Lecture 18- Presynaptic Events 2

Question 1

What is Botulinum toxin?

Answer 1

-One of the most poisonous biological substances known,
-It is a neurotoxin produced by the bacterium Clostridium
botulinum.
-8 types: (A, B, C1, C2, D, E, F and G).
-Blocks release of the neurotransmitter acetylcholine, at the
neuromuscular junction
-Resulting in muscle paralysis.

Question 2

How long do the effects of botulinum toxin A usually last?

Answer 2

3 months (different types last different periods)

Question 3

Is botulinum toxin used for anything good?

Answer 3

-Is used in the management of a wide variety of medical
conditions
-In 2002, the FDA approved the use of Botox® (Botulinum
toxin-A) for the cosmetic purpose of temporarily reducing glabeller forehead frown lines.

Question 4

How is SNAP25 (t-SNARE) invovled in the functioning of botulinum A?

Answer 4

-Heavy chain of the toxin binds selectively and irreversibly to presynaptic receptors on cholinergic neurons
-Endocytosis
-Light chain is cleaved and released and
-Binds to SNAP25
-Preventing exocytosis/ fusion of synaptic
vesicles with the membrane

-Overall SNAP25 is cleaved by Botulinum Toxin A

Question 5

Other than SNAP25 what else is effected by botulinum toxins?

Answer 5

-Synaptobrevin is also affected by botulinum toxins
-The cleavage of the VAMP (Snyaptobrevin is an example of a V-SNARE) protein by the toxin inhibits vesicle fusion and neurotransmitter release into
the synapse.

Question 6

How does Tetanus toxin effect release mechanisms and how is this different to the effect Botulinum toxin has?

Answer 6

-Botulinum effects SNARE proteins typically which normally result in the release of acetylcholine and muscle contraction. When Botulinum toxin in applied acetylcholine is not secreted which results in muscle contraction not happening and flaccid paralysis.

-Tetanus toxin on the other hand effects Synaptobrevin. Typically Synaptobrevin results in the release of glycine or GABA and mean acetylcholine is not secreted and muscle contraction is halted. Tetanus toxin acts by breaking Synaptobrevin meaning glycine/ GABBA is not secreted and therefore that too much acetylcholine is. Muscle contraction is therefore not halted and this leads to spastic paralysis (muscles tighten).

Question 7

What does a-latrotoxin interact with? Where is it made/ how does it have it’s effect?

Answer 7

-The synaptic protein neurexin
-a-latrotoxin is derived from widow spiders.
-And makes Calcium pores (forms ca2+ channels)
-Works via neurexin binding
-It binding leads to the phosphorylation of SNARE proteins
-This causes massive release of SSV including acetylcholine from sensory and motor neurons
-Release of LDCV remains the same (not effected)

Question 8

Is the release of LDCV and SVV controlled in the same way?

Answer 8

-Release of small synaptic vesicles and large dense core vesicles are controlled in different ways.

Question 9

When compared to SVV what do LDCV require for vesicle fusion?

Answer 9

Dense core vesicles require repetitive and more prolonged stimulation for vesicle fusion

Question 10

In what ways does stimulation strength change neurotransmitter release?

Answer 10

-Both qualitatively and quantitatively
- When stimulation strength is increased it is known as neuromodulation

Question 11

What does weak stimulation strength cause in terms of vesicle/ neurotransmitter release?

Answer 11

-Low frequency action potentials
-Limited release of small synaptic vesicles containing classical neurotransmitters

Question 12

What does strong stimulation strength cause in terms of vesicle/ neurotransmitter release?

Answer 12

-High frequency action potentials
-Increased release of small synaptic vesicles containing classical neurotransmitters
and
-release from large dense core vesicles containing neuropeptides

Question 13

In general how can toxins act to alter release of neurotransmitters/ vesicle fusion?

Answer 13

Toxins can act by interfering with proteins and/ or via altering ion flux

Question 14

What does a calcium influx (as caused by a-latrotoxin) cause?

Answer 14

-Ca2+ influx not only stimulates exocytosis but also releases vesicles from the reserve pool
-Thus rapidly replacing neurotransmitter supply

Question 15

What is some background information about SYNAPSIN?

Answer 15

-Is an abundant evolutionary conserved phosphoprotein found at nearly all
synapses in the nervous system.
-Three distinct SYN genes (SYN1, SYN2, and SYN3)
-Multiple isoforms: I/IIA, IIB, IIIA, and IIIB
-SynI the most abundant isoform in mature neurons

Question 16

What does synapsin control?

