Frontal Cortex Homogenates Research Articles

Apple juice concentrate maintains acetylcholine levels following dietary compromise

Oxidative stress contributes to age-related cognitive decline. In some instances, consumption of fruits and vegetables rich in antioxidant can provide superior protection than supplementation with purified antioxidants. Our prior studies have shown that supplementation with apple juice concentrate (AJC) alleviates oxidative damage and cognitive decline in adult (9-12 months) mice lacking ApoE (as a model of increased oxidative stress) and in normal aged (2-2.5 years) mice when challenged with a vitamin-deficient, oxidative stress-promoting diet. Here, we demonstrate that AJC, administered in drinking water, maintains acetylcholine levels that otherwise decline when adult and aged mice are maintained on the above deficient diet. Normal mice aged either 9-10 months or 2-2.5 years and ApoE-/- mice aged 9-10 months were maintained for 1 month on a complete diet or a diet lacking folate and vitamin E and containing iron as a pro-oxidant, and additional groups received 0.5% AJC ad libitum in drinking water. Spectrophotometric assay of acetylcholine levels revealed a significant decline in homogenates of combined frontal cortex and hippocampus for all mice maintained on the deficient diet, and a prevention of this decline in mice maintained on the deficient diet when supplemented with AJC. These findings provide a likely mechanism by which consumption of antioxidant-rich foods such as apples can prevent the decline in cognitive performance that accompanies dietary and genetic deficiencies and aging.

Low molecular weight species of tau in Alzheimer's disease are dependent on tau phosphorylation sites but not on delayed post-mortem delay in tissue processing

Gel electrophoresis and Western blotting of sarkosyl-insoluble fractions enriched in hyper-phosphorylated tau in Alzheimer disease (AD) have been used to analyze the pattern of phospho-tau by using different antibodies directed to the amino-terminal, core and carboxyl terminus of tau, and by using samples with increased artificial post-mortem delay in order to gain understanding on the characteristics of the band pattern and its vulnerability to post-mortem degradation. In addition to the typical profile of three major bands of 68, 64 and 60 kDa, several bands of lower molecular weight have been distinguished in frontal cortex homogenates in four AD cases stage V of Braak and Braak in optimal samples with 2 h of post-mortem delay. Lower bands, ranging from 60 to 22 kDa, are best seen with antibodies directed to the core of tau protein and, particularly, to the carboxy-terminus, thus suggesting the presence of truncated or cleaved forms of tau containing the C-terminal region. This pattern is not the result of post-mortem degradation, as artificial post-mortem delay of the same sample does not reveal the appearance of new bands with time. On the contrary, tau degradation, manifested as a reduction in the number and intensity of the bands, may occur between 8 and 26 h post-mortem and is universal in samples with post-mortem delays of 50 h.

Oxidation, glycoxidation, lipoxidation, nitration, and responses to oxidative stress in the cerebral cortex in Creutzfeldt–Jakob disease

Gel electrophoresis and Western blotting of frontal cortex homogenates have been carried out in sporadic Creutzfeldt–Jakob disease (CJD) cases and age-matched controls to gain understanding of the expression of glycation-end products (AGEs). N-Carboxymethyl-lysine (CML) and N-carboxyethyl-lysine (CEL) were used as markers of glycoxidation; 4-hydroxynonenal (4-HNE) and malondialdehyde-lysine (MDAL) as markers of lipoxidation; and nitrotyrosine (N-tyr) and neuronal, endothelial and inducible nitric oxide synthase (nNOS, eNos and iNos) as markers of protein nitration and as sources of NO production, respectively. Age receptor (RAGE) and Cu/Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) expression levels were also examined. The results showed a significant increase in the expression levels of AGE ( p < 0.05), CEL ( p < 0.001), RAGE ( p < 0.05), HNE-modified proteins ( p < 0.01), nNOS, iNOS and eNOS ( p < 0.01 and p < 0.05, respectively), N-tyr ( p < 0.05), and SOD1 ( p < 0.05) and SOD2 ( p < 0.05). No relationship was observed between PrP genotype, PrP type, PrP burden, and expression levels of oxidative stress markers. The present findings demonstrate oxidative, glycoxidative, lipoxidative and nitrative protein damage, accompanied by increased oxidative responses, in the cerebral cortex in sporadic CJD. These results provide support for the concept that oxidative stress may have important implications in the pathogenesis of prion diseases.

