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Depletion of Molecular Chaperones from the Endoplasmic Reticulum and Fragmentation of the Golgi Apparatus Associated with Pathogenesis in Pelizaeus-Merzbacher Disease*

  • Yurika Numata
    Affiliations
    Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-machi, Kodaira-shi, Tokyo 187-8502

    Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryomachi, Aobaku, Sendai 980-8574
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  • Toshifumi Morimura
    Affiliations
    Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-machi, Kodaira-shi, Tokyo 187-8502

    Unit for Neurobiology and Therapeutics, Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta-Tsukinowa-cho, Otsu, Shiga 520-2192, Japan
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  • Shoko Nakamura
    Affiliations
    Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-machi, Kodaira-shi, Tokyo 187-8502
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  • Eriko Hirano
    Affiliations
    Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-machi, Kodaira-shi, Tokyo 187-8502
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  • Shigeo Kure
    Affiliations
    Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryomachi, Aobaku, Sendai 980-8574
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  • Yu-ich Goto
    Affiliations
    Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-machi, Kodaira-shi, Tokyo 187-8502
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  • Ken Inoue
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-42-346-1713; Fax: 81-42-346-1743;
    Affiliations
    Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-machi, Kodaira-shi, Tokyo 187-8502
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  • Author Footnotes
    * This work was supported in part by grants from the Health and Labor Sciences Research Grants, Research on Intractable Diseases H24-Nanchitou-Ippan-072 (to K. I.), a grant from Takeda Science Foundation (to K. I.), and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, KAKENHI: 21390103 and 23659531 (to K. I.) and 23580417 (to T. M.).
Open AccessPublished:January 23, 2013DOI:https://doi.org/10.1074/jbc.M112.435388
      Missense mutations in the proteolipid protein 1 (PLP1) gene cause a wide spectrum of hypomyelinating disorders, from mild spastic paraplegia type 2 to severe Pelizaeus-Merzbacher disease (PMD). Mutant PLP1 accumulates in the endoplasmic reticulum (ER) and induces ER stress. However, the link between the clinical severity of PMD and the cellular response induced by mutant PLP1 remains largely unknown. Accumulation of misfolded proteins in the ER generally leads to up-regulation of ER chaperones to alleviate ER stress. Here, we found that expression of the PLP1-A243V mutant, which causes severe disease, depletes some ER chaperones with a KDEL (Lys-Asp-Glu-Leu) motif, in HeLa cells, MO3.13 oligodendrocytic cells, and primary oligodendrocytes. The same PLP1 mutant also induces fragmentation of the Golgi apparatus (GA). These organelle changes are less prominent in cells with milder disease-associated PLP1 mutants. Similar changes are also observed in cells expressing another disease-causing gene that triggers ER stress, as well as in cells treated with brefeldin A, which induces ER stress and GA fragmentation by inhibiting GA to ER trafficking. We also found that mutant PLP1 disturbs localization of the KDEL receptor, which transports the chaperones with the KDEL motif from the GA to the ER. These data show that PLP1 mutants inhibit GA to ER trafficking, which reduces the supply of ER chaperones and induces GA fragmentation. We propose that depletion of ER chaperones and GA fragmentation induced by mutant misfolded proteins contribute to the pathogenesis of inherited ER stress-related diseases and affect the disease severity.

      Introduction

      A number of inherited human diseases are caused by missense mutations. These mutations in the membrane and secretary proteins often lead to improper protein folding and accumulation in the endoplasmic reticulum (ER),
      The abbreviations used are: ER
      endoplasmic reticulum
      PLP1
      proteolipid protein 1
      PMD
      Pelizaeus-Merzbacher disease
      msd
      myelin synthesis deficit
      SPG2
      spastic paraplegia type 2
      msd
      myelin synthesis deficit
      GA
      Golgi apparatus
      PDI
      protein-disulfide isomerase
      CALR
      calreticulin
      GRP78
      glucose-regulated protein of 78 kDa
      CANX
      calnexin
      MBP
      myelin basic protein
      UPR
      unfolded protein response
      ATF6
      activating transcription factor 6
      IRE1
      inositol-requiring kinase 1
      XBP1
      X-box protein 1
      CHOP
      C/EBP homologous protein
      ALS
      amyotrophic lateral sclerosis
      MPZ
      myelin protein zero
      PMP22
      peripheral myelin protein 22
      CMT
      Charcot-Marie-Tooth disease
      SC
      spinal cords
      TUNEL
      terminal deoxynucleotidyl transferase dUTP nick end labeling
      MGC
      mixed glial culture
      BFA
      brefeldin A
      MOG
      myelin oligodendrocyte glycoprotein
      GFP
      green fluorescent protein
      luc
      luciferase
      Rluc
      Renilla luciferase
      Igκ
      immunoglobulin κ light chain
      Cluc
      cytoplasmic luciferase
      DMSO
      dimethyl sulfoxide.
