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Journal ArticleDOI

Pathological manifestations of Farber disease in a new mouse model.

TL;DR: A novel acid ceramidase mutant mouse model is reported that enables the study of pathogenic mechanisms of FD and ceramide accumulation and offers further insights into the pathogenesis of this disease.
Abstract: Farber disease (FD) is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments are clinically available and affected patients have a severely shortened lifespan. Due to the low incidence, the pathogenesis of FD is still poorly understood. Here, we report a novel acid ceramidase mutant mouse model that enables the study of pathogenic mechanisms of FD and ceramide accumulation. Asah1tmEx1 mice were generated by deletion of the acid ceramidase signal peptide sequence. The effects on lysosomal targeting and activity of the enzyme were assessed. Ceramide and sphingomyelin levels were quantified by liquid chromatography tandem-mass spectrometry (LC-MS/MS) and disease manifestations in several organ systems were analyzed by histology and biochemistry. We show that deletion of the signal peptide sequence disrupts lysosomal targeting and enzyme activity, resulting in ceramide and sphingomyelin accumulation. The affected mice fail to thrive and die early. Histiocytic infiltrations were observed in many tissues, as well as lung inflammation, liver fibrosis, muscular disease manifestations and mild kidney injury. Our new mouse model mirrors human FD and thus offers further insights into the pathogenesis of this disease. In the future, it may also facilitate the development of urgently needed therapies.

Summary (4 min read)

Introduction

  • Farber disease (FD) is a rare lysosomal storage disorder resulting from acid ceramidase (human AC, murine Ac) deficiency (Sugita et al., 1972).
  • FD histology is characterized by granulomas with lipid-laden macrophages (Farber et  al., 1957).
  • Severely affected patients (subtype I and IV) rarely survive 2 years of age and exhibit lung involvement (type I and IV), neurological deficits (type I) and/or hepatosplenomegaly (type IV).
  • The authors have generated an acid ceramidase mutant mouse model to mirror FD.
  • Ac deficiency, new insights into the pathogenesis of FD and other ceramide-related diseases can be obtained.

Results

  • Deletion of acid ceramidase signal peptide disrupts lysosomal targeting and activity of acid ceramidase.
  • To confirm the deletion of the signal peptide sequence on the RNA level, total RNA was isolated from several tissues, reverse transcribed and subjected to polymerase chain reaction (PCR) using a primer binding to the deleted sequence and a second primer bridging the Exon2/Exon3 junction.
  • Ac colocalized with the lysosomal marker protein Lamp1 in Wt cells, no such colocalization was detected in cells derived from Asah1tmEx1 mutants .
  • Asah1tmEx1 mutants exhibit reduced ceramidase activity in a tissue-specific manner, with significant reductions in ceramidase activity in liver, spleen, thymus and bone marrow .
  • Varying degrees of residual activity were observed in all organs .

Ceramide and sphingomyelin accumulate

  • The authors quantified ceramide levels in several tissues of Asah1tmEx1 mice by liquid chromatography tandem-mass spectrometry (LC-MS/MS).
  • The authors also tested for potential changes upstream of ceramide by analyzing sphingomyelin levels.
  • Similarly, spleens of Asah1tmEx1 mice had elevated C16-, C22-, C24- and total sphingomyelin levels, but reduced C20-sphingomyelin and in Asah1tmEx1 kidneys, all sphingomyelin species were elevated except for C24- and C24-1  sphingomyelin, which were reduced .
  • Asah1tmEx1 mice fail to thrive, have severely shortened survival and show histopathological signs of Farber disease MCP-1, MIP-1α, VEGF and IP-10  were significantly elevated , whereas IL-6 was not significantly different and IL-12 remained below the detection limit of the assay (not shown).

Macrophage accumulation in the central

  • Nervous system of Asah1tmEx1 mice Severe FD typically presents with substantial neurological deficits.
  • A recent publication using a mouse model for the disease has provided first insights into the central nervous system (CNS) component of the disease: lipid accumulation due to acid ceramidase deficiency results in storage compounds in the CNS and an accumulation of microglia and/or macrophages, as well as some neurodegeneration late in the disease (Sikora et al., 2017).
  • Perivascular macrophage cuffs were especially present in the cerebellum and in the meninges.
  • Neurons of the first two cortical layers exhibited more intense cathepsin D-immunoreactivity compared to controls.
  • The intraparenchymal macrophage infiltrates were accompanied by astroglial activation, as demonstrated by glial acid fibrillary protein (GFAP) immunoreactivity .

Hematological disease manifestations

  • Some case reports have indicated hematological manifestations of FD (Antonarakis et al., 1984; Fujiwaki et al., 1992; Mondal et al., 2009), but without any clear consistency.
  • The authors prepared blood counts to gain further insights and observed a significant reduction of leukocytes in Asah1tmEx1 mice .
  • Upon further differentiation, the number of circulating lymphocytes were reduced, whereas the numbers of granulocytes and monocytes remained unchanged .
  • Histological analyses revealed that the enlargement of the spleen, lymph node and thymus was due to foamy macrophage infiltration, which disturbed the normal tissue architecture of the immune organs .

