3. Neuromuscular junction

Question 1

How do we prevent an over-excitation of the post-synaptic membrane?

Answer 1

1. Desensitization: the receptor changes its conformation, not binding Sodium ions anymore, although capable of binding of ACh.
2. The enzyme acetylcholine esterase within the synaptic cleft causes the degradation of the transmitter and thus keeps its level constant.

Question 2

allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme’s active site

Answer 2

allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme’s active site

Question 3

The evoked post-synaptic potential (EPSP) is determined by three factors

Answer 3

1. The total number of ACh-receptors.
2. The open-state probability of each receptor.
3. The refractory period

Question 4

Antagonists of ACh

Answer 4

Curare (reversable) and Bungarotoxin (permanent)

Question 5

Calcium channel types relevant for ACh release

Answer 5

P/Q and N

Question 6

The ESPS is preceded by?

Answer 6

A Ca2+ influx in the presynaptic neuron and ACh release

Question 7

Explain exocytosis

Answer 7

Mobilization: the big increase in ca2+ activates CAM kinase 2, which phosphorylysis synapsin. Synpasin becomes inactive and the vesicle is released
Trafficking: the vesicle moves along the membrane. We need GTP Rab3A for this
Docking-priming: vesicle markers such as synaptobrevin associate with their membrane partner (here SNAP-25). The vesicle is then bound to the membrane and the fusion can happen

When the vesicle approaches the presynaptic membrane it’s called priming, when the presynaptic membrane catches the vesicle it’s called super priming and when the fusion pore is set up and NTs are released it’s fusion pore opening

Question 8

What are important proteins/thing in keeping the vesicle close the the Ca2+ receptors?

Answer 8

The SNARE complex and RIM and RIM binding proteins supported by Munc13 and Rab/27

Question 9

What protein is responsible for endocytosis

Answer 9

clathrin

Question 10

What shapes does the Neuromuscular junction take under development

Answer 10

moves form a plug to prezel like structure. The pretzel structure is done after 3 weeks

Question 11

synaptic elimination

Answer 11

when all but one synapse is eliminated 3 weeks after birth between the pre- and postsynaptic neurons in the neuromuscular junction

Question 12

Process of a synapsis being formed in the neuromuscular junction

Answer 12

Agrin attaches MUSK (muscle specific tyrosine kinase) and thus interacts with Rapsyn to create ACh-R Clustering

Question 13

What determines the amount of AChR in synaptic formation?

Answer 13

neuregulin levels (it binds to its post-synaptic receptor the erb-kinase. Activation of the erb-kinase initiates the MAPK pathway and starts gene expression of the ACh-receptor)

Question 14

What’s the importance of laminin-221 and it’s beta-2 chain?

Answer 14

The matrix protein laminin-221 plays a major role in correct pre-synaptic differentiation of the NMJ.
The Beta-2 channel are localized in the synaptic cleft at the place at which pre- and post-synaptic compartments meet. This association leads to accumulation of the pre-synaptic voltage-gated calcium channes (N- as well as P/Q-type). The cluster formation proceeds an enhanced Ca2+ influx which is recognized as a differentiation signal. The signal implies stop of axonal elongation and start of pre-synaptic differentiation

Question 15

Congenital myathenic syndromes (CMS)

Answer 15

Disorder develops at birth or in childhood (the earlier the worse). Takes the pre, post or synaptic form (fast/slow channel)

Question 16

fast vs slow channel postsynaptic CMS

Answer 16

fast channel: missing receptors or don’t stay open long enough
Slow channel: channels stay open too long

Question 17

treatment for presynaptic CMS

Answer 17

3.4 DAP will lead to more Ach, AChE blockers will lead to a longer stay of Ach in the synapse

Question 18

treatment for slow-channel CMS

Answer 18

Quinitine, Fluoxetine. Both drugs are long-lived, open-channel blockers of AChR that shorten theduration of channel opening events in a concentration dependent manner.

Question 19

autophagy

Answer 19

Autophagy is the body’s way of cleaning out damaged cells, in order to regenerate newer, healthier cells, according to Priya Khorana, PhD, in nutrition education from Columbia University. “Auto” means self and “phagy” means eat. So the literal meaning of autophagy is “self-eating.”

Question 20

Myasthenia Gravis (MG)

Answer 20

MG is an autoimmune and genetic neuromuscular junction disorder. Autoantibodies against the ACh-receptor lead to enhance autophagy of the receptor. This, in turn, causes a widening of the synaptic cleft with less foldings. The distance between pre- and post-synapse increases, thus delaying the ligand binding to the receptor

Question 21

Treatment of Myasthenia Gravis

Answer 21

AChE inhibitor, especially pyridostigmine, are used to provide symptomatic relief but this is rarely complete and does not alter the basic disease.

Immunosuppressive therapies include corticosteroids and azathioprine or related drugs that suppress antibody synthesis.

Plasmapheresis, removing the plasma and the antibodies to the ACh receptor, often ameliorates symptoms within days or a few weeks, but the benefit is transient.

Intravenous administration of immunoglobulins also reduces the titer of antibodies to the ACh receptor by mechanisms that are not clear.

The temporary benefit may be sufficient to prepare a patient for thymectomy or to support the patient through more severe episodes.

Question 22

Lambert Eaton Syndrom

Answer 22

LES is an autoimmune disease with antibodies against the P/Q-type calcium channels

Question 23

Treatement of Lambert Eaton Syndrom

Answer 23

potassium channels blockers are used to prolong the depolarization of the membrane to keep the calcium channels longer open and enhance the vesicle release.

