David J. Smith - US grants

The research will describe the neuroanatomical and neurochemical bases of ketamine's (Ketalar; Ketaject) analgesic effect. It will be accomplished by an evaluation of the presence and function of opiate receptor sub-types in neuroanatomical loci (i.e., sites known to be loci of morphine's analgesic action); a description of opiate receptor sub-types with which ketamine interacts; and a determination of the consequences of an interaction of ketamine at these receptors. The research is divided into five scientific approaches. Each is designed to provide component information important to the project as a whole and/or to test the same proposal under different conditions. The methods are: 1) a pharmacological comparison of the analgesic action in rats of various sub-classes of opiate drugs presumed to be specific for sub-types of opiate receptors, and an evaluation of the participation of monoaminergic and opiate neuronal processes in these analgesias; 2) an in vitro comparison of the receptor binding characteristics of ketamine and other opiate drugs, specific for different receptors, using opiate binding assays; 3) an in vivo evaluation of the ability of ketamine to interact with opiate receptors that may mediate its special pharmacological actions; 4) local administration of ketamine and opiate drugs, specific for different opiate receptors, into central nervous system loci (i.e., those sites responsible for morphine analgesia) and an analysis of the neurochemical and neuroanatomical bases for the resulting analgesia and 5)*an in vitro and in vivo analysis of the participation of opiate or non-opiate mechanism(s) in the spinal analgesic action of ketamine, a site of action of established differences between ketamine and morphine. The study should define ketamine's opiate receptor preference and action (agonistic-antagonistic) on various sub-types of receptors found in areas of the central nervous system associated with morphine's analgesic action. The possibility that ketamine uses neuronal circuitry activated by morphine is being tested. Ketamine may be found to be dissimilar to morphine and share components of analgesic mechanisms associated with other classes of opiate drugs. Differences in the analgesic mechanisms associated with various types of analgesic drugs will begin to be defined which is consistent with our long term goal to determine pathways and neurotransmitters of analgesia.*

PROJECT SUMMARY/ABSTRACT The Ubiquitin Proteasome System (UPS) regulates the degradation of the majority of proteins in the cell and, as such, it is involved in essentially every cellular process. Because of its central role, misregulation within the UPS can potentiate or cause diseases, such as neurodegeneration and cancer. It is now well understood that protein misfolding and accumulation, which are intimately associated with neurodegenerative disease, can impair the UPS, exacerbating the disease. In fact, there is great interest to find ways of activating proteasome function as possible treatments for neurodegenerative disorders. To the contrary, in neoplastic disease the UPS is often exploited and even upregulated; due to this, a first line treatment for multiple myeloma is proteasome inhibition. The UPS thus sits at a shared and critical position in these two major human diseases. The proteasome?the central degradative machinery of the UPS?is regulated by very different regulatory complexes (e.g. 19S, PA28??, PA28?, PA200, and putatively P97). The job of these complexes is to regulate the function of the core particle of the proteasome, the 20S, which isolates its protein degradation chamber from the cellular milieu. A commonality shared by these regulators is that they all function to induce opening of the 20S proteasome substrate gate, which exposes substrates to the interior degradation chamber. The proteasome, and its regulators, provide a rich regulatory landscape to develop therapies that could profoundly impact these two large fields of study. This will require a deep biochemical understanding of the involved molecular mechanisms. The recent barrage of proteasomal structures facilitate this effort, but structures without an understanding of the dynamic mechanisms that underlie their functions are limited. Therefore, this proposal is primarily focused on understanding the biochemical function of three of these diverse proteasomal complexes and defining how they regulate protein degradation. We will focus on three specific questions: 1) How do the N-terminal domains of the proteasomal ATPases affect proteasome function?, 2) How does the mammalian P97 function to stimulate protein degradation by the 20S proteasome?, and 3) How does PA28? regulate 20S function to catalyze nuclear protein degradation? We have chosen to focus on these three regulators because they each play unique roles in the types of substrates that they degrade, and they each play key roles in specific human diseases. We implement a variety of approaches and systems to address these questions including studying function of proteasomal regulators from archaea, yeast, nematodes, mammals, and humans. Furthermore, we are using C. elegans as an animal model system to test our biochemically derived models and genetically test therapeutic concepts. The successful completion of this study will produce a sustained impact in the field by defining the central mechanisms of these three different cellular strategies for regulating protein degradation, each of which play different but critical roles in biology and disease.

At the cellular level Alzheimer?s disease (AD) is characterized by the accumulation of misfolded and damaged proteins. Prominent species that accumulate early and play fundamental roles in disease pathogenesis are Amyloid ? (A?), Tau, and sometimes ?-synuclein (?-syn). A vast body of literature supports the notion that the cell?s protein degradation systems do not function sufficiently enough in AD to clear these misfolded proteins. The cell?s primary system for the degradation of such misfolded or damaged proteins is the Ubiquitin Proteasome System (UPS). We have recently found that pathologically relevant oligomeric forms of A?, Tau, and ?-syn can potently and directly inhibit isolated 20S and 26S proteasomes, even inhibiting ubiquitin- dependent protein degradation in vitro. Based on our preliminary data we hypothesize that such pathological oligomers contribute to AD pathogenesis by directly inhibiting proteasome function in neurons. What we do not know is if proteasome inhibition by such oligomers can cause AD related neuronal dysfunction, nor do we know the molecular mechanisms involved. We propose to fill this gap in knowledge by 1) elucidating the precise mechanism of proteasome inhibition by these oligomers in vitro and in vivo 2) generating proteasomes that are hyper-active or resistant to inhibitory oligomers and 3) testing if hyper-active or oligomer resistant proteasomes can rescue neuronal function in cellular and animal models of AD. Our proposal is innovative because we have generated highly novel animal model and preliminary data that supports a novel mechanistic hypotheses, which addresses a fundamental component of AD. Extending these studies will allow us to generate disease resistant proteasomes allowing us to conclusively determine if direct proteasome impairment by AD related oligomers can cause neuronal dysfunction. This contribution is significant because it will fill a gap in our knowledge by demonstrating that the pathological oligomers associated with AD cause neuronal dysfunction, at least in part, by directly inhibiting the proteasome. In addition, this study will also demonstrate if proteasome activation can protect neurons from AD related proteotoxicities. These outcomes are expected to have a positive impact because they demonstrate that the proteasome is a prime therapeutic target to treat Alzheimer?s disease and provides a precise molecular mechanism that can be exploited for pharmacological development.