Passive immunization against phosphorylated tau improves features of Huntington's disease pathology

Abstract

Huntington's disease is classically described as a neurodegenerative disorder of monogenic aetiology. The disease is characterized by an abnormal polyglutamine expansion in the huntingtin gene, which drives the toxicity of the mutated form of the protein. However, accumulation of the microtubule-associated protein tau, which is involved in a number of neurological disorders, has also been observed in patients with Huntington's disease. In order to unravel the contribution of tau hyperphosphorylation to hallmark features of Huntington's disease, we administered weekly intraperitoneal injections of the anti-tau pS202 CP13 monoclonal antibody to zQ175 mice and characterized the resulting behavioral and biochemical changes. After 12 weeks of treatment, motor impairments, cognitive performance and general health were improved in zQ175 mice along with a significant reduction in hippocampal pS202 tau levels. Despite the lack of effect of CP13 on neuronal markers associated with Huntington's disease pathology, tau-targeting enzymes and gliosis, CP13 was shown to directly impact mutant huntingtin aggregation such that brain levels of amyloid fibrils and huntingtin oligomers were decreased, while larger huntingtin protein aggregates were increased. Investigation of CP13 treatment of Huntington's disease patient-derived induced pluripotent stem cells (iPSCs) revealed a reduction in pS202 levels in differentiated cortical neurons and a rescue of neurite length. Collectively, these findings suggest that attenuating tau pathology could mitigate behavioral and molecular hallmarks associated with Huntington's disease.

Keywords: antibody; huntingtin; hyperphosphorylation; iPSC; mHTT; neurodegenerative diseases; pS202 tau; tauopathy; zQ175.

Graphical abstract

Figure 1

CP13 antibody treatment improves motor and cognitive performance in zQ175 mice (A) Timeline of antibody treatment and behavioral evaluations. (B–D) Cognitive performance was assessed as the total number of entries (B), the total number of correct entries (C) and the percent of correct entries (D) in the Y maze. (E–G) Motor performance was assessed as the distance traveled in the open field (E). Short- and long-term memory was evaluated by measuring intrasession (F) and intersession (G) habituation in the open field. Data are presented as mean ± SEM with individual animal results indicated as data points. WT (S) n = 8–9; WT (CP13) n = 6–7; zQ175 (S) n = 7–10; zQ175 (CP13) n = 9–10. For all graphs, statistics were performed using a two-way ANOVA with Sidak's post-hoc test. ^#For (E), we confirmed a genotype difference between saline-treated WT and zQ175 mice by performing a Student's t test. ∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. m, meters; S, saline; WT, wild type.

Figure 2

CP13 antibody treatment decreases levels of phosphorylated tau in the cortex and hippocampus of zQ175 mice (A, F, and K) Representative immunoblots depicting protein levels of tau phosphorylated at serine 202 (pS202), serine 199 (pS199), serines 396/404 (p396/404), total tau (t-tau) and total protein (tot prot) in the cortex (A), hippocampus (F) and striatum (K) of WT or zQ175 mice treated with CP13 or saline. (B–E) Quantification of protein levels in cortical homogenates and calculated ratios of t-tau normalized over tot prot (B), pS202 (C), pS199 (D) or pS396/404 (E) normalized to t-tau levels. (G–J) Quantification of protein levels in hippocampal homogenates and calculated ratios of t-tau normalized over tot prot (G), pS202 (H), pS199 (I) or pS396/404 (J) normalized to t-tau levels. (L–O) Quantification of protein levels in striatal homogenate and calculated ratios of t-tau normalized over tot prot (L), pS202 (M), pS199 (N) or pS396/404 (O) normalized to t-tau levels. Data are presented as mean ± SEM with individual animal results indicated as data points. WT (S) n = 3–4; WT (CP13) n = 3–4; zQ175 (S) n = 4–6; zQ175 (CP13) n = 5–6. For all graphs, statistical analyses were performed using a two-way ANOVA with Sidak's post-hoc tests. ∗p ≤ 0.05 and ∗∗p < 0.01. kDa, kilodalton; t-tau, total tau.

Figure 3

CP13 antibody treatment does not induce a glial response (A) Representative images of cortical astrocytes labeled by immunohistochemistry with the GFAP marker. (B–D) Quantification of the number of GFAP-positive astrocytes in brain sections prepared from the cortex (B), hippocampus (C)and striatum (D). (E–G) Quantification of GFAP protein levels by western blot using homogenates prepared from the cortex (E), hippocampus (F) and striatum (G). (H) Representative images of cortical microglia labeled by immunohistochemistry with the Iba1 marker. (I–K) Quantification of the number of Iba1-positive microglia in brain sections prepared from the cortex (I), hippocampus (J) and striatum (K). (L–N) Quantification of Iba1 protein levels by western blot using homogenates prepared from the cortex (L), hippocampus (M) and striatum (N). Data are expressed as mean ± SEM with individual animal results indicated with data points. WT (S) n = 3–4; WT (CP13) n = 3–4; zQ175 (S) n = 3–6; zQ175 (CP13) n = 3–6. For all graphs, statistics were performed using a two-way ANOVA with Sidak's post-hoc test. ∗p < 0.05 and ∗∗p < 0.01. Scale bars: 20 μm. GFAP, glial fibrillary acidic protein; Iba1, ionized calcium binding adaptor molecule; k, thousands; N, number; Tot prot, total protein.

