Cell Calcium 62 (2017) 1–15

Contents lists available at ScienceDirect

Cell Calcium
journal homepage: www.elsevier.com/locate/ceca

The endoplasmic reticulum-mitochondria coupling in health and

disease: Molecules, functions and significance
Riccardo Filadi a , Pierre Theurey a , Paola Pizzo a,b,∗
a
Department of Biomedical Sciences, University of Padova, Italy
b
Neuroscience Institute, National Research Council (CNR), Padova, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The close apposition between endoplasmic reticulum (ER) and mitochondria represents a key plat-
Received 5 December 2016 form, capable to regulate different fundamental cellular pathways. Among these, Ca2+ signaling and lipid
Received in revised form 9 January 2017 homeostasis have been demonstrated over the last years to be deeply modulated by ER-mitochondria
Accepted 9 January 2017
cross-talk. Given its importance in cell life/death decisions, increasing evidence suggests that alterations
Available online 12 January 2017
of the ER-mitochondria axis could be responsible for the onset and progression of several diseases, includ-
ing neurodegeneration, cancer and obesity. However, the molecular identity of the proteins controlling
Keywords:
this inter-organelle apposition is still debated. In this review, we summarize the main cellular pathways
Calcium (Ca2+ )
Mitochondria
controlled by ER-mitochondria appositions, focusing on the principal molecules reported to be involved
Endoplasmic reticulum (ER) in this interplay and on those diseases for which alterations in organelles communication have been
Inter-organelle contacts reported.
ER-mitochondria tethering © 2017 Elsevier Ltd. All rights reserved.
Mitochondria-Associated Membranes
(MAMs)
Mitofusin 2

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Molecules: proteinaceous structures connecting the ER to mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Physical and functional tethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2. The elusive role of Mfn2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Functions: cellular pathways dependent on ER-mitochondria coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.1. Lipid homeostasis, mitochondria dynamics, autophagy, inflammation and mtDNA distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.2. ER-mitochondria Ca2+ cross-talk functionalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. Significance: dysfunctions in ER-mitochondria coupling and diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.1. Metabolic syndrome and inflexibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.2. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.3. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.4. Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.5. Amyotrophic lateral sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

∗ Corresponding author at: Department of Biomedical Sciences, University of

Padova, Via U. Bassi 48, 35121 Padova, Italy.
E-mail address: paola.pizzo@unipd.it (P. Pizzo).

http://dx.doi.org/10.1016/j.ceca.2017.01.003
0143-4160/© 2017 Elsevier Ltd. All rights reserved.
2 R. Filadi et al. / Cell Calcium 62 (2017) 1–15
1. Introduction
Inter-organelle communication represents an emerging aspect
in cell biology: indeed, cellular organelles do not function as iso-
lated structures but rather form dynamic interconnected networks
which can be modulated according to cellular needs. In particular,
the endoplasmic reticulum (ER)-mitochondria physical/functional
coupling plays a central role in a variety of cell pathways and
increasing evidence highlights its alteration in several diseases,
including diabetes and obesity, cancer and neurodegenerative dis-
eases, such as Alzheimer’s Disease (AD), Parkinson’s Disease (PD)
and Amyotrophic Lateral Sclerosis (ALS).
Physical contacts between ER and mitochondria have been
firstly observed by EM in the 50’s in rat tissues [1] but such features
have been considered for long time to be artifacts of fixation. More
recent experiments in living cells expressing GFP variants within
the two organelles [2] and electron micrograph images of quickly
frozen samples [3] have demonstrated conclusively that such phys-
ical interactions between the two organelles indeed exist (Fig. 1A-B
). Similar structures were then described in yeast and, recently, also
in plants [4,5]. Their composition and thickness, however, are not
constant and the distance between ER and the outer mitochondrial
membrane (OMM) can be extremely variable, ranging from ∼10 nm
up to 80–100 nm [6]. Usually, at smooth ER-mitochondria contact
sites the gap between the two opposing membranes is smaller
(10–15 nm) than in the case of rough ER (typically 20–30 nm) [7],
probably to allow ribosomes accommodation. Regions in which
OMM and ER membranes proceed in parallel at larger distances
(50–100 nm) have been also reported (see for instance [8]), and can
be continuous to sites in which the two organelles are closer or, on
the contrary, exist as separate, independent units. However, while
in the case of the closer contacts (below ∼30 nm) [7] electron-dense
filamentous structures, proteinaceous in their nature, clearly pro-
trude from the two opposing membranes (Fig. 1A), such structures
have never been observed for the more distant organelles apposi-
tions. Thus, whether these latter are randomly occurring or tightly
regulated, physiologically relevant events, is still unknown.
This contribution aims to update ER-mitochondria connections
from multiple points of view: i) the molecules involved in the for-
mation/modulation of ER-mitochondria inter-organelle structures;
ii) the specific cellular functions dependent on ER-mitochondria
coupling, especially those relying on their Ca2+ cross-talk; iii) the
alterations of the ER-mitochondria platform described in different
diseases and reported to be pathogenic.
2. Molecules: proteinaceous structures connecting the ER
to mitochondria
2.1. Physical and functional tethers
The molecular identity of the structures mediating ER-
mitochondria tethering is more defined in yeast, where two
principal protein complexes are involved: the ER-mitochondria
encounter structure (ERMES) and the ER membrane protein com-
plex (EMC) (Fig. 2). Both structures were identified by genetic
screens in S. cerevisiae mutant cells whose growth/lipid metabolism
defects could be rescued by artificial ER-mitochondria tethers.
ERMES is formed by the cytosolic protein Mdm12, the ER mem-
brane protein Mmm1 and the OMM proteins Mdm34 and Mdm10
[9]. In addition to the four core components, the mitochondrial
Rho-like GTPase Gem1 represents a facultative regulatory sub-
unit of the ERMES complex [10]. ERMES has been functionally
implicated in ER-mitochondria lipid transport (though different
groups failed to find defects in lipid metabolism in cells lacking
the complex), as well as in several other cellular pathways, such
as mitochondrial dynamics, inheritance, protein import, mitochon-
drial DNA (mtDNA) inheritance and mitophagy (see [11] for a recent
review). Importantly, ERMES homologs have not been identified in
mammals, while the mitochondrial proteins Miro1/2, involved in
Ca2+ -dependent mitochondrial transport mediated by the kinesin
adapter Milton [12], are the metazoan orthologue of Gem1.
The more recently identified tethering complex EMC contains
in yeast six proteins, Emc1-6 [13] and is also required for phos-
phatidylserine (PS) import into mitochondria and assembly of
multi-pass ER-membrane proteins [14]. Emc proteins interact with
the mitochondrial protein Tom5 of the translocase of the outer
membrane (TOM) complex, forming a tether between the two
organelles [13]. EMC is a highly conserved protein complex, present
in every major eukaryotic lineage, and in mammalian cells contains
four additional proteins (Emc7–10) [15].
Recently, a proteomic analysis identified the ER transmembrane
protein Ltc1/Lam6 as a potential additional tether in yeast, thanks
to its interaction with the OMM proteins TOM70/71 [16]. Ltc1/Lam6
has also been observed to mediate the transfer of sterols between
the two membranes.
Less clear is the molecular nature of the physical tether between
the two organelles in metazoan cells. Several proteins have been
demonstrated to modulate ER-mitochondria coupling: the major-
ity of them appears to be located on either the OMM or specific ER
membrane domains, called mitochondria-associated membranes
(MAM) [17]. Despite the growing number of these proteins, the for-
mal and univocal demonstration that the lack of a given molecule
abolishes ER-mitochondria contacts has never been provided. Most
likely, different and independent tethering complexes may exist
and compensate one for the lack of the others, increasing the com-
plexity of the analysis.
One multi-protein structure that strongly correlates with func-
tional ER-mitochondria coupling is the IP3R-Grp75-VDAC complex
(Fig. 2). In particular, it has been shown that ER resident inosi-
tol trisphosphates receptors, IP3Rs, (especially the MAM enriched
IP3R3) physically interact with the cytosolic fraction of the mito-
chondrial chaperone Grp75 and the voltage-dependent anion
channel 1 (VDAC1) of the OMM [18], functionally favoring mito-
chondrial Ca2+ uptake upon IP3-dependent ER Ca2+ release (see
below). The role of such complex as a physical tether between the
two organelles is however questionable, since DT40 cells knock-out
(KO) for the three IP3R isoforms show, by EM analysis, unmodified
ER-mitochondria physical association [7].
The molecular scaffold formed by the ER resident vesicle-
associated membrane protein-associated protein B, VAPB, and
the OMM protein tyrosine phosphatase-interacting protein-51,
PTPIP51, has been also demonstrated to be involved in physical
and functional ER-mitochondria tethering [19,20]. Genetic manip-
ulation of these proteins are linked to variations in organelles
juxtaposition and altered ER-mitochondria Ca2+ cross-talk. Inter-
estingly, these functionalities are also affected by the expression of
both VAPB mutants linked to Amyotrophic Lateral Sclerosis (ALS)
[19] or the ALS-associated proteins TDP43 [20] and FUS [21], that
disrupt the endogenous VAPB-PTPIP51 interaction, indicating a key
role for the mitochondria-ER axis in ALS pathogenesis (see below).
In mammalian cells, the mitochondrial protein PTPIP51 has been
recently reported to physically interact also with the ER-located
oxysterol-binding protein (OSBP)-related proteins ORP5 and ORP8
[22] (see also below), two molecules previously shown to be impor-
tant for cortical ER-plasma membrane contacts [23].
Another example of protein regulating ER-mitochondria jux-
taposition is the phosphofurin acidic cluster sorting 2 protein,
PACS-2, a cytosolic multifunctional sorting protein which controls
organelles apposition in a B-cell receptor-associated protein 31,
BAP31 (an ER cargo receptor), −dependent manner [24]. However,
the role of PACS-2 on ER-mitochondria juxtaposition is not com-
 R. Filadi et al. / Cell Calcium 62 (2017) 1–15 3

