Modeling Sporadic Progressive Supranuclear Palsy in 3D Midbrain Organoids: Recapitulating Disease Features for In Vitro Diagnosis and Drug Discovery

Abstract

Objective: Progressive Supranuclear Palsy (PSP) is a severe neurodegenerative disease characterized by tangles of hyperphosphorylated tau protein and tufted astrocytes. Developing treatments for PSP is challenging due to the lack of disease models reproducing its key pathological features. This study aimed to model sporadic PSP-Richardson's syndrome (PSP-RS) using multi-donor midbrain organoids (MOs).

Methods: The MOs were generated by pooling induced pluripotent stem cells (iPSCs) from 4 patients with sporadic probable PSP-RS and compared them with MOs from 3 healthy control (HC) subjects. We performed comprehensive analyses of MOs over 120 days to assess neuronal death, reactive gliosis, and the accumulation of 4R-tau and hyperphosphorylated tau forms (pThr231, pSer396, pThr181, and pSer202/pThr205 [AT8]) using immunofluorescence microscopy and Western blot. On day 90, immunohistochemical analysis using pSer396 and AT8 antibodies was conducted to assess disease pathology.

Results: PSP-derived MOs showed progressive size reduction compared with HC-derived MOs, linked to upregulated apoptosis-related mRNA markers. Dopaminergic neuron degeneration was marked by decreased tyrosine hydroxylase (TH) and increased neurofilament light chain (NfL). Immunofluorescence and Western blot revealed accumulation of all investigated tau forms with a peak at 90 days, along with a significant rise in GFAP-positive cells in PSP-derived MOs. Immunochemistry confirmed typical PSP histological alterations, such as neurofibrillary tangles and tufted-shaped astrocytes, absent in HC-derived organoids.

Interpretation: We developed a robust in vitro PSP model reproducing the key molecular and histologic features of the disease. This result holds promise for advancing basic and clinical research in PSP, paving the way for in vitro molecular diagnosis and identification of novel therapeutic targets. ANN NEUROL 2025;97:845-859.

FIGURE 1

Generation of multi‐donor MOs. (A) A graphical overview illustrating the timeline and progression of multi‐donor MO differentiation (strategy adapted from Ref. 32). (B) Representative bright‐field images depict the progression of hiPSC culture and MOs differentiation from days −3 to 90. Scale bar = 100 μm. Additionally, a bright‐field image of a neuromelanin‐pigmented MO from the control group at day 120 is presented, with a scale bar of 100 μm. Fontana‐Masson staining of sections from control MOs highlights neuromelanin release (scale bar = 60 μm, bottom right). (C, D) The qRT‐PCR analysis of selected early midbrain and pluripotency markers at day 20 of differentiation, along with late midbrain markers at day 60. Values are expressed as fold change relative to undifferentiated pooled iPSCs. Statistical analysis was conducted using a 2‐tailed unpaired t test (n = 3 MOs). (E) Immunostaining results show FOXA2/LMX1A and OTX2/MAP2 at day 20 (scale bar = 200 μm, upper panel); FOXA2/MAP2 and TH/LMX1A at day 30 (middle panel); and GIRK2/CALB and DDC at day 60 and day 90, respectively (scale bar = 50 μm, bottom panel). Nuclei were stained with DAPI. DAPI = 4′,6‐diamidino‐2‐phenylindole; hiPSC = human‐induced pluripotent stem cell; MO = midbrain organoid; qRT‐PCR = quantitative real‐time polymerase chain reaction. [Color figure can be viewed at www.annalsofneurology.org]

FIGURE 2

Genetic background of patients with PSP‐RS and cell proliferation analysis. (A) Genotyping of the MAPT H1/H2 haplotype using end point PCR, with GAPDH as the loading control. (B) The qRT‐PCR analysis of genes associated with PSP neurodegeneration at day 120. Values are shown as fold change relative to healthy control MOs, with statistical analysis performed using a 2‐tailed unpaired t test. **p ≤ 0.01, ***p ≤ 0.001. Data are presented as mean ± SEM. (C) Proliferation assay on iPSCs derived from healthy controls and patients with PSP‐RS. Data are presented as the mean ± SEM of 3 independent experiments, performed in duplicate at each time point. (D) Cell cycle analysis of iPSCs from healthy donors and patients with PSP‐RS, showing the percentage of cells in G1, S, and G2/M phases 48 hours after nocodazole synchronization (12 hours). (E) Contribution of individual cell lines to the mosaic MO was assessed by demultiplexing single‐cell RNA sequencing data (from day 90 mosaic MO) based on genetic variants (SNPs) identified via whole‐genome sequencing (data not shown). iPSCs = induced pluripotent stem cells; MAPT = microtubule‐associated protein tau; MO = midbrain organoid; PCR = polymerase chain reaction; PSP‐RS = progressive supranuclear palsy Richardson's syndrome; qRT‐PCR = quantitative real‐time PCR; SNPs = single nucleotide polymorphisms. [Color figure can be viewed at www.annalsofneurology.org]

