This study investigates the effects of ethanol on neuronal and astroglial metabolism using 1H‐[13C]‐NMR spectroscopy in conjunction with infusion of [1,6‐13C2]/[1‐13C]glucose or [2‐13C]acetate, respectively. A three‐compartment metabolic model was fitted to the 13C turnover of GluC3, GluC4, GABAC2, GABAC3, AspC3, and GlnC4 from [1,6‐13C2]glucose to determine the rates of tricarboxylic acid (TCA) and neurotransmitter cycle associated with glutamatergic and GABAergic neurons. The ratio of neurotransmitter cycle to TCA cycle fluxes for glutamatergic and GABAegic neurons was obtained from the steady‐state [2‐13C]acetate experiment and used as constraints during the metabolic model fitting. 1H MRS measurement suggests that depletion of ethanol from cerebral cortex follows zero order kinetics with rate 0.18 ± 0.04 μmol/g/min. Acute exposure of ethanol reduces the level of glutamate and aspartate in cortical region. GlnC4 labeling was found to be unchanged from a 15 min infusion of [2‐13C]acetate suggesting that acute ethanol exposure does not affect astroglial metabolism in naive mice. Rates of TCA and neurotransmitter cycle associated with glutamatergic and GABAergic neurons were found to be significantly reduced in cortical and subcortical regions. Acute exposure of ethanol perturbs the level of neurometabolites and decreases the excitatory and inhibitory activity differentially across the regions of brain.
Munc13‐1 is a pre‐synaptic active‐zone protein essential for neurotransmitter release and involved in pre‐synaptic plasticity in brain. Ethanol, butanol, and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13‐1 with EC50s of 52 mM, 26 mM, and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13‐1 C1 exclusively at Glu‐582, which was identified by mass spectrometry. Mutation of Glu‐582 to alanine, leucine, and histidine reduced the alcohol binding two‐ to five‐fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild‐type Munc13‐1 compared with the mutants. If Munc13‐1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss‐of‐function mutation in the conserved Dunc‐13 in Drosophila melanogaster. The Dunc‐13P84200/+ heterozygotes have 50% wild‐type levels of Dunc‐13 mRNA and display a very robust increase in ethanol self‐administration. This phenotype is reversed by the expression of the rat Munc13‐1 protein within the Drosophila nervous system. The present studies indicate that Munc13‐1 C1 has binding site(s) for alcohols and Munc13‐1 activity is sufficient to restore normal self‐administration to Drosophila mutants deficient in Dunc‐13 activity.
Energy metabolism supports both inhibitory and excitatory neurotransmission processes. This study investigated the specific contribution of astrocytic metabolism to γ‐aminobutyric acid (GABA) synthesis and inhibitory GABAergic neurotransmission that remained to be ilucidated in vivo. Therefore, we measured 13C incorporation into brain metabolites by dynamic 13C nuclear magnetic resonance spectroscopy at 14.1 T in rats under α‐chloralose anaesthesia during infusion of [1,6‐13C]glucose. The enhanced sensitivity at 14.1 T allowed to quantify incorporation of 13C into the three aliphatic carbons of GABA non‐invasively. Metabolic fluxes were determined with a mathematical model of brain metabolism comprising glial, glutamatergic and GABAergic compartments. GABA synthesis rate was 0.11 ± 0.01 μmol/g/min. GABA‐glutamine cycle was 0.053 ± 0.003 μmol/g/min and accounted for 22 ± 1% of total neurotransmitter cycling between neurons and glia. Cerebral glucose oxidation was 0.47 ± 0.02 μmol/g/min, of which 35 ± 1% and 7 ± 1% was diverted to the glutamatergic and GABAergic tricarboxylic acid cycles, respectively. The remaining fraction of glucose oxidation was in glia, where 12 ± 1% of the TCA cycle flux was dedicated to oxidation of GABA. 16 ± 2% of glutamine synthesis was provided to GABAergic neurons. We conclude that substantial metabolic activity occurs in GABAergic neurons and that glial metabolism supports both glutamatergic and GABAergic neurons in the living rat brain.
