Adaptive homeostasis is defined as the transient expansion or contraction of the homeostatic range following exposure to subtoxic, non-damaging, signaling molecules or events, or the removal or cessation of such molecules or events (Mol. Aspects Med. (2016) 49, 1–7). Adaptive homeostasis allows us to transiently adapt (and then de-adapt) to fluctuating levels of internal and external stressors. The ability to cope with transient changes in internal and external environmental stress, however, diminishes with age. Declining adaptive homeostasis may make older people more susceptible to many diseases. Chronic oxidative stress and defective protein homeostasis (proteostasis) are two major factors associated with the etiology of age-related disorders. In the present paper, we review the contribution of impaired responses to oxidative stress and defective adaptive homeostasis in the development of age-associated diseases.
Stress resistance is the phenomenon in which a mild stress enables cells, tissues, or whole organisms to withstand future toxic levels of that stress. The ability to cope with fluctuating levels of stressors such as temperature, pH, oxidative stress, food deprivation, hypoxia, osmolarity, and heavy metals to name a few, is not a fixed characteristic of cells, tissues, or organisms. Cells and organisms make transient and reversible changes to cope with fluctuating internal and external conditions, including many forms of stress. These changes occur through adaptive homeostasis. Adaptive homeostasis is defined as ‘the transient expansion or contraction of the homeostatic range in response to exposure to subtoxic, non-damaging, signaling molecules or events, or the removal or cessation of such molecules or events’ . Adaptive responses to stress depend on biochemical changes to existing enzymes and significant alterations in patterns of gene expression, such as specific stress response factors, including proteases, molecular chaperones, antioxidants, and heat-shock proteins (HSPs) [2–4]. As will be shown in this review, adaptive homeostasis and stress resistance decline with age (Figure 1).
A graphic representation of the decline in adaptive homeostasis that occurs with increasing age
Cells and organisms are constantly exposed to oxidative stress. Oxidative stress is a major type of stress which is defined on the basis of a pro-oxidizing shift in the thiol-redox state and the resulting dysfunction of redox-sensitive proteins. Oxidative stress occurs when the equilibrium of oxidant/antioxidant balance is disrupted, and there is a shift toward an oxidative status, that is accompanied by detrimental effects on cell survival including lipid peroxidation and oxidative modification of DNA, RNA, and proteins . Free radicals play a vital role in physiological processes in signaling pathways, gene regulation, and cellular differentiation .
During periods of high oxidative stress, the ability of cells and organisms to cope with such stress can be transiently altered to meet changing demands through a process called oxidative stress adaptation [7–9]. Studies in Caenorhabditis elegans (nematode worms) and Drosophila melanogaster (fruit flies) show that adaptation to oxidative stress involves an increase in proteolytic activity, and increased expression of the 20S proteasome mediated by the SKN-1 and CNC-C orthologs of the mammalian nuclear factor erythroid-2-like factor 2 (Nrf2) transcription factor, respectively . However, the ability to mount an adaptive response to oxidative stress declines with age. Defective SKN-1 signaling in aged C. elegans impairs 20S proteasome-dependent adaptation to oxidative stress . When proteolytic capacity declines below a critical threshold of activity required to cope with oxidative stress, aggregates of damaged macromolecules impair normal cell function and lead to further complications resulting in disease.
Adaptive responses to oxidative stress decline with ageing. Ageing is characterized by loss of oxidant/antioxidant homeostasis and an impaired ability to mount an adaptive response against oxidative stress, which leads to an increase in protein oxidation and accumulation of protein aggregates [12–14]. An overproduction of reactive oxygen species and a diminished antioxidant capacity is implicated in ageing and age-related diseases. Oxidative damage to mitochondrial enzymes and the mitochondrial genome plays major roles in various age-related degenerative processes. Recent studies in D. melanogaster have shown that adaptive responses to oxidative stress are sex- and age-dependent. Increased expression and activity of the 20S proteasome, and resistance to oxidative stress upon exposure to low non-toxic H2O2 concentrations was lost in aged female flies. In contrast, male flies showed no adaptation to H2O2, regardless of age .
The mitochondrial Lon protease is a key enzyme involved in the degradation of oxidized proteins within the mitochondrial matrix, key regulator of mitochondrial metabolism, and essential factor for maintenance and repair of mtDNA. Under conditions of acute oxidative stress, Lon is up-regulated. However, Lon levels decline in ageing and aberrant Lon expression is involved in age-related diseases [16,17]. Oxidative stress adaptation in D. melanogaster flies requires Lon protease expression. However, stress adaptation requires sex-specific Lon isoform expression. Both aged female and male flies exhibited decrease in basal Lon protease activity and proteolytic activity compared with their young counterparts, despite similar Lon protein levels between both age groups . These studies suggest that stress adaptation is sex-biased and declines with age. There are multiple human diseases involving chronic oxidative stress that show a marked sex bias such as cardiovascular disease, neurological disorders, and diabetes, and the results revealed by these studies will aid in understanding these differences.
The etiology of ageing and various disorders is multifactorial; however re-occurring themes linking ageing and disease are compromised response to stress and impaired proteostasis. Our discussion presented here attempts to integrate two major factors that are associated with diminished stress resistance and defective adaptive homeostasis in age-related diseases: oxidative stress and protein turnover. Specifically, we will focus on the role of the Keap1-Nrf2 (Kelch-like erythroid cell derived protein with CNC homology associated protein 1) pathway in regulating an antioxidant response against oxidative stress and the contribution of impaired protein degradation in onset and progression of a subset of age-related disorders, in particular neurological disorders. Discussion on the role of mitochondrial Lon protease in this review will be limited as its role in human disease and ageing has been reviewed [17,19].
Stress resistance and adaptive homeostasis pathways
Nrf2/electrophile response element signaling
The ‘cap‘n’collar’ (CNC) basic leucine zipper transcription factor Nrf2 is the master regulator of cellular redox homeostasis. Nrf2 is ubiquitously expressed in all the tissues in humans. Under homeostatic conditions, Nrf2 is inhibited from translocating into the nucleus by its negative regulator Keap1. Keap1 plays a role in regulating the localization and degradation of Nrf2 and in sensing oxidative stimuli through its Kelch domain. Keap1 binds Nrf2 and sequesters it to the actin or myosin cytoskeleton. Keap1 also acts as a Cullin-3 ubiquitin ligase E3 complex substrate adaptor, which targets Nrf2 for polyubiquitinylation and subsequent degradation by the 26S proteasome. Lastly, Keap1 also has cysteine residues in the cysteine-rich intervening region (Cys151, Cys273, and Cys288) that are required for Nrf2 binding. Upon oxidative modification of these cysteine residues, Keap1 loses its Nrf2 binding ability and releases Nrf2 .
During periods of oxidative stress, Keap1 is also phosphorylated, further enhancing its dissociation from Nrf2. Oxidant-induced dissociation of the 26S proteasome (mediated by Ecm29 and HSP70 ) prevents further Nrf2 degradation and increases the Nrf2 pool size. Following phosphorylation by AKT and/or PKCγ, Nrf2 enters the nucleus where it binds to various electrophile response elements (EpREs), also known as the antioxidant response elements (AREs) of target genes, thereby acting as an ‘on’ switch that increases their transcription . By activating the expression of genes whose products reduce oxidative stress, detoxify and eliminate toxins, Nrf2 regulates the adaptive response to various stressors. The Keap1-Nrf2 regulatory system is graphically described in Figure 2.
The Keap1-Nrf2 regulatory system
Nrf2/EpRE signaling regulates the basal and stress-inducible expression of over 250 cytoprotective genes, including enzymes involved in biosynthesis, utilization, and recycling of reduced glutathione (GSH). These include glutathione reductase (GR) and glutathione peroxidase (GPx), detoxification enzymes such as glutathione S-transferase (GST), NADPH:quinone oxidoreductase (NQO1), heme-oxygenase (HO-1), isoforms of superoxide dismutase (SOD) 1–3 (SOD1–3), as well as proteins involved in proteostasis [21,22].
The 20S proteasome
The 20S proteasome is one of the cell’s primary ATP-independent mechanisms to degrade oxidatively damaged proteins in the cytoplasm, endoplasmic reticulum, and nucleus of eukaryotes . The 20S proteasome is an approximately 670 kDa core tube-like complex, consisting of four stacked rings. The two external rings are made of seven α subunits, and the two internal rings each consist of seven β subunits – three of which are responsible for distinct proteolytic activities including chymotrypsin-like, trypsin-like, and caspase-like activities [23,24]. In the center of the ring, there is a narrow pore where proteins targeted for degradation can enter in an unfolded state.