      resulting in an induction of ER stress. In cells under ER stress, accumulation of mutant proteins in the ER activates the unfolded protein response (UPR), which initiates a block in translation, increases retrotranslocation and degradation of ER-localized proteins, and bolsters the protein-folding capacity of the ER (
      • Kaufman R.J.
      Orchestrating the unfolded protein response in health and disease.
      ,
      • Lindholm D.
      • Wootz H.
      • Korhonen L.
      ER stress and neurodegenerative diseases.
      ). Through these processes, the UPR functions as a cellular quality control system that essentially protects cells from the toxicity of accumulated proteins in the ER. The UPR is activated by three distinct pathways, activating transcription factor 6 (ATF6), inositol-requiring kinase 1 (IRE1), and protein kinase-like ER kinase (
      • Szegezdi E.
      • Logue S.E.
      • Gorman A.M.
      • Samali A.
      Mediators of endoplasmic reticulum stress-induced apoptosis.
      ), all of which are negatively regulated by interaction with the 78-kDa glucose-regulated protein (GRP78, also referred to as BiP/HSPA5). On accumulation of unfolded protein, GRP78 binds to unfolded proteins and dissociates from the ER stress sensors, which trigger the UPR (
      • Szegezdi E.
      • Logue S.E.
      • Gorman A.M.
      • Samali A.
      Mediators of endoplasmic reticulum stress-induced apoptosis.
      ). ATF6 induces transcription of major ER chaperones and X box-binding protein 1 (XBP1) (
      • Yamamoto K.
      • Sato T.
      • Matsui T.
      • Sato M.
      • Okada T.
      • Yoshida H.
      • Harada A.
      • Mori K.
      Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6α and XBP1.
      ). The endonuclease activity of IRE1 splices XBP1 (
      • Calfon M.
      • Zeng H.
      • Urano F.
      • Till J.H.
      • Hubbard S.R.
      • Harding H.P.
      • Clark S.G.
      • Ron D.
      IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA.
      ), which functions as a transcription factor that drives the expression of UPR-related genes (
      • Yamamoto K.
      • Sato T.
      • Matsui T.
      • Sato M.
      • Okada T.
      • Yoshida H.
      • Harada A.
      • Mori K.
      Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6α and XBP1.
      ). The ATF6 and IRE1-XBP1 axes promote the expression of ER chaperones that facilitate the correct folding or assembly of ER proteins and prevent their aggregation, thereby improving cell survival (
      • Szegezdi E.
      • Logue S.E.
      • Gorman A.M.
      • Samali A.
      Mediators of endoplasmic reticulum stress-induced apoptosis.
      ,
      • Yamamoto K.
      • Sato T.
      • Matsui T.
      • Sato M.
      • Okada T.
      • Yoshida H.
      • Harada A.
      • Mori K.
      Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6α and XBP1.
      ,
      • Yoshida H.
      • Matsui T.
      • Yamamoto A.
      • Okada T.
      • Mori K.
      XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.
      ). However, when ER stress overwhelms the capacity of this intrinsic quality control, apoptosis is induced by up-regulation of the C/EBP homologous protein (CHOP).
      In inherited diseases associated with ER stress, different mutations in the causative genes result in various phenotypes. One representative example, Pelizaeus-Merzbacher disease (PMD), is an X-linked recessive leukodystrophy characterized by diffuse hypomyelination in the central nervous system (CNS) (
      • Inoue K.
