Physio Axonal Transport (7)

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  1. ALS (Amyotrophic Lateral Sclerosis)
    • progressive/selective degeneration & death of motor neurons in the brain & spinal cord that causes paralysis of voluntary muscles
    • initial symptoms appear in the arms, hands, & legs as cramping, twitching, & exaggerated reflexes
    • in 75% of classic cases bulbar muscles involved in swallowing, chewing, & speech are affected
  2. What muscles don't tend to be affected in ALS?
    sensory neurons and muscles affecting eye movements & the urinary tract are typically NOT affected
  3. What typically causes death in a patient with ALS?
    • respiratory failure which typically occurs within 3-5 years after symptom onset
    • adult onset is between 40-60 years old (median age: 55)
    • it's more frequent in males
    • sporadic ALS accounts for the majority of cases, while familial ALS is inherited & accounts for 10% of cases
  4. 2 Major Functions of the Axon
    • 1. propagating the action potential from the cell body to the presynaptic terminals
    • 2. providing a physical conduit for the transport of material between the cell body & the synapses
  5. Growing Axon
    • a growing axon uses transmembrane Axon Guidance (Outgrowth) Receptors on its Growth Cone (filopodia-like) to navigate complex guidance cues in its extracellular environment to find its synaptic targets
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  6. What are some necessary entities that are transported from a neuron's cell body down to its synapse via the axon?
    • Synaptic vesicle precursors
    • Neuropeptide-containing dense-core vesicles
    • Ion channels (Na+, K+, & Ca2+ channels)
    • Neurotransmitter receptors
    • Growth factor receptors
    • Organelles (mitochondria, endosomes)
    • Golgi outposts
    • Active zone components
  7. What are items transported from a neuron's synaptic terminal back to its cell body via its axon?
    Axonal injury signals

    Signaling endosomes activated by pro-survival growth factors

    Damaged or misfolded proteins targeted for lysosomal degradation
  8. Vesicle Formation
    • 1. transmembrane proteins or those destined for secretion are synthesized in the ER
    • 2. cargo destined for secretion is loaded into vesicles that bud from the ER at exit sites & traffic to the golgi complex where further processing occurs
    • 3. vesicles containing items being transported bud from the trans-golgi network (TGN) & move to other membrane organelles (endosomes, lysosomes) or to the plasma membrane
    • 4. at the cell surface vesicles fuse w/ the PM via exocytosis & contents are either released into the extracellular space or added to the PM
  9. Coat Proteins
    • assist in the pinching off of vesicles in the secretory pathway
    • eg. COPI, COPII, clathrin, & dynamin (a GTPase)
  10. Clathrin
    • coat protein responsible for vesicle formation at membrane compartments like the trans-golgi network or the plasma membrane during endocytosis
    • SPONTANEOUSLY (no ATP) forms a 3 pronged triskelia structure that can self-assemble into a stable cage around the vesicle as it pinches off from a membrane
  11. How does clathrin associate with membrane protein cargo?
    • via adapter complexes (adaptins) that confer binding specificity
    • how only a specific subset of membrane protein cargo is selected to undergo clathrin-mediated trafficking
    • adaptins bind to specific cargo and facilitate their transport
  12. How is a newly-formed vesicle pinched off from the membrane it originates from?
    via the GTPase Dynamin, which forms a ring at the neck of the budding vesicle & prevents leakage at the membrane during fission
  13. How does a clathrin-coated vesicle fuse with other membranes?
    • a clathrin-coated vesicle is very stable & cannot fuse with other membranes until the clathrin coat is REMOVED by a set of cytoplasmic ATPases (uncoating proteins)
    • the assembly of the clathrin cage is spontaneous, therefore its disassembly requires ATP hydrolysis
  14. COP Proteins (coatamers)
    • coat proteins involved in trafficking between the ER & the Golgi complex
    • COP II: involved in vesicle formation & trafficking from the ER to the golgi
    • COP I: involved in vesicle formation & trafficking from the Golgi back to the ER (retrieve ER proteins back from the Golgi)