The 26S proteasome is formed by the addition of a 19S regulatory particle to each of the α rings of the 20S proteasome. The 19S regulatory particle is an approximately 700 kDa multisubunit complex that recognizes, de-ubiquitinylates, and unfolds substrate proteins. The de-ubiquitinylated protein substrate then enters the 20S proteasome core where it is hydrolyzed by the catalytic β subunits of the core, generating peptides that are subsequently degraded to their constituent amino acids. Key structural and functional aspects of the proteasome are shown in Figure 3.
Structure of the 26S proteasome found in the cytoplasm, nucleus, and endoplasmic reticulum
Protein synthesis and protein degradation are important processes involved in cellular homeostasis. During periods of homeostasis, the cell relies on the ubiquitin-proteasome system (UPS), with the 26S proteasome as the predominant form for protein degradation. As noted above, the 26S proteasome recognizes and degrades substrate proteins tagged with a polyubiquitin recognition signal. The ubiquitinylation of a substrate protein is regulated by a multienzyme system. Ubiqutin (a small protein of 76 amino acids (8.5 kDa)) is activated by the E1 enzyme. The activated ubiquitin is then transferred on to an E2 enzyme, which then associates with E3 ubiquitin ligases. The E3s ligate the ubiquitin to a lysine residue on the target protein. A polyubiquitin chain is then formed through the covalent attachment of a second ubiquitin to the first. The polyubiquitinated protein substrate is then recognized by the 26S proteasome and degraded into small peptides .
During conditions of high oxidative stress, critical sulphydryl groups in the 19S regulatory subunits may lose their proteolytic capacity as they are highly susceptible to oxidative damage. Most of the remaining 26S proteasomes are disassembled by Ecm29, and the 19S caps are sequestered by HSP70 [4,5]. 20S core proteasomes are freed to quickly degrade oxidized proteins. Approximately 3–5 h after the initial stress, 26S Proteasome reassembly is catalyzed by HSP70. During periods of high oxidative stress, when the cellular proteome is most susceptible to oxidative damage, it is critical that there is a functioning pool of 20S proteasome. Induction of the 20S proteasome is regulated by the stress-responsive transcription factor Nrf2 . When proteasomal activity is low, the Nrf2 signal transduction pathway is used to increase expression of the 20S proteasome.
Mitochondrial Lon protease
The ATP-stimulated mitochondrial Lon P1 protease is a major regulator of mitochondrial metabolism and response to free radical damage. Mitochondria contain an electron transport chain (ETC), that transfers high energy electrons to a series of membrane protein complexes, before final acceptance by oxygen. Unfortunately, this process also generates reactive oxygen species as by-products, including sueperoxide and hydrogen peroxide, which can cause damage to surrounding macromolecules. Superoxide is dismutated to hydrogen peroxide by SOD. Hydrogen peroxide is converted into water by GPx with the concomitant oxidation of GSH to GSSG. As mitochondria are one of the major producers of cellular free radicals, removal of mitochondrial oxidatively damaged proteins is crucial for mitochondrial homeostasis and normal cellular function [16,19].
Lon P1 is the primary mitochondrial protease found in the matrix that degrades oxidatively damaged mitochondrial proteins, including aconitase . Oxidized mitochondrial proteins must be rapidly degraded or else they will aggregate, cross-link, and cause subsequent oxidative toxicity. During periods of acute increased cellular stress, Lon is transiently increased to cope with elevated mitochondrial damage [16,27]. However, during ageing and prolonged periods of oxidative stress, Lon protease protein levels are down-regulated, and this Lon deficiency is associated with increased levels of carbonylated proteins that accumulate within the mitochondria . Key functional roles of the Lon protease are shown in Figure 4. Under non-stressful conditions, Lon migrates to the inner surface of the inner mitochondrial membrane where it binds to mtDNA and is crucial for its maintenance and repair as well as for mitochondrial proliferation .
Schematic representation of antioxidant responses to mitochondrial superoxide and hydrogen peroxide production, and Lon involvement in mitochondrial biology
The brain is highly susceptible to damage induced by oxidative stress and reactive oxygen species production, as most of neuronal ATP is generated by mitochondrial oxidative phosphorylation (OXPHOS), only 10% of brain ATP is produced by glycolysis . In addition, its high lipid content makes the brain vulnerable to lipid peroxidation. Mitochondrial dysfunction reduces neuronal capacity to respond to bioenergetic challenges, which impair brain metabolism, and may contribute to neuronal death. Oxidative stress lesions in mtDNA, along with mitochondrial respiratory dysfunction, apoptosis, and impaired antioxidant defense systems are involved in the pathology of neurodegenerative disorders .
Alzheimer’s disease (AD) is the main cause of dementia in older adults, and is currently ranked as the sixth leading cause of death in the United States. Two major hallmarks of AD pathology are the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs) which comprise the hyperphosphorylated τ protein . Studies of AD rodent models and brains from AD patients have shown that accumulation of Aβ plaques and NFTs causes synaptic dysfunction, oxidative stress, calcium dysregulation, and neurodegeneration [32,33].
Oxidative stress is associated with AD, in which Nrf2/EpRE signaling and proteasomal activity are reduced . The proteasome is essential for the degradation of both Aβ and τ. In studies using the 3xTg murine model of AD, young mice exhibited increased phosphorylation of Nrf2, whereas aged mice show a sex-dependent decrease in Nrf2 phosphorylation . These findings suggest that in the early stages of AD, there is an attempt by the Nrf2 system to combat oxidative damage. As the disease progresses, however, Nrf2 phosphorylation decreases, and is targeted for degradation by the 26S proteasome. Disruption of Nrf2 signaling in AD prevents up-regulation of many Nrf2 antioxidant targets and up-regulation of the 20S proteasome – two critical defenses against the increased oxidative stress involved in the AD progression [22,35]. Notably, assessment of Nrf2 expression in the frontal cortex and the hippocampus of AD patients has found that Nrf2 is predominantly expressed in the cytoplasm, in contrast with age-matched normal hippocampal and frontal cortex tissues, in which it is expressed in both the nucleus and cytoplasm. In addition, Nrf2 does not co-localize with Aβ plaques or τ in AD . These findings suggest that in AD, Nrf2 is not translocating to the nucleus despite increased oxidative stress and misfolded proteins in these regions, therefore the Nrf2 pathway may be dysfunctional. Additional studies are warranted to understand how the Nrf2 pathway is altered by AD progression.
Aβ is a peptide that is produced by aberrant proteolytic processing of amyloid precursor protein (APP) into two isoforms which are 40 and 42 amino acids in length (Aβ40 and Aβ42). The Aβ42 isoform contains two additional hydrophobic residues, which makes it aggregate readily and is therefore more toxic to neurons than the Aβ40 isoform . Under normal physiological conditions, low (picomolar) amounts of Aβ can modulate synaptic activity and act as a metal ion chelator due to its high binding affinity for Cu, Fe, and Zn [37–39]. Aβ clearance and degradation is modulated by astrocytes and microglia . Although the mechanisms involved in the transition from normal physiological function to pathological disease are unclear, studies suggest that dysregulation of protein degradation is involved . As the disease progresses, prolonged and widespread activation of microglia and astrocytes increase neuroinflammation and oxidative stress, and the severity of glial activation correlates with the extent of neuronal death and cognitive decline [41,42]. The inability to maintain a homeostatic balance between amyloid production and its degradation results in Aβ accumulation, which directly inhibits proteasomal activity [43,44], and elevates oxidative stress, resulting in exacerbated protein malfunction [45,46]. The proteolytic activity of the 20S proteasome and the ATP-ubiquitin-dependent 26S proteasome is greatly reduced in the AD brain [47,48]. It is important to note that the reduced proteolytic activity may be the result of accumulation of damaged, aggregated, and cross-linked cytosolic proteins that impede proteolytic degradation (including Aβ and hyperphosphorylated τ) rather than a decrease in proteasome expression.
Under homeostatic conditions, endogenous τ is turned over by the proteasome . However in AD, accumulation of hyperphosphorylated τ blocks proteolysis by the 20S proteasome in vitro. Studies using τ aggregates isolated from human AD brains show that 20S proteasome co-immunoprecipitates with Ser212/Thr214 hyperphosporylated τ, and low proteasomal activity correlated with increased τ aggregates in these samples, notably there was no significant difference in proteasomal protein expression between AD and age-matched controls, which suggested that τ aggregates inhibit proteasomal activity in AD .
Parkinson’s disease (PD) is the second most common neurodegenerative disease after AD, and is characterized by a progressive accumulation of α-synuclein, mitochondrial dysfunction, impaired protein degradation in the substantia nigra (SN), and loss of dopaminergic neurons in the SN pars compacta (SNpc) causing severe motor impairments. PD affects over 1% of the population over the age of 60 in the U.S.A., with an average age of diagnosis of 62 years; however PD prevalence reaches 5% in individuals ages over 85 .
