- Myasthenia gravis is an autoimmune disorder where antibodies target acetylcholine receptors at the neuromuscular junction, impairing signal transmission. - Three proposed mechanisms for receptor impairment are: accelerated receptor degradation, blockade of acetylcholine binding sites, and complement-mediated membrane damage. - Early studies found a reduction in acetylcholine receptors at the neuromuscular junction in myasthenia gravis patients, correlating with impaired signal transmission. This helped establish the autoimmune nature and antibody-mediated mechanisms of the disorder.

1. Brad Christman
5/13/2014
Prof. Walter
Bio 341
Mechanism of Neuromuscular Junction Inhibition
in Myasthenia gravis
Intro to Myasthenia Gravis:
Myasthenia gravis is an autoimmune disorder that interferes with normal
transmission of the neurotransmitter acetylcholine in the neuromuscular
junction. The physiology of the disorder is well documented. Acetylcholine
receptor antibodies (AchR-ab) produced by specific mature B cells circulate in
the blood and target sensitized nicotinic acetylcholine receptors (AchR) on the
postsynaptic membrane of the neuromuscular junction (Lindstrom 1973). In
Myasthenia Gravis the mechanism and pathogenesis of neuromuscular
transmission impairment is still under study however a reduction in the number
of functional AchR’s on the postsynaptic membrane has been documented
(Drachman et. al 1973). At least three antibody-mediated mechanisms have
been proposed to explain AchR impairment: accelerated endocytosis and
degradation of AChR’s, functional blockade of Ach-binding sites, and
complement-mediated damage of the postsynaptic membrane (Vincent 2005).
Adaptive Immune Response & Autoimmunity:
In the adaptive immune response, specific B cells that circulate in the serum of
the blood engulf specific antigens and incorporate a part of them onto their
surface membrane. The display of this antigen fragment bound to its specific
MHC molecule recruits a matching mature Helper T-cell. The binding of these
two cells stimulates the Helper T-cell to secrete specific cytokines that induce
the B cell to multiply and mature into antibody producing plasma cells. These
plasma cells produce specific antibodies that can circulate in the blood
targeting the antigen. This process of the adaptive immune response results in
chemical signaling that allows other immune system cells such as macrophages
and killer T-cells to locate and destroy the targeted antigen (Lodish et al. 2013).

2. The adaptive immune system may take a period of time before it can adapt and
multiply the antibodies necessary to neutralize a new antigen. However, once
the B cells and Helper T-cells have been adapted, they can stay dormant in
memory cells of the lymph ready to quickly bind to its specific antigen and
multiply if it reappears. This fighting off of specific foreign bodies such as
specific bacteria and virus’s is a vital aspect of our immune system.
Autoimmunity is defined when the antigens being attacked by antibodies of the
adaptive immune response are not foreign antigens but tissues and cells of the
organism (Rose & Bona 1993). Because of this, if Myasthenia Gravis goes
untreated, the immune response of the antibodies can continue attacking the
tissues until the organism as a whole is compromised. As a result of the
pathology of the adaptive immune response and the specific nature of
antibodies, autosensitization of tissue has been recognized as the initial trigger
in autoimmunity; autosensitization has to occur before autoimmune antibodies
are produced. Autosensitization is when the immune response recognizes the
self as a foreign body and initiates the adaptive immune response against it
(Goodnow et al. 2005). Little molecular research has been done on the factors
involved in triggering autosensitization, although environmental factors,
viruses and gene expression have been thought to be possible contributors
(Vincent 2005). In Myasthenia gravis the Thymus is suspected to be a main
player involved in the autosensitization of Ach-R. Ach-R specific T cell lines
can be cloned from the thymus, and cultured thymic lymphocytes
produce AChR-specific autoantibodies. The thymus is the site of T-Cell
maturation. Thymic abnormalities are frequently present and specifically
associated with MG. 30-45% of people with thymomas have MG and 10-15%
of MG patients have thymomas. Because of this a thymectomy has been
employed as a effective treatment option (Meriggioli 2009).
The AChR is an oligomeric membrane protein consisting of five subunits α2, β,
δ and ɛ at the adult end plate. The dominat AchR-ab epitope is directed toward
the main immunogenic region (MIR) on the alpha subunits. However, other
studies suggest that some patients do not have MIR antibodies and some have
antibodies that are directed against regions on different subunits (Vincent
2005). The epitope is the antigen specific binding site of an antibody. Due to
the adaptive/specific nature of antibody production, not everyone’s epitopes are

