**2.3.2.1 Acetylcholinesterase inhibitors (AChEIs)**

AChEIs interfere with the catalytic breakdown of the neurotransmitter ACh, rendering ACh available for a longer period of time at the NMJ and the nicotinic AChRs (nAChRs). The nonselective AChEIs affect both the nAChRs and the muscarinic receptors in exocrine glands. Common adverse effects include muscarinic symptoms, such as increased gut

Myasthenia Gravis: New Insights into the Effect

modified strains permit genetic analysis.

**2.4.1 AChR+ EAMG: Clinical phenotype** 

(Berman and Patrick 1980).

**2.4.2 MuSK+ EAMG: Clinical phenotype** 

**(EAMG)** 

of MuSK Antibodies and Acetylcholinesterase Inhibitors 445

**2.4 Establishment and phenotype of murine models of experimental autoimmune MG** 

Naturally occurring MG in animals is present in canine MG. This canine form of MG has a natural course of clinical and immunological remission in the majority of dogs, even without initiation of immunosuppressive treatment (Shelton and Lindstrom 2001). Therefore, the use of the canine MG in determining the effect of immunosuppressant medication is limited. EAMG can be induced in rabbits, rats and mice either by passive transfer of human antibodies/sera or by immunization of the antigen in adjuvant. In 1978 it was first shown that active immunization with AChR from Torpedo (ray fish) electric organ, in rabbits leads to flaccid paralysis and an MG-like disease. Since in rabbits the disease has an acute action and shortly leads to death, these animals are not routinely used for EAMG studies. Some studies have used guinea pigs and even primates, but the current most widely used model is EAMG induced in female young mice or rats. The disease in rats has a short acute phase and a chronic phase, which mimics the human disease, excluding the involvement of the thymus (Meinl, Klinkert et al. 1991). In the mice only a chronic phase is induced. EAMG is more difficult to induce in mice and usually requires multiple immunizations and only a fraction of the mice develop disease symptoms. However, the murine model has an advantage of a plethora of mouse-specic reagents and knock-out mouse strains, which enable analyses that cannot be performed in rats. One further advantage of using the mouse is that its immune system is well characterized and the availability of inbred and genetically

Immunization with purified denatured *Torpedo* AChR α1, β1, γ, or δ subunits can cause EAMG, but it is inefficient compared to native AChR (Lindstrom, Einarson et al. 1978). Purified α1-subunit is the most potent, as might be expected since its sequence is the most conserved. Also, there are two α1 in an AChR which permit cross-linking by antibodies, which may utilize renaturation to provoke antibodies to the main immunogenic region (MIR) (Lindstrom, Luo et al. 2008). It is of outmost importance to use a mouse strain which possesses IL-6, since mice deficient in IL-6 have been shown to be resistant to the development of EAMG upon immunization (Deng, Goluszko et al. 2002). In the acute phase of active EAMG or EAMG passively transmitted with serum, binding of AChR antibody and its complement target the postsynaptic membrane for attack by macrophages (Lindstrom 2000). Mice immunized with AChR in Complete Freund´s Adjuvant (CFA) develop a flaccid paralysis (Figure 4a), a drooping of the head and tail and weakness primarily of the forelimbs, which may rapidly progress to respiratory failure (Berman and Patrick 1980). The myasthenic phenotype in the AChR+ EAMG mice has been reported to be restored in all cases to nearly normal after treatment with neostigmine intraperitoneally

The antibodies in MuSK+ MG are directed against the extracellular domain of MuSK, which contains IgG-like domains. The first study which proved the pathogenic role of MuSKantibodies in animals was done in 2006, when New Zealand White rabbits were repeatedly injected with 100-400 μg of purified chimeric protein composed of the MuSK ectodomain

motility, which may lead to stomach pain and diarrhea. Other muscarinic effects are increased gastric acid secretion, hyperhidrosis, increased sweating, salivation, and lacrimation. The presence of daily muscarinic adverse effects correlates with the baseline inhibition of AChE activity in the blood (Punga, Sawada et al. 2008). The most common form of nonselective AChEI is pyridostigmine bromide (PB Mestinon®), which is especially effective at the onset of MG. In some patients, typically those with purely ocular weakness, this treatment may be sufficient to manage the fatigue. Overdose of AChEIs can cause a cholinergic crisis, which is characterized by increasing muscle weakness, which results in dysphagia and respiratory insufficiency in severe cases. The distinction between a cholinergic and a myasthenic crisis, the latter being caused by myasthenic weakness, is important for the medical treatment of the patient. These two conditions have different clinical reactions related to the initial intake of the drug. During incipient overdose in cholinergic crisis, symptoms of weakness increase shortly after ingestion and wane before the next dose, a situation opposite to that seen in myasthenic crisis. This typical pattern may not always be easy to detect; therefore, other markers are useful. For example, the EDs observed on motor nerve stimulation are sometimes observed in MG patients who are receiving high doses of PB and may signify impending cholinergic crisis (Punga and Stalberg 2009).

