Nrf2—a therapeutic target for the treatment of neurodegenerative diseases

Highlights

• Oxidative stress in neurodegenerative diseases

• The Keap1-Nrf2-ARE pathway in brain

• Changes in Nrf2 and Nrf2-dependent genes in diseased brain tissue

• Transgenic, viral, and chemical-mediated activation of Nrf2 in animal models

• Effect of Nrf2 activation on progression of neurodegeneration in animal models

Abstract

The brain is very sensitive to changes in redox status; thus maintaining redox homeostasis in the brain is critical for the prevention of accumulating oxidative damage. Aging is the primary risk factor for developing neurodegenerative diseases. In addition to age, genetic and environmental risk factors have also been associated with disease development. The primary reactive insults associated with the aging process are a result of oxidative stress (OS) and nitrosative stress (NS). Markers of increased oxidative stress, protein and DNA modification, inflammation, and dysfunctional proteostasis have all been implicated in contributing to the progression of neurodegeneration. The ability of the cell to combat OS/NS and maintain a clearance mechanism for misfolded aggregating proteins determines whether or not it will survive. A critical pathway in this regard is the Nrf2 (nuclear factor erythroid 2-related factor 2)- antioxidant response element (ARE) pathway. Nrf2 activation has been shown to mitigate a number of pathologic mechanisms associated with Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease, and multiple sclerosis. This review will focus on the role of Nrf2 in these diseases and the potential for Nrf2 activation to attenuate disease progression.

Introduction

The brain is very sensitive to changes in redox status; thus maintaining redox homeostasis in the brain is critical for the prevention of accumulating oxidative damage. Aging is the primary risk factor for developing neurodegenerative diseases. In addition to age, genetic and environmental risk factors have also been associated with disease development. The primary reactive insults associated with the aging process are a result of oxidative stress (OS) and nitrosative stress (NS). OS/NS are produced endogenously via enzymatic and spontaneous reactions through a variety of sources and normal physiological functions [1], [2].

Downstream markers of OS/NS have been identified clearly in all neurodegenerative diseases. Signs of lipid peroxidation, such as aldehydes including 4-hydroxynonenol (4-HNE), are increased in Parkinson’s disease (PD) [3], [4], [5], Alzheimer’s disease (AD) [6], [7], [8], and amyotrophic lateral sclerosis (ALS) [9], [10]. There is also a correlation between the affected brain regions and 4-HNE adducted proteins [11], [12], [13]. Another major marker of OS production is protein carbonyls, representing protein oxidation. Carbonyls are present in the substantia nigra (SN) in PD [14], [15], AD [16], and ALS [17] and in affected brain regions in other diseases [18], [19], [20], [21]. Oxidative damage to DNA/RNA has been evaluated by measuring 8-hydroxy-2-deoxyguanosine (8-OHDG) and is increased in central and peripheral nervous systems of patients with neurodegenerative diseases [22], [23], [24], [25], [26], [27], [28], [29], [30]. Post-translational modification proteins modify protein structure and function. Protein structure and function can be altered by phosphorylation, nitration, ubiquitination, and glycosylation. Such modifications have been observed in alpha-synuclein (SYN) in patients with synucleinopathies including PD and dementia with Lewy bodies (DLB) [31], [32], [33]. Nitration and phosphorylation of tau protein has been found in the hippocampus and neocortex of patients with AD and other tau pathologies [34], [35]. Nitrotyrosine-modified proteins are elevated eight-fold in the hippocampus and neocortex of AD brains [36], [37]. These region-specific protein modifications correlate with areas of increased OS/NS in the brain. Furthermore, such modifications are thought to contribute to protein misfolding and subsequent aggregate/inclusion formation. Because most neurodegenerative diseases have characteristic misfolded protein aggregates, such as SYN in PD, beta-amyloid (Aβ) plaques and hyperphosphorylated tau neurofibrillary tangles (NFTs) in AD, huntingtin (Htt) in Huntington’s disease (HD), and superoxide dismutase 1 (SOD1) in ALS, protein aggregation and regulation of misfolded protein clearance by the proteasome and autophagy appear to be vital to pathogenesis.