Angiotensin‐(1‐7) [Ang‐(1‐7)] is an alternative product of the brain renin‐angiotensin system that exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We previously identified a peptidase that metabolizes Ang‐(1‐7) to the inactive metabolite product Ang‐(1‐4) in CSF of adult sheep. This study purified the peptidase 1445‐fold from sheep brain medulla and characterized this activity. The peptidase was sensitive to the chelating agents o‐phenanthroline and EDTA, as well as the mercury compound p‐chloromercuribenzoic acid (PCMB). Selective inhibitors to angiotensin‐converting enzyme, neprilysin, neurolysin, and thimet oligopeptidase did not attenuate activity; however, the metallopeptidase agent JMV‐390 was a potent inhibitor of Ang‐(1‐7) hydrolysis (Ki = 0.8 nM). Kinetic studies using 125I‐labeled Ang‐(1‐7), Ang II, and Ang I revealed comparable apparent Km values (2.6, 2.8, and 4.3 μM, respectively), but a higher apparent Vmax for Ang‐(1‐7) (72 vs. 30 and 6 nmol/min/mg, respectively; p < 0.01). HPLC analysis of the activity confirmed the processing of unlabeled Ang‐(1‐7) to Ang‐(1‐4) by the peptidase, but revealed < 5% hydrolysis of Ang II or Ang I, and no hydrolysis of neurotensin, bradykinin or apelin‐13. The unique characteristics of the purified neuropeptidase may portend a novel pathway to influence actions of Ang‐(1‐7) within the brain.
Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo13C NMR spectroscopy coupled with the infusion labeled glial‐specific substrate, such as acetate. In this study, we infused alpha‐chloralose anesthetized rats with [2‐13C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using 1H‐[13C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two‐compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (Vg) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (VNT) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (VPC) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (Vxg and Vxn) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations.
Although numerous positron emission tomography (PET) studies with 18F‐fluoro‐deoxyglucose (FDG) have reported quantitative results on cerebral glucose kinetics and consumption, there is a large variation between the absolute values found in the literature. One of the underlying causes is the inconsistent use of the lumped constants (LCs), the derivation of which is often based on multiple assumptions that render absolute numbers imprecise and errors hard to quantify. We combined a kinetic FDG‐PET study with magnetic resonance spectroscopic imaging (MRSI) of glucose dynamics in Sprague–Dawley rats to obtain a more comprehensive view of brain glucose kinetics and determine a reliable value for the LC under isoflurane anaesthesia. Maps of Tmax/CMRglc derived from MRSI data and Tmax determined from PET kinetic modelling allowed to obtain an LC‐independent CMRglc. The LC was estimated to range from 0.33 ± 0.07 in retrosplenial cortex to 0.44 ± 0.05 in hippocampus, yielding CMRglc between 62 ± 14 and 54 ± 11 μmol/min/100 g, respectively. These newly determined LCs for four distinct areas in the rat brain under isoflurane anaesthesia provide means of comparing the growing amount of FDG‐PET data available from translational studies.
Depression is one of the most debilitating neuropsychiatric disorders. Most of the current antidepressants have long remission time and low recovery rate. This study explores the impact of ketamine on neuronal and astroglial metabolic activity in prefrontal cortex in a social defeat (SD) model of depression. C57BL/6 mice were subjected to a social defeat paradigm for 5 min a day for 10 consecutive days. Ketamine (10 mg/kg, intraperitoneal) was administered to mice for two consecutive days following the last defeat stress. Mice were infused with [1,6‐13C2]glucose or [2‐13C]acetate to assess neuronal and astroglial metabolic activity, respectively, together with proton‐observed carbon‐edited nuclear magnetic resonance spectroscopy in prefrontal cortex tissue extract. The 13C labeling of amino acids from glucose and acetate was decreased in SD mice. Ketamine treatment in SD mice restored sucrose preference, social interaction and immobility time to control values. Acute subanesthetic ketamine restored the 13C labeling of brain amino acids from glucose as well as acetate in SD mice to the respective control values, suggesting that rates of neuronal and astroglial tricarboxylic acid (TCA) cycle and neurotransmitter cycling were re‐established to normal levels. The finding of improved energy metabolism in SD mice suggests that fast anti‐depressant action of ketamine is linked with improved neurotransmitter cycling.
