Eva L. Feldman - US grants

Insulin, insulin growth factor-I (IGF-I) and insulin growth factor-II (IGF-11) are known to support neuronal survival as well as stimulate cell proliferation and vigorous neuritic outgrowth. While the concept has emerged that insulin and the insulin-like growth factors are neuronotrophic factors, where in the nervous system the factors are produced and what transmembrane signal they employ remain unknown. We plan to use immunohistochemistry to determine the distribution of IGF-I and II in normal and regenerating rat sciatic nerve. Later, IGF-I and II production sites will be localized with in situ hybridization techniques. In vitro specific IGF-I and Il receptor binding sites will be determined in a model system, the SH-SY5Y neuroblastoma clone, using iodinated ligands, competitive binding curves and affinity crosslinking. IGF-I and Il mRNA will be measured in the neuroblastomas as well as a defined Schwann cell line. We believe the transmembrane signal of the insulin-like growth factors may be mediated through the activity of phosphoinositide (PI) turnover. We have preliminary results which show an IGF-II stimulation of PI turnover in the SH-SY5Y neuroblastoma cells. The kinetics of IGF-I and II stimulated formation of 3H-inositol phosphates will be measured along with a comparison between receptor occupancy and PI turnover. Intracellular calcium and protein kinase C activity will be assayed. Our long term goal is to understand how the insulin-like growth factors regulate growth in the nervous system and to bridge the gap between receptor activation, signal induction and neurite outgrowth.

Carcinogenesis is a multistep process (1). Cellular transformation can result from interactions between oncogenes, growth factors, growth factor receptors and growth suppressor genes (2). Autocrine production of growth factors, secondary to a loss of tumor growth suppressor gene activity, is one mechanism which allows malignant cells to maintain an unregulated proliferative state. One candidate growth factor, insulin-like growth factor II (IGF-II), can mediate autocrine growth of several malignancies (3,4), including Wilms tumor (5). Indeed, research on Wilms tumor has given insight to a potential mechanism underlying constitutive IGF-II production. The major fetal IGF-II promoter contains multiple binding domains for wt1, a zinc finger protein and member of the family of tumor growth suppressor genes (6). wt1, present in urogenital tissues (7), is a potent repressor of IGF-II transcription (8). Loss of IGF-II repression secondary to wt1 dysfunction is associated with the development of Wilms tumor (9,10). The objective of this proposal is to examine the role of IGF-II and potential IGF-II dysfunction in the growth regulation of a different cancer: human neuroblastoma (NBL). NBL is the second most common solid tumor in childhood (11) and accounts for 10% of all juvenile cancer (12). Histopathological examination of tumors reveals multiple neoplastic neural crest cell types, including neuroblasts, melanocytes, glial cells and chrondrocytes (13). Research on NBL has focused on the development of cell lines established from different NBL tumors (14). The SK-N-SH NBL cell line was derived from a 4 year old girl one month before her death (15). Analysis of clonal sublines of SK-N-SH reveal two distinct cell types: the SH-SY5Y clone consisting of N cells, or presumptive neuroblasts with small round cell bodies and neuritic processes (16) and the SHEP clone comprised of flattened substrate adherent cells with either glial, epithelial or melanocyte-like properties (16). SH-SY5Y cells exhibit serum independent growth, multiple in soft agar and form tumors in nude mice (16). In contrast cells, SHEP cells require serum and are unable to multiple in agar or form murine tumors(16). The current proposal focuses on the autocrine production of insulin-like growth factor-II (IGF-II) by NBL cells as one event in carcinogenesis . In this model, N cells (SH-SY5Y cells) produce IGF-II which acts as an autocrine N cell growth factor as well as a paracrine growth factor for S cells (SHEP cells), which do not produce IGF-II. These putative autocrine and paracrine loops are mediated via the type I IGF receptor and represent unregulated production of IGF-II secondary to loss of tumor growth suppressor gene activity. This proposal has three specific aims: 1. Determine if IGF-II, acting via the type I IGF receptor, can support the growth requirements of SHEP cells. 2. Determine whether deregulated expression of IGF-II alters the phenotype of SHEP cells. 3. Identify potential nervous system specific growth suppressor genes. Results gained from these studies are of definite clinical importance. Therapies aimed at interrupting the NBL IGF-II autocrine and paracrine loops may stop growth in all NBL cell types (17). Strategies include inhibiting both the ligand and receptor, using neutralizing antibodies, blocking antibodies, modified oligonucleotides or compounds which indirectly block IGF-II action, such as suramin (18). Clearly, anti-growth factor therapy, targeted at specific genes, has both theoretical and practical appeal in the treatment of NBL.

DESCRIPTION (provided by applicant): The most common complication of diabetes is neuropathy, which occurs, in approximately 60 percent of all diabetic patients. Our work has generated a new theory: that glucose-mediated apoptosis contributes to the development of diabetic neuropathy and that interrupting the death pathway with insulin-like growth factor-I (IGF-I) could afford a new means of therapy. This application aims to understand how glucose kills and IGF-I rescues neurons in both cell culture and animal models of diabetic neuropathy. We speculate high glucose promotes the formation of reactive oxygen species (ROS). Mt membrane depolarization (MMD) then occurs in direct response to glucose, ROS or dimerization of the anti-apoptotic protein Bcl-xL with the pro-apoptotic proteins Bax/Bak. In each case, there is release of Mt cytochrome c into the cytosol and activation of the caspase cascade. It is unknown if caspases can be directly cleaved by ROS, or which initiator (-8 or -9) or downstream effector (-3 through -7) caspases are involved in the cascade. IGF-I may interrupt cell death at one or more points in the pathway. Human neuroblastoma cells, primary sensory neurons and mouse models are used to test this model. We have 4 aims: 1. Characterize ROS formation and the point of IGF-I protection following glucose exposure 2. Characterize the roles of Bcl proteins and determine the point of IGF-I protection following glucose exposure 3. Characterize the caspase cascade and determine the point of IGF-I protection following glucose exposure 4. Use genetically altered mice to characterize the death pathway in clinical diabetic neuropathy and determine the point of IGF-I protection

