Mitochondria-dependent phase separation of disease-relevant proteins drives pathological features of age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) damages the retinal pigment epithelium (RPE), the tissue that safeguards photoreceptor health, leading to irreversible vision loss. Polymorphisms in cholesterol and complement genes are implicated in AMD, yet mechanisms linking risk variants to RPE injury remain unclear. We sought to determine how allelic variants in the apolipoprotein E cholesterol transporter modulate RPE homeostasis and function. Using live-cell imaging, we show that inefficient cholesterol transport by the AMD risk-associated ApoE2 increases RPE ceramide, leading to autophagic defects and complement-mediated mitochondrial damage. Mitochondrial injury drives redox state-sensitive cysteine-mediated phase separation of ApoE2, forming biomolecular condensates that could nucleate drusen. The protective ApoE4 isoform lacks these cysteines and is resistant to phase separation and condensate formation. In Abca4 -/-Stargardt macular degeneration mice, mitochondrial dysfunction induces liquid-liquid phase separation of p62/SQSTM1, a multifunctional protein that regulates autophagy. Drugs that decrease RPE cholesterol or ceramide prevent mitochondrial injury and phase separation in vitro and in vivo. In AMD donor RPE, mitochondrial fragmentation correlates with ApoE and p62 condensates. Our studies demonstrate that major AMD genetic and biological risk pathways converge upon RPE mitochondria, and identify mitochondrial stress-mediated protein phase separation as an important pathogenic mechanism and promising therapeutic target in AMD.

Analysis of mitochondrial dynamics and function in the retinal pigment epithelium by high-speed high-resolution live imaging

“…In addition to dyes such as Mitotracker, viral transduction using baculovirus to express fluorescently-tagged pyruvate dehydrogenase (CellLight Mitochondria, Thermo Fisher) can be used for live imaging ( Table 1 ). Transducing primary polarized RPE with CellLight Mitochondria at ∼14 virus particles/cell for 18 h yielded transduction efficiencies of ≥ 40% with no observed toxicity and labeled mitochondria comparable to that seen with Mitotracker probes ( Tan et al, 2016 ; La Cunza et al, 2021 ). Compared to transgene expression, fluorescent probes that label the mitochondria uniformly label almost all the cells in the monolayer.…”

“…Investigating mitochondrial dynamics in the intact living mouse retina is essential for understanding how RPE mitochondrial function and dysfunction contributes to retinal disease. Mice, which are widely used to model and study retinal degenerations, are generated on different genetic backgrounds such as the pigmented 129S1/SvlmJ and C57BL/6J mice, the BALB/c albino mice, as well as mixed backgrounds ( Zhao et al, 2011 ; Mao et al, 2014 ; Biswal et al, 2018 ; Brown et al, 2019 ; La Cunza et al, 2021 ). The RPE in its native state is pigmented due to the presence of melanosomes, which protect the RPE by scavenging free radicals ( Rózanowski et al, 2008 ).…”

“…The unique features of the RPE–a postmitotic, polarized tissue that deals with the immense metabolic burden of daily photoreceptor outer segment clearance in a highly oxidative environment–places unique stresses on its mitochondria. Recent studies show that mitochondrial function influences RPE cell fate ( Hazim et al, 2022 ), and that AMD-associated stressors such as smoking and complement activation promote mitochondrial fragmentation and dysfunction ( Woodell et al, 2013 ; Tan et al, 2016 ; La Cunza et al, 2021 ). Given that RPE mitochondrial function directly impacts photoreceptor health and vision ( Handa et al, 2019 ; Ferrington et al, 2021 ; Hurley, 2021 ), insight into mechanisms and machinery that maintain mitochondrial homeostasis in the RPE in vivo and how they are disrupted in disease is essential for developing sight-saving therapies.…”

“…Increased mtDNA damage and OXPHOS defects ( Ferrington et al, 2017 ; Zhang et al, 2020 ) have been documented in RPE cultures established from human donors with AMD. Using high-resolution imaging, we recently demonstrated that RPE mitochondria in retinal cryosections from AMD donors are highly fragmented, in contrast to the interconnected, healthy mitochondria seen in the RPE of unaffected donors ( La Cunza et al, 2021 ). High-speed live imaging of cell-based and mouse model of macular degeneration helped us identify a novel biophysical mechanism that directly connects RPE mitochondrial dysfunction to drusen formation: redox state changes in fragmented mitochondria drive liquid-liquid phase separation of proteins with reactive cysteines, which nucleate drusen-like aggregates within the RPE ( La Cunza et al, 2021 ).…”

Potential Role of Artemisinin And Its Derivatives In The Treatment of Age-related Macular Degeneration

“…The retina is one of the places with high oxygen consumption and high metabolism in the human body, which is related to the presence of a large number of mitochondria in RPE cells and a large number of selective ion channels in the RPE cell membrane. Mitochondria are important sites of oxidative metabolism in the human body, and mitochondrial dysfunction caused by hypoxia, in ammation and other factors is one of the important factors for oxidative damage in RPE cells [14,37,40,41]. The change of RPE cells membrane will lead to abnormal function of PRE transport and metabolism system.…”