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  • A dibenzoylmethane derivative inhibits lipopolysaccharide-induced NO production in mouse microglial cell line BV-2
    Neurochem. Int. (IF 3.262) Pub Date : 2017-04-05
    Katsura Takano, Natsumi Ishida, Kenji Kawabe, Mitsuaki Moriyama, Satoshi Hibino, Tominari Choshi, Osamu Hori, Yoichi Nakamura

    Microglial activation has been suggested to play important roles in various neurodegenerative diseases by phagocytosis and producing various factors such as nitric oxide (NO), proinflammatory cytokines. Excessive production of NO, as a consequence of increased inducible nitric oxide synthase (iNOS) in microglia, contributes to the neurodegeneration. During a search for compounds that regulate endoplasmic reticulum (ER) stress, a dibenzoylmethane derivative, 2,2’-dimethoxydibenzoylmethane (DBM 14–26) was identified as a novel neuroprotective agent (Takano et al., Am. J. Physiol. Cell Physiol. 293, C1884-1894, 2007). We previously reported in cultured astrocytes that DBM 14–26 protected hydrogen peroxide-induced cell death and inhibited lipopolysaccharide (LPS)-induced NO production (Takano et al., J. Neurosci. Res. 89, 955–965, 2011). In the present study, we assessed the effects of DBM 14–26 on microglia using the mouse cell line BV-2 and found that DBM 14–26 inhibited LPS-induced iNOS expression and NO production also in microglia. DBM 14–26 also suppressed LPS-induced IL-1β expression. Conditioned medium of BV-2 cells stimulated by LPS significantly decreased cell viability of neuron (human neuroblastoma SH-SY5Y cells) compared with the absence of LPS. Conditioned medium of BV-2 cells stimulated by LPS in the presence of DBM 14–26 did not significantly decreased cell viability of neuron. These results indicate that microglial activation by LPS causes neuronal cell death and DBM 14–26 protect neuron through the inhibition of microglial activation. Functional regulation of microglia by DBM 14–26 could be a therapeutic candidate for the treatment of neurodegenerative diseases.

  • Genetic overexpression of glutathione peroxidase-1 attenuates microcystin-leucine-arginine-induced memory impairment in mice
    Neurochem. Int. (IF 3.262) Pub Date : 2018-06-13
    Eun-Joo Shin, Yeong Gwang Hwang, Duc Toan Pham, Ji Won Lee, Yu Jeung Lee, Dongjin Pyo, Xin Gen Lei, Ji Hoon Jeong, Hyoung-Chun Kim
  • The protective effect of α-lipoic acid against bisphenol A-induced neurobehavioral toxicity
    Neurochem. Int. (IF 3.262) Pub Date : 2018-06-13
    Jasim Khan, Shikha Salhotra, Shahzad Ahmad, Shikha Sharma, Sayed Aliul Hasan Abdi, Basu Dev Banerjee, Suhel Parvez, Sarika Gupta, Sheikh Raisuddin
  • The neurotoxin diethyl dithiophosphate impairs glutamate transport in cultured Bergmann glia cells
    Neurochem. Int. (IF 3.262) Pub Date : 2018-06-13
    Tatiana N. Olivares-Bañuelos, Isabel Martínez-Hernández, Luisa C. Hernández-Kelly, Donají Chi-Castañeda, Libia Vega, Arturo Ortega

    Glutamate, the main excitatory neurotransmitter in the vertebrate Central Nervous System, is involved in almost every aspect of brain physiology, and its signaling properties are severely affected in most neurodegenerative diseases. This neurotransmitter has to be efficiently removed from the synaptic cleft in order to prevent an over-stimulation of glutamate receptors that leads to neuronal death. Specific sodium-dependent membrane transporters, highly enriched in glial cells, elicit the clearance of glutamate. Once internalized, it is metabolized to glutamine by the glia-enriched enzyme Glutamine synthetase. Accumulated glutamine is released into the extracellular space for its uptake into pre-synaptic neurons and its conversion to glutamate that is packed into synaptic vesicles completing the glutamate/glutamine cycle. Diverse chemical compounds, like organophosphates, directly affect brain chemistry by altering levels of neurotransmitters in the synaptic cleft. Organophosphate compounds are widely used as pesticides, and all living organisms are continuously exposed to these substances, either in a direct or indirect manner. Its metabolites, like the diethyl dithiophosphate, are capable of causing brain damage through diverse mechanisms including perturbation of neuronal-glial cell interactions and have been associated with attention-deficit disorders and other mental illness. In order to characterize the neurotoxic mechanisms of diethyl dithiophosphate, we took advantage of the well characterized model of chick cerebellar Bergmann glia cultures. A significant impairment of [3H] d-Aspartate transport was found upon exposure to the metabolite. These results indicate that glia cells are targets of neurotoxic substances such as pesticides and that these cells might be critically involved in the associated neuronal death.

  • Modulation of GABA and glycine receptors in rat pyramidal hippocampal neurones by 3α5β-pregnanolone derivatives
    Neurochem. Int. (IF 3.262) Pub Date : 2018-06-07
    Julia V. Bukanova, Elena I. Solntseva, Sergey N. Kolbaev, Eva Kudova

    The ability of pregnanolone glutamate (PA-Glu), pregnanolone hemisuccinate (PA-hSuc) and pregnanolone hemipimelate (PA-hPim), neuroactive steroids with a negative modulatory effect on excitatory N-methyl-d-aspartate receptors, to influence the functional activity of inhibitory γ-aminobutyric acid and glycine receptors was estimated. The GABA- and glycine-induced chloride currents (IGABA and IGly) were measured in isolated pyramidal neurons of the rat hippocampus using the patch-clamp technique. Compound PA-Glu was found to potentiate IGABA and to inhibit IGly, while PA-hSuc and PA-hPim inhibited both IGABA and IGly. Moreover, PA-Glu, PA-hSuc, and PA-hPim had a greater effect on desensitization than on the peak amplitude of IGly. At a high concentration of glycine (500 μM), the effect of neurosteroids on the peak amplitude of IGly disappeared, and the acceleration of desensitization remained. The conversion of PA-Glu into androstane glutamate (AND-Glu), an analogue that lacks the C-17 acetyl moiety, completely eliminated the effects on these receptors. Our results indicate that the C-17 acetyl moiety is crucial for the action on IGABA and IGly. Our results indicate that the pregnanolone derivatives, in contrast to the androstane analogues, modulate IGABA and IGly at low micromolar concentrations and this family of neurosteroids can be useful for future structure-activity relationship studies of the steroid modulation of other receptor types.

  • Human dihydrolipoamide dehydrogenase (E3) deficiency: Novel insights into the structural basis and molecular pathomechanism
    Neurochem. Int. (IF 3.262) Pub Date : 2017-06-02
    Attila Ambrus, Vera Adam-Vizi

    This review summarizes our present view on the molecular pathogenesis of human (h) E3-deficiency caused by a variety of genetic alterations with a special emphasis on the moonlighting biochemical phenomena related to the affected (dihydro)lipoamide dehydrogenase (LADH, E3, gene: dld), in particular the generation of reactive oxygen species (ROS). E3-deficiency is a rare autosomal recessive genetic disorder frequently presenting with a neonatal onset and premature death; the highest carrier rate of a single pathogenic dld mutation (1:94–1:110) was found among Ashkenazi Jews. Patients usually die during acute episodes that generally involve severe metabolic decompensation and lactic acidosis leading to neurological, cardiological, and/or hepatological manifestations. The disease owes its severity to the fact that LADH is the common E3 subunit of the alpha-ketoglutarate (KGDHc), pyruvate (PDHc), and branched-chain α-keto acid dehydrogenase complexes and is also part of the glycine cleavage system, hence the malfunctioning of LADH simultaneously incapacitates several central metabolic pathways. Nevertheless, the clinical pictures are usually not unequivocally portrayed through the loss of LADH activities and imply auxiliary mechanisms that exacerbate the symptoms and outcomes of this disorder. Enhanced ROS generation by disease-causing hE3 variants as well as by the E1-E2 subcomplex of the hKGDHc likely contributes to selected pathogeneses of E3-deficiency, which could be targeted by specific drugs or antioxidants; lipoic acid was demonstrated to be a potent inhibitor of ROS generation by hE3 in vitro. Flavin supplementation might prove to be beneficial for those mutations triggering FAD loss in the hE3 component. Selected pathogenic hE3 variants lose their affinity for the E2 component of the hPDHc, a mechanism which warrants scrutiny also for other E3-haboring complexes.

  • Mitochondria/metabolic reprogramming in the formation of neurons from peripheral cells: Cause or consequence and the implications to their utility
    Neurochem. Int. (IF 3.262) Pub Date : 2017-06-13
    Gary E. Gibson, Ankita Thakkar

    The induction of pluripotent stem cells (iPSC) from differentiated cells such as fibroblasts and their subsequent conversion to neural progenitor cells (NPC) and finally to neurons is intriguing scientifically, and its potential to medicine is nearly infinite, but unrealized. A better understanding of the changes at each step of the transformation will enable investigators to better model neurological disease. Each step of conversion from a differentiated cell to an iPSC to a NPC to neurons requires large changes in glycolysis including aerobic glycolysis, the pentose shunt, the tricarboxylic acid cycle, the electron transport chain and in the production of reactive oxygen species (ROS). These mitochondrial/metabolic changes are required and their manipulation modifies conversions. These same mitochondrial/metabolic processes are altered in common neurological diseases so that factors related to the disease may alter the cellular transformation at each step including the final phenotype. A lack of understanding of these interactions could compromise the validity of the disease comparisons in iPSC derived neurons. Both the complexity and potential of iPSC derived cells for understanding and treating disease remain great.

  • PQBP1, an intrinsically disordered/denatured protein at the crossroad of intellectual disability and neurodegenerative diseases
    Neurochem. Int. (IF 3.262) Pub Date : 2017-06-13
    Hitoshi Okazawa

    PQBP1 (polyglutamine binding protein-1) is the earliest identified molecule among the group of disease-related intrinsically disordered/denatured proteins. PQBP1 interacts with splicing-related factors via the disordered/denatured domain and regulates post-transcriptional gene expression. The mutations cause intellectual disability due to decreased dendritic spines and abnormal expression of synapse molecules in neurons, and microcephaly due to elongated cell cycle time and abnormal expression of cell cycle proteins in neural stem progenitor cells. Meanwhile, PQBP1 interacts with polyglutamine tract sequences translated from CAG triplet disease genes via their disordered/denatured structures. The second hit on PQBP1 by such neurodegenerative disease proteins is supposed to similarly impair synapse functions in neuron and proliferation of stem cells. The alteration of gene expression profile and consequently induced phenotypes of neuron and stem cells via secondary impairment of the intrinsically disordered/denatured protein PQBP1, which are similar to developmental disorders by PQBP1 gene mutations, could be a part of the main pathologies shared by multiple neurodegenerative diseases.

  • Mitochondrial permeability transition pore: Back to the drawing board
    Neurochem. Int. (IF 3.262) Pub Date : 2017-06-21
    Christos Chinopoulos

    Current models theorizing on what the mitochondrial permeability transition (mPT) pore is made of, implicate the c-subunit rings of ATP synthase complex. However, two very recent studies, one on atomistic simulations and in the other disrupting all genes coding for the c subunit disproved those models. As a consequence of this, the structural elements of the pore remain unknown. The purpose of the present short-review is to (i) briefly review the latest findings, (ii) serve as an index for more comprehensive reviews regarding mPT specifics, (iii) reiterate on the potential pitfalls while investigating mPT in conjunction to bioenergetics, and most importantly (iv) suggest to those in search of mPT pore identity, to also look elsewhere.

