In vivo quasi-elastic light scattering detects molecular changes in the lenses of adolescents with Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder in humans. DS is associated with increased prevalence of several ocular sequelae, including characteristic blue-dot cerulean cataract. DS is accompanied by age-dependent accumulation of Alzheimer's disease (AD) amyloid-ß (Aß) peptides and amyloid pathology in the brain and comorbid early-onset Aß amyloidopathy and colocalizing cataracts in the lens. Quasi-elastic light scattering (QLS) is an established optical technique that noninvasively measures changes in protein size distributions in the human lens in vivo. In this cross-sectional study, lenticular QLS correlation time was decreased in adolescent subjects with DS compared to age-matched control subjects. Clinical QLS was consistent with alterations in relative particle hydrodynamic radius in lenses of adolescents with DS. These correlative results suggest that noninvasive QLS can be used to evaluate molecular changes in the lenses of individuals with DS.

Insights into the biochemical and biophysical mechanisms mediating the longevity of the transparent optics of the eye lens.

In the human eye, a transparent cornea and lens combine to form the "refracton" to focus images on the retina. This requires the refracton to have a high refractive index "n," mediated largely by extracellular collagen fibrils in the corneal stroma and the highly concentrated crystallin proteins in the cytoplasm of the lens fiber cells. Transparency is a result of short-range order in the spatial arrangement of corneal collagen fibrils and lens crystallins, generated in part by post-translational modifications (PTMs). However, while corneal collagen is remodeled continuously and replaced, lens crystallins are very long-lived and are not replaced and so accumulate PTMs over a lifetime. Eventually, a tipping point is reached when protein aggregation results in increased light scatter, inevitably leading to the iconic protein condensation-based disease, age-related cataract (ARC). Cataracts account for 50% of vision impairment worldwide, affecting far more people than other well-known protein aggregation-based diseases. However, because accumulation of crystallin PTMs begins before birth and long before ARC presents, we postulate that the lens protein PTMs contribute to a "cataractogenic load" that not only increases with age but also has protective effects on optical function by stabilizing lens crystallins until a tipping point is reached. In this review, we highlight decades of experimental findings that support the potential for PTMs to be protective during normal development. We hypothesize that ARC is preventable by protecting the biochemical and biophysical properties of lens proteins needed to maintain transparency, refraction, and optical function.

Alzheimer's disease amyloid-ß pathology in the lens of the eye.

