Marie W. Wooten - US grants

The proposed study involves synthesis of antisense S-oligonucleotides specific to alpha, beta and gamma protein kinase C mRNA's. These oligonucleotides will be used as a tool for selective inhibition of gene expression by inhibiting PKC translation. Experiments will be conducted to establish that the synthetic oligonucleotides inhibit translation of a specific PKC-mRNA without affecting translation of the other PKC-mRNA's or overall translation. Detailed experiments will be conducted in HL60 cells to determine the turnover of alpha/ beta PKC-mRNA's during differentiation in response to PMA, DMSO, Vitamin D3 and retinoic acid. Optimal conditions for introducing specific PKC oligonucleotides into HL60 cells in culture will be conducted and their effect on translation determined. Lastly, we will examine whether inhibiting translation of either of the three PKC- mRNA's has an effect on the ability of HL60 cells to enter a differentiation program in response to a specific inducer. This study will provide information regarding differential regulation of PKC isoform expression and a means to examine the functional heterogeneity of this gene family.

PC12 cells have been used as a model system to examine the cellular and molecular mechanisms that govern neuronal differentiation. These cells respond to nerve growth factor (NGF) and acquire the properties of sympathetic neurons. Using PC12 cells, I have observed that ethanol possesses the capability of synergizing with suboptimal concentrations of NGF to induce early neurite extension and subsequent differentiation. Preliminary studies indicate alterations in both protein kinase C and cAMP systems as targets for the effects of ethanol. The long term objective is to develop an understanding of the mechanism by which ethanol interacts with transmembrane signalling pathways to promote gene expression and subsequent differentiation. The specific aims are therefore to define conditions by which ethanol synergizes to promote differentiation by use of pharmacological agents with known specificity and correlate the presence or absence of neurites with indices for differentiation and expression of NGF-specific genes. I propose to detail the contribution of signal transducing pathways which interact to communicate the ethanol transmembrane signal by: a) examining protein kinase C and specifically explore alterations in the isoforms of this kinase brought about by both acute and chronic ethanol exposure; b) analyze activation pathways which either precede PKC activation, ie 'PI-turnover', are a result of PKC activation, i.e. coactivation of other kinase systems such as MAP2 and S6; c) correlate the activation of the adenylate cyclase system as a potential 'cross-talk' regulator between signal transducing pathways; and lastly, d) examine the phosphorylation of tyrosine hydroxylase-as a convergent substrate for multiple kinases.

Protein kinase C (PKC) has been implicated as a component of the Nerve growth factors (NGF) signalling pathway in PC12 cells; although, disagreement regarding the role that PKC plays in this process has persisted in the literature. We provide novel information demonstrating that activation of atypical zeta-PKC relates to the signalling paradox surrounding NGF and PKC isoforms. In addition, we also document that zeta- PKC is activated by NGF in a trk-dependent manner and modulated in a positive fashion by increases in cAMP. We hypothesize that zeta-PKC lies in parallel with raf kinases and is a major constituent of a newly described signalling pathway serving as a MAP kinase kinase kinase (MEK kinase). The goal of this project is to precisely define the role which zeta-PKC plays in mediating signal-coupling and neurite outgrowth. In order to fulfill this goal, two specific aims will be undertaken. We are proposing to; 10 establish the means by which activation of zeta-PKC modulates an existing or newly defined pathway by studying its interaction with proteins which comprise athe classical NGF signalling cascade involving ras / raf yieldsMAP kinase and 2) overexpress zeta-PKC to upregulate its signal and study both biochemical and morphological aspects of overexpression. The long-term goal of these studies is to establish the mechanism whereby zeta-PKC regulates NGF dependent and independent signalling involving neuronal differentiation. To accomplish these aims we will employ PC12 cells deficient in zeta-PKC or those overexpressing this isoform. The regulation exerted by zeta-PKC will be studied using a range of techniques: in situ activity assays, immunoprecipitation of individual signaling components, reconstitution, protein purification, peptide mapping and phosphoamino acid analysis. The outcome of these studies stand to change our conceptual framework of NGF-signal coupling and neurite outgrowth. Moreover, the findings generated by this study will have a direct impact upon Alzheimer's research, since PC12 cells have been used as a model system to elucidate the role of amyloid precursor protein (APP). Previous work in PC12 cells has demonstrated that APP is capable of enhancing neurite outgrowth and acts as a mediator of NGF's effects. Hence, elucidating the signalling pathway(s) leading to neurite extension will bear directly into the etiology and treatment of Alzheimer's, as well as, other human disease in which alterations in PKC and NGF are hypothesized to play a role.

