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1.
Mol Neurobiol ; 59(9): 5452-5475, 2022 Sep.
Article in English | MEDLINE | ID: mdl-35715683

ABSTRACT

Autism spectrum disorders (ASDs) are increasingly diagnosed as developmental disabilities of unclear etiology related to genetic, epigenetic, or environmental factors. The diagnosis of ASD in children is based on the recognition of typical behavioral symptoms, while no reliable biomarkers are available. Rats in whom ASD-like symptoms are due to maternal administration of the teratogenic drugs valproate or thalidomide on critical day 11 of pregnancy are widely used models in autism research. The present studies, aimed at detecting changes in the levels of hydrophilic and hydrophobic metabolites, were carried out on 1-month-old rats belonging to the abovementioned two ASD models and on a control group. Analysis of both hydrophilic and hydrophobic metabolite levels gives a broader view of possible mechanisms involved in the pathogenesis of autism. Hippocampal proton magnetic resonance (MRS) spectroscopy and ex vivo nuclear magnetic resonance (NMR) analysis of serum and urine samples were used. The results were analyzed using advanced statistical tests. Both the results of our present MRS studies of the hippocampus and of the NMR studies of body fluids in both ASD models, particularly from the THAL model, appeared to be consistent with previously published NMR results of hippocampal homogenates and data from the literature on autistic children. We detected symptoms of disturbances in neurotransmitter metabolism, energy deficit, and oxidative stress, as well as intestinal malfunction, which shed light on the pathogenesis of ASD and could be used for diagnostic purposes. These results confirm the usefulness of the noninvasive techniques used in ASD studies.


Subject(s)
Autism Spectrum Disorder , Autistic Disorder , Body Fluids , Animals , Female , Hippocampus , Magnetic Resonance Spectroscopy , Metabolomics/methods , Pregnancy , Rats
2.
Mol Neurobiol ; 57(7): 3089-3105, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32468248

ABSTRACT

Autism spectrum disorders (ASDs) are increasingly being diagnosed. Hypotheses link ASD to genetic, epigenetic, or environmental factors. The role of oxidative stress and the imbalance between excitatory and inhibitory neurotransmission in the pathogenesis of ASD has been suggested. Rats in which ASD symptoms are induced by valproate (VPA) or thalidomide (THAL) application in utero are useful models in ASD studies. Our study investigated whether rats in ASD models show changes in metabolite levels in the brain consistent with the hypothetical pathomechanisms of ASD. Female rats were fed one dose of 800 mg/kg VPA or 500 mg/kg THAL orally on the 11th day of gestation, and 1-month offspring were used for the experiments. Metabolic profiles from proton nuclear magnetic resonance spectroscopy of hydrophilic and hydrophobic extracts of rat hippocampi were subjected to OPLS-DA statistical analysis. Large differences between both models in the content of several metabolites in the rat hippocampus were noticed. The following metabolic pathways were identified as being disturbed in both ASD models: steroid hormone biosynthesis; fatty acid biosynthesis; the synthesis and degradation of ketone bodies; glycerophospholipid metabolism; cholesterol metabolism; purine metabolism; arginine and proline metabolism; valine, leucine, and isoleucine biosynthesis and degradation. These results indicate disorders of energy metabolism, altered structure of cell membranes, changes in neurotransmission, and the induction of oxidative stress in the hippocampus. Our data, consistent with hypotheses of ASD pathomechanisms, may be useful in future ASD studies, especially for the interpretation of the results of metabolomics analysis of body fluids in rat ASD models.


Subject(s)
Autistic Disorder/metabolism , Hippocampus/metabolism , Metabolic Networks and Pathways/physiology , Metabolome/physiology , Animals , Disease Models, Animal , Female , Lipid Metabolism/physiology , Magnetic Resonance Spectroscopy , Metabolomics , Rats
3.
J Mol Neurosci ; 69(1): 94-105, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31134532

