Fig 2: Mitochondrial associated tau is not membrane integrated. (A,B) Mitochondria isolated from 8-month-old htau mice were incubated on ice in a sodium carbonate extraction buffer and fractionated by ultracentrifugation to differentiate membrane associated from membrane-integrated proteins. The resulting fractions were assessed by immunoblot for the indicated proteins (P = pellet/insoluble (membrane-integrated), S = soluble (membrane associated). (A) Blot for Hsp60 and OxPhos as representative controls for the assay. (B) Blot of total tau. (C) Synaptic mitochondria isolated from 8-month-old htau mice were digested with the indicated concentration of trypsin on ice for 30 min, and then solubilized with SDS buffer and analyzed by immunoblot for total Tau, Sdha, and Vdac1 to determine the extent of protease digestion. (D) Diagram illustrating mitochondrial sub-localization of the proteins probed for in (A–C). Red asterisks on Vdac1, Tau, and Sdha represent relative epitope location for the antibodies used in immunoblotting following protease treatment.

Fig 3: Effect of single and multiple exposure of gamma irradiation on phosphorylation of JNK, ERK and Tau proteins. Brain homogenates from various experimental mice were studied to identify the phosphorylation of stress related proteins such as JNK, ERK and Tau. Representative western blot and densitometric analysis of (A) pJNK normalized using total JNK (Wt; n = 5, Tg; n = 5 per group) (B) pERK normalized using total ERK (Wt; n = 5, Tg; n = 5 per group) and (C) pTau (ser 199) normalized using β-actin (Wt; n = 5, Tg; n = 5 per group). Different groups are indicated as Wt (Wild type), Tg (Untreated Transgenic), Tg+0.5 (S) (Transgenic mice received single dose of 0.5 Gy radiation), Tg+0.5 (M) (Transgenic mice received 0.5 Gy radiation twice a week for one month), Tg+4 (S) (Transgenic mice received single dose of 4 Gy radiation). Values are expressed as mean ± SEM. ∗p < 0.05 compared to wild type and #p < 0.05 compared to untreated APP/PS1 mice (Tg).

Fig 4: Loss of PS function causes hippocampal-dependent memory deficits in Tau transgenic mice. Morris water maze and CFC was performed with 6 month-old WT, Tau, PS1 cKO;Tau and PS cKO;Tau mice. a Visible platform phase consisted in six trials and platform latency in seconds was measured. b During learning phase, which consisted in six trials during five consecutive days, latency to the platform in seconds was measured. c Latency to the first entry to the target quadrant in seconds, number of crossings to the target quadrant and percentage of time in the target quadrant in the first 30 s of the test were analyzed. d Representative heat map of each analyzed group during the probe test. e CFC showing the percentage of freezing time immediately and 24 h after conditioning. Values represent mean % freezing time ± s.e.m. (n = 7–10 mice/group). Statistical analysis was determined by one or two-way ANOVA followed by Tukey’s post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 5: Increased pathological PKA-mediated tau phosphorylation in Tau mice lacking PS. Left: Representative immunohistochemical images of phosphorylated tau (PG5: pSer409) in 6 month-old non- and tau transgenic mice. Inset: magnified images showing somatic PG5 staining in PS cKO;Tau brains. Scale bar: 100 μm. Right: Quantitative analysis of number of PG5-positive cells/section in the indicated brain region in littermate control (WT), Tau, PS1 cKO;Tau and PS cKO;Tau mice. Values represent mean PG5-positive cells ± s.e.m. (3 slices per mouse, 4–5 mice/group). Statistical analysis was determined by two-way ANOVA followed by Tukey’s post hoc tests. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: Amygdala: basolateral amygdala; CA1/CA3 hippocampus; DG, dentate gyrus; EC, entorhinal cortex; RSC, retrosplenial cortex