  • The broad spectrum of signaling pathways regulated by unfolded protein response in neuronal homeostasis
    Neurochem. Int. (IF 3.262) Pub Date : 2017-06-28
    Atsushi Saito, Kazunori Imaizumi

    The protein folding capabilities in the endoplasmic reticulum (ER) are disturbed by alternations in the cellular homeostasis such as the disruption of calcium ion homeostasis, the expression of mutated proteins and oxidative stress. In response to these ER dysfunctions, eukaryotic cells activate canonical branches of signal transduction cascades to restore the protein folding capacity and avoid irreversible damages, collectively termed the unfolded protein response (UPR). Prolonged ER dysfunctions and the downregulation of UPR signaling pathways have been accepted as a crucial trigger for the pathogenesis of various neurodegenerative diseases. Furthermore, recent studies have revealed that the UPR has a wide spectrum of signaling pathways for unique physiological roles in the diverse developmental, differential and lipidomic processes. A developed and intricate ER network exists in the neurites of neurons. Neuronal ER functions and ER-derived signaling mediate efficient communication between cell soma and distal sites through local protein synthesis, sorting and lipogenesis. However, relevant of ER-derived UPR signaling pathways in the elaborate mechanisms regulating neuronal activities, synaptic functions and protective responses against injury is not fully elucidated. In this review, we summarized our current understanding of how the UPR functions provide the appropriate signals for neuronal capabilities. We also reviewed how UPR dysfunctions lead to the pathogenesis of neurodegenerative diseases, and the possibilities ameliorating their toxic effects by targeting UPR components.

  • Pathophysiological role of prostaglandin E2-induced up-regulation of the EP2 receptor in motor neuron-like NSC-34 cells and lumbar motor neurons in ALS model mice
    Neurochem. Int. (IF 3.262) Pub Date : 2017-07-04
    Yasuhiro Kosuge, Hiroko Miyagishi, Yuki Yoneoka, Keiko Yoneda, Hiroshi Nango, Kumiko Ishige, Yoshihisa Ito

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective degeneration of motor neurons. The primary triggers for motor neuronal death are still unknown, but inflammation is considered to be an important factor contributing to the pathophysiology of ALS both clinically and in ALS models. Prostaglandin E2 (PGE2) and its corresponding four E-prostanoid receptors play a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. It has also been shown that PGE2-EP2 signaling in glial cells (astrocytes or microglia) promotes motor neuronal death in G93A mice. The present study was designed to investigate the levels of expression of EP receptors in the spinal motor neurons of ALS model mice and to examine whether PGE2 alters the expression of EP receptors in differentiated NSC-34 cells, a motor neuron-like cell line. Immunohistochemical staining demonstrated that EP2 and EP3 immunoreactivity was localized in NeuN-positive large cells showing the typical morphology of motor neurons in mice. Semi-quantitative analysis showed that the immunoreactivity of EP2 in motor neurons was significantly increased in the early symptomatic stage in ALS model mice. In contrast, the level of EP3 expression remained constant, irrespective of age. In differentiated NSC-34 cells, bath application of PGE2 resulted in a concentration-dependent decrease of MTT reduction. Although PGE2 had no effect on cell survival at concentrations of less than 10 μM, pretreatment with 10 μM PGE2 significantly up-regulated EP2 and concomitantly potentiated cell death induced by 30 μM PGE2. These results suggest that PGE2 is an important effector for induction of the EP2 subtype in differentiated NSC-34 cells, and that not only EP2 up-regulation in glial cells but also EP2 up-regulation in motor neurons plays a pivotal role in the vulnerability of motor neurons in ALS model mice.

  • Traffic jam hypothesis: Relationship between endocytic dysfunction and Alzheimer's disease
    Neurochem. Int. (IF 3.262) Pub Date : 2017-07-08
    Nobuyuki Kimura, Katsuhiko Yanagisawa

    Membrane trafficking pathways, like the endocytic pathway, carry out fundamental cellular processes that are essential for normal functioning. One such process is regulation of cell surface receptor signaling. A growing body of evidence suggests that β-amyloid protein (Aβ) plays a key role in Alzheimer's disease (AD) pathogenesis. Cleavage of Aβ from its precursor, β-amyloid precursor protein (APP), occurs through the endocytic pathway in neuronal cells. In early-stage AD, intraneuronal accumulation of abnormally enlarged endosomes is common, indicating that endosome trafficking is disrupted. Strikingly, genome-wide association studies reveal that several endocytosis-related genes are associated with AD onset. Also, recent studies demonstrate that alteration in endocytosis induces not only Aβ pathology but also the propagation of tau protein pathology, another key pathological feature of AD. Endocytic dysfunction can disrupt neuronal physiological functions, such as synaptic vesicle transport and neurotransmitter release. Thus, “traffic jams” in the endocytic pathway may be involved in AD pathogenesis and may serve as a novel target for the development of new therapeutics.

  • Involvement of endoplasmic reticulum stress and neurite outgrowth in the model mice of autism spectrum disorder
    Neurochem. Int. (IF 3.262) Pub Date : 2017-07-12
    Koichi Kawada, Seisuke Mimori, Yasunobu Okuma, Yasuyuki Nomura

    Neurodevelopmental disorders are congenital impairments, impeding the growth and development of the central nervous system. These disorders include autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder in Diagnostic and Statistical Manual of Mental Disorders-5. ASD is caused by a gene defect and chromosomal duplication. Despite numerous reports on ASD, the pathogenic mechanisms are not clear. The optimal methods to prevent ASD and to treat it are also not clear. Other studies have reported that endoplasmic reticulum (ER) stress contributes to the pathogenesis of neurodegenerative diseases. In this study, we have investigated ER stress condition and neuronal maturation in an ASD mice model employing male ICR mice. An ASD mice model was established by injecting with valproic acid (VPA) into pregnant mice. The offspring born from VPA-treated mothers were subjected to the experiments as the ASD model mice. The cerebral cortex and hippocampus of ASD model mice were found to be under high ER stress. The mRNA levels of Hes1 and Pax6 were decreased in the cerebral cortex of the ASD model mice, but not in the hippocampus. In addition, the mRNA level in Math1 was increased in the cerebral cortex. ER stress inhibited dendrite and axon extension in primary culture derived from the cerebral cortex of E14.5 mice. Furthermore, dendrite outgrowth was suppressed in primary culture derived from the cerebral cortex of ASD model mice by the same method. These results indicated the possibility that ER stress induces abnormal neuronal maturation in the embryonal cerebral cortex of ASD model mice employing male ICR mice. Therefore, ER stress may contribute to the pathogenesis of ASD.

  • Ubiquitination at the mitochondria in neuronal health and disease
    Neurochem. Int. (IF 3.262) Pub Date : 2017-07-12
    Christian Covill-Cooke, Jack H. Howden, Nicol Birsa, Josef T. Kittler

    The preservation of mitochondrial function is of particular importance in neurons given the high energy requirements of action potential propagation and synaptic transmission. Indeed, disruptions in mitochondrial dynamics and quality control are linked to cellular pathology in neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Here, we will discuss the role of ubiquitination by the E3 ligases: Parkin, MARCH5 and Mul1, and how they regulate mitochondrial homeostasis. Furthermore, given the role of Parkin and Mul1 in the formation of mitochondria-derived vesicles we give an overview of this area of mitochondrial homeostasis. We highlight how through the activity of these enzymes and MDV formation, multiple facets of mitochondrial biology can be regulated, ensuring the functionality of the mitochondrial network thus preserving neuronal health.

  • Pharmacologic modeling of primary mitochondrial respiratory chain dysfunction in zebrafish
    Neurochem. Int. (IF 3.262) Pub Date : 2017-07-18
    James Byrnes, Rebecca Ganetzky, Richard Lightfoot, Michael Tzeng, Eiko Nakamaru-Ogiso, Christoph Seiler, Marni J. Falk

    Mitochondrial respiratory chain (RC) disease is a heterogeneous and highly morbid group of energy deficiency disorders for which no proven effective therapies exist. Robust vertebrate animal models of primary RC dysfunction are needed to explore the effects of variation in RC disease subtypes, tissue-specific manifestations, and major pathogenic factors contributing to each disorder, as well as their pre-clinical response to therapeutic candidates. We have developed a series of zebrafish (Danio rerio) models that inhibit, to variable degrees, distinct aspects of RC function, and enable quantification of animal development, survival, behaviors, and organ-level treatment effects as well as effects on mitochondrial biochemistry and physiology. Here, we characterize four pharmacologic inhibitor models of mitochondrial RC dysfunction in early larval zebrafish, including rotenone (complex I inhibitor), azide (complex IV inhibitor), oligomycin (complex V inhibitor), and chloramphenicol (mitochondrial translation inhibitor that leads to multiple RC complex dysfunction). A range of concentrations and exposure times of each RC inhibitor were systematically evaluated on early larval development, animal survival, integrated behaviors (touch and startle responses), organ physiology (brain death, neurologic tone, heart rate), and fluorescence-based analyses of mitochondrial physiology in zebrafish skeletal muscle. Pharmacologic RC inhibitor effects were validated by spectrophotometric analysis of Complex I, II and IV enzyme activities, or relative quantitation of ATP levels in larvae. Outcomes were prioritized that utilize in vivo animal imaging and quantitative behavioral assessments, as may optimally inform the translational potential of pre-clinical drug screens for future clinical study in human mitochondrial disease subjects. The RC complex inhibitors each delayed early embryo development, with short-term exposures of these three agents or chloramphenicol from 5 to 7 days post fertilization also causing reduced larval survival and organ-specific defects ranging from brain death, behavioral and neurologic alterations, reduced mitochondrial membrane potential in skeletal muscle (rotenone), and/or cardiac edema with visible blood pooling (oligomycin). Remarkably, we demonstrate that treating animals with probucol, a nutrient-sensing signaling network modulating drug that has been shown to yield therapeutic effects in a range of other RC disease cellular and animal models, both prevented acute rotenone-induced brain death in zebrafish larvae, and significantly rescued early embryo developmental delay from either rotenone or oligomycin exposure. Overall, these zebrafish pharmacologic RC function inhibition models offer a unique opportunity to gain novel insights into diverse developmental, survival, organ-level, and behavioral defects of varying severity, as well as their individual response to candidate therapies, in a highly tractable and cost-effective vertebrate animal model system.

  • Oxytocin release via activation of TRPM2 and CD38 in the hypothalamus during hyperthermia in mice: Implication for autism spectrum disorder
    Neurochem. Int. (IF 3.262) Pub Date : 2017-07-20
    Haruhiro Higashida, Teruko Yuhi, Shirin Akther, Sarwat Amina, Jing Zhong, Mingkun Liang, Tomoko Nishimura, Hong-Xiang Liu, Olga Lopatina

    Oxytocin (OT) is a critical molecule for social recognition that mediates social and emotional behaviors. OT is released during stress and acts as an anxiolytic factor. To know the precise molecular mechanisms underlying OT release into the brain during stress is important. It has been reported that intracellular concentrations of free calcium in the hypothalamic neurons are elevated by simultaneous stimulation of cyclic ADP-ribose (cADPR) and heat. We have reported in vitro and in vivo data that supports the idea that release of OT in the brain of male mice is regulated by cADPR and fever in relation to stress conditions. 1) Significantly higher levels of OT release were observed in hypothalamus cultures isolated from subordinate mice in group-housed males compared to dominant males after cage-switch stress; 2) OT concentrations in micro-perfusates at the paraventricular nucleus upon perfusion stimulation with cADPR were enhanced in subordinate mice compared to dominant mice; 3) The OT concentration in the cerebrospinal fluid (CSF) was higher in endotoxin-shock mice with fever compared to controls with no body temperature increase; and 4) In mice exposed to new environmental stress, the CSF OT level transiently increased 5 min after exposure, while the rectal temperature increased from 36.6 °C to 37.8 °C from 5 to 15 min after exposure. In this review, we examine whether or not cADPR and hyperthermia co-regulate hypothalamic OT secretion during social stress through the elevation of intracellular free Ca2+ concentrations involved in CD38-dependent Ca2+ mobilization and TRPM2-dependent Ca2+ influx. Finally, we propose that the interaction between CD38 and TRPM2 seems to be a new mechanism for stress-induced release of OT, which may result in anxiolytic effects for temporal recovery from social impairments in children with autism spectrum disorder during hyperthermia.