ABSTRACT

The lack of a single predictive or diagnostic test in multiple sclerosis (MS) remains a major obstacle in the patient's care. The aim of this study was to investigate metabolic profiles, especially lipids in cerebrospinal fluid (CSF) using 1H-NMR spectroscopy and metabolomics analysis to discriminate MS patient group from the control ones. In this study, 19 MS patients and 19 controls, without neurological problems, patients were enrolled. To obtain the CSF metabolic profiles, NMR spectroscopy was used. Hydrophilic and hydrophobic compounds were analyzed using univariate and multivariate supervised analysis orthogonal partial least square discriminant analysis (OPLS-DA). Targeted OPLS-DA analysis of 32 hydrophilic and 17 hydrophobic compounds obtained 9 hydrophilic metabolites and 8 lipid functional groups which had the highest contribution to patient's group separation. Lower concentrations of CSF hydrophilic and hydrophobic compounds were observed in MS patients as compared to control group. Acetone, choline, urea, 1,3-dimethylurate, creatinine, isoleucine, myo-inositol, leucine, and 3-OH butyrate; saturated and monounsaturated acyl groups of ω-9, ω-7, ω-6, ω-3, and fatty acid, triglycerides, 1,3-DG, 1-MG, and unassigned component signal at 3.33 ppm were the most important signal compounds in group separation. Analysis of metabolic profile of raw CSF and their lipid extract shows decreased levels of many compounds and led to the conclusion that MS patients could have a disturbance in many metabolic pathways perhaps leading to the decreased level of acetyl-CoA and/or inflammation. CSF metabolic profile analyses could be used as a fingerprint for early MS diagnosis.


Subject(s)
Metabolome , Multiple Sclerosis/cerebrospinal fluid , Acetone/cerebrospinal fluid , Adult , Amino Acids/cerebrospinal fluid , Biomarkers/cerebrospinal fluid , Case-Control Studies , Choline/cerebrospinal fluid , Creatinine/cerebrospinal fluid , Female , Humans , Lipids/cerebrospinal fluid , Male , Middle Aged , Urea/cerebrospinal fluid
4.
J Physiol Pharmacol ; 65(5): 679-86, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25371527

ABSTRACT

Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant with recognized neuro- and cytotoxic properties that are presumably mediated by intracellular Ca(2+) release. Other studies have demonstrated that ryanodine is able to inhibit Ca(2+) efflux from skeletal sarcoplasmic reticulum (SR) membranes in response to the known Ca(2+) releaser thapsigargin, provided that the macrocyclic brominated tyrosine derivative bastadin 5 is also present. Similar effects supporting the role of ryanodine receptors in thapsigargin-evoked Ca(2+) release have been observed in primary cultures of rat cerebellar granule cells (CGCs). Here, we used CGCs and the fluorescent intracellular Ca(2+) probe fluo-3 to test the following hypotheses: (1) TBBPA shares Ca(2+) releasing properties with thapsigargin, and (2) synthetic bastadin 12 can replace bastadin 5 as a pharmacological tool to identify these similarities. The results demonstrated that either 200 nM thapsigargin or 30 µM bastadin 12 alone induced an increase in the intracellular Ca(2+) level in CGCs, whereas 2.5 and 10 µM bastadin 12 had no effect on the basal Ca(2+) concentration. The thapsigargin-induced Ca(2+) release was partially reduced by co-administration of either 2.5 µM bastadin 12 or 200 µM ryanodine, and the release of Ca(2+) was nearly completely attenuated by these compounds when they were given together. TBBPA (5, 10 and 25 µM) administration caused a concentration-dependent increase in CGC Ca(2+) levels. Administration of 2.5 µM bastadin 12 with 200 µM ryanodine blocked the increase in intracellular Ca(2+) evoked by 10 µM TBBPA, although these compounds were ineffective when applied separately. These results indicate that bastadin 12 may replace bastadin 5 when testing the ability of ryanodine to inhibit Ca(2+) release from the intracellular stores of cultured neurons, and our findings support the hypothesis that TBBPA and thapsigargin induce intracellular Ca(2+) release through a common mechanism.


Subject(s)
Calcium/metabolism , Halogenated Diphenyl Ethers/pharmacology , Peptides, Cyclic/pharmacology , Ryanodine/pharmacology , Animals , Cells, Cultured , Cerebellum/cytology , Polybrominated Biphenyls , Rats, Wistar , Thapsigargin
5.
Toxicology ; 276(3): 154-63, 2010 Oct 29.
Article in English | MEDLINE | ID: mdl-20696200

