Editorial

2 pages, 165 KiB

Open AccessEditorial

Retinal Cell Biology in Health and Disease

by Hossein Ameri

Cells 2024, 13(4), 297; https://doi.org/10.3390/cells13040297 - 06 Feb 2024

Viewed by 497

The intricate network of cells and processes that govern retinal health has long been a subject of fascination and intensive study within the scientific community [...] Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

Research

Jump to: Editorial, Review

15 pages, 5045 KiB

Open AccessArticle

Non-Invasive Evaluation of Retinal Vascular Alterations in a Mouse Model of Optic Neuritis Using Laser Speckle Flowgraphy and Optical Coherence Tomography Angiography

by Seth E. Buscho, Fan Xia, Shuizhen Shi, Jonathan L. Lin, Bartosz Szczesny, Wenbo Zhang, Massoud Motamedi and Hua Liu

Cells 2023, 12(23), 2685; https://doi.org/10.3390/cells12232685 - 22 Nov 2023

Cited by 1 | Viewed by 993

Abstract

Optic neuritis, a characteristic feature of multiple sclerosis (MS), involves the inflammation of the optic nerve and the degeneration of retinal ganglion cells (RGCs). Although previous studies suggest that retinal blood flow alterations occur during optic neuritis, the precise location, the degree of [...] Read more.

Optic neuritis, a characteristic feature of multiple sclerosis (MS), involves the inflammation of the optic nerve and the degeneration of retinal ganglion cells (RGCs). Although previous studies suggest that retinal blood flow alterations occur during optic neuritis, the precise location, the degree of impairment, and the underlying mechanisms remain unclear. In this study, we utilized two emerging non-invasive imaging techniques, laser speckle flowgraphy (LSFG) and optical coherence tomography angiography (OCTA), to investigate retinal vascular changes in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). We associated these changes with leukostasis, RGC injury, and the overall progression of EAE. LSFG imaging revealed a progressive reduction in retinal blood flow velocity and increased vascular resistance near the optic nerve head in the EAE model, indicating impaired ocular blood flow. OCTA imaging demonstrated significant decreases in vessel density, number of junctions, and total vessel length in the intermediate and deep capillary plexus of the EAE mice. Furthermore, our analysis of leukostasis revealed a significant increase in adherent leukocytes in the retinal vasculature of the EAE mice, suggesting the occurrence of vascular inflammation in the early development of EAE pathology. The abovechanges preceded or were accompanied by the characteristic hallmarks of optic neuritis, such as RGC loss and reduced visual acuity. Overall, our study sheds light on the intricate relationship between retinal vascular alterations and the progression of optic neuritis as well as MS clinical score. It also highlights the potential for the development of image-based biomarkers for the diagnosis and monitoring of optic neuritis as well as MS, particularly in response to emerging treatments. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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22 pages, 9527 KiB

Open AccessFeature PaperArticle

Mitophagy in Astrocytes Is Required for the Health of Optic Nerve

by Meysam Yazdankhah, Sayan Ghosh, Haitao Liu, Stacey Hose, J. Samuel Zigler, Jr. and Debasish Sinha

Cells 2023, 12(20), 2496; https://doi.org/10.3390/cells12202496 - 20 Oct 2023

Cited by 1 | Viewed by 1088

Abstract

Mitochondrial dysfunction in astrocytes has been implicated in the development of various neurological disorders. Mitophagy, mitochondrial autophagy, is required for proper mitochondrial function by preventing the accumulation of damaged mitochondria. The importance of mitophagy, specifically in the astrocytes of the optic nerve (ON), [...] Read more.

Mitochondrial dysfunction in astrocytes has been implicated in the development of various neurological disorders. Mitophagy, mitochondrial autophagy, is required for proper mitochondrial function by preventing the accumulation of damaged mitochondria. The importance of mitophagy, specifically in the astrocytes of the optic nerve (ON), has been little studied. We introduce an animal model in which two separate mutations act synergistically to produce severe ON degeneration. The first mutation is in Cryba1, which encodes βA3/A1-crystallin, a lens protein also expressed in astrocytes, where it regulates lysosomal pH. The second mutation is in Bckdk, which encodes branched-chain ketoacid dehydrogenase kinase, which is ubiquitously expressed in the mitochondrial matrix and involved in the catabolism of the branched-chain amino acids. BCKDK is essential for mitochondrial function and the amelioration of oxidative stress. Neither of the mutations in isolation has a significant effect on the ON, but animals homozygous for both mutations (DM) exhibit very serious ON degeneration. ON astrocytes from these double-mutant (DM) animals have lysosomal defects, including impaired mitophagy, and dysfunctional mitochondria. Urolithin A can rescue the mitophagy impairment in DM astrocytes and reduce ON degeneration. These data demonstrate that efficient mitophagy in astrocytes is required for ON health and functional integrity. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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20 pages, 7270 KiB

Open AccessArticle

Disheveled-1 Interacts with Claudin-5 and Contributes to Norrin-Induced Endothelial Barrier Restoration

by Mónica Díaz-Coránguez, Laura González-González, Amy Wang, Xuwen Liu and David A. Antonetti

Cells 2023, 12(19), 2402; https://doi.org/10.3390/cells12192402 - 04 Oct 2023

Cited by 2 | Viewed by 1384

Abstract

Previous studies have revealed that norrin can reverse vascular endothelial-growth-factor (VEGF)-induced permeability in a β-catenin-dependent pathway. Here, we have explored the contribution of disheveled-1 (DVL1) in norrin-induced blood-retinal barrier (BRB) restoration. We provide evidence that in addition to canonical signaling, DVL1 promotes tight [...] Read more.

