Daria Mochly-rosen - US grants

Protein kinase C (C-Kinase) is a key enzyme in signal transduction. In the heart, C-Kinase affects contractility, secretion, cell hypertophy and myofibril structure. There are seven C-Kinase Isozymes, each of which is likely to be involved in a different function. Activation of C-Kinase isozymes results in their translocation to intracellular structures such as the cell membrane and cytoskeletal elements. Identifying which isozymes are activated in the heart and determining the intracellular location of each activated C-Kinase will lead to understanding the functional role of each isozyme. Using biochemical, immunological and immunohistochemical techniques I will determine: (1) the cellular structures to which individual C-Kinase isozymes translocate and whether C-Kinase isozymes vary in their sensitivity to activation by different hormones and neurotransmitters. (2) the protein receptor for activated C-Kinase (RACKs) in these structures and whether they are isozyme specific and (3) the sites on the C-Kinase molecule that are responsible for binding RACKs. C-Kinase is implicated in pathological conditions in the heart including hypertrophy and dysrhythmia. Thus, elucidating the functional divergence of the various C-Kinase isozymes will enable therapeutic intervention resulting from isozyme-specific modulation.

Protein kinase C (PKC) isozymes comprise a family of related cytosolic kinases that translocate to the cell particulate fraction on stimulation. There are at least nine different PKC isozymes, six of which are found in the heart. PKC involvement in regulation of cardiac contractility, hypertrophy, organization of myofibrils and gene expression has been indicated by numerous studies. However, since isozyme-specific inhibitors are currently unavailable, the role of individual PKC isozymes in mediating each of these cardiac functions has not yet been determined. Activation of PKC isozymes in cardiac myocytes caused translocation of individual PKC isozymes to distinct subcellular sites. Some of the sequences in different PKC isozymes that are required for this specific translocation have already been identified, and others will be identified as described in the following proposal. Using peptides, homologous to these sequences, the translocation of specific PKC isozymes will be inhibited. We have previous demonstrated that inhibition of translocation of PKC also inhibits PKC mediated functions. Therefore, using these isozyme-specific translocation inhibitory peptides, it will be possible to determine the role of individual PKC isozymes in cardiac contractility, hypertrophy and organization of myofibrils. PKC is implicated in pathological conditions in the heart including hypertrophy and dysrhythmia. Our work will elucidate the role of various PKC isozymes using a new family of isozyme-specific inhibitors that is being developed. These data will lead to generation of therapeutic agents that interfere only with the activity of individual malfunctioning isozymes and not with the normal activity of other PKC isozymes.

DESCRIPTION (APPLICANT'S ABSTRACT): Both acute and chronic ingestion of ethanol result in multiple effects in the central nervous system (CNS) as well as in a variety of other organs such as liver and heart. In the CNS, there are both short and long term effects of chronic alcohol consumption such as intoxication, memory loss, tolerance to the acute intoxicating effects of ethanol, addiction, and dependence. The pleiotropic effects of ethanol exposure can be also observed at a molecular level; amounts and activities of components of several signal transduction systems are altered as a result of exposure to ethanol in a time- and dose-dependent manner. The molecular targets for ethanol and the mechanism by which ethanol-induced effects occur are largely unknown. However, studies from several laboratories indicate that the effects of ethanol are specific. This proposal is focused on protein kinase C (PKC) signaling pathway, a key pathway in the CNS. Short- and long-term exposures to ethanol specifically alter the level and activity of components of this key signal transduction system. There are multiple PKC isozymes that regulate important cellular functions and preliminary studies using a model of neuronal cells in culture suggest that ethanol exposure also results in changes in the subcellular localization of a specific PKC isozyme and its anchoring protein, RACK. The study of ethanol-induced changes in the CNS and their functional consequences is a particular challenge because individual brain regions and various cell types in each region express different PKC isozymes and have different sensitivities to ethanol. Here the investigator describes her plan to devise tools to identify the effects of ethanol in individual cells in situ. They will raise novel monoclonal antibodies (mcAbs) that will distinguish between states of activity of individual PKC isozymes as well as mcAbs to RACK. These mcAbs will be used in immunocytochemical studies of model neuronal cell culture systems as well as in brain of control and ethanol-treated mice. Such mcAbs will allow, for the first time, the determination of how ethanol affects the localization and activity of the components of this key signal transduction system in specific regions and cells in the brain. These studies will help elucidate the molecular basis of ethanol effects and alcoholism in man.

