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ER stress and diseasesHiderou Yoshida1,2 1 Department of Biophysics, Graduate School of Science, Kyoto University, Japan 2 PRESTO-SORST, Japan Science and Technology Agency, Japan Keywor

ER stress and diseases

Hiderou Yoshida1,2

1 Department of Biophysics, Graduate School of Science, Kyoto University, Japan

2 PRESTO-SORST, Japan Science and Technology Agency, Japan

Keywords

conformational disease; cytoplasmic splicing;

ER stress response; ER-associated protein

degradation (ERAD); Golgi stress response

Correspondence

H Yoshida, Department of Biophysics,

Graduate School of Science, Kyoto

(Received 11 September 2006, revised

14 November 2006, accepted 8 December

Abbreviations

AIGP, axotomy-induced glyco ⁄ Golgi protein; APP, amyloid precursor protein; ASK1, apoptosis signal-regulating kinase 1; ATF, activating transcription factor; BAK, Bcl-2 homologous antagonist ⁄ killer; BAP, BiP-associated protein; Bap31, B cell receptor-associated protein 31; Bax, Bcl2-associated X protein; Bcl2, B cell leukemia 2; BI-1, Bax inhibitor 1; Bim, Bcl2-interacting mediator of cell death; BiP, binding protein; bZIP, basic leucine zipper; c-Abl, Abelson murine leukemia viral oncogene homolog 1; C ⁄ EBP, CCAAT ⁄ enhancer-binding protein; CHOP, C ⁄ EBP- homologous protein; CREB, cAMP response element-binding protein; CREBH, cAMP response element-binding protein H; CReP, constitutive repressor of eIF2a phosphorylation; DAP, death-associated protein; Der1, degradation in the endoplasmic reticulum protein 1; Derlin-1, Der1- like protein 1; Doa10, degradation in the endoplasmic reticulum protein 10; DR5, death receptor 5; EDEM, ER degradation enhancing

a )mannosidase-like protein; eIF2 a, a-subunit of eukaryotic translational initiation factor 2; ER, endoplasmic reticulum; ERAD, ER-associated degradation; ERdj, ER dnaJ; ERO1, ER oxidoreductin; ERp72, ER protein 72; ERSE, ER stress response element; FKBP13, FK506-binding protein 13; GADD, growth arrest and DNA damage; gp78, glycoprotein 78; GRP, glucose-regulated protein; HEDJ, human ER-associated dnaJ; HIAP2, human inhibitor of apoptosis 2; HRD1, HMG-CoA reductase degradation protein 1; HSP, heat shock protein; IAP, inhibitor of apoptosis; IDDM, insulin-dependent diabetes mellitus; IRE1, inositol requirement 1; JNK, Jun kinase; Keap1, Kelch-like Ech-associated protein 1; LZIP, basic leucine zipper protein; NIDDM, noninsulin-dependent diabetes mellitus; NOXA, neutrophil NADPH oxidase factor; Npl4, nuclear protein localization 4; NRF, nuclear respiratory factor; ORP150, oxygen-regulated protein 150; OS9, osteosarcoma 9; p58IPK, 58 kDa-inhibitor of protein kinase; pATF6(N), the nuclear form of ATF6 protein; PDI, protein disulfide isomerase; PERK, PRKR-like endoplasmic reticulum kinase; PKR, double stranded RNA-dependent protein kinase; PLP1, proteolipid protein 1; polyQ, polyglutamine; PrP, pion protein; PrP c , cellular PrP; PrPSc, scrapie PrP; PS1, presenillin 1; PUMA, p53 up-regulated modulator of apoptosis; pXBP1(S), the spliced form of XBP1 protein; pXBP1(U), the unspliced form of XBP1 protein; RIP, regulated intramembrane proteolysis; RseA, regulator of s E ; S1P, site 1 protease; S2P, site 2 protease; SAPK, stress-activated protein kinase; SEL1, suppressor of lin12-like; SREBP, sterol response element-binding protein; TDAG51,

T cell death-associated gene 51; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TRAF2, TNF receptor-associated factor 2; TRB3, Tribbles homolog 3; UBC6, ubiquitin conjugase 6; UBC7, ubiquitin conjugase 7; UBE1, ubiquitin-activating enzyme 1; UBE2G2, ubiquitin-activating enzyme 2G2; UBX2, UBX domain-containing protein 2; UCH-L1, ubiquitin C-terminal esterase L1; Ufd1, ubiquitin fusion degradation protein 1; UPRE, unfolded protein response element; VCP, valocin-containing protein; WFS1, Wolfram syndrome 1; XBP1, x-box binding protein 1; XIAP, inhibitor of apoptosis, x-linked; XTP3B, XTP3-transactivated gene B.

The endoplasmic reticulum (ER) is an organelle where

secretory or membrane proteins are synthesized

Nas-cent proteins are folded with the assistance of

molecu-lar chaperones and folding enzymes located in the ER

(collectively called ER chaperones), and only correctly

folded proteins are transported to the Golgi apparatus

(Fig 1) Unfolded or malfolded proteins are retained

in the ER, retrotranslocated to the cytoplasm by the

machinery of ER-associated degradation (ERAD), and

degraded by the proteasome ER chaperones and

ERAD components are constitutively expressed in the

ER to deal with nascent proteins When cells

synthes-ize secretory proteins in amounts that exceed the

capa-city of the folding apparatus and ERAD machinery,

unfolded proteins are accumulated in the ER

Unfol-ded proteins expose hydrophobic amino-acid residues

that should be located inside the protein and tend to

form protein aggregates Protein aggregates are so

toxic that they induce apoptotic cell death and cause

‘conformational diseases’ such as neurodegenerative

disorders and diabetes mellitus To alleviate such a

stressful situation (ER stress), eukaryotic cells activate

a series of self-defense mechanisms referred to

collec-tively as the ER stress response or unfolded

pro-tein response [1–4]

The mammalian ER stress response consists of fourmechanisms The first is attenuation of protein synthe-sis, which prevents any further accumulation of un-folded proteins The second is the transcriptionalinduction of ER chaperone genes to increase foldingcapacity, and the third is the transcriptional induction

of ERAD component genes to increase ERAD ability.The fourth is the induction of apoptosis to safely dis-pose of cells injured by ER stress to ensure the survival

of the organism

In this article, I will describe the basics of the malian ER stress response that are essential to under-standing conformational diseases I will review hottopics such as ERAD, regulated intramembrane pro-teolysis (RIP) and cytoplasmic splicing, and brieflysummarize the ER stress-related diseases

mam-ER stress-inducing chemicals

Chemicals such as tunicamycin, thapsigargin, anddithiothreitol are usually used to evoke ER stress incultured cells or animals for experimental purposes Iwill briefly summarize the ER stress-inducing chemicalsbelow

