1. Cell Biology
  2. Genetics and Genomics
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A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency

  1. Dana M Talsness
  2. Katie G Owings
  3. Emily Coelho
  4. Gaelle Mercenne
  5. John M Pleinis
  6. Raghavendran Partha
  7. Kevin A Hope
  8. Aamir R Zuberi
  9. Nathan L Clark
  10. Cathleen M Lutz
  11. Aylin R Rodan
  12. Clement Y Chow  Is a corresponding author
  1. Department of Human Genetics, University of Utah School of Medicine, United States
  2. Department of Internal Medicine, Division of Nephrology and Hypertension, and Molecular Medicine Program, University of Utah, United States
  3. Department of Computational and Systems Biology, University of Pittsburgh, United States
  4. Genetic Resource Science, The Jackson Laboratory, United States
  5. Medical Service, Veterans Affairs Salt Lake City Health Care System, United States
Research Article
Cite this article as: eLife 2020;9:e57831 doi: 10.7554/eLife.57831
6 figures, 2 tables and 6 additional files

Figures

Figure 1 with 1 supplement
Lethality phenotype of NGLY1 knockdown is highly modifiable by strain background.

(A) Drosophila cross for NGLY1 knockdown in each Drosophila genetic reference panel (DGRP) strain. (B) Proportion of NGLY1 knockdown flies surviving for each cross was calculated based on the number eclosing compared to the expected number. Expected number was based on the largest control balancer class for each cross.

Figure 1—figure supplement 1
Ncc69 expression is not correlated with survival.

Survival upon knockdown of NGLY1 was plotted versus baseline expression levels of Ncc69 in the DGRP (r = −0.022, N = 154, p=0.78). DGRP gene expression data was taken from Huang et al., 2014.

Figure 2 with 1 supplement
Gene set enrichment analysis.

Top significant ontological categories identified by GSEA. p-values are indicated by red-to-blue gradient, with red the lowest p-values and blue the highest p-values. Gene number identified in each category is indicated by the size of the circle.

Figure 2—figure supplement 1
NGLY1 knockdown causes circadian rhythm defect.

Activity monitor was used to analyze 2- to 5-day-old flies for 1 week in complete darkness after 3 days of entrainment in a 12 hr light, dark cycle. At least 15 flies were analyzed for each genotype. Period length was calculated from activity using ClockLab. One-way ANOVA gave an overall p<0.0001. Subsequently Tukey’s test was used to calculate individual adjusted p-values between genotypes shown on the graph.

NGLY1 and Ncc69 interact genetically in Drosophila.

(A) Proportion of flies surviving to eclosion in ubiquitous knockdowns. NGLY1 knockdown (KD) are UAS-PnglRNAi/+; Tubulin-GAL4/+. Ncc69 KD are UAS-Ncc69RNAi/+; Tubulin-GAL4/+. NGLY1 Ncc69 double knockdown (DKD) are UAS-PnglRNAi/+ UAS-Ncc69RNAi/Tubulin-GAL4/+. Four separate matings were performed for each cross with at least 40 offspring generated for the balancer control for each. Fraction surviving is calculated compared to balancer offspring. Chi-square analysis was performed for the total number of flies compared to expected Mendelian numbers. NGLY1 KD χ2 = 109.7, p<0.0001; Ncc69 KD χ2 = 1.002, p=0.3168, and NGLY1 Ncc69 DKD χ2 = 186, p<0.0001. (B) Bang sensitivity assay to assess seizures in glial knockdown flies. WT flies are attP2 and attP40. NGLY1 KD are UAS-PnglRNAi/+; repo-GAL4/+. Ncc69 KD are UAS-Ncc69RNAi/+; repo-GAL4/+. NGLY1 Ncc69 DKD are UAS-PnglRNAi/+; UAS-Ncc69RNAi/ repo-GAL4. For each genotype, at least 45 4- to 7-day-old females were used to calculate the percent seizing at a given time after vortexing. Repeated measures ANOVA p-value=0.000176.