Suckling Rat Brain Regional Distribution of Acetylcholinesterase Activity in Galactosaemia In Vitro

We aimed to evaluate the effect of in vitro galactosaemia on acetylcholinesterase (AChE) activity in different suckling rat brain regions. Various concentrations of galactose (Gal), galactose-1-phosphate (Gal-1-P) and/or galactitol (Galtol) were preincubated for 1 h with homogenates from frontal cortex, hippocampus and for 1-3 h with hypothalamus homogenates at 37( composite function)C. AChE activity was determined spectrophotometrically. Mixture A (Gal-1-P (2 mM), Galtol (2 mM), and Gal (4 mM) (=brain concentrations in classical galactosaemia)) or mixture B (Galtol (2 mM) and Gal (1 mM) (=brain concentrations in galactokinase deficiency galactosaemia)) inhibited by 18-20% (P < 0.01) AChE activity in frontal cortex or hippocampus homogenates. Gal-1-P (2-8 mM) reduced AChE activity by 20% (P < 0.01) on frontal cortex and hippocampus homogenates. Galtol (2-8 mM) resulted in an AChE inhibition (20-22% (P < 0.01)) in hippocampus, 2 mM of the substance had the same effect (20%, P < 0.01) on frontal cortex, whereas higher concentrations (4-8 mM) failed to decrease the enzyme activity anymore. Gal (1-8 mM) did not change AChE activity in the studied areas. Additionally, the hypothalamus enzyme activity was measured considerably high and remained unaltered in the presence of the above compounds. In conclusion, AChE activity was significantly higher in hypothalamus compared with those in frontal cortex and hippocampus. Frontal cortex and hippocampus AChE was significantly inhibited by Gal derivatives, whereas hypothalamus AChE activity remained unaltered possibly due to the histologically different innervation of this area.

Suckling Rat Brain Regional Distribution of Na+,K+-ATPase Activity in the In Vitro Galactosaemia: The Effect of L-Cysteine and Glutathione

Inhibition of Na+,K+-ATPase activity causes edema and cell death in central nervous system. We determined the in vitro effects of galactose-l-phosphate (Gal-1-P), galactitol (Galtol) and galactose (Gal) (mix A = classical galactosaemia) or Galtol and Gal (mix B = galactokinase deficiency galactosaemia), on Na+,K+-ATPase activity in suckling rat brain frontal cortex, hippocampus or hypothalamus homogenates. Gal-1-P or Galtol alone at different concentrations, significantly inhibited Na+,K+-ATPase whereas Gal activated the enzyme in all investigated brain regions. Both mix A and mix B inactivated the enzyme by 20-30% (p < 0.001) in all studied areas. L-Cysteine (Cys) and glutathione (GSH) supplementation in mix B not only reversed the enzyme inhibition but also resulted in an activation of 50-60%, (p < 0.001) in all brain areas. Their presence in mix A also activated the inhibited Na+,K+-ATPase in hippocampus and hypothalamus to a lower degree, whereas Cys reversed the frontal cortex enzyme activity to control value only. These findings indicate that oxidation of the enzyme critical groups may be involved in galactosaemia, producing inhibitory effect. This phenomenon is reversed by antioxidants Cys and GSH, implying that free radicals may be implicated in the observed enzyme inactivation.

