James W. Russell, M.D. - US grants

Diabetic neuropathy (DN) is the most common cause of peripheral neuropathy in the United States, yet the pathogenesis remains unknown. Although diabetes can affect all peripheral neurons, sensory neurons are most commonly affected, possibly because dorsal root ganglion (DRG) neurons reside outside the blood-nerve barrier. Hyperglycemia has been implicated in both animal and human studies in the pathogenesis of DN and recent clinical trials have shown a reduction in the progression of DN with careful control of blood glucose an intensive insulin therapy. However even excellent glycemic control fails to prevent or reverse DN. Insulin-alike growth factor I (IGF-I) can improve glycemic control in diabetes and is able to promote neuronal growth, development, and regeneration of neurons. In ongoing preliminary studies, we find that hyperglycemia leads to impaired rat DRG sensory neuronal growth and programmed cell death (PCD). In both paradigms, IGF-I is neuroprotective. Our initial investigations of IGF-I neuroprotection reveal that 1) IGF-I acts trough the type I IGF receptor (IGF-IR activation results in downstream phosphorylation of focal adhesion proteins involved in organization of the actin cytoskeleton and neurite formative, and 3) IGF-IR activation of phosphatidylinositol-3 kinase (PI-3K) is essential for rescue of neuronal cells from PCD. We have developed a novel hypothesis to explain hyperglycemic coupled neurotoxicity. We speculate that high glucose alters IGF-IR activation in DRG neurons. This result in changes in the phosphorylation of focal adhesion proteins which results in disruption of the actin cytoskeleton and impairs DGR neurite growth. We believe subsequent cytoskeletal changes alone, or in conjunction with direct glucose toxicity, induce PCD in DRG neurons. Activation of IGF-IR 1) prevents PCD by enhancing focal adhesion protein phosphorylation and stabilizing the cytoskeleton, and/or 2) blocks PCD by activating PI 3K pathways, which may effect PCD regulatory proteins like bcl-2 and/or death proteases. In this proposal we will test each component of the model. We have 2 aims: 1. Examine the effect of high glucose on DRG neurons. In DRG neurons, in response to high glucose, examine: a) DRG neuronal morphology and neurite growth b) IGF-IR transcription, cell surface abundance, and autophosphorylation c) Phosphorylation of focal adhesion proteins and the DRG cytoskeleton and d) PCD in DRG 2. Characterize IGF-IR protection of DRG neurons following glucose exposure. In DRG neuron, in response to high glucose, examine the effect of IGR-I on: a) DRG neuronal morphology and neurite growth b) IGF-IR transcription, cell surface abundance, and autophosphorylation c) Phosphorylation of focal adhesion proteins and the DRG cytoskeleton 3. Investigate the components underlying IGF-IR rescue of DRG from glucose-induced PCD. a) Determine the association between the observed changes in focal adhesion proteins, the cytoskeleton, an PCD pathways in response to high glucose b) Examine the effect of high glucose and IGF-I on IGF-IR activation of PI-3K c) Ascertain if IGF-IR activation prevents PCD by promoting expression of regulatory proteins that suppress cell death, like bcl-2 d) Determine if high glucose promotes PCD by activation of death proteases, and the role of IGF-IR activation in modulating this PCD pathway. IGF-I is currently undergoing evaluation in clinical trails of diabetic neuropathy. The current proposal will help elucidate the mechanisms underlying the role of IGF-I in preventing changes in neuronal morphology and PCD in diabetic neuropathy.