We developed the novel positron emission tomography (PET) ligand 2‐[5‐(4‐[11C]methoxyphenyl)‐2‐oxo‐1,3‐benzoxazol‐3(2H)‐yl]‐N‐methyl‐N‐phenylacetamide ([11C]MBMP) for translocator protein (18 kDa, TSPO) imaging and evaluated its efficacy in ischemic rat brains. [11C]MBMP was synthesized by reacting desmethyl precursor ( 1 ) with [11C]CH3I in radiochemical purity of ≥ 98% and specific activity of 85 ± 30 GBq/μmol (n = 18) at the end of synthesis. Biodistribution study on mice showed high accumulation of radioactivity in the TSPO‐rich organs, e.g., the lungs, heart, kidneys, and adrenal glands. The metabolite analysis in mice brain homogenate showed 80.1 ± 2.7% intact [11C]MBMP at 60 min after injection. To determine the specific binding of [11C]MBMP with TSPO in the brain, in vitro autoradiography and PET studies were performed in an ischemic rat model. In vitro autoradiography indicated significantly increased binding on the ipsilateral side compared with that on the contralateral side of ischemic rat brains. This result was supported firmly by the contrast of radioactivity between the ipsilateral and contralateral sides in PET images. Displacement experiments with unlabelled MBMP or PK11195 minimized the difference in uptake between the two sides. In summary, [11C]MBMP is a potential PET imaging agent for TSPO and, consequently, for the up‐regulation of microglia during neuroinflammation.
The 13C‐labeling patterns in glutamate and glutamine from brain tissue are quite different after infusion of a mixture of 13C‐enriched glucose and acetate. Two processes contribute to this observation, oxidation of acetate by astrocytes but not neurons, and preferential incorporation of α‐ketoglutarate into glutamate in neurons, and incorporation of α‐ketoglutarate into glutamine in astrocytes. The acetate:glucose ratio, introduced previously for analysis of a single 13C NMR spectrum, provides a useful index of acetate and glucose oxidation in the brain tissue. However, quantitation of relative substrate oxidation at the cell compartment level has not been reported. A simple mathematical method is presented to quantify the ratio of acetate‐to‐glucose oxidation in astrocytes, based on the standard assumption that neurons do not oxidize acetate. Mice were infused with [1,2‐13C]acetate and [1,6‐13C]glucose, and proton decoupled 13C NMR spectra of cortex extracts were acquired. A fit of those spectra to the model indicated that 13C‐labeled acetate and glucose contributed approximately equally to acetyl‐CoA (0.96) in astrocytes. As this method relies on a single 13C NMR spectrum, it can be readily applied to multiple physiologic and pathologic conditions.
Neuro‐2a (N2a) neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine polyphosphates. The ApnA‐cleaving activity has been analysed with the use of the fluorogenic compound BODIPY® FL guanosine 5′‐O‐(3‐thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a Km value of 4.9 ± 1.3 μM. Diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, and the nucleoside monophosphate AMP behaved as an inhibitor of BODIPY® FL guanosine 5′‐O‐(3‐thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide pyrophosphatase/phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn2+ ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal‐like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation.
Most ingested ethanol is metabolized in the liver to acetaldehyde and then to acetate, which can be oxidized by the brain. This project assessed whether chronic exposure to alcohol can increase cerebral oxidation of acetate. Through metabolism, acetate may contribute to long‐term adaptation to drinking. Two groups of adult male Sprague–Dawley rats were studied, one treated with ethanol vapor and the other given room air. After 3 weeks the rats received an intravenous infusion of [2‐13C]ethanol via a lateral tail vein for 2 h. As the liver converts ethanol to [2‐13C]acetate, some of the acetate enters the brain. Through oxidation the 13C is incorporated into the metabolic intermediate α‐ketoglutarate, which is converted to glutamate (Glu), glutamine (Gln), and GABA. These were observed by magnetic resonance spectroscopy and found to be 13C‐labeled primarily through the consumption of ethanol‐derived acetate. Brain Gln, Glu, and, GABA 13C enrichments, normalized to 13C‐acetate enrichments in the plasma, were higher in the chronically treated rats than in the ethanol‐naïve rats, suggesting increased cerebral uptake and oxidation of circulating acetate. Chronic ethanol exposure increased incorporation of systemically derived acetate into brain Gln, Glu, and GABA, key neurochemicals linked to brain energy metabolism and neurotransmission.
