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KU-32 (2) and KU-596 (3), are first and second generation cytoprotective novologues that are derivatives of novobiocin (1), a heat shock protein 90 (Hsp90) C-terminal inhibitor. Although 2 and 3 improve mitochondrial bioenergetics and have demonstrated considerable cytoprotective activity, they contain a synthetically demanding noviose sugar. This issue was initially addressed by creating noviomimetics, such as KU-1202 (4), which replaced the noviose sugar with ether-linked cyclohexyl derivatives that retained some cytoprotective potential due to their ability to increase mitochondrial bioenergetics. Based on structure-activity relationship (SAR) studies of KU-1202 (4), the current study investigated 3'- and 4'-substituted cyclohexyl scaffolds as noviomimetics and determined their efficacy at increasing mitochondrial bioenergetic as a marker for cytoprotective potential.

Diabetic peripheral neuropathy (DPN) is a complication of diabetes whose pathophysiology is linked to altered mitochondrial bioenergetics (mtBE). KU-596 is a small molecule neurotherapeutic that reverses symptoms of DPN, improves sensory neuron mtBE, and decreases the pro-oxidant protein, thioredoxin-interacting protein (Txnip) in a heat shock protein 70 (Hsp70)-dependent manner. However, the mechanism by which KU-596 improves mtBE and the role of Txnip in drug efficacy remains unknown. Mitophagy is a quality-control mechanism that selectively targets damaged mitochondria for degradation. The goal of this study was to determine if KU-596 therapy improved DPN, mtBE, and mitophagy in an Hsp70- and Txnip-dependent manner. Mito-QC (MQC) mice express a mitochondrially targeted mCherry-GFP fusion protein that enables visualizing mitophagy. Diabetic MQC, MQC × Hsp70 knockout (KO), and MQC × Txnip KO mice developed sensory and nerve conduction dysfunctions consistent with the onset of DPN. KU-596 therapy improved these measures, and this was dependent on Hsp70 but not Txnip. In MQC mice, diabetes decreased mtBE and increased mitophagy and KU-596 treatment reversed these effects. In contrast, KU-596 was unable to improve mtBE and decrease mitophagy in MQC × Hsp70 and MQC × Txnip KO mice. These data suggest that Txnip is not necessary for the development of the sensory symptoms and mitochondrial dysfunction induced by diabetes. KU-596 therapy may improve mitochondrial tolerance to diabetic stress to decrease mitophagic clearance in an Hsp70- and Txnip-dependent manner.

Heat shock protein 90 (Hsp90) is a chaperone under investigation for the treatment of cancer and neurodegenerative diseases. Neuroprotective Hsp90 C-terminal inhibitors derived from novobiocin (novologues) include KU-32 and KU-596. These novologues modulate molecular chaperones and result in an induction of Heat Shock Protein 70 (Hsp70). “Noviomimetics” replace the synthetically complex noviose sugar with a simple cyclohexyl moiety to maintain biological efficacy as compared to novologues KU-596 and KU-32. In this study, we further explore the development of noviomimetics and evaluate their efficacy using a luciferase refolding assay, immunoblot analysis, a c-jun assay, and an assay measuring mitochondrial bioenergetics. These new noviomimetics were designed and synthesized and were found to induce Hsp70 and result in improved biological activity. Noviomimetics 39e and 40a were found to potently induce Hsp70 and in very promising effects in cellular assays.

Increased expression of the c-jun transcription factor occurs in a variety of human neuropathies and is critical in promoting Schwann cell (SC) dedifferentiation and loss of the myelinated phenotype. Using cell culture models, we previously identified KU-32 as a novobiocin-based C-terminal heat shock protein 90 (Hsp90) inhibitor that decreased c-jun expression and the extent of demyelination. Additional chemical optimization has yielded KU-596 as a neuroprotective novologue whose mechanistic efficacy to improve a metabolic neuropathy requires the expression of Hsp70. The current study examined whether KU-596 therapy could decrease c-jun expression and improve motor function in an inducible transgenic model of a SC-specific demyelinating neuropathy (MPZ-Raf mice). Treating MPZ-Raf mice with tamoxifen activates the MAPK kinase pathway, increases c-jun expression and produces a profound demyelinating neuropathy characterized by a loss of motor function and paraparesis. KU-596 therapy did not interfere with MAPK activation but reduced c-jun expression, significantly improved motor performance, and ameliorated the extent of peripheral nerve demyelination in both prevention and intervention studies. Hsp70 was necessary for the drug’s neuroprotective efficacy since MPZ-Raf × Hsp70 knockout mice did not respond to KU-596 therapy. Collectively, our data indicate that modulating Hsp70 may provide a novel therapeutic approach to attenuate SC c-jun expression and ameliorate the onset of certain demyelinating neuropathies in humans.

Mitochondria damage plays a critical role in acetaminophen (APAP)-induced necrosis and liver injury. Cells can adapt and protect themselves by removing damaged mitochondria via mitophagy. PINK1-Parkin pathway is one of the major pathways that regulate mitophagy but its role in APAP-induced liver injury is still elusive. We investigated the role of PINK1-Parkin pathway in hepatocyte mitophagy in APAP-induced liver injury in mice. Wild-type (WT), PINK1 knockout (KO), Parkin KO, and PINK1 and Parkin double KO (DKO) mice were treated with APAP for different time points. Liver injury was determined by measuring serum alanine aminotransferase (ALT) activity, H&E staining as well as TUNEL staining of liver tissues. Tandem fluorescent-tagged inner mitochondrial membrane protein Cox8 (Cox8-GFP-mCherry) can be used to monitor mitophagy based on different pH stability of GFP and mCherry fluorescent proteins. We overexpressed Cox8-GFP-mCherry in mouse livers via tail vein injection of an adenovirus Cox8-GFP-mCherry. Mitophagy was assessed by confocal microscopy for Cox8-GFP-mCherry puncta, electron microscopy (EM) analysis for mitophagosomes and western blot analysis for mitochondrial proteins. Parkin KO and PINK1 KO mice improved the survival after treatment with APAP although the serum levels of ALT were not significantly different among PINK1 KO, Parkin KO and WT mice. We only found mild defects of mitophagy in PINK1 KO or Parkin KO mice after APAP, and improved survival in PINK1 KO and Parkin KO mice could be due to other functions of PINK1 and Parkin independent of mitophagy. In contrast, APAP-induced mitophagy was significantly impaired in PINK1-Parkin DKO mice. PINK1-Parkin DKO mice had further elevated serum levels of ALT and increased mortality after APAP administration. In conclusion, our results demonstrated that PINK1-Parkin signaling pathway plays a critical role in APAP-induced mitophagy and liver injury., Graphical abstract Image 1, Highlights • Cox8-GFP-mCherry is a novel molecular probe to monitor mitophagy in cultured hepatocytes and in mouse livers. . • Double deletion of PINK1 and Parking exacerbates acetaminophen-induced liver injury and mortality in mice.

