Journal of Neurochemistry - Current Research Articles
Current research articles: Neurochemistry
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Journal of Neurochemistry - Editor: Wiley
JNC continues to be a leading source for research into all aspects of neuroscience, with a particular focus on molecular and cellular aspects of the nervous system, the pathogenesis of neurological disorders and the development of disease specific biomarkers.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07778.xAbstractSpermidine (SPD) is an endogenous aliphatic amine with polycationic structure that modulates NMDA receptor activity and improves memory. Recent evidence suggests that cAMP-dependent protein kinase (PKA) and cAMP response element-binding protein (CREB) play a role in SPD-induced improvement of memory. In the current study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in SPD-induced facilitation of memory of inhibitory avoidance task in adult rats. The post-training administration of the PKC inhibitor, 3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride [GF 109203X, 2.5 ?mol, intrahippocampal (ih)] with SPD (0.2 nmol, ih) prevented memory improvement induced by SPD. Intrahippocampal administration of SPD (0.2 nmol) facilitated PKC phosphorylation in the hippocampus, 30 min after administration. GF 109203X prevented not only the stimulatory effect of SPD on PKC but also PKA and CREB phosphorylation. These results suggest that memory enhancement induced by the ih administration of SPD involves the cross-talk between PKC and PKA/CREB, with sequential activation of PKC and PKA/CREB pathways, in rats.Spermidine improves memory by facilitating crosstalk between protein kinases C (PKC) and A (PKA)Spermidine (SPD) facilitates memory consolidation and NMDA receptor (NMDAr) functioning. This study shows that SPD sequentially activates PKC and PKA/CREB signaling in the hippocampus, probably by direct and indirect PKC-induced activation of adenylyl cyclases (AC). Indirect mechanisms may include neurogranin (Ng) phosphorylation and consequent increased calmodulin availability. Downstream mechanisms involved in memory facilitation by SPD, an endogenous polyamine, are proposed.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07772.xAbstractDietary lipids modify brain fatty acid profile, but evidence of their direct effect on neuronal function is sparse. The enthorinal cortex (EC) neurons connecting to the hippocampus play a critical role in learning and memory. Here, we have exposed mice to diets based on canola:soybean oils (40 : 10, g/kg) or safflower : corn oils (25 : 25, g/kg) to investigate the relationship between the lipid profile of brain fatty acids and the intrinsic properties of EC neurons. Consumption of canola : soybean oil-enriched diet led to the increase of the monounsaturated fatty acid oleic acid and to a decrease of arachidonic acid in ethanolamine glycerophospholipids of the white matter. We also found an important rise in docosahexaenoic acid (DHA) within ethanolamine glycerophospholipids and phosphatidylserine of gray matter. The canola:soybean oil treatment led to a shorter duration of action potential (?21%), a reduction in the duration of postsynaptic response (?21%) and increased firing activity (+43%). Data from additional experiments with animals fed DHA alone or DHA with canola oil suggested that dietary monounsaturated fatty acid may have contributed to these effects on EC neuron physiology. Since neuronal function within the enthorhinal-hippocampal loop is critical to learning and memory processes, the present data may provide a functional basis for the beneficial cognitive effects of canola oil-based diets.High-MUFA intake increased oleic acid (OLA) and decreased arachidonic acid (ARA) in phospholipids of myelinated axons in the brain white matter. Such diet-induced changes were associated with narrower action potentials and shorter postsynaptic responses in pyramidal neurons from the entorhinal cortex, which could have profound impact on brain function.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07767.xAbstractLight-induced damage is a widely used model to study retinal degeneration. We examined whether bacterial lipopolysaccharide (LPS) protects the retina against light-induced injury. One day before intense light exposure for 24 h, rats were intravitreally injected with LPS in one eye and vehicle in the contralateral eye. At several time points after light exposure, rats were subjected to electroretinography and histological analysis. Bax, Bcl-xL, p-Akt, and p-Stat3 levels were assessed by Western blotting, and retinal thiobarbituric acid reactive substances levels were measured as an index of lipid peroxidation. One group of animals received injections of dexamethasone, aminoguanidine (an inducible NOS inhibitor), 5-hydroxydecanoic acid (a mitochondrial K+/ATP channel blocker), or wortmannin [a phosphoinositide-3-kinase (PI3K) inhibitor] in order to analyze their effect on the protection induced by LPS. LPS afforded significant morphologic and functional protection in eyes exposed to intense light. Light damage induced an increase in mitochondrial Bax/cytoplasmic Bax ratio, and lipid peroxidation which were prevented by LPS. Dexamethasone and wortmannin (but not aminoguanidine or 5-hydroxydecanoic acid) prevented the effect of LPS. Moreover, wortmannin prevented the effect of LPS on p-Akt levels. These results indicate that LPS provides retinal protection against light-induced stress, probably through a PI3K/Akt-dependent mechanism.A moderate inflammation protects the retina from light damageIntense light induces retinal degeneration. We examined whether bacterial lipopolysaccharide (LPS) protects the retina against light-induced injury. LPS affords significant morphologic and functional protection in eyes exposed to intense light. The retinal protection against light damage induced by a moderate dose of LPS could constitute a future fertile avenue for promoting the survival of retinal cells.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07738.x.AbstractCocaine/heroin combinations (speedball) exert synergistic neurochemical and behavioral effects that are thought to contribute to the increased abuse potential and subjective effects reported by polydrug users. In vivo fast-scan cyclic voltammetry was used to examine the effects of chronic intravenous self-administration (25 consecutive sessions) of cocaine (250 ?g/inf), heroin (4.95 ?g/inf) and speedball (250/4.95 ?g/inf cocaine/heroin) on changes in electrically evoked dopamine (DA) efflux, maximal rate of DA uptake (Vmax) and the apparent affinity (Km) of the DA transporter in the nucleus accumbens. The increase in electrically evoked DA was comparable following cocaine and speedball injection; however, heroin did not increase evoked DA. DA transporter Km values were similarly elevated following cocaine and speedball, but unaffected by heroin. However, speedball self-administration significantly increased baseline Vmax, while heroin and cocaine did not change baseline Vmax, compared with the baseline Vmax values of drug-naïve animals. Overall, elevated DA clearance is a likely consequence of synergistic elevations of nucleus accumbens extracellular DA concentrations by chronic speedball self-administration, as reported previously in microdialysis studies. The present results indicate neuroadaptive processes that are unique to cocaine/heroin combinations and cannot be readily explained by simple additivity of changes observed with cocaine and heroin alone.Speedball Self-administration Increases DAT-mediated Reuptake Rate Fast-scan cyclic voltammetry was used to investigate the effects of chronic cocaine, heroin and speedball self-administration on the dynamics of dopamine neurotransmission within the nucleus accumbens. Speedball self-administration significantly increased baseline rates of DAT-mediated reuptake (Vmax). The present results indicate neuroadaptive processes that are unique to cocaine/heroin combinations and cannot be readily explained by simple additivity of changes observed with cocaine or heroin alone.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07779.xAbstractMidbrain dopaminergic neurons are implicated in various neurological and psychiatric diseases as well as drug addiction. Thus, the study of their generation and maintenance is pivotal to further our understanding of these disease-underlying mechanisms and development of novel therapeutics. Here, using an embryonic stem cell in vitro differentiation system and mutant dreher mouse, we showed that Lmx1a, an early regulator of midbrain dopamine neural progenitor phenotype specification, is also involved in the regulation of midbrain dopaminergic maturation by regulating gene expression of the dopamine transporter. Forced expression of Lmx1a induced dopamine transporter expression precociously in immature dopaminergic neurons, accompanied by significant increase in specific dopamine uptake. Lmx1a binds to well-conserved sequences in the dopamine transporter promoter region, and this binding sequence directs Lmx1a-dependent activation of reporter gene expression. Furthermore, during mouse embryonic development, dopamine transporter was more severely affected by Lmx1a mutation compared to other dopamine markers such as tyrosine hydroxylase and dopa decarboxylase, again supporting the role of Lmx1a in midbrain dopaminergic maturation in vivo. Thus, this study demonstrates that dopamine transporter is a direct target of Lmx1a and emphasizes a novel role of Lmx1a as one of regulators of mature midbrain dopaminergic neurotransmitter phenotypes.Lmx1a, an early regulator of midbrain dopamine neural progenitor, is also expressed in post-mitotic dopaminergic neurons, implicating a role for dopaminergic maturation/maintenance. Here, we show that Lmx1a critically regulates the expression of a marker of mature dopamine neurons, the dopamine transporter. Thus, this study emphasizes a novel role of Lmx1a as a regulator of mature midbrain dopaminergic neurotransmitter phenotypes.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07771.xAbstractWhile free radicals and inflammation constitute major routes of neuronal injury occurring in amyotrophic lateral sclerosis (ALS), neither antioxidants nor non-steroidal anti-inflammatory drugs have shown significant efficacy in human clinical trials. We examined the possibility that concurrent blockade of free radicals and prostaglandin E2 (PGE2)-mediated inflammation might constitute a safe and effective therapeutic approach to ALS. We have developed 2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobenzoic acid] (AAD-2004) as a derivative of aspirin. AAD-2004 completely removed free radicals at 50 nM as a potent spin-trapping molecule and inhibited microsomal PGE2 synthase-1 (mPGES-1) activity in response to both lipopolysaccharide-treated BV2 cell with IC50 of 230 nM and recombinant human mPGES-1 protein with IC50 of 249 nM in vitro. In superoxide dismutase 1G93A transgenic mouse model of ALS, AAD-2004 blocked free radical production, PGE2 formation, and microglial activation in the spinal cords. As a consequence, AAD-2004 reduced autophagosome formation, axonopathy, and motor neuron degeneration, improving motor function and increasing life span. In these assays, AAD-2004 was superior to riluzole or ibuprofen. Gastric bleeding was not induced by AAD-2004 even at a dose 400-fold higher than that required to obtain maximal therapeutic efficacy in superoxide dismutase 1G93A. Targeting both mPGES-1-mediated PGE2 and free radicals may be a promising approach to reduce neurodegeneration in ALS and possibly other neurodegenerative diseases.Targeting both free radicals and mPGES-1 in ALS This study was carried out to examine the pathologic roles of both free radicals and mPGES-1-mediated inflammation in a murine model of ALS. AAD-2004 prevents free radical production and mPGES-1-mediated PGE2 formation at nanomolar concentrations and shows better efficacy than ibuprofen and riluzole without causing gastric damage. Targeting both free radicals and mPGES-1-mediated inflammation sheds light on novel medications for ALS patients.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07769.xAbstractAlpha-synuclein (?-syn) is a synaptic protein that mutations have been linked to Parkinson’s disease (PD), a common neurodegenerative disorder that is caused by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc). How ?-syn can contribute to neurodegeneration in PD is not conclusive but it is agreed that mutations or excessive accumulation of ?-syn can lead to the formation of ?-syn oligomers or aggregates that interfere with normal cellular function and contribute to the degeneration of dopaminergic neurons. In this study, we found that ?-syn can impair the normal dynamics of mitochondria and this effect is particular prominent in A53T ?-syn mutant. In mice expressing A53T ?-syn, age-dependent changes in both mitochondrial morphology and proteins that regulate mitochondrial fission and fusion were observed. In the cellular model of PD, we found that ?-syn reduces the movement of mitochondria in both SH-SY5Y neuroblastoma and hippocampal neurons. Taken together, our study provides a new mechanism of how ?-syn can contribute to PD through the impairment of normal dynamics of mitochondria.This study was performed to determine how alpha-synuclein can impair normal dynamics of mitochondria. Alpha-synuclein impairs normal dynamics of mitochondria through Mfn1 and Mfn2. We provide a novel mechanism of how alpha-synuclein contributes to PD through impairment of mitochondrial dynamics.