscience research is essential. Faithful small animal models of human neurological and psychiatric function and dysfunction must be developed and capitalized upon to enhance our understanding of the nervous system and to aid the development of new disease prevention and treatment strategies. The University of Alabama at Birmingham (UAB) has a long history of establishing and supporting thematic cores and centers and as part of a recently completed UAB School of Medicine Strategic Planning process, the development of a truly innovative and interdisciplinary Comprehensive Neuroscience Center has been identified as a top institutional priority. UAB has experienced a dramatic influx of accomplished neuroscience investigators in numerous departments over the last several years and has committed substantial resources to the recruitment of both junior and senior level neuroscience investigators over the next five years. In this application we propose to establish a series of inter-related core facilities that will facilitate ongoing studies of genetically modified rodents and other small animals and enhance future generation, characterization, and mechanistic analyses of small animal models of neurological and psychiatric function and dysfunction. In addition to an Administrative Core (Core A), five new research cores will be established: Core B, Molecular Engineering; Core C, Cellular and Molecular Neuropathology; Core D, Neuroimaging; Core E, In Vivo Physiology &Phenotyping; and Core F, Cellular and Synaptic Physiology. These cores are specifically designed to provide new research capabilities and will complement existing facilities to provide neuroscience investigators unparalleled ability to develop and study unique animal models of neurobiology and neuropathology. In addition to the large neuroscience community at UAB, the Alabama Neuroscience Blueprint Core Center will support the research activities of Neuroscience Blueprint funded investigators throughout the State of Alabama and at institutions in neighboring states. Combined with ongoing efforts to establish a Comprehensive Neuroscience Center at UAB, this application will dramatically enhance neuroscience research capabilities in the Deep South. Finally, a major goal of this application is to provide junior investigators and previously funded Blueprint Institute and Center neuroscientists a mechanism by which they can be scientifically productive during an exceedingly tight NIH budget period.

DESCRIPTION (provided by applicant): Alterations in the neuromuscular junction (NMJ) have recently been reported in motor neuron diseases such as Spinal muscular atrophy (SMA);however, little is known about the pathways that regulate synaptic activity and development in motor neurons. Although transcriptional mechanisms have been shown to regulate critical steps in the development of the nervous system, recent studies have highlighted the importance of the ubiquitin proteasome system (UPS) in the development and maintenance of synaptic connections. By regulating ubiquitin signaling pathways, such as kinase activation and the trafficking and abundance of cellular proteins, the UPS can control developmental transition points during the maturation of the nervous system. However, it is not known how the cell regulates available ubiquitin pools required for these processes. Given the distance that separates the motor neuron cell body and endplate, specialized mechanisms must ensure the stable expression of ubiquitin necessary for axon path finding, synaptic targeting and motor endplate maturation. Our studies now demonstrate that the proteasomal deubiquitinating enzyme Usp14 is required for the postnatal development of the motor neuron endplate. Homozygous axJ mice, which are deficient for Usp14, display a resting tremor, hind limb rigidity, reduced muscle mass and die by 8 weeks of age. These mice do not have ubiquitinated protein aggregates or accelerated neuronal cell death, but instead show ubiquitin loss that correlates with impaired motor endplate maturation during the first two weeks of postnatal development. Restoration of ubiquitin levels in the axJ mice increases body mass and motor function and prevents postnatal lethality, indicating that ubiquitin loss can be a major contributor to neuromuscular disease. Our recent studies also demonstrate ubiquitin loss in a mouse model of SMA, which displays impaired NMJ maturation and function similar to the axJ mice, validating the importance of identifying the developmental pathways regulated by ubiquitin. Our working hypothesis is that Usp14 functions to maintain ubiquitin levels required for the development and activity of mammalian synapses. The first aim of this proposal will determine the contribution of ubiquitin loss in the axJ mice to the development and activity of the NMJ. In the second aim, we will investigate a newly proposed catalytic-independent function of Usp14 on the proteasome and determine if it is required for development and synaptic transmission at the NMJ. The third aim is designed to determine the role of motor neurons and motor endplates in the disease process in the axJ mice. The final aim will examine the ubiquitin-dependent pathways that control synaptic maturation and function of the NMJ. This proposal will use a combination of genetics and biochemistry to investigate the essential enzymatic functions of Usp14 on the proteasome and determine how changes in the activity of Usp14 alter signaling pathways required for synaptic development and function. PUBLIC HEALTH RELEVANCE: The ubiquitin proteasome system functions to control cellular pathways by regulating protein levels within cells. Since alterations in protein turnover are believed to be central to several chronic neurological diseases, the identification and analysis of components of the ubiquitin proteasome system will provide new insights into the mechanisms of neurological disease and identify potential targets for therapeutic intervention.

