Drosophila melanogaster Mutated in its GBA1b Ortholog Recapitulates Neuronopathic Gaucher Disease
Abstract
:1. Introduction
2. Materials and Methods
2.1. Antibodies
2.2. Construction of Plasmids
2.3. Cells and Transfection
2.4. Fly Strains
2.5. MG132 (Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) Treatment
2.6. Ambroxol Treatment
2.7. RNA Preparation
2.8. RT-PCR
2.9. Quantitative Real Time PCR (qRT-PCR)
2.10. Whole Transcriptome Sequencing and Analysis
2.11. Detection of Spliced Xbp1 mRNA Processing
2.12. SDS–PAGE and Western Blotting
2.13. Total Lipid Extraction and Quantification of GlcCer and Ceramide
2.14. GlcCer and GlcSph Determination by LC-MS/MS
2.15. GCase Activity Assay
2.16. GCase Labeling with Activity-Based Probes
2.17. Endoglycosidase H (Endo-H) Sensitivity
2.18. Lysotracker and Confocal Imaging
2.19. Isolation of Hemolymph and FACS Analysis
2.20. Climbing Assay
2.21. Survival Assay
2.22. Statistics
3. Results
3.1. Expression of the Fly Normal and Mutant GBA1 Genes
3.2. GCase Activity and Substrate Accumulation
3.3. Changes in Lysosome Morphology in GBA1bm/m Mutant Flies
3.4. ERAD and UPR Activation in GBA1bm/m flies
3.5. Development of Inflammation and Neuroinflammation in GBA1bm/m flies
3.6. Partial Rescue of the GBA1bm/m Phenotype by Ambroxol
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Name | Primers for Construction of Plasmids |
GBA1a | F: 5′-CTGAATAGGGAATTGGGAATTCGTATGGGAAAAATGTTCGC-3′ |
R: 5′-GAGGTACCCTCGAGCATTTCAAGCACTTATTGAAGAGAACGGCGG-3′ | |
GBA1b | F: 5′-CTGAATAGGGAATTGGGAATTCGTATGCCAGATATGAAGACAC-3′ |
R: 5′-CCCTCTAGAGGTACCCTCGAGTAGGCCCTCATATAGCTTTCACC-3′ | |
Name | Primers for qRT-PCR |
GBA1a | F: 5′-GAGTGGTTCCTTATCTCCAGTT-3′ |
R: 5′-ACGGTAAACTTCTCCTCCTTAC-3′ | |
GBA1b | F: 5′-AAGAACTTCCGGTGGAGCTA-3′ |
R: 5′-CAATTCATTGTATGCCCAGGGT-3′ | |
sXbp1 | F: 5′-CCGAACTGAAGCAGCAACAGC-3′ |
R: 5′-GTATACCCTGCGGCAGATCC-3′ | |
HSC-70-3 | F: 5′-GCTGGTGTTATTGCCGGTCTGC-3′ |
R: 5′-GATGCCTCGGGATGGTTCCTTGC-3′ | |
ATF4 | F: 5′-AGACGCTGCTTCGCTTCCTTC-3′ |
R: 5′-GCCCGTAAGTGCGAGTACGCT-3′ | |
ATTC | F: 5′-CTGCACTGGACTACTCCCACATCA-3′ |
R: 5′-CGATCCTGCGACTGCCAAAGATTG-3′ | |
Cec | F: 5′-CATTGGACAATCGGAAGCTGGGTG-3′ |
R: 5′-TAATCATCGTGGTCAACCTCGGGC-3′ | |
Drs | F: 5′-AGTACTTGTTCGCCCTCTTCGCTG-3′ |
R: 5′-CCTTGTATCTTCCGGACAGGCAGT-3′ | |
Mtk | F: 5′-CATCAATCAATTCCCGCCACCGAG-3′ |
R: 5′-AAATGGGTCCCTGGTGACGATGAG-3′ | |
RP49 | F: 5′-TAAGAAGCGCACAAAGCACT-3′ |
R: 5′-GGGCATCAGATATTGTCCCT-3′ |
Biological Process | All Known Participating Genes (D. Mel.) | Expected | Observed | Fold Enrichment | p-Value | FDR |
---|---|---|---|---|---|---|
Response to external biotic stimulus | 363 | 1.37 | 18 | 13.13 | 1.03 × 10−15 | 8.04 × 10−12 |
Response to bacterium | 256 | 0.97 | 15 | 15.57 | 3.88 × 10−14 | 7.56 × 10−11 |
Defense response | 367 | 1.39 | 15 | 10.82 | 5.65 × 10−12 | 8.81 × 10−9 |
Defense response to Gram-positive bacterium | 56 | 0.21 | 8 | 37.82 | 8.63 × 10−11 | 1.12 × 10−7 |
