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Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression

  1. Edward Pym
  2. Nikhil Sasidharan
  3. Katherine L Thompson-Peer
  4. David J Simon
  5. Anthony Anselmo
  6. Ruslan Sadreyev
  7. Qi Hall
  8. Stephen Nurrish
  9. Joshua M Kaplan  Is a corresponding author
  1. Massachusetts General Hospital, United States
  2. Harvard Medical School, United States
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Cite this article as: eLife 2017;6:e18931 doi: 10.7554/eLife.18931

Abstract

Shank is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. Here we show that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression.

https://doi.org/10.7554/eLife.18931.001

Introduction

Shank is a synaptic scaffolding protein (containing SH3, Ankyrin, PDZ, proline-rich and SAM domains) (Grabrucker et al., 2011). Because Shank is highly enriched in the post-synaptic densities of excitatory synapses, prior studies have focused on the idea that Shank proteins regulate some aspect of synapse formation or function. Through its various domains, Shank proteins bind hundreds of other synaptic proteins (Lee et al., 2011; Sakai et al., 2011) thereby potentially altering diverse cellular functions. Shank proteins have been implicated in synaptic transmission, synapse formation, synaptic plasticity, and cytoskeletal remodeling (Jiang and Ehlers, 2013).

Mammals have three Shank genes, each encoding multiple isoforms (Jiang and Ehlers, 2013). Several mouse Shank knockouts have been described but these mutants exhibit inconsistent (often contradictory) synaptic and behavioral defects (Jiang and Ehlers, 2013), most likely resulting from differences in which Shank isoforms are impacted by each mutation. The biochemical mechanism by which Shank mutations alter synaptic function and behavior has not been determined.

In humans, Shank mutations and CNVs are linked to Autism Spectrum Disorders (ASD), schizophrenia, and mania (Durand et al., 2007; Peça et al., 2011). Haploinsufficiency for 22q13 (which spans the Shank3 locus) occurs in Phelan-McDermid syndrome (PMS), a syndromic form of ASD (Phelan and McDermid, 2012). PMS patients exhibit autistic behaviors accompanied by hypotonia, delayed speech, and intellectual disability (ID) (Bonaglia et al., 2011). Heterozygous inactivating Shank3 mutations are found in sporadic ASD and schizophrenia (Durand et al., 2007; Peça et al., 2011). These genetic studies suggest that decreased Shank3 function likely plays an important role in the pathophysiology of these psychiatric disorders.

A parallel set of genetic studies suggest that increased Shank3 function also contributes to psychiatric diseases. 22q13 duplications spanning Shank3 are found in ASD, schizophrenia, ADHD, and bipolar disorder (Durand et al., 2007; Failla et al., 2007; Han et al., 2013). These 22q13 duplications involve multiple genes; consequently, the contribution of increased Shank3 to these psychiatric disorders was uncertain. To address this issue, a transgenic mouse model was developed that selectively over-expresses Shank3 (Han et al., 2013). This transgenic mouse exhibited hyperactive behavior and susceptibility to seizures. Taken together, these studies suggest that too little or too much Shank3 is associated with several psychiatric disorders.

If Shank3 mutations and CNVs are causally associated with these psychiatric disorders, cellular and circuit phenotypes should also be sensitive to Shank3 copy number. Consistent with this idea, several defects have been reported in Shank3+/- heterozygotes, including: decreased mEPSC frequency and spine density (Zhou et al., 2016), decreased Ih current density (Yi et al., 2016), decreased TRPV1 current density (Han et al., 2016), increased tactile sensitivity (Orefice et al., 2016), and decreased post-synaptic Homer abundance (Wang et al., 2016). Increased Shank expression was associated with increased spine density in hippocampus and decreased inhibitory synapses (Han et al., 2013). In many cases (Han et al., 2013; Orefice et al., 2016; Wang et al., 2016; Zhou et al., 2016), it was not determined if these phenotypes are a cell autonomous consequence of altered Shank3 copy number. While these studies identify cellular deficits associated with Shank3 CNVs, it remains unclear which Shank binding partners and cellular functions are responsible for psychiatric traits, nor why these traits are sensitive to both increased and decreased Shank gene dosage.

To further investigate how Shank proteins regulate nervous system development and function, we analyzed Shank function in an invertebrate genetic model. Here we show that C. elegans SHN-1 is a dose-sensitive regulator of Cav1 calcium current and CREB induced gene expression in C. elegans body muscles.

Results

The SHN-1 PDZ domain binds EGL-19/Cav1 channels

C. elegans has a single Shank gene, shn-1. The SHN-1 protein lacks an SH3 domain but has all other domains found in mammalian Shank proteins (Figure 1A). Many protein ligands have been identified for the Shank PDZ domain (Lee et al., 2011). Of these potential binding partners, we focused on EGL-19/Cav1 because human CACNA1C (which encodes a Cav1 α-subunit) is mutated in Timothy Syndrome (TS), a rare monogenic form of ASD (Splawski et al., 2005, 2004), and polymorphisms linked to CACNA1C are associated with multiple psychiatric disorders (Cross-Disorder Group of the Psychiatric Genomics Consortium, 2013). We confirmed that SHN-1’s PDZ domain binds the EGL-19 carboxy-terminal motif (-VTTLCOOH) by both yeast two-hybrid and GST-pull down assays (Figure 1—figure supplement 1A and B). Thus, like their mammalian counterparts, SHN-1 binds to EGL-19/ Cav1 (Zhang et al., 2005).

Figure 1 with 1 supplement see all
SHN-1 promotes EGL-19/Cav1 channel function.

(A) The protein domains found in SHN-1 and rat Shank3A are compared. SHN-1 lacks an SH3 domain but contains all other domains found in mammalian Shank proteins. Homology between the worm and mammalian protein is shown for each domain. (B–F) Voltage-activated Ca+2 currents were recorded from adult body wall muscles of the indicated genotypes at holding potentials of −60 to +40 mV. Averaged traces (B), mean current density as a function of holding potential (C), normalized conductance as a function of holding potential (D), mean current density at 10 mV (E), and mean deactivation time constants (F) are shown. shn-1 mutants had significantly decreased Ca+2 current-density and this defect was rescued by a single copy transgene expressing SHN-1 in body muscles (nuSi26) (D). No significant differences were observed for voltage-dependence of current activation and de-activation kinetics. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1.

https://doi.org/10.7554/eLife.18931.002

EGL-19/Cav1 calcium currents are diminished in shn-1 mutants

The impact of Shank binding on Cav1.3 function has been assessed by over-expression in Xenopus oocytes and in cultured neurons (Stanika et al., 2016; Zhang et al., 2005) but has not been tested in mutant or transgenic animals. Because shn-1 is expressed in muscles (Stefanakis et al., 2015), we assayed EGL-19/Cav1 channel function by recording voltage-activated calcium currents in body muscle, using salines containing potassium channel blockers (TEA and 4AP). In these conditions, the remaining voltage-activated inward current is entirely blocked by the EGL-19 antagonist nemadipine (Lainé et al., 2011). In shn-1(tm488) null mutants, calcium current density was significantly decreased (Figure 1B,C,E). Neither the voltage-dependence of calcium current activation (Figure 1D) nor the deactivation kinetics (Figure 1F) were altered in shn-1(tm488) null mutants. The shn-1 calcium current defect was rescued by a transgene restoring SHN-1 expression in body muscles (Figure 1E), confirming that SHN-1 has a cell autonomous effect on EGL-19/Cav1 currrent. To determine if SHN-1’s effects on calcium currents were specific, we measured voltage-activated potassium currents in body muscles (Figure 1—figure supplement 1C). Neither the voltage-dependence nor the current density of fast and slow potassium currents were significantly altered in shn-1 mutants. Collectively, these results suggest that SHN-1 specifically regulates the expression or function of EGL-19/Cav1 channels.

SHN-1 binding to EGL-19 increases Cav1 current density

SHN-1 effects on calcium current density could result from direct binding of SHN-1 to EGL-19/Cav1 or indirectly via other SHN-1 binding partners. We did several experiments to distinguish between these possibilities. First, we utilized CRISPR to isolate two deletion alleles that alter the EGL-19 carboxy-terminus (nu495 and nu496) (Figure 2A). Both deletion mutants exhibited decreased calcium current density (similar to the defect observed in shn-1 null mutants) (Figure 2B–C). The egl-19(nu496) mutation had no effect on the voltage-dependence of calcium current activation nor on deactivation kinetics (Figure 2—figure supplement 1A and B). The egl-19(nu496) and shn-1 null mutations did not have additive effects on calcium current density in double mutants, as would be predicted if SHN-1’s effects on calcium current require direct binding to EGL-19’s carboxy-terminus (Figure 2D and E). To further examine the functional impact of the SHN-1 PDZ interaction with EGL-19, we analyzed shn-1(ok1241) mutants, which have an in-frame deletion spanning exons encoding the PDZ domain and part of the proline-rich domain (Figure 1—figure supplement 1D). The shn-1(ok1241) mutants exhibited a decrease in calcium current density similar to those observed in shn-1 null and the egl-19 carboxy-terminal deletion mutants and had no effect on voltage-dependence of current activation nor on deactivation kinetics (Figure 2—figure supplement 1C–F). Collectively, these results suggest that SHN-1 binding to EGL-19’s carboxy-terminus promotes the expression or function of L-type calcium channels.

Figure 2 with 1 supplement see all
SHN-1 binding to EGL-19’s carboxy-terminus promotes the expression or function of L-type calcium channels.

(A) Predicted c-terminal sequences of mutant EGL-19 proteins are shown. egl-19(nu496) is a 22 bp deletion and egl-19(nu495) is a 5 bp deletion, both resulting in frame shifts that delete the carboxy-terminal PDZ ligand of EGL-19 (-VTTLCOOH). Residues in blue represent the PDZ ligand. Residues in red represent those introduced by the frame shift mutations. (B–E) Voltage-activated Ca+2 currents were recorded from adult body wall muscles of the indicated genotypes at holding potentials of −60 to +40 mV. Representative traces (B), mean current density at 0 mV (C, E), and mean current density as a function of holding potential (D) are shown. The egl-19(nu496) and shn-1(tm488) single mutants had similar decreases in Ca+2 current-density, and additive defects were not observed in the double mutant. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1.

https://doi.org/10.7554/eLife.18931.004

SHN-1 promotes trafficking of EGL-19 channels to the cell surface

SHN-1 effects on calcium current could result from a change in EGL-19 delivery to the cell surface. We performed two further experiments to test this idea. First, we measured the gating currents of voltage-activated channels in body muscles (Figure 3A–B). The activation of Cav channels is mediated by depolarization-induced movements of positively charged residues in membrane-spanning S4 helices, which are termed gating charges. When there is no net calcium current (by holding the muscle membrane at the reversal potential), gating charge movement can be measured as a small voltage-activated current. The magnitude of gating currents can be used as a measure of Cav channel surface abundance (Fu et al., 2011; Hulme et al., 2006). The total voltage activated gating charge in body muscles was significantly reduced in shn-1 null mutants (Figure 3B). This shn-1 mutant defect in gating charge is unlikely to result from decreased surface delivery of other voltage-activated channels because voltage-activated potassium currents were unaltered in shn-1 mutants (Figure 1—figure supplement 1C). Thus, analysis of gating currents suggests that the decreased calcium current exhibited in shn-1 mutants arises from decreased trafficking of EGL-19/Cav1 channels to the cell surface.

