Hydrogen sulfide (H2S) functions as a physiological gas transmitter in both normal and pathophysiological cellular events. H2S is produced from substances by three enzymes: cystathionine β‐synthase (CBS), cystathionine γ‐lyase (CSE), and 3‐mercaptopyruvate sulfurtransferase (MST). In human tissues, these enzymes are involved in tissue‐specific biochemical pathways for H2S production. For example, CBS and cysteine aminotransferase/MST are present in the brain, but CSE is not. Thus, we examined the expression of H2S production‐related enzymes in peripheral nerves. Here, we found that CSE and MST/cysteine aminotransferase, but not CBS, were present in normal peripheral nerves. In addition, injured sciatic nerves in vivo up‐regulated CSE in Schwann cells during Wallerian degeneration (WD); however, CSE was not up‐regulated in peripheral axons. Using an ex vivo sciatic nerve explant culture, we found that the inhibition of H2S production broadly prevented the process of nerve degeneration, including myelin fragmentation, axonal degradation, Schwann cell dedifferentiation, and Schwann cell proliferation in vitro and in vivo. Thus, these results indicate that H2S signaling is essential for Schwann cell responses to peripheral nerve injury.
By using two structurally unrelated hydrogen sulfide (H2S) donors 5‐(4‐methoxyphenyl) ‐3H‐1, 2‐dithiole‐3‐thione (ADT) and sodium hydrosulfide (NaHS), this study investigated if H2S protected blood–brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO). ICR mice underwent MCAO and received H2S donors at 3 h after reperfusion. Infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression were examined at 48 h after MCAO. We also investigated if ADT protected BBB integrity by suppressing post‐ischemic inflammation‐induced Matrix Metalloproteimase‐9 (MMP9) and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ADT increased blood H2S concentrations, decreased infarction, and improved neurological deficits. Particularly, ADT reduced EB extravasation, brain edema and preserved expression of tight junction proteins in the ischemic brain. NaHS also increased blood H2S levels and reduced EB extravasation following MCAO. Moreover, ADT inhibited expression of pro‐inflammatory markers induced Nitric Oxide Synthase (iNOS) and IL‐1β while enhanced expression of anti‐inflammatory markers arginase 1 and IL‐10 in the ischemic brain. Accordingly, ADT attenuated ischemia‐induced expression and activity of MMP9. Moreover, ADT reduced NOX‐4 mRNA expression, NOX activity, and inhibited nuclear translocation of Nuclear Factor Kappa‐B (NF‐κB) in the ischemic brain. In conclusion, H2S donors protected BBB integrity following experimental stroke possibly by acting through NF‐κB inhibition to suppress neuroinflammation induction of MMP9 and NOX4‐derived free radicals.
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.
The gene encoding leucine‐rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon‐γ (IFN‐γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN‐γ‐mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal‐regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN‐γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP‐1 cells and human peripheral blood monocytes stimulated the ERK5‐LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5‐dependent IFN‐γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages.
Human TPH2 (hTPH2) catalyzes the rate‐limiting step in CNS serotonin biosynthesis. We characterized a single‐nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased Vmax with unchanged Km values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild‐type enzyme). The S41Y polymorphism decreased cyclic AMP‐dependent protein kinase A‐mediated phosphorylation ~ 50% relative to wild‐type hTPH2, suggesting that the S41Y mutation may disrupt the post‐translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y‐transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression.
Vitamin C is an essential factor for neuronal function and survival, existing in two redox states, ascorbic acid (AA), and its oxidized form, dehydroascorbic acid (DHA). Here, we show uptake of both AA and DHA by primary cultures of rat brain cortical neurons. Moreover, we show that most intracellular AA was rapidly oxidized to DHA. Intracellular DHA induced a rapid and dramatic decrease in reduced glutathione that was immediately followed by a spontaneous recovery. This transient decrease in glutathione oxidation was preceded by an increase in the rate of glucose oxidation through the pentose phosphate pathway (PPP), and a concomitant decrease in glucose oxidation through glycolysis. DHA stimulated the activity of glucose‐6‐phosphate dehydrogenase, the rate‐limiting enzyme of the PPP. Furthermore, we found that DHA stimulated the rate of lactate uptake by neurons in a time‐ and dose‐dependent manner. Thus, DHA is a novel modulator of neuronal energy metabolism by facilitating the utilization of glucose through the PPP for antioxidant purposes.
