Drosophila melanogaster Mutated in its GBA1b Ortholog Recapitulates Neuronopathic Gaucher Disease

Abstract

Gaucher disease (GD) results from mutations in the GBA1 gene, which encodes lysosomal glucocerebrosidase (GCase). The large number of mutations known to date in the gene lead to a heterogeneous disorder, which is divided into a non-neuronopathic, type 1 GD, and two neurological, type 2 and type 3, forms. We studied the two fly GBA1 orthologs, GBA1a and GBA1b. Each contains a Minos element insertion, which truncates its coding sequence. In the GBA1a^m/m flies, which express a mutant protein, missing 33 C-terminal amino acids, there was no decrease in GCase activity or substrate accumulation. However, GBA1b^m/m mutant flies presented a significant decrease in GCase activity with concomitant substrate accumulation, which included C14:1 glucosylceramide and C14:0 glucosylsphingosine. GBA1b^m/m mutant flies showed activation of the Unfolded Protein Response (UPR) and presented inflammation and neuroinflammation that culminated in development of a neuronopathic disease. Treatment with ambroxol did not rescue GCase activity or reduce substrate accumulation; however, it ameliorated UPR, inflammation and neuroinflammation, and increased life span. Our results highlight the resemblance between the phenotype of the GBA1b^m/m mutant fly and neuronopathic GD and underlie its relevance in further GD studies as well as a model to test possible therapeutic modalities.

Keywords: Gaucher disease; GlcCer; GlcSph; glucocerebrosidase; inflammation; unfolded protein response.

Figure 1

Expression of the two normal and the two mutant Drosophila GBA1 genes. (A) Schematic representation of the GBA1 genes locus. GBA1a is located 2 kb upstream of GBA1b. CG31413 is a non-relevant gene between them. Triangle represents the Minos element insertion site in each gene. Exons of GBA1a appear in dark grey and those of GBA1b-in light grey. (B) Expression of normal (GBA1a and GBA1b) and mutant (GBA1a^m and GBA1b^m) GBA1 alleles in bodies and heads of control (w1118), GBA1a^m/m and GBA1b^m/m flies as analyzed by quantitative Real Time-PCR (qRT-PCR). Presented is the average ± standard error of five independent experiments. Expression of GBA1a in w1118 was considered 100%. * p < 0.05, ** p < 0.01. (C) FlyBase deep sequencing data summary (expression by tissue). Only the two highest expressed exons were counted.

Figure 2

Protein products of the fly mutant alleles. (A) Mutant protein sequences (red) of GBA1a (GBA1a^m) and GBA1b (GBA1b^m) compared to the wt protein sequence (black). Red lines represent the deletion in each mutant protein (33 C-terminal amino acids in GBA1a^m and 133 C-terminal amino acids in GBA1b^m including W408 that stabilizes the substrate). Blue color highlights amino acids comprising the active site. Green color highlights amino acids associated with substrate recognition [33]. (B) Protein expression of normal and mutant GBA1a and GBA1b alleles, coupled to a myc-tag in transgenic (Tg.) flies, expressed under the daughterless (Da)-GAL4 driver. (C) Known glycosylation sites in human GCase and predicted ones in Drosophila GBA1a and GBA1b-encoded GCases. Common sites in fly and human GCases are highlighted with gray. (D) Protein lysates, prepared from transgenic (Tg.) flies expressing wt or mutant myc-tagged GBA1a or GBA1b cDNAs, under the Da-GAL4 driver, were incubated with or without endo-H. The lysates were electrophoresed through SDS–PAGE and the corresponding blot was interacted with anti-myc antibody. Peptides 1 and 2 represent GBA1b-encoded GCase before and after endo-H treatment, respectively. Peptides 3 and 4 represent mutant GBA1b^m-encoded GCase before and after endo-H treatment, respectively. Peptides 5 (71.8 kDa) and 6 (67.4 kDa) represent GBA1a-encoded GCase before and after endo-H treatment, respectively.

Figure 3

GCase activity and substrate accumulation in mutant flies. (A) A TLC plate presenting GCase activity as measured in lysates prepared from bodies and heads of control (w1118) and mutant flies. The value obtained for w1118+CBE (Conduritol-B-Epoxide, a GCase inhibitor) was considered zero. Shown below the plate is the average activity, obtained from three independent experiments. (B) ME569 epoxide ABP-labeling of active GCase in bodies and heads of control (w1118), GBA1a^m/m and GBA1b^m/m flies at 12 days post-eclosion. The gel was blotted with anti actin specific antibody. (C) TLC plates showing GlcCer accumulation in bodies and heads of 2-, 12- and 18-day-old flies. St, standards. (D) GlcCer species, extracted from bodies and heads of 12-day-old GBA1b^m/m flies, were analyzed by LC-MS/MS. GlcCer 14:1 is the most common GlcCer species in flies. (E) LC-MS/MS analysis for C14:1 GlcCer extracted from bodies and heads of 12-day-old flies. (F) LC-MS/MS analysis for C14:0 GlcSph extracted from heads of 12-day-old flies. (G) Rescue of GCase activity in GBA1b^m/m flies, expressing different GBA1 orthologs as transgenes: human GCase (hGCase), GBA1a- or GBA1b-encoded fly GCases. Transgenes were expressed under the Da-GAL4 driver. Activity was tested in fly bodies as described in (A). Shown below the plate is the average activity, obtained from three independent experiments.

