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. 2024 Jun 7;10(23):eadj4735.
doi: 10.1126/sciadv.adj4735. Epub 2024 Jun 5.

Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2

Mohammed R Shaker  1   2 , Andrii Slonchak  3 , Bahaa Al-Mhanawi  1 , Sean D Morrison  1 , Julian D J Sng  3 , Justin Cooper-White  1   2   4 , Alexander A Khromykh  3   5 , Ernst J Wolvetang  1   2
Affiliations

Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2

Mohammed R Shaker et al. Sci Adv. .

Abstract

Why individuals with Down syndrome (DS) are more susceptible to SARS-CoV-2-induced neuropathology remains elusive. Choroid plexus (ChP) plays critical roles in barrier function and immune response modulation and expresses the ACE2 receptor and the chromosome 21-encoded TMPRSS2 protease, suggesting its substantial role in establishing SARS-CoV-2 infection in the brain. To explore this, we established brain organoids from DS and isogenic euploid iPSC that consist of a core of functional cortical neurons surrounded by a functional ChP-like epithelium (ChPCOs). DS-ChPCOs recapitulated abnormal DS cortical development and revealed defects in ciliogenesis and epithelial cell polarity in ChP-like epithelium. We then demonstrated that the ChP-like epithelium facilitates infection and replication of SARS-CoV-2 in cortical neurons and that this is increased in DS. Inhibiting TMPRSS2 and furin activity reduced viral replication in DS-ChPCOs to euploid levels. This model enables dissection of the role of ChP in neurotropic virus infection and euploid forebrain development and permits screening of therapeutics for SARS-CoV-2-induced neuropathogenesis.

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Figures

Fig. 1.
Fig. 1.. Generation of human self-organizing multiple ChPCOs in 3D from neuroectoderm.
(A) Schematic representation of the strategy used to generate ChPCOs from hPSCs. Below brightfield images showing the developmental stages of ChPCO overtime in vitro. Scale bar, 500 μm. (B) Graph showing ChPCO growth in vitro. Data are presented as means ± SD. N = 13; total organoids N = 156 summarized in table S8. (C) qRT-PCR of MSX1/2, AQP1, TTR, and KLOTHO. Values were normalized to GAPDH levels and expressed relative to day 7 values. Data are means ± SD; n = 4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 via one-way analysis of variance (ANOVA). (D) Immunostaining of section organoids showing the protein expression of PAX6 (green), LMX1A (red), MSX1/2 (green), LMX1A (red), TTR (green), and LMX1A (red) and counterstained with Hoechst 33342 (blue). Scale bar, 72 μm. (E) Whole-mount immunostaining of ChPCO on day 42 of differentiation. Left images showing the multiple ChP-like epithelia stained with KLOTHO (red) and ZO1 (cyan), Scale bar, 110 μm. Right images are 100× magnification of a single ChP-like epithelium stained with ZO1 (cyan), KLOTHO (red), and TTR (green), Scale bar, 30 μm. (F) Graph representing the average z score per column (pool of three replicates), showing distribution of genes for LVChP and 4VChP obtained from bulk RNA-seq. Data are presented as means ± SD. *P < 0.05, **P < 0.01 via Student’s t test. (G) Box blot showing distribution of marker genes for LVChP obtained from bulk RNA-seq of ChPCOs and COs. Data are presented as minimum to maximum. ****P < 0.0001 via one-way ANOVA. See fig. S2E for individual genes used. (H) Heatmap comparing the Spearman correlation coefficient of the bulk RNA-seq of ChPCOs and COs to adult human ChP obtained from 44- to 70-year-old donors (104).
Fig. 2.
Fig. 2.. Development and functional analysis of ChP-like epithelium in ChPCOs.
