Fei-ya LI,Raymond WONG,Ekaterina TURLOVA,Hong-shuo SUN,2,3,4

(1.Department of Physiology,2.Department of Surgery,3.Department of Pharmacology,4.Institute of Medical Science,Faculty of Medicine,University of Toronto,Toronto,Canada M5S 1A8)

Pathological role of transient receptor potential melastatin member 2 channel in neurodegenerative diseases and Alzheimer disease

Fei-ya LI1,Raymond WONG1,Ekaterina TURLOVA1,Hong-shuo SUN1,2,3,4

(1.Department of Physiology,2.Department of Surgery,3.Department of Pharmacology,4.Institute of Medical Science,Faculty of Medicine,University of Toronto,Toronto,Canada M5S 1A8)

Neurodegenerative diseases refer to incurable conditions that result in progressive degeneration or death of nerve cells.This causes functional movement deficits and cognitive problems such as dementias.Among different types of neurodegenerative diseases,Alzheimer disease(AD) accounts for majority of the cases.The transient receptor potential melastatin member 2(TRPM2) channel is a Ca2+-permeable non-selective cation channel which has been studied and implicated in the pathological process of AD through different pathways including inflammation.This review summarizes the contribution of TRPM2 in AD pathology and recent advances in pharmacology of TRPM2,with a focus on relationships between β-amyloid,oxidative stress and Ca2+.We also discuss the potential future research direction for neurodegenerative diseases.

transient receptor potential melastatin member 2;neurodegenerative diseases; Alzheimer disease;β-amyloid;oxidative stress;Ca2+;inflammation

Neurodegenerative disease is an umbrella term for conditions that mainly affect the neurons in the human brain.Neurons are the fundamen⁃talelementsofthe centralnervoussystem (CNS)and normally do not have the ability to re⁃generate themselves.When dead neurons accu⁃mulate,severe diseases may arise.Neurodegen⁃erative diseases such as Parkinson disease (PD),Alzheimer disease(AD),and Huntington disease(HD)are responsible for the greatest burden on families,our society and healthcare system.Within the neurodegenerative diseases, AD represents approximately 60%-70%of cases. Cures for the aforementioned diseases have remained elusive.In recent years,ion channels became the third largest target for drug development and potential therapeutic target for neurodegen⁃erative diseases.Among the ion channels,the transient receptor potential melastatin-subfamily member(TRPM)channels,one of the six subfamilies in the TRP family,show strong trend in having roles in neurodegenerative diseases.Scientists aim to confront the growing challenge posed by neurodegenerative diseases in our aging population via unraveling the relationship between the physi⁃ological functions of the TRPM protein family and the pathophysiology of the neurodegenera⁃tive diseases.Studies to date already indicated that the altered TRPM expressions in mammalian cells could lead to human ion channel diseases, also known as channelopathies[1].

The TRP channel was first identified as a protein inDrosophilamelanogaster[2-6].TRP channels are non-selective cation channels;somehave high selectivity to Ca2+,others are more permeable for Mg2+ions or other trace metal ions[7-9].Studies have shown that more than 30 different TRP genes have been identified and the TRP channels are grouped into 6 families named as TRPC(canonical),TRPM(melastatin), TRPV(vanilloid),TRPA(ankyrin),TRPP(polycystin) and TR PML(mucolipin)[10-12]. All these TRP channels consist of six transmembrane domains arranged in a tetrameric structure and are widely expressed in various cell types in the body,including neurons[13-16].Among the TRP channels,TRPM channels are implicated in brain diseases and neurodegenerative diseases[15].

