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• W2143326415 abstract "Deletion of serine 63 from P0 glycoprotein (P0S63del) causes Charcot-Marie-Tooth 1B neuropathy in humans, and P0S63del produces a similar demyelinating neuropathy in transgenic mice. P0S63del is retained in the endoplasmic reticulum and fails to be incorporated into myelin. Here we report that P0S63del is misfolded and Schwann cells mount a consequential canonical unfolded protein response (UPR), including expression of the transcription factor CHOP, previously associated with apoptosis in ER-stressed cells. UPR activation and CHOP expression respond dynamically to P0S63del levels and are reversible but are associated with only limited apoptosis of Schwann cells. Nonetheless, Chop ablation in S63del mice completely rescues their motor deficit and reduces active demyelination 2-fold. This indicates that signaling through the CHOP arm of the UPR provokes demyelination in inherited neuropathy. S63del mice also provide an opportunity to explore how cells can dysfunction yet survive in prolonged ER stress—important for neurodegeneration related to misfolded proteins. Deletion of serine 63 from P0 glycoprotein (P0S63del) causes Charcot-Marie-Tooth 1B neuropathy in humans, and P0S63del produces a similar demyelinating neuropathy in transgenic mice. P0S63del is retained in the endoplasmic reticulum and fails to be incorporated into myelin. Here we report that P0S63del is misfolded and Schwann cells mount a consequential canonical unfolded protein response (UPR), including expression of the transcription factor CHOP, previously associated with apoptosis in ER-stressed cells. UPR activation and CHOP expression respond dynamically to P0S63del levels and are reversible but are associated with only limited apoptosis of Schwann cells. Nonetheless, Chop ablation in S63del mice completely rescues their motor deficit and reduces active demyelination 2-fold. This indicates that signaling through the CHOP arm of the UPR provokes demyelination in inherited neuropathy. S63del mice also provide an opportunity to explore how cells can dysfunction yet survive in prolonged ER stress—important for neurodegeneration related to misfolded proteins. Myelin protein zero (P0) is the major protein produced by myelinating Schwann cells (Kirschner et al., 2004Kirschner D.A. Wrabetz L. Feltri M.L. The P0 Gene.in: Lazzarini R.A. Myelin Biology and Disorders. Elselvier Academic Press, San Diego2004: 523-545Google Scholar). P0 is a single-pass transmembrane protein, with a large amino-terminal extracellular domain (ECD) and a short carboxy-terminal intracellular tail. The protein is encoded by the Mpz gene and synthesized in the endoplasmic reticulum (ER), where it undergoes N-glycosylation and formation of one intramolecular disulfide bond. In myelin, P0 is present as tetramers that compact the extracellular appositions of myelin wraps via in trans homophilic interactions (D'Urso et al., 1990D'Urso D. Brophy P.J. Staugaitis S.M. Gillespie C.S. Frey A.B. Stempak J.G. Colman D.R. Protein zero of peripheral nerve myelin: biosynthesis, membrane insertion, and evidence for homotypic interaction.Neuron. 1990; 4: 449-460Abstract Full Text PDF PubMed Scopus (243) Google Scholar, Filbin et al., 1990Filbin M. Walsh F. Trapp B. Pizzey J. Tennekoon G. Role of myelin Po protein as a homophilic adhesion molecule.Nature. 1990; 344: 871-872Crossref PubMed Scopus (302) Google Scholar, Shapiro et al., 1996Shapiro L. Doyle J.P. Hensley P. Colman D. Hendrickson W.A. Crystal structure of the extracellular domain from Po, the major structural protein of peripheral nerve myelin.Neuron. 1996; 17: 435-449Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Accordingly, mice with homozygous disruption of Mpz show uncompaction of myelin in development (Giese et al., 1992Giese K.P. Martini R. Lemke G. Soriano P. Schachner M. Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons.Cell. 1992; 71: 565-576Abstract Full Text PDF PubMed Scopus (437) Google Scholar), and mice with heterozygous disruption show altered myelin maintenance in the adult (Martini et al., 1995Martini R. Zielasek J. Toyka K.V. Giese K.P. Schachner M. Protein zero (P0)-deficient mice show myelin degeneration in peripheral nerves characteristic of inherited human neuropathies.Nat. Genet. 