Aromatic-Turmerone Analogs Protect Dopaminergic Neurons in Midbrain Slice Cultures through Their Neuroprotective Activities.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. The inflammatory activation of microglia participates in dopaminergic neurodegeneration in PD. Therefore, chemicals that inhibit microglial activation are considered to have therapeutic potential for PD. Aromatic (ar)-turmerone is a main component of turmeric oil extracted from Curcuma longa and has anti-inflammatory activity in cultured microglia. The aims of the present study are (1) to investigate whether naturally occurring S-enantiomer of ar-turmerone (S-Tur) protects dopaminergic neurons in midbrain slice cultures and (2) to examine ar-turmerone analogs that have higher activities than S-Tur in inhibiting microglial activation and protecting dopaminergic neurons. R-enantiomer (R-Tur) and two analogs showed slightly higher anti-inflammatory effects in microglial BV2 cells. S- and R-Tur and these two analogs reversed dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. Unexpectedly, this neuroprotection was independent of the inhibition of microglial activation. Additionally, two analogs more potently inhibited dopaminergic neurodegeneration triggered by a neurotoxin, 1-methyl-4-phenylpyridinium, than S-Tur. Taken together, we identified two ar-turmerone analogs that directly and potently protected dopaminergic neurons. An investigation using dopaminergic neuronal precursor cells suggested the possible involvement of nuclear factor erythroid 2-related factor 2 in this neuroprotection.

Therapeutic potential of d-cysteine against in vitro and in vivo models of spinocerebellar ataxia.

Spinocerebellar ataxia (SCA) is a group of autosomal-dominantly inherited ataxia and is classified into SCA1-48 by the difference of causal genes. Several SCA-causing proteins commonly impair dendritic development in primary cultured Purkinje cells (PCs). We assume that primary cultured PCs expressing SCA-causing proteins are available as in vitro SCA models and that chemicals that improve the impaired dendritic development would be effective for various SCAs. We have recently revealed that D-cysteine enhances the dendritic growth of primary cultured PCs via hydrogen sulfide production. In the present study, we first investigated whether D-cysteine is effective for in vitro SCA models. We expressed SCA1-, SCA3-, and SCA21-causing mutant proteins to primary cultured PCs using adeno-associated viral serotype 9 (AAV9) vectors. D-Cysteine (0.2 mM) significantly ameliorated the impaired dendritic development commonly observed in primary cultured PCs expressing these three SCA-causing proteins. Next, we investigated the therapeutic effect of long-term treatment with D-cysteine on an in vivo SCA model. SCA1 model mice were established by the cerebellar injection of AAV9 vectors, which express SCA1-causing mutant ataxin-1, to ICR mice. Long-term treatment with D-cysteine (100 mg/kg/day) significantly inhibited the progression of motor dysfunction in SCA1 model mice. Immunostaining experiments revealed that D-cysteine prevented the reduction of mGluR1 and glial activation at the early stage after the onset of motor dysfunction in SCA1 model mice. These findings strongly suggest that D-cysteine has therapeutic potential against in vitro and in vivo SCA models and may be a novel therapeutic agent for various SCAs.

Nicotine promotes angiogenesis in mouse brain after intracerebral hemorrhage.

Here we examined the effect of nicotine on angiogenesis in the brain after intracerebral hemorrhage (ICH), as angiogenesis is considered to provide beneficial effects on brain tissues during recovery from injury after stroke. Nicotine was administered to C57BL/6 mice suffering from collagenase-induced ICH in the striatum, either by inclusion in drinking water or by daily intraperitoneal injection. Nicotine administration by both routes enhanced angiogenesis within the hematoma-affected regions, as revealed by increased CD31-immunopositive area at 7 and 14 d after ICH. Double immunofluorescence histochemistry against CD31 and proliferating cell nuclear antigen revealed that nicotine increased the number of newly generated vascular endothelial cells within the hematoma. In spite of enhanced angiogenesis, nicotine did not worsen vascular permeability after ICH, as assessed by Evans Blue extravasation. These effects of nicotine were accompanied by an increased number of surviving neurons in the hematoma at 7 d after ICH. Unexpectedly, nicotine did not increase expression of vascular endothelial growth factor mRNA in the brain and did not enhance recruitment of endothelial progenitor cells from the bone marrow. These results suggest that nicotine enhances angiogenesis in the brain after ICH, via mechanisms distinct from those involved in its action on angiogenesis in peripheral tissues.