Protein kinase C (PKC) has been implicated in a number of cardiac functions. These include regulation of the strength and rate of contraction, expression of myofibril proteins, organization of cytoskeleton, regulation of cell size during normal development, and protection from hypoxia-induced cell death. We found that there are at least six different PKC isozymes in neonatal heart. These isozymes share long stretches of sequence homologies. In addition, each isozyme has unique sequences that are quite well conserved in evolution. It is therefore likely that individual PKC isozymes play specific roles in cardiac function, and that the isozyme-unique sequences determine the specificity of individual isozymes for each function. In this proposal, we plan to focus on determining the role of delta and epsilonPKC isozymes. We found that on activation, deltaPKC associates with fibrillar and perinuclear structures, and epsilonPKC isozyme associates with the myofibrils, perinucleus and cell-cell contact structures. Our recent data suggest that one of the unique regions in epsilonPKC contains the binding site for epsilonPKC, specific binding proteins at the perinucleus, myofibril and cell-cell contact. Similarly, we predict that the deltaPKC-specific binding site is in the corresponding unique region of deltaPKC. Here, we plan to identify the sequences within the above isozyme-unique regions that are required for isozyme-specific association of their sites of translocation. We will identify PKC fragments and peptides that inhibit the translocation of specific PKC isozymes to these sites. We have previously demonstrated that inhibition of translocation of PKC inhibits PKC-mediated functions. Therefore, using these delta and epsilon-specific translocation inhibitors, we will determine the role of the respective isozymes in protection of cardiac myocytes from hypoxia-induced cell death, cardiac contraction, organization of cytoskeletal elements, and regulation of cell size. PKC modulates pathological heart conditions including protection from ischemic damage, dysrhythmia and hypertrophy. By identifying isozyme- specific translocation inhibitors, the role of individual PKC isozymes in normal and malfunctioning heart can be determined. Moreover, this approach may lead to the generation of very specific and novel therapeutic agents.

Protein kinase C (PKC) has been implicated in a number of cardiac functions, including regulation of the strength and rate of contraction and protection from hypoxia-induced cell death. We found that there are at least six different PKC isozymes in neonatal heart. These isozymes share long stretches of sequence homologies. In addition, each isozyme has unique sequences that are quite well conserved in evolution. It is therefore expected that individual PKC isozymes play specific roles in cardiac function and that the isozyme-unique sequences mediate this specificity. Our research has focused on identifying PKC isozyme-selective inhibitors. We have capitalized on observations that individual isozymes translocate to different subcellular sites following activation. We suggested that this isozyme-selective subcellular localization reflects the association of individual isozymes with their selective anchoring proteins, and that inhibition of isozyme translocation selectively inhibits the function mediated by each isozyme. To this end, we have designed translocation inhibitors that interfere with the association of each PKC isozyme and its selective anchoring protein. Recently, we have also identified activators of individual isozymes. We now have selective inhibitors for all the isozymes present in heart and selective activators for some of them. With the exception of one inhibitor, all were developed in our laboratory. In this proposal, we plan to apply these novel tools to identify the role of different PKC isozymes in cardiac functions that are altered in specific disease states. We will test a new working hypothesis that individual PKC isozymes have opposing effects in cardiac function. If correct, it may explain the current controversy in the field. Using isolated adult cells, intact heart and whole animal studies, we plan to determine the role of PKC isozymes in the response of cardiac myocytes and fibroblasts to hypoxia (AIM A) and in the regulation of chronotrophy (AIM B). We will also determine the role of PKC isozymes in the regulation of proliferation of cardiac fibroblasts (AIM C). Our published and preliminary data demonstrate the feasibility of these studies as well as our substantial progress in sorting out these complex events. Our overall goal is to relate these findings to human disease; identification of the individual PKC isozyme whose activity is required and sufficient to induce the tested cardiac function may reveal novel targets for therapy of heart diseases.

