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. 2019 Dec 10;116(50):25322-25328.
doi: 10.1073/pnas.1906565116. Epub 2019 Nov 25.

DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

Yi Liu  1   2 , Xiaopin Ma  2 , Hisashi Fujioka  3 , Jun Liu  4 , Shengdi Chen  4 , Xiongwei Zhu  5
Affiliations

DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

Yi Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson's disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

Keywords: DJ-1; ER-mitochondria association; IP3R3; Parkinson’s disease; mitochondrial dysfunction.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
DJ-1 ablation reduced ER-mitochondria association and disrupted mitochondrial calcium upload and ATP production. (A) Representative electron micrographs of ER-mitochondria association in control and DJ-1 KO M17 cells. M, mitochondria, ER, endoplasmic reticulum. (Scale bar, 500 nm.) (B and C) Quantitative analysis of percentage of the mitochondrial surface closely apposed to ER (B) and average length (C) of ER-mitochondria association in control (n = 9) and DJ-1 KO M17 cells (n = 6). (D and E) Representative curves of the time scan of mitochondrial Ca2+ (D) and ER Ca2+ (E) after histamine (His) treatment in control and DJ-1 KO M17 cells of 4 independent experiments. (Inset graph) Maximal mitochondrial (D) or ER (E) Ca2+ peak fluorescence. (F) Intracellular ATP concentration in control and DJ-1 KO cells. Data are means ± SD based on 3 independent experiments, Student t test; *P < 0.05; ***P < 0.001.
Fig. 2.
Fig. 2.
DJ-1 is a MAM protein that plays a critical role in ER-mitochondria association. (A–C) Subcellular fractionation and immunoblot characterization of DJ-1 in the MAM fraction in normal M17 cells (A), WT C57BL6 mouse brain (B), and human cortex (C). Homo, total cell lysates or brain homogenates; CANX, calnexin; Sig 1R, Sigma 1R. (D) In vitro ER-mitochondria contact formation assay demonstrated that DJ-1 KO decreased ER binding to mitochondria. Representative immunoblot (Left) and quantitative analysis (Right) of ER bound to mitochondria in the pellets after incubation of purified mitochondria (mito) and microsome (micro) fraction from control or DJ-1 KO M17 cells. Data are means ± SD of 3 independent experiments. One-way ANOVA, *P < 0.05; **P < 0.01.
Fig. 3.
Fig. 3.
DJ-1 is part of the IP3R3-Grp75-VDAC1 complex at the MAM. (A) In situ close association between DJ-1/Grp75 (Top), DJ-1/IP3R3 (Middle), and DJ-1/VDAC1 (Bottom) were determined by PLA in normal M17 cells. (Scale bar, 5 μm.) (B–D) Immunoblot analysis of IP3R3, Grp75, and/or VDAC1 in the DJ-1 immunoprecipitates of total lysates (B) and MAM fraction (C) from normal M17 cells or MAM fraction prepared from C57BL6 mouse brain (D); WL, whole lysate. (E and F) Representative BN-PAGE and immunoblot analysis of the total cell lysates (E) and crude mitochondrial fraction (F) prepared from normal M17 cells. (G) Representative 2D separation and immunoblot analysis of total cell lysates from normal M17 cells. In E–G, green shading highlights the macrocomplex that contained all 4 components. Data are representative of 3 independent experiments.
Fig. 4.
Fig. 4.
DJ-1 ablation disrupted the IP3R3-Grp75-VDAC1 complex. (A and B) Representative images and quantification of PLA signals revealed decreased IP3R3-Grp75 (127 to 200 cells) and IP3R3-VDAC1 (67 to 86 cells) in DJ-1 KO cells. (Scale bar, 5 μm.) (C) Representative immunoblot and quantitative analysis of Grp75 coimmunoprecipitated with IP3R3 antibody in control and DJ-1 KO cells. (D) Representative Blue-native PAGE and immunoblot images and quantitative analysis of the macrocomplex in total cell lysates from control and DJ-1 KO cells. (A–D) Data are means ± SD based on 3 independent experiments, Student t test, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 5.
Fig. 5.
DJ-1 ablation caused IP3R3 accumulation at MAM. (A) Representative immunoblot and quantitative analysis of MAM proteins in total cell lysates and MAM fractions from control and DJ-1 KO M17 cells. Data are normalized to calnexin. WL, whole lysate; CANX, calnexin; Sig 1R, Sigma 1R. (B) Representative immunoblot and quantitative analysis of IP3R3 in the detergent-soluble (S) and -insoluble (I) fractions of total cell lysates from control and DJ-1 KO M17 cells. (C) Representative immunoblot and quantitative analysis of time-dependent degradation of IP3R3 after treatment of CCh in control and DJ-1 KO M17 cells. Data are means ± SD from 3 independent experiments, Student t test; *P < 0.05; **P < 0.01; ****P < 0.0001.
Fig. 6.
Fig. 6.
DJ-1 ablation caused MAM deficits in vivo. (A) Representative images and quantification of IP3R3-VDAC1 PLA signals revealed decreased IP3R3-VDAC1 association in TH+ neurons in the substantia nigra of DJ-1 KO mice. IP3R3-VDAC1 PLA (red) were developed in brain sections and costained with tyrosine hydroxylase (TH, green) (n = 3/group). (Scale bar, 20 μm.) (B) Representative immunoblot and quantitative analysis of Grp75 coimmunoprecipitated with IP3R3 antibody in the brain homogenates of DJ-1 KO and WT control mice (n = 3/group). (C) Representative BN-PAGE/immunoblot and quantitative analysis of the macrocomplex detected by IP3R3 in the crude mitochondria fraction from mouse brain homogenates (n = 4/group). (D) Representative immunoblot and quantitative analysis of MAM proteins, normalized to calnexin, in the brain homogenates (Homo) and MAM fractions from DJ-1 KO mice. CANX, calnexin; Sig 1R, Sigma 1R; β-Tub, β-tubulin. Data are means ± SD, Student t test; *P < 0.05; **P < 0.01; ***P < 0.001.
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