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TORC1 regulation of dendrite regrowth after pruning is linked to actin and exocytosis

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 polarised 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. Author Summary During development, neurons grow axons and dendrites that they use to make synaptic connections. Such connections are often fine-tuned through pruning and regrowth of axons and dendrites, but the coordination of the two processes is not well understood. It had previously been shown that hormone signaling suppresses the TORC1 growth pathway during pruning of Drosophila sensory neuron dendrites. We found that TORC1 is required for the subsequent regrowth of these dendrites. TORC1 activates protein biosynthesis, and our analyses suggest that it primarily targets neurite growth pathways, but not degenerative pathways. These growth pathways include the actin cytoskeleton and the secretion machinery with the small GTPase RalA. Thus, the TORC1 growth pathway is a major hub coordinating neurite pruning and regrowth.


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 polarised 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). 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.
Growth signaling pathways, and in particular the target of rapamycin complex 1 (TORC1) pathway has been implicated in axon regrowth after pruning (3,4). TORC1 is a multi-subunit kinase that can be stimulated by diverse stimuli including amino acid sensing through the RAG complex, phosphatidylinositol-3-kinase (PI3K) signaling downstream of growth factor receptors (5), and through transcriptional activation of the TOR gene (4). Interestingly, activation of TORC1 signaling also promotes axon regrowth after injury (6), suggesting that developmental regrowth pathways may be particularly well suited to stimulate growth also after injury. Thus, an understanding of regrowth pathways is also desirable in the medical context. TORC1 mainly promotes growth by enhancing the rate of protein synthesis. This is achieved through either of two targets, RpS6 kinase (S6K) and eIF4E-binding protein (4E-BP) (5). S6K is activated by TORC1 and promotes ribosome biogenesis. 4E-BP binds to and inhibits the translation initiation factor eIF4E, and TORC1-mediated phosphorylation inhibits it and thus derepresses eIF4E. Different mRNAs depend to varying degrees on eIF4E for translation initiation, and thus, TORC1 inhibition or activation has differential effects on the translatome (7)(8)(9). The degree to which an mRNA depends on eIF4E for translation seems to be encoded in part in its 5' untranslated region (5' UTR). For example, ribosomal protein mRNAs often have short, so-called 5' terminal oligopyrimidine (TOP) motifs in their 5'UTRs that confer a strong dependence on eIF4E, and hence TORC1, for maximal translation. Other motifs governing TORC1 dependence also exist but are not well understood (8).
Whether TORC1 signaling confers any specificity during regrowth after pruning is not clear.
While several signaling pathways involved in axon regrowth after pruning are known, less is known for dendrite regrowth after pruning. In Drosophila, the peripheral class IV dendritic arborization neurons (c4da) prune and then regrow their sensory dendrites (10). Recently, transcriptional (11) and posttranscriptional (12) gene regulatory pathways as well as specific cytoskeletal regulators (13) were identified that contribute to dendrite regrowth, but how they contribute to temporal regulation and specificity is still unclear. The TORC1 pathway has previously been shown to be downregulated during c4da neuron dendrite pruning (15), opening up the possibility of differential regulation. Furthermore, we recently provided evidence that translation initiation may be differentially regulated during dendrite pruning (16).
Here, we show that TORC1 is indeed required for dendrite regrowth after pruning.
Experiments with 5' UTR reporter genes suggest that TORC1 selectively stimulates translation of regrowth, but not pruning factors linked to the actin cytoskeleton.
Furthermore, we find that exocytosis via RalA and the exocyst is also required for dendrite regrowth, and we provide evidence that TORC1 is involved in the regulation of this pathway as well.

