I1 - pre-syn mechanisms Flashcards
(55 cards)
1
Q
what are the properties of NT release probability?
A
imperfect, variable, can be spontaneous
2
Q
what are the key requirements of the pre-synaptic machinery?
A
- need pool of vesicle ready to be released on demand (RRP)
also have recyclable pool and reserve pool (~85%) - need a fast, local siognal to trigger exocytosis on demand in response to AP (VGCCs)
- machinery for exocytosis needs to respond to Ca: SNARE + Ca2+ sensor
- release machinery needs to be in close proximity to Ca2+ channels for speed + sensitivity: active zone, Ca channels tethered to synaptic machinery
- renewable machinery: vesicle capture + endocytosis of proteins + membranes + local repackaging of vesicles
3
Q
what is the quantal theory?
A
there is EM evidence for NTs being secreted from vesicles in discrete packages (quanta)
4
Q
what are some methods of experimentally measuring NT secretion?
A
- fluorescent loaded labels: OQA: vesicles caused to load fluoro dye, solution washed off + vesicle visualised during AP, as cell destains
- electrophysiology: plasma membrane store charge, vesicle fusion -> more membrane -> patch electrode onto exocytosing cell -> chunks of fusion = steps in cell capacitance
- electrochemical methods to detect NTs directly
- genetically encoded fluoro reporters to visualise real time release
5
Q
what are the modes of NT release?
not synch/asynch/spont
A
- synaptic vesicle release – classic
- dense-core vesicle release – bulkier NTs like neuropeptides
- transporter reversal – uptake transmitter reversed e.g. drug induced (SERTs for SSRIs + DATs for amphetamines), mb in glial cells
- diffusible NTs – lipids/gaseous NTs, control synthesis via Ca2+ sensitivity
6
Q
what are the 3 key SNAREs that form the SNAREpin complex
A
- synaptobrevin (v-SNARE) (VAMP): on vesicle membrane
- syntaxin + SNAP-25 (t-SNARE): on target membrane
synaptobrevin + syantxin contribue 1 a-helix & SNAP-25 contributes 2 to form core 4 SNARE motif complex required for fusion
7
Q
what are R and Q-SNAREs?
A
R = arginine, tends to correspond to v-SNARE
Q = glutamine, tends to correspond to t-SNARE
8
Q
how large is a SNARE motif?
A
60-70aas
9
Q
What was the Weber et al 1998 expt on v and t-SNAREs?
A
- made recombinant v- and t-SNAREs, reconstituted them into separate lipid membrane + found out which combinations would fuse
- fluorescent receptor in the membrane, quenched within membrane but fluoro when membrane fusion occurred due to membrane mixing
- measured spontaneous associations of vesicular + plasma membrane
- if add v-SNAREs to v-SNAREs, nothing, if add to t-SNAREs –> fluorescence
10
Q
describe the process of SNARE docking
A
high energy state, then SNAREs dock -> loose trans-SNARE complex -> primed -> tight trans-SNARE complex (SNAREpin)
-> SNARE motifs align, then SNARE proteins bring membranes together, at this point there are very few energetic barriers -> membrane zippering + fusion pore formation -> cis-SNARE complex -> i.e. membranes are fused
11
Q
how exactly does a trans-SNARE complex (SNAREpin) become a cis-SNARE complex?
A
- 3 helices of t-SNAREs bind at N terminals, assemble with 4th helix (anchored in vesicle membrane)
- as the membranes get closer, SNAREs zip together, and when the binding reaches the C-terminal end, they zip tighter together until the inward force causes all SNAREs to snap flush together
- generates a fusion pore, and causes the membranes to snap together
- this is sterically prevented until fusion occurs by physical protein obstacles
12
Q
how does BOTOX work?
A
- protease, cleaves VAMP (synaptobrevin), syntaxin or SNAP-25 -> stops spontanesou fusion (relevantly, at NMJs)
- clinical uses: blepharospasm, upper motor neuron syndromes, chronic migraine
13
Q
what are some additional proteins in regulation of docking?
A
- Munc18 controls SNARE complex assembly by binding syntaxin + stabilising its conformation -> promotes complex formation + other SNARE binding
- Munc13: initiates priming by displacing Munc18 from SNARE, promotes zippering though AP still needed for release
- Rab: GTP-binding proteins, bring vesicles into active zone + catalyse interactions between vesicles + other proteins
- Rab effectors (RIM): scaffolding proteins, recruit Munc13 to active zone
- complexin - fusion clamp in absence of Ca2+, functions as a brake via steric hindrance
14
Q
how are synucleins implicated in modulating synaptic vesicle fusion?
