Edward M. Mills

Chondroitin-4-sulfation negatively regulates axonal guidance and growth

Glycosaminoglycan (GAG) side chains endow extracellular matrix proteoglycans with diversity and complexity based upon the length, composition and charge distribution of the polysaccharide chain. Using cultured primary neurons, we show that specific sulfation in the GAG chains of chondroitin sulfate mediates neuronal guidance cues and axonal growth inhibition. Chondroitin-4-sulfate (CS-A), but not chondroitin-6-sulfate (CS-C), exhibits a strong negative guidance cue to mouse cerebellar granule neurons. Enzymatic and gene-based manipulations of 4-sulfation in the GAG side chains alter their ability to direct growing axons. Furthermore, 4-sulfated chondroitin sulfate GAG chains are rapidly and significantly increased in regions that do not support axonal regeneration proximal to spinal cord lesions in mice. Thus, our findings show that specific sulfation along the carbohydrate backbone carries instructions to regulate neuronal function.

Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading.

ABSTRACT Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine608-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.

Cyanide-Induced Apoptosis and Oxidative Stress in Differentiated PC12 Cells

Abstract: Terminally differentiated PC12 cells are a useful neuron‐like model for studying programmed cell death in response to nerve growth factor (NGF) deprivation. This in vitro model was used to investigate the mechanism by which cyanide‐induced histotoxic hypoxia produces neuronal degeneration. Treatment of undifferentiated PC12 cells with 0.1 mM KCN for 24 h did not produce cell death. In contrast, treatment of differentiated PC12 cell cultures with 0.1 mM KCN for 24 h increased cell death by 43% when compared with control cultures, as measured by trypan blue dye exclusion and lactate dehydrogenase release assays. The Ca2+/Mg2+‐dependent endonuclease inhibitor aurintricarboxylic acid and the transcriptional inhibitor actinomycin D partially attenuated hypoxic toxicity, suggesting roles for endonuclease activation and transcription in this model of neuronal death. Extracted DNA from cyanide‐treated neurons demonstrated cleavage into oligonucleosomal fragments on gel electrophoresis. Transmission electron microscopic analysis showed morphological changes consistent with apoptotic cell death, including membrane blebbing and convolution, as well as chromatin condensation and margination to the nuclear membrane. Addition of either ascorbate or catalase to the cultures partially attenuated the loss of cell viability induced by cyanide, and decreased the incidence of apoptotic cells after treatment, based on the in situ detection of DNA strand breaks. The ability of cyanide to elevate intracellular oxidant species was determined by microfluorescence in differentiated PC12 cells loaded with the oxidant‐sensitive dye 2′,7′‐dichlorofluorescin. Exposure of cells to 0.1 mM KCN produced a rapid generation of oxidants that was blocked ∼50% by ascorbate or catalase. These observations indicate that cyanide induces apoptosis in terminally differentiated, and not undifferentiated, PC12 cells, and that antioxidants significantly reduce the incidence of cyanide‐induced apoptosis.

Nerve growth factor treatment prevents the increase in superoxide produced by epidermal growth factor in PC12 cells.

Stimulation of pheochromocytoma (PC12) cells with the mitogen epidermal growth factor (EGF) produced a rapid and robust accumulation of intracellular reactive oxygen species (ROS), an accumulation which, in other systems, has been shown to be essential for mitogenesis. Brief pretreatment of the cells with nerve growth factor (NGF) suppressed the EGF-mediated ROS increase. EGF failed to produce elevations in ROS in a PC12 variant stably expressing a dominant-negative p21 ras construct (PC12-N17) or in cells pretreated with the MEK inhibitor PD098059. NGF failed to suppress the increase in ROS in the PC12 variant nnr5, which lacks p140 trk receptors. The suppression of the increase in ROS by NGF was restored in nnr5 cells stably expressing p140 trk (nnr5-trk), but NGF failed to prevent the increase in ROS in nnr cells expressing mutant p140 trk receptors that lack binding sites for Shc and phospholipase Cγ. Among several inhibitors of superoxide-generating enzymes, only the lipoxygenase inhibitor, nordihydroguaiaretic acid reduced EGF-mediated ROS accumulation. The inhibitory action of NGF on ROS production was mimicked by the nitric oxide donor, sodium nitroprusside, and was blocked by an inhibitor of nitric-oxide synthetase, l-nitroarginine methyl ester. These results suggest a novel mechanism for the rapid interruption of mitogenic signaling by the neurotrophin NGF.

