Orai mediated Calcium entry determines activity of central dopaminergic neurons by regulation of gene expression

Joint Public Review:

The manuscript by Mitra and coworkers analyses the functional role of Orai in the excitability of central dopaminergic neurons in Drosophila. The authors show that a dominant-negative mutant of Orai (OraiE180A) significantly alters the gene expression profile of flight-promoting dopaminergic neurons (fpDANs). Among them, OraiE180A attenuates the expression of Set2 and enhances that of E(z) shifting the level of epigenetic signatures that modulate gene expression. The present results also demonstrate that Set2 expression via Orai involves the transcription factor Trl. The Orai-Trl-Set2 pathway modulates the expression of VGCC, which, in turn, are involved in dopamine release. The topic investigated is interesting and timely and the study is carefully performed and technically sound.

The reviewers appreciate the authors' efforts to revise the manuscript in order to address many of their concerns. Nevertheless, there remain a few important issues:

1. The main issue relates to Set2, and how STIM1 expression rescues Set2-dependent functions in Set2 KO flies. If Set2 is downstream of STIM1, how would STIM1 over-expression rescue a Set2-dependent effect?

2. There is still no characterization of SOCE in fpDANs from flies expressing native Orai or the dominant negative OraiE180A mutant.

3. The revised version does not include an analysis of the STIM:Orai stoichiometry, which has been demonstrated to be essential for SOCE.

Author Response

The following is the authors’ response to the current reviews.

1. The main issue relates to Set2, and how STIM1 expression rescues Set2-dependent functions in Set2 KO flies. If Set2 is downstream of STIM1, how would STIM1 over-expression rescue a Set2-dependent effect?

STIM rescue is of Set2 knockdown (RNAi) and NOT Set2 Knockout flies. Over expression of STIM raises SOCE in primary cultures of Drosophila neurons (as demonstrated in previous publications from our group: Agrawal et al., 2010; Chakraborty et al, 2016; Deb et al., 2016). The higher SOCE drives greater expression of Set2 from the endogenous locus thus reducing the efficacy of Set2 RNAi. Hence the rescue by STIM of Set2 KD flies in Figure S2E. We have explained this in lines 227-234.

1. There is still no characterization of SOCE in fpDANs from flies expressing native Orai or the dominant negative OraiE180A mutant.

Measurement of SOCE is not technically feasible in ex-vivo preps due to the presence of extracellular calcium in the brain milieu. In the past we have measured SOCE from primary cultures of central dopaminergic neurons expressing either native Orai OR OraiE180A mutant (Pathak et al., 2015) where we found that all dopaminergic neurons expressing OraiE180A exhibit very low SOCE. This is the reason we have not measured SOCE in the fewer cells of the fpDAN subset marked by THD' GAL4. This point has been specifically mentioned and explained in the section on “limitations of the study” at the end of the manuscript.

1. The revised version does not include an analysis of the STIM:Orai stoichiometry, which has been demonstrated to be essential for SOCE.

To measure such stoichiometry we would need to perform direct measurements of STIM and Orai levels by protein extraction from the fpDANs of all appropriate genotypes. This is not feasible due to the small number of cells available from each brain.

I confirm that there are no changes to the text OR figures from the previous version of the manuscript.

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The following is the authors’ response to the original reviews.

[…]

The manuscript by Mitra and coworkers analyses the functional role of Orai in the excitability of central dopaminergic neurons in Drosophila. The authors show that a dominant-negative mutant of Orai (OraiE180A) significantly alters the gene expression profile of flight-promoting dopaminergic neurons (fpDANs). Among them, OraiE180A attenuates the expression of Set2 and enhances that of E(z) shifting the level of epigenetic signatures that modulate gene expression. The present results also demonstrate that Set2 expression via Orai involves the transcription factor Trl. The Orai-Trl-Set1 pathway modulates the expression of VGCC, which, in turn, are involved in dopamine release. The topic investigated is interesting and timely and the study is carefully performed and technically sound; however, there are several major concerns that need to be addressed:

1. In Figure S2E, STIM is overexpressed in the absence of Set2 and this leads to rescue. It is presumed that STIM overexpression causes excess SOCE, yet this is rarely the case. Perhaps the bigger concern, however, is how excess SOCE might overcome the loss of SET2 if SET2 mediates SOCE-induced development of flight. These data are more consistent with something other than SET2 mediating this function.

