The highest levels of dystrophin gene products are in areas in which neurons maintain a high degree of synaptic plasticity: olfactory bulb, hippocampus, neocortex and cerebellum (Gorecki et al. 1997, 1998; Lidov et al. 1990). Synaptic plasticity has been examined in both the hippocampus and cerebellum and found to be altered in the *mdx* mouse in both of these brain regions (Anderson et al. 2004, 2010; Vaillend et al. 1998, 1999, 2004). Vaillend's group examined the CA1 dendritic layer of the hippocampus and found that NMDA-receptor dependent short-term and long-term potentiation, and long-term depression were abnormally enhanced in *mdx* mice (Perronnet & Vaillend 2010; Vaillend et al. 1998, 1999, 2004). Both our and Vaillend's group have investigated the function of the GABA receptor in the *mdx* cerebellum and hippocampus respectively as it is known to colocalise with dystrophin (Knuesel et al. 1999). In *mdx* mice hippocampus and cerebellum there is a marked reduction of GABAA clusters, with the number (but not size) of GABAA clusters being reduced by ~ 50% (Knuesel et al. 1999). In both the cerebellum and hippocampus the GABAA antagonist had a decreased effect (Anderson et al. 2003; Vaillend et al. 2002). When miniature inhibitory postsynaptic potentials were examined in the cerebellum a decrease in frequency and amplitude was found in contrast to the hippocampus where an increase in frequency was demonstrated (Anderson et al. 2003; Kueh et al. 2008; Graciotti et al. 2008). We have noted however that the reduction in amplitude may lead to a falsely lowered frequency due to lowered amplitude IPSCs being below counting threshold. These results have led to the 'dysfunctional inhibition' hypothesis being postulated as the cause of the cognitive impairment seen in DMD (Perronnet & Vaillend 2010). Dallerac et al. (2011) demonstrated that *mdx* mice treated with U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral vectors to induce skipping of the premature stop codon at exon 23 of the dystrophin gene normalises hippocampal synaptic plasticity. The vector was injected into the hippocampus and months after two months CA1 hippocampal LTP, which is normally enhanced compared to wildtype, was no longer different from wildtype. In studies of seizure induction in mice, dystrophin deficiency has been found to alter the neuronal excitability of AMPA/kainictype glutamate receptors suggesting a dysfunctional excitatory-inhibitory balance (De Sarro et al. 2004; Perronnet & Vaillend 2010; Yoshihara et al. 2003). Basolateral nucleus of the amygdale pyramidal neurons have also been studied in the *mdx* mouse and have been demonstrated to have decreased inductions of GABA-ergic IPSCs by noradrenalin. These authors note that in the *mdx* there were cells sensitive to noradrenalin (~40%) and a larger subset of cells insensitive to noradrenalin (~60%). Whilst the regular-spiking non-pyramidal basolateral nucleus of the amygdale neurons had similar proportions of noradrenalininduced depolarisation and AP firing (~50%) in wildtype and *mdx* demonstrating that the mechanism by which noradrenalin depolarises interneurons beyond AP threshold is not impaired in *mdx* mice. Rather it is more likely that a decrease in the number of normal functioning GABAergic synapses between the noradrenalin-responsive interneurons and pyramidal neurons underlies their findings (Sekiguchi et al. 2009).

Duchenne Muscular Dystrophy and Brain Function 105

interneurons are normally dystrophin negative. Firstly we examined a presynaptic form of synaptic plasticity in the cerebellum – short-term synaptic plasticity mediated at the parallel-fibre to Purkinje cell synapse. We found no difference between wildtype and *mdx* mice in this form of plasticity as expected due to the post-synaptic localisation of

Fig. 4. Long-term depression (LTD) in cerebellar Purkinje cells of wild-type and *mdx* mice. Upper graph shows the average slope of evoked EPSPs in wild-type and *mdx* Purkinje cells before and after LTD induction. Open circles are wildtype Purkinje cells (n=11) and closed circles are *mdx* Purkinje cells (n=12). Slopes are normalised to the average slope for wildtype and *mdx* cells respectively recorded in the 10 minutes preceding LTD induction. Thin horizontal bars above the abscissa represent the two 10 minute time periods during which the EPSP slopes were averaged and represent the magnitude of early and late phases of LTD. Vertical bars are SEM and for clarity are shown on one side of the data points only. Lower bar graph displays the average slope for all wild-type and all *mdx* cells in the early

and late phases following LTD induction (Modified from Anderson et al. 2004)

Work from our laboratory has similarly demonstrated altered GABA-ergic function in the cerebellum of *mdx* mice. We found that evoked excitatory post-synaptic potentials in cerebellar Purkinje cells show a decreased response (approx 50%) to bicuculline (a GABA antagonist) compared to wildtype (Anderson et al. 2003). We also demonstrated that miniature inhibitory postsynaptic currents had a significantly decreased amplitude in *mdx* cerebellar Purkinje cells compared to wildtype. This difference in amplitude was principally due to the absence of large amplitude miniature inhibitory postsynaptic currents in *mdx* mice. We postulate that this decrease is the result of a decrease in the number of postsynaptic GABA-ergic receptors (Nusser et al. 1997). This finding was reproduced in later studies (Fig. 3; Kueh et al. 2008, 2011). We further went on to investigate the number of GABAA channels located at the GABAergic synapse of cerebellar Purkinje cells. We found a significant reduction in the number of receptors at the PSD in *mdx* compared with littermate controls determined by non-stationary noise analysis of spontaneous miniature inhibitory postsynaptic currents. Single unitary conductance, rise and decay times of the currents were no different from littermate controls demonstrating that although there is a reduction in the number of channels at the postsynaptic membrane in *mdx* cerebellar Purkinje cells, the GABAA channels that are present are functioning normally. Further, Gaboxadol, an extrasynaptic GABA agonist, was applied inducing an increase in the holding current of the Purkinje cell. In *mdx* mice this increase was significantly greater (~200%) compared to littermate controls (Kueh et al. 2011), indicating that in *mdx* mice the number of extrasynpatic GABAA receptors is increased.

Fig. 3. A scatter plot of rise time versus peak amplitude, showing the distribution of mIPSCs in a (i) *mdx* and a (ii) littermate control mouse. The inserts show a section of the recording in aCSF and when TTX was added to the bath. iii) Cumulative probability of mIPSC amplitudes (average) in *mdx* and littermate control cells. There was a significant difference between mIPSC amplitudes between *mdx* and litermate control cells (Mann Whitney test, *p=*0.0001) (Modified from Kueh et al. 2011).

Our group has also investigated synaptic plasticity in the *mdx* cerebellum which is uniquely suited to these investigations as the Purkinje cell – the major output neuron of the cerebellum normally expresses a specific full-length dystrophin – P-dystrophin, which is absent in *mdx* mice. The major inputs to the Purkinje cells – parallel fibres and inhibitory

Work from our laboratory has similarly demonstrated altered GABA-ergic function in the cerebellum of *mdx* mice. We found that evoked excitatory post-synaptic potentials in cerebellar Purkinje cells show a decreased response (approx 50%) to bicuculline (a GABA antagonist) compared to wildtype (Anderson et al. 2003). We also demonstrated that miniature inhibitory postsynaptic currents had a significantly decreased amplitude in *mdx* cerebellar Purkinje cells compared to wildtype. This difference in amplitude was principally due to the absence of large amplitude miniature inhibitory postsynaptic currents in *mdx* mice. We postulate that this decrease is the result of a decrease in the number of postsynaptic GABA-ergic receptors (Nusser et al. 1997). This finding was reproduced in later studies (Fig. 3; Kueh et al. 2008, 2011). We further went on to investigate the number of GABAA channels located at the GABAergic synapse of cerebellar Purkinje cells. We found a significant reduction in the number of receptors at the PSD in *mdx* compared with littermate controls determined by non-stationary noise analysis of spontaneous miniature inhibitory postsynaptic currents. Single unitary conductance, rise and decay times of the currents were no different from littermate controls demonstrating that although there is a reduction in the number of channels at the postsynaptic membrane in *mdx* cerebellar Purkinje cells, the GABAA channels that are present are functioning normally. Further, Gaboxadol, an extrasynaptic GABA agonist, was applied inducing an increase in the holding current of the Purkinje cell. In *mdx* mice this increase was significantly greater (~200%) compared to littermate controls (Kueh et al. 2011), indicating that in *mdx* mice the number of

Fig. 3. A scatter plot of rise time versus peak amplitude, showing the distribution of mIPSCs in a (i) *mdx* and a (ii) littermate control mouse. The inserts show a section of the recording in

amplitudes (average) in *mdx* and littermate control cells. There was a significant difference between mIPSC amplitudes between *mdx* and litermate control cells (Mann Whitney test,

Our group has also investigated synaptic plasticity in the *mdx* cerebellum which is uniquely suited to these investigations as the Purkinje cell – the major output neuron of the cerebellum normally expresses a specific full-length dystrophin – P-dystrophin, which is absent in *mdx* mice. The major inputs to the Purkinje cells – parallel fibres and inhibitory

aCSF and when TTX was added to the bath. iii) Cumulative probability of mIPSC

extrasynpatic GABAA receptors is increased.

