朱 丽，王国庆，李戴弟，张 锋

(遵义医学院 基础药理教育部重点实验室暨特色民族药教育部国际合作联合实验室，贵州 遵义 563099)

Neurodegenerative diseases are caused by the loss of neurons and/or myelin sheaths.They are characterized by the presence of abnormal protein aggregates in neurons and glial cells,which are divided into acute and delayed neurodegenerative diseases.Most of these diseases are delayed[1].Neurodegenerative diseases mainly include Alzheimer's disease (AD),Parkinson's disease (PD),Huntington's disease (HD),amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS)[2].

Oxidative stress is considered to be one of the major pathogenesis of various neurodegenerative diseases,and the nervous system is very sensitive to changes in the redox state,so that maintaining the redox balance is essential to prevent accumulated oxidative damage[3].Free radical-mediated oxidative stress formation of reactive oxygen species (ROS) (such as superoxide radical O2-hydrogen peroxide H2O2) and reactive nitrogen species (RNS) (such as NO,N2O4,HNO3,ONOO-) can damage proteins and lipids.The source of these free radicals may be exogenous as endogenous biochemical and enzymatic processes,such as ionizing radiation,photochemical reactions,and environmental toxins[4].Some current antioxidant include antioxidase (e.g.superoxide,glutathione peroxidase),antioxidant proteins (e.g.thioredoxin,peroxidoreductin) and antioxidant molecules (e.g.lipoic acid,GSH,vitamin E) etc[5].In the brain,superoxide and glutathione peroxidase seem particularly important.

Once the body is attacked by the disease,the oxidation balance in the body is broken and the levels of ROS / RNS are increased and then oxidative stress occurs.The neurons at this time are likely to be damaged[6].After antioxidant administration,antioxidant genes coordinate the up-regulation of glutathione S-transferase (GSTπ),catalase (Cat),glutathione-peroxidase (GPxs),and heme oxygenase 1 (HO-1) and peroxide-reducing enzymes (Prdxs) to reduce the oxidative load and restore cellular homeostasis.This process is regulated by the combination of Nuclear factor erythroid 2 related factor 2 (Nrf2) with this consensus binding sequence.

Nrf2 is a member of the Cap-n-collar (CNC) transcription factor family.Nrf2 has become one of the most well-known family members of cell protection in oxidative stress and electrophilic stress.The human Nrf2 protein consists of 605 amino acids and contains several highly conserved Nrf2-ECH homologous (Neh) domains.The Neh1 region contains a C-terminal leucine zipper structure,Bzip,which binds to a small nuclear Maf protein to form a heterodimer and binds to an anti-oxidant response element (ARE).The Neh2 region contains two binding sites for DLG and ETGE that can negatively regulate binding to Kelch-like ECH-associated protein 1 (Keap1).Neh3 is located at the C-terminus of Nrf2 and is closely related to the transcriptional activity of cells.Neh4 and Neh5 are located between Neh1 and Neh2 and are important structures involved in initiating transcription of downstream genes.The Neh3-5 domain is thought to play a pivotal role in transactivation,binding to the various components of the transcription device.The Neh6 region is a non-Keap1-dependent serine-rich Nrf2 degradation regulatory region associated with the negative regulation of Nrf2[7].

Keap1 is a multi-domain repressor protein of the Kelch family and consists of 50 members.They were named Kelch-like 1- 42 (KLHL1-42) or 1-14 (KBTBD1-14) containing Kelch and BTB domains,respectively.They are the major upstream regulators of Nrf2 and contain five domains,namely the N-terminal domain (NTR),the intervening region (IVR),the BTB region,the double glycine repeat region (DGR),and the C-terminal domain (CTR).The DGR region is also known as the Kelch region,which binds Keap1 to Nrf2 in the envelope and also binds to intracytoplasmic actin.The IVR region is rich in cysteine and is a functional regulatory region of the entire protein.The BTB region is related to homodimerization of Keap1[8].

