Dynamin-related protein 1 regulates substrate oxidation in skeletal muscle by stabilizing cellular and mitochondrial calcium dynamics.

Mitochondria undergo continuous cycles of fission and fusion to promote inheritance, regulate quality control, and mitigate organelle stress. More recently, this process of mitochondrial dynamics has been demonstrated to be highly sensitive to nutrient supply, ultimately conferring bioenergetic plasticity to the organelle. However, whether regulators of mitochondrial dynamics play a causative role in nutrient regulation remains unclear. In this study, we generated a cellular loss-of-function model for dynamin-related protein 1 (DRP1), the primary regulator of outer membrane mitochondrial fission. Loss of DRP1 (shDRP1) resulted in extensive ultrastructural and functional remodeling of mitochondria, characterized by pleomorphic enlargement, increased electron density of the matrix, and defective NADH and succinate oxidation. Despite increased mitochondrial size and volume, shDRP1 cells exhibited reduced cellular glucose uptake and mitochondrial fatty acid oxidation. Untargeted transcriptomic profiling revealed severe downregulation of genes required for cellular and mitochondrial calcium homeostasis, which was coupled to loss of ATP-stimulated calcium flux and impaired substrate oxidation stimulated by exogenous calcium. The insights obtained herein suggest that DRP1 regulates substrate oxidation by altering whole-cell and mitochondrial calcium dynamics. These findings are relevant to the targetability of mitochondrial fission and have clinical relevance in the identification of treatments for fission-related pathologies such as hereditary neuropathies, inborn errors in metabolism, cancer, and chronic diseases.

Hindbrain cytoglucopenia-induced increases in systemic blood glucose levels by 2-deoxyglucose depend on intact astrocytes and adenosine release.

The hindbrain contains critical neurocircuitry responsible for generating defensive physiological responses to hypoglycemia. This counter-regulatory response (CRR) is evoked by local hindbrain cytoglucopenia that causes an autonomically mediated increase in blood glucose, feeding behavior, and accelerated digestion; that is, actions that restore glucose homeostasis. Recent reports suggest that CRR may be initially triggered by astrocytes in the hindbrain. The present studies in thiobutabarbital-anesthetized rats show that exposure of the fourth ventricle (4V) to 2-deoxyglucose (2DG; 15 µmol) produced a 35% increase in circulating glucose relative to baseline levels. While the 4V application of the astrocytic signal blocker, fluorocitrate (FC; 5 nmol), alone, had no effect on blood glucose levels, 2DG-induced increases in glucose were blocked by 4V FC. The 4V effect of 2DG to increase glycemia was also blocked by the pretreatment with caffeine (nonselective adenosine antagonist) or a potent adenosine A1 antagonist (8-cyclopentyl-1,3-dipropylxanthine; DPCPX) but not the NMDA antagonist (MK-801). These results suggest that CNS detection of glucopenia is mediated by astrocytes and that astrocytic release of adenosine that occurs after hypoglycemia may cause the activation of downstream neural circuits that drive CRR.

IL-1ß reciprocally regulates chemokine and insulin secretion in pancreatic ß-cells via NF-κB.

Proinflammatory cytokines impact islet ß-cell mass and function by altering the transcriptional activity within pancreatic ß-cells, producing increases in intracellular nitric oxide abundance and the synthesis and secretion of immunomodulatory proteins such as chemokines. Herein, we report that IL-1ß, a major mediator of inflammatory responses associated with diabetes development, coordinately and reciprocally regulates chemokine and insulin secretion. We discovered that NF-κB controls the increase in chemokine transcription and secretion as well as the decrease in both insulin secretion and proliferation in response to IL-1ß. Nitric oxide production, which is markedly elevated in pancreatic ß-cells exposed to IL-1ß, is a negative regulator of both glucose-stimulated insulin secretion and glucose-induced increases in intracellular calcium levels. By contrast, the IL-1ß-mediated production of the chemokines CCL2 and CCL20 was not influenced by either nitric oxide levels or glucose concentration. Instead, the synthesis and secretion of CCL2 and CCL20 in response to IL-1ß were dependent on NF-κB transcriptional activity. We conclude that IL-1ß-induced transcriptional reprogramming via NF-κB reciprocally regulates chemokine and insulin secretion while also negatively regulating ß-cell proliferation. These findings are consistent with NF-κB as a major regulatory node controlling inflammation-associated alterations in islet ß-cell function and mass.