*p=*0.0001) (Modified from Kueh et al. 2011).

interneurons are normally dystrophin negative. Firstly we examined a presynaptic form of synaptic plasticity in the cerebellum – short-term synaptic plasticity mediated at the parallel-fibre to Purkinje cell synapse. We found no difference between wildtype and *mdx* mice in this form of plasticity as expected due to the post-synaptic localisation of

Fig. 4. Long-term depression (LTD) in cerebellar Purkinje cells of wild-type and *mdx* mice. Upper graph shows the average slope of evoked EPSPs in wild-type and *mdx* Purkinje cells before and after LTD induction. Open circles are wildtype Purkinje cells (n=11) and closed circles are *mdx* Purkinje cells (n=12). Slopes are normalised to the average slope for wildtype and *mdx* cells respectively recorded in the 10 minutes preceding LTD induction. Thin horizontal bars above the abscissa represent the two 10 minute time periods during which the EPSP slopes were averaged and represent the magnitude of early and late phases of LTD. Vertical bars are SEM and for clarity are shown on one side of the data points only. Lower bar graph displays the average slope for all wild-type and all *mdx* cells in the early and late phases following LTD induction (Modified from Anderson et al. 2004)

Duchenne Muscular Dystrophy and Brain Function 107

alterations seen in found in *mdx* CNS, as well as the plethora of biochemical alterations in seen in both humans and animal models of DMD has yet to emerge. The role of the smaller dystrophin gene products in the brain are now beginning to be appreciated with a recent report demonstrating that a loss of the smallest of these – Dp71, can lead to changes at the behavioural, cell membrane and synaptic levels (Daoud et al. 2009). The role of dystrophin and the dystrophin-glycoprotein complex in the CNS is slowly being elucidated. With a greater understanding of the function of this protein useful therapies may be able to ameliorate the CNS manifestations of Duchenne muscular dystrophy as well as aid in the

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**6. References**

dystrophin. We then went on to examine a post-synaptic mediated form of synaptic plasticity – long-term depression. As postulated the extent of depression was decreased in *mdx* cerebellar Purkinje cells compared to wildtype (Fig, 4; Anderson et al. 2004). We next examined homosynaptic longterm depression at this synapse and found that there was no difference between wildtype and *mdx* cerebellar Purkinje cells in the initial observation period, however the depression was significantly greater in the latter part of the observation period in *mdx* compared to wildtype (Anderson et al. 2010). The three most compelling explanations for the differences demonstrated above are i) an alteration in calcium homeostasis, ii) an indirect effect of altered GABAA receptor localisation and/or trafficking and iii) an alteration in putative AMPA-receptor localisation/trafficking. The most recent investigations of the multiplicity of effects of the known GABAA receptor dysfunction on the neurophysiology of the *mdx* mouse is compelling.

Further unpublished data from our laboratory has demonstrated that that short term synaptic plasticity (inhibitory interneuron to Purkinje cell synapse) is no different between *mdx* and wildtype. However rebound potentiation – a form of longterm synaptic plasticity expressed at this synapse is significantly different. Wildtype cerebellar Purkinje cells demonstrated potentiation of the inhibitory postsynaptic potential as previously reported, however the *mdx*  cerebellar Purkinje cells depressed. In a pilot study 5/12 wildtype cerebellar Purkinje cells demonstrated rebound potentiation (0 depressed) compared to 0/5 *mdx* cells demonstrating potentiation and 4/5 cells demonstrating depression. Although preliminary, these findings are the first to demonstrate a deficit in GABA-mediated synaptic plasticity. These findings also locate the problem at the post-synaptic (dystrophin-deficient Purkinje cell) locus as the presynaptically-mediated short-term synaptic plasticity of this synapse is preserved. (Anderson 2009).

## **5. Conclusion**

The role of dystrophin in the CNS is complex and incompletely understood. It is clear that the absence of this protein leads to profound functional deficits at the macro-level (behavioural alterations and cognitive impairment) as well as the micro-level (alterations in synaptic plasticity, GABA-ergic functioning). Morphologically there are alterations in cellular architecture and organisation as well as channel localisation. The increase in investigations of the role of dystrophin in the CNS has led to a rapid appreciation of the consequence of its absence in this tissue, however a clear mechanism by which these alterations occur has yet to emerge. The majority of current evidence points convincingly to a link between a lack of dystrophin and alterations in the localisation of GABAA receptors. Furthermore there is evidence that a lack of dystrophin is associated with abnormal functioning of GABAA receptor-mediated cellular activity as measured by changes in amplitude of IPSCs. The well-established cognitive-impairment seen in the boys with dystrophinopathies may be due to an underlying abnormality in synaptic plasticity as demonstrated in the *mdx* animal models. However evidence linking the alteration in GABAergic localisation and function with alterations in synaptic plasticity is speculative. The postulated dysfunctional excitatory-inhibitory balance – perhaps mediated by chronic alteration of calcium-handling by these neurons, is the most compelling hypothesis to date linking these two major findings (alterations in GABA clustering and function with alterations in synaptic plasticity). Further how a lack of dystrophin leads to the structural alterations seen in found in *mdx* CNS, as well as the plethora of biochemical alterations in seen in both humans and animal models of DMD has yet to emerge. The role of the smaller dystrophin gene products in the brain are now beginning to be appreciated with a recent report demonstrating that a loss of the smallest of these – Dp71, can lead to changes at the behavioural, cell membrane and synaptic levels (Daoud et al. 2009). The role of dystrophin and the dystrophin-glycoprotein complex in the CNS is slowly being elucidated. With a greater understanding of the function of this protein useful therapies may be able to ameliorate the CNS manifestations of Duchenne muscular dystrophy as well as aid in the effort to arrest the devastating muscular degeneration.

#### **6. References**

106 Muscular Dystrophy

dystrophin. We then went on to examine a post-synaptic mediated form of synaptic plasticity – long-term depression. As postulated the extent of depression was decreased in *mdx* cerebellar Purkinje cells compared to wildtype (Fig, 4; Anderson et al. 2004). We next examined homosynaptic longterm depression at this synapse and found that there was no difference between wildtype and *mdx* cerebellar Purkinje cells in the initial observation period, however the depression was significantly greater in the latter part of the observation period in *mdx* compared to wildtype (Anderson et al. 2010). The three most compelling explanations for the differences demonstrated above are i) an alteration in calcium homeostasis, ii) an indirect effect of altered GABAA receptor localisation and/or trafficking and iii) an alteration in putative AMPA-receptor localisation/trafficking. The most recent investigations of the multiplicity of effects of the known GABAA receptor dysfunction on the

Further unpublished data from our laboratory has demonstrated that that short term synaptic plasticity (inhibitory interneuron to Purkinje cell synapse) is no different between *mdx* and wildtype. However rebound potentiation – a form of longterm synaptic plasticity expressed at this synapse is significantly different. Wildtype cerebellar Purkinje cells demonstrated potentiation of the inhibitory postsynaptic potential as previously reported, however the *mdx*  cerebellar Purkinje cells depressed. In a pilot study 5/12 wildtype cerebellar Purkinje cells demonstrated rebound potentiation (0 depressed) compared to 0/5 *mdx* cells demonstrating potentiation and 4/5 cells demonstrating depression. Although preliminary, these findings are the first to demonstrate a deficit in GABA-mediated synaptic plasticity. These findings also locate the problem at the post-synaptic (dystrophin-deficient Purkinje cell) locus as the presynaptically-mediated short-term synaptic plasticity of this synapse is preserved.