The melastatin(TRPM)subfamily of TRP family has been extensively studied.TRPM family has 8 isoforms named as TRPM1-TRPM8.Partic⁃ularly,TRPM1 is required for photoresponse and human TRPM1 mutationsare associated with congenitalstationarynightblindness(CSNB) where patients get night blindness and lack rod function at an early age[2].TRPM2 is a sensor for oxidative stress and it also plays a role in insulin secretion[17].TRPM3 is a sensor for noxious heat[18-20],while TRPM8 is a sensor for cold temperature[21-22].TRPM4,like TRPM2,is related to insulin secretion[23].TRPM5 is related to trans⁃mitter secretion in taste bud cells[24].TRPM6 is permeable to Mg2+and it functions in the distal convoluted tubule of the kidney as a doorsill for modulating Mg2+homeostasis by maintaining a threshold for Mg2+entry[25].TRPM7 is known to play an importantpathophysiologicalrole in stroke[26]and neonatal hypoxic-ischemic brain injury[27];TRPM7 also regulates axonal outgrowth and maturation ofneurons[28].Lastly,some studies show that the TRPM2 subfamily may play a role in the neurodegenerative diseases including AD[1,15,29-30],and the focus of this review is on our current understanding of the TRPM2 channel and its pathophysiological role in the neurodegenerative diseases,as well as its thera⁃peutic potential for AD.

1 TRPM2 STRUCTURE AND TRANS⁃MEMBRANE TOPOLOGY

TRPM2 is one of the eight members in TRPM subfamily.It is a calcium-permeable nonselective cation channel and it is activated by intracellular adenosine diphosphoribose(ADPR) through a unique NUDT9-H domain[31].TRPM2 channels are expressed in many tissues including brain,lung,liver,heart,and various cell types, including neurons,microglia immune cells and pancreatic β-cells[9,32-33].

As it possesses bidirectional function between the ion channel itself and the C-terminal enzymatic NUDT9-H domain,TRPM2 is also known as a″chanzyme″,similar to that of TRPM6andTRPM7[31,34].TRPM2channelis encoded by TRPM2 gene,which in humans,is located in 21q22.3 region on chromosome with 32 exons and spans 90kb[31,35].In mice,this gene consists of 34 exons and spans 61kb[31,35].More⁃over,in human,there is an additional exon located at the 5′-terminus with a CgG island in the TRPM2 gene[35].TRPM2 protein in human consistsofapproximately1503aminoacids while in rodents each protein consists of 1507 amino acids[31,36].To form a channel,TRPM proteins typically assemble into homotetramers, each containing six transmembrane segments (S1-S6)with both intracellular N-and C-termini flanking the sides and a putative pore loop between the fifth and sixth segments.At the N-terminus,there are 4 homologous regions as well as a calmodulin(CaM)binding domain,a vital region that plays a role in regulating the channel opening property[37-39].At the C-terminus,there is a TRP box as well as a coiled-coil domain, both ofwhich have been assumed to be essential for the TRPM2 homogenous tetrameric assembling[16,31].

ADPR has been reported and accepted as the activation molecule of TRPM2 channel.It regulates the opening of the channel by binding to the NUDT9-H homology domain and subse⁃quenthydrolysis of ribose 5-phosphate and adenosine monophosphate(AMP)and calcium entry into the cell.Since the channels are not selective to Ca2+,Na+and K+ions can also pass through the channel during the opening.The currents recorded from the TRPM2 channels differ from both Na+channels and K+channels. Unlike Na+and K+channels,TRPM2 channels show a linearⅠ-Ⅴrelationship and the channels could stay in the open stage for a long time[31,40].

Regulation of TRPM2 via ADPR includes both facilitation and inhibition.Facilitation factors include Ca2+,hydrogen peroxide(H2O2),cyclic ADPR (Cadpr)[41]and nicotinic acid adenine dinucleotide phosphate(NAADP)[31],while inhibition factors include protons and AMP[41].These regu⁃latory factors could either directly or indirectly modulate TRPM2 channels.We will discuss modulatory effects of Ca2+and H2O2in details in the next section.

2ROLEOFTRPM2INALZHEIMER DISEASE

TRPM2 has been implicated in Ca2+-mediated functional processes of insulin secretion,cyto⁃kineproduction,cellmotility,aswellascelldeath[31]. Its reactive oxygen species(ROS)sensitivity has implicated TRPM2 as a potential therapeutic targetfor neurodegenerative diseases and pathologies related to oxidative stress[31].It also makes TRPM2 a potential target for chronic inflammation[31,42].AD,the most common form of dementia,causes problems in cognitive functions. Symptoms ofmemory loss and intellectual disabilities develop slowly initially,but gradually exacerbate becoming serious enough to affect daily tasks.AD has three disease stages(mild, moderate and severe)and it is an irreversible brain disorder with no effective treatment.Based on current understanding,TRPM2 may contribute to AD through β-amyloid(Aβ),oxidative stress and Ca2+entry[29,43-45].Recent studies continue to show the importance of the aforementioned mechanisms in causing AD and consequently the therapeutic potential of intervention within these pathways.

