Rick T. Dobrowsky - US grants

Abstract 9513596 Dobrowsky The objectives of this proposal are to determine the structural components and the molecular mechanisms which regulate the coupling of the low affinity neurotrophin receptor, p75NTR, to the activation of a novel lipid second messenger system, the sphingomyelin (SM) cycle. Activation of the SM cycle produces ceramide which has been implicated in mediating the growth suppressing or apoptotic effects of specific cytokines and growth factors. Neurotrophins are growth factors which are necessary for the differentiation and maintenance of neuronal cells. The PI has recently demonstrated that neurotrophins induce SM hydrolysis through p75NTR. The cellular location of this SM pool will be determined. The structural requirements for the interaction of sphingomyelinase with the the p75 NTR will be determined, and their interaction will be studied in the presence and absence of the neurotrophin ligand. Since activation may also occur through intermediary molecules, the effect of neurotrophins on p75NTR dependent phospholipase A2 activation and arachidonic acid production a potential physiologic activator of SMase) will be examined. Since there are some indications of crosstalk between this pathway and tyrosine kinase, p75NTR dependent SM hydrolysis will be examined in tyrosine kinase receptor mutants, or by using inhibitors to uncouple tyrosine kinase from downstream signaling proteins. These studies will provide mechanistic insights into factors which regulate the coupling of p75NTR to SMase and elucidate the functional role of crosstalk pathways in the coordinate regulation of neurotrophin signaling pathways. They will also provide information on the role of lipids in intracellular signaling. ***

DESCRIPTION (Adapted from applicant's abstract): Neurotrophins are a family of growth factors that help to regulate the survival and differentiation of neurons. They influence cellular behavior through their interaction with two distinct receptors, the Trk tyrosine kinase family of receptors and the low affinity p75NTR receptor. The latter receptor can signal independently of the Trk family, regulating cell death via the generation of ceramide. In addition, it appears that p75 can increase the activity of Trk, and in contrast, that Trk can silence p75 signaling; that is, there seems to be reciprocal interactions between these receptors. There is evidence that these signaling events are initiated in caveolae and caveolae related domains (CRD), lipid-rich domains enriched in cholesterol, glycolipids and specific proteins, including the key protein component caveolin. The general hypothesis to be tested is that "compartmentalization of neurotrophin receptors within caveolae/CRDs and the interaction of these receptors with structural proteins in these domains is critical for the regulation neurotrophin signaling." To test this hypothesis, three specific aims are proposed: In the first aim, experiments are proposed to determine how structural proteins of CRDs interact with Trk receptors and regulate signaling. The investigators will ask if a putative binding domain in Trk receptors in fact regulate interactions with caveolin; if so-called scaffolding domains in caveolin regulates interactions between Trk and p75 receptors; if other structural proteins present in CRDs interact with caveolin, in particular, flottlin; and ask about the mechanism of inhibition of tyrosine activation by caveolin. In the second aim, investigations will be made regarding the molecular mechanism of Trk inhibition by p75 signaling in CRDs. In particular, the mechanism of a ligand-activated sphingomyelinase localized in caveolae will be investigated by determining if Trk activation of the PI3K/PKB (Akt) pathway, and phosphorylation of certain sequences in acid sphingomyelinase, regulate acid sphingomyelinase activity. In aim three, the effect of the lipid composition in regulating the partitioning and signaling of neurotrophin receptors in CRDs will be assessed. These studies will determine if compartmentalization of the receptors into CRDs is in fact necessary, and if the CRD content of cholesterol and sphingomyelin affects the localization of Trk to CRDs, and compromises signaling.

Binding of nerve growth factor (NGF) to the low affinity neurotrophin receptor, p75/NTR, induces the hydrolysis of sphingomyelin (SM) and generation of the pro-apoptotic lipid metabolite, ceramide. However, a significant gap exists in our understanding of how this p75/NTR- dependent signaling pathway is organized is organized and regulated. The long range goal of this research is to develop a more comprehensive understanding of the molecular mechanisms of P75/NTR-dependent signaling. The rationale for investigating the mechanisms of P75/NTR signaling is underscored by the emerging role of p75/NTR-dependent ceramide production in mediating neuronal apoptosis. Evidence has been obtained that the p75/NTR-SM signaling pathway is organized in caveolae, invaginations of the plasma membrane enriched in SM and other signaling molecules. Significantly, despite the presence of p75/NTR in non- caveolar regions of the membrane, only the caveolar pool of p75/NTR is involved in hydrolyzing SM and generating ceramide via the activation of an acid sphingomyelinase (SMase). Further, p75/NTR specifically associates with caveolin, the main protein constituent of caveolae which serves to organize and sequester signaling molecules within caveolae. The central hypothesis to be tested is that specific factors regulate the interaction of p75/NTR-dependent activation of an acid SMase. The specific aims of this project are: 1) to determine how p75/NTR interacts with structural proteins of caveolae; and 2) to identify the mechanism of p75/NTR-dependent activation of acid sphingomyelinase in caveolae. The expected outcome of these studies is that we will provide important and novel information relevant to our understanding of signal transduction pathways operative in neurodegenerative processes.