Nrf2-ARE binding results in the Keap1-Nrf2 pathway,which induces the expression of more than 100 cytoprotective genes.Under physiological conditions,Keap1 binds Cul3 through its BTB region,and Kelch region binds Nrf2,linking Nrf2 to the E3 complex.It transfers ubiquitin from E3 to the lysine residue of Nrf2 (between the ETGE motif and the DLG motif),and the ubiquitinated Nrf2 is rapidly degraded[9].When the body receives oxidative stress,the specific cysteine residue of Keap1 is modified,resulting in a conformational change.The affinity of the DLG motif to Keap1 is attenuated and isolated,thereby protecting Nrf2 from degradation by ubiquitination,maintaining stable expression of Nrf2 and retaining in an inactive state.However,after Nrf2 phosphorylation,it enters the nucleus and binds to the small Maf protein to induce the expression of downstream genes.Finally,the level of Nrf2 in the cells is returned to the initial state.

Here,this review summarized the role of Nrf2 on the pathogenesis of neurodegenerative diseases.To regulate the expression of Nrf2 might be one of the directions to prevent and treat neurodegenerative diseases.

1 Nrf2 and AD

AD is the most common neurodegenerative disease and is characterized by memory deficits and cognitive decline caused by loss of synapses and neurons in the hippocampus and cerebral cortex[10].The major neuropathological hallmark of AD is the abnormal deposition of amyloid-beta peptide (Aβ) in extracellular senile plaques and intracellular neurofibrillary tangles formed by hyperphosphorylated tau[11].Oxidative stress associated with mitochondrial dysfunction is an important contributor to neuronal death in AD[12].The pathogenesis of AD is still largely unknown.Thus,the discovery of effective treatment remains a key goal of modern medicine[13].Studies on AD have demonstrated that oxidative stress is closely related to the inflammatory process.ROS and RNS can damage the nerve endings and cause dysfunction and loss of synapses,and further induce the development of diseases.In the early stage of AD,Aβ production increases and with the accumulation of Aβ,which further induces and exacerbates oxidative damage,and promotes detoxification and neurodegeneration[14].Furtherly,Aβ1-40 and Aβ1-42 are the dominant forms in several different lengths of Aβ,whereas the level of Aβ1-40 is the highest and Aβ1-42 is more neurotoxic and oligomerized.A battery of evidence indicates that Aβ1-42 is the key event to AD pathogenesis by eliciting neuronal apoptosis through oxidative stress[15].

There is an increasing evidence that the AD brain is in a state of great oxidative stress[16].Delivery of Nrf2 directly to the hippocampus using a lentiviral vector encoding human Nrf2 (LV-Nrf2) effectively improved spatial learning and memory in APP/PS1 mice[17].In addition,the increased ROS levels were recognized as an early factor in the development of AD,and the level of HO-1 oxidation was significantly reduced in the cerebral cortex and hippocampus of AD patients[18].The detection results of related enzymes showed that the peroxidase-superoxide dismutase,catalase and glutathione peroxidase in the brain of oxidative stress mice were significantly reduced.The level of lipid oxidation was significantly higher and its expression was significantly higher than that in patients without dementia.The expression of NAD(P)H dehydrogenase 1 (NQO1) in neurons and astrocytes of AD brain was also significantly higher than that in healthy brain[19].

At present,the treatment of AD is limited to symptomatic treatment.The drugs used to improve cognitive impairment include memantine and acetylcholinesterase (AChE) inhibitors.More than 100 drugs for AD treatment have been declared failure since 2002[20].Since oxidative stress occurs early in AD,the anti-oxidative therapies have long been considered a new hope[21].Although the anti-oxidant therapy in AD clinical trials encountered a temporary failure,the activation/overexpression of autologous endogenous anti-oxidant pathways by Nrf2 did show a significant protection in various AD animal models[22].Therefore,the optimistic perspective for future uses of anti-oxidative potential treatment for AD is emerged.

2 Nrf2 and PD

PD is the second most common neurodegenerative disorder with the main clinical symptoms of static tremor,muscle rigidity,slow movement,and abnormal gait.The pathological changes were the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta,and the reduction of striatal dopamine (DA) content[23].Although the underlying pathogenesis of PD has not been fully elucidated,there is an overwhelming evidence that both oxidative damage and mitochondrial dysfunction caused degeneration of dopaminergic neurons.