The role of dystrophin in the CNS is complex and incompletely understood. It is clear that the absence of this protein leads to profound functional deficits at the macro-level (behavioural alterations and cognitive impairment) as well as the micro-level (alterations in synaptic plasticity, GABA-ergic functioning). Morphologically there are alterations in cellular architecture and organisation as well as channel localisation. The increase in investigations of the role of dystrophin in the CNS has led to a rapid appreciation of the consequence of its absence in this tissue, however a clear mechanism by which these alterations occur has yet to emerge. The majority of current evidence points convincingly to a link between a lack of dystrophin and alterations in the localisation of GABAA receptors. Furthermore there is evidence that a lack of dystrophin is associated with abnormal functioning of GABAA receptor-mediated cellular activity as measured by changes in amplitude of IPSCs. The well-established cognitive-impairment seen in the boys with dystrophinopathies may be due to an underlying abnormality in synaptic plasticity as demonstrated in the *mdx* animal models. However evidence linking the alteration in GABAergic localisation and function with alterations in synaptic plasticity is speculative. The postulated dysfunctional excitatory-inhibitory balance – perhaps mediated by chronic alteration of calcium-handling by these neurons, is the most compelling hypothesis to date linking these two major findings (alterations in GABA clustering and function with alterations in synaptic plasticity). Further how a lack of dystrophin leads to the structural

neurophysiology of the *mdx* mouse is compelling.

(Anderson 2009).

**5. Conclusion**

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**1. Introduction**

Dystrophy (DMD).

animal models.

**6**

*France*

**Proteomic Analysis**

**in Dystrophic Dog Muscle**

*1University of Nantes CNRS UMR6204,*

*2ONIRIS/INRA UMR 703,*

**of Signalling Pathway Deregulation**

During recent years, considerable effort has been made to develop proteomics technologies, with the aim of providing a complementary approach to the genomics tools already used in biomedical settings. This development has been extremely fast, and a number of emerging methodological proteomics tools now allow scientists to study the variable aspects of proteins in particular cell types, tissues or disease states. These tools include antibody arrays, two-dimensional-gel electrophoresis (2D-GE) and mass spectrometry (MS), the latter knowing an increasing use. In particular, candidate or non-candidate-based analyses of cell signalling represent powerful approaches for the investigation of the answers developed by cells in response to genetic modifications. Signalling molecules are key players in the regulation of the numerous and various biological processes occurring in a cell, and the alteration of signalling pathways has been associated with multiple diseases. Alterations in individual signalling pathways have been described in neuromuscular disorders, however, little information is available regarding their putative implication in Duchenne Muscular

DMD is an X-linked neuromuscular disorder that affects 1 newborn in 3500. This recessive disease represents the most common and severe form of muscular dystrophy. Although the genetic basis of the disease is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. Increasing evidence suggests that mechanisms secondary to the dystrophin deficiency at the basis of the disease, such as alterations in key signalling pathways, may play an important role. Proteomic profiling of dystrophic *vs* healthy skeletal muscle can help to generate a DMD-specific proteomic signature. Understanding which particular signal transduction pathways are involved in muscular dystrophy might provide a basis for new target and therapeutic agents discovery. This chapter examines signalling pathways status in skeletal muscles from the Golden Retriever Muscular Dystrophy (GRMD) dog, the only clinically relevant animal model for DMD (Valentine et al., 1988; Cooper et al., 1988). More specifically, we will describe how proteomic studies were successfully used to identify reliable biomarkers of the disease in

Marie Féron1, Karl Rouger2 and Laetitia Guével1,2

## **Proteomic Analysis of Signalling Pathway Deregulation in Dystrophic Dog Muscle**

Marie Féron1, Karl Rouger2 and Laetitia Guével1,2 *1University of Nantes CNRS UMR6204, 2ONIRIS/INRA UMR 703, France*

## **1. Introduction**

122 Muscular Dystrophy

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During recent years, considerable effort has been made to develop proteomics technologies, with the aim of providing a complementary approach to the genomics tools already used in biomedical settings. This development has been extremely fast, and a number of emerging methodological proteomics tools now allow scientists to study the variable aspects of proteins in particular cell types, tissues or disease states. These tools include antibody arrays, two-dimensional-gel electrophoresis (2D-GE) and mass spectrometry (MS), the latter knowing an increasing use. In particular, candidate or non-candidate-based analyses of cell signalling represent powerful approaches for the investigation of the answers developed by cells in response to genetic modifications. Signalling molecules are key players in the regulation of the numerous and various biological processes occurring in a cell, and the alteration of signalling pathways has been associated with multiple diseases. Alterations in individual signalling pathways have been described in neuromuscular disorders, however, little information is available regarding their putative implication in Duchenne Muscular Dystrophy (DMD).

DMD is an X-linked neuromuscular disorder that affects 1 newborn in 3500. This recessive disease represents the most common and severe form of muscular dystrophy. Although the genetic basis of the disease is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. Increasing evidence suggests that mechanisms secondary to the dystrophin deficiency at the basis of the disease, such as alterations in key signalling pathways, may play an important role. Proteomic profiling of dystrophic *vs* healthy skeletal muscle can help to generate a DMD-specific proteomic signature. Understanding which particular signal transduction pathways are involved in muscular dystrophy might provide a basis for new target and therapeutic agents discovery. This chapter examines signalling pathways status in skeletal muscles from the Golden Retriever Muscular Dystrophy (GRMD) dog, the only clinically relevant animal model for DMD (Valentine et al., 1988; Cooper et al., 1988). More specifically, we will describe how proteomic studies were successfully used to identify reliable biomarkers of the disease in animal models.

Proteomic Analysis of Signalling Pathway Deregulation in Dystrophic Dog Muscle 125

Sarcoglycan complex

**PTEN PP2A**

**PI3K PiP3 PDK1 PDK1 <sup>P</sup>**

**Akt Akt P**

**GSK3ß GSK3ß**

**P**

compensatory hypertrophy.

**IRS**

Insulin receptor

> **Protein Synthesis Hypertrophy**

**PP2A**

**Rictor mTOR**

decreased in GRMD dog; increased in GRMD dog; identical; non determined

Akt directly phosphorylates glycogen synthase kinase-3 (GSK3 at Ser9, thereby repressing its activity (Cross et al., 1995), and catalyses, *via* the mammalian target of rapamycin (mTOR), 70-kDa ribosomal protein S6 kinase (p70S6K) phosphorylation and activation (Chung et al., 1992; Glass, 2005; Inoki et al., 2002; Inoki et al., 2005; Price et al., 1992) (Figure 1). The PI3K/Akt/GSK3 and PI3K/Akt/mTOR/p70S6K pathways have been implicated in the regulation of skeletal muscle mass. Akt/mTOR signals were found to be upregulated during hypertrophy and downregulated during atrophy and the activation of Akt or p70S6K (or inactivation of GSK3appeared to be sufficient to induce hypertrophy. Moreover, in addition to acting as an inductive cue for hypertrophy, activation of the Akt/mTOR pathway could also prevent muscle atrophy *in vivo* (Bodine et al., 2001; Rommel et al., 2001). Furthermore, it has been shown *in vitro* that the overexpression of Src homology 2 (SH2) domain-containing inositol-5'-phosphatase 2 (SHIP-2), which, like PTEN, decreases PIP3 level, led to atrophy whereas the overexpression of a dominant negative mutant, which increases PIP3 level, induced hypertrophy (Rommel et al., 2001). The overexpression of

Fig. 1. Schematic diagram of signalling in the PI3K/Akt pathway and deregulations detected in GRMD skeletal muscle. Proteins shown in red, green and yellow indicate decreased, increased and unchanged level of expression or activity of these enzymes in GRMD *vs*. healthy muscle, respectively. As protein phosphatase 2A (PP2A) activity was not statistically modulated in GRMD muscle, the protein is shown in yellow. The deregulations detected in GRMD skeletal muscle could lead to decreased protein synthesis and block

**PDK1 P**

Dystrophin

X

**p70S6K p70S6K P**

F-actin

**mTOR**

**Grb2 FAK**

Dystroglycan complex

## **2. Signalling pathways and DMD**

In dystrophic muscles, the absence of dystrophin, and the consequent destabilization of the dystrophin-glycoprotein associated complex DGC (a multiprotein transmembrane complex), lead to the loss of sarcolemma integrity, calcium overload, calpains activation and finally, necrosis of the myofibers (Muntoni et al., 2003). Besides providing mechanical stability, the DGC interacts with several proteins, including growth factor receptor-bound protein 2 (Grb2) (Yang et al., 1995), neuronal nitric oxide synthase (nNOS) (Brenman et al., 1995), calmodulin (Madhavan et al., 1992), focal adhesion kinase (FAK) (Cavaldesi et al., 1999) and caveolin-3 (Crosbie et al., 1998), that play a role in cell signalling. Grb2 has been identified as a component of the Ras/ mitogen-activated protein kinases (MAPK) signalling pathway and both FAK and Grb2 function as mediators of survival signalling in various cell types, often working through the phosphatidyl inositol 3-kinase (PI3K)/Akt pathway (Langenbach&Rando, 2002). Even if this mechanism can, in part, account for the degenerative phenotype observed in DMD, it seems increasingly obvious that the deregulation of intracellular signalling pathways also plays a role. These pathways, which are implicated in the regulation of crucial processes such as the balance between apoptosis and cell survival or the equilibrium between atrophy and hypertrophy, involve cascades of phosphorylation/dephosphorylation events. Protein kinases represent key enzymes responsible for the phosphorylation of specific targets. Moreover, altered cell signalling is thought to increase the susceptibility of muscle fibers to secondary triggers, such as functional ischemia and oxidative stress, and free-radical scavengers can have a direct impact on the activity/phosphorylation of some components of the MAPK cascades (Hnia et al., 2007). In progressive muscular dystrophy, muscles are characterized by hypertrophy in the early phase, while atrophic changes are observed with aging (Noguchi, 2005).