ABSTRACT

Thimerosal (TH), an ethylmercury complex of thiosalicylic acid has been used as preservative in vaccines. In vitro neurotoxicity of TH at high nM concentrations has been reported. Although a number of toxicological experiments demonstrated high affinity of mercury to thiol groups of the extracellular amino acids and proteins that may decrease concentration of free TH in the organism, less is known about the role of interactions between proteins and amino acids in protection against TH neurotoxicity. In the present study we examined whether the presence of serum proteins and of l-cysteine (Cys), d,l-homocysteine (Hcy), N-acetyl cysteine (NAC), l-methionine (Met) and glutathione (GSH) in the incubation medium affects the TH-induced changes in the viability, the intracellular levels of calcium and zinc and mitochondrial membrane potential in primary cultures of rat cerebellar granule cells. The cells were exposed to 500 nM TH for 48 h or to 15-25 µM TH for 10 min. Our results demonstrated a decrease in the cells viability evoked by TH, which could be prevented partially by serum proteins, albumin or in a dose-dependent manner by 60, 120 or 600 µM Cys, Hcy, NAC and GSH, but not by Met. This neuroprotection was less pronounced in the presence of proteins. Incubation of neurons with TH also induced the rise in the intracellular calcium and zinc concentration and decrease in mitochondrial membrane potential, and these effects were abolished by all the sulfur containing compounds studied and administered at 600 µM concentration, except Met. The loss of the ethylmercury moiety from TH as a result of interaction with thiols studied was monitored by (1)H NMR spectroscopy. This extracellular process may be responsible for the neuroprotection seen in the cerebellar cell cultures, but also provides a molecular pathway for redistribution of TH-derived toxic ethylmercury in the organism. In conclusion, these results confirmed that proteins and sulfur-containing amino acids applied separately reduce TH neurotoxicity, while their combination modulates in more complex way neuronal survival in the presence of TH.


Subject(s)
Blood Proteins/physiology , Cerebellum/drug effects , Cerebellum/physiology , Sulfhydryl Compounds/physiology , Thimerosal/antagonists & inhibitors , Thimerosal/toxicity , Animals , Animals, Newborn , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Cerebellum/pathology , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/physiology , Molecular Weight , Rats , Rats, Wistar , Serum Albumin, Bovine/physiology
6.
Neurochem Res ; 29(11): 2121-3, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15662846

ABSTRACT

Rat cerebral nonsynaptic mitochondria were incubated in medium containing 2 mM glutamine (Gln) or 2 mM glutamate (Glu), in the presence of a Gln uptake inhibitor histidine (His) as well as other basic amino acids, lysine and arginine (Lys, Arg) not inhibiting Gln uptake. Subsequently, the mitochondrial contents of Glu and Gln were determined by HPLC. Incubation in the presence of Glu alone increased the Glu content from approximately 3.5 to 15 nmol/mg protein, without affecting the Gln content. On the other hand, incubation with Gln increased the content of Gln from approximately 1.5 to approximately 12 nmol/mg, and that of Glu to 10 nmol/mg. As expected, addition of His did not alter the Glu and Gln content resulting from incubation with Glu. However, His significantly decreased to almost the preincubation level the content of Glu in mitochondria incubated with Gln, without affecting the content of Gln. No other amino acid had any effect on these parameters. The results point to the existence of distinct Gln pools, one of which is accessible to external Gln via a His-sensitive transporter and is accessible for deamidation in the mitochondria.


Subject(s)
Amides/metabolism , Brain Chemistry/drug effects , Glutamine/metabolism , Histidine/pharmacology , Mitochondria/metabolism , Animals , Arginine/metabolism , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Glutamic Acid/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Lysine/metabolism , Male , Mitochondria/drug effects , Rats
7.
Neurotoxicology ; 21(3): 295-300, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10894118

ABSTRACT

Exposure of rat cerebral mitochondria to 2-10 mM glutamine (Gln) for 20 min, produced a concentration-dependent, gradual decrease of light scattering reflecting mitochondrial swelling. The light scattering decreasing effect of 5 mM Gln was attenuated by 0.5 microM cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition (mPT) induction. Histidine (His), which is a potent inhibitor of high affinity Gln uptake to mitochondria, attenuated Gln-induced decrease of mitochondrial light scattering when added at equimolar concentration, and abolished the decrease when added at 15 mM concentration shortly before addition of Gln. His inhibited the uptake of 5 mM [14C]Gln in a concentration-dependent manner as measured during 3 min incubation. CsA did neither affect [14C]Gln uptake nor modified its inhibition by His. The effects of 5 mM His and 0.5 microM CsA on mitochondrial light scattering were additive, indicating that mitochondrial swelling represents a cumulative effect of Gln -driven entry of osmotically obligated water and induction of mPT. Addition of ammonium ions at neurotoxic concentrations neither influenced the decrease of light scattering induced by Gln, nor produced any change in light scattering when added alone. The results point to mitochondrial swelling and subsequent activation of mPT, as one of the potential mechanisms by which Gln induces metabolic disturbances in the brain in hyperammonemic conditions.


