Heat shock proteins and hormesis in the diagnosis and treatment of neurodegenerative diseases

Modulation of endogenous cellular defense mechanisms via the vitagene system represents an innovative approach to therapeutic intervention in diseases causing chronic tissue damage, such as in neurodegeneration. The possibility of high-throughoutput screening using proteomic techniques, particularly redox proteomics, provide more comprehensive overview of the interaction of proteins, as well as the interplay among processes involved in neuroprotection. Here by introducing the hormetic dose response concept, the mechanistic foundations and applications to the field of neuroprotection, we discuss the emerging role of heat shock protein as prominent member of vitagene network in neuroprotection and redox proteomics as a tool for investigating redox modulation of stress responsive vitagenes. Hormetic mechanisms are reviewed as possibility of targeted therapeutic manipulation in a cell-, tissue- and/or pathway-specific manner at appropriate points in the neurodegenerative disease process.     

Hormesis, cellular stress response and vitagenes as critical determinants in aging and longevity

Understanding mechanisms of aging and determinants of life span will help to reduce age-related morbidity and facilitate healthy aging. Average lifespan has increased over the last centuries, as a consequence of medical and environmental factors, but maximal life span remains unchanged. Extension of maximal life span is currently possible in animal models with measures such as genetic manipulations and caloric restriction (CR). CR appears to prolong life by reducing reactive oxygen species (ROS)-mediated oxidative damage. But ROS formation, which is positively implicated in cellular stress response mechanisms, is a highly regulated process controlled by a complex network of intracellular signaling pathways. By sensing the intracellular nutrient and energy status, the functional state of mitochondria, and the concentration of ROS produced in mitochondria, the longevity network regulates life span across species by co-ordinating information flow along its convergent, divergent and multiply branched signaling pathways, including vitagenes which are genes involved in preserving cellular homeostasis during stressful conditions. Vitagenes encode for heat shock proteins (Hsp) Hsp32, Hsp70, the thioredoxin and the sirtuin protein systems. Dietary antioxidants, such as carnosine, carnitines or polyphenols, have recently been demonstrated to be neuroprotective through the activation of hormetic pathways, including vitagenes. The hormetic dose-response, challenges long-standing beliefs about the nature of the dose-response in a lowdose zone, having 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. In this review we discuss the most current and up to date understanding of the possible signaling mechanisms by which caloric restriction, as well hormetic caloric restriction-mimetics compounds by activating vitagenes can enhance defensive systems involved in bioenergetic and stress resistance homeostasis with consequent impact on longevity processes.     

Cellular Stress Responses,Mitostress and Carnitine Insufficiencies as Critical Determinants in Aging and Neurodegenerative Disorders: Role of Hormesis and Vitagenes

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.     

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. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Sex hormonal regulation and hormesis in aging and longevity: role of vitagenes

Aging process is accompanied by hormonal changes characterized by an imbalance between catabolic hormones, such as cortisol and thyroid hormones which remain stable and hormones with anabolic effects (testosterone, insulin like growth factor-1 (IGF-1) and dehydroepiandrosterone sulphate (DHEAS), that decrease with age. Deficiencies in multiple anabolic hormones have been shown to predict health status and longevity in older persons.Unlike female menopause, which is accompanied by an abrupt and permanent cessation of ovarian function (both folliculogenesis and estradiol production), male aging does not result in either cessation of testosterone production nor infertility. Although the circulating serum testosterone concentration does decline with aging, in most men this decrease is small, resulting in levels that are generally within the normal range. Hormone therapy (HT) trials have caused both apprehension and confusion about the overall risks and benefits associated with HT treatment. Stress-response hormesis from a molecular genetic perspective corresponds to the induction by stressors of an adaptive, defensive response, particularly through alteration of gene expression. Increased longevity can be associated with greater resistance to a range of stressors. During aging, a gradual decline in potency of the heat shock response occur and this may prevent repair of protein damage. Conversely, thermal stress or pharmacological agents capable of inducing stress responses, by promoting increased expression of heat-shock proteins, confer protection against denaturation of proteins and restoration of proteome function. If induction of stress resistance increases life span and hormesis induces stress resistance, hormesis most likely result in increased life span. Hormesis describes an adaptive response to continuous cellular stresses, representing a phenomenon where exposure to a mild stressor confers resistance to subsequent, otherwise harmful, conditions of increased stress. This biphasic dose–response relationship, displaying low-dose stimulation and a high-dose inhibition, as adaptive response to detrimental lifestyle factors determines the extent of protection from progression to metabolic diseases such as diabetes and more in general to hormonal dysregulation and age-related pathologies. Integrated responses exist to 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. Vitagenes encode for heat shock proteins (Hsps), thioredoxin and sirtuin protein systems. Nutritional antioxidants, have recently been demonstrated to be neuroprotective through the activation of hormetic pathways under control of Vitagene protein network. Here we focus on possible signaling mechanisms involved in the activation of vitagenes resulting in enhanced defense against functional defects leading to degeneration and cell death with consequent impact on longevity processes.     

