Abstract
In tuberous sclerosis complex (TSC), Tsc2 mutations are associated with more severe disease manifestations than Tsc1 mutations and the role of extracellular vesicles (EVs) in this context is not yet studied. We report a comparative analysis of EVs derived from isogenic renal cells except for Tsc1 or Tsc2 gene status and hypothesized that in spite of having similar physical characteristics, EVs modulate signaling pathways differently, thus leading to TSC heterogenicity. We used mouse inner medullary collecting duct (mIMCD3) cells with the Tsc1 (T1G cells) or Tsc2 (T2J cells) gene disrupted by CRISPR/CAS9. EVs were isolated from the cell culture media by size-exclusion column chromatography followed by detailed physical and chemical characterization. Physical characterization of EVs was accessed by tunable resistive pulse sensing and dynamic light scattering, revealing similar average sizes and zeta potentials (at pH 7.4) for EVs from mIMCD3 (123.5 ± 5.7 nm and −16.3 ± 2.1 mV), T1G cells (131.5 ± 8.3 nm and −19.8 ± 2.7 mV), and T2J cells (127.3 ± 4.9 nm and −20.2 ± 2.1 mV). EVs derived from parental mIMCD3 cells and both mutated cell lines were heterogeneous (>90% of EVs < 150 nm) in nature. Immunoblotting detected cilial Hedgehog signaling protein Arl13b; intercellular proteins TSG101 and Alix; and transmembrane proteins CD63, CD9, and CD81. Compared to Tsc2 deletion, Tsc1 deletion cells had reduced EV production and release rates. EVs from Tsc1 mutant cells altered mTORC1, autophagy, and β-catenin pathways differently than EVs from Tsc2-mutated cells. Quantitative PCR analysis revealed the down regulation of miR-212a-3p and miR-99a-5p in EVs from Tsc2-mutated cells compared to EVs from Tsc1-mutant cells. Thus, EV-derived miR-212-3p and mIR-99a-5p axes may represent therapeutic targets or biomarkers for TSC disease.
Highlights
Tuberous sclerosis complex (TSC) affects over one million people worldwide, and the disease manifests from abnormalities in embryonic and postpartum cell growth control that can impact every organ system
Extracellular vesicles were isolated from the conditioned media of murine renal collecting duct cells and Tsc1deleted mIMCD3 designated as T1G, as per our previous paper (Bissler et al, 2019)
At physiological pH 7.4, the zeta potentials of mIMCD3, T1G, and T2J-derived extracellular vesicles (EVs) were recorded in a range of −15 to −20 mV (Figure 2B)
Summary
Tuberous sclerosis complex (TSC) affects over one million people worldwide, and the disease manifests from abnormalities in embryonic and postpartum cell growth control that can impact every organ system. Given that the TSC gene products interact to regulate the mTORC1 pathway, the human disease phenotype is unexpectedly different for patients with TSC1-associated disease compared to those with TSC2-associated disease (Dabora et al, 2001; Sancak et al, 2005; Aronow et al, 2012; Kothare et al, 2014). This genotype–phenotype difference is observed in Tsc mouse models of neurological disease. Loss of either gene leads to increased mTORC1 activity, but the mechanism for the disease severity difference is unknown
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