Structural changes in lenses of mice lacking the gap junction protein connexin43

PURPOSE: To investigate the role of the gap junction protein connexin43 (Cx43), which is predominantly expressed in lens epithelial cells in the control of lens development and organization. METHODS: Newborn mice in which the Cx43 gene was disrupted by homologous recombination were used. Lenses from Cx43 (-/-) mice and wild-type littermates were processed by using 2% glutaraldehyde fixation for light and transmission electron microscopy and by freezing in liquid nitrogen for light and confocal microscopy of immunofluorescence in cryosections. RESULTS: In wild-type mice, Cx43 was immunolocalized to apical and lateral regions of lens epithelial cells and throughout the cornea, iris, ciliary body, and retina. In the bow, or equatorial, region of the lens, Cx43 disappeared gradually at the margins of the epithelial layer, whereas major intrinsic polypeptide, MP26, and alpha-crystallins were only detected in differentiated fiber cells. Ultrastructural studies revealed that epithelial cells and epithelial fiber cells were connected by large gap junctions. Lens fiber cells were closely apposed to apical boundaries of epithelial cells and apposed to one another along their entire lengths. In Cx43 (-/-) mice, epithelial cells were connected more loosely. The distribution of MP26 and alpha-crystallin in bow region fiber cells in Cx43 (-/-) lenses was not distinguishable from that in the lenses of wild-type mice. Cx46 and Cx50 were also expressed in superficial and cortical fiber cells, with similar distributions in Cx43 (-/-) and wild-type mice. However, organization of appositional membranes between lens fiber cells and between fiber and epithelial cells differed dramatically in the Cx43 (-/-) lens. In contrast to the close apposition of cells in lenses of normal mice, fiber cells in Cx43 (-/-) lenses were largely separated from apical surfaces of epithelial cells, and large vacuolar spaces were apparent between fiber cells, most prominently in deeper cortical regions. CONCLUSIONS: The normal differentiation of lens fiber cells in the bow region in lenses of Cx43 (-/-) mice, evidenced by similar distributions of Cx46, Cx50, MP26, and alpha-crystallin, suggests that the expression of Cx43 is not required for this process. However, these lenses exhibit grossly dilated extracellular spaces and intracellular vacuoles, indicative of early stages of cataract formation. These changes suggest that osmotic balance within the lens is markedly altered in Cx43 (-/-) animals, highlighting the importance of intercellular communication mediated by lens epithelial Cx43 gap junctions in the function of this tissue.     

Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens

SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin/BM40) is a secreted Ca2+-binding glycoprotein that interacts with a range of extracellular matrix molecules, including collagen IV. It is widely expressed during embryogenesis, and in vitro studies have suggested roles in the regulation of cell adhesion and proliferation, and in the modulation of cytokine activity. In order to analyse the function of this protein in vivo, the endogenous Sparc locus was disrupted by homologous recombination in murine embryonic stem cells. SPARC-deficient mice (Sparctm1Cam) appear normal and fertile until around 6 months of age, when they develop severe eye pathology characterized by cataract formation and rupture of the lens capsule. The first sign of lens pathology occurs in the equatorial bow region where vacuoles gradually form within differentiating epithelial cells and fibre cells. The lens capsule, however, shows no qualitative changes in the major basal lamina proteins laminin, collagen IV, perlecan or entactin. These mice are an excellent resource for further studies on how SPARC affects cell behaviour in vivo.     

A proteinase K inhibitor using alpha,beta-unsaturated (dehydro) residues: a presumptive model

PURPOSE: To quantify age-related changes in products of lipid oxidation in human lenses and to relate these changes to membrane hydrocarbon chain structure. Deviation from a well-defined membrane-lipid composition and structure could result in alterations in membrane function and disruption of the homeostasis of the cell. METHODS: Infrared spectroscopy was used to detect lipid compositional and structural changes in human lens membranes associated with age and cataracts. RESULTS: Lipid oxidation increased linearly threefold relative to total phospholipids in subjects ranging in age between 1 and 85 years, as was evident by increases in trans double bonds, lipid carbonyls, and secondary products. There was no statistical difference between the levels of lipid oxidation in the cortex or nucleus. Lipid hydrocarbon chain order (rigidity) increased from approximately 40% at birth to 70% at 80 years of age. Changes in lipid order correlated with changes in the relative content of membrane phosphatidylcholine and sphingomyelin, and with the level of lipid oxidation. CONCLUSIONS: Lipid oxidation increased linearly and uniformly throughout the human lens with age. The change in lipid oxidation with age correlated to a change in lipid order.     

