Cellular Stress Response (Hormesis) in Response to Bioactive Nutraceuticals with Relevance to Alzheimer Disease

Abstract

Significance:

Alzheimer's disease (AD) is the most common form of dementia associated with aging. As the large Baby Boomer population ages, risk of developing AD increases significantly, and this portion of the population will increase significantly over the next several decades.

Recent Advances:

Research suggests that a delay in the age of onset by 5 years can dramatically decrease both the incidence and cost of AD. In this review, the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in AD is examined in the context of heme oxygenase-1 (HO-1) and biliverdin reductase-A (BVR-A) and the beneficial potential of selected bioactive nutraceuticals.

Critical Issues:

Nrf2, a transcription factor that binds to enhancer sequences in antioxidant response elements (ARE) of DNA, is significantly decreased in AD brain. Downstream targets of Nrf2 include, among other proteins, HO-1. BVR-A is activated when biliverdin is produced. Both HO-1 and BVR-A also are oxidatively or nitrosatively modified in AD brain and in its earlier stage, amnestic mild cognitive impairment (MCI), contributing to the oxidative stress, altered insulin signaling, and cellular damage observed in the pathogenesis and progression of AD. Bioactive nutraceuticals exhibit anti-inflammatory, antioxidant, and neuroprotective properties and are potential topics of future clinical research. Specifically, ferulic acid ethyl ester, sulforaphane, epigallocatechin-3-gallate, and resveratrol target Nrf2 and have shown potential to delay the progression of AD in animal models and in some studies involving MCI patients.

Future Directions:

Understanding the regulation of Nrf2 and its downstream targets can potentially elucidate therapeutic options for delaying the progression of AD. Antioxid. Redox Signal. 38, 643–669.

Introduction

Alzheimer's disease (AD) is a progressive, age-associated neurodegenerative disease in which brain changes may occur 20 or more years before the onset of symptoms. AD is the most common cause of dementia currently estimated to affect 6.2 million Americans (Alzheimer's Association, 2021) and is the seventh leading cause of death in the United States (Murphy et al, 2021). Age is the greatest risk factor for the development of AD, increasing significantly in the population aged 65 years or older.

Lifespan extending medical advances coupled with the aging Baby Boomer population are expected to significantly increase the prevalence of AD, as the population most at risk of developing the disease is projected to increase from 56.1 million in 2020 to 85.7 million in 2050 (Vespa et al, 2020). Increase in AD prevalence will be accompanied by increased costs estimated to reach $1.1 trillion in current dollars by 2050 (Alzheimer's Association, 2021).

Several studies have indicated that an intervention that would delay the onset of AD by 5 years could decrease total health care payments by 33%–39% (Alzheimer's Association, 2015; Zissimopoulos et al, 2014).

The pathological hallmarks of AD include extra-neuronal deposition of amyloid β-peptide (Aβ), intraneuronal neurofibrillary tangles comprising hyperphosphorylated tau (htau), inflammation, neurite and synapse loss, glucose hypometabolism, and neuronal atrophy (Chandra et al, 2019; Schilling et al, 2019; Schilling et al, 2016). The progressive development of AD, or the AD continuum, consists of preclinical AD (PCAD), mild cognitive impairment (MCI) due to AD, and dementia due to AD further characterized by mild, moderate, and severe stages (Masters et al, 2015; Sun et al, 2018).

PCAD is associated with changes in brain chemistry, including abnormal levels of Aβ and glucose hypometabolism but no cognitive or behavioral symptoms (Dubois et al, 2016; Sperling et al, 2011). MCI due to AD is characterized by evidence of brain changes and changes in memory that do not interfere with everyday tasks (Albert et al, 2011). In dementia due to AD, cognitive or behavioral symptoms interfere with everyday activities to an increasing extent reflecting the degree of cumulative damage as the disease advances from mild to moderate and finally to severe (McKhann et al, 2011). Notably, the length of each phase varies, and all individuals do not go on to convert from PCAD to MCI and from MCI to AD (Bennett et al, 2006; Jack et al, 2016).

Aβ is a 39- to 42-amino acid product of the concerted proteolytic cleavage of amyloid precursor protein (APP) by β-secretase (BACE) on the amino-terminus and γ-secretase on the carboxy-terminus (Selkoe, 2001). Aβ(1–42) is the more neurotoxic species and comprises the majority of Aβ found in senile plaques in AD brain (Butterfield and Boyd-Kimball, 2018; Selkoe, 2001; Walsh and Teplow, 2012). Genetic mutations in presenilin-1 or presenilin-2, which comprise the catalytic element of γ-secretase, or in APP result in familial AD (FAD) and all subsequently increase the production of Aβ(1–42) (Goate and Hardy, 2012; Selkoe, 2001).

Elevated oxidative stress is implicated in the pathogenesis of AD (Swomley and Butterfield, 2015). Aβ(1–42) induces oxidative stress both in vitro and in vivo and is proposed to form small oligomers that are inserted into the lipid bilayer initiating lipid peroxidation (Butterfield, 2020; Butterfield and Boyd-Kimball, 2019; Butterfield and Boyd-Kimball, 2018). Extraneuronal deposits of Aβ measured by positron emission tomography (PET) precede and positively correlate with cognitive decline and cerebral atrophy (Villemagne et al, 2013).

Redox proteomic analyses of brain tissue across the AD continuum confirm oxidative stress is an early event in disease pathogenesis (Butterfield, 2020; Butterfield and Boyd-Kimball, 2019; Butterfield and Boyd-Kimball, 2018). Biomarkers of oxidative stress include protein carbonyls (Butterfield and Stadtman, 1997), 3-nitrotyrosine (3-NT) (Beckman et al, 1994), and protein-bound 4-hydroxy-2-trans-nonenal (HNE) (Esterbauer et al, 1991; Schaur et al, 2015).

HNE, a product of lipid peroxidation, is a highly reactive α, β-unsaturated alkenal that reacts with lysine, histidine, and cysteine residues (Castro et al, 2017). Such irreversible oxidative protein modifications initiate conformation changes, resulting in altered or lost function with consequences toward disease progression (Butterfield and Boyd-Kimball, 2019; Butterfield and Boyd-Kimball, 2018).

Oxidative Stress and Hormesis

In aerobic organisms that are dependent on molecular oxygen, reactive oxygen species (ROS) are produced as part of normal metabolism. Homeostasis is maintained by endogenous antioxidants that are capable of scavenging free radicals before irreparable damage occurs to lipids, proteins, and nucleic acids; however, oxidative stress occurs when pro-oxidants, such as ROS or reactive nitrogen species (RNS), exceed antioxidants at the cellular level (Halliwell, 2007). Hormesis is a phenomenon observed at the cellular and organismal level of processes that exhibit a biphasic dose response.

Generally, low concentrations are associated with favorable biological response whereas high concentrations are associated with damaging or toxic effects (Mehdi et al, 2021). Redox and stress signaling plays a central role in hormetic responses through induction of antioxidant response elements (ARE) in the DNA by nuclear factor erythroid 2-related factor 2 (Nrf2), resulting in the compensatory upregulation of transcription of genes coding for endogenous antioxidants, antioxidant enzymes, and heat shock proteins (Berry and López-Martínez, 2020; Mehdi et al, 2021; Rattan, 2019).

Hormesis may be a promising method for modulating or delaying aging and age-related diseases such as AD (Mehdi et al, 2021). With that in mind, this review is broadly divided into two sections: one section on the transcriptional effects of Nrf2 in AD and MCI including the induction of heme oxygenase-1 (HO-1) and biliverdin reductase-A (BVR-A) and another section on the effect of selected bioactive nutraceuticals on the induction of Nrf2 relevant to AD and MCI.

Transcriptional effects of Nrf2 in AD and MCI

Transcriptional regulation of the ARE by Nrf2

Transcriptional regulation by Nrf2 has been extensively reviewed (He et al, 2020; Tonelli et al, 2018). Briefly, Nrf2 is a ubiquitous cap “n” collar (CNC) basic-region leucine zipper (bZIP) transcription factor that modulates oxidative stress by binding ARE enhancer sequences in regulatory regions of target genes (Fão et al, 2019; Sihvola and Levonen, 2017). Nrf2 protein in humans contains 605 amino acids and 7 conserved Nrf2-ECH homology (Neh) domains with Neh2, Neh4, and Neh5 located in the N-terminus and Neh1, Neh3, and Neh6 located in the C-terminus (Itoh et al, 1999).

Neh7 lies between Neh5 and Neh6 (Tonelli et al, 2018). Two highly conserved motifs named by their one letter amino acid code, DLG (high affinity) and ETGE (low affinity), are contained in the Neh2 domain and are critical for interaction with Kelch-like erythroid cell-derived protein with CNC homology associated protein 1 (Keap1), a dimeric, redox-sensitive adaptor protein of cullin 3 (Cul3), the latter an E3 ubiquitin ligase that ubiquitinylates and targets cytosolic Nrf2 for proteasomal degradation (Canning et al, 2015).

Under unstressed conditions, low levels of intracellular Nrf2 are maintained by proteasomal degradation with a half-life of 20 min (Sihvola and Levonen, 2017). Oxidative stress induces structural changes and diminished activity in Keap1, allowing for nuclear accumulation of Nrf2 and transcription of over 250 target genes on Nrf2 dimerization with small musculoaponeurotic fibrosarcoma oncogene homolog protein (Maf) and binding to the ARE (Fig. 1) (Canning et al, 2015).