Heat shock protein 90 (HSP90) is a molecular chaperone that is up-regulated in cancer and is required for the folding of numerous signaling proteins. Consequently, HSP90 represents an ideal target for the development of new anti-cancer agents. The human HSP90 isoform, glucose-regulated protein 94 (GRP94), resides in the endoplasmic reticulum and regulates secretory pathways, integrins, and Toll-like receptors, which contribute to regulating immunity and metastasis. However, the cellular function of GRP94 remains underinvestigated. We report that GRP94 knockdown cells are defective in intracellular transport and, consequently, negatively impact the trafficking of F-actin toward the cellular cortex, integrin α2 and integrin αL toward the cell membrane and filopodia, and secretory vesicles containing the HSP90α-AHA1-survivin complex toward the leading edge. As a result, GRP94 knockdown cells form a multipolar spindle instead of bipolar morphology and consequently manifest a defect in cell migration and adhesion.

The mammalian imprinted Dlk1-Gtl2 locus produces multiple non-coding RNAs (ncRNAs) from the maternally inherited allele, including the largest miRNA cluster in the mammalian genome. This locus has characterized functions in some types of stem cell, but its role in hematopoietic stem cells (HSCs) is unknown. Here, we show that the Dlk1-Gtl2 locus plays a critical role in preserving long-term repopulating HSCs (LT-HSCs). Through transcriptome profiling in 17 hematopoietic cell types, we found that ncRNAs expressed from the Dlk1-Gtl2 locus are predominantly enriched in fetal liver HSCs and the adult LT-HSC population and sustain long-term HSC functionality. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. This regulation in turn inhibits mitochondrial biogenesis and metabolic activity and protects LT-HSCs from excessive reactive oxygen species (ROS) production. Our data therefore show that the imprinted Dlk1-Gtl2 locus preserves LT-HSC function by restricting mitochondrial metabolism.

Neuronal mitochondrial dysfunction and oxidative stress are key pathophysiologic mechanisms of diabetic peripheral neuropathy (DPN). KU-596 is a small molecule modulator of heat shock protein 90 (Hsp90) that can reverse clinically relevant measures of DPN in diabetic animal models. Mechanistically, drug efficacy requires Hsp70 and correlates with improving mitochondrial maximal respiratory capacity (MRC) and decreasing oxidative stress in diabetic sensory neurons. The goal of this study was to determine if ex vivo treatment of diabetic neurons with KU-596 improves MRC by decreasing glucose-induced oxidative stress in an Hsp70-dependent manner. Sensory neurons were isolated from non-diabetic or diabetic mice wild type (WT) or Hsp70 knockout (Hsp70 KO) mice and treated with KU-596 in the presence of low or high glucose concentrations. In diabetic WT and Hsp70 KO neurons, hyperglycemia significantly increased superoxide levels, but KU-596 only decreased superoxide in WT neurons. Similarly, KU-596 significantly improved MRC in diabetic WT neurons maintained in high glucose but did not improve MRC in diabetic Hsp70 KO neurons under the same conditions. Since manganese superoxide dismutase (MnSOD) is the main mechanism to detoxify mitochondrial superoxide radicals, the cause and effect relationship between improved respiration and decreased oxidative stress was examined after knocking down MnSOD. Downregulating MnSOD in diabetic WT neurons increased hyperglycemia-induced superoxide levels, which was still significantly decreased by KU-596. However, KU-596 did not improve MRC following MnSOD knockdown. These data suggest that the ability of KU-596 to improve MRC is not necessarily dependent on decreasing mitochondrial superoxide in a MnSOD-dependent manner.

KU-596 is a second-generation C-terminal heat shock protein 90 KDa (Hsp90) modulator based on the natural product, novobiocin. KU-596 has been shown to induce Hsp70 levels and manifest neuroprotective activity through induction of the heat shock response. A ring-constrained analog of KU-596 was designed and synthesized to probe its binding orientation and ability to induce Hsp70 levels. Compound 2 was found to exhibit comparable or increased activity compared to KU-596, which is under clinical investigation for the treatment of neuropathy.

Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure-function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors.

NIDDK, JDRF, and the Diabetic Neuropathy Study Group of EASD sponsored a meeting to explore the current status of animal models of diabetic peripheral neuropathy. The goal of the workshop was to develop a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy. The discussion was divided into five areas: (1) status of commonly used rodent models of diabetes, (2) nerve structure, (3) electrophysiological assessments of nerve function, (4) behavioral assessments of nerve function, and (5) the role of biomarkers in disease phenotyping. Participants discussed the current understanding of each area, gold standards (if applicable) for assessments of function, improvements of existing techniques, and utility of known and exploratory biomarkers. The research opportunities in each area were outlined, providing a possible roadmap for future studies. The meeting concluded with a discussion on the merits and limitations of a unified approach to phenotyping rodent models of diabetic neuropathy and a consensus formed on the definition of the minimum criteria required for establishing the presence of the disease. A neuropathy phenotype in rodents was defined as the presence of statistically different values between diabetic and control animals in 2 of 3 assessments (nocifensive behavior, nerve conduction velocities, or nerve structure). The participants propose that this framework would allow different research groups to compare and share data, with an emphasis on data targeted toward the therapeutic efficacy of drug interventions.

Impaired neuronal mitochondrial bioenergetics contributes to the pathophysiologic progression of diabetic peripheral neuropathy (DPN) and may be a focal point for disease management. We have demonstrated that modulating heat shock protein (Hsp) 90 and Hsp70 with the small-molecule drug KU-32 ameliorates psychosensory, electrophysiologic, morphologic, and bioenergetic deficits of DPN in animal models of type 1 diabetes. The current study used mouse models of type 1 and type 2 diabetes to determine the relationship of changes in sensory neuron mitochondrial bioenergetics to the onset of and recovery from DPN. The onset of DPN showed a tight temporal correlation with a decrease in mitochondrial bioenergetics in a genetic model of type 2 diabetes. In contrast, sensory hypoalgesia developed 10 weeks before the occurrence of significant declines in sensory neuron mitochondrial bioenergetics in the type 1 model. KU-32 therapy improved mitochondrial bioenergetics in both the type 1 and type 2 models, and this tightly correlated with a decrease in DPN. Mechanistically, improved mitochondrial function following KU-32 therapy required Hsp70, since the drug was ineffective in diabetic Hsp70 knockout mice. Our data indicate that changes in mitochondrial bioenergetics may rapidly contribute to nerve dysfunction in type 2 diabetes, but not type 1 diabetes, and that modulating Hsp70 offers an effective approach toward correcting sensory neuron bioenergetic deficits and DPN in both type 1 and type 2 diabetes.