FIGURE 3

PSP‐RS MOs underwent atrophy and neuronal degeneration. (A) PSP‐RS MOs showed a significant reduction in size by day 60, with a diameter of 0.4 cm compared to 1 cm in HC‐derived MOs. (B) Growth rates of PSP‐RS organoids at various stages, with error bars representing mean ± SEM (n = 5). (C) The qRT‐PCR analysis of apoptosis‐related genes at day 120. Values are expressed as fold change relative to control MOs, with statistical analysis via two‐tailed unpaired t test, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. (D) Cryosections of HC‐ and PSP‐derived MOs at day 120 stained for TH (scale bar 50 μm). (E) Quantification of TH protein levels in HC‐ and PSP‐derived MOs at day 120, assessed by OD in immunoblot analysis. (F) Cryosections of HC‐ and PSP‐RS MOs at day 120 stained for NfL (scale bar = 50 μm). (G) Immunoblot quantification of NfL protein levels in HC‐ and PSP‐RS MOs at day 120 using OD. Western blot data are presented as mean ± SEM. Significance versus HC MOs was calculated using t‐test, *p ≤ 0.05, with GAPDH as the loading control. HC = healthy control; MOs = midbrain organoids; NfL = neurofilament light chain; OD = optical density; PSP‐RS = progressive supranuclear palsy Richardson's syndrome; TH = tyrosine hydroxylase. [Color figure can be viewed at www.annalsofneurology.org]

FIGURE 4

PSP‐RS MOs accumulated hyperphosphorylated tau protein. (A–D) Quantification of pThr231 (Invitrogen, MN1040) (A), pSer396 (Invitrogen, 44752G) (B), pThr181 (Cell Signaling, 12,885) (C), and AT8 (pSer202/Thr205) (Invitrogen, MN1020) (D) protein levels at different time points (day 60, day 90, and day 120) assessed using immunoblot analysis in HC and PSP‐RS MOs. Expression levels of p‐Tau were quantified using OD measurement. Data are presented as mean ± standard error (SEM). Significance was calculated versus relative HC MOs using the t test, *p ≤ 0.05, **p ≤ 0.01. GAPDH was used as loading control. (E) Representative immunofluorescence images of pThr231, pS396, pThr181, and AT8 labeling in HC and PSP‐RS MOs at day 90. DAPI (blue) was used to stain nuclei. Scale bar 50 μm. (F) Immunoblot analysis of 4R tau isoform in HC and PSP‐RS MOs at day 90 detected with a specific antibody (Cell Signaling, 79327S). (G) Quantification of 4R‐tau Western blot. Data are presented as mean ± standard error (SEM). Significance was calculated versus relative HC MOs using the t test, **p ≤ 0.01. GAPDH was used as loading control. HC = healthy control; MOs = midbrain organoids; OD = optical density; PSP‐RS = progressive supranuclear palsy Richardson's syndrome. [Color figure can be viewed at www.annalsofneurology.org]

FIGURE 5

PSP‐RS MOs accumulate GFAP‐immunoreactivity and exhibit the presence of NFTs and TAs. (A) Immunofluorescence for GFAP in HC and PSP‐RS cryosections at day 90. DAPI (blue) was used for nuclear staining. Scale bar = 50 μm. (B) Immunoblot analysis of GFAP protein expression level in PSP‐RS and HC MOs at day 120 and relative quantification conducted using OD measurements. Data are presented as mean ± standard error (SEM). Significance was calculated versus relative HC MO using the t test, *p ≤ 0.05. GAPDH was used as loading control. (C, D) Immunohistochemistry for S396 and AT8 (Ser202/Ser205) in MO sections of HC confirming the absence of pathological tau aggregates. (E, F) Immunohistochemistry with DAB, counterstained with hematoxylin, for S396 and AT8 in PSP‐RS MOs highlighting the presence of NFTs (left) and TAs (right). Scale bar = 24 μm. (G) Left: Representative immunofluorescence staining for 4R tau reveals dramatic increased expression in mosaic PSP MOs. DNA was counterstained with DAPI (blue). Scale bar = 25 μm. Right: Quantification of mean fluorescence intensity of 4R tau (green channel). A total of 10 images over 3 different biological replicates for both conditions were analyzed using ImageJ software. Data are presented as mean ± SEM and significance was calculated using the t test, ****p ≤ 0.0001. HC = healthy control; MOs = midbrain organoids; NFTs = neurofibrillary tangles; OD = optical density; PSP‐RS = progressive supranuclear palsy Richardson's syndrome; TAs = tufted astrocytes. [Color figure can be viewed at www.annalsofneurology.org]