3. structurally identical. In this way antigen-antibody binding affinity can change
from one MG case to the other. However, the highly conserved genetic code
for AchR of skeletal muscles results in nearly identical AchR molecular
structure from person to person. In addition
to epitope variability, ChR antibody titres are highly variable among MG
patients, ranging from 0 to more than 1000 nm/L. These titer variations may or
may not correlate with the location and severity of the disease. For example,
the disease can remain localized (e.g. Ocular MG), involve mainly specific
muscle groups (e.g. Bulbar MG) or spread to include most muscles
(Generalized MG). There is still a lot that is not fully understood about the
variability in the pathogenesis of the adaptive immune response in MG.
(Newson-Davis et al. 1978).
Physiology & Diagnosing:
In muscle innervation, when an action potential (AP) reaches the nerve terminal
of a motor neuron, the depolarization causes an influx of calcium that results in
the release of Ach from thepresynaptic terminal. Ach diffuses across the
synaptic cleft and binds to Ach-R found on the edges of junction folds of the
postsynaptic membrane, resulting in a graded end-plate potential (EPP). Ach-
R’s are transmembrane ligand gated Ion channels that undergo a
conformational change when Ach binds to it. This conformational change
opens an ion channel that allows sodium and potassium ions to diffuse across
the postsynaptic membrane causing a graded potential. If this graded potential
reaches threshold, it creates an action potential in the sarcolemma that goes on
to contract a motor unit via excitation-contraction coupling. Almost always, the
end plate potential triggers an action potential (Plomp et al. 1995).
Normal EPP’s depolarize to a greater level than the AP threshold level. The
difference between these two levels is the safety factor. For Myasthenia gravis
patients, this safety factor is reduced. Worst case, the EPP fails to reach AP
threshold, resulting in no muscle contraction. In diagnosing Myasthenia gravis,
repetitive nerve stimulation (RNS) is one of the first diagnostics employed to
test for MG. This diagnostic test rapidly innervates nerves and takes data on
the EPP’s. In Myasthenia Gravis patients the EPP is decremental, meaning that
the depolarization each graded potential decreases with every innervation. This
is due to the fact that there is a reduced number of active AchR that cannot
handle the rapid influx of Ach that result from rapid innervation (Kothari

4. 2004). AchR’s are found at a normal density of10,000 receptors/micrometer2.
The amount of sodium and potassium the channels allow through their pores,
conductance varies from 50–110 pS, with the conductance depending on the
specific subunit composition as well as the permeant ion (Miyazawa
& Fujiyoshi 2004).
The most specific diagnostic test for Myasthenia Gravis is testing for the
acetylcholine receptor antibodies (AchR-ab). Three libratory studies are
commercially available and may be used when testing for the presence
of AchR-Ab: binding, modulating, and blocking (Plomp et al. 1995). Although
the antibodies circulate throughout the body, often time’s impairments in ocular
movement, speech, swallowing and breathing are most noticeable. Usually
ocular impairment is the first symptom of Myasthenia Gravis. No sensory,
autonomic, or cognition impairment results with the disease. This is due to the
fact that the blood brain barrier prevents the AchR-ab from inhibiting neural
synapses in the central nervous system. The onset of the disorder can vary
greatly between cases. Onset of severe life threatening symptoms also known
as a Myasthenic crisis also varies greatly between cases.
Current Treatments:
There is no specific protocol for the treatment of Myasthenia gravis. Although
in a crises, acetylcholineesterase inhibitor is first used to prevent the breakdown
of acetylcholine in the NMJ. This treatment leads to an increase in the level and
duration of acetylcholine in the NMJ and an increase in the strength of
neuromuscular transmission. In addition, MG patients are placed on
immunosuppressant drugs and a prednisone steroid therapy that decreased the
titer of autoimmune antibodies. This was only a temporary treatment. The next
step in treatment, a long-term solution is plasmapheresis, thymectomy or
intravenous immunoglobulins. The goal of plasmapheresis or plasma exchange
is to remove the circulating immune complexes and AchR-Ab. Patients
undergo a 2-week course of 5 to 6 exchanges. Removing on average 1-Pint of
plasma per exchange. Although the number of exchanges and interval between
them often times must be tailored to each patient, taking into account the
general clinical conditions, severity of MG, and potential side effects (Vincent
2005). Open studies showed that PE was effective in at least 60% to 70% of
treated patients, however this treatment option is temporary, usually lasting

