A Centralized Data Mining and Analysis Portal for Diabetic Neuropathy Research
Diabetic neuropathy (DN) is the most common complication of diabetes with significant morbidity, mortality and cost. 60% - 70% of diabetic patients have neuropathy often resulting in poor quality of life. Better understanding of the molecular mechanism of development and progression of DN is crucial for designing mechanism based therapies. Genome-wide molecular studies in animal models are integral to understanding human disease pathogenesis. Diabetic Complications Consortium (DCC) has extensively characterized mouse models of human DN. Integrating high-throughput gene expression data in human DN with those in the animal models is critical in delineating species-specific as well as shared mechanisms between human and mouse. Our group has developed a database system that integrates transcriptomics data from our studies in mouse models and human DN. Goal of this proposal is to extend the existing system to form a centralized repository for publicly available transcriptomics data sets of DN and to provide a data-mining and data-analysis portal to the diabetes complications research community. Specific aims of the project are: Aim 1. Identify and annotate human and mouse DN gene expression data sets in the DCC data repository, National Center for Biotechnology Information (NCBI) Gene Expression Omnibus and European Bioinformatics Institute (EBI) ArrayExpress; process data using our established data processing pipeline to achieve consistency. Aim 2. Extend the existing database to efficiently store large transcriptomics data sets and upload processed data into the database; develop and implement data mining and data analysis tools with a user friendly web-based interface. Making this centralized data repository and analysis portal available to the research community at large will aid investigators in generating hypotheses and designing future experiments. Comparing gene regulation in the well characterized DCC murine models and human DN will facilitate selection of models that best recapitulate human disease mechanism for further exploration.

Identifying Alterations in Mitochondrial Dynamics Associate
Diabetes is a growing epidemic, affecting more than 387 million individuals worldwide. Up to 60% of diabetic patients have diabetic neuropathy (DN), a debilitating microvascular complication that results in the progressive loss of sensory nerve function in the extremities. Despite the deleterious impact of DN, therapies for the disease are limited to symptomatic relief. To develop effective treatments that specifically target DN, a mechanistic understanding of molecular pathways that result in neurological dysfunction associated with diabetes is needed. Recent evidence suggests that dyslipidemia, rather than hyperglycemia, is the clinical parameter that correlates with the progression of DN. Since metabolic pathways converge on the mitochondria (Mt), these organelles play a central role in maintaining neuronal cellular function and energy homeostasis through mitochondrial (Mtl) trafficking mechanisms and endoplasmic reticulum (ER)-mediated calcium signaling pathways. However, metabolic overload associated with diabetes may result in aberrant calcium dynamics in the primary sensory neurons of the nervous system, the dorsal root ganglia (DRG), resulting in diminished Mtl trafficking and cell death induced by ER-Mt contact sites. We hypothesize that hyperlipidemia increases the level of ER-Mt contact sites in DRG neurons, creating a localized calcium flux which triggers Mtl apoptosis and halts Mtl trafficking. We will test this hypothesis by 1) evaluating the role of ER-Mt interactions in Mtl dysfunction and neuronal cell death in the DRG and sural nerve of a high fat (HF)-fed mouse model, and 2) identifying changes in calcium dynamics that impair Mtl trafficking in hyperlipidemic DRG neurons. These studies will provide important insight into the role of Mtl dynamics in DN and thereby support our long-term goal of identifying therapeutic targets that specifically improve Mtl function and restore nerve function to patients with DN.

Role of NADPH Oxidase 5 (NOX5) in Diabetic Neuropathy
Peripheral neuropathy (PN) is a common complication of diabetes, prediabetes, and obesity. There is no effective treatment for PN, and approaches that slow or reverse disease progression are urgently needed. Although the pathogenesis of PN is poorly understood, evidence has shown that reactive oxygen species (ROS) mediate in part the cellular and molecular injury observed in PN. NADPH oxidase (NOX) enzymes generate ROS, and we are interested in the role of NOX5, one of the 7 NOX isoforms, in PN. NOX5 is present only in man and absent in rats and mice, and our preliminary results demonstrate that the NOX5 gene is regulated by hypomethylation in peripheral nerves of diabetic subjects with PN, and that this hypomethylation promotes increased NOX5 gene and protein expression and enhanced ROS generation. Our objective in this proposal is to extend these observations to validate the role of NOX5 as a major player in the development and progression of PN. We hypothesize that NOX5 upregulation contributes to the development of PN. To test this hypothesis, we will: 1) generate and characterize a transgenic (Tg) mouse model expressing human NOX5 specifically in Schwann cells and evaluate the effects of NOX5 expression on PN progression in the setting of type 2 diabetes (T2D), and 2) determine the therapeutic efficacy of NOX5 inhibition using a specific NOX inhibitor (GKT137831; Cayman Chemical, Michigan) on PN in our Tg mouse model with T2D. These studies will provide insight into the therapeutic potential of a new mechanism-based approach and thereby support our long-term goal of identifying therapeutic targets that restore nerve function in prediabetic, diabetic, and obese subjects with PN.

Role of Microbiota in the Pathogenesis of Diabetic Neuropathy
Metabolic disruption represented by diabetes, dyslipidemia, prediabetes and metabolic syndrome leads to serious neurological complications such as peripheral neuropathy (PN). Much effort has been undertaken to understand the pathophysiology of PN and the link between metabolic dysregulation and peripheral nerve injury. Work by our group and others using the high fat diet (HFD)-mouse model have shown that the microbiota of the HFD-fed mice is significantly different than those of their control littermates. This disruption in microbiota is referred to as dysbiosis which has been correlated with PN. Moreover, dietary reversal of the HFD by a standard diet or oleate-rich diet succeeded in rectifying the disruption of the microbiota and reversal of PN. Apart from a handful of association studies, the role of microbiota in PN has not been well studied or characterized. There is also a gap in our understanding of the mechanisms by which HFD-associated dysbiosis imparts nerve injury and predisposes PN. In the current study, we aim at (1) investigating the role of microbiota in mediating PN and (2) determine the effect of microbiota on fatty acid absorption and metabolism in gut and nerves as a candidate pathway for nerve injury. To achieve our aims, we are going to use C57BL/6J mice (4-week-old) that will be depleted from their microbiota by an antibiotic cocktail (administered for 2 weeks) followed by fecal microbial transplant (FMT) from different sources and phenotyped for any PN abnormalities. After 10 weeks of FMT inoculation (16 weeks of age), feces will be collected from all mice groups for analysis of microbiota by 16S rRNA sequencing and determination of fecal fatty acid content followed by the sacrifice of mice and measurement of metabolic parameters (glucose, lipid profile, fatty acids, fatty acid metabolites). Nerves, colons, ileum will be harvested for the determination of protein expression of signaling proteins involved in fatty acid absorption and metabolism (Mogat2, PLA2g2e, Cyp2c). This study will provide a novel insight into the role of microbiota in PN and the mechanism by which it imparts nerve injury or protection. This will ultimately allow us to optimally target the microbiota to restore nerve function in prediabetic, diabetic, and obese subjects with PN.

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