A
- synucleins enhance rate of fusion pore dilation during secretory vesicle exocytosis
- inferred from observations that exocytosis of neuropeptides from dense core vesicles was slowed in synTKO mice (a, b and y KO) [Somayaji 2020] – a-syn oligomers can inhibit SNARE-complex formation by binding synaptobrevin 2
15
Q
how is the vesicle membrane retrieved from the presynaptic membrane post-fusion?
A
via endocytosis
- endophilin promotes invagination , recruitrs dynamin (GTPase) -> promotes fission of clathrin-coated vesicles from membrane
- synaptojanin-1 (Dephosph lipids to release adaptor proteins + allows auxilin binding) -> auxilin stimulates removal of clathrin coat
16
Q
mutations in what proteins have been associated with EOPD?
A
endophilin : SH3GL2 (GWAS)
auxilin: DNAJC6
bc recycling of vesicles needs to occur immedaitely after exocytosis to allow for further NTission
17
Q
what are fusion regulators?
A
- Ca2+: the rise of intracell Ca through VGCCs drives fusion + NT release [Katz 1967]
- synaptotagmin (Ca2+ sensor) [Takamori 2008]
18
Q
where have synaptic experiments typically been conducted?
what did they show?
A
Calyx of Held (auditory system) – is big
- load pre-syn terminal with caged Ca compound (light sensitive cage), when shine light -> Ca is released -> intracell Ca increases locally, transient current occurs and EPSC is detected
- proves you don’t actually need an AP, just need Ca2+ release
19
Q
what is synaptotagmin?
A
vesicular protein, 16 mammalian isoforms, 1, 2, 9 are common
2 Ca2+ binding domains: C2A and C2B = a Ca-sensing unit
exhibits cooperative CA binding: 4 or 4 Ca needed per synaptotagmin
C2B inserts into CSM in Ca-dependent manner, but only PIP2-containing membranes (i.e. CSM only)
20
Q
how does synaptotagmin work?
A
as Ca enters cell it binds synaptotagmin domains -> S does conformational change -> bends pre-syn membrane towards SV -> removes complexin - a steric hindrance -> overcomes energetic barriers -> fusion pore created
Silva 2021: synaptotagmin KO prevents synchronous release upon stimulus
21
Q
whats the point of synaptotagmin?
A
gives the cell an extra way to regulate NT release probability, means that AP arriving does not always lead to NT release
in some synapses you want release probability to be 1 to prevent energy wastage
but maybe you want a burst of activity to regulate NT release – artificially lower fidelity – if only a little bit of Ca enters, synaptotagmin is not activated as needs 4/5 Cas, but if another AP arrives in quick succession -> threshold met + NT released: RESIDUAL CA HYPOTHESIS
+ can be dynamically adapted w short-term plasticity
22
Q
what is NSF?
A
a fusion protein, ATPase
ATP hydrolysis dissembles the SNARE complex into individual SNAREs
23
Q
what does alpha-SNAP do?
A
adapts SNARE and NSF to let them bind to each other
24
Q
brief describe the SNARE cycle
A
high energy state, vesicle + membrane unbound -> docking -> loose trans-SNARE complex -> priming -> tight trans-SNARE complex
-> Ca2+ influx + synaptotagmin -> zippering + fusion pore formation -> cis-SNARE complex -> dilation -> clathrin coat
-> auxilin stimulates removal of coat -> a-SNAP and NSF dissemble SNARE complex -> high energy state
25
what are the 3 modes of vesicular release?
* synchronous: rapid, large (>2nA), shortlived
* asynchronous: rapid after AP, small (~0.2nA), persists beyond AP
* spontaneous: nothing to do w AP, termed 'minis' (<50pA), reflects release of individual quanta?, gets Ca from from VGCC sources
26
what are the Ca sensors for the 3 types of vesicular release?
synch: synaptotagmin 1, 2, 9
asynch: syt7?
spontaneous: syt1? doc2?
27
what is the function of spontaneous vesicular release?
stabilises synapse/receptor function or development
28
what was the experiment detailing the differences between modes of vesicular release?
Pang & Sudhof 2010
got asynch firing when KO'd synaptotagmin-1 i.e. the synaptotagmins that mediate fast, synchronous release and complexins
spontaneous: seen when VGCCs are blocked i.e. it depends on extracell + intracell Ca2+ and Ca2+ release from stores
29
what are some expts done to investigate spontaneous release (mEPSCs)?