      PLP1-related inherited dysmyelinating disorders. Pelizaeus-Merzbacher disease and spastic paraplegia type 2.
      ). Missense mutations in the proteolipid protein 1 (PLP1) gene cause a wide spectrum of clinical phenotypes from a mild allelic disease, spastic paraplegia type 2 (SPG2) to severe connatal PMD (
      • Inoue K.
      PLP1-related inherited dysmyelinating disorders. Pelizaeus-Merzbacher disease and spastic paraplegia type 2.
      ). In these diseases, mutant proteins are misfolded and accumulate in the ER, leading to induction of ER stress and apoptosis of oligodendrocytes in the CNS (
      • Southwood C.M.
      • Garbern J.
      • Jiang W.
      • Gow A.
      The unfolded protein response modulates disease severity in Pelizaeus-Merzbacher disease.
      ,
      • Gow A.
      • Southwood C.M.
      • Lazzarini R.A.
      Disrupted proteolipid protein trafficking results in oligodendrocyte apoptosis in an animal model of Pelizaeus-Merzbacher disease.
      ). However, little is known about how different mutations in the same gene induce ER stress differently and affect clinical severity. Factors, such as retention of misfolded proteins or the extent of UPR activation, may influence phenotypic variation (
      • Gow A.
      • Southwood C.M.
      • Lazzarini R.A.
      Disrupted proteolipid protein trafficking results in oligodendrocyte apoptosis in an animal model of Pelizaeus-Merzbacher disease.
      ,
      • Gow A.
      • Lazzarini R.A.
      A cellular mechanism governing the severity of Pelizaeus-Merzbacher disease.
      ,
      • Roboti P.
      • Swanton E.
      • High S.
      Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein.
      ). However, can any other factors contribute to the pathology of such ER stress-related disease? Here we focused on ER chaperones as a potential player. ER chaperones are highly conserved proteins that assist in protein folding. Therefore, it is generally believed that accumulation of misfolded proteins in the ER up-regulates chaperones to alleviate ER stress. In terms of its association with disease pathology, interaction between the mutant PLP1 and a major ER chaperone, calnexin (CANX), was shown to inhibit degradation of the misfolded mutant proteins (
      • Swanton E.
      • High S.
      • Woodman P.
      Role of calnexin in the glycan-independent quality control of proteolipid protein.
      ). In the mutant superoxide dismutase model of amyotrophic lateral sclerosis (ALS), an ER stress-associated neurodegenerative disease, down-regulation of another chaperone, calreticulin (CALR), was shown to induce ER stress and trigger the death of mutant superoxide dismutase motoneurons (
      • Bernard-Marissal N.
      • Moumen A.
      • Sunyach C.
      • Pellegrino C.
      • Dudley K.
      • Henderson C.E.
      • Raoul C.
      • Pettmann B.
      Reduced calreticulin levels link endoplasmic reticulum stress and Fas-triggered cell death in motoneurons vulnerable to ALS.
      ). A recent study reported up-regulation of protein-disulfide isomerase (PDI, also referred to as P4HB), which is a chaperone in the ER catalyzing the formation and breakage of protein disulfides bonds, in microglia of transgenic mutant superoxide dismutase 1 mice (
      • Jaronen M.
      • Vehvilainen P.
      • Malm T.
      • Keksa-Goldsteine V.
      • Pollari E.
      • Valonen P.
      • Koistinaho J.
      • Goldsteins G.
      Protein disulfide isomerase in ALS mouse glia links protein misfolding with NADPH oxidase-catalyzed superoxide production.
      ). Therefore, we sought to determine whether changes in the expression of ER chaperones alter the accumulation of misfolded protein and ER stress, potentially modifying the cellular and clinical phenotypes.
      For this purpose, we used PMD as a model and investigated missense PLP1 mutations (
      • Southwood C.M.
      • Garbern J.
      • Jiang W.
      • Gow A.
      The unfolded protein response modulates disease severity in Pelizaeus-Merzbacher disease.
      ,
      • Roboti P.
      • Swanton E.
      • High S.
      Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein.
      ). PLP1 with an A243V substitution (PLP1msd) is representative of the severe hypomyelination in myelin synthesis deficit (msd) mice (
      • Gencic S.
      • Hudson L.D.