DESCRIPTION (provided by applicant): Ethanol has paradoxical effects on the human myocardium; consumption of large amounts over a lifetime can lead to alcoholic cardiomyopathy, whereas consumption of ethanol in moderation can prevent or reduce the risk of coronary artery disease (CAD) and cardiac ischemia. In addition to the preventive effect of moderate consumption of ethanol on CAD, acute exposure to ethanol induces a direct protective effect on the myocardium. This latter mode of protection by ethanol is thought to mimic a natural form of cardioprotection, termed ischemic preconditioning. However, the cardioprotective effect of ethanol appears to have a narrow therapeutic window; ethanol exerts both negative and positive effects on the myocardium. Identifying the mediators of ethanol's effects on the heart and identifying means to selectively activate those that are cardioprotective and inhibit those that contribute to cardiac damage may enable the use of ethanol in prevention of cardiac damage during or after acute MI. The research proposal described above focuses on one scenario - the effect of acute exposure to ethanol on the response of the heart to ischemia and reperfusion. We plan to determine the role of ethanol on the activities of mitochondrial enzymes that correlate with the cardioprotective effect of ethanol (AIM 1), identify ethanol-induced PKC-selective substrates and their role in cardioprotection (AIM 2) and determine whether acute treatment with ethanol can be used as a cardioprotective agent in a large animal model of transient ischemia and reperfusion in vivo (AIM 3). Together, our studies will assess the molecular basis and potential use of ethanol as an agent to protect the heart from ischemic damage.

DESCRIPTION (provided by applicant): The present application for support of the Chemical and Molecular Pharmacology Training Program represents a new venture undertaken by the Stanford Department of Molecular Pharmacology in response to the extraordinarily rapid advances in molecular and structural biology over the past ten years. While the Department has had a very successful graduate program over the past decades--several of its graduates can now be found in leading positions in academic institutions and in industry--the recent explosive growth of knowledge in biological sciences has made it clear that a new set of concepts and skills will be needed for a successful career in Pharmacology in the future. The sequencing of whole genomes and the initial characterization of myriad proteins and signal transduction pathways is defining the pharmacology of the future: the study of intricate and delicately-balanced cell regulatory networks, and of how drugs can and do impinge upon them. To fully bring this future to fruition will require the application of rigorous quantitative methods and concepts--those of physics, chemistry, systems analysis and informatics. It will therefore also require the attracting of a different type of student to the field, a student with interests and abilities in quantitative science. If the discipline of Pharmacology is to meet this challenge, it must develop graduate training programs that anticipate and convey the knowledge required to advance the field from its present stage. Building on its strong tradition of innovation the Stanford Department of Molecular Pharmacology is therefore implementing a two-track graduate program. The first track emphasizes molecular biology, cell biology, and medical pharmacology, in recognition of the fact that established molecular biological methods still hold a great deal of promise for discovery and analysis of life's processes. The second track places greater emphasis on rigorous quantitative methods of analysis, in recognition of the fact that this is the type of analysis required to solve the next generation of problems and fulfill the mission of Pharmacology as the science concerned with mechanisms of drug action.

