Axonal Transport

Question 1

Axonal Transport

Answer 1

transport of proteins and organelles essential in all cells
more extreme in neurons
highly polarised
syntheisied in cell body and required in different locations

Question 2

Challenge in Axonal transport

Answer 2

Big journey – long distances to cover

Question 3

• Microtubules

Answer 3

Rails at which long distance axonal transport occurs
Composed of tubulin dimers of alpha and beta tubulin
Polymerise to assemble micro tubule
Plus end quick growth minus end slow
Protofilament composed of head-to-tail arrays of tubulin dimers, are arranged in parallel.

Question 4

Polarity-

Answer 4

In axons MT uniformly orientated plus end pointing away from cell body and toward growth cone and synapse

Question 5

Anterograde

Answer 5

Toward plus end

Question 6

Retrograde

Answer 6

Towards minus end

Question 7

MT base Molecular motors

Answer 7

Mechanincal enz 
ATPases – ATP > ADP to generate force 
2 families : 
Kinesin
Cytoplasmic Dynamin

Question 8

Kinesin 1

Answer 8

Anterograde
Plus end directed towards the synapse
Heterotetromeric protein tail stalk and motor domain
2 heavy chains and 2 light chain
Light chain regulates heavy chain – inhibits activity of motor unless carrying something
Vesicles huge and motor proteins tiny

Question 9

Kinesin superfamily :

Answer 9

14 kinesin families
41 in humans
Majority plus end directed

Question 10

Cytoplasmic Dyneins-

Answer 10

MT based
Retrograde – minus end directed
Multi subunit complex
Dynactin component involved in binding cargo

Question 11

Axon transport is divided into what two categories?- how was it discovered

Answer 11

Slow and fast
-Found using radioactive methionine – shows any new protein synthesis – radioactive methionine shown in the protein
slice up
Gel
See which proteins labelled and how far have they travelled along optic nerve or sciatic nerve

Question 12

Fast

Answer 12

40Cm per day

Membrane bound organelles

Question 13

Slow

Answer 13

1mm per day

Proteins

Question 14

How does the difference in speed arise ?

Answer 14

Both fast and slow axonal transport are mediated by the same “fast” molecular motors kinesin and cytoplasmic dynein. The slower overall rate of slow axonal transport is due to prolonged pauses between movements – “stop and go” model (express vs. local train)

Question 15

Molecular components of MT base axonal transport

Answer 15

• Engines- Dynein and Kinesins
• Fuel – mitochondria
• Carriages – Neurofilament
• Rails- Microtubules

Question 16

Defects in neurodegenerative disease :

Answer 16

Length dependent neuronal disease – degeneration of long motor neruons e.g CMT/ MND
Pathologies – Axonal and cell body accumulation of organelles and proteins
Axonal swelling filled with card (should be transported down axons )
Axonal transport is disrupted

Question 17

Neurodegeneration examples and pathologies

Answer 17

Soma filled with neurofilament in ALS < Pathology
TDP-4 accumulation in MND < things not being trafficked
Incorrect accumulation – incorrect location
Shown using transfection – GFP labelled mitochondria accumulate in the cell body
Less mitochondria in the axon in mutant

Question 18

HSP-

Answer 18

affect upper motor neuron s
Cell bodies in the motor cortex cross the spinal cord – connects brain to LMN
Large accumulation of proteins that should be transported

Question 19

Prime evidence comes from MND

Answer 19

• ALs
• Distal hereditary motor neuropathy
• Spinal Muscular atrophy
• Hereditary Spastic paraplegia (HSP)

Question 20

Upstream effect

Answer 20

Axonal transport defects are observed before the onset of neurodegeneration/ suggesting that they are the cause rather than consequence
Failure to supply essential components

Question 21

Molecular mechanisms of Axonal transport defects

Issues with

Answer 21

1. Motor proteins
2. MT
3. cargo
4. Mitochondria

Question 22

Mutations in what part of the molecular mechanism have been identified ?

Answer 22

Mutations in molecular motor identified in a number of disease
LOF mutations
HSP – mutation in Kinesin
ALS- part of dynactin complex

Question 23

Hallmark pathologies for many human neurodegenerative diseases:

Answer 23

Axonal and cell body accumulations of organelles and proteins
Axonal swellings filled with cargo

Question 24

How Do Axonal Transport Defects Cause/Contribute to Neurodegeneration?