Broad action of Hsp90 as a host chaperone required for viral replication

Viruses are intracellular pathogens responsible for a vast number of human diseases. Due to their small genome size, viruses rely primarily on the biosynthetic apparatus of the host for their replication. Recent work has shown that the molecular chaperone Hsp90 is nearly universally required for viral protein homeostasis. As observed for many endogenous cellular proteins, numerous different viral proteins have been shown to require Hsp90 for their folding, assembly, and maturation. Importantly, the unique characteristics of viral replication cause viruses to be hypersensitive to Hsp90 inhibition, thus providing a novel therapeutic avenue for the development of broad-spectrum antiviral drugs. The major developments in this emerging field are hereby discussed. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Acetylcarnitine and cellular stress response: roles in nutritional redox homeostasis and regulation of longevity genes

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.     

Oxidative Stress,Redox Homeostasis and Cellular Stress Response in Ménière’s Disease: Role of Vitagenes

Ménière’s disease (MD) is characterized by the triad of fluctuating hearing loss, episodic vertigo and tinnitus, and by endolymphatic hydrops found on post-mortem examination. Increasing evidence suggests that oxidative stress is involved in the development of endolymphatic hydrops and that cellular damage and apoptotic cell death might contribute to the sensorineural hearing loss found in later stages of MD. While excess reactive oxygen species (ROS) are toxic, regulated ROS, however, play an important role in cellular signaling. The ability of a cell to counteract stressful conditions, known as cellular stress response, requires the activation of pro-survival pathways and the production of molecules with anti-oxidant, anti-apoptotic or pro-apoptotic activities. Among the cellular pathways conferring protection against oxidative stress, a key role is played by vitagenes, which include heat shock proteins (Hsps) as well as the thioredoxin/thioredoxin reductase system. In this study we tested the hypothesis that in MD patients measurable increases in markers of cellular stress response and oxidative stress in peripheral blood are present. This study also explores the hypothesis that changes in the redox status of glutathione, the major endogenous antioxidant, associated with abnormal expression and activity of carbonic anhydrase can contribute to increase oxidative stress and to disruption of systemic redox homeostasis which can be associated to possible alterations on vulnerable neurons such as spiral ganglion neurons and consequent cellular degeneration. We therefore evaluated systemic oxidative stress and cellular stress response in patients suffering from Meniere’s disease (MD) and in age-matched healthy subjects. Systemic oxidative stress was estimated by measuring protein oxidation, such as protein carbonyls (PC) and 4-hydroxynonenal (HNE) in lymphocytes of MD patients, as well as ultraweak luminescence (UCL) as end-stable products of lipid oxidation in MD plasma and lymphocytes, as compared to age-matched controls, whereas heat shock proteins Hsp70 and thioredoxin (Trx) expression were measured in lymphocytes to evaluate the systemic cellular stress response. Increased levels of PC (P < 0.01) and HNE (P < 0.05) have been found in lymphocytes from MD patients with respect to control group. This was paralleled by a significant induction of Hsp70, and a decreased expression of Trx (P < 0.01), whereas a significant decrease in both plasma and lymphocyte ratio reduced glutathione GSH) vs. oxidized glutathione (GSSG) (P < 0.05) were also observed. In conclusion, patients affected by MD are under condition of systemic oxidative stress and the induction of vitagenes Hsp70 is a maintained response in counteracting the intracellular pro-oxidant status generated by decreased content of GSH as well as expression of Trx. The search for novel and more potent inducers of vitagenes will facilitate the development of pharmacological strategies to increase the intrinsic capacity of vulnerable ganglion cells to maximize antidegenerative mechanisms, such as stress response and thus cytoprotection.     