Fig. 1. ER-mitochondria structural apposition. (A) EM image of a FAD-PS2-T122R expressing wt MEF showing some mitochondria (mt) in close apposition to ER membranes.
Scale bar: 500 nm. The inset highlights a close contact in which several proteinaceous structures connecting the two organelles are visible (red arrows). Image acquired by
G. Turacchio, Institute of Protein Biochemistry, CNR, Naples. (B) Confocal microscopy image of a HeLa cell expressing a mitochondrial matrix RFP and an ER targeted GFP, in
which several appositions between the two organelles are visible (yellow spots). Scale bar: 10 ␮m. The inset highlights some specific points in which ER tubules seem to wrap
mitochondria (white arrows), likely marking sites for future mitochondrial fission (see text for details). (C) The cartoon schematically represents an ER-mitochondria contact
site, with some proteins proposed to play a structural role at this interface (see text for details). Traces on the right box represent simulated changes in [Ca2+ ] over time in
the bulk cytosol (black, dotted trace), in the mitochondrial matrix (red trace) and at regions of ER-mitochondria interface (black, continuous trace), upon an IP3-mediated
ER Ca2+ release. Note the different scales and the almost immediate generation of Ca2+ hot-spots on mitochondrial surface after IP3Rs opening. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Cell Functionalities relying on ER-mitochondria interactions. The cartoon summarizes the major reported functions modulated by ER-mitochondria juxtaposition,
and the principal molecules involved in these cross-talks, as described in the text or in other contributions (see for example [60,82]). For all the abbreviations used and the
description of specific functions, refer to the text. In italic, yeast proteins.

