Charleen T. Chu, MD, Duke University, PhD, Duke University - US grants

DESCRIPTION (applicant's abstract): Parkinson's disease is the most common debilitating movement disorder of the aging human population. The neurons that degenerate in Parkinson's disease are subject to increased oxidative stress because superoxide and other reactive species are generated during dopamine metabolism. 6-hydroxydopamine (6-OHDA) is a redox cycling dopamine analog, which can be targeted to selectively damage the nigrostriatal system that degenerates in Parkinson's disease. Phosphotyrosine signaling pathways activated by neuroprotective factors, such as brain derived neurotrophic factor and glial cell line-derived neurotrophic factor, are important for dopaminergic neuron function and survival. This proposal is designed to investigate the hypothesis that oxidant-mediated alterations in phosphotyrosine signaling contribute to degeneration of dopaminergic neurons in Parkinson's disease. Nitrotyrosine, a marker of oxidative stress involving peroxynitrite formation, is increased in both the 6-OHDA rodent model and in human Parkinsonian brain tissues. Peroxynitrite is formed from the reaction of superoxide with nitric oxide, implicating these free radicals in the pathogenesis of Parkinson's disease. In this proposal, mechanisms by which 6-OHDA, superoxide, and nitric oxide affect phosphotyrosine signaling cascades will be investigated using immortalized dopaminergic neuron lines and mice with genetically altered levels of extracellular superoxide dismutase. This comprehensive set of studies will yield important insights concerning mechanisms by which oxidative stress affects neurotrophic signaling in dopaminergic neurons, potentially contributing to development of combined antioxidant-neurotrophic factor therapies for Parkinson's disease.

