bims-mideyd 2023-05-07 papers

bims-mideyd

Biomed News

on Mitochondrial dysfunction in eye diseases

Issue of 2023‒05‒07
six papers selected by
Raji Shyam
Indiana University Bloomington

Crosstalk of protein clearance, inflammasome, and Ca2+ channels in retinal pigment epithelium derived from age-related macular degeneration patients.
Aging induces cell loss and a decline in phagosome processing in the mouse retinal pigment epithelium.
CRISPR editing demonstrates rs10490924 raised oxidative stress in iPSC-derived retinal cells from patients with ARMS2/HTRA1-related AMD.
Altered zinc homeostasis in a primary cell culture model of the retinal pigment epithelium.
Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.
Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures.

J Biol Chem. 2023 May 01. pii: S0021-9258(23)01798-2. [Epub ahead of print] 104770

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

Viivi Karema-Jokinen, Ali Koskela, Maria Hytti, Heidi Hongisto, Taina Viheriälä, Mikko Liukkonen, Tommi Torsti, Heli Skottman, Anu Kauppinen, Soile Nymark, Kai Kaarniranta.

  Degeneration and/or dysfunction of retinal pigment epithelium (RPE) is generally detected as the formation of intra- 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.

Keywords:  Age-related macular degeneration; Autophagy; Calcium; Heat shock protein (HSP); Induced pluripotent stem cell (iPS cell) (iPSC); Inflammasome; L-type Ca(2+) channels; Retinal pigment epithelium

DOI:  https://doi.org/10.1016/j.jbc.2023.104770
Neurobiol Aging. 2023 Mar 13. pii: S0197-4580(23)00048-9. [Epub ahead of print]128 1-16

Aging induces cell loss and a decline in phagosome processing in the mouse retinal pigment epithelium.

Jessica Y W Ma, Ursula Greferath, Josephine H C Wong, Linda J Fothergill, Andrew I Jobling, Kirstan A Vessey, Erica L Fletcher.

  Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss and dysfunction in the retinal pigment epithelium (RPE) with age is known to contribute to disease development. The aim of this study was to investigate how the C57BL/6J mouse RPE changes with age. RPE structure was found to change with age and eccentricity, with cell size increasing, nuclei lost, and tight junctions altered in the peripheral retina. Phagocytosis of photoreceptor outer segments (POS) by the RPE was investigated using gene expression analysis and histology. RNA-Seq transcriptomic gene profiling of the RPE showed a downregulation of genes involved in phagosome processing and histological analysis showed a decline in phagosome-lysosome association in the aged tissue. In addition, failures in the autophagy pathway that modulates intracellular waste degradation were observed in the aged RPE tissue. These findings highlight that RPE cell loss and slowing of POS processing contribute to RPE dysfunction with age and may predispose the aging eye to AMD development.

Keywords:  Age-related macular degeneration; Aging; LC3-associated phagocytosis; Lysosomes; Mouse; Murine; Phagocytosis; Retinal pigment epithelium; Senescence

DOI:  https://doi.org/10.1016/j.neurobiolaging.2023.03.003
Proc Natl Acad Sci U S A. 2023 May 09. 120(19): e2215005120

CRISPR editing demonstrates rs10490924 raised oxidative stress in iPSC-derived retinal cells from patients with ARMS2/HTRA1-related AMD.

Ya-Ju Chang, Laura A Jenny, Yong-Shi Li, Xuan Cui, Yang Kong, Yao Li, Janet R Sparrow, Stephen H Tsang.

  Genome-wide association studies (GWAS) have identified genetic risk loci for age-related macular degeneration (AMD) on the chromosome 10q26 (Chr10) locus and are tightly linked: the A69S (G>T) rs10490924 single-nucleotide variant (SNV) and the AATAA-rich insertion-deletion (indel, del443/ins54), which are found in the age-related maculopathy susceptibility 2 (ARMS2) gene, and the G512A (G>A) rs11200638 SNV, which is found in the high-temperature requirement A serine peptidase 1 (HTRA1) promoter. The fourth variant is Y402H complement factor H (CFH), which directs CFH signaling. CRISPR manipulation of retinal pigment epithelium (RPE) cells may allow one to isolate the effects of the individual SNV and thus identify SNV-specific effects on cell phenotype. Clustered regularly interspaced short palindromic repeats (CRISPR) editing demonstrates that rs10490924 raised oxidative stress in induced pluripotent stem cell (iPSC)-derived retinal cells from patients with AMD. Sodium phenylbutyrate preferentially reverses the cell death caused by ARMS2 rs10490924 but not HTRA1 rs11200638. This study serves as a proof of concept for the use of patient-specific iPSCs for functional annotation of tightly linked GWAS to study the etiology of a late-onset disease phenotype. More importantly, we demonstrate that antioxidant administration may be useful for reducing reactive oxidative stress in AMD, a prevalent late-onset neurodegenerative disorder.

