Jer R. Kuszak

Alzheimer's Disease Amyloid-β 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 Alzheimers disease (AD) amyloid precursor protein (APP). Triplication of the APP gene accelerates APP expression leading to cerebral accumulation of APP-derived amyloid-β peptides (Aβ), 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 Aβ 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 Aβ 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 Aβ accumulation, co-localizing amyloid pathology, and fiber cell cytoplasmic Aβ aggregates (∼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 Aβ in DS lenses. Incubation of synthetic Aβ 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 Aβ accumulation as a key pathogenic determinant linking lens and brain pathology in both DS and AD.

Knockout of the intermediate filament protein CP49 destabilises the lens fibre cell cytoskeleton and decreases lens optical quality, but does not induce cataract

In this report, the phenotype associated with the first targeted knockout of the lens specific intermediate filament gene CP49 is described. Several surprising observations have been made. The first was that no cataract was observed despite the fact that the beaded filaments of the lens fibre cells had been disrupted. Light scatter and the lens optical properties had, however, deteriorated in the CP49 knockout lenses compared to litter mate controls. These changes were accompanied by dramatic changes in plasma membrane organisation of the fibre cells as revealed by detailed morphological examinations and providing the second surprising result. The CP49 knockout mouse is therefore an important model to study the functional link between lens transparency, the cytoskeleton and plasma membrane organisation.

A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. I: Transparency and epithelial cell layer

Short-term photochemical insult of cultured rat lenses caused by the generation of H2O2, O2<--and OH. was found to lead to rapid irreversible damage to the epithelial cell layer. This irreversible damage was measured by Trypan blue staining, terminal deoxyribonucleotidyl transferase labeling, DNA laddering and morphological analyses. There appears to be an inverse relationship between the period of photochemical insult and the post-insult time required to observe epithelial cell damage. Insulting periods of a few hours require post-insult intervals of days to observe significant cell damage and weeks before complete cortical cataracts are found. Epithelial cell damage precedes the loss of transparency.

Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0-LTR protein.

Aquaporin‐0 (AQP0) is the major intrinsic protein of lens fiber cells and the founder member of the water channel gene family. Here we show that disruption of the AQP0 gene by an early transposon (ETn) element results in expression of a chimeric protein, comprised of ∼75% AQP0 and ~25% ETn long terminal repeat (LTR) sequence, in the cataract Fraser (CatFr) mouse lens. Immunoblot analysis showed that mutant AQP0‐LTR was similar in mass to wild‐type AQP0. However, immunofluorescence microscopy revealed that AQP0‐LTR was localized to intracellular membranes rather than to plasma membranes of lens fiber cells. Heterozygous CatFr lenses were similar in size to wild‐type but displayed abnormal regions of translucence and light scattering. Scanning electron microscopy further revealed that mature fiber cells within the core of the heterozygous Cat Fr lens failed to stratify into uniform, concentric growth shells, suggesting that the AQP0 water channel facilitates the development of the unique cellular architecture of the crystalline lens.—Shiels, A., Mackay, D., Bassnett, S., Al‐Ghoul, K., Kuszak, J. Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0‐LTR protein. FASEB J. 14, 2207–2212 (2000)

An ultrastructural analysis of the epithelial-fiber interface (EFI) in primate lenses

The purpose of this study was to conduct a comprehensive ultrastructural analysis of the epithelial-fiber interface (EFI) in normal adult primate (Macaque nemestrina and fascicularis; 6-9 years old, n = 10) lenses. Scanning electron microscopy (SEM) was used to initially characterize the gross size, shape and three-dimensional organization of central zone (cz) epithelial cells and the anterior ends of elongating fibers beneath these cells. This fiducial information was essential to properly orient lens pieces in freeze fracture specimen carriers for the production of replicas with unambiguously identifiable EFI. Transmission electron microscopy (TEM) of replicas and thin-sectioned material were used to ultrastructurally analyse the cz EFI. TEM thin-sectioned material was also used to ultrastructurally analyse the pregerminative (pgz), germinative (gz) and transitional zone (tz) EFI. Correlative SEM and TEM of cz EFI components revealed that the apical membrane of both epithelial and elongating fiber cells were irregularly polygonal in shape, and aligned in parallel as smooth, concave-convex surfaces. However, whereas epithelial cell apical surfaces had minimal size variation, elongating fibers were larger and considerably variable in size. Quantitative analysis of > 10000 micron2 cz elongating fiber apical surfaces failed to detect any gap junctions defined in freeze fracture replicas as complementary aggregates of transmembrane proteins (connexons) conjoined across a narrowed extracellular space. However, a comparable frequency of vesicular events was noted in this region as quantified previously in adult and embryonic chick lens. Correlative TEM analysis > 1500 linear micrometers of thin-sectioned EFI from this region confirmed the presence of epithelial-epithelial gap junctions, elongating fiber-elongating fiber gap junctions, and an extreme paucity of epithelial-elongating fiber gap junctions. In contrast, TEM analysis of > 1000 linear micrometers of thin-sectioned pgz, gz and tz EFI, confirmed the presence of epithelial-epithelial gap junctions, elongating fiber-elongating fiber gap junctions, numerous epithelial-elongating fiber adherens junctions and a few epithelial-elongating fiber gap junctions. Thus, the results of this and previous quantitative morphological and physiological studies (electronic and dye coupling) demonstrate that there is limited coupling between cz epithelial cells and underlying elongating fibers. Furthermore, the absence of gap junctional plaques in cz EFI freeze-fracture replicas and either pentalaminar or septalaminar profiles in correlative thin-sections, suggests that this limited coupling could be mediated via isolated gap junction channels. However, the results of this and previous quantitative studies further show that a greater degree of coupling exists across the pgz, gz and tz regions of the EFI and that this coupling is likely to be mediated by gap junction plaques. Finally, this and other studies continue to demonstrate that transcytotic processes play a role in lens physiology at the EFI.