1995; 11: 281-286Crossref PubMed Scopus (281) Google Scholar). More than 100 mutations of P0, many in the ECD, are associated with hereditary motor and sensory neuropathies (Shy, 2005Shy M.E. HMSN Related to MPZ (P0) Mutations.in: Dyck P.J. Thomas P.K. Peripheral Neuropathy. W.B. Saunders, Philadelphia2005: 1681-1706Crossref Scopus (12) Google Scholar, Wrabetz et al., 2004Wrabetz L. Feltri M.L. Kleopa K.A. Scherer S.S. Inherited Neuropathies: Clinical, Genetic and Biological Features.in: Lazzarini R.A. Myelin Biology and Disorders. Elselvier Academic Press, San Diego2004: 905-951Google Scholar). Although the specific pathogenetic mechanisms have not been fully elucidated, dominant inheritance and widely varying phenotypes suggest that many P0 mutations act through gain of abnormal function (Martini et al., 1995Martini R. Zielasek J. Toyka K.V. Giese K.P. Schachner M. Protein zero (P0)-deficient mice show myelin degeneration in peripheral nerves characteristic of inherited human neuropathies.Nat. Genet. 1995; 11: 281-286Crossref PubMed Scopus (281) Google Scholar, Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar). For example, deletion of serine 63 (S63del) or its mutation to a cysteine (S63C) results in Charcot-Marie-Tooth (CMT) type 1B and Dèjèrine-Sòttas syndrome (DSS) in humans, respectively (Hayasaka et al., 1993Hayasaka K. Himoro M. Sawaishi Y. Nanao K. Takahashi T. Takada G. Nicholson G. Ouvrier R. Tachi N. De novo mutation of the myelin Po gene in Dejerine-Sottas disease (hereditary motor and sensory neuropathy type III).Nat. Genet. 1993; 5: 266-268Crossref PubMed Scopus (197) Google Scholar, Kulkens et al., 1993Kulkens T. Bolhuis P.A. Wolterman R.A. Kemp S. te Nijenhuis S. Valentijn L.J. Hensels G.W. Jennekens F.G. de Visser M. Hoogendijk J.E. Bass F. Deletion of the serine 34 codon from the major peripheral myelin protein P0 gene in Charcot-Marie-Tooth disease type 1B.Nat. Genet. 1993; 5: 35-39Crossref PubMed Scopus (180) Google Scholar). Transgenic mice expressing both mutant and normal P0 confirm that these mutations produce a gain of abnormal function (Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar). The pathomechanisms are likely to differ between the two mutations, for although S63C is incorporated into myelin, producing packing defects, S63del is retained in the ER. In the ER, membrane proteins are subject to stringent quality control (Ron and Walter, 2007Ron D. Walter P. Signal integration in the endoplasmic reticulum unfolded protein response.Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4415) Google Scholar). Accumulation of misfolded proteins in the ER sequesters chaperones like binding immunoglobulin protein (BiP) and activates the unfolded protein response (UPR). As an adaptive response, UPR upregulates transcription of chaperones, temporarily attenuates new translation, and activates protein degradation via the proteasome. However, at high levels of ER stress, UPR signaling contributes to apoptosis (Ron and Walter, 2007Ron D. Walter P. Signal integration in the endoplasmic reticulum unfolded protein response.Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4415) Google Scholar). For example, deletion of the gene encoding CHOP (CAATT enhancer-binding protein homologous protein, a transcription factor active in the UPR) protects cells against apoptosis (Marciniak et al., 2004Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1343) Google Scholar, Zinszner et al., 1998Zinszner H. Kuroda M. Wang X. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum.Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1610) Google Scholar). Here we report that P0S63del activates a canonical and dose-dependent UPR, including CHOP, and consequentially, demyelination in Schwann cells. ER retention and UPR depend on alteration of the pattern of hydrophobic residues in P0 β strand C, not changes at residue 63, a mechanism potentially shared by another CMT1B mutation, deletion of phenylalanine 64 (Ikegami et al., 1996Ikegami T. Nicholson G. Ikeda H. Ishida A. Johnston H. Wise G. Ouvrier R. Hayasaka K. A novel homozygous mutation of the myelin Po gene producing Dejerine- Sottas disease (hereditary motor and sensory neuropathy type III).Biochem. Biophys. Res. Commun. 