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07775.xAbstractImmunophilin is the collective name given to a family of proteins that bind immunosuppressive drugs: Some immunophilins are Hsp90-binding cochaperones that affect steroid receptor function. Mood and anxiety disorders are stress-related diseases characterized by an impaired function of the mineralocorticoid and glucocorticoid receptors, two of the major regulatory elements of the hypothalamus-pituitary-adrenocortical axis. Genetic variations of the FK506-binding protein of 51-kDa, FKBP51, one of the immunophilins bound to those steroid receptor complexes, were associated with the effectiveness of treatments against depression and with a major risk-factor for the development of post-traumatic stress disorders. Interestingly, immunophilins show polymorphisms and some polymorphic isoforms of FKBP51 correlate with a greater impairment of steroid receptor functions. In this review, we discuss different aspects of the role of FKBP51 in such steroid receptor function and the impact of genetic variants of the immunophilin on the dysregulation of the stress response.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07773.xAbstractProtective effects of statins have been well documented for stroke therapy. Here, we used a systematic review and meta-analysis to assess these evidences. We identified 190 studies using statin treatment in stroke animal models by electronic searching. From those, only studies describing ischemic occlusive stroke and reporting data on infarct volume and/or neurological outcome were included in the analysis (41 publications, 1882 animals). The global estimate effect was assessed by Weighted Mean Difference meta-analysis. Statins reduced infarct volume by 25.12% (20.66%–29.58%, P < 0.001) and consistently, induced an improvement on neurological outcome (20.36% (14.17%–26.56%), P < 0.001). Stratified analysis showed that simvastatin had the greatest effect on infarct volume reduction (38.18%) and neurological improvement (22.94%), whereas bigger infarct reduction was observed giving the statin as a pre-treatment (33.5%) compared with post-treatment (16.02%). The use of pentobarbital sodium, the timing of statin administration, the statement of conflict of interest and the type of statin studied were found to be independent factors in the meta-regression, indicating their influence on the results of studies examining statin treatment. In conclusion, this meta-analysis provides further evidences of the efficacy of statins, supporting their potential use for human stroke therapy.Statins for acute stroke: Pooled data from the bench gives clues for clinical trials design. Although the protective effect of statins on experimental stroke is not novel, a peer evaluation is needed to provide faithful translation into the clinics. This systematic review and meta-analysis supports the evidence of statin benefit to reduce infarct growth and improve neurological outcome in experimental stroke. Moreover, the analysis establishes the main factors influencing statin neuroprotective effect, such as type of statin, routes and timing of administration that might be used in clinical trials.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07770.xAbstractThe effects of intracerebroventricular administration of neuropeptide Y (NPY), which is believed to play an important role in neuroprotection against excitotoxicity and in the modulation of adult neurogenesis, were evaluated in an animal model of hippocampal neurodegeneration and temporal lobe epilepsy represented by trimethyltin (TMT) intoxication. A single TMT injection (8 mg/kg) causes, in the rat brain, massive neuronal death, selectively involving pyramidal neurons, accompanied by glial activation and enhanced hippocampal neurogenesis. Our data indicate that intracerebroventricular administration of exogenous NPY (at the dose of 2 ?g/2 ?L, 4 days after TMT-administration), in adult rats, exerts a protective role in regard to TMT-induced hippocampal damage and a proliferative effect on the hippocampal neurogenic niche through the up-regulation of Bcl-2, Bcl2l1, Bdnf, Sox-2, NeuroD1, Noggin and Doublecortin genes, contributing to delineate more clearly the role of NPY in in vivo neurodegenerative processes.Neuropeptide Y: neuroprotection and pro-neurogenic effects in an animal model of hippocampal neurodegeneration and temporal lobe epilepsy. With regard to the intriguing issue of neuroprotection, the study was performed to investigate the possibility to counteract neuronal death during neurodegeneration and to modulate injury induced neurogenesis, possibly directing the process towards a reparative outcome, using NPY, which is a candidate for an adjuvant role in neurodegeneration and epilepsy.The most relevant novel finding is the direct demonstration that NPY administration induces the early activation of antiapoptotic pathways and neurogenic genes.This study adds useful information concerning the role of in vivo NPY administration on mechanisms that counteract neurodegenerative processes, such as cellular events leading to neuroprotection and the stimulation of endogenous neurogenesis.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07768.xAbstractFibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson’s disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.FGF-2-deficient mice contain supernumerary dopaminergic neurons in substantia nigra. During late embryogenesis ventral midbrain neurogenesis and levels of nuclear FgfR1 are increased, which is accompanied by reduced postnatal apoptosis. Our study on the developmental consequence of FGF-2 deficiency for dopaminergic neuron development emphasizes the multifaceted properties of FGF-2, which could have implications on neurological diseases with a developmental origin.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07766.xAbstractType I interferons (IFNs) have been shown to act on neurons and to cause neuronal damage through mechanisms not completely defined. Here, we investigated the effects of type I IFNs on brain-derived neurotrophic factor (BDNF)-induced TrkB receptor signaling and neurotrophic activity. In retinoic acid-treated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons, long-term exposure to IFNs curtailed BDNF-induced activation of phosphatidylinositol 3-kinase, phospholipase C? and extracellular-regulated kinases 1 and 2 signaling. Moreover, IFN-? inhibited BDNF-induced cell survival, neurite outgrowth, and expression of neuronal markers, such as neurofilament proteins, growth-associated protein-43 and glutamate ?-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR1. The IFN inhibitory effects were associated with down-regulation of TrkB and inhibition of TrkB autophosphorylation. In SH-SY5Y cells, blockade of either Janus kinase with pyridone 6 or signal transducer and activator of transcription (STAT) 1 with siRNA transfection attenuated IFN-?-induced TrkB down-regulation. Quantitative real time RT-PCR indicated that IFN-? significantly reduced TrkB mRNA levels. Moreover, blockade of protein kinase R counteracted IFN-?-induced inhibition of TrkB expression and signaling. These data indicate that in neuronal cells IFNs negatively regulate BDNF signaling and neurotrophic activity through inhibition of TrkB activation and Janus kinase/Signal transducer and activator of transcription-dependent down-regulation of TrkB.Type I interferons (IFNs) have been shown to cause neuronal damage through mechanisms not completely defined. Here, we show that in differentiated SH-SY5Y neuroblastoma cells and mouse primary cortical neurons long-term exposure to IFNs impairs BDNF signaling and neurotrophic activity through down-regulation of TrkB autophosphorylation and expression. The negative regulation of BDNF signaling may contribute to type I IFN neurotoxicity.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07765.xAbstractThe goal of this project was to characterize deoxypyrimidine salvage pathways used to maintain deoxynucleoside triphosphate pools in isolated brain mitochondria and to determine the extent that antiviral pyrimidine analogs utilize or affect these pathways. Mitochondria from rat brains were incubated in media with labeled and unlabeled deoxynucleosides and deoxynucleoside analogs. Products were analyzed by HPLC coupled to an inline UV monitor and liquid scintillation counter. Isolated mitochondria transported thymidine and deoxycytidine into the matrix, and readily phosphorylated both of these to mono-, di-, and tri-phosphate nucleotides. Rates of phosphorylation were much higher than rates observed in mitochondria from heart and liver. Deoxyuridine was phosphorylated much more slowly than thymidine and only to dUMP. 3?-azido-3?-deoxythymidine, zidovudine (AZT), an antiviral thymidine analog, was phosphorylated to AZT-MP as readily as thymidine was phosphorylated to TMP, but little if any AZT-DP or AZT-TP was observed. AZT at 5.5 ± 1.7 ?M was shown to inhibit thymidine phosphorylation by 50%, but was not observed to inhibit deoxycytidine phosphorylation except at levels > 100 ?M. Stavudine and lamivudine were inert when incubated with isolated brain mitochondria. The kinetics of phosphorylation of thymidine, dC, and AZT were significantly different in brain mitochondria compared to mitochondria from liver and heart.Fishing in the brain mitochondrial deoxynucleotide pools.Our goal was to characterize deoxypyrimidine salvage pathways used to maintain deoxynucleoside triphosphate pools in brain mitochondria and to determine the extent that antiviral pyrimidine analogs utilize or affect these pathways. We discovered very high thymidine kinase 2 activity with novel kinetics. Understanding the activation of pyrimidine analogs in the brain is crucial to understanding toxicity.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07764.xAbstractHIV-1-associated neurocognitive disorder (HAND) is a syndrome that ranges clinically from subtle neuropsychological impairments to profoundly disabling HIV-associated dementia. Not only is the pathogenesis of HAND unclear, but also effective treatments are unavailable. The HIV-1 transactivator of transcription protein (HIV-1 Tat) is strongly implicated in the pathogenesis of HAND, in part, because of its well-characterized ability to directly excite neurons and cause neurotoxicity. Consistent with previous findings from others, we demonstrate here that HIV-1 Tat induced neurotoxicity, increased intracellular calcium, and disrupted a variety of mitochondria functions, such as reducing mitochondrial membrane potential, increasing levels of reactive oxygen species, and decreasing bioenergetic efficiency. Of therapeutic importance, we show that treatment of cultured neurons with ketone bodies normalized HIV-1 Tat induced changes in levels of intracellular calcium, mitochondrial function, and neuronal cell death. Ketone bodies are normally produced in the body and serve as alternative energy substrates in tissues including brain and can cross the blood–brain barrier. Ketogenic strategies have been used clinically for treatment of neurological disorders and our current results suggest that similar strategies may also provide clinical benefits in the treatment of HAND.The pathogenesis of HIV-1-associated neurocognitive disorder (HAND) is unclear and effective treatments are unavailable. We found that pre-treatment of neurons with ketone bodies attenuated HIV-1 Tat-induced neuronal injury, calcium overload, and alterations in mitochondrial function and ATP levels. Ketogenic strategies might be considered a potential therapeutic intervention against HAND.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07763.xAbstractSpreading depression (SD), the likely cause of migraine aura and perhaps migraine, is triggered by widespread and unfettered neuronal hyperexcitability. Migraine and the initiating hyperexcitability of seizure, which involve oxidative stress (OS), are likely interrelated. Environmental enrichment (EE) decreases seizure and can reduce migraine. EE’s well-characterized neuroprotective effect involves insulin-like growth factor-1 (IGF-1). Accordingly, we asked if IGF-1 could mitigate the hyperexcitability that initiates SD using rat hippocampal slice cultures. We demonstrate that IGF-1 significantly decreased SD susceptibility and related OS. We mimicked OS of SD and observed that IGF-1 abolished hyperexcitability from OS. Application of an antioxidant significantly decreased SD susceptibility and co-administration of an antioxidant with IGF-1 produced no additive effect, whereas an oxidizer significantly increased SD, and this effect was abrogated by IGF-1. Moreover, IGF-1 significantly decreased baseline OS, despite seemingly paradoxically increasing CA3 bursting. These results suggest that IGF-1 increased endogenous antioxidants to levels sufficient to buffer against the OS of SD. Insulin similarly mitigated SD susceptibility, but required a far greater dose. Since brain IGF-1 increases with EE, and, like insulin, independently functions as an EE mimetic, we suggest that EE mimetics are a novel source of therapeutics for SD, and by extension, migraine.IGF-1 may prevent migraine Environment enrichment increases learning and memory and is neuroprotective –interdependent adaptive changes involving IGF-1. Here we show that IGF-1 reduced spreading depression, the likely cause of migraine, by decreasing oxidative stress while also increasing neuronal activity, consistent with learning. Our data indicate that IGF-1, an environmental enrichment-mimetic, may be a novel therapeutic for frequent and chronic migraine.