Protective effect of carnosine during nitrosative stress in astroglial cell cultures.

Formation of nitric oxide by astrocytes has been suggested to contribute, via impairment of mitochondrial function, to the neurodegenerative process. Mitochondria under oxidative stress are thought to play a key role in various neurodegenerative disorders; therefore protection by antioxidants against oxidative stress to mitochondria may prove to be beneficial in delaying the onset or progression of these diseases. Carnosine has been recently proposed to act as antioxidant in vivo. In the present study, we demonstrate its neuroprotective effect in astrocytes exposed to LPS- and INFgamma-induced nitrosative stress. Carnosine protected against nitric oxide-induced impairment of mitochondrial function. This effect was associated with decreased formation of oxidatively modified proteins and with decreased up-regulation oxidative stress-responsive genes, such as Hsp32, Hsp70 and mt-SOD. Our results sustain the possibility that carnosine might have anti-ageing effects to brain cells under pathophysiological conditions leading to degenerative damage, such as aging and neurodegenerative disorders.

Increased expression of heat shock proteins in rat brain during aging: relationship with mitochondrial function and glutathione redox state.

It is generally recognized that lipid peroxides play an important role in the pathogenesis of several diseases and that sulfhydryl groups are critically involved in cellular defense against endogenous or exogenous oxidants. Recent evidence indicates that lipid peroxides directly participate in induction of cytoprotective proteins, such as heat shock proteins (Hsps), which play a central role in the cellular mechanisms of stress tolerance. Oxidative damage plays a crucial role in the brain aging process and induction of Hsps is critically utilized by brain cells in the repair process following various pathogenic insults. In the present study, we investigated, in rats 6, 12, and 28 months old, the role of heat shock expression on aging-induced changes in mitochondrial and antioxidant redox status. In the brain expression of Hsp72 and Hsc70 increased with age up to 28 months; at this age the maximum induction was observed in the hippocampus and substantia nigra followed by cerebellum, cortex, septum and striatum. Hsps induction was associated with significant changes in glutathione (GSH) redox state and HNE levels. Interestingly, a significant positive correlation between decrease in GSH and increase in Hsp72 was observed in all brain regions examined during aging. Analysis of mitochondrial complexes showed a progressive decrease of Complex I activity and mRNA expression in the hippocampus and a significant decrease of Complex I and IV activities in the substantia nigra and septum. Our results sustain a role for GSH redox state in Hsp expression. Increase of Hsp expression promotes the functional recovery of oxidatively damaged proteins and protects cells from progressive age-related cell damage. Conceivably, heat shock signal pathway by increasing cellular stress resistance may represent a crucial mechanism of defence against free radical-induced damage occurring in aging brain and in neurodegenerative disorders.

Redox regulation of heat shock protein expression in aging and neurodegenerative disorders associated with oxidative stress: a nutritional approach.

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Alzheimer's disease (AD) is a progressive disorder with cognitive and memory decline, speech loss, personality changes and synapse loss. Many approaches have been undertaken to understand AD, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to AD pathogenesis. Brains of AD patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress.Recently, the involvement of the heme oxygenase (HO) pathway in anti-degenerative mechanisms operating in AD has received considerable attention, as it has been demonstrated that the expression of HO is closely related to that of amyloid precursor protein (APP). HO induction occurs together with the induction of other HSPs during various physiopathological conditions. The vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, products of HO-catalyzed reaction, represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. Increasing interest has been focused on identifying dietary compounds that can inhibit, retard or reverse the multi-stage pathophysiological events underlying AD pathology. Alzheimer's disease, in fact, involves a chronic inflammatory response associated with both brain injury and beta-amyloid associated pathology. All of the above evidence suggests that stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms. Spice and herbs contain phenolic substances with potent antioxidative and chemopreventive properties, and it is generally assumed that the phenol moiety is responsible for the antioxidant activity. In particular, curcumin, a powerful antioxidant derived from the curry spice turmeric, has emerged as a strong inducer of the heat shock response. In light of this finding, curcumin supplementation has been recently considered as an alternative, nutritional approach to reduce oxidative damage and amyloid pathology associated with AD. Here we review the importance of the heme oxygenase pathway in brain stress tolerance and its significance as an antidegenerative mechanism potentially important in AD pathogenesis. These findings have offered new perspectives in medicine and pharmacology, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms such as the heat shock response, through nutritional antioxidants or pharmacological compounds, represents an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. Consistent with this notion, maintenance or recovery of the activity of vitagenes, such as the HO gene, conceivably may delay the aging process and decrease the occurrence of age-related neurodegenerative diseases.

Disruption of thiol homeostasis and nitrosative stress in the cerebrospinal fluid of patients with active multiple sclerosis: evidence for a protective role of acetylcarnitine.

