Multiomics Analysis Reveals Novel Genetic Determinants for Lens Differentiation, Structure, and Transparency.

Recent advances in next-generation sequencing and data analysis have provided new gateways for identification of novel genome-wide genetic determinants governing tissue development and disease. These advances have revolutionized our understanding of cellular differentiation, homeostasis, and specialized function in multiple tissues. Bioinformatic and functional analysis of these genetic determinants and the pathways they regulate have provided a novel basis for the design of functional experiments to answer a wide range of long-sought biological questions. A well-characterized model for the application of these emerging technologies is the development and differentiation of the ocular lens and how individual pathways regulate lens morphogenesis, gene expression, transparency, and refraction. Recent applications of next-generation sequencing analysis on well-characterized chicken and mouse lens differentiation models using a variety of omics techniques including RNA-seq, ATAC-seq, whole-genome bisulfite sequencing (WGBS), chip-seq, and CUT&RUN have revealed a wide range of essential biological pathways and chromatin features governing lens structure and function. Multiomics integration of these data has established new gene functions and cellular processes essential for lens formation, homeostasis, and transparency including the identification of novel transcription control pathways, autophagy remodeling pathways, and signal transduction pathways, among others. This review summarizes recent omics technologies applied to the lens, methods for integrating multiomics data, and how these recent technologies have advanced our understanding ocular biology and function. The approach and analysis are relevant to identifying the features and functional requirements of more complex tissues and disease states.

Autophagy-prominent cell clusters among human lens epithelial cells: integrated single-cell RNA-sequencing analysis.

BACKGROUND: Autophagy is an important process that maintains the quality of intracellular proteins and organelles. There is extensive evidence that autophagy has an important role in the lens. Human lens epithelial cells (HLECs) play a key role in the internal homeostasis of the lens. HLEC subtypes have been identified, but autophagy-prominent cell clusters among HLECs have not been characterized. PURPOSE: To explore the existence of autophagy-prominent cell clusters in HLECs. METHODS: Three donated lenses (HLECs from two whole lenses and HLECs from one lens without the anterior central 6-mm zone) were used for single-cell RNA sequencing (scRNA-seq). AUCell and AddModuleScore analysis were used to identify potential autophagy-prominent cell clusters. Transmission electron microscopy (TEM) was used to confirm the results. RESULTS: High-quality transcripts from 18,120 cells were acquired by scRNA-seq of the two intact lenses. Unsupervised clustering classified the cells into four clusters. AUCell and AddModuleScore analysis revealed cluster 1 is autophagy-prominent. scRNA-seq analysis of HLECs from the lens capsule lacking the central zone confirmed the cluster 1 HLECs was located in the central capsule zone. The TEM result showed that greater autophagy activity was observed in the HLECs in central capsule zone, which further supported the above conclusions based on scRNA-seq analysis that autophagy was prominent in the central zone where the cluster 1 HLECs located. CONCLUSIONS: We identified an autophagy-prominent cell cluster among HLECs and revealed that it was localized in the central zone of the lens capsule. Our findings will aid investigations of autophagy in HLECs and provide insights to guide related research.

BMP-4 and BMP-7 Inhibit EMT in a Model of Anterior Subcapsular Cataract in Part by Regulating the Notch Signaling Pathway.

Purpose: The proliferation, migration, and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) are believed to be the pathological mechanisms underlying anterior subcapsular cataract (ASC). Bone morphogenetic proteins (BMPs) inhibit transforming growth factor-beta (TGF-ß)-induced fibrosis in the lens. Herein, we aimed to further clarify the roles of BMP-4/BMP-7 in the progression and the underlying mechanisms of fibrotic cataract. Methods: BMP-4/BMP-7, TGF-ß2, jagged-1 peptide, or DAPT were applied in a mouse injury-induced ASC model and in the human LEC cell line SRA01/04. The volume of opacity was examined by a slit lamp and determined by lens anterior capsule whole-mount immunofluorescence. Global gene expression changes were assessed by RNA sequencing, and the levels of individual mRNAs were validated by real-time PCR. Protein expression was determined by the Simple Western sample dilution buffer. Cell proliferation was examined by CCK8 and EdU assays, and cell migration was measured by Transwell and wound healing assays. Results: Anterior chamber injection of BMP-4/BMP-7 significantly suppressed subcapsular opacification formation. RNA sequencing of the mouse ASC model identified the Notch pathway as a potential mechanism involved in BMP-mediated inhibition of ASC. Consistently, BMP-4/BMP-7 selectively suppressed Notch1 and Notch3 and their downstream genes, including Hes and Hey. BMP-4/BMP-7 or DAPT suppressed cell proliferation by inducing G1 cell cycle arrest. BMP-4/BMP-7 also inhibited TGF-ß2-induced cell migration and EMT by modulating the Notch pathway. Conclusions: BMP-4/BMP-7 attenuated ASC by inhibiting proliferation, migration, and EMT of LECs via modulation of the Notch pathway, thereby providing a new avenue for ASC treatment.