Neuropathological hallmarks of Alzheimer's disease (AD) include pathogenic accumulation of amyloid-ß (Aß) peptides and age-dependent formation of amyloid plaques in the brain. AD-associated Aß neuropathology begins decades before onset of cognitive symptoms and slowly progresses over the course of the disease. We previously reported discovery of Aß deposition, ß-amyloidopathy, and co-localizing supranuclear cataracts (SNC) in lenses from people with AD, but not other neurodegenerative disorders or normal aging. We confirmed AD-associated Aß molecular pathology in the lens by immunohistopathology, amyloid histochemistry, immunoblot analysis, epitope mapping, immunogold electron microscopy, quantitative immunoassays, and tryptic digest mass spectrometry peptide sequencing. Ultrastructural analysis revealed that AD-associated Aß deposits in AD lenses localize as electron-dense microaggregates in the cytoplasm of supranuclear (deep cortex) fiber cells. These Aß microaggregates also contain αB-crystallin and scatter light, thus linking Aß pathology and SNC phenotype expression in the lenses of people with AD. Subsequent research identified Aß lens pathology as the molecular origin of the distinctive cataracts associated with Down syndrome (DS, trisomy 21), a chromosomal disorder invariantly associated with early-onset Aß accumulation and Aß amyloidopathy in the brain. Investigation of 1249 participants in the Framingham Eye Study found that AD-associated quantitative traits in brain and lens are co-heritable. Moreover, AD-associated lens traits preceded MRI brain traits and cognitive deficits by a decade or more and predicted future AD. A genome-wide association study of bivariate outcomes in the same subjects identified a new AD risk factor locus in the CTNND2 gene encoding δ-catenin, a protein that modulates Aß production in brain and lens. Here we report identification of AD-related human Aß (hAß) lens pathology and age-dependent SNC phenotype expression in the Tg2576 transgenic mouse model of AD. Tg2576 mice express Swedish mutant human amyloid precursor protein (APP-Swe), accumulate hAß peptides and amyloid pathology in the brain, and exhibit cognitive deficits that slowly progress with increasing age. We found that Tg2576 trangenic (Tg+) mice, but not non-transgenic (Tg-) control mice, also express human APP, accumulate hAß peptides, and develop hAß molecular and ultrastructural pathologies in the lens. Tg2576 Tg+ mice exhibit age-dependent Aß supranuclear lens opacification that recapitulates lens pathology and SNC phenotype expression in human AD. In addition, we detected hAß in conditioned medium from lens explant cultures prepared from Tg+ mice, but not Tg- control mice, a finding consistent with constitutive hAß generation in the lens. In vitro studies showed that hAß promoted mouse lens protein aggregation detected by quasi-elastic light scattering (QLS) spectroscopy. These results support mechanistic (genotype-phenotype) linkage between Aß pathology and AD-related phenotypes in lens and brain. Collectively, our findings identify Aß pathology as the shared molecular etiology of two age-dependent AD-related cataracts associated with two human diseases (AD, DS) and homologous murine cataracts in the Tg2576 transgenic mouse model of AD. These results represent the first evidence of AD-related Aß pathology outside the brain and point to lens Aß as an optically-accessible AD biomarker for early detection and longitudinal monitoring of this devastating neurodegenerative disease.

Age-related loss of auditory sensitivity in the zebrafish (Danio rerio).

Age-related hearing loss (ARHL), also known as presbycusis, is a widespread and debilitating condition impacting many older adults. Conventionally, researchers utilize mammalian model systems or human cadaveric tissue to study ARHL pathology. Recently, the zebrafish has become an effective and tractable model system for a wide variety of genetic and environmental auditory insults, but little is known about the incidence or extent of ARHL in zebrafish and other non-mammalian models. Here, we evaluated whether zebrafish exhibit age-related loss in auditory sensitivity. The auditory sensitivity of adult wild-type zebrafish (AB/WIK strain) from three adult age subgroups (13-month, 20-month, and 37-month) was characterized using the auditory evoked potential (AEP) recording technique. AEPs were elicited using pure tone stimuli (115-4500 Hz) presented via an underwater loudspeaker and recorded using shielded subdermal metal electrodes. Based on measures of sound pressure and particle acceleration, the mean AEP thresholds of 37-month-old fish [mean sound pressure level (SPL) = 122.2 dB ± 2.2 dB SE re: 1 µPa; mean particle acceleration level (PAL) = -27.5 ± 2.3 dB SE re: 1 ms-2] were approximately 9 dB higher than that of 20-month-old fish [(mean SPL = 113.1 ± 2.7 dB SE re: 1 µPa; mean PAL = -37.2 ± 2.8 dB re: 1 ms-2; p = 0.007)] and 6 dB higher than that of 13-month-old fish [(mean SPL = 116.3 ± 2.5 dB SE re: 1 µPa; mean PAL = -34.1 ± 2.6 dB SE re: 1 ms-2; p = 0.052)]. Lowest AEP thresholds for all three age groups were generally between 800 Hz and 1850 Hz, with no evidence for frequency-specific age-related loss. Our results suggest that zebrafish undergo age-related loss in auditory sensitivity, but the form and magnitude of loss is markedly different than in mammals, including humans. Future work is needed to further describe the incidence and extent of ARHL across vertebrate groups and to determine which, if any, ARHL mechanisms may be conserved across vertebrates to support meaningful comparative/translational studies.

In Vivo Quasi-Elastic Light Scattering Eye Scanner Detects Molecular Aging in Humans.