(A) Schematic diagram outlining the apicobasal polarity of ChP. (B) Box blot showing distribution of polarity genes [listed in (C) and fig. S3A] obtained from bulk RNA-seq. Data are minimum to maximum. ***P < 0.001, ****P < 0.0001 via one-way ANOVA. (C) Heatmap of representative apicobasal polarity genes within bulk RNA-seq. Values are shown as z score. (D) qRT-PCR of apicobasal polarity genes in ChPCOs. All values were normalized to GAPDH and expressed relative to day 7 values. Data are means ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001 via one-way ANOVA. N = 3. (E) TEM of ChPCOs on day 28 showing the high density of mitochondria, tight junction at the apical side, microvilli (light brown), and nucleus (light purple). Scale bar, 250 nm. (F) ChPCO sections stained with LAMININ (red) and protein ZO1 (green) and counterstained with Hoechst 33342. Scale bar, 20 μm. Dotted white line represents the average intensity of LAMININ and ZO1 expression along the apicobasal of ChP-like epithelium plotted in the graph. (G) Box blot showing distribution of CSF secretome genes [listed in (H)] obtained from bulk RNA-seq. Data are presented as minimum to maximum. ****P < 0.0001 via one-way ANOVA. (H) Heatmap of CSF secretome genes obtained from bulk RNA-seq. Values are z score. Red asterisks represent the genes selected for ELISA experiment shown in (J). (I) ChPCO sections immunostained with water channel aquaporin 1 (AQP1) in red and TTR in green in ChP-like epithelium and counterstained with Hoechst 33342. Scale bar, 20 μm. (J) Luminex multiplex/ELISA showing CSF secretome protein markers in medium of ChPCOs. Data are shown as means ± SD; N = 3. *P < 0.05, **P < 0.01, ****P < 0.0001 via one-way ANOVA.
Fig. 3.
Fig. 3.. Development and functional analysis of ciliogenesis in ChP-like epithelium.
(A) Box blot showing distribution of marker genes (listed below in the heatmap) associated with cilia obtained from bulk RNA-seq of ChPCOs on days 28 and 56 and COs on day 56. Data are presented as minimum to maximum, with notches centered on the median. ****P < 0.0001 via one-way ANOVA. Below heatmap expression of marker genes related to cilia. Values are shown as z score. (B) Analysis of immunostained ChPCO sections on day 28 showing ARL13B (red) protein expression in cilia of ChP-like epithelium marked with TTR (green). The section was counterstained with Hoechst 33342. Scale bar, 20 μm; magnified image scale bar = 5 μm. Yellow arrow indicates a multiciliated cell; yellow arrowhead indicates a mono-ciliated cell. (C) Quantification of cilia length in ChPCOs on days 1, 21, 28, and 56 of differentiation. Data are presented as means ± SD; ****P < 0.0001 via one-way ANOVA. Number of independent experiments = 3. Individual dots represent a cilium length. (D) Stacked bar graph showing the percentage of cells with a single cilium and multiple cilia in human ChP-like epithelial cells in ChPCOs on days 7, 21, 28, and 56 of differentiation. Data are presented as means ± SD. Number of independent experiments = 3. (E) Distribution of cilium number in ChPCOs cultured for days 14, 21, 28, and 56. The data are presented as a percentage of ChP cells with a single primary cilium or multicilia. Minimum n = 3 per time point. Total number of experiments = 16; total number of analyzed organoids = 40. Data are presented as means ± SD. Kolmogorov-Smirnov test, *P < 0.05. The number of organoids analyzed and the number of experiments from each time point are summarized in table S8.
Fig. 4.
Fig. 4.. ChPCOs contain cortical neurons with progress maturation over time in culture.