[unreadable] DESCRIPTION (provided by applicant): Parkinson's disease is a debilitating movement disorder characterized by death of dopaminergic midbrain neurons. Mutations in PTEN-induced putative kinase 1 (PINK1) have been recently identified in familial parkinsonism, and some populations of early onset sporadic Parkinson's disease. In order to assess potential causative effects of mutations, it is necessary to first understand the regulation and function of wild type PINK1. One key unresolved issue is whether PINK1 is a functional kinase. This proposal is designed to investigate the feasibility of a novel research direction focused upon the regulation of PINK1 phosphorylation, localization, and function under conditions of stress elicited by parkinsonian neurotoxins. Our previous studies suggest that mitochondrially targeted kinase pathways play a central role in toxin-induced dopaminergic cell death. Thus, we hypothesize that PINK1 is a functional kinase that interacts with a set of cell death- regulatory kinases that localize to mitochondria. To address this hypothesis, we will determine if PINK1 shows kinase activity that is regulated by and participates in known mitochondrially targeted signaling pathways, using both cell free and culture systems. We will determine whether subcellular localization of PINK1 is regulated in neurotoxin models of dopaminergic cell death, and compare the effects of PINK1 and a kinase dead PINK1 mutant on mitochondrial cell death in dopaminergic cell lines and primary midbrain cultures. Completion of these studies will lead to a better understanding of the role of PINK1 during dopaminergic neuronal cell injury, and how it participates in signaling networks that influence whether these cells live or die. Following this exploratory/developmental phase, more extensive studies will be aimed at identifying downstream targets of PINK1 activation and the effects of disease-associated mutations on PINK1 regulation, as these may lead to novel therapeutic approaches. In addition, a group of immunochemical and molecular reagents will have been created that can facilitate the work of other investigators studying mechanisms of Parkinson's and related diseases. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Dopaminergic (DA) neurons are sensitive to oxidative insults and degenerate in age-related neurodegenerative diseases. A morphologic form of regulated cell death characterized by prominent autophagic vacuoles (AVs) has been identified in neurons. Autophagy is normally a highly regulated process sequestering cytoplasmic components for lysosomal degradation. However, dysregulated or excessive autophagy can be harmful to cells, producing a condition that can be conceptualized as "autophagic stress." Although AVs are observed in degenerating DA neurons in Parkinson disease and its in vitro and in vivo models, the role of autophagy in DA neuronal injury remains to be elucidated. Our studies indicate that oxidative neurotoxins elicit increased mitochondrial autophagy in DA neurons. Moreover, the regulation of this injury-induced autophagy is different from that of nutrient-deprivation systems. This proposal investigates the hypotheses that: autophagy contributes to neurite retraction and cell death in injured DA neurons, and that reactive oxygen species and MARK signals regulate injury-induced autophagy. We will use the complex I inhibitor MPP+ to produce mitochondria-targeted injury, and the redox cycling 6- hydroxydopamine to model generalized oxidative stress, comparing acute and chronic treatments. A combination of molecular, biochemical, live cell imaging and transgenic approaches will be applied to DA cell lines, primary midbrain cultures and mice to determine the role of autophagy in DA neurite retraction and cell death, and to study MAPK and oxidative phospholipid signals involved in its regulation. Completion of these studies will yield important insights into mechanisms by which autophagic responses regulate DA neurite degeneration and cell death during oxidative neuronal injuries. Relevance: Mitchondrial impairment and autophagic stress are prominent features of Parkinson/Lewy body disease. In contrast to physiologic conditions, inducing autophagy in the presence of dysregulating pathologic forces may promote cell death. A better understanding of mechanisms that contribute to autophagic stress will help focus future research efforts to restore balance to the system. Thus, studying the role and regulation of autophagic responses in oxidatively-injured neurons may enhance development of novel therapies applicable to age-related neurodegenerative diseases and other brain disorders involving oxidative stress. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Parkinson disease (PD) is a debilitating neurodegenerative disease for which there are no effective neuroprotective therapies. Converging data from post-mortem brain studies, oxidative toxin models, and PD genetics all implicate altered mitochondrial kinase networks in PD pathogenesis. Mutations in the mitochondrial PTEN-induced kinase 1 (PINK1) occur in familial and possibly sporadic PD. The candidate is an established and productive investigator in the signaling regulation of neuronal fate in culture and animal models of PD pathogenesis. The candidate's published and preliminary work implicate mitochondrial targeting of extracellular signal regulated protein kinase 2 (ERK2) and decreased PINK1 signaling in promoting dopaminergic neuronal cell death. Overexpression of wild type PINK1 confers neuroprotection in cell death models. Thus, identifying downstream targets of PINK1, and of mitochondrial targets of redox-activated ERK2, would represent important steps in understanding mechanisms that regulate neuronal survival and death. Identification of substrates for novel kinases such as PINK1, and of context specific ERK2 targets, can be accomplished using the powerful techniques of phospho-proteomics and mass spectroscopy. This proposal capitalizes on the strong cell signaling, proteomic/metabolomic, pharmaceutical, and neurodegeneration research environments at the University of Pittsburgh. The candidate's immediate goals are to obtain training in mass spectrometry and phospho-proteomics under the mentorship of Billy Day, Director of the Pittsburgh Proteomic Core Facility. Co-mentor Bruce Freeman, expert in free radical biochemistry, will provide complementary training in basic redox proteomics. In addition to short courses, seminars, and technical workshops, practical experience will be derived from a pilot project involving ATP-binding pocket mutagenesis and mass spectrometry to identify substrates of PINK1. Lay summary: This career enhancement award will provide an established physician-scientist studying neurodegenerative diseases with essential cross-training in highly specialized target identification technologies. Solidifying interdisciplinary collaborations will enable the principal investigator to more rapidly translate knowledge on factors that determine whether or not neurons can successfully adapt to disease promoting stresses into novel targets for designing neuroprotective PD therapies. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

The overall goal of this program project is to investigate mechanisms by which alterations in mitochondrial protein function(s) contribute to neuronal degeneration related to Parkinson's disease (PD). Thus, a central Neuropathology Core to examine alterations in subcellular protein distribution, post-translational modification, and association with neuronal injury/death markers in PD/Lewy body disease (LBD) patient brain tissues and related model systems forms an essential component interfacing with each of the individual projects. The Core will be directed by a practicing neuropathologist with expertise in Lewy body diseases and subcellular protein trafficking in PD models, and draw upon extensive resources of the University of Pittsburgh Brain Bank. The neuropathology core will apply multi-label fluorescence or biochemical techniques to study protein localization, phosphorylation and macromolecular interactions in appropriately fixed or frozen tissues, in defining selection criteria to obtain pertinent regions of brain from diseased and matched control subjects, and in standardizing quantitative image analysis protocols for experimental material and human brain tissue studies across the projects. The core personnel have ongoing, productive collaborations with the individual project leaders, as reflected in shared manuscripts and preliminary data. Given the success of these interactions, we expect a continued expansion of translational efforts between disease models and human pathology.