Answer 24

Axonal transport defects are observed before the onset of neurodegeneration/disease suggesting that they are part of the cause of disease rather than a consequence!
Failure to supply essential components/energy to the axon/synapse
Failure to clear waste (e.g. misfolded proteins, non-functional mitochondria)
Impaired signalling – pro-survival and/or stress signals

Question 25

mutations in molecular motors :

Answer 25

All LOF Motor neuron disorders HSP (SPG10): Kinesin-1 Reid, et al. Am. J. Hum. Genet. 2002; J. Med. Genet. 2003 ALS: p150Glued (a component of the dynactin complex) Puls, et al. Nat. Genet. 2003 CMT: KIF1Bß (Kinesin-3 family member); transports vesicles. Zhao, et al. Cell 2001

Question 26

Phosphorylation of molecular motors

Answer 26

Molecular motor activity and interaction with cargo can be regulated by phosphorylation Abnormal/Excessive activation of kinases is a hallmark of neurodegenerative diseases Stress kinases: p38 MAPkinase, JNK ALS, Huntington’s Disease GSK3ß Alzheimer’s Disease

Question 27

Stress kinases: p38 MAPkinase

Answer 27

P38 MAPK is activated in ALS Phosphorylation of kinesin light chain by p38 MAPkinase halts kinesin-1 mediated transport of mitochondria. De Vos et.al

Question 28

Stress kinases: c-Jun N-terminal kinase (JNK)

Answer 28

Huntington’s Disease, SBMA (poly-Q androgen receptor) | Phosphorylation of the kinesin-1 motor domain by JNK prevents its interaction with microtubules

Question 29

Alzheimer’s Disease: GSK3ß and CK2

Answer 29

- Phosphorylation of kinesin light chain by GSK3ß prevents attachment of kinesin-1/KLC to vesicles - Casein kinase 2 (CK2) activation by amyloid beta induces phosphorylation of KLC and disrupts axonal transport

Question 30

Steric hindrance by Tau

Answer 30

Main MT binding protein High levels of tau in aLzheimers MT bound tau prevents kinesin 1 transport Trap and causes release kinesin molecules detach upon encountering tau

Question 31

Stabilization of Microtubules: Role of Tau

Answer 31

Tau stabilizes axonal microtubules | Phosphorylation of tau by e.g. GSK3ß (activated in AD) releases tau from microtubules -> destabilisation

Question 32

MT Bundling -

Answer 32

MT bundling – mutations in spastin – cause Mt to bundle so don’t form usual network

Question 33

Spastin

Answer 33

Spastin most common gene mutated in HSP LOF of spastin which usually severs MT into pieces Causes reduction in anterograde transport of mitochondria

Question 34

PTM of Tubulin

Answer 34

- Acetylation of tubulin cause increase binding of kinesin 1 and transport - Histone deacetylase 6 inhibition compensated for transport deficit in HD – increase rates of transport

Question 35

Are hallmark for pathology of ALS

Answer 35

Phosphorylation of Neurofilaments

Question 36

Phosphorylation of Neurofilaments

Answer 36

Accumulation axons not in the cell body – disease \NF heavy phosphorylated in cell body

Question 37

Mitochondria

Answer 37

Mitochondria are selectively targeted in many neurodegenerative disease that involve axonal transport defects: Motor neuron disorders ALS: SOD1 HSP: paraplegin Alzheimer’s Disease: Aβ Parkinson’s Disease: Pink, DJ1 Huntington’s Disease CMT: mitofusin 2 Reduced mitochondrial function leads to ATP deficit which will affect axonal transport Dysfunctional mitochondria show reduced anterograde transport (Miller and Sheetz, JCS, 2004)

Question 38

Miss-assembly of Neurofilaments

Answer 38

Mutant NFL in CMT | Mutant HSPB1 in Distal Hereditary Motor Neuropathy

Question 39

De Vos

Answer 39

Familial amyotrophic lateral sclerosis-linked SOD1mutants perturb fast axonal transport to reduce axonal mitochondria content

Question 40

Mitochondria in parkinsons

Answer 40

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin - Mitochondrial dysfunction in Parkinson’s disease - The Parkinson’s disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila

Question 41

ALS mutations inhibit what?

Answer 41

ALS mutations inhibit anterograde transport of mitochondria

Question 42

Defective axonal transport leads to what??

Answer 42

Defective axonal transport leads to depletion of axonal mitochondria

Question 43

Defective axonal transport leads to what??

Answer 43

Defective axonal transport leads to depletion of axonal mitochondria And increased mitochondrial spacing

Question 44

The Mitochondrial Hypothesis

Answer 44

Dysfunction of mitochondria leads to reduced axonal ATP. Depletion of mitochondria from axons causes/exacerbates axonal transport defects Age-related damage to mitochondria may amplify any primary defects to axonal transport. In this way, disruption to mitochondria may explain why many neurodegenerative diseases are diseases of old age.