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Translational studies in Zellweger spectrum disorders
Berendse, K.
Publication date
2016
Document Version
Final published version
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Citation for published version (APA):
Berendse, K. (2016). Translational studies in Zellweger spectrum disorders.
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Chapter 1
1
Peroxisomes
Peroxisomes are multipurpose organelles surrounded by a membrane containing
different transport systems that control in- and efflux of metabolites, are present
in almost all eukaryotic cells
1and are involved in multiple different metabolic
pathways, both catabolic and anabolic
2. The name of the organelle originates
from the description by De Duve and Baudhuin
3, after Rhodin first described the
discovery of a new organelle in mouse kidney cells in 1954
4. In 1966, De Duve
and Baudhuin were the first to describe the presence of catalase and hydrogen
peroxide generating enzymes in specific microbodies and therefore named these
organelles “peroxisomes”. In humans, peroxisomes are found in every cell, except
red blood cells. Peroxisomes are approximately 1µm in width and have a half-life of
approximately 2 days in cultured mammalian cells
5. The number of peroxisomes
varies considerably among species and in different cell types, and also depends
on the developmental stage and environmental conditions. Peroxisomes adapt to
metabolic needs as they can proliferate and multiply or be degraded in response to
various stimuli
67. Because this thesis only comprises research in humans and mice,
yeast peroxisomes will not be discussed in detail, although yeast is an excellent
model to study more fundamental aspects of peroxisomal function. For a detailed
review see
89.
Peroxisome biogenesis
Peroxins are essential for the biogenesis of normal peroxisomes. There are 14
human peroxins, encoded by 14 different
PEXgenes
10. These peroxins are involved
in either import of peroxisomal membrane proteins (PMPs, i.e.
PEX3, PEX11, PEX16
and PEX19) or import of peroxisomal matrix proteins (i.e.
PEX1,
PEX2, PEX5, PEX6,
PEX7, PEX10, PEX12, PEX13, PEX14, PEX26) (figure 1). Within the PMP import
process, PEX3 is located in the peroxisomal membrane and acts as a docking site
for PEX19, which is a protein located in the cytosol and responsible for binding
PMPs
11. The exact function of PEX16 is unknown, but it has been hypothesized
that it functions as a receptor for PEX19
10. When defects involve these proteins,
peroxisome biogenesis is disturbed and often no peroxisomal remnants can be
found in the cell
12.
Transportation of peroxisomal matrix proteins into peroxisomes is mediated by two
peroxisomal targeting sequences (i.e. PTS-1 and PTS-2)
1314, which are recognized
by specific cytosolic receptor proteins, being the PEX5 small isoform (PEX5S) for
C-terminal tripeptide PTS1 motifs and the PEX5 long form (PEX5L)- PEX7 complex
for N-terminal octapeptide PTS2 proteins, respectively
15 16 17(figure 1). PEX5L
contains 37 additional amino acids that allows it to bind to PEX7, transporting PEX7
to the peroxisomal membrane. Only three proteins are currently known to have a
PTS2 signal in humans, i.e. thiolase (for
β-oxidation), phytanoyl-CoA 2-hydroxylase
(for
α-oxidation) and alkyl-dihydroxyacetone-phosphate synthase (involved in the
ether lipid synthesis pathway)
2. The import machinery of peroxisomal matrix proteins
consists of the PEX14- PEX13 complex, which is the docking complex for PEX5 and
PEX7
1819. Thereafter the targeted proteins are transported across the peroxisomal
membrane via PEX2, PEX10 and PEX12
20. Notably, it was recently reported that
the import of PTS-1 and PTS-2 proteins is performed by distinct pores in yeast
21.
