Giovanni Pennisi

Oxidative stress, mitochondrial dysfunction and cellular stress response in Friedreich's ataxia

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinsons disease, Alzheimers disease, Friedreichs ataxia (FRDA), multiple sclerosis and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) associated with mitochondrial dysfunction. The mitochondrial genome may play an essential role in the pathogenesis of these diseases, and evidence for mitochondria being a site of damage in neurodegenerative disorders is based in part on observed decreases in the respiratory chain complex activities in Parkinsons, Alzheimers, and Huntingtons disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant imbalance, are thought to underlie defects in energy metabolism and induce cellular degeneration. The precise sequence of events in FRDA pathogenesis is uncertain. The impaired intramitochondrial metabolism with increased free iron levels and a defective mitochondrial respiratory chain, associated with increased free radical generation and oxidative damage, may be considered possible mechanisms that compromise cell viability. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols, and in addition, that decreased expression of frataxin protein is associated with FRDA. Many approaches have been undertaken to understand FRDA, 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 FRDA pathogenesis. Brains of FRDA 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. In the central nervous system, heat shock protein (HSP) synthesis is induced not only after hyperthermia, but also following alterations in the intracellular redox environment. The major neurodegenerative diseases, Alzheimers disease (AD), Parkinsons disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntingtons disease (HD) and FRDA are all associated with the presence of abnormal proteins. Among the various HSPs, HSP32, also known as heme oxygenase I (HO-1), has received considerable attention, as it has been recently demonstrated that HO-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. 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. This may open up new perspectives in medicine, 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, pharmacological compounds or gene transduction, may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.

Cellular stress response: a novel target for chemoprevention and nutritional neuroprotection in aging, neurodegenerative disorders and longevity

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.

Redox regulation of cellular stress response in aging and neurodegenerative disorders: role of vitagenes

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, AβP oligomerization and consequent increased apoptosis are of particular interest, with respect to a chronic inflammatory response, brain injury and β-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.

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

Summary. 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 β-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.

Traumatic Brain Injury: Oxidative Stress and Neuroprotection

SIGNIFICANCE A vast amount of circumstantial evidence implicates high energy oxidants and oxidative stress as mediators of secondary damage associated with traumatic brain injury. The excessive production of reactive oxygen species due to excitotoxicity and exhaustion of the endogenous antioxidant system induces peroxidation of cellular and vascular structures, protein oxidation, cleavage of DNA, and inhibition of the mitochondrial electron transport chain. RECENT ADVANCES Different integrated responses exist in the brain to detect oxidative stress, which is controlled by several genes termed vitagens. Vitagens encode for cytoprotective heat shock proteins, and thioredoxin and sirtuins. CRITICAL ISSUES AND FUTURE DIRECTIONS This article discusses selected aspects of secondary brain injury after trauma and outlines key mechanisms associated with toxicity, oxidative stress, inflammation, and necrosis. Finally, this review discusses the role of different oxidants and presents potential clinically relevant molecular targets that could be harnessed to treat secondary injury associated with brain trauma.

The mismatch negativity and the P3a components of the auditory event-related potentials in autistic low-functioning subjects

OBJECTIVE In order to understand better the psychophysiological basis of auditory processing abnormalities in autism, we decided to study two automatic components of the auditory event-related potentials (ERPs): the mismatch negativity (MMN)--a component of the ERP which is recorded when, during repetitive auditory stimulation, rare changes are introduced--and the novelty-related P3a which is recorded as a response to unexpected novel events occurring in a sequence of repetitive stimuli. METHODS Ten male subjects, mean age 12.3 years (SD 4.95), affected by autism and mental retardation were admitted to this study. All patients were also mentally retarded. Ten normal male subjects, mean age 12.2 years (SD 3.94), were used as controls. Auditory evoked potentials were recorded from 19 scalp electrodes (10-20 system), and stimuli were presented in sequences consisting of 2000 tones (70 dB, ISI=800 ms). Three types of stimuli were presented: (1) standard stimuli (1000 Hz tones, 80% of total stimuli), (2) deviant stimuli (1300 Hz tones, 10% of total stimuli), and (3) novel stimuli (complex and non-monotonal, 10% of total stimuli). To quantify the MMN, the evoked response to the standard tones was subtracted from the corresponding deviant stimulus response and its amplitude and latency at peak were measured over Fz, Cz and Pz; similarly, the P3a component of the ERP was obtained by subtracting the response to the standard tone from that to the novel stimuli and its amplitude and latency at peak were measured over Fz, Cz and Pz. Also, the amplitude and latency at peak for the N1 component of the auditory evoked potential obtained with the standard stimuli were measured over Fz, Cz and Pz. The correlation between age and MMN and P3a amplitude was also analyzed. RESULTS N1 showed significantly shorter latencies in the autistic groups. MMN elicited by deviant stimuli, but not that elicited by novel stimuli, was found to be significantly larger in autistic children than in normal controls. P3a showed higher amplitude in autistic subjects than in normal controls during childhood; the opposite was observed during young adulthood. DISCUSSION Our findings indicate that significant changes in ERPs can also be seen in non-cooperative individuals with autism and mental retardation, which might be different from the changes already reported for high-functioning autistic subjects and deserve further insight. These changes show developmental modifications that should be taken into consideration when analyzing data from autistic subjects.

