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Understanding Nrf2 and its Impact on Neurodegenerative Diseases

by Dr Alex Jimenez | Detoxification, Functional Medicine, Health, Holistic Medicine, Natural Health, Nutrition, Oxidative Stress, Remedies, Research Studies, Treatments, Wellness

Neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, affect millions of individuals worldwide. A variety of treatment options are available to treat the symptoms of several neurodegenerative diseases although the results are often limited. Research studies have found that oxidative stress caused by both internal and external factors can be a cause for the development of neurodegenerative diseases. The transcription factor, Nrf2, has been determined to function as a major defense mechanism against oxidative stress. The purpose of the article below is to show the effects of Nrf2 on neurodegenerative diseases.

Modulation of Proteostasis by Transcription Factor NRF2

Neurodegenerative diseases are linked to the accumulation of specific protein aggregates, suggesting an intimate connection between injured brain and loss of proteostasis. Proteostasis refers to all the processes by which cells control the abundance and folding of the proteome thanks to a wide network that integrates the regulation of signaling pathways, gene expression and protein degradation systems. This review attempts to summarize the most relevant findings about the transcriptional modulation of proteostasis exerted by the transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2). NRF2 has been classically considered as the master regulator of the antioxidant cell response, although it is currently emerging as a key component of the transduction machinery to maintain proteostasis. As we will discuss, NRF2 could be envisioned as a hub that compiles emergency signals derived from misfolded protein accumulation in order to build a coordinated and perdurable transcriptional response. This is achieved by functions of NRF2 related to the control of genes involved in the maintenance of the endoplasmic reticulum physiology, the proteasome and autophagy.

Keywords: Neurodegenerative diseases, Unfolded protein response, Proteasome, Ubiquitin, Autophagy, Oxidative stress

Abbreviations

Sciencedirect.com/science/article/pii/S2213231716304050

Introduction

Nuclear Factor (erythroid-derived 2)-like 2 (NRF2) is a basic-leucine-zipper protein considered nowadays as a master regulator of cellular homeostasis. It controls the basal and stress-inducible expression of over 250 genes that share in common a cis-acting enhancer termed the antioxidant response element (ARE) [1], [2], [3], [4], [5]. These genes participate in phase I, II and III detoxification reactions, glutathione and peroxiredoxin/thioredoxin metabolism, NADPH production through the pentose phosphate pathway and malic enzyme, fatty acid oxidation, iron metabolism, and proteostasis [6]. Given these wide cytoprotective functions, it is possible that a single pharmacological hit in NRF2 might mitigate the effect of the main culprits of chronic diseases, including oxidative, inflammatory and proteotoxic stress. The role of NRF2 in the modulation of the antioxidant defense and resolution of inflammation have been addressed in numerous studies (reviewed in [7]). Here, we will focus on its role in proteostasis, i.e., the homeostatic control of protein synthesis, folding, trafficking and degradation. Examples will be provided in the context of neurodegenerative diseases.

Loss of Proteostasis Influences NRF2 Activity in Neurodegenerative Diseases

A general hallmark of neurodegenerative diseases is the occurrence of aberrant aggregation of some proteins. Thus, misfolded protein aggregates of ?-synuclein (?-SYN) are found in Parkinson’s disease (PD), ?-amyloid (A?) plaques and hyper-phosphorylated TAU neurofibrillary tangles in Alzheimer’s disease (AD), huntingtin (Htt) in Huntington’s disease (HD), superoxide dismutase 1 (SOD1) and TAR DNA binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS), prion protein (PrP) in spongiform encephalopathies, etc. Protein aggregates can have an impact on several cellular pathways, which in turn may affect NRF2 levels and activity.

Different Layers of Regulation Tightly Control NRF2 Activity

Under physiological conditions, cells exhibit low NRF2 protein levels because of its rapid turnover. In response to different stimuli, NRF2 protein is accumulated, enters the nucleus and increases the transcription of ARE-containing genes. Therefore, management of NRF2 protein levels is a key point that should integrate positive and negative input signals. As we will discuss further, NRF2 is activated by diverse overlapping mechanisms to orchestrate a rapid and efficient response but on the other hand NRF2 could be inhibited, probably in a second phase, in order to switch off its response.

From the classic point of view, activation of NRF2 has been considered as a consequence of the cellular response to oxidant or electrophilic compounds. In this regard, the ubiquitin E3 ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1) plays a crucial role. Molecular details will be further addressed in Section 4.1. In brief, KEAP1 acts as a redox sensor due to critical cysteine residues leading to NRF2 ubiquitination and proteasomal degradation. In addition to this classic modulation, NRF2 is profoundly regulated by signaling events. Indeed, different kinases have been shown to phosphorylate and regulate NRF2. For instance, NRF2 can be phosphorylated by mitogen activated protein kinases (MAPKs), although its contribution to NRF2 activity remains unclear [8], [9], [10], [11]. PKA kinase as well as some PKC isozymes [12], CK2 [13] or Fyn [14] phosphorylate NRF2 modifying its stability. Previous work from our group reported that glycogen synthase kinse-3? (GSK-3?) inhibits NRF2 by nuclear exclusion and proteasomal degradation [15], [25], [26], [27], [28], [29], [30]. The molecular details will be discussed in the Section 4.1. Furthermore, NRF2 is submitted to other types of regulation. For instance, NRF2 acetylation by CBP/p300 increases its activity [17], while it is inhibited by miR153, miR27a, miR142-5p, and miR144 [16], or by methylation of cytosine-guanine (CG) islands within the NRF2 promoter [18].

Impact of Protein Aggregates on NRF2 Regulatory Mechanisms

In this section we will focus in how accumulation of misfolded protein could impact NRF2 activity providing some of the pathways mentioned above as illustrative examples. Firstly, we need to consider that protein accumulation has been tightly linked with oxidative damage. Indeed, misfolded protein accumulation and aggregation induce abnormal production of reactive oxygen species (ROS) from mitochondria and other sources [19]. As mentioned above, ROS will modify redox-sensitive cysteines of KEAP1 leading to the release, stabilization and nuclear localization of NRF2.

Regarding proteinopathies, an example of dysregulated signaling events that may affect NRF2 is provided by the hyperactivation of GSK-3? in AD. GSK-3?, also known as TAU kinase, participates in the phosphorylation of this microtubule-associated protein, resulting in its aggregation, formation of neurofibrillary tangles and interruption of axonal transport (reviewed in [20]). On the other hand, GSK-3? dramatically reduces NRF2 levels and activity as mentioned above. Although not widely accepted, the amyloid cascade proposes that toxic A? oligomers increase GSK-3? activity together with TAU hyper-phosphorylation and neuron death [21], [22]. There are different models to explain how A? favors GSK3-? activity. For instance, A? binds to the insulin receptor and inhibits PI3K and AKT signaling pathways, which are crucial to maintain GSK-3? inactivated by phosphorylation at its N-terminal Ser9 residue [23]. On the other hand, extracellular A? interacts with Frizzled receptors, blocking WNT signaling [24] and again resulting in release of active GSK-3?. In summary, A? accumulation leads to abnormal hyperactivation of GSK-3?, thus impairing an appropriate NRF2 response.

As discussed in the following section, misfolded proteins lead to activation of PERK and MAPKs, which in turn up-regulate NRF2 [31], [8], [9], [10], [11]. Moreover, dysregulated CBP/p300 activity has been reported in several proteinopathies [32] and a global decrease in DNA methylation in AD brains was also shown [33], therefore providing grounds to explore the relevance of these findings in NRF2 regulation.

We and others have observed in necropsies of PD and AD patients an increase in NRF2 protein levels and some of its targets, such as heme oxygenase 1 (HMOX1), NADPH quinone oxidase 1 (NQO1), p62, etc., both by immunoblot and by immunohistochemistry [34], [35], [36], [37], [38], [39]. The up-regulation of NRF2 in these diseases is interpreted as an unsuccessful attempt of the diseased brain to recover homeostatic values. However, another study indicated that NRF2 is predominantly localized in the cytoplasm of AD hippocampal neurons, suggesting reduced NRF2 transcriptional activity in the brain [40]. It is conceivable that the disparity of these observations is related to changes in the factors that control NRF2 along the progressive stages of neurodegeneration.

Three major systems contribute to proteostasis, namely the unfolded protein response (UPR), the ubiquitin proteasome system (UPS) and autophagy. Next, we present evidence to envision NRF2 as a hub connecting emergency signals activated by protein aggregates with the protein derivative machinery.

NRF2 Participates in the Unfolded Protein Response (UPR)

NRF2 Activation in Response to the UPR

Oxidative protein folding in the ER is driven by a number of distinct pathways, the most conserved of which involves the protein disulfide-isomerase (PDI) and the sulfhydryl oxidase endoplasmic oxidoreductin 1 (ERO1? and ERO1? in mammals) as disulfide donor. Briefly, PDI catalyzes the formation and breakage of disulfide bonds between cysteine residues within proteins, as they fold, due to the reduction and oxidation of its own cysteine aminoacids. PDI is recycled by the action of the housekeeping enzyme ERO1, which reintroduces disulfide bonds into PDI [41]. Molecular oxygen is the terminal electron acceptor of ERO1, which generates stoichiometric amounts of hydrogen peroxide for every disulfide bond produced [42]. Peroxidases (PRX4) and glutathione peroxidases (GPX7 and GPX8) are key enzymes to reduce hydrogen peroxide in the ER. When this oxido-reductive system does not work properly, abnormal accumulation of misfolded proteins occurs in the ER and a set of signals named the unfolded protein response (UPR) is transmitted to the cytoplasm and nucleus to reestablish the ER homeostasis [43]. Three membrane-associated proteins have been identified for sensing ER stress in eukaryotes: activating transcription factor 6 (ATF6), pancreatic ER eIF2? kinase (PERK, also double-stranded RNA-activated protein kinase-like ER kinase), and inositol-requiring kinase1 (IRE1). The luminal domain of each sensor is bound to a 78 kDa chaperone termed glucose-regulated protein (GRP78/BIP). BIP dissociates upon ER stress to bind unfolded proteins, leading to the activation of the three sensors [44].

NRF2 and its homologue NRF1, also related to the antioxidant response, participate in the transduction of the UPR to the nucleus. In the case of NRF1, this protein is located at the ER membrane and undergoes nuclear translocation upon deglycosylation or cleavage. Then, UPR activation leads to the processing of NRF1 and nuclear accumulation of the resulting fragment in the nuclear compartment. However, the ability to transactivate ARE-containing genes of this NRF1 fragment is still under discussion [45].

Glover-Cutter and co-workers showed activation of the NRF2 orthologue of C. elegans, SKN-1, with different ER stressors. Increased SKN-1 expression was dependent on different UPR mediators, including IRE1 or PERK worm orthologues [46]. In PERK-deficient cells, impaired protein synthesis leads to accumulation of endogenous peroxides and subsequent apoptosis [47]. The effector used by PERK to protect the ER from these peroxides might be NRF2, since it has been reported that PERK phosphorylates NRF2 at Ser40, thus preventing its degradation by KEAP1 [31]. The induction of ASK1 is also likely to play a role in this route through the TRAF2-mediated kinase action of IRE1 [48]. Although the role of MAPKs in the regulation of NRF2 is still controversial, it was recently suggested that the IRE1-TRAF2-ASK1-JNK pathway might activate NRF2 [49] (Fig. 1). Interestingly, in C. elegans and human cells, new evidence suggests that cysteine sulfenylation of IRE1 kinase at its activation loop inhibits IRE1-mediated UPR and initiates a p38 antioxidant response driven by NRF2. The data suggest that IRE1 has an ancient function as a cytoplasmic sentinel that activates p38 and NRF2 [50].