(A) Diagram of the development of the cortical plate layers: VZ is ventricle zone. (B) qRT-PCR of ventricular zone gene PAX6 and cortical neuronal layer genes (TBR1, CTIP2, SATB2, and CUX1/2). All values were normalized to GAPDH and expressed relative to day 7 values. Data are means ± SD; **P < 0.01, ***P < 0.001, ****P < 0.0001 via one-way ANOVA. N = 3. (C) Immunostaining of ChPCO sections showing PAX6 (red), TBR1 (red), CTIP2 (red), SATB2 (red), and CUX1 (red) and counterstained with Hoechst 33342. Scale bars, 10 μm (PAX6), 20 μm (TBR1, CTIP2, and SATB2), and 30 μm (CUX1). (D) Box blot showing distribution of astrocyte genes (listed in fig. S4B) obtained from bulk RNA-seq. Data are presented as minimum to maximum. *P < 0.05 via one-way ANOVA. Below image shows ChPCO section immunostained with GFAP (red) and counterstained with Hoechst 33342. Scale bar, 30 μm. (E) Immunostaining of ChPCO section showing myelinated neurons marked by neurofilament (green) and MBP (red) and counterstained with Hoechst 33342 (blue). Scale bar, 100 μm. Dotted white box indicates zoomed images below. Scale bar, 2 μm. (F) Immunostaining of serial sections of ChPCO showing DCX (gray), SOX2 (red), and TTR (green). Scale bar, 200 μm. Dotted square white box indicates zoomed images with scale bar = 50 μm. (G) Immunostaining of serial sections of ChPCO showing TBR2 (gray) and SOX2 (red). Scale bar, 50 μm. (H) Immunostaining of serial sections of ChPCO showing SATB2 (gray) and SOX2 (red). Scale bar, 200 μm. Dotted square white box indicates zoomed images with scale bar = 50 μm. (I) Schematic diagram outlining the architectural structure of ChPCO with neural compartment.
Fig. 5.
Fig. 5.. Functional analysis of cortical neurons in ChPCOs.
(A) Analysis of immunostaining of ChPCO sections on day 56 showing neurons with post- and presynapses marked by neurofilament (gray), HOMER1 (red), and SYNT1 (green). Scale bar, 10 μm. Dotted rectangular white box indicates zoomed images of a neuron shown below with scale bar = 1 μm. Doted square white box indicates zoomed images of connected pre- and post-synapse with scale bar = 1 μm. (B) Schematics of extracellular recordings from ChPCOs on MEA. Below are the representative transient plots from neural activities recorded in ChPCOs on day 56. Scale bar, 100 ms (horizontal) and 100 μV (vertical). (C to F) Bar graphs show the changes in the patterns of neural activity in ChPCOs on days 28 and 56 compared to COs on day 56. Bar graphs represent means ± SD in firing rate (C), mean bursting rate (D), mean burst duration (E), and mean interburst interval (F). *P < 0.05 via one-way ANOVA. Total number of experiments = 3; total number of analyzed organoids = 9. The number of organoids analyzed and the number of experiments from each time point are summarized in table S8. (G) Magnified image of sectioned ChPCOs on day 56 immunostained for glutamatergic (GLUR3, green) and GABAergic (GABA, red) neurons on day 56. All sections were counterstained with Hoechst 33342 (blue). Scale bar, 20 μm; scale bar for magnified image = 10 μm. (H) Bar graphs showing the changes in the patterns of mean firing rate before and after drug treatments. The following drugs were used: 50 μM glutamate, 10 μM NMDAA, and 50 μM bicuculline. *P < 0.05, **P < 0.01, ***P < 0.001 via Student’s t test.
Fig. 6.
Fig. 6.. Transcriptional profile of ChPCO modeling DS.
(A) Representative bright-field images of euploid- and DS-iPSC–derived ChPCOs on day 28. Scale bar, 500 μm. (B) Representative image of euploid-iPSC–derived ChPCOs on day 28 immunostained with LMX1A (red) and TTR (green). The section was counterstained with Hoechst 33342 (blue). Scale bar, 100 μm. (C) Quantification of ChP-like epithelium and cortical tissues compartments in at least three different euploid (EU79) and DS (DS18) hiPSC-derived ChPCOs. Individual dots represent a single organoid. Total number of experiments = 8; total number of analyzed organoids = 29. The number of organoids analyzed and the number of experiments from each time point are summarized in table S8. (D) Volcano plot highlighting DEGs in euploid and DS ChPCOs on day 28. Significant up-regulated genes are shown in red, and down-regulated genes are shown in cyan. Top-most DEGs as well as TMPRSS2, FURIN, and NRP1 genes are labeled. (E) Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in DS ChPCOs compared to euploid organoids. Z scores indicate the cumulative increase or decrease in expression of the genes associated with each term. Size of the bubbles is proportional to the number of DEGs associated with respective GO term. BP, biological processes; CC, cellular components; MF, molecular functions.
Fig. 7.
Fig. 7.. Analysis of ciliogenesis and apicobasal polarity in ChPCO derived from DS.