Abnormal eye development associated with Cat4a, a dominant mouse cataract mutation on chromosome 8

PURPOSE: Cat4a, one of four mutant alleles at the mouse Cat4 locus, causes central corneal opacity and anterior polar cataract in heterozygotes and microphthalmia in homozygotes. The Cat4 locus has been mapped to chromosome 8, 31 cM from the centromere. In this study ocular development of Cat4a mutant mice was investigated to characterize the defects in eye morphogenesis. METHODS: Serial sections from eyes of wild-type, heterozygous, and homozygous littermates were examined by means of light microscopy at selected intervals from embryonic day 11 to postnatal day 1. Eyes of adult heterozygous and homozygous mice also were evaluated histologically. RESULTS: Failure of separation of the lens vesicle from the surface ectoderm was the earliest structural defect observed. In heterozygous embryos, the abnormality was limited to persistent connection of the anterior pole of the lens to the cornea. Adult heterozygotes had defects in the central corneal stroma and endothelium and anterior polar cataracts with or without keratolenticular adhesion. In homozygous embryos, the persistent connection of lens to surface ectoderm was associated with aborted lens development, failure of closure of the optic fissure, and impairment of growth of the eyecup. Microphthalmic eyes of adult homozygous mice had a poorly developed cornea, and the anterior chamber and vitreous compartment were absent. An extensively folded retina and remnants of a degenerated lens filled the interior of the globe. CONCLUSIONS: A developmental defect inhibits separation of the lens vesicle from surface ectoderm in mice heterozygous or homozygous for the Cat4a mutation. In homozygotes subsequent lens and eye morphogenesis are also severely affected. Cat4a shows phenotypical similarity to several other independent mouse mutations including Small eye, a mutation of the Pax6 gene. Cat4 may be one of several genes involved in a common developmental path and may be part of the Pax6-regulated gene cascade governing eye morphogenesis.     

The effect of light deprivation on the mouse lens

This work was undertaken to test the hypothesis that first exposure of the eye to light is responsible for the changes in lens protein expression patterns observed around the time of birth. The effect of light deprivation on lens properties was examined in Balb c mice which were bred, reared and maintained in complete darkness for up to 7 months. Data were collected on body weight, lens weight, lens protein contents and crystallin distributions. The data were compared with those obtained from age matched mice maintained in natural light/dark conditions. No significant differences were observed in body weight between animals maintained in the light and dark. However, animals kept in the dark had significantly smaller lenses. After 6 months in the dark, the lens represented 0.02% of body weight compared with 0.031% in the light reared animals (P < 0.001). Lens protein concentration, insoluble protein contents and crystallin synthesis patterns were indistinguishable for the two groups of animals. It is concluded that light stimulation of the eye is required for optimal lens growth but does not affect the production of specific crystallins.     

Do routine antibiotics after loop diathermy excision reduce morbidity?

AIMS: To determine functional results after unilateral and bilateral cataract surgery in children with different aphakic optical correction. METHODS: In this retrospective study, we evaluated visual acuity and binocular vision in 107 children who underwent cataract surgery during the 10 year period from 1985 to 1995. Aphakia was corrected by an intracapsular intraocular lens (IOL), spectacles or contact lenses. RESULTS: Mean visual acuity was > 20/40 (< 0.3 log MAR) with normal binocular vision in 58 children over 7 months of age operated on for bilateral cataracts. Pseudophakic eyes regained visual acuity > 20/63 (< 0.5 log MAR) more often (90%) than aphakic eyes (46%) (p < 0.001). Binocular vision was also achieved more often after IOL implantation (p < 0.001). Visual outcome of early bilateral cataracts was less satisfactory in children with abnormal foveolar function. For 49 children who had surgery for unilateral cataracts, prognosis was poor when surgery was performed before the age of 7 months. For cataract surgery in older children (> or = 7 months) mean visual acuities were better with IOL implantation (p < 0.05). CONCLUSION: Cataract surgery with unilateral and bilateral IOL implantation can provide a beneficial effect on final visual outcome in children who are operated on before abnormal foveolar function develops.     