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07762.xAbstractThe mechanisms that regulate synapse formation and maintenance are incompletely understood. In particular, relatively few inhibitors of synapse formation have been identified. Receptor protein tyrosine phosphatase ? (RPTP?), a transmembrane tyrosine phosphatase, is widely expressed by neurons in developing and mature mammalian brain, and functions as a receptor for chondroitin sulfate proteoglycans that inhibits axon regeneration following injury. In this study, we address RPTP? function in the mature brain. We demonstrate increased axon collateral branching in the hippocampus of RPTP? null mice during normal aging or following chemically induced seizure, indicating that RPTP? maintains neural circuitry by inhibiting axonal branching. Previous studies demonstrated a role for pre-synaptic RPTP? promoting synaptic differentiation during development; however, subcellular fractionation revealed enrichment of RPTP? in post-synaptic densities. We report that neurons lacking RPTP? have an increased density of pre-synaptic varicosities in vitro and increased dendritic spine density and length in vivo. RPTP? knockouts exhibit an increased frequency of miniature excitatory post-synaptic currents, and greater paired-pulse facilitation, consistent with increased synapse density but reduced synaptic efficiency. Furthermore, RPTP? nulls exhibit reduced long-term potentiation and enhanced novel object recognition memory. We conclude that RPTP? limits synapse number and regulates synapse structure and function in the mature CNS.The mechanisms regulating synaptic connections in the mature brain are complex and remain to be elucidated. Our study reveals a novel role for receptor protein tyrosine phosphatase ? (RPTP?), a transmembrane tyrosine phosphatase, in the regulation of dendritic spine morphology and synaptic plasticity in the adult CNS. These findings suggest that inhibitors of RPTP? might be beneficial to manipulate synaptic plasticity, promote recovery of function and enhance learning and memory after injury and in neurodegenerative diseases. RPTP? function at synapses: (a) Pre-synaptic RPTP? promotes synaptic differentiation through interactions with post-synaptic TrkC and NGL-3. (b) Increased dendritic spine density and spine length in the brains of RPTP? null mice. (c) Increased frequency of spontaneous excitatory mini-excitatory post-synaptic currents in RPTP? null mice, consistent with increased numbers of synapses. (d) Mice lacking RPTP? exhibit enhanced performance of in tests of novel object recognition.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07761.xThe amyloid precursor protein (APP) is thought to be neuroprotective following traumatic brain injury (TBI), although definitive evidence at moderate to severe levels of injury is lacking. In the current study, we investigated histological and functional outcomes in APP?/? mice compared with APP+/+ mice following a moderate focal injury, and whether administration of sAPP? restored the outcomes in knockout animals back to the wildtype state. Following moderate controlled cortical impact injury, APP?/? mice demonstrated greater impairment in motor and cognitive outcome as determined by the ledged beam and Barnes Maze tests respectively (p < 0.05). This corresponded with the degree of neuronal damage, with APP?/? mice having significantly greater lesion volume (25.0 ± 1.6 vs. 20.3 ± 1.6%, p < 0.01) and hippocampal damage, with less remaining CA neurons (839 ± 245 vs. 1353 ± 142 and 1401 ± 263). This was also associated with an impaired neuroreparative response, with decreased GAP-43 immunoreactivity within the cortex around the lesion edge compared with APP+/+ mice. The deficits observed in the APP?/? mice related to a lack of sAPP?, as treatment with exogenously added sAPP? post-injury improved APP?/? mice histological and functional outcome to the point that they were no longer significantly different to APP+/+ mice (p < 0.05). This study shows that endogenous APP is potentially protective at moderate levels of TBI, and that this neuroprotective activity is related to the presence of sAPP?. Importantly, it indicates that the mechanism of action of exogenously added sAPP? is independent of the presence of endogenous APP.This study examined the effects of knockout of the amyloid precursor protein (APP) on outcome following moderate focal traumatic brain injury (TBI). Knockout of APP led to greater impairment in motor and cognitive outcome following TBI than in wildtype mice and was accompanied by a significant increase in the amount of cortical and neuronal damage. These deficits relate to lack of the APP metabolite, sAPP?, as treatment with exogenously added sAPP? post-injury improved outcome in APP knockout mice so that they were no longer significantly different to wildtype mice. Thus, delineating the mechanism of action of sAPP? will assist in its development as a potential therapeutic agent following TBI.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07759.xAbstractMouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [125I]-Epibatidine competition-binding studies revealed that the ?3?2* and ?6?2* nicotinic subtype-selective peptide ?-conotoxin MII-blocked binding to 40 ± 5% of SuSC nAChRs. Acetylcholine-evoked [3H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [3H]-GABA release is inhibited by ?-conotoxin MII. However, the highly ?6?2*-subtype-selective ?-conotoxin PIA did not affect [3H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [3H]-GABA release is entirely ?2 subunit-dependent. ?4 subunit deletion decreased total function by >90%, and eliminated ?-conotoxin MII-resistant release. ACh-stimulated SuSC [3H]-GABA release was unaffected by ?3, ?5 or ?6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, ?-conotoxin MII-sensitive ?3?4?2 nAChR. The remaining ?-conotoxin MII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via ?4?2* subtype nAChRs.Novel ?3?4?2 nicotinic subtype modulates superior colliculus GABA release.This study examined nicotinic acetylcholine receptor (nAChR) subtypes modulating GABA release in the superior colliculus, a region highly enriched in ?-conotoxin MII-sensitive nAChRs. A novel functional ?3?4?2 nAChR subtype was found to mediate superficial superior colliculus GABA release. This study confirms the expression of functional, non-?6?2-subtype, ?-conotoxin MII-sensitive nAChRs in mammalian brain, and uncovers a new level of modulation of superior colliculus GABA release.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07758.xAbstractMany genes associated with familial Parkinson’s disease contribute to mitochondrial morphology and function. Some of these genes, for example, Pink1 and Parkin, are part of a common pathway. The presenilin-associated rhomboid-like (PARL) gene was recently linked to familial Parkinson’s disease. The PARL gene product is found in the inner mitochondrial membrane and cleaves the optic atrophy 1 protein, involved in mitochondrial morphology and apoptosis. In Drosophila, the PARL-related rhomboid-7 gene acts upstream of pink1 and parkin. However, such a genetic relationship is still unknown in vertebrates. Here, we show that the zebrafish genome comprises two parl paralogs: parla and parlb. Morpholino-mediated loss of parla and/or parlb function resulted in mild neurodegeneration, as evidenced by a lower density of dopaminergic neurons. Patterning of dopaminergic neurons was also perturbed in the ventral diencephalon. Morphants exhibited extensive cell death throughout the entire body as well as increased larval mortality. The morphant phenotype could be rescued by injection of human PARL mRNA, but not catalytically inactive PARL, suggesting functional conservation between the human and zebrafish proteins. More importantly, the zebrafish pink1 mRNA as well as the human PINK1 mRNA, but not kinase-dead nor Parkinson’s disease-linked mutant PINK1 mRNA, also rescued the morphant phenotype, providing evidence that Parl genes may function upstream of Pink1, as part of a conserved pathway in vertebrates.A mutation in the PARL gene has been found in Parkinson’s disease patients, but the effect of this mutation on dopaminergic (DA) neurons is unclear. Our knockdown of the parl genes in zebrafish resulted in dopaminergic neuron patterning defects and larval mortality. Zebrafish pink1 mRNA rescued the morphant phenotype, suggesting that parl and pink1 act in a common genetic pathway in vertebrates, as they do in Drosophila.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07757.xAbstractThe potassium (K+) analogue thallium (Tl+) can be used as a tracer for mapping neuronal activity. However, because of the poor blood–brain barrier (BBB) K+-permeability, only minute amounts of Tl+ enter the brain after systemic injection of Tl+-salts like thallium acetate (TlAc). We have recently shown that it is possible to overcome this limitation by injecting animals with the lipophilic chelate complex thallium diethyldithiocarbamate (TlDDC), that crosses the BBB and releases Tl+ prior to neuronal or glial uptake. TlDDC can thus be used for mapping CNS K+ metabolism and neuronal activity. Here, we analyze Tl+-kinetics in the rodent brain both experimentally and using simple mathematical models. We systemically injected animals either with TlAc or with TlDDC. Using an autometallographic method we mapped the brain Tl+-distribution at various time points after injection. We show that the patterns and kinetics of Tl+-redistribution in the brain are essentially the same irrespective of whether animals have been injected with TlAc or TlDDC. Data from modeling and experiments indicate that transmembrane Tl+-fluxes in cells within the CNS in vivo equilibrate at similar rates as K+-fluxes in vitro. This equilibration is much faster than and largely independent of the equilibration of Tl+-fluxes across the BBB. The study provides further proof-of-concept for the use of TlDDC for mapping neuronal activity and CNS K+-metabolism. A theoretical guideline is given for the use of K+-analogues for imaging neuronal activity with general implications for the use of metal ions in neuroimaging.The potassium (K+) analogue thallium (Tl+) can be used as a tracer for mapping neuronal activity. Neurons increase the rate of Tl+-uptake with increasing activity, but Tl+-efflux increases in return, and insight into Tl+-kinetics is crucial when interpreting Tl+-uptake patterns. In the present study, we analyze, both experimentally and using simple mathematical models, Tl+-kinetics in the rodent brain and provide a theoretical guideline for the use of K+-analogues for imaging neuronal activity with general implications for the use of metal ions in neuroimaging.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07756.xAbstractParkinson’s disease is characterized by a deficiency in motor cortex modulation due to degeneration of pigmented dopaminergic neurons of the substantia nigra projecting to the striatum. These neurons are particularly susceptible to oxidative stress, perhaps because of their dopaminergic nature. Like all catecholamines, dopamine is easily oxidized, first to a quinone intermediate and then to dopaminochrome (DAC), a 5-dihydroxyindole tautomer, that is cytotoxic in an oxidative stress-dependent manner. Here we show, using the murine mesencephalic cell line MN9D, that DAC causes cell death by apoptosis, illustrated by membrane blebbing, Annexin V, and propidium iodide labeling within 3 h. In addition, DAC causes oxidative damage to DNA within 3 h, and positive terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescence by 24 h. DAC, however, does not induce caspase 3 activation and its cytotoxic actions are not prevented by the pan-caspase inhibitor, Z-VAD-fmk. DAC-induced cytotoxicity is limited by the PARP1 inhibitor, 5-aminoisoquinolinone, supporting a role for apoptosis-inducing factor (AIF) in the apoptotic process. Indeed, AIF is detected in the nuclear fraction of MN9D cells 3 h after DAC exposure. Taken together these results demonstrate that DAC induces cytotoxicity in MN9D cells in a caspase-independent apoptotic manner, likely triggered by oxidative damage to DNA, and involving the translocation of AIF from the mitochondria to the nucleus.Death by dopaminochrome: a role for AIF but not caspase Dopaminochrome (DAC) may play a role in neurodegeneration observed in Parkinson’s disease (PD). Using MN9D cells we show that DAC causes caspase-independent apoptosis through activation of poly(ADP)ribose polymerase 1 (PARP1), and subsequent translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. These results provide a mechanism by which DAC could contribute to neurodegeneration in PD.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07755.xAbstractSynaptic glycine receptors (GlyRs) are hetero-pentameric chloride channels composed of ? and ? subunits, which are activated by agonist binding at subunit interfaces. To examine the pharmacological properties of each potential agonist-binding site, we substituted residues of the GlyR ?1 subunit by the corresponding residues of the ? subunit, as deduced from sequence alignment and homology modeling based on the recently published crystal structure of the glutamate-gated chloride channel GluCl. These exchange substitutions allowed us to reproduce the ??, ?? and ?? subunit interfaces present in synaptic heteromeric GlyRs by generating recombinant homomeric receptors. When the engineered ?1 GlyR mutants were expressed in Xenopus oocytes, all subunit interface combinations were found to form functional agonist-binding sites as revealed by voltage clamp recording. The ??-binding site displayed the most distinct pharmacological profile towards a range of agonists and modulators tested, indicating that it might be selectively targeted to modulate the activity of synaptic GlyRs. The mutational approach described here should be generally applicable to heteromeric ligand-gated ion channels composed of homologous subunits and facilitate screening efforts aimed at targeting inter-subunit specific binding sites.The pharmacological profiles of the distinct subunit interfaces found in heteromeric synaptic GlyRs (scheme, left) have not been characterized in detail. By exchange substitutions of the agonist binding loops, the ??, ?? and ?? interfaces present in native synaptic GlyRs were individually generated in recombinant homomeric receptors (scheme, right). This approach should be generally applicable to the analysis of ligand binding sites located at subunit interfaces of heteromeric receptors composed of homologous subunits.