chapter 11 - NMJ Flashcards
11.1. [nmj] explain and schematically draw the structural organization of the NMJ (cellular and molecular)
> Neuromuscular junction (NMJ):
* synapse between motor neurons and skeletal muscle fibers
* allows posture, movement and respiration
* NMJ dysfunction/degeneration occurs in several neuromuscular diseases, e.g. myasthenia gravis, ALS, SMA,
Neurotransmitter: ACh
Postsynaptic receptor: nicotinic ACh receptor (nAChR)
member of the Cys-loop receptor family
nAchR: 5 subunit families (a1-7, 9, 10; B1-4; gama; ohm; epsilon); subunit compositions at NMJ:
embryonic: a2By ohm; adult: a2b epsilon ohm
Organization of NMJ
A: motor neurons whose soma and dendrites are located in the spinal cord send their
axons to the periphery and form neuromuscular junctions to innervate skeletal
muscle fibers. Axons are wrapped by myelin sheaths formed by Schwann cells. At the
site of neuromuscular contact, axons ramify into branches and form presynaptic
nerve terminals that are capped by terminal Schwann cells and covered by
kranocytes. Note the accumulation of fundamental myonuclei (black) and
postsynaptic folds at the site of the nerve-muscle contact.
B: high magnification of the neuromuscular junction. In addition to ACh-filled vesicles,
local specializations in the presynaptic motor nerve terminal include active zones
(where vesicles fuse with the terminal membrane) and a high number of
mitochondria (brown). Postsynaptic specializations in the skeletal muscle fibers
include folds that form opposite the active zones, aggregates of AChRs (red) at the
crest, and high concentrations of NaV1.4 (green) in the troughs of the folds. The
localization and high concentration of AChR and NaV1.4 are important for efficient
neuromuscular transmission.
C: whole mount view of the synaptic band of a mouse diaphragm. Motor axons are
labeled with antibodies to growth-associated protein 43 (GAP-43; green), and
postsynaptic AChRs are visualized by BTX (red).
11.2. [nmj] describe the different stages of NMJ development, including the cellular and molecular interactions involved
(A) At early embryonic stages (E11 to E13.5 in mice), primary myotubes are formed
before innervation by the motor nerve
(Aa) A central zone of AChR clusters without contact to motor axons is formed (a
process called ‘pre-patterning’). Some myonuclei in the center of the muscle
express higher levels of Musk than those in the periphery.
(Ab) At the beginning of motor innervation of the primary myotubes, the number of
myonuclei with high Musk levels increases, and myoblasts proliferate in the
center inside the basal laminae of the primary myotubes.
(Ba) During late embryonic development (E14.5 to postnatal stages), muscle size
increases due to formation of secondary myotubes through fusion of proliferating
myoblasts.
In primary myotubes, innervation initiates transcription of synaptic genes in
myonuclei underlying the neuromuscular contact. The nuclei are called
‘fundamental’ or ‘subsynaptic’ nuclei. AChR cluster size, number and shape are
refined with innervation (E14) that disperses non-synaptic AChR clusters and
induces new neural AChR synaptic clusters through the secretion of Agrin.
Initially, the secondary myotubes are electrically coupled to the primary myotubes
through gap junctions.
(Bb) Subsequently, secondary myotubes segregate from the primary myotubes and
secrete their own basal lamina. At this stage, myotubes are multiply innervated,
and electrical activity leads to condensation of AChRs to the site of innervation.
Furthermore, expression of synaptic genes is suppressed in nonsynaptic nuclei and selectively stimulated in subsynaptic nuclei.
(C) In mature muscle, each fiber is innervated by one motor neuron. Excessive
innervation is removed during the process of synapse elimination.
Molecular mechanisms
Agrin binds to a receptor complex called LRP4-MuSK complex. Agrin is secreted by the motor neuron terminal and binds to LRP4. LRP4 is a co-receptor of MuSk. When Agrin binds, MuSK is activated. It’s a kinase so phosphorylates downstream signal transduction proteins. One of those is Dok7 (?). when MuSK is activated, it results in clustering of AChRs by a protein called rapsin. And also a stimulation of expression of the AChR subunit genes along with other synaptic genes. This is through signalling pathways like Rac and JNK. Sequential phosphorylation of proteins that are kinases themselves, so when phosphorylated, they phosphorylate other proteins, etc. Ultimately phosphorylation of a TF called Erm (ET5 (?)). It is activated, binds to promotor region of target genes and induces transcription of those genes. Genes like AChR subunit genes, MuSK, LRP4, Rapsin, etc.
