The many faces of SIRT6 in the retina and retinal pigment epithelium.

Sirtuin 6 (SIRT6) is a member of the mammalian sirtuin family of NAD+-dependent protein deacylases, homologues of the yeast silent information regulator 2 (Sir2). SIRT6 has remarkably diverse functions and plays a key role in a variety of biological processes for maintaining cellular and organismal homeostasis. In this review, our primary aim is to summarize recent progress in understanding SIRT6's functions in the retina and retinal pigment epithelium (RPE), with the hope of further drawing interests in SIRT6 to increase efforts in exploring the therapeutic potential of this unique protein in the vision field. Before describing SIRT6's role in the eye, we first discuss SIRT6's general functions in a wide range of biological contexts. SIRT6 plays an important role in gene silencing, metabolism, DNA repair, antioxidant defense, inflammation, aging and longevity, early development, and stress response. In addition, recent studies have revealed SIRT6's role in macrophage polarization and mitochondrial homeostasis. Despite being initially understudied in the context of the eye, recent efforts have begun to elucidate the critical functions of SIRT6 in the retina and RPE. In the retina, SIRT6 is essential for adult retinal function, regulates energy metabolism by suppressing glycolysis that affects photoreceptor cell survival, protects retinal ganglion cells from oxidative stress, and plays a role in Müller cells during early neurodegenerative events in diabetic retinopathy. In the RPE, SIRT6 activates autophagy in culture and protects against oxidative stress in mice. Taken together, this review demonstrates that better understanding of SIRT6's functions and their mechanisms, both in and out of the context of the eye, holds great promise for the development of SIRT6-targeted strategies for prevention and treatment of blinding eye diseases.

Retinal ganglion cell repopulation for vision restoration in optic neuropathy: a roadmap from the RReSTORe Consortium.

Retinal ganglion cell (RGC) death in glaucoma and other optic neuropathies results in irreversible vision loss due to the mammalian central nervous system's limited regenerative capacity. RGC repopulation is a promising therapeutic approach to reverse vision loss from optic neuropathies if the newly introduced neurons can reestablish functional retinal and thalamic circuits. In theory, RGCs might be repopulated through the transplantation of stem cell-derived neurons or via the induction of endogenous transdifferentiation. The RGC Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) Consortium was established to address the challenges associated with the therapeutic repair of the visual pathway in optic neuropathy. In 2022, the RReSTORe Consortium initiated ongoing international collaborative discussions to advance the RGC repopulation field and has identified five critical areas of focus: (1) RGC development and differentiation, (2) Transplantation methods and models, (3) RGC survival, maturation, and host interactions, (4) Inner retinal wiring, and (5) Eye-to-brain connectivity. Here, we discuss the most pertinent questions and challenges that exist on the path to clinical translation and suggest experimental directions to propel this work going forward. Using these five subtopic discussion groups (SDGs) as a framework, we suggest multidisciplinary approaches to restore the diseased visual pathway by leveraging groundbreaking insights from developmental neuroscience, stem cell biology, molecular biology, optical imaging, animal models of optic neuropathy, immunology & immunotolerance, neuropathology & neuroprotection, materials science & biomedical engineering, and regenerative neuroscience. While significant hurdles remain, the RReSTORe Consortium's efforts provide a comprehensive roadmap for advancing the RGC repopulation field and hold potential for transformative progress in restoring vision in patients suffering from optic neuropathies.

Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Optic Nerve Crush and Experimental Glaucoma.

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor ß pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes present three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Experimental Glaucoma and Optic Nerve Crush.

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and in human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and on separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor ß pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

Alternative RNA Splicing in the Retina: Insights and Perspectives.

Alternative splicing is a fundamental and highly regulated post-transcriptional process that enhances transcriptome and proteome diversity. This process is particularly important in neuronal tissues, such as the retina, which exhibit some of the highest levels of differentially spliced genes in the body. Alternative splicing is regulated both temporally and spatially during neuronal development, can be cell-type-specific, and when altered can cause a number of pathologies, including retinal degeneration. Advancements in high-throughput sequencing technologies have facilitated investigations of the alternative splicing landscape of the retina in both healthy and disease states. Additionally, innovations in human stem cell engineering, specifically in the generation of 3D retinal organoids, which recapitulate many aspects of the in vivo retinal microenvironment, have aided studies of the role of alternative splicing in human retinal development and degeneration. Here we review these advances and discuss the ongoing development of strategies for the treatment of alternative splicing-related retinal disease.

