Hydrogen sulfide (H2S) is a gaseous neuromodulator produced from L‐cysteine. H2S is generated by three distinct enzymatic pathways mediated by cystathionine γ‐lyase (CSE), cystathionine β‐synthase (CBS), and mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT). This study investigated the relative contributions of these three pathways to H2S production in PC12 cells (rat pheochromocytoma‐derived cells) and the rat dorsal root ganglion. CBS, CAT, and MPST, but not CSE, were expressed in the cells and tissues, and appreciable amounts of H2S were produced from L‐cysteine in the presence of α‐ketoglutarate, together with dithiothreitol. The production of H2S was inhibited by a CAT inhibitor (aminooxyacetic acid), competitive CAT substrates (L‐aspartate and oxaloacetate), and RNA interference (RNAi) against MPST. Immunocytochemistry revealed a mitochondrial localization of MPST in PC12 cells and dorsal root ganglion neurons, and the amount of H2S produced by CAT/MPST at pH 8.0, a physiological mitochondrial matrix pH, was comparable to that produced by CSE and CBS in the liver and the brain, respectively. Furthermore, H2S production was markedly increased by alkalization. These results indicate that CAT and MPST are primarily responsible for H2S production in peripheral neurons, and that the regulation of mitochondrial metabolism may influence neuronal H2S generation.
Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and β‐hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β‐hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of β‐hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β‐hydroxybutyrate was present in these neurons. In addition, the NMDA receptor‐induced calcium responses in the neurons were diminished in the presence of β‐hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of β‐hydroxybutyrate augmented transmitter release induced by the KATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of KATP channels in the effects of ketone bodies on transmitter release.
For our nervous system to function properly, each neuron must generate a single axon and elongate the axon to reach its target. It is known that actin filaments and their dynamic interaction with microtubules within growth cones play important roles in inducing axon extension. However, it remains unclear how cytoskeletal dynamics is controlled in growth cones. In this study, we report that Rufy3, a RUN domain‐containing protein, is a neuron‐specific and actin filament‐relevant protein. We find that the appropriate expression of Rufy3 in mouse hippocampal neurons is required for the development of a single axon and axon growth. Our results show that Rufy3 specifically interacts with actin filament‐binding proteins, such as Fascin, and colocalizes with Fascin in growth cones. Knockdown of Rufy3 impairs the distribution of Fascin and actin filaments, accompanied by an increased proportion of neurons with multiple axons and a decrease in the axon length. Therefore, Rufy3 may be particularly important for neuronal axon elongation by interacting with Fascin to control actin filament organization in axonal growth cones.
The cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. Here, we show that stimulation of cultured mouse cerebellar neurons by a function‐triggering L1 antibody leads to cathepsin E‐mediated generation of a sumoylated 30 kDa L1 fragment (L1‐30) and to import of L1‐30 into the nucleus. Mutation of the sumoylation site at K1172 or the cathepsin E cleavage site at E1167 abolishes generation of L1‐30, while mutation of the nuclear localization signal at K1147 prevents nuclear import of L1‐30. Moreover, the aspartyl protease inhibitor pepstatin impairs the generation of L1‐30 and inhibits L1‐induced migration of cerebellar neurons and Schwann cells as well as L1‐dependent in vitro myelination on axons of dorsal root ganglion neurons by Schwann cells. L1‐stimulated migration of HEK293 cells expressing L1 with mutated cathepsin E cleavage site is diminished in comparison to migration of cells expressing non‐mutated L1. In addition, L1‐stimulated migration of HEK293 cells expressing non‐mutated L1 is also abolished upon knock‐down of cathepsin E expression and enhanced by over‐expression of cathepsin E. The findings of the present study indicate that generation and nuclear import of L1‐30 regulate neuronal and Schwann cell migration as well as myelination.