Our statement that STIM overexpression overcomes deficits in SOCE is based on the following published work, which has been highlighted in the revised version of the manuscript (see Lines 226-233):

1. Studies of SOCE in wildtype cultured larval Drosophila neurons demonstrated that overexpression of STIM raised SOCE to the same extent as co-expression of STIM and Orai in the WT background (Chakraborty et al, 2016; Figure 1D).

2. Both Carbachol-induced IP3-mediated Ca2+ release and SOCE (measured by Ca2+ add back after Thapsigargin-induced store depletion) were rescued in primary cultures of IP3R hypomorphic mutant (itprku) Drosophila neurons by overexpression of STIM (Agrawal et al., 2010; Figure 8A-G).

3. Deb et al., 2016 (Supplementary Figure 2h,i) reaffirmed that overexpression of STIM significantly improves SOCE after Thapsigargin-induced passive store-depletion in Drosophila neurons expressing IP3RRNAi.

4. Consistent with the cellular rescue of SOCE, defects in flight initiation and physiology observed in the heteroallelic IP3R hypomorphic background (itprku) could be rescued by overexpression of STIM (Agrawal et al., 2010; Figure 3A-E) as well as Orai (Venkiteswaran and Hasan, 2009; Figure 3).

5. In Figure S2E, we show that flight deficits arising from THD’> Set2RNAi are rescued upon overexpression of STIM (i.e. THD’>Set2RNAi; STIMOE). Here and in another recent publication (Mitra et al., 2021) we show that neurons expressing Set2RNAi exhibit reduced expression of the IP3R and reduced ER-Ca2+ release presumably leading to reduced SOCE. As mentioned above we have consistently found that STIM overexpression raises both IP3-mediated Ca2+ release and SOCE in Drosophila neurons.

In this study, we propose that Ca2+ release through the IP3R followed by SOCE are part of a positive feedback loop (described in the revised manuscript- see Lines 302-307) driving expression of Set2 which in turn upregulates expression of mAChR and IP3R (Figure 3F) to regulate dopaminergic neuron function. Our observation that loss of Set2 (THD’>Set2RNAi) can be rescued by STIM overexpression is consistent with this model because:

1. Loss of Set2 (THD’>Set2RNAi) results in downregulation of several genes including mAChR and IP3R leading to decreased SOCE.

2. As evident from our previous studies increased STIM expression in the Set2RNAi background (THD’>Set2RNAi; STIMOE) is expected to enhance SOCE which we predict would rescue Set2 expression leading to rescue of other Set2 dependent downstream functions like flight (Figure 2D).

1. In Figure 3, data is provided linking SET2 expression and Cch-induced Ca2+ responses. The presentation of these data is confusing. In addition, the results may be a simple side effect of SET2-dependent expression of IP3R. Given that this article is about SOCE, why isn't SOCE shown here? More generally, there are no measurements of SOCE in this entire article. Measuring SOCE (not what is measured in response to Cch) could help eliminate some of this confusion.

This section has been re-written in the revised version for better clarity and we have explained how Set2-dependent IP3R expression is an important component of Orai-mediated Ca2+ entry in fpDANs (see Lines 302-307). Here, we propose that IP3-mediated Ca2+ release and SOCE, through Orai, are together part of a positive feedback loop (see Lines 286-307) driving transcription of Set2 which in turn upregulates mAChR and IP3R expression (Figure 3F). We hypothesized that the observed loss of CCh-induced Ca2+ response in the Set2RNAi background (Figure 3B-D; THD’>Set2RNAi) results from decreased itpr and mAChR expression and verified this in Figure 3E. This is further validated by the rescue of CCh-induced Ca2+ response and itpr/mAChR expression in the OraiE180A background upon Set2 overexpression (Figure 3B-E; THD’>OraiE180A; Set2OE). We were constrained to measure CCh-induced Ca2+ responses in OraiE180A expressing neurons for the following reasons (highlighted in the revised version of the manuscript- (See Lines 307-313; ‘Limitations of the study’-Lines 719-735):

1. SOCE measurements through Tg mediated store Ca2+ release followed by Ca2+ add back require a 0 Ca2+ environment that can only be achieved in culture. The Drosophila brain is bathed in hemolymph which contains Ca2+ and there do not exist any methods to readily deplete Ca2+ from the tissue to create a 0 Ca2+ environment without also effecting the health of the neurons.

