Fig 2: PPAR? activation is required for anti-amyloidogenic effect of NPD1 but not for activation of ADAM10.HNG cells over-expressing ßAPPsw were co-transfected with 2.0 µg of pEGFP or PPAR? cDNA or incubated with 0.5 µM of NPD1 or rosiglitazone (RGZ) in the absence or presence of 2 µM of GW9662 for 48 h before being harvested for assay. (A) ELISA assay of total Aß and Aß42 peptides (ng/ml) from conditioned media of HNG cells with different treatments; (B) Western blot analysis of ßAPP fragments as well as ADAM10 and BACE1 from HNG cells under different conditions as indicated in the lower part of panel (B) in these Western images; (C) Western analysis of sAPPßsw or CTFs from HNG cells over-expressing ßAPPsw in the absence or presence of 2 µM of GW9662; (D–F) Quantification of ßAPP fragments, ADAM10 and BACE1 levels based on Western results (n = 3). *p<0.01 vs. ßAPPsw control; **p<0.01 vs ßAPPsw + pEGFP co-transfection; *N p<0.01 vs. ßAPPsw + NPD1; *R p<0.01 vs. ßAPPsw + rosiglitazone; **p<0.01 vs. ßAPPsw + PPAR? co-transfection.

Fig 3: NPD1 promotes non-amyloidogenic, neurotrophic bioactivity via pleiotrophic mechanisms.Membrane esterified DHA is excised by phospholipase A2 (PLA2) to yield free DHA; in turn free DHA is 15-lipoxygenated to generate NPD1 which then enters a neuroprotective cycle. These events are mediated, in part, by inhibiting apoptosis, by blocking inflammatory signaling, by promoting cell survival and by shifting ßAPP processing from an amyloidogenic into a neurotrophic, non-amyloidogenic pathway. BACE1 activity is suppressed and a-secretase (ADAM10) activity is stimulated, thus down-regulating Aß42 peptide release from membranes. Augmentation of BACE1 and ADAM10 by NPD1 may be mediated via other neuromolecular factors. We note that the ADAM10 cleavage product sAPPa further induces the conversion of free DHA into NPD1, thus constituting a positive, neurotrophic feedback loop.

Fig 4: BACE1 expression and Aß accumulation in the brain of the obesity model and the alteration of the gut microbiota composition in the obesity model.a Brains of 20 weeks old ICR mice, which were fed with HFD for 14 weeks were extracted. The extracted brains were cryo-sectioned coronally, and separated into hippocampus and cortex. The expression levels of APP, BACE1, and ß-actin were analyzed by western blot. ß-actin was used as a loading control. Data are presented as a mean ± S.E.M. n = 4. b Brain samples for immunohistochemistry were immunostained with APP, BACE1, and Aß and PI. Images shown in result are representative. All scale bars, 25 µm (magnification, ×100) c Alpha diversity between control and high-fat; left panel: Observed OTUs, right panel: Shannon’s diversity index. d Beta diversity between control (•) and high-fat (•); Bray–Curtis, unweighted UniFrac distance, weighted UniFrac distance. e–g STAMP analysis; e Differences in relative abundance of firmicutes between groups at the phylum level, f Differences in relative abundance of clostridiales between groups at the order level, g Differences in relative abundance of ruminococcaceae and lachnospiraceae between groups at the family level. h The concentration of NaB in plasma of mice was measured by GC–MS. NaB concentration data are presented as a mean ± S.E.M. n = 4. *p < 0.05 versus control. All blot and immunofluorescence images shown are representative.

Fig 5: Changes in APP processing and Aß secretion in SH-SY5Y cells under AGE stimulation. Cells were received 1 h pretreatment with hesperetin (hesp; 20–40 µmol/L) or rosiglitazone (Ros; 10 µmol/L), continued to 24 h AGE (200 µg/mL) exposure. (A) A representative Western blot analyzes the relative levels of amyloid precursor protein (APP), ß-site APP-cleaving enzyme (BACE), insulin degrading enzyme (IDE), and neprilysin (NEP). Band densities were normalized with ß-actin. (B) ELISA assay was used to determine the level changes in Aß1-40 and Aß1-42. The results are presented as the mean ± SD of five independent experiments performed triplicate (n = 5). a p < 0.05 and b p < 0.01 when compared to the data from the untreated control group (control). c p < 0.05 and d p < 0.01 when compared to the data from the vehicle (Veh)-treated cells.