Michael John Palladino - US grants

DESCRIPTION (provided by applicant): The long-term goal of this research program is to understand the biochemical pathways that regulate age-dependent neuronal viability in metazoans. Using a tractable genetic system to elucidate these pathways and uncover the mechanisms by which they contribute to neurodegenerative diseases and normal senescence, are important goals of my research. We have isolated a number of mutants in an unbiased forward genetic screen that provide us a novel perspective with which to study neuronal senescence and neurodegeneration in vivo. This proposal focuses on mutations that affect ATPalpha (Na/K ATPase), which cause progressive neurodegeneration in Drosophila. In humans loss-of-function Na/K ATPase mutations cause Rapid-onset Dystonia Parkinsonism (RDP) and familial hemiplegic migraines (FHM). Na/K ATPase activity is reduced after ischemia and traumatic brain injury, and is associated with Alzheimer's disease. We hypothesize that neuropathogenesis is mechanistically similar in our mutants and patients with neurological diseases, including RDP and FHM, and propose experiments to elucidate these mechanistic details. Additionally, we propose to identify mutations capable of suppressing the underlying dysfunction and neurodegeneration. We will study these mutants using genetic, molecular, cell biological, and biochemical techniques to elucidate the mechanisms by which the mutations lead to cellular dysfunction and discover how this dysfunction manifests as neurological phenotypes, such as paralysis, seizures and progressive neurodegeneration. Together these specific experiments will elucidate the mechanisms of neuropathogenesis in ATPalpha mutants and lead to a better understanding of age-related neurodegeneration and senescence.

[unreadable] DESCRIPTION (provided by applicant): The overall goal of the Pittsburgh Summer Program for Undergraduate Research Growth (Pitt-SPURG) is to encourage entry of undergraduates majoring in quantitative sciences into careers in biomedical research by introducing them to contemporary problems in biological signal transduction and receptor function that benefit from inter-disciplinary approaches. Pitt-SPURG will exploit the mentor focus on nuclear and membrane-delimited receptors and cell signaling, where there is extensive application of physical, biochemical and structural approaches to addressing these biological problems. During the 10-week program, students will conduct original research in an area of receptors, G proteins, or signal transduction under the mentored supervision of Molecular Pharmacology Program faculty. Students will work on problems at the frontier of biomedical science using state-of-the art techniques. Students will also follow a dedicated series of lectures to introduce physical and quantitative science majors to nuclear and membrane-delimited receptors and biological signaling problems and technical approaches to introduce them to this subject. Additionally, they will participate in a seminar program designed to further their understanding of the scientific process and critical analysis, and workshop forums on career paths within biomedical research. The long-term goal is to promote scientific training of new investigators that exploits interdisciplinary approaches to address significant research problems in these disciplines. Prospective students will be selected from a large and outstanding pool of candidates that historically apply to summer research programs at the University of Pittsburgh. The program will be widely advertised on internet websites, in student newspapers, mailings to college and universities with high numbers of students pursuing graduate scientific training, and by word of mouth. A core of NIH-funded, experienced investigators have been chosen as mentors. Applicants will be asked to submit a curriculum vita including personal data, academic major, grade point average, previous experience, and their top choices of summer research. Summer students will be selected based on merit and appropriateness of fit and diversity by the Executive Committee. Ancillary activities will include laboratory safety, and scientific ethics. At the end of the program, each student will present the results of his/her work at a colloquium of participating students and mentors. A certificate will be awarded to all students successfully completing the Program. Longitudinal follow-up will monitor graduate training and career choices elected by student participants in this program and will be compared with that of entering biomedical graduate students, who were quantitative science majors but did not participate in Pitt-SPURG or similar programs. This tracking will provide an assessment of the ability of the program to achieve its goals. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The ATP6 protein functions as a hydrogen ion channel that couples ion transport with rotary ATP catalysis. Missense mutations in the human ATP6 gene are believed to cause at least three related and devastating syndromes characterized by progressive muscle impairment and neurological symptoms: NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh's syndrome) and FBSN (familial bilateral striatal necrosis) and contribute to the pathogenesis of several other age-related diseases. We have isolated a missense mutation within the mitochondrial ATP6 gene of Drosophila and developed this as model of mitochondrial encephalomyopathy. We propose to utilize this genetic animal model system to elucidate the pathophysiological basis for progressive encephalomyopathies in vivo. PUBLIC HEALTH RELEVANCE: Mitochondrial disease affect ~ 1 in 4000 individuals. Currently there exists no effective pharmacotherapy for these devastating diseases. Our understanding of disease pathogenesis of these progressive conditions is limited, in large part due to a lack of animal models of these diseases. Our model system captures many relevant features of these diseases in an amenable genetic system and this proposal aims to study pathogenesis of these diseases.