NMDA receptors control vagal afferent excitability in the nucleus of the solitary tract.

Previous behavioral studies have demonstrated that presynaptic N-methyl-d-aspartate (NMDA) receptors expressed on vagal afferent terminals are involved in food intake and satiety. Therefore, using in vitro live cell calcium imaging of prelabeled rat hindbrain slices, we characterized which NMDA receptor GluN2 subunits may regulate vagal afferent activity. The nonselective NMDA receptor antagonist d,l-2-amino-5-phosphonopentanoic acid (d,l-AP5) significantly inhibited vagal terminal calcium influx, while the excitatory amino acid reuptake inhibitor d,l-threo-ß-benzyloxyaspartic acid (TBOA), significantly increased terminal calcium levels following pharmacological stimulation with ATP. Subunit-specific NMDA receptor antagonists and potentiators were used to identify which GluN2 subunits mediate the NMDA receptor response on the vagal afferent terminals. The GluN2B-selective antagonist, ifenprodil, selectively reduced vagal calcium influx with stimulation compared to the time control. The GluN2A-selective antagonist, 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl] benzyl]benzenesulfonamide (TCN 201) produced smaller but not statistically significant effects. Furthermore, the GluN2A/B-selective potentiator (pregnenolone sulfate) and the GluN2C/D-selective potentiator [(3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone; (CIQ)] enhanced vagal afferent calcium influx during stimulation. These data suggest that presynaptic NMDA receptors with GluN2B, GluN2C, and GluN2D subunits may predominantly control vagal afferent excitability in the nucleus of the solitary tract.

CXCL12 sensitizes vago-vagal reflex neurons in the dorsal medulla.

Previous studies from our laboratory illustrated the potential for stromal cell-derived factor one [CXCL12; also referred to as SDF-1] to act on its receptor [CXCR4] within the dorsal vagal complex [DVC] of the hindbrain to suppress gastric motility (Hermann et al., 2008). While CXCR4 receptors are essential for normal brain development, they also play a critical role in the proliferation of the HIV virus and initiation of metastatic cell growth in the brain. Anorexia, nausea, and failed autonomic regulation of gastrointestinal function are significant causes of morbidity and are contributory factors in the mortality associated with these disease states. The implication of our previous study was that CXCL12 caused gastric stasis by acting on gastric reflex circuit elements in the DVC. This hindbrain complex includes vagal afferent terminations in the solitary nucleus, neurons in the solitary nucleus (NST) and visceral efferent motorneurons in the dorsal motor nucleus (DMN) that are responsible for the regulation of digestive functions from the oral cavity to the transverse colon. In the current study, in vivo single-unit neurophysiological recordings from physiologically-identified NST and DMN components of the gastric accommodation reflex show that while injection of femtomole doses of CXCL12 onto NST or DMN neurons has no effect on their basal activity, CXCL12 amplifies the effect of gastric vagal mechanosensory input to activate the NST and, in turn, inhibit DMN motor activity.

Tumor necrosis factor activation of vagal afferent terminal calcium is blocked by cannabinoids.

