# Contacting the gut: Mitochondria-associated Endoplasmic Reticulum Membranes in the Enteric Nervous System

Jenny Greig, Giada Delfino, Pascal Derkinderen, Sébastien Paillusson

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Jenny Greig, Giada Delfino, Pascal Derkinderen, Sébastien Paillusson. Contacting the gut: Mitochondria-associated Endoplasmic Reticulum Membranes in the Enteric Nervous System. Contact, 2024, 7, 10.1177/25152564241244941. inserm-04587199

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# Contacting the gut: Mitochondria-associated Endoplasmic Reticulum Membranes in the Enteric Nervous System

Jenny Greig<fnref n="1" />*, Giada Delfino<fnref n="2" />*, Pascal Derkinderen<fnref n="2" />, and Sébastien Paillusson<fnref n="2" /> ID

## Abstract

Changes in the connections between the endoplasmic reticulum (ER) and mitochondria, as well as alterations in mitochondria-associated ER membrane (MAM) signalling, have been documented in various neurodegenerative diseases affecting the brain. Despite the growing recognition of the significance of the gut-brain axis in neurodegenerative conditions, there has been no prior investigation into the biology of MAM within the enteric nervous system (ENS). Our recent research reveals, for the first time, the existence of connections between the ER and mitochondria within enteric neurons. Additionally, we observed alterations in the dynamics of these connections in the enteric neurons from a mouse model exhibiting age-related neurodegeneration. These findings provide the first detailed characterization of MAM in the ENS under physiological conditions and in a mouse model of age-associated neurodegeneration and shed new light on the potential role of enteric MAM in the context of neurodegenerative disorders.

## Keywords

enteric nervous system, mitochondria-associated ER membranes, Parkinson's disease, Alzheimer's disease, ageing

Over the past decade, changes in both mitochondrial-associated membrane (MAM) dynamics and signalling have consistently been linked to age-related neurodegenerative disorders such as Parkinson's disease (PD) and Alzheimer's disease (AD). Neuronal damage in these disorders often coincides with cellular functions regulated by contacts between the endoplasmic reticulum (ER) and mitochondria, as well as MAM signalling (Markovinovic et al., 2022).

A growing body of research indicates that the gut plays a significant role in neurodegeneration, as seen in PD, AD, and age-related neurodegeneration. The enteric nervous system (ENS), a key component of the gut-brain axis, governs major gastrointestinal functions (Markovinovic et al., 2022). Despite extensive research on MAM in the central nervous system (CNS), no data are available regarding MAM in the ENS, and this forms the focal point of the current study.

In our recently published study (Delfino et al., 2024), we have, for the first time, identified the presence of ER-mitochondria contacts in enteric neurons. Utilizing electron microscopy, we conducted a detailed characterization of these interactions. Our initial findings demonstrated that mitochondria naturally form MAM with ER membranes in enteric neurons of the myenteric plexus in young adult C57BL/6J mice. Approximately 14% of the total mitochondrial surface was found to be in contact with ER membranes. Notably, these connections exhibit similarities and are found

in the same order of magnitude to what is found in the neurons in the CNS, where 10 to 15% of the total mitochondrial surface is occupied by ER (Stoica et al., 2014, 2016). We further characterized the length, spatial distribution, and surface occupation of ER on mitochondria, revealing the dynamic nature of MAM in enteric neurons. Key MAM proteins were expressed in the gut, such as Sigma1R and mitofusin 2 and tethers such as VAPB and PTPIP51 were actively interacting in enteric neurons.

These observations prompted an exploration of ER-mitochondria associations in enteric neurons in a pathological context, using SAMP8 mice — a non-transgenic model of age-related neurodegeneration displaying both

## Corresponding Author:

Sébastien Paillusson, Nantes Université, INSERM, CHU Nantes, The Enteric Nervous System in Gut and Brain Disorders, Nantes, France.

Email: sebastien.paillusson@univ-nantes.fr

<figure><img src="image_2.png" /></figure>

<footnote n="1">¹INSERM, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France</footnote>

<footnote n="2">²Nantes Université, INSERM, CHU Nantes, The Enteric Nervous System in Gut and Brain Disorders, Nantes, France</footnote>

<footnote>*These authors have equally contributed to this work</footnote>

<footnote>Received February 1, 2024. Revised March 12, 2024. Accepted March 18, 2024</footnote>
CNS and GI dysfunctions. Our investigation revealed consti-
pation in 8-month-old SAMP8 mice compared to control
animals. Concurrently, SAMP8 enteric neurons exhibited
an increased number of ER-mitochondria contacts compared
to control mice, accompanied by, a reduction in PGP9.5 and
in HuD positive cells by 75% and 53%, respectively, indica-
tive of enteric neuronal loss. Interestingly, no difference in
ER-mitochondria contacts was observed in longitudinal
smooth muscle cells. We also found that calcium homeosta-
sis we altered in SAMP8 mice enteric neurons. Because the
non-transgenic SAMP8 model recapitulates some of the
complexity found in patients, it is not easy to draw a direct
link between MAM dysfunction and slow transit as symp-
toms cannot be solely attributed to MAM alteration.
However, our findings are rather suggesting that the observed
slow transit constipation in SAMP8 mice is the result of neur-
onal dysfunction leading to the loss of enteric neurons, rather
than a functional defect in smooth muscle cells. This indi-
cates that damage made to MAM in the enteric neurons
could contribute to altered gut function.

