TORC1 Regulation of Dendrite Regrowth After Pruning is Linked to Actin and Exocytosis

Abstract

Neurite pruning and regrowth are important mechanisms to adapt neural circuits to distinct developmental stages. Neurite regrowth after pruning often depends on differential regulation of growth signaling pathways, but their precise mechanisms of action during regrowth are unclear. Here, we show that the PI3K/TORC1 pathway is required for dendrite regrowth after pruning in Drosophila peripheral neurons during metamorphosis. TORC1 impinges on translation initiation, and our analysis of 5’ untranslated regions (UTRs) of remodeling factor mRNAs linked to actin suggests that TOR selectively stimulates the translation of regrowth over pruning factors. Furthermore, we find that dendrite regrowth also requires the GTPase RalA and the exocyst complex as regulators of polarized secretion, and we provide evidence that this pathway is also regulated by TOR. We propose that TORC1 coordinates dendrite regrowth after pruning by coordinately stimulating the translation of regrowth factors involved in cytoskeletal regulation and secretion.

Introduction

Developmental neurite remodeling is an important mechanism that serves to specify neuronal circuits, e. g., to adapt them to specific developmental stages. During remodeling, specific axons and dendrites can be removed through pruning, while others are maintained [1]. Cell biological pathways underlying neurite pruning therefore must involve mechanisms for spatial regulation of neurite destruction and maintenance. This spatial aspect of pruning may in part be due to cytoskeleton regulation [2] and tissue mechanical aspects [3]. Following pruning, remodeling neurons often regrow new neurites with a morphology adapted to subsequent developmental stages. The specific mechanisms underlying neurite regrowth after pruning are only beginning to be understood. In particular, it is interesting to ask how regrowth is coordinated with pruning at the signaling level, and if it uses different mechanisms than initial neurite growth.

Materials and method

Fly strains

All crosses were done at 25°C. For expression labeling of c4da neurons, we used ppk-GAL4 insertions on the second and third chromosomes [38]. MARCM clones of the TORdeltaP mutant allele (BL 7014) were induced with SOP-FLP [39] and labeled by tdtomato expression under nsyb-GAL4R57C10 [40] and a ppk-eGFP promotor fusion [41]. ppk-MApHS [30] was used to express pHluorin-CD4-tdtomato. UAS lines were UAS-tdtomato [30], UAS-Rac1V12 (BL 6291), UAS-cdc42V12 (BL 4854), UAS-RalAG20V (BL 81049), UAS-lifeact::GFP (BL 35544), UAS-4E-BP LL [42], UAS-GFP::RalA [27]. UAS-dsRNA lines were: TOR (BL 34639), raptor (#1: BL 31528, #2: 31529), rictor (#1: BL 31388, #2: BL 31527), sin1 (BL 36677), Trc (BL 28326), Act5C (VDRC 7139), RpL13 (VDRC 101369), Rac1 (VDRC 49246), RalA (#1: BL 29580, #2: VDRC 105296), Sec6 (VDRC 105836), Sec10 (BL 27483), Sec15 (#1: VDRC 105126, #2: VDRC 35161), Exo70 (VDRC 103717), Exo84 (VDRC 108650), Pdk1 (#1: BL 27725, #2: VDRC 109812), PI3K92E (VDRC 107390), InR (VDRC 992), E75 (BL 35780), Hr51 (BL 39032), Rag A-B (BL 34590), Rag C-D (BL 32342), Cdc42 (VDRC 100794). UAS-dsRNA lines were coexpressed with UAS-dcr2 [43], and Orco dsRNA (BL 31278) or mcherry dsRNA (BL 35785) were used as controls. Lines for CRISPR-mediated knockdowns were: PI3K92E sgRNA (BL 80898), Akt1 sgRNA #1 (BL 83097), Akt1 sgRNA #2 (BL 83502). UAS-Cas9P2 (BL 58986) was used for conditional CRISPR.

Results

TORC1 is required for c4da neuron dendrite regrowth after pruning

To visualize regrowth of c4da neuron dendrites after pruning, we first established a timecourse of regrowth. We chose to study the ventrolateral c4da neuron v’ada, because it remodels its dendrites with a similar timecourse as the dorsal c4da neuron ddaC [11,16], but is easier to visualize due to a lower number of autofluorescent bristles in its dendritic field. C4da neuron dendrite pruning is terminated at approximately 16–20 h APF [11,16]. We assessed v’ada neuron morphology at 24, 48, 72 and 96 h APF. As a control for dsRNA-mediated knockdowns, we expressed a dsRNA construct directed against the odorant coreceptor Orco, which is not expressed in these neurons, and does not affect dendrite regrowth (S1 Fig). In these control neurons, dendrite regrowth started shortly before 48 h APF (Fig 1A and 1C). Long branched dendrites had formed at 72 h APF, and full dendritic field coverage was reached at approximately 96 h APF (Fig 1A and 1C).

Discussion

Here we investigated a potential role and of TOR signaling in dendrite regrowth after pruning. PI3K/TOR signaling has previously been implicated in neurite (re-)growth, but both upstream signaling and its downstream targets have not been extensively explored. TORC1 had not previously been implicated in dendrite regrowth, but it had been shown that it must be downregulated during the pruning process preceding dendrite regrowth [17]. We found that the TORC1 complex is specifically required for dendrite regrowth after pruning in Drosophila sensory neurons, but not for initial growth of larval dendrites. We can envisage two possible explanations for this surprising specificity. For one, protein persistence and maternal contribution are known to mask early developmental phenotypes in Drosophila. However, the larval stage is a phase of intense growth and an abundance of nutrients. It is therefore interesting to speculate that another growth regulatory pathway may predominate—or be redundant with TOR—during the larval stage. For example, dMyc has been shown to regulate growth via transcription of ribosomal RNAs [32].

Citation: Sanal N, Keding L, Gigengack U, Michalke E, Rumpf S (2023) TORC1 regulation of dendrite regrowth after pruning is linked to actin and exocytosis. PLoS Genet 19(5): e1010526. https://doi.org/10.1371/journal.pgen.1010526

Editor: Yan Song, Peking University, CHINA

Received: November 14, 2022; Accepted: April 14, 2023; Published: May 11, 2023

Copyright: © 2023 Sanal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by grant RU1673/6-1 from the Deutsche Forschungsgemeinschaft (DFG) and the Collaborative Research Center CRC1348 (project B04) to SR. NS was supported by the Cells-in-Motion (EXC1003) graduate school and Cells-in-Motion bridging funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.