Project summary: Archetypical mitochondrial diseases are common and devastating conditions with an extremely poor prognosis. Gene therapies have been proposed including allotopic expression of recoded mitochondrial genes from the nucleus. The extreme hydrophobicity of mitochondrial-encoded proteins remains a technical hurdle to such therapies. We propose a novel allotopic gene therapy approach where the RNA is actively transported into mitochondria for translation. We have developed vectors that express stable, transportable RNAs and will test their efficacy in vivo. Any serious investigation aimed at developing allotopic expression as a gene therapy would require a well-characterized, pathogenic, endogenous mitochondrial mutation in an amenable genetic system where feasibility can be demonstrated and optimized in vivo. We propose a rigorous test of mitochondrial RNA transport using both biochemical and phenotypic assays of function using a well-characterized model animal system.

DESCRIPTION (provided by applicant): Protein quality control mechanisms are required for normal cellular health to prevent disease and avoid premature senescence. Unique mutant proteins are substrates of these pathways, are recognized by protein quality control pathway components and typically degraded by the proteasome. Proteins with subtle missense mutations can retain function yet degradation by these pathways can underlie pathogenesis. The molecular and physical basis of protein quality control substrate recognition are poorly understood, especially for soluble cytosolic proteins. This application proposes to use a powerful multi disciplinary approach to define novel proteins in the protein quality control pathway and elucidate the physical and structural basis of substrate recognition.

? DESCRIPTION (provided by applicant): Mutations in the mitochondrially-encoded ATP6 subunit of the ATP synthase protein complex cause human mitochondrial encephalomyopathies including maternally inherited Leigh Syndrome (MILS), neuropathy, ataxia, and retinitis pigmentosa (NARP), and familial bilateral striatal necrosis (FBSN). Symptoms of these devastating progressive disorders include shortened lifespan, neurodegeneration, organ system failure, and seizures. No cures have been found, and effective treatment of the symptoms has proven difficult. For example, the seizures present in these diseases are often refractory to common anti-epileptic drugs (AEDs). Recently, a point mutation affecting the ATP6 subunit has been identified and characterized in Drosophila melanogaster. This mutant line, termed ATP6[1], shares the progressive pathological features of the above disorders, thus providing one of the only stable animal models of mitochondrial disease that can be studied throughout the animal's lifetime. Using patch clamp electrophysiology on whole-brain explants from adult ATP6[1] flies, we have found that the well-characterized large lateral ventral neurons (l-LNv) of the Drosophila circadian and sleep circuits exhibit hyperexcitability, including elevated interspik membrane potential and firing frequency, and increased responses to excitatory stimuli including direct light exposure and depolarizing current injections. This makes them an excellent model neuron for the study of the seizures associated with mitochondrial diseases. Our preliminary data suggests the altered excitability may be due to multiple factors, which include changes in both membrane channels present in the l-LNv and in synaptic inputs. We propose to use these well- characterized and electrophysiologically-accessible neurons to identify these channel subunits and synaptic contributions, so that upon completion of this work we will know the relevant molecular contributors which may ultimately become therapeutic targets for the seizures of mitochondrial disease.

? DESCRIPTION (provided by applicant): Mitochondrial diseases are common and devastating conditions with an extremely poor prognosis. Gene therapies have been proposed involving allotopic expression of recoded mitochondrial genes from the nucleus, however, the viability of such an approach remains controversial. We have discovered that the major technical hurdles to such an approach that limit the development of a novel gene therapy are competition from the endogenous mutant protein in the complex and the hydrophobicity of these proteins. We have discovered a novel mitochondrial translation inhibition (TLI) approach to prevent expression of the mutant protein within mitochondria. We propose to test the combination of mitochondrial TLI with a novel therapy approach that targets coding RNAs to mitochondria. Such an approach directly addresses both technical hurdles using novel and previously untested methods. Any serious investigation aimed at developing a novel mitochondrial gene therapy would require a well-characterized, pathogenic, endogenous mitochondrial mutation in an amenable genetic system where feasibility can be demonstrated and optimized in vivo. We propose a rigorous test of mitochondrial TLI and mitochondrial-targeted RNA expression using several biochemical and phenotypic assays of function in a well-characterized animal model system.

Project Summary/Abstract: Genetic control of mitochondria is nearly completely lacking, however, the utility of such tools would be transformative to numerous fields of biomedical science. Developing genetic methods to knockdown or express/rescue endogenous mitochondrial genes would be a powerful research tool but would also immediately enable the development of novel therapies. We have developed novel vectors (mtTRES) that targeted RNAs to mitochondria and shown their ability to reduce the expression of endogenous genes in animals and human cells. We have also developed vectors capable of expressing long, coding RNAs to mitochondria allowing us to perform overexpression/rescue experiments. We propose to utilize mitochondrial-targeted riboendonucleases as an alternative method to reduce expression of endogenous genes but also to willfully process mtTRES expressed RNAs and facilitate their efficient expression. The development of novel methods to genetically manipulate mitochondrial gene expression in animals, which will be validated in human cells will have a broad impact on the fields of aging, mitochondrial biology and neurodegenerative disease research.