5. only 4-5 weeks. Con’s to the use of chronic plasma exchange are the need for
good vascular accesses and the obvious effects on several plasma components
being removed. The long-term benefit of plasma exchange is still under study
(Gadjos et. al 2002).
Mechanism of Myasthenia gravis Inhibition:
The fundamental mechanism and pathogenesis affecting the neuromuscular
junction of MG patients is not yet fully understood. However, the reduction of
available AchR has been documented to cause the defecit in neuromuscular
junction transmission in Myasthenia gravis (Fambrough et al. 1973). Although
proposed Mechanisms behind NMJ inhibition bounced around for over a
century,Fambrough and his colleagues conducted the first studies to verify the
mechanism behind NMJ impairment in Myasthenia gravis. To do this, they
took “motor point” biopsies from MG patients. They incubated the samples
with radio labeled I-a-BuTx, a neurotoxin that binds to AchR at the Ach
binding site. After scintillation counting and autoradiography they found an
80% reduction inAchR’s in MG patients n comparison to non-MG patient
controls. They correlated this reduction in AchR with the marked reductions in
the Motor Endplate potential exhibited by these patients. Following this data,
autoimmunity was proposed and the hunt for the antibody began. “In the most
sensitive radioimmunoassay, which detects antibodies bound to AchR labeled
with 125I-α-bungarotoxin (125I-α-BuTx), elevated titers have were found in 80-
90% of patients with MG”. However, the antibody titer corresponded only
approximately to the clinical status of the patients (Drachmen et. al 1973). The
isolation of these antibodies, experimental employment of 125I-α-BuTx, and the
growing knowledge immunology and physiology allowed researchers to make
great progress in the study of this disease. For example, it was found that the
disease can be passively transferred to experimental animals by daily injections
of purified MG IgG (Toyka et al. 1975). The pathogenic role of AchR-ab was
also verified by the drastic improvement of MG symptoms observed following
plasma exchange (Newsom-Davis et. al 1978). With these discoveries, today
MG meets all the criteria of Witbesky to categorize it as an autoimmune
disease (Rose & Bona 1993). Once the antibody was isolated, studies were
done to test the specific mechanism of AchRreduction in MG patients. The first
mechanism studied and verified was the acceleration of AchR degradation
via endocytosis in response to antibody crosslinking of the receptors known as

6. antigenic modulation. The receptors linked to antibodies are internalized and
degraded by lysosomal enzymes. This crosslinking was found to reduce the
half-life of AchR’s at the NMJ from about 10 days to about 5 days
(Drachman et. al 1978). The second mechanism studied was complementation
and activation of the membrane attack complex resulting in the destructive
changes in the morphology of the postsynaptic membrane. This mechanism is
most likely the most important because it results in a reduction of the
postsynaptic folds and a reduction in the functional AchR’s and ion channels
required for signal transduction (Arahata 1987). It has also been verified that
there is a compensatory response to the reduction of AchR’s. AchR synthesis
has been shown to increase in AchR turnover studies (Wilson et. al 1983). Also
interesting is the presynaptic motor neurons ability to recognize impaired
transmission and try and compensate by increasing the number of Ach packets
released (Plomp et. al). A functional block of AchR by the antibody may also
play some role in the mechanism of inhibition however this mechanism has
been a matter of controversy.AchR-ab almost never binds specifically to the
Ach binding site however the antibodies have been determined to block I-a-
BuTx, which binds to the Ach binding site (Drachman et. al 1973). There is no
reliable evidence for involvement of the cellular mediated immunity in the
pathogenic mechanisms at the neuromuscular junction in MG. The majority of
electron microscopic studies have not identified apoptosis, macrophages or
natural killer cells indicative of cell initiated immune response (Vincent 2005).
Research Proposal & Approach: What is the
Mechanism of Neuromuscular Junction Inhibition in
Myasthenia gravis?
At least three antibody-mediated mechanisms have been proposed to
explain AchR impairment: accelerated endocytosis and degradation of AChR, functional
blockade of Ach-binding sites, and complement-mediated damage of the postsynaptic
membrane. (Vincent 2005).