Figure 4

CP13 antibody treatment does not alter neuronal markers (A and B) Quantification of protein levels and representative immunoblots detecting markers of axon damage (SMI32) in the cortex (A) and hippocampus (B). (C) Quantification of DARPP32 protein levels in striatal tissue homogenate by western blot. (D–F) Quantification of protein levels and representative immunoblots detecting the glutamatergic neuron marker VGlut1 in the cortex (D), hippocampus (E) and striatum (F). (G–I) Quantification of protein levels and representative immunoblots detecting the post-synaptic protein PSD95 in homogenates prepared from the cortex (G), hippocampus (H) and striatum (I). (G–I) Data are expressed as mean ± SEM with individual animal results indicated with data points. WT (S) n = 3–4; WT (CP13) n = 3–4; zQ175 (S) n = 4–6; zQ175 (CP13) n = 4–6. For all graphs, statistics were performed using a two-way ANOVA with Sidak's post-hoc test. ∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. PSD95, postsynaptic density protein 95; VGLT1, vesicular glutamate transporter 1.

Figure 5

CP13 antibody treatment does not affect soluble HTT and mHTT levels in the brain (A, D, and G) Representative confocal photomicrographs of brain slices labeled by immunofluorescence to detect HTT. (B–I) Quantification of protein levels and representative immunoblots showing soluble HTT (MAB2166 antibody) and soluble mHTT (MAB1574 antibody) in cortical (B and C), hippocampal (E and F) and striatal (H and I) brain homogenates. Data are expressed as mean ± SEM with individual animal results indicated with data points. WT (S) n = 3–4; WT (CP13) n = 3–4; zQ175 (S) n = 4–6; zQ175 (CP13) n = 4–6. For all graphs, statistics were performed using a two-way ANOVA with Sidak's post-hoc test;∗p < 0.05 and ∗∗∗∗p < 0.0001. Scale bars: 10 μm. HTT, huntingtin; mHTT; mutant huntingtin.

Figure 6

Reduced tau phosphorylation is associated with increased cortical and striatal insoluble HTT and mHTT (A–C) Representative immunoblots and quantification of filter retardation assays targeted to the detection of insoluble HTT and mHTT in tissue homogenates prepared from the cortex (A), hippocampus (B) and striatum (C) using N18, EM48, 1C2 and D7F7 antibodies. (D–F) Graphs show a stereological-based quantification for the number of HTT aggregates and their distribution across the 0–6 μm³ size range in the cortex (D), hippocampus (E) and striatum (F). The aggregates display a range of volumes and are categorized as very small (0.25–0.5 μm³), small (0.5–2 μm³), medium (2–4 μm³) or large (>4 μm³). (G–I) Graphs showing single data points in the cortex (G), hippocampus (H) and striatum (I) extracted from the respective aggregate distribution (D–F). Data are expressed as mean ± SEM with individual animal results indicated with data points. WT (S) n = 4–5; WT (CP13) n = 3–4; zQ175 (S) n = 3–5; zQ175 (CP13) n = 3–6. Statistical analyses were performed using a two-way ANOVA with Sidak's post-hoc test (A–C) or a Student's t test (G–I). ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Est, estimated.

Figure 7

CP13 antibody treatment decreases brain levels of oligomers and amyloid fibrils (A–F) Quantification of protein oligomers detected by dot blot and representative membranes probed using an oligomer- (A11) or amyloid-fibril-specific (OC) antibody in tissue homogenates prepared from the cortex (A and D), hippocampus (B and E) and striatum (C and F). (G–I) Quantification of HTT aggregates by SDD-AGE and representative membranes shown for the cortex (G), hippocampus (H) and striatum (I). Data are expressed as mean ± SEM with individual animal results indicated with data points. WT (S) n = 3–4; WT (CP13) n = 3–4; zQ175 (S) n = 4–6; zQ175 (CP13) n = 4–6. For all graphs, statistical analyses were performed using a two-way ANOVA with Sidak's post-hoc test. ∗p ≤ 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Figure 8

CP13 antibody treatment reduces tau hyperphosphorylation in human iPSC-derived cortical neurons (A) Graphical representation of the experimental procedure implemented to differentiate iPSCs into cortical neurons. (B) Representative confocal images of control and HD iPSC-derived neurons immunostained for MAP2 (red), VGlut1 (green)and nuclear stain DAPI (blue). (C) Quantification of the proportion of iPSC-derived neurons immunopositive for VGlut1 and MAP2 neuronal markers. Data are from three biological replicates and a total of 600 control and 572 HD neurons were counted. For both controls and HD, two independent iPSC lines each were analyzed (control lines, 17/18 CAG repeats; HD lines, 180 CAG repeats and 60 CAG repeats). (D) Representative confocal images of HD iPSC-derived neurons immunostained for MAP2 (green), anti-mouse secondary antibody (red) and nuclear stain DAPI (blue). (E) Quantification of pS202 tau normalized to total tau and beta actin protein levels as well as representative immunoblots showing pS202 tau, total tau and the loading control β-actin in iPSC-derived cortical neurons. Data are from four biological replicates prepared using two independent control iPSC lines (17/18 CAG repeats) and one HD line (180 CAG repeats). (F) Quantification of neurite length after 14 days in culture in absence or presence of 15 μg/mL IgG or CP13. Data are from two biological replicates and more than 200 neurites were measured in each experimental condition (control lines, 17/18 CAG repeats; HD lines, 180 CAG repeats and 60 CAG repeats). Statistical analysis was performed using a one-way ANOVA with Dunnett's multiple comparisons test (E and F); ∗p ≤ 0.05 and ∗∗p < 0.01. Outliers in (E) were identified using the Grubbs test (alpha = 0.05) and excluded from analysis. Scale bars: 25 μm (B) and 10 μm (D). IF, immunofluorescence; iPSCs, induced pluripotent stem cells; NPCs, neural progenitor cells; ns, not significant.