SHN-1 promotes EGL-19/Cav1 delivery to the cell surface.

(A–B) Voltage-activating gating currents were significantly decreased in shn-1 null mutants. Averaged trace of gating current in wild type adult body muscles (A) and mean gating charge (normalized to capacitance) (B) are shown. (C–G) Surface delivery of the Terrier fusion protein is significantly reduced in shn-1 null mutants. (C) A schematic illustrating the structure of the Terrier fusion protein is shown. (D) A schematic illustrating the imaged region (left) and representative images of Terrier pHluorin fluorescence in the nerve ring are shown. Mean pHluorin puncta intensity (E), pHluorin puncta area (F), and total pHluorin puncta fluorescence (G) are shown. Regions of interest utilized to quantify Terrier fluorescence are indicated (D). The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (**p<0.01, *p<0.05). Error bars indicate SEM.

https://doi.org/10.7554/eLife.18931.006

To further investigate SHN-1’s effects on EGL-19 trafficking, we designed a fluorescent reporter construct containing a trans-membrane domain fused to EGL-19’s cytoplasmic tail domain (504 amino acids), which includes the carboxy-terminal PDZ ligand (Figure 3C). This chimeric protein (designated Terrier to indicate the presence of EGL-19’s cytoplasmic tail) was expressed in body muscles. The Terrier protein contains tagRFP in the cytoplasmic domain, and pHluorin (a pH-sensitive GFP) in the ectodomain (Figure 3C). Thus, red fluorescence reports total Terrier protein while green fluorescence reports surface Terrier molecules. When expressed in body muscles, Terrier exhibits a punctate green and red fluorescence in the nerve ring, where body muscles receive synaptic input (Figure 3C–D). In shn-1 null mutants, the intensity and size of green Terrier puncta were significantly decreased (Figure 3D–G), indicating decreased surface Terrier protein in the nerve ring. Thus, decreased calcium current density (20%) in shn-1 mutants was mirrored by similar decreases in gating charge (26%) and total surface Terrier fluorescence (36%). Collectively, these results suggest that the decreased calcium current in shn-1 null mutants arises from decreased delivery of EGL-19/Cav1 channels to the cell surface.

gem-4 is an activity-induced CRH-1/CREB target expressed in muscles

Increased cytoplasmic calcium activates expression of a large number genes, hereafter designated activity-induced gene expression. Although Cav1 channels account for a small fraction of bulk calcium entry in neurons, Cav1 channels account for the majority of activity-induced gene expression (Ma et al., 2012). This privileged ability of Cav1 channels to activate gene expression is thought to be mediated by direct physical coupling of Cav1 channels to the calcium sensors responsible for activating CREB (Deisseroth et al., 1996; Wheeler et al., 2008).

Because shn-1 mutations alter EGL-19/Cav1 current, we hypothesized that SHN-1 may also play a role in activity-induced gene expression. To test this idea, we first identified activity-induced muscle genes. We analyzed gene expression following depolarization of body muscles with a nicotinic acetylcholine (ACh) agonist (levamisole, Lev). Lev-induced genes were identified using the Affymetrix C. elegans gene chip. This analysis identified 427 genes whose expression was significantly increased following muscle depolarization (>2 fold change, FDR p<0.05) (Figure 4Supplementary file 2). 67% (287/427) of Lev-induced genes contain binding sites for the myogenic transcription factor HLH-1 (<5 kb from the transcriptional start site, TSS) in chromatin-immunoprecipitation experiments (http://www.modencode.org), suggesting that these genes are expressed in body muscles. Of the HLH-1 binding genes, 81% (233/287) contain predicted CREB binding sites (<5 kb from the TSS). These results suggest that C. elegans body muscles (like other excitable cells) have a large number of activity-induced genes, many of which are potential CREB transcriptional targets.

Using this Lev-induced gene list, we devised a simple reporter assay for CREB induced gene expression. For this purpose, we focused on the gem-4/Copine gene, which was the top hit from our analysis of Lev-induced genes (induced ~30 fold) (Figure 4). The gem-4 promoter contains multiple CRH-1/CREB binding sites, implying that it could be a direct CRH-1 transcriptional target. Quantitative RT-PCR confirmed that Lev treatment increased gem-4 mRNA levels 8-fold (Figure 5A). We designed a transcriptional reporter (Figure 5B) that compares expression of the gem-4 promoter (expressing NLS-GFP) with a control promoter unaffected by depolarization (the myo-3 promoter, expressing NLS-mCherry) in individual muscle cells. Using this reporter, we found that Lev treatment increased gem-4 expression 8–12-fold (Figure 5C–D) while myo-3 expression was unaltered (Figure 5—figure supplement 1A). Lev-induction of the gem-4 reporter was eliminated by mutations inactivating a Lev receptor subunit (UNC-29), indicating that gem-4 induction was not mediated by an off target effect of Lev (Figure 5C). Lev-induction of gem-4 was also blocked in mutants lacking CRH-1/CREB and this defect was rescued by a transgene expressing CRH-1 in body muscles (Figure 5D). These results identify gem-4 as a CRH-1/CREB target expressed in body muscles.

Analysis of mRNA abundance following muscle depolarization.

mRNA abundance in Lev (200 µM, 1 hr) versus mock treated synchronized L4 larvae is plotted. Fold change (x-axis) is plotted against the statistical significance (y-axis) for each probeset. Fold changes are shown in log2 scale. Adjusted P values are shown in - log10 scale. Genes with increased (red dots) and decreased (green dots) expression are indicated (>2 Fold-change, FDR p<0.05). Probe sets corresponding to gem-4 and cex-1 are indicated. All genes that are differentially expressed following Lev treatment are listed in Supplementary file 2.

https://doi.org/10.7554/eLife.18931.007
Figure 5 with 1 supplement see all
gem-4 Copine expression in body muscle is induced by depolarization.

Induction of gem-4 expression was analyzed by qPCR (A) and using a transcriptional reporter (B–H). (A) The abundance of gem-4 mRNA (assessed by qPCR) was increased following 1 hr levamisole (Lev) exposure. The number of biological replicates is indicated. (B) A schematic diagram of the gem-4 reporter construct (left) and representative images of muscle nuclei (right) before and after a 20 min Lev exposure, and 2 hr recovery. (C–D) The mean fold induction of the gem-4 reporter (Pgem-4) after Lev treatment is shown. Lev-induced gem-4 expression was abolished in mutants lacking UNC-29, an essential subunit of the Lev receptor (C) and in mutants lacking the transcription factor CRH-1 (D). The crh-1 mutant defect in gem-4 induction was rescued by a transgene expressing CRH-1 in body muscles (D). (E) Expression of the gem-4 reporter was measured following photo-stimulation of transgenic animals that express ChR2 in cholinergic motorneurons. Expression of gem-4 was significantly increased by 2, 5, 10, and 20 Hz photo-stimulation (for 20 min). Photo-evoked gem-4 expression was not observed when animals were not cultured with ATR. (F–G) The fold induction of the gem-4 reporter following Lev exposure was significantly reduced in shn-1 null mutants (F) but not in shn-1(ok1241) mutants, which lack the PDZ domain (G). Lev-induced gem-4 expression was significantly increased in egl-19(nu496) mutants, which lack the carboxy-terminal PDZ ligand (H). (I–J) Lev induction of the cex-1 reporter is significantly reduced in shn-1 mutants. (I) Schematics of the cex-1 and myo-3 reporters are shown. (J) Expression of the cex-1 reporter (normalized to myo-3 expression in the same nucleus) was significantly increased by Lev treatment. The Lev-induced expression of the cex-1 reporter was significantly reduced in shn-1 mutants. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001; **p<0.01; *p<0.05). Error bars indicate SEM.

https://doi.org/10.7554/eLife.18931.008

Lev treatment is a non-physiological stimulus that produces prolonged muscle depolarization. To determine if synaptic activity induces the gem-4 reporter, we evoked excitatory synaptic input to muscles by photo-stimulating transgenic animals expressing channel rhodopsin in cholinergic motor neurons (Figure 5E). In patch clamp recordings of body muscles, spontaneous action potentials are observed at ~1 Hz (Gao and Zhen, 2011). Therefore, to mimic a realistic pattern of synaptic input, we photo-stimulated motor neurons (25 ms light pulse) at 1–20 Hz for 20 min. The gem-4 reporter was significantly induced following 2, 5, 10, and 20 Hz photo-stimulation (Figure 5E). Induction was not observed at lower frequencies (0.5 and 1 Hz) nor when animals were cultured without all trans-retinal (ATR) (Figure 5E). Photo-induction of gem-4 expression was eliminated in unc-13 mutants (Figure 5—figure supplement 1B), which have dramatically reduced synaptic vesicle exocytosis (Richmond et al., 1999). By contrast, unc-13 mutations did not prevent Lev-induced gem-4 expression (Figure 5—figure supplement 1C). Similar levels of gem-4 induction were produced by 20 Hz photo-stimulation and Lev treatment (Figure 5—figure supplement 1B–C). Thus, the gem-4 reporter was induced by both synaptic and Lev-evoked muscle depolarization. Expression of a mouse gem-4 paralog (N-copine) in hippocampal neurons is also induced by high frequency stimulation of acute brain slices (Nakayama et al., 1998); consequently, activity-induced copine expression is observed in both muscles and neurons and is conserved across phylogeny.

SHN-1 is required for gem-4 induction

Using this gem-4 reporter construct, we next asked if SHN-1 is required for CRH-1/CREB-induced gene expression. Lev-induced gem-4 expression was significantly decreased in shn-1(tm488) null mutants (Figure 5F). To determine if SHN-1 controls expression of other activity-induced genes, we developed a transcriptional reporter for a second Lev-induced gene (cex-1) (Figures 4 and 5I). Expression of the cex-1 reporter in body muscles was dramatically induced following Lev treatment (Figure 5J). Because baseline cex-1 expression in untreated muscles could not be reliably detected, we were unable to accurately measure the fold-induction of the cex-1 reporter following Lev treatment. As seen with the gem-4 reporter, we found that Lev-induced cex-1 expression in muscles was dramatically reduced in shn-1 null mutants (Figure 5J). Taken together, these results support the idea that SHN-1 promotes activity-induced gene expression in body muscles.