2.1RelationshipbetweenTRPM2and Alzheimer disease viaβ -amyloid

Aβ is a peptide consisting of 39-43 amino acids among which the Aβ-fragment 25-35 (Aβ25-35)is actually the biologically active region and is crucially involved in AD[46].The peptide resultsfrom the breakdown ofthe amyloid precursor protein(APP)[47].Which is cleaved by β-secretase and γ-secretase,yielding Aβ[47-48].Aβ molecules can aggregate together and form soluble oligomers,accumulation of which is known to be highly toxic to nerve cells[49].Advanced brain imaging techniques allow researchers to study the development and spread of abnormal amyloid proteins in the living brain,as well as changes in brain structure and function.The level of Aβ could be determined and used for predicting AD in the early stage[50-51].

Recently,a research has illustrated that TRPM2 can be positively regulated by Aβ.A recent study[44]used electrophysiological techniques, animal models and other molecular techniques to first reveal whether TRPM2 channel function could be modulated by Aβ oligomers and then test the effect of TRPM2 channels on Aβ oligomers toxicity.The electrophysiology results showed that TRPM2 currents〔induced byN-methyl-D-aspartic acid(NMDA)〕were magnified after the treatment of Aβ oligomers for 24 h.Therefore, augmentation of the recorded currents indicates that Aβ oligomers can positively modulate the opening property of TRPM2 channel.The APP/ PS1 mouse model used in the study was shown that when compared to wildtype mice,TRPM2 KO mice have reduced levels of endoplasmic reticulum(ER)stress in their brains,which is a pathophysiological change that could be used as a marker for AD[44-45].Normally in AD patients or AD mousemodels,ER stresswillincrease due to protein misfolding.Therefore the results of the study showed that symptoms of AD disease were improved with deletion of the TRPM2 channels.

In summary,Aβ has been viewed as a pivotalfactor involved in the pathology of AD,and its toxicity is elicited,atleastpartially,by the TRPM2 channel.Therefore,it is quite possible that TRPM2 is a downstream transducer of Aβ toxicity during the development of AD.

2.2 Relationship between TRPM2 and Alzheimer disease via oxidative stress

Oxidative stress leads to many pathophysio⁃logical conditions in the body including neurode⁃generative diseases like AD[52].Oxidative stress is essentially an imbalance between the overpro⁃duction of ROS and the reduced ability of the body to lower the levels of these free radicals through anti-oxidants[52-53]. ROS is naturally generated during the process of respiration via the mitochondrial electron transport chain and sometimes they could be beneficial.

H2O2is one typical chemical that generates oxidative stress in cells.TRPM2 channel activation has also been linked to oxidative stress.For example,the activation of TRPM2 channel after stimuli of oxidative stress release is regulated as follows:① H2O2activates production of ADPR and mobilizes ADPR from the mitochondria;② sufficient ADPR causes the TRPM2 channel opening via binding the NUDT9-H domain in the C-terminus.Following channelopening,Ca2+influx results,which increases the possibility of CaM binding to TRPM2 and therefore enhances the channel′s open probability(Fig 1).As[Ca2+]irises,itactivates the extrinsic and intrinsic apoptotic pathways,leading to the activation of caspase 3.Oxidative stress is known to have role in free ADPR generation via poly(ADP-ribose) polymerases(PARPs).The PARPscanbe degraded by poly(ADP-ribose)glycohydrolases, and then lead to the formation of ADPR[54]. Therefore,H2O2could activate TRPM2 channels through ADPR formation.An alternative hypothesis is that there may be a direct mechanism for H2O2regulating TRPM2 gating that is ADPR-independent[31].

In summary,oxidative stress is shown to be involved in the pathology of AD,and it is also a factor in TRPM2 channel gating.Thus,there is possibility that TRPM2 is a factor for the develop⁃ment of AD through H2O2-induced oxidative stress.