Endoplasmic Reticulum Stress and the Lysosomal Pathway Play Crucial Roles in the Progression of ßB2-Crystallin Mutation-Induced Congenital Cataracts in Mice.

Purpose: Congenital cataract is a major cause of visual impairment and childhood blindness; however, its underlying mechanism remains unclear. Here, we aimed to identify the roles of endoplasmic reticulum stress (ERS), lysosomal pathway, and lens capsule fibrosis during the progression of ßB2-crystallin mutation-induced congenital cataract in mice. Methods: BetaB2-W151C knock-in mice were generated using the CRISPR/Cas9 system. Lens opacity was assessed with a slit-lamp biomicroscopy and dissecting microscope. Transcriptional profiles of the lenses in W151C mutant and wild-type (WT) control mice were detected at 3 months of age. Immunofluorescence of lens anterior capsule was photographed with a confocal microscope. Real-time PCR and immunoblot were used to detect gene mRNA and protein expressions, respectively. Results: BetaB2-W151C knock-in mice developed progressive bilateral congenital cataracts. At 2 to 3 months of age, lens opacity rapidly progressed to complete cataracts. Additionally, multilayered LEC plaques developed beneath the lens anterior capsule in homozygous mice at 3 months of age, and severe fibrosis was observed in the whole lens capsule at 9 months of age. Microarray analysis of whole genome transcriptomics and the validation results of real-time PCR revealed that genes of ERS, the lysosomal pathway, apoptosis, and cell migration and fibrosis were significantly upregulated in ßB2-W151C mutant mice during the accelerated development of cataract. Moreover, the syntheses of various crystallins stagnated in ßB2-W151C mutant mice. Conclusions: ERS, the lysosomal pathway, apoptosis, and fibrosis all contributed to the accelerated development of congenital cataract. The inhibition of ERS and lysosomal cathepsins may be promising therapeutic strategies for congenital cataract.

High-Throughput Transcriptomics of Celf1 Conditional Knockout Lens Identifies Downstream Networks Linked to Cataract Pathology.

Defects in the development of the ocular lens can cause congenital cataracts. To understand the various etiologies of congenital cataracts, it is important to characterize the genes linked to this developmental defect and to define their downstream pathways that are relevant to lens biology and pathology. Deficiency or alteration of several RNA-binding proteins, including the conserved RBP Celf1 (CUGBP Elav-like family member 1), has been described to cause lens defects and early onset cataracts in animal models and/or humans. Celf1 is involved in various aspects of post-transcriptional gene expression control, including regulation of mRNA stability/decay, alternative splicing and translation. Celf1 germline knockout mice and lens conditional knockout (Celf1cKO) mice develop fully penetrant cataracts in early postnatal stages. To define the genome-level changes in RNA transcripts that result from Celf1 deficiency, we performed high-throughput RNA-sequencing of Celf1cKO mouse lenses at postnatal day (P) 0. Celf1cKO lenses exhibit 987 differentially expressed genes (DEGs) at cut-offs of >1.0 log2 counts per million (CPM), ≥±0.58 log2 fold-change and <0.05 false discovery rate (FDR). Of these, 327 RNAs were reduced while 660 were elevated in Celf1cKO lenses. The DEGs were subjected to various downstream analyses including iSyTE lens enriched-expression, presence in Cat-map, and gene ontology (GO) and representation of regulatory pathways. Further, a comparative analysis was done with previously generated microarray datasets on Celf1cKO lenses P0 and P6. Together, these analyses validated and prioritized several key genes mis-expressed in Celf1cKO lenses that are relevant to lens biology, including known cataract-linked genes (e.g., Cryab, Cryba2, Cryba4, Crybb1, Crybb2, Cryga, Crygb, Crygc, Crygd, Cryge, Crygf, Dnase2b, Bfsp1, Gja3, Pxdn, Sparc, Tdrd7, etc.) as well as novel candidates (e.g., Ell2 and Prdm16). Together, these data have defined the alterations in lens transcriptome caused by Celf1 deficiency, in turn uncovering downstream genes and pathways (e.g., structural constituents of eye lenses, lens fiber cell differentiation, etc.) associated with lens development and early-onset cataracts.