DESCRIPTION (provided by applicant): While proteasome inhibitors are an effective cancer treatment option, the idea of accelerating the activity of the proteasome for medical purposes represents a novel treatment option for neurological disorders where harmful proteins accumulate and cause disease. Chronic neurological diseases such as Parkinson's, Alzheimer's, Huntington's, Frontotemporal dementia and Spinocerebellar ataxias are characterized by the presence of ubiquitinated protein aggregates and reduced proteasome function. Therefore, while these mutant proteins can be targeted for proteasomal degradation, they are not effectively eliminated by the ubiquitin proteasome system. We recently determined that either genetic or pharmacological inhibition of the ubiquitin hydrolase activity of Usp14 is sufficient to accelerate protein degradation by the proteasome. The levels of several proteins, including tau, TDP-43 and ataxin-3, were significantly decreased following the inhibition of Usp14's ubiquitin- hydrolase activity, indicating that Usp14 can function as an inhibitor of the proteasome. Our working hypothesis is that Usp14 functions to edit the ubiquitin side chains of proteins prior to their commitment to proteasomal degradation, resulting in release of the substrate from the proteasome. The focus of this proposal is to determine if loss of Usp14's ubiquitin hydrolase-activity can reduce the levels of aggregate- prone proteins produced in animal models of Huntington's disease, Frontotemporal dementia and Parkinson's disease. This novel approach to enhancing proteasome function for the clearance of aggregate- prone proteins may lead to a powerful new treatment option for patients suffering from chronic neurological diseases.

Peripheral neuropathies affect more than 20 million people in the United States. Patients with peripheral neuropathies suffer from debilitating motor and sensory deficits that can cause severe pain and paralysis. Many forms of inherited peripheral neuropathies impair Schwann cell function and result in abnormal myelin production or demyelination, which is thought to be the underlying cause of the motor and sensory deficits. Schwann cells intimately associate with axons to organize peripheral nerves during development and to insulate axons with myelin. The NRG1/ERBB signaling pathway allows for Schwann cells and axons to communicate with each other and provides essential instructions that regulate Schwann cell proliferation, migration and myelination. Modulation of the NRG1/ERBB signaling pathway can restore function to several rodent models of Charcot-Marie-Tooth disease (CMT). Therefore, identifying the mechanisms that control NRG1/ERBB signaling has important implications for the treatment of peripheral neuropathies. Our data from mice and cell culture experiments indicate that the endosome is a critical regulator of ERBB2/3 function during myelination. To investigate the endosomal pathways controlling ERBB2/3 signaling, we have developed mouse models that impair endosomal sorting of internalized cell surface receptors. We have focused on hepatocyte growth factor regulated tyrosine kinase substrate (HGS), which directs the sorting of internalized receptors through the endosome. HGS expression is diminished in two different mouse models of CMT, implicating defective endosomal sorting as a cause for demyelinating neuropathies. Our data now indicate that loss of HGS in Schwann cells replicates many features of inherited peripheral nerve disorders, including motor and sensory deficits and dysmyelination of sciatic nerves. Impairing endosomal function by deleting the Hgs gene specifically in Schwann cells also showed that ERBB2/3 receptor signaling is dependent upon endosomal sorting to activate its downstream signaling pathways. In addition, we have identified a novel endosomal protein complex in Schwann cells that occurs during myelination. To test the hypothesis that endosomal sorting regulates ERBB2/3 function during myelination, Aim 1 will determine the role of endosomal sorting in Schwann cells for the development and function of peripheral nerves, and Aim 2 will determine how endocytic trafficking controls the sorting and signaling of the ERBB2/3 receptors in Schwann cells. To investigate the mechanism regulating ERBB2/3 function in Schwann cells, Aim 3 will determine which HGS interacting protein complexes are required for ERBB2/3 sorting and signaling in Schwann cells. The completion of this proposal is expected to provide novel insights on the endosomal biology of Schwann cells and further our understanding of how endosomal sorting controls ERBB receptor signaling during myelination. Our long-range goals are to determine the regulatory pathways that control endosomal function in Schwann cells in order to identify targets for the treatment of demyelinating diseases such as CMT.