  • Spatial organization of genome architecture in neuronal development and disease
    Neurochem. Int. (IF 3.262) Pub Date : 2017-07-28
    Yuki Fujita, Toshihide Yamashita

    Although mammalian genomes encode genetic information in their linear sequences, their fundamental function with regard to gene expression depends on the higher-order structure of chromosomes. Current techniques for the evaluation of chromosomal structure have revealed that genomes are arranged at several hierarchical levels in three-dimensional space. The spatial organization of genomes involves the formation of chromatin loops that bypass a wide range of genomic distances, providing a connection between enhancers and chromosomal domains. Furthermore, they form chromatin domains that are arranged into chromosome territories in the three-dimensional space of the cell nucleus. Recent studies have shown that the spatial organization of the genome is essential for normal brain development and function. Activity-dependent alterations in the spatial organization of the genome can regulate transcriptional activity related to neuronal plasticity. Disruptions in the higher-order chromatin architecture have been implicated in neuropsychiatric disorders, such as cognitive dysfunction and anxiety. Here, we discuss the growing interest in the role of genome organization in brain development and neurological disorders.

  • Mitochondrial dysfunction in the neuro-degenerative and cardio-degenerative disease, Friedreich's ataxia
    Neurochem. Int. (IF 3.262) Pub Date : 2017-08-04
    Shannon Chiang, Danuta S. Kalinowski, Patric J. Jansson, Des R. Richardson, Michael L.-H. Huang

    Mitochondrial homeostasis is essential for maintaining healthy cellular function and survival. The detrimental involvement of mitochondrial dysfunction in neuro-degenerative diseases has recently been highlighted in human conditions, such as Parkinson's, Alzheimer's and Huntington's disease. Friedreich's ataxia (FA) is another neuro-degenerative, but also cardio-degenerative condition, where mitochondrial dysfunction plays a crucial role in disease progression. Deficient expression of the mitochondrial protein, frataxin, is the primary cause of FA, which leads to adverse alterations in whole cell and mitochondrial iron metabolism. Dys-regulation of iron metabolism in these compartments, results in the accumulation of inorganic iron deposits in the mitochondrial matrix that is thought to potentiate oxidative damage observed in FA. Therefore, the maintenance of mitochondrial homeostasis is crucial in the progression of neuro-degenerative conditions, particularly in FA. In this review, vital mitochondrial homeostatic processes and their roles in FA pathogenesis will be discussed. These include mitochondrial iron processing, mitochondrial dynamics (fusion and fission processes), mitophagy, mitochondrial biogenesis, mitochondrial energy production and calcium metabolism.

  • Mitophagy in neurodegenerative diseases
    Neurochem. Int. (IF 3.262) Pub Date : 2017-08-08
    Carlo Rodolfo, Silvia Campello, Francesco Cecconi

    Neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and Amyotrophic Lateral Sclerosis (ALS), are a complex “family” of pathologies, characterised by the progressive loss of neurons and/or neuronal functions, leading to severe physical and cognitive inabilities in affected patients. These syndromes, despite differences in the causative events, the onset, and the progression of the disease, share as common features the presence of aggregate-prone neuro-toxic proteins, in the form of aggresomes and/or inclusion bodies, perturbing cellular homeostasis and neuronal function (Popovic et al., 2014), and the presence of dysfunctional mitochondria. The removal of protein aggregates and of damaged organelles, through the ubiquitin-proteasome system (UPS) and/or the autophagy/lysosome machinery, is a crucial step for the maintenance of neuronal homeostasis. Indeed, their impairment has been reported as associated with the development of these diseases. In this review, we focus on the role played by mitophagy, a specialised form of autophagy, in the onset and progression of major neurodegenerative diseases, as well as on possible therapeutic approaches involving mitophagy modulation.

  • Alzheimer's disease as oligomeropathy
    Neurochem. Int. (IF 3.262) Pub Date : 2017-08-16
    Kenjiro Ono

    Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder and is characterized by pathological aggregates of amyloid β-protein (Aβ) and tau protein. On the basis of genetic evidence, biochemical data, and animal models, Aβ has been suggested to be responsible for the pathogenesis of AD (the amyloid hypothesis). Aβ molecules tend to aggregate to form oligomers, protofibrils, and mature fibrils. Although mature fibrils in the final stage have been thought to be the cause of AD pathogenesis, recent studies using synthetic Aβ peptides, a cell culture model, Aβ precursor protein transgenic mice models, and human samples, such as cerebrospinal fluids and postmortem brains of AD patients, suggest that pre-fibrillar forms (oligomers of Aβ) are more deleterious than are extracellular fibril forms. Based on this recent evidence showing that oligomers have a central role in the pathogenesis of AD, the term “oligomeropathy” could be used to define AD and other protein-misfolding diseases. In this review, I discuss recent developments in the “oligomer hypothesis” including our research findings regarding the pathogenesis of AD.

  • Analysis of lipid raft molecules in the living brain slices
    Neurochem. Int. (IF 3.262) Pub Date : 2017-08-24
    Norihiro Kotani, Takanari Nakano, Yui Ida, Rina Ito, Miki Hashizume, Arisa Yamaguchi, Makoto Seo, Tomoyuki Araki, Yasushi Hojo, Koichi Honke, Takayuki Murakoshi

    Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the “Enzyme-Mediated Activation of Radical Sources (EMARS)” method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405–7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called “EMARS products” distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices.

  • Alterations in the E3 ligases Parkin and CHIP result in unique metabolic signaling defects and mitochondrial quality control issues
    Neurochem. Int. (IF 3.262) Pub Date : 2017-08-26
    Britney N. Lizama, Amy M. Palubinsky, BethAnn McLaughlin

    E3 ligases are essential scaffold proteins, facilitating the transfer of ubiquitin from E2 enzymes to lysine residues of client proteins via isopeptide bonds. The specificity of substrate binding and the expression and localization of E3 ligases can, however, endow these proteins with unique features with variable effects on mitochondrial, metabolic and CNS function. By comparing and contrasting two E3 ligases, Parkin and C-terminus of HSC70-Interacting protein (CHIP) we seek to highlight the biophysical properties that may promote mitochondrial dysfunction, acute stress signaling and critical developmental periods to cease in response to mutations in these genes. Encoded by over 600 human genes, RING-finger proteins are the largest class of E3 ligases. Parkin contains three RING finger domains, with R1 and R2 separated by an in-between region (IBR) domain. Loss-of-function mutations in Parkin were identified in patients with early onset Parkinson's disease. CHIP is a member of the Ubox family of E3 ligases. It contains an N-terminal TPR domain and forms unique asymmetric homodimers. While CHIP can substitute for mutated Parkin and enhance survival, CHIP also has unique functions. The differences between these proteins are underscored by the observation that unlike Parkin-deficient animals, CHIP-null animals age prematurely and have significantly impaired motor function. These properties make these E3 ligases appealing targets for clinical intervention. In this work, we discuss how biophysical and metabolic properties of these E3 ligases have driven rapid progress in identifying roles for E3 ligases in development, proteostasis, mitochondrial biology, and cell health, as well as new data about how these proteins alter the CNS proteome.

  • Sex differences in the mitochondrial bioenergetics of astrocytes but not microglia at a physiologically relevant brain oxygen tension
    Neurochem. Int. (IF 3.262) Pub Date : 2017-09-06
    Sausan M. Jaber, Evan A. Bordt, Niraj M. Bhatt, Daniel M. Lewis, Sharon Gerecht, Gary Fiskum, Brian M. Polster

    Biological sex is thought to influence mitochondrial bioenergetic function. Previous respiration measurements examining brain mitochondrial sex differences were made at atmospheric oxygen using isolated brain mitochondria. Oxygen is 160 mm Hg (21%) in the atmosphere, while the oxygen tension in the brain generally ranges from ∼5 to 45 mm Hg (∼1–6% O2). This study tested the hypothesis that sex and/or brain physiological oxygen tension influence the mitochondrial bioenergetic properties of primary rat cortical astrocytes and microglia. Oxygen consumption was measured with a Seahorse XF24 cell respirometer in an oxygen-controlled environmental chamber. Strikingly, male astrocytes had a higher maximal respiration than female astrocytes when cultured and assayed at 3% O2. Three percent O2 yielded a low physiological dissolved O2 level of ∼1.2% (9.1 mm Hg) at the cell monolayer during culture and 1.2–3.0% O2 during assays. No differences in bioenergetic parameters were observed between male and female astrocytes at 21% O2 (dissolved O2 of ∼19.7%, 150 mm Hg during culture) or between either of these cell populations and female astrocytes at 3% O2. In contrast to astrocytes, microglia showed no sex differences in mitochondrial bioenergetic parameters at either oxygen level, regardless of whether they were non-stimulated or activated to a proinflammatory state. There were also no O2- or sex-dependent differences in proinflammatory TNF-α or IL-1β cytokine secretion measured at 18 h activation. Overall, results reveal an intriguing sex variance in astrocytic maximal respiration that requires additional investigation. Findings also demonstrate that sex differences can be masked by conducting experiments at non-physiological O2.

  • Vesicular movements in the growth cone
    Neurochem. Int. (IF 3.262) Pub Date : 2017-09-27
    Motohiro Nozumi, Michihiro Igarashi

    Growth cones, which are the highly motile tips of extending neuronal processes in developing neurons, have many vesicles. These vesicles are likely essential for the membrane expansion that is required for nerve growth, and probably coordinate with rearrangement of the cytoskeletons. Such mechanisms are poorly understood from molecular and cell biological aspects. Recently, we used superresolution microscopic approaches and described new mechanisms that are involved in the interaction between the vesicles and F-actin in the leading edge of the peripheral domain. Vesicles mainly accumulate in the central domain of growth cones. However, the dynamics of vesicles in each domain, for example, clathrin dependency, are totally distinct from each other. Here, we discuss the diversity of the dynamics of vesicular and related proteins that play different roles in nerve growth.

  • Involvement of neuronal and glial activities in control of the extracellular d-serine concentrations by the AMPA glutamate receptor in the mouse medial prefrontal cortex
    Neurochem. Int. (IF 3.262) Pub Date : 2017-09-28
    Sayuri Ishiwata, Asami Umino, Toru Nishikawa

    It has been well accepted that d-serine may be an exclusive endogenous coagonist for the N-methyl-d-aspartate (NMDA)-type glutamate receptor in mammalian forebrain regions. We have recently found by using an in vivo dialysis method that an intra-medial prefrontal cortex infusion of S-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (S-AMPA), a selective AMPA-type glutamate receptor agonist, causes a reduction in the extracellular levels of d-serine in a calcium-permeable AMPA receptor antagonist-sensitive manner. The inhibitory influence by the AMPA receptor on the extracellular d-serine, however, contradicts the data obtained from in vitro experiments that the AMPA receptor stimulation leads to facilitation of the d-serine liberation. This discrepancy appears to be due to the different cell setups between the in vivo and in vitro preparations. From the viewpoints of the previous reports indicating (1) the neuronal presence of d-serine synthesizing enzyme, serine racemase, and d-serine-like immunoreactivity and (2) the same high tissue concentrations of d-serine in the glia-enriched white matter and in the neuron-enriched gray matter of the mammalian neocortex, we have now investigated in the mouse medial prefrontal cortex, the effects of attenuation of neuronal and glial activities, by tetrodotoxin or fluorocitrate, respectively, on the S-AMPA-induced downregulation of the extracellular d-serine contents. In vivo dialysis studies revealed that a local infusion of tetrodotoxin or fluorocitrate eliminated the ability of S-AMPA given intra-cortically to cause a significant decrease in the dialysate concentrations of d-serine without affecting the elevating effects of S-AMPA on those of glycine, another intrinsic coagonist for the NMDA receptor. These findings suggest that the control by the AMPA receptor of the extracellular d-serine levels could be modulated by the neuronal and glial activities in the prefrontal cortex. It cannot be excluded that fluorocitrate would indirectly alter the modulation by changing synaptic neurotransmission via glial activity attenuation as previously reported.

  • Roles of CSGalNAcT1, a key enzyme in regulation of CS synthesis, in neuronal regeneration and plasticity
    Neurochem. Int. (IF 3.262) Pub Date : 2017-10-05
    Michihiro Igarashi, Kosei Takeuchi, Sayaka Sugiyama

    Chondroitin sulfate (CS) is a sulfated glycosaminoglycan composed of a long chain of repeating disaccharide units that are attached to core proteins, resulting in CS proteoglycans (CSPGs). In the mature brain, CS is concentrated in perineuronal nets (PNNs), which are extracellular structures that surround synapses and regulate synaptic plasticity. In addition, CS is rapidly synthesized after CNS injury to create a physical and chemical barrier that inhibits axon growth. Most previous studies used a bacterial CS-degrading enzyme to investigate the physiological roles of CS. Recent studies have shown that CS is synthesized by more than 15 enzymes, all of which have been characterized in vitro. Here we focus on one of those enzymes, CSGalNAcT1 (T1). We produced T1 knockout mice (KO), which show extensive axon regeneration following spinal cord injury, as well as the loss of onset of ocular dominance plasticity. These results from T1KO mice suggest important roles for extracellular CS in the brain regarding neuronal plasticity and axon regeneration.

