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E.A. Blakely, P.Y. Chang, K.A. Bjornstad, M.P. McNamara, C.J. Rosen, L.T. Chylack, L.E. Goldstein; Profile of ß–amyloid precursor protein (ß–APP) synthesis and processing during human lens cell differentiation . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2627.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Amyloid precursor proteins (ß–APP) and their homologs APLP1/2 are highly conserved membrane–spanning proteins. Proteolytic processing of ß–APP generates splice fragments, including the Alzheimer's disease (AD) ß–amyloid (Aß) peptides, the major constituent of AD neuritic plaques. Goldstein and colleagues have recently reported the identification of cytosplasmic Aß, amyloid pathology, and co–localizing supranuclear (SN) cataracts in lenses from AD patients (Lancet 361:1258, 2003). To understand the unique cytosolic localization of Aß in lens fiber cells (LFC), we investigated ß–APP processing as a function of lens cell differentiation. Methods: A cultured human lens cell model (Blakely et al., IOVS 41:3898, 2000) and human lens tissue were used for these studies. Aß/ß–APP expression at sequential differentiation stages was studied using western blot (WB), immunohistofluorescence (IHF), and immunogold EM (IEM). Transcriptional regulation of ß–APP was evaluated with quantitative RT–PCR. Results: IEM analysis of whole human lens confirms cytosolic Aß localization with very low levels of intracellular Aß in central lens epithelium as compared to fiber cells. The epithelium appears to rapidly secrete and internalize Aß, a finding consistent with our identification of this peptide in human primary aqueous humor. IHF analysis of the cultured cells demonstrates that Aß is expressed in early exponential LEC, with increased expression during differentiation. WB analysis of cell lysates reveals 110/130 kDa ß–APP with differential ß–APP/C–terminal fragment processing in LEC compared to differentiating LFC. Quantitative RT–PCR of ß–APP messages confirms differential gene expression as a function of lens cell terminal differentiation. Conclusions: We have demonstrated that human LEC and LFC differentially process and express ß–APP and Aß. These findings are significant in light of the unique cytosolic localization of Aß in equatorial LFC in AD lenses and consistent with the recent identification of ß–APP processing enzymes in rodent lens (Li et al., Mol Vis 9:179, 2003). In addition, we have confirmed and validated our findings in lens tissue that this in vitro model system can be used to study ß–APP expression and processing and may yield important information in the understanding of pathogenic generation of Aß during lens differentiation.
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