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. 2006 Feb 22;26(8):2358-68.
doi: 10.1523/JNEUROSCI.3819-05.2006.

Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice

Lawrence Wrabetz  1 , Maurizio D'Antonio, Maria Pennuto, Gabriele Dati, Elisa Tinelli, Pietro Fratta, Stefano Previtali, Daniele Imperiale, Jurgen Zielasek, Klaus Toyka, Robin L Avila, Daniel A Kirschner, Albee Messing, M Laura Feltri, Angelo Quattrini
Affiliations

Different intracellular pathomechanisms produce diverse Myelin Protein Zero neuropathies in transgenic mice

Lawrence Wrabetz et al. J Neurosci. .

Abstract

Missense mutations in 22 genes account for one-quarter of Charcot-Marie-Tooth (CMT) hereditary neuropathies. Myelin Protein Zero (MPZ, P0) mutations produce phenotypes ranging from adult demyelinating (CMT1B) to early onset [Déjérine-Sottas syndrome (DSS) or congenital hypomyelination] to predominantly axonal neuropathy, suggesting gain of function mechanisms. To test this directly, we produced mice in which either the MpzS63C (DSS) or MpzS63del (CMT1B) transgene was inserted randomly, so that the endogenous Mpz alleles could compensate for any loss of mutant P0 function. We show that either mutant allele produces demyelinating neuropathy that mimics the corresponding human disease. However, P0S63C creates a packing defect in the myelin sheath, whereas P0S63del does not arrive to the myelin sheath and is instead retained in the endoplasmic reticulum, where it elicits an unfolded protein response (UPR). This is the first evidence for UPR in association with neuropathy and provides a model to determine whether and how mutant proteins can provoke demyelination from outside of myelin.