Studies from the postmortem brain analysis of PD patients presented an increased level of 4-hydroxy-2-nonenal (HNE),a byproduct of lipids,and the high expressions of HO-1 and NQO1 in glial cells[24].In PD patients,there was a decrease in innermost dopa in the central nervous system in addition to the decreased antioxidant levels and increased iron levels.These findings implied that the increased oxidative stress in the central nervous system of PD patients may cause the interference with transferrin-mediated iron transport and also contribute to the production of ROS[25].

Moreover,DA neurons were more easily oxidized than other neurons in the brain[26].DA may also be spontaneously oxidized to electron-deficient DA (DAQ) in the presence of unsaturated metal ions or enzyme-catalyzed.DAQ contains a partial positive charge that is primarily located on the carbon atom of the catechol ring and is easily attacked by the thiolate (-S-) ion contained in the cysteine residues of GSH and proteins.The main product of cysteine thiolate and the DAQ reaction,5-cysteinyl-DA,has been detected in the human central nervous system and its level in PD brains was increased[27].Furtherly,the cysteine residues play a key role in protein function,modifying these residues by irreversible covalent reactions between DAQ and cysteine may have an adverse effect on cell health.Because the striatum contains a large amount of DA,it is also more susceptible to oxidative stress.However,Nrf2 not only regulates the expression of antioxidant enzymes and increases the ability to resist oxidative stress,but also modulates the gene glutathione (GSH) pathway to avoid thiolate ion attack and effectively protect cells.Therefore,Nrf2 activation would become an effective potential for the treatment of PD.

At present,some antioxidants (especially Nrf2 activators) have long-term potential for the protection against DA neurodegeneration in PD patients.These drugs include monomethyl fumarate (MMF),dimethyl fumarate (DMF),and triterpenoid RTA-408,as well as some Chinese herbal medicines.Among them,synthetic triterpenoids (TP),such as 2-cyano-3,12-dioxine-1,9(11)- diene-28-acid (CDDO) and its derivatives are effective activators of the Nrf2/ARE pathway[28].The clinical efficacy of DMF in the treatment of PD has attracted considerable attention.The antioxidant capacity,cytoprotection and immunomodulatory effects of DMF and MMF are mainly attributed to their ability to activate the Nrf2 pathway.Recently,it has been reported that the high-affinity iron chelator deferiprone (3-hydroxy-1,2-dimethyl-4-pyridone) provides a protective effect in phase 2 randomized clinical trials in patients with PD[29].In addition,several Chinese herbal medicines including flavonoids and non-flavonoids,and various antioxidants,such as glutathione peroxidase,have been observed to produce neuroprotection in various animal models of PD[30].These studies indicate that Nrf2 is a feasible drug target for PD treatment.

3 Nrf2 and HD

HD is an autosomal dominant neurodegenerative disease with abnormal amplification of cytosine,adenine and guanine (CAG) trinucleotide repeats in its genes[31].The number of pathogenic CAG repeats is related to the severity and age of the onset of the disease.The most common clinical manifestation of HD is chorea,manifested as spontaneous and transient muscle contractions.However,the damage is far more than these,including mood and personality changes,depression,cognitive impairment,weight loss and bioenergetic deficiency[32].Although HD could damage many organ tissues,it appears to be particularly harmful to brain tissue.Neuropathologically,the disease is characterized by the loss of moderately multiple spiny neurons in the other cerebral regions including the neocortex,cerebral cortex,globus pallidus,thalamus,and cerebellum[33].There is currently no delay in preventing the development of HD,and recent studies have shown that a synthetic mitochondrial-targeted antioxidant can prevent oxidative damage to mitochondria in the HdhQ1 knock-in mouse model of HD and delay disease progression.

