Reduced ER-mitochondrial contact sites and mitochondrial Ca2+ flux in PRKN-mutant patient tyrosine hydroxylase reporter iPSC lines

Mutsumi Yokota; Yutaro Yoshino; Mitsuko Hosoi; Ryota Hashimoto; Soichiro Kakuta; Takahiro Shiga; Kei-Ichi Ishikawa; Hideyuki Okano; Nobutaka Hattori; Wado Akamatsu; Masato Koike

doi:10.3389/fcell.2023.1171440

Reduced ER-mitochondrial contact sites and mitochondrial Ca²⁺ flux in PRKN-mutant patient tyrosine hydroxylase reporter iPSC lines

Front Cell Dev Biol. 2023 Sep 8:11:1171440. doi: 10.3389/fcell.2023.1171440. eCollection 2023.

Authors

Mutsumi Yokota^#¹, Yutaro Yoshino^#¹, Mitsuko Hosoi¹, Ryota Hashimoto², Soichiro Kakuta³, Takahiro Shiga⁴, Kei-Ichi Ishikawa^{4

5}, Hideyuki Okano⁶, Nobutaka Hattori^{5

7}, Wado Akamatsu⁴, Masato Koike¹

Affiliations

¹ Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo, Japan.
² Laboratory of Cell Biology, Biomedical Research Core Facilities, Juntendo University Graduate School of Medicine, Tokyo, Japan.
³ Laboratory of Morphology and Image Analysis, Biomedical Research Core Facilities, Juntendo University Graduate School of Medicine, Tokyo, Japan.
⁴ Center for Genomic and Regenerative Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan.
⁵ Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan.
⁶ Department of Physiology, Keio University School of Medicine, Tokyo, Japan.
⁷ Neurodegenerative Disorders Collaborative Laboratory, RIKEN Center for Brain Science, Saitama, Japan.

^# Contributed equally.

Abstract

Endoplasmic reticulum-mitochondrial contact sites (ERMCS) play an important role in mitochondrial dynamics, calcium signaling, and autophagy. Disruption of the ERMCS has been linked to several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, the etiological role of ERMCS in these diseases remains unclear. We previously established tyrosine hydroxylase reporter (TH-GFP) iPSC lines from a PD patient with a PRKN mutation to perform correlative light-electron microscopy (CLEM) analysis and live cell imaging in GFP-expressing dopaminergic neurons. Here, we analyzed ERMCS in GFP-expressing PRKN-mutant dopaminergic neurons from patients using CLEM and a proximity ligation assay (PLA). The PLA showed that the ERMCS were significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control under normal conditions. The reduction of the ERMCS in PRKN-mutant patient dopaminergic neurons was further enhanced by treatment with a mitochondrial uncoupler. In addition, mitochondrial calcium imaging showed that mitochondrial Ca²⁺ flux was significantly reduced in PRKN-mutant patient dopaminergic neurons compared to the control. These results suggest a defect in calcium flux from ER to mitochondria is due to the decreased ERMCS in PRKN-mutant patient dopaminergic neurons. Our study of ERMCS using TH-GFP iPSC lines would contribute to further understanding of the mechanisms of dopaminergic neuron degeneration in patients with PRKN mutations.

Keywords: ER-mitochondrial contact sites; PRKN; dopaminergic neurons; iPSC; tyrosine hydroxylase reporter.

Grants and funding

This work was funded by the Japan Society for the Promotion of Science (Grant-in-Aid for Early-Career Scientists, 18K15466 to MY, and Grants-in-Aid for Scientific Research, 22K07523 to MY and 17K08522, 20K07743, and 23K06323 to MK) and the Ministry of Education, Culture, Sports, Science and Technology of Japan (the Private School Branding Project to MK, WA, and NH, Japan-Supported Programs for the Strategic Research Foundation at Private Universities (S1411007), and the program for women researchers from Juntendo University in 2019–2023, funded by “Initiative for Realizing Diversity in the Research Environment”).