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07754.xAbstractIntravenous immunoglobulin (IVIg) preparations obtained by fractionating blood plasma, are increasingly being used increasingly as an effective therapeutic agent in treatment of several inflammatory diseases. Its use as a potential therapeutic agent for treatment of stroke and Alzheimer’s disease has been proposed, but little is known about the neuroprotective mechanisms of IVIg. In this study, we investigated the effect of IVIg on downstream signaling pathways that are involved in neuronal cell death in experimental models of stroke and Alzheimer’s disease. Treatment of cultured neurons with IVIg reduced simulated ischemia- and amyloid ?peptide (A?)-induced caspase 3 cleavage, and phosphorylation of the cell death-associated kinases p38MAPK, c-Jun NH2-terminal kinase and p65, in vitro. Additionally, A?-induced accumulation of the lipid peroxidation product 4-hydroxynonenal was attenuated in neurons treated with IVIg. IVIg treatment also up-regulated the anti-apoptotic protein, Bcl2 in cortical neurons under ischemia-like conditions and exposure to A?. Treatment of mice with IVIg reduced neuronal cell loss, apoptosis and infarct size, and improved functional outcome in a model of focal ischemic stroke. Together, these results indicate that IVIg acts directly on neurons to protect them against ischemic stroke and A?-induced neuronal apoptosis by inhibiting cell death pathways and by elevating levels of the anti-apoptotic protein Bcl2.Intravenous immunoglobulin for Stroke Intravenous immunoglobulin(IVIg) is a therapeutic modality approved for the treatment of various condition. This study was performed in order to understand the mechanism ofhow IVIg elicits its neuroprotective effect in stroke and amyloid beta induced neuronal apoptosis. The findings from this study showed that IVIg elicits its neuroprotective effects by not only inhibiting the cell death pathways but also elevating the anti-apoptotic protein Bcl2. This study provides a better understanding of how IVIg plays a role in neuroprotection and therefore provides more evidence to encourage the use of IVIg as therapeutic modality in stroke and Alzheimer’s disease.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07752.xAbstractThe metabotropic glutamate receptors (mGluRs) fine-tune the efficacy of synaptic transmission. This unique feature makes mGluRs potential targets for the treatment of various CNS disorders. There is ample evidence to show that the ubiquitin proteasome system mediates changes in synaptic strength leading to multiple forms of synaptic plasticity. The present study describes a novel interaction between post-synaptic adaptors, long Homer-3 proteins, and one of the 26S proteasome regulatory subunits, the S8 ATPase, that influences the degradation of the metabotropic glutamate receptor 1? (mGluR1?). We have shown that the two human long Homer-3 proteins specifically interact with human proteasomal S8 ATPase. We identified that mGluR1? and long Homer-3s immunoprecipitate with the 26S proteasome both in vitro and in vivo. We further found that the mGluR1? receptor can be ubiquitinated and degraded by the 26S proteasome and that Homer-3A facilitates this process. Furthermore, the siRNA mediated silencing of Homer-3 led to increased levels of total and plasma membrane-associated mGluR1? receptors. These results suggest that long Homer-3 proteins control the degradation of mGluR1? receptors by shuttling ubiquitinated mGluR-1? receptors to the 26S proteasome via the S8 ATPase which may modulate synaptic transmission.Miserable mortals who, like leaves, at one moment flame with life.......and at another moment weakly perish (Homer, The Iliad). Synaptic complexity dictates that a sub-family of Homer proteins act as adaptors to deliver metabotropic glutamate receptors to neuronal 26S proteasomes for degradation. The present study was performed to identify new interactors with each of the non-redundant six ATPases in the base of the 19S regulator of the 26S proteasome to control the degradation of novel protein substrates. This study demonstrates that long Homer proteins act to deliver metabotropic glutamate receptor 1? to neuronal 26S proteasomes via the S8 ATPase for degradation. The findings serve to emphasise the importance of the ubiquitin proteasome system in controlling neuroreceptor activity in the nervous system.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07750.xAbstractThe spinal cord of the freshwater turtle Trachemys dorbignyi regenerates after complete transection (Rehermann et al. J. Comp. Neurol. 515, 2009, 197–214). This remarkable ability may be related to the persistence around the central canal (CC) of progenitors functionally clustered via connexin 43 (Cx43) that express brain lipid binding protein (BLBP) and the transcription factor Pax6 (Russo et al. J. Neurosci. 28, 2008, 8510–8516). Indeed, because BLBP+ cells appear in the bridge joining the rostral and caudal stumps, we speculated that progenitors contacting the central canal may play a key part in spinal cord regeneration. To test this hypothesis, we designed degenerated primers pairing conserved regions for key proteins synthesized in progenitors (BLBP, Cx43, and Pax6) and the neuronal protein HuB. Fragments of these proteins were amplified, cloned, and sequenced. Based on these sequences, we analyzed the changes in the expression levels using quantitative real-time RT-PCR with specific primers, comparing the injured spinal cord at different times after injury (4, 12, 20, and 60 days) with uninjured spinal cords. We found a transient, early increase of BLBP, Cx43 and HuB mRNA, with Pax6 remaining unchanged. These results suggest that the selected genes – active in progenitor cells – play an important part in early mechanisms of spinal cord regeneration.Early mechanisms for endogenous repair of the injured spinal cordUnlike mammals, turtles display a substantial degree of endogenous repair after spinal cord injury. We find that brain lipid bind protein and connexin 43, but not Pax6 genes -active in multi-potent progenitors- are upregulated during early stages of regeneration. These findings suggest that progenitors react to injury by favoring the production of glia to support the navigation of regenerating axons. Immunohistochemistry for brain lipid binding protein (BLBP) in a stump of the spinal cord 4 days after complete transection.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07745.xAbstractPurkinje cell protein 4-like 1 (Pcp4l1) is a small neuronal IQ motif protein closely related to the calmodulin-binding protein Pcp4/PEP-19. PEP-19 interacts with calmodulin via its IQ motif to inhibit calmodulin-dependent enzymes and we hypothesized Pcp4l1 would have similar properties. Surprisingly, full-length Pcp4l1 does not interact with calmodulin in yeast two-hybrid or pulldown experiments yet a synthetic peptide constituting only the IQ motif of Pcp4l1 binds calmodulin and inhibits calmodulin-dependent kinase II. A nine-residue glutamic acid-rich sequence in Pcp4l1 confers these unexpected properties. This element lies outside the IQ motif and its deletion or exchange with the homologous region of PEP-19 restores calmodulin binding. Conversion of a single isoleucine (Ile36) within this motif to phenylalanine, the residue present in PEP-19, imparts calmodulin binding onto Pcp4l1. Moreover, only aromatic amino acid substitutions at position 36 in Pcp4l1 allow binding. Thus, despite their sequence similarities PEP-19 and Pcp4l1 have distinct properties with the latter harboring an element that can functionally suppress an IQ motif. We speculate Pcp4l1 may be a latent calmodulin inhibitor regulated by post-translational modification and/or co-factor interactions.Here we characterize the biochemical properties of Purkinje cellprotein 4-like 1 (Pcp4l1) a small IQ motif protein closely related to the calmodulin regulators PEP-19 and neurogranin. We identify a motif within Pcp4l1 that prevents binding to calmodulin and through detailed mutagenesis identify isoleucine 36 as a critical residue for this inhibitory function. Collectively our experiments demonstrate an unexpected and previously uncharacterized mechanism for regulation of IQ motif to calmodulin binding.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07743.xAbstractThe actin cytoskeleton in dendritic spines is organized into microdomains, but how signaling molecules that regulate actin are spatially governed is incompletely understood. Here we examine how the localization of the RacGEF kalirin-7, a well-characterized regulator of actin in spines, varies as a function of post-synaptic density area and spine volume. Using serial section electron microscopy, we find that extrasynaptic, but not synaptic, expression of kalirin-7 varies directly with synapse size and spine volume. Moreover, we find that overall expression levels of kalirin-7 differ in spines bearing perforated and non-perforated synapses, due primarily to extrasynaptic pools of kalirin-7 expression in the former. Overall, our findings indicate that kalirin-7 is differentially compartmentalized in spines as a function of both synapse morphology and spine size.An immuno-electron microscopy image showing a perforated post-synaptic density (PSD) in which kalirin-7 is exclusively localized extrasynaptically. Although kalirin-7 is known to regulate dendritic spine morphology, how its localization within individual spine domains contributes to the regulation of spine morphology is unknown. Using immuno-electron microscopy, we paradoxically find that increased expression of kalirin-7 extrasynaptically rather than synaptically is most directly related to synapse and spine size. These findings indicate that there are multiple kalirin-7 microdomains within spines that are likely to differentially influence spine morphology, and this study extends the current scientific knowledge of the spatial regulation of actin-regulatory molecules within individual spines.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07742.xAbstractThe extension of processes of oligodendrocyte (OLG) and their precursor cells are crucial for migration, axonal contact and myelination. Here we show that a non-lethal oxidative stress induced by 3-nitropropionic acid (3-NP) elicited a rapid shortening of processes (?24%) in primary OLGs and in oligodendroglial cell line (OLN-93) cells (?36%) as compared with vehicle-exposed cells. This was reversible and prevented by antioxidants. Proteomics of OLG lysates with and without 3-NP treatment yielded collapsin response mediator protein 2 (CRMP-2) as a candidate effector molecule. Inhibition of rho kinase was sufficient to prevent process retraction in both OLGs and OLN-93 cells. Oxidative stress increased phosphorylation of CRMP-2 at T555 that was completely prevented by Y27632. Moreover, transfection of OLN-93 cells with the mutant CRMP-2 T555A which cannot be phosphorylated by rho kinase, prevented process shortening induced by 3-NP as compared with wild-type CRMP-2. Our results suggest a role for endogenous reactive oxygen species in a pathway that regulates OLG process extension. The vulnerability of late myelinated neurons in the adult brain and the presence of white matter pathology in human dementias warrant the study of this oligodendroglial pathway in the early stages of neurodegenerative conditions characterized by oxidative stress.Reactive oxygen species modulate oligodendroglial process extension The branching and extension of oligodendrocyte (OLG) processes, crucial for axon myelination, are regulated by extracellular signals. Yet, the role of reactive oxygen species (ROS) in such a dynamic mechanism is not known. Mild oxidative stress in mature OLG triggered a rapid process shortening mediated by phosphorylation of CRMP-2 by ROCK. We propose a novel level of process growth modulation by ROS that may help to understand early OLG dysfunction in neurodegeneration.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07751.xAbstractApart from its hematopoietic activity, erythropoietin (EPO) is also known as a tissue-protective cytokine. In the brain, EPO and its receptor are up-regulated in response to insult and exert pro-survival effects. EPO binds to its receptor (EPOR) via high- and low-affinity binding sites (Sites 1 and 2, respectively), inducing conformational changes in the receptor, followed by the activation of downstream signaling cascades. Based on the crystal structure of the EPO:EPOR2 complex, we designed a peptide, termed Epobis, whose sequence encompassed amino acids from binding Site 1. The present study shows that the Epobis peptide specifically binds to EPOR and induces neurite outgrowth from primary neurons in an EPOR-expression dependent manner. Furthermore, Epobis promoted the survival of hippocampal and cerebellar neuronal cultures after kainate treatment and KCl deprivation, respectively. Thus, we identified a new functional agonist of EPOR with the potential to promote neuroregeneration and neuroprotection.A novel functional agonist of the erythropoietin receptor with the potential to promote neuroregeneration and neuroprotectionErythropoietin (EPO) is a cytokine with both hematopoietic and neuroprotective properties, and the development of neuroprotective EPO mimetics lacking hematopoietic activity is an important issue. This study describes the identification and characterization of a novel agonist of the EPO receptor with neuritogenic and neuronal survival promoting properties. This finding has important implications for the development of treatments for neurodegenerative disorders.