Fig.1 Activation mechanisms of TRPM2 and the subsequent downstream signaling cascade.Neurons expressTRPM2 channels,which are activated byADP-ribose (ADPR),and potentiated by nicotinamide adenine dinucleotide(NAD+)and H2O2.TRPM2 activation promotes Ca2+influx,which can lead to Ca2+imbalance.Extracellular H2O2can permeate through the cell membrane,and H2O2-induced Ca2+release from intracellular Ca2+stores further contributes to the Ca2+imbalance.This can induce neuronal cell death,which ultimately,can accumulate into the onset of neurodegenerative diseases.

2.3 Relationship between TRPM2 and Alzheimer disease via Ca2+

Ca2+,both extra-and intracellularly,plays a critical role in activating TRPM2 channels[39,55-56]. Even though TRPM2 is a″chanzyme″as previ⁃ously discussed,it is the binding of ADPR to the C-terminus that is critical for TRPM2 channel activation instead of enzymatic activity of the NUDT9-H domain[5].Intracellular Ca2+facilitates TRPM2 activation by strengthening the channel sensitivity to ADPR[57].Interaction between CaM and the IQ motif in the N-terminus of TRPM2 strengthens the positive feedback for TRPM2 activation,which subsequently causes increased Ca2+influx and enhanced intracellular calcium concentration[37].ADPR alone cannot trigger the activation of TRPM2 currents in the absence of Ca2+and a minimum of 30 nmol·L-1intracellular Ca2+is necessary to activate TRPM2 currents[55]. This facilitation is specific for Ca2+as studies showed that other ions such as Ba2+,or Zn2+,or Mg2+do not have such facilitation property[31,55]. Other than intracellular Ca2+,extracellular Ca2+also plays a critical role in TRPM2 activation and the facilitation function has a high efficiency:200 μmol·L-1of extracellular Ca2+could generate the same effect as 1 mmol·L-1external Ca2+[55]. Varying concentrations of Ca2+may differently modulate the gating property of TRPM2.The high concentration of intracellular Ca2+could promote the production or release of ADPR from mitochondria.Recentevidence concurs that TRPM2 channel can still be activated by[Ca2+]ieven when the ADPR binding sites are mutated. According to this result,a different signaling pathway for TRPM2 activation may lead into an unexplored mechanism.

Under physiological conditions,Ca2+is a secondary messengerand plays a role in maintaining the normal functions of neurons.Under pathophysiological conditions,Ca2+overload will trigger the activation of multiple apoptotic signaling pathways,such as cytochrome/caspase 3[58], c-Jun/JNK[59]and protein kinase C（PKC）/NF-κB[60]pathways,all of which have been implicated in AD.

3 INVOLVEMENT OF TRPM2 IN INFLAM⁃MATION

As a newly identified Ca2+-permeable nonselective cation channel and the sensor of ROS, TRPM2 channel has recently been indicated to be involved in inflammation.Since TRPM2 channels are abundantly expressed in various tissue types,TRPM2 channels are also expressed and functional[61],and can act as a sensor for ROS in microglia,the resident immune cells of the CNS[54,62-63].There is evidence that suggests a role of TRPM2 channels as an oxidative stress and metabolic sensor regulating immune and inflammatory responses[64].TRPM2 contributes to lipopolysaccharides(LPS)/interferon γ-induced production of nitric oxide through the proinflam⁃matory p38/JNK pathway in microglia[65].In addition,TRPM2activationhasbeensuggested to be the link between oxidative stress and nod-like receptor family,pyrin domain containing 3(NLRP3) inflammasome activation[66].Mori,et al[67-68]recently discovered that deficiency of TRPM2 reduced inflammatory and neuropathic pain in several mouse models,including carrageenan or Formalininduced inflammatory pain,and peripheral nerve injury-induced neuropathic pain models.However, TRPM2 had no effect on basal mechanical and thermal nociceptive sensitivities[67-68].Furthermore, this group also discovered that TRPM2 channels are involved in pathological pain models based on centraland peripheralneuroinflammation, rather than physiologicalnociceptive pain[69]. Although the involvement of TRPM2 in ischemic stroke is not yet fully understood,in striatal neurons,TRPM2 activation induced death through oxidative stress[70].Specifically,TRPM2 channel gating is linked to NMDA channel activation. NMDA channel activity elicits a proinflammatory signaling cascade that begins with the production of nitric oxide and reactive nitrogen species,as well as generation of ADP-ribose.In ischemia,the nuclear enzyme PARP-1 is highly activated, which in turn phosphorylates and thus activates ADP-ribose.Consequently,ADP-ribose opens TRPM2 channels.Because TRPM2 activation is downstream ofNMDA channels, TRPM2 channels make an exciting therapeutic target.

