Parkinson’s disease-associated PLA2G6 protects IP3R1 protein to control ER-mitochondria tethering and Ca^2+ transfer

Domain: biology
Published: 2026-03-19
Editorial outcome: major_revision

Abstract
Mutations in the phospholipase A2 group VI (PLA2G6) gene have been linked to autosomal recessive
Parkinson's disease (PD), yet the molecular mechanisms remain poorly understood. This study provides
the in vitro and in vivo evidence, specifically in dopaminergic neurons derived from patients with PD,
that PLA2G6 loss-of-function disrupts the mitochondria-associated endoplasmic reticulum (ER) membrane
(MAM), a critical regulator of Ca2+ transfer and energy homeostasis. This study demonstrates that the
PLA2G6 protein localizes to the MAM and physically associates with the IP3R1-GRP75-VDAC1
complex. PLA2G6 deficiency destabilizes this complex, accelerating IP3R1 degradation, which in turn
reduces ER-mitochondria contacts and impairs Ca2+ transfer. Notably, introducing a MAM linker restores
the phenotypes caused by PLA2G6 loss. In iPSCs-derived dopaminergic neurons from patients with PD
harboring PLA2G6 mutations, the structural and functional disruption of the MAM is further confirmed,
underscoring its role in PD pathogenesis. These findings uncover the pivotal function of PLA2G6 within
the MAM and suggest that modulating inter-organelle contacts could be a therapeutic strategy for
correcting PD's ion channel dysfunction and energy imbalances.

Introduction
Parkinson's disease (PD), one of the most prevalent neurodegenerative disorders, is marked by the
progressive loss of dopaminergic neurons in the substantia nigra pars compacta and remains without a
curative treatment1. While most PD cases are sporadic, 5-10% are familial, linked to mutations in the
PARK loci. However, both sporadic and familial forms exhibit similar phenotypes, suggesting potential
shared underlying mechanisms2. A critical cellular feature in PD is the simultaneous dysfunction of the
endoplasmic reticulum (ER) and mitochondria3-6, with mutations in several PD-related genes contributing
to these organelle disturbances7,8. Existing therapies targeting either the ER or mitochondria have shown
limited success, prompting the hypothesis that ER-mitochondria contact sites may play a pivotal role in
PD pathogenesis9.

The mitochondria-associated ER membrane (MAM) is a specialized subdomain of the ER membrane
that is closely juxtaposed (within 30 nm) to the mitochondrial outer membrane10. Several molecular
complexes, such as IP3R-GRP75-VDAC1, ERMIT2-MPFN2, VAPB-PTPIP51, and BAP31-Fis1, are
responsible for tethering the MAM10-14. MAM regulates key cellular processes, including phospholipid
synthesis, Ca2+ transfer, ER stress, mitochondrial quality control, autophagy, and ferroptosis10,15,16.

Genetic studies have identified at least 54 mutations in the phospholipase A2 group VI gene
(PLA2G6, also referred to as PARK14), which encodes calcium-independent phospholipase A2β (iPLA2β,
also known as iPLA2-VIA or PLA2G6), as contributors to PLA2G6-related dystonia-parkinsonism17-19.
Previous studies have shown that PLA2G6 localizes to both the ER and mitochondria, playing a pivotal
role in maintaining neuronal ER and mitochondrial homeostasis20,21. While MAM dysfunction has been
implicated in neurodegenerative diseases involving α-Synuclein, DJ-1, and Parkin-linked PD22-24, whether
PLA2G6 specifically localizes to the MAM and facilitates ER-mitochondria communication has yet to be
determined.

This study identified MAM dysfunction in Pla2g6 knockout (KO) N2a cells, mouse brains, and
dopaminergic neurons derived from patients with PD harboring PLA2G6 mutations, leading to impaired
mitochondrial Ca2+ uptake and ATP production. This dysfunction arises from the disruption of the IP3R1-
GRP75-VDAC1 complex and accelerated IP3R1 degradation due to PLA2G6 deficiency. These results
reveal a molecular mechanism contributing to PLA2G6-linked PD pathology.

Results
PLA2G6 protein is localized in the MAM
To explore PLA2G6's potential role in ER-mitochondria contacts, we first determined whether
PLA2G6 is present in the MAM. Subcellular organelles, including mitochondria, ER, and MAM, were
isolated from N2a cells, mouse ventral midbrains, and human brain tissues using Percoll-based
ultracentrifugation. The identity and purity of these fractions were confirmed through immunoblotting
with organelle-specific markers: Sigma1R and calreticulin for ER/MAM, VDAC1 and GRP75 for
mitochondria/MAM, and COX IV for mitochondria. Endogenous PLA2G6 is localized in the MAM
fraction (Fig. 1a–c). In addition, as previously reported, the PLA2G6 protein was also partially detected
in the mitochondria and ER fractions20. Further validation of PLA2G6 localization was achieved through
immunofluorescence using highly intelligent and sensitive structured illumination microscopy (HIS-SIM),
revealing that some puncta-like staining of PLA2G6 (magenta) protein was specifically at ER (green)-
mitochondria (blue) contact points (Fig. 1d, e; Supplementary Movie 1). Additionally, immuno-electron
microscopy in N2a cells confirmed PLA2G6's presence at ER-mitochondria junctions (Fig. 1f).
Collectively, these complementary experiments provide robust evidence of PLA2G6's localization within
the MAM.

The ER-mitochondria contacts were decreased after PLA2G6 absence
Given PLA2G6's localization in the MAM, it was hypothesized that the protein may contribute to
preserving MAM integrity and function. HIS-SIM analysis revealed that colocalization of the
mitochondria (green) and ER (magenta) was reduced in PLA2G6 KO U2OS cells compared to control
cells (Fig. 2a, b, and Supplementary Fig. 1a). To further corroborate this finding, quantitative transmission
electron microscopy (TEM) was employed to assess the MAM length of individual MAM (defined as the
proximity between ER and mitochondrial membranes within 30 nm) and the ratio of MAM length to
mitochondrial perimeter. In control N2a cells, the average MAM length was 189.9 ± 8.9 nm, which
decreased to 145.2 ± 6.6 nm in Pla2g6 KO cells (Fig. 2c, d, and Supplementary Fig. 1b). Additionally,
the ratio of MAM length to mitochondrial perimeter was reduced from 4.92% ± 0.28% in control cells to
3.35% ± 0.24% in Pla2g6 KO cells (Fig. 2c, e).

To further examine ER-mitochondria contacts at the three-dimensional (3D) level, focus ion beam-
scanning electron microscopy (FIB-SEM) was employed. This approach provided a 3D view of the
interactions between mitochondria (green) and the ER (magenta) (Fig. 2f; Supplementary Movies 2, 3).
In Pla2g6 KO N2a cells, the surface of ER-mitochondria contacts decreased by 38.5% compared to
control cells (Fig. 2g). The MAM surface-to-mitochondrial surface ratio in control cells was 4.96% ±
0.52%, while in Pla2g6 KO cells, this ratio dropped to 1.62% ± 0.37% (Fig. 2h). Since mitochondrial
membrane proteins may influence ER-mitochondria contacts, further investigation into the effects of
PLA2G6 deficiency on mitochondrial membrane protein expression was conducted. Western blot (WB)
analysis revealed no significant changes in the levels of MFN2, TOM40, VADC1, TIM23, or TOM20
between control and Pla2g6 KO N2a cells (Supplementary Fig. 1c, d). Notably, ER-lysosome and
mitochondria-lysosome contacts remained unaffected in PLA2G6 KO cells (Supplementary Fig. 1e–h).
These results demonstrate that PLA2G6 deficiency specifically disrupts ER-mitochondria contacts.

In vivo analysis further corroborated these results (Supplementary Fig. 2a). HIS-SIM assays showed
a reduction in the colocalization of mitochondria and ER in the dopaminergic neurons of Pla2g6 KO mice
compared to wild-type (WT) mice (Supplementary Fig. 2b, c). To specifically examine the role of
PLA2G6 in regulating ER-mitochondria contacts within dopaminergic neurons, we performed tyrosine
hydroxylase (TH) immunolabeling to identify dopaminergic neurons in the substantia nigra, followed by
comparative analysis of ER-mitochondria interaction between WT and KO mice. We revealed that MAM
length in the substantia nigra dopaminergic neurons of WT mice averaged 247.1 ± 9.3 nm, while this was
reduced to 130.7 ± 3.9 nm in Pla2g6 KO mice (Supplementary Fig. 2d, e). Additionally, the MAM length-
to-mitochondrial perimeter ratio decreased from 7.69% ± 0.38% in WT mice to 4.77% ± 0.21% in Pla2g6
KO mice (Supplementary Fig. 2d, f). Collectively, these results indicate that PLA2G6 is essential for
stabilizing ER-mitochondria contacts both in vitro and in vivo.

The PLA2G6 protein is composed of ankyrin repeat (ANK) domains and patatin-like phospholipase
(PNPLA) domain (refer to the UniProt). To investigate which domain is involved in maintaining the
stability of MAM, we generated ANK and PNPLA truncated mutants. Overexpression of WT or PNPLA
truncated mutant (ΔPNPLA) in PLA2G6 KO cells restores the ER-mitochondria contacts, but this
interaction was not rescued by ANK truncated mutant (Supplementary Fig. 3a–e). These results indicate
that the ANK domain of the PLA2G6 protein is essential for MAM formation.

Ca2+ transfer from the ER to mitochondria and mitochondrial ATP production were repressed after
PLA2G6 silencing
MAM is recognized as playing a key role in Ca2+ transfer between the ER and mitochondria25-27.
Given that PLA2G6 deficiency impairs ER-mitochondria contacts, its impact on Ca2+ transfer from the
ER to mitochondria was further explored. N2a cells were transfected with Ca2+-specific fluorescent
indicators targeting the mitochondria (CEPIA4mt) and ER (R-CEPIA1er)28. Stimulation with
extracellular histamine (200 μM), known to generate inositol 1,4,5-trisphosphate (IP3) and activate the
IP3 receptor 1 (IP3R1), facilitated Ca2+ transfer from the ER to mitochondria29. Notably, mitochondrial
Ca2+ import was significantly reduced in Pla2g6 KO N2a cells compared to controls following histamine
stimulation (Fig. 2i, j). Additionally, Ca2+ release from the ER was significantly reduced in Pla2g6 KO
N2a cells (Supplementary Fig. 4a, b). To determine whether the transfer of Ca2+ mediated by polycystic
kidney disease 2 (PKD2) channels or ryanodine receptors within the MAM is influenced by PLA2G6, the
PKD2-specific agonist triptolide30 and the ryanodine receptor agonist caffeine31 were utilized to stimulate
Ca2+ transfer from the ER to mitochondria. Notably, no significant changes in mitochondrial Ca2+ import
were detected following triptolide or caffeine stimulation in both Pla2g6 KO N2a cells and control cells
(Supplementary Fig. 4c–f). These results indicate that PLA2G6 predominantly regulates Ca2+ transfer
from the ER to mitochondria through the IP3R1 channel. Previous studies have indicated that
mitochondrial respiration and ATP production are dependent on mitochondrial Ca2+ levels32-34.
Consequently, the impact of PLA2G6 deficiency on cellular bioenergetics was further examined by
measuring the oxygen consumption rate (OCR). The results revealed that PLA2G6 deficiency led to
decreased basal respiration, ATP production, and maximal respiration in comparison to control cells (Fig.
2k, l).