Ataxic phenotype and neurodegeneration are triggered by the impairment of chaperone-mediated autophagy in cerebellar neurons.

AIMS: Chaperone-mediated autophagy (CMA) is a pathway involved in the autophagy lysosome protein degradation system. CMA has attracted attention as a contributing factor to neurodegenerative diseases since it participates in the degradation of disease-causing proteins. We previously showed that CMA is generally impaired in cells expressing the proteins causing spinocerebellar ataxias (SCAs). Therefore, we investigated the effect of CMA impairment on motor function and the neural survival of cerebellar neurons using the micro RNA (miRNA)-mediated knockdown of lysosome-associated protein 2A (LAMP2A), a CMA-related protein. METHODS: We injected adeno-associated virus serotype 9 vectors, which express green fluorescent protein (GFP) and miRNA (negative control miRNA or LAMP2A miRNA) under neuron-specific synapsin I promoter, into cerebellar parenchyma of 4-week-old ICR mice. Motor function of mice was evaluated by beam walking and footprint tests. Immunofluorescence experiments of cerebellar slices were conducted to evaluate histological changes in cerebella. RESULTS: GFP and miRNA were expressed in interneurons (satellite cells and basket cells) in molecular layers and granule cells in the cerebellar cortices, but not in cerebellar Purkinje cells. LAMP2A knockdown in cerebellar neurons triggered progressive motor impairment, prominent loss of cerebellar Purkinje cells, interneurons, granule cells at the late stage, and astrogliosis and microgliosis from the early stage. CONCLUSIONS: CMA impairment in cerebellar interneurons and granule cells triggers the progressive ataxic phenotype, gliosis and the subsequent degeneration of cerebellar neurons, including Purkinje cells. Our present findings strongly suggest that CMA impairment is related to the pathogenesis of various SCAs.

Glucocorticoids negatively regulates chaperone mediated autophagy and microautophagy.

Glucocorticoids are released from the adrenal cortex and are important for regulating various physiological functions. However, a persistent increase in glucocorticoids due to chronic stress causes various dysfunctions in the central nervous system which can lead to mental disorders such as depression. Macroautophagy, one of the pathways of the autophagy-lysosome protein degradation system, is dysregulated in psychiatric disorders, implicating a disturbance of protein degradation in the pathogenesis of psychiatric disorders. In the present study, we investigated whether glucocorticoids affect the activity of chaperone-mediated autophagy (CMA) and microautophagy (mA), the other two pathways of the autophagy-lysosome system. Treatment of human-derived AD293 cells and primary cultured rat cortical neurons with dexamethasone, a potent glucocorticoid receptor agonist, and endogenous glucocorticoids decreased both CMA and mA activities. However, this decrease was significantly suppressed by treatment with RU-486, a glucocorticoid receptor antagonist. In addition, dexamethasone significantly decreased lysosomal Hsc70. These findings suggest that glucocorticoids negatively regulate CMA and mA in a glucocorticoid receptor-dependent manner, and provide evidence for CMA and mA as novel therapeutic targets for depression.

Laquinimod and 3,3'-diindolylemethane alleviate neuropathological events and neurological deficits in a mouse model of intracerebral hemorrhage.

We examined the effects of compounds shown to activate aryl hydrocarbon receptor (AhR) signaling on a mouse model of intracerebral hemorrhage (ICH). Daily oral administration of laquinimod (25 mg/kg) or 3,3'-diindolylmethane (250 mg/kg) from 3 h after ICH induction improved motor functions, prevented the decrease of neurons within the hematoma, and attenuated activation of microglia/macrophages and astrocytes in the perihematomal region as well as infiltration of neutrophils into the hematoma. Elevated expression of AhR was detected in microglia and neutrophils, and both drugs inhibited upregulation of interleukin-6 and CXCL1. These results propose AhR as a therapeutic target for ICH.

Interactions between rat cortico-striatal slice cultures and neutrophil-like HL60 cells under thrombin challenge: Toward elucidation of pathological events in intracerebral hemorrhage.