The early proinflammatory cytokine tumor necrosis factor (TNF) is released in significant quantities by the activated immune system in response to infection, leukemia, autoimmune disorders, and radiation sickness. Nausea, emesis, and anorexia are common features of these disorders. TNF action on vagal afferent terminals in the brainstem is a likely cause of the malaise associated with these disorders. Our previous work has shown that TNF action to excite vagal afferents occurs as a result of sensitization of ryanodine channels in afferent nerve terminals. For millennia, cannabinoids (CB) have been used to combat the visceral malaise associated with chronic disease, although the mechanism of action has not been clear. Previous work in culture systems suggests that CB1 agonists can suppress neurotransmission by downregulating ryanodine channels through a protein kinase A (PKA)-dependent mechanism. Laser confocal calcium imaging methods were used to directly examine effects of CB1 cannabinoid agonists and TNF on visceral afferent signaling in the rat hindbrain. CB1 agonists blocked the effects of TNF to amplify vagal afferent responsiveness; blockade of PKA with H89 also eliminated the TNF amplification effect. These results help to explain the effectiveness of cannabinoids in blocking the malaise generated by TNF-releasing disease processes by opposing effects on ryanodine channels.

Hydrogen sulfide augments synaptic neurotransmission in the nucleus of the solitary tract.

Within the brain stem, the nucleus tractus solitarii (NTS) serves as a principal central site for sensory afferent integration from the cardiovascular and respiratory reflexes. Neuronal activity and synaptic transmission in the NTS are highly pliable and subject to neuromodulation. In the central nervous system, hydrogen sulfide (H2S) is a gasotransmitter generated primarily by the enzyme cystathionine-ß-synthase (CBS). We sought to determine the role of H2S, and its generation by CBS, in NTS excitability. Real-time RT-PCR, immunoblot, and immunohistochemistry analysis identified the presence of CBS in the NTS. Patch-clamp electrophysiology in brain stem slices examined excitatory postsynaptic currents (EPSCs) and membrane properties in monosynaptically driven NTS neurons. Confocal imaging of labeled afferent synaptic terminals in NTS slices monitored intracellular calcium. Exogenous H2S significantly increased the amplitude of evoked solitary tract (TS)-EPSCs, frequency of miniature (m)EPSCs, and presynaptic terminal calcium fluorescence in the NTS. H2S did not alter action potential discharge or postsynaptic properties. On the other hand, the CBS inhibitor aminooxyacetate (AOA) significantly reduced the amplitude of TS-EPSCs and presynaptic terminal calcium fluorescence in the NTS without altering postsynaptic properties. Taken together, these data support a presynaptic role for endogenous H2S in modulation of excitatory neurotransmission in the NTS.

TNF activates astrocytes and catecholaminergic neurons in the solitary nucleus: implications for autonomic control.

Tumor necrosis factor [TNF] produces a profound anorexia associated with gastrointestinal stasis. Our work suggests that the principal site of action of TNF to cause this change in gastric function is via vagal afferents within the nucleus of the solitary tract [NST]. Excitation of these afferents presumably causes gastric stasis by activating downstream NST neurons that, in turn, suppress gastric motility via action on neurons in the dorsal motor nucleus of the vagus that project to the stomach. Results from our parallel studies on gastric vago-vagal reflexes suggest that noradrenergic neurons in the NST are particularly important to the generation of reflex gastroinhibition. Convergence of these observations led us to hypothesize that TNF action in the NST may preferentially affect putative noradrenergic neurons. The current study confirms our observations of a dose-dependent TNF activation of cells [as indicated by cFOS production] in the NST. The phenotypic identity of these TNF-activated neurons in the NST was approximately 29% tyrosine hydroxylase [TH]-positive [i.e., presumably noradrenergic neurons]. In contrast, less than 10% of the nitrergic neurons were activated after TNF exposure. Surprisingly, another 54% of the cFOS-activated cells in the NST were phenotypically identified to be astrocytes. Taken together with previous observations, the present results suggest that intense or prolonged vagal afferent activity [induced by visceral pathway activity, action of gut hormones or cytokines such as TNF] can alter local astrocyte immediate early gene expression that, in turn, can provoke long-term, perhaps permanent changes in the sensitivity of vagal-reflex circuitry.