Muscular disease manifestations

  • In Asah1tmEx1 mice Despite reports that acid ceramidase expression and activity are very low in skeletal muscle (Li et al., 1998), the authors observed a significant increase of ceramide in muscle tissue .
  • Upon observation, Asah1tmEx1 mice showed some potential indicators of muscular dystrophy .
  • The authors also measured creatinine phosphokinase (CPK)- and lactate dehydrogenase (LDH) levels, two markers for tissue damage.
  • The authors observed no histological abnormalities in Asah1tmEx1 mice , despite significant ceramide accumulation in Asah1tmEx1 kidneys .
  • Serum creatinine levels were unaltered in the mutants , although mutant mice weighed significantly less than their wildtype littermates .

Discussion

  • FD is a severe genetic disorder with very limited treatment options.
  • Pathophysiologically, little is known other than that ceramide accumulates due to mutations in the ASAH1 gene.
  • To gain further insights into the disease and to potentially enable the search for new treatment options, the authors have generated an Asah1 mutant mouse model through EIIa-cre mediated excision of Exon1 .
  • The authors also report CNS, lung and liver involvement in their model , which is typically present in the more severe forms of FD (Moser et al., 2001).
  • The authors observed a marked reduction in lymphocyte counts in spleen, thymus and lymph nodes relative to the total body weight .

Animal husbandry

  • All mice were maintained on the C57BL/6-J background.
  • Mice were bred and housed in the vivarium of the University Hospital Essen, Germany under pathogen-free conditions as defined by the Federations of European Laboratory Animal Science Associations .
  • Generation of the Asah1tmEx1 mouse model A conditional Asah1 mutant model was generated by GenOway (Lyon, France) through flanking Exon1 with loxP sites and removal of the neomycin selection cassette via crossing to Flp recombinase deleter mice.
  • Subsequently, a PCR was conducted using a forward primer binding to the sequence encoding the signal peptide and a reverse primer bridging the Exon2/Exon3 junction, yielding a 220 bp fragment for the Wt allele or no fragment upon Cre-mediated excision in the Asah1tmEx1 samples.

Intracellular localization of Ac

  • Mesenchymal stem cells were generated as described before (Peister et al., 2004) and maintained in IMDM medium (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (GE Healthcare, Freiburg, Germany) and 10% horse serum (Thermo Fischer Scientific).
  • Bone marrow was flushed from both femurs and tibias with PBS, pelleted by centrifugation and Bereitgestellt von | Universitätsbibliothek Bern Angemeldet Heruntergeladen am | 20.02.19 10:38 snap-frozen.
  • Thin layer chromatography was conducted with ethyl acetate:acetic acid (100:1, v/v, ceramidase assays) or chloroform:methanol (80:20, v/v, sphingomyelinase assays) as running buffer.

Ceramide and sphingomyelin quantification

  • Ceramides and sphingomyelin were extracted from indicated tissues and quantified as recently described (Huston et al., 2016).
  • Briefly, lipid extraction was performed using C17-ceramide and C16-d31-sphingomyelin as internal standards.
  • Sample analysis was carried out by rapid-resolution LC-MS/MS using a Q-TOF 6530  mass spectrometer (Agilent Technologies, Waldbronn, Germany) operating in the positive ESI mode.
  • Quantification was performed with MassHunter Software (Agilent Technologies).

Electron microscopy analysis

  • Samples were cut to tissue blocks of approx.
  • 3–6  mm, fixed and processed for semi-thin sectioning and ultrastructural analysis by electron microscopy.
  • All samples were fixed in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.4) and embedded in epoxy resin as previously described (Zaugg et al., 1999).
  • Ultrathin sections were viewed with a Philips CM 12 transmission electron microscope and micrographs obtained using a Morada camera (12 Megapixel) with AnalySIS ITEM software.

Histopathological assessment

  • Mice were sacrificed by CO2 exposure and perfused with 0.9% sodium chloride followed by 4% paraformaldehyde (PFA) via the right ventricle.
  • Organs were dissected and fixed further in 4% PFA, dehydrated with ethanol, embedded in paraffin and trimmed to 4–6 μm thin sections (peripheral organs) or 1–5 μm thin sections .
  • These were stained as indicated with hematoxylin and eosin, SafraninO/FastGreen, Luxol blue/PAS or Masson Trichrome staining kit (Sigma-Aldrich, St Louis MO, USA).
  • For immunhistochemical analyses, the following antibodies were used: polyclonal rabbit anti-GFAP (#Z0334, Dako, Glostrup, Denmark) and anti-Cathepsin D (#1029, R&D Systems, Minneapolis MN, USA).

