Age-related Macular Degeneration (AMD), a blinding eye disease, is characterized by pathological protein- and lipid-rich drusen deposits underneath the retinal pigment epithelium (RPE) and atrophy of the RPE monolayer in advanced disease stages - leading to photoreceptor cell death and vision loss. Currently, there are no drugs that stop drusen formation or RPE atrophy in AMD. Here we provide an iPSC-RPE AMD model that recapitulates drusen and RPE atrophy. Drusen deposition is dependent on AMD-risk-allele CFH(H/H) and anaphylatoxin triggered alternate complement signaling via the activation of NF-κB and downregulation of autophagy pathways. Through high-throughput screening we identify two drugs, L-745,870, a dopamine receptor antagonist, and aminocaproic acid, a protease inhibitor that reduce drusen deposits and restore RPE epithelial phenotype in anaphylatoxin challenged iPSC-RPE with or without the CFH(H/H) genotype. This comprehensive iPSC-RPE model replicates key AMD phenotypes, provides molecular insight into the role of CFH(H/H) risk-allele in AMD, and discovers two candidate drugs to treat AMD.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder caused by a missense substitution in CTRP5. Distinctive clinical features include sub-retinal pigment epithelium (RPE) deposits, choroidal neovascularization, and RPE atrophy. In induced pluripotent stem cells-derived RPE from L-ORD patients (L-ORD-iRPE), we show that the dominant pathogenic CTRP5 variant leads to reduced CTRP5 secretion. In silico modeling suggests lower binding of mutant CTRP5 to adiponectin receptor 1 (ADIPOR1). Downstream of ADIPOR1 sustained activation of AMPK renders it insensitive to changes in AMP/ATP ratio resulting in defective lipid metabolism, reduced Neuroprotectin D1(NPD1) secretion, lower mitochondrial respiration, and reduced ATP production. These metabolic defects result in accumulation of sub-RPE deposits and leave L-ORD-iRPE susceptible to dedifferentiation. Gene augmentation of L-ORD-iRPE with WT CTRP5 or modulation of AMPK, by metformin, re-sensitize L-ORD-iRPE to changes in cellular energy status alleviating the disease cellular phenotypes. Our data suggests a mechanism for the dominant behavior of CTRP5 mutation and provides potential treatment strategies for L-ORD patients.

Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.

In cells undergoing apoptosis phosphatidylserine, a major component of the plasma membrane, translocates to the outer leaflet where it provides eat-me signals for phagocytic recognition and is bound by annexin-V, an apoptotic marker. The need to track retinal ganglion cell death (RGC) in response to glaucomatous damage or optic neuropathy has led to the development of DARC (detection of apoptosing retinal cells) imaging, providing non-invasive, in vivo assessment of RGC death. Although the eye is an immune privileged site, resident and infiltrating immune cells are known to respond quickly to trauma or infection. Some immune cells have binding sites for annexin homologs; thus, their presence may confound estimates of apoptosis measured by annexin-V labeling. The purpose of this study was to re-examine the accuracy of annexin-V apoptotic labeling in the posterior eye and to temporally characterize contributions of non-apoptotic labeling in response to optic nerve (ON) injury. Here, we found annexin-V labeling consists of two phases. Initially, there is a rapid phase matching the time course of apoptotic cell death indicated by cleaved caspase-3 immunostaining observed ex vivo. This is followed by a sustained plateau phase that persists long after the peak of degeneration. We demonstrate that annexin-V binds to a specific subpopulation of myeloid cells in the retina, which were identified using simultaneous confocal scanning laser ophthalmoscopy. Optical coherence tomography and confocal imaging reveal these cells occupy the posterior hyaloid space above the retinal nerve fiber layer and at various retinal depths. Our results highlight the cellular morphological heterogeneity of non-apoptotic annexin-V labeling of retinal microglia. Accordingly, pharmacological depletion of microglia abolishes annexin-V labeling of elongated microglia in vivo revealing fainter labeling of round RGCs. Thus, consideration should be given to the time course of the immune response when interpreting fluorescently labeled annexin-V to visualize retinal cell apoptosis for clinical diagnosis.