FIG. 1.

Structure and regulation of Nrf2. Nrf2 contains seven conserved Neh domains (A). The Neh2 domain contains two conserved motifs, DLG and ETGE that are critical for interaction with Keap1. Keap1 is a redox-sensitive adaptor protein of Cul3 an E3 ubiquitin ligase that targets cytosolic Nrf2 for proteasomal degradation. Under stress conditions, modification of and structural changes in Keap1 allow for the nuclear translocation of Nrf2, which dimerizes with small Maf and binds to ARE enhancer sequences in the DNA promoting upregulation of 250 target genes, including HO-1, SOD1, CAT, NQO1, GPx, GR, GST, TR, GS, GCLC, and GCLM. GS, GCLC, and GCLM are all involved in the synthesis of glutathione (B). ARE, antioxidant response element; CAT, catalase; CNC, cap “n” collar; Cul3, cullin 3; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase modifier subunit; GPx, glutathione peroxidase; GR, glutathione reductase; GS, glutathione synthetase; GST, glutathione-S-transferase; HO-1, heme oxygenase-1; Keap1, Kelch-like erythroid cell-derived protein with CNC homology associated protein 1; Maf, musculoaponeurotic fibrosarcoma oncogene homolog protein; Neh, Nrf2-ECH homology; NQO1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear factor erythroid 2-related factor 2; SOD1, superoxide dismutase-1; TR, thioredoxin reductase.

Upregulated antioxidant enzymes include superoxide dismutase-1 (SOD1), catalase (CAT), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), thioredoxin reductase (TR), HO-1, and enzymes involved in the synthesis of glutathione (GSH) including glutathione synthetase (GS), glutamate-cysteine ligase catalytic subunit (GCLC), and glutamate-cysteine ligase modifier subunit (GCLM) (Fão et al, 2019; He et al, 2020).

Nrf2 also regulates metabolism by increasing expression of genes involved in glycolysis, pentose phosphate pathway, amino acid metabolism, fatty acid synthesis, heme metabolism, glycogen metabolism, nucleotide biosynthesis, one carbon metabolism, glutaminolysis, and decreasing expression of genes involved in gluconeogenesis (He et al, 2020). The cytoprotective effects of Nrf2 also include modulating expression of proinflammatory cytokines and maintaining mitochondrial integrity and regulating mitochondrial biogenesis (Brandes and Gray, 2020). Nuclear accumulation and degradation of Nrf2 is also regulated by phosphorylation mediated by multiple kinases, including glycogen synthase kinase 3β (GSK-3β) (Rojo et al, 2012).

Nrf2 is expressed throughout the body, but differentially with especially high levels found in the lungs and intestines (Osama et al, 2020). Age-related decrease in Nrf2 protein expression and activity has been reported in primary astrocyte culture of murine spinal cord (Duan et al, 2009). Nrf2 is expressed in glial cells and to a lesser extent in neurons (Lee et al, 2003; Shih et al, 2003).

The protein level of Nrf2 is significantly decreased in the inferior parietal lobule (IPL) of AD brain (Bahn et al, 2019), and nuclear translocation of Nrf2 is significantly decreased in AD hippocampus compared with the age-matched control (Ramsey et al, 2007). Transgenic mouse models of AD deficient in Nrf2 show an increase in amyloidogenic processing of APP, phosphorylated tau, inflammation, oxidative stress, glial activation, autophagic disfunction, and exacerbation of cognitive deficits (Branca et al, 2017; Joshi et al, 2015; Rojo et al, 2018; Rojo et al, 2017). Collectively, these findings suggest that alterations in Nrf2 and its downstream responses may be involved in the pathogenesis of AD and identify Nrf2 as a potential therapeutic target.

HO: an introduction

HO catalyze the rate-determining step in concert with NADPH-cytochrome P450 reductase in the degradation of endogenous heme (iron protoporphyrin IX) to biliverdin IXα, ferrous iron (Fe²⁺), and carbon monoxide (CO) in equimolar amounts (Fig. 2) (Dunn et al, 2014). There are three known isoforms of HO: HO-1, also known as heat shock protein 32 (HSP32), a redox-sensitive inducible isoform, and HO-2 and HO-3, both of which are constitutive isoforms.

FIG. 2.

Reaction and protective effects of HO-1. HO-1 acts in concert with NADPH:cytochrome p450 reductase to convert iron protoporphyrin IX into biliverdin IXα, CO, and Fe²⁺. Endogenous CO alters glucose utilization to increase PPP production of NADPH, which provides reduction power for antioxidants and increases mitophagy and mitochondrial biogenesis. Iron is sequestered in ferritin to reduce the occurrence of Fenton chemistry and minimize the production of subsequent reactive oxygen species. Biliverdin IXα is converted into bilirubin by BVR-A. BVR-A, biliverdin reductase-A; CO, carbon monoxide; Fe²⁺, ferrous iron; PPP, pentose phosphate pathway.

HO-2 plays a major role in iron recycling and homeostasis, whereas HO-1 plays a major role in the production of the antioxidant biliverdin (more on this below). HO-1 is a type 1 integral membrane protein in the smooth endoplasmic reticulum situated such that most of the protein, including the active site, is in the cytoplasm. HO-1 protein expression, positively regulated by Nrf2, can be induced by multiple stressors and stimuli, including ultraviolet radiation, nitric oxide, cytokines, heavy metals, infections, and oxidized low-density lipoprotein as a cytoprotective and adaptive response (Consoli et al, 2021).

Under normal physiological conditions and low heme concentrations, HO-1 expression is repressed by BTB and CNC homology 1 (BACH1) in complex with Mafs that compete with Nrf2/Maf heterodimers for binding to ARE enhancer regions; however, an increase in heme levels blocks BACH1 from binding to the DNA and enhances nuclear export and degradation of BACH1 (Suzuki et al, 2004). However, under stress conditions, HO-1 truncated at the C-terminus translocates to the nucleus and activates oxidant response-dependent transcription independent of enzymatic activity in vitro (Lin et al, 2007).

This suggests that HO-1 may also play a cytoprotective role via cellular signaling separate from its enzymatic activity, but further studies are needed to confirm this notion.

In addition to biliverdin, the direct products of HO-1 including Fe²⁺ and CO exert hormetic effects at the cellular level. The most abundant transition metal in the brain, iron is required for normal physiological functions (Peng et al, 2021); however, excess Fe²⁺ leads to the potential for Fenton chemistry inducing lipid peroxidation and cell death (Winterbourn, 1995). Consequently, for normal physiological function, a balance must be maintained between iron availability and storage.

In the brain, iron is primarily stored in ferritin with Fe³⁺ comprising the core of the structure surrounded by a protein shell consisting of ferritin heavy chain (FTH) and ferritin light chain (FTL). Sequestration of iron in ferritin protects against free excess iron that may induce oxidative stress (Zhang et al, 2021c); however, brain iron and ferritin increase with age (Connor et al, 1990) and both are elevated in the AD brain (Connor et al, 1992; Raven et al, 2013; van Rooden et al, 2015), accumulating with aggregated Aβ in senile plaques (Everett et al, 2018) and correlating with accelerated cognitive decline (Ayton et al, 2020).

The structure of ferritin is altered in AD brain. The iron core of ferritin contains more of the redox-active Fe²⁺ rather than Fe³⁺ (Quintana et al, 2004), and the protein level of FTH is increased (Sultana et al, 2007). Both FTH and FTL are under the transcriptional activation of Nrf2, and BACH1 acts as a repressor (Kerins and Ooi, 2018). Aβ has been shown to cause reduction of Fe³⁺ in vitro and has been proposed to play a role in the conversion of Fe³⁺ to Fe²⁺ in the AD brain (Everett et al, 2018).

Mitochondrial ferritin (MtFt) protein levels also are increased in AD temporal cortex (Wang et al, 2011). MtFt exhibits high homology with FTH and is proposed to play a role in decreasing cytosolic iron (Gao and Chang, 2014). The increase in iron sequestration proteins in AD brain potentially indicates a cytoprotective upregulation in response to iron dysregulation.

HO-1 is a main source of endogenous CO. HO-1 production of CO reportedly protects against Aβ toxicity in vitro (Hettiarachchi et al, 2014). Low concentrations of CO inhibit cytochrome c oxidase (complex IV) in the mitochondrial electron transport chain, decreasing mitochondrial respiration, and resulting in the production of ROS (Zuckerbraun et al, 2007). This phenomenon is accompanied by a shift in glucose utilization to the pentose phosphate pathway to regenerate NADPH required for antioxidant defenses (Stucki et al, 2020).

CO may also regulate the activity of cytochrome P450-dependent monooxygenases (CYPs). The CYPs function in detoxification and can convert xenobiotics into electrophiles that may activate Nrf2 transcription of HO-1 producing endogenous CO that can bind to the heme moiety of CYPs inhibiting activity and the formation of additional electrophiles. Low concentrations of CO also promote cell survival via mitophagy and stimulation of mitochondrial biogenesis. Conversely, high concentrations of endogenous CO induce apoptosis (Stucki and Stahl, 2020).

