Brain, Vol. 124, No. 11, 2177-2187,
November 2001
© 2001 Oxford University Press
Regulation of Schwann cell proliferation and apoptosis in PMP22-deficient mice and mouse models of Charcot–Marie–Tooth disease type 1A
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Institute of Cell Biology, Department of Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich, Switzerland
Correspondence to:
Dr Ueli Suter, Institute of Cell Biology, ETH Hönggerberg CH-8093 Zürich E-mail: usuter{at}cell.biol.ethz.ch
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Charcot–Marie–Tooth disease type 1A (CMT1A) is caused by an increased dosage of the peripheral myelin protein 22 (PMP22) gene or by point mutations affecting the same gene. Based on in vitro data, PMP22 might be involved, besides in its proven role in the regulation of myelination and myelin maintenance, in the control of Schwann cell proliferation and programmed cell death. In this report, we have used mice lacking PMP22 and mouse models for CMT1A to analyse Schwann cell proliferation and apoptosis in vivo during postnatal sciatic nerve development. Our results show that there is no significant change in the number of Schwann cells at postnatal day 1 in the analysed PMP22 mutants compared with the corresponding wild-type animals. Furthermore, the rate of proliferation also was not changed at this early developmental time point. In contrast, cell density and proliferation rates were increased, albeit with different kinetics, in all PMP22 mutants later in development. The increase in proliferation is paralleled by a higher number of apoptotic Schwann cells found in the nerves. Thus, increased Schwann cell proliferation and apoptosis, but only in later development and in adults, are hallmarks of PMP22 mutant mice, regardless of whether increased or decreased PMP22 gene dosage or point mutations affecting the PMP22 gene are responsible for the resulting demyelinating, dysmyelinating or amyelinating phenotypes.
Schwann cell; PMP22; proliferation; apoptosis; CMT
BrdU = bromodeoxyuridine; CMT = Charcot–Marie–Tooth disease; HNPP = hereditary neuropathy with liability to pressure palsies; P = postnatal day; PMP22 = peripheral myelin protein 22; PCR = polymerase chain reaction; Tr = Trembler; TUNEL = d-UTP-digoxigenin nick end-labelling
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Introduction |
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The inherited peripheral neuropathy syndrome Charcot–Marie–Tooth disease (CMT) is the most common genetic disorder in neurology, with an estimated prevalence of 1 : 2500 (Skre, 1974
). CMT patients suffering from the most frequently observed subtype CMT1A show slowed nerve conduction velocities, reduced compound motor and sensory nerve action potentials, progressive distal weakness, sensory loss and decreased reflexes (Dyck et al., 1993; Krajewski et al., 2000). Pathological hallmarks are segmental demyelination of motor and sensory nerves with incomplete remyelination. Genetically, the autosomal-dominant CMT1A is due to an intrachromosomal duplication on the short arm of chromosome 17p12 including the disease-causing, dose-sensitive peripheral myelin protein 22 (PMP22) gene (Raeymaekers et al., 1991; Lupski and Garcia, 1992; Patel et al., 1992). Intriguingly, the reciprocal deletion of the same DNA fragment is associated with hereditary neuropathy with liability to pressure palsies (HNPP) (Chance et al., 1993), which is usually not progressive but rather characterized by temporary palsies after pressure trauma (Windebank et al., 1993). Rare cases of more severe forms of CMT1A can also be due to missense mutations in PMP22 (Roa et al., 1993; Naef and Suter, 1999). Furthermore, HNPP has been found in association with PMP22 frameshift mutations (Nicholson et al., 1994; Young et al., 1997).
Several animal models for CMT1A are available. These include the spontaneous mouse mutants Trembler (Tr) and Tr-J which carry point mutations in the PMP22 gene (Suter et al., 1992a, 1992b). The same mutations have also been found in severely affected CMT1A patients (Valentijn et al., 1992; Ionasescu et al., 1997). Models for CMT1A due to increased PMP22 gene dosage have been generated in mice (Huxley et al., 1996; Magyar et al., 1996) and rats (Sereda et al., 1996; Niemann et al., 2000). Interestingly, the neurological and pathological features of mutant mice correlated well with the findings in human patients and led to a pronounced reappraisal of the axonal defects in CMT1A (Sancho et al., 1999; Krajewski et al., 2000). Mice lacking PMP22 develop a demyelinating peripheral neuropathy reminiscent of severe CMT1 (Adlkofer et al., 1995) while, as expected from human genetics, heterozygous PMP22 knock-out mice revealed a pathology comparable with HNPP (Adlkofer et al., 1997a).