      Conservative amino acid substitution in the myelin proteolipid protein of jimpy(msd) mice.
      ) and humans (
      • Yamamoto T.
      • Nanba E.
      • Zhang H.
      • Sasaki M.
      • Komaki H.
      • Takeshita K.
      Jimpy(msd) mouse mutation and connatal Pelizaeus-Merzbacher disease.
      ), whereas two other mutations, W163L and I187T, are representative of the milder condition found in mild PMD/SPG2 patients (
      • Koizume S.
      • Takizawa S.
      • Fujita K.
      • Aida N.
      • Yamashita S.
      • Miyagi Y.
      • Osaka H.
      Aberrant trafficking of a proteolipid protein in a mild Pelizaeus-Merzbacher disease.
      ,
      • Kobayashi H.
      • Hoffman E.P.
      • Marks H.G.
      The rumpshaker mutation in spastic paraplegia.
      ): the latter is also the mutation found in an SPG2 mouse model, rumpshaker (
      • Schneider A.
      • Montague P.
      • Griffiths I.
      • Fanarraga M.
      • Kennedy P.
      • Brophy P.
      • Nave K.A.
      Uncoupling of hypomyelination and glial cell death by a mutation in the proteolipid protein gene.
      ). We also employed mutants in two other genes responsible for peripheral myelin disorders, a myelin protein zero (MPZ) mutant associated with a severe neuropathy, Dejerine-Sottas neuropathy (
      • Khajavi M.
      • Inoue K.
      • Wiszniewski W.
      • Ohyama T.
      • Snipes G.J.
      • Lupski J.R.
      Curcumin treatment abrogates endoplasmic reticulum retention and aggregation-induced apoptosis associated with neuropathy-causing myelin protein zero-truncating mutants.
      ), and two peripheral myelin protein 22 (PMP22) mutants that are associated with a clinically mild neuropathy, Charcot-Marie-Tooth disease (
      • D'Antonio M.
      • Feltri M.L.
      • Wrabetz L.
      Myelin under stress.
      ,
      • Gow A.
      • Sharma R.
      The unfolded protein response in protein aggregating diseases.
      ,
      • Warner L.E.
      • Garcia C.A.
      • Lupski J.R.
      Hereditary peripheral neuropathies. Clinical forms, genetics, and molecular mechanisms.
      ).
      In this study, we examined the expression of ER chaperones in response to mutants of PLP1 and two other genes. Unexpectedly, we found that some ER chaperones were depleted rather than up-regulated. In addition, these mutant proteins induced fragmentation of the Golgi apparatus (GA). We also found an association between these changes and phenotypic severity. Furthermore, we proposed potential mechanisms underlying these cellular phenotypes. The results of this study suggest that changes in these subcellular organelles may contribute to the cellular pathogenesis and phenotypic severity of inherited ER stress-related diseases caused by mutant proteins.

      DISCUSSION

      Involvement of ER stress and the subsequent UPR has been implicated in pathogenesis of multiple human inherited diseases, including cystic fibrosis (
      • Ward C.L.
      • Omura S.
      • Kopito R.R.
      Degradation of CFTR by the ubiquitin-proteasome pathway.
      ), retinitis pigmentosa (
      • Shinde V.M.
      • Sizova O.S.
      • Lin J.H.
      • LaVail M.M.
      • Gorbatyuk M.S.
      ER stress in retinal degeneration in S334ter Rho rats.
      ), CMT (
      • Gow A.
      • Sharma R.
      The unfolded protein response in protein aggregating diseases.
      ), and PMD (
      • Southwood C.M.
      • Garbern J.
      • Jiang W.
      • Gow A.
      The unfolded protein response modulates disease severity in Pelizaeus-Merzbacher disease.
      ,
      • Roboti P.
      • Swanton E.
      • High S.
      Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein.
