Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies

Abstract

Pathological tau aggregation leads to filamentous tau inclusions and characterizes neurodegenerative tauopathies such as Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Tau aggregation coincides with clinical symptoms and is thought to mediate neurodegeneration. Transgenic mice overexpressing mutant human P301S tau exhibit many neuropathological features of human tauopathies including behavioral deficits and increased mortality. Here, we show that the di-phenyl-pyrazole anle138b binds to aggregated tau and inhibits tau aggregation in vitro and in vivo. Furthermore, anle138b treatment effectively ameliorates disease symptoms, increases survival time and improves cognition of tau transgenic PS19 mice. In addition, we found decreased synapse and neuron loss accompanied by a decreased gliosis in the hippocampus. Our results suggest that reducing tau aggregates with anle138b may represent an effective and promising approach for the treatment of human tauopathies.

Keywords: Alzheimer’s disease; Anle138b; Protein aggregation; Tau; Tau aggregation inhibitor; Tauopathy.

Fig. 1

Anle138b binds to aggregated tau and reduces tau aggregates in vitro. a Fluorescence spectrometry of anle138b (250 nM) excited at 300 nm with tau monomers (left panel) and pre-aggregated tau protein (right panel). Tau monomers did not change the fluorescence spectra of anle138b, whereas addition of pre-aggregated tau protein changed the fluorescence properties of anle138b and increased fluorescence at 340 nm. b Exemplary SIFT measurement (SIFT scanning for intensely fluorescent targets). c The formation of tau aggregates in vitro is significantly inhibited by the application of anle138b (p < 0.05, t test). d Correlation between tau anti-aggregative effect and compound brain concentration. Anle138b shows the best correlation of tau aggregation inhibition and bioavailability

Fig. 2

Anle138b treatment reduces tau pathology in PS19 mice. a AT8 stainings of non-transgenic (ntg), untreated and anle138b-treated PS19 mice of coronal brain sections (upper panel) and hippocampal CA1 area (lower panel). b, c Immunohistochemical staining of brain sections with the conformation-specific tau antibody MC1 (b) and Gallyas silver staining (c) in treated and untreated PS19 mice. d–f Quantification of AT8, MC1 and silver-positive area in treated and untreated PS19 mice (n = 9 mice/group; 10–12 months). Asterisks indicate significant differences relative to untreated PS19 mice (*p < 0.05; **p < 0.01; t test). Scale bars 500 µm in low-power images and 50 µm in high-power images

Fig. 3

Anle138b reduces insoluble tau levels in PS19 mice a Immunoblots of brain homogenates of non-transgenic (ntg), untreated and anle138b-treated PS19 mice probed with HT7 and E178 antibody detecting total tau. b Densitometric analysis of immunoblots showing the brain protein levels of total tau (HT7 and E178) in untreated and anle138b-treated PS19 mice. (n = 7 mice/group; 10-12 months). c Immunoblots of brain homogenates of ntg, untreated and anle138b-treated PS19 mice probed with the phosphorylation-specific antibodies (AT8 and AT180) and phosphorylation-independent antibody E178. d Densitometric analysis of immunoblots showing phosphorylated tau (AT8, AT180) in untreated and anle138b-treated PS19 mice. (n = 10–11 mice/group; 10–12 months). Immunoblots using the antibodies HT7 and AT100 (e) and densitometric analysis (f) of sarkosyl-insoluble aggregated tau protein in the brain of untreated PS19 transgenic mice in comparison to anle138b-treated animals. (n = 9–10 mice/group; 10–12 months). Immunoblots of sucrose-gradient centrifugation fractions from untreated and anle138b-treated mouse brain homogenates, to separate tau aggregates according to their molecular weight (g) and densitometric analysis (h). Anle138b-treated PS19 mice showed a significant reduction of high molecular weight species in the 50 % sucrose fraction in comparison to untreated animals (n = 7 mice/group; 10–12 months). Asterisks indicate significant differences relative to untreated PS19 mice (*p < 0.05; t test)

Fig. 4

Anle138b treatment ameliorates neuropathology of PS19 mice NeuN staining (a) and quantification (b) of the hippocampal CA3 region of ntg, untreated and anle138b-treated PS19 mice. Staining with the synaptic marker synaptophysin in the stratum lucidum (c) and quantification (d) of ntg, untreated and anle138b-treated PS19 mice. Microglia Iba1 and astrocyte GFAP staining (e, g) and quantification (f, h) of ntg and PS19 mice treated and untreated with anle138b. a–h (n = 8–9 PS19 mice/group; n = 4–5 ntg mice/group; 10–12 months). Scale bars 25 µm. Asterisks indicate significant differences relative to untreated PS19 mice (*p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni’s multiple-comparisons test)

Fig. 5

Effect of anle138b treatment on survival and behavior in PS19 transgenic mice a Kaplan–Meier plots of PS19 transgenic and ntg mice. Anle138b treatment significantly prolonged survival time of PS19 mice (**p < 0.01; n = 37 mice/group; Log-Rank test) and attenuated 12-month survival rate compared with untreated PS19 littermates (p < 0.01, Log-Rank test). b Learning and memory were analyzed at 9 months of age using an object–place recognition task. Gray line indicates chance level. Untreated PS19 mice did not show significantly higher exploration of the object in the novel location relative to the object in the known location (discrimination ratio n.s.), which is consistent with memory impairments. In contrast, Anle138b-treated PS19 mice spent significantly more time exploring the object in the novel location, indicating that treatment restored learning and memory in PS19 mice (**p < 0.01; ***p < 0.001; n = 19–24 mice/group; Significantly different from a hypothetical 0.50, one-sample t test). c Total distance traveled in the open-field test was similar in untreated and anle138b-treated mice independent of the genotype, indicating that motor behavior was unaffected in these animals (n = 19–24 mice/group)