pletely elucidated and there is no direct evidence to suggest that
it acts as an ER-mitochondria tether: although depletion of the
protein causes mitochondria uncoupling from the ER, a BAP31-
dependent mitochondria fragmentation is also observed in these
conditions [24], which could indirectly influence ER-mitochondria
association. Moreover, PACS-2 has a complex role in modulating
endomembranes traffic and apoptosis [25]. For example, the PACS-
2/BAP31 complex is involved in Bid translocation to mitochondria
and apoptosis induction (Fig. 2), and a mitochondria-ER platform
formed by the OMM protein Fis-1 and the ER resident protein BAP31
has been reported to be involved in the recruitment/activation of
pro-caspase 8 and in the subsequent cell death pathway [26]. Again,
4 R. Filadi et al. / Cell Calcium 62 (2017) 1–15
whether Fis-1/BAP31 is just a functional complex or is endowed
with an additional tethering capacity has not been established.
The Familial Alzheimer’s Disease (FAD) related proteins
Presenilin-1 and −2 (PS1/2) have been also reported to be enriched
in MAM [27]. However, only PS2, but not PS1, is able to favor physi-
cal and functional ER-mitochondrial coupling, with FAD-linked PS2
mutants more effective than the wild type (wt) protein [28,29] (see
also below). Recently, it has been demonstrated that PS2 is able to
increase the number of ER-mitochondria close contacts only in the
presence of mitofusin 2 (Mfn2), a master regulator of organelles
tethering whose action has been recently debated (see below): by
sequestering the latter protein and removing its negative effect
on organelles tethering, PS2 enhances ER-mitochondria association
and Ca2+ transfer [30] (Fig. 1C). Whether PS2 represents one of the
possible multiple tethers between ER and mitochondria, or sim-
ply works as a pure bait for the stumbling block Mfn2 (see below),
remains to be clarified.
2.2. The elusive role of Mfn2
Among the different proteins proposed to have a role in the
formation/modulation of ER-mitochondria tether structures, Mfn2
deserves a specific issue.
In mammalian cells, the OMM resident Mfn2, involved in
mitochondrial fusion process, has been originally proposed as an
ER-mitochondria tether [31]. Indeed, part of the protein resides in
ER membranes, particularly in MAM, and has been demonstrated by
crossed co-immunoprecipitation experiments to physically inter-
act with the OMM resident Mfn2, or with its homologue Mfn1.
The observation, by confocal microscopy, that Mfn2−/− mouse
embryonic fibroblasts (MEFs) display a reduced ER-mitochondria
co-localization [31], as well as the previously reported role of Mfns
in tethering adjacent mitochondria during their fusion [32,33], ini-
tially argued in favor of the proposed role of Mfn2 as a tether.
Functionally, Mfn2−/− MEFs were demonstrated to have a reduced
mitochondrial Ca2+ uptake upon ER Ca2+ release, further suggest-
ing that the two organelles could be more distant and creating
consensus on the fact that Mfn2 connects them. Based on this
model, a number of studies used genetic manipulation of Mfn2 as
a tool to modulate ER-mitochondria coupling, further reinforcing
the thinking of this protein as a bona fide tether. However, many of
these studies just assumed the original model and interpreted the
obtained results as its consequence, lacking a direct evaluation of
the effects of Mfn2 modulation on inter-organelle association. For
instance, Mfn2 down-regulation was found to have an impact on
autophagosomes formation at ER-mitochondria contact sites [34],
but the effects of Mfn2 modulation on inter-organelle tethering was
not directly investigated. Similarly, the E3 ubiquitin ligase MITOL
was found to modulate ER-mitochondria apposition, and this was
correlated to its effect on Mfn2 ubiquitination, but the direct effects
of Mfn2 on organelles coupling were only evaluated by Mander’s
colocalization coefficient [35], a method that has been later demon-
strated to be misleading whenever marked changes in organelle’s
morphology occur (see below and [8]). Moreover, the effects of
MITOL were not verified in Mfn2−/− cells, and thus they may, or
may not, be related to Mfn2. On the same line, Mfn2−/− MEFs were
observed to form less starvation-induced autophagosomes, due to
an impairment of lipid transfer from mitochondria [36]; also in this
case, Mfn2 ablation effects on ER-mitochondria tethering were just
assumed and not measured. Moreover, a more recent paper from
the same group showed a strong correlation between up-regulated
mitochondrial fusion processes and autophagy [37], potentially
explaining the results they have previously obtained in Mfn2−/−
MEFs (where mitochondrial fusion is impaired).
Another study [38] found that conditional Mfn2−/− mice show,
in the heart, a 30% reduction in junctional sarcoplasmic reticulum
(jSR)-mitochondria close contacts length, but the number of con-
tacts was not evaluated. However, given that in cardiomyocytes
the structures of both SR and mitochondria are highly ordered and
continuously rearranged with age, making the jSR-mitochondria
apposition a peculiar case significantly different from that of the
ER-mitochondria connection (for a recent review, see [39]), a
direct comparison between the two structures should be avoided,
especially considering the dramatic changes in mitochondrial and
potentially SR morphology induced by Mfn2 ablation.
A remarkable exception is the elegant paper of Schneeberger
et al. [40], where a careful quantification of ER-mitochondria
juxtaposition was performed in POMC neurons. The authors
demonstrated that in obese mice there is a reduction in ER-
mitochondria contacts in POMC neurons and a reduction in Mfn2
expression. Moreover, they demonstrated that the selective Mfn2
ablation in POMC neurons induces obesity and, in 18 weeks old
mice, a reduction in ER-mitochondria coupling. It is, however, dif-
ficult to establish whether this decreased inter-organelle contact
is a direct consequence of Mfn2 ablation or a consequence of the
resulting obesity (see also below).
In the attempt to investigate the effects of Mfn2 on ER-
mitochondria tethering, a breaking point came from a quantitative
electron microscopy (EM) approach, demonstrating that in
Mfn2−/− MEFs (the same cell line used in [31]) the number of close
contacts between the two organelles (distance <20 nm), as well as
the percentage of OMM engaged in the formation of contacts, is
doubled, compared to wt MEFs [41]. Notably, Mfn2 re-expression
in Mfn2−/− cells rescued these parameters, suggesting that the
observed phenotypes were not due to clonal adaptation. However,
in the same study, the reduced ER-mitochondria co-localization
index, previously found in Mfn2−/− MEFs by a confocal microscopy
analysis [31], was confirmed [41], casting doubts on the validity of
the latter method to quantitatively analyze inter-organelle apposi-
tion.
Recently, it has been confirmed that Mfn2 ablation, or its acute
down-regulation, increases ER-mitochondria coupling, reconciling
EM and confocal microscopy analysis [8]. In particular, it has been
demonstrated that the paradoxically lower ER-mitochondria co-
localization, obtained by fluorescence microscopy, is due to the
fact that Mfn2 ablation deeply affects mitochondrial morphol-
ogy, fragmenting the organelles and, at the same time, swelling
them. By computational simulations, it has been shown that any
mitochondrial swelling, even in the presence of a higher per-
centage of OMM in contact with the ER, induces an apparent
and artifactual reduction in the classical Manders’ and Pearson’s
co-localization coefficients. The problem was tackled by consid-
ering only the perimeter of mitochondria, instead of their entire
area/volume: by this method, even by confocal microscopy, the
percentage of mitochondrial perimeter in contact with the ER was
found higher upon Mfn2 ablation/down-regulation, in accordance
with EM data. Regarding the reduced mitochondrial Ca2+ uptake
originally observed in Mfn2−/− MEFs upon ER Ca2+ release (a feature
interpreted as a consequence of a looser ER-mitochondria associa-
tion in these cells [31]), it has been found that this clone shows a
∼50% reduction in MCU expression level, compared to wt MEFs [8].
Thus, the reduced mitochondrial Ca2+ rises cannot be considered in
these cells a readout of a decreased organelles apposition. Indeed,
acute Mfn2 knock-down in wt MEFs, that does not change MCU
expression, resulted in increased mitochondrial Ca2+ peaks, when-
ever an IP3-mediated ER Ca2+ release was induced, and increased
sensitivity to Ca2+ -mediated apoptosis [8].
A higher ER-mitochondria tethering, measured by EM, was also
associated with lower levels of Mfn2 and Mfn1 by another inde-
pendent group [42,43]. Interestingly, a different role for Mfn1 and
Mfn2 in the modulation of mitochondria contacts with smooth
and rough ER, respectively, was proposed [42]. More extended
 R. Filadi et al. / Cell Calcium 62 (2017) 1–15
ER-mitochondria juxtapositions (thickness < 25 nm), but not a vari-
ation in their number, were reported in HEK cells in which Mfn2
was acutely down-regulated and, functionally, an increased Ca2+
shuttling between the two organelles was observed [44]. Finally,
the Alzheimer’s disease related protein Presenilin-2, by binding
and inhibiting the antagonistic role of Mfn2, has been shown to
favor ER-mitochondria physical and functional coupling [30]. Thus,
accordingly to all these new data, Mfn2 can be still considered a
master regulator of ER-mitochondria coupling, but with an antag-
onistic role.
Very recently, another paper came out by the same authors
that originally proposed the ER-mitochondria tether role for Mfn2
[45]. This latter claimed “definitive” evidence for the specific issue
and argued against the published findings showing the contrary
[8,41]. The decreased ER-mitochondria co-localization, originally
observed by confocal microscopy in Mfn2−/− MEFs by the same
authors [31], is here justified by EM data showing that, upon Mfn2
deletion, ER and mitochondria are 1–3 nm more apart compared
to controls (while no difference in this parameter was found previ-
ously [8,41] in the same cell lines, wt and Mfn2−/− MEFs). Moreover,
upon Mfn2 depletion, a robust reduction in mitochondrial Ca2+
uptake, upon IP3-linked stimulations, was found, confirming their
previous results [31]. The latter effect was proposed to be indepen-
