John J. Mieyal - US grants

The program goal is to understand and limit ischemia-reperfusion injury in the elderly heat. Aging associated effects in mitochondrial electron transport result in increased generation of free radicals that propagate molecular, cellular, and tissue injury. Oxyradicals modify glutathione (GSH) and critical sylfhydryl groups on proteins that are vital for energy production, ion transport, gene regulation, membrane integrity, etc. The net effect of ischemia-reperfusion injuries is likely contributed by decrease in antioxidant protection and repair systems. The thiol-disulfide oxidoreductase (TDOR) enzymes thioltransferase and thioredoxin are important defense systems that maintain critical sulfhydryl proteins, yet they have not been featured in studies on aging, nor well characterized in mitochondria. This project is aimed at understanding age-related changes in protection and repair capacity of the TDOR enzymes, and at testing potential therapeutic agents that counteract the age-dependent changes. It is hypothesized that advanced age is associated with decline in capacity to protect viral sulfhydryl proteins, which could be due to diminution in contents of enzymes involved in sulfhydryl homeostasis or a decrease in the inducibility of these enzymes that is a natural part of the cellular response to oxidative stress. The primary focus will be cardiac myocytes, and mitochondria in particular. The primary animal model will be Fischer 344 rats. Specifically, this project will measure total and subcellular distribution of thioltransferase and thioredoxin contents and activities in heart tissues from 6, 18, 24 and 28 m.o. rats to determine changes in sulfhydryl protein defense capacity with aging and in response to ischemia- reperfusion, in the absence and presence of cell permeable antioxidant agents. The activities, thiol status, and nature of oxidative modification of key functional mitochondrial and cytosolic enzymes with sensitive sulfhydryl moieties will be studied to assess cytotoxic relevance of age- dependent changes in response to ischemia-reperfusion and antioxidant therapy. The same proteins will be studied in vitro to characterize the mechanism(s) of their oxidative modification and the relative efficiency of the thioltransferase and thioredoxin systems to catalyze repair. This study will provide new insights into protein-SH oxidation as a contribution mechanism of ischemia-reflow injury, and it will test the utility of cell-permeable antioxidants in mitigating that injury, especially in elderly animals that are more susceptible.

DESCRIPTION (provided by applicant): The long-term goal is to understand why hearts of elderly humans and animal models are more prone to irreversible damage from ischemia-reperfusion (I/R). I/R generates reactive oxygen species (ROS) that modify thiol groups on proteins, leading to mixed disulfides between glutathione (GSH) and protein-SH (protein-SSG). Our previous studies characterized glutaredoxin (GRx) as the enzyme that specifically catalyzes protein-SH/-SSG exchange in cells. Using an established animal model of aging, Fischer 344 rats, our preliminary studies revealed that GRx activity in cytosol and mitochondria was decreased in heart cells from elderly rats compared to young adults. The operating hypothesis is that such age-dependent changes in glutaredoxin activity perturb the thiol-disulfide steady-state and corresponding activity of specific proteins that are intermediates in redox signaling pathways that determine whether a cell survives an oxidative insult or commits to apoptosis. Thus, cardiomyocytes of the elderly are more susceptible to oxidant-induced apoptosis. Accordingly, this project focuses on GRx and its regulation of the S-glutathionylation status of apoptotic signaling proteins. Mechanisms of change in cytosolic and mitochondrial GRx activities will be studied as a function of age and ischemia-reperfusion. The protein-SH/-SSG status and activities of apoptotic signaling intermediates in cardiomyocytes of young adult and elderly rat hearts will be studied in relationship to GRx activity. Results will be correlated with the content and distribution of GRx in natural cardiomyocytes and in cardiomyocytes in which the content and/or form of GRx have been manipulated by adenoviral gene transfer in vitro and in vivo. These studies will enhance our general understanding of the role of glutaredoxin in apoptotic regulation. Moreover they will address potential mechanisms for age-dependent predisposition of hearts to irreversible injury and provide insights for novel therapeutic interventions.