DESCRIPTION: (adapted from the investigator's abstract) Cancer is the major cause of death in children between the ages of 1 and 15 years. Neuroblastoma (NBL), the second most common solid tumor in childhood, accounts for 10percent of all juvenile cancer. Hematogenous dissemination of NBL with local invasion into bone marrow, leptomeninges and other organs is largely refractory to conventional radiation and chemotherapy. They are interested in the role of insulin-like growth factor (IGF)-I and II and the type I IGF receptor (IGF-IR) in the carcinogenic and metastatic potential of NBL. In the work, they utilize cell lines established from different human NBL tumors. The SH-SY5& NBL line was subcloned from a tumor of a 4 year old girl one month prior to her death. They find that SH-SY5Y cells secrete IGF-II which acts via IGF-IR to promote both autocrine growth and resistance to programed cell death. The most recent studies demonstrate IGF-I and II are also potent NBL motility factors. IGF treatment of SH-SY5Y cells results in redistribution of the actin cytoskeleton with the formation of rapidly moving membrane reffles. Ruffling is followed by protrusion of lamellipodia which adhere to specific extracellular matrix molecules and form stable adhesion foci. They have developed a novel hypothesis centered on understanding the mechanism which underlies NBL motility. They believe IGFs bind to IGF-IR, stimulating receptor autophosphorylation, insulin receptor substrate-1 (IRS-1) phosphorylation, and activation of phsophatidylinositol-3 kinase (PI-3K). The results in the activation of a GTP-binding protein rac which, in turn, promotes actin polymerization followed by membrane ruffling and protrusion of the leading tumor edge. Protruding membranes form lamellipodia which adhere to the extracellular matrix and are stabilized by focal adhesions. Repetition of the cycle coupled with release of old adhesions allows continued lamellipodial advance and NBL migration. The purpose of the current proposal is to test the initial components of the hypothesis. They have 4 aims: 1) Characterize the morphological effects of IGF-I on NBL; 2) Determine the role of IGF-IR signaling cascades in IGF-I mediated morphological changes; 3) Determine the role of PI-3K in IGF-I mediated morphological changes and rac activation; 4) Examine the role of rac in IGF-I mediated membrane ruffling, lamellipodial formation and cellular motility. Results gained from these studies are of definite clinical importance. Therapies aimed at interrupting IGF mediated NBL motility may alter NBL metastatic potential. Strategies include inhibiting both the ligand and receptor, using neutralizing antibodies, blocking antibodies, or medified oligonucleotides. Clearly, anti-growth factory therapy, targeted at specific genes, has both theoretical and practical appear in the treatment of NBL.

DESCRIPTION (provided by applicant): This training program in basic and clinical neuroscience has been funded for the past 24 years, and involves the Departments of Neurology, Pediatrics, Neurosurgery, Internal Medicine and Anesthesia at the University of Michigan (U-M) and the Ann Arbor VA Medical Centers. 35,000 square feet of laboratory space are available for basic research, and 30 faculty members, most of whom are both clinical neurologists and basic or clinician scientists, serve as preceptors. We train medical and graduate students, physicians, and basic scientists to conduct basic and clinical neuroscience research. We offer basic science training in cell and molecular neuroscience, neurochemistry, neurophysiology, neuropharmacology, neurogenetics, and clinical science training in the dementias, neuromuscular diseases, movement disorders, sleep disorders and neurogenetics. Clinical training involves evaluation of clinical disorders, clinical-pathological correlations, neuropharmacological interventions, anatomical and functional imaging, including positron emission tomography studies, epidemiological studies, and experimental therapeutics. Training is in the individual laboratory or clinical program but is supplemented by interdisciplinary and collaborative project meetings, seminars and appropriate course work. All trainees in clinical science programs are required to enroll in the School of Public Health's Masters Program in Clinical Research Design and Statistical Analysis. We propose to increase our trainee number from 4 to 5 postdoctoral fellows per year. Postdoctoral trainees are neurologists or physicians in related fields who have completed clinical training and select a basic or clinical research career, and biomedical scientists who seek training in basic neuroscience research. Trainees are selected competitively by the program's Executive Committee. While the Executive Committee monitors progress of trainees, individual preceptors are responsible for guiding their trainees in generating research proposals, supervising the trainees'work, and evaluating the trainees'performance. Efforts are made to recruit qualified women and minority students for training at all levels.

insulin dependent diabetes mellitus; person with disability

DESCRIPTION (provided by applicant): In response to the request for applications DK-05-011, entitled Animal Models of Diabetic Complications Consortium (AMDCC), the Investigators from the current AMDCC Neuropathy Phenotyping Core are proposing to develop 2 new mouse models of diabetic neuropathy (DN) targeting the biochemical pathways of oxidative stress. Our general strategic approach is to accelerate glucose-mediated oxidative injury in neurons in genetic models of type 2 diabetes. While many gene products participate in this process, we will concentrate on targeting 2 enzymes involved in superoxide detoxification: mitochondrial superoxide dismutase 2 (SOD2) and catalase. Our initial approach will concentrate on developing 2 Cre-loxP models on a susceptible genetic background. In parallel, we propose 2 hypothesis-driven specific aims for discovering the basic pathophysiologic mechanisms underlying DN. Aim 1 will test the hypothesis that decreased catalase activity in sensory neurons will make these neurons more susceptible to glucose-mediated injury. Aim 2 will test the hypothesis that animal models with DN have morphological and biochemical markers of increased oxidative stress in the peripheral nervous system. Information gained from this application will lead to new insights into the pathogenesis of DN and allow for the development of more relevant murine models of this disabling complication. Relevance to Public Health: 20 million Americans are diabetic and the incidence is increasing by 5% each year. Although DN is a common and highly morbid condition, there are no treatments for DN outside of control of the diabetic condition itself. Our studies will identify cellular targets for treatment of DN and have the potential to benefit all patients with diabetes.