1996; 222: 107-110Crossref PubMed Scopus (50) Google Scholar). Ablation of Chop in S63del mice reverses behavioral, electrophysiological, and morphological abnormalities, indicating the UPR as a novel pathogenetic mechanism in demyelinating peripheral neuropathies. Finally, UPR activation is rapidly reversed by reducing mutant protein levels in mutant nerves, suggesting its dosage as a logical therapeutic target. Given that Schwann cell death follows, not precedes demyelination, we propose that the maladaptive UPR and CHOP in S63del nerves produces a novel cellular dysfunction in Schwann cells. P0S63del causes a demyelinating neuropathy via gain of abnormal function, but does not arrive to the myelin sheath (Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar). To characterize potential toxic mechanisms operative outside of myelin, we surveyed protein quality control in S63del nerves. We considered autophagy (lysosomes), proteasomes, and UPR in S63del nerves. LAMP1 staining was slightly expanded but did not reveal the lysosomal aggregates that have been reported for PMP22 mutants, where aggresomes are cleared by autophagy (see Figure S1 available online) (Fortun et al., 2003Fortun J. Dunn Jr., W.A. Joy S. Li J. Notterpek L. Emerging role for autophagy in the removal of aggresomes in Schwann cells.J. Neurosci. 2003; 23: 10672-10680Crossref PubMed Google Scholar). Electron microscopy did not reveal aggregates or swollen ER (Tsang et al., 2007Tsang K.Y. Chan D. Cheslett D. Chan W.C. So C.L. Melhado I.G. Chan T.W. Kwan K.M. Hunziker E.B. Yamada Y. et al.Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function.PLoS Biol. 2007; 5: e44Crossref PubMed Scopus (139) Google Scholar) in S63del Schwann cells (Figure S2A). Also, EDEM1, a marker of endoplasmic reticulum activated degradation (ERAD), was expressed at normal levels, preliminarily suggesting limited ERAD and proteasomal involvement (Figure S2B). When expressed in mice of the Mpz null background, P0S63del is retained in the endoplasmic reticulum (Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar). However, CMT1B patients have both MPZ WT and S63del alleles. Even when P0S63del was coexpressed with P0wt (Figure 5C), immunostaining on teased fibers showed that P0 was still retained in the ER (Figure 1A). These data, taken together with preliminary evidence that the transcription of BiP and CHOP is upregulated in S63del nerves (Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar), suggest that the UPR is an important form of quality control for P0S63del. Therefore, we asked whether P0S63del elicited a full UPR and how the UPR correlated with disease. In mouse, disease severity correlates with MpzS63del mRNA expression (Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar). To examine dose dependence, we exploited mice expressing S63del mRNA at levels of one (S63del-L) or two (S63del-H) endogenous alleles. BiP and CHOP mRNA levels were increased by 5.2- and 2.7-fold in S63del-L and 5.7- and 14.6-fold in S63del-H sciatic nerves, respectively (Figures 1B and 1C). Induction of BiP and CHOP mRNA was tissue and mutation specific. It was not detected in the brain (data not shown) or in nerves of S63C, P0Myc (pathological model of CMT1B; Previtali et al., 2000Previtali S.C. Quattrini A. Fasolini M. Panzeri M.C. Villa A. Filbin M.T. Li W. Chiu S.Y. Messing A. Wrabetz L. Feltri M.L. Epitope-tagged P(0) glycoprotein causes Charcot-Marie-Tooth-like neuropathy in transgenic mice.J. Cell Biol. 2000; 151: 1035-1046Crossref PubMed Scopus (44) Google Scholar), or P0 overexpressor mice (Wrabetz et al., 2000Wrabetz L. Feltri M. Quattrini A. Inperiale D. Previtali S. D'Antonio M. Martini R. Yin X. Trapp B. Zhou L. et al.P0 overexpression causes congenital hypomyelination of peripheral nerve.J. Cell Biol. 2000; 148: 1021-1033Crossref PubMed Scopus (126) Google Scholar, Yin et al., 2000Yin A. Kidd G. Wrabetz L. Feltri M. Messing A. Trapp B. Schwann cell myelination requires timely and precise targeting of P0 protein.J. Cell Biol. 2000; 148: 1009-1020Crossref PubMed Scopus (62) Google Scholar) (Figure 1B). The level of induction was robust, comparable to the levels of BiP and CHOP mRNA acutely induced by tunicamycin (glycosylation inhibitor) in liver (2.5- and 19-fold) (Figures 1B and 1C) or cultured sciatic nerve segments (4- and 10-fold) (Figures 5A and 5B) of normal mice. Induction of BiP and CHOP in S63del and tunicamycin-treated nerves was confirmed by western analysis (Figure 1D). As for mRNA levels, CHOP protein induction was higher in S63del-H mice than in S63del-L mice, in parallel with the ratio