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07748.xAbstractSignal regulatory protein ? (SIRP?) is a neuronal membrane protein that undergoes tyrosine phosphorylation in the brain of mice in response to forced swim (FS) stress in cold water, and this response is implicated in regulation of depression-like behavior in the FS test. We now show that subjection of mice to the FS in warm (37°C) water does not induce the tyrosine phosphorylation of SIRP? in the brain. The rectal temperature (Trec) of mice was reduced to 27° to 30°C by performance of the FS for 10 min in cold water, whereas it was not affected by the same treatment in warm water. The level of tyrosine phosphorylation of SIRP? in the brain was increased by administration of ethanol or picrotoxin, starvation, or cooling after anesthesia, all of which also induced hypothermia. Furthermore, the tyrosine phosphorylation of SIRP? in cultured hippocampal neurons was induced by lowering the temperature of the culture medium. CD47, a ligand of SIRP?, as well as Src family kinases or SH2 domain-containing protein phosphatase 2 (Shp2), might be important for the basal and the hypothermia-induced tyrosine phosphorylation of SIRP?. Hypothermia is therefore likely an important determinant of both the behavioral immobility and tyrosine phosphorylation of SIRP? observed in the FS test.SIRP? is a membrane protein that undergoes tyrosine phosphorylation in the brain and is implicated in regulation of depression-like behavior in the forced swim test in cold water. We show that lowering the temperature in vivo and in vitro increased the level of tyrosine phosphorylation of SIRP?. Tyrosine phosphorylation of SIRP? is a novel cellular signal induced by low temperature.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07737.xAlzheimer’s disease (AD) is the most common form of dementia and cognitive impairment usually characterized by widespread neurodegeneration throughout the association cortex, limbic system and hippocampus. Aberrant protein phosphorylation is a defining pathological hallmark of AD and implicated in the dysregulation of major cellular processes through highly dynamic and complex signaling pathways. Here in, we demonstrate 81 proteins, of 600 spots selected, unambiguously identified as phosphorylated, providing a partial phosphoproteome profile of AD substantia nigra and cortex and respective control brain regions. More importantly, abnormal phosphorylation signal intensity of nine physiologically important proteins observed can profoundly affect cell metabolism, signal transduction, cytoskeleton integration, and synaptic function and accounts for biological and morphological alterations. Our studies employed two-dimensional gel electrophoresis for protein separation, Pro-Q® Diamond phosphoprotein staining and electrospray ionization quadrupole time of flight tandem MS for protein identification. NetPhosk 1.0 is used for the confirmation of protein modification status as well known/putative phosphoproteins. A further insight into the links among the identified phosphoproteins and functional roles STRING 8.3, KEGG and REACTOME pathway databases were applied. The present quantitative phosphoproteomic analysis can be supportive in establishing a broad database of potential protein targets of abnormal phosphorylation in AD brain.Aberrant phosphorylation and Alzheimer’s disease The molecular mechanisms underlying aberrant response to phosphorylation leading to AD pathology are not fully understood. The study facilitated the identification of nine proteins aberrantly phosphorylated in cortex and substantia nigra targeting cell metabolism, signal transduction, cytoskeletal integration and synaptic function. These findings may aid in developing effective therapeutic strategies and establishing broad database of potential proteins involved in AD.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07741.xAbstractWe have recently reported that Presenilin 1 (PS1), a causative gene of familial Alzheimer disease (AD), down-regulates the expression level of insulin receptor (IR) as well as its signaling through a ?-secretase-independent pathway. PS1 is phosphorylated by glycogen synthase kinase 3 ? at the serine 353 and 357 residues. The main purpose of the present study was to clarify the effect of PS1 phosphorylation on IR/insulin signaling. Here, we demonstrate that the pseudo-phosphorylation mutant of PS1 inhibited IR transcription and reduced IR expression compared with wild-type PS1. Importantly, there was a decrease in expression of IR in AD brains, and the phosphorylation ratio of PS1 was negatively correlated with IR level in human brain samples. In the data from mouse models of AD, IR reduction was not observed at the pre-A? deposition stage but became apparent in that of post-A? deposition. Together with our previous reports, these results suggest that phosphorylated PS1 can promote the down-regulation of insulin signaling, which may be a positive feed-forward mechanism inhibiting insulin signaling. As insulin resistance is reported to be a risk factor for sporadic AD, this PS1-mediated regulatory mechanism of brain insulin signaling may be causally associated with AD pathology.The function of phosphorylated Presenilin 1 has not been elucidated. We demonstrate that phosphorylated Presenilin 1 at the serine 353 and 357 residues down-regulated the expression of insulin receptor compared with wild-type Presenilin 1, thus suggesting that phosphorylated PS1 may play the crucial role in a positive feed-forward cycle inhibiting insulin signaling. From our findings, insulin signaling dysfunction may be associated with the pathogenesis of Alzheimer disease.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07709.xAbstractAlzheimer’s disease (AD) affects about 35.6 million people worldwide, and if current trends continue with no medical advancement, one in 85 people will be affected by 2050. Thus, there is an urgent need to develop a cost-effective, easy to use, sensor platform to diagnose and study AD. The measurement of peptide amyloid beta (A?) found in CSF has been assessed as an avenue to diagnose and study the disease. The quantification of the ratio of A?1–40/42 (or A? ratio) has been established as a reliable test to diagnose AD through human clinical trials. Therefore, we have developed a multiplexed, implantable immunosensor to detect amyloid beta (A?) isoforms using triple barrel carbon fiber microelectrodes as the sensor platform. Antibodies act as the biorecognition element of the sensor and selectively capture and bind A?1–40 and A?1–42 to the electrode surface. Electrochemistry was used to measure the intrinsic oxidation signal of A? at 0.65 V (vs. Ag/AgCl), originating from a single tyrosine residue found at position 10 in its amino acid sequence. Using the proposed immunosensor A?1–40 and A?1–42 could be specifically detected in CSF from mice within a detection range of 20–50 nM and 20–140 nM respectively. The immunosensor enables real-time, highly sensitive detection of A? and opens up the possibilities for diagnostic ex vivo applications and research-based in vivo studies.Amyloid-? (A?) peptide accumulates in Alzheimer’s disease patients and is believed to precipitate disease onset. We developed an electrical immuno microsensor for highly sensitive and specific detection of different isoforms of A?. The label-free detection is based on quantitatively measuring redox signals of A? using a carbon fiber microelectrode array. Real-time detection capabilities and miniature size of sensor may allow for clinical diagnostic and potential in vivo animal study.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07697.xAbstractVitamin A (VA) is important for postnatal brain development, and VA deficiency (VAD) can cause learning and spatial memory deficits in rats. Most of the biological functions of VA are mediated by retinoic acid (RA). To investigate the mechanisms underlying VA deficits, mother rats were fed elemental diets to achieve blood VA levels classified as normal, deficient or severely deficient. Shuttle box and Morris water maze tests revealed impairments in learning ability and spatial memory, respectively, in adolescent VAD rats (p 30–35). Electrophysiology showed weaker long-term potentiation in VAD rats compared to VA normal rats. Examination of NMDA-induced calcium (Ca2+) excitability revealed decreased excitability in hippocampal slices from VAD rats during postnatal development. Relative to VA normal rats, VAD rats also had decreased NMDA receptor NR1 mRNA and protein expression in later stages of postnatal development (p 10–30), as well as differences in retinoic acid receptor (RAR?) mRNA and protein expression. Furthermore, primary hippocampal neurons in culture showed increased neuronal Ca2+ excitability in response to all-trans-RA or 9-cis-RA, coupled with increases in RAR? and NR1 expression similar to those observed in vivo. We also found weaker calcium excitability and lower expression of NR1 mRNA and protein after specific silencing of RAR?. Finally, we found that RA signals affected the expression of NR1 do not directly through transcriptional regulation. These data support the new idea that continuous postnatal VAD inhibits RAR? expression, which decreases NR1 expression via no direct transcriptional regulation and then inhibits hippocampal neuronal Ca2+excitability which affects long-term potentiation, finally producing deficits in active learning and spatial memory in adolescence.Unravelling the mechanisms of memory in rats with Vitamin A deficiency Vitamin A deficiency (VAD) can cause learning and spatial memory deficits in rats. Most of the biological functions of vitamin A (VA) are mediated by retinoic acid (RA). We show that in VAD rats, long-term potentiation (LTP) was weak and NMDA-induced Ca2+ excitability in hippocampal CA1 neurons was suppressed compared to rats with normal VA levels. VAD rats also differed in retinoic acid receptor (RAR?) mRNA and protein expression. Our data support the new idea that continuous postnatal VAD inhibits RAR? expression, which decreases NR1 expression via no direct transcriptional regulation and then inhibits hippocampal neuronal Ca2+ excitability which affects LTP, finally producing deficits in active learning and spatial memory in adolescence.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07740.xAbstractMammals employ large numbers of odorant receptors to sample and identify volatile chemicals in the environment. These receptors are thought to vary not only in specificity for particular odorants, but also in breadth of tuning. That is, some odorant receptors are narrowly focused on a few closely related structures, while other odorant receptors may be ‘broadly tuned’, responding to a wide variety of odorant structures. In this study, we have performed a detailed examination the mouse odorant receptor MOR256-17, demonstrating that this receptor is broadly tuned. This receptor responds to odorant structures that span a significant portion of a multi-dimensional odor space. However, we found that broad tuning was not a defining characteristic of other members the MOR256 subfamily. Two additional members of this odorant receptor subfamily (MOR256-8 and MOR256-22) were more narrowly focused on small sets of odorant structures. Interestingly, the receptive range of MOR256-17 encompassed a variety of nitrotoluenes, including various trinitrotoluene synthesis intermediates, degradation products and trinitrotoluene itself, suggesting the potential utility of odorant receptors in the development of sensing technologies for the detection of explosives and other forms of contraband.Smelling a bit of everything: A broadly tuned olfactory receptor The mammalian olfactory receptor repertoire is thought to include receptors with broad specificity. A mouse olfactory receptor is shown to be broadly tuned, recognizing chemical structures scattered across a large portion of odor space and yet making subtle distinctions among closely related structures. Understanding the molecular basis for such broad, yet discriminating, odorant recognition is a major challenge in olfaction.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07749.xAbstractThe concept that the immune system plays a central role in the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica (NMO) was indisputable. However, neurodegenerative pathological features including loss of axons and neurons were also found in the lesions of these diseases. ?-Synuclein is one of the most abundant proteins in pre-synaptic terminals. Recently, many research show ?-synuclein level in CSF may reflect the progression of synaptic dysfunction and neuronal apoptosis. Whether the levels of CSF ?-synuclein are changed in MS and NMO patients remain unknown. In this study, CSF ?-synuclein was measured by an enzyme-linked immunosorbent assay (ELISA) in MS (n = 18) patients, NMO (n = 22) patients, Parkinson’s disease patients (n = 9), and the controls (n = 11). We found concentration of ?-synuclein in MS and NMO patients were significantly higher than Parkinson’s disease subgroup and the controls. Both MS and NMO revealed an increased disease disability with increased CSF ?-synuclein. Thus, CSF ?-synuclein may be reflect injure axons and neurons in inflammatory demyelinating diseases.