Since PLA2G6 is also involved in the regulation of α-synuclein (α-Syn) aggregation and lipid
peroxidation19, it is highly questionable whether these phenotypes can be explained solely by the reduction
in Ca2+ import into mitochondria. To address this issue, we used HEK293 cells stably transfected with α-
Syn (α-Syn-HEK293 cells) as reporter cells. Transduction of α-Syn-HEK293 cells with α-Syn preformed
fibrils (PFFs) induced α-Syn aggregation and phosphorylation (pS129). This effect was potentiated by
mitochondrial calcium uniporter (MCU) inhibitors such as ruthenium red (RR) or MCU-i4, which reduced
mitochondrial calcium import and consequently enhanced both α-Syn aggregation and phosphorylation
(Supplementary Fig. 5a, b). A similar phenomenon was found in cultured primary neurons
(Supplementary Fig. 5c–f). To determine the effect of reduced mitochondrial Ca2+ import on lipid
peroxidation, we stained cells using the BODIPY 581/591 C11 probe. The result indicated that reduced
mitochondrial Ca2+ import did not affect the lipid peroxidation (Supplementary Fig. 5g, h).

ER-mitochondria Linker expression rescues MAM and mitochondrial dysfunction in PLA2G6-
deficient cells
To determine whether the reduced mitochondrial Ca2+ import and ATP production in PLA2G6-
deficient cells were indeed caused by disrupted ER-mitochondria associations, the ER-mitochondria
Linker was overexpressed to promote MAM formation35-37. The specificity of the Linker was validated
by confirming its localization at ER-mitochondria contact sites (Supplementary Fig. 6a, b).
Overexpression of the Linker successfully restored ER-mitochondria contacts in Pla2g6 KO N2a cells to
levels observed in control cells (Supplementary Fig. 6c–e). Previous studies have implicated PLA2G6 in
the regulation of mitochondrial morphology and membrane potential21,38, and consistent with these results,
mitochondria in Pla2g6 KO N2a cells appeared enlarged, with reduced membrane potential compared to
controls (Supplementary Fig. 6c, f–h). Notably, the ER-mitochondria Linker partially reversed
mitochondrial enlargement and improved membrane potential in Pla2g6 KO cells by enhancing ER-
mitochondria interactions (Supplementary Fig. 6c, f–h). Moreover, the Linker restored mitochondrial Ca2+
import in Pla2g6 KO N2a cells (Supplementary Fig. 6i, j). Overexpression of the Linker plasmid also
significantly improved basal respiration, ATP production, and maximal respiration in Pla2g6 KO N2a
cells (Supplementary Fig. 6k, l). These results confirm that the disruption of ER-mitochondria integrity
due to PLA2G6 deficiency leads to impaired mitochondrial morphology and function.

PLA2G6 interacts with the IP3R1-GRP75-VDAC1 complex
To further explore the mechanism by which PLA2G6 regulates ER-mitochondria contacts, mass
spectrometric (MS) analysis was conducted to determine whether PLA2G6 directly binds to ER-
mitochondria tethering proteins. The analysis identified GRP75 as a potential PLA2G6-interacting protein
within the MAM (Fig. 3a). This interaction was confirmed through co-immunoprecipitation (co-IP)
experiments, where endogenous PLA2G6 and GRP75 were reciprocally pulled down, demonstrating their
binding (Fig. 3b, c). Since our results of Fig. 1a–c showed that GRP75 coexists with PLA2G6 protein in
the MAM and mitochondria, we isolated mitochondria and analyzed the submitochondrial localization of
PLA2G6 and GRP75 to determine their precise location within mitochondria. When mitochondria remain
intact, only proteins localized to the mitochondrial outer membrane (MOM), such as TOM20, are
accessible to Protease K digestion. However, disrupting the MOM with a hypotonic swelling buffer
exposes proteins of the mitochondrial inner membrane (MIM), like TIM23, allowing their subsequent
degradation by the Protease. Only after complete membrane lysis with Triton X-100, which led to the
digestion of all the mitochondrial proteins by Protease K, including matrix proteins (NDUFA9). Our
results showed that most of the endogenous PLA2G6 is digested by Protease K in mitochondria treated
with hypotonic swelling buffer, but all the endogenous GRP75 is digested by Protease K in mitochondria
treated with Triton X-100 (Supplementary Fig. 7a). Furthermore, we found endogenous PLA2G6 co-
immunoprecipitated with IP3R1 in the MAM fraction (Supplementary Fig. 7b). These results suggest that
most of the PLA2G6 proteins coexist and interact with GRP75 in the MAM, but not in the
submitochondrial compartments.

Given previous findings that GRP75 forms a complex with IP3R1 and VDAC1 in the MAM to
maintain its integrity and function39, this study assessed whether PLA2G6 interacts with the IP3R1-
GRP75-VDAC1 complex. An in situ proximity ligation assay (PLA), which detects protein interactions
within a 40 nm range, was used to evaluate the proximity of PLA2G6 to the components of this tripartite
complex. The assay confirmed colocalization between PLA2G6 and GRP75 (Fig. 3d, middle), as well as
PLA2G6 with both IP3R1 and VDAC1 (Fig. 3d, top and bottom). These interactions were also observed
in human brain samples (Supplementary Fig. 8a). To ensure specificity, control experiments using a single
antibody or Pla2g6 KO models yielded no PLA signals, ruling out non-specific binding (Supplementary
Fig. 8b–h). These results indicate close spatial proximity of PLA2G6 with the IP3R1-GRP75-VDAC1
complex. To further confirm the physical interaction between PLA2G6 and the IP3R1-GRP75-VDAC1
complex, co-IP assays were performed on crude mitochondrial fractions (MAM + mitochondria) both in
vitro and in vivo. Endogenous PLA2G6 co-immunoprecipitated with IP3R1, GRP75, and VDAC1 from
the mitochondrial fractions of N2a cells (Fig. 3e). Similarly, co-IP analysis of crude mitochondrial
fractions obtained from mouse ventral midbrains revealed that endogenous PLA2G6 can co-
immunoprecipitate with IP3R1, GRP75, and VDAC1 proteins (Fig. 3f). Previous studies have reported
that DJ-1 and GSK3β also interacted with IP3R1-GRP75-VDAC1 complex23,31, thus we performed co-IP
experiment to determine whether PLA2G6 physically associated with DJ-1 and GSK3β. We found that
endogenous PLA2G6 also co-immunoprecipitates with DJ-1 and GSK3β, but not with lysosomal protein
LAMP1 (Supplementary Fig. 7c).

To evaluate whether PLA2G6 forms a macromolecular complex with IP3R1, GRP75, and VDAC1,
blue native polyacrylamide gel electrophoresis (BN-PAGE) was conducted using crude mitochondrial
fractions in both in vitro and in vivo conditions. Consistent with previous reports31,39,40, IP3R1, GRP75,
and VDAC1 were detected as part of a high molecular weight complex (Fig. 3g, h). Notably, despite
PLA2G6 existing at multiple molecular weights, a portion of it was also found in the same high molecular
weight complex as IP3R1, GRP75, and VDAC1 (Fig. 3g, h). Furthermore, a two-dimensional (2D) BN-
PAGE analysis of crude mitochondrial fractions from N2a cells confirmed the co-presence of PLA2G6,
IP3R1, GRP75, and VDAC1 in the same macromolecular complex (Fig. 3i). Collectively, these results
demonstrate that PLA2G6, along with IP3R1, GRP75, and VDAC1, forms a functional macromolecular
complex within the MAM under physiological conditions.

PLA2G6 deficiency disrupts the integrity of the IP3R1-GRP75-VDAC1 complex
Given the established association of PLA2G6 with the IP3R1-GRP75-VDAC1 complex in the MAM
and the observed disruption of ER-mitochondria contacts in PLA2G6-deficient cells, further investigation
was conducted to determine if PLA2G6 deficiency destabilizes this complex, thereby impairing ER-
mitochondria interactions. PLA was initially performed in both control and Pla2g6 KO N2a cells to assess
interactions between IP3R1 and VDAC1, as well as IP3R1 and GRP75. Results indicated a significant
reduction in PLA signal dots, corresponding to these interactions, in Pla2g6 KO cells compared to controls
(Fig. 4a–d). Similarly, a decrease in IP3R1-GRP75 and IP3R1-VDAC1 PLA signals was observed in TH-
positive neurons from Pla2g6 KO mice relative to WT mice (Fig. 4e–h). To ensure specificity, control
experiments using IP3R1 KO models yielded no PLA signals, ruling out non-specific binding
(Supplementary Fig. 8i–k). Moreover, co-IP assays demonstrated a diminished co-precipitation of
VDAC1 and GRP75 by IP3R1 in Pla2g6 KO N2a cells compared to control cells (Fig. 4i, j). These results
were mirrored in the ventral midbrains of WT and Pla2g6 KO mice (Fig. 4k, l). Consistent with co-IP
results, BN-PAGE of crude mitochondrial fractions from both N2a cells and ventral midbrain tissues
revealed a reduction in the macromolecular complex recognized by IP3R1 in Pla2g6 KO cells and mice
(Fig. 4m–p). Collectively, these results confirm that Pla2g6 KO disrupts the IP3R1-GRP75-VDAC1
complex, leading to impaired ER-mitochondria contacts.