  15. In contrast to clathrin, how does a COP protein coat form around a vesicle?
    COP proteins REQUIRE ATP to form the cage around a budding membrane & retain their coats until they dock at a target membrane
  16. Once they reach the membrane of the presynaptic terminal, how do vesicles dock & fuse?
    • they must dock onto receptors in the membrane then fuse to release their contents into the synaptic cleft
    • specialized SNARE proteins are required for this process b/c lipid bilayers are negatively charged & do not fuse spontaneously
    • SNAREs provide specificity for membrane fusion events by targeting specific vesicles to their correct membrane
  17. Synaptobrevin
    the v-SNARE on synaptic vesicles
  18. SNAP-25 & Syntaxin
    • the two proteins that compose the t-SNARE on the plasma membrane of a neuron
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  19. SNAREs & the Synaptic Vesicle Cycle
    • • the vesicle remains in a docked position until an action potential arrives at the synaptic terminal & opens voltage-gated Ca2+ channels
    • • the rise in intracellular Ca2+ triggers synaptic vesicle fusion with the plasma membrane
    • • after fusion, the membrane flattens & the SNARE complexes are disassembled by NSF, an ATPase, & α-SNAP (which attaches NSF to the SNARE)
    • • v-SNAREs & other synaptic vesicle membrane proteins are retrieved by clathrin-mediated endocytosis so that the synaptic vesicle components can be reused

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  20. Fusion is regulated by small GTPases called ____. Disassembly of SNARE complexes requires associated proteins called ___ & ______.
    • Rabs: regulate vesicle & membrane fusion
    • NSF & α-SNAP: disassemble SNARE complexes
  21. Rab proteins
    • small GTPases that regulate vesicle trafficking by binding & slowly hydrolyzing GTP
    • act as molecular switches that assemble in their GTP-bound state & disassemble when bound by GDP
  22. The Synaptic Vesicle Cycle
    • 1. vesicle buds off early endosome
    • 2. imports NTs
    • 3. docks on neuron's plasma membrane
    • 4. is primed using ATP
    • 5. Ca2+ influx causes vesicle & membrane fusion → NTs (contents) are released into synaptic cleft
    • 6. the vesicle - merged with the plasma membrane - is endocytosed so proteins can be recycled
    • 7. ATPases pump protons (H+) into the vesicle, lowering its internal pH
    • 8. it re-fuses/turns back into an early endosome
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  23. How do vesicles move in axons?
    • along microtubules (cytoskeletal tracks), hollow tubes 25nm in diameter composed of long tracks of polymeric α & β tubulin dimers
    • in an axon, the microtubule + end faces away from the cell body (in the soma & dendrites, microtubule orientation is mixed)
  24. Microtubule-associated Proteins (MAPs)
    • MAPs bind to the tubulin subunits of microtubules & regulate their stability ("traffic signals"...)
    • are distributed differentially between axons & dendrites
    • eg. Tau is found only in axons, whereas MAP2 is found only in dendrites
  25. Fast Anterograde Transport
    • how membrane channels, synaptic vesicles, dense-core vesicles (DCVs), and organelles such as mitochondria, endosomes, & multivesicular bodies (MVBs) are transported away from the cell body to presynaptic terminals
    • fast means they move at 0.5-10 μm/sec
  26. Slow Anterograde Transport
    • how actin, cytoskeletal elements like neurofilaments (tubulin + actin), soluble proteins, & clathrin are transported away from the cell body to presynaptic terminals
    • slow means they move at 0.01-0.001 μm/sec
  27. Kinesin
    • the molecular motor for anterograde transport
    • uses microtubule tracks to transport vesicles
    • Tail: interacts with receptors on cargo (light chain)
    • Stalk: responsible for dimerization
    • Head: the motor domain which consists of a catalytic core (ATPase) & a linker (heavy chain)
    • *ATP is required for movement
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  28. Kinesin Tail Domain
    a unique domain responsible for interacting with cargo & various adaptors providing cargo specificity for MT transport
