Defects in the Neuroendocrine Axis Contribute to Global Development Delay in a Drosophila Model of NGLY1 Deficiency

Abstract

N-glycanase 1 (NGLY1) Deficiency is a rare monogenic multi-system disorder first described in 2014. NGLY1 is evolutionarily conserved in model organisms. Here we conducted a natural history study and chemical-modifier screen on the Drosophila melanogaster NGLY1 homolog, Pngl We generated a new fly model of NGLY1 Deficiency, engineered with a nonsense mutation in Pngl at codon 420 that results in a truncation of the C-terminal carbohydrate-binding PAW domain. Homozygous mutant animals exhibit global development delay, pupal lethality and small body size as adults. We developed a 96-well-plate, image-based, quantitative assay of Drosophila larval size for use in a screen of the 2,650-member Microsource Spectrum compound library of FDA approved drugs, bioactive tool compounds, and natural products. We found that the cholesterol-derived ecdysteroid molting hormone 20-hydroxyecdysone (20E) partially rescued the global developmental delay in mutant homozygotes. Targeted expression of a human NGLY1 transgene to tissues involved in ecdysteroidogenesis, e.g., prothoracic gland, also partially rescues global developmental delay in mutant homozygotes. Finally, the proteasome inhibitor bortezomib is a potent enhancer of global developmental delay in our fly model, evidence of a defective proteasome "bounce-back" response that is also observed in nematode and cellular models of NGLY1 Deficiency. Together, these results demonstrate the therapeutic relevance of a new fly model of NGLY1 Deficiency for drug discovery and gene modifier screens.

Keywords: Drosophila; N-glycanse 1; NGLY1; Pngl; disease model.

Figure 1

Genotyping and phenotyping Pngl^PL allele. A) We used CRISPR to create an allele of Pngl with a stop codon and white + transgene inserted upstream of the PAW domain. Sequencing confirmed the integration of the stop codon and mw+ immediately 3′ to bp 1906699 (Release 5.57) in chromosome 2R (NT_033778.3). B) The fraction of Pngl^PL/+ heterozygote, Pngl^PL/PL homozygote, or Pngl^PL/PL;Actin > NGLY1 flies that eclose over time normalized to the total surviving flies for each genotype. Flies were grown on standard untreated media. The ratio of flies emerging in this experiment for Pngl^PL/PL, Pngl^PL/PL ; Act > hNGLY1, and Pngl^PL/CyO is 15:40:137 individuals. Ubiquitous expression of a human NGLY1 transgene rescued the 2-day development delay to eclosion observed in Pngl^PL homozygotes. C) Pngl^PL homozygous adult flies are smaller than homozygous siblings expressing the human NGLY1 transgene. D) Pngl^PL homozygotes are 25% smaller than their heterozygous siblings (P < 0.01; Student’s t-test).

Figure 2

Pngl^PL homozygotes are hypersensitive to dimethyl sulfoxide (DMSO). A) An image of Pngl^PL homozygous larvae grown in 96-well plate format on food dosed with different concentrations (0–0.4%) of DMSO. Pngl^PL homozygous larvae are hypersensitive to DMSO, a decrease in larval size is observed starting at 0.025% DMSO and larval size is decrease as the dose of DMSO is increased. The time to pupation of B) Pngl^Ex20 (n = 3 replicates, 20-30 individuals/replicate) or C) Pngl^PL (n = 5 replicates, 20-30 individuals/replicate) homozygous larvae reared on food treated with DMSO. Homozygous mutant larvae exhibit hypersensitivity to DMSO showing a delayed time to pupariate and increased lethality.

Figure 3

Layout of an Pngl^PL assay plate with examples of controls and pre-hit compound. Larvae were cultured in 96-well plates with negative controls on the left most wells (column 1) which carried Pngl^PL homozygous with vehicle DMSO at 0.1%. 80 testing wells were in the middle (columns 2-11) which carried Pngl^PL homozygous larvae with a compound at 1μM, and DMSO at 0.1%. Right most top wells (12A-12F) contain positive control group 1 with Pngl^PL heterozygous larvae and 0.1% DMSO. Right most bottom wells (12G-12H) contain positive control group 2 with Pngl^PL homozygous larvae without DMSO. The compound in well G4 was considered a pre-hit compound.

Figure 4

The assay had a consistent difference between positive and negative controls. A) A consistent separation between positive and negative controls with Z’Factor >0, indicated an assay that could significantly identify chemical suppressors. B) Three replicates of a 32 plate library were analyzed and 73 of 96 plates had a Z’Factor >0.

