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Poly(ADP-ribose) polymerases (PARPs) are recognized as key regulators of cell survival or death. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. The enzyme may be overactivated in response to inflammatory cues, thus depleting cellular energy pools and eventually causing cell death. Accordingly, PARP-1 inhibitors, acting by competing with its physiological substrate NAD(+), have been proposed to play a protective role in a wide range of inflammatory and ischemia/reperfusion-associated diseases. Recently, it has also been reported that PARP-1 regulates proinflammatory mediators, including cytokines, chemokines, adhesion molecules, and enzymes (e.g., iNOS). Furthermore, PARP-1 has been shown to act as a coactivator of NF-κB- and other transcription factors implicated in stress/inflammation, as AP-1, Oct-1, SP-1, HIF, and Stat-1. To further substantiate this hypothesis, we tested the biomolecular effects of PARP-1 inhibitors DPQ and PJ-34 on human glioblastoma cells, induced to a proinflammatory state with lipopolysaccharide and Interferon-γ. PARP-1 expression was evaluated by laser scanning confocal microscopy immunofluorescence (LSM); nitrite production, LDH release and cell viability were also determined. LSM of A-172, SNB-19 and CAS-1 cells demonstrated that DPQ and PJ-34 downregulate PARP-1 expression; they also cause a decrease of LDH release and nitrite production, while increasing cell viability. Similar effects were caused in all three cell lines by N-mono-methyl-arginine, a well known iNOS inhibitor, and by L-carnosine and trehalose, two antioxidant molecules. These results demonstrate that, similar to other well characterized drugs, DPQ and PJ-34 reduce cell inflammation and damage that follow PARP-1 overexpression, while they increase cell survival: this suggests their potential exploitation in clinical Medicine.

The predominant molecular symptom of aging is the accumulation of altered gene products. Moreover, several conditions including protein, lipid or glucose oxidation disrupt redox homeostasis and lead to accumulation of unfolded or misfolded proteins in the aging brain. Alzheimer's and Parkinson's diseases or Friedreich ataxia are neurological diseases sharing, as a common denominator, production of abnormal proteins, mitochondrial dysfunction and oxidative stress, which contribute to the pathogenesis of these so called "protein conformational diseases". The central nervous system has evolved the conserved mechanism of unfolded protein response to cope with the accumulation of misfolded proteins. As one of the main intracellular redox systems involved in neuroprotection, the vitagene system is emerging as a neurohormetic potential target for novel cytoprotective interventions. Vitagenes encode for cytoprotective heat shock proteins (Hsp) Hsp70 and heme oxygenase-1, as well as thioredoxin reductase and sirtuins. Nutritional studies show that ageing in animals can be significantly influenced by dietary restriction. Thus, the impact of dietary factors on health and longevity is an increasingly appreciated area of research. Reducing energy intake by controlled caloric restriction or intermittent fasting increases lifespan and protects various tissues against disease. Genetics has revealed that ageing may be controlled by changes in intracellular NAD/NADH ratio regulating sirtuin, a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Recent findings suggest that several phytochemicals exhibit biphasic dose responses on cells with low doses activating signaling pathways that result in increased expression of vitagenes encoding survival proteins, as in the case of the Keap1/Nrf2/ARE pathway activated by curcumin and NAD/NADH-sirtuin-1 activated by resveratrol. Consistently, the neuroprotective roles of dietary antioxidants including curcumin, acetyl-l-carnitine and carnosine have been demonstrated through the activation of these redox-sensitive intracellular pathways. Although the notion that stress proteins are neuroprotective is broadly accepted, still much work needs to be done in order to associate neuroprotection with specific pattern of stress responses. In this review the importance of vitagenes in the cellular stress response and the potential use of dietary antioxidants in the prevention and treatment of neurodegenerative disorders is discussed. © 2008 Springer Science+Business Media, LLC.

Age-related impairment of functionality of the central nervous system (CNS) is associated with increased susceptibility to develop many neurodegenerative diseases. Increased oxidative stress in the CNS of aged animals is manifested by increased protein oxidation, which is believed to contribute to the age-related learning and memory deficits. Glutamate dysregulation, mitochondrial dysfunction and impaired protein synthesis are observed in aged brains, along with increased protein oxidation. Interestingly, all of these age-related cellular alterations can be improved by caloric restriction (CR), which can also improve the plasticity and recovery of the CNS. Although the beneficial effects of CR on brains are well established, the mechanism(s) of its action remains unclear. In order to gain insight into the mechanism of CR in the brain, we located the brain regions that are benefited the most from reduced oxidative stress by CR. Along with other brain regions, striatum (ST) showed significantly decreased bulk protein carbonyl levels and hippocampus (HP) showed decreased bulk protein 3-nitrotyrosine (3-NT) levels in CR aged rats when compared to those of age matched controls. To determine which proteins were oxidatively modified in these brain regions, we used parallel proteomics approach to identify the proteins that are altered in oxidation and expression. The specific carbonyl levels of pyruvate kinase M2 (PKM2), alpha-enolase (ENO1), inositol monophosphatase (INSP1), and F1-ATPase Chain B (ATP-F1B) were significantly decreased in ST of aged CR rats. In contrast, the expression levels of phosphoglycerate kinase 1 (PKG1), inosine monophosphate cyclohydrolase (IMPCH) and F1-ATPase Chain A (ATP-F1A) were significantly increased in the ST of CR rats. In the hippocampus of CR rats, the specific 3-NT levels of malate dehydrogenase (MDH), phosphoglycerate kinase 1 (PKG1) and 14-3-3 zeta protein were significantly decreased and expression levels of DLP1 splice variant 1 (DLP1), mitochondrial aconitase (ACO2), dihydrolipoamide dehydrogenase (DLDH), neuroprotective peptide H3 (NPH3), and eukaryotic translation initiation factor 5A (eIF-5A) are increased. Moreover, an unnamed protein product (UNP1) with similar sequence to initiation factor 2 (IF-2) was decreased in the HP of CR rats. Our data support the hypothesis that CR induces a mild metabolic stress response by increasing the production of neurotrophic proteins, therefore, priming neurons against apoptosis. Moreover, our study shows that the improvement of glutamate dysregulation, mitochondrial dysfunction and protein synthesis by CR is, at least partially, due to the CR-mediated alteration of the oxidation or the expression of PKM2, ENO1, INSP1, ATP-F1B, PKG1, IMPCH, ATP-F1A MDH, PKG1 and 14-3-3 zeta protein, DLP1, ACO2, DLDH, NPH3, eIF-5A and UNP1. This study provides valuable insights into the mechanisms of the beneficial factors on brain aging by CR.

