Fig 2: HNG cells transfected with ßAPPsw and treated with NPD1 - effects on precursor a-secretase (pro-ADAM10), mature ADAM10 (m-ADAM10), ß-secretase (BACE1) and ?-secretase (PS1).(A) Control or HNG cells over-expressing ßAPPsw were incubated with increasing doses (0, 50, 100 and 500 nM, respectively) of NPD1 before cell lysates were harvested and subjected to Western blotting for the precursor of ADAM10 (pro-ADAM10), mature ADAM10 (m-ADAM10), BACE1 and PS1 using the levels of ß-actin as a control in the same sample; (B) Quantitation of gel bands in (A); NPD1 activates m-ADAM10 while inhibiting BACE1 expression; quantification of m-ADAM10 and BACE1 expression by Western blotting analysis after normalization to ß-actin; results are means ± SEM (n = 3); *p<0.01 vs. ßAPPsw control.

Fig 3: Speculative model of Aß generation by protease-containing pEV(1) Somatic mutations induced by age, metal ions or other stress stimuli may stimulate liver and/or cells of other organs or the immune system (e.g. PBMC)46 for an inflammatory response that includes the secretion of pEV containing CCF and active proteases. These pEV possibly function to alert or activate the immune defense in the periphery, for example by differentiating precursor cells.65(2) Depending on their ligands on the vesicle surface, in this case Hyaluronan (HA) and/or HA fragments, pEV are incorporated by specific target cells like monocytes, CP cells or neurons in the hippocampus (Figs. 2,3 and S4c). A single vesicles may contain only one type of protease or inflammatory factor. (3) Once incorporated, these incoming vesicles may fuse with early endosomes (18) containing various substrates including APP. (4) Endosomal compartments may fuse with vesicles containing different proteases, including ADAM10/17, cleaving APP in a physiological (non-amyloidogenic) manner, but also non-physiological (amyloidogenic) manner that includes BACE activity. (5) Non-amyloidogenic peptides, like P3, may be secreted or degraded in lysosomes. Conversely, 6) amyloidogenic Aß withstands degradation, may accumulate (not shown) and/or be released by secretion to function as an anti-microbial peptide. (7) Likely, the cleavage of APP leads to a feedback loop to the ER/Golgi inducing the translation of more APP.

Fig 4: Gas6 inhibits the decrease in BACE1 protein level induced by Tyro3 overexpression in 293APPswe cells.(A, B) Western blot statistical analysis showed that BACE1 levels were significantly lower in Tyro 3-CFP transfected cells than in GFP controls. Gas6 treatment was found to reverse this effect. *P<0.05 versus GFP control (two way ANOVA).

Fig 5: Hypoxia treatment alters the post-translational modification of APP. A Western blot analysis of BACE1 levels from 4 mo wild-type (WT) and APP/PSEN1 mice treated in normoxia or hypoxia for 6 weeks. B Quantification of the relative BACE1 relative protein levels in cortex and hippocampus of 4 mo APP/PSEN1 mice. C qPCR analysis of the Bace1 mRNA levels in cortex and hippocampus of 4 mo APP/PSEN1 mice. D Western blot analysis of the levels of C-terminal factors (CTFs) of APP from 4 mo APP/PSEN1 mice. E Quantification of the relative CTF levels in cortex and hippocampus of 4 mo mice. F Ratio of APP CTFs in cortex and hippocampus of 4 mo APP/PSEN1 mice. Data are means ± SEM. *P < 0.05, ***P < 0.005 in T-test. n = 8 APP/PSEN1 N 4 mo, n = 9–10 APP/PSEN1 H 4 mo. CTF C-terminal factor, hippo hippocampus, H hypoxia, mo months old, N normoxia