Figure 4 with 1 supplement
Endogenous NKCC1 is altered in NGLY1-deficient MEFs.

(A) Control (+/+) and NGLY1 null (-/-) MEFs were grown to confluency and then lysed to isolate the membrane and cytoplasmic fractions. Three separate membrane lysates for both genotypes were analyzed by immunoblotting for NKCC1 compared to a molecular weight marker (MWM). Blot was used for molecular weight calculations of the upper-most limit (see un-cropped blot in Figure 4—figure supplement 1) (B) And the lower-most limit (C) of the protein band. Red bar represents the mean. Two-tailed t-test was used to calculate p-values. (D) Membrane lysates from MEFs were treated with N-Glycosidase F (PNGase F), O-Glycosidase (O-Gly), or Endoglycosidase H (Endo H) for 1 hr then analyzed by immunoblot. Control (C)Samples were treated in all the same conditions but without the added enzyme. (E) MEFs were treated with 500 nM bortezomib (Bz) or equal volume of vehicle control (DMSO) for 4 hr then lysed to collect membrane and cytoplasmic fractions. Lysates were analyzed by immunoblotting for NKCC1. NRF1 was analyzed as a positive control of proteasome inhibition. Dark band at about 140 kDa in both (A), (D), and (E) is believed to be non-specific.

Figure 4—figure supplement 1
Full western blot of NKCC1 in NGLY1 +/+ and -/- MEFs.

Tris-acetate gel was run at 150V for 2.5 hr to sufficiently distinguish between the different molecular weights of NKCC1, and therefore the 75 kDa band of the protein marker was the bottom-most band. The 250, 150, 100, and 75 kDa molecular weights were used for a standard curve in the Li-cor software Image Studio, so that the molecular weight of the NKCC1 bands could be calculated.

Figure 5 with 1 supplement
NGLY1 -/- MEFs show decreased NKCC1-specific ion flux.

(A) Bumetanide-sensitive 86Rb flux was measured in NGLY1 +/+ and NGLY1 -/- MEFs to measure NKCC1 activity. Flux was examined in three bath conditions, isotonic (iso), hypotonic (hypo), and hypertonic (hyper). There was a significant effect of genotype (p<0.0001) in two-way ANOVA, with no significant effect of condition (p=0.5756) or interaction (p=0.8075). Adjusted p-values for Sidak’s multiple comparisons test between NGLY1 +/+ and NGLY1 -/- are shown in the figure. (B) Ouabain-sensitive 86Rb flux was measured in NGLY1 +/+ and NGLY1 -/- MEFs to measure Na+/K+-ATPase activity in the same three conditions as in A. There were no significant effects of genotype (p=0.0516), condition (p=0.3047) or interaction (p=0.4711) by two-way ANOVA, indicating the NGLY1 knockout has a specific effect on NKCC1 activity without affecting Na+/K+-ATPase activity.

Figure 5—figure supplement 1
86Rb uptake in MEFs occurs through bumetanide-sensitive and ouabain-sensitive pathways.

Either NGLY1 +/+ (A) or NGLY1 -/- (B) MEFs were incubated with DMSO as a vehicle control or with the NKCC1 inhibitor, bumetanide, or the Na+/K+-ATPase inhibitor, ouabain. 86Rb flux was measured in three conditions: isotonic (iso), hypotonic (hypo), and hypertonic (hyper). The sum of bumetanide-sensitive and ouabain-sensitive flux was compared to vehicle control. There were no significant effects of genotype (p=0.3267), condition (p=0.3602) or an interaction (p=0.6244) in NGLY1 +/+ cells, nor of genotype (p=0.9422), condition (p=0.4987) or an interaction (p=0.9909) in NGLY1 -/- cells by two-way ANOVA, indicating that in both cell types 86Rb flux was comprised of the bumetanide-sensitive and ouabain-sensitive activities.