DESCRIPTION (provided by applicant): The most common complication of diabetes is neuropathy, which occurs in more than 50% of diabetic patients. Previous research shows that diabetic hyperglycemia is associated with apoptosis in neurons. This proposal aims to understand how glucose kills and IGF-I rescues neurons in both cell culture and animal models of diabetic neuropathy. Our work has resulted in a novel theory. In diabetic neurons, high glucose up-regulates reactive oxygen species (ROS) including nitric oxide (NO) and peroxinitrites. This results in depolarization of the inner mitochondrial (Mt) membrane, release of cytochrome c into the cytosol, and induction of caspase mediated programmed cell death (PCD). In contrast, insulinlike growth factor I (IGF-I), activates the IGF-I receptor and regulates uncoupling proteins 2 and 3 (UCP2 and UCP3) through a phosphatidylinositol 3kinase (PI3K)-mediated pathway. Regulation of UCP2 or UCP3 results in stabilization of the Mt membrane potential, and inhibits activation of initiator caspases, including caspase-9, and effector caspases, such as caspase-3. Interrupting hyperglycernic ROS induced PCD may offer new therapy for diabetic neuropathy. This model will be tested both in vitro and in vivo, using primary sensory neurons, PC12 cells, and a rat model of type II diabetes. We have 3 Aims: 1) Characterize glucose and IGFI control of ROS induced PCD, 2) characterize IGF-I up-regulation of UCPs in preventing ROS induced mitochondrial dysfunction and PCD, and 3) characterize the role of ROS, NO, and UCPs in diabetic neuropathy.

DESCRIPTION (provided by applicant): Oxidative stress and mitochondrial dysfunction have been associated with a wide range of neurodegenerative diseases and metabolic disorders such as diabetes. A major public health problem is the increase in the incidence of obesity-related diseases, such as diabetes and its complications and the increased incidence of neurodegenerative diseases, for example Parkinson's and Alzheimer's diseases in the aging population. Therapeutic and preventive strategies to reduce the complications of diabetes and to treat neurodegenerative diseases are urgently needed. Several lines of evidence indicate that a common link in these diseases is diminished mitochondrial oxidative phosphorylation and response to oxidative injury. Key regulators of mitochondrial function, the nuclear respiratory coactivators help to regulate mitochondrial oxidative phosphorylation and prevent cellular and neuronal injury. SIRT1 is a member of the sirtuin family of NAD+dependent deacetylases, which is proposed to be responsible for health benefits provided by caloric restriction. Furthermore, resveratrol found in the skin of red grapes increases the activity SIRT1, prolongs life-span in mice, and may prevent neurodegeneration. A key component of the protective response mediated by SIRT1 is deacetylation and activation of the transcription factor PGC-11 leading to increased mitochondrial regeneration and improved cellular oxidative energy metabolism. The role of SIRT1 and its mechanism/s of action at cellular level are uncertain;however investigators with a wide spectrum of research foci have an interest in understanding the biological actions of SIRT1 in different tissues. To study this, generation of transgenic mice that conditionally expresses SIRT1 is needed. In response to an PA from NCRR (PA-07-336) to develop animal models of human disease that are applicable to the research interests of two or more categorical NIH Institutes/Centers, we will develop a transgenic mouse that expresses mouse SIRT1 and mitochondrial targeted enhanced yellow fluorescent protein under the control of tetracycline responsive element (TRE-SIRT1/mito-eYFP). Co-expression of mito-eYFP with SIRT1 will be used to identify, isolate and study the influence of SIRT1 expression on mitochondrial function. Then, SIRT1 expression will be targeted to central and peripheral neurons by crossing it with CamKII-1 tTA mice. The transgenic mice developed in this study will aid investigators from NINDS, NIDDK, NIA and other institutes to test disease mechanisms and develop SIRT1 mediated therapies. Specifically in this proposal, we will investigate the mechanism by which SIRT1 protects central and peripheral neurons against diabetes-induced neuronal injury. The proposal also describes how the animal models produced can be developed by other investigators to study SIRT1 biology in non-neuronal cells. We have two aims: (1) To develop a transgenic mouse that expresses mouse SIRT1 under the control of tetracycline responsive element (TRE;TRE-SIRT1/mito-eYFP). (2) To phenotype the bigenic SIRT1 neuron specific mouse model. Lay description: Obesity, diabetes, and neurodegenerative diseases affect large numbers of people. The SIRT1 protein is considered to be a master regulator of the body's defense against disease and is activated by resveratrol found in red grapes. We aim to uncover the mechanism by which S1RT1 protects neurons from diabetes induced neurological complications. PUBLIC HEALTH RELEVANCE: This proposal is submitted in response to a PA from NCRR PA-07-336 "Development of Animal models and Related Biological Materials for Research". The research objective of this PA is to "develop, characterize or improve animal models for human disease and that models to be considered must be applicable to the research interests of two or more categorical NIH Institutes/Centers". A major public health problem is the increase in the incidence of obesity-related diseases, such as diabetes and its complications and the increased incidence of neurodegenerative diseases, for example Parkinson's and Alzheimer's diseases in the aging population. We will develop a transgenic mouse that expresses mouse SIRT1 and mitochondrial targeted enhanced yellow fluorescent protein under the control of tetracycline responsive element (TRE-SIRT1/mito-eYFP). Co-expression of mito-eYFP with SIRT1 will be used to identify, isolate and study the influence of SIRT1 expression on mitochondrial function. Then, SIRT1 expression will be targeted to central and peripheral neurons by crossing it with CamKII-1 tTA mice. The transgenic mice developed in this study will aid investigators from NINDS, NIDDK, NIA and other institutes to test disease mechanisms and develop SIRT1 mediated therapies. Our overall hypothesis is that activation of SIRT1 in the central (CNS) and peripheral nervous system (PNS) would reduce oxidative stress and improve regulation of Mt function in neurons and other tissues that may be important in neurodegenerative diseases, diabetes and its complications, and in delaying or reducing the effect of aging in the nervous system. Activation of the sirtuins offers the potential for a novel treatment of several human diseases that are related to oxidative injury and defects of mitochondrial function.

PROJECT SUMMARY/ABSTRACT The rationale for this proposal is that there is currently no specific medication that prevents or reverses diabetic neuropathy in humans and this is a major gap in scientific knowledge. Oxidative stress and mitochondrial (Mt) dysfunction are recognized as important causative factors in neurodegenerative disease and in diabetic neuropathy. Our central hypothesis is that nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) are precursors that in the presence of nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) increase tissue NAD+ levels in the peripheral nervous system. This in turn activates SIRT1, and in turn downstream transcription factors, which differentially regulate specific Mt complexes to optimize Mt respiration and prevent Mt degeneration. Our objectives are to determine if NR or NMN can be used as a therapy for experimental diabetic neuropathy, determine if the SIRT1- PGC-1? signaling pathway provides molecular targets for treatment of neuropathy, identify potential Mt respiratory chain targets that may respond to treatment, determine if the axonal enzyme that converts NMN to NAD, Nmnat2, is present in regenerating axons in skin biopsies from diabetic animals and human subjects and if measurement of Nmnat2 may be useful as a marker for response to NR. In the Methods, we will use a variety of molecular, electrophysiology, and pathology tools to achieve the aims of the study. Animal models of type 1 and 2 diabetes will be used to determine the effect of NAD+ and SIRT1 overexpression on neuropathy. In isolated Mt or whole DRG neurons we will manipulate NAD+ and SIRT1 to assess the effect on overall Mt function and specific Mt respiratory complexes. Nmnat2 levels will be determined in control and age and gender matched skin biopsies from subjects with different severity of diabetes and diabetic neuropathy. The preliminary findings support the overall objectives of the proposal and provide promising evidence that NR and NMN would provide a potential therapy for diabetic neuropathy and that NAD+ activation of the SIRT1- PGC-1? signaling pathway is important in regulating Mt respiration. The status of the project based on our recent manuscripts shows that PGC-1? has a key role in regulating Mt function in diabetic neuropathy and that knockdown of PGC-1? intensifies the neuropathy. This novel research will examine the upstream activator of PGC-1?, namely SIRT1 in diabetic neuropathy and help further explore a new potential therapy (NR) that can be taken into clinical studies in a timely manner.