Expressional alterations in myelin-specific transcripts in human
Charcot-Marie-Tooth disease (CMT) is the most common inherited neurologic disorder. Reportedly 10-28 cases exist per 100,000 in Western societies. CMT patients suffer from a variable degree of motor dysfunction which adversely affects locomotion and balance. These deficits explain by a slowing of impulse conduction in motor and sensory nerves. This affects the recruitment of muscle fibres for contraction whereby slow motor units are preferentially affected (Gale et al., 1982; Roy et al., 1996). This results in muscle weakness and wasting in the extremities of the body.
The inefficient propagation of excitatory signals in moto neurons towards peripheral muscle in CMT disease is caused by a myelination defect which has a genetic origin. Some 20 genes for axon proteins and myelin have been implicated in CMT, and allow the classification into specific subtypes of the disease. Frequently affected of these genes are peripheral myelin protein 22 (pmp22) and myelin protein zero (MPZ) which are mutated in a majority of cases (Braathen et al., 2011). CMT subtype 1A (CMT1A) is the most predominant disease type being associated with a duplication of the pmp22 gene (Wise et al., 1993; Magyar et al. 1996). The pmp22 gene encodes for a factor that is incorporated into the myelin sheet of neurons and is thought to control myelin thickness. The pathology on the CMT1A disease types is explained by slowed saltatory transduction in moto neurons due to the aberrant nerve insulation (Sereda & Griffiths, 1996).
Currently there is no cure for CMT. There are however a number of occupational interventions that can be used to effectively manage its symptoms (
Gene transfer therapy is a promising route for future cures of muscular disorders. However major limitations appear regarding the treatment of neurological diseases such as CMT. In this case one would normally expect that the therapy must directly target moto neurons to initiate a compensatory mechanism that can correct the missing myelination. This is currently not feasible. In this regard the important positive influence of exercise on motor performance in CMT patients demands consideration as this may offers an indirect means to improve neuromuscular function. This relates to the well established feed forward control of the muscle phenotype by muscle use (Fluck & Hoppeler, 2003). It has been pointed out that the effects of increased muscle recruitment with exercise involve the promotion of the slow-oxidative expression program (Schmutz et al., 2010; Baumann et al., 1987). Recently, has been established that electrically imposed muscle recruitment can re-establish muscle functionality in denervated muscle of tetraplegic patients through the remodelling of the contractile and metabolic makeup (Boncompagni et al., 2007). The positive influence of exercise in CMT patients suggests that the use-dependent pathway importantly affects neuro-muscular function.
We reasoned that altered muscle use combined with gene therapy for focal adhesion kinase (FAK), an enzyme that regulates recruitment dependent slow oxidative gene expression, would be a suitable venue to promote pmp22 expression in skeletal muscle. Towards this end we investigated the use dependence of pmp22 expression in human skeletal muscle and assessed the nature of contractile defects in muscles of a mouse model for CMT at the molecular, cellular and functional level. Subsequently we tested whether overexpression of FAK can enhance pmp22 expression in skeletal muscle (Durieux et al., 2009).
Hindlimb suspension of mice was performed for 3 days as previously described at the University of Berne (Switzerland) (Dapp et al., 2004). This was achieved by attaching the tail via a swivel hook to a movable X-Y system; thereby preventing the mouse from touching the ground with its hindlimbs while permitting free movement within the entire cage. Soleus muscles were extracted from anesthetized animals (5% isoflurane).
The experiments were performed at the Universities of Berne (Switzerland) and Lyon (France) with the permission of the local Animal Care Committee of the Canton of Berne (Switzerland) and following the recommendations provided by the European Convention for the protection of Vertebrate Animals used for Experimental and Scientific purposes (Strasbourg, 18.III.1986).
Subsequently RNA was labelled during reverse-transcription and hybridized to different platforms dependent on the species:
For human samples this involved the use of a full human genome Affimetrix platform through a commercial provider (DNA vision, Gosselies, Belgium). For statistical analysis raw signals were normalized to total mRNA signal on the array and assessed with statistical analysis of microarrays between pre and 8-hours post samples with T-tests (SAM).
For studies with rat and mouse soleus muscle, 3 micrograms of total RNA were reverse transcribed with SuperScript II (Life Technologies, Basel, Switzerland) using [α-32P]dATP and gene-specific primers. Radio-labelled target cDNA was hybridised in ExpressHyb solution overnight at 68°C to nylon membranes which spotted cDNA probes (BD Biosciences, Allschwil, Switzerland). The membranes were washed (four times for 1 h in 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate), 1% SDS and once for 30 min in 0.1x SSC, 0.5% SDS at 68°C and exposed for 5 days to a phosphoimager (Molecular Dynamics, Sunnyvale, CA) for quantification with AIDA Array Easy software (Raytest Schweiz, Urdorf, Switzerland).
