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Jiangyuan Gao, Jing Z Cui, Aikun Wang, Joanne A Matsubara; Different Structures of Aβ1-40 Assemblies Induce Distinctive Primary RPE Cell Responses in vitro. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3439.
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Aβ1-40 is a pathological drusen component whose role in AMD etiology is under investigation. However, little is known about its conformational changes and the associated effects on RPE during AMD pathogenesis. This study aims to compare and elucidate the differential impact of high and low molecular weight Aβ1-40 assemblies on RPE cells.
High molecular (>170 KDa, fibrillar) Aβ1-40 species was collected after 1 week incubation in Tris-buffered saline (TBS) at 37 Celsius following initial suspension in sterile deionized water. Similarly, low molecular (<15 KDa, oligomeric/monomeric) Aβ1-40 species was obtained after 24 hour incubation following initial suspension in hexafluoroisopropanol. Both Aβ1-40 preparations were subjected to electrophoresis separation and detected by immunoblot to verify proper structures. Human primary RPE cells at passage 9 were treated with either form of Aβ1-40 at various concentrations for MTT and LDH assays. Quantitative RT-PCR (qRT-PCR) was applied to quantify gene expression in oxidative stress, for example, NAD(P)H dehydrogenase, quinone 1 (NQO1) and in inflammatory responses, such as complement factor H (CFH) and Caspase-1 (CASP1), after Aβ1-40 treatment.
At 24 hour, both forms of Aβ1-40 increased LDH release and decreased MTT colorimetric reaction when compared to non-treated RPE cells. At the same concentration, fibrillar Aβ1-40 presented equivocal cytotoxicity and concomitantly a dose-dependent decrease of intracellular reductants compared to oligomeric/monomeric Aβ1-40. In qRT-PCR, 5.0 μM fibrillar Aβ1-40 downregulated CFH by 1.5 fold compared to non-treated RPE cells. Both CASP1 and NQO1 were decreased by 1.5 fold in 0.3 μM fibrillar Aβ1-40 treated cells compared to 0.3 μM oligomeric/monomeric Aβ1-40 treated ones.
The current study addresses an important topic that different forms of Aβ1-40 assemblies may affect primary RPE differentially. Fibrillar Aβ1-40 seems more likely to cause oxidative stress than its low molecular counterpart. High concentration of fibrillar Aβ1-40 sequesters RPE’s self-protection from complement activation and therefore may play a role in AMD pathogenesis. Given the presence of both forms of Aβ1-40 in drusen deposits, understanding their distinctive biological effects on RPE may provide insights on AMD disease progression and also shed light on the future development of focused treatment strategies.
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