Heme oxygenase (HO) and cyclooxygenase (COX) are two hemeproteins involved in the regulation of several functions in the nervous system. Heme oxygenase is the enzyme responsible for the degradation of heme into ferrous iron, carbon monoxide (CO), and biliverdin, the latter being further reduced in bilirubin (BR) by biliverdin reductase. Heme oxygenase-derived CO is a gaseous neuromodulator and plays an important role in the synaptic plasticity, learning and memory processes, as well as in the regulation of hypothalamic neuropeptide release, whereas BR is an endogenous molecules with antioxidant and anti-nitrosative activities. Cyclooxygenase is considered a pro-inflammatory enzyme as free radicals and prostaglandins (PGs) are produced during its catalytic cycle. Although PGs are also involved in a variety of physiologic conditions including angiogenesis, hemostasis, or regulation of kidney function, upregulation of COX and increase in PGs levels are a common feature of neuroinflammation. In the brain, a functional interplay exists between HO and COX. Heme oxygenase regulates COX activity by reducing the intracellular heme content or by generating CO, which stimulates PGE2 release. Increased levels of PGs, free radicals, and the associated oxidative stress serve in the brain as a trigger for the induction of HO isoforms which increases cellular antioxidant defenses to counteract oxidative damage. The importance of the interaction between HO and COX in the regulation of physiologic brain functions, and its relevance to neuroprotective or neurodegenerative mechanisms are discussed. © 2006 Wiley-Liss, Inc.

A great deal of increasing evidence designs PARP as a multifunctional protein implicated in many cellular functions. Much interest is emerging to understand the precise mechanisms by which PARP mediates genome stabilization and protection against damage, as well as its involvement in cell death, either apoptotic or necrotic. Aside from the clearly established role of PARP hyperactivation in necrotic cell death, after excessive DNA damage and energy failure, it appears to be actively involved in the phenomenon of apoptosis. However, its exact role is still controversial. The identification of several enzymes sharing the poly(ADP-ribose) polymerase catalytic domain (PARPs), but with different features and subcellular localization, has opened a new perspective in the field of poly(ADP-ribosyl)ation. The picture of the role of PARP in the control of cell homeostasis became even more complex after demonstration of its implication in the regulation of gene transcription. The notion that energy failure is the sole mechanism by which PARP promotes cell death is therefore under reevaluation.

Neuronal nitric oxide synthase (nNOS) is a constitutively expressed and calcium-dependent enzyme. Despite predominantly expressed in neurons, nNOS has been also found in astrocytes, although at lower expression levels. We have studied the regulation of nNOS expression in cultured rat astrocytes from cortex and spinal cord by Western blotting and immunocytochemistry. nNOS was not detectable in cultured astrocytes grown in serum-containing medium (SCM), but was highly expressed after serum deprivation. Accordingly, calcium-dependent NOS activity and both intracellular nitrite levels and nitrotyrosine immunoreactivity after glutamate stimulation were higher in serum-deprived astrocytes than in cells grown in SCM. Serum deprivation induced a modification of astrocytes morphology, from flat to stellate. nNOS upregulation was also observed in reactive astrocytes of rat hippocampi after electrically induced status epilepticus, as demonstrated by double-labeling experiments. Thus, nNOS upregulation occurs in both in vitro stellate and in vivo reactive astrocytes, suggesting a possible involvement of glial nNOS in neurological diseases characterized by reactive gliosis.

In the present study, we investigated the effect of Ginkgo biloba extract, EGb 761, and one of its components, bilobalide, on gene expression of subunit 1 of mitochondrial NADH dehydrogenase (ND1) in PC12 cells. By Northern blot analysis we found a ∼2-fold significant increase in ND1 mRNA level, after 48 and 72 h exposure to 100 μg/ml EGb 761 and to 10 μg/ml bilobalide. We also evaluated, by oxygraphy measurements, mitochondrial respiration during state 3 and state 4. In cells treated with EGb 761 and bilobalide for 48 and 72 h, state 4 respiration was significantly decreased, and the respiratory control ratio was increased. These results provide evidence that EGb 761 and bilobalide exert their protective effects by up-regulating mitochondrial ND1 gene expression and by decreasing state 4 respiration, whose increase is thought to be responsible for oxidative damage.

Nitric oxide and other reactive nitrogen species appear to play several crucial roles in the brain. These include physiological processes such as neuromodulation, neurotransmission and synaptic plasticity, and pathological processes such as neurodegeneration and neuroinflammation. There is increasing evidence that glial cells in the central nervous system can produce nitric oxide in vivo in response to stimulation by cytokines and that this production is mediated by the inducible isoform of nitric oxide synthase. Although the etiology and pathogenesis of the major neurodegenerative and neuroinflammatory disorders (Alzheimer's disease, amyothrophic lateral sclerosis, Parkinson's disease, Huntington's disease and multiple sclerosis) are unknown, numerous recent studies strongly suggest that reactive nitrogen species play an important role. Furthermore, these species are probably involved in brain damage following ischemia and reperfusion, Down's syndrome and mitochondrial encephalopathies. Recent evidence also indicates the importance of cytoprotective proteins such as heat shock proteins (HSPs) which appear to be critically involved in protection from nitrosative and oxidative stress. In this review, evidence for the involvement of nitrosative stress in the pathogenesis of the major neurodegenerative/ neuroinflammatory diseases and the mechanisms operating in brain as a response to imbalance in the oxidant/antioxidant status are discussed.

A study was undertaken in rats to evaluate the effects of short-term oral ethanol administration on the levels of fatty acid ethyl esters (FAEE) in brain and peripheral organs in the presence and absence of pretreatment with L-carnitine. Administration of ethanol to rats for seven days resulted in fatty acid ethyl ester formation, particularly in the heart and brain, but also in the kidney and liver. FAEE generation was associated with a significant increase of GSH transferase activity. Treatment with L-carnitine significantly reduced both FAEE and GSH transferase activity, and these effects were associated with a significant decrease in alcohol blood concentrations. The present evidence supports the hypothesis that fatty acid ethyl esters could be mediators involved in the production of alcohol-dependent syndromes. Administration of L-carnitine through an increment in lipid metabolism and turnover, and by the modulation of cellular antioxidant enzymes, greatly reduces these metabolic abnormalities supporting its potential usefulness as a pharmacological tool in alcoholism management.

Recent studies suggest a functional diversity of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)- and calretinin (CR)-positive cells, which represent different populations of non-principal cells. To this end, non-radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double-immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV-containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR-A and GluR-D mRNAs, moderate levels of GluR-C and low levels of GluR-B mRNAs in all the examined regions of hippocampus. The vast majority of CR-containing cells have a much lower expression of GluR-A, -C and -D mRNA than PV-positive neurons, although similarly featuring low levels of GluR-B mRNA. Only a subpopulation of CR-containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR-A and -D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR-B subunit expression, PV- and CR-containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.