Figure 5

Positive correlation between 3X replicates. A) The three pairwise comparisons of Z-scores show positive correlations indicating that a set of small molecules could modify the small larval size phenotype B) The positive correlations between replicates is more evident when only plotting Z-scores of < -2 or > 2. C) Larval size is partially rescued when 20E is fed to Pngl^PL homozygous larvae.

Figure 6

20-hydroxyecdysone (20E), but not an earlier ecdysone pathway precursor (7-dehydrocholesterol), partially rescues developmental delay and lethality in Pngl^PL mutants. The time to pupation of A) Pngl^PL/+ heterozygous or B) Pngl^PL homozygous larvae reared on food treated with different concentration of 20E (n = 10 replicates, 20-30 individuals/replicate). 20E did not impact developmental rate of heterozygous Pngl^PL larvae to pupation, but partially rescued development delay of homozygous Pngl^PL larvae to pupation at 200µM. The time to pupation of C) Pngl^PL/+ heterozygous or D) Pngl^PL homozygous larvae reared on food treated with different concentration of 7-d (n = 3 replicates, 25-30 individuals/replicate). 7-d did not impact developmental rate of heterozygous or homozygous Pngl^PL larvae to pupation. E) The fraction of animals surviving to eclosion. 20E, but not 7-d, rescued larval lethality of Pngl^PL homozygous at 200µM. (n = 10 replicates, 20-30 individuals/replicate; n = 3 replicates, 25-30 individuals/replicate, respectively).

Figure 7

Pngl is necessary for normal function of the ring gland. The fraction of Pngl^PL / Pngl^Ex20 compound heterozygotes eclosed with human NGLY1 driven by the ring gland driver (blue) A) 2_286-GAL4 B) spookier-GAL4, or C) phantom-GAL4 compared to sibling controls lacking a driver (red). The reported values are normalized to the total number of eclosed Pngl^PL / Pngl^Ex20 compound heterozygotes for each experiment. A) Compound heterozygous Pngl^PL / Pngl^Ex20 larvae expressing 2_286 > NGLY1 eclosed earlier and had lower lethality than control flies (64 Pngl^PL / Pngl^Ex20; 2_286-GAL4 individuals compared to 120 Pngl / CyO siblings; 74 Pngl^PL / Pngl^Ex20; 2_286 > hNGLY1 individuals compared to 185 Pngl / CyO siblings). Compound heterozygous Pngl^PL / Pngl^Ex20 larvae expressing A) spookier > NGLY1 or B) phantom > NGLY1 had lower lethality at eclosion than control flies (23 Pngl^PL / Pngl^Ex20; spok-GAL4 individuals compared to 337 Pngl / CyO siblings; 43 Pngl^PL / Pngl^Ex20; spok > hNGLY1 individuals compared to 398 Pngl / CyO siblings); and 19 Pngl^PL / Pngl^Ex20; phm-GAL4 individuals compared to 235 Pngl / CyO siblings; 63 Pngl^PL / Pngl^Ex20; phm > hNGLY1 individuals compared to 459 Pngl / CyO siblings, respectively).

Figure 8

Pngl larvae are ≥10X more sensitive to the proteasome inhibitor bortezomib. Images A) and quantification of larval size B) of 3 day Pngl^PL/+ heterozygous or age matched Pngl^PL/PL mutant larvae raised on food treated with 0, 1, 5, 10 or 25µM bortezomib. Black points report the size (in arbitrary units) of individual larvae. Blue lines report the mean and SE of these populations. Bortezomib delays larval developmental progression in Pngl^PL homozygous larvae more severely than Pngl^PL/+ heterozygous larvae leading to smaller sized larvae, and is lethal for homozygotes at concentrations equal to or greater than 5 µM. For these experiments, Bortezomib was solubilized in DMSO, resulting in a final concentration of DMSO in treated food of 0.025%, which is below the threshold of effect on Pngl^PL homozygous larvae. (** significant at P < 0.01; Student’s t-test) C) Quantification of larval size for Pngl^PL homozygous larvae raised on untreated food, or food treated with 0.1% DMSO, 1 µM bortezomib, 0.1% DMSO and 1 µM bortezomib, or 0.1% DMSO, 1 µM bortezomib, and 133 µM 20E. 20E partially rescued the effect of bortezomib treatment on larval size in Pngl^PL homozygotes. (** significant at P < 0.01; Student’s t-test).