cells

Review
Myasthenia Gravis: Pathogenic Effects of
Autoantibodies on Neuromuscular Architecture
Inga Koneczny 1, * and Ruth Herbst 2, *
1 Institute of Neurology, Medical University of Vienna, 1090 Vienna, Austria
2 Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna,
1090 Vienna, Austria
* Correspondence: inga.koneczny@meduniwien.ac.at (I.K.); ruth.herbst@meduniwien.ac.at (R.H.);
Tel.: +43-1-40400-55690 (I.K.); +43-1-40160-33276 (R.H.)




Received: 7 June 2019; Accepted: 28 June 2019; Published: 2 July 2019


Abstract: Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ).
Autoantibodies target key molecules at the NMJ, such as the nicotinic acetylcholine receptor (AChR),
muscle-specific kinase (MuSK), and low-density lipoprotein receptor-related protein 4 (Lrp4), that
lead by a range of different pathogenic mechanisms to altered tissue architecture and reduced densities
or functionality of AChRs, reduced neuromuscular transmission, and therefore a severe fatigable
skeletal muscle weakness. In this review, we give an overview of the history and clinical aspects of
MG, with a focus on the structure and function of myasthenic autoantigens at the NMJ and how they
are affected by the autoantibodies’ pathogenic mechanisms. Furthermore, we give a short overview
of the cells that are implicated in the production of the autoantibodies and briefly discuss diagnostic
challenges and treatment strategies.

Keywords: myasthenia gravis; neuromuscular junction; AChR; MuSK; Lrp4; Agrin; autoimmunity;
autoantibodies; history; immunopathogenesis

1. Clinical Aspects of Myasthenia Gravis

Myasthenia gravis (MG; from Greek: myos = muscle, asthenos = weakness, and Latin: gravis =
severe) is an autoimmune disease of the neuromuscular junction (NMJ), and is considered a classic
example of an antibody-mediated autoimmune disease. MG is a rare disorder, with an estimated
prevalence of 70–163 per million for acetylcholine receptor (AChR) MG, and around 1.9–2.9 per million
for muscle specific kinase (MuSK) MG [1–3]. Women are more often affected than men, with a female
to male ratio of 3:1 for AChR MG and a ratio of 9:1 for MuSK MG [4]. The characterizing symptom is
fatigable skeletal muscle weakness. Initial weakness often affects only ocular muscles, manifesting
as ptosis (hanging of the eyelid) or diplopia (double vision). Most patients progress to generalized
weakness, e.g., of limb muscles, within the first two years after disease onset. Other muscles that can
be involved are bulbar muscles, which are necessary for speaking (leading to dysarthria), chewing
and swallowing (causing dysphagia). Respiratory muscles can also be affected in up to 20% of cases
with AChR MG, leading to a myasthenic crisis where patients need to be ventilated artificially. AChR
MG can be further divided into several subgroups (see also Table 1): (1) Early-onset MG (EOMG)
defines patients with an age of onset below 50 years, and are predominantly females with an onset
in the 2nd and 3rd decade, frequently present with thymic hyperplasia; (2) late-onset MG (LOMG)
with a higher fraction of male patients, often with an additional presence of striational antibodies;
(3) thymoma-associated MG (TAMG), which affects approximately 10% of AChR MG patients; (4) ocular
MG (OMG) with predominantly ocular symptoms; and (5) fetal or neonatal forms in which maternal
autoantibodies pass the placenta. The passive transfer of antibodies against the adult AChR towards

Cells 2019, 8, 671; doi:10.3390/cells8070671 www.mdpi.com/journal/cells

Cells 2019, 8, 671 2 of 35

the fetus leads to a mild form of transient MG that passes weeks after birth. The symptoms include
hypotonia, impaired sucking, swallowing, and breathing. Patients go into remission after days to
months [5–7]. Antibodies against the fetal form of the AChR cause severe developmental defects and
are a cause of arthrogryposis multiplex congenita [8–11].

Table 1. Clinical subgroups of myasthenia gravis (modified from [4]).

MG Subgroup Clinical Characteristics Antigen Thymus Pathology IgG Subclass

Age of onset <50, sex ratio (F:M) Thymic
Early onset MG
3:1, genetic association with AChR lymphofollicular IgG1, IgG
(EOMG)
HLA-B8, A1, and DRw3 hyperplasia
Age of onset >50, sex ratio (F:M) Normal thymus
Late onset MG
1:1.5, genetic association with AChR (age-related thymus IgG1, IgG3
(LOMG)
HLA-A3, B7, and DRw2 atrophy)
Paraneoplastic MG,
Thymoma associated non-pathogenic antibodies against
AChR Thymoma IgG1, IgG3
MG (TAMG) striated muscle, titin, ryanodine
receptor
Restricted to ocular muscles, low Variable, no
Ocular MG (OMG) AChR IgG1, IgG3
AChR titres lymphoid follicles
Severe phenotype, respiratory,
and bulbar muscle weakness, sex
MuSK MG MuSK Normal thymus IgG4
ratio (F:M) up to 9:1, genetic
association with HLA-DR14-DQ5
Variable (normal,
Mild phenotype, sex ratio (F:M) thymoma, thymic
Lrp4 MG Lrp4 IgG1, IgG2
2.5:1 lymphofollicular
hyperplasia)
Generalized weakness, often also
additional AChR, MuSK, or Lrp4 No thymoma (few
Agrin MG Agrin N/A
antibodies, associated with severe studies)
weakness
Transient neonatal Mild symptoms, onset at birth,
AChR, MuSK maternal IgG
MG (TNMG) remission after days to months
Reduced fetal mobility,
Fetal myasthenia Fetal AChR γ
arthrogryposis congenital (AMC), maternal IgG
gravis subunit
very severe, risk of fetal death

A diagnosis of MG is primarily based on the observation of fluctuating skeletal muscle weakness.

Initially, the patients often present with ocular muscle weakness with ptosis and diplopia that is not
associated with sensory deficits. The diagnosis is then verified by (1) testing the serum for the presence
of known myasthenic autoantibodies (discussed in Section 7 in more detail), (2) electrophysiological
tests including single-fiber electromyography (SFEMG), and repetitive nerve stimulation (RNS), and
(3) inhibition of acetylcholinesterase (AChE) e.g., with pyridostigmine or ice pack test, which usually
improves the symptoms [12–15]. Although this is not a clinical review (excellent reviews focusing
on clinical aspects and diagnosis can be found here: [16–19]), we would like to briefly discuss the
electrophysiology of the NMJ in MG and its relevance for disease diagnosis.
ACh is released from synaptic vesicles upon the influx of Ca2+ into the nerve terminal. It is
estimated that each vesicle contains 5000–10,000 ACh molecules and this amount is referred to
as the “quantum” of neurotransmitter, which was first described by Fatt, Katz, and Miledi in
1952 [20,21]. A presynaptic action potential (AP) induces the release of about 20–200 quanta (in humans
around 20 quanta compared to 200 in frogs) [22], which give rise to a depolarization of the muscle
membrane, known as end-plate potential (EPP). The number of quanta released at a given NMJ during
neuromuscular transmission is referred to as the “quantal content” of the EPP. The release of a single
quantum results in small depolarizations (0.5–1 mV), the miniature EPPs (mEPPs). During a nerve
impulse, mEPPs sum up to generate an EPP, which triggers an AP in the muscle fiber when the peak
of the EPP reaches the critical threshold. In normal muscle, this happens with every nerve impulse.
Cells 2019, 8, 671 3 of 35