There is now mounting evidence showing that slow transit
is an enteric neuropathy rather than a functional disorder
(Bassotti et al., 2013), with many studies showing that
severely constipated patients exhibit abnormalities in the
ENS, such as changes in the neurochemical coding
(Wattchow et al., 2008). MAM plays a critical role in regu-
lating keys cellular functions in neurons, which include
calcium homeostasis, mitochondria biogenesis and oxidative
phosphorylation needed for ATP production, neurite growth,
synaptic activity and autophagy amongst others
(Markovinovic et al., 2022). Therefore, a defect in MAM sig-
nalling could trigger neuronal dysfunctions in the ENS
leading to gastrointestinal dysfunction. Despite current
knowledge, whether and how MAM regulate digestives func-
tions (e.g. intestinal motility and barrier permeability)
remains unexamined until our work. One way to understand
the direct role that MAM could play in regulating digestive
functions through enteric neurons is to modulate (up or
down) the amount of tethering between ER and mitochondria
in a neuronal-specific manner. In a recent publication, Lu
et al. have linked a reduction in ER-mitochondria associa-
tions with cardiac dysfunctions during ageing suggesting
that damage made to MAM during ageing could affect
other key organs like the heart (Lu et al., 2022).

From the literature (Markovinovic et al., 2022), both
VAPB-PTPIP51 and mitofusin 2 seem to be the most reliable
and interesting tethering proteins to modulate, since they
seem to be involved in short and large contact spacing
respectively which regulate different MAM functions.
Moreover, knock-out animal models for VAPB, PTPIP51
and mitofusin 2 are already available but haven't been
studied for gastrointestinal features yet.

Gastrointestinal disorders, in particular constipation, is
now considered as a characteristic feature in neurodegenera-
tive disorders such as PD or AD (Rao and Gershon, 2016);

however, the mechanistic make up behind this is still
poorly understood as no obvious neuronal loss seems to
occur. In the case of PD, growing evidence show that the
aggregation of alpha-synuclein, one of the major PD
drivers, occurs early in enteric neurons and closely mirrors
the synucleinopathy found in central neurons (de Guilhem
de Lataillade et al., 2020). Our hypothesis is that pathological
drivers like accumulation of alpha-synuclein or Aβ triggers
damage to MAM homeostasis in enteric neurons, which
alters neuronal function even without any neuronal loss in
a similar fashion to the CNS. In order to confirm this hypoth-
esis, a first critical step would be to perform an in-depth char-
acterization of MAM in the ENS of PD patients at different
disease stages. To do so, submucosal biopsies from PD
patients would be an ideal place to start as they offer fresh
tissue without any post-mortem artefacts, and multiple
samples could be taken from the same patient to perform a
comprehensive study, as one biopsy could be used for elec-
tronic microscopy, one for proximity ligation assay, one for
IHC and a last one for western blotting, giving a multiple
range of assays to draw a reliable conclusion. Aiming in
the same direction, more research is needed to characterize
whether MAM biology is affected both in the CNS and the
ENS during neurodegenerative disorders like PD, and if so
in what time scale. Therefore restoring the MAM in neurode-
generative disorders seems to be a way forward in alleviating
disease symptoms, and some clinical trials have even reached
phase II for PD with this approach (Payne et al., 2020).
However, because of the complexity of the damage to
MAM in a given disease, it is often difficult to determine
whether ER-mitochondria interactions would benefit from
being tightened or loosened, making it important to under-
stand the nature of the dysfunction in a particular patient.
Using enteric biopsies to monitor MAM status and changes
in tethering proteins in a patient could help to determine
the individual requirements for a modulating treatment for
MAM dysfunction.

Acknowledgements

We acknowledge the graphical abstract was partly generated using
BioRender software.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Fédération pour la Recherche sur le Cerveau (FRC), Association AMADYS, Conseil Régional des Pays de la Loire, Agence Nationale de la Recherche, (grant number PO CCI 2014 FR16M20P008, ANR-16-IDEX-0007).
ORCID iD

Sébastien Paillusson https://orcid.org/0000-0001-7013-5582

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