  • Quantitative temporal changes in DTI values coupled with histological properties in cuprizone-induced demyelination and remyelination
    Neurochem. Int. (IF 3.262) Pub Date : 2017-10-10
    Ryutaro Yano, Junichi Hata, Yoshifumi Abe, Fumiko Seki, Keitaro Yoshida, Yuji Komaki, Hideyuki Okano, Kenji F. Tanaka

    Diffusion tensor imaging (DTI) is widely used to evaluate microstructural variations in brain tissue. In particular, fractional anisotropy (FA), reflecting the magnitude and orientation of anisotropic water diffusion, allows us to detect pathological events in white matter. An ex vivo DTI study coupled with histological assessment is an efficient strategy to evaluate the myelination process, i.e. demyelination and remyelination. The relationship between DTI values and myelin content or the individual cellular components such as oligodendrocytes, microglia, and astrocytes during both processes of demyelination and remyelination are not well-understood. To address this issue, we employed a cuprizone-inducible demyelination mouse model. Demyelination can be induced in this model during cuprizone exposure and termination of cuprizone exposure induces remyelination. We fed the mice cuprizone-containing chow for 4 weeks and then normal chow for an additional 4 weeks. The ex vivo DTI was performed to evaluate the white matter profiles observed by FA, mean diffusivity (MD), and radial diffusivity (RD) at both demyelinating and remyelinating time points, and then we evaluated histological properties at the same time points. The results indicated a gradual FA decrease during the cuprizone treatment (0, 2, 3, 4 weeks). A lower peak was seen at 1 week after the normal chow was resumed, with recovery to baseline at 2 and 4 weeks. MD and RD showed an opposing pattern to that of FA. These DTI values were positively or negatively correlated with myelin content regardless of the status of the white matter. The RD value was more sensitive to myelination status than FA and MD. We have clarified the temporal changes in the DTI values coupled with histological properties over both the demyelination and remyelination processes.

  • Calcium uptake and cytochrome c release from normal and ischemic brain mitochondria
    Neurochem. Int. (IF 3.262) Pub Date : 2017-10-16
    Alexander Andreyev, Pratistha Tamrakar, Robert E. Rosenthal, Gary Fiskum

    At abnormally elevated levels of intracellular Ca2+, mitochondrial Ca2+ uptake may compromise mitochondrial electron transport activities and trigger membrane permeability changes that allow for release of cytochrome c and other mitochondrial apoptotic proteins into the cytosol. In this study, a clinically relevant canine cardiac arrest model was used to assess the effects of global cerebral ischemia and reperfusion on mitochondrial Ca2+ uptake capacity, Ca2+ uptake-mediated inhibition of respiration, and Ca2+-induced cytochrome c release, as measured in vitro in a K+-based medium in the presence of Mg2+, ATP, and NADH-linked oxidizable substrates. Maximum Ca2+ uptake by frontal cortex mitochondria was significantly lower following 10 min cardiac arrest compared to non-ischemic controls. Mitochondria from ischemic brains were also more sensitive to the respiratory inhibition associated with accumulation of large levels of Ca2+. Cytochrome c was released from brain mitochondria in vitro in a Ca2+-dose-dependent manner and was more pronounced following both 10 min of ischemia alone and following 24 h reperfusion, in comparison to mitochondria from non-ischemic Shams. These effects of ischemia and reperfusion on brain mitochondria could compromise intracellular Ca2+ homeostasis, decrease aerobic and increase anaerobic cerebral energy metabolism, and potentiate the cytochrome c-dependent induction of apoptosis, when re-oxygenated mitochondria are exposed to abnormally high levels of intracellular Ca2+.

  • Motoneuron degeneration in the trigeminal motor nucleus innervating the masseter muscle in Dystonia musculorum mice
    Neurochem. Int. (IF 3.262) Pub Date : 2017-10-21
    M. Ibrahim Hossain, Masao Horie, Nozomu Yoshioka, Masayuki Kurose, Kensuke Yamamura, Hirohide Takebayashi

    Dystonia musculorum (dt) mice, which have a mutation in the Dystonin (Dst) gene, are used as animal models to investigate the human disease known as hereditary sensory and autonomic neuropathy type VI. Massive neuronal cell death is observed, mainly in the peripheral nervous system (PNS) of dt mice. We and others have recently reported a histopathological feature of these mice that neurofilament (NF) accumulates in various areas of the central nervous system (CNS), including motor pathways. Although dt mice show motor disorder and growth retardation, the causes for these are still unknown. Here we performed histopathological analyses on motor units of the trigeminal motor nucleus (Mo5 nucleus), because they are a good system to understand neuronal responses in the mutant CNS, and abnormalities in this system may lead to problems in mastication, with subsequent growth retardation. We report that motoneurons with NF accumulation in the Mo5 nuclei of DstGt homozygous mice express the stress-induced genes CHOP, ATF3, and lipocalin 2 (Lcn2). We also show a reduced number of Mo5 motoneurons and a reduced size of Mo5 nuclei in DstGt homozygous mice, possibly due to apoptosis, given the presence of cleaved caspase 3-positive Mo5 motoneurons. In the mandibular (V3) branches of the trigeminal nerve, which contains axons of Mo5 motoneurons and trigeminal sensory neurons, there was infiltration of Iba1-positive macrophages. Finally, we report atrophy of the masseter muscles in DstGt homozygous mice, which showed abnormal nuclear localization of myofibrils and increased expression of atrogin-1 mRNA, a muscle atrophy-related gene and weaker masseter muscle strength with uncontrolled muscle activity by electromyography (EMG). Taken together, our findings strongly suggest that mastication in dt mice is affected due to abnormalities of Mo5 motoneurons and masseter muscles, leading to growth retardation at the post-weaning stages.

  • Insulin expression in cultured astrocytes and the decrease by amyloid β
    Neurochem. Int. (IF 3.262) Pub Date : 2017-11-03
    Katsura Takano, Keisuke Koarashi, Kenji Kawabe, Masanori Itakura, Hidemitsu Nakajima, Mitsuaki Moriyama, Yoichi Nakamura

    Insulin resistance in brain has been reported in Alzheimer's diseases (AD). Insulin signaling is important for homeostasis in brain function and reported to be disturbed in neurons leading to tau phosphorylation and neurofibrillary tangles. Many investigations of insulin in neurons have been reported; however, it has not been reported whether astrocytes also produce insulin. In the present study, we assessed the expression of insulin in astrocytes cultured from rat embryonic brain and the effects of amyloid β1-42 (Aβ) and lipopolysaccharide (LPS) on the expression. We found that astrocytes expressed preproinsulin mRNAs and insulin protein, and that Aβ or LPS decreased these expressions. Antioxidants, glutathione and N-acetylcysteine, restored the decreases in insulin mRNA expression by Aβ and by LPS. Insulin protein was detected in astrocyte conditioned medium. These results suggest that astrocytes express and secrete insulin. Oxidative stress might be involved in the decreased insulin expression by Aβ or LPS. The insulin decrease by Aβ in astrocytes could be a novel disturbing mechanism for brain insulin signaling in AD.

  • Strong sonic hedgehog signaling in the mouse ventral spinal cord is not required for oligodendrocyte precursor cell (OPC) generation but is necessary for correct timing of its generation
    Neurochem. Int. (IF 3.262) Pub Date : 2017-11-06
    Hirokazu Hashimoto, Wen Jiang, Takeshi Yoshimura, Kyeong-Hye Moon, Jinwoong Bok, Kazuhiro Ikenaka

    In the mouse neural tube, sonic hedgehog (Shh) secreted from the floor plate (FP) and the notochord (NC) regulates ventral patterning of the neural tube, and later is essential for the generation of oligodendrocyte precursor cells (OPCs). During early development, the NC is adjacent to the neural tube and induces ventral domains in it, including the FP. In the later stage of development, during gliogenesis in the spinal cord, the pMN domain receives strong Shh signaling input. While this is considered to be essential for the generation of OPCs, the actual role of this strong input in OPC generation remains unclear. Here we studied OPC generation in bromi mutant mice which show abnormal ciliary structure. Shh signaling occurs within cilia and has been reported to be weak in bromi mutants. At E11.5, accumulation of Patched1 mRNA, a Shh signaling reporter, is observed in the pMN domain of wild type but not bromi mutants, whereas expression of Gli1 mRNA, another Shh reporter, disappeared. Thus, Shh signaling input to the pMN domain at E12.5 was reduced in bromi mutant mice. In these mutants, induction of the FP structure was delayed and its size was reduced compared to wild type mice. Furthermore, while the p3 and pMN domains were induced, the length of the Nkx2.2-positive region and the number of Olig2-positive cells decreased. The number of OPCs was also significantly decreased in the E12.5 and E14.5 bromi mutant spinal cord. In contrast, motor neuron (MN) production, detected by HB9 expression, significantly increased. It is likely that the transition from MN production to OPC generation in the pMN domain is impaired in bromi mutant mice. These results suggest that strong Shh input to the pMN domain is not required for OPC generation but is essential for producing a sufficient number of OPCs.

  • Trophic modulation of gamma oscillations: The key role of processing protease for Neuregulin-1 and BDNF precursors
    Neurochem. Int. (IF 3.262) Pub Date : 2017-12-09
    Hideki Tamura, Sadao Shiosaka, Shota Morikawa

    Gamma oscillations within the cerebral cortex and hippocampus are associated with cognitive processes, including attention, sensory perception, and memory formation; a deficit in gamma regulation is a common symptom of neurologic and psychiatric disorders. Accumulating evidence has suggested that gamma oscillations result from the synchronized activity of cell assemblies coordinated mainly by parvalbumin-positive inhibitory interneurons. The modulator molecules for parvalbumin-positive interneurons are major research targets and have the potential to control the specific oscillatory rhythm and behavior originating from neural coordination. Neuregulin-1 and brain-derived neurotrophic factor have been focused on as synaptic trophic factors that are associated with gamma oscillations. Synaptic activity converts precursor trophic factors into their biologically active forms by proteolytic cleavage, which could, in turn, modulate cell excitability and synaptic plasticity through each receptor's signaling. From these findings, the processing of trophic factors by proteases in a synaptic microenvironment might involve gamma oscillations during cognition. Here, we review the trophic modulation of gamma oscillations through extracellular proteolysis and its implications in neuronal diseases.

  • Neuropathic pain inhibitor, RAP-103, is a potent inhibitor of microglial CCL1/CCR8
    Neurochem. Int. (IF 3.262) Pub Date : 2017-12-14
    Mami Noda, Daichi Tomonaga, Kota Kitazono, Yusaku Yoshioka, Jiadai Liu, Jean-Philippe Rousseau, Richard Kinkead, Michael R. Ruff, Candace B. Pert

    Chemokine signaling is important in neuropathic pain, with microglial cells expressing chemokine (C-C motif) receptor CCR2, CCR5 and CCR8, all playing key roles. In the previous report (Padi et al., 2012), oral administration of a short peptide, RAP-103, for 7 days fully prevents mechanical allodynia and inhibits the development of thermal hyperalgesia after partial ligation of the sciatic nerve in rodents. As for the mechanism of the inhibiting effect of RAP-103, it was speculated to be due to dual blockade of CCR2 and CCR5. We report here that RAP-103 exhibits stronger antagonism for CCR8 (half maximal inhibitory concentration [IC50] 7.7 fM) compared to CCR5 (IC50 < 100 pM) in chemotaxis using primary cultured mouse microglia. In addition, RAP-103 at a concentration of 0.1 pM completely inhibits membrane ruffling and phagocytosis induced by chemokine (C-C motif) ligand 1 (CCL1), an agonist for CCR8. It has been shown that CCL1/CCR8 signaling is important in tactile allodynia induced by nerve ligation. Therefore, CCR8, among other chemokine receptors such as CCR2/CCR5, could be the most potent target for RAP-103. Inhibitory effects of RAP-103 on plural chemokine receptors may play important roles for broad clinical use in neuropathic pain treatment.

