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Immune Deficiency in Mouse Models for Inherited Peripheral The Journal of Neuroscience, January 15, 2000, 20(2):729–735 Immune Deficiency in Mouse Models for Inherited Peripheral Neuropathies Leads to Improved Myelin Maintenance Christoph D. Schmid,1,2 Martina Stienekemeier,1 Stephan Oehen,3 Frank Bootz,4 Ju¨ rgen Zielasek,1 Ralf Gold,1 Klaus V. Toyka,1 Melitta Schachner,5 and Rudolf Martini,1,2 1Department of Neurology, University of Wu¨ rzburg, D-97080 Wu¨ rzburg, Germany, 2Department of Neurobiology, Swiss Federal Institute of Technology, CH-8093 Zu¨ rich, Switzerland, Institutes of 3Experimental Immunology and 4Laboratory Animal Science, University of Zu¨ rich, CH-8092 Zu¨ rich, Switzerland, and 5Zentrum fu¨ r Molekulare Neurobiologie, University of Hamburg, D-20246 Hamburg, Germany The adhesive cell surface molecule P0 is the most abundant duction properties is less severe when the immune system is 1/2 glycoprotein in peripheral nerve myelin and fulfills pivotal func- deficient. Moreover, isolated T-lymphocytes from P0 mice tions during myelin formation and maintenance. Mutations in show enhanced reactivity to myelin components of the periph- the corresponding gene cause hereditary demyelinating neu- eral nerve, such as P0 ,P2 , and myelin basic protein. We 1/2 ropathies. In mice heterozygously deficient in P0 (P0 mice), hypothesize that autoreactive immune cells can significantly an established animal model for a subtype of hereditary neu- foster the demyelinating phenotype of mice with a primarily ropathies, T-lymphocytes are present in the demyelinating genetically based peripheral neuropathy. nerves. To monitor the possible involvement of the immune 1/2 system in myelin pathology, we cross-bred P0 mice with null Key words: Charcot-Marie-Tooth disease; myelin degenera- mutants for the recombination activating gene 1 (RAG-1) or with tion; Schwann cell; P0 ; myelin protein zero; immune system; mice deficient in the T-cell receptor a-subunit. We found that in immune deficiency; T-lymphocytes; macrophages; electron mi- 1/2 P0 mice myelin degeneration and impairment of nerve con- croscopy; electrophysiology Myelin protein zero (MPZ or P0), the most abundant glycopro- (Thomas et al., 1994; Gabree¨ls-Festen et al., 1996; Tachi et al., tein of the myelin sheath of peripheral nerves, is a transmem- 1997). Recently, we have shown that heterozygous P0 null mutant 1/2 brane adhesion molecule of the Ig superfamily that fulfills mul- mice (P0 ) are appropriate models for mild CMT1B forms tiple functions during myelin development and maintenance (for with an initially normal myelin formation followed by myelin review, see Martini and Schachner, 1997). Its pivotal role in the decompaction and demyelination starting at 4 months of life peripheral nervous system is reflected by the fact that mutations (Martini et al., 1995a; for review, see Martini, 1997). Although it in the corresponding gene cause inherited neuropathies, such as is well accepted that various heterozygous mutations or the re- Charcot-Marie-Tooth (CMT) disorder, type 1B, the De´je´rine- duction in gene dosage of P0 lead to demyelination (for review, Sottas syndrome, and congenital hypomyelination (for review, see see Martini et al., 1998), the molecular and cellular mechanisms De Jonghe et al., 1997; Martini et al., 1998; Nelis et al., 1999). that lead to myelin degeneration are not yet understood. Based on These disorders are typically associated with muscular weakness the observation that nerves of patients suffering from inherited 1/2 and atrophy, sensory dysfunction, and skeletal deformities. Biop- neuropathies (Schmidt et al., 1996) or nerves of P0 mice sies from CMT1B patients revealed that, dependent on the mu- contain endoneurial T-lymphocytes (Schmid et al., 1996; Shy et tation in the P0 gene, divergent pathological hallmarks can be al., 1997), we tested the hypothesis that the immune system might found. Such hallmarks comprise either formation of myelin be involved in the development of the demyelinating phenotype. 1/2 tomacula, i.e., focally thickened myelin sheaths of reduced stabil- We, therefore, cross-bred P0 mice with homozygous null ity or, alternatively, myelin decompaction and demyelination mutants for the recombination activating gene 1 (RAG-1) or with mice deficient in the T-cell receptor a-subunit (TCRa), i.e., with Received June 14, 1999; revised Sept. 10, 1999; accepted Oct. 22, 1999. mutants that are deficient in mature T- and B-lymphocytes or This study was supported by the Swiss National Science Foundation (NF 31- 45890.95 to R.M.), Gemeinnu¨tzige Hertie-Stiftung (GHS 2/3378/96 to R.M. and only T-lymphocytes, respectively. We found that, in the absence 1/2 M. Sch.), the Deutsche Forschungsgemeinschaft (Priority Program “Microglia,” of an intact immune system, myelin degeneration in P0 mice is MA1053/3-1, to R.M.), Research Funds of the University of Wu¨rzburg European 1/2 significantly reduced in comparison to P0 mice with normal Union (Clinical, genetic and functional analysis of peripheral neuropathies), and by 1/2 the German Ministry for Education and Research. We are grateful to Professor