DESCRIPTION (provided by applicant): Experimental approaches to improve our understanding and treatment of major diabetic complications have focused on single mechanisms or pathways and resulted in the identification of specific mechanisms that drive diabetic damage. With the recent emergence of genome-wide profiling capatiilities and comprehensive data integration strategies, biomedical research is at a point where it can move toward a more holistic view of tissue responses to complex chronic diseases. This is of particular relevance to diabetic end-organ damage since multiple mechanisms converge to slowly alter the cellular milieu in target tissues in diabetes, mandating the integration of separate pathways to elucidate the complex pattern of responses in the treatment or prevention of diabetic complications. Indeed, therapies that have worked best to prevent progression of diabetic nephropathy (DN) and polyneuropathy (DPN) affect multiple pathways and mechanisms, whereas those that target a single, "critical" pathway have often yielded disappointing results. Our team of scientists will use a systems biology approach to achieve 3 goals, to: 1) efficiently identify the essential cellular responses that lead to DN and DPN, 2) identify those responses that are most amenable to conventional and novel therapies, and 3) discover biomarkers for the critical cellular alterations that lead to complications and respond to effective therapies. Our strategy relies on information-rich sequential and reciprocal transcriptomic, protein and metabolite comparisons between humans with DN and DPN and the best extant murine models of these complications. Our hypothesis is that a complex network of responses, including but not limited to those altered by oxidant stress, leads to the onset and progression of diabetic microvascular complications. These critical responses will be identified by performing genome-wide RNA and metabolite profiles from kidney and nerve of humans with DN and DPN. The expression data sets of human end-organ damage from untreated and treated animals will be compared to data sets obtained from kidney and nerve from murine models with DN and DPN. Three different treatment paradigms known to ameliorate DN and DPN will be used as independent tools in the mouse models to identify new critical responses that lead to complications and are responsible for effective treatment in humans. This reciprocal cross-species approach will identify candidate pathways and molecules whose regulation alters disease progression. Finally, we will return to the murine models of DN and DPN to discover new biomarkers that will be useful in the diagnosis and therapeutic management of human DN and DPN. PUBLIC HEALTH RELEVANCE: Two of the most devastating complications of diabetes are kidney disease and nerve disease which both result in increased illness and death. The multifactorial causes of these complications remain poorly understood and treatments are limited. Therefore, we have devised a team approach to use "systems biology" to study diabetic humans with these complications, with the help of mouse models, and to identify the critical molecular responses that cause kidney and nerve damage and that can be reversed by therapy.

DESCRIPTION (provided by applicant): Challenge Area: (03) Biomarker Discovery and Validation Challenge Topic: Discovery of biomarkers for disease risk, progression or response to therapy in diseases of interest to NIDDK. 03-DK-101 Title: Biomarkers in Diabetic Neuropathy Twenty million Americans have diabetes and the incidence is increasing by 5% per year. The most common complication of diabetes is diabetic neuropathy (DN). Current methods used to confirm DN and measure its progression include nerve conduction studies, quantitative sensory measures and decreased sural nerve myelinated fiber density (MFD). The identification of DN biomarkers would greatly enhance our understanding of early events in this complication and could be used to predict its development and rate of progression. No biomarkers have been established for DN leaving this complication to develop unchecked. We hypothesize that a complex network of metabolic changes in type 2 diabetes may predict the onset and progression of DN. Bioinformatics protocols applied to metabolic, neuroanatomical and neurophysiology data from a clinical trial of DN indicate that dyslipidemia, specifically elevated triglyceride levels, is associated with rapid progression of DN. The current proposal employs microarray analyses to examine differentially expressed genes involved in lipid metabolism in both human sural nerve samples and in peripheral nerves from a relevant animal model, the BKS-db/db mouse. Our membership in the National Center for Integrated Biomedical Informatics (NCIBI) at the University of Michigan provides us with a high-powered computing environment and the expertise for computationally intensive analyses. We have two Specific Aims: Hypothesis 1: The application of bioinformatics will identify targets (genes, proteins and metabolic markers) involved in the initiation and progression of DN. Specific Aim 1: Identify genes of interest regulated across species in DN. a. Use microarrays to identify biomarker pathways and targets in human sural nerve samples from patients with type 2 diabetes and DN. b. Perform cross-species validation between sciatic nerve microarrays from mice with type 2 diabetes and the human sural nerve microarrays in Aim 1a. Hypothesis 2: Verified transcriptomics will predict changes in encoded proteins (in peripheral nerve) and metabolites (in plasma and urine) critical to the initiation and progression of DN in human patients and animal models. These functional responses will lead to the identification of biomarkers useful in the diagnosis and therapeutic management of human DN. Specific Aim 2: Validate the biological relevance of identified gene targets in a type 2 murine model with DN a. Localize and quantify target gene products (proteins) in sciatic nerve using immunolocalization and western blotting b. Identify and examine metabolite levels in biological fluids (plasma and urine) PUBLIC HEALTH RELEVANCE: The most common complication of diabetes is peripheral neuropathy (DN). The identification of DN biomarkers would greatly enhance our understanding of early events in this complication and could be used to predict its development and rate of progression. We hypothesize that diabetes directly affects peripheral nerve gene expression and that these data will aid in the identification of useful DN biomarkers. Microarray analyses will compare changes in gene expression between human sural nerve biopsies and BKS-db/db mice, a well-researched model of type 2 diabetes. These data will be used to identify dysregulated intracellular pathways that would result in detectable biomarkers of DN in serum or urine and changes in expression following treatment in animal models.

DESCRIPTION (provided by applicant): The 2009 Peripheral Nerve Society (PNS) meeting will be held from July 4th - 8th 2009 at Congress Centrum W[unreadable]rzburg in W[unreadable]rzburg, Germany. The PNS is an international organization of physicians and scientists dedicated to forwarding our understanding of peripheral nerve biology and disease, with the ultimate aim of developing treatments for patients with various forms of peripheral nerve disease. The PNS facilitates both basic and clinical research, physician and scientific training, and consensus development of clinical standards of care. PNS members come from a broad spectrum of academic interests and departments. The PNS pursues its goals at biennial meetings that include an international and multidisciplinary audience. The meetings focus on translating basic science discoveries into clinical treatments for patients. The PNS develops collaborative guidelines for treatment of peripheral nerve diseases. Development and mentorship of the next generation of peripheral nerve scientists and clinicians is an important role of the PNS, and junior member attendance is encouraged and interaction with senior investigators facilitated. Meetings are held in cloistered settings to encourage informal interactions. Meeting results are published in the Journal of the Peripheral Nervous System. PUBLIC HEALTH RELEVANCE: Peripheral nerve diseases are a common and important cause of disability. The biennial Peripheral Nerve Society meeting brings together scientists and clinicians from across the world in order to share research findings and to develop new treatments. The meeting directly impacts public health and patient care by disseminating important new ideas (both research and therapeutic) and by developing consensus guidelines.