This reliable neurotransmission at the NMJ is the result of the ACh amount released being greater
than that required to excite a muscle fiber. The release of an excess of neurotransmitter makes sure
that neuromuscular transmission occurs under various physiological conditions. The term “safety
factor” has been used to describe this excess [23]. Different definitions of the safety factor have been
proposed and used (for more details see Wood & Slater [23]) but in general it defines the ratio of actual
nerve-induced transmitter release to the amount of transmitter required to trigger an AP. Factors that
affect the safety factor are the amount of released ACh, as well as the density of AChRs and Na+
channels at the postsynaptic membrane and the secondary synaptic folds [24]. The safety factor differs
between species and muscles. Human intercostal muscle was reported to have a safety factor of 2 [25].
Neuromuscular disorders such as MG negatively affect neuromuscular transmission. Amplitudes
of mEPPs and EPPs are greatly reduced, resulting in sub-threshold EPPs that fail to elicit muscle
fiber excitation [25]. Recordings of compound motor action potentials (CMAPs) measures the sum
of all APs triggered within particular muscle fibers and a failure of muscle fiber excitation can be
detected by a decline of the amplitude. In addition to reduced EPPs, a decreased level of voltage gated
sodium channels leads to an increased threshold to induce an AP in MG [26]. Upon RNS, this in turn
leads to a reduced response to motor neuron stimulus by muscle APs, which can be measured as
CMAP decrement [27]. In contrast, normal muscle shows no change in CMAP amplitude with RNS.
In AChR-MG, the mEPPs are reduced, but the EPPs are still higher than expected, due to an increased
quantal content [28]. This suggests a compensatory mechanism to overcome the reduced postsynaptic
depolarization [29,30]. This has, however, not been observed in MuSK MG patients and MuSK MG
animal models [31–35], as both the mEPP and EPP amplitudes were similarly reduced. Furthermore,
MuSK antibodies also elicit presynaptic effects by blocking a retrograde signaling to the motor neuron,
perhaps via disturbed anchoring of Lrp4.
MG (at least the subtypes with autoantibodies against AChR and MuSK) fulfils the Witebsky
postulates that determine whether a disease is of autoimmune origin [36,37]. These include: (1) The
presence of pathogenic autoantibodies or self-reactive T cells, (2) the reproduction of disease by the
passive transfer of patient antibodies or T-cells to experimental animals, and (3) circumstantial evidence
from clinical cues. Pathogenic mechanisms of serum or purified IgG from MG patients with AChR
or MuSK autoantibodies have been identified, the passive transfer of patient serum or purified IgG
has reproduced myasthenic weakness in experimental animals, and clinical cues for the autoimmune
nature of the disease is the improvement of the symptoms after immunosuppression and after the
removal of antibodies by plasmapheresis or B-cell depletion (discussed below in detail). MG with
autoantibodies to Lrp4, Agrin, or ColQ do not yet fulfill the Witebsky postulates, since the pathogenic
mechanisms of the antibodies are not entirely clear yet and, importantly, passive transfer animal
models are still lacking.
MG is known as a classic example of a type II hypersensitivity reaction (following the Gell and
Coombs classification). This means that IgG class autoantibodies target antigens expressed at the
cell-surface or in the extracellular matrix and cause organ-specific damage. Most interestingly, MG
can be caused by autoantibodies of different IgG subclasses. IgG1 and IgG3 subclass antibodies
target the AChR, IgG1 and IgG2 target Lrp4, and interestingly, autoantibodies of the atypical IgG4
subclass target MuSK [38]. AChR and MuSK MG are considered archetypes for their respective kind
of antibody-mediated autoimmune diseases. Pathogenic effects directly correlate to changes in NMJ
structure and function, which greatly broadened our understanding of the physiology of the healthy
NMJ, as well as the functioning of the immune system.
Patients with AChR MG frequently have thymic abnormalities, such as thymic hyperplasia (this
is frequently associated with an early onset of disease) or thymoma. It is thought that the thymus
plays a role in pathogenesis (discussed in Section 5.4). These patients respond well to the surgical
removal of the thymus gland [39,40]. Patients with MuSK MG do not show thymic abnormalities and
there does not seem to be a clinical benefit of thymectomy [41–43]. MuSK MG patients are also more
severely affected by more frequent myasthenic crises (up to 40% of cases) [44–47]. Lrp4 MG patients
Cells 2019, 8, 671 4 of 35

are predominantly female (female to male ratio of 2.5:1) and have usually a generalized mild muscular
weakness, unless additional antibodies to MuSK or AChR are present [48]. In an exceptional case,
Lrp4 MG with severe muscle weakness manifested during infection with a new sequence isotype of
Leptospira interrogans [49]. The role of the thymus in Lrp4 MG is unclear; one epidemiological study
suggests the occasional presence of thymic hyperplasia [48]. A pilot study with four Lrp4 MG thymi
showed no thymic abnormalities, but the positive clinical response in two of the patients still suggests
a potential benefit of thymectomy [50].

2. History of MG
In 1672, myasthenia gravis was described for the first time by Thomas Willis, a physician from
Oxford (Box 1) [51]. Further clinical studies in the 19th century by Samuel Wilks, Wilhelm Erb, and
Samuel Goldflam led to the description of a disease characterized by fatigable skeletal muscle weakness.
The very detailed reports contain a range of historically interesting observations. In one paragraph,
a patient was described to have fallen in a shallow pond while cleaning the dishes, and needing
rescue due to weakness. Treatment suggestions included taking tonics, specifically those of arsenic,
chinin, iodine, iron, and digitalis, as well as warm baths, cold showers, remedial exercises, and daily
“galvanisation” of the spinal cord with a current of 2–3 milliampere. The reports also contained very
astute clinical observations, such as the fluctuating nature of the weakness and the patterns of muscle
weakness [52–54]. The disease was thus named Erb–Goldflam disease, until 1895 when Friedrich
Jolly coined the term “myasthenia gravis pseudoparalytica” [55], and showed by repetitive nerve
stimulation that the innervated muscle had a decrease in response, thus proving that neuromuscular
transmission was affecting these patients. Ground-breaking work by Otto Loewi [56] demonstrated
the chemical nature of the synapse and in 1934, Henry Dale discovered that acetylcholine (ACh)
release is essential for neuromuscular transmission [57,58]. In 1934, Mary Walker discovered that an
injection of physostigmine and neostigmine, back then already known to prevent the break-down of
ACh, improved facial weakness in a MG patient. This led to the conclusion that MG is a “curare-like
poisoning of the motor end-organs” [59].

Box 1. Original quote by Thomas Willis (translated by the English poet Samuel Pordage in 1688).

“ . . . those labouring with a want of Spirits, who will exercise local motions, as well as they can, in the
morning are able to walk firmly, to fling their Arms hither and thither, or to take up any heavy thing; before
noon the stock of the Spirits being spent, which had flowed into the Muscles, they are scarce able to move Hand
or Foot. At this time I have under my charge an Honest Woman, who for many years hath been obnoxious to
this sort of spurious Palsie, not only in her Members, but also in her tongue; she for some time can speak freely
and readily enough, but after she has spoke long, or hastily, or eagerly, she is not able to speak a word, but she
becomes mute as a Fish, nor can she recover the use of her voice under an hour or two.”

The characteristic histological malformations of the NMJ were first described by Coers and
Desmedt in 1959 [60]. The idea that autoantibodies could damage tissues and cells belonging to their
own body was first formulated at the end of the 19th century, when Paul Ehrlich first hypothesized that
so-called “anti-toxins” could be directed against self-structures in the body (reviewed by [61]). In 1960,
Simpson and Nastuk hypothesized that MG might be an autoimmune disease [62,63]. This theory
was proven true in 1973, as Patrick and Lindstrom discovered (by accident no less) the pathogenic
effect of AChR antibodies, when they immunized rabbits with the aim to produce AChR antibodies.
The resulting antibodies cross-reacted with the rabbit’s own AChRs, and the animals developed a
MG-like muscle weakness. Soon afterwards, AChR antibodies were found in MG patients using
a radioimmunoprecipitation assay [64,65]. The passive transfer of patient antibodies to mice also
reproduced the disease, proving the pathogenicity of AChR autoantibodies [66].
These studies were made possible by the isolation of α-bungarotoxin, which is a snake toxin
that binds AChR irreversibly [67,68] and the isolation of AChRs from the electric organs of Torpedo
fish [69]. AChR antibodies were found to block the function of AChRs [70] and Fambrough and
Cells 2019, 8, 671 5 of 35

Drachman discovered a markedly reduced number of AChRs in the muscle biopsies of MG patients [71].
Furthermore, IgG and complement depositions were found at the NMJ of these patients [72,73]. AChR
MG and its three key pathogenic mechanisms are well studied today and are discussed in more detail
in Section 5.1. New extracellular autoantibody targets were only discovered in the new millennium,
and these are to date MuSK [74], Lrp4 [75–77], Agrin [78,79], and ColQ [80]. It seems that the discovery
of new antibody targets in MG is traditionally met with disbelief and doubt. Initial studies in MuSK
MG showed an absence of complement deposition and a loss of AChR in patient biopsies (taken
from intercostal muscles), which led to doubt that these antibodies are pathogenic. However, the
pathogenicity of MuSK autoantibodies has been demonstrated in experimental animals by active
immunization with MuSK antigen and by passive transfer of patient serum and IgG [33,81–83],
including the passive transfer of purified IgG4 from patient serum [84]. Similarly, Lrp4 and Agrin
antibodies as new pathogenic entities in MG are met with skepticism, since key experiments to prove
their pathogenicity are still lacking (as discussed in Section 5.3). To date, there are active immunization
models for Lrp4 MG [85–87] and Agrin MG [88] available, but unfortunately a passive transfer of patient
serum or IgG to experimental animals, which would prove the pathogenicity of these autoantibodies,
is still lacking. The pathogenic mechanisms have been investigated, but remain unclear. There are no
studies on the pathogenicity of ColQ antibodies, neither in vitro nor in vivo.
Autoantibodies against intracellular antigens were also discovered in MG patients, among these are
autoantibodies to three striational antigens, ryanodine receptor, titin and Kv1.4, and cortactin [89–92].
These antibodies are considered as non-pathogenic but have diagnostic value as biomarkers [38,93–96],
with striational antibodies being associated with the presence of a thymoma [97,98]. Despite these
advances, a population of seronegative MG patients with no known autoantibodies remains, and
further studies in antigen discovery are required to identify the pathogenic autoantibodies in this
population. An excellent review on the history of MG for further reading can be found here [99].