The absence of clinical tools to evaluate individual variation in the pace of aging represents a major impediment to understanding aging and maximizing health throughout life. The human lens is an ideal tissue for quantitative assessment of molecular aging in vivo. Long-lived proteins in lens fiber cells are expressed during fetal life, do not undergo turnover, accumulate molecular alterations throughout life, and are optically accessible in vivo. We used quasi-elastic light scattering (QLS) to measure age-dependent signals in lenses of healthy human subjects. Age-dependent QLS signal changes detected in vivo recapitulated time-dependent changes in hydrodynamic radius, protein polydispersity, and supramolecular order of human lens proteins during long-term incubation (~1 year) and in response to sustained oxidation (~2.5 months) in vitro. Our findings demonstrate that QLS analysis of human lens proteins provides a practical technique for noninvasive assessment of molecular aging in vivo.

The occhiolino (occ) mutant Zebrafish, a model for development of the optical function in the biological lens.

BACKGROUND: Zebrafish visual function depends on quality optics. An F3 screen for developmental mutations in the Zebrafish nervous system was conducted in wild-type (wt) AB Zebrafish exposed to 3 mM of N-ethyl-N-nitrosourea (ENU). RESULTS: Mutant offspring, identified in an F3 screen, were characterized by a small pupil, resulting from retinal hypertrophy or hyperplasia and a small lens. Deficits in visual function made feeding difficult after hatching at approximately 5-6 days postfertilization (dpf). Special feeding conditions were necessary for survival of the occhiolino (occ) mutants after 6 dpf. Optokinetic response (OKR) tests measured defects in visual function in the occ mutant, although electroretinograms (ERGs) were normal in the mutant and wt. Consistent with the ERGs, histology found normal retinal structure in the occ mutant and wt Zebrafish. However, lens development was abnormal. Multiphoton imaging of the developmental stages of live embryos confirmed the formation of a secondary mass of lens cells in the developing eye of the mutant Zebrafish at 3-4 dpf, and laminin immunohistochemistry indicated the lens capsule was thin and disorganized in the mutant Zebrafish. CONCLUSIONS: The occ Zebrafish is a novel disease model for visual defects associated with abnormal lens development. Developmental Dynamics 246:915-924, 2017. © 2017 Wiley Periodicals, Inc.

Functional sequences in human alphaB crystallin.

BACKGROUND: Human alphaB crystallin (HspB5) contains the alpha crystallin core domain, a series of antiparallel beta-strands organized into the characteristic beta sandwich of small heat shock proteins (sHsps). The full 3-dimensional structure for alpha crystallin has not been determined and the mechanism for the biological activity remains elusive because sHsps participate in multiple interactions with a broad range of target proteins that favor self-assembly of polydisperse fibrils and complexes. We selected human alphaB crystallin to study interactive sequences because it is involved in many human condensation, amyloid, and aggregation diseases and it is very sensitive to the destabilization of unfolding proteins. Sophisticated methods are being used to analyze and complete the structure of alphaB crystallin with the expectation of understanding sHsp function. This review considers the identification of interactive sites on the surface of the alphaB crystallin, which may be the key to understanding the multifunctional activity of human alphaB crystallin. SCOPE OF REVIEW: This review summarizes the research on the identification of the bioactive interactive sequences responsible for the function of human alphaB crystallin, an sHsp with chaperone-like activity. MAJOR CONCLUSIONS: The multifunctional activity of human alphaB crystallin results from the interactive peptide sequences exposed on the surface of the molecule. The multiple, non-covalent, interactive sequences can account for the selectivity and sensitivity of alphaB crystallin to the initiation of protein unfolding. GENERAL SIGNIFICANCE: Human alphaB crystallin may be an important part of an endogenous protective mechanism in aging cells and tissues. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging.