Neutrophils constitute the major population of infiltrating leukocytes after stroke including intracerebral hemorrhage (ICH), and these cells may exhibit pro-inflammatory and anti-inflammatory phenotypes depending on the external stimuli. Here we constructed an experimental system to evaluate how the properties of neutrophils were influenced by the injured brain tissues. HL60 cells differentiated into neutrophils were added to the culture medium of neonatal rat cortico-striatal slices maintained at liquid-air interface. Thrombin was applied to the cultures to mimic the pathogenic events associated with ICH. HL60 cells responded to thrombin by increasing mRNA expression of pro-inflammatory IL-1ß and anti-inflammatory IL-10 with a different time course. Co-presence of cortico-striatal slice cultures significantly enhanced IL-1ß mRNA expression, whereas attenuated IL-10 mRNA expression, in HL60 cells. Toll-like receptor 4 (TLR4) agonist lipopolysaccharide synergistically enhanced IL-1ß mRNA expression with thrombin, and TLR4 inhibitor TAK-242 abolished thrombin-induced IL-1ß mRNA expression in the presence of slice cultures. On the other hand, thrombin-induced cell death in cortico-striatal cultures was attenuated by the presence of HL60 cells. This experimental system may provide a unique platform to elucidate complex cell-to-tissue interactions during ICH pathogenesis.

Chronic memantine administration prevents ouabain-induced hyperactivity in mice via maintenance of Na+, K+-ATPase activity in the hippocampus.

We have previously reported that mice received intracerebroventricular injection of ouabain, an inhibitor of Na+, K+-ATPase, exhibited hyperactivity via overactivation of glutamatergic neurons. Here we investigated the effects of memantine, a blocker of N-methyl-d-aspartate receptors, on ouabain-induced hyperactivity. In mice that received ouabain injection, chronic memantine administration prevented the hyperactivity and the decrease in the Na+, K+-ATPase activity in the hippocampus. Memantine also protected neurons without affecting glial activation in the hippocampus of these mice. Our results suggest that memantine improves hyperactivity via the maintenance of Na+, K+-ATPase activity and neurons in the hippocampus in this mouse model.

Reciprocal Regulation of Chaperone-Mediated Autophagy/Microautophagy and Exosome Release.

Autophagy-lysosome proteolysis is involved in protein quality control and classified into macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA), by the routes of substrate delivery to lysosomes. Both autophagy-lysosome proteolysis and exosome release are strongly associated with membrane trafficking. In the present study, we investigated how chemical and small interfering RNA (siRNA)-mediated activation and inhibition of these autophagic pathways affect exosome release in AD293 cells. Activation of MA and mA by rapamycin and activation of CMA by mycophenolic acid significantly decreased exosome release. Although lysosomal inhibitors, NH4Cl and bafilomycin A1, significantly increased exosome release, a MA inhibitor, 3-methyladenine, did not affect. Exosome release was significantly increased by the siRNA-mediated knockdown of LAMP2A, which is crucial for CMA. Inversely, activity of CMA/mA was significantly increased by the prevention of exosome release, which was induced by siRNA-mediated knockdown of Rab27a. These findings indicate that CMA/mA and exosome release are reciprocally regulated. This regulation would be the molecular basis of extracellular release and propagation of misfolded proteins in various neurodegenerative diseases.

Rapamycin activates mammalian microautophagy.

Autophagy-lysosome proteolysis is classified into macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA). In contrast to MA and CMA, mA have been mainly studied in yeast. In 2011, mammalian mA was identified as a pathway to deliver cytosolic proteins into multivesicular bodies. However, its molecular mechanism is quite different from yeast mA. Using a cell-based method to evaluate mA and CMA, we revealed that rapamycin, an activator of yeast mA, significantly activated mammalian mA. Although rapamycin activates MA, mA was also activated by rapamycin in MA-deficient cells. These findings suggest that rapamycin is a first-identified activator of mammalian mA.

A Nurr1 agonist amodiaquine attenuates inflammatory events and neurological deficits in a mouse model of intracerebral hemorrhage.

Inflammatory responses are considered to play pivotal roles in the pathogenesis of intracerebral hemorrhage (ICH). Here we show that a nuclear receptor Nurr1 (NR4A2) was expressed prominently in microglia/macrophages and astrocytes in the perihematomal region in the striatum of mice after ICH. Daily administration of a Nurr1 agonist amodiaquine (40 mg/kg, i.p.) from 3 h after ICH induction diminished perihematomal activation of microglia/macrophages and astrocytes. Amodiaquine also suppressed ICH-induced mRNA expression of IL-1ß, CCL2 and CXCL2, and ameliorated motor dysfunction of mice. These results suggest that Nurr1 serves a novel target for ICH therapy.