The first group of ER stressors comprises tion inhibitors Most of the proteins synthesized inthe ER are N-glycosylated, and the N-glycosylation is

glycosyla-cytoplasm

ER

ER chaperone

degraded ribosome

often essential for protein folding Thus, chemicals that

disturb N-glycosylation have the potential to induce

ER stress Tunicamycin is an antibiotic produced by

Streptomyces lysosuperificus that inhibits

N-glycosyla-tion by preventing UDP-GlcNAc–dolichol phosphate

GlcNAc-phosphate transferase activity [5,6]

2-Deoxy-d-glucose is also used to inhibit N-glycosylation [7],

but is less efficient than tunicamycin

Another class of ER stressors is Ca2+ metabolism

disruptors As the concentration of Ca2+ ion in the

ER is kept at a high level and ER chaperones such as

BiP require Ca2+ ions, chemicals that perturb Ca2+

metabolism in the ER induce ER stress Ca2+

ionoph-ores such as A23187 and the Ca2+ pump inhibitor,

thapsigargin, are often used to evoke ER stress [5,8]

The third category of ER stressors is reducing

agents As the lumen of the ER is highly oxidative,

proteins synthesized there can form intermolecular or

intramolecular disulfide bonds between their cysteine

residues As the formation of disulfide bonds is

important for the folding of secretory proteins,

redu-cing agents that disrupt disulfide bonds evoke ER

stress Dithiothreitol and 2-mercaptoethanol are often

used to this end [9,10]

Hypoxia is also known to induce ER stress,

although the underlying mechanism is unknown It is

speculated that a decrease in glucose concentration

induced by hypoxia (because hypoxia induces

glyco-lytic enzymes to sustain ATP production and then cells

consume glucose) inhibits N-glycosylation, leading to

ER stress [11]

ER chaperones

ER chaperones include molecular chaperones and

fold-ing enzymes located in the ER, which are responsible

for the folding of nascent proteins [4,12] They are also

involved in the unfolding of malfolded proteins in

ERAD In this section, I will review mammalian ER

chaperones, focusing on recent discoveries

Binding protein (BiP)⁄ glucose-regulated protein (GRP)78

is a well-known ER chaperone that belongs to the heat

shock protein (HSP)70 family BiP binds to the

hydro-phobic region of unfolded proteins via a

substrate-binding domain and facilitates folding through

conformational change evoked by the hydrolysis of

ATP by the ATPase domain Oxygen-regulated

pro-tein (ORP)150⁄ GRP170 is an ER chaperone belonging

to the HSP110 family (a HSP70 subfamily), and

facili-tates protein folding via a mechanism similar to

that for BiP It was originally identified as a

pro-tein expressed in response to hypoxia ER dnaJ

(ERdj)1, ERdj3⁄ human ER-asociated dnaJ (HEDJ),

ERdj4, ERdj5, SEC63, and p58IPK are ER ones belonging to the HSP40 family, and modulate thefunctions of BiP by regulating its ATPase activity as acochaperone BiP-associated protein (BAP), which is amember of the GrpE family, also modulates the func-tions of BiP by enhancing nucleotide exchange.GRP94 is an ER chaperone belonging to the HSP90family, and facilitates folding through the hydrolysis

chaper-of ATP FKBP13 is a peptidyl-prolyl isomerasebelonging to the FKBP family These ER chaperonesare involved in the general folding process of secretoryproteins

Calnexin and calreticulin are ER chaperones ically involved in the folding of glycoprotein High-mannose type oligosaccharide is attached en bloc tomost proteins synthesized in the ER, and then trimmedsequentially (Fig 2) When two glucose residues aretrimmed by glucosidase I or II and the protein con-tains only one glucose residue, calnexin and calreticulinbind and fold the client protein When the last glucoseresidue is trimmed by glucosidase II, the client isreleased from calnexin and calreticulin, and binds toUDP-glucose–glycoprotein glucosyltransferase If theprotein is folded, it is released from the enzyme andtransported to the Golgi apparatus If it is not folded,UDP-glucose–glycoprotein glucosyltransferase attachesone glucose residue and returns it to calnexin and cal-reticulin This folding process is called the calnexincycle [13] Calnexin and calreticulin share a similarmolecular structure and function, although they aretransmembrane and luminal proteins, respectively.Numerous folding enzymes are involved in the forma-tion of disulfide bonds in the ER, such as protein disul-fide isomerase (PDI), ERp72, ERp61, GRP58⁄ ERp57,ERp44, ERp29, and PDI-P5 These folding enzymesoxidize cysteine residues of nascent proteins and helpproteins to form correct disulfide bonds Reduced fold-ing enzymes are reoxidized by ER oxidoreductin(ERO1), which can use molecular oxygen as a terminalelectron acceptor [14]

specif-ERAD

Unfolded or malfolded proteins are trapped by theERAD machinery and transported to the cytoplasm[15–17] Retrotranslocated proteins are ubiquitinatedand degraded by the proteasome in the cytosol.Thus, the process of ERAD can be divided into foursteps, recognition, retrotranslocation, ubiquitination,and degradation (Fig 3) As ERAD is one of thehottest topics in the study of ER stress, I will sum-marize our current understanding of mammalianERAD systems

During the calnexin cycle, the oligosaccharide of nascent

polypeptides contains nine mannose residues When one

mannose residue is trimmed by a-mannosidase I,

nas-cent polypeptides with eight mannose residues are

released from calnexin or calreticulin and bind to

ER degradation-enhancing a-mannosidase-like tein (EDEM) (Fig 2), which discriminates unfoldedproteins from folded proteins [18–22] There are threegenes for EDEM, and both EDEM1 and EDEM2are involved in ERAD EDEM1 is an ER membraneprotein, whereas EDEM2 and EDEM3 are luminal pro-teins [23–25] All EDEMs contain the mannosidase-like

pro-Fig 3 Mammalian ERAD machinery Unfolded proteins released from the calnexin cycle are captured by a recognition complex containing EDEM and OS9, moved to the cytosol through retrotranslocation machinery, polyubiquitinated by the E1–E2–E3 system, and degraded by the proteasome The precise function of each ERAD component is described in the text.