MALDI imaging requires careful sample preparation to obtain reliable, high-quality images of small molecules, peptides, lipids, and proteins across tissue sections. Poor crystal formation, delocalization of analytes, and inadequate tissue adherence can affect the quality, reliability, and spatial resolution of MALDI images. We report a comparison of tissue mounting and washing methods that resulted in an optimized method using conductive carbon substrates that avoids thaw mounting or washing steps, minimizes protein delocalization, and prevents tissue detachment from the target surface. Application of this method to image ocular lens proteins of small vertebrate eyes demonstrates the improved methodology for imaging abundant crystallin protein products. This method was demonstrated for tissue sections from rat, mouse, and zebrafish lenses resulting in good-quality MALDI images with little to no delocalization. The images indicate, for the first time in mouse and zebrafish, discrete localization of crystallin protein degradation products resulting in concentric rings of distinct protein contents that may be responsible for the refractive index gradient of vertebrate lenses.

Genome sequencing of idiopathic pulmonary fibrosis in conjunction with a medical school human anatomy course.

Even in cases where there is no obvious family history of disease, genome sequencing may contribute to clinical diagnosis and management. Clinical application of the genome has not yet become routine, however, in part because physicians are still learning how best to utilize such information. As an educational research exercise performed in conjunction with our medical school human anatomy course, we explored the potential utility of determining the whole genome sequence of a patient who had died following a clinical diagnosis of idiopathic pulmonary fibrosis (IPF). Medical students performed dissection and whole genome sequencing of the cadaver. Gross and microscopic findings were more consistent with the fibrosing variant of nonspecific interstitial pneumonia (NSIP), as opposed to IPF per se. Variants in genes causing Mendelian disorders predisposing to IPF were not detected. However, whole genome sequencing identified several common variants associated with IPF, including a single nucleotide polymorphism (SNP), rs35705950, located in the promoter region of the gene encoding mucin glycoprotein MUC5B. The MUC5B promoter polymorphism was recently found to markedly elevate risk for IPF, though a particular association with NSIP has not been previously reported, nor has its contribution to disease risk previously been evaluated in the genome-wide context of all genetic variants. We did not identify additional predicted functional variants in a region of linkage disequilibrium (LD) adjacent to MUC5B, nor did we discover other likely risk-contributing variants elsewhere in the genome. Whole genome sequencing thus corroborates the association of rs35705950 with MUC5B dysregulation and interstitial lung disease. This novel exercise additionally served a unique mission in bridging clinical and basic science education.

Self-assembly of protein aggregates in ageing disorders: the lens and cataract model.

Cataract, neurodegenerative disease, macular degeneration and pathologies of ageing are often characterized by the slow progressive destabilization of proteins and their self-assembly to amyloid-like fibrils and aggregates. During normal cell differentiation, protein self-assembly is well established as a dynamic mechanism for cytoskeletal organization. With the increased emphasis on ageing disorders, there is renewed interest in small-molecule regulators of protein self-assembly. Synthetic peptides, mini-chaperones, aptamers, ATP and pantethine reportedly regulate self-assembly mechanisms involving small stress proteins, represented by human αB-crystallin, and their targets. Small molecules are being considered for direct application as molecular therapeutics to protect against amyloid and protein aggregation disorders in ageing cells and tissues in vivo. The identification of specific interactive peptide sites for effective regulation of protein self-assembly is underway using conventional and innovative technologies. The quantification of the functional interactions between small stress proteins and their targets in vivo remains a top research priority. The quantitative parameters controlling protein-protein interactions in vivo need characterization to understand the fundamental biology of self-assembling systems in normal cells and disorders of ageing.

Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens.

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α2ß2-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.

Hemoglobin interactions with αB crystallin: a direct test of sensitivity to protein instability.

