Aging is the greatest risk factor for chronic human diseases, including many eye diseases. Geroscience aims to understand the effects of the aging process on these diseases, including the genetic, molecular, and cellular mechanisms that underlie the increased risk of disease over the lifetime. Understanding of the aging eye increases general knowledge of the cellular physiology impacted by aging processes at various biological extremes. Two major diseases, age-related cataract and age-related macular degeneration (AMD) are caused by dysfunction of the lens and retina, respectively. Lens transparency and light refraction are mediated by lens fiber cells lacking nuclei and other organelles, which provides a unique opportunity to study a single aging hallmark, i.e., loss of proteostasis, within an environment of limited metabolism. In AMD, local dysfunction of the photoreceptors/retinal pigmented epithelium/Bruch\'s membrane/choriocapillaris complex in the macula leads to the loss of photoreceptors and eventually loss of central vision, and is driven by nearly all the hallmarks of aging and shares features with Alzheimer\'s disease, Parkinson\'s disease, cardiovascular disease, and diabetes. The aging eye can function as a model for studying basic mechanisms of aging and, vice versa, well-defined hallmarks of aging can be used as tools to understand age-related eye disease.

Clouding of the transparent eye lens, or cataract(s), is a leading cause of visual impairment that requires surgical replacement with a synthetic intraocular lens to effectively restore clear vision. Most frequently, cataract is acquired with aging as a multifactorial or complex trait. Cataract may also be inherited as a classic Mendelian trait-often with an early or pediatric onset-with or without other ocular and/or systemic features. Since the early 1990s, over 85 genes and loci have been genetically associated with inherited and/or age-related forms of cataract. While many of these underlying genes-including those for lens crystallins, connexins, and transcription factors-recapitulate signature features of lens development and differentiation, an increasing cohort of unpredicted genes, including those involved in cell-signaling, membrane remodeling, and autophagy, has emerged-providing new insights regarding lens homeostasis and aging. This review provides a brief history of gene discovery for inherited and age-related forms of cataract compiled in the Cat-Map database and highlights potential gene-based therapeutic approaches to delay, reverse, or even prevent cataract formation that may help to reduce the increasing demand for cataract surgery.

Tunicate orthologs in the human genome comprise just 84 genes of the 19,872 protein-coding genes and 23 of the 16,528 non-coding genes, yet they stand at the base of the Olfactores clade, which radiated to generate thousands of tunicate and vertebrate species. What were the powerful drivers among these genes that enabled this process? Many of these orthologs are present in gene families. We discuss the biological role of each family and the orthologs\' quantitative contribution to the family. Most important was the evolution of a second type of cadherin. This, a Type II cadherin, had the property of detaching the cell containing that cadherin from cells that expressed the Type I class. The set of such Type II cadherins could now detach and move away from their Type I neighbours, a process which would eventually evolve into the formation of the neural crest, \"the fourth germ layer\", providing a wide range of possibilities for further evolutionary invention. A second important contribution were key additions to the broad development of the muscle and nerve protein and visual perception toolkits. These developments in mobility and vision provided the basis for the development of the efficient predatory capabilities of the Vertebrata.

Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within ∼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S → N acetyl transfer mechanism.