CXCR4 receptors in the dorsal medulla: implications for autonomic dysfunction.

The chemokine receptor, CXCR4, plays an essential role in guiding neural development of the CNS. Its natural agonist, CXCL12 [or stromal cell-derived factor-1 (SDF-1)], normally is derived from stromal cells, but is also produced by damaged and virus-infected neurons and glia. Pathologically, this receptor is critical to the proliferation of the HIV virus and initiation of metastatic cell growth in the brain. Anorexia, nausea and failed autonomic regulation of gastrointestinal (GI) function cause morbidity and contribute to the mortality associated with these disease states. Our previous work on the peripheral cytokine, tumor necrosis factor-alpha, demonstrated that similar morbidity factors involving GI dysfunction are attributable to agonist action on neural circuit elements of the dorsal vagal complex (DVC) of the hindbrain. The DVC includes vagal afferent terminations in the solitary nucleus, neurons in the solitary nucleus (NST) and area postrema, and visceral efferent motor neurons in the dorsal motor nucleus (DMN) that are responsible for the neural regulation of digestive functions from the oral cavity to the transverse colon. Immunohistochemical techniques demonstrate a dense concentration of CXCR4 receptors on neurons throughout the DVC and the hypoglossal nucleus. CXCR4-immunoreactivity is also intense on microglia within the DVC, though not on the astrocytes. Physiological studies show that nanoinjection of SDF-1 into the DVC produces a significant reduction in gastric motility in parallel with an elevation in the numbers of cFOS-activated neurons in the NST and DMN. These results suggest that this chemokine receptor may contribute to autonomically mediated pathophysiological events associated with CNS metastasis and infection.

TNFalpha: a trigger of autonomic dysfunction.

During disease, infection, or trauma, the cytokine tumor necrosis factor alpha (TNF alpha) causes fever, fatigue, malaise, allodynia, anorexia, gastric stasis associated with nausea, and emesis via interactions with the central nervous system. Our studies have focused on how TNF alpha produces a profound gastric stasis by acting on vago-vagal reflex circuits in the brainstem. Sensory elements of this circuit (i.e., nucleus of the solitary tract [NST] and area postrema) are activated by TNF alpha. In response, the efferent elements (i.e., dorsal motor neurons of the vagus) cause gastroinhibition via their action on the gastric enteric plexus. We find that TNF alpha presynaptically modulates the release of glutamate from primary vagal afferents to the NST and can amplify vagal afferent responsiveness by sensitizing presynaptic intracellular calcium-release mechanisms. The constitutive presence of TNF alpha receptors on these afferents and their ability to amplify afferent signals may explain how TNF alpha can completely disrupt autonomic control of the gut.

Tumor necrosis factor potentiates central vagal afferent signaling by modulating ryanodine channels.

Disease processes such as infection, leukemia, and autoimmune disorders are often associated with nausea, emesis, and anorexia. A common denominator of these rather disparate states is the production of the early, proinflammatory cytokine tumor necrosis factor-alpha (TNF) in significant quantities. Recent studies have shown that TNF may act as a neuromodulator in the hindbrain to produce malaise by potentiating visceral afferent signaling at the central processes of the vagus nerve. However, the mechanism by which TNF produces this signal amplification is not known. Our time-lapse calcium imaging studies of individual central vagal afferent varicosities in the caudal brainstem slice preparation show that, although TNF has minimal direct effects to elevate terminal intracellular calcium levels, TNF does potentiate the terminal afferent responses to other stimuli through a ryanodine-based, calcium-induced calcium release mechanism. Such a scheme may explain how TNF sensitizes visceral as well as somatosensory primary afferents.

Immunocytochemical localization of TNF type 1 and type 2 receptors in the rat spinal cord.