Knockout of miR-184 in zebrafish leads to ocular abnormalities by elevating p21 levels.

miR-184 is one of the most abundant miRNAs expressed in the lens and corneal tissue. Mutations in the seed region of miR-184 are responsible for inherited anterior segment dysgenesis. Animal models recapitulating miR-184-related anterior segment dysgenesis are still lacking, and the molecular basis of ocular abnormalities caused by miR-184 dysfunction has not been well elucidated in vivo. In the present study, we constructed a miR-184-/- zebrafish line by destroying both two dre-mir-184 paralogs with CRISPR-Cas9 technology. Although there were no gross developmental defects, the miR-184-/- zebrafish displayed microphthalmia and cataract phenotypes. Cytoskeletal abnormalities, aggregation of γ-crystallin, and lens fibrosis were induced in miR-184-/- lenses. However, no obvious corneal abnormalities were observed in miR-184-/- zebrafish. Instead of apoptosis, deficiency of miR-184 led to aberrant cell proliferation and a robust increase in p21 levels in zebrafish eyes. Inhibition of p21 by UC2288 compromised the elevation of lens fibrosis markers in miR-184-/- lenses. RNA-seq demonstrated that levels of four transcriptional factors HSF4, Sox9a, CTCF, and Smad6a, all of which could suppress p21 expression, were reduced in miR-184-/- eyes. The predicted zebrafish miR-184 direct target genes (e.g., atp1a3a and nck2a) were identified and verified in miR-184-/- eye tissues. The miR-184-/- zebrafish is the first animal model mimicking miR-184-related anterior segment dysgenesis and could broaden our understanding of the roles of miR-184 in eye development.

Cellular activity of autophagy and multivesicular bodies in lens fiber cells during early lens development in rbm24a mutant of zebrafish: Ultrastructure analysis.

Use of zebrafish as animal model for various diseases during early developmental stages has been exponentially increased with the aim to achieve the best representative results in this transparent fish. Recent studies documented that Rbm24a mutant causes cataract formation and resulted in blindness using the zebrafish model. Therefore, correct interpretation of studies that aimed for molecular approaches, a description of comparative and in-depth analysis of development of lens in wildtype and mutant is crucial to obtain the correct conclusion. In this study, we use a gold standard method the Transmission Electron Microscopy (TEM) to analysis the lens development in rbm24a mutant zebrafish. Firstly, we compare the cellular structures at 16-20 h post fertilization (hpf), the lens placode in ectoderm indicated delay lens development in rbm24a mutant than wildtype (siblings) zebrafish. At 33 hpf, loosely appeared lens fiber cells showed heterogenous electron density with numbers of mitochondria in lens of rbm24a mutant, revealed the influence of gene mutation in lens development. A detail ultrastructure of lens of rbm24a mutant also presented at 33 hpf. Comparatively in wildtype (siblings) at 33 hpf, lens exhibited homogenous electron density in tightly packed lens fiber cells with few mitochondria. Furthermore, to characterize the lens in rbm24a mutant we obtained data of cellular structures on 25 hpf and 1.5 days' post fertilization (dpf). At 25 hpf in mutant zebrafish, the detached solid sphere lens mass from ectoderm showed karyorrhexis, mitophagy and vesicles (also multivesicular bodies), these cellular structures supposed to hamper the development of future fiber cells. Moreover, at 1.5 dpf in mutant, nuclear excisosome, multilamellar bodies and irregular shaped mitochondria in heterogenous electron dense cytoplasm of lens fiber cells, collectively shown affected lens transparency. In summary the ultrastructure results of lens of rbm24a mutant zebrafish expand our knowledge and give reflection of different cellular activities like autophagy, apoptosis, vesicles (multivesicular bodies) and nuclear excisosomes which play their role in transparency achievement.

Impacts of autophagy on the formation of organelle-free zone during the lens development.

