June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Role of PI3K Signaling Inhibition in Inducing Autophagy-Dependent Organelle Elimination
Author Affiliations & Notes
  • Rifah Gheyas
    Anatomy, Cell Biology, and Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
  • A Sue Menko
    Anatomy, Cell Biology, and Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Rifah Gheyas, None; A Sue Menko, None
  • Footnotes
    Support  NIH Grant EY026478
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 2084. doi:
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      Rifah Gheyas, A Sue Menko; Role of PI3K Signaling Inhibition in Inducing Autophagy-Dependent Organelle Elimination. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2084.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : Blocking of PI3K/Akt signaling is known as a canonical autophagy-inducing pathway. Previous studies performed in our lab have linked autophagy mechanisms to the removal of mitochondria and ER from the center of the developing lens to form the Organelle Free Zone (OFZ). Our new studies have investigated the role of PI3K pathways in autophagy-dependent elimination of mitochondria, ER, and nuclei during lens development.

Methods :
At E12, prior to formation of the OFZ, chick embryo lenses are exposed for 24 hrs in organ culture to the pan-PI3K inhibitors, LY294002 or CH5132799, or the PI3K/Akt-specific inhibitor, MK-2206, with DMSO as the vehicle control. Differentiation state-specific regions were microdissected for immunoblot analysis. Whole lenses either cryosectioned for immunostaining with antibodies to organelles, autophagy markers, or signaling molecules, or labeled live for mitochondrial potential with Rhodamine123 prior to confocal image analysis. Development-stage-specific studies were also performed.

Results : In studies of the lysosomal protein LAMP1 we provide evidence that lysosomes are induced as OFZ formation begins at E13. Exposure of E12 lenses to either LY294002 or MK-2206 induces LAMP1, along with other autophagy-specific proteins, and the premature elimination of mitochondria and ER in lens fiber cells. While mitochondrial depolarization is known to occur before mitochondrial removal, blocking PI3K signaling with LY294002 had no impact on mitochondrial polarity, demonstrating that the link between suppression of PI3K signaling and mitochondrial loss was a direct result of autophagy induction. The two distinct pan-PI3K inhibitors lead to premature elimination of organelles. Pan-PI3K inhibition induced removal of nuclei, with nuclear loss associated with DNA cleavage as demonstrated by TUNEL assay, without impacting cell survival. In contrast, specifically blocking just the PI3K/Akt signaling axis alone induced premature loss of mitochondria and ER within 24 hrs without inducing DNA cleavage or nuclear loss.

Conclusions : The loss of mitochondria and ER to form the OFZ is by an autophagic-dependent mechanism activated by suppression of the PI3K/Akt signaling axis. While the elimination of nuclei is also PI3K-regulated, its induction involves suppression of multiple downstream effectors of PI3K.

This is a 2021 ARVO Annual Meeting abstract.


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