DESCRIPTION (provided by applicant): Over 500,000 strokes occur in this country annually, causing significant disability and death. Only one treatment, recombinant tissue plasminogen activator (rt-PA), is currently approved for stroke. However, just about 3% of the patients receive it, probably because the time-window for therapy is brief (3 hours or less). Therefore, there is a great need for effective neuroprotective agents that could be given alone or in conjunction with thrombolytic agents. Furthermore, prophylactic neuroprotective use may be indicated in certain clinical settings. Each year, in the United States alone, 600,000 adults and 12,000 children undergo open heart operations utilizing cardiopulmonary bypass, during which the brain is subjected to periods of ischemia. Despite advances in surgical procedures and shortening of the ischemic event, CNS dysfunction remains a leading cause of morbidity and mortality in these patients. Because the exact timing of the ischemic insult is known ahead of time in these patients, the potential exists to significantly reduce myocardial damage by pre-treatment with agents that protect the brain from ischemia-reperfusion damage prior to surgery. Our HYPOTHESIS is that select protein kinase C (PKC) isozymes play an important (positive and/or negative) role in the various stages of response of the brain to ischemic insult. Using isozyme-specific inhibitor and activator peptides that we have developed, we plan to; determine which PKC isozymes are activated following cerebral ischemia and when does that occur, and determine whether inhibition or activation of these isozymes can provide protection from ischemic reperfusion damage to the brain. These studies will demonstrate the effectiveness of in vivo delivery of PKC regulating peptides for the treatment of ischemic reperfusion injury of the CNS. Should the treatment be found efficacious and safe, these peptide regulators of PKC activity may be useful for the treatment of stroke and brain ischemia in humans.

Protein kinase C (PKC) isozymes play a key role in insult-induced cardiac remodeling and the progression to heart failure (HF). Conflicting data on the role of individual PKC isozymes in modulating these functions have been reported, in part due to the use of PKC isozyme non-selective pharmacological tools. Our research identified PKC isozyme-selective inhibitors and activators, which we apply to determine the role of individual isozymes in normal and diseased heart. We identified peptide inhibitors and activators for each isozyme and found that the peptides can be effectively delivered into the heart, in vivo. Using these PKC regulating peptides, we previously showed opposing roles for specific PKC isozymes in various functions; we showed that ePKC activation or delta PKC inhibition provides cardioprotection from ischemia in vitro, ex vivo and recently in vivo. We also found that our isozyme-specific peptide regulators of PKC remain effective when continuously delivered in vivo, for 10 days. Therefore, for the first time, we can determine whether these PKC-regulating peptides can prevent, enhance or reduce cardiac remodeling and transition to heart failure, a study that requires regulation of PKC in a sustained fashion. Due to the limitations of animal models for cardiac remodeling and HF, we plan to use 3 different models, and use the peptides as selective pharmacological tools to determine the role of each PKC isozyme in the development of HF. The first model involves pressure overload using transverse aortic constriction in mice. The second uses pressure overload in hypertensive Dahl rats, which develop reliable cardiac remodeling and heart failure after initiation of a high salt diet. The third model follows post myocardial infarction-induced cardiac remodeling and HF in mice. Peptide regulators of individual isozymes (activators or inhibitors) will be delivered in a sustained fashion at different times during the course of the disease to determine the role of each PKC isozyme in the development of adaptive and maladaptive remodeling and the transition to heart failure. Together, these studies will identify the PKC isozyme(s) that should be targeted for the development of new therapeutics for human heart failure, especially if PKC-based pharmacotherapy is considered