Recent findings emphasize the crucial interplay between axons and Schwann cells in the cellular and molecular basis of CMT1A (for a review, see Naef and Suter, 1998). This conceptual framework originally has been derived from normal development of peripheral nerves where neurones regulate proliferation, survival and differentiation of Schwann cells (reviewed by Adlkofer and Lai, 2000). Schwann cells, in turn, support the suvival of neurones during development (reviewed by Jessen and Mirsky, 1999). In addition, Schwann cells are key determinants of the structure of myelinated fibres, in particular the establishment and maintenance of the nodes of Ranvier (reviewed by Arroyo and Scherer, 2000). CMT1 animal models have revealed that the alteration of Schwann cell physiology associated with demyelination and remyelination leads to both altered protein localization in Schwann cell membranes and loss of juxtaparanodal clustering of potassium channels in the axolemma (Neuberg et al., 1999). Furthermore, severe axonal atrophy was found in these mouse mutants (Sancho et al., 1999).
Our current understanding of the regulation of Schwann cell proliferation in CMT1A is less clear and appears somewhat confusing at this time (reviewed by Naef and Suter, 1998; Muller, 2000). Data obtained from in vitro studies suggest a direct role for PMP22 in regulating cell growth. Retroviral PMP22 gene transfer in cultured Schwann cells caused growth suppression, while reduced PMP22 expression had the opposite effect (Zoidl et al., 1995). Consistent with these findings, cultivated Schwann cells isolated from nerve biopsies of CMT1A patients carrying the duplication showed decreased proliferation (Hanemann et al., 1998). Overexpression of PMP22 in Schwann cells and NIH-3T3 cells using microinjection of expression constructs, however, induced cell death in both cell types (Fabbretti et al., 1995; Brancolini et al., 1999). If cell death was blocked by co-expression of Bcl-2, these experiments also revealed a potential function for PMP22 in controlling cell morphology, possibly through the modulation of Rho small GTPase (Brancolini et al., 1999). Overexpression of PMP22 point mutations, including the Tr mutation, in the same in vitro paradigm led to a strongly reduced apoptotic response and the proteins behaved in a dominant-negative manner when co-expressed with wild-type PMP22 (Fabbretti et al., 1995).
The molecular and cellular function of PMP22 is of cardinal importance for our understanding of CMT1A. In this report, we have examined the role of PMP22 in Schwann cell proliferation and cell death in early development and disease. To this end, we have analysed and compared peripheral nerves of homozygous PMP22 knock-out mice (Adlkofer et al., 1995), the CMT1A mouse model Tr (PMP22 missense point mutation; for a review, see Suter et al., 1993) and PMP22 transgenic mice that carry extra copies of the PMP22 gene (Magyar et al., 1996).
