Aaron Bowman - US grants

[unreadable] DESCRIPTION (provided by applicant) [unreadable] [unreadable] The long-term objective of the proposed work is to understand how environmental and genetic factors interact to influence selective neuropathology. The work contained within this proposal focuses on the influence of manganese (Mn) exposure on the pathophysiology of Huntington's disease (HD). Mn over-exposure has been associated with changes in iron homeostasis and energy metabolism, and has been shown to promote the aggregation of intrinsically amyloidogenic proteins. Furthermore, a major site of Mn accumulation in the brain is the corpus striatum, which contains the neurons most vulnerable in HD. The chronic neurotoxic stress rendered by the mutant HD gene has been associated with alterations in iron homeostasis, deficits in cellular energy metabolism, and accumulation of the disease protein into amyloid-like inclusions. These similarities in the pathophysiology of HD and Mn neurotoxicity suggest a potential for Mn exposure to modulate HD neuropathology. Using pilot project resources provided by the National Institute of Environmental Health Sciences (NIEHS) Core Center in Molecular Toxicology at Vanderbilt University, the investigators tested the influence of increased Mn exposure on a striatal cell model of HD. These pilot experiments revealed a surprising and exciting result, that mutant HD striatal cells are resistant to Mn toxicity and pathophysiologically relevant exposures to Mn suppress mutant HD phenotypes. This proposal will utilize cellular and mouse models of disease to examine the molecular basis of this gene-environment neuroprotective interaction and evaluate the potential of Mn exposure to modulate HD pathogenesis. These studies are organized around three specific aims. In the first of these the investigators will define the contribution of specific HD protein domains and specific cellular mediators of Mn action to the Mn-HD gene-environment interaction by functional domain mapping and evaluating other metals with similar neurotoxic properties. In the second aim the investigators will determine if Mn ions alter the conformational or functional properties of the HD protein by biochemical and biophysical protein assays utilizing cellular and animal models of HD. Then, in the third aim the investigators will evaluate known pathological endpoints of Mn toxicity and HD neuropathology to elucidate the physiological processes that underlie the Mn-HD interaction in vivo. These specific aims are aligned with the mission of the NIEHS in that they examine the impact of a specific environmental toxicant on the pathophysiological processes of human disease. Finally, by exploring a gene-environment interface that moderates the onset and progression of HD, this study seeks to reveal mechanistic detail for how convergent genetic and environmental factors can enhance or suppress disease. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Manganese (Mn) is a potent neurotoxin. We hypothesize that PARK2, a strong Parkinson's disease (PD) genetic risk factor, alters neuronal vulnerability to modifiers of cellular Mn status, particularly at the level of mitochondrial dysfunction and oxidative stress. The long-term goal of this research is to elucidate the basis of Mn-induced neurotoxicity and to identify mechanistic-based neuroprotective strategies to mitigate human Mn exposure risk. Our approach will utilize a novel high-throughput assay of intracellular Mn levels to identify small molecule modifiers of cellular Mn status and neurotoxicity. Genetic modifiers of Mn transport and toxicity will be defined and translational studies of existing and newly identified genetic and small molecule modifiers of Mn toxicity will be performed utilizing a primary human neuronal model system based upon human induced pluripotent stem cell (hiPSC) technology. Aim 1 will identify lead compounds that alter neuronal Mn transport and toxicity in vitro using striatal and mesencephalic murine neuronal cell lines and in vivo using C. elegans. Aim 2 will delineate functional pathways that regulate Mn transport and toxicity in vivo and in vitro. Specific Aim 3 will test the hypothesis that human neuronal models of PD exhibit increased sensitivity to perturbations of cellular Mn status. These specific aims hold the promise of delineating common initiator signals for the modulation of Mn neurotoxicity, shedding light on mechanisms and susceptibility associated with exposure to this metal. This dual-PI proposal is bolstered by its use of innovative state-of-the-art complimentary approaches in diverse model systems.