  • A refined concept: α-synuclein dysregulation disease
    Neurochem. Int. (IF 3.262) Pub Date : 2018-01-02
    Hideki Mochizuki, Chi-Jing Choong, Eliezer Masliah

    α-synuclein (αSyn) still remains a mysterious protein even two decades after SNCA encoding it was identified as the first causative gene of familial Parkinson's disease (PD). Accumulation of αSyn causes α-synucleinopathies including PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Recent advances in therapeutic approaches offer new antibody-, vaccine-, antisense-oligonucleotide- and small molecule-based options to reduce αSyn protein levels and aggregates in patient's brain. Gathering research information of other neurological disease particularly Alzheimer's disease, recent disappointment of an experimental amyloid plaques busting antibody in clinical trials underscores the difficulty of treating people who show even mild dementia as damage in their brain may already be too extensive. Prodromal intervention to inhibit the accumulation of pathogenic protein may advantageously provide a better outcome. However, treatment prior to onset is not ethically justified as standard practice at present. In this review, we initiate a refined concept to define early pathogenic state of αSyn accumulation before occurrence of brain damage as a disease criterion for αSyn dysregulation disease.

  • Circadian modification network of a core clock driver BMAL1 to harmonize physiology from brain to peripheral tissues
    Neurochem. Int. (IF 3.262) Pub Date : 2018-01-03
    Teruya Tamaru, Ken Takamatsu

    Circadian clocks dictate various physiological functions by brain SCN (a central clock) -orchestrating the temporal harmony of peripheral clocks of tissues/organs in the whole body, with adaptability to environments by resetting their timings. Dysfunction of this circadian adaptation system (CAS) occasionally causes/exacerbates diseases. CAS is based on cell-autonomous molecular clocks, which oscillate via a core transcriptional/translational feedback loop with clock genes/proteins, e.g., BMAL1: CLOCK circadian transcription driver and CRY1/2 and PER1/2 suppressors, and is modulated by various regulatory loops including clock protein modifications. Among mutants with a single clock gene, BMAL1-deficient mice exhibit the most drastic loss of circadian functions. Here, we highlight on numerous circadian protein modifications of mammalian BMAL1, e.g., multiple phosphorylations, SUMOylation, ubiquitination, acetylation, O-GlcNAcylation and S-nitrosylation, which mutually interplay to control molecular clocks and coordinate physiological functions from the brain to peripheral tissues through the input and output of the clocks.

  • Pathological role of lipid interaction with α-synuclein in Parkinson's disease
    Neurochem. Int. (IF 3.262) Pub Date : 2018-01-03
    Mari Suzuki, Kazunori Sango, Keiji Wada, Yoshitaka Nagai

    Alpha-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In sporadic PD and DLB, normally harmless αSyn proteins without any mutations might gain toxic functions by unknown mechanisms. Thus, it is important to elucidate the factors promoting the toxic conversion of αSyn, towards understanding the pathogenesis of and developing disease-modifying therapies for PD and DLB. Accumulating biophysical and biochemical studies have demonstrated that αSyn interacts with lipid membrane, and the interaction influences αSyn oligomerization and aggregation. Furthermore, genetic and clinicopathological studies have revealed mutations in the glucocerebrosidase 1 (GBA1) gene, which encodes a degrading enzyme for the glycolipid glucosylceramide (GlcCer), as strong risk factors for PD and DLB, and we recently demonstrated that GlcCer promotes toxic conversion of αSyn. Moreover, pathological studies have shown the existence of αSyn pathology in lysosomal storage disorders (LSDs) patient’ brain, in which glycosphingolipids (GSLs) is found to be accumulated. In this review, we focus on the lipids as a key factor for inducing wild-type (WT) αSyn toxic conversion, we summarize the knowledge about the interaction between αSyn and lipid membrane, and propose our hypothesis that aberrantly accumulated GSLs might contribute to the toxic conversion of αSyn. Identifying the trigger for toxic conversion of αSyn would open a new therapeutic road to attenuate or prevent crucial events leading to the formation of toxic αSyn.

  • CDC42EP4, a perisynaptic scaffold protein in Bergmann glia, is required for glutamatergic tripartite synapse configuration
    Neurochem. Int. (IF 3.262) Pub Date : 2018-01-09
    Natsumi Ageta-Ishihara, Kohtarou Konno, Maya Yamazaki, Manabu Abe, Kenji Sakimura, Masahiko Watanabe, Makoto Kinoshita

    Configuration of tripartite synapses, comprising the pre-, post-, and peri-synaptic components (axon terminal or bouton, dendritic spine, and astroglial terminal process), is a critical determinant of neurotransmitter kinetics and hence synaptic transmission. However, little is known about molecular basis for the regulation of tripartite synapse morphology. Previous studies showed that CDC42EP4, an effector protein of a cell morphogenesis regulator CDC42, is expressed exclusively in Bergmann glia in the cerebellar cortex, that it forms tight complex with the septin heterooligomer, and that it interacts indirectly with the glutamate transporter GLAST and MYH10/nonmuscle myosin ΙΙB. Scrutiny of Cdc42ep4−/− mice had revealed that the CDC42EP4-septins-GLAST interaction facilitates glutamate clearance, while the role for CDC42EP4-septins-MYH10 interaction has remained unsolved. Here, we find anomalous configuration of the tripartite synapses comprising the parallel fiber boutons, dendritic spines of Purkinje cells, and Bergmann glial processes in Cdc42ep4−/− mice. The complex anomalies include 1) recession of Bergmann glial membranes from the nearest active zones, and 2) extension of nonactive synaptic contact around active zone. In line with the recession of Bergmann glial membranes by the loss of CDC42EP4, overexpression of CDC42EP4 in heterologous cells promotes cell spreading and partitioning of MYH10 to insoluble (i.e., active) fraction. Paradoxically, however, Cdc42ep4−/− cerebellum contained significantly more MYH10 and N-cadherin, which is attributed to secondary neuronal response mainly in Purkinje cells. Given cooperative actions of N-cadherin and MYH10 for adhesion between neurons, we speculate that their augmentation may reflect the extension of nonactive synaptic contacts in Cdc42ep4−/− cerebellum. Transcellular mechanism that links the absence of CDC42EP4 in Bergmann glia to the augmentation of N-cadherin and MYH10 in neurons is currently unknown, but the phenotypic similarity to GLAST-null mice indicates involvement of the glutamate intolerance. Together, the unique phenotype of Cdc42ep4−/− mice provides a clue to novel molecular network underlying tripartite synapse configuration.

  • A role for KCC3 in maintaining cell volume of peripheral nerve fibers
    Neurochem. Int. (IF 3.262) Pub Date : 2018-01-31
    Bianca Flores, Cara C. Schornak, Eric Delpire

    The potassium chloride cotransporter, KCC3, is an electroneutral cotransporter expressed in the peripheral and central nervous system. KCC3 is responsible for the efflux of K+ and Cl− in neurons to help maintain cell volume and intracellular chloride levels. A loss-of-function (LOF) of KCC3 causes Hereditary Motor Sensory Neuropathy with Agenesis of the Corpus Callosum (HMSN/ACC) in a population of individuals in the Charlevoix/Lac-Saint-Jean region of Quebec, Canada. A variety of mouse models have been created to understand the physiological and deleterious effects of a KCC3 LOF. Though this KCC3 LOF in mouse models has recapitulated the peripheral neuropathy phenotype of HMSN/ACC, we still know little about the development of the disease pathophysiology. Interestingly, the most recent KCC3 mouse model that we created recapitulated a peripheral neuropathy-like phenotype originating from a KCC3 gain-of-function (GOF). Despite the past two decades of research in attempting to understand the role of KCC3 in disease, we still do not understand how dysfunction of this cotransporter can lead to the pathophysiology of peripheral neuropathy. This review focuses on the function of KCC3 in neurons and its role in human and health and disease.

  • New roles of reactive astrocytes in the brain; an organizer of cerebral ischemia
    Neurochem. Int. (IF 3.262) Pub Date : 2018-02-02
    Schuichi Koizumi, Yuri Hirayama, Yosuke M. Morizawa

    The brain consists of neurons and much higher number of glial cells. They communicate each other, by which they control brain functions. The brain is highly vulnerable to several insults such as ischemia, but has a self-protective and self-repairing mechanisms against these. Ischemic tolerance or preconditioning is an endogenous neuroprotective phenomenon, where a mild non-lethal ischemic episode can induce resistance to a subsequent severe ischemic injury in the brain. Because of its neuroprotective effects against cerebral ischemia or stroke, ischemic tolerance has been widely studied. However, almost all studies have been performed from the viewpoint of neurons. Glial cells are structurally in close association with synapses. Recent studies have uncovered the active roles of astrocytes in modulating synaptic connectivity, such as synapse formation, elimination and maturation, during development or pathology. However, glia-mediated ischemic tolerance and/or neuronal repairing have received only limited attention. We and others have demonstrated that glial cells, especially astrocytes, play a pivotal role in regulation of induction of ischemic tolerance as well as repairing/remodeling of neuronal networks by phagocytosis. Here, we review our current understanding of (1) glial-mediated ischemic tolerance and (2) glia-mediated repairing/remodeling of the penumbra neuronal networks, and highlight their mechanisms as well as their potential benefits, problems, and therapeutic application.

  • Brain bioenergetics in rats with acute hyperphenylalaninemia
    Neurochem. Int. (IF 3.262) Pub Date : 2018-02-14
    Nádia Weber Dimer, Bruna Klippel Ferreira, Jotele Fontana Agostini, Maria Luiza Gomes, Luiza Wilges Kist, Fernanda Malgarin, Milena Carvalho-Silva, Lara Mezari Gomes, Joyce Rebelo, Marisa Jádna Silva Frederico, Fátima Regina Mena Barreto Silva, Eduardo Pacheco Rico, Mauricio Reis Bogo, Emilio Luiz Streck, Gustavo Costa Ferreira, Patrícia Fernanda Schuck

    Phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism caused by deficient phenylalanine hydroxylase (PAH) activity. The deficiency results in increased levels of Phe and its metabolites in fluids and tissues of patients. PKU patients present neurological signs and symptoms including hypomyelination and intellectual deficit. This study assessed brain bioenergetics at 1 h after acute Phe administration to induce hyperphenylalaninemia (HPA) in rats. Wistar rats were randomized in two groups: HPA animals received a single subcutaneous administration of Phe (5.2 μmol/g) plus p-Cl-Phe (PAH inhibitor) (0.9 μmol/g); control animals received a single injection of 0.9% NaCl. In cerebral cortex, HPA group showed lower mitochondrial mass, lower glycogen levels, as well as lower activities of complexes I-III and IV, ATP synthase and citrate synthase. Higher levels of free Pi and phospho-AMPK, and higher activities of LDH, α-ketoglutarate dehydrogenase and isocitrate dehydrogenase were also reported in cerebral cortex of HPA animals. In striatum, HPA animals had higher LDH (pyruvate to lactate) and isocitrate dehydrogenase activities, and lower activities of α-ketoglutarate dehydrogenase and complex IV, as well as lower phospho-AMPK immunocontent. In hippocampus, HPA rats had higher mRNA expression for MFN1 and higher activities of α-ketoglutarate dehydrogenase and isocitrate dehydrogenase, but decreased activities of pyruvate dehydrogenase and complexes I and IV. In conclusion, our data demonstrated impaired bioenergetics in cerebral cortex, striatum and hippocampus of HPA rats.