Cytokine analysis

  • Cytokine levels were quantified by enzyme-linked immunosorbent assay.
  • Blood counts and clinical chemistry analysis Blood was drawn and anti-coagulated with EDTA for blood counts using a VetABC™ (Scil).
  • For single cell suspension organs were passed through a 70 μm cell strainer (BD, Heidelberg, Germany).
  • For the analysis of granulomonocytic cells, a lineage cocktail containing antibodies against CD3, CD4, CD8, TCRβ, CD19, B220, Nk1.1 and Ter119  was used to distinguish lymphoid and erythroid cells.
  • Information regarding antibody concentrations, fluorochromelabels and manufacturers are summarized in Table 1.

Characterization of lung inflammatory infiltrate

  • Immune cell infiltration into the lung was characterized and quantified by flow cytometry.
  • Absolute cell counts were obtained from the live cell gate using forward- and sideward scatter.
  • The analysis was conducted on a FACS calibur (BD, Heidelberg, Germany).
  • Information regarding antibody concentrations, fluorochromelabels and manufacturers are summarized in Table 2.

Statistical analyses

  • Data are presented as arithmetic means ± standard deviation.
  • For two-group comparisons, the authors used Student’s t-test and for multiple comparisons they used analysis of variance with Bonferroni posttests after confirming normal distribution with D’Agostino & Pearson omnibus normality test.
  • All data were obtained from independent measurements.
  • Acknowledgments: We thank S. Keitsch, C. Müller and S. Harde for their excellent help with the animal experiments and D. Herrmann for his excellent technical assistance with the LC-MS/MS analyses.the authors.the authors.
  • The authors thank Beat Haenni for the electron microscopical preparations.

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Biol. Chem. 2018; 399(10): 1183–1202
Nadine Beckmann, Stephanie Kadow, Fabian Schumacher, Joachim R. Göthert,
StefanieKesper, Annette Draeger, Walter J. Schulz-Schaeffer, Jiang Wang, Jan U. Becker,
Melanie Kramer, Claudine Kühn, Burkhard Kleuser, Katrin Anne Becker, Erich Gulbins
andAlexander Carpinteiro*
Pathological manifestations of Farber disease
inanew mouse model
https://doi.org/10.1515/hsz-2018-0170
Received February 27, 2018; accepted May 7, 2018; previously
published online June 14, 2018
Abstract: Farber disease (FD) is a rare lysosomal storage
disorder resulting from acid ceramidase deficiency and
subsequent ceramide accumulation. No treatments are
clinically available and affected patients have a severely
shortened lifespan. Due to the low incidence, the patho-
genesis of FD is still poorly understood. Here, we report a
novel acid ceramidase mutant mouse model that enables
the study of pathogenic mechanisms of FD and ceramide
accumulation. Asah1
tmEx1
mice were generated by dele-
tion of the acid ceramidase signal peptide sequence. The
effects on lysosomal targeting and activity of the enzyme
were assessed. Ceramide and sphingomyelin levels were
quantified by liquid chromatography tandem-mass
spectrometry (LC-MS/MS) and disease manifestations in
several organ systems were analyzed by histology and
biochemistry. We show that deletion of the signal pep-
tide sequence disrupts lysosomal targeting and enzyme
activity, resulting in ceramide and sphingomyelin accu-
mulation. The affected mice fail to thrive and die early.
Histiocytic infiltrations were observed in many tissues,
as well as lung inflammation, liver fibrosis, muscu-
lar disease manifestations and mild kidney injury. Our
new mouse model mirrors human FD and thus offers fur-
ther insights into the pathogenesis of this disease. In the
future, it may also facilitate the development of urgently
needed therapies.
Keywords: acid ceramidase; ceramide; Farber disease;
lysosomal storage disorders.
Introduction
Farber disease (FD) is a rare lysosomal storage disorder
resulting from acid ceramidase (human AC, murine Ac)
deficiency (Sugita etal., 1972). AC is encoded by the ASAH1
gene (murine Asah1), located on 8p21.3-p22 (Li et al.,
1999). Functionally, AC is a lipid hydrolase and deacylates
ceramide to sphingosine. Ceramide is an important lipid
mediator and has been implicated in many pathologies,
including apoptosis and inflammation (reviewed in Coant
etal., 2017; Schuchman and Desnick, 2017). AC deficiency
in FD results in an accumulation of lysosomal ceramide
(Levade etal., 1995), which is considered the cause of the
disease, although the molecular details are unknown.
FD clinically presents with deformed joints, sub-
cutaneous nodules and progressive hoarseness (Moser
etal., 2001). It can resemble juvenile idiopathic arthritis
*Corresponding author: Alexander Carpinteiro, Department of
Molecular Biology, University of Duisburg-Essen, Hufelandstraße 55,
D-45147 Essen, Germany; and Department of Hematology, University
Hospital Essen, Hufelandstraße 55, D-45147 Essen, Germany,
e-mail: alexander.carpinteiro@uk-essen.de
Nadine Beckmann, Stephanie Kadow, Melanie Kramer,
ClaudineKühn and Katrin Anne Becker: Department of Molecular
Biology, University of Duisburg-Essen, Hufelandstraße 55, D-45147
Essen, Germany
Fabian Schumacher: Department of Molecular Biology, University of
Duisburg-Essen, Hufelandstraße 55, D-45147 Essen, Germany; and
Department of Toxicology, Institute of Nutritional Science, University
of Potsdam, Arthur-Scheunert-Allee 114-116, D-14558Nuthetal,
Germany
Joachim R. Göthert and Stefanie Kesper: Department of
Hematology, University Hospital Essen, Hufelandstraße 55, D-45147
Essen, Germany
Annette Draeger: Institute of Anatomy, University of Bern, Baltzerstr.
2, CH-3012 Bern, Switzerland
Walter J. Schulz-Schaeffer: Insitute of Neuropathology,
University of the Saarland, Kirrberger Str. 100, D-66421 Homburg,
Germany
Jiang Wang: Department of Pathology and Laboratory Medicine,
UC Health University Hospital, 234 Goodman Street, Cincinnati, OH
45219, USA
Jan U. Becker: Institute of Pathology, University Hospital Cologne,
Kerpener Straße 62, D-50937 Cologne, Germany
Burkhard Kleuser: Department of Toxicology, Institute of Nutritional
Science, University of Potsdam, Arthur-Scheunert-Allee 114-116,
D-14558Nuthetal, Germany
Erich Gulbins: Department of Molecular Biology, University of
Duisburg-Essen, Hufelandstraße 55, D-45147 Essen, Germany; and
Department of Surgery, University of Cincinnati, 231 Albert Sabin
Way, ML 0558, Cincinnati, OH 45229, USA
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1184 