3. Antigens and Their Structure

3.1. AChRs
More than 60 years ago, Del Castillo and Katz discovered that AChRs were localized to the
NMJ [100]. Ultrastructural investigations of membrane preparations of the Torpedo californica electric
organ led to a description of the AChR as a hexagonal structure, proposed to be a ring composed
of six receptor subunits [101], which later turned out to be a pentamer composed of five receptor
subunits [102]. AChRs were purified and biochemically characterized by a great many of studies, again
mostly in the Torpedo electric organ (extensively reviewed in [103,104]). AChR genes were cloned
subsequently in the 1980s, again first from Torpedo and later from rodents and humans [105,106].
AChRs are member of a superfamily of neurotransmitter-gated ion channels, each comprised of
five homologous subunits arranged around a central ion channel. Seventeen AChR subunits have
been cloned and are subdivided into four classes. Class I–III represent neuronal AChR subunits and
class IV include muscle AChRs.
AChR subunits show 35–50% sequence homology in the N-terminal region, are glycosylated, and
share structural features (Figure 1A) [107]. Three highly conserved and mainly α-helical transmembrane
domains (M1–M3) encompass between the large extracellular domain and the cytoplasmic domain
(containing one α-helix). A fourth α-helical transmembrane domain (M4) crosses back to the
extracellular space creating a short (10–20 amino acids) extracellular sequence. The N-terminal
extracellular portion is organized around a β-sandwich core and the cytoplasmic domains of AChR β
and δ contain a regulated phosphotyrosine site, which is important for cytoskeletal anchorage [108,109].
Muscle AChRs have the composition α2 βδγ in embryonic muscle or α2 βδε in adult muscle [103].
AChR subunits are organized like barrel staves around the central ion channel in the order αγαδβ
(Figure 1B). ACh binding sites are present at the subunit interfaces: αγ -γ or ε and αδ -δ. The pore-lining
helices of the closed channel create a hydrophobic narrowing in the membrane, which acts as the gate.
Cells 2019, 8, 671 6 of 35

The transition to an open, active state is facilitated by the occupation of both agonist binding sites.
Cells 2019, 8, x 6 of 36
This triggers a reorganization of the ligand-binding domain that displaces the extracellular region
of the agonist
β subunit.bindingThesites.
β-subunit helices
This triggers tilt outward and
a reorganization of thedestabilize the arrangement
ligand-binding of pore-lining
domain that displaces the
helices, expanding the pore asymmetrically and enhancing its polarity in the
extracellular region of the β subunit. The β-subunit helices tilt outward and destabilize gate region [110–112].
the
This paired allosteric
arrangement transition helices,
of pore-lining changesexpanding
the structure fromasymmetrically
the pore a tense (closed)
andstate towarditsapolarity
enhancing more relaxed
in
(open)the
state.
gate region [110–112]. This paired allosteric transition changes the structure from a tense (closed)
state toward a more relaxed (open) state.

1. Schematic
FigureFigure presentation
1. Schematic of the
presentation AChR
of the AChR andanditsits
cross-linking
cross-linkingviaviathe
the MIR.
MIR. (A) Ribbondiagram
(A) Ribbon diagram of
a single
ofAChR subunit
α AChR
a single viewed viewed
α subunit in an orientation such that
in an orientation suchthe central
that axis ofaxis
the central theofpentamer is to is
the pentamer thetoside
(right).the side
The (right). The
membrane membraneas
is indicated is aindicated as a grey
grey rectangle. rectangle.
α-helices α-helices
M1–M4 areM1–M4 are membrane- and
membrane-spanning
spanning
form the and formgate
ion-permeable the ion-permeable
of the AChR. The gateMIR
of the(shown
AChR. in The MIR
red) (shown in
is located red) is located
between β2- andbetween
β3-strands.
β2- and
(B) Ribbon β3-strands.
diagram of the(B) Ribbon
whole diagram
receptor of thefrom
viewed whole thereceptor viewed
synaptic cleft. from
AChR theαsynaptic cleft.the
in blue with AChRMIR in
α in blue with the MIR in red, AChR β in magenta, AChR δ in cyan, and AChR γ in grey. The MIR is
red, AChR β in magenta, AChR δ in cyan, and AChR γ in grey. The MIR is shown in red. (C) Schematic
shown in red. (C) Schematic presentation of autoantibody-induced inter-molecular cross-linking via
presentation of autoantibody-induced inter-molecular cross-linking via the MIR. The molecular models
the MIR. The molecular models were drawn using the molecular coordinates from the density maps
were drawn using the molecular coordinates from the density maps deposited in the Protein Data Bank
deposited in the Protein Data Bank (PDB file accession number 3BG9 [112]) and the Web-based 3D
(PDB file accession number 3BG9 [112]) and the Web-based 3D Structure Viewer iCn3D.
Structure Viewer iCn3D.

The N-terminal region

The N-terminal of AChR
region of AChR α αrepresents
represents the
the main immunogenic
main immunogenic region
region (MIR)
(MIR) [113–115].
[113–115].
The MIRTheis MIR
a cluster
is a of overlapping
cluster epitopesepitopes
of overlapping rather than one than
rather singleone
epitope
singleand epitopes
epitope andare conformation
epitopes are
dependent. Half of
conformation all MG patients
dependent. Half of allgenerate
MG patientsautoantibodies against the
generate autoantibodies MIR.the
against The MIR
MIR. TheisMIR
angled
outwardis angled outward
from the centralfrom
axisthe central
of the AChR,axis which
of the prevents
AChR, which prevents the cross-linking
the cross-linking of twowithin
of two α subunits α
subunits within an AChR, and instead induces the cross-linking of adjacent AChRs (Figure
an AChR, and instead induces the cross-linking of adjacent AChRs (Figure 1C). MIR-specific antibodies 1C). MIR-
do notspecific antibodies
interfere with the do binding
not interfere with the
of ACh binding of ACh ortoα-bungarotoxin
or α-bungarotoxin the ACh binding to thesite,
AChnorbinding
do they
site, nor do they allosterically affect the AChR function.
allosterically affect the AChR function.
3.2. Agrin
3.2. Agrin
In the 1970s, Jack McMahan proposed that regulatory signals for forming and maintaining
In the 1970s, Jack McMahan proposed that regulatory signals for forming and maintaining synaptic
synaptic differentiation may be present within the synaptic extracellular matrix, or basal lamina of
differentiation mayhypothesis
the NMJ. This be presentwaswithin the synaptic
confirmed by a set extracellular
of regeneration matrix, or basal
experiments lamina
using of the NMJ.
the musculus
This hypothesis was
pectoralis of theconfirmed by a set
frog. McMahan andof colleagues
regeneration experiments
demonstrated thatusing
pre- the musculus
as well pectoralis of
as postsynaptic
the frog. McMahanisand
differentiation colleagues
controlled demonstrated
by factors localized in that
thepre- as well
synaptic basalaslamina
postsynaptic differentiation
[116–118]. The electric is
controlled
organby factors californica
of Torpedo localizedwasin the
usedsynaptic
to purifybasal lamina
an activity, [116–118].
which Thethe
stimulated electric organ
clustering of Torpedo
of AChRs
in cultured
californica was usedmuscle cells [119].
to purify This led
an activity, to thestimulated
which identification
theofclustering
a protein of
named
AChRs Agrin (from Greek
in cultured muscle
“ageirein”,
cells [119]. to gather
This led to thetogether) [120]. of a protein named Agrin (from Greek “ageirein”, to gather
identification
The Agrin gene encodes for a ~200 kDa protein with multiple protein binding domains (Figure
together) [120].
2A). The protein is expressed in many tissues and cell types, exists in different splice variants, and is
The Agrin gene encodes for a ~200 kDa protein with multiple protein binding domains (Figure 2A).
post-translationally modified by glycosylation [121]. The Agrin isoform, which is important for NMJ
The protein is expressed in many tissues and cell types, exists in different splice variants, and is
development, is made by motor neurons, hence being called neuronal Agrin, and contains a N-
post-translationally
terminal lamininmodified by glycosylation
binding domain required for[121]. The Agrin
its anchorage to isoform,
the basal which
lamina is important
and for19NMJ
an 8, 11, or
development, is made by motor neurons, hence being called neuronal Agrin, and contains a N-terminal
Cells 2019, 8, 671 7 of 35

laminin binding
Cells 2019, 8, x domain required for its anchorage to the basal lamina and an 8, 11, or 19 amino acid
7 of 36
insert in the B/Z splice site within the C-terminal portion of the protein, which is essential for AChR
amino acid
clustering insertThe
[122]. in the B/Z splice site within
carboxy-terminal thethe
half of C-terminal
protein portion
with threeof the protein, which(LG)
laminin-G-like is essential
domains
forfour
and AChR clustering
epidermal [122]. factor-
growth The carboxy-terminal
(EGF) like domainshalf of
is the protein
fully activewith
in AChRthreeclustering
laminin-G-like
when(LG) added
todomains
culturedand four cells
muscle epidermal
[123].growth factor-B/Z
The crucial (EGF) likesite
splice domains is fully
is located in active in AChR clustering
the N-terminus of the most
when added
C-terminal LGtodomain
cultured(LG3).
muscle LG3
cells [123].
adopts The crucial B/Zβsplice
a 14-strand site is
jellyroll located
fold that in
is the N-terminustoofthe
homologous
the most C-terminal LG domain (LG3). LG3 adopts a 14-strand β jellyroll fold
LNS (laminin, neurexin, and sex hormone-binding globulin-like) domain [124,125]. An 8 amino-acid that is homologous to
the LNS (laminin, neurexin, and sex hormone-binding globulin-like) domain
B/Z region was found to form a long loop, which represents the primary interface between Agrin [124,125]. An 8 amino-
acid B/Z region was found to form a long loop, which represents the primary interface between Agrin
and Lrp4. A lack of the B/Z loop or mutation of critical residues at the tip of the loop interferes
and Lrp4. A lack of the B/Z loop or mutation of critical residues at the tip of the loop interferes with
with Lrp4 binding [125]. Although Agrin LG3 is monomeric in solution, it dimerizes in a tetrameric
Lrp4 binding [125]. Although Agrin LG3 is monomeric in solution, it dimerizes2+in a tetrameric Agrin–
Agrin–Lrp4 complex. The Agrin–Agrin dimer interface lies close to a Ca -binding site in Agrin,
Lrp4 complex. The Agrin–Agrin dimer interface lies close to a Ca2+-binding site in Agrin, which may
which may enforce the local conformation of Agrin and foster dimer formation, thereby enhancing the
enforce the local conformation of Agrin and foster dimer formation, thereby enhancing the Agrin–
Agrin–Lrp4 interaction.
Lrp4 interaction.