  • Axon-terminals expressing EAAT2 (GLT-1; Slc1a2) are common in the forebrain and not limited to the hippocampus
    Neurochem. Int. (IF 3.262) Pub Date : 2018-03-09
    Yun Zhou, Bjørnar Hassel, Tore Eid, Niels Christian Danbolt

    The excitatory amino acid transporter type 2 (EAAT2) represents the major mechanism for removal of extracellular glutamate. In the hippocampus, there is some EAAT2 in axon-terminals, whereas most of the protein is found in astroglia. The functional importance of the neuronal EAAT2 is unknown, and it is debated whether EAAT2-expressing nerve terminals are present in other parts of the brain. Here we selectively deleted the EAAT2 gene in neurons (by crossing EAAT2-flox mice with synapsin 1-Cre mice in the C57B6 background). To reduce interference from astroglial EAAT2, we measured glutamate accumulation in crude tissue homogenates. EAAT2 proteins levels were measured by immunoblotting. Although synapsin 1-Cre mediated gene deletion only reduced the forebrain tissue content of EAAT2 protein to 95.5 ± 3.4% of wild-type (littermate) controls, the glutamate accumulation in homogenates of neocortex, hippocampus, striatum and thalamus were nevertheless diminished to, respectively, 54 ± 4, 46 ± 3, 46 ± 2 and 65 ± 7% of controls (average ± SEM, n = 3 pairs of littermates). GABA uptake was unaffected. After injection of U-13C-glucose, lack of neuronal EAAT2 resulted in higher 13C-labeling of glutamine and GABA in the hippocampus suggesting that neuronal EAAT2 is partly short-circuiting the glutamate-glutamine cycle in wild-type mice. Crossing synapsin 1-Cre mice with Ai9 reporter mice revealed that Cre-mediated excision occurred efficiently in hippocampus CA3, but less efficiently in other regions and hardly at all in the cerebellum. Conclusions: (1) EAAT2 is expressed in nerve terminals in multiple brain regions. (2) The uptake catalyzed by neuronal EAAT2 plays a role in glutamate metabolism, at least in the hippocampus. (3) Synapsin 1-Cre does not delete floxed genes in all neurons, and the contribution of neuronal EAAT2 is therefore likely to be larger than revealed in the present study.

  • X-ray irradiation induces disruption of the blood–brain barrier with localized changes in claudin-5 and activation of microglia in the mouse brain
    Neurochem. Int. (IF 3.262) Pub Date : 2018-03-12
    Yukari Yoshida, Yukihiko Sejimo, Masashi Kurachi, Yasuki Ishizaki, Takashi Nakano, Akihisa Takahashi

    X-ray irradiation (X-irradiation) induces disruption of the blood–brain barrier (BBB). However, the mechanisms underlying the permeability changes are unclear. Therefore, in the present study, we examined the cellular and molecular changes produced by X-irradiation of the brain. Male ICR mice were irradiated locally on their head, posterior to the bregma, except for the eyes, with a single dose of 60 Gy. BBB permeability was assessed using Evans blue dye. We also examined vascular endothelial growth factor (VEGF) expression, microglial morphology, and the expression of the tight junction protein claudin-5 from 0.5 to 7 days after irradiation. An increase in BBB permeability and a decrease in the expression of VEGF protein occurred in a time-dependent manner. In addition, the number of activated microglia (CD68+/Iba-1+ double-positive cells), the amount of tumor necrosis factor-α protein and immunoreactivity of nuclear factor-kappaB increased by irradiation, while the expression of claudin-5 on vascular endothelial cells diminished markedly in the cerebral cortex starting 0.5 days after irradiation. These results suggest that the downregulation of claudin-5 expression mediated by activated microglia may contribute to the BBB disruption induced by X-irradiation.

  • Current perspective of mitochondrial biology in Parkinson's disease
    Neurochem. Int. (IF 3.262) Pub Date : 2018-03-14
    Navneet Ammal Kaidery, Bobby Thomas

    Parkinson's disease (PD) is one of the most common neurodegenerative movement disorder characterized by preferential loss of dopaminergic neurons of the substantia nigra pars compacta and the presence of Lewy bodies containing α-synuclein. Although the cause of PD remains elusive, remarkable advances have been made in understanding the possible causative mechanisms of PD pathogenesis. An explosion of discoveries during the past two decades has led to the identification of several autosomal dominant and recessive genes that cause familial forms of PD. The investigations of these familial PD gene products have shed considerable insights into the molecular pathogenesis of the more common sporadic PD. A growing body of evidence suggests that the etiology of PD is multifactorial and involves a complex interplay between genetic and environmental factors. Substantial evidence from human tissues, genetic and toxin-induced animal and cellular models indicates that mitochondrial dysfunction plays a central role in the pathophysiology of PD. Deficits in mitochondrial functions due to bioenergetics defects, alterations in the mitochondrial DNA, generation of reactive oxygen species, aberrant calcium homeostasis, and anomalies in mitochondrial dynamics and quality control are implicated in the underlying mechanisms of neuronal cell death in PD. In this review, we discuss how familial PD-linked genes and environmental factors interface the pathways regulating mitochondrial functions and thereby potentially converge both familial and sporadic PD at the level of mitochondrial integrity. We also provide an overview of the status of therapeutic strategies targeting mitochondrial dysfunction in PD. Unraveling potential pathways that influence mitochondrial homeostasis in PD may hold the key to therapeutic intervention for this debilitating neurodegenerative movement disorder.

  • Long noncoding RNA DANCR mediates cisplatin resistance in glioma cells via activating AXL/PI3K/Akt/NF-κB signaling pathway
    Neurochem. Int. (IF 3.262) Pub Date : 2018-03-21
    Yangang Ma, Guanghua Zhou, Mengyou Li, Dianfeng Hu, Lianqun Zhang, Peng Liu, Kai Lin

    Malignant glioma is an aggressive type of brain tumor with poor prognosis and mostly incurable. Although cisplatin is used for adjuvant chemotherapy against glioma, intrinsic and acquired resistance restricts the application of cisplatin. Long noncoding RNA (lncRNA) DANCR is reported to regulate the differentiation and progression of several cancers. However, whether DANCR participates in cisplatin resistance of glioma is still unknown. In this study, we found that DANCR expression was negatively correlated with cisplatin sensitivity in glioma cells. Gain-of and loss-of function assays revealed that DNACR attenuated cisplatin-induced cell proliferation inhibition in vitro and xenograft growth suppression in vivo. Furthermore, DNACR also attenuated cisplatin-induced cell apoptosis in vitro and in vivo. Mechanistically, we found that DANCR upregulated AXL via competitively binding miR-33a-5p, miR-33b-5p, miR-1-3p, miR-206, and miR-613. Through upregulating AXL, DANCR activated PI3K/Akt/NF-κB signaling pathway in glioma cells. Inhibiting AXL/PI3K/Akt/NF-κB signaling pathway reversed the effects of DANCR on cisplatin resistance. In conclusion, we identified a cisplatin-resistance associated lncRNA DANCR. DANCR promotes cisplatin resistance via activating AXL/PI3K/Akt/NF-κB signaling pathway in glioma. Our data suggested that DANCR would be a potential biomarker for predicting cisplatin sensitivity and a therapeutic target for enhancing cisplatin efficacy in glioma.

  • Activity dependent internalization of the glutamate transporter GLT-1 requires calcium entry through the NCX sodium/calcium exchanger
    Neurochem. Int. (IF 3.262) Pub Date : 2018-03-21
    Ignacio Ibáñez, David Bartolomé-Martín, Dolores Piniella, Cecilio Giménez, Francisco Zafra

    GLT-1 is the main glutamate transporter in the brain and its trafficking controls its availability at the cell surface, thereby shaping glutamatergic neurotransmission under physiological and pathological conditions. Extracellular glutamate is known to trigger ubiquitin-dependent GLT-1 internalization from the surface of the cell to the intracellular compartment, yet here we show that internalization also requires the participation of calcium ions. Consistent with previous studies, the addition of glutamate (1 mM) to mixed primary cultures (containing neurons and astrocytes) promotes GLT-1 internalization, an effect that was suppressed in the absence of extracellular Ca2+. The pathways of Ca2+ mobilization by astrocytes were analyzed in these mixed cultures using the genetically encoded calcium sensor GCaMP6f. A complex pattern of calcium entry was activated by glutamate, with a dramatic and rapid rise in the intracellular Ca2+ concentration partially driven by glutamate transporters, especially in the initial stages after exposure to glutamate. The Na+/Ca2+ exchanger (NCX) plays a dominant role in this Ca2+ mobilization and its blockade suppresses the glutamate induced internalization of GLT-1, both in astrocytes and in a more straightforward experimental system like HEK293 cells transiently transfected with GLT-1. This regulatory mechanism might be relevant to control the amount of GLT-1 transporter at the cell surface in conditions like ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated and they promote rapid Ca2+ mobilization.

  • Altered release and uptake of gamma-aminobutyric acid in the cerebellum of dystrophin-deficient mice
    Neurochem. Int. (IF 3.262) Pub Date : 2018-06-01
    Janyerson Dannys Pereira da Silva, Diego Vannucci Campos, Fabiana Moreira Nogueira-Bechara, Roberta Sessa Stilhano, Sang Won Han, Rita Sinigaglia-Coimbra, Maria Teresa R. Lima-Landman, Antônio José Lapa, Caden Souccar

    Dystrophin deficiency caused by mutations of the related gene leads to muscle wasting in Duchenne muscular dystrophy (DMD). Some patients with DMD also present with intellectual disability and various degrees of neurological disorders, which have been related to a decreased number of postsynaptic gamma-aminobutyric acid type A receptors (GABAARs) in the hippocampus (HPC) and cerebellum (CBL). The aim of this study was to examine the relevance of dystrophin in the presynaptic GABAergic function in brain regions in which this protein is normally abundant. [3H]-GABA release, induced by nicotinic receptor (nAChR) activation or K+ depolarization, and [3H]-GABA uptake were determined using synaptosomes extracted from the cortex (CTX), HPC, and CBL of littermate control and mdx mice. Superfusion of the synaptosomes with nicotine or high K+ solutions led to a concentration-dependent and Ca2+-dependent [3H]-GABA release in control and mdx synaptosomes. [3H]-GABA release induced by 10 μM nicotine in mdx CBL synaptosomes was 47% less than that in control mice. K+-induced [3H]-GABA release did not differ between control and mdx synaptosomes. α7-containing and β2-containing nAChRs were involved in nicotine-induced [3H]-GABA release in control and mdx synaptosomes. Kinetic analysis of [3H]-GABA uptake in mdx CBL synaptosomes showed a reduced (50%) half-maximal uptake time (t1/2) and increased (44%) rate of [3H]-GABA uptake (Vmax) compared to controls. The apparent transporter affinity (Km) for GABA was not altered. Our findings show that dystrophin deficiency in mdx mice is associated with significant changes in the release and uptake of GABA in the CBL. These presynaptic alterations may be related to the reported decrease in postsynaptic GABAAR in the same brain region. The results indicate possible dysfunction of GABAergic synapses associated with dystrophin deficiency in the CBL, which may contribute to the cognitive and neurobehavioral disorders in mdx mice and patients with DMD.

  • Hyperekplexia-associated mutations in the neuronal glycine transporter 2
    Neurochem. Int. (IF 3.262) Pub Date : 2018-05-30
    Beatriz López-Corcuera, Esther Arribas-González, Carmen Aragón

    Hyperekplexia or startle disease is a dysfunction of inhibitory glycinergic neurotransmission characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. Although rare, this disorder can have serious consequences, including sudden infant death. One of the most frequent causes of hyperekplexia are mutations in the SLC6A5 gene, encoding the neuronal glycine transporter 2 (GlyT2), a key component of inhibitory glycinergic presynapses involved in synaptic glycine recycling though sodium and chloride-dependent co-transport. Most GlyT2 mutations detected so far are recessive, but two dominant missense mutations have been described. The detailed analysis of these mutations has revealed structural cues on the quaternary structure of GlyT2, and opens the possibility that novel selective pharmacochaperones have potential therapeutic effects in hyperekplexia.

  • Regulation of GABAA receptors by prolonged exposure to endogenous and exogenous ligands
    Neurochem. Int. (IF 3.262) Pub Date : 2018-05-31
    María Clara Gravielle

    GABAA receptors mediate most of the fast inhibitory transmissions in the central nervous system. These receptors are pentameric complexes that exhibit high structural and pharmacological heterogeneity, as they can be constructed from 19 distinct subunits. GABAA receptors are the targets of numerous clinically relevant drugs used to treat various disorders such as anxiety, insomnia and epilepsy. These receptors are also the targets of many volatile anesthetics and drugs of abuse, such as alcohol. This review is focused on the effect of long-term treatment with GABA, and the positive allosteric modulators benzodiazepines, neurosteroids and ethanol on GABAA receptors. Prolonged exposure of GABAA receptors to these compounds triggers several adaptive mechanisms that lead to changes in the structure, function and localization of receptors. These changes include GABAA receptor subunit expression, intracellular trafficking and phosphorylation. These adaptations are relevant to different physiological, pathological and pharmacological conditions and, in most cases, are associated with the development of tolerance. Understanding the molecular mechanisms underlying these regulatory processes will be relevant for therapeutic benefits.