Fatality assessment and variant risk monitoring for COVID-19 using three new hospital occupancy related metrics.

BACKGROUND: Though case fatality rate (CFR) is widely used to reflect COVID-19 fatality risk, its use is limited by large temporal and spatial variation. Hospital mortality rate (HMR) is also used to assess the severity of COVID-19, but HMR data is not directly available globally. Alternative metrics are needed for COVID-19 severity and fatality assessment. METHODS: We introduce new metrics for COVID-19 fatality risk measurements/monitoring and a new mathematical model to estimate average hospital length of stay for deaths (Ldead) and discharges (Ldis). Multiple data sources were used for our analyses. FINDINGS: We propose three, new metrics: hospital occupancy mortality rate (HOMR), ratio of total deaths to hospital occupancy (TDHOR), and ratio of hospital occupancy to cases (HOCR), for dynamic assessment of COVID-19 fatality risk. Estimated Ldead and Ldis for 501,079 COVID-19 hospitalizations in 34 US states between 7 August 2020 and 1 March 2021 were 18·2(95%CI:17·9-18·5) and 14·0(95%CI:13·9-14·0) days, respectively. We found the dramatic changes in COVID-19 CFR observed in 27 countries during early stages of the pandemic were mostly caused by undiagnosed cases. Compared to the first week of November 2021, the week mean HOCRs (mimics hospitalization-to-case ratio) for Omicron variant (58·6% of US new cases as of 25 December 2021) decreased 65·16% in the US as of 16 January 2022. INTERPRETATION: The new and reliable measurements described here could be useful for COVID-19 fatality risk and variant-associated risk monitoring. FUNDING: No specific funding was associated with the present study.

Aquaporin 4 is not present in normal porcine and human lamina cribrosa.

Aquaporin 4 is absent from astrocytes in the rodent optic nerve head, despite high expression in the retina and myelinated optic nerve. The purpose of this study was to quantify regional aquaporin channel expression in astrocytes of the porcine and human mouse optic nerve (ON). Ocular tissue sections were immunolabeled for aquaporins 1(AQP1), 4(AQP4), and 9(AQP9), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and alpha-dystroglycan (αDG) for their presence in retina, lamina, myelin transition zone (MTZ, region just posterior to lamina) and myelinated ON (MON). Semi- quantification of AQP4 labeling & real-time quantitative PCR (qPCR) data were analyzed in retina and ON tissue. Porcine and control human eyes had abundant AQP4 in Müller cells, retinal astrocytes, and myelinated ON (MON), but minimal expression in the lamina cribrosa. AQP1 and AQP9 were present in retina, but not in the lamina. Immunolabeling of GFAP and αDG was similar in lamina, myelin transition zone (MTZ) and MON regions. Semi-quantitative AQP4 labeling was at background level in lamina, increasing in the MTZ, and highest in the MON (lamina vs MTZ, MON; p≤0.05, p≤0.01, respectively). Expression of AQP4 mRNA was minimal in lamina and substantial in MTZ and MON, while GFAP mRNA expression was uniform among the lamina, MTZ, and MON regions. Western blot assay showed AQP4 protein expression in the MON samples, but none was detected in the lamina tissue. The minimal presence of AQP4 in the lamina is a specific regional phenotype of astrocytes in the mammalian optic nerve head.

The role of aquaporin-4 in optic nerve head astrocytes in experimental glaucoma.

PURPOSE: To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null). METHODS: C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression, and transport block were also analyzed. RESULTS: B6 mice had abundant AQP4 expression in Müller cells, astrocytes of retina and myelinated ON (MON), but minimal AQP4in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p≤0.05). Bead-injection led to comparable 3D-IOP elevation (p = 0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6). CONCLUSION: Lack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head.

Gene regulatory networks controlling vertebrate retinal regeneration.

Injury induces retinal Müller glia of certain cold-blooded vertebrates, but not those of mammals, to regenerate neurons. To identify gene regulatory networks that reprogram Müller glia into progenitor cells, we profiled changes in gene expression and chromatin accessibility in Müller glia from zebrafish, chick, and mice in response to different stimuli. We identified evolutionarily conserved and species-specific gene networks controlling glial quiescence, reactivity, and neurogenesis. In zebrafish and chick, the transition from quiescence to reactivity is essential for retinal regeneration, whereas in mice, a dedicated network suppresses neurogenic competence and restores quiescence. Disruption of nuclear factor I transcription factors, which maintain and restore quiescence, induces Müller glia to proliferate and generate neurons in adult mice after injury. These findings may aid in designing therapies to restore retinal neurons lost to degenerative diseases.