Author response image 1

Tables

Table 1
Candidate modifier genes identified from GWA.

Rank order of candidate genes was established based on the most significant associated SNP in the respective gene.

Rank orderGeneFBgnHuman orthologPeriphery/membraneProteostasis
1expFBgn0033668---nono
2Ncc69FBgn0036279NKCC1/2yesno
3CG5888FBgn0028523---yesno
4CG16898FBgn0034480---nono
5bru3FBgn0264001CELF2/3/4/5/6nono
6CG31690FBgn0051690TMTC2noyes
7CG7227FBgn0031970SCARB1nono
8CR44997FBgn0266348---nono
9rgnFBgn0261258Manynono
10M6FBgn0037092GPM6Ayesno
11Rab26FBgn0086913RAB26yesyes
12Obp56iFBgn0043532---nono
135-HT1AFBgn0004168HTR1Ayesno
14CG33012FBgn0053012ERMP1noyes
15rstFBgn0003285---yesno
16CR43926FBgn0264547---nono
17CG7337FBgn0031374WDR62nono
18hiwFBgn0030600MYCBP2yesyes
19fidFBgn0259146TRMT9Bnono
20nmoFBgn0011817NLKnono
21SirupFBgn0031971SDHAF4nono
22tstFBgn0039117SKIV2Lnono
23Mdr50FBgn0010241manyyesno
24Cpr49AaFBgn0050045---nono
25COX7CFBgn0040773COX7Cnono
26Eip63EFBgn0005640CDK14/15yesno
27CG30048FBgn0050048PKD1nono
28CG15040FBgn0030940---nono
29SP2353FBgn0034070EGFLAMnono
30MfFBgn0038294---nono
31omeFBgn0259175manynono
32esnFBgn0263934PRICKLE1-3nono
33hafFBgn0261509manynono
34dallyFBgn0263930GPC3/5yesno
35robo2FBgn0002543ROBO1/2/3/4nono
36Gyc32EFBgn0010197NPR1/2yesno
37CG8170FBgn0033365manynono
38CG8405FBgn0034071TMEM259noyes
39scafFBgn0033033---yesno
40borrFBgn0032105CDCA8yesno
41Syx7FBgn0267849STX7/12yesno
42DIP-deltaFBgn0085420manyyesno
43cv-cFBgn0285955DLC1yesno
44Snmp2FBgn0035815CD36/SCARB1nono
45MerFBgn0086384NF2yesno
46sbaFBgn0016754---nono
47HsromegaFBgn0001234---noyes
48CCAP-RFBgn0039396NPSR1yesno
49Hrd3FBgn0028475SEL1Lnoyes
50blueFBgn0283709NEURL4noyes
51CG6262FBgn0034121TREHnono
52CG45186FBgn0266696SVILnono
53SpnFBgn0010905PPP1R9Ayesno
54dncFBgn0000479PDE4A/B/C/Dnono
55CG4374FBgn0039078manynono
56sffFBgn0036544manynoyes
57CG42383FBgn0259729NSFL1Cnoyes
58DybFBgn0033739DTNByesno
59CG34371FBgn0085400---nono
60CG4341FBgn0028481TMTC2noyes
61CG30043FBgn0050043ERMP1noyes
Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Drosophila melanogaster)PnglGenBankID:35527
Dmel_CG7865
Gene (Drosophila melanogaster)Ncc69GenBankID: 39410
Dmel_CG4357
Gene (Mus musculus)NGLY1GenBankID: 59007
Gene (Mus musculus)NKCC1GenBankID: 20496Slc12a2
Genetic reagent (Drosophila melanogaster)Pngl-RNAiBloomington Drosophila Stock CenterRRID:BDSC_54853y1 v1; P{y+t7.7 v+t1.8=TRiP.HMJ21590}attP40
Genetic reagent (Drosophila melanogaster)Tubulin-GAL4Bloomington Drosophila Stock CenterRRID:BDSC_5138y1 w*;
P{w+mC = tubP-GAL4}LL7/TM3, Sb1 Ser1
Genetic reagent (Drosophila melanogaster)Tubulin-GAL80Bloomington Drosophila Stock CenterRRID:BDSC_5190y1 w[*]; P{w[+mC]=tubP-GAL80}LL9 P{w[+mW.hs]=FRT(w[hs])}2A/TM3, Sb1
Genetic reagent (Drosophila melanogaster)Drosophila Genetics Reference PanelBloomington Drosophila Stock CenterSet of 194 strains, example strain: BDSC:55014, RRID:BDSC_55014
Genetic reagent (Drosophila melanogaster)Pdf-GAL4Bloomington Drosophila Stock CenterRRID:BDSC_6899P{w[+mC]=Pdf-GAL4.P2.4}X, y1 w[*]
Genetic reagent (Drosophila melanogaster)UAS-Pngl-RNAiBloomington Drosophila Stock CenterRRID:BDSC_42592y1 sc* v1 sev21; P{y+t7.7 v+t1.8=TRiP.HMS02424}attP40
Genetic reagent (Drosophila melanogaster)UAS-Ncc69-RNAiBloomington Drosophila Stock CenterRRID:BDSC_28682y1 v1; P{y+t7.7 v+t1.8=TRiP.JF03097}attP2
Cell line (Mus musculus)Mouse embryonic fibroblasts (MEF)Jackson LabsPrimary line from mouse #027060,https://www.jax.org/strain/027060
AntibodyAnti-NKCC1 (Rabbit polyclonal)Cell SignalingCat#14581, RRID:AB_2798524IB: 1:1000
AntibodyAnti-TCF11/NRF1 (Rabbit monoclonal)Cell SignalingCat#8052, RRID:AB_11178947IB: 1:1000
AntibodyIRDye 800CW Goat-anti-rabbitAbcamCat#216773IB: 1:10,000
Commercial assay or kitCell Fractionation KitCell SignalingCat#9038
Chemical compound, drugBortezomibEMD MilliporeCat# 179324-69-7
Software, algorithmGenome Wide AssociationChow et al., 2016
Software, algorithmGene Set Enrichment AnalysisSubramanian et al., 2005
Software,
algorithm
Evolution Rate CovariationClark et al., 2012
Software, algorithmRhttps://www.r-project.org/