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 of the ventrolateral c4da neuron v'ada. As c4da neuron dendrite pruning is terminated at approximately 16 h APF, we assessed c4da 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. In these control neurons, dendrite regrowth started shortly before 48 h APF ( Fig. 1 A, C). Long branched dendrites had formed at 72 h APF, and full dendritic field coverage was reached at approximately 96 h APF ( Fig. 1 A, C).
In a candidate screen for factors that affected v'ada dendrite regrowth, we identified TOR, a kinase involved in growth regulation. v'ada neurons expressing TOR dsRNA displayed a strong delay in dendrite regrowth, such that only a fraction of the dendritic field was covered at 72 h APF ( Fig. 1 B, C). In order to confirm these results, we next used CRISPR/Cas9. Compared to control neurons only expressing Cas9, neurons expressing two sgRNAs targeting TOR had much shorter dendrites at 72 h APF ( Fig. 1 D -F). Furthermore, v'ada neuron MARCM clones homozygous for a TOR null mutation also displayed strongly reduced dendrite growth at this stage when compared to heterozygous control neurons in the same animals ( Fig. 1 G -I, the seemingly shorter dendrite length in control animals in these experiments is due to the weaker fluorescence of the used c4da neuron marker).
To address whether TOR is generally required for dendrite growth, we assessed the effects of TOR knockdown on elaboration of larval v'ada dendrites. Consistent with a previous report (17), loss of TOR only led to a mild -if any -decrease in dendrite length at the larval stage (Fig. S1). TOR knockdown had also no effect on dendrite pruning (Fig. S1).
TOR exists in two complexes, TORC1 and TORC2, that impinge on translation and the cytoskeleton, respectively. TORC2 has previously been shown to regulate dendritic tiling in larval c4da neurons (17). To distinguish between these complexes during dendrite regrowth after pruning, we knocked down specific TORC1 and TORC2 subunits. Knockdown of raptor, a TORC1 subunit, caused strong dendrite regrowth defects, while knockdown of the TORC2 subunits rictor and sin1 only had mild effects (Fig. S2). Furthermore, overexpression of a constitutively active form of the TORC1 downstream target S6K led to a significant rescue of the regrowth defects caused by TOR knockdown (Fig. S2). Thus, TOR, mostly via the TORC1 complex, is specifically required for dendrite regrowth after pruning.
Dendrite regrowth requires the InR/PI3K/PDK1/Akt1 pathway TORC1 can be activated by several upstream mechanisms, including the levels of specific amino acids and phosphatidylinositol-3 kinase (PI3K) signaling downstream of receptor tyrosine kinases (5). In Drosophila mushroom body  neurons, TOR has been reported to be activated by transcriptional upregulation through the transcription factors UNF and E75 (18). In order to distinguish between these possibilities, we assessed the effects of dsRNAs or sgRNAs targeting these pathways on dendrite regrowth. Knockdown of UNF or E75 did not affect dendrite regrowth ( An assay to assess TORC1 sensitivity of remodeling factor mRNAs TORC1 promotes growth by upregulating translation through activation of S6K and inhibition of 4E-BP. Specificity in this system arises through the differential sensitivity of various mRNAs to 4E-BP inhibition. As no TORC1 targets are known in the context of neuronal remodeling, we aimed to establish an assay for 4E-BP sensitivity of different remodeling factor mRNAs. 4E-BP sensitivity is often encoded in the 5' UTRs of an mRNA. We therefore designed UAS transgenes driving expression of nuclear-localized, short-lived tdtomato (19) under the control of 5' UTRs from regrowth and pruning factor mRNAs (Fig. 3A, B). To be able to account for effects of GAL4 strength, we additionally co-expressed UAS-CD8::GFP without a specific 5' UTR for normalization (Fig. 3A). As ribosomal protein mRNAs are strongly regulated by TOR (7,9), we chose the 5' UTR of the Drosophila RpL13 mRNA as a bona fide candidate factor for a TOR/4E-BP target. Consistent with the importance for protein production during dendrite growth (20), RpL13 knockdown caused dendritic defects already at the larval stage and was strongly required for dendrite regrowth (Fig. S3).
The small GTPase Rac1 had recently been shown to be required for dendrite regrowth via actin regulation (13). As some mammalian actin isoforms are regulated by TOR (21) and the Drosophila actin isoform Act5C was also required for dendrite regrowth (Fig. S3), we chose the 5' UTRs of the Rac1 and Act5C mRNAs as regrowth factor candidates. The actin severing enzyme Mical is required for c4da neuron dendrite pruning (22). As Mical knockdown does not cause any apparent dendrite regrowth defects (