Studies of the X chromosome-linked muscular dystrophy (*mdx*) mouse model of DMD (Bulfield et al., 1984) revealed modulations in MAPK signalling cascades, as dystrophic animals exhibited increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (Kumar et al., 2004; Lang et al., 2004) and c-jun N-terminal kinases 1 and 2 (JNK1/2) (Kolodziejczyk et al., 2001; Nakamura et al., 2005; St-Pierre et al., 2004), and decreased phosphorylation of p38 (Lang et al., 2004). The PI3K/Akt signalling pathway has also been shown to be affected in the *mdx* mouse, with an increased synthesis and phosphorylation of Akt observed (Dogra et al., 2006; Peter&Crosbie, 2006). Studies finally demonstrated that directly modulating signalling pathways activity could improve *mdx* muscle function (Kim et al., 2010; Tang et al., 2010). More specifically, increasing Akt activity by transgenic overexpression of the activated kinase itself has been shown to be able to reverse the dystrophic phenotype (Blaauw et al., 2009; Peter et al., 2009).

Moreover, the phosphorylation status of Akt was shown to be altered in human and canine dystrophic biopsies (Peter&Crosbie, 2006; Feron et al., 2009). Enhanced expression and activity of the phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN) has been observed in dystrophin-deficient dog muscle, and proposed to be at the origin of Akt inactivation (Feron et al., 2009). Indeed, PTEN opposes PI3K action by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P3) (Maehama&Dixon, 1998) and the increased activity detected in GRMD muscle would presumably lead to a decreased level of the phosphoinositide, which should limit the recruitment and activation of Akt (see Figure 1 for a schematic representation of the signalling pathway deregulation in dystrophic dog muscle).

In dystrophic muscles, the absence of dystrophin, and the consequent destabilization of the dystrophin-glycoprotein associated complex DGC (a multiprotein transmembrane complex), lead to the loss of sarcolemma integrity, calcium overload, calpains activation and finally, necrosis of the myofibers (Muntoni et al., 2003). Besides providing mechanical stability, the DGC interacts with several proteins, including growth factor receptor-bound protein 2 (Grb2) (Yang et al., 1995), neuronal nitric oxide synthase (nNOS) (Brenman et al., 1995), calmodulin (Madhavan et al., 1992), focal adhesion kinase (FAK) (Cavaldesi et al., 1999) and caveolin-3 (Crosbie et al., 1998), that play a role in cell signalling. Grb2 has been identified as a component of the Ras/ mitogen-activated protein kinases (MAPK) signalling pathway and both FAK and Grb2 function as mediators of survival signalling in various cell types, often working through the phosphatidyl inositol 3-kinase (PI3K)/Akt pathway (Langenbach&Rando, 2002). Even if this mechanism can, in part, account for the degenerative phenotype observed in DMD, it seems increasingly obvious that the deregulation of intracellular signalling pathways also plays a role. These pathways, which are implicated in the regulation of crucial processes such as the balance between apoptosis and cell survival or the equilibrium between atrophy and hypertrophy, involve cascades of phosphorylation/dephosphorylation events. Protein kinases represent key enzymes responsible for the phosphorylation of specific targets. Moreover, altered cell signalling is thought to increase the susceptibility of muscle fibers to secondary triggers, such as functional ischemia and oxidative stress, and free-radical scavengers can have a direct impact on the activity/phosphorylation of some components of the MAPK cascades (Hnia et al., 2007). In progressive muscular dystrophy, muscles are characterized by hypertrophy in

the early phase, while atrophic changes are observed with aging (Noguchi, 2005).

reverse the dystrophic phenotype (Blaauw et al., 2009; Peter et al., 2009).

Studies of the X chromosome-linked muscular dystrophy (*mdx*) mouse model of DMD (Bulfield et al., 1984) revealed modulations in MAPK signalling cascades, as dystrophic animals exhibited increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (Kumar et al., 2004; Lang et al., 2004) and c-jun N-terminal kinases 1 and 2 (JNK1/2) (Kolodziejczyk et al., 2001; Nakamura et al., 2005; St-Pierre et al., 2004), and decreased phosphorylation of p38 (Lang et al., 2004). The PI3K/Akt signalling pathway has also been shown to be affected in the *mdx* mouse, with an increased synthesis and phosphorylation of Akt observed (Dogra et al., 2006; Peter&Crosbie, 2006). Studies finally demonstrated that directly modulating signalling pathways activity could improve *mdx* muscle function (Kim et al., 2010; Tang et al., 2010). More specifically, increasing Akt activity by transgenic overexpression of the activated kinase itself has been shown to be able to

Moreover, the phosphorylation status of Akt was shown to be altered in human and canine dystrophic biopsies (Peter&Crosbie, 2006; Feron et al., 2009). Enhanced expression and activity of the phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN) has been observed in dystrophin-deficient dog muscle, and proposed to be at the origin of Akt inactivation (Feron et al., 2009). Indeed, PTEN opposes PI3K action by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P3) (Maehama&Dixon, 1998) and the increased activity detected in GRMD muscle would presumably lead to a decreased level of the phosphoinositide, which should limit the recruitment and activation of Akt (see Figure 1 for a schematic representation of the signalling pathway deregulation in dystrophic dog muscle).

**2. Signalling pathways and DMD**

Fig. 1. Schematic diagram of signalling in the PI3K/Akt pathway and deregulations detected in GRMD skeletal muscle. Proteins shown in red, green and yellow indicate decreased, increased and unchanged level of expression or activity of these enzymes in GRMD *vs*. healthy muscle, respectively. As protein phosphatase 2A (PP2A) activity was not statistically modulated in GRMD muscle, the protein is shown in yellow. The deregulations detected in GRMD skeletal muscle could lead to decreased protein synthesis and block compensatory hypertrophy.

Akt directly phosphorylates glycogen synthase kinase-3 (GSK3 at Ser9, thereby repressing its activity (Cross et al., 1995), and catalyses, *via* the mammalian target of rapamycin (mTOR), 70-kDa ribosomal protein S6 kinase (p70S6K) phosphorylation and activation (Chung et al., 1992; Glass, 2005; Inoki et al., 2002; Inoki et al., 2005; Price et al., 1992) (Figure 1). The PI3K/Akt/GSK3 and PI3K/Akt/mTOR/p70S6K pathways have been implicated in the regulation of skeletal muscle mass. Akt/mTOR signals were found to be upregulated during hypertrophy and downregulated during atrophy and the activation of Akt or p70S6K (or inactivation of GSK3appeared to be sufficient to induce hypertrophy. Moreover, in addition to acting as an inductive cue for hypertrophy, activation of the Akt/mTOR pathway could also prevent muscle atrophy *in vivo* (Bodine et al., 2001; Rommel et al., 2001). Furthermore, it has been shown *in vitro* that the overexpression of Src homology 2 (SH2) domain-containing inositol-5'-phosphatase 2 (SHIP-2), which, like PTEN, decreases PIP3 level, led to atrophy whereas the overexpression of a dominant negative mutant, which increases PIP3 level, induced hypertrophy (Rommel et al., 2001). The overexpression of

Proteomic Analysis of Signalling Pathway Deregulation in Dystrophic Dog Muscle 127

Fig. 2. Schematic diagram of the proteomic analysis of dystrophic dog muscle. H&E

purified from 4-month-old healthy and GRMD dog muscle.

