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

David A Merle; Francesca Provenzano; Mohamed Ali Jarboui; Ellen Kilger; Simon J Clark; Michela Deleidi; Angela Armento; Marius Ueffing

doi:10.3390/antiox10121944

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

Antioxidants (Basel). 2021 Dec 3;10(12):1944. doi: 10.3390/antiox10121944.

Authors

David A Merle^{1

2}, Francesca Provenzano³, Mohamed Ali Jarboui^{1

4}, Ellen Kilger¹, Simon J Clark^{1

5

6}, Michela Deleidi³, Angela Armento¹, Marius Ueffing^{1

3}

Affiliations

¹ Institute for Ophthalmic Research, Department for Ophthalmology, Eberhard Karls University of Tübingen, 72076 Tübingen, Germany.
² Department of Ophthalmology, Medical University of Graz, 8036 Graz, Austria.
³ German Center for Neurodegenerative Diseases (DZNE), 72076 Tübingen, Germany.
⁴ Core Facility for Medical Bioanalytics, Institute for Ophthalmic Research, Eberhard-Karls University of Tuebingen, 72076 Tübingen, Germany.
⁵ Department for Ophthalmology, University Eye Clinic, Eberhard Karls University of Tübingen, 72076 Tübingen, Germany.
⁶ Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK.

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.

Keywords: age-related macular degeneration (AMD); complement factor H (CFH/FH); interactome; mammalian target of rapamycin (mTOR); mitochondrial respiration; retinal pigment epithelium (RPE) cells; ubiquitin-proteasomal pathway.