Animal models of hereditary neuropathies

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Introduction

On the basis of new insights into the genetic basis of different types of hereditary neuropathies, several animal models have been generated using transgenic and gene targeting techniques in mice and rats. Animal models are excellent tools to learn more about the biological function of specific proteins involved in myelin maintenance, glia/axon interactions and, in general, about cellular mechanisms of inherited neuropathies. Furthermore, these models reflect the human disease and provide the opportunity to es- tablish therapeutic strategies. Up to now, the available animals are mainly models for the group of hereditary motor and sensory neuropathies (Char- cot-Marie-Tooth disease; CMT).

The scientific and therapeutical value of each of theses models must be carefully evaluated since transgenic mice and rats carrying comparable mutations or genetic defects as in humans may not always reflect the human disease per se in all facets. In this chapter, a list of available animal models of hereditary neuropathies and their specific clinical and histopathological features are presented. From a histopathologic and electrophysiologic point of view, two main different types of CMT can be distinguished. Primary demyelination is the basis of CMT1, CMT4 and Djerine Sottas syndrome (DSS), while in CMT2 primary axonal degeneration causes the clinical phenotype [28].

In this chapter, gene symbols are given in capitalized italics for the human genes. Mice or rat genes are indicated in non-capitalized italics.

15.1 Models for demyelinating CMT1A:

peripheral myelin 22 (pmp22)

15.1.1 pmp22 transgenic rats

z Genetic defect: These rats overexpress pmp22 protein (pmp22) because they carry extra copies of the pmp22 gene (pmp22) [24].

of hereditary neuropathies

P. Young, U. Suter

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z Phenotype: Behavioral symptoms appear at about 60 days after birth.

Heterozygous pmp22 overexpressing rats show deficits in their front limb grip force and in their capability to walk on a round horizontal bar (bar test). Nerve conduction velocities of peripheral nerves are decreased. De- myelinated and amyelinated large axons are detectable within the second postnatal month. Homozygous pmp22 overexpressing rats are affected more severely than the heterozygous animals. For unknown reasons they show severe spasticity and seizures. Histopathologically these homozygous animals completely lack myelinated fibers.

z Corresponding CMT syndrome:Heterozygous pmp22 overexpressing rats reflect the clinical, electrophysiological and histopathological changes in CMT1A patients. Homozygous pmp22 overexpressing rats do not have a di- rect human counterpart but it should be noted that a homozygous duplication on chromosome 17p11.2 causes a severe hereditary neuropathy in humans [14]. With the aid of this animal model, the positive therapeutic ef- fect of the progesterone antagonist onapristone has been shown (see also chapter 13) [25].

15.1.2 pmp22 transgenic mice

z Genetic defect:Transgenic mice carrying varying copy numbers of pmp22 were created by different groups [9, 15].

z Phenotype:pmp22 overexpressing transgenic mice show histopathological changes that are correlated with the gene copy number and severity of the clinical phenotype [10]. Mice with a high copy number of the pmp22 transgene show demyelinated and amyelinated nerve fibers within the PNS ear- lier than mice with low copy numbers. There is strong evidence that the phenotypic expression in these transgenic mice results not only from Schwann cell pathology but also from axonal and neuronal changes [19, 21, 22].

z Corresponding CMT syndrome: pmp22 overexpressing mice reflect some clinical and histopathological features of CMT1A.

15.1.3 Inducible pmp22 transgenic mice

z Genetic defect: Transgenic mice carrying the pmp22 transgene under a regulatory tetracycline inducible promoter. In this animal model pmp22 expression ceases when tetracycline is administered with the food [20].

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z Phenotype: These mice show the same phenotype as straight pmp22 overexpressing mice (as long as they are not fed tetracycline with their chow). This phenotype is reverted when tetracycline is given and a reduction of the demyelination of the peripheral nerves can be observed.

z Corresponding CMT syndrome: In the case of pmp22 overexpression, mice are comparable to human CMT1A. Reduction of pmp22 expression after addition of tetracycline to the food results in decreased demyelination, but complete reversion of the CMT1A-like phenotype is not achieved.