Tumor necrosis factor-alpha (TNF-alpha) is secreted in numerous pathophysiological situations by a variety of cell types. Tactile hypersensitivity (allodynia) is one component of a constellation of "illness behaviors" triggered by TNF-alpha. TNF-alpha is also implicated in neuropathic pain after peripheral nerve injury and apoptosis after spinal cord injury (SCI). It is possible that SCI, illness- and peripheral injury-induced hypersensitivity may share a similar spinal mediated etiology. These studies identify the locus of type-1 TNF (TNFR1 or p55) and type-2 TNF (TNFR2 or p75) receptors within the spinal cord. At all spinal levels, TNFR1 receptor immunoreactivity (TNFR1-ir) was constitutively expressed on cells and afferent fibers within the dorsal root ganglia, afferent fibers of the dorsal root, dorsal root entry zone (REZ) and within lamina I and II of the dorsal horn. Unilateral dorsal rhizotomy eliminated the characteristic pattern of TNFR1-ir at the rhizotomized REZ. In contrast, TNFR2-ir was consistently absent from dorsal root fibers and the region of the root entry zone. Consistent with our previous report, medullary afferent fibers in the solitary tract and spinal trigeminal tract labelled for TNF1-ir, but did not express TNFR2-ir. The presence TNFR1-ir on dorsal horn afferents, suggests that TNF-alpha may be a mechanism responsible for tactile hypersensitivity during illness. The presence of TNFR1 receptors, and perhaps their long-term activation or plasticity, may also play a critical role in the chronic allodynia and hyperreflexia observed after SCI or peripheral nerve damage.

TNF alpha-p55 receptors: medullary brainstem immunocytochemical localization in normal and vagus nerve-transected rats.

Tumor necrosis factor alpha (TNF(alpha)) is a potent modulator of autonomic reflex mechanisms that control the stomach. Evidence suggests that TNF(alpha) action directly on vago-vagal reflex control circuits causes the autonomic misregulation of digestion manifested as gastrointestinal stasis, nausea, and emesis associated with illness. Neurophysiological studies indicated that TNF(alpha) may have effects on vagal afferents in the solitary nucleus, as well as neurons of the solitary nucleus (NST) and dorsal motor nucleus (DMN) of the vagus. The aim of this study was to determine the location of the TNFR1 receptor (p55) in the medulla using immunocytochemical methods. We devised a technique for localizing the p55 receptor using heat-induced antigen recovery in fixed tissue sections. This protocol allowed us to demonstrate that dense p55-immunoreactivity (p55-ir) is constitutively present on central (but not peripheral) vagal afferents in the solitary tract (ST) and nucleus; p55-ir is also present on afferents entering the spinal trigeminal nucleus. Unilateral supra-nodose vagotomy eliminated p55-ir from ipsilateral central vagal afferents. Virtually all neurons in the brainstem appeared to express p55-ir at a low level, i.e., just above background. However, vagotomy caused a dramatic up-regulation of p55-ir in vagal motor neurons. This increase in p55-ir in axotomized neurons may play a pivotal role in the connection between the occurrence of the injury and the initiation of apoptotic processes resulting in elimination of damaged neurons.

TNFalpha-stimulation of cFos-activation of neurons in the solitary nucleus is suppressed by TNFR:Fc adsorbant construct in the dorsal vagal complex.

The cytokine tumor necrosis factor alpha (TNF(alpha)) may act within the neural circuitry of the medullary dorsal vagal complex (DVC) to affect changes in gastric function such as gastric stasis, loss of appetite, nausea, and vomiting. The definitive demonstration that endogenously generated TNF(alpha) is acting within the DVC circuitry to affect these changes has been impeded by the lack of an antagonist for TNF(alpha). The present studies used localized central nervous system microinjections of the TNF-adsorbant construct (TNFR:Fc) to specifically neutralize the ability of endogenously produced TNF(alpha) to activate NST neurons. Our studies reveal that TNFR:Fc suppresses induction of cFos normally evoked by TNF(alpha). These results validate our hypothesis that circulating TNF(alpha) may act directly within the DVC to affect gastric function in a variety of pathophysiological states.