7. Intro: What is the mechanism of Acyteylcholine antibodies (Ach-ab) inhibition of
Acetylcholine Receptors (AchR) receptivity and function? Based on the literature,
inhibition of AchRcould be due to any off the three mechanisms proposed above. My
research proposal includes a comprehensive protocol aimed at better understanding the
mechanism by which the Ach-ab inhibits and destroys acetylcholine receptors in the
neuromuscular junction.
Hypothesis:
I think the mechanism of Acetylcholine Receptor (AchR) inhibition is somewhat
dependent on the specific form of the Acetylcholine antibodies (Ach-ab). I think the
mechanism of AchRinhibition is dependent on the ratio of receptor bound Ach-ab to
total AchR in neuromuscular junction (NMJ) and therefore the mechanism dynamic
throughout the course of the disease and pathogenesis can vary from case to case.
ResearchQuestions:
-What happens when antibodies bind to receptors?
-Does it stop Ach from binding or does it stop AchR from functioning (opening)?
Or does it result in a loss of receptors by endocytosis or the compliment mediated
immune response?
Approach:
Experiment 1: Is Ach-ab bound to AchR?
Yes: Go to experiment 2&3.
No: Go to alternate explanation.
To determine if AchR-ab’s are bound to AchR’s I want to conduct a binding assay on a
reconstituted frog oocyte. I will use a frog oocyte with AchR’s expressed on its surface. I
will follow the protocol for AchR specific mRNA preparation and insertion. I will

8. implant these AchR specific mRNA into the oocyte for expression. Once the
frog oocyte with AchR’s is prepared I will do a binding assay using acetylcholine and
fluorescently tagged AchR-ab’s purified from human serum. I will follow the protocol for
fluorescent tagging of antibodies. I will expose the prepared oocytes to the tagged Ach-
ab’s. After each trial, spin down the preparation, remove the soup and wash
the pelleted oocytes two times to remove any excess AchR-ab’s. Remove washed pellet
and conduct microscopy and fluorescent spectroscopy. I will look for AchR-ab binding to
the AchR’s. This binding level will tell us whether or not AchR-ab is binding or not
to AchR’s.
Experiment 2: Does AchR-ab binding inhibit Ach binding?
In this experiment we want to see if Ach-ab binding to Ach affects the ability of Ach to
bind to AchR. To do this, a similar experiment to Experiment 1 will be conducted. The
difference between these experiments is that in experiment 2 I will use 125I-α-
bungarotoxin (125I-α-BuTx), which has been experimentally tested to bind to the Ach
binding site (Drachman et. Al 1973). This will be a two-part experiment. Part 1 will
test 125I-α-BuTx’s ability to bind to AchR’s on its own. Part 2 will test 125I-α-BuTx’s
ability to bind to AchR’s in the presence of Ach-ab’s. Binding ability will be quantified
using liquid scintillation spectrometer and autoradiography. A decrease in isotope return
from part 1 to part 2 following the addition of 125I-α-BuTx’s will indicate Ach-
ab’s inhibition of Ach binding to Ach-R. We will be able to calculate mg of bound 125I-α-
BuTx per Oocyte. Based on recent studies and what is known about MG, a decrease in
isotope detection from part 1 to part 2 should be the result. If not see alternate
explanation. This experiment may need to be conducted w/ 14 C radio labeled Ach as
there might be some steric and affinity differences between Ach and 125I-α-BuTx.
Experiment 3: Does AchR-ab binding decrease Ach binding affinity?
To do this experiment I will conduct an antibody-affinity chromatography experiment. To
do this, I will covalently attach the AchR onto beads. I will then conduct three trials. In

9. one trial I will add Ach via a neutral buffer to the cylinder. The next trial I will add Ach
and AchR-ab into the cylinder. The last trial I will add the AchR-ab first followed by
Ach. I will conduct this experiment at different flow rates and with different AchR-
ab’s epitopes from different MG patients. After each trial I will quantify the amount and
the type of protein that flowed through using SDS-Page and Coomassie blue staining
with Prism software. Then I will then wash and elucidate the cylinder and quantify the
amount and type of elucidated protein. Based on this experiment, I will be able to see the
change in binding affinity between AchR and Ach when AchR-ab is not bound,
competing for binding and bound. (Lodish et al. 2013).This assay could give you good
results on the competitive nature of Ach and the AchR-ab. It could also give you
interesting results on how Achr to Ach binding affinity is affected by different epitopes of
the antibody.
Experiment 4: Does AchR-ab binding inhibit functionality of AchR’s in signal
transduction?
Yes: Go to Experiment #5
No: Go to alternate explanation.
Does the presence of AchR-ab completely inhibit AchR functioning? Or does it only
partially inhibit due to changes in binding affinity, binding duration or conduction time?
To answer these questions, I will do a patch clamping experiment with the reconstituted
frog Oocyte from experiments 1-3. This experiment will allow me to measure the effects
of Ach-ab’s binding on the functionality AchR. AchR is a ligand gated ion channel that is
known to undergo a conformational change and open when bound to 2 Ach’s. To conduct
a patch clamp experiment, I will have to apply a patch electrode with slight suction to a
region of the Oocyte cell membrane. In addition, I will add an intercellular electrode.
This experiment will be conducted in 2 parts. Part 1 will constitute filling the patch
electrode with a current conducting saline solution and a known concentration of Ach.
Part 2 will constitute filling the patch electrode with a current conducting saline solution
along with a known concentration of Ach and Ach-ab. This patch clamp device will
maintain constant voltage across the membrane and measure current flow across