15.1.4 pmp22 knockout mice

z Genetic defect:pmp22 deficient mice were generated by classical gene targeting techniques [1, 2].

z Phenotype: Homozygous pmp22 deficient mice show early onset of demyelination within the peripheral nervous system (PNS) resulting in an early onset of paresis and muscular atrophy in the hind limbs [2]. Clini- cally these mice also exhibit inducible myotonia-like symptoms [30]. Elec- trophysiologically, reduced nerve conduction velocities (NCVs) of the peripheral nerves are observed. Histopathologically, focally hypermyelinated fibers (so called tomacula) are found in younger animals followed by demyelination (Fig. 15.1) [2].

Clinically, heterozygous pmp22 deficient mice show a very mild phenotype without significant paresis or muscular atrophy [1]. Microscopic changes in the PNS resemble the human condition of hereditary neuropathy with liability to pressure palsy (HNPP) with longitudinally oriented hypermyelinated stretches (tomacula) as histopathological hallmarks [1].

z Corresponding CMT syndrome: Homozygous pmp22 deficient mice can be seen as a model for severe forms of demyelinating neuropathies. Histo- pathologically, heterozygous pmp22 deficient mice resemble the human HNPP phenotype. As in HNPP, these mice develop a mild progressive demyelinating neuropathy in later ages.

15.1.5 Mice carrying point mutations in pmp22 trembler, trembler J, Tr-m1H, Tr-m2H

z Genetic defect:A spontaneous point mutation in the pmp22 gene causing an amino acid exchange from glycine to aspartic acid at codon 150 was found in trembler mice [29]. Trembler-J mice carry a point mutation in pmp22 causing an amino acid exchange from leucine to proline at codon 16 [27]. Tr-m1H and Tr-m2H mice were generated in the course of a ge- nome-wide, phenotype-driven, large-scale N-ethyl-N-nitrosourea (ENU)

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mutagenesis screen [11]. In Tr-m1H, the mutation causes an amino acid exchange at codon 12 (histidine to arginine), while in Tr-m2H the amino acid exchange is a codon 153 tyrosine substitution for a stop codon. These four different mutations in mice are dominant.

z Phenotype:All four mice lines exhibit a severe form of hypo- and demyelination. Most nerve fibers are found to be hypomyelinated while few fibers show demyelination. Focal ineffective remyelination ± causing so called onion bulb myelin formations ± is rare. A typical feature is the trembling observed from early postnatal stages onwards. Muscle wasting starts as early as six weeks of age [16].

z Corresponding CMT syndrome: Mice carrying point mutations reflect the situation of severely affected CMT1A patients carrying PMP22 point mutations. Mutations corresponding to the trembler-J mutation and the trembler mutation in mice were also found in very severely affected CMT1 pa- Fig. 15.1. Cross section of sciatic nerves from a PMP22 wildtype mouse (a, PMP22 wt/wt), PMP22 heterozygous deficient mouse (b, PMP22 0/wt) and a PMP22 overexpressing mouse mutant (c, PMP22 over). In b most prominent is a fiber with thickened myelin caused by supernu- merous myelin lamellae and reduced axon size (asterisk). This structure corresponds to the so called tomacula in HNPP. The arrow indicates a fiber which is almost void of myelin. c shows a significant loss of myelinated nerve fibers. Myelin thickness is reduced in most fibers. (Courtesy of Dr. Sara Sancho, Fribourg, Switzerland)

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tients [31]. Thus, trembler-J and trembler mice are accepted as animal models for CMT1A caused by PMP22 point mutations. Tr-m2H also serves as a model for CMT1A, however, extended histopathological analysis is not available [11, 12]. Analogous to the Tr-m1H mutation, a mutation of codon 12 (histidine to glutamine) has been identified in a patient with a severe form of CMT (Djerine-Sottas syndrome) [32] (see also: Inherited Periph- eral Neuropathies Mutation Database (IPNMD) at: http://molgen-www.uia.

ac.be/CMTMutations/ for human mutations).