UDP-GP Glc1Man9GlcNAc2-unfolded protein Man9GlcNAc2-unfolded protein

Man9GlcNAc2-folded protein

Man8GlcNAc2-folded protein Glc1Man8GlcNAc2-unfolded protein Man8GlcNAc2-unfolded protein

ER

EDEM

mannosidase I mannosidase I

Fig 2 Folding and degradation of glycoprotein Sugar chains of nascent glycoproteins synthesized in the ER are trimmed by glucosidase I or

II, and polypeptides containing one glucose residue are folded by the calnexin cycle One mannose residue of polypeptides that is unable to

be folded by the calnexin cycle is removed by mannosidase I, and then the polypeptides are recognized by EDEM and degraded by ERAD.

domain, which may be responsible for recognition of

mannose residues

Osteosarcoma 9 (OS9) and XTP3-transactivated

gene B (XTP3B) are other ERAD components

respon-sible for the recognition of unfolded proteins [26–28]

OS9 specifically binds to unfolded glycoproteins

con-taining eight (or five) mannose residues OS9 also

binds to unglycosylated unfolded proteins, suggesting

that it plays a critical role in the recognition of both

glycosylated and unglycosylated proteins OS9 and

XTP3B [29] contain the mannose-6-phosphate

recep-tor-like domain, which may be critical to the

recogni-tion of mannose residues

Retrotranslocation

Nascent glycoproteins recognized by EDEM and OS9

as malfolded are destined for the retrotranslocation

machinery [30,31] Before their retrotranslocation,

nas-cent proteins associate with PDI and BiP to cleave

disulfide bonds and to unfold the partially folded

struc-ture, respectively [32–34] Although unfolded ER

pro-teins were previously speculated to be retrotranslocated

through the translocon containing Sec61, the molecular

structure of the retrotranslocation machinery remains

elusive Derlin-1 is a mammalian homolog of yeast

Der1, and thought to be a critical component of the

machinery Derlin-1 may form a retrotranslocation

channel in the ER membrane and associates with p97

through an adaptor protein, valocin-containing

protein (VCP)-interacting membrane protein 1

(VIMP1) [35] Derlin-2 and Derlin-3, other Der1

homo-logs, are also involved in ERAD [35–37], although the

exact underlying mechanism is still unclear

p97⁄ cdc48 ⁄ VCP is a cytosolic AAA-ATPase and

recruits unfolded ER proteins to the cytosol [38,39]

Ubiquitin fusion degradation protein 1 (Ufd1) and

nuclear protein localization 4 (Npl4) bind to p97 as a

cofactor and help p97 to extract unfolded proteins

The polypeptide portion of unfolded proteins interacts

with p97, whereas the polyubiquitin chains attached to

them are recognized by both p97 and Ufd1 and may

activate the ATPase activity of p97 [40–42]

Ubiquitination

Retrotranslocated (or retrotranslocating) proteins are

ubiquitinated by the E1–E2–E3 ubiquitin system

Ubiquitin is first conjugated to enzyme E2 by enzyme

E1, and then transferred to ERAD substrates by

enzyme E3 HMG-CoA reductase degradation

pro-tein 1 (HRD1), gp78, and TEB4⁄ Doa10 are

mem-brane-anchored E3 ligases involved in ERAD [43–46],

whereas ubiquitin conjugase (UBC)6 and G2⁄ UBC7 are E2 conjugase involved in ERAD UBE1

UBE2-is an E1 ubiquitin-activating enzyme that UBE2-is tously involved in protein degradation by the protea-some HRD1 shows a preference for substrates thatcontain misfolded luminal domains, whereas Doa10prefers transmembrane proteins containing misfoldedcytosolic domains (Doa10 also ubiquitinates cytosolicproteins) These two distinctive ERAD systems arecalled ERAD-L (luminal ERAD) and ERAD-C (cyto-solic ERAD) [47,48] EDEM and OS9 are thought tospecifically recognize ERAD-L substrates Actually,they form distinct ubiquitin–ligase complexes: theHRD1 complex contains HRD1, OS9, HRD3, Derlin-

ubiqui-1, USAubiqui-1, UBX2 and p97, whereas the Doa10 complexconsists of Doa10, UBX2 and p97 [49–51] Substratescontaining misfolded transmembrane domains skip theinteraction to OS9 and HRD3, and directly associatewith the HRD1 complex, which is called the ERAD-Mpathway [49]

However, there are a lot of other E3 ligases involved

in the ERAD, and they preferentially recognize distinctERAD substrates FBX2 (F-box only protein 2) isanother E3 ligase that specifically recognizes N-glycos-ylated proteins located in the cytosol [52,53] Parkin is

an E3 involved in Parkinson’s disease (see below) Inthe case of cystic fibrosis transmembrane conductanceregulator, its folding status is sequentially monitored

by the two E3 ligase complexes, such as the RMA1complex and the CHIP (C-terminus of Hsc70-interact-ing protein) complex [54]

Molecules other than E1–E2–E3 enzymes are alsoinvolved in ubiquitination UBX2 binds to both p97and E3 ligases such as HRD1 and Doa10 to recruitE3 to p97 [55], whereas gp78 directly associates withp97 [56] The ubiquitin-domain protein, Herp (homo-cysteine-induced endoplasmic reticulum protein),associates with a complex containing HRD1, p97,Derlin-1, and VCP-interacting membrane pro-tein [57,58]

DegradationRetrotranslocated and ubiquitinated proteins aredeglycosylated by peptide–N-glycanase before theirdegradation by the proteasome, because bulky glycanchains may hamper the entrance of substrates intothe proteasome pore As peptide–N-glycanase is asso-ciated with Derlin-1, it is possible that deglycosylationoccurs coretrotranslocationally [59] Deglycosylatedsubstrates are then delivered to the proteasome Dsk2and Rad23 facilitate this delivery of ERAD substrates[60]

Response pathways for ER stress

The mammalian ER stress response has four

mecha-nisms: (1) translational attenuation; the enhanced

expression of (2) ER chaperones and (3) ERAD

components; (4) induction of apoptosis These four

responses are regulated by the regulatory pathways as

described below (Fig 4)

PERK pathway

PERK is a type I transmembrane protein located in the

ER, which senses the accumulation of unfolded

pro-teins in the ER lumen [61–63] The luminal portion of

PERK is involved in sensing unfolded proteins,

whereas the cytoplasmic portion contains a kinase

domain In the absence of ER stress, BiP binds to the

luminal domain of PERK and keeps it from being

acti-vated (Figs 4 and 5A) In response to ER stress, BiP is

released from PERK, and PERK is activated through

oligomerization and trans-phosphorylation [64]

