Mechanism of Neuromuscular Junction Inhibition in Myasthenia

gravis

By Brad Christman

Bio 341

Prof. Anne Walter

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

Adaptive Immune Response

Acetylcholine Receptors

Neuromuscular Junction

Anatomy & Physiology

Na+

Na+

Open Na+

ChannelClosed K+

Channel

K+

Na+ K+Action potential

+ ++++

++ ++++

+

Axon terminal

Synapticcleft

ACh

ACh

Sarcoplasm of muscle fiber

K+

Graded Potential->Action Potential

Action potential is

propagated along the

sarcolemma and down the

T tubules.

Steps in E-C Coupling:

TroponinTropomyosin blocking active sites

Myosin

Actin

Active sites exposed and ready for myosin binding

Ca2+

Terminal cisternaof SR

Voltage-sensitivetubule protein

T tubule

Ca2+releasechannel

Myosincross 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-ContractionCoupling

Symptoms

Diagnostic Tests

Blood Tests for MG antibodies

Diagnostic Tests Cont.

Treatment

• Acetylcholineesterase inhibitors

• Immunosuppressant’s

• Plasmapheresis

• Intravenous immunoglobulin (IVIG)

• Thymectomy

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?

Research Proposal

• Aim: Identify the mechanism of AchR-abinhibition of NMJ activity in Myasthenia gravis

• Literature: At least three antibody-mediated mechanisms have been proposed to explain AchRimpairment: accelerated endocytosis and degradation of AChR, functional blockade of Ach-binding sites, and complement-mediated damage of the postsynaptic membrane. (Lindstrom).

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.

Protocol: Experiment 1: Is Ach-abbound to AchR?

• Binding assay on a reconstituted frog oocyte

• AchR specific mRNA

• Expression

• Binding assay w/ fluorescently tagged AchR-ab’sfrom human serum

• Microscopy and fluorescent spectroscopy

• Verify Binding

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

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

Testing the Mechanisms of AchRinhibition:

• 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

Mechanism #1Mechanism #2

Mechanism #3

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?

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

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

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

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

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Thank You!