Figure2.2.Structure
Figure Structure of
of Agrin,
Agrin,Lrp4,
Lrp4,and
andMuSK.
MuSK.(A)(A)
Neuronal Agrin
Neuronal is anchored
Agrin in thein
is anchored basal
the lamina via
basal lamina
its laminin-binding NtA-domain. The third LG domain contains the B/Z splice insert (in red),
via its laminin-binding NtA-domain. The third LG domain contains the B/Z splice insert (in red), which
forms a loop critical for Lrp4 binding. (B) Lrp4 is comprised of a large extracellular domain, one
which forms a loop critical for Lrp4 binding. (B) Lrp4 is comprised of a large extracellular domain,
transmembrane domain and a short cytoplasmic tail. The BP domains 1–3 and LDLa repeats 4–8 are
one transmembrane domain and a short cytoplasmic tail. The BP domains 1–3 and LDLa repeats 4–8
sufficient for binding to MuSK. The first BP domain is critical for Agrin binding with supporting
are sufficient for binding to MuSK. The first BP domain is critical for Agrin binding with supporting
function of LDLa repeats 6–8 (dotted line). (C) MuSK interacts with Lrp4 via the Ig1 domain. Ig1 is
function of LDLa repeats 6–8 (dotted line). (C) MuSK interacts with Lrp4 via the Ig1 domain. Ig1
also important for homodimerization. The kinase domain is phosphorylated and activated upon
is also important for homodimerization. The kinase domain is phosphorylated and activated upon
Agrin-induced binding of Lrp4. MuSK autoantibodies are predominantly directed against the Ig1
Agrin-induced binding of Lrp4. MuSK autoantibodies are predominantly directed against the Ig1
domain, less against the Ig2 domain (shown as red arrowhead), and occasionally against the CRD
domain, less against the Ig2 domain (shown as red arrowhead), and occasionally against the CRD
(dotted line). BP, β-propeller; CRD, cysteine-rich domain; LDLa, low-density lipoprotein receptor
(dotted line). BP, β-propeller; CRD, cysteine-rich domain; LDLa, low-density lipoprotein receptor
domain class A; KD, kinase domain; Lam EGF-like, laminin EGF-like; LG, laminin globular-like; NtA,
domain classAgrin.
N-terminal A; KD, kinase domain; Lam EGF-like, laminin EGF-like; LG, laminin globular-like; NtA,
N-terminal Agrin.
Cells 2019, 8, 671 8 of 35

3.3. Lrp4
It took more than 20 years to identify Lrp4 as the Agrin receptor at the NMJ. After the
discovery of MuSK, researchers very quickly realized that Agrin does not directly bind to MuSK [126].
The hypothetical molecule MASC (myotube-associated specificity component) was introduced being
either a muscle-specific modification, co-receptor, or co-ligand. A recessive screen for genes involved
in early mouse development identified two mutant alleles of Lrp4, a member of the low-density
lipoprotein receptor family [127]. These Lrp4−/− mice had a variety of developmental malformations
including digit and craniofacial defects, kidney hypoplasia, and agenesis, but in addition, the Lrp4
mutants died at birth due to respiratory failure.
Lrp4 is expressed in multiple tissues in the mouse, and is therefore important for the proper
development and morphogenesis of different organs such as limbs, lungs, and kidneys [127]. Lrp4 is a
single-pass transmembrane protein with a large extracellular domain that contains eight low-density
lipoprotein receptor domain class A (LDLa) repeats, two EGF-like domains, and four β-propeller
(BP) domains, each of which is fused together with an EGF-like domain (Figure 2B). Neuronal
Agrin binds predominantly to the first BP domain, although the last few LDLa repeats contribute to
Agrin-binding [128]. The crystal structure of the Agrin-Lrp4 complex showed that the central part
of BP1 represents a six-bladed β-propeller domain (conserved in the LDLR family) with two slightly
concave surfaces perpendicular to the β-propeller’s central axis [125]. One surface is enclosed by the
two flanking EGF-like domains, the second surface is involved in Agrin binding via the B/Z loop (as
described above). Crystal structures of close relatives of Lrp4 such as Lrp6 demonstrated the use
of the same surface for ligand binding. The Agrin–Lrp4 dimers further assemble into a tetrameric
complex via interfaces between Agrin and Lrp4 (no Lrp4 dimers were observed), which is required for
MuSK activation. The third BP domain as well as the fourth and fifth LDLa repeats are critical for
binding of Lrp4 to MuSK (via the first Ig-like domain, described below) [128]. The transmembrane and
intracellular domains are not required for MuSK activation by Agrin.

3.4. MuSK
MuSK was first identified as a novel Trk-related receptor tyrosine kinase (RTK) enriched in the
electric organ of Torpedo californica [129]. Later, it was isolated as a gene product that was highly
induced during muscle denervation in mice [130]. Its expression was initially thought to be restricted
to skeletal muscle, hence the name muscle-specific kinase, but MuSK was subsequently also found in
other tissues and cells [131–134].
MuSK presents a single member subfamily within the family of RTKs. MuSK contains three Ig-like
(Ig) domains and one cysteine-rich domain (CRD), also known as Frizzled (FZ)-like domain in the
extracellular region. The transmembrane region is followed by a short juxtamembrane region, a tyrosine
kinase domain, and an eight amino-acid C-terminal sequence. With the exception of mammals, all other
vertebrate species, including zebrafish and chicken, carry a Kringle domain between the CRD and the
single transmembrane helix. The Ig1 domain is crucial for MuSK homodimerization as well as Lrp4
binding [128,135]. In particular, a solvent-exposed region in Ig1 of MuSK (around Ile 96) is required
for MuSK to bind Lrp4 (Figure 2C). The function of the CRD is unresolved, as it is not required for
Agrin-induced MuSK activation, but was shown to mediate Wnt-induced MuSK activation and AChR
clustering in cultured muscle cells as well as muscle pre-patterning in zebrafish [136–138]. The MuSK
CRD binds Wnt proteins in vitro, but MuSK activation and AChR clustering by Wnt requires Lrp4
expression in muscle cells [137,139]. Similarly, genetic studies using mice expressing MuSK ∆CRD
produced conflicting results [140,141]. Likewise, the role of the Kringle domain in non-mammalian
MuSK is unclear. The kinase domain is composed of a N-terminal lobe with a five-stranded β sheet
and one α helix, and a predominantly α helical C-terminal lobe [142]. The N-terminal lobe contains
residues important for ATP binding and the C-terminal lobe includes the activation loop and the
catalytic loop. Mutation of tyrosines in the activation loop as well as the mutation of Lys608 in the
C-terminal lobe interfere with MuSK activation [136,143,144]. Tyr553 is located in the juxtamembrane
Cells 2019, 8, 671 9 of 35

region and represents a major phosphorylation site within a NPYX motif, which, upon phosphorylation
of Tyr553, recruits Dok-7 [142,145,146]. The mutation of Tyr553 and failure of autophosphorylation
inhibits MuSK activation and blocks Dok-7 binding [144]. The C-terminal sequence is disordered in
the crystal structure and is therefore accessible for protein binding, but functional studies have shown
that this region is dispensable for the MuSK function [136].

3.5. ColQ
ColQ is localized to the extracellular matrix and is crucial for the anchoring of AChE to the
NMJ [147]. It is a collagen in which the central collagenous domain is flanked by specific N- and
C-terminal peptides. Trimers of ColQ conjoin in a triple helix tail. There are three forms of AChE
generated by different splicing and posttranslational modifications: AChET, which is the main isoform
found in the muscle, brain, and other tissues that is a relevant splice variant at the NMJ, AChEH,
which is expressed in erythrocytes, and AChER, which is not expressed in humans. Tetramers of
catalytic AChET subunits associate with the collagen tails according to levels of AChE present [148].
At the NMJ, most of the enzyme is present as A12 forms, which have all three collagen tails filled with
tetramers. ColQ-deficient mutant mice lack clusters of AChE at the NMJ and congenital myasthenic
syndromes associated with AChE deficiency are caused by recessive mutations in ColQ [149,150]. More
recently it was demonstrated that MuSK interacts with ColQ, thereby conferring synaptic localization
of AChE [151].

