Axon Guidance Flashcards
Neuronal differentiation
Follows neural precursor (neuroblast) migration
Layer IV neurons differentiate before Layer III neurons migrate through
Stepwise:
* Neurite outgrowth
* Axon and dendrite specification
* Target selection and stabilization
* Synapse formation
Differentiation is regulated by intracellular and extracellular signals
Cultured hippocampal neuron after 8 days in vitro (DIV) expressing green fluorescent protein (GFP)
Neurite extension and axon specification
Stage 2 neurons
Neurite extension
No discernable axon
Neurite extension and axon specification
Stage 3 neurons
Axon specified
Establishment of neuronal polarity
Symmetry breaking
Neuron structure and function depend on structural polarity
Dendritic polarization
- Receptors
- Mixed polarity microtubule structure
- Organelle distribution
Axonal polarization
- Synaptic vesicles
- Unipolar microtubule structure
- Organelle distribution
Microtubule structure
Microtubules are made of a-b-tubulin heterodimers
* Alternate in single protofilament
* GTP in pocket
* hydrolyzed upon dimer- dimer binding
Microtubule is made of 13 protofilaments
Slight angle of protofilament interaction yields a helical tube
Plus end – fast growing
Minus end – slow growing
Microtubule Dynamic Instability
Loss of cap OR slow growth that leads to GTP-GDP conversion before new heterodimer
addition leads to depolymerization/shrinking
GTP cap keeps microtubule stable/growing
Stages of microtubule dynamics
Growth
Constant addition of new
heterodimers and presence of GTP cap
Stages of microtubule dynamics
Shrinking
Hydrolysis of GTP to GDP leading to instability of the polymer and heterodimer release
Stages of microtubule dynamics
Catastrophe
Direct conversion from growth to shrinking
Stages of microtubule dynamics
Rescue
Direct conversion from shrinking to growing
Microtubule structure differs
between neuronal compartments
- Dendrites have mixed microtubule polarity
- Axons have unipolar microtubules (plus end facing distal process)
- Growth cones have highly dynamic microtubules
Implications for function
Polarized localization of proteins requires sorting and transport
NgCAM
Cell adhesion molecule that is axonally polarized
Polarized localization of proteins requires sorting and transport
TfR
Receptor that is dendritically polarized
Polarized localization of proteins requires sorting and transport
Vamp2
Synaptic vesicle protein
Active transport moves cargos into axons or dendrites
What moves the cargos?
Microtubule-based motors move cargos into axons and/or dendrites
- Microtubules are plus end out in the axon
- Microtubules have “mixed polarity” in dendrites (~half are + end out and ~half are – end out)
- Microtubule polarity dictates direction of motor movement
- Kinesins move cargo towards microtubule plus ends
- Dynein moves cargo towards microtubule minus ends
Do the motors steer or do the cargos tell the motors where to go?
Smart motor
Motor selects axon or dendrite
Do the motors steer or do the cargos tell the motors where to go?
Cargo steering
- Address label on cargo dictates where motor moves
- Address label likely applied during protein processing/vesicle formation in the Golgi apparatus
Still an active area of research
Secretory Pathway
Soma: Mitochondria, Nucleus, rough ER, Golgi apparatus
- Newly synthesized proteins are oftentimes translated into the rough ER
- Vesicles containing these proteins bud from the rough ER and move into the Golgi for processing
and packaging for transport
Vesicle coats direct steering of cargo in the cell body
CopII coated vesicles
ER to Golgi
CopI coated vesicles
Golgi to ER (retrieval)
Clathrin coated vesicles
Golgi to plasma membrane
Clathrin coat assembly:
* Adaptor proteins bind cargo receptor
* Clathrin binds adaptor proteins
* Vesicle buds
* Clathrin coat removed, adaptor proteins remain
Adaptor protein complexes may steer
cargo into neuronal compartments
- AP4 complexes concentrate cargoes at TGN
- Vesicles bud
- Bind kinesin motors
- Transport to axon terminals
- In C. elegans AP1 has been shown to coat vesicles for
dendritic transport and AP3 for axonal - Different cargos in different AP coated vesicles
The Axon Growth Cone
- Large, flat lamellipodia
- Spikes emerging from lamellipodia are filopodia
- Microtubules invade center of the lamellipodia
- Actin underlies filopodia
Growth cones are highly motile
Growth cone can be divided into regions
P Domain
Peripheral domain
Contains actin networks
Growth cone can be divided into regions
T Zone
Transitional zone in the
middle
Growth cone can be divided into regions
C Domain
Central domain
Contains bundled microtubules with dynamic ends
Mouse hippocampal neuron
- Actin labeled by phalloidin (white in A and green in merges)
- Microtubules labeled by antibody label against tyrosinated tubulin (white in B and
red in merges)
Intracellular growth cone actions
to guide axon outgrowth - actin
- Filopodia can reach 5-50um or more, allowing growth cone to sample large area of the environment
- Receptors on filopodial tips allow environmental factors to guide the growth cone
Chick growth cone: - Left label with fluorescent phalloidin to mark polymerized actin
- Right electron micrograph showing branched and
bundled actin networks in the growth cone
Actin Protofilament
- Individual actin subunit – G (globular) actin
- 375 amino acid polypeptide
- Binds ATP/ADP
- ATP bound when not in filament
- Hydrolyzes to ADP when filamentous
- alpha-actin - muscle
- Beta and gamma actin non- muscle cells
- Subunits assemble into filaments (F-actin)
- Filaments have a + and – end
- = fast growing; also known as barbed end
- = slow growing; also known as pointed end
How does actin influence the growth and dynamics of the growth cone?