Rolf immune cells. Moreover, isolated T-lymphocytes from P0 Zinkernagel (University of Zu¨rich) for valuable advice and discussions, Heinrich mice show enhanced reactivity to myelin components of the Blazyca and Kathrin Rohner for excellent technical assistance, and Dr. Stefano peripheral nerve, such as P ,P , and myelin basic protein (MBP). Carenini for discussions. We are indebted to Dr. Imme Haubitz (Department of 0 2 Mathematics and Statistics, University of Wu¨rzburg) for performing the statistical We conclude that the immune system may be functionally in- analysis of the quantification of immune cells in peripheral nerves and of the volved in the demyelinating phenotype of mice with a primarily splenocyte proliferation assay. We also thank Dr. M. Koltzenburg for the possibility to use electronic equipment. genetically mediated peripheral neuropathy. Correspondence should be addressed to Rudolf Martini, Department of Neurol- ogy, Section of Developmental Neurobiology, University of Wu¨rzburg, D-97080 MATERIALS AND METHODS Wu¨rzburg, Germany. E-mail: [email protected]. Animals and determination of genotypes. Mice deficient in the gene for 2/2 Copyright © 2000 Society for Neuroscience 0270-6474/00/200729-07$15.00/0 protein zero (P0 ; Giese et al., 1992) or the recombination activating 730 J. Neurosci., January 15, 2000, 20(2):729–735 Schmid et al. • Improved Myelin in Immune-Deficient Myelin Mutants 2 2 gene 1 (RAG-1 / ; Mombaerts et al., 1992) were generated as described were not or only thinly myelinated (less than five turns) was determined previously and backcrossed to the C57BL/6 strain for six to eight gener- by electron microscopy. Analysis of morphometric parameters such as ations. Double mutants were generated by cross-breeding heterozygous the g-ratio (myelin thickness; Friede, 1972), the diameter of axons 1/2 2/2 P0-mutants (P0 mice) with RAG-1 mice under specific pathogen- (Friede, 1972), and the endoneurial space was done by electron micros- free conditions (Institute of Laboratory Animal Science, University of copy on randomly selected parts of ultrathin cross sections of the quad- Zu¨rich) in analogy to the breeding protocol described previously (Mar- riceps branch of the femoral nerve. The endoneurial space was deter- tini et al., 1995b; Carenini et al., 1997). Mice deficient for the T-cell mined as the fraction of total nerve area not occupied by fibers. Electron 2 2 receptor a subunit (TCRa / ; Philpott et al., 1992) were bred on a mixed micrographs of two randomly selected parts per nerve cross section were 1 2 background (129/Ola/Hsd, BALB/c, C57Bl/6) and cross-bred with P / digitized with a scanner (HP 4C). Morphometric parameters were eval- 0 3 mice as described above. Genotypes of P0-mutants were determined by uated at a final magnification of 8000 using the Image Tool Applica- conventional PCR using oligonucleotides 59-TCAGTTCCTTGTC- tion, version 1.28 program (University of Texas UTHSCSA). Statistical 1/2 2/2 CCCCGCTCTC-39,59-GGCTGCAGGGTCGCTCGGTGTTC-39, and significance of differences between mean values of P0 /RAG1 1/2 1/2 59-ACTTGTCTCTTCTGGGTAATCAA-39 leading to 334 or 500 bp versus P0 /RAG1 mice was determined by double-sided Student’s products for the P0 null mutation or wild-type allele, respectively. The t test using Microsoft Excel software. phenotype of the RAG-1 or the TCRa null mutation was determined by Culture of splenocytes and proliferation assay. Splenocytes from four 1/1 1/2 fluorescence activated cell sorting (FACS) analysis of peripheral blood myelin wild-type (P0 ) and four P0 mice at the age of 8 months 6 cells stained with phycoerythrin (PE)-coupled anti-CD4 and fluorescein- were seeded into 96-well microtiter plates at a density of 1.5 3 10 /ml isothiocyanate-coupled anti-CD8 antibodies (PharMingen, Hamburg, (Nunc, Wiesbaden, Germany). Splenocytes were exposed to recombinant m Germany) using a FACScan instrument (Becton Dickinson, Heidelberg, human (rh) P0, rhP2 (Weishaupt et al., 1995), rat MBP (10 g/ml each), m Germany). To rule out the possibility that the effects observed were a or concanavalin A (Con A; 2.5 g/ml), as described (Pette et al., 1990; consequence of differences in the genetic background, all pathological Stienekemeier et al., 1999) except that we used 1% autologous serum and 3 25 alterations were investigated in littermates. For all pathological studies, added 5 10 M 2-mercaptoethanol. After 3 d, murine interleukin the femoral quadriceps nerve was chosen that contains ;500 myelinated (IL)-2 (2.5 ng/ml;R&DSystems, Wiesbaden, Germany) was added. On axons in wild-type mice (Lindberg et al., 1999), thus being an ideal day 8, the nonadherent cells from each plate were split into two new paradigm for quantitative analyses. All experiments described here on microtiter plates. Cells were reactivated on day 14 by addition of irradi- animals were approved by the Veterinary Administration of Zu¨rich, ated, syngeneic splenocytes to both subcultures, whereas the respective Switzerland and by the Regierung von Unterfranken (Wu¨rzburg, antigens were added to only one as described for the split well technique Germany).
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