N. Beckmann etal.: New mouse model of Farber disease
in infancy, for which it is commonly misdiagnosed at first
(Kostik etal., 2013; Sólyom etal., 2014; Torcoletti et al.,
2014). FD diagnosis is obtained by demonstration of
reduced AC activity and/or abnormally high ceramide
levels in cultured cells, biopsy samples or urine (Sugita
etal., 1975; Dulaney etal., 1976; Fensom etal., 1979; Kudoh
and Wenger, 1982; Antonarakis et al., 1984; Ben-Yoseph
etal., 1989; Levade etal., 1996). FD histology is character-
ized by granulomas with lipid-laden macrophages (Farber
et al., 1957). Depending on disease severity and organ
involvement, seven subtypes have been classified (Moser
et al., 2001). Severely affected patients (subtype I and
IV) rarely survive 2 years of age and exhibit lung involve-
ment (type I and IV), neurological deficits (type I) and/or
hepatosplenomegaly (type IV). Patients with a less severe
phenotype (type II–III, VVII) may survive into adulthood.
So far, the disease variability cannot be explained and
the pathophysiology is still poorly understood. Several
mutations in different domains of the enzyme have been
described, including two single base pair mutations
(Gln22His and His23Asp) in the signal peptide sequence
(Zhang etal., 2000), but reports on genotype-phenotype
correlations are missing for most of the identified ASAH1
mutations.
Currently, no cure for FD exists and treatment options
are limited to supportive care. Favorable results on joint
manifestations have been reported in response to allo-
genic hematopoetic stem cell transplantation (Ehlert
etal., 2006; Torcoletti etal., 2014) and AC enzyme replace-
ment therapy is currently being developed (He etal., 2017).
Unfortunately, both options are unsuitable for patients
with pulmonary disease and neurological involvement,
which are hallmarks of severe FD (Yeager etal., 2000).
Apart from its role in FD, ceramide accumulation has
also been attributed a pathogenic role in a number of other
diseases, including for instance, respiratory diseases (i.e.
cystic fibrosis; Teichgräber etal., 2008), neurological dis-
orders (i.e. major depression, Gulbins et al., 2013, and
neurodegeneration, Filippov etal., 2012), metabolic and
cardiovascular disease (reviewed in Iqbal etal., 2017) and
(auto-)inflammation [i.e. rheumatoid arthritis (Kosinska
etal., 2014) and multiple sclerosis (Singh etal., 1998)].
We have generated an acid ceramidase mutant
mouse model to mirror FD. Two further acid ceramidase
loss-of-function models exist. The first deleted a large
portion of the catalytic domain of acid ceramidase and
was reported to be early embryonic lethal (Li etal., 2002).
The second model is viable and carries a point-mutation
(P361R) outside of the catalytic domain, which is puta-
tively involved in co-factor binding (Alayoubi etal., 2013).
By studying disease manifestations in viable models of
systemic Ac deficiency, new insights into the pathogen-
esis of FD and other ceramide-related diseases can be
obtained.
Results
Deletion of acid ceramidase signal peptide
disrupts lysosomal targeting and activity of
acid ceramidase
We generated an Asah1 mutant mouse model by flanking
Exon1, which encodes the enzyme’s signal peptide, with
loxP sites and removed the neomycin selection cassette by
crossing to Flp recombinant deleter mice. To generate the
constitutive model, the floxed mice were mated with EIIa-
cre deleter mice and the resulting heterozygous offspring
were subsequently mated among each other, result-
ing in Wt controls and homozygous Asah1
tmEx1
mutants
(Figure1A).
To confirm the deletion of the signal peptide sequence
on the RNA level, total RNA was isolated from several
tissues, reverse transcribed and subjected to polymerase
chain reaction (PCR) using a primer binding to the deleted
sequence and a second primer bridging the Exon2/Exon3
junction. Primers directed against the housekeeping
protein RPS6were used as positive controls. As expected,
tissues obtained from Asah1
tmEx1
mice show no signal for
the Asah1-Exon1sequence (Figure1B).
Deletion of Exon1should result in either a complete
knock-out phenotype due to the deletion of the start
codon or lead to a mutant protein initiated from an alter-
native start codon. The later should lack lysosomal target-
ing because of the deletion of the signal peptide sequence.
We studied this by analyzing the intracellular localization
of Ac by confocal immunofluorescence analysis. Whereas
Ac colocalized with the lysosomal marker protein Lamp1
in Wt cells, no such colocalization was detected in cells
derived from Asah1
tmEx1
mutants (Figure 1C). Instead, Ac
was distributed diffusely throughout the cytosol in these
cells (Figure 1C).
We assessed the effect of disrupted lysosomal tar-
geting on Ac activity in a ceramidase activity assay. We
used organ lysates from Asah1
tmEx1
mice and NBD-labeled
ceramide as the substrate and conducted the experiment
at acidic pH. Asah1
tmEx1
mutants exhibit reduced cerami-
dase activity in a tissue-specific manner, with significant
reductions in ceramidase activity in liver, spleen, thymus
and bone marrow (Figure 1D). Varying degrees of residual
activity were observed in all organs (Figure 1D).
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N. Beckmann etal.: New mouse model of Farber disease