Recent studies suggest that NO and its reactive derivative peroxynitrite are implicated in the pathogenesis of multiple sclerosis (MS). Patients dying with MS demonstrate increased astrocytic inducible nitric oxide synthase activity, as well as increased levels of iNOS mRNA. Peroxynitrite is a strong oxidant capable of damaging target tissues, particularly the brain, which is known to be endowed with poor antioxidant buffering capacity. Inducible nitric oxide synthase is upregulated in the central nervous system (CNS) of animals with experimental allergic encephalomyelitis (EAE) and in patients with MS. We have recently demonstrated in patients with active MS a significant increase of NOS activity associated with increased nitration of proteins in the cerebrospinal fluid (CSF). Acetylcarnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, in the present study, MS patients were treated for 6 months with acetylcarnitine and compared with untreated MS subjects or with patients noninflammatory neurological conditions, taken as controls. Western blot analysis showed in MS patients increased nitrosative stress associated with a significant decrease of reduced glutathione (GSH). Increased levels of oxidized glutathione (GSSG) and nitrosothiols were also observed. Interestingly, treatment of MS patients with acetylcarnitine resulted in decreased CSF levels of NO reactive metabolites and protein nitration, as well as increased content of GSH and GSH/GSSG ratio. Our data sustain the hypothesis that nitrosative stress is a major consequence of NO produced in MS-affected CNS and implicate a possible important role for acetylcarnitine in protecting brain against nitrosative stress, which may underlie the pathogenesis of MS.

Molecular chaperones and their roles in neural cell differentiation.

During the development of the nervous system, a large number of neurons are eliminated through naturally occurring neuronal death. Many morphological and biochemical properties of such dying neurons are reminiscent not only of apoptosis, a type of death involving the action of genetically programmed events, but also of epigenetic phenomena such as oxidative stress. Increasing evidence demonstrates that oxidative stress alters the expression of antioxidant enzymes and enhances expression and/or DNA binding of numerous transcription factors, including heat shock factor. The latter is a transcription factor for specific promoter elements located upstream of the heat shock genes. Heat shock proteins (Hsps) are essential, highly conserved proteins that are needed for normal cell growth and maintenance, and expression of Hsps has been detected during embryogenesis in various organisms. Developmental profiles of Hsps indicate that they are differentially regulated during neural maturation, suggesting a role for Hsps in neural cell differentiation. Their putative function in cell remodeling during migration and differentiation, as well as during postnatal development, a time of extensive cell differentiation, is considered in the present review. Moreover, the function of Hsps in cell signaling, protein transport and the effect of heat shock on neural plate induction and brain development are discussed.

Regional distribution of heme oxygenase, HSP70, and glutathione in brain: relevance for endogenous oxidant/antioxidant balance and stress tolerance.

It is generally recognized that lipid peroxides play an important role in the pathogenesis of several diseases and that sulfhydryl groups are critically involved in cellular defense against endogenous or exogenous oxidants. Recent evidence indicates that lipid peroxides directly participate in induction of cytoprotective proteins, such as heat shock proteins (Hsps), which play a central role in the cellular mechanisms of stress tolerance. Heme oxygenase (HO) is a stress protein that has been implicated in defense mechanisms against agents that may induce oxidative injury, such as endotoxins, cytokines and heme and its induction represents a common feature in a number of neurodegenerative diseases. In the present report we studied regional distribution of heme oxygenase (HO) activity and protein expression, together with that of Hps70, in brain of C57BL6 mice. Endogenous lipid peroxidation was investigated on the basis of the analysis of ultra weak chemiluminescence, hydro peroxides and lipid soluble fluorescent products, and compared to the regional distribution of thiols, antioxidant enzymes and trace metals. Our results show that levels of HO activity and expression of inducible Hsp70 and the ratio of GSH/GSSG in the different brain regions examined were positively correlated with the content of peroxides. Substantia Nigra was the brain area exhibiting the highest levels of HO-2, constitutive and inducible Hsp70, GSSG, peroxides, iron, and calcium, in contrast with the lowest content in GSH, GSH/GSSG ratio and glutathione reductase activity, compared to the other cerebral regions examined. Among these, cortex showed the lowest levels of HO-2, Hsp70, GSSG and peroxides that were associated with the highest levels of GSH and GSH/GSSG ratio. These data support the hypothesis that the glutathione redox state and basal peroxides can directly participate in the signaling pathways of heat shock protein expression and hence of stress tolerance.

Long-term ethanol administration enhances age-dependent modulation of redox state in brain and peripheral organs of rat: protection by acetyl carnitine.

Evidence is accumulating that intermediates of oxygen reduction may be associated with the development of alcoholic disease. Free radical-induced perturbation of the oxidant/antioxidant balance in the cell is widely recognized as the main causative factor of age-related disorders. In the present study we investigated the effects of 20 months of ethanol consumption on the antioxidant defense system in different rat organs compared with normal aging in the absence and presence of treatment with L-acetyl carnitine. We demonstrate that aged rats underwent significant perturbation of the antioxidant defense system, as indicated by depletion of reduced glutathione (GSH) content, increased oxidized GSH, free radical-induced luminescence associated with increased hydroxynonenal content and decreased GSH reductase activity. These modifications, observed particularly in brain and liver compared with other organs, were enhanced by long-term alcohol exposure and, interestingly, were significantly reduced with acetyl carnitine supplements. Our results indicate that decreased GSH reductase activity and thiol depletion are important factors in effecting a pathogenic role for oxidative stress in aging and in all situations in which age-correlated and oxidant-induced changes occur, such as in alcoholism. Administration of acetyl carnitine greatly reduces these metabolic abnormalities. Our findings support its pharmacological potential in the management of alcoholic disturbances.