Actin is actively treadmilling in the growth cone to extend filopodia and facilitate growth cone motility.
Disrupting actin dynamics significantly slow outgrowth and makes the growth cone insensitive to directional cues.
Actin Assembly
- Actin subunits can assemble spontaneously but are highly unstable
- Rapidly disassemble
- For filamentous actin formation, nucleation must occur
Actin Nucleation
Initial aggregate stabilized by multiple subunit-subunit contacts that forms the stable base of an assembling filament
Arp2/3 complex
Arp: Actin related protein
- Nucleates actin at the minus end allowing plus end growth/minus end stability
- Arp2/3 bind to side of filamentous actin to seed new filament
- Forms a branched actin network
Arp2/3 Actin Nucleation
- Protein structures of actin, Arp2 and Arp3
- Arp2/3, stimulated by an activating factor, change conformation
- This allows binding of actin monomers and subsequent filament growth
- Branched actin is nucleated by Arp2/3
Formin Actin Nucleation
Family of proteins
- Work as dimers
- Bind to actin plus end
- Recruit two actin monomers to grow filament
- Functions to build straight/unbranched actin filaments
Regulators of actin assembly
- Actin monomer concentration can regulate polymerization
- More actin monomers = more likely to have polymer growth
- Cellular concentration of actin monomers high enough that without regulation there would be uncontrolled actin filament growth
- Regulation of monomer availability
Regulators of actin assembly
Thymosin
- Binds actin monomers
- Prevents their ability to incorporate into filamentous actin
Regulators of actin assembly
Profilin
- Binds actin monomers
- Enhances their ability to integrate into a filament
- Formin-mediated assembly is augmented by Profilin presence on actin monomer
Filamin
- Allows filaments to be bound at roughly right angles
- Forms gel-like actin essential for lamellipodia
Essential for cell migration
Mutations in Filamin cause
periventricular heterotopia
- Instead of migrating into the cortex, newly born neurons stay where they are born in the periventricular region
- Form nodules (arrows to the left)
- Associated with epilepsy that is resistant to medication and intellectual disabilities
Two aspects particularly important for motility of the growth cone
- Polymerization and recycling of actin filaments
- In the P domain, myosin II motor walks on actin to create traction forces that physically pull the growth cone forward towards adhesion sites
Actin-mediated growth cone
motility
- Growth cone extends towards attractive cues and away from repulsive ones
- Mix of actin polymerization in the leading edge
- Actomyosin-based contraction in the Tzone
- Actin depolymerization between the peripheral and T-zone
3-6um/min
Mix of actin polymerization in the leading edge
Regulated by profilin and thymosin
Actomyosin-based contraction in the T-zone
Regulated by myosin which is an actin-based motor protein
Actin depolymerization between the peripheral and T-zone
Regulated by cofilin and actin depolymerizing factor (ADF)
The “Molecular Clutch”
Actin retrograde flow does not exert force on its own. Force requires connection to substrate
* Substrate = extracellular matrix
* Connection is through focal adhesions
Microtubule regulation and growth cone motility
Microtubules are contained in the C domain
Highly dynamic: Lots of growth and retraction
What happens when dynamics are disrupted?
Effect of microtubule drugs on growth cone cytoskeletal structure is loss of dynamic microtubule ends. Decreasing microtubule dynamics makes growth cones unable to respond to extracellular cues for guidance
Actin and microtubule dynamics are essential for growth cone guidance.
While axons will continue to grow (very slowly), they cannot
respond to directional cues without cytoskeletal regulation.