The thorough degeneration of organelles in the core of the lens is certainly a hallmark event during the lens development. Organelles degradation in the terminal differentiation process of lens fiber cells to form an organelle-free zone is critical for lens maturation and transparency. Several mechanisms have been proposed to expand our understanding of lens organelles degradation, including apoptotic pathways, the participation of ribozyme, proteolytic enzyme and phospholipase A and acyltransferase, and the newly discovered roles for autophagy. Autophagy is a lysosome-dependent degradation reaction during which the "useless" cellular components are degraded and recycled. These cellular components, such as incorrectly folded proteins, damaged organelles and other macromolecules, are first engulfed by the autophagosome before being further delivered to lysosomes for degradation. Although autophagy has been recognized involving in organelle degradation of the lens, the detailed functions remain to be discovered. Recent advances have revealed that autophagy not only plays a vital role in the intracellular quality control of the lens but is also involved in the degradation of nonnuclear organelles in the process of lens fiber cell differentiation. Herein, we first review the potential mechanisms of organelle-free zone formation, then discuss the roles of autophagy in intracellular quality control and cataract formation, and finally substantially summarize the potential involvement of autophagy in the development of organelle-free zone formation.

Reconstructions of individual fish trophic geographies using isotopic analysis of eye-lens amino acids.

Fish eye lenses are a proteinaceous structure that grows by accumulating layers in a chronological manner. Each layer becomes metabolically inert, capturing the ratio of heavy/light carbon and nitrogen isotopes at time of formation. Therefore, eye lenses contain chronological isotopic records and can be used to create a temporal isotopic history throughout an individual's lifetime. We analyzed eye lens amino-acid δ15N to address spatio-temporal baseline variability and to reconstruct trophic histories of 10 individual Red Snapper. Proteins from sequential eye lens laminae were derivatized to measure 10 amino acids, from which glutamic acid (trophic) and phenylalanine (source) were used to estimate trophic positions at different points in life. Best-fitting regressions were generated to represent individual (R2 ≥ 0.89) and generalized (R2 = 0.77) trophic trajectory for Red Snapper. The resulting trophic trajectories indicated an increase in trophic position with increasing length. Until recently, there has not been a lifetime isotopic structure with enough organic nitrogen to recreate geographic histories using compound-specific stable isotope analysis of amino acids (CSIA-AA). This study confirms that eye-lens laminae can be used to reconstruct trophogeographic histories via CSIA-AA.

Phase separation of α-crystallin-GFP protein and its implication in cataract disease.

Cataract, the leading cause of blindness worldwide, is caused by crystallin protein aggregation within the protected lens environment. Phase separation has been implicated as an important mechanism of protein aggregation diseases, such as neurodegeneration. Similarly, cataract has been proposed to be a protein condensation disease in the last century. However, whether crystallin proteins aggregate via a phase separation mechanism and which crystallin protein initiates the aggregation remain unclear. Here, we showed that all types of crystallin-GFP proteins remain soluble under physiological conditions, including protein concentrations, ion strength, and crowding environments. However, in age or disease-induced aberrant conditions, α-crystallin-GFP, including αA- and αB-crystallin-GFP, but not other crystallin-GFP proteins, undergo phase separation in vivo and in vitro. We found that aging-related changes, including higher crystallin concentrations, increased Na+, and decreased K+ concentrations, induced the aggregation of α-crystallin-GFP. Furthermore, H2O2, glucose, and sorbitol, the well-known risk factors for cataract, significantly enhanced the aggregation of αB-crystallin-GFP. Taken together, our results revealed that α-crystallin-GFP forms aggregates via a phase transition process, which may play roles in cataract disease. Opposite to the previously reported function of enhancing the solubility of other crystallin, α-crystallin may be the major aggregated crystallin in the lens of cataract patients.

Lens-specific ßA3/A1-conditional knockout mice: Phenotypic characteristics and calpain activation causing protein degradation and insolubilization.