DESCRIPTION (provided by applicant): Leishmaniasis is an endemic disease in tropical and subtropical regions of over 80 countries with more than 2 million new cases occurring annually http://www.who.int/tdr/diseases/leish/diseaseinfo.htm. Receptor for activated C kinase 1 (RACK1), a ubiquitous and highly conserved scaffold protein, binds to proteins involved in many signaling processes leading to cell survival, growth and differentiation. We have successfully generated short peptide regulators that either disrupt or enhance the interactions of RACK1 with these signaling proteins and thus regulate their biological effects. Little is known about the cellular function of the Leishmania homolog of RACK1, Leishmania homolog of receptors for activated C kinase, LACK. We believe that LACK, due to its highly conserved nature, is also a key scaffolding protein involved in essential signaling processes of the Leishmania parasite. We propose a novel approach, utilizing our well established methods of peptide design, to identify and characterize LACK- binding partners and to target protein-protein interactions between LACK and these Leishmaniasis- signaling enzymes. These peptides may serve as leads for the development of new therapeutics for the treatment of Leishmaniasis. Specifically, in this early feasibility proposal, we plan to: 1. Identify and characterize Leishmania signaling proteins that are LACK-binding partners (LACK-BP). 2. Rationally design peptides that selectively inhibit the interactions between the scaffold LACK and LACK-BP and test their activity towards Leishmania sp. proliferation and in vitro infection. To identify and characterize LACK-BP, we will utilize several methodologies including protein overlays (Far Western), in vitro pull-down assays, and expression interaction cloning. To design peptide modulators of protein-protein interactions, we will use sequences in RACK-BP that are required for RACK binding and identify homologous sequences in LACK-BP. Additionally, we will identify evolutionary conserved sequences in homologs of RACK-BP and use structural modeling to look for regions of homology for peptide design. Finally, we will identify short peptides derived from LACK that are likely to disrupt the binding and hence the signaling of LACK-BPs. The peptide modulators will be tethering to cell-permeable vectors and tested for their biological activity on proliferation of Leishmania promastigotes in culture and on Leishmania-infected murine macrophages. The peptides developed as part of the proposed project are likely to exert significant biological effects. Importantly, identification of LACK-binding proteins and peptides that interfere with their biological activities can help guide the development of new therapeutics for patients suffering from Leishmania infection. PROJECT NARRATIVE: Two million new cases of Leishmania infection are reported annually, with a resultant death rate of 3%. The current treatment for Leishmaniasis, chemotherapy, is highly toxic and ineffective. Our proposed studies will use a new approach to identify key players involved in Leishmania viability, allowing us to identify and create highly specific therapeutics that disrupt these cellular processes. This therapeutic approach is likely to provide a less toxic, highly specific and effective way of treating Leishmaniasis.

DESCRIPTION (provided by applicant): Our lab has studied the role of protein kinase C (PKC) isozymes in cardiac ischemia for almost 20 years, using unique tools that we have developed and continue to develop. The work was initially triggered by our hypothesis that anchoring of activated PKC to subcellular sites via binding to select protein substrates, RACKs, is critical for PKC function. To prove the original RACK hypothesis, we generated new tools, short peptides that interfere with protein-protein interactions within PKC and between PKC and RACKs. These highly selective first generation peptide regulators of PKC have been instrumental in elucidating opposing (yin/yang) roles for two PKC isozymes, delta and epsilon PKC, in the responses to cardiac injury by ischemia and reperfusion (I/R). Here, we plan to expand this work while maintaining the focus on delta and epsilon PKC in models of myocardial infarction (MI). These studies will be carried out at six different `levels': in vivo, ex vivo, at the cellular level, at the subcellular, the molecular and the sub- molecular levels. Specifically, in AIM 1, we plan to identify selective substrates of each isozyme in each subcellular fraction. In AIM 2, we plan to determine the molecular basis for the opposing roles of delta and epsilon PKC during I/R. In AIM 3, we plan to use novel (second generation) PKC regulating peptides to selectively inhibit I/R-induced interaction of delta and epsilon PKC with distinct protein substrates and not with others at each particular subcellular site. The proposed study will provide a better understanding of how activation of these two homologous isozymes during I/R exerts opposing effects on outcome;I/R- induced delta PKC activation leads to cardiac infarction and activation of epsilon PKC to cardioprotection. Clearly, complex responses are elicited to both protect the cells from ischemic injury as well as to induce cell death. Our studies will continue to elucidate these processes using unique pharmacological tools that we have designed - short peptides that regulate isozyme-specific functions, selectively. Current treatment for acute myocardial infarction (AMI) is aimed at inducing reperfusion by either mechanical means (balloon catheters) or by the use of enzymatic means (thrombolytics) to disrupt the occlusion. While these treatments are effective in limiting the duration of ischemia, no therapeutic treatment is currently available to prevent ischemic injury and to reduce reperfusion injury (I/R injury) associated with these interventions. Our proposed studies will continue to elucidate these fundamentally important processes and our long term goal is that this work will identify new and highly specific therapeutics to treat patients subjected to cardiac ischemia and reperfusion. PUBLIC HEALTH RELEVANCE: Current treatment for acute myocardial infarction (AMI) is aimed at inducing reperfusion by either mechanical means (balloon catheters) or by the use of enzymatic means (thrombolytics) to disrupt the occlusion. While these treatments are effective in limiting the duration of ischemia, no therapeutic treatment is currently available to prevent ischemic injury and to reduce reperfusion injury associated with these interventions. Our proposed studies will continue to elucidate these fundamentally important processes and our long term goal is that this work will identify new and highly specific therapeutics to treat patients subjected to cardiac ischemia and reperfusion.