Previous studies have revealed that norrin can reverse vascular endothelial-growth-factor (VEGF)-induced permeability in a β-catenin-dependent pathway. Here, we have explored the contribution of disheveled-1 (DVL1) in norrin-induced blood-retinal barrier (BRB) restoration. We provide evidence that in addition to canonical signaling, DVL1 promotes tight junction (TJ) stabilization through a novel, non-canonical signaling pathway involving direct claudin-5 (CLDN5) binding. Immunofluorescence staining of rat retinal cross-sections showed enriched expression of DVL1 and 3 at endothelial capillaries and co-localization with CLDN5 and ZO-1 at the TJ complex in primary bovine retinal endothelial cells (BRECs). Barrier properties of BRECs were determined via measurements of trans-endothelial electrical resistance (TEER) or permeability to 70 kDa RITC-dextran. These studies demonstrated that norrin restoration of barrier properties after VEGF treatment required DVL1 as an siRNA knockdown of Dvl1 but not Dvl2 or Dvl3, reduced basal barrier properties and ablated norrin-induced barrier restoration. However, loss of Dvl1 did not decrease β-catenin signaling activity as measured by Axin2 mRNA expression, suggesting the contribution of a non-canonical pathway. DVL and TJ protein interactions were analyzed via co-immunoprecipitation of endogenous protein in BRECs, which demonstrated that DVL1 interacts with both CLDN5 and ZO-1, while DVL3 interacts only with ZO-1. These interactions were most abundant after inducing BRB restoration by treating BRECs with VEGF and norrin. DVL has previously been shown to form intramolecular bindings between the C-terminal PDZ-binding motif (PDZ-BM) with an internal PDZ domain. Co-transfection of HEK293 cells with DVL1 and CLDN5 or relevant mutants revealed that DVL1 interacts with CLDN5 through the DVL PDZ domain binding, CLDN5 PDZ-BM, in competition with DVL1 PDZ-BM, since DVL/CLDN5 interaction increases with deletion of the DVL1 PDZ-BM and decreases by co-expressing the C-terminal fragment of DVL1 containing the PDZ-BM or through deletion of CLDN5 PDZ-BM. In BREC cells, transfection of the C-terminal fragment of DVL1 downregulates the expression of CLDN5 but does not affect the expression of other proteins of the TJs, including ZO-1, occludin, CLDN1 or VE-cadherin. Blocking DVL1/CLDN5 interaction increased basal permeability and prevented norrin induction of barrier properties after VEGF. Combined with previous data, these results demonstrate that norrin signals through both a canonical β-catenin pathway and a non-canonical signaling pathway by which DVL1 directly binds to CLDN5 to promote barrier properties. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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23 pages, 80012 KiB

Open AccessArticle

Polarized RPE Secretome Preserves Photoreceptors in Retinal Dystrophic RCS Rats

by Kabir Ahluwalia, Juan-Carlos Martinez-Camarillo, Biju B. Thomas, Aditya Naik, Alejandra Gonzalez-Calle, Dimitrios Pollalis, Jane Lebkowski, Sun Young Lee, Debbie Mitra, Stan G. Louie and Mark S. Humayun

Cells 2023, 12(13), 1689; https://doi.org/10.3390/cells12131689 - 22 Jun 2023

Cited by 3 | Viewed by 1674

Abstract

Retinal degenerative diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa, lack effective therapies. Conventional monotherapeutic approaches fail to target the multiple affected pathways in retinal degeneration. However, the retinal pigment epithelium (RPE) secretes several neurotrophic factors addressing diverse cellular pathways, potentially preserving [...] Read more.

Retinal degenerative diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa, lack effective therapies. Conventional monotherapeutic approaches fail to target the multiple affected pathways in retinal degeneration. However, the retinal pigment epithelium (RPE) secretes several neurotrophic factors addressing diverse cellular pathways, potentially preserving photoreceptors. This study explored human embryonic stem cell-derived, polarized RPE soluble factors (PRPE-SF) as a combination treatment for retinal degeneration. PRPE-SF promoted retinal progenitor cell survival, reduced oxidative stress in ARPE-19 cells, and demonstrated critical antioxidant and anti-inflammatory effects for preventing retinal degeneration in the Royal College of Surgeons (RCS) rat model. Importantly, PRPE-SF treatment preserved retinal structure and scotopic b-wave amplitudes, suggesting therapeutic potential for delaying retinal degeneration. PRPE-SF is uniquely produced using biomimetic membranes for RPE polarization and maturation, promoting a protective RPE secretome phenotype. Additionally, PRPE-SF is produced without animal serum to avoid immunogenicity in future clinical development. Lastly, PRPE-SF is a combination of neurotrophic factors, potentially ameliorating multiple dysfunctions in retinal degenerations. In conclusion, PRPE-SF offers a promising therapeutic candidate for retinal degenerative diseases, advancing the development of effective therapeutic strategies for these debilitating conditions. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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18 pages, 5015 KiB

