Jan Klaschka

Expression of the hippocampal NMDA receptor GluN1 subunit and its splicing isoforms in schizophrenia: postmortem study.

There is accumulating evidence that disturbances in N-methyl-d-aspartate receptor (NMDA-R) functioning are associated with the pathogenesis of schizophrenia. To assess actual changes in the expression of the GluN1 subunit and its isoforms, we measured absolute differences in the levels of mRNA/protein for panGluN1 (eight isoforms altogether) as well as the mRNA individual isoforms in the postmortem left/right hippocampus of patients with schizophrenia in comparison with non-psychiatric subjects. There were no significant differences in the panGluN1 subunit mRNA expression, but the absolute left/right differences were much more pronounced in the patients with schizophrenia. Protein levels of the GluN1 subunit in the left hippocampus in male schizophrenic patients were lower than controls. The expression of the NR1-4b isoform was attenuated in the left, whereas the NR1-2b was reduced in the right hippocampus of schizophrenic patients. Isoforms associated with the efficiency of NMDA-induced gene expression and with phosphorylation occurred more commonly in schizophrenic hippocampi. In summary, our study suggests that NMDA-R hypofunction in schizophrenia might be selectively dependent on the dysregulation of GluN1 subunit expression, which exhibits a somewhat different expression in the left/right hippocampus of psychotic patients.

Age- and sex-dependent laterality of rat hippocampal cholinergic system in relation to animal models of neurodevelopmental and neurodegenerative disorders.

Studies suggest age- and sex-dependent structural and functional patterns of human cerebral lateralization underlie hemisphere specialization and its alterations in schizophrenia. Recent works report sexual dimorphism of neurons in the hippocampal formation and specialization of hemispheres in rats. Our experiments indicate for the first time functional lateralization of the high-affinity choline uptake (HACU) system directly associated with a synthesis of acetylcholine in the hippocampus of Wistar rats. The markedly increased HACU activity was found in the left compared to the right hippocampus of adult male but not female animals. Lineweaver-Burk plot analysis revealed a statistically significant increase of Vmax in the left hippocampus of 14-day-old when compared to 7-day-old males. It appears that laterality of HACU occurs during late postnatal maturation, and its degree is markedly enhanced after puberty and attenuated during aging. Quinolinic acid (QUIN), an endogenous agonist of N-methyl-d-aspartate type glutamate receptors, was used in this study to evaluate the neurodevelopmental hypothesis of schizophrenia. It is known that elevated levels of QUIN accompany viral infections, increasing the risk of developing schizophrenia. Bilateral intracerebroventricular application of QUIN (250 nmoles/ventricle) to pups aged 12 days significantly impaired the cholinergic hippocampal system of adolescent male and female rats and reversed lateralization of male HACU. Morphological analysis indicated marked changes in brain lesion sizes (extensive 24 h and moderate 38 days after the operation). Asymmetry of lesions was observed in the majority of cases, but the left hemisphere was not generally more vulnerable to QUIN effects than the right side. Moreover, no lateral differences were found between lesioned hippocampi in the specific binding of [3H]hemicholinium-3 (10%–15% loss of binding sites when compared to sham-operated animals). In summary, our results indicate a symmetrical drop in the number of choline carriers of lesioned male rats but a asymmetrical decrease in the activity of remaing carriers, suggesting defects in processes of sexual brain differentiation, leading under normal conditions to the higher activity of carriers in the left hippocampus. The data demonstrate viral infection–mediated alterations in normal patterns of brain asymmetry and are discussed in relation to animal models of neurodevelopmental and neurodegenerative diseases.

Enhanced levels of mitochondrial enzyme 17β-hydroxysteroid dehydrogenase type 10 in patients with Alzheimer disease and multiple sclerosis

The multifunctional mitochondrial enzyme 17beta-hydroxysteroid dehydrogenase type 10 might play a role in the development of Alzheimer disease via its high-affinity binding to amyloid beta peptides and its neuronal over-expression. It is suggested that the cerebrospinal fluid levels of the enzyme, free or bound to amyloid beta peptides, are a potential specific biomarker of Alzheimer disease. However, mitochondrial dysfunction seems to play a role in many neurological diseases including multiple sclerosis. In this study, the specificity of changes in relation to the enzyme over-expression was evaluated using enzyme-linked immunosorbent and surface plasmon resonance sensors. The data indicated pronounced increases in the enzyme levels, specifically to 179% in multiple sclerosis and to 573% in Alzheimer disease when compared to the age-matched controls. Although the differences between both diseases were statistically significant, enzyme levels do not appear to be a highly specific biomarker of Alzheimer disease. On the other hand, enhancement in levels of the enzyme bound to amyloid beta peptides was only observed in people with Alzheimer disease, which suggests that the complex should be further considered as a possible biomarker. In patients with multiple sclerosis, our results are the first to demonstrate significant changes in enzyme expression and to suggest possible alterations in amyloid beta peptides.