Activa-ted PERK phosphorylates and inactivates the a-subunit

of eukaryotic translational initiation factor 2 (eIF2a),

leading to translational attenuation The tion of PERK is transient as the protein is dephosphor-ylated by specific phosphatases such as CReP(constitutive repressor of eIF2a phosphorylation), pro-tein phosphatase 2C-GADD34, and p58IPK CReP isconstitutively expressed, whereas the expression ofGADD34 and p58IPK is induced on ER stress byPERK and activating transcription factor (ATF)6pathways, respectively

phosphoryla-Interestingly, translation of the transcription factorATF4 is up-regulated by eIF2a-mediated translationalattenuation There are several small ORFs in the5¢-UTR of ATF4 mRNA (Fig 5B) The ribosome firstbinds to a 5¢-cap structure, slides on the ATF4mRNA, and then starts translation at the small ORFswith unphosphorylated (active) eIF2a As the ribosome

is released from the ATF4 mRNA upon the ation of translation at the stop codon of small ORFs,the ATF4 ORF cannot be translated in the absence of

termin-ER stress In contrast, as phosphorylated (inactive)eIF2a cannot start translation, the probability that theribosome reaches the ATF4 ORF is increased in thepresence of ER stress Thus, the translation of ATF4

ATF6 PERK

DBD-AD mature mRNA

NF-Y

pXBP1(S) pXBP1(U)

Fig 4 Mammalian response pathways for ER stress Three response pathways (PERK, ATF6, and IRE1 pathways) regulate the mammalian

ER stress response PERK, a transmembrane kinase, phosphorylates eIF2a to attenuate translation, and to up-regulate expression of ATF4, leading to enhanced transcription of target genes such as CHOP ATF6, a transmembrane transcription factor, is translocated to the Golgi apparatus and cleaved by proteases such as S1P and S2P, leading to enhanced transcription of ER chaperone genes IRE1, a transmem- brane RNase, splices XBP1 pre-mRNA, and pXBP1(S) translated from mature XBP1 mRNA activates transcription of ERAD component genes.

is remarkably enhanced in response to ER stress The

targets of ATF4 include CHOP (C⁄ EBP homology

protein), a transcription factor involved in the

induc-tion of apoptosis, and proteins involved in amino-acid

metabolism such as asparagine synthetase or those

involved in resistance to oxidative stress [65]

eIF2a is also phosphorylated by other kinases, such

as dsRNA-dependent protein kinase (PKR), GCN2

(general control of amino-acid synthesis 2) and

heme-regulated translational inhibitor These kinases are

activated by viral infections, amino-acid starvation,

and heme deficiency, respectively, indicating that

trans-lational attenuation and ATF4 induction is induced by

not only ER stress but also these physiological tions Thus, the cellular response mediated by thephosphorylation of eIF2a is called the integrated stressresponse and is essential for cell survival [66]

situa-ATF6 pathwayThere is another sensor molecule, ATF6, on the ERmembrane [67–70] ATF6 is a type II transmembraneprotein, the luminal domain of which is responsible forthe sensing of unfolded proteins The cytoplasmicportion of ATF6 has a DNA-binding domain con-taining the basic-leucine zipper motif (bZIP) and a

small ORFs CAP

- ER stress

ATF4 mRNA CAP

eIF2α

ATF4 mRNA CAP

to the release of ribosomes before they reach the ATF4 ORF Upon ER stress, most

of the eIF2a becomes inactive ated), and translation rarely starts at the small ORFs, thus ribosomes can reach the ATF4 ORF and induce translation of ATF4 protein.

(phosphoryl-transcriptional activation domain In the absence of

ER stress, BiP binds to the luminal domain of ATF6

and hinders the Golgi-localization signal, leading to

inhibition of ATF6 translocation (Fig 5A) [71–75] In

response to the accumulation of unfolded proteins, BiP

dissociates from ATF6, and ATF6 is moved to the

Golgi apparatus by vesicular transport (Fig 4) In the

Golgi apparatus, ATF6 is sequentially cleaved by a

pair of processing proteases called site 1 protease (S1P)

and site 2 protease (S2P), and the resultant

cytoplas-mic portion of ATF6 [pATF6(N)] translocates into the

nucleus In the nucleus, pATF6(N) binds to a

cis-act-ing element, the ER stress response element (ERSE),

and activates the transcription of ER chaperone genes

such as BiP, GRP94 and calreticulin [68] The

consen-sus sequence of the ER stress response element is

CCAAT-(N9)-CCACG, and ATF6 binds to the

CCACG portion, whereas a general transcription

fac-tor, NF-Y (nuclear factor Y), binds to the CCAAT

portion

The cleavage of ATF6 is unique, especially as the

second cleavage by S2P occurs in the transmembrane

region [75] This process is called regulated

intramem-brane proteolysis (RIP), which is well conserved from

bacteria to mammals (Fig 6) The most characterized

substrate of RIP is sterol response element-binding

protein (SREBP) [75] SREBP is a transcription factor

that is located in the ER membrane like ATF6 Upon a

deficiency of sterol, SREBP is transported to the Golgiapparatus, cleaved by S1P and S2P, and activates thetranscription of genes involved in the biosynthesis ofsterol Thus, the activation of ATF6 and SREBP ismainly regulated at the level of vesicular transport Theregulation of the transport of SREBP has been wellcharacterized, and regulatory components such as thesensor-escort protein SCAP (SREBP cleavage-activa-ting protein) and the anchor protein INSIG (insulin-induced gene 1) have been identified [76]

There are two genes for ATF6, called ATF6a andATF6b, which have a similar function and are ubiqui-tously expressed [68,77] Recently, several bZIP tran-scription factors located in the ER and regulated byRIP have been reported cAMP response element-bind-ing protein H (CREBH) is specifically expressed inliver, and processed by S1P and S2P in response to

ER stress [78] CREBH activates the transcription ofacute-phase response genes involved in acute inflam-matory responses OASIS (old astrocyte specificallyinduced substance) is also cleaved by S1P and S2P inresponse to ER stress in astrocytes and activates thetranscription of BiP [79] A spermatid-specific tran-scription factor, Tisp40 (transcript induced in spermio-genesis 40), is also severed by S1p and S2P andactivates the transcription of EDEM [80] These tissue-specific ATF6-like molecules may contribute to the ERstress response

Fig 6 Molecules regulated by RIP RIP is conserved from bacteria to mammals, and is involved in various biological processes SREBP ses a sterol deficiency and activates the transcription of genes involved in sterol synthesis Cleavage of APP by RIP results in the production

sen-of antibody, which is responsible for the onset sen-of Alzheimer’s disease Notch is a cell surface protein that is cleaved by RIP upon binding Delta, leading to the activation of target genes involved in differentiation Bacterial RseA protein anchors a transcription factor, r E , to keep it inactive In response to accumulation of unfolded proteins in the periplasm, RseA is cleaved by RIP, leading to transcriptional activation of periplasmic chaperones.

