Ktorza Etan

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SERAC1 is a component ofthe mitochondrial serine transporter complex required

forthe maintenance of mitochondrial DNA.

Abstract:
Mitochondrial diseases constitute a heterogeneous group of genetic diseases 1. These diseases are
linked to deficiencies in intracellular energy production, particularly in the oxidative phosphorylation
(OXPHOS) system2, and are expressed mainly in energy-dependent tissues.
Among these diseases, Serine deficiency site-containing protein 1(protein found at the interface of
mitochondria) is associated with the mitochondrial 3-methylglutaconic aciduria with deafness,
(hepatopathy), encephalopathy, Leigh-like syndrome disease and MEGDEL syndrome. We would
study here one article highlighting the importance Of SERAC1 as a component of the mitochondrial
serine transporter complex that required for the maintenance of mitochondrial DNA.

Introduction:
Several studies have shown that dNTP (The deoxyribonucleoside triphosphates (dNTPs) are the
essential building blocks for DNA replication and repair) plays an important role for the mtDNA
maintenance. In fact, nine genes responsible for mitochondrial diseases are also involved in the
regulation of mitochondrial dNTP3 homeostasis, and mutations in these genes have been associated
with a subtype of OXPHOS disorders. In addition, nucleotides derived from the one-carbon cycle
(One-carbon (1C) metabolism comprises a series of interlinking metabolic pathways that include the
methionine and folate cycles that are central to cellular function) have also been shown to be essential
for DNA replication. All these informations suggest the existence of a link between mtDNA
replication and one-carbon cycle metabolism 4 including a hypothesized role of one-carbon cycle to
dNTPs.
In this study, the research demonstrated that SERAC1: a protein found at the interface of
mitochondria, is an indispensable component of the one-carbon cycle machinery. In fact,
in addition to the known functions of SERAC1 they found that SERAC1 facilitates the transport of
cytosolic serine into the mitochondria by interacting with and stabilizing SFXN1, (a mitochondrial

1
S. Rahman, Mitochondrial disease in children. J. Intern. Med. 287, 609–633 (2020)

2
O. M. Russell, G. S. Gorman, R. N. Lightowlers, D. M. Turnbull, Mitochondrial diseases: Hope for the future. Cell 181, 168–188 (2020).

3
A. E. Frazier, D. R. Thorburn, A. G. Compton, Mitochondrial energy generation disorders: Genes, mechanisms, and clues to pathology. J. Biol.
Chem. 294, 5386–5395 (2019).

4
. A. Suomalainen, B. J. Battersby, Mitochondrial diseases: The contribution of organelle stress responses to pathology. Nat. Rev. Mol. Cell Biol. 19,
77–92 (2018).
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serine transporter)5. SERAC1 depletion in cells and mouse models blocked the one-carbon cycle and
disrupted the cellular nucleotide pool, leading to the loss of mtDNA and compromised OXPHOS,
which the administration of dNTP precursors partially reversed.

Results:
In order to determine what role plays SERAC1, the researchers first determined the effects of
SERAC1 deletion on mice.
For this, the researchers created a mouse mutant for the SERAC gene by deleting exons 3 to 7: a
Serac1-/- constitutive mouse (fig. S1, A to D). This SERAC1 mutation induces neither sex bias nor
causes embryonic lethality. By observing these mice over a period of 6.5 months the researchers
observed several interesting results: In fact, the deletion of Serac1 did not affect body weight. (Fig. 1,
A and B)

In juvenile and adult mice, these Serac1−/− mice exhibited weaker forelimb grip strength than wild-
type littermates (Fig. 1C).

similar results were observed in males and females. Thus, these results revealing a decrease in muscle
strength in Serac1−/− mice.
The researchers also performed an MRI of the brain of the mice and revealed lesions in the basal
ganglia (a brain structure responsible for motor coordination) of these mice. (Fig. 1E)
The study of the abundance of the abundance of N-acetyl aspartate (NAA), a quantitative marker of
functional neurons, was lower in Serac1−/− mice than in their wild-type littermates (Fig. 1F).