Cataract mutations and lens development

The lens plays an essential role for proper eye development. Mouse mutants affecting lens development are excellent models for corresponding human disorders. Moreover, using mutations in particular genes the process of eye and lens development can be dissected into distinct steps. Therefore, three mouse mutants will be described in detail and discussed affecting three essential stages: formation of the lens vesicle, initiation of secondary lens fiber cell formation, and terminal differentiation of the secondary fiber cells. The mutant aphakia (ak) has been characterized by bilaterally apakic eyes [Varnum and Stevens (1968) J. Hered. 59, 147-150], and the corresponding gene was mapped to chromosome 19 [Varnum and Stevens (1975) Mouse News Letters 53, 35]. Recent investigations in our laboratory refined the linkage 0.6 +/- 0.3 N cm proximal to the microsatellite marker D19Mit10. The linked gene Pax2, responsible for proper development of the posterior part of the eye and the optic nerve, was excluded as candidate gene by sequence analysis. Histological analysis of the homozygous ak mutants revealed a persisting lens stalk and subsequently the formation of lens rudiments. The lens defects led to irregular iris development and retinal folding. Congenital aphakia is known as a rare human anomaly. Besides a corneal dystrophy (CDTB), no corresponding disease is localized at the homologous region of human chromosome 10q23. The Cat3 mutations are characterized by vacuolated lenses caused by alterations in the beginning of secondary lens fiber cell differentiation at embryonic day 12.5. Secondary malformations develop at the cornea and the iris, but the retina remains unaffected. Two mutant alleles of the Cat3 locus have been mapped to mouse chromosome 10 very close to the microsatellite markers D10Mit41 and D10Mit95 (less than 0.3 cM). Since Cat3 is mapped to a position, which is homologous to human chromosome 12q21-24, the disorder cornea plana congenita can be considered as a candidate disease. The series of Cat2 mutations have been mapped close to the locus encoding the gamma-crystallin gene cluster Cryg [L?ster et al. (1994) Genomics 23, 240-242]. The Cat2nop mutation is characterized by a deletion of 11 bp and an insertion of 4 bp in the 3rd exon of Crygh leading to a truncated gamma B-crystallin. The defect in the Crygh gene is causative for the stop of lens fiber cell differentiation from embryonic day 15.5 onward. Besides the lens, no further ocular tissue is affected. The Cat2 mouse mutants are interesting models for human cataracts caused by mutations in the gamma-crystallin genes at human chromosome 2q32-35. The ak, Cat3 and Cat2 mutants are discussed in the context of other mutants affecting early eye and lens development. Additionally, human congenital cataracts are discussed, which have been characterized similar to the mouse models. The overview of the three types of mutants demonstrates that genes, which affect the early eye development, e.g. at the lens vesicle stage, have consequences for the development of the whole eye. In contrast, if the mutation influences later steps of lens differentiation, the consequences are restricted to the lens only. These data indicate a decreasing effect of the lens for the regulation of eye development during embryogenesis.     

Lens-specific expression of PDGF-A alters lens growth and development

The vertebrate lens provides an in vivo model to study the molecular mechanisms by which growth factors influence development decisions. In this study, we have investigated the expression patterns of platelet-derived growth factor (PDGF) and PDGF receptors during murine eye development by in situ hybridization. Postnatally, PDGF-A is highly expressed in the iris and ciliary body, the ocular tissues closest to the germinative zone of the lens, a region where most proliferation of lens epithelial cells occurs. PDGF-A is also present in the corneal endothelium anterior to the lens epithelium in embryonic and early postnatal eyes. PDGF-B is expressed in the iris and ciliary body as well as in the vascular cells which surround the lens during early eye development. In the lens, expression of PDGF-alpha receptor (PDGF-alphaR), a receptor that can bind both PDGF-A and PDGF-B, is restricted to the lens epithelium throughout life. The expression of PDGF-alphaR in the lens epithelial cells and PDGF (A- and B-chains) in the ocular tissues adjacent to the lens suggests that PDGF signaling may play a key role in regulating lens development. To further examine how PDGF affects lens development in vivo, we generated transgenic mice that express human PDGF-A in the lens under the control of the alphaA-crystallin promoter. The transgenic mice exhibit lenticular defects that result in cataracts. The percentage of surface epithelial cells in S-phase is increased in transgenic lenses compared to their nontransgenic littermates. Higher than normal levels of cyclin A and cyclin D2 expression were also detected in transgenic lens epithelium. These results together suggest that PDGF-A can induce a proliferative response in lens epithelial cells. The lens epithelial cells in the transgenic mice also exhibit characteristics of differentiating fiber cells. For example, the transgenic lens epithelial cells are slightly elongated, contain larger and less condensed nuclei, and express fiber-cell-specific beta-crystallins. Our results suggest that PDGF-A normally acts as a proliferative factor for the lens epithelial cells in vivo. Elevated levels of PDGF-A enhance proliferation, but also appear to induce some aspects of the fiber cell differentiation pathway.     