Increased expression and activity of HO-1 is initially a protective stress response. Voluntary running has been shown to increase HO-1 activity in the hippocampus and frontal cortex in aged Wistar rats accompanied by a decrease in cerebral amyloid angiopathy and 11C-Pittsburgh Compound-B (PIB) retention (Kurucz et al, 2018). Induction of HO-1 by the statin atorvastatin decreased oxidative stress and increased GSH levels in the parietal cortex of aged beagles (Barone et al, 2011a; Butterfield et al, 2012). However, overexpression of HO-1 induces oligomerization of Aβ and Tau and contributes to synaptic damage in vivo (Li et al, 2015; Si et al, 2018).

HO in MCI and AD and models thereof

The protein expression level of HO-1 is significantly increased in temporal cortex, IPL, and hippocampus in patients with AD compared with age-matched controls (Barone et al, 2014; Barone et al, 2012a; Calabrese et al, 2006; Schipper et al, 1995). No significant change in HO-1 protein expression in the hippocampus or cerebellum from patients with MCI, nor in the cerebellum from patients with AD has been observed (Barone et al, 2012a; Di Domenico et al, 2010). HO-1 colocalizes with senile plaques and neurofibrillary tangles in AD temporal cortex and hippocampus (Schipper et al, 1995; Smith et al, 1994). Microglial HO-1 protein expression is significantly increased in AD cortex (Fernández-Mendívil et al, 2020).

In addition, HO-1 positive astrocytes are significantly increased in the MCI and AD temporal cortex and hippocampus and correlate with the extent of cognitive decline (Schipper et al, 2006). HO-2 is significantly decreased in the hippocampus of patients with MCI and in both the hippocampus and IPL of patients with AD (Barone et al, 2012a; Calabrese et al, 2006). HO-2 protein expression is not altered in MCI or AD cerebellum (Barone et al, 2012a).

In addition to protein expression level changes and post-translational modification, HO-1 is also susceptible to oxidatively modification in the MCI and AD brain. HO-1 is significantly modified by protein-bound HNE in the hippocampus of MCI and AD in addition to the AD cerebellum. Significant protein carbonylation of HO-1 is also present in the AD hippocampus and MCI cerebellum (Barone et al, 2012a). Activity of HO-1 is increased by Akt-mediated phosphorylation of Ser188 (Salinas et al, 2004). Increased serine phosphorylation of HO-1 has been observed in the MCI and AD cerebellum and in AD, but not the MCI hippocampus (Barone et al, 2012a).

Alterations in HO-1 and the effects of overexpression of HO-1 are also observed in models of AD. HO-1 protein levels are significantly increased in microglia of 5xFAD transgenic mice aged 12 and 18 months compared with wild-type mice in the hippocampus, cortex, thalamus, and amygdala. In addition, these microglia were found to be localized surrounding Aβ plaques (Fernández-Mendívil et al, 2020).

Glial HO-1 hyperactivity, likely stimulated in response to Aβ-induced oxidative stress, has been proposed to play a transducer role in the development of AD; thus, modulation of glial HO-1 activity has been explored as a potential therapeutic target (Schipper et al, 2019). Competitive inhibitors of HO-1 significantly decreased protein carbonyls in cultured rat astrocytes overexpressing HO-1 (Gupta et al, 2014; Song et al, 2006) and significantly reduced astroglial activation in APP_swe/PS1_ΔE9 transgenic mice (Gupta et al, 2014).

HO-1 conceivably is increased in the AD brain as a protective mechanism to combat oxidative stress; however, HO-1 becomes a target of oxidative modification that can alter its structure and function. Phosphorylation of Ser188, which increases HO-1 activity, is also a protective mechanism; however, prolonged overexpression or hyperactivity may lead to tau phosphorylation and the deleterious effects of Fe²⁺ and CO (Barone et al, 2012a). Collectively, these findings reflect a compensatory mechanism that has become deleterious, and a fine balance is required to maintain the neuroprotective benefits of HO-1.

BVR-A: an introduction

BVR has two isoforms, BVR-A and BVR-B. A multifunctional enzyme, BVR-A's primary function is to convert biliverdin IXα to the antioxidant, bilirubin IXα (Fig. 3). Conversely, high levels of bilirubin are neurotoxic to premature infants and newborns (Mireles et al, 1999). Bilirubin has demonstrated reduction of ROS and inflammation and activation of the nuclear transcription factor, peroxisome proliferator-activated receptor α, PPARα (Thomas et al, 2022).

FIG. 3.

Reaction and functionality of BVR. BVR-A reduces biliverdin to bilirubin, which is facilitated by the cofactor NADH or NADPH. The product, bilirubin, is found to reduce oxidative stress and inflammation. The enzyme itself has been found to impact cellular signaling in MAP kinase, ERK 1/2 Ser/Thr/Tyr kinases, and insulin. In addition, BVR-A impairment promotes amyloid beta-peptide production and insulin resistance.

Nanomolar levels of bilirubin were found to be neuroprotective against oxidative insult (Dore and Snyder, 1999) and scavenge ROS more effectively than alpha tocopherol (Stocker et al, 1987). Mancuso (2017) showed that bilirubin also has denitrification capacity, as it scavenges RNS. PPARα has been shown to promote neuronal survival by attenuating Aβ accumulation (Fidaleo et al, 2014) and modulating neuroinflammation and neurotransmission (Cheng et al, 2015).

BVR-B uses other isoforms of biliverdin, including biliverdin beta, biliverdin gamma, and biliverdin delta (Kapitulnik and Maines, 2009; Pereira et al, 2001). BVR-B does not reduce biliverdin IXα but plays a pivotal role in fetal development, whereas BVR-A predominates in adulthood. BVR-B generates the byproduct, bilirubin Ixβ, which is conjugated with glucuronic acid before it is excreted as bile (Pereira et al, 2001) and does not undergo internal hydrogen bonding (Cunningham et al, 2000).

BVR-A acts as a serine/threonine/tyrosine kinase involved in cell signaling and heme metabolism. The Ser/Tyr kinase activity is stimulated by oxidative insult, which results in translocation of the protein into the nucleus in response to oxidative stress and cGMP (Lerner-Marmarosh et al, 2008; Maines et al, 2001). Heme metabolism is facilitated by the HO-1/BVR-A system, which enhances the cellular stress response system.

On autophosphorylation, BVR-A interacts with the MAP kinase protein family, including ERK1, ERK2 and regulates the expression of HO-1 and iNOS as it inhibits toll-like receptor 4 expression (Salim et al, 2001; Wegiel et al, 2011). Phosphorylated BVR-A binds to phosphoinositol-3-kinase (PI3-K) and activates Akt signaling downstream of insulin receptor kinases. It is activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T³⁰⁸ before S⁴⁷³ autophosphorylation (Lerner-Marmarosh et al, 2005). BVR-A acts as a scaffolding protein regulating GSK-3β phosphorylation and subsequent inhibition by Akt.

This, in turn, marks the enzyme's role as mediator of the Akt1/GSK-3β pathway and its function in activating Akt1 by PDK1 (Miralem et al, 2016). This severely impacts insulin signaling, which will be further discussed later. BVR-A and HO-1 levels are also raised during exercise (Thomas et al, 2022). The increases in BVR-A/HO-1 during exercise contribute to increased bilirubin levels, which downregulates proinflammatory cytokines and M1 macrophages (Takei et al, 2019).

Animal models and subjects in human experiments presented a significant increase in bilirubin when participating in high intensity training (Neubauer et al, 2010). This study also showed that elevated bilirubin levels are protective against DNA damage, ROS-induced inflammation, and production of ROS.

BVR-A contains two conserved regions that contribute to its functionality, including several key binding motifs such as a leucine zipper, adenine dinucleotide, Src binding domain (Lerner-Marmarosh et al, 2005), Akt (Miralem et al, 2016), PDK1 (Miralem et al, 2016), and Zn/metal binding motif (Maines et al, 1996). Structurally, BVR has a five leucine-repeat motif separated by a hexapeptide sequence, Leu¹²⁹(xxxxxx)-Leu¹³⁶(xxxxxx)-Lys¹⁴³(xxxxxx)-Leu¹⁵⁰(xxxxxx)-Leu¹⁵.

The leucine zipper motif contributes to the dimerization of the protein and its DNA binding sites in its basic region. As a homodimer, BVR binds to DNA via the leucine repeat motif. The dimer form of BVR forms a helix-turn-helix motif. The phosphoprotein, BVR, is a leucine zipper-like DNA binding protein, which contributes to the activator protein (AP-1) signaling cascade and the generation of cAMP regulated genes in the HO-1 stress response system (Florczyk et al, 2008).

As a zinc metalloprotein, bilirubin reductase is NADPH-dependent; BVR-A is co-factor dependent based on pH, as NADH is used under acidic conditions (Maines and Trakshel, 1993), whereas NADPH is used under basic conditions (Yamaguchi et al, 1994). Exogenous zinc inhibits NADPH-dependent activity of the enzyme (Maines et al, 1996). Both NADH and NADPH binding regions are present in BVR due to the predominance of Cys and His residues in the enzyme.