Abstract/Project summary: Protein quality control (PQC) mechanisms are required for cellular health, to prevent age-related diseases and to avoid premature aging and senescence. PQC substrates are recognized by specialized components and are typically degraded by the ubiquitin proteasome system (UPS). To explore the relationship between PQC and age-related disease, the applicant?s lab identified a novel substrate for this pathway, mutant Triosephosphate Isomerase (TPI). Numerous subtle amino acid substitutions in TPI are pathogenic, and result in progressive multisystem disease. Importantly, the mutant protein retains function and it is now known that increased turnover of the functioning protein by the UPS underlies disease pathogenesis. Because the molecular and physical basis of PQC substrate recognition is poorly understood, especially for soluble cytosolic proteins such as mutant TPI, a genome-wide genetic screen was performed that led to the identification of numerous novel regulators of mutant TPI turnover. The immediate goal of the screen is to define how mutant TPI is pathologically selected for disposal by the UPS and to identify pharmacologic targets for a therapy. The screen identified all of the known and predicted regulators of TPI (HSP70, HSP90, proteasome subunits, ligases, etcetera) as well as many additional modifiers of turnover. Importantly, several of these regulators are highly conserved proteins of currently ?unknown? function. We propose here to rigorously validate these regulators of mutant TPI and extend our analysis of the novel regulators by examining their role in the turnover of panel of other biomedically relevant substrates including alpha-synuclein, estrogen receptor and cystic fibrosis transmembrane conductance regulator. Overall this project will identify and validate conserved human PQC proteins that are critical to healthy animal aging and therapeutic targets for various progressive disease states.

Abstract/Project summary: TPI Df is a devastating untreatable childhood metabolic disease resulting in anemia, paralysis, irreversible brain damage and premature death. Numerous subtle amino acid substitutions in Triosephosphate Isomerase (TPI) are pathogenic and result in rapidly progressing multisystem disease. Importantly, pathogenic TPI Df mutations have been shown to result in protein that retains function but are unstable. Pathogenesis of numerous TPI DF mutations is known to result from increased turnover of the functioning protein by Protein Quality Control pathways (PQC). We have developed a human cellular TPI Df model of the ?common? mutation and validated its use in optical screening. We are utilizing this model in an automated compound screening platform to identify first in class TPI Df small molecule therapies. To validate small molecule therapies there is a desperate need for a mammalian model of TPI Df. This proposed research will meet this need by using CRISPR to create a TPI Df model with the ?common? mutation. A mouse model with construct validity for TPI Df that demonstrates analogous behavioral, physiological, and metabolic phenotypes is an important resource for the research community.

Abstract/Project summary: TPI Df is a devastating untreatable childhood metabolic disease resulting in anemia, paralysis, irreversible brain damage and premature death. Numerous single amino acid substitutions in Triosephosphate Isomerase (TPI) are pathogenic and result in rapidly progressing multisystem disease. Importantly, all known pathogenic TPI Df mutations result in a protein that retains function and pathogenesis is known to result from increased turnover of the functioning protein by Protein Quality Control pathways (PQC). We have developed a human cellular TPI Df assay based on a cellular model of the ?common? E104D mutation and implemented it for high-content, high-throughput imaging. We have used this model in a pilot screen and validated its utility to identify novel compounds that modulate mutant TPI protein levels in human cells. We propose to develop the assay to full HTS standards, conduct a screen of several relevant compound libraries, and identify first-in-class TPI Df small molecule therapies. We will validate hits in secondary assays for TPI stability and activity in TPI Df patient cells, prioritize them in a panel of in vitro toxicology and metabolic stability assays, examine structure activity relationships (SARs) of the lead compounds and substantially validate them in vivo using a recently developed mouse TPI Df model. Overall this project will discover and validate the first ever treatments for TPI Df that will provide the basis for clinical trials.

Abstract/Project summary: TPI Df is a devastating untreatable childhood metabolic disease resulting in anemia, paralysis, irreversible brain damage and premature death. Numerous subtle amino acid substitutions in Triosephosphate Isomerase (TPI) are pathogenic and result in rapidly progressing multisystem disease. Importantly, all known pathogenic TPI Df mutations result in a protein that retains function and pathogenesis is known to result from increased turnover of the functioning protein by Protein Quality Control pathways (PQC). We have developed a human cellular TPI Df assay based on a cellular model of the ?common? E104D mutation and implemented it for high-content, high-throughput imaging. We have used this model in a pilot screen and validated its utility to identify novel compounds that modulate mutant TPI protein levels in human cells. We have developed the assay to full HTS standards, and propose to screen the 225,000 member NIH MLSMR compound library to identify hit-to-lead compounds to develop into TPI Df small molecule therapies. We will validate hits in secondary assays for TPI stability and activity in TPI Df patient cells, prioritize them in a panel of in vitro toxicology and metabolism assays, examine structure activity relationships (SARs) of the lead compounds and substantially validate them in vivo using established Drosophila and mouse models that reflect the entire range of TPI Df disease severities. Overall, this project will discover initial therapies for development and test efficacy of our lead compounds in TPI Df models, including a newly validated mouse model.