Can Motor Recovery in Stroke Patients Be Predicted by Early Transcranial Magnetic Stimulation

BACKGROUND AND PURPOSE We used transcranial magnetic stimulation of the motor cortex to evaluate the functional state of corticospinal pathways innervating the first dorsal interosseous muscle of the hand in 26 patients suffering from a first-ever ischemic stroke in the middle cerebral artery territory. METHODS All patients had complete hand palsy and were tested within the first 24 hours from stroke onset. Patients were also tested clinically with the MRC, Rankin, and National Institutes of Health (NIH) stroke scales at day 1 and with MRC and NIH scales and the Barthel Index at day 14. Electrophysiological testing was repeated at day 14. Patients were divided into three subgroups according to the amplitude of the maximal response (motor evoked potential [MEP]) evoked at day 1. RESULTS After 2 weeks, all 6 patients with initial MEPs > 5% maximum motor response (Mmax) showed some first dorsal interosseous muscle motor function recovery, whereas 19 of 20 patients with initially absent or small (< 5% Mmax) MEPs were left with complete hand palsy. There were strong positive correlations between MEP amplitude at day 1 and MRC and Barthel Index scores at day 14. However, measurement of central motor conduction time proved to be of little prognostic value. CONCLUSIONS We conclude that early-performed transcranial magnetic stimulation is a valuable prognostic tool for motor recovery from stroke and that relatively preserved MEP amplitude shortly after stroke is a better prognostic factor than normal central motor conduction time.

Proteomics analysis provides insight into caloric restriction mediated oxidation and expression of brain proteins associated with age-related impaired cellular processes: Mitochondrial dysfunction, glutamate dysregulation and impaired protein synthesis

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.

Post-stroke reorganization of hand motor area: a 1-year prospective follow-up with focal transcranial magnetic stimulation

OBJECTIVE Focal transcranial magnetic stimulation was used to test prospectively corticospinal excitability changes and reorganization of first dorsal interosseous (FDI) motor cortical representation in 31 patients who experienced a first ischemic stroke in the middle cerebral artery territory. All had severe hand palsy at onset. METHODS Patients were assessed clinically with the Medical Research Council, Rankin, the National Institutes of Health stroke scales and Barthel Index at days 1, 8, 30, 90, 180 and 360 after stroke. The following parameters of FDI motor evoked potential (MEPS) to focal transcranial magnetic stimulation were measured at the same delays: motor threshold, MEP amplitude, excitable cortical area, hot spot and center of gravity of FDI motor maps on affected and unaffected hemispheres. Correlations were sought between clinical and electrophysiological parameters. RESULTS In patients whose affected motor cortex remained excitable at day 1, motor thresholds were not significantly different between sides and were similar to those of controls. Persistence of MEP on the affected side at day 1 was a strong predictor of good recovery. If present at day 1, MEPs recorded in affected FDI were significantly smaller than of the opposite side or in normals and progressively recovered up to day 360. In these patients, area of excitable cortex remained stable throughout the entire study. At day 1, amplitudes of MEPs obtained in unaffected FDI were significantly larger than later. Between days 1 and 360, we observed a significant displacement of center of gravity of motor maps towards more frontal regions on the affected side while no change was noted on the unaffected side. CONCLUSIONS Our data confirm the early prognosis value of transcranial magnetic stimulation in stroke. They indicate that the brain insult induces a transient hyperexcitability of the unaffected motor cortex. The evolution of FDI motor maps along the course of recovery mostly reflect corticospinal excitability changes but might also reveal some degree of brain plasticity. Most modifications observed occurred within 3 months of stroke onset.

Nitric oxide synthase is present in the cerebrospinal fluid of patients with active multiple sclerosis and is associated with increases in cerebrospinal fluid protein nitrotyrosine and S-nitrosothiols and with changes in glutathione levels

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.