Response to external stimulus | 912 | 3.44 | 19 | 5.52 | 4.18 × 10−10 | 4.66 × 10−7 |
Defense response to bacterium | 224 | 0.85 | 11 | 13 | 8.90 × 10−10 | 8.68 × 10−7 |
Multi-organism process | 1431 | 5.41 | 22 | 4.07 | 3.10 × 10−9 | 2.69 × 10−6 |
Response to stress | 1122 | 4.24 | 19 | 4.48 | 1.24 × 10−8 | 8.78 × 10−6 |
Defense response to other organism | 291 | 1.1 | 11 | 10.01 | 1.23 × 10−8 | 9.61 × 10−6 |
Humoral immune response | 87 | 0.33 | 7 | 21.3 | 5.60 × 10−8 | 3.64 × 10−5 |
Antimicrobial humoral response | 72 | 0.27 | 6 | 22.06 | 4.41 × 10−7 | 2.64 × 10−4 |
Immune response | 192 | 0.73 | 8 | 11.03 | 7.30 × 10−7 | 4.06 × 10−4 |
Biological Process | All Known Participating Genes (D. Mel.) | Expected | Observed | Fold Enrichment | p-Value | FDR |
---|---|---|---|---|---|---|
Antibacterial humoral response | 28 | 0.09 | 6 | 68.61 | 7.99 × 10−10 | 6.23 × 10−6 |
Antimicrobial humoral response | 72 | 0.22 | 7 | 31.13 | 4.17 × 10−9 | 1.08 × 10−5 |
Immune system process | 316 | 0.99 | 11 | 11.14 | 3.24 × 10−9 | 1.26 × 10−5 |
Humoral immune response | 87 | 0.27 | 7 | 25.76 | 1.42 × 10−9 | 1.59 × 10−5 |
Response to biotic stimulus | 363 | 1.13 | 11 | 9.7 | 1.31 × 10−8 | 1.70 × 10−5 |
Defense response to Gram-positive bacterium | 56 | 0.17 | 6 | 34.3 | 3.42 × 10−8 | 3.34 × 10−5 |
Response to bacterium | 256 | 0.8 | 9 | 11.26 | 9.82 × 10−8 | 8.51 × 10−5 |
Immune response | 192 | 0.6 | 8 | 13.34 | 1.58 × 10−7 | 1.23 × 10−4 |
Defense response | 367 | 1.15 | 10 | 8.72 | 1.75 × 10−7 | 1.24 × 10−4 |
Defense response to other organism | 291 | 0.91 | 9 | 9.9 | 2.82 × 10−7 | 1.83 × 10−4 |
Defense response to bacterium | 224 | 0.7 | 8 | 11.43 | 4.91 × 10−7 | 2.95 × 10−4 |
Response to external stimulus | 912 | 2.85 | 13 | 4.56 | 2.78 × 10−6 | 1.55 × 10−3 |
Response to stimulus | 2640 | 8.25 | 21 | 2.55 | 1.14 × 10−5 | 5.94 × 10−3 |
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Cabasso, O.; Paul, S.; Dorot, O.; Maor, G.; Krivoruk, O.; Pasmanik-Chor, M.; Mirzaian, M.; Ferraz, M.; Aerts, J.; Horowitz, M. Drosophila melanogaster Mutated in its GBA1b Ortholog Recapitulates Neuronopathic Gaucher Disease. J. Clin. Med. 2019, 8, 1420. https://doi.org/10.3390/jcm8091420
Cabasso O, Paul S, Dorot O, Maor G, Krivoruk O, Pasmanik-Chor M, Mirzaian M, Ferraz M, Aerts J, Horowitz M. Drosophila melanogaster Mutated in its GBA1b Ortholog Recapitulates Neuronopathic Gaucher Disease. Journal of Clinical Medicine. 2019; 8(9):1420. https://doi.org/10.3390/jcm8091420
Chicago/Turabian StyleCabasso, Or, Sumit Paul, Orly Dorot, Gali Maor, Olga Krivoruk, Metsada Pasmanik-Chor, Mina Mirzaian, Maria Ferraz, Johannes Aerts, and Mia Horowitz. 2019. "Drosophila melanogaster Mutated in its GBA1b Ortholog Recapitulates Neuronopathic Gaucher Disease" Journal of Clinical Medicine 8, no. 9: 1420. https://doi.org/10.3390/jcm8091420