**Figure 1 Regulation of NRF2 by the UPR.** Accumulation of unfolded or misfolded proteins inside the endoplasmic reticulum can initiate the unfolded protein response (UPR). First, the chaperone BIP is released from the intraluminal domain of the ER sensors IRE1 and PERK to bind unfolded/misfolded proteins. This enables dimerization and trans-auto-phosphorylation of their cytosolic domains. PERK activation results in direct NRF2 phosphorylation at Ser40, leading to NRF2 translocation to the nucleus and activation of target genes. IRE1 activation induces the recruitment of TRAF2 followed by ASK1 and JNK phosphorylation and activation. As JNK has been reported to phosphorylate and activate NRF2, it is reasonable to think that IRE1 activation would lead to increased NRF2 activity.

Many studies on the induction of the UPR have been conducted with the inhibitor of protein glycosylation tunicamycin. NRF2 appears to be essential for prevention of tunicamycin-induced apoptotic cell death [31] and its activation under these conditions is driven by the autophagic degradation of KEAP1 [51]. Accordingly, shRNA-mediated silencing of NRF2 expression in ?TC-6 cells, a murine insulinoma ?-cell line, significantly increased tunicamycin-induced cytotoxicity and led to an increase in the expression of the pro-apoptotic ER stress marker CHOP10. On the other hand, NRF2 activation by 1,2-dithiole-3-thione (D3T) reduced tunicamycin cytotoxicity and attenuated the expression of CHOP10 and PERK [52]. Interestingly, olfactory neurons submitted to systemic application of tunicamycin increased NRF2 in parallel with other UPR-members such as CHOP, BIP, XBP1 [53]. These results have been extended to in vivo studies, as lateral ventricular infusion of tunicamycin in rats induced expression of PERK and NRF2 in the hippocampus accompanied by significant cognitive deficits, increased TAU phosphorylation and A?42 deposits [54].

NRF2 Up-Regulates Key Genes for the Maintenance of the ER Physiology

The ER lumen needs an abundant supply of GSH from the cytosol in order to maintain disulfide chemistry. NRF2 modulates crucial enzymes of the GSH metabolism in the brain, such as cystine/glutamate transport, ?-glutamate cysteine synthetase (?-GS), glutamate-cysteine ligase catalytic and modulator subunits (GCLC and GCLM), glutathione reductase (GR) and glutathione peroxidase (GPX) (reviewed in [55]). The relevance of NRF2 in the maintenance of GSH in the ER is supported by the finding that pharmacological or genetic activation of NRF2 results in increased GSH synthesis via GCLC/GCLM, while inhibiting the expression of these enzymes by NRF2-knockdown caused an accumulation of damaged proteins within the ER leading to the UPR activation [56].

In C. elegans several components of the UPR target genes regulated by SKN-1, including Ire1, Xbp1 and Atf6. Although NRF2 up-regulates the expression of several peroxidase (PRX) and glutathione peroxidase (GPX) genes in mammals (reviewed in [57]), only GPX8 is a bona fide ER-localized enzyme, harboring the KDEL retrieval signal [58]. Loss of GPX8 causes UPR activation, leakage of ERO1?-derived hydrogen peroxide to the cytosol and cell death. Hydrogen peroxide derived from ERO1? activity cannot diffuse from the ER to the cytosol owing to the concerted action of GPX8 and PRX4 [59]. In this regard, an analysis of the antioxidant defense pathway-gene expression array using RNA from wild type and NRF2-null mice tissue, revealed that the expression of GPX8 was down-regulated in the absence of NRF2 [60]. In line with this, transcriptome analysis from patient samples suffering from myeloproliferative neoplasms, polycythemia or myelofibrosis, diseases also associate with oxidative stress and low-grade chronic inflammation, show lower expression levels of both NRF2 and GPX8 compared with control subjects [61]. There are not yet studies that specifically involve GPX8 in human brain protection but a transcriptome analysis in mice indicates a compensatory GPX8 increase in response to the Parkinsonian toxin MPTP [62].

Impact of NRF2 on the UPR Dysregulation in Neurodegenerative Diseases

Malfunction of PDI enzymes and chronic activation of the UPR might subsequently initiate or accelerate neurodegeneration. Disease-affected neurons, animal models of neurodegenerative disease as well as post-mortem human tissues evidenced up-regulation of several UPR-markers in most of these disorders. The alteration of PDI/UPR pathway in neurodegenerative diseases has been nicely reviewed in [63] but the following highlights from brain post-mortem samples should be considered. PDI levels are increased in tangle-bearing neurons and in Lewy Bodies of AD and PD patients, respectively [64], [65]. PDI and ERP57 are up-regulated in CSF from ALS patients and in brains from CJD subjects [66], [67], [68]. BIP, PERK, IRE1 and ATF6 are elevated in samples from patients with AD, PD or ALS [69], [70], [71], [67]. BIP, CHOP and XBP1 are elevated in post-mortem brain samples from HD [72], [73]. Moreover, up-regulation of ERP57, GRP94 and BIP was found in cortex tissues from CJD patients [74]. Altogether, this evidence reveals that the accumulation of misfolded proteins in the brain parenchyma leads to a deleterious and chronic activation of the UPR. Interestingly, there is a recent study linking activation of NRF2 by PERK in early AD. In this study, the authors analyzed whether oxidative stress mediated changes in NRF2 and the UPR may constitute early events in AD pathogenesis by using human peripheral blood cells and an AD transgenic mouse model at different disease stages. Increased oxidative stress and increased pSer40-NRF2 were observed in human peripheral blood mononuclear cells isolated from individuals with mild cognitive impairment. Moreover, they reported impaired ER calcium homeostasis and up-regulated ER-stress markers in these cells from individuals with mild cognitive impairment and mild AD [75].

Mutual Regulation of NRF2 and the Ubiquitin Proteasome�System (UPS)

The UPS Modulates NRF2 Protein Levels

The UPS participates in the degradation of damaged or misfolded proteins and controls the levels of key regulatory molecules in the cytosol and the nucleus. The central core of this system is a large multisubunit enzyme that contains a proteolytic active complex named 20S. The 20S core proteasome degrades unfolded proteins, but binding to different regulatory protein complexes changes its substrate specificity and activity. For instance, the addition of one or two 19S regulatory subunits to the 20S core constitutes the 26S proteasome and changes its specificity towards native folded proteins [76], [77]. Proteasomal degradation needs covalent binding of ubiquitin. Conjugation of ubiquitin proceeds via a three-step cascade mechanism. First, the ubiquitin-activating enzyme E1 activates ubiquitin in an ATP-requiring reaction. Then, one E2 enzyme (ubiquitin-carrier protein or ubiquitin-conjugating enzyme) transfers the activated ubiquitin from E1 to the substrate that is specifically bound to a member of the ubiquitin-protein ligase family, named E3. Although the exact fate of the ubiquitinated-protein will depend on the nature of the ubiquitin chain, this process generally results in the degradation by the 26S proteasome [78].

The E3-ligase KEAP1 is the best known inhibitor of NRF2. The mechanism of KEAP1 regulation elegantly explains how NRF2 levels adjust to oxidant fluctuations. Under basal conditions, newly synthesized NRF2 is grabbed by the homodimer KEAP1, which binds one NRF2 molecule at two amino acid sequences with low (aspartate, leucine, glycine; DLG) and high (glutamate, threonine, glycine, glutamate; ETGE) affinity. The interaction with KEAP1 aids to present NRF2 to the CULLIN3/RBX1 protein complex, resulting in its ubiquitination and subsequent proteasomal degradation. However, redox modification of KEAP1 impedes presentation of NRF2 to the UPS represented by CULLIN3/RBX1. As a result, newly synthetized NRF2 escapes KEAP1-dependent degradation, accumulates in the nucleus and activates ARE-containing genes [79], [80], [81], [82].

The E3-ligase adaptor ?-TrCP is also a homodimer that participates in the signaling events related to the phosphorylation of NRF2 by GSK-3?. This kinase phosphorylates specific serine residues of NRF2 (aspartate, serine, glycine, isoleucine serine; DSGIS) to create a degradation domain that is then recognized by ?-TrCP and tagged for proteasome degradation by a CULLIN1/RBX1 complex. The identification of the specific amino acids that are phosphorylated by GSK-3? in this degron was conducted by a combination of site-directed mutagenesis of the Neh6 domain, 2D-gel electrophoresis [15], [26] and mass spectroscopy [83]. Consequently, inhibition of GSK-3? by highly selective drugs or siRNAs against GSK-3 isoforms resulted in an increase in NRF2 protein levels. Similar results were found with siRNAs against ?-TrCP isoforms 1 and 2. Stabilization of NRF2 following GSK-3? inhibition occurred in KEAP1-deficient mouse embryo fibroblasts and in an ectopically expressed NRF2 deletion mutant lacking the critical ETGE residues for high-affinity binding to KEAP1, further demonstrating a KEAP1-independent regulation.

In the context of neurodegenerative diseases, we can envision the modulation of NRF2 by the UPS in two different ways. On the one hand, the KEAP1 system would sense redox imbalance derived from misfolded protein accumulation, while GSK-3/?-TrCP axis would act as an active participant in signaling transduction altered by loss of proteostasis (Fig. 2).

**Figure 2 The UPS tightly controls NRF2 levels.** Under homeostatic conditions, low NRF2 levels are maintained by the action of the E3 ligases adaptors KEAP1 and ?-TrCP. Left, NRF2 binds to the Kelch domains of a KEAP1 homodimer through a low (DLG) and a high (ETGE) affinity motifs. Through its BTB domain, KEAP1 simultaneously binds to a CULLIN3/RBX1 complex, enabling NRF2 ubiquitination and degradation by the 26 S proteasome. Moreover, GSK-3? phosphorylates Ser335 and Ser338 residues of NRF2 to create a degradation domain (DpSGIpSL) that is then recognized by the ubiquitin ligase adaptor ?-TrCP and tagged for proteasome degradation by a CULLIN3/RBX1 complex. Right, Upon exposure to reactive oxygen species or electrophiles critical Cys residues in KEAP1 are modified, rendering KEAP1 unable of interacting efficiently with NRF2 or CULLIN3/RBX1 and then this transcription factor increases its half-life and transcriptional activity towards ARE-genes. Signaling pathways that result in inhibition of GSK-3?, such AKT phosphorylation at Ser9, result in NRF2 impaired degradation by the proteasome, accumulation and induction of target genes.