In the present study we evaluated the effects of NO synthase (NOS) induction on the regulation of cytochrome c oxidase (CO) and F0F1-ATPase subunit expression in astroglial and mixed cortical cell cultures. In mixed cortical cell cultures, 18 h of treatment with lipopolysaccharide (LPS, 0.1 microgram/mL) plus interferon-gamma (INF-gamma, 10 U/mL) caused an increase of mRNAs for CO-I, F0F1-ATPase 6 and also for iNOS at 20 DIV. The induction of both CO-I and F0F1-ATPase 6 was abolished by the NOS inhibitor N-monomethyl-L-arginine (NMMA) or by the enzymatic scavenger superoxide dismutase/catalase (SOD/CAT). In primary astroglial cell cultures, treatment for 18 h with increasing concentrations of LPS and INF gamma, produced an increase in the amount of mitochondrial encoded CO-I and -II subunits, with no significant modifications of nuclear encoded subunit IV. An increase was also observed at level of transcription for CO-I and -II, and F0F1-ATPase 6 mRNAs. These effects were abolished by addition of NMMA or SOD/CAT. mRNA induction of CO-I was higher in mixed cortical than in astroglial cell cultures while that of F0F1-ATPase 6 was similar in both cell types. These results suggest that the expression of mitochondrial encoded subunits (CO-I, CO-II and F0F1-ATPase 6) is up-regulated in response to oxygen and NO reactive species. The activity of cytochrome c oxidase decreased after LPS/INF gamma treatment in both astroglial and mixed cortical cultures. The activity of ATP synthase was unmodified, while ATP content drastically decreased after LPS/INF gamma treatment, in both astroglial and mixed cortical cultures. The enzymatic activities of catalase and Mn-SOD (mitochondrial) showed a significant increase after LPS/INF gamma treatment, which was abolished by NMMA.

In the present study we analyzed the age-dependent changes of mRNA levels for cytochrome c oxidase and F0F1-ATP synthase subunits in rat cerebral cortex and cerebellum. To establish whether the regulation of expression is transcriptional or post-transcriptional, the results were compared to those related to protein subunits levels, of the same enzymatic complexes, previously observed. The different patterns of age-related changes of mRNA subunits, in particular the lower increments, compared with those related to protein subunits, indicate that post-transcriptional mechanisms of regulation might be involved in the coordinated expression of the various subunits of each complex. Northern blotting analyses of RNA from the cerebellum of rats at the various ages, showed also differences in age-dependent patterns of transcription between cerebral cortex and cerebellum. Moreover, the major age-dependent changes of mitochondrial-encoded subunits, compared with the nuclear-encoded ones, previously observed at proteins level, occur also during transcription.

The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. A prediction of this theory is that, among species, differential rates of aging may be apparent on the basis of intrinsic differences in oxidative damage accrual. Although widely accepted, there is a growing number of exceptions to this theory, most contingently related to genetic model organism investigations. Proteins are one of the prime targets for oxidative damage and cysteine residues are particularly sensitive to reversible and irreversible oxidation. The adaptation and survival of cells and organisms requires the ability to sense proteotoxic insults and to coordinate protective cellular stress response pathways and chaperone networks related to protein quality control and stability. The toxic effects that stem from the misassembly or aggregation of proteins or peptides, in any cell type, are collectively termed proteotoxicity. Despite the abundance and apparent capacity of chaperones and other components of homeostasis to restore folding equilibrium, the cell appears poorly adapted for chronic proteotoxic stress which increases in cancer, metabolic and neurodegenerative diseases. Pharmacological modulation of cellular stress response pathways has emerging implications for the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. A critical key to successful medical intervention is getting the dose right. Achieving this goal can be extremely challenging due to human inter-individual variation as affected by age, gender, diet, exercise, genetic factors and health status. The nature of the dose response in and adjacent to the therapeutic zones, over the past decade has received considerable advances. The hormetic dose-response, challenging long-standing beliefs about the nature of the dose-response in a lowdose zone, has the potential to affect significantly the design of pre-clinical studies and clinical trials as well as strategies for optimal patient dosing in the treatment of numerous diseases. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing stress responses, including carnitines. This paper describes in mechanistic detail how hormetic dose responses are mediated for endogenous cellular defense pathways, including the possible signaling mechanisms by which the carnitine system, by interplaying metabolism, mitochondrial energetics and activation of critical vitagenes, modulates signal transduction cascades that confer cytoprotection against chronic degenerative damage associated to aging and neurodegenerative disorders.

Poly(ADP-ribose) polymerases (PARPs) play a crucial role in DNA damage surveillance through their nick sensor functions. Since PARPs' over activation leads to an excessive consumption of NAD(+) and ATP depletion, these enzymes also are involved in the early events of programmed cell death as well as in necrosis. In order to verify the protective action of L: -carnosine and trehalose against NO induced cell death, in the present study we examined their effects on the expression of PARP-1, PARP-2 and iNOS in primary rat astrocyte and oligodendrocyte cells, treated with lipopolysaccharide (LPS) and interferon gamma (INFγ), through semi-quantitative PCR and western analysis. To further characterize the molecular mechanisms underlying L-carnosine and trehalose action, we measured cell viability, nitrite production and LDH release. The data obtained clearly demonstrate that in the stress model employed L-carnosine and trehalose down regulate PARP-1 and PARP-2 expression in both cell phenotypes, thus suggesting their possible application in clinical trials.

The enzyme poly(ADP-ribose)polymerase (PARP) has a leader role in the DNA damage survey mechanisms by its nick-sensor function, but it is also involved in the early events of the programmed cell death, particularly during inflammatory injury, as a coactivator of NF-kB. In the present study, we evaluated the PARP involvement in the mechanisms of protection and/or cell death in rat astroglial cell cultures during the early phase of proinflammatory commitment after lipopolysaccharide and interferon gamma treatment. According with the recent findings that PARP-1 phosphorylation by MAPK/ERK-2 pathway seems to modulate PARP activation, in time course experiments we demonstrated that a very early PARP activation and expression is able to trigger a cell death pathway, DNA damage independent, during strong proinflammatory insults, maintaining its role of guardian of the genome stability only during the normal cell cycling.

Oxidative stress has been suggested to play a main role in the pathogenesis of type 2 diabetes mellitus and its complications. As a consequence of this increased oxidative status, a cellular-adaptive response occurs requiring functional chaperones, antioxidant production, and protein degradation. This study was designed to evaluate systemic oxidative stress and cellular stress response in patients suffering from type 2 diabetes–induced nephropathy and in age-matched healthy subjects. Systemic oxidative stress has been evaluated by measuring advanced glycation end-products (pentosidine), protein oxidation (protein carbonyls [DNPH]), and lipid oxidation (4-hydroxy-2-nonenal [HNE] and F2-isoprostanes) in plasma, lymphocytes, and urine, whereas the lymphocyte levels of the heat shock proteins (Hsps) heme oxygenase-1 (HO-1), Hsp70, and Hsp60 as well as thioredoxin reductase-1 (TrxR-1) have been measured to evaluate the systemic cellular stress response. We found increased levels of pentosidine (P < 0.01), DNPH (P < 0.05 and P < 0.01), HNE (P < 0.05 and P < 0.01), and F2-isoprostanes (P < 0.01) in all the samples from type 2 diabetic patients with nephropathy with respect to control group. This was paralleled by a significant induction of cellular HO-1, Hsp60, Hsp70, and TrxR-1 (P < 0.05 and P < 0.01). A significant upregulation of both HO-1 and Hsp70 has been detected also in lymphocytes from type 2 diabetic patients without uraemia. Significant positive correlations between DNPH and Hsp60, as well as between the degree of renal failure and HO-1 or Hsp70, also have been found in diabetic uremic subjects. In conclusion, patients affected by type 2 diabetes complicated with nephropathy are under condition of systemic oxidative stress, and the induction of Hsp and TrxR-1 is a maintained response in counteracting the intracellular pro-oxidant status.