**3.1 Protein array profiling**

(hematoxylin and eosin) staining showing classical pathological changes of DMD, including fiber size variation, fiber splitting, and central nucleation in skeletal muscle. 2D-GE, isotopecoded affinity tag (ICAT) quantitative proteomic analysis and antibody array of proteins

Biomedical research of the 21st century will largely be based on the results of studies focusing on the evaluation of gene expression and performed in order to develop molecular tools for the diagnosis and treatment of human diseases. Currently, DNA arrays represent the most commonly used means to follow gene expression in health and disease, and in muscular dystrophies in particular (Chen et al., 2000; Rouger et al., 2002). However, gene expression studies are limited by several aspects: i) gene expression levels do not necessarily reflect the level of proteins (that can also be regulated by degradation), ii) the activity of some proteins is regulated by posttranslational modifications (PTM) such as phosphorylation, glycosylation, carbonylation, acetylation and ubiquitylation, or by allosteric modifications

SHIP-2 in healthy mice muscle had no effect on fiber size but the overexpression of the phosphatase in a model of compensatory hypertrophy completely blocked the hypertrophy response (Bodine et al., 2001). It is thereby likely that the overexpression and increased activity of PTEN detected in GRMD muscle (by decreasing Akt activity and p70S6K phosphorylation, and by activating GSK3 could prevent compensatory muscle hypertrophy. More recently, the peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1) and PTEN inhibitor DJ-1/Parkinson disease (autosomal recessive early-onset) 7 (PARK7) appeared substantially reduced in GRMD *vs* healthy muscle (Feron et al., 2009; Guevel et al., 2011). Given the role of DJ-1 in the regulation of PTEN, this suggests that PTEN activation in GRMD dog muscle may originate from the under-expression of DJ-1. Noteworthy, in addition to its role in PTEN's regulation, DJ-1 also promotes the activity of PGC-1 (Zhong&Xu, 2008). As such, DJ-1 sensitive signalling pathways may provide high priority targets for the development of novel drug therapies for DMD.

Thus, compelling evidence suggest that alterations in signal transduction pathways may represent significant contributing factors to the progression of DMD. Proteomic profiling performed on the *mdx* mouse (Doran et al., 2006; Lewis et al., 2009; Ohlendieck, 2011) and GRMD dog models (Feron et al., 2009; Guevel et al., 2011) identified signalling proteins and reliable biomarkers of the secondary changes taking place in dystrophic muscles.

## **3. Proteomic analysis of dystrophic dog muscle**

Proteomic approaches have been developed in order to try to identify putative biomarkers of DMD. The proteome-wide investigation of proteins requires technological efforts in three essential steps: the separation, the identification and the quantification of multiple proteins. Reversible protein phosphorylation is arguably the most common and significant mechanism for the dynamic control of biological processes. Phosphorylation can dramatically alter a protein's biological location and/or activity and recent studies clearly highlighted the involvement of phosphoproteins and kinases in DMD (Kolodziejczyk et al., 2001; Kumar et al., 2004; Lang et al., 2004; Peter&Crosbie, 2006; Feron et al., 2009). Although up to one-third of the total proteome might be phosphorylated, the absolute levels of any single protein specie might be very low. In contrast to the traditional biochemical study of single proteins or isolated pathways in the context of muscular dystrophy, technical advances in the high-throughput screening by MS and array-based technology have established new ways of identifying entire cellular proteins populations in one shift analytical approach.

Global identification of signalling proteins can be done by a dedicated approach using antibody arrays. Antibody arrays also serve as an attractive option to carry out phosphoproteomic profiling in disease (Feron et al., 2009; Gembitsky et al., 2004; Kingsmore, 2006). Phospho-specific antibody arrays commercially available facilitate the investigation of specific activated pathways in muscular disorders. On the other hand, a considerable number of proteomic studies have employed unbiased technology such as 2D-GE and stable isotope-labelling techniques combined with MS. To construct an accurate model of the proteome variations occurring in dystrophic dog muscle, complementary proteomic screenings have been done (Figure 2).

Fig. 2. Schematic diagram of the proteomic analysis of dystrophic dog muscle. H&E (hematoxylin and eosin) staining showing classical pathological changes of DMD, including fiber size variation, fiber splitting, and central nucleation in skeletal muscle. 2D-GE, isotopecoded affinity tag (ICAT) quantitative proteomic analysis and antibody array of proteins purified from 4-month-old healthy and GRMD dog muscle.

#### **3.1 Protein array profiling**

126 Muscular Dystrophy

SHIP-2 in healthy mice muscle had no effect on fiber size but the overexpression of the phosphatase in a model of compensatory hypertrophy completely blocked the hypertrophy response (Bodine et al., 2001). It is thereby likely that the overexpression and increased activity of PTEN detected in GRMD muscle (by decreasing Akt activity and p70S6K phosphorylation, and by activating GSK3 could prevent compensatory muscle hypertrophy. More recently, the peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1) and PTEN inhibitor DJ-1/Parkinson disease (autosomal recessive early-onset) 7 (PARK7) appeared substantially reduced in GRMD *vs* healthy muscle (Feron et al., 2009; Guevel et al., 2011). Given the role of DJ-1 in the regulation of PTEN, this suggests that PTEN activation in GRMD dog muscle may originate from the under-expression of DJ-1. Noteworthy, in addition to its role in PTEN's regulation, DJ-1 also promotes the activity of PGC-1 (Zhong&Xu, 2008). As such, DJ-1 sensitive signalling pathways may provide high priority targets for the

Thus, compelling evidence suggest that alterations in signal transduction pathways may represent significant contributing factors to the progression of DMD. Proteomic profiling performed on the *mdx* mouse (Doran et al., 2006; Lewis et al., 2009; Ohlendieck, 2011) and GRMD dog models (Feron et al., 2009; Guevel et al., 2011) identified signalling proteins and

Proteomic approaches have been developed in order to try to identify putative biomarkers of DMD. The proteome-wide investigation of proteins requires technological efforts in three essential steps: the separation, the identification and the quantification of multiple proteins. Reversible protein phosphorylation is arguably the most common and significant mechanism for the dynamic control of biological processes. Phosphorylation can dramatically alter a protein's biological location and/or activity and recent studies clearly highlighted the involvement of phosphoproteins and kinases in DMD (Kolodziejczyk et al., 2001; Kumar et al., 2004; Lang et al., 2004; Peter&Crosbie, 2006; Feron et al., 2009). Although up to one-third of the total proteome might be phosphorylated, the absolute levels of any single protein specie might be very low. In contrast to the traditional biochemical study of single proteins or isolated pathways in the context of muscular dystrophy, technical advances in the high-throughput screening by MS and array-based technology have established new ways of identifying entire cellular proteins populations in one shift

Global identification of signalling proteins can be done by a dedicated approach using antibody arrays. Antibody arrays also serve as an attractive option to carry out phosphoproteomic profiling in disease (Feron et al., 2009; Gembitsky et al., 2004; Kingsmore, 2006). Phospho-specific antibody arrays commercially available facilitate the investigation of specific activated pathways in muscular disorders. On the other hand, a considerable number of proteomic studies have employed unbiased technology such as 2D-GE and stable isotope-labelling techniques combined with MS. To construct an accurate model of the proteome variations occurring in dystrophic dog muscle, complementary proteomic

reliable biomarkers of the secondary changes taking place in dystrophic muscles.

development of novel drug therapies for DMD.

analytical approach.

screenings have been done (Figure 2).

**3. Proteomic analysis of dystrophic dog muscle**

Biomedical research of the 21st century will largely be based on the results of studies focusing on the evaluation of gene expression and performed in order to develop molecular tools for the diagnosis and treatment of human diseases. Currently, DNA arrays represent the most commonly used means to follow gene expression in health and disease, and in muscular dystrophies in particular (Chen et al., 2000; Rouger et al., 2002). However, gene expression studies are limited by several aspects: i) gene expression levels do not necessarily reflect the level of proteins (that can also be regulated by degradation), ii) the activity of some proteins is regulated by posttranslational modifications (PTM) such as phosphorylation, glycosylation, carbonylation, acetylation and ubiquitylation, or by allosteric modifications

Proteomic Analysis of Signalling Pathway Deregulation in Dystrophic Dog Muscle 129

perturbations. In order to characterize the complete dystrophic proteome, the use the recent

A

Healthy GRMD

1

2 3 4

5 <sup>6</sup> <sup>7</sup> 1. control 2. ERK 3. JNK 4. p38 5. GSK3 6. p70S6K 7. Akt

2D-GE technology coupled to MS became favorable.