NRF2 Increases UPS Activity Through the Transcriptional Control of Proteasome Subunits

NRF2 up-regulates the expression of several proteasome subunits, thus protecting the cell from the accumulation of toxic proteins. Twenty proteasome- and ubiquitination-related genes appear to be regulated by NRF2, according to a wide microarray analysis from liver RNA that was set up with the NRF2 inducer D3T [84]. In a posterior study, the same authors evidenced that the expression of most subunits of the 26S proteasome were enhanced up to three-fold in livers from mice treated with D3T. Subunit protein levels and proteasome activity were coordinately increased. However, no induction was seen in mice where the transcription factor NRF2 was disrupted. Promoter activity of the PSMB5 (20S) proteasome subunit increased with either NRF2 overexpression or treatment with activators in mouse embryonic fibroblasts, and AREs were identified in the proximal promoter of PSMB5 [85]. Pharmacological activation of NRF2 resulted in elevated expression levels of representative proteasome subunits (PSMA3, PSMA6, PSMB1 and PSMB5) only in non-senescent human fibroblasts containing functional NRF2 [86]. NRF2 activation during adaptation to oxidative stress results in high expression of the PSMB1 (20S) and PA28? subunits (or S11, proteasome regulator) [87]. Moreover, results from human embryonic stem cells revealed that NRF2 controls the expression of the proteasome maturation protein (POMP), a proteasome chaperone, which in turn modulates the proliferation of self-renewing human embryonic stem cells, three germ layer differentiation and cellular reprogramming [88]. All together, these studies indicate that NRF2 up-regulates the expression of key components of the UPS and therefore actively contributes to the clearance of proteins that otherwise would be toxic.

The NRF2-UPS Axis in Neurodegenerative Diseases

The role of the UPS in neurodegenerative diseases is a field of intensive debate. Initial studies reported decreased proteasome activity in human necropsies of patients affected from several neurodegenerative diseases. However, other studies employing in vitro and in vivo approaches found unchanged or even increased proteasome activity (reviewed in [89]). One possible explanation for this discrepancy is that the levels of the UPS components might change during disease progression and in different brain regions as is has been suggested for NRF2-targets.

Despite this controversy, it should be noted that up-regulation of ARE-containing proteasome genes will reinforce the UPS by increasing the clearance of toxic proteins in the brain. Indeed, ablation of NRF1, also modulator of the antioxidant response, in neuronal cells leads to impaired proteasome activity and neurodegeneration. Chromatin immunoprecipitation experiments and transcriptional analysis demonstrated that PSMB6 is regulated by NRF1. In addition, gene expression profiling led to the identification of NRF1 as a key transcriptional regulator of proteasome genes in neurons, suggesting that perturbations in NRF1 may contribute to the pathogenesis of neurodegenerative diseases [90]. Interestingly, NRF1 and its long isoform called TCF11 were shown to up-regulate ARE-containing proteasome genes upon proteasome inhibition in a feedback loop to compensate for reduced proteolytic activity [91], [92].

Regarding NRF2, there is a correlation among reduction of NRF2, RPT6 (19 S) and PSMB5 (20 S) levels in the midbrain of DJ-1-deficient mice treated with the neurotoxin paraquat [93]. Moreover, the naturally-occurring compound sulforaphane (SFN) gives a more robust image of NRF2 as a crucial modulator of the UPS. In vitro experiments with murine neuroblastoma Neuro2A cells evidenced an enhanced expression of the catalytic subunits of the proteasome, as well as its peptidase activities in response to SFN. This drug protected cells from hydrogen peroxide-mediated cytotoxicity and protein oxidation in a manner dependent on proteasome function [94]. In addition, Liu and co-workers employed a reporter mouse to monitor the UPS activity in response to SFN in the brain. These mice ubiquitously express the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation by the UPS (GFPu). In cerebral cortex, SFN reduced the level of GFPu with a parallel increase in chymotrypsin-like (PSMB5), caspase-like (PSMB2), and trypsin-like (PSMB1) activities of the 20 S proteasome. In addition, treatment of Huntington-derived cells with SFN revealed that NRF2 activation enhanced mHtt degradation and reduced mHtt cytotoxicity [95]. The major mechanism of SFN action is through induction of NRF2 [96]. The specific contribution of NRF2 should be addressed employing NRF2-null systems in further studies.

Functional Connection Between NRF2 and Macroautophagy

NRF2 Protein Levels are Modulated by the Adaptor Protein P62

Autophagy refers to the degradation of cytosolic components inside lysosomes. This process is used for the clearance of long-lived and misfolded proteins as well as damaged organelles. A direct link between NRF2 and autophagy was first observed in connection with the adaptor protein p62, also termed SQSTM1 [97], [98], [99], [100], [101]. This protein shuttles ubiquitinated proteins to the proteasomal and lysosomal degradation machineries and sequesters damaged proteins into aggregates prior to their degradation. P62 presents an ubiquitin-associated (UBA) domain, for binding to ubiquitinated proteins, and a LC3-interacting region (LIR) for integration with the autophagosomal membrane through the autophagy receptor LC3.

Although the p62-mediated induction of NRF2 and its target genes was first reported in 2007 [102], the molecular mechanism was not fully understood until the discovery of its interaction with KEAP1 [103], [98], [99], [100], [101]. Komatsu and coworkers identified a KEAP1 interacting region (KIR) in p62 that bound KEAP1 in the same basic surface pocket as NRF2 and with a binding affinity similar to the ETGE motif in NRF2, suggesting competition between p62 and NRF2. The phosphorylation of Ser351 in the KIR motif in p62 (349-DPSTGE-354) was shown to increase its affinity for KEAP1, competing with NRF2 binding and allowing its accumulation and transcriptional activation of its target genes [98], [99]. In fact, p62 overexpression led to reduced NRF2 ubiquitination and consequent stabilization as well as induction of its target genes [104]. Some kinases have been suggested to participate in p62 phosphorylation. The mammalian target of rapamycin complex 1 (mTORC1) may be implicated, as treatment with the mTOR inhibitor rapamycin suppressed the phosphorylation of p62 and the down-regulation of KEAP1 upon arsenite treatment. Recently, it was demonstrated that TGF-?-activated kinase 1 (TAK1) could also phosphorylate p62, enhancing KEAP1 degradation and NRF2-up-regulation. The authors of this study suggest this is a way to regulate cellular redoxtasis under steady-state conditions, as TAK1-deficiency up-regulates ROS in the absence of any exogenous oxidant in different mouse tissues in parallel with a reduction in NRF2 protein levels [105].

A p62 construct lacking the UBA domain was still capable of binding KEAP1, implying that the interaction did not depend on ubiquitinated KEAP1 [101]. However, the p62 homologue in Drosophila melanogaster, named Ref(2), does not contain a KIR motif and does not directly interact with DmKEAP1, although it can bind to ubiquitinated DmKEAP1 through the UBA domain. Moreover, DmKEAP1 can directly interact with Atg8 (homologue to mammalian LC3). KEAP1 deficiency results in Atg8 and autophagy induction dependent on the NRF2 orthologue CncC and independent on TFEB/MITF [106]. The relationship between NRF2 and autophagy seems to be conserved though, highlighting its functional relevance.

The induction of NRF2 by p62 is the result of both the competition to bind KEAP1 and degradation of KEAP1 in the lysosome. Silencing of p62 with siRNA doubled KEAP1 half-life in parallel with a decrease in NRF2 and its target genes [101]. In agreement, ablation of p62 expression evidenced increased levels of KEAP1 compared with wild type mice. Very relevant, the increment in KEAP1 levels was not affected by proteasome inhibitors but was reduced under starvation-inducing autophagy [107]. In fact, KEAP1 is present in mammalian cells in autophagic vesicles decorated with p62 and LC3 [99], [100], [103]. All these data suggest that KEAP1 is a substrate of the macroautophagy machinery, but this issue should be analyzed with more detail because of the existence of some controversial results. KEAP1 protein levels were increased in Atg7-null mice, a key effector of macroautophagy [107], but pharmacological inhibition of macroautophagy with torin1, E64/pepstatin or bafilomycin failed to accumulate KEAP1 [107], [100]. Overall, these results suggest that increased p62 levels sequester KEAP1 into autophagic vacuoles and probably these results in KEAP1 autophagic degradation allowing NRF2 activation (Fig. 3). Two different studies reported that the sulfinic acid reductases SESTRINS play an important role in this context. SESTRIN 2 interacts with p62, KEAP1 and RBX1 and facilitates p62-dependent degradation of KEAP1 and NRF2 activation of target genes [108]. Another study showed that SESTRIN 2 interacted with ULK1 and p62, promoting phosphorylation of p62 at Ser403 which facilitated degradation of cargo proteins including KEAP1 [109].

**Figure 3 NRF2 levels are regulated by the adaptor protein p62.** The phosphorylation of Ser 351 in the KIR motif of p62 (349-DPSTGE-354) by mTORC1, TAK1 or other kinases results in increased affinity for binding to KEAP1 due to resemblance to the ETGE motif in NRF2. As a consequence, phosphorylated p62 displaces NRF2 and binds KEAP1. The LIR motif in p62 enables interaction with LC3 in the autophagosomal membrane, so that p62-KEAP1 complex is eventually degraded in the lysosome. As a consequence NRF2 is able to accumulate, translocate to the nucleus and increase the transcription of ARE-containing genes, including p62. This regulatory mechanism provides a perdurable NRF2 response, as KEAP1 has to be newly synthesized in order to inhibit NRF2 activity.

Modulation of Macroautophagy Genes by NRF2

NRF2 regulates the expression of relevant genes for macroautophagy as well as it does for the UPR and the UPS. The first evidence came from studies in which p62 expression was shown to be induced upon exposure to electrophiles, ROS and nitric oxide [110], [111], [112]. The mechanism of induction was described some years later with the finding that p62 contains a functional ARE in its gene promoter [99]. In a recent study, several other functional AREs were found and validated following bioinformatics analysis and ChIP assays. Moreover, mouse embryonic fibroblasts and cortical neurons from Nrf2-knockout mice exhibited reduced p62 expression, which could be rescued with an NRF2-expressing lentivirus. Similarly, NRF2 deficiency reduced p62 levels in injured neurons from mice hippocampus [36]. Therefore, it has been suggested that NRF2 activation increases p62 levels, resulting in KEAP1 degradation and favoring further NRF2 stabilization in a positive feedback loop. This non-canonical mechanism of NRF2 induction requires changes in gene expression and might be a relevant response to prolonged cellular stress.

The cargo recognition protein NDP52 was shown to be transcriptionally regulated by NRF2. NDP52 works in a similar way to p62, recognizing ubiquitinated proteins and interacting with LC3 through a LIR domain, so that cargoes are degraded in lysosomes. Five putative AREs were found in Ndp52 promoter DNA sequence. Three of them were identified with different mutant constructs and ChIP assays as indispensable for NRF2-mediated Ndp52 transcription [113]. Of note, Ndp52 mRNA levels were reduced in the hippocampus of Nrf2-knockout mice. One of these sequences was also validated in an independent study as an NRF2-regulated ARE [36].

However, the role of NRF2 in the modulation of autophagy is not limited to the induction of these two cargo-recognition proteins. In order to gain deeper insight in the role of NRF2 in the modulation of additional autophagy-related genes, our group screened the chromatin immunoprecipitation database ENCODE for two proteins, MAFK and BACH1, which bind the NRF2-regulated AREs. Using a script generated from the JASPAR’s consensus ARE sequence, we identified several putative AREs in many autophagy genes. Twelve of these sequences were validated as NRF2 regulated AREs in nine autophagy genes, whose expression was diminished in mouse embryo fibroblasts of Nrf2-knockout mice but could be restored by an NRF2-expressing lentivirus. Our study demonstrated that NRF2 activates the expression of some genes involved in different steps of the autophagic process, including autophagy initiation (ULK1), cargo recognition (p62 and NDP52), autophagosome formation (ATG4D, ATG7 and GABARAPL1), elongation (ATG2B and ATG5), and autolysosome clearance (ATG4D). Consequently, autophagy flux in response to hydrogen peroxide was impaired when NRF2 was absent [36].

