Cis P-tau underlies vascular contribution to cognitive impairment and dementia and can be effectively targeted by immunotherapy in mice

Abstract

Compelling evidence supports vascular contributions to cognitive impairment and dementia (VCID) including Alzheimer's disease (AD), but the underlying pathogenic mechanisms and treatments are not fully understood. Cis P-tau is an early driver of neurodegeneration resulting from traumatic brain injury, but its role in VCID remains unclear. Here, we found robust cis P-tau despite no tau tangles in patients with VCID and in mice modeling key aspects of clinical VCID, likely because of the inhibition of its isomerase Pin1 by DAPK1. Elimination of cis P-tau in VCID mice using cis-targeted immunotherapy, brain-specific Pin1 overexpression, or DAPK1 knockout effectively rescues VCID-like neurodegeneration and cognitive impairment in executive function. Cis mAb also prevents and ameliorates progression of AD-like neurodegeneration and memory loss in mice. Furthermore, single-cell RNA sequencing revealed that young VCID mice display diverse cortical cell type-specific transcriptomic changes resembling old patients with AD, and the vast majority of these global changes were recovered by cis-targeted immunotherapy. Moreover, purified soluble cis P-tau was sufficient to induce progressive neurodegeneration and brain dysfunction by causing axonopathy and conserved transcriptomic signature found in VCID mice and patients with AD with early pathology. Thus, cis P-tau might play a major role in mediating VCID and AD, and antibody targeting it may be useful for early diagnosis, prevention, and treatment of cognitive impairment and dementia after neurovascular insults and in AD.

Fig. 1.. Robust cis P-tau in axons with no evidence of tau tangles in patients with VaD and BCAS mice.

(A to C) Immunofluorescence (IF) staining of brain sections from patients with VaD and age-matched controls with AT8 antibody for early tangle-like structures (A), AT100 antibody for late tangle-like structures (B), and with thioflavin S for NFT-like structure (C). DAPI, 4API, with thioflavin S for NF. (D and E) Near-infrared (NIR) fluorescence imaging of cis P-tau in brain sections from patients with VaD and age-matched controls (D). Yellow arrows indicated the cortex overlying corpus callosum. Quantitation and statistics for the staining intensity in the cingulate cortex overlying corpus callosum (CCtx-CC) are shown in (E). (F and G) IF staining of cis P-tau in the CCtx-CC and neocortex (Neo-Ctx) from patients with VaD and age-matched controls with cis P-tau mAb. A representative image is shown in (F). Quantitation and statistics for the staining intensity is shown in (G). (H and I) IF costaining of cis P-tau with neurofilament (H) or MBP (I) in the CCtx-CC and CC from patients with VaD. Insets show higher magnifications. (J to L) Two-month-old WT mice were subjected to sham or BCAS operation using external microcoils in blue to reduce cerebral blood flow by ~50% (J), followed by IF of cis P-tau in the cortex overlying corpus callosum (Ctx-CC) at different post-surgery time points (green arrows) (K and L). The data were presented as means ± SEM. The P values in (E) and (G) were calculated using unpaired two-tailed Student’s t test, and the P values in (L) were calculated using one-way analysis of variance (ANOVA) with post hoc Dunnett’s multiple comparison test. *P < 0.05 and ****P < 0.0001.

Fig. 2.. Cis mAb treatment of BCAS mice blocks the development of VCID-like pathology and brain dysfunction at 1 and 6 months after the surgery.

(A) Experimental setup for treating BCAS mice with cis mAb for 28 days. Two-month-old WT mice underwent either sham or BCAS operation, followed by treatment with either cis mAb or IgG isotype control for up to 28 days [300 μg per mouse, intraperitoneally (i.p.), every 3 days for four times, and then 150 μg per mouse every week afterward]. Black arrows, mAb injection; green arrows, functional or pathological assays. (B to E) Ctx-CC from sham, BCAS + IgG, or BCAS + cis mAb mice were examined by immunoblotting for cis P-tau and total tau (C), immunostaining and quantitation for cis P-tau (B), or GFAP (D) or MBP (E). (F) Long-term potentiation (LTP) recording of hippocampal slides. fEPSP slopes over time are shown in dot graph (left), and normalized fEPSP slope for the last 10 min is shown in bar graph (right). (G) Discrimination ratio (left) and distance traveled (right) in novel object recognition assays. (H) Experimental setup for treating BCAS mice with cis mAb for 6 months. Two-month-old WT mice were subjected to either sham or BCAS operation and treated with cis mAb or IgG isotype control for 6 months (300 μg, i.p., every 3 days for four times, and then 200 μg every week afterward). (I) Quantitation of cis P-tau IF intensity from different mouse brain regions. (J) Discrimination ratio (left) and distance traveled (right) in novel object recognition assays. (K) The time mice stay in open arm (left) and distance traveled (right) in elevated plus maze assays. The data were presented as means ± SEM, and the P value was calculated using one-way ANOVA with post hoc Dunnett’s multiple comparison test. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.. Pin1 is inhibited in BCAS, whereas brain-specific Pin1 overexpression blocks VCID-like pathology and brain dysfunction in BCAS mice.

