Distinct microRNA signatures define sporadic PSP-RS and PD in patient-derived midbrain organoids

Abstract

Progressive supranuclear Palsy-Richardson syndrome (PSP-RS) is a rare, rapidly progressive tauopathy often misdiagnosed as Parkinson's disease (PD) due to overlapping clinical features and the lack of reliable molecular biomarkers. To address this need, we generated human midbrain organoids from induced pluripotent stem cells (iPSCs) derived from individuals with sporadic PSP-RS, PD, and healthy controls (HCs), and performed longitudinal small RNA sequencing to profile microRNA (miRNA) signatures. These 3D organoids recapitulated disease-relevant pathologies, including tau hyperphosphorylation in PSP-RS and α-synuclein aggregation in PD. Transcriptomic analysis revealed dynamic, disease-specific miRNA signatures. Notably, miR-5683, miR-873-5p, miR-219b-5p, and miR-219a-2-3p were enriched in PSP-RS, while PD organoids showed increased expression of miR-1-3p and miR-133b. Differential expression analysis identified miR-5683, miR-3085-3p, and miR-124-3p as robust classifiers distinguishing PSP-RS from controls. Our findings support iPSC-derived midbrain organoids as a relevant platform for modeling atypical parkinsonian syndromes and uncovering candidate miRNA biomarkers for early and differential diagnosis.

Keywords: Biological sciences; Cellular neuroscience; Natural sciences; Neuroscience.

Graphical abstract

Figure 1

Generation of midbrain organoids (MOs) (A) Representative brightfield images showing morphological development of midbrain organoids derived from healthy controls (HC), Parkinson’s disease (PD), and progressive supranuclear Palsy-Richardson syndrome (PSP-RS) iPSCs at days 4, 8, and 20. Scale bars, 200 μm. (B) Quantitative RT-PCR analysis of early midbrain progenitor markers at day 20. Data are expressed as fold change relative to pooled undifferentiated iPSCs (mean ± SEM, n = 3). (C and D) Immunofluorescence staining at day 20 confirms midbrain regional identity and neuroepithelial organization, showing co-expression of FOXA2 with LMX1A (C) and ZO-1 (D). Nuclei are counterstained with DAPI (blue). Scale bars: 50 μm (C), 10 μm (D). (E) RT-qPCR analysis of mature midbrain neuronal markers at day 60 demonstrates ongoing MO maturation across all conditions. Data are presented as fold change relative to undifferentiated iPSCs (mean ± SEM, n = 3). (F) Immunofluorescence at day 60 confirms specification of dopaminergic neuronal subtypes, showing co-expression of TH with GIRK2 (A9 lineage, upper panels) and with CALB (A10 lineage, lower panels). Scale bars, 50 μm. (G) Immunostaining for DDC (dopaminergic marker) and NFL (neuronal marker) at day 60 further validates midbrain neuronal identity. Scale bar, 50 μm. Nuclei are counterstained with DAPI (blue).

Figure 2

PD-derived MOs recapitulate hallmark pathological features (A) Western blot analysis at day 90 reveals increased levels of phosphorylated α-synuclein at Ser129 (pS129) in PD midbrain organoids (MOs) compared to healthy controls (HC). GAPDH was used as a loading control. (B) Densitometric quantification of pS129-α-synuclein levels normalized to GAPDH. Data are presented as mean ± SEM (n = 3). p < 0.05, Welch’s t test. (C) Immunoblot showing elevated levels of oligomeric and tetrameric α-synuclein in PD MOs relative to controls. Molecular weights are indicated for α-synuclein multimers. (D) Quantification of oligomeric α-synuclein signal intensity normalized to GAPDH. Data represent mean ± SEM (n = 3). ∗∗p < 0.001, Welch’s t test. (E) Immunofluorescence analysis of PD MO cryosections stained for MAP2 and pS129-α-synuclein reveals intracellular inclusions resembling Lewy body-like structures (white arrowhead, enlarged in right panel). Nuclei were counterstained with DAPI. Scale bars, 50 μm; 10 μm higher magnification. (F) Immunostaining for tyrosine hydroxylase (TH) and quantification of mean fluorescence intensity (MFI) show reduced dopaminergic marker expression in PD MOs compared to HC. Data are presented as mean ± SEM from at least 12 regions of interest (ROIs) across 3 biological replicates. p < 0.05, Welch’s t test. Scale bars, 25 μm.

