Axon guidance Flashcards
Define axon guidance
The process by which neurons are able to find their synaptic targets
The growth cone of an axon is made of primarily this cytoskeletal element
Filopodia
How do axons grow?
By extension and retraction of filopodia
Axons in their immature state are often referred to as…
Neurites
What kinds of glycoproteins and proteoglycans are present on the extracellular matrix? (5)
- laminin
- fibronectin
- collagen
- tenascin
- heparin sulfate PG
Notably, the growth cone contains receptors for…
Members of the integrin family (a and b subunits)
What are integrins?
The primary proteins that animal cells use to bind to the ECM
Filopodia are rich in this protein
Actin
What proteins are responsible for mediating an interaction between the ECM and the cytoskeleton (specifically, actin)
Integrins
ECm glycoproteins and proteoglycans are (secreted/stationary)
Secreted
Where are integrins found?
On cell surfaces (transmembrane)
What is the role of talin?
Bridges actin and integrin
What is the role of FAK and Src?
They are kinases which mediate the binding of actin and integrin
What are vinculin and paxilin?
Adaptor proteins that aid in the correct spatial binding of actin and integrin
Describe what is happening here, what is the assay?
Assay: looking for growth of neurites along the laminin strip.
Laminin strips applied to a developing neuron and see where the axons start growing – testing to see if laminin is an appropriate axon growth substrate
Immunostaining reveals that the soma and axon of developing neurons are rich in (actin/microtubules) and the growth cone is rich in (actin/microtubules)
- Microtubules
- Actin
Why is aplysia a good model organism for studying axon guidance? (3)
- They have large neurons
- Well-characterized nervous system
- Easy to examine in culture
If you wanted to knock out the growth of axons, what kinds of molecules might you want to target?
Actin inhibitors – inhibit the formation of filopodia
What is cytochalasin B?
An inhibitor of actin polymerization
What happens when cytochalasin B is applied to growing axons?
There is a complete retraction of filopodia
What are the key molecular switches that regulate the cytoskeleton?
Rho family GTPases
What are the 3 key Rho GTPases studied in this lecture?
Rho
Rac
Cdc42
Rho GTPases are active when bound to…
GTP
Rho GTPases are inactive when bound to…
GDP
Rho-bound GTP is converted to GDP by…
GTPases activating proteins (GAPs)
Rho-bound GDP is activated by…
Gunanine nucleotide exchange factor (GEF)
When bound to Rho GTPases, GDP is phosphorylated to GTP…
NEVER
Active Rho-GTPases commonly interact with (2)
Protein kinases
Actin-binding proteins
Activated Rho-GTPases directly or indirectly affect local assembly and disassembly of…
F-actin (filamentous actin, including filopodia)
What are focal adhesions?
Actin aggregates which anchor the growth cone to the base of the developing axon
What are stress fibers?
Actin filaments within the growth cone which interact with non-muscle myosin – have a contractile function
Rac mediates the formation of…
Focal complexes
What are focal complexes?
Actin and integrin complexes – Interaction area with ECM
Rac impacts the formation of this actin structure
Lamellopodia (flat sheets of actin at the edges of the growth cone)
How does Cdc42 impact growth cone movement?
Controls the outgrowth of filopodia, which interact with non-muscle myosin to have contractile functions
Who was one of the first individuals to propose the existence of molecular mechanisms controlling axon guidance?
Roger Sperry (1939)
Roger Sperry postulated that (1) controlled axon guidance
Embryonic gradients
What would happen to these neurons if the limb bud were to be truncated?
Neurons would die
Describe how Rita Levi-Montalcini “got lucky” with her discoveries
The lab directly next door to her just happened to be a tumour lab and they shared tumours with her. Had she not had easy access to these she may not have made her discoveries
Don’t get me wrong she slays and wasn’t “lucky” she deserves all the credit she gets
What inspired Rita to use tumours to see if truncated limb buds could still have live neurons?
She knew tumours had unique growth properties and growth factors which might “inspire” neurons not to die
What occurred to peripheral neurons in Rita’s experiments when tumours were placed near the truncated limb bud?
Neurons did not die and grew to innervate the tumour
Rita hypothesized that truncated limb buds in the presence of tumours could grow to be innervated due to the presence of (1) in the tumours
Neuron survival factors/signals
Describe the assay that Rita developed to isolate “nerve growth factor”
Isolated sensory and sympathetic ganglia from chick embryos (which are relatively easy to work with) and combine with fragments or extracts of a tumour
Readout: Observation of neuron growth
Describe the contribution of Stanley Cohen to Rita’s work
He fractionated tumour extract and applied it to growing neurons to isolate the compound which induced significant neuron growth
Who were the two people who shared in the nobel prize in physiology and medicine for the isolation of nerve growth factor?