(A) Quantitation of active Pin1 immunoreactivity in the CCTx-CC of human VaD brain and normal control. (B) Top disease association with a putative Pin1 enhancer SNP E06-21879. VaD, vascular dementia; DD, Hodgkin’s disease; TCL, T cell lymphomas; PVD, peripheral vascular disease; RD, respiratory disorders; TC, thyroid cancer; SBH, subarachnoid hemorrhage; CVD, cerebrovascular disease; MSD, multisystem degeneration. (C and D) Immunoblotting of cortical lysate of 2-month-old Pin1 TG mice with Pin1 antibody (C) and quantified (D). (E to I) Indicated mouse brain sections were analyzed by IF cis P-tau (E), GFAP (F), MBP (G), GST-pi (H), and Iba1 (I) antibodies, followed by quantified intensity in the Ctx-CC at 28 days after surgery. (J) Bar graphs showing the percentage of correct choices in T maze. (K) Bar graphs showing discrimination ratio (left) and distance traveled (right) in novel object recognition assays. The data in (D) to (K) were presented as means ± SEM. The P value in (D) was calculated using unpaired two-tailed Student’s t test. The P values in (E) to (K) were calculated using one-way ANOVA with post hoc Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.. Cis mAb prevents and attenuates the development of NFT-like pathology and functional impairments in htau mice.

(A) Experimental setup. Three-month-old htau mice underwent treatment of cis mAb or IgG controls for 8 months, followed by functional and pathological examinations. Black arrows, antibody injection; green lines, functional or pathological assays. (B) Effect of cis mAb treatment on behavioral deficits in htau mice, as assayed by the Morris water maze in htau mice escape latency (left) in the acquisition trials and trajectories and time spent in target quadrant in the probe trial (right). (C to F) Effects of cis mAb treatment on the accumulation of cis P-tau and the development of tangle-like pathology in htau mice, as assayed by immunoblotting of cis P-tau and total tau (D); IF staining of cis P-tau (C) in the neocortex and hippocampus; IF with AT8 antibody (E); and Gallyas silver staining (F) to detect tangle-like pathology in neocortex and hippocampus after 8 months of treatment. (G) Experimental setup. Thirteen-month-old htau mice were subjected to treatment of cis mAb or IgG isotype control for 6 months. Black arrows, antibody injection; green lines, functional or pathological assays. (H and I) Effects of cis mAb treatment on the novel object recognition and T maze. Dot graph showing longitudinal assessment of discrimination ratio before and after treatment with either cis mAb (red) or control (black) (H). Bar graph showing the percentage of correct choices in T maze after treatment (I). (J and K) Effects of cis mAb treatment of aged htau mice on the accumulation of cis P-tau, neuronal loss, and tangle-like pathology, as shown by IF for cis P-tau (J), with AT8 mAb (L), Gallyas silver staining (M) to detect tangle-like pathology, and NeuN antibody (K) to detect neuronal loss in neocortex and hippocampus. Inset images are high magnifications of representative areas. ND, not detectable. The data were presented as means ± SEM. The P values in (H) (left) and (I) are computed using unpaired two-tailed Student’s t test. The P value in (H) (right) is computed using paired two-tailed Student’s t test. The P values in (B) to (E) and (J) to (K) were calculated using one-way ANOVA with post hoc Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 5.. BCAS induces diverse cortical cell type–specific transcriptomic changes, and the vast majority of the global alterations are recovered by cis mAb.