Natural iminosugar (+)-lentiginosine inhibits ATPase and chaperone activity of hsp90

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and cell viability. The relevance of Hsp90 as a therapeutic target for numerous diseases states has prompted the identification and optimization of novel Hsp90 inhibitors as an emerging therapeutic strategy. We performed a screening aimed to identify novel Hsp90 inhibitors among several natural compounds and we focused on the iminosugar (+)-lentiginosine, a natural amyloglucosidases inhibitor, for its peculiar bioactivity profile. Characterization of Hsp90 inhibition was performed using a panel of chemical and biological approaches, including limited proteolysis, biochemical and cellular assays. Our result suggested that the middle domain of Hsp90, as opposed to its ATP-binding pocket, is a promising binding site for new classes of Hsp90 inhibitors with multi-target anti-cancer potential.     

Nutritional antioxidants and adaptive cell responses: an update

Many plant antioxidants, intaken through the daily diet or plant-derived dietary supplements, have been shown able to prevent free radical-related diseases by counteracting cell oxidative stress. However, it is now considered that the in vivo beneficial effects of these phytochemicals are unlikely to be explained just by their antioxidant capability. Several plant antioxidants exhibit hormetic properties, by acting as 'low-dose stressors' that may prepare cells to resist more severe stress. In fact, low doses of these phytochemicals activate cell signaling pathways (being the most prominent examples the modulation of the Nrf2/Keap1 pathway, the NF-κB pathway and the Sirtuin-FOXO pathway) but high doses are cytotoxic. Herein we review the adaptive responses induced by the most known plant hormetic antioxidants, which are sulforaphane, resveratrol, curcumin, flavonoids, green tea catechins and diallylsulphides, as well as the molecular mechanisms involved in such responses. Furthermore, this review outlines that the hormetic properties of these bioactive plant antioxidants might be successfully employed for realizing health-promoting dietary interventions especially in the field of neurodegenerative diseases and cancer.     

Analyzing and engineering cell signaling modules with synthetic biology

Signaling pathways lie at the heart of cellular responses to environmental cues. The ability to reconstruct specific signaling modules ex vivo allows us to study their inherent properties in an isolated environment, which in turn enables us to elucidate fundamental design principles for such motifs. This synthetic biology approach for analyzing natural, well-defined signaling modules will help to bridge the gap between studies on isolated biochemical reactions-which can provide great mechanistic detail but do not capture the complexity of endogenous signaling pathways-and those on entire networks of protein interactions-which offer a systems-level view of signal transduction but obscure the mechanisms that underlie signal transmission and processing. Additionally, minimal signaling modules can be tractably engineered to predictably alter cellular responses, opening up possibilities for creating biotechnologically and biomedically useful cellular devices.     

Cellular stress response,sirtuins and UCP proteins in Alzheimer disease: role of vitagenes

Alzheimer’s Disease (AD) is a neurodegenerative disorder affecting up to one third of individuals reaching the age of 80. Different integrated responses exist in the brain to detect oxidative stress which is controlled by several genes termed Vitagenes. Vitagenes encode for cytoprotective heat shock proteins (Hsp), as well as thioredoxin, sirtuins and uncouple proteins (UCPs). In the present study we evaluate stress response mechanisms in plasma and lymphocytes of AD patients, as compared to controls, in order to provide evidence of an imbalance of oxidant/antioxidant mechanisms and oxidative damage in AD patients and the possible protective role of vitagenes.We found that the levels of Sirt-1 and Sirt-2 in AD lymphocytes were significantly higher than in control subjects. Interestingly, analysis of plasma showed in AD patients increased expression of Trx, a finding associated with reduced expression of UCP1, as compared to control group.This finding can open up new neuroprotective strategies, as molecules inducing this defense mechanisms can represent a therapeutic target to minimize the deleterious consequences associated to oxidative stress, such as in brain aging and neurodegenerative disorders.     