As a small stress response protein, human αB crystallin, detects protein destabilization that can alter structure and function to cause self assembly of fibrils or aggregates in diseases of aging. The sensitivity of αB crystallin to protein instability was evaluated using wild-type hemoglobin (HbA) and hemoglobin S (HbS), the glutamate-6-valine mutant that forms elongated, filamentous aggregates in sickling red blood cells. The progressive thermal unfolding and aggregation of HbA and HbS in solution at 37°C, 50°C and 55°C was measured as increased light scattering. UV circular dichroism (UVCD) was used to evaluate conformational changes in HbA and HbS with time at the selected temperatures. The changes in interactions between αB crystallin and HbA or HbS with temperature were analyzed using differential centrifugation and SDS PAGE at 37°C, 50°C and 55°C. After only 5 minutes at the selected temperatures, differences in the aggregation or conformation of HbA and HbS were not observed, but αB crystallin bound approximately 6% and 25% more HbS than HbA at 37°C, and 50°C respectively. The results confirmed (a) the remarkable sensitivity of αB crystallin to structural instabilities at the very earliest stages of thermal unfolding and (b) an ability to distinguish the self assembling mutant form of HbS from the wild type HbA in solution.

New insights into the mechanism of lens development using zebra fish.

On the basis of recent advances in molecular biology, genetics, and live-embryo imaging, direct comparisons between zebra fish and human lens development are being made. The zebra fish has numerous experimental advantages for investigation of fundamental biomedical problems that are often best studied in the lens. The physical characteristics of visible light can account for the highly coordinated cell differentiation during formation of a beautifully transparent, refractile, symmetric optical element, the biological lens. The accessibility of the zebra fish lens for direct investigation during rapid development will result in new knowledge about basic functional mechanisms of epithelia-mesenchymal transitions, cell fate, cell-matrix interactions, cytoskeletal interactions, cytoplasmic crowding, membrane transport, cell adhesion, cell signaling, and metabolic specialization. The lens is well known as a model for characterization of cell and molecular aging. We review the recent advances in understanding vertebrate lens development conducted with zebra fish.

Evolution of the vertebrate beaded filament protein, Bfsp2; comparing the in vitro assembly properties of a "tailed" zebrafish Bfsp2 to its "tailless" human orthologue.

In bony fishes, Bfsp2 orthologues are predicted to possess a C-terminal tail domain, which is absent from avian, amphibian and mammalian Bfsp2 sequences. These sequences, are however, not conserved between fish species and therefore questions whether they have a functional role. For other intermediate filament proteins, the C-terminal tail domain is important for both filament assembly and regulating interactions between filaments. We confirm that zebrafish has a single Bfsp2 gene by radiation mapping. Two transcripts (bfsp2α and bfsp2ß) are produced by alternative splicing of the last exon. Using a polyclonal antibody specific to a tridecameric peptide in the C-terminal tail domain common to both zebrafish Bfsp2 splice variants, we have confirmed its expression in zebrafish lens fibre cells. We have also determined the in vitro assembly properties of zebrafish Bfsp2α and conclude that the C-terminal sequences are required to regulate not only the diameter and uniformity of the in vitro assembly filaments, but also their filament-filament associations in vitro. Therefore we conclude zebrafish Bfsp2α is a functional orthologue conforming more closely to the conventional domain structure of intermediate filament proteins. Data mining of the genome databases suggest that the loss of this tail domain could occur in several stages leading eventually to completely tailless orthologues, such as human BFSP2.

Multiple sites in αB-crystallin modulate its interactions with desmin filaments assembled in vitro.

The ß3- and ß8-strands and C-terminal residues 155-165 of αB-crystallin were identified by pin arrays as interaction sites for various client proteins including the intermediate filament protein desmin. Here we present data using 5 well-characterised αB-crystallin protein constructs with substituted ß3- and ß8-strands and with the C-terminal residues 155-165 deleted to demonstrate the importance of these sequences to the interaction of αB-crystallin with desmin filaments. We used electron microscopy of negatively stained samples to visualize increased interactions followed by sedimentation assays to quantify our observations. A low-speed sedimentation assay measured the ability of αB-crystallin to prevent the self-association of desmin filaments. A high-speed sedimentation assay measured αB-crystallin cosedimentation with desmin filaments. Swapping the ß8-strand of αB-crystallin or deleting residues 155-165 increased the cosedimentation of αB-crystallin with desmin filaments, but this coincided with increased filament-filament interactions. In contrast, substitution of the ß3-strand with the equivalent αA-crystallin sequences improved the ability of αB-crystallin to prevent desmin filament-filament interactions with no significant change in its cosedimentation properties. These data suggest that all three sequences (ß3-strand, ß8-strand and C-terminal residues 155-165) contribute to the interaction of αB-crystallin with desmin filaments. The data also suggest that the cosedimentation of αB-crystallin with desmin filaments does not necessarily correlate with preventing desmin filament-filament interactions. This important observation is relevant not only to the formation of the protein aggregates that contain both desmin and αB-crystallin and typify desmin related myopathies, but also to the interaction of αB-crystallin with other filamentous protein polymers.

