Stac protein regulates release of neuropeptides

Abstract

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca²⁺ However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca²⁺ channels (CaChs) and excitation-contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac, which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca²⁺ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides.

Keywords: Drosophila melanogaster; L-type voltage-gated calcium channel; neuropeptide; stac.

Fig. 1.

Dstac and Dmca1D are expressed by proctolin⁺ motor boutons. (A) Anti-proctolin and anti- horseradish peroxidase (anti-HRP) labeling of motor nerves in larvae selectively expressing membranous GFP in proctolin⁺ motor neurons (Proct:GAL4 > UAS:mCD8GFP) showed expression of mCD8GFP and proctolin at type Ib and type Is branches. The anti-HRP labels an unknown protein that is present on the plasma membrane of motor nerves and boutons. Shown are the type Is branch of the RP2 (arrow) and the type Ib branch of the MN4-Ib motor neuron (asterisk) on muscle 4 (45). Merge is Proct:GAL4 > UAS:mCD8GFP and anti-Proctolin. The images are a single focal plane. (Scale bar: 3 μm.) (B) Anti-Dstac and anti-HRP labels type 1b (asterisk) and 1s (arrow) boutons. Left Inset shows higher magnification view of type 1b boutons. Right Inset shows higher magnification view of type 1s boutons. Shown are type 1s boutons of RP2 and type 1b boutons of MN4-1b on muscle 4. (C) Anti-Dmca1D and anti-HRP labels type 1b (asterisk) and 1s (arrow) boutons. Left Inset shows higher magnification view of type 1b boutons. Right Inset shows higher magnification view of type 1s boutons. Shown are type 1s boutons of RP2 and type 1b boutons of MN4-1b on muscle 4. Some of the labeling by both anti-Dstac and anti-Dmca1D beyond the motor nerves may represent muscle labeling since body wall muscles also express Dstac and Dmca1D (38, 46). The images show a single focal plane. (Scale bar: 3 μm.) See also SI Appendix, Fig. S1.

Fig. 2.

Dstac mutants generated by CRISPR-Cas9 showed reduced locomotion. (A) Schematic of two Dstac protein variants (CG43729-RU and CG43729-RV) is shown. Arrows denote the location where early stop codons occurred due to the indels (triangles) created by CRISPR-Cas9. Half arrows denote primer sites used to sequence the mutations. All primers used are listed in SI Appendix, Table S1 and Supplementary Materials and Methods. The cDNA PCR fragments of Dstac^ΔSH3.1 are smaller than wt for both RU and RV variants, suggesting the deletions of the SH3 domain in the Dstac cDNA. The deletions of SH3 were confirmed by sequencing the wt and Dstac^ΔSH3.1 cDNA bands (SI Appendix, Supplementary Text). The translated mutant cDNA sequences showed that an early stop codon was produced in Dstac^ΔSH3.1 (SI Appendix, Supplementary Text). The schematic is not to scale. (B) Offsprings of founder 1 after five generations of outcrossing (Dstac^ΔSH3.1) showed decreased locomotion compared with wt (wt, n = 35; Dstac^ΔSH3.1, n = 36; one-tailed, unpaired t test). In this histogram and all subsequent ones, SEMs are shown, and triangles denote the median. (C) Offsprings of founder 2 after 10 generations of outcrossing (Dstac^ΔSH3.2, n = 21) showed decreased locomotion compared with wt (n = 120) and heterozygous siblings (n = 78) (one-way ANOVA, Tukey’s multiple comparisons test). ns denotes not significant. (D) Expression of Dstac^wt by heterozygous Dstac^ΔSH3.1 larvae (n = 43) increased locomotion compared with heterozygous controls (n = 104), and expression of Dstac^wt by homozygous Dstac^ΔSH3.1 (n = 33) rescued Dstac^ΔSH3.1 (n = 29) locomotion (Kruskal–Wallis test, Dunn’s multiple comparisons test). The larvae were genotyped by PCR after the locomotion assay.

Fig. 3.

Dstac isoforms that contain the SH3 domain are affected in Dstac^ΔSH3.1. (A) Schematic of domain composition of nine Dstac protein variants. Not all variants contain an SH3 domain. (B) Quantification of Dstac transcript levels in wt third instar larval CNS and body wall assayed by RT-PCR. Error bars represent SEM. RT-PCR primers for Dstac (CG43729) transcripts and GAPDH used for normalization can be found in SI Appendix, Table S1 and Supplementary Materials and Methods. (C) Two isoforms without the SH3 domain (CG43729-RC and CG43729-RP) and two isoforms that contain the SH3 domain (CG43729-RU and CG43729-RV) were PCR amplified from wt and Dstac^ΔSH3.1 cDNA. Half arrows denote the primer sites. The levels of isoforms that don’t have SH3 domain (RC and RP) were comparable between wt and Dstac^ΔSH3.1 (Mann–Whitney U test), but the isoforms that normally contained the SH3 domain (RU and RV) were diminished in Dstac^ΔSH3.1 (Mann–Whitney U test) (RC: wt n = 5, Dstac^ΔSH3.1 n = 5; RP: wt n = 8, Dstac^ΔSH3.1 n = 8; RU: wt n = 5, Dstac^ΔSH3.1 n = 5; RV: wt n = 9, Dstac^ΔSH3.1 n = 9; each dot represents one cDNA gel band).

