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iPSC modeling of young-onset Parkinson’s disease reveals a molecular signature of disease and novel therapeutic candidates

Abstract

Young-onset Parkinson’s disease (YOPD), defined by onset at <50 years, accounts for approximately 10% of all Parkinson’s disease cases and, while some cases are associated with known genetic mutations, most are not. Here induced pluripotent stem cells were generated from control individuals and from patients with YOPD with no known mutations. Following differentiation into cultures containing dopamine neurons, induced pluripotent stem cells from patients with YOPD showed increased accumulation of soluble α-synuclein protein and phosphorylated protein kinase Cα, as well as reduced abundance of lysosomal membrane proteins such as LAMP1. Testing activators of lysosomal function showed that specific phorbol esters, such as PEP005, reduced α-synuclein and phosphorylated protein kinase Cα levels while increasing LAMP1 abundance. Interestingly, the reduction in α-synuclein occurred through proteasomal degradation. PEP005 delivery to mouse striatum also decreased α-synuclein production in vivo. Induced pluripotent stem cell-derived dopaminergic cultures reveal a signature in patients with YOPD who have no known Parkinson’s disease-related mutations, suggesting that there might be other genetic contributions to this disorder. This signature was normalized by specific phorbol esters, making them promising therapeutic candidates.

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Fig. 1: YOPD-derived iPSCs can be differentiated into mDA neural cultures that accumulate α-synuclein.
Fig. 2: Paired RNA-seq and proteomic analyses from mDA cultures.
Fig. 3: Lysosomal α-synuclein degradation is specifically impaired in YOPD mDA cultures.
Fig. 4: Treatment of YOPD mDA cultures with a PKC agonist reduces intracellular α-synuclein levels.
Fig. 5: YOPD signatures across 10 control individuals and 12 patients with YOPD.
Fig. 6: Confirmation of the effects and mechanism of PEP005.

Data availability

All requests for raw and analyzed data and materials are promptly reviewed by the Cedars-Sinai Board of Governor’s Regenerative Medicine Institute to verify whether the request is subject to any intellectual property or confidentiality obligations. Patient-related data not included in the paper may be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement. All transcriptomic data from this study are available in the GEO repository under GSE120746. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD011326.

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Acknowledgements

We thank S. Svendsen for critical input and editing of the manuscript and T. Pierson for consultation on protein degradation and additional editing of the manuscript. We also thank A. Singleton and D. Hernandez for NeuroX analysis and WGS of patient samples, R. Holewinski for assistance with proteomic analysis and sample acquisition, V. Mattis and H. Park for assisting with striatal dissections, D. Torolina for sectioning and staining of mouse brains and G. Lawless for assistance with ELISA assays. We also thank the Cedars-Sinai Proteomics and Metabolomics Core. The majority of this work was supported by the Joseph Drown Foundation (C.N.S.) and the Board of Governors Regenerative Medicine Institute (C.N.S.) and a National Institutes of Health grant, 5UG3NS105703-02 (C.N.S.). Support for the clinical sample collection and patient information came from the Widjaja Family Foundation (M.T.). Support for the proteomics analysis was from the Advanced Clinical Biosystems Institute (J.E.V.E.).

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Contributions

A.H.L., S.S., N.Y. and C.N.S. designed experiments. A.H.L., S.S., N.Y., V.J.D., V.J.G. and A.N.F. performed experiments. A.H.L., S.S. and C.N.S. wrote the manuscript. S.S. and R.H. performed transcriptomic analysis. P.A. performed all animal work and K.M.R. processed brain tissue. S.S., A.H.L., D.W. and M.G.B. generated YOPD iPSC lines. R.M. performed in silico modeling. M.R.J. performed WGS analysis. Z.S. and N.T.M. performed dopamine release experiments. J.E.V.E. supervised the proteomic data analysis. M.T. provided patient samples and clinical data, and helped supervise the project. C.N.S. supervised the project.

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Correspondence to C. N. Svendsen.

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An intellectual protection patent is pending for diagnostic and drug screening for molecular signatures of early-onset sporadic Parkinson’s disease.

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Extended data

Extended Data Fig. 1 Generation of YOPD iPSCs.

(a) SSEA4 (green) and OCT-4 immunostaining in undifferentiated iPSCs from YOPD patients. (b) Normal karyotypes from YOPD patient iPSCs, 20 metaphase spreads were analyzed for each line.

Extended Data Fig. 2 Additional characterization of mDA cultures.

(a) Expression of dopaminergic neuron genes in day 30 mDA cultures from 6 iPSC lines. 3 biological replicates per line were averaged. Data are normalized to average expression in control lines and presented as 3 CTR vs 3 PD lines. No significant differences were detected using multiple t-tests with Holm-Sidak correction for multiple comparisons. (b) HPLC detection of total dopamine in d30 mDA culture lysates. n=9 CTR n=9 PD not significant p=0.29 two-tailed t-test with Welch’s correction. (c) HPLC detection of released dopamine in aCSF following a 15 min incubation at 37oC. n=9 CTR n=9 not significant p=0.14 two-tailed t-test with Welch’s correction. (d) MEA recording of spontaneous activity from 02iCTR mDA neurons at day 21 of differentiation. (e) MEA recordings of control and YOPD mDA neurons at d30 of differentiation. (f) Average sorted spikes per neuron at d30. Points represent an average of 4 independent wells, n=6 CTR n=6 PD colors indicate iPSC lines. Bar graphs represent mean, error bars represent standard deviation (s.d.).