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07746.xAbstractGlutamate excitotoxicity causes neuronal dysfunction and degeneration. It is implicated in chronic disorders, including Alzheimer’s disease, and in acute CNS insults such as ischemia. These disorders share prominent morphological features, including axon degeneration and cell body death. However, the molecular mechanism underlying excitotoxicity-induced neurodegeneration remains poorly understood. A key molecular feature of neurodegeneration is deficits in microtubule-based cargo transport that plays a pivotal role in maintaining the balance of survival and stress signaling in the axon. We developed an excitotoxicity-induced neurodegeneration system in primary neuronal cultures. We find that excitotoxicity generates a C-terminal truncated form of p150Glued, a major component of the dynactin complex, which exacerbates axon degeneration. This p150Glued truncated form was identified in brain tissues of patients with Alzheimer’s disease. Overexpression of wild-type (WT) dynein intermediate chain (DIC), a dynein component that interacts with p150Glued and links dynein and dynactin complexes, DIC (S84D) mutant, and WT p150Glued suppressed axon degeneration. These modulating effects of p150Glued and DIC on excitotoxicity-induced axon degeneration are also observed in apoptosis and cell body death. Thus, our findings identify retrograde transport proteins, p150Glued and DIC, as novel modulators of neurodegeneration induced by glutamate excitotoxicity.p150Glued dynactin and dynein intermediate chain (DIC) modulate excitotoxic neuronal death. Our findings provide a new insight into the molecular mechanism underlying excitotoxic neuronal death and include the identification of a C-terminal truncated p150Glued that contributes to the process of neuronal death. Importantly, this truncated form was identified in brain tissues of patients with Alzheimer’s disease, providing relevance to the pathogenesis of human neurological disorders.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07739.xAbstractNeuropsychiatric disorders characterized by behavioral disinhibition, including disorders of compulsivity (e.g. obsessive–compulsive disorder; OCD) and impulse-control (e.g. impulsive aggression), are severe, highly prevalent and chronically disabling. Treatment options for these diseases are extremely limited. The pathophysiological bases of disorders of behavioral disinhibition are poorly understood but it has been suggested that serotonin dysfunction may play a role. Mice lacking the gene encoding brain tryptophan hydroxylase 2 (Tph2?/?), the initial and rate-limiting enzyme in the synthesis of serotonin, were tested in numerous behavioral assays that are well known for their utility in modeling human neuropsychiatric diseases. Mice lacking Tph2 (and brain 5HT) show intense compulsive and impulsive behaviors to include extreme aggression. The impulsivity is motor in form and not cognitive because Tph2?/? mice show normal acquisition and reversal learning on a spatial learning task. Restoration of 5HT levels by treatment of Tph2?/? mice with its immediate precursor 5-hydroxytryptophan attenuated compulsive and impulsive–aggressive behaviors. Surprisingly, in Tph2?/? mice, the lack of 5HT was not associated with anxiety-like behaviors. The results indicate that 5HT mediates behavioral disinhibition in the mammalian brain independent of anxiogenesis.Serotonin dysfunction has been implicated in a number of neuropsychiatric diseases such as depression and anxiety but its precise role in these disorders is poorly understood. In the present study, mice were genetically depleted of brain serotonin by knocking out the gene for tryptophan hydroxylase 2 and tested in numerous validated tests of behavioral disorders. Mice lacking brain serotonin are highly compulsive, impulsive and aggressive. These results indicate that brain serotonin mediates a common pathway of maladaptive behaviors that is best characterized as behavioral disinhibition.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07733.xAbstractProper development of neuronal networks relies on the polarization of the neurons, thus the establishment of two compartments, axons and dendrites, whose formation depends on cytoskeletal rearrangements. Rnd proteins are regulators of actin organization and they are important players in several aspects of brain development as neurite formation, axon guidance and neuron migration. We have recently demonstrated that mice lacking RhoE/Rnd3 expression die shortly after birth and have neuromotor impairment and neuromuscular alterations, indicating an abnormal development of the nervous system. In this study, we have further investigated the specific role played by RhoE in several aspects of neuronal development by using hippocampal neuron cultures. Our findings show that neurons from a mice lacking RhoE expression exhibit a decrease in the number and the total length of the neurites. We also show that RhoE-deficient neurons display a reduction in axon outgrowth and a delay in the process of neuronal polarization. In addition, our results suggest an involvement of the RHOA/ROCK/LIMK/COFILIN signaling pathway in the neuronal alterations induced by the lack of RhoE. These findings support our previous report revealing the important role of RhoE in the normal development of the nervous system and may provide novel therapeutic targets in neurodegenerative disorders.Has RhoE/Rnd3 a role in neuronal development? Rnd proteins are important players in several aspects of brain development. Our results demonstrate that hippocampal neurons from a mice lacking RhoE expression exhibit a decrease in both neurite and axon outgrowth and also a delay in the process of neuronal polarization. We have also found that the RHOA/ROCK/LIMK/COFILIN pathway is involved in those alterations and its modulation may be very valuable to address neurodegenerative therapy.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07736.xAbstractAbnormal hyperexcitability of primary sensory neurons contributes to neuropathic pain development after nerve injury. Nerve injury profoundly reduces the expression of big conductance Ca2+ -activated K+ (BK) channels in the dorsal root ganglion (DRG). However, little is known about how nerve injury affects BK channel activity in DRG neurons. In this study, we determined the changes in BK channel activity in DRG neurons in a rat model of neuropathic pain and the contribution of brain-derived neurotrophic factor (BDNF) to reduced BK channel activity. The BK channel activity was present predominantly in small and medium DRG neurons, and ligation of L5 and L6 spinal nerves profoundly decreased the BK current density in these neurons. Blocking BK channels significantly increased neuronal excitability in sham control, but not in nerve-injured, rats. The BDNF concentration in the DRG was significantly greater in nerve-injured rats than in control rats. BDNF treatment largely reduced BK currents in DRG neurons in control rats, which was blocked by either anti-BDNF antibody or K252a, a Trk receptor inhibitor. Furthermore, either anti-BDNF antibody or K252a reversed reduction in BK currents in injured DRG neurons. BDNF treatment reduced the mRNA levels of BK?1 subunit in DRG neurons, and anti-BDNF antibody attenuated the reduction in the BK?1 mRNA level in injured DRG neurons. These findings suggest that nerve injury primarily diminishes the BK channel activity in small and medium DRG neurons. Increased BDNF levels contribute to reduced BK channel activity in DRG neurons through epigenetic and transcriptional mechanisms in neuropathic pain.Ca2+-activated K+ channels: a key regulator of pain-sensing neurons. Stimulation of Ca2+-activated K+ channels normally acts as a “brake” to restrain neurons’ firing activity. Nerve injury suppresses the activity of Ca2+-activated K+ channels in sensory neurons to facilitate pain transduction through a neurotrophic factor. The Ca2+-activated K+ channels represent a new target to treat nerve pain.
J. Neurochem. (2012) 121, 793–805.AbstractAlthough enhanced calpain activity is well documented after traumatic brain injury (TBI), the pathways targeting specific substrate proteolysis are less defined. Our past work demonstrated that calpain cleaves voltage gated sodium channel (NaCh) ?-subunits in an in vitro TBI model. In this study, we investigated the pathways leading to NaCh cleavage utilizing our previously characterized in vitro TBI model, and determined the location of calpain activation within neuronal regions following stretch injury to micropatterned cultures. Calpain specific breakdown products of ?-spectrin appeared within axonal, dendritic, and somatic regions 6 h after injury, concurrent with the appearance of NaCh ?-subunit proteolysis in both whole cell or enriched axonal preparations. Direct pharmacological activation of either NMDA receptors (NMDArs) or NaChs resulted in NaCh proteolysis. Likewise, a chronic (6 h) dual inhibition of NMDArs/NaChs but not L-type voltage gated calcium channels significantly reduced NaCh proteolysis 6 h after mechanical injury. Interestingly, an early, transient (30 min) inhibition of NMDArs alone significantly reduced NaCh proteolysis. Although a chronic inhibition of calpain significantly reduced proteolysis, a transient inhibition of calpain immediately after injury failed to significantly attenuate NaCh proteolysis. These data suggest that both NMDArs and NaChs are key contributors to calpain activation after mechanical injury, and that a larger temporal window of sustained calpain activation needs consideration in developing effective treatments for TBI.Signaling pathways for calpain activation after Traumatic Brain Injury Enhanced calpain activity is a hallmark consequence of Traumatic Brain Injury, whereas the pathways leading to proteolysis of specific substrates are less well defined. Here, we demonstrate how early NMDA receptor activation underlies calpain mediated proteolysis of voltage gated sodium channels following an in vitro mechanical insult. Propagating within the first several hours after injury, this signaling pathway may be amenable to new therapeutic approaches.
J. Neurochem. (2012) 121, 830–839.AbstractMitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson’s disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H2O2) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.Abnormal mitochondrial dynamics is implicated in neurodegenerative diseases. We found that expression of PD-associated DJ-1 mutants resulted in excessive mitochondrial fragmentation via increased DLP1 expression, which in turn leads to mitochondrial dysfunction and increased neuronal susceptibility to H2O2 or MPP+. Expression of WT DJ-1 caused mitochondrial elongation and protection. These studies suggest that impaired mitochondrial dynamics underlies neuronal dysfunction/degeneration caused by DJ-1 mutations.
J. Neurochem. (2012) 121, 738–750.AbstractDocosahexaenoic acid (DHA, 22 : 6) and eicosapentaenoic acid (EPA, 20 : 5) are omega-3 polyunsaturated fatty acids (n-3 PUFAs) with distinct anti-inflammatory properties. Both have neuroprotective effects acutely following spinal cord injury (SCI). We examined the effect of intravenous DHA and EPA on early inflammatory events after SCI. Saline, DHA or EPA (both 250 nmol/kg) were administered 30 min after T12 compression SCI, to female Sprague-Dawley rats. DHA significantly reduced the number of neutrophils to some areas of the injured epicentre at 4 h and 24 h. DHA also reduced C-reactive protein plasma levels, whereas EPA did not significantly reduce neutrophils or C-reactive protein. Laminectomy and SCI elicited a sustained inflammatory response in the liver, which was not reversed by the PUFAs. The chemokine KC/GRO/CINC and the cytokine IL-6 provide gradients for chemotaxis of neutrophils to the epicentre. At 4 h after injury, there was a significant increase in IL-6, KC/GRO/CINC, IL-1? and tumour necrosis factor-? in the epicentre, with a return to baseline at 24 h. Neither DHA nor EPA returned their levels to control values. These results indicate that the acute neuroprotective effects of n-3 PUFAs in rat compression SCI may be only partly attributed to reduction of some of the early inflammatory events occurring after injury.Omega-3 fatty acids and the early inflammatory response after spinal cord injury The omega-3 fatty acids docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) have potent neuroprotective effects after spinal cord injury (SCI) but their mechanisms of action are poorly understood. Here, we discovered that the acute administration of DHA leads to a modest reduction in neutrophil infiltration at the injury site, but EPA does not. Therefore, the reduction of the early neuroinflammation by DHA and EPA is likely to play a minor role in their neuroprotective properties when given acutely after SCI.
J. Neurochem. (2012) 121, 806–817.AbstractOur previous studies have indicated that de novo ceramide synthesis plays a critical role in ethanol-induced apoptotic neurodegeneration in the 7-day-old mouse brain. In this study, we examined whether the formation of sphingosine 1-phosphate (S1P), a ceramide metabolite, is associated with this apoptotic pathway. Analyses of basal levels of S1P-related compounds indicated that S1P, sphingosine, sphingosine kinase 2, and S1P receptor 1 increased significantly during postnatal brain development. In the 7-day-old mouse brain, sphingosine kinase 2 was localized mainly in neurons. Subcellular fractionation studies of the brain homogenates showed that sphingosine kinase 2 was enriched in the plasma membrane and the synaptic membrane/synaptic vesicle fractions, but not in the nuclear and mitochondrial/lysosomal fractions. Ethanol exposure in 7-day-old mice induced sphingosine kinase 2 activation and increased the brain level of S1P transiently 2–4 h after exposure, followed by caspase 3 activation that peaked around 8 h after exposure. Treatment with dimethylsphingosine, an inhibitor of sphingosine kinases, attenuated the ethanol-induced caspase 3 activation and the subsequent neurodegeneration. These results indicate that ethanol activates sphingosine kinase 2, leading to a transient increase in S1P, which may be involved in neuroapoptotic action of ethanol in the developing brain.The link between ethanol-induced sphingosine 1-phosphate elevation and neuroapoptosis. Both pro-apoptotic and anti-apoptotic actions of S1P in the brain have been reported. Our results indicated that ethanol-induced caspase 3 activation was preceded by the transient elevation of sphingosine 1-phosphate (S1P) via sphingosine kinase 2 (SphK2) activation in the neonatal mouse brain. The neuroprotective effects of a SphK inhibitor, dimethylsphingosine, suggest the pro-apoptotic effect of S1P in this mouse model for fetal alcohol spectrum disorder.
