Abstract
Purpose :
Lowe syndrome is a rare congenital disease that presents with renal failure, developmental delays, congenital cataracts, and glaucoma. The coordination between endocytosis dysfunction and the Akt/ mTORC1 signaling in Lowe syndrome patient cells is unclear. The present study tests the hypothesis that microtubule-based transport proteins are involved in the trafficking of lysosomes and maintenance of subsequent basal mTORC1 and Akt signaling. The relationship between endocytosis dysfunction and mTORC1/Akt signaling was also examined.
Methods :
We examined mTORC1 and Akt signaling in normal human fibroblasts (NHF558) treated with nocodazole, which blocks microtubule polymerization, in complete culture medium. Activation of mTORC1signaling was assayed by the phosphorylation of p70 S6 Kinase (Thr389); activation of Akt signaling, by the p-ser473 Akt level. We also examined lysosome distribution in noncancerous cells in which Arl8a, Arl8b, SKIP, Rab7a or RLIP was knockdown by siRNA. Lysosome distribution was quantified by using average LAMP1 intensity.
Results :
Knockdown of anterograde transport proteins small GTPase Arl8 or SKIP in normal human fibroblasts leads to perinuclear lysosome positioning and decreased mTORC1 signaling. However, Arl8 or SKIP knockdown in Lowe 1676 cells did not cause perinuclear lysosome accumulation and decreased mTORC1-mediated phosphorylation of p70 S6 Kinase (Thr389). Furthermore, we found that knockdown of retrograde transport proteins caused lysosomes to be scattered throughout the cytoplasm and decreased the perinuclear lysosome accumulation in normal human fibroblasts. However, we did not observe similar disruption in Lowe patient-derived fibroblasts with Rab7 or RLIP knockdown. Thus, these experiments show that mTORC1 and AKT signaling is sensitive to lysosome dynamics and that OCRL plays a role in the crosstalk between these pathways.
Conclusions :
Whereas previous studies mainly focused on how transporters such as the kinesins and dynein motors regulate lysosome positioning, here we showed that the microtubule and actin systems themselves also play important roles in the transporting of lysosomes. These data suggested that under pathological conditions (e.g. congenital glaucoma), there are alternative pathways to regulate the mTORC1 and Akt signaling response to nutrient stimulus.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.