The long-term program goal is to understand why elderly hearts of humans and animal models are more prone to irreversible ischemic injury. In conjunction with other projects of this PPG that are aimed at characterizing specific age-and ischemia-dependent changes in cardiomyocytes due to increases in mitochondrial-generated radical species, this project focuses on increased susceptibility of the cardiomyocytes to apoptosis as a contributing mechanism. Oxyradicals modify glutathione (GSH) and critical sulfhydryl groups on proteins that are vital for many cell functions including energy production, ion transport, gene regulation, membrane integrity, and regulation of the balance between cell survival and controlled cell death (apoptosis). The operating hypothesis is that the net effect of cardiovascular damage associated with ischemia-reperfusion injuries is likely exacerbated by age-related changes in homeostatic regulation of protein thiol-disulfide status. Therefore this project focuses on the relationship between oxidative-stress-induced changes in glutaredoxin-mediated redox regulation of reversible S-glutathionylation of critical proteins and the susceptibility of the cells to undergo apoptosis. Using the primary animal model Fischer 344 rats during the current funding period, glutaredoxin (GRxl) content and activity were found to be decreased in cytosol, and the net glutaredoxin activity (GRX1 + GRx2) appeared to be decreased also in mitochondria of heart cells from elderly rats compared to young adult rats. In contrast thioredoxin and thioredoxin reductase were unchanged in cytosol and mitochondria with age. Accordingly the focus for continuation of this project is on the glutaredoxin system and its role in regulation of S-glutathionylation of cellular proteins (protein-SSG). The mechanisms of changes in cytosolic and mitochondrial glutaredoxin activities will be investigated, and the role of glutaredoxin in regulating the activities of key mediators of apoptotic signaling in cardiomyocytes of young adult and elderly rat hearts will be studied to ascertain how this control network contributes to increased myocardial damage after ischemicreperfusion injury. In particular, changes in the protein-SSG status of key mediators of apoptotic regulation in the mitochondria and the cytosol will be studied as a function of age and ischemia / reperfusion, and correlated with the content and distribution of glutaredoxin in natural hearts and in hearts in which the content and/or form of glutaredoxin have been manipulated by in vivo gene transfer. The animals with genetically altered contents of heart glutaredoxin will be studied collaboratively by all of the projects. These studies will enhance our general understanding of the role of glutaredoxin in apoptotic regulation while addressing potential mechanisms for age-dependent predisposition of hearts to irreversible injury.

[unreadable] DESCRIPTION (provided by applicant): This is a revised application for competitive renewal of the Molecular Therapeutics Training Program (MTTP) at Case Western Reserve University (CWRU). The application requests support for three predoctoral students in the first year of the grant, and six students in subsequent years, with a total funding period of five years. The global objective of the MTTP is to provide predoctoral students with the necessary knowledge base and research skills to begin independent investigative and teaching careers, thus increasing the supply of pharmacology-based skilled scientists and educators who will pursue independent careers in academia as well as research-based industry. [unreadable] [unreadable] The program itself is designed with a three-tiered progression. First, a didactic foundation in cell and molecular biology is established along with three meaningful research rotations to facilitate mentor selection. Secondly a foundation in physiology and pharmacology is achieved via an intensive two-part core course. Thirdly, students acquire advanced understanding in their area of specialization via advanced courses and thesis research. To facilitate this advanced stage, the training faculty and advanced courses are organized according to four tracks, namely Molecular Pharmacology & Cell Regulation, Membrane Biology & Pharmacology, Cancer Therapeutics, and Translational Therapeutics. This multifaceted, approach provides students with a strong foundation in fundamental pharmacology and the associated sciences, coupled with individualized advanced training in modern pharmacology. The interdisciplinary design of the program fosters productive interactions among students and faculty in basic and clinical departments throughout the School of Medicine, around the common theme of therapeutics. The priority outcome of the program is to develop students with the scientific maturity to address new research questions through hypothesis-driven experimental designs. [unreadable] [unreadable] The Program is focused and administered in the Department of Pharmacology, and it benefits from strong interdisciplinary interactions among basic science and clinical faculty as trainers in the advanced tracks. Eleven primary faculty of Pharmacology collaborate with 27 faculty members from other primary disciplines to provide a rich diversity of research expertise concentrated on a common theme of innovative training in therapeutics. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The overall objective of our research is to identify and validate novel targets for the treatment of Parkinson's disease (PD). PD is characterized by loss of dopaminergic neurons, which are especially vulnerable to oxidative stress. Glutaredoxin (Grx) is an antioxidant defense enzyme that counteracts oxidative stress and maintains thiol homeostasis by catalyzing the reversible formation of protein-glutathione mixed disulfides on cysteine residues (protein-SSG). This oxidative modification is implicated in change of function for many proteins involved in cell survival, including proteins that have been previously identifie with familial forms of PD. Using C. elegans as a model organism, we have found that deficiency in the nematode homolog of Grx1, the major isoform of Grx in mammals, leads to exacerbation of dopaminergic degeneration elicited by overexpression of mutant LRRK2 (G2019S or R1441C), ¿-synuclein, or tyrosine hydroxylase. These findings indicate that Grx1 deficiency can predispose to PD-relevant phenotype in an animal model, suggesting that Grx1 deficiency contributes to PD pathogenesis. Supporting the relevance of these genetic models, we have obtained preliminary evidence that Grx1 content is decreased in postmortem brains of PD patients. Accordingly, we propose to characterize and validate the neuroprotective role of Grx1 in mammalian models of PD, and to investigate whether changes in glutathionylation status of key proteins implicated in familial PD also contribute importantly to the disease pathogenesis. To examine if Grx1 deficiency confers vulnerability to dopaminergic degeneration in mammals, we propose in Aim 1 to characterize a novel mouse model generated from crossing existing transgenic human LRRK2 mutant animals with Grx1-knockout animals. The transgenic LRRK2 mutant mice with Grx1-knockout represent a genetically engineered mammalian animal model of elevated oxidative stress and specific deficiency in thiol homeostasis. We will determine whether this novel mouse model manifests loss of dopaminergic neurons characteristic of PD. The outcome of this study would validate if Grx1 serves as a critical regulator of PD pathogenesis. In Aim 2, we will identify oxidative cysteine modifications and changes in function of the proteins implicated in PD pathogenesis including ¿-synuclein, parkin, UCH-L1, DJ-1, PINK1, and LRRK2. The contribution of cysteine modifications to PD-like phenotype will be examined by the use of non-oxidizable mutant forms of the proteins in mammalian cell and C. elegans models of PD. Correspondence between susceptibility to neurodegeneration and oxidative modification of proteins implicated in familial PD would provide an important advance in understanding pathogenic mechanisms underlying sporadic PD.

DESCRIPTION (provided by applicant): The global objective of the NIH-funded Molecular Therapeutics Training Program (MTTP) is to provide highly qualified predoctoral students with the knowledge base and research skills to begin independent investigative and teaching careers in the pharmacological sciences. The MTTP provides a uniform conceptual framework and research environment through which students obtain the Ph.D. degree in Pharmacology at Case Western Reserve University (CWRU). Designed with a three-tiered progression, the MTTP first establishes a didac- tic foundation in cell and molecular biology coupled with research rotations to facilitate mentor selection. Secondly, a foundation in molecular and physiological pharmacology is achieved via an intensive two-part core course which emphasizes both quantitative analysis and disease-focused study of drugs. Thirdly, students specialize via advanced courses and research focus, organized according to four tracks: Molecular Pharmacology & Cell Regulation, Membrane & Structural Biology & Pharmacology, Cancer Therapeutics, and Translational Therapeutics. The interdisciplinary design fosters productive interactions among students and faculty in basic and clinical departments around the common theme of thera- peutics. Our priority outcome is to develop students with the scientific maturity to address new research questions through hypothesis-driven experimental designs. There is a need in the academic and private sectors for well-trained, highly qualified scientists with core training in the principles of pharmacology. This demand is widespread, including educators, researchers, and industry leaders. The long-term goal of this program is to increase the supply of pharmacology-based skilled scientists and educators by providing a rigorous training program that yields Ph.D. graduates who will pursue more advanced postdoctoral training on their way to independent careers in academia and research-based industry. Special features/activities of the MTTP for trainees: By establishing a common foundation in both cell and mo- lecular biology and the molecular and physiological bases of pharmacology all students share a common scientific lan- guage. As students diversify into the four Advanced Training Tracks (emphasizing cell regulation, structural biology, cancer biology, or translational studies) they remain united b many functions. The Graduate Student Organization (GSO) fosters educational as well as social interactions on a monthly basis, and the Departmental Journal Club provides oppor- tunities for the students to present current research findings to the entire department. All students fulfill th same re- quirements for advancement to candidacy, Ph.D. dissertation, and publications regardless of the area of specialization of their mentor. The GSO represents a partnership in education whereby students participate on the MTTP Steering Com- mittee, Curriculum Committee, and Prelim I Committee, providing student perspective on functions and proposed renova- tions of the Program. The Departmental Journal Club is structured for students in a progressive fashion where they attend and submit feedback on presentations by others before they make their own presentations and receive feedback from both their peers and a faculty committee. This experience has led to our students perennially winning a disproportionately greater number of presentation awards compared to other programs. In addition, the Annual Departmental Retreat show- cases the scientific accomplishments of the entire program and