15.2 Models for demyelinating CMT1B:

myelin protein zero (mpz) knockout mice

z Genetic defect: Disruption of mpz encoding the p0 protein by gene targeting techniques. Homozygous and heterozygous animals have been generated resulting in different levels of p0 expression [7].

z Phenotype: Homozygous p0 deficient mice exhibit a severe muscle atrophy at early stages of postnatal development [17]. Most nerve fibers are amyelinated. Heterozygous mice show mild motor behavioral deficits and mild tremor [17]. Heterozygous animals show much milder alterations of myelinated nerve fibers such as dysmyelination, onion bulb formation and mild axonal loss starting at four months of age [17]. In these mice a pecu- liar type of nerve fibers is found showing patchy demyelination leading to the formation of so called heminodes along the axon. Heminodes are characterized by the histopathological feature of nodes of Ranvier which show paranodal organization on the side of the nodal and paranodal region while the paranodal formations are lacking on the other side.

z Corresponding CMT phenotype: Homozygous p0 deficient mice do not reflect the broad spectrum of human MPZ associated CMT phenotypes. They are comparable to congenital hypomyelination or very severe cases of CMT, also called Djerine-Sottas syndrome (DSS).

Heterozygous p0 deficient mice resemble the human demyelinating phenotype of CMT1B associated with point mutations in MPZ. However, some CMT1B patients show a more axonal phenotype which is not reflected by the accompanying axonal loss caused by demyelination in heterozygous p0 deficient mice. These patients can be diagnosed as CMT2 or dominant in- termediate CMT (DI-CMT) by clinical, electrophysiological and genetic measures [5, 33].

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15.3 Models for demyelinating and axonal CMTX:

gap junction protein beta 1 (gjb1) knockout mice

z Genetic defect:Gene disruption by gene targeting techniques resulting in deficiency for the protein Cx32. Homozygous and heterozygous mice have been generated [18, 23].

z Phenotype:Homozygous Cx32 deficient mice exhibit a peripheral neuropathy after approximately five months [23]. Histopathologically demyelination is a typical feature with thinning of compacted myelin layers and onion bulb formation. Strikingly, periaxonal collars are thickened, a feature not observed in other demyelinating CMT syndromes [3, 23].

z Corresponding CMT syndrome:In CMTX, clinical and histopathological features are heterogeneous [33] due to the fact that some patients present with a predominantly demyelinating neuropathy while others suffer from a predominantly axonal neuropathy. A third group presents a mixture of demyelination and axonal pathology [8, 33]. In contrast to the human disease, axonal degeneration is less prominent in Cx32 deficient mice [3, 23].

15.4 Model for demyelinating CMT4F:

periaxin (prx) knockout mice

z Genetic defect:prx is a constituent of the dystroglycan-dystrophin-related protein-2 complex, which links the Schwann cell cytoskeleton to the extra- cellular matrix. prx null mutants have been generated by gene targeting techniques [26].

z Phenotype: prx deficient mice develop difficulties in walking at six to nine months of age accompanied by neuropathic pain [26]. Demyelination and tomacula-like structures can be found in these mutant animals although myelination initially is normal.

z Corresponding CMT syndrome:CMT4F is an autosomal recessive CMT syndrome, which is characterized by demyelination and excessive neuropathic pain. The similarity between the histopathological and clinical features of prx deficient mice and the corresponding features of CMT4F patients lead to the identification of PRX as a candidate gene for CMT4F. Therefore, prx deficient mice represent an excellent model of CMT4F.

