Abstract The human macula, a high-acuity retinal region essential for central vision, emerges early in development, but the cellular basis of its regional specialisation remains incompletely resolved. Building on recent studies that identified CYP26A1-mediated retinoic acid (RA) suppression in the presumptive macula, we define a CRYAA-positive progenitor-glial compartment in the temporal human retina from post-conception week (pcw) 7 onward. Single-cell RNA sequencing, immunohistochemistry and spatial morphometry indicate that this compartment is linked to regional gliogenic maturation and later corresponds to a specialised macular Müller glial population, rather than simply reflecting uniform pan-retinal developmental timing. Spatial mapping shows that the CYP26A1-positive domain remains delimited as the surrounding retina expands. Adult tissue analysis shows that CYP26A1 expression is retained in primate macular Müller glia and in corresponding acute-zone regions of visual-streak species. These findings extend current models of CYP26A1-mediated RA modulation in human acute zone/macular development by linking the early CYP26A1 domain to a regionally specialised CRYAA-positive progenitor-glial compartment. We propose that early macular patterning includes a spatially restricted glial programme that may contribute to later regional architecture and disease vulnerability.

Neurodegenerative diseases of the retina result from diverse insults, including genetic mutations, metabolic deficiencies, vascular compromise, and inflammatory injury. These processes converge on dysfunction of the neurovascular unit, where neurons, glia, and vascular cells cooperate to maintain retinal health. Thus, neuroprotection must be considered in a broader context that incorporates support of glial and vascular elements in addition to neurons. In this chapter, we review both classical and emerging neuroprotective strategies in retinal disease. We summarize preclinical and clinical studies of trophic factor-based approaches, including ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), fibroblast growth factor 2 (FGF2), insulin-like growth factor-1 (IGF-1), and transforming growth factor-β (TGF-β), outlining mechanisms, efficacy, limitations, and safety. We also highlight nonclassical agents such as mesencephalic astrocyte-derived neurotrophic factor (MANF) and the lipid mediator erucamide, which act through distinct pathways to modulate stress responses and neurovascular stability. Additional approaches, including stem cell-based therapies, extracellular vesicles, metabolic supplementation, and lifestyle interventions, are discussed. Finally, we emphasize the importance of human-derived models such as retinal explants and organoids to overcome translational barriers. Collectively, these studies suggest that multimodal strategies may offer meaningful neuroprotection and preserve vision in progressive retinal disease.

Neurovasculoglial cross-talk underlying breakdown of the neurovascular unit is a central, yet poorly understood, component of many neurodegenerative disorders of the CNS, including retinal disease. Primary fatty acid amides have been identified to regulate this cross-talk between vasculature and neuronal tissues, but specific molecules and mechanisms remain unresolved. Here we show, using an unbiased high-resolution metabolomics screen, that erucamide, a 22:1 monounsaturated omega-9 fatty acid amide, is highly dysregulated during photoreceptor degeneration in mice. In vivo delivery of erucamide using organosilane-modified porous silicon nanoparticles activated retinal myeloid cells, leading to the upregulation of angiogenic and neurotrophic cytokines that limited vascular and neuronal degeneration. We identified TMEM19 as a binding protein for erucamide that is crucial for myeloid cell activation and subsequent neuroprotection. These findings reveal a previously unknown primary fatty acid amide pathway that modulates neuroimmune interactions during retinal degenerative diseases. We propose erucamide and analogs as candidate therapeutics.

Neurotrophic factors are a family of proteins that promote the growth and survival of both developing and mature neurons. Extensive preclinical studies have demonstrated neuroprotective properties conferred by ciliary neurotrophic factor (CNTF) in a variety of neuron types across several species. Neuroprotection that CNTF confers slows or prevents neuron loss and appears to be agnostic to the nature of the neurodegenerative mutation or injury. However, translation of these studies to the clinic remains a challenge due in part to delivery barriers inherent to the central nervous system and the short half-life of CNTF. The molecular effect of CNTF delivered by a variety of strategies in model systems has been extensively studied in the neural retina. Long-term retinal neuroprotection has been documented using encapsulated cells that have been genetically modified to produce a stable source of CNTF. Clinical trials have shown that CNTF is well tolerated for use in the human retina. This review focuses on the mechanism of action of CNTF and its potential as a therapeutic agent in retinal disease, with a focus on macular telangiectasia type 2 where CNTF has shown efficacy in slowing the rate of ellipsoid zone loss.