Next we asked if gem-4 induction requires binding of SHN-1’s PDZ domain to EGL-19’s carboxy-terminus. Deleting EGL-19’s PDZ ligand [in egl-19(nu496) mutants] significantly increased gem-4 induction while deleting SHN-1’s PDZ domain [in shn-1(ok1241) mutants] had no effect on gem-4 induction (Figure 5G–H). These results indicate that SHN-1 regulates EGL-19/Cav1 current and CRH-1/CREB activation by distinct mechanisms, since the former requires PDZ binding to EGL-19 while the latter does not.

Calcium current and gem-4 induction are sensitive to shn-1 gene dose

Deletion and duplication of human shank genes are both associated with ASD, schizophrenia, and mania (Bonaglia et al., 2006; Durand et al., 2007; Failla et al., 2007; Gauthier et al., 2010; Han et al., 2013). If Shank CNVs are causally associated with these psychiatric disorders, cellular phenotypes should be similarly sensitive to Shank copy number. To test this idea, we analyzed the effect of shn-1 gene dosage on calcium currents and Lev-induced gem-4 expression (Figure 6). We analyzed animals with 0 (tm488 homozygotes), 1 (tm488/+ heterozygotes), 2 (WT), and 4 (WT +2 single copy shn-1 transgenes) copies of shn-1. Compared to wild type controls, muscle calcium current density was significantly increased in animals containing 1 and 4 copies of shn-1 and was significantly decreased in animals containing 0 copies of shn-1 (Figure 6A–C). The kinetics of calcium current deactivation were not significantly altered by changes in shn-1 dosage (Figure 6D). Similarly, when compared to wild type controls, gem-4 induction was significantly diminished in animals containing 0, 1, and 4 shn-1 copies (Figure 6E). Thus, L-type calcium current density and gem-4 induction were both sensitive to shn-1 copy number. Interestingly, increased and decreased shn-1 gene dosage produced similar defects in calcium current and gem-4 induction.

shn-1 gene dosage regulates calcium current density and gem-4 induction.

The effect of varying shn-1 gene dosage on calcium current density (A–B) and gem-4 induction (C) was analyzed. The following genotypes were analyzed: 0 copies of shn-1 [shn-1(tm488) homozygotes], 1 copy of shn-1 [shn-1(tm488)/+ heterozygotes], 2 copies of shn-1 (wild-type) and 4 copies of shn-1 (nuSi26 homozygotes in wild-type). (A–B) Muscle Ca+2 current was sensitive to changes in shn-1 gene dose, with decreased (0 shn-1 copies) and increased (1 and 4 shn-1 copies) current density observed in the indicated genotypes. Mean current density as a function of holding potential (A), mean current density at 10 mV (B), and mean current deactivation time constants (C) are shown. (D) Lev-induced gem-4 expression was significantly reduced in animals with 0, 1, and 4 copies of shn-1. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (***p<0.001; **p<0.01). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for panels A-C are shown in Supplementary file 1.

https://doi.org/10.7554/eLife.18931.010

Cholinergic transmission is not sensitive to shn-1 gene dosage

To determine SHN-1’s role in synaptic transmission, we recorded excitatory post-synaptic currents (EPSCs) from body muscles. Evoked responses were significantly larger in shn-1 null mutants (Figure 7A,C). The shape of evoked responses in wild type and shn-1 mutants were indistinguishable, indicating that the kinetics of evoked release was unaltered (Figure 7B). The rate and amplitude of spontaneous miniature EPSCs (mEPSCs) were unaltered in shn-1 mutants (Figure 7—figure supplement 1A–C). The change in evoked EPSC amplitude combined with unaltered mEPSC amplitudes indicates a pre-synaptic change in cholinergic transmission. The shn-1 null EPSC defect was rescued by transgenes expressing SHN-1 in body muscles, implying that SHN-1 functions post-synaptically (Figure 7B). The evoked EPSCs (Figure 7C) and mEPSCs (Figure 7—figure supplement 1D–E) observed in animals with 1, 2, and 4 shn-1 copies were not significantly different, indicating that synaptic transmission is not sensitive to shn-1 copy number. Similar results were recently reported in mice where glutamatergic transmission in the striatum was enhanced in Shank3B-/- homozygotes but this effect was not observed in Shank3B+/- heterozygotes (Peixoto et al., 2016).

Figure 7 with 1 supplement see all
Synaptic transmission at the NMJ is not sensitive to shn-1 gene dosage.

Stimulus-evoked EPSCs were recorded from adult body wall muscles. Representative traces of evoked responses (A), averaged shn-1 and WT evoked responses (normalized to equal amplitudes) (B), and mean evoked charge transfer (C) are shown. Evoked charge was significantly increased in shn-1 homozygotes and this defect was rescued by a single copy transgene (nuSi26) that restored SHN-1 expression in body muscles (C). Averaged peak normalized shn-1 and WT evoked responses have indistinguishable rise and decay times, indicating that rise and decay kinetics were unaltered (B). Evoked charge transfer (D) and peak amplitudes (E) did not differ significantly in animals containing 1, 2, and 4 shn-1 copies. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type controls are indicated (**p<0.01; ns, not significant). Error bars indicate SEM. Mean, standard errors, sample sizes, and p values for this figure are shown in Supplementary file 1.

https://doi.org/10.7554/eLife.18931.011

Discussion

Because Shank proteins are highly enriched at post-synaptic densities, prior studies focused on the idea that Shank regulates synapse formation or function in some manner (Jiang and Ehlers, 2013). Here we provide evidence that Shank regulates Cav1 calcium current density and activity-induced gene expression, and that these two cellular functions are mediated by distinct dosage-sensitive mechanisms.

SHN-1 promotes EGL-1/Cav1 channel delivery to the cell surface

We find that SHN-1’s PDZ domain binds the EGL-19/Cav1 carboxy-terminus (like their mammalian counterparts) and that disrupting this interaction decreased muscle calcium current. Mutations deleting SHN-1’s PDZ domain and those deleting EGL-19’s PDZ ligand both decreased EGL-19 calcium current and additive defects were not observed in double mutants. These results suggest that SHN-1’s effects on calcium current are mediated by binding of its PDZ domain to EGL-19’s carboxy-terminus. Analysis of gating currents and Terrier reporter fluorescence suggest that the decreased muscle calcium currents in shn-1 mutants arises primarily from decreased trafficking of EGL-19/Cav1 channels to the plasma membrane.

Shank effects on Cav1 current in mammalian neurons or muscles have not been reported. The carboxy-terminus of mammalian Cav1.3 (-ITTLCOOH) and Cav1.2 (-VSSLCOOH) channels are both predicted to bind PDZ domains (Zhang et al., 2005). When mutant constructs lacking the carboxy-terminal PDZ ligands of Cav1.3 or Cav1.2 channels were expressed in hippocampal neurons, no changes in calcium current were observed; however, the Cav1.3 truncation mutant exhibited decreased synaptic localization (Weick et al., 2003; Zhang et al., 2005). The rat Shank 1 and 3 PDZ domains exhibit strong binding to Cav1.3 (but not Cav1.2). Thus, Shank proteins could promote synaptic targeting of Cav1.3; however, the PDZ protein responsible for Cav1.3 targeting has not been identified. These studies relied on over-expression of mutant Cav1 channels (rather than knockin mutations altering the endogenous genes); consequently, it remains possible that Shank proteins could also regulate Cav1 current in mammalian neurons or muscles.

SHN-1 promotes activity-induced expression of gem-4

Prior studies of mammalian neurons proposed that Cav1 channel binding to a PDZ scaffolding protein promotes activity-induced CREB phosphorylation (Weick et al., 2003; Zhang et al., 2005). These studies showed that deleting the carboxy-terminal PDZ ligand in Cav1.2 or 1.3, or over-expressing peptides containing the PDZ-ligands diminish activity-induced CREB phosphorylation and transcription of CREB targets. The PDZ protein responsible for these effects was not identified in these studies. Here we show that SHN-1 promotes expression of a CRH-1/CREB transcriptional target in muscles but that this effect does not require binding of SHN-1’s PDZ domain to EGL-19’s carboxy-terminus. In fact, deleting EGL-19’s PDZ ligand significantly enhanced depolarization induced CRH-1/CREB target expression, perhaps by disrupting EGL-19 interaction with another protein.

Our results suggest that SHN-1’s effects on calcium current density and gem-4 induction are mediated by distinct mechanisms. Increased calcium current is mediated by SHN-1’s PDZ domain binding to EGL-19’s carboxy-terminus whereas another SHN-1 domain (most likely the Ankyrin or Proline-rich domains) enhances CREB activation.

A puzzling aspect of our results is that calcium current density and induced gem-4 expression were poorly correlated. For example, reduced gem-4 induction was observed in mutants with decreased (shn-1 null mutants) and increased (shn-1/+ heterozygotes) EGL-19/Cav1 current density. Similar results were also reported in cultured mammalian neurons, where CREB phosphorylation was poorly correlated with calcium current density and instead was better correlated with Cav1 open probability (Wheeler et al., 2008). Based on these results (and others), these authors proposed that CREB phosphorylation is mediated by a calcium sensor that is spatially very close to (and possibly physically associated with) activated Cav1 channels (Deisseroth et al., 1996; Wheeler et al., 2008). In this scenario, CREB phosphorylation would be strongly correlated with calcium levels in the nanodomain of a Cav1 channel but less correlated with global cytoplasmic calcium. Thus, our results suggest that CRH-1 activation may also be mediated by tight coupling of a calcium sensor to activated EGL-19/Cav1 channels.