      ). Although there is wide phenotypic variation in each of these diseases, even among the mutations in same genes, little is known about the factors that determine the difference in ER stress and the severity of disease. In this study, we investigated the organelle changes in cells expressing different PLP1 missense mutations associated with a wide-range of clinical severities in PMD. We demonstrated that accumulation of the ER stress-associated mutant PLP1 leads to depletion of some important ER chaperones and GA fragmentation, both of which are more profound in cells expressing mutants associated with more severe phenotypes. We also found that an ER stress-related MPZ mutant also induces these cellular phenotypes; however, two PMP22 mutants, which cannot induce ER stress despite their ER retention, do not induce them. Based on these findings, we suggest that the cellular phenotypes of ER chaperone depletion and GA fragmentation may be involved in the pathogenesis of particular mutations in certain genes in ER stress-related diseases.
      We observed that PDI, CALR, and GRP78 were depleted in the ER of HeLa cells transfected with the PLP1msd gene, whereas CANX remained unaffected. Similar phenomenon was also observed in endogenous Pdi and Calr in the SCs of msd mice (Fig. 6, C and D). By contrast, we could not find an obvious decrease of endogenous Grp78 in the mutant SCs, possibly due to the large enhancement of mRNA up-regulation. We considered the features that were either functionally or structurally common among the depleted chaperones. Functionally, each of these chaperones has a distinct role in protein folding and maintenance of ER homeostasis. For example, PDI catalyzes the formation and rearrangement of molecular disulfide bonds for protein folding (
      • Higa A.
      • Chevet E.
      Redox signaling loops in the unfolded protein response.
      ). CALR and CANX are calcium-binding proteins implicated in the trimming of N-glycosylation and storage of calcium in the ER (
      • Ellgaard L.
      • Frickel E.M.
      Calnexin, calreticulin, and ERp57. Teammates in glycoprotein folding.
      ). GRP78 controls activation of the UPR, acting as a sensor for misfolded proteins in the ER (
      • Bertolotti A.
      • Zhang Y.
      • Hendershot L.M.
      • Harding H.P.
      • Ron D.
      Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.
      ). Based on this evidence, it is unlikely that the depletion is linked to a particular function of these chaperones.
      Structurally, PDI, CALR, and GRP78 contain a carboxyl-terminal retrieval signal KDEL motif (
      • Munro S.
      • Pelham H.R.
      A C-terminal signal prevents secretion of luminal ER proteins.
      ), which is recognized by the KDEL receptor to transport them back to the ER (
      • D'Souza-Schorey C.
      • Chavrier P.
      ARF proteins. Roles in membrane traffic and beyond.
      ). This retrieval mechanism by the KDEL receptor contributes to quality control at the ER (
      • Yamamoto K.
      • Fujii R.
      • Toyofuku Y.
      • Saito T.
      • Koseki H.
      • Hsu V.W.
      • Aoe T.
      The KDEL receptor mediates a retrieval mechanism that contributes to quality control at the endoplasmic reticulum.
      ). We observed that the KDEL receptor was localized in the ER in cells expressing PLP1msd, whereas the same protein was localized in the GA in PLP1wt and control cells (Fig. 9J). Moreover, depletion of PDI, CALR, and GRP78 was also observed in HeLa cells treated with the chemical ER stressor, BFA, which inhibits retrograde transport from the GA to the ER (
      • Anders N.
      • Jürgens G.
      Large ARF guanine nucleotide exchange factors in membrane trafficking.
      ,
      • Citterio C.
      • Vichi A.
      • Pacheco-Rodriguez G.
      • Aponte A.M.
      • Moss J.
      • Vaughan M.
      Unfolded protein response and cell death after depletion of brefeldin A-inhibited guanine nucleotide-exchange protein GBF1.
      ). Interestingly, the other chemical ER stressors tested, thapsigargin and tunicamycin, did not recapitulate the findings. These results suggest that misfolded mutant proteins may induce ER chaperone depletion by inhibition of their KDEL receptor-mediated retrograde transport of these chaperones by mis-localizing KDEL receptor.
      Our results suggested that PLP1 and MPZ mutants, and possibly other mutant proteins that evoke ER stress, specifically deplete chaperones containing a KDEL motif from the ER. These proteins were unlikely degraded by the ERAD-proteasome system (Figs. 2, B and C, and 3, E and F). We further demonstrated that the proportion of these chaperone proteins in the digitonin-soluble fraction, which contains the plasma membrane and cytosolic proteins, increased in cells expressing PLP1msd (Fig. 2, D and E). However, we found no change in the amounts of these chaperone proteins on the cell surface (Fig. 3, B and D). These results suggest that KDEL-containing ER chaperones mainly translocate from the ER to the cytosol in cells expressing ER stress proteins. However, we could not rule out a possibility that small populations may translocate to the plasma membrane, as described previously (
      • Zhang Y.
