Constitutive Activation of Mechanistic Target of Rapamycin Complex 1 (mTORC1) Caused Degeneration of the Retinal Pigment Epithelium (RPE)

Abstract

Purpose : Signaling pathways mediated by mTORC1 play key roles in regulating cell metabolism, growth, autophagy, and immune responses. Our previous work demonstrated that RPE aging is associated with elevated mTORC1 activity. The causative link between aberrant mTORC1 activation and RPE degeneration has not been established. The goals of this study are to characterize the retinal phenotype of a conditional knockout mice with RPE-specific mTORC1 hyperactivation and to explore the underlying mechanisms.

Methods : Mice with constitutive mTORC1 activation in the RPE were generated by depleting its upstream suppressor protein tuberous sclerosis complex 1 (TSC1), through crossing TSC1^flox/flox mice with BEST1-Cre transgenic C57BL/6J mice. The RPE-specific knockout of TSC1 and the resulting mTORC1 hyperactivation were confirmed by western blot or immunostaining of RPE flat mounts. The ocular phenotype was examined by fundus imaging, optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) in live animals, and was further validated by histopathology and transmission electron microscopy (TEM). Visual acuity was assessed by measuring the optokinetic responses. Selected downstream targets of mTORC1 were further analyzed by quantitative RT-PCR and western blot analyses.

Results : TSC1^ΔRPE mice displayed mTORC1 hyperactivation specifically in the RPE, without affecting the mTORC2 pathway. RPE pathology was evident in 5-week-old homozygous knockouts and progressed with age. Between 3 to 6 months, TSC1^ΔRPE mice showed extensive RPE hypo/hyperpigmentation, lipid droplet accumulation and subretinal cell infiltration. TEM detected membrane whorls between photoreceptor outer segments and apical surface of the RPE, fragmented mitochondria and other signs of defective vesicular trafficking in the RPE. The mRNA level of TFEB and VPS11 was reduced in TSC1^ΔRPE mice compared to TSC1^flox/flox, BEST1-Cre or wild type mice.

Conclusions : Hyperactivated mTORC1 can lead to RPE and photoreceptor degeneration, by mechanisms that involve disrupted membrane trafficking.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.