Implications for understanding psychiatric disorders

In humans, Shank deletions and duplications both confer risk for ASD and schizophrenia (Bonaglia et al., 2006; Durand et al., 2007; Failla et al., 2007; Gauthier et al., 2010; Han et al., 2013). Thus, too little or too much Shank is associated with psychiatric traits. If Shank CNVs are causally linked to these psychiatric disorders, cellular defects linked to ASD should exhibit similar sensitivity to Shank gene dosage. Consistent with this idea, several studies previously reported cellular and behavioral defects in Shank3m/+ heterozygotes (Han et al., 2016; Orefice et al., 2016; Wang et al., 2016; Yi et al., 2016). Increased Shank3 copy number has been analyzed in a single study (Han et al., 2013), which reported several behavioral and synaptic phenotypes. None of these studies identified phenotypes that were shared between decreased and increased Shank3 dosage. In most cases, it was not determined if cellular defects were cell autonomous consequences of altered Shank3 dosage (Han et al., 2013; Orefice et al., 2016; Wang et al., 2016; Zhou et al., 2016). Here we find that changes in shn-1 gene dosage alter two cell autonomous phenotypes in muscles: L-channel calcium current and expression of CREB target genes. For both phenotypes, similar defects were observed in muscles with decreased and increased shn-1 dosage. Thus, we identify two cellular phenotypes that exhibit the same pattern of dose sensitivity that is observed for Shank3 in schizophrenia and ASD. Based on these results, we propose that human Shank CNVs also cause cellular phenotypes, which may include altered calcium current and CREB target expression. By contrast, other shn-1 mutant phenotypes (e.g. evoked EPSCs) were not sensitive to shn-1 gene dosage. Thus, sensitivity to Shank copy number provides a useful criterion to determine which phenotypes are more likely to contribute to the psychiatric traits associated with Shank CNVs. Two recent studies showed that Shank3 binds directly to Ih and TRPV1 channels in neurons and that the corresponding currents densities are significantly reduced in Shank3+/- heterozygotes (Han et al., 2016; Yi et al., 2016). These results (together with those reported here) suggest that an important function of Shank proteins is to regulate ion channel densities.

How does shn-1 dose alter L-current and CREB target expression? In general, dosage sensitive phenotypes are thought to occur by disrupting the function of multimeric protein complexes. In such cases, under and over-expression of individual subunits alter the stoichiometry of assembled holo-complexes, leading to decreased activity. For example, increased and decreased expression of the yeast histones H2A and H2B results in similar loss of function phenotypes (chromosome loss and altered gene expression) (Clark-Adams et al., 1988; Meeks-Wagner and Hartwell, 1986). Shank binds many other synaptic proteins (Lee et al., 2011; Sakai et al., 2011) and undergoes zinc induced polymerization into large complexes (Baron et al., 2006; Hayashi et al., 2009). Thus, increased and decreased Shank abundance could disrupt the stoichiometry of post-synaptic complexes. Our results suggest that L-current and CREB activation are sensitive to subtle changes in the stoichiometry of Shank protein complexes. Our results further suggest that dynamic changes in the composition of Shank complexes provides a mechanism to regulate circuit function and plasticity (and potentially psychiatric traits).

Shank regulation of L-currents and CREB activation could both contribute to the pathophysiology of ASD. Human Shank mutations are particularly linked to ASD associated with intellectual disability (Leblond et al., 2014). CREB has long been linked to learning and memory in several model organisms (Bourtchuladze et al., 1994; Dash et al., 1990; Yin et al., 1994); consequently, Shank’s CREB activation function could directly contribute to cognitive deficits associated humans Shank CNVs. CREB-induced BDNF expression promotes development of inhibitory synapses in the cortex (Hong et al., 2008). Thus, Shank’s CREB activation function could alter synaptic inhibition, a phenotype found in several ASD models (Dani et al., 2005; Rubenstein and Merzenich, 2003). Finally, L-channels play an important role in calcium signaling in dendrites and spines (Higley and Sabatini, 2012). Thus, Shank’s Cav1 current density function could contribute to cognitive or developmental defects in ASD by adjusting dendritic calcium signaling in CNS neurons. For these reasons, we propose that Shank effects on Cav1 currents and CREB target expression could play an important role in the pathophysiology of ASD (and potentially other psychiatric disorders).

Materials and methods

Experimental procedures

Strains

Strain maintenance and genetic manipulation were performed as described (Brenner, 1974). Animals were cultivated at room temperature (~22°C) on agar nematode growth media seeded with OP50 bacteria. The following strains were used in this study:

KP7624 nuIs525[Pgem-4::NLS-GFP; Pmyo-3::NLS-mCherry] V

KP7598 unc-29(x29) I; nuIs525 V

KP7583 crh-1(tz2) III; nuIs525 V

KP7601 oxSi91[Punc17::ChIEF::mCherry] II; nuIs525 V

KP7618 unc-13(s69) I; nuIs525 V

KP7896 unc-13(s69) I; oxSi91 II; nuIs525 V

KP7032 shn-1(tm488) II

KP7272 shn-1(ok1241) II

KP7461 nuSi26[Pmyo-3::shn-1] shn-1(tm488) II

KP7573 shn-1(tm488) II; nuIs525 V

KP7574 bli-2(e768) shn-1(tm488) II; nuIs525 V

KP7567 nuSi26 II; nuIs525 V

KP7493 nuSi26 II

KP7212 bli-2(e768) shn-1(tm488) II

KP7992 egl-19(nu496) IV

KP7997 egl-19(nu496) IV; nuIs525 V

KP8046 shn-1(tm488) II; egl-19(nu496) IV

KP8047 shn-1(tm488) II; egl-19(nu496) IV; nuIs525 V

KP7991 egl-19(nu495) IV

KP8303 nuSi74 [Pmyo-3::Terrier]

KP8304 nuSi74; shn-1(tm488)

KP8274 nuSi66 [Pmyo-3::NLS::TagBFP2]; nuSi67 [Pcex-1::NLS::mNeonGreen]; nuSi70[Pgem-4::NLS::tagRFPt]

KP8301 nuSi66;nuSi67;nuSi70;shn-1(tm488)

Transgenic animals were prepared by microinjection, and integrated transgenes were isolated following UV irradiation, as described (Dittman and Kaplan, 2006). Single copy transgenes were isolated by the MoSCI and miniMoS techniques (Frøkjaer-Jensen et al., 2008; Frøkjær-Jensen et al., 2014).

shn-1 dosage experiments

Animals with different shn-1 copy numbers were constructed as follows: 0 copies, shn-1(tm488) homozygotes; 1 copy, WT males were crossed with bli-2 shn-1(tm488) homozygotes [KP7212 (for electrophysiology) and KP7574 (for gem-4 induction)] and non-Blister F1 hermaphrodites were analyzed; 2 copies, WT males were crossed with bli-2 homozygotes and non-Blister F1 hermaphrodites were analyzed; 4 copies, WT animals homozygous for the single copy transgene nuSi26. gem-4 reporter expression in bli-2/+ heterozygotes and WT controls did not differ significantly and were pooled for 2 copies of shn-1.

Constructs and transgenes

Pgem-4 reporter

2 kb of 5’ non-coding sequences from the gem-4 gene was cloned into a vector expressing NLS-GFP (pPD122.56). A myo-3 promoter region (2.3 kb) was sub-cloned into a vector expressing NLS-mCherry. Both were injected into WT animals at 5 ng/µl and stable arrays picked. A single extrachromosomal array was integrated by UV irradiation and outcrossed six times (nuIs525).

Pcex-1 reporter 

KP#3310 has the cex-1 promoter (1038 bp), a single SV40 NLS, mNeonGreen (Shaner et al., 2013) (codon optimized for C. elegans) followed by the EGL-13 NLS and the cex-1 3’ UTR (2097bp) inserted between the SacII and PstI sites of pCFJ1662. A single hygromycin resistant integrant, nuSi67 was obtained as described (Frøkjær-Jensen et al., 2014). cex-1 reporter expression in each muscle nucleus was normalized to BFP expressed in the same nucleus (using the myo-3 promoter).

myo-3 expression of BFP

KP#3309 has the myo-3 promoter (2386 bp), a single SV40 NLS, mTagBFP2 (Subach et al., 2011) (codon optimized for C. elegans) followed by the EGL-13 NLS and the unc-54 3’ UTR inserted between the SbfI and SnaBI sites of pCFJ901. A single G418 resistant integrant, nuSi66 was obtained and mapped within the abch-1 gene on Chromosome II as described (Frøkjær-Jensen et al., 2014).

Terrier

KP#3308 has the myo-3 promoter, PAT-4 signal sequence (from pPD122.36 Addgene), super-ecliptic pHluorin (Dittman and Kaplan, 2006), PAT-3 transmembrane domain (pPD122.36) followed by the cDNA of EGL-19B coding for residues 1374–1872, tagRFP-T (codon optimized for C. elegans), and the cDNA of EGL-19B coding for residues 1873–1877, followed by the let-858 3’ UTR (pPD122.36) inserted between the HindIII and AflII sites in the polylinker of the miniMOS vector pCFJ910 (Addgene).

Single copy insertions of Terrier were obtained using the miniMOS technique as described (Frøkjær-Jensen et al., 2014).

shn-1 rescue

A C33B4.3a cDNA was cloned using gateway into DONR221. Multisite gateway was used to assemble Pmyo-3::shn-1::unc54UTR into pCFJ150 and a single copy transgene (nuSi26) was obtained by injecting EG6699. The unc-119(ed3) allele was outcrossed from nuSi26 prior to analysis.

Fluorescence imaging

Confocal imaging was performed using an Olympus 60x objective (NA 1.45) on an Olympus FV-1000 confocal microscope at 5x digital zoom. For Pgem-4 imaging, ~15 worms were exposed to 1 mM Lev for 20 mins. Two hours after Lev stimulation, worms were immobilized on 10% agarose pads with 0.3 µl of 0.1 µm diameter polystyrene microspheres (Polysciences 00876–15, 2.5% w/v suspension). Individual muscle nuclei were imaged next to the terminal bulb of the pharynx and analysed using FIJI (https://fiji.sc). The ratio was obtained of green (Pgem-4) to red (Pmyo-3) for each nucleus. A same day WT control (+/- Lev) was analyzed for all genotypes. For terrier imaging ~6–10 worms were immobilized on 10% agarose pads with 0.3 µl of 0.1 µm diameter polystyrene microspheres and for each worm the closest neuromuscular junction to the surface was imaged. Worms were only accepted for analysis if they had clearly identifiable red and green puncta. Both intensity and area were analysed using FIJI.

Retinal plates

4 µl of all trans-retinal (ATR, 100 mM dissolved in ethanol) was mixed with 250 µl of OP50 E. coli and spread on 60 mm NGM plates. Plates were allowed to dry for 24 hr and approx. 40 L4 animals were added and allowed to grow in the dark for 16–24 hr before the assay. Control plates used 4 µl of ethanol mixed with 250 µl of OP50.

