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. 2025 Jan 17;43(1):sxae068.
doi: 10.1093/stmcls/sxae068.

A de novo missense mutation in PPP2R5D alters dopamine pathways and morphology of iPSC-derived midbrain neurons

Jasmine L Carter  1   2   3   4   5 , Julian A N M Halmai  1   2   3   4   5 , Jennifer J Waldo  1   2   3   4   5 , Paula A Vij  1   2   3   4   5 , Maribel Anguiano  6 , Isaac J Villegas  1   2   3   4   5 , Yu Xin Du  1   2   3   4   5 , Jan Nolta  3   4 , Kyle D Fink  1   2   3   4   5
Affiliations

A de novo missense mutation in PPP2R5D alters dopamine pathways and morphology of iPSC-derived midbrain neurons

Jasmine L Carter et al. Stem Cells. .

Abstract

Induced pluripotent stem cell (iPSC) models of neurodevelopmental disorders (NDDs) have promoted an understanding of commonalities and differences within or across patient populations by revealing the underlying molecular and cellular mechanisms contributing to disease pathology. Here, we focus on developing a human model for PPP2R5D-related NDD, called Jordan syndrome, which has been linked to Early-Onset Parkinson's Disease (EOPD). Here we sought to understand the underlying molecular and cellular phenotypes across multiple cell states and neuronal subtypes in order to gain insight into Jordan syndrome pathology. Our work revealed that iPSC-derived midbrain neurons from Jordan syndrome patients display significant differences in dopamine-associated pathways and neuronal architecture. We then evaluated a CRISPR-based approach for editing heterozygous dominant G-to-A mutations at the transcript level in patient-derived neural stem cells. Our findings show that site-directed RNA editing is influenced by sgRNA length and cell type. These studies support the potential for a CRISPR RNA editor system to selectively edit mutant transcripts harboring G-to-A mutations in neural stem cells while providing an alternative editing technology for those suffering from NDDs.

