Rick T. Dobrowsky

Sphingolipid signalling domains Floating on rafts or buried in caves

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.

Diminished Superoxide Generation Is Associated With Respiratory Chain Dysfunction and Changes in the Mitochondrial Proteome of Sensory Neurons From Diabetic Rats

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.

Neurotrophic Modulation of Myelinated Cutaneous Innervation and Mechanical Sensory Loss in Diabetic Mice

Human diabetic patients often lose touch and vibratory sensations, but to date, most studies on diabetes-induced sensory nerve degeneration have focused on epidermal C-fibers. Here, we explored the effects of diabetes on cutaneous myelinated fibers in relation to the behavioral responses to tactile stimuli from diabetic mice. Weekly behavioral testing began prior to streptozotocin (STZ) administration and continued until 8 weeks, at which time myelinated fiber innervation was examined in the footpad by immunohistochemistry using antiserum to neurofilament heavy chain (NF-H) and myelin basic protein (MBP). Diabetic mice developed reduced behavioral responses to non-noxious (monofilaments) and noxious (pinprick) stimuli. In addition, diabetic mice displayed a 50% reduction in NF-H-positive myelinated innervation of the dermal footpad compared with non-diabetic mice. To test whether two neurotrophins nerve growth factor (NGF) and/or neurotrophin-3 (NT-3) known to support myelinated cutaneous fibers could influence myelinated innervation, diabetic mice were treated intrathecally for 2 weeks with NGF, NT-3, NGF and NT-3. Neurotrophin-treated mice were then compared with diabetic mice treated with insulin for 2 weeks. NGF and insulin treatment both increased paw withdrawal to mechanical stimulation in diabetic mice, whereas NT-3 or a combination of NGF and NT-3 failed to alter paw withdrawal responses. Surprisingly, all treatments significantly increased myelinated innervation compared with control-treated diabetic mice, demonstrating that myelinated cutaneous fibers damaged by hyperglycemia respond to intrathecal administration of neurotrophins. Moreover, NT-3 treatment increased epidermal Merkel cell numbers associated with nerve fibers, consistent with increased numbers of NT-3-responsive slowly adapting A-fibers. These studies suggest that myelinated fiber loss may contribute as significantly as unmyelinated epidermal loss in diabetic neuropathy, and the contradiction between neurotrophin-induced increases in dermal innervation and behavior emphasizes the need for multiple approaches to accurately assess sensory improvements in diabetic neuropathy.

Phosphoinositide 3‐kinase regulates crosstalk between Trk A tyrosine kinase and p75NTR‐dependent sphingolipid signaling pathways

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.

Stabilization of the cyclin‐dependent kinase 5 activator, p35, by paclitaxel decreases β‐amyloid toxicity in cortical neurons

One hallmark of Alzheimers disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid‐β (Aβ) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Aβ toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Aβ peptides, inhibits Aβ‐induced activation of cyclin‐dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Aβ‐induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Aβ‐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 Aβ 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 Aβ‐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 Aβ toxicity and neurofibrillary pathology in AD and other tauopathies.

Tau neurofibrillary pathology and microtubule stability.

We previously reported that nonomolar concentrations of Taxol and several structurally diverse microtubule (MT)-stabilizing agents significantly enhanced the survival of neurons in the presence of fibrils of amyloid β peptide (Aβ). Pretreatment of neurons with MT-stabilizing drugs also blocked Aβ-induced activation of tau hyperphosphorylation. Although tau is a substrate for several kinases, we initially focused on cdk5, as this tau kinase has been shown to be activated in Aβ-treated neurons and Alzheimer’s disease (AD) brain. In an in vitro kinase assay, Taxol inhibited activation of cdk5 by Aβ. In addition, the proposed cellular cascade in which calpain activation leads to cleavage of the cdk5 regulator, p35, to the strong kinase activator p25 was also prevented. Taxol did not directly inhibit the activity of either cdk5 or calpain, indicating that other cellular components are required for the effect of the drug on Aβ activation of tau phosphorylation. Our results suggest that drugs that interact with MTs can alter signaling events in neurons, possibly because some MTs play a role in organizing protein complexes involved in responses to Aβ. Thus the cytoskeletal network may serve as a biosensor of cellular well-being.

p75 neurotrophin receptor signaling: mechanisms for neurotrophic modulation of cell stress?

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 p75NTR signaling remain to be fully characterized, many of the currently identified pathways activated by p75NTR may be generally characterized as stress response signals. This review describes recent advances in identifying the molecular components involved in p75NTR signal transduction and suggests that p75NTR signaling may more aptly serve as a general mechanism for the transduction and modulation of stress signals. J. Neurosci. Res. 61:237–243, 2000.

The Dlk1-Gtl2 Locus Preserves LT-HSC Function by Inhibiting the PI3K-mTOR Pathway to Restrict Mitochondrial Metabolism

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.

Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice

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-[(2R,3R,4S,5R)-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.

Nutrient excess and altered mitochondrial proteome and function contribute to neurodegeneration in diabetes

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.