Abstract: PO1520
Tsc Gene Locus Disruption and Differences in Renal Epithelial Extracellular Vesicles
Session Information
- Cystic Kidney Diseases: Mechanisms, Genetics, and Treatment
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1001 Genetic Diseases of the Kidneys: Cystic
Authors
- Kumar, Prashant, The University of Tennessee Health Science Center College of Medicine, Memphis, Tennessee, United States
- Al-Zadjali, Fahad, The University of Tennessee Health Science Center College of Medicine, Memphis, Tennessee, United States
- Yao, Ying, The University of Tennessee Health Science Center College of Medicine, Memphis, Tennessee, United States
- Bissler, John J., The University of Tennessee Health Science Center College of Medicine, Memphis, Tennessee, United States
Background
The severity of tuberous sclerosis complex (TSC) manifestations seem to be different depending which TSC locus is affected. This is a puzzling finding, given that the gene product of both loci heterodimerize to regulate mTORC1 activity, so loss of either one releases the repression and results in constitutive mTORC1 activation.
Methods
To begin to understand possible mechanisms for this difference, we have used mouse inner medullary collecting duct (mIMCD) cells with either the Tsc1 or the Tsc2 gene disrupted by a CRISP/CAS9 strategy. We have previously characterized the Tsc2-mutant cell line derived EVs, and present here intriguing differences between the extracellular vesicles (EVs) derived from cells with mutant Tsc1 or Tsc2 genes. To characterize the EVs, we used tunable resistive pulse sensing, dynamic light scattering, transition electron microscopy and mmunoblot analysis.
Results
To characterize the size of the EVs, we used tunable resistive pulse sensing and dynamic light scattering. The parental cell line had an average size of 123.5 ± 5.7 nm and mutant Tsc1-derived EVs had an average size 131.5 ± 8.3 nm. The surface charge for the two cell lines were -16.3 ± 2.1mV and -19.8 ± 2.7mV respectively. The isolated nanosized vesicle had excellent purity as assayed using transmission electron microscope. Both cell lines had a heterogenous population of EVs based on size, and more than 90% of the EVs were smaller than 150nm. Immunoblot analysis revealed by the presence of the cilial Hedgehog signaling protein Arl13b and the intercellular proteins TSG101 and Alix, as well as the transmembrane proteins CD63, CD9, and CD81. Tsc1 deletion resulted in less EVs production and synthesis rate compared to Tsc2 deletion. RNA and protein transfection studies were done to evaluate the role of EVs in disease pathology. Quantitative PCR analysis showed the downregulation of miR-212a-3p and miR-99a-5p in EVs derived from Tsc2 which are sought to contribute the more TS severity as compared to TSC1. In addition, miR-212-3p/mTORC1 and mIR-99a-5p/mTORC1 axis are could be a novel therapeutic and biomarker strategy for TSC disease.
Conclusion
We have found that the intercellular communication of EVs has significant differences depending upon which TSC locus is affected, and this difference may be involved in the different phenotypes expressed.
Funding
- Other U.S. Government Support