DESCRIPTION (provided by applicant): Diabetic neuropathy (DN) is the most common chronic complication of diabetes, ultimately affecting half of patients with type 1 diabetes (T1DM) and leading to severe morbidity, high mortality, major physical disability, poor quality of life and estimated total annual costs of $22 billion. Due to the complex structure and anatomy of the peripheral nervous system, DN presents with a very broad spectrum of clinical symptoms and deficits, including severe pain, sensory deficits, foot ulcers and amputations. Despite the high morbidity associated with DN, most randomized clinical trials evaluating therapies for established DN have been disappointing. To date there is no pathogenetic treatment for this condition. The Diabetes Control and Complications Trial (DCCT) demonstrated that intensive control designed to achieve near-normal glycemia is essential in reducing the risk of DN development in type 1 diabetes. However, attainable intensive glycemic control, although necessary, is insufficient to prevent adverse nervous system effects, justifying a therapeutic need to identify new drug targets to treat DN early in its course. Evidence for an important role of low-grade inflammation and of nuclear factor kappa B (NF-kB) activation in the pathogenesis of DN and in the pain syndrome associated with DN is emerging from both experimental and clinical studies. This suggests that agents with known anti-inflammatory properties, such as salicylates, may prevent the development of DN and the pain associated with DN. Salsalate (Disalcid), a pro-drug form of salicylate, is an FDA approved treatment for osteoarthritis and other rheumatologic conditions. It is a highly effective drug in blocking the IKK2/NF-:B pathway, with a large margin of safety, and low cost. Salsalate has recently been shown to have glucose lowering effects. We propose to develop a clinical trial to evaluate the effect of Salsalate on DN. Our specific aims are: Aim 1: Develop a proposal for a multi-center randomized clinical trial to evaluate the effects of salsalate on measures of DN in patients with T1DM Aim 2: Develop the supporting materials needed to conduct the trial in Aim 1, develop the criteria of site selection, reach out to the sites of interest and finalize the selection process, and develop the general and site specific recruitment strategies. The accomplishment of these aims will allow implementing the proposed clinical trial protocol on a sample of subjects to test documents and procedures during the planning grant phase R34 and allow submission of an application for a multi-center trial grant. PUBLIC HEALTH RELEVANCE: This study is relevant to public health because diabetic neuropathy (DN) is the most common chronic complication of diabetes, ultimately affecting half of patients with diabetes and leading to severe morbidity, high mortality, major physical disability, and poor quality of life. This is relevant to the NIH mission because to date there is still no specific treatment for DN. We propose to use a FDA-approved drug commonly used to treat osteoarthritis, which has recently been shown to have glucose lowering effects, to address inflammation - a critical mediator in the development and progression of peripheral nerve damage in diabetes, and pain- the most common and cumbersome symptom for patients with DN.

DESCRIPTION (provided by applicant): Type 1 diabetes (T1DM) alters carbohydrate, amino acid, and fatty acid metabolism contributing to nephropathy, retinopathy and peripheral neuropathy, three of the most debilitating complications of diabetes. The prevailing view suggests that systemic hyperglycemia drives complications by similar biochemical mechanisms in all tissues. However, this has not been specifically tested, especially in vivo. In these studies, we propose that the normal cellular nutrient utilization in cells of complication-prone tissues is distinct and therefore reaction to insulin deficiency, hyperglycemia and other changes of T1DM will also be distinct. Indeed, our preliminary data demonstrate tissue-specific changes in cellular metabolite levels that are not driven by mass-action alone but appear to be due to selective metabolic reprogramming of the target tissues. In contrast to increased levels in kidney, glycolysis and tricarboxylic acid cycle (TCA) cycle intermediates are decreased in diabetic mouse nerve and retina despite ambient hyperglycemia. Importantly, the increased mouse kidney and human urinary levels of TCA metabolites predict progression of diabetic nephropathy, suggesting that metabolic reprogramming may play a pathogenic role in the progression of complications. These findings lead to our hypothesis that diabetic complications arise from tissue-specific metabolic reprogramming resulting in alterations in fuel utilization which lead to dysfunction of the tissue. To test this hypothesis, we will use sensitive and specific mass spectrometer based metabolomic analysis in models of diabetes to define changes in metabolite levels and flux in three complications-prone tissues, retina, kidney and peripheral nerves. We will extend these studies to humans with T1DM to understand intrinsic differences from non-diabetics in metabolite levels and flux in the kidney. We will define the changes in mRNA and protein expression and post-translational protein modification to determine the basis for altered metabolite levels. Finally, we will utilize appropriately engineered mouse animals to directly test the refined hypotheses arising from these studies. Our specific aims are to: Aim 1: Identify the alterations in steady state abnormalities of intermediary metabolism in kidney, nerve and retina in the best murine models of diabetic complications using state-of-the-art metabolomic approaches Aim 2: Determine metabolite flux in all 3 tissues from the murine models and identify the key regulatory reactions that contribute to the metabolite abnormalities. Aim 3: Assess steady state and dynamic metabolite changes in humans with type 1 diabetes with and without microvascular complications. Aim 4: Define regulatory mechanisms of altered cellular metabolism in complication-prone tissues and test their effect in murine models. PUBLIC HEALTH RELEVANCE: This proposal will test the hypothesis that diabetic complications arise from specific changes in cellular substrate metabolism which can be defined using modern molecular phenotyping techniques in both animal models and in humans and that interventions to modulate specific metabolic pathways may mitigate or prevent development and progression of these complications. Our study is designed to gain a better understanding of the changes in metabolite levels and flux in complications-prone tissues in animal models and patients with type 1 diabetes mellitus and to determine how the metabolite changes reflect altered levels or activities of specific proteins and lipids which contribute to 'microvascular'complications. We will utilize in vivo and in vitro analysis of mouse models and human patients to achieve these goals.