The SN is particularly susceptible to loss of dopaminergic neurons with increasing age and prominently accumulates oxidative lesions in PD . Interestingly, the SN seems to be more susceptible to neuronal loss with ageing compared with other brain regions. The SN is a highly oxidative environment, partially due to neuronal dopamine metabolism, which generates superoxide and H2O2 and also reduced reuptake of dopamine into synaptic terminals as expression of the dopamine transporter (DAT) declines with age in the ventral SN [53,54]. α-synuclein increases in the SN during normal ageing, and in PD, α-synuclein accumulates within Lewy bodies and mitochondria . Mutations in the gene encoding α-synuclein (PARK1) are associated with familial forms of PD . Numerous genes involved in maintenance of mitochondrial homeostasis have been linked to PD. PARK1, Pink1 (PARK6), Parkin (PARK2), and DJ-1 (PARK7) genes have functions linked to maintaining mitochondrial homeostasis, including membrane potential, calcium homeostasis, cristae structure, respiratory activity, mtDNA integrity, and mitophagy – all processes which have been implicated in PD pathogenesis . PTEN-induced kinase 1 (Pink1) is a mitochondria-targeted serine/threonine (Ser/Thr) kinase linked to autosomal recessive familial form and early onset PD. In damaged mitochondria, Pink1 recruits the E3 ubiquitin ligase Parkin from the cytosol to the outer mitochondrial membrane to initiate mitophagy. Decreased expression and activity of mitochondrial ETC complexes I and IV have been found in SN tissue from PD patients .
DJ-1 is an oxidative stress-regulated chaperone and transcriptional modulator that translocates from the cytosol to mitochondria and the nucleus upon oxidation. DJ-1 is involved in the Keap1-Nrf2 pathway by stabilizing Nrf2, following its release from Keap1 under oxidative conditions. Loss of DJ-1 function renders mitochondria more sensitive to oxidative stress and complex I inhibitors . DJ-1 inhibits the aggregation and toxicity of α-synuclein. α-synuclein accumulates in mitochondria during PD and is associated with impaired complex I activity, decreased mitochondrial membrane potential, and increased oxidative stress . Mutations in DJ-1 and alterations in DJ-1 levels or isoforms have been documented in early onset of familial and sporadic PD, suggesting a common role in the pathogenesis of PD [59–61].
Under homeostatic conditions, α-synuclein is degraded by the UPS and autophagy, however impaired proteolytic activity of the 20S proteasome has been detected in both familial and sporadic forms of PD within the SN. McNaught et al.  detected a selective loss of 20S proteasome α-subunits in dopaminergic neurons of the SN in sporadic PD. Altered Nrf2 expression in the SN has also been implicated in PD. Nrf2 expression patterns in neuromelanin-containing neurons in the SN, as assessed by immunohistochemistry, revealed that Nrf2 predominantly localizes in the cytoplasm whereas a strong nuclear Nrf2 staining is found in age-matched PD brains. Nuclear Nrf2 localization in PD suggests the activation of endogenous antioxidant responses in dopaminergic neurons within the SN, however this response may be insufficient to protect surviving neurons from chronic oxidative insult .
Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by an autosomal dominant mutation in the huntingtin (HTT) gene resulting in an expansion of polyglutamine repeats (CAG)n in the HTT protein. It is characterized by chorea, dementia, and neuronal death in the cortex and striatum. Mutant HTT forms intraneuronal inclusion bodies in the nucleus and cytoplasm, especially in the perinuclear area and in dendrites .
The onset of HD is inversely correlated with the length of the CAG trinucleotide expansion that codes for polyglutamine repeats; longer CAG repeats are associated with an earlier and more severe presentation of HD . Repeats of 40 or more are associated with disease expression, and on an average HD symptoms typically appear in midlife (40 years). Late-onset HD (age 50 years or more) tends to be associated with 36–39 CAG repeats [64,65]. HD is not an ageing-related disorder per se, however age-related changes in proteostasis and oxidative stress may augment the damage caused by the mutant HTT. As HD patients age, the ability to cope with the mutant HTT aggregates, UPS inhibition, and mitochondrial dysfunction diminishes, resulting in oxidative damage, neuronal death, and massive degeneration of the cortex and striatum . DNA methylation analysis of HD brain samples revealed accelerated epigenetic ageing in the frontal lobe, parietal lobe, and cingulate gyrus with more broad changes in brain methylation levels . A multivariate model analysis of the DNA methylation data in the present study suggests that HD status increases the biological age by 3.2 years .
Mutant HTT expression is associated with increased oxidative stress and reactive oxygen species production. Damage to mtDNA is implicated as a potential mechanism by which mutant HTT leads to mitochondrial dysfunction and contributes to HD progression . Assessment of oxidative stress in postmortem brain tissue samples of HD patients have found increased oxidative mtDNA lesions (8-OHdG) in various regions including the caudate putamen, striatum, and parietal cortex compared with age-matched controls [68–70]. Gene expression studies on animal models and patient tissue samples have provided valuable information regarding the cellular processes and pathways involved in HD pathology [71,72], but the direct effects of mutant HTT may be masked by age-related changes and other secondary processes in the advanced stages of the disease, therefore cell culture HD models have been used to study early HD pathogenesis. Rat pheochromocytoma 12 (PC12) cells induced to express the mutant HTT gene have been used to characterize the effects of mutant HTT in the initial stages of HD. Gene expression analysis of PC12 cells, 5 days after induction revealed up-regulation of a subset of Nrf2-responsive transcripts in mutant HTT expressing cells including NQO1 and GST. The present study also identified a few Nrf2-responive genes that were down-regulated in mutant HTT expressing cells: HO-1, Taldo1, and Prdx6 . In agreement with the present in vitro study, some antioxidant proteins such as GPx, catalase, and SOD (but not all Nrf2/EpRE-driven gene products) are increased in human HD brain . These studies suggest that although the Nrf2 pathway is activated early in HD pathogenesis in response to oxidative stress, Nrf2 signaling may not be fully activated or is impaired. Recently, Quinti et al. found that neural stem cells (NSCs) differentiated from HD patient-derived induced pluripotent stem cells (iPSCs) had impaired Nrf2 signaling, which was ameliorated pharmacologically, by inducing Nrf2 using a selective Keap1-modifying small molecule (MIND4-17) [75,76]. These studies are indicative of an impaired Nrf2/EpRE antioxidant response in HD and highlight Nrf2 as an attractive potential therapeutic target for HD.
Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS) is an adult onset, severe neurodegenerative disorder characterized by progressive weakness, atrophy, and spasticity of muscles due to upper and lower motor neuron death in the cortex, brainstem, and spinal cord, which leads to paralysis. As ALS progresses, motor neurons show axonal degeneration and myelin loss. Surviving motor neurons contain cytoplasmic ubiquitinylated protein aggregates and oxidative stress lesions. Three of the commonly found proteins in these inclusions are TAR DNA-binding protein-43 (TDP-43), SOD1, and fused in sarcoma (FUS) . In the later stages, ALS affects the motor neurons controlling the respiratory muscles and throat, which culminates in lethal respiratory failure. ALS is classified as either familial or sporadic, depending on whether it contains an inherited genetic component. The onset for sporadic ALS (sALS) occurs between ages 50–60 years and 40–60 years for familial ALS (fALS) .
The first causative mutation to be identified in ALS is mutation in SOD1. Mutations in copper-zinc SOD1 play a causal role in approximately 20% of fALS cases. fALS etiology is characterized by mitochondrial dysfunction, increasing oxidative stress, and accumulation of mutant SOD1 which impairs proteasomal activity and induces endoplasmic reticulum (ER) stress via dysfunction of the ER associated degradation (ERAD) process . The role of mutant SOD1 in ALS has been studied primarily in transgenic rodent models overexpressing mutant SOD1. Overexpression of the mutant G93A-SOD1 gene in transgenic mice impaired mitochondrial respiration and ATP synthesis, in addition to oxidative damage to mitochondrial proteins and lipids in the brain and spinal cord at the time of disease onset . Mutant SOD1 accumulates in vacuolated mitochondria in G93A-SOD1 mutant mice, where it co-localizes with cytochrome c. Notably, mitochondrial vacuolization is preceded by motor neuron death, implicating mitochondrial dysfunction as a causal factor in fALS [81,82]. Accumulation of mutant SOD1 is associated with impaired proteasomal function. A progressive decrease in proteolytic activity of the 20S proteasome but not a decrease in proteasomal levels in the lumbar spinal cord of mutant G93A-SOD1 transgenic mice was observed prior to significant motor neuron death which suggests that proteasomal dysfunction may be an early event that contributes to ALS progression .