Additional files

Supplementary file 1

NGLY1 DGRP cross progeny counts.

The number of eclosed flies were scored for each resulting genotype. The ‘no marker’ column represents flies expressing the NGLY1 RNAi. The largest balanced genotype was used as ‘expected’ for percent survival.

https://cdn.elifesciences.org/articles/57831/elife-57831-supp1-v3.xlsx
Supplementary file 2

GWA analysis for survival in NGLY1 DGRP screen.

Single-nucleotide polymorphisms (SNPs) are listed by chromosome position and rs ID.

https://cdn.elifesciences.org/articles/57831/elife-57831-supp2-v3.zip
Supplementary file 3

Top associated SNPs.

The top 125 variants. SNPs are listed in rank order of significance.

https://cdn.elifesciences.org/articles/57831/elife-57831-supp3-v3.xlsx
Supplementary file 4

Gene set enrichment analysis (GSEA).

Gene Ontology (GO) terms are listed by rank significance. Individual genes within each category are listed with the FBgn#.

https://cdn.elifesciences.org/articles/57831/elife-57831-supp4-v3.xlsx
Supplementary file 5

Evolutionary rate covariance (ERC).

Co-evolving genes are listed by rank significance (sumnlogpvbest). Genes that are known to cause a Congenital Disorder of Glycosylation (CDG) are highlighted in red.

https://cdn.elifesciences.org/articles/57831/elife-57831-supp5-v3.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/57831/elife-57831-transrepform-v3.docx

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