Disturbances in protein phase transitions promote protein aggregation─a neurodegeneration hallmark. The modular Ran-binding protein 2 (Ranbp2) is a cytosolic molecular hub for rate-limiting steps of phase transitions of Ran-GTP-bound protein ensembles exiting nuclear pores. Chaperones also regulate phase transitions and proteostasis by suppressing protein aggregation. Ranbp2 haploinsufficiency promotes the age-dependent neuroprotection of the chorioretina against phototoxicity by proteostatic regulations of neuroprotective substrates of Ranbp2 and by suppressing the buildup of polyubiquitylated substrates. Losses of peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities of the cyclophilin domain (CY) of Ranbp2 recapitulate molecular effects of Ranbp2 haploinsufficiency. These CY impairments also stimulate deubiquitylation activities and phase transitions of 19S cap subunits of the 26S proteasome that associates with Ranbp2. However, links between CY moonlighting activity, substrate ubiquitylation, and proteostasis remain incomplete. Here, we reveal the Ranbp2 regulation of small heat shock chaperones─crystallins in the chorioretina by proteomics of mice with total or selective modular deficits of Ranbp2. Specifically, loss of CY PPIase of Ranbp2 upregulates αA-Crystallin, which is repressed in adult nonlenticular tissues. Conversely, impairment of CY\'s chaperone activity opposite to the PPIase pocket downregulates a subset of αA-Crystallin\'s substrates, γ-crystallins. These CY-dependent effects cause age-dependent and chorioretinal-selective declines of ubiquitylated substrates without affecting the chorioretinal morphology. A model emerges whereby inhibition of Ranbp2\'s CY PPIase remodels crystallins\' expressions, subdues molecular aging, and preordains the chorioretina to neuroprotection by augmenting the chaperone capacity and the degradation of polyubiquitylated substrates against proteostatic impairments. Further, the druggable Ranbp2 CY holds pan-therapeutic potential against proteotoxicity and neurodegeneration.

BACKGROUND: Nuclear organization of interphase chromosomes involves individual chromosome territories, \"open\" and \"closed\" chromatin compartments, topologically associated domains (TADs) and chromatin loops. The DNA- and RNA-binding transcription factor CTCF together with the cohesin complex serve as major organizers of chromatin architecture. Cellular differentiation is driven by temporally and spatially coordinated gene expression that requires chromatin changes of individual loci of various complexities. Lens differentiation represents an advantageous system to probe transcriptional mechanisms underlying tissue-specific gene expression including high transcriptional outputs of individual crystallin genes until the mature lens fiber cells degrade their nuclei.
RESULTS: Chromatin organization between mouse embryonic stem (ES) cells, newborn (P0.5) lens epithelium and fiber cells were analyzed using Hi-C. Localization of CTCF in both lens chromatins was determined by ChIP-seq and compared with ES cells. Quantitative analyses show major differences between number and size of TADs and chromatin loop size between these three cell types. In depth analyses show similarities between lens samples exemplified by overlaps between compartments A and B. Lens epithelium-specific CTCF peaks are found in mostly methylated genomic regions while lens fiber-specific and shared peaks occur mostly within unmethylated DNA regions. Major differences in TADs and loops are illustrated at the ~ 500 kb Pax6 locus, encoding the critical lens regulatory transcription factor and within a larger ~ 15 Mb WAGR locus, containing Pax6 and other loci linked to human congenital diseases. Lens and ES cell Hi-C data (TADs and loops) together with ATAC-seq, CTCF, H3K27ac, H3K27me3 and ENCODE cis-regulatory sites are shown in detail for the Pax6, Sox1 and Hif1a loci, multiple crystallin genes and other important loci required for lens morphogenesis. The majority of crystallin loci are marked by unexpectedly high CTCF-binding across their transcribed regions.
CONCLUSIONS: Our study has generated the first data on 3-dimensional (3D) nuclear organization in lens epithelium and lens fibers and directly compared these data with ES cells. These findings generate novel insights into lens-specific transcriptional gene control, open new research avenues to study transcriptional condensates in lens fiber cells, and enable studies of non-coding genetic variants linked to cataract and other lens and ocular abnormalities.

