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R. D. McCarty, M. J. Nolan, R. M. Beverley, K. Quinn, B. Y. J. T. Yue, J. R. Samples, P. A. Knepper; Gamma Secretase and Trabecular Meshwork Stress. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4870.
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Primary open-angle glaucoma (POAG) and Alzheimer's disease share many similarities in pathogenic mechanisms. γ-secretase is a presenilin dependent enzyme which mediates the proteolytic cleavage of several transmembrane proteins. Examples of γ-secretase substrates include CD44 to form soluble sCD44, a potential biomarker of POAG, amyloid precursor to form amyloid β-peptide, a biomarker of Alzheimer’s disease, LDL receptor-related protein, E-cadherin and Erb-4. The intracellular domains (ICD) of γ-secretase substrates are liberated and function as nuclear signals. The purpose of this study was to determine whether metabolic stress alters γ-secretase, β-secretase, and MT1-MMP in trabecular meshwork (TM) cells.
Human TM cells were grown in modified Eagle’s medium containing 10% fetal bovine serum (FBS) until confluent, washed twice with PBS, and incubated in MEM containing 0.1% FBS with 1, 10, and 40 mM lactate for 3, 12, and 24 hrs. The media was aspirated; the cells were washed with cold PBS, subjected to lysis buffer (Sigma CS0750) containing 1% Triton X-100, and separated by an Optiprep density gradient (Sigma D1556). The preparation was centrifuged at 200,000 x g for 18 hrs; nine 1.0 ml fractions were pipetted from the top (lightest) to bottom (heaviest). Each fraction was analyzed for protein content, resolved by SDS polyacrylamide electrophoresis, and immunoblotted with anti- γ-secretase, β-secretase, MT1-MMP, and caveolin-1.
The distribution of γ-secretase in lactate stressed TM cells was in fractions 3 through 9 and a decreased intensity was observed in fractions 6 through 9 compared to controls. In contrast, the distribution of β-secretase was observed in fractions 5 through 7 with a robust intensity (increased more than three-fold) in fractions 6 and 7 compared to controls. There was no demonstrable change in the distribution of MT1-MMP in metabolic stressed TM cells. The content of caveolin-1 was markedly decreased in fractions 4 and 5 compared to controls.
This is a first demonstration of γ- and β-secretase, and their modulation by metabolic stress, in TM cells. Both γ- and β-secretase are concentrated in caveolin lipid rafts, and the decrease in γ-secretase and increase in β-secretase may be important in the TM response to metabolic stress.
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