Members of the nerve growth factor (NGF) family promote the survival of neurons during development. NGF specifically activates the receptor trkA, initiating a signal transduction cascade which ultimately blocks cell death. Here we show that NGF can have the opposite effect, inducing the death of mature oligodendrocytes cultured from postnatal rat cerebral cortex. This effect was highly specific, because NGF had no effect on oligodendrocyte precursors and astrocytes. Other neurotrophins such as brain-derived neurotrophin factor (BDNF) and neurotrophin-3 (NT-3) did not induce cell death. NGF binding to mature oligodendrocytes expressing the p75 neurotrophin receptor, but not trkA, resulted in a sustained increase of intracellular ceramide and c-Jun amino-terminal kinase (JNK) activity, which are thought to participate in a signal transduction pathway leading to cell death. Taken together, these results indicate that NGF has the ability to promote cell death in specific cell types through a ligand-dependent signalling mechanism involving the p75 neurotrophin receptor.

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.The peptide hormone insulin stimulates the uptake and storage of glucose in skeletal muscle and adipose tissue while simultaneously inhibiting its efflux from the liver. In certain pathological conditions, including Type 2 diabetes mellitus (1) and metabolic syndrome X (2), these tissues become resistant to insulin such that a maximal dose of the hormone is unable to elicit these anabolic responses. Numerous studies suggest that the oversupply of lipid to peripheral tissues might contribute to the development of this insulin resistance. First, insulin-resistant subjects frequently display signs of abnormal lipid metabolism including obesity (3), increased circulating free fatty acid (FFA) 1 concentrations (4, 5), and elevated intramyocellular lipid levels (6). In fact, the size of the intramyocellular lipid depot correlates more tightly with the severity of insulin resistance than most known risk factors (6). Second, experimentally exposing peripheral tissues to lipids decreases their sensitivity to insulin. For example, (a) incubating isolated muscle strips or cultured muscle cells with FFAs (7-11), (b) infusing lipid emulsions into rodents or humans (12-15), or (c) expressing lipoprotein lipase in skeletal muscle of transgenic mice (16, 17) promotes intramyocellular lipid accumulation and compromises insulin-stimulated glucose uptake. These observations have prompted investigators to hypothesize that increased availability of lipids to peripheral tissues causes insulin resistance, perhaps by promoting the accumulation of one or more fat-derived metabolites capable of inhibiting insulin action (6, 18). The insulin receptor is a heterotetrameric tyrosine kinase receptor that mediates all of the anabolic effects of insulin (19). The activated receptor phosphorylates intracellular docking molecules (termed insulin receptor substrates, or IRS proteins) that r...

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.

SUMMARY 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.

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OBJECTIVEImpairments 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 METHODSProteomic 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).RESULTSProteins 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.CONCLUSIONSAbnormal 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.

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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.