Lecture 3 - Axonal Growth - Tropism Flashcards
What is the first step in neuronal identity and the formation of connections?
Polarization (and is a primary property of all cells).
A neuron starts round with
no obvious processes (neurites).
Over time the neuron (neurite) begins to
extend multiple processes and becomes multipolar.
What defines the polarity of the neuron?
One of the multiple processes become the axon, which then defines the polarity of the neuron.
What is at the tip of the axons?
a specialized structure (or structures) called growth cones.
Where is the growth cone located?
specialized motile structure at the tip of extending axon
What does the growth cone do?
explores extracellular environment, determines direction of growth, and guides extension of axon; key decision-making structure in axon pathfinding morphological characteristics
lamellapodium –
fan-shaped sheet at tip of axon; contains actin filaments and microtubules
filopodia –
fine processes extending out from lamellapodium; contain actin filaments; form and disappear rapidly
How does the leading edge of filopodium respond to environmental cues?
Gobular actin (G-actin) can be incorporated into filamentous actin (F-actin)
A key to growth cone turning is
the binding of F-actin binding proteins to F-actin, which regulates retrograde flow.
In growth cone tuning what regulates retrograde flow?
The binding of F-actin binding proteins to F-actin
What happens when encountering an attractive cue?
assembly is increased and retrograde flow slowed causing turning towards the attractive cue (repulsion is the opposite)
What is the role of microtubules in growth cone tuning?
1) It makes the core of the cytoskeleton in the axon extremely stable and strong 2) Modulation of interactions with microtubules modulate stability and turning of the axons.
What are primarily responsible for axon elongation?
Microtubules
In growth cone signaling what dictates direction?
F-actin
How can discrete regions of the growth cones be detected?
by different types of actin and tubulin
F-actin is in
lamellipodium and filopodia
Tyrosinated microtubules are enriched in
lamellipodia
Acetylated microtubules are only in
the axons.
In axonal growth, what happens at decision points?
The growth cone shape changes
What are the axon guidance signals?
There are 4 types - non-diffusible (short-range) signals: 1) contact attraction 2) contact repulsion diffusible (long-range) signals: 3) chemoattraction 4) chemorepulsion
What is the function of the axon guidance signals?
all 4 types may act in concert to guide axon to appropriate target; ensures accurate guidance
What does the binding of axon guidance molecules to receptors do?
on growth cones binding activates signaling cascades that result in reorganization of the growth cone cytoskeleton which controls the direction and rate at which the growth cone moves.
What do attractive growth cone interactions do?
they promote actin polymerization in the direction of the attractive molecules
What do repellent growth cone interactions do?
promote actin depolymerization and growth cone collapse.
In the peripheral nervous system what are the non diffusible guidance molecules
laminins, collagens and fibronectin are attracive substrates for growth cones
In the peripheral nervous system, what happens to the non diffusible substrates?
these substrates bind to growth cone receptors called integrins, which triggers a series of signaling cascades leading to axon growth and elongation
Describe the ECM molecules in the CNS.
They are expressed at low levels
Matrix of the CNS is mostly composed of
hyaluronan, proteoglycans and glycoproteins but it lacks many of the “typical” matrix molecules (laminins, collagens, fibronectin)
For non-diffusible guidance molecules, how would you describe the CNS extracellular matrix molecules
they are largely repulsive
Describe the nondiffusible guidance molecules, CAMs.
They are attractive
Where are the CAMs of the CNS located?
Located on surface of growing axons, growth cones, and surrounding cells or targets
What do CAMs act as?
ligands and receptors typically via homophilic binding; attractive interaction
What is the relationship between CAM and Calcium?
Calcium independent
Ligand/receptor CAM interaction does what?
induces interaction with cytoplasmic kinases in growth cone
L1 CAM
has been associated with fasciculation (bundling) of groups of axons as they grow
Non-diffusible guidance molecules; cadherins
they are attractive
Where are cadherins located?
On the surface of growing axons, growth cones and surrounding cells or targets
What do cadherins act as?
Act as ligands and receptors typically via homophilic binding; attractive interaction
What is the relationship between calcium and cadherin?
Calcium dependent
What does the Ligand/receptor interaction for cadherin trigger?
intracellular signaling pathways that lead to actin binding and organization
Describe the non diffusible guidance molecules, semaphorins
mostly repellent
Are Semaphorins diffusible or non diffusible?
non-diffusible
Where are semaphorins located?