For the rat experiment cDNA microarrays (Atlas Rat 1.2, no. 7854; Clontech Laboratories, Ozyme, France) were used. More details to this cDNA array can be found on Gene Expression Omnibus (GEO,
For the mouse experiment custom-designed low-density Atlas cDNA expression arrays with 229 double-spotted probes of mouse cDNAs associated with skeletal muscle form and function were employed. More details to this cDNA array can be found on GEO under GPL1097. For statistical analysis raw signals were normalized to total mRNA signal on the array and assessed with SAM.
Micrographs were taken from corresponding fields and the fibre types were classified. Slow and fast type fibres were differentiated on the basis of the presence of immunoreactivity for either type I or type II myosin heavy chain isoforms. Subsequently the percentages of each fiber population were counted.
The overexpression of FAK in muscle fibres was detected out with a Leica TCS SP5 confocal microscope on a DMI6000 stage powered by Argon laser and He–Ne lasers (Leica Microsystem CMS, Mylton Keynes, UK). In brief, 12 micometer cryosections were reacted with a 1 : 100 dilution of rabbit FAK antibody A-17 (Santa Cruz) and MHC2 antibody in 0.3% BSA in phosphate-buffered saline (PBS) and reacted with fluorescent-labelled secondary antibodies (Alexa488-conjugated anti-rabbit IgG, and Alexa555-conjugated anti-mouse IgG, Molecular Probes/Invitrogen). Sections were embedded in fluorescence-compatible mounting medium (DAKO, Denmark). Signal for FAK and the co-detected myosin protein was inspected with fluorescence after excitation at 458 nm, 476 nm and 488 nm with sampling in channels between 510–533 nm (Alexa 488) and 593–614 nm (Alexa 555).
2.1.1. Regulation of myelin-specific transcripts by muscle activity
Muscle fibres of anti-gravitational muscles are active for a considerable duration during free movement and quiet standing (Hennig & Lomo, 1985). We wished to investigate transcript regulation of factors of the myelin sheet by muscle activity. Towards this end we carried out an exploratory microarray analysis with the slow oxidative soleus muscle of rats that was subjected to changes in muscle activity by hindlimb suspension. The model allows reducing load bearing activity to slow oxidative soleus muscle by non-invasive means with the suspension of hindlimbs with the option to increase muscle activity with subsequent reloading (Morey Holten model as referred by Dapp et al., 2004).
Transcript profiling of rat soleus muscle showed lowered expression of the myelin-specific transcripts, peripheral myelin protein 22 (pmp22), myelin protein zero (MPZ), myelin basic protein (mbp) as well as myelin proteolipid protein and myelin-associated glycoprotein with reduced muscle activity (Fig. 1). With reloading the abundance of all of these transcripts were sizably increased.
We assessed the response of myelin-specific factors in human knee extensor muscle to exhaustive exercise. It has been pointed out before that this exercise stress induces a compensatory expression response during the first 24 hours of recovery from exercise (Pilegaard et al., 2000; Fluck, 2006). The analysis in the mixed
|myelin basic protein||MBP||1.7||0.3||2%|
|myelin expression factor 2||MYEF2||1.4||0.2||3%|
|peripheral myelin protein 22||PMP22||1.8||0.4||5%|
|myelin associated glycoprotein||MAG||1.1||0.1||5%|
|myelin protein zero||MPZ||1.2||0.1||21%|
|myelin oligodendrocyte glycoprotein||MOG||1.2||0.1||25%|
2.2. Contractile defects in a mouse model of pmp22-dependent CMT
Pmp22-tg mice show reduced pmp22 protein expression, lack myelination, and demonstrate neurogenic muscle atrophy (Magyar et al., 1996).
We compared the muscles of pmp22-tg mice vs. strain matched controls to identify contractile defects with pmp22-dependent moto neuron aberration. Soleus muscle of pmp22-tg mice demonstrated a shift towards an atrophic, slow phenotype. This was indicated by the reduced muscle weight (Fig. 2D) and elevated percentage of slow type muscle fibres (Fig. 2A-C). This became manifest in prolonged time-to-peak and half time of muscle relaxation (Fig. 2D). The aberrations resulted in a reduction in RNA messengers for the slow oxidative expression program concomitantly with the elevated abundance of gene transcripts for fast type myogenesis (Fig. 3). The only exceptions were GAPDH and CA3 being involved in pH regulation of slow type muscle fibres.