2. Cultures of the subset of dopaminergic neurons (THD’) we have focused on in this study were not feasible due to the small number of neurons being studied from the total number of dopaminergic neurons in the brain (~35/400). In previous studies we have shown that SOCE post-Tg induced store depletion is abrogated in cultured dopaminergic neurons from Drosophila upon expression of OraiE180A (Pathak et al., 2015). Furthermore, Carbachol-induced IP3-mediated Ca2+ release is tightly coupled to SOCE in Drosophila neurons (Venkiteswaran and Hasan, 2009) and Ca2+ release from the IP3R is physiologically relevant for flight behavior in THD’ neurons (Sharma and Hasan, 2020).

1. A significant gap in the study relates to the conclusion that trl is a SOCE-regulated transcription factor. This conclusion is entirely based on genetic analysis of STIMKO heterozygous flies in which a copy of the trl13C hypomorph allele is introduced. While these results suggest a genetic interaction between the expression of the two genes, the evidence that expression translates into a functional interaction that places trl immediately downstream of SOCE is not rigorous or convincing. All that can be said is that the double mutant shows a defect in flight which could arise from an interruption of the circuit. Further, it is not clear whether the trl13C hypomorph is only introduced during the critical 72-96 hour time window when the Orai1E180E phenotype shows up. The same applies to the over-expression of Set2 and the other genes. If the expression is not temporally controlled, then the phenotype could be due to the blockade of an entirely different aspect of flight neuron function.

The idea that Trl functions downstream of Orai-mediated Ca2+ entry in THD’ neurons is based on the following genetic evidence (highlighted in the revised version; see Lines 339-341; 351-367; 647-65; ‘Limitations of the study’: 736-739)

1. In Figure 4D, we show evidence of genetic interaction between trl-STIM and trl-Set2. The rescue of trl13c/STIMKO with STIM overexpression in THD’ neurons indicates that excess SOCE (driven by STIMOE) may activate the residual Trl (there exists a WT Trl copy in this genetic background) to rescue THD’ flight function. This is further supported by the rescue of trl/STIMKO with Set2 overexpression in THD’ neurons, which is consistent with the feedback loop model proposed in Figure 5C (see Lines 390-396) where we propose that reduced SOCE leads to reduced ‘activated’ Trl and thus reduced Set2 expression, and the latter is rescued by SET2OE . The manner in which SOCE ‘activates’ Trl is the subject of ongoing investigations.

2. The trl hypomorphic alleles (including trl13C) exist as genetic mutants and they affect Trl function in all tissues throughout development. While we concede that these mutant alleles would affect multiple functions at other stages of development, which may impinge on the phenotypes noted in Figure S4B, we have used a targeted RNAi approach to validate Trl function specifically in the THD’ neurons (see Figure 4C; Lines 339-341).

3. Overexpression mediated rescues (including Set2) were not induced only during the critical 72-96 hrs APF developmental window. Having established that Orai function drives critical gene expression during this window (Figure 1), it is reasonable to assume that Set2 rescue of loss of flight in OraiE180A occurs in the same time window where flight is disrupted (see Lines 221-224).

1. In Figure 4, data is shown that SOCE compensates for the loss of Trl, the presumed mediator of SOCE-dependent flight. The fact that flight deficits are rescued by raising SOCE in the absence of Trl is very inconsistent with this conclusion.

We apologise for this confusion and have clarified in the revision (see Lines 346-367). trl13c is a recessive allele of Trl and has been written as such throughout the text and in the figures (i.e trl13c and NOT Trl13c). In all cases of Trl mutant rescue by STIMOE and Set2OE there exists residual Trl that can be activated by excess SOCE thus leading to the rescue. This is true for trl13C/ STIMKO where each mutant is present as a heterozygote (the complete genotype of this strain is STIMKO/+; trl13c/+; this has been corrected in the revision). Similarly, for TrlRNAi we expect reduced levels (but not complete loss) of Trl. Thus the SOCE rescue of loss of Trl occurs in conditions where Trl levels are reduced but NOT absent. Homozygous trl null mutants are lethal.