J. Neurochem. (2012) 121, 700–704.AbstractTyrosine hydroxylase (TH) is the rate-limiting enzyme in norepinephrine synthesis, and its expression and activity are regulated by many factors in sympathetic neurons. Cytokines that act through gp130, such as ciliary neurotrophic factor (CNTF) decrease norepinephrine production in sympathetic neurons by suppressing TH mRNA and stimulating degradation of TH protein, leading to the loss of enzyme. Their effect on the activity of TH is unclear, but recent in vivo observations suggest that cytokines may stimulate TH activity. We investigated this issue by quantifying TH protein levels and activity in cultured sympathetic neurons. We also examined the state of TH phosphorylation on serine 31 and 40, sites known to affect TH activity and degradation. We found that CNTF, acting through gp130, stimulated the rate of l-3,4-dihydroxyphenylalanine production while at the same time decreasing TH enzyme levels, thereby increasing the specific activity of the enzyme. We also found that phosphorylation of TH on Ser31 was increased, and phosphorylation on Ser40 was decreased, after four days of CNTF exposure. Our data are consistent with previous findings that Ser31 phosphorylation stimulates TH activity, whereas Ser40 phosphorylation can target TH for proteasomal degradation.Inflammatory cytokines have contradictory effects on tyrosine hydroxylase Inflammatory cytokines like ciliary neurotrophic factor (CNTF), suppress tyrosine hydroxylase (TH) content in sympathetic neurons, but the loss of TH in vivo is not always accompanied by a similar loss of catecholamines. We found that while CNTF decreased TH levels in sympathetic neurons, it also stimulated enzyme activity. Thus, cytokines trigger the loss of TH protein while increasing the activity of remaining enzyme.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07724.xAbstractWe have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancer-driven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders.We have developed a simple method to differentiate ES and iPS cells into serotonergic neurons. A significant population of the cells became serotonergic by culturing on solidified Matrigel with noggin. ES cells carrying Pet-1-enhancer-driven EGFP were differentiated and sorted into ?80% pure cultures of serotonergic neurons. We believe that the present method will help to study the nature of serotonergic neurons and therapeutic perspectives for psychiatric disorders.
J. Neurochem. (2012) 121, 818–829.AbstractThe disruption of blood–spinal cord barrier (BSCB) after spinal cord injury (SCI) elicits an intensive local inflammation by the infiltration of blood cells such as neutrophils and macrophages, leading to cell death and permanent neurological disability. SCI activates matrix metalloprotease-9 (MMP-9), which is known to induce BSCB disruption. Here, we examined whether valproic acid (VPA), a histone deacetylase inhibitor, would attenuate BSCB disruption by inhibiting MMP-9 activity, leading to improvement of functional outcome after SCI. After moderate spinal cord contusion injury at T9, VPA (300 mg/kg) were immediately injected subcutaneously and further injected every 12 h for 5 days. Our data show that VPA inhibited MMP-9 activity after injury, and attenuated BSCB permeability and degradation of tight junction molecules such as occludin and ZO-1. In addition, VPA reduced the expression of inflammatory mediators including tumor necrosis factor-?. Furthermore, VPA increased the levels of acetylated histone 3, pAkt, and heat-shock protein 27 and 70, which have anti-apoptotic functions after SCI. Finally, VPA inhibited apoptotic cell death and caspase 3 activation, reduced the lesion volume and improved functional recovery after injury. Thus, our results demonstrated that VPA improves functional recovery by attenuating BSCB disruption via inhibition of MMP-9 activity after SCI.Matrix metalloprotease-9 (MMP-9), which is known to induce blood–spinal cord barrier (BSCB) disruption, is activated after spinal cord injury (SCI). In this study, we show that valproic acid (VPA), a histone deacetylase inhibitor, inhibited MMP-9 activity and thereby attenuated BSCB permeability, tight junction disruption, and blood infiltration after SCI. Our results suggest that VPA may provide effective therapeutic interventions for improving functional outcome by preventing BSCB disruption after SCI.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07729.xAbstractVesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein–protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, ?III and acetylated ?-tubulin, but not neurofilaments or the actin-binding protein drebrin, in in vitro-binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light-chain motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post-synaptic density. Moreover, in vitro-binding assays indicate that Nesca directly binds KIF5B, kinesin light-chain and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or the synaptosome-associated protein 25 kDa (SNAP-25) in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein receptors that are essential to exocytosis.Nesca is a novel adapter protein that is exclusively expressed in the nervous system. Until now, however, a functional role for Nesca has been unknown. Using cell biology, confocal microscopy and in vitro-binding studies, we show that Nesca is a novel microtubule-binding protein, that it binds the kinesin motor KIF5 and the synaptic membrane precursor protein syntaxin-1. These studies suggest that Nesca serves a role, similar to syntabulin, in the anterograde transport of synaptic membrane precursor proteins essential to exocytosis.
J. Neurochem. (2012) 121, 785–792.AbstractDelayed cerebral ischemia resulting from extracellular hemoglobin is an important determinant of outcome in subarachnoid hemorrhage. Hemoglobin is scavenged by the CD163-haptoglobin system in the circulation, but little is known about this scavenging pathway in the human CNS. The components of this system were analyzed in normal cerebrospinal fluid and after subarachnoid hemorrhage. The intrathecal presence of the CD163-haptoglobin–hemoglobin scavenging system was unequivocally demonstrated. The resting capacity of the CD163-haptoglobin–hemoglobin system in the normal CNS was 50 000-fold lower than that of the circulation. After subarachnoid hemorrhage, the intrathecal CD163-haptoglobin–hemoglobin system was saturated, as shown by the presence of extracellular hemoglobin despite detectable haptoglobin. Hemoglobin efflux from the CNS was evident, enabling rescue hemoglobin scavenging by the systemic circulation. Therefore, the CNS is not capable of dealing with significant intrathecal hemolysis. Potential therapeutic options to prevent delayed cerebral ischemia ought to concentrate on augmenting the capacity of the intrathecal CD163-haptoglobin–hemoglobin scavenging system and strategies to encourage hemoglobin efflux from the brain.This study was performed to examine the CD163-haptoglobin–hemoglobin system in the healthy human CNS and after subarachnoid hemorrhage. Compared with the circulation, the healthy human CNS has a very limited hemoglobin-scavenging capacity, which is clearly overwhelmed in subarachnoid hemorrhage. Results identify two potential therapeutic strategies to prevent delayed cerebral ischemia: inhibition of CD163 shedding and augmentation of Hb efflux from the brain.
J. Neurochem. (2012) 121, 751–762.AbstractAlzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognition. One of the hallmarks of AD is the accumulation of beta-amyloid (A?). Although endoplasmic reticulum stress, mitochondrial dysfunction, and oxidative stress have been implicated in A? toxicity, the molecular mechanism(s) of A?-induced neurotoxicity are not fully understood. In this study, we present evidence that the glia-derived stress protein metallothionein (MT) attenuates A?-induced neurotoxicity by unique mechanisms. MT expression was increased in brain astrocytes of a NSE-APPsw transgenic mouse model of AD. Astrocyte-derived MT protected N2a neuroblastoma cells and primary cortical neurons against A? toxicity with concurrent reduction of reactive oxygen species levels. MT reversed A?-induced down-regulation of Bcl-2 and survival signaling in neuroblastoma cells. Moreover, MT inhibited A?-induced proinflammatory cytokine production from microglia. The neurotoxicity of A?-stimulated microglia was significantly attenuated by MT-I. The results indicate that MT released from reactive astrocytes may antagonize A? neurotoxicity by direct inhibition of A? neurotoxicity and indirect suppression of neurotoxic microglial activation. These findings broaden the understanding of neurotoxic mechanisms of A? and the crosstalk between A? and MT in AD.Metallothionein-I (MT-I) modulates A?-induced neurotoxicity. This study demonstrates that astrocyte-derived MT-I under neurodegenerative conditions such as Alzheimer’s disease (AD) exerts neuroprotective effects via neuron-glia crosstalk. MT-I seems to be neuroprotective by modulating multiple events associated with A? pathology, such as inflammation, oxidative stress, and apoptosis. These findings may contribute to understanding of neurotoxic mechanisms of A? and suggest the therapeutic use of MT-I against AD.
J. Neurochem. (2012) 121, 774–784.AbstractPyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer’s disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-? protein A?37, A?38, A?40, A?42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-A?3-x, pGlu-ADan) or possess the intact N-terminus (A?1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified A?38, ??40 and ??42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-A?3-42 showing the highest effect. Among the unmodified A? peptides, only A?1-42 exhibites such propensity, which was similar to pGlu-A?3-38 and pGlu-A?3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of A?1-x, A?3-x or N-truncated pGlu-A?3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.Neurodegenerative disorders such as Alzheimer’s disease (AD) are characterized by progressive accumulation of N-truncated and pyroglutamate (pGlu)-modified amyloid peptides. Oligomers formed from pGlu-modified A? display significantly enhanced potential to influence hippocampal LTP. The effect correlates with velocity of oligomerization and surface hydrophobicity. We conclude: oligomers of N-terminally modified A?38, 40 and 42 potently influence neuronal function. Thereby, pGlu-modified A? probably contributes to AD symptomatology.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07705.xAbstractAlthough multiple biochemical pathways produce adenosine, studies suggest that the 2?,3?-cAMP-adenosine pathway (2?,3?-cAMP?2?-AMP/3?-AMP?adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2?,3?-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2?,3?-cAMP, 3?,5?-cAMP, 2?-AMP, 3?-AMP, or 5?-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2?,3?-cAMP increased 2?-AMP, 3?-AMP and adenosine, and 3?,5?-cAMP increased 5?-AMP and adenosine. In both brain regions, 2?-AMP, 3-AMP and 5?-AMP were converted to adenosine. Microdialysis experiments in 2?,3?-cyclic nucleotide-3?-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (controlled cortical impact model) activated the brain 2?,3?-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2?,3?-cAMP to 2?-AMP and to adenosine. In CSF from traumatic brain injury patients, 2?,3?-cAMP was significantly increased in the initial 12 h after injury and strongly correlated with CSF levels of 2?-AMP, 3?-AMP, adenosine and inosine. We conclude that in vivo, 2?,3?-cAMP is converted to 2?-AMP/3?-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans.New insights into the neurochemistry of brain injury Although ‘cAMP’ usually refers to ‘3?,5?-cAMP’, recent studies identify a positional isomer of 3?,5?-cAMP, namely 2?,3?-cAMP. We performed this study to determine whether the brain converts 2?,3?-cAMP to its corresponding AMPs (2?-AMP and 3?-AMP) and metabolizes these AMPs to adenosine. Our results support the concept of a brain ‘2?,3?-cAMP-adenosine pathway’ that is activated by brain injury, mediated in part by CNPase and provides protection against brain injury. These findings provide new insights regarding a neurochemical mechanism that protects brains from injury.
J. Neurochem. (2012) 121, 763–773.Abstract?-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step in the proteolysis of the APP to the amyloid ?-protein (A?), a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, the regulation of which is not well understood. We identified a G-rich sequence within exon 3 of BACE1 involved in controlling splice site selection. Mutation of the G-rich sequence decreased use of the normal 5? splice site of exon 3, which leads to full-length and proteolytically active BACE1, and increased use of an alternative splice site, which leads to a shorter, essentially inactive isoform. Nuclease protection assays, nuclear magnetic resonance, and circular dichroism spectroscopy revealed that this sequence folds into a G-quadruplex structure. Several proteins were identified as capable of binding to the G-rich sequence, and one of these, heterogeneous nuclear ribonucleoprotein H, was found to regulate BACE1 exon 3 alternative splicing and in a manner dependent on the G-rich sequence. Knockdown of heterogeneous nuclear ribonucleoprotein H led to a decrease in the full-length BACE1 mRNA isoform as well as a decrease in A? production from APP, suggesting new possibilities for therapeutic approaches to Alzheimer’s disease.Messenger Origami: BACE1 mRNA Structure Controls Splicing Alternative splicing of pre-mRNA of BACE1 (aka, beta-secretase) can affect levels of active protease and, thereby, production of the amyloid ?-protein (A?). A G-quadruplex structure in exon 3 and interaction with hnRNP H protein helps regulate BACE1 splicing. Targeting this regulatory mechanism can lower A? levels, a major therapeutic goal for Alzheimer’s disease.