Expression of WT but not PD-associated PLA2G6 D331Y mutant rescues Pla2g6 KO-induced
MAM deficits
The homozygous c.991G > T (p.D331Y) missense mutation in the PLA2G6 gene has been implicated
in early-onset autosomal recessive PD41-43. To assess whether the PLA2G6 D331Y mutant similarly
disrupts MAM structure and function by impairing the IP3R1-GRP75-VDAC1 complex, CRISPR-
resistant human WT PLA2G6 and the PLA2G6 D331Y mutant were re-expressed in Pla2g6 KO N2a cells
(Supplementary Fig. 9a). Initial co-IP analysis revealed that, despite comparable expression levels of WT
PLA2G6 and the PLA2G6 D331Y mutant in Pla2g6 KO N2a cells, re-expression of human WT PLA2G6
significantly enhanced its interaction with IP3R1 compared to the D331Y mutant (Supplementary Fig. 9b,
c). Furthermore, WT PLA2G6 promoted the physical association of IP3R1 with GRP75 and VDAC1,
whereas the PLA2G6 D331Y mutant failed to do so (Supplementary Fig. 9b, c).

Mitochondrial Ca2+ import and OCR were subsequently evaluated following the re-expression of
WT and D331Y mutant PLA2G6 in Pla2g6 KO N2a cells. Re-expression of WT PLA2G6 successfully
restored mitochondrial Ca2+ import in Pla2g6 KO N2a cells upon histamine stimulation (Supplementary
Fig. 9d, e). Additionally, the reductions in basal respiration, ATP production, and maximal respiration
observed in Pla2g6 KO N2a cells were alleviated by WT PLA2G6 re-expression (Supplementary Fig. 9f,
g), confirming its functional activity in mouse N2a cells. In contrast, the PLA2G6 D331Y mutant was
unable to rescue these deficiencies (Supplementary Fig. 9f, g). Overall, these results demonstrate that the
re-expression of WT PLA2G6, but not the D331Y mutant, mitigates the impairments in mitochondrial
Ca2+ import and ATP production caused by Pla2g6 KO.

PLA2G6 ablation promotes the excessive degradation of IP3R1
To further investigate whether PLA2G6 influences the expression of IP3R1-VDAC1 channels, WB
analysis was performed on whole-cell lysates and MAM fractions from N2a cells and mouse ventral
midbrains. A notable reduction in IP3R1 levels was observed in both whole-cell lysates and MAM
fractions of Pla2g6 KO N2a cells and mouse brains (Fig. 5a–f). However, no significant changes were
detected in the levels of GRP75, VDAC1, and Sigma1R in either the whole-cell lysates or MAM fractions
of Pla2g6 KO cells and mice (Fig. 5a–f). Furthermore, to determine whether the phospholipase activity
of the PLA2G6 protein is essential for IP3R1 stabilization, we re-expressed the WT PLA2G6 and
catalytically impaired PLA2G6 (S519A) in Pla2g6 KO N2a cells and showed no significant difference in
IP3R1 protein expression levels between the WT group and S519A group (Supplementary Fig. 10a, b).
This data suggests that the phospholipase activity of PLA2G6 is not essential for IP3R1 stabilization.
Since previous studies indicated that the catalytic function of PLA2G6 is impaired by mutations
associated with infantile neuroaxonal dystrophy (INAD), but not PD44, we assessed whether PD-
associated mutants (D331Y, R741Q) and INAD-associated mutants (G517C, G638R) impact Ca²⁺ transfer
and IP3R1 levels. Results demonstrated that re-expressing the D331Y mutant in KO cells showed no
significant difference in Ca²⁺ transfer or IP3R1 levels compared to empty vector-transfected KO controls,
with both being lower than KO cells expressing WT PLA2G6. In contrast, re-expression of INAD-
associated mutants (G517C, G638R) or the PD-associated mutant (R741Q) upregulated Ca²⁺ transfer and
IP3R1 levels to levels comparable to those observed in KO cells expressing WT PLA2G6 (Supplementary
Fig. 10c–f). These findings is potentially attributable to mutations of G517C, G638R or R741Q do not
affect the ANK domain. This indicates that mutations at different sites within the PLA2G6 gene may lead
to distinct pathogenic mechanisms.

In addition, to explore whether the regulation between PLA2G6 and IP3R1 proteins is unidirectional
or reciprocal, we constructed stable IP3R1 KO N2a cells and transiently overexpressed IP3R1-GFP in
control cells. Our finding demonstrates that IP3R1 does not regulate PLA2G6 expression (Supplementary
Fig. 10g–j). To elucidate the mechanisms underlying the downregulation of IP3R1 protein, mRNA levels
of IP3R1 were assessed in control and Pla2g6 KO N2a cells using quantitative real-time polymerase chain
reaction (qRT-PCR). No significant differences in IP3R1 mRNA levels were detected (Fig. 5g), suggesting
that the observed reduction in IP3R1 protein is likely due to post-translation regulation.

To investigate whether PLA2G6 deficiency leads to the downregulation of IP3R1 through an
accelerated protein degradation pathway, carbachol (CCh), a widely used activator of IP3R1 that induces
its activation and subsequent degradation, was administered at different time points to both control and
Pla2g6 KO N2a cells to assess the effect on IP3R1 homeostasis45. Immunoblotting analysis showed a
more rapid degradation of IP3R1 in Pla2g6 KO N2a cells compared to controls (Fig. 5h, i),
indicating that PLA2G6 deficiency promotes accelerated IP3R1 degradation. Furthermore, basal IP3R1 protein
levels were lower in Pla2g6 KO cells under normal conditions (Fig. 5a, d, h), highlighting PLA2G6's role
in maintaining IP3R1 stability in resting cells. Therefore, we treated both control and Pla2g6 KO N2a
cells with cycloheximide (CHX), a protein synthesis inhibitor, at different time points to assess the
degradation efficiency of IP3R1 protein under normal conditions. The result demonstrated that Pla2g6
KO led to accelerated degradation of IP3R1 under normal conditions (Supplementary Fig. 10k, l). In
addition, we used MG-132 and 3-MA to block proteasomal and lysosomal degradation pathways,
respectively. The result showed that MG-132, but not 3-MA, restored the degradation of IP3R1 in Pla2g6
KO cells under normal conditions, suggesting that the degradation of IP3R1 due to Pla2g6 KO is through
the proteasomal pathway (Supplementary Fig. 10m, n).

Previous studies have also indicated that activated IP3R1 undergoes degradation via the ubiquitin-
proteasome pathway, facilitated by ER lipid raft-associated protein 1/2 (ERLIN1/2) and the E3 ubiquitin
ligase Ring Finger Protein 170 (RNF170)46-48. To further elucidate the mechanism by which PLA2G6
deficiency enhances IP3R1 degradation, both control and Pla2g6 KO N2a cells were stimulated with CCh
for 20 minutes. A reduction in IP3R1 expression and increased ubiquitination of IP3R1 were observed in
Pla2g6 KO cells (Fig. 5j). Additionally, an enhanced interaction between IP3R1 and ERLIN2 was
detected following PLA2G6 depletion (Fig. 5j). PLA assay was conducted to assess the physical
association between IP3R1 and the ERLIN1/2-RNF170 complex in CCh-treated cells. Increased number
of PLA signals for IP3R1-ERLIN1, IP3R1-ERLIN2, and IP3R1-RNF170 was observed in Pla2g6 KO
cells compared to controls (Fig. 5k, l). Similarly, the number of PLA signals for IP3R1-ERLIN1, IP3R1-
ERLIN2, and IP3R1-RNAF170 also increased in Pla2g6 KO cells compared to controls under basal
conditions (Supplementary Fig. 10o, p), suggesting that PLA2G6 deficiency promotes the binding of the
ERLIN1/2-RNF170 complex to IP3R1 protein, thereby accelerating its degradation..

Mutant PLA2G6 leads to MAM dysfunction in the dopaminergic neurons derived from patients
with PD
Given that loss-of-function mutations in the PLA2G6 gene are linked to autosomal recessive PD41,42
and that PLA2G6 plays a pivotal role in regulating ER-mitochondria contacts, further investigation was
conducted to assess these contacts, as well as mitochondrial Ca2+ import and ATP production, in
dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) of patients with PD. iPSCs
from two patients with PD harboring PLA2G6 mutations and two healthy controls were differentiated into
dopaminergic neurons using established protocols (Supplementary Fig. 11a–c)49,50. Immunostaining of
these neurons with TH, TOM20, and PDI as markers for dopaminergic neurons, mitochondria, and ER,
respectively, followed by colocalization analysis using HIS-SIM, revealed a reduction in ER-
mitochondria contacts in patient-derived neurons compared to controls (Fig. 6a, b). This observation was
corroborated by TEM, which showed a decrease in both the length of individual MAMs and the MAM
length-to-mitochondrial perimeter ratio in neurons from PLA2G6-mutant patients (Fig. 6c–e).
Mitochondrial Ca2+ uptake was assessed using the indicator dye Rhod2, followed by histamine treatment
(200 μM), revealing a significant reduction in Ca2+ import in PLA2G6-mutant neurons compared to
controls (Fig. 6f, g). OCR measurements also indicated lower basal respiration, ATP production, and
maximal respiration in patient-derived neurons relative to healthy controls (Fig. 6h, i). Notably, the
application of the ER-mitochondria Linker successfully restored mitochondrial Ca2+ import, basal
respiration, ATP production, and maximal respiration in PLA2G6-mutant neurons (Supplementary Fig.
12a–d). In addition, we also demonstrated a diminished co-precipitation of PLA2G6, VDAC1, and GRP75
by IP3R1 in PLA2G6-mutant neurons compared to controls (Supplementary Fig. 13a, b). BN-PAGE of
crude mitochondrial fractions from dopaminergic neurons revealed a reduction in the macromolecular
complex recognized by IP3R1 in PLA2G6-mutant neurons compared to controls (Supplementary Fig. 13c,
d). These results closely parallel those observed in Pla2g6 KO N2a cells and mouse brains, suggesting
that loss-of-function mutations in PLA2G6 contribute to PD pathogenesis by disrupting ER-mitochondria
contacts, leading to impaired mitochondrial Ca2+ import and reduced ATP production.

