L11 - Neuromuscular junction and motor unit

Question 1

What are the pre- and post- synaptic specializations of peripheral chemical synapses?

Answer 1

1) Presynaptic specialization: clustering of synaptic vesicles (filled with neurotransmitters) at the active zone of presynaptic terminals
2) Postsynaptic specialization: clustering of numerous acetylcholine neurotransmitter receptors (AChR) at the motor end-plate region

Question 2

What causes AchR to cluster?

Answer 2

Clustering signals induced by agrin (secreted proteoglycan) incorporated into the extracellular matrix

Question 3

Describe the cycle of Ach as a neurotransmitter.

Answer 3

1) Choline and Acetyl CoA from mitochondria are combined by Choline acetyltransferase to make Ach
2) Ach released into synaptic celft by exocytosis from presynaptic neuron
3) Ach activate Cholinergic receptor at Postsynaptic neuron
4) Ach broken down by acetylcholinesterase (AchE) into Acetate and Chloine
5) Cholineistransported backtotheaxonterminal andisre‐usedtomake moreACh.

Question 4

Describe the activation of AchR?

Answer 4

2 Ach molecules bind to activate AChR (nicotinic cholinergic receptor) enriched at motor end plate of skeletal muscle fiber

> > inducesconformationalchanges andopening ofnonspecific monovalent cation channel

> > ResultsinaninfluxofNa+anda smalleffluxofK+

> > Induce membranedepolarization, andmusclecontraction

Question 5

Describe the experimental evidence for AchR clustering after nerve damage?

Answer 5

When motor axons were cut and muscle cells were damaged in adult frogs, they degenerated, leaving behind the basal lamina

New myofibers formed beneath this basal lamina

AChRclusteringsignals= secretedproteoglycanthatbecomesincorporatedintothe extracellularmatrix at original synaptic site, even though motor nerve terminals were absent

> > AChR do not have to be activated to cluster, AChR ClusteringInducedbyContactwithNeurites

Question 6

What is a marker that shows AchR clustering to muscle cells?

Answer 6

fluorescent α‐bungarotoxin

> > can bind to potently inhibit AChR α1 subunit

> > Gives fluorescentsignalsatAChR clusteringsites

Question 7

Explain the mechanism of Agrin-MuSK signaling?

Answer 7

Agrin activates a receptor complex that includes binding protein (Lrp4) and muscle-specific kinase (MuSK):

> > phosphorylate/ activate MuSK instantly

> > leads to AChRphosphorylation andclustering over next few hours

Question 8

List some symptoms of Myasthenia gravis?

Answer 8

progressive disease:

1. Ocular, oral symptoms:
   - Eyelid drooping
   - Double / blurred vision
   - Opthalmoplegia
   - Affects talking, chewing, swallowing
2. Generalized MG
   - facial paralysis
   - difficulty breathing
   - problems walking up stairs or lifting objects

Question 9

Describe the 3 pathogenesis pathways for MG?

Answer 9

Autoimmune dysfunction: auto-antibodies destroy postysnaptic AchRs in 3 ways:

1) Activate complement cascade to form membrane attack complex (MAC) to lyse / disrupt NMJ structure
2) Antigenic modulation: crosslinks AChR to induce endocytosis and degradation of postsynaptic AChRs by lysosomes = decrease postsynaptic AChR density
3) Inhibit AChR functions (i.e. channel properties): e.g. bind to α subunit so ACh cannot bind = Affects communication between nerve cell and muscle cell = no muscle activation

Question 10

What is the overall effect of Anti‐AChRantibodies in MG?

Answer 10

inducetheendocytosisand degradationofAChRs leadingtoareductionof postsynapticAChRdensity andmuscleweakness

Question 11

What approach can be taken to Dx MG?

Answer 11

Screen for autoimmune antibodies: seropositive MG (>85%), seronegative MG

Question 12

What are the 3 serological approaches to Dx MG?

Answer 12

1. Enzyme-linked immunosorbent assay (ELISA)
2. Radioimmunoprecipitation assay (RIPA) / radioimmunoassay (RIA)
3. Cell-based assay (CBA)

Question 13

What does ELISA detect in diagnosing MG?

Answer 13

Sensitive, quantitative assay to detect, measure circulating IgG autoantibodies in patient’s serum sample

Question 14

Describe the simplified procedures of ELISA in Dx of MG?

Answer 14

Coat a solid surface with recombinant proteins (e.g. AChR) as antigen

Then incubate with:

1. Patient’s serum sample (primary antibody), wash away unbound Ab
2. Enzyme-conjugated (horseradish peroxidase (HRP)) secondary antihuman antibody (from animals)
3. Colorimetric substrate

Question 15

What is the positive result for ELISA in Dx of MG?

Answer 15

Positive result: color detection (activation of the Coorimetric substrate)

Question 16

Give one disadvantage of ELISA in the Dx of MG?

Answer 16

Falsepositivereactionscausedbyhydrophobicbindingof immunoglobulincomponentsinsamplespecimenstosolidsurfaces

Question 17

What is the principal of Radioimmunoprecipitation assay (RIPA) in Dx of MG?

