Cholesterol depletion modifies nasal retinal ganglion cells axonal growth cone attraction to EphA3

SPELZINI, GONZALO; FIORE, LUCIANO; MEDORI, MARA; SCICOLONE, GABRIEL

belem

Congreso; 3er FALAN Congress; 2022

Federación de Asociaciones Latinoamericanas y del Caribe de Neurociencias (FALAN)

INTRODUCTIONThroughout the formation of the retino-tectal ordered connection, EphA3gradient take part in nasal retina ganglion cellsaxonal growth and guidance to the rostraltectum bythe interaction with ephrinA2. The precisedistribution of ephrinA2 into cholesterolenriched plasma membrane domains, known as lipid rafts, is essential for the suitable transduction of the recognized signals. However, whether such lipid rafts are required for theappropriate axonal guidance mediated by EphA3 gradient remains unknown.OBJECTIVESIn this work, using a pharmacological approach in retinal neuronalcultures and retinal explants we further demonstrate that disrupting lipidrafts interferes with the chemoattractive response of growth cones toEphA3.METHODSBy the use of live-cell EphA3 gradient analysis by Dunn chamber weobserved variances in axon guidance mediated by EphA3. We also characterize by immunocytochemistry the distribution of ephrinA2 along the axon membrane after lipid rafts disruption and cholesterol determination by HPLC and by staining with philipin.RESULTSBy performing live-cell microscopy in retinal cell cultures, we present a fullcharacterization of the axonal guidance and growth cones dynamics dueto EphA3 gradients.Using a pharmacological approach in primary neuronal cultures andretinal explants, we demonstrate that the membrane cholesterol integrity of membrane raftmicrodomains is of crucial importance to translate the EphA3-dependent ephrinA2 responseinto growth cone expansion and axonal chemorattraction. Furthermore, we show thatmembrane cholesterol depletion alters axonal structure by producing varicosities and that EphA3partially protect against this effectCONCLUSIONSIn the present study, we have shown that 5 mM of β-MCD for 30 minutesits sufficient to reduce cholesterol from cell plasma membrane that produces a decrease inaxonal length, an increase in the proportion of collapsed growth cones, formation of axonalvaricosities, inhibition of EphA3 gradient-mediated axonal guidance, and a change in the pattern ofephrin A2 expression.