Neurovasculoglial crosstalk is critical in establishing and maintaining a functional neurovascular unit. Breakdown in the unit is central to many neurodegenerative disorders of the CNS of which the retina is a component. A growing literature indicated that primary fatty acid amides (PFAMs) can regulate this crosstalk between vasculature and neuronal tissues. In this study we describe a central role for erucamide, a 22:1 mono-unsaturated omega-9 fatty acid amide, in degenerating retinal tissues. Using high-resolution global mass spectrometry-based metabolomics, we cataloged metabolites in murine models of retinal degeneration and show that while PFAMs, in general, are highly dysregulated, erucamide is the one most significantly diminished during photoreceptor atrophy. Using rodent models of retinal degeneration and novel organosilane-modified porous silicon nanoparticles (pSiNPs) for the in vivo delivery of erucamide, we demonstrate that erucamide activates CD11b+ myeloid cells, leading to the upregulation of angiogenic and neurotrophic cytokines that stabilize retinal degeneration. We identified TMEM19 as a novel binding protein for erucamide that is crucial for human iPSC-derived macrophage precursor cells activation and subsequent neurotrophic and angiogenic factor production. These findings reveal a previously unknown PFAM pathway that is modulated during retinal degenerative diseases, demonstrating that erucamide or functional analogues and their action through TMEM19 may be useful as a therapeutic alternative to neuroprotective and stem cell-based approaches for the treatment of retinal degenerative diseases.

In multiple neurodegenerative diseases, including age-related macular degeneration, retinitis pigmentosa, and macular telangiectasia type 2 (MacTel), retinal pigment epithelial (RPE)-cells proliferate and migrate into the neuroretina, forming intraretinal pigment plaques. Though these pigmentary changes are hallmarks of disease progression, it is unknown if their presence is protective or detrimental. Here, we first evaluated the impact of pigment plaques on vascular changes and disease progression in MacTel. In a retrospective, longitudinal study, we analyzed multimodal retinal images of patients with MacTel and showed that pigment plaques were associated with decreased vascular leakage and stabilized neovascular growth. We then modeled the underlying pathomechanisms of pigment plaque formation in aberrant neovascular growth using the very-low-density lipoprotein receptor mutant (Vldlr−/−) mouse. Our data indicated that during RPE-proliferation, migration and accumulation along neovessels RPE-cells underwent epithelial-mesenchymal transition (EMT). Pharmacologic inhibition of EMT in Vldlr−/− mice decreased pigment coverage, and exacerbated neovascular growth and vascular leakage. Our findings indicate that the proliferation, migration and perivascular accumulation of RPE-cells stabilize vascular proliferation and exudation, thereby exerting a protective effect on the diseased retina. We conclude that interfering with this “natural repair mechanism” may have detrimental effects on the course of the disease and should thus be avoided.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO), account for ∼25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (1) show advanced retinal degeneration in a male patient (60-70 year old) harboring four transcriptionally active intact copies of rhodopsin, (2) recapitulated the clinical phenotypes using retinal organoids, and (3) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (qRT-PCR and bulk RNA-sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300-days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that adRP due to RHO-CNV develops due protein overexpression overloading the photoreceptor post-translational modification machinery.