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Ceramide and sphingomyelin accumulate
inAsah1
tmEx1
mice
A key feature of FD is the accumulation of ceramide,
which is thought to be the cause of the disease. We
quantified ceramide levels in several tissues of Asah1
tmEx1
mice by liquid chromatography tandem-mass spectro-
metry (LC-MS/MS). Total ceramide was significantly ele-
vated in all tested Asah1
tmEx1
tissues. This also applied to
each individual ceramide species tested (Figure2A–F).
We also tested for potential changes upstream of cera-
mide by analyzing sphingomyelin levels. We observed an
increase of C16- and C24:1-sphingomyelins in the lungs of
Asah1
tmEx1
mice, whereas C18-, C20-, C22-, and C24-sphin-
gomyelins were reduced (Figure 2G). In Asah1
tmEx1
livers, all
sphingomyelin species were significantly elevated except
for C22- and C24-sphingomyelins (Figure 2H). Similarly,
spleens of Asah1
tmEx1
mice had elevated C16-, C22-, C24- and
total sphingomyelin levels, but reduced C20-sphingomye-
lin (Figure 2I) and in Asah1
tmEx1
kidneys, all sphingomyelin
species were elevated except for C24- and C24-1 sphin-
gomyelin, which were reduced (Figure 2J). In Asah1
tmEx1
muscle and brain tissues, all sphingomyelin levels and
total sphingomyelin were significantly increased without
exception (Figure 2K, L).
Sphingosine, but not sphingosine-1-phos-
phate is elevated in Asah1
tmEx1
mice
To test if the accumulation of ceramide impacts the
activity of other sphingolipid-metabolizing enzymes, we
quantified sphingomyelinase activity at acidic pH and
ceramidase activity at neutral pH. Both were not signifi-
cantly altered in Asah1
tmEx1
liver samples (Figure3A, B). We
also quantified sphingosine and sphingosine-1-phosphate
(S1P) levels in the livers of Asah1
tmEx1
mice. Liver sphingo-
sine levels showed a four-fold elevation in Asah1
tmEx1
mice
(Figure 3C), whereas S1P levels were not significantly dif-
ferent (Figure 3D).
Asah1
tmEx1
mice fail to thrive, have severely
shortened survival and show histopathologi-
cal signs of Farber disease
We tested the biological relevance of the increased cera-
mide and sphingomyelin levels in the Ac mutant mice
by monitoring body weight and survival. Asah1
tmEx1
mice
weighed significantly less than their littermates already at
the age of weaning, barely gained weight afterwards and
started to lose weight from week 5 onwards (Figure4A).
The earliest death occurred on day 47 and no Asah1
tmEx1
mouse survived for more than 63days (Figure 4B). No sig-
nificant differences were seen between male and female
Asah1
tmEx1
mice regarding weight changes and survival.
Figure 1:Deletion of acid ceramidase signal peptide disrupts lyso-
somal targeting and activity of acid ceramidase.
(A) The structure of the murine Asah1 gene in the Asah1
fl/fl
EIIa-cre
tg
constitutive knock-out line is schematically shown. LoxP sites were
added flanking Exon1. Upon EIIa-cre mediated excision Exon1 is
deleted in the acid ceramidase mutant mice (Asah1
tm1Ex1
). Image
adapted from Moser etal. (2001). (B) Total RNA was isolated from
different tissues and reverse transcribed. The absence of Exon1 in
Asah1
tmEx1
mice was analyzed by PCR using a forward primer binding
in the targeted region of Exon1. RPS6-PCR was conducted in parallel
as a positive control. (C) Immunofluorescence staining of mesen-
chymal stem cells with antibodies directed against acid ceramidase
(Ac, red) and the lysosomal marker lysosomal-associated membrane
protein1 (Lamp1, green) to determine cellular localization of Ac in
the knock-out model. Representative images of three independent
experiments are shown. Scale bar: 10 μm. (D) Assessment of acid
ceramidase activity upon deletion of the signal peptide. Organ
lysates were prepared and incubated in the presence of NBD-labeled
ceramide. Data are presented as mean±SD (n=4–7mice for each
group). Multiplicity adjusted p-values are indicated (two-way ANOVA
with Bonferroni posttests): **p<0.01; ***p<0.001.
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N. Beckmann etal.: New mouse model of Farber disease
Lysosomal storage diseases such as FD are char-
acterized by storage bodies (Sikora et al., 2017). In
