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Cell Bio Mechanisms > neuronal cytoskeleton > Flashcards

neuronal cytoskeleton Flashcards

1
Q

homeostasis in axons

A

after neurogeneis in development
the same neurons remain throughout life
so maintaining homeostasis in them is v important

cytoskeleton plays role in this

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2
Q

Neuromuscular junction

A

v large
see distinct structure in microscopy

terminus of motor neuron contains cluster of synaptic vesicles
lots of fusion of membranes - so many mitochondria present to provide ATP

lots of cytoskeleton organises synaptic structure

myelin secreted by glial cell to protect axon

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3
Q

synapse types

A

terminal:
-synapse formed at end of axon
-common for neuromuscular junction

en passant:
-littered along axon’s length
-branch out slightly (not to extent of dendrite)
-many synapses in brain are en passant

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4
Q

Axonal cytoskeleton components

A

acin
MT (^conserved)
neurofilaments (neuron specficic)

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5
Q

importance of axonal transport

A

neurons are non dividing
cannot regenerate
machinery used in them needs repleneshing

axonal transport does this
many neurodegenerative diseases (eg motor neuron disease) result from transport machinery malfunction

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6
Q

axonal transport cargo types

A

lots of cargo are cyroskeletal proteins (tubulin, G-actin, neurofilament subunits)
lots are synaptic vesicle precursors or components of synapse

-NT receptors
-mitochondria
-SV precursors
-lysosomes, autophagosomes - degradation machinery
-endosomes

lots of transport synapse -> cell body too

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7
Q

fast v slow axonal transport

A

fast - 50-400mm/day
>vesicles
>membrane bound organelles

slow - 0.2-10mm/day
>cytoskeletal components
>harder to observe - less well understood

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8
Q

identification of slow and fast transport

A

radiolabel AA
inject into dorsal ganglion of Spinal cord
labelled AAs incorporate into axons
follow pulse over time period

segment nerve along its length
look at presence of labelled proteins in that section
bands corresponding to some proteins were detected in later segments earlier than other proteins
FASTER transport

could identify proteins present in segments by using IP for known proteins (from the mWt on the autoradiograph)
and using mass spec for unknown ones

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9
Q

axonal transport machinery

A

same essential machinery used for fast and slow
act in diff way

MTs - the track
gives directional polarity for transport
minus ends towards body
plus ends facing axon tip - more dynamic closer to synaptic structure

motors:
kinesin (anterograde soma-> synapse) towards plus end
cytoplasmic dyenin - retrograde - towards minus end

this grants directionality to the cargo

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10
Q

axonal cargo machinery and disease

A

mutatiosn in kinesins in vertebrate axons
give neurodevelopmental or neurodegeneration disease phenotypes

dyenin mutations linked too

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11
Q

motor types in axonal transport

A

only one cytoplasmic dyenin

many kinesins
some primerily dendritic (excluded from axons)
some kinesins are shared
some exclusively axonal

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12
Q

kinesin superfamily

A

can be + or - end directed (most + directed)
bind diff cargoes

primarily are transporters

kinesin-1 present in axons
but is used generally in many transport processes in many cell types

Kinesin-3 (KIF1A)
exclusive to axons
transports SV structures

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13
Q

Kinesin-3 (KIF1A) discovery screen

A

first discovered in C. elegans genetic screen
look for mutants with specific uncoordinated movement phenotype
curl up instead of move in sinusoidal wave

Unc mutants (uncoordinated)

Unc-104 protein - conserved across organisms
known as Kinesin-3

transports mature AND precursor SVs

mutant was uncoordinated because cargo wasnt reaching synapses properly

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14
Q

Visualising axonal transport w Kinesin-3

A

GFP-Rab-3
Rab-3 part of SV
can see GFP fluorescence

use Kymograph to plot fluorescence distance along axon (x-axis) against time (y-axis)
diagonal lines indicate movement along axons (steeper=faster?)

do this with Kinesin-3 mutant known to cause disease phenotype
see synaptic vesicles not moveing
kymograph shows straight vertical lines

shows that kinesin-3 is transporting SVs

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15
Q

KIF1A (kinesin-3) associated neurological disorder KAND

A

many diseases associated with KIF1A mutations
dont know what the mutations are doing specifically
can test them using model systems

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16
Q

Testing KIF1A human disease mutations in model systems

A

PH - membrane binding domain

human and C elegans KIF1A relatively conserved
mutated residues in C. elegans that are associated w human KIF1A-associated disorders
replicate them in model
when we do this - generates loss of function uncoordinated phenotype seen in unc-104 mutations

molecular level:
look at c. elegans tail motor neuron
en passant synapses
make R254Q mutation in unc-104 (KIF1A)
get uncoordinated phenotype
synapses seen to be formed in wrong place - in the dendrite - flips the conformation
either because Unc-104 id tranasporting vesicles to the wrong place and not at all
get no snyaptic transmission as they are in dendrite and not at NM junction

can mimic mutations in human disease
and see effects on model systems to get more info than could in a vertebrate system