1. In Figure 5 (A-C), data is provided that Trl transcripts are unaffected by loss of SOCE and that overexpression cannot rescue flightlessness. From this, the authors conclude that this gene "must" be calcium responsive. While that is one possibility, it is also possible that these genes are not functionally linked.

The idea that Trl is functionally linked to SOCE is based on the following evidence (included in the revised version- see Lines 339-341; 346-367; 391-396)

1. In Figure 4C we show that flight defects caused by partial loss of Trl (THD’>TrlRNAi) were rescued by STIM overexpression (THD’>TrlRNAi; STIMOE). As mentioned above we have found that STIM overexpression raises SOCE.

2. Heteroalleles of the trl13C hypomorph exhibit a strong genetic interaction with a single copy of the null allele of STIMKO as shown by the flight deficit of trl13c/+; STIMKO/+ (trl13C/STIMKO ) flies (Figure 4D). The genotypes will be corrected in the revision.

3. Flight defects in trl13C/STIMKO flies could be rescued by STIM overexpression in the THD’ neurons (trl13C/STIMKO; THD’>STIMOE)

4. In Figure 4E, we show that partial loss of Trl in THD’ neurons (THD’>TrlRNAi) leads to decreased expression of the Ca2+ responsive genes mAChR, itpr, and Set2 genes indicating that Trl is a constituent of the SOCE-driven transcriptional feedback loop (see Figure 5C).

Since we could not detect a well-defined Ca2+ binding domain in Trl, we hypothesize that it could be activated by a Ca2+ dependent post-translational modification. Phosphoproteome analysis of Trl demonstrated that it does indeed undergo phosphorylation at a Threonine residue (T237; Zhai et al., 2008), which lies within a potential site for CaMKII. Independently, CaMKII has been identified as a binding partner of Trl from a Trl interactome study (Lomaev et al., 2018). Past work from our group (Ravi et al., 2018) identified a role for CaMKII in THD’ neurons in the context of flight. We are currently testing if CaMKII functions downstream of SOCE in THD’ neurons to mediate flight and will update this information in the next version of the manuscript.

Now included in the revised version of the manuscript as Figure S5; Lines 397-424)

1. There is no characterization of SOCE in fpDANs from flies expressing native Orai or the dominant negative OraiE180A mutant. While the authors refer to previous studies, as the manuscript is essentially based on Orai function thapsigargin-induced SOCE should be tested using the Ca2+ add-back protocol in order to assess the release of Ca2+ from the ER in response to thapsigargin as well as the subsequent SOCE.

The fpDANs consist of 16-19 neurons in each hemisphere (PPL1 are 10-12 and PPM3 are 6-7 cells; Pathak et al., 2015). Measuring SOCE from these neurons in vivo is not possible due to the presence of abundant extracellular Ca2+ in the brain. Given their sparse number, it proved technically challenging to isolate the fpDANs in culture to perform SOCE measurements using the Ca2+ add back protocol. Due to these reasons, we have relied upon using Carbachol to elicit IP3-mediated Ca2+ release and SOCE as a proxy for in vivo SOCE. In previous studies we have shown that Carbachol treatment of cultured Drosophila neurons elicits IP3-mediated Ca2+ release and SOCE (Agrawal et al., 2010; Figure 8). Moreover, expression of OraiE180A completely blocks SOCE as measured in primary cultures of dopaminergic neurons (Pathak et al., 2015; Figure 1E). Hence we have not repeated SOCE measurements from all dopaminergic neurons in this work. In the revised version we have explicitly stated this weakness of our study and the reasons for it (See Lines 307-313; ‘Limitations of the study’-Lines 719-735).

1. In the experiments performed to rescue flight duration in Set2RNAi individuals the authors overexpress STIM and attribute the effect to "Excess STIM presumably drives higher SOCE sufficient to rescue flight bout durations caused by deficient Set2 levels.". This should be experimentally tested as the STIM:Orai stoichiometry has been demonstrated as essential for SOCE.