NADH adapts a folded conformation due to a Lys209 residue, whereas NADPH induces a conformational change at Lys219 in the crystalline structure that provides stability for the catalysis mechanism via the γ -methene bridge (Kikuchi et al, 2001; Whitby et al, 2002). This structure contributes to the tight binding of Zn to two regions of the protein in its native conformation. One site has a high avidity for zinc, whereas the other is thought to be in a hydrophobic loop between Cys281 and Cys292 (Maines et al, 1996). This hypothesized metal coordination is plausible as it would provide a tetrahedral coordination between histidine's imidazole ring and Zn, resulting in tertiary folding facilitating the interaction between oppositely charged residues (Maines et al, 1999).

BVR-A in animal models and human subjects of AD

Higher mammalian models are necessary to continue the propagation of scientific study for neurodegenerative disorders. For AD, feline, canine, porcine, and bovine models have been used to study this neurological disorder using genetic modifications that mirror those used in rodent models. A feline model that demonstrated β-amyloid pathology, phosphorylated tau, and tau aggregation in the hippocampus and entorhinal cortex in aging cats was consistent with AD progression in humans; however, neuritic plaques were not evident (Fiock et al, 2020). As β-amyloid antibody has not been validated in cats, levels of Aβ(1–40) and Aβ(1–42) cannot yet be determined.

Aβ deposition was first discovered in aged canines in 1993 by Cummings et al (1993). As observed in humans, this correlated with cognitive decline seen in humans with Aβ oligomers (Cummings et al, 1996; Head et al, 2002; Head et al, 2000; Satou et al, 1997). This discovery led to additional studies demonstrating a correlation between htau (Abey et al, 2021), cognitive decline (Ozawa et al, 2016), and neuroinflammation (Schutt et al, 2016) in aged dogs.

BVR-A dysfunction exacerbates Aβ production by Ser phosphorylation of BACE1 in aged beagles, a higher mammalian model for AD, which deposits canine APP that has a 98% homology to human APP (Barone et al, 2012b; Triani et al, 2018). Currently, transgenic canine models expressing human APP containing the Indiana (V717F) and Swedish (670N and M671L) mutations resulting in Aβ accumulation, enlarged ventricles, and an atrophied hippocampus are being used to study AD (Lee et al, 2014).

The APP6₉₅sw transgene to induce human APP expression and atypical cleavage of BACE cleavage resulting in the production of Aβ(1–40) and Aβ(1–42) has been observed in 3 month hemizygous minipigs (Kragh et al, 2009). A double transgenic model in minipigs having APP6₉₅sw and PSEN1M146I mutations demonstrates an increase in Aβ(1–42) (Jakobsen et al, 2016). A triple transgenic pig model for AD in which elevated tau, APP, and presenilin 1 (PS1) mimic the progression of AD in humans has been established using a multi-cistronic vector system (Lee et al, 2017).

BVR-A is found primarily in porcine gastric fundus, which contributes to the downstream production of bilirubin that provides gastrointestinal defense in pigs and antioxidant protection of oxidative stress by superoxide anions (Colpaert et al, 2002).

Our laboratory demonstrated that BVR-A is upregulated in plasma, cerebrospinal fluid, and hippocampus of subjects with MCI and AD, but its activity is significantly reduced (Barone et al, 2011b). In addition, we showed that BVR-A is oxidatively modified and nitrated in the hippocampus of subjects with amnestic MCI and AD (Barone et al, 2011c).

No changes were detected in the cerebellum, which could be attributed to the fact that bilirubin and BVR-A are regionally localized. There is no evidence of elevated oxidative stress nor significant AD pathology in the cerebellum (Ewing and Maines, 1995; Hensley et al, 1995). These modifications of the enzyme can contribute to cellular stress response in the pathogenesis and progression of AD.

Although Aβ homologous with human Aβ (Johnstone et al, 1991) has been observed in cattle, plaque deposits have not been found (Costassa et al, 2016). Aged cattle of multiple breeds showed Aβ deposits in the hippocampus and cortex. Older cows demonstrated an increased Aβ load compared with younger cows. Moreno-Gonzalez et al injected amyloid containing cow tissue into APP/PS1 transgenic mice to study amyloidosis.

The results showed a significant Aβ load and larger plaques compared to control (Moreno-Gonzalez et al, 2021). Polar bears have exhibited aggregated Abeta deposits in the dentate gyrus similar to that found in early-stage AD (Tekirian et al, 1996). These deposits were similar to those observed in AD brain, thereby providing a higher mammal model to study this neurodegenerative disorder.

Bovine BVR-B and human flavin reductase share a nearly identical amino acid sequence (Shalloe et al, 1996). Bovine BVR-A shows limited cross-reactivity with BVR-B in bovines. BVR-A activity is reduced in comparison to bilirubin reductase activity in rabbits and sheep models (George et al, 1989; Rigney and Mantle, 1988).

BVR-A: a link to insulin signaling

Under normal conditions, the hormone insulin binds to the insulin receptor, a tyrosine receptor kinase, to maintain blood glucose levels. Glucose metabolism has been shown to be disrupted in MCI (Arnaiz et al, 2001) and AD using 18Fluoro-2-deoxyglucose positron-emitting tomography and 18Fluoro-2-deoxyglucose/functional magnetic resonance imaging scans (De Santi et al, 2001; Zhang et al, 2021b). These scans also demonstrate global changes in the entorhinal neocortex and multiple subregions of the precuneus that may contribute to cognitive decline associated with this neurodegenerative disorder (Zhang et al, 2021b).

Insulin signaling is impaired in those with diabetes and AD (Barone et al, 2021). Type II diabetes mellitus (T2DM) has significant pathophysiological connections to AD such as neuroinflammation (Stozicka et al, 2007), mitochondrial dysfunction (Moreira et al, 2007), oxidative stress (Butterfield and Halliwell, 2019; Stohs, 1995), metabolic syndrome (Grunblatt et al, 2011), insulin resistance (de la Monte, 2012), and advanced glycation end products (Smith et al, 1996).

Insulin resistance has been shown to trigger early onset AD in people with Down syndrome (DS), a condition caused by triplication of chromosome 21. Brains in persons with DS with AD share multiple neuropathological features such as reduction of energy related enzymes, including hexokinase, NADH-Q oxidoreductase (Complex I), succinate-Q reductase (Complex II), cytochrome c oxidase (Complex III), and ATP synthase (Tramutola et al, 2020). These protein alterations contribute to the reduction energy metabolism hypothesis associated with neurodegeneration.

Pancreatic beta cells secrete insulin as well as amylin, a polypeptide that is a major component of amyloid deposits found in people with T2DM (Luskey, 1992). Amylin and Aβ, the latter as noted earlier as a pathological hallmark of AD, share multiple similarities, including a β-sheet secondary structure, binding to the same receptor, and are degraded by insulin degrading enzyme. These similarities bolster the significance of insulin signaling in both MCI and AD.

Specifically, amylin shows a positive correlation with Aβ(1–42) and Aβ(1–40) in plasma from patients in the population-based nutrition, aging, and memory in the elderly study in which a large scale investigation was conducted to examine relationships among plasma amylin, Aβ, and ApoE allele status (Qiu et al, 2014).

Brain-resident htau is a second pathological hallmark of AD. Amylin analogs such as pramlintide (Zhu et al, 2017) show a reduction in tau phosphorylation in the 5xFAD and 3xTgAD transgenic mouse models of AD, thereby demonstrating a potential therapeutic agent for AD. Htau has been associated with diabetes; however, synaptic and cognitive deficits were observed in Type I diabetes (T1D) mice more often compared with T2DM mice (Trujillo-Estrada et al, 2019). This could be attributed to the reduction of the overall number of dendritic spines and hippocampal mushroom dendritic spines, thereby facilitating the disruption of memory formation and neuronal communication in T1DM mice.

Insulin regulates tau phosphorylation, which is crucial to the mechanism of insulin signaling as this leads to GSK-3β inactivation. Under diabetic conditions, GSK-3β is activated, thereby resulting in an increase in phosphorylated tau (Clodfelder-Miller et al, 2006). GSK-3β is significantly activated in the T1DM model, and an increase in tau phosphorylation was observed (Abbondante et al, 2014; Dey et al, 2017). In addition, GSK-3β is inactivated as a function of age in the parietal cortex of beagles (Triani et al, 2018).

This insulin resistance is similar to that observed in AD. BVR-A serves as a substrate for insulin tyrosine kinase activity and a kinase for Ser phosphorylation of insulin receptor substrate (IRS)-1. Ser phosphorylation of IRS-1 inhibits the insulin signaling pathway, whereas tyrosine phosphorylation of IRS-1 stimulates this pathway. Insulin increases BVR-A tyrosine phosphorylation (Fig. 4) (Cimini et al, 2019). If bilirubin reductase activity is knocked down by small interfering RNA (siRNA), glucose uptake is increased in response to insulin, thereby evincing a functional role for BVR in insulin signaling (Lerner-Marmarosh et al, 2005).

FIG. 4.