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17
Q

Dyenin basic

A

megadalton enzyme
only one kind of them in cells
instead uses accessory subunits to confer specificity - uses ADAPTERs
is main minus oriented motor (apart from v specific minus directed kinesins)
transports all minus end directed cargo

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18
Q

dyenin adapter proteins role

A

confer cargo specificity to dyenin

not just interactor between motor and cargo
also are activators of motility
need right adapter on right protein to activate motility

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19
Q

in vitro molecular motor assay

A

hard to do with recombinant dyenin as it just wouldnt move

instead can use TIRF microscopy to watch individual fluorescently labelled motors move in cells

Dyenin on own - see binding and unbinding of MTs
but no movement

add in adapter proteins - allowed dyenin movement to begin

adapters need to be present to activate movement
prevents dyenin from wasting energy with no cargo bound

20
Q

dyenin dysfunction in axon trafficking

A

mutations in dyenin v rare
as is important in many cellular processes (as is present in all cells - only one dyenin)

DYNC1H1 cytoplasmic dyenin 1 heavy chain 1
mutant leads to neurodevelopmental/degeneration symptoms

hypothesised from this that inhibition of dyenin mediated axonal transport is sufficient to cause motor neuron degeneration

21
Q

targeted inhibition of dyenin in neurons

A

engineer cell specific KO of dyenin in neuron subset of cells after brain has developed in transgenic mice

KO dyenin
shows neurodegenerative symptoms

these mice displayed Gait abnormalities
dyenin absence = decrease in movement parameters

dyenin mediated transport important to keep homeostasis of neuron and prevent neurodegeneration

22
Q

Dyenin and huntington’s disease

A

mutation in HTT protein
HTT interacts w Dyenin activating adapter HaP1
adapter for lysosomes
so cant do retrograde lysosome transport

no clearing of degraded products
this could be causing neurodegeneration

23
Q

Niemann-Pick disease and dyenin

A

mutations in NCP1/2
change in lipid composition of lysosome membranes
excess lipids esp. cholesterol accumulating in lysosome membrane

effect not clear
suggestes that this change in composition causes kinesins and dyenins to no longer bind them properly
affecting where lysosome ends up in cell

-increased dyenin clustering
-anterograde transport reduced
-causes childhood neurodegeneration

24
Q

growth cone cues

A

1 can move
2 can respond to external cues - repulsive and attractive

attractive cues trigger growth cone to move there
repulsive cause contraction of cone

25
Q

growth cone cytoskeleton

A

v enriched for actin and MTs

Actin throughout lamellipodium like structure

MTs v dense in the shaft and get sparse towards the growth cone structure
MTs invade the spiky Filopodia regions of the growth cone

26
Q

actin dynamics and growth cone protrusion

A

actin is v polar polymer
pointed and barbed end (more growth from barbed??)

filopodia = linear F-actin bundled together by actin bundling proteins
lamellipodia = branched actin network

actun depolymerisation collapses growth cones
drugs eg Cytochalasin D/Latrunculin
see no F-actin staining, see no growth cone movement
growth cone motility similar to cell migration

27
Q

MT instruction of F-actin remodelling in G cone

A

aren’t necessary for movement
important for telling the actin structures where to go -give direction to actin growth
also stabilise actin structures
also allow trafficking of machinery required for growth cone propulsion

so 2 diff jobs here

28
Q

microtubule drugs effect on growth cone direction

A

Taxol - stabilises MT filaments
>control: G cone moves towards cue
>Taxol+: growth cone movement strays from path of cue
- so MT turnover dynamics are essential for axon growth/guidance

perturb dynamics w MT stabiliser
=cones dont move in right direction
so axonal tips dont go right place

29
Q

Growth cone MT dynamics modulation by MAPs

A

MTs undergo dynamic instability
grow and shrink
tightly regulated by many diff proteins that help to stabilise or depolymerise
can control the growing and shrinking

MTs are a lot less dynamic in the axon shaft than in the growth cone as the MTs need to remain there as tracks

can create more MT turnover by either
-changing plus end dynamics
-or generating more MT ends by cutting MTs

30
Q

MT severing enzymes

A

Spastin
Katanin
Fidgetin

hexamers that can slice MTs
can create dynamic ends where tehre werent before (ie by moving the tubulin-GTP cap)

in vitro these proteins fragment the MTs up
doesnt happen like this in growth cones
these proteins are locally regulated in growth cone tips to generate new structures

31
Q

MT severing enzymes and disease

A

most mutated gene in hereditary spastic Paraplegia
katanin involved in autism related disorders