Subsequently, PEX5 and PEX7 are released from the membrane for another import
cycle (via mono-ubiquitination) or targeted to the proteasome for degradation (via
poly-ubiquitination)
22, so called pexophagy
23. Pexophagy is a process that has
extensively been studied in yeast
24. The PEX1, PEX6 and PEX26 complex releases
PEX5 from the peroxisomal membrane
25. When a defect is located in peroxins
involved in the matrix protein import, remnants of peroxisomes, either missing a
group of matrix proteins or without any content (“ peroxisomal ghosts”) can be
observed
12. PEX11, which consists of three isoforms, including PEX11
α, PEX11
βand PEX11
γ, is involved in the elongation and constriction of peroxisomes. It is
hypothesized that PEX11
αis involved in the proliferation and PEX11
βin growth
and division of peroxisomes
26. The exact function of these isoforms remains to be
elucidated and will not be discussed further.
In recent years, several theories were proposed regarding the formation/division
of peroxisomes, mainly based on research in yeast models
2728. The current view
Peroxisomal lumen Cytosol 7 7 5S 5S 5L 5L pts-1 pts-1 pts-2 pts-2 7 14 13 2 12 10 26 6 1 16 3 PMP 19 PMP 19 PMP Matrix protein import Membrane protein import
1
is that peroxisomes are formed
de novofrom the endoplasmic reticulum, but they
can also be formed from pre-existing peroxisomes via division
6 29. Peroxisomal
division occurs via three different processes, including elongation, constriction
and fission. Several proteins, i.e. PEX11, dynamin-like protein 1/dynamin-related
protein 1 (DLP1/DRP1)
30, fission 1 (FIS1)
31, mitochondrial fission factor (Mff)
32and ganglioside-induced differentiation associated protein 1 (GDAP1)
33have been
discovered to play a role in this process in humans. Notably, all these proteins,
except for PEX11, are also involved in the fission of mitochondria.
Several co-factors, substrates and products, such as NADH and ATP are necessary
for proper peroxisomal function. Transportation of small solute molecules occurs
via the channel forming protein PXMP2
34, whereas transport of large molecules
(i.e. so called “bulky” solutes), but also ATP and cofactors, requires active transport
35
. Among others, this active transport is mediated by three ATP-binding cassettes
(ABC) transporters, classified as “subfamily D”; ABCD-1, -2 and -3
36 37. These
transporters have a hydrophobic transmembrane domain and hydrophilic region,
and are also known as adrenoleukodystrophy protein
38, adrenoleukodystrophy
related protein
39and peroxisomal membrane protein 70
40, respectively. Research
in yeast, mice and humans revealed that very-long chain acyl-CoAs are transported
via ABCD1
41 42, whereas ABCD2 is believed to play a role in the
β-oxidation of
polyunsaturated fatty acids (e.g. DHA), as illustrated by the
ABCD2
knockout
mice
43. ABCD2 also accepts certain substrates for ABCD1
44. ABCD3 is the most
abundant and mediates the transport of branched-chain acyl-CoAs
45.
Peroxisomal functions
As mentioned before, peroxisomes play an important role in several metabolic
processes. Knowledge of many of these processes was gained from the discovery
of patients with peroxisomal defects and associated biochemical alterations in
plasma, urine and skin fibroblasts. Below, the main peroxisomal metabolic pathways
in humans will be described briefly.
Fatty acid
α-oxidation
Fatty acids with a methyl group at the third position have to be shortened by one
carbon atom via the peroxisomal
α-oxidation system to produce 2-methyl fatty
acids. Phytanic acid (3,7,11,14-tetramethylhexadecanoic acid) is a typical example
of a 3-methyl branched chain fatty acid that is converted into the corresponding
2-methyl fatty acid pristanic acid (2,4,6,10-tetramethylpentadecanoic acid)
46, which
subsequently can be degraded via the peroxisomal
β-oxidation system
4748. Before
α
-oxidation can occur, the substrate needs to be activated to an acyl-CoA ester.