Cell death in models of spinal cord injury.

Current treatments for acute spinal cord injury are based on animal models of human spinal cord injury (SCI). These models have shown that the initial traumatic injury to cord tissue is followed by a long period of secondary injury that includes a number of cellular and biochemical cascades. These secondary injury processes are potential targets for therapies. Continued refinement of rat and mouse models of SCI, along with more detailed analyses of the biology of the lesion in these models, points to both necrotic and apoptotic mechanisms of cell death after SCI. In this chapter, we review recent evidence for long-term apoptotic death of oligodendrocytes in long tracts undergoing Wallerian degeneration following SCI. This process appears to be related closely to activation of microglial cells. It is has been thought that microglial cells might be the source of cytotoxic cytokines, such as tumor necrosis factor-alpha (TNF-alpha), that kill oligodendrocytes. However, more recent evidence in vivo suggests that TNF-alpha by itself may not induce necrosis or apoptosis in oligodendrocytes. We review data that suggests other possible pathways for apoptosis, such as the neurotrophin receptor p75 which is expressed in both neurons and oligodendrocytes after SCI in rats and mice. In addition, it appears that microglial activation and TNF-alpha may be important in acute SCI. Ninety minutes after a moderate contusion lesion, microglia are activated and surround dying neurons. In an 'atraumatic' model of SCI, we have now shown that TNF-alpha appears to greatly potentiate cell death mediated by glutamate receptors. These studies emphasize that multiple mechanisms and interactions contribute to secondary injury after SCI. Continued study of both contusion models and other new approaches to studying these mechanisms will be needed to maximize strategies for acute and chronic therapies, and for neural repair.

Tumor necrosis factor-alpha inhibits physiologically identified dorsal motor nucleus neurons in vivo.

Our previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) activates solitary nucleus neurons involved in vago-vagal reflex control of gastric motility. Here, we describe the dual role of TNF-alpha as also modulating neurons in the dorsal motor nucleus of the vagus (DMN) that respond to gastric distention. A large majority of physiologically identified DMN neurons are rapidly and completely inhibited by exposure to TNF-alpha, suggesting that TNF-alpha may induce gastric stasis by functioning as a hormone that modulates both portions of this reflex pathway during illness.

LPS-induced suppression of gastric motility relieved by TNFR:Fc construct in dorsal vagal complex.

Our previous studies suggested that the cytokine tumor necrosis factor-alpha (TNF-alpha) may act within the neural circuitry of the medullary dorsal vagal complex (DVC) to affect changes in gastric function, such as gastric stasis, loss of appetite, nausea, and vomiting. The definitive demonstration that endogenously generated TNF-alpha is capable of affecting gastric function via the DVC circuitry has been impeded by the lack of an antagonist for TNF-alpha. The present studies used localized central nervous system applications of the TNF-adsorbant construct (TNFR:Fc; TNF-receptor linked to the Fc portion of the human immunoglobulin IgG1) to attempt to neutralize the suppressive effects of endogenously produced TNF-alpha. Gastric motility of thiobutabarbital-anesthetized rats was monitored after systemic administration of lipopolysaccharide (LPS) to induce TNF-alpha production. Continuous perfusion of the floor of the fourth ventricle with TNFR:Fc reversed the potent gastroinhibition induced by LPS, i.e., central thyrotropin-releasing hormone-induced increases in motility were not inhibited. This disinhibition of gastric stasis was not seen after intravenous administration of similar doses of TNFR:Fc nor ventricular application of the Fc fragment of human immunoglobulin. These results validate our previous studies that suggest that circulating TNF-alpha may act directly within the DVC to affect gastric function in a variety of pathophysiological states.