Neuroscience

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

Rishav Mitra
Shlesha Richhariya
Gaiti Hasan author has email address

National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India
Department of Biology, Brandeis University, Waltham, USA

https://doi.org/10.7554/eLife.88808.3

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Figures and data

Orai-mediated Ca²⁺ entry sets the gene expression profile of flight-promoting dopaminergic neurons in late development and early adulthood.
A. A schematic of Ca²⁺ release through the IP₃R and SOCE through STIM/Orai (upper panel) followed by representation of the wildtype (Ca²⁺ permeable) and mutant (Ca²⁺ impermeable) Orai channels (lower panel). B. Anatomical location of THD’ DANs in the fly central brain immunolabelled for mCD8GFP (upper panel) followed by a cartoon of central brain DAN clusters. Scale bar indicates 20 μm. PPL1 and PPM3 clusters are labelled in red and green respectively. C. Measurement of flight bout durations demonstrate a requirement for Orai-mediated Ca²⁺ entry in THD’ DANs and in 2 pairs of PPL1 DANs marked by the MB296B driver. D. THD’ DANs require Ca²⁺ entry through Orai at 72-96 hours APF and 0-2 days post eclosion to promote flight. In C and D flight bout durations in seconds (s) are represented as a swarm plot where each genotype is represented by a different color, and each fly as a single data point. The Δ Flight parameter shown below indicates the mean difference for comparisons against the shared Canton S control and is shown as a Cumming estimation plot. Mean differences are plotted as bootstrap sampling distributions. Each 95% confidence interval is indicated by the ends of the vertical error bars. The same letter beneath each distribution refers to statistically indistinguishable groups after performing a Kruskal-Wallis test followed by a post hoc Mann-Whitney U-test (p<0.005). At least 30 flies were tested for each genotype. E. THD’ DANs were labelled with cytosolic eGFP (10 μm scale), isolated using FACS and validated for enrichment of GFP mRNA by qRT-pCR (lower panel). The qRT-pCR results are from 4 biological replicates with different letters representing statistically distinguishable groups after performing a two-tailed t test (p<0.05). F. RNA-seq comparison of FAC sorted populations of GFP labelled THD’ DANs from THD’>GFP and THD’>GFP;Orai^E180A pupal dissected CNSs. The RNA-Seq data is represented in the form of a volcano plot of fold change vs FDR. Individual dots represent genes, coloured in red (upregulated) or blue (downregulated) by >1 fold. G. Downregulated genes were identified by 3 different methods of DEG analysis, quantified and compared as a Venn diagram. H. Gene expression trajectories of SOCE-induced DEGs plotted as a function of developmental time (modENCODE Consortium et al., 2010) and clustered into 3 groups using k-means analysis. I. The relative proportion of downregulated, upregulated genes, and a random set of genes found in the clusters described in (H) indicate that 75% of downregulated genes exhibit a pupal peak of expression.