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Animals and genotype analysis
Homozygous PMP22 knock-out, heterozygous PMP22 transgenic and Tr mice were obtained from our own breeding colonies. Wild-type mice from the corresponding genetic backgrounds were used as controls (PMP22 knock-out, Agouti SV129EV/C57BL/6; PMP22 transgenic, B6C3; Tr, CBA). We have taken special care to analyse mice with comparable genetic backgrounds within each group with different PMP22 mutations at each time point. Comparisons between the three groups have to be viewed considering the fact that these strains do not have an identical genetic background. The genotypes were assessed by either Southern blot or polymerase chain reaction (PCR) analysis of mouse tail genomic DNA. Initially, PMP22 knock-out and PMP22 transgenic mice were genotyped by Southern blot as previously described (Adlkofer et al., 1995
; Magyar et al., 1996). PCR protocols were later designed to genotype PMP22 knock-out and PMP22 transgenic mouse progeny. Two PCRs were carried out to distinguish the wild-type from the PMP22 knock-out allele. Oligonucleotide primer 5'-CACGTCTTGCCTGCTCTGAC-3', located upstream of the homologous region of the targeting construct, was common to both PCRs. This common primer in combination with the neo gene internal oligonucleotide primer 5'-CGCAGCGCATCGCCTTCTATC-3' was used to identify the PMP22 knock-out allele. Conditions for hot-start PCRs were 4 min initial denaturation, followed by 35 cycles of 95°C for 30 s, 66°C for 1 min and 72°C for 2 min using a thermal cycler (Perkin-Elmer, GeneAmp PCR System 9600). Oligonucleotide primer 5'-GAGACGAAGAGCAACAC-3' within exon 1 of the PMP22 gene together with the common primer allowed the identification of the wild-type allele. Hot-start PCR conditions were as follows: 4 min initial denaturation, followed by 35 cycles of 95°C for 1 min, 52°C for 1 min and 72°C for 2 min. PMP22 transgenic progeny were identified using the pTCFupper oligonucleotide primer 5'-CCTCAACCTACTATGG-3' and the PMP22 internal oligonucleotide primer 5'-AGGGCAATAAACACACTG-3' (Magyar et al., 1996). Hot-start PCR conditions were as follows: 4 min initial denaturation, followed by 35 cycles of 94°C for 30 s, 60°C for 1 min and 72°C for 1.5 min. PCR analysis of Tr mice has been reported elsewhere (Adlkofer et al., 1997b). Experiments were performed in accordance with the legal requirements of the Eidgenössische Technische Hochschule and Kanton Zürich (Switzerland).
d-UTP-digoxigenin nick end-labelling (TUNEL) assay
PMP22 mutant mice (PMP22 knock-out, PMP22 transgenic and Tr mice) and wild-types of the corresponding genetic background at postnatal days 1, 4, 10 and 21, and 10 weeks of age were sacrificed with a lethal dose of sodium pentobarbital (Nembutal®, Abbott, Ill., USA) and their sciatic nerves were removed. Nerves were fixed in 4% paraformaldehyde in PBS (phosphate-buffered saline) for 4 h at 4°C, dehydrated and embedded in paraffin. Longitudinal sections, 5 µm thick, were mounted on slides, dewaxed and rehydrated. Initially, slides were incubated in methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase, followed by a 20 min digestion with proteinase K (20 µg/ml) at 37°C. Sections were pre-equilibrated in TdT buffer (30 mM Tris–HCl pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride) prior to the addition of the TUNEL reaction mix (0.2 U/µl TdT enzyme, 6 nM biotin-16-dUTP, 6 nM dATP in TdT buffer). The enzymatic reaction was carried out at 37°C for 1.5 h and was terminated by incubating in 2x SSC (standard saline citrate) for 15 min at room temperature. After incubation of the sections in ABComplex (avidin–biotin complex) tagged to horseradish peroxidase (HRP) (Dako, Denmark) for 1 h at 37°C, incorporation of the labelled deoxynucleotide was visualized by applying the diaminobenzidine colour substrate solution for 12 min at room temperature in the dark. Nuclei were counterstained with methyl green (Elias, 1969). Positive and negative controls were run in parallel. Positive controls for the TUNEL reaction were paraffin sections of a E13.5 mouse embryo and sections of mouse small intestine previously digested with DNase I. As negative control, a section of an E13.5 mouse embryo was incubated in TUNEL reaction mix without adding the TdT enzyme.
To quantify apoptosis, sciatic nerve longitudinal sections were visualized in a Zeiss Axiophot with the x100 lens. A grid with 100 divisions mounted in one of the microscope objectives helped to define the field of analysis. The nuclei counting proceeded from the proximal to the distal end of the sciatic nerve sections until 1000 or 500 nuclei, depending on the age of the animals, were counted in consecutive microscopic fields. All TUNEL-labelled cells from the total number of counted nuclei were recorded at the same time. The cell death index was calculated as the percentage of nuclei that were TUNEL positive. At least four animals per genotype and age group were analysed. Statistical analysis was performed with the Mann–Whitney U-test (two-side test paradigm) using Statview, version 4.0 software. P values 
0.05 were considered to be statistically significant.