Fatty acid
β-oxidation
Fatty acid
β-oxidation (i.e. the removal of the carboxyl end of the fatty acid) is one of
the main functions of peroxisomes. Similar to the mitochondrial fatty acid
β-oxidation,
the oxidation consists of four different steps, including dehydrogenation, subsequent
hydration, dehydrogenation and thiolytic cleavage
49. In each cycle, 2 carbon atoms
of different substrates are removed. Because the peroxisomal
β-oxidation system
cannot degrade fatty acids fully into acetyl-CoA units, the shortened fatty acids
are shuttled to the mitochondria for further degradation, which may occur via two
different routes including a carnitine dependent route and a free fatty acid route
(see
2for detailed discussion). One of the most abundant omega-3 fatty acids is
docosahexaenoic acid (DHA). DHA is considered to be essential for normal brain
and retinal development and function
50 5152. Its precursor tetracosahexanaenoic
acid undergoes one cycle of peroxisomal
β-oxidation to form DHA
53. In addition,
the peroxisomal
β-oxidation system can handle other substrates such as
branched chain fatty acids like pristanic acid and the bile acid intermediates di-
and trihydroxycholestanoic acid, long chain dicarboxylic acids, the side chains of
eicosanoids and other substrates described below.
Bile acid synthesis
Liver peroxisomes are involved in the production of bile acids. The bile acid
intermediates dihydroxycholestanoic acid (DHCA) and trihydroxycholestanoic acid
(THCA) undergo one cycle of peroxisomal
β-oxidation to form the CoA esters of the
primarily bile acids chenodeoxycholic acid (CDCA) and cholic acid (CA), respectively
54
. These bile acids are then conjugated to their tauro- or glyco-conjugated forms
by the peroxisomal/cytosolic enzyme bile acid-CoA:amino acid N-acyltransferase
(BAAT)
55. Subsequently these bile acids are exported from the peroxisomes into bile
canaliculi to be further excreted into the bile.
Glyoxylate detoxification
Glycolate is consumed via vegetables or formed in the mitochondria after
catabolism of hydroxyl proline, derived from meat or endogenous collagen turnover.
Glycolate is metabolized to glyoxylate in peroxisomes. The end point of glyoxylate
detoxification in liver and kidney, as catalysed by the peroxisomal enzyme
alanine-glyoxylate aminotransferase (AGT), is glycine
56. When peroxisomes are absent and
AGT is localized in the cytosol, glyoxylate is oxidized to oxalate by cytosolic lactate
dehydrogenase. Oxalate cannot be degraded further in vertebrate species and can
readily precipitate as calcium oxalate thus forming insoluble crystals. Because of its
insolubility, oxalate will deposit in tissue and body fluids, with detrimental effects on
many organs, including the kidneys.
56.
1
Ether phospholipids synthesis
Phospholipids are major components of the cell membrane and play an important
role in a variety of processes
57 58. Phospholipids have a glycerol or sphingosyl
backbone with a polar or hydrophilic head group and nonpolar or hydrophobic
fatty acid side chains
59. The majority of phospholipids have an ester bond at the
sn-1 position of the glycerol backbone. Ether phospholipids, however, have an
ether bond at this latter position. In mammals, ether phospholipids usually occur
in their plasmalogen form, as characterized by an unsaturated 1-0-alkenyl (vinyl
ether) group at the sn-1 position. Plasmalogens are found in myelin, heart muscle,
erythrocytes and retina
60. Formation of this characteristic ether bond, requires the
obligatory participation of peroxisomes by virtue of the fact that the two enzymes
dihydroxyacetonephosphate transferase (DHAPAT) and alkylglycerone phosphate
synthase (AGPS) are strictly peroxisomal enzymes. The additional steps for full
synthesis of plasmalogens are performed in the endoplasmic reticulum
60.
Other peroxisomal functions
Other metabolic processes in which peroxisomes play a role include: cellular
redox metabolism, with pro-oxidant enzymes (FAD-linked oxidases) and the
anti-oxidant enzymes catalase and Cu/Zn-superoxide dismutase
61. Other functions of
peroxisomes include: the oxidation of L-pipecolic acid and D-amino acids, fatty acid
chain elongations and the oxidation of polyamines, although these are all (minor)
peroxisomal processes. For a detailed review see
2.
