D1/NMDA receptors and concurrent methamphetamine+ HIV-1 Tat neurotoxicity.

The interactive effects of HIV-1 infection and methamphetamine (METH) abuse in producing cognitive dysfunction represent a serious medical problem; however, the neural mechanisms underlying this interactive neurotoxicity remain elusive. In this study, we report that a combination of low, sub-toxic doses of METH + HIV-1 Tat 1-86 B, but not METH + HIV-1 gp120, directly induces death of rodent midbrain neurons in vitro. The effects of D1- and NMDA-receptor specific antagonists (SCH23390 and MK-801, respectively) on the neurotoxicity of different doses of METH or HIV-1 Tat alone and on the METH + HIV-1Tat interaction in midbrain neuronal cultures suggest that the induction of the cell death cascade by METH and Tat requires both dopaminergic (D1) and N-methyl D-aspartate (NMDA) receptor-mediated signaling. This interactive METH+Tat neurotoxicity does not occur in cultures of hippocampal neurons, which are predominately glutamatergic, express very low levels of dopamine receptors, and have no functional dopamine transporter (DAT). Thus, the presence of a subpopulation of neurons capable of dopamine release/uptake is essential for METH+Tat induction of the cell death cascade. Overall, our results support the hypothesis that METH and HIV-1 Tat disrupt the normal conjunction of signaling between D1 and NMDA receptors, resulting in neural dysfunction and death.

HIV-1 protein-mediated amyloidogenesis in rat hippocampal cell cultures.

Since the beginning of the highly active antiretroviral therapy (HAART) era, epidemiological evidence indicates an increasing incidence of Alzheimer's (AD)-like brain pathology in aging HIV patients. Emerging evidence warns of potential convergent mechanisms underlying HIV- and Abeta-mediated neurodegeneration. We found that HIV-1 Tat B and gp120 promote the secretion of Abeta 1-42 in primary rat fetal hippocampal cell cultures. Our results demonstrate that the variant of Tat expressed by the neurotropic subtype of HIV-1 virus (HIV-1 clade B) specifically induces both the release of amyloidogenic Abeta 1-42 and the accumulation of cell-bound amyloid aggregates. The results of the research rationalize testing of the ability of beta-amyloid aggregation inhibitors to attenuate HIV protein-mediated cognitive deficits in animal models of NeuroAIDS. The long-term goal of the study is to evaluate the potential benefits of anti-amyloidogenic therapies for management of cognitive dysfunction in aging HIV-1 patients.

Protein oxidation in the brain in Alzheimer's disease.

In this study we used immunohistochemistry and two-dimensional fingerprinting of oxidatively modified proteins (two-dimensional Oxyblot) together to investigate protein carbonyl formation in the Alzheimer's disease brain. Increased protein oxidation was detected in sections from the hippocampus and parahippocampal gyrus, superior and middle temporal gyri of six Alzheimer's disease and six age-matched control human subjects, but not in the cerebellum. In two brain regions severely affected by Alzheimer's disease pathology, prominent protein carbonyl immunoreactivity was localized in the cytoplasm of neurons without visual pathomorphological changes and degenerating neurons, suggesting that intracellular proteins might be significantly affected by oxidative modifications. Following two-dimensional electrophoresis the positions of some individual proteins were identified using specific antibodies, and immunoblot analysis for protein carbonyls was performed. These studies demonstrated the presence of protein carbonyl immunoreactivity in beta-tubulin, beta-actin and creatine kinase BB in Alzheimer's disease and control brain extracts. Protein carbonyls were undetectable in spots matching glial fibrillary acidic protein and tau isoforms. Specific protein carbonyl levels in beta-actin and creatine kinase BB were significantly higher in Alzheimer's disease than in control brain extract. beta-Tubulin did not demonstrate a significant increase in specific protein carbonyl content in Alzheimer's disease brains. We suggest that oxidative stress-induced injury may involve the selective modification of different intracellular proteins, including key enzymes and structural proteins, which precedes and may lead to the neurofibrillary degeneration of neurons in the Alzheimer's disease brain.

Aging in a dish: age-dependent changes of neuronal survival, protein oxidation, and creatine kinase BB expression in long-term hippocampal cell culture.