Similarly,oxidative stress also plays an important role in HD[34].There is further evidence that the up-regulation of Nrf2-ARE-transcripts may induce improved locomotor performance and reduce striatal degeneration and oxidative stress and improve the survival rate of transgenic HD mice[35].Hace1 KO mice showed increased oxidative stress in the brains of Hace1 WT and Hace1 knockout mice and were impaired in antioxidative stress[36].In addition,E3 ubiquitin protein ligase 1 (HACE1) was found to be essential for optimizing Nrf2 activation in oxidative stress challenged cells[36],since HACE1 depletion resulted in the decreased Nrf2 activity.Remarkably,a decrease in HACE1 level was found in the striatum of HD patients[35].In addition,the ectopic expression of HACE1 in striatal neuronal progenitors provided protection against mutant huntingtin-induced redox imbalances and oxidative stress hypersensitivity by enhancing Nrf2 function.Defects in mitochondrial complex II,III,and IV in the striatum of HD patients caused mitochondrial dysfunction[37],and therefore,oxidative stress might be involved in the pathogenesis of HD.

In a few treatment options for HD,tetrabenazine has been approved by the U.S.Food and Drug Administration (FDA) for the treatment of HD[38].An evidence that neurons were increased after oral administration of the Nrf2 activator dimethyl fumarate (DMF) in HD model mice with R6/2 and YAC 128,and neurons in the motor cortex and striatum were also improvedwas demonstrated[39].Both DMF and MMF can be used as inducers of oxidative stress in HD models[40].Under DMF treatment,the neurons remain relatively intact in morphology and do not affect the pathogen of HD,which has a significant protective effect on the affected tissues.Until now,treatment options for HD have been limited to symptomatic treatment.These studies have shown that activation of the Nrf2 pathway through small molecule compound interventions or physical therapy is a viable option for future treatment of HD.

4 Nrf2 and ALS

ALS is the most common adult-onset motor neuron disease and is a debilitating disease characterized by the loss of more than one inferior motor neuron[41].The disease causes motor neurons in the primary motor cortex,brainstem,and spinal cord to undergo progressive degeneration.Patients usually develop rapid numbness of skeletal muscle and eventually die due to respiratory failure within 3 to 5 years after the onset of the disease[42].Most cases of ALS are spontaneously acquired (sporadic ALS; sALS),and only 10% to 15% of cases of ALS are hereditary (familial ALS,fALS)[41].Genetic studies have shown that in addition to the prominent role of SOD1 in familial ALS,some components of cellular oxidative stress responses are associated with ALS (including variants encoding NfFe2L2 and KEAP1 encoding the Nrf2 and Keap1 proteins,respectively)[43].

Because high brain oxygen consumption leads to production of ROS,Nrf2-ARE activators provide powerful neuroprotection.It is noteworthy that activation of Nrf2 produced a protective effect in hSOD1G93A ALS model mice[44].Collectively,these findings support the oxidative stress that Nrf2 pathway-mediated neuroprotection may protect neurons against the progression of ALS disease.Although there is still a lot of knowledge gap from the dissociation of Nrf2 to nuclear localization and the role as a transcription factor,the activation of the Nrf2 signaling cascade represents a novel and unique neuronal defense mechanism that seeks to treat ALS and other neurodegenerative diseases by enhancing intrinsic factors.

Riluzole is also the only neuroprotective treatment approved for ALS.It is a glutamate-resistant drug and also has antioxidant properties[45].There is evidence that RBM45 can modulate the novel effect of the antioxidant response inhibitor KEAP1 through direct protein-protein interactions,thereby inhibiting neuroprotective antioxidant responses[46].Manganin Porphyrin (AEOL10150),an antioxidant and free radical scavenger,accumulates in the mitochondria and nucleus,crosses the blood-brain barrier,effectively scavenges active substances,and significantly prolongs the survival of mSOD1G93A mice at the onset of symptoms[47].The multifunctional iron-chelating drugs M30 and HLA20 reduce the neurotoxicity induced by peroxynitrite ion generators SIN-1 and H2O2and increase the expression of Tfr,an iron metabolism-associated protein in NSC-34 cells[48].Antioxidant vitamin E (alpha-tocopherol) slowed the onset and progression of sputum in ALS mice.These results suggest that antioxidants may play a significant role in ALS prevention in the future[49].In summary,although antioxidant-based therapies for ALS have not shown efficacy in clinical trials to date,antioxidants might possess a promising therapeutic potential based on current research results.