Open AccessFeature PaperArticle

Rabconnectin-3α/DMXL2 Is Locally Enriched at the Synaptic Ribbon of Rod Photoreceptor Synapses

by Alina Dittrich, Girish Ramesh, Martin Jung and Frank Schmitz

Cells 2023, 12(12), 1665; https://doi.org/10.3390/cells12121665 - 19 Jun 2023

Cited by 1 | Viewed by 1267

Abstract

Ribbon synapses reliably transmit synaptic signals over a broad signalling range. Rod photoreceptor ribbon synapses are capable of transmitting signals generated by the absorption of single photons. The high precision of ribbon synapses emphasizes the need for particularly efficient signalling mechanisms. Synaptic ribbons [...] Read more.

Ribbon synapses reliably transmit synaptic signals over a broad signalling range. Rod photoreceptor ribbon synapses are capable of transmitting signals generated by the absorption of single photons. The high precision of ribbon synapses emphasizes the need for particularly efficient signalling mechanisms. Synaptic ribbons are presynaptic specializations of ribbon synapses and are anchored to the active zone. Synaptic ribbons bind many synaptic vesicles that are delivered to the active zone for continuous and faithful signalling. In the present study we demonstrate with independent antibodies at the light- and electron microscopic level that rabconnectin-3α (RC3α)—alternative name Dmx-like 2 (DMXL2)—is localized to the synaptic ribbons of rod photoreceptor synapses in the mouse retina. In the brain, RC3α-containing complexes are known to interact with important components of synaptic vesicles, including Rab3-activating/inactivating enzymes, priming proteins and the vesicular H⁺-ATPase that acidifies the synaptic vesicle lumen to promote full neurotransmitter loading. The association of RC3α/DMXL2 with rod synaptic ribbons of the mouse retina could enable these structures to deliver only fully signalling-competent synaptic vesicles to the active zone thus contributing to reliable synaptic communication. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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16 pages, 5143 KiB

Open AccessArticle

Peptide Lv Promotes Trafficking and Membrane Insertion of K_Ca3.1 through the MEK1–ERK and PI3K–Akt Signaling Pathways

by Dylan L. Pham, Autumn Niemi, Ria Blank, Gabriella Lomenzo, Jenivi Tham, Michael L. Ko and Gladys Y.-P. Ko

Cells 2023, 12(12), 1651; https://doi.org/10.3390/cells12121651 - 17 Jun 2023

Cited by 1 | Viewed by 1074

Abstract

Peptide Lv is a small endogenous secretory peptide that is proangiogenic through hyperpolarizing vascular endothelial cells (ECs) by enhancing the current densities of K_Ca3.1 channels. However, it is unclear how peptide Lv enhances these currents. One way to enhance the current [...] Read more.

Peptide Lv is a small endogenous secretory peptide that is proangiogenic through hyperpolarizing vascular endothelial cells (ECs) by enhancing the current densities of K_Ca3.1 channels. However, it is unclear how peptide Lv enhances these currents. One way to enhance the current densities of ion channels is to promote its trafficking and insertion into the plasma membrane. We hypothesized that peptide Lv-elicited K_Ca3.1 augmentation occurs through activating the mitogen-activated protein kinase kinase 1 (MEK1)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)–protein kinase B (Akt) signaling pathways, which are known to mediate ion channel trafficking and membrane insertion in neurons. To test this hypothesis, we employed patch-clamp electrophysiological recordings and cell-surface biotinylation assays on ECs treated with peptide Lv and pharmaceutical inhibitors of ERK and Akt. Blocking ERK or Akt activation diminished peptide Lv-elicited EC hyperpolarization and increase in K_Ca3.1 current densities. Blocking PI3K or Akt activation decreased the level of plasma membrane-bound, but not the total amount of K_Ca3.1 protein in ECs. Therefore, the peptide Lv-elicited EC hyperpolarization and K_Ca3.1 augmentation occurred in part through channel trafficking and insertion mediated by MEK1–ERK and PI3K–Akt activation. These results demonstrate the molecular mechanisms of how peptide Lv promotes EC-mediated angiogenesis. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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18 pages, 7426 KiB

Open AccessArticle

Isolation and Characterization of the Primary Marmoset (Callithrix jacchus) Retinal Pigment Epithelial Cells

by Ha Young Jang, Chang Sik Cho, Young Mi Shin, Jina Kwak, Young Hoon Sung, Byeong-Cheol Kang and Jeong Hun Kim

Cells 2023, 12(12), 1644; https://doi.org/10.3390/cells12121644 - 16 Jun 2023

Cited by 1 | Viewed by 1393

Abstract

Marmosets have emerged as a valuable primate model in ophthalmic research due to their similarity to the human visual system and their potential for generating transgenic models to advance the development of therapies. In this study, we isolated and cultured primary retinal pigment [...] Read more.