Lewy bodies, mainly composed of α‐synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low‐order αS oligomers are neurotoxic and critical species in the pathogenesis of α‐synucleinopathies. To develop disease modifying therapies for α‐synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo‐induced cross‐linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N‐terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long‐term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers.
Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α‐synuclein (α‐Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α‐Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co‐cultures of neurons and astrocytes. We found that oligomeric but not monomeric α‐Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre‐incubation of cells with isotope‐reinforced polyunsaturated fatty acids (D‐PUFAs) completely prevented the effect of oligomeric α‐Syn on lipid peroxidation. Inhibition of lipid peroxidation with D‐PUFAs further protected cells from cell death induced by oligomeric α‐Syn. Thus, lipid peroxidation induced by misfolding of α‐Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease.
Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose‐ and time‐dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK‐Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation‐dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat‐shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant‐induced activation of the MEK‐Erk pathway. These findings provide important insights into the mechanisms of activity‐driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation‐mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure.
Dysregulated metabolism and consequent extracellular accumulation of amyloid‐β (Aβ) peptides in the brain underlie the pathogenesis of Alzheimer's disease. Extracellular Aβ in the brain parenchyma is mainly secreted from the pre‐synaptic terminals of neuronal cells in a synaptic activity‐dependent manner. The p24 family member p24α2 reportedly attenuates Aβ generation by inhibiting γ‐secretase processing of amyloid precursor protein; however, the pattern of expression and localization of p24α2 in the brain remains unknown. We performed immunohistochemical staining and subcellular fractionation for p24α2 in the mouse brain. Immunostaining showed that p24α2 is broadly distributed in the gray matter of the central nervous system and is predominantly localized to synapses. Subcellular fractionation revealed prominent localization of p24α2 in the pre‐synaptic terminals. Immunoisolation of synaptic vesicles (SV) indicated that p24α2 is condensed at active zone‐docked SV. During development, p24α2 expression is highest in the post‐natal period and gradually decreases with age. We also confirmed that amyloid precursor protein and γ‐secretase components are localized at active zone‐docked SV. Our results suggest a novel functional role for p24α2 in the regulation of synaptic transmission and synaptogenesis, and provide evidence for the participation of p24α2 in the regulation of Aβ generation and secretion in the brain.
Gain‐of‐toxic‐function mutations in Seipin (Asparagine 88 to Serine (N88S) and Serine 90 to Leucine (S90L) mutations, both of which disrupt the N‐glycosylation) cause autosomal dominant motor neuron diseases. However, the mechanism of how these missense mutations lead to motor neuropathy is unclear. Here, we analyze the impact of disruption of N‐glycosylation of Seipin on synaptic transmission by over‐expressing mutant Seipin in cultured cortical neurons via lentiviral infection. Immunostaining shows that over‐expressed Seipin is partly colocalized with synaptic vesicle marker synaptophysin. Electrophysiological recordings reveal that the Seipin mutation significantly decreases the frequency, but not the amplitudes of miniature excitatory post‐synaptic currents and miniature inhibitory post‐synaptic currents. The amplitude of both evoked excitatory post‐synaptic currents and inhibitory post‐synaptic current is also compromised by mutant Seipin over‐expression. The readily releasable pool and vesicular release probability of synaptic vesicles are both altered in neurons over‐expressing Seipin‐N88S, whereas neither γ‐amino butyric acid (GABA) nor α‐Amino‐3‐hydroxy‐5‐methyl‐4‐ isoxazolepropionic acid (AMPA) induced whole cell currents are affected. Moreover, electron microscopy analysis reveals decreased number of morphologically docked synaptic vesicles in Seipin‐N88S‐expressing neurons. These data demonstrate that Seipin‐N88S mutation impairs synaptic neurotransmission, possibly by regulating the priming and docking of synaptic vesicles at the synapse.