Retinitis pigmentosa (RP) is a prevalent inherited retinal degenerative disease worldwide, affecting 1 in 4,000 people. The disease is characterized by an initial loss of night vision followed by a loss of daylight and color vision. Many of the RP disease genes are expressed in the rod photoreceptors, the cell type that initiates dim light vision. Following loss of rods, the cone photoreceptors, which initiate daylight vision, also are affected and can die leading to total loss of vision. The reasons for loss of cone vision are not entirely clear, but appear to be due to loss of the rods. Previously we showed that overexpressing Txnip, an α-arrestin protein, in mouse models of RP using AAV gene therapy prolonged the survival of RP cones (). At least part of the mechanism for cone survival was a switch in the fuel source, from glucose to lactate. In addition, the mitochondria of cones were both morphologically and functionally improved by delivery of Txnip. We have gone on to test several alleles of Txnip for the ability to prolong cone survival in rd1, a mouse model of RP. In addition, proteins that bind to Txnip and/or have homology to Txnip were tested. Five different deletion alleles of Txnip were expressed in cones or the retinal pigmented epithelium (RPE). Here we show that the C-terminal half of Txnip (149-397aa) is sufficient to remove GLUT1 from the RPE cell surface, and improved rd1 cone survival when expressed specifically in the RPE. Overexpressing Arrdc4, an α-arrestin that shares 60% similar protein sequence to Txnip, reduced rd1 cone survival. Reduction of the expression of HSP90AB1, a protein that interacts with Txnip and regulates metabolism, improved the survival of rd1 cones alone and was additive for cone survival when combined with Txnip. However, full length Txnip with a single amino acid change, C247S, as we tested in our original study, remains the most highly efficacious form of the gene for cone rescue. The above observations suggest that only a subset of the hypothesized and known activities of Txnip play a role in promoting RP cone survival, and that the activities of Txnip in the RPE differ from those in cone photoreceptors.

The accumulation of atypical, cytotoxic 1-deoxysphingolipids (1-dSLs) has been linked to retinal diseases such as diabetic retinopathy and Macular Telangiectasia Type 2. However, the molecular mechanisms by which 1-dSLs induce toxicity in retinal cells remain poorly understood. Here, we integrate bulk and single-nucleus RNA-sequencing to define biological pathways that modulate 1-dSL toxicity in human retinal organoids. Our results demonstrate that 1-dSLs differentially activate signaling arms of the unfolded protein response (UPR) in photoreceptor cells and Müller glia. Using a combination of pharmacologic activators and inhibitors, we show that sustained PERK signaling through the integrated stress response (ISR) and deficiencies in signaling through the protective ATF6 arm of the UPR are implicated in 1-dSL-induced photoreceptor toxicity. Further, we demonstrate that pharmacologic activation of ATF6 mitigates 1-dSL toxicity without impacting PERK/ISR signaling. Collectively, our results identify new opportunities to intervene in 1-dSL linked diseases through targeting different arms of the UPR.

In the retina, microglia are resident immune cells that are essential for development and function. Retinal microglia play a central role in mediating pathological degeneration in diseases such as glaucoma, retinitis pigmentosa, age-related neurodegeneration, ischemic retinopathy, and diabetic retinopathy. Current models of mature human retinal organoids (ROs) derived from iPS cell (hiPSC) do not contain resident microglia integrated into retinal layers. Increasing cellular diversity in ROs by including resident microglia would more accurately represent the native retina and better model diseases in which microglia play a key role. In this study, we develop a new 3D in vitro tissue model of microglia-containing retinal organoids by co-culturing ROs and hiPSC-derived macrophage precursor cells (MPCs). We optimized the parameters for successful integration of MPCs into retinal organoids. We show that while in the ROs, MPCs migrate to the equivalent of the outer plexiform layer where retinal microglia cells reside in healthy retinal tissue. While there, they develop a mature morphology characterized by small cell bodies and long branching processes which is only observed in vivo. During this maturation process these MPCs cycle through an activated phase followed by a stable mature microglial phase as seen by the down regulation of pro-inflammatory cytokines and upregulation of anti-inflammatory cytokines. Finally, we characterized mature ROs with integrated MPCs using RNAseq showing an enrichment of cell-type specific microglia markers. We propose that this co-culture system may be useful for understanding the pathogenesis of retinal diseases involving retinal microglia and for drug discovery directly in human tissue.

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.