Marmosets have emerged as a valuable primate model in ophthalmic research due to their similarity to the human visual system and their potential for generating transgenic models to advance the development of therapies. In this study, we isolated and cultured primary retinal pigment epithelium (RPE) cells from marmosets to investigate the mechanisms underlying RPE dysfunction in aging and age-related macular degeneration (AMD). We confirmed that our culture conditions and materials supported the formation of RPE monolayers with functional tight junctions that closely resembled the in vivo RPE. Since serum has been shown to induce epithelial–mesenchymal transition (EMT) in RPE cells, we compared the effects of fetal bovine serum (FBS) with serum-free supplements B27 on transepithelial electrical resistance (TER), cell proliferation, and morphological characteristics. Additionally, we assessed the age-related morphological changes of in vivo and primary RPE cells. Our results indicate that primary marmoset RPE cells exhibit in vivo-like characteristics, while cells obtained from an older donor show evidence of aging, including a failure to form a polarized monolayer, low TER, and delayed cell cycle. In conclusion, our primary marmoset RPE cells provide a reliable in vitro model for developing novel therapeutics for visual-threatening disorders such as AMD, which can be used before animal experiments using marmosets. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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28 pages, 7387 KiB

Open AccessFeature PaperArticle

Injury to Cone Synapses by Retinal Detachment: Differences from Rod Synapses and Protection by ROCK Inhibition

by Ellen Townes-Anderson, Éva Halász, Ilene Sugino, Amy L. Davidow, Laura J. Frishman, Luke Fritzky, Fawad A. K. Yousufzai and Marco Zarbin

Cells 2023, 12(11), 1485; https://doi.org/10.3390/cells12111485 - 27 May 2023

Cited by 3 | Viewed by 2008

Abstract

Attachment of a detached retina does not always restore vision to pre-injury levels, even if the attachment is anatomically successful. The problem is due in part to long-term damage to photoreceptor synapses. Previously, we reported on damage to rod synapses and synaptic protection [...] Read more.

Attachment of a detached retina does not always restore vision to pre-injury levels, even if the attachment is anatomically successful. The problem is due in part to long-term damage to photoreceptor synapses. Previously, we reported on damage to rod synapses and synaptic protection using a Rho kinase (ROCK) inhibitor (AR13503) after retinal detachment (RD). This report documents the effects of detachment, reattachment, and protection by ROCK inhibition on cone synapses. Conventional confocal and stimulated emission depletion (STED) microscopy were used for morphological assessment and electroretinograms for functional analysis of an adult pig model of RD. RDs were examined 2 and 4 h after injury or two days later when spontaneous reattachment had occurred. Cone pedicles respond differently than rod spherules. They lose their synaptic ribbons, reduce invaginations, and change their shape. ROCK inhibition protects against these structural abnormalities whether the inhibitor is applied immediately or 2 h after the RD. Functional restoration of the photopic b-wave, indicating cone-bipolar neurotransmission, is also improved with ROCK inhibition. Successful protection of both rod and cone synapses with AR13503 suggests this drug will (1) be a useful adjunct to subretinal administration of gene or stem cell therapies and (2) improve recovery of the injured retina when treatment is delayed. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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12 pages, 2215 KiB

Open AccessArticle

Mitochondrial Dysfunction and Impaired Antioxidant Responses in Retinal Pigment Epithelial Cells Derived from a Patient with RCBTB1-Associated Retinopathy

by Zhiqin Huang, Dan Zhang, Shang-Chih Chen, Di Huang, David Mackey, Fred K. Chen and Samuel McLenachan

Cells 2023, 12(10), 1358; https://doi.org/10.3390/cells12101358 - 10 May 2023

Cited by 2 | Viewed by 1577

Abstract

Mutations in the RCBTB1 gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we investigated the effect of RCBTB1 deficiency on mitochondria and oxidative stress responses in induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial [...] Read more.

Mutations in the RCBTB1 gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we investigated the effect of RCBTB1 deficiency on mitochondria and oxidative stress responses in induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial (RPE) cells from control subjects and a patient with RCBTB1-associated retinopathy. Oxidative stress was induced with tert-butyl hydroperoxide (tBHP). RPE cells were characterized by immunostaining, transmission electron microscopy (TEM), CellROX assay, MitoTracker assay, quantitative PCR and immunoprecipitation assay. Patient-derived RPE cells displayed abnormal mitochondrial ultrastructure and reduced MitoTracker fluorescence compared with controls. Patient RPE cells displayed increased levels of reactive oxygen species (ROS) and were more sensitive to tBHP-induced ROS generation than control RPE. Control RPE upregulated RCBTB1 and NFE2L2 expression in response to tBHP treatment; however, this response was highly attenuated in patient RPE. RCBTB1 was co-immunoprecipitated from control RPE protein lysates by antibodies for either UBE2E3 or CUL3. Together, these results demonstrate that RCBTB1 deficiency in patient-derived RPE cells is associated with mitochondrial damage, increased oxidative stress and an attenuated oxidative stress response. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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13 pages, 13747 KiB

Open AccessCommunication

Distribution of Copper, Iron, and Zinc in the Retina, Hippocampus, and Cortex of the Transgenic APP/PS1 Mouse Model of Alzheimer’s Disease

by Seyed Mostafa Hosseinpour Mashkani, David P. Bishop, Newsha Raoufi-Rad, Paul A. Adlard, Olga Shimoni and S. Mojtaba Golzan

Cells 2023, 12(8), 1144; https://doi.org/10.3390/cells12081144 - 13 Apr 2023

Cited by 2 | Viewed by 1902

Abstract

A mis-metabolism of transition metals (i.e., copper, iron, and zinc) in the brain has been recognised as a precursor event for aggregation of Amyloid-β plaques, a pathological hallmark of Alzheimer’s disease (AD). However, imaging cerebral transition metals in vivo can be extremely challenging. [...] Read more.