Rita Levi-Montalcini and Stanley Cohen
What are the primary receptors for NGF? (2)
TRKA
p75
Downstream effects of NDF/TRKA activation impact this important molecular switch
Rho GTPases
Describe the general steps of the NDF pathway leading to neurite growth
- NGF binds to surface receptor
- Activation of Rho GTPases
- Increase in actin polymerization
- Neurite outgrowth and differentiation
Name 5 neurotrophins
NGF
neurotrophin-3
BDNF
GDNF
CNTF
Define a neurotrophin
A molecule which promotes axon outgrowth, survival, and differentiation of neurons
True or false: NGF is capable of specifying the correct synaptic targets for an axon
False: it helps guide the axons to the correct general area but NGF alone is not capable of specifying targets
Name 4 non-neurotrophin axon guidance molecules
Ephrins, netrins, slit, and semaphorins
The primary intracellular interaction of ephrins and netrins are with…
GEFs
The primary intracellular interaction of Slit and Semaphorins are with…
GAPs
Downstream of Ephrins and Netrins, GEFs activate…
RhoA and Rac1
Downstream of Slit and Semaphorins, GAPs inhibit…
RhoA, Rac1, and Cdc42
Ephrins and Netrins act as (attractive/repulsive) cues for axons
Repulsive
Slit and Semaphorins act as (attractive/repulsive) cues for axons
Attractive
Describe the “stop and go” mechanism of axon growth
Different attractive and repulsive signals dictate whether axon growth into a region will stop or go
Describe why NT-3 responsive neurons can project to the ventral spinal cord, but NGF-responsibe mechanoreceptors and nociceptors cannot
The ventral region expresses Semaphorin-3, which is an attrative cue for NT-3 neurons, but which causes NGF-responsive neurons to halt their growth
What is happening in this image?
Looking at NGF-responsive neurons, when NGF present get robust outgrowth of neurites
What is happening in this image?
NGF-responsive neurons are exposed to sema-3, and when this is done there is robust repulsion of neurites around the source of sema-3
NGF RESPONSIVE NEURONS CONTAIN SEMA RECEPTORS, SEMA IS A REPULSIVE CUE WHICH IS WHY WE SEE RETRACTION FROM THE AREA OF SEMA
What is happening in these images?
NT3 responsive neurons are being cultured in the presence (right) and absence (left) of Sema 3, when Sema-3 is present, there is an attraction of neurites towards the source
NT3 RESPONSIVE NEURONS DO NOT CONTAIN SEMA RECEPTORS, SEMA IS A REPULSIVE CUE WHICH IS WHY WE SEE GROWTH INTO THE AREA OF SEMA
Who discovered the somatosensory cortical map?
Wilder Penfield
Three examples of sensory maps in the brain include
The somatosensory cortex
The auditory map on the basillary membrane
Retina
Describe the geniculate visual pathway in rodents
Projections to the LGN (lateral geniculate nucleus) to the V1 cortex
Describe the extrageniculate pathway in rodents
Projections to the superior colliculus to the V2 cortex
What is the primary role of the geniculate pathway?
Processing info about static and moving objects, good at pattern recognition
What is the primary role of the extrageniculate pathway?
Directs behavioural responses toward specific points in “body-centered” space (eye movements, for example)
The focus of the following discussion paper examines these projections
ganglion cell axons from the retina projecting to the superior colliculus (start of the extrageniculate pathway)
Eph is the (receptor/ligand) and ephrin is the (receptor/ligand)
Receptor
Ligand
Ephrin A’s are linked to the membrane via…
GPI linkages (re: these are LIGANDS)
Ephrin B’s are linked to the membrane via…
Transmembrane domains (re: these are LIGANDS)
What is the full name for a GPI anchor? What are they?
Glycophosphatidilinositol
Glycolipid that can be attached to the C-term of a protein thru post-translational modification
EphA5 and A6 are highest in the (nasal/temporal) retina
Temporal
Ephrin A’s are highest in the (posterior-caudal/anterior-rostral) superior colliculus
Posterior-caudal
Ephs and ephrins are expressed in this motif
Concentration gradients
