mTOR Inhibition via Rapamycin Treatment Partially Reverts the Deficit in Energy Metabolism Caused by FH Loss in RPE Cells

Abstract

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including aging, genetics, and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation, and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), which leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. Although the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress, and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of which are associated with UPS. As inhibition of mTORC1 was previously shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.

Nutritional Factors: Benefits in Glaucoma and Ophthalmologic Pathologies

“…General strategies targeting the mTOR signaling pathway have been determined to possess viable therapeutic potentials for managing ocular neurodegenerative disorders [ 126 ]. The in vivo application of RAPA was correlated with the partial reversal of metabolic failure via the loss of complement factor H in retinal pigment epithelial cells, which is an important pathophysiological contributor to photoreceptor degeneration in ARMD [ 127 ]. mTOR inhibition via systemic administration of RAPA also has been shown to suppress VEGF expression and retinal oxidative stress response to hyperglycemia in murine models [ 128 ].…”

“…Regarding glaucoma, topical application of RAPA has been associated with decreased intraocular pressure via the added inhibition of Rho-associated protein kinase in a murine aqueous humor [ 127 ]. mTOR inhibition by RAPA has also been found to provide neuroprotection for retinal ganglion cells (RGCs) [ 130 ].…”

Characterization of the Basal and mTOR-Dependent Acute Pulmonary and Systemic Immune Response in a Murine Model of Combined Burn and Inhalation Injury

“…Specifically, we utilized systemic administration of rapamycin (daily, starting 7 days before the injury) to inhibit mTOR activity at the time of burn or B+I injury. We and others published that a 7-day protocol of high-dose rapamycin (4–10 mg/kg/day) inhibits intracellular phosphorylation of the downstream molecule S6 via flow cytometry after burn injury in immune cells [ 41 , 46 , 49 , 50 ]. As we previously reported, we did not observe a significant difference in mortality between vehicle-treated burn-only and rapamycin-treated burn-only mice (data not shown [ 41 ]).…”

CFH Haploinsufficiency and Complement Alterations in Early-Onset Macular Degeneration

“…Other future opportunities include studying the effects of decreased CFH expression on its non-canonical roles in chemostasis, 25 cellular attachment, 46 , 47 modulation of lipoprotein binding, 48 upregulation of the pro-inflammatory pathway, 33 and cellular metabolism. 49 Finally, Müller glia, microglia, and retinal vascular cells are major contributors of complement activators in the neural retina, and apically secreted CFH may play a role in regulating subretinal complement activity. 50 Overcoming the technical challenges of including the neural retina and choroidal endothelium, which contributes significantly to local CFH and FHL-1, 50 , 51 would improve in vitro disease modeling.…”