Figure 3

Differential expression of miRNAs in midbrain organoids across disease conditions and time points (A–C) Volcano plots showing differentially expressed miRNAs at days 60, 90, and 120 for: (A) PSP-RS vs. healthy controls (HC), (B) PD vs. HC, and (C) PSP-RS vs. PD. Upregulated miRNAs (log₂ fold change >1, adjusted p value <0.05) are shown in red; downregulated miRNAs (log₂ fold change < −1, adjusted p value <0.05) are shown in green. (D–F) Venn diagrams representing overlapping differentially expressed miRNAs across all three time points for: (D) PSP-RS vs. HC (n = 69), (E) PD vs. HC (n = 102), and (F) PSP-RS vs. PD (n = 34).

Figure 4

Identification and validation of differentially expressed miRNAs and their predicted target genes (A) Heatmap displaying the expression profiles (TPM: transcripts per million) of selected differentially expressed miRNAs across healthy control (HC), Parkinson’s disease (PD), and PSP-Richardson syndrome (PSP-RS) midbrain organoids at different time points. Unsupervised clustering reveals disease- and condition-specific miRNA expression patterns. (B–D) Temporal expression trends of representative miRNAs in: (B) PSP-RS vs. HC, (C) PD vs. HC, (D) PSP-RS vs. PD. Expression values are shown as log₂ fold change at days 60, 90, and 120. (E–G) RT-qPCR validation of predicted target genes of selected differentially expressed miRNAs: (E) PSP-RS vs. HC, (F) PD vs. HC, (G) PSP-RS vs. PD. Bar graphs represent fold changes in gene expression relative to controls. Data are shown as mean ± SEM (n = 3). Statistical significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; Welch’s t-test.

Figure 5

Gene ontology (GO) enrichment analysis of predicted target genes of differentially expressed miRNAs (A) GO enrichment analysis of target genes for miR-124-3p (left) and miR-5683 (right), showing significantly enriched biological processes in PSP-RS vs. HC and PSP-RS vs. PD comparisons, respectively. (B) GO analysis of predicted targets for miR-133b, miR-199a-5p, miR-10b-5p, and miR-1-3p, differentially expressed in PD vs. HC comparisons, highlighting key pathways related to oxidative stress, neuronal apoptosis, autophagy, and synaptic organization. (C) GO terms enriched among predicted targets of miR-219a-2-3p and miR-219b-5p, which were differentially expressed in PSP-RS vs. PD. Enriched terms include RNA processing, transcription regulation, and protein binding functions. Each dot represents a significantly enriched GO term, with dot size indicating the number of genes (Count) associated with the term and color representing the adjusted p value (log₁₀padj). GeneRatio refers to the proportion of target genes associated with a given GO term.

Figure 6

Mitochondrial dysfunction, oxidative stress, and DNA damage response impairments in PD and PSP-RS midbrain organoids (A) RT-qPCR analysis of oxidative stress response genes (SOD2, GPX1, and NRF2) in PSP-RS and PD organoids compared to healthy controls (HC). (B) Western blot quantification of NRF2 expression in PD and PSP-RS patient-derived samples relative to healthy controls. (C) Expression of genes involved in mitochondrial dynamics (MFN2, FIS1, OPA1, and DRP1) in PSP-RS and PD organoids vs. HC. (D) Western blot quantification of NDUFS1, PGC-1ɑ, and PARKIN expression in PD and PSP-RS patient-derived samples relative to healthy controls. (E) Representative images of TOM20 immunofluorescence and quantification of mitochondrial morphology, showing mitochondrial puncta and interconnected networks expressed as normalized mitochondrial aspect ratio. At least 10 ROI were analyzed for each condition. Scale bar 10 μm (F) Elevated expression of the pro-apoptotic protein BAX and reduced levels of the anti-apoptotic protein BCL-XL in patient-derived samples compared to HC. (G) Representative TUNEL staining images and quantification showing increased apoptosis in PD and PSP-RS organoids vs. HC. Nuclei counterstained with DAPI (blue). Scale bar, 25 μm. Quantification based on manual counting of at least 10,000 cells per condition. All data are presented as mean ± SEM from three biological replicates. Statistical significance: ns = not significant; p < 0.05, p < 0.01, ∗p < 0.001, ∗∗p < 0.0001. Welch’s t test was used unless otherwise specified; Tukey’s one-way ANOVA test was applied for (E) and (G).