4 PHARMACOLOGY OF TRPM2

As TRPM2 is highly related to neurodegen⁃erative diseases,the pharmacology of TRPM2 has attracted major interest in research field but currently is far from reaching a satisfactory state. However,different TRPM2 channels blockers have been widely studied and discovered but none of them possess a desirable specificity. Compounds that reported to have effect on TRPM2 channel also affected other targets.

Flufenamic acid(FFA)was the first to be identified as TRPM2 blocker.It belongs to class of non-steroidal anti-inflammatory drugs(NSAIDs). Such fenamates are capable of producing antiinflammatory effects in the CNS.Studies have been done on TRPM2-expressing HEK-293 cells, and FFA evoked a pH-dependent inhibition of ADPR-or H2O2-induced cation currents[71].However, FFA has inhibitory effect not only on TRPM2channel;it has similar effect on other channels in TRP family such as TRPM4,TRPM5,TRPC3 and TRPC5[72-73].It also has an activation effect on TRPC6[74]and TRPA1[75].Therefore,fenamates like FFA are hardly satisfactory tools for study on clarification of role of TRPM2 channel.

Other known blockers(but non-specific)of TRPM2 are the anti-fungals clotrimazole and econazole,and 2-aminoethoxydiphenylborate (2-APB).The anti-fungal compounds exert an open-channel block of TRPM2 channels activated by ADPR in HEK-293 cells but the inhibitory effect is irreversible[71].2-APB was first identified as an inositol 1,4,5-trisphosphate(IP3)receptor antagonist[76].Again,2-APB has been reported to exert inhibitory effect on certain TRPC channels and TRPM channels while also showing activation effect on some TRPV channels[77].Some heavy metal ions such as La3+and Gd3+[78-79]are known to have inhibitory effect on most TRP channel but TRPM2 seems to be an exception[73].A more recent study shows that divalent copper(Cu2+)is a potent TRPM2 channel blocker[80].

Recently,AG490 is identified to have inhibitory effect on TRPM2 channel[81].The study shows that AG490 almost thoroughly blocked H2O2-induced intracellular Ca2+increase and significantly reduces H2O2-induced TRPM2 currents.H2O2can also activate the TRPA1 channel,however, AG490 has no significant effect on the H2O2-induced Ca2+influx mediated by TRPA1 channels. ADPR is anotherendogenous activatorfor TRPM2,however,in the patch-clamp study on TRPM2/HEK cells,AG490 only inhibits the H2O2-induced but not ADPR-induced TRPM2 inward current[81].Structurally,AG490 belongs to tyrphostin family and was synthesized in the early 1990s. Since AG490 was initially found to be a Janus kinase 2(JAK2)inhibitor,the same study examined whetherthe inhibitory effectofAG490 on TRPM2 activity was depending on the inhibition of JAK2.The study also tested the effects of other JAK2 inhibitors and none of them had effect on H2O2-induced Ca2+increase by TRPM2 channels.Thus,the results suggest that the inhibitory effect of AG490 on TRPM2 activation be independent from JAK2 inhibition.AG490 may manifest its inhibitory effect by scavenging intracellular hydroxyl radicals[81].

Besides channel blocker discovery studies, the potentialpharmacologicalpossibilities of agonists of TRPM2 channels currently allow researchers to manipulate the channel activation. H2O2and ADPR are well known TRPM2 channel activator and binding of ADPR to TRPM2 may be a pharmacological target in the future.Several studies have suggested that other than H2O2and ADPR,TRPM2 channels can be also activated by nucleotides including cyclic ADPR(cADPR)[41], nicotinamide-adenine dinucleotide(NAD)[82],nicotinic acid-adenine dinucleotide(NAAD),NAAD-phosphate (NAADP)[83-84].However,whether these nucleotides play a direct role on TRPM2 activation remains controversial.

