Homeostatic plasticity fails at the intersection of autism-gene mutations and a novel class of common genetic modifiers
- Department of Biochemistry and Biophysics Kavli Institute for Fundamental Neuroscience University of California, San Francisco, United States
- Department of Psychiatry UCSF Weill Institute for Neurosciences University of California, San Francisco, United States
- School of Biosystem and Biomedical Science, College of Health Science, Korea University, Republic of Korea
Figures
Figure 1 with 2 supplements
Heterozygous ASD gene mutations do not affect baseline transmission or PHP.
(A) Schematic of the Drosophila locus for CHD8, ASH1L, CHD2, WDFY3 and RIMS1 with gene disruptions indicated. (B) Representative EPSP and mEPSP traces for indicated genotypes (+ / - PhTx for each …
Figure 1—figure supplement 1
Patch-Seq analysis of gene expression in type 1b and type 1 s motoneurons.
(A) Image of the larval central nervous system with expression of UAS-CD8-GFP driven by MN1-GAL4. Inset, a rhodamine filled patch electrode targets a single identified motoneuron for excision and …
Figure 1—figure supplement 2
Double-heterozygous gene mutation combinations impair homeostatic plasticity.
(A–D) Scatter plots of quantal content (y axis) versus mEPSP amplitude (x axis) for A) wild type; B) ASH1L/+, RIMS1/+ double heterozygous mutant (red) and ASH1L/+ heterozygous mutant (grey); C) CHD2/…
Figure 2
Screen for common genomic modifiers of ASD-associated gene mutations.
(A) Diagram of genetic screen. (B) Screen results are shown with yellow circles representing average data per genotype. Fit (solid blue line) and confidence interval (dotted lines encompassing 95% …
Figure 3
Absence of an additive effect of gene heterozygosity on synaptic transmission or PHP.
(A) Scatter plot showing the number of genes deleted (y axis) versus quantal content (x axis) in the presence of PhTx for all deficiencies tested. Each circle represents average data from an …
Figure 4 with 1 supplement
Identification of common modifiers of diverse ASD-associated mutations.
(A) Genetic interaction matrix showing average mEPSP (top two matrix) and EPSP (bottom two matrix) amplitudes in the absence (left) and presence (right) of PhTx, as indicated. Values are according …
Figure 4—figure supplement 1
One-way ANOVA with Dunnett’s multiple comparisons test (compared to w1118).
Genetic interaction matrix showing color-coded p-values from One-way ANOVA with Dunnett’s multiple comparisons test. Each individual box represents p-values for the comparison of percent change in …
Figure 5 with 3 supplements
Single genes are common modifiers of diverse ASD-associated mutations.
(A) Schematic of the PPP2R5D gene locus and the PPP2R5D104 deletion mutation (red horizontal bar). (B) Representative traces for indicated genotypes. Bar graph (right) shows percent change in mEPSP …
Figure 5—figure supplement 1
Analysis of the NMJ morphology.
(A) Structured illumination microscopy (SIM) images of neuromuscular junction for indicated genotypes. Insets show single confocal sections. Staining for anti-Brp (green) to mark active zones and …
Figure 5—figure supplement 2
A PPP2R5D loss-of-function mutation disrupts PHP, but PDPK1 does not.
(A) Representative mEPSP and EPSP traces for PPP2R5D104 homozygous mutant (-/+ PhTx) (B) Percent change in mEPSP amplitude (gray bars) and quantal content (red bars) with PhTx compared to baseline …
Figure 5—figure supplement 3
Firing properties of motoneurons are not different in a double heterozygous mutant.
(A) Representative traces for motoneuron firing upon injection of 200 pA step current across indicated genotypes. (B), Action potential frequency is plotted against current injection amplitude for …
Figure 6 with 1 supplement
ASD gene-modifier interaction causes impaired synaptic membrane organization.
(A–C) Representative electron microscopy images of individual boutons inclusive of (A) PPP2R5D/+, (B) CHD8/+ and (C) CHD8/+; PPP2R5D/+ double heterozygous mutant. Insets (C) show individual active …
Figure 6—figure supplement 1
Analysis of short-term depression in CHD8/+; PPP2R5D/+ double heterozygote.
(A) Representative traces for EPSCs following 50 Hz stimulation (40 stimuli, 1st four shown for purposes of display) from wild type and the CHD8/+;PPP2R5D/+ double heterozygous mutant. The first …
Figure 7
Differential gene expression analysis identifies CREG.
(A) Volcano plot display of differentially expressed genes (DEX) for each heterozygous mutant versus wild type. Candidate ASD-gene modifiers are indicated (orange dots). Horizontal dashed line …
Figure 8 with 3 supplements
CREG is a homeostatic repressor that blocks PHP and regulates synapse ultrastructure.
(A) Quantification of transcriptional changes calculated by RNAseq for four genes (CREG, Ect3, PEPCK2 and Cyp6a23) in CHD8/+; PPP2R5D/+ double heterozygous mutant versus wild-type. (B) …
Figure 8—figure supplement 1
Expression levels of CREG during Drosophila larval development.
(A) CREG expression levels measured in larval CNS by microarray (log2) across different developmental time points with three different probes (see from which data are derived). (B) Quantification of …
Figure 8—figure supplement 2
CREG overexpression does not substantively alter NMJ anatomy.
(A) Immunostaining of wild type (left) and Tub-Gal4 >UAS Creg (right; CregOE) larval NMJ for Brp, DLG and HRP as indicated. (B) Cumulative distribution plot of BRP area for wild type (black) and Creg…
Figure 8—figure supplement 3
Ultrastructure analysis of the CHD2/+; PPP2R5D/+ double heterozygous mutant.
