Tau seeds from patients induce progressive supranuclear palsy pathology and symptoms in primates

Abstract

Progressive supranuclear palsy is a primary tauopathy affecting both neurons and glia and is responsible for both motor and cognitive symptoms. Recently, it has been suggested that progressive supranuclear palsy tauopathy may spread in the brain from cell to cell in a 'prion-like' manner. However, direct experimental evidence of this phenomenon, and its consequences on brain functions, is still lacking in primates. In this study, we first derived sarkosyl-insoluble tau fractions from post-mortem brains of patients with progressive supranuclear palsy. We also isolated the same fraction from age-matched control brains. Compared to control extracts, the in vitro characterization of progressive supranuclear palsy-tau fractions demonstrated a high seeding activity in P301S-tau expressing cells, displaying after incubation abnormally phosphorylated (AT8- and AT100-positivity), misfolded, filamentous (pentameric formyl thiophene acetic acid positive) and sarkosyl-insoluble tau. We bilaterally injected two male rhesus macaques in the supranigral area with this fraction of progressive supranuclear palsy-tau proteopathic seeds, and two other macaques with the control fraction. The quantitative analysis of kinematic features revealed that progressive supranuclear palsy-tau injected macaques exhibited symptoms suggestive of parkinsonism as early as 6 months after injection, remaining present until euthanasia at 18 months. An object retrieval task showed the progressive appearance of a significant dysexecutive syndrome in progressive supranuclear palsy-tau injected macaques compared to controls. We found AT8-positive staining and 4R-tau inclusions only in progressive supranuclear palsy-tau injected macaques. Characteristic pathological hallmarks of progressive supranuclear palsy, including globose and neurofibrillary tangles, tufted astrocytes and coiled bodies, were found close to the injection sites but also in connected brain regions that are known to be affected in progressive supranuclear palsy (striatum, pallidum, thalamus). Interestingly, while glial AT8-positive lesions were the most frequent near the injection site, we found mainly neuronal inclusions in the remote brain area, consistent with a neuronal transsynaptic spreading of the disease. Our results demonstrate that progressive supranuclear palsy patient-derived tau aggregates can induce motor and behavioural impairments in non-human primates related to the prion-like seeding and spreading of typical pathological progressive supranuclear palsy lesions. This pilot study paves the way for supporting progressive supranuclear palsy-tau injected macaque as a relevant animal model to accelerate drug development targeting this rare and fatal neurodegenerative disease.

Keywords: non-human primate; parkinsonism; prion-like; progressive supranuclear palsy; tau; tauopathy.

Figure 1

Extraction, purification and in vitro characterization of tau aggregates. (A) Schematic summary of the extraction/purification protocol. (B) Filter retardation assay probed with tau monoclonal antibody (HT7) to assess the final total tau concentration in CTL-tau and PSP-tau samples. (C) Quantitative results of the high-throughput seeding assay using the tau P301S-Venus cell line transfected with patient-derived tau seeds. ****P < 0.0001; **P = 0.0008; two-way ANOVA with Tukey’s multiple comparison test. (D) Representative fluorescent images of the seeding assay. Arrowheads represent the tau-venus-positive punctae/aggregates typically taken into account to measure the relative level of seeding 48 h after adding seeds or control material. Because this assay revealed a higher in vitro seeding activity of the PSP-tau-MES sample compared to PSP-tau-CTX, the PSP-tau-MES sample was selected for further in vitro characterization and future injections in macaques. (E) Immunofluorescence performed on PSP-tau seeded and control cells with AT8 antibody and pFTAA staining (probe of filamentous proteins). All scale bars = 150 µm. (F) Western blot of soluble and sarkosyl-insoluble proteins from PSP-tau seeded and controls cells using the pan-tau KJ9A and AT8 antibodies.

Figure 2

Injections of patient-derived PSP-tau aggregates induced parkinsonism in macaques. (A) Schematic representation of the experimental design and of the kinematics recording session. Macaques were equipped with markers for the extraction of whole-body kinematics in 3D (57 kinematic features for each gait cycle were computed and analysed). Recordings were performed in three recording sessions that occurred 6, 12 and 18 months post-injection. (B) Principal component analysis (PCA) 6 months post-injection (first session). Each dot on the left panel plot represents one gait cycle. Different colours represent different macaques. Larger dots represent the means across all gait cycles of each macaque. The right panel plot shows the same data represented by ellipsoids with the centre and principal semi-axis as the mean and SD calculated across all the gait cycles for that condition and macaque—in a space spanned by the three leading principal components (PCs). (C) We performed the PCA analysis over the concatenated gait parameter data of three recording sessions m6, m12 and m18. The plot shows the points that represent average of gait parameters for each session and each macaque projected into the spanned by the three leading PCs. Note that CTL-tau No. 2 macaque declined to perform m18 session. (D) Bar plots represent the mean values of gait features suggestive of parkinsonism. Difference limb elevation angle describes the amplitude of limb oscillation in degrees around the hip joint; Difference shank elevation angle describes the amplitude of limb oscillation in degrees around the knee joint; Difference knee angle describes the difference in degrees between maximal flexion and maximal extension of the limb (number of gait cycles: CTL-tau 1: 43, CTL-tau 2: 33, PSP-tau 1: 36 and PSP-tau 2: 36). *P < 0.05, **P < 0.01, ***P < 0.001; Kruskal–Wallis test with Tukey–Kramer correction for multiple comparisons.

Figure 3

Injections of patient-derived PSP-tau aggregates induced the progressive occurrence of a dysexecutive syndrome in macaques. (A) Schematic representation of the longitudinal assessment using the object retrieval task that assesses inhibition, planning and perseverations. (B) Longitudinal analysis of macaques’ performance with hard items. Thin dotted lines: longitudinal performance of individual macaques. Wide and full lines: mean trajectory of PSP and CTL macaques. **P < 0.01 and ***P < 0.001; two-way ANOVA.

Figure 4

Injections of patient-derived PSP-tau aggregates induced typical neuropathological PSP lesions. (A) Anatomical representation showing the schematic distribution of tau pathology in PSP macaques along the antero-posterior (rostro-caudal) axis. (B–J) Representative images of AT8 immunostaining. (B and C) Tufted astrocytes, (D) globose tangles (E) coiled bodies and (F) neurofibrillary tangles and threads were observed close to the injection sites. (G–I) Tau inclusions were also found in anterior remote brain areas, in the putamen (Pu), caudate (Cd), Globus pallidus (GPe) and antero-ventral thalamus (VA). Scale bars = 20 µm. (K) Representative double-stained immunofluorescence images confirming astrocytic (GFAP-S100), oligodendroglial (Olig2) and neuronal (NeuN) phospho-tau (AT8) inclusions. (L) Representative images of three-repeat isoform RD3 and four-repeat isoform RD4 immunostaining in PSP and control macaques. Scale bars = 20 µm.

Figure 5

Injections of patient-derived PSP-tau aggregates induce dopaminergic neuronal loss in the substantia nigra of macaques. (A) Representative images of thyroxine hydroxylase (TH) immunohistochemistry on the substantia nigra of CTL-tau (left) and PSP-tau macaques (right). (B) Stereological quantification of the number of TH+ neurons in the substantia nigra of macaques. Data are shown as mean ± SEM (horizontal lines). Each dot represents one NHP of the control (grey) and PSP-injected macaques (blue). The bootstrapped mean difference with 95% CI (error bar) is shown on the right side of this graph. Scale bars = 10 µm (inset, 20 µm).