The chaperome constitutes a broad family of molecular chaperones and co-chaperones that facilitate the folding, refolding, and degradation of the proteome. Heat shock protein 90 (Hsp90) promotes the folding of numerous oncoproteins to aid survival of malignant phenotypes, and small molecule inhibitors of the Hsp90 chaperone complex offer a viable approach to treat certain cancers. One therapeutic attribute of this approach is the selectivity of these molecules to target high affinity oncogenic Hsp90 complexes present in tumor cells, which are absent in nontransformed cells. This selectivity has given rise to the idea that disease may contribute to forming a stress chaperome that is functionally distinct in its ability to interact with small molecule Hsp90 modulators. Consistent with this premise, modulating Hsp90 improves clinically relevant endpoints of diabetic peripheral neuropathy but has little impact in nondiabetic nerve. The concept of targeting the "diabetic chaperome" to treat diabetes and its complications is discussed.

The etiology of diabetic peripheral neuropathy (DPN) involves an interrelated 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. In a complex, chronic neurodegenerative disease such as 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 sufficiently beneficial to pharmacologically enhance the activity of endogenous cytoprotective pathways to aid neuronal tolerance to and recovery from glucotoxic stress. In pursuing this paradigm shift, we have shown that modulating the activity and expression of molecular chaperones such as heat shock protein 70 (Hsp70) may provide translational potential for the effective medical management of insensate DPN. Considerable evidence supports that modulating Hsp70 has beneficial effects in improving inflammation, oxidative stress, and glucose sensitivity. Given the emerging potential of modulating Hsp70 to manage DPN, the current review discusses efforts to characterize the cytoprotective effects of this protein and the benefits and limitations that may arise in drug development efforts that exploit its cytoprotective activity.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes that is associated with axonal atrophy, demyelination, blunted regenerative potential, and loss of peripheral nerve fibers. The development and progression of DPN is due in large part to hyperglycemia but is also affected by insulin deficiency and dyslipidemia. Although numerous biochemical mechanisms contribute to DPN, increased oxidative/nitrosative stress and mitochondrial dysfunction seem intimately associated with nerve dysfunction and diminished regenerative capacity. Despite advances in understanding the etiology of DPN, few approved therapies exist for the pharmacological management of painful or insensate DPN. Therefore, identifying novel therapeutic strategies remains paramount. Because DPN does not develop with either temporal or biochemical uniformity, its therapeutic management may benefit from a multifaceted approach that inhibits pathogenic mechanisms, manages inflammation, and increases cytoprotective responses. Finally, exercise has long been recognized as a part of the therapeutic management of diabetes, and exercise can delay and/or prevent the development of painful DPN. This review presents an overview of existing therapies that target both causal and symptomatic features of DPN and discusses the role of up-regulating cytoprotective pathways via modulating molecular chaperones. Overall, it may be unrealistic to expect that a single pharmacologic entity will suffice to ameliorate the multiple symptoms of human DPN. Thus, combinatorial therapies that target causal mechanisms and enhance endogenous reparative capacity may enhance nerve function and improve regeneration in DPN if they converge to decrease oxidative stress, improve mitochondrial bioenergetics, and increase response to trophic factors.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in which hyperglycemia-induced mitochondrial dysfunction and enhanced oxidative stress contribute to sensory neuron pathology. KU-32 is a novobiocin-based, C-terminal inhibitor of the molecular chaperone, heat shock protein 90 (Hsp90). KU-32 ameliorates multiple sensory deficits associated with the progression of DPN and protects unmyelinated sensory neurons from glucose-induced toxicity. Mechanistically, KU-32 increased the expression of Hsp70, and this protein was critical for drug efficacy in reversing DPN. However, it remained unclear if KU-32 had a broader effect on chaperone induction and if its efficacy was linked to improving mitochondrial dysfunction. Using cultures of hyperglycemically stressed primary sensory neurons, the present study investigated whether KU-32 had an effect on the translational induction of other chaperones and improved mitochondrial oxidative stress and bioenergetics. A variation of stable isotope labeling with amino acids in cell culture called pulse SILAC (pSILAC) was used to unbiasedly assess changes in protein translation. Hyperglycemia decreased the translation of numerous mitochondrial proteins that affect superoxide levels and respiratory activity. Importantly, this correlated with a decrease in mitochondrial oxygen consumption and an increase in superoxide levels. KU-32 increased the translation of Mn superoxide dismutase and several cytosolic and mitochondrial chaperones. Consistent with these changes, KU-32 decreased mitochondrial superoxide levels and significantly enhanced respiratory activity. These data indicate that efficacy of modulating molecular chaperones in DPN may be due in part to improved neuronal mitochondrial bioenergetics and decreased oxidative stress.

KU-32 is a novel, novobiocin-based Hsp90 inhibitor that protects against neuronal glucotoxicity and reverses multiple clinical indices of diabetic peripheral neuropathy in a rodent model. However, any drug with potential for treating diabetic complications must also have no adverse effects on the function of pancreatic islets. Thus, the goal of the current study was to assess the effect of KU-32 on thein vitroviability and function of human islets. Treating human islets with KU-32 for 24 hours showed no toxicity as assessed using the alamarBlue assay. Confocal microscopy confirmed that with a minimum of 2-day exposure, KU-32 improved cellular viability by blocking apoptosis. Functionally, isolated human islets released more glucose-stimulated insulin when preincubated in KU-32. However, diabetic BKS-db/db mice, a model for type 2 diabetes, administered KU-32 for 10 weeks did not show any significant changes in blood glucose and insulin levels, despite having greater insulin staining/beta cell in the pancreas compared to untreated BKS db/db mice. In summary, KU-32 did not harm isolated human islets and may even be protective. However, the effect does not appear significant enough to alter thein vivometabolic parameters of diabetic mice.

Previous studies have shown that herpes simplex virus type 1 (HSV-1) replication is inhibited by the cyclin-dependent kinase (cdk) inhibitor roscovitine. One roscovitine-sensitive cdk that functions in neurons is cdk5, which is activated in part by its binding partner, p35. Because HSV establishes latent infections in sensory neurons, we sought to determine the role p35 plays in HSV-1 replication in vivo. For these studies, wild-type (wt) and p35#x2212;/#x2212; mice were infected with HSV-1 using the mouse ocular model of HSV latency and reactivation. The current results indicate that p35 is an important determinant of viral replication in vivo.

OBJECTIVE Impairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome. RESEARCH DESIGN AND METHODS Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in mitochondria from dorsal root ganglia (DRG) of control, 22-week-old streptozotocin (STZ)-diabetic rats, and diabetic rats treated with insulin. Rates of oxygen consumption and complex activities in mitochondria from DRG were measured. Fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix-specific reactive oxygen species (ROS). RESULTS Proteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. For example, cytochrome c oxidase subunit IV (COX IV; a complex IV protein) and NADH dehydrogenase Fe-S protein 3 (NDUFS3; a complex I protein) were reduced by 29 and 36% (P < 0.05), respectively, in diabetes and confirmed previous Western blot studies. Respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin-induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control. CONCLUSIONS Abnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. Alternative pathways involving polyol pathway activity appear to contribute to raised ROS in axons of diabetic neurons under high glucose concentration.