of mutant P0S63del to P0wt. CHOP activates transcription of the Downstream Of CHOP genes (DOCs) (Wang et al., 1998Wang X.Z. Kuroda M. Sok J. Batchvarova N. Kimmel R. Chung P. Zinszner H. Ron D. Identification of novel stress-induced genes downstream of chop.EMBO J. 1998; 17: 3619-3630Crossref PubMed Scopus (260) Google Scholar). Accordingly, DOCs 1 and 6, although not 4, were induced in S63del nerve. Tunicamycin treatment activated DOC4 in NIH 3T3 cells, but not in sciatic nerve, raising the possibility that Schwann cells respond differently to CHOP (Figure 1E). Altogether, these data show that BiP, CHOP, and its target genes are upregulated in S63del nerves to levels comparable to those found when UPR is acutely induced by tunicamycin. Of these, the level of CHOP correlated best with mutant protein level and disease severity. To characterize the UPR in S63del nerves more fully, we analyzed activation of the three arms of the UPR: IRE-1 and PERK kinases and cleavage of ATF6 transcription factor (Ron and Walter, 2007Ron D. Walter P. Signal integration in the endoplasmic reticulum unfolded protein response.Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4415) Google Scholar). Activated IRE-1 noncanonically splices the mRNA of the transcription factor X-box-binding protein 1 (XBP-1), an event specific to UPR (Yoshida et al., 2001Yoshida 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.Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2706) Google Scholar). The spliced form of XBP-1 was increased by 2.5-, 2.7-, and 5-fold in S63del-L, S63del-H, and tunicamycin-treated nerves, respectively (Figure 2A). Activated PERK phosphorylates eIF2α and upregulates translation of the transcription factor ATF4 and transcription of ATF3, CHOP, and the oxidoreductin ERO1-Lβ (Jiang et al., 2004Jiang H.Y. Wek S.A. McGrath B.C. Lu D. Hai T. Harding H.P. Wang X. Ron D. Cavener D.R. Wek R.C. Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response.Mol. Cell. Biol. 2004; 24: 1365-1377Crossref PubMed Scopus (364) Google Scholar, Pagani et al., 2000Pagani M. Fabbri M. Benedetti C. Fassio A. Pilati S. Bulleid N.J. Cabibbo A. Sitia R. Endoplasmic reticulum oxidoreductin 1-lbeta (ERO1-Lbeta), a human gene induced in the course of the unfolded protein response.J. Biol. Chem. 2000; 275: 23685-23692Crossref PubMed Scopus (216) Google Scholar, Ron and Walter, 2007Ron D. Walter P. Signal integration in the endoplasmic reticulum unfolded protein response.Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4415) Google Scholar). Indeed, the levels of eIF2α phosphorylation increased 1.8-fold, and ATF4 3-fold in S63del-L nerves (Figure 2B). Similarly, ATF3 mRNA was induced 2.5-fold in S63del-L, 12.4-fold in S63del-H nerves, and 6.1-fold in tunicamycin-treated liver (Figure 2C). Likewise, ERO1-Lβ was induced in S63del-H nerves and tunicamycin-treated liver to a similar extent (Figure S2C and data not shown). The UPR was activated in Schwann cells, as the transcription factor CHOP, which was undetectable in normal nerves, was localized to the nucleus of S63del Schwann cells (Figure 2D). Finally, dose-dependent ATF6 cleavage was detected in S63del-L and -H nerves (Figure 2E). Thus, P0S63del provokes a dose-dependent activation of IRE-1, PERK, and ATF6 pathways in Schwann cells of S63del nerves, indicating a canonical UPR. In S63del nerves, ER retention of P0S63del and UPR activation suggested protein misfolding. Thus, we performed western analysis for the intramolecular disulfide bond of P0 (Figure 3A). The mobility of P0wt was increased by 3–4 kD under nonreducing conditions, consistent with the presence of a disulfide bond. Nonreduced P0S63del showed a similar shift (arrowhead), but in addition produced a smear, which could represent alternatively folded intermediates. In contrast, another neuropathic mutation at the same residue, P0S63C, which is not retained in the ER, migrated similarly to P0wt. Serine 63 lies in β strand C, characterized by an alternating hydrophobic and hydrophilic pattern of residues (Figure 3B) (Shapiro et al., 1996Shapiro L. Doyle J.P. Hensley P. Colman D. Hendrickson W.A. Crystal structure of the extracellular domain from Po, the major structural protein of peripheral nerve myelin.Neuron. 