Methods
Plasmids and reagents
The plasmid pCMV CEPIA4mt was obtained from MiaoLingBio (P18478), and pCMV R-CEPIA1er was
sourced from Addgene (#58216). The OMM-mRFP-ER and SOMM-GFP-ER plasmids, generously
provided by Xing-Guo Liu, served as MAM Linkers36,78. The pTagBFP-Mito, pEGFP-Mito, pEGFP-ER,
pDsRed-ER, pDsRed-PLA2G6, LAMP1-pDsRed, and PIP3R1-pEGFP plasmids were purchased from
Youbio Biological Technology. pcDNA-PLA2G6 (human)-3×Flag, pcDNA-D331Y PLA2G6 (human)-
3×Flag, pcDNA-G517C PLA2G6 (human)-3×Flag, pcDNA-G638R PLA2G6 (human)-3×Flag, pcDNA-
R741Q PLA2G6 (human)-3×Flag, pcDNA-S519A PLA2G6 (human)-3×Flag, pcDNA-ΔPNPLA, and
pcDNA-ΔANK plasmids were acquired from WZ Biosciences. Additional reagents used in the study
include histamine (Selleck, S3968), Oligomycin (Sigma, O4876), FCCP (Sigma, C2920), rotenone
(Sigma, R8875), Antimycin A (Sigma, A8674), carbachol (Selleck, S4359), triptolide (Selleck, S3604),
caffeine (Chengdu Must Bio-Technology, A0470), RR (Selleck, E2993), MCU-i4 (Selleck, S9842), CHX
(Selleck, S7418), MG-132 (Selleck, S2619), and 3-MA (Selleck, S2767). The α-Syn PFFs were kindly
gifted by Zhen-Tao Zhang from Renmin Hospital of Wuhan University.

Cell cultures, transfection, and treatments.
Mouse neuroblastoma N2a cells were obtained from the Cell Bank of the Chinese Academy of Sciences
(Beijing, China, catalog number SCSP-5035). U2OS cells were obtained from Procell Life Science &
Technology (Wuhan, China, catalog number CL-0236). The α-Syn-HEK293 cells were donated by Zhen-
Tao Zhang from Renmin Hospital of Wuhan University. These cells were cultured in DMEM (Gibco,
11960044), supplemented with 10% fetal bovine serum (Gibco, 10099141C) and 1%
penicillin/streptomycin (Gibco, 15140122), and maintained at 37°C in a 5% CO₂ environment.
Transfection of N2a, U2OS cells with plasmids was performed using Lipofectamine 3000 (Invitrogen,
L3000075) following the manufacturer's protocol. N2a cells were stimulated with 10 μM carbachol or
100μg/mL CHX for various time intervals.

The α-Syn-HEK293 cells were transduced with PFFs according to previous methods79. Briefly, cells were
seeded in 24-well plates. First, 5 μg of PFFs was diluted in Opti-MEM (Gibco, 11058021) to achieve a
final volume of 100 μL. In a separate tube, 96 μL of Opti-MEM was combined with 4 μL of Lipofectamine
2000 (Invitrogen, 11668030). The two solutions were then mixed and incubated at room temperature for
20 minutes. Finally, the resulting mixture was added to the culture medium. α-Syn-HEK293 cells were
treated with 5 μM RR or 2 μM MCU-i4 to inhibit the MCU.

Primary cortical neurons were prepared from E18 embryonic mice and cultured in Neurobasal media
(Gibco, 21103049) supplemented with B-27 (Gibco, 17504044), glutamax (Gibco, 35050061), and 1%
penicillin/streptomycin (Gibco, 15140122), and maintained at 37°C in a 5% CO₂ environment. Primary
neurons were subjected to 1 μg of PFFs, 5 μM RR, or 2 μM MCU-i4 on days in vitro (DIV) 7 and used
for immunofluorescence or WB experiments on DIV 14.

Dopaminergic neuron differentiation and virus infection
Primary skin fibroblasts were obtained from one 30-year-old male and one 41-year-old female patients
with PLA2G6-mutant PD, as well as two male healthy controls (aged 32 and 35 years). This was done
with written informed consent (approval No. 2015-048). The fibroblasts were cultured in DMEM (Gibco,
11960044) supplemented with 10% fetal bovine serum (Gibco, 10099141C) and 1%
penicillin/streptomycin (Gibco, 15140122) at 37°C in 5% CO . Patient 1 carried compound heterozygous
variants: c.991G > T (p.D331Y) and c.1631T > C (p.M544T). Patient 2 had compound heterozygous
variants: c.991G > T (p.D331Y) and c.1915delG (p.A639Qfs*27). Both patients were previously
reported80. Healthy control fibroblasts were kindly provided by Professor Zhi-Ying Wu from the Second
Affiliated Hospital of Zhejiang University School of Medicine. Genomic DNA was extracted from
fibroblasts, and mutations were validated through Sanger sequencing, with primers listed in
Supplementary Table 2. Informed consent was obtained from all participants.

Fibroblasts were reprogrammed into iPSCs using the CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit
(Invitrogen, A16517). Briefly, fibroblasts were thawed and cultured for 4 days in 24-well plates using a
fibroblast culture medium. Fibroblasts were then transduced with Sendai viral particles expressing SOX2,
c-MYC, OCT4, and KLF4, according to the manufacturer’s instructions. By day 21, iPSC colonies were
ready for transfer, and undifferentiated iPSCs were manually picked and expanded on fresh culture dishes.
The selected iPSCs were passaged every 4 days until no residual Sendai virus remained. The pluripotency
of the iPSCs was confirmed by staining for markers TRA-1-60 (Millipore, MAB4360, 1:100), SOX2
(Invitrogen, 14-9811-82, 1:100), SSEA4 (CST, 4755S, 1:100), and OCT3/4 (Santa Cruz, sc-5279, 1:50).

Dopaminergic neuron differentiation from iPSCs followed previously established protocols49,50. Briefly,
healthy control and PLA2G6-PD iPSCs were dissociated using EDTA (Invitrogen, 15575-020) and seeded
on Matrigel-coated (Corning, 354277) 10 cm dishes. The differentiation protocol included exposure to 10
μM β-mercaptoethanol (Invitrogen, 21985-023) from days 1–7, 200 nM LDN193189 (Selleck, S2618)
from days 1–11, and 10 μM SB431542 (Tocris, 1614) from days 1–7. Cells were also exposed to 100
ng/mL human sonic hedgehog (Peprotech, 100-45) from days 2–9, 2 μM purmorphamine (TargetMol,
T1810) from days 2–9, 100 ng/mL FGF8 (Peprotech, 100-25) from days 2–9, and 1 μM CHIR99021
(Sigma, SML1046) from days 3–14. On day 9, 120 μM was added to eliminate undifferentiated iPSCs.
On day 15, cells were dissociated using Accutase (Gibco, A1110501) and re-plated on 6 cm dishes coated
with Poly-L-ornithine (Sigma, P4957), Fibronectin (Sigma, F0895), and Laminin (Sigma, L2020), using
differentiation medium supplemented with 200 μM ascorbic acid (Sigma, A4403), 20 ng/mL BDNF
(Peprotech, 450-02), 20 ng/mL GDNF (Peprotech, 450-10), 500 μM dbcAMP (Sigma, D0627), and 1
ng/mL TGF-β3 (Peprotech, 100-36E). For the characterization of human dopaminergic neurons, the
differentiated cells were stained with the following specific markers: TH (dopaminergic neuronal marker,
CST, 58844 or Sigma, MAB318, 1:500), MAP2 (neuronal marker, Abcam, ab32454, 1:500), TUJ1
(neuronal marker, Sigma, T8578, 1:500), FOXA2 (ventral midbrain neuronal marker, abnova, H00003170,
1:100), NURR1 (dopaminergic neuronal marker, Proteintech, 10975-2-AP, 1:100), Girk2 (dopaminergic
neuronal marker, Proteintech, 21647-1-AP, 1:100). Dopaminergic neurons were infected with lentivirus
expressing OMM-GFP-ER or an empty vector (WZ Biosciences) at a multiplicity of infection of 20, and
cells were analyzed 72 hours post-infection.

Generation knockout (KO) cell lines
KO cells were generated using the CRISPR-Cas9 gene-editing system. The mouse Pla2g6 guide RNA
sequence (WZ Biosciences): 5’ CTCAAGTGAACGTGTCCGGG 3’, the mouse IP3R1 guide RNA
sequence (IGE Biotechnology): 5’ CCGTCAACTGTAATACAAGC 3’, the human PLA2G6 guide RNA
sequence (IGE Biotechnology): 5’ GAGGGGGCTGGCTCCGTAACG 3’. The sequence was cloned into
the PX459 vector, which carries the pSpCas9. This construct was then transfected into N2a cells using
Lipofectamine 3000 (Invitrogen, L3000075), following the manufacturer’s instructions. After 36 hours,
cells were subjected to puromycin selection for an additional 36 hours. Surviving cells were diluted into
single-cell populations in 96-well plates, and gene knockout was confirmed via immunoblotting.

Animals and animal care
Pla2g6 KO C57BL/6Smoc mice (Cat. No. NM-KO-200413) were obtained from Shanghai Model
Organisms Center, Inc. Mice were housed in a temperature- and humidity-controlled environment, under
a 12-hour light/dark cycle, with access to water and food. Three-month-old Pla2g6 KO male mice and
age-matched WT male littermates were used for experiments. Genotyping was performed using PCR,
following the protocol provided by Shanghai Model Organisms Center, Inc. All experimental procedures
were approved and overseen by the Institutional Ethics Committee of the Second Affiliated Hospital of
Zhejiang University School of Medicine (approval No. 2022-0240).

Human brain samples
Human brain samples were collected from one 54-year-old male and one 70-year-old female patients who
underwent deep brain stimulation. They provided written informed consent. Experiments involving
human brain samples were conducted in accordance with the Declaration of Helsinki and were approved
by the Institutional Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of
Medicine (approval No. 2024-0134).

MAM fractionation
MAM fractions were isolated from N2a cells and mouse ventral midbrains using a previously established
protocol23,81. Cells or tissues were homogenized in lysis buffer (225 mM mannitol, 75 mM sucrose, and
30 mM Tris-Cl, pH 7.4) using a Dounce homogenizer. Lysates were first centrifuged at 600 g for 5 minutes
to remove unbroken cells and nuclei. The resulting supernatant was centrifuged at 7000 g and then at
10,000 g for 10 minutes to separate the crude mitochondrial pellet from the ER-containing supernatant.
To purify the ER, the supernatant was centrifuged at 20,000 g for 30 minutes and then at 100,000 g for 1
hour. The crude mitochondrial pellet was resuspended in mitochondrial suspension buffer (MRB, 250 mM
mannitol, 5 mM HEPES pH 7.4, and 0.5 mM EGTA), and the suspension was layered onto a Percoll
medium (225 mM mannitol, 25 mM HEPES pH 7.4, 1 mM EGTA, and 30% (v/v) Percoll). After
centrifugation at 95,000 g for 30 minutes, the MAM fraction was separated from the upper third of the
centrifuge tube, while the mitochondrial fraction was isolated from the lower third. The mitochondrial
fraction was centrifuged at 6300 g for 10 minutes to obtain a pure mitochondrial pellet. The MAM fraction
was then diluted in MRB and subjected to centrifugation at 6300 g for 10 minutes. Subsequently, the
supernatant was centrifuged at 100,000 g for 1 hour to isolate the MAM pellet.