The assumption that STIM overexpression drives higher SOCE is based upon previously published work from Drosophila neurons (Agrawal et al., 2010; Chakraborty et al, 2016; Deb et al., 2016) which demonstrates that excess WT STIM overcomes IP3R deficiencies (RNAi or hypomorphic mutants) to rescue SOCE. We agree that STIM-Orai stoichiometry is essential for SOCE, and propose that the rescue backgrounds possess sufficient WT Orai, which is recruited by the excess STIM to mediate the rescue. We have referenced the earlier work to validate our use of STIMOE for rescue of SOCE (See Lines 226-233).

Here, we propose that Set2 is part of a positive feedback loop (see Lines 286-307) driving transcription of mAChR and IP3R (Figure 3F). In keeping with this hypothesis, we posit that the phenotypes observed in the Set2RNAi background (Figure 2D) result from decreased itpr and mAChR expression (validated in Figure 3E). This is further validated by the Set2 overexpression mediated rescue of OraiE180A (Figure 2D) and rescue of itpr/mAChR expression in the OraiE180A background (Figure 3B-E; THD’>OraiE180A; Set2OE).

1. The authors show that overexpression of OraiE108A results in Stim downregulation at a mRNA level. What about the protein level? And more important, how does OraiE108A downregulate Stim expression? Does it promote Stim degradation? Does it inhibit Stim expression?

We hypothesize that changes in STIM mRNA observed in the THD’ > OraiE180A neurons stems from an overall reduction in IP3-mediated Ca2+ release and SOCE due to loss of Trl-Set2 driven gene expression detailed in our transcriptional feedback loop model (Figure 5C; see Lines 286-307; 581-591). We have attempted to explain this aspect more clearly in the revised version of the manuscript. While we agree that measuring levels of STIM protein would be helpful, estimation of protein levels from a limited number of neurons (~35 cells per brain) is technically challenging. The STIM antibody does not work well in immunohistochemistry. In the absence of any experimental evidence we cannot comment on how expression of OraiE180A might affect STIM protein turnover (see Lines 307-313).

1. Lines 271-273, the authors state "whereas overexpression of a transgene encoding Set2 in THD' neurons either with loss of SOCE (OraiE180A) or with knockdown of the IP3R (itprRNAi), lead to significant rescue of the Ca2+ response". This is attributed to a positive effect of Set2 expression on IP3R expression and the authors show a positive correlation between these two parameters; however, there is no demonstration that Set2 expression can rescue IP3R expression in cells where the IP3R is knocked down (itprRNAi). This should be further demonstrated.

The rescue of IP3R expression by Set2 overexpression in itprRNAi was demonstrated in a different set of Drosophila neurons in an earlier study (Mitra et al., 2021) and has not been repeated specifically in THD’ neurons (see Lines 286-307). Similar to the previous study, here we tested CCh stimulated Ca2+ responses of THD’ neurons with itprRNAi and itprRNAi; SetOE (Fig S3), which are indeed rescued by SET2OE see Lines 280-285)

1. The data presented in Figure 3E should be functionally demonstrated by analyzing the ability of CCh to release Ca2+ from the intracellular stores in the absence of extracellular Ca2+.

CCh-mediated Ca2+ release from the intracellular stores in the absence of extracellular Ca2+ has been described in primary cultures of Drosophila neurons in previously published work (Venkiteswaran and Hasan, 2009; Agrawal et al., 2010) This work focuses on a set of 16-19 dopaminergic neurons in a hemisphere of the Drosophila central brain. It is technically challenging to generate a 0 Ca2+ environment in vivo, which is essential for measuring store Ca2+ release. Given their meagre numbers, primary cultures of these neurons is not readily feasible. (see Lines 307-313; ‘Limitations of the study’-Lines 719-735)

1. The conclusion that SOCE regulates the neuronal excitability threshold is based entirely on either partial behavioral rescue of flight, or measurements of KCl-induced Ca2+ rises monitored by GCaMP6m in DAN neurons. The threshold for neuronal excitability is a precise parameter based on rheobase measurements of action potentials in current-clamp. Measurements of slow calcium signals using a slow dye such as GCaMp6m should not be equated with neuronal excitability. What is measured is a loss of the calcium response in high K depolarization experiments, which occurs due to the loss of expression of Cav channels. Hence, the use of this term is not accurate and will confuse readers. The use of terms referring to neuronal excitability needs to be changed throughout the manuscript. As such, the conclusions regarding neuronal excitability should be strongly tempered and the data reinterpreted as there are no true measurements of neuronal excitability in the manuscript. All that can be said is that expression of certain ion channel genes is suppressed. Since both Na+ channels and K+ channel expression is down-regulated, it is hard to say precisely how membrane excitability is altered without action potential analysis.