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Extended Data Fig. 3 Whole-cell patch clamp measurements from control and YOPD mDA cultures.

(a) Capacitance is similar between control and YOPD. (b) No difference in resting membrane potential (RMP) is observed. (c) Voltage-gated sodium current density is decreased in YOPD compared to control. No difference is observed in either the (d) inwards rectifying potassium current density, or (e) delayed rectifier potassium current density. * denotes significance p = 0.018 two tailed t-test with Welch’s correction. Bar graphs represent mean, error bars represent standard deviation (s.d.).

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Extended Data Fig. 4 Western blots of soluble and insoluble lysate fractions.

Western blot of D30 mDA cultures under non-denaturing conditions for α-synuclein and loading control of β-actin. Fractionation experiment was conducted once in 5 independent iPSC lines.

Extended Data Fig. 5 Western blots of YOPD markers in undifferentiated iPSCs.

(a) Western blot of p-PKCα and α-Syn in undifferentiated iPSCs and (b) relative quantification of α-Syn levels (n=4 CTR n=5 PD p=0.87 two-tailed t-test with Welch’s correction); no quantification of p-PKCα was possible in the iPSCs as no bands were detected. Bar graphs represent mean, error bars represent standard deviation (s.d.).

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Extended Data Fig. 6 Paired transcriptomic and proteomic analysis.

Pearson correlation plots of (a) transcriptomic and (b) proteomic data. (c) PCA plot of all detected proteins. (d) PCA plot of matching RNA-Seq transcripts. (e) PCA plot of matching proteins. (f) PCA plots of filtered data with 190iPD line omitted. (g) Matched GSEA terms conducted on 190iPD omitted data set n=9 CTR n=6 PD term significance determined by FDR <0.1.

Extended Data Fig. 7 Testing of additional Proteosomal inhibitors.

(a) Western blot of D30 mDA cultures in the presence of indicated proteasomal inhibitors. Quantification of blots from multiple differentiations (n=4 CTR, n=4 PD) with each point representing a band intensity from a separate differentiation. (b) P53 one-way ANOVA with Tukey multiple comparisons test (F 17.53 DF 20 p=0.0005 CTR Lac p=0.0003 CTR Epox p=0.0009 PD Lac p=0.001 PD Epox). (c) α-Syn one-way ANOVA with Tukey multiple comparisons test (F 1.6 DF 20 p=0.0.21). Bar graphs represent mean, error bars represent standard deviation (s.d.).

Source data

Extended Data Fig. 8 Additional characterization of PEP005 treatment.

(a) Immunocytochemistry showing TH and α-synuclein (α-Syn) in 200iPD d30 mDA cultures with and without PEP005 treatment. Images are representative of 2 additional lines tested. (b) Day 30 mDA neurons treated with PEP005 from multiple YOPD and control lines. (c) Quantification of LC3I/II and α-Syn band intensities relative to untreated cells from the same line. (d) Time-course of PEP005 treatment in YOPD and control mDA neurons. (e) Quantification of α-Syn, p-PKCα, LAMP1, and TH band intensities in YOPD and control mDA neurons in timecourse study. qPCR from paired samples (n=3 wells 02iCTR n=3 wells 190iPD) over PEP005 time-course showing (f) SNCA and (g) TH expression. Bar graphs represent mean, error bars represent standard deviation (s.d.).

Source data

Extended Data Fig. 9 mDA differentiation across multiple lines and clones.

(a) Tyrosine Hydroxylase (TH) production by western blot across 8 control and 12 YOPD patients. TH levels were first normalized to β-actin, then to compare across blots the levels were normalized to the average signal (TH/ β-actin) of each gel. (b) Western blots of TH and α-synuclein (α-Syn) levels in day 30 mDA cultures across 3 unique clonal lines from ED044iCTR and from 192iPD. (c) Quantification of band intensities for TH and α-Syn normalized to β-actin. Bands represent independent biological replicates from 3 separate wells differentiated in the same experiment. *indicates p=0.0002 via one-way ANOVA (F 51.42, DF 8) with Tukey multiple comparisons test compared to other clones of the same line. Bar graphs represent mean, error bars represent standard deviation (s.d.).

Source data

Extended Data Fig. 10 Dose-response and in silico analysis of PEP005 and related molecules.

(a) Structures of Phorbol esters similar to PEP005 tested in mDA cultures. (b) Western blots of α-synuclein (α-Syn) and p-PKCα in response to varying PEP005 doses. (c) Western blots of α-Syn and p-PKCα in response to varying Prostratin (PRO) doses in both YOPD and control mDA cultures. Dose ranging studies were repeated twice. (d) Predictive modeling of PEP005 binding sites on PKCα and similar affinity sites on additional proteins. (e) Three dimensional model of PEP005 binding sites on PKCα, PKCδ, and Ras overlaid to show similarity.

Supplementary information

Supplementary Information

Supplemental Tables 1–5 and example flow cytometry gating.

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Laperle, A.H., Sances, S., Yucer, N. et al. iPSC modeling of young-onset Parkinson’s disease reveals a molecular signature of disease and novel therapeutic candidates. Nat Med 26, 289–299 (2020). https://doi.org/10.1038/s41591-019-0739-1

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