We have generated a mouse knock-in model of the human inherited neuropathy CMT1B that is caused by an R98C mutation in MPZ
. This mutation causes a severe hypomyelinating neuropathy in which the onset of ambulation is delayed and in which impairment is severe prior to adulthood (Gabreels-Festen et al., 1996
; Shy et al., 2004
; Bai et al., 2006
). The neuropathy is also severe in mice. R98C/+ mice are weak at an early age, have slow nerve conduction velocities and have reduced myelin in their PNS. R98C/R98C mice are extremely disabled in infancy, have minimal PNS myelin and have nerve conduction velocities of ~5
m/s. It is interesting to compare nerve morphology from Mpz
R98C mice and MPZ
R98C patients. All reports of R98C sural nerve biopsies identified reduced numbers of myelinated axons and regions of segmental demyelination (Gabreels-Festen et al., 1996
; Kirschner et al., 1996
; Komiyama et al., 1997
; Hattori et al., 2003
; Bai et al., 2006
), both of which we also observed in R98C/+ mice. However, there were differences between the mice and patients. We did not observe onion bulbs in the mice, whereas these were frequent in the patient we had observed (Bai et al., 2006
). Similarly, we did not observe areas of non-compact myelin in R98C/+ mice whereas Gabreels-Festen et al. (1996)
and Kirschner et al. (1996)
detected uncompacted myelin in 23–68% of myelinated fibres, beginning at the major dense line in a sural nerve biopsy from a 14-month old. However, we did not detect non-compact myelin in either of the two biopsies performed on our adult patient with the R98C mutation (Bai et al., 2006
). It may be that some morphological features are variable with patients having the same mutations, that certain findings differ depending on the specific nerve analysed or that there are differences between mice and humans related to onion bulb formation and thresholds for myelin decompaction.
Mutant MpzR98C is retained within the endoplasmic reticulum in mouse Schwann cells. In heterozygous animals there is Mpz in myelin as well as in the endoplasmic reticulum. We presume that the Mpz in myelin is predominantly from the wild-type allele because minimal Mpz and myelin are detectable around ensheathed axons from R98C/R98C mice. Taken together, these results suggest that MPZR98C is unable to be incorporated into compact myelin and that at least some normal MPZ remains able to be incorporated into myelin in the presence of one allele expressing MPZR98C.
Despite the paucity of Mpz in myelin, our data demonstrate that the R98C/+ neuropathy is not caused simply by a lack of Mpz. Heterozygous Mpz
-null mice, in which there is haplo-insufficiency of Mpz, have no clinical phenotype or abnormalities of nerve conduction velocities until they are 4–6 months of age (Martini et al., 1995
; Shy et al., 1997
). Morphologically, Mpz+/−
peripheral nerves also appear normal until several months of age when they develop asymmetric demyelination suggestive of chronic inflammatory demyelinating polyneuropathy (Shy et al., 1997
) or immune-mediated neuropathies (Martini, 1999
). Moreover, the coordinate programme of myelin gene expression during development is similar in wild-type and Mpz+/−
mice (Shy et al., 1997
; Menichella et al., 2001
). R98C/+ mice differ in all of these aspects. They are abnormal clinically, have slow nerve conduction velocities, abnormal myelin and have abnormal myelin gene expression—all before 6-weeks of age.
Although homozygous Mpz
null mice (Mpz−/−
) also have severe neuropathy in infancy with nerve conduction velocities of ~5
m/s, there are also significant differences between these animals and R98C/R98C knock-in mice. Mpz−/−
mice have axons ensheathed by multiple layers of loosely compacted myelin (Giese et al., 1992
) with a disorganized programme of myelin gene expression such that some genes like Mag
are upregulated with others like Pmp22
downregulated (Giese et al., 1992
; Xu et al., 2000
). In contrast, R98C/R98C nerves have scarcely any wraps ensheathing axons and their programme of myelin gene expression is coordinately decreased compared to normal mice of the same age. Differences between Mpz
knock out and R98C knock-in nerves were also detected by X-ray diffraction. Compared with the R98C/+, we found that sciatic nerve segments from the Mpz+/−
nerves showed a greater relative amount of myelin and a wider period than R98C/+ nerves. Finally, R98Cneo/R98Cneo nerves (in which loxP-neoR-loxP has not been excised) express half of Mpz
messenger RNA compared to R98C/R98C nerves, which would predict worse hypomyelination if MR98C acted through loss of function. Instead, R98Cneo/R98Cneo nerves manifest much milder hypomyelination, suggesting that MR98C provokes a dose-dependent toxic gain of function (C.F. and L.W., unpublished). Taken together, all of these data demonstrate that the neuropathy in R98C mutant mice occurs by abnormalities caused by the mutant Mpz
rather than by the loss of Mpz
itself. These differences also suggest important issues for therapy with CMT1B mutations that are similar to R98C. Since mutant R98C causes toxicity without being transported to myelin, therapeutic approaches might best be focused on detoxifying the mutant protein rather than on increasing levels of wild type Mpz in myelin.