Luman⁄ LZIP ⁄ CREB3 can be cut by S1P and S2P

and activates the transcription of EDEM through a

cis-acting element, unfolded protein response element

(UPRE), although ER stress cannot induce Luman

RIP [80–82] CREB4 is transported to the Golgi

apparatus in response to ER stress, is cleaved by S1P

and S2P, and activates the transcription of BiP,

although cleavage is not observed upon ER stress [83]

These ATF6-like molecules, which are insensitive to

ER stress, might be activated in situations other than

ER stress and activate transcription of ER chaperones

IRE1 pathway

The third sensor molecule in the ER membrane is

IRE1 (inositol requirement 1) [84–86] The luminal

domain of IRE1 is similar to that of PERK and

involved in the sensing of unfolded proteins, whereas

the cytoplasmic domain contains a kinase domain and

an RNase domain There are two genes for IRE1,

IRE1a and IRE1b Upon ER stress, BiP suppression

of IRE1 activation is released, and IRE1 is activated

through dimerization and transphosphorylation (Figs 4

and 5A) [64] Activated IRE1a converts XBP1 (x-box

binding protein 1) pre-mRNA into mature mRNA by

an unconventional splicing mechanism [69,87] As the

DNA-binding domain and the activation domain are

encoded in ORFs in XBP1 pre-mRNA, a

pro-tein translated from pre-mRNA [pXBP1(U)] cannot

activate transcription In contrast, a protein translated

from mature mRNA [pXBP1(S)] activates the

tran-scription of ERAD component genes such as EDEM,

HRD1, Derlin-2, and Derlin-3 through a cis-acting

ele-ment, unfolded protein response eleele-ment, as these two

ORFs are joined in mature mRNA [37,88,89]

pXBP1(S) also induces the expression of proteins

involved in lipid synthesis and ER biogenesis, as well

as the expression of ER chaperones such as BiP,

p58IPK, ERdj4, PDI-P5 and HEDJ [90,91] Thus,

XBP1 is essential to the function of cells that produce

large amounts of secretory proteins such as pancreatic

b-cells, hepatocytes, and antibody-producing plasma

cells [92–95]

The splicing of XBP1 pre-mRNA by IRE1a is quite

different from conventional mRNA splicing (Fig 7A)

[69] Conventional splicing is catalyzed by the

spliceo-some, and the consensus sequence at the exon–intron

border is GU-AG or AU-AC (Chambon’s rule) The

splicing reaction is sequential: the 5¢ site is cleaved first,

then the 3¢ site after a lariat structure is formed In

con-trast, unconventional splicing of XBP1 pre-mRNA is

catalyzed by IRE1a and RNA ligase, and there is a pair

of stem–loop structures at the exon–intron border

instead of GU-AG or AU-AC Moreover, the splicingreaction is not sequential but random

The most important difference between conventionaland unconventional splicing is where the reactionoccurs (Fig 7B) Conventional splicing (nuclear spli-cing) takes place in the nucleus, whereas unconven-tional splicing (cytoplasmic splicing) occurs in thecytoplasm The biological significance of cytoplasmicsplicing is that pre-mRNA used for translation in thecytoplasm can be spliced when it is necessary tochange the nature of the protein translated from themRNA, in response to extracellular or intracellularsignaling In contrast, as nuclear splicing cannot splicemRNA exported to the cytoplasm, it is necessary forpre-mRNA to be transcribed de novo and spliced.Thus, cytoplasmic splicing would be a very rapid, ver-satile, and energy-efficient mechanism with minimalwaste as compared with conventional mRNA splicing.Recently, it was found that pXBP1(U) encoded inXBP1 pre-mRNA is a negative feedback regulator ofpXBP1(S) Thus, in the case of XBP1, pre-mRNA andmature mRNA encode negative and positive regula-tors, respectively, and their expression is switched bycytoplasmic splicing in response to the situation in the

ER [96]

IRE1b is specifically expressed in epithelial cells

of the gastrointestinal tract, and thought to cleaverRNA to attenuate translation in response to ERstress [84] When IRE1b–⁄ – mice were exposed to aninducer of inflammatory bowel disease, they actuallydeveloped colitis, possibly because of the enhanced

ER stress [97]

Recently, the crystal structure of the luminal domain

of IRE1a was solved [98] The luminal domain is ilar in structure to the peptide-binding domain ofmajor histocompatibility complexes, suggesting theinteresting possibility that it directly senses ER stress

sim-by directly binding unfolded proteins

Apoptosis-inducing pathwaysThe accumulation of unfolded proteins in the ER istoxic to cells Thus, if the PERK, ATF6, and IRE1pathways cannot suppress ER stress, an apoptoticpathway is triggered to ensure survival of the organism

as a last line of defense A number of pathways havebeen reported to be involved in ER stress-inducedapoptosis, and the full induction of apoptosis seems torequire the concomitant activation of several deathpathways, although there remain many arguments over

ER stress-induced apoptosis [99–105] In this section, Iwill briefly summarize the known death pathways,focusing on recent progress (Fig 8)

The most characterized pathway is the CHOP

path-way CHOP⁄ GADD153 (growth arrest and DNA

damage 153) is a transcription factor, the expression of

which is induced by the ATF6 and PERK pathways

upon ER stress [70,106,107] CHOP–⁄ – cells exhibit

less programmed cell death when faced with ER stress

[108], suggesting that the CHOP pathway is a major

regulator of ER stress-induced apoptosis As for the

target genes of CHOP, CHOP activates the

transcrip-tion of GADD34, ERO1, DR5 (death receptor 5), and

carbonic anhydrase VI, which seem to be responsible

for apoptosis GADD34 associated with protein

phos-phatase 2C enhances dephosphorylation of eIF2a and

promotes ER client protein biosynthesis [109], whereas

ERO1, which encodes an ER oxidase, makes the ER a

more hyper-oxidizing environment [110] DR5, which

encodes a cell surface death receptor, may activate

caspase cascades [111] Carbonic anhydrase VI maychange the cellular pH, affecting various cellular pro-cesses [112,113] However, the exact signaling mechan-ism from CHOP to apoptosis is still unclear

The second apoptotic pathway is the IRE1–TRAF2–ASK1 pathway The cytoplasmic part of IRE1 binds

to an adaptor protein, TRAF2 (tumor necrosis factorreceptor-associated factor 2), which couples plasmamembrane death receptor to Jun kinase (JNK) andstress-activated protein kinase (SAPK) [114] IRE1 andTRAF2 form a complex with a mitogen-activatedprotein kinase kinase kinase, ASK1 (apoptosis signal-regulating kinase 1), and this IRE1–TRAF2–ASK1complex is responsible for the phosphorylation andactivation of JNK [115] Actually, IRE1–⁄ – cells aswell as ASK1–⁄ – cells are impaired in the activation

of JNK and apoptosis by ER stress In contrast,

A

B

Fig 7 Cytoplasmic splicing (A) Comparison

between nuclear and cytoplasmic splicing.