Can be secreted or anchored to the cell surface; secreted forms are probably attached to the cell surface or the extracellular matrix, so they are not really diffusible
Are semphorins attractive or repellent?
Mostly repellent, but have also been shown to be attractive in some situations
What happens to semaphorins receptors?
Receptors on growth cones are members of the plexin family; cell surface forms bind directly to plexins, while secreted forms bind neuropilins which then complex with plexins
What do cell surface semaphorins bind to?
The bind directly to plexins
What do secreted semaphorins bind to?
Bind neuropilins which then complex with plexins
What does semaphorin ligand/receptor interaction result in?
growth cone collapse and inhibition of axon extension via receptor interaction with intracellular signaling molecules
What do semaphorins promote?
Growth cone collapse
Are ephrins diffusible or non-diffusible?
Non-diffusible
Are ephrins attractive or repellent?
repellent
What are ephrins?
Cell surface signaling molecules similar to cell adhesion molecules
What are the classes of ephrin?
1) ephrin-A = GPI-linked to cell surface 2) ephrin-B = single pass transmembrane proteins
What does each class of ephrine have?
Each has its own set of receptors on growth cones, eph A receptors and eph B receptors, that are receptor tyrosine kinases
What type of receptors are ephrin receptors?
Receptor tyrosine kinases
What does ephrin interaction with its receptor on the growth cone results in?
a repellent interaction that collapses the growth cone
In the developing brain axons from the retina make
precise connections with the optic tectum (in frog and chick).
What are the connections from the retina to the optic tectum?
1) Temporal retina to anterior tectum 2) Nasal retina to posterior tectum
What is the expression of ephrins in the optic tectum?
Ephrins (A2 and A5) are expressed on the optic tectum in an anterior to posterior gradient with the highest concentration at the posterior side.
Axons from temporal retina are
repulsed by ephrin in posterior tectum because they express high levels of an Eph receptor (EphA3).
Axons from the nasal retina are
blind to ephrin because the lack the eph receptor.
Netrins
diffusible guidance molecules (attractive or repellent)
Slits
diffusible guidance molecules (repellent)
Netrins are secreted by
target cells in midline of embryo
Netrins can be both attractive and repellent depending on what?
the receptors expressed on the growth cone
Attractive receptors are
members of the DCC family
Repellent receptors are
members of the UNC5 family
Slits are
diffusible, Secreted proteins
Receptors on growth cones are
members of the Robo family
Slit interaction with its receptor on the growth cone results in
a repellent interaction
Knockout of netrin
eliminates crossing of commissural axons
Netrin and Slit at the midline
they act in concert
Commisural axons
cross in the spinal cord and then make contact with cells on the opposite side.
What is the biologically challenging problem that commissural axons present?
1) Cells have to first avoid its target cell on the same side of the spinal cord 2) Has to cross at a very specific site in the cord 3) Once crossed has to not re-cross 4) Has to find its target cell on the other side of the cord.
Comissural axons express what?
DCC and are originally attracted to the midline which produces high levels of netrin.
What happens as commissural axons cross the midline
they then upregulate Robo which keeps them from recrossing because of the high level of slit at the midline
Why keeps commissural axons from recrossing?
The upregualtion of Robo and the high level of slit
As commissural axons cross the midline what happens?
There is cross talk between Robo and DCC, where Robo signaling inhibits DCC signaling
Axon guidance cues
1) extracellular matrix adhesion 2) cell surface adhesion 3) fasciculation 4) chemoattraction 5) contact inhibition 6) chemorepulsion
How do Axon Guidance Molecules Work in Concert to Guide Growth Cones?
Multiple guidance cues, both attractive and repellent, line the pathways followed by axons, so the growth cone must integrate the information provided by these guidance molecules to reach the appropriate target
What happens in selective synapse formation?
1) after reaching correct final target region, axons must decide which cells to innervate
Synaptogenesis
best understood in the peripheral nervous system (neuromuscular junction in particular)
Synaptogenesis at the neuromuscular junction
1) A motor axon approaches makes contact with a myotube (seemingly at random). 2) Both the nerve terminal and myotube differentiate after contact has been made (with the nerve becoming a motor terminal and the muscle forming postsynaptic apparatus.)