DESCRIPTION (Applicant's abstract): A frequent and often debilitating consequence of diabetes mellitus is the development of diabetic neuropathy (DN). One of the metabolic abnormalities associated with the onset of DN in both human and animal models is altered signaling through neurotrophic factors and an upregulation of the p75 NTR neurotrophin receptor (p75 NTR) in Schwann cells (SC). Insulin-like growth factor-1 (IGF-1) may act directly on SC through activation of its tyrosine kinase receptor but p75 NTR is the only receptor expressed by SC which can bind nerve growth factor (NGF). Additionally, mature SC express caveolin-1, the primary structural protein present in caveolae. Caveolin-1 can regulate growth factor signaling and genetic evidence supports that changes in caveolin-1 expression is associated with the development of several disease phenotypes. Interestingly, the levels of caveolin-1 are down-regulated in SC following cell stress, a time when p75NTR is upregulated. A significant gap exists in our understanding of the role of caveolin-1 in DN and its relevance to regulating growth factor signaling in SC. Our preliminary data indicates that altered expression of caveolin-1 may regulate cellular responses to glucose and signaling by neurotrophic factors in immortalized Schwann cells. Therefore, the objective of this R21 proposal is to test the innovative idea that changes in caveolin-1 expression may contribute to the altered neurotrophism associated with the development of DN by affecting the cellular response to glucose and signaling through p75 NTR and IGF receptors. Our overall hypothesis is that hyperglycemia decreases the expression of caveolin-1 in SC leading to enhanced p75 NTR-dependent signal transduction and decreased signaling through IGF receptors. Our specific aims are: 1) to determine if changes in caveolin-1 expression directly effects p75 NTR and IGF-1 signal transduction in immortalized SC and 2) to determine the effect of in vivo hyperglycemia on caveolin-1 expression in sciatic nerve. The expected outcome of our studies is that we will identify a putative physiological role for caveolin-1 in mediating neurotrophic signals in DN. Understanding the molecular parameters which regulate neurotrophic signaling in SC may provide insight to help increase the therapeutic efficacy of neurotrophic factors in the treatment of DN.

Diabetic peripheral neuropathy (DPN) is a common complication in diabetic individuals that results from a progressive degeneration of neurons and Schwann cells (SCs). Although much attention has focused on neuronal loss in DPN, SCs also undergo substantial degeneration and are critical for re-establishing axon- glial interaction necessary for regeneration. Analysis of the inter-related metabolic insults induced by hyper- glycemia have identified that increased production of superoxide anion may be a focal event that contributes to mitochondria! dysfunction and apoptosis of SCs. On the other hand, insulin-like growth factor-1 (IGF-1) decreases mitochondrial dysfunction and apoptosis of SCs. To date, the role of oxidative stress and IGF-1 in regulating mitochondrial function in SCs have focused only on a small subset of proteins that contribute to apoptosis. However, it is unclear that SCs undergo extensive apoptosis in DPN. We hypothesize that the opposing effects of superoxide production and IGF-1 signaling in SCs may be more critical in balancing changes in both the expression and post-translational modification of mitochondrial proteins that affect aspects of organellar homeostasis central to regulating SC regeneration. Since a significant gap exists with regard to the broad effect of glucose-induced superoxide production and IGF-1 signaling in maintaining the mitochondrial proteome, our specific aims are to: 1) Identify the role of glucose-induced superoxide production in altering the mitochondrial proteome of SCs using pharmacological, molecular and quantitative proteomic approaches. 2) Identify the role of glucose-induced superoxide production in enhancing the level of tyrosine nitration in the mitochondrial proteome. 3) Identify the sufficiency/necessity of phosphatidyl- inositol 3 kinase in attenuating glucose-induced superoxide production by IGF-1. Collectively, our studies will identify mitochondrial proteins that are susceptible to glucose-induced oxidative stress and improve our understanding of how growth factor signaling may improve mitochondrial function in diabetic nerve.