At the end of the 1980s, it was clearly demonstrated that cells produce nitric oxide and that this gaseous molecule is involved in the regulation of the cardiovascular, immune and nervous systems, rather than simply being a toxic pollutant. In the CNS, nitric oxide has an array of functions, such as the regulation of synaptic plasticity, the sleep-wake cycle and hormone secretion. Particularly interesting is the role of nitric oxide as a Janus molecule in the cell death or survival mechanisms in brain cells. In fact, physiological amounts of this gas are neuroprotective, whereas higher concentrations are clearly neurotoxic.

Increasing evidence suggests a critical role for oxidative and nitrosative stress in the pathogenesis of most important neurodegenerative disorders. Parkinson's disease (PD) is a neurodegenerative disease characterized by a severe depletion in number of dopaminergic cells of the substantia nigra (SN). Administration of L-DOPA (LD) is the more effective treatment for patients with PD. However, the vast majority of patients suffer LD-related complications, which represent the major problem in the clinical management of PD. In the present study, LD administration to rats resulted in a significant dose-dependent increase in Hsp70 synthesis which was specific for the SN. The amount of 70 kDa protein increased after 6 h treatment reaching the maximal induction after 24-48 h. Induction of Hsp70 in the SN was associated with a significant increase in constitutive Hsc70 and mitochondrial Hsp60 stress proteins, and with increased expression of mitochondrial complex I whereas no significant changes were found in the activity of complex IV. In the same experimental conditions, a significant decrease in reduced glutathione was observed, which was associated with an increased content of oxidized glutathione content as well as nitric oxide (NO) synthase activity, NO metabolites and nitrotyrosine immunoreactivity. Interestingly, Hsp70 induction, iNOS up-regulation and nitrotyrosine formation have been confirmed also in SN and striatum of rats treated with LD and carbidopa, this latter being an inhibitor of the peripheral DOPA decarboxylase. Our data are in favor of the importance of the heat shock signal pathway as a basic mechanism of defense against neurotoxicity elicited by free radical oxygen and nitrogen species produced in aging and neurodegenerative disorders.

The term “nitrosative stress” is used to indicate the cellular damage elicited by reactive nitrogen species (RNS), including nitric oxide (NO) and its congeners peroxynitrite (OONO − ), N 2 O 3 , nitroxyl anion, and nitrosonium. Nitrosative stress has been implicated in the pathogenesis of neurodegenerative disorders. In this chapter, the importance of nitrosative stress in the pathogenesis of brain aging and neurodegenerative disorders as well as the key role played by HO-1 in modulating the onset and progression of Alzheimer's disease (AD) and Parkinson's disease (PD) is discussed. The mechanism(s) by which nitrosative stress is involved in the pathogenesis of neurodegenerative diseases is manifold. In fact, NO itself has been shown to regulate selected proteins—such as parkin, metalloproteinase-9 (MMP-9), and glyceraldehyde-3-phosphate dehydrogenase (GADPH) —via an S -nitrosylation reaction, thereby triggering the onset and progression of PD and other neurodegenerative disorders. On the other hand, NO can react with reactive oxygen species (ROS), in particular superoxide, derived from mitochondrial impairment and form peroxynitrite, which nitrosates proteins and forms 3-NT-modified proteins, which cause an impairment in cellular metabolic pathways and therefore cell death. Therefore, it is possible to hypothesize that drugs which are able to counteract iNOS induction and therefore excess NO formation can be very useful in limiting the consequences of oxidative and nitrosative stress associated with neurodegenerative disorders. Furthermore, the compelling evidence of the vitagene network as a defense system operating in the brain during times of oxidative and nitrosative stress opens new perspectives in the treatment of neurodegenerative diseases.

Reduced expression and/or activity of antioxidant proteins lead to oxidative stress, accelerated aging and neurodegeneration. However, while excess reactive oxygen species (ROS) are toxic, regulated ROS play an important role in cell signaling. Perturbation of redox status, mutations favoring protein misfolding, altered glyc(osyl)ation, overloading of the product of polyunsaturated fatty acid peroxidation (hydroxynonenals, HNE) or cholesterol oxidation, can disrupt redox homeostasis. Collectively or individually these effects may impose stress and lead to accumulation of unfolded or misfolded proteins in brain cells. Alzheimer's (AD), Parkinson's and Huntington's disease, amyotrophic lateral sclerosis and Friedreich's ataxia are major neurological disorders associated with production of abnormally aggregated proteins and, as such, belong to the so-called "protein conformational diseases". The pathogenic aggregation of proteins in non-native conformation is generally associated with metabolic derangements and excessive production of ROS. The "unfolded protein response" has evolved to prevent accumulation of unfolded or misfolded proteins. Recent discoveries of the mechanisms of cellular stress signaling have led to new insights into the diverse processes that are regulated by cellular stress responses. The brain detects and overcomes oxidative stress by a complex network of "longevity assurance processes" integrated to the expression of genes termed vitagenes. Heat-shock proteins are highly conserved and facilitate correct protein folding. Heme oxygenase-1, an inducible and redox-regulated enzyme, has having an important role in cellular antioxidant defense. An emerging concept is neuroprotection afforded by heme oxygenase by its heme degrading activity and tissue-specific antioxidant effects, due to its products carbon monoxide and biliverdin, which is then reduced by biliverdin reductase in bilirubin. There is increasing interest in dietary compounds that can inhibit, retard or reverse the steps leading to neurodegeneration in AD. Specifically any dietary components that inhibit inappropriate inflammation, AbetaP oligomerization and consequent increased apoptosis are of particular interest, with respect to a chronic inflammatory response, brain injury and beta-amyloid associated pathology. Curcumin and ferulic acid, the first from the curry spice turmeric and the second a major constituent of fruit and vegetables, are candidates in this regard. Not only do these compounds serve as antioxidants but, in addition, they are strong inducers of the heat-shock response. Food supplementation with curcumin and ferulic acid are therefore being considered as a novel nutritional approach to reduce oxidative damage and amyloid pathology in AD. We review here some of the emerging concepts of pathways to neurodegeneration and how these may be overcome by a nutritional approach.