Hyperglycemia-induced mitochondrial dysfunction contributes to sensory neuron pathology in diabetic neuropathy. Although Schwann cells (SCs) also undergo substantial degeneration in diabetic neuropathy, the effect of hyperglycemia on the SC mitochondrial proteome and mitochondrial function has not been examined. Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantify the temporal effect of hyperglycemia on the mitochondrial proteome of primary SCs isolated from neonatal rats. Of 317 mitochondrial proteins identified, about 78% were quantified and detected at multiple time points. Pathway analysis indicated that proteins associated with mitochondrial dysfunction, oxidative phosphorylation, the TCA cycle, and detoxification were significantly increased in expression and over-represented. Assessing mitochondrial respiration in intact SCs indicated that hyperglycemia increased the overall rate of oxygen consumption but decreased the efficiency of coupled respiration. Although a glucose-dependent increase in superoxide production occurs in embryonic sensory neurons, hyperglycemia did not induce a substantial change in superoxide levels in SCs. This correlated with a 1.9-fold increase in Mn superoxide dismutase expression, which was confirmed by immunoblot and enzymatic activity assays. These data support that hyperglycemia alters mitochondrial respiration and can cause remodeling of the SC mitochondrial proteome independent of significant contributions from glucose-induced superoxide production.

OBJECTIVE Evaluate if Erb B2 activation and the loss of caveolin-1 (Cav1) contribute to the pathophysiological progression of diabetic peripheral neuropathy (DPN). RESEARCH DESIGN AND METHODS Cav1 knockout and wild-type C57BL/6 mice were rendered diabetic with streptozotocin, and changes in motor nerve conduction velocity (MNCV), mechanical and thermal hypoalgesia, Erb B2 phosphorylation (pErb B2), and epidermal nerve fiber density were assessed. The contribution of Erb B2 to DPN was assessed using the Erb B2 inhibitors PKI 166 and erlotinib and a conditional bitransgenic mouse that expressed a constitutively active form of Erb B2 in myelinated Schwann cells (SCs). RESULTS Diabetic mice exhibited decreased MNCV and mechanical and thermal sensitivity, but the extent of these deficits was more severe in diabetic Cav1 knockout mice. Diabetes increased pErb B2 levels in both genotypes, but the absence of Cav1 correlated with a greater increase in pErb B2. Erb B2 activation contributed to the mechanical hypoalgesia and MNCV deficits in both diabetic genotypes because treatment with erlotinib or PKI 166 improved these indexes of DPN. Similarly, induction of a constitutively active Erb B2 in myelinated SCs was sufficient to decrease MNCV and induce a mechanical hypoalgesia in the absence of diabetes. CONCLUSIONS Increased Erb B2 activity contributes to specific indexes of DPN, and Cav1 may be an endogenous regulator of Erb B2 signaling. Altered Erb B2 signaling is a novel mechanism that contributes to SC dysfunction in diabetes, and inhibiting Erb B2 may ameliorate deficits of tactile sensitivity in DPN.

Neuregulins (NRGs) are growth factors which bind to Erb receptor tyrosine kinases that localize to Schwann cells (SCs). Although NRGs can promote cell survival, mitogenesis, and myelination in undifferentiated SCs, they also induce demyelination of myelinated co-cultures of SCs and dorsal root ganglion (DRG) neurons. We have shown previously that Erb B2 activity increased in premyelinating SCs in response to hyperglycemia, and that this correlated with the downregulation of the protein caveolin-1 (Cav-1). As myelinated SCs undergo substantial degeneration in diabetic neuropathy, we used myelinated SC/DRG neuron co-cultures to determine if hyperglycemia and changes in Cav-1 expression could enhance NRG-induced demyelination. In basal glucose, NRG1 caused a 2.4-fold increase in the number of damaged myelin segments. This damage reached 3.8-fold under hyperglycemic conditions, and was also associated with a robust decrease in the expression of Cav-1 and compact myelin proteins. The loss of Cav-1 and compact myelin proteins following hyperglycemia and NRG treatment was not due to neuronal loss, since the axons remained intact and there was no loss of PGP 9.5, an axonal marker protein. To examine if changes in Cav-1 were sufficient to alter the extent of NRG-induced demyelination, SC/DRG neurons co-cultures were infected with antisense or dominant-negative Cav-1(P132L) adenoviruses. Either antisense-mediated downregulation or mis-localization of endogenous Cav-1 by Cav-1(P132L) resulted in a 1.5- to 2.4-fold increase in NRG-induced degeneration compared to that present in control cultures. These data support that hyperglycemia and changes in Cav-1 are sufficient to sensitize myelinated SC/DRG co-cultures to NRG-induced demyelination.

Ceramide is a bioactive sphingolipid that can prevent calpain activation and beta-amyloid (A beta) neurotoxicity in cortical neurons. Recent evidence supports A beta induction of a calpain-dependent cleavage of the cyclin-dependent kinase 5 (cdk5) regulatory protein p35 that contributes to tau hyperphosphorylation and neuronal death. Using cortical neurons isolated from wild-type and p35 knockout mice, we investigated whether ceramide required p35/cdk5 to protect against A beta-induced cell death and tau phosphorylation. Ceramide inhibited A beta-induced calpain activation and cdk5 activity in wild-type neurons and protected against neuronal death and tau hyperphosphorylation. Interestingly, A beta also increased cdk5 activity in p35-/- neurons, suggesting that the alternate cdk5 regulatory protein, p39, might mediate this effect. In p35 null neurons, ceramide blocked A beta-induced calpain activation but did not inhibit cdk5 activity or cell death. However, ceramide blocked tau hyperphosphorylation potentially via inhibition of glycogen synthase kinase-3beta. These data suggest that ceramide can regulate A beta cell toxicity in a p35/cdk5-dependent manner.

KU-32 and KU-596 are novobiocin-derived, C-terminal heat shock protein 90 (Hsp90) modulators that induce Hsp70 levels and manifest neuroprotective activity. However, the synthetically complex noviose sugar requires 10 steps to prepare, which makes translational development difficult. In this study, we developed a series of “noviomimetic” analogues of KU-596, which contain noviose surrogates that can be easily prepared, while maintaining the ability to induce Hsp70 levels. Both sugar and sugar analogues were designed, synthesized, and evaluated in a luciferase reporter assay, which identified compound 37, a benzyl containing noviomimetic, as the most potent inducer of Hsp70.

We have previously demonstrated that modulating molecular chaperones with KU-32, a novobiocin derivative, ameliorates physiologic and bioenergetic deficits of diabetic peripheral neuropathy (DPN). Replacing the coumarin core of KU-32 with a meta-fluorinated biphenyl ring system created KU-596, a novobiocin analogue (novologue) that showed neuroprotective activity in a cell-based assay. The current study sought to determine whether KU-596 offers similar therapeutic potential for treating DPN. Administration of 2-20 mg/kg of KU-596 improved diabetes induced hypoalgesia and sensory neuron bioenergetic deficits in a dose-dependent manner. However, the drug could not improve these neuropathic deficits in diabetic heat shock protein 70 knockout (Hsp70 KO) mice. To gain further insight into the mechanisms by which KU-596 improved DPN, we performed transcriptomic analysis of sensory neuron RNA obtained from diabetic wild-type and Hsp70 KO mice using RNA sequencing. Bioinformatic analysis of the differentially expressed genes indicated that diabetes strongly increased inflammatory pathways and that KU-596 therapy effectively reversed these increases independent of Hsp70. In contrast, the effects of KU-596 on decreasing the expression of genes regulating the production of reactive oxygen species were more Hsp70-dependent. These data indicate that modulation of molecular chaperones by novologue therapy offers an effective approach toward correcting nerve dysfunction in DPN but that normalization of inflammatory pathways alone by novologue therapy seems to be insufficient to reverse sensory deficits associated with insensate DPN.