Retinal ganglion cell innervation of LGN
- Axons from retinal ganglion cells in the eye enter CNS through optic nerve
- Axons then branch and go either ipsi- or contra-laterally
- Project to LGN or superior colliculus
- Synapse to visual cortex
Multiple rounds of selection guide axon outgrowth
Pathway Selection
Choosing the correct path
Axons from retinal ganglion cells must choose the ipsilateral or contralateral pathway at the optic chiasm
Multiple rounds of selection guide axon outgrowth
Target Selection
Choosing the correct area to innervate
Once axons in the optic tract reach the thalamus, where do they innervate?
* Lateral geniculate nucleus
Multiple rounds of selection guide axon outgrowth
Address Selection
Choosing the correct cells to synapse with
Once in the LGN, retinotopy must be established and the correct layer of the LGN innervated
Extracellular cues guide axon outgrowth
- cell - cell contacts
- cell - local extracellular cue
- cell - diffusible signal
Axon Growth Cone Function
- Interacts with extracellular components to guide
growth - If signals are permissive, filopodia are stabilized and
growth cone advances - If signals are repulsive filopodia retract and growth cone does not advance
Three stages of growth cone advance
Protrusion
Extension of filopodia and lamellipodia
Three stages of growth cone advance
Engorgement
Microtubules in the C-domain extend closer to the peripheral region fixing direction of growth
Three stages of growth cone advance
Consolidation
Actin filaments in the growth cone neck depolymerize and the membrane shrinks to form a cylindrical shaft
Actin filaments in the neck depolymerize to shrink the axon shaft
Permissive signals for axon outgrowth originate from:
- Extracellular matrix
- Other axons
- Other cells long distances away
- Tend to be chemoattractants or chemorepellents
Extracellular Matrix (ECM)
ECM factors regulate neurite outgrowth and axon guidance
ECM is composed of a mix of glycoproteins and proteoglycans
Glycoprotein
Protein that has an oligosaccharide chain covalently attached to an amino acid side chain
* Oligosaccharide attachment is a modification either during or postprotein translation
Proteoglycan
Subclass of glycoprotein
- Has a polysaccharide (larger string of sugars than oligosaccharide)
- Side chain is an amino sugar (specific replacement of amino group for hydroxyl group)
Examples of ECM components that contribute to axon guidance
- Laminin
- Fibronectin
- Collagen
- Tenascin
- Heparin sulfate proteoglycans
Both laminin and fibronectin can alter the effect of classic guidance cues on axon direction
Laminin
A glycoprotein critical for axon outgrowth
- Heterotrimeric glycoprotein
- Composed of alpha, beta, and gamma subunits
- Takes on a cruciform shape
- Major component of extracellular matrix in developing and mature CNS
- 15 laminin subtypes based on differential subunit
usage
Mutation of Laminin
Causes axon
outgrowth and guidance defects
Mutations in laminin alpha 1 gene causes defects in CNS axon pathways including
* Retinal ganglion cell axons
* Early forebrain axons
* Hindbrain reticulospinal axons
Peripheral axons are normal
Fibronectin
Another glycoprotein critical for axon outgrowth
- Expressed in dynamic patterns in regions of active morphogenesis (spinal cord and cortical subplate)
- Expressed in ventricular zone during earliest stages of CNS development
- FN is also distributed along radial glial processes in association with preplate neuron and is produced by migrating neurons that target specific cortical domains
- FN may help neurons discriminate between adjacent guides
- In cell culture, Fibronectin can: promote proliferation and migration
Knockdown of fibronectin decreases axon outgrowth in cultured neurons
Classic Guidance Cues
Ephrin and netrin
Their effect on axonal outgrowth (inhibition vs attraction) depends on whether the ECM is primarily Laminin or Fibronectin
How do ECM components signal to the growth cone?