Carnosine, a b-alanyl-histidine dipeptide, is present in quite high concentration in brain, it seems to exert a protective effect against cell damage caused by oxidants. In the present study we investigated the effect of carnosine addition (20 mM) against NO-induced oxidative stress in rat astroglial cell cultures. We measured cell viability (MTT), nitrite production, LDH release into culture medium and the effect of a specific inhibitor of iNOS, the N-mono-metil-arginine (NMMA 1 mM). After LPS (4 lg/ml) and INF-c (400U/ml) treatment, a decrease of cell viability and an increase of LDH and nitrite release was observed. Interestingly all these effects were prevented by carnosine addition. Moreover, as determined by ESI Mass spectra, carnosine was able to trap NO forming NO-carnosine adducts. Furthermore an increase of cell survival and a decrease of LDH and nitrite release were also observed after NMMA addition demonstrating that oxidative stress in these conditions is due to iNOS induction. The effects of carnosine treatment in all the experimental conditions tested, demonstrate a marked protection of this compound against NO-induced oxidative stress. The data obtained strongly suggest an antioxidant action of carnosine, however further investigations are necessary to better clarify its role for possible clinical application.

Alzheimer's disease (AD) is a neurodegenerative disorder with cognitive and memory decline, personality changes, and synapse loss. Increasing evidence indicates that factors such as oxidative and nitrosative stress, glutathione depletion, and impaired protein metabolism can interact in a vicious cycle, which is central to AD pathogenesis. In the present study, we demonstrate that brains of AD patients undergo oxidative changes classically associated with a strong induction of the so-called vitagenes, including the heat shock proteins (HSPs) heme oxygenase-1 (HO-1), HSP60, and HSP72, as well as thioredoxin reductase (TRXr). In inferior parietal brain of AD patients, a significant increase in the expression of HO-1 and TRXr was observed, whereas HO-2 expression was decreased, compared with controls. TRHr was not increased in AD cerebellum. Plasma GSH was decreased in AD patients, compared with the control group, and was associated with a significant increase in oxidative stress markers (i.e., GSSG, hydroxynonenal, protein carbonyl content, and nitrotyrosine). In AD lymphocytes, we observed an increased expression of inducible nitric oxide synthase, HO-1, Hsp72, HSP60, and TRXr. Our data support a role for nitrative stress in the pathogenesis of AD and indicate that the stress-responsive genes, such as HO-1 and TRXr, may represent important targets for novel cytoprotective strategies.

Efficient functioning of maintenance and repair processes seem to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, under control of several genes termed vitagenes. These include members of the heat shock protein system, and there is now evidence that the heat shock response contributes to establishing a cytoprotective state in a wide variety of human conditions, including inflammation, neurodegenerative disorders, and aging. Among the various heat shock proteins, heme oxygenase-1 has received considerable attention; it has been recently demonstrated that heme oxygenase-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Acetyl-L-carnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, we report here that treatment of astrocytes with acetyl-L-carnitine induces heme oxygenase-1 in a dose- and time-dependent manner and that this effect was associated with up-regulation of heat shock protein 60 as well as high expression of the redox-sensitive transcription factor Nrf2 in the nuclear fraction of treated cells. In addition, we show that addition of acetyl-L-carnitine to astrocytes, prior to proinflammatory lipopolysaccharide- and interferon-gamma-induced nitrosative stress, prevents changes in mitochondrial respiratory chain complex activity, protein nitrosation and antioxidant status induced by inflammatory cytokine insult. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly, manipulation of endogenous cellular defense mechanisms via acetyl-L-carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age-related diseases. Copyright (c) 2005 Wiley-Liss, Inc.

Fragile X (FRAX) syndrome is a common inherited form of mental retardation resulting from the lack of fragile X mental retardation protein (FMRP) expression. The consequences of FMRP absence in the mechanism underlying mental retardation are unknown. Here, we tested the hypothesis that glutamate receptor (GluR) expression might be altered in FRAX syndrome. Initialin situhybridization and Western blotting experiments did not reveal differences in mRNA levels and protein expression of AMPA and NMDA subunits and metabotropic glutamate subtype 5 (mGlu5) receptors between control andFmr1knock-out (KO) mice during postnatal development. However, a detergent treatment (1% Triton X-100) revealed a selective reduction of mGlu5 receptor expression in the detergent-insoluble fraction of synaptic plasma membranes (SPMs) from KO mice, with no difference in the expression of NR2A, GluR1, GluR2/3, GluR4, and Homer proteins. mGlu5 receptor expression was also lower in Homer immunoprecipitates fromFmr1KO SPMs. Homer, but not NR2A, mGlu5, and GluR1, was found to be less tyrosine phosphorylated inFmr1KO than control mice. Our data indicate that, in FRAX syndrome, a reduced number of mGlu5 receptors are tightly linked to the constituents of postsynaptic density and, in particular, to the constitutive forms of Homer proteins, with possible consequent alterations in synaptic plasticity.

Aging is associated with a reduced ability to cope with physiological challenges. Although the mechanisms underlying age-related alterations in stress tolerance are not well defined, many studies support the validity of the oxidative stress hypothesis, which suggests that lowered functional capacity in aged organisms is the result of an increased generation of reactive oxygen and nitrogen species. Increased production of oxidants in vivo can cause damage to intracellular macromolecules, which can translate into oxidative injury, impaired function and cell death in vulnerable tissues such as the brain. To survive different types of injuries, brain cells have evolved networks of responses, which detect and control diverse forms of stress. This is accomplished by a complex network of the so-called longevity assurance processes, which are composed of several genes termed vitagenes. Among these, heat shock proteins form a highly conserved system responsible for the preservation and repair of the correct protein conformation. The heat shock response contributes to establishing a cytoprotective state in a wide variety of human diseases, including inflammation, cancer, aging and neurodegenerative disorders. Given the broad cytoprotective properties of the heat shock response, there is now a strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. Acetylcarnitine is proposed as a therapeutic agent for several neurodegenerative disorders, and there is now evidence that it may play a critical role as modulator of cellular stress response in health and disease states. In the present review, we first discuss the role of nutrition in carnitine metabolism, followed by a discussion of carnitine and acetyl-l-carnitine in mitochondrial dysfunction, in aging, and in age-related disorders. We then review the evidence for the role of acetylcarnitine in modulating redox-dependent mechanisms leading to up-regulation of vitagenes in brain, and we also discuss new approaches for investigating the mechanisms of lifetime survival and longevity.