dent from variations in MCU expression level. Finally, employing
a previously reported FRET-based ER-mitochondria artificial teth-
ers [46], a reduced FRETratio in Mfn2−/− cells compared to controls
has been reported and interpreted as a proof of decreased inter-
organelle contacts [45].
Several points are however debatable in this contribution and,
although a quantitative EM analysis was employed to investigate
the issue at higher resolution, the paper does not provide any con-
vincing new evidence in favor of the original hypothesis. First of
all, the key parameter, i.e., the number of contacts between the
organelles, that was previously found doubled in Mfn2−/− cells
[8,41] was not measured. Instead, only ER-mitochondria inter-
face length, i.e., the averaged extension of each single contact, is
reported to be decreased in Mfn2−/− cells (from 253 nm in wt to
195 nm in Mfn2−/− cells; a feature not observed in previous studies
[8,41]). However, the quantification of this parameter is not enough
to sustain a reduced ER-mitochondria juxtaposition in Mfn2−/−
cells: if the contacts in these cells are more frequent (as it has been
previously observed), the percentage of mitochondrial perimeter in
contact with the ER could be increased. Thus, it is critical to exactly
know the number of contacts, in addition to their extension, to
solve this debated point. Furthermore, the reduced interface length,
observed only in this study, cannot be chosen as an unequivocal
proof of ER-mitochondria apposition since the same paper showed
that: Mfn1−/− cells (which present an unchanged ER-mitochondria
tethering [31]) display a similar reduction in this parameter, and
Mfn2 re-expression in Mfn2−/− cells does not recover it, thus inval-
idating also the bizarre ERMICC coefficient used [45]. Importantly,
the difference in ER-mitochondria distance, found by EM analysis,
between controls and Mfn2−/− cells (+1–3 nm in these latter cells)
does not explain the substantial apparent reduction in organelles’
co-localization, as previously observed by confocal microscopy
[8,31,41]. Indeed, differences in this range of values are incompat-
ible with the resolution of confocal microscopy (at best ∼200 nm,
i.e., 100 fold lower), and does not produce any outcome observable
by this technique.
Moreover, the interpretation of the FRET data reported for
Mfn2−/− cells, and presented as an additional proof of a reduced
ER-mitochondria juxtaposition, is questionable and argue against
the proposed conclusion. Firstly, raw values of basal and maximal
FRET are missing. These parameters are indeed crucial: to allow, by
this method, an indirect comparative quantification of organelle
contacts in wt and Mfn2−/− cells, maximal FRET values, artificially
5
induced by a rapamycin treatment, previously shown by EM to
force ER-mitochondria contacts formation [46], should be similar
in the two conditions. In fact, morphological alterations of both
ER and mitochondria (as occurring in Mfn2−/− cells [31]) have a
dramatic impact on protein mobility within the two organelles,
as previously demonstrated by FRAP experiments [31]. This effect
could potentially alter the rapamycin-induced reconstitution of the
artificial tether (FRETmax ), which is based on a zippering effect
allowed by the free lateral diffusion within organelles’ membranes
of the two modified GFP-variants forming the tether [46]. Secondly,
the reported reduction in Mfn2−/− cells of the FRETratio , calcu-
lated as (FRETmax -FRETbasal )/FRETbasal [45], means that in these cells
FRETbasal is closer to FRETmax . Thus, the presented data, showing
that Mfn2−/− cells display a lower FRETratio , indicate that in these
cells there is an increased, and not a decreased, ER-mitochondria
tethering, as previously demonstrated [8,41].
From the functional point of view as well, some data appear
inconsistent. The very small difference reported in organelles dis-
tance (∼2 nm) would have a marginal effect (<1%) on the [Ca2+ ]
generated at the ER-mitochondria interface, as it can be calculated
[47], thus hardly explaining the reported marked reduction in mito-
chondrial Ca2+ uptake, upon cell stimulation [45]. The latter results
would be more likely explained by a mitochondrial depolarization
(reported in Mfn2−/− MEFs [48,49]) or by a lower MCU expression
in Mfn2 ablated cells, compared to controls [8]. Regarding this latter
result, it has been argued that it was due to different cell densities,
showing, however, that only in control cells MCU level decreases
at high density, but not in Mfn2−/− cells [45], thus not explaining
the previous finding. On the contrary, in the same contribution,
changes in MCU complex expression levels were reported: rep-
resentative Western blots for MCU, MICU1 and MICU2 revealed
major variations in the expression of these proteins in cells ablated
of Mfn2. Moreover, the experimental approach of permeabilized
cells perfused with different media containing fixed [Ca2+ ], to study
the intrinsic capability of mitochondria to take up Ca2+ , was here
criticized. It has been stated that results obtained by this protocol
(showing that in Mfn2−/− cells there is a decreased mitochondrial
Ca2+ uptake independently from the ER-mitochondria association
[8]) are difficult to interpret because the presence of the ER, with its
high Ca2+ affinity SERCA, could “contaminate” the outcome [45]. To
investigate the same issue, isolated mitochondria have been used
instead. However, it must be noted that the classical permeabi-
lization protocol, applied in multiple occasions by several groups
(1 min in 100 ␮M digitonin-containing medium without ATP, fol-
lowed by other 2 min washing with an intracellular-like medium
without digitonin and ATP; [8]), does not allow any SERCA activ-
ity. Instead, the presented protocol, isolating mitochondria from
mice liver without any specific purification step [45], leads to a
mitochondrial fraction highly contaminated by ER vesicles.
Finally, Ca2+ data obtained in intact cells are also puzzling: it
has been claimed that, upon cell stimulation, Mfn2 ablated cells
exhibit a lower mitochondrial Ca2+ transient, compared to controls
(as a proof of a reduced ER-mitochondria juxtaposition). However,
the presented data showed that the mitochondrial [Ca2+ ] peaks
obtained in controls, under a specific condition, can be variable till
two order of magnitude [45], making any comparison with those of
Mfn2−/− cells very difficult. Moreover, upon MCU overexpression
in wt and Mfn2−/− MEF cells, the reported mitochondrial [Ca2+ ]
peaks, obtained upon cell stimulation, were very similar to those
previously found by the same authors in the very same cells with
endogenous levels of MCU [31]: given the well-known dramatic
impact of the relative amounts of each MCU-complex component
on the process of mitochondrial Ca2+ uptake [50], these results
appear difficult to explain.
Thus, since this latter contribution does not significantly modify
previous multiple conclusions that sustain a negative role of Mfn2
6 R. Filadi et al. / Cell Calcium 62 (2017) 1–15
in ER-mitochondria tethering [8,30,41–44], the specific mechanism
by which this protein modulates organelles’ coupling remains elu-
sive. Likely, Mfn2 physically interacts with and inhibits the proper
assembly of still unknown tethering proteins (Figure 1C), though
further investigations will be necessary to address this issue.
3. Functions: cellular pathways dependent on
ER-mitochondria coupling
3.1. Lipid homeostasis, mitochondria dynamics, autophagy,
inflammation and mtDNA distribution
ER-mitochondria contacts, in both yeast and mammals, repre-
sent important structures linked to multiple pathways, ranging
from different mitochondria functionalities to several cell pro-
cesses, such as proliferation, death, autophagy, lipid metabolism,
Ca2+ signaling, bioenergetics, unfolded protein response (UPR)
and inflammation [4]. While for some of them the role of ER-
mitochondria coupling has been extensively studied (for example,
on lipid synthesis, Ca2+ homeostasis, mitochondria metabolism
and cell death; see below), for others, evidence has started to
accumulate only very recently. Thus, the ER-mitochondria axis is
progressively emerging as a complex signaling platform fundamen-
tal for cell survival.
The first cellular function demonstrated to rely on ER-
mitochondria juxtaposition was the maintenance of membrane
lipid composition. Indeed, lipid synthesis and transfer are the more
prominent features of ER-mitochondria contact domains: MAM
were originally defined by Jean Vance as fraction X, biochemi-
cally distinct from the bulk ER membranes because particularly
enriched in enzymes catalyzing phosphatidylserine (PS), phos-
phatidylethanolamine (PE) and phosphatidylcholine (PC) synthesis
[51,52]. In fact, although the ER is the master organelle for lipid syn-
thesis, certain phospholipids synthetic pathways require enzymes
that are located in both the ER and the mitochondria: for exam-
ple, the biosynthesis cycle of PC and PE starts from PS synthesis at
ER-MAM domains by PS synthase [53,54], an then follows its decar-
boxylation into PE at the inner mitochondrial membrane (IMM),
and back to the ER for conversion into PC [51,55,56]. Linked to their
synthesis, there is also an extensive lipid exchange between ER
and mitochondria membranes: PC is the most abundant mitochon-
drial phospholipid and, together with PS, must be imported from
the ER through yet uncharacterised mechanisms, likely involv-
ing ER membranes and OMM close juxtapositions [57]. Moreover,
ceramides synthesis has been shown to occur at MAM [58] and
these latter have been also described as microdomains enriched in
cholesterol and gangliosides, modified sphingolipids, similarly to
the detergent-resistant lipid rafts at the plasma membrane [59].
The molecular mechanisms allowing ER-mitochondria lipid
exchange (Fig. 2) are mostly unknown [60] and insights come
almost exclusively from yeast: both the ERMES and the ECM com-
plex are involved in lipid trafficking between the organelles [61,62];
ERMES has Synaptotagmin-like Mitochondrial lipid-binding Pro-
tein (SMP) domains [63] that favor the transfer of lipids (especially
PC) through tubular, hydrophobic structures [64]. Some yeast
members of the oxysterol-binding homology (Osh) proteins fam-
ily are also necessary for proper PS homeostasis [13]. Recently,