4. Antigen Function during NMJ Development

4.1. The Agrin/Lrp4/MuSK Signaling Complex

Agrin, Lrp4 and MuSK represent a functional unit that constitutes the primary scaffold of the
developing NMJ that initiates post- as well as presynaptic differentiation. MuSK is activated by the
neuron-specific isoform of Agrin [126], a heparansulfate proteoglycan made by motor neurons and
deposited in the basal lamina of the synaptic cleft [152]. Agrin does not bind MuSK directly, but interacts
with Lrp4 [153,154]. Lrp4 and MuSK are pre-assembled in the absence of Agrin but the activation of
MuSK is only induced in the presence of Agrin, which forms a tetrameric complex with Lrp4 [125].
Agrin binding to Lrp4 increases MuSK-Lrp4 interaction by a yet unknown mechanism. Upon binding,
the Lrp4/MuSK tetramer presumably rearranges in a way that leads to dimerization and subsequent
autophosphorylation of MuSK. Recruitment of Dok-7 further enhances MuSK dimerization resulting
in the full activation of the MuSK kinase [145,155,156]. The downstream signaling cascade induces
the formation of the NMJ, including postsynaptic differentiation characterized by the accumulation
of AChRs at synaptic sites and presynaptic differentiation illustrated by the development of active
zones [157] (Figure 3A). In accordance with this model, mice lacking Agrin, MuSK, or Lrp4 fail to form
NMJs and consequently die at birth due to respiratory failure [127,158,159]. The AChR expression
is normal, but very few nerve contacts (in Agrin-/- mice) or no contacts (in MuSK-/- and Lrp4-/- mice)
are associated with AChRs or other postsynaptic specializations. In addition, motor axons keep on
growing and do not form arborized nerve terminals. Synaptic proteins like AChE, rapsyn, neuregulin-1,
and its receptors, the ErbBs, which are usually clustered at NMJs, are uniformly distributed in muscle
from mice lacking Agrin, MuSK, or Lrp4 [127,158,159]. Similarly, synapse-specific transcription in
nuclei underlying NMJs is absent in these mice since genes, such as the AChR subunits genes, which
are transcribed equally in synaptic and non-synaptic nuclei.
 develop MG. When a healthy, pregnant woman produces anti-γ subunit antibodies, they can be
transferred through the placenta and to the embryo. Here, the antibodies cause a fetal AChR
inactivation syndrome, which leads to the reduced movement of the fetus. This has dire
developmental consequences, the new-born children present with arthrogryposis multiplex
congenita, a developmental disorder hallmarked by multiple joint contractures and profound
Cells 2019, 8, 671 10 of 35
respiratory impairment that may lead to severe disabilities or fetal death [8–11,209].

Figure 3. Pathogenic mechanisms of MG autoantibodies at the NMJ. (A) At the healthy NMJ, neural
Figure 3. Pathogenic mechanisms of MG autoantibodies at the NMJ. (A) At the healthy NMJ, neural
Agrin stimulation induces interaction between Lrp4 and MuSK, leading to MuSK autophosphorylation
Agrin stimulation induces interaction between Lrp4 and MuSK, leading to MuSK
and activation and the phosphorylation and clustering of AChRs. A retrograde signal for presynaptic
autophosphorylation and activation and the phosphorylation and clustering of AChRs. A retrograde
development is sent via Lrp4. (B) MG antibodies of IgG1 and IgG3 subclass against AChR have three
signal for presynaptic development is sent via Lrp4. (B) MG antibodies of IgG1 and IgG3 subclass
pathogenic mechanisms: (1) Cross-linking and increased turnover of AChR lead to reduced AChR
against AChR have three pathogenic mechanisms: (1) Cross-linking and increased turnover of AChR
levels at the NMJ, (2) activation of the classical complement cascade, formation of the membrane attack
lead to reduced AChR levels at the NMJ, (2) activation of the classical complement cascade, formation
complex (MAC) and complement-mediated damage of the postsynaptic membrane, and (3) direct block
of the membrane attack complex (MAC) and complement-mediated damage of the postsynaptic
of function by preventing the binding of acetylcholine. (C) Bispecific IgG4 antibodies of IgG4 subclass
membrane, and (3) direct block of function by preventing the binding of acetylcholine. (C) Bispecific
against MuSK bind monovalently to MuSK and block Lrp4-MuSK interaction, thus interrupting the
IgG4 antibodies of IgG4
agrin-Lrp4-MuSK-Dok7 subclass
signaling against
axis MuSK reduced
and causing bind monovalently to MuSK
densities of AChR and
at the block Lrp4-MuSK
synapse. A further
interaction,
effect thus interrupting
is the disruption the agrin-Lrp4-MuSK-Dok7
of a retrograde signal from Lrp4 to the signaling axis and
motor neuron. causing
Divalent reduced
binding of
densities of AChR at the synapse. A further effect is the disruption of a retrograde signal
MuSK IgG leads to dimerization, autophosphorylation, and activation of MuSK independent of Agrin from Lrp4
to the motor
stimulation neuron.
and causesDivalent binding
the formation of MuSK
of ectopic IgG leads
AChR to dimerization,
clusters. Created withautophosphorylation,
BioRender. and
activation of MuSK independent of Agrin stimulation and causes the formation of ectopic AChR
4.2. Prepatterning and NMJ
clusters. Created Formation
with BioRender.

Aneural AChR clusters were first described 20 years ago [160–162]. This so-called pre-patterning of
5.2. Antibodies to MuSK
the muscle depends on MuSK and Lrp4 and happens prior to the arrival of motor neurons. In contrast,
MuSK antibodies
Agrin mutant mice develop behave fundamentally
aneural different
AChR clusters, fromare
but these AChR antibodies.
dispersed upon They belong [160].
innervation to the
ItIgG4
was subclass
therefore[210,211],
postulated which
that does not activate theMuSK
Agrin-independent classical complement
activation system
induces or immune and
pre-patterning cells,
duenerve-derived
that to structural differences
Agrin acts in as the Fc regionof(see
a stabilizer Box 2). Furthermore,
pre-existing AChR clusters.IgG4Agrin
can undergo Fab-arm
is anchored to
exchange,
the which
basal lamina is the exchange
through of IgG4laminin
the N-terminal half-molecules and the and
binding domain generation ofLrp4,
binds to bi-specific
whichantibodies
induces
(reviewed
the in detail
dimerization andhere [212]). This of
autoactivation hasMuSK.
also been
Thedemonstrated
MuSK signaling for MuSK autoantibodies
then induces [213].asIn
pre- as well
fact, an estimate
postsynaptic of 99% of Upon
differentiation. IgG4 and MuSK IgG4
innervation, MuSKis therefore bi-specific,
expression is rapidlyor functionally monovalent,
downregulated and MuSK
which is
protein means they cannot
specifically cross-link
localized to thean antigen of the
postsynaptic samemembrane
muscle kind, excluding
[130].antigenic modulation
In addition, tyrosineas
phosphates such as Shp2 counterbalance MuSK activation [163]. These regulatory mechanisms and
the spatially restricted activation by Agrin are thought to ensure localized signaling at synaptic sites.

4.3. NMJ Maturation and Maintenance

For precise and refined muscle contraction, NMJ maturation plays a critical role. The size of the
NMJ increases and its morphology and molecular composition changes to warrant a rapid and efficient
Cells 2019, 8, 671 11 of 35

neurotransmission [157]. At the pre-synapse, multiple inputs are eliminated to one nerve terminal per
postsynaptic site, and a number of synaptic vesicles and active zones increase. The muscle membrane
invaginates to form post-junctional folds, proteins become compartmentalized along these folds,
the AChR subunit composition changes from α2 βδγ to α2 βδε, and AChRs become greatly enriched at
the crest of postsynaptic folds (more than 10,000 receptors/µm2 ). Morphology and high AChR density
are maintained throughout most of an organism’s adult life. The molecular mechanisms that regulate
maturation and maintenance are not well understood. However, lasting Agrin/Lrp4/MuSK signaling
is also required for NMJ maturation and maintenance. The elimination of Agrin, MuSK, or Lrp4 in
adult mice leads to NMJ disassembly [164–167]. Proteolytic cleavage of Agrin was shown to influence
NMJ remodeling and maturation [168]. In vitro, it was shown that the ECM-induced topologically
complex AChR cluster depends both on MuSK kinase activity and the integrity of the extracellular
domain of MuSK [169]. Furthermore, mutations in MUSK, AGRN, or LRP4 cause congenital myasthenic
syndromes associated with NMJ fragmentation, loss of AChRs, and impaired neurotransmission and
muscle function [170,171].

4.4. NMJ Aging

During aging, muscle fiber number, size, and types change and become infiltrated with adipocytes
and connective tissue [172]. Associated with these alterations, NMJ fragmentation, reduced AChR
density, and denervation have been observed [173]. In aged human subjects, motor neurons are lost,
whereas in mice the presynaptic phenotype represents more variables (depending on the muscle
type) [172–175]. Further controversial questions are whether changes in NMJ morphology are associated
with a decline in neurotransmission and whether NMJ degeneration contributes to aging or results from
it [176]. Given the changes in NMJ morphology, in particular fragmentation and the loss of AChRs,
it is tempting to speculate that these changes might contribute to LOMG. The mechanisms that lead to
NMJ decline in aging organisms are not well understood. As described above, NMJ fragmentation
and disassembly was observed in adult mice lacking Agrin or Lrp4 [164,167]. Furthermore, enhanced
proteolytic cleavage of Agrin destabilizes NMJs and induces denervation [177].