2.3. Aberrant muscle plasticity in CMT mice with altered muscle use
Towards understanding the functional implication of motor unit recruitment for control of muscle gene expression we subjected pmp22-tg mice to 3 days of hindlimb suspension. Microarray experiments identified important alterations in transcript expression in
skeletal muscle. Thereby transcript levels of factors of myogenesis, fast contraction, glycolysis and oxidative metabolism altered in opposite ways between wildtype and pmp22-tg muscle.
2.4. Muscle-targeted overexpression of the mechano-sensor FAK elevates myelin factor expression
We assessed whether overexpression of the governor of slow-oxidative gene expression, focal adhesion kinase (FAK), can control myelin factor expression and pmp22-dependent gene transcripts. Using gene electro transfer we introduced a constitutively active expression plasmid for FAK, pCMV-FAK, in soleus muscle of the right leg of Wistar rats. Transfection of the contralateral muscle with empty pCMV plasmid served as intra-animal control. Immunofluorescence experiments showed that overexpression of FAK was confined to muscle fibres but was not observed in contralateral controls (Fig. 5). Microarray analysis demonstrated that FAK overexpression increased transcript levels of pmp22 along with the one of MBP in soleus muscle of active rats. Concurrently the expression of a battery of slow oxidative genes was increased (Durieux et al., 2009).
Abbreviations: Glut 4, facilitative glucose transporter member 4; IGF-BP5, Insulin-like growth factor binding protein 5. For others consult legend to figure 3.
2.5. FAK dependent elevation of pmp22 expression is use dependent
FAK signals mechanical stimuli in muscle to gene expression and protein synthesis (Durieux et al. 2009; Klossner et al., 2009). We subjected the FAK-transfected soleus muscles to 7 days of hindlimb suspension and subsequent one day of reloading to assess the interaction of muscle activity with gene therapy. Pmp22 transcript expression was little affected by reduced load bearing. With subsequent reloading however pmp22 levels were reduced. This effect was transient and was gone 5 days after reloading
Skeletal muscle is the largest tissue of vertebrate species and most muscle groups rely on muscle activity to maintain their phenotype (Booth & Thomason, 1991). This is most pronounced in slow oxidative muscle types which are typically involved in control of gait and posture (Roy et al., 1996). This resembles the preferential affection of oxidative muscles
in the trembler model of CMT disease (Gale et al., 1982). This use-dependent control of gene expression and protein synthesis (Fluck & Hoppeler, 2003; Wilkinson et al., 2008) is not taken into consideration in current gene therapy of neurological muscle disorders.
Here we prove the concept that muscle fibre targeted somatic transgenesis can affect nerve-related gene expression. The results point out that muscle activity importantly modifies the pre-translational effects of the somatic overexpression of the governor of the slow oxidative muscle program, FAK, on the expression of myelin-specific transcripts. This was shown by a similar regulation of pmp22, MPZ and MBP transcripts by muscle disuse with reduced load-bearing (down-regulation) in rats, increased load-bearing muscle activity in rats and with endurance exercise (up-regulation) in man (table 1, Fig. 1). The direction of the transcript levels points out that that myelin-specific transcript expression is related to fibre recruitment. Such a mutual control of the muscle-nerve interaction is known from the action of neurotrophic factors during normal development of the slow muscle fiber phenotype (Carrasco & English, 2003). These findings support the novel concept that in addition to feed forward control of muscle gene expression by muscle use (Calvo et al., 2001) a feedback mechanism exists whereby muscle activity in return regulates moto neurons (Fig. 6).
The functional consequences of use dependent expressional alterations in myelin sheet-specific factors in skeletal muscle remain to be explored. Pmp22 is a major component of myelin expressed in the compact portion of essentially all myelinated fibres in the peripheral nervous system and is produced predominantly by Schwann cells. Pmp22 is expressed in cranial nerves but not in the mature central nervous system. Studies in injured nerve suggested a role during Schwann cell growth and differentiation and maturation of the neuromuscular junction (Spreyer et al., 1991; Patel et al., 1992; Magyar et al., 1996). Interestingly the identified transcript level changes in our study are in line with the degeneration of NMJ with disuse (Deschenes et al., 2001) and neurological adaptation which improves fibre recruitment early with exercise (Roy et al., 1996). This supports a hypothesis whereby elevated myelin-specific transcript expression may allow the preservation and improvement of neuromuscular control of muscle fibres.