Results from different experimental systems demonstrate that increased oxidative damage plays a role in normal aging and age-associated pathology. In the current study, long-term cultures of hippocampal neurons were examined as a model system. It was established that neuronal survival in long-term culture decreases according to the Gompertz law and that neuronal "aging in the dish" is associated with increased oxidative damage of cell proteins. The increase of protein carbonyl formation in aged neurons was demonstrated both by Western blot analysis for oxidized proteins and by in situ immunocytochemical method, which was developed to analyze protein oxidation in fixed cells. In aging neuronal cultures, a gradual increase in creatine kinase (CK) content but decreased activity of enzyme per immunoreactive protein was found, suggesting the accumulation of inactive CK molecules. The increase in CK content was not a result of generalized protein elevation, since analysis of beta-actin content showed a time-dependent loss, probably reflecting decreased number of cellular processes with aging. These findings, showing "aging in a dish," consistent with the notion that aging is associated with increased protein oxidation, provide a system for study of age-related neurodegenerative disorders associated with oxidative stress.

The expression of several mitochondrial and nuclear genes encoding the subunits of electron transport chain enzyme complexes, cytochrome c oxidase, and NADH dehydrogenase, in different brain regions in Alzheimer's disease.

In this study, changes of the expression of two mitochondrial and two nuclear genes encoding the subunits of cytochrome c oxidase (CO) and NADH dehydrogenase (ND) were studied in the hippocampus, inferior parietal lobule, and cerebellum of 10 Alzheimer's disease (AD) and 10 age-matched control subjects. The altered proportion between CO II and CO IV mRNAs was observed in the AD brain. Changes of the proportion between CO II and CO IV transcripts may contribute to the kinetic perturbation of CO documented in AD. A coordinated decrease of ND4 and ND15 mRNAs was found in the AD hippocampus and inferior parietal lobule, but not in cerebellum. The decrease of ND4 gene expression may lead to the inhibition of normal ubiquinone oxidoreductase activity of ND. This study suggests that changes of the expression of mitochondrial and nuclear genes, encoding parts of ND and CO enzyme complexes, may contribute to alterations of oxidative metabolism in AD.

Adriamycin-induced changes of creatine kinase activity in vivo and in cardiomyocyte culture.

Adriamycin (ADM) is an anthracycline anti-neoplastic agent, whose clinical effectiveness is limited by severe side effects, including cardiotoxicity. The toxic effects of ADM are likely to be the consequence of the generation of free radicals. This study demonstrates that ADM induces significant changes in the activity of the oxidative sensitive enzyme creatine kinase (CK) in the heart in vivo and in a cardiomyocyte culture model. The changes observed are likely to reflect the ability of ADM to damage the plasma membrane of cardiac cells and to induce the direct inactivation of CK. The role for ADM-derived free radicals is one of the possible mechanisms for the CK inactivation observed during the ADM treatment.

Antioxidants protect against reactive oxygen species associated with adriamycin-treated cardiomyocytes.

Adriamycin (ADM) is a broad-spectrum antineoplastic antibiotic used to treat cancer patients. However, the usefulness of this drug is presently limited by the development of a dose-dependent cardiotoxicity. A current hypothesis for the ADM-induced cardiotoxicity is the production of reactive oxygen radicals by the drug. We utilized the fluorescent indicator 2',7'-dichlorodihydrofluorescein diacetate (DCFH/DA), in which fluorescence appears if reactive oxygen species (ROS) are present, to investigate the ability of ADM to generate reactive oxygen species and the potential protective effect of antioxidants in a cultured cardiomyocyte model. All three of the antioxidants (alpha-phenyl-tert-butyl nitrone (PBN), trolox, and 5-aminosalicylic acid (5-ASA)) tested in our ADM-treated myocytes provided protection against the oxidative stress induced by the drug. These findings suggest that antioxidants modulate ADM-induced oxidative stress, and they are discussed in terms of a possible therapeutic strategy in the prevention of cardiotoxicity resulting from ADM administration.

Oxidation of cytosolic proteins and expression of creatine kinase BB in frontal lobe in different neurodegenerative disorders.

The presence of the biomarkers of oxidative damage, protein carbonyl formation and the inactivation of oxidatively sensitive brain creatine kinase (CK BB, cytosolic isoform), were studied in frontal lobe autopsy specimens obtained from patients with different age-related neurodegenerative diseases: Alzheimer's disease (AD), Pick's disease (PkD), diffuse Lewy body disease (DLBD), Parkinson's disease (PD), and age-matched control subjects. The CK activity was significantly reduced in the frontal lobe of AD, PkD and DLBD subjects, and CK BB-specific mRNA was significantly reduced in AD and DLBD. Protein carbonyl content was significantly increased in AD, PkD and DLBD. The results of this study confirm that the presence of biomarkers of oxidative damage is related to the presence of histopathological markers of neurodegeneration. Our data suggest that oxidative damage contributes to the development of the symptoms of frontal dysfunction in AD, PkD and DLBD. The development of frontal dysfunction in idiopathic PD might be secondary to oxidative damage and neuronal loss primarily located in the nigrostriatal system. The results of CK BB expression analysis demonstrate that the loss of the isoenzyme in different neurodegenerative diseases is likely the consequence of its posttranslational modification, possibly oxidative damage. Changes in CK BB expression may be an early indicator of oxidative stress in neurons.