  • Blockade and reversal of swimming-induced paralysis in C. elegans by the antipsychotic and D2-type dopamine receptor antagonist azaperone
    Neurochem. Int. (IF 3.262) Pub Date : 2018-05-22
    Osama Refai, Randy D. Blakely

    The catecholamine neurotransmitter dopamine (DA) exerts powerful modulatory control of physiology and behavior across phylogeny. Perturbations of DA signaling in humans are associated with multiple neurodegenerative and behavioral disorders, including Parkinson's disease, attention-deficit/hyperactivity disorder, addiction and schizophrenia. In the nematode C. elegans, DA signaling regulates mating behavior, learning, food seeking and locomotion. Previously, we demonstrated that loss of function mutations in the dat-1 gene that encodes the presynaptic DA transporter (DAT-1) results in a rapid cessation of movement when animals are placed in water, termed Swimming Induced Paralysis (Swip). Mutations in genes supporting DA biosynthesis, vesicular packaging and DA signaling suppresses Swip in dat-1 animals, consistent with paralysis as arising from excessive DA signaling at the extrasynaptic D2-type DA receptor DOP-3. Although animals grown on the vesicular monoamine transporter antagonist reserpine diminish Swip, the drug must be applied chronically, can impact the signaling of multiple biogenic amines, and has been reported to have penetrant, off-target actions. Here, we demonstrate that the antipsychotic drug azaperone potently and rapidly suppresses Swip behavior in either dat-1 mutants, as well as in wildtype animals treated with the DAT-1 antagonist nisoxetine, with genetic experiments consistent with DOP-3 antagonism as the mechanism of Swip suppression. Reversal of Swip in previously paralyzed dat-1 animals by azaperone application demonstrates an otherwise functionally-intact swimming circuit in these mutants. Finally, whereas azaperone suppresses DA-dependent Swip, the drug fails to attenuate the DA-independent paralysis induced by βPEA, aldicarb or genetic disruption of γ-aminobutyric acid (GABA) signaling. We discuss our findings with respect to the use of azaperone as a potent and selective tool in the identification and analysis of presynaptic mechanisms that regulate DA signaling.

  • Fisetin alleviates oxidative stress after traumatic brain injury via the Nrf2-ARE pathway
    Neurochem. Int. (IF 3.262) Pub Date : 2018-05-22
    Li Zhang, Handong Wang, Yali Zhou, Yihao Zhu, Maoxin Fei

    Fisetin, a natural flavonoid, has neuroprotection properties in many brain injury models. However, its role in traumatic brain injury (TBI) has not been fully explained. In the present study, we aimed to explore the neuroprotective effects of fisetin in a mouse model of TBI. We found that fisetin improved neurological function, reduced cerebral edema, attenuated brain lesion and ameliorated blood-brain barrier (BBB) disruption after TBI. Moreover, the up-regulation of malondialdehyde (MDA) and the activity of glutathione peroxidase (GPx) were reversed by fisetin treatment. Furthermore, administration of fisetin suppressed neuron cell death and apoptosis, increased the expression of B-cell lymphoma 2 (Bcl-2), while decreased the expression of Bcl-2-associated X protein (Bax) and caspase-3 after TBI. In addition, fisetin activated the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway following TBI. However, fisetin only failed to suppress oxidative stress in Nrf2−/− mice. In conclusion, our data provided the first evidence that fisetin played a critical role in neuroprotection after TBI partly through the activation of the Nrf2-ARE pathway.

  • Increased brain docosahexaenoic acid has no effect on the resolution of neuroinflammation following intracerebroventricular lipopolysaccharide injection
    Neurochem. Int. (IF 3.262) Pub Date : 2018-05-22
    Marc-Olivier Trépanier, Kathryn E. Hopperton, Vanessa Giuliano, Mojgan Masoodi, Richard P. Bazinet

    Resolution of inflammation in the periphery was once thought to be a passive process, but new research now suggests it is an active process mediated by specialized pro-resolving lipid mediators (SPM) derived from omega-3 polyunsaturated fatty acids (n-3 PUFA). However, this has yet to be illustrated in neuroinflammation. The purpose of this study was to measure resolution of neuroinflammation and to test whether increasing brain docosahexaenoic acid (DHA) affects the resolution of neuroinflammation.C57Bl/6 mice, fat-1 mice and their wildtype littermates, fed either fish oil or safflower oil, received lipopolysaccharide (LPS) in the left lateral ventricle. Animals were then euthanized at various time points for immunohistochemistry, gene expression, and lipidomic analyses.Peak microglial activation was observed at 5 days post-surgery and the resolution index was 10 days. Of the approximately 350 genes significantly changed over the 28 days post LPS injection, 130 were uniquely changed at 3 days post injection. No changes were observed in the bioactive mediator pools. However, a few lysophospholipid species were decreased at 24hr post surgery. When brain DHA is increased, microglial cell density did not resolve faster and did not alter gene expression.In conclusion, resolution of neuroinflammation appears to be independent of SPM. Increasing brain DHA had no effect in this model.

  • Protective effect of S-nitrosoglutathione administration against hyperglycemia induced disruption of blood brain barrier is mediated by alterations in tight junction proteins and cell adhesion molecules
    Neurochem. Int. (IF 3.262) Pub Date : 2018-05-21
    Aanchal Aggarwal, Inderjit Singh, Rajat Sandhir

    Diabetes is associated with increased blood brain barrier (BBB) permeability resulting in neurological deficits. The present study investigated the role of S-nitrosoglutathione (GSNO) on tight junction proteins and cell adhesion molecules in streptozotocin-induced diabetic mice. Diabetes was induced by intraperitoneal injection of streptozotocin (40 mg/kg body weight) for 5 days in mice. GSNO was administered daily (100 μg/kg body weight, orally) for 8 weeks after the induction of diabetes. A significant decline was observed in the cognitive ability of diabetic animals assessed using radial arm maze test. A significant increase was observed in nitrotyrosine levels in cortex and hippocampus of diabetic mice. Relative mRNA and protein expression of tight junction proteins viz; zona occludens-1 (ZO-1) and occludin were significantly lower in the microvessels isolated from cortex and hippocampus of diabetic animals, whereas expression of claudin-5 mRNA and protein was unaltered. Immunofluorescence of tight junction proteins confirmed loss of ZO-1 and occludin in the diabetic brain. Furthermore, significant increase in interstitial cell adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 mRNA and protein expression was observed in diabetic animals. Ultrastructure of microvessels from diabetic brain was also altered thereby confirming BBB disruption. GSNO administration to diabetic animals, on the other hand, was able to ameliorate loss of ZO-1 and occludin as well as normalize ICAM-1 and VCAM-1 expression, restore BBB integrity and vascular inflammation, and improve cognitive deficits. Therefore, our findings clearly suggest that GSNO is a therapeutic with potential to protect BBB and thereby preventing diabetes induced neurological deficits.

  • Tumor necrosis factor receptor 2 is required for ischemic preconditioning-mediated neuroprotection in the hippocampus following a subsequent longer transient cerebral ischemia
    Neurochem. Int. (IF 3.262) Pub Date : 2018-05-17
    Jae-Chul Lee, Chan Woo Park, Myoung Cheol Shin, Jun Hwi Cho, Hyang-Ah Lee, Young-Myeong Kim, Joon Ha Park, Ji Hyeon Ahn, Jeong Hwi Cho, Hyun-Jin Tae, In Koo Hwang, Tae-Kyeong Lee, Moo-Ho Won, Il Jun Kang

    Tumor Necrosis Factor-α (TNF-α) is a proinflammatory cytokine implicated in neuronal damage in response to cerebral ischemia. Ischemic preconditioning (IPC) provides neuroprotection against a subsequent severer or longer transient ischemia by ischemic tolerance. Here, we focused on the role of TNF-α in IPC-mediated neuroprotection against neuronal death following a subsequent longer transient cerebral ischemia (TCI). Gerbils used in this study were randomly assigned to eight groups; sham group, TCI operated group, IPC plus (+) sham group, IPC + TCI operated group, sham + etanercept (an inhibitor of TNF-a) group, TCI + etanercept group, IPC + sham + etanercept group, and IPC + TCI + etanercept group. IPC was induced by a 2-min sublethal transient ischemia, which was operated 1 day prior to a longer (5-min) TCI. A significant death of neurons was found in the stratum pyramidale (SP) in the CA1 area (CA1) of the hippocampus 5 days after TCI; however, IPC protected SP neurons from TCI. We found that TNF-α immunoreactivity was significantly increased in CA1 pyramidal neurons in the TCI and IPC + TCI groups compared to the sham group. TNF-R1 expression in CA1 pyramidal neurons of the TCI group was also increased 1 and 2 days after TCI; however, in the IPC + TCI group, TNF-R1 expression was significantly lower than that in the TCI group. On the other hand, we did not detect TNF-R2 immunoreactivity in CA1 pyramidal neurons 1 and 2 days after TCI; meanwhile, in the IPC + TCI group, TNF-R2 expression was significantly increased compared to TNF-R2 expression at 1 and 2 days after TCI. In addition, in this group, TNF-R2 was newly expressed in pericytes, which are important cells in the blood brain barrier, from 1 day after TCI. When we treated etanercept to the IPC + TCI group, IPC-induced neuroprotection was significantly weakened. In brief, this study indicates that IPC confers neuroprotection against TCI by TNF-α signaling through TNF-R2 and suggests that the enhancement of TNF-R2 expression by IPC may be a legitimate strategy for a therapeutic intervention of TCI.

  • Agmatine potentiates neuroprotective effects of subthreshold concentrations of ketamine via mTOR/S6 kinase signaling pathway
    Neurochem. Int. (IF 3.262) Pub Date : 2018-05-12
    Mauren K. Tavares, Suellen dos Reis, Nicolle Platt, Isabella A. Heinrich, Ingrid A.V. Wolin, Rodrigo B. Leal, Manuella P. Kaster, Ana Lúcia S. Rodrigues, Andiara E. Freitas

    Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is one of the most robust neurobiological findings in the pathophysiology of major depressive disorder (MDD) over the last 40 years. The persistent increase in glucocorticoids levels induces morphological and anatomical changes in the brain, especially in the hippocampus. Ketamine represents a major advance for the treatment of MDD, however the psychotomimetic effects of this compound limit its widespread use. Agmatine is a neuromodulator that has been shown to be a putative novel and well-tolerated antidepressant/augmenter drug. In this study, the exposure of HT22 hippocampal neuronal cell line to corticosterone (50 μM) induced a significant neuronal cell death. Interestingly, the incubation of HT22 cells with the fast-acting antidepressant drug ketamine (1 μM) prevented the corticosterone-induced toxicity. Similarly, agmatine caused a significant cytoprotection at the concentration of 0.1 μM against corticosterone (50 μM) cell damage. Notably, the incubation with a subthreshold concentration of ketamine (0.01 μM) in combination with a subthreshold concentration of agmatine (0.001 μM) prevented the neuronal damage elicited by corticosterone (50 μM). A 24 h co-incubation with subthreshold concentrations of ketamine (0.01 μM) and agmatine (0.001 μM) was able to cause a significant increase in the phosphorylation levels of Akt (Ser473) and p70S6 kinase (Thr389) as well as PSD95 immunocontent. Neither glycogen synthase kinase-3β (Ser9) phosphorylation nor β catenin immunocontent were altered by a 24 h co-incubation period. Finally, the co-incubation of cells for 30 min did not produce any effect in the phosphorylation or immunocontent of any protein investigated. Taken together, our results support the notion that the combination of subthreshold concentrations of ketamine and agmatine has cytoprotective effects against corticosterone-induced cell death. This effect is accompanied by its ability to activate Akt and mTOR/S6 kinase signaling pathway, and increase the expression of synaptic proteins.