Interplay with other organelles
Peroxisomes are known to interact with different organelles
62. For example,
because peroxisomes cannot degrade fatty acids to completion, due to the lack
of a respiratory chain, the products of peroxisomal β-oxidation have to be shuttled
to mitochondria for full oxidation to CO2 and H2O
2. Moreover, peroxisomes and
mitochondria interplay in the lipid homeostasis, the production and scavenging of
reactive oxygen species, maintenance of redox states, thermogenesis and they share
similar proteins for division and fission, which will not be discussed in detail
63 64 65. An
example is the fission protein DLP1. When defective it results in both a peroxisomal
and mitochondrial fission defect with devastating results on clinical phenotype
66.
There are also enzymes that are located in both mitochondria and peroxisomes such
as 3-hydroxy-3-methylglutaryl-CoA lyase
67and alpha- methylacyl-CoA racemase
(AMACR)
68. It is important to note that since the discovery of peroxisomal diseases,
concomitant mitochondrial alterations and/or dysfunction have also been reported
69 70 71
.
Peroxisomal disorders
After the initial discovery of peroxisomes in 1954, Goldfischer was the first to
associate disease with peroxisomal dysfunction/absence in 1973
69. Since 1973,
multiple inherited metabolic diseases of varying extent, related to peroxisomal
dysfunction, were discovered. The peroxisomal disorders can be grouped into two
different classes based on the underlying defect in (1) the biogenesis of peroxisomes
or (2) defects in single peroxisomal enzymes (table 1).
Peroxisome biogenesis disorders
The group of peroxisome biogenesis disorders (PBDs) is comprised of two
main entities, including the Zellweger spectrum disorders (ZSDs) and rhizomelic
chondrodysplasia punctata (RCDP, type 1 and 5). Other PBDs include defects
in the genes for peroxisomal fission, i.e. mutations in
DLP1
66,
MFF
72,
GDAP1
33and
PEX11
β 73. Details about ZSDs will be described in Chapter 2. Briefly, the
ZSD are characterized by a spectrum of disease severity ranging from the very
mild Heimler syndrome
74and relatively mild infantile Refsum disease (IRD)
75to
neonatal adrenoleukodystrophy (NALD)
76and the most severe phenotype, classical
Zellweger syndrome (ZS)
77. As a result of peroxisomal dysfunction of varying extent,
several biochemical markers can be measured in plasma, urine and erythrocytes,
including among others elevated levels of very-long chain fatty acids (VLCFAs) and
bile acid intermediates DHCA and THCA.
The pathology within the RCDP spectrum is due to defects in the plasmalogen
synthesis, leading to reduced levels of plasmalogens. Clinically these patients have
cataract, skeletal dysplasia (i.e. shortening of proximal long bones, rhizomelia) and
calcification of cartilage (chondrodysplasia punctata) on X-ray
78. The life expectancy
is usually short
79. However, milder phenotypes are also described
80. RCDP 1 is
caused by mutations in
PEX7 and is the most common form of RCDP
80. RCPD
type 5 was recently added to the RCDP spectrum and results from a mutation in
PEX5L
81.
Single peroxisomal enzyme deficiencies
Because this thesis is primarily focused on the peroxisome biogenesis disorders,
the single peroxisomal enzyme deficiencies will only be described briefly. For a
detailed review see
26.