Differentiation of the muscle (at the NMJ) is induced by what?
agrin,
AT the NMJ, what does agrin activate?
It activates MuSK, causing the clustering and increased local expression of acetylcholine receptor (AchR) through rapsyn
At the NMJ, what stabilizes the synaptic structure?
Both the motor nerve and the muscle make ECM components to form a basal lamina, which stabilizes the synaptic structure.
In synaptogenesis, what does the motor axon do?
it approaches a newly formed myotube.
In synaptogenesis, what happens at the area of contact between the motor axon and myotube?
the axon differentiates into a motor nerve terminal that is specialized for transmitter release,
In synaptogenesis, what is the role of the Schwann cell
its processes cap the terminal, and the muscle forms a complex postsynaptic apparatus.
Acetylcholine receptors (AChRs) in synaptogenesis
they are initially present at a moderate level throughout the myotube surface.
Contrast the myotube to adult muscle.
by contrast, AChRs are highly concentrated in the postsynaptic membrane and virtually absent extrasynaptically.
What accounts for the difference in the distribution of the AChRs in the myotube versus the adult muscle?
This clustering involves both redistribution of AChR proteins, and localized synaptic synthesis of AChRs.
In synaptogenesis, for the clustering of AChRs what does the local synthesis results from?
enhanced transcription of AChR genes by subsynaptic nuclei and by repression of extrasynaptic nuclei.
The agrin– muscle-specific kinase (MuSK)–rapsyn–AChR pathway.
1) z+ agrin is released from the nerve terminal and becomes stabilized in the basal lamina of the synaptic cleft. 2) Agrin activates MuSK to cluster AChRs through the cytoplasmic linker protein rapsyn
Synaptogenesis in the superior cervical ganglion
1) T1 and T4 axons use the same long-range guidance cues to reach ganglion, but each innervates a different set of neurons 2) Synapse formation must be selective – the correct pre and post synaptic neurons have higher affinity for each other
In the Superior cervical ganglion what happens to axons arriving from level T1?
They form synapses on cell bodies of neurons that project to targets in eye
In the Superior cervical ganglion what happens to axons arriving from level T4?
they form synapses on cell bodies of neurons that project to targets in ear
In the Superior cervical ganglion what happens to both T1 and T4 axons?
They use the same guidance cues to reach the ganglion via the cervical sympathetic trunk, so differential innervation of ganglionic neurons must occur at the level of synapse formation
How do the “correct” pre- and postsynaptic neurons form?
they have higher affinity for one another, so the incoming axons preferentially form synapses on the correct targets.
In the Superior cervical ganglion what happens to the system of bias in synapse formation?
it guides innervation throughout the nervous system during development without limiting it in any absolute way. This phenomena has been known for more that 30 years and yet the precise molecular mechanisms are still unclear.
The molecular mechanisms driving the formation of synapses in the CNS
are just beginning to be elucidated.
What are the basic steps that are thought to apply to all synapses?
1) The nascent presynaptic process, derived from the growth cone, recognizes an appropriate site on the target cell via cadherin/protocadherin family of adhesion molecule 2) Synaptic vesicles and active zone components begin to accumulate 3) Additional ad
What is the function of neurexin (in synaptogenesis)?
it helps localize cytoskeletal elements, synaptic vesicles, active zone proteins and voltage gated Ca2+ channels to the presynaptic membrane
What is the function of neuroligin (in synaptogenesis)?
It recruits neurotransmitter receptors and other postsynaptic proteins to postsynaptic membrane
Diversity in synaptogenesis
Despite the common molecular events described above, there is considerable diversity among developing synapses, which may be due to the large number of family members or splice variants of each type of molecule that could be involved.
What are the trophic interactions between neurons and their target cells?
1) Target dependency for differentiation and survival 2) target cells secrete neurotrophic factors 3) neuronal competition for neurotrophic factors 4) no trophic factors = death via apoptosis
What happens to the neurons after synaptogenesis?
Dependency on targets for survival and differentiation
Post synaptogenesis, what happens to target cells?
They secrete neurotrophic factors
Post synaptogenesis, why would neurons undergo cell death?
Competition for limited resources, insufficient neurotrophic support from their targets degenerate and die via apoptosis
Post synaptogenesis, what is the role of target cells?
They play a role in determining the number of cells that innervate them
What happens to synaptic rearrangement during postnatal life?
modulation of synaptic connections via trophic interactions
What do trophic interactions ensure?