Long-term ethanol administration enhances age-dependent modulation of redox state in different brain regions in the rat: protection by acetyl carnitine.

Chronic alcoholism is a major public health problem and causes multiorgan diseases and toxicity. Although the majority of ethanol ingested is metabolized by the liver, it has intoxicating effects in the brain. Evidence is accumulating that intermediates of oxygen reduction may be associated with the development of alcoholic disease. Several studies have shown the capacity of carnitine and its derivatives to influence ethanol metabolism. We have previously demonstrated that preadministration of L-carnitine to rats receiving ethanol significantly reduced fatty acid ethyl esters in different organs and that the carnitine/acylcarnitine system is crucial for maintaining a functional acetyl-CoA/CoA ratio under conditions in which cellular homeostasis is exposed to the deleterious effects of accumulating organic acids. Ethanol, administered to rats for 20 months, induced significant changes in the status of glutathione, primarily in the brain regions of hippocampus and cerebellum, followed by cortex and striatum, where a decrease in reduced glutathione (GSH) and the GSH/oxidized glutathione ratio was found. The same brain regions showed a significant increase in free radical-induced luminescence and hydroxynonenal (HNE), which were associated with decreased GSH reductase activity. Long-term supplementation with acetyl carnitine significantly reduced GSH depletion, particularly in the brain regions of hippocampus, an effect associated with decreased luminescence and HNE formation. In addition, acetyl carnitine treatment increased GSH reductase and arginase activities. Our results indicate that decreased GSH reductase activities associated with thiol depletion are important factors sustaining a pathogenic role in alcohol-related pathologies. Administration of acetyl carnitine greatly reduces these metabolic abnormalities. This evidence supports the pharmacological potential of acetyl carnitine in the management of alcoholic disturbances.

Nitric oxide synthase induction in astroglial cell cultures: effect on heat shock protein 70 synthesis and oxidant/antioxidant balance.

Glial cells in the nervous system can produce nitric oxide in response to cytokines. This production is mediated by the inducible isoform of nitric oxide synthase. Radical oxygen species (ROS) and nitric oxide (NO) derivatives have been claimed to play a crucial role in many different processes, both physiological such as neuromodulation, synaptic plasticity, response to glutamate, and pathological such as ischemia and various neurodegenerative disorders. In the present study we investigated the effects of NO synthase (iNOS) induction in astrocyte cultures on the synthesis of heat shock proteins, the activity of respiratory chain complexes and the oxidant/antioxidant balance. Treatment of astrocyte cultures for 18 hr with LPS and INFgamma produced a dose dependent increase of iNOS associated with an increased synthesis of hsp70 stress proteins. This effect was abolished by the NO synthase inhibitor L-NMMA and significantly decreased by addition of SOD/CAT in the medium. Time course experiments showed that iNOS induced protein expression increased significantly by 2 hr after treatment with LPS and INFgamma and reached a plateau at 18 hr; hsp70 protein synthesis peaked around 18 and 36 hr after the same treatment. Addition to astrocytes of the NO donor sodium nitroprusside resulted in a dose dependent increase in hsp70 protein that was comparable to that found after a mild heat shock. Additionally, a decrease in cytochrome oxidase activity, a marked decrease in ATP and protein sulfhydryl contents, an increase in the activity of the antioxidant enzymes mt-SOD and catalase were found which were abolished by L-NMMA. These findings suggest the importance of mitochondrial energy impairment as a critical determinant of the susceptibility of astrocytes to neurotoxic processes and point to a possible pivotal role of hsp70 in the signalling pathways of stress tolerance.

HSP70 induction in the brain following ethanol administration in the rat: regulation by glutathione redox state.

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.

Long-term ethanol administration enhances urinary ultraweak luminescence and age-dependent modulation of redox in central and peripheral organs of the rat.

Numerous experimental evidence sustains a pathogenic role for oxidative stress in aging. Acute and chronic ethanol metabolism is also known to be associated with oxidative perturbation of cellular oxidant/antioxidant balance. In the present work we investigated the effects of 25 months of ethanol consumption on the antioxidant defense system in different organs of rats, in comparison with normal and aged animals. We show that aged rats underwent a significant perturbation of the antioxidant defense system, as indicated by depletion of reduced glutathione content, increases in oxidized glutathione and free radical-induced urinary luminescence associated with a decrease of glutathione reductase and increase of glutathione transferase activities. These modifications, observed particularly in the liver and brain, were enhanced by long-term alcohol exposure. Our results indicate that increased glutathione transferase activity and decreased glutathione reductase activity, followed by thiol depletion, are important factors sustaining a pathogenic role for oxidative stress in aging and in all situations where age-correlated changes occur. They also reinforce the oxidative potential of toxic compounds, such as ethanol intoxication.

Effect of nitric oxide synthase induction on the expression of mitochondrial respiratory chain enzyme subunits in mixed cortical and astroglial cell cultures.

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.