X-linked adrenoleukodystrophy (X-ALD): X-ALD is the peroxisomal disease with the
highest prevalence and affects both men and women
82. In males, the first disease
1
Table 1 Classification of peroxisomal disorders
Disorder Gene
Peroxisome biogenesis disorders
Dynamin-like protein 1 (DLP1) deficiency DLP1 Ganglioside-induced differentiation associated protein 1
(GDAP1) deficiency GDAP1
Mitochondrial fission factor (Mff) deficiency MFF Rhizomelic chondrodysplasia punctata type 1 (RCDP1) PEX7
Rhizomelic chondrodysplasia punctata type 5 (RCDP5) PEX5 (long isoform) Zellweger spectrum disorders (ZSDs) PEX1, 2, 3, 5, 6, 10, 11β, 12, 13, 14, 16,
19, 26
Single peroxisomal enzyme deficiencies
70-kDa peroxisomal membrane protein (PMP70)
deficiency ABCD3
α-methylacyl-CoA racemase (AMACR) deficiency AMACR
Acatalasemia CAT
Acyl-CoA oxidase 1 (ACOX1) deficiency ACOX1 Bile acid-CoA:amino acid N-acyltransferase (BAAT)
deficiency BAAT
Classic Refsum disease PHYH/PEX7
D-bifunctional protein (DBP) deficiency HSD17B4
Isolated glycolic aciduria HAO1
Primary hyperoxaluria type I AGXT
Rhizomelic chondrodysplasia punctata type 2 (RCDP2) GNPAT Rhizomelic chondrodysplasia punctata type 3 (RCDP3) AGPS Rhizomelic chondrodysplasia punctata type 4 (RCDP4) FAR1 Sterol carrier protein X (SCPx) deficiency SCP2
X-adrenoleukodystrophy (X-ALD) ABCD1
progressive cerebral demyelination and/or myelopathy. Female X-ALD carriers
develop signs of myelopathy and/or peripheral neuropathy
83. Overall, the symptoms
are progressive with age. The disease is caused by a mutation in the
ABCD1 gene
84
and characterised by accumulation of VLCFAs in tissues and plasma. Recently,
X-ALD was implemented in the newborn screening in New York, USA
85and it
will soon be implemented in The Netherlands for male neonates. Because levels
of C26:0-lysophosphatidylcholine (C26:0-lysoPC) in bloodspots are determined for
this newborn screening, ZSD patients will also be diagnosed, as they also present
with elevated levels of C26:0-lysoPC.
Peroxisomal acyl-CoA oxidase 1 (ACOX1) deficiency is caused by mutations in
the
ACOX1 gene
86. Because
ACOX1 is involved in the
β-oxidation of VLCFAs,
patients present with elevated levels of VLCFAs in plasma and fibroblasts. Clinical
presentations range from a fatal, early onset form with hypotonia, frequent seizures,
impaired vision and hearing to a late-onset form with cerebellar abnormalities.
To date multiple patients with ACOX deficiency have been reported
8788. Proper
diagnosis requires enzyme testing followed by mutation analysis.
2-Methyl-CoA racemase (AMACR) processes fatty acids with a methyl group in
the (R)-configuration to the (S)-configuration, making them ready for peroxisomal
β
-oxidation. The AMACR enzyme is located in both peroxisomes and mitochondria,
and the two forms are produced from the same gene
8968. Patients with AMACR
deficiency accumulate the (R)-configuration of pristanic acid and bile acid
intermediates
90. Clinical symptoms include, among others, liver disease, retinopathy,
peripheral neuropathy and epilepsy in old and/or young
90919293.
D-bifunctional protein (DBP) deficiency is caused by a defect in the enzyme
catalysing the second and third step of the peroxisomal
β-oxidation. Mutations in the
HSD17B4 gene result in DBP deficiency. The clinical phenotype is broad
94ranging
from patients dying in the first year of life to patients surviving into adolescence,
albeit with severe handicaps (Prof. Poll-The, unpublished data). Recently, several
adult patients, with only minor clinical symptoms were diagnosed using whole
exome sequencing
9596. Because DBP is involved in the
β-oxidation of straight chain
VLCFAs as well as branched-chain FAs, patients accumulate VLCFAs, pristanic and
bile acid intermediates in plasma. The enzyme activity can be measured in skin
fibroblasts, differentiating DBP from ZSD.