Diabetic peripheral neuropathy (DPN) is a frequent and potentially traumatic complication in diabetic individuals. The chronic nature of diabetes and its associated hyperglycemic episodes initiate a complex and inter-related series of metabolic and vascular insults that contribute to the polygenic etiology of DPN. One contributing factor in DPN is an altered neurotrophism that results from changes in the synthesis and expression of neurotrophins, insulin-like growth factor, and various cytokine-like growth factors that can directly act upon distinct subpopulations of sensory and motor neurons. Although considerable effort has focused upon examining growth factor signaling in hyperglycemically stressed neurons, myelin-forming Schwann cells also undergo substantial degenerative changes in DPN. However, scant attention has been devoted to understanding the effect of hyperglycemia on the response of Schwann cells to growth factors critical to their function. Neuregulins are gliotrophic growth factors that signal through members of the Erb B receptor-tyrosine kinase family. The neuregulin/Erb B ligand-receptor cassette can differentially influence the response of Schwann cells throughout their development by regulating cell survival, mitogenesis, and differentiation. The activity of Erb B receptors may also be affected by their interaction with caveolin-1, a protein marker of caveolae ("little caves"). However, whether neuregulin signaling may be directly or indirectly altered under conditions of hyperglycemic stress and contribute to the physiological progression of DPN is unknown. This brief review will provide a perspective on a putative role of changes in the caveolar proteome of Schwann cells in contributing to an "altered neuregulinism" in DPN.

Multiple studies suggest that lipid oversupply to skeletal muscle contributes to the development of insulin resistance, perhaps by promoting the accumulation of lipid metabolites capable of inhibiting signal transduction. Herein we demonstrate that exposing muscle cells to particular saturated free fatty acids (FFAs), but not mono-unsaturated FFAs, inhibits insulin stimulation of Akt/protein kinase B, a serine/threonine kinase that is a central mediator of insulin-stimulated anabolic metabolism. These saturated FFAs concomitantly induced the accumulation of ceramide and diacylglycerol, two products of fatty acyl-CoA that have been shown to accumulate in insulin-resistant tissues and to inhibit early steps in insulin signaling. Preventing de novo ceramide synthesis negated the antagonistic effect of saturated FFAs toward Akt/protein kinase B. Moreover, inducing ceramide buildup recapitulated and augmented the inhibitory effect of saturated FFAs. By contrast, diacylglycerol proved dispensable for these FFA effects. Collectively these results identify ceramide as a necessary and sufficient intermediate linking saturated fats to the inhibition of insulin signaling.

One hallmark of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid-beta (Abeta) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Abeta toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Abeta peptides, inhibits Abeta-induced activation of cyclin-dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Abeta-induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Abeta-induced activation of the cytosolic cdk5-p25 complex, but not the membrane-associated cdk5-p35 complex. MT-stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood-brain barrier, we assessed the potential of taxanes to attenuate Abeta toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood-brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Abeta-induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT-stabilizing agents may provide a therapeutic approach to decrease Abeta toxicity and neurofibrillary pathology in AD and other tauopathies.

The mechanism of crosstalk between signaling pathways coupled to the Trk A and p75NTR neurotrophin receptors in PC12 cells was examined. In response to nerve growth factor (NGF), Trk A activation inhibited p75NTR-dependent sphingomyelin (SM) hydrolysis. The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, LY294002, reversed this inhibition suggesting that Trk A activation of PI 3-kinase is necessary to inhibit sphingolipid signaling by p75NTR. In contrast, SM hydrolysis induced by neurotrophin-3 (NT-3), which did not activate PI-3 kinase, was uneffected by LY294002. However, transient expression of a constituitively active PI 3-kinase inhibited p75NTR-dependent SM hydrolysis by both NGF and NT-3. Intriguingly, NGF induced an association of activated PI 3-kinase with acid sphingomyelinase (SMase). This interaction localized to caveolae-related domains and correlated with a 50% decrease in immunoprecipitated acid SMase activity. NGF-stimulated PI 3-kinase activity was necessary for inhibition of acid SMase but was not required for ligand–induced association of the p85 subunit of PI 3-kinase with the phospholipase. Finally, this interaction was specific for NGF since EGF did not induce an association of PI 3-kinase with acid SMase. In summary, our data suggest that PI 3-kinase regulates the inhibitory crosstalk between Trk A tyrosine kinase and p75NTR-dependent sphingolipid signaling pathways and that this interaction localizes to caveolae-related domains.

Ceramide is a novel lipid mediator involved in regulating cell growth, cell differentiation and cell death. Many studies have focused on characterizing the stimulus-induced production of ceramide and identifying putative downstream molecular targets. However, little remains known about the localization of the regulated production of ceramide through sphingomyelin metabolism in the plasma membrane. Additionally, it is unclear whether a localized increase in ceramide concentration is necessary to facilitate downstream signalling events initiated by this lipid. Recent studies have suggested that detergent-insoluble plasma membrane domains may be highly localized sites for initiating signal transduction cascades by both tyrosine kinase and sphingolipid signalling pathways. These domains are typically enriched in both sphingolipids and cholesterol and have been proposed to form highly ordered lipid rafts floating in a sea of glycerophospholipids. Alternatively, upon integration of the cholesterol binding protein caveolin, these domains may also form small cave-like structures called caveolae. Emerging evidence suggests that the enhanced sphingomyelin content of these lipid domains make them potential substrate pools for sphingomyelinases to produce a high local concentration of ceramide. The subsequent formation of ceramide microdomains in the plasma membrane may be a critical factor in regulating downstream signalling through this lipid messenger.

The recent recognition that the p75 neurotrophin receptor, p75((NTR)), can induce apoptotic signals has contributed to the perception that it acts primarily as a death receptor. Although the molecular mechanisms of p75(NTR) signaling remain to be fully characterized, many of the currently identified pathways activated by p75(NTR) may be generally characterized as stress response signals. This review describes recent advances in identifying the molecular components involved in p75(NTR) signal transduction and suggests that p75(NTR) signaling may more aptly serve as a general mechanism for the transduction and modulation of stress signals.