5
N. Kory, G. A. Wyant, G. Prakash, J. Uit de Bos, F. Bottanelli, M. E. Pacold, S. H. Chan, C. A. Lewis, T. Wang, H. R. Keys, Y. E. Guo, D. M.
Sabatini, SFXN1 is a mitochondrial serine transporter required for one-carbon metabolism. Science 362, eaat9528 (2018).
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These data which correspond to the human symptoms of the syndrome of MEGD(H)EL demonstrated
that Serac1−/− mice can develop a Leigh-like syndrome based on the diagnostic criteria of Leigh and
Leigh-like syndrome 6.
The researchers then, tried to characterize the pathological alterations of the organs of the
SERAC1−/− Mouse. For this, they performed histological analyzes of brain and muscle tissue of
SERAC1−/− Mouse. The researchers observed that, compared with wild-type mice, the percentage of
microglia in basal ganglia of 6-month-old SERAC1−/− mice was significantly lower than the normal
range. They supposed that increased apoptosis may be responsible for decreasing the number of
neurons in SERAC1−/− mice compared with controls. Because enough neurons and 10 to 15%
microglia are required (in mutated mice this number was significantly lower) for optimal brain
function, these results provide evidence that deletion of Serac1 induces brain lesions in mice.
Moreover, histological features of MEGD(H)EL syndrome as cardiomyopathy were also observed in
mutated mice.
Liver disease being one of the main characteristics of the syndrome, and the fact that hepatic failure
frequently manifests in the infantile MEGD(H)EL syndrome 7. Researchers performed a histological
analysis on mutated baby mice aged 1, 3, 6 and 12 months. Morphological analysis revealed the
presence of hepatic cell edema in SERAC1−/− mice from the age of 1 month, as well as they
observed an advanced phenotype with damaged liver cells in 12-month-old SERAC1-/- mice (Fig.
2A)

6
F. Baertling, R. J. Rodenburg, J. Schaper, J. A. Smeitink, W. J. Koopman, E. Mayatepek, E. Morava, F. Distelmaier, A guide to diagnosis and
treatment of Leigh syndrome. J. Neurol. Neurosurg. Psychiatry 85, 257–265 (2014).

7
O. Sarig, D. Goldsher, J. Nousbeck, D. Fuchs-Telem, K. Cohen-Katsenelson, T. C. Iancu, I. Manov, A. Saada, E. Sprecher, H. Mandel, Infantile
mitochondrial hepatopathy is a cardinal feature of MEGDEL syndrome (3-methylglutaconic aciduria type IV with sensorineural deafness,
encephalopathy and Leigh-like syndrome) caused by novel mutations in SERAC1. Am. J. Med. Genet. A 161A, 2204–2215 (2013).
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figure 2A:

Moreover, other characteristics indicating a damage of the liver, such as the increased accumulation
of glycogen (Fig. 2B) and increased hepatic cholesterol accumulation (Fig. 2C) were observed in
SERAC1−/− mice aged 1 to 6 months. According to a study which shows that SERAC1 is responsible
for
cholesterol transfer 8, the researchers concluded that deletion of SERAC1 in mouse impairs the
transfer of cholesterol from the liver to other organs.

Other histological characteristics like altered CL (Cardiolipin is a unique phospholipid which is

localized and synthesized in the inner mitochondrial membrane) and PG (Phosphatidylglycerol is a
phospholipid) remodeling in SERAC1−/− were also observed, thus, the researchers concluded that
that the SERAC1−/− mice generated in this study mimicked the major phenotypes observed inpatients
with the MEGD(H)EL syndrome.
The researchers then looked at the molecular changes caused by a mutation of SERAC1. Previous
studies have shown that CL imbalance due to SERAC1 deficiency is responsible for impaired
OXPHOS because CL is vital for the successful assembly of mitochondrial OXPHOS complexes 9,
other Studies have shown that SERAC1 deficiency is often associated with OXPHOS disorders 10,
however the conditions under which SERAC1 influences OXPHOS remain unclear.