Our study was aimed at investigating whether complement, a complex of soluble and membrane‐associated serum proteins, could, in addition to its well‐documented post‐synaptic activity, also pre‐synaptically affect the release of classic neurotransmitters in central nervous system (CNS). Complement (dilution 1 : 10 to 1 : 10000) elicited the release of preloaded [3H]‐d ‐aspartate ([3H]d ‐ASP) and endogenous glutamate from mouse cortical synaptosomes in a dilution‐dependent manner. It also evoked [3H]d ‐ASP release from mouse hippocampal, cerebellar, and spinal cord synaptosomes, as well as from rat and human cortical nerve endings, but left unaltered the release of GABA, [3H]noradrenaline or [3H]acetylcholine. Lowering external Na+ (from 140 to 40 mM) or Ca2+ (from 1.2 to 0.1 mM) ions prevented the 1 : 300 complement‐evoked [3H]d ‐ASP release from mouse cortical synaptosomes. Complement‐induced releasing effect was unaltered in synaptosomes entrapped with the Ca2+ ions chelator 1,2‐bis‐(2‐aminophenoxy) ethane‐N,N,N’,N’, tetra‐acetic acid or with pertussis toxin. Nifedipine,/ω‐conotoxin GVIA/ω‐conotoxin MVIIC mixture as well as the vesicular ATPase blocker bafilomycin A1 were also inefficacious. The excitatory amino acid transporter blocker DL‐threo‐ß‐benzyloxyaspartic acid, on the contrary, reduced the complement‐evoked releasing effect in a concentration‐dependent manner. We concluded that complement‐induced releasing activity is restricted to glutamatergic nerve endings, where it was accounted for by carrier‐mediated release. Our observations afford new insights into the molecular events accounting for immune and CNS crosstalk.
Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia‐induced seizures on adenosine A1 and A2A receptors and 5′‐nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real‐time PCR, and 5′‐nucleotidase activity assays. Hyperthermic seizures were induced in 13‐day‐old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [3H]DPCPX as radioligand, and mRNA coding A1 was observed 48 h after hyperthermia‐induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [3H]ZM241385 as radioligand, 48 h after hyperthermia‐evoked convulsions. These short‐term changes in A1 and A2A receptors were also accompanied by a loss of 5′‐nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5′‐nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A1 and A2A receptors and loss of 5′‐nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures.
Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)‐hydroxycholesterol, 24(S)OH‐C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin‐cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH‐C. Proteoliposomes containing apolipoprotein A‐I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A‐I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH‐C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH‐C esterification. When highly neurotoxic 24(S)OH‐C was treated with enzyme and proteoliposomes before incubation with differentiated SH‐SY5Y cells, the neuron survival improved. The esters of 24(S)OH‐C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH‐C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH‐C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol‐induced neurotoxic injuries to neurons in culture.
G protein‐coupled estrogen receptor (GPER) is a relatively recently identified non‐nuclear estrogen receptor, expressed in several tissues, including brain and blood vessels. The mechanisms elicited by GPER activation in brain microvascular endothelial cells are incompletely understood. The purpose of this work was to assess the effects of GPER activation on cytosolic Ca2+ concentration, [Ca2+]i, nitric oxide production, membrane potential and cell nanomechanics in rat brain microvascular endothelial cells (RBMVEC). Extracellular but not intracellular administration of G‐1, a selective GPER agonist, or extracellular administration of 17‐β‐estradiol and tamoxifen, increased [Ca2+]i in RBMVEC. The effect of G‐1 on [Ca2+]i was abolished in Ca2+‐free saline or in the presence of a L‐type Ca2+ channel blocker. G‐1 increased nitric oxide production in RBMVEC; the effect was prevented by NG‐nitro‐l ‐arginine methyl ester. G‐1 elicited membrane hyperpolarization that was abolished by the antagonists of small and intermediate‐conductance Ca2+‐activated K+ channels, apamin, and charibdotoxin. GPER‐mediated responses were sensitive to G‐36, a GPER antagonist. In addition, atomic force microscopy studies revealed that G‐1 increased the modulus of elasticity, indicative of cytoskeletal changes and increase in RBMVEC stiffness. Our results unravel the mechanisms underlying GPER‐mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature.