Involvement of exosomes in dopaminergic neurodegeneration by microglial activation in midbrain slice cultures.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopamine neurons in the substantia nigra. Microglial activation is frequently observed in the brains of patients with PD and animal models. Interferon-γ (IFN-γ)/lipopolysaccharide (LPS) treatment triggers microglial activation and the reduction of dopamine neurons in midbrain slice cultures. We have previously reported that nitric oxide (NO) is mainly involved in this dopaminergic degeneration. However, this degeneration was not completely suppressed by the inhibition of NO synthesis, suggesting that factors other than NO also contribute to dopaminergic neurodegeneration. Exosomes are extracellular vesicles with diameters of 40-200 nm that contain various proteins and micro RNAs and are regarded as a novel factor that mediates cell-to-cell interactions. Previous studies have demonstrated that exosome release is enhanced by microglial stimulation and that microglia-derived exosomes increases neuronal apoptosis. In the present study, we investigated whether exosomes are involved in dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. IFN-γ/LPS treatment to the midbrain slice cultures activated microglia, increased exosomal release, and decreased dopamine neurons. GW4869, an inhibitor of a neutral sphingomyelinase 2, decreased exosomal release and significantly prevented dopaminergic neurodegeneration by IFN-γ/LPS without affecting NO production. In contrast, D609, an inhibitor of sphingomyelin synthase and NO synthase, did not affect dopaminergic neurodegeneration, although it strongly inhibited NO production. The protective effect mediated by inhibition of NO synthase would be counteracted by enhanced exosomal release caused by D609 treatment. In addition, dopaminergic neurodegeneration is triggered by the treatment of exosomes isolated from culture media of IFN-γ/LPS-treated slices. These results suggest that exosomes are involved in dopaminergic neurodegeneration by microglial activation.

Propranolol prevents cerebral blood flow changes and pain-related behaviors in migraine model mice.

Propranolol, a ß-adrenergic receptor blocker, is one of the most commonly used prophylactic drugs for migraines. Cortical spreading depression (CSD) is the propagation wave of neuronal excitation along with cerebral blood flow (CBF) changes over the cerebral cortex and has been implicated in the pathological process of migraine auras and its pain response. However, the effect of propranolol on CSD-related CBF changes and behavioral responses remains poorly understood. In this study, we measured CSD-related CBF responses using a micro-device with a green light emitting diode (LED) and micro-complementary-metal-oxide-semiconductor (CMOS) image sensor and evaluated pain-related reduced locomotor activity in mice. An injection of KCl into the visual cortex led to CSD-related CBF changes; however, propranolol prevented the increase in CBF as well as delayed the propagation velocity in KCl-induced CSD. Furthermore, an injection of KCl reduced locomotor activity and induced freezing behavior in awake and freely moving mice, which were prevented by propranolol treatment. These results suggest that the modulation of CSD-related CBF responses by the blockade of ß-adrenergic receptor contributes to its prophylactic effects on migraines.

Na+, K+-ATPase inhibition causes hyperactivity and impulsivity in mice via dopamine D2 receptor-mediated mechanism.

Hyperactivity and impulsivity are common symptoms in several psychiatric disorders. Although dysfunction of Na+, K+-ATPase has been reported to be associated with the psychiatric disorders, it is not clear whether inhibition of Na+, K+-ATPase causes behavioral effects, including hyperactivity and impulsivity, in mice. Here, we evaluated the effect of intracerebroventricular (icv) injection of ouabain, an inhibitor of Na+, K+-ATPase, on hyperactivity and impulsivity in mice. At seven days after icv injection, ouabain-injected mice displayed the increase in the distance traveled in the open field arena in the open field test and the increase in the number of head-dipping behavior in the cliff avoidance test. Chlorpromazine or haloperidol, typical antipsychotics, reduced the hyperactivity and impulsivity in ouabain-injected mice. On the other hand, neither lithium carbonate nor valproate, established mood-stabilizing drugs, improved hyperactivity and impulsivity in our mouse model. Furthermore, ouabain-injected mice exhibited the increase in the number of c-fos-positive cells in the nucleus accumbens and the prefrontal cortex but not in the ventral tegmental area, which was reduced by haloperidol. These results suggest that the dysfunction of Na+, K+-ATPase causes hyperactivity and impulsivity via hyperactivation of dopamine D2 receptor-mediated signaling pathway, causing disturbed neuronal circuits in mice.