Recent reports imply that patients with EDs are more prone to have daily nicotinic side effects, including muscle fasciculations and fatigue as a possible sign of overtreatment (Punga, Sawada et al. 2008). In this study, elderly MG patients were more prone to develop cholinergic side effects, as well as EDs. Additionally, MuSK+ MG patients often have a negative edrophonium test and are also reported to clinically benefit less from pyridostigmine bromide (Evoli, Bianchi et al. 2008). Instead, MuSK+ patients may worsen or develop pronounced nicotinic side effects including muscle cramps and fasciculations in response to PB treatment (Evoli, Tonali et al. 2003; Punga, Flink et al. 2006). Based on these observations of hypersensitivity to increased amounts of acetylcholine in MuSK+ patients, the general guidelines do not recommend AChEIs as a form of treatment in this group of patients (Skeie, Apostolski et al. 2010).

EN101 is a selective AChEI, an antisense oligodeoxynucleotide that acts at the mRNA level and selectively reduces the production of the enzymatic isoform of stress-related "readthrough" (AChE-R) through destruction of AChE-R mRNA. This compound selectively lowers the levels of AChE-R in both blood and muscle, yet leaves the synaptic variant of AChE-S unaffected. EN101 treatment in rats with EAMG, in which daily oral or intravenous administration of EN101 reduced AChE in blood and muscle and improved survival, muscle strength and disease severity (Brenner, Hamra-Amitay et al. 2003). In this study, stabilization of the CMAP decrement on RNS and muscle strength over the entire course of treatment was also observed. Interestingly, a Phase 1b open-label trial with oral EN101 (Monarsen) was recently conducted in 16 MG patients who were receiving at least 180 mg ofpyridostigmine bromide daily (Argov, McKee et al. 2007). This study reported an overall clinical improvement in approximately 47% of patients, as well as an improvement in the swallowing time component. Further, the effects of EN101 lasted for greater than 24 hours, indicating the possibility of a reduction in multiple dosing through the use of antisense therapy. Further studies are needed to conclude whether EN101 may also have immunomodulatory effects through an effect on the immune cholinergic system and thus mediation of neuroimmune interactions.

motility, which may lead to stomach pain and diarrhea. Other muscarinic effects are increased gastric acid secretion, hyperhidrosis, increased sweating, salivation, and lacrimation. The presence of daily muscarinic adverse effects correlates with the baseline inhibition of AChE activity in the blood (Punga, Sawada et al. 2008). The most common form of nonselective AChEI is pyridostigmine bromide (PB Mestinon®), which is especially effective at the onset of MG. In some patients, typically those with purely ocular weakness, this treatment may be sufficient to manage the fatigue. Overdose of AChEIs can cause a cholinergic crisis, which is characterized by increasing muscle weakness, which results in dysphagia and respiratory insufficiency in severe cases. The distinction between a cholinergic and a myasthenic crisis, the latter being caused by myasthenic weakness, is important for the medical treatment of the patient. These two conditions have different clinical reactions related to the initial intake of the drug. During incipient overdose in cholinergic crisis, symptoms of weakness increase shortly after ingestion and wane before the next dose, a situation opposite to that seen in myasthenic crisis. This typical pattern may not always be easy to detect; therefore, other markers are useful. For example, the EDs observed on motor nerve stimulation are sometimes observed in MG patients who are receiving high doses of PB and may signify impending cholinergic crisis (Punga and