Although oxidative stress has been implicated in the pathogenesis of ALS, the role of Nrf2 antioxidant defense in ALS is more complex. Nrf2 knockout in G93A-SOD1 mice had modest effects on EpRE/ARE-driven gene induction. NQO1 induction was impaired by Nrf2−/−, whereas the induction of other EpRE/ARE-driven genes, such as HO-1 and genes involved in the GSH pathway (GCLC and GCLM), and tissue GSH levels do not differ between Nrf2−/− G93A-SOD1 and Nrf2+/+ G93A-SOD1 mice . Selective overexpression of Nrf2 in neurons or type II skeletal muscle fibers delayed disease onset, increased EpRE/ARE-driven gene expression and total GSH content, but did not extend survival in G93A-SOD1 mice . These findings suggest that there are additional mediator(s) besides Nrf2 that may be regulating protection against oxidative stress in ALS.
Mutations in sequestrome-1 (SQSTM1), which codes for p62, are also linked to ALS and impaired expression/regulation of oxidative stress response genes. Nrf2 regulates SQSTM1 gene expression via a positive-feedback loop , and Nrf2 expression is induced when SQSTM1 binds to Keap1, via its Keap1-interaction region (KIR) . Mutations in the KIR of SQSTM1 are associated with a loss of Keap1 binding, and impaired Nrf2 signaling . p62 deficiency has also been associated with impaired complex 1 mitochondrial respiration, elevated GSH levels, and reactive oxygen species production in patient fibroblasts carrying SQSTM1 mutations .
Approximately 25% of ALS patients also experience subtle cognitive deficits. In particular, up to 15% of ALS patients are diagnosed with frontotemporal dementia (FTD), with up to 50% showing some FTD symptoms . Mutations in the same disease-causing genes have been reported for both disorders, these include the expanded GGGGCC hexanucleotide repeat expansion in chromosome 9 ORF 72 (C9orf72)  and mutations in the p62 gene (SQSTM1). FTD is the second most common type of dementia after AD, and is characterized by degeneration of the frontal and temporal lobes of the brain which results in progressive changes in behavior, personality, and language difficulties while perception and memory remain relatively intact [92,93]. The C9orf72 mutation is the most common genetic abnormality in both familial FTD (25%) and fALS (30–50%). iPSC-derived motor neurons from patients with the C9orf72 repeat expansion show an age-dependent increase in DNA damage, reactive oxygen species production, and induction of the p53 pathway. Interactome analysis of the C9orf72 dipeptide repeat protein indicate that the expanded repeat, (GR)80, preferentially binds to ribosomal proteins, two-thirds of which are mitochondrial ribosomal proteins required for translation of the mitochondrial ETC subunits, which compromises mitochondrial respiratory function .
The blood–brain barrier (BBB) maintains the homeostatic environment in the brain, and age-induced changes in the microvasculature increase BBB permeability [95,96]. The incidence of ischemic stroke is higher in the elderly than in younger age groups particularly in females, and is associated with age-related health complications such as type II diabetes, hypertension, and cardiovascular disease. The endothelial cells that form the BBB have greater mitochondrial content than non-barrier forming endothelial cells, and mitochondrial content is decreased with age [97,98]. Reduced mitochondrial respiration and reduced expression of ETC complexes in cerebrovascular endothelial cells is associated with increased blood–brain permeability and worsened outcomes after transient middle cerebral artery occlusion (MCAO) in mice . In ageing, increased production of H2O2 and superoxide in endothelial cells impairs the vasodilatory activity of nitric oxide [99,100]. Deletion of endothelial nitric oxide synthase in mice is associated with larger brain infarcts after MCAO compared with controls . Endothelial nitric oxide acts as a vasodilator, and its inactivation reduces vasodilating capacity and tissue perfusion, which suggest that oxidative stress-induced vascular deterioration in ageing may contribute to ischemic stroke .
Brain GSH levels play a crucial role in protection against ischemia/stroke and preserving the integrity of the BBB. Depletion of GSH is associated with increased BBB permeability, and GSH levels are significantly decreased in response to ischemic injury in rat models of hypoxia-induced BBB breakdown [103,104]. Decreased levels of brain GSH following hypoxia/reperfusion may involve membrane GSH transport mediated by multidrug resistance proteins (Mrps). GSH and GSSG are transport substrates for Mrp1, Mrp2, and Mrp4. Ibbotson et al. , recently found increased Nrf2 nuclear translocation following hypoxia/reperfusion in an ischemia rodent model. Nrf2 signaling mediated increased transcription of the genes encoding the GSH-transporting Mrp isoforms (Abcc1, Abbc2, and Abcc4) in brain microvessels. These findings indicate that the Mrp transporters may be novel therapeutic targets for modulating GSH transport and restoring redox balance in disorders involving a compromised BBB, such as stroke. Systemic administration of sulphoraphane increased the expression of Nrf2/EpRE-driven genes in rat models of traumatic brain injury  and intracerebral hemorrhage . Nrf2 induction and subsequent expression of antioxidant genes, including GST, GPx, and HO-1, attenuated loss of tight junction proteins and endothelial cells after brain injury, thus protecting the integrity of the BBB .
Brain ischemia-induced oxidative stress results in production of misfolded and oxidized proteins, and impairs protein degradation pathways, which results in impaired proteasomal function and accumulation of ubiquitin-containing protein aggregates . Transient global ischemia in a gerbil model resulted in a progressive accumulation of ubiquitinylated protein aggregates in neurons, which preceded neuronal death in the CA1 region of the hippocampus . Transient global ischemia impairs 26S proteasome function by promoting proteasome disassembly during the early period of reperfusion in rat and gerbil models [110,111]. The down-regulation of proteasomal activity is associated with increased oxidative stress, and ATP depletion following ischemia prevents 26S proteasome reassembly [111,112]. These results suggest that brain ischemia induces an accumulation of ubiquitinylated protein aggregates and impairs proteasomal activity, which is associated with apoptotic neuronal death.
Hypertension is seen in approximately 70% of elderly stroke patients . In particular, elderly women are at a higher risk for stroke and hypertension, and tend to have worse outcomes than men . Diminished insulin-like growth factor-1 (IGF-1) levels and estrogen–IGF-1 interaction contribute to the development of hypertension in post-menopausal women [114,115]. In addition, estrogen increases cerebral blood flow by enhancing endothelial-derived nitric oxide production, and angiogenesis. Estrogen depletion in post-menopausal women may also contribute to the sex differences in stroke incidence amongst the elderly .
Emotional stress, depression, and anxiety
Stress is a common reaction to adversity, but chronic stress exposure and diminished stress resiliency can lead to major depressive disorder (MDD) and anxiety disorders, amongst other psychiatric illnesses. Depression is prominent amongst older people, with a significant impact on well being and quality of life. Oxidative stress is implicated in the development of anxiety disorders, emotional stress, and depression. A link between oxidative stress and emotional stress is not surprising considering the evidence that depression and anxiety is often experienced by patients with disorders linked to oxidative stress (chemotherapy-related cognitive impairment (CRCI), AD, PD, HD, cancer). A recent review of the research on depression spanning the past 20 years suggest that the prevalence of depression does not increase with age, but depression in older adults is more chronic and more difficult to treat as antidepressants are less effective .
Many reports have also found an association between depression and diminished antioxidant levels in human clinical studies and animal models of depression. Analysis of oxidative stress markers in depressed patients revealed an association between depression, higher plasma levels of malondialdehyde (MDA) an end product of lipid peroxidation, diminished antioxidant capacity, and increased SOD activity in red blood cells of patients with MDD [118,119]. Similar results have been reported by meta-analysis studies [120,121]. Antioxidant supplementation, with N-acetylcysteine (NAC) augments antidepressant efficacy in clinical studies for depressive symptoms [122,123].
MDD is associated with alterations in the UPS pathway. A few studies have linked genetic variants in proteasomal subunit proteins and down-regulation of genes involved in ubiquitinylation of synaptic proteins in patients with psychiatric disorders [124–126]. Single-nucleotide polymorphisms (SNPs) in proteasome subunit α7 (PSMA7), proteasome 26S non-ATPase subunit 9 (PSMD9), and proteasome 26S non-ATPase subunit 13 (PSDM13) are associated with poor antidepressant treatment response in depressed patients [124,126,127]. The intronic SNP PSMD13 rs3817629 is associated with lower PSMD13 expression in fibroblasts of patients with MDD. PSMD9 missense SNP rs1043307 is associated with anxiety disorder in type II diabetes and MDD. PSMD9 mRNA expression is down-regulated in peripheral blood cells of MDD patients, specifically in non-responders to antidepressants. Notably, anxiety in MDD patients is a negative predictor of antidepressant treatment response . Under oxidative conditions, the damage to proteins by reactive oxygen species results in misfolded proteins and altered proteasomal activity. The SNPs in proteasomal subunits described above, and low expression levels proteasomal subunits may result in the inability of the proteasome to effectively degrade damaged proteins in oxidative conditions such as those implicated in MDD, resulting in impaired proteostasis.
