Altered ER-mitochondria contact impacts mitochondria calcium homeostasis and contributes to neurodegeneration in vivo in disease models

Abstract

Calcium (Ca²⁺) homeostasis is essential for neuronal function and survival. Altered Ca²⁺ homeostasis has been consistently observed in neurological diseases. How Ca²⁺ homeostasis is achieved in various cellular compartments of disease-relevant cell types is not well understood. Here we show in Drosophila Parkinson's disease (PD) models that Ca²⁺ transport from the endoplasmic reticulum (ER) to mitochondria through the ER-mitochondria contact site (ERMCS) critically regulates mitochondrial Ca²⁺ (mito-Ca²⁺) homeostasis in dopaminergic (DA) neurons, and that the PD-associated PINK1 protein modulates this process. In PINK1 mutant DA neurons, the ERMCS is strengthened and mito-Ca²⁺ level is elevated, resulting in mitochondrial enlargement and neuronal death. Miro, a well-characterized component of the mitochondrial trafficking machinery, mediates the effects of PINK1 on mito-Ca²⁺ and mitochondrial morphology, apparently in a transport-independent manner. Miro overexpression mimics PINK1 loss-of-function effect, whereas inhibition of Miro or components of the ERMCS, or pharmacological modulation of ERMCS function, rescued PINK1 mutant phenotypes. Mito-Ca²⁺ homeostasis is also altered in the LRRK2-G2019S model of PD and the PAR-1/MARK model of neurodegeneration, and genetic or pharmacological restoration of mito-Ca²⁺ level is beneficial in these models. Our results highlight the importance of mito-Ca²⁺ homeostasis maintained by Miro and the ERMCS to mitochondrial physiology and neuronal integrity. Targeting this mito-Ca²⁺ homeostasis pathway holds promise for a therapeutic strategy for neurodegenerative diseases.

Keywords: ER–mitochondria contact site; Miro; PINK1; Parkinson’s disease; calcium homeostasis.

Fig. 1.

Mito-Ca²⁺ analysis in DA neurons of PD model flies. (A) Mito-GCaMP signals in central brain DA neurons (outlined with dashed line) of third instar larvae grown on normal food or food containing BAPTA or Ca²⁺. Quantification of relative GCaMP signal is shown in bar graph. (B and C) Images of Mito-GCaMP signals and quantification showing elevated mito-Ca²⁺ in PINK1^B9 adult DA neurons. Bar graph shows mito-GCaMP signal normalized with mito-DsRed, which measures mitochondrial mass. WB in C shows mito-GCaMP protein level detected with anti-calmodulin antibody. (D) Rhod2-AM staining of mito-Ca²⁺ in PINK1^B9 adult DA neurons, with mitochondria labeled by mito-GFP. Bar graph shows Rhod2-AM signal normalized with mito-GFP, which measures mitochondrial mass. Arrows and circles mark mitochondrial units with similar mass. * marks Rhod2-AM signal in non-TH⁺ neurons. Error bar: SEM; *P < 0.05, ***P < 0.001 versus control in one-way ANOVA. n = 5. (Scale bars in A, B, and D: 10 μm.)

Fig. 2.

Miro mediates the elevation of mito-Ca²⁺ in PINK1^B9 DA neurons. (A and B) Attenuation of mito-Ca²⁺ increase in PINK1^B9 adult DA neurons by Miro-RNAi. Representative images of mito-GCaMP and quantification of signal intensity are shown in A. Signals were normalized relative to TH-Gal4 > mito-GCaMP control. WB in B shows comparable expression of mito-GCaMP in the genotypes analyzed. (C and D) Representative images of mito-GCaMP and quantification of signal intensity showing attenuation of elevated mito-Ca²⁺ in Miro-OE (C) or PINK1^B9 (D) adult DA neurons after knocking down IP3R, Porin, and MCU. (E and F) Representative images of mito-GCaMP and quantification of signal intensity showing attenuation of mito-Ca²⁺ increase in Miro-OE (E) or PINK1^B9 (F) adult DA neurons after treatment with 2 µM RU360 or 50 µM 2-APB. The DA neurons are outlined with white dashed line. Error bar: SEM; **P < 0.01, ***P < 0.001 versus control in one-way ANOVA. n = 5. (Scale bars in A and C–F: 5 μm.)