(A) Box blot showing distribution of cilia genes (listed in heatmap) obtained from bulk RNA-seq of ChPCOs on day31. Data minimum to maximum. ****P < 0.0001 via one-way ANOVA. Below heatmap of cilia genes, values are shown as z score. (B) Immunostaining of day 56 ChPCOs with ARL13B (red) and TTR (green) and counterstained with Hoechst 33342. Scale bar, 20 μm. Dotted white boxes denote areas that are magnified. (C) Stacked bar graph demonstrating the percentage of cells with a single cilium and multiple cilia in ChP-like epithelial cells in ChPCOs. Data are means ± SD. N = 3. *P < 0.05; **P < 0.01 via one-way ANOVA. Total number of experiments = 13; total number of analyzed organoids = 35 as summarized in table S8. (D) Distribution of cilia number in ChPCOs. Total number of experiments = 14; total number of analyzed organoids = 29 summarized in table S8. Data are presented as means ± SD. Kolmogorov-Smirnov test, *P < 0.05. (E) Immunostaining of day 56 ChPCO sections with β-catenin (red) and ZO1 (green) and TTR (magenta) and counterstained with Hoechst 33342 (blue). Scale bar, 25 μm. Dotted white boxes denote areas that are magnified. White arrows indicate ZO1 distribution at basolateral domain. Scale bar, 10 μm. (F) Immunostaining of day 56 ChPCOs with E-CADHERIN (red) and ZO1 (green) and counterstained with Hoechst 33342 (blue). Scale bar, 25 μm. Dotted white boxes denote areas that are magnified. Scale bar, 10 μm. White arrows indicate ZO1 distribution at basolateral domain. Yellow arrow indicates E-CADHERIN enrichment at the apical domain. (G) Graph showing the percentage of mislocalized ZO1 in ChP-like epithelium of day 56 ChPCOs. Data are presented as means ± SD. ****P < 0.0001 via Student’s t test. N = 3.
Fig. 8.
Fig. 8.. SARS-CoV-2 productively infects ChPCO but not CO organoids.
(A) Abundance of ACE2-, FURIN-, and TMPRSS2-derived RNA-seq reads in day 31 ChPCOs. Data are means ± SD. *P < 0.05, ****P < 0.0001 via Student’s t test. N = 3. (B) Western blots showing the levels of ACE2, FURIN, TMPRSS2, and ACTIN in day 31 ChPCOs. (C) Section of euploid ChPCOs stained with TMPRSS2 (red), FURIN (red), and TTR (green) and counterstained with Hoechst 33342. Scale bar, 20 μm. (D) Schematic diagram of the SARS-CoV-2 infection protocol. (E) Graphs showing SARS-CoV-2 nucleocapsid and RdRp in day 31 organoids. Data are means ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001 via two-way ANOVA. N = 3. (F) Viral titers from ChPCO supernatants after SARS-CoV-2 (106 FFUs) treatment. Data are means ± SEM. N = 3; *P < 0.05, **P < 0.01 via Student’s t test. (G) ChPCO sections stained with TTR (green) and SARS-CoV-2 spike (red) after SARS-CoV-2 (106 FFUs) or mock (SARS-CoV-2 at 104 FFUs) and counterstained with Hoechst 33342. Scale bar, 60 μm. (H) Graph showing the SARS-CoV-2–positive cells in ChPCOs. Data are means ± SEM. *P < 0.05 via Student’s t test. Fifteen ChPCO sections from n = 3. (I) Graph showing the percentages of SARS-CoV-2–positive cells within ChPCOs. Data are means ± SEM. *P < 0.05, ***P < 0.001 via Student’s t test. Fifteen ChPCO sections from n = 3. (J) ChPCO sections stained with TTR (green), SARS-CoV-2 spike (red), and NEUN (gray). Scale bar, 100 μm. Orthogonal projections are confocal z-stacks stained with MAP2 (green), SARS-CoV-2 nucleocapsid (red), and NEUN (gray) and counterstained with Hoechst 33342. Scale bar, 10 μm. Yellow arrows indicate infected neurons. (K) 3D-reconstruction SARS-CoV-2–positive syncytia with maximum Z-plane projection. White numbers mark individual nucleus. Scale bar, 10 μm. (L) Graph showing the percentage of SARS-CoV-2–positive syncytia after 72 hpi. Data are means ± SEM. *P < 0.05 via Student’s t test. Fifteen ChPCO sections from n = 3.