Chronic stress exposure can increase susceptibility to depression and is associated with oxidative stress. Bouvier et al. , demonstrated that Nrf2 is a key regulator of oxidative stress in the chronic stress (social defeat) rat model. Animals who developed a depression-like phenotype displayed down-regulation of nuclear Nrf2 in the hippocampus, lipid peroxidation, DNA oxidation, and an increase in the inactive form of peroxiredoxin-2 (Prx-2), an antioxidant enzyme involved in H2O2 reduction to H2O. In addition to the alterations in hippocampal redox homeostasis, neurons of the CA3 hippocampal region had a significant reduction in apical dendritic length and spine loss. Investigation of the upstream mechanism regulating Nrf2, revealed that brain-derived neurotrophic factor (BDNF) is involved in the nuclear translocation of Nrf2. BDNF down-regulation in rat hippocampi decreased Nrf2 translocation to the nucleus and induced oxidative stress. Rescue of Nrf2 function with the antioxidant tert-Butylhydroquinone or BDNF augmentation via 7,8-DHF, a selective TrkB agonist, prevented susceptibility to depression . Separate studies examining the influence of the antidepressant fluoxetine, a commonly prescribed selective serotonin reuptake inhibitor, on Nrf2 signaling in rodent models of stress  and anxiety/depression  have revealed that fluoxetine up-regulates HO-1 through an Nrf2-dependent pathway, and increases cortical and hippocampal BDNF expression levels. These findings open up the potential for development of oxidative stress targeted therapies for various neurodegenerative and psychiatric disorders that are associated with alteration in redox homeostasis.
Anxiety is an aversive emotional response to a threat or a perceived threat. The feeling of fear is accompanied by emotional stress, and when this emotion is extreme and persistent, it is classified as pathological . Most research on anxiety had focussed on neurotransmitter dysregulation including the γ-aminobutyric acidergic (GABAergic) and serotoninergic systems, until Hovatta et al.  identified a correlation between expression of two genes involved in antioxidant response to oxidative stress and anxiety. Gene expression profiling of mice strains expressing anxiety-like behavior identified two genes glyoxalase 1 (Glo1) and glutathione reductase 1 (GR 1) to play a causal role in the anxiety-like phenotype . GR catalyzes the reduction of GSSG to the sulphydryl form of glutathione (GSH). Glo1 uses GSH as a cofactor to detoxify methylglyoxal – a highly reactive dicarbonyl compound formed as a glycolysis by-product. This was the first study that demonstrated a link between altered oxidative stress response and anxiety. A separate study examining the oxidative status in the brains of Swiss albino male mice with contrasting levels of anxiety reported elevated reactive oxygen species levels in neuronal and glial cells in the cerebellum, hippocampus, cerebral cortex, and in peripheral leukocytes of anxious mice. Nrf2 knockdown by siRNA injected into the dorsal third ventricle of the brains of adult male Wistar rats induced anxiety-like behavior, increased expression of cleaved caspase-3, and quite unexpectedly increased respiratory activity of the mitochondrial ETC complexes, perhaps as a compensatory mechanism in response to oxidative stress-induced neuronal damage . Together these findings suggest an imbalance of the redox system in anxiety-related disorders [131,134].
Chemotherapy-related cognitive impairments (CRCIs) are commonly reported during and after completion of chemotherapy treatment. CRCI includes changes across various cognitive domains such as working memory, executive function, and processing speed. While 33% of cancer survivors experience long-term CRCI, older adults may have an increased likelihood of developing CRCI [135–137]. Although studies focussing solely on the prevalence of CRCI in the ageing population are limited, two of the most commonly affected groups by CRCI – the breast cancer and ovarian cancer populations are composed of patients with a median age of 60–63 years . Studies examining cognitive function of women aged 60+ receiving adjuvant chemotherapy for breast cancer have found reductions in performance in neurocognitive testing 6 months after the completion of chemotherapy treatment [139–141]. Notably, cognitive dysfunction in the studies of breast cancer patients correlate with reductions in gray and white matter density [142,143] and reduced hippocampal volume . In addition, Heflin et al. found that elderly cancer survivors have an increased risk for developing persistent long-term cognitive impairments and dementia .
The mechanisms by which chemotherapy can cause long-term cognitive changes have not been well characterized; however multiple candidate mechanisms for CRCI have been proposed. Amongst the CRCI candidate mechanisms, DNA damage and oxidative stress have been described for various chemotherapeutic agents including cisplatin, doxorubicin, carmustine, methotrexate, and cyclophosphamide [146–149]. Doxorubicin results in oxidative stress by producing superoxide ions that damage complex I of the ETC, in addition it increases levels of tumor necrosis factor-α (TNF-α) in the plasma, which crosses the BBB and activates apoptotic pathways resulting in neural death . Rodent studies examining the effects of cisplatin on neurotoxicity and cognition have found that cisplatin causes neural free radical production and mtDNA mutations which impair mitochondrial respiratory capacity and result in neuronal dendritic spine loss and apoptotic cell death in hippocampal neurons and NSCs [146,151–153]. Diminished antioxidant capacity and low levels of key antioxidants (GSH, SOD) have been observed in the blood of chemotherapy-treated cancer patients [154,155] as well as in the blood and brain tissue of chemotherapy-treated rodents [156,157].
Cancers (a brief overview)
Cancers are an extremely heterogenous group of diseases about which it is hard to make generalizations. In addition, the importance of oxidative stress and proteostasis (or deviations from proteostasis) in various forms of cancer have been widely studied. For these reasons, we have made the topic of oxidative stress, adaptive homeostasis, and proteolysis in various cancers its own review (to be published separately). In the present monograph, therefore, we have limited the discussion of cancers to just a bare minimum.
For most cancer types, incidence increases with age. Adults aged 50–74 account for 53% of all new cancer cases, and elderly people aged 75+ account for 36%, with more cancer cases in males than females in both age groups. The elderly population is more susceptible to developing cancers . A few factors involved in this age discrepancy in cancer incidence include accumulation of genomic damage (mtDNA and nuclear DNA lesions), prolonged exposure to environmental carcinogens, chronic inflammation, weakened immune response, and increased oxidative stress .
Mitochondrial dysfunction, oxidative stress, and deregulation of cellular energetics are hallmarks of cancer biology. To fuel uncontrolled cell proliferation under fluxing oxygen conditions, cancer cells must adapt, or reprogram their energy production by limiting their energy production largely to glycolysis (Warburg effect) . Under homeostatic conditions, Nrf2 levels are kept low due to Keap1 inhibition. Nrf2 plays a protective role by regulating the cellular adaptive response to oxidative stress and maintaining cellular redox homeostasis. However in many cancers, aberrant Nrf2/EpRE regulation results in Nrf2 constitutive activation, which accelerates cancer progression in pancreatic cancer and in non-small-cell lung carcinoma, [161,162] and is associated with chemoresistance and radioresistance .
Nrf2 activation in cancer cells facilitates metabolic programming by redirecting glucose and glutamine into anabolic pathways through the PI3K/Akt pathway . Nrf2 activation has also been shown to increase the expression of anti-apoptotic genes Bcl-2, Bcl-XL which enhances cancer cell survival and drug resistance in the Hepa-1 and Hepa-G2 hepatocarcinoma cell lines [165,166]. Pharmacological inhibition of Nrf2 has been shown to reverse the chemoresistance in several human cancer cell lines including human breast adenocarcinoma, myelogenous leukemia, and liver cancer [167–169]. While Nrf2 represents a potential target for novel adjuvant therapies in cancer treatment, careful consideration must be made to possible toxicities and additional diseases associated with oxidative stress and antioxidant depletion.
A dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts is required for the maintenance, strength, and integrity of the human skeleton. In osteoporosis, bone homeostasis is lost, and bone resorption exceeds bone formation leading to low bone mass and deterioration of bone tissue and an increased susceptibility to fractures, especially of the hip, spine, and wrist. Post-menopausal women are particularly susceptible to osteoporosis as estrogen deficiency negatively affects bone homeostasis . Estrogen deficiency leads to increased levels of TNF and receptor activator of NF-κB ligand (RANKL)  and enhanced oxidative stress  which promotes osteoclast formation. GPx, the primary antioxidant enzyme expressed by osteoclasts is up-regulated by estrogen , and estrogen deficiency in ovariectomized rodent models is associated with impaired antioxidant expression .