Recent reports imply that patients with EDs are more prone to have daily nicotinic side effects, including muscle fasciculations and fatigue as a possible sign of overtreatment (Punga, Sawada et al. 2008). In this study, elderly MG patients were more prone to develop cholinergic side effects, as well as EDs. Additionally, MuSK+ MG patients often have a negative edrophonium test and are also reported to clinically benefit less from pyridostigmine bromide (Evoli, Bianchi et al. 2008). Instead, MuSK+ patients may worsen or develop pronounced nicotinic side effects including muscle cramps and fasciculations in response to PB treatment (Evoli, Tonali et al. 2003; Punga, Flink et al. 2006). Based on these observations of hypersensitivity to increased amounts of acetylcholine in MuSK+ patients, the general guidelines do not recommend AChEIs as a form of treatment in this group of

EN101 is a selective AChEI, an antisense oligodeoxynucleotide that acts at the mRNA level and selectively reduces the production of the enzymatic isoform of stress-related "readthrough" (AChE-R) through destruction of AChE-R mRNA. This compound selectively lowers the levels of AChE-R in both blood and muscle, yet leaves the synaptic variant of AChE-S unaffected. EN101 treatment in rats with EAMG, in which daily oral or intravenous administration of EN101 reduced AChE in blood and muscle and improved survival, muscle strength and disease severity (Brenner, Hamra-Amitay et al. 2003). In this study, stabilization of the CMAP decrement on RNS and muscle strength over the entire course of treatment was also observed. Interestingly, a Phase 1b open-label trial with oral EN101 (Monarsen) was recently conducted in 16 MG patients who were receiving at least 180 mg ofpyridostigmine bromide daily (Argov, McKee et al. 2007). This study reported an overall clinical improvement in approximately 47% of patients, as well as an improvement in the swallowing time component. Further, the effects of EN101 lasted for greater than 24 hours, indicating the possibility of a reduction in multiple dosing through the use of antisense therapy. Further studies are needed to conclude whether EN101 may also have immunomodulatory effects through an effect on the immune cholinergic system and thus

Stalberg 2009).

patients (Skeie, Apostolski et al. 2010).

mediation of neuroimmune interactions.

#### **2.4 Establishment and phenotype of murine models of experimental autoimmune MG (EAMG)**

Naturally occurring MG in animals is present in canine MG. This canine form of MG has a natural course of clinical and immunological remission in the majority of dogs, even without initiation of immunosuppressive treatment (Shelton and Lindstrom 2001). Therefore, the use of the canine MG in determining the effect of immunosuppressant medication is limited. EAMG can be induced in rabbits, rats and mice either by passive transfer of human antibodies/sera or by immunization of the antigen in adjuvant. In 1978 it was first shown that active immunization with AChR from Torpedo (ray fish) electric organ, in rabbits leads to flaccid paralysis and an MG-like disease. Since in rabbits the disease has an acute action and shortly leads to death, these animals are not routinely used for EAMG studies. Some studies have used guinea pigs and even primates, but the current most widely used model is EAMG induced in female young mice or rats. The disease in rats has a short acute phase and a chronic phase, which mimics the human disease, excluding the involvement of the thymus (Meinl, Klinkert et al. 1991). In the mice only a chronic phase is induced. EAMG is more difficult to induce in mice and usually requires multiple immunizations and only a fraction of the mice develop disease symptoms. However, the murine model has an advantage of a plethora of mouse-specic reagents and knock-out mouse strains, which enable analyses that cannot be performed in rats. One further advantage of using the mouse is that its immune system is well characterized and the availability of inbred and genetically modified strains permit genetic analysis.

#### **2.4.1 AChR+ EAMG: Clinical phenotype**

Immunization with purified denatured *Torpedo* AChR α1, β1, γ, or δ subunits can cause EAMG, but it is inefficient compared to native AChR (Lindstrom, Einarson et al. 1978). Purified α1-subunit is the most potent, as might be expected since its sequence is the most conserved. Also, there are two α1 in an AChR which permit cross-linking by antibodies, which may utilize renaturation to provoke antibodies to the main immunogenic region (MIR) (Lindstrom, Luo et al. 2008). It is of outmost importance to use a mouse strain which possesses IL-6, since mice deficient in IL-6 have been shown to be resistant to the development of EAMG upon immunization (Deng, Goluszko et al. 2002). In the acute phase of active EAMG or EAMG passively transmitted with serum, binding of AChR antibody and its complement target the postsynaptic membrane for attack by macrophages (Lindstrom 2000). Mice immunized with AChR in Complete Freund´s Adjuvant (CFA) develop a flaccid paralysis (Figure 4a), a drooping of the head and tail and weakness primarily of the forelimbs, which may rapidly progress to respiratory failure (Berman and Patrick 1980). The myasthenic phenotype in the AChR+ EAMG mice has been reported to be restored in all cases to nearly normal after treatment with neostigmine intraperitoneally (Berman and Patrick 1980).