10. membrane at the tip of patch electrode. This method prevents changing voltage gradients
from inhibiting sodium influx (Lodish et al 2013). This experiment will test the effect
Ach-ab has on sodium influx and thus signal transduction in the postsynaptic membrane.
If the current flow (sodium influx) decreases across the membrane in the presence
ofAchR-ab’s than it can be concluded that AchR-ab’s inhibit the functionality of signal
transduction. For each trial it should be ensured that AchR quantity within the patch
electrode is equal to one. This could be a good experiment to test how different ratios
of AchR-ab to Ach effect signal transduction. Another focus of this study might be to see
whether there is a change in ionic channel open time when AchR-ab is bound. Taking
short interval measurements of depolarization time for each test part will give you this
result.
Alternate Explanation:
No binding of AchR-ab to AchR observed
Based on current research and understanding of MG, it is known that the autoantibody
Ach-ab binds to the autoantigen AchR. So it is expected that Ach-ab present in the
solution of Experiment 1&2 will inhibit Ach binding. There are multiple reasons for these
unexpected results. First, a AchR specific mRNA translation mistake could lead to Ach-
ab’s inability recognizing the expressed AchR’s. Secondly, patients expressing MG
symptoms don’t always have AchR-ab’s. MusK is inhibited by MusK-ab antibodies in
the NMJ. MusK is a signaling protein involved with the development and maintenance of
the NMJ. Without proper signaling by MusK the patency of the NMJ decreases. In 20 %
of MG cases, MG symptoms are caused by auto MuSK antibodies instead of
auto AchR antibodies. If auto MuSK antibodies were taken from a human host instead
of AchR-ab’s and used in experiment 1 or 2 most likely no inhibition of Ach binding
would be seen. Auto MusK antibodies cause MG symptoms by a similar but different
mechanism. I chose to focus on AchR-ab MG in this proposal.
Testing the Mechanisms of AchR inhibition:

11. Now that we know Ach-ab binding inhibits AchR functionality in vitro study, we need to
determine the mechanism of AchR inhibition in living tissue?
There are 3 proposed mechanisms of Ach-ab inhibition of AchR’s. The first mechanism
proposes that Ach-ab simply blocks the binding site of Ach, preventing Ach binding and
signal transduction. If this were the case Ach-ab binding would occur but no reduction
of AchR’s would be observed. The second mechanism proposes that Ach-ab binding
to AchR’s inducesendocytosis of the postsynaptic membrane. If this were the case, we
would see an increased rate of endocytosis when Ach-ab is bound to Ach-R. The third
mechanism proposes that Ach-ab binding to AchR results in complement mediated
damage of the postsynaptic membrane. If this were the case, Reduction in the number
of AchR’s and overall organization of the postsynaptic membrane would decrease.
Experiment 5:Testing for mechanism #1 (Blocking of binding site)
To test for this mechanism, I will create a primary cell culture of the NMJ cells cooled at
4 degrees Celsius to eliminate degradation and minimize possible antibody
dissociation. They will be treated overnight in the cold with AchR-ab’s and then are
saturated with 125I-α-BuTx’s. The loss of 125I-α-BuTx binding sites in the cultures treated
with AchR-ab is attributable to AchR blockade (Drachman et. al 1973).
Experiment 6: Testing for Mechanism #2 (Induction of Endocytosis)
To test whether or not muscle cells induce endocytosis in response to Ach-
ab bound to AchR, I would again use a primary muscle/nerve cell culture experiment. I
would set it up similar to Experiment #5 only I would culture the cells in fluorescent
media to follow endocytosis. I would microinject the Ach-ab to the NMJ and then
stimulate the release of Ach into the NMJ with a microelectrode. I would conduct
multiple trials of this experiment with varying intervals and time spans of nerve
innervations’. I might try random fast, random slow, repetitive fast, repetitive slow and
no innervations. I would take fluorescent microscopic images of the muscle cells at
standard time intervals and calulate the rate of fluorescent vacuole formation. This would