PROJECT SUMMARY/ABSTRACT. In recent years, mutations in PTEN-induced kinase 1 (PINK1) have been associated with autosomal recessive Parkinsonism. Early studies have uniformly shown a neuroprotective role for PINK1, indicating that studying this familial form of PD may offer valuable insights into potential therapeutic strategies. Dysregulation of mitochondria and autophagy are both centrally implicated in toxin, genetic and environmental approaches to modeling PD. Our preliminary data implicate PINK1 in the upstream regulation of autophagy and in maintenance of mitochondrial interconnectivity, neurite health and synaptic function. Using differentiated neuronal cell lines and primary neuron cultures from control and PINK1 knockout mice, we will determine mechanisms by which PINK1 protects from toxin and genetic PD models, focusing on: 1) the role of subcellular localization in PINK1-mediated neuroprotection, 2) mechanisms regulating PINK1 localization, and 3) identification and characterization of downstream components of the PINK1 signaling pathway, using both candidate and nonbiased proteomic approaches. Achieving the goals of this project to obtain a better understanding of PINK1 regulation, subcellular compartmentalization and downstream pathways will provide insight into new therapeutic strategies to reduce neuronal dysfunction and cell death in PD.

DESCRIPTION (provided by applicant): The maintenance of well-functioning mitochondria plays a key role in neuronal health. In the previous project period, we found that neuronal injury in several neurotoxin and genetic models of parkinsonian neurodegeneration converged on eliciting increased mitochondrial turnover by autophagy (mitophagy). While mitophagy in some models is neuroprotective, in other models, inhibiting autophagy reduces neurite retraction and cell death. We hypothesize that the capacity to replace damaged/degraded mitochondria through mitochondrial biogenesis is important in determining survival-death outcomes in this context. Preliminary data indicate a key role for extracellular signal-regulated protein kinase 2 (ERK2), which shows an altered mitochondrial distribution in Parkinson's disease midbrain neurons, in regulating both mitophagy and mitochondrial biogenesis. We will utilize differentiated neuroblastoma cells, primary embryonic mouse neurons and in vivo mouse models to study the mechanism(s) leading to the observed decreases in mitochondrial content and function, study the role of phosphorylation in regulating biogenesis, and determine the neuroprotective potential for strategies to modulate mitochondrial content in toxin and dominant genetic models of Parkinson's disease.

Dendrite regulation by the mitochondrial kinase PINK1: Implications for PD/LBD PROJECT SUMMARY. Synaptic loss is a major structural correlate of dementia, and reduced spine density is observed in the Parkinson's disease (PD)-Lewy body dementia (LBD) disease spectrum. Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) cause early-onset PD and PD with dementia (PDD). Heterozygous carriers also exhibit cognitive-executive dysfunction and limbic-cortical degeneration. As PINK1 is neuroprotective in a wide range of genetic and toxin-based models of neurodegeneration, studying its function in neurons may offer insights into potential therapeutic strategies. Endogenous PINK1 exists in both mitochondrial and cytosolic compartments. Our prior studies show that these pools of PINK1 play divergent roles in regulating mitochondrial fission-fusion, mitophagy, calcium homeostasis and dendritic morphogenesis. Moreover, loss of PINK1 results in dendritic simplification in cortical and midbrain neurons. We hypothesize that PINK1 interacts with cytosolic targets to regulate neuron differentiation and synaptodendritic complexity. Using mass spectrometry, we identified novel PINK1-interacting proteins, which preliminary studies implicate in neurite extension or neuronal transport. We will study the role of these novel PINK1 interactions in regulating dendritogenesis and mitochondrial transport into dendrites using primary cortical and midbrain neurons, differentiated neuronal cell lines and PINK1 knockout and control mice. The potential role of phosphorylation and the impact of PD-linked mutations on these neuron-specialized functions of PINK1 will be analyzed. The neuroprotective potential of upregulating downstream pathway components will be tested in vitro and in Pink1-/- mice. A better understanding of novel PINK1-driven mechanisms that act to prevent dendritic simplification may yield valuable insights for neuroprotection in the PD-LBD disease spectrum.

