2. Intro to Myasthenia gravis
• Autoimmune disease
• Acetycholine receptors (AchR) targeted by
acetycholine receptor antibodies (AchR-ab)
• Inhibition of neuromuscular junction activity
• 200-400 cases of MG per million people
• AchR-ab’s are Immunoglobulin antibodies G
(IgG) produced via the adaptive immune
response
8. Action potential is
propagated along the
sarcolemma and down the
T tubules.
Steps in E-C Coupling:
Troponin
Tropomyosin blocking active sites
Myosin
Actin
Active sites exposed and
ready for myosin binding
Ca2+
Terminal cisterna
of SR
Voltage-sensitive
tubule protein
T tubule
Ca2+release
channel
Myosin
cross
bridge
Ca2+
Sarcolemma
Calcium ions are
released.
Calcium binds to
troponin and removes
the blocking action of
tropomyosin.
Contraction
begins
1
2
3
4
Excitation
Contraction
Excitation-
Contraction
Coupling
13. Research Questions
• 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?
• What role does AchR-ab concentration and
type play in all this?
14. Research Proposal
• Aim: Identify the mechanism of AchR-ab
inhibition of NMJ activity in Myasthenia gravis
• Literature: 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. (Lindstrom).
15. Hypothesis
• I think the mechanism of Acetylcholine Receptor
(AchR) inhibition is not entirely dependent on the
specific form of the Acetylcholine antibodies
(Ach-ab). Nor do I think that there is one specific
mechanism of inhibition. Instead, I believe the
mechanism of AchR inhibition is dependent on
the percentage of AchR’s bound with Ach-ab in
the neuromuscular junction (NMJ) and therefore
the mechanism can change throughout the
course of the disease or from case to case.
16. Protocol: Experiment 1: Is Ach-ab
bound to AchR?
• Binding assay on a reconstituted frog oocyte
• AchR specific mRNA
• Expression
• Binding assay w/ fluorescently tagged AchR-ab’s
from human serum
• Microscopy and fluorescent spectroscopy
• Verify Binding
17. Experiment 2: Does AchR-ab binding
inhibit Ach binding?
• Reconstituted frog oocyte
• Binding assay w/ 14C Ach
• Trial 1: 14C Ach’s ability to bind to AchR’s
• Trial 2: 14C Ach’s ability to bind to AchR’s in
the presence of Ach-ab’s
• Binding of Ach quantified w/ liquid
scintillation spectrometer
18. Experiment 3: Does AchR-ab binding
inhibit functionality of AchR’s in
signal transduction?
• Does complete or partial inhibition exist?
• Patch clamping experiment w/ reconstituted frog
Oocyte
• Patch electrode w/ current conducting saline
solution and Ach suctioned to a single AchR
• Run a test w/ and w/ out AchR-ab in solution
• Intracellular electrode will measure current flow
at tip of patch electrode
• Record changes of current flow at varying
intervals for each test
19. Testing the Mechanisms of AchR
inhibition:
• Experiment #4: Testing for mechanism #1 (Blocking of
binding site)
- Expectation: Ach-ab binding would result in no reduction of
AchR’s
• Experiment # 5: Testing for Mechanism #2 (Induction of
membrane Endocytosis)
- Increased rate of endocytotic vesicle formation and
reduction AchR’s
• Experiment #6: Testing for Mechanism #3 (Complement
mediated damage of the postsynaptic membrane)
- Reduction in the number of AchR’s and decreased
organization of the postsynaptic membrane
21. Experiment #4
• Primary Cell Culture of motornuerons & muscle cells
• Add AchR-ab to NMJ
• Repetitive nerve stimulation w/ microelectrode to
induce Ach release
• Vary time period/intervals for stimulation
• Conduct pull down assay: wash, isolate membrane
proteins and quantify AchR bound AchR-ab via western
blotting techniques
• Quantify change in AchR bound AchR-ab before and
after stimulation period
• No change?
22. Experiment #5
• Primary Cell Culture of muscle and nerve cells
• Cultured in Fluorescent dye to follow endocytosis
• Ach-ab to the NMJ and then stimulate the release
of Ach into the NMJ
• Fluorescent microscopy images of the NMJ over
time
• Observe the rate of fluorescent vacuole
formation
• Repeat w/ immunohistochemistry techniques for
AchR-ab bound AchR within vacuoles
23. Experiment #6
• Mice w/ Experimentally Induced Myasthenia
gravis (EIMG)
• Observational histology experiment
• Remove and fix NMJ tissue samples from EIMG
mice
• Microscope observations of
integrity/organization of postsynaptic membrane
• Fluorescently tag for AchR’s & membrane attack
complexes
• Decreased organization, reduction of AchR’s and
presence of membrane attack complexes indicate
complement mediated NMJ inhibition
24. Conclusion
• Current research suggests that all three of the
proposed mechanisms can and do occur
-Blocking
-Endocytosis
-Complement
• What factors influence the mechanism of NMJ
inhibition is still under question
25. Future Directions
• Doing the experiment!
• Changing AchR-ab concentration or type
• Adapting experiment for testing inhibition
mechanisms in humans
• Adapting/personalizing treatment options to
match inhibition mechanism
26. References
1. Patrick, J.; Lindstrom, J. (1973). "Autoimmune response to acetylcholine
receptors”.Science180 (4088): 871–872. doi:10.1126/science.180.4088.871.
2. Lodish, Harvey; Berk, A., Amon, A., Bretscher, A., Kaiser, C., Kriefer, M., et al. (2013).
Molecular cell biology (7th ed.). New York: W.H. Freeman and Co. ISBN 978-1-429-3413-
9.
3. Christopher C. Goodnow, Jonathon Sprent, B.F.d.S. Barbara, Carola G. Vinuesa. “Cellular
and genetic mechanisms of self tolerance and autoimmunity”. Nature 435 (7042): 590-
597. doi:10.1038/nature03724
4. Meriggioli MN, Sanders DB. Autoimmune myasthenia gravis: emerging clinical and
biological heterogeneity. Lancet Neurol. 2009;8(5):475–490.
5. Penn AS, Low BW, Jaffe IA, Luo L, Jacques J. Drug-induced autoimmune myasthenia
gravis [review]. Ann N Y Acad Sci. 1998;841:433-449.
6. Antozzi C, Gemma M, Regi B, et al. A short plasma exchange protocol is effective in
severe myasthenia gravis. J Neurol. 1991;238(2):103–107.
7. Skeie GO, Apostolski S, Evoli A, et al. Guidelines for the treatment of autoimmune
neuromuscular transmission disorders. Eur J Neurol. 2006;13(7):691–699.