Ca²⁺ entry through Orai regulates gene expression by Set2-mediated histone modification.
A. Scatterplot of GO categories enriched in SOCE-responsive genes. Individual GO terms are represented as differently colored circles, with radius size indicating number of genes enriched in that category. B. Fold change of SET domain containing genes as indicated. Individual circles on the Y-axis for each gene represent transcript variants pertaining to that gene. C. Transcripts of the H3K36 methyltransferase (left) are significantly diminished (right) in THD’ DANs either upon loss of Orai function (THD’>Orai^E180A) or by knockdown of Set2 (THD’>Set2^RNAi). qRT-PCRs were performed from FAC sorted THD’ DANs with 3 biological replicates. Individual 2^-ΔΔCT values are shown as points. Letters represent statistically distinguishable groups after performing an ANOVA and post hoc Tukey test (p<0.05). D. Significant rescue of flight bout durations seen in THD’ > Orai^E180A flies by overexpression of Set2 and by knockdown of the Kdm4B demethylase indicating a net requirement for H3K36me3. Flight durations of single flies are depicted as swarm plots and the Δ flight parameter is shown below. Both were measured as described in the legend to Figure 1. N = 30 or more flies for each genotype. Letters represent statistically distinguishable groups after performing an ANOVA and post hoc Tukey test (p<0.005) E. Representative images (upper panel) and quantification (lower panel) from immunostaining of H3K36me3 and H2BmRFP in nuclei of THD’ DANs from at least 10 brains. Scale bar represents 5 μm. The boxplot represents individual H3K36me3/H2BmRFP ratios from each THD’ DAN for each genotype. Letters represent statistically distinguishable groups after performing an ANOVA and post hoc Tukey test (p<0.05) F. H3K36me3 enrichment over the gene bodies of SOCE-responsive genes in wildtype fly heads represented in the form of a tag density plot. G. H3K36me3 signal is enriched on WT SOCE-responsive genes with greater downregulation upon loss of Orai function. Individual data points represent WT H3K36me3 ChIP-Seq signals from adult fly heads represented as a boxplot (left) and a regression plot (right; Pearson’s correlation coefficient = 0.11) indicating a correlation between extent of downregulation upon loss of SOCE and greater enrichment of H3K36me3. H. SOCE-responsive genes are enriched in H3K36me3 signal as compared to H3K27me3 signal as measured from relevant ChIP-Seq datasets. Adult fly head ChIP-Seq datasets for measurements in F to H were obtained from modEncode (modENCODE Consortium et al., 2010). I. Schematic representation of how Orai mediated Ca²⁺ entry regulates a balance of two opposing epigenetic signatures in developing DANs. J. Pharmacological inhibition of H3K27me3 using GSKS343 (left) in THD’>Orai^E180A flies results in a dose-dependent rescue of flight bout durations (right). Flight assay measurements from N> 30 flies, are represented as described earlier. Letters represent statistically distinguishable groups after performing an ANOVA and post hoc Tukey test (p<0.005).

Orai-mediated Ca²⁺ entry potentiates cellular Ca²⁺ responses to cholinergic inputs through Set2 and a transcriptional feedback loop
A. A schematic of intracellular Ca²⁺ signaling downstream of neuromodulatory signaling where activation of mAChR stimulates intracellular Ca²⁺ release through the IP₃R followed by SOCE through STIM/Orai. B. Cholinergic inputs by addition of carbachol (CCh) evoke Ca²⁺ signals as measured by change in the fluorescence of GCaMP6m in THD’ DANs of ex-vivo brains. Representative GCaMP6m images of THD’ DANs are shown with baseline and peak evoked responses in the indicated genotypes. Scale bar = 10 μM. C. Median GCaMP6m responses plotted as a function of time. A shaded region around the solid line represents the 95% confidence interval from 4-5 cells imaged per brain from 10 or more brains per genotype. D. Individual cellular responses depicted as a paired plot where different letters above indicate statistically distinguishable groups after performing a Kruskal Wallis Test and a Mann-Whitney U-test (p<0.05). E. qRT-PCR measurements of itpr, mAChR, Stim and Orai from FAC sorted THD’ DANs obtained from three biological replicates of appropriate genetic backgrounds. The genotypes include DANs with loss of cellular Ca²⁺ responses (THD’>Orai^E180A; and THD’>Set2^RNAi-1), rescue of cellular Ca²⁺ response by overexpression of Set2 (THD’>Orai^E180A; Set2^OE) and Set2 overexpression. Bar plots indicate mean expression levels in comparison to rp49, with individual data points represented as hollow circles. The letters above indicate statistically indistinguishable groups after performing a Kruskal Wallis Test and a Mann-Whitney U-test (p<0.05). F. Schematic representation of a transcriptional feedback loop downstream of cholinergic stimulation in THD’ DANs.