  • Serum 25-hydroxyvitamin D deficiency predicts poor outcome among acute ischemic stroke patients without hypertension
    Neurochem. Int. (IF 3.262) Pub Date : 2018-05-03
    Bingjun Zhang, Yuge Wang, Yi Zhong, Siyuan Liao, Zhengqi Lu

    25-Hydroxyvitamin D (25(OH)D) deficiency is a frequent condition in patients who suffer acute ischemic stroke (AIS), and several studies suggested that it may be associated with a poorer prognosis. Whether this association is affected by hypertension is unclear. Our aim was to investigate the association between 25(OH)D levels and both clinical severity and outcome after 3 months in AIS patients stratified by the history of hypertension. Consecutive first-ever AIS patients admitted to the Third Affiliated Hospital of Sun Yat-sen University, China were identified. Clinical information was collected. Serum 25(OH)D levels were measured at baseline. Stroke severity was assessed at admission using the National Institutes of Health Stroke Scale (NIHSS) score. Functional outcome was evaluated after 3 months of onset using the modified Rankin Scale (mRS). Multivariate analyses were performed using logistic regression models. During the study period, 377 patients were diagnosed as AIS and were included in the analysis. 25(OH)D deficiency was not associated with the risk of NIHSS at admission and 3 months mRS both in total patients and the hypertension subgroup. Among AIS without hypertension, 25(OH)D deficiency subjects had a significantly higher of NIHSS at admission and 3 months mRS compared with those with 25(OH)D ≥ 50 nmol/L. The odds ratios (95% confidence interval) were 5.51(1.83–16.60) and 4.63(1.53–14.05) in the multivariable adjusted model (P for linear trend < 0.05). Serum lower 25(OH)D levels can be seen as an independent prognostic factor of functional outcome in AIS without hypertension. Additional studies about improving prognosis of AIS by vitamin D supplementation could be first applied to these patients.

  • Huntington's disease pattern of transcriptional dysregulation in the absence of mutant huntingtin is produced by knockout of neuronal GLT-1
    Neurochem. Int. (IF 3.262) Pub Date : 2018-04-27
    Robert B. Laprairie, Geraldine T. Petr, Yan Sun, Kathryn D. Fischer, Eileen M. Denovan-Wright, Paul A. Rosenberg

    GLT-1 is the major glutamate transporter in the brain, and is expressed in astrocytes and in axon terminals in the hippocampus, cortex, and striatum. Neuronal GLT-1 accounts for only 5–10% of total brain GLT-1 protein, and its function is uncertain. In HD, synaptic dysfunction of the corticostriate synapse is well-established. Transcriptional dysregulation is a key feature of HD. We hypothesized that deletion of neuronal GLT-1, because it is expressed in axon terminals in the striatum, might produce a synaptopathy similar to that present in HD. If true, then some of the gene expression changes observed in HD might also be observed in the neuronal GLT-1 knockout. In situ hybridization using 33P labeled oligonucleotide probes was carried out to assess localization and expression of a panel of genes known to be altered in expression in HD. We found changes in the expression of cannabinoid receptors 1 and 2, preproenkaphalin, and PDE10A in the striatum of mice in which the GLT-1 gene was inactivated in neurons by expression of synapsin-Cre, compared to wild-type littermates. These changes in expression were observed at 12 weeks of age but not at 6 weeks of age. No changes in DARPP-32, PDE1B, NGFIA, or β-actin expression were observed. In addition, we found widespread alteration in expression of the dynamin 1 gene. The changes in expression in the neuronal GLT-1 knockout of genes thought to exemplify HD transcriptional dysregulation suggest an overlap in the synaptopathy caused by neuronal GLT-1 deletion and HD. These data further suggest that specific changes in expression of cannabinoid receptors, preproenkephalin, and PDE10A, considered to be the hallmark of HD transcriptional dysregulation, may be produced by an abnormality of glutamate homeostasis under the regulation of neuronal GLT-1, or a synaptic disturbance caused by that abnormality, independently of mutation in huntingtin.

  • Endogenous acetylcholine regulates neuronal and astrocytic vascular endothelial growth factor expression levels via different acetylcholine receptor mechanisms
    Neurochem. Int. (IF 3.262) Pub Date : 2018-04-26
    Kyoko Kimura, Kinzo Matsumoto, Hironori Ohtake, Jun-Ichiro Oka, Hironori Fujiwara

    Vascular endothelial growth factor (VEGF), a signaling molecule involved in angiogenesis, plays an important role in neuroprotection and neurogenesis. In the present study, we aimed to elucidate the mechanisms underlying endogenous acetylcholine (ACh)-induced VEGF expression in neurons and astrocytes, and identify the neuronal cells contributing to its expression in the medial septal area, a nuclear origin of cholinergic neurons mainly projecting to the hippocampus. The mRNA expression and secretion of VEGF were measured by RT-PCR and ELISA using mouse primary cultured cortical neurons and astrocytes. VEGF expression in the medial septal area was assessed by RT-PCR and immunostaining using mice treated with tacrine [9-amino-1,2,3,4-tetrahydro-acridine HCl (THA); 2.5 mg/kg, i.p.] once daily for 7 days. The THA treatment increased VEGF mRNA expression in neurons in a manner that was reversed by mecamylamine, a nicotinic ACh receptor (AChR) antagonist, whereas in mouse primary cultured astrocytes, carbachol, but not THA dose-dependently increased VEGF mRNA expression and secretion in a manner that was inhibited by scopolamine, a muscarinic AChR inhibitor. In in vivo studies, the administration of THA significantly increased the expression of VEGF in medial septal cholinergic neurons and the effects of THA were significantly blocked by mecamylamine. THA also significantly increased the expression levels of a phosphorylated form of VEGF receptor 2 (p-VEGFR2), an activated form of VEGFR2. The present results suggest that endogenous ACh plays an up-regulatory role for VEGF expression in neurons and astrocytes via different mechanisms. Moreover, endogenous ACh-induced increases in VEGF levels appear to activate VEGFR2 on medial septal cholinergic neurons via an autocrine mechanism.

  • Mitochondrial alterations in Parkinson's disease human samples and cellular models
    Neurochem. Int. (IF 3.262) Pub Date : 2018-04-26
    Mara Zilocchi, Giovanna Finzi, Marta Lualdi, Fausto Sessa, Mauro Fasano, Tiziana Alberio

    Mitochondrial impairment is one of the most important hallmarks of Parkinson's disease (PD) pathogenesis. In this work, we wanted to verify the molecular basis of altered mitochondrial dynamics and disposal in Substantia nigra specimens of sporadic PD patients, by the comparison with two cellular models of PD. Indeed, SH-SY5Y cells were treated with either dopamine or 1-methyl-4-phenylpyridinium (MPP+) in order to highlight the effect of altered dopamine homeostasis and of complex I inhibition, respectively. As a result, we found that fusion impairment of the inner mitochondrial membrane is a common feature of both PD human samples and cellular models. However, the effects of dopamine and MPP+ treatments resulted to be different in terms of the mitochondrial damage induced. Opposite changes in the levels of two mitochondrial protein markers (voltage-dependent anion channels (VDACs) and cytochrome c oxidase subunit 5β (COX5β)) were observed. In this case, dopamine treatment better recapitulated the molecular picture of patients' samples. Moreover, the accumulation of PTEN-induced putative kinase 1 (PINK1), a mitophagy marker, was not observed in both PD patients samples and cellular models. Eventually, in transmission electron microscopy images, small electron dense deposits were observed in mitochondria of PD subjects, which are uniquely reproduced in dopamine-treated cells. In conclusion, our study suggests that the mitochondrial molecular landscape of Substantia nigra specimens of PD patients can be mirrored by the impaired dopamine homeostasis cellular model, thus supporting the hypothesis that alterations in this process could be a crucial pathogenetic event in PD.

  • Protective influences of N-acetylcysteine against alcohol abstinence-induced depression by regulating biochemical and GRIN2A, GRIN2B gene expression of NMDA receptor signaling pathway in rats
    Neurochem. Int. (IF 3.262) Pub Date : 2018-04-25
    Rutuja Yawalkar, Harish Changotra, Girdhari Lal Gupta

    Evidences have indicated a high degree of comorbidity of alcoholism and depression. N-acetylcysteine (NAC) has shown its clinical efficiency in the treatment of several psychiatric disorders and is identified as a multi-target acting drug. The ability of NAC to prevent alcohol abstinence-induced depression-like effects and underlying mechanism(s) have not been adequately addressed. This study was aimed to investigate the beneficial effects of NAC in the alcohol abstinence-induced depression developed following long-term voluntary alcohol intake. For evaluation of the effects of NAC, Sprague–Dawley rats were enabled to voluntary drinking of 4.5%, 7.5% and 9% v/v alcohol for fifteen days. NAC (25, 50, and 100 mg/kg) and fluoxetine (5 mg/kg) were injected intraperitoneally for three consecutive days during the alcohol abstinence period on the days 16, 17, 18. The behavioral studies were conducted employing forced swim test (FST), and tail suspension test (TST) on day 18 to determine the effects of N-acetylcysteine and fluoxetine in the ethanol withdrawal induced-depression. Blood alcohol concentration, alcohol biomarkers like SGPT, SGOT, ALP, GGT, and MCV were estimated by using commercially available kits. Serotonin concentrations were measured in the plasma, hippocampus and pre-frontal cortex using the rat ELISA kit. The expression of GRIN1, GRIN2A, GRIN2B genes for the N-methyl d-aspartate receptors (NMDAR) subunits in the hippocampus and the prefrontal cortex were also examined by reverse-transcription quantitative polymerase chain reaction. The results revealed that alcohol abstinence group depicted increased immobility time in FST and TST. Further, NAC exerted significant protective effect at the doses 50 mg/kg and 100 mg/kg, but 25 mg/kg showed insignificant protection against alcohol abstinence-induced depression. The increased level of biochemical parameters following ethanol abstinence were also reversed by NAC at the dose of 100 mg/kg. The significant reversal effect of NAC on the serotonin level following alcohol abstinence was greater in the hippocampus as compared to the third-day alcohol withdrawal group. The increased expression levels of GRIN2A and GRIN2B following ethanol abstinence were reversed with a higher dose of NAC (100 mg/kg) treatment. In conclusion, the results of the study reveal that NAC has remarkable protective effects in the alcohol abstinence-induced depression by modulating alcohol markers, serotonin levels and GRIN2A, GRIN2B gene expression of NMDAR signaling pathway in rats.

  • Regulation of the ARE-binding proteins, TTP (tristetraprolin) and HuR (human antigen R), in inflammatory response in astrocytes
    Neurochem. Int. (IF 3.262) Pub Date : 2018-04-24
    Alina A. Astakhova, Dmitry V. Chistyakov, Marina G. Sergeeva, Georg Reiser

    Control of decay of mRNA containing the adenine-uridine rich elements (AREs) is an important post-transcriptional mechanism involved in the regulation of inflammatory gene expression. Two widely recognized proteins in this machinery are HuR (human antigen R) – a protein that stabilizes ARE-containing mRNA and TTP (tristetraprolin) – a protein that shortens half-lives of ARE-containing mRNA. Although HuR and TTP regulation mechanisms have been well studied in cells of hematopoietic origin, there are no respective data in astrocytes, cells of ectodermal origin which play an important role in neuroinflammation. Therefore we evaluated the existence of TTP and HuR in primary astrocytes and characterized the features of their regulation after stimulation by the proinflammatory stimuli thrombin, ATP, and agonists of TLR4, TLR2. All proinflammatory stimuli increased levels of TTP mRNA, but not HuR mRNA. Transcripts of both HuR and TTP underwent stabilization upon lipopolysaccharide (LPS) treatment, measured with the actinomycin D protocol. This effect was abolished by treatment with SB203580, an inhibitor of р38 МАРК. Both TTP and HuR transcripts were sensitive to modulation by anisomycin and cycloheximide. LPS induced translocation of HuR protein from nucleus to cytoplasm. TTP is localized in the cytosolic fraction and localization is not sensitive to LPS treatment. Our data for the first time reveal specificity of regulation of ARE-binding proteins in astrocytes. We propose possibilities to manipulate brain inflammatory processes via post-transcription regulatory steps in astrocytes.

Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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