Relevance of NRF2-Mediated Macroautophagy Genes Expression in Neurodegenerative Disorders

Defective autophagy has been shown to play an important role in several neurodegenerative diseases [114] and ablation of autophagy leads to neurodegeneration in mice [115], [116]. Atg7-knockout mice revealed that autophagy deficiency results in p62 accumulation in ubiquitin-positive inclusion bodies. KEAP1 was sequestered in these inclusion bodies, leading to NRF2 stabilization and induction of target genes [103]. Importantly, excessive accumulation of p62 together with ubiquitinated proteins has been identified in neurodegenerative diseases, including AD, PD and ALS [117]. In fact, neurons expressing high levels of APP or TAU of AD patients also expressed p62 and nuclear NRF2, suggesting their attempt to degrade intraneuronal aggregates through autophagy [36].

NRF2 deficiency aggravates protein aggregation in the context of AD. In fact, increased levels of phosphorylated and sarkosyl-insoluble TAU are found in Nrf2-knockout mice, although no difference in kinase or phosphatase activities could be detected comparing with the wild-type background [113]. Importantly, NDP52 was demonstrated to co-localize with TAU in murine neurons and direct interaction between phospho-TAU and NDP52 was shown by co-immunoprecipitation experiments both in mice and AD samples, pointing to its role in TAU degradation. Interestingly, silencing of NDP52, p62 or NRF2 in neurons resulted in increased phospho-TAU [113], [118]. Moreover, increased intraneuronal APP aggregates were found in the hippocampus of APP/PS1?E9 mice when NRF2 was absent. This correlated with altered autophagy markers, including increased phospho-mTOR/mTOR and phospho-p70S6k/p70S6k ratios (indicative of autophagy inhibition), augmented levels of pre-cathepsin D and a larger number of multivesicular bodies [119]. In mice co-expressing human APP (V717I) and TAU (P301L), NRF2 deficiency led to increased levels of total and phospho-TAU in the insoluble fraction and increased intraneuronal APP aggregates, together with reduced neuronal levels of p62, NDP52, ULK1, ATG5 and GABARAPL1. Co-localization between the adaptor protein p62 and APP or TAU was reduced in the absence of NRF2 [36]. Overall, these results highlight the importance of NRF2 in neuronal autophagy.

Different Transcription Factors Act Coordinately to Modulate Proteostasis

Under steady state conditions, proteostasis is controlled via protein-protein interactions and post-translational modifications obtaining a rapid response. However, cellular adaptation requires the transcriptional regulation of the UPR, UPS and autophagy genes. Considering that nerve cells are continuously submitted to low-grade toxic insults, including oxidative and proteotoxic stress, a reinforcement of proteostasis induced by transcriptional modulation might help preventing brain degeneration.

In the case of the UPR, the activation of each of the three arms will finally result in the transcriptional induction of certain genes (reviewed in [43]). For instance, an ATF6-derived fragment (ATF6f) binds to ER-stress response elements (ERSE) and induces the expression of several genes, including XBPI, BIP and CHOP. In addition, PERK signaling leads to the activation of the transcription factor ATF4, which controls the expression of multiple UPR-related genes and some others including the NRF2 target genes Hmox1 and p62. Finally, IRE1 activation results in the generation of an active transcription factor, spliced XBP1 (XBP1s), which controls the transcription of genes encoding proteins involved in protein folding.

On the other hand, NRF1 was shown to be necessary for proteasomal gene expression in the brain, as Nrf1-knockout mice exhibited reduced expression of genes encoding various subunits of the 20S core, as well the 19S regulatory complex together with impaired proteasomal function [90]. Both NRF1 and NRF2 bind to ARE sequences in the promoter regions of its target genes, which suggests they have overlapping transcriptional activities, although they differ in their regulatory mechanisms and cellular localization [120].

Transcription factors of the Forkhead box O (FOXO) family control the expression of multiple autophagy-related genes. Similar to what occurs with NRF2, there are multiple layers of regulation of the activity of FOXO members, which can be induced upon nutritional or oxidative stress [121]. Finally, the transcription factor TFEB, considered the master regulator of lysosomal biogenesis, plays a crucial role in regulation of autophagy under nutritional stress conditions. Thus, inhibition of mTORC1 leads to nuclear translocation of TFEB and induction of the expression of autophagy genes [122].

Overall, the existence of different transcriptional regulators of these machineries also suggests crosstalk and partially redundant mechanisms that may assure proteostasis under different circumstances. Accordingly, NRF2 may have a relevant role in tissues that support high levels of oxidative stress. For instance, oxidative stress-induced NRF2 may function under nutrient-rich conditions to transcriptionally up-regulate autophagy, similar to what has been found for TFEB under starvation conditions. Moreover, the brain functions largely under nutrient-rich conditions, posing NRF2 as a relevant mechanism to activate autophagy in neurons.

Promising�Therapeutic Potential for NRF2 in Proteinopathies

In the past few years, a great progress has been made in the knowledge of the regulatory roles of the UPR, UPS and autophagy on NRF2 activity, as well as the reciprocal NRF2-mediated transcription of components of these three systems. Therefore, new therapeutic possibilities may arise based on the exploitation of NRF2 as a crucial regulator of protein clearance in neurodegenerative diseases.

However, a key remaining question is whether it will be useful or deleterious to increase NRF2 levels in brain. Analysis of epidemiological data may provide a partial answer, as it indicates that the NFE2L2 gene is highly polymorphic and some single nucleotide polymorphisms found in its promoter regulatory region may provide a range of �physiological� variability in gene expression at the population level and some haplotypes were associated with decreased risk and/or delayed onset of AD, PD or ALS [123]. Moreover, as discussed by Hayes and colleagues [124], NRF2 effect might have an U-shaped response, meaning that too low NRF2 levels may result in a loss of cytoprotection and increased susceptibility to stressors, while too much NRF2 might disturb homeostatic balance towards a reductive scenario (reductive stress), which would favor protein misfolding and aggregation. Low NRF2 levels in the brain support the idea that a slight up-regulation may be sufficient to achieve a benefit under pathological conditions. In fact, the protective role of pharmacological NRF2-mediated activation of protein clearance has been shown in different neurodegeneration cell culture and in vivo models.

SFN is a pharmacological NRF2 activator that was demonstrated to induce proteasomal and autophagy gene expression [95], [36]. Interestingly, Jo and colleagues demonstrated that SFN reduced the levels of phosphorylated TAU and increased Beclin-1 and LC3-II, suggesting NRF2 activation may facilitate degradation of this toxic protein through autophagy [113]. Moreover, degradation of mHtt was enhanced with SFN, and this was reverted with the use of MG132, indicating proteasomal degradation of this toxic protein [95]. Autophagy-mediated degradation of phospho- and insoluble-TAU was reported with the organic flavonoid fisetin. This compound was able to induce autophagy by simultaneously promoting the activation and nuclear translocation of both TFEB and NRF2, along with some of its target genes. This response was prevented by TFEB or NRF2 silencing [125]. Bott and colleagues reported beneficial effects of a simultaneous NRF2, NRF1 and HSF1 activator on protein toxicity in spinal and bulbar muscular atrophy, a neurodegenerative disorder caused by expansion of polyglutamine-encoding CAG repeats in which protein aggregates are present [126]. The potential of NRF2 activation for the treatment of neurodegenerative disorders has been demonstrated with the approval of BG-12, the oral formulation of the NRF2 inducer dimethyl fumarate (DMF), for the treatment of multiple sclerosis [127], [128]. The success of DMF with autoimmune diseases with a strong inflammatory component suggests that neurodegenerative diseases might benefit from repositioning this drug. In a recent preclinical study of an ?-synucleinopathy model of PD, DMF was shown to be neuroprotective due, in part, to its induction of autophagy [129]. Studies reporting beneficial effects of NRF2 on neurodegeneration but not focusing on its effect on protein clearance are even more abundant (for a comprehensive review, see [7]). This is quite relevant, as it highlights the multiple damaging processes that can be simultaneously targeted by a single hit in NRF2, also including oxidative stress, neuroinflammation or mitochondrial dysfunction. However, future work will be needed to definitely determine if pharmacological activation of NRF2 may be a valid strategy to facilitate degradation of toxic proteins in the brain.

As explained before, exacerbated GSK-3? activity was reported in neurodegenerative diseases and it has been speculated that consequent NRF2 reduction can be partially responsible for the deleterious outcome. Under these pathological conditions, GSK-3 inhibitors could also cooperate to increase NRF2 levels and proteostasis. The beneficial effects of GSK-3 inhibitors have been reported in different models of neurodegeneration and, more interesting, GSK-3 repression was shown to reduce the levels of toxic proteins [130], [131], [132], [133]. Although no direct links between GSK-3 inhibition and NRF2-transcriptional regulation of genes promoting proteostasis have been observed yet, it is reasonable to speculate that down-regulation of GSK-3 activity would result in increased NRF2 levels, which eventually will result in reinforced proteostasis.

The transcriptional activity of NRF2 as well as the cellular capacity to maintain proteostasis decrease with age, the main risk factor for the development of neurodegenerative diseases. It is reasonable to think that the reinforcement of NRF2 and, consequently, proteostasis would, at least, delay the accumulation of protein aggregates and neurodegeneration. Indeed, treatment of human senescent fibroblasts with 18?-glycyrrhetinic acid (18?-GA) triterpenoid promoted NRF2 activation, leading to proteasome induction and enhanced life span. This study suggests that pharmacological activation of NRF2 is possible even in late life [86]. Moreover, a later study showed that this compound mediated SKN-1 and proteasome activation in C.elegans with beneficial effects on AD progression in relevant nematode models [134].

All things considered, NRF2-mediated induction of proteostasis-related genes seems to be beneficial in different proteinopathies.

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Isothiocyanates are some of the most important plant compounds you can get in your diet. In this video I make the most comprehensive case for them that has ever been made. Short attention span? Skip to your favorite topic by clicking one of the time points below. Full timeline below.