TOR affects actin regulation during dendrite regrowth after pruning
The 5' UTR reporter assay indicated that TOR might be involved in actin regulation during dendrite regrowth. To test this idea, we expressed the F-actin marker lifeact::GFP in c4da neurons and measured lifeact::GFP levels in the dendrites. To control for potential differences in reporter transcription levels, we normalized the lifeact::GFP levels to tdtomato. In control animals, lifeact::GFP could be readily detected in dendrites at both 48 and 72 h APF (Fig. 4 A, C, D, F). While the overall fluorescence intensity was lower in neurons lacking TOR, the lifeact::GFP/tdtomato ratio was similar to that of the control at 48 h APF (Fig. 4 B, C). In contrast, the lifeact::GFP/tdtomato ratio was significantly lower in the absence of TOR at 72 h APF ( Fig. 4 E, F), indicating a reduction in dendritic F-actin during this period of dendrite regrowth.
To further corroborate this effect of TOR on the actin cytoskeleton, we asked if TOR interacted genetically with Rac1 during dendrite regrowth. In control neurons, expression of GTPase-deficient, constitutively active Rac1 V12 did not lead to a significant increase in the total dendrite length at 72 h APF (Fig. 4 G, H, K). In contrast, Rac1 V12 caused a significant dendrite length increase in the TOR knockdown background when compared to TOR knockdown alone, indicating a partial rescue (Fig. 4 I -K). Importantly, this rescue was not common to small GTPases in general, as expression of a constitutively active form of Cdc42 did not lead to a rescue of the dendrite growth defects, even though Cdc42 was also required for c4da neuron dendrite regrowth (Fig. S4). These data suggest that reduced actin polymerization via specific pathways contributes to the regrowth defects upon loss of TOR.

The small GTPase RalA is required for dendrite regrowth after pruning
In our candidate screen for dendrite regrowth regulators, we also found evidence for an involvement of Drosophila RalA in dendrite regrowth. Specifically, RalA knockdown with two independent dsRNA constructs caused strong dendrite regrowth defects at 72 h APF (Fig. 5 A -D). In contrast to the strong effect on dendrite regrowth after pruning, RalA knockdown did not cause apparent defects in larval c4da neuron dendrite elaboration (Fig. S5), indicating that it might be specifically required at the regrowth stage.
Ral GTPases are small GTPases that regulate membrane trafficking and various signaling pathways. In mammals, there are two closely related Ral GTPases, RalA and RalB. Of these, RalB has been described as an upstream regulator of the TOR pathway (23) To test whether RalA was also linked to TOR signaling, we asked again if constitutively active, GTPase-deficient RalA G20V could rescue the dendrite regrowth defects induced by loss of TOR. C4da neurons expressing RalA G20V alone did not have significantly longer dendrites than controls at 72 h APF (Fig. 5 G, H, K). In contrast, RalA G20V led to significant rescue of the dendrite regrowth defects in neurons expressing TOR sgRNA (Fig. 5 I -K). Thus, RalA might act in a TOR downstream pathway during dendrite regrowth.

Polarized exocytosis is required for dendrite regrowth
As our data suggested that RalA might act at the plasma membrane, we next asked whether the exocyst complex could be involved in dendrite regrowth as well. This complex links exocytic vesicles to the plasma membrane via an interaction with Rab proteins and plasma membrane phospholipids (26). It consists of the Sec5 subcomplex (containing Sec3, Sec5, Sec6, Sec10) and the Exo84 subcomplex (containing Exo70, Exo84, Sec8 and Sec15). Loss of the exocyst had previously been shown to cause defects in larval dendrite branching (27). We knocked down subunits from both subcomplexes using RNAi and found that loss of Sec6, Sec10, Exo70, Exo84 or Sec15 caused dendrite regrowth defects at 72 h APF compared to control neurons expressing Orco dsRNA (Fig 6 A -G). To verify these results with an independent method, we generated sgRNAs targeting Sec5 and Exo84 and coexpressed them with Cas9 in c4da neurons. Again, tissue-specific CRISPR knockdown of Sec5 and Exo84 caused strong regrowth defects at 72 h APF (Fig. 6 H -K). Thus, polarized exocytosis via the exocyst is required for dendrite regrowth after pruning, strongly suggesting the involvement of the RalA-exocyst pathway.