A mis-metabolism of transition metals (i.e., copper, iron, and zinc) in the brain has been recognised as a precursor event for aggregation of Amyloid-β plaques, a pathological hallmark of Alzheimer’s disease (AD). However, imaging cerebral transition metals in vivo can be extremely challenging. As the retina is a known accessible extension of the central nervous system, we examined whether changes in the hippocampus and cortex metal load are also mirrored in the retina. Laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) was used to visualise and quantify the anatomical distribution and load of Cu, Fe, and Zn in the hippocampus, cortex, and retina of 9-month-old Amyloid Precursor Protein/Presenilin 1 (APP/PS1, n = 10) and Wild Type (WT, n = 10) mice. Our results show a similar metal load trend between the retina and the brain, with the WT mice displaying significantly higher concentrations of Cu, Fe, and Zn in the hippocampus (p < 0.05, p < 0.0001, p < 0.01), cortex (p < 0.05, p = 0.18, p < 0.0001) and the retina (p < 0.001, p = 0.01, p < 0.01) compared with the APP/PS1 mice. Our findings demonstrate that dysfunction of the cerebral transition metals in AD is also extended to the retina. This could lay the groundwork for future studies on the assessment of transition metal load in the retina in the context of early AD. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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19 pages, 2432 KiB

Open AccessArticle

In Vivo Efficacy and Safety Evaluations of Therapeutic Splicing Correction Using U1 snRNA in the Mouse Retina

by Sebastian Swirski, Oliver May, Malte Ahlers, Bernd Wissinger, Martin Greschner, Christoph Jüschke and John Neidhardt

Cells 2023, 12(6), 955; https://doi.org/10.3390/cells12060955 - 21 Mar 2023

Cited by 1 | Viewed by 1683

Abstract

Efficacy and safety considerations constitute essential steps during development of in vivo gene therapies. Herein, we evaluated efficacy and safety of splice factor-based treatments to correct mutation-induced splice defects in an Opa1 mutant mouse line. We applied adeno-associated viruses to the retina. The [...] Read more.

Efficacy and safety considerations constitute essential steps during development of in vivo gene therapies. Herein, we evaluated efficacy and safety of splice factor-based treatments to correct mutation-induced splice defects in an Opa1 mutant mouse line. We applied adeno-associated viruses to the retina. The viruses transduced retinal cells with an engineered U1 snRNA splice factor designed to correct the Opa1 splice defect. We found the treatment to be efficient in increasing wild-type Opa1 transcripts. Correspondingly, Opa1 protein levels increased significantly in treated eyes. Measurements of retinal morphology and function did not reveal therapy-related side-effects supporting the short-term safety of the treatment. Alterations of potential off-target genes were not detected. Our data suggest that treatments of splice defects applying engineered U1 snRNAs represent a promising in vivo therapeutic approach. The therapy increased wild-type Opa1 transcripts and protein levels without detectable morphological, functional or genetic side-effects in the mouse eye. The U1 snRNA-based therapy can be tailored to specific disease gene mutations, hence, raising the possibility of a wider applicability of this promising technology towards treatment of different inherited retinal diseases. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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22 pages, 5457 KiB

Open AccessFeature PaperArticle

Membrane Attack Complex Mediates Retinal Pigment Epithelium Cell Death in Stargardt Macular Degeneration

by Eunice Sze Yin Ng, Nermin Kady, Jane Hu, Arpita Dave, Zhichun Jiang, Jacqueline Pei, Michael B. Gorin, Anna Matynia and Roxana A. Radu

Cells 2022, 11(21), 3462; https://doi.org/10.3390/cells11213462 - 02 Nov 2022

Cited by 9 | Viewed by 2855

Abstract

Recessive Stargardt disease (STGD1) is an inherited retinopathy caused by mutations in the ABCA4 gene. The ABCA4 protein is a phospholipid-retinoid flippase in the outer segments of photoreceptors and the internal membranes of retinal pigment epithelial (RPE) cells. Here, we show that RPE [...] Read more.