Progressive juvenile-onset punctate cataracts caused by mutation of the gammaD-crystallin gene

Cataracts are a significant public health problem. Here, we describe the genetic alteration responsible for a progressive form of cataract, segregating as an autosomal dominant trait in a three-generation pedigree. Unlike most autosomal dominant cataracts, these are not clinically apparent at birth but are initially observed in the first year or two of life. The opacification evolves relatively slowly, generally necessitating removal of the lens in childhood or early adolescence. A genome-wide search in our kindred revealed linkage at 2q33-35 where the gamma-crystallin gene cluster resides. A single base alteration resulting in an Arg- 14 --> Cys (R14C) substitution in gammaD-crystallin was subsequently identified. Protein modeling suggests that the effect of this mutation is a subtle one, affecting the surface properties of the crystallin molecule rather than its tertiary structure, consistent with the fact that the patients' lenses are normal at birth. This is the first gene defect shown to be responsible for a noncongenital progressive cataract, and studying the defective protein should teach us more about the mechanisms underlying cataract formation.     

Purification of biologically active SPARC expressed in Saccharomyces cerevisiae

SPARC (secreted protein, acidic and rich in cysteine) is a secreted, Ca+2-binding glycoprotein that modulates interactions between cells and their immediate extracellular matrix. Traditional sources of SPARC have been mammalian bone, platelets, a basement membrane tumor, and cultured cells; most if not all preparations, however, contain platelet-derived growth factor and one or more serum proteins that bind specifically to purified SPARC. To avoid these contaminants, as well as the toxic lipid moiety associated with endotoxin, we expressed recombinant wild-type and a mutated murine SPARC in two strains of Saccharomyces cerevisiae: one strain was transfected with an expression vector encoding a proprietory signal peptide that directed the secretion of the recombinant protein. Recombinant SPARC was also purified from cell lysates of a different, nonreverting strain of S. cerevisiae that was optimized for large-scale fermentation runs. A mutant murine SPARC lacking the single glycosylation site was also expressed following substitution of Asn98 with Asp98 in the wild-type sequence. Purification of SPARC was achieved by copper-affinity and hydrophobic-interaction chromatography. Both the wild-type and the glycosylation-defective recombinant proteins exhibited high levels of activity in two bioassays with endothelial cells: inhibition of cell spreading/disruption of actin microfilaments and competition for the binding of nonrecombinant 125I-labeled SPARC to the cell surface. The availability of biologically active, recombinant SPARC will facilitate investigation of the structural and functional properties of this protein, which is expressed at high levels in healing wounds, atherosclerotic plaque, and several cancers and diseases of connective tissue.     

Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts

In the ocular lens, gap junctional communication is a key component of homeostatic mechanisms preventing cataract formation. Gap junctions in rodent lens fibers contain two known intercellular channel-forming proteins, connexin50 (Cx50) and Cx46. Since targeted ablation of Cx46 has been shown to cause senile-type nuclear opacities, it appears that Cx50 alone cannot meet homeostatic requirements. To determine if lens pathology arises from a reduction in levels of communication or the loss of a connexin-specific function, we have generated mice with a targeted deletion of the Cx50 gene. Cx50-null mice exhibited microphthalmia and nuclear cataracts. At postnatal day 14 (P14), Cx50-knockout eyes weighed 32% less than controls, whereas lens mass was reduced by 46%. Cx50-knockout lenses also developed zonular pulverulent cataracts, and lens abnormalities were detected by P7. Deletion of Cx50 did not alter the amounts or distributions of Cx46 or Cx43, a component of lens epithelial junctions. In addition, intercellular passage of tracers revealed the persistence of communication between all cell types in the Cx50-knockout lens. These results demonstrate that Cx50 is required not only for maintenance of lens transparency but also for normal eye growth. Furthermore, these data indicate that unique functional properties of both Cx46 and Cx50 are required for proper lens development.     