(R)‐3‐[2,6‐cis‐Di(4‐methoxyphenethyl)piperidin‐1‐yl]propane‐1,2‐diol (GZ‐793A) inhibits methamphetamine‐evoked dopamine release from striatal slices and methamphetamine self‐administration in rats. GZ‐793A potently and selectively inhibits dopamine uptake at the vesicular monoamine transporter‐2 (VMAT2). This study determined GZ‐793A's ability to evoke [3H]dopamine release and inhibit methamphetamine‐evoked [3H]dopamine release from isolated striatal synaptic vesicles. Results show GZ‐793A concentration‐dependent [3H]dopamine release; nonlinear regression revealed a two‐site model of interaction with VMAT2 (High‐ and Low‐EC50 = 15.5 nM and 29.3 μM, respectively). Tetrabenazine and reserpine completely inhibited GZ‐793A‐evoked [3H]dopamine release, however, only at the High‐affinity site. Low concentrations of GZ‐793A that interact with the extravesicular dopamine uptake site and the High‐affinity intravesicular DA release site also inhibited methamphetamine‐evoked [3H]dopamine release from synaptic vesicles. A rightward shift in the methamphetamine concentration‐response was evident with increasing concentrations of GZ‐793A, and the Schild regression slope was 0.49 ± 0.08, consistent with surmountable allosteric inhibition. These results support a hypothetical model of GZ‐793A interaction at more than one site on the VMAT2 protein, which explains its potent inhibition of dopamine uptake, dopamine release via a High‐affinity tetrabenazine‐ and reserpine‐sensitive site, dopamine release via a Low‐affinity tetrabenazine‐ and reserpine‐insensitive site, and a low‐affinity interaction with the dihydrotetrabenazine binding site on VMAT2. GZ‐793A inhibition of the effects of methamphetamine supports its potential as a therapeutic agent for the treatment of methamphetamine abuse.
The nucleus accumbens is highly heterogeneous, integrating regionally distinct afferent projections and accumbal interneurons, resulting in diverse local microenvironments. Dopamine (DA) neuron terminals similarly express a heterogeneous collection of terminal receptors that modulate DA signaling. Cyclic voltammetry is often used to probe DA terminal dynamics in brain slice preparations; however, this method traditionally requires electrical stimulation to induce DA release. Electrical stimulation excites all of the neuronal processes in the stimulation field, potentially introducing simultaneous, multi‐synaptic modulation of DA terminal release. We used optogenetics to selectively stimulate DA terminals and used voltammetry to compare DA responses from electrical and optical stimulation of the same area of tissue around a recording electrode. We found that with multiple pulse stimulation trains, optically stimulated DA release increasingly exceeded that of electrical stimulation. Furthermore, electrical stimulation produced inhibition of DA release across longer duration stimulations. The GABAB antagonist, CGP 55845, increased electrically stimulated DA release significantly more than light stimulated release. The nicotinic acetylcholine receptor antagonist, dihydro‐β‐erythroidine hydrobromide, inhibited single pulse electrically stimulated DA release while having no effect on optically stimulated DA release. Our results demonstrate that electrical stimulation introduces local multi‐synaptic modulation of DA release that is absent with optogenetically targeted stimulation.