Impact of BVR on the insulin signaling pathway. Insulin binds to the insulin receptor, a tyrosine receptor kinase. On phosphorylation, IRSs, particularly (IRS-1), which bind to the receptor and recruit the tyrosine phosphatases, SHP1 and SHP-2, both of which impact ligand associated insulin receptor signaling. A phosphorylated serine residue at position 307 in IRS-1 leads to insulin resistance, thereby influencing insulin activity and diabetes status. On activation of insulin cell signaling, BVR-A is increased. BVR-A modulates IRS-1 functions. If BVR-A is phosphorylated at Tyr¹⁹⁸, glucose uptake is elevated, thereby impacting insulin activation. IRS, insulin receptor substrate.

Selected bioactive nutraceuticals that modulate Nrf2

Polyphenols: an introduction

By definition, polyphenolic compounds (polyphenols) are plant-based compounds containing multiple phenolic rings that have dietary benefits. Flavonoids, a class of polyphenols, are primarily found in colorful fruits and vegetables (Table 1). There are six major subclasses of flavonoid biomolecules based on chemical structure, including isoflavones, anthocyanins, chalcones, flavones, flavan-3-ols (catechins), and flavonols that will be discussed in greater detail (Fig. 5).

Table 1.

Classification of Flavonoids

Flavonoid sub class	Example	Dietary food source
Isoflavones	Daidzein and genistein	Soybeans and peanuts
Anthocyanins	Cyanidin and malvidin	Berries, currants, grapes
Chalcones	Quercetin and naringenin	Citrus fruits
Flavones	Apigenin and luteolin	Artichokes, celery, parsley
Catechins (flavan-3-ols)	Epicatechin and epigallocatechin	Berries, chocolate, teas
Flavonols	Fisetin and kaempferol	Kiwi, onions, spinach

FIG. 5.

Basic structure of a flavonoid and their subclasses. The basic structure of a flavonoid (center image) can form multiple subclasses of flavonoid biomolecules, which have been shown to play a key role in signal transduction, antioxidant status, aging, angiogenesis, and cell cycle regulation.

Bioflavonoids play a pivotal and ever-expanding role in angiogenesis (Liu et al, 2009), aging (Clayton et al, 2021), cell cycle regulation (Wu et al, 2002), cell signaling (Chen et al, 2019a; Singh and Agarwal, 2006), and antioxidants (Counet and Collin, 2003). These compounds are inhibitors to key anti-inflammatory enzymes, including cyclooxygenase, lipoxygenase, phosphoinositide-3-kinase, and xanthine oxidase. Therefore, a diet rich in fruits and vegetables reportedly contributes to a significant association between cancer prevention and overall health (Singh and Agawarl, 2006; Singh et al, 2014).

Anthocyanins are key antioxidants found in berries and leafy vegetables. These compounds are protective against free radical damage (Kalin et al, 2002) and inflammation (Rice-Evans et al, 1995). Structurally, anthocyanins differ from all flavonoids in the B ring component. Structurally, isoflavones contain a bond between the third carbon (C3) of B ring and the C ring of the basic flavonoid structure. Isoflavones have been extensively studied for their properties in relation to estrogen, as they bind to estrogen receptors and exhibit anti-estrogenic or reduced estrogen activity.

These findings present an important link between nutrition and estrogen metabolism, bolstering their role as a potential chemotherapeutic agent (Wu, 2000). Primarily, isoflavones are found in legumes and nuts. Chalcones are intermediates in the flavonoid biosynthetic pathway. Similar to other flavonoids, they are cytoprotective (Sikander et al, 2011) and possess anti-tumor activity (Lai et al, 2013). Structurally, these are the most unique of the six major classes of flavonoids as there are only two cyclic rings.

Chalcones are found in foods such as citrus fruits, apples, tomatoes, and licorice. Flavones contain a ketone group at fourth carbon (C4) of the B ring, and a bond between second carbon (C2) of the B ring and the C ring. Flavones possess anti -inflammatory (Borghi et al, 2013) and anti-mutagenic (Huang et al, 1983) effects, making them powerful agents in their role as anti-tumor agents and pain modulators (Zhang et al, 2021a; Zhu et al, 2016). These biomolecules are found in herbs such as oregano and parsley.

Compared with the other flavonones, flavan-3-ols or catechins have the least reactive B ring structure. This could correlate to their unique anti-obesity properties (Klaus et al, 2005; Nomura et al, 2008) along with those found in other flavanones, which could support the beneficial health effects of incorporating catechins into diet (Yoshitomi et al, 2021). Catechins span a large subclassification that includes components found in green tea, including epicatechin, gallocatechin, epigallocatechin, and their 3-O-gallate derivatives.

Catechins are abundant in tea, berries (strawberries, blackberries, blueberries, raspberries), apples, grapes, chocolate, and red wine. Higher concentrations are found in outer skins of fruits food peels (Lattanzio et al, 2001). The last subset of flavonoids, flavonols are hydroxylated at the third carbon (C3) found in the B ring. These biomolecules have anti-inflammatory (Goh et al, 2012), neuroprotective (Roth et al, 1999), and antioxidant proprieties.

Flavonols are the most abundant flavonoids in plant foods and are mainly present in leafy vegetables, apples, onions, broccoli, and berries. Overall, the role of flavonoids are promising targets and beneficial nutraceuticals in future research direction involving the role of Nrf2 in neurodegeneration (Nassar et al, 2015). Although the list of bioactive nutraceuticals is expansive, this review will focus on ferulic acid ethyl ester (FAEE), sulforaphane, epigallocatechin-3 gallate (EGCG), and resveratrol in their relationships to Nrf2.

These specific compounds were selected based on their (1) relevance to health benefits, (2) growing evidence of positively impacting inhibition of neurodegeneration as demonstrated by Nrf-2 and HO-1 signaling, and (3) potential hormetic role in delaying the progression of AD in animal models and in human subjects. The structures of these biomolecules are shown in Figure 6.

FIG. 6.

Structures of selected bioactive nutraceuticals. The structures of FAEE, sulforaphane, EGCG, and resveratrol are shown. EGCG, epigallocatechin-3 gallate; FAEE, ferulic acid ethyl ester.

Effect of ferulic acid, FAEE, and caffeic acid phenyl ester on Nrf2 in MCI/AD

Ferulic acid (FA), FAEE, and caffeic acid phenyl ester are all phenolic compounds that may reduce the risk of disease by combating oxidative stress (Fig. 7) (Kulkarni et al, 2021; Sultana, 2012). FA (4-hydroxy-3-methoxycinnamic acid) is a naturally occurring antioxidant and anti-inflammatory synthesized from caffeic acid that is found in several fruits, vegetables, and grains.

FIG. 7.

Structures of FA, FAEE, CAPE, and CAPE analogues. The structure of FA and its ethyl ester derivative FAEE are shown, in addition to the structure of CAPE and its synthetic derivatives FA-97 and VP961. CAPE, caffeic acid phenethyl ester; FA, ferulic acid.

The antioxidant properties of FA are due to the phenolic nucleus that allows for the formation of a highly resonance stabilized phenoxy radical that can delocalize the unpaired electron throughout the molecule including the unsaturated side chain (Fig. 8) (Srinivasan et al, 2007). FA not only protects against ROS and lipid peroxidation in vitro (Trombino et al, 2004), but also decreases neuroinflammation (Yan et al, 2013), apoptosis (Jin et al, 2006), and BACE activity (Mori et al, 2013) in vivo. FA inhibits Aβ fibril formation and destabilizes preformed Aβ fibrils in vitro (Ono et al, 2005).

FIG. 8.

Resonance stabilization of FA. FA acts as an antioxidant by forming a highly resonance stabilized phenoxy radical that can delocalize the unpaired electron throughout the molecule, including the unsaturated side chain.

Structure-activity relationship studies indicate that phenolic groups may interfere with aromatic amino acid residue stacking driving self-assembly of Aβ β-sheets and stabilization of the complex by hydrogen bonding (Porat et al, 2006). Structurally, FA has both the required phenolic group and hydrogen bond donor. In silico docking studies indicate that FA interacts with Aβ deposits through hydrogen bonding and inhibits Aβ aggregation by interfering with β-sheet formation (Zhang et al, 2013). Chronic oral administration of FA (5.3 mg/kg/day) significantly decreased Aβ(1–42) deposition in 12-month-old APP_swe/PS1_ΔE9 transgenic mice (Yan et al, 2013). A systemic review of FA in animal models of AD has recently been published (Wang et al, 2021).

FAEE (ethyl-4-hydroxy-3-methoxycinnamic acid) is a phenolic compound and naturally occurring alkyl ester derivative of FA with increased antioxidant scavenging properties and ability to cross the blood-brain barrier due to greater hydrophobicity (Sultana, 2012). Loss of phospholipid asymmetry resulting in prolonged presentation of phosphatidylserine in the outer leaflet is a known trigger of apoptosis and is observed in both MCI and AD brain (Bader Lange et al, 2008) and in APP^NLh/APP^NLh x PS1^P264L/PS1^P24L human double mutant knock-in mice, a knock-in, not transgenic, model of AD (Bader Lange et al, 2010).