DESCRIPTION (Adapted from applicant's abstract): Atypical PKC isoforms (iota/zeta) have been shown to play a critical role in regulation of various neuronal processes including, differentiation, survival and long-term potentiation. In PC-12 cells a PKCs, as well as their second messenger activator, P13 polyphosphoinositides, are required for both differentiation and survival responses. We have recently shown that aPKCs are tyrosine phosphorylated in response to nerve growth factor (NGF). ZIP/p62 and Par4 are binding proteins that regulate the localization and activation of aPKCs. Preliminary studies document an NGF receptor-regulated phosphotyrosine dependent interaction of src with sPKC. We hypothesize that tyrosine phosphorylation of atypical PKC by src moduates activity, as well as, its ability to interact with its protein regulators, Par4 and ZIP. Therefore, one specific aim is to study the mechanisms by which aPKC-iota is regulated by tyrosine phsophorylation to include regulation by NGF receptor components, p75 and TrkA, as well as, second messenger. A second aim is to unravel the mechanismwhereby aPKC is regulated by src kinase to include regulation of aPKC localization, signal-complex formation and interaction with protein regulators, as well as to examine the specific tyrosine residues that regulate NGF/aPKC signal-coupling. Various biochemical and functional assays will be used to define the function of src in regulation of aPKC signaling. Overall, a comprehensive set of experiments using enzyme activity assays, subcellular fractionation, coimmunoprecipitation and site-directed mutagenesis will be employed. The outcome of this work will continue to alter the conceptual framework of neurotrophic signal coupling and the role that aPKC plays in this process. These findings will contribute to further understanding of the mechanism whereby aPKC is regulated and may provide new therapeutic targets for neurodegenerative disorders.

DESCRIPTION (provided by applicant): The aim of this project will be to test the feasibility of developing and testing a novel peptide inhibitor that will specifically block Par4 interaction with the atypical protein kinase C isoform zeta. Par4 is a protein well established as a mediator of neuronal degeneration associated with the C pathogenesis of Alzheimer's disease. Clear evidence exists demonstrating that apoptotic stimuli cause Par4 to bind zetaPKC followed by cell death. Moreover, elevated Par4 suppresses the activity of the transcription factor nuclear factor kappa B, whose activity is required for transcription of prosurvival genes. The binding domain, which mediates the interaction between Par4 and zetaPKC has been mapped to the leucine zipper (LZ) domain (amino acids 265-332) of Par4. Moreover, deletion of Par4's LZ inhibits Par4's ability to induce apoptosis. Thus, the specific aims for this exploratory proposal will be: 1) to employ "peptide walking" to identify a specific peptide within the leucine zipper domain of Par4 that can be used to effectively disrupt interaction of zetaPKC with Par4; 2) to establish uptake of the peptide by cells in culture and delivery of the peptide to brain in vivo; 3) to establish functional inhibition of Par4 activity by the peptide inhibitor, and last; 4) to test the ability of the peptide to block neuronal cell death and restore normal function. Collectively these findings will lead to a significant breakthrough in the development of a designer therapeutic agent, which will protect neurons from the effects of Alzheimer's and other neurodegenerative diseases.

ADVANCE Auburn: SEM Transformation through "Small Wins" will develop, adapt, and implement the "small wins" approach for producing lasting change for increasing the representation, participation, advancement and success rate of women faculty in science, engineering and mathematics (SEM) disciplines. The "small wins" approach (Meyerson and Fletcher, 2000) advocates transforming a workplace through a series of small positive changes used to improve the working environment for those who are disproportionately affected by unsupportive and oftentimes inconsiderate practices in the workplace. Since the changes produced by "small wins" are incremental and locally driven, the approach is non-threatening and more easily accepted by an entrenched culture such as often exists in the SEM academic disciplines. Successive "small wins" build upon themselves such that substantive and lasting changes in the work environment and culture are achieved and assimilated over time. This award will develop and apply the "small wins" approach to transform the SEM disciplines at Auburn University and, subsequently, at other institutions.