BrdU incorporation assay
Mice of the same genotype and age as for the TUNEL assay were injected intraperitoneally with bromodeoxyuridine (BrdU; 100 µg/g of body weight) in 0.9% NaCl/7 mM NaOH. One hour after BrdU injection, animals were sacrificed and tissues of interest were removed. Tissue-Tek-embedded sciatic nerves were frozen in liquid nitrogen-cooled isopentene and sectioned with a cryostat. Six consecutive longitudinal sections, 5 µm thick, were mounted alternatively onto two Superfrost slides, air dried and fixed for 5 min with 2% paraformaldehyde in PBS. Because our DNA denaturation protocol precluded the simultaneous identification of BrdU-labelled cells and staining of nuclei in the same section, one slide was stained with haematoxylin to count nuclei whereas the second slide was processed to reveal BrdU-positive cells. After blocking endogenous peroxidase activity, DNA denaturation was achieved by incubating the sections in 2 M HCl for 15 min at 37°C followed by neutralization in 0.1 M sodium borate pH 8.5 for 10 min. Nuclei that incorporated BrdU were labelled with a biotinylated anti-BrdU monoclonal antibody (Caltag Laboratories, Calif., USA) diluted 1 : 20 and detected by the ABComplex–horseradish peroxidase method as described above. Sections of small intestine from the same animals were used as positive controls.
BrdU-labelled cells were counted in three alternated consecutive longitudinal sections. Starting from the proximal end of the sciatic nerve, 4–6 consecutive microscopic fields, as defined by the grid mounted in the microscope objective, were examined per section with a x100 lens. All BrdU-labelled cells present within the defined fields were counted. An identical quantification method was used to assess the number of haematoxylin-stained nuclei in the other three alternated sections. The proliferation index was calculated as the percentage of BrdU-labelled cells in relation to the total number of haematoxylin-stained nuclei present in the same area. Cell density was defined as the total number of haematoxylin-stained nuclei per mm2. Usually five animals per genotype and age group were analysed. Statistical analysis was performed with the Mann–Whitney U-test (two-side test paradigm) using Statview, version 4.0 software. P values 0.05 were considered to be statistically significant.
Immunohistochemistry
To demonstrate that the BrdU- and TUNEL-positive cells were Schwann cells, selected nerves were immunostained with a polyclonal rabbit antiserum against S100 protein (Dako, Denmark) combined with either BrdU or TUNEL labelling. For these experiments, sciatic nerve sections were embedded in Tissue-Tek and 5 µm thick cryosections obtained. Thereafter, identical protocols to those described above were used to process sections for either BrdU or TUNEL labelling, except that Texas red-tagged streptavidin (Jackson ImmunoResearch Laboratories, Pa., USA) was used as detection system. S100-labelled cells were identified with a fluorescein-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories).
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Results |
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Earlier analysis of PMP22 mouse mutants indicated that PMP22 plays a key role in the initial steps of myelination and in the maintenance of the myelin sheath (reviewed by Carenini et al., 1999
; D'Urso et al., 1999; Naef and Suter, 1998). Since demyelination induces proliferation of Schwann cells in various mouse mutants, regardless of the underlying molecular defect (Giese et al., 1992; Anzini et al., 1997; Neuberg et al., 1999), we decided to start our analysis at postnatal day (P) 1, prior to the onset of appreciable myelination. Since PMP22 is expressed by peripheral glia, motor neurones and sensory neurones before birth (Baechner et al., 1995; Parmantier et al., 1995, 1997; Hagedorn et al., 1999), this strategy should allow the detection of alterations in Schwann cell proliferation and cell death in early development without the interference of demyelination. We analysed three groups of PMP22 mutants to assess the influence of PMP22 gene dosage (PMP22 transgenic mice and PMP22 knock-out mice) and the effect of altered PMP22 protein (Tr mutant) on the proliferation and apoptosis of Schwann cells in comparison with wild-type controls. To follow the development and to evaluate the probable onset of alteration of proliferation and/or apoptosis, we examined sciatic nerves of P1, P4, P10 and P21 animals. In addition, 10-week-old young adult mice with completed peripheral myelination were also examined.