Catechin polyphenols: neurodegeneration and neuroprotection in neurodegenerative diseases

Neurodegeneration in Parkinson's, Alzheimer's, and other neurodegenerative diseases seems to be multifactorial, in that a complex set of toxic reactions including inflammation, glutamatergic neurotoxicity, increases in iron and nitric oxide, depletion of endogenous antioxidants, reduced expression of trophic factors, dysfunction of the ubiquitin-proteasome system, and expression of proapoptotic proteins leads to the demise of neurons. Thus, the fundamental objective in neurodegeneration and neuroprotection research is to determine which of these factors constitutes the primary event, the sequence in which these events occur, and whether they act in concurrence in the pathogenic process. This has led to the current notion that drugs directed against a single target will be ineffective and rather a single drug or cocktail of drugs with pluripharmacological properties may be more suitable. Green tea catechin polyphenols, formerly thought to be simple radical scavengers, are now considered to invoke a spectrum of cellular mechanisms of action related to their neuroprotective activity. These include pharmacological activities like iron chelation, scavenging of radicals, activation of survival genes and cell signaling pathways, and regulation of mitochondrial function and possibly of the ubiquitin-proteasome system. As a consequence these compounds are receiving significant attention as therapeutic cytoprotective agents for the treatment of neurodegenerative and other diseases.     

Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity

The molecular chaperone and cytoprotective activities of the Hsp70 and Hsp40 chaperones represent therapeutic targets for human diseases such as cancer and those that arise from defects in protein folding; however, very few Hsp70 and no Hsp40 modulators have been described. Using an assay for ATP hydrolysis, we identified and screened small molecules with structural similarity to 15-deoxyspergualin and NSC 630668-R/1 for their effects on endogenous and Hsp40-stimulated Hsp70 ATPase activity. Several of these compounds modulated Hsp70 ATPase activity, consistent with the action of NSC 630668-R/1 observed previously (Fewell, S. W., Day, B. W., and Brodsky, J. L. (2001) J. Biol. Chem. 276, 910-914). In contrast, three compounds inhibited the ability of Hsp40 to stimulate Hsp70 ATPase activity but did not affect the endogenous activity of Hsp70. Two of these agents also compromised the Hsp70/Hsp40-mediated post-translational translocation of a secreted pre-protein in vitro. Together, these data indicate the potential for continued screening of small molecule Hsp70 effectors and that specific modulators of Hsp70-Hsp40 interaction can be obtained, potentially for future therapeutic use.     

Geldanamycin induces heat shock protein 70 and protects against MPTP-induced dopaminergic neurotoxicity in mice

As key molecular chaperone proteins, heat shock proteins (HSPs) represent an important cellular protective mechanism against neuronal cell death in various models of neurological disorders. In this study, we investigated the effect as well as the molecular mechanism of geldanamycin (GA), an inhibitor of Hsp90, on 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity, a mouse model of Parkinson disease. Neurochemical analysis showed that pretreatment with GA (via intracerebral ventricular injection 24 h prior to MPTP treatment) increased residual dopamine content and tyrosine hydroxylase immunoreactivity in the striatum 24 h after MPTP treatment. To dissect out the molecular mechanism underlying this neuroprotection, we showed that the GA-mediated protection against MPTP was associated with a reduction of cytosolic Hsp90 and an increase in Hsp70, with no significant changes in Hsp40 and Hsp25 levels. Furthermore, in parallel with the induction of Hsp70, striatal nuclear HSF1 levels and HSF1 binding to heat shock element sites in the Hsp70 promoter were significantly enhanced by the GA pretreatment. Together these results suggested that the molecular cascade leading to the induction of Hsp70 is critical to the neuroprotection afforded by GA against MPTP-induced neurotoxicity in the brain and that pharmacological inhibition of Hsp90 may represent a potential therapeutic strategy for Parkinson disease.     

Mitochondrial Involvement in Brain Function and Dysfunction: Relevance to Aging,Neurodegenerative Disorders and Longevity

It is becoming increasingly evident that the mitochondrial genome may play a key role in neurodegenerative diseases. Mitochondrial dysfunction is characteristic of several neurodegenerative disorders, and evidence for mitochondria being a site of damage in neurodegenerative disorders is partially based on decreases in respiratory chain complex activities in Parkinson's disease, Alzheimer's disease, and Huntington's disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant balance perturbation, are thought to underlie defects in energy metabolism and induce cellular degeneration. Efficient functioning of maintenance and repair process seems 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, which are composed of genes termed vitagenes. A promising approach for the identification of critical gerontogenic processes is represented by the hormesis-like positive effect of stress. In the present review, we discuss the role of energy thresholds in brain mitochondria and their implications in neurodegeneration. We then review the evidence for the role of oxidative stress in modulating the effects of mitochondrial DNA mutations on brain age-related disorders and also discuss new approaches for investigating the mechanisms of lifetime survival and longevity.