Recessive Stargardt disease (STGD1) is an inherited retinopathy caused by mutations in the ABCA4 gene. The ABCA4 protein is a phospholipid-retinoid flippase in the outer segments of photoreceptors and the internal membranes of retinal pigment epithelial (RPE) cells. Here, we show that RPE cells derived via induced pluripotent stem-cell from a molecularly and clinically diagnosed STGD1 patient exhibited reduced ABCA4 protein and diminished activity compared to a normal subject. Consequently, STGD1 RPE cells accumulated intracellular autofluorescence-lipofuscin and displayed increased complement C3 activity. The level of C3 inversely correlated with the level of CD46, an early negative regulator of the complement cascade. Persistent complement dysregulation led to deposition of the membrane attack complex on the surface of RPE cells, decrease in transepithelial resistance, and subsequent cell death. These findings are strong evidence of complement-mediated RPE cell damage in STGD1, in the absence of photoreceptors, caused by reduced CD46 regulatory protein. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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15 pages, 2759 KiB

Open AccessArticle

Visual Function and Survival of Injured Retinal Ganglion Cells in Aged Rbfox1 Knockout Animals

by Lei Gu, Jacky M. K. Kwong, Joseph Caprioli and Natik Piri

Cells 2022, 11(21), 3401; https://doi.org/10.3390/cells11213401 - 27 Oct 2022

Cited by 3 | Viewed by 1503

Abstract

Rbfox1 is a multifunctional RNA binding protein that regulates various aspects of RNA metabolism important for neuronal differentiation and normal physiology. Rbfox1 has been associated with neurodevelopmental and neurological conditions as well as age-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s. We have [...] Read more.

Rbfox1 is a multifunctional RNA binding protein that regulates various aspects of RNA metabolism important for neuronal differentiation and normal physiology. Rbfox1 has been associated with neurodevelopmental and neurological conditions as well as age-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s. We have shown that in mammalian retinas Rbfox1 is expressed in retinal ganglion cells (RGCs) and in amacrine cells (ACs). This study investigates the effect of advanced age (22-month-old mice) on visual function, retinal morphology and survival of injured retinal ganglion cells (RGC) in Rbfox1 knockout (KO) animals. A visual cliff test, which was used to evaluate visual function, showed that 22-month old Rbfox1 KO mice have profound depth perception deficiency. Retinal gross morphology in these animals appeared to be normal. Optic nerve crush (ONC) induced axonal injury resulted in approximately 50% of RGC loss in both Rbfox1 KO and age-matched control animals: the average RGC densities in uninjured control and Rbfox1 KO animals were 6274 ± 1673 cells/mm² and 6004 ± 1531 cells/mm², respectively, whereas 1 week after ONC, RGC numbers in the retinas of control and Rbfox1 KO mice were reduced to 2998 ± 858 cells/mm² and 3036 ± 857 cells/mm², respectively (Rbfox1 KO vs. Rbfox1 KO + ONC, p < 0.0001 and control vs. control + ONC, p < 0.0001). No significant difference between RGC numbers in Rbfox1 KO + ONC and age-matched control + ONC animals was observed, suggesting that Rbfox1 has no effect on the survival of injured RGCs. Interestingly, however, contrary to a commonly accepted view that the number of RGCs in old (18 month of age) compared to young animals is reduced by approximately 40%, the RGC densities in 22-month-old mice in this study were similar to those of 4-month-old counterparts. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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17 pages, 14605 KiB

Open AccessArticle

Assessment of Choroidal Vasculature and Innate Immune Cells in the Eyes of Albino and Pigmented Mice

by Ismail S. Zaitoun, Yong-Seok Song, Hammam B. Zaitoun, Christine M. Sorenson and Nader Sheibani

Cells 2022, 11(20), 3329; https://doi.org/10.3390/cells11203329 - 21 Oct 2022

Cited by 4 | Viewed by 2393

Abstract

The visualization of choroidal vasculature and innate immune cells in the eyes of pigmented mice has been challenging due to the presence of a retinal pigment epithelium (RPE) layer separating the choroid and retina. Here, we established methods for visualizing the choroidal macrophages, [...] Read more.

The visualization of choroidal vasculature and innate immune cells in the eyes of pigmented mice has been challenging due to the presence of a retinal pigment epithelium (RPE) layer separating the choroid and retina. Here, we established methods for visualizing the choroidal macrophages, mast cells, and vasculature in eyes of albino and pigmented mice using cell type-specific staining. We were able to visualize the choroidal arterial and venous systems. An arterial circle around the optic nerve was found in mice similar to the Zinn–Haller arterial circle that exists in humans and primates. Three different structural patterns of choriocapillaris were observed throughout the whole choroid: honeycomb-like, maze-like, and finger-like patterns. Choroidal mast cells were relatively few but dense around the optic nerve. Mast cell distribution in the middle and periphery was different among strains. Macrophages were found in all layers of the choroid. Thus, utilizing the simple and reliable methods described herein will allow the evaluation of transgenic and preclinical mouse models of ocular diseases that affect the choroid, including age-related macular degeneration (AMD), diabetic choroidopathy, and retinopathy of prematurity. These studies will advance our understanding of the pathophysiology, and molecular and cellular mechanisms that can be targeted therapeutically, in these diseases. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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16 pages, 4006 KiB

Open AccessArticle

Intraocular RGD-Engineered Exosomes and Active Targeting of Choroidal Neovascularization (CNV)

by Dimitrios Pollalis, Dongin Kim, Gopa Kumar Gopinadhan Nair, Changsun Kang, Arjun V. Nanda and Sun Young Lee

Cells 2022, 11(16), 2573; https://doi.org/10.3390/cells11162573 - 18 Aug 2022

Cited by 12 | Viewed by 3105

Abstract

Purpose: To assess the transretinal penetration of intravitreally injected retinal multicell-derived exosomes and to develop exosome-based active targeting of choroidal neovascularization (CNV) by bioengineering with ASL, which is composed of a membrane Anchor (BODIPY), Spacer (PEG), and targeting Ligands (cyclic RGD peptide). Methods: [...] Read more.