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15.5 Model for axonal CMT2A2:

kinesin motor protein 1 beta (kif1b) knockout mice

z Genetic defect:Classical gene targeting and disruption of the kif1b locus.

Homozygous mice are not viable. Heterozygous mice are viable and show reduced kif1b protein levels [36].

z Phenotype:Heterozygous kif1b deficient mice develop progressive muscle weakness and atrophy of the hind limbs [36]. Axonal degeneration is not a striking phenotypic feature of kif1b deficient mice but axonal transport via synaptic vesicles is disturbed.

z Corresponding CMT syndrome:CMT2A2 is characterized by primary axonal loss and degeneration. Heterozygous kif1b deficient mice mimic the human CMT2A2 phenotype clinically and histopathologically.

15.6 Model for axonal CMT2E: neurofilament light chain (nefl) knockout mice

z Genetic defect: Mouse lines have been generated in which nefl has been disrupted by gene targeting strategies [13, 37].

z Phenotype:Mice deficient for nefl show no striking axonal loss or degeneration. Axonal regeneration is delayed which is not observed in the human situation [37].

z Corresponding CMT syndrome:CMT2E caused by mutations in NEFL is not reflected by nefl deficient mice.

15.7 Model for recessive CMT4C1:

lamin A/C (lmna) knockout mice

z Genetic defect: lmna deficient mice were generated by gene targeting techniques [6].

z Phenotype:Ultrastructural exploration of sciatic nerves of lmna deficient mice revealed reduction of axon density, axonal enlargement, and the pres- ence of non myelinated axons [6].

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z Corresponding CMT phenotype: Autosomal recessive CMT2 (CMT2B1) is caused by mutations in LMNA. Histopathological and clinical findings in these patients resemble the changes found in lmna deficient mice [6].

15.8 Conclusions

Animal models exist for some demyelinating and some axonal CMT syndromes. With the aid of these models, approaches for therapeutic strategies can be further developed in the future. Additional models have to be and will be generated to mimic human CMT syndromes that have no appropriate model.

Newly discovered hereditary neuropathy genes and animal models will provide the basis for a deeper view into the molecular mechanisms which lead to the clinical phenotype and syndrome. The establishment of causa- tive treatment strategies is critically dependent on appropriate models in which axonal and glial function can be dissected (see also for review [4, 16, 28, 34, 35].

References

1. Adlkofer K, Frei R, Neuberg DH, Zielasek J, Toyka KV, Suter U (1997) Heterozy- gous peripheral myelin protein 22-deficient mice are affected by a progressive demyelinating tomaculous neuropathy. J Neurosci 17:4662±4671

2. Adlkofer K, Martini R, Aguzzi A, Zielasek J, Toyka KV, Suter U (1995) Hypermye- lination and demyelinating peripheral neuropathy in Pmp22-deficient mice. Nat Genet 11:274±280

3. Anzini P, Neuberg DH, Schachner M, Nelles E, Willecke K, Zielasek J, Toyka KV, Suter U, Martini R (1997) Structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin 32. J Neurosci 17:4545±4551

4. Berger P, Young P, Suter U (2002) Molecular cell biology of Charcot-Marie-Tooth disease. Neurogenetics 4:1±15

5. Chapon F, Latour P, Diraison P, Schaeffer S, Vandenberghe A (1999) Axonal phenotype of Charcot-Marie-Tooth disease associated with a mutation in the myelin protein zero gene. J Neurol Neurosurg Psychiatry 66:779±782

6. De Sandre-Giovannoli A, Chaouch M, Kozlov S, Vallat JM, Tazir M, Kassouri N, Szepetowski P, Hammadouche T, Vandenberghe A, Stewart CL, Grid D, Levy N (2002) Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie- Tooth disorder type 2) and mouse. Am J Hum Genet 70:726±736

7. Giese KP, Martini R, Lemke G, Soriano P, Schachner M (1992) Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons. Cell 71:565±576

(9)