Lateralization of hippocampal nitric oxide mediator system in people with Alzheimer disease, multi-infarct dementia and schizophrenia.

There is evidence that brain lateralization underlying hemispheric specialization can be observed also at biochemical level. However, hemispheric differences in nitric oxide mediator system have not yet been evaluated. The hippocampus and planum temporale are highly asymmetrical regions but the degree of their laterality is altered in demented or psychotic people. In the study, l-glutamate/l-arginine/l-citrulline concentrations, nitric oxide synthase activities/expressions and nitrites/nitrates levels were estimated in autoptic hippocampi. Right/left laterality in endothelial synthase activity and in nitrites/nitrates was observed in controls. Lateral changes were estimated in patients with Alzheimer disease (a marked increase in activities of constitutive synthases and in expression of inducible enzyme in the left side) and schizophrenia (an increase in activities of all enzymes especially in the right side). Significant shifts from positive to negative correlations were found between laterality of some components of nitric oxide pathway and of planum temporale volumetry under pathological conditions. The hippocampal nitric oxide system appears to be globally right/left lateralized, especially via actions of highly asymmetrical endothelial synthase. The results suggest a specific involvement of all synthases in the development of selected diseases and show that lateral analyses are of sufficient sensitivity to reveal subtle links. The volumetric asymmetry of the planum temporale as a marker of handedness is not probably simply linked to brain laterality at biochemical level but reflects alterations due to pathological processes.

Amyloid Beta Peptide 1-40 and the Function of Rat Hippocampal Hemicholinium-3 Sensitive Choline Carriers: Effects of a Proteolytic Degradation In Vitro

Effects of amyloid beta peptide 1-40 (Abeta) and of plant cysteine proteases bromelain and papain on the high-affinity uptake of choline (HACU) and the specific binding of [3H]hemicholinium-3 ([3H]HC-3) have been investigated on hippocampal synaptosomes from young adult male Wistar rats under basal and stimulated conditions (55 mM KCl). Depolarization increased significantly the HACU levels (the changes were predominantly in Vmax) and mildly the [3H]HC-3 binding (the changes especially in KD). Nonaggregated Abeta at low nM concentrations suppressed the depolarization effects but was ineffective under basal conditions during a short-term incubation. Higher μM concentrations decreased the HACU and binding under basal conditions in a time-dependent manner. The binding changes were firstly associated with alterations in KD and secondarily were accompanied also by a drop in Bmax. The results suggest that Abeta directly influences high-affinity carriers, inhibits their transport activity and enhances their sensitivity to proteolytic cleavage. Stimulation increases the sensitivity of carriers to the interaction with Abeta.

(3H)hemicholinium-3 binding sites in postmortem brains of human patients with Alzheimer's disease and multi-infarct dementia.

(3H)Hemicholinium-3 ((3H)HCh-3), a potent, selective, and competitive inhibitor of the high-affinity choline uptake process was used for the detection of high-affinity choline carriers in the hippocampus (gyrus parahippocampalis), neocortex (gyrus frontalis medius), and cerebellum (lobulus semilunaris inferior) in autopsy samples of people with Alzheimers disease, multi-infarct dementia and from other psychiatric and nonpsychiatric patients. The effect of postmortem delay was eliminated by means of the cerebellum used as an individual standard. The density of (3H)HCh-3 binding sites was decreased in the hippocampus and neocortex from individuals with multi-infarct dementia and unchanged in the brain tissue from people with Alzheimers disease in comparison with control patients. No changes in dissociation constants were found. In Alzheimers disease, high-affinity choline transport appears to be reduced by a dysfunction of cholinergic neuronal membrane rather than by a significant decrease in the number of presynaptic cholinergic nerve terminals. Results provide evidence of a decrease in the number of nerve endings in people with multi-infarct dementia and suggest different vulnerability of particular brain areas to vascular disorders.

