Maria Sapienza

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.

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.

Randomised double-blind placebo-controlled study of the effect of inhibition of nitric oxide synthesis in bradykinin-induced asthma

BACKGROUND Bronchoconstriction induced by bradykinin is reduced by the release of nitric oxide (NO) in the airways of guinea pigs. Inhaled NO is known to cause bronchodilatation in asthmatic patients. To find out the role of endogenous NO in airway response to bradykinin in asthma, we examined the effect of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) on broncho-constriction after bradykinin challenge in ten patients with mild asthma. METHODS The study had a randomised, double-blind, placebo-controlled, cross-over design. Participants were studied during two phases, each consisting of 2 study days. After baseline measurements of forced expiratory volume in 1 s (FEV1) participants inhaled an aerosol of L-NMMA or saline (placebo). After 5 min, saline and doubling doses of bradykinin (from 0.25 nmol) were inhaled until FEV1 fell by at least 20% of the post-saline value. The effect of L-NMMA and placebo on airway response to doubling concentrations of methacholine (from 0.03 mg/mL) was then examined. We also assessed the effect of the inactive enantiomer of L-NMMA, D-NMMA, and placebo on bronchoconstriction after bradykinin or methacholine challenge in six of the participants. FINDINGS The geometric mean of the provocative dose producing a 20% fall in FEV1 to bradykinin was 138.0 nmol (range 48.2-475.2 nmol) after placebo and 11.2 nmol (range 0.9-51.3 nmol) after L-NMMA (p < 0.01). L-NMMA also caused a decrease in the provocative concentration of methacholine producing a 20% fall in FEV1 from 0.93 mg/mL (range 0.12-2.55 mg/mL) to 0.38 mg/mL (range 0.06-0.92 mg/mL; p < 0.01). In contrast, D-NMMA did not affect airway response to bradykinin or methacholine. INTERPRETATION The results suggest that bronchoconstriction after bradykinin inhalation is greatly inhibited by the formation of NO in airways of asthmatic patients and that NO could have a bronchoprotective role in asthma.

Oxidative stress and cellular stress response in diabetic nephropathy

Abstract  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.

Redox regulation of cellular stress response by ferulic acid ethyl ester in human dermal fibroblasts: role of vitagenes

Skin is one of the main targets for reactive oxygen species; thus, reactive oxygen species-induced damage and protein and lipid modifications occur, and skin can undergo a wide array of diseases, from photosensitivity to cancer. In this study, human dermal fibroblasts exposed to hydrogen peroxide (0-1000 micromol/L) exhibited a marked increase in both protein carbonyls and 4-hydroxy-2-nonenal, which are indices of protein and lipid oxidation, respectively. An amount of 25 micromol/L ferulic acid ethyl ester, a well-known nutritional antioxidant, significantly counteracted both protein and lipid oxidation and reduced the loss in cell viability elicited by 500 micromol/L of hydrogen peroxide. A common way for cells to react to oxidative stress is up-regulation of vitagenes. To the vitagene family belong the heat shock proteins heme oxygenase-1 and heat shock protein-70, which are involved in the cellular defense against oxidative stress by different mechanisms. The administration of 25 micromol/L ferulic acid ethyl ester significantly decreased hydrogen peroxide-induced protein and lipid oxidation. Dermal fibroblasts exposed to 25 micromol/L ferulic acid ethyl ester in the presence of 500 micromol/L hydrogen peroxide showed an increased level of both heme oxygenase-1 and heat shock protein-70 compared with dermal fibroblasts treated with hydrogen peroxide alone. These findings provide evidence for the protective role of vitagenes in free radical-induced skin damage and highlight the potential protective use of nutritional antioxidants, such as ferulic acid and its derivatives.

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 INFγ-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.

Cellular stress response in aging brain: A redox proteomics approach

Objectives: Neurotrophic factors play key roles in the developing auditory pathway including the sensory epithelium of the inner ear, and structures involved in the central nervous processing of auditory stimuli. In the present investigation, we explored a possible implication of variant BDNF in the susceptibility to chronic tinnitus. Methods: 222 subjects complaining of chronic tinnitus were recruited from a tinnitus clinic and underwent detailed neurootological examinations including otoscopy, stapedius reflexes, middle ear pressure measurements, pure tone audiometry, tinnitus pitch and loudness matches. Subjects were genotyped for a biallelic BDNF missense variant (Val66Met). Prevalence of the substitution was compared to the prevalence in an ethnically homogenous group of healthy controls (N = 317). Results: Carriers of the Met variant were significantly less likely to develop chronic tinnitus with comorbid hearing impairment (p = .02, OR = 1.62, 95% CI = 1.1–2.5). When no assumptions of dominance were made for the minor allele, the Met allele still conferred protection against tinnitus with hearing impairment (p = .05, OR = 1.42, 95% CI = 1.0–2.0). Conclusions: The present study suggests a role of variant BDNF in modulating the genetic susceptibility to chronic tinnitus with hearing impairment. Possible implications of this finding include a differential response to the pharmacological treatment of tinnitus, and specifically, to the neurotrophic effects of antidepressants.