#### **2.4.2 MuSK+ EAMG: Clinical phenotype**

The antibodies in MuSK+ MG are directed against the extracellular domain of MuSK, which contains IgG-like domains. The first study which proved the pathogenic role of MuSKantibodies in animals was done in 2006, when New Zealand White rabbits were repeatedly injected with 100-400 μg of purified chimeric protein composed of the MuSK ectodomain

Myasthenia Gravis: New Insights into the Effect

fast-synapsing muscles (Xu, Jha et al. 2006).

**EAMG** 

(Figure 6c).

of MuSK Antibodies and Acetylcholinesterase Inhibitors 447

MuSK+ EAMG has been accomplished by i.p. injections of plasma (Benveniste, Jacobson et al. 2005) or purified IgG from MuSK+ MG patients (Cole, Reddel et al. 2008) in C57BL6 mice. Cole et al also reported that mice injected with IgG from two of three anti-MuSKpositive patients lost weight, developed myasthenic muscle weakness and a prominent

**2.4.3 Morphological changes at the neuromuscular junction and muscle atrophy in** 

In experimental mice injected with anti-MuSK-positive patient IgG, postsynaptic AChR staining is reduced to as little as 22% of that seen in control mice in both the tibial and diaphragm muscles (Cole, Reddel et al. 2008). The mice which develop MuSK+ EAMG following this injection show reduced apposition of the nerve terminal and the postsynaptic AChR cluster. In later studies, mice injected with MuSK+ patient IgG have also been found to have reductions in postsynaptic MuSK staining and this loss preceded the impairment of postsynaptic AChRs (Cole, Ghazanfari et al. 2010). In this study, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. The sera obtained from mice immunized with MuSK inhibit agrin-induced AChR aggregation in C2C12 myotubes (Jha, Xu et al. 2006). Further, disruption of neuromuscular junctions have been observed and it has been proposed that so called delayed synapsing muscles, including the diaphragm, tibalis posterior and sternomastoid are more severely affected than the so called

In a recent study, morphological changes presynaptically and postsynaptically in whole mount preparations of muscle fibers from the bulbar sternomastoid and omohyoid muscles were examined in control mice and in mice immunized with MuSK (Punga, Lin et al, 2011). In the control mice, the postsynaptic clusters (labeled with α-bungarotoxin) were closely aligned with the presynaptic motor nerve terminal (Figure 5a). However, in the MuSK+ EAMG mice, a severe disruption of the NMJ morphology was observed, especially prominent in the facial and neck muscles (Punga, Lin et al, 2011). The AChR clusters were fragmented and dispersed along the muscle fiber (Figure 5b). Except for disruption of the postsynaptic area with less clustering of AChRs, the nerve terminal area was found to be smaller than in the control mice (Figure 5b), suggesting a secondary presynaptic effect of reduced MuSK signalling. When comparing the morphology of the NMJs in the bulbar omohyoid muscle, the AChR clusters were arranged in the junctional folds in the control mice (Figure 6a). In parallell rounds of mice immunized with MuSK or AChR confocal images revelaled that the AChR clusters were severely fragmented in the MuSK+ mice (Figure 6b), whereas in the AChR+ EAMG mice a similar fragmentation of the AChRs was not observed, although the AChR clusters and the folding of the NMJs were simplified

Benveniste et al found increased protein levels of the muscle RING-finger protein 1 (MuRF-1), a marker for skeletal muscle atrophy, in the masseter muscle, but not in the gastrocnemius muscle, of mice injected with plasma from MuSK+ MG patients (Benveniste, Jacobson et al. 2005). Increased mRNA levels of MuRF-1 and atrogin-1 have also been found in the masseter of MuSK+ EAMG mice, but not in the limb muscles, further in support of a atrophy process localized to the facial muscles (Punga, Lin et al, 2011; Punga, unpublished data). There is a difference in the reaction to disturbed or exaggerated agrin-MuSK signalling in different skeletal muscles in the sense that muscles with high MuSK levels have

cervicothoracic hump, which may reflect cervical extensor weakness.

