The Relationship And Mechanism Of Astrocytes Lysosomal Function And Intracelluar β-amyloid Protein Containing

Objective: 1. To study the effects of Aβ on the pH, volume and number of lysosome in cultured astrocyts of mice. 2. To study the effect of H+-pumping inhibitor bafilomycin A1 on the pH, volume of lysosome and the degradation of Aβ in astrocytes lysosome of mice. 3. To study the effect of NH4 Cl on the degradation of Aβ in lysosome of cultured astrocytes. 4. To study the effect of forskolin on the pH, volume and number of lysosome in cultured astrocyts of mice, and find out the potential effect of forskolin on lysosomal function in astrocytes. 5. To study the effect of forskolin on lysosomal degradation of exogenous Aβ in astrocytes. 6. To study the effect of forskolin on the content of intracellular Aβ, and find out the effect of forskolin on the content of intracellular Aβ in astrocytes.Methods: Primary cortex astrocytes were cultured from postnatal 1-3 days old mice. Purifed cells were used anti-GFAP(Glial fibrillary acidic protein, GFAP) monoclonal antibody to identify cultured astrocytes. We used LysoTracker Red DND-99 for living cell staining to observe the dynamic change of lysosomes. Cells were incubated with 200 nM Aβ for 30 min, and incubated with 250 nM bafilomycin A1 for 30 min(Or incubated with 200 μM forskolin for 45 min), then incubated with 1 μM DND-99 for 30 min, we imaged living cells to observe the change of pH, volume and number of lysosome in cultured astrocyts of mice. Cells were incubated with 200 nM Fluor 488-labbed Aβ for 30 min, and then with 250 nM bafilomycin A1 or 25 mM NH4 Cl for 30 min respectively(Or incubated with 200 μM forskolin for 45 min), we imaged living cells to observe the content of intracellular Aβ in astrocytes. Cells were incubated with 200 nM Aβ for 30 min, and incubated with 200 μM forskolin for 45 min, lysis of cells and used the method of ELISA to detect the intracellular Aβ42 contents.Results: 1. Exogenous Aβ42 was located in lysosomes through endocytosis by astrocytes. 2. Aβ42 treatment decreased the pH of lysosome, increased the volume of lysosome, and reduced the number of lysosome. 3. The number of lysosome was reduced by bafilomycin A1 via basification of lysosome, the degradation of intracellular Aβ42 was also decreased by the treatment of bafilomycin A1. 4. Exogenous Aβ42 was decreased by NH4 Cl via acidification of lysosome. 5. Forskolin treatment decreased the pH of lysosome, increased the volume of lysosome, and reduced the number of lysosome; exogenous Aβ42 was accumulated within cells by treatment of forskolin; and the content of intracellular Aβ was increased by treatment of forskolin.Conclusion: Aβ42 was located in lysosomes within 30 min. The accumulation of Aβ in lysosome damaged the normal function of lysosome, cause a decline in the number of lysosomes. Forskolin has the similar effects as Aβ, and facilitate exogenous Aβ accumulate in the lysosome, eventually leads to the reduction of lysosome number. Forskolin induced activation of PKA signaling pathways which facilitated endogenous Aβ accumulate in lysosome. This may be the underlysing mechanism of forskolin causing the damage of lysosomal function.