Right picture: focusses on functional domains in proteins
Agrin is a protein that’s glycosylated. It binds to Lrp4. Two agrin molecules bind to two Lrp4 moleules. Bring them together. Results in activation of MuSK. MuSK then phosphorylates the other MuSK molecule. Which results in activation of MuSK. Then further phosphorylate other proteins like Dok7 which then results in further signal transduction.
11.3. [nmj] explain and schematically draw the molecular mechanism underlying selective expression of AChR subunit genes in subsynaptic nuclei
In primary myotubes, innervation initiates transcription of synaptic genes in
myonuclei underlying the neuromuscular contact. The nuclei are called
‘fundamental’ or ‘subsynaptic’ nuclei. AChR cluster size, number and shape are
refined with innervation (E14) that disperses non-synaptic AChR clusters and
induces new neural AChR synaptic clusters through the secretion of Agrin.
Initially, the secondary myotubes are electrically coupled to the primary myotubes
through gap junctions.
-Agrin is a proteoglycan synthesized by the MN and the muscle cell, but only the
nerve-secreted isoform is active and induces AChR clustering. Agrin binds LRP4 but its
post-synaptic effect is transduced through MuSK activation.
-Dok-7 is a muscle-specific adapter protein that is recruited by MuSK once
phosphorylated and induces a cascade of phosphorylation that leads to AChR
clustering.
-In adult/mature NMJs, action potential activity suppresses synaptic genes in
nonsynaptic myonuclei, while their expression is maintained in the fundamental nuclei
by agrin-Lrp4-MuSK signaling and the subsequent activation of transcription factor
Erm, which drives expression of AChR-encoding genes (Chrn) and Musk.
11.4. [nmj] describe the cellular mechanisms that regulate AChR surface expression levels
Parallel signaling pathways at the NMJ are involved in the coordinated maturation of
the presynaptic terminal (green), the postsynaptic muscle fibre (red) and the
perisynaptic Schwann cells (PSCs; grey); dotted lines indicate pathways that are still
being debated. Agrin, which is released by the nerve terminal, muscle fibre and
surrounding PSCs, acts on the LRP4–MUSK complex, which comprises low-density
lipoprotein receptor-related protein 4 (LRP4) and muscle-specific tyrosine kinase
receptor (MUSK). The phosphorylation of MUSK leads to rapsyn-mediated clustering
of ionotropic acetylcholine receptors (AChRs) and postsynaptic maturation. AChR
clustering can also be enhanced by WNT ligands (associated with MUSK in the figure),
whereas release of ACh inhibits AChR clustering. LRP4 is mainly of postsynaptic origin,
although neuronal LRP4 may also have a role. LRP4 acts as a co-receptor for agrin and
stimulates AChR clustering as well as presynaptic maturation by clustering synaptic
vesicles and active-zone proteins. Neuregulin 1 (NRG1) can be released by the nerve
terminal and/or surrounding PSCs, and binds to PSC-expressed or postsynaptic ERBB
receptors (ERBB2 or ERBB3). The binding of NRG1 to ERBB receptors on PSCs
promotes PSC survival and maturation. Although the exact signaling pathway is
controversial, the activation of postsynaptic ERBB receptors by NRG1 may increase
levels of postsynaptic proteins such as rapsyn, MUSK and AChR, and could have a role
in AChR clustering, which overall leads to postsynaptic maturation. Synaptic laminins,
such as laminins β2, α4 and α5, are released by the muscle fibre. They form
heterotrimeric glycoproteins that are included in the basal lamina and are important
for proper pre- and postsynaptic alignment and maturation, as well as PSC maturation (not shown). Laminin β2 binds to presynaptic calcium channels and regulates activezone proteins (not shown). Postsynaptically, laminins interact with integrin β1, which
increases AChR clustering. Fibroblast growth factors (specifically FGF7, FGF10 and
FGF22) are released by the muscle fibre and activate mainly presynaptic type 2B FGF
receptors (FGFR2B); thus, they are important for vesicle clustering and presynaptic
maturation, as are type IV collagen α chains (α2, α3 and α6). PSC-derived
transforming growth factor-β (TGFβ) induces presynaptic maturation and
postsynaptic differentiation by upregulating the expression of agrin. Synaptically
released ATP is detected by PSC-expressed purinergic type 2Y receptors (P2YRs) and
triggers increases in intra-PSC Ca2+ concentrations. PSCs also express muscarinic
AChRs (mAChRs), and their activation by the local application of ACh triggers
increases in intra-PSC Ca2+ concentrations. However, mAChRs are not activated by
endogenous ACh release. Matrix metalloproteinases (MMPs) in the extracellular
matrix (ECM) that surrounds PSCs regulate the composition of the ECM and cleave
matrix proteins such as agrin, triggering its removal from the ECM.