Neuronal nitric oxide synthase (nNOS) is a constitutively expressed and calcium-dependent enzyme. Despite predominantly expressed in neurons, nNOS has been also found in astrocytes, although at lower expression levels. We have studied the regulation of nNOS expression in cultured rat astrocytes from cortex and spinal cord by Western blotting and immunocytochemistry. nNOS was not detectable in cultured astrocytes grown in serum-containing medium (SCM), but was highly expressed after serum deprivation. Accordingly, calcium-dependent NOS activity and both intracellular nitrite levels and nitrotyrosine immunoreactivity after glutamate stimulation were higher in serum-deprived astrocytes than in cells grown in SCM. Serum deprivation induced a modification of astrocytes morphology, from flat to stellate. nNOS up-regulation was also observed in reactive astrocytes of rat hippocampi after electrically induced status epilepticus, as demonstrated by double-labeling experiments. Thus, nNOS upregulation occurs in both in vitro stellate and in vivo reactive astrocytes, suggesting a possible involvement of glial nNOS in neurological diseases characterized by reactive gliosis.

Increasing evidence supports the notion that reduction of cellular expression and activity of antioxidant proteins and the resulting increase of oxidative stress are fundamental causes in the aging processes and neurodegenerative diseases. In the present study, we evaluated, in the brains of young and aged rats, the gene expression profiles of two inducible proteins critically involved in the cellular defense against endogenous or exogenous oxidants: heme oxygenase-1 (HO-1) and manganese superoxide dismutase-2 (SOD-2). SOD-2 is an essential antioxidant and HO-1 has been reported to be very active in regulating cellular redox homeostasis. Deregulation of these enzymes has been extensively reported to play a crucial role in the pathogenesis of neurodegenerative disorders. To measure the regional distribution of HO-1 and SOD-2 transcript levels in the rat brain, we have developed a real time quantitative reverse transcription-polymerase chain reaction protocol. Although these two genes presented a highly dissimilar range of expression, with SOD-2 >HO-1, both transcripts were highly expressed in the cerebellum and the hippocampus, showing in a different scale a strikingly parallel distribution gradient. To further investigate the regional brain expression of these mRNAs, we performed in situ hybridization using specific riboprobes. In situ hybridization results showed that both transcripts were highly concentrated in the hippocampus, the cerebellum and some specific regions of the brain cortex. We have also quantified, by reverse transcription-polymerase chain reaction, the brain expression of HO-1 and SOD-2 mRNAs in middle aged (12 months) and aged (28 months) rats. We found that the hippocampus of aged rats presents a significant down regulation of SOD2 mRNA expression and a parallel upregulation of HO-1 mRNA compared with young (6 months) and middle-aged rats. Furthermore, in the cerebellum of the aged rats, we detected a parallel significant upregulation of both HO-1 and SOD-2 transcripts. These regional age-dependent differences may help to explain the increased susceptibility to oxidative damage in these two brain areas during aging.

Nitric oxide (NO) is hypothesized to play a role in the immunopathogenesis of multiple sclerosis (MS). Increased levels of NO metabolites have been found in patients with MS. Peroxynitrite, generated by the reaction of NO with superoxide at sites of inflammation, is a strong oxidant capable of damaging tissues and cells. Inducible NO synthase (iNOS) is up-regulated in the CNS of animals with experimental allergic encephalomyelitis (EAE) and in patients with MS. In this study, Western blots of cerebrospinal fluid (CSF) from patients with MS demonstrated the presence of iNOS, which was absent in CSF from control subjects. There was also NOS activity present in both MS and control CSF. Total NOS activity was increased (by 24%) in the CSF from MS patients compared with matched controls. The addition of 0.1 mM ITU (a specific iNOS inhibitor) to the samples did not change the activity of the control samples but decreased the NOS activity in the MS samples to almost control levels. The addition of 1 mM L-NMMA (a nonisoform specific NOS inhibitor), completely inhibited NOS activity in CSF from control and MS subjects. Nitrotyrosine immunostaining of CSF proteins was detectable in controls but was greatly increased in MS samples. There were also significant increases in CSF nitrate + nitrite and oxidant-enhanced luminescence in MS samples compared with controls. Additionally, a significant decrease in reduced glutathione and significant increases in oxidized glutathione and S-nitrosothiols were found in MS samples compared with controls. Parallel changes in NO metabolites were observed in the plasma of MS patients, compared with controls, and accompanied a significant increase of reduced glutathione. These data strongly support a role for nitrosative stress in the pathogenesis of MS and indicate that therapeutic strategies focussed on decreasing production of NO by iNOS and/or scavenging peroxynitrite may be useful in alleviating the neurological impairments that occur during MS relapse.

Estrogens are recognized as neuroprotective and neurotrophic agents in the central nervous system. They are involved in neuronal differentiation and survival and promote neural development. Estrogen receptors alpha (ER-alpha) and beta (ER-beta) are predominantly expressed in neurons, whereas their presence in glial cells in vivo is more controversial. Changes in their expression during development have been described in different brain areas, but little is known about their presence in the spinal cord. We have carried out an immunohistochemical study in an attempt to analyze the expression of both ERs in astrocytes and oligodendrocytes of the rat spinal cord and their modifications during postnatal development. RT-PCR analysis of whole spinal cord extracts from 4-, 12-, and 25-day-old and adult rats indicated changes in the expression of both receptors during maturation. Immunohistochemistry of slices of the lumbar tract revealed that in an area of the ventral spinal cord that does not contain neuronal cell bodies, but mainly fibers and glial cells, both ER-alpha and ER-beta can be detected. Immunostaining is clearly nuclear, and, in the case of ER-alpha, both markedly positive and weakly labeled cells can be identified. ER-alpha is expressed during early development to progressively decline in the adult stage. In contrast, the ER-beta signal is low and peaks at postnatal day 25, whereas it is almost undetectable at other ages. Colocalization studies revealed that, at postnatal day 25, ER-alpha and ER-beta are expressed in astrocytes (identified by the specific marker glial fibrillar acidic protein) and oligodendrocytes (labeled by antimyelin 2',3'-cyclic nucleotide 3'-phosphodiesterase). The present results confirm the expression of ER-alpha and ER-beta in glial cells in vivo and suggest that, also in the spinal cord, glial cells may contribute to the effects of estrogen during development.

In the present study, we investigated the effect of Ginkgo biloba extract, EGb 761, and one of its components, bilobalide, on gene expression of subunit 1 of mitochondrial NADH dehydrogenase (ND1) in PC12 cells. By Northern blot analysis we found a approximately 2-fold significant increase in NDI mRNA level, after 48 and 72 h exposure to 100 microg/ml EGb 761 and to 10 microg/ml bilobalide. We also evaluated, by oxygraphy measurements, mitochondrial respiration during state 3 and state 4. In cells treated with EGb 761 and bilobalide for 48 and 72 h, state 4 respiration was significantly decreased, and the respiratory control ratio was increased. These results provide evidence that EGb 761 and bilobalide exert their protective effects by up-regulating mitochondrial ND1 gene expression and by decreasing state 4 respiration, whose increase is thought to be responsible for oxidative damage.