8
22. S. B. Wortmann, F. M. Vaz, T.Gardeitchik, L. E. Vissers, G.H. Renkema, J.H. Schuurs-Hoeijmakers, W. Kulik, M. Lammens, C. Christin, L. A.
Kluijtmans, R. J. Rodenburg, L. G. Nijtmans, A. Grunewald, C. Klein, J. M. Gerhold, T. Kozicz, P. M. van Hasselt, M. Harakalova, W. Kloosterman,
I. Baric, E. Pronicka, S. K. Ucar, K. Naess, K. K. Singhal, Z. Krumina, C. Gilissen, H. van Bokhoven, J. A. Veltman, J. A. Smeitink, D. J. Lefeber, J.
N. Spelbrink, R. A. Wevers, E. Morava, A. P. de Brouwer, Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function
and intracellular cholesterol trafficking and cause dystonia and deafness. Nat. Genet. 44, 797–802 (2012).

9,10
S. B. Wortmann, F. M. Vaz, T.Gardeitchik, L. E. Vissers, G.H. Renkema, J.H. Schuurs-Hoeijmakers, W. Kulik, M. Lammens, C. Christin, L. A.
Kluijtmans, R. J. Rodenburg, L. G. Nijtmans, A. Grunewald, C. Klein, J. M. Gerhold, T. Kozicz, P. M. van Hasselt, M. Harakalova, W. Kloosterman,
I. Baric, E. Pronicka, S. K. Ucar, K. Naess, K. K. Singhal, Z. Krumina, C. Gilissen, H. van Bokhoven, J. A. Veltman, J. A. Smeitink, D. J. Lefeber, J.
N. Spelbrink, R. A. Wevers, E. Morava, A. P. de Brouwer, Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function
and intracellular cholesterol trafficking and cause dystonia and deafness. Nat. Genet. 44, 797–802 (2012).

10
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To elucidate this mechanism the researchers focused on the liver and observed fragmented and
shrunken mitochondria in mice with deletion of SERAC1.
They noticed (Fig. 3, A and E) that the number of mitochondria was significantly higher in the liver of
mutated mice than in the liver of wild type mice of the same litter.

An assessment of mitochondrial marker protein expression confirmed these observations (Fig. 3F).
In the mutant mice, the researchers observed an alteration in mitochondrial respiration (Fig. 3H). The
OXPHOS complex enzyme activity assay further revealed a significant reduction in mitochondrial
activities in the livers of SERAC1-/- mice. However, the activity of complexes I and II were not
affected by SERAC1 deletion (Fig. 3I). Thus, in mutated mice the number of mitochondria is greater,
but they are less functional.

In addition, the researchers detected a lower mtDNA content in the liver, skeletal muscles, and brain
in the mutated mice.
The researchers then tried to determine the possible interactions of SERAC1. SERAC1 has been
shown to be localized in the endoplasmic reticulum (ER) and the ER-mitochondrial interface 11. In this
article the researchers found that most SERAC1 are colocalized in the mitochondria.
To reveal the role of SERAC1 in mtDNA homeostasis, researchers co-immunoprecipitated

11
S. B. Wortmann, F. M. Vaz, T.Gardeitchik, L. E. Vissers, G.H. Renkema, J.H. Schuurs-Hoeijmakers, W. Kulik, M. Lammens, C. Christin, L. A.
Kluijtmans, R. J. Rodenburg, L. G. Nijtmans, A. Grunewald, C. Klein, J. M. Gerhold, T. Kozicz, P. M. van Hasselt, M. Harakalova, W. Kloosterman,
I. Baric, E. Pronicka, S. K. Ucar, K. Naess, K. K. Singhal, Z. Krumina, C. Gilissen, H. van Bokhoven, J. A. Veltman, J. A. Smeitink, D. J. Lefeber, J.
N. Spelbrink, R. A. Wevers, E. Morava, A. P. de Brouwer, Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function
and intracellular cholesterol trafficking and cause dystonia and deafness. Nat. Genet. 44, 797–802 (2012).
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(co-IP) SERAC1 and performed mass analysis by spectrometry. They discovered that SFNX1, a
mitochondrial serine transporter localized to the inner mitochondrial membrane 12, can
interact with SERAC1 (Fig. 4G)

Volcano plots showing candidate SERAC1-interacting proteins obtained from co-IP/mass spectrometry analyses. Candidate proteins with
mitochondrial localization (UniProt) were selected on the basis of a standard log2FC and –log10 (P value) greater than 1. Fold change (FC)