it has been suggested that sterols are directly transported from
the ER to the mitochondria through Ltc1/Lam6, an ER-located
sterol transporter enriched in MAM [16] and interacting with the
ERMES complex [65,66]. Ltc1/Lam6 also likely functions as a tether
between ER and mitochondria by interacting with the OMM pro-
teins Tom71/70 [65].
In mammalian cells, ORP5 and ORP8, which are present at ER-
mitochondria contact sites and interact with the mitochondrial
membrane protein PTPIP51, have been reported to be involved in PS
transfer [22] (Fig. 2). As far as mitochondrial sterols import is con-
cerned, recent studies have provided the first possible molecular
identity in the OMM translocator protein (TSPO) [67]. TSPO seems
to be enriched in MAM by interacting with VDAC [68] (Fig. 2).
ER-mitochondria connections play also a key role in controlling
the morphology and the dynamics of the mitochondrial network
(Fig. 2). Indeed, mitochondrial fission occurs at positions where
ER contacts mitochondria [69], mediating organelle constriction
before the recruitment of dynamin-related protein 1 (DRP1), form-
ing helical oligomers that wrap around the organelles and divide
them in a GTP-dependent manner [70]. In metazoan cells, mito-
chondrial division mediated by ER constrictions involves also actin
polymerization through the ER protein inverted formin-2 (INF2),
a promoter of actin polymerization/depolarization [71,72]. Mito-
chondrial fission is an essential event involved in mitochondrial
network shaping and is required to generate organelles that are
at the right size to be transported by molecular motors along the
cytoskeleton. Moreover, mitochondrial fission is important for the
release of cytochrome c and other pro-apoptotic factors from the
mitochondrial inter-membrane space to trigger cell death, and
it is thought to facilitate the removal of damaged organelles by
mitophagy [73].
Moreover, it has been recently shown that autophagosomes
can originate at ER-mitochondria contact sites. Key components
of the autophagy process (Atg5, Atg14, Beclin-1, Vps15, Vps34)
were found in MAM under starved conditions (and this localization
was stable throughout all the autophagosome formation process)
and alterations in ER-mitochondria tethering led to decreased
autophagy [34]. In addition, fundamental processes, such as lipida-
tion of LC3-I into LC3-II and the recruitment of calnexin to raft-like
microdomains containing glanglioside-3 (GD3) [74,75], have been
shown to depend on ER-mitochondria connection (Fig. 2), placing
this inter-organelle interface at the heart of the autophagy pro-
cess. Very recently, the OMM protein FUNDC1, which functions
as LC3 receptor during hypoxia-induced mitophagy [76], has been
also found to be enriched in MAM and function as an adaptor
protein coordinating mitochondrial dynamics and quality control,
in response to different signaling pathways and mitochondrial
stresses. Depending on its phosphorylation pattern, FUNDC1 can
interact with calnexin, DRP1 and OPA1, mediating either mito-
chondria elongation in response to hypoxic stimuli or mitochondria
fragmentation and mitophagy [77,78].
A further cell process that could involve ER-mitochondria
contacts is the formation and regulation of inflammasomes, multi-
molecular complexes that work as a platform for the processing
and release of pro-inflammatory cytokines [79]. One of these com-
plexes, the Nod-like receptor NLRP3, that mediates the activation
of interleukins IL1␤ and IL18, localizes, when inactive, to the ER but,
upon inflammosome activation, redistributes to ER-mitochondria
clusters comprising MAM [80]. Since one of the physiological trig-
gers of NLRP3 activation is ROS, and given that mitochondria and
ER are major intracellular locations for ROS production, MAM
represent a privileged sites enriched in ROS [81] where the inflam-
masome becomes active (Fig. 2). Interestingly, upon knock-down
of VDAC1, which is enriched in MAM and mediates mitochon-
drial Ca2+ entry (see below), inflammasome activation is reduced
[80], indicating that ER-mitochondria Ca2+ cross-talk and conse-
quent mitochondrial ROS production play a crucial role in this
phenomenon (see [82–84] for recent reviews). Moreover, in cystic
fibrosis cell models, it has been recently shown that mitochon-
drial Ca2+ uptake integrates pro-inflammatory signals initiated
by pathogen-associated molecules and relays the information to
NLRP3 [85], acting as a synergic stress stimulus in triggering exac-
erbated inflammatory response.
 R. Filadi et al. / Cell Calcium 62 (2017) 1–15
Physical ER-mitochondria juxtapositions have been recently
reported to be spatially linked to mtDNA synthesis and distribution
in dividing mitochondria in both yeast [60,86] and human cells [87],
supporting the idea that ER-mitochondria contacts are involved in
the distribution of mtDNA copies to daughter mitochondria during
the division process.
3.2. ER-mitochondria Ca2+ cross-talk functionalities
Ca2+ uptake into mitochondrial matrix is known to occur since
the pivotal studies on isolated mitochondria in the 60 s [88]. It was
early clear that the electrochemical gradient (, ∼ -180 mV, neg-
ative on the side of the matrix), generated by the activity of the
respiratory chain across the IMM [89], represents the ultimate ther-
modynamic basis responsible for the import of the cation. However,
in the 70 s, the accurate definition of the characteristics/kinetics
of the specialized transport mechanisms that strictly control the
process [90], whose molecular identity has been recently clarified
[91,92], revealed that the so called “Mitochondrial Ca2+ uniporter”
(MCU) is activated and allows an appreciable uptake of the cation
into the matrix only in the presence of relatively high [Ca2+ ].
The interested readers are referred to some recent reviews for an
overview on MCU complex and other transporters identity/activity
[93–95].
In particular, the low MCU affinity for Ca2+ (Kd ∼ 15–20 ␮M)
does not match the physiological [Ca2+ ] reached in the cytosol
of living cells, ranging from 50 to 100 nM in resting conditions
to peaks of 1–3 ␮M, upon stimulation. In the early 90 s, the find-
ing that, upon cell stimulation, mitochondria take up Ca2+ with an
amplitude much higher than the predicted one was thus initially
puzzling [96]. These surprising results were obtained in living cells
thanks to the introduction of genetically encoded Ca2+ indicators
(firstly aequorin [96]) specifically targeted to mitochondrial matrix
(see [97] for a recent review on different mitochondria-targeted
Ca2+ probes). The puzzle was solved by the observation that part
of mitochondria is strategically located in very close apposition
to the sites of Ca2+ release from the ER [2,98] (see also above).
In these regions, the fast opening of specific Ca2+ channels on ER
membranes (such as IP3Rs and/or RyRs) induces the generation
of high [Ca2+ ] microdomains in proximity of their mouths. Juxta-
posed mitochondria experience these Ca2+ hot-spots (i.e., they are
locally exposed to a [Ca2+ ] one order of magnitude higher than that
of the bulk cytoplasm [46,99]), allowing to overcome the thresh-
old for a prompt MCU activation and a fast mitochondrial Ca2+
uptake (Fig. 1C). On the other hand, the latter process locally buffers
cytosolic Ca2+ rises, deeply affecting the allosteric modulation of
ER Ca2+ -releasing channels. The details of the process that leads
to the generation of Ca2+ microdomains and their relationship to
mitochondria has been recently reviewed [39], as well as the role
and significance of mitochondrial Ca2+ uptake in cell functionality
[100]. This contribution will be limited to a brief summary of the
main aspects of mitochondrial functionality regulated by Ca2+ , tak-
ing into particular consideration the signaling events that are more
directly affected by the close proximity with the ER.
On the consequences linked to [Ca2+ ] rises within mitochon-
dria, two possible and opposite effects can be achieved: stimulation
of the Krebs cycle, NADH formation and fueling of the respira-
tory chain activity, with increased ATP production; or sensitization
towards the intrinsic apoptosis pathway, due to mitochondrial Ca2+
overload, opening of the mitochondrial Permeability Transition
Pore (mPTP) and release of cytochrome c (Fig. 2).
As to the former, it is well established that transient increases
in matrix [Ca2+ ], such as those obtained upon an IP3-dependent
ER Ca2+ release, stimulate NADH production and ATP synthe-
sis [101,102]. Indeed, three different enzymes, one immediately
upstream of, and the other two directly taking part in the Krebs
7
cycle, are modulated by matrix Ca2+ [103]. The first one (Pyru-
vate Dehydrogenase, PDH) converts pyruvate into acetyl-CoA, that
enters into the Krebs cycle. PDH is active when de-phosphorylated
and Ca2+ activates its phosphatase. The other two enzymes (Isoc-
itrate Dehydrogenase, ICDH, and Oxoglutarate Dehydrogenase,
OGDH) are both regulated by Ca2+ through its direct binding and
allosteric modulation (Kd of 20–30 ␮M and 1 ␮M, respectively
[104]), inducing an increased affinity for their substrates. A fourth
Ca2+ -sensitive mitochondrial dehydrogenase, the FAD-dependent
Glycerol-phosphate Dehydrogenase (GPDH), is located in the IMM
and senses the cation concentration in the intermembrane space
(IMS), thanks to a here located Ca2+ binding motif (Kd for Ca2+
of 0.1 ␮M). GPDH transfers reducing equivalents from glycolytic
NADH to the electron transport chain (ETC) as FADH2 and favors
ATP production. Moreover, it has been reported in isolated mito-
chondria that Ca2+ can directly stimulate the ETC and the F0 F1
ATP synthase activity [105,106]. In addition to the above described
matrix Ca2+ effects, specific IMM-located mitochondrial carriers
for different nucleotides/metabolites/co-factors are modulated by
IMS [Ca2+ ]. These Ca2+ -binding mitochondrial carriers (CaMCs) are
usually endowed with N-terminal EF Ca2+ -binding domains facing
the IMS [107,108]. Recently, in a very elegant study, a constitutive,
low-level IP3-mediated ER Ca2+ release has been demonstrated to
be essential for the stimulation of mitochondrial ATP production,
likely through mitochondrial uptake of the released ER Ca2+ and
the above described stimulatory effects [109]. Indeed, the block of
this ER-mitochondria Ca2+ shuttling (through the IP3Rs inhibitor
Xestospongin B or by genetic IP3Rs deletion) deeply impaired