Keywords: EOPD; Jordans Syndrome; NDD; RNA editing and CRISPR/Cas13; disease modeling; iPSC-derived neuron; midbrain neuron; stem cell.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Generation of patient-derived E198K and isogenic iPSC lines. (A) Graphic of E198K fibroblast line reprogrammed to iPSCs to create E198K iPSC line and CRISPR/Cas9 editing of E198K variant to create isogenic line. (B) Sanger sequencing of PPP2R5D exon 5 confirms heterozygous guanosine and adenosine peaks in E198K line (left) and correction in the isogenic line (right). (C) Representative 10× images of isogenic and E198K iPSCs positive for pluripotent markers. Scale bar represents 100 µM. (D) Relative expression of canonical pluripotent genes in isogenic and E198K iPSCs determined by RT-qPCR. *** Significantly different from isogenic iPSCs, 2-way ANOVA P < .05. (E) Relative expression of cMYC in PPP2R5D allele iPSC series determined by RT-qPCR. * Significantly different from healthy PGP iPSCs, n = 3 biological replicates (dot) per line, one-way ANOVA P = .0048. (F) Wild-type and mutant PPP2R5D in isogenic and E198K iPSCs determined by RT-qPCR with allele-specific primers. ***Significantly different from isogenic iPSC, t test P = .0007. **Significantly different from isogenic PPP2R5D, t test P = .0021. (G) Western blot of PPP2R5D (B56δ) in isogenic and E198K iPSCs with Histone 3 (H3) housekeeping.
Figure 2.
Figure 2.
Neural stem cell marker expression and proliferation is reduced in Jordan syndrome E198K iPSCs induced into a neural fate. (A) Graphic of iPSC induction to a neural stem cell fate. (B) Representative 20× images of isogenic and E198K NSCs positive for neural stem cell markers. Scale bar represents 100 µM. (C) Relative expression of neural cell fate genes in isogenic and E198K iPSCs determined by RT-qPCR. *Significantly different from isogenic NSCs, 2-way ANOVA P < .05. (D) Wild-type and mutant PPP2R5D in isogenic and E198K NSCs determined by RT-qPCR with allele-specific primers. **Significantly different from isogenic PPP2R5D, t test P = .0064. (E) Western blot of PPP2R5D (B56δ) in isogenic and E198K NSCs with Histone 3 (H3) housekeeping. (F) Proliferation analysis on isogenic and E198K NSCs (n = 22 biological replicates (dots) isogenic and n = 20 biological replicates (dots) per line. *Significantly different from isogenic neurons, n = 3 independent experiments, t-test P = .0352.
Figure 3.
Figure 3.
E198K midbrain neurons are more abundant and complex than isogenic midbrain neurons. (A) Graphic of timeline to create midbrain NSCs and midbrain neurons. (B-D) Representative 20× images of isogenic and E198K neurons positive for neuronal markers and midbrain markers. Scale bar represents 100 µM. (E) Neuronal morphology analysis on isogenic and E198K neurons at day 13 (n = 36 biological replicates (dots) per line over 3 independent experiments. ****Significantly different from isogenic midbrain neurons, n = 3 independent experiments, t-test P < .05. (F) Western blot of PPP2R5D in midbrain neurons.
Figure 4.
Figure 4.
Transcriptome-wide changes across disease context and cell state. (A) Volcano plot of FDR adjusted P value vs fold change for differential DESeq2 expression analysis between the isogenic and E198K in iPSC, NSC, midbrain NSCs (MNSC), and midbrain neurons (MN). Differentially expressed genes are highlighted in blue (downregulated) or red (upregulated) based on FDR < 1%, log fold change > 0.5. (B-C) Venn diagram showing the overlap of differentially expressed genes between the iPSC, NSC, MNSC, and MN cell states. (D) Top biological processes upregulated or downregulated in isogenic or E198K iPSCs, NSCs, mNSC, or MN based on FDR adjusted P values are shown (E) STRING analysis of genes associated with the regulation of dopamine secretion in MN.
Figure 5.
Figure 5.
Markers of cell state expression and PP2A Components. (A-B) Heatmap of genes associated with cell state and the PP2A heterotrimeric complex. (C-H) Relative expression based on the number of reads per gene normalized to actin of (C) TH **** significantly different from isogenic midbrain neurons, one-way ANOVA P < .0001. (D) PPP2R1A **** Significantly different from isogenic NSCs, one-way ANOVA P < .0001 and different from isogenic midbrain neuron P = .0035. (E) PPP2R1B **significantly different from isogenic iPSC, one-way ANOVA P = .0038 and different from isogenic midbrain neuron P < .001. (F) PPP2CA *significantly different from isogenic NSCs, one-way ANOVA P < .0110. (G) PPP2CB. (H) PPP2R5D **significantly different from isogenic NSCs, one-way ANOVA P < .0001 and different from isogenic midbrain NSC P = .0023.
Figure 6.
Figure 6.
50nt PspdCas13b sgRNA with Tiled A-C Mismatch Do Not Edit the Endogenous E198K site in patient-derived neural stem cells. (A) Amplicon sequencing results of the relative read number of wild-type (GAA) and E198K (AAA) reads in E198K NSCs transfected with sgRNA and dPspCas13b--ADAR2DD(E488Q/T375G)-delta-984-1090 plasmid construct. (B) Amplicon sequencing results of E198K NSCs transfected with PPIB sgRNA and dPspCas13b--ADAR2DD(E488Q/T375G)-delta-984-1090 plasmid construct. Percent editing was determined as the number of edited transcript reads over not edited transcripts in the treated group and normalized to not-treated E198K NSCs. *significantly different from not treated E198K NSCs, t-test P < .05. (C-F) Amplicon sequencing results of percent editing of the E198K site in E198K NSCs following screen with sgRNA of increasing length and PspdCas13b-ADAR2DD constructs. Percent editing was determined as the number of edited transcript reads over not edited transcripts in treated group and normalized to not-treated E198K. * ** and **** significantly different from respective construct with no gRNA control, one-way ANOVA P < .05. (G—J) Amplicon sequencing results of percent editing of the E198K site in E198K NSCS following screen with sgRNA 190 and variable A-C mismatch position and PspdCas13b-ADAR2DD constructs. Percent editing was determined as the number of edited transcript reads over not edited transcripts in treated group and normalized to not-treated E198K. * ** and **** significantly different from respective construct with no gRNA control, one-way ANOVA P < .05.
Figure 7.
Figure 7.
Top DEGs are not rescued by PspdCas13b-ADAR2DD constructs and sgRNA 190. Relative expression of the 3 top DEGs between the isogenic and E198K NSC cell states in isogenic, not-treated E198K NSCs, E198K NSCs treated with PspdCas13b-ADAR2DD(E488Q), PspdCas13b-ADAR2DD(E488Q)-delta-984-1090, PspdCas13b-ADAR2DD(E488Q/T375G), or PspdCas13b-ADAR2DD(E488Q/T375G)-delta-984-1090. (A) RWDD28 relative expression determined by RT-qPCR. * Significantly different from total isogenic, one-way ANOVA P < .05. (B) ZNF717 relative expression determined by RT-qPCR. * Significantly different from total isogenic, one-way ANOVA P < .05. (C) CTSF relative expression determined by RT-qPCR. * Significantly different from total isogenic, one-way ANOVA P < .05.
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