Retinal homeostasis and metformin-induced protection are not affected by retina-specific Pparδ knockout.

Peroxisome proliferator-activated receptors (PPARs) are a family of three nuclear hormone receptors (PPARα, PPARδ, and PPARγ) that are known to regulate expression of lipid metabolism and oxidative stress genes. Given their role in reducing oxidative stress in a variety of tissues, these genes are likely important for retinal homeostasis. This hypothesis has been further supported by recent studies suggesting that PPAR-activating drugs are protective against retinal degenerations. The objective of the present study was to determine the role of PPARδ in the neuroretina. RNA-seq data show that Pparα and Pparδ are both expressed in the retina, but that Pparδ is expressed at 4-fold higher levels. Single-cell RNAseq data show that Pparδ is broadly expressed in all retinal cell types. To determine the importance of Pparδ to the retina, we generated retina-specific Pparδ knockout mice. We found that deletion of Pparδ had a minimal effect on retinal function or morphology out to 12 months of age and did not increase retinal sensitivity to oxidative stress induced by exposure to bright light. While data show that PPARδ levels were increased by the drug metformin, PPARδ was not necessary for metformin-induced protection from light damage. These data suggest that Pparδ either has a redundant function with Pparα or is not essential for normal neuroretina function or resistance to oxidative stress.

Infectability of human BrainSphere neurons suggests neurotropism of SARS-CoV-2

Reports from Wuhan suggest that 36% of COVID-19 patients show neurological symptoms, and cases of viral encephalitis have been reported, suggesting that the virus is neurotropic under unknown circumstances. This is well established for other coronaviruses. In order to understand why some patients develop such symptoms and others do not, we address herein the infectability of the central nervous system (CNS). Reports that the ACE2 receptor ­ critical for virus entry into lung cells ­ is found in different neurons support this expectation. We employed a human induced pluripotent stem cell (iPSC)- derived BrainSphere model, which we used earlier for Zika, Dengue, HIV and John Cunningham virus infection studies. We detected the expression of the ACE2 receptor, but not TMPRSS2, in the model. Incubating the BrainSpheres for 6 hours with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 led to infection of a fraction of neural cells with replication of the virus evident at 72 hpi. Virus particles were found in the neuronal cell body extending into apparent neurite structures. PCR measurements corroborated the replication of the virus, suggesting at least a tenfold increase in virus copies per total RNA. Leveraging state-of-the-art 3D organotypic cell culture, which has been shown to allow both virus infection and modeling of (developmental) neurotoxicity but is at the same time simple enough to be transferred and used in a BSL-3 environment, we demonstrate, for the first time, the potential critically important neurotropism of SARS-CoV-2.

Disrupted Blood-Retina Lysophosphatidylcholine Transport Impairs Photoreceptor Health But Not Visual Signal Transduction.

Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood-retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using the Mfsd2a knock-out mouse as animal model. Our lipidome analyses of Mfsd2a-/- retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated in Mfsd2a-/- retinas. Mfsd2a-/- retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructure Mfsd2a-/- photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENT Phospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood-retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.

A Drug-Tunable Gene Therapy for Broad-Spectrum Protection against Retinal Degeneration.

Retinal degenerations are a large cluster of diseases characterized by the irreversible loss of light-sensitive photoreceptors that impairs the vision of 9.1 million people in the US. An attractive treatment option is to use gene therapy to deliver broad-spectrum neuroprotective factors. However, this approach has had limited clinical translation because of the inability to control transgene expression. To address this problem, we generated an adeno-associated virus vector named RPF2 that was engineered to express domains of leukemia inhibitory factor fused to the destabilization domain of bacterial dihydrofolate reductase. Fusion proteins containing the destabilization domain are degraded in mammalian cells but can be stabilized with the binding of the drug trimethoprim. Our data show that expression levels of RPF2 are tightly regulated by the dose of trimethoprim and can be reversed by trimethoprim withdrawal. We further show that stabilized RPF2 can protect photoreceptors and prevent blindness in treated mice.