Adriamycin induces protein oxidation in erythrocyte membranes.

Adriamycin is an anthracycline antineoplastic agent whose clinical effectiveness is limited by severe side effects, including cardiotoxicity. A current hypothesis for adriamycin cardiotoxicity involves free radical oxidative stress. To investigate this hypothesis in a model system, we applied the technique of immunochemical detection of protein carbonyls, known to be increased in oxidized proteins, to study the effect of adriamycin on rat erythrocyte membranes. Erythrocytes obtained from adriamycin-treated rats demonstrated an increase of carbonyl formation in their membrane proteins. Yet, in separate experiments when adriamycin was incubated with rat erythrocyte ghosts, there was no significant increase of membrane protein carbonyls detected. In contrast, isolated erythrocytes incubated with an adriamycin-Fe3+ complex exhibited a robust carbonyl incorporation into their membrane proteins in a time-dependent manner. The level of carbonyl formation was dependent upon the concentration of Fe3+ known to form the adriamycin-Fe3+ complex. When the time course between protein carbonyl formation and lipid peroxidation was compared, protein carbonyl detection occurred earlier than lipid peroxidation as assayed by thiobarbituric acid reactive substances formation. These results are consistent with the notion that oxidative modification of membrane proteins may contribute to the development of the acute adriamycin-mediated toxicity.

Amyloid beta-peptide (1-40)-mediated oxidative stress in cultured hippocampal neurons. Protein carbonyl formation, CK BB expression, and the level of Cu, Zn, and Mn SOD mRNA.

Mechanism of amyloid beta-peptide (A beta) toxicity in cultured neurons involves the development of oxidative stress in the affected cells. A significant increase in protein carbonyl formation was detected in cultured hippocampal neurons soon after the addition of preaggregated A beta(1-40), indicating oxidative damage of proteins. We report that neurons, subjected to A beta(1-40), respond to A beta oxidative impact by activation of antioxidant defense mechanisms and alternative ATP-regenerating pathway. The study demonstrates an increase of Mn SOD gene expression and the restoration of Cu, Zn SOD gene expression to a normal level after temporary suppression. Partial loss of creatine kinase (CK) BB activity, which is the key enzyme for functioning of the creatine/phosphocreatine shuttle, was compensated in neurons surviving the A beta oxidative attack by increased production of the enzyme. As soon as the oxidative attack triggered by the addition of preaggregated A beta (1-40) to rat hippocampal cell cultures has been extinguished, CK BB expression and SOD isoenzyme-specific mRNA levels in surviving neurons return to normal. We propose that the maintenance of a constant level of CK function by increased CK BB production together with the induction of antioxidant enzyme gene expression in A beta-treated hippocampal neurons accounts for at least part of their adaptation to A beta toxicity.

Protein oxidation and enzyme activity decline in old brown Norway rats are reduced by dietary restriction.

The effect of aging and diet restriction (DR) on the activity of creatine kinase (CK), glutamine synthetase (GS) and protein carbonyl formation in the cerebellum, hippocampus and cortex of male and female brown Norway (BN) rats has been investigated. It was demonstrated that CK activity in three different regions of the rat brain declines with age by 30%. Age-related decrease of GS activity was only 10-13% and did not reach statistical significance. Consistent with previously published studies, age-related increase of protein carbonyl content in each brain area studied has been observed. Preventive effects of a caloric restricted diet on the age-associated protein oxidation and changes of the activity of CK and GS in the brain was observed for both aging male and female BN rats. DR delayed the accumulation of protein carbonyls. Age-related changes of CK activity in rat brain were abrogated by DR. The activity of GS in the brain of old rats subjected to the caloric restricted diet was higher than that in the brain of young animals fed ad libitum. The results are consistent with the notion that DR may relieve age-associated level of oxidative stress and lessen protein damage.

The expression of key oxidative stress-handling genes in different brain regions in Alzheimer's disease.