The broad, long-range objective of the Translational Incubator Core (Core C) is to enhance and accelerate the translational aspects of the Center's goals to develop new classes of host-targeting antiviral therapeutics that are capable of treating multiple NIAID Emerging and Re-emerging Priority Pathogen viruses, when used alone or in combination with other available agents. The Translational Incubator Core will be responsible for providing strategic scientific, regulatory, preclinical and clinical guidance to all U19 research projects. The Core will assist projects in establishing Target Product Profiles and Development Plans for advancing their therapeutics towards the clinic. The Core will provide guidance in areas such as appropriate use of in vitro assays and in vivo models to demonstrate efficacy, pharmacokinetics, ADME (absorption, distribution, metabolism, and excretion), and preclinical safety. When appropriate, the Translational Incubator Core will provide advice regarding commercialization options when this is required to further advance the project. In addition, each year the Core will select and provide guidance to pilot projects to develop therapeutics or supportive technologies that address emerging or re-emerging infectious diseases and are both consonant with the translational research theme of the Center and capable of enhancing existing projects. The Translational Incubator Core will seek to achieve these objectives via the following specific aims: 1) To provide regulatory science expertise and support to all participating research projects. 2) To provide strategic advice, expertise and practical support on the translational aspects of the Center's projects, thereby accelerating progress in accomplishment of preclinical development activities. 3) To identify research projects and technologies within the greater Stanford community that could directly enhance the Center's projects and translational efforts to develop new classes of host-targeting antiviral therapies

? DESCRIPTION (provided by applicant): This T32 application request is to support our Molecular Pharmacology predoctoral training program by providing funding for 10 predoctoral students, 5 in year 2 and 5 in year 3. The purpose of this training program is to train students to carry out rigorous basic and translational research. The program will train the students to become experts in modern pharmacology and in drug discovery and development. These efforts will prepare the students for careers in academic research and biotechnology companies, and for teaching, consulting or other pharmacology related positions. This new Training Program builds on the strength of SPARK, the school's translational research effort. SPARK was created eight years ago at Stanford and is now duplicated in numerous institutions in the US and throughout the world. Taught together with industry veterans and experts, SPARK provides hands-on and student team approach that leads to a real-world experience in drug development. This unique graduate student training program is integrated with formal course work that includes not only the mandatory core pharmacology disciplines, cell signaling and drug discovery, but also chemistry, systems biology and math. The interdepartmental training program draws upon an outstanding group of 30 collaborative faculty members, with excellent track record of student training. They include faculty members in the Departments of Chemical and Systems Biology, Biochemistry, Bioengineering, Biology, Chemistry, Dermatology, Medicine, Microbiology and Immunology, Molecular and Cellular Physiology, Otolaryngology, Pathology, and Pediatrics. Students' training includes two years of advanced coursework followed by up to three years of independent research. The training is integrated with drug development efforts through SPARK, as well as courses in the responsible conduct of research, animal care, and safety. Students are required to present their research work to the program faculty four times every year: at our Program's science lunch seminar, at the Program's annual retreat and at two graduate research committee meetings. In addition to the faculty mentors and the above committee members, we have created a detailed structure, tracking mechanism and a network of faculty members who follow and assist students with their research and career development. The overarching goal of the training program is to develop the skills, imagination, critical thinking and knowledge in sciences to enable the students to become outstanding scholars and leaders in Academia or Industry.