FAEE (25 μM) protects against Aβ(1–42)-induced loss of phospholipid asymmetry in vitro (Abdul and Butterfield, 2005) and prevents Aβ(1–42)-induced protein oxidation, protein nitration, and lipid peroxidation in vitro (10 μM) (Sultana et al, 2005) and ex vivo (150 mg/kg, i.p.) (Perluigi et al, 2006). FAEE induces HO-1 protein expression in vitro (Scapagnini et al, 2004) and in vivo (Joshi et al, 2006). Higher concentrations of FAEE (50 μM) are cytotoxic and did not induce HO-1 in vitro, whereas lower concentrations induce HO-1 protein expression in astrocytic (15 μM) and neuronal (5 μM) cultures (Scapagnini et al, 2004).

Caffeic acid phenethyl ester (2-phenylethyl (2E)-3-(3,4-dihydroxyphenyl)prop-2-enoate; CAPE) is an ester of caffeic acid and phenethyl alcohol that is a hydrophobic, polyphenolic compound naturally occurring in beehive propolis. CAPE is also found in the resinous exudate of buds and leaves of species of Populus. Like FA and FAEE, CAPE also has antioxidant and anti-inflammatory properties (Kulkarni et al, 2021) and crosses the blood-brain barrier (Silva et al, 2016).

CAPE induces increased HO-1 protein expression and activity in astrocytes at 30 μM, but it results in cytotoxicity at higher concentrations (50–100 μM) (Scapagnini et al, 2002). CAPE administered i.p. for 10 days (10 mg/kg) following i.c.v. injection of soluble oligomeric Aβ(1–42) in 9-week-old C57Bl/6 mice significantly decreased oligomeric Aβ(1–42)-induced oxidative stress as measured by dichlorofluorescein (DCF) fluorescence and reduced GSH content, cell death, caspase-9 activation, and cognitive effects in the hippocampus.

In addition, CAPE was shown to significantly activate Nrf2 and increase HO-1 protein expression and reduce glial activation in the hippocampus 10 days post-injection of Aβ. Basal levels of Nrf2 were observed 20 days post-injection (Morroni et al, 2018). Long-term administration of CAPE in aging rats (15 mg/kg/day, i.p., 95 days) resulted in decreased lipid peroxidation as indexed by malondialdehyde and increased SOD2 and CAT activities in the cerebral cortex (Eşrefoělu et al, 2010).

The therapeutic use of CAPE is limited by water solubility, low bioavailability, and structural instability. Thus, CAPE derivatives are being explored to circumvent these limitations. One such example is FA-97, CAPE 4-O-glucoside, which shows dose-dependent protection against H₂O₂-induced apoptosis, lipid peroxidation, and protein oxidation in vitro. In addition, FA-97 increased SOD2 activity, GSH levels, and HO-1 and NQO1 protein expression levels by inducing nuclear translocation and transcriptional activity of Nrf2.

This was confirmed with the use of Nrf2 siRNA, which blocked the effects of FA-97. Taken together, these findings indicate that FA-97 functions as an antioxidant by activating Nrf2. To explore the effects of FA-97 in vivo, scopolamine (SCOP; 3 mg/kg i.p.) was used to induce learning and memory impairment in a mouse model. FA-97 (5 or 10 mg/kg; oral gavage; 30 days) prevented SCOP-induced impairment in spatial learning and memory in a Morris water maze (training and tests occurred during days 21–30).

FA-97 (10 mg/kg) reportedly protects against oxidative stress in vivo by promoting nuclear translocation and transcriptional activity of Nrf2 in both hippocampus and cortex. Molecular docking simulation suggests that FA-97 could activate Nrf2 by binding directly via hydrogen bonding interactions, completing with Keap1, to allow release of Nrf2 for nuclear translocation (Wan et al, 2019). VP961, a derivative of CAPE, which differs by two methoxy groups, protects against 100 μM H₂O₂-induced cell death in SHSY-5Y neuroblastoma cells (5 μM) and induced nuclear translocation and transcriptional activity of Nrf2, resulting in increased HO-1 gene expression and protein levels.

Notably, VP961 induced the same effects as CAPE (10 μM) but at half the concentration. The potency of VP961 was confirmed in human lymphoblastoid cell lines derived from pediatric DS and healthy donors (Pagnotta et al, 2022). Oxidative stress is an early event in DS (Butterfield et al, 2014; Cenini et al, 2012; Perluigi et al, 2011); thus, this model allowed for testing of the antioxidant effects of VP961. CAPE (10 μM) had no effect on the level of oxidative stress as indexed by HNE; however, VP961 (5 μM) significantly decreased HNE levels (Pagnotta et al, 2022). Collectively, this study suggests that VP961 is a more potent analogue of CAPE with potential therapeutic effects; however, further study is needed to elucidate the neuroprotective effects of the compound.

Effect of sulforaphane on Nrf2 in MCI/AD

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane] is an aliphatic isothiocyanate derived from glucoraphanin that is found mainly in sprouts of cruciferous vegetables. Sulforaphane is lipophilic, exhibits high bioavailability, antioxidant, anti-inflammatory, and anti-apoptotic effects (Schepici et al, 2020), and crosses the blood-brain barrier intact (Jazwa et al, 2011). The antioxidant effects of sulforaphane are manifested through activation of Nrf2, as sulforaphane stimulates a dose-dependent increase in HO-1 and NQO1 protein expression and protects against superoxide radical induced oxidative stress in astrocytes (Bergström et al, 2011).

These results were confirmed in vivo in a traumatic brain injury mouse model in which 5 mg/kg, i.p. induced nuclear translocation and activation of Nrf2 transcriptional activity resulting in upregulation of HO-1 and NQO1 and reduced protein oxidation and lipid peroxidation. Further evidence was provided by repeating this study in Nrf2 knockout mice where sulforaphane failed to protect against oxidative stress (Hong et al, 2010).

Thiols react with sulforaphane by attack on the electrophilic isothiocyanate carbon, resulting in thionoacyl adducts (Kensler et al, 2012). Sulforaphane may accumulate in cells as a conjugate of GSH (Zhang, 2000) and initiates Nrf2 nuclear translocation by modifying Keap1 Cys151, thereby blocking CuI3 interaction and subsequent proteasomal degradation (Hu et al, 2011; Zhang and Hannink, 2003). Mutation of Cys151 to a serine residue blocks sulforaphane-induced activation of Nrf2 and restores basal level proteasomal degradation (Zhang and Hannink, 2003).

These results provide a hypothesis for the nuclear translocation and transcriptional activation of Nrf2 but does not account for the sulforaphane-induced increase in Nrf2 protein expression. DNA methylation affects gene expression and has been shown to repress transcription of Nrf2 in N2a cells expressing human Swedish mutant APP (N2a/APPswe) (Cao et al, 2016). In the same cellular model, sulforaphane (1.25 or 2.5 μM) was shown to increase Nrf2 levels by decreasing expression of DNA methyltransferases (DNMT) and, subsequently, DNA methylation of CpG sites in the Nrf2 promoter (Zhao et al, 2018).

The neuroprotective effects of sulforaphane in AD were recently reviewed (Kim, 2021). Sulforaphane decreases Aβ levels in vitro (Zhang et al, 2021d) and in vivo (Bahn et al, 2019; Hou et al, 2018). Sulforaphane is a potent, non-competitive inhibitor of BACE1, the rate limiting enzyme in the production of Aβ (Youn et al, 2020). Sulforaphane administered every other day for 2 months induced Nrf2 repression of Bace1 in both 9-month-old 5xFAD (10 mg/kg, i.p.) and 7-month-old 3xTg-AD (5 or 10 mg/kg, i.p.) mice, resulting in decreased cortical and hippocampal Aβ levels and improved cognitive function (Bahn et al, 2019). Treatment with sulforaphane for 4 months also inhibited Aβ generation and oligomerization in 6-month-old PS1V97L transgenic mice (Hou et al, 2018).

Microglia can play a beneficial role in the clearance of Aβ by phagocytosis or a detrimental role by releasing proinflammatory cytokines. In the AD brain, a decline in Aβ phagocytosis is observed as contributing to the decrease in Aβ clearance (Guo et al, 2022). Sulforaphane (5 μM) ameliorated decreased microglial phagocytosis induced by low concentrations (100 and 500 ng/mL) of soluble oligomeric Aβ, suggesting that sulforaphane may shift microglia to an M2 phenotype, which is associated with repair and anti-inflammatory properties (Chilakala et al, 2020).

Taken together, these studies are consistent with the notion that sulforaphane not only acts as an antioxidant indirectly by upregulating transcription and nuclear translocation of Nrf2 allowing for Nrf2 mediated gene transcription (Fig. 9), but also may shift reactive microglia to reparative M2 forms and thereby inhibit the accumulation of Aβ.

FIG. 9.

Sulforaphane-induced nuclear translocation and activation of Nrf2. Sulforaphane is attacked by the Cys151 thiol of Keap1 at the electrophilic isothiocyanate carbon, resulting in a thionoacyl adduct that causes release of Nrf2, allowing for nuclear translocation of Nrf2 and activation of gene transcription.

Epigallocatechin 3-gallate and its link to the Nrf-2 pathway

As noted earlier, epigallocatechin 3-gallate (EGCG) is a catechin commonly found in green tea. EGCG is reported to efficiently alleviate symptoms and body fat from a high fat, Western diet that caused obesity and metabolic syndrome in rats (Chen et al, 2011). This polyphenolic biomolecule has demonstrated antioxidant (Stull et al, 2002), neuroprotective (Weinreb et al, 2009; Weinreb et al, 2004), cardioprotective (Zheng et al, 2011), cytoprotective, and metal chelating properties (Weinreb et al, 2009) and is suggested to be effective as an adjuvant against cancer and inflammation (Lee et al, 2013).