The substitution of leucine to proline at position 39 (p.P39L) in human αB-crystallin (αB-Cry) has been associated with conflicting interpretations of pathogenicity in cataracts and cardiomyopathy. This study aimed to investigate the effects of the p.P39L mutation on the structural and functional features of human αB-Cry. The mutant protein was expressed in Escherichia coli (E. coli) and purified using anion exchange chromatography. We employed a wide range of spectroscopic analyses, gel electrophoresis, transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques to investigate the structure, function, stability, and fibrillation propensity of the mutant protein. The p.P39L mutation caused significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry and increased the thermal stability of the protein. The mutant αB-Cry exhibited an increased chaperone activity and an altered oligomeric size distribution, along with an increased propensity to form amyloid aggregates. It is worth mentioning, increased chaperone activity has important positive and negative effects on damaged cells related to cataracts and cardiomyopathy, particularly by interfering in the process of apoptosis. Despite the apparent positive nature of the increased chaperone activity, it is also linked to adverse consequences. This study provides important insights into the effect of proline substitution by leucine at the N-terminal region on the dual nature of chaperone activity in human αB-Cry, which can act as a double-edged sword.

Exploring the mechanisms underlying the toxicity of amyloid oligomers (AOs) presents a significant opportunity for discovering cures and developing treatments for neurodegenerative diseases. Recently, using a combination of ion mobility spectrometry-mass spectrometry (IMS-MS) and X-ray crystallography (XRC), we showed that the peptide KVKVLWDVIEV, which is the G95W mutant of αB-Crystallin (90-100) and abbreviated as G6W, self-assembles up to a dodecamer that structurally resembles lipid transport proteins. The glycine to tryptophan mutation promotes not only larger oligomers and enhanced cytotoxicity in brain slices than the wild type but also a narrow hydrophobic cavity suitable for fatty acid or phospholipid binding. Here, we determine the plausibility of a novel cytotoxic mechanism where the G6W\'s structural motif could perturb lipid homeostasis by determining its lipid binding selectivity and specificity. We show that the G6W oligomers have a strong affinity toward unsaturated phospholipids with a preference toward phospholipids containing 16-C alkyl chains. Molecular dynamics simulations demonstrate how an unsaturated, 16-C phospholipid fits tightly inside and outside G6W\'s hydrophobic cavity. This binding is exclusive to the G6W peptide, as other amyloid oligomers with different atomic structures, including its wildtype αB-Crystallin (90-100) and several superoxide dismutase 1 (SOD1) peptides that are known to self-assemble into amyloid oligomers (SOD1P28K and SOD1WG-GW), do not experience the same strong binding affinity. While the existing chaperone-lipid hypothesis on amyloid toxicity suggests amyloid-lipid complexes perforate cell membranes, our work provides a new outlook, indicating that soluble amyloid oligomers disrupt lipid homeostasis via selective protein-ligand interactions. The toxic mechanisms may arise from the formation of unique amyloid oligomer structures assisted by lipid ligands or impaired lipid transports.

Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined structural changes in αAc, αBc and native heteromeric lens α-crystallins (αLc) in their apo-states and at varying degree of chaperone saturation leading to co-aggregation, using lysozyme and insulin as model clients. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP oligomeric scaffold, whereby the native cage-like sHSP assembly displays a directional growth to accommodate saturating conditions of client sequestration. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP oligomers with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across αAc, αBc and αLc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of α-crystallins, carrying potential implications for a pathway toward cataract formation.

Congenital cataract is a major cause of childhood blindness worldwide, with crystallin mutations accounting for over 40 % of gene-mutation-related cases. Our research focused on a novel R114C mutation in a Chinese family, resulting in bilateral coronary cataract with blue punctate opacity. Spectroscopic experiments revealed that βA3-R114C significantly altered the senior structure, exhibiting aggregation, and reduced solubility at physiological temperature. The mutant also displayed decreased resistance and stability under environmental stresses such as UV irradiation, oxidative stress, and heat. Further, cellular models confirmed its heightened sensitivity to environmental stresses. These data suggest that the R114C mutation impairs the hydrogen bond network and structural stability of βA3-crystallin, particularly at the boundary of the second Greek-key motif. This study revealed the pathological mechanism of βA3-R114C and may help in the development of potential treatment strategies for related cataracts.