(A) Representative example of the CHD2/+ single heterozygous mutant NMJ. (B), Two representative examples of the double heterozygous mutant. The membranes of the synaptic cleft are clearly defined …
Figure 9
Summary and Model.
(A) Summary of genetic interactions. RIMS1 interacts with three of four ASD gene orthologues, impairing PHP. RIMS1 interactions identified in a genetic screen as modifiers are shown below in green. …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | w1118 | Bloomington Drosophila Stock Center | BDSC:3605 | |
| Genetic reagent (D. melanogaster) | rim103 | PMID:23175813 | ||
| Genetic reagent (D. melanogaster) | kis1 | Bloomington Drosophila Stock Center | BDSC:431 | |
| Genetic reagent (D. melanogaster) | chd1[1] and chd1 [1], chd1[wt] | PMID:21177652 | ||
| Genetic reagent (D. melanogaster) | ash1-mimic | Bloomington Drosophila Stock Center | BDSC:23524 | |
| Genetic reagent (D. melanogaster) | bchs58 | Bloomington Drosophila Stock Center | BDSC:9887 | |
| Genetic reagent (D. melanogaster) | UAS-CREG | this paper | Fly carrying UAS-Creg transgene | |
| Genetic reagent (D. melanogaster) | wrd104 | PMID:16957085 | ||
| Genetic reagent (D. melanogaster) | pdk1 | PMID:21930778 | ||
| Genetic reagent (D. melanogaster) | ok371-gal4 | PMID:16378756 | ||
| Genetic reagent (D. melanogaster) | tubulin-gal4 | PMID:21930778 | ||
| Genetic reagent (D. melanogaster) | 3rd chromosome deficiency collection | Bloomington Drosophila Stock Center | ||
| Genetic reagent (D. melanogaster) | Creg-m1 | Bloomington Drosophila Stock Center | BDSC:42140 | |
| Genetic reagent (D. melanogaster) | Creg-m2 | Bloomington Drosophila Stock Center | BDSC:22800 | |
| Genetic reagent (D. melanogaster) | MN1b-gal4 | Bloomington Drosophila Stock Center | BDSC:40701 | |
| Genetic reagent (D. melanogaster) | MN1s-gal4 | Bloomington Drosophila Stock Center | BDSC:49227 | |
| Genetic reagent (D. melanogaster) | uas-cd8:gfp | PMID:10197526 | ||
| Chemical compound, drug | Philanthotoxin-433 | Santa Cruz Biotechnology | GH28782 | |
| Antibody | Anti-brp (Mouse monoclonal) | Developmental Studies Hybridoma Bank | RRID:AB_2314866 | IF (1:100) |
| Antibody | Anti-dlg (Rabbit monoclonal) | PMID:29303480 | IF (1:1000) | |
| Antibody | Cy3 anti-rabbit | Jackson Immuno-research Laboratories | RRID:AB_2338000 | IF (1:500) |
| Antibody | Alexa488 anti-mouse | Jackson Immuno-research Laboratories | RRID:AB_2338840 | IF (1:500) |
| Software, algorithm | Igor Pro 8.03 | Wavemetrics | RRID:SCR_000325 | |
| Software, algorithm | Graphpad PRISM 7.04 | Graphpad | RRID:SCR_002798 | |
| Software, algorithm | Adobe Illustrator CC 2018 | Adobe | RRID:SCR_010279 | |
| Software, algorithm | MiniAnalysis 6.0.7 | SynaptoSoft | RRID:SCR_002184 | |
| Software, algorithm | SlideBook 6 | Intelligent Imaging | RRID:SCR_014300 | |
| Sequence-based reagent | CREG primer | Applied Biosystems | Dm02135967_g1 | |
| Sequence-based reagent | Ect3 primer | Applied Biosystems | Dm02139373_g1 | |
| Sequence-based reagent | Pepck2 | Applied Biosystems | Dm02366462_s1 | |
| Sequence-based reagent | Cyp6a23 | Applied Biosystems | Dm01824231_g1 | |
| Sequence-based reagent | rpl32 | Applied Biosystems | Dm02151827_g1 | |
| Commercial assay or kit | RNeasy Plus Mini kit | QIAGEN | ID:74134 | |
| Commercial assay or kit | SuperScript III First-Strand synthesis system | Invitrogen | Cat# 18080051 | |
| Commercial assay or kit | TURBO DNA-free kit | ThermoFisher | Cat# AM1907 | |
| Commercial assay or kit | TaqMan Fast Universal PCR Master Mix | Applied Biosystem | Cat# 4352042 | |
| Commercial assay or kit | Lexogen’s Split RNA Extraction Kit | Lexogen | Cat# 008 | |
| Commercial assay or kit | 3’mRNA-Seq Library Prep Kit | Lexogen | Cat# 015 | |
| Commercial assay or kit | Single Cell/Low Input RNA Library Prep Kit | New England Biolabs | Cat# E6420S | |
| Recombinant DNA reagent | pTW (Gateway vector) | DGRC | Cat# 1129 | |
| Recombinant DNA reagent | pENTR-dTOPO Cloning Kit | Invitrogen | Cat# K240020 | |
| Recombinant DNA reagent | Creg cDNA | Drosophila Genomics Resource Center | GH28782 |
Additional files
-
Supplementary file 1
Supplementary Tables 1-7 are presented.
Each table is referred to independently in the text.
- https://cdn.elifesciences.org/articles/55775/elife-55775-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/55775/elife-55775-transrepform-v2.docx