12. be possible because the fluorescent die on the outside of the cell would be brought into
the cell and easily observed following edocytotic vacuole formation. This experiment
would be conducted with and without Ach-ab added. If the fluorescent vacuole formation
increased in response to Ach-ab in the NMJ then induction of endocytosis could be a
potential mechanism for AchR signal transduction inhibition. To get further accuracy in
your results, you could repeat the study using C14 tagged anti-AchR-ab antibodies.
Thisimmunohistochemistry technique allows you to track the Achr-abs and see if they are
being internalized via endocytosis. This would show you if membrane sections
containing Ach-abbound to AchR were specifically targeted for endocytosis or not. This
experiment might also provide you with insight into the effect of
motor nueron stimulation on the induction ofendocytosis.
Experiment 7: Testing for Mechanism #3 (Complement mediated damage of the
postsynaptic membrane)
Because of the complex/dynamic/heterogeneous environment of the mammalian
body, I think it is only appropriate to study the adaptive immune response/complement in
a live animal study. To test the third mechanism of AchR inhibition, I want to conduct a
study using mice with experimentally induced Myasthenia gravis. I will follow
documented literature on inducing MG in mice (Toyka et al. 1975). Once MG is induced,
I will conduct an observational histology experiment. This procedure for this experiment
will constitute removing thin tissue samples of muscle and nerve cells. These tissue
samples will be fixed via the snap freezing protocol and examined under a microscope.
Each tissue sample would be examined for the number of AchR’s, the
integrity/organization of the postsynaptic membrane, the presence of immune cells
(Helper T-cells, B cells, Macrophages etc.) and most importantly the presence
of cytolytic membrane attack complexes. The cytolytic membrane attack complex is a
direct indication of immune system complementation. Complement is the process by
which a group of constitutive serum proteins bind to microbial or fungal surfaces, thereby
activating a proteolytic cascade that culminates in the formation of
the cytolytic membrane attack complex (Lodish et. al 2013). Disorganization in the

13. postsynaptic membrane folds, reduction in the number of AchR’s, and the presence of
immune cells in particular cytolyticmembrane attack complexes are all indicators of
complement mediated damage of the postsynaptic membrane. If these indicators are
identified compliment mediated damage could be concluded as the primary mechanism
of AchR signal transduction inhibition. It might be useful to dye or tag specific aspects of
the tissue samples in order to better observe/quantify specific molecules of interest such
as Ach-ab, AchR or the membrane attack complex. Observations on tissue samples from
experimental induced MG mice will be cross-referenced with tissue samples of normal
mice. This experiment sets up well to look at the NMJ at various stages of the disease.
These different stages in the disease often correlate with differing titers of the antibody.
Therefore the effect different titers have on the mechanism of NMJ inhibition can be
observed. A specific epitope of AchR-ab can be induced in mice, and the changes in
these specific epitopes can be studied in regards to the NMJ inhibition mechanism.
Observing different combinations of AchR-ab epitopes may also be enlightening.
Conclusion:
In conclusion, this series of experiments will seek to discover the mechanism by which
MG antibodies inhibit NMJ signal transmission. Much is already known about the
mechanisms of inhibition that can and do exist in MG patients. However, the variation in
the pathogenesis of the disease between MG patients is still not fully understood. The
dynamic nature of the mechanism of NMJ inhibition may be an explanation for the
variations in MG pathogenesis. Variations in the affinity of AchR-ab epitopes and
variations in the ratio of receptor bound Ach-ab to total AchR in the NMJ I hypothesize
to be the determining factors in the NMJ inhibition mechanism. My research is set up to
see the effects that changes in antibody affinity and antibody concentration have on
verified mechanism of NMJ inhibition. My research proposal could result in clinical trials
for the study of MG pathogenesis if strong correlations were made between specific
inhibitory mechanisms and specific AchR-ab epitopes and antibody concentrations. If
these correlations remained consistent following clinical trials, this relationship between
the mechanism and the variable factors of the disease could lead to the personalization of

14. the MG treatment plan. For example, the epitope could be determined, the ratio of
receptor bound Ach-ab to total AchR in the NMJ could be quantified and just like that
you know the mechanism of NMJ inhibition that is causing that patients symptoms.
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