Mg 2+-ATPase activity in suckling rat brain regions in galactosaemia in vitro. l-Cysteine and glutathione effects

Mg 2+-ATPase activity is implicated with Mg 2+ homeostasis, maintaining high brain intracellular Mg 2+ content. We determined the in vitro effects of galactose-1-phosphate (Gal-1-P), galactitol (Galtol) and galactose (Gal) {mix A = Gal-1-P(2 mM) + Galtol(2 mM) + Gal(4 mM) concentrations commonly found in patients with classical galactosaemia} or Galtol and Gal {mix B = Galtol(2 mM) + Gal(1 mM) concentrations usually measured in patients with galactokinase deficiency galactosaemia} on Mg 2+-ATPase activity in suckling rat brain frontal cortex, hippocampus or hypothalamus homogenates. Gal-1-P significantly ( p < 0.001) enhanced enzyme activity in all the brain areas measured, whereas Galtol and Gal failed to cause any effect in the same regions. Mix A remarkably ( p < 0.001) stimulated Mg 2+-ATPase in the studied areas. On the contrary, mix B had no effect. The supplementation of antioxidant l-cysteine (Cys) or reduced Glutathione (GSH) in mix A failed to reverse to normal the activated enzyme in frontal cortex and hypothalamus, while they significantly reduced Mg 2+-ATPase activation in hippocampus. Conclusions: (a) Gal-1-P enormously activated Mg 2+-ATPase in all the studied brain regions, (b) Mix A, also, excessively activated the enzyme in the same areas, (c) the production of free radicals may be implicated with the enzyme activation and (d) Cys or GSH significantly decreased the activated hippocampal Mg 2+-ATPase.

Synthesis and evaluation of 11C-labeled 6-substituted 2-arylbenzothiazoles as amyloid imaging agents.

The synthesis and evaluation of a series of neutral analogues of thioflavin-T (termed BTA's) with high affinities for aggregated amyloid and a wide range of lipophilicities are reported. Radiolabeling with high specific activity [(11)C]methyl iodide provided derivatives for in vivo evaluation. Brain entry in control mice and baboons was high for nearly all of the analogues at early times after injection, but the clearance rate of radioactivity from brain tissue varied by more than 1 order of magnitude. Upon the basis of its rapid clearance from normal mouse and baboon brain tissues, [N-methyl-(11)C]2-(4'-methylaminophenyl)-6-hydroxybenzothiazole (or [(11)C]6-OH-BTA-1) was selected as the lead compound for further evaluation. The radiolabeled metabolites of [(11)C]6-OH-BTA-1 were polar and did not enter brain. The binding affinities of [N-methyl-(3)H]6-OH-BTA-1 for homogenates of postmortem AD frontal cortex and synthetic Abeta(1-40) fibrils were similar (K(d) = 1.4 nM and 4.7 nM, respectively), but the ligand-to-Abeta peptide binding stoichiometry was approximately 400-fold higher for AD brain than Abeta(1-40) fibrils. Staining of AD frontal cortex tissue sections with 6-OH-BTA-1 indicated the selective binding of the compound to amyloid plaques and cerebrovascular amyloid. The encouraging in vitro and in vivo properties of [(11)C]6-OH-BTA-1 support the choice of this derivative for further evaluation in human subject studies of brain Abeta deposition.

Region selective alterations of soluble guanylate cyclase content and modulation in brain of cirrhotic patients

Modulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) is altered in brain from experimental animals with hyperammonemia with or without liver failure. The aim of this work was to assess the content and modulation of sGC in brain in chronic liver failure in humans. Expression of the α-1, α-2, and β-1 subunits of sGC was measured by immunoblotting in autopsied frontal cortex and cerebellum from cirrhotic patients and controls. The contents of α-1 and α-2 subunits of guanylate cyclase was increased both in cortex and cerebellum, whereas the β-1 subunit was not affected. Addition of the NO-generating agent S-nitroso- N-acetyl-penicillamine (SNAP) to homogenates of frontal cortex from controls increased the activity of sGC 87-fold, whereas, in homogenates from cirrhotic patients, the increase was significantly higher (183-fold). In contrast, in cerebellum, activation of guanylate cyclase by NO was significantly lower in patients (156-fold) than in controls (248-fold). A similar regional difference was found in rats with portacaval anastomosis. In conclusion, these findings show that the NO-guanylate cyclase signal transduction pathway is strongly altered in brain in patients with chronic liver failure and that the effects are different in different brain areas. Given that activation of sGC by NO in brain is involved in the modulation of important cerebral processes such as intercellular communication, learning and memory, and the sleep-wake cycle, these changes could be implicated in the pathogenesis of hepatic encephalopathy in these patients. (H EPATOLOGY 2002;36:1155-1162.)

