975B Poster - 15. Models of human disease
Friday April 08, 2:00 PM - 4:00 PM

Authors:
Konrad Lang; Helena Heinkele; Julian Milosavljevic; Lea Gerstner; Tobias Hermle

Affiliation: Renal Division, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany

Keywords:
s. renal disease; g. CRISPR/Cas9

Introduction:
Mutations in the gene GAPVD1 cause nephrotic syndrome in humans. GAPVD1 interacts with the early endosomal regulator RAB5 but the subcellular localization of GAPVD1 remains unclear. Silencing of Gapvd1 in the podocyte-like Drosophila nephrocytes resulted in mistrafficking of fly nephrin.
Methods: We generated conditional knockdowns and a stable genetic deletion of Drosophila Gapvd1 by CRISPR/Cas9 and used microhomology-mediated end joing to introduce a c-terminal HA-tag into the genomic locus of Gapvd1. We performed a functional analysis of the novel fly models.
Results:
Using coexpression of tandem Gapvd1-gRNAs and Cas9 in the germline we generated twin frameshift mutations at the second and third exons of the Drosophila Gavpd1 gene. Flies carrying these mutations were homozygous viable without any overt phenotype. In contrast, the podocyte-like nephrocytes of these animals showed a severely altered slit diaphragm architecture with mislocalization of fly nephrin and the orthologue of NEPH1 as well as a partial loss of both proteins from the surface. This phenotype was similar but considerably stronger than the phenotype observed when using RNAi-mediated silencing. This suggests that the homozygous frameshift mutations result in a null allele. The phenotype was confirmed by conditional CRISPR/Cas-mediated knock down using two independent gRNAs. Deletion of Gapvd1 in the Drosophila model thus results in a phenotype that manifests exclusively in podocyte-like nephrocytes. This recapitulates the disease manifestation in human patients with GAPVD1 mutations who presented exclusivey with nephrotic syndrome, supporting a role for Drosophila as a model for this genetic disease. To study the subcellular localization of Drosophila Gapvd1, we introduced an HA-tag into the c-terminus of the Gapvd1 locus. Immunofluorescence of nephrocytes derived from the resultant knock-in lines showed co-localization of the Gapvd1-HA protein with Drosphila Rab5, supporting that Gapvd1 primarily resides in early endosomes. We overexpressed human GAPVD1 in nephrocytes, that equally localized in early endosomes. Gain-of-function of the human gene entailed reduced tracer endocytosis in nephrocytes, suggesting a dominant negative effect.
Conclusion: We established suitable new Drosophila models to study the function of Gapvd1 in nephrocytes as an invertebrate podocyte model and observed colocalization with Rab5 and a nephrocyte-restricted loss-of-function phenotype.