Identification of Trl as an SOCE responsive transcription factor
1. Schematic of motif enrichment analysis for identification of putative SOCE dependent transcription factors (TFs). B. Candidate TFs identified (upper panel) and their expression through development (lower panel; modEncode(modENCODE Consortium et al., 2010)) suggests Trl as a top SOCE-responsive candidate TF. C. Genetic depletion of Trl in THD’ DANs results in significant flight defects, that can be rescued by overexpression of Set2. D. Heteroallelic combination of the Trl hypomorphic allele (trl^13C) with a STIM deficiency causes significant flight deficits, that can be rescued by overexpression of STIM or Set2. E.qRT-PCRs measured relative to rp49 show reduced Set2, itpr, and mAChR levels in THD’ DANs upon Trl knockdown with Trl^RNAi-1. Individual data points of 4 biological replicates are shown as circles and mean expression level as a bar plot (+ SEM). The letters above indicate statistically indistinguishable groups after performing a Kruskal Wallis Test and a Mann-Whitney U-test (p<0.05). F. A representative western blot (left) showing reduced H3K36me3 levels in a Trl mutant combination. Quantification of H3K36me3 from 3 biological replicates of WT (control) and Trl mutant brain lysates (panel on the right). Letters indicate statistically indistinguishable groups after performing a two-tailed t-test (p<0.05).G. Knockdown of Trl in THD’ neurons attenuate Ca²⁺ response to CCh as shown in representative images of GCamP6m fluorescence quantified in (H-I). Scale bar = 10 μM. Set2 overexpression in the background of THD’>Trl^RNAi rescues the cholinergic response (G-I). Quantification of Ca²⁺ responses are from 10 or more brains per genotype and were performed as described in the legend to Figure 3. The letters above indicate statistically indistinguishable groups after performing a Kruskal Wallis Test and a Mann-Whitney U-test (p<0.05).

Trl activity downstream of SOCE is required for THD’ DAN activity.
A. Overexpression of WT Trl is insufficient to rescue flight deficits in THD’>Orai^E180A flies as evident from flight bout measurements in the indicated genotypes. Flight assay measurements are as described earlier. B. Trl transcript levels are not altered in THD’>Orai^E180A neurons with loss of SOCE. qRT-PCR data are measured relative to rp49. The bar plot indicates mean expression levels, with individual data points represented as circles. The letters above indicate statistically indistinguishable groups from 3 independent biological replicates after performing a two-tailed t-test (p<0.05). C. Schematic with possible Ca²⁺ - mediated activation of Trl downstream of Orai-mediated Ca²⁺ entry (SOCE). D. Genes encoding ion channels are enriched among SOCE responsive genes in THD’ DANs as determined by GO analysis. Circles of varying radii are scaled according to the number of genes enriched in that category. E. Downregulation of individual ion channel genes depicted as a heatmap. F. Number of Trl binding sites (GAGA repeats) shown as a bar plot in the regulatory regions (2 kb upstream or 1^st intron) of key SOCE-responsive ion channel genes compared to known Trl targets (En and Ubx). The dashed line indicates the average expected number of Trl targets. G. Heatmap of 2^-ΔΔCT values measured using qRT-PCRs from sorted THD’ DANs with knockdown of Trl. H. Representative images of KCl-induced depolarizing responses in THD’ DANs with knockdown of Trl. (Scale bar = 10 μM) quantified in I and J. Quantification of Ca²⁺ responses are from 10 or more brains per genotype and were performed as described in the legend to Figure 3. The letters above indicate statistically indistinguishable groups after performing a Kruskal Wallis Test and a Mann-Whitney U-test (p<0.05).