ßA3/A1-crystallin is a lens structural protein that plays an important role in maintaining lens transparency via interactions with other crystallins. While the function of ßA3/A1-crystallin in the retina is well studied, its functions in the lens, other than as a structural protein, remain unclear. In the current study, we generated the lens-specific ßA3/A1-crystallin conditional knockout mouse (named ßA3/A1ckO) and explored phenotypic changes and the function of the crystallin in the lens. The ßA3/A1ckO mice showed congenital cataract at birth and exhibited truncation of lens proteins. Several truncated protein fragments were recovered as a pellet during a low-speed centrifugation (800 rpm, 70 x g) followed by a relatively higher speed centrifugation (5000 rpm, 2744 x g). Mass spectrometric analysis of pellets recovered following the two centrifugations showed that among the fragments with Mr < 20 kDa, the majority of these were from ß-tubulin, and some from phakinin, αA-crystallin, and calpain-3. Further, we observed that in vitro activation of calpain-3 by calcium treatment of the wild-type-lens homogenate resulted in the degradation of calpain-3, αA-crystallin and ß-tubulin and insolubilization of these proteins. Based on these results, it was concluded that the activation of calpain 3 resulted in proteolysis of ß-tubulin, which disrupted cellular microtubular structure, and caused proteolysis of other lens proteins (αA-crystallin and phakinin). These proteolyzed protein fragments become insoluble, and together with the disruption of microtubular structure, and could be the causative factors in the development of congenital nuclear cataract in ßA3/A1cKO mice.

Early Stage UV-B Induced Molecular Modifications of Human Eye Lens γD-Crystallin.

In the human eye lenses, the crystallin proteins facilitate transparency, light refraction, as well as UV light protection. A deregulated balanced interplay between α-, ß-, and γ-crystallin can cause cataract. γD-crystallin (hγD) is involved in the energy dissipation of absorbed UV light by energy transfer between aromatic side chains. Early UV-B induced damage of hγD with molecular resolution is studied by solution NMR and fluorescence spectroscopy. hγD modifications are restricted to Tyr 17 and Tyr 29 in the N-terminal domain, where a local unfolding of the hydrophobic core is observed. None of the tryptophan residues assisting fluorescence energy transfer is modified and hγD is remained soluble over month. Investigating isotope-labeled hγD surrounded by eye lens extracts from cataract patients reveals very week interactions of solvent-exposed side chains in the C-terminal hγD domain and some remaining photoprotective properties of the extracts. Hereditary E107A hγD found in the eye lens core of infants developing cataract shows under the here used conditions a thermodynamic stability comparable to the wild type but an increased sensitivity toward UV-B irradiation.

Aquaporin 5 maintains lens transparency by regulating the lysosomal pathway using circRNA.

The lens is transparent, non-vascular, elastic and wrapped in a transparent capsule. The lens oppacity of AQP5-/- mice was increased more than that of wild-type (AQP5+/+ ) mice. In this study, we explored the potential functional role of circular RNAs (circRNAs) and transcription factor HSF4 in lens opacity in aquaporin 5 (AQP5) knockout (AQP5-/- ) mice. Autophagy was impaired in the lens tissues of AQP5-/- mice. Autophagic lysosomes in lens epithelial cells of AQP5-/- mice were increased compared with AQP5+/+ mice, based on analysis by transmission electron microscopy. The genetic information of the mice lens was obtained by high-throughput sequencing, and then the downstream genes were analysed. A circRNA-miRNA-mRNA network related to lysosomal pathway was constructed by the bioinformatics analysis of the differentially expressed circRNAs. Based on the prediction of the TargetScan website and the validation by dual luciferase reporter assay and RNA immunoprecipitation-qPCR, we found that circRNA (Chr16: 33421321-33468183+) inhibited the function of HSF4 by sponging microRNA (miR-149-5p), and it downregulated the normal expression of lysosome-related mRNAs. The accumulation of autophagic lysosome may be one of the reasons for the abnormal development of the lens in AQP5-/- mice.

Aggrelyte-2 promotes protein solubility and decreases lens stiffness through lysine acetylation and disulfide reduction: Implications for treating presbyopia.