Previous examinations of soleus and EDL muscle in the trembler mouse model of CMT revealed that the deep/oxidative and soleus muscles are particularly affected by CMT but fibre type differences were not described (Gale et al., 1982). Our measures in the pmp22-tg model of CMT emphasize that in addition to altered muscle contractility, slow fibre size and transcript expression in the anti-gravitational muscle, m. soleus, is anomalous. The reduction in RNA messengers for the slow oxidative expression program (Fig. 3) is possibly explained by volumetric alterations in fibre size as suggested by visually larger CSA ratio between fast and slow type fibres reflecting atrophy of slow type fibres. Smaller type I fibres in animal models for pmp22-dependent motor and sensory neuropathy type has been noted before (Schuierer et al., 2005). This would increase the contribution of transcripts in fast type muscle fibres. The concomitantly elevated abundance of transcripts for fast type myogenesis between pmp22-tg and wildtype mice likely reflects altered regulation in fast muscle fibres (Deschenes et al., 2001).
A main finding of our investigation was that differences between pmp22 and wildtype mice were preserved in an ‘opposite’ transcript response of soleus muscle to reduced load-bearing. Altered transcript expression is a potentially important indicator of pathological changes with reduced muscle activity (Bey et al. 2003; Chen et al., 2007). In this regard it is important that the response of soleus muscle from wildtype mice to 3 days of unloading reproduced the changes reported in a different mouse strain after 7 days of reduced load-bearing (Dapp, Schmutz et al. 2004). This overlap concerned contractile (MHCIIB) and metabolic factors (COXVb, COXVIa2, LDH2). Exceptions concerned those involved in myogenesis which were not altered after 3 days in this study. Strikingly, the transcript response of soleus muscle of pmp22-tg mice to suspension “mirrored” the differences seen between the pmp22-tg mice and wildtype mice at baseline. These observations imply the existence of use-dependent mechanism that “inverts” expression responses in model of CMT.
This observation relates to the effects of current occupational therapy by exercise. We have shown before that FAK overexpression controls contractile performance of soleus muscle in a activity-dependent manner via the regulation of transcript expression (Durieux et al., 2009). Intriguingly with FAK overexpression and the resumption of loading bearing muscle activity, the transcript level of myelination factors were reduced compared to controls (Fig. 5). Our previous findings in the suspension model show that reduced myogenic factor RNA expression after 24 hours of the reloading stimulus is related to a concomitant increase is the encoded protein (Fluck et al., 2008). This relates to the observation that FAK acts as a molecular switch for transition between anabolic and catabolic reactions in skeletal muscle and controls the protein synthetic pathway via p70S6K (Durieux et al., 2009; Klossner et al., 2009). This suggests that transcript level changes in the early phase of reloading may reflect inverse alterations in protein synthesis. These relationships imply that the ’muscle activity’ is a confounding variable which would be valuable to be considered in somatic gene therapy of skeletal muscle. The fact that activity dependent gene regulation is reflected by the mechano-regulation of FAK activity by pTyr-397 phosphorylation (Durieux et al., 2009) indicates possible venues to stimulate this directly in situations where muscle activity is not an option.
The extent to which differences between slow and fast motor neurons are involved in the coupling between muscle excitation and gene expression (excitation-transcription coupling) is currently not well understood (Schiaffino & Serrano, 2002; Wakeling & Syme, 2002). Based on a preferential atrophy of slow type muscle fibres (Roy et al., 1996; Dapp et al., 2004), hindlimb suspension is expected to affect slow motor units more that fast type units. Our findings on the distinction of basal and disuse-induced transcript expression with slowed signal propagation down the motoneuron (Magyar et al., 1996) highlight the importance of correct excitation for control of gene expression (Calvo et al., 2001).
Our observations indicate that muscle dysfunction with Charcot-Marie-Tooth (CMT) is not due to a single, central mechanism. A novel, contraction-dependent feedback mechanism is identified that controls myelin-specific transcripts via a muscle fibre-related pathway. Somatic transfection of muscle fibres with the mechanosensor FAK prove the concept that pmp22 expression which is lowered in CMT can be stimulated when combined with skeletal muscle use. This indicates that muscle activity is a confounding variable that warrants exploration in future gene therapeutic strategies to treat and manage neuromuscular disease.
The study was financially supported by grants from the Région Rhône-Alpes, the Association Française contre les myopathies, and the Swiss National Science Foundation. The experiments were performed at the University of Berne (Switzerland), the University of Lyon (France), and Manchester Metropolitan University. The assistance of Dominique Desplanches, Anne-Cécile Durieux, Damien Freyssenet and Prof. Hans Hoppeler during the suspension experiments is greatly acknowledged.
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