Different mutations in the
PHYH gene result in Refsum disease
97, leading to a
dysfunctional phytanoyl-CoA 2-hydroxylase, an enzyme involved in the first step
of the peroxisomal
α-oxidation
98. Furthermore, specific mutations in
PEX7 are also
reported to cause Refsum disease
99. As phytanic acid is present in daily food,
1
levels in plasma
100. Therefore the therapy for this disease is relatively simple: a
phytanic acid restricted diet. The clinical phenotype is broad ranging from retinitis
pigmentosa, deafness, polyneuropathy and anosmia to liver disease
101.
Currently three different types of RCDP, caused by defects in single peroxisomal
enzymes, exist. A mutation in the DHAPAT gene results in RCDP type 2
102. RCDP
type 3 is caused by mutations in the alkyl-dihydroxyacetone phosphate synthase
gene
103. Specific mutations in fatty acyl-CoA reductase 1 are described to cause a
deficiency in the production of fatty acid alcohols for the plasmalogens biosynthesis
104
, which is referred to as RCDP type 4
105. In general, these diseases cannot
be distinguished based on clinical phenotype and patients present with similar
symptoms as the RCDP type 1 phenotype
57, except that no rhizomelia appears to
be present in RCDP type 4 despite the very low plasmalogen levels.
Other enzyme deficiencies with divergent clinical phenotypes include; primary
hyperoxaluria type I
106, isolated glycolic aciduria
107, sterol carrier protein X (SCPx)
deficiency
108, 70-kDa peroxisomal membrane protein (PMP70) deficiency
45, bile
acid-CoA:amino acid N-acyltransferase (BAAT) deficiency
109and acatalasemia
110 111, which will not be discussed in detail.
Mouse models
In order to study the pathogenic mechanism of peroxisomal dysfunction and the
consequences of different
PEX mutations and associated biochemical alterations,
several mouse models have been generated. Because this thesis is aimed at PBDs,
mice with defects in single peroxisomal enzymes will not be discussed here. For a
detailed review see
112113. The main symptoms of mice with selective inactivation
of peroxisomes in several tissues (e.g. brain and liver);
Pex5-alfp
114,
Pex5-albumin
115116117
,
Pex5-Nestin
114118,
Pex5-NEX
119120,
Pex5-gfap
119,
Pex5-Cnp
121122123,
Pex13-Nestin
124,
Pex5-AMH
125,
Pex5-aP2
126, are summarized in table 2. Mouse
models for PBD, except the RCDP type 1
127, will be introduced briefly. It should be
noted that the majority of mice had a severe phenotype with mice dying soon after
birth.
Mice with a defect in
PEX2 (global knockout) were created in different backgrounds
(i.e. C57BL/6 and Swiss Webster)
128129. Marked embryonic lethality was reported
in the C57BL/6 background. When pups were born alive, they suffered from
growth retardation, hypotonia, impaired feeding and eventually death within 24
hours. Swiss mice, however, sporadically survived for approximately 1-2 weeks.
Biochemically, the most pronounced abnormalities were increased VLCFAs, altered
bile acid metabolism and decreased levels of DHA and plasmalogens. Both models
resemble the human ZS phenotype.
The
PEX5 mouse model (global knockout) was the first mouse model for ZSD and
also mimics the severe ZS phenotype
130. Mice were born with intra-uterine growth
retardation, were severely hypotonic, and died shortly after birth. Biochemically
these mice had increased levels of VLCFAs and depletion of plasmalogens.
A mouse model with a defect in
PEX10 was created using N-ethyl-N-nitrosourea
(ENU) mutagenesis
131. The majority of mice died within a few hours after birth,
but some mice did survive. These mice were severely growth retarded and had an
ataxic gait. Biochemical results, obtained from E18.5 and 2 adult mice, showed
significantly increased levels of the bile acid intermediates and low plasmalogens
at E18.5.
Two different models were made with a global knockout of
PEX11
α. In the model
generated by Li et al
132, all peroxisomal biomarkers in plasma were normal and
the same is true for the mouse model developed by Weng et al
133. Only a reduced
number of peroxisomes in livers of
PEX11
αmice were found. When stressed with
fibrates or a high fat diet, the phenotype, such as fatty liver, was more pronounced.