Purpose: To assess the transretinal penetration of intravitreally injected retinal multicell-derived exosomes and to develop exosome-based active targeting of choroidal neovascularization (CNV) by bioengineering with ASL, which is composed of a membrane Anchor (BODIPY), Spacer (PEG), and targeting Ligands (cyclic RGD peptide). Methods: Retinal multicell-derived exosomes were recovered from a whole mouse retina using differential ultracentrifugation. Their size, number, and morphology were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosome markers were confirmed using an exosome detection antibody array. Intravitreal injection of fluorescent (PKH-26)-labeled or engineered ASL exosomes (1 × 10⁶ exosomes/μL) were given to the wild-type mouse or laser-induced CNV mouse model. Retinal uptake of exosomes was assessed by in vivo retinal imaging microscopy and histological staining with DAPI, GSA, and anti-integrin α_v for retinal sections or choroid/RPE flat mounts. Active targeting of CNV was assessed by comparing retinal uptake between areas with and without CNV and by colocalization analysis of ASL exosomes with integrin α_v within CNV. Staining with anti-F4/80, anti-ICAM-1, and anti-GFAP antibodies on retinal sections were performed to identify intracellular uptake of exosomes and immediate reactive retinal gliosis after exosome treatment. Results: An average of 2.1 × 10⁹ particles/mL with a peak size of 140 nm exosomes were recovered. Rapid retinal penetration of intravitreally injected exosomes was confirmed by retinal imaging microscopy at 3 and 24 h post-injection. Intravitreally delivered PKH-26-labeled exosomes reached inner and outer retinal layers including IPL, INL, OPL, and ONL at 1 and 7 days post-injection. Intravitreally injected ASL exosomes were predominantly delivered to the area of CNV including ONL, RPE, and choroid in laser-induced CNV mouse models with 89.5% of colocalization with integrin α_v. Part of exosomes was also taken intracellularly to vascular endothelial cells and macrophages. After intravitreal injection, neither naive exosomes nor ASL exosomes induced immediate reactive gliosis. Conclusions: Intravitreally delivered retinal multicell-derived exosomes have good retinal penetration, and ASL modification of exosomes actively targets CNV with no immediate reactive gliosis. ASL exosomes have a great potential to serve as an intraocular drug delivery vehicle, allowing an active targeting strategy. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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15 pages, 2519 KiB

Open AccessArticle

Retinal Vascular Physiology Biomarkers in a 5XFAD Mouse Model of Alzheimer’s Disease

by Nathanael Matei, Sophie Leahy, Norman P. Blair, James Burford, Mansour Rahimi and Mahnaz Shahidi

Cells 2022, 11(15), 2413; https://doi.org/10.3390/cells11152413 - 04 Aug 2022

Cited by 3 | Viewed by 2280

Abstract

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder that affects the brain and retina and lacks reliable biomarkers for early diagnosis. As amyloid beta (Aβ) manifestations emerge prior to clinical symptoms and plaques of amyloid may cause vascular damage, identification of retinal vascular [...] Read more.

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder that affects the brain and retina and lacks reliable biomarkers for early diagnosis. As amyloid beta (Aβ) manifestations emerge prior to clinical symptoms and plaques of amyloid may cause vascular damage, identification of retinal vascular biomarkers may improve knowledge of AD pathophysiology and potentially serve as therapeutic targets. The purpose of the current study was to test the hypothesis that retinal hemodynamic and oxygen metrics are altered in 5XFAD mice. Methods: Thirty-two male mice were evaluated at 3 months of age: sixteen 5XFAD transgenic and sixteen wild-type mice. Spectral-domain optical coherence tomography, vascular oxygen tension, and blood flow imaging were performed in one eye of each mouse. After imaging, the imaged and fellow retinal tissues were submitted for histological sectioning and amyloid protein analysis, respectively. Protein analysis was also performed on the brain tissues. Results: Retinal physiological changes in venous diameter and blood velocity, arterial and venous oxygen contents, coupled with anatomical alterations in the thickness of retinal cell layers were detected in 5XFAD mice. Moreover, an increase in Aβ42 levels in both the retina and brain tissues was observed in 5XFAD mice. Significant changes in retinal oxygen delivery, metabolism, or extraction fraction were not detected. Based on compiled data from both groups, arterial oxygen content was inversely related to venous blood velocity and nerve fiber/ganglion cell layer thickness. Conclusions: Concurrent alterations in retinal hemodynamic and oxygen metrics, thickness, and tissue Aβ42 protein levels in 5XFAD mice at 3 months of age corresponded to previously reported findings in human AD. Overall, these results suggest that this mouse model can be utilized for studying pathophysiology of AD and evaluating potential therapies. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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12 pages, 8472 KiB

Open AccessArticle

Insights into Metabolic Activity and Structure of the Retina through Multiphoton Fluorescence Lifetime Imaging Microscopy in Mice

by Niranjana Kesavamoorthy, Jason A. Junge, Scott E. Fraser and Hossein Ameri

Cells 2022, 11(15), 2265; https://doi.org/10.3390/cells11152265 - 22 Jul 2022

Cited by 2 | Viewed by 1656

Abstract

Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the fundus of the eyes, but cannot detect NAD(P)H. We used multiphoton FLIM [...] Read more.

Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the fundus of the eyes, but cannot detect NAD(P)H. We used multiphoton FLIM to study the metabolic state of the retina in fixed eyes of wild-type mice C57BL6/J. We sectioned the eye using a polyacrylamide gel-embedding technique and estimated the percentage of bound NAD(P)H. We found that oxidative phosphorylation was the predominant metabolic state, particularly in the inner retina, when a fixed retina was used. We also demonstrated the feasibility of FAD imaging of the retina. In addition, we demonstrated that autofluorescence and various FLIM channels, such as hemoglobin, melanin and collagen, can be used to evaluate the structure of the retina and other parts of the eye without any special staining. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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Review

Jump to: Editorial, Research

21 pages, 3127 KiB

Open AccessReview

Current and Future Landscape in Genetic Therapies for Leber Hereditary Optic Neuropathy

by Hoda Shamsnajafabadi, Robert E. MacLaren and Jasmina Cehajic-Kapetanovic

Cells 2023, 12(15), 2013; https://doi.org/10.3390/cells12152013 - 07 Aug 2023

Cited by 2 | Viewed by 2096

Abstract

Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial genetic disease that causes blindness in young adults. Over 50 inherited mitochondrial DNA (mtDNA) variations are associated with LHON; however, more than 95% of cases are caused by one of three missense [...] Read more.

Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial genetic disease that causes blindness in young adults. Over 50 inherited mitochondrial DNA (mtDNA) variations are associated with LHON; however, more than 95% of cases are caused by one of three missense variations (m.11778 G > A, m.3460 G > A, and m.14484 T > C) encoding for subunits ND4, ND1, and ND6 of the respiration complex I, respectively. These variants remain silent until further and currently poorly understood genetic and environmental factors precipitate the visual loss. The clinical course that ensues is variable, and a convincing treatment for LHON has yet to emerge. In 2015, an antioxidant idebenone (Raxone) received European marketing authorisation to treat visual impairment in patients with LHON, and since then it was introduced into clinical practice in several European countries. Alternative therapeutic strategies, including gene therapy and gene editing, antioxidant and neurotrophic agents, mitochondrial biogenesis, mitochondrial replacement, and stem cell therapies are being investigated in how effective they might be in altering the course of the disease. Allotopic gene therapies are in the most advanced stage of development (phase III clinical trials) whilst most other agents are in phase I or II trials or at pre-clinical stages. This manuscript discusses the phenotype and genotype of the LHON disease with complexities and peculiarities such as incomplete penetrance and gender bias, which have challenged the therapies in development emphasising the most recent use of gene therapy. Furthermore, we review the latest results of the three clinical trials based on adeno-associated viral (AAV) vector-mediated delivery of NADH dehydrogenase subunit 4 (ND4) with mitochondrial targeting sequence, highlighting the differences in the vector design and the rationale behind their use in the allotopic transfer. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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13 pages, 839 KiB

Open AccessReview

Retinal Cell Damage in Diabetic Retinopathy

by Jing Zhou and Bo Chen

Cells 2023, 12(9), 1342; https://doi.org/10.3390/cells12091342 - 08 May 2023

Cited by 13 | Viewed by 3142

Abstract

Diabetic retinopathy (DR), the most common microvascular complication that occurs in diabetes mellitus (DM), is the leading cause of vision loss in working-age adults. The prevalence of diabetic retinopathy is approximately 30% of the diabetic population and untreated DR can eventually cause blindness. [...] Read more.

Diabetic retinopathy (DR), the most common microvascular complication that occurs in diabetes mellitus (DM), is the leading cause of vision loss in working-age adults. The prevalence of diabetic retinopathy is approximately 30% of the diabetic population and untreated DR can eventually cause blindness. For decades, diabetic retinopathy was considered a microvascular complication and clinically staged by its vascular manifestations. In recent years, emerging evidence has shown that diabetic retinopathy causes early neuronal dysfunction and neurodegeneration that may precede vascular pathology and affect retinal neurons as well as glial cells. This knowledge leads to new therapeutic strategies aiming to prevent dysfunction of retinal neurons at the early stage of DR. Early detection and timely treatment to protect retinal neurons are critical to preventing visual loss in DR. This review provides an overview of DR and the structural and functional changes associated with DR, and discusses neuronal degeneration during diabetic retinopathy, the mechanisms underlying retinal neurodegeneration and microvascular complications, and perspectives on current and future clinic therapies. Full article

(This article belongs to the Special Issue Retinal Cell Biology in Health and Disease)

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