Keap1-Nrf2 regulates RANKL-dependent osteoclastogenesis through modulation of intracellular reactive oxygen species signaling . Nrf2 deficiency in bone marrow derived macrophages derived from Nrf2−/− mice augments RANKL-induced osteoclast differentiation and bone resorption which is associated with diminished antioxidant production [176,177]. In separate studies, stimulation of mouse osteoclast precursors with RANKL up-regulates Keap1, decreases Keap1-Nrf2 ratio, and down-regulates cytoprotective enzymes including HO-1 and NQO1 . Kanzaki et al. recently found that RANKL attenuates Nrf2-mediated expression of cytoprotective enzymes by inducing the nuclear translocation of BTB and CNC homology 1 (Bach1), a repressor of Nrf2 . Interestingly, pharmacological induction of Bach1 export from the nucleus induced Nrf2 translocation to the nucleus, increased the expression of antioxidant enzymes, and resulted in reduction in osteoclastogenesis and bone damage in a bone destruction mouse model .
Furthermore, other groups have shown that reactive oxygen species production via a non-mitochondrial pathway, NADPH oxidases (NOXs) 1–4 (NOX1–4) is implicated in osteoclast differentiation. Although the relative contributions of each NOX isoform to reactive oxygen species production and role in osteoclasts are disputed in the field, the involvement of NOX4 reactive oxygen species production in osteoclastogenesis has been characterized . NOX4 is a constitutively active enzyme, found on intracellular organelle membranes and produces H2O2. NOX4 knockout in mice increased bone density, reduced osteoclast numbers and circulating bone resorption markers. Loss of NOX4 also down-regulated H2O2 and Ca2+ levels during osteoclast differentiation. Pharmacological inhibition of NOX4 in an ovariectomy-induced osteoporosis murine model attenuated bone loss . Bone from patients with untreated osteoporosis exhibited increased NOX4 expression and protein levels compared with age-matched controls .
Osteoarthritis (OA) is one of the most common degenerative diseases of the joints amongst the elderly population. OA is characterized by an age- or time-dependent progressive loss of joint homeostasis due to degeneration of the articular cartilage and elevated chondrocyte death affecting individuals over the age of 50 . Chondrocytes are responsible for the synthesis and breakdown of the cartilaginous extracellular matrix . Under homeostatic conditions, chondrocytes are quiescent cells. However in OA, chondrocytes take on an activated phenotype resulting in increased secretion of proinflammatory cytokines including IL-6, IL-1β, and TNF-α. OA chondrocytes also produce extracellular matrix degrading proteins, such as matrix metalloproteinases (MMPs) [183,184]. The anabolic activity of chondrocytes in articular cartilage diminishes with age. IGF-1 is the main anabolic growth factor for articular cartilage and plays a key role in cartilage homeostasis by balancing proteoglycan synthesis and breakdown by chondrocytes. Chondrocyte responsiveness to IGF-1 decreases with age and in OA, which may contribute to cartilage deterioration in OA [185,186].
Human articular chondrocytes actively produce reactive oxygen species and reactive nitrogen species [187–189]. Enhanced reactive oxygen species and reactive nitrogen species production, mitochondrial dysfunction, and diminished activity of GR and thioredoxin reductase  occurs in ageing chondrocytes and contributes to the activated chondrocyte phenotype in OA [183,191]. Increased reactive oxygen species levels may contribute to IGF-1 loss of responsiveness in aged OA chondrocytes by interfering with cartilage matrix synthesis [192,193]. Excessive reactive oxygen species production triggers the activation of signaling pathways leading to apoptosis or necrosis of chondrocytes in OA .
Nrf2 activation in human OA chondrocytes suppresses IL-1β-induced MMP-1, MMP-3, and MMP-13 mRNA synthesis and protein levels, and induces the expression of antioxidant enzymes NQO1 and HO-1 [195,196]. Studies using the monosodium iodoacetate (MIA) articular injection and destabilization of the medial meniscus (DMM) models of OA have revealed more severe cartilage damage in Nrf2−/− mice compared with wild-type controls. In both, MIA and DMM models, Nrf2 downstream proteins HO-1 and NQO1 are up-regulated . These results suggest that the Nrf2 pathway exerts a protective function in OA development. Recently, Nrf2 has been recognized as a protein acetylation target, and its acetylation increases Nrf2 signaling and EpRE/ARE-driven gene expression [198,199]. Nrf2 acetylation using histone deacetylase (HDAC) inhibitor trichostatin A promotes Nrf2 nuclear translocation, increased expression of HO-1 and NQO1 in chondrocytes, and reduced levels of proinflammatory cytokines in MIA and DMM mouse models of OA .
The UPS plays a crucial role in regulating NF-κB activation . The NF-κB pathway is activated and contributes to the proinflammatory environment in rheumatic disorders including OA. NF-κB activation mediates MMP-1, MMP-3, MMP-13 mRNA and protein expression induced by the proinflammatory cytokines TNF-α and IL-1β in human OA articular chondrocytes . NF-κB is present in the cytoplasm in most mammalian cells in an inactive state through its association with the inhibitory κB proteins (IκBs). The IκBs are degraded by the 26S proteasome following polyubiquitinylation. Upon degradation of IκB, the NF-κB complex translocates to the nucleus where it regulates inflammatory signaling . UPS inhibition via proteasomal inhibitor MG132 in the DMM OA mouse model protected cartilage from cytokine-mediated resorption and degradation and MMP-13 expression. Although the molecular mechanism by which proteasomal inhibition exerts a protection against OA cartilage damage in this model remains unclear, it was suggested that it limits IκB degradation by impeding NF-κB signal transduction .
The ageing kidney
A normal kidney contains approximately 1 million nephrons, each of which contributes to the total glomerular filtration rate (GFR). Under homeostatic conditions, the kidney is able to maintain GFR, despite renal injury, as the remaining nephrons adapt through hyperfiltration and compensatory hypertrophy to continue normal clearance of plasma solutes. Kidney function declines with age. Some notable age-related changes in the kidney are decreased number of functional glomeruli and tubules, decreased GFR and renal blood flow, nephron loss, glomerulosclerosis, and tubulointerstitial fibrosis . In addition to age-related decline in kidney function, other factors that affect vascular health such as hypertension, diabetes, cardiovascular disease, and smoking may increase susceptibility to nephrotoxicity in advanced age .
Oxidative stress is a major mediator of age-related loss of renal function and renal diseases such as chronic kidney disease. The prevalence of chronic kidney disease is higher in older people, and it is a major co-morbidity associated with diabetes, hypertension, heart disease, and stroke . In a rodent model of chronic kidney disease, rats subjected to a 5/6 nephrectomy exhibited a progressive increase in oxidative stress, NF-κB activation, decrease in Nrf2 nuclear expression, and down-regulation of Nrf2 target genes including SOD isoforms, GPx, NQO1, and HO-1 in the remaining kidney. GSH depletion and down-regulation of GSH-related enzymes (GST, GPx, and glutamate cysteine ligase subunits) were also observed in this model, indicating a maladaptive response by the remnant kidney to cope with oxidative stress and inflammation . These changes were associated with marked increase in Keap1, suggesting that Nrf2 activation was repressed, accounting for the impaired antioxidant response. Nrf2 knockout intensifies oxidative stress and renal injury in mice models, including models of ischemia–reperfusion and cisplatin chemotherapy-induced nephrotoxicity . Renal function and survival is significantly worse in Nrf2−/− mice compared with the wild-type control mice. Induction of Nrf2 by antioxidant supplementation with NAC or GSH improves renal function .
Several studies suggest that deficiencies in antioxidant capacity may play a role in the ageing kidney. In aged rats, GST activity is decreased. Although renal basal GSH levels were not lower in aged rats compared with younger rats, basal renal GSH levels were depleted more rapidly when aged rats were challenged with acute ischemia exposure .
Kidney androgen regulated protein (KAP) is expressed in proximal tubule epithelial cells and is critical for maintaining cardiovascular–renal homeostasis, although the exact function of the KAP protein is unknown . Overexpression of KAP in the kidneys of male transgenic mice induced hypertension and enhanced the excretion of urinary markers of oxidative stress (8-iso-prostaglandin F2α, 8-OHdG), augmented mtDNA damage and MDA levels, and diminished catalase and GPx activity in the kidneys. These observations suggest an association between sex-dependent androgen differences in the development of hypertension and renal damage [211,212].
Pathogenic bacteria, leukocyte infiltration, increased levels of proinflammatory cytokines, and elevated reactive oxygen species production are associated with chronic prostatitis, which is persistent inflammation of the prostate gland . The most common type of prostatitis is chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) which accounts for 90% of cases, and affects up to 15% of men . The risk for developing prostatitis increases with age , CP/CPPS is characterized by pain or discomfort in the abdomen, pelvis, and genitals, for a period of 3 months, as well as irritation and obstructive lower urinary tract symptoms in the absence of urinary tract infection. The causes for CP/CPPS are not well understood.