Submitochondrial localization analysis
N2a cells were harvested from six 15-cm dishes, and then isolated mitochondria. The mitochondrial
pellets were resuspended in homogenization buffer (20 mM HEPES/KOH, pH 7.4, 220 mM mannitol, 70
mM sucrose, 1 mM EDTA, 0.5% BSA) supplemented with 1 mM PMSF and Protease inhibitor cocktail,
then divided for different treatments: two aliquots in homogenization buffer (control), two in hypotonic
buffer (10 mM HEPES/KOH, pH 7.4, 1 mM EDTA), and two in buffer containing 0.5% Triton X-100
(incubated on ice for 10 minutes). One aliquot from each group was treated with 66.7 μg/ml Protease K
on ice for 20 minutes, while the other remained untreated. Samples were precipitated with 300 μl of 30%
(w/v) TCA on ice for 10 minutes, centrifuged at 18,000 g for 10 minutes at 4°C, washed with 100%
ethanol, and dissolved in 100 μl sample buffer (60 mM Tris-HCl, pH 6.8, 7% glycerol, 2% 2-
mercaptoethanol, 2% SDS, 0.02% bromophenol blue). After boiling at 95°C for 10 minutes, 10 μl of each
sample was subjected to immunoblotting.

Immunofluorescent imaging
To determine the subcellular localization of PLA2G6, U2OS cells were seeded on the confocal dish
(cellvis, D35-20-1.5H) and transfected with pDsRed2-Mito, pEGFP-ER, and pTagBFP-PLA2G6
plasmids at 37°C for 24 hours. Super-resolution imaging was conducted using high intelligent and
sensitive structured illumination microscopy (HIS-SIM) with a 100 × 1.5 NA oil immersion objective for
three-dimensional analysis. Image processing is done using Wiener Reconstruction. The specific
parameter information is as follows: defocus elimination 5, notch filter 10, 3D Wiener parameter 0.5. The
3D reconstruction utilized Imaris (Bitplane).

To assess the mitochondria-ER, mitochondria-lysosome, and ER-lysosome colocalization in U2OS cells,
the cells were seeded on the confocal dish (cellvis, D35-20-1.5H) and transfected with fluorescent
plasmids of organelles for 24 hours. Dopaminergic neurons were grown on poly-D-lysine (PDL; Beyotime,
ST508)-coated glass coverslips, followed by fixation, permeabilization, and blocking. Cells were
incubated overnight at 4°C with primary antibodies against TOM20 (CST, 42406S, 1:200), PDI (Abcam,
ab2792, 1:100), and TH (Abcam, ab76442, 1:100). Secondary antibodies conjugated with Alexa Fluor
(Invitrogen) were applied for one hour at room temperature the following day. 3D super-resolution images
were captured by HIS-SIM and processed using Wiener Reconstruction (defocus elimination 5, notch
filter 10, 3D Wiener parameter 0.5). Then we use Imaris software (Bitplane) to segment mitochondria and
ER by Surface Creation and Filament Diameter, respectively. Finally, Pearson's Correlation Coefficient
was used to quantify the co-localization between ER and mitochondria.

For assessing the specificity of the Linker, N2a cells were cultured on glass slides and transfected with
OMM-mRFP-ER, pTagBFP-Mito, and pEGFP-ER plasmids for 24 hours. Super-resolution images were
captured using HIS-SIM.

For assessing the intensity of pS129, the α-Syn-HEK293 or primary neurons were fixed with 4% PFA for
20 min, and then permeabilized with 1% TX-100 for 15 min at room temperature. After washing, cells
were incubated overnight at 4°C with primary antibodies against pS129 (Biolegend, 825701). Secondary
antibodies conjugated with Alexa Fluor (Invitrogen) were applied for one hour at room temperature the
following day. Images were captured by Leica TCS SP8 confocal microscope Leica TCS SP8 confocal
microscopy and analyzed using ImageJ.

Immuno-electron microscopy
Cells and mouse brains were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde for 15 minutes at
room temperature, followed by permeabilization with 0.1% saponin for 40 minutes and blocking with 0.1%
BSA for 1 hour at room temperature. Afterward, cells were incubated overnight at 4°C with primary
antibody PLA2G6 (Proteintech, 22030-1-AP, 1:50) or TH (CST, 58844, 1:100). Following PBS washes,
cells were treated with a FluroNanogold-labeled anti-rabbit secondary antibody for 1 hour at room
temperature. Subsequent fixation was performed with 2.5% glutaraldehyde, followed by silver
enhancement. Ultrathin sections were obtained using an ultramicrotome after 48 hours of polymerization
at 60°C and examined via TEM (Tecnai G2 spirit 120 kV).

WB
Protein extraction from cells and mouse ventral midbrains was carried out using RIPA lysis buffer
(Beyotime, P0013B) supplemented with a protease inhibitor cocktail (MCE, HY-K0010) on ice for 30
minutes. After centrifugation at 18407 g for 10 minutes, the supernatant was collected, and protein
concentration was quantified using the BCA Assay Kit (Invitrogen, 23227). Samples were mixed with
loading buffer and boiled for 8 minutes before loading 20 μg of denatured protein onto 4–20% SDS-PAGE
gels and transferring to PVDF membranes. The membranes were blocked with 5% milk for 1 hour at room
temperature, followed by overnight incubation with primary antibodies at 4°C. The next day, membranes
were treated with horseradish peroxidase-conjugated secondary antibodies. Immunoblotting signals were
detected using a chemiluminescence reagent (Invitrogen, 32106) and quantified via ImageJ. The
antibodies used for immunoblotting included PLA2G6 (Abcam, ab259950, 1:1000), GRP75 (Santa Cruz,
sc-133137, 1:500), VDAC1 (Proteintech, 55259-1-AP, 1:1000), Sigma1R (CST, 61994, 1:1000), COX IV
(ABclonal, A11631, 1:1000), Calreticulin (ABclonal, A20986, 1:1000), IP3R1 (ABclonal, A21471,
1:1000), MFN2 (Abcam, ab124773, 1:1000), TOM40 (ABclonal, A3213, 1:1000), TOM20 (CST, 42406S,
1:1000), TIM23 (Proteintech, 11123-1-AP, 1:1000), ubiquitin (Santa Cruz, sc-8017, 1:500), ERLIN2
(ABclonal, A0781, 1:1000), NDUFA9 (ABclonal, A3196, 1:1000), pS129 (CST, 23706, 1:1000), GSK3β
(ABclonal, A2081, 1:1000), DJ-1 (Proteintech, 68915-6-Ig, 1:1000), GFP (HUABIO, ET1607-31,
1:2000), LAMP1 (Santa Cruz, sc-20011, 1:500) and GAPDH (Proteintech, 60004-1-Ig, 1:5000).

Co-immunoprecipitation (Co-IP)
For Co-IP assays, proteins were extracted from cells and mouse ventral midbrains using 1% NP-40 lysis
buffer (Beyotime, P0013F) supplemented with a protease inhibitor cocktail (MCE, HY-K0010) on ice for
30 minutes. Primary antibodies or IgG were incubated with 25 μL of protein G magnetic beads (Invitrogen,
10004D) at room temperature for 1 hour. The protein lysates were then incubated with the antibody-bound
magnetic beads overnight at 4°C. The beads were washed four times with PBS the following day. Pulled-
down proteins were eluted by boiling in 1× SDS-PAGE sample buffer and analyzed by WB. Antibodies
used for IP included: PLA2G6 (Proteintech, 22030-1-AP, 1 μg), GRP75 (Santa Cruz, sc-133137, 1 μg),
and IP3R1 (Santa Cruz, sc-271197, 1 μg). The precipitated proteins were in-gel digested by adding 1%
trypsin and incubating at 37 °C overnight to peptides.

Mass spectrometry (MS) analysis
For the IP experiment, PLA2G6 antibody (Proteintech, 22030-1-AP, 1 μg) was added to N2a cell lysates,
with IgG serving as a negative control. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
analysis was subsequently conducted by Hangzhou Cosmos Wisdom Biotech Co., Ltd (IP, n = 2; IgG, n
= 2 from 2 biologically independent experiments). The brief experimental protocols are as follows: the
precipitated proteins were in-gel digested with 2 μg trypsin in 50 mM NH4HCO3 at 37 ℃ overnight to
peptides. After digestion, peptides were extracted from the gel piece with 50% acetonitrile/0.1% formic
acid, dried, and resuspended in 0.1% formic acid. The extracted peptides were dried in SpeedVacuum
concentrator (Thermo Scientific) and resuspended in 0.1% formic acid for LC-MS/MS analysis.
Peptides were analyzed by LC-MS/MS using an EASY nLC-1200 system (Thermo Scientific) at a
constant flow rate of 400 nl/min with the following gradient program: 2–7% mobile phase B (0.1% formic
acid in 80% acetonitrile) over 1 min, 7–35% B over 35 min, 35–55% B over 9 min, 55–100% B over 7
min, and held at 100% B for 8 min. Eluates were analyzed using a Q Exactive HF-X mass spectrometer
(Thermo Scientific) with an electrospray voltage of 2.0 kV; full MS scans were acquired at a resolution
of 60,000, the top 20 most abundant precursor ions were fragmented by higher-energy collisional
dissociation with a normalized collision energy of 27, MS/MS scans were acquired at a resolution of
30,000, and dynamic exclusion was set to 20 s.

Raw data were processed using Proteome Discoverer software (v2.4, Thermo Scientific) with database
searches against the Swiss-Prot mouse protein database (release 2022_01). Trypsin/P was used as the
cleavage enzyme with up to 2 missed cleavages; precursor ion mass tolerance was set to 10 ppm and
fragment ion mass tolerance to 0.01 Da. Carbamidomethylation of cysteine was specified as a fixed
modification, while oxidation of methionine and N-terminal acetylation were set as variable modifications.
Only high-confidence peptides were retained for protein identification. Candidate proteins need to fulfill
the following conditions: unique peptides ≥ 2, IP abundances/ IgG abundances ≥ 10. Candidate proteins
have all been listed in the Source Data, and PLA2G6, GRP75, and VDAC1 have been marked in red.