Endogenous Nitric Oxide Inhibits, Whereas Awakening Stimuli Increase, the Activity of a Subset of Orexin Neurons.

The lateral hypothalamic area contains neurons expressing neuronal nitric oxide synthase (nNOS), in addition to orexin neurons. Here we examined whether the activity of orexin neurons was regulated by endogenous nitric oxide (NO) in male C57BL/6 mice. Caffeine (30 mg/kg, intraperitoneally (i.p.)) increased the number of orexin neurons positive for c-Fos, a marker of neuronal activity, and also increased the number of NOS/c-Fos-positive cells as identified by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and c-Fos immunohistochemistry. Diphenhydramine hydrochloride (10 mg/kg. i.p.) decreased c-Fos-positive orexin neurons but had no significant effect on the number of c-Fos-positive NOS neurons. nNOS inhibitor 7-nitroindazole (25 mg/kg, i.p.) alone increased c-Fos-positive orexin neurons, and combined treatment with caffeine and 7-nitroindazole did not show additive effect in the number of c-Fos-positive orexin neurons. In contrast, 7-nitroindazole decreased c-Fos-positive NOS neurons and attenuated caffeine-induced increase in c-Fos-positive NOS neurons. Sleep deprivation increased c-Fos-positive cells in both orexin neurons and NOS neurons, and 7-nitroindazole did not show additive effect with sleep deprivation in the activation of orexin neurons. Together, these results suggest that endogenous NO negatively regulates the activity of a subset of orexin neurons, and this subset of orexin neurons overlaps with that activated by awakening stimuli.

d-Cysteine promotes dendritic development in primary cultured cerebellar Purkinje cells via hydrogen sulfide production.

Hydrogen sulfide and reactive sulfur species are regulators of physiological functions, have antioxidant effects against oxidative stresses, and are endogenously generated from l-cysteine. Recently, a novel pathway that generates hydrogen sulfide and reactive sulfur species from d-cysteine has been identified. d-Amino acid oxidase (DAO) is involved in this pathway and, among the various brain regions, is especially abundant in the cerebellum. d-Cysteine has been found to be a better substrate in the generation of hydrogen sulfide in the cerebellum than l-cysteine. Therefore, d-cysteine might be a novel neuroprotectant against cerebellar diseases such as spinocerebellar ataxia (SCA). However, it remains unknown if d-cysteine affects cerebellar Purkinje cells (PCs), which are important for cerebellar functions and are frequently degenerated in SCA patients. In the present study, we investigated whether the production of hydrogen sulfide from d-cysteine affects the dendritic development of cultured PCs. d-Cysteine was found to enhance the dendritic development of PCs significantly, while l-cysteine impaired it. The effect of d-cysteine was inhibited by simultaneous treatment with DAO inhibitors and was reproduced by treatment with 3-mercaptopyruvate, a metabolite of d-cysteine produced by the action of DAO, and disodium sulfide, a donor of hydrogen sulfide. In addition, hydrogen sulfide was immediately produced in cerebellar primary cultures after treatment with d-cysteine and 3-mercaptopyruvate. These findings suggest that d-cysteine enhances the dendritic development of primary cultured PCs via the generation of hydrogen sulfide.

Na+, K+-ATPase inhibition induces neuronal cell death in rat hippocampal slice cultures: Association with GLAST and glial cell abnormalities.

Na+, K+-ATPase is a highly expressed membrane protein. Dysfunction of Na+, K+-ATPase has been implicated in the pathophysiology of several neurodegenerative and psychiatric disorders, however, the underlying mechanism of neuronal cell death resulting from Na+, K+-ATPase dysfunction is poorly understood. Here, we investigated the mechanism of neurotoxicity due to Na+, K+-ATPase inhibition using rat organotypic hippocampal slice cultures. Treatment with ouabain, a Na+, K+-ATPase inhibitor, increased the ratio of propidium iodide-positive cells among NeuN-positive cells in the hippocampal CA1 region, which was prevented by MK-801 and d-AP5, specific blockers of the N-methyl-d-aspartate (NMDA) receptor. EGTA, a Ca2+-chelating agent, also protected neurons from ouabain-induced injury. We observed that astrocytes expressed the glutamate aspartate transporter (GLAST), and ouabain changed the immunoreactive area of GFAP-positive astrocytes as well as GLAST. We also observed that ouabain increased the number of Iba1-positive microglial cells in a time-dependent manner. Furthermore, lithium carbonate, a mood-stabilizing drug, protected hippocampal neurons and reduced disturbances of astrocytes and microglia after ouabain treatment. Notably, lithium carbonate improved ouabain-induced decreases in GLAST intensity in astrocytes. These results suggest that glial cell abnormalities resulting in excessive extracellular concentrations of glutamate contribute to neurotoxicity due to Na+, K+-ATPase dysfunction in the hippocampal CA1 region.

