Presentation
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6th Internet World Congress for Biomedical Sciences |
Albert Sun(1), Bozena Draczynska-Lusiak(2), Grace Sun(3)
(1)(2)(3)Department of Pharmacology. University of Missouri - Columbia. United States
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Uptake of native and oxidized BLP by primary cortical neurons
Using native and oxidized BLPs enriched with apoE3 or apoE4 and labeled with the fluorescent probe Di-I, we examined uptake of these BLP by primary cortical neurons. Micrographs in Fig 1 show a small uptake of native BLPs into neurons, regardless whether they are enrichment with apoE3 (A) or apoE4 (C) (see pink color). However, when oxidized BLP were presented to the neurons, large amounts of the BLPs were taken up regardless whether they are enriched with apoE3 or apoE4.
Uptake of BPLs by neurons was also examined using BLPs constructed with brain lipids containing [14C]cholesterol. Data in Fig 2 showed a time-dependent increase in uptake of labeled BLPs by neurons. In agreement with the uptake of Di-I BPL, oxidized BLPs were taken up more readily by the neurons than native BLPs.
Neuronal cell death induced by oxidized BLPs
Experiments were carried out to determine whether increased uptake of oxidized BLPs by neurons was associated with enhanced neuronal cell death. Data in Fig 3 show the results using the MTT reduction assay. In this experiment, native BLPs (enriched with apoE4) exposed to neurons for 48 hrs showed no decrease in neuronal cell survivability as compared with controls. However, when oxidized BLPs were added to neurons for the same period of time, neuronal survivability was significantly reduced by 40% (Fig 3). We also tested the effect of resveratrol, a polyphenolic compound known to ameliorate oxidant stress in cells (Chanvitayapongs et al., 1997). When neurons were pretreated with resveratrol (0.5 mM) prior to exposure to oxidized BLPs, resveratrol was able to protect the neurons completely from cell death induced by oxidized BLP.
Similar results were obtained using lactate dehydrogenase (LDH) release was use as an index of cell death. As shown in Fig 4, exposure of neurons to oxidized but not native BLP resulted in an increase in cell death. Furthermore, pretreatment of neurons with resveratrol could similarly reduce cell death due to oxidized BLPs (Fig. 4).
Neuronal cell death mediated by Aß and BLPs
In this study, we examined the effects of Aband oxidized BLPs on neuron cell death. When aggregated Ab were added to the neurons, honeycomb-like structures could be seen in the medium as well as attached to the neurons. Based on results of MTT assay, exposure of neurons to aggregated Aß (1-42, 2 mM) for 24 hr resulted in a decrease in MTT by 23% (Fig 5). Native BLP did not alter cell viability and did not decrease cell viability when incubated with Ab. However, addition of Fe2+/DTP (50 mM) to Ab together with native BLP could give slightly greater reduction of MTT as compared to that mediated by Ab alone. However, cells exposed to oxidized BLP showed MTT reduction (24%) similar to that mediated by Ab alone. Nevertheless, when neurons were exposed to Ab together with oxidized BLPs, MTT reduction (40%) was greater than those due to Ab and oxidized BLP alone. These results indicate that Aß and Fe2+ may cause additional oxidative stress to the cells, and Aß may potentiate the damaging effects of oxidized BLP.
Representative micrographs of primary cortical neurons exposed to Ab and/or oxidized BLP are shown in Fig 6. In panel A, control neurons are bright and show strong connections. When neurons were exposed to Ab (Panel B) or oxidized BLP (Panel D) for 24 hr, cells became dull and neurites became thin and started to disintegrate (see arrows). The combined treatment of Aß and oxidized BLP (Panel C) resulted in complete disappearance of neurites and extensive cell death.
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