GSK3α/β

Log2

from healthy muscle.

biomarkers.

ratio GRMD/Healthy

B

0 0,5 11

0.5-0.5-1

GSK3β

Akt pan

Akt1

**3.2 Mass spectrometry-based proteomic analyses**

\*

Akt2

p70S6K

ERK1

\* \* \*

ERK2

5 7 62 3 4

JNK pan

Fig. 3. Antibody array analysis revealed PI3K/Akt and MAPK signalling pathway modulation in GRMD skeletal muscle. Healthy and GRMD muscle extracts from 4-month-

**3.2.1 Separation of muscle proteins by two-dimensional gel electrophoresis**

old dogs were incubated with two antibody arrays. A ratio of signal intensity (GRMD/healthy) was calculated, and log transformed (base2). A cutoff value was

JNK1

JNK2

determined by ANOVA analysis at 95% confidence level (p<0.05). \* - significantly different

MS-based proteomics represents an unbiased approach allowing the comprehensive cataloging of the whole protein alterations associated with a specific disease. This chapter outlines the findings from recent applications of MS-based proteomics for studying alterations in dystrophic dog muscle, and examines novel strategies to establish DMD-specific

2D-GE represents a highly reproducible and discriminatory technique that allows the analysis of the accessible (meaning soluble and abundant) muscle proteins. The proteins that appear differentially expressed between the different samples analyzed are then identified by high-throughput MS (matrix-assisted laser desorption/ionization time-of-flight - MALDI-ToF or electrospary ionization - ESI). Proteomics dataset are finally interlinked with international web-based gel electrophoretic and protein sequence databanks for comparative analysis. Modern mass spectrometers produce and separate ions according to their mass-to-charge ratio (m/z) with an extraordinary resolving power. MS-based analysis of the skeletal muscle proteome has already been successfully used in the context of muscle development, fiber type specification, fast-to-slow transformation, muscle growth and aging, and in the context of denervation-induced fiber damage, atrophy, obesity, diabetes and muscular dystrophies (Ohlendieck, 2010). Detailed 2D maps of the major soluble muscle proteome, including proteins involved in actomyosin apparatus, regulation of contraction, ion homeostasis, signalling, cytosolic and mitochondrial metabolism and stress response,

JNK3

p38α

p38δ

\*

p38γ

\*

and iii) localization changes can also play roles in this regulation. On the other hand, a lot of studies have been focusing on single proteins, protein complexes or isolated pathways, limiting the understanding of the pathogenesis of DMD at the organism level. In order to obtain this information and to be able to measure in parallel the expression and the state of activation of several hundreds of proteins, it was important to develop new approaches. Protein array-based approaches can provide not only data complementary to DNA microarrays but also provide unique information about the functional state of proteins under normal and pathological conditions (Hanash, 2003; MacBeath, 2002). Miniaturized protein array technology has opened a new chapter in biotechnology due to its ability to compare, characterize and quantify simultaneously a large number of proteins in the form of spots, thus replacing numerous individual protein by protein tests.It also allows parallel evaluation of several parameters in complex biological solutions. Moreover, a minute spot with immobilized protein sample on an array slide provides greater sensitivity for the detection of molecular interactions compared to other binding assays (Ekins&Chu, 1999). For the first time, antibody arrays were used by Anderson group to look for protein expression changes in spinal muscular atrophy (Anderson&Davison, 1999). A relatively small number of differences were found within a group of proteins that function as both RNA binding proteins and transcription factors. A second group used microarrays to profile the level of proteins associated with calcium regulation in sarcoplasmic reticulum isolated from muscle (Schulz et al., 2006). They used a reverse-phase protein array printed with proteins from genotyped animals and probed with seven target proteins important in calcium regulation. Reverse-phase arrays have been used for profiling phosphorylated proteins in various cancers (Grubb et al., 2003; Sheehan et al., 2005), and it was hoped that these arrays would become a powerful clinical tool for diagnosis and therapy guidance in different diseases.

More recently, the antibody array technology was used to assess the phosphorylation status of key proteins of the MAPK and PI3K/Akt signalling pathways in the *Vastus lateralis* muscle from 4-month old GRMD *vs* healthy dogs. The antibody array technology represents a powerful tool for the semi-quantitative comparison of the expression and/or phosphorylation level of a high number of proteins in a limited number of samples (Sakanyan, 2005; Yeretssian et al., 2005). The main advantage resides in the gain of time that it provides, as a high number of proteins can be studied in just one experiment. Moreover (and in contrary to the ICAT technology for example), antibody arrays give access to information about PTM, such as phosphorylation, which is of course crucial in the context of cell signalling. Though it represents a biased technique (data is obtained only for the antibodies initially spotted on the membrane) but, as hundreds of different antibodies can be spotted onto the same membrane, it can easily be used for screening purposes such asfor disease diagnosis using disease biomarkers. This study indicated that Akt1, GSK3 and p70S6K, as well as ERK1/2 and the p38 and kinases all displayed a decreased phosphorylation level in canine dytrophic muscle (Figure 3). Antibody arrays allow the detection of the presence of specific proteins, and the level of expression of phospho-proteins in disease tissue (Cahill, 2001), thus having a potential for biomedical and diagnostic applications. However, it had not been possible to address the systematic analysis of proteins using this dedicated approach.

A lot of evidence now indicates that various signalling and metabolic pathways are altered in DMD, and a global, unbiased, proteomics study was necessary to identify these

and iii) localization changes can also play roles in this regulation. On the other hand, a lot of studies have been focusing on single proteins, protein complexes or isolated pathways, limiting the understanding of the pathogenesis of DMD at the organism level. In order to obtain this information and to be able to measure in parallel the expression and the state of activation of several hundreds of proteins, it was important to develop new approaches. Protein array-based approaches can provide not only data complementary to DNA microarrays but also provide unique information about the functional state of proteins under normal and pathological conditions (Hanash, 2003; MacBeath, 2002). Miniaturized protein array technology has opened a new chapter in biotechnology due to its ability to compare, characterize and quantify simultaneously a large number of proteins in the form of spots, thus replacing numerous individual protein by protein tests.It also allows parallel evaluation of several parameters in complex biological solutions. Moreover, a minute spot with immobilized protein sample on an array slide provides greater sensitivity for the detection of molecular interactions compared to other binding assays (Ekins&Chu, 1999). For the first time, antibody arrays were used by Anderson group to look for protein expression changes in spinal muscular atrophy (Anderson&Davison, 1999). A relatively small number of differences were found within a group of proteins that function as both RNA binding proteins and transcription factors. A second group used microarrays to profile the level of proteins associated with calcium regulation in sarcoplasmic reticulum isolated from muscle (Schulz et al., 2006). They used a reverse-phase protein array printed with proteins from genotyped animals and probed with seven target proteins important in calcium regulation. Reverse-phase arrays have been used for profiling phosphorylated proteins in various cancers (Grubb et al., 2003; Sheehan et al., 2005), and it was hoped that these arrays would become a powerful clinical tool for diagnosis and therapy guidance in

More recently, the antibody array technology was used to assess the phosphorylation status of key proteins of the MAPK and PI3K/Akt signalling pathways in the *Vastus lateralis* muscle from 4-month old GRMD *vs* healthy dogs. The antibody array technology represents a powerful tool for the semi-quantitative comparison of the expression and/or phosphorylation level of a high number of proteins in a limited number of samples (Sakanyan, 2005; Yeretssian et al., 2005). The main advantage resides in the gain of time that it provides, as a high number of proteins can be studied in just one experiment. Moreover (and in contrary to the ICAT technology for example), antibody arrays give access to information about PTM, such as phosphorylation, which is of course crucial in the context of cell signalling. Though it represents a biased technique (data is obtained only for the antibodies initially spotted on the membrane) but, as hundreds of different antibodies can be spotted onto the same membrane, it can easily be used for screening purposes such asfor disease diagnosis using disease biomarkers. This study indicated that Akt1, GSK3 and p70S6K, as well as ERK1/2 and the p38 and kinases all displayed a decreased phosphorylation level in canine dytrophic muscle (Figure 3). Antibody arrays allow the detection of the presence of specific proteins, and the level of expression of phospho-proteins in disease tissue (Cahill, 2001), thus having a potential for biomedical and diagnostic applications. However, it had not been possible to address the

A lot of evidence now indicates that various signalling and metabolic pathways are altered in DMD, and a global, unbiased, proteomics study was necessary to identify these

systematic analysis of proteins using this dedicated approach.

different diseases.

perturbations. In order to characterize the complete dystrophic proteome, the use the recent 2D-GE technology coupled to MS became favorable.

Fig. 3. Antibody array analysis revealed PI3K/Akt and MAPK signalling pathway modulation in GRMD skeletal muscle. Healthy and GRMD muscle extracts from 4-monthold dogs were incubated with two antibody arrays. A ratio of signal intensity (GRMD/healthy) was calculated, and log transformed (base2). A cutoff value was determined by ANOVA analysis at 95% confidence level (p<0.05). \* - significantly different from healthy muscle.

