Physio Axonal Transport (7)

• 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

sensory neurons and muscles affecting eye movements & the urinary tract are typically NOT affected

• 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

• 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

• 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
• 

• 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

Axonal injury signals

Signaling endosomes activated by pro-survival growth factors

Damaged or misfolded proteins targeted for lysosomal degradation

• 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

• assist in the pinching off of vesicles in the secretory pathway
• eg. COPI, COPII, clathrin, & dynamin (a GTPase)

• 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

• 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

via the GTPase Dynamin, which forms a ring at the neck of the budding vesicle & prevents leakage at the membrane during fission

• 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

• 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)

COP proteins REQUIRE ATP to form the cage around a budding membrane & retain their coats until they dock at a target membrane

• 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

the v-SNARE on synaptic vesicles

• the two proteins that compose the t-SNARE on the plasma membrane of a neuron
• 

• • 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

• Rabs: regulate vesicle & membrane fusion
• NSF & α-SNAP: disassemble SNARE complexes

• 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

• 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
• 

• 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)

• 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

• 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

• 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

• 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
• 

a unique domain responsible for interacting with cargo & various adaptors providing cargo specificity for MT transport

• the KIFC subfamily
• however the majority of kinesins move along microtubules towards the plus-end (anterograde transport in an axon)

• 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"

• 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

• 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

• 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

• 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

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.

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

• 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

• 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

• 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