Fig. 4. A) One mouse with typical flaccid paralysis, especially of the limbs, after immunization with torpedo AChR. The clinical course in the AChR+ mice is progessive, however the response to intraperitoneal injection of neostigmine induces an improvement in clinical weakness. B) The phenotype of the mice immunized with rat MuSK shows a severe weakness of the neck extensor muscles, demonstrating a prominent cervical kyphosis and inability to raise the head. The mice are not as affected in the hind limbs as in the forelimbs and faciobulbar area, with difficulties ingesting food and water and subsequent significant weight loss. The MuSK+ mice do not show any clinical improvement from neostigmine injection, on the contrary muscle fasciculations and twitches are seen.

and the Fc region of human IgG1 (MuSK-Fc) (Shigemoto, Kubo et al. 2006). All of the 4 recipient rabbits manifested flaccid weakness after 3 or 4 repeated injections with MuSK-Fc. The actively induced murine model can be produced produced by injection of the extracellular domain of 10 g of recombinant rat MuSK (aa 21-491) (Jones, Moore et al. 1999) in a mix with CFA in emulsion (Jha, Xu et al. 2006). The prominent features of MuSK+ EAMG in mice resemble the human MuSK+MG phenotype with kyphosis, indicating weakness in the cervical extensor muscles and the thoracic paraspinal muscles (Figure 4b). Additionally, one prominent clinical feature of the MuSK+ mice is the weight loss, which is significant compared to the control mice (Punga, Lin et al, 2011). This finding further supports the involvement of faciobulbar weakness, preventing the MuSK+ EAMG mice to chew and swallow and therefore explaining the irreversible weight loss. Passively induced

Fig. 4. A) One mouse with typical flaccid paralysis, especially of the limbs, after

injection, on the contrary muscle fasciculations and twitches are seen.

immunization with torpedo AChR. The clinical course in the AChR+ mice is progessive, however the response to intraperitoneal injection of neostigmine induces an improvement in clinical weakness. B) The phenotype of the mice immunized with rat MuSK shows a severe weakness of the neck extensor muscles, demonstrating a prominent cervical kyphosis and inability to raise the head. The mice are not as affected in the hind limbs as in the forelimbs and faciobulbar area, with difficulties ingesting food and water and subsequent significant weight loss. The MuSK+ mice do not show any clinical improvement from neostigmine

and the Fc region of human IgG1 (MuSK-Fc) (Shigemoto, Kubo et al. 2006). All of the 4 recipient rabbits manifested flaccid weakness after 3 or 4 repeated injections with MuSK-Fc. The actively induced murine model can be produced produced by injection of the extracellular domain of 10 g of recombinant rat MuSK (aa 21-491) (Jones, Moore et al. 1999) in a mix with CFA in emulsion (Jha, Xu et al. 2006). The prominent features of MuSK+ EAMG in mice resemble the human MuSK+MG phenotype with kyphosis, indicating weakness in the cervical extensor muscles and the thoracic paraspinal muscles (Figure 4b). Additionally, one prominent clinical feature of the MuSK+ mice is the weight loss, which is significant compared to the control mice (Punga, Lin et al, 2011). This finding further supports the involvement of faciobulbar weakness, preventing the MuSK+ EAMG mice to chew and swallow and therefore explaining the irreversible weight loss. Passively induced MuSK+ EAMG has been accomplished by i.p. injections of plasma (Benveniste, Jacobson et al. 2005) or purified IgG from MuSK+ MG patients (Cole, Reddel et al. 2008) in C57BL6 mice. Cole et al also reported that mice injected with IgG from two of three anti-MuSKpositive patients lost weight, developed myasthenic muscle weakness and a prominent cervicothoracic hump, which may reflect cervical extensor weakness.

#### **2.4.3 Morphological changes at the neuromuscular junction and muscle atrophy in EAMG**

In experimental mice injected with anti-MuSK-positive patient IgG, postsynaptic AChR staining is reduced to as little as 22% of that seen in control mice in both the tibial and diaphragm muscles (Cole, Reddel et al. 2008). The mice which develop MuSK+ EAMG following this injection show reduced apposition of the nerve terminal and the postsynaptic AChR cluster. In later studies, mice injected with MuSK+ patient IgG have also been found to have reductions in postsynaptic MuSK staining and this loss preceded the impairment of postsynaptic AChRs (Cole, Ghazanfari et al. 2010). In this study, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. The sera obtained from mice immunized with MuSK inhibit agrin-induced AChR aggregation in C2C12 myotubes (Jha, Xu et al. 2006). Further, disruption of neuromuscular junctions have been observed and it has been proposed that so called delayed synapsing muscles, including the diaphragm, tibalis posterior and sternomastoid are more severely affected than the so called fast-synapsing muscles (Xu, Jha et al. 2006).