DESCRIPTION (provided by applicant): This proposal outlines a plan for a Phase 1b clinical trial for the injection of human spinal cord derived stem cells (HSSCs) into the cervical spinal cord of patients with ALS. This trial is a follow up to a trial already underway at Emory University, where 12 ALS patients have been injected with the same HSSCs into the lumbar spinal cord. In order to move this therapeutic approach closer to a clinical trial to determine if t is effective in ameliorating disease, we are proposing to test the safety of HSSC injection into the cervical spinal cord. Motor neurons in the cervical spinal cord innervate the respiratory diaphragm, the loss of which is typically the cause of death in ALS patients. We propose that the protection of these neurons is likely to prolong life by preserving respiratory function. This safety trial will employ progressive dose escalation to determine the maximum tolerated dose that can be used for the long term goal of performing Phase 2 and Phase 3 efficacy trials. There are two specific aims. In Aim 1 we propose to sequentially escalate the dose of delivery as defined by 1) the number of cells/injection, 2) the number of injections into the cord, and 3) either unilateral or bilateral injections. This dose escalation scheme is designed to safely and efficiently test our ability to achieve a pre-defined target therapeutic dose, which can be used in the next phase of testing therapeutic efficacy. Aim 2 of this proposal is to examine several exploratory endpoints that may be used to test the efficacy of this therapy in future Phase 2 and Phase 3 trials. These include measures of respiratory function, diaphragm function, muscle strength, and electrical characteristics of muscle (Electrical Impedance Myography). Successful completion of this Phase 1b trial will allow for testing of this highly innovative approach to the treatment of ALS. The impact will extend beyond patients with ALS since this novel trial will provide data on surgical approach and safety, as well as trial design that will be highly relevant to cellular therapeutics for spinal cord injury, multiple sclerosis, spinal muscular atrophies, as well as other neurological diseases. PUBLIC HEALTH RELEVANCE: ALS is a devastating neurodegenerative disease with no effective treatments. Introduction of human spinal cord derived stem cells (HSSCs) into the spinal cord of ALS patients represents a potentially powerful way to prevent motor neuron death and prolong life. This Phase 1b trial will determine the safety of providing a Target Therapeutic Dose of HSSC injections into the cervical spinal cord of ALS patients, directed at preserving the motor neurons that control breathing. This Phase 1b trial is a necessary stepping stone toward more definitive therapeutic trials of HSSCs in patients with ALS.

Project Summary / Abstract Core E - Microvascular Complications Core The microvascular complications of type 1 and 2 diabetes are major causes of morbidity and mortality. Adequate murine models of these complications are critical to their understanding, treatment and prevention. Development and testing of murine models of diabetic microvasular complications has been slowed by the relative paucity of uniform phenotyping standards and methods. The Complications Core will help to overcome this barrier by providing validated, reproducible and standardized phenotyping of the 3 major microvascular complications: diabetic polyneuropathy (DPN), nephropathy (DN) and retinopathy (DR). The Microvascular Complications Core will make the phenotyping of mouse models of diabetic complications available, expeditious, affordable, effective, and convenient for individual investigators. In addition to providing education, consultation and advice regarding the analysis of mouse models of complications, the Core will provide phenotyping services using the specialized equipment that it operates. Specifically, the Core will provide assessment of murine DPN, DN and DR using clinical and anatomical assays for each complication and will also offer advanced phenotyping of models exhibiting each complication. DPN advanced testing will include phenotyping of models exhibiting neuropathy such as measures of cell death and oxidative stress in dorsal root ganglion (DRG) and peripheral nerve. DN advanced phenotyping will include measures of podocyte number, precise morphometric analysis of glomerular expansion, glomerular volume and tubulointerstitial fibrosis, EM morphometry of podocyte foot processes, immunohistochemical analysis of podocyte specific proteins, and glomerular isolation. DR advanced testing will include measures of retinal vascular permeability, retinal cell death and non-lethal measures of retinal morphology using optical coherence tomography and visual function using optokinetic response. A significant strength of the Complications Core is that all testing methods will be housed within one laboratory and that all three complications can be assessed in a single mouse. This will allow for the efficient flow and coordination of animal testing, data collection and ultimately tissue harvest. Many assays performed in conscious animals may be repeated over the course of the experiment while others will be performed immediately prior to euthanasia. All components of the Complications Core will work closely together and with client investigators to coordinate the phenotypic characterization of microvascular complications. This comprehensive approach will reveal commonalties and differences among complications as well as addressing the impact of specific gene alterations and treatment regimens.

? DESCRIPTION (provided by applicant): Diabetic neuropathy (DN) is the most common chronic complication of diabetes, affecting up to 50% of individuals with type 1 diabetes (T1DM). DN is a progressive disease, leading to severe morbidity and staggering health care costs ($22 billion/year www.diabetes.org). Patients experience poor quality of life due to pain, loss of sensation leading to poor balance, falls and eventual foot deformities with high rates of ulcerations and amputations. While not as commonly diagnosed as DN, cardiovascular autonomic neuropathy (CAN) carries equal morbidity with patients experiencing orthostasis, arrhythmias and premature death. Evidence for an important role of low-grade inflammation in the pathogenesis of DN and CAN is emerging from both experimental and clinical studies. We recently completed a series of microarray analyses of sural nerve biopsies from subjects with stable versus progressive DN and discovered a highly significant increase in the expression of inflammatory response genes in subjects with progressive DN. Salsalate is highly effective anti- inflammatory therapy, with a large margin of safety and low cost. In a prior proof of concept pilot study (R03 DK 094499), we obtained the IND (IND 113650) for the use of salsalate and completed clinical and safety assessments in 8 subjects with T1DM and DN, treated with 3 gram/day salsalate for 3 months, with evidence of early clinical efficacy and drug safety. Our long-term goal is to identify mechanism based therapies for the treatment of DN and CAN. The objective here is to determine if salsalate is an effective and safe therapy for DN and CAN. Our central hypothesis is that inflammation has a role in the development and progression of DN and CAN and that blocking inflammation with salsalate can positively affect disease progression. The rationale for the proposed research is that a pilot clinical trial of salsalate therapy will: 1) test the role of inflammation in DN and CAN, 2) allow for biomarker development for both disorders, and 3) provide the justification for a large scale clinical trial of salsalate n the treatment of DN and CAN. We plan to test our central hypothesis, and thereby accomplish the objective of this application, by pursuing two specific aims: Aim 1: Examine the therapeutic efficacy of 3 gram/day salsalate for 12 months on measures of DN and CAN in subjects with T1DM and mild DN Aim 2: Determine the safety profile of 3 gram/day salsalate in patients with T1DM and mild DN.