5. Pathogenic Effects and Origin of Autoantibodies

5.1. AChR Antibodies

Approximately 85% of patients have autoantibodies against the AChR. These antibodies mainly
belong to the IgG1 and IgG3 subclass (see additional information in Box 2) [178–180], and many
recognize the MIR [181–184]. The AChR antibodies induce pathogenicity by three main mechanisms
(Figure 3B): (1) Activation of the classical complement cascade [185]. Here, C1q binds to the Fc
region of the AChR antibodies, followed by the breakdown of C3 into C3a and C3b, which, as
downstream products of the complement cascade, initiate the formation of the membrane attack
complex (MAC). C3 and patient IgG, as well as MAC, can be found colocalized with AChR at the NMJ
of patients [72,73,186,187]. The patient serum is depleted of early complement components C3 and
C4 while terminal complement components are increased [188,189]. The complement attack leads to
membrane lysis and severe damage of the postsynaptic apparatus, including simplified junctional
folds, debris in the intrasynaptic space, and a loss of AChRs, as well as voltage-gated sodium channels
from the synapse [186,190,191]. Furthermore, patient serum induced a complement-mediated lysis of
rat myotubes in vitro. [192]. Complement mediated damage is a major cause of the pathogenicity of
AChR antibodies, which can be demonstrated in animal models of MG. Soluble complement receptors
improved symptoms in a passive transfer rat model [193], the inhibition of the complement cascade by
suppressing the expression of C2 or C5 significantly improved clinical symptoms in a mouse (C2) [194]
and rat (C5) [195] model of MG as did the linking of a complement regulator to the motor endplate via
coupling to a recombinant AChR antibody [196]. Inhibitors of late complement component C5 are by
now in clinical use (Section 6.1). (2) Binding and cross-linking of AChRs by antibodies (many of which
Cells 2019, 8, 671 12 of 35

target the MIR [197]) may lead to endocytosis and therefore a loss of AChR densities. The normal
half-life of AChRs at the mature synapse is eight to 11 days [198]. Patient IgG cause an increased
turnover and degradation of AChRs in skeletal muscle cells [199–203]. The mechanism requires the
divalent binding of an antibody, as Fab fragments (derived from patient IgG) were unable to induce
endocytosis, unless a secondary antibody was used to cross-link the Fabs [201]. (3) The direct inhibition
of the function of the AChRs by preventing the binding of ACh or blocking the channel [70,204–207].

Box 2. Antibody structure and function.

Cells 2019, 8, x 11 of 37

MG antibodies belong to the IgG class, which can also be divided into the Fc and the
Fab region. Historically these names derive from the two breakdown products
generated when IgG is digested with papain. The “Fab” part, for “Fragment, antigen
binding”, is the part of the antibody that contains the variable regions of the heavy
and light chains (VL , VH ), which binds the antigen, as well as the first constant
domain (CH 1, CL ). Fc stands for “fragment, crystallized”, and comprises most of the
constant region of the two heavy chains of an antibody (CH 2-CH 3), and determines
the antibody class and subclass, whether the antibody is membrane bound or soluble,
and the effector mechanisms of the antibody. These may include the activation of the
classical complement cascade, antibody-dependent cellular cytotoxicity, opsonization,
blocking enzymes or receptors or formation of immune complexes. Many
autoantibodies belong to the IgG1 and IgG3 subclasses that bind C1q and activate the
classical complement pathway, and that also bind to activating Fcγ receptors on
immune cells leading to their activation. IgG1, 2 and 3 also cross-link antigens,
forming either immune complexes with soluble antigen, or causing endocytosis of
transmembrane proteins. In recent years a range of autoantibodies associated with
IgG4 subclass antibodies were discovered. Due to structural differences in the Fc
region, IgG4 does not bind C1q or activating Fcγ receptors and is bi-specific, and
therefore has an “anti-inflammatory” effect thought to counteract overshooting
immune responses after chronic antigen exposure. IgG4 autoantibodies are usually
pathogenic by a blocking mechanism that affects enzyme or receptor function or
disrupts protein-protein interaction. Figure created with BioRender.

The loss ofwhich

antibodies, AChRs bydemonstrated
can be (1) and (2)inhas animalconsequences for neuromuscular
models of MG. Soluble complement receptors transmission as reduced
AChR improved symptoms
densities leadintoa passive
reduced transfer rat model [193],
sensitivity the inhibition of the
for acetylcholine, complement
reduced cascadeand reduced EPPs, and
mEPPs,
by suppressing the expression of C2 or C5 significantly improved clinical symptoms in a mouse (C2)
an overall reduction of the safety factor (see Section 1). Interestingly, it was found that patients with
[194] and rat (C5) [195] model of MG as did the linking of complement regulator to the motor endplate
AChR antibodies
via coupling show anAChR
to a recombinant increased
antibody quantal content
[196]. Inhibitors of late[28], whichcomponent
complement suggests C5 a
areretrograde signal from
the muscle to the nerve in an attempt to compensate for reduced AChR levelsof by an increase of ACh
by now in clinical use (Chapter 6.1). (2) Binding and cross-linking of AChRs by antibodies (many
which target the MIR [197]) may lead to endocytosis and therefore a loss of AChR densities. The
release,
normalthough it AChRs
half-life of remains unclear
at the by which
mature synapse retrograde
is eight signal
to eleven days [198].this occurs
Patient [29,30].
IgG cause an
While turnover
increased most AChR antibodies
and degradation target
of AChRs in the AChR
skeletal muscle subunit,
α cells there
[199–203]. Theare individuals with antibodies
mechanism
requires
against thedivalent
fetal γbinding of antibody,
subunit. The γ as Fab fragments
subunit is only (derived from patient
expressed during IgG) were
the unable
first to
30 weeks of life, after which
induce endocytosis, unless a secondary antibody was used to cross-link the Fabs [201]. (3) Direct
it isinhibition
exchanged by the adult ε subunit with the exception of the extraocular
of the function of the AChRs by preventing binding of ACh or blocking of the channel muscle, where AChR γ
subunit expression is maintained [208]. Therefore, adults with these antibodies do not develop MG.
[70,204–207].
When a healthy, pregnant woman produces anti-γ subunit antibodies, they can be transferred through
the placenta and to the embryo. Here, the antibodies cause a fetal AChR inactivation syndrome, which
leads to the reduced movement of the fetus. This has dire developmental consequences, the new-born
children present with arthrogryposis multiplex congenita, a developmental disorder hallmarked by
multiple joint contractures and profound respiratory impairment that may lead to severe disabilities or
fetal death [8–11,209].

5.2. Antibodies to MuSK

MuSK antibodies behave fundamentally different from AChR antibodies. They belong to the IgG4
subclass [210,211], which does not activate the classical complement system or immune cells, due to
structural differences in the Fc region (see Box 2). Furthermore, IgG4 can undergo Fab-arm exchange,
which is the exchange of IgG4 half-molecules and the generation of bi-specific antibodies (reviewed in
detail here [212]). This has also been demonstrated for MuSK autoantibodies [213]. In fact, an estimate
Cells 2019, 8, 671 13 of 35

of 99% of IgG4 and MuSK IgG4 is therefore bi-specific, or functionally monovalent, which means
they cannot cross-link an antigen of the same kind, excluding antigenic modulation as pathogenic
mechanism. Epitope mapping studies [74,210,214–216] suggest that MuSK antibodies bind to the first
two Ig-like domains, and in a subset of patients also to the CRD domain, but the first Ig-like domain
is considered as the predominant target (Figure 3C). This is interesting because this domain covers
the region around Ile96 [128], which is required for the interaction of MuSK with Lrp4. And indeed,
it was found that MuSK antibodies of the IgG4 subclass exclusively led to a block of Lrp4-MuSK
interaction [211,217]. This in turn caused a reduction of MuSK phosphorylation [217]. Moreover,
purified IgG4 from MuSK MG patients, as well as monovalent Fab fragments derived from patient
IgG, led to a reduction of newly formed Agrin-induced AChR clusters [211]. Taken together, this
demonstrates that the MuSK antibodies interrupt the Agrin-Lrp4-MuSK-Dok-7 signaling axis, causing
reduced densities of AChRs at the synapse and therefore defects in neuromuscular transmission.
Notably, IgG1–3 antibodies do not have the same effect on MuSK-Lrp4 interaction, but were
still able to reduce Agrin-induced clustering of AChR in C2C12 myotubes [211], suggesting an
additional pathogenic mechanism. One effect could be a block of MuSK dimerization, as the first Ig-like
domain also contains a hydrophobic patch around Leu38, which is essential for MuSK dimerization
and activation [135]. However, one study did not find reduced MuSK dimerization by patient
antibodies [217], and divalent commercial antibodies against MuSK were found to rather induce MuSK
dimerization and activation by cross-linking [218]. A recent study showed that, interestingly, the effect
depends on antibody valency [219]. Recombinant divalent MuSK antibodies, derived from MuSK MG
patient B-cells, led to the phosphorylation of MuSK and induced Agrin-independent AChR clustering,
presumably by dimerization, while monovalent Fab fragments from the same antibody clone reduced
MuSK phosphorylation and AChR clustering. This suggests that overall there may be (at least) two
competing mechanisms at work: 1) MuSK IgG4 (monovalent) blocks Lrp4-MuSK interaction and
MuSK activation, while 2) divalent MuSK IgG1–3 antibodies induce ectopic, Agrin-independent MuSK
dimerization and activation thereby recruiting AChRs to extrasynaptic sites (Figure 3C).
Patient-derived MuSK antibodies, as well as Fab fragments, are also able to disperse pre-existing
AChR clusters that were induced by overexpression of Dok-7 [211,220]. It is unclear how Dok-7-induced
AChR clusters can be affected, since Agrin, and probably also Lrp4, are not involved in the formation of
these AChR clusters. Possible mechanisms could be a disruption of MuSK-Dok-7 interaction, perhaps
induced by conformational changes in MuSK, or a disruption of Dok-7-induced MuSK dimers, either
by preventing MuSK-MuSK interaction itself, or by inducing new MuSK dimers by cross-linking, and
in this process also disrupting pre-existing MuSK dimers.
Interestingly, MuSK antibodies also affect the retrograde signaling to the motor neuron. In patients
and animal models, presynaptic abnormalities were found, as well as a reduced quantal release of
AChE [31–35]. It is unclear which retrograde signaling pathway could be modified by the antibodies,
but it is suspected that the disruption of Lrp4-MuSK interaction may play a role, perhaps by a loss of
anchoring of Lrp4 to the synapse, as Lrp4 contributes to a retrograde signal from the muscle to the
motor neuron [221,222].

