Optical control of ERK and AKT signaling promotes axon regeneration and functional recovery of PNS and CNS inDrosophila

Abstract

15Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple 16 signaling circuits. Channelrhodopsin-based optogenetics shows feasibility of stimulating neural 17 repair but does not pin down specific signaling cascades. Here, we utilized optogenetic systems, 18 optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to 19 neuroregeneration in live Drosophila larvae. We showed that optoRaf or optoAKT activation not 20 only enhanced axon regeneration in both regeneration competent and incompetent sensory 21 neurons in the peripheral nervous system, but also allowed temporal tuning and proper guidance 22 of axon regrowth. Furthermore, optoRaf and optoAKT differ in their signaling kinetics during 23 2 regeneration, showing a gated versus graded response, respectively. Importantly in the central 24 nervous system, their activation promotes axon regrowth and functional recovery of the 25 thermonociceptive behavior. We conclude that non-neuronal optogenetics target damaged 26 neurons and signaling subcircuits, providing a novel strategy in the intervention of neural 27 damage with improved precision. 28 29 92 5 the previous optogenetic AKT system (Ong et al. 2016) with two tandom CIBNs (referred to as 93 optoAKT in this work) (Supplemental Fig. S1A). Consistent with previous studies, the association 94 of CIBN and CRY2 took about 1 second and the CIBN-CRY2 complex dissociated in the dark 95 within 10 minutes (Kennedy et al. 2010; Zhang et al. 2014). The fusion of Raf or AKT does not 96 affect the association and dissociation kinetics of CIBN and CRY2 and multiple cycles of CRY2-97 CIBN association and dissociation can be triggered by alternating light-dark treatment 98 (Supplemental Fig. S1B-S1D, Movie S1, S3). Activation of optoRaf and optoAKT resulted in 99 nuclear translocation of ERK-EGFP (Fig. 1A, Movie S2) and nuclear export of FOXO3-EGFP 100 (Fig. 1B, Movie S4) resolved by live-cell fluorescence imaging, indicative of activation of the 101 ERK and AKT signaling pathways, respectively.102Western blot analysis on pERK (activated by optoRaf) in HEK293 cells showed that 103 pERK activity (Fig. 1C) increased within 10 min blue light stimulation and returned to the basal 104 level 30 min after the blue light was shut off (Fig. 1D). There was a slight decrease of pERK 105 activity upon optoRaf activation for over 10 min, likely due to a negative feedback, which has 106 been consistently observed in previous studies (Zhou et al. 2017). On the other hand, continuous 107 light illumination maintained a sustained activation of pCRY2-mCh-AKT (referred to as 108 optoAKT in this work) within an onset of 10 min (Fig. 1E). The inactivation kinetics of pAKT 109 was 30 min, similar to that of pERK ( Fig. 1F, 1G). Note we use only the phosphorylated and 110 total forms of CRY2-mCh-AKT to quantify the light response of optoAKT because the 111 endogenous AKT does not respond to light.112 113 optoRaf and optoAKT do not show crosstalk activity at the pERK and pAKT level 114 ...

Membrane-associated shortened Trk receptors promote neuroprotection and robust axon regeneration without ligands

“…The kinase activity is required for F-iTrkB transphosphorylation at Tyr515, causing F-iTrkB to interact with the adaptor proteins Shc and GRB2. This interaction is known to induce multiple complex formations with Ras and PI3K, followed by activation of the Ras-ERK and PI3K-AKT signaling, both of which play crucial roles in neuroprotection and axon regeneration (23)(24)(25)(26)(27). Indeed, both F-iTrkB and F-iTrkA successfully activated these signals in vitro, and optic nerve regeneration was observed in vivo with intraocular injection of AAV-F-iTrkB and AAV-F-iTrkA.…”

Gliotransmission orchestrates neuronal type-specific axon regeneration

“…It is thus plausible that growth factors, neurotransmitters, and peptides can regulate axon regeneration through regulation of Ras activity. Among the many Ras targets, the canonical MAPK pathway has been shown to regulate axon regeneration, as activation of this pathway promotes axon regeneration in both mammals and flies (Hollis et al, 2009;O'Donovan et al, 2014;Perlson et al, 2005;Wang et al, 2020), and activated ERK has been found to transport retrogradely from the injury site to the cell body (Perlson et al, 2005).…”

“…To investigate how Ca 2+ spikes determine axon regeneration, we focused on Ras GTPase as a potential downstream effector based on the findings that intracellular Ca 2+ activates Ras and that mitogen-activated protein kinase (MAPK) pathways regulate axon regeneration in both mammals and flies (Hollis et al, 2009;O'Donovan et al, 2014;Perlson et al, 2005;Rosen et al, 1994;Wang et al, 2020). We found that reducing Ras activity in C4da neurons, by expressing the Drosophila or mouse version of dominant negative Ras (Ras DN ), significantly impaired axon regeneration (Figure 6A-6B).…”

A kinase translocation reporter reveals real-time dynamics of ERK activity in Drosophila

“…Egfr and Heartless in glia both signal through ERK to promote different outcomes: inducing differentiation of the photoreceptor target field and ensheathment of photoreceptor axons, respectively (Fernandes et al, 2017; Franzdóttir et al, 2009). Recent advances in the development of optogenetic tools to manipulate ERK signalling in flies have enabled a fine dissection of the temporal requirements for ERK activity during development, and refined our understanding of the signalling dynamics in cell fate decisions (Bunnag et al, 2020; Johnson and Toettcher, 2019; Johnson et al, 2017; Patel et al, 2019; Wang et al, 2020; Yadav et al, 2021). Thus, the ability to monitor ERK activity dynamics in Drosophila, in combination with the tools to manipulate it, would provide an unparalleled understanding of how temporal signalling dynamics regulate fate decisions.…”