Neurotrophins signal through Trk tyrosine kinase receptors and the low-affinity neurotrophin receptor p75(NTR). We have shown previously that activation of Trk A tyrosine kinase activity can inhibit p75(NTR)-dependent sphingomyelin hydrolysis, that caveolae are a localized site for p75(NTR) signaling, and that caveolin can directly interact with p75(NTR). The ability of caveolin to also interact with tyrosine kinase receptors and inhibit their activity led us to hypothesize that caveolin expression may modulate interactions between neurotrophin signaling pathways. PC12 cells were transfected with caveolin that was expressed efficiently and targeted to the appropriate membrane domains. Upon exposure to nerve growth factor (NGF), caveolin-PC12 cells were unable to develop extensive neuritic processes. Caveolin expression in PC12 cells was found to diminish the magnitude and duration of Trk A activation in vivo. This inhibition may be due to a direct interaction of caveolin with Trk A, because Trk A co-immunoprecipitated with caveolin from Cav-Trk A-PC12 cells, and a glutathione S-transferase-caveolin fusion protein bound to Trk A and inhibited NGF-induced autophosphorylation in vitro. Furthermore, the in vivo kinetics of the inhibition of Trk A tyrosine kinase activity by caveolin expression correlated with an increased ability of NGF to induce sphingomyelin hydrolysis through p75(NTR). In summary, our results suggest that the interaction of caveolin with neurotrophin receptors may have functional consequences in regulating signaling through p75(NTR) and Trk A in neuronal and glial cell populations.

We examined neurotrophin-induced sphingomyelin hydrolysis in cells expressing solely the low affinity neurotrophin receptor, p75NTR, and in PC12 cells that co-express p75NTR and Trk receptors. Nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and NT-5 stimulated sphingomyelin hydrolysis with similar kinetics in p75NTR-NIH-3T3 cells. Although brain-derived neurotrophic factor (10 ng/ml) was slightly more potent than NGF at inducing sphingomyelin hydrolysis, NT-3 and NT-5 induced significant hydrolysis (30-35%) at 0.1 to 1 ng/ml in p75NTR-NIH-3T3 cells. NT-3 did not induce sphingomyelin hydrolysis in Trk C-NIH-3T3 cells nor in cells expressing a mutated p75NTR containing a 57-amino acid cytoplasmic deletion, thus demonstrating the role of p75NTR in this signal transduction pathway. In p75NTR-NIH-3T3 cells, neurotrophin-induced sphingomyelin hydrolysis 1) localized to an internal pool of sphingomyelin, 2) was not a consequence of receptor internalization, and 3) showed no specificity with respect to the molecular species of sphingomyelin hydrolyzed. In contrast to cells expressing solely p75NTR, NGF (100 ng/ml) did not induce sphingomyelin hydrolysis in PC12 cells. Interestingly, NT-3 (10 ng/ml) induced the same extent of sphingomyelin hydrolysis in PC12 cells as was apparent in p75NTR-NIH-3T3 cells. However, in the presence of NGF, NT-3 was unable to induce sphingomyelin hydrolysis, raising the possibility that Trk was modulating p75NTR-dependent sphingomyelin hydrolysis. Inhibition of Trk tyrosine kinase activity with 200 nM K252a enabled both NGF and NT-3 in the presence of NGF to induce sphingomyelin hydrolysis. These data support that p75NTR serves as a common signaling receptor for neurotrophins through induction of sphingomyelin hydrolysis and that cross-talk pathways exist between Trk and p75NTR-dependent signaling pathways.

Modulating molecular chaperones is emerging as an attractive approach to treat neurodegenerative diseases associated with protein aggregation, DPN (diabetic peripheral neuropathy) and possibly, demyelinating neuropathies. KU-32 [ N-(7-((2 R,3 R,4 S,5 R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2 H-pyran-2-yloxy)-8-methyl-2-oxo-2H-chromen-3-yl)acetamide] is a small molecule inhibitor of Hsp90 (heat shock protein 90) and reverses sensory deficits associated with myelinated fibre dysfunction in DPN. Additionally, KU-32 prevented the loss of myelinated internodes induced by treating myelinated SC (Schwann cell)-DRG (dorsal root ganglia) sensory neuron co-cultures with NRG1 (neuregulin-1 Type 1). Since KU-32 decreased NRG1-induced demyelination in an Hsp70-dependent manner, the goal of the current study was to clarify how Hsp70 may be mechanistically linked to preventing demyelination. The activation of p42/p44 MAPK (mitogen-activated protein kinase) and induction of the transcription factor c-Jun serve as negative regulators of myelination. NRG1 activated MAPK, induced c-Jun expression and promoted a loss of myelin segments in DRG explants isolated from both WT (wild-type) and Hsp70 KO (knockout) mice. Although KU-32 did not block the activation of MAPK, it blocked c-Jun induction and protected against a loss of myelinated segments in WT mice. In contrast, KU-32 did not prevent the NRG1-dependent induction of c-Jun and loss of myelin segments in explants from Hsp70 KO mice. Overexpression of Hsp70 in myelinated DRG explants prepared from WT or Hsp70 KO mice was sufficient to block the induction of c-Jun and the loss of myelin segments induced by NRG1. Lastly, inhibiting the proteasome prevented KU-32 from decreasing c-Jun levels. Collectively, these data support that Hsp70 induction is sufficient to prevent NRG1-induced demyelination by enhancing the proteasomal degradation of c-Jun.

Compound 2 (KU-32) is a first-generation novologue (a novobiocin-based, C-terminal, heat shock protein 90 (Hsp90) inhibitor) that decreases glucose-induced death of primary sensory neurons and reverses numerous clinical indices of diabetic peripheral neuropathy in mice. The current study sought to exploit the C-terminal binding site of Hsp90 to determine whether the optimization of hydrogen bonding and hydrophobic interactions of second-generation novologues could enhance neuroprotective activity. Using a series of substituted phenylboronic acids to replace the coumarin lactone of 2, we identified that electronegative atoms placed at the meta-position of the B-ring exhibit improved cytoprotective activity, which is believed to result from favorable interactions with Lys539 in the Hsp90 C-terminal binding pocket. Consistent with these results, a meta-3-fluorophenyl substituted novologue (13b) exhibited a 14-fold lower ED(50) for protection against glucose-induced toxicity of primary sensory neurons compared to 2.

Quantification of intra-epidermal nerve fibers (iENFs) is an important approach to stage diabetic peripheral neuropathy (DPN) and is a promising clinical endpoint for identifying beneficial therapeutics. Mechanistically, diabetes decreases neuronal mitochondrial function and enhancing mitochondrial respiratory capacity may aid neuronal recovery from glucotoxic insults. We have proposed that modulating the activity and expression of heat shock proteins (Hsp) may be of benefit in treating DPN. KU-32 is a C-terminal Hsp90 inhibitor that improved thermal hypoalgesia in diabetic C57Bl/6 mice but it was not determined if this was associated with an increase in iENF density and mitochondrial function. After 16 weeks of diabetes, Swiss Webster mice showed decreased electrophysiological and psychosensory responses and a >30% loss of iENFs. Treatment of the mice with ten weekly doses of 20 mg/kg KU-32 significantly reversed pre-existing deficits in nerve conduction velocity and responses to mechanical and thermal stimuli. KU-32 therapy significantly reversed the pre-existing loss of iENFs despite the identification of a sub-group of drug-treated diabetic mice that showed improved thermal sensitivity but no increase in iENF density. To determine if the improved clinical indices correlated with enhanced mitochondrial activity, sensory neurons were isolated and mitochondrial bioenergetics assessed ex vivo using extracellular flux technology. Diabetes decreased maximal respiratory capacity in sensory neurons and this deficit was improved following KU-32 treatment. In conclusion, KU-32 improved physiological and morphologic markers of degenerative neuropathy and drug efficacy may be related to enhanced mitochondrial bioenergetics in sensory neurons.