Key sections:

00:01:14 – Cancer and mortality
00:19:04 – Aging
00:26:30 – Brain and behavior
00:38:06 – Final recap
00:40:27 – Dose

Full timeline:

00:00:34 – Introduction of sulforaphane, a major focus of the video.
00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
00:02:12 – Prostate cancer risk.
00:02:23 – Bladder cancer risk.
00:02:34 – Lung cancer in smokers risk.
00:02:48 – Breast cancer risk.
00:03:13 – Hypothetical: what if you already have cancer? (interventional)
00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
00:04:38 – Sulforaphane and cancer.
00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
00:08:32 – Inhibition of breast cancer stem cells.
00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
00:19:04 – Beginning of aging section.
00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
00:20:34 – Importance of low inflammation for longevity.
00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
00:23:40 – Mid-video recap: cancer, aging sections
00:24:14 – Mouse studies suggest sulforaphane might improve adaptive immune function in old age.
00:25:18 – Sulforaphane improved hair growth in a mouse model of balding. Picture at 00:26:10.
00:26:30 – Beginning of brain and behavior section.
00:27:18 – Effect of broccoli sprout extract on autism.
00:27:48 – Effect of glucoraphanin on schizophrenia.
00:28:17 – Start of depression discussion (plausible mechanism and studies).
00:31:21 – Mouse study using 10 different models of stress-induced depression show sulforaphane similarly effective as fluoxetine (prozac).
00:32:00 – Study shows direct ingestion of glucoraphanin in mice is similarly effective at preventing depression from social defeat stress model.
00:33:01 – Beginning of neurodegeneration section.
00:33:30 – Sulforaphane and Alzheimer’s disease.
00:33:44 – Sulforaphane and Parkinson’s disease.
00:33:51 – Sulforaphane and Hungtington’s disease.
00:34:13 – Sulforaphane increases heat shock proteins.
00:34:43 – Beginning of traumatic brain injury section.
00:35:01 – Sulforaphane injected immediately after TBI improves memory (mouse study).
00:35:55 – Sulforaphane and neuronal plasticity.
00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
00:37:19 – Sulforaphane and duchenne muscular dystrophy.
00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
00:41:01 – Anecdotes on sprouting at home.
00:43:14 – On cooking temperatures and sulforaphane activity.
00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
00:44:56 – Cooking techniques and cruciferous vegetables.
00:46:06 – Isothiocyanates as goitrogens.

The nuclear factor erythroid-derived 2 (NF-E2)-related factor 2, otherwise known as Nrf2, is a transcription factor which regulates the expression of a variety of antioxidant and detoxifying enzymes. Research studies have also demonstrated its role in controlling oxidative stress. Most neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, are characterized by oxidative stress and chronic inflammation, the common targets of Nrf2 treatment approaches. Dr. Alex Jimenez D.C., C.C.S.T. Insight

Concluding Remarks

Transcription factor NRF2 orchestrates a proteostatic response by sensing to and modulating changes in the UPR, UPS and autophagy (Fig. 4). Consequently, the lack of NRF2 has been shown to aggravate proteinopathy, suggesting that NRF2 is necessary for optimal protein clearance. All together, we can speculate that NRF2 might be an interesting therapeutic target for proteinopathies.

**Figure 4 NRF2 as a hub connecting proteotoxic-derived emergency signals to a protective transcriptional response.** The accumulation of unfolded/misfolded proteins will lead to the activation of the unfolded protein response (UPR) in the ER. Activation of PERK or MAPK may result in the transcriptional induction of the ER-resident Gpx8 and several enzymes regulating GSH levels, critical to ensure correct protein folding. Protein aggregates inhibit proteasome activity (UPS), probably avoiding NRF2 degradation. NRF2 has been shown to specifically modulate the transcription of Psma3, Psma6, Psmb1, Psmb5 and Pomp genes. Several other subunits were up-regulated in an NRF2-dependent manner in response to D3T, probably enlarging the list of proteasome subunits regulated by NRF2. Autophagy is the main pathway for the degradation of protein aggregates. Autophagy also regulates NRF2, connecting this degradation pathway with NRF2 transcriptional induction of p62, Ndp52, Ulk1, Atg2b, Atg4c, Atg5, Atg7 and Gabarapl1.

Acknowledgements

Sciencedirect.com/science/article/pii/S2213231716304050

According to the article above, while the symptoms of neurodegenerative diseases can be treated through a variety of treatment options, research studies have demonstrated that Nrf2 activation can be a helpful treatment approach. Because Nrf2 activators target broad mechanisms of disease, all neurodegenerative diseases can benefit from the use of the Nrf2 transcription factor. The findings of Nrf2 have revolutionized the treatment of neurodegenerative diseases. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.

Curated by Dr. Alex Jimenez

Referenced from:�Sciencedirect.com

Additional Topic Discussion: Relieving Knee Pain without Surgery

Knee pain is a well-known symptom which can occur due to a variety of knee injuries and/or conditions, including�sports injuries. The knee is one of the most complex joints in the human body as it is made-up of the intersection of four bones, four ligaments, various tendons, two menisci, and cartilage. According to the American Academy of Family Physicians, the most common causes of knee pain include patellar subluxation, patellar tendinitis or jumper’s knee, and Osgood-Schlatter disease. Although knee pain is most likely to occur in people over 60 years old, knee pain can also occur in children and adolescents. Knee pain can be treated at home following the RICE methods, however, severe knee injuries may require immediate medical attention, including chiropractic care. �

EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

***

Nrf2 Explained: The Keap1-Nrf2 Pathway

by Dr Alex Jimenez | Cancer Health, Detoxification, Functional Medicine, Health, Holistic Medicine, Natural Health, Nutrition, Oxidative Stress, Research Studies, Supplements, Treatments, Wellness

Oxidative stress is described as cell damage caused by free radicals, or unstable molecules, which can ultimately affect healthy function. The human body creates free radicals to neutralize bacteria and viruses, however, external factors, such as oxygen, pollution, and radiation, can often also produce free radicals. Oxidative stress has been associated with numerous health issues.

Oxidative stress and other stressors turn on internal protective mechanisms which can help regulate the human body’s antioxidant response. Nrf2 is a protein which senses levels of oxidative stress and enables the cells to protect themselves from internal and external factors. Nrf2 has also been demonstrated to help regulate genes involved in the production of antioxidant enzymes and stress-response genes. The purpose of the article below is to explain the effects of Nrf2 in cancer.

Abstract

The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. Although cell signaling pathways triggered by the transcription factor Nrf2 prevent cancer initiation and progression in normal and premalignant tissues, in fully malignant cells Nrf2 activity provides growth advantage by increasing cancer chemoresistance and enhancing tumor cell growth. In this graphical review, we provide an overview of the Keap1-Nrf2 pathway and its dysregulation in cancer cells. We also briefly summarize the consequences of constitutive Nrf2 activation in cancer cells and how this can be exploited in cancer gene therapy.

Keywords: Nrf2, Keap1, Cancer, Antioxidant response element, Gene therapy

Introduction

The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to endogenous and exogenous stresses caused by reactive oxygen species (ROS) and electrophiles [1]. The key signaling proteins within the pathway are the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) that binds together with small Maf proteins to the antioxidant response element (ARE) in the regulatory regions of target genes, and Keap1 (Kelch ECH associating protein 1), a repressor protein that binds to Nrf2 and promotes its degradation by the ubiquitin proteasome pathway (Fig. 1). Keap1 is a very cysteine-rich protein, mouse Keap1 having a total of 25 and human 27 cysteine residues, most of which can be modified in vitro by different oxidants and electrophiles [2]. Three of these residues, C151, C273 and C288, have been shown to play a functional role by altering the conformation of Keap1 leading to nuclear translocation of Nrf2 and subsequent target gene expression [3] (Fig. 1). The exact mechanism whereby cysteine modifications in Keap1 lead to Nrf2 activation is not known, but the two prevailing but not mutually exclusive models are (1) the �hinge and latch� model, in which Keap1 modifications in thiol residues residing in the IVR of Keap1 disrupt the interaction with Nrf2 causing a misalignment of the lysine residues within Nrf2 that can no longer be polyubiquitinylated and (2) the model in which thiol modification causes dissociation of Cul3 from Keap1 [3]. In both models, the inducer-modified and Nrf2-bound Keap1 is inactivated and, consequently, newly synthesized Nrf2 proteins bypass Keap1 and translocate into the nucleus, bind to the ARE and drive the expression of Nrf2 target genes such as NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HMOX1), glutamate-cysteine ligase (GCL) and glutathione S transferases (GSTs) (Fig. 2). In addition to modifications of Keap1 thiols resulting in Nrf2 target gene induction, proteins such as p21 and p62 can bind to Nrf2 or Keap1 thereby disrupting the interaction between Nrf2 and Keap1 [1], [3] (Fig. 3).

Fig. 1. Structures of Nrf2 and Keap1 and the cysteine code. (A) Nrf2 consists of 589 amino acids and has six evolutionarily highly conserved domains, Neh1-6. Neh1 contains a bZip motif, a basic region � leucine zipper (L-Zip) structure, where the basic region is responsible for DNA recognition and the L-Zip mediates dimerization with small Maf proteins. Neh6 functions as a degron to mediate degradation of Nrf2 in the nucleus. Neh4 and 5 are transactivation domains. Neh2 contains ETGE and DLG motifs, which are required for the interaction with Keap1, and a hydrophilic region of lysine residues (7 K), which are indispensable for the Keap1-dependent polyubiquitination and degradation of Nrf2. (B) Keap1 consists of 624 amino acid residues and has five domains. The two protein�protein interaction motifs, the BTB domain and the Kelch domain, are separated by the intervening region (IVR). The BTB domain together with the N-terminal portion of the IVR mediates homodimerization of Keap1 and binding with Cullin3 (Cul3). The Kelch domain and the C-terminal region mediate the interaction with Neh2. (C) Nrf2 interacts with two molecules of Keap1 through its Neh2 ETGE and DLG motifs. Both ETGE and DLG bind to similar sites on the bottom surface of the Keap1 Kelch motif. (D) Keap1 is rich in cysteine residues, with 27 cysteines in human protein. Some of these cysteines are located near basic residues and are therefore excellent targets of electrophiles and oxidants. The modification pattern of the cysteine residues by electrophiles is known as the cysteine code. The cysteine code hypothesis proposes that structurally different Nrf2 activating agents affect different Keap1 cysteines. The cysteine modifications lead to conformational changes in the Keap1 disrupting the interaction between the Nrf2 DLG and Keap1 Kelch domains, thus inhibiting the polyubiquitination of Nrf2. The functional importance of Cys151, Cys273 and Cys288 has been shown, as Cys273 and Cys288 are required for suppression of Nrf2 and Cys151 for activation of Nrf2 by inducers [1], [3].

Fig. 2. The Nrf2-Keap1 signaling pathway. (A and B) in basal conditions, two Keap1 molecules bind to Nrf2 and Nrf2 is polyubiquitylated by the Cul3-based E3 ligase complex. This polyubiquitilation results in rapid Nrf2 degradation by the proteasome. A small proportion of Nrf2 escapes the inhibitory complex and accumulates in the nucleus to mediate basal ARE-dependent gene expression, thereby maintaining the cellular homeostasis. (C) Under stress conditions, inducers modify the Keap1 cysteines leading to the inhibition of Nrf2 ubiquitylation via dissociation of the inhibitory complex. (D) According to the hinge and latch model, modification of specific Keap1 cysteine residues leads to conformational changes in Keap1 resulting in the detachment of the Nrf2 DLG motif from Keap1. Ubiquitination of Nrf2 is disrupted but the binding with the ETGE motif remains. (E) In the Keap1-Cul3 dissociation model, the binding of Keap1 and Cul3 is disrupted in response to electrophiles, leading to the escape of Nrf2 from the ubiquitination system. In both of the suggested models, the inducer-modified and Nrf2-bound Keap1 is inactivated and, consequently, newly synthesized Nrf2 proteins bypass Keap1 and translocate into the nucleus, bind to the Antioxidant Response Element (ARE) and drive the expression of Nrf2 target genes such as NQO1, HMOX1, GCL and GSTs [1], [3].