structural components involved in NMJ function
AchE is localized to the synaptic basal lamina and is essential to inactivate ACh. The
homotetrameric subunits co-assemble with the triple helical collagen tail, termed
ColQ, which tethers the entire enzyme to the synaptic basal lamina.
The dystrophin glycoprotein complex (DGC) contains dystroglycan, which is
posttranslationally cleaved into α-dystroglycan (αDG) and the transmembrane
component β-dystroglycan (βDG), the sarcoglycans (α through δ) and sarcospan (ss).
βDG associates with rapsyn to link AchRs to the DGC and connects to α-dystrobrevin
(αDB), to α-syntrophin (syn) and utrophin. Utrophin links the entire complex to the Factin cytoskeleton.
The voltage-gated sodium channels (Nav 1.4) are localized to the throughs of the
synaptic cleft.
Note: Rapsyn (receptor associated protein of the synapse) is a membrane-bound
cytoplasmic molecule that binds AChR and is necessary for AChR clustering
dynamic of the AchR at NMJ
Nicotinic acetylcholine receptors (nAChR) subunits are synthesized in the ER and
exported to the muscle plasma membrane. From the ER, instead of being targeted to
the cell surface, most nAChR subunits are degraded by the ER-associated ubiquitinproteasome degradation pathway. In the postsynaptic membrane, there is significant
lateral diffusion between the synaptic and perisynaptic membrane spaces. Lateral
diffusion of nAChRs from the perisynapse into the NMJ contributes significantly to
maintain the synaptic receptor density. Conversely, when receptors escape from the
postsynaptic density into the perisynaptic space, there is significant internalization of
nAChRs into endosomal compartments. Trafficking through the endosomal pathway,
a fraction of internalized nAChRs is targeted for degradation. However, a significant
portion of those nAChRs actually recycle back into the synaptic membrane,
contributing to the maintenance of the synaptic nAChR pool. Most of these dynamics
are tightly regulated in the NMJ by several stimuli, such as synaptic activity or
association of dystrophin glycoprotein complex components.
11.5. [nmj] discuss the functional properties different muscle fiber types and their differential vulnerability in neuromuscular disease
muscle metabolism
Scheme showing some differences in glucose, lactate, and fatty acid metabolism
between fast and slow muscle fibers. Pathways prevalent in fast or slow muscle fibers
are shown as red or green arrows, respectively. DHAP, dihydroxyacetone phosphate;
GLUT4, glucose transporter 4; F-6-P, fructose-6-phosphate; FAT/CD36, fatty acid
translocase; FFA, free fatty acids; F-1,6-P, fructose-1,6-bisphosphate; F-2,6-P, fructose-
2,6-bisphosphate; G-3-P, glyceraldehyde-3-phosphate; G-6-P, glucose-6-phosphate;
GPD1, glycerolphosphate dehydrogenase 1 (cytoplasmic); GPD2, glycerolphosphate
dehydrogenase 2 (mitochondrial); HK, hexokinase; LDH, lactate dehydrogenase;
MCT1, monocarboxilic acid transporter 1; MCT4, monocarboxilic acid transporter 4;
PDH, pyruvate dehydrogenase; PFK, phosphofructokinase; PFKFB3,
phosphofructokinasefructose bisphosphatase 3; TG, triglycerides.
Muscle fiber type-selective vulnerability of motor axons in ALS
In SOD1-ALS mouse models:
-axons of fast-fatigable MNS:
degenerate presymtomatically
-axons of fast fatigue-resistant
MNs. degenerate at symptom onset
-axons of slow MNs: resistant to degeneration