Cerebrospinal fluid (CSF) α‐synuclein (ASYN) levels are emerging as a possible biomarker in a number of neurodegenerative conditions; however, there has been little study of such levels in demyelinating conditions with neurodegeneration such as multiple sclerosis (MS). In this study, we aimed to assess CSF ASYN levels in MS spectrum [clinically isolated syndrome (CIS) and MS] patients and compare them to those obtained in control subjects with benign neurological conditions (BNC). We used a recently developed, ultra‐sensitive sandwich enzyme‐linked immunosorbent assay to measure and compare CSF ASYN levels in three categories of subjects: BNC (n = 38), CIS (n = 36) and MS [Relapsing Remitting (RRMS, n = 22) and Primary Progressive (PPMS, n = 15)]. We also performed secondary analyses, including relationship of CSF ASYN levels to aging, gender, presence of CSF oligoclonal bands (OB) and gadolinium (Gd)‐enhancing demyelinating lesions on T1‐weighted MRIs. CSF ASYN levels were found to be significantly lower in the CIS (78.2 ± 7.5 pg/mL), RRMS (76.8 ± 5.1 pg/mL), and PPMS (76.3 ± 6.7 pg/mL) groups compared to the BNC (125.7 ± 13.6 pg/mL) group. Secondary analyses did not reveal additional correlations. Our results suggest that in a cohort of CIS and MS patients, CSF ASYN levels are decreased, thus providing another possible link between MS and neurodegeneration. Future studies will need to be performed to confirm and extend these findings, to lead to a fuller understanding of the possible biological link between ASYN and MS.
Choroidal neovascularization (CNV) is a leading cause of blindness in age‐related macular degeneration. Production of vascular endothelial growth factor (VEGF) and macrophage recruitment by retinal pigment epithelial cells (RPE) significantly contributes to the process of CNV in an experimental CNV model. Serine racemase (SR) is expressed in retinal neurons and glial cells, and its product, d ‐serine, is an endogenous co‐agonist of N‐methyl‐d ‐aspartate receptor. Activation of the receptor results in production of nitric oxide (.NO), a molecule that promotes retinal and choroidal neovascularization. These observations suggest possible roles of SR in CNV. With laser‐injured CNV mice, we found that inactivation of SR‐coding gene (Srrnull) significantly reduced CNV volume, neovascular density, and invading macrophages. We exploited the underlying mechanism in vivo and ex vivo. RPE from wild‐type (WT) mice expressed SR. To explore the possible downstream target of SR inactivation, we showed that choroid/RPE homogenates extracted from laser‐injured Srrnull mice contained less inducible nitric oxide synthase and decreased phospho‐VEGFR2 compared to amounts in WT mice. In vitro, inflammation‐primed WT RPEs expressed more inducible NOS, produced more.NO and VEGF than did inflammation‐primed Srrnull RPEs. When co‐cultured with inflammation‐primed Srrnull RPE, significantly fewer RF/6A‐a cell line of choroidal endothelial cell, migrated to the opposite side of the insert membrane than did cells co‐cultured with pre‐treated WT RPE. Altogether, SR deficiency reduces RPE response to laser‐induced inflammatory stimuli, resulting in decreased production of a cascade of pro‐angiogenic cytokines, including.NO and VEGF, and reduced macrophage recruitment, which contribute synergistically to attenuated angiogenesis.
For over the last 50 years, the molecular mechanism of anti‐psychotic drugs' action has been far from clear. While risperidone is very often used in clinical practice, the most efficient known anti‐psychotic drug is clozapine (CLO). However, the biochemical background of CLO's action still remains elusive. In this study, we performed comparative proteomic analysis of rat cerebral cortex following chronic administration of these two drugs. We observed significant changes in the expression of cytoskeletal, synaptic, and regulatory proteins caused by both antipsychotics. Among other proteins, alterations in collapsin response mediator proteins, CRMP2 and CRMP4, were the most spectacular consequences of treatment with both drugs. Moreover, risperidone increased the level of proteins involved in cell proliferation such as fatty acid‐binding protein‐7 and translin‐associated factor X. CLO significantly up‐regulated the expression of visinin‐like protein 1, neurocalcin δ and mitochondrial, stomatin‐like protein 2, the calcium‐binding proteins regulating calcium homeostasis, and the functioning of ion channels and receptors.
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