EGCG has been proposed to be a plausible treatment strategy for neurological disorders such as AD (Chesser et al, 2016; Choi et al, 2001), Parkinson disease (Lorenzen et al, 2014; Mandel et al, 2004), and Huntington disease (Cheng et al, 2013). EGCG reduces Aβ load and phosphorylated tau in AD models and inhibits α-synuclein oligomer toxicity in Parkinson disease models (Weinreb et al, 2004). EGCG has been reported to be more effective as a radical scavenger when compared with the classical antioxidants alpha-tocopherol and ascorbic acid (Nanjo et al, 1996).

The bioavailability of EGCG is poor but can be enhanced by several nutritional factors, including ascorbic acid, omega-3 fatty acid supplementation in the absence of caffeine on overnight fasting, and royal jelly protein (Han et al, 2020). The use of nanoparticles to optimize the oral bioavailability of EGCG have been studied as a method for delivery enhancement (Smith et al, 2010; Zhang and Zhang, 2018).

This catechin demonstrates ability to scavenge ROS and RNS. EGCG reportedly inhibits the formation of hydroxyl radicals through a Fenton reaction mechanism (Jomova et al, 2010). EGCG has been studied as a strong Nrf2 activator since it has been shown to activate HO-1 (He et al, 2018), PI3-K (Na et al, 2008), and ERK (Zipper and Mulcahy, 2000), inhibit the arylhydrocarbon receptor (Ma et al, 2004), and modulate the NLRP3 inflammasome (Tsai et al, 2011) and the TrkA/p75 signaling pathway (Valdovinos-Flores et al, 2019).

As mentioned earlier, this induction of Nrf2 is thought to be due to the conjugation of EGCG to GSH. Although this conjugation lowers levels of available GSH, MAP kinases are activated, thereby phosphorylating and activating Nrf2 (Miao et al, 2005). This activation can lead to increased overall antioxidant levels through Nrf2-ARE interactions. These profound impacts regulate and positively affect signal transduction, inflammation, and memory and learning.

Arsenic (III), as sodium arsenite, activates Nrf2 evinced by increased ROS levels in multiple tissues. Chronic arsenic exposure via environmental pollution promotes liver damage such as cirrhosis, hepatomegaly, and abnormal liver function. Oxidative damage and inflammation are consequences of As(III) exposure, which negatively affects antioxidant balance.

EGCG also activates the Nrf2 signaling pathway by reducing malondialdehyde and transaminase levels and increasing SOD2, CAT, and GSH levels in arsenic-induced hepatotoxicity, and EGCG was shown to reduce effects of liver damage (Han et al, 2017). EGCG appears to enhance the antioxidant response of Nrf2 via arsenic-induced oxidative stress protection against liver damage (Jiang et al, 2009). Nrf2 overexpression has been shown to promote tumorigenesis and resistance to chemotherapies, however this is reversed on gene silencing via siRNA mediation of Nrf2 and concomitant treatment with EGCG.

This compound causes a partial reversal of sensitization of tamoxifen treatment in MCF-7/TAM cells, thereby bolstering the antioxidant potential as a novel chemotherapeutic treatment (Esmaeili, 2016). In addition, in studies in which Nrf2 is knocked down, the protective effect of EGCG is lost against oxalate-induced epithelial mesenchymal transition in renal cells. This shows an expansion of EGCG's role as an anti-fibrotic agent (Kanlaya and Thongboonkerd, 2019).

As a result of its association with the Nrf2 pathway, EGCG has demonstrated a therapeutic effect on pro-inflammatory species such as TNF alpha and IL-1 beta inflammation (Ahmed et al, 2017; Leonardo and Dore, 2011). All these studies noted earlier, taken together, suggest that EGCG plays a pivotal role in the Nrf2 pathway. Continued efforts in research will provide insight to new avenues of association between these key biochemical partners.

EGCG can easily pass through the blood-brain barrier. On entering the blood-brain barrier, this molecule is metabolized to gallic acid and epigallocatechin. Epigallocatechin is further decomposed into the metabolite, 5-(3′,5′-dihydroxyphenyl)-γ-valerolactone (EGC-M5) and its conjugates (Fig. 10). In human neuroblastoma SH-SY5Y cells, the number of neurites increases as well as their length after EGCG supplementation.

FIG. 10.

Metabolism of EGCG. EGCG is metabolized to gallic acid and epigallocatechin on encountering the blood-brain barrier. Epigallocatechin is further catabolized to EGC-M5, an anti-diabetic and antioxidant metabolite. EGC-M5, 5-(3′,5′-dihydroxyphenyl)-γ-valerolactone.

By modulating EGCG through its metabolism, a positive outcome regarding the promotion of neurogenesis has been established (Unno et al, 2017). This outcome can contribute to the neuroprotective properties such as pro-autophagy and suppression of Aβ(1–42) aggregation, thereby becoming a promising therapy for neurodegenerative disorders such as AD.

EGCG and AD

EGCG has been extensively evaluated as a potential treatment strategy for AD based on its influence on Aβ remodeling (Engel et al, 2012; Palhano et al, 2013), tau (Rezai-Zadeh et al, 2008), and reduction of mitochondrial dysfunction (Butterfield and Boyd-Kimball, 2020; Dragicevic et al, 2011; Pradeepkiran and Reddy, 2020; Swerdlow, 2018) associated with AD. Although Aβ aggregation at the phospholipid interface is reduced via EGCG treatment, this inhibition is less efficient and amyloid aggregation occurs (Engel et al, 2012).

Although EGCG possesses strong antioxidant properties, this biomolecule undergoes autooxidation on introduction to air; however, by binding EGCG to human serum albumin, the antioxidant becomes stable and is less likely to decompose (Ishii et al, 2011). This air-oxidation of EGCG can impact its ability to remodel Aβ(1–40) fibrils as SOD1-mediated pre-oxidized EGCG delayed appearance of Thioflavin T fluorescence. Reduced EGCG oxidation demonstrates decreased amyloid remodeling activity (Palhano et al, 2013), thereby bolstering the need to continue studying EGCG and its role in amyloidosis and amyloid related disorders.

The APP_sw (APP KM670/671NL—Swedish) transgenic mouse model demonstrates overexpression of Aβ(1–42) by 12 months and phosphorylated tau, but no amyloid plaques. Reduction of Aβ deposition after 6 months of oral (50 mg/kg) EGCG treatment (Rezai-Zadeh et al, 2008) was observed in the cingulate cortex, entorhinal cortex, and hippocampus. Tau treated with EGCG inhibits aggregation in a time-dependent manner.

Paired helical filaments are inhibited after tau treatment with EGCG after 24 h. This could be due to the increase in hydrophobicity as aggregation occurs, which is reduced in a dosage- and time-dependent course. Mature tau fibrils treated with EGCG are disintegrated and cleared (Sonawane et al, 2020). This ground-breaking work in mice showed that EGCG increased cell viability by reducing tau toxicity and inhibiting tau aggregation, both exhibited in AD pathology.

Dragicevic et al showed that EGCG restored mitochondrial function in the APP/PS1 transgenic mouse model of AD. This model expresses human APP and PS1, mutations that have been associated with early-onset AD. Aβ load is high and plaque accumulation is observed by 6 months. The results from this study showed a lowering of ROS, significant increase in ATP, and restoration of mitochondrial membrane potential in the hippocampus, striatum and cortex (Dragicevic et al, 2011).

EGCG administration over 12 months combined with social interaction, healthy diet, exercise, and cognitive training aims at showing a delay in cognitive decline in APOE4 carriers (Forcano et al, 2021). Due to the high association between apolipoprotein E4 and AD risk, the results from this study may show promise to the therapeutic potential of epigallocatechin-3-gallate as a plausible treatment for the prevention of cognitive decline in those with AD.

Resveratrol and its link to the Nrf-2 pathway

Resveratrol (3,4′,5′-trihydroxystilbene) is a natural product highly concentrated in skins of red grapes and red wine, which has been well documented to show health benefits. Resveratrol has been found to have multiple biological properties as an antioxidant (Olas et al, 2001; Virgili and Contestabile, 2000), an anti-inflammatory agent (Wang et al, 2013), an anti-cancer agent (Dorrie et al, 2001; ElAttar and Virji, 1999; Hsieh and Wu, 2000), a vasoprotectant (Ungvari et al, 2010), a cardioprotective compound (Mokni et al, 2007), a neuroprotective biomolecule (Gao and Hu, 2005; Virgili and Contestabile, 2000), and an anti-bacterial compound (Morales et al, 2002).

Most notably, it is a well-established lipid peroxide scavenger, thereby inhibiting lipid peroxidation and the NF-kappaB pathway (Fig. 11). Resveratrol demonstrated a reduction in thiobarbituric acid reactive substances and malondialdehyde, both indices of lipid peroxidation, in cerebral ischemic injured animals (Lin et al, 2021).

FIG. 11.

Resveratrol as a lipid radical scavenger. On oxidative insult, the unpaired electron can become delocalized within the aromatic rings and conjugated double bonds of resveratrol, thereby increasing stabilization.