In vitro vs in vivo Pb effects on brain protein kinase C activity

Alteration of normal protein kinase C (PKC) function by environmental Pb exposure during neurodevelopment is hypothesized to be an important mechanism of toxicity underlying neurologic impairment. Previous studies have reported widely varying effects of Pb on PKC, possibly in part because of differences in in vitro and in vivo models used in those studies. Therefore, we tested the hypothesis that, with comparable tissue Pb levels, the effects of in vitro Pb exposure on brain PKC are the same as the effects caused by in vivo Pb exposure of intact animals. For chronic in vivo Pb exposure, female Long–Evans rats were exposed to Pb or vehicle from postnatal days 1 to 34–36 ( n=10/treatment). For in vitro Pb exposure, homogenate of the frontal cortex region was exposed directly to Pb in an amount comparable to that accumulated in brain during chronic in vivo Pb exposure. Brain Pb levels were measured using ultraclean techniques and inductively coupled plasma mass spectrometry. PKC activity was subsequently determined in cytosolic and membrane subcellular fractions in the frontal cortex, hippocampus, and remaining brain regions. Results indicate that brain Pb levels following in vivo Pb exposure were increased ∼20-fold above those of nonexposed animals (vehicle group [Pb] ∼130 ng Pb/g dry wt.). However, in vivo Pb exposure did not measurably alter brain PKC activity in the regions tested. In contrast, in vitro Pb exposure significantly increased PKC activity by ∼20% in the frontal cortex homogenate membrane subcellular fraction. These results indicate that Pb added in vitro caused more dramatic effects than those produced by a comparable amount of Pb in the tissue from in vivo exposure. While the mechanisms underlying these outcomes are not clear, they suggest that in vitro models might not accurately reflect effects of chronic low-level in vivo Pb exposure.

Synthesis and evaluation of 2-(3'-iodo-4'-aminophenyl)-6-hydroxybenzothiazole for in vivo quantitation of amyloid deposits in Alzheimer's disease.

A potent and brain permeable amyloid ligand has been identified as a lead compound capable of I-123/125-labelling for single photon emission computed tomography (SPECT) imaging. In this study, we report the synthesis and I-125-radiolabelling of Compound 6 and its in vitro and in vivo properties. Compound 6 [2-(3'-iodo-4'-aminophenyl)-6-hydroxybenzothiazole] bound to synthetic A beta(1-40) fibrils in a saturable manner, exhibiting an affinity (Ki) of 11+/-1.1 nM in a competitive binding assay using a tritiated thioflavin T analog ([3H]BTA-1) as radioligand. [125I]6 binding to synthetic A beta(1-40) fibrils fit a single-site model. [125I]6 exhibited several-fold higher binding to homogenates of frontal cortex from post-mortem Alzheimer's disease brain relative to age-matched control brain homogenates. No difference in binding was observed in cerebellum. The ratio of radioactivity concentration between frontal cortex and cerebellum was 6-fold higher in AD brain homogenates than the age-matched control. [125I]6 also readily penetrated the blood-brain barrier in normal control mice with an average radioactivity concentration of 6.43+/-0.62%ID/g detected in the whole brain at 2 min post i.v. injection. At 30 min, the radioactivity concentration decreased to 0.40+/-0.05%ID/g, indicating good clearance in the absence of amyloid deposits in the brain.