ABSTRACT Diabetes is associated with altered carbohydrate, amino acid, and fatty acid metabolism, contributing to diabetic complications, including diabetic peripheral neuropathy (PN). Diabetic PN affects ~60% of diabetic patients and is characterized by progressive loss of peripheral nerves in a stocking and glove pattern (extremities affected first), with pain and eventual loss of sensation. Despite extensive research over recent decades, the pathogenesis of DN remains unclear, and there is no treatment beyond traditional glucose control, which hardly affects PN development and progression in type 2 diabetes (T2D). Therefore, there is a critical need to determine the specific metabolic mechanisms contributing to the onset and progression of DN in order to identify mechanism-based intervention strategies. Our long-term goal is to meet this need and develop much-needed therapies that impact PN before the onset of disease in order to significantly improve quality of life of diabetic patients. In the current proposal, our objective is to leverage human subjects enrolled in an NIH-funded clinical study at the Investigational Weight Management Clinic (IWMC) at the University of Michigan and a mouse model of high fat diet (HFD)-induced obesity, prediabetes, and PN to identify the mechanisms that contribute to PN pathogenesis. We hypothesize that distinct metabolic alterations occur in obesity, prediabetes, and T2D which induce metabolic reprogramming within the peripheral nerve, altering fuel utilization and ultimately leading to tissue dysfunction. We will test this hypothesis in two aims. First, we will use sensitive and specific mass spectrometry-based metabolomic analysis on plasma from obese, prediabetic human subjects with PN in the IWMC study and from mouse models with PN. Mice will be fed standard diet (SD) or HFD from 5-16 wk of age. We will compare the metabolomic profiles between humans and mice with PN, with the goal of identifying both common and distinct signatures that associate with PN. This cross-species approach will allow us to discover pathogenic metabolomic signatures to act as biomarker(s) of PN in man and mouse, and to identify candidate pathways and molecules whose regulation play crucial roles in the pathogenesis of PN. Second, we will measure changes in mitochondrial function and fuel substrate utilization in peripheral nerve from the mice with PN before and after weight loss. We will use five groups of mice: mice fed SD or HFD from 5-16 wk of age, mice fed a SD or HFD from 5-24 wk, and mice fed a HFD from 5-16 wk then switched to SD from 16-24 wk [HFD- dietary reversal (HFD-DR)]. Notably, HFD-DR mice show significant regression of all PN parameters at 24 wk. These studies will link nerve-specific bioenergetic abnormalities and PN phenotypes to identify candidate bioenergetic pathways whose regulation play crucial roles in the pathogenesis of PN. Together, the proposed studies using obese, prediabetic patients and mice with PN will increase our understanding of how the peripheral nerve adapts to chronic and specific changes in substrate availability beyond excess glucose.

ABSTRACT ALS is a progressive and fatal neurodegenerative disease with complex unknown pathogenesis. Recent evidence supports a gene-time-environment hypothesis whereby environmental exposures trigger neurodegeneration when superimposed on a genetic risk profile. Supporting this premise, long-term adverse environmental exposures are linked to ALS risk, environmental pollutant exposures strongly correlate with ALS prevalence, and we have shown that persistent organic pollutants (POPs), in particular measured organochlorine pesticide and reported pesticides, exposures strongly increase ALS risk in a subset of ALS patients from Michigan. Therefore, there is a critical need to understand how the ?ALS exposome,? defined as the lifetime of environmental exposures, contributes to ALS risk and drives disease pathogenesis. In this proposal, we will harness the power of advanced metabolomics analyses to gain insight into the effect that environmental exposures, such as residential and occupational exposures, have on the metabolome. As metabolites reflect the impact of exogenous exposures on cellular processes, metabolomics has emerged as the new frontier in exposome research. Our objective is to identify the metabolomics signatures that associate with POP exposures and historical exposure risk factors, and associate with ALS progression. Our central hypothesis is that POP exposures will lead to conserved metabolomic signatures in both plasma and central nervous system (CNS) tissues. In Aim 1, we will use longitudinal plasma from ALS participants from our unique University of Michigan ALS Patient Repository (UMAPR) with high versus low concentrations of POPs, as well as plasma from geographically dispersed healthy control subjects, to better characterize how POP exposures impact the metabolome. In Aim 2, we will evaluate whether metabolomic signatures are shared in ALS subjects with similar occupational and residential exposure risks and whether these signatures diverge in subjects with disparate risks in order to yield insights into causal mechanisms from prior epidemiologic studies. Finally, in Aim 3, we will determine whether metabolomic signatures in ALS subject plasma are present in post-mortem brain and spinal cord tissue and correlate with exposures. Overall, completion of these aims will establish a comprehensive and rigorous dataset of metabolomic signatures associated with exposures to POPs, as well as residential and occupational exposure risk histories across the disease course, to provide insight into the influence of exposures on the onset and progression of ALS. These outcomes will have an important positive translational impact by identifying modifiable factors that can mitigate the risk of developing ALS, uncovering associated metabolic changes that represent biomarkers, and guiding future studies on new pathophysiologic disease mechanisms.

Program Summary/Abstract This U01 proposal is designed to provide the preclinical framework required to advance a novel stem cell therapy to human clinical trials as an effective treatment for Alzheimer's disease (AD). AD is the most prevalent age-related neurodegenerative disorder and leading cause of dementia, affecting an estimated 5.3 million people in the U.S. There is no cure and no means of prevention. To date, a handful of traditional, single-target pharmacological approaches have produced only marginal clinical improvements - there is a critical need for more effective therapies. Therefore, the long-term goal of our research is to develop a disease-modifying cellular therapy for AD that will have a meaningful impact on patients' lives. Cellular therapies target multiple disease mechanisms and provide a multifaceted approach to treat the complex pathologies associated with AD. In collaboration with Neuralstem, Inc., we have developed a unique line of human cortex-derived neural stem cells (NSCs) that produce several neuroprotective growth factors. Our findings to date, as well as proof- of- concept studies by others, show the benefit of cell therapies in AD models and indicate that efficacy is enhanced when coupled with delivery of trophic factors. In our proposed approach, NSC transplantation will combine the multifactorial therapeutic potential of a cellular therapy with sustained and directed delivery of neurotrophic factors, providing increased benefit compared to traditional approaches and improving outcomes in AD. Our preliminary data in a mouse model demonstrate that NSC transplantation is safe and effective, significantly impacting cognition and reducing A? plaque burden. In this proposal, we will determine the maximum tolerated dose and assess NSC bio-distribution and tissue tropism in two well-established and highly relevant mouse models: 5XFAD and rTg4510. We will then perform large-scale efficacy testing of NSCs in these mouse models and complete a dose-response feasibility study in non-human primates, which are anatomically and cognitively more relevant to human clinical testing. Overall, our proposal will have a significant impact on AD by providing proof-of-concept efficacy data for a well-characterized cellular therapy in two relevant mouse models and safety data in a large animal with a brain structure that is more analogous to humans. Completion of our proposed IND-enabling studies, as well as our laboratory's unique track record of translating proof-of-principle animal studies to human trials, will enable this stem cell therapy to progress into an attainable disease-modifying intervention for AD patients.