Fig. 4.

Knockdown of Dstac, Dmca1D, and proctolin selectively in proctolin⁺ motor neurons reduced larval locomotion. (A) Dstac^RNAi larvae in which Dstac was knocked down in proctolin neurons (proct:GAL4 > UAS:Dstac^RNAi) showed decreased locomotion compared with control Luciferase^RNAi larvae (proct:GAL4 > UAS:Luciferase^RNAi) (control n = 156, Dstac^RNAi n = 156; one-tailed Mann–Whitney U test). (B) Dmca1D^RNAi in which Dmca1D was knocked down in proctolin neurons (proct:GAL4 > UAS:Dmca1D^RNAi) showed decreased locomotion compared with control Luciferase^RNAi larvae (control n = 150, Dmca1D^RNAi n = 143; one-tailed, unpaired t test). (C) proctolin^RNAi larvae in which proctolin was knocked down in proctolin neurons (proct:GAL4 > UAS:proctolin^RNAi) showed decreased locomotion compared with control Luciferase^RNAi larvae (control n = 144, proctolin^RNAi n = 75; one-tailed, unpaired t test). See also SI Appendix, Fig. S4.

Fig. 5.

Deficiencies in Dmca1D and Dstac decrease Ca²⁺ transients in motor boutons. (A) GCaMP6f expressed selectively in proctolin⁺ motor boutons of a wt control larva (Proct:GAL4 > UAS:GCaMP6f;UAS:mCD8tdTomato;UAS:Luciferase^RNAi). Asterisk denotes type Is motor branch on muscle 4. (Scale bar: 3 μm.) (B) Example of Ca²⁺ transients from a type Is bouton on muscle 4 in a wt control larva (Top) and in a Dmca1D KD (proct:GAL4 > UAS: Dmca1D^RNAi) larva (Bottom). See Movie S1. (C and D) The peaks and area under the peaks of Ca²⁺ transients (ΔF/F) in the boutons of Dmca1D KD larvae (Proct:GAL4 > UAS:GCaMP6f;UAS:Dmca1D^RNAi) were smaller compared with wt control. Peaks (C), one-tailed, unpaired t test. Area under peaks (D), one-tailed Mann–Whitney U test. (E) The number of Ca²⁺ transients over 5 min in Dmca1D KD and wt control boutons were comparable (Mann–Whitney U test). Data in C–E were from 24 boutons in 19 wt control larvae and 21 boutons in 16 Dmca1D KD larvae. (F) Example of Ca²⁺ transients from a type Is bouton on muscle 4 in a wt control larva (Top) and in a Dstac^ΔSH3.1 larva (Bottom). (G and H) The peaks and area under the peaks of Ca²⁺ transients in the boutons of Dstac^ΔSH3.1 larvae (Dstac^ΔSH3.1;Proct:GAL4 > UAS:GCaMP6f) are smaller compared with wt control (one-tailed Mann–Whitney U test). (I) The number of Ca²⁺ transients over 5 min in Dstac^ΔSH3.1 and wt boutons are comparable (Mann–Whitney U test). Data in G–I were from 25 boutons in 25 wt control larvae and 18 boutons in 18 Dstac^ΔSH3.1 larvae. Each data point represents the averaged peaks or area under peaks of Ca²⁺ transients per bouton.

Fig. 6.