Mice with a defect in
PEX11
β(global knockout) were born in a normal Mendelian
ratio but displayed neonatal lethality, hypotonia and growth retardation. Only a minor
increase in the hepatic VLCFAs levels and decreased brain plasmalogens were
reported
134.
Mice with defective
PEX13 (global knockout) presented with severe hypotonia and
growth retardation and died soon after birth. No intra-uterine death was documented.
VLCFAs were elevated and plasmalogens were decreased
135.
Similar to our model (see Chapter 4), another group created a mouse model with a
homozygous missense mutation in Pex1
136, which is the most common mutation in
ZSD patients
137and is associated with a relatively mild phenotype
138with survival
into adulthood (see Chapter 7). Mice with this defect were born in normal Mendelian
ratios. Some survived into adulthood, but the majority died of a natural cause before
reaching adulthood. All mice were growth retarded. Biochemical abnormalities
comprised increased plasma levels of VLCFAs, DHCA and THCA, C26:0-lysoPC
in bloodspots and low levels of plasmalogens in bloodspots. Overall, this model
represents the milder end of the ZSD spectrum.
1
Mouse gene/model (loxP)
Cell type with per
oxisomal deficiency Main symptoms/pathology Biochemical abnormalities Remarks Liver Pex5 -alfp-cr e
Inactivation of hepatocytes during fetal development
Gr
owth r
etar
dation, cholestasis,
steatosis, steatorrhea and mild tremor
Not r
eported
Mitochondrial abnormalities, 80% died within 8 days
Pex5
-albumin-cr
e
Hepatocytes >1week postnatal
Gr
owth r
etar
dation untill 3
months of age. Hepatomegaly
, steatosis, fibr osis and car cinogenesis C24 BA, glycogen ↓, C 24
BA, phytanic-, pristanic-acid, lactate
↑
Mitochondrial abnormalities, impair
ed de novo lipogenesis, PP AR α activation, perturbed carbohydrate metabolism Brain Pex5 -Nestin-cr e Neur ons, astr ocytes, oligodendr ocytes
Survival into adulthood. Motor and coor
dination deficits and
cognitive impairment, evolving in lethargy and death befor
e
the age of 6 months. Delays in neocortex and cer
ebellum
formation, impairment in the formation and maintenance of myelin, axonal degeneration
VLCF A ↑, plasmalogens ↓ Astr o- and micr ogliosis, accumulation of lipid dr oplets Pex5 -NEX-cr e For ebrain pr ojection neur ons No obvious symptoms None Pex5 -gfap-cr e Astr ocytes, pr ecursor cells of neur
ons and oligodendr
ocytes No obvious symptoms VLCF A ↑, plasmalogens ↓ Accumulation of lipid dr oplets Table 2
Overview of the main symptoms/pathology of mice with selective inactivation of per
oxisomes in several tissues
BA bile acids, VLCF
A very long-chain fatty acids
Mouse gene/model (loxP)
Cell type with per
oxisomal deficiency Main symptoms/pathology Biochemical abnormalities Remarks Pex5 -Cnp Oligodendr ocytes
Myelin and axonal abnormalities, late-onset progr
essive neur
odegenerative
disease, peripheral neur
opathy with muscle
weakness VLCF A ↑, plasmalogens ↓
91% died within 12 months, r
eactive gliosis and
neur oinflammation, paranodal swellings Pex13 -nestin-cr e Neur ons, astr ocytes, oligodendr ocytes Gr owth r etar dation, impair ed
reflex and motor behaviour development, abnormal cer
ebellar formation
Plasmalogens
↓
70% died within 5 weeks, mitchondrial dysfunction, oxidative str
ess, glyosis
Testis Pex5
-AMH-cr
e
Sertoli
Testicular degeneration and infertility
Lipid dr
oplets
White adipose tissue Pex5-aP2 White adipose tissue and non-adipogenic tissues, such as chondr
ocytes, myocytes,
neur
ons and osteocytes.