Carvedilol reverses hyperthermia and attenuates rhabdomyolysis induced by 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) in an animal model*

Objective:Hyperthermia is a potentially fatal manifestation of severe 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) intoxication. No proven effective drug treatment exists to reverse this potentially life-threatening hyperthermia, likely because mechanisms of peripheral thermogenesis are poorly understood. Using a rat model of MDMA hyperthermia, we evaluated the acute drug-induced changes in plasma catecholamines and used these results as a basis for the selection of drugs that could potentially reverse this hyperthermia. Design:Prospective, controlled, randomized animal study. Setting:A research institute laboratory. Subjects:Male, adult Sprague-Dawley rats. Interventions:Based on MDMA-induced changes in plasma catecholamine levels, rats were subjected to the nonselective (&bgr;1 + &bgr;2) adrenergic receptor antagonists propranolol or nadolol or the &agr;1- + &bgr;1,2,3-adrenergic receptor antagonist carvedilol before or after a thermogenic challenge of MDMA. Measurement and Main Results:Plasma catecholamines levels 30 mins after MDMA (40 mg/kg, subcutaneously) were determined by high-pressure liquid chromatography and electrochemical detection. Core temperature was measured by a rectal probe attached to a thermocouple. Four hours after MDMA treatment, blood was drawn and serum creatine kinase levels were measured as a marker of rhabdomyolysis using a Vitros analyzer. MDMA induced a 35-fold increase in norepinephrine levels, a 20-fold increase in epinephrine, and a 2.4-fold increase in dopamine levels. Propranolol (10 mg/kg, intraperitoneally) or nadolol (10 mg/kg, intraperitoneally) administered 30 mins before MDMA had no effect on the thermogenic response. In contrast, carvedilol (5 mg/kg, intraperitoneally) administered 15 mins before or after MDMA prevented this hyperthermic response. Moreover, when administered 1 hr after MDMA, carvedilol completely reversed established hyperthermia and significantly attenuated subsequent MDMA-induced creatine kinase release. Conclusion:These data show that &agr;1 and &bgr;3-adrenergic receptors may contribute to the mediation of MDMA-induced hyperthermia and that drugs targeting these receptors, such as carvedilol, warrant further investigation as novel therapies for the treatment of psychostimulant-induced hyperthermia and its sequelae.

The role of the sympathetic nervous system and uncoupling proteins in the thermogenesis induced by 3,4-methylenedioxymethamphetamine.

Body temperature regulation involves a homeostatic balance between heat production and dissipation. Sympathetic agents such as 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) can disrupt this balance and as a result produce an often life-threatening hyperthermia. The hyperthermia induced by MDMA appears to result from the activation of the sympathetic nervous system (SNS) and the hypothalamic-pituitary-thyroid/adrenal axis. Norepinephrine release mediated by MDMA creates a double-edged sword of heat generation through activation of uncoupling protein (UCP3) along with α1- and β3-adrenoreceptors and loss of heat dissipation through SNS-mediated vasoconstriction. This review examines cellular mechanisms involved in MDMA-induced thermogenesis from UCP activation to vasoconstriction and how these mechanisms are related to other thermogenic conditions and potential treatment modalities.

Roles of Norepinephrine, Free Fatty Acids, Thyroid Status, and Skeletal Muscle Uncoupling Protein 3 Expression in Sympathomimetic-Induced Thermogenesis

Thyroid hormone (TH) plays a fundamental role in thermoregulation, yet the molecular mediators of its effects are not fully defined. Recently, skeletal muscle (SKM) uncoupling protein (UCP) 3 was shown to be an important mediator of the thermogenic effects of the widely abused sympathomimetic agents 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy) and methamphetamine. Expression of UCP3 is regulated by TH. Activation of UCP3 is indirectly regulated by norepinephrine (NE) and is dependent upon the availability of free fatty acids (FFAs). We hypothesized that UCP3 may be a molecular link between TH and hyperthermia, requiring increased levels of both NE and FFAs to accomplish the thermogenic effect. Here, we demonstrate that MDMA (40 mg/kg s.c.) significantly increases plasma FFA levels 30 min after treatment. Pharmacologically increasing NE levels through the inhibition of phenylethanolamine N-methyltransferase with ±2,3-dichloro-α-methylbenzylamine potentiated the hyperthermic effects of a 20 mg/kg dose of MDMA. Using Western blots and regression analysis, we further illustrated that chronic hyperthyroidism in rats potentiates the hyperthermic effects of MDMA and increases levels of SKM UCP3 protein in a linear fashion according to levels of circulating plasma TH. Conversely, chronic hypothyroidism results in a hypothermic response to MDMA that is directly proportionate to decreased UCP3 expression. Acute TH supplementation did not change the skeletal muscle UCP3 expression levels or temperature responses to MDMA. These findings suggest that, although MDMA-induced hyperthermia appears to result from increased NE and FFA levels, susceptibility is ultimately determined by TH regulation of UCP3-dependent thermogenesis.