Integrin receptors bind to laminin and fibronectin to transduce signal
- Alpha and beta integrin receptors on growth cone membrane
- Form heterodimers: 1 alpha + 1 beta
- 24 heterodimers can be made in human neurons
- Highest expression in the brain during development
- Decline in levels of integrins in mature brain
- Integrin expression can promote axon extension in neurons that normally are not able to extend neurites
- Powerful modulator of axon outgrowth
Upstream factors controlling laminin-integrin signaling
Upstream example: Talin
Internal factors, i.e. other proteins, regulate the upstream and downstream activities of integrin receptor function
- Talin binds to beta subunit of integrin receptors
- Loss of Talin prevents integrin receptor activation
- Data suggests talin binding alters the angle of the transmembrane segment of beta integrins which is necessary to bind ligand (laminin or fibronectin)
Downstream factors controlling laminin-integrin signaling
Downstream example: FAK and Src
- Kinases – phosphorylate things
- Activated by growth factor signaling (depicted as “guidance cue receptor”) and integrin receptors
- Modulates axon pathfinding through interaction with actin and integrin receptors
Downstream factors controlling laminin-integrin signaling
Downstream example 2: Vinculin and Paxillin
- Focal adhesion proteins
- Proteins involved in the direct or indirect linkage between actin filaments and integrin receptors
There is a physical link between ECM (e.g. laminin) and actin
Focal adhesions in the growth cone
Growth cone labeled with antibodies to recognize
paxcillin –focal adhesion protein (green) and actin (red)
Focal adhesions downstream of integrin receptors turnover to control directional growth
Permissive signals for axon
outgrowth originate from:
- Extracellular matrix
- Other axons
- Other cells long distances away which tend to be chemoattractants or chemorepellents
Chemoattractants and Chemorepellents
Guide growth cones
- Challenge in wiring the brain
- Distances between connected structures
- In early stages, nervous system is only a few centimeters long.
- Pioneer axons stretch as nervous system expands.
- Guide neighbor axons to same targets
- Pioneer neurons grow in the correct direction by “connecting the dots.”
Pioneer Neurons
Use attractants and repellents to pathfind
Classic examples include:
* Netrins
* Slits
* Semaphorins
* Ephrins
Chemoattractant
First chemoattractant identified: Netrin
Diffusible molecule that acts over a distance to attract a growing axon
Netrin
First chemoattractant
- Netrin is secreted by neurons in the ventral midline of the spinal cord
- Attracts axons of neurons from the dorsal horn to join the spinothalamic tract
- These axons have netrin receptors that work with growth cone machinery to promote directed outgrowth
- In the example of the dorsal horn neurons, after being guided to the spinothalamic tract by netrin, netrin signaling must be countered for growth cones to complete crossing.
Chemorepellent
- A diffusible molecule that repels axons
- Axons must have receptors to recognize this cue
In this example, the chemorepellent is slit
* Receptor is Robo
* Robo is upregulated after growth cones cross midline
* Leads to continued growth now away from midline
Attraction and repulsion work to pattern the visual system
- As you have seen before, there is a tract from the retina to the lateral geniculate nucleus
- Once at the LGN, the axons must find their “address”
- i.e. the correct part of the LGN to synapse on
- Similarly, axons that instead go to the superior colliculus must do the same
- Ephrins pattern the visual system at the level of axon target finding in the superior colliculus
- Called the tectum in frogs
- Ephrin gradient across the tectum/SC with high levels in the posterior region
- Ephrin is a repellent
- Axons that have the ephrin receptor will not grow where there are high levels of ephrin
- Axon that leave the temporal retina have ephrin receptors
Downstream of chemoattractants and repellents
- Chemoattractants and repellents modulate directional growth cone advance through regulation of the cytoskeleton
Can work through:
* activation/inactivation ofsmall GTPases such as Rho, Rac, and Cdc42
* Receptor-mediated phosphorylation of cytoskeletal regulators
* Direct binding to microtubules or microtubule binding proteins
Growth Cone Function
Growth cone advance/axon outgrowth can be facilitated by fasciculation
Fasciculation
- Adhesion of axons together
- Caused by surface cell adhesion molecules (CAMs)
- E.g. cadherins, dscams, etc.
Homotypic CAM interactions
Interaction between the same cell adhesion molecule
Heterotypic CAM interactions
Interactions between different cell adhesion molecules
L1CAM
Some proteins can perform homotypic or heterotypic interactions
L1CAM inactivation causes abnormal muscle innervation
* L1CAM function was blocked in developing chick thigh muscle
* This results in axons sprouting from the nerve due to loss of adhesion
NCAM
Loss of another cell adhesion molecule (NCAM) causes abnormal retinal axon pathfinding
* Retina of chicken embryo
* Axons are normally in a clear, fasciculated track
* Blocking NCAM causes axon misrouting in the retina
* Loss of NCAM also affects axon fasciculation tectum
* Tectum is known as superior colliculus in mammals
Cadherins
Another type of cell adhesion protein that regulate axon fasciculation
Protocadherin17 (pcdh17)
Important for homotypic fasciculation of amygdala axons as they extend to the hypothalamus and ventral striatum
Loss of this cell adhesion molecule causes abnormal
axon outgrowth of amygdala axons on route to the hypothalamus.
Interacts with actin to regulate outgrowth