Optogenetic stimulation

ACh release at NMJs was evoked in animals expressing the Channelrhodopsin variant ChIEF in cholinergic neurons (oxSi93) (Watanabe et al., 2013). Animals were photo-stimulated with seven 470 nm Rebel LEDs mounted on a 40 mm SinkPadII fitted with a Round Concentrator Lens and powered with a 700 mA DC Driver (Luxeon Star LEDs). 20 min of 25 ms light pulses were generated at the indicated frequencies using an Arduino Uno. Pulse frequency and duration were confirmed using a photodiode and oscilloscope.

qPCR

Total RNA was purified from a synchronized population of young adult worms treated with 200 μM Levamisole for 1 hr and mock-treated samples. RNA was isolated using standard Trizol-bromochloropropane extraction methods in combination with the Qiagen RNeasy Kit. RNA was DNase treated using the Qiagen on-column RNase free DNase set. Samples were prepared from the following genotypes: wild-type (N2 Bristol) and shn-1 (tm488) on two separate days. One micrograms of total RNA was used to synthesize cDNA using RETROscript (Ambion). Real-time PCR was performed using iTaq Universal SYBR Green Supermix (BioRad) and a 7500 Fast Real-Time PCR System (Applied Biosystems). All reactions were run in triplicate and on at least two biological replicates. All the values are normalized to rpl-32 as internal control as well as to the transcript levels in untreated samples. Statistical significance was determined using the two-tailed Student’s t test.

Microarray analysis

RNA isolation and cDNA synthesis was performed as described for qPCR. 6 Affymetrix C. elegans GeneChip were used (3 × 1 hr 200 μM Levamisole exposure and 3x mock-treated samples). Expression values were determined using the Robust Multi-chip Average (RMA) method. Probe sets that showed a > 2 fold change between mock and levamisole treated, with an unadjusted p-value of <0.0001 were considered to be Levamisole responsive. HLH-1 chip-seq data was taken from modencode (http://www.modencode.org). The PWM used for analysis of crh-1 binding sites was from Homer (http://homer.ucsd.edu/homer/).

Electrophysiology

Whole-cell patch-clamp measurements were performed using a Axopatch 200B amplifier with pClamp 10 software (Molecular Devices). The data were sampled at 10 kHz and filtered at 5 kHz. All recordings were performed at room temperature (~19–21°C)

Evoked EPSCs

 Worms were superfused in an extracellular solution containing (in mM) 127 NaCl, 5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 20 glucose, 1 CaCl2 and 4 MgCl2, bubbled with 5% CO2, 95% O2 at 20°C. Whole cell recordings were carried out at –60 mV using an internal solution containing 105 mM CH3O3SCs, 10 mM CsCl, 15 mM CsF, 4 mM MgCl2, 5 mM EGTA, 0.25 mM CaCl2, 10 mM HEPES and 4 mM Na2ATP, adjusted to pH 7.2 using CsOH. Under these conditions, we only observed endogenous acetylcholine EPSCs. For endogenous GABA IPSC recordings the holding potential was 0 mV. All recording conditions were as described (McEwen et al., 2006). Stimulus-evoked EPSCs were stimulated by placing a borosilicate pipette (5–10 µm) near the ventral nerve cord (one muscle distance from the recording pipette) and applying a 0.4 ms, 30 µA square pulse using a stimulus current generator (WPI).

Ca+2 current recordings 

The pipette solution contained (in mM): 140 CsCl; 10 TEA-Cl; 5 MgCl2; 5 EGTA; 10 Hepes, pH 7.2, with ~320 mosM CsOH. The extracellular solution contained (in mM): 140 TEA-Cl; 5 CaCl2; 1 MgCl2; 3 4-aminopyridine; 10 glucose; five sucrose; 15 Hepes, pH 7.4, with ~330 mosM CsOH. The voltage-clamp protocol consisted of −60 mV for 50 ms, −90 mV for 50 ms, test voltage (from −60 mV to +4 mV) 200 ms. Access resistance was continuously monitored, and ranged between 7 and 15 MΩ. Series resistance was not compensated. The voltage dependence of the Ca+2 current density were fitted with the equation: I(V) = Gmax(V – Vrev) ⁄ ({1 + exp[(V0.5 – V) ⁄ k]}), where I(V) is the density of the current measured, V is the test pulse, Gmax is the maximum conductance, Vrev is the apparent reversal potential, V0.5 is the half-activation voltage, and k is a steepness factor. The decay tau was well fit by a single exponential function and calculated from a test potential of 0 mV fitting the curve from the peak of the current till the end of the pulse.

Gating currents

 The pipet and bath solutions were as described for the calcium current recordings. To resolve gating currents leak and capacitive transients were subtracted using a p/4 protocol, and measured by applying a series of test pulses at 5s intervals from the holding potential of −90 mV to potentials between +40 mV and +50 mV in 2 mV increments and integrating the gating charge movement at the reversal potential for the ionic current.

K+ current recordings

The bath solution contained (in mM): NaCl 140, KCl 5, CaCl2 5, MgCl2 5, dextrose 11 and HEPES 5 (pH 7.2, 320 mOsm); and the pipette solution contained (in mM): KCl 120, KOH 20, Tris 5, CaCl2 0.25, MgCl2 4, sucrose 36, EGTA five and Na2ATP 4 (pH 7.2, 323 mOsm). The voltage-clamp protocol consisted of −60 mV for 50 ms, −90 mV for 50 ms, test voltage (from −60 mV to +60 mV) 1000 ms. IKfast was defined as the peak current after the capacitance transients, and IKslow was defined as the average current of the last 100 ms of each voltage step.

Statistics

Data was assessed for a normal distribution using the D'Agostino-Pearson normality test and equality of variances using the F-Test. For comparisons of normally distributed data with equal variances a two-tailed Student’s t-test was used. For all other comparisons the Mann–Whitney U test was used. For analysis of calcium current-voltage relationships, two-way ANOVA with Sidak’s correction for multiple comparisons was utilized. All statistics were performed in GraphPad Prism 6 and significant differences are indicated as follows: *p<0.05, **p<0.01, and ***p<0.001.

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Decision letter

  1. Graeme W Davis
    Reviewing Editor; University of California, San Francisco, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for submitting your work entitled "Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

Summary of reviewer comments and criticism:

The manuscript by Pym and colleagues presents phenotypic and molecular/genetic insight into the function of Shank with potential relevance to the pathophysiology of disease. In particular, these insights include potential regulation of calcium currents, downstream transcription and Shank gene dosage effects, a property of human disease biology that has yet to be well-connected to any particular cellular phenotype. For all of these reasons, the reviewers share enthusiasm for the study. However, there are several areas of concern. First, there are technical improvements that should be made and which are clearly specified in the reviews. Second, all three reviewers felt that additional insight into the regulation of the calcium channels was warranted. The extent to which this is possible remains unclear, but should be addressed at some level. Third, reviewers expressed concern by lack of mechanism. After further discussion this focused on several issues. On the one hand, the reviewers expressed concern regarding the broader cellular context for the phenotypic observations. Specifically, to what extent are the observed changes in gem-4 specific to this gene, versus part of much broader regulatory disruptions? Likewise, to what extent are other channels or ionic currents altered? This is something that may be known to the authors based on their electrophysiological and gene profiling experiments that have been performed to date. If not, these are straightforward additions that could be pursued. On the other hand, there were concerns regarding the connection of Shank gene dosage to the cellular phenotypes. It may not be possible, in this system, to take this analysis to the level of changes in protein abundance, in vivo. But additional information to connect gene dosage to phenotypic severity would go a long way to establishing a model. Finally, the paper reads as a series of phenotypic observations and lacks coherence. It is not obvious to the reviewers how to address this issue. But, this is clearly something that could clearly benefit from additional experimentation as well as a more lengthy treatment in the text.

Reviewer #1:

This paper begins with the prior observation that the C. elegans orthologue of Shank is expressed in muscle, enabling the authors to examine the effects of this disease associated protein on the synaptic transmission properties and gene expression profile of the C. elegans neuromuscular junction (NMJ). The NMJ is a powerful system in the worm, allowing a combination of powerful genetics (dose-dependence of shank) with analysis of ionic currents, in vivo. The authors provide evidence (stated largely in their own words) that "SHN-1's effects on calcium current are mediated by binding of its PDZ domain to EGL-19's carboxy-terminus." Their results suggest, "that SHN-1's effects on calcium current density and gem-4 induction are mediated by distinct mechanisms. Increased calcium current is mediated by SHN-1's PDZ domain binding to EGL-19's carboxy-terminus whereas another SHN-1 domain (most likely the Ankyrin or Proline-rich domains) enhances CREB activation." They find that, "changes in shn-1 gene dosage alter two cellular phenotypes: L-channel calcium current and expression of CREB target genes. Thus, their "results support the idea that human Shank CNVs cause cellular phenotypes that include altered calcium current and CREB target expression." When taken together, these data amount to an interesting advance with relevance to the cellular underpinnings of disorders caused by dose-dependent effects on expression of Shank that could reasonably be related to the cause or expression of disease related neurological symptoms in human. The manuscript suffers a bit from the brevity of style, presumable a consequence of prior submission at a different journal. Inclusion of additional information, already in the authors possession, or readily attained, could add an important dimension to this work that would increase the study's impact (see below).

Major issues:

1) The authors identify gem-4 in an expression-array analysis of Lev-treated NMJ. Since the analysis has been completed, it seems reasonable to request that the entire transcriptional profiling data set be deposited in eLife as part of this manuscript. Is gem-4 one of several hundred up-regulated genes, or one of only a few? The implications are important for understanding the degree to which the identified signaling is a simplification of a much broader effect. For example, a major conclusion of this paper is that Shank regulates both Cav1 function and, simultaneously, gene expression. But, the way the paper is written, the effects on gene expression are limited to a single gene.

2) Are the mechanisms identified by the authors part of a larger ion channel disruption process, or might these data point to differential effects in different cell types? The authors cite recent work from the Sudhof lab linking Shank to Ih. The authors make an effective argument that their observed effects on calcium channels are conserved in other species, and potentially relevant to disease. I suspect that the authors have analyzed additional currents and my have an indication, yes or no, that other channels are also dis-regulated. It may not be necessary to define which channels are dis-regulated, but it would be important to know whether there is a change in outward currents, A-type or delayed rectifiers. Conversely, if there is no change in these additional macroscopic currents, it would underscore the specificity of the effects being observed.

3) A notable limitation of this system is the inability to associate changes in calcium currents with the synaptic localization and/or function of the calcium channel. It is not immediately clear if this limitation can be solved. However, the authors do suggest that the calcium micro-domain may be relevant in their Discussion section, making this a relevant issue even at these small muscle cells.