mitochondrial ATP synthesis, leading to an increased intracellular
AMP/ATP ratio, AMPK activation and a potent autophagy induction.
Similarly, stable MCU/MCUR1 knockdown, that dampens mito-
chondrial Ca2+ uptake, reduces cellular oxygen consumption rate,
activates AMPK and induces autophagy [110]. The pro-survival
effect of a physiological ER-mitochondria Ca2+ transfer has been
also suggested by a transient increased ER-mitochondria coupling
during the early phases of ER-stress and unfolded protein response
(UPR) [111]. This strengthened physical apposition has been inter-
preted as an attempt to overcome ER stress, energizing the cells
by stimulating mitochondrial ATP production via increased matrix
[Ca2+ ] (Fig. 2).
While physiological mitochondrial [Ca2+ ] stimulate ATP pro-
duction, excessive accumulation of the cation in the matrix (Ca2+
overload) leads to the so called “permeability transition”. This
latter, mediated by a Ca2+ -sensitive, high-conductance and voltage-
dependent channel (mPTP) represents an increased permeability of
the IMM to small molecules (up to 1500 Da), that in turn induces
mitochondrial depolarization, impairs oxidative phosphorylation,
increases ROS production, leads to mitochondrial swelling and
OMM rupture, with release of IMS-resident pro-apoptotic factors
(reviewed in [112,113]). Among them, of particular importance are
cytochrome c, apoptosis induction factor (AIF), Smac/DIABLO, pro-
caspase 9 and endonuclease G. The release of these factors induces
three different events (reviewed in [113]). The first is the assem-
bly in the cytosol of the “apoptosome” (induced by cytochrome c
interaction with the apoptosis protease activating factor 1, APAF1),
which activates pro-caspase 9 and effector caspases (caspase-3, −6,
−7). The second is the inhibition, by Smac/DIABLO, of caspase’s
inhibitors, such as inhibitor of apoptosis protein (IAP). The third is
represented by DNA fragmentation and chromatin condensation,
by AIF and endonuclease G.
The molecular identity of the mPTP remained elusive up to
few years ago. It was firstly demonstrated that the c subunit of
the ATP synthase is a major regulator of mPTP activity [114];
then, dimers of the F0 F1 ATP synthase have been proposed to
form the mPTP [115]. Though there is consensus on the fact
8 R. Filadi et al. / Cell Calcium 62 (2017) 1–15
that the ATP synthase is an essential component of the mPTP,
more debated is the exact definition of which ATP synthase sub-
units directly take part in its formation [116]. Moreover, recently,
a different mechanism was proposed to mediate mitochondrial
Ca2+ -induced cell death, represented by the Ca2+ binding to the
IMM-located cardiolipin, cardiolipin coalescence, disassembling of
the cardiolipin-associated respiratory complex II, release of a ROS
generating sub-complex and cell death triggering [117]. Finally, the
importance of the ER-mitochondria platform in determining cell
life or death decisions is further strengthened by multiple evidence
indicating that different oncogenes and tumor suppressors actively
shape ER to mitochondria Ca2+ transfer (see below and [118,119]).
ER-mitochondria coupling is also crucial in modulating cell
metabolism. Indeed, at cellular level, metabolism relies on differ-
ent complementary and exclusive pathways, compartmentalized
within the eukaryotic cell. In this regard, the regulation of metabolic
pathways, in order to maintain proper cell energy homeostasis,
depends on the activity of different organelles. In particular, ER
and mitochondria are two major organelles controlling cellular
metabolism and energy production. On one hand, mitochondria
are the conductor in adapting energy production to energy require-
ments. By hosting the Krebs cycle, which is the end-point of lipid,
glucose and glutamine catabolism, as well as the ETC responsible
for the production of ATP through the consumption of reductive
power, mitochondria have a major impact on metabolite fluxes,
energy charge and redox state of the cell. On the other hand, criti-
cal steps of glucose and lipid anabolism take place within the ER, as
well as protein synthesis and sorting, making the organelle a cru-
cial actor in metabolic homeostasis. Hence, proper cell metabolism
is directly linked to the proper activity of these two compartments,
which in turn is relying on their mutual interaction.
As described above, Ca2+ transfer from the ER to mitochondria
stimulates, and is necessary for, mitochondrial activity, regulat-
ing cell redox state and ATP production [101,102]. However, the
importance of Ca2+ homeostasis in the control of metabolism is not
limited to mitochondria. Indeed, cytosolic Ca2+ directly activates
glucose metabolism by stimulating the activity of gluconeogene-
sis and glycogenolysis enzymes in the liver [120]. Moreover, the
action of glucagon and catecholamines, hormones participating
in the control of glucose metabolism, is mediated by increases in
cytosolic Ca2+ and subsequent positive modulation of calmodulin-
dependent kinases and calcineurin, leading to the activation of
the transcription factor FOXO [121]. In this pathway, ER-located
IP3Rs play a central role, mediating cytosolic Ca2+ oscillations,
through the release of the cation from the ER but also through
its direct interaction with the cAMP response element-binding
protein-regulated transcription coactivator 2 (CRTC2) promoting
its calcineurin-mediated phosphorylation/activation [122]. Thus,
the importance of the ER-mitochondria axis in the control of
cellular Ca2+ homeostasis gives to this interface a major role
in the control of metabolism, not only by stimulating mito-
chondrial activity but also regulating the action of different
hormones. Interestingly, insulin, a major actor in the control
of the entire body metabolism, induces an inhibitory phospho-
rylation of IP3Rs by activating the MAM-located enzyme AKT
[123]. Moreover, mTORC2 and PTEN, two proteins which are
part of the insulin signaling cascade, are also present at the
ER-mitochondria interface [124,125], and mTORC2 and phospho-
rylated AKT are recruited to MAM upon insulin stimulation in the
liver [124,126].
By this dual role in Ca2+ -mediated control of mitochondrial
activity, cytosolic enzymes and insulin signaling, ER-mitochondria
platform appears as a crucial element in the maintenance and
adaptation of cell metabolism to external stimuli and energy
requirements.
4. Significance: dysfunctions in ER-mitochondria coupling
and diseases
Since communication between ER and mitochondria regulates
a multitude of physiological processes (see above), it is not surpris-
ing that recently alterations in organelles’ physical and functional
tethering have been proposed as a common hallmark in differ-
ent pathologies, such as neurodegenerative diseases, metabolism
syndrome and cancer. A comprehensive and detailed dissertation
on this aspect is far away from the scope of the present contribu-
tion. We here present a brief summary of the main studies finding
alterations in ER-mitochondria pathways that can be linked to the
pathogenesis of specific human diseases (Table 1). The interested
reader is also referred to additional reviews on this topic [127–129].
4.1. Metabolic syndrome and inflexibility
Mitochondria and ER activity are essential in the control of
metabolic homeostasis (see above) and, in general, impaired func-
tions of the ER [130] and mitochondria [131] have been associated
with obesity, insulin resistance (IR) and metabolic defects. Given
the inter-dependency of these two organelles, the ER-mitochondria
axis has recently emerged as a potential central actor in the
pathophysiology of metabolic syndrome [132]. Indeed, genetically
or diet-induced obesity and IR have been associated with dis-
rupted ER-mitochondria interactions in liver and neurons [40],
even though a debate remains whether organelles’ juxtaposition
is increased [133] or decreased [126,134] in models of hepatic IR.
However, growing evidence indicates that experimental disrup-
tion of ER-mitochondria interactions can induce IR. For example,
the knock-out of a subunit of mTORC2, which induces a reduced
ER-mitochondria association in MEF cells [124], has been associ-
ated with IR in the liver [135]. Moreover, Mfn2 ablation (to alter,
in a positive or negative manner (see above), the physiological ER-
mitochondria interaction) has been associated in POMC neurons
with leptin-resistance, leading to obesity [40] (but see also above),
and with IR in hepatocytes and myotubes [136].
Ca2+ is likely to represent the mechanistic link between
ER-mitochondria interaction and metabolic impairment. Indeed,
disruption of Ca2+ homeostasis has been shown to mediate the
suppression of hepatic insulin signaling through Ca2+ /calmodulin-
dependent protein kinase II hyper-activation [137] and to link
mitochondrial dysfunction to ER stress and hepatic IR [138]; the
later phenomenon was in fact counteracted by SERCA overex-
pression to restore ER Ca2+ homeostasis in the liver of obese
mice [139]. Moreover, specific alteration of mitochondrial Ca2+
uptake can disrupt insulin signaling in cardiomyocytes [140],
and IP3R1 heterozygous mutant mice develop glucose intoler-
ance and hypersensitivity to diet-induced metabolic syndrome
[141]. Likewise, mice knock-out for CypD, a protein that modulates
ER-mitochondria coupling in liver and cardiomyocytes [126,142],
have been shown to develop hepatic IR through MAM impair-
ment and subsequent disruption of Ca2+ homeostasis [143], while
ablation of the MAM-located protein cisd2 induces cytosolic Ca2+
de-regulation, mitochondrial dysfunction and IR in the adipose tis-
sue [144]. If these studies provide convincing proofs that MAM
impairment can lead to disrupted metabolism, it is likely than an
excess of ER-mitochondria communication can have similar con-
sequences, as shown by the fact that liver specific overexpression
of PACS-2 or IP3R1 in mice is linked to IR [133].
On this line, tools to modulate ER-mitochondria interactions
could represent an efficient approach to restore metabolic homeo-
stasis. Indeed, hepatic insulin sensitivity can be rescued in models
of obesity and IR, in which MAM have been shown to be decreased,
through the overexpression of CypD [126]. An interesting ele-
ment is that there seems to be an interdependency between
 R. Filadi et al. / Cell Calcium 62 (2017) 1–15 9