J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07676.xAbstractParkinson’s disease (PD) is the second most common neurodegenerative disorders with a variable combination of motor and non-motor symptoms. Mutations in several genes including ATP13A2 (PARK9) are reported to be associated with PD. The underlying mechanism of PD is not well defined, however, both genetic and environmental causes contribute to it. ATP13A2 gene locates in chromosome 1 and contains 29 exons encoding for a protein of 1180 amino acids with 10 transmembrane domains. Abnormal gene expression has been implicated in neurodegenerative disorders. The transcriptional regulation of the ATP13A2 gene is unknown. In this report, we cloned and functionally characterized the human ATP13A2 gene promoter. We showed that the promoter region of the human ATP13A2 gene contains hypoxia response elements which can bind to transcription factor hypoxia-inducible factor 1? (HIF-1?). Hypoxia up-regulated ATP13A2 transcription via HIF-1? in HEK293 and dopaminergic MN9D cells. Our study indicates that hypoxia signaling plays a very important role in the regulation of human ATP13A2 gene expression. Further study is needed to determine the role of hypoxia in the pathogenesis of PD and its interaction with other PD causative genes, which will provide insights to the role of hypoxia and dysregulation of gene expression in Parkinson’s disease.This study aimed to define the molecular mechanism underlying transcriptional regulation of a Parkinson disease-associated ATP13A2 gene. We cloned and functionally characterized the human ATP13A2 gene promoter, and found that hypoxia up-regulated ATP13A2 transcription via HIF-1? in HEK293 and dopaminergic MN9D cells. Our study indicates that hypoxia signaling plays a very important role in the regulation of human ATP13A2 gene expression, and further study is needed to determine the role of hypoxia in the pathogenesis of PD and its interaction with other PD causative genes.
J. Neurochem. (2012) 121, 717–729.AbstractCerebellin precursor protein (Cbln1) is essential for synapse integrity in cerebellum through assembly into complexes that bridge pre-synaptic ?-neurexins (Nrxn) to post-synaptic GluR?2. However, GluR?2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluR?2 and Nrxns1-3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, (deleted in colorectal cancer (DCC) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln null mice. Thus, Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less well-characterized role of netrin/DCC in adult brain.Cbln1 binds to neurexins and GluR?2 on pre- and post-synaptic membranes, respectively to stabilize synapses. We show that another family member, Cbln4 binds weakly or not at all to these receptors and cbln4-null mice lack the synaptic defects seen in cbln1-knockout mice. However, Cbln4 binds uniquely to DCC in a netrin-displaceable manner suggesting it functions via this receptor in brain.
J. Neurochem. (2012) 121, 705–716.AbstractGlutamate receptor (GluR) ?1 is widely expressed in the developing forebrain, whereas GluR?2 is selectively expressed in cerebellar Purkinje cells. Recently, we found that trans-synaptic interaction of postsynaptic GluR?2 and pre-synaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum. Thus, a question arises whether GluR?1 regulates synapse formation in the forebrain. In this study, we showed that the N-terminal domain of GluR?1 induced inhibitory presynaptic differentiation of some populations of cultured cortical neurons. When Cbln1 or Cbln2 was added to cultures, GluR?1 expressed in HEK293T cells induced preferentially inhibitory presynaptic differentiation of cultured cortical neurons. The synaptogenic activity of GluR?1 was suppressed by the addition of the extracellular domain of NRXN1? or NRXN1? containing splice segment 4. Cbln subtypes directly bound to the N-terminal domain of GluR?1. The synaptogenic activity of GluR?1 in the presence of Cbln subtypes correlated well with their binding affinities. When transfected to cortical neurons, GluR?1 stimulated inhibitory synapse formation in the presence of Cbln1 or Cbln2. These results together with differential interactions of Cbln subtypes with NRXN variants suggest that GluR?1 induces preferentially inhibitory presynaptic differentiation of cortical neurons by interacting with NRXNs containing splice segment 4 through Cbln subtypes.Precise synaptic connections between neurons in the brain provide the basis of perception, learning, memory and cognition. Trans-synaptic interaction of glutamate receptor (GluR) ?2 and neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum. Here, we show that GluR?1 preferentially induces inhibitory pre-synaptic differentiation of some populations of cortical neurons by interacting with NRXNs through Cblns.
J. Neurochem. (2012) 10.1111/j.1471-4159.2011.07622.xAbstractGABAergic transmission in the neostriatum plays a central role in motor coordination, in which a plethora of GABA-A receptor subunits combine to modulate neural inhibition. GABA? receptors were originally described in the mammalian retina. These receptors possess special electrophysiological and pharmacological properties, forming a characteristic class of ionotropic receptors. In previous studies, we suggested that GABA? receptors are expressed in the neostriatum, and in this report we show that they are indeed present in all the calretinin-positive interneurons of the neostriatum. In addition, they are located in calbindin-positive interneurons and projection neurons that express the dopamine D2 receptor. GABA? receptors were also located in 30% of the glial fibrillary acidic protein-positive cells, and may therefore also contribute to gliotransmission. Quantitative reverse transcription-PCR suggested that the mRNAs of this receptor do not express as much as in the retina, and that GABA?2 is more abundant than GABA?1. Electrophysiological recordings in brain slices provided evidence of neurons expressing a cis-4-aminocrotonic acid-activated, 1,2,5,6-tetrahydropyridine-4-yl methylphosphinic acid-sensitive ionotropic GABA receptor, indicating the presence of functional GABA? receptors in the neostriatum. Finally, electron-microscopy and immunogold located the receptors mainly in perisynaptic as well as in extrasynaptic sites. All these observations reinforce the importance of GABA? receptors in the neostriatum and contribute to the diversity of inhibitory regulation in this area.GABA? receptors in neurons and astrocytes of striatum GABA transmission plays a central role in the interneurons of the striatum. We aimed to investigate whether GABA? receptors, which are bicuculline-resistant and do not desensitize are expressed in this area. We found that GABA? are present in calretinin neurons and astrocyes, and although the receptors are sensitive to TPMPA, they most likely form heteromeric complexes with other GABA subunits.
J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07524.xAbstractFOXP2, a forkhead box-containing transcription factor, forms homo- or hetero-dimers with FOXP family members and localizes to the nucleus, while FOXP2(R553H), which contains a mutation related to speech/language disorders, features reduced DNA binding activity and both cytoplasmic and nuclear localization. In addition to being a loss-of-function mutation, it is possible that FOXP2(R553H) also may act as a gain-of-function mutation to inhibit the functions of FOXP2 isoforms including FOXP2Ex10+ lacking forkhead domain. Foxp2(R552H) knock-in mouse pups exhibit impaired ultrasonic vocalization and poor dendritic development in Purkinje cells. However, expressions of Foxp2 isoforms in the developing Purkinje are unclear. The appearance of ‘apical cytoplasmic swelling’ (mitochondria-rich regions that are the source of budding processes) correlates with dendritic development of Purkinje cells. In the present study, we focused on Foxp2 isoforms localizing to the apical cytoplasmic swelling and identified two isoforms lacking forkhead domain: Foxp2Ex12+ and Foxp2Ex15. They partly localized to the membrane fraction that includes mitochondria. Foxp2Ex12+ mainly localized to the apical cytoplasmic swelling in early developing Purkinje cells at the stellate stage (P2–P4). Mitochondrial localization of Foxp2Ex12+ in Purkinje cells was confirmed by immune-electron microscopic analysis. Foxp2Ex12+ may play a role in dendritic development in Purkinje cells.Foxp2 isoform lacking the forkhead domain are important for developing Purkinje cells Expressions of Foxp2 isoforms in developing Purkinje cells are unclear. Here, two isoforms lacking forkhead domain, Foxp2Ex12+ and Foxp2Ex15+, were identified. Foxp2Ex12+ localized to perisomatic protrusions in early developing Purkinje cells and to the apical cytoplasmic swelling, mitochondria-rich regions that are the source of budding processes. These findings suggest that Foxp2Ex12+ may play a role in dendritic development in Purkinje cells.
J. Neurochem. (2012) 121, 730–737.AbstractHomeostasis of the brain is dependent on the blood–brain barrier (BBB). This barrier tightly regulates the exchange of essential nutrients and limits the free flow of immune cells into the CNS. Perturbations of BBB function and the loss of its immune quiescence are hallmarks of a variety of brain diseases, including multiple sclerosis (MS), vascular dementia, and stroke. In particular, diapedesis of monocytes and subsequent trafficking of monocyte-derived macrophages into the brain are key mediators of demyelination and axonal damage in MS. Endothelin-1 (ET-1) is considered as a potent pro-inflammatory peptide and has been implicated in the development of cardiovascular diseases. Here, we studied the role of different components of the endothelin system, i.e., ET-1, its type B receptor (ETB) and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis of a human brain endothelial cell barrier. Our pharmacological inhibitory and specific gene knockdown studies point to a regulatory function of these proteins in transendothelial passage of monocytes. Results from this study suggest that the endothelin system is a putative target within the brain for anti-inflammatory treatment in neurological diseases.The endothelin system, a potential target for anti-inflammation treatment of the brain: the brain endothelial cell barrier is implicated in several neurological diseases, including multiple sclerosis (MS), vascular dementia and stroke. The endothelin system in brain endothelial cells is distributed over different cellular compartments and regulates the passage of monocytes. The results of this study yield new insights into monocyte diapedesis over the barrier, pointing to a set of putative therapeutic targets for anti-inflammation treatment in neurological diseases such as MS, vascular dementia and stroke.
We have developed a thin layer chromatography (TLC)-Blot system that makes possible the direct analysis of blotted glycosphingolipids on a PVDF membrane from a high-performance TLC (HPTLC) plate by immunological staining, chemical staining, enzymatic treatment and mass spectrometric (MS) analysis. An ion trap type matrix-assisted laser desorption/ionization-time of flight (MALDI-QIT-TOF) MS apparatus improved not only the molecular identification but also the analysis of molecular species of lipids on the PVDF membrane. A new approach for glyco- and lipidomics, molecular scanning technology by a combination of TLC-Blot and MALDI-QIT-TOF MS, was developed and applied to human brain gangliosides separated from the tissues of patients with neural diseases and control patients. The results clearly showed a change of ganglioside composition, in addition to identifying individual ganglioside molecular species, in the hippocampus gray matter of patients with Alzheimer’s disease. The results strongly suggested that metabolic changes of gangliosides played an important role in the progression of this disease. The present technology with molecular imaging should provide valuable information for elucidating the significance of molecular species in neuronal functions such as neural transmission, memory, and learning.
Lysophosphatidic acid receptor (LPA1) signaling initiates neuropathic pain through demyelination of the dorsal root (DR). Although LPA is found to cause down-regulation of myelin proteins underlying demyelination, the detailed mechanism remains to be determined. In the present study, we found that a single intrathecal (i.t.) injection of LPA evoked a dose- and time-dependent down-regulation of myelin-associated glycoprotein (MAG) in the DR through LPA1 receptor. A similar event was also observed in ex vivo DR cultures. Interestingly, LPA-induced down-regulation of MAG was significantly inhibited by calpain inhibitors (calpain inhibitor X, E-64 and E-64d) and LPA markedly induced calpain activation in the DR. The pre-treatment with calpain inhibitors attenuated LPA-induced neuropathic pain behaviors such as hyperalgesia and allodynia. Moreover, we found that sciatic nerve injury activates calpain activity in the DR in a LPA1 receptor-dependent manner. The E-64d treatments significantly blocked nerve injury-induced MAG down-regulation and neuropathic pain. However, there was no significant calpain activation in the DR by complete Freund’s adjuvant treatment, and E-64d failed to show anti-hyperalgesic effects in this inflammation model. The present study provides strong evidence that LPA-induced calpain activation plays a crucial role in the manifestation of neuropathic pain through MAG down-regulation in the DR.
Nicotinic acetylcholine receptors (nAChRs) transmit the agonist signal to the channel gate through a number of extracellular domains. We have previously shown that particular details of the process of coupling binding to gating could be quantitative and qualitatively different in muscle and neuronal type nAChRs. We have extended previous studies on homomeric ?7 nAChRs to heteromeric ?3?4 nAChRs, by mutating residues located at loops 2 and 7, and M2-M3 linker of both ?3 and ?4 subunits which, in order to monitor surface expression, were modified to bind ?-bungarotoxin, and expressed in Xenopus oocytes. We show that, in general, mutations in these domains of both ?3 and ?4 subunits affect the gating function, although the effects are slightly larger if they are inserted in the ?3 subunit. However, the involvement of a previously reported intrasubunit interaction in coupling (Gln48-Ile130) seems to be restricted to the ?4 subunit. We also show that mutations at these domains, particularly loop 2 of the ?3 subunit, change the pharmacological profile of ?3?4 nAChRs, decreasing nicotine’s and increasing cytisine’s effectiveness relative to ACh. It is concluded that, unlike muscle nAChRs, the non-? subunits play a relevant role in the coupling process of neuronal ?3?4 nAChRs.
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