Question 24

Motor neuron disease

Answer 24

A disease group where the dysfunction of the neuromuscular end-palte leads to degradation of the motor neuron

Question 25

Motor neuron 1/2

Answer 25

Motor neuron 1 (upper motor neuron) is in the cortex and motor neuron 2 (lower motor neuron) is in the spinal cord - damage to the upper motor neuron gives upper motor neuron syndrome (e.g. spasticity) and damage to the lower motor neuron gives lower motor neuron syndrome (e.g. paralysis).

Question 26

culturing of motor neuons

Answer 26

They need to be in a toti-potent state (embryonic) from mice. Embryos are prepared at day 13,5 post conception. At that time point motor axons already innervated the target muscle. After preparation of the spinal cord the “enriched” primary motoneurons re-growth their axons again. This process is termed differentiation. Can live up to a week in a dish

Question 27

monogenetic

Answer 27

relating to or involving the origin of diverse individuals or kinds by descent from a single ancestral individual or kind the entire present human population is monogenetic.

Question 28

Spinal muscular atrophy

Answer 28

is a fatal monogenetic disorder in childhood. It’s characterized by weakness and wasting (atrophy) in muscles used for movement (skeletal muscles). The weakness tends to be more severe in the muscles that are close to the center of the body (proximal) compared to muscles away from the body’s center (distal)

Question 29

explain SMN genes

Answer 29

SMN1 and SMN2. SMN 1 encodes 100% SMN protein. The human SMN2 carries a point mutation in exon 7, which affects a splicing enhancer site. This in turn causes skipping of exon 7 leading to a truncated and non-functional protein in 80-90 % of all cases. SMA patients do not have enough full-functioning SMN protein to survive. The higher the SMN2 copy number the higher the SMN protein level, the less severe is their phenotype. The phenomenon is termed “gene dose effect”.

Question 30

What’s the main function of the SMN complex?

Answer 30

To assemble SNURP (small nuclear ribonucleoprotein particle) and transport to the nucleus to setup the spliceosomal complex.

Question 31

Why are mouse models of SMN difficult/unnatrual?

Answer 31

Only the primates carry two SMN copies. On human level one SMN gene copy harbor the splice enhancer mutation (SMN2). Mice have only one SMN copy which encodes 100 % SMN protein. Depletion of the murine Smn gene is embryonic lethal. The expression of the human SMN2 in mice overcomes embryonic lethality and leads to a SMA mouse model. The higher the SMN2 level the less severe is the phenotype comparable to SMA patients

Question 32

What’s the pathology behind Spinal muscular atrophy ?

Answer 32

While we observe paralysis, this is not caused by loss of motor neurons, but rather functional loss at the neuromuscular junction. We see a decreased level of vesicles and the motor neuron is growing out of control. The growth cones on the motor neurons are smaller with less ß-actin signal intensity as well as reduced ß-actin mRNA. hnRNPR might be implicated

Question 33

What does less beta-actin mean for the motor neuron?

Answer 33

This indirectly caused less Ca2+ channel activity and there by less exocytosis

Question 34

How do we treat lacking Ca2+ channels in SMN?

Answer 34

To bypass the decreased Ca2+ influx, blockers of the Cav2.2/2.1 channels were applied (R-Roscovitine). R-Roscovitine reduced the closing kinetics of the channels to keep them longer open.

Question 35

How can we genetically treat SMA?

Answer 35

We increase SMN2 transcription in one of 2 ways: 1) antisense oligonucleotides bind to the enhancer splice site to prevent exon 7 splicing leading higher SMN protein level or 2) infecting SMA patients with a AAV9-SMN cDNA (adenoassociated viruses carrying SMN cDNA) the SMN protein level also increased

Question 36

Spinal muscular atrophy with respiratory distress type 1 (SMARD1)

Answer 36

Symptoms of SMA starts proximally, SMARD1 starts distally. respiratory distress comes up earlier than in SMA. Two variants: Infantile (lethal within the first year) and juvenile (lifespan up to 10 years) form

Question 37

Genetic background of SMARD1

Answer 37

Recessive mutations of the Immunoglobulin µ-binding protein 2 gene (IGHMBP2) on chromosome 11

Question 38

Ighmbp2

Answer 38

Ighmbp2 is a ATP-dependent 5’ 3’ helicase, which unwinds RNA duplices in vitro. Ighmbp2 is a ribosome-associated helicase primarily binds to the 60S ribosome subunit

Question 39

The IGHMBP2 mouse model

Answer 39

The neuromuscular disorder (Nmd) mouse model. Unlike the SMA mouse model, the one coding for SMARD1 is “natrual”. Two weeks after birth the mouse develop a hind limb paralysis. 4-6 weeks after birth a paralysis of the diaphragm is detectable. The paralysis of the diaphragm is due to a myopathy as the nervus phrenicus is not affected (thus no atrophy)

Question 40

Do we think that the SMARD1 starts at the neuromuscular endplate?

Answer 40

No, because in contrast to SMA, motoneuron loss is pre-symptomatically detectable (we see motor neuron loss before issues with the presynaptic area). We don’t see any presynaptic defects in the motor neurons that are still alive

Question 41

IGF-1

Answer 41

IGF-1 is a growth factor which supports nerve as well as muscle cells and works as treatment for mouse models of SMARD1. PEG_IGF-1 fixes:
- digaphram regrowth
- decrease in muscle deficits
- Reduced IGF-I blood serum levels
- stimulates IGF-IR activation and Akt phosphorylation
- increases terminal sprouting
However, it does not compensate for motoneuron and motor axon loss in SMARD1 mice

Question 42

Ighmbp2 deficiency leads to?

Answer 42

enhanced axonal branching behavior and reduced β-actin protein level in growth cones. however, there is no changes of ß-actin mRNA in the growth-cone of SMARD motor neurons
There is no alteration of spontaneous Ca2+ transients