Fig. 9.
Fig. 9.. Transcriptional analysis in euploid and trisomy 21 ChPCOs upon SARS-CoV-2 infection.
(A) Abundance of SARS-CoV-2–derived RNA-seq reads in euploid (EU79) and DS (DS18) ChPCOs. Read counts were normalized to the library sizes. Error bars indicate SD. Statistical analysis was performed by Student’s t test. (B) Distribution of SARS-CoV-2–derived RNA-seq reads across viral genome. Data for DS (DS18) ChPCOs are shown in red, and euploid (EU79) ChPCOs are in blue. (C) Distribution multidimensional scaling analysis of RNA-seq read counts derived from uninfected and SARS-CoV-2–infected human ChPCOs of euploid (EU79) and DS (DS18). (D and E) Volcano plots highlight DEGs in SARS-CoV-2–infected DS (D) and euploid (E) ChPCOs compared to mock. (F) Scatterplot indicating the differences in SARS-CoV-2–induced gene expression changes between DS18 and EU79 ChPCOs. In (D) to (F), the significantly (FDR-adjusted P < 0.05) up- and down-regulated genes with at least twofold change in expression levels are shown in red and cyan, respectively. Most DEGs are labeled. (G) Distribution of the SARS-CoV-2–responsive DEGs identified in (F) across individual chromosomes. (H) GO and (I) KEGG pathway enrichment analysis of DEGs in (F). Z scores indicate the cumulative increase or decrease in expression of the genes associated with each term. Size of the bubbles is proportional to the number of DEGs associated with respective GO term. (J) Expression of the chromosome 21–associated genes identified in (F) in SARS-CoV-2–infected and uninfected DS18 and euploid (EU79) ChPCOs.
Fig. 10.
Fig. 10.. Drug screening of FDA-approved inhibitors against TMPRSS2 activity in ChPCOs.
(A) Diagram of the SARS-CoV-2 infection protocol. Remdesivir was added immediately after infection and for 72 hours during infection. (B) Quantification of viral titers from infected ChPCO supernatants treated with 100 μM TMPRSS2 inhibitors. Data are means ± SEM. *P < 0.05, **P < 0.01 via Student’s t test. N = 20; organoid number = 60 summarized in table S8. (C) Quantification of viral titers from infected ChPCO supernatants treated with 50 μM furin inhibitor and 100 μM remdesivir. Data are means ± SEM. N = 3 summarized in table S8; ****P < 0.0001 via two-way ANOVA. (D) Quantification of viral titers from infected ChPCO supernatants treated with 50 μM furin inhibitor and 100 μM remdesivir. Data are means ± SEM. N = 3 summarized in table S8; **P < 0.01, ***P < 0.0001, ****P < 0.0001 via two-way ANOVA. (E) Graphs showing the SARS-CoV-2 Envelop, nucleocapsid, and RdRp in ChPCOs on day 31. Data are means ± SD. **P < 0.01, ****P < 0.0001 via one-way ANOVA. N = 3 summarized in table S8. (F) Quantification of viral titers from infected ChPCO supernatants treated with 50 μM furin inhibitor, 100 μM remdesivir, and 100 μM nafamostat. Data are presented as means ± SEM. N = 3 summarized in table S8. ***P < 0.0001, ****P < 0.0001 via two-way ANOVA. (G) Quantification of viral titers from ChPCO supernatants treated with 50 μM furin inhibitor, 100 μM remdesivir, and 100 μM nafamostat after SARS-CoV-2 Omicron (106 FFUs) infection. Data are means ± SEM. N = 3 summarized in table S8. ***P < 0.001, ****P < 0.0001 via two-way ANOVA. (H) Graphs showing the SARS-CoV-2 Omicron genes Envelop, nucleocapsid, and RdRp in EU79 and DS18 ChPCOs on day 31. Data are means ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001 via one-way ANOVA. N = 3 summarized in table S8.
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