DESCRIPTION (provided by applicant): Oxidative stress, ubiquitination defects, and mitochondrial dysfunction are well recognized markers of various neurodegenerative diseases. This proposal is designed to elucidate the mechanism whereby the signaling scaffold, p62, regulates trafficking and removal of ubiquitinated proteins and damaged mitochondria. The specific aims of this proposal are based upon the CENTRAL HYPOTHESIS that p62 is involved in the transport of proteins to the proteasome or autophagosome, employing a common tag, K63-polyubiquitin. Sequestosome 1/p62 is predominantly expressed in the hippocampus, the center of learning and memory, where it serves as a scaffold to regulate the trafficking of K63-polyubiquitinated proteins and phosphorylation of atypical protein kinase C iota/lambda substrates. Gene-targeted deletion of p62 in mice causes oxidative stress leading to Alzheimer-like characteristics such as tau-phosphorylation, loss of short term memory and synaptic plasticity, reduced serum BDNF, depression, anxiety, along with accumulation of highly insoluble polyubiquitinated proteins in the adult mice. Humans with AD likewise exhibit reduced p62 levels in hippocampus that correlate with accumulation of insoluble polyubiquitinated tau. These findings suggest that a reduced amount of p62 may act in conjunction with other environmental or genetic factors to influence susceptibility to development of AD. Our long term goal is to understand the mechanism whereby removal of p62 in the brain results in oxidative stress that contributes to development of mouse AD. Our preliminary findings reveal that p62 converges upon HDAC6 to regulate it activity and thereby influences tubulin acetylation, leading to impaired clearance of aggregated proteins. Along with p62's role in autophagy the elimination of damaged mitochondria is impaired, which together with appearance of ubiquitin aggregates results in oxidative stress. The following specific aims are proposed to further elucidate the p62 regulatory pathway. Specific Aim 1 will examine the mechanism whereby p62 cooperates with HDAC6 to regulate clearance of aggregated polyubiquitinated proteins. Specific Aim 2 will examine the mechanism whereby p62 regulates the trafficking, clearance and energetics of mitochondria. Specific Aim 3 will define p62's role in mediating oxidative stress resistance. An innovative approach employing various complementary methods will be undertaken within the scope of the project. These findings are likely to provide insight into mechanism whereby p62 function impinges upon energetics and form of mitochondria, trafficking of interacting proteins, as well as, accumulation of polyubiquitin aggregates. Altogether, further knowledge of p62 may contribute to new treatments for AD, as well as, other neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: This project will tease apart the function of the scaffolding protein p62. The innovative aspect of this project is that the role of p62 in trafficking of tau and other interacting proteins is largely undefined. P62 is a multifunctional protein involved in both of the two major protein degradation mechanisms, ubiquitin proteasome system and autophagy-lysosome pathway. The specific aims of this proposal are based upon the CENTRAL HYPOTHESIS that p62 is a novel shuttling factor that participates in the transport of proteins to the proteasome, autophagosome and removal of damaged mitochondria. Three synergistic but non-overlapping specific aims are proposed to further unravel the mechanistic details regarding the role of p62 in vivo. Aim 1 will illuminate the means by which p62 regulates clearance of aggregated proteins. Aim 2 will elucidate the mechanism whereby p62 regulates the trafficking and clearance of mitochondria. Aim 3 will define p62's role in mediating oxidative stress resistance. The results from this study will provide invaluable insights into the role of p62 in clearance of misfolded protein aggregates and neurodegeneration. These findings may provide an avenue to develop new therapeutics for the treatment of Alzheimer's disease as well as other neurodegenerative diseases, which possess disturbances in p62 expression as a component of their underlying pathophysiology.