Subject(s)
Brain/drug effects , Glutamine/pharmacology , Mitochondria/drug effects , Mitochondrial Swelling/drug effects , Animals , Brain/metabolism , Female , Hyperammonemia/metabolism , Mitochondria/metabolism , Mitochondrial Swelling/physiology , Rats , Rats, Wistar
8.
Acta Neurobiol Exp (Wars) ; 60(4): 427-35, 2000.
Article in English | MEDLINE | ID: mdl-11200170

ABSTRACT

The aim of this in vivo microdialysis study was to characterise the regulation of prostaglandin D2 (PgD2) synthesis by NMDA receptors in the rabbit hippocampus in relation to changes in extracellular Ca2+ concentration ([Ca2+]e) and nitric oxide (NO) levels. Samples of dialysate were analysed for changes in PgD2 concentration, in [Ca2+]e and in the level of NO. The results demonstrated that a 20-min pulse application of 0.1-2.5 mM NMDA via a microdialysis probe induced a prolonged stimulation of PgD2 release that was sensitive to competitive NMDA receptor antagonists. An inhibitor of voltage-sensitive Na+ channels, tetrodotoxin, did not influence this effect but significantly suppressed basal PgD2 production, whereas a NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), prevented NMDA-evoked NO release and inhibited NMDA-induced PgD2 release in an L-arginine-sensitive manner. NO donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, stimulated PgD2 release. NMDA-evoked decrease in [Ca2+]e was insensitive to TTX and L-NAME. These results demonstrate an in vivo NMDA receptor-mediated modulation of PgD2 synthesis in the brain, in which NO participates.


Subject(s)
Calcium/physiology , Hippocampus/physiology , N-Methylaspartate/pharmacology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/physiology , Penicillamine/analogs & derivatives , Prostaglandin D2/biosynthesis , Receptors, N-Methyl-D-Aspartate/physiology , Animals , Extracellular Space/physiology , Hippocampus/drug effects , Nitric Oxide Donors/pharmacology , Penicillamine/pharmacology , Rabbits , S-Nitroso-N-Acetylpenicillamine , Tetrodotoxin/pharmacology
9.
Brain Res Bull ; 53(6): 813-9, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11179848

ABSTRACT

In vivo microdialysis combined with the measurement of (45)Ca(2+) efflux from prelabelled hippocampus demonstrated a pronounced N-methyl-D-aspartate (NMDA)-evoked (45)Ca(2+) release to the dialysate in the rat dentate gyrus (DG) and CA1, whereas in rabbit a slight release of (45)Ca(2+) was observed only in the DG. In vitro, we noticed that the NMDA-evoked increase in Fura-2 detected intracellular Ca(2+) concentration in synaptoneurosomes from the rat, but not from the rabbit hippocampus, was strongly inhibited by the ryanodine receptor (RyR) antagonists dantrolene and ryanodine. To establish the mechanism of these differences, we characterised their possible dependence on the expression of RyR and their co-localisation with the calcium binding protein calbindin D(28k). A pronounced expression of [(3)H]ryanodine binding sites in the rat DG, which is only slight in the CA1, was demonstrated whereas in rabbit they were only found in the DG. The pattern of expression of calbindin D(28k) immunoreactivity and RyR in the rat and rabbit hippocampus was similar. These results suggest that the functional role of RyR in the generation of the NMDA receptor-mediated intracellular Ca(2+) signalling in the rabbit hippocampal neurones is marginal when compared to the rat. These differences reflect a diverse expression of RyR in both species. The corresponding differences in calbindin D(28k) immunoreactivity are most probably secondary in nature.