? DESCRIPTION (provided by applicant): Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common genetic disorder in humans, with more than 160 point mutations in this gene, and over 400 million people affected. Although most research relates to G6PD deficiency and increased hemolysis (breakdown of red blood cells) and accumulation of bilirubin in adults, epidemiological studies indicate that G6PD deficiency is also a major cause of pathologic neonatal bilirubin accumulation (jaundice) and a contributor to morbidity, including neurological injury (kernicterus). The hypothesis to be tested here is that correcting the activity and stability of G6PD mutants as well as increasing the activity of the wild-type enzyme will decrease bilirubin-induced neurotoxicity in infants. In this proposal, the plan to begin developing a treatment for kernicterus using a totally novel approach, by developing activators of wild-type (Wt) and mutant G6PDs is described. The project includes four aims: AIM 1: Identify small molecule chaperones that increase the catalytic activity of Wt and common G6PD mutants using an in vitro screen of a library of small molecules. AIM 2: Determine the X-ray crystal structure of the Wt and mutant G6PD enzymes in complex with the G6PD chaperone(s). Aim 3: Evaluate the ability of the chaperone(s) to protect cultured cells expressing Wt or the mutant G6PDs from bilirubin-induced cytotoxicity and elucidate the molecular basis of their effects. AIM 4: Evaluate the ability of the small molecule chaperone(s) to reduce bilirubin-induced neurotoxicity in rodent models of newborn hyperbilirubinemia, in vivo. Two established rat models of kernicterus (Gunn rats and bilirubin-injected Wt rats) and three new mouse models [transgenic mice, mimicking three common G6PD mutations with kernicterus] will be used. Small molecules, identified in Aim 3 to protect neurons from bilirubin-induced toxicity, will be tested for their ability to prevent or reduce neurological injury (measured as loss of motor and auditory skills) due to hyperbilirubinemia in one or more of these rodent models, in vivo. The experience of Dr. Stevenson (co-PI) in basic research related to hyperbilirubinemia and in clinical care of such newborns, the expertise of Dr. Wakatsuki in crystallography, together with our expertise in drug discovery and development and in neuroscience research, and the expertise of other advisors in drug screening, medicinal chemistry, in gene editing and in assessing auditory and motor dysfunctions, place the team in a unique position to address the above goals.

PROJECT SUMMARY Fanconi anemia (FA) is caused by mutations in one of at least 19 FA complementation (FANC) group genes, which are required to repair DNA interstrand cross links (ICL). Patients with FA have congenital anomalies, hematopoietic stem cell (HSC) injury resulting in increased susceptibility to bone marrow failure (severe aplastic anemia: SAA) and leukemia, increased sensitivity to chemotherapy and radiation, and high risk of secondary cancers, even if they are successfully treated by HSC transplantation for their aplastic anemia or leukemia. Because the mutations underlying FA affect every cell in the body, there is currently no cure for FA. Recent studies have demonstrated that ICL are caused by reactive aldehydes, such as acetaldehyde (MeCHO). Oxidation by the aldehyde dehydrogenase (ALDH) family of enzymes detoxify aldehydes. In East Asia, a highly prevalent point mutation in the Aldehyde Dehydrogenase 2 (ALDH2) gene causes a semidominant loss of function, and decreased ability to oxidize highly reactive MeCHO. Exposure to MeCHO occurs through generation by endogenous pathways, exogenous ingestion or inhalation, or as the major product of ethanol (EtOH) metabolism. The ALDH2*2 mutation results in the well-known ?Asian flushing syndrome? marked by a disulfiram-like response to ingestion of small amounts of EtOH. The ALDH2*2 mutation also results in increased susceptibility to cancer, especially esophageal cancer. Thus, the ALDH2 and FA DNA repair pathways confer two tiers of genome protection from the toxic effects of MeCHO. Humans and mice doubly mutated for both pathways rapidly develop bone marrow failure due to the loss of HSC. Our analyses of mice with a knock-in of the human ALDH2*2 mutation at the murine ALDH2 allele show that, even in a basal environment, (without exposure to EtOH) HSC are 1) progressively lost, 2) at a competitive disadvantage to normal HSC, and 3) have a gene expression profile typical of a response to interferons, which are known inhibitors of stem cell self-renewal. Preliminary results also indicate that EtOH exposure causes significant decline in HSC numbers in wildtype (ALDH2*1/*1) mice. We have developed ALDH activators (Aldas), small molecules which increase the activity of both ALDH2*1 and ALDH2*2, or re-direct other ALDH family members, e.g., ALDH3a1, to also oxidize MeCHO. To measure the cellular aldehydic load, we have also made unique fluorescence based sensors, which can be used in flow cytometric analyses of individual cells, e.g., HSC. In this grant we will test the hypothesis that Aldas can protect FA HSC from genotoxic injury by decreasing the load of reactive aldehydes. We will study murine models doubly mutated for FANCD2 and ALDH2*2 to determine the protective effect of ALDH2 activation, measure the effects of Aldas on aldehydic load in HSC, and determine whether recruitment of ALDH3a1 to MeCHO metabolism protects HSC. These pre-clinical studies have the potential to rapidly translate to much needed clinical trials aimed at prevention of SAA, leukemia and cancer in patients with FA.

