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

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

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 &amp; 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.</jats:p

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

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.publishe

Primer sequences and amplification efficiencies for qPCR.

Primer sequences and amplification efficiencies for qPCR.</p

Relative mRNA expression of <i>AQP4</i> (A), <i>GFAP</i> (B), and <i>CD68</i> (C) in regions of the porcine retina and optic nerve head.

All samples were normalized to the geometric mean of the corresponding housekeeping gene values. R, Retina; L, Lamina; MTZ, myelin transition zone; MON, myelinated optic nerve. N = 4 replicates per region. Standard deviation error bars. Data was considered statistically significant if p < 0.05; * (0.033), ** (0.0021), *** (0.0002), **** (0.0001).</p

Longitudinal sections of porcine optic nerve head labeled for aquaporin 1 (AQP1, red) and aquaporin-9 (AQP9, green) and DAPI (blue).

Labeling of AQP1 and AQP9 was substantial at the internal limiting membrane (A), but minimal throughout the lamina and myelinated optic nerve, except in the walls of larger blood vessel walls as seen in higher power images (B, C). Scale Bar: 100 μm (A), 50 μm (B, C).</p

Longitudinal cryosections of immunolabeled aquaporin-4 and phalloidin on human (A-E), and porcine (B) optic nerve head tissue.

Minimal label for AQP4 (green) is visible in the lamina cribrosa (between dotted white lines in A, B; seen at higher power in C and E. AQP4 label is prominent in the retinal nerve fiber layer, prelaminar area and myelinated optic nerve in each species. Phalloidin labeling of F-actin (red) is prominent in the prelamina, lamina and myelinated ON (D, E). AQP4 labeling is visible within axon bundles and pronounced along the edge of axons bundles and at the pia surface. DAPI (blue) identifies cell nuclei. Scale Bar: 200 μm (A, D), 50 μm (B, C, E).</p