Abstract Despite overwhelming evidence that the immune system plays a significant role in amyotrophic lateral sclerosis (ALS) pathogenesis, information about the role of the immune system in human patients is lacking. Our preliminary data show that natural killer (NK) cells are upregulated in the blood of ALS patients. NK cells function as the innate immune analogs of adaptive CD8 T cells. These cells can also skew immune polarization or directly lyse infected and/or dying cells (NK) without the aid of antigen presenting cells. NK cell numbers are increased in the spinal cord of ALS mice, and other innate lymphoid cells have been previously shown to contribute to ALS progression. In addition, recent studies have demonstrated that the motor neurons of ALS mice lack major histocompatibility complex 1 (MHCI) expression; this is important as MHCI expression protects cells from NK cell-mediated death. Unlike anti-inflammatory cell types that are currently being studied for use in ALS therapeutics, such as regulatory T cells, NK are purely pro-inflammatory and can be targeted in the blood using methods of depletion; thus, the purpose of this proposal is to determine the role of NK cells in driving ALS, with the long-term goal of developing future therapeutics. We hypothesize that NK cells contribute to ALS pathology by accelerating motor neuron death and by producing pro-inflammatory cytokines such as interferon-gamma, tumor necrosis factor-?, and granulocyte macrophage colony-stimulating factor that can skew downstream immune responses in the CNS. We therefore propose two Specific Aims which will assess the role of NK cells in both mouse and man. In the first Aim, we will deplete NK cells in ALS mice using commercially available antibodies to determine whether the absence of NK cells delays disease onset and slows progression. In the second Aim we will isolate NK and innate lymphoid cells from the blood of ALS patients and show using flow cytometry and qPCR that these cells are more prone to inflammatory cytokine production. We will also analyze the kinetics of NK cell accumulation and cytokine production by taking monthly blood draws from a cohort of ALS patients. These data will then be matched to clinical metrics of disease progression to demonstrate correlation between NK cells and ALS. Overall, completion of these studies will have considerable impact on both our understanding of the role of these cells in ALS pathogenesis and their potential as a therapeutic target.

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with complex unknown pathogenesis. Recent evidence supports a gene-time-environment hypothesis whereby environmental exposures trigger neurodegeneration when superimposed on a genetic risk profile. Supporting this premise, long-term adverse environmental exposures are linked to ALS risk and progression; we have shown that measured and reported pesticide exposures strongly increase ALS risk and that high levels of persistent organic pollutants (POPs) decrease ALS survival in ALS subjects in Michigan. Therefore, there is a need to delineate the ?ALS exposome,? defined as the lifetime of environmental exposures that contributes to ALS risk. In this proposal, our objectives are to improve our ALS exposome model by enhancing insight into pollutant mixtures associated with ALS accounting for genetic risk, identifying periods of susceptibility to exposures, correlating toxin measurements in easily assessable biofluids with epidemiologic data, and identifying whether these environmental toxins are absorbed into the central nervous system (CNS) in order to improve insight into the gene-time-environment hypothesis in ALS. Our central hypothesis is that identifying environmental pollutants in biofluids and CNS tissues will advance models of ALS pathogenesis. In Aim 1, we will better characterize the ALS exposome by measuring environmental toxins in biological samples obtained longitudinally from ALS subjects from the University of Michigan ALS Patient Repository and age- and sex-matched controls across the State of Michigan to yield insight into the pollutant mixtures that contribute to disease risk and survival, accounting for genetic susceptibility via polygenic risk scores. In Aim 2, we will evaluate residential and occupational histories for association with ALS risk and survival, while also correlating exposure histories to toxin measures from Aim 1, to gain comprehensive insight into exposure mixtures and time windows critical for ALS risk. Finally, in Aim 3, we will quantitate environmental toxins and heavy metals in ALS and control CNS tissues, and link peripheral alterations with observed changes in ALS CNS tissue and critical exposure windows to thereby ascertain environmental risk factors that potentially contribute to ALS pathogenesis. Overall, successful completion of these aims will have an important positive translational impact by identifying ALS disease risk factors associated with occupational and environmental exposures, while accounting for genetic susceptibility. This proposal will therefore expand our understanding of the ALS exposome in the context of genetic risk, identify toxins that pose a public health risk, identify occupations linked to exposures, and establish a framework to test for these exposures in other neurodegenerative diseases. This understanding of the ALS exposome will support much-needed public health interventions to target modifiable disease risk factors in this lethal disorder.

ABSTRACT Air pollution is an understudied amyotrophic lateral sclerosis (ALS) risk factor. ALS and air pollution cause in- flammation, yet no studies link these two critical ALS topics. The long-term goals are to prevent, slow, or stop ALS progression. The overall objective is to determine how long-term air pollution exposure alters ALS risk, progression, and survival and gain insight into the role of inflammation on the path to neuronal damage. The central hypothesis is that air pollution triggers peripheral immune profiles that are an important pathophysio- logic agent of ALS risk, progression and survival. Guided by preliminary data showing that i) ALS cases have higher particulate matter (PM)2.5 exposure compared to controls, ii) the cases with the highest PM2.5 exposures have increased eosinophils, natural kill cell markers, and cytokine activation, and iii) existing literature connect- ing air pollution exposure to ALS disease severity, the rationale is that linking air pollution to ALS inflammatory profiles will strengthen the association between ALS and air pollution to inform critical and needed preventative strategies, as well as define new disease biomarkers and therapeutic targets. The central hypothesis will be tested by pursuing two specific aims: 1) Characterize air pollution exposures for cases and controls in the Uni- versity of Michigan ALS patient biorepository and determine how these exposures associate with ALS risk, pro- gression, and survival; and 2) Evaluate immune profiles as a mediators of the associations between air pollu- tion exposures and ALS risk, progression, and survival using data from cases and controls in the University of Michigan ALS patient biorepository. In Aim 1a, air pollution exposures for cases and controls are estimated using well-established prediction models for key ambient air pollutants including PM2.5, NO2, and traffic related air pollutants (TRAPs). Exposures will span the years 2000 through 2020 and account for the residential his- tory for all subjects and symptom onset date for ALS cases. Aim 1b will investigate how these exposures asso- ciate with ALS risk and, in Aim 1c, with ALS progression and survival. Aim 2a will determine the immune pro- files that associate with long-term, spatiotemporal, air pollution exposures developed in Aim 1. As the literature links air pollution to inflammation and inflammation is linked to ALS progression and survival, Aim 2b, will in- vestigate whether immune profiles play a role in the causal pathway by performing a mediation analysis be- tween air pollution and ALS risk, progression, and survival. The research proposed in this application is innova- tive, in the applicants? opinion, because it rigorously develops thorough spatiotemporal air pollution data that captures temporal and spatial air pollution trends and analyzes a deeply phenotyped ALS and control cohort from the industrial Midwest. The proposed research is significant because it will provide critical data on the dis- tinct types of air pollution, immune profiles, and immune pathways associated with ALS risk, progression, and survival. Ultimately, as air pollution is a modifiable risk factor, knowledge from this proposal can guide preven- tion efforts, therapeutic targets, and blood-based biomarkers for use in clinical studies.