Lower body weight, incr
eased
fat mass, r
educed lipolysis,
impair
ed adr
energic tone which
impacts on muscle function, impair
ed thermogenesis of
skeletal muscle when fasted, degr
ee of insulin r esistance Plasmalogens ↓ Table 2 Part 2 BA bile acids, VLCF
1
Outline of this thesis
This thesis focusses on translational studies in patients with a ZSD, with the aim to
discover and test new treatments and to create a new mouse model to study the
pathogenesis of the milder ZSD phenotype in more detail. The first part of the thesis,
Chapter 1, briefly introduces peroxisomes, peroxisomal biogenesis and function,
diseases associated with peroxisomal dysfunction and lastly existing PBD mouse
models are described. The second part of the introduction (Chapter 2) provides insight
in the history of ZSDs, different clinical phenotypes, diagnosis, clinical management
and therapeutic options. In general, no treatment for ZSDs exists and management
is mainly based on supportive therapy.
To accomplish the original goal of the thesis we tested several promising compounds
in skin fibroblasts of patients with a mild ZSD. When immunofluorescent staining
for the PTS-1 protein catalase is performed, these skin fibroblasts of mild patients
with a specific mutation in
PEX1, display a mixed population of cells either with
or without import competent peroxisomes, the so called “peroxisomal mosaicism”
139
. These skin fibroblasts regain catalase import competent peroxisomes when
cultured at 30°C, resembling healthy control cells lines. In contrast, when cells are
cultured at 40°C, all cells lose import competent peroxisomes. One compound (i.e.
arginine) was able to (partially) restore peroxisomal function in skin fibroblasts of
mild ZSD patients and was tested in more detail. Hence, detailed results of these
experiments are described in Chapter 3, of part II laboratory studies. As described in
Chapter 1, several mouse models for PBDs have been created. The majority of these
models, however, represent the severe end of the ZSD spectrum, whereas only
limited data is available on the first mouse model for mild ZSD patients. In Chapter 4,
the results of a comprehensive characterisation of our Pex1 mouse model, a model
that represents the milder end of the ZSD spectrum, is presented.
Cholic acid, marketed as Cholbam® in the United States, was recently approved by
the FDA for treatment in patients with a ZSD under a ‘rare paediatric disease priority
review voucher’. Until now, cholic acid therapy has not been studied in a large
cohort of ZSD patients and approval was based on case reports only. In Chapter
5, we report the results of the first large clinical trial of nine months cholic acid
supplementation in 19 patients with a ZSD. Briefly, our results indicate that cholic
acid does suppress the formation of bile acids in the majority of patients but it can
be harmful for patients suffering from severe liver disease.
ZSDs are generally associated with the severe classical ZS phenotype with patients
presenting symptoms such as hypotonia, prolonged jaundice, severe cognitive
impairment and death within the first year of life. In Chapter 6, part IV, we focus on
extension of the clinical phenotype with some important implications for diagnostics
and general knowledge for all those involved in the care of ZSD patients. We describe
the clinical phenotype of patients with a ZSD surviving into adulthood, and present
age specific symptoms such as peripheral neuropathy with or without pyramidal
signs. Moreover, we show that some patients with prolonged survival present an
insidiously progressive disease course, despite normalization of biomarkers for
peroxisomal disease measured in plasma and erythrocytes. The disease should be
considered as a slowly progressive disease with a subgroup of patients surviving
into adulthood and not solely as a devastating paediatric disease with death within
the first years of life. In addition, recent years many patients were extensively
examined during several visits at our outpatient clinic. During these visits we noted
that a subgroup of patients developed adrenal insufficiency. Therefore we started a
cohort study to determine the prevalence of adrenal insufficiency in (mild) ZSD. The
results of this study are described in Chapter 7. Finally, all findings are discussed and
future prospectives are provided in Chapter 8.
1
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