Ethanol is metabolized by alcohol dehydrogenase to acetaldehyde, a known toxic agent that can form adducts on macromolecules. The levels of acetaldehyde and other aldehydes increase under oxidative stress, and the increase in aldehydic load is associated with a number of common human pathologies. Relevant to this proposal, increased aldehydic load contributes to neurodegeneration. A major defense from acetaldehyde and from other aldehydes that we generate or are exposed to from the environment is the mitochondrial matrix enzyme, aldehyde dehydrogenase 2 (ALDH2). Ten years ago, our research (supported by this grant) demonstrated a critical cytoprotective role for ALDH2: direct activation with a small molecule that we discovered, Alda-1, reduces oxidative damage in a number of cell and animal models of human diseases. A common human inactivating point mutation in ALDH2 (ALDH2*2), present in ~560 million East Asians, increases the cell injury and cell death by aldehydic load induced by high levels of ethanol or by oxidative stress, and Alda-1 treatment reduces this injury. Because ALDH2*2 mutation is so common, we propose the HYPOTHESIS that additional ALDH2 deficiencies may exist in significantly large numbers in populations other than East Asians. Supported by our preliminary data, in AIM 1 we plan to identify and characterize new common ALDH2 variants, determine the effect of these variants on ethanol-induced acetaldehyde metabolism in human cells and identify new small molecules (ALDH2 activators; Aldas) that correct these newly identified inactive variants. Mounting evidence connects aldehydic load and neurodegenerative diseases, such as Alzheimer?s disease (AD). There is also a significant increased risk for AD among carriers of ALDH2*2 genotype. Because mitochondrial dysfunction contributes to neurodegeneration and aldehydes cause mitochondrial dysfunction, and because there is a correlation between frequent alcohol consumption, increased risk of dementia and a potential role for acetaldehyde in neurodegeneration, we plan to test our second HYPOTHESIS that inactivating variants of ALDH2 and the higher acetaldehyde levels following ethanol consumption contribute to mitochondrial dysfunction and thus to neurodegeneration. In AIM 2, we will characterize new ALDH2-inactive variants and their effect on mitochondrial structure and function in the presence of ethanol in cultured neuronal cells, in AD patient-derived cells, and in an AD mouse model expressing inactivating ALDH2 variants, in the presence or absence of chronic exposure to moderate levels of ethanol. Over 80% of the population in the US consumes alcoholic beverages. As ~1-2% of people over the age of 65 and ~30% of people over 80 develop AD, the possibility that ethanol consumption contributes to the progression of the disease in the general population and in patients deficient in ALDH2 activity, and the identification of a potential therapeutic intervention, are the subjects of this proposal.