The abnormal ‘toxic’ gain of function caused by MR98C either arrests Schwann cell development in a promyelinating stage or slows their differentiation beyond this stage in a dose-dependent fashion. Myelinating Schwann cells in mice normally establish a 1:1 relationship with axons at the time of birth (Trapp et al., 1988
). R98C/R98C Schwann cells have established this 1:1 relationship at birth, but never synthesize more than rudimentary myelin even by 6-months of age. Thus, electron micrographs at post-natal Day 2 resemble those at 6-months of age in the homozygous mice. R98C/+ Schwann cells do myelinate as the animals age but the myelin never reaches its normal thickness, particularly for large calibre axons and myelin gene expression always remains reduced compared with wild-type levels.
We do not completely understand how the MpzR98C arrests or slows myelination but it most likely involves regulation of the transcription factors c-Jun and Krox-20 as the proportion of cells expressing these is abnormally elevated and reduced, respectively, in R98C mice. c-Jun expression actively inhibits myelination when present in the nucleus of premyelinating Schwann cells (Parkinson et al., 2008
). Additionally, Schwann cells with sustained heterologous expression of c-Jun are unable to myelinate in dorsal root ganglia co-cultures and in such cells Krox-20 expression is suppressed (Parkinson et al., 2008
). Conversely, Krox-20 has been shown to inhibit c-Jun expression in Schwann cells. In cultured Schwann cells, enforced expression of Krox-20 is sufficient to decrease c-Jun expression (Parkinson et al., 2008
). In R98C mice, c-Jun expression remains high in R98C/R98C Schwann cells, even at 6-months of age, suggesting that many Schwann cells are actively prevented from entering a myelinating state by their expression of c-Jun. c-Jun is also detected in R98C/+ Schwann cell nuclei but not in wild-type nuclei.
Schwann cells ensheathing axons in R98C mice can be separated into two groups based on c-Jun and MBP expression: c-Jun+
. Likewise, we can identify two groups of Schwann cells based on Krox-20 and MBP expression: Krox-20−
. We presume that those cells that express MBP (c-Jun−
) have committed to myelination, while those that don’t express MBP (c-Jun+
) are in a premyelinating state. A recent in vitro
study showed that Schwann cells can undergo multiple transitions between de-differentiated (premyelinating) and differentiated (myelinating) states without cell cycle re-entry. De-differentiation to a premyelinating state required JNK activity and the upregulation of c-Jun expression (Monje et al., 2010
). In R98C mice we occasionally observe c-Jun+
Schwann cells ensheathing an axon. This raises the intriguing possibility that these represent a transition between myelinating and premyelinating Schwann cells. We hypothesize that UPR activation in myelinating cells could drive this transition.
We have strong evidence for a canonical response of the UPR in nerves of R98C mice. One possibility is that the IRE1 arm of the UPR alters the regulation of c-Jun and Krox20. Endoplasmic reticulum stress is known to induce binding of TRAF2 to the cytoplasmic domain of IRE1 and in combination with signalling mediated by ASK1 to activate JNK (Nishitoh et al., 1998
). JNK expression is increased in R98C Schwann cells. Activated JNK phosphorylates c-Jun and can stimulate and maintain its expression (Jessen and Mirsky, 2008
). Induction of the UPR could thus promote the increased expression of c-Jun in R98C Schwann cells and drive their de-differentiation. Additional crossing of R98C or R98C-Chop null mice with Ire1-null animals could test the hypothesis. We would predict that less activation of the UPR would enable increased myelination to occur in these mice.
Activation of the UPR has also been detected in S63del mice. In S63del animals, ablation of Chop
from the Perk arm of the UPR ameliorated the clinical phenotype, decreased F-wave latencies and reduced the numbers of demyelinated fibres in sciatic nerves (Pennuto et al., 2008
). Conversely, ablation of Chop
did not significantly alter the phenotype of the R98C mice. In patients, the R98C MPZ
mutation causes a severe phenotype in which they do not walk independently until ~3 years of age and typically cannot walk independently in adulthood (Shy et al., 2004
; Bai et al., 2006
). In contrast, the S63del phenotype is milder with most patients walking by 1 year of age and ambulating independently as adults (Miller et al., in press
). Myelination is developmentally delayed in R98C patients (congenital hypomyelination) where it is able to occur relatively normally in S63del patients. In S63del, demyelination at later stages appears to be the predominant mechanism causing neuropathy. Thus, endoplasmic reticulum stress and UPR activation are not limited to mutations that cause severe, early onset phenotypes. Rather, our results suggest that patients with CMT1B with various levels of clinical impairment may have UPR activation; however, the mechanisms through which the UPR affects myelination are likely to be distinct. Identifying which CMT mutations activate the UPR and the mechanisms by which it alters myelination will be likely to have therapeutic importance as treatments emerge that ameliorate endoplasmic reticulum stress and UPR activation.