1996; 17: 435-449Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). We hypothesized that deletion of serine 63 altered the disposition of the 4 hydrophobic residues (FWYP) in β strand C (Figure 3B, compare WT and S63del). Therefore, we compared the effect of deleting serine 63 to either deletion of phenylalanine 64 (F64del), conversion of both serine 63 and threonine 65 into phenylalanines (S63F/T65F), substitution of serine 63 with phenylalanine (S63F), or substitution of serine 63 with cysteine (S63C), which alter the disposition of 3, 2, 1, or 0 hydrophobic residues, respectively. P0 was fused at its carboxyl terminus in frame with the discosoma red (DsRed) fluorescent protein. Modification of P0 at the carboxyl terminus does not impair targeting of P0 to the plasma membrane in cells (Shames et al., 2003Shames I. Fraser A. Colby J. Orfali W. Snipes G.J. Phenotypic differences between peripheral myelin protein-22 (PMP22) and myelin protein zero (P0) mutations associated with Charcot-Marie-Tooth-related diseases.J. Neuropathol. Exp. Neurol. 2003; 62: 751-764Crossref PubMed Scopus (44) Google Scholar) or to myelin in transgenic sciatic nerve (M.P., P. Fratta, and L.W., unpublished data). The chimeras were transiently expressed in COS-7 (Figure 3D) or Chinese hamster ovary (CHO) cells (data not shown), and the cells were immunostained for the ER resident lectin, calnexin (CNX) (Figure 3D). Both P0wt and P0S63C were targeted to the plasma membrane, whereas P0S63del was confined to the ER, consistent with prior analysis of these mutant P0s in transgenic mice (Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar). In addition, transfected P0MycDsRed was trafficked to the cell membrane (data not shown), just as P0Myc arrives to myelin in transgenic sciatic nerve (Previtali et al., 2000Previtali S.C. Quattrini A. Fasolini M. Panzeri M.C. Villa A. Filbin M.T. Li W. Chiu S.Y. Messing A. Wrabetz L. Feltri M.L. Epitope-tagged P(0) glycoprotein causes Charcot-Marie-Tooth-like neuropathy in transgenic mice.J. Cell Biol. 2000; 151: 1035-1046Crossref PubMed Scopus (44) Google Scholar), further validating the transfected cells as a model. S63F was trafficked to the cell membrane, whereas F64del and S63F/T65F were mostly retained in the ER. Deletion of glutamic acid 71 in the loop connecting β strands C and C′ did not impair protein trafficking (data not shown). S63del and F64del also induced BiP transcription and XBP-1 splicing in COS-7 cells to levels similar to those obtained upon tunicamycin treatment (Figure 3C). These data suggest that it is not alteration of the side chain at residue 63 but rather a perturbed alignment of hydrophobic residues in β strand C that results in ER retention and activation of the UPR, providing further support for the notion that P0S63del is globally misfolded. S63del nerves are characterized by hypomyelination, demyelinated fibers, and onion bulbs (Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar). A few nascent onion bulbs are detectable at postnatal day 28 (P28) and become evident by 6 months of age. To determine whether the UPR activation preceded or followed the appearance of pathological findings in diseased nerves, we analyzed P0, BiP, and CHOP expression across development. In WT and S63del-L mice, BiP and CHOP mRNAs were immediately upregulated during development, mirroring the robust induction of P0 expression that occurs immediately after birth and peaks around P28 (Figure 4), although CHOP rapidly leveled off and was maintained at a plateau, suggesting more complicated regulation. Similar results were obtained for S63del-H mice (data not shown). Thus, the robust UPR as measured by peak BiP and CHOP levels is associated with the onset of myelin instability, destruction, and failed remyelination marked by onion bulbs. The relative amounts of BiP and CHOP differed between S63del-L and S63del-H mice (Figure 1). Given that BiP chaperone helps cells to fold aberrant proteins (Gething, 1999Gething M.J. Role and regulation of the ER chaperone BiP.Semin. Cell Dev. Biol. 1999; 10: 465-472Crossref PubMed Scopus (413) Google Scholar), whereas CHOP may kill overwhelmed cells (Marciniak et al., 2004Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1343) Google Scholar, Zinszner et al., 1998Zinszner H. Kuroda M. Wang X. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum.Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1610) Google Scholar), the increased level of CHOP in the face of stable amounts of BiP could represent a transition from a cell survival response in S63del-L to an apoptotic reponse in S63del-H. One prediction of this hypothesis would be that in S63del-L nerves the level of BiP is near maximal and cannot be augmented. Therefore, segments of sciatic nerves were explanted and maintained in culture for 20 hr with or without tunicamycin. At time zero, BiP and CHOP were increased by 3.3- and 3.8-fold in S63del as compared to WT nerves (Figures 5A and 5B). Surprisingly, 20 hr after axotomy, BiP and CHOP expression decreased to levels similar to WT mice, although parallel treatment with tunicamycin induced BiP/CHOP to 3.9/10.4- and 3.8/6.9-fold in WT and mutant mice, respectively. These data indicate that, after axotomy, UPR is almost completely reversed in mutant nerves and is reinduced to similar levels by tunicamycin. Unfortunately, additive effects cannot be addressed after axotomy. One possible explanation was that axotomy of the cultured nerve segments produced acutely decreased mutant P0 (maintainence of Mpz expression depends on axonal signals; Trapp et al., 1988Trapp B.D. Hauer P. Lemke G. Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells.J. Neurosci. 1988; 8: 3515-3521Crossref PubMed Google Scholar). In fact, 20 hr after axotomy, the level of P0wt was partially reduced (Figure 5C, arrow), whereas P0S63del was almost undetectable, also suggesting that P0S63del is less stable (Figure 5C, arrowhead). Similar results were obtained in sciatic nerve segments from S63del mice deleted of endogenous P0, where only mutant P0 is present (S63del−/−). As expected, this effect was transcriptionally mediated, as the level of P0 mRNA was very low at 20 hr after axotomy (Figure 5D), and had fallen significantly already by 6 hr after axon cut (data not shown). Thus, the UPR in S63del nerves is continuously activated by sustained expression of mutant protein. Transcriptional induction of BiP and CHOP can be halted by simply discontinuing mutant transgene expression, indicating that the UPR is reversible in vivo. Of the UPR mediators, the level of CHOP expression correlated best with disease severity. To demonstrate a direct role for the CHOP-dependent arm of the UPR in the pathogenesis of demyelination in S63del nerves, we crossed S63del-L and Chop null mice (Zinszner et al., 1998Zinszner H. Kuroda M. Wang X. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum.Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1610) Google Scholar) and analyzed behavior, neurophysiology, and morphology in WT, S63del, Chop null, and S63del/Chop null littermates. Chop null mice show no overt neuromuscular defects (Zinszner et al., 1998Zinszner H. Kuroda M. Wang X. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum.Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1610) Google Scholar; see below). Four-month-old S63del mice had lost one-third to one-half of their motor capacity as compared to WT or Chop null littermates in analysis by accelerating rotarod (Figure 6A) (Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar). Strikingly, S63del/Chop null mice regained completely normal motor capacity in rotarod analysis (Figure 6A). This was accompanied by a partial rescue of electrophysiological and morphological abnormalities. In particular, S63del mice manifest reduced nerve conduction velocity (NCV) and increased F-wave latency (Wrabetz et al., 2006Wrabetz L. D'Antonio M. Pennuto M. Dati G. Tinelli E. Fratta P. Previtali S. Imperiale D. Zielasek J. Toyka K. et al.Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice.J. Neurosci. 2006; 26: 2358-2368Crossref PubMed Scopus (108) Google Scholar). In 6-month-old S63del/Chop null mice, the F-wave latency was significantly improved, even if the NCV did not change. In addition, there were half the number of demyelinated fibers and a trend toward reduction of onion bulbs as compared to S63del mice (Figure 6B and Table 1). Consistent with unchanged NCV, hypomyelination remained (Figure 6B), as we have documented previously in other transgenic mice that overexpress total P0 (Wrabetz et al., 2000Wrabetz L. Feltri M. Quattrini A. Inperiale D. Previtali S. D'Antonio M. Martini R. Yin X. Trapp B. Zhou L. et al.P0 overexpression causes congenital hypomyelination of peripheral nerve." @default.
• W2143326415 created "2016-06-24" @default.
• W2143326415 creator A5024143813 @default.
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