5.3. Antibodies to Lrp4, Agrin, and ColQ

Lrp4 itself is also a target for autoantibodies [48,75–77]. It is not clear yet whether Lrp4, Agrin,
and ColQ antibodies are pathogenic, since relevant experiments are still missing. The pathogenic
mechanisms of Lrp4 are under investigation, but one might speculate that Lrp4 autoantibodies could
interfere either with binding to Agrin or with binding to MuSK. Indeed, a recent study showed a
disruption of the Lrp4-Agrin interaction by patient serum using immunoprecipitation of tagged Agrin
and co-immunoprecipitation and quantification of Lrp4 [76], and a second study showed a disruption
of Agrin binding to immobilized Lrp4 on ELISA plates by Lrp4 MG patient serum [75]. In line with a
non-functional Agrin-Lrp4-MuSK signaling axis, Lrp4 MG patient serum also reduced Agrin-induced
AChR clustering in C2C12 mouse myotubes [75,77,85]. Reduced Agrin-induced AChR clustering and
Cells 2019, 8, 671 14 of 35

reduced Agrin-induced MuSK phosphorylation could be reproduced in active immunization mouse

models [85,86].
Since the antibodies are of an IgG1/2 subclass, further mechanisms might be possible. They might
activate the complement system, and Lrp4 antibodies from immunized mice or rabbits do activate
complement [86,87], but this mechanism has not been demonstrated in MG patients yet. Active
immunization of mice with extracellular Lrp4 led to the production of complement-fixing mouse IgG2
antibodies to Lrp4 (equivalent to human IgG1). The mice had IgG and complement C3 deposits at
the NMJ, and cells derived from the lymph nodes produced pro-inflammatory cytokines in response
to immunogen stimulation, similar to an active immunization model of AChR MG [87]. These data
require validation by studies with human patient serum, since the active immunization of the mice
may not reflect the physiological situation in the patient. The patient antibodies could theoretically also
cross-link Lrp4 and cause endocytosis and degradation of autoantibodies, similar to AChR antibodies.
Fragmented AChR clusters, distorted axon terminals, and an abnormal NMJ ultrastructure have been
shown in an active immunization mouse model, but not yet in experiments with patient antibodies [86].
Active immunization models also showed postsynaptic abnormalities and reduced mEPPs, as well
as a CMAP decrement similar to MG patients, myasthenic weakness, and interestingly, presynaptic
changes [85–87]. This (taken together with presynaptic changes in MuSK MG) is further evidence that
Agrin-Lrp4-MuSK interaction is relevant for retrograde signaling to the motor neuron, though it is
not understood how. Interestingly, Lrp4 antibodies from an active immunization mouse model also
affected Agrin-independent AChR clustering, which is an observation that was also made with patient
derived MuSK antibodies [85,211] and suggests that there may be further mechanisms at work in the
development and stabilization of AChR clusters, perhaps by yet unknown binding partners of MuSK
and Lrp4 that act independent of Agrin.
There are only a few studies on Agrin autoantibodies, but in one study, patient serum was
found to inhibit Agrin-induced MuSK phosphorylation in C2C12 myotubes, suggesting a blocking
of Lrp4-Agrin interaction [78], and in line with these findings the active immunization of mice with
neural (but not muscle) Agrin-induced myasthenic weakness and fragmented synapses [88]. These
results suggest that the autoantibodies may be directed against the B/Z loop located in LG3, which
would explain the observed lack of MuSK activation due to an impairment of Agrin-LRP4 interaction.
The pathogenic mechanisms of antibodies against ColQ have not been studied yet. Importantly, to
prove the pathogenicity of Lrp4, Agrin, and ColQ autoantibodies, it is necessary to passively transfer
patient derived serum or IgG to experimental animals and to show that myasthenic weakness could be
reproduced in the animal model.

5.4. Autoantibody Production in MG

The identification of the autoantibodies’ source is essential as antibody-producing cells are key
therapeutic targets. Antibodies are produced by antigen-specific B-cell subsets, that is, short-lived
plasmablasts and long-lived plasma cells. The B-cells also require T-cell help for activation, and thus
MG is also classified as a B-cell mediated, T-cell dependent autoimmune disease. Nevertheless, our
understanding of the cellular immunology in MG is still incomplete. Every B-cell undergoes V(D)J
recombination that stochastically generates one type of B-cell receptor (BCR)/antibody, resulting in up
to 1011 unique B-cell clones, of which some will recognize self-antigens and may cause autoimmunity.
Therefore, during maturation, B-cells undergo negative selection for self-antigens in the bone marrow
at two “tolerance checkpoints” to remove poly- and autoreactive B-cells by clonal deletion, induction
of anergy, or receptor editing [223–225]. The BCR repertoire in MG was found to be disturbed in a
small number of patients [226], suggesting that aberrant B-cell receptor editing might contribute to
the formation of autoreactive B-cells. Furthermore, it was shown that in MG patients, defects in the
negative selection lead to an escape of autoreactive B-cells from checkpoint controls [227]. In line
with this observation, patients with MG have a high risk in developing further autoimmune diseases
Cells 2019, 8, 671 15 of 35

(poly-autoimmunity) [228]. At the same time, the patients also have reduced numbers of B10 cells,
which is a subset of regulatory B-cells (Bregs) [229,230].
Nevertheless, B-cells depend on activation by T-cells that recognize the same antigen, and
the negative selection of autoreactive T-cells is considered as a more stringent process. T-cells
undergo a negative selection for self-antigens in the thymus, specifically in the thymic medulla. Here,
the autoimmune regulator (AIRE) transcription factor, expression of which itself is regulated by
estrogen [231,232], induces the ectopic expression of tissue-restricted self-antigens in stromal cells
such as medullary thymic epithelial cells (mTECs). These antigens are then presented by mTECs
directly or after antigen “handover” by antigen-presenting cells to the developing T-cells [233,234].
Upon a strong binding of T-cells via the T-cell receptor to self-antigens, the cells are removed from
the repertoire, by clonal deletion, induction of anergy, or by a transformation to regulatory T-cells
(Tregs) that have an immune regulatory and anti-inflammatory function. It is thought that thymic
abnormalities such as follicular hyperplasia or thymoma contribute to defects in central tolerance
mechanisms and the escape of AChR specific T-cells [235]. These include: (1) a defective AIRE
expression in mTECs [236,237]; (2) the downregulation of AIRE transcription by estrogen, which
may explain the female predominance [232,238]; (3) an absence of thymic medulla and cells involved
in the negative selection in thymoma [239–242]; (4) hyperplastic or neoplastic mTECs presenting
autoantigen [243,244] or downregulating MHC II expression [239,245,246], and (5) aberrant expression
of pro-inflammatory cytokines (reviewed by [235,247]).
These mechanisms may contribute to the escape of AChR-specific T helper (Th) cells (CD4+ cells)
from tolerance that then activate AChR-specific B-cells to produce autoantibodies. Thymic myoid
cells express muscle antigens, among these is the whole intact AChR. Antibody and complement
attack on thymic myoid cells may lead to the formation of antigen-antibody complexes that stimulate
antigen-presenting cells and lead to the formation of ectopic germinal centers (GC) in the thymus [248–
252]. Ectopic GCs in the EOMG thymus promote antigen-driven affinity maturation and differentiation
to autoreactive memory B-cells and plasma cells [253]. B-cells in the thymus of MG patients with
hyperplasia showed an increase in memory B-cells and potentially autoreactive B-cells associated with
autoimmunity [242,254]. B-cells in the MG thymus were found to be activated [255], clonally expanded,
and also phenotypically more different compared to B-cells in healthy control thymi [256,257]. Ectopic
GCs in the thymus contained AChR-specific long-lived plasma cells [258–260] that spontaneously
produced high levels of AChR antibodies in vitro [260–266]. Plasmablast levels were also elevated in
the blood of patients with AChR MG, though it was not tested whether these produced myasthenic
antibodies [242]. Thymic cells derived from patients with Lrp4 MG also produced high levels of IgG,
but Lrp4-specific antibodies could not be detected [50].
AChR-specific Th cells are important in MG to induce the production of the pathogenic AChR
antibodies [267,268]. The altered thymic function causes an increase in the release of autoreactive CD4+
and CD8+ T-cells and leads to altered T-cell subset balance [239,241,268–271]. T follicular helper cells,
which play a role in B-cell selection and survival in GCs, were increased in numbers [272,273]. Th17
cells play a role in inflammation and tissue injury in autoimmunity [274], and may also play a role in
MG [275–280]. Furthermore, an increase in the Th1/Th17 cells was observed in MG patients, as well as
a change in the balance of cytokines, with increased levels of Th1/Th17 cytokines and reduced levels of
IL-10 [229,278,281–283]. Tregs are key players in the maintenance of peripheral self-tolerance [284–286].
In MG, Tregs were functionally impaired and unable to suppress T-cell activation in vitro [277,287–290]
or they were found to be reduced in numbers [271,291–293].
In addition to the abnormalities in the immune cells, other factors may contribute to the early stages
of the pathogenesis, including virus infection, genetic predisposition, environmental factors (e.g., stress,
vitamin D levels), dysregulation of pro-inflammatory cytokines, chemokines and miRNAs, aberrant
expression of Toll-like receptors, and others, but they exceed the limits of this review. The following
excellent reviews are suggested for further reading: [48,235,247,294–297].
Cells 2019, 8, 671 16 of 35