Table 1
Main disease-related proteins, and relative interactors, reported to modulate ER-mitochondria coupling. See text for details.

PROTEINS FUNCTIONS INTERACTORS ASSOCIATED DISEASES REFERENCES

VAPB Physically/functionally tethers ER to mitochondria by PTPIP51 Amyotrophic Lateral 19; 20

interacting with the OMM-located PTPIP51 Sclerosis (ALS)
PTPIP51 Physically/functionally tethers mitochondria to ER by VAPB ALS 19; 20
interacting with the ER-located VAPB
TDP-43 Disrupts ER-mitochondria apposition by a GSK-3␤-mediated GSK-3␤ ALS 20
disassembly of the VAPB-PTPIP51 complex (VAPB/PTPIP51)
FUS Disrupts ER-mitochondria apposition by a GSK-3␤-mediated GSK-3␤ ALS 21
disassembly of the VAPB-PTPIP51 complex (VAPB/PTPIP51)
SOD1 Expression of hSOD1-G93A mutant alters ER-mitochondria ? ALS 183
Ca2+ homeostasis
SIGMA1-R Loss of SIGMA1-R or expression of loss-of-function mutants IP3Rs ALS; Distal Hereditary 184; 185
impair ER-mitochondria communication Motor Neuropathies
(dHMNs)
PS2 Expression of PS2, especially of FAD-mutants, favors Mfn2 Alzheimer’s disease 28; 29; 30
ER-mitochondria physical/functional coupling, by sequestering (AD)
Mfn2
Mfn2 Modulates either positively or negatively ER-mitochondria Mfn2/Mfn1; PS2; Charcot-Marie-Tooth 31; 35; 41; 8
physical/functional juxtaposition MITOL Neuropathy Type 2A 40; 136
(CMT2A)
Obesity, Insulin
resistance (IR)
AKT/PKB Inhibits ER Ca2+ release and negatively affects Ca2+ -dependent IP3Rs Cancer 123; 155; 156
apoptotis by phosphorylating IP3Rs
PTEN Sensitizes cells to Ca2+ -dependent apoptosis by IP3Rs Cancer 125
de-phosphorylating IP3Rs and promoting its activity
PML Promotes ER-mitochondria Ca2+ transfer and apoptosis by IP3R3/AKT/PP2A Cancer 157
forming a complex containing IP3R3, AKT and PP2A. PP2A
suppresses AKT-mediated IP3R3 phosphorylation, favoring its
activity
BRCA1 It is recruited to ER during apoptosis, sensitizing the IP3Rs to IP3Rs Cancer 158
low [IP3]
FATE1 Uncouples ER from mitochondria, protecting from Ca2+ - and ? Cancer 159
drug-induced cell death
PARKIN Favors ER-mitochondria coupling, increasing mitochondrial ? Parkinson’s disease 174
Ca2+ uptake and sustaining ATP synthesis (PD)
␣-synuclein Favors mitochondrial Ca2+ uptake by enhancing ? PD 177; 178
ER-mitochondria apposition; disease-associated mutants
uncouple the two organelles
DJ-1 Promotes ER-mitochondria coupling by counteracting p53 p53 PD 179; 180
activity; wt, but not pathogenic mutants, sustains ATP
production
mTORC2 Promotes ER-mitochondria coupling and has been proposed to IP3R-Grp75-VDAC1 IR 124; 135
be an MAM signaling hub by controlling AKT mediated complex
phosphorylation of IP3R, HK2 and PACS-2 AKT
PACS-2
HK2
IP3R1 ER Ca2+ release channel Grp75 IR 133; 141; 18;
VDAC1 Metabolic syndrome 123; 125
AKT
PTEN
PACS-2 Favors physical/functional ER-mitochondria juxtaposition and Bid IR 24; 26; 133
controls Bid-mediated apotosis Fis-1/BAP31
CypD Promotes ER-mitochondria coupling through an unknown IP3R-Grp75-VDAC1 IR 126; 142; 143
mechanism complex Hypoxia-
reoxygenation
injury
cisd2 Although its function remains unclear, this protein is crucial in ? Wolfram syndrome 144
the maintenance of Ca2+ homeostasis and ER/mitochondria IR
function