Alzheimer's disease (AD) has been hypothesized to be associated with oxidative stress. In this study, the expression of key oxidative stress-handling genes was studied in hippocampus, inferior parietal lobule, and cerebellum of 10 AD subjects and 10 control subjects using reverse transcriptase-polymerase chain reaction (RT-PCR). The content of Mn-, Cu,Zn-superoxide dismutases (Mn- and Cu,Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GSSG-R) mRNAs, and the "marker genes" (beta-actin and cyclophilin) mRNAs was determined. This study suggests that gene responses to oxidative stress can be significantly modulated by the general decrease of transcription in the AD brain. To determine if the particular oxidative stress handling gene transcription was induced or suppressed in AD, the "oxidative stress-handling gene/beta-actin" ratios were quantified and compared with control values in all brain regions studied. The Mn-SOD mRNA/beta-actin mRNA ratio was unchanged in all regions of the AD brain studied, but an increase of the Cu,Zn-SOD mRNA/beta-actin mRNA ratio was observed in the AD inferior parietal lobule. The levels of peroxidation handling (CAT, GSHPx, and GSSG-R) mRNAs normalized to beta-actin mRNA level were elevated in hippocampus and inferior parietal lobule, but not in cerebellum of AD patients, which may reflect the protective gene response to the increased peroxidation in the brain regions showing severe AD pathology. The results of this study suggest that region-specific differences of the magnitude of ROS-mediated injury rather than primary deficits of oxidative stress handling gene transcription are likely to contribute to the variable intensity of neurodegeneration in different areas of AD brain.

Oxidative modification of glutamine synthetase by amyloid beta peptide.

beta-Amyloid peptide (A beta), the main constituent of senile plaques and diffuse amyloid deposits in Alzheimer's diseased brain, was shown to initiate the development of oxidative stress in neuronal cell cultures. Toxic lots of A beta form free radical species in aqueous solution. It was proposed that A beta-derived free radicals can directly damage cell proteins via oxidative modification. Recently we reported that synthetic A beta can interact with glutamine synthetase (GS) and induce inactivation of this enzyme. In the present study we present the evidence that toxic A beta(25-35) induces the oxidation of pure GS in vitro. It was found that inactivation of GS by A beta, as well as the oxidation of GS by metal-catalyzed oxidation system, is accompanied by an increase of protein carbonyl content. As it was reported previously by our laboratory, radicalization of A beta is not iron or peroxide-dependent. Our present observations consistently show that toxic A beta does not need iron or peroxide to oxidize GS. However, treatment of GS with the peptide, iron and peroxide together significantly stimulates the protein carbonyl formation. Here we report also that A beta(25-35) induces carbonyl formation in BSA. Our results demonstrate that beta-peptide, as well as other free radical generators, induces carbonyl formation when brought into contact with different proteins.

The expression of creatine kinase isoenzymes in neocortex of patients with neurodegenerative disorders: Alzheimer's and Pick's disease.

Creatine kinase (CK) activity was found decreased in the brains of patients with Alzheimer's disease (AD) and Pick's disease (PD). However, the decrease of total CK activity in AD was more pronounced than in PD. Analysis of the activity of two CK isoforms, BCK and ubiquitous mitochondrial CK, demonstrated that the decrease of total CK activity in AD and PD was related to the decrease of BCK activity. The decline of CK activity both in AD and PD correlated well with the decline of the content of immunoreactive BCK in brain extracts. The BCK mRNA level in AD and PD was not significantly different from control patients and could not be the primary reason for decreases in CK content and activity. The decreased level of BCK in AD and PD brains might be caused by posttranscriptional events, which could affect the translation of BCK mRNA and/or lead to the inactivation and degradation of the enzyme. Because CK is sensitive to oxidative modification, it is possible that the changes observed in this study result from free radical damage.

Alpha 1-antichymotrypsin interaction with A beta (1-42) does not inhibit fibril formation but attenuates the peptide toxicity.

alpha 1-Antichymotrypsin (ACT) is intimately associated with senile plaques in the Alzheimer's diseased (AD) brain. It was reported that ACT can promote the polimerization of A beta (1-42) into amyloid filaments. It was suggested that neurotoxic amyloid deposits arise when beta-peptide is induced to form fibrils by ACT or other amyloid-promoting factors (pathological chaperones) expressed in AD brain. However, it was reported recently that ACT can inhibit fibrillization of A beta (1-40) and disaggregate pre-formed beta-amyloid fibrils of this synthetic A beta peptide. Our previous study [Aksenova et al., Neurosci. Lett., 411 (1996) 43-48] confirmed that ACT is able to inhibit A beta (1-40) aggregation into fibrils, but it was shown that at the same time ACT does not change the peptide cytotoxicity. In this report we have observed that interaction of ACT with A beta (1-42), unlike that for ACT-A beta (1-40) interaction, does not prevent the formation of insoluble A beta (1-42) aggregates, but completely blocks the peptide's toxicity in rat hippocampal cell cultures. These results are discussed in terms of the potential double role of peptide-protein interactions on A beta aggregation and neurotoxicity.

alpha-1-antichymotrypsin interaction with A beta (1-40) inhibits fibril formation but does not affect the peptide toxicity.