Proximity ligation assay (PLA)
PLA was performed using Duolink® reagents (Sigma, DUO92008) to assess proteins located within < 40
nm proximity. Briefly, cells or ventral midbrain sections were fixed, permeabilized, blocked, and
incubated overnight at 4°C with paired primary antibodies (Supplementary Table 1). After three washes,
cells and sections were treated with oligonucleotide-conjugated secondary antibodies (anti-rabbit PLUS
and anti-mouse MINUS). Ventral midbrain sections were incubated with the TH primary antibody (Abcam,
ab76442, 1:400) following oligonucleotide-conjugated secondary antibody treatment. On Day 3, the
sections were incubated with the corresponding secondary antibody (Invitrogen). A negative control was
included for all PLA experiments. Images were captured using a Leica TCS SP8 confocal microscope,
and PLA signal dots were quantified via ImageJ.

Blue native-polyacrylamide gel electrophoresis (BN-PAGE)
Crude mitochondria were isolated from cells and ventral midbrains and lysed in 1% NP-40 on ice for 30
minutes. Supernatants were collected following centrifugation at 18407 g for 10 minutes, and protein
concentrations were determined with the BCA Assay Kit (Invitrogen, 23227). For the first dimension, 30
μg of protein mixed with 0.25% G-250 was loaded onto a 3–12% Bis-Tris gel (Invitrogen, BN1001BOX)
and transferred to PVDF membranes. The gel was excised for the second dimension, reduced with 1 ×
NuPage reducing agent in 1 × NuPage LDS sample buffer for 15 minutes, and cysteine alkylation was
performed with N,N-Dimethylacrylamide for 15 minutes at room temperature. The reaction was quenched
with 20% ethanol in 1 × LDS and 0.1 × reducing agent for 15 minutes, after which the gel strip was
transferred onto a 4–20% Tris-Glycine gel (Invitrogen, EC6026BOX) for immunoblotting analysis.

TEM
N2a cells and dopaminergic neurons were fixed overnight at 4°C in 2.5% glutaraldehyde, followed by
refixation with 1% osmium tetroxide for 1 hour, staining with 2% uranyl acetate for 30 minutes, and
ethanol-gradient dehydration. Samples were embedded in 100% acetone for two hours and sectioned into
90 nm ultrathin slices. Sections were subsequently stained with 4% uranyl acetate and 1% lead citrate,
and the ultrastructure of N2a cells was analyzed via TEM (Tecnai G2 Spirit 120 kV). The term "MAM
length" refers to the measurement of the outer mitochondrial membrane specifically at the region where
it contacts the ER membrane. And the criteria of ER-mitochondria contacts are defined as: the parallel
membrane alignment ≤ 30 nm. The term "ratio of MAM length to mitochondrial perimeter" refers to the
ratio of the total length of all MAMs to the sum of all mitochondrial perimeters in a single cell. To quantify
the MAM lengths and mitochondrial perimeters, we first use Image J to convert the pixels of the image
into lengths according to the scale bar. Then use the Image J manual tracing of the mitochondrial outer
membrane to measure the length of MAM and the perimeter of the mitochondria. Measurements were
carried out by investigators blinded to the experimental conditions.

FIB-SEM
N2a cells were fixed in 2.5% glutaraldehyde for 24 hours, followed by washing with 0.1 M phosphate
buffer. Cells were then scraped and collected in 0.2% BSA/0.1 M phosphate buffer, and centrifuged at
845 g. Post-fixation was performed with 2% OsO4 and 3% potassium ferricyanide in 0.1 M phosphate
buffer for 1 hour on ice. After rinsing with distilled water, cells were incubated in 1% thiocarbohydrazide
for 20 minutes, followed by 30-minute incubation with 1% aqueous uranyl acetate at room temperature.
Samples were rinsed with distilled water, dehydrated in a graded ethanol series, and embedded in EPON
812 resin.

Resin blocks containing leaf tissue were meticulously trimmed using a trimmer. These blocks were
mounted on a 90° stub and coated with platinum for 300 seconds using an ion-sputtering apparatus. To
target specific areas, SEM equipped with an ultramicrotome was employed, facilitating simultaneous
trimming of the resin blocks and imaging of the samples. Once the target area was identified, resin blocks
were transferred to a 45° pre-tilted SEM stub and coated with platinum for 300 seconds. Imaging was
performed with a dual beam SEM (FIB Helios G3 UC) in serial surface view mode with a 10 nm slice
thickness at 30 kV and 0.79 nA. Each surface was imaged with a 2 kV acceleration voltage and 0.2 nA
current in backscatter electron mode using an in-column backscatter electron detector (ICD). During
milling, the sample stage was tilted at 7°, and for imaging, the tilt angle was adjusted to 45°. Images were
captured at a resolution of 4096 × 3536 pixels with a dwell time of 2 μs per pixel.

The term "MAM surface" specifically denotes the region of the outer mitochondrial membrane that
directly contacts the ER membrane. And the criteria of ER-mitochondria contacts are defined as: the
parallel membrane alignment ≤ 30 nm. The term "MAM surface-to-mitochondrial surface ratio" refers to
the proportion between the total contact surface of a single mitochondrial outer membrane with the ER
membrane and the surface of that mitochondrion. The FIB-SEM slices stack is loaded by the Amira
software. Then we run the Align Slices tool to correct the spatial misalignment between consecutive slices
and run the Box Filter to reduce the noise of images. We segmented images by manual tracing of the
mitochondrial outer membrane and ER membrane to generate surface reconstructions. To quantify the
ER-mitochondria contacts, we first convert length (nm) to pixels according to voxel size (2.4847 × 2.4847
× 10 nm) and use the Arithmetic module to create a mask of overlapping proximity regions. Finally, we
use Material Statistics to quantify the surface area of ER-mitochondria contacts (MAM surface) and the
mitochondria surface. Measurements were carried out by investigators blinded to the experimental
conditions.

Subcellular Ca2+ monitoring
For Ca2+ monitoring, N2a cells were transfected with pCMV CEPIA4mt or pCMV R-CEPIA1er to assess
mitochondrial or ER luminal Ca2+ levels. Dopaminergic neurons were stained with Rhod2 (Invitrogen,
R1244) for mitochondrial Ca2+ level measurement, according to the manufacturer's protocol. Following
transfection or staining, cells were placed in Ca2+-free Hank's buffered salt solution (HBSS) supplemented
with 20 mM HEPES. Cells were stimulated with 200 μM histamine, 200 nM triptolide, or 5 mM caffeine,
and imaged at 1.27-second intervals using an inverted confocal laser scanning microscope (ZEISS LSM
900) with a 40 × objective. Image analysis was performed with ImageJ using the time series analyzer V3
plugin. F values were calculated by averaging 20 frames prior to stimulation, and ΔF was defined as
F -F .
max 0

Oxygen consumption rate (OCR) measure
To measure the OCR, two million cells were added to each chamber of the Oxygraph-2k (OROBOROS
Instruments). Sequential injections of the ATP synthase inhibitor Oligomycin (2.5 μM), the uncoupling
reagent FCCP (2 μM), and a combination of rotenone (0.5 μM) and antimycin A (2.5 μM) were
administered to block complexes I and III. These tests were utilized to determine various parameters of
mitochondrial function, including basal respiration, ATP production, and maximal respiration. The basal
respiration was calculated as the difference between the basal OCR (recorded before the addition of any
modulators) and the non-mitochondrial OCR (recorded after the addition of antimycin A and rotenone).
The ATP production was determined as the difference between basal respiration and the OCR recorded
after the addition of oligomycin. The maximal respiration was calculated as the difference between the
maximal OCR (recorded after the optimal concentration of FCCP addition) and the non-mitochondrial
OCR.

Mitochondrial membrane potential
Mitochondrial membrane potential was assessed in N2a cells using TMRM (Invitrogen, T668). Living
cells were incubated with 100 nM TMRM dye for 30 minutes at 37°C, followed by washing with PBS.
The fluorescence of these living cells was measured using an inverted confocal laser scanning microscope
(ZEISS LSM 900) with a 63 × objective.

Lipid Peroxidation
The levels of lipid peroxidation were measured using the BODIPY 581/591 C11 reagent (Invitrogen,
D3861) following the manufacturer's instructions. Briefly, The N2a cells were seeded in six-well plates
and treated with RR or MCU-i4 for 24 hours. They were then washed with PBS twice and incubated with
5 μM BODIPY 581/591 C11 reagent for 30 min at 37℃. Cells were harvested and washed twice with
PBS and then resuspended in 500 μL of PBS. 10000 single cells were acquired from each group and
measured by flow cytometer (Beckman CytoFLEX) equipped with a 488 nm laser. Data were analyzed
using the FlowJo software.

RNA isolation and qRT-PCR
Total RNA was isolated from N2a cells using TRIzol reagent (Takara, 9109) in accordance with the
manufacturer's instructions. Complementary deoxyribonucleic acid (cDNA) was synthesized using the 5
× Evo M-MLV RT Premix (Accurate Biology, AG11706). The qRT-PCR assay was then conducted using
the 2 × SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, AG11701) on the StepOnePlus™
Real-Time PCR System (Invitrogen). The primers were purchased from Sangon Biotech and listed in
Supplementary Table 2.