Fig. 3. Mechanisms for constitutive nuclear accumulation of Nrf2 in cancer. (A) Somatic mutations in Nrf2 or Keap1 disrupt the interaction of these two proteins. In Nrf2, mutations affect ETGE and DLG motifs, but in Keap1 mutations are more evenly distributed. Furthermore, oncogene activation, such as KrasG12D[5], or disruption of tumor suppressors, such as PTEN [11] can lead to transcriptional induction of Nrf2 and an increase in nuclear Nrf2. (B) Hypermethylation of the Keap1 promoter in lung and prostate cancer leads to reduction of Keap1 mRNA expression, which increases the nuclear accumulation of Nrf2 [6], [7]. (C) In familial papillary renal carcinoma, the loss of fumarate hydratase enzyme activity leads to the accumulation of fumarate and further to succination of Keap1 cysteine residues (2SC). This post-translational modification leads to the disruption of Keap1-Nrf2 interaction and nuclear accumulation of Nrf2 [8], [9]. (D) Accumulation of disruptor proteins such as p62 and p21 can disturb Nrf2-Keap1 binding and results in an increase in nuclear Nrf2. p62 binds to Keap1 overlapping the binding pocket for Nrf2 and p21 directly interacts with the DLG and ETGE motifs of Nrf2, thereby competing with Keap1 [10].

Mechanisms of Activation and Dysregulation of Nrf2 in Cancer

Although cytoprotection provided by Nrf2 activation is important for cancer chemoprevention in normal and premalignant tissues, in fully malignant cells Nrf2 activity provides growth advantage by increasing cancer chemoresistance and enhancing tumor cell growth [4]. Several mechanisms by which Nrf2 signaling pathway is constitutively activated in various cancers have been described: (1) somatic mutations in Keap1 or the Keap1 binding domain of Nrf2 disrupting their interaction; (2) epigenetic silencing of Keap1 expression leading to defective repression of Nrf2; (3) accumulation of disruptor proteins such as p62 leading to dissociation of the Keap1-Nrf2 complex; (4) transcriptional induction of Nrf2 by oncogenic K-Ras, B-Raf and c-Myc; and (5) post-translational modification of Keap1 cysteines by succinylation that occurs in familial papillary renal carcinoma due to the loss of fumarate hydratase enzyme activity [3], [4], [5], [6], [7], [8], [9], [10] (Fig. 3). Constitutively abundant Nrf2 protein causes increased expression of genes involved in drug metabolism thereby increasing the resistance to chemotherapeutic drugs and radiotherapy. In addition, high Nrf2 protein level is associated with poor prognosis in cancer [4]. Overactive Nrf2 also affects cell proliferation by directing glucose and glutamine towards anabolic pathways augmenting purine synthesis and influencing the pentose phosphate pathway to promote cell proliferation [11] (Fig. 4).

Fig. 4. The dual role of Nrf2 in tumorigenesis. Under physiological conditions, low levels of nuclear Nrf2 are sufficient for the maintenance of cellular homeostasis. Nrf2 inhibits tumor initiation and cancer metastasis by eliminating carcinogens, ROS and other DNA-damaging agents. During tumorigenesis, accumulating DNA damage leads to constitutive hyperactivity of Nrf2 which helps the autonomous malignant cells to endure high levels of endogenous ROS and to avoid apoptosis. Persistently elevated nuclear Nrf2 levels activate metabolic genes in addition to the cytoprotective genes contributing to metabolic reprogramming and enhanced cell proliferation. Cancers with high Nrf2 levels are associated with poor prognosis because of radio and chemoresistance and aggressive cancer cell proliferation. Thus, Nrf2 pathway activity is protective in the early stages of tumorigenesis, but detrimental in the later stages. Therefore, for the prevention of cancer, enhancing Nrf2 activity remains an important approach whereas for the treatment of cancer, Nrf2 inhibition is desirable [4], [11].

Given that high Nrf2 activity commonly occurs in cancer cells with adverse outcomes, there is a need for therapies to inhibit Nrf2. Unfortunately, due to structural similarity with some other bZip family members, the development of specific Nrf2 inhibitors is a challenging task and only a few studies of Nrf2 inhibition have been published to date. By screening natural products, Ren et al. [12] identified an antineoplastic compound brusatol as an Nrf2 inhibitor that enhances the chemotherapeutic efficacy of cisplatin. In addition, PI3K inhibitors [11], [13] and Nrf2 siRNA [14] have been used to inhibit Nrf2 in cancer cells. Recently, we have utilized an alternative approach, known as cancer suicide gene therapy, to target cancer cells with high Nrf2 levels. Nrf2-driven lentiviral vectors [15] containing thymidine kinase (TK) are transferred into cancer cells with high ARE activity and the cells are treated with a pro-drug, ganciclovir (GCV). GCV is metabolized to GCV-monophosphate, which is further phosphorylated by cellular kinases into a toxic triphosphate form [16] (Fig. 5). This leads to effective killing of not only TK containing tumor cells, but also the neighboring cells due to the bystander effect [17]. ARE-regulated TK/GCV gene therapy can be further enhanced via combining a cancer chemotherapeutic agent doxorubicin to the treatment [16], supporting the notion that this approach could be useful in conjuction with traditional therapies.

Fig. 5. Suicide gene therapy. Constitutive Nrf2 nuclear accumulation in cancer cells can be exploited by using Nrf2-driven viral vector for cancer suicide gene therapy [16]. In this approach, lentiviral vector (LV) expressing thymidine kinase (TK) under minimal SV40 promoter with four AREs is transduced to lung adenocarcinoma cells. High nuclear Nrf2 levels lead to robust expression of TK through Nrf2 binding. Cells are then treated with a pro-drug, ganciclovir (GCV), which is phosphorylated by TK. Triphosphorylated GCV disrupts DNA synthesis and leads to effective killing of not only TK containing tumor cells, but also the neighboring cells due to the bystander effect.

Nrf2 is a master regulator which triggers the production of powerful antioxidants in the human body which help eliminate oxidative stress. Various antioxidant enzymes, such as superoxide dismutase, or SOD, glutathione, and catalase, are also activated through the Nrf2 pathway. Furthermore, certain phytochemicals like turmeric, ashwagandha, bacopa, green tea, and milk thistle, activate Nrf2. Research studies have found that Nrf2 activation can naturally enhance cellular protection and restore balance to the human body.

Dr. Alex Jimenez D.C., C.C.S.T. Insight

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Key sections:

00:01:14 – Cancer and mortality
00:19:04 – Aging
00:26:30 – Brain and behavior
00:38:06 – Final recap
00:40:27 – Dose

Full timeline:

00:00:34 – Introduction of sulforaphane, a major focus of the video.
00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
00:02:12 – Prostate cancer risk.
00:02:23 – Bladder cancer risk.
00:02:34 – Lung cancer in smokers risk.
00:02:48 – Breast cancer risk.
00:03:13 – Hypothetical: what if you already have cancer? (interventional)
00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
00:04:38 – Sulforaphane and cancer.
00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
00:08:32 – Inhibition of breast cancer stem cells.
00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
00:19:04 – Beginning of aging section.
00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
00:20:34 – Importance of low inflammation for longevity.
00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
00:23:40 – Mid-video recap: cancer, aging sections
00:24:14 – Mouse studies suggest sulforaphane might improve adaptive immune function in old age.
00:25:18 – Sulforaphane improved hair growth in a mouse model of balding. Picture at 00:26:10.
00:26:30 – Beginning of brain and behavior section.
00:27:18 – Effect of broccoli sprout extract on autism.
00:27:48 – Effect of glucoraphanin on schizophrenia.
00:28:17 – Start of depression discussion (plausible mechanism and studies).
00:31:21 – Mouse study using 10 different models of stress-induced depression show sulforaphane similarly effective as fluoxetine (prozac).
00:32:00 – Study shows direct ingestion of glucoraphanin in mice is similarly effective at preventing depression from social defeat stress model.
00:33:01 – Beginning of neurodegeneration section.
00:33:30 – Sulforaphane and Alzheimer’s disease.
00:33:44 – Sulforaphane and Parkinson’s disease.
00:33:51 – Sulforaphane and Hungtington’s disease.
00:34:13 – Sulforaphane increases heat shock proteins.
00:34:43 – Beginning of traumatic brain injury section.
00:35:01 – Sulforaphane injected immediately after TBI improves memory (mouse study).
00:35:55 – Sulforaphane and neuronal plasticity.
00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
00:37:19 – Sulforaphane and duchenne muscular dystrophy.
00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
00:41:01 – Anecdotes on sprouting at home.
00:43:14 – On cooking temperatures and sulforaphane activity.
00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
00:44:56 – Cooking techniques and cruciferous vegetables.
00:46:06 – Isothiocyanates as goitrogens.

Acknowledgments

This work was supported by the Academy of Finland, the Sigrid Juselius Foundation and the Finnish Cancer Organisations.

In conclusion, nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or Nrf2, is a protein which increases the production of antioxidants which protect the human body against oxidative stress. As described above, the stimulation of the Nrf2 pathway are being studies for the treatment of diseases caused by oxidative stress, including cancer. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.

Curated by Dr. Alex Jimenez

Referenced from:�Sciencedirect.com

Additional Topic Discussion: Relieving Knee Pain without Surgery

EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

***

An Integrative Holistic Approach To Migraine Headaches

by Dr Alex Jimenez | Headaches & Treatments, Holistic Medicine, Integrative Functional Wellness

Holistic: Migraine headaches are typically debilitating, and require a comprehensive approach for successful treatment. It is helpful to consider migraine headache as a symptom of an underlying imbalance, rather than simply a diagnosis. A holistic approach is a satisfying way to think about and treat migraine headache. Physicians trained in this approach will consider a broad array of features that may contribute to the experience of migraine headache, including disturbances within the following key areas:

Nutrition
Digestion
Detoxification
Energy production
Endocrine function
Immune system function/inflammation
Structural function
Mind-body health

Migraine headache is an excellent example of biologic uniqueness; the underlying factors participating in each individual�s outcome may differ quite a bit from person to person. The journey of identifying and addressing these factors often results in an impressive improvement in frequency and intensity of the expression of migraine. Committed individuals will find the added benefit of better general health along the way.

Nutritional Considerations: Holisitic

Food Allergy/Intolerance

Numerous well-designed studies have demonstrated that detection and removal of foods not tolerated will greatly reduce or eliminate migraine manifestations. True allergy may not be associated with migraine in most individuals, but food intolerance is more common. Migraine frequency and intensity have been demonstrated to respond well to elimination diets, in which commonly offending foods are removed for several weeks. Elimination diets are easy to perform (although they do require a high degree of commitment and education), and can help in identifying foods that are mismatched to an individual. The majority of patients who undergo an elimination diet learn that their diets were contributing to chronic symptoms, and they typically feel much better during the elimination phase. Common foods that act as migraine triggers include: chocolate, cow�s milk, wheat/gluten grains, eggs, nuts, and corn. In children specifically, common migraine triggers include cheese, chocolate, citrus fruits, hot dogs, monosodium glutamate, aspartame, fatty foods, ice cream, caffeine withdrawal, and alcoholic drinks, especially red wine and beer.

There are several methods which may be used to detect food allergies. Laboratory testing can be convenient, but is not always a reliable means of detecting food intolerance. (See Summary of Recommendations for information on how to implement the elimination diet).