Abnormalities of SNARE Mechanism Proteins in Anterior Frontal Cortex in Severe Mental Illness

A fundamental molecular component of neural connectivity is the SNARE (SNAP receptor) protein complex, which consists of three proteins, syntaxin, SNAP-25 and VAMP. Under appropriate conditions, the SNARE complex can be formed in vitro. To investigate the hypothesis that dysregulation of SNARE proteins or their interactions could be abnormal in severe mental disorders, the three SNARE proteins and the complex were studied in post-mortem anterior frontal cortex homogenates. An ELISA was used to quantify SNARE protein immunoreactivities in cortical homogenates from four groups: patients with schizophrenia who died of causes other than suicide (n = 6), patients with schizophrenia and suicide (n = 7), patients with depression and suicide (n = 11), and controls (n = 11). Differences between groups in patterns of SNARE protein immuno-reactivities were demonstrated [Wilks' Lambda F(9,68) = 3.57, P = 0.001]. Protein-by-protein analyses indicated a significant reduction in SNAP-25 immunoreactivity in the schizophrenia non-suicide group [28% decrease relative to controls, F(3,31) = 6.45, P = 0.002, Student-Newman-Keuls test, P < 0.01]. The intercorrelations between SNARE protein and synaptophysin immunoreactivities were high in controls, but lower in the other groups, further indicating disturbances in relationships between these proteins. The extent of SNARE complex formation in vitro was studied using immuno-blotting. Significant differences related to group membership were observed for the SNARE complexes identified by SNAP-25 [Wilks' Lambda F(3,31) = 4.76, P = 0.008] and by syntaxin immunostaining [Wilks' Lambda F(3,31) = 9.16, P = 0.0002]. In both groups with suicide as a cause of death, relatively more SNAP-25 and syntaxin was present in the heterotrimeric SNARE complex than in other molecular forms. These abnormalities in the SNARE complex could represent a molecular substrate for abnormalities of neural connectivity in severe mental disorders.

Specific labelling of serotonin 5-HT 1B receptors in rat frontal cortex with the novel, phenylpiperazine derivative, [ 3H]GR125,743 : A pharmacological characterization

Although several tritiated agonists have been used for radiolabelling serotonin (5-hydroxytryptamine, 5-HT) 1B receptors in rats, data with a selective, radiolabelled antagonist have not been presented. Inasmuch as [ 3H]GR125,743 specifically labels cloned, human and native guinea pig 5-HT 1B receptors and has been employed for characterization of cerebral 5-HT 1B receptor in the latter species [Eur. J. Pharmacol. 327 (1997) 247.], the present study evaluated its utility for characterization of native, cerebral 5-HT 1B sites in the rat. In homogenates of frontal cortex, [ 3H]GR125,743 (0.8 nM) showed rapid association ( t 1/2=3.4 min), >90% specific binding and high affinity ( K d=0.6 nM) for a homogeneous population of receptors with a density ( B max) of 160 fmol/mg protein. In competition binding studies, affinities were determined for 15 chemically diverse 5-HT 1B agonists, including 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1 H-indole-3-yl]ethylamine (L694,247; p K i, 10.4), 5-carboxamidotryptamine (5-CT; 9.7), 3-[3-(2-dimethylamino-ethyl)-1 H-indol-6-yl]- N-(4-methoxybenzyl)acrylamide (GR46,611; 9.6), 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1 H-indole (RU24,969; 9.5), dihydroergotamine (DHE; 8.6), 5- H-pyrrolo[3,2- b]pyridin-5-one,1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl (CP93,129; 8.4), anpirtoline (7.9), sumatriptan (7.4), 1-[2-(3-fluorophenyl)ethyl]-4-[3-[5-(1,2,4-triazol-4-yl)-1 H-indol-3-yl]propyl]piperazine (L775,606; 6.4) and (−)-1( S)-[2-[4-(4-methoxyphenyl)piperazin-1-yl]ethyl]- N-methyl-3,4-dihydro-1 H-2-benzopyran-6-carboxamide (PNU109,291; <5.0). Similarly, affinities were established for 13 chemically diverse antagonists, including N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR125,743; p K i, 9.1), (−)-cyanopindolol (9.0), (−)-tertatolol (8.2), N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiozol-3-yl)biphenyl-4-carboxamide (GR127,935; 8.2), N-[3-(1,4-benzodioxan-5-yl)piperidin-4-yl] N-(indan-2yl)amine (S18127; 7.9), metergoline (7.8), (−)-pindolol (7.6), 1′-methyl-5-[2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-biphenyl-4-ylcarbonyl]-2,3,6,7-tetrahydro-5 H-spiro[furo[2,3- f]indole-3,4′-piperidine] (SB224,289; 7.5) and ketanserin (<5.0). These rank orders of affinity correspond to the binding profile of 5-HT 1B rather than 5-HT 1D receptors. The low affinities of L775,066 and PNU109,291 versus L694,247 should be noted, as well as the low affinity of ketanserin as compared to SB224,289. Finally, in line with species differences, the affinities of several ligands including CP93,129, RU24,969, (−)-pindolol and (−)-propanolol in rat 5-HT 1B sites were markedly different to guinea pig 5-HT 1B sites labelled with [ 3H]GR125,743. In conclusion, [ 3H]GR125,743 is an appropriate tool for the radiolabelling of native, rat 5-HT 1B receptors and permitted determination of the affinities of an extensive series of ligands at these sites.