One of the primary endogenous sources of OS is the mitochondrial electron transport chain. Increased mitochondrial dysfunction associated with neurodegenerative diseases leads to increased OS generation and reduction in the production of ATP. In addition and associated with neuroinflammation, the enzymes of the NADPH oxidase system generate superoxide anions. The combination of superoxide anion with nitric oxide, produced by nitric oxide synthase, generates the highly reactive NS peroxynitrite. Combating OS/NS is dependent upon the cell’s ability to maintain cellular redox homeostasis. A critical pathway in this regard is the Nrf2 (nuclear factor erythroid 2-related factor 2)- antioxidant response element (ARE) pathway. The ARE is an enhancer element having the consensus sequence RTGACnnnGC, which is located in the 5’ flanking region of many phase II detoxifying and antioxidant genes [38], [39]. Nrf2 is a cytoplasmic protein sequestered by the actin-bound protein Keap1 (Kelch ECH associating protein) [40], [41]. Keap1, a Cul3-based E3 ligase, polyubiquitinates Nrf2, targeting it for subsequent proteasomal degradation [42], [43]. Oxidative stress or exposure to electrophilic agents that react with Keap1 stabilize Nrf2, leading to increased Nrf2 protein levels and nuclear accumulation of Nrf2. Once in the nucleus, Nrf2 dimerizes with small Maf proteins and binds to the ARE, transcriptionally driving expression of several detoxifying and antioxidant genes [44], [45].

Nrf2 contains six well-conserved Nrf2-ECH homologous (Neh) domains that support molecular functions. The CNC (cap’n’ collar) and DNA binding regions are located in the Neh1 domain, as is the Maf dimerization site. Neh4 and Neh5 are necessary for recruitment of transcription factors and other canonical proteins required for gene expression [46]. Molecular studies have determined that the Neh2 domain is required for the cytoplasmic localization of Nrf2, because deletion of Neh2 leads to continuous nuclear translocation of Nrf2 to the nucleus. Yeast two-hydridization screening using the Neh2 domain from Nrf2 as bait identified Keap1 as an Nrf2 binding protein. Eighty percent of the independently isolated clones screened were Keap1 positive, suggesting specificity of the Keap1–Nrf2 interaction [40]. Keap1 has two canonical domains, the Kelch domain and the bric-a-brac, tramtrack, broad-complex (BTB) domain. The Kelch domain binds actin and thus tethers the Keap1–Nrf2 complex to the cytoskeleton. The BTB domain is important for protein dimerization of Keap1 molecules. There are many cysteine residues in the Keap1 protein that potentially function as sensors of oxidants and electrophiles; humans have 27 and rat and mouse have 25 [44], [47]. The “hinge and latch” model proposes that two Keap1 molecules bind Nrf2 at high- and low-affinity sites located in the Neh2 domain [48]. The hinge domain, EGTE, supports high affinity and the latch domain, DLG, low affinity. When Keap1 senses oxidative or electrophilic stress, the low-affinity domain binding Nrf2 is abolished and proteosomal degradation of Nrf2 is disrupted [49]. In addition to Keap1, Nrf2 turnover can be regulated by GSK3β/β-TrCP- and Hrd1-dependent mechanisms in different pathological states [50], [51]. GSK3β phosphorylates the Neh6 domain of Nrf2. This facilitates binding of the β-TrCP/Cul1 E3 ligase complex to Nrf2. Nrf2 is then ubiquitinated and degraded through β-TrCP-mediated proteasomal degradation. Hrd1 is another E3 ubiquitin ligase that resides in the endoplasmic reticulum membrane. Hrd1 directly interacts with Nrf2 at the Neh4-5 domains by binding to the cytoplasmic C-terminal region of Hrd1, leading to Nrf2 ubiquitination and degradation.

In addition, there are endogenous proteins that have been shown to interact with Keap1 and activate the Nrf2 pathway. An initial screen using Nrf2-dependent ARE-luciferase activity identified seven activating proteins: sequestosome 1 (SQSTM1 or p62), d-site of albumin promoter binding protein (DBP), dipeptidylpeptidase 3 (DPP3), BCL2-like 1 (BCL2L1 or Bcl-x_L), the kinesin family member 26B (KIF26B), cAMP-responsive element binding protein-regulated transcription coactivator 1 (TORC1), myeloid cell leukemia sequence 1 (Mcl1), and the splicing factor arginine/serine-rich 10 (SFRS10) [52]. Subsequently, both SQSTM1 and DPP3 were shown to interact with Keap1 [53], [54]. The Wilms tumor gene on the X chromosome (WTX) and PALB2, a major BRCA2 binding partner, also bind to Keap1, whereas p21 directly binds to Nrf2, leading to inhibition of Nrf2 ubiquitination and increased Nrf2-dependent gene expression [55], [56], [57]. This review will focus on the role of Nrf2 in neurodegeneration and the potential for Nrf2 activation to mitigate progression of neurological diseases.