Fig 2: BCAA restriction lowers brain pathology and restores neurotransmitter content in APP/PS1 mice. 11-month-old WT or APP/PS1 mice without cognitive impairment were placed on a control or BCAA-restricted (50%) diet that is iso-caloric and iso-nitrogenous for two months. (A) Western blots for proteins involved in BCAA degradation in liver and cortex of the brain. (B) pBCKDH and total BCKDH in liver at the end of two months. (C) BCKDH inactivity index (pBCKDH/BCKDH) in the cortex. (D) Cortical Aβ-42 levels normalized to protein by ELISA. (E) Insulin-degrading enzyme (IDE) in the cortex of the brain. (F) Western blots for IDE, PSD95, and phosphorylated Tau in the cortex. (G) Phosphorylated state of Tau at threonine residue 205, (H) Serine residue 202, and (I) Serine residue 396. (J) Protein expression of PSD95. (K) Analysis of genes involved in neuroinflammation and amyloid production and degradation, normalized to B2M. (L) Monoamine neurotransmitter concentrations in the hippocampus and (M) Cortex. NE—Norepinephrine; DA—Dopamine; DOPAC—Dopamine metabolite; 5-HT—Serotonin; 5-HIAA—Serotonin metabolite. * p < 0.05. Groups with different letters are significantly different from each other with p < 0.05.

Fig 3: Tau protein isoforms and phosphorylation in EEC lines. (A) Hippocampus (2-month-old mice, Hippo) and GLUTag cell lysates were subjected to immunoblot analysis using the pan-Tau antibody A0024. (B) Lysates of GLUTag cell were treated (+) or not (–) with lambda phosphatase (λP) before immunoblotting with the pan-tau antibodies A0024, D1M9X, and Tau-1. (C) GLUTag cells lysates were treated (+) or not (–) with lambda phosphatase (λP) before immunoblotting with the tau isoform-specific antibodies 3R and 4R and the pan-tau antibody A0024. Hippocampus lysate from 2-month old mice dephosphorylated with lambda phosphatase (λP+), which contains all 4R isoforms and 0N3R isoform (McMillan et al., 2008; Liu and Götz, 2013) was used as a ladder to determine mouse tau isoform profile in GLUTag cells; the red lines show comigration. (D) NCI-H716 cell lysates were treated (+) or not (–) with lambda phosphatase (λP) before immunoblotting with the tau isoform-specific antibody 3R and the pan-tau antibody A0024. Tau ladder, which contains all 6 dephosphorylated human tau isoforms was used to determine tau isoform profile. (E) GLUTag cells lysates were subjected to immunoblot analysis using Tau-1 antibody. Rat sciatic nerve lysates were used as positive control to detect big tau. In all experiments, β-actin immunoblot was used as a loading control. The results shown in panels (A,C) are representative of 3 and 2 independent experiments, respectively. The results shown in panel (B) are representative of 7, 2, and 3 independent experiments for A0024, DM19X, and Tau-1 antibodies, respectively. The results shown in panels (D,E) are representative of 3 and 2 independent experiments, respectively.