Statistical analysis
All data presented were obtained from distinct samples, as specified in the figure legends, with no data
excluded. Results are expressed as mean ± SEM for at least n = 3. Group differences were analyzed using
a two-tailed unpaired Student's t-test, while comparisons among multiple groups were assessed using one-
way ANOVA with Tukey’s multiple comparison tests via Prism 9.5.1 software (GraphPad). A p-value of
≤ 0.05 was established as the threshold for statistical significance.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors present evidence suggesting that PLA2G6 is a component of the IP3R1-GRP75-VDAC1 complex and is localized to the ER-mitochondria contact sites (MAMs). They propose that PLA2G6 may play a role in regulating the transfer of Ca2+ from the ER to mitochondria, highlighting its potential involvement in MAM function. They further demonstrate that loss of PLA2G6 leads to degradation of IP3R1 and destabilizes the IP3R1-GRP75-VDAC1 complex, which in turn disrupts ER-to-mitochondria Ca2+ transfer. This model is further supported by experiments in iPSC-derived dopaminergic neurons from Parkinson’s disease patients harboring PLA2G6 variants.\n\nThe manuscript's primary strength lies in its report of a potential interaction between PLA2G6 and the IP3R1-GRP75-VDAC1 complex. However, several major concerns affect the novelty and robustness of their conclusions. The manuscript does not clearly establish a causal link between the observed reduction in MAM length and the disruption of mitochondrial/ER morphology and functon(See 1). Moreover, the evidence suggesting that PLA2G6 loss leads to a reduction in MAM length is not compelling (see 2). Furthermore, while PLA2G6 is in the IP3R1-GRP75-VDAC1 complex, the authors do not address PLA2G6’s role in maintaining MAM integrity. Since the authors' model hinges on these three points, the absence of critical supporting data, along with several other issues (outlined below), raises many issues. Therefore, the manuscript is not ready for publication. Here is a detailed list of the specific concerns.\n\n1. Defects in PLA2G6 have been widely associated with ER and mitochondrial abnormalities across various models, including flies, cultured cells and iPSC-derived dopaminergic neurons. In this manuscript, the authors propose that loss of PLA2G6 disrupts MAMs. However, a key issue is that the structural abnormalities observed in MAMs could be a secondary consequence of changes to the ER and/or mitochondria. To assess this, it is essential to investigate the role of PLA2G6 in both the ER and mitochondria, examine potential alterations in structure (e.g., area or length) and function. For instance, the observed reduction in mitochondrial Ca2+ transfer could result from impaired Ca2+ release from the ER or reduced mitochondrial Ca2+ uptake.\n\n2. In Fig. 2d, the change in \"Length of each MAM\" between control and KO cells is subtle, and only two sample points are different in the control group. Similarly, the \"Size of each MAM\" (Fig. 2g) also has a subtle reduction. These small differences raise concerns about the robustness of the MAM phenotype and its role in the Ca2+ transfer in PLA2G6 KO cells.\n\n3. Building on point 2, the \"MAM length/mitochondrial perimeter (%)\" (Fig. 2e) and \"MAM surface/mitochondrial surface (%)\" (Fig. 2h) show a more pronounced reduction than the \"Length of each MAM\" (Fig. 2d) and \"Size of each MAM\" (Fig. 2g). This suggests that the MAM defect is primarily driven by the expansion of the mitochondrial perimeter, rather than changes in the length of individual MAMs. These findings further support the concern that the observed MAM defects are secondary to broader ER and mitochondrial abnormalities.\n\n4. Figures 1a-c show that PLA2G6 is not only present in MAM, but predominantly found in crude mitochondrial and ER fractions. Additionally, the staining data in Fig. 1d reveals that PLA2G6 is homogeneously distributed across both the ER and mitochondria. These findings indicate that PLA2G6 is broadly localized to the ER and mitochondria, rather than being specifically enriched at the MAM. BTW, if PLA2G6 were truly localized to the MAM, one would expect puncta-like staining in Fig. 1d, which is not observed. These observations strengthen concerns that the MAM phenotypes are secondary to defects in the ER and mitochondria, thereby diminishing the novelty of the manuscript's central claim.\n\n5. In Fig. S2, the PLA2G6 KO N2a and HEK293T cells do not exhibit the lysosome expansion phenotype. This contradicts previous reports showing expanded lysosomes in PLA2G6 KO cells, mice, and patient-derived neurons. This discrepancy raises concerns about the specificity of their KO methodology and, consequently, the robustness of their data.\n\n6. Previous studies have shown that PLA2G6[ko/ko] mice typically exhibit detectable defects at around 1 year of age. At 3 months, no obvious mitochondrial morphological defects have been reported (PMID: 27030050). However, in Fig. 2j, the authors show obvious mitochondrial morphological defects in the dopaminergic neurons of the 3-month-old PLA2G6[ko/ko] mice. Can the authors provide an explanation for this discrepancy? Are mitochondria in dopaminergic neurons more sensitive to PLA2G6 loss?", "reviewer_2": "This manuscript demonstrates that PLA2G6 is localized to the mitochondria-associated membranes (MAMs) and plays a critical role in MAM maintenance, as shown in cultured cells, mouse models, and iPSC-derived neurons. Additionally, the study suggests that MAM destabilization reduces Ca2+ influx into mitochondria, resulting in mitochondrial dysfunction, as observed in cultured cells and patient-derived neurons.\n\nAs a mechanism by which PLA2G6 maintains MAM, the study reveals that the stability of IP3R1 in the IP3R1-GRP75-VDAC1 complex within the MAM is enhanced in the presence of PLA2G6. Conversely, in the pathogenic mutant D331Y, MAM stability is compromised. Upon activation, IP3R1 is ubiquitinated by ERLINs/RNF170 and subsequently degraded. The authors speculate that PLA2G6 might inhibit the binding of ERLINs/RNF170 to IP3R1, thereby stabilizing IP3R1.\n\nThe role of PLA2G6 proposed in this study is straightforward and clear. However, cells from patients with PLA2G6 mutations exhibit a variety of severe pathologies, including increased lipid membrane oxidation and enhanced aggregation of alpha-synuclein. It is highly questionable whether these severe phenotypes can be explained solely by the reduction in Ca2+ influx into mitochondria. Notably, phospholipase activity is reportedly diminished in PLA2G6 D331Y; is phospholipase activity essential for IP3R1 stabilization? If so, through what mechanism? This question could be addressed using a phospholipase-dead mutant.\n\nAdditional concerns are listed below:\n• The super-resolution images show fragmented mitochondria and ER, which do not appear in normal morphology. What image processing techniques were used? The super-resolution images suggest a deviation from normal morphology, as indicated by comparison with FIB-SEM-reconstructed ER and mitochondria. Meanwhile, in Fig. 2c, the mitochondria in wild-type N2a cells appear swollen, suggesting potential fixation artifacts.\n• In Fig. 2, please define the metrics for \"length,\" \"size,\" and \"surface of MAM.\" Additionally, what does \"the ratio of MAM length to mitochondrial perimeter\" indicate?\n• What evidence supports that the EM images in Fig. 2j are from dopaminergic neurons?\n• In the legend of Fig. 2i, the sentence \"The ER surface was quantified as the region surrounding mitochondria.\" is unclear.\n• For Fig. 3a, where interacting proteins were identified by mass spectrometry using anti-PLA2G6 immunoprecipitates, could the authors provide the full dataset? Were IP3R1, GRP75, VDAC1, ERLIN1/2, and RNF170 detected? Was GRP75, a known sticky protein, absent from control IgG immunoprecipitates?\n• In Fig. 3b, PLA2G6 and GRP75 should be detected separately to validate antibody specificity.\n• In Fig. 3e and f, IP was performed on mitochondrial crude fractions, but in comparison to the results of Fig. 3b and c, GRP75's co-immunoprecipitation appears significantly reduced relative to its presence in input. What does this suggest?\n• The data in Fig. 3i do not provide sufficient evidence to convincingly conclude that these proteins form a complex.\n• Why are signals concentrated near the nuclei in the PLA images of Fig. 4 and Fig. S6?\n• Is the observed reduction in IP3R1 expression in PLA2G6 KO consistent? Fig. S7b does not show a reduction in IP3R1. Also, why does VDAC1 appear as a doublet in some instances and a single band in others?\n• If PLA2G6 regulates IP3R1 stability under basal conditions, PLA data for ERLIN2-IP3R1 and RNF170-IP3R1 without CCh stimulation would be necessary. Are data available for ERLIN1? Were cells treated with CCh in Fig. 5k and l?\n\nMinor Points:\n• Fig. 2 and Fig. S7 present redundant KO data, and the figure layout could be optimized.\n• Why does the introduced PLA2G6 in Fig. S7a differ in molecular weight from the endogenous protein?\n• The citations in Lines 185 and 196 referring to Fig. S6 are incorrect and should reference Fig. S5.", "reviewer_3": "The study explores the role of PLA2G6 in regulating ER-mitochondrial contact sites, mitochondrial function, and its implications for Parkinson's disease (PD). Employing complementary approaches such as advanced imaging techniques (HIS-SIM, TEM, FIB-SEM), biochemical fractionation, and functional assays, the study investigates PLA2G6 localization and function across in vitro and in vivo models.\n\nThe presented data are interesting and relevant.\n\nWhile the relationship between PLA2G6, mitochondria, and ER-mitochondrial contact sites has been previously documented—particularly the role of PLA2G6 at mitochondria-associated membranes (MAMs) in Drosophila (PMID: 31548400)—the current study should clearly articulate how its findings extend or differ from this prior work to underscore its novel contributions. This study strengthens translational relevance by using several complementary models, including human induced pluripotent stem cell-derived systems. To further solidify the findings, additional experiments are recommended, particularly validation of the role of the GRP75/IP3R/VDAC complex in human PD patient-derived cells. This would provide deeper insights and enhance the study’s contribution to understanding PLA2G6’s role in PD pathogenesis.\n\nThe authors present evidence supporting a role for PLA2G6 in regulating mitochondria-associated membrane (MAM) integrity by modulating the IP3R-GRP75-VDAC1 complex through interactions with GRP75. To strengthen the mechanistic understanding, the authors should elaborate on how IP3R1 degradation impacts calcium homeostasis and its downstream effects on neuronal function.\n\n1. The authors propose structural and functional associations between PLA2G6 and GRP75; however, additional experimental validation is necessary to substantiate this claim. Performing co-immunoprecipitation experiments to confirm the interaction between PLA2G6 and GRP75, while including known interactors of the GRP75/IP3R/VDAC complex as controls, would provide stronger evidence and significantly enhance the robustness of these findings.\n\n2. A detailed cellular localization analysis of PLA2G6 and GRP75 within specific ER and mitochondrial subcompartments (e.g., ER lumen, ER membrane, MAM, MOM, MIM, and mitochondrial matrix) would provide critical insights into the spatial basis of these interactions. This analysis could clarify the precise sites of functional relevance. Additionally, Figure 1d is of poor quality, and the claimed finding is not evident. The figure should be improved, with higher resolution and clear annotations, to support the stated results effectively.\n\n3. An additional figure where IP3R/VDAC1 PLA is performed in an IP3R or VDAC1 knock-out or knock-down cell line should be included as a negative control.\n\n4. A blot against a non-PLAG2A6 interacting protein should be included to highlight that the IP only detects PLAG2A6 interacting proteins.\n\n5. To determine whether reduced IP3R1 levels stem from increased degradation, the authors could consider blocking degradation pathways to clarify if degradation of IP3R1 overexpression impacts PLA2G6 or if there is a reciprocal regulatory mechanism.\n\n6. While the study emphasizes IP3R1-mediated Ca²⁺ transfer, the lack of significant changes in PKD2- or ryanodine receptor-mediated Ca²⁺ flux could be better discussed to refine PLA2G6’s specificity in regulating IP3R1-dependent processes.