1.1 Diabetic peripheral neuropathy Diabetes Mellitus (DM) is estimated to affect 347 million patients globally as of 2008 (Danaei et al., 2011) and this number is expected to double by 2030 (Wild et al., 2004). DM results from the failure of our body to generate and/or respond to insulin, the principal hormone regulating uptake of glucose from the blood stream, leading to abnormally high blood glucose levels, termed hyperglycemia. Sensory neurons absorb glucose as a direct function of extracellular glucose concentration instead of insulin-mediated glucose uptake and are particularly susceptible to the cumulative metabolic insults imposed by chronic hyperglycemia (Tomlinson and Gardiner, 2008). This damage leads to secondary microvascular complications that contribute to the progression of diabetic neuropathy. Diabetic peripheral neuropathy (DPN) is the most prevalent complication of DM (Tahrani et al., 2010), also comprises the most common form of peripheral neuropathy globally, surpassing leprosy in this aspect (Harati, 2010; Martyn and Hughes, 1997). Depending on the case definitions used, 30-70% of patients with either type 1 or type 2 DM are diagnosed with some form of peripheral neuropathy (National Institutes of Diabetes and Digestive and Kidney Diseases). A distal symmetric sensorimotor polyneuropathy is the most frequent manifestation of DPN. Patients with this form of DPN are predisposed to foot ulceration and increased risk of amputation; ulcerative complications from DPN account for approximately 87% of non-traumatic lower extremity amputations. In addition to this traumatic medical event, DPN is also a major contributing factor to the development of joint deformities, limb threatening ischemia as well as other various neurological dysfunctions (Harati, 2010). This greatly reduces the quality of life in people with DM through increased disability and is assuming more hospitalizations than all other diabetic complications combined (Mahmood et al., 2009). As a consequence, approximately $15 billion are spent on DPN annually in the US, causing a major drain on healthcare expenditure (Rathmann and Ward, 2003). With the staggering individual, social and economic burden brought about by DPN-associated morbidity and mortality, development of effective treatments that prevent and/or reverse DPN are needed but currently unmet.

The role of the low-affinity neurotrophin receptor (p75NTR) in signal transduction is undefined. Nerve growth factor can activate the sphingomyelin cycle, generating the putative-lipid second messenger ceramide. In T9 glioma cells, addition of a cell-permeable ceramide analog mimicked the effects of nerve growth factor on cell growth inhibition and process formation. This signaling pathway appears to be mediated by p75NTR in T9 cells and NIH 3T3 cells overexpressing p75NTR. Expression of an epidermal growth factor receptor-p75NTR chimera in T9 cells imparted to epidermal growth factor the ability to activate the sphingomyelin cycle. These data demonstrate that p75NTR is capable of signaling independently of the trk neurotrophin receptor (p140trk) and that ceramide may be a mediator in neurotrophin biology.

Extracellular agonists such as tumor necrosis factor-alpha (TNF-alpha) activate the sphingomyelin cycle leading to the generation of ceramide. Ceramide has been suggested as an important mediator of the effect of TNF-alpha on growth inhibition, c-myc down-regulation, apoptosis, and the activation of the nuclear factor kappa B. Although there is no clearly defined intracellular target for ceramide activity, previous studies have demonstrated the existence of a ceramide-activated protein phosphatase (CAPP) in vitro. Since c-myc is an early downstream cellular target for TNF-alpha, we examined the role of ceramide and CAPP in c-myc down-regulation. In intact HL-60 cells ceramide induced down-regulation of c-myc RNA levels. C2-ceramide was active at 1-10 microM and caused 40-80% inhibition of c-myc RNA levels at 30-120 min of treatment. In nuclear run-on studies, C2-ceramide induced a block to transcription elongation of the c-myc transcript without affecting transcription through the first exon. Therefore, ceramide appeared to inhibit c-myc expression via a mechanism identical with that of TNF-alpha. HL-60 cells contained CAPP which was inhibited by okadaic acid (0.1-10 nm). CAPP in HL-60 cells was activated by D-erythro-ceramide but not D-erythro-dihydroceramide. The specificity of activation of CAPP by ceramide in vitro was matched by a similar specificity of c-myc down-regulation in cells. Moreover, okadaic acid inhibited the effects of ceramide and TNF-alpha on c-myc down-regulation. On the other hand, okadaic acid did not inhibit the ability of phorbol 12-myristate 13-acetate to down-regulate c-myc, demonstrating the existence of at least two distinct pathways in the regulation of c-myc expression. These results demonstrate that CAPP is important for ceramide-induced down-regulation of c-myc in myeloid leukemia cells. The implications of these findings in further delineating a sphingomyelin signaling pathway with important anti-proliferative effects are discussed.

Dysfunctional insulin and insulin-like growth factor-I (IGF-I) signaling contributes to the pathological progression of diabetes, diabetic peripheral neuropathy (DPN), Alzheimer's (AD), Parkinson's (PD) and Huntington's diseases (HD). Despite their prevalence, there are limited therapeutic options available for the treatment of these neurodegenerative disorders. Therefore, establishing a link between insulin/IGF-I and the pathoetiology of these diseases may provide alternative approaches toward their management. Many of the heat shock proteins (Hsps) are well-known molecular chaperones that solubilize and clear damaged proteins and protein aggregates. Recent studies suggest that modulating Hsps may represent a promising therapeutic avenue for improving insulin and IGF-I signaling. Pharmacological induction of the heat shock response (HSR) may intersect with insulin/IGF-I signaling to improve aspects of neurodegenerative phenotypes. Herein, we review the intersection between Hsps and the insulin/IGF systems under normal and pathological conditions. The discussion will emphasize the potential of non-toxic HSR inducers as viable therapeutic agents.

Diabetic neuropathy is a major complication of diabetes that results in the progressive deterioration of the sensory nervous system. Mitochondrial dysfunction has been proposed to play an important role in the pathogenesis of the neurodegeneration observed in diabetic neuropathy. Our recent work has shown that mitochondrial dysfunction occurs in dorsal root ganglia (DRG) sensory neurons in streptozotocin (STZ) induced diabetic rodents. In neurons, the nutrient excess associated with prolonged diabetes may trigger a switching off of AMP kinase (AMPK) and/or silent information regulator T1 (SIRT1) signaling leading to impaired peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α) expression/activity and diminished mitochondrial activity. This review briefly summarizes the alterations of mitochondrial function and proteome in sensory neurons of STZ-diabetic rodents. We also discuss the possible involvement of AMPK/SIRT/PGC-1α pathway in other diabetic models and different tissues affected by diabetes.