ABSTRACT Type 1 diabetes (T1D) is associated with brain and cognition changes as well as well-documented longer-term micro- and macrovascular complications. The impact of brain changes and cognitive deficits on functional out- comes, however, is less clear, and most current data relates to T1D youth. Thus, T1D is associated with signif- icant health and functional morbidity, and in addition to personal costs, community and public health burden is significant and likely increasing in line with the rising global T1D prevalence. Much current information about T1D outcomes is derived from cross-sectional or longitudinal studies with short follow-up periods. These have provided rich data about the short-term T1D impact on individuals, but have an inability to discern causal asso- ciations and longer-term effects. Longitudinal studies examining within-individual changes in brain and cogni- tion over time are required to better define specific risk and resilience factors that influence long-term out- comes. The Royal Children?s Hospital (RCH) diabetes cohort study is the only prospective study of individuals with T1D from childhood diagnosis through adulthood, with four previous waves of data collection. At baseline, participants with T1D did not differ from healthy controls on IQ, specific cognitive skills, or emotional well-being. Subsequent waves of data collection documented structural and functional brain changes, cognitive decre- ments, and poorer functional outcome in T1D compared to healthy controls into early adulthood. We now pro- pose a further follow-up of this cohort, the CLARiFY study (The Cognition and Longitudinal Assessments of Risk Factors over 30 Years (CLARiFY) Diabetes Complications Study), to document brain, cognition, and func- tional outcomes in mid-adult life ~30 years after T1D onset. We will use longitudinal data from the current study and from previous waves of data collection to identify which glycemic insults are most detrimental to the brain/cognition and at which age/stage of neurodevelopment. The following specific hypotheses will be tested: 1) in cross-sectional analyses, T1D subjects will have lower brain volumes, lower cognitive scores, and poorer functional outcomes at middle adulthood than non-T1D subjects, and in longitudinal analyses, T1D subjects will exhibit a greater decline in cognitive performance that associates with greater MRI differences at middle adulthood, and greater change in MRI brain volumes from 12 years to 30 years post-diagnosis; and 2) hyper- glycemia in childhood (<18 years) will be the strongest dysglycemic determinant of brain health in mid-adult life. Further, the association between pre-pubertal hyperglycemia and brain outcomes will be most pronounced in those with (a) T1D onset at <6 years of age; (b) severe hypoglycemia and/or DKA episodes after early expo- sure to hyperglycemia, and (c) evidence of extra-cerebral microvascular and/or macrovascular disease in mid- adult life. An empirically based stratification of glycemic neurotoxic ?risk? as it varies across the life cycle would allow clinicians to offer optimal clinical management designed to both achieve good metabolic outcomes while at the same time protecting the developing brain and preserving cognitive function.

ABSTRACT Genetic heritability incompletely explains amyotrophic lateral sclerosis (ALS), and the pace of ALS genetic dis- coveries has slowed, meaning entirely new research directions are needed to unravel disease mechanisms and identify therapies. Our goal is to understand, cure, and prevent ALS. Our overall approach is to identify the intersection of exposures, genomics, epigenomics, transcriptomics, metabolomics, and inflammation on ALS. Our rationale is that prior environmental risk scores (ERS) based on even crude plasma measures of limited classes of pollutants associate with a 7-fold increase in ALS risk and 2-fold decrease in survival, therefore a detailed understanding of the exposome with other omics can immediately provide new, much needed strate- gies for both ALS treatment and prevention. We propose 3 aims: 1) comprehensively assess environmental exposures and polygenic factors in ALS versus control subjects to identify synergistic environment-polygenic associations that increase ALS risk; 2) define exposome signatures in the ALS epigenome, transcriptome, and metabolome; and 3) determine how environmental exposures alter ALS immune profiles and identify drug tar- gets. First, we account for the complex exposure data from self-reports, geospatial analysis, and biospecimens using component-ERS (cERS) for specific exposure types (e.g. pesticides, metals, air pollution) and a poly- ERS for combined exposures. We account for genetic risk using polygenic risk scores (PRS) and C9ORF72 status. We will build ALS risk and prediction models based on ERS and PRS. Next, using cERS, poly-ERS, and PRS, we determine the environmental signature on the DNA methylome, mRNA and microRNA, to identify exposures that associate with differentially expressed genes and target pathways. Expression quantitative trait loci (eQTL) analyses will define the relative contribution of polymorphisms vs exposures on gene expression. High resolution untargeted metabolomics will reveal the environmental signature of the ALS metabolome and identify new toxicants. All datasets will be integrated using pan-omics techniques to identify gene-metabolite networks that are disease targets. Finally, we will classify immune profiles that associate with cERS and poly- ERS to identify therapeutic targets using existing FDA approved drugs. Our proposal is highly innovative; it de- fines for each patient, (i) their exposome, summarized with cERS/poly-ERS; (ii) their genome summarized by PRS and its association with ERS to understand the combined gene/environment risk; (iii) their multi-omic en- vironmental signatures from the epigenome, transcriptome, metabolome, and inflammasome; (iv) their dysreg- ulated pathways, ranked by their association to ALS risk and progression to identify personalized mechanism- based drug-targets; and (v) ALS prediction models and preventative strategies via risk factor modification. Our parallel, multi-omics approach is significantly faster than serial, single-toxicant/omic approaches, and its inte- grated nature captures the full spectrum of omics intersections, accelerating scientific discovery. We will make significant strides in finding completely new therapeutic targets and public health preventative strategies.