\n\nMinor\n- The Methods section should clarify the methods used to quantify mitochondrial morphology and MAM.\n- The authors should provide a more comprehensive characterization of iPSC differentiation across the different lines by including additional relevant markers for validation. Furthermore, presenting low-magnification images would help assess the overall differentiation efficiency and uniformity, thereby enhancing the rigor and clarity of the study.", "reviewer_4": "The authors used transmission electron microscopy (TEM) and focused ion beam-scanning electron microscopy (FIB-SEM) to observe and analyze inter-organelle contacts, which is a fascinating area of research. However, there are several aspects where I would appreciate further clarification.\n\nMajor comments:\n\n1. FIB-SEM Imaging and Segmentation Methodology: The manuscript mentions the image processing steps of \"alignment, filtering, and manual segmentation\" but lacks a detailed explanation of the specific methods or algorithms employed. It is unclear whether automated segmentation algorithms were used or if the segmentation process relied on manual intervention. If manual segmentation was utilized, how was consistency ensured across different datasets? Additionally, the resolution of the images in terms of lateral (horizontal) resolution should be clarified to provide a better understanding of the imaging quality.\n\n2. Super-resolution Imaging and Co-localization Analysis: While the manuscript describes the use of super-resolution imaging for co-localization analysis, the description of the software and the specific analysis methods employed could be strengthened. It would be helpful to provide more detail on the computational tools or algorithms used to quantify co-localization, as well as how potential issues such as background noise and image artifacts were addressed during the analysis.\n\n3. ER-mitochondria Contact Sites: What detailed methods were used to calculate the ER-mitochondria (also including ER-lysosome and mitochondria-lysosome) contact sites? Specifically, what hyperparameters of the contact site calculator in the Amira software did the authors implement? For example, how close must two objects be to be considered a contact site? If this threshold is changed, will the results change? Multiple softwares (Amira, Imaris, ImageJ) were used. Did they use the same quantification method or criterion? Understanding the methodology behind these calculations is crucial for interpreting the results accurately.\n\n4. The Morphology of ER: As shown in Figures 2 c and f, it seems that there are not only differences between the ER-mitochondria contact sites, but also differences between ER morphology. For example, the diameter of the tubular structure formed by the ER (or MAM).\n\n5. Results: The article appears to focus solely on the area of contact surfaces, noting that “in Pla2g6 KO N2a cells, the size of ER-mitochondria contacts decreased by 38.5% compared to control cells (Fig. 2g).” However, Figure 2f indicates that individual mitochondria have multiple contact sites. How might the analysis of changes in the number of contact sites contribute to the understanding of this phenomenon? Could it occur that, despite a potential reduction in the overall contact area (i.e., the sum of all contact surfaces), the area of each individual contact site may increase? This phenomenon could arise as a result of a decrease in the number of contact points. An analysis of both surface area and contact site quantity could provide deeper insights.\n\nMinor comments:\n\nFinally, a minor discussion point: the morphology of mitochondria is closely related to both health and disease states [1]. Do the authors anticipate that variations in mitochondrial shape could influence the contact area and the number of contact sites? Exploring this relationship may add an interesting dimension to the current study.\n\nI look forward to the authors' responses to these queries, which I believe will enhance the clarity and depth of the manuscript.\n\n[1] Yapa N M B, Lisnyak V, Reljic B, et al. Mitochondrial dynamics in health and disease[J]. FEBS letters, 2021, 595(8): 1184-1204."}, "author_response": "Dear Reviewers,\n\nWe are sincerely grateful for your invaluable comments, which have greatly enhanced the quality of this work. Enclosed with this letter are your review comments and our point-by-point responses, demonstrating our thorough consideration of all feedback received during the peer review process.\n\nThe original comments appear in italics, with our corresponding responses in standard font and blue highlighting. The edits in the manuscript are presented in regular text with red highlights.\n\nThank you for your time and thoughtful review. We trust you will find our responses adequately address every concern raised, and respectfully request your continued engagement through re-evaluation of this substantially strengthened version.\n\nBest regards,\n\nBao-Rong Zhang, Professor/MD\nThe Second Affiliated Hospital, School of Medicine, Zhejiang University.\nPhone: +86-571-87784752; FAX: +86-571-87784752\nEmail: brzhang@zju.edu.cn\n\n[... Author responses to Reviewers 1-4 ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "There are three major concerns that remain unaddressed. The original concerns are listed under \"Unaddressed Concern\" sections and my new comments are listed in the \"Comment on the authors’ response to this concern\" section.\n\nUnaddressed Concern 1:\nER and mitochondrial abnormalities have previously been associated with PLA2G6 loss-of-function models. Hence, the structural changes observed at the mitochondria-associated membranes (MAMs) could be secondary to broader disruptions in ER and/or mitochondrial architecture. Two unresolved issues underscore this concern. First, in the original Fig. 1d, PLA2G6 was shown to colocalize predominantly with mitochondrial markers and only partially with ER markers, rather than being specifically enriched at the MAM. This finding directly contradicts the authors’ claim of a specific role for PLA2G6 at the MAM.\n\nComment on the authors’ response to this concern:\nTo address this concern, the authors replaced the images in Fig. 1d. The original figure displayed a relatively broad distribution of PLA2G6 across mitochondria and some ER, rather than a specific enrichment at MAMs. However, in the revised images, PLA2G6 staining is confined to the MAM, with no apparent signal in the surrounding ER or mitochondria. This not only contradicts their original data but also conflicts with the published literature, raising serious concerns.\n\nUnaddressed Concern 2:\nTo follow up on the previous concern, the changes presented in Fig. 2 regarding MAM structure are subtle. For example: the difference in 'Length of each MAM' between control and knockout cells is minimal, with only two data points showing variation in the control group. Similarly, the reported reduction in 'Size of each MAM' is modest. These marginal differences raise concerns about the robustness and biological relevance of the proposed MAM phenotype resulting from PLA2G6 loss.\n\nComment on the authors’ response to this concern:\nIn the revised manuscript, the authors addressed the role of MAMs in Ca²⁺ transfer. However, they did not provide a clear explanation or additional data to support a biological role for PLA2G6 in regulating MAM morphology. The reported increase in 'Length of each MAM' appears to be driven primarily by two outliers, while the majority of data points show no significant difference. Similarly, the reported reduction in 'Size of each MAM' is modest.\n\nUnaddressed Concern 3:\nPrevious studies have shown that PLA2G6[ko/ko] mice typically exhibit detectable defects around 1 year of age. At 3 months, no obvious mitochondrial morphological defects have been reported (PMID: 27030050). However, in Fig. 2j, the authors show obvious mitochondrial morphological defects in the dopaminergic neurons of the 3-month-old PLA2G6[ko/ko] mice. Can the authors provide an explanation for this discrepancy? Are mitochondria in dopaminergic neurons more sensitive to PLA2G6 loss?\n\nComment on the authors’ response to this concern:\nIn this revised version, the authors also have not provided any explanation or additional data to address this.\n\nIn summary, given these concerns, which significantly compromise the conclusions and biological relevance of their arguments, I recommend rejection.", "reviewer_2": "The revised manuscript presents significantly improved organelle imaging, which in turn enhances the persuasiveness of the authors’ claims.\n\nAn important new finding in this revision is that the lipase activity of PLA2G6 does not appear to be related to the phenotypes observed by the authors. Due to the lack of definitive information on the physiological substrates of PLA2G6, the relationship between its lipase activity and disease-associated mutations remains unclear. Although previous reports have assessed lipase activity of disease-associated mutants using artificial substrates, the correlation between missense mutations and enzymatic activity has been inconsistent (PMID: 20886109, PMID: 21700586). To clarify the relationship between IP3R1 stability and the disease relevance, this reviewer recommends that the authors investigate the correlation between the observed Ca²⁺ dynamics (and/or IP3R1 levels) and several known pathogenic PLA2G6 variants. In particular, it would be valuable to determine whether there are differences between INAD/NBIA-related mutations and PD-associated mutations. Furthermore, clarifying which specific domain(s) of PLA2G6 are essential for MAM formation would substantially improve the quality of the manuscript.\n\nBelow are some minor points:\n•Regarding the raw data for the mass spectrometry analysis, I was able to confirm the Excel dataset included in this manuscript; however, I could not locate PXD057620 in the PRIDE website.\n•In the legend of Fig. S6, it states “n = 3 independent experiments.” Could the authors clarify whether this refers to representative results from three independent experiments?\n•In the legend of Fig. S6b, it reads “Immunoblotting analysis of GRP7.” Please confirm whether this is a typographical error.", "reviewer_3": "The Authors have address all our previous concerns.", "reviewer_4": "I appreciate the authors' efforts in revising the manuscript. The additional technical details and discussions adequately address my concerns raised in the previous round. I find the current version improved and more complete. I have no further major comments."}, "author_response": "First of all, we sincerely express our gratitude to the editor and reviewers for their time, positive comments and helpful advices. Specifically, we have addressed these comments and concerns in a point-by-point manner as follows. The original comments appear in italics, with our corresponding responses in standard font and blue highlighting. The edits in the manuscript are presented in regular text with red highlights.\n\n[... Author responses to Reviewers 1-4 ...]"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns,", "reviewer_2": "The authors have made efforts to elucidate the correlation between pathogenic variants of PD and NBIA and Ca²⁺ dynamics, and this reviewer believes the overall quality of the manuscript has improved as a result. However, in Figure S3c–f, it remains impossible to compare the calcium dynamics of each mutant with those of wild-type PLA2G6. In other words, a \"KO + WT\" experimental group should have been included within the same experimental system. Considering the potential effects of disease-associated variants, it is entirely plausible that the results could show “partially impaired\" rather than completely abolished function."}, "author_response": "First of all, we sincerely express our gratitude to the editor and reviewers for their time, positive comments and helpful advices. Specifically, we have addressed these comments and concerns in a point-by-point manner as follows. The original comments appear in italics, with our corresponding responses in standard font and blue highlighting. The edits in the manuscript are presented in regular text with red highlights.\n\n[... Author responses to Reviewers 1-2 ...]"}, {"round": 4, "reviewer_comments": {"reviewer_2": "All concerns have been resolved."}, "author_response": ""}]

Decision letter