The claim that SOCE influences neuronal excitability is based on the following observations:

1. Interruption of the transcriptional feedback loop involving SOCE, Trl, and Set2 through loss of any of its constituents, results in the downregulation of VGCCs (Figure 5G, 6H), which are essential components of action potentials.

2. OraiE180A mediated loss of SOCE in THD’ neurons abrogates the KCl-evoked depolarization response (Figure 6B, C) measured using GCaMP6m. We verified that this response requires VGCC function using pharmacological inhibition of L-type VGCCs (Figure 6E, F).

3. SOCE deficient THD’ neurons, which were presumably compromised in their ability to evoke action potentials could be rescued to undergo KCl-evoked depolarisation by expression of NachBac, which lowers the depolarization threshold (Figure 7C, D) or through optogenetic stimulation using CsChrimson (Figure 7F).

We agree that ‘neuronal excitability threshold’ is a precise electrophysiological parameter that has not been directly investigated here by measurement of action potentials. Therefore, references to neuronal excitability have been tempered throughout the revised manuscript and be replaced with a more generic reference to ‘neuronal activity’. In this context we have included further evidence supporting reduced activity of THD’ neurons upon loss of SOCE in the revision.

Since one of the key functional outcomes of activity during critical developmental periods such as the 72-96 hrs APF developmental window identified in this study, is remodelling of neuronal morphology, we decided to investigate the same in our context. Neuronal activity can drive changes in neurite complexity and axonal arborization (Depetris-Chauvin et al., 2011) especially during critical developmental periods (Sachse et al., 2007). To understand if Orai mediated Ca2+ entry and downstream gene expression through Set2 affects this activity-driven parameter, we investigated the morphology of fpDANs, and specifically measured the complexity of presynaptic terminals within the 2’1 lobe MB using super-resolution microscopy. We found striking changes in the neurite volume upon expression of OraiE180A which could be rescued by restoring either Set2 (OraiE180A; Set2OE) or by inducing hyperactivity through NachBac expression (OraiE180A ; NachBacOE). These data have been included in the revised manuscript (Figure 8 B, C, D; see Lines 481-482; 519-534; 584-591; 701-704).

1. Related, since trl does not contain any molecular domains that could be regulated by Ca2+ signaling, it is unclear whether trl is directly regulated by SOCE or the regulation is highly indirect. Reporter assays evaluating trl activation upon Ca2+ rises would provide much stronger and more direct evidence for the conclusion that trl is a SOCE-regulated TF. As such the evidence is entirely based on RNAi downregulation of trl which indicates that trl is essential but has no bearing on exactly what point of the signaling cascade it is involved.

We agree that luciferase Trl reporters would provide a direct method to test SOCE-mediated activation. Future investigations will be targeted in this direction. Regarding possible mechanisms of Trl activation - since we could not detect a well-defined Ca2+ binding domain in Trl, we hypothesize that it may be phosphorylation by a Ca2+ sensitive kinase. Phosphoproteome analysis of Trl indicates that it does indeed undergo phosphorylation at a Threonine reside (T237; Zhai et al., 2008), which may be mediated by the Ca2+ sensitive kinase-CaMKII based on binding partners identified in the Trl interactome (Lomaev et al., 2018; Past work (Ravi et al., 2018) has indeed demonstrated a requirement for CaMKII in THD’ neurons for flight. We are currently testing whether CaMKII functions downstream of SOCE in these neurons to mediate flight, and will be updating this information in the next version of the manuscript.

New data and analysis has been included - see Figure S5; ‘Limitations of the study’- Lines 397-424; 736-739).