Dmca1D currents in RP2 motor neuron cell bodies were largely decreased in Dstac^ΔSH3.1. (A) Voltage-clamp recording in response to a single voltage step of a wt control RP2 (Left) and a Dstac^ΔSH3.1 RP2 (Right). The RP2 motor neuron cell bodies were held at −70 mV and stepped to −10 mV for 200 ms in four different solutions (basal: 0 Ca²⁺ solution plus TTX; Ca²⁺: normal Ca²⁺ solution; Ba²⁺: Ba²⁺ solution; Cd²⁺: Ba²⁺ solution plus Cd²⁺). See SI Appendix, Supplementary Materials and Methods for details. The wt control RP2 cell bodies showed an inward Ca²⁺ current that inactivated and a larger Ba²⁺ current that barely inactivated whereas Dstac^ΔSH3.1 RP2 cell bodies showed significantly smaller Ca²⁺ and Ba²⁺ currents. Addition of Cd ²⁺ to the Ba²⁺ solution eliminated the Ba²⁺ current. (B) The peak Ba²⁺ current densities from Dstac^ΔSH3.1 RP2 cell bodies were reduced compared with control (control n = 7 cells from seven larvae, Dstac^ΔSH3.1 n = 6 cells from six larvae, one-tailed Mann–Whitney U test). The RP2 motor neuron cell bodies were held at −70 mV and stepped to −10 mV for 200 ms. (C) Full I/V curves from control and Dstac^ΔSH3.1 RP2 cell bodies were created by holding the membrane voltage (Vm) at −70 mV and stepping from −90 mV to +50 mV with 10-mV increments for 100 ms. Ca²⁺ current peaked at +10 mV in both control (n = 8) and Dstac^ΔSH3.1 (n = 11) larvae, and the currents were significantly decreased in Dstac^ΔSH3.1 compared to controls at −10 mV, 0 mV, +10 mV, and +20 mV (one-tailed Mann–Whitney U test; P value: 0.0034, 0.0004, 0.0003, and 0.0018, respectively). (D) Voltage-clamp Ca²⁺ currents at +10 mV of a control and Dstac^ΔSH3.1 RP2 cell body. (E) The peak Ca²⁺ current density from the voltage steps of Dstac^ΔSH3.1 RP2 cell bodies were reduced significantly compared with control. (Control n = 8 cells from eight larvae, Dstac^ΔSH3.1 n = 11 cells from 11 larvae, one-tailed Mann–Whitney U test).

Fig. 7.

Release of neuropeptides is diminished in Dstac and Dmca1D deficient motor boutons. (A) Expression of Dilp2-GFP (fluorescence in arbitrary units [a.u.]) by type Is motor boutons on muscle 4 of Dmca1D KD (proct:GAL4 > UAS:Dmca1D^RNAi; n = 15) and control (proct:GAL4 > UAS:Luciferase^RNAi; n = 15) was comparable (Left; unpaired t test). Release of Dilp2-GFP was decreased in Dmca1D KD compared with control (Middle; one-tailed Mann–Whitney U test). Examples of Dilp2-GFP expression before and after exposure to high potassium of control (Upper Right) and Dmca1D KD (Lower Right). The boutons are shown in pseudocolor. (Scale bar: 2 μm.) (B) Expression of Dilp2-GFP by motor boutons of Dmca1D^AR66 (nysb:Dilp2-GFP;Dmca1D^AR66; n = 14) was higher than that by control (nysb:Dilp2-GFP; n = 17) (Left; unpaired t test). Release of Dilp2-GFP was decreased in Dmca1D^AR66 compared with control (Middle; one-tailed Mann–Whitney U test). Examples of Dilp2-GFP expression before and after exposure to high KCl of control (Upper Right) and Dmca1D^AR66 (Lower Right). (Scale bar: 2 μm.) (C) Expression of Dilp2-GFP by motor boutons of Dstac KD (proct:GAL4 > UAS:Dstac^RNAi; n = 6) and control (n = 6) was comparable (Left; Mann–Whitney U test). Release of Dilp2-GFP was decreased in Dstac KD compared with control (Middle; one-tailed Mann–Whitney U test). Examples of Dilp2-GFP expression before and after exposure to high KCl of control (Upper Right) and Dstac KD (Lower Right). (Scale bar: 2 μm.) (D) Expression of Dilp2-GFP by motor boutons of Dstac^ΔSH3.1 (Dstac^ΔSH3.1;proct:GAL4 > UAS:Dilp2-GFP; n = 7) and control (proct:GAL4 > UAS:Dilp2-GFP; n = 7) was comparable (Left; Mann–Whitney U test). Release of Dilp2-GFP was decreased in Dstac^ΔSH3.1 compared with control (Middle; one-tailed Mann–Whitney U test). Examples of Dilp2-GFP expression before and after exposure to high KCl of control (Upper Right) and Dstac^ΔSH3.1 (Lower Right). (Scale bar: 2 μm.) (E) Nerve evoked synaptic potentials of muscle 4 were comparable between wt (n = 11) and Dstac^ΔSH3.1 (n = 9) (unpaired t test).

Fig. 8.

Model for the role of Dstac in neuropeptide release from RP2 motor neuron boutons. In wt (Left) Dstac regulates Ca²⁺ influx (circles) through Dmca1D channels (thick dashed arrow) to initiate CICR from the RyR Ca²⁺ release channel in the ER into the cytosol (thick arrow) and subsequent release of proctolin neuropeptide (hexagon) from motor boutons. In Dstac^∆SH3.1 (Right) Ca²⁺ influx through Dmca1D channels is decreased (thin dashed arrow) resulting in less CICR (thin arrow), and thus reduced release of proctolin neuropeptide.