Dynamic subunit exchange and the regulation of microtubule assembly by the stress response protein human alphaB crystallin.

BACKGROUND: The small heat shock protein (sHSP), human alphaB crystallin, forms large, polydisperse complexes that modulate the tubulin-microtubule equilibrium using a dynamic mechanism that is poorly understood. The interactive sequences in alphaB crystallin for tubulin are surface exposed, and correspond to interactive sites for the formation of alphaB crystallin complexes. METHODOLOGY/PRINCIPAL FINDINGS: There is sequence homology between tubulin and the interactive domains in the beta8-strand of the core domain and the C-terminal extension of alphaB crystallin. This study investigated the hypothesis that the formation of tubulin and alphaB crystallin quaternary structures was regulated through common interactive domains that alter the dynamics of their assembly. Size exclusion chromatography (SEC), SDS-PAGE, microtubule assembly assays, aggregation assays, multiple sequence alignment, and molecular modeling characterized the dynamic response of tubulin assembly to increasing concentrations of alphaB crystallin. Low molar ratios of alphaB crystallin:tubulin were favorable for microtubule assembly and high molar ratios of alphaB crystallin:tubulin were unfavorable for microtubule assembly. Interactions between alphaB crystallin and unassembled tubulin were observed using SEC and SDS-PAGE. CONCLUSIONS/SIGNIFICANCE: Subunits of alphaB crystallin that exchange dynamically with the alphaB crystallin complex can interact with tubulin subunits to regulate the equilibrium between tubulin and microtubules.

Alzheimer's disease amyloid-beta links lens and brain pathology in Down syndrome.

Down syndrome (DS, trisomy 21) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans. In DS, triplication of chromosome 21 invariably includes the APP gene (21q21) encoding the Alzheimer's disease (AD) amyloid precursor protein (APP). Triplication of the APP gene accelerates APP expression leading to cerebral accumulation of APP-derived amyloid-beta peptides (Abeta), early-onset AD neuropathology, and age-dependent cognitive sequelae. The DS phenotype complex also includes distinctive early-onset cerulean cataracts of unknown etiology. Previously, we reported increased Abeta accumulation, co-localizing amyloid pathology, and disease-linked supranuclear cataracts in the ocular lenses of subjects with AD. Here, we investigate the hypothesis that related AD-linked Abeta pathology underlies the distinctive lens phenotype associated with DS. Ophthalmological examinations of DS subjects were correlated with phenotypic, histochemical, and biochemical analyses of lenses obtained from DS, AD, and normal control subjects. Evaluation of DS lenses revealed a characteristic pattern of supranuclear opacification accompanied by accelerated supranuclear Abeta accumulation, co-localizing amyloid pathology, and fiber cell cytoplasmic Abeta aggregates (approximately 5 to 50 nm) identical to the lens pathology identified in AD. Peptide sequencing, immunoblot analysis, and ELISA confirmed the identity and increased accumulation of Abeta in DS lenses. Incubation of synthetic Abeta with human lens protein promoted protein aggregation, amyloid formation, and light scattering that recapitulated the molecular pathology and clinical features observed in DS lenses. These results establish the genetic etiology of the distinctive lens phenotype in DS and identify the molecular origin and pathogenic mechanism by which lens pathology is expressed in this common chromosomal disorder. Moreover, these findings confirm increased Abeta accumulation as a key pathogenic determinant linking lens and brain pathology in both DS and AD.