8. Hattori N, Yamamoto M, Yoshihara T, Koike H, Nakagawa M, Yoshikawa H, Oh- nishi A, Hayasaka K, Onodera O, Baba M, Yasuda H, Saito T, Nakashima K, Kira J, Kaji R, Oka N, Sobue G (2003) Demyelinating and axonal features of Charcot- Marie-Tooth disease with mutations of myelin-related proteins (PMP22, MPZ and Cx32): a clinicopathological study of 205Japanese patients. Brain 126:134±151 9. Huxley C, Passage E, Manson A, Putzu G, Figarella-Branger D, Pellissier JF, Fontes

M (1996) Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNA. Hum Mol Genet 5:563±569 10. Huxley C, Passage E, Robertson AM, Youl B, Huston S, Manson A, Saberan-Djo-

niedi D, Figarella-Branger D, Pellissier JF, Thomas PK, Fontes M (1998) Correla- tion between varying levels of PMP22 expression and the degree of demyelination and reduction in nerve conduction velocity in transgenic mice. Hum Mol Genet 7:449±458

11. Isaacs AM, Davies KE, Hunter AJ, Nolan PM, Vizor L, Peters J, Gale DG, Kelsell DP, Latham ID, Chase JM, Fisher EM, Bouzyk MM, Potter A, Masih M, Walsh FS, Sims MA, Doncaster KE, Parsons CA, Martin J, Brown SD, Rastan S, Spurr NK, Gray IC (2000) Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy. Hum Mol Genet 9:1865±1871 12. Isaacs AM, Jeans A, Oliver PL, Vizor L, Brown SD, Hunter AJ, Davies KE (2002)

Identification of a new Pmp22 mouse mutant and trafficking analysis of a Pmp22 allelic series suggesting that protein aggregates may be protective in Pmp22-associated peripheral neuropathy. Mol Cell Neurosci 21:114±125

13. Lee MK, Cleveland DW (1994) Neurofilament function and dysfunction: involve- ment in axonal growth and neuronal disease. Curr Opin Cell Biol 6:34±40 14. LeGuern E, Gouider R, Mabin D, Tardieu S, Birouk N, Parent P, Bouche P, Brice A

(1997) Patients homozygous for the 17p11.2 duplication in Charcot-Marie-Tooth type 1A disease. Ann Neurol 41:104±108

15. Magyar J, Martini R, Ruelicke T, Aguzzi A, Adlkofer K, Dembic Z, Zielasek J, Toy- ka K, Suter U (1996) Impaired differentiation of Schwann cells in transgenic mice with increased PMP22 gene dosage. J Neurosci 16:5351±5360

16. Maier M, Berger P, Suter U (2002) Understanding Schwann cell-neurone interactions: the key to Charcot- Marie-Tooth disease? J Anat 200:357±366

17. Martini R, Zielasek J, Toyka K, Giese P, Schachner M (1995) Protein zero (P0)-deficient mice show myelin degeneration in peripheral nerves characteristic of inherited human neuropathies. Nat Genet 11:281±286

18. Nelles E, Butzler C, Jung D, Temme A, Gabriel HD, Dahl U, Traub O, Stumpel F, Jungermann K, Zielasek J, Toyka KV, Dermietzel R, Willecke K (1996) Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice. Proc Natl Acad Sci USA 93:9565±9570

19. Norreel JC, Vinay L, Fontes M, Clarac F (2003) Close relationship between motor impairments and loss of functional motoneurons in a Charcot-Marie-Tooth type 1A model. Neuroscience 116:695±703

20. Perea J, Robertson A, Tolmachova T, Muddle J, King RH, Ponsford S, Thomas PK, Huxley C (2001) Induced myelination and demyelination in a conditional mouse model of Charcot-Marie-Tooth disease type 1A. Hum Mol Genet 10:1007±1018 21. Sancho S, Magyar JP, Aguzzi A, Suter U (1999) Distal axonopathy in peripheral

nerves of PMP22-mutant mice. Brain 122:1563±1577

22. Sancho S, Young P, Suter U (2001) Regulation of Schwann cell proliferation and apoptosis in PMP22-deficient mice and mouse models of Charcot-Marie-Tooth disease type 1A. Brain 124:2177±2187