#### **3.2 Mass spectrometry-based proteomic analyses**

MS-based proteomics represents an unbiased approach allowing the comprehensive cataloging of the whole protein alterations associated with a specific disease. This chapter outlines the findings from recent applications of MS-based proteomics for studying alterations in dystrophic dog muscle, and examines novel strategies to establish DMD-specific biomarkers.

#### **3.2.1 Separation of muscle proteins by two-dimensional gel electrophoresis**

2D-GE represents a highly reproducible and discriminatory technique that allows the analysis of the accessible (meaning soluble and abundant) muscle proteins. The proteins that appear differentially expressed between the different samples analyzed are then identified by high-throughput MS (matrix-assisted laser desorption/ionization time-of-flight - MALDI-ToF or electrospary ionization - ESI). Proteomics dataset are finally interlinked with international web-based gel electrophoretic and protein sequence databanks for comparative analysis. Modern mass spectrometers produce and separate ions according to their mass-to-charge ratio (m/z) with an extraordinary resolving power. MS-based analysis of the skeletal muscle proteome has already been successfully used in the context of muscle development, fiber type specification, fast-to-slow transformation, muscle growth and aging, and in the context of denervation-induced fiber damage, atrophy, obesity, diabetes and muscular dystrophies (Ohlendieck, 2010). Detailed 2D maps of the major soluble muscle proteome, including proteins involved in actomyosin apparatus, regulation of contraction, ion homeostasis, signalling, cytosolic and mitochondrial metabolism and stress response,

Proteomic Analysis of Signalling Pathway Deregulation in Dystrophic Dog Muscle 131

(GO) annotation of the altered proteome led to several key findings which might reflect the ongoing muscle regeneration taking place in dystrophic muscle. Among the proteins altered in the intracellular signalling category in the dystrophic muscle, protein phosphatase 1 (PP1) and DJ-1 appear particularly interesting. PP1, which is present in skeletal muscle, is known to regulate both glycogen and fatty metabolism, while promoting the dephosphorylation of myosin The protein DJ-1 (also called PARK7) was recently described as a negative regulator of PTEN (Kim et al., 2005; Villa-Moruzzi et al., 1996). Interestingly, the underexpressed proteins primarily composed of metabolic proteins, many of which have been shown to be regulated by PGC-1. Interestingly, among the several PGC-1 targets identified to be under-expressed in dystrophic dog muscle, 5 (namely 6 phosphofructokinase, phosphoglucomutase-1, aconitase 2, cytochrome c1 and fatty acid binding protein 3) have already been identified in a different transcriptomic study as underexpressing in DMD compared to healthy human biopsies (Pescatori et al., 2007). PGC-1 has been described as a potent regulator of mitochondrial biogenesis and oxidative metabolism in skeletal muscle (Wu et al., 1999; Lin et al., 2002). In addition, activation of the peroxysome proliferator-activated receptor (PPAR)/PGC-1 pathway has been shown, by preventing the bioenergetic deficit observed, to efficiently improve a mitochondrial myopathy phenotype (Wenz et al., 2008), suggesting that PGC-1 mediated improvement of dystrophic muscle may rely (in part) on the restoration of PGC-1 mitochondrial targets (Handschin et al., 2007). Interestingly, a recent study has shown that pharmacologic activation of PPAR/ also leads to an upregulation in the expression of utrophin A, which was concurrent with a partial correction of the dystrophic phenotype (Miura et al., 2009). Taken together, these results provide compelling new evidence that defects in PTEN and PGC-1contribute to profound signalling pathway deregulation in the canine model of DMD as well as to the disease progression. In addition, they demonstrate that proteomics tools are of particular interest for the study of muscular disorders. Recently, the combination of proteomics, metabolomics and fluoximics has confirmed the existence in the *mdx* mouse of perturbations that reflect mitochondrial energetic alterations (Griffin&Des Rosiers, 2009). The broad aim of these studies has been two-fold, first the identification of co-founding factors that promote or limit the disease progression and second, the identification of new biomarkers that could

be used to more accurately define the disease status.

**4. Reliable biomarkers of DMD, with a special focus on signalling proteins**

As previously mentioned, the absence of a single protein (dystrophin) in muscle has devastating consequences. Despites the tremendous efforts that have been made for more than 20 years in order to try to understand how this initial genetic defect could lead to the progressive and irreversible muscle wasting observed, the pathogenesis of DMD has not been fully characterized. Furthermore, no curative treatment is yet available and DMD patients are still dying during early adulthood. The identification, at the proteome level, of the alterations associated with DMD is important for at least five reasons. By providing a better understanding of the pathogenesis of DMD, they should i) improve diagnosis, ii) enable a better monitoring of disease progression, iii) lead to the proposal of new therapeutic targets (in the perspective of a pharmacological treatment – alone or in combination with a gene or cell therapy approach), iv) enable the fast and efficient evaluation of the benefits provided by the treatments currently under study (are they able to

have been established for various mammals (Doran et al., 2009a). The results obtained by this high-throughput technology should then be confirmed by more classical techniques such as western immunoblotting and immunohistochemistry. For example, the *mdx* mouse model was employed in recent proteomics profiling studies which revealed new disease markers in dystrophin-deficient fibres (Doran et al., 2004; Doran et al., 2006a, 2006b; Gardan-Salmon et al., 2011). A differential in-gel analysis (DIGE) analysis of *mdx vs* normal diaphragm muscle revealed a drastic differential expression pattern of 35 proteins, with 21 proteins being decreased (including the F-box only protein 11 - Fbxo11, adenylate kinase 1 – AK1, and the calcium-binding protein regucalcin) and 14 proteins being increased, including the small cardiovascular heat shock protein cvHSP and muscle proteins such as vimentin, desmin and myosin heavy chain (MHC) (Doran et al., 2006a).

More recently, the GRMD dog model was used to profile changes in protein abundance associated with DMD using 2D-GE. To eliminate the structural and contractile proteins that are over-abundant in crude protein extracts prepared from skeletal muscle, and to enrich the samples in signalling proteins, the study restricted the analysis to the cytosolic and phospho-enriched proteins of the *Vastus lateralis* muscle removed from 4-month-old healthy and dystrophic dogs. Among the differentially expressed proteins , 8 were chosen, according to their high level of dysregulation, for further identification by MS. This led to the identification of skeletal muscle markers involved in the contractile function and mitochondrial proteins involved in energy metabolism (Guevel et al., 2011). Although 2D-GE analysis represents an efficient technique to identify relative changes in protein expression, it is not well suited for studying low-abundant proteins, which are often important regulators of cell signalling. The high abundance of cytoskeletal, contractile and chaperone proteins identified in the phospho-enriched sample combined with dynamic range issues associated with the 2D-GE approach hampered studies on skeletal muscle analysis. Recently, Hojlund and colleague used 1D-GE and high-performance liquid chromatography (HPLC)-ESI-MS/MS to characterize the proteome of human skeletal muscle (Hojlund et al., 2008). The proteins identified in this study provide a representation of the major biological function of healthy human skeletal muscle. To elucidate changes in the proteome associated with DMD, and to overcome disadvantages of 2D-GE, peptidecentric approach can be used, which allows quantitative comparison of two samples. Numerous stable isotope-labelling techniques have been employed in quantitative shotgun proteomics, including isobaric tag for relative and absolute quantification (iTRAQ); isotopecoded affinity tag (ICAT); and stable isotope labelling by amino-acids in cell culture (SILAC) (Ohlendieck, 2011). In dystrophic skeletal muscle, the ICAT labelling approach has been used for the quantitative proteomic profiling of healthy and GRMD dog muscles.