Cell fate and differentiation of the developing ocular lens.

PURPOSE: Even though zebrafish development does not include the formation of a lens vesicle, the authors' hypothesis is that the processes of cell differentiation are similar in zebrafish and mammals and determine cell fates in the lens. METHODS: Two-photon live embryo imaging was used to follow individual fluorescently labeled cells in real-time from the placode stage at 16 hours postfertilization (hpf) until obvious morphologic differentiation into epithelium or fiber cells had occurred at approximately 28 hpf. Immunohistochemistry was used to label proliferating, differentiating, and apoptotic cells. RESULTS: Similar to the mammal, cells in the teleost peripheral lens placode migrated to the anterior lens mass and differentiated into an anterior epithelium. Cells in the central lens placode migrated to the posterior lens mass and differentiated into primary fiber cells. Anterior and posterior polarization in the zebrafish lens mass was similar to mammalian lens vesicle polarization. Primary fiber cell differentiation was apparent at approximately 21 hpf, before separation of the lens from the surface ectoderm, as evidenced by cell elongation, exit from the cell cycle, and expression of Zl-1, a marker for fiber differentiation. TUNEL labeling demonstrated that apoptosis was not a primary mechanism for lens separation from the surface ectoderm. CONCLUSIONS: Despite the absence of a lens vesicle in the zebrafish embryo, lens organogenesis appears to be well conserved among vertebrates. Results using three-dimensional live embryo imaging of zebrafish development showed minimal differences and strong similarities in the fate of cells in the zebrafish and mammalian lens placode.

The zebrafish lens proteome during development and aging.

PURPOSE: Changes in lens protein expression during zebrafish development results in a smooth gradient of refractive index necessary for excellent optical function. Age-related changes in crystallin expression have been well documented in mammals but are poorly understood in the zebrafish. METHODS: In the zebrafish lens, a systematic analysis of protein content with age was performed using size exclusion chromatography (SEC) combined with linear trap quadrupole Fourier transform tandem mass spectrometry (LTQ-FT LC-MS/MS; rank-order shotgun) proteomics in lenses of larval, juvenile, and adult zebrafish. RESULTS: alpha-Crystallins, previously shown to have low abundance in the zebrafish lens, were found to increase dramatically with maturation and aging. SEC determined that beta-crystallin was predominant at 4.5 days. With age, the alpha- and gamma-crystallins increased, and a high molecular weight fraction appeared between six weeks and six months to become the dominant component by 2.5 years. Similarly, shotgun proteomics determined that beta-crystallins were the predominant proteins in the young lens. With age, the proportion of alpha- and gamma-crystallins increased dramatically. After crystallins, calpain 3, membrane, and cytoskeletal proteins were most abundant. Five new beta-crystallins and 13 new gamma-crystallins were identified. CONCLUSIONS: As expected, SEC and proteomics demonstrated changing levels of protein expression with age, especially among the crystallins. The results also confirmed the existence of novel crystallins in the zebrafish genome.

Early lens development in the zebrafish: a three-dimensional time-lapse analysis.

In vivo, high-resolution, time-lapse imaging characterized lens development in the zebrafish from 16 to 96 hr postfertilization (hpf). In zebrafish, the lens placode appeared in the head ectoderm, similar to mammals. Delamination of the surface ectoderm resulted in the formation of the lens mass, which progressed to a solid sphere of cells separating from the developing cornea at approximately 24 hpf. A lens vesicle was not observed and apoptosis was not a major factor in separation of the lens from the future cornea. Differentiation of primary fibers began in the lens mass followed by formation of the anterior epithelium after delamination was complete. Secondary fibers differentiated from elongating epithelial cells near the posterior pole. Quantification characterized three stages of lens growth. The study confirmed the advantages of live-cell imaging for three-dimensional quantitative structural characterization of the mechanism(s) responsible for cell differentiation in formation of a transparent, symmetric, and refractile lens.