Effects of L-phenylalanine on acetylcholinesterase and Na+,K+-ATPase activities in suckling rat frontal cortex, hippocampus and hypothalamus.

The effect of different L-phenylalanine (Phe) concentrations (0.12-12.1 mM) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+-ATPase activities was evaluated in homogenates of suckling rat frontal cortex, hippocampus and hypothalamus. Phe, at high concentrations, reduced AChE activity in frontal cortex and hippocampus by 18%-20%. On the contrary, the enzyme activity was unaltered in the hypothalamus. Na+,K+-ATPase was stimulated by high levels of the amino acid, both in the frontal cortex and the hypothalamus by 60%, whereas it was inhibited in the hippocampus by 40%. Mg2+-ATPase was not influenced by Phe. It is suggested that: a) In the frontal cortex, the improper acetylcholine (ACh) release, due to AChE inhibition by Phe, combined with the stimulation of Na+,K+-ATPase, possibly explain tremor and the hyperkinetic behaviour in patients with classical phenylketonuria (PKU). b) In the hippocampus, inhibition of AChE by Phe could lead to problems in memory, while Na+,K+-ATPase inhibition by Phe may induce metabolic disorders and electrical instability of the synaptosomal membrane. c) In the hypothalamus, the behavioral problems in PKU "off diet" may be related to noradrenaline (NA) levels, which are probably correlated with the modulated Na+,K+-ATPase by Phe.

Cotinine binding to nicotinic acetylcholine receptors in bovine chromaffin cell and rat brain membranes.

Cotinine is the major metabolite of nicotine. It has nicotine-like biological activity, but its potency is low. We studied cotinine binding to nicotinic receptors labelled with [3H]epibatidine. In membranes from cultured bovine chromaffin cells [3H]epibatidine bound to two apparent sites with K(d) values of 93 and 1400 pM. The low-affinity binding represented two-thirds of the binding sites. In rat frontal cortex and hippocampus homogenate membranes, only one apparent binding site was detected. The Kd values were 40 and 62 pM, in frontal cortex and hippocampus, respectively. Nicotine displaced [3H]epibatidine 10 times more potently from the brain than from the chromaffin cell membranes, and cotinine had over two orders of magnitude lower affinity than nicotine. In addition, the competitive nicotinic receptor antagonists methyllycaconitine and dihydro beta-erythroidine displaced [3H]epibatidine (100 pM and 1 nM) from the chromaffin cell membranes. Alpha-bungarotoxin did not affect the binding of 100 pM [3H]epibatidine. However, upon labelling with 1 nM [3H]epibatidine alpha-bungarotoxin (10 nM to 10 microM) displaced one-sixth of the bound radioligand. Our results demonstrate that 100 pM to 1 nM [3H]epibatidine labels mostly neuronal heteropentameric nicotinic receptors in bovine chromaffin cell membranes, and that cotinine is a low-affinity nicotinic ligand both in the adrenal chromaffin cell and in the brain receptors.

Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electrophoresis approach prior to mass spectrometry.

A preparative proteomic approach, involving liquid phase isoelectric focusing (IEF) in combination with one-dimensional electrophoresis and electroelution followed by mass spectrometry and database searches, was found to be an important tool for identifying low-abundant proteins (microgram/L) in human cerebrospinal fluid (CSF) and membrane proteins in human frontal cortex. Several neuron-related proteins, such as amyloid precursor-like protein, chromogranins A and B, glial fibrillary acid protein, beta-trace, transthyretin, ubiquitin, and cystatin C, were identified in CSF. Several types of proteins were also characterized from a detergent-solubilized human frontal cortex homogenate including membrane proteins such as synaptophysin, syntaxin and Na+/K+ ATPase. One-third of the identified proteins have not previously been identified in human CSF or human frontal cortex using proteomic techniques. The absence of these proteins in two-dimensional electrophoresis maps might be due to insufficient amounts or low solubility. The advantages of using preparative liquid phase electrophoretic separations for identifying proteins from complex biological mixtures are speed of analysis, high loadability in the IEF separation, nondiscrimination of membrane proteins or low abundance proteins, yielding sufficient amounts for characterization by mass spectrometry. The use of this strategy in proteome studies of CSF/brain tissue is expected to offer new perspectives in studies of the pathology of neurodegenerative diseases, and reveal new potential markers for brain disorders.

Effects of L-phenylalanine on acetylcholinesterase, (Na+,K+)-ATPase and Mg2+-ATPase activities in adult rat whole brain and frontal cortex.

The effect of different L-phenylalanine (Phe) concentrations (0.12-12.1 mM) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+-ATPase activities was investigated in homogenates of adult rat whole brain and frontal cortex at 37 degrees C. AChE, (Na+,K+)-ATPase and Mg2+-ATPase activities were determined after preincubation with Phe. AChE activity in both tissues showed a decrease up to 18% (p<0.01) with Phe. Whole brain Na+,K+-ATPase was stimulated by 30-35% (p<0.01) with high Phe concentrations, while frontal cortex Na+,K+-ATPase was stimulated by 50-55% (p<0.001). Mg2+-ATPase activity was increased only in frontal cortex with high Phe concentrations. It is suggested that: a) The inhibitory effect of Phe on brain AChE is not influenced by developmental factors, while the stimulation of Phe on brain Na+,K+-ATPase is indeed affected; b) The stimulatory effect of Phe on rat whole brain Na+,K+-ATPase is decreased with age; c) Na+,K+-ATPase is selectively more stimulated by high Phe concentrations in frontal cortex than in whole brain homogenate; d) High (toxic) Phe concentrations can affect Mg2+-ATPase activity in frontal cortex, but not in whole brain, thus modulating the amount of intracellular Mg2+.

Neuronal Subclass-Selective Loss of Pyruvate Dehydrogenase Immunoreactivity Following Canine Cardiac Arrest and Resuscitation