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Figures

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Characterization of S63 transgenic mice. A, Ribbon diagram of P0 ECD demonstrates the location of S63 at the beginning of β-strand C [arrow; adapted from Shapiro et al. (1996) with permission of Elsevier]. B, Diagram of the six exons of wild-type Mpz, TgS63del, and S63C with nucleotide alterations indicated by asterisks. C, Semiquantitative RT-PCR analysis of transgene expression. RT product was amplified by a primer pair that recognized identically either endogenous Mpz or TgS63 cDNAs. DpnII digestion distinguished endogenous (Endog; 326 nt band) from TgS63 (244 and 82 nt bands). Overexpression (—fold OE) is the ratio Tg/Endog signals.
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Phenotypes of S63 mutant mice in a wild-type (Mpz+/+) background. A, Table describing the features of S63 phenotypes. Exp, Expression from Figure 1. B–H, Morphological analysis of 1.5- to 2-year-old S63 mutant mice reveals myelin alterations. S63del sciatic nerves contain florid onion bulbs [indicated by arrows and magnified 2.5× in insets; lines 129.1 (B), 129.2 (C), 129.4 (D)] compared with WT (F). Ultrastructural analysis of S63del129.2 sciatic nerve (C) shows two types of onion bulbs, one with multiple Schwann cell processes in various relationships with multiple small axons (G), the other with multiple Schwann cell processes interspersed in collagen fibers, surrounding a single thinly myelinated axon (H). Endoplasmic reticulum and Golgi appeared normal. In contrast, S63C sciatic nerves [130.2 (E)] show more hypomyelination (compare arrowheads in E–D) and less onion bulb formation. a, Axon; m, myelin; sc, Schwann cell; cb, collagen bundle; scp, Schwann cell process. Scale bar: (in H) B–F, 20 μm; G–H, 3 μm.
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The S63del demyelination phenotype is not rescued by reduced Mpz dosage. TgS63del (A) was bred into the Mpz+/− background (S63del//+/−; B) and compared with Mpz+/− (C) and WT (D) animals. Semithin section analysis of sciatic nerves from 16-month-old mice show that S63del//+/− nerves continue to manifest onion bulbs (arrows; magnified 2.5× in insets) and thin myelin sheaths (arrowheads) despite near normal Mpz dosage. The images are representative of multiple, randomly selected fields in sciatic nerve for each genotype. Scale bar: (in D) 30 μm.
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S63del//Mpz+/− mice provide a good model of human CMT1B neuropathy. A, Rotarod analysis shows that S63del//+/− remain on the accelerating cylinder about one-half the time of wild type. Error bars represent SEM; n = 10. B, CMAPs were recorded from the small foot muscles after supramaximal proximal sciatic nerve stimulation. Shown are redrawn original recordings from 1-year-old mice. The filled triangles indicate the onset of the M-response; the open triangles indicate the onset of the F-wave. Note the different amplitude scales. Semithin section analysis of mutant (C/C′–E/E′) and wild-type (F, F′) sciatic (C–F) or digital (C′–F′) nerves reveals onion bulbs (arrows) and hypomyelination, which are more evident distally and progress with age. m, Month. Scale bar: (in F′) 25 μm.
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Biochemical and expression analysis of P0S63 mutants shows that P0S63del has altered post-translational modification and markedly reduced steady-state protein levels. A, Western analysis for P0S63 mutant proteins in WT, Mpz+/− (P0+/−), or Mpz-null (P0−/−) backgrounds. Note that P0S63del migrates faster than P0WT (arrow), at a size similar to a minor band seen in all lanes (arrowhead). Tubulin (Tub) detection confirmed equal loading of protein. The numbers indicate relative molecular weights. B, Semiquantitative RT-PCR analysis for P0 mRNA (P0) relative to GAPDH in sciatic nerves from Mpz+/− (P0+/−), S63del (S63delWT), or S63del in the Mpz-null background (S63delP0−/−) quantified in C relative to wild type defined as 1. The numbers in B refer to cycles of PCR amplification. D, Western analysis for P0 on sciatic nerve lysates from WT or S63 mutants in the Mpz-null background (e.g., S63delP0−/−) untreated (C) or after digestion with EndoH (H) or PNGaseF (F). Untreated P0WT migrated with a relative molecular weight of 28.5 kDa. EndoH treatment of WT revealed ∼60% high-type (sensitive) and 40% complex-type (resistant) P0 glycosylation as reported previously in adult rat nerve homogenates (Brunden, 1992). PNGaseF digestion of P0WT completely removed oligosaccharide and produced migration at 24.8 kDa (D’Urso et al., 1990). Glycosylation of S63C was similar to that of WT. In contrast, most P0S63del was insensitive to both EndoH and PNGaseF digestion, indicating that the majority of the protein was not glycosylated at the steady state. Note that the relative molecular weight of S63del protein [S63delP0−/− (C); estimated to be 20.5 kDa] was less than that of deglycosylated P0WT [WT (F)], suggesting further post-translational differences.
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P0S63 mutant proteins arrive to diverse intracellular locations. Immunofluorescence staining for P0 (A, D, G) or KDEL (B, E, H) or overlay of the two (C, F, I) in WT (A–C), S63del (D–F), or S63C (G, H) sciatic nerves. P0 and KDEL staining were almost mutually exclusive in wild-type nerves, with most P0 staining in circular myelin sheaths. S63C nerves were similar, except for occasional yellow overlay staining (arrows in inset; magnified 2×; asterisk in I indicates the same fiber). In contrast, almost all P0 and KDEL staining coincided in S63del nerves. Ultrastructural analysis of periodicity (J–M) showed that both wild-type and S63C myelin sheaths were compacted but that the periodicity (white circles indicate 11 major dense lines equal to 10 periods) was 21% larger in S63C myelin. In contrast, S63del, like Mpz −/− (P0 −/−) myelin, was uncompacted. Note that all S63 mutants have been bred into the Mpz-null background in this figure. Scale bar: (in I) A–I, 20 μm; J, K, 50 nm; L, M, 160 nm.
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S63del sciatic nerves manifest a UPR. A, Quantitative real-time RT-PCR analysis for BiP and CHOP mRNA levels relative to WT set arbitrarily to 1. All values (mean ± SEM for n = 5 mice, all in Mpz+/+ background) were first normalized to 18S rRNA levels. Note that BiP and CHOP levels were significantly increased in S63del compared with S63C, P0OE, or WT (p < 0.01 by Student's t test). Liver dissected from animals 72 h after intraperitoneal injection of tunicamycin (TUN) showed a comparable induction of BiP and CHOP mRNA levels. UT, Intraperitoneal saline injection. B, RT-PCR analysis for induction of XBP1 splicing shows that the XBP1 splice variant (arrowhead) appears below XBP1 (arrow) in sciatic nerves from two S63del mice and from animals at 48 h (TUN48) or 72 h (TUN72) after injection of tunicamycin, but not from wild-type or S63C mice.
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References

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