Conventional splicing is catalyzed by the

spliceosome in the nucleus, and there is a

consensus sequence at the exon–intron

boundary such as GU-AG or AU-AC The

splicing reaction is sequential: the 5¢ site is

cleaved first, the lariat structure is formed,

and then the 3¢ site is cleaved In contrast,

unconventional splicing is catalyzed by IRE1

and RNA ligase in the cytoplasm, there is a

characteristic stem–loop structure at the

boundary, and the splicing reaction is

ran-dom without forming a lariat structure (B)

Biological significance of cytoplasmic

spli-cing As nuclear splicing cannot splice

pre-mRNA exported to the cytoplasm, de novo

transcription is required to change the

char-acter of the protein encoded in the

pre-mRNA In contrast, as cytoplasmic splicing

can splice pre-mRNA that is translated in

the cytoplasm, it can rapidly change the

character of a protein in response to

exter-nal or interexter-nal stimuli, without de novo

transcription.

TRAF2–⁄ – cells are more susceptible to apoptosis

trig-gered by ER stress, which might be inconsistent with

the above model [116] TRAF2 also associates with

caspase-12 and regulates its activation [117] IRE1–

TRAF2 activates the transcriptional repressor ATF3

as well, leading to the activation of apoptosis [118]

These suggest that the IRE1–TRAF2–ASK1 pathway

is a major regulator of ER stress-induced apoptosis

TNFR1 (tumor necrosis factor receptor 1), a receptor

for TNF-induced cell death, associates with IRE1a

upon ER stress, and the activation of JNK by ER

stress is impaired in TNFR1–⁄ – cells This suggests

that TNFR1 mediates the ER stress-induced activation

of JNK [119], possibly forming a complex with IRE1a,

TRAF2, and ASK1 The expression of TNFa is

up-regulated by the IRE1 pathway during ER stress

[120,121], which may contribute to the activation of

TNFR1

Caspases are well-known pro-apoptotic components,

and caspases 2, 3, 4, 7, 9 and 12 are reported to be

involved in ER stress-induced cell death [122–131]

Caspase-12 is associated with the ER membrane, and

activated by ER stress, possibly by calpain [132] Then

caspase-12 activates caspase-9, which in turn activatescaspase-3 [133], leading to cell death Caspase-12–⁄ –mice are resistant to ER stress-induced apoptosisbut sensitive to other death stimuli, suggesting thatcaspase-12 is a regulator specific to ER stress-inducedapoptosis [134] The activation of caspase-12 by ERstress is observed in models of various diseases such asAlzheimer’s disease, polyglutamine disease, ischemia,and viral infection [135–139], suggesting that ERstress-induced apoptosis is closely involved in thesediseases (see below) However, the involvement ofcaspase-12 in apoptosis of human cells is still open toquestion, as the human caspase-12 gene contains sev-eral mutations critical to function [140] It is possiblethat an unidentified caspase other than caspase-12 isresponsible in human cells

Bcl2 family proteins are well-known components ofthe programmed cell death machinery, and some keycomponents are involved in ER stress-induced apopto-sis In general, pro-apoptotic members of the Bcl2family seem to be recruited to the ER surface and

to activate caspase-12, whereas the anti-apoptoticmembers inhibit this recruitment, although the exact

Fig 8 ER stress-induced apoptotic pathways The subcelluar distribution of factors involved in ER stress-induced cell death is shown apoptotic and anti-apoptotic factors are indicated in black and white letters, respectively Pro-apoptotic Bcl2 proteins positively regulate the IRE1 and caspase pathways, whereas anti-apoptotic Bcl2 proteins negatively regulate the latter p53 enhances expression of pro-apoptotic Blc2 family proteins such as PUMA and NOXA The IRE1 pathway activates JNK and SAPK, leading to apoptosis c-Abl translocates from the ER to the mitochondria in response to ER stress A fuller explanation is given in the text.

Pro-relationship between these factors is still unclear So I

will briefly describe our current understanding of Bcl2

family proteins The anti-apoptotic factor Bcl2 is

down-regulated by the transcription factor CHOP

upon ER stress, which leads to enhanced oxidant

injury and apoptosis [141] Overexpression of Bcl2

inhibits the activation of caspase-12 and apoptosis

during ER stress [142] The pro-apoptotic factor, BAD

(Bcl2 antagonist of cell death), is dephosphorylated

and activated in response to ER stress [143], whereas

other pro-apoptotic factors, Bax (Bcl2-associated X

protein) and Bak (Bcl-2 homologous antagonist⁄

kil-ler), are present in the ER membrane as well as the

mitochondrial membrane [144,145] During ER stress,

Bax and Bak oligomerize and activate caspase-12

Interestingly, Bax and Bak associate with IRE1a and

modulate IRE1a function during ER stress [146] Bax

and Bak are required for most forms of apoptosis

[147] The transcription of PUMA (p53 up-regulated

modulator of apoptosis) and NOXA (neutrophil

NADPH oxidase factor), pro-apoptotic members of

the BH3 (homology domain-3) domain-only family, is

up-regulated by p53 during ER stress, and PUMA–⁄ –

cells and NOXA–⁄ – cells are resistant to ER

stress-induced apoptosis [148,149] Another pro-apoptotic

component, Bim (Bcl2-interacting mediator of cell

death), translocates from the dynein-rich compartment

to the ER membrane and activates caspase-12 in

response to ER stress, whereas an anti-apoptotic

fac-tor, Bcl-xL (Bcl-2-like 1), binds to Bim and inhibits its

translocation [150] Bim-knockdown cells are resistant

to ER stress The ER-localized anti-apoptotic factor

BI-1 (Bax inhibitor-1) inhibits the activation of Bax

during ER stress, and BI-1–⁄ – mice are sensitive to

ER stress, whereas mice overexpressing BI-1 are

resist-ant [151] BIK (Bcl2-interacting killer) is an

ER-locali-zed pro-apoptotic component which enhances the

recruitment of BAX and BAK to the ER [152] Bap31

(B cell receptor-associated protein 31) is a

pro-apop-totic factor that is cleaved and activated upon ER

stress, its cleavage being dependent on calnexin [153]