Chronic impairment of aerobic energy metabolism accompanies global cerebral ischemia and reperfusion and likely contributes to delayed neuronal cell death. Reperfusion-dependent inhibition of pyruvate dehydrogenase complex (PDHC) enzyme activity has been described and proposed to be at least partially responsible for this metabolic abnormality. This study tested the hypothesis that global cerebral ischemia and reperfusion results in the loss of pyruvate dehydrogenase immunoreactivity and that such loss is associated with selective neuronal vulnerability to transient ischemia. Following 10 min canine cardiac arrest, resuscitation, and 2 or 24 h of restoration of spontaneous circulation, brains were either perfusion fixed for immunohistochemical analyses or biopsy samples were removed for Western immunoblot analyses of PDHC immunoreactivity. A significant decrease in immunoreactivity was observed in frontal cortex homogenates from both 2 and 24 h reperfused animals compared to samples from nonischemic control animals. These results were supported by confocal microscopic immunohistochemical determinations of pyruvate dehydrogenase immunoreactivity in the neuronal cell bodies located within different layers of the frontal cortex. Loss of immunoreactivity was greatest for pyramidal neurons located in layer V compared to neurons in layers IIIc/IV, which correlates with a greater vulnerability of layer V neurons to delayed death caused by transient global cerebral ischemia.

Synaptic and plasticity-associated proteins in anterior frontal cortex in severe mental illness

Abnormalities of proteins involved in neurotransmission and neural plasticity at synapses are reported in schizophrenia, and may be markers of dysregulated neural connectivity in this illness. Studies of brain development and neural regeneration indicate a dynamic interplay between neural and oligodendroglial mechanisms in regulating synaptic plasticity and axonal sprouting. In the present study, markers of synapses (synaptophysin), plasticity (growth-associated protein-43) and oligodendrocytes (myelin basic protein) were investigated in anterior frontal cortex homogenates from individuals with schizophrenia and depression. Synaptophysin immunoreactivity was reduced in schizophrenics who died of natural causes relative to controls. Myelin basic protein immunoreactivity was decreased in both schizophrenics and depressed individuals who died by suicide. Overall, no changes were observed in growth-associated protein-43 immunoreactivity. However, a slight increase in immunoreactivity in depressed suicides relative to control was observed. These findings support the hypothesis that synaptic abnormalities are a substrate for disordered connectivity in severe mental illness, and suggest that synaptic–oligodendroglial interactions may contribute to the mechanism of dysregulation in certain cases.

Activities of key glycolytic enzymes in the brains of patients with Alzheimer's disease.

The activities of hexokinase, aldolase, pyruvate kinase, lactate dehydrogenase and glucose 6-phosphate dehydrogenase were determined in brains of patients with Alzheimer's disease (AD) and in age matched controls. For pyruvate kinase and lactate dehydrogenase a significant increase in specific activity was found in frontal and temporal cortex of AD brains, while the activities of aldolase and hexokinase are not changed. Glucose 6-phosphate dehydrogenase activity was significantly reduced in hippocampus. The increase of some glycolytic enzyme activities is correlated with increased contents of lactate dehydrogenase and glial fibrillary acidic protein (GFAP) in homogenates of frontal and temporal cortex and elevated phosphofructokinase (PFK) and GFAP in astrocytes from the same brain areas. The data extend previous findings on an increase in brain PFK specific activity in AD and suggest that the increased activity of some glycolytic enzymes may be, at least in part, the result of the reactive astrocytosis developing in the course of AD.

Castration differentially alters [3H]nisoxetine binding to norepinephrine uptake sites in olfactory bulb and frontal cortex of male rats.

In the present study, [3H]nisoxetine binding to norepinephrine (NE) uptake sites and [3H]norepinephrine uptake were investigated within olfactory bulb (OB) and frontal cortex homogenates from intact and castrated male rats. Statistically significant reductions in the number of [3H]nisoxetine binding sites (Bmax) were found in OB from the castrates, while significantly increased Bmax values were obtained in the frontal cortex. Castration also significantly altered the affinity (Kd) of [3H]nisoxetine binding in the frontal cortex, but not in the OB. Assessment of [3H]norepinephrine uptake showed that in neither brain regions were there any statistically significant differences in Km nor Vmax between the castrated and intact male rats, indicating that the basal uptake process is not changed following castration in either of these brain areas. These results demonstrate the differential effects of castration upon [3H]nisoxetine binding sites between the OB and frontal cortex. Such findings provide new evidence for one of the mechanisms by which androgens may modulate central noradrenergic activity.