Analysis of nuclear fiber cell compaction in transparent and cataractous diabetic human lenses by scanning electron microscopy

BackgroundCompaction of human ocular lens fiber cells as a function of both aging and cataractogenesis has been demonstrated previously using scanning electron microscopy. The purpose of this investigation is to quantify morphological differences in the inner nuclear regions of cataractous and non-cataractous human lenses from individuals with diabetes. The hypothesis is that, even in the presence of the osmotic stress caused by diabetes, compaction rather than swelling occurs in the nucleus of diabetic lenses.MethodsTransparent and nuclear cataractous lenses from diabetic patients were examined by scanning electron microscopy (SEM). Measurements of the fetal nuclear (FN) elliptical angles (anterior and posterior), embryonic nuclear (EN) anterior-posterior (A-P) axial thickness, and the number of EN fiber cell membrane folds over 20 μm were compared.ResultsDiabetic lenses with nuclear cataract exhibited smaller FN elliptical angles, smaller EN axial thicknesses, and larger numbers of EN compaction folds than their non-cataractous diabetic counterparts.ConclusionAs in non-diabetic lenses, the inner nuclei of cataractous lenses from diabetics were significantly more compacted than those of non-cataractous diabetics. Little difference between diabetic and non-diabetic compaction levels was found, suggesting that diabetes does not affect the degree of compaction. However, consistent with previous proposals, diabetes does appear to accelerate the formation of cataracts that are similar to age-related nuclear cataracts in non-diabetics. We conclude that as scattering increases in the diabetic lens with cataract formation, fiber cell compaction is significant.

Perturbing the Ubiquitin Pathway Reveals How Mitosis Is Hijacked to Denucleate and Regulate Cell Proliferation and Differentiation In Vivo

Background The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6. Methodology and Principal Findings We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27kip, a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues. Conclusions and Significance K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.

The Intermediate Filament Systems in the Eye Lens

Publisher Summary This chapter describes the basic methods to purify important lens fractions and to track the distribution of IF proteins in the eye lens and their associated proteins. It also provides the description of the methods used for the preparation, sectioning, and examination of eye lenses. The IF proteins of the lens help in determining its optical properties. Whereas, a transparent lens is normal, a lens with either a reduced level of transparency or a localized opacity is abnormal and/or pathological. One of the pitfalls in the purification of lens proteins is proteolysis, especially when urea buffers are used, but the methods in the chapter describe the effective purification of CP49, filensin, and vimentin for use in vitro assembly and structural studies. The animal models can help to understand the mechanisms of cataractogensis, and in combination with the optical studies the futuristic possibility of regenerating superlenses for combating cataract may be expected.

Intralenticular implant study in pigmented rabbits: Opacity lensmeter assessment

PURPOSE To quantitatively analyze the clarity of regenerated lens material after endocapsular lens extraction and restoration of the lens capsular bag with and without implantation of an intralenticular disc lens. SETTING Shepherd Research Center, Allergan, Irvine, California, USA. METHOD The clarity of regenerated lens material was evaluated by Interzeag Opacity Lensmeter 701 (OLM) recordings after endocapsular lens extraction in New Zealand/Dutch Belt pigmented rabbits with (n = 21) and without (n = 16) placement of a disc-shaped intralenticular implant in the capsular bag. Postoperative objective measurements were performed at 1, 2, and 3 weeks and 1, 2, 3, and 6 months. Comparisons were made between young and adult rabbits. RESULTS Mean OLM results were similar in both groups at weeks 1, 2, 3, and 4. After 1 month, progressive central compaction of early irregular regenerated lens fibers was associated with increased OLM readings that were higher in the intralenticular implant group than in the control group. Regenerated lens opacification was greater in tissue posterior to the intralenticular lens than in that anterior to the disc lens. CONCLUSION Insertion of an intralenticular disc lens into the lens capsule bag was associated with poor optical clarity primarily of the posterior regenerated lens tissue. The OLM was useful in assessing the degree of opacification of the regenerated lenses.