electron-micrographs of liver and brain sections, we
identified storage bodies of various shapes and sizes,
the most prominent being lamellated, zebra-like bodies
in hippocampal sections of the Asah1
tmEx1
mutants
(Figure4C).
Clinically, the hallmark feature of FD is joint deforma-
tion. We analyzed knee joints of Asah1
tmEx1
mice and noted
marked synovial hyperplasia due to the presence of foamy
Figure 2:Ceramide and sphingomyelin accumulation in Asah1
tmEx1
mice.
(A–L) The effect of Asah1 knock-out on ceramide (A–F) and sphingomyelin (G–L) levels were analyzed by rapid resolution LC-MS/MS of
snap-frozen organ samples of 6-week-old mice. Data are presented as mean±SD (n=10–13mice for each group). Asterisks indicate
significant differences of the respective lipid species compared to wildtype (Student’s t-test): *p<0.05; **p<0.01; ***p<0.001.
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N. Beckmann etal.: New mouse model of Farber disease
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Figure 2(continued)
macrophages (Figure 4D). No other arthritic markers, such
as cartilage loss, bone erosion or exudation into the joint
cavity were present (Figure 4D).
Granulomas with lipid-laden macrophages are gener-
ally considered a key feature of FD and these have been
reported in several tissues. Asah1
tmEx1
mice also showed
such histiocytic infiltrations in the lymphoid organs,
which even disrupted the underlying tissue architecture
(Figure 4E).
Asah1
tmEx1
share the characteristic inflamma-
tory cytokine profile of FD
Monocyte chemoattractant protein-1 (MCP-1) has been
suggested to be the driver of macrophage infiltration in
FD and a characteristic cytokine profile of FD patients has
recently been published (Dworski etal., 2017). To test if
our mouse model matches this patient cytokine profile,
we analyzed the serum levels of MCP-1, MIP-1 alpha, IL-6,
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Citations
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Journal ArticleDOI
TL;DR: It is shown that lysosomal ceramide plays a critical role in phenotype change and sEV release in SMCs, which may contribute to the arterial stiffness during the development of AMC, and amitriptyline prevented changes in Pi‐treated CASMCs.
Abstract: Arterial medial calcification (AMC) is associated with crystallization of hydroxyapatite in the extracellular matrix and arterial smooth muscle cells (SMCs) leading to reduced arterial compliance. The study was performed to test whether lysosomal acid sphingomyelinase (murine gene code: Smpd1)‐derived ceramide contributes to the small extracellular vesicle (sEV) secretion from SMCs and consequently leads to AMC. In Smpd1 trg/SMcre mice with SMC‐specific overexpression of Smpd1 gene, a high dose of Vit D (500 000 IU/kg/d) resulted in increased aortic and coronary AMC, associated with augmented expression of RUNX2 and osteopontin in the coronary and aortic media compared with their littermates (Smpd1 trg/SMwt and WT/WT mice), indicating phenotypic switch. However, amitriptyline, an acid sphingomyelinase (ASM) inhibitor, reduced calcification and reversed phenotypic switch. Smpd1 trg/SMcre mice showed increased CD63, AnX2 and ALP levels in the arterial wall, accompanied by reduced co‐localization of lysosome marker (Lamp‐1) with multivesicular body (MVB) marker (VPS16), a parameter for lysosome‐MVB interaction. All these changes related to lysosome fusion and sEV release were substantially attenuated by amitriptyline. Increased arterial stiffness and elastin disorganization were found in Smpd1 trg /SMcre mice as compared to their littermates. In cultured coronary arterial SMCs (CASMCs) from Smpd1 trg/SMcre mice, increased Pi concentrations led to markedly increased calcium deposition, phenotypic change and sEV secretion compared with WT CASMCs, accompanied by reduced lysosome‐MVB interaction. However, amitriptyline prevented these changes in Pi‐treated CASMCs. These data indicate that lysosomal ceramide plays a critical role in phenotype change and sEV release in SMCs, which may contribute to the arterial stiffness during the development of AMC.

