Functional study of Drosophila β-glucuronidase for developing a fly model of Mucopolysaccharidosis Type VII

Bar, Sudipta (2018) Functional study of Drosophila β-glucuronidase for developing a fly model of Mucopolysaccharidosis Type VII. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Sly syndrome or Mucopolysaccharidosis VII (MPSVII) is an autosomal recessive lysosomal storage disorder caused by the deficiency of β-glucuronidase (β-GUS), a lysosomal enzyme required for degradation of glycosaminoglycans. Lysosomal accumulation of undegraded or partially degraded glycosaminoglycans results in cellular and multiple organ dysfunction leading to premature deaths in most cases. The most prominent clinical features include mental retardation, bone and facial deformities, restricted mobility hepatosplenomegaly, heart and respiratory problems and hydrops fetalis. The only approved treatment for this fatal disease is enzyme replacement therapy (ERT). In November 2017, Ultragenix pharmaceutical, after the first successful clinical trial, got the approval for ERT in the USA from Food and Drug Administration (FDA). ERT is a very expensive treatment (expenses can reach up to 350,000 dollars per year for a patient) and the patient is required to be treated with the enzyme regularly for the entire life.Therefore, extensive molecular level knowledge about the disease pathogenesis is required for developing an alternative and cheaper therapy for MPS VII. One major bottleneck towards this is lack of a suitable animal model for this disease. Although MPS VII mouse model is available, given the severity of the phenotype those animals are extremely difficult to breed and maintain and cannot be widely used by the research community for large-scale experimentations. To circumvent this problem, we decided to develop a simpler Drosophila model of this disease that can be easily propagated for routine experiments and also used for genetic and drug screening studies. Therefore, the aim of this Ph.D. thesis was to identify the functional β-GUS orthologue in Drosophila followed by knocking out the corresponding gene and characterization of the mutant fly. Our overall goal was to establish the β-GUS knockout fly as an alternative animal model of MPSVII for in-depth analysis of the disease pathogenesis. We analyzed the Drosophila genome and identified the CG2135 gene as a putative β-GUS orthologue in Drosophila. Sequence alignment revealed that the CG2135 protein has ~60% sequence similarity with that of human β-GUS. All the active site residues of human β-GUS and the frequently mutated residues in the MPSVII patients were mostly conserved in the CG2135. Homology modeling revealed that CG2135 possessed overall 3D structural similarity having similar domains and motif like human β-GUS. Encouraged by these initial findings, we performed an RT-PCR-based expression analysis and found that the CG213 mRNA is ubiquitously expressed in all developmental stages and in all major tissues of the adult fly. To confirm that CG2135 gene produced an active protein, we cloned the corresponding cDNA, expressed and purified the protein to homogeneity. Our assay results revealed that the specific activity of purified CG2135 is 3.7x10⁶ units/mg, which is comparable to the activity of human β-GUS. Other biochemical properties of the CG2135 protein, like, thermal stability, pH profile and inhibitor susceptibility, are quite similar to that of human β-GUS. The CG2135 protein was also found to be localized in the lysosome, as expected. In sum, we have identified the CG2135 gene as an authentic β-GUS orthologue in Drosophila and also confirmed it as a functional lysosomal enzyme. We then moved on to creating a CG2135 knockout fly. Next, we generated the CG2135 knockout flyby homologous recombination. The knockout construct was designed such a way that upon recombination it would delete 212 bp CG2135 coding region (along with start codon) and would also cause a frameshift. After targeted recombination, the homozygous knockout (CG2135⁻/⁻) fly was generated, which was viable with no obvious abnormalities. Genomic DNA PCR confirmed the desired deletion in the genome, which was replaced with the red-eye and GAL4 markers. The RT-PCR result showed complete absence of the CG2135 mRNA in the CG2135⁻/⁻ fly, as expected of a knockout fly. However, in contrary to our expectations, the β-GUS activity in the CG2135⁻/⁻ fly although was reduced by 2.4 folds but the activity was not completely abolished. This residual β-GUS activity in the CG2135⁻/⁻ fly indicated the presence of another β-GUS like protein in Drosophila. Our bioinformatics study and experimental results confirmed the presence of a gene of unassigned function in the Drosophila genome (annotated as CG15117), that has high sequence homology with that of CG2135 but is ~6 times less active than CG2135. The CG15117 lacks the ER targeting sequence that is the characteristics of all lysosomal acid hydrolase and hence it appears to be non-lysosomal enzyme playing some non-conventional role. We also generated a UASCG2135 transgenic fly using germline transformation. The UAS CG2135 transgenic line was used to overexpressthe CG2135 protein in the background of CG2135⁻/⁻ and this transgenic rescue fly was used to check for the phenotype reversal. Detail phenotypic characterization of the CG2135⁻/⁻ fly is described in the following chapter. The extensive behavioural and pathological characterization of the CG2135⁻/⁻ fly was undertaken. The phenotypes of the CG2135⁻/⁻ fly were compared with the wild type fly as well as the CG2135 transgenic rescue fly to arrive at a meaningful conclusion. The CG2135⁻/⁻ fly showed ~30% embryonic lethality as compared to wild type fly suggesting a moderate developmental defect in this fly. Similar developmental defect at the neonatal stage was earlier reported in the MPSVII mouse model. The CG2135⁻/⁻fly had shortened lifespan and exhibited progressive decline in locomotor ability, which is typical of neurodegenerative diseases and also observed in MPSVII patients and the mouse model. Pathological analysis of the brain and muscle was undertaken since these two tissues are often associated with movement disorders. The CG2135⁻/⁻ fly brain showed increased accumulation of enlarged lysosomes, polyubiquitinated proteins and mitochondria, as expected in a storage disease with defected cellular clearance machinery. Histopathological study of the brain sections revealed extensive vacuolations with loss of dopaminergic neurons in the CG2135⁻/⁻ fly as compared to age-matched wild type fly. As opposed to the wild type fly, we also found loss of muscle integrity in the CG2135⁻/⁻ fly, which was caused by extensive muscle cell apoptosis. Most of these abnormal phenotypes seen in the CG2135⁻/⁻ fly were reverted in the CG2135 transgenic rescue fly, confirming the functional importance of the fly β-GUS. The neuromuscular degeneration observed in this fly provides important mechanistic insights for better understanding the disease pathogenesis. Having established the CG2135⁻/⁻ fly as a new animal model of MPSVII we utilized it for testing the efficacy of resveratrol, a natural polyphenol with multiple health benefits, in reverting the disease phenotypes. We found that dietary administration of resveratrol provided significant protection against neuromuscular degeneration and it also restored normal locomotor ability in the CG2135⁻/⁻ fly. Thus, we have not only developed a new animal model for MPSVII but also uncovered the therapeutic potential of resveratrol for managing the disease. This work has opened up the possibility of utilizing the CG2135⁻/⁻ fly for large scale genetic screening for identification of genetic modifiers of the disease phenotype. Also, our work has highlights the usefulness of the CG2135⁻/⁻ fly as a convenient platform for rapid drug screening, similar to our study with resveratrol. This may provide important lead molecules, which can be further explored for alternative drug development against MPSVII.

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