      • Liu R.
      • Ni M.
      • Gill P.
      • Lee A.S.
      Cell surface relocalization of the endoplasmic reticulum chaperone and unfolded protein response regulator GRP78/BiP.
      ).
      In contrast, the GA fragmentation observed in cells treated with BFA was also observed in cells treated with thapsigargin (data not shown). GA fragmentation has been reported in another ER stress-related disorder, ALS (
      • Nakagomi S.
      • Barsoum M.J.
      • Bossy-Wetzel E.
      • Sütterlin C.
      • Malhotra V.
      • Lipton S.A.
      A Golgi fragmentation pathway in neurodegeneration.
      ). These findings suggest that GA fragmentation may be a common pathology in ER stress-related diseases.
      An association between cellular pathology and clinical severity for PLP1 mutations has been reported. Gow and Lazzarini (
      • Gow A.
      • Lazzarini R.A.
      A cellular mechanism governing the severity of Pelizaeus-Merzbacher disease.
      ) reported a cellular mechanism that the amount of mutant PLP1 gene product accumulated in the ER accounts for disease severity in PMD. Recent studies showed that differences in the UPR (
      • Gow A.
      • Southwood C.M.
      • Lazzarini R.A.
      Disrupted proteolipid protein trafficking results in oligodendrocyte apoptosis in an animal model of Pelizaeus-Merzbacher disease.
      ) and ER quality control (
      • Roboti P.
      • Swanton E.
      • High S.
      Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein.
      ) have the potential to modulate disease severity. These reports suggest that retention of PLP1 mutants determines the severity of ER stress and clinical outcome. Consistent with these findings, the depletion of ER chaperones and GA fragmentation are closely linked to clinical severity (Figs. 5C and 9D), indicating that these cellular phenotypes are associated with disease pathology. In addition, we also demonstrated that PLP1msd not only induces ER stress, but also inhibits secretion and cell surface expression of proteins, probably due to impairment of ER chaperone transport from the GA to the ER and/or GA fragmentation. These trafficking defects may also contribute to the pathogenesis of disease by preventing cell-to-cell and cell-to-environment communications. Additional studies are required to elucidate how these mutants affect the maturation and trafficking of other membrane and secretory proteins.
      Based on our findings, we propose a novel model for mechanisms to explain how mutant misfolded proteins affect intracellular homeostasis, as summarized in Fig. 10. When misfolded proteins accumulate in the ER, they inhibit GA to ER retrograde transport by KDEL receptor mis-localization in the ER (Fig. 10B). Ultimately, inhibition of retrograde transport results in depletion of KDEL-containing ER chaperones from the ER. Blockage of GA to ER retrograde transport also contributes to abnormal accumulation of ER chaperones in the cis-Golgi, which may, in part, contribute to GA fragmentation (Fig. 10C). As a consequence, the ER to GA transport of membrane/secretory proteins is disturbed, and misfolded proteins and other membrane/secretory proteins accumulate in the ER, resulting in a trafficking defect and further acceleration of ER stress (Fig. 10D). This unexpected discovery and new model for the disease mechanism may promote our understanding of how different mutations in the same gene differently evoke ER stress and affect disease phenotype. Our findings may have further implications for ER stress-related diseases in which the UPR modulates pathology. Because practically no effective treatment is available for these diseases, ER chaperone and GA may serve as potential targets for therapeutic intervention.

      Acknowledgments

      We thank Dr. W. B. Macklin (Cleveland Clinic Foundation) for providing msd mice, Dr. H. Osaka (Kanagawa Children's Medical Center) for the human PLP1 genes, Dr. J. R. Lupski (Baylor College of Medicine) for the human PMP22 genes and MPZ genes, Dr. J. Miyazaki (Osaka University) for pCAGGS, and Dr. M. Itoh (NCNP) for anti-PLP1 antibody, respectively.

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