In calnexin-deficient cells, the cleavage of Bap31 and

ER stress-induced apoptosis are inhibited

The inhibitor of apoptosis (IAP) family has also

been reported to be involved in ER stress-induced

apoptosis Human inhibitor of apoptosis 2 (HIAP2) is

an IAP that inhibits caspase-3 and caspase-7

Expres-sion of HIAP2 is induced upon ER stress at the level

of translation: caspases activated by ER stress cleave

eukaryotic initiation factor, p97⁄ DAP5 ⁄ NAT1, and

the cleavage product specifically activates the HIAP2

internal ribosome entry site, leading to enhanced

trans-lation of HIAP2 [131] Transcription of IAP-2 and

XIAP (inhibitor of apoptosis, X-linked), two otherIAPs, is up-regulated during ER stress, and cells inwhich these IAPs have been knocked down are sensi-tive to ER stress-induced apoptosis [154] Cells overex-pressing XIAP or HIAP1 are resistant to ER stress[122,155] These results suggest involvement of IAPproteins in ER stress-induced apoptosis

c-Abl (Abelson murine leukemia viral oncogenehomolog 1) is a protein tyrosine kinase distributed inthe nucleus and cytoplasm, and c-Abl activated by adeath signal induces phosphorylation and activation ofpro-apoptotic JNK and SAPK Interestingly, c-Abl isalso located on the ER surface, and translocates to themitochondria upon ER stress, where it induces therelease of cytochrome c [156] c-Abl–⁄ – cells are resist-ant to ER stress-induced apoptosis A c-Abl-interact-ing protein, Aph2 (anterior phalynx defective 2), isalso located on the ER and shows pro-apoptotic acti-vity [157], suggesting that c-Abl forms a distinct path-way leading to ER stress-induced apoptosis

PKR is a dsRNA-dependent protein kinase which isactivated upon viral infection, and its phosphorylation

in the nucleus is up-regulated in response to ER stress.Interestingly, PKR-knockdown cells or PKR mutantcells are resistant to ER stress-induced apoptosis, sug-gesting that PKR is a pro-apoptotic factor during ERstress [158]

TDAG51 (T-cell death-associated gene 51) is a ber of the pleckstrin homology-related domain family,and its transcription is induced by ER stress throughthe PERK pathway [159] Overexpression of TDAG51induces apoptosis, suggesting that TDAG51 is involved

mem-in ER stress-mem-induced apoptosis and the development ofatherosclerosis (see below)

Nuclear respiratory factor (NRF)1 and NRF2 aretranscription factors that regulate the oxidative stressresponse NRF2 is distributed in the cytoplasmthrough its association with the microtubule-associatedprotein Keap1 (Kelch-like Ech-associated protein 1).Upon ER stress, PERK phosphorylates NRF2 anddissociates it from Keap1, leading to the nuclearrecruitment of NRF2 [160] Remarkably, NRF2–⁄ –cells are sensitive to ER stress-induced apoptosis,whereas NRF1 is located in the ER membrane, andtranslocates to the nucleus upon ER stress [161], sug-gesting that these proteins are involved in ER stress-specific apoptosis

ATF6 is also involved in the apoptotic process ing myogenesis In differentiating myoblast, the ATF6pathway is activated, and expression of BiP and CHOP

dur-is up-regulated, which may activate caspase-12 over, AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoridehydrochloride], an inhibitor of ATF6 activation, blocks

More-apoptosis, suggesting that the ATF6 pathway

contri-butes to apoptosis during myogenesis [162]

TRB3 (Tribbles homolog 3) is a human ortholog of

Drosophila tribble, and its transcription is induced by

ER stress through the PERK–ATF4–CHOP pathway

Interestingly, TRB3-knockdown cells are resistant to

ER stress-induced apoptosis, suggesting that TRB3 is

a pro-apoptotic factor during ER stress [163,164]

p53 is a transcription factor that induces growth

arrest and apoptosis in response to various forms of

cellular stress, such as DNA damage There are several

reports suggesting a connection between p53 and ER

stress Upon ER stress, p53 is phosphorylated by

gly-cogen synthase kinase 3B, leading to the distribution

of p53 in the cytoplasm and destabilization of p53,

and attenuation of p53-dependent apoptosis [165–167]

Interestingly, Scotin and SCN3B (sodium channel

sub-unit beta 3), p53-inducible pro-apoptotic proteins, are

located in the ER [168,169] Moreover, transcription of

PUMA and NOXA, which are involved in ER

stress-induced apoptosis, is up-regulated by p53 during ER

stress [148], suggesting that the p53 pathway regulates

the apoptotic pathway during ER stress

Other components such as VCP and ALG-2

(apop-tosis-linked-gene 2) [104], AIGP1 [170], elongation

factor-1a [171], and NRADD (neurotrophin receptor

associated death domain) [172] are also reported to be

involved in ER stress-induced apoptosis Their precise

functions and working mechanisms are still to be

clar-ified

ER stress-related diseases

Unfolded or malfolded proteins readily form

aggre-gates in the ER as well as the cytosol Recent reports

suggested that small aggregates are highly toxic, as

they impair the ubiquitin proteasome pathway [173]

and sequester transcription factors such as

CREB-binding protein and TATA-CREB-binding protein [174,175],

whereas large aggregates such as the aggresome and

the inclusion body are cytoprotective [176]

Interest-ingly, molecular chaperones such as HSP70 and TRiC

(TCP1- ring complex) can suppress the formation as

well as toxicity of protein aggregates [174,177–181]

The diseases caused by the malfolding of cellular

pro-teins are collectively called ‘conformational diseases’ or

‘folding diseases’ As malfolded proteins and protein

aggregates can evoke ER stress, it has been speculated

that ER stress is involved in most conformational

dis-eases, particularly Alzheimer’s disease and Parkinson’s

disease, although it is still heavily disputed whether

ER stress (or protein aggregates) is a major cause of

these diseases In this section, I will briefly summarize

recent findings on how ER stress is involved in formational diseases

con-Neurodegenerative diseasesNeurons are thought to be sensitive to protein aggre-gates, and there are many reports that ER stress isinvolved in neurodegenerative diseases [182–184] Infact, disruption of SIL1⁄ BAP, a cochaperone of BiP,results in the accumulation of protein aggregates andneurodegeneration [184] Most of these diseases arecaused by aging or genetic background, althoughseveral are assumed to be infectious, especially priondisease