Changes in glutathione (GSH) and glutathione disulfide (GSSG) levels and/or redox status have been suggested to mediate the induction of heat shock proteins (HSPs) that follows exposure to oxidizing agents such as ethanol. Here we report the effects of ethanol administration to rats at intracellular levels of GSH, GSSG, HSP70, and protein carbonyls in brain and liver. Following 7 days of ethanol administration, there was a significant decrease in GSH, a significant induction of HSP70, and a significant increase in protein carbonyls in all brain regions studied and in liver. In cortex, striatum, and hippocampus there was a significant correlation between (a) the decrease in GSH, (b) the increase in GSSG, and (c) the decrease in GSH/GSSG ratio and HSP70 levels induced in response to ethanol. These data support the hypothesis that a redox mechanism may be involved in the heat-shock signal pathway responsible for HSP70 induction in the brain.

The effect of tumor necrosis factor-alpha (TNF-alpha) on neuronal viability has been investigated in the SK-N-BE neuroblastoma cell line. These cells undergo differentiation upon chronic treatment with retinoic acid. Exposure of SK-N-BE cells to TNF-alpha produced a proliferative response in undifferentiated cells, whereas a reduced cell number was observed in retinoic acid (RA)-differentiated cultures. This biphasic response may be related to the different expression of TNF-alpha receptors (TNFRs); a significant increase in the density of TNFR1 was in fact observed following RA-induced differentiation. Under these conditions, a pronounced increase in the formation of ceramide-1-phosphate (which was prevented by the selective inhibitor of phosphatidylcholine-specific phospholipase C, D609) and an activation of caspase-3 upon TNF-alpha challenge were evident. Selective blockade of each TNFR subtype allowed a more detailed analysis of the effect observed. Preincubation with an anti-TNFR1 antibody prevented the cytotoxic effect of TNF-alpha in RA-differentiated SK-N-BE cells, whereas the anti-TNFR2 antibody blocked the proliferative activity of the cytokine in undifferentiated cultures.

Platelet-derived growth factor (PDGF) and ciliary neurotrophic factor (CNTF) have pleiotropic actions on many cell types. In the presence of these factors, oligodendroglia respond by enhanced survival when deprived of trophic factors or in the presence of the cytotoxic cytokine, tumor necrosis factor-alpha (TNF-alpha). To determine whether these two oligodendroglial survival factors converge in their signaling cascades, we examined their JAK/STAT pathways in enriched oligodendrocyte (OL) progenitors and in the progenitor OL cell line, central glia-4 (CG-4). Cytokine pathways such as JAK/STAT have been characterized extensively in hematopoietic cells; however, it is increasingly evident that the same cytokines that play a role in hematopoiesis also play a role during development and injury of the central nervous system. This is the first study that clearly defines the presence and activation of JAK/STAT proteins in OL progenitors and compares the signal transduction pathway of two well-known oligodendroglial survival factors. In this study, we report that PDGF- and CNTF-induced OL progenitors responded with a rapid tyrosine phosphorylation of JAK1, JAK2, STAT1alpha/beta, and STAT3. We feel that these identified JAK/STAT signaling molecules play a large role in the cellular response to these factors. Because both PDGF and CNTF enhance OL progenitor survival, these JAK/STATs may play a role in regulating this important cellular process.

The levels of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of F(0)F(1)-ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, 26 months of age were analyzed by Western blot. Age-related changes in the content of subunits, encoded either in mitochondrial or nuclear DNA, were observed.

In the present investigation we address the question of whether one of the responses to increased neuronal activity is a modification of the expression of the different subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptors (GluR-1, GluR-2, GluR-3). Thus, we used two different models of generalized status epilepticus, as widespread elevated neuronal activity, to study in vivo responses of the AMPA receptor mRNA expression in rat forebrain. By Northern blot analysis and in situ hybridization, we show that one of the delayed responses to LiCl/pilocarpine-induced status epilepticus is a dramatic change in the mRNA level of some subunits of AMPA-selective glutamate receptors. These effects, which appear between 6 and 12 h after the drug treatment, are subunit and brain region specific. The most striking example of differential expression of the three examined GluR mRNAs can be observed in the dentate gyrus of the hippocampus. In this specific brain subregion an increase of GluR-3 mRNA level is induced 12 h after LiCl/pilocarpine treatment, while a clear decrease in GluR-1 mRNA level and no significant change in GluR-2 mRNA level can be observed in the same area under these experimental conditions. Both the GluR-1 decrease and the GluR-3 increase are transient effects and a return to basal level can be observed after 48-72 h. In the CA1 layer of the hippocampus, a parallel decrease of both GluR-1 and GluR-3 expression is found 12-24 h after drug treatment, followed by a recovery of the expression to control values at 48 h. In kainate-induced epilepsy we could reproduce the late increase (12-24 h) in GluR-3 mRNA in the dentate gyrus; however, under this experimental condition, no clear decrease of GluR-1 expression can be observed in this area. A general decrease in mRNA level for the AMPA receptor subunits (GluR-1-3) in the hippocampal layers, in particular in CA3 and CA4 subfields, was also observed. In conclusion the results reported in the present paper reveal a specific regulation of GluR gene expression in the granule cells of the hippocampal dentate gyrus and stimulate further investigation on the functional role of the GluR-3 subunit in the receptor-channel complex.

We have used primary neuronal cultures prepared from fetal cerebral hemispheres to investigate the effects of different glutamate receptor agonists and antagonists on the expression of transcription factor encoding genes, such as c-fos, fosB, c-jun, junB, junD, c-myc, and zif/268. The addition of glutamate (100 microM) to the culture medium rapidly activated c-fos, fosB, c-jun, junB and zif/268 gene expression, reaching the maximal level at 30-60 minutes for zif/268 and at 60 minutes for the other genes. The onset of fosB mRNA accumulation was slightly delayed in comparison to the other genes. No clear induction was found for junD and c-myc. Different glutamate receptor agonists, such as NMDA, kainate, quisqualate, trans-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) were able to increase c-fos, c-jun, and zif-268 mRNA levels with rapid and transient kinetics similar to those observed after glutamate treatment. Similar results were obtained for junB and fosB after kainate and quisqualate stimulation. Pretreatment with MK-801, a non competitive NMDA antagonist, produced an almost complete inhibition of glutamate-driven expression of transcription factor genes, thus suggesting that NMDA receptor plays a major role in glutamate induced-gene expression. On the contrary the kainate/AMPA receptor antagonist, DNQX, did not influence glutamate induced-gene expression. Under the conditions used in the present study, NMDA was effective in inducing the simultaneous activation of several IEGs even when added to the culture medium containing millimolar concentration of magnesium. When experiments were performed in Krebs solution, NMDA was effective in stimulating zif/268 and c-fos mRNAs only in the absence of Mg2+, while glutamate activated c-fos and zif/268 both in the presence and absence of magnesium ions. As expected, NMDA effect was fully inhibited by MK-801. The level of AP-1 DNA binding activity, as measured by electrophoretic mobility shift assay, increased after addition of glutamate and NMDA to cultured neurons and such increase was antagonized by the pretreatment with MK-801.