DESCRIPTION (provided by applicant): The etiology of diabetic peripheral neuropathy (DPN) is complex and involves the degeneration of both neurons and Schwann cells (SCs). Although much attention has focused on how altered growth factor signaling contributes to neuronal dysfunction, a significant gap exists in our understanding of how hyper-glycemia affects gliotrophic factors. Neuregulin-1 (NRG1) is a gliotrophic growth factor that promotes cell survival, mitogenesis and myelination by activating Erb B receptor tyrosine kinases in developing SCs. In contrast, and relevant to the etiology of DPN, pathologic activation of Erb B2 in myelinated SCs can induce demyelination and the onset of peripheral neuropathies. Our broad hypothesis is that diabetes induces an altered neuregulinism that contributes to SC degeneration and the progression of DPN. In support of this hypothesis, we provide evidence that diabetes stimulates Erb B2 activity in peripheral nerve and that this correlates with the downregulation of a negative regulator of Erb B2, caveolin-1 (Cav-1). Using myelinated SC/sensory neuron co-cultures, we demonstrate that hyperglycemia decreases Cav-1 levels and enhances NRG1-induced demyelination. Cav-1 may contribute to the degeneration of myelinated axons in vivo as the rate of onset of a mechanical hypoalgesia was faster in diabetic Cav-1 knockout versus wild type mice. Similarly, we show that Erb B2 activity is sufficient to cause a decrease in motor nerve conduction velocity and induce a mechanical hypoalgesia using a novel SC-specific conditional transgenic mouse that upregulates a constitutively-active Erb B2 in response to doxycycline. Thus, our goal is to integrate findings from animal and cellular models to gain mechanistic insight into how pathologic activation of Erb B2 affects SCs and contributes to the onset of sensory dysfunctions in DPN. Our objectives are to: 1) determine the mechanism by which Cav- 1 enhances the degenerative effects of NRG1 under hyperglycemic conditions using myelinated SC/sensory neuron explants from wild type and Cav-1 null mice, 2) determine the necessity/sufficiency of Cav-1 in contributing to Erb B2 activation and the onset of DPN using Cav-1 null mice and 3) determine the effect of diabetes on NRG expression in diabetic nerve and ascertain the sufficiency of Erb B2 in contributing to sensory deficits using novel Erb B2 conditional transgenic mice. This work will provide a new paradigm toward understanding the effect of NRGs in modulating axo-glial interactions in DPN. PUBLIC HEALTH RELEVANCE: Diabetic peripheral neuropathy (DPN) results from the degeneration of nerves that transmit sensations from the legs and arms. Schwann cells (SCs) are specialized cells that closely associate with many nerves and also undergo profound changes in DPN. Our hypothesis is that prolonged hyperglycemic stress alters the response of SCs to growth factors called neuregulins. In adult myelinated nerve, neuregulins can induce demyelination, which contributes to DPN. Using a cell culture model of myelinated nerve, we have identified that glucose increases the degenerative effects of neuregulins. Thus, the objectives of this research are to determine if diabetes affects the expression and activity of neuregulins in diabetic nerve from mice and to identify the molecular events by which neuregulins may induce nerve degeneration. The expected outcome of these studies is that we will identify molecular interactions that may enhance the therapeutic benefit of growth factors in the treatment of DPN.

