The Mechanism Of Astrocyte-mediated Degradation Of β-amyloid Protein

Objective: In this study, we used primary cell culture and confocal microscopy, to investigatewhether the degradation of extracelluar β-amyloid protein(Aβ) is in lysosomes of astrocyte.APPswe and PS1ΔE9double transgenic mice is the model mice of Alzheimer’s disease(AD), weused it to study whether the efficiency of degradation between APP/PS1transgenic mice and non-transgenic mice is different; whether the the efficiency of degradation between young astrocytesand elderly astrocytes is different; whether NH4Cl has effect on the degradation of Aβ, Throughthese experiments to explain the underlying pathogenesis of AD.Methods: APP/PS1double transgenic mice which were hybrided were identified by polymerasechain reaction(PCR). One-day-old mice were prepared for primary astrocytes culture. Cells weretreated at the day7in vitro(DIV7) and at the day21in vitro(DIV21) with200nM Hilyte Fluor488-labled Amyloid-β protein42(Aβ42) and1μM Lysotracker DND Red99(DND-99), Aβ42andDND-99were incubated30min, and then astrocytes were incubated at37℃with different time.The photos were obtained from confocal microscopy. Live cell imaging was used to study thedifference of co-localization rate and fluorescence power between APP/PS1double transgenicmice and non-transgenic mice.25mM NH4Cl was used after the astrocytes were incubated withAβ42and DND-99in order to investigate whether the efficiency of degradation between NH4Cltreated and non-treated is different.Results:1. Extracelluar Aβ42were located to lysosomes in astrocytes.2. The co-localization rate between Aβ42and DND-99treated lysosomes was more higher in APP/PS1transgenic mice than that in non-transgenic mice in DIV7astrocytes. After incubated2hours with37℃, The fluorescence of Aβ42is higher in DIV7astrocytes in AD mice than that inDIV7astrocytes in normal mice. After incubated2hours with37℃, The fluorescence oflysosomes is lower in DIV7astrocytes in AD mice than that in DIV7astrocytes in normal mice.3. The fluorescence of Aβ42decreased in DIV21astrocytes than that in DIV7astrocytes. Thefluorescence of lysosomes decreased in DIV21astrocytes than that in DIV7astrocytes. Thefluorescence of Aβ42and lysosomes both decreased in DIV21AD astrocytes than that in DIV21normal astrocytes.4. The co-localization rate decreased after treated with NH4Cl in APP/PS1transgenic mice inDIV7astrocytes. Conclusions:1. Extracelluar Aβ42was located to lysosomes in astrocytes after the cells were incubated withAβ42and DND-99after30min.2. It is more effective to degrade extracelluar Aβ42in DIV7astrocyte in non-transgenic mice thanthat in APP and PS1double transgenic mice, the activity of lysosomes seems higher in DIV7astrocytes than that in APP/PS1transgenic mice.3. It is more effective to degrade extracelluar Aβ42in DIV21astrocyte in non-transgenic mice thanthat in APP/PS1double transgenic mice, but in non-transgenic mice, the efficiency ofdegradation decreased in DIV21astrocytes than that in DIV7astrocytes.4. Acidic environment can increase the efficiency of degradation of extracelluar Aβ42in astrocytes.