* measured in the presence of VGCC and VGSC inhibitors, but P/Q-type, N- and P/Q-type or R-type VGCC blockers didn't block NT release
* but is Ca sensitive: in purkinje cells:
- if add intracell Ca buffer BAPTA-AM, depol event decrease in frequency
- if extracell Ca removed and intracell Ca buffered, mEPSCs almost entirely disappear. if add thapsigargin (mobilises intracell Ca stores) -> depol occurs [Xu 2009]
- and if extracell Ca is increased from 2 -> 5 mM, see more spontaneous release [Yamasaki 2006]
30
through what mechanism does spontaneous release of glut signal?
through eEF2 kinase
- ketamine-mediated suppression of resting NMDAR activity -> inhibition of eEF2 kinase, dephsoph of eEF2 and augmentation of BDNF synthesis
this happens through spontaneous transmission?
31
what key mechanisms do the 3 modes of release appear to share?
fusion mech mediated by synaptobrevin-2, SNAP-25 and syntaxin-1
supported by strong impairment of spontaneous release when t-SNAREs are KO'd or KD'd, and complete loss in absence of Munc-18
32
what are the 4 critical mechanisms for synchronous release?
* generation + maintenance of RRP that can be quickly exocytosed upon Ca2+ entry
* pre-syn VGCCs opening briefly with minimal delay upon AP arrival
* release machinery must respond quickly to sharp AP-gated Ca signals + to sharp decay of signal
* pre-syn Ca channel must be spatially coupled to Ca2+-sensing mechanism, so [Ca] incr and decr quickly when channels open/close
these steps in combination SYNCHRONISE release, asynch + spontaneous may differ in 1 of 4 steps
33
what proteins are involved in the priming step of vesicle fusion?
Munc13 - acts after docking, before fusion
RIM - possibly recruits Munc13 to active zone + activates it. docks vesicles via Rab3, though additional mechs are involved (found by KO)
synaptotagmin + CAPS
34
what is the current model of SNARE complex assembly?
RRP vesicle arrested in fusion-ready state with a partially-zippered SNARE complex
complexin inhibits zippering until Ca binds to synaptotagmin
OR
SNARE zippering just proceeds after Ca2+ triggering and partially zippered state is not needed
35
what is experimental evidence for Syt1 being the Ca2+ sensor for synchronous release?
* abolishing Syt1 in flies, worms, mice impairs synchronous release + causes a shift to asynchronous release [observed 1990s/2000s]
* subtle Syt1 mutations altering Ca-dependent PPL binding in vitro altered release probability -> Syt1 = Ca-dependent switch
36
describe the actions of RIM proteins
central, tether Ca2+ channels to presyn release sites
form tripartite complexes w RIM-binding proteins and Ca2+ channels
RIM N-terminals bind Rab3 and Munc13 -> tether vesicles close to Ca channels to promote fast, synchronous release
additional parallel mechs: involve ELKS, SNARE proteins, bassoon and neurexins, contributions not well understood
37
where is the delayed release due to asynchronous release commonly seen?
cerebellar granule cells, dorsal horn synapses and deep cerebellar nuclei-> inferior olive synapses (>90% of NT release)
38
what is the physiological relevance of asynchronous release?
provides smooth and graded inhibition during high-freq activation e.g. in cochlear nucleus and inferior olive. may contribute to coincidence detection
at climbing fibre-purkinje cell synapse: desynch of vesicle fusion may be more effective at triggering multiple APs in PCs
generally: may elevate overall transmission, activating high-affinity receptors as NT stays around, but less effective at triggering spiking
39
what may be the disease involvement of asynchronous release?
more prominent in spinal muscular atrophy, AD and epilepsy
40
how have we studied asynchronous release? why is this bad?
typically done by attributing long-lasting components of average synaptic currents to asynch: but complicated by factors e.g. NT accumulation, spillover, receptor desensitisation etc
preferable: detect quantal events + use multiple trials to construct histogram to identify a component of release corresponding to AR
must avoid expt conditions where intrinsic neuronal firing properties/reverberating circuit produces appearance of AR
41
how are we starting to study asynch release now?