DESCRIPTION (provided by applicant): Huntington Disease (HD) is a neurodegenerative disorder pathologically characterized by selective degeneration of neurons within the striatum, cortex and hypothalamus. HD is caused by a CAG repeat expansion within the HTT gene, with longer repeats being strongly associated with earlier age-of-onset. Although repeat length explains over half of the variability in age of onset, a landmark genetic study attributed the majority of residual variability to unknown environmental factors. Metal ions with neurotoxic properties are strong candidates for environmental agents that may modulate selective neurodegenerative process like HD because, (1) the differential accumulation of various metals across neuronal subtypes, (2) the similarities between metal ion cytotoxicity and cellular pathways of neurodegeneration, and (3) our research in the previous funding cycle demonstrating altered vulnerability in mouse models of HD to both manganese and cadmium. The long-term goal of this research program is to reveal the pathogenic mechanisms underlying gene-environment interactions in neurodegenerative disease, focusing on HD given its clearly defined genetic etiology, to inform environmental health strategies to delay disease onset or slow the progression of disease. Our highly innovative approach combines (a) a novel high-throughput method to quantify cellular Mn status, (b) a state-of-the-art high throughput screen (HTS) facility at the Vanderbilt Institute of Chemical Biology (VICB), and (c) the clinical relevance of a patient-specific neuronal model system based on human induced pluripotent stem cell (hiPSC) technology. Aim 1 will test the hypothesis that an HD striatal Mn handling deficit discovered in the previous funding cycle will enable a HTS to find small molecules that mitigate the actions of HD environmental risk factors. Aim 2 will test the hypothesis that human striatal neuroprogenitors (NPs) from HD patients have increased sensitivity to non-cytotoxic levels of metal toxicants impinging upon specific stress response pathways. Aim 3 will test the clinical potential of small molecule modifiers of environmental risk factors in HD and whether the magnitude of HD-specific toxicant vulnerability will correlate by patient with established disease-modifiers such as neural lineage specificity, CAG-repeat length and clinical variation in age-of-onset. These specific aims will reveal disease-relevant environmental stress responses and identify small molecules to mitigate vulnerabilities and restore neuronal homeostasis in HD. Furthermore, discovery of toxicant interactions and patient-specific responses may inform environmental health strategies to delay disease onset or slow the progression of HD using a personalized medicine approach.

DESCRIPTION (provided by applicant) Funds are requested to support eight pre-doctoral (Ph.D. candidates) and six postdoctoral trainees in the Training Program in Environmental Toxicology, the long-standing training component of the Center in Molecular Toxicology at Vanderbilt University. This interdisciplinary program provides research career training in molecular aspects of toxicology related to environmental health. Because the field is inherently interdisciplinary, research training in the program spans chemistry, biochemistry, chemical biology, structural biology, analytical technology, functional genomics, pathogen-host interactions, disease pathology, and exposure science. The faculty preceptors have appointments in the departments of Biochemistry, Biological Sciences, Biomedical Informatics, Chemistry, Medicine, Neurology, Pathology/Microbiology/Immunology, Pediatrics, and Pharmacology, all of whom train doctoral students and postdoctoral fellows. Training is achieved through basic and specialized coursework, research rotations, dissertation research, and participation in seminars, journal clubs, and joint research meetings. A distinctive feature of the Program is hands-on training on diverse technology platforms through a highly developed and open system of research facility cores at Vanderbilt. Graduate students are recruited to the Department of Chemistry through departmental mechanisms, with assistance from the Center in Molecular Toxicology. In the medical school departments, graduate students are initially recruited into either the Interdisciplinary Graduate Program in Biomedical and Biological Sciences or the Quantitative and Chemical Biology Program, where they spend the first 9 months in a common core curriculum and do laboratory rotations. Graduate students are supported for the first year by these programs. Students then are recruited into the Training Program in Environmental Toxicology from these first-year pools, and training program support begins in the second year. Both pre-doctoral and postdoctoral trainees are selected by the Training Program Advisory Committee, with guidelines to ensure distribution of trainees and monitoring of progress. The list of preceptors includes 18 faculty members who are all Investigators in the Center in Molecular Toxicology. Major research areas in the Center include oxidative damage, DNA damage and repair, maintenance of genomic integrity, enzymatic biotransformation and reactions of electrophiles, neurotoxicology, respiratory disease pathophysiology, systems biology, and pathogen-host interactions. Graduates from the program have been highly successful in academia, industry, and other professional settings and include leaders in the field.

