Lecture 3 - Molecular Presynaptic Plasticity Flashcards
What are 3 mechanical ways the presynapse could possibly change?
Probability of release
Filling of vesicle
Transmitter conc in cleft
How else could the presynapse change and give examples
Retrograde messages
NO, CO, neurotrophins, endocannabinoids
How can the postsynapse change mechanically?
Insertion of new receptors
Gating of existing receptors (phosphorylating NMDAR means it gates more)
What are the 4 proposed ways a synapse ‘learns’?
Presynaptic mechanisms
Retrograde messages
Postsynaptic mechanisms
Number of synapses
What experiment did Zucker and Regehr (2002) perform?
Paired-pulse facilitation
- Gave 2 pulses spaced at a set time
- Plotted interval between them and size of response
What was the result of Zucker and Regehr’s (2002) experiment?
Response to second pulse was bigger because residual calcium still in synapse
Short term placticity
What is post tetanic potentiation (PTP)?
The stable response carrying on a few minutes after pulses have stopped due to calcium signalling activating CAMKII which phosphorylates proteins
Why do you get depression that lasts as long as the stimulus?
Presynapse runs out of synaptic vesicles
What protein is involved in mobilisation of synaptic vesicles?
Synapsins
What proteins are involved in docking and priming of synaptic vesicles?
Rab3A/RIM1a/14-3-3
What proteins are involved in synaptic vesicle fusion?
SNAREs, munc18
What animal has a very large synapse and so is good for looking at vesicles?
Lamparay
What does RRP stand for and what is it?
Readily releasable pool
Vesicles that are bound to the membrane ready to be released
What is the recycling pool?
Vesicles being turned over ready to attach to the membrane
What is the reserve pool?
Vesicles tethered to actin cytoskeleton
What is synapsin and what is a feature of it?
The bridge between the synaptic vesicle and actin cytoskeleton
Heavily phosphorylated
What happens to make the vesicle fall off the cytoskeleton?
Protein kinase A phosphorylates synapsin
What did Paul Greengard do and what was the result?
Knocked out synapsin 1 and looked for memory defects.
No effect on the mouse at all.
What did George Augustine do and what was the result?
Knocked out all 3 synapsins.
Mouse was fine however it did not recover from synaptic depression.
How many are there and what do Rab proteins generally do?
60
Like passports for membrane fusion events
What Rab protein is involved in synaptic vesicle exocytosis and what happens?
Rab3
Binds to GTP and binds to an effector bound to the membrane allowing fusion.
What are the SNARE proteins?
Syntaxin
SNAP-25-C
VAMP (Synaptobrevin)
What protein is involved in calcium sensing on the vesicle?
Synaptotagmin
How did Fernandez-Chacon (2001) show that synaptotagmin was the calcium sensor if knocking it out is lethal?
- Mutated residues in the Ca binding pocket but don’t chelate Ca.
- Meant synaptotagmin didn’t bind Ca quite as well
- R233Q was most effective residue
- Measured response, less neurotransmitter release for mutant, reduced EPSC
What else did Fernandez-Chacon do and what was strange about the results?
- Did a high frequency pulse test
- Mutant had an increased response to short term placticity
Why does the synaptotagmin mutant have an increased response for short term plasticity
As the residual calcium increases it catches up
What is the protein process of vesicle fusion?
1 - SNARE-independent vesicle tethering (Rab3)
2 - t-SNARE activation
3 - SNAREpin formation and synaptotagmin binds
4 - SNAREpins zip up mediating fusion
5 - SNAP and NSF use ATP to disassemble SNARE complex
How did Sudhof try to find the molecular mechanism for mfLTP?
- Knocked out several presynaptic PKA substrates (synapsins, Rab3A, Rabphilin) but all left normal mfLTP.
What is RIM1 alpha and what happened when Sudhof knocked it out?
- Rab interacting molecule Sudhof knocked out
- Required for SV priming
- Knock out mouse had no mfLTP
What did Sudhof then discover about RIM1 alpha and how?
- Made a knock in mutant by putting an alanine instead of a serine in
- Mouse had mfLTP showing the knock out not having it was caused by something else
What are the features of synaptotagmin 12 (syt12)
- Does not bind Ca
- PKA phosphorylates Ser97
- Binds Syt1 (not PKA-dependent) to regulate its interaction with SNARE complex
What are phospho-specific antibodies?
Inject a peptide into a rabbit and hope it will make antibodies against the region that is phosphorylated.
This antibody can then tell you if the protein if phosphorylated or not.
How can you stimulate mfLTP?
Add Forskolin which activates adenyl cyclase
What is the model for mfLTP?
1 strong stimulation = loads of calcium
2 Ca activates AC = loads of cAMP
3 cAMP activates PKA which phosphorlyates synaptotagmin 12 (Syt12)