- inhibit dynamin or incr presyn activity -> reduces synch and asynch release
- Ca channel blockade may incr asynch as fewer vesicles are released synchronously
- Sr2+ replacement of extracell Ca can demonstrate asynch dependence on Ca --> Sr2+ enhances asynch due to less effiicent buffering/extrusion [Goda 1994]
42
is asynchronous release dependent on intracellular Ca2+?
yes
Ca triggers asynch release, though through different mechanisms
intro of slow Ca2+ chelator EGTA eliminates AR, but not SR
suggests Ca2+ sensor for AR is further from the Ca2+ source, responding to bulk cytosolic Ca not high local Ca
43
how was the crayfish used to investigate asynch release?
crayfish NMJ: linear relationship between freq of quantal events + presyn Ca levels revealed when [Ca] is less than 600nM
much steeper dependence on Ca2+ found for higher level
suggests AR is mediated by specialised Ca sensor w linear dependence on Ca
then, Ca2+ photolysis was used to determine Ca dependence of AR at calyx of Held
44
what was expt on AR in calyx of Held? and in hippocampus?
* Ca2+ photolysis in Syt2 KO mice where release prominently AR
* found asynch sensor had surprisingly low affinity but lower Ca cooperativity than Syt2
* in H: expt in Syt1 KO hippocampal neurons found glut release shifted from steep to ~linear
suggests specialised Ca2+ sensor mediates AR with a cooperativity of 1-2, acting when presyn Ca is <0.5um
45
what are the hypothesised Ca sensors for AR? why?
- Syt7: slow kinetics: Morpholino KD at zebrafish NMJ supports this, reduces AR while Syt2 KD reduces SR
- in syt7 KO inhib cortical synapses, AR unaltered, so cannot be sole mediator
OR
-Doc2: have 2 C2 domains similar to synaptotagmin
- some evidence that Doc2 acts as a Ca sensor but also some that AR is unaffected by reducing Doc2. controversial hypothesis.
46
what are Ca sources for AR?
- regulation of presyn Ca signalling, due to buffers and prolonged Ca decay following sustained activity, could regulate AR
- activity-dependent activation of Ca-permeable TRPV1 and P2X2 receptors may promote AR
- VGCCs provide longer-lasting phase of Ca2+ entry contributing to AR after prolonged depol but not a single stimulus
- CICR can promote vesicle fusion
- mitochondria influence AR by sequestering Ca and reducing intracell [] during high activity
47
what other random proteins are involved in AR?
- synapsin 2 desynchronises NT release at inhibitory synapses + can regulate AR
- SAP97 regulates AR at some synapses via N-cadherin control
48
how is spontaneous release measured?
as mini postsyn current (mPSCs) in the presence of TTX to block AP
49
what are the proposed functions of spontaneous release?
* regulate neuron excitability: contribute to basal extracell NT levels + activation of high-affinity receptors
* single quanta can trigger APs in small interneurons
* synaptic stabilisation + LTP: disruption can induce pre-syn homeostatic plasticity
* spontaneous glut release maintains dendritic spines by activating AMPARs and restricting GluR1 AMPAR diffusion
* regulates protein synthesis in dendrites + plasticity by activating NMDARs, despite Mg2+ block
50
how is spontaneous release regulated?
complexin: KO/KD in drosophila, c. elegans and cultured veterbrate neurons increased spontaneous event frequency
canonical SNAREs
51
what is the role of Ca in spontaneous release?
* can be Ca independent or dependent on bulk/local Ca
* less dependent on extracell Ca than evoked release
* blocking TRPV1 eliminates >90% spontaneous release at some brain stem neurons, suggests TRPV1 tonic activation causes Ca influx driving spontaneous release
* at cerebellar inhibitory synapses, spontaneous release evoked by Ca2+ transients from intracell stores
significant fraction remains after Ca buffering or channel blockade, is this really Ca independent or driven by bulk Ca?
52
what are proposed Ca sensors for spontaneous release?
* syt1: seen in cultured hippocampal cells
* doc2: KO or KD reduces spontaneous release >50% without altering, but reduction rescued by Ca2+-binding Doc2, suggests Doc2 regulates spontaneous release but may not be Ca2+ sensor itself
53
does spontaneous release use the same vesicle pool as the others?
fluorescent dye studies provide conflicting results
most likely mediated by shared vesicular pool employing synaptobrevin 2
54
how did Somayaji et al use FSCV to detect electrochemically active NTs?
* recording electrode placed in dorsal striatum, stimulating electrode in ventral midbrain
* evoked DA release from SNc projections
* 30 pulses at 20Hz, 50Hz and 90Hz
* also 20, 30 and 60 pulses at constant 50Hz
* evoked DA release measured: correlated with stimulus frequency or pulse number
* demonstrates synchronous release
55
what are downsides of electrochemical methods of measuring NTssion?
* only measures bulk concs of NTs in extracell space: can be influenced by reuptake and diffusion
* cannot directly visualise single fusion events or provide spatial info
* could use fluoroscent sensors (genetic) to allow visualisation of NT release in real time in vitro