Answer 17

Using Radioactiveα- bungarotoxintoindicatethepresenceof pathogenicautoantibodiesagainst AChRsinatesttube

Question 18

Describe the simplified procedure for Radioimmunoprecipitationassay in Dx of MG?

Answer 18

1. Incubate a mixture of radioactive α-bungarotoxin (or other antigens), detergent extracts of human muscle, and patient’s serum sample
2. Incubate and pull down with secondary anti-human antibody by centrifugation and wash pellet

Question 19

What is a positive result in RIPA test for Dx of MG?

Answer 19

Positive result: radioactive signals in the pellet

Question 20

Give one disadvantage in using RIPA in Dx of MG?

Answer 20

require safety precautions when using radioactive isotopes

Question 21

What is the principal of using Cell-based assay (CBA) in Dx of MG?

Answer 21

Express postsynaptic proteins (AChR or MuSK) in heterologous cells to test the binding of pathogenic autoantibodies

Question 22

Describe the simplified procedures for using CBA in Dx of MG?

Answer 22

1. Transfect cells with DNA plasmid which encodes the protein of interest (e.g. AChR or MuSK)
2. Incubate with patient’s serum sample, then with fluorescent secondary anti-human antibody

Question 23

What is the positive result for using CBA in Dx of MG?

Answer 23

Positive result: fluorescence signals

Activation of the fluorescent secondary anti-human antibody

Question 24

Give 2 disadvantages in using CBA for the Dx of MG?

Answer 24

Time-consuming (need to transfect, wait for expression)

Labor-intensive

Question 25

What are 2 current treatment options for MG?

Answer 25

1) Anti-cholinesterase therapy&raquo_space; Pyridostigmine ( acetylcholinesterase (AChE) inhibitor)
2) Immunosuppressant therapy – corticosteroids

Question 26

How does Pyridostigmine treat MG?

Answer 26

acetylcholinesterase (AChE) inhibitor):

Decrease degradation of neurotransmitter ACh

strengthen neuromuscular transmission

Question 27

How does immunosuppressant therapy treat MG?

Answer 27

corticosteroids:
- Induce steroid hormones
- suppress body’s immune system

Question 28

Give a disadvantage for each of the 2 treatment options for MG?

Answer 28

For Pyridostigmine: effects start to wear off after a few hours

For corticosteroids: risk of infection in patients with chronic disease (due to blockage of production of antibodies)

Question 29

Define a motor unit in a skeletal muscle?

Answer 29

Motorunitsinaskeletalmuscle:

• Containhundredsofmusclefibers
• Contractatthesametime
• Controlledbyasinglemotorneuron

Question 30

Explain how tension applied to a muscle tendon remain balanced/ constant during prolonged muscle contraction?

Answer 30

muscle fibers of different motor units are intermingled

Overall force applied remain relatively constant even though individual motor units cycle between contraction and relaxation

Question 31

Describe the concurrent mechanisms in increasing the strength of voluntary muscle contraction?

Answer 31

Successive activation of the same / additional motor units by increasing both:

1. Number of active motor units (spatial recruitment)
2. Firing frequency (temporal recruitment: could lead to tetanus)

Question 32

What is the difference between unfused and complete tetanus?

Answer 32

Unfused/ incomplete tetanus: successive stimuli to muscle fiber is far apart enough to allow slight relaxation in- between

Complete tetanus: Successive stimuli are close enough to achieve steady maximum tension, until fatigue causes the muscle to lose tension despite continuing stimuli

Question 33

Explain how Clostridium tetani can cause tetanus?

Answer 33

Release toxin in tissue that suppresses motor neuron inhibition&raquo_space; Sustained, powerful contraction of skeletal muscles throughout body

Question 34

What are some symptoms of Clostridium tetani infection?

Answer 34

Early: headache, muscle stiffness, difficulty in swallowing, lockjaw, widespread muscle spasms

Late: Severe tetanus with 40 - 60 % mortality rate

Question 35

What is the diagnostic procedure to assess the health of muscles, motoneurons?

Answer 35

Electromyography (EMG):

Measures voltage change during muscle contraction (depolarization)

Translates raw EMG electrical signals between motor neurons and muscles intoindividual motor unit action potential (MUAP)

Question 36

What are the 2 types of EMG?

Answer 36

Surface and Intramuscular signals

Question 37

Compare the advantages and disadvantages between 2 types of EMG?

Answer 37

1) Surface:
- Advantage: Non-invasive
- Disadvantage: limited assessment of muscle activity, restricted to superficial muscles

2) Intramuscular:
- Adv: Better assessment of deep muscles
- Disadv: Invasive insertion of needle through skin, Involves voluntary contraction and less informativeinpatientsunwillingorunabletocooperate

Question 38

How is motor unit recruitment demonstrated?

Answer 38

Start at minimal effort muscle contraction

Gradual increase in muscle strength shows Recruitment of a second motor unit with increase in EMG frequency

Further increase in muscle strength recruits a third motor unit

Question 39

With the same load, will you see higher EMG signals recorded from the muscle of a trained or an untrained person?

Answer 39

• In a trained muscle, individual muscle fibres contain more myofilaments

> > stronger than those in untrained muscle

> > less motor units have to be activated to develop a given force

> > therefore the measured EMG signal will be smaller in a trained person