Resveratrol is found to be protective against experimental stroke and ischemic brain damage by reducing levels of matrix metalloprotease-9 (MMP-9) and activating PPARα (Gao et al, 2006; Inoue et al, 2003; Li et al, 2012; Sakata et al, 2010). Studies from Leonardo and Dore (2011) also demonstrate a reduction of oxidative stress and lipid peroxidation thereby showing neuroprotective properties in a rat transient MCA occlusion model.

As stated in earlier sections of this review article, under normal conditions Keap1 isolates Nrf2 in the cytoplasm, where Nrf2 regulates cellular defenses against oxidative stress. However, during oxidative stress conditions, Nrf2 and Keap1 are dissociated. Nrf-2 translocates to the nucleus where it regulates HO-1 expression via binding with the ARE of DNA.

Resveratrol is reported to increase GSH levels as a protective factor by upregulating glutamate-cysteine ligase (GCL), the rate-determining step of GSH biosynthesis, against oxidative stress by activating Nrf2 (Kode et al, 2008) in patients with lung injury from cigarette smoke-induced oxidative stress. Resveratrol also reportedly scavenged ROS and restored GSH levels by inducing nuclear translocation of Nrf2 and increasing GCL and GST activity (Rubiolo et al, 2008). Kode et al (2008) supported these findings by providing similar evidence that showed that the knockdown of Nrf2 demonstrated increased Keap1 and an accompanying decrease in antioxidant levels by reduced GCL messenger RNA (mRNA) expression.

Resveratrol restores GCL activity after 24 h, thereby exhibiting the strong antioxidant activity of resveratrol and its association with Nrf2. Multiple studies have been conducted to show resveratrol's potential as a treatment in fetal alcohol syndrome (Kumar et al, 2011), diabetes (Bhatt et al, 2012; Ramadori et al, 2009), cancer (Singh et al, 2014), hypertension (Zhou et al, 2018), and brain and blast injuries (Cong et al, 2021; Gao et al, 2018) as a strong Nrf2 activator.

Resveratrol and AD

In addition to the skins of red grapes, blackberries, pomegranates, and peanut products are natural sources of resveratrol. Due to its role as a potent antioxidant, resveratrol has been widely investigated as a plausible therapy in the progression of MCI to AD to late-stage AD. In vivo and in vitro studies have been conducted to validate resveratrol's function as a neuroprotectant by protecting against Aβ toxicity, enhancing GSH levels, and overall antioxidant levels (Savaskan et al, 2003), promoting Aβ clearance (Marambaud et al, 2005), potentiating sirtuin 1 (SIRT1) proteins that contribute to the inhibition of NF-kappa B expression (Yeung et al, 2004), inhibiting lipid peroxidation (Rege et al, 2015), lowering oxidative stress (Kumar et al, 2007), and stimulating p53 deacetylation via SIRT1 (Howitz et al, 2003).

The interactions between resveratrol and the overexpression of SIRT1 can provide protection against neuronal dysfunction observed in AD. In the Tg19959 transgenic mouse model of AD, which incorporates both Swedish (KM670/671NL) and Indiana (V717F) mutations, exhibiting increased Aβ production and an Aβ(1–42)/Aβ(1–40) ratio, respectively, dietary supplementation with resveratrol demonstrated reduced plaque formation in the medial cortex, striatum, and hypothalamus, but it did not alter APP fragmentation (Karuppagounder et al, 2009).

In the triple transgenic mouse model (3xTg-AD), which contains mutations in the APP, Swedish KM670/671NL mutation, PSEN1M146V, and MAPT P301L genes, mice develop progressive Aβ deposition starting at 3 months and tau aggregates at 12 months. Learning and memory deficits occur before plaque and tangle formation. Nutritional supplementation with resveratrol reduced markers of inflammation, autophagy, ubiquitinylation, and Aβ toxicity, yet increased expression of SIRT1 (Broderick et al, 2020). The 5xTg-AD (Tg6799) transgenic mouse model contains five FAD mutations, including the Swedish, Florida (I716V), London (V717I), and two PSEN mutations (M146L and L286V).

This is an excellent model to study AD, as it exhibits early amyloid pathology in the form of amyloid plaques and detectable Aβ(1–42) levels within the first 2 months of age. Aβ burden and Aβ(1–42)/Aβ(1–40) ratio increase exponentially over time. After resveratrol administration, Aβ(1–42) levels, amyloid plaque formation, and BACE levels were lowered in these transgenic mice (Chen et al, 2019b). Most notably, learning and memory were significantly improved, further supporting the role of antioxidants in neurodegenerative disease, neuroprotection, and reduction of cognitive decline.

Although resveratrol supplementation for 6 months resulted in reduced hippocampal loss and glycated A1C concentrations in patients suffering from MCI, memory impairment was not improved (Kobe et al, 2017). Kobe et al showed that resting-state functional connectivity was significantly increased due to resveratrol supplementation. This is the first study to show positive outcomes of resveratrol on these measures, which may correlate to the known association between diabetes, AD risk, and antioxidant status (Butterfield and Halliwell, 2019).

Resveratrol has also been shown to improve overall innate immunity in MCI patients by increasing phagocytosis of Aβ(1–42) by monocytes and macrophages when given in a nutritional supplement containing docosahexaenoic acid, eicosapentaenoic acid, Vitamin D₃ and other antioxidants, thereby demonstrating the impact of nutrition on amyloidogenesis, immunity, and disease progression (Famenini et al, 2017; Fiala et al, 2015).

Yearlong treatment, in the form of a daily 500 mg dose of trans-resveratrol in patients with mild and moderate AD, showed a significant decrease in MMP-9 and progressive decline in Aβ(1–40) levels in people with advancing dementia (Gu et al, 2021). These results contribute to the reduction in neuroinflammation and neurotoxicity that is well established during the progression of AD.

Conclusions

Hormesis can be defined as the beneficial effect of upregulation of protective genes on exposure to low doses of agents that have been found to be toxic at higher doses. The responses of multiple polyphenolic compounds have proved to show pleiotropic effects regarding reducing oxidative stress, neuroinflammation, and providing neuroprotection to delay the progression of AD in animal models and in some studies involving human subjects.

Data from prior and current studies give insights into the role of nutrition and natural products in prevention of oxidative stress-associated neurodegeneration and Aβ pathology. Taken together, the use of polyphenols has had a transformative effect and a promising future in potential therapeutic interventions in the devasting dementing disorder of AD. That said, better bioavailability of these agents will be required in our opinion to achieve disease modifying effects in MCI and AD.

Abbreviations Used

Aβ

amyloid β-peptide

AD

Alzheimer's disease

APP

amyloid precursor protein

ARE

antioxidant response elements

BACE

β-secretase

BACH1

BTB and CNC homology 1

BVR

biliverdin reductase

CAPE

caffeic acid phenethyl ester

CAT

catalase

CNC

cap “n” collar

CO

carbon monoxide

Cul3

cullin 3

CYPs

cytochrome P450-dependent monooxygenases

DS

Down syndrome

EGCG

epigallocatechin gallate

EGC-M5

5-(3′',5′-dihydroxyphenyl)-γ-valerolactone

FA

ferulic acid

FAD

familial Alzheimer's disease

FAEE

ferulic acid ethyl ester

Fe²⁺

ferrous iron

FTH

ferritin heavy chain

FTL

ferritin light chain

GCL

glutamate-cysteine ligase

GCLC

glutamate-cysteine ligase catalytic subunit

GCLM

glutamate-cysteine ligase modifier subunit

GPx

glutathione peroxidase

GR

glutathione reductase

GS

glutathione synthetase

GSH

glutathione

GSK-3β

glycogen synthase kinase 3β

GST

glutathione-S-transferase

HNE

4-hydroxy-2-trans-nonenal

HO

heme oxygenase

Htau

hyperphosphorylated tau

IPL

inferior parietal lobule

IRS

insulin receptor substrate

Keap1

Kelch-like erythroid cell-derived protein with CNC homology associated protein 1

Maf

musculoaponeurotic fibrosarcoma oncogene homolog protein

MCI

mild cognitive impairment

MMP-9

matrix metalloprotease-9

MtFt

mitochondrial ferritin

Neh

Nrf2-ECH homology

NQO1

NAD(P)H:quinone oxidoreductase 1

Nrf2

nuclear factor erythroid 2-related factor 2

PCAD

preclinical Alzheimer's disease

PDK1

phosphatidylinositol-dependent kinase 1

PET

positron emission tomography

PI3-K

phosphoinositol-3-kinase

PPP

pentose phosphate pathway

PS1

presenilin 1

RNS

reactive nitrogen species

ROS

reactive oxygen species

SCOP

scopolamine

siRNA

small interfering RNA

SIRT1

sirtuin 1

SOD 1

Cu/Zn-superoxide dismutase

SOD2

Mn-superoxide dismutase

T1DM

type I diabetes

T2DM

type II diabetes

TR

thioredoxin reductase

Authors' Contributions

D.A.B.: principal author, conceptualized, wrote, reviewed and edited the article; D.B.-K. wrote aspects of the article and prepared some of the figures; and T.T.R. wrote some of the articles and prepared some of the figures.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported in part by an NIH grant (AG060056).