That each target cell is innervated by the right number of axons and that each axon innervates the right number of cells
Where is the postsynaptic rearrangement during postnatal life?
Its best understood in the peripheral nervous system (NMJ and autonomic ganglia)
Once neuronal populations are established, what is the function of trophic interactions?
they help ensure that each target cell is innervated by the right number of axons and that each axon innervates the right number of target cells.
Where is synaptic rearrangement best understood?
this process is best understood in the peripheral nervous system at the neuromuscular junction and in the autonomic ganglia where a single neuron innervates the target cell:
In synaptic rearrangement initially what happens?
1) skeletal muscle fibers and some parasympathetic neurons are innervated by multiple neurons (polyneuronal innervation)
In synaptic rearrangement what happens during postnatal development?
synaptic inputs are gradually eliminated until only a single one remains
In synaptic rearrangement what is the role of competition?
the process appears to involve competition between the different neurons for “ownership” of the target cell
For synapse elimination at the neuromuscular junction, describe competion.
competition is dependent on electrical activity in both pre- and postsynaptic cells since blocking either results in persistence of polyneuronal innervation
For synapse elimination at the neuromuscular junction what happens to competing axons?
they gradually segregate at synaptic sites, and the losing axon atrophies and retracts.
What happens at the same time the losing axon atrophies and retracts at the NMJ?
the postsynaptic specializations under the losing axon are lost
What happens to the single remaining nerve terminal at the NMJ?
it enlarges as the endplate region expands during postnatal muscle growth
What directs axons to target cells?
Guidance molecules
What happens when the axons get to the target cells?
They form synapses on selected target cells
What do target cells secrete?
Limiting amounts of neutrophins to regulate the number of innervating neurons
How are axonal branching patterns redefined?
Via competition and synapse elimination
What are the functions of neurotrophins?
1) the survival of subsets of neurons 2) formation and maintenance of the appropriate numbers of connections (number of target cells contacted) 3) elaboration of axonal and dendritic branches to support connections (number of synapses formed)
What is nerve growth factor?
Member of the neurotrophin family, NGF acts on a few specific populations of peripheral neurons
In addition to neurotrophins, what are some other neurotrophins?
BDNF, NT-3, NT-4/5, difficult to identify because it’s present in small quantities
What is the evidence for the trophic function of NGF?
1) neuronal death in absence of NGF 2) survival of excess neurons with increased levels of NGF 3) presence and production of NGF in target cells 4) presence of NGF receptors in innervating nerve terminals
Give an example of the sensitivity of neurons to growth factors.
Chick sensory ganglion show not a single axon in the absence of NGF but robust axon extension in the presence of NGF
What is the effect of neurotrophins on neurite outgrowth?
Different Neurons are differentially sensitive to growth factors
DRGs respond to
all growth factors
Nodose ganglia (NG) neurons respond most robustly to
NT3
Sympathetic ganglia (SG) most robustly to
NGF and not at all to BDNF
Neurotrophin receptors
Trk receptors and p75 receptor
Trk receptors
family of receptor tyrosine kinases (TrkA, TrkB, and TrkC)
What do Trk receptors bind to?
each binds a distinct subset of the neurotrophins
What do all three types of neurotrophin receptors have high affinity for?
processed (cleaved) neurotrophins only
p75 receptor
can be activated by all neurotrophins
What do p75 receptors have a high affinity for?
unprocessed neurotrophins
What do p75 receptors have a low affinity for?
processed neurotrophins
What allows the neurons to respond selectively to different neurotrophins being secreted by different target cells?
The expression of distinct receptors by subsets of neurons
What does activation of each class of neurotrophin receptors (Trk or p75) by neurotrophins trigger
distinct intracellular signaling cascades
Each class of receptor can?
activate a variety of intracellular signaling cascades
In neurotrophin signaling the specific cascade will do what?
The cascade that is activated will determine the cellular response: 1) cell survival or death 2) cell and process growth or differentiation 3) activity dependent synaptic stabilization or elimination
Neurotrophic interactions depend on?
1) neurotrophins secreted by target cells 2) neurotrophin receptors on neuron 3) Intracellular signaling cascades present in neuron
Neurotrophic interactions determine what?
1) number of neurons 2) shape of neurons (degree of arborization) 3) patterns of neuronal connections (number of synapses)