The hydroxyl radical in lens nuclear cataractogenesis

Cataract is the major cause of blindness; the most common form is age-related, or senile, cataract. The reasons for the development of cataract are unknown. Here we demonstrate that nuclear cataract is associated with the extensive hydroxylation of protein-bound amino acid residues, which increases with the development of cataract by up to 15-fold in the case of DOPA. The relative abundance of the oxidized amino acids in lens protein (assessed per parent amino acid) is DOPA > o- and m-tyrosine > 3-hydroxyvaline, 5-hydroxyleucine > dityrosine. Nigrescent cataracts, in which the normally transparent lens becomes black and opaque, contain the highest level of hydroxylated amino acids yet observed in a biological tissue: for example, per 1000 parent amino acid residues, DOPA, 15; 3-hydroxyvaline, 0.3; compared with dityrosine, 0.05. The products include representatives of the hydroperoxide and DOPA pathways of protein oxidation, which can give rise to secondary reactive species, radical and otherwise. The observed relative abundance corresponds closely with that of products of hydroxyl radical or metal-dependent oxidation of isolated proteins, and not with the patterns resulting from hypochlorite or tyrosyl-radical oxidation. Although very little light in the 300-400-nm range passes the cornea and the filter compounds of the eye, we nevertheless also demonstrate that photoxidation of lens proteins with light of 310 nm, the part of the spectrum in which protein aromatic residues have residual absorbance, does not give rise to the hydroxylated aliphatic amino acids. Thus the post-translational modification of crystallins by hydroxyl radicals/Fenton systems seems to dominate their in vivo oxidation, and it could explain the known features of such nuclear cataractogenesis.     

Evidence for normal aging of the septo-hippocampal cholinergic system in apoE (-/-) mice but impaired clearance of axonal degeneration products following injury

The association of the epsilon4 allele of apoE with increased risk for Alzheimer's disease (AD) and with poor clinical outcome after certain acute brain injuries has sparked interest in the neurobiology of apoE. ApoE (-/-) mice provide a tool to investigate the role of apoE in the nervous system in vivo. Since integrity of the basal forebrain cholinergic system is severely compromised in AD, with severity of dysfunction correlating with apoE4 gene dosage, the present study tested the hypothesis that apoE is required to maintain the normal integrity of basal forebrain cholinergic neurons (BFCNs). Histological and biochemical analyses of the septo-hippocampal cholinergic system were performed in apoE (-/-) mice during aging and following injury. Using unbiased quantitative methods, there was little or no evidence for defects in the septo-hippocampal cholinergic system, as assessed by p75(NTR)-immunoreactive neuron number and size in the medial septum, cholinergic fiber density in the hippocampus, and choline acetyltransferase activity in the hippocampus, cortex, and striatum in aged apoE (-/-) mice (up to 24 months of age) as compared to age-matched wild-type mice of the same strain. In addition, cholinergic neuronal survival and size following fimbria-fornix transection in apoE (-/-) mice did not differ from controls. However, following entorhinal cortex lesion, there was persistence of degeneration products in the deafferented hippocampus in apoE (-/-) mice. These data suggest that although apoE is not required for the maintenance of BFCNs in vivo, it may play a role in the clearance of cholesterol-laden neurodegeneration products following brain injury.     

Mental rotation and temporal contingencies

The physiological role of prion protein (PrP) remains unknown. Mice devoid of PrP develop normally but are resistant to scrapie; introduction of a PrP transgene restores susceptibility to the disease. To identify the regions of PrP necessary for this activity, we prepared PrP knockout mice expressing PrPs with amino-proximal deletions. Surprisingly, PrP lacking residues 32-121 or 32-134, but not with shorter deletions, caused severe ataxia and neuronal death limited to the granular layer of the cerebellum as early as 1-3 months after birth. The defect was completely abolished by introducing one copy of a wild-type PrP gene. We speculate that these truncated PrPs may be nonfunctional and compete with some other molecule with a PrP-like function for a common ligand.     