Ceramide activates a cytosolic protein phosphatase present in rat T9 glioma cells and rat brain. Ceramide-activated protein phosphatase (CAPP) was found to share several properties with protein phosphatase 2A (PP2A) leading to the hypothesis that ceramide may directly activate PP2A. PP2A was isolated as a heterotrimer (AB'C, AB alpha C), heterodimer (AC), or free C subunit, and the effect of ceramide on the catalytic activity was assessed. C2-ceramide, 5-20 microM, activated heterotrimeric PP2A up to 3.5-fold but had no effect on the activity of AC or C. Ceramides possessing hexanoyl, decanoyl, and myristoyl but not stearoyl acyl chains also activated heterotrimeric PP2A. Ceramide activation of heterotrimeric PP2A required the presence of a B subunit since trypsinization or heparin treatment abolished ceramide activation. Activation of heterotrimeric PP2A was specific for ceramide because related sphingolipids had no effect. Moreover, dihydro-C2-ceramide, which lacks the trans double bond in the sphingoid base, inhibited AB'C activity by > 90% at 10 microM. The specificity of activation of AB'C and AB alpha C by stereoisomers of C2-ceramide was found to differ. Whereas activation of AB'C by either DL-erythro- or threo-C2-ceramide was similar, AB alpha C was activated by either D- or L-erythro-C2-ceramide but not by the threo isomers. CAPP isolated from T9 cells was most effectively activated by D-erythro-C2-ceramide. CAPP was found to possess two peaks of ceramide activated phosphatase activity. The initial peak of activity was coincident with the elution of AB'C and was stimulated 1.8-fold by 20 microM C2-ceramide. A second peak of phosphatase activity was negligible in the absence of ceramide but was stimulated 5.5-fold by 20 microM C2-ceramide. These results support the hypothesis that ceramide is a specific lipid second messenger modulating heterotrimeric PP2A activity.

Ceramide is emerging as a potential physiologic regulator of growth and differentiation in mammalian cells. This regulation may be mediated through the action of a serine/threonine ceramide-activated protein phosphatase (CAPP). In this study, the existence of a ceramide-mediated pathway of cell regulation in Saccharomyces cerevisiae was investigated. Incubating exponentially growing S. cerevisiae cells with 1-20 microM cell-permeable ceramide (C2-ceramide) produced a dose-dependent inhibition of proliferation. A number of other lipids and detergents, such as arachidonate, oleate, Triton X-100, dioctanoylglycerol, and phenylaminoalcohol ceramide analogs, were largely ineffective, demonstrating the specificity of the response. Stereospecificity was demonstrated, in that the D enantiomer of erythro-C2-ceramide was more potent than the L enantiomer. More dramatically, a highly specific structural requirement for C2-ceramide was demonstrated, in that 1-12 microM C2-dihydroceramide was completely ineffective at inhibiting growth. Since C2-dihydroceramide lacks the 4-5 trans double bond present in C2-ceramide, this suggests that the antiproliferative properties of C2-ceramide depend upon the presence of the double bond. This raises an interesting possibility; the dehydrogenase responsible for introduction of the double bond during endogenous ceramide synthesis may regulate cell growth by controlling the cellular concentrations of dihydroceramide and ceramide. The oxygenase responsible for introduction of the final hydroxyl group in phytoceramide could provide a similar regulatory function in yeast. The potential role of CAPP in ceramide action in yeast was investigated next. Crude extracts of S. cerevisiae also contained a ceramide-dependent serine/threonine phosphatase activity, which was sensitive to inhibition by okadaic acid. This enzyme exhibited stereospecificity and structural requirements identical to that of the ceramide-induced growth inhibition. We conclude that both the growth-inhibitory response to ceramide and CAPP activity are conserved in S. cerevisiae. The identical stereochemical and structural requirements in both the biological and phosphatase assays suggest that the anti-proliferative effects of ceramide in yeast may be mediated in part through the action of CAPP.

Increasing the expression of Hsp70 (heat-shock protein 70) can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy) is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2 R, 3 R, 4S, 5 R)-3, 4-dihydroxy-5-methoxy-6, 6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide) was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia) neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type) mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout) mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity) and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin) or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.

Previous research suggests that brain oxidative stress and altered rodent locomotor behavior are linked. We observed bio-behavioral changes in methionine sulfoxide reductase A knockout mice associated with abnormal dopamine signaling. Compromised ability of these knockout mice to reduce methionine sulfoxide enhances accumulation of sulfoxides in proteins. We examined the dopamine D(2)-receptor function and expression, which has an atypical arrangement and quantity of methionine residues. Indeed, protein expression levels of dopamine D(2)-receptor were higher in knockout mice compared with wild-type. However, the binding of dopamine D(2)-receptor agonist was compromised in the same fractions of knockout mice. Coupling efficiency of dopamine D(2)-receptors to G-proteins was also significantly reduced in knockout mice, supporting the compromised agonist binding. Furthermore, pre-synaptic dopamine release in knockout striatal sections was less responsive than control sections to dopamine D(2)-receptor ligands. Behaviorally, the locomotor activity of knockout mice was less responsive to the inhibitory effect of quinpirole than wild-type mice. Involvement of specific methionine residue oxidation in the dopamine D(2)-receptor third intracellular loop is suggested by in vitro studies. We conclude that ablation of methionine sulfoxide reductase can affect dopamine signaling through altering dopamine D(2)-receptor physiology and may be related to symptoms associated with neurological disorders and diseases.

Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(amino-methyl)benzenesulfonic acid (ABS) in the presence of K(3)Fe(CN)(6) to form a highly fluorescent 2-phenylbenzoxazole product. Synthetic 3-NT peptide (0.005-1 μM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 μM K(3)Fe(CN)(6) in 0.1 M Na(2)HPO(4) buffer (pH 9.0) was converted with yields95% to a single fluorescent product incorporating two ABS molecules per 3-NT residue, with fluorescence excitation and emission maxima at 360 ± 2 and 490 ± 2 nm, respectively, and a quantum yield of 0.77 ± 0.08, based on reverse-phase LC with UV and fluorescence detection, fluorescence spectroscopy and LC-MS-MS analysis. This protocol was successfully tested for quantitative analysis of in vitro Tyr nitration in a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from C2C12 cultured cells exposed to peroxynitrite, with a detection limit of ca. 1 pmol 3-NT by fluorescence spectrometry, and an apparent LOD of 12 and 40 pmol for nitropeptides alone or in the presence of 100 μg digested cell proteins, respectively. LC-MS-MS analysis of ABS tagged peptides revealed that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration in proteomic studies. Fluorogenic tagging with ABS also can be instrumental for detection and visualization of protein 3-NT in LC and gel-based protein separations.