Schwann cell proliferation
S100 was used as a Schwann cell marker in combination with BrdU incorporation (for example, see Fig. 1A–C). BrdU-positive cells without S100 immunoreactivity were not observed (data not shown).
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First, we compared the cell density in sciatic nerves of wild-type and mutants at P1. No significant differences between mutant mice and their corresponding control animals were observed within the three paired groups that were analysed (Fig. 2A
, C and E). In all three wild-type control groups, cell density in the sciatic nerve decreased from P1 until the age of 10 weeks, to approximately one-third of the initial value (Fig. 2A, C and E). In the mutant animals, we observed a significant increase in cell density in Tr animals from P21 onwards (Fig. 2A) and in the transgenic PMP22 animals as early as P4 (Fig. 2E).
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Next, we determined the proliferation rate by calculating the number of cells with BrdU incorporation in relation to the total cell number (BrdU index). In wild-type mice, this index ranged from 15.8 ± 2.5% to 18.9 ± 1.5% at P1 and was reduced to undetectable levels in 10-week-old animals. In the Tr mutants, we found a strong increase of Schwann cell proliferation at P10 and in 10-week-old animals, while proliferation of Schwann cells at P21 did not reach significance in comparison with the age-matched wild-type controls with comparable genetic background (Fig. 2B
). These findings are consistent with pathology data indicating that significant myelination occurs initially in these mice (Ayers and Anderson, 1976) followed by demyelination and, as shown here and suggested earlier (Perkins et al., 1981), accompanied by Schwann cell proliferation leading to cellular onion bulb structures. In PMP22 knock-out animals, a significant increase in the rate of proliferation was found only in 10-week-old animals (Fig. 2D). This result is consistent with the described pathology in these mice which are affected by focal hypermyelination (tomacula formation) early in life, followed by demyelination including onion bulb cell formation later (Adlkofer et al., 1995; Sancho et al., 1999). PMP22 transgenic mice showed a constant, significantly elevated proliferation index from P4 onwards in relation to the wild-type control group, consistent with the observed almost complete amyelination of the peripheral nerves of these animals throughout development (Fig. 2F; Magyar et al., 1996; Sancho et al., 1999). In contrast to wild-type animals, proliferating cells were detectable in the sciatic nerves of all three mutants at 10 weeks of age, but the rate was reduced to 1–2%.
Cell death of Schwann cells
Cell death was analysed by TUNEL staining in combination with S100 immunohistochemistry. Examples of dying Schwann cells in the sciatic nerves of various PMP22 mutant mice, also characterized by fragmented nuclei, are shown in Fig. 3. Furthermore, a representative overview of apoptosis in a sciatic nerve of a Tr mutant at 10 weeks of age and the corresponding wild-type animal is shown (Fig. 4). In wild-type mice, considerable programmed cell death was observed from P1 onwards, peaking between P4 and P10, with very low levels at P21, and no detectable dying cells at the age of 10 weeks (Fig. 5). These findings are consistent with earlier suggestions that axon–Schwann cell interactions regulate the correct Schwann cell number during early postnatal development through apoptosis (Grinspan et al., 1996; Syroid et al., 1999).
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The rate of apoptosis was similar in the three mutant groups and in the corresponding wild-type animals at P1 (Fig. 5A
–C). In Tr animals, significantly increased cell death was observed starting at P21 and continuing at 10 weeks of age (Figs 4A and 5A). The results in PMP22 knock-out animals were qualitatively similar to Tr (Fig. 5B). In PMP22 transgenic mice, the rate of apoptosis was already doubled compared with wild-type at P4, and this strongly increased apoptosis continued up to adulthood (Fig. 5C). Earlier studies have indicated that the low affinity neurotrophin receptor p75 is involved in the induction of apoptosis in transected nerves (Syroid et al., 2000). Interestingly, p75 is also upregulated in peripheral nerves of PMP22 transgenic mice (Magyar et al., 1996). However, a potential causative function for p75 in the increased Schwann cell apoptosis of these animals remains to be established. In summary, all three groups of PMP22 mutant animals showed a significant increase of apoptotic Schwann cells at P21. At the age of 10 weeks, none of the corresponding wild-type groups showed any apoptotic Schwann cells, while apoptosis was still prominent in the mutant mice.
