Abstract
Purpose :
RNA localization and translation in axons contributes to promoting axon regeneration. 3’ untranslated regions (3’UTRs) of mRNAs regulate the specificity of RNA localization and local translation in highly compartmentalized neurons, that can act precisely to various stimuli at long distances. In this study, we examined growth cone RNA translation and dependence on 3’UTRs in neurons expressing different targeting 3’UTRs under various conditions, to explore mechanisms involved in regional growth cone translation.
Methods :
Constructs carrying photoswitchable fluorescent myrDendra with a non-specific-targeting γ-actin 3’UTR, an axon-targeting Gap43 3’UTR, a dendrite-targeting neurogranin 3’UTR, or no 3’UTR were transfected into hippocampal neurons. Dendra signals in distal growth cones were recorded before and after photoswitching to measure fluorescence recovery dependent on local translation. Regulation of local protein synthesis by brain-derived neurotrophic factor (BDNF), forskolin, and KCl-induced depolarization and repolarization was measured. 3’UTR fragments responsive to BDNF and forskolin were screened in growth cone protein synthesis assay.
Results :
Neurons expressing myrDendra with a non-specific-targeting γ-actin 3’UTR showed significant recovery of Dendra intensity in distal growth cones at 20 and 40 minutes after photoswitching; the recovery was disrupted by anisomycin, confirming recovery was due to local protein synthesis. No significant recovery was seen in neurons expressing myrDendra 3’Gap43, myrDendra 3’Neurogranin or no 3’UTR control, but BDNF or BDNF plus forskolin strongly promoted local synthesis of myrDendra 3’Gap43. Significant Dendra recovery was detected in myrDendra 3’Gap43-expressing neurons after washing out from an 18 hr treatment with KCl, although neither acute depolarization or longer term hyperpolarization induced by 2 hr or 20 hr KCl, respectively, promoted growth cone synthesis. In a follow-up screen of 3’UTR fragments derived from axon-localized mRNAs ranked for their minimum free energy values of the predicted axonal RNA structure, 9 3’UTR fragments enhanced local protein synthesis in growth cones when treated with BDNF and forskolin.
Conclusions :
These data provide a promising in vitro platform to screen candidate 3’UTR sequences that promote local translation in growth cones, and identify molecular mechanisms that may be leveraged to promote axon regeneration after injury or in degenerative diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.