(A) Experimental setup of the single-nucleus RNA-seq for BCAS mice treated with IgG or cis mAb, with sham littermate as controls. (B) tSNE (t-distributed stochastic neighbor embedding) plots of color-coded cortical cell types. (C) Normalized expression of marker genes for different cell types: Slc17a7 (glutamatergic neurons, Ex), Gad1 and Gad2 (GABAergic neurons, In), Aqp4 and Slc1a3 (astrocytes, As), Olig1 and Pdgfra (oligodendrocytes and their progenitor cells, Ol), and Cldn5 and Flt1 (endothelia, En). (D to F) Top up-regulated (D) or down-regulated DEGs (E) in different cell types in BCAS mice with or without cis mAb treatment, and a list of up-regulated excitatory neuronal DEGs that have not been clearly linked to neurodegeneration or stroke (F). Dot plots are color coded with average expression and sized with percentage of cells expressing the gene. (G to J) Validation of two DEGs Caprin2 (G and I) and Hsd3b2 (H and J) with IF staining (G and H), with quantitations (I and H). The data in (I) and (J) were presented as means ± SEM, and the P values were calculated using one-way ANOVA with post hoc Dunnett’s multiple comparison test. (K) Top: Log₂-transformed relative normalized expression of each gene compared to sham. Cell types are labeled on the top. Bottom: Number of DEGs are shown in bar graphs. Genes that are differentially expressed in BCAS + IgG but not in BCAS + cis mAb mice (referencing to sham mice) are defined as recovered genes. (L) Xyplot showing correlation between average tau expression and the recovered DEG percentage in five major cell types.

Fig. 6.. BCAS in young mice induces the global transcriptomic changes resembling those in patients with AD, most of which are recovered by cis mAb.

(A) Enriched Gene Ontology (GO) terms with different classes color coded. (B) Heatmap showing average expression z score. (C to G) Dot plots (C) and IF staining validations (D to G) for four microtubule-related down-regulated DEGs. (H) IF staining validations of down-regulated hemoglobin genes in BCAS mouse and recovery in cis mAb–treated mice. (I and J) Gene set enrichment analyses of down-regulated gene sets in patients with incipient AD (I) and AD (J), within BCAS mouse DEGs in excitatory neurons. (K and L) Heatmaps of the shared DEGs in excitatory neurons in young BCAS mice and patients with AD (K) and bar graphs showing the cis mAb recovery percentage of shared DEGs (L).

Fig. 7.. Stereotactic cortical injection of purified cis P-tau is sufficient to induce prion-like progressive neurodegeneration and brain dysfunction.

(A) Experimental setup. Red arrows, stereotaxic injections; black arrows, mAb injections; green lines, functional and pathological assays. (B to F) Behavioral assessment of mice at different time (labeled in blue) after cis P-tau injection and mAb treatment. Bar graphs showing time spent in center in bright-light OF (open field) assays (B), time in open arms in elevated plus maze (C), latency to fall in accelerating rotatord (D), discrimination ratio (E) in novel object recognition, and freezing time in fear conditioning (F). (G to I) Cortical LTP (H) and ultrastructural pathologies of axonal microtubules and mitochondria (I) were determined in mPFC away from the injection site (G) at 10 months after the injection. The data were presented as means ± SEM, and the P values were calculated one-way ANOVA with post hoc Dunnett’s multiple comparison test. *P < 0.05 and **P < 0.01.

Fig. 8.. Injected cis P-tau induces the conserved transcriptomic changes that are not only highly relevant to cistauosis and axonopathy but are also found in early VCID and AD.

(A) The setup of the single-nucleus RNA-seq experiments for cis P-tau–injected mice, with vehicle controls. (B) tSNE plots of different color-coded cortical cell types from vehicle or cis P-tau–injected mice. (C) Normalized expression of marker genes for different cell types: Slc17a7 (glutamatergic neurons, Ex), Gad1, Gad2 (GABAergic neurons, In), Aqp4, Slc1a3 (astrocytes, As), Olig1, Pdgfra (oligodendrocytes and their progenitor cells, Ol), Cldn5, and Flt1 (endothelia, En). (D) Enriched GO terms with different classes color coded. (E and F) Venn diagram of the shared up-regulated (E) and down-regulated excitatory neuronal DEGs (F) between the cis P-tau—injected mice and BCAS mice. Two-tailed Fisher’s exact test is implemented to compute the P values for the overlap. (G and H) Validation of two up-regulated DEGs Caprin2 (G) and Hsd3b2 (H) and their response to cis mAb by IF staining. (I) Twenty-four conserved genes that are commonly down-regulated in the excitatory neurons of cis P-tau–injected mice, BCAS mice, and human patients only with early, but not late, AD pathology (35).