Investigation of the pathogenic mechanisms of neurodegenerative diseases

Recent breakthrough in genetic studies of Parkinson's disease has provided the first clue that mutations in α-synuclein (a synaptic vesicle-associated protein), parkin (an ubiquitin-protein ligase), UCH-L1 (a deubiquitinating enzyme), and DJ-1 (an ubiquitously expressed protein of unknown function) may be involved in the pathogenesis of Parkinson's disease. We are using an interdisciplinary approach to delineate the molecular pathways by which the mutations in these proteins lead to neurodegeneration and to identify other molecular players in the pathogenic pathways. For example, our recent work provides first evidence that DJ-1 may function as a cysteine protease and this function is completely abolished by familial Parkinson's disease-associated L166P mutation. Our proteomic analyses reveal that UCH-L1 is a major target of oxidative damage in idiopathic Parkinson's and Alzheimer's disease brains, raising the possibility that pathogenic effects similar to those caused by UCH-L1 genetic mutations might be achieved by the identified oxidative modifications of UCH-L1 in sporadic forms of the diseases. In addition, we are very interested in understanding the role of abnormal vesicular trafficking in neurodegenerative diseases. We recently found that HAP1, a novel protein that is associated with the Huntington's disease protein huntingtin, functions in endosomal trafficking, suggesting that abnormal vesicular trafficking may contribute to the pathogenic mechanism of Huntington's disease. Through our studies, we hope to define new protein targets for understanding and treating neurodegenerative disorders, such as Parkinson's, Alzheimer's, and Huntington's diseases.

2. Elucidation of the molecular basis of neurotransmitter release and its regulation

Another major goal of our research is to elucidate the molecular mechanisms underlying neurotransmitter release in both normal and pathological states. As a first step towards this goal, we have identified two novel neuronal proteins, called SNIP and Spring, that interact with SNAP-25, an essential component of the neurotransmitter release machinery. Our preliminary studies suggest that these novel proteins may function in the spatial organization and/or temporal coordination of the exocytotic and endocytotic components. We are characterizing these new proteins complexes at the nerve terminals, and determining the functional importance of these novel proteins and their protein-protein interactions using a combination of biochemical, cell biological, and molecular genetic approaches, including targeted gene disruption.

3. To study protein ubiquitination and degradation at synaptic terminals

Protein ubiquitination and degradation have emerged as a crucial mechanism in regulating synaptic development and function, and disturbances in the ubiquitin-proteasome pathway have been implicated in the pathogenesis of a variety of neurodegenerative diseases. Surprisingly, very little is presently known about the ubiquitination machinery and its substrate proteins in the nervous system. Our recent work reveals that synaptophysin (a synaptic vesicle-associated protein) is targeted for degradation via the ubiquitin-proteasome pathway by Siah E3 ubiquitin-protein ligases. Furthermore, we have discovered a novel E3 ubiquitin-protein ligase called Staring that regulates the ubiquitination and proteasome-mediated degradation of syntaxin 1, an essential component of neurotransmitter release machinery. We are doing experiments to further identify and characterize E3 ubiquitin-protein ligases and study their role in regulation of protein ubiquitination and degradation at synaptic terminals.

4. To understand the coordinated regulation of vesicular trafficking and signal transduction

It is becoming increasingly clear that neurotransmitter release shares similar mechanisms and many of the same or closely related proteins with other vesicular trafficking pathways. In our effort to search for proteins that regulate neurotransmitter release, we isolated hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) as a ubiquitously expressed regulator of vesicular trafficking. We have shown that Hrs regulates the lysosomal trafficking of epidermal growth factor (EGF) receptors via its interaction with sorting nexin 1 (SNX1), a mammalian homologue of yeast Vps5p that recognizes the lysosomal targeting signal of the EGF receptor. Since trafficking of EGF receptors controls the diversity, intensity and duration of EGF signaling, it is possible that Hrs and its associated proteins may be involved in the coordinated regulation of receptor trafficking and signaling. Currently, we are testing this hypothesis by studying the dual functions of these proteins in vesicular trafficking and signal transduction. The long-term goal of this research is to understand, at the molecular level, how endocytosed proteins are sorted and transported to lysosomes for degradation, and how this process becomes dysregulated in neurological diseases