Patients with chronic prostatitis have higher seminal oxidative stress and diminished antioxidant levels compared with healthy men . Symptoms in patients with chronic prostatitis may last for several weeks or recur periodically for many years, which can negatively impact sperm function, and result in infertility in some patients. Expression of inflammatory mediators IL-8, IL-1β, and ICAM-1 is associated with endothelial cell damage, and negatively correlates with erectile dysfunction in CP/CPPS patients . In particular, higher levels of reactive oxygen species were detected in chronic prostatitis patients with leukocytospermia [216,218]. Leukocytes are the primary source of reactive oxygen species production in semen [216,219]. A separate study in patients with chronic bacterial prostatitis, found increased nitric oxide and MDA levels, and decreased levels of the antioxidant enzymes SOD, catalase, and GPx in the blood . Oxidative stress in the chronic non-bacterial prostatitis (CNP) rat model is associated with down-regulation of Nrf2 target genes NQO1, GST, and HO-1, and a decrease in the Nrf2 to Keap1 ratio. Antioxidant NAC administration ameliorated prostatitis and increased Nrf2/EpRE-driven gene expression .
Non-alcoholic fatty liver disease
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in Western countries. The prevalence rate of NAFLD is between 15% and 30% in adults, and increases to 35.1% in people aged greater than 65 . NAFLD is characterized by defective fatty acid metabolism – an imbalance between lipid uptake and synthesis that exceeds oxidation and removal, resulting in an excess accumulation of fat deposits in hepatocytes . NAFLD is commonly associated with obesity, type II diabetes, and insulin resistance. NAFLD ranges from simple steatosis (fatty infiltration in liver) to non-alcoholic steatohepatitis (NASH), which can progress to fibrosis and ultimately liver cirrhosis ; 20%–30% of individuals with NAFLD develop NASH, which consists of lipid buildup and inflammation. Lipid metabolism is increased as a response to the accumulation of fatty acids and triglycerides in the liver, which causes oxidative stress through overproduction of reactive oxygen species. Oxidative stress also triggers production of inflammatory cytokines, which causes inflammation via activation of NF-κB and c-Jun N-terminal kinase (JNK), ultimately resulting in the development of NASH .
Mitochondrial dysfunction plays a central role in the pathogenesis of NAFLD. Depletion of mtDNA, decreased respiratory activity, and impaired mitochondrial β-oxidation are associated with NAFLD . Impairment of β-oxidation increases fatty acid accumulation in hepatocytes which combined with elevated reactive oxygen species levels augments lipid peroxidation which results in a vicious cycle of hepatic oxidative stress-induced damage. Reactive oxygen species and by-products of lipid peroxidation further impair respiratory chain function in hepatocytes and increase generation of proinflammatory cytokines (TNF-α, Fas ligand). Impaired mitochondrial function in hepatocytes resulting in reduced ATP synthesis and increased reactive oxygen species production has been reported in patients with NASH [226–228].
Several studies indicate that antioxidant defense mechanisms are altered in NAFLD. Hepatocyte GSH content and SOD activity was decreased in NAFLD patients, this depletion is further exacerbated in patients with steatohepatitis – inflammation and lipid accumulation in the liver . Hepatic GSH, as well as SOD, catalase, and GPx activity were decreased in the livers of obese fa/fa rats fed a high-fat diet . Lipid peroxidation and protein carbonyls and NOX activity were elevated which was associated with fibrosis . In a separate study, Nrf2−/− mice fed a high-fat diet developed more severe NASH with cirrhosis, than wild-type Nrf2+/+ mice. The livers of Nrf2−/− mice fed a high-fat diet had increased induction of lipogenesis genes and suppression of β-oxidation genes compared with the wild-type livers, which suggests greater fatty acid synthesis in the Nrf2−/− livers. Administration of the high-fat diet stimulated hepatic oxidative stress as indicated by increased levels of GSSG, MDA, protein carbonyls, and increased levels of apoptosis and DNA damage. Unlike the Nrf2+/+ mice, Nrf2−/− mice had decreased expression of EpRE/ARE-driven genes, including genes involved in GSH homeostasis and NQO1. This attenuated antioxidant response in Nrf2−/− mice may stimulate inflammation, contributing to the rapid development of NASH . This study suggests that Nrf2 protects the liver against steatosis and NASH by suppressing lipogenesis and promoting fatty acid oxidation, and enabling adaptation to high-fat diet-induced oxidative stress. Notably, studies on aged Ldlr−/− mice, which models human NASH and atherosclerosis, fed high-fat diets show a greater decline in antioxidant gene expression which correlates with a decrease in Nrf2 expression compared with young Ldrl−/− animals . Aged Ldlr−/− mice also displayed more severe steatosis, along with NASH, in contrast with the young Ldrl−/− mice which only developed fatty livers .
Alcoholic liver disease
Alcoholic liver disease (ALD) complications in the elderly are higher due to impaired ethanol metabolism. An elderly person’s liver is more susceptible to the toxic effects of alcohol, and prognosis of ALD in the elderly is poor usually due to hepatic damage induced by heavy lifelong consumption of alcohol . The severity of ALD ranges from steatosis to cirrhosis. Hepatic ethanol is processed by two major pathways: alcohol dehydrogenase (ADH), which resides in the cytosol and cytochrome P450 2E1 (CYP2E1) which is a component of the ER. The activity of alcohol metabolizing enzymes ADH, acetaldehyde dehydrogenase (ALDH), and CYP2E1 diminishes with age . In addition, body water content also decreases with age, these two factors lead to elevated concentrations of ethanol in the blood .
Acetaldehyde is a highly reactive intermediate of alcohol metabolism that accounts for most of the ethanol-induced toxicity. Acetaldehyde promotes hepatic lipid accumulation. Nrf2−/− mice fed an ethanol diet, displayed increased mortality associated with liver failure at doses that were tolerated by wild-type control animals. Loss of Nrf2 resulted in significantly lower ADH activity and accumulation of acetaldehyde in the liver, leading to steatosis and up-regulation of proinflammatory cytokines TNF-α and IL-6. Nrf2−/− mice also had impaired GSH homeostasis; mitochondrial GSH levels were significantly lower and associated with mitochondrial swelling .
In ALD, inclusions of damaged intermediate filaments, known as Mallory–Denk bodies (MDBs) accumulate within hepatocytes. MDBs have been found to contain ubiquitin, HSPs 70, 90, and 25, as well as the β5 catalytic subunit of the 20S proteasome, and Tbp7 (an ATPase subunit of the 26S proteasome) in an ALD mouse model . Serum concentrations of free ubiquitin and polyubiquitin chains are higher in subjects with alcoholic liver cirrhosis than in normal subjects . These studies suggest that the UPS is unable to degrade the protein components of the MDBs. Formation of MDB aggregates in ethanol-exposed HepG2 cells correlates with an ethanol-induced reduction in proteasomal activity . Ethanol administration in rats results in decreased proteolytic activity of the 20S proteasome, which is associated with increased hepatic content of liver peroxides, suggesting that ethanol-induced oxidative stress may impair proteasomal activity .
Insulin resistance and type II diabetes
Diabetes is characterized by dysfunction in the body’s ability to maintain glucose and insulin homeostasis. Diabetes increases the risks for multiple age-related diseases including cancer, stroke, cardiovascular diseases, atherosclerosis, NAFLD, PD, and AD [241,242]. Diabetes is divided into two categories, type I and type II. Type I is a genetic disorder where autoimmune destruction of pancreatic β cells leads to insufficient production of insulin, whereas type II diabetes occurs when insufficient insulin is produced or insulin secretion is impaired resulting in high blood glucose levels. Type II diabetes accounts for more than 90% of individuals diagnosed with diabetes. The elderly are at a higher risk for developing type II diabetes, due to the increase in insulin resistance and pancreatic β cell dysfunction with age. Glucose homeostasis requires fasting blood glucose levels to be maintained between 70 and 100 mg/dl. Higher fasting glucose blood levels between 100 and 125 mg/dl are indicative of insulin resistance, and levels above 126 mg/dl are an indicator of diabetes . Under homeostatic conditions, pancreatic β cells adapt to insulin resistance by increasing mass and insulin secretion. However, failure of β cells to properly compensate results in hyperglycemia .