32
Q

Spastin depletion

A

RNAi KO of spastin translation
reduces axonal branches

dendrite architecture remains normal

no. of primary axon branches is greatly diminished

primary branches are a result of the growth cone being stabilised
and this growth cone stabilisation isnt occurring in absence of spastin

the MT dynamics involved in g cone stabilisation is being impaired in spastin KO
-need constant MT turnover to stabilise growth cone
-perturb protein that affects MT turnover
-so g cone less stable
-so reduced primary branches (rely on stable growth cone)

correlated evidence saying that this is possible mechanism for how dynamics affect growth cone guidance

33
Q

KIF21A and disease

A

ocular synkinesis linked to mutations in kinesin KIF21A
Congenital fibrosis of extraocular muscles type 1 - CFEOM1

patients with this mutation have issues w eye blinking movement pattern
many mutations associated w this are in KIF21A
most of these in either motor domain or coiled coil region

34
Q

KIF21A mutant study in mice

A

recapitulate the KIF21A motor domain and coiled coil domain mutations in mouse model

ocular nerves found to have an axon guidance defect
leaves no coordinated movement between ocular muscles and neuron

reduce number of neurons involved in eye movement
because if neurons dont reach right target they can degenerate

in KIF21A mutant knock in mice
saw this reduced no.

35
Q

KIF21A deletion, and MT dynamics

A

if delete this kinesin completely
there is not this reduce neuron phenotype
so this mutation is a dominant effect on patient phenotype

KIF21A regulates MT dynamicls
labelled KIF21A seen by the PM
links the MT to the cell cortex allowing local regulation of cell dynamics

KIF21A mutations in CFEOM1 generate OVERACTIVE Kif21A
WT Kif21A dont move very well
these mutants move a lot more (gain of function)

36
Q

effect of overactive Kif21A

A

in WT - motors in inhibited state
has to bind particular structure or be in particular location to be activated

this inhibition is not occuring in the GOF CFEOM1 mutants

knock in animals have larger growth cone
and more filopodia
so overactive growth cone
gives lots of unregulated movement
makes it hard for ocular nerves to reach target
so get uncoordinated ocular muscles

37
Q

axon regeneration

A

brain/CNS not well
PNS/sCord - better

factors:
- neuronal survival
-axon re-extension
-synapse reformation
-myelination
-experience dependent refinement of newly formed circuits

axons stay in same place for long time
when sever axon
need to reactivate the extension machinery to re-extend

38
Q

Aplysia model system for axon regen

A

has good regen capacity

cut neuron
growth cone like structure appears
propels axon growth
regeneration needs to bring back growth cone dynamics

39
Q

effect of severing axon

A

massive restructureing of MT and actin cytoskeleton after axon severing
lots of filament polymerisation/growth
lots of vesicles delivering new membrane to growth cone

40
Q

non regenerating neuron phenotype

A

retraction bulb appears
axon dies :(

need to sever MTs w Severing proteins to get growth cone reactivation and avoid this i think

41
Q

identifying genes involved in axon regen

A

invertebrate models more suited to regeneration (vertebrates regen less) and are also more genetically tractable

C. elegans genetic screen
-has axonal branches across body’s width
-sever one of these
-grows back to other side after developing g cone like structure
- mutagenesis screen to identify mutations that cant generate this g cone-like structure
-mutants have retraction bulb-like structure instead

42
Q

DLK-1 pathway

A

conserved pathway across species

DLK-1 MAPK activated after axon is cut
activates signalling pathway
activation of TFs
a lot of MT associated proteins - MAPs
control MT turnover and dynamics at axonal tips
allows extension of axonal growth cone

43
Q

investigating DLK-1 effect on MT dynamics during axon regeneration

A

track MT plus ends with GFP-EBP2

sever axon
3hrs-not many EBP2 tracks
6hrs-lot more EBP2 tracks

in absence of DLK-1
3hrs-not much change from control
6hrs-fewer GFP-EBP2 tracks at axonal tip

DLK-1 mutant has reduced no. of plus ends and MT length at later time point

so DLK-1 important for burst of axonal growth after Axotomy
can push axons to regenerate by affecting MT dynamics

44
Q

DLK-1 specific target to give axon regen

A

DLK-1 KO
cut axon
no regrowth

DLK-1, KLP-7(MT depolymerising kinesin) KO
rescues axonal growth in teh DLK-1 deletion strain

DLK-1 is promoting MT polymerisation by directly/indirectly inhibiting Klp-7
OR otherwise affecting MT dynamics by promotion of some other factor
this is needed for MT growth forwards

45
Q

DLK-1 mechanism use in regenerative medecine

A

create sCord injury in rat
use MT stabilising epoB drug (like taxol but it can pass blood-brain barrier)
gave greater sCord axon regeneration

kind of stabilising the MTs in the axons after injury - cna regain ability to regen part of axons

controlling MT dynamics is one way to promote regen mechanisms
these drugs dont just target one particular MT type so dont know MT specificity
but can say that affecting dynamics is sufficient to increase sCord regen

Cell Bio Mechanisms (11 decks)

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