### **3.2.2 Quantitative proteomic analysis**

ICAT labelling followed by LC-MS/MS was used to analyze the quantitative variations of the proteome in both a cytoplasmic and a phospho-enriched fraction prepared from the *Vastus lateralis* muscle of 4-month old healthy and GRMD dogs (Guevel et al., 2011). A total of 84 proteins appeared significantly altered (61 proteins from the cytosolic fraction and 36 proteins from the phospho-enriched fraction, with an overlap of 13 proteins). These proteins were classified into 7 major categories including: i) muscle development and contraction, ii) glycolytic metabolism, iii) oxidative metabolism, iv) calcium ion homeostasis, v) intracellular signalling, vi) regulation of apoptosis, and vii) other functions. Gene Ontology

have been established for various mammals (Doran et al., 2009a). The results obtained by this high-throughput technology should then be confirmed by more classical techniques such as western immunoblotting and immunohistochemistry. For example, the *mdx* mouse model was employed in recent proteomics profiling studies which revealed new disease markers in dystrophin-deficient fibres (Doran et al., 2004; Doran et al., 2006a, 2006b; Gardan-Salmon et al., 2011). A differential in-gel analysis (DIGE) analysis of *mdx vs* normal diaphragm muscle revealed a drastic differential expression pattern of 35 proteins, with 21 proteins being decreased (including the F-box only protein 11 - Fbxo11, adenylate kinase 1 – AK1, and the calcium-binding protein regucalcin) and 14 proteins being increased, including the small cardiovascular heat shock protein cvHSP and muscle proteins such as

More recently, the GRMD dog model was used to profile changes in protein abundance associated with DMD using 2D-GE. To eliminate the structural and contractile proteins that are over-abundant in crude protein extracts prepared from skeletal muscle, and to enrich the samples in signalling proteins, the study restricted the analysis to the cytosolic and phospho-enriched proteins of the *Vastus lateralis* muscle removed from 4-month-old healthy and dystrophic dogs. Among the differentially expressed proteins , 8 were chosen, according to their high level of dysregulation, for further identification by MS. This led to the identification of skeletal muscle markers involved in the contractile function and mitochondrial proteins involved in energy metabolism (Guevel et al., 2011). Although 2D-GE analysis represents an efficient technique to identify relative changes in protein expression, it is not well suited for studying low-abundant proteins, which are often important regulators of cell signalling. The high abundance of cytoskeletal, contractile and chaperone proteins identified in the phospho-enriched sample combined with dynamic range issues associated with the 2D-GE approach hampered studies on skeletal muscle analysis. Recently, Hojlund and colleague used 1D-GE and high-performance liquid chromatography (HPLC)-ESI-MS/MS to characterize the proteome of human skeletal muscle (Hojlund et al., 2008). The proteins identified in this study provide a representation of the major biological function of healthy human skeletal muscle. To elucidate changes in the proteome associated with DMD, and to overcome disadvantages of 2D-GE, peptidecentric approach can be used, which allows quantitative comparison of two samples. Numerous stable isotope-labelling techniques have been employed in quantitative shotgun proteomics, including isobaric tag for relative and absolute quantification (iTRAQ); isotopecoded affinity tag (ICAT); and stable isotope labelling by amino-acids in cell culture (SILAC) (Ohlendieck, 2011). In dystrophic skeletal muscle, the ICAT labelling approach has been

vimentin, desmin and myosin heavy chain (MHC) (Doran et al., 2006a).

used for the quantitative proteomic profiling of healthy and GRMD dog muscles.

ICAT labelling followed by LC-MS/MS was used to analyze the quantitative variations of the proteome in both a cytoplasmic and a phospho-enriched fraction prepared from the *Vastus lateralis* muscle of 4-month old healthy and GRMD dogs (Guevel et al., 2011). A total of 84 proteins appeared significantly altered (61 proteins from the cytosolic fraction and 36 proteins from the phospho-enriched fraction, with an overlap of 13 proteins). These proteins were classified into 7 major categories including: i) muscle development and contraction, ii) glycolytic metabolism, iii) oxidative metabolism, iv) calcium ion homeostasis, v) intracellular signalling, vi) regulation of apoptosis, and vii) other functions. Gene Ontology

**3.2.2 Quantitative proteomic analysis**

(GO) annotation of the altered proteome led to several key findings which might reflect the ongoing muscle regeneration taking place in dystrophic muscle. Among the proteins altered in the intracellular signalling category in the dystrophic muscle, protein phosphatase 1 (PP1) and DJ-1 appear particularly interesting. PP1, which is present in skeletal muscle, is known to regulate both glycogen and fatty metabolism, while promoting the dephosphorylation of myosin The protein DJ-1 (also called PARK7) was recently described as a negative regulator of PTEN (Kim et al., 2005; Villa-Moruzzi et al., 1996). Interestingly, the underexpressed proteins primarily composed of metabolic proteins, many of which have been shown to be regulated by PGC-1. Interestingly, among the several PGC-1 targets identified to be under-expressed in dystrophic dog muscle, 5 (namely 6 phosphofructokinase, phosphoglucomutase-1, aconitase 2, cytochrome c1 and fatty acid binding protein 3) have already been identified in a different transcriptomic study as underexpressing in DMD compared to healthy human biopsies (Pescatori et al., 2007). PGC-1 has been described as a potent regulator of mitochondrial biogenesis and oxidative metabolism in skeletal muscle (Wu et al., 1999; Lin et al., 2002). In addition, activation of the peroxysome proliferator-activated receptor (PPAR)/PGC-1 pathway has been shown, by preventing the bioenergetic deficit observed, to efficiently improve a mitochondrial myopathy phenotype (Wenz et al., 2008), suggesting that PGC-1 mediated improvement of dystrophic muscle may rely (in part) on the restoration of PGC-1 mitochondrial targets (Handschin et al., 2007). Interestingly, a recent study has shown that pharmacologic activation of PPAR/ also leads to an upregulation in the expression of utrophin A, which was concurrent with a partial correction of the dystrophic phenotype (Miura et al., 2009). Taken together, these results provide compelling new evidence that defects in PTEN and PGC-1contribute to profound signalling pathway deregulation in the canine model of DMD as well as to the disease progression. In addition, they demonstrate that proteomics tools are of particular interest for the study of muscular disorders. Recently, the combination of proteomics, metabolomics and fluoximics has confirmed the existence in the *mdx* mouse of perturbations that reflect mitochondrial energetic alterations (Griffin&Des Rosiers, 2009). The broad aim of these studies has been two-fold, first the identification of co-founding factors that promote or limit the disease progression and second, the identification of new biomarkers that could be used to more accurately define the disease status.

#### **4. Reliable biomarkers of DMD, with a special focus on signalling proteins**

As previously mentioned, the absence of a single protein (dystrophin) in muscle has devastating consequences. Despites the tremendous efforts that have been made for more than 20 years in order to try to understand how this initial genetic defect could lead to the progressive and irreversible muscle wasting observed, the pathogenesis of DMD has not been fully characterized. Furthermore, no curative treatment is yet available and DMD patients are still dying during early adulthood. The identification, at the proteome level, of the alterations associated with DMD is important for at least five reasons. By providing a better understanding of the pathogenesis of DMD, they should i) improve diagnosis, ii) enable a better monitoring of disease progression, iii) lead to the proposal of new therapeutic targets (in the perspective of a pharmacological treatment – alone or in combination with a gene or cell therapy approach), iv) enable the fast and efficient evaluation of the benefits provided by the treatments currently under study (are they able to

Proteomic Analysis of Signalling Pathway Deregulation in Dystrophic Dog Muscle 133

pyrophosphatase enzymes was detected, highlighting the general mitochondrial dysfunction and metabolism crisis taking place in dystrophic muscles. Concerning the serine protease inhibitor family (ii), an up-regulation was detected for protease inhibitor member 1a and serine protease inhibitors 3, 6 and 1-5, suggesting a partial inhibition of proteolysis in *mdx* muscles. Concerning growth and differentiation (iii), an increase was detected in the expression level of PP1, cofilin 2 (CFL2) and 14-3-3, indicating active proliferation and differentiation. Of interest, an increase in CFL2 had also been observed in human DMD biopsies. As far as calcium homeostasis is concerned (iv), the up-regulation of PP1 can also be cited, as the phosphatase binds to the ryanodine-sensitive calcium release channel protein to regulate calcium flux. The calcium-binding protein annexin V was also increased. Finally, and concerning cytoskeleton reorganization and biogenesis (v), RhoGDI-1, -actin and tropomyosin 1 were found to be up-regulated in *mdx* muscles, indicating cytoskeleton remodeling. At one and six months, 62 and 48 differentially expressed proteins were detected, respectively. At one month, most of the proteins detected were downregulated whereas at six months (as it was the case at three months), most of them were upregulated. These results confirmed the specificity of the one month stage ("DMD-like" crisis) in the evolution of the disease in the *mdx* mouse. Six proteins were detected as differentially expressed in *mdx vs* healthy muscles at the three stages tested: AK1 (down-regulated), CK (up-regulated), myosin light chain 2 (MLC2, up-regulated), annexin V, tropomyosin and 14-3-3 (all down-regulated at one month and up-regulated at three and six months). Some of these changes thus appear directly linked to the absence of dystrophin whereas some others appear more dependent of the phenotype on the muscle. The elevation detected in MLC2 levels could reflect the proliferation/differentiation processes occurring in *mdx* muscles, as the protein is involved in muscle differentiation, and its consistent elevation during the progression of the disease suggests that *mdx* muscles may assume a chronic or abnormal