Answer 16

-Associated with cytosolic face of small synaptic vesicles (not LDCVs)
-Controls synaptic vesicle mobility
-and post-docking steps of exocytosis (priming and fusion with the membrane)
-Ca2+-dependent phosphorylation following strong depolarization

Question 17

What does synapsin bind to and how do we know this?

Answer 17

-Synapsin binds actin cytoskeleton
-Know because of images which show Actin + purified rat brain synaptic vesicles with and without synapsin. Without the vesicles are just scattered everywhere, whereas with they align with the cytoskeleton

Question 18

What does K+ depolarization result in?

Answer 18

Movement of SV and synapsin to the active zone

Question 19

What results in phosphorylation of Synapsin? What is the effect of phosphorylation?

Answer 19

-CaMKII
-The phosphorylation state of synapsin regulates synaptic vesicle availability

Question 20

What is the effect of injection of a peptide that competes with synapsin binding?

Answer 20

-Disperses the distal cluster of SVs, while docked SVs remain intact.
-Synaptic depression is hastened following peptide injection. Basically this means that inhibiting/ opposing synapsin impairs the activity of the synapse. A readily releasable reserve pool disappears.

Question 21

What cellular changes to excitatory synapses happen in Synapsin Knockout?

Answer 21

-SYN deletion selectively reduces the size of the Reserve Pool of SVs
-This results in:
! Impairment in glutamate release and
! Delayed recovery from synaptic transmission
! with relatively normal basal transmission.

Question 22

What cellular changes happen to inhibitory synapses in synapsin knockout? How does this differ to the excitatory synapses?

Answer 22

-Note that evidence suggesting that synapsins regulate synaptic vesicle pools differently in excitatory and inhibitory synapses
-Specifically, knockout of synapsins in inhibitory neurons results in a loss of
synaptic vesicles from both the readily releasable pool and the reserve pool
-While knockout of synapsins in excitatory neurons has a selective effect on the reserve pool of synaptic vesicles.

Question 23

For synapsin what does the knock out phenotype look like?

Answer 23

-Genetic deletion of single or multiple SYN genes produces a severe epileptic phenotype. This occurs after 2–3 months of age and becomes progressively more intense
-Schizophrenia-like phenotypes
-An impairment in the inhibitory network and a shift towards excitability has a behavioural effect of cognitive impairments.
-These cognitive impairments take the form of emotional and spatial memory deficits. This means impaired object recognition and fear memory.

Question 24

What human diseases does alterations to synapsin result in?

Answer 24

-Schizophrenia and other psychoses
-Aggressive behaviour
-Learning disabilities
-Epilepsy
-Autism

Mutation result in:
-mRNA degradation and reduced levels
-Impaired phosphorylation

Question 25

How does Synapsin relate to autism spectrum disorder?

Answer 25

-Synapsin knockout mice as a model of human diseases such as epilepsy and autism.
(impairments in social behaviour, social communication and repetitive behaviours)
-There a lot of different molecules and neurotransmitters effected in what is known as the synaptic autism pathway and synapsin is one of them.

Question 26

What is a knock out mouse?

Answer 26

-Genetically modified mouse

-Where a particular gene has been inactivated (knocked out). By disrupting it with an artificial piece of DNA

-This can occur throughout development, or “conditional”- only i.e. the gene is knocked out in a particular cell type or stage of development

-Can compare behaviour with and without the gene of interest (littermate controls)

Why do this?
-To determine function of a gene
-Model a disease

Question 27

What do the results show from the study that used knockout mice to disrupt synapsin?

Answer 27

Controls: No significant difference in….
-Motor function
-Olfactory impairments
-Anxiety-related behaviour
(checking that Synapsin knockout has not effected processes that where it should not have an effect)

-Non-control: Evaluated measure of social interaction and various behaviours.
-Using a social approach test
-young SynII−/− mice had reduced interest for both cups
-Basically, mice that have had the Synapsin gene knocked out have impairments in their social behaviour mimicking ASD
-The results demonstrate an involvement of synapsins in the development of the
behavioral traits of Autism Spectrum Disorders.

By cups mean this…
-cup without mouse (exploring objects)
-mouse in cup (exploring socially)
Preference talks to preference for