Reviewer #2:

The manuscript "Shank is a dose-dependent regulator of Cav1 Calcium current and CREB target expression" by Pym et al. using C. elegans as a model system attempts to uncover how Shank interactions and copy number variations impact psychiatric disorders. Based on the data and analysis presented here, the authors make the following conclusions

1) The C. elegans homolog of Shank, SHN-1 interacts with the C. elegans homolog of Cav1.3 ELG19 though a direct PDZ interaction to regulate Cav1.3 current density

2) SHN-1 PDZ interaction with ELG19 is not required for CREB based actvation of gem-4.

3) One copy of SHN-1 or 4 copies of SHN-1 lead to identical changes in SHN-1 effects on ELG19 currents and gem-4 induction.

4) Loss of SHN-1 leads to increases in cholinergic transmission compared to wild-type animals, while one copy of SHN-1 or 4 copies of SHN-1 have no effect on cholinergic transmission.

While this manuscript is interesting in its current form and the findings that a single copy of SHN-1 phenocopies 4 copies of SHN- 1/Shank and that the ELG19/Cav1.3 PDZ interaction with SHN-1/Shank interaction regulates ELG19/Cav1.3 density are very novel there are major concerns that need to be addressed before the paper is suitable for publication in eLife.

Major concerns:

1) The authors demonstrate that SHN-1 regulates Calcium current density and claim that this is through the PDZ interaction. However, the authors have no insight to whether this is due to decreases in Cav1.3 levels or due open probability. Therefore the authors should provide some mechanistic insight how SHN-1 regulates ELG19 current density through this PDZ interaction. This is critical as it has been previously shown by Zhang et al., 2005 that the PDZ ligand in the mammalian Cav1.3 is dispensable for interacting with mammalian Shank.

2) There is no consistency between Figure 1 and Figure 2 in regards to wt Calcium current activation and densities. The maximum current in wild-type in Figure 1 is at 0 mV in Figure 2 is +10 mV while in Figure 2 while in Figures 1 and 2 the maximum Calcium currents for the mutant is 0 mV. So based on the data presented in the manuscript, it remains unknown if loss of SHN-1 or the EGL19 mutants impact Calcium current activation. In addition, in Figure 2 average Calcium density is two-fold higher than in Figure 1. Why the disparity? Furthermore, the holding voltages are different between the two. This can lead the reader to believe that hyperpolarizing the terminal to -90 mV relieves some sort of inhibition. The authors should redo Figure 2 with the same holding potential as Figure 1.

3) The authors look at copy number of SHN-1 but never give insight into the mechanisms of copy number regulation and it remains largely a phenomenological finding. Getting a mechanistic insight in how SHN-1 copy number leads to various changes that are observed in the manuscript is critical. In particular, how does one copy of SHN-1 or four copies of SHN-1 lead to the identical phenotype in Calcium current density? At minimum, the authors should correlate SHN-1 protein expression levels to copy number. What happens if you put in 3 copies, or 8 copies of SHN-1?

4) The authors demonstrate that loss of SHN-1 leads to an increase in cholinergic transmission. This is interesting since this there is no change in mEPSC amplitude or frequency. Some insight into how SHN-1 loss effects only evoked release should be done. Does the loss of SHN-1 also impact GABAergic transmission in the same manner?

5) The mutant ok1241 is not an acceptable mutant to just look at PDZ function. This mutant deletes much of the Proline domain in SHN-1.

6) Based on the IV recordings, it appears the SHN-1 copy number and EGL19 PDZ mutants impact EGL19 inactivation. The authors should go back it look at the Calcium current deactivation kinetics and see if there is a difference.

Reviewer #3:

In this manuscript, Pym and colleagues have used C. elegans to study the Autism linked gene Shank-3. In their work, they decided to focus on the interaction between the Shank-3 ortholog, Shn-1, and the sodium channel subunit Egl-19, as its mammalian ortholog was shown to play a role in a rare form of ASD. Pym and colleagues first show that Egl-19 and Shn-1 can indeed bind, just like their mammalian orthologs. They find that diminished currents in muscles in Shn-1 mutants are likely mediated by defects in Egl-19 expression, localization or function. Then, the authors focus on another Shn-1 mediated process, which is activity regulated expression. They perform a screen to identify the genes upregulated upon neuronal activation of muscle and decided to focus on gem-4. They confirm that gem-4 activation requires Shn-1 activity but not its binding to Egl-19 thereby suggesting both processes are mediated by independent mechanisms. Finally, they determine that both Egl19 dependent currents as well as gem-4 expression are sensitive to Shn-1 gene dose, something that is know from human patients. While the findings are of interest, especially due to the importance of Shank-3 in autism, I am not sure this paper represents a major step forward in our understanding. The Egl-19 and gem-4 parts don't fit together and the story remains unfocused. Importantly, the mechanisms by which Shn-1 affects either Egl-19 dependent current or gem-4 transcription remain unanswered. Finally, how all this is or is not relevant to autism is unclear.

Specific comments:

1) Yeast two hybrid and GST pull down assays can, at best, indicate that two proteins CAN bind to each other (in these very artificial contexts) and definitely do not prove interaction in vivo. I would use more careful words. Probably beyond the scope of this paper but to show that they interact in vivo, even in the non-biochemical organisms such as C. elegans, one can perform experiments with tagged transgenic proteins and test their proximity in a wide range of strategies… Also, the experiments shown in Sup 1A+B are quite poorly controlled. So Egl19 does not bind the beads but does it bind other PDZ domains? In the well-referenced paper (Zhang et al., 2005) they performed many required controls to be able to suggest that the mammalian orthologs interact.

2) I was surprised that the authors did not attempt to describe the sub cellular localization of Egl-19 in WT and mutant worms… The best experiment would be to endogenously tag Egl-19 but even the second best – to follow the localization of transgenic Egl-19 was not performed. The PDZ binding motif has to remain on the C-terminal so one can N-terminally tag or tag C-terminally with the addition of the PDZ binding motif afterwards. This lies at the heart of how Shn-1 affects Egl-19 and is therefore a key question that remains unanswered in this work.

3) The activity dependent transcription experiment – I was surprised that the raw data are not shown. Is gem-4 the top hit but one out of many or is it the only one? Ideally one would like to view this.

4) Figure 3—figure supplement 2 does not exist (or at least I could not find it).

Taken together, I think this work represents two interesting stories that are currently incomplete and perhaps unfortunately do not fit very well together.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for resubmitting your work entitled "Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression" for further consideration at eLife. Your revised article has been evaluated by Richard Aldrich (Senior editor) and a Reviewing editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The study has been greatly strengthened by the addition of new data. The authors have included data acquired but not previously included in the manuscript. The authors have added new analyses of ion channel currents that may have co-varied and have improved the analysis of calcium channel electrophysiology as requested in the initial round of review. In addition, the authors have extended their study by providing new information regarding the mechanism of calcium channel modulation. Taken together, these advances address the major outstanding concerns raised in the initial round of review. There was considerable enthusiasm in the initial round of review for the ideas and approach.

A remaining issue is one of clarifying new Figure 3. The figure could be clarified to make it easier to understand the region of interest that is being visualized in panel D. Although there is a cartoon, the transition from the cartoon to the images in panel D could be improved.

https://doi.org/10.7554/eLife.18931.015

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

We are pleased that the reviewers and editors found our results interesting and significant. The review provides many helpful suggestions, which have greatly improved our paper. In response to these comments, we added several new experiments: 1) we repeated our analysis calcium current densities using a single protocol (responding to Review #2, comment 2); 2) we show that voltage-activated gating currents in body muscles are significantly reduced in shn-1 null mutants (Figure 3A-B), suggesting that SHN-1 regulates delivery of EGL-19/Cav1 to the cell surface; 3) we show that surface delivery of a chimeric membrane protein that contains EGL-19’s cytoplasmic tail (Figure 3C) is also significantly reduced in shn-1 null mutants (Figure 3D-G); 4) we show that SHN-1 mutations do not alter other voltage-activated currents in body muscles (Figure 1—figure supplement 1C), indicating that SHN-1 selectively regulates calcium current density; 5) we provide a full description of Levamisole-induced gene expression (Figure 4, Table S2); and 6) we show that SHN-1 is required for activity-induced expression of a second muscle gene (cex- 1), which supports the idea that SHN-1 controls expression of multiple activity-induced genes (Figure 5I-J). These results, as well as complete responses to all reviewer comments, are detailed below.

Reviewer #1:

[…] Major issues:

1) The authors identify gem-4 in an expression-array analysis of Lev-treated NMJ. Since the analysis has been completed, it seems reasonable to request that the entire transcriptional profiling data set be deposited in eLife as part of this manuscript. Is gem-4 one of several hundred up-regulated genes, or one of only a few? The implications are important for understanding the degree to which the identified signaling is a simplification of a much broader effect. For example, a major conclusion of this paper is that Shank regulates both Cav1 function and, simultaneously, gene expression.

As requested, we now provide a complete description of the Lev-induced gene set:

Results section:

“We analyzed gene expression following depolarization of body muscles with a nicotinic acetylcholine (ACh) agonist (levamisole, Lev). […] These results suggest that C. elegans body muscles (like other excitable cells) have a large number of activity-induced genes, many of which are potential CREB transcriptional targets.”

But, the way the paper is written, the effects on gene expression are limited to a single gene.

Prompted by this comment, we now show that SHN-1 is also required for expression of a second activity-induced gene:

Results:

“To determine if SHN-1 controls expression of other activity-induced genes, we developed a transcriptional reporter for a second Lev-induced gene (cex-1) (Figures 4 and 5I). Expression of the cex-1 reporter in body muscles was dramatically induced following Lev treatment (Figure 5J). Because baseline cex-1 expression in untreated muscles could not be reliably detected, we were unable to accurately measure the fold-induction of the cex-1 reporter following Lev treatment. As seen with the gem-4 reporter, we found that Lev-induced cex-1 expression in muscles was dramatically reduced in shn-1 null mutants (Figure 5J).”

2) Are the mechanisms identified by the authors part of a larger ion channel disruption process, or might these data point to differential effects in different cell types? The authors cite recent work from the Sudhof lab linking Shank to Ih. The authors make an effective argument that their observed effects on calcium channels are conserved in other species, and potentially relevant to disease. I suspect that the authors have analyzed additional currents and my have an indication, yes or no, that other channels are also dis-regulated. It may not be necessary to define which channels are dis-regulated, but it would be important to know whether there is a change in outward currents, A-type or delayed rectifiers. Conversely, if there is no change in these additional macroscopic currents, it would underscore the specificity of the effects being observed.

Thanks for pointing this out. As requested we now provide analysis of voltage-activated outward currents, finding that they are not significantly altered in shn-1 mutants. As the reviewer indicates, these results are important because they indicate that SHN-1 selectively alters muscle Cav1 current and has no effect on voltage-activated outward currents:

“To determine if SHN-1’s effects on calcium currents were specific, we measured voltage- activated potassium currents in body muscles (Figure 1—figure supplement 1C). Neither the voltage- dependence nor the current density of fast and slow potassium currents were significantly altered in shn-1 mutants.”