21 citations

Journal ArticleDOI
TL;DR: In cultured coronary arterial smooth muscle cells from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction.
Abstract: Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.

19 citations

Journal ArticleDOI
TL;DR: It is suggested that lysosomal acid ceramidase (AC) determines the fate of multivesicular bodies (MVBs) to control the exosome-mediated release of NLRP3 inflammasome products in CECs, which is enhanced by AC deficiency leading to aggravated arterial inflammatory response during hyperglycemia.

16 citations

Journal ArticleDOI
TL;DR: A review of existing literature that investigated sphingolipids as biomarkers for metabolic disease prediction is presented in this article, where the authors explore the advantages and disadvantages of these biomarkers.
Abstract: Biomarkers are important tools for describing the adequacy or inadequacy of biological processes (to allow for the early and accurate diagnosis) and monitoring the biological effects of intervention strategies (to identify and develop optimal dose and treatment strategies). A number of lipid biomarkers are implicated in metabolic disease and the circulating levels of these biomarkers are used in clinical settings to predict and monitor disease severity. There is convincing evidence that specific circulating ceramide species can be used as biological predictors and markers of cardiovascular disease, atherosclerosis and type 2 diabetes mellitus. Here, we review the existing literature that investigated sphingolipids as biomarkers for metabolic disease prediction. What are the advantages and disadvantages? Are circulating ceramides predominantly produced in the liver? Will hepatic sphingolipid inhibitors be able to completely prevent and treat metabolic disease? As sphingolipids are being employed as biomarkers and potential metabolic disease treatments, we explore what is currently known and what still needs to be discovered.

14 citations

Journal ArticleDOI
TL;DR: In this paper, the authors found that atherogenic stimulation by oxysterol 7-ketocholesterol (7-Ket) induced inflammasome formation and activation, reduced lysosome-multivesicular bodies (MVBs) fusion, and increased secretion of EVs that contain IL-1β.
Abstract: Recent studies reported that vascular endothelial cells (ECs) secrete NLR family pyrin domain-containing 3 (NLRP3) inflammasome products such as interleukin-1β (IL-1β) via extracellular vesicles (EVs) under various pathological conditions. EVs represent one of the critical mechanisms mediating the cell-to-cell communication between ECs and vascular smooth muscle cells (VSMCs). However, whether or not the inflammasome-dependent EVs directly participate in the regulation of VSMC function remains unknown. In the present study, we found that in cultured carotid ECs, atherogenic stimulation by oxysterol 7-ketocholesterol (7-Ket) induced NLRP3 inflammasome formation and activation, reduced lysosome-multivesicular bodies (MVBs) fusion, and increased secretion of EVs that contain inflammasome product IL-1β. These EC-derived IL-1β-containing EVs promoted synthetic phenotype transition of co-cultured VSMCs, whereas EVs from unstimulated ECs have the opposite effects. Moreover, acid ceramidase (AC) deficiency or lysosome inhibition further exaggerated the 7-Ket-induced release of IL-1β-containing EVs in ECs. Using a Western diet (WD)-induced hypercholesterolemia mouse model, we found that endothelial-specific AC gene knockout mice (Asah1fl/fl/ECCre) exhibited augmented WD-induced EV secretion with IL-1β and more significantly decreased the interaction of MVBs with lysosomes in the carotid arterial wall compared to their wild-type littermates (WT/WT). The endothelial AC deficiency in Asah1fl/fl/ECCre mice also resulted in enhanced VSMC phenotype transition and accelerated neointima formation. Together, these results suggest that NLRP3 inflammasome-dependent IL-1β production during hypercholesterolemia promotes VSMC phenotype transition to synthetic status via EV machinery, which is controlled by lysosomal AC activity. Our findings provide novel mechanistic insights into understanding the pathogenic role of endothelial NLRP3 inflammasome in vascular injury through EV-mediated EC-to-VSMC regulation.