23. Scherer SS, Xu YT, Nelles E, Fischbeck K, Willecke K, Bone LJ (1998) Connex- in32-null mice develop demyelinating peripheral neuropathy. Glia 24:8±20

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24. Sereda M, Griffiths I, Puhlhofer A, Stewart H, Rossner MJ, Zimmerman F, Magyar JP, Schneider A, Hund E, Meinck HM, Suter U, Nave KA (1996) A transgenic rat model of Charcot-Marie-Tooth disease. Neuron 16:1049±1060

25. Sereda MW, Meyer zu Horste G, Suter U, Uzma N, Nave KA (2003) Therapeutic administration of progesterone antagonist in a model of Charcot-Marie-Tooth disease (CMT-1A). Nat Med 9:1533±1537

26. Sherman DL, Fabrizi C, Gillespie CS, Brophy PJ (2001) Specific disruption of a schwann cell dystrophin-related protein complex in a demyelinating neuropathy.

Neuron 30:677±687

27. Suter U, Moskow JJ, Welcher AA, Snipes GJ, Kosaras B, Sidman RL, Buchberg AM, Shooter EM (1992) A leucine-to-proline mutation in the putative first trans- membrane domain of the 22-kDa peripheral myelin protein in the trembler-J mouse. Proc Natl Acad Sci USA 89:4382±4386

28. Suter U, Scherer SS (2003) Disease mechanisms in inherited neuropathies. Nat Rev Neurosci 4:714±726

29. Suter U, Welcher AA, Ozcelik T, Snipes GJ, Kosaras B, Francke U, Billings-Gagliar- di S, Sidman RL, Shooter EM (1992) Trembler mouse carries a point mutation in a myelin gene. Nature 356:241±244

30. Toyka KV, Zielasek J, Ricker K, Adlkofer K, Suter U (1997) Hereditary neuromyo- tonia: a mouse model associated with deficiency or increased gene dosage of the PMP22 gene. J Neurol Neurosurg Psychiatry 63:812±813

31. Valentijn L, Baas F, Wolterman R, Hoogendijk J, Van den Bosch H (1992) Identical point mutations of PMP-22 in Trembler-J mouse and Charcot-Marie-Tooth disease type 1A. Nat Genet 2:288±290

32. Valentijn LJ, Ouvrier RA, van den Bosch NH, Bolhuis PA, Baas F, Nicholson GA (1995) Dejerine-Sottas neuropathy is associated with a de novo PMP22 mutation.

Hum Mutat 5:76±80

33. Young P, Grote K, Kuhlenbaumer G, Debus O, Kurlemann H, Halfter H, Funke H, Ringelstein EB, Stogbauer F (2001) Mutation analysis in Chariot-Marie Tooth disease type 1: point mutations in the MPZ gene and the GJB1 gene cause comparable phenotypic heterogeneity. J Neurol 248:410±415

34. Young P, Suter U (2003) The causes of Charcot-Marie-Tooth disease. Cell Mol Life Sci 60:2547±2560

35. Young P, Suter U (2001) Disease mechanisms and potential therapeutic strategies in Charcot-Marie-Tooth disease. Brain Res Brain Res Rev 36:213±221

36. Zhao C, Takita J, Tanaka Y, Setou M, Nakagawa T, Takeda S, Yang HW, Terada S, Nakata T, Takei Y, Saito M, Tsuji S, Hayashi Y, Hirokawa N (2001) Charcot-Marie- Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta.

Cell 105:587±597

37. Zhu Q, Couillard-Despres S, Julien JP (1997) Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp Neurol 148:299±316