GABAA receptors are pentameric ligand‐gated ion channels that mediate inhibitory fast synaptic transmission in the central nervous system. Consistent with recent pentameric ligand‐gated ion channels structures, sequence analysis predicts an α‐helix near the N‐terminus of each GABAA receptor subunit. Preceding each α‐helix are 8–36 additional residues, which we term the N‐terminal extension. In homomeric GABAC receptors and nicotinic acetylcholine receptors, the N‐terminal α‐helix is functionally essential. Here, we determined the role of the N‐terminal extension and putative α‐helix in heteromeric α1β2γ2 GABAA receptors. This role was most prominent in the α1 subunit, with deletion of the N‐terminal extension or further deletion of the putative α‐helix both dramatically reduced the number of functional receptors at the cell surface. Conversely, deletion of the β2 or γ2 N‐terminal extension had little effect on the number of functional cell surface receptors. Additional deletion of the putative α‐helix in the β2 or γ2 subunits did, however, decrease both functional cell surface receptors and incorporation of the γ2 subunit into mature receptors. In the β2 subunit only, α‐helix deletions affected GABA sensitivity and desensitization. Our findings demonstrate that N‐terminal extensions and α‐helices make key subunit‐specific contributions to assembly, consistent with both regions being involved in inter‐subunit interactions.
A major cause of alcohol toxicity is the production of reactive oxygen species generated during ethanol metabolism. The aim of this study was to compare the effect of binge drinking‐like alcohol exposure on a panel of genes implicated in oxidative mechanisms in adolescent and adult mice. In adolescent animals, alcohol decreased the expression of genes involved in the repair and protection of oxidative DNA damage such as atr, gpx7, or nudt15 and increased the expression of proapoptotic genes such as casp3. In contrast, in the adult brain, genes activated by alcohol were mainly associated with protective mechanisms that prevent cells from oxidative damage. Whatever the age, iterative binge‐like episodes provoked the same deleterious effects as those observed after a single binge episode. In adolescent mice, multiple binge ethanol exposure substantially reduced neurogenesis in the dentate gyrus and impaired short‐term memory in the novel object and passive avoidance tests. Taken together, our results indicate that alcohol causes deleterious effects in the adolescent brain which are distinct from those observed in adults. These data contribute to explain the greater sensitivity of the adolescent brain to alcohol toxicity.
Chronic neuroinflammation may be a critical component of intractable inflammatory diseases, including neuropathic pain. Because angiogenesis as a result of vascular endothelial growth factor (VEGF) signaling plays a pivotal role in inflammation, we focused on the mechanisms of VEGF‐regulated neuropathic pain in mice. The mRNA and protein expression of VEGFA were up‐regulated in the injured sciatic nerve after partial sciatic nerve ligation (PSL). VEGFA was localized to accumulated macrophages and neutrophils derived from bone marrow. Up‐regulation of VEGFA was mediated by histone H3 acetylation and trimethylation in its promoter region. VEGF receptors (VEGFR1 and VEGFR2) were localized to vascular endothelial cells or macrophages. By ex vivo fluorescence imaging and immunohistochemistry using DiI fluorescence, progression of angiogenesis was observed in the injured sciatic nerve after PSL. Perineural administration of pharmacological inhibitors of VEGFA and VEGFR tyrosine kinases prevented tactile allodynia and thermal hyperalgesia caused by PSL. Moreover, we determined the contribution of VEGF‐ and CXC‐chemokine receptor 4‐expressing angiogenic macrophages to neuropathic pain. Taken together, VEGFA is up‐regulated in injured peripheral nerves and participates in angiogenesis and prolonged pain behaviors through its receptors. We propose that VEGFA‐related components may underlie peripheral sensitization leading to neuropathic pain.
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