High-affinity choline uptake and muscarinic receptors in rat brain during aging

The aim of this study was to determine the effect of aging on the high-affinity choline uptake (HACU) and the muscarinic acetylcholine receptors (mAChR) in the brain of Wistar male rats and to define more precisely the steps of the brain cholinergic degeneration in the course of the whole animal life. In 24-month-old rats, a substantial decrease in HACU values in the hippocampus (to 65-75%) and in the density of mAChR in the cortex (to 76%) was found in comparison with 3-month-old controls. The interaction of muscarinic receptor antagonist pirenzepine with [(3)H]QNB indicated a decrease in low-affinity sites (M(2)) in 24-month-old rats. The first slight changes due to aging manifested themselves by the reduction in HACU values very early (between 6 and 12 months), the decrease of the muscarinic receptor density was observed in a later stage (19-month-old animals). Regression analysis indicated considerable dependence of the HACU values on age (the correlation coefficient r = -0.689, the slope b = -0.279 pmol/4 min per mg(prot) per month, P < 0.001) while the density of muscarinic receptors does not correlate with age so markedly (r = -0.415, b = -6.316 fmol/mg(prot) per month, P = 0.018).

Age- and Sex-Dependent Effects of Ethanol on Hippocampal Hemicholinium-3 Sensitive Choline Carriers During Postnatal Development of Rats

Vulnerability of hippocampal hemicholinium-3 (HC-3)-sensitive carriers to ethanol was evaluated in vitro during rat postnatal development. The high-affinity uptake of [3H]choline (HACU) and the specific binding of [3H]HC-3 were measured on synaptosomes from 7-, 14-, and 60-day- and 3-month-old male and female Wistar rats. Marked increases of basal (between 7 and 60 days of age) and of stimulated HACU levels via K+-depolarization (between 14 days and 3 months) but only a mild elevation in [3H]HC-3 binding (between 7 days and 3 months) associated with alterations in the binding site number were found. On the mature tissue, ethanol at high concentrations (5%) moderately inhibited the choline transport under basal conditions but totally eliminated depolarization effects. However, both age- and sex-dependent alterations in basal HACU mediated by high or low pharmacologically relevant alcohol concentrations (50–100 mM) were observed in the immature tissue. Namely, the dose- and incubation time–dependent inhibition of HACU associated with changes in the transport velocity was found in postnatal male but not female tissue. [3H]HC-3 binding site was not markedly sensitive to ethanol actions. Anisotropy measurements in the region of the hydrophilic heads of phospholipid bilayers and in the membrane hydrocarbon core indicated penetration of 100 mM ethanol to immature female but not male tissue. Our results suggest the noncompetitive binding of alcohol to choline carriers from immature male tissue and correspond with data reporting significant sexual dimorphism of postnatal hippocampal neurons. The direct effects of ethanol on male choline carriers can contribute to the inhibition of acetylcholine synthesis and to sex-dependent neurotoxic effects of alcohol applied in vivo during early and late postnatal period.

Effect of aging on lipid peroxide levels induced by L-glutamic acid and estimated by means of a thiobarbituric acid test in rat brain tissue

Studies of age-related changes based on the thiobarbituric acid (TBA) test appear to be inconsistent and contradictory. In our work, real basal (hypothetical, corrected to the zero concentration of atmospheric oxygen), basal (atmospheric oxygen-stimulated) and stimulated (L-glutamic and hydrochloric acids) levels of TBA-reactive products (TBARP) were estimated in the brain (hippocampus--HPC, cortex--COR, cerebellum--CRB) from young (3 months) and old (28 months) male and female Wistar rats. The values of basal levels of TBARP were different in young (HPC > COR > CRB) as well as in old animals (COR > CRB > HPC). Thus, the process of aging caused a significant decrease in the HPC (to 72%) and no change in the COR and CRB. Levels stimulated by L-glutamic acid were significantly decreased (COR, CRB) and unchanged (HPC) in old compared to young animals. The real basal levels estimated by regression analysis seem to be higher in the brain tissue damaged by aging. We discuss the usefulness of the TBA test applied to aged tissue, the effect of acidosis, the effectivity of L-glutamic acid to generate free radicals and the differences between individual brain areas.

Effects of Aging and Dementia on the Levels of Thiobarbituric-Acid- Reactive Products Stimulated by L-Glutamic Acid in Human Autopsy and Biopsy Brain Tissue

Basal and stimulated (by L-glutamic acid, GA) levels of thiobarbituric-acid-reactive products were estimated in the brain tissue (hippocampus, cortex and cerebellum) from autopsy samples of people with Alzheimer disease (AD), multi-infarct dementia (MID) and from nondemented control patients. The experiment was also performed on biopsy brain tissue (cortex) of nondemented controls. The biopsy brain tissue influenced by normal aging in vivo showed a limited susceptibility to undergo lipid peroxidation stimulated by GA in vitro in comparison with the younger tissue. A significant decrease in the ratio of stimulated to basal levels was found in the cerebellum of MID patients in comparison with nondemented controls and AD patients.