Notes 20100409.120037 Guest Lecture Retinoic Acid Chemoattractant Neurites
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Retinoic Acid, Chemoattractant, Neurites
- Gaynor E Spencer, Biological Sciences, Brock
- developing nervous system
- small lipophilic molecule
- neurite outgrowth
- patterning of anterior-posterior axis of neural tube
- insufficiency: missing posterior
- excess: teratogenic
- plays a role in adult nervous system
- learning and memory
- regeneration of PNS and CNS
- implicated in decline of cognition in aging
- linked to ALS and Alzheimer's disease
- emerging as an important molecule in development and in adult brain
- RA can pass into nucleus and binds into two kinds of receptors
- RAR retinoic acid receptor
- RXR retinoid X receptor
- three subtypes in each of the receptors (alpha, beta, gamma)
- RAR, RXR both act as transcription factors for target genes
- isomers--
- retinal -- all trans-retinoic acid studied the most
- 9-cis-retinoic acid -- difficult to show in vivo
- 13-cis-retinoic acid (mentioned)
- chick embryonic neurons, NGF signals neuron growth-- enhanced when supplemented with RA
- limb regeneration for newt spinal cord explants
- outgrowth is directed toward RA bead
- outgrowth hence directs outgrowth (not only promote, enhance)
Neurons
- growth cones -- filopodia do sensing, finds appropriate target
- guidance cues -- substrate bound, diffusible factors
- negative cue: turn away or collapse
- positive cue: turn toward growth cone
Palm Snail
- Lymnaea stagnalis
- model organism
- neurons are well mapped
- PeA cells: maps to the foot of the snail
- VF cells: putative motor neurons
- selection of single neuron cell body from VF ganglion
- not a lot of information in previous work for retinoic acid
- growth cone was not original concern
- let's look at the behaviour of RA in the invertebrate brain
- does the Lymnaea brain actually contain RA?
- RXR reported in Lymnaea -- generally binds the 9-cis isomer
- CNS and Hemolymph in Lymnaea contained both all-trans and 9-cis RA
- outgrowth response and turns now verified for both all-trans and 9-cis RA
Media
- DM: defined medium, no trophic factors, no neurites
- EtOH: control medium, some neurite formation
- EtOH+RA: much growth
- significant enhancement over 2 to 4 days
- the first evidence RA activity in brain is conserved between vert/invertebrates.
- crush sites ( ... )
RA as a chemoattractant for growth cones
- growth cone significantly tuns toward RA
- controls had significant turns away from pressure cone -- mechanical?
Mechanisms
- ((9-cis RA + RXR + RAR + transRA) + RARE) enters the nucleus, starts transcription
- is transcription actually involved with the growth and turning?
- actinomycin -- transcription blocker -- does not stop neurite growth
- resected neurites can continue to grow in direction of RA -- does not require transcription?
- non-transcriptional mechanisms
- RA binds to PKC
- RA ( ... )
- does the growth require local protein synthesis?
- anisomycin -- translation inhibitor -- blocks neurite growth
- checked for both the intact and the resected neurite
- role of RA, PKC?
- direct binding of RA to PKC
- PKC modulates Ca++ influx with steroid hormones (non-genomic)
- Ca++ influx?
- PKC inhibitor (requires DMSO -- requires control studies with DMSO)
- Cadmium: Calcium channel blocker-- growth cone turning abolished
- DMSO, PKC -- inconclusive
RXR
- stained with antibody -- western blot conducted -- nucleus did not contain RXR
- RXR was found in neurites and axons only
- RXR are not found in nucleus (but it's usually a transcription factor)
- neurite branches may contain no RXR as well
- RXR found in growth cones
- determine if RXR induces growth cone turning
- if we puff on RXR-specific agonist, growth cone turning detected -- repeat with resected neurites and DMSO
- RXR is thought to be the 9-cis RA native receptor
- need to show RXR-specific antagonist is capable of causing turning cone away
- a separate antagonist worked correctly
- suggests a novel non-transcriptional role of RXR
Conclusion
- RA induced neurite behaviour-- conserved
- mechanism local protein synthesis, calcium influx, translation
- non-genomic actions of RA -- RXR agonists also worked
Glossary
- Dunn chamber
- filopodia: sensor in growth cone for growth cues
- RARE: retinoic acid response element
- resected neurite: cell body is removed from petry dish