Fig. 3.

Mito-Ca²⁺ influx through ERMCS mediates Miro-OE effect on mitochondrial morphology and DA neuron maintenance. (A and B) Immunostaining of the PPL1 cluster of DA neurons of adult animals (A) and data quantification (B) showing the effects of ERMCS component RNAi, Milton-RNAi, or Drp1-OE on DA neuron number in Miro-OE condition. (C and D) Mito-GFP reporter staining in the PPL1 cluster of adult DA neurons (C), and data quantification (D) showing the effects of ERMCS component RNAi, Milton-RNAi, or Drp1-OE on mitochondrial morphology in Miro-OE condition. (E and F) Data quantification showing the effects of Ca²⁺ chelation with BAPTA or EGTA/EDTA, and IP3R inhibition with 2-APB, on DA neuron number (E) and mitochondrial morphology (F) in Miro-OE flies. (G) Representative immunostaining and data quantification showing increased mitochondria–ER contact in Miro-OE DA neurons. Arrows point to areas of contact. Error bar: SEM; *P < 0.05, **P < 0.01, ***P < 0.001 versus control in one-way ANOVA. n = 10. (Scale bars in A, C, and G: 10 μm.)

Fig. 4.

Pathogenic role of mito-Ca²⁺ homeostasis mediated by ERMCS in PINK1 PD model. (A and B) Immunostaining of the PPL1 cluster of DA neurons of adult animals (A) and data quantification (B) showing the effects of ERMCS component RNAi on DA neuron number in PINK1 mutant. (C and D) Mito-GFP reporter staining in the PPL1 cluster of adult DA neurons (C) and data quantification (D) showing the effects of ERMCS component RNAi on mitochondrial enlargement in PINK1 mutant. (E) Representative immunostaining and quantification of mitochondria–ER colocalization in PINK1 mutant DA neurons. Arrows point to areas of contact. Error bar: SEM; *P < 0.05, **P < 0.01, ***P < 0.001 versus control in one-way ANOVA. n = 10. (Scale bars in A, C, and E: 10 μm.)

Fig. 5.

Effect of Miro-regulated ER–mitochondria Ca²⁺ signaling on NMJ synaptic morphogenesis. (A and B) Immunostaining of third instar larvae with anti-HRP showing effects of Polo heterozygosity or Polo-RNAi on NMJ morphology (A) in wild-type or dMiro-OE condition, and quantification of bouton number (B). Green, HRP. N, number of animals analyzed. (C and D) Immunostaining of third instar larvae with anti-HRP showing effects of OE of Miro WT or phosphovariants on NMJ morphology (C) and bouton number (D). (E and F) Effects of RNAi of ERMCS components on NMJ morphology (E) and bouton number (F). (G) Effects of Miro-RNAi and Miro-OE on NMJ bouton number in hLRRK2-G2019S OE condition. Error bar: SEM; *P < 0.05, **P < 0.01, ***P < 0.001 versus control in one-way ANOVA. n.s, nonsignificant. (Magnification: A, 195×; C and E, 280×.)

Fig. 6.

Mito-Ca²⁺ dyshomeostasis in the Drosophila PAR-1 model of neurodegeneration. (A and B) Mito-GCaMP signals in third instar larval eye discs of control and GMR-Gal4 > PAR-1 animals grown on normal food or food containing 50 μM 2-APB. Quantification of relative mito-GCaMP signal intensity from A is shown in bar graph in B. (C and D) Effects of IP3R inhibition with 2-APB (C) or by RNAi (D) on the small eye phenotype caused by PAR-1 OE in the photoreceptors. (Magnification: 20×.) (E and F) Data quantification showing the effects of RNAi (E) or OE (F) of ERMCS components on NMJ bouton number in Mhc-Gal4 > PAR-1 condition. Error bar: SEM; *P < 0.05, **P < 0.01, ***P < 0.001 versus control in one- and two-way ANOVA. (Scale bars: 5 μm.)