Foods such as chocolate, cheese, beer, and red wine are believed to cause migraine through the effect of �vasoactive amines� such as tyramine and beta-phenylethylamine. These foods also contain histamine. Individuals who are sensitive to dietary histamine seem to have lower levels of diamine oxidase, the vitamin B6-dependent enzyme that metabolizes histamine in the small bowel. The use of vitamin B6 improves histamine tolerance in some individuals, presumably by enhancing the activity of this enzyme.

Other diet-related triggers associated with migraine headache include: glucose/insulin imbalances, excessive salt intake, and lactose intolerance. Aspartame, commonly used as a sweetener, may also trigger migraines. Each of these factors may be readily avoided by adopting more conscious eating habits, and by carefully reading labels.

Magnesium

An estimated 75% of people consuming the standard American diet (SAD) are not getting adequate magnesium, and it is felt to represent one of the most common micronutrient deficiencies, manifested by a diverse range of problems. Though many elements can contribute to magnesium depletion, stress is among them, and both acute and chronic stress are associated with increased episodes of migraine. Daily doses of magnesium should be first line considerations for migraine sufferers (caution if kidney function is impaired), and intravenous magnesium can be very helpful in an emergency room setting, but probably only works to terminate an acute migraine if the individual is truly magnesium deficient.

Essential Fatty Acids

It is important to remember that the brain is largely composed of fat. Although essential fatty acids have not received much research attention relative to migraine, there may be a significant role of fatty acids and their metabolites in the pathogenesis of migraine headache. Two small placebo-controlled studies demonstrated that omega-3 fatty acids significantly outperformed placebo in reducing headache frequency and intensity. High quality fish oil should always be used. A good frame of reference is that each capsule should contain at least 300 mg of EPA and 200 mg of DHA. A reasonable starting dose would be two to four capsules twice daily with meals.

Digestive Function: Holistic

Holistic practitioners are generally sensitive to the centrality of the gastrointestinal tract in producing overall health. Though we utilize a reductionistic approach to understanding human anatomy and physiology, we might consider that no system functions as an independent entity (GI, endocrine, cardiovascular, immune, etc.), and that a complex symphony of interrelated functions cuts across organ systems. For example, much of the immune system is found in the Peyer�s patches of the GI tract; in this light, we can see how food, chemicals, and unhealthy microbes might produce immune system activation from gastrointestinal exposure. We also recognize the importance of a balanced ecosystem of intestinal microbes; intestinal dysbiosis, or disordering of the gastrointestinal ecology, may readily produce symptoms, both within and distant from the GI tract. Some colonic bacteria act upon dietary tyrosine to produce tyramine, a recognized migraine trigger for some individuals. H. pylori infection is a probable independent environmental risk factor for migraine without aura, especially in patients not genetically or�hormonally susceptible. A high percentage of migraine patients experienced relief from migraines when H. Pylori infection was eradicated.

Detoxification: Holistic

Patients with migraine headache sometimes report that strong chemical odors such as tobacco smoke, gasoline, and perfumes may act as triggers. It is not uncommon for migraineurs to report that they are triggered by walking down the laundry soap aisle in the grocery store. Support for phase 1 and especially phase 2 detoxification may be beneficial for these individuals, as toxic overload or impaired enzymes of detoxification could theoretically be a significant mediator of headaches. Susceptibility to toxicity may be potentiated by a combination of excessive toxic exposures, genetic polymorphisms leading to inadequate detoxification enzyme production, or depletion of nutrient cofactors that drive phase two detoxification conjugation reactions Support for detoxification function is particularly important in modern life, given our exposure to unprecedented high levels of toxic chemicals. Some nutrients that supply support for detoxification function include: n-acetyl cysteine (NAC), alpha lipoic acid, silymarin (milk thistle), and many others.

Energy Production: Holistic

Riboflavin (Vitamin B2)

Energy production within the parts of the cell called mitochondria can be impaired in some migraine sufferers. Riboflavin is a key nutrient that is involved in energy production at this level. Riboflavin at 400 mg/day is an excellent therapeutic choice for migraine headache because it is well tolerated, inexpensive, and provides a protective effect from oxidative toxicity. Its use in children has been investigated, leading to similar conclusions,suggesting that, for pediatric and adolescent migraine prophylaxis, 200 mg per day was an adequate dose, but four months were necessary for optimal results.

Coenzyme Q10

CoenzymeQ10 (CoQ10) is also a critical component of energy function, and is an important antioxidant. Evidence supports the administration of CoQ10 in reducing the frequency of migraines by 61%. After three months of receiving 150 mg of CoQ10 at breakfast, the average number of headache days decreased from seven to three per month. Another study, using 100 mg of water soluble CoQ10 3x/day, revealed similar results. CoQ10 deficiency appears to be common in the pediatric and adolescent population, and can be an important therapeutic consideration in these age groups. Like riboflavin, CoQ10 is well tolerated (though expensive), with little risk of toxicity. It must be used with extreme caution in patients who also take warfarin, as CoQ10 may counteract the anticoagulation effects of warfarin. It is also noteworthy that many medications can interfere with CoQ10 activity, including statins, beta-blockers, and certain antidepressants and antipsychotics.

Endocrine (Hormone) Function

Female Hormones

It does not appear coincidental that migraine onset correlates with the onset of menstruation and that episodes are linked to menstruation in roughly 60% of female migraineurs. Although there is no universal agreement over the precise relationship between female hormones and migraine headache, it is apparent that the simultaneous fall of estrogen and progesterone levels before the period correlates with menstrual migraine. Estrogen gel used on the skin can reduce headaches when used premenstrually. Some researchers have found that continuous use of estrogen may be necessary to control menstrual migraines, which tend to be more severe, frequent, longer lasting, and debilitating than general migraines. Although published studies are lacking, many practitioners have used transdermal or other bioidentical forms of progesterone premenstrually with success. Of course, the risks of using hormones must be weighed against the benefits. Interestingly, administration of magnesium (360 mg/day) during second half of the menstrual cycle in 20 women with menstrually related migraines resulted in a significant decrease of headache days.

Melatonin

Melatonin, the next downstream metabolite of serotonin, is important in the pathogenesis of migraines. Decreased levels of plasma and urinary melatonin have been observed in migraine patients, and melatonin deficiency appears to increase risk for migraine. Melatonin has been used with some success, presumably via a restorative effect on circadian rhythms. A small study in children demonstrated significant improvement in their migraine or tension headache frequency with a 3 mg nightly dose of melatonin Melatonin appears to modulate inflammation, oxidation, and neurovascular regulation in the brain, and in one study, a dose of 3 mg/day was shown to be effective in reducing migraine headache frequency by at least 50% in 25 of 32 individuals. Ironically, some patients anecdotally report an increase of headaches (generally not migraine) when administered melatonin. The brains of migraineurs do not seem adaptable to extremes; a regular schedule of sleep and meals and avoidance of excessive stimulation are advisable to reduce excessive neural activation.

Immune Function/Inflammation: Holistic

Medications that produce an anti-inflammatory effect, such as aspirin and nonsteroidal agents, frequently produce an improvement in migraine symptoms during an acute attack. The herbs described below also play a role in reducing inflammation. Inflammation and oxidative stress can be identified in many conditions and disease states. It is important to acknowledge that the standard �modern� lifestyle is pro-inflammatory; our bodies are constantly reacting to one trigger after another (foods mismatched to our physiology, toxic burden, emotional stressors, excessive light and other stimulation) that activate our inflammatory cytokines (messengers of alarm). Providing broad-based support through lifestyle change and targeted nutrients may improve outcomes substantially, and this may be achieved foundationally by simplifying our�ingestions/exposures and supporting metabolic terrain. Herbal therapies are included in this section because of their relevant effects upon inflammation.

Feverfew (Tanacetum parthenium)

The precise mechanism of action of feverfew as a migraine preventive is unknown Though at least three studies found no benefit with feverfew, several controlled studies have revealed favorable results in improving headache frequency, severity, and vomiting when feverfew was compared to placebo. There are several caveats that should accompany the use of this herb:

Because of its anti-platelet effects, feverfew must be used with caution in patients on blood thinning products; avoid in patients on warfarin/Coumadin.
Feverfew does not have a role in managing acute migraine headache.
When withdrawing feverfew, do so with a slow taper, since rebound headache may occur.
Feverfew is not known to be safe during pregnancy and lactation.
Proceed with caution if an individual has an allergy to other members of the Asteraceae family (yarrow, chamomile, ragweed).
Most commonly reported adverse effects are oral ulceration (particularly for those chewing the leaves raw), and GI symptoms, reversible with discontinuation.

Feverfew is otherwise well tolerated. The typical dosage range is 25-100 mg 2x/day of encapsulated dried leaves with meals.

Butterbur (Petasites hybridus)

Butterbur is another effective herbal therapy for migraine headache. Butterbur is well tolerated, with no known interactions. Some individuals have reported diarrhea when using butterbur. In one study, its efficacy was demonstrated in children and adolescents between the ages of 6 and 17 years. Its safety is unknown during pregnancy and lactation. The plant�s pyrrolizidine alkaloids can toxic to the liver and carcinogenic, so only extracts that have specifically removed these compounds should be utilized. Many of the studies on Butterbur utilized the product Petadolex� because it is a standardized extract that has removed these alkaloids of concern. The usual dosage is 50 mg, standardized to 7.5 mg petasin and isopetasin, 2-3x/day with meals (although recent studies show that higher doses appear to be more effective1,2 ). Interestingly, butterbur�s diverse qualities make it useful for other conditions, including seasonal allergic rhinitis, and possibly painful menstrual cramps.

Ginger (Zingiber officinalis)

Ginger root is a commonly used botanical, known to suppress inflammation and platelet aggregation. Little clinical investigation has been performed relative to ginger use in migraine headache, but anecdotal reports and speculation based on its known properties make it a safe and appealing choice for migraine treatment. Some practitioners advise patients with acute migraine to sip a cup of warm ginger tea. Though evidence for this practice is lacking, it is a low-risk, pleasant, and relaxing intervention, and ginger is known to have anti-nausea effects. The most anti-inflammatory support is found in fresh preparations of ginger and in the oil.

Structural Considerations: Holistic

Practitioners of manual medicine seem to achieve success in reducing headache through various techniques such as spinal manipulation, massage, myofascial release, and craniosacral therapy Manual medicine practitioners frequently identify loss of mobility in the cervical and thoracic spine in migraineurs. While many forms of physical medicine seem helpful in shortening the duration and intensity of an episode of migraine, literature support is sparse with regard to manipulation as a modality to prevent recurrent migraine episodes. However, a randomized controlled trial of chiropractic spinal manipulation performed in 2000 revealed a significant improvement in migraine frequency, duration, disability, and medication use in 83 treatment group participants. Tension headache may also respond favorably to these techniques because of the structural component involved in muscular tension. The incidence of migraine in patients with TMJ dysfunction is similar to that in the general population, whereas the incidence of tension headache in patients with TMJ dysfunction is much higher than in the general population. Craniosacral therapy is a very gentle manipulative technique that may also be safely attempted with migraine.

Mind-Body Health: Holistic

There are few things more insulting than to be told by a medical professional to �Just reduce your stress.� Though the total load of stress experienced by an individual can be reduced through paring down unnecessary obligations, many everyday life stressors are unavoidable and cannot be simply eradicated. Thus, the answer to reducing stress for unavoidable contributors lies in two important areas: enhancing physical and mental resilience to stress, and modifying the emotional response to stress.