ER-mitochondria coupling and metabolic homeostasis since high-
fat diet and CypD knock-out mice, two models in which hepatic
IR have been linked to reduced interactions between ER and
mitochondria, exhibited a restored MAM quantity after insulin sen-
sitivity improvement upon specific pharmacological treatments
[126]. However, these findings are still under intensive debate,
since a major study showed opposite results, i.e., in the liver of a
genetic mouse model of obesity and IR (ob/ob mice), an increased
interaction between the two organelles, leading to Ca2+ -mediated
mitochondrial dysfunction, and an ameliorated insulin sensitivity,
upon inhibition of organelle juxtaposition by the knocking-down
of PACS-2 or IP3R1, have been observed [133]. The fact that both
increasing and decreasing organelles coupling lead to metabolic
dysfunctionalities can be re-conciliated within the idea that for cell
metabolism, the strength of ER-mitochondria coupling are neither
good nor bad. The right amount needs to be suited to the cellu-
lar state, and both Ca2+ overload and impaired Ca2+ uptake could
lead to mitochondrial dysfunctions. However, the understanding of
why similar experimental models and tissues can produce opposite
outcomes modulating the MAM state will require further investi-
gation. Part of the answer probably resides in the highly dynamic
characteristic of both metabolism and ER-mitochondria interac-
tions, in which adaptive responses to metabolic challenges and
environmental stimuli can influence MAM formation/stability.
10 R. Filadi et al. / Cell Calcium 62 (2017) 1–15
IR is usually seen as a disruption of a cellular signaling pathway.
A more recent and complete way of approaching metabolic dis-
eases is to picture metabolism as a flexible network of enzymes
and metabolites that adapts to external nutritional stimuli, a
phenomenon called metabolic flexibility [145]. Thus, metabolic
impairment can be seen as the inability of the organism to switch
nutrient sources between fatty acid and glucose in different ener-
getic contexts: fasting and fed states, physical activity or hormone
stimulation [145]. Given their importance in energy homeostasis,
ER and mitochondrial functions are crucial in adapting metabolism
to nutrient availability and have been shown to be key actors in
metabolic flexibility at the cellular level [146].
ER-mitochondria coupling has recently emerged as a new actor
in the modulation of metabolism. Indeed, the adaptation of this
functional relationship participates in the control of mitochon-
drial activity regarding energy requirements and metabolic context
[134]. In particular, MAM quantity increases during fasting in the
liver [147], a phenomenon that was shown to be controlled by
the modulation of the pentose phosphate-protein phosphatase
2 A (PP2A) pathway by glucose availability, leading to subsequent
mitochondrial fusion and increased Ca2+ uptake and metabolism
of the organelle [148]. Interestingly, mitochondrial dysfunction
has been implicated in metabolic inflexibility [146], and a recent
study demonstrated an impairment of ER-mitochondria coupling
modulation upon fasting in the liver of IR mice, associated with
mitochondrial fragmentation and decreased oxygen consumption
[148]. This observation is supported by the finding that a chronic
hyper-activation of PP2A, a negative pathway for MAM formation,
has been found in the IR liver [149,150].
4.2. Cancer
Over the last years, increasing evidence has been provided on
the capacity of cancer cells to remodel intracellular Ca2+ signaling,
favoring their survival and proliferation. Among multiple mech-
anisms, enhanced resistance to apoptosis of cancer cells involves
modulation of the ER-mitochondria Ca2+ cross-talk. Several tumors
display an altered expression of proteins, including oncogenes and
tumor suppressors, that directly affect MAM functionality and in
particular ER-mitochondria Ca2+ transfer [151,152] (Fig. 2). Below,
we provide few examples, and the interested readers are referred
to a recent review for a more complete list of the proteins that have
been involved in these pathways [119].
In general, tumor suppressors enhance ER-mitochondria pro-
apoptotic Ca2+ transfer, while oncogenes dampen it. For instance,
the oncogenes Mcl-1, Bcl-2 and Bcl-XL decrease ER Ca2+ con-
tent, likely by increasing the basal IP3R-dependent Ca2+ leak,
avoiding ER-derived mitochondrial Ca2+ overload upon stress con-
ditions (reviewed in [119]). On the contrary, a MAM resident
fraction of the tumor suppressor p53 physically interacts with,
and enhances the activity of, the Ca2+ -ATPase SERCA, increasing
ER [Ca2+ ] and sensitizing cells to apoptosis [153]. In addition to
the above mentioned effects on IP3Rs, Bcl-2 has also been reported
to interact with SERCA and limit its activity [154]. The oncogene
AKT (also known as PKB) has been reported to phosphorylate
IP3Rs [155], inhibiting ER Ca2+ release and negatively affecting
the Ca2+ -dependent apoptotic response [123,156]. On the contrary,
the tumor suppressor protein phosphatase and tensin homolog
(PTEN), commonly lost or mutated in human cancers, is one of the
most important negative regulator of the AKT pathway. Indeed, not
only it dephosphorylates phosphatidylinositol 3,4,5-trisphosphate
(PIP3) to phosphatidylinositol 4,5-bisphosphate (PIP2), via its lipid
phosphatase activity (thus blocking PIP3-dependent AKT recruit-
ment/activation to plasma membrane), but also directly interacts
with IP3Rs and removes their AKT-dependent phosphorylation
[125]. This latter effect is exerted specifically by an ER-resident
fraction of PTEN, that increases after apoptosis stimulation with
arachidonic acid: the resulting increased ER Ca2+ release induces
mitochondrial Ca2+ overload, sensitizing cells to apoptosis [125].
The tumor suppressor PML has been shown to act on the same path-
way as well: by interacting with IP3Rs, it promotes the formation
of a complex containing IP3R3, AKT and the protein phosphatase
PP2A. In turn, PP2A suppresses AKT-mediated IP3R3 phosphoryla-
tion, thus promoting ER-mitochondria Ca2+ transfer and apoptosis
[157]. The tumor suppressor proteins BRCA1 was found to be
recruited to the ER during apoptosis in an IP3R-dependent manner,
sensitizing the IP3R to low [IP3], thus favoring the Ca2+ -dependent
apoptotic response [158]. Recently, the cancer-testis antigen FATE1
has been reported to uncouple ER from mitochondria, protect-
ing from Ca2+ - and drug-induced cell death [159]. Though cancer
cells avoid massive mitochondrial Ca2+ uptake, as a mechanism
to escape from Ca2+ -overload and apoptosis, a constitutive, low
level ER to mitochondria Ca2+ transfer is essential for their viabil-
ity. Indeed, very recently, it has been demonstrated that a basal
mitochondrial Ca2+ uptake is necessary to sustain tumor cells
bioenergetics and to provide them with essential metabolites [160].
Overall, these recent findings highlight the importance of the mod-
ulation of ER-mitochondria interface in cancer cells.
4.3. Alzheimer’s disease
Alzheimer’s disease (AD) is an irreversible neurodegenerative
disorder, whose pathogenesis has been classically considered as
strictly dependent on an altered production of the amyloid beta
(A␤) peptide. The idea that A␤ generation is the key event, whose
perturbation is at the basis of AD onset, found strong genetic
supports in the middle of the 90’s, with the discovery that the
familial forms of AD (FAD) are linked to autosomal dominant muta-
tions in three genes, encoding for amyloid precursor protein (APP),
Presenilin-1 (PS1) and Presenilin-2 (PS2). Indeed, A␤ is generated
from APP, thanks to its cleavage by the ␤- and the ␥-secretases;
PS1 and PS2 (as alternative catalytic core of ␥-secretase) are fun-
damental in the process. Though the pathogenicity of A␤ has
been well established, so far many efforts aimed at controlling the
generation or the accumulation of the different species of the pep-
tide (typically, A␤40 and A␤42) failed to convincingly impact on
the onset/progression of the pathology, suggesting that additional
molecular mechanisms may be altered since the early phases of the
disease. In addition to anomalies in A␤ generation, AD patients dis-
play different early intracellular alterations, such as Ca2+ and lipids
dyshomeostasis, increased ROS levels, impairments in autophagy,
axonal transport and mitochondrial dynamics. Interestingly, MAM
are the central hub where many of these activities are controlled or
directly take place. Thus, very recently, the hypothesis that AD may
be a disease associated to alterations of MAM has started to emerge
as an interesting possibility. The discovery that both PS1 and PS2
are enriched in MAM [27], as well as APP, was the first hit in sup-
port of this idea. The presence of PSs, APP and ␥-secretase activity
in MAM has been independently confirmed by others [161], and
we recently reported that FAD-PS2 mutants are enriched in MAM,
compared to the wt protein [30].
More debated is the role of PSs in ER-mitochondria con-
tact sites. We firstly reported that the expression of the only
PS2 (wt and, more potently, FAD-mutants), but not of PS1,
favors ER-mitochondria physical and functional coupling, result-
ing in an increased ER-mitochondria Ca2+ transfer [28]. Later,
both endogenous PS1 and PS2 have been suggested to negatively
modulate ER-mitochondria coupling, while their FAD-mutants
increase organelles’ apposition [162], affecting phospholipids and
cholesterol metabolism. In this latter paper, an increased ER-
mitochondria coupling has been observed in fibroblasts from both
FAD and sporadic AD patients. Though the observation is poten-
 R. Filadi et al. / Cell Calcium 62 (2017) 1–15
tially interesting, in our opinion it is not clear how sporadic AD
cases may converge on MAM alterations similarly to those mecha-
nistically caused by PSs or APP mutations, as it has been proposed
[162]. Moreover, we have recently confirmed that only PS2 is able to
increase ER-mitochondria tethering, by binding and sequestering
Mfn2 [30], thus removing the inhibitory effect of the latter protein
on organelles’ apposition [8]. In addition, in another study, neu-
ronal FAD-PS1 expression has been associated with a decreased
ER-mitochondria physical and functional coupling [163].
Several other evidence supports the link between ER-
mitochondria association and AD. For example, acute exposure to
pathogenic levels of A␤ has been reported to strengthen ER to mito-
chondria Ca2+ transfer [164] and an increased ER-mitochondria
coupling has been observed in different AD models [29,164]. Very
recently, it has been shown that Mfn2 down-regulation, likely by
increasing ER-mitochondria association, as verified by quantita-
tive EM, results in a substantial alteration in the process of A␤
generation [44]. This defect was associated to an impairment of
␥-secretase maturation.
The relationship between A␤ peptides and mitochondria
remains also unclear. A␤ accumulation into mitochondria has been
reported and suggested to increase ROS production and impair
mitochondrial respiration [165]. A multitude of mitochondrial
functions have been reported to be affected by A␤ [166,167]. Pro-
vided that the machinery for A␤ production is not present in
mitochondria, A␤ has been proposed to be taken up by mitochon-
dria via a TIM/TOM-mediated (but -independent) mechanism
and observed, by immunoelectron microscopy, to localize to mito-
chondrial cristae [168]. A specific effect of A␤ on the processing
of most N-terminal presequences of mitochondrial proteins has
been reported and suggested to induce an imbalanced organel-
lar proteome, resulting in mitochondrial dysfunctions [169]. Very
recently, an elegant study failed to find an A␤ import activity into
mitochondria, but confirmed A␤ association (especially of A␤42)
with the organelle, likely due to OMM interactions [170]. Though
the study was performed on isolated mitochondria, potentially los-
ing some additional molecular components that may impact on the
in vivo outcome, A␤ was not found to directly affect mitochondrial
functionalities, but to impair mitochondrial pre-proteins import
within the organelle, because of its aggregation with these pro-
teins [170]. However, beyond the specific effects on mitochondria,
it is still unclear how A␤ reaches these organelles. Indeed, after its
generation from plasma membrane-located APP, A␤ is released in
the extracellular environment or, in case of intracellular genera-
tion, as it has been suggested [171], into the lumen of the involved
organelles. Even in the case of A␤ endocytosis from the extracel-
lular milieu [172], how peptides can reach the cytosol before to
interact with mitochondria remains elusive. The fact that A␤ can
be produced in MAM (i.e., in close proximity to mitochondria) may
help to elucidate this aspect, but the topological problem discussed
above remains, to date, an open question. Overall, we believe that,
though it is difficult to reconcile all these recent findings in one
mechanistic model, it starts to be clear that AD and MAM alter-
ations are associated. In particular, reports from different groups
create consensus on the fact that ER-mitochondria coupling is early
increased in AD pathogenesis. Whether this increased association
has a direct impact on AD onset/progression, or it is just an epiphe-
nomenon, or an attempt to protect cells from other insults, is still
unclear. In both cases, however, MAM modulation appears as a
promising novel therapeutic target to exploit for pharmacologi-
cal purpose. To this aim, the clarification of the precise molecular
mechanism, that leads to an increased ER-mitochondria coupling,
appears of fundamental importance. For instance, while FAD-PS2
mutants expression seems able to directly affect the tethering, it
is still not clear how other FAD-mutants, or still unknown molecu-
lar mechanisms in sporadic AD, may converge on ER-mitochondria
11
platform. A long term A␤ exposure appears as a possible path-
way, though further investigations will be necessary to address this
crucial point.
4.4. Parkinson’s disease
Among neurodegenerative diseases, Parkinson’s Disease (PD) is
probably the first one for which genetic studies suggested, since the
beginning, a strong and quite immediate correlation with mito-
chondrial alterations. Indeed, some of the proteins mutated in
familial forms of PD, such as PINK-1 and Parkin, are targeted to
or dynamically associated with mitochondria [173]. More recent
observations suggest that some of the PD-related proteins (not
only those classically associated with mitochondrial functionality,
see below) may be involved in the modulation of ER-mitochondria
apposition, thus suggesting the intriguing possibility that, similarly
to AD, even some of the alterations observed in PD could be linked
to MAM anomalies. In many cases, however, though the functions
of wt endogenous proteins have been elucidated, studies on the
effects of PD-related mutants are still lacking.
Endogenous Parkin, for instance, has been reported to favor
ER-mitochondria physical and functional coupling, increasing
mitochondrial Ca2+ uptake and sustaining ATP synthesis [174].
Recently, however, a strengthened ER-mitochondria apposition has
been described in fibroblasts from Parkin (PARK-2) knock-out mice
and from PD patients harboring PARK-2 mutations [175]. Higher
levels of Mfn2 (a target protein ubiquitinated by Parkin) were
observed and interpreted as responsible for the increased inter-
organelle association. In our opinion, being the list of Parkin’s
targets quite wide, the effects of Parkin ablation on the tether-
ing may, or may not, be Mfn2-dependent. Similarly, drosophila
PINK1/Parkin mutants displayed defective mitochondria that acti-
vate the PERK branch of UPR, a neurotoxic process potentially
modulated by Mfn2 [176]. ␣-synuclein has been also reported to
favor mitochondrial Ca2+ uptake, by enhancing ER-mitochondria
physical association [177]. Recently, ␣-synuclein has been reported
to be localized at MAM [178]: the disease-linked mutants are less
associated with MAM and induce ER-mitochondria uncoupling.
Overexpression of wt DJ-1 (a protein usually localized in the cyto-
sol − but see also below − whose mutant forms are associated with
familial PD) has been also shown to increase ER-mitochondria cou-
pling [179]. The final outcome is that DJ-1 is able to counteract the
negative effects of the tumor suppressor protein p53 on organelles
juxtaposition [179]. Recently, it has been shown that, upon star-
vation, a fraction of DJ-1 translocates into mitochondrial matrix to
sustain ATP synthesis, an ability which is lost by pathogenic DJ-1
mutants [180].
4.5. Amyotrophic lateral sclerosis
Amyotrophic Lateral Sclerosis (ALS) is the most common form
of motor neuron disease and is clinically and genetically associated
to Fronto-Temporal Dementia (FTD) [181]. Mutations in the genes
encoding for TDP-43, FUS and C9ORF72-derived protein are associ-
ated to ∼ 50% of the familial forms of the disease [181]. In neurons
and glia of patients, cytosolic and nuclear inclusions of these pro-
teins are observed. A key role for ER-mitochondria axis in ALS/FTD
onset/progression has been recently started to emerge (reviewed
in [182]). In particular, overexpression of both wt and mutated
TDP-43 decreases ER-mitochondria physical and functional cou-
pling [20], by disrupting VAPB-PTPIP51 interaction (see above)
through activation of GSK-3␤. With a very similar molecular mech-
anism, converging on a GSK-3␤-dependent disassembling of the
VAPB-PTPIP51 tethering complex, FUS (wt and ALS-linked mutants)
overexpression has been reported to impair ER-mitochondria jux-
taposition, Ca2+ transfer and, consequently, mitochondrial ATP
12 R. Filadi et al. / Cell Calcium 62 (2017) 1–15
production [21]. Importantly, expression of VAPB mutant forms
(associated with some familial forms of ALS type-8) increases
ER-mitochondria Ca2+ shuttling [19]. In mouse embryonic motor
neurons, overexpression of the ALS-related hSOD1 mutant G93A,
has been observed to alter ER and mitochondrial Ca2+ homeostasis
[183]. Moreover, loss of the MAM protein SIGMA1-R (a condition
linked to some familial forms of ALS/FTD) has been reported to
impair ER-mitochondria communication [184], similarly to Dis-
tal Hereditary Motor Neuropathies (dHMNs)-associated SIGMA1-R
loss-of-function mutations [185]. Overall, these studies strongly
suggest that alterations of ER-mitochondria platform could be an
early event at the basis of ALS/FTD.
5. Conclusion
ER-mitochondria coupling is increasingly suggested to play
essential roles in different fundamental cellular pathways. The
functional significances of this apposition have started to be
elucidated and are constantly growing. Given that alterations of ER-
mitochondria juxtaposition and/or cross-talk have been reported
to be early observed in different pathological conditions, the mod-
ulation of this key intracellular axis appears as a good candidate
for possible therapeutic strategies, aimed at hindering the onset
and progression of devastating diseases. For this purpose, the exact
knowledge of the molecules that control inter-organelle apposi-
tion appears of fundamental importance, and basic-science studies
seeking at defining their identity are encouraged.
Acknowledgements
The Authors thank grants from the University of Padova; the
Italian Ministry of University and Scientific Research; Fondazione
Cassa di Risparmio di Padova e Rovigo (CARIPARO Foundation;
Progetti d’eccellenza 2011/2012) and EU Joint Programme in
Neurodegenerative Disease, 2015-2018, “Cellular Bioenergetics in
Neurodegenerative Diseases: A system-based pathway and target
analysis” for their research work support. R.F. and P.T. are Research
fellows of the CARIPARO and EU Joint Programme in Neurodegen-
erative Disease grant, respectively.
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