In a recent study, morphological changes presynaptically and postsynaptically in whole mount preparations of muscle fibers from the bulbar sternomastoid and omohyoid muscles were examined in control mice and in mice immunized with MuSK (Punga, Lin et al, 2011). In the control mice, the postsynaptic clusters (labeled with α-bungarotoxin) were closely aligned with the presynaptic motor nerve terminal (Figure 5a). However, in the MuSK+ EAMG mice, a severe disruption of the NMJ morphology was observed, especially prominent in the facial and neck muscles (Punga, Lin et al, 2011). The AChR clusters were fragmented and dispersed along the muscle fiber (Figure 5b). Except for disruption of the postsynaptic area with less clustering of AChRs, the nerve terminal area was found to be smaller than in the control mice (Figure 5b), suggesting a secondary presynaptic effect of reduced MuSK signalling. When comparing the morphology of the NMJs in the bulbar omohyoid muscle, the AChR clusters were arranged in the junctional folds in the control mice (Figure 6a). In parallell rounds of mice immunized with MuSK or AChR confocal images revelaled that the AChR clusters were severely fragmented in the MuSK+ mice (Figure 6b), whereas in the AChR+ EAMG mice a similar fragmentation of the AChRs was not observed, although the AChR clusters and the folding of the NMJs were simplified (Figure 6c).

Benveniste et al found increased protein levels of the muscle RING-finger protein 1 (MuRF-1), a marker for skeletal muscle atrophy, in the masseter muscle, but not in the gastrocnemius muscle, of mice injected with plasma from MuSK+ MG patients (Benveniste, Jacobson et al. 2005). Increased mRNA levels of MuRF-1 and atrogin-1 have also been found in the masseter of MuSK+ EAMG mice, but not in the limb muscles, further in support of a atrophy process localized to the facial muscles (Punga, Lin et al, 2011; Punga, unpublished data). There is a difference in the reaction to disturbed or exaggerated agrin-MuSK signalling in different skeletal muscles in the sense that muscles with high MuSK levels have

Myasthenia Gravis: New Insights into the Effect

**2.4.4 Cholinergic hyperactivity after AChEIs in EAMG** 

EAMG grade 2 and 3 (Berman and Patrick 1980).

and in worst cases also cholinergic crisis.

**3. Conclusion** 

unbeneficial effects.

**4. Acknowledgment** 

of MuSK Antibodies and Acetylcholinesterase Inhibitors 449

Evaluation of the response to AChEIs is usually performed by i.p. injection of a mix of neostigmine bromide (0.0375 mg/kg) and atropine sulfate (0.015 mg/kg) in mice with

In 3 MuSK+ EAMG mice, the opposite response to the common restoration of weakness in AChR+ EAMG was seen with more pronounced weakness which manifested itself as chin down even more along with nicotinic side effects including muscle fasciculations in the back- and limb muscles and abnormal twitches of the tail (Punga et al, unpublished observations). The observed fragmentation and dispersion of AChR clusters could explain why MuSK+ MG patients do not respond beneficially to AChEIs, since an increased acetylcholine level would not be able to induce a synchronous endplate potential due to the temporal dispersion of AChRs. Additionally, the reason for the cholinergic hyperactivity in MuSK+ MG, here also displayed in the MuSK+ EAMG mice, may be explained by the loss of MuSK at the NMJ, which in turn also diminishes the binding between MuSK-ColQ and AChE. This means that in the MuSK+ EAMG mice where MuSK antibodies disrupt the NMJ and reduce the amount of MuSK, the AChE is also down-regulated (Punga, Lin et al, 2011). Then, when exogenous AChEI is administered, a further blocking of AChE is taking place and consequently this mimics an overdose of AChEIs which causes the nicotinic side effects