DESCRIPTION (provided by applicant): The maintenance of well-functioning mitochondria plays a key role in neuronal health. In the previous project period, we found that neuronal injury in several neurotoxin and genetic models of parkinsonian neurodegeneration converged on eliciting increased mitochondrial turnover by autophagy (mitophagy). While mitophagy in some models is neuroprotective, in other models, inhibiting autophagy reduces neurite retraction and cell death. We hypothesize that the capacity to replace damaged/degraded mitochondria through mitochondrial biogenesis is important in determining survival-death outcomes in this context. Preliminary data indicate a key role for extracellular signal-regulated protein kinase 2 (ERK2), which shows an altered mitochondrial distribution in Parkinson's disease midbrain neurons, in regulating both mitophagy and mitochondrial biogenesis. We will utilize differentiated neuroblastoma cells, primary embryonic mouse neurons and in vivo mouse models to study the mechanism(s) leading to the observed decreases in mitochondrial content and function, study the role of phosphorylation in regulating biogenesis, and determine the neuroprotective potential for strategies to modulate mitochondrial content in toxin and dominant genetic models of Parkinson's disease.

Synaptic loss is a major structural correlate of dementia, and reduced spine density is observed in the Parkinson?s disease (PD)-Lewy body dementia (LBD) disease spectrum. Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) cause early-onset PD and PD with dementia (PDD). Heterozygous carriers also exhibit cognitive-executive dysfunction and limbic- cortical degeneration. As PINK1 is neuroprotective in a wide range of genetic and toxin-based models of neurodegeneration, studying its function in neurons may offer insights into potential therapeutic strategies. Endogenous PINK1 exists in both mitochondrial and cytosolic compartments. Our prior studies show that these pools of PINK1 play divergent roles in regulating mitochondrial fission-fusion, mitophagy, calcium homeostasis and dendritic morphogenesis. Moreover, loss of PINK1 results in dendritic simplification in cortical and midbrain neurons. We hypothesize that PINK1 interacts with cytosolic targets to regulate neuron differentiation and synaptodendritic complexity. Using mass spectrometry, we identified novel PINK1- interacting proteins, which preliminary studies implicate in neurite extension or neuronal transport. We will study the role of these novel PINK1 interactions in regulating dendritogenesis and mitochondrial transport into dendrites using primary cortical and midbrain neurons, differentiated neuronal cell lines and PINK1 knockout and control mice. The potential role of phosphorylation and the impact of PD-linked mutations on these neuron-specialized functions of PINK1 will be analyzed. The neuroprotective potential of upregulating downstream pathway components will be tested in vitro and in Pink1-/- mice. A better understanding of novel PINK1-driven mechanisms that act to prevent dendritic simplification may yield valuable insights for neuroprotection in the PD-LBD disease spectrum. This supplement enhances the goals of the parent R01 to study mechanisms by which PINK1 regulates mitochondrial transport in neurons, while providing mentorship and rigorous training opportunities for a Latino graduate student aspiring to a future independent career studying neurological diseases.! ! !

Dendritic simplification and synaptic loss represent major structural correlates of dementia in Alzheimer?s disease (AD) and frontotemporal dementia (FTD). Mutations in the microtubule-associated protein tau are a major cause of FTD and increase the risk of developing AD. Mutations in valosin-containing protein (VCP) have also been linked to familial FTD. In the prior project period, we discovered that dendritic calcium dyshomeostasis, dysregulated mitophagy and altered protein phosphorylation contribute to dendritic shrinkage in several models of neurodegeneration. We also discovered a novel interaction between VCP and the neuroprotective kinase PTEN-induced kinase 1 (PINK1). VCP is a multifunctional protein implicated in protein degradation, vesicular transport and Golgi remodeling, and these functions are mediated by distinct VCP cofactors. Based on preliminary data, we hypothesize that PINK1 interacts with VCP to prevent tau- mediated dendritic shrinkage. We will utilize primary rodent cortical neurons and human iPSC-derived neurons to study quality control mechanisms that contribute to neuroprotection against tau-mediated degeneration of dendritic arbors and spines. We will also assess the neuroprotective potential of upregulating PINK1 signaling in vitro and in vivo. Completion of these studies will reveal how proteins implicated in different neurodegenerative diseases function together to protect against tau-mediated neurodegeneration.