1. Are NFAT levels altered in the Orai1 loss of function mutant? If not, this should be explicitly stated. It would seem based on previous literature that some gene regulation may be related to the downregulation of this established Ca2+-dependent transcription factor. Same for NFkb.

As mentioned in the revised version of the manuscript (see Lines 315-326), Drosophila NFAT lacks a calcineurin binding site and is therefore not sensitive to Ca2+ (Keyser et al., 2007). In the past we tested if knockdown of NF-kB in dopaminergic neurons gave a flight phenotype and did not observe any measurable deficit. From the RNAseq data we find a slight downregulation of NFAT (0.49 fold, p value=0.048) and NF-kb (0.26 fold, p value =0.258) the significance of which is unclear at this point. We did not find any consensus binding sites for these two factors in the regulatory regions of downregulated genes from THD’ neurons.

1. Does over-expression of Set2 restore ion channel expression especially those of the VGCCs? This would provide rigorous, direct evidence that SOCE-mediated regulation of VGCCs through Set2 controls voltage-gated calcium channel signaling.

Set2 overexpression in the OraiE180A background indeed restores the expression of VGCC genes (see Figure 6H; Lines 461-468).

1. All 6 representative panels from Figure 3B are duplicated in Figure 4G. Likewise, 2 representative panels from Figure 5H are duplicated in Figure 6D. Although these panels all represent the results from control experiments, the relevant experiments were likely not conducted at the same time and under the same conditions. Thus, control images from other experiments should not be used simply because they correspond to controls. This situation should be clarified.

We regret the confusion caused by the same representative images for the control experiments. These have been replaced by new representative images for Figure 4G and 6D in the updated version of the manuscript.

1. The figures are unusually busy and difficult to follow. In part this is because they usually have many panels (Fig. 1: A-I; Fig. 2, A-J, etc) but also because the arrangement of the panels is not consistent: sometimes the following panel is found to the right, other times it is below. It would help the reader to make the order of the panels consistent, and, if possible, reduce the number of panels and/or move some of the panels to new figures (eLife does not limit the number of display items).

The image panels have been rearranged for ease of reading in the updated version of the manuscript.

1. As a final recommendation, the reviewers suggest that the authors a- Reword the text that refers to membrane excitability since membrane excitability was not directly measured here. b-Explain why STIM1 rescues the partial loss of flight in Set2 RNAi flies (Fig. S2E); and c- Explain how/why trl is calcium regulated and test using luciferase (or other) reporter assays whether Orai activation leads to trl activation.

a. Textual references to membrane excitability have been appropriately modified and some new data has been included in this regard (see Figure 8 B, C, D; Lines 481-483; 519-534; 584-591; 701-704).

b. We have provided a detailed explanation for how STIM overexpression might rescue the phenotypes caused by Set2RNAi in Point 1 (see Lines 226-233). In short, these phenotypes depend upon IP3R mediated Ca2+ entry driving a transcriptional feedback loop. We relied upon past reports that STIM overexpression upregulates IP3R-mediated Ca2+ release and SOCE in Drosophila itpr mutant neurons (Agrawal et al., 2010; Chakraborty et al, 2016; Deb et al, 2016). We therefore propose that STIM overexpression in the Set2RNAi background rescues IP3R mediated Ca2+ release followed by SOCE, which drives enhanced Set2 transcription, counteracting the effects of the RNAi. We will explain this more clearly with past references in the next revision.

c. We have provided a detailed response to this comment in Point 12. Briefly, we agree that building luciferase reporters for Trl could be an ideal strategy to test for its responsiveness to SOCE and needs to be done in future. As an alternate strategy, we have looked at data from existing studies of interacting partners of Trl (Lomaev et al., 2017) and identified CamKII, which is both Ca2+ responsive (Braun and Schulman, 1995; Yasuda et al., 2022), and thus might activate Trl through a phosphorylation-switch like mechanism (see Figure S5; ‘Limitations of the study’-736-739; Lines 397-424). Moreover, a previous publication identified a requirement for CamKII in THD’ neurons for Drosophila flight (Ravi et al., 2018). We have tested the ability of a dominant active version of CamKII to rescue THD’>E180A flight deficits and have included this information in the next version of the manuscript.

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