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Discussion |
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In this study, we have examined Schwann cell proliferation and apoptosis in three different mouse strains with altered expression of the PMP22 gene. The spontaneous mouse mutant Tr carries a point mutation in the PMP22 gene which leads to intracellular retention of the mutated protein in vitro and in vivo (Naef et al., 1997
; Colby et al., 2000). Since PMP22 appears to form dimers, it is conceivable that wild-type PMP22 is also retained, at least partially, within the cell, suggesting a dominant–negative disease mechanism (Naef et al., 1997; Naef and Suter, 1999; Tobler et al., 1999; Brancolini et al., 2000). However, a contribution by a gain-of-function effect, possibly due to overloading of intracellular compartments, has also been demonstrated by genetic analysis (Adlkofer et al., 1997b). Similar to observations in animal models of Pelizaeus–Merzbacher disease where disrupted proteolipid protein trafficking appears to result in oligodendrocyte apoptosis (Gow et al., 1998), we show that a substantial increase in Schwann cell apoptosis is observed in Tr at P21 and at the age of 10 weeks. In support of a causative action of the Tr protein in the induction of apoptosis, we have also observed strongly increased programmed cell death in adult homozygous Tr mice compared with the heterozygous animals used in our study (preliminary observation). In addition, increased levels of apoptosis have also been described in the Tr-Ncnp mouse which carries an in-frame deletion in the PMP22 gene (Suh et al., 1997). The most straightforward interpretation of these data is that excessive intracellular accumulation of mutated PMP22 cannot be tolerated by Schwann cells in vivo and, consequently, the cells die. A similar mechanism may also explain the increased apoptosis in PMP22 transgenic mice, consistent with the abundant empty basal lamina present in the nerves of these animals (Magyar et al., 1996). The substantially earlier onset of apoptosis in PMP22 transgenic mice compared with Tr could be due to the high PMP22 gene dosage in these animals which may lead to fast intracellular accumulation of large amounts of misfolded PMP22 and immediate activation of the apoptosis pathway (Pareek et al., 1993). Alternative interpretations, however, are also possible. In the process of this study, we have confirmed that Schwann cell apoptosis occurs naturally in developing nerves of wild-type mice during the first two postnatal weeks (Grinspan et al., 1996; Syroid et al., 2000). It has been suggested that axon–Schwann cell interactions in this way regulate the correct Schwann cell numbers by competition for axonally derived neuregulins (Grinspan et al., 1996; Syroid et al., 1996). Sciatic nerves of PMP22 transgenic animals contain virtually no myelin and generate an excess of Schwann cells starting already at P4, presumably since they are exposed continuously to mitogenic axolemma proteins. Thus, the early phase of increased Schwann cell apoptosis in PMP22 transgenic mice from P4 to P10, which is not present in Tr, may also involve processes regulating correct Schwann cell numbers in early development, including the availability of and spatial exposure to limited amounts of survival factors (reviewed by Garratt et al., 2000). Importantly, there is a window of susceptibility of Schwann cells to apoptosis during development since programmed cell death could be enhanced by axotomy only during early development (Grinspan et al., 1996). Thus, the Schwann cell death observed from P21 onwards in PMP22 mutants is likely to be due to other mechanisms.
Toxification through intracellular accumulation may contribute to the late phase of apoptosis in Tr and PMP22 transgenic mice, but the increased levels of Schwann cell apoptosis seen in PMP22 knock-out mice require other explanations. Based on the fact that this increase is correlated with morphological signs of abundant deymelination, remyelination and onion bulb formation (Adlkofer et al., 1995; Sancho et al., 1999), it is likely that dying supernumerary Schwann cells are responsible for our findings. The same mechanisms may also contribute to the apoptosis observed in Tr late in development since onion bulb formation associated with increased cell density is found within peripheral nerves (Ayers and Anderson, 1976; Perkins et al., 1981).
