Data Availability StatementAll relevant data are within the paper. at 10M exhibited IC50 activity towards -secretase. Western Blot analysis to detect protein expression showed significantly decreased expression of -secretase at both the doses (Fig 6B). Since there is another possibility that Honokiol inhibits APP -processing and A generation through promoting -secretase activity, we also assayed the activity of TACE, a major -secretase in PS70 cells treated with Honokiol. We found that Honokiol did not affect TACE activity (Fig 6E), suggesting that Honokiol does not affect -secretase activity. Honokiol treatment dose-dependently decreased the secreted level of sAPP, an amino-terminal fragment of APP generated by -secretase cleavage by 48% and 40% respectively (Fig 6C). Consistently, the level of APP -CTF (a carboxyl-terminal fragment of APP generated by -secretase cleavage) was also decreased upon Honokiol treatment by 23% and 40% (Fig 6D). These results suggest that Honokiol inhibits -cleavage of APP. In addition, Honokiol (10M) increased the level of secreted sAPP, the major extracellular fragment of APP released by -secretase cleavage Flavopiridol irreversible inhibition (Fig 6C). Moreover, we found that Honokiol treatment did not affect protein levels TFR2 of APP, and -secretase ADAM10 (Fig 6D). These results suggest that Honokiol reduces APP amyloidogenic processing not through affecting but through -secretase levels Flavopiridol irreversible inhibition [64]. Open in a separate windows Fig 6 Honokiol treatment reduces amyloidogenic pathway by inhibits -secretase activity and reducing APP -CTF and sAPP levels.PS70 cells were treated with DMSO (negative control), Insulin (10nM) or indicated doses of Honokiol for 24h. (A) Cell lysates were assayed for -secretase activity by using a commercial kit from Biovision and subjected to comparison. (B) Cell lysates were processed and examined for BACE expression in Western blots with anti-BACE1 antibodies. -Actin was used as a loading control. (C) Conditioned media and (D) cell lysates were analyzed by WB using respective antibodies. The Western blots shown are representative of at least three impartial experiments. Densitometric quantification was performed and expressed as percentage switch. (E) PS70 cells treated with DMSO, insulin 10nM, Honokiol (5 & 10M) and -secretase inhibitor TAPI-1 (10M) for 24 hours. Cell lysates were assayed for -secretase activity for comparison (One-way ANOVA followed by Dunnetts post hoc test, n = 3, *: p 0.05, **: p 0.01, ***: p 0.001). Honokiol increases SIRT3 and activates AMPK-CREB-PGC1 pathway To confirm that high insulin levels predispose to increased A formation, we used increasing concentrations of Insulin (0, 1, 5 and 10nM) on PS70 cells and found that A levels (normalized to -actin) increased significantly (1.12-, 1.28- and 1.46-fold) compared to control (Fig 7A). Both doses (5 and 10M) of Honokiol increased SIRT3 levels by nearly twofold and optimum SIRT3 activation was found to be at 24h (Fig 7B). Furthermore, we explored the molecular signaling pathway related to the reduction of A levels by Honokiol. Honokiol (5Mand 10M) increased the phosphorylation of AMPK by (1.37- and 1.5- fold) compared to total AMPK at 24 hours. Similarly, phosphorylation of CREB was increased by (1.42- and 1.61-fold) with respect to total CREB (Fig 7C). These phosphorylation changes of AMPK and CREB in turn are found to increase the levels of PGC1. Similarly, we found a statistically significant increase in the levels of PGC1 (1.75) fold at 10M normalized to GADPH, but no effect Flavopiridol irreversible inhibition was noticed at 5M (Fig 7C, n = 3, p 0.05). Insulin (10nM) decreased but did not show a statistically significant switch in the phosphorylation of AMPK, CREB and PGC1. Open in a separate windows Fig 7 Effect of Honokiol on SIRT3 activation and AMPK-CREB-PGC1 pathway.Effect of increasing concentrations of insulin (0, 1, 5 and 10nM) on TsA (Total secreted A) along with the densitometric analysis of band intensity normalized to -Actin. (B) Representative Western blot of SIRT3 performed on whole cell lysates from PS70 cells exposed to either vehicle (DMSO) or 5 and 10M Honokiol for 24 and 48 h. The graph displays the densitometric analysis of band intensity of the SIRT3 normalized to the corresponding GADPH level, used as loading control. (C) Effect of Insulin 10nM and Honokiol (5 and 10M) on p-AMPK/AMPK ratio. Representative Western blot of total AMPK and phosphorylated-AMPK (p-AMPK) levels, total CREB and phosphorylated-CREB (p-CREB) levels and PGC-1 performed on whole cell lysates from PS70 cells exposed to either vehicle, Insulin or Honokiol for 24 h. The graph displays the statistical analysis of the p-AMPK/AMPK and p-CREB/CREB ratio calculated by densitometric analysis of band intensity normalized to the corresponding -Actin used as loading control; PGC-1 normalized to corresponding GAPDH level, used as loading control. Data, means SEM are expressed as percentage of vehicle-treated control; n = 3 under each condition. Significance was calculated with Student’s test, *p 0.05, vs. vehicle-treated cells. Discussion In this study, we statement that Honokiol, an activator of SIRT3 attenuated oxidative stress and beta amyloid secretion in PS70 cells, in addition to improving mitochondrial.