5 Nrf2 and MS

MS is an autoimmune-mediated neurodegenerative disorder characterized by demyelination of neuronal axons[50].There are four clinical types of multiple sclerosis:relapsing remitting,secondary progressive,primary progressive and progressive recurrent.The former account for about 85% of cases,but as the symptoms change over time,changes can occur between patients[51].Pathological changes were pathogenic attacks on myelin and oligodendrocytes by autoreactive CD4+Th1 and Th17 cells,infiltrating macrophages and resident microglia,causing axon retraction and astrogliosis.Reactive microglial cells and astrocytes then secrete inflammatory mediators that include not only cytokines and chemokines,but also produce reactive oxygen and nitrogen substances that contribute to oxidative stress,leading to neuroinflammation-related tissue damage[52].The lesions are located around the ventricles,especially in the subventricular veins around the ventricle (mainly near the ventricular body and lateral ventricle corners).Clinical manifestations are neurological deficits in vision loss,incoordination,weakness,loss of sensation and cognitive changes,and heterogenic disorders of bladder and bowel dysfunction.However,the etiology of MS is unclear.Recent studies have shown that oxidative damage plays a major role in the pathogenesis of demyelination and neurodegeneration in MS.Oxidative damage is associated with the presence of microglia-expressing enzymes (such as NADPH oxidase and myeloperoxidase) involved in the production and activation of oxygen free radicals[53].

Experimental autoimmune encephalomyelitis (EAE) is the most widely used MS model[54].Since it has many clinical,histological,immunological,and genetic features with MS,EAE has been used to gain important insights into the pathogenesis of MS and to validate new targets for MS therapy.The autopsy study showed that the process of neuronal Nrf2 activation is similar to the development of EAE.In the cuprizo multiple sclerosis model,both Nrf2 and target protein are reduced[55].It is well known that MS can cause oxidative stress through the effector molecule,reactive nitrogen species (RNS),which releases pro-inflammatory cytokines and free radicals after localization in glial cells to help confer host immune protection.Free radicals,such as superoxide,could react to produce RNS,thereby inducing oxidative damage,demyelination and more pronounced axonal loss in Nrf2-knockout mouse EAE model mice[54].

FDA approved the use of dimethyl fumarate (DMF) in March,2013,which is a recently approved oral therapy for the treatment of relapsing remitting multiple sclerosis (RRMS).DMF can directly activate Nrf2,protect oligodendrocytes,myelin,axons and neurons,and reduce oxidative stress through protein nitrosylation.In 2006,an exploratory study of the first oral fumarate was performed on 10 MS patients.The study confirmed the safety and efficacy of DMF[54].In the EAE model,DMF produced a neuroprotective effect by modulating the anti-oxidation response element Nrf2,but this effect was absent in Nrf2-/-mice[56].Treatment of primary cultures of animals or CNS cells with DMF resulted in an increased nuclear localization of Nrf2.Autopsy results also indicated an increase in Nrf2,and in its high expression areas,HO-1 and NQO1 expressions were also increased.However,no expression of antioxidant target genes was observed in Nrf2 knockout mice.Several lines of studies using natural compounds,such as sulphoraphane,matrine,and CDDO-TFEA,have been identified as treatment of EAE by Nrf2 modulation[57].These studies indicate that the Nrf2-ARE pathway participates in the pathogenesis of MS and holds vast promise for the treatment of MS.

In summary,over the past decade,statistical data show that redox imbalance is a common condition in the pathologies of several neurodegenerative disorders,and maintaining cellular redox homeostasis is essential to prevent neurodegeneration.Since Nrf2-ARE plays a pivotal role in the pathogenesis of many neurodegenerative diseases,and DMF has been formally approved by the FDA for the treatment of neurodegenerative diseases.Moreover,Sulforaphane (SFN) is an isothiocyanate obtained from cruciferous vegetables and is one of the most effective natural Nrf2 activators.SFN demonstrated neuroprotective effects in various neurodegenerative diseases models,mainly due to its ability to increase glutathione levels and glutathione-dependent enzymes.The knowledge gained from these studies will further assist in the clinical determination of relevant methods of activating Nrf2 in the central nervous system and may be effective in treating neurodegenerative diseases.