Neuromuscular Junction

Question 1

why do we care about the NMJ?

Answer 1

it’s easy to study - very large, and simple while being accessible on peripheral tissues

Question 2

what 3 cell types are involved in the NMJ?

Answer 2

• motoneuron
• muscle cell
• Schwann cell

Question 3

basal lamina

Answer 3

aka synaptic basement membrane
covers the muscle fiber and Schawnn cells
- has a different composition in the NMJ synapse than extrasynaptically

Question 4

explain AP signal passage through the NMJ

Answer 4

• nerve AP reaches presynaptic terminal and opens voltage gated Ca2+ channels
• Ca2+ entry triggers ACh vesicle fusion to the presynaptic membrane and ACh release
• ACh diffuses across synapse through basement membrane
• ACh bind to AChR which opens ligand-gated ion channels (Na+ moves in, K+ moves out)
• local postsynaptic membrane depolarization occurs (EPP)
• if threshold endplate voltage is reached, voltage gated Na+ channels open and muscle AP follows

Question 5

how to pre and post synaptic membrane components align themselves?

Answer 5

• axon terminals on presynaptic membrane perfectly overlay AChRs on postsynaptic membrane

Question 6

endplate potential (EPP)

Answer 6

local post-synaptic depolarization of the muscle fibre surrounding the AChRs

• only open AChRs
• are observed only when muscle AP is blocked (lab setting)

Question 7

miniature endplate potential (mEPP)

Answer 7

electrical signal induced when a single ACh vesicle is released

Question 8

explain electrical events that occur in neuromuscular transmission

Answer 8

• 1st have an EPP
• if EPP is large enough to reach AP threshold voltage, it triggers opening of voltage-gated Na+ channels
• in healthy individuals, EPP will surpass AP threshold every time

Question 9

safety factor

Answer 9

EPP amplitude-AP theshold

• ensures EPP reliably activates myofiber
  ie. some leeway to be sure we reach threshold

Question 10

Novichok

Answer 10

a Russian poison synthesized artificial version neurotoxin

• targets ACh esterase (breaks down ACh) and blocks enzyme from working
• was deployed in the UK by spies, targets were hospitalized but survived, though the toxin can have long term effects, one bystander was killed

Question 11

choline acetyl transferase (ChAT)

Answer 11

an enzyme that catalyzes ACh synthesis in the intracellular space

• takes choline + acetyl-CoA to make ACh
• is highly expressed in MNs so is often used as a marker in immunofluorescence

Question 12

where do the components of ACh comes from for its synthesis

Answer 12

Acetyl CoA comes from mitochondria

choline comes from extracellular fluid

Question 13

vesicular ACh transporter (VAChT)

Answer 13

packs ACh into secretory vesicles

Question 14

immediately releasable pool of ACh

where is it found

Answer 14

• 2-10% of ACh vesicles come from here
• localized at active zones of presynaptic membrane
• is first to be released upon arrival of AP and Ca2+ influx

Question 15

active zones

Answer 15

discrete spots of ACh vesicles

- when Ca2+ enters the cell, vesicles fuse to the presynaptic membrane

Question 16

reserve pool of ACh

Answer 16

cycles into active zones to replete stores when depleted

Question 17

acetylcholine esterase (AChE)

Answer 17

breaks down ACh into choline and acetate

• choline gets cycled back
• found in basement membrane of primary and secondary junctional folds

Question 18

cytomatrix of active zones (CAZ)

Answer 18

dense collections of proteins that make up active zones

Question 19

where are AChRs found?

Answer 19

in peaks of secondary junctional folds

Question 20

synapsin

Answer 20

an intermembrane protein that helps vesicle move from reserve pool to the immediately releasable pool
- works when immediately releasable pool is depleted

Question 21

synaptobrevin

Answer 21

found in presynaptic membrane

- works with SNARE complex to be responsible for nucleation and zippering

Question 22

zippering vs nucleation

Answer 22

both require interaction/weaving of synaptobrevin and SNARE protein complex

• nucleation keeps ACh vesicles from leaving the active zone
• zippering brings vesicle into contact with presynaptic membrane, preparing it to be fused to it

Question 23

SNARE protein complex

Answer 23

proteins expressed all over the body that are important for docking vesicles at the membrane

Question 24

synaptogamin

Answer 24

• activated by Ca2+ to caused synaptogamin-mediated fusion of ACh vesicle and presynaptic membrane to allow for exocytosis

Question 25

positive signals at the NMJ/muscle fiber

Answer 25

• are released from nerve terminal and result in triggering gene expression of synaptic proteins at the NMJ
• supersede negative signals in local regions
• descend onto sub-synaptic nuclei which are responsible for generating the proteins involved at the NMJ

Question 26

negative signals at the NMJ/muscle fiber

Answer 26

• elicit electronegative pathways when released which turn on proteins which inhibit expression of NMJ proteins like AChRs
• suppress protein expression everywhere in the muscle fiber and prevent formation of other NMJs

Question 27

if positive and negative signals have such opposing functions, how do things get done?

Answer 27

they are balanced, positive overrule in local regions where the signals are at the NMJ

Question 28

what positive signals are there?

Answer 28

• agrin

- neuregulin

Question 29

agrin mechanism

Answer 29

• is released by MN terminal and bind to its receptor Lrp4-MuSK complex
• MuSK recruits Dok-7 to stabilize the AChR cluster and rapsyn anchors AChRs to the postsynaptic membrane

Question 30

neuregulin mechanism

Answer 30

NGR-1 protein is released by MN terminal which bind to Erbβ receptors on motor endplate

• Erbβ and MuSK stimulate MAPK pathway that converges on subsynaptic myonuclei
• when Erbβ binds to its receptor, it activates GABP transcription factor which dimerizes and binds to N Box synapse specific gene promoters
• GABP is only expressed in sub-synaptic nuclei

Question 31

overall negative signal mechanisms

Answer 31

global AChR transcription is inhibited via:

• myogenin
• calcium sensitive proteins CaMKII and PKC

Question 32

myogenin and negative signalling

Answer 32

when the AP goes down the muscle fiber it releases Ca2+ which inhibits myogenin
- myogenin usually activates AChR gene expression by binding to E box promoter region of AChRs

Question 33

calcium sensitive proteins CaMKII and PKC involvement in negative signalling

Answer 33

• both proteins are activated by Ca2+

- signalling from CaMKII and PKC result in transcriptional repression of myogenin (suppress its promoter)

Question 34

can mice survive without any of the following:
MuSK receptor
Lrp4
Dok-7
agrin

Answer 34

nope. they die prematurely bc of respiratory failure