Keywords:  CRISPR; age-related macular degeneration; stem cells

DOI:  https://doi.org/10.1073/pnas.2215005120
Front Nutr. 2023 ;10 1124987

Altered zinc homeostasis in a primary cell culture model of the retinal pigment epithelium.

Ana Álvarez-Barrios, Lydia Álvarez, Enol Artime, Montserrat García, Imre Lengyel, Rosario Pereiro, Héctor González-Iglesias.

  The retinal pigment epithelium (RPE) is progressively degenerated during age-related macular degeneration (AMD), one of the leading causes of irreversible blindness, which clinical hallmark is the buildup of sub-RPE extracellular material. Clinical observations indicate that Zn dyshomeostasis can initiate detrimental intracellular events in the RPE. In this study, we used a primary human fetal RPE cell culture model producing sub-RPE deposits accumulation that recapitulates features of early AMD to study Zn homeostasis and metalloproteins changes. RPE cell derived samples were collected at 10, 21 and 59 days in culture and processed for RNA sequencing, elemental mass spectrometry and the abundance and cellular localization of specific proteins. RPE cells developed processes normal to RPE, including intercellular unions formation and expression of RPE proteins. Punctate deposition of apolipoprotein E, marker of sub-RPE material accumulation, was observed from 3 weeks with profusion after 2 months in culture. Zn cytoplasmic concentrations significantly decreased 0.2 times at 59 days, from 0.264 ± 0.119 ng·μg-1 at 10 days to 0.062 ± 0.043 ng·μg-1 at 59 days (p < 0.05). Conversely, increased levels of Cu (1.5-fold in cytoplasm, 5.0-fold in cell nuclei and membranes), Na (3.5-fold in cytoplasm, 14.0-fold in cell nuclei and membranes) and K (6.8-fold in cytoplasm) were detected after 59-days long culture. The Zn-regulating proteins metallothioneins showed significant changes in gene expression over time, with a potent down-regulation at RNA and protein level of the most abundant isoform in primary RPE cells, from 0.141 ± 0.016 ng·mL-1 at 10 days to 0.056 ± 0.023 ng·mL-1 at 59 days (0.4-fold change, p < 0.05). Zn influx and efflux transporters were also deregulated, along with an increase in oxidative stress and alterations in the expression of antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. The RPE cell model producing early accumulation of extracellular deposits provided evidences on an altered Zn homeostasis, exacerbated by changes in cytosolic Zn-binding proteins and Zn transporters, along with variations in other metals and metalloproteins, suggesting a potential role of altered Zn homeostasis during AMD development.

Keywords:  AMD in vitro model; cell culture; physical barrier; retinal pigment epithelium; sub-RPE deposits; zinc dyshomeostasis

DOI:  https://doi.org/10.3389/fnut.2023.1124987
Proc Natl Acad Sci U S A. 2023 May 09. 120(19): e2221045120

Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Jennings C Luu, Aicha Saadane, Henri Leinonen, Elliot H Choi, Fangyuan Gao, Dominik Lewandowski, Maximilian Halabi, Christopher L Sander, Arum Wu, Jacob M Wang, Rupesh Singh, Songqi Gao, Emma M Lessieur, Zhiqian Dong, Grazyna Palczewska, Robert F Mullins, Neal S Peachey, Philip D Kiser, Marcin Tabaka, Timothy S Kern, Krzysztof Palczewski.

  Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Keywords:  eye; phosphodiesterase; retina; retinal degeneration; rhodopsin

DOI:  https://doi.org/10.1073/pnas.2221045120
J Vis Exp. 2023 Apr 14.

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures.

Qitao Zhang, Gillian Autterson, Jason M L Miller.

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

DOI: https://doi.org/10.3791/65242