6. Therapies Targeting the NMJ

6.1. Therapies Targeting the Immune System

MG is an autoimmune disease caused by autoantibodies and as such, treatment relies on
targeting the immune system to reduce pathogenic antibodies [298–301]. This includes general
immunosuppression, removal of antibodies by plasmapheresis [302], modulating the immune system
by intravenous IgG [303], and the removal of the thymus, where antibody producing cells reside [40].
More recent approaches include the use of complement inhibitors, specifically monoclonal antibodies
targeting C5 (eculizumab), have been widely studied as a potential treatment and are considered as
clinically beneficial for MG patients [304–307], and there are efforts to develop new monoclonal antibodies
or small interfering RNAs (siRNAs) targeting the complement system [195,308–310]. A different approach
is the direct linking of a complement regulator to the motor endplate by fusion to single chain AChR
antibodies [196]. Another line of treatment focuses on the depletion of antigen producing cells. Current
models propose that in AChR MG antibody production relies mostly on long-lived plasma cells.
Removal of the thymus, which is the site where long-lived plasma cells reside, is clinically beneficial
for the patients [40]. Another strategy to remove plasma cells is the use of proteasome inhibitors like
Bortezomib, which was proven to be efficient in vitro and in vivo [311,312].
In contrast, B-cell depletion therapy with monoclonal antibodies against CD20 (Rituximab) is less
efficient for the removal of long-lived plasma cells because CD20 is only expressed on B-cells and
precursors for short-lived plasma cells [313], but not long-lived plasma cells [314,315]. Patients with
MuSK MG benefit from B-cell depletion more than patients with AChR MG, which suggests that MuSK
antibodies are produced by short-lived plasmablasts [316–329]. In addition to Rituximab, a range of
new monoclonal antibodies targeting B-cells, antibodies and cytokines are being developed [300,330].
Taken together these therapies do, however, not target antigen-specific antibodies or antibody
producing cells, but target whole parts of the immune system. New, bold approaches are necessary
for tailored, antigen-specific therapy. This includes immunization with cytoplasmic parts of the
AChR (similar to a vaccination), to induce the production of antibodies that are non-pathogenic [331],
which was found to be beneficial in a MG animal model. Apheresis of antigen-specific antibodies or
antigen-specific plasma cells are further tailored therapies [332,333], as is a competitive block of the
binding site for the AChR antibody with a monoclonal antibody (mAB). The mAb 637 was cloned from
an AChR MG patient, and with IgG1 subclass-induced MG-like symptoms in rhesus monkeys, but
its immunologically inert and hinge-deleted IgG4 isoform competed with pathogenic antibodies and
ameliorated the symptoms in the animals [334].

6.2. Therapies Targeting the NMJ

A different approach is to target the structure and function of the NMJ. Most patients are treated
routinely with AChE inhibitors (AChEi) [335], which prolong the effector time of ACh at the synapse
and compensate for reduced AChR densities at the synapse. Notably this is not the case in MuSK MG,
where patients experience exacerbation with AChEi [336,337], which suggests an increased sensitivity
to ACh in MuSK MG. Presynaptic changes can be found in MuSK MG patients and, unlike in AChR
MG patients, their quantal release of ACh is not increased [31,32], perhaps due to a block of retrograde
signaling by the MuSK antibodies. It might be possible that other factors that are released by the
motor neuron, such as Agrin, might also be reduced, depleting positive signals for NMJ maintenance.
The prolonged dwell of ACh in the synaptic cleft could in theory then destabilize AChR clusters, as
ACh is also a signal for AChR cluster dissipation [338,339]. There are also observations that MuSK
MG patients respond with repeated action potentials to AChEi, probably due to a prolonged EPP
and lack of Na channel inhibition [24,340]. Furthermore, MuSK also has a role for anchoring ColQ
to the synapse and one group found an effect of MuSK antibodies on ColQ-MuSK interaction [341],
suggesting reduced levels of AChE at the MuSK MG synapse.
Cells 2019, 8, 671 17 of 35

For patients with MuSK MG, strengthening the NMJ architecture, and specifically the dense
clustering of AChR, could be a new treatment strategy. MuSK antibodies block phosphorylation
of MuSK, which in turn leads to reduced MuSK activation and signal transduction. Experiments
by Huda et al. showed that the inhibition of the tyrosine phosphatase Shp2 overcame the effects of
MuSK antibodies on AChR clusters in vitro [342]. In line with this observation is that a loss of Dok-7,
which is also important for MuSK activation, might increase the susceptibility to MG in experimental
animals [343]. Casein kinase 2 (CK2) was also found to induce serine phosphorylation in the MuSK
kinase domain, which promotes AChR clustering [344] and may also be a target for therapies. Another
attempt to fortify the NMJ against the effects of MG antibodies is the overexpression of rapsyn, which
stabilized the AChRs and successfully prevented the loss of AChRs in animal models [345,346]. In a
different approach, a recombinant Agrin consisting of the C-terminal region of mouse neural Agrin
was found to improve MG symptoms and neurotransmission and to promote AChR clustering in a rat
active immunization model of MG [347]. These studies, though still in early experimental phases, show
great promise, and it would be important to improve research into these NMJ-specific therapies. To this
end, it would be important to develop a physiological model of the human NMJ such as in vitro NMJs
generated from human myoblasts and motor neurons. Efforts to develop such a model system are being
made using human-induced pluripotent stem cells (iPSCs). Although these studies have demonstrated
proper differentiation into motor neurons and myotubes, as well as nerve-dependent contraction and
calcium influx, characteristic features of pre-and postsynaptic development are still lacking [348–351].
Therefore, reliable in vitro models recapitulating functional human NMJs are still unavailable.

7. Challenges of Antibody Diagnostics

There are several commercially available reliable diagnostic tests to detect autoantibodies against
AChR and MuSK (these include radioimmunoassays and ELISA). In a subgroup of patients, however,
autoantibodies against AChR cannot be detected by these assays. These antibodies recognize clustered
AChR and can only be detected by cell-based assays (CBAs) [38,352–354]. Overall, CBAs are perhaps the
most sensitive method of choice for the detection of antibodies against cell-surface expressed structures,
if appropriate controls (mock transfected cells, control sera) are included in the assay [38,355–358].
These are semi-quantitative by fluorescence microscopy, but also quantitative if binding of antibodies to
the antigen-transfected cells is measured by flow-cytometry [211]. Diagnostic methods for the detection
of autoantibodies against AChR, MuSK, and clustered AChR are well established. This is unfortunately
not yet the case for all myasthenic antibodies. The frequency of Lrp4 MG varies between 2–50% in
the group of patients without AChR and MuSK antibodies, depending on the method of choice for
detecting autoantibodies [38,48,75,359]. One difficulty is the expression of Lrp4 and specifically the
transport to the cell surface for CBAs, which might be slightly enhanced by co-expression of chaperones
such as Mesdc2 [360], or by using fixed and permeabilized cells. The former approach might still lead
to low levels of antigen expression and an underestimation of antibody levels, while the latter approach
cannot exclude the binding of antibodies to irrelevant intracellular antigen, such as Lrp4s (and other
proteins) still in the endoplasmic reticulum, that have not been properly folded and/or glycosylated.
The variation of frequencies with live and fixed CBAs as well as ELISA demonstrates that specificity
and sensitivity of these assays need optimization and cross-validation over several centers.
Lrp4 and Agrin antibodies have also been detected in patients with amyotrophic lateral sclerosis
(ALS) [361–363], and their pathogenic role in MG and ALS is unclear. Notably, AChR antibodies were
also detected in ALS patients [363–365]. AChR antibody pathogenicity in the context of MG is well
documented, but it remains unclear in ALS, since passive transfer models using serum or IgG from
AChR, as well as Lrp4 and Agrin antibody-positive ALS patients, have not yet been reported. It is
possible that these antibodies are epiphenomena or they play a role in both diseases, and perhaps they
have diagnostic value as biomarkers.
In any case, we need improved methods to reliably detect the full range of
myasthenic autoantibodies.
Cells 2019, 8, 671 18 of 35

8. Concluding Remarks
Our understanding of MG has greatly improved in the last few years and decades, especially
regarding the pathogenic mechanisms of AChR and MuSK IgG4 antibodies, but it is far from complete.
The etiology and immunopathogenesis are unclear, as is the contribution of MuSK IgG1–3 and
antibodies against Lrp4, Agrin, and ColQ for disease. Most importantly, a fraction of 5–10% of
patients have no known autoantibodies and are still considered as seronegative. The discovery of new
autoantigens at the NMJ in these patients is essential for the understanding of their pathophysiology.
New and improved diagnostic assays for Lrp4, Agrin, and any new autoantigens that may be identified
are necessary for correct diagnosis and clinical management. Most importantly, we will require an
improved in vitro model of the NMJ to recreate the physiological environment in MG, for antigen
discovery studies, but also to study the pre- and postsynaptic pathogenic mechanisms of MG antibodies
and for the development of new therapeutic strategies at the patient NMJ.

Funding: This work was funded by the Austrian Science Fund (FWF), T996-B30 (I.K.) and P28485-B27 (R.H.).
Acknowledgments: Open Access Funding by the Austrian Science Fund (FWF).
Conflicts of Interest: The authors declare no conflict of interest.

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