The goal of this proposal is to establish in vitro tissue chip models of the closely related neurological disorders tuberous sclerosis complex (TSC) epilepsy, DEPDC5-associated epilepsy, and their associated cardiac dysfunction. The proposed research leverages emerging bioengineering technology for microphysiological systems developed at the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) with human induced pluripotent stem cell tools in regular use at Vanderbilt University Medical Center to ask probing questions about genetic disorders that afflict the heart and brain and about the drugs to treat them. The VIIBRE neurovascular unit (NVU)/blood-brain barrier and cardiac I-Wire organ-on-chip models will test the hypothesis that mTORC1 and mTORC2 signaling differentially affect neural and cardiac dysfunction in TSC- and DEPDC5-associated epilepsy. The primary and shared abnormality in patients with TSC and DEPDC5- associated epilepsy is dysregulation of the mTOR kinase complex 1 (mTORC1) signaling pathway. TSC also has abnormalities in mTORC2 signaling not seen in DEPDC5-associated epilepsy. A focus on mTOR signaling in these human mTORopathies has several advantages. First, rapamycin and related compounds are FDA- approved mTORC1 inhibitors and have been shown to have efficacy in some aspects of the disease manifestations of TSC. Second, TSC- and DEPDC5-associated epilepsy are both associated with neural and cardiac dysfunction. Third, the role for compensatory or differential mTORC2 activity is unclear and controversial. For patients with TSC, drugs targeting the mTORC1 signaling pathway have been associated with shrinkage of brain tumors, reduced seizures, and improved cardiac function. Thus, drug development for this group of diseases is well suited for study using both the NVU and I-Wire cardiac-tissue chips. In its first two years, the project will develop the NVU and I-Wire disease models, aimed at refining the TSC and DEPDC5 NVU model; applying the I-Wire model to TSC and DEPDC5 cardiomyocytes; and validating outcome methodologies in control and patient-derived NVU and I-Wire chips. The next three years aim to evaluate, for biomarker identification in control, TSC, and DEPDC5 NVU and I-Wire chips, changes in mTORC1 and mTORC2 signaling, protein markers of cellular health and toxicity, metabolites, functional measures and electrophysiological activity; and, use ion mobility-mass spectrometry to evaluate NVU and I-Wire outcome measures plus drug metabolites after treatment with mTORC1 inhibitor rapamycin, the seizure drug vigabatrin, and novel pre-clinical mTOR drug candidates. The NVU and I-Wire will assess the efficacy and toxicity of these agents and define TSC/DEPDC5 shared vs disease-specific effects. With this organ-on-chip/human induced pluripotent stem cell platform, it will be possible to address currently confounding mechanisms of pathogenesis, identify new disease biomarkers, quantify how drugs cross the normal and diseased blood-brain barrier, and ultimately develop effective therapies and hence enable bench-to-bedside translation.

SUMMARY There is a fundamental gap in the knowledge base about how chronic manganese exposures impacts develop- ment of Alzheimer?s disease. The neurotoxic effects of manganese poisoning are known, as well as the motor impairments that are its behavioral sequelae. However, chronic lower-level exposures have not been studied. The neuropathology of Alzheimer?s disease develops over decades prior to onset of severe cognitive and be- havioral change (dementia) and thus its development is particularly susceptible to influence from environmental factors. Manganese represents an environmental toxin with high likelihood of importance since exposure occurs through multiple sources (contaminated water, food, inhalation from pollution and industrial complexes). Further, exposure directly targets many of the primary mechanisms involved in Alzheimer?s disease pathology: ?-amyloid accumulation, oxidative stress and glial changes relating to neuroinflammation. Our central hypothesis is that Chronic elevated manganese (Mn) exposure drives cognitive decline through impaired glutamate homeostasis. Our long-term objectives are to isolate the direct link(s) between Mn and cognitive decline by demonstrating how chronic Mn exposure affects altered glutamate clearance and other pathologies to a greater extent in mouse and human stem cell models of AD than in controls. We will do this by: (1) Demonstrating the extent to which chronic Mn exposure accelerates AD neuropathology. Following 3 months treatment with Mn to significantly elevate brain Mn we will assess multiple markers of AD-related neuropathology, oxidative stress and neuroin- flammation at the gene, protein and cellular level incorporating direct hypothesis testing and hypothesis gener- ating approaches. Changes will be assessed prior to- and after onset of significant ?-amyloid accumulation (6- and 12 months of age), and in ?-amyloid positive (APP/PSEN1, familial AD model) and negative mice (APOE4/TREM2, sporadic AD model; and wild-type mice). (2) Demonstrating the extent to which chronic Mn exposure impacts cognitive decline. We will assess learning and memory at the two age points using a com- prehensive battery of behavioral tests for cognitive and motor changes. We will directly assess the potential for Mn to impact the molecular basis of memory, synaptic strengthening through long term potentiation. Human stem cell models will be utilized to validate these findings. (3) Establishing the role of brain Mn levels in synaptic glutamate homeostasis. We will address the hypothesis that Mn directly impacts synaptic glutamate homeostasis through primary cell culture and stem cell models and assess glutamate uptake and release. We will functionally test the glutamatergic system by electrophysiological recordings. Finally we will utilize GLT-1 knockout mice to further probe the role of GLT-1 in particular in this relationship. Together these data will confirm the role of chronic Mn exposure in AD neuropathology and cognitive decline, and specifically address its impact on glutamatergic dyshomeostasis. Understanding these mechanisms will highlight an under-studied role for al- tered Mn handling in Alzheimer?s disease, and provide a new target for disease prevention and interventions.