Several age-dependent modifications of inner mitochondrial membrane and synaptosomal plasma membrane proteins from different brain regions of 4-, 12-, 18- and 24-month-old male Wistar rats, were observed. Some proteins, identified by immunoblotting assay as various subunits of mitochondrial respiratory chain complexes and calmodulin, were particularly impaired. Chronic treatment with CDP-choline at a dose of 20 mg/kg body weight per day for 28 days caused significant changes in the amounts of several of the above mentioned proteins. Most of the proteins, which decreased during aging, showed a significant increase after CDP-choline treatment compared with the corresponding control values at the same age. The effect of CDP-choline might be due to: — the increased availability of cytidylic nucleotides, which in the brain are present in limited amounts compared to the other nucleotides; — the increased content of total adenine nucleotides; —the improvement of brain energy metabolism.

We have characterized the genomic response of astroglial cells to excitatory amino acids by using selective agonists and antagonists for the various receptor subtypes and by analyzing different primary response genes, such as members of the Fos (c-fos and fosB) and Jun (c-jun, junB, and junD) families, zif/268, and c-myc. A rapid and transient elevation of mRNA levels for c-fos, fosB, c-jun, junB, and zif/268 was observed after addition of glutamate to cultured astrocytes, whereas junD and c-myc expression was not affected. The level of AP-1 DNA binding activity, as measured by the electrophoretic mobility shift assay, also increased after addition of glutamate to cultured astrocytes. Glutamate-induced c-fos expression was not affected by the N-methyl-D-aspartate receptor antagonists MK-801 and D-2-amino-5-phosphonopentanoate, by the kainate/alpha-amino-3-hydroxy-5- methylisoxazole-4-propionate (AMPA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or by the broad-spectrum antagonist kynurenate. Kainate and AMPA were also effective in inducing primary response gene expression, and their actions were antagonized by kynurenate and DNQX but not by MK-801. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid, a selective agonist for the metabotropic glutamate receptor, induced primary response gene expression, but its action was not antagonized by different glutamate antagonists, including L-2-amino-3-phosphonopropionate. In conclusion, our data suggest that cultured astrocytes express both kainate/AMPA ionotropic receptors and metabotropic receptors coupled to the rapid and coordinated activation of different classes of transcriptional factor genes.

The effect of hypoxia on the protein composition of mitochondria from cerebral cortex of rats at 4, 12, and 24 months of age was investigated. The proteins were separated by electrophoresis on SDS polyacrylamide gels and the percent content was evaluated by measuring the optical density of the stained gels. The results demonstrate that hypoxic treatment causes a decrease in the amount of some proteins as follows: the 90 and the 16 kDa Mw proteins at 4 months; the 82 and the 79 kDa Mw proteins at 24 months; the 52-49, 35 and 20 kDa at all ages investigated; the 44 kDa protein at 4 and 12 months and the 28 kDa protein at 4 and 24 months of age. Our results show that hypoxic conditions affect mitochondrial protein composition to a greater extent than aging alone.

The effect of hypoxia on the protein composition of synaptic plasma membranes (SPM) isolated from cerebral cortex of rats at 4, 12, and 24 months of age was investigated. The proteins were separated by SDS polyacrylamide gel electrophoresis and the percent content was evaluated by measuring the optical density of the stained gels. After hypoxic treatment various proteins showed significant changes. Some proteins were only affected at 4 and 12 months of age and not at 24 months. The various modified proteins may be identified according to their molecular weight, as follows: the 18 kDa protein with calmodulin; the 23 kDa protein with D3 subunits; the 28 kDa protein could contain the delta subunit of the Ca2+ channel. The changes in the amount of some SPM proteins during hypoxia is consistent with the alteration in membrane polarization and neurotransmission observed in this condition. The effect of aging at the synaptosomal level seems to be a selective process; after hypoxia the age-related changes of many proteins are more pronounced.

Several studies suggest that a decline in transcriptional and translational events may play a crucial role in the molecular mechanism underlying the aging process (for a review see Giuffrida and Lajtha, 1987).

Results of experiments in cell cultures suggested that epidermal growth factor might influence an early stage of astroglial or neuronal cell differentiation. In order to evaluate this hypothesis the effects of subcutaneous and intracerebral treatment with epidermal growth factor on glutamine synthetase, an astroglial marker enzyme, and glutamate decarboxylase activity, a marker enzyme of GABAergic neurons, were investigated during postnatal development of mouse brain. Epidermal growth factor, at the dose used, induced the well-known effects of the in vivo treatment, such as a decrease in body weight and a precocious incisor eruption and eyelid opening. A decrease in forebrain and cerebellum wet weight was also observed. However, repeated epidermal growth factor treatment, during early postnatal life, failed to influence glutamine synthetase activity in forebrain or cerebellum, while a significant decrease was observed in the brain stem. No effect of epidermal growth factor on forebrain glutamate decar-boxylase activity was observed. Although epidermal growth factor receptors have been detected in the newborn rodent brain, the role of this growth factor in brain development remains to be elucidated.

Protein conformational diseases, such as Alzheimer's, Parkinson's and Huntington's, affect a large portion of aging population. The pathogenic dysfunctional aggregation of proteins in non-native conformations is associated with metabolic derangements and excessive production of reactive oxygen species. Reduction of cellular expression and activity of antioxidant proteins result in increased oxidative stress. Free-radicals derived from mitochondrial dysfunction and from the cyclooxygenase enzyme activity play a role in oxidative damage of brain. Cyclooxygenase also mediates in neuro-inflammation by the production of pro-inflammatory prostaglandins which contribute to brain injury. The pathogenic role of cyclooxygenase has been demonstrated in Alzheimer and Parkinson diseases. The brain responses to detect and control diverse forms of stress are accomplished by a complex network of "longevity assurance processes" integrated to the expression of genes termed vitagenes. Heat shock proteins are a highly conserved system responsible for the preservation and repair of correct protein conformation. Heme oxygenase-1, a inducible and redox-regulated enzyme, is currently considered as having an important role in cellular antioxidant defense. A neuroprotective effect, due to its heme degrading activity, and tissue-specific pro-oxidant effects, due to its products CO and free iron, are under debate. There is a current interest in dietary compounds that can inhibit, retard or reverse the multi-stage pathophysiology of Alzheimer disease, with a chronic inflammatory response, brain injury and beta-amyloid associated pathology. Curcumin and ferulic acid, two powerful antioxidants, the first from the curry spice turmeric and the second a major constituent of fruit and vegetables, have emerged as strong inducers of the heat shock response. Food supplementation with curcumin and ferulic acid is considered a nutritional approach to reduce oxidative damage and amyloid pathology in Alzheimer disease.