Figure 4

Changes in lysosomes morphology in GBA1b^m/m flies. (A) Hemocytes were extracted from hemolymph of 12-day-old control (w1118) and GBA1b^m/m flies, stained with LysoTracker-Red and analyzed by FACS. LysoTracker signal was measured by side scatter using 530 nm excitation (SSC:FITC_530) and cell size was measured by forward light scatter (FSC:LinH). LysoTracker-labeled hemocytes, larger than those extracted from w1118 flies, were encircled. (B). Quantification of encircled cells from (A). Presented is the average ± standard error of three independent assays. (C) Representative confocal images of the subesophageal ganglion of brains dissected from control (w1118) and GBA1b^m/m flies at three different time points. (D) Quantification of LysoTracker in images as shown in (C). Presented is the average ± standard error of 30 images from each line. (E) Representative confocal images of Fat body dissected from third instar larvae of control (w1118) and GBA1b^m/m flies, stained with Lysotracker and anti GlcCer antibodies. (F) Quantification of GlcCer signal as analyzed form confocal images as shown in (E). Presented is the average ± standard error of 30 images from each line. *** p < 0.0005.

Figure 5

ERAD and UPR activation in GBA1b^m/m flies. (A) Protein lysates prepared from HEK293T cells, transfected with plasmids expressing normal or mutant GBA1b-encoded proteins (mycHispcDNAGBA1b-GBA1b, mycHispcDNAGBA1b^m-GBA1b^m), were treated or untreated for 18 h with MG132. The corresponding blot was interacted with anti-myc and anti-Erk antibodies. (B) To quantify the results, WT and mutant myc-GBA1b intensity was divided by that of Erk in the same lane, and the number obtained for GBA1b without treatment was considered 1. The results represent the mean ± standard error of three independent experiments. (C,D) mRNA levels of UPR markers: activating transcription factor 4 (ATF4), Heat shock-70-3 (HSC-70-3) and spliced x-box binding protein (sXBP1), in bodies (C) and heads (D) of control (w1118), and GBA1b^m/m flies at 2 and 18 days post-eclosion, as analyzed by qRT-PCR. mRNA level of each marker was normalized to that of RP49. mRNA levels of the different tested genes in control flies were considered 1. * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 6

Inflammation and neuroinflammation in GBA1b^m/m flies. (A) A schematic representation of the IMD and the Toll innate immunity pathways in Drosophila. (B,C) mRNA levels of Attacin-C (ATTC), Cecropin (Cec), Drosomycin (Drs) and Metchnikowin (Mtk) in bodies (B) and heads (C) of control (w1118) and GBA1b^m/m flies at three different time points (2, 12 and 18 days post-eclosion), as analyzed by qRT-PCR. mRNA levels of the different tested genes in control flies were considered 1. (D,E) mRNA enrichment of gene expression in bodies (D) and heads (E) of 12-day-old GBA1b^m/m flies compared to GBA1b^m/+ and control (w1118) flies. The right heat map is an enlargement of GBA1b^m/m elevated mRNAs (boxed in the left heat map). Genes that participate in immune response are highlighted in orange. The genes analyzed by qRT-PCR in B and C are boxed. (F) mRNA levels of ATTC, Cec, Drs and Mtk in hemolymph of control (w1118) and GBA1b^m/m flies at 12 days post-eclosion, as analyzed by qRT-PCR. (G) mRNA levels of chitinase 4 (Cht4) in bodies of w1118 and GBA1b^m/m flies at 2, 12 and 18 days post-eclosion, as analyzed by qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 7

Partial rescue of GBA1b^m/m pathologies by ambroxol. (A) A TLC plate showing GCase activity as measured in lysates prepared from bodies and heads of control and GBA1b^m/m flies, treated or untreated for 12 days with ambroxol. (B,C) mRNA levels of UPR markers: ATF4, HSC-70-3 and sXBP1, in bodies (B) and heads (C) of control (w1118), and GBA1b^m/m flies, untreated or treated for 18 days with ambroxol. * p < 0.05, ** p < 0.01, *** p < 0.005. (D,E) mRNA levels of inflammation markers: ATTC, Cec, Drs and Mtk, in bodies (D) and heads (E) of GBA1b^m/m flies in comparison to age matched controls (w1118), untreated or treated for 12 days with ambroxol. mRNA levels of the tested genes in untreated control flies were considered 1. (F) Thirty flies from each line were untreated or treated for 12 days with ambroxol and analyzed for locomotion behavior at Days 2, 7, 12 and 18 post eclosion. (G) A curve showing the overall survival of w1118 and GBA1b^m/m flies untreated or treated for 12 days with ambroxol. The numbers represent three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.005. Amb.-ambroxol.