Crosstalk of protein clearance, inflammasome, and Ca2+ channels in retinal pigment epithelium derived from age-related macular degeneration patients

Degeneration and/or dysfunction of retinal pigment epithelium (RPE) is generally detected as the formation of intracellular and extracellular protein aggregates, called lipofuscin and drusen, respectively, in patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. These clinical hallmarks are linked to dysfunctional protein homeostasis and inflammation and furthermore, are both regulated by changes in intracellular Ca2+ concentration. While many other cellular mechanisms have been considered in the investigations of AMD-RPE, there has been relatively little work on understanding the interactions of protein clearance, inflammation, and Ca2+ dynamics in disease pathogenesis. Here we established induced pluripotent stem cell–derived RPE from two patients with advanced AMD and from an age- and gender-matched control subject. We studied autophagy(definition) and inflammasome activation under disturbed proteostasis(definition) in these cell lines and investigated changes in their intracellular Ca2+ concentration and L-type voltage-gated Ca2+ channels. Our work demonstrated dysregulated autophagy and inflammasome activation in AMD-RPE accompanied by reduced intracellular free Ca2+ levels. Interestingly, we found currents through L-type voltage-gated Ca2+ channels to be diminished and showed these channels to be significantly localized to intracellular compartments in AMD-RPE. Taken together, the alterations in Ca2+ dynamics in AMD-RPE together with dysregulated autophagy and inflammasome activation indicate an important role for Ca2+ signaling in AMD pathogenesis, providing new avenues for the development of therapeutic approaches. Degeneration and/or dysfunction of retinal pigment epithelium (RPE) is generally detected as the formation of intracellular and extracellular protein aggregates, called lipofuscin and drusen, respectively, in patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. These clinical hallmarks are linked to dysfunctional protein homeostasis and inflammation and furthermore, are both regulated by changes in intracellular Ca2+ concentration. While many other cellular mechanisms have been considered in the investigations of AMD-RPE, there has been relatively little work on understanding the interactions of protein clearance, inflammation, and Ca2+ dynamics in disease pathogenesis. Here we established induced pluripotent stem cell–derived RPE from two patients with advanced AMD and from an age- and gender-matched control subject. We studied autophagy and inflammasome activation under disturbed proteostasis in these cell lines and investigated changes in their intracellular Ca2+ concentration and L-type voltage-gated Ca2+ channels. Our work demonstrated dysregulated autophagy and inflammasome activation in AMD-RPE accompanied by reduced intracellular free Ca2+ levels. Interestingly, we found currents through L-type voltage-gated Ca2+ channels to be diminished and showed these channels to be significantly localized to intracellular compartments in AMD-RPE. Taken together, the alterations in Ca2+ dynamics in AMD-RPE together with dysregulated autophagy and inflammasome activation indicate an important role for Ca2+ signaling in AMD pathogenesis, providing new avenues for the development of therapeutic approaches. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly population, and it has become a major public health and financial burden. The most prevalent form of AMD, dry AMD (80–85% of cases), is currently untreatable (1Kaarniranta K. Uusitalo H. Blasiak J. Felszeghy S. Kannan R. Kauppinen A. et al.Mechanisms of mitochondrial dysfunction(definition) and their impact on age-related macular degeneration.Prog. Retin. Eye Res. 2020; 79: 100858Google Scholar). Wet AMD in turn (affecting 10–15% of patients) is defined by the growth of blood vessels from choriocapillaris into the retina, a process called neovascularization. Intravitreal injections of anti-vascular endothelial growth factor and drugs (i.e. bevacizumab, ranibizumab, aflibercept, brolucizumab) have proven to be effective to some extent (2Hollaus M. Bühl W. Schmidt-Erfurth U. Sacu S. The challenges of treating neovascular age-related macular degeneration.Klin. Monbl. Augenheilkd. 2021; 239: 1033-1042Google Scholar). AMD etiology is known to be multifactorial. Aging, smoking, arteriosclerosis, obesity, hypertension, hypercholesterolemia, and unhealthy diet predispose to AMD (1Kaarniranta K. Uusitalo H. Blasiak J. Felszeghy S. Kannan R. Kauppinen A. et al.Mechanisms of mitochondrial dysfunction and their impact on age-related macular degeneration.Prog. Retin. Eye Res. 2020; 79: 100858Google Scholar). In addition, the genetic factors seem comprising up to 71% of the disease severity (3Warwick A. Lotery A. Genetics and genetic testing for age-related macular degeneration.Eye (Lond). 2018; 32: 849-857Google Scholar, 4Masuda T. Shimazawa M. Hara H. Retinal diseases associated with oxidative stress and the effects of a free radical scavenger (Edaravone).Oxid. Med. Cell. Longev. 2017; 20179208489Google Scholar, 5Wimmers S. Karl M.O. Strauss O. Ion channels in the RPE.Prog. Retin. Eye Res. 2007; 26: 263-301Google Scholar), which is strongly linked to increased oxidative stress (4Masuda T. Shimazawa M. Hara H. Retinal diseases associated with oxidative stress and the effects of a free radical scavenger (Edaravone).Oxid. Med. Cell. Longev. 2017; 20179208489Google Scholar). The increased oxidative stress may induce dysfunction of retinal pigment epithelium (RPE) leading to AMD. RPE is a tight monolayer of cells under the photoreceptors that has many important roles in maintaining retinal homeostasis and the function of photoreceptors, such as providing metabolic support, controlling ionic homeostasis, absorbing excessive light, regenerating visual pigment, and helping photoreceptor renewal through phagocytosis (5Wimmers S.