3) A notable limitation of this system is the inability to associate changes in calcium currents with the synaptic localization and/or function of the calcium channel. It is not immediately clear if this limitation can be solved. However, the authors do suggest that the calcium micro-domain may be relevant in their Discussion section, making this a relevant issue even at these small muscle cells.

We agree that this was an important limitation of our original submission. In response to this comment (and related comments in Reviews 2 and 3), we provide two new experiments (analysis of muscle gating currents and trafficking analysis of a chimeric membrane protein). Both of these new experiments support the conclusion that SHN-1 promotes the delivery of EGL-19/Cav1 channels to the plasma membrane.

These results greatly enhance the significance our findings and are described in the revised text as follows:

“SHN-1 effects on calcium current could result from a change in EGL-19 delivery to the cell surface. […] Collectively, these results suggest that the decreased calcium current in shn-1 null mutants arises from decreased delivery of EGL-19/Cav1 channels to the cell surface.”

Reviewer #2:

[…] Major concerns:

1) The authors demonstrate that SHN-1 regulates Calcium current density and claim that this is through the PDZ interaction. However, the authors have no insight to whether this is due to decreases in Cav1.3 levels or due open probability. Therefore the authors should provide some mechanistic insight how SHN-1 regulates ELG19 current density through this PDZ interaction. This is critical as it has been previously shown by Zhang et al., 2005 that the PDZ ligand in the mammalian Cav1.3 is dispensable for interacting with mammalian Shank.

We agree that this was a significant limitation in our original submission. Prompted by this concern (and related comments in Reviews 1 and 3), we add two new experiments to address the mechanism for Shank alteration of L-current. First, we record voltage-activated gating currents in muscles, finding that the decreased calcium current in shn-1 results from a decrease in the number of channels in the plasma membrane (Figure 3B). Second, we find that shn-1 null mutants have decreased trafficking of a chimeric reporter protein to the plasma membrane, again suggesting that SHN-1 promotes delivery of EGL- 19/Cav1 channels to the cell surface (Figure 3C-D). These data are now described in the revised text, as follows:

“SHN-1 effects on calcium current could result from a change in EGL-19 delivery to the cell surface. […] Collectively, these results suggest that the decreased calcium current in shn-1 null mutants arises from decreased delivery of EGL-19/Cav1 channels to the cell surface.”

2) There is no consistency between Figure 1 and Figure 2 in regards to wt Calcium current activation and densities. The maximum current in wild-type in Figure 1 is at 0 mV in Figure 2 is +10 mV while in Figure 2 while in Figures 1 and 2 the maximum Calcium currents for the mutant is 0 mV. So based on the data presented in the manuscript, it remains unknown if loss of SHN-1 or the EGL19 mutants impact Calcium current activation. In addition, in Figure 2 average Calcium density is two-fold higher than in Figure 1. Why the disparity? Furthermore, the holding voltages are different between the two. This can lead the reader to believe that hyperpolarizing the terminal to -90 mV relieves some sort of inhibition. The authors should redo Figure 2 with the same holding potential as Figure 1.

Recordings in these two figures were obtained with different protocols. Prompted by this comment, we repeated all recording using a single protocol (which yields larger current densities in all genotypes). All of the original findings were replicated with these new recordings. We apologize for the confusion caused by our use of multiple protocols in the original submission.

3) The authors look at copy number of SHN-1 but never give insight into the mechanisms of copy number regulation and it remains largely a phenomenological finding. Getting a mechanistic insight in how SHN-1 copy number leads to various changes that are observed in the manuscript is critical. In particular, how does one copy of SHN-1 or four copies of SHN-1 lead to the identical phenotype in Calcium current density? At minimum, the authors should correlate SHN-1 protein expression levels to copy number. What happens if you put in 3 copies, or 8 copies of SHN-1?

We agree that it would be very exciting to provide a mechanism to explain how calcium current and CREB target expression are similarly disrupted by decreased and increased Shank gene dosage. Even without a mechanistic explanation for shn-1 dosage effects, we believe that our paper represents a significant advance for the field. To our knowledge, these phenotypes are the only examples where the same cell autonomous defects have been linked to increased and decreased Shank copy number (which parallels the effects of Shank3 CNVs on risk for Autism and schizophrenia). Now that we have established specific cellular traits that exhibit sensitivity to shn-1 copy number, it is possible to design experiments asking how this dose sensitivity arises. However, I think it is fair to say that this is a difficult problem that will require many additional experiments. In fact, I am not aware of any good examples where the detailed biochemical mechanism for a haplo-insufficient or duplication phenotype has been determined. (Please let me know if you know of good examples!) Thus, while I agree that this is an important future goal, I hope that the reviewers will agree that describing a mechanism to explain shn-1 dosage sensitivity could be addressed in future publications.

Prompted by this comment, we made several changes in our revised paper. First, we now clearly list examples where cellular defects were identified in Shank3+/- heterozygotes and in transgenic animals containing an extra copy of Shank3:

Introduction:

“If Shank3 mutations and CNVs are causally associated with these psychiatric disorders, cellular and circuit phenotypes should also be sensitive to Shank3 copy number. […] While these studies identify cellular deficits associated with Shank3 CNVs, it remains unclear which Shank binding partners and cellular functions are responsible for psychiatric traits, nor why these traits are sensitive to both increased and decreased Shank gene dosage.”

Discussion:

“In humans, Shank deletions and duplications both confer risk for ASD and schizophrenia (Bonaglia et al., 2006; Durand et al., 2007; Failla et al., 2007; Gauthier et al., 2010; Han et al., 2013). […] In most cases, it was not determined if cellular defects were cell autonomous consequences of altered Shank3 dosage (Han et al., 2013; Orefice et al., 2016; Wang et al., 2016; Zhou et al., 2016).”

Second, we provide a model for how increased and decreased shn-1 copy number could result in the same phenotypes in the revised Discussion:

Discussion:

“How does shn-1 dose alter L-current and CREB target expression? In general, dosage sensitive phenotypes are thought to occur by disrupting the function of multimeric protein complexes. […] Our results further suggest that dynamic changes in the composition of Shank complexes provides a mechanism to regulate circuit function and plasticity (and potentially psychiatric traits).”

4) The authors demonstrate that loss of SHN-1 leads to an increase in cholinergic transmission. This is interesting since this there is no change in mEPSC amplitude or frequency. Some insight into how SHN-1 loss effects only evoked release should be done.

We agree that the increased evoked release in shn-1 mutants is interesting and merits further study; however, this evoked release defect does not exhibit sensitivity to shn-1 dosage. The NMJ transmission phenotype is included here to demonstrate that sensitivity to shn-1 dosage can be utilized to distinguish between different Shank phenotypes. Further experiments analyzing this recessive synaptic defect (although interesting) would not alter our conclusion that some phenotypes are dose sensitive while others are not. Consequently, we hope that the reviewers will agree that this phenotype can be further analyzed in a future study.

Does the loss of SHN-1 also impact GABAergic transmission in the same manner?

The mIPSC frequency was unaltered while mIPSC amplitudes were modestly decreased in shn-1 null mutants. As detailed in our response to comment #4, we prefer to focus on phenotypes sensitive to shn-1 copy number in this paper. For this reason, we respectfully request that more detailed studies of NMJ defects in shn-1 mutants could be addressed in future studies.

5) The mutant ok1241 is not an acceptable mutant to just look at PDZ function. This mutant deletes much of the Proline domain in SHN-1.

We agree that an allele that selectively deletes the PDZ domain would be preferable and are attempting to isolate such an allele with Cas9 (no luck thus far). Although the ok1241 allele deletes part of the proline rich domain, we do not believe that this undermines any of our conclusions. Voltage-clamp recordings of body muscles in ok1241 exhibit a similar decrease in calcium current density to those observed in shn-1 null mutants and in egl-19(nu496) mutants (which deletes the c-terminal ligand for the SHN-1 PDZ domain). And shn-1(null) mutations and the egl-19(nu496) PDZ ligand mutation did not have additive effects on calcium current in double mutants, implying that SHN-1 alters calcium current via its effects on EGL-19’s c-terminus. Collectively, these results strongly support our conclusion that binding of SHN-1’s PDZ domain to EGL-19’s c-terminal PDZ ligand promotes expression of EGL-19 current. By contrast gem-4 activation was not decreased after deleting EGL-19’s c-terminal PDZ ligand (i.e. nu496) nor after deleting SHN-1’s PDZ domain and part of its Pro-rich domain (i.e. ok1241). Thus, the CREB activation function of SHN-1 clearly does not require either the SHN-1 PDZ domain or its ligand (EGL-19’s c- terminus). Does the reviewer suggest that disrupting the proline-rich domain could somehow obscure an effect of the PDZ domain on gem-4 induction?

We hope that the revised text explains these results and our interpretations more clearly:

“SHN-1 effects on calcium current density could result from direct binding of SHN-1 to EGL-19/Cav1 or indirectly via other SHN-1 binding partners. […] Collectively, these results suggest that SHN-1 binding to EGL-19’s carboxy-terminus promotes the expression or function of L-type calcium channels.”

6) Based on the IV recordings, it appears the SHN-1 copy number and EGL19 PDZ mutants impact EGL19 inactivation. The authors should go back it look at the Calcium current deactivation kinetics and see if there is a difference.

As requested, we analyzed deactivation kinetics and found no difference, as shown in: Figures 1F; Figure 2—figure supplement 1F; and Figure 6C.

Reviewer #3:

[…] Finally, they determine that both Egl19 dependent currents as well as gem-4 expression are sensitive to Shn-1 gene dose, something that is know from human patients.

I am not aware of any publication showing that Shank gene dosage alters either CaV1 current or CREB activated gene expression (in any organism including humans). If the reviewer is aware of such studies, please let us know. Identifying these as cell autonomous phenotypes sensitive to Shank gene dosage is the central and most significant finding of our study.

While the findings are of interest, especially due to the importance of Shank-3 in autism, I am not sure this paper represents a major step forward in our understanding.

We apologize if our original text did not clearly state the significance of our findings. Our principal claims for significance are as follows:

1) Shank gene copy number had not been previously linked to either CaV1 current or expression of CREB target genes (both of which are thought to be linked to Autism and other psychiatric disorders).

2) No prior studies identified cellular phenotypes that exhibit the unusual pattern of sensitivity to Shank copy number whereby too little and too much Shank produce similar phenotypes (which mirrors the effects of Shank copy number on risk for Autism and schizophrenia).

