1. A Study On Degradation Pathway Of APH-1 And Nicastrin Prrotein In Gamma-Secretase Complex 2. A Study On The Therapeutic Effect Of Valproic Acid On Alzheimer's Disease

Objectives: To investigate whether degradation of Aph-1 goes through ubiquitin-proteasome pathway or lysosomal pathway ; The relationships between the Aph-1 protein level and gamma-secretase activity and Abeta generation. The outcome of this study will have significant impact on AD pathogenesis and drug development.Methods Various methods such as Cell culture, Western blot, pulse-chase metabolic labeling technique, immunoprecipitation, double immuno- fluoresecnt staining, ELISA assay, combined with proteasomal and lysosomal inhibition were used to check Aph-1 expression level in N2a and HEK293 cells.Results: By treating cells with proteasome specific inhibitors, we found that expression of endogenous and exogenous Aph-1 was significantly increased both in neuronal and non-neuronal cells(p<0.001). The effect of the proteasome inhibitors on Aph-1 showed a dose- and time-dependent manner. Lysosomal pathway is not involved in Aph-1 degradation(p>0.05). Pulse-chase metabolic labeling experiment showed that the turnover of newly-synthesized radiolabeled Aph-1 protein was blocked by Lactacystin. Immunoprecipitation and double immunofluorescent staining analysis revealed that Aph-1 and ubiquitin colocalize and interact with each other in cells. Proteasomal inhibitor Lactacystin treatment in Aph-1 transfected PS1/2-KO and PS1/2-Wt cells reavealed that degradation of Aph-1 was not affected by PS. ELISA analysis indicated that overexpression of Aph-1 by Lactacystin treatment can increase the levels of both Abeta40 and Abeta42(p<0.001).Conclusion:Our data indicate that degradation of Aph-1 protein is mediated by the ubiquitin-proteasome pathway in neuronal and non-neuronal cells, and is not related to lysosome; Proteasomal degradation of Aph-1 is independent of PS; overexpression of Aph-1 or proteasomal inhbition of Aph-1 can promote APP processiong and Abeta production. Objective: The present study is aimed at exploring the degradation pathway of Nicastrin and its related molecular mechanism; the relationship between Nicastrin protein level and gamma-secretase activity and Abeta generation. The outcome of this study will have significant impact on AD pathogenesis and drug development.Methods: Following generation of NCT stable cell lines, various methods such as Western blot, pulse-chase metabolic labeling technique, cell fractionation, immunoprecipitation, double immunofluoresecnt staining, ELISA assay, combined with proteasomal and lysosomal inhibition were used to check NCT expression level in NCT stable cell line or non- transfected neuronal(SH-SY5Y, primary neuron) and non-neuronal (HEK293) cells.Results: We found that treatment of cells with either proteasomal or lysosomal inhibitors can significantly increase both endogenous and exogenous NCT in various cell lines(p<0.001), and effect of proteasomal and lysosomal inhibitors on NCT was time- and dose-dependent. Pulse-chase metabolic labeling experiment showed that the turnover of newly-synthesized radiolabeled Nicastrin protein was blocked by Lactacystin and Chloroquine. Cell fractionation experiment showed that NCT distributes primarily in ER and Golgi apparatus, few NCT located in lysosme; Immunofluorescent microscopic analysis showed that NCT accumulates in the ER and Golgi apparatus after proteasomal inhibition, while lysosomal inhibition leads to the accumulation of NCT in lysosomal apparatus. Immunohistochemical staining showed that NCT colocalizes with ubiquitin and co-immunoprecipitation can pull down both NCT and ubiquitin, ELISA analysis indicated that overexpression of NCT can faciliate APP processiong and levels of both Abeta40 and Abeta42(p<0.001).Conclusion: Taken together, our study demonstrates that degradation of NCT involves both the proteasome and lysosome in neuronal and non-neuronal cells, and NCT is ubiquitinated before degradation,; Proteasomal degradation of NCT is independent of PS; overexpression of Nicastrin can facilitate APP processiong and Abeta production . Objective: To investigate the effect of Valproic acid (VPA), a selective GSK-3βinhibitor, on APP processing and Abeta production in HEK293 cells stably transfected Swedish mutant APP751 and in the brains of APP23+/-(670/671) Alzheimer's disease transgenic mouse model, as well as to explore its possible molecular mechanisms underlying therapeutic action of VPA. The present study will provide a novel therapeutic interventions in Alzheimer's disease.Methods: (1) In Vitro study: VPA with various concetrations were used to treat stable APP751swe cells for 48h; then collecting media for ELISA assay and harvesting cells for Western blot, to examine the effect of VPA on APP processing and Abeta production in stable APP751swe cells; (2)In vivo study: 30mg/kg.d of VPA and same amount of saline were peritoneally injected into APP23+/- transgenic mice for 4 weeks. Morris water maze and Fear conditioning test were conducted to check the effect of VPA on the capability of spatial learning and memory of APP23+/- transgenic mice; Immunohistochemical staining, Thisoflavin S staining, Immunofluorescent staining and silver staining were performed to examine the effect of VPA on the morphological changes in the brains of APP23+/- transgenic mice; Western blot and ELISA analysis were used to investigate the effect of VPA on APP processing and Abeta production in the brains of APP23+/- transgenic mice.Results: (1)In vitro study: Western blot showed that C99 and C83 proteins, s ubstrates ofγ-secretase, were signficantly increased, while the production ofγ-secretase, Aβ40 and Aβ42 were notably decreased following VPA treatment. But VPA has no effect on the holoprotein of APP and PS.(2) In vivo study:①Behavioral test: Visible platform test showed that VPA-treated and saline-treated mice had similar escape latency (P>0.05) and path length (P>0.05);Hidden platform test showed that VPA-treated mice had a significantly lesser latency(P<0.01) and fewer pathlength(P<0.01) to reach the platform compared with saline-treated mice; Furthermore, VPA-treated mice had significantly much more platform-passing times in the probe trial(P<0.01); In Fear conditioning test, VPA-treated mice had much more Freeze times immediately or 24h after electrical shock, in comparison to saline-treated mice(P<0.001).②Morphological test: Immuohisto- chemical staining and Thioflavin S staining showed that the numbers of senile plaques in the cortex and hippocampus of VPA-treated APP23 mice were significantly decreased, compared with saline-treated mice(P<0.001); furthmore, Immunofluorescent staining and Silver staining revealed that the numbers of cell bodies, drendrites and axons of neurons in the cortex and hippocampus of VPA-treated APP23 mice were significantly increased, and neuronal fibers arranged orderly;③Protein level test: Western blot showed that C99 and C83 were signficantly increased, GSK-3β-pser9 protein was also increased greatly, while Aβ40 and Aβ42 were remarkably decreased in VPA- treat mice (P<0.001). But VPA has no effect on the holoprotein of APP, PS and total GSK-3βprotein. These results were consistant with data got in cells.Conclusion: VPA notably decreases Abeta generation by inhibition gamma-secretase activity in stable APP751swe cells and in the brains of APP23 AD transgenic mouse model; VPA significantly lowers the Abeta deposition and formation of SP, while increases the numbers of neurons in the brains of APP23 mice; VPA can remarkably improves the capability of spatial learning and memory of APP23 transgenic mice. VPA exerts its therapeutic effect through inhibiting GSK-3βactivity.