Synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer's-like tauopathy

Abstract

Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with tau pathologies, are neurodegenerative diseases characterized by neurofibrillary tangles (NFTs) comprising filamentous tau protein. Although emerging evidence suggests that tau pathology may be transmitted, we demonstrate here that synthetic tau fibrils are sufficient to transmit tau inclusions in a mouse model. Specifically, intracerebral inoculation of young PS19 mice overexpressing mutant human tau (P301S) with synthetic preformed fibrils (pffs) assembled from recombinant full-length tau or truncated tau containing four microtubule binding repeats resulted in rapid induction of NFT-like inclusions that propagated from injected sites to connected brain regions in a time-dependent manner. Interestingly, injection of tau pffs into either hippocampus or striatum together with overlaying cortex gave rise to distinct pattern of spreading. Moreover, unlike tau pathology that spontaneously develops in old PS19 mice, the pff-induced tau inclusions more closely resembled AD NFTs because they were Thioflavin S positive, acetylated, and more resistant to proteinase K digestion. Together, our study demonstrates that synthetic tau pffs alone are capable of inducing authentic NFT-like tau aggregates and initiating spreading of tau pathology in a tauopathy mouse model.

Figure 1.

Injection of T40/PS and K18/PL pffs into hippocampus induces tauopathy in young PS19 Tg mice. A, Schematic illustration of injection site. Top and bottom show coronal and sagittal planes, respectively. Red line indicates injection path and red dot indicates injection site. B and C are microscopic images of mouse brains 3 months after injection with PBS and synthetic human α-synuclein pffs, respectively, immunostained with mAb MC1. The three columns display low-power images of injected hemispheres, high-power images of CA1 region, and lateral CA3 region of injected hippocampus, in this order. Scale bars: left column, 1 mm; right column, 100 μm. D, Microscopic images of brain sections immunostained with MC1 showing, from left to right, the ipsilateral hemisphere of CA1 and lateral CA3, as well as the caudal part of entorhinal cortex (Caudal Ent Cxt) and the contralateral CA3, from PS19 mice injected with T40/PS fibrils survived for 1 month (n = 3), 3 months (n = 3), and 6 months (n = 2) after injections. E, Same as D except that K18/PL pffs were inoculated into the hippocampus of PS19 mice. n = 5 for each survival period. Scale bar, 100 μm.

Figure 2.

Effects of tau pff injections into hippocampus are dose and time dependent. A, Different amounts of K18/PL pffs (0.05, 0.5, and 5 μg) (top) or T40/PS pffs (0.05, 0.5, and 5 μg) (bottom) were injected into hippocampus, and mice were killed at 1, 2, and 4 weeks after injection. Each panel shows medial part of CA3 and dentate gyrus. Brain sections were stained with MC1 (left) and AT8 (right). Scale bar, 100 μm. B–E, Quantitative analyses of MC1-positive neurons in ipsilateral hippocampus (B), contralateral hippocampus (C), ipsilateral entorhinal cortex (D), and contralateral entorhinal cortex (E). Animals used for quantitative analyses were combined from both T40/PS and K18/PL injections. n = 5 for 1 week, n = 4 for 2 weeks, n = 8 for 1 month, n = 8 for 3 months, and n = 7 for 6 months. Significant differences between pairwise comparisons were marked with stars. *p < 0.05, **p < 0.01, ***p < 0.001. F, Quantitative analysis of NeuN-positive neurons at different time points after injection. There is no significant difference between any postinjection periods. The number of animals used for this analysis was the same as B–E. Ent Cortex, Entorhinal cortex.

Figure 3.

Injection of K18/PL pffs into striatum and overlaying cortex induces tauopathy. A, Schematic illustrations of injection sites with coronal and sagittal planes. Red line indicates injection path, and red dot indicates injection site. B, Microscopic images of ipsilateral cortex, thalamus, substantia nigra, corpus callosum, and ipsilateral striatum (from left to right) from PS19 mice immunostained with MC1 after injection with K18/PL pffs and survived for 1 (n = 3), 3 (n = 6), and 9 (n = 3) months. C, Different amounts of K18/PL pffs (0.05, 0.5, and 5 μg) were injected into striatum and overlaying cortex, and mice were killed at 1 week, 2 weeks, and 1 month after injection. Each panel shows injected cortex. Two mice were used for each concentration of tau pffs injection and each survival period. Brain sections were stained with MC1. Scale bars, 100 μm.

