bims-mideyd 2022-02-20 papers

Redox Biol. 2022 Feb 09. pii: S2213-2317(22)00033-7. [Epub ahead of print]51 102261

REV-ERBα regulates age-related and oxidative stress-induced degeneration in retinal pigment epithelium via NRF2.

Shuo Huang, Chi-Hsiu Liu, Zhongxiao Wang, Zhongjie Fu, William R Britton, Alexandra K Blomfield, Theodore M Kamenecka, Joshua L Dunaief, Laura A Solt, Jing Chen.

Retinal pigment epithelium (RPE) dysfunction and atrophy occur in dry age-related macular degeneration (AMD), often leading to photoreceptor degeneration and vision loss. Accumulated oxidative stress during aging contributes to RPE dysfunction and degeneration. Here we show that the nuclear receptor REV-ERBα, a redox sensitive transcription factor, protects RPE from age-related degeneration and oxidative stress-induced damage. Genetic deficiency of REV-ERBα leads to accumulated oxidative stress, dysfunction and degeneration of RPE, and AMD-like ocular pathologies in aging mice. Loss of REV-ERBα exacerbates chemical-induced RPE damage, and pharmacological activation of REV-ERBα protects RPE from oxidative damage both in vivo and in vitro. REV-ERBα directly regulates transcription of nuclear factor erythroid 2-related factor 2 (NRF2) and its downstream antioxidant enzymes superoxide dismutase 1 (SOD1) and catalase to counter oxidative damage. Moreover, aged mice with RPE specific knockout of REV-ERBα also exhibit accumulated oxidative stress and fundus and RPE pathologies. Together, our results suggest that REV-ERBα is a novel intrinsic protector of the RPE against age-dependent oxidative stress and a new molecular target for developing potential therapies to treat age-related retinal degeneration.

Keywords: Age-related macular degeneration; Aging; NRF2; Oxidative damage; REV-ERBα; Retinal pigment epithelium

DOI: https://doi.org/10.1016/j.redox.2022.102261

Int J Mol Sci. 2022 Jan 30. pii: 1606. [Epub ahead of print]23(3):

SARM1 Ablation Is Protective and Preserves Spatial Vision in an In Vivo Mouse Model of Retinal Ganglion Cell Degeneration.

Laura K Finnegan, Naomi Chadderton, Paul F Kenna, Arpad Palfi, Michael Carty, Andrew G Bowie, Sophia Millington-Ward, G Jane Farrar.

The challenge of developing gene therapies for genetic forms of blindness is heightened by the heterogeneity of these conditions. However, mechanistic commonalities indicate key pathways that may be targeted in a gene-independent approach. Mitochondrial dysfunction and axon degeneration are common features of many neurodegenerative conditions including retinal degenerations. Here we explore the neuroprotective effect afforded by the absence of sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1), a prodegenerative NADase, in a rotenone-induced mouse model of retinal ganglion cell loss and visual dysfunction. Sarm1 knockout mice retain visual function after rotenone insult, displaying preservation of photopic negative response following rotenone treatment in addition to significantly higher optokinetic response measurements than wild type mice following rotenone. Protection of spatial vision is sustained over time in both sexes and is accompanied by increased RGC survival and additionally preservation of axonal density in optic nerves of Sarm1-/- mice insulted with rotenone. Primary fibroblasts extracted from Sarm1-/- mice demonstrate an increased oxygen consumption rate relative to those from wild type mice, with significantly higher basal, maximal and spare respiratory capacity. Collectively, our data indicate that Sarm1 ablation increases mitochondrial bioenergetics and confers histological and functional protection in vivo in the mouse retina against mitochondrial dysfunction, a hallmark of many neurodegenerative conditions including a variety of ocular disorders.

Keywords: NAD+; NADase; axon degeneration; retinal degeneration; sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1)

DOI: https://doi.org/10.3390/ijms23031606

Exp Eye Res. 2022 Feb 12. pii: S0014-4835(22)00062-8. [Epub ahead of print] 108981

Quantification of mitophagy using mKeima-mito in cultured human primary retinal pigment epithelial cells.

Cody R Fisher, Mara C Ebeling, Deborah A Ferrington.

The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of visual disorders, including age-related macular degeneration (AMD). Utilizing primary RPE cultures to investigate disease is an important step in understanding disease mechanisms. However, the use of primary RPE cultures presents a number of challenges, including the limited number of cells available and the presence of auto-fluorescent pigment that interferes with quantifying fluorescent probes. Additionally, primary RPE are difficult to transfect with exogenous nucleic acids traditionally used for fluorescent imaging. To overcome these challenges, we used an adeno-associated viral (AAV) vector to express a pH sensitive fluorescent protein, mKeima, fused to the mitochondrial targeting sequence of cytochrome oxidase subunit 8A (mKeima-mito). mKeima-mito allows for quantification of mitochondrial autophagy (mitophagy) in live cell time lapse imaging experiments. We also developed an image analysis pipeline to selectively quantify mKeima-mito while removing the signal of auto-fluorescent pigment from the dataset by utilizing information from the mKeima fluorescent channels. These techniques are demonstrated in primary RPE cultures expressing mKeima-mito treated with 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), an uncoupler that depolarizes the mitochondrial membrane and leads to mitochondrial fragmentation and mitophagy. The techniques outlined provide a roadmap for investigating disease mechanisms or the effect of treatments utilizing fluorescent probes in an important cell culture model.

DOI: https://doi.org/10.1016/j.exer.2022.108981

BMC Biol. 2022 Feb 15. 20(1): 47

Hyaluronidase-1-mediated glycocalyx impairment underlies endothelial abnormalities in polypoidal choroidal vasculopathy.

Kan Xing Wu, Natalie Jia Ying Yeo, Chun Yi Ng, Florence Wen Jing Chioh, Qiao Fan, Xianfeng Tian, Binxia Yang, Gunaseelan Narayanan, Hui Min Tay, Han Wei Hou, N Ray Dunn, Xinyi Su, Chui Ming Gemmy Cheung, Christine Cheung.

BACKGROUND: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis.
RESULTS: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples.
CONCLUSIONS: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.

Keywords: Age-related macular degeneration; Endothelial dysfunction; Glycocalyx; Hyaluronidase-1; Polypoidal choroidal vasculopathy

DOI: https://doi.org/10.1186/s12915-022-01244-z