Recent studies have shown that senile plaque-associated or glial-derived proteins can prevent fibril formation of beta-amyloid peptide (A beta), while increasing the neurotoxicity of the latter (in the case of glutamine synthetase, apolipoprotein J or thrombin). alpha-1-Antichymotrypsin (ACT) is a glial-derived protein associated with senile plaques in the Alzheimer's brain. In this report we show that ACT, a minor protein component of beta-amyloid deposits, is able to inhibit A beta (1-40) aggregation into fibrils, but unable to modulate the toxicity of A beta (1-40) in primary rat hippocampal cell cultures. These results are discussed in terms of the potential role of glial-derived proteins on A beta aggregation and neurotoxicity.

Glutamine synthetase-induced enhancement of beta-amyloid peptide A beta (1-40) neurotoxicity accompanied by abrogation of fibril formation and A beta fragmentation.

beta-Amyloid peptide (A beta) is the main constituent in both senile plaques and diffuse deposits in Alzheimer's diseased brains. It was previously shown that synthetic A beta s were able to form free radical species in aqueous solution and cause both oxidative damage to cell proteins and inactivation of key metabolic enzymes. We also previously demonstrated that an interaction of A beta (1-40) with the oxidatively sensitive enzyme glutamine synthetase (GS) resulted in both inactivation of GS and an increase of A beta toxicity to hippocampal cell cultures. In the present study the enhancement of A beta toxicity during interaction with GS was found to be accompanied by abrogation of fibril formation and partial fragmentation of A beta (1-40). HPLC elution profiles demonstrated the production of several peptide fragments. Analysis of the amino acid sequence of the major fragments identified them as the first 15 and the last six amino acids of A beta (1-40). The fragmentation of A beta was inhibited by immunoprecipitation of GS.

Enhancement of beta-amyloid peptide A beta(1-40)-mediated neurotoxicity by glutamine synthetase.

The beta-amyloid peptide (A beta), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of A beta(1-40) and its C-end fragment, A beta(25-35). Toxic forms of A beta rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that A beta radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for A beta-derived oxidizing species, the present study was conducted to determine if GS could protect against A beta neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of A beta(1-40). The A beta-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of A beta(1-40) neurotoxicity.

Drastic reduction of the zinc- and magnesium-stimulated protein tyrosine kinase activities in Alzheimer's disease hippocampus.

Tyrosine phosphorylation of proteins from postmortem hippocampi of five Alzheimer's disease and five control cases have been compared. It was found that addition of Zn2+ or Mg2+ to membrane fractions of control hippocampi caused the phosphorylation of 32-, 40-, 55-, 60-, 80- and 100-kDa proteins or 43-, 55-, 60- and 90-KdA proteins, respectively. The phosphorylation of all these proteins is shown to be drastically reduced in Alzheimer's disease hippocampi. Vanadate, an inhibitor of protein tyrosine phosphatases, had no influence on the level of protein phosphorylation. Western blot analysis did not reveal any differences in the anti-phosphotyrosine immunoreactive membrane proteins from Alzheimer's disease and control hippocampi. Tyrosine kinase activity of immunoprecipitated p60c-src from Alzheimer's disease and control hippocampi were the same. In conclusion, the Zn(2+)- and Mg(2+)-stimulated tyrosine kinase activities, distinct from activity of p60c-src, are decreased in Alzheimer's disease hippocampus.

[The detection of cytoplasmic creatinine phosphokinase BB in nerve cell nuclei under normal conditions, in schizophrenia, and in Alzheimer's disease patients]. / Vyiavlenie tsitoplzmaticheskoi KFK BB v iadrakh nervnykh kletok v norme, pri shizofrenii i bolezni Al'tsgeimera.

This work is devoted to the study of CPK BB content in nuclear fraction of nervous cells in normal brain and in brains of mental (schizophrenia and Alzheimer disease) patients. With the help of the immunocytochemistry and immunoblotting was detected, that the nuclear membrane fraction of brain contains significant amount of CPK BB. On the contrary, in the nuclear membrane fractions of schizophrenic and Alzheimer disease brains the content of this isoenzyme decreased. Therefore in the brain of schizophrenic and Alzheimer disease patients the content of CPK BB decreased not only in cytosolic fractions, but also in the fractions of nuclear membranes. We demonstrated also, that cytosolic CPK BB associates not only with nuclear membranes, but with synaptosomal and microsomal fractions and mitochondrias of normal brain cells.