Aging proteins in the lens become increasingly aggregated and insoluble, contributing to presbyopia. In this study, we investigated the ability of aggrelyte-2 (N,S-diacetyl-L-cysteine methyl ester) to reverse the water insolubility of aged human lens proteins and to decrease stiffness in cultured human and mouse lenses. Water-insoluble proteins (WI) of aged human lenses (65-75 years) were incubated with aggrelyte-2 (500 µM) for 24 or 48 h. A control compound that lacked the S-acetyl group (aggrelyte-2C) was also tested. We observed 19%-30% solubility of WI upon treatment with aggrelyte-2. Aggrelyte-2C also increased protein solubility, but its effect was approximately 1.4-fold lower than that of aggrelyte-2. The protein thiol contents were 1.9- to 4.9-fold higher in the aggrelyte-2- and aggrelyte-2C-treated samples than in the untreated samples. The LC-MS/MS results showed Nε -acetyllysine (AcK) levels of 1.5 to 2.1 nmol/mg protein and 0.6 to 0.9 nmol/mg protein in the aggrelyte-2- and aggrelyte-2C-treated samples. Mouse (C57BL/6J) lenses (incubated for 24 h) and human lenses (incubated for 72 h) with 1.0 mM aggrelyte-2 showed significant decreases in stiffness with simultaneous increases in soluble proteins (human lenses) and protein-AcK levels, and such changes were not observed in aggrelyte-2C-treated lenses. Mass spectrometry of the solubilized protein revealed AcK in all crystallins, but more was observed in α-crystallins. These results suggest that aggrelyte-2 increases protein solubility and decreases lens stiffness through acetylation and disulfide reduction. Aggrelyte-2 might be useful in treating presbyopia in humans.

Promotion of Protein Solubility and Reduction in Stiffness in Human Lenses by Aggrelyte-1: Implications for Reversing Presbyopia.

With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for presbyopia. In this study, we tested aggrelyte-1 (S,N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58-75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44-66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia.

The second extracellular domain of connexin 50 is important for in cell adhesion, lens differentiation, and adhesion molecule expression.

Connexin (Cx)-forming channels play essential roles in maintaining lens homeostasis and transparency. We showed here channel-independent roles of Cx50 in cell-cell adhesion and confirmed the second extracellular (E2) domain as a critical domain for cell adhesion function. We found that cell adhesion decreased in cells expressing chimeric Cx50 in which the E2 domain was swapped with the E2 domain of either Cx43 or Cx46. In contrast, adhesion increased in cells expressing chimeric Cx43 and Cx46 with the Cx50 (E2) domain. This function is Cx channel-independent and Cx50 E2 domain-dependent cell adhesion acting in both homotypic and heterotypic manners. In addition, we generated eight site mutations of unique residues between Cx50 and the other two lens Cxs and found that mutation of any one of the residues abolished the adhesive function. Moreover, expression of adhesive-impaired mutants decreased adhesion-related proteins, N-cadherin and ß-catenin. Expression of the adhesion-impaired Cx50W188P mutant in embryonic chick lens caused enlarged extracellular spaces, distorted fiber organization, delayed nuclear condensation, and cortical cataracts. In summary, the results from both in vitro and in vivo studies demonstrate the importance of the adhesive function of Cx50 in the lens.

Autophagy Requirements for Eye Lens Differentiation and Transparency.

Recent evidence points to autophagy as an essential cellular requirement for achieving the mature structure, homeostasis, and transparency of the lens. Collective evidence from multiple laboratories using chick, mouse, primate, and human model systems provides evidence that classic autophagy structures, ranging from double-membrane autophagosomes to single-membrane autolysosomes, are found throughout the lens in both undifferentiated lens epithelial cells and maturing lens fiber cells. Recently, key autophagy signaling pathways have been identified to initiate critical steps in the lens differentiation program, including the elimination of organelles to form the core lens organelle-free zone. Other recent studies using ex vivo lens culture demonstrate that the low oxygen environment of the lens drives HIF1a-induced autophagy via upregulation of essential mitophagy components to direct the specific elimination of the mitochondria, endoplasmic reticulum, and Golgi apparatus during lens fiber cell differentiation. Pioneering studies on the structural requirements for the elimination of nuclei during lens differentiation reveal the presence of an entirely novel structure associated with degrading lens nuclei termed the nuclear excisosome. Considerable evidence also indicates that autophagy is a requirement for lens homeostasis, differentiation, and transparency, since the mutation of key autophagy proteins results in human cataract formation.

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin.

Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR ("Critical sequence") contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

Aquaporins in Eye.

The major part of the eye consists of water. Continuous movement of water and ions between the ocular compartments and to the systemic circulation is pivotal for many physiological functions in the eye. The movement of water facilitates removal of the many metabolic products of corneal-, ciliary body-, lens-, and retinal metabolism, while maintaining transparency in the optical compartments. Transport across the corneal epithelium and endothelium maintains the corneal transparency. Also, aqueous humor is continuously secreted by the epithelia of the ciliary body and maintains the intraocular pressure. In the retina, water is transported into the vitreous body and across the retinal pigment epithelium to regulate the extracellular environment and the hydration of the retina. Aquaporins are a major contributor in the water transport throughout the eye.