Experiments in cell culture have suggested that PMP22 is directly involved in the regulation of the cell cycle (Zoidl et al., 1995, 1997) and the induction of cell death (Fabbretti et al., 1995; Brancolini et al., 1999). To address this issue, we have analysed the PMP22 mutants at P1. We reasoned that the function of PMP22 in myelination and myelin maintenance is not a substantially interfering factor at this time point since the sciatic nerve is still largely unmyelinated. Our analysis revealed no significant alterations in Schwann cell density, proliferation index or the rate of apoptosis in any PMP22 mutant compared with wild-type mice at this early time point. Thus, normal PMP22 expression is dispensable for correct Schwann cell proliferation and cell death up to P1. An influence of PMP22 in these processes later in development, however, cannot be excluded. This is difficult to assess due to the underlying myelin breakdown (demyelination) or amyelination pathologies which alter axon–Schwann cell interactions and, consequently, proliferation and apoptosis in ways that are not fully understood.
How do our data compare with the human disease? In nerve biopsies of young CMT1A patients carrying the PMP22 duplication on chromosome 17p.12, Schwann cells show no signs of increased proliferation (Hanemann et al., 1997). Consistent with our observations in PMP22 transgenic mice, the authors concluded that there is no evidence for altered initial Schwann cell proliferation prior to the process of de- and remyelination. Intriguingly, however, human Schwann cells obtained from CMT1A patients carrying the chromosomal duplication show a decrease in proliferation in vitro (Hanemann et al., 1998). This difference between in vitro and in vivo studies may reflect age-related changes and/or the course of disease. Alternatively, culturing CMT1A Schwann cells may unmask a difference in their potential for decreased proliferation that cannot be observed in vivo, a common emerging theme in developmental biology (Anderson, 2001).
Evidence for apoptosis of Schwann cells in CMT1A and HNPP nerve biopsies from patients with proven PMP22 duplications and deletions has been provided by Erdem and colleagues using the TUNEL method (Erdem et al., 1998). Quantitative studies showed a significantly reduced number of total Schwann cells in CMT1A compared with controls indicating a loss of Schwann cells. In HNPP, the number of total Schwann cells was increased and significant Schwann cell apoptosis was observed. The authors suggested on the basis of further morphological analysis that this Schwann cell apoptosis might be related to the regenerative state of the nerve resulting from the process of sprout pruning (Erdem et al., 1998).
In summary, we show that altered expression of PMP22 does not affect the generation of the correct number of early Schwann cells. Furthermore, the proliferation index is not changed in PMP22 mutants in early postnatal development. However, increased proliferation and apoptosis are prominent features in all mutants at later developmental stages. Based on the differences in the kinetics of these alterations during the development of the different mutants, it is likely that overloading of intracellular compartments and altered access to trophic factors due to myelination deficiencies are responsible for the increased cell density, proliferation and apoptosis observed in PMP22 mutant peripheral nerve.
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* These two authors contributed equally to this paper
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We wish to thank Annette Kolar for excellent technical assistance. This work was supported by the Swiss National Science Foundation, the Swiss National Research Program for Neurodegenerative Disorders (NFP38), the Swiss Muscle Disease Foundation, the Wolfermann–Nägeli Stiftung and the Swiss Bundesamt for Science related to the Commission of the European Communities, specific RTD programme `Quality of life and Management of Living Resources', QLK6-CT-2000-00179, `The role of neurosteroids in healthy aging: therapeutical perspectives'. It does not necessarily reflect its views and in no way anticipates the Commission's future policy in this area. P.Y. is a recipient of a grant from the Deutsche Forschungsgemeinschaft (YO48/1-1) and is on sabbatical from the Department of Neurology, University of Münster, Germany.
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Received March 2, 2001. Revised April 10, 2001. Accepted July 2, 2001.
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