Other analyzes (PLA and co-IP/immunoblot analyzes) carried out have also confirmed the interaction
between SERAC1 and SFXN1. The researchers then tried to determine the molecular role of SERAC1
in the regulation of mitochondrial function. For this they deleted SERAC1 in HEK293T cells.
They observed in SERAC1−/− HEK293T cells (Consistent with findings in Serac1−/- mouse liver):
-an increase of mitochondrial marker proteins
-altered mitochondrial morphology
-a weaker basal mitochondrial respiration and a lower mtDNA content.
Moreover, they found that that SERAC1 migrated with SFXN1 and formed several complexes
ranging from 130 to 690 kDa, which reinforce the previous results concerning the interaction of
SERAC1 and SFXN1. In addition, deletion of SERAC1 altered the formation of complexes
containing SFXN1,
especially high molecular weight complexes. Furthermore, SERAC1 was required for the maintaining
the stability of SFXN1 indeed, SFXN1 unassembled at SERAC is unstable.
The researchers then wanted to determine the role of SERAC1 and SFXN1 for serine metabolism in
the one-carbon cycle.
Several studies shown that the one-carbon cycle is one of the main sources of dNTPs for the
DNA synthesis 13. The researchers used this data to hypothesize that SERAC1 regulates the
availability of dNTPs for mtDNA replication by altering the one-carbon cycle.
To verify this hypothesis, the researchers measured the nucleotide content and found an overall
decrease in cellular nucleotides as well as an increase in the serine/glycine ratio in mouse liver and
HEK293T cells with SERAC1 deletion.

12
N. Kory, G. A. Wyant, G. Prakash, J. Uit de Bos, F. Bottanelli, M. E. Pacold, S. H. Chan, C. A. Lewis, T. Wang, H. R. Keys, Y. E. Guo, D. M.
Sabatini, SFXN1 is a mitochondrial serine transporter required for one-carbon metabolism. Science 362, eaat9528 (2018)

13
G. S. Ducker, J. D. Rabinowitz, One-carbon metabolism in health and disease. Cell Metab. 25, 27–42 (2017).
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Finally, the researchers investigated whether nucleoside/nucleotide supplementation can restore
SERAC1 deficiency-mediated mtDNA depletion by increasing mtDNA content in SERAC1−/−
HEK293T cells, immortalized lymphocytes derived from MEGD(H)EL syndrome, and in a Mouse
model of MEGD(H)EL syndrome.
As can be seen in Figure 6A and 6B, In SERAC1−/− HEK293T cells, low mtDNA due to impaired
serine metabolism was restored by deoxynucleoside (dN) supplementation for 1 day and by
addition of formate. Moreover, endogenous mitochondrial respiration was also increased after the
addition of dNs to SERAC1−/− HEK293T cells.

To validate these results in the human context, the researchers used a 9-year-old patient
whose symptoms indicated the early onset of the MEGD(H)EL syndrome. The administration of dNs
restored both mtDNA content and mitochondrial respiration.
Additionally, to assess the efficacy of nucleotide supplementation therapy (dNTP supplementation
therapy) for MEGD(H)EL syndrome, the researchers used Serac1−/− mice as an in vivo model.
The Serac1−/− mice treated with dNTPs showed an improved beam walking performance by looking
at the time to walk across the beam, whereas wild-type mice were not affected by dNTP
administration, indicating a partial alleviation of motor coordination. Further analyzes confirmed
these results. Furthermore, at the liver level, the administration of dNTPs reversed the increase in
hepatic glycogen and cholesterol in Serac1−/− mice.
These, results indicate that dNTP supplementation is potentially an effective treatment for
MEGD(H)EL syndrome.