Subject(s)
Calcium Signaling/physiology , Hippocampus/metabolism , Neurons/metabolism , Rabbits/metabolism , Rats/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Binding Sites/drug effects , Binding Sites/physiology , Calbindins , Calcium/metabolism , Calcium Signaling/drug effects , Excitatory Amino Acid Agonists/pharmacology , Female , Hippocampus/cytology , Hippocampus/drug effects , Male , Microdialysis , N-Methylaspartate/pharmacology , Neurons/cytology , Neurons/drug effects , Radioligand Assay , Rats, Wistar/metabolism , Receptors, N-Methyl-D-Aspartate/drug effects , Ryanodine/pharmacology , Ryanodine Receptor Calcium Release Channel/drug effects , Ryanodine Receptor Calcium Release Channel/metabolism , S100 Calcium Binding Protein G/metabolism , Synaptosomes/drug effects , Synaptosomes/metabolism , Tritium
10.
Acta Pol Pharm ; 57 Suppl: 129-33, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11293243

ABSTRACT

The mitochondrial permeability transition (MPT) resulting from calcium-induced opening of cyclosporin A (CsA)-sensitive megachannels, leading to deenergisation of mitochondria and release of pro-apoptotic cytochrome c, has been implicated in the pathomechanism of excitotoxic neurodegeneration. The aim of this work was to test neuroprotective potential of CsA in the model of N-methyl-D-aspartate-(NMDA)-induced excitotoxicity in vivo, and to verify utility of microdialysis of the rabbit hippocampus in vivo for these mechanistic studies. In vitro experiments demonstrated that the early rapid phase of Ca(2+)-induced swelling of isolated brain mitochondria, and of accompanying cytochrome c release, was strongly inhibited by 0.5 microM CsA. In the in vivo experiments 1 mM NMDA was applied for 20 min to the hippocampus in a control, or 5 microM CsA-containing dialysis medium via transhippocampal microdialysis probes, and changes in extracellular Ca2+ concentration and in NO release were monitored. Application of NMDA induced a prolonged decrease in the extracellular concentration of Ca2+, reflecting influx of Ca2+ to stimulated neurones. CsA only slightly enhanced this effect. NMDA induced also release of NO to the dialysis medium. Morphological examination 30 min after NMDA application visualised swelling of dendritic mitochondria and cisternae of endoplasmatic reticulum of pyramidal neurones in the CA1 sector of the hippocampus in the vicinity of microdialysis probes. CsA prevented mitochondrial swelling. After 24 h degeneration of the CA1 pyramidal neurones close to a microdialysis probes was observed, which was partially prevented in CsA-treated rabbits. These results indicate that the mechanism of CsA nuroprotection may be at least in part ascribed to prevention of MPT. Microdialysis of the rabbit hippocampus combined with NMDA excitotoxicity appeared to be useful in mechanistic studies of CsA neuroprotection.


Subject(s)
Cyclosporine/pharmacology , Hippocampus/drug effects , Neuroprotective Agents/pharmacology , Animals , Calcium/metabolism , Calcium/pharmacology , Hippocampus/pathology , Microdialysis , N-Methylaspartate/toxicity , Nitric Oxide/biosynthesis , Rabbits , Rats , Rats, Wistar
11.
Ross Fiziol Zh Im I M Sechenova ; 85(1): 212-9, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10389178

ABSTRACT

In vivo microdialysis combined with measurements of 45Ca efflux from pre-labelled rat hippocampus has been utilised in our laboratory to demonstrate NMDA-evoked 45Ca2+ release to dialysate, reflecting calcium-induced calcium release (CICR) via ryanodine receptors (RyR). In the present study we attempted to reproduce this phenomenon in the rabbit hippocampus. Application of 1 mM NMDA to dialysis medium induced a decrease in Ca2+ concentration in dialysate, as a result of extracellular Ca2+ influx to neurones. The release of 45Ca2+ was not observed, instead a decrease in 45Ca2+ efflux rate from the NMDA treated rabbit hippocampus was noted, along with release to dialysate of prostaglandin D2, taurine and phosphoethanolamine. All these effects, reflecting different steps of intracellular calcium signalling, were insensitive to 100 microM dantrolene and 50 microM ryanodine, RyR modulators known to interfere with NMDA-evoked 45Ca2+ release in the rat hippocampus. Thus, although the results of this study demonstrate the role of extracellular Ca2+ influx to neurones in NMDA-evoked generation of Ca2+ signal in the rabbit hippocampus, the activity of CICR was not detected.


Subject(s)
Calcium/metabolism , Excitatory Amino Acid Agonists/pharmacology , Hippocampus/metabolism , N-Methylaspartate/pharmacology , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Ethanolamines/metabolism , Microdialysis , Prostaglandin D2/metabolism , Rabbits , Taurine/metabolism
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