Figure 4.

Injections of tau pffs transmit tau pathology to the LC. A, T40/PS pffs (top 2 panels) or K18/PL pffs (bottom 2 panels) injection into hippocampus. Lower-power microscopic images of brainstem (left), medial region of LC of contralateral (middle) and ipsilateral (right) to injection site, at 1 month (top) and 3 months (bottom) after pff inoculation are shown stained with MC1. Scale bars: left, 200 μm; right, 100 μm. B, K18/PL injection into striatum and overlaying cortex at 1 week (wk), 2 weeks, 1 month (mo), and 3 months after the injection. Scale bars: left, 200 μm; right, 100 μm. C, MC1 (green) and TH (red) in two-color immunofluorescence staining, which revealed that many MC1-positive neurons are colocalized with TH cells in LC (merge). Scale bar, 100 μm. D, Quantitative analysis of MC1-positive LC neurons. n = 2–9 mice for each group. Black bars represent ipsilateral, and white bars represent contralateral.

Figure 5.

Heat maps of tau pathology in coronal sections stained with MC1 for hippocampus injection. Semiquantitative analyses of tau pathology graded using MC1 antibody. Each panel represents heat map pathology distribution on one of the six different coronal planes (bregma, −0.98, −1.22, −2.18, −2.92, −4.48, and −5.52 mm) at 1 month (n = 8), 3 months (n = 8), or 6 months (n = 7) after tau pff injection into hippocampus. Left column shows sagittal view of the selected coronal planes used to generate the heat maps. Blue star indicates injection site.

Figure 6.

Heat maps of tau pathology in coronal sections stained with MC1 for striatum and overlaying cortex injection. Semiquantitative analyses of tau pathology graded using MC1 antibody. Each panel represents heat-map pathology distribution on one of the six different coronal planes (bregma, 1.98, 0.38, −2.18, −2.92, −4.48, and −5.52 mm) after 1 month (n = 6), 3 months (n = 9), and 9 months (n = 5) injection into striatum and overlaying cortex. Left column shows sagittal view of the selected coronal planes. Blue stars indicate injection sites.

Figure 7.

Properties of tau tangles in tau pff-injected versus uninjected aged PS19 mice. A, Brain sections were immunostained with mAbs AT8 to detect hyperphosphorylated tau, T49 to detect mouse tau, TG3 to detect specific conformation and phosphorylation changes, anti-ac-K280 rabbit antibody to detect acetylation at K280, and ThS histochemistry to detect mature NFT-like structures after the injection of K18/PL pffs into hippocampus at 1 week, 2 weeks, 1 month, and 3 months after injection. Comparison was made with a 14-month-old uninjected PS19 mouse. Scale bars, 100 μm. B, Double immunofluorescence with T49 (green) and 17025 (red) (top) or MC1 (green) and K280 (red) (bottom). Scale bars, 50 μm.

Figure 8.

Comparison between T40/PS pff-injected and normally aged PS19 mice in different brain regions. Brain sections were immunostained with MC1, anti-ac-K280 antibody, and ThS. Three different brain regions are shown, i.e., medial part of CA3 in hippocampus, caudal part of the cortex, and LC of T40/PS pff-injected PS19 (A) and uninjected aged PS19 (B). Scale bars, 100 μm. C, Double immunofluorescence with MC1 (red) and ThS (green). Scale bar, 50 μm.

Figure 9.

Comparison of the effects of PK treatment on pff-injected and uninjected aged PS19 mice. A, MC1 stained hippocampus (lateral CA3) and LC of K18/PL pff-injected mouse (top panels) and uninjected aged PS19 mouse (bottom panels) with no treatment or with 10 μg/ml PK treatment. Scale bar, 100 μm. B, Quantitative analyses of MC1-positive neurons in CA3 and LC. Data represents MC1 immunoreactivity normalized to no PK treatment conditions for both injected (n = 5) and uninjected aged (n = 4) PS19 mice. White bars represent no treatment, and black bars represent PK treatment. **p < 0.01, ***p < 0.001. C, Immunoblot analysis of cortex and hippocampus extracted sequentially with RIPA buffer, followed by 2% SDS. On each blot, the leftmost lane is uninjected age-matched control, and the other two lanes are two different injected mice (Injected) at 3 months after injection. mAb PHF1 detects hyperphosphorylated tau, and GAPDH was used as a loading control.