3) Most prior studies suggest that Shank’s principal role is to regulate synapse formation or function (whereas our data suggest that non-synaptic functions could be equally important).

4) Our study represents a significant advance in identifying the cellular consequences of increased Shank copy number, which was analyzed in only a single prior study (the transgenic Shank3 mouse in Han et al., 2013).

5) Altered CaV1 current and CREB activation are cell autonomous defects associated with Shank copy number changes. By contrast, cell autonomy was not demonstrated in several prior studies of dose sensitive Shank phenotypes (Han, Nature 2013; Wang, Nat. Comm. 2016; Zhou, Neuron 2016; Orefice, Cell 2016).

6) Shank is linked to CaV1 current and CREB activation by direct biochemical interactions (unlike many other Shank associated phenotypes).

7) Linking Shank copy number to changes in CREB activation could provide an explanation for why Shank3 mutations are disproportionately found in Autism associated with intellectual disability.

Prompted by this comment, we revised our paper to highlight the significance of our findings:

Discussion):

“Consistent with this idea, several studies previously reported cellular and behavioral defects in Shank3m/+ heterozygotes (Han et al., 2016; Orefice et al., 2016; Wang et al., 2016; Yi et al., 2016). […] Thus, we identify two cellular phenotypes that exhibit the same pattern of dose sensitivity that is observed for Shank3 in schizophrenia and ASD.”

The Egl-19 and gem-4 parts don't fit together and the story remains unfocused.

For several reasons, we argue that Shank effects on CaV1 current and CREB activated gene expression should be described in a single paper. First, the primary source of calcium activating CREB is mediated by activated CaV1 channels; consequently, it makes sense to analyze both phenotypes in a single paper. Second, changes in CaV1 channels and activity-induced gene expression have both been linked to Autism (and other psychiatric disorders), but neither has been directly linked to Shank copy number. Third, both phenotypes exhibit the same unusual sensitivity to Shank copy number that is observed for risk of Autism and Schizophrenia (i.e. too much and too little Shank cause the same defect). Fourth, no other phenotypes have been described that exhibit this same pattern of sensitivity to Shank copy number. And fifth, our analysis suggests that these two Shank functions can be dissociated by certain mutations, implying that they are mediated by distinct biochemical mechanisms. This contrast would not be as evident if the stories were separated.

Prompted by this comment, we revised our text to explain why CREB activation is linked to changes in L- channels function:

“Increased cytoplasmic calcium activates expression of a large number genes, hereafter designated activity-induced gene expression. Although Cav1 channels account for a small fraction of bulk calcium entry in neurons, Cav1 channels account for the majority of activity- induced gene expression (Ma et al., 2013). This privileged ability of Cav1 channels to activate gene expression is thought to be mediated by direct physical coupling of Cav1 channels to the calcium sensors responsible for activating CREB (Deisseroth et al., 1996; Wheeler et al., 2008).”

Specific comments:

1) Yeast two hybrid and GST pull down assays can, at best, indicate that two proteins CAN bind to each other (in these very artificial contexts) and definitely do not prove interaction in vivo. I would use more careful words. Probably beyond the scope of this paper but to show that they interact in vivo, even in the non-biochemical organisms such as C. elegans, one can perform experiments with tagged transgenic proteins and test their proximity in a wide range of strategies… Also, the experiments shown in Sup 1A+B are quite poorly controlled. So Egl19 does not bind the beads but does it bind other PDZ domains? In the well-referenced paper (Zhang et al., 2005) they performed many required controls to be able to suggest that the mammalian orthologs interact.

We agree that the Y2H and pull down assays show that a direct interaction is plausible and that direct binding studies would be a stronger argument. Binding (i.e. coIP) assays from worm extracts are possible (and we have performed such experiments in several prior papers). However, biochemical analysis of CaV1 channels is difficult in any prep. In fact, no prior study has attempted to comprehensively identify proteins associated with CaV1 channels. CaV1 channels are huge, poorly expressed proteins that are difficult to solubilize. We have attempted to detect tagged EGL-19 channels in worm extracts by IP’s and western blots with no success.

Given the difficulty associated with biochemical analysis of CaV1 channels, we instead relied on genetic results to support our conclusion that SHN-1 binds to and regulates EGL-19 channels in vivo. Our results are as follows: 1) very similar calcium current defects are observed in shn-1(null) mutants, shn-1(ok1241) mutants which lack the PDZ domain, and egl-19(nu496) mutants which lack the c-terminal ligand for SHN-1’s PDZ domain; 2) the calcium current defects observed in shn-1(null) and in egl-19(nu496) mutants are both cell autonomous (i.e. both proteins must be present in muscle cells to obtain normal calcium currents), consistent with these proteins altering calcium current by directly binding to each other; 3) shn-1(null) mutations and egl-19(nu496) mutations do not have additive effects on calcium current in double mutants, implying that SHN-1 alters calcium current in a manner that requires EGL-19’s c-terminal ligand for SHN-1’s PDZ domain; and 4) shn-1 null mutations cause similar decreases in EGL- 19 calcium current and in the surface abundance of a chimeric membrane protein containing EGL-19’s cytoplasmic tail sequence (Terrier), indicating that EGL-19’s cytoplasmic tail domain is sufficient to confer dependence on SHN-1 for membrane trafficking. We hope that the reviewer will agree that the strength of these genetic results offsets the absence of more direct biochemical analysis of SHN-1 binding to EGL-19.

2) I was surprised that the authors did not attempt to describe the sub cellular localization of Egl-19 in WT and mutant worms… The best experiment would be to endogenously tag Egl-19 but even the second best – to follow the localization of transgenic Egl-19 was not performed. The PDZ binding motif has to remain on the C-terminal so one can N-terminally tag or tag C-terminally with the addition of the PDZ binding motif afterwards. This lies at the heart of how Shn-1 affects Egl-19 and is therefore a key question that remains unanswered in this work.

We agree that this was an important limitation of our original submission. In response to this comment (and related comments in Review 1), we provide two new experiments (analysis of muscle gating currents and trafficking analysis of a chimeric membrane protein). Both of these new experiments support the conclusion that SHN-1 promotes the delivery of EGL-19/Cav1 channels to the plasma membrane. These results greatly enhance the significance our findings and are described in the revised text as follows:

“SHN-1 effects on calcium current could result from a change in EGL-19 delivery to the cell surface. We performed two further experiments to test this idea. […] Collectively, these results suggest that the decreased calcium current in shn-1 null mutants arises from decreased delivery of EGL-19/Cav1 channels to the cell surface.”

3) The activity dependent transcription experiment – I was surprised that the raw data are not shown. Is gem-4 the top hit but one out of many or is it the only one? Ideally one would like to view this.

As requested, we now provide a summary of the genome wide expression profile associated with Lev- induced muscle depolarization. The complete set of activity induced genes is listed in a supplemental excel file, and is summarized in the revised text as follows:

Results:

“We analyzed gene expression following depolarization of body muscles with a nicotinic acetylcholine (ACh) agonist (levamisole, Lev). […] These results suggest that C. elegans body muscles (like other excitable cells) have a large number of activity-induced genes, many of which are potential CREB transcriptional targets.”

4) Figure 3—figure supplement 2 does not exist (or at least I could not find it).

Thanks for pointing out this error. We deleted the citation of this figure in the text.

[Editors’ note: the author responses to the re-review follow.]

[…] A remaining issue is one of clarifying new Figure 3. The figure could be clarified to make it easier to understand the region of interest that is being visualized in panel D. Although there is a cartoon, the transition from the cartoon to the images in panel D could be improved.

As request by the Reviewing Editor, we have modified the schematic in Figure 3D, to illustrate the location of the imaged region for our Terrier reporter construct.

https://doi.org/10.7554/eLife.18931.016

Article and author information

Author details

  1. Edward Pym

    1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    2. Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    EP, Conceptualization, Investigation, Writing—original draft, Writing—review and editing
    Competing interests
    The authors declare that no competing interests exist.
  2. Nikhil Sasidharan

    1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    2. Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    NS, Investigation, Methodology
    Competing interests
    The authors declare that no competing interests exist.
  3. Katherine L Thompson-Peer

    1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    2. Department of Neurobiology, Harvard Medical School, Boston, United States
    Present address
    Department of Physiology, University of California, San Francisco, San Francisco, United States
    Contribution
    KLT-P, Investigation
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4200-3870
  4. David J Simon

    1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    2. Department of Neurobiology, Harvard Medical School, Boston, United States
    3. Program in Neuroscience, Harvard Medical School, Boston, United States
    Present address
    Department of Neurobiology, Stanford School of Medicine, Palo Alto, United States
    Contribution
    DJS, Investigation, Methodology
    Competing interests
    The authors declare that no competing interests exist.
  5. Anthony Anselmo

    Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    Contribution
    AA, Formal analysis, microarray data analysis
    Competing interests
    The authors declare that no competing interests exist.
  6. Ruslan Sadreyev

    Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    Contribution
    RS, Formal analysis, microarray data analysis
    Competing interests
    The authors declare that no competing interests exist.
  7. Qi Hall

    1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    2. Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    QH, Investigation, Methodology
    Competing interests
    The authors declare that no competing interests exist.
  8. Stephen Nurrish

    1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    2. Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    SN, Resources, Investigation
    Competing interests
    The authors declare that no competing interests exist.
  9. Joshua M Kaplan

    1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    2. Department of Neurobiology, Harvard Medical School, Boston, United States
    3. Program in Neuroscience, Harvard Medical School, Boston, United States
    Contribution
    JMK, Conceptualization, Supervision, Writing—original draft, Writing—review and editing
    For correspondence
    kaplan@molbio.mgh.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7418-7179

Funding

National Institute of Neurological Disorders and Stroke (NS32196)

  • Joshua M Kaplan

Simons Foundation (SF273555)

  • Joshua M Kaplan

Nancy Lurie Marks Family Foundation

  • Edward Pym

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank the following for strains, advice, reagents, and comments on the manuscript: C. elegans stock center, S Mitani, and members of the Kaplan lab. This work was supported by a postdoctoral fellowship from the Nancy Lurie Marks Family Foundation (EP), and by research grants to JK from the NIH (NS32196) and from the Simons Foundation for Autism Research (SF273555). Additional data described in the manuscript are presented in the supporting online material.

Reviewing Editor

  1. Graeme W Davis, University of California, San Francisco, United States

Publication history

  1. Received: June 19, 2016
  2. Accepted: April 18, 2017
  3. Accepted Manuscript published: May 6, 2017 (version 1)
  4. Version of Record published: May 15, 2017 (version 2)

Copyright

© 2017, Pym et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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