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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
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What muscles don't tend to be affected in ALS?
sensory neurons and muscles affecting eye movements & the urinary tract are typically NOT affected
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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
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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
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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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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
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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
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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
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Coat Proteins
- assist in the pinching off of vesicles in the secretory pathway
- eg. COPI, COPII, clathrin, & dynamin (a GTPase)
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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
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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
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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
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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
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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)
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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
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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
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Synaptobrevin
the v-SNARE on synaptic vesicles
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SNAP-25 & Syntaxin
- the two proteins that compose the t-SNARE on the plasma membrane of a neuron

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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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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
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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
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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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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)
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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
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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
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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
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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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Kinesin Tail Domain
a unique domain responsible for interacting with cargo & various adaptors providing cargo specificity for MT transport
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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)
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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"
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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