Additional file 1 of Human tau-overexpressing mice recapitulate brainstem involvement and neuropsychiatric features of early Alzheimer’s disease
Creators: name: Khan, Kanza M.
name: Balasubramanian, Nagalakshmi
name: Gaudencio, Gabriel
name: Wang, Ruixiang
name: Selvakumar, Govindhasamy Pushpavathi
name: Kolling, Louis
name: Pierson, Samantha
name: Tadinada, Satya M.
name: Abel, Ted
name: Hefti, Marco
name: Marcinkiewcz, Catherine A.
Publisher: name: figshare
Year: 2023
Subjects: subject: Neuroscience
subject: 111714 Mental Health
subject: FOS: Health sciences

description: Additional file 1: Supplementary Methods: RT-PCR quantification and tau splice isoforms; Detection of tau isoforms in whole brain lysates. Supplementary Figure 1: Validation of htau mice showing presence of insoluble 3R tau isoform in whole brain lysates at 12 months of age. (A) Mouse and human tau splice isoforms in C57BL/6J, htau +/- and MAPT -/- mice. The human-specific primers only amplified products in htau +/- while mouse-specific primers only amplified products in C57BL/6J mice. (B) (i) Protein expression of human tau (HT7) in total protein extracts and (ii) 3R and 4R tau isoforms in Sarkosyl soluble and insoluble fractions from C57BL/6J, htau +/- and MAPT -/- mice. Only htau +/- mice contained human-specific tau (HT7) and the 3R tau isoform in detergent insoluble fractions. Both htau +/- and C57 mice contained the 4R isoform in soluble fractions. Supplementary Figure 2: Orthogonal images confirming hyperphosphorylated tau in 5-HT neurons in the DRN. (A) Atlas plate depicting the mid region of the DRN that was used to obtain orthogonal planes. (B-D) Representative orthogonal planes showing colocalization between 5-HT (cyan) and ptau (red) in the DRN. (E) Atlas plate depicting the mid region of the LC that was used to obtain orthogonal planes. (F-H) Representative orthogonal planes showing colocalization between TH (cyan) and ptau (red) in the LC. Supplementary Figure 3: Hyperphosphorylated tau and monoaminergic depletion in the brainstem of female htau mice at 4 months. (A) Representative confocal images of 5-HT immunostaining (20X; scale bar = 200 µm) and 5-HT/AT8 co-staining (60X; scale bar = 50 µm) in the DRN of C57BL/6J and htau +/- mice. (B) Atlas plate depicting one of the DRN regions analyzed (mid DRN) (C) Representative orthogonal image showing colocalizaton of ptau with 5-HT neurons in the DRN (100X; scale bar = 20 µm). (D) Histogram of 5-HT cell counts/mm2, (E) 5-HT immunoreactive area (%), (F) ptau (AT8) optical density in subregions of the DRN and (G) Correlation analysis between 5-HT IR area and AT8 optical density in the DRN (H) Representative confocal images of TH immunostaining (20X; scale bar = 200 µm) and TH/AT8 colocalization (60X; scale bar = 50 µm) in the LC of C57BL/6J and htau +/- mice. (I) Atlas plate depicting one of the LC regions analyzed (mid LC) (J) Representative orthogonal image showing colocalization of ptau with TH neurons in the LC (100X; scale bar = 20 µm). (K) Histogram of TH cell counts/mm2, (L) TH immunoreactive area, (M) ptau (AT8) optical density in subregions of the LC, and (N) Correlation analysis between TH immunoreactive area and AT8 optical density in the mid LC. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Supplementary Figure 4: Glial activation in the DRN and LC in htau mice at 4 months. (A) Representative confocal images of Iba-1 and GFAP immunostaining in the DRN of C57 and htau +/- mice (60X; scale bar = 50 µm). (B) Iba-1+ cell counts/mm2, (C) Iba-1 optical density, and (D) Iba-1 immunoreactive area in subregions of the DRN. (E) GFAP+ cells/mm2, (F) GFAP optical density, and (G) GFAP immunoreactive area in subregions of the DRN. (H) Representative confocal images of Iba-1 and GFAP immunostaining in the LC of C57 and htau +/- mice. (I) Iba-1+ cells/mm2, (J) Iba-1 optical density, and (K) Iba-1 immunoreactive area in subregions of the LC. (L) GFAP+ cells/mm2, (M) GFAP optical density, and (N) GFAP immunoreactive area in subregions of the LC. Data are represented as mean ± SEM. *p &lt; 0.05, **p &lt; 0.01. White arrows denote Iba-1 +or GFAP+ cell bodies. Supplementary Figure 5: Reduced 5-HT innervation of the entorhinal cortex in htau mice at 4 months. Representative confocal images of 5-HT fiber immunostaining (40x; scale bar = 50 um) in C57 and htau mice. (A) Atlas representation of a section of the EC used for analysis (in red). (B) Representative confocal images of 5-HT staining in the EC of C57BL/6J and htau mice at 40X. (C) Histogram of % 5-HT IR area in subregions of the EC. (D) Representative confocal images of 5-HT staining in the EC at 100X. (E) Atlas representation of CA1 region of the hippocampus used in the analysis (in red). (F) Representative confocal image of SERT staining in the CA1 region of the hippocampus in C57BL/6J and htau mice. (G) Histogram of % SERT IR area in subregions of the hippocampus (H) Representative confocal images of SERT staining in the CA1 region of the hippocampus at 100X. Data are represented as mean ± SEM. *p &lt; 0.05, **p &lt; 0.01. Supplementary Table 1: List of fold change values obtained from RT-qPCR of genes tested in the dorsal raphe nucleus, locus coeruleus, dorsal and ventral hippocampus, and entorhinal cortex of C57 and htau (*p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001).