5 CONCLUSION AND FUTURE DIREC⁃TIONS

Based on what has been discussed,there are several lines of evidence that highlight the roles of TRPM2 channels in the AD.Both the mechanism underlying the cause of AD and the regulatory mechanism of TRPM2 channel are not fully understood.Firstly,both Akt and GSK3-β are pivotal protein kinases that confer the prosurvival effect in neurons,and it has been verified that TRPM2 inhibition activates Akt and promotes the phosphorylation of GSK3-β,thus enhancing the survival of neurons subjected to ischemic insult.However,whether JAK/STAT and other signaling pathways are involved is still unknown. On the other hand,it has been shown that TRPM2 is involved in neuronal toxicity mediated by Aβ.Although aggregation of Aβ and overphos⁃phorylation of tau proteins are two pathological characteristics of AD disease,it is uncertain if tau proteins that are located inside the cells would have any regulatory roles on TRPM2 channels as well.All these mechanisms still need more clarification.

As discussed,the opening of TRPM2 chan⁃nel regulates oxidative stress,calcium ion influx and Aβ toxicity,all of which may cause neuronal cell death.Such assumptions lead to some future directions.First,TRPM2 could be a biomarker in the future for therapeutic diagnosis.Based on current understanding,increased TRPM2 activity increases the risk of neuronal cell death which could lead to dementia development.Neurode⁃generative diseases like AD do not have typical symptoms at the early stage;it is a slow and pro⁃gressive brain disorder process that takes place many years before the appearance of cognitive deficits.TRPM2 can potentially be used as a clinical biomarker to detect the onset of dementia at an earlier stage,ultimately resulting in a larger window frame for therapeutic action.Second,a specific TRPM2 blocker needs to be discovered. A drug needs to be designed that has a certain degree of specificity and efficacy for inhibition of TRPM2.

Furthermore,as TRPM2 is shown to be related to neurodegenerative diseases,human tissue samples are needed for further evaluation. Samples from patients that suffer from AD have not yet collected for testing TRPM2 expression. Doing so,TRPM2 may be a biomarker for clinical diagnosisin the future.Researcheson the biological roles of TRPM2 in various cells and tissues have been performed in many laboratories, but the current progress is limited partially due to unavailable specific TRPM2 blockers.Modulation of TRPM2 channels may become an important pharmacological therapeutic target in the near future that is expected to be of value in a wide range of pathological conditions.

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瞬时受体电位M2型离子通道在神经退行性病变和阿尔茨海默病病理学中的作用

李菲娅1，Raymond WONG1,Ekaterina TURLOVA1,孙宏硕1,2,3,4

(1.Department of Physiology,2.Department of Surgery,3.Department of Pharmacology,4.Institute of Medical Science,Faculty of Medicine,University of Toronto,Toronto,Canada M5S 1A8)

神经退行性病变是一种可导致神经细胞渐进性退变或死亡的不可治愈性疾病。该疾病可诱发功能性运动障碍以及精神问题，比如痴呆。在诸多种类的神经退行性病变中，阿尔茨海默病占据最大比例。瞬时受体电位M2型（TRPM2）离子通道是一个可通过钙离子的非选择性阳离子通道。研究表明，TRPM2离子通道有可能通过不同路径（包括炎症路径）参与阿尔茨海默病的病理过程。本综述主要通过对TRPM2与Aβ、氧化应激以及钙离子之间关系的讨论，总结概括了TRPM2在阿尔茨海默病病理学中的作用，也对近期TRPM2药理学领域的研究进展进行了探讨，并对如何进一步研究TRPM2在阿尔茨海默病中的作用进行了展望。

瞬时受体电位M2型；神经退行性病变；阿尔茨海默病；Aβ；氧化应激；钙离子；炎症

孙宏硕，E-mail：hss.sun@utoronto.ca

2016-05-04 接受日期：2016-06-14）

R966

:A

:1000-3002-(2016)06-0656-11

10.3867/j.issn.1000-3002.2016.06.005

Biography：Fei-ya LI,female,graduate student,research focus on the role of ion channels in neuroprotection and drug development for stroke and hypoxia.E-mail:feiya.li@ mail.utoronto.ca

Hong-shuo SUN,E-mail:hss.sun@ utoronto.ca

（本文编辑：乔 虹）