Links between TOR and membrane regulation during dendrite regrowth
The above data opened up the possibility that exocytosis may be a downstream target of TOR signaling during c4da neuron dendrite regrowth. To test whether TOR manipulation affects membrane traffic during c4da neuron dendrite regrowth, we made use of a pHluorin-CD4-tdtomato reporter (28). Here, the extracellularly exposed pHluorin moiety of the reporter only fluoresces in a neutral pH environment, as is present on the cell surface (Fig. 7 A). The pHluorin-CD4-tdtomato reporter can therefore be used to monitor endocytosis (29), exocytosis (27) and phagocytosis (29). In control c4da neurons at 48 h APF, pHluorin and tdtomato were clearly detectable in the growing dendrites (Fig. 7 B -B'', D). tdtomato fluorescence intensity was comparable in dendrites of control c4da neurons and neurons expressing TOR sgRNA. However, the pHluorin signal was significantly weaker in neurons expressing TOR sgRNA, resulting in a significant decrease of the pHluorin/tdtomato ratio (Fig. 7 C -D), suggesting that TOR downregulation may lead to defective or delayed membrane traffic during dendrite regrowth in c4da neurons.
How could TOR be linked to exocytosis during dendrite regrowth? One possibility is that the 5' UTRs of membrane traffic factor mRNAs might also be sensitive to 4E-BP and disinhibition by TORC1. To test this idea, we generated a 5' UTR reporter using the 5'UTR of RalA mRNA. Indeed, the RalA 5' UTR rendered reporter expression partially sensitive to 4E-BP (Fig. 7 E -E''). Thus, our results suggest that TOR promotes exocytic membrane traffic during dendrite regrowth through translational upregulation of membrane trafficking regulators.

Discussion
Here we investigated a potential role and of TOR signaling in dendrite regrwoth 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 (15). We found that the TORC1 complex is specifically is required for dendrite regrowth after pruning in Drosophila sensory neurons and assessed its target specificity. Our results suggest that TORC1 likely broadly stimulates the translation of regrowth factors, including those linked to actin regulation and polarized exocytosis (Fig. 7 F). The involvement of polarized secretion factors potentially points to a specific spatial regulation of dendrite regrowth, as the axons of c4da neurons are not pruned during the early pupal stage and therefore do not need to regrow (10). In line with this idea, the exocyst has recently also been implicated in neurite regrowth after injury (30). Despite the possibly very broad range of TORC1 targets, our analyses suggest that TORC1 also imposes specificity in that it does not promote the translation of the pruning factor Mical, and that its targets might have opposing functions to those regulated by the pruning trigger ecdysone (Fig. 7 F).
We recently found that the metabolically regulated AMP-dependent protein kinase (AMPK) is required for dendrite pruning in the fly PNS through adaptation of cellular metabolism (31). AMPK is sometimes described as a TOR antagonist, and a recent study found that AMPK also antagonizes TOR during dendrite pruning (32). It is therefore interesting to speculate that TOR may also act on neuronal metabolism during regrowth.
While a clear mechanism has been proposed that inhibits TOR during dendrite pruning in response to ecdysone (15), it is less clear what activates it during regrowth. Our results suggest that InR/PI3K/Akt1 signaling is upstream of TOR here, thus potentially linking dendrite regrowth to extrinsic hormonal cues. In order to better understand the temporal regulation of the process, it will be interesting to identify the ligand(s) involved. Overall, our results provide a rationale as to why TOR must be differentially regulated during neuronal remodeling and suggest pathways by which neuronal remodeling is linked to systemic developmental signals.

Dissection, Microscopy and Live imaging
For analysis of regrowth defects, appropriately staged pupae were dissected out of the pupal case at 24 -96 h APF and v'ada in segments A2 -A4 was visualized in live animals using a Zeiss LSM710 confocal microscope. All images were analyzed using Fiji (39), the NeuronJ plugin (40) was used for dendrite length measurements. NLStdtomato PEST /CD8::GFP intensity ratios of 5' UTR reporters were measured in the soma of third instar lateroventral (v'ada) c4da neurons. lifeact::GFP/tdtomato and pHluorin/tdtomato ratios were measured in primary dendrites at the indicated timepoints.

Quantification and statistical analysis
Dendrite regrowth phenotypes were analyzed by measuring dendrite length at the indicated timepoints using the Fiji NeuronJ plugin, these data were analyzed with Prism6 software using a two-tailed Mann-Whitney U test. Intensity ratios for NLStdtomato PEST /CD8::GFP (5' UTR reporters), dendritic lifeact::GFP/tdtomato and pHluorin/tdtomato ratios were also compared with a two-tailed Mann-Whitney U test.

Acknowledgements
We would like to thank C. Klämbt for support, M. Krahn for comments on the