Discussion:
The aim of this study was to elucidate the mechanism of action of the SERAC1 gene in MEGD(H)EL
syndrome, also known as MEGDEL. Indeed, a deficiency of this gene is associated with the
appearance of this syndrome.This is why understanding the mechanism of action of this gene would
make it possible to propose a way of treatment for the patient suffering from this disease. Thus, to
better understand the pathogenesis of this mitochondrial disease, the researchers set up an in vivo
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model. For this, they generated a mouse model by constitutively deleting SERAC1, which mimics the
main diagnostic clinical features of patients with MEGD(H)EL syndrome. In this mouse model, most
of the diagnostic phenotypes of MEGD(H)EL syndrome were detected.
However, although clinical and molecular pathological evaluations support a MEGD (H) EL-like
syndrome in Serac1−/− mice , one of the limitations of this study would be that, Serac1−/− mice have
less severe pathology than MEGD(H)EL patients syndrome. Indeed, for example, 50% of patients
with this syndrome die in adolescence, and many affected children are unable to walk and have
complete absence of speech14 , whereas no death was observed in Middle-aged Serac1−/− mice in this
study.
Additionally, the lipidomic profile of the liver Serac1−/− mice did not reproduce altered CL and PG
profiles observed in fibroblasts from patients with SERAC1 deficiency.
Several explanations may explain these observed differences, such as the fact that fibroblasts and
HEK293T cells were cultured under optimized artificial conditions 15, while mice were fed a standard
diet, which may also contribute to the difference in lipidomic profile between liver cells in vivo and
cells cultured in vitro. Thus, additional and complementary models to the one used in this study may
be useful to better understand the pathogenic role of SERAC1 in humans.
However, despite the limitation mentioned above, the establishment of the mouse model of the
MEGD(H)EL syndrome made it possible to study the molecular mechanism of SERAC1 and to
develop a therapeutic strategy for treatment of MEGD(H)EL syndrome. In fact, in this study, the
researchers demonstrated the function of SERAC1 in cholesterol trafficking, by observing that the
livers of
Serac1−/− mice exhibited cholesterol accumulation.Furthermore, in this study, scientists have
expanded the functional repertoire of SERAC1 and discovered that it is a component of the serine
transporter complex necessary for the one-carbon cycle. Indeed, they discovered that SERAC1
interacts with SFXN1 and is required for SFXN1-mediated serine transport. However, further studies
are needed to reveal the structure and precise function of complexes containing SERAC1 and SFXN1.
This study also demonstrated that SERAC1 is a component of the serine transporter and facilitates
mtDNA replication by providing a carbon cycle-derived nucleotide. A continuation of the study
would be to study whether patients with MEGD(H)EL syndrome have a decrease in mtDNAncontent
and share clinical phenotypes with mtDNA depletion syndrome. On the other hand, in this study, the
researchers also showed that the administration of exogenous nucleosides/nucleotides restored

14 ,15
.
R. R. Maas, K. Iwanicka-Pronicka, S. Kalkan Ucar, B. Alhaddad, M. AlSayed, M. A. Al-Owain, H. I. Al-Zaidan, S. Balasubramaniam, I.
Baric, D. K. Bubshait, A. Burlina, J. Christodoulou, W. K. Chung, R. Colombo, N. Darin, P. Freisinger, M. T. Garcia Silva, S. Grunewald, T. B.
Haack, P. M. vanHasselt, O. Hikmat, F. Horster, P. Isohanni, K. Ramzan, R. Kovacs-Nagy, Z. Krumina, E. Martin-Hernandez, J. A. Mayr, P.
McClean, L. De Meirleir, K. Naess, L. H. Ngu, M. Pajdowska, S. Rahman, G. Riordan, L. Riley, B. Roeben, F. Rutsch, R. Santer, M. Schiff, M.
Seders, S. Sequeira, W. Sperl, C. Staufner, M. Synofzik, R. W. Taylor, J. Trubicka, K. Tsiakas, O. Unal, E. Wassmer, Y. Wedatilake, T. Wolff, H.
Prokisch, E. Morava, E. Pronicka, R. A. Wevers, A. P. de Brouwer, S. B. Wortmann, Progressive deafness-dystonia due to SERAC1 mutations: A
study of 67 cases. Ann. Neurol. 82, 1004–1015 (2017).

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mtDNA depletion induced by SERAC1 deficiency in both cells and mouse models. These data
suggest that MEGD(H)EL syndrome is caused, at least partially, by primary mtDNA depletion due to
SERAC1 deficiency and that SERAC1 deficiency can also be called a deficiency in a carbon cycle.

In conclusion, we can say that this study has made it possible to highlight the mechanism of action
caused by a deficiency in SERAC1 in the MEGD(H)EL syndrome. Moreover, it appears that
nucleotide supplementation may be an effective treatment for MEGD(H)EL syndrome. But other
treatments can be considered such as gene therapy to allow the re-expression of SERAC1.