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Subhasree Basu, Suren Rajakaruna, Beverly Reyes, Elisabeth Van Bockstaele, A Menko; JNK inactivation suppresses mTOR (Mammalian Target of Rapamycin) pathway to induce loss of nuclei and organelles in the lens through Autophagy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4042.
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© ARVO (1962-2015); The Authors (2016-present)
Organelle Free Zone (OFZ) formation in the lens is a tightly regulated event crucial for lens clarity, yet the signaling pathways and the mechanism that induce the loss of nuclei and organelles remain unknown. Here, we examined how the inactivation of JNK leads to the formation of the OFZ through an mTOR-regulated autophagic pathway.
Studies used primary chick lens cell cultures that formed lentoids or an E13 whole chick embryo lens organ-culture system. Cells/lenses were exposed to JNK inhibitor (SP600125), the autophagy inducer/mTOR inhibitor (Rapamycin) or vehicle DMSO for 24hrs. Cells were fixed and labeled for Golgi/ER markers, nuclei and/or the autophagy markers Beclin-1 and LC3B and imaged by confocal microscopy. Immunoblot analysis examined expression of LC3BII, and activation of mTOR/Raptor and p70S6K. Immunoprecipitation was performed on microdissected E15 lenses. Presence of autophagic vesicles in treated lenses was analyzed by Transmission Electron Microscopy (EM).
At E15, a time in chick lens development when nuclei and organelles are removed from the central lens fiber cells (FC), the ER marker calreticulin co-localized in vesicular structures with Beclin-1 and LC3B. This result suggested that organelles are removed from these lenses by an autophagic process. We also examined expression of mTOR, a major component of the mTORC1 signaling complex, which negatively regulates autophagy. mTOR expression was suppressed in the FC region and weakly associated with Raptor, a necessary intermediate step in formation of the mTORC1 complex. This finding further suggested that OFZ formation requires inactivation of mTORC1. Raptor is a known target of JNK. Blocking JNK activation induced expression of Beclin-1 and LC3BII and loss of nuclei and organelles within 24hrs. JNK inactivation also suppressed activation of mTOR, Raptor and p70S6K (a direct target of mTORC1), showing that JNK is an upstream regulator of the mTORC1 signal. EM studies confirmed that JNK induced the formation of autophagic vesicles. Rapamycin, which also inactivates mTORC1 also upregulated LC3BII, induced autophagy and the subsequent loss of nuclei and organelles, confirming a role for autophagy in OFZ formation in the lens.
JNK inactivation suppresses the mTORC1 signal to induce loss of nuclei and organelles in the lens by an autophagic process.
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