DESCRIPTION (provided by applicant): The etiology of diabetic peripheral neuropathy (DPN) initiates from an inter-related series of metabolic and vascular insults that ultimately contribute to sensory neuron degeneration. In the quest to pharmacologically manage DPN, small molecule inhibitors have been developed to target proteins regarded as diabetes specific as well as those that increase in multiple disease states. Such efforts have not proven successful suggesting the identification of novel targets that play a fundamental role in regulating protein integrity and preserving nerve function in the diabetic state may represent a new paradigm. Heat shock protein 90 (Hsp90) is a molecular chaperone that binds client proteins and promotes their folding into biologically active structures. It is also the master regulator of a cytoprotective heat shock response, which aids the refolding of aggregated and damaged proteins that occur upon cell stress. Both the N- and C-terminal ATP binding domains of Hsp90 regulate its interaction with proteins. N-terminal inhibitors of Hsp90 exhibit potent cytotoxicity against tumor cells and are in clinical trials, but these compounds also induce a cytoprotective heat shock response at concentrations necessary for cytotoxicity. In contrast, we have developed potent small molecule inhibitors of the Hsp90 C-terminal domain whose neuroprotective efficacy is manifested at concentrations far below those necessary to induce neuro-toxicity. The lead compound for these inhibitors, KU- 32, is based upon novobiocin. KU-32 protects against hyperglycemia-induced death of sensory neurons and can attenuate several physiologic indices of DPN in mice through induction of the heat shock response. Unfortunately, this molecule requires significant synthetic preparation, thus preventing full elucidation of structure-activity relationships and limiting its use in animals/humans. Thus, the goal of this proposal is to provide new compounds derived from KU-32 that exhibit better neuroprotective activity and can be prepared in a minimal number of synthetic procedures. An initial screen will identify compounds with increased efficacy relative to KU-32 and lead candidates will be tested for protection against glycemic stress of sensory neurons, followed by animal studies of DPN in both wild-type and Hsp70 knockout mice. The outcome of this work will further develop and identify small molecule C-terminal Hsp90 inhibitors that decrease neurodegeneration in the absence of significant neurotoxicity. PUBLIC HEALTH RELEVANCE: Diabetic neuropathy is a common complication of diabetes that develops in about 60% of the approximately 24 million Americans afflicted with diabetes. Despite the impact of diabetic neuropathy on decreasing the quality of life, the existing FDA approved treatments are limited to drugs originally targeted to treat depression (Cymbalta) and convulsions (Lyrica). This project focuses on optimizing the effectiveness of a new class of therapeutics for the direct treatment of diabetic neuropathy.

DESCRIPTION (provided by applicant): The etiology of diabetic peripheral neuropathy (DPN) involves an inter-related series of metabolic and vascular insults that ultimately contribute to sensory neuron degeneration. In the quest to pharmacologically manage DPN, small molecule inhibitors have targeted proteins and pathways regarded as diabetes specific as well as others whose activity are altered in numerous disease states. These efforts have not yielded any significant therapies, due in part to the complicating issue that the biochemical contribution of these targets/pathways to the progression of DPN does not occur with temporal and/or biochemical uniformity between individuals. Thus, we have pursued the rational identification of a new molecular paradigm that offers a druggable target and provides translational potential for effective medical management of DPN at various stages of disease progression. In complex, chronic neurodegenerative diseases such as Alzheimer's disease and DPN, it is increasingly appreciated that effective disease management may not necessarily require targeting a pathway or protein considered to contribute to disease progression. Alternatively, it may prove beneficial to pharmacologically enhance the activity of endogenous neuroprotective pathways to aid neuronal recovery and stress tolerance. To this end, we have synthesized a novel small molecule that activates an endogenous cytoprotective response by inhibiting the molecular chaperone, heat shock protein 90 (Hsp90). Hsp90 is the master regulator of the cytoprotective heat shock response, which upregulates expression of Hsp70 and antioxidant genes. Our lead compound is a non-toxic, bioavailable molecule called KU-32 and we provide evidence that it reverses multiple clinical indices of DPN, promotes the recovery and reinnervation of damaged sensory fibers into the epidermis, increases mitochondrial bioenergetics and decreases oxidative stress in models of Type 1 and Type 2 diabetes. Mechanistically, inhibiting Hsp90 with KU-32 induces other chaperones such as cytosolic Hsp70 and mitochondrial Hsp70 (mtHsp70) in diabetic dorsal root ganglia. Importantly, the efficacy of KU-32 requires Hsp70 since the drug is ineffective in reversing DPN in diabetic Hsp70 KO mice. In response to a comprehensive set of preliminary data gathered from animal and primary cell models, our broad hypothesis is that modulating Hsp70 and its paralogs can rescue sensory neurons from hyperglycemic stress by antagonizing aspects of glucose-induced mitochondrial dysfunction. To address this hypothesis, aim one will identify if reversing the clinical indices of DPN by KU-32 requires an Hsp70-dependent increase in mitochondrial bioenergetics of sensory neurons. Aim 2 will determine if Hsp70 enhances mitochondrial bioenergetics by decreasing oxidative stress in adult sensory neurons. Aim 3 will identify if Hsp70 and mtHsp70 augment mitochondrial function by increasing protein import in diabetic neurons. The outcomes of our work will provide fundamental molecular insight into how Hsp70 paralogs improve sensory neuron bio- energetics and validate that modulating molecular chaperones is a viable approach to medically manage DPN.