Attenuation of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy)-induced rhabdomyolysis with α1- plus β3-adrenoreceptor antagonists

Studies were designed to examine the effects of α1 (α1AR)‐ plus β3‐adrenoreceptor (β3AR) antagonists on 3,4‐methylenedioxymethamphetamine (MDMA, Ecstasy)‐induced hyperthermia and measures of rhabdomyolysis (creatine kinase (CK)) and renal function (blood urea nitrogen (BUN) and serum creatinine (sCr)) in male Sprague–Dawley rats. MDMA (40 mg kg−1, s.c.) induced a rapid and robust increase in rectal temperature, which was significantly attenuated by pretreatment with the α1AR antagonist prazosin (100 μg kg−1, i.p.) plus the β3AR antagonist SR59230A (5 mg kg−1, i.p.). CK levels significantly increased (peaking at 4 h) after MDMA treatment and were blocked by the combination of prazosin plus SR59230A. At 4 h after MDMA treatment, BUN and sCr levels were also significantly increased and could be prevented by this combination of α1AR‐ plus β3AR‐antagonists. The results from this study suggest that α1AR and β3AR play a critical role in the etiology of MDMA‐mediated hyperthermia and subsequent rhabdomyolysis.

Reactive oxygen species generated by cyanide mediate toxicity in rat pheochromocytoma cells.

Peroxide formation has been implicated in impairment of motor function by cyanide which occurs in both animals and man. The present study employs the neuronal model, rat pheochromocytoma (PC12) cells to evaluate peroxidation as a toxic mechanism of cyanide. Confocal imaging shows that peroxides form within a few seconds in cell cytoplasm after cyanide exposure and continue to accumulate over a period of several minutes. Peroxide generation by cyanide is decreased to about 50% by phospholipase A2 inhibitors indicating involvement of arachidonic acid in the oxidative process. Also antioxidant defense enzymes (CuZn superoxide dismutase and especially catalase) in PC12 cells are inhibited by cyanide. It appears that peroxide accumulation after cyanide treatment involves both inhibition of breakdown and increased production. Furthermore, both peroxide accumulation and cell death induced by cyanide in PC12 cells are blocked by an antioxidant (ascorbate). These data support the hypothesis that the cytotoxic action of cyanide is related in part to an oxidative process.

Caenorhabditis elegans UCP4 protein controls complex II-mediated oxidative phosphorylation through succinate transport

Background: The function of the C. elegans mitochondrial uncoupling protein 4 (ceUCP4) has not been characterized. Results: Worms deficient in ceUCP4 displayed hypometabolic phenotypes and complex II dysfunction that corresponded with a significant loss of mitochondrial succinate import. Conclusion: ceUCP4 plays a novel role in the regulation of complex II by controlling succinate transport into mitochondria. Significance: Understanding how extramitochondrial succinate and ceUCP4 regulate complex II-mediated metabolism is critical for understanding the mechanisms of cellular respiration. The novel uncoupling proteins (UCP2–5) are implicated in the mitochondrial control of oxidant production, insulin signaling, and aging. Attempts to understand their functions have been complicated by overlapping expression patterns in most organisms. Caenorhabditis elegans nematodes are unique because they express only one UCP ortholog, ceUCP4 (ucp4). Here, we performed detailed metabolic analyzes in genetically modified nematodes to define the function of the ceUCP4. The knock-out mutant ucp4 (ok195) exhibited sharply decreased mitochondrial succinate-driven (complex II) respiration. However, respiratory coupling and electron transport chain function were normal in ucp4 mitochondria. Surprisingly, isolated ucp4 mitochondria showed markedly decreased succinate uptake. Similarly, ceUCP4 inhibition blocked succinate respiration and import in wild type mitochondria. Genetic and pharmacologic inhibition of complex I function was selectively lethal to ucp4 worms, arguing that ceUCP4-regulated succinate transport is required for optimal complex II function in vivo. Additionally, ceUCP4 deficiency prolonged lifespan in the short-lived mev1 mutant that exhibits complex II-generated oxidant production. These results identify a novel function for ceUCP4 in the regulation of complex II-based metabolism through an unexpected mechanism involving succinate transport.