A multitude of programs to reduce the impact of stress on our physical and emotional well-being are rapidly becoming mainstream. For example, mindfulness meditation programs by Jon KabatZinn, PhD and many others are being offered to communities by hospitals around the country. This technique is simple to perform and has demonstrated positive outcomes in heart disease, chronic pain, psoriasis, hypertension, anxiety, and headaches. Breathwork and guided imagery techniques are likewise effective in producing a relaxation response and helping patients to feel more empowered about their health.

Biofeedback and relaxation training have been used with mixed success for migraine headache. Thermal biofeedback uses the temperature of the hands to help the individual learn that inducing the relaxation response will raise hand temperature and facilitate other positive physiologic changes in the body. Learning how to take more active control over the body may reduce headache frequency and severity. The effectiveness of biofeedback and relaxation training in reducing the frequency and severity of migraine headaches has been the subject of dozens of clinical studies, revealing that these techniques can be as effective as medication for headache prevention, without the adverse effects. Other relevant modalities to consider in this light include cognitive behavioral therapy, neurolinguistic programming, hypnosis, transcutaneous electrical nerve stimulation, and laser therapy.

Exercise should not be overlooked as a modality helpful in migraine headache. Thirty-six patients with migraine who exercised 3x/week for 30 minutes over six weeks experienced significant improvement in headache outcomes. Pre-exercise beta-endorphin levels in these individuals were inversely proportional to the degree of improvement in their post-exercise headache parameters. All patients should understand the critical importance of exercise on general health.

Acupuncture: Holistic

A discussion about a holistic integrative approach to migraine headache would be incomplete without acupuncture, which is an effective treatment modality for acute and recurrent migraine. A qualified/licensed practitioner of Traditional Chinese Medicine or a physician trained in medical acupuncture should be consulted.

Holistic: Summary Of Recommendations

Since initiators of migraine headache may be cumulative, identify and avoid them when possible. Consider the basic areas of dysfunction bulleted on the first page of this syllabus.
The incidence of food intolerance is high in patients with migraine headache; consider a comprehensive elimination diet for four to six weeks, during which time the following foods are eliminated: dairy products, gluten-containing grains, eggs, peanuts, coffee/black tea, soft drinks, alcohol, chocolate, corn, soy, citrus fruits, shellfish, and all processed foods. Careful reintroduction of one food at a time, no more often than every 48 hours, may help identify a food culprit. Meticulous recording of foods reintroduced is necessary. Most patients feel improved vitality during the elimination phase. Foods that clearly produce migraine (or other) symptoms should be avoided or used on a rotation schedule of not more than once every four days. If multiple foods introduced back into the diet seem to produce migraine headache, consider the possibility of altered intestinal permeability (leaky gut syndrome).
Consider the following supplements (Consult a qualified practitioner for advice):
Magnesium glycinate: 200-800 mg/day in divided doses (decrease to tolerance if diarrhea occurs)
Vitamin B6 (pyridoxine): 50-75 mg/day, balanced with B complex o 5-HTP: 100-300 mg 2x/day, with or without food, if clinically appropriate
Vitamin B2 (riboflavin): 400 mg/day, balanced with B complex
Coenzyme Q10: 150 mg/day
Consider hormonal therapies
Trial of melatonin: 0.3-3 mg at bedtime
Trial of progesterone or estradiol, carefully individualized, under medical supervision.
Botanical medicines
Feverfew: 25-100 mg 2x/day with meals
Butterbur: 50 mg 2-3x/day with meals
Ginger root
Fresh ginger, approximately 10 gm/day (6 mm slice)
Dried ginger, 500 mg 4x/day
Extract standardized to contain 20% gingerol and shogaol; 100-200 mg 3x/day for prevention, and 200 mg every 2 hours (up to 6 x/day) for acute migraine
Manual medicine may be helpful for some individuals.
Acupuncture
Mind-body support
Thermal biofeedback
Read The Relaxation Response by Herbert Benson, MD
Mindfulness meditation programs
Centering prayer
Breathwork
Guided imagery
Yoga, tai chi, qi gong, etc.
Many other modalities to consider!

Conclusion: Holistic Medicine

Patients will often request a more natural and self directed approach to health care. The recommendations above are typically very safe to implement, and are often welcomed by migraine sufferers. A practitioner with an integrative holistic focus will investigate an extensive array of predisposing factors to determine the underlying features most likely involved in a given individual�s condition. In this way, we treat the individual, rather than his or her diagnosis, and we will generate a favorable impact upon his/her overall health in the process.

Chiropractic Care & Headaches

Spring Allergies: How to Diagnose, Treat, and Prevent Them

by Dr Alex Jimenez | Holistic Medicine, Natural Health

Do you have the sniffles? Are your eyes watering? If so, you may be a victim of spring’s abundance of pollen that’s triggering your runny nose and watery eyes. A warm February caused trees and plants to bloom early, with many blossoming a full month ahead of schedule, allowing for a longer pollen season. As a result, experts say we’re in for a monster of an allergy season.

Before you blame your misery on pollen, however, make sure you don’t have a cold instead. Symptoms of both allergies and colds include stuffy noses, post-nasal drip, and headaches, but if you have fever, muscle aches, and a loud, violent cough, you probably have a cold. Also, if you’ve been suffering for more than about two weeks, you can place the blame on seasonal allergies, which typically last two or more months.

Although you can’t escape pollen, you can use these 10 tips to minimize your exposure:

• Stay indoors during peak pollen times — usually between 10 a.m. and 4 p.m., according to the American Academy of Allergy, Asthma & Immunology.

• Keep pollen from entering your home by keeping doors and windows shut, and don’t use fans that will bring in pollen-bearing air.

• Run air conditioning or air cleaning systems to remove allergens from indoor air. Use high-efficiency filters (HEPA) for best results, but replace them regularly or they become a breeding ground for bacteria.

• When gardening, wear a mask to reduce breathing in pollen and mold, and wash your face and hands when you come inside.

• Shower and wash your hair at night instead of in the morning to remove the pollen your body has collected during the day.

• Keep car windows closed.

• Monitor pets. If your indoor pets go outside, keep them off furniture and bathe them frequently to reduce the pollen they bring inside.

• Wear sunglasses to help shield your eyes from pollen.

• Watch your diet. Certain foods can make your seasonal allergies worse. Those allergic to ragweed could have problems with bananas, melons, zucchini, cucumber, and chamomile tea.

• Use salt lamps. Salt lamps are made from a chunk of salt that has been hollowed out to make room for a small light bulb or candle. Heating the salt produces negative ions which help purify the air of pollen, dust, and other pollutants.

You can also use these five natural remedies to fight the symptoms of allergies:

• Probiotics. An analysis of 23 randomized studies at Vanderbilt University Medical Center found that probiotics, the “good” bacteria found in yogurt, improved the symptoms of people with seasonal allergies, such as sneezing and a stuffy nose, in 17 of the studies. Researchers theorize probiotics change the composition of bacteria in the intestines in ways that modulate the body’s immune response and stop it from reacting to pollen and other allergens.

• Butterbur. A Swiss study published in the British Medical Journal found that one tablet four times a day (32 mg total) of this European herb relieved hay fever symptoms as effectively as the drug cetirizine, the active component of Zyrtec, with none of the drowsiness. Another study compared butterbur to Allegra with similar results. (Caution: Do not combine a drug for allergy relief with butterbur — you may overdose.)

• Quercetin. An antioxidant found in many fruits and vegetables, including apples and onions, quercetin acts as an anti-inflammatory that helps ease allergic symptoms. A double-blind, placebo-controlled Japanese study found that taking quercetin daily for eight weeks significantly reduced itching and irritation of the eyes in people with pollen allergies. Other research has found that quercetin reduces the amount of histamine produced by and released from cells.

• Stinging nettle. In a double-blind trial published in Planta Medica, 57 percent of patients found the herb was better at reducing the sneezes and sniffles of allergy than a placebo. Researchers believe the herb reduces the amount of histamine the body produces in response to an allergen, such as pollen.

• Acupuncture. A study published in the American Journal of Chinese Medicine found that acupuncture reduced the hay fever symptoms of all study participants. Another study found that acupuncture eliminated allergy symptoms in more than half of participants after only two treatments.

Of course, you can always take over-the-counter non-sedating antihistamines, such as Allegra, Claritin or Zyrtec Take them 30 minutes before going outside.

If your misery doesn’t ease or if your symptoms worsen, see your doctor. Physicians can prescribe cortisone nasal sprays, and immunotherapy in the form of shots or drops under the tongue may be prescribed for those with the most severe symptoms.

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Understanding Nrf2 and its Impact on Neurodegenerative Diseases

Modulation of Proteostasis by Transcription Factor NRF2

Abbreviations

Introduction

Loss of Proteostasis Influences NRF2 Activity in Neurodegenerative Diseases

Different Layers of Regulation Tightly Control NRF2 Activity

Impact of Protein Aggregates on NRF2 Regulatory Mechanisms

NRF2 Participates in the Unfolded Protein Response (UPR)

NRF2 Activation in Response to the UPR

NRF2 Up-Regulates Key Genes for the Maintenance of the ER Physiology

Impact of NRF2 on the UPR Dysregulation in Neurodegenerative Diseases

Mutual Regulation of NRF2 and the Ubiquitin Proteasome�System (UPS)

The UPS Modulates NRF2 Protein Levels

NRF2 Increases UPS Activity Through the Transcriptional Control of Proteasome Subunits

The NRF2-UPS Axis in Neurodegenerative Diseases

Functional Connection Between NRF2 and Macroautophagy

NRF2 Protein Levels are Modulated by the Adaptor Protein P62

Modulation of Macroautophagy Genes by NRF2

Relevance of NRF2-Mediated Macroautophagy Genes Expression in Neurodegenerative Disorders

Different Transcription Factors Act Coordinately to Modulate Proteostasis

Promising�Therapeutic Potential for NRF2 in Proteinopathies

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Concluding Remarks

Acknowledgements

Additional Topic Discussion: Relieving Knee Pain without Surgery

EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

Nrf2 Explained: The Keap1-Nrf2 Pathway

Abstract

Introduction

Mechanisms of Activation and Dysregulation of Nrf2 in Cancer

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Acknowledgments

Additional Topic Discussion: Relieving Knee Pain without Surgery

EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

An Integrative Holistic Approach To Migraine Headaches

Nutritional Considerations: Holisitic

Food Allergy/Intolerance

Magnesium

Essential Fatty Acids

Digestive Function: Holistic

Detoxification: Holistic

Energy Production: Holistic

Riboflavin (Vitamin B2)

Coenzyme Q10

Endocrine (Hormone) Function

Female Hormones

Melatonin

Immune Function/Inflammation: Holistic

Feverfew (Tanacetum parthenium)

Butterbur (Petasites hybridus)

Ginger (Zingiber officinalis)

Structural Considerations: Holistic

Mind-Body Health: Holistic

Acupuncture: Holistic

Holistic: Summary Of Recommendations

Conclusion: Holistic Medicine

Chiropractic Care & Headaches

Spring Allergies: How to Diagnose, Treat, and Prevent Them

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Clinic Locations

Additional Areas Of Care Offered

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