13 citations

References
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TL;DR: In this paper, the authors present a list of disorders of MITOCHONDRIAL FUNCTION, including the following: DISORDERS OF MIOCHONDRIC FERTILITY XIX, XVI, XIX.
Abstract: I. INTRODUCTION II. PERSPECTIVES III. GENERAL THEMES IV. CANCER V. CHROMOSOMES VI. DIAGNOSTIC APPROACHES VII. CARBOHYDRATES VIII. AMINO ACIDS IX. ORGANIC ACIDS X. DISORDERS OF MITOCHONDRIAL FUNCTION XI. PURINES AND PYRIMIDINES XII. LIPIDS XIII. PORPHYRINS XIV. METALS XV. PEROXISOMES XVI. LYSOSOMAL DISORDERS XVII. VITAMINS XVIII. HORMONES XIX. BLOOD XX. IMMUNE AND DEFENSE SYSTEMS XXI. MEMBRANE TRANSPORT DISORDERS XXII. CONNECTIVE TISSUE XXIII. CARDIOVASCULAR SYSTEM XXIV. KIDNEY XXV. MUSCLE XXVI. LUNG XXVII. SKIN XXVIII. NEUROGENETICS XXIX. EYE XXX. MULTISYSTEM INBORN ERRORS OF DEVELOPMENT

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01 Mar 2004-Blood
TL;DR: A protocol that provides rapidly expanding MSCs from 5 strains of inbred mice with differences in their media requirements for optimal growth, rates of propagation, and presence of the surface epitopes CD34, stem cell antigen-1 (Sca-1), and vascular cell adhesion molecule 1 (VCAM-1).

1,023 citations

Journal ArticleDOI
TL;DR: Inhibition of Asm as a new treatment strategy for cystic fibrosis is suggested because it shows that ceramide age-dependently accumulates in the respiratory tract of uninfected Cftr-deficient mice and prevents all pathological findings, including susceptibility to infection.
Abstract: Microbial lung infections are the major cause of morbidity and mortality in the hereditary metabolic disorder cystic fibrosis, yet the molecular mechanisms leading from the mutation of cystic fibrosis transmembrane conductance regulator (CFTR) to lung infection are still unclear. Here, we show that ceramide age-dependently accumulates in the respiratory tract of uninfected Cftr-deficient mice owing to an alkalinization of intracellular vesicles in Cftr-deficient cells. This change in pH results in an imbalance between acid sphingomyelinase (Asm) cleavage of sphingomyelin to ceramide and acid ceramidase consumption of ceramide, resulting in the higher levels of ceramide. The accumulation of ceramide causes Cftr-deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi and high susceptibility to severe Pseudomonas aeruginosa infections. Partial genetic deficiency of Asm in Cftr(-/-)Smpd1(+/-) mice or pharmacological treatment of Cftr-deficient mice with the Asm blocker amitriptyline normalizes pulmonary ceramide and prevents all pathological findings, including susceptibility to infection. These data suggest inhibition of Asm as a new treatment strategy for cystic fibrosis.

517 citations

Journal ArticleDOI
TL;DR: Comb complementary DNA cloning and functional expression of the enzyme termed “N-acylethanolamine-hydrolyzing acid amidase (NAAA)” from human, rat, and mouse demonstrated that NAAA is a novel N-acyleddylamine-Hydrolyzed enzyme that shows structural and functional similarity to acid ceramidase.

309 citations

Journal ArticleDOI
TL;DR: Lowering ceramide abundance may be a central goal for the future development of antidepressants as the role of the acid sphingomyelinase-ceramide system as a target for antidepressants is investigated.
Abstract: Major depression is a highly prevalent severe mood disorder that is treated with antidepressants. The molecular targets of antidepressants require definition. We investigated the role of the acid sphingomyelinase (Asm)-ceramide system as a target for antidepressants. Therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduced Asm activity and ceramide concentrations in the hippocampus, increased neuronal proliferation, maturation and survival and improved behavior in mouse models of stress-induced depression. Genetic Asm deficiency abrogated these effects. Mice overexpressing Asm, heterozygous for acid ceramidase, treated with blockers of ceramide metabolism or directly injected with C16 ceramide in the hippocampus had higher ceramide concentrations and lower rates of neuronal proliferation, maturation and survival compared with controls and showed depression-like behavior even in the absence of stress. The decrease of ceramide abundance achieved by antidepressant-mediated inhibition of Asm normalized these effects. Lowering ceramide abundance may thus be a central goal for the future development of antidepressants.

299 citations

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Q1. What are the contributions in this paper?

Here, the authors report a novel acid ceramidase mutant mouse model that enables the study of pathogenic mechanisms of FD and ceramide accumulation. The authors show that deletion of the signal peptide sequence disrupts lysosomal targeting and enzyme activity, resulting in ceramide and sphingomyelin accumulation. Their new mouse model mirrors human FD and thus offers further insights into the pathogenesis of this disease. 

Instead, sphingosine accumulation may be the result of enhanced activities of other enzymes with a ceramidase activity, i. e. adiponectin receptors, but further studies are necessary to elucidate which enzyme is Bereitgestellt von | Universitätsbibliothek Bern Angemeldet Heruntergeladen am | 20. Further studies are required to determine the exact mechanism. However, further studies are necessary to elucidate this in more detail and to determine the definite cause of CPK elevations in their model, since the authors can not exclude damage to other tissues ( i. e. heart, brain, kidney ) at this time. In the future, it may also facilitate the development of urgently needed therapies in these debilitating diseases.