bring the community of faculty, students and research staff together to enjoy the science as well as recreational activities. These periodic functions are reinforced on a weekly basis by the universally attended Seminar Series in the Pharmacological Sciences which features frontier research perti- nent to novel therapeutic developments. Furthermore both faculty and students are engaged in the recruiting efforts for new students and new faculty, again in both a professional and social manner. The camaraderie and cooperation among the MTTP students is manifest in many ways, including their participation in special events such as the annual food drive, their service as mentors for high school and undergrad research interns, their invitations of special seminar speakers each year, and their organization of the annual Graduate Student Symposium at the School of Medicine. Graduates of the Program have pursued postdoctoral work at prestigious institutions, and the majority has continued in research careers. Collaborative and interdisciplinary features of the MTTP - MTTP Faculty represent six major research centers in Cleveland: CWRU, University Hospitals, the CWRU Comprehensive Cancer Center, the Cleveland Clinic, the Cleveland VA Medical Center, and MetroHealth Medical Center. This rich diversity of research and training settings is accomplished while maintaining a united focus on pharmacology. A majority (34/42) of the training faculty hold formal appointments in the Department of Pharmacology, and all faculty share common program and research interests. Four- teen of the trainers have appointments in clinical departments, adding an important human disease-oriented dimension to the training experience. Research projects range from the molecular and cellular analysis of signal transduction and tran- scriptional regulation to the structural analysis of integral membrane receptor and transport molecules, and include com- plex, integrated transgenic models of human disease. The common thread is the focus of research on modern therapeutics. There is a continuum provided from molecular to clinical research, and the choice of focus is given to each student according to the Advanced Training Tracks and the specialty of the mentor. Collectively the MTTP trainers are a highly interactive group of experienced, well-funded investigators, and there are many examples of collaborative scientific inte- ractions among the program faculty. Junior faculty trainers have either obtained national funding already or show high promise for obtaining that funding in the near future, and most are already mentoring predoctoral students in their laboratories. There is strong tangible support provided on an annual basis directly for the MTTP by the School of Medicine.

The global objective of the NIH-funded Molecular Therapeutics Training Program (MTTP) is to provide highly qualified predoctoral students with the knowledge base and research skills to begin independent investigative and teaching careers in the pharmacological sciences. Rapid advances in biotechnology coupled to the ever- expanding development of new and repurposed therapeutics has created a growing need for highly qualified scientists with core training in the principles and practice of pharmacology. The MTTP has had sustained excellent outcomes and an excess of excellent training grant eligible predoctoral students. Therefore, support is requested for an expansion of the program from four to six predoctoral positions for all years. The goal of the program is to develop professional pharmacologists with the maturity to address new research questions through creativity and collaboration and to communicate their findings to a wide audience documented by publications in premier scientific journals and effective communication with the lay public. We expect our graduates to be fully capable of contributing to the design and evaluation of therapeutic strategies in a range of careers. The MTTP provides a uniform conceptual framework and research environment through which students obtain the PhD degree in Pharmacology at Case Western Reserve University (CWRU). Designed with a three-tiered progression, the MTTP first establishes a didactic foundation in cell and molecular biology coupled with research rotations to facilitate mentor selection. Secondly, a foundation in molecular and physiological pharmacology is achieved via an intensive two-part core course which emphasizes both quantitative analysis and disease- focused study of drugs. Thirdly, students specialize via advanced courses and thesis research. In addition, rigor and reproducibility is a longitudinal component of the curriculum, which emphasizes unbiased experimental approaches that take into account group size, blinded experiments, and sex as a biological variable. These steps ensure that the importance of rigor and reproducibility is at the forefront from year 1 through preparation and defense of the thesis. The thirty-eight MTTP faculty preceptors represent six major research centers in Cleveland: CWRU, University Hospitals, the CWRU Comprehensive Cancer Center, the Cleveland Clinic, the Cleveland VA Medical Center, and MetroHealth Medical Center. This rich diversity of research and training settings is accomplished while maintaining a united focus on pharmacology. The interdisciplinary design fosters productive interactions among students and faculty in basic and clinical departments around the common theme of therapeutics. Collectively the MTTP trainers are a highly interactive group of experienced, well-funded investigators, and there are many examples of collaborative scientific interactions among the program faculty. The environment at CWRU is excellent for research and training PhD students. Finally, The CWRU SOM is committed to graduate education and covers all first-year costs of all PhD students entering the MTTP.