Alzheimer’s disease is the most common generative disease, and characterized by cerebral neu-ritic plaques of amyloid b-peptide [185–191] Recentfindings strongly suggest that one of the major causes

neurode-of Alzheimer’s disease is an accumulation neurode-of amyloidb-peptide, although tau also seems to be involved inthe disease Studies of patients with autosomal-domin-ant familial Alzheimer’s disease have identified threegenes responsible for the disease, amyloid precursorprotein (APP), PS1 and PS2 The amyloid precursorprotein encoded by APP is a transmembrane protein,the function of which is still unknown, whereas bothPS1 and PS2 encode a protein called presenilin, which

is an essential component of a protease called ase APP is sequentially cleaved by a b-secretase calledBACE (b-site amyloid b A4 precursor protein-cleavingenzyme 1) and c-secretase, leading to the accumulation

c-secret-of amyloid b-peptide Interestingly, cells expressingPS1 mutants show a waned ER stress response and aresensitive to ER stress [192] The activation of ATF6,IRE1, and PERK is also disturbed in these mutantcells [193] Moreover, proteins involved in ER stress-induced apoptosis, such as PKR and caspase-4, areinvolved in the onset of Alzheimer’s disease [125,158].Actually, the ER stress response is activated in patientswith Alzheimer’s disease [192,194,195], and poly-morphism of SEL1, a component of ERAD, is linked

to Alzheimer’s disease [196] These findings stronglysuggest a strong causal relationship between ER stressand Alzheimer’s disease, and it is highly possible that

ER stress invoked by the accumulation of amyloidb-peptide is one of the key mechanisms of Alzheimer’sdisease

Parkinson’s disease is the second most commonneurodegenerative disease, which is characterized by aloss of dopaminergic neurons [197] Analyses of patientswith familial Parkinson’s disease have revealed threegenes responsible for the disease, encoding a-synuc-lein, Parkin, and ubiquitin C-terminal esterase L1

(UCH-L1) a-synuclein is a cytoplasmic protein which

forms aggregates, called Lewy bodies, characteristic of

Parkinson’s disease, but the link between a-synuclein

and ER stress is unclear In contrast, Parkin is a

ubiqu-itin-protein ligase (E3) involved in ERAD [198] One of

the substrates of ERAD ubiquitinated by Parkin is Pael

receptor, a homolog of endothelin receptor type B [199]

Interestingly, expression of Parkin is induced by ER

stress, and neuronal cells overexpressing Parkin are

resistant to ER stress [200] As for UCH-L1, it is an

abundant protein in neurons and stabilizes a monomeric

ubiquitin [201,202] It has been shown that UCH-L1

ubiquitinates unfolded proteins and might be involved

in ERAD [203] These findings strongly suggest the

involvement of ER stress in Parkinson’s disease In

addition, there are several other reports supporting the

link between ER stress and Parkinson’s disease First,

Parkinson’s disease mimetics, such as

6-hydroxydopam-ine, specifically induce ER stress in neuronal cells

[204,205] Second, expression of ER chaperones such as

PDI is up-regulated in the brain of Parkinson’s disease

patients, and PDI is accumulated in Lewy bodies

[206] The incidence of sporadic Parkinson’s disease

increases with age, but it is still unclear whether the ER

stress response wanes in patients with Parkinson’s

disease

Polyglutamine (polyQ) diseases are

neurodegenera-tive disorders caused by duplications of the

CAG-repeat in certain genes, and include Huntington’s

disease, spinobulbar muscular atrophy (Kennedy

dis-ease), Machado-Joseph disease,

dentatorubral-pallido-luysian atrophy (Haw River Syndrome), and

spinocerebellar ataxia Large polyQ stretches translated

from the CAG-repeat form insoluble protein

aggre-gates, which are toxic to cells Although the exact

mechanism behind the toxicity of polyQ remains to be

clarified, one possibility is that polyQ aggregates

sequester other normal proteins, such as transcription

factors, which are indispensable to cell function [174]

Another possibility is that the polyQ aggregate itself is

toxic to cells All known polyQ proteins causing

neu-rodegenerative diseases are cytosolic, but they evoke

ER stress, as polyQ proteins suppress the function of

the proteasome, which is an essential component of

ERAD [115,136,207] Interestingly, p97, a component

of the ERAD machinery, enhances degradation of

po-lyQ proteins and suppresses popo-lyQ protein-induced

neurodegeneration [208] Judging from these findings,

it is probable that ER stress is involved in the onset of

polyQ diseases

Pelizaeus-Merzbacher disease is a progressive

neuro-degenerative disorder characterized by a loss of

coordi-nation, motor abilities, and intellectual function [209]

Currently, it is thought that the disease is caused by amutation of the PLP1 gene which encodes a transmem-brane proteolipid essential for the maintenance of mye-lin sheaths, as well as in oligodendrocyte developmentand axonal survival [210,211] As a missense mutationcauses a more severe phenotype than a null mutation,

it is speculated that the missense mutant of PLP1forms aggregates in the ER and evokes ER stress-induced cell death [212] Actually, expression ofCHOP, BiP, ERp59, and ERp72 is up-regulated in thebrains of mice expressing missense PLP and the brains

of patients with Pelizaeus-Merzbacher disease

Prion disease, also called transmissible form encephalopathy, encompasses Creutzfeldt-Jakobdisease, Gerstmann–Straussler–Scheinker syndrome,fatal familial insomnia, Kuru, Alpers syndrome,bovine spongiform encephalopathy, transmissible milkencephalopathy, chronic wasting disease, and scrapie[213] Characteristic features of the disease are a loss

spongi-of motor control and dementia The only gene to beidentified so far as being responsible for prion dis-ease is PrP, which encodes a protein anchored to thecell surface There is no difference in amino-acidsequence between the normal protein PrPc and itspathological form PrPSc PrPSc is rich in b-sheets,converts PrPc into PrPSc, and forms amyloid fibrilsthat are thought to be toxic to cells Interestingly, inmurine cells infected with PrPSc, the expression of

ER chaperones such as GRP58 and GRP94 as well

as caspase-12 is up-regulated [214] Moreover, theexpression of these chaperones is considerablyincreased in patients with Creutzfeldt-Jakob disease.Finally, overexpression of GRP58 protects cells fromPrPSc-induced cell death, whereas inhibition ofGRP58 expression with small interfering RNA results

in a severe phenotype [215] These findings stronglysuggest that ER stress is involved in the pathology

of prion disease

Amyotrophic lateral sclerosis, also called Lou rig’s disease, is a progressive neuromuscular diseaseand shows characteristic pathological features such as

Geh-a loss of motor neurons in the cerebrGeh-al cortex Geh-and nal cord Analysis of patients with familial amyotroph-

spi-ic lateral sclerosis has revealed that superoxidedismutase-1 is responsible for the disease Mutantsuperoxide dismutase-1 forms aggregates in the ER,evokes ER stress, induces the expression of BiP, andactivates caspase-12, leading to neuronal cell death.These findings support the notion that ER stressinduced by superoxide dismutase aggregates is a majorcause of amyotrophic lateral sclerosis [216–218].GM1 gangliosidosis is an autosomal recessivelysosomal storage disorder characterized by an