Fig. 6

Fig. 6 Methazolamide treatment reduces tau levels and neuron loss and improves object recognition in PS19 tau transgenic mice.

a, Scheme for methazolamide dosing in PS19 mice. Tau PS19 mice at 8–9 months were implanted with subcutaneous methazolamide-loaded osmotic minipumps (20 mg per kg (body weight) per day; ALZET Model 2002) for 28 days in total. Pumps were replaced once at day 14. Behavioral testing was performed once before implanting the minipump and subsequently at days 26 (habituation to arena) and 27 (novel object recognition testing) after treatment. b,c, Representative images (b) and quantification (c) of western blots to evaluate the effects of 20 mg per kg (body weight) per day methazolamide treatment on levels of tau (Tau5) in sarkosyl-soluble and sarkosyl-insoluble fractions from the cerebral cortex of PS19 mice compared with mice treated with vehicle only. The same soluble fraction from a vehicle control-treated mouse was run in parallel to insoluble fractions on every gel (b); HE, high exposure; LE, low exposure. GAPDH was used as a loading control. Methazolamide treatment significantly reduced the levels of soluble tau (soluble Tau5/GAPDH) compared with vehicle control treatment and diminished insoluble tau (insoluble Tau5/GAPDH) to a lesser extent (c). Insoluble fractions were normalized to control (n = 21 methazolamide-treated mice and n = 19 vehicle control-treated mice). Values are shown as mean ± s.e.m.; *P ≤ 0.05 versus vehicle control. Data were analyzed by two-tailed unpaired t-test; VC, vehicle control. d, A novel object recognition task was performed using PS19 mice (untreated) and wild-type littermates (untreated) in parallel to PS19 mice treated with methazolamide or vehicle control. All mice were assessed at 34 weeks and were reassessed at day 27. Wild-type and PS19 littermates that were not implanted with minipumps were assessed in parallel (n = 20 wild-type mice; n = 15 PS19 mice; n = 22 PS19 mice treated with methazolamide; n = 21 vehicle control-treated PS19 mice). The plot shows changes in scores from 34 weeks to 34 weeks + 27 days (after treatment) as the percentage of novelty preference analyzed using a Wilcoxon matched-pairs signed-rank test (nonparametric method) to compare before and after treatment novel object recognition task scores; *P ≤ 0.05 versus vehicle control. e, Unbiased stereological estimates of numbers of NeuN⁺ CA1 neurons in the hippocampus of PS19 mice and vehicle-treated and methazolamide-treated PS19 mice. Wild-type littermate brains were used as a positive control. Data in the plots represent mean ± s.e.m. (n = 8 vehicle-treated mice and n = 6 methazolamide-treated mice per group). Data were analyzed by one-tailed unpaired t-test. f,g, Effects of 20 mg per kg (body weight) per day methazolamide treatment on levels of phosphorylated tau, as detected by antibodies to AT8 and PHF1 in the hippocampus (CA1) and entorhinal cortex regions of brains of PS19 mice. Methazolamide treatment reduces hyperphosphorylated tau levels compared with vehicle control treatment. Data in the plots represent mean ± s.e.m. (n = 8 vehicle-treated mice and n = 6 methazolamide-treated mice per group). Data were analyzed by two-tailed unpaired t-test.

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