Finger-like projections of plasma membrane in the most senescent fiber cells of human lenses

PURPOSE: To corroborate the findings of finger-like membrane projections in monkey and baboon lenses, in human lens fiber cells. METHODS: Normal human lenses, two months to 76 years old, as well as age-related nuclear cataracts, were immersion fixed in 2% paraformaldehyde-0.2% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2 for 24 h at room temperature, cut into 200-500 microm thick sections along the equatorial axis, fixed for an additional 12-18 h at room temperature, dehydrated in an ascending ethanol series and embedded in Lowicryl K4M. Semi-thick sections (0.25-1.0 microm) were cut, absorbed onto 75 or 100 nickel slotted grids, labeled with anti-MIP 26 or phalloidin, stained with 2% uranyl acetate and viewed by transmission electron microscopy at 100 kV. RESULTS: Transmission electron microscopy micrographs demonstrated the presence of finger-like plasma membrane projections measuring 0.16-0.25 microm in diameter and 1.0-6.5 microm in length with bulbous terminal tips in the most senescent fiber cells in two-month and older normal lenses, as well as, in nuclear cataracts. These projections appeared to overlie furrowed membrane domains in the extracellular space, as well as project into the cytosol along the cytosolic leaflet of plasma membrane. CONCLUSIONS: The results extend the findings in monkey and baboon lenses, to the human lens, and demonstrate that these projections, which sparsely label with antiserum against MIP 26, but not filamentous actin, not only extend into the extracellular space, but also project inward into the cytosol.     

Tumorigenicity by human papillomavirus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation

The human papillomavirus type 16 (HPV-16) E6 and E7 oncogenes are thought to play a role in the development of most human cervical cancers. These E6 and E7 oncoproteins affect cell growth control at least in part through their association with and inactivation of the cellular tumor suppressor gene products, p53 and Rb. To study the biological activities of the HPV-16 E6 and E7 genes in epithelial cells in vivo, transgenic mice were generated in which expression of E6 and E7 was targeted to the ocular lens. Expression of the transgenes correlated with bilateral microphthalmia and cataracts (100% penetrance) resulting from an efficient impairment of lens fiber cell differentiation and coincident induction of cell proliferation. Lens tumors formed in 40% of adult mice from the mouse lineage with the highest level of E6 and E7 expression. Additionally, when lens cells from neonatal transgenic animals were placed in tissue culture, immortalized cell populations grew out and acquired a tumorigenic phenotype with continuous passage. These observations indicate that genetic changes in addition to the transgenes are likely necessary for tumor formation. These transgenic mice and cell lines provide the basis for further studies into the mechanism of action of E6 and E7 in eliciting the observed pathology and into the genetic alterations required for HPV-16-associated tumor progression.     

The molecular chaperone alphaA-crystallin enhances lens epithelial cell growth and resistance to UVA stress

alphaA-Crystallin (alphaA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of alphaA, but in differentiated fiber cells, alphaA is the most abundant soluble protein. The lenses of alphaA-knock-out mice develop opacities at an early age, implying a critical role for alphaA in the maintenance of fiber cell transparency. However, the function of alpha-crystallin in the lens epithelium is unknown. To investigate the physiological function of alphaA in lens epithelial cells, we used the following two systems: alphaA knock-out (alphaA(-/-)) mouse lens epithelial cells and human lens epithelial cells that overexpress alphaA. The growth rate of alphaA(-/-) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of alphaA(-/-) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the alphaA(-/-) cultures was increased 40-fold over wild type. In cells lacking alphaA, UVA exposure modified F-actin, but actin was protected in cells expressing alphaA. Stably transfected cell lines overexpressing human alphaA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoalphaA. Cells overexpressing alphaA were resistant to UVA stress, as determined by clonogenic survival. alphaA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of alphaA was not associated with its relocalization to the nucleus. These results indicate that alphaA has important cellular functions in the lens over and above its well characterized role in refraction.     

Mouse cortical neurones lacking APP show normal neurite outgrowth and survival responses in vitro

We have cultured neurones from the developing cortex of mice that have had the amyloid precursor protein gene deleted (APP-null). Neurones cultured for a period of 24 h show similar neurite outgrowth and survival responses to wild-type neurones. Similar neurite outgrowth responses were also seen when neurones from APP-null mice were treated with a neurotrophic peptide derived from the APP sequence and compared with wild-type neurones. Finally, cortical cultures derived from APP-null mice showed similar survival responses to the toxic amyloid-beta peptide.