In summary, studies in the recent years of the murine EAMG model provide further insights regarding the action of MuSK antibodies at the NMJ and give evidence for their pathogenetic role, especially in facial and bulbar muscles. The results of the MuSK antibody attack is fragmentation and dispersion of nicotinic AChRs, postsynaptic perturbation and a subsequent impaired neuromuscular transmission. Since MuSK+ MG is very focal in its clinical manifestations it is very important to examine the clinically weak muscles also neurophysiologically to confirm the diagnosis, and for morphological purpose when examining the NMJ pathophysiology. The findings of dispersion of AChRs may also indicate irreparable changes at the NMJ, explaining muscle atrophies in these patients. It is therefore of importance to identify MG patients as early as possible, and especially MuSK+ MG, since delayed treatment may result in muscle atrophies and even functional denervation due to long time of blocked neuromuscular transmission. Immunosuppressive treatment should always be the main medication in MG and AChEIs is not receommended as symptomatic treatment in MuSK+ patients due to the cholinergic hypsersensitivity and

I thank the group of Professor Markus A Rüegg in the department of Neurobiology/Pharmacology in Basel, Switzerland, for collaboration on the AChR+ and MuSK+ EAMG mouse model, especially professor Rüegg for the scientific discussions, Dr Shuo Lin for the work on the muscle morphology and Filippo Oliveri for production of recombinant rat MuSK. Professor emeritus Erik V Stålberg is acknowledged for guiding and supporting the initial MG studies, for teaching me SFEMG, for the scientific discussions and for the great friendship. I genuinely thank my family for their never-ending love and support.

Fig. 5. The effect of MuSK-antibodies at the neuromuscular junction (NMJ) in mice immunized with MuSK. Confocal microscopy images with 100x magnification of immunostained whole mount muscle fibers from the sternomastoid muscle. A) A normal NMJ, where α-bungarotoxin labels the AChR clusters (green) and antibodies against synaptophysin and neurofilament labels the nerve terminal (red). Note the close alignment between the motor nerve terminal and clustered postsynaptic AChRs. B) In MuSK+ MG, the presynaptic nerve terminal area is significantly smaller and the AChR clusters are fragmented and scattered along the muscle fiber. Scale bar is 10 μm.

Fig. 6. Immunolabeling of whole mount muscle fibers from the omohyoid muscle, where postsynaptic acetylcholine receptors (AChRs) are labelled with α-bungarotoxin (white). In the control mice (A) a normal pattern with postsynaptic AChR clusters are seen in the junctional folds, adjacent to the motor nerve. In MuSK+ EAMG mice (B), the AChRs are very faint, with a subsequent reduction in postsynaptic AChR cluster area. In AChR+ EAMG mice (C), the staining intensity of the α-bungarotoxin was less reduced than in the MuSK+ EAMG mice, however there is a disruption in AChR cluster morphology with simplification of the postsynaptic morphology and less folding. Scale bar is 10 µm.

an increased plasticity (Punga, Maj et al, 2011). n the contrary, low muscle-intrinsic MuSK levels render some muscles, such as the masseter, more vulnerable to the postsynaptic perturbation of MuSK antibodies with subsequent denervation and atrophy (Punga, Lin et al, 2011). This is hypothesized to play a role for the muscle selectivity also in MuSK+ MG and EAMG.

## **2.4.4 Cholinergic hyperactivity after AChEIs in EAMG**

Evaluation of the response to AChEIs is usually performed by i.p. injection of a mix of neostigmine bromide (0.0375 mg/kg) and atropine sulfate (0.015 mg/kg) in mice with EAMG grade 2 and 3 (Berman and Patrick 1980).

In 3 MuSK+ EAMG mice, the opposite response to the common restoration of weakness in AChR+ EAMG was seen with more pronounced weakness which manifested itself as chin down even more along with nicotinic side effects including muscle fasciculations in the back- and limb muscles and abnormal twitches of the tail (Punga et al, unpublished observations). The observed fragmentation and dispersion of AChR clusters could explain why MuSK+ MG patients do not respond beneficially to AChEIs, since an increased acetylcholine level would not be able to induce a synchronous endplate potential due to the temporal dispersion of AChRs. Additionally, the reason for the cholinergic hyperactivity in MuSK+ MG, here also displayed in the MuSK+ EAMG mice, may be explained by the loss of MuSK at the NMJ, which in turn also diminishes the binding between MuSK-ColQ and AChE. This means that in the MuSK+ EAMG mice where MuSK antibodies disrupt the NMJ and reduce the amount of MuSK, the AChE is also down-regulated (Punga, Lin et al, 2011). Then, when exogenous AChEI is administered, a further blocking of AChE is taking place and consequently this mimics an overdose of AChEIs which causes the nicotinic side effects and in worst cases also cholinergic crisis.
