Fig 2: Identification of inactivated and activated APP expression cell lines using PCR.(A) Genomic locations of PCR primers and Southern blot probes for the inactivated and activated APP expression construct. Schematic illustration of the targeting constructs with locations of primers used to detect recombination events. To identify recombination events in the inactive APP expression cell lines, we use primer pairs primer1 and primer2. Cells with recombination events in both chromosomes will have PCR products of the following expected sizes: primer1: 0 bp; primer2: 215bp; In cells with a recombination event in only one of the two chromosomes then the PCR products will have the following expected sizes: primer1: 204 bp; primer2: 215bp; In cells with no recombination events the PCR products will have the expected sizes of: primer1: 204 bp; primer2: 0 bp; To detect deletion of the stop cassette in activated APP expression cell lines, we use primer pairsprimer3 and primer4. In cells with recombination events in both chromosomes then the PCR products will have the following expected sizes: primer3: 0 bp; primer4: 216bp; In cells with recombination events in only one of the two chromosomes then PCR products will have the following expected sizes: primer3: 801 bp; primer4: 216 and 2516 bp; In cells with no recombination events the PCR products will have the expected sizes: primer3: 801 bp; primer4: 2516 bp. (B) PCR analysis of inactivated/activated APP expression cell lines using primer pairs primer1, primer2, primer3 and primer4. Results using primer pairs primer1, primer2, primer3 and primer4 are shown. In the upper two panels (primer1 and primer2), lanes 1 to 5 are wildtype APP coding sequence knock-in cell lines and lanes 7 to 12 are mutant APP coding sequences knock-in cell lines. Lane 6 is PCR from untransfected Balb/c3T3 cell genomic DNA. In the lower two panels (primer3 and primer4), lanes 1 to 5 are wildtype APP coding sequence knock-in cell lines and lanes 8 to 11 are mutant APP coding sequences knock-in cell lines. Lanes 6 and 7 are PCR results of cell lines w5 and s12, respectively. For primer1 and primer2 recombination events were detected for both chromosomes in lanes 1, 7, 9, 10 and 12, which we named cell lines w5, s7, s9, s10 and s12. Cell lines w5 and s12 were then used for Cre transfection for removal of the stop cassette, as they the best GFP expression. Primer3 and primer4 were used to detect stop cassette deletion. Lane 1 shows that both stop cassettes were deleted from cell line w5c1. In lanes 2, 3, 5 and 8, stop cassette deletion was detected in only one of the two chromosomes, and these lines are named the cell lines w5c2, w5c3, w5c5 and s12c8. No stop cassette deletion was detected in negative controls w5 and s12 in lanes 6 and 7.

Fig 3: Brivaracetam, but not ethosuximide, reverses impairments in spatial memory in APP/PS1 mice. Aged APP/PS1 mice were administered brivaracetam by osmotic minipump or continuous delivery of ethosuximide (ETX) via drinking water. Four-week administration of brivaracetam (Briva) fully reversed memory impairments in APP/PS1 mice in the Morris water maze (A) and Probe Trial (B). Chronic (7-week) administration of ethosuximide did not improve performance in the Morris water maze or probe trial in APP/PS1 mice (C, D). For the Briva cohort, wild-type (WT) + vehicle: n = 11; WT + Briva: n = 11, APP/PS1 + vehicle: n = 15, APP/PS1 + Briva: n = 16. For chronic ETX therapy, WT + vehicle: n = 7, WT + ETX: n = 7, APP + vehicle: n = 6, APP + ETX: n = 7. *P < 0.05, ***P < 0.001, repeated-measures analysis of variance with post hoc comparisons. V1 through V4 indicate visible platform swim trials. + Indicates drug therapy; - indicates vehicle.

Fig 4: Abnormal cerebral blood flow increases amyloid pathology. A. and C. Representative images of CAA (A) and plaque (C) pathologies in anterior areas of control, sham-operated and transverse aortic constriction surgery (TAC)-operated APP DSL. TAC was performed on the left common carotid arteries of 6-month old APP DSL mice that terminated for analysis 3 months post-surgery. B. and D. Quantification of CAA (B) and plaque (D) load in left and right part of anterior (Ant.) or posterior (Post.) brain regions. Scale bars: 50 µm in A; 200 µm in C. N = 4 animals/group and 10 sections/animal. Coronal sections spanning from Bregma -2.58 ~ -0.58 mm were counted as anterior region;-0.94 ~ 3.64 mm were counted as posterior region. E-H, ELISA analysis of soluble (E and F) and insoluble (G and H) Aß40 (E and G) and 42 (F and H) on anterior and posterior cortical areas in both left and right hemispheres. Each sample was measured in duplicates on ELISA plates. N = 3 animals/treatment. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Expression of proteins associated with AD is altered by eIF4A inhibition. A Western analysis was performed on lysates from SH-SY5Y cells treated with 10 µM hippuristanol (+) or DMSO control (-) for 4 or 24 hours with antibodies specific to APP, TAU and TXN (bands of around 70 kDa, 60 kDa and 12–14 kDa respectively). The 70 kDa band was detected by the APP specific antibody representing a cleavage product specific to K+ isoforms [53]. Secreted A-beta was measured by western analysis of cell culture media. The 56 kDa product is interpreted as an aggregate of A-beta, as has previously been reported for western analysis of brain tissue [51]. Protein levels were normalized to actin, and mean relative proportions of protein with standard errors are indicated beneath each gel image. Significant differences (p=0.05 by t-test) are indicated by asterisks. B. Immunoprecipitation following 35S-methionine labeling demonstrates a reduction in novel synthesis of APP, TAU but not TXN when SH-SY5Y cells are treated with 10 µM hippuristanol. Secreted A-beta levels are also reduced, as determined by immunoprecipitation from cell culture medium. Mean relative proportions of protein with standard errors are indicated beneath each gel image. Significant differences (p=0.05 by t-test) are indicated by asterisks. C. Secreted A-beta levels in the medium of 9×105 SH-SY5Y cells were assayed by ELISA 24 hours following treatment with 10 µM hippuristanol (+), demonstrating a modest but reproducible decrease in comparison to control cells (-). Means and standard errors of 6 replicates are shown p = 0.05.