fpDAN excitability requires Orai-mediated Ca²⁺ entry acting through Set2 -mediated VGCC gene expression.
KCl-induced depolarizing responses in fpDANs of the indicated genotypes measured using GCaMP6m indicate a requirement for Orai and Set2. Representative images A. (scale bar = 10 μM), quantified in B. and C. KCl evoked responses are modulated upon treatment with 10 μM TTX (magenta), 10 μM Nimodipine (orange), and upon cac^RNAⁱ (purple)-representative images (scale bar = 10 μM), (D), quantified in E. Median KCl-evoked GCaMP6m responses plotted as a function of time. The solid line indicates the time point of addition of 70 mM KCl. KCl responses are quantified as a paired plot of peak responses and F. with letters representing statistically indistinguishable groups as measured using a Kruskall Wallis test and post hoc Mann-Whitney U test (p<0.05). Quantification of Ca²⁺ responses are from 10 or more brains per genotype and were performed as described in the legend to Figure 3. G. Schematic of a typical VGCC and its constituent subunits. H. Heatmap of 2^-ΔΔCT values measured using qRT-PCRs from sorted THD’ DANs show a reduction in the expression of VGCC subunit genes upon loss of Orai function and a rescue by Set2 overexpression. I. Flight assays representing the effect of various VGCC subunit gene RNAis showing flight defects to varying extents. Overexpression of the key VGCC subunit gene-cac is required (I) but not sufficient (J) for restoring flight defects caused by loss of Orai function. Flight assay measurements are as described earlier.

SOCE-mediated gene expression sets the excitability threshold during pupal development
A. Altering excitability in THD’ DANs using Kir2.1 or GtACR2 mediated inhibition or CsChrimson or TrpA1 during the critical 72-96 hour APF developmental window results in significant flight defects. Orai loss of function phenotypes can be rescued by overexpression of NachBac or CsChrimson in terms of flight bout durations (B, E) and depolarizing KCl responses (C, D, D, G). H. Schematic for Orai-mediated Ca²⁺ entry regulating expression of key genes that regulate the excitability threshold of dopaminergic neurons regulating flight during a critical developmental window. Flight assays are represented as described earlier, (n>30). Ca²⁺ responses were quantified as described in the legend to Figure 3 and were from 10 or more brains per genotype. Letters above each genotype represent statistically indistinguishable groups as measured using a Kruskall-Wallis test and post hoc Tukey test (p<0.05).

THD’ DANs require SOCE-mediated gene expression for axonal arborization and neuromodulatory dopamine release in the mushroom body γ lobe.
A. Schematic of a KC-DAN-MBON tripartite synapse at the γ2α’1 MB lobe (upper), innervated by fpDANs (lower). The γ2α’1 MB lobe is marked by a red circle and arrow and the fpDAN clusters are marked with a green arrow (PPL1) and blue arrow (PPM3). Scale bar=50 μM. B. Representative images of the mushroom body γ lobe marked with THD’ driven mCD8GFP (green) and immunolabelled with anti-nc82 (Brp) antibody (magenta) to mark the presynaptic terminals. Scale bar = 5 μm. C. Representative optical sections through the mushroom body γ lobe from the indicated genotypes. Changes in axonal arborization observed were quantified as total projection volume in D. E. CCh-evoked DA release measured at the γ2α’1 MB lobe using the GRAB-DA sensor expressed in THD’ DANs. Representative GRAB-DA images of THD’ DANs are shown with baseline and peak evoked responses in the indicated genotypes. Scale bar = 10 μM. F. Median GRAB-DA responses plotted as a function of time. A shaded region around the solid line represents the 95% confidence interval from 10 or more brains per genotype. G. Individual cellular responses depicted as a paired plot of peak responses where different letters above indicate statistically distinguishable groups after performing a Kruskal Wallis Test and a Mann-Whitney U-test. H. Schematic describing how Orai-mediated Ca²⁺ entry regulates expression of key genes that control neuronal activity in fpDANs.

Schematic outline of the role of SOCE in determining excitability and function of dopaminergic neurons required for flight circuit development.
SOCE acts through a transcriptional feedback loop including the transcription factor Trl, and the H3K36 methyltransferase Set2 to induce the expression of genes required for neuronal excitability and flight.

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