Ageing in humans and rodents is associated with a decline in glucose-stimulated insulin secretion from pancreatic β cells. Impaired insulin secretion and insulin resistance are two hallmarks of type II diabetes . Multiple factors in ageing contribute to the development of diabetes including decrease in β cell mass and impaired function, altered glucose transport as indicated by decreased expression of glucose transporters in skeletal muscle (GLUT4) , increased oxidative stress, and mitochondrial dysfunction [247–249]. Oxidative stress impairs insulin signaling, which plays a role in the development of insulin resistance and type II diabetes. Low levels of H2O2 are required for β-cell insulin secretion in response to glucose, however chronic elevation of reactive oxygen species decreases β-cell mass and function by decreasing transcription factor (Pdx2 and MafA) binding to the insulin gene . Islets from type II diabetes patients’ exhibit lower ATP content and diminished glucose-stimulated insulin secretion. In addition, other studies have demonstrated augmented release of proinflammatory cytokines (TNF-α, IL-6) from leukocytes which may further impair insulin signaling and glucose uptake [247,251].
Decreased Nrf2 expression and activity is associated with diabetes and is also involved in diabetic complications. At the onset of diabetes, Nrf2 protein expression and Nrf2-gene products NQO1 and HO-1 are up-regulated due to acute hyperglycemic conditions, but decreased in late stages of type II diabetes in patients and mouse models [252,253].
Age-related macular degeneration
Age-related macular degeneration (AMD) is the leading cause of blindness in people over the age of 65. AMD is a progressive degeneration of the retina, the retinal pigment epithelium (RPE), Bruch’s membrane, and underlying choroid. Macula damage results in loss of central vision. Mitochondria are a major source reactive oxygen species in the photoreceptors and RPE. Increased mtDNA lesions [254,255] and down-regulation of the DNA repair enzymes 8-oxoguanine-DNA glycosylase 1 (OGG1) [255–257] in the macula and RPE correlate with AMD progression. Mice deficient in SOD1 and SOD2 developed age-dependent degeneration of the retina, with features typical of AMD in humans [207,258]. Nrf2 knockout mice develop age-dependent degeneration of the retina with similarities with human AMD pathology including drusen deposition, RPE degeneration, and deregulated autophagy . These studies strongly implicate oxidative stress as a causal factor in AMD.
Ageing is accompanied by a higher incidence of cardiovascular disease due to age-related changes in heart morphology, impaired proteolysis, and mitochondrial dysfunction. In ageing, there is an increase in maladaptive cardiac hypertrophy which contributes to hypertension and development of heart failure. In rodents, cardiomyocyte number decreases and remaining cardiomyocytes become enlarged with age . Nrf2 protects against maladaptive cardiac responses to mechanical stress by suppressing oxidative stress. Nrf2−/− mice in a mouse model of pressure overload after transverse aortic constriction (TAC) developed cardiac hypertrophy, myocardial fibrosis and apoptosis, resulting in overt heart failure and diminished expression of antioxidant genes including GPx, HO-1, SOD2, and SOD3 . Nrf2 signaling and mitochondrial manganese SOD2 are also depleted in aged vessels, and disruption of Nrf2 signaling impairs angiogenesis in human coronary arterial endothelial cells .
The heart is especially prone to oxidative damage with increasing age. Mitochondrial complex III and IV respiratory activity is impaired in interfibrillar mitochondria isolated from aged (24 months) male Fisher rats. This decline in respiratory activity was associated with a significant increase in oxidative stress levels as measured by 4-hydroxy-2-nonenal-modified proteins, protein carbonyls, and MDA . In addition cardiac mitochondria isolated from elderly rats had increased mtDNA damage as assessed by an increased content of 8-OHdG lesions . Cardiomyocyte mitochondrial respiratory dysfunction and impaired protein turnover is associated with heart failure [265–267]. Accumulation of ubiquitinated proteins has been reported in human heart failure, which suggests impaired UPS function [268,269].
Summary and conclusion
Ageing is characterized by increase in oxidative stress and diminished adaptive homeostatic responses which lead to an accumulation of oxidatively damaged proteins, DNA, and lipids in cells, especially in the last third of lifespan. Proteasomal dysfunction and accumulation of misfolded proteins further impair protein turnover, also weakening resistance to multiple stressors. Oxidative stress and impaired proteostasis are common amongst various diseases that afflict the ageing population. Multiple age-related human diseases show a significant sex bias including neurodegenerative diseases, kidney diseases, and cardiovascular diseases. Age- and sex-related hormonal changes have been associated with this disparity in disease incidence between male and females. Recent studies have elucidated a potential molecular mechanism for sex bias in adaptation to oxidative stress involving the mitochondrial Lon protease . Here, we reviewed the involvement of impaired response to oxidative stress and defective adaptive homeostasis in a subset of age-associated diseases, a summary of which is presented in Figure 5.
A graphic representation of age-related diseases associated with diminished stress responses and defective adaptive homeostasis
Diminished Nrf2 expression and subsequent impaired Nrf2/EpRE signaling are the key features of ageing that contribute to high levels of oxidative stress in various age-related disorders. In particular, Nrf2/EpRE signaling is impaired or insufficient to attenuate severe oxidative stress in many disorders described in this review. As a master regulator of cellular homeostasis, activation of Nrf2 is widely considered as a potential target for the treatment of various neurodegenerative disorders. Nrf2 overexpression has prevented neuronal pathology in rodent models of AD, PD, HD, and ALS. In clinical trials, antioxidant supplementation via NAC increased dopamine transporter binding in the caudate and putamen in the brains of PD patients . Antioxidant clinical trials in AD, have shown limited therapeutic efficacy in ameliorating cognitive deficits . Limited efficacy or conflicting results of antioxidant supplementation for various disorders may be the result of limited ability to cross the BBB.
Potent activators of Nrf2 have been shown to be neuroprotective, however unregulated Nrf2 expression is detrimental as Nrf2 overexpression may be oncogenic and is associated with resistance to chemotherapy . Recent studies suggest that selective Keap1 inhibition, using a selective Keap1-modifying small molecule (MIND4-17) [75,76], activated Nrf2 signaling and was protective against oxidative damage in NSCs differentiated from HD patient-derived iPSCs.
Impaired proteostasis is also a common feature of ageing and various age-related disorders discussed in this review. Oxidative stress is often accompanied by impaired proteasomal activity and accumulation of protein aggregates in neurological disorders. Overexpression of the proteasomal catalytic β5 subunit of the 20S core proteasome in C. elegans enhances proteasomal activity, resulting in lifespan extension and increased resistance to oxidative stress . Notably, in this same study, β5 subunit overexpression protected against the aggregate-related pathology progression and polyQ or Aβ proteotoxicity in C. elegans models of AD and HD . Proteasomal activation using 18α-glycrrhetinic acid, decreased accumulation of Aβ deposits in an AD C. elegans model . These studies suggest that modulation of proteolytic activity in neurodegenerative disorders may be a considerable therapeutic option for the future.
An understanding of the role of defective adaptive homeostasis in ageing and disease is critical for the development of new therapeutic approaches to treat various disorders, especially those in which oxidative stress and impaired proteostasis contribute to the pathology.
The authors declare that there are no competing interests associated with the manuscript.
This work was supported by the National Institute of Environmental Health Sciences of the US National Institutes of Health [grant number ES 003598 (to K.J.A.D.)]; the National Institute on Aging of the US National Institutes of Health [grant number AG 052374 (to K.J.A.D.)]; the National Institute for Neurological Diseases and Stroke Award [grant number NS072234 (to D.A.B.)]; the National Cancer Institute of the National Institutes of Health [grant number P30CA062203 (to D.A.B.)]; the NIH MBRS-IMSD Training Grant [grant number GM055246 (to N.L.)]; and the NINDS/NIH predoctoral fellowship [grant number NS082174 (to N.L.)].
N.L., D.A.B., and K.J.A.D. contributed equally to the conception, initial writing, and revisions of this review paper.
serine/threonine kinase 1
alcoholic liver disease
amyotrophic lateral sclerosis
age-related macular degeneration
antioxidant response element
BTB and CNC homology 1
B-cell lymphoma 2
B-cell lymphoma-extra large
brain-derived neurotrophic factor
Broad-complex, Tramtrack, and Bric-à-brac
chronic prostatitis/chronic pelvic pain syndrome
chemotherapy-related cognitive impairment
cytochrome P450 2E1
chromosome 9 ORF 72
destabilization of the medial meniscus
electrophile response element
electron transport chain
Extracellular mutant 29
glomerular filtration rate
insulin-like growth factor 1
induced pluripotent stem cell
inhibitory κB protein
intracellular adhesion molecule 1
interleukin 1 beta
kidney androgen regulated protein
Kelch-like erythroid cell derived protein with CNC homology associated protein 1
middle cerebral artery occlusion
major depressive disorder
non-alcoholic fatty liver disease
nuclear factor erythroid-2-like factor 2
nuclear factor kappa-light-chain-enhancer of activated B cells
neural stem cell
PTEN-induced kinase 1
protein kinase C gamma
receptor activator of NF-κB ligand
retinal pigment epithelium
tumor necrosis factor α
tropomyosin receptor kinase B