Amyotrophic lateral sclerosis is a fatal neurodegenerative disease that affects motor neurons. The recruitment of autophagy (macroautophagy) and mitochondrial dysfunction are documented in amyotrophic lateral sclerosis patients and experimental models expressing mutant forms of Cu, Zn superoxide dismutase (SOD1) protein, but their impact in the disease remains unclear. Hypoxia is a stress closely related to the disease in patients and mutant SOD1 mice; in individual cells, hypoxia activates autophagy and regulates mitochondrial metabolism as fundamental adaptive mechanisms. Our aim was to examine whether mutant SOD1 changed this response. Hypoxia (1% O2 for 22 h) caused greater loss of viability and more marked activation of caspase 3/7 in the motor neuronal NSC‐34 cell line stably transfected with the G93A mutant human SOD1 (G93A‐NSC) than in the one with the wild‐type SOD1 (WT‐NSC) or in untransfected NSC‐34. In the G93A‐NSC cells, there was a more marked accumulation of the LC3‐II autophagy protein, attributable to autophagic stress; 3‐methyladenine, which acts on initiation of autophagy, fully rescued G93A‐NSC viability and reduced the activation of caspase 3/7 indicating this was a secondary event; the metabolic handling of hypoxia was inappropriate possibly contributing to the autophagic stress. Our findings evidentiate that the G93A mutation of SOD1 profoundly altered the adaptive metabolic response to hypoxia and this could increase the cell susceptibility to this stress.
Lysophosphatidic acid (LPA) is a signaling molecule that binds to six known G protein‐coupled receptors: LPA1–LPA6. LPA evokes several responses in the CNS, including cortical development and folding, growth of the axonal cone and its retraction process. Those cell processes involve survival, migration, adhesion proliferation, differentiation, and myelination. The anatomical localization of LPA1 is incompletely understood, particularly with regard to LPA binding. Therefore, we have used functional [35S]GTPγS autoradiography to verify the anatomical distribution of LPA1 binding sites in adult rodent and human brain. The greatest activity was observed in myelinated areas of the white matter such as corpus callosum, internal capsule and cerebellum. MaLPA1‐null mice (a variant of LPA1‐null) lack [35S]GTPγS basal binding in white matter areas, where the LPA1 receptor is expressed at high levels, suggesting a relevant role of the activity of this receptor in the most myelinated brain areas. In addition, phospholipid precursors of LPA were localized by MALDI‐IMS in both rodent and human brain slices identifying numerous species of phosphatides and phosphatidylcholines. Both phosphatides and phosphatidylcholines species represent potential LPA precursors. The anatomical distribution of these precursors in rodent and human brain may indicate a metabolic relationship between LPA and LPA1 receptors.
As an endogenous gaseous molecule, hydrogen sulfide (H2S) has attracted extensive attention because of its multiple biological effects. However, the effect of H2S on amygdala‐mediated emotional memory has not been elucidated. Here, by employing Pavlovian fear conditioning, an animal model widely used to explore the neural substrates of emotion, we determined whether H2S could regulate emotional memory. It was shown that the H2S levels in the amygdala of rats were significantly elevated after cued fear conditioning. Both intraamygdala and systemic administrations of H2S markedly enhanced amygdala‐dependent cued fear memory in rats. Moreover, it was found that H2S selectively increased the surface expression and currents of NMDA‐type glutamate receptor subunit 2B (GluN2B)‐containing NMDA receptors (NMDARs) in lateral amygdala of rats, whereas blockade of GluN2B‐containing NMDARs in lateral amygdala eliminated the effects of H2S to enhance amygdalar long‐term potentiation and cued fear memory. These results demonstrate that H2S can regulate amygdala‐dependent emotional memory by promoting the function of GluN2B‐containing NMDARs in amygdala, suggesting that H2S‐associated signaling may hold potential as a new target for the treatment of emotional disorders.
Neuro‐2a (N2a) neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine polyphosphates. The ApnA‐cleaving activity has been analysed with the use of the fluorogenic compound BODIPY® FL guanosine 5′‐O‐(3‐thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a Km value of 4.9 ± 1.3 μM. Diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, and the nucleoside monophosphate AMP behaved as an inhibitor of BODIPY® FL guanosine 5′‐O‐(3‐thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide pyrophosphatase/phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn2+ ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal‐like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation.
The 19‐transmembrane, multisubunit γ‐secretase complex generates the amyloid β‐peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β‐amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen‐2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high‐resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen‐2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N‐terminal maltose binding protein tag on Pen‐2 still permits incorporation into the complex and subsequent presenilin‐1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen‐2 and the γ‐secretase complex.
Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein‐regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth‐associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.
The administration of pan histone deacetylase (HDAC) inhibitors reduces ischemic damage to the CNS, both in vitro and in animal models of stroke, via mechanisms which we are beginning to understand. The acetylation of p53 is regulated by Class I HDACs and, because p53 appears to play a role in ischemic pathology, the purpose of this study was to discover, using an in vitro white matter ischemia model and an in vivo cerebral ischemia model, if neuroprotection mediated by HDAC inhibition depended on p53 expression. Optic nerves were excised from wild‐type and p53‐deficient mice, and then subjected to oxygen–glucose deprivation in the presence and absence of a specific inhibitor of Class I HDACs (MS‐275, entinostat) while compound action potentials were recorded. Furthermore, transient focal ischemia was imposed on wild‐type and p53‐deficient mice, which were subsequently treated with MS‐275. Interestingly, and in both scenarios, the beneficial effects of MS‐275 were most pronounced when p53 was absent. These results suggest that modulation of p53 activity is not responsible for MS‐275‐mediated neuroprotection, and further illustrate how HDAC inhibitors variably influence p53 and associated apoptotic pathways.
Cu/Zn‐superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER‐Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER‐Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER‐Golgi transport by over‐expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER‐Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.
Retinal degenerative diseases (RDs) are a group of inherited diseases characterized by the loss of photoreceptor cells. Selective photoreceptor loss can be induced in mice by an intraperitoneal injection of N‐methyl‐N‐nitrosourea (MNU) and, because of its selectivity, this model is widely used to study the mechanism of RDs. Although it is known that calcium‐calpain activation and lipid peroxidation are involved in the initiation of cell death, the precise mechanisms of this process remain unknown. Heat shock protein 70 (HSP70) has been shown to function as a chaperone molecule to protect cells against environmental and physiological stresses. In this study, we investigated the role of HSP70 on photoreceptor cell death in mice. HSP70 induction by valproic acid, a histone deacetylase inhibitor, attenuated the photoreceptor cell death by MNU through inhibition of apoptotic caspase signals. Furthermore, HSP70 itself was rapidly and calpain‐dependently cleaved after MNU treatment. Therefore, HSP70 induction by valproic acid was dually effective against MNU‐induced photoreceptor cell loss as a result of its anti‐apoptotic actions and its ability to prevent HSP70 degradation. These findings might help lead us to a better understanding of the pathogenic mechanism of RDs.
Angiotensin‐(1‐7) [Ang‐(1‐7)] is an alternative product of the brain renin‐angiotensin system that exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We previously identified a peptidase that metabolizes Ang‐(1‐7) to the inactive metabolite product Ang‐(1‐4) in CSF of adult sheep. This study purified the peptidase 1445‐fold from sheep brain medulla and characterized this activity. The peptidase was sensitive to the chelating agents o‐phenanthroline and EDTA, as well as the mercury compound p‐chloromercuribenzoic acid (PCMB). Selective inhibitors to angiotensin‐converting enzyme, neprilysin, neurolysin, and thimet oligopeptidase did not attenuate activity; however, the metallopeptidase agent JMV‐390 was a potent inhibitor of Ang‐(1‐7) hydrolysis (Ki = 0.8 nM). Kinetic studies using 125I‐labeled Ang‐(1‐7), Ang II, and Ang I revealed comparable apparent Km values (2.6, 2.8, and 4.3 μM, respectively), but a higher apparent Vmax for Ang‐(1‐7) (72 vs. 30 and 6 nmol/min/mg, respectively; p < 0.01). HPLC analysis of the activity confirmed the processing of unlabeled Ang‐(1‐7) to Ang‐(1‐4) by the peptidase, but revealed < 5% hydrolysis of Ang II or Ang I, and no hydrolysis of neurotensin, bradykinin or apelin‐13. The unique characteristics of the purified neuropeptidase may portend a novel pathway to influence actions of Ang‐(1‐7) within the brain.
Reversible post‐translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remain unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α‐ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans‐succinylase that mediates succinylation in an α‐ketoglutarate‐dependent manner. Inhibition of KGDHC reduced succinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl‐CoA suggests that the catalysis owing to the E2k succinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC.
下载免费PDF全文