  29. Which family of Kinesins can move towards the minus-end of microtubules?
    • the KIFC subfamily
    • however the majority of kinesins move along microtubules towards the plus-end (anterograde transport in an axon)
  30. Kinesin Uses ATP to 'Walk' on Microtubules
    • 1. in solution, both heads of kinesin are bound to ADP (it is NOT attached to the MT)
    • 2. when one head binds to a MT β-tubulin subunit, its bound ADP is released
    • 3. in this open spot ATP binds, causing a conformational change that thrusts the trailing head forward
    • 3. the new forward head is induced to bind to the MT & in the process releases its ADP
    • 4. now the lagging head hydrolyzes its ATP to ADP + Pi
    • 5. Pi is released, leaving the head bound only to ADP, causing it to dissociate from the MT
    • 6. the head still MT bound has an open spot for ATP, which binds & continues the "walking"
  31. Adaptors
    • proteins that connect cargo with kinesin motor tail domains for MT transport
    • association of cargo w/ adaptors or with the motor can be regulated by post-translational modifications like phosphorylation
    • motors have redundant functions & one type of cargo is often transported by multiple motor family members
    • eg. KIF5 can transport many different types of cargo
  32. Cytoplasmic Dynein
    • an ATPase that moves toward the minus-end of microtubules
    • consists of a large 2 MDa complex of proteins w/ 2 heavy chains & multiple intermediate & diverse light chains
    • is activated by dynactin
  33. What does the large number of proteins in the cytoplasmic dynein complex suggest?
    • that the motor may be able to interact w/ diverse cargo through different associated proteins
    • there's a lot of redundancy built into the transport system
  34. Dynein Retrograde Transport
    • 1. injury signaling – after axon injury dynein transmits signals from the site of injury to the cell body
    • 2. transports lysosomes & vesicles targeted to the lysosome back to the cell body – required for proper degradation of misfolded/damaged proteins in the axon
    • 3. transports neurotrophic factors (BDNF, NGF) & signaling endosomes from target tissues back to the cell bodies – important for neuronal differentiation & survival
  35. Mechanism for Dynein Movement
    The globular heads of the heavy chain have a ring-structure composed of AAA repeats that project MT-binding stalks. ATP binding alters the AAA repeat domain of one head domain, which frees up its MT binding while ATP hydrolysis allows the head to rebind MTs taking a 8 nm step toward the minus-end. The other head binds ATP and repeats the process leading to processive movement along microtubules. A family of dynein motors distinct from cytoplasmic dynein function in intraflagellar transport in cilia.
  36. Amyotrophic Lateral Sclerosis (ALS)
    dominant mutations (G59S) in the microtubule binding domain of the p150/Glued subunit of dynactin result in protein misfolding & aggregation in axons when caused by the inherited form of ALS
  37. Hereditary Spastic Paraplegia (HSP)
    • a neurodegenerative disorder characterized by progressive spastic motor neuron degeneration of the lower extremities starting with a degeneration of synapses
    • various point mutations in kinesin 1/KIF5A results in reduced axonal cargo transport in an inherited form of HSP
  38. Huntington’s Disease (HD)
    • a progressive neurodegenerative disorder caused by expansion of CAG triplets in the gene for huntingtin (htt)
    • HTT & Huntington Associated protein (HAP1) are transported bidirectionally in axons
    • HAP1 binds to both Kinesin light chain & the p150/glued subunit of dynactin
    • overexpression of htt in animal disease models results in defects in axonal transport
  39. Charcot-Marie-Tooth Disease (CMT)
    • a rare progressive neuropathy
    • genetic studies of one family w/ an inherited form of CMT (type 2A1) identified a point mutation in the Kinesin KIF1β that resulted in a defective motor function & peripheral neuropathy in mouse models
Card Set:
Physio Axonal Transport (7)
2014-01-23 00:40:29
MBS Physiology
Exam 1
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