Lysosomal dysfunction and early glial activation are involved in the pathogenesis of spinocerebellar ataxia type 21 caused by mutant transmembrane protein 240.

Spinocerebellar ataxia type 21 (SCA21) is caused by missense or nonsense mutations of the transmembrane protein 240 (TMEM240). Molecular mechanisms of SCA21 pathogenesis remain unknown because the functions of TMEM240 have not been elucidated. We aimed to reveal the molecular pathogenesis of SCA21 using cell and mouse models that overexpressed the wild-type and SCA21 mutant TMEM240. In HeLa cells, overexpressed TMEM240 localized around large cytoplasmic vesicles. The SCA21 mutation did not affect this localization. Because these vesicles contained endosomal markers, we evaluated the effect of TMEM240 fused with a FLAG tag (TMEM-FL) on endocytosis and autophagic protein degradation. Wild-type TMEM-FL significantly impaired clathrin-mediated endocytosis, whereas the SCA21 mutants did not. The SCA21 mutant TMEM-FL significantly impaired autophagic lysosomal protein degradation, in contrast to wild-type. Next, we investigated how TMEM240 affects the neural morphology of primary cultured cerebellar Purkinje cells (PCs). The SCA21 mutant TMEM-FL significantly prevented the dendritic development of PCs, in contrast to the wild-type. Finally, we assessed mice that expressed wild-type or SCA21 mutant TMEM-FL in cerebellar neurons using adeno-associated viral vectors. Mice expressing the SCA21 mutant TMEM-FL showed impaired motor coordination. Although the SCA21 mutant TMEM-FL did not trigger neurodegeneration, activation of microglia and astrocytes was induced before motor miscoordination. In addition, immunoblot experiments revealed that autophagic lysosomal protein degradation, especially chaperone-mediated autophagy, was also impaired in the cerebella that expressed the SCA21 mutant TMEM-FL. These dysregulated functions in vitro, and induction of early gliosis and lysosomal impairment in vivo by the SCA21 mutant TMEM240 may contribute to the pathogenesis of SCA21.

Polysulfide protects midbrain dopaminergic neurons from MPP+-induced degeneration via enhancement of glutathione biosynthesis.

Polysulfides are endogenous sulfur-containing molecular species that may regulate various cellular functions. Here we examined the effect of polysulfides exogenously applied to rat midbrain slice cultures, to address their potential neuroprotective actions. Na2S3 at concentrations of 10 µM or higher prevented 1-methyl-4-phenylpyridinium (MPP+)-induced loss of dopaminergic neurons. Na2S4 at 10 µM also protected dopaminergic neurons from MPP+ cytotoxicity, whereas Na2S and Na2S2 at the same concentration had no significant effect. We also found that Na2S3 (10 µM) prevented MPP+-induced increase in intracellular reactive oxygen species as detected by 2',7'-dichlorofluorescein fluorescence. In addition, the protective effect of Na2S3 was abolished by l-buthionine sulfoximine, an inhibitor of glutathione synthesis. In cellular models of neurons (SH-SY5Y cells) and glial cells (C6 cells), Na2S3 (30 and 100 µM) increased expression of mRNAs encoding the subunits of glutamate cysteine ligase, the rate-limiting enzyme for glutathione biosynthesis. Consistently, the cellular content of total glutathione was increased by Na2S3, and the effect was more prominent in SH-SY5Y cells than in C6 cells. These results suggest that polysulfides are efficient neuroprotectants superior to monosulfur species such as H2S and HS-, and that the neuroprotective effect of polysulfides is mediated by upregulation of glutathione biosynthesis.

Cystamine-mediated inhibition of protein disulfide isomerase triggers aggregation of misfolded orexin-A in the Golgi apparatus and prevents extracellular secretion of orexin-A.

Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy.