University of Veterinary Medicine Hannover
Department of Pathology
Center for Systems Neuroscience Hannover
Pathogenetic role of cholesterol biosynthesis and STAT3 signalling in chronic
demyelinating diseases
Thesis
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
(PhD)
awarded by the University of Veterinary Medicine Hannover
by
Wenhui Sun
Shandong, P.R. China
Hannover, Germany (2014)
Supervisor:
Prof. Dr. Wolfgang Baumgärtner, Ph.D.
Supervision Group:
Prof. Dr. Wolfgang Baumgärtner, Ph.D.
Prof. Dr. Martin Stangel
Prof. Dr. Rita Gerardy-Schahn
1st Evaluation:
Prof. Dr. Wolfgang Baumgärtner, Ph.D.
Department of Pathology,
University of Veterinary Medicine Hannover, Germany
Prof. Dr. Martin Stangel
Clinic for Neurology,
Medical School Hannover, Germany
Prof. Dr. Rita Gerardy-Schahn
Institute for Cellular Chemistry,
Medical School Hannover, Germany
2nd Evaluation:
Prof. Dr. Peter Axel Schmidt
Institute of Pathology and Forensic Veterinary Medicine,
University of Veterinary Medicine Vienna, Austria
Date of final exam: 10.10.2014
Sponsorship: Wenhui Sun has received a scholarship from the China Scholarship
Council.
To my family
Parts of the thesis have already been published:
Publication:
Sun, Y., Lehmbecker, A., Kalkuhl, A., Ulrich, D., Sun, W., Rohn, K., Tzvetanova,
I.D., Nave, K.A., Baumgärtner, W., Ulrich, R. (2014). STAT3 represents a molecular
switch possibly inducing astroglial instead of oligodendroglial differentiation of
oligodendroglial progenitor cells in Theiler’s murine encephalomye litis. Neuropathol
Appl Neurobiol 2014 Mar 10. doi: 10.1111/nan.12133.
Contents
Contents
1. Aims ......................................................................................................................... 1
2. Introduction ............................................................................................................ 3
2.1 Paigen diet........................................................................................................... 3
2.1.1 General comments about the Paigen diet ...................................................... 3
2.1.2 Pathologic changes induced by Paigen diet ................................................... 5
2.1.3 Paigen diet induced lesions in mice ............................................................... 7
2.2 Lipids ................................................................................................................... 7
2.2.1 Physiological and pathophysiology roles of fatty acids................................. 9
2.2.2 Cholesterol biosynthesis in the central nerves system (CNS) .................... 13
2.3 Methods for cholesterol analysis..................................................................... 14
2.4 Cholesterol in the Theiler’s murine encephalomyelitis (TME) ................... 17
3. STAT3 represents a molecular switch possibly inducing astroglial instead of
oligodendroglial differentiation of oligodendroglial progenitor cells in
Theiler’s murine encephalomyelitis ................................................................... 21
4. Central nervous system de- and remyelination is independent from the
systemic cholesterol level in Theiler’s murine encephalomyelitis .................. 23
5. Discussion.............................................................................................................. 79
5.1 Role of JAK-STAT signalling in TME ........................................................ 79
5.2 Influence of dietary cholesterol supple mentation in TME........................ 81
5.3 Inte raction between STAT3 and cholesterol biosynthesis in TME .......... 84
6. Summary............................................................................................................... 87
7. Zusammenfassung................................................................................................ 89
8. References ............................................................................................................. 93
9. Acknowledge ments ............................................................................................ 105
I
Aims
1. Aims
Multiple sclerosis (MS) is an immune- mediated demyelinating disease of the central
nervous system (CNS) in humans with unknown and presumably multiple etiologies,
possibly initiated by a virus or virus-triggered immunopathology (Lucchinetti et al.
2004; Lassmann et al. 2001; Owens et al. 2011). The pathologic hallmarks of MS
include inflammation, demyelination, gliosis and axonal damage.
Theiler’s murine encephalomyelitis virus (TMEV) strains may induce demyelinating
Theiler’s murine encephalomyelitis (TME) in susceptible SJL mouse strains that
represents a model to study the pathogenesis especially of the chronic progressive
course of MS and to apply potential treatments (Olesyak et al. 2004; Ulrich et al. 2008
and 2010). The failure of remyelination in MS is thought to be contributed to axonal
injury and degeneration, a major cause for the neurological disability in humans
(Irvine and Blakemore 2008). Recent evidence suggests that a block of
oligodendroglial differentiation causes remyelination failure in MS (Franklin and
Ffrench-Constant 2008; Kuhlmann et al. 2008). Similar results were encountered in
TME. Here an increased number of oligodendroglial precursor cells (OPC) and a lack
of differentiation to mature oligodendrocytes was present (Ulrich et a l. 2008). The
cause for this arrest in OPC differentiation is unknown. The objectives of this research
project were therefore to clarify the mechanisms leading to the endogenous blockade
of OPC differentiation and to identify whether this differentiation a rrest can be
overcome with therapeutic interventions in this TME model for MS.
1
Introduction
2. Introduction
2.1 Paigen diet
2.1.1 General comments about the Paigen diet
Numerous studies have found an association between the consumption of certain
lipid-enriched diets and the rate of atherosclerotic lesion development in murine
models of atherosclerosis (Getz and Reardon 2006). A variety of different lipidenriched diets have been used for studies of atherosclerosis (Getz and Reardon 2006).
Diets vary in the amount of cholesterol, the type and amount of fatty acids, and the
absence or presence of cholate. Each of these components as well as the protein
source have been shown to influence the lipoprotein level and/or the development of
atherosclerosis, however, dietary cholesterol seems to represent the major atherogenic
component (Getz and Reardon 2006).
A lipid-rich diet that is widely applied in murine models of atherosclerosis is called
Paigen diet (Paigen et al. 1985, 1987 and 1990; Paigen 1995). This diet originated
from the Thomas-Hartroft diet which contained 30% cocoa butter, 5% cholesterol, 2%
sodium cholate, 30% casein, 5% alphacel, 4% vitamin mixture, 4% salt mixture, 6.5%
sucrose, 6.5% dextrose, 6.5% dextrin, and 0.5% choline chloride (Morrisett et al.
1982). Thomas and Hartroft (1959) used this high- fat diet to induce arterial damage
and myocardial infarction in rats. A significant number of myocardial and renal
infarcts were induced in rats fed large amounts of either butter or lard (Thomas and
Hartroft 1959). Along with this cholesterol-elevating regimen, thrombosis occurred
before the appearance of severe structural changes in the arterial walls compared to a
diet with high level of less saturated fat, e.g. corn oil (Thomas and Hartroft 1959).
A modified diet based on the Thomas-Hartroft diet was applied by Paigen et al.
(1985), however, this diet caused considerable mortalities (Paigen et al. 1985). Deaths,
due to respiratory infections only occurred in mice fed the atherogenic diet (Paigen et
al. 1985). It was assumed that the atherogenic diet caused a suppression of the
immune response (Paigen et al. 1985). To overcome the mortality problem, a diet
3
Introduction
containing 1 part Thomas-Hartroft high- fat diet and 3 parts breeding chow, the socalled Paigen diet was created containing 15% fat, 1.25% cholesterol, and 0.5%
sodium cholate with a ratio of polyunsaturated to saturated fatty acids of 0.69 (Paigen
et al. 1985; Nishina et al. 1990; Paigen 1995; Getz and Reardon 2006). The
predominant fat in the Paigen diet are saturated fatty acids derived from either cocoa
butter or butter fat. 1% corn oil is often added to avoid a polyunsaturated fatty acid
deficiency (Getz and Reardon 2006). Feeding such a diet reproducibly induces lesions
within 10-14 weeks in susceptible mouse strains without any increased mortality
(Ishida et al. 1991).
Saturated fatty acids are derived from either cocoa butter or butter fat ( Getz and
Reardon 2006). The predominant lipid composition of cocoa butter is triacylglycerol
(TAG) (Table 1). Most cocoa butters have similar triacylglycerol compositions.
Palmitic-oleic-palmitic acid (POP), palmitic-oleic-stearic acid (POS) and stearicoleic-stearic acid (SOS) are the major triacylglycerols in cocoa butter (Table 2;
Chaiseri and Dimick 1989; Hernandez and Castellote 1989; Lipp et al 2001; Guyon et
al. 2003). The main fatty acids of cocoa butter are palmitic acid (range 22.5%-23.1%),
stearic acid (range 24.51%-37.4%) and oleic acid (range 28.74%-38.4%).
Compositions of the fatty acids of the cocoa butter are shown in Table 3 (Lipp et al.
2001; Rao and Lokesh 2003).
Cholesterol is necessary to elevate plasma cholesterol levels in laboratory mice fed
Paigen diet (Paigen et al. 1985). Cholate is not used in atherogenic diets, unless its
complex effect is being investigated specifically (Getz and Reardon 2006). As a bile
salt, cholate can facilitate cholesterol absorption and exert a feedback control on
cholesterol transformation to bile acid within the hepatobiliary system (Getz and
Reardon 2006). Thus, a diet with cholate will increase cholesterol loading and hence
hypercholesterolemia (Getz and Reardon 2006). In addition, cholate induced hepatic
gene expression was particularly related to fibrosis (Boisvert et al. 1999). Moreover,
4
Introduction
feeding of a diet containing 0.5% cholate for 5 to 7 months resulted in skin
xanthomatas in LDLR-/ - mice (Ishibashi et al. 1994).
Table 1: Range of lipid composition of cocoa butter from different countries*
Lipid classes
Range (wt%)
Mean (wt%)
Triacylglycerols
96.21-97.30
96.97
Diacylglycerols
0.80-1.79
1.30
Monoacylglycerols
0.02-0.04
0.03
Free fatty acids
0.88-1.46
1.17
Sterols
0.10-0.14
0.12
*according to Widlak 1999; wt% = weight percentage
Table 2:
Average of triacylglycerol compositions of cocoa butter from different
countries*
Triacylglyce rol
PLiP
POO
PLiS
POP
SOO
SLiS
POS
SOS
SOA
0.68
2.65
3.33
17.99
4.78
2.54
39.04
25.65
0.50
(%)
Average
* according to Chaiseri and Dimick 1989
PLiP = palmitic- linoleic-palmitic acid;
PLiS = palmitic- linoleic-stearic acid;
POO = palmitic-oleic-oleic acid;
POP = palmitic-oleic-palmitic acid;
POS = palmitic-oleic-stearic acid;
SLiS = stearic-linoleic-stearic acid;
SOA = stearic-oleic-arachidic acid;
SOO = stearic-oleic-oleic acid;
SOS = stearic-oleic-stearic acid
2.1.2 Pathologic changes induced by Paigen diet
5
Introduction
Susceptible C57BL/6 mice fed with Paigen diet developed quite reproducible small
atherosclerotic lesions in the aortic arch after 14 weeks (Paigen et al. 1985). At week
14, large accumulation of intracellular fat in many foam cells was observed within
lesion and they bulged slightly into the lumen of the aorta (Paigen et al. 1985).
Massive atherosclerotic lesions were found in the coronary arteries after 6 months and
in the aortic arch after 9 months in susceptible mice (Paigen et al. 1985, 1987 and
1990). No lesions were found in control mice from different strains maintained on
breeder chow which contains 10-11% fat, and no added cholesterol and cholate
(Paigen et al. 1985 and 1990).
Table 3: Fatty acid composition of cocoa butter (individual fatty acid
composition % / total fatty acid of cocoa butter)*
Fatty acid
%
Capric C10:0
trace-12.5
Lauric C12:0
trace
Myristic C14:0
trace-4.32
Myristoleic C14:1 (ω-5)
1.29
Palmitic C16:0
22.5-23.1
Palmitoleic C16:1 (ω-7)
0.95
Margaric C17:0
trace
Stearic C18:0
24.51-37.4
Oleic C18:1 (ω-9)
28.74-38.4
Linoleic C18:2 (ω-6)
1.5-3.93
*according to El-Saied et al. 1981 and Rao and Lokesh 2003
6
Introduction
Typical fatty streaks were also observed in other animal species such as rabbits and
rats fed an atherogenic diet (Paigen et al. 1985). In the rabbit, foam cell- filled fatty
streak type lesions progressed to fibrous plaques, reminiscent of human lesions, if the
atherogenic diet was alternated weekly with normal chow (Constantinides 1960;
Paigen et al. 1985).
The total plasma cholesterol level was elevated in mice after 5 weeks feeding of the
Paigen diet. However, there was little correlation between plasma cholesterol levels
and presence of aorta lesions (Paigen et al. 1985; Vergnes 2003).
2.1.3 Paigen diet induced lesions in mice
Inbred mouse strains show considerable differences in the rate of lesion formation
following an atherogenic diet (Paigen et al. 1985). So far, most experiments that used
the Paigen diet as an atherogenic diet employed the particularly atherosclerosis
susceptible mouse strain C57BL/6 (Paigen et al. 1985; Getz and Reardon 2006). This
strain, however, does not develop chronic-demyelinating demyelination after infection
with Theiler’s murine encephalomyelitis virus (Lipton and Dal Canto 1979). The
Theiler’s murine encephalomyelitis susceptible mouse strain SJL/J mice fed Paigen
diet developed, however, almost no atherosclerotic plaques in the aortic arch after 18
weeks compared to the C57BL/6 mice. Interestingly 20% of these SJL/J mice had
gallstones compared to only 14% of Paigen diet fed C57BL/6 mice (Paigen et al.
1990).
2.2 Lipids
Lipids are water- insoluble biomolecules that are highly soluble in organic solvents
such as chloroform (Berg et al. 2002; Stockhamand Scott 2002). Lipids have many
functions in the body, particularly as an energy source e.g. triglycerides and fatty
acids, as structural components of cell membranes e.g. phospholipids and cholesterol,
and as substrates for hormones and second messengers (Stockham and Scott 2002).
7
Introduction
Based on the chemical composition, lipids are classified in the seven major lipid
classes fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids,
prenol lipids, saccharolipids, and polyketides (Fahy et al. 2005; Fahy et al. 2009).
Fatty acids are particular components of lipids and possess the most biologically
significant properties of lipids in cell membranes (Berg et al. 2002). Fatty acids are
divided into either saturated fatty acids which do not contain any double bonds or
unsaturated fatty acids which contain 1-6 double bonds with carbon chains varying
between 2 and 36 carbon atoms. Thus fatty acid chains differ by length, often
categorized as short-, medium-, and long-chain fatty acids, and prostaglandins (fattyacid derivatives; Stockham and Scott 2002; Fahy et al. 2005; Fahy et al. 2009;
Tvrzicka et al. 2011).
Glycerolipids are esters of glycerol and fatty acids, divided into TAG,
diacylglycerols and monoacylglycerols (Stockham and Scott 2002; Fahy et al. 2005;
Fahy et al. 2009).
Glycerophospholipids and sphingolipids are abundant in all biological membranes
(Fahy et al. 2005; Fahy et al. 2009). A glycerophospholipid molecule normally
contains four components: fatty acids, a backbone (glycerol) to which the fatty acids
are attached, a phosphate and a small hydrophilic compound such as choline, serine,
ethanolamine or inositol linked to the phosphate (Berg et al. 2002; Fahy et al. 2005;
Fahy et al. 2009). Sphingomyelin (ceramide phosphocholine), which represents a
prototypic sphingolipid is composed of two fatty acids bound to a sphingoid base
backbone which is coupled to a hydrophilic choline by phosphate (Fahy et al. 2005;
Fahy et al. 2009).
Cerebrosides (ceramide monosaccharide), such as cerebroside sulphate, ceramide
oligosaccharide and ganglioside also belong to the group of sphingolipids (Berg et al.
2002; Fahy et al. 2005; Fahy et al. 2009). The common structural component of
glycosphingolipids
is
ceramide
(N-acylsphingosine).
They
are
similar
to
sphingomyelin, both are derivatives of a ceramide, but they lack the phosphordiesterbound polar head groups (Berg et al. 2002; Fahy et al. 2005; Fahy et al. 2009).
8
Introduction
Sterols are cholesterol, cholesterol esters, bile acids, steroid hormones, and vitamin D
(Stockham and Scott 2002; Fahy et al. 2005; Fahy et al. 2009).
Other lipids include terpenes (Vitamins A, E and K) and wax aliphatic alcohols
(Stockham and Scott 2002; Fahy et al. 2005; Fahy et al. 2009).
Lipoproteins are lipids combined with other classes of compounds including verylow-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density
lipoproteins (HDL).
Derived lipids are molecules, soluble in lipid solvents that are produced by
hydrolysis of natural lipids.
2.2.1 Physiological and pathophysiology roles of fatty acids
Fatty acids are substantial components of cell membranes (Tvrzicka et al. 2011). Not
all fatty acids can be produced endogenously in humans and animals because of the
lack of certain desaturases (Tvrzicka et al. 2011). Dietary sources of fatty acids
include animal products (butter, lard) and tropical plant oils (coconut, palm) for
saturated fatty acids, as well as vegetable oils (olive, sunflower and soybean oils) and
marine products (algae and fish oils) for unsaturated fatty acids (Tvrzicka et al. 2011).
Fatty acids take part in complex metabolic pathways and play an important role in
humans and other species (Tvryicka et al. 2011; Kremmyda et al. 2011). Fatty acids in
form of TAG from dietary fat are a principal source of energy (Kremmyda et al. 2011).
TAG are a substantial component of the adipose tissue, which serves multiple
metabolic as well as structural and functional roles such as temperature (e.g. in marine
mammals) and mechanical isolator (Kremmyda et al. 2011).
Fatty acids are also structural components of all cell membranes in the form of
glycerophospholipids, which can influence the thickness and fluidity of the membrane
and the activity of membrane associated proteins including enzymes, ion channels,
receptors and transporters (Nelson and Cox 2005; Kremmyda et al. 2011). Fatty acids
also form second messengers. Phosphatidylinositol and phosphatidylcholine may play
a role as sources of intracellular signals (Kremmyda et al. 2011).
9
Introduction
Fatty acids of the cell membrane, especially longer-chain polyunsaturated fatty acids
including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and
arachidonic acid are also precursors of lipid mediators, such as eicosanoids
(prostaglandins, thromboxanes and leukotrienes), resolvins and neuroprotectins
(Smith et al. 2008; Kremmyda et al. 2011).
Fatty acids influence cell signalling by acting as ligands of receptors or as modulators
of gene transcription in signal transcription pathways (Chapkin et al. 2008; Tvryicka
et al. 2011). Fatty acids are also involved in receptor interactions in signal
transduction pathway. Amide derivatives of fatty acid such as ethanolamine, ammonia
and some bioactive amines interact with cannabinoid as well as vaniloid receptors.
The brain cannabinoid receptors takes part in signal pathways of memory, movement,
emotional and nociceptive processes. The peripheral cannabinoid receptors are
involved in the modulation of immunocompetent cells (Pertwee 2008; Kremmyda et
al. 2011).
The fatty acid status and the fatty acid composition of blood plasma and adipose
tissue can be influenced by the dietary fat. Moreover, variation of fatty acid
composition due to increased intake of animal fat and decreased intake of vegetable
and marine origin oils may induce a variety of pathological processes (Kunesováet al.
2006; Vecka et al. 2006; Hlavatýet al. 2008; Asp et al. 2011; Gillingham et al. 2011;
Teng et al. 2010 & 2011).
Essential fatty acids (EFA) are specific fatty acids which need to be taken from the
diet, e.g. linoleic, alpha- linolenic (Tvrzicka et al. 2011). A number of disorders are
induced by a deficiency of EFA, summarized in Table 4. The pathophysiological and
clinical differences between n-3 and n-6 polyunsaturated fatty acids deficiencies are
also shown in Table 5.
10
Introduction
Table 4: Essential fatty acid deficiency syndrome*
Essential fatty acid deficiency syndrome
Growth disturbances
Increased transepidermal losses of water (increased skin permeability)
Increased predisposition to bacterial infections
Male and female infertility
Decreased status of AA/ increased status of mead acid (20:3 n-9)
Disturbed stability of biomembranes
Disturbed cholesterol transport
Increased fragility of capillaries
Kidney failure (hematuria. hypertension)
Lower contractility of myocardium
Abnormal QRS in electrocardiogram
Lowered production of ATP (myocardium. liver)
Dysopsia (lowered visual acuity. disturbed adaptation to darkness)
Neurological disturbances (sensor and motor neuropathies)
Increased food demand with negative nitrogen balance
Disturbed synthesis of eicosanoids
*according to Kremmyda et al. 2011
AA = Arachidonic acid; ATP = Adenosine triphosphate; QRS = QRS complex
Excess intake of fat leads to higher risk of cardiovascular disease (Bysted et al. 2005;
Astrup et al. 2011; Kremmyda et al. 2011). Observational studies have found that
increased saturated fatty acids intake is linked to an increased plasma low-density
lipoprotein cholesterol (LDL-cholesterol) level and increased cardiovascular disease.
Moreover high monounsaturated fatty acid intake from olive oil results in increased
high-density lipoprotein cholesterol (HDL-cholesterol) and decreased
LDL-
cholesterol (Astrup et al. 2011; Kremmyda et al. 2011). Furthermore, unsaturated
fatty acids may also be able to modulate the immune system through several
mechanisms including reduction of T lymphocyte proliferation, reduction of cytokine
synthesis, such as IL-1 and IL-2, increased phagocytic activity, and modification of
11
Introduction
Natural Killer (NK) cell activity (Wang et al. 1994; De Pablo and De Cienfuegos
2000; Gupta et al. 2012). The influence of different families of fatty acids on immune
system functions in both experimental animals and humans is shown in Table 6.
Table 5: n-3 and n-6 polyunsaturated fatty acids (PUFAs) deficiencies in human
and animals*
Deficiency
n-3 PUFAs
n-6 PUFAs
Clinic
Normal skin, growth and
Growth retardation;
reproduction;
Skin lesions;
Abnormal
Disturbed reproduction;
electroretinogram;
Steatosis;
Dysopsia;
Polydipsia
Polydipsia
Biochemical parameters
18: 3n-3 and 22:6n-3
18:2n-6 and 20:4n-6
22:4n-6 and 22:5n-6
20:3n-9
20:3n-9
(only
(only
with
with parallel of PUFAs n-3)
parallel of PUFAs n-6)
*according to Kremmyda et al. 2011
Fatty acid can cross the blood-brain barrier via penetration or through direct uptake of
lipoprotein particles mediated by lipoprotein receptors (Dhopeshwarkar and Mead
1973; Smith and Nagura 2001). High fat diets might increase the uptake of fatty acids
into the brain from the plasma (Patil et al. 2005 and 2006; Laine et al. 2007; Gupta et
al. 2012). Alterations or elevations of saturated fatty acids can disrupt and undermine
brain function (Patil et al. 2005 and 2006; Laine et al. 2007; Gupta et al. 2012).
Saturated free fatty acids (palmitic acid, lauric acid and stearic acid) could trigger
inflammation in cultured macrophages and release TNFα and IL-6 in cultured
astrocytes. Furthermore, they modulate amyloid processing in neurons and astrocytes.
However unsaturated fatty acids were unable to induce cytokine release from cultured
astrocytes (Patil et al. 2005 and 2006; Laine et al. 2007; Gupta et al. 2012). To
12
Introduction
conclude, animal models with increased serum lipids might be associated with
increased brain inflammation (Mattson et al. 2003; Perry et al. 2003).
2.2.2 Cholesterol biosynthesis in the central nerves system (CNS)
In healthy mice, rats and humans, cholesterol is produced in the CNS by local de novo
synthesis and it is not derived from the blood plasma due to the closed blood-brain
barrier (Adibhatla and Hatcher 2008; Hayashi 2011). While astrocytes synthetize
cholesterol and release it together with apolipoprotein E, neurons degrade cholesterol
to 24(S)- hydroxycholesterol (Chen et al 2013). Investigations conducted by Saher et
al. (2005) and Verheijen et al. (2009) indicated that oligodendrocytes and Schwann
cells are primarily responsible for the synthesis of cholesterol required for
myelination. However, in an experimentally- induced conditional transcriptional
blockade of key enzymes of cholesterol biosynthesis, these cells used cholesterol from
the extracellular space for the synthesis of myelin membranes (Saher et al. 2005;
Verheijen et al. 2009). Saher et al. (2005) showed that the delayed myelination
observed in the oligodendrocyte-specific squalene-synthase knock-out mice cannot be
influenced positively by a cholesterol enriched diet. However, other studies suggested
that an experimental change in serum cholesterol concentration may have an effect on
the cholesterol metabolism of the brain (Eckert et al. 2001). Thus, for example the
statin “Lovasatin” can induce a reduction of cholesterol in the brain of C57BL/6-mice
(Eckert et al. 2001). It is also known that elevated serum cholesterol levels positively
correlate with an increased expression of apolipoprotein E (ApoE) in the brain
(Petanceska et al. 2003). ApoE is the major cholesterol- transporting protein in the
CNS. It is mainly expressed by astrocytes and ApoE is responsible for the formation
of cholesterol- rich lipoproteins (high-density lipoprotein, HDL). This is mediated by
ATP-binding transporter molecules (Hayashi 2011). HDL is responsible for the
transport of cholesterol to neighboring cells (Hayashi 2011). An up-regulation of the
transcription of the Apoe- and the ATP-binding cassette, sub- family G (WHITE),
member 1 (Abcg1) genes was show in the spinal cord of TMEV- infected mice (Ulrich
et al. 2010).
13
Introduction
2.3 Methods for cholesterol analysis
Detection for cholesterol analysis mainly includes histological, chromatographic and
colorimetric methods. To visualize cholesterol in tissues and cells, various previous
reports used filipin staining as the standard method for histology (Kruth 1984; Kruth
and Fry 1984; Lee and Kruth 2003). Filipin is a polyene antifungal antibiotic
produced naturally by the bacteria Streptomyces filipinensis (Arthur et al. 2011).
Filipin has been shown to mediate a cellular leakage in membrane permeability in
natural and artificial biological membranes, when an interaction between filipin and
sterol, preferably cholesterol, occurred (Norman et al. 1972). Filipin specifically binds
3β-hydroxysterols, e.g. free cholesterol within artificial phospholipid bilayers and
biological membranes. Due to this feature, filipin has been used as a histochemical
stain for cholesterol in biomembranes (Williamson 1969; van Leeuwen et al. 2008).
The filipin stain appears blue when viewed under the fluorescence microscope in the
UV range between 360-460nm (Reid et al. 2004). However, detection of filipin
staining under confocal microscopy is difficult, because filipin staining exhibits rapid
photobleaching (Reid et al. 2004). In addition, filipin staining detects only
unesterified cholesterol but does not react with cholesteryl ester (Kruth 1984; Kruth
and Fry 1984).
The lipid-soluble dye oil red O, which does not stain unesterified cholesterol, was
used to detect hydrophobic lipids, including esterified cholesterol (Kruth 1984; Kruth
and Fry 1984; Lee and Kruth 2003). Dye specificities are shown in Table 7.
14
Introduction
Table 6: Influence of different families of dietary fatty acids on immune system
functions in both experimental animals and humans*
Immune
n-3
function
Lymphocyte
Decreased in humans
proliferation
and animals
n-6
n-9
No effect in rats
Decreased in rats
Decreased
IL-1
production
Decreased in patients
patients
suffering
from
from No effect in rats
rheumatoid arthritis
in
suffering
rheumatoid
arthritis, decreased
in Balb/c mice
IL-2
Decreased in humans
production
and animals
No effect in rats
Decreased in humans.
TNF
decreased
production
NK
Increased in mice
No effect in Balb/c
in Decreased in rats
mice
macrophages of mice
cell Decreased in rats
activity
and humans
Decreased in Balb/c
Decreased in rats
mice and rats
*according to De Pablo and Cienfuegos 2000
IL = interleukin. NK = natural killer cells. TNF = tumor necrosis factors
Table 7: Dye specificities for detection different lipid classes*
Lipids
Oil Red O
Filipin
Free fatty acids
+
-
Triglyceride
+
-
Unesterified cholesterol
-
+
Esterified cholesterol
+
-
Phospholipids
-
-
*according to Rudolf and Curcio 2009
+: reliably detectable, -: not detectable
15
Introduction
To detect lipids, thin-layer chromatography (TLC) and gas chromatography (GS)
are commonly used chromatographic analysis techniques (Hamilton and Hamilton
1992). In the last twenty years, high-performance liquid chromatography (HPLC)
replaced preparative gas chromatography as a method for lipid analysis (Volin 2001).
Several advantages make TLC particularly useful for lipid analysis. TLC is an easy to
perform technique, inexpensive and easily established (Hamilton and Hamilton 1992).
Analyses are performed rapidly and many samples can be screened simultaneously
together with standards (Hamilton and Hamilton 1992; Fuchs 2011). In addition,
samples can be recovered for further analysis (Hamilton and Hamilton 1992; Fuchs
2011). An overview of important techniques of lipid analysis, and the advantages and
drawbacks of the various methods are listed in Table 8 (Fuchs et al. 2011).
The princple of TLC is the separation of different lipid classes. This is achieved on a
stationary phase due to polarity differences of the analytes (Fuchs et al. 2011). The
most popular stationary phases for lipid separations are silica gel, alumina or
kieselgur (Fuchs et al. 2011). Generally, the lipid mixture is applied to the stationary
phase, and then the mixture is resolved by differential migration of its components, as
a stream of solvent passes through the layer of the adsorbent by capillary action. Each
lipid component has a characteristic mobility which can be described as its retention
factor (Rf) value. The Rf value is defined as the distance travelled b y the component
divided by the distance travelled by the solvent front.
Rf = distance traveled by sample/distance traveled by solvent
Since lipids are generally colorless, the separated lipid components have to be
rendered visible by chemical reagents. For quantitative analysis, the proportions of the
individual components are then determined by various techniques available (Hamilton
and Hamilton 1992).
TLC is currently used for two different methods of lipid analysis (Lederkremer and
Johnson 1965; White et al. 1998). Firstly, the different classes of lipids are separately
extracted and then each class of lipid is analyzed via unique TLC methodology.
Alternatively, a complex mixture of lipids is separated on TLC plates and then further
16
Introduction
characterized. The lipid classes are divided into neutral lipids such as triglycerides,
polar
lipids
such as phospholipids,
and
cholesterol.
Ideally,
lipids
are
chromatographed on a single alumina or silica gel TLC plate using sequential solvent
systems running in the same dimension. Relatively nonpolar lipids such as neutral
lipids, fatty acids and cholesterol migrate to unique positions in the upper half of the
chromatogram, whereas relatively polar lipids like phospholipids and sphingolipids
are separated on the lower half of the chromatogram (Lederkremer and Johnson 1965;
White et al. 1998).
Serum cholesterol and triglyceride are the most frequently measured lipid compounds
in blood or other body fluids (Stockham and Scott 2002). The colorimetric assay
technique is assumed to be a fast and economic method to determine cholesterol
concentrations in serum and other tissues (Viturro 2010; Li et al. 2012). Furthermore,
the colorimetric assay has its advantages in its simplicity in both the instrumentation
and performance (Jiang et al. 2010).
2.4 Cholesterol in the Theiler’s murine encephalomyelitis
Cholesterol is an important constituent of most mammalian cell membranes and its
concentration in various cellular membranes is tightly controlled by homeostatic
processes (Maxfield and Wüstner 2012). Cholesterol plays an important role in
determining the biophysical properties of biological membranes, as well as a
precursor for the synthesis of steroid hormones, bile acids, and lipoproteins (Liscum
and Underwood 1995). Cholesterol is present in tissues and plasma as free cholesterol
or as cholesterol ester, which can either be a degradation form or a storage form of
cholesterol (Mukherjee et al. 1998). Cholesterol belongs to the family of steroids, is
composed of an approximately planar steroid ring system, with a 3β-hydroxysterol
function on one end and a hydrophobic alkyl tail on the other (Mukherjee et al. 1998).
The cholesterol metabolism of the CNS has many unique features, such as a
predominantly local de novo synthesis and intercellular transport with high-density
lipoproteins (HDL; Adibhatla and Hatcher 2008; Hayashi 2011). However, many
brain-specific aspects of cholesterol biosynthesis and metabolism such as the
17
Introduction
excretion of excess cholesterol from the brain are poorly understood (Hayashi 2011).
Due to the high lipid and cholesterol levels in the myelin sheaths, it is not surprising
that a reduced concentration of phospholipids and total cholesterol, and an increase of
cholesterol esters have been seen in the foci of demyelination in multiple sclerosis
(MS; Cumings 1955; Wender et al. 1974). Notably, there is a lower but similar
reduction of cholesterol in the normal appearing white matter of MS patients
(Cumings 1955; Wender et al. 1974). The analysis of transcriptional changes in MS
and models of virus- induced murine myelin loss such as Theiler's murine
encephalomyelitis (TME) and experimental autoimmune encephalomyelitis (EAE),
showed a down-regulation of cholesterol biosynthesis with a particularly close
association to demyelination (Lock et al. 2002; Muelller et al. 2008; Ulrich et al.
2010). A primary transcriptional change of cholesterol biosynthesis can be postulated
as an important factor in the pathogenesis of demyelinating diseases (Lock et al. 2002;
Ulrich et al. 2010). In particular, the initially unexplained therapeutic failure of statins
in clinical trials in MS patients provided further evidence of an association between
cholesterol metabolism and demyelination (Weber et al. 2007; Klopfleisch et al. 2008;
Miron et al. 2009). By now, no biochemical basic data regarding cholesterol
metabolism exist in the model of TMEV- induced demyelination. In contrast to the
toxic
cuprizone- induced
model
of
MS
and
experimental
autoimmune
encephalomyelitis (EAE), the TME model is characterized by a progressive chronic
course with poor remyelination and thus provides ideal conditions for the analysis of
the degradation of cholesterol and phospholipids (Ulrich et al. 2008). The effect of a
statin therapy with cholesterol biosynthesis- inhibiting, anti- inflammatory and
immunomodulatory effects has been described in other models of MS (Klopfleisch et
al. 2008; Miron et al. 2009; Paintlia et al. 2005; Youssef et al. 2002). However, so far
an opposite cholesterol- increasing effect on the metabolism has not yet been studied
in any MS model. Cholesterol increasing diets such as the Paigen diet represent
standard models of atherosclerosis (Getz and Reardon 2006) and will be transmitted
to an MS model for the first time in the planned investigations of this thesis.
18
Introduction
Table 8: Overview of important techniques of lipid analysis. The advantages and drawbacks of the various methods are listed
(according to Fuchs et al 2011)
Technique
Thin-layer chromatography
(TLC)
Principle
Separation is achieved on a
“stationary phase”
(normally silica gel) due to
polarity differences of the
analyses.
Advantages
TLC is quite inexpensive
and fast. Variations of the
mobile phase enable
separation of even complex
mixtures. Different
stainings can be easily
performed.
Drawbacks
Oxidation (of unsaturated lipids)
may occur if the TLC plate is
stored for a while since a large
(lipid) surface is exposed to
atmospheric oxygen. Preparative
applications are limited.
Gas chromatography (GC)
Separation of volatile
compounds on a carrier
gas. Detection often
performed by means of
mass spectrometry.
Highly established in fatty Only volatile compounds can be
acid analysis. Automated analyzed. Thus, derivatization of
devices are commercially the analyte is required.
available.
High-performance liquid
chromatography (HPLC)
Separation on a “stationary High quality separations are More time-consuming and
phase” under high pressure achievable. Also applicable expensive than TLC. Detection of
by elution with different on a preparative scale.
saturated lipids (lack of UV
solvents.
absorptions) is difficult. Postcolumn derivatization is
challenging.
Remarks
Often used as initial method if
a complex lipid mixture has
to be analyzed.
Most widely applied
technique for examination
and quantifying fatty acyl
compositions of lipids.
However, increasingly
replaced by soft ionization
mass spectrometry
techniques.
Routine method of lipid
isolation in many
laboratories. However, “finetuning” of the composition of
the mobile phase to the lipid
mixture of interest is
challenging.
19
Hypercholesterolemia in TME
3. STAT3 represents a molecular switch possibly inducing astroglial instead of
oligodendroglial differentiation of oligodendroglial progenitor cells in Theiler’s murine
encephalomyelitis
Yanyong Sun*†, Annika Lehmbecker*†, Arno Kalkuhl‡ , Ulrich Deschl‡ , Wenhui Sun*†, Karl
Rohn§, Iva D. Tzvetanova¶, Klaus-Armin Nave¶, Wolfgang Baumgärtner*†, Reiner Ulrich*†
*
Department of Pathology, University of Veterinary Medicine Hannover, Germany, †Centre
for Systems Neuroscience Hannover, Germany, ‡ Department of Non-Clinical Drug Safety,
Boehringer Ingelheim Pharma, Biberach (Riß), Germany, and §Department of Biometry,
Epidemiology and Information Processing,
University of Veterinary Medicine Hannover, Germany, ¶Department of Neurogenetics, Max
Planck Institute of Experimental Medicine, Göttingen, Germany
Abstract
Aims: Insufficient oligodendroglial differentiation of oligodendroglial progenitor cells
(OPCs) is suggested to be responsible for remyelination failure and astroglial scar formation
in Theiler's murine encephalomyelitis (TME). The aim of the present study is to identify
molecular key regulators of OPC differentiation in TME, and to dissect their mechanism of
action in vitro. Methods: TME virus (TMEV) infected SJL/J-mice were evaluated by rotarod
analysis, histopathology, immunohistology, and gene expression microarray analysis. The
STAT3 pathway was activated using meteorin and inhibited using STAT3 inhibitor VII in the
glial progenitor cell line BO -1 and in primary rat OPCs in vitro. Results: As expected,
immunohistology demonstrated progressively decreasing myelin basic protein-positive white
matter in TME. In contrast, intralesional NG2 -positive OPCs as well as GFAP-positive
21
STAT3 in TME
astrocytes were increased. Gene Set Enrichment Analysis revealed 26 Gene Ontology terms
including “JAK -STAT cascade” to be significantly positively correlated with the density of
NG2-positive OPCs. Immunohistology revealed an increased amount of activated,
phosphorylated STAT3-expressing astrocytes, OPCs, and microglia/macrophages within the
lesions. Meteorin-induced activation of STAT3-signalling in BO-1 cells and primary rat OPCs
resulted in an enhanced GFAP- and reduced CNPase-expression. In contrast, an oppositional
result was observed in BO-1 cells treated with STAT3 inhibitor VII. Conclusions: The STAT3
pathway is a key regulator of OPC-differentiation, suggested to shift their differentiation from
an oligodendroglial towards an astrocytic fate, thereby inducing astrogliosis and insufficient
remyelination in TME.
Neuropathol Appl Neurobiol 2014 Mar 10.
doi: 10.1111/nan.12133.
22
Hypercholesterolemia in TME
4.
Central nervous system de- and remyelination is independent from systemic
cholesterol level in Theiler´s murine encephalomyelitis.
Barbara B.R. Raddatz1,2,* , Wenhui Sun1,2,* , Graham Brogden3,4 , Yanyong Sun1,2 , Patricia
Kammeyer1 , Arno Kalkuhl5 , Florian Colbatzky5 , Ulrich Deschl5 , Hassan Y. Naim4 , Wolfgang
Baumgärtner1,2 and Reiner Ulrich1,2
1
Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany
2
Center for Systems Neuroscience, University of Veterinary Medicine Hannover, Hannover,
Germany
3
Fish Disease Research Unit, University of Veterinary Medicine Hannover, Hannover,
Germany
4
Department of Physiological Chemistry, University of Veterinary Medicine Hannover,
Hannover, Germany
5
Department of Non-Clinical Drug Safety, Boehringer Ingelheim Pharma GmbH & Co KG,
Biberach (Riß), Germany
* These authors contributed equally to this project and should be considered co-first authors
Submitted
Corresponding author:
Dr. med. vet. Reiner Ulrich. Ph.D.
Department of Pathology, University of Veterinary Medicine Hannover
Bünteweg 17
D-30559 Hannover, Germany
Tel.: 0511-953-8670
Fax.: 0511-953-8675
E-mail: Reiner.Ulrich@tiho- hannover.de
23
Hypercholesterolemia in TME
Abstract
High fat intake has been described as a risk for developing multiple sclerosis (MS). Since then,
no final conclusion about the effect of high fat diets and hypercholesterolemia on MS was
drawn. Cholesterol biosynthesis is a key pathway during myelination and disturbances of the
cholesterol biosynthesis are described in demyelinating diseases. To address the possible
interaction of cholesterol and dyslipidemia in MS, cholesterol biosynthesis, lipid composition
of the major lipid repositories in the body and experimentally- induced, systemic
hypercholesterolemia were examined in Theiler’s murine encephalomyelitis (TME), a model
for MS, using DNA microarrays, histology, immunohistochemistry, serum clinical chemistry,
and high performance thin layer chromatography.
An overall down-regulation of genes associated with cholesterol biosynthesis was present on
the transcriptional level. The levels of galactocerebroside and sphingomyelin in the spinal
cord were reduced in the advanced TME stage. De- and remyelination were independent from
serum cholesterol levels. Serum hypercholesterolemia exhibits no negative effect on virusinduced, inflammatory demyelination of the central nervous system in the atherosclerosisresistant SJL/J mouse strain.
The findings indicate that the inconclusive reports regarding dyslipidemia and MS could
result from an indirect pathomechanistic relation and/or confounding influence of genetic
predisposition towards atherosclerosis.
24
Hypercholesterolemia in TME
Introduction:
Half a century ago a positive correlation between high fat intake and the risk for developing
multiple sclerosis (MS) was described (Swank, 1950). Since then numerous studies have been
conducted
investigating
the
relationship
between
high
fat
diet,
obesity
and
hypercholesterolemia as possible etiological factors for MS (Alter et al., 1974; Esparza et al.,
1995; Ghadirian et al., 1998; Lauer, 1997; Ozay et al., 2014; Swank, 1954; Warren et al.,
1982; Westlund and Kurland, 1953; Zhang et al., 2000). However, conflicting findings of
various studies allowed no final conclusion about the possible beneficial or detrimental effect
of high fat diets on initiation and progression of demyelinating diseases. Increasing evidence
suggests that obesity and subsequent dyslipidemia is an important comorbidity in MS (Marrie
and Horwitz, 2010). Changes in lifestyle due to physical and mental impairment were
suggested as possible contributing factors (Marrie and Horwitz, 2010). In addition, recent
studies revealed an association between an adverse lipid profile (high serum levels of total
cholesterol, low density lipoproteins (LDL) and triglycerides) and progressing disease
severity (Giubilei et al., 2002; Tettey et al., 2014a; Tette y et al., 2014b; Weinstock-Guttman
et al., 2013; Weinstock-Guttman et al., 2011). Adverse lipid profiles, especially low HDL and
high LDL levels, are known to act as pro- inflammatory mediators either initiating or
exacerbating inflammatory diseases such as atherosclerosis, cardiovascular disease, metabolic
syndrome and obesity (Esteve et al., 2005).
The hallmark of the progressive form of MS is ongoing myelin destruction and a failure of
sufficient remyelination (Ferguson et al., 1997; Podbielska et al., 2012; Trapp and Nave, 2008;
Trapp et al., 1999). Only about 20% of MS patients display prominent remyelination
(Patrikios et al., 2006). Paradoxically, the presence of oligodendrocyte precursor cells (OPCs),
predecessors of myelin- forming oligodendrocytes, was repeatedly described in MS lesions
(Chang et al., 2000; Franklin, 2002; Horner et al., 2002; Scolding et al., 1998). The
differentiation and maturation process of OPCs seems to be dysregulated by unknown factors
leading to a failure of remyelination. This has been shown for MS and in some related animal
models (Chang et al., 2000; Franklin, 2002; Kotter et al., 2006; Kuhlmann et al., 2008; Sun et
al., 2014; Ulrich et al., 2008).
Theiler´s murine encephalomyelitis (TME) is an experimentally, virus- induced, inflammatory,
demyelinating disease of the spinal cord. Observations in TME indicate a robust association
of down-regulated cholesterol biosynthesis with demyelination and deterioration of the
25
Hypercholesterolemia in TME
clinical score (Ulrich et al., 2010). Since cholesterol biosynthesis is described as a ratelimiting step in myelin synthesis (Saher et al., 2011), an altered cholesterol biosynthesis is
suggested as a possible pathomechanistic factor inhibiting remyelination (Ulrich et al., 2010).
Imbalances of the lipid and cholesterol metabolism in MS and several animal models of
demyelinating diseases have been frequently been described (Confaloni et al., 1988; Cumings,
1955; Gerstl et al., 1961; Wender et al., 1974). Decreased cholesterol levels were observed in
MS lesions as well as in the normal appearing white and grey matter of MS patients (Cumings,
1955; Gerstl et al., 1961; Wender et al., 1974). Under physiological conditions cholesterol is
synthesized locally de novo in the central nervous system (CNS) and the blood brain barrier
shields the brain cholesterol pool from the circulatory cholesterol pool. Nonetheless, brain
endothelial cells have the possibility of an LDL uptake through luminal receptors (Bjorkhem
and Meaney, 2004; Heverin et al., 2005). Interestingly, under pathological conditions the
interaction between the CNS and circulatory cholesterol seems to be enhanced (Balazs et al.,
2004; Baron and Hoekstra, 2010; Chrast et al., 2011; de Preux et al., 2007; Karasinska et al.,
2009; Leoni and Caccia, 2013; Saher et al., 2005; Saher and Simons, 2010; Zhao et al., 2007).
Moreover, a physiologic hypercholesterolemia is observed during the peak of the myelination
process (Dietschy and Turley, 2004; Uranga and Keller, 2010). Similarly, administration of
dietary lipids during pregnancy and lactation apparently had an accelerating effect on the
myelination of the CNS (Salvati et al., 1996). Furthermore, feeding high levels of cholesterol
resulted in an increased brain cholesterol level in animal models (Dufour et al., 2006; Sparks
et al., 1994).
The aforementioned diverse observations display the complex and currently still elusive
interactions between MS and cholesterol metabolism. Due to the controversial discussion
about the efficacy of statins, 3-hydroxy-3 methylglutaryl-coenzyme-A reductase (HMG-CoAreductase)- inhibitors (Weber et al., 2007), as possible cholesterol- lowering drugs for the
treatment of MS, further studies investigating the interaction between demyelinating diseases
such as MS and cholesterol biosynthesis are required. Especially studies that address the role
of cholesterol biosynthesis on disease progression of MS, with particular emphasis on its
potential beneficial effect on remyelination are required. To further elucidate the interaction
between initiation and progression of myelin loss and remyelination, the aims of the present
study were threefold and included (i) a detailed analysis of the cholesterol biosynthesis
pathway on the transcriptional level, (ii) a quantitative analysis of the lipid composition of
26
Hypercholesterolemia in TME
spinal cord, blood serum, and liver, and (iii) a determination of the effect of experimentallyinduced, systemic hypercholesterolemia on de- and remyelination in a virus model of MS.
27
Hypercholesterolemia in TME
Results
Analysis of the cholesterol biosynthesis pathway on the transcriptional level
In order to focus on cholesterol biosynthesis, a subset of 21 genes representing the
MetacoreT M cholesterol biosynthesis pathway was analyzed in detail (Figure 1; Supplemental
table 1). The low level and pathway analysis of the complete dataset has been described in
detail in a previous publication (Ulrich et al., 2010). Notably, nearly all genes involved in
cholesterol biosynthesis showed a mild down-regulation in TMEV- infected animals compared
to mock- infected animals (Figure 1; Supplemental table 1). 8, 18, 20, and 18 differentially
regulated genes were detected at 14, 42, 98 and 196dpi, respectively (Figure 1; Supplemental
table 1). 7 genes were differentially regulated at all time-points from 14-196dpi.
When ranked according to their fold change, these 7 genes were among the genes with the
most severe down-regulated transcripts of the analyzed subset in animals with advanced TME
and histological prominent demyelination at 98 and 196dpi. The most severe do wn-regulation
was detected for isopentenyl-diphosphate delta isomerase (Idi1). Additional analysis of single,
manually selected genes involved in cholesterol metabolism and transport showed, that 7dehydrocholesterol reductase (Dhcr7), and cytochrome P450, family 46, subfamily a,
polypeptide 1 (Cyp46a1) were significantly down-regulated beginning with day 42, the first
day of significant demyelination in TME. In contrast, apolipoprotein E (Apoe) and ATPbinding cassette, sub-family A (ABC1), member 1 (Abca1) were significantly up-regulated.
Quantitative analysis of the lipid composition of blood serum, liver and spinal cord
The lipid composition of liver, blood serum, and spinal cord was measured in order to detect
the influence of TMEV- infection on the lipid composition of the major storage pools of lipids
in the body. No significant influence of infection on the serum levels of total cholesterol, LDL,
HDL, triglycerides and FFA was observed (Figure 2). Similarly, TMEV infection did not
influence
levels
of
phosphatidylethanolamine,
triglycerides,
cardiolipin,
FFA,
cholesterol,
phosphatidylinositol,
monoacylglycerol,
phosphatidylserine,
phosphatidylcholine and sphingomyelin compared to mock- infected animals in the liver
(Figure 2). In the spinal cord, galactocerebroside and sphingomyelin levels were significantly
decreased in TMEV- infected animals at 196dpi (Figure 2). Free fatty acids, cholesterol,
monoacylglycerol,
28
cardiolipin,
phosphatidylethanolamine
phosphatidylinositol,
Hypercholesterolemia in TME
phosphatidylserine, and phosphatidylcholine were not affected by TMEV infection at any
time-point (Figure 2).
Influence of hypercholesterolemia on TMEV-infection
Peripheral metabolic and pathomorphological changes
Paigen diet significantly increased the body weight in TMEV-infected animals beginning
from -7dpi till 140dpi compared to the control diet group, and mock- infected animals
receiving the Paigen diet had a significant higher body weight compared to TMEV- infected
animals at all time-points post infection (Figure 3).
In order to detect the influence of the Paigen diet on the systemic cholesterol repositories
level, blood serum and liver were analyzed. Over the entire investigated period (7dpi till
196dpi), mice fed the Paigen diet displayed a significant increase in serum total cholesterol
levels compared with control diet mice (Figure 3). TMEV- infected, Paigen diet mice showed
a trend towards lower total cholesterol levels compared to mock- infected. TMEV- infected,
Paigen diet mice displayed a trend towards lower total cholestero l levels beginning at 42dpi
with a significant difference observed at 98dpi (Figure 3). LDL serum levels were also
significantly increased in TMEV- and mock- infected mice fed the Paigen diet. A significant
increase in serum HDL levels was measured on day 21, 42, 98dpi in TMEV- and mockinfected mice on Paigen diet (Figure 3). Paigen diet, TMEV–infected mice showed a trend
towards lower serum triglyceride levels with a statistical significance at 98 and 196dpi
compared to Paigen diet, mock- infected mice (Figure 3). Additionally, a trend towards
decreased serum triglyceride levels with statistical significance at 21dpi, 42dpi and 196dpi
was observed in Paigen diet, TMEV-infected mice compared to control diet, TMEV- infected
animals (Figure 3). Paigen diet, TMEV-infected mice showed a trend towards lower free fatty
acids serum levels as compared to Paigen diet, mock- infected mice, with a statistical
significance at 42 and 98dpi. Serum albumin levels showed a trend to lower levels in Paigen
diet, TMEV- infected mice compared to control diet, TMEV- infected mice with a statistical
significance at 7 and 98dpi (Figure 4). No differences were detected between feeding groups
or between TMEV- and mock-infected mice for ALT, GLDH (Figure 4), GGT, total bilirubin,
and direct bilirubin. GGT, total bilirubin, and direct bilirubin were under the detection limit of
3 IU/L, 1.7µmol/L, and 1.5µmol/L respectively, in the majority of animals.
29
Hypercholesterolemia in TME
Paigen diet induced a significantly higher liver weight compared to the control diet in TMEVand mock- infected animals. Control diet, TMEV- infected mice exhibited a decreased liver
weight at 98 and 196dpi compared to control diet, mock- infected mice (Figure 4).
Histological examination of the liver revealed a moderate to severe, multifocal to diffuse,
centrolobularly accentuated, microvesicular hepatic lipidosis in 96.6% respectively 83.0% of
all mock- infected respectively TMEV-infected, Paigen diet mice (Figure 4). In addition, the
oil red O positive area was significantly higher in TMEV- and mock- infected, Paigen diet
mice compared to control diet mice (Figure 4). A mild, multifocal, periportally accentuated,
hepatic fibrosis was associated with the fatty degeneration in Paigen diet mice in 31% of the
mock-infected and 13.3% of TMEV- infected mice, beginning with 98dpi (Figure 4). The
inflammatory response in the liver showed no significant difference between the feeding
groups. No necrosis or cholestasis was detectable in any of the groups. HPTLC analysis of the
lipid content of the liver revealed a significant increase in the amount of triglycerides and
cholesterol in TMEV-, and mock- infected, Paigen diet mice at 98dpi. Phosphatidylinositol
was increased only in TMEV- infected, Paigen diet fed mice compared to TMEV- infected
control
diet
fed
mice.
Sphingomyelin,
phosphatidylserine,
phosphatidylcholine,
monoacylglycerol, phosphatidylethanolamine and cardiolipin showed comparable levels in
the Paigen diet and control diet groups (Figure 4).
Histological examination of the heart, aorta and large pulmo nary arteries was performed to
exclude Paigen diet induced atherosclerotic changes. In HE-stained sections of the heart, 6%
of all animals showed a mild, multifocal lymphohistioplasmacytic infiltration with no
significant influence due to TMEV infection or the feeding regimen. A mild, focal,
lymphohistiocytic, subintimal or intramural infiltration was found in the aorta of 3% of the
animals and in large pulmonary arteries in 1% of the animals with no significant influence of
infection or feeding regimen.
CNS metabolic and pathomorphological changes
The feeding regimen had no influence on the motoric performance of the animals as
determined by the Rotarod assay. Mock- infected, Paigen diet mice attained a significant
higher number of rpm compared to TMEV- infected, Paigen diet mice beginning with 28dpi.
Rotarod performance in TMEV-infected, Paigen diet mice was reduced by about 75.5 % at
196dpi compared to their performance at 0dpi (Figure 5).
30
Hypercholesterolemia in TME
Scoring of the degree of meningitis and leukomyelitis revealed no significant differences
between the feeding groups. TMEV-infection induced a mild to moderate, mononuclear
infiltration in the meninges and the perivascular space of the white matter beginning at 7dpi
(Figure 5). Mock- infection caused minimal meningitis at all time-points, and a minimal
leukomyelitis was observed only at day 7dpi. The infiltrates were composed of CD3-positive
T- lymphocytes, IgG producing B-lymphocytes and few CD107b-positive macrophages in the
meninges and perivascular spaces (Figure 5). The inflammation in the parenchyma of the
white matter was dominated by CD107b-positive macrophages and to a lesser extent by CD3positive T- lymphocytes and IgG producing B- lymphocytes. Single CD3-, CD107b- and IgGpositive cells were detectable in mock- infected animals (Figure 5). CD3-positive Tlymphocytes represented the first cellular response to TMEV- infection with a significantly
higher cell density as early as 7dpi in TMEV- infected animals compared to mock- infected
animals (Figure 5). A significantly higher amount of IgG producing B- lymphocytes and
CD107b-positive macrophages was detectable beginning with 42dpi in all TMEV- infected
mice compared to mock- infected mice. The feeding regimen had no influence on the amount
and quality of the inflammatory response (Figure 5).
A severe progressive demyelination was present in all TMEV- infected animals beginning at
42dpi as assessed in LFB-CV-stained spinal cord sections with no difference between the
feeding groups (Figure 6). No demyelination was observed in mock-infected animals.
Evaluation of HE- and toluidine blue stained specimens confirmed the results obtained in
LFB-CV-stained spinal cord sections with a strong correlation of the three independent
evaluations (Spearman’s correlation, HE, r = 0.95, p0.05; toluidine blue, r= 0.89, p0.05).
The amount of demyelination, as determined by the MBP- immunopositive white matter area
was in accordance with the semi-quantitative histological evaluation using LFB-CV.
Beginning at 98dpi, TMEV- infected animals showed a progressively decreasing MBPimmunoreactivity of the white matter compared to mock- infected animals. No significant
difference was detectable between the two feeding groups (Figure 7). NG2-positive cell
density was significantly increased in TMEV- infected animals compared to mock- infected
animals, starting at 42dpi in Paigen and control diet group. At 196dpi TMEV- infected, Paigen
diet mice had a significantly decreased number of NG2- immunopositive cells compared to
TMEV- infected, control diet mice (Figure 7). Remyelination, semi-quantitatively assessed in
semi-thin toluidine blue-stained spinal cord sections, was progressively increasing from 42dpi
to 196dpi in TMEV- infected animals (Figure 6). PRX-immunohistochemistry indicated an
31
Hypercholesterolemia in TME
involvement of Schwann cell remyelination in this process by an increasing number of PRXimmunopositive cells at 98 and 196dpi (Figure 7). The feeding regiment had no influence on
the amount and timing of remyelination or the amount of Schwann cell remyelination. The
quantitative analysis of the lipid content of the spinal cord detected a significantly higher level
of sphingomyelin only at 98dpi in TMEV- infected, Paigen diet fed mice. No effect of the
feeding regimen was detectable on 21, 42, 196dpi (Figure 7). Similarly, the quantity of
cholesterol, free fatty acid, monoacylglycerol, galactocerebroside, phosphatidylethanolamine,
cardiolipin, phosphatidylinositol, phosphatidylserine, and phosphatidylcholine was not
influenced by the feeding regimen (Figure 7).
The amount of astrogliosis assessed by immunohistochemistry showed an increase in the
GFAP- immunopositive area with a statistical significance at 42dpi and 196dpi (Figure 7). No
difference was observed between the two feeding groups. Immunohistochemistry confirmed
the presence of viral antigen in the spinal cords of the TMEV-infected animals in association
to inflammatory and demyelinating changes with a prominent expression starting at 42dpi. In
mock-infected animals no virus was present in the spinal cords (Figure 7).
32
Hypercholesterolemia in TME
Discussion
Previous studies reported inconsistent results concerning the role of cholesterol as a
pathomechanistic factor (Swank, 1950) or comorbidity of MS (Marrie and Horwitz, 2010).
However, cholesterol biosynthesis is also a key pathway during the physiologic myelination
process (Saher et al., 2005), and transcriptional down-regulation of cholesterol biosynthesis
was the most important biological function associated with demyelination in TME (Ulrich et
al., 2010). Here, we addressed the question, whether dietary factors might contribute to
increased remyelination and examined potential changes along the cholesterol biosynthetic
pathway at the transcriptional level. Furthermore, the lipid composition of the main
cholesterol repositories of the body as well as the effect of experimentally- induced systemic
hypercholesterolemia on de- and remyelination in the TME model of MS were assessed.
Transcriptional profiling of Cholesterol biosynthesis
On the transcriptional level, we observed an overall down-regulation of genes associated with
cholesterol biosynthesis, comparable to observations in myelin oligodendrocyte glycoprotein
(MOG)- induced experimental autoimmune encephalomyelitis (EAE) in rats (Mueller et al.,
2008) and in MS patients (Lock et al., 2002). This down-regulation was suggested to be a
transcriptional representation of a reduced capacity for myelin repair (Lock et al., 2002). The
majority of the examined genes showed progressive down-regulation, indicating a continuous
decline in the ability to synthesize cholesterol. This correlates with the chronic progressive
clinical course of TME. Seven genes of the cholesterol biosynthesis pathway were downregulated at all examined time-points. The rather early decrease in their expression, already at
14dpi, indicates their regulatory importance or a special vulnerability of oligodendrocytes
triggered by the viral infection or the inflammatory changes in the environment. Idi1 was
identified as the gene with the strongest down-regulation. It encodes an enzyme that catalyzes
the conversion of isopentenyl diphosphate to dimethylallyl diphosphate, a substrate for
farnesyl synthesis (Maglott et al., 2011). Reduced activity of the enzyme is known to be the
main pathomechanism in the Zellweger syndrome or neonatal adrenoleukodystrophy (Maglott
et al., 2011). The neuropathological lesions in these disorders include an inflammatory
demyelination, non- inflammatory dysmyelination and non-specific reduction of the myelin
volume in the white matter. Mutations in Dhcr24 diminish the reduction of the delta-24
double bond of sterol intermediates during cholesterol biosynthesis (Waterham et al., 2001).
33
Hypercholesterolemia in TME
Mutations in Sc5d diminish the transformation of lanosterol into 7-dehydrocholesterol
(Kanungo et al., 2013). Both result in a phenotype similar to the human Smith–Lemli–Opitz
syndrome (SLOS; Saher et al., 2005). SLOS is elicited by an inherited mutation in DHCR7, a
gene responsible for the conversion of 7-dehydrocholesterol to cholesterol. The disorder is
characterized by dysmyelinogenesis (Caruso et al., 2004; Elias et al., 1997; Irons et al., 1997).
Dietary cholesterol supplementation is one of the standard therapies in this disease (Caruso et
al., 2004; Elias et al., 1997; Irons et al., 1997). Down-regulation of DHCR7 as observed in the
present study, was described not only in MS lesions, but also in the normal appearing white
matter of affected patients. This finding suggests a down-regulation of cholesterol
biosynthesis prior to demyelination (Lindberg et al., 2004). Similarly, a mutation in Hmgcs1,
a gene important for the conversion of (S)-3-hydroxy-3-methylglutaryl-CoA and CoA to
acetyl-CoA, seems to be responsible for a misdirected migration of OPCs causing a lack of
interaction between glial cells and axons resulting in a failure to express myelin genes
(Mathews et al., 2014). Changes in CNS cholesterol homeostasis occur in all likelihood due to
the down-regulation of Cyp46a1, encoding an important cholesterol-removing enzyme, and
the increased gene expression of Apoe and Abca1, both of which are involved in the efflux
and transport of cholesterol (Martin et al., 2010).
Based on these observations, it can be concluded that the observed failure of sufficient
remyelination might be caused by a change in the genes described above causing a severe
dysregulation during myelination. Observations during developmental myelination suggested
the presence of specific checkpoints to ensure sufficient production of cholesterol to precede
brain myelination (Fünfschilling et al., 2012; Herz and Farese, 1999). Considering the concept
of an orchestrated remyelination process, it seems plausible that the suggested dysregulation
of OPCs in MS and its animal models may be caused by insufficient cholesterol biosynthesis.
However, a down-regulation of the genes associated with cholesterol biosynthesis secondary
to demyelination or loss of oligodendrocytes cannot be excluded. To further elucidate the
underlying processes, the lipid composition of the main cholesterol repositories in the body
was analyzed in the second part of the study.
Changes in the lipid composition of blood serum, liver, and spinal cord
Recent studies indicate that serum dyslipidemia could be a comorbidity of MS (Marrie and
Horwitz, 2010). Different MRI studies in MS patients showed an association with an adverse
34
Hypercholesterolemia in TME
lipid profile and disability progression (Giubilei et al., 2002; Tettey et al., 2014a; Tettey et al.,
2014b; Weinstock-Guttman et al., 2013; Weinstock-Guttman et al., 2011), an effect we could
not reproduce in the present study. It can eventually be related to the fact that mice have
relatively high HDL and low LDL levels under physiological conditions (Getz and Reardon,
2006). Therefore, the mouse is generally not as susceptible as humans to a disruption of this
lipoprotein balance (Getz and Reardon, 2006). In one study, MS patients’ plasma levels of
triglycerides were increased, whereas LDL levels decreased in comparison to healthy controls
(Palavra et al., 2013). Another study reported increased total cholesterol, HDL, LDL, and
triglyceride serum levels in MS patients (Sternberg et al., 2013).
In the spinal cord, galactocerebroside (galactosylceramide; GalC) and sphingomyelin levels
were significantly decreased in TMEV-infected animals compared to mock- infected animals
at 196dpi. GalC is the most typical myelin lipid and is used as a marker for mature
oligodendrocytes (Baumann and Pham-Dinh, 2001). In addition, GalC is proportional to the
amount of myelin during development (Norton and Poduslo, 1973). GalC-deficient mice
show impaired insulator function of the myelin sheath (Baumann and Pham-Dinh, 2001).
Sphingomyelin is involved in cell adhesion and forms lipid rafts with cholesterol for signal
transduction (Podbielska et al., 2012; for a review see Lindner and Naim, 2009). Interestingly,
cholesterol levels were not different between TMEV- and mock infected animals, although a
decrease in cholesterol is described in lesions and normal appearing white matter in MS
patients (Cumings, 1955; Gerstl et al., 1961; Wender et al., 1974). However, our findings are
in line with a study in EAE. In the latter, transcriptional changes in cholesterol transporters,
but no alterations in spinal cord cholesterol levels were detected (Mueller et al., 2008). A
plausible explanation could be the different degradation capacities for various lipid
components in the myelin sheath and the sustained duration of the disease in human patients.
Under physiological circumstances, cholesterol has by far the longest half- life of about 300
days. In contrast, cerebrosides have a half- life of about 20 days (Ando et al., 2003).
Furthermore, hyperactivities of degradation enzymes as described for the hydrolysis of
sphingomyelin may play a central role (Wheeler et al., 2008). However, an impaired response
or clearing activity of macrophages might represent a possible cause for the poor clearance of
myelin debris leading to dysregulation of OPC differentiation (Kotter et al., 2006; Robinson
and Miller, 1999).
35
Hypercholesterolemia in TME
Influence of hypercholesterolemia on TMEV-infection
Since we did not detect serum dyslipidemia in a lipid balanced diet, we were interested in the
influence of hypercholesterolemia in TMEV- infected animals. A possible beneficial effect of
cholesterol supplementation appeared to be plausible in view of our microarray study and an
in vitro study, in which treatment with statins that lead to the formation of abnormal myelin
membranes (Maier et al., 2009) or retraction of processes and cell death of OPCs and
oligodendrocytes, that could be rescued by cholesterol supplementation (Miron et al., 2007).
Cholesterol supplementation has never been offered as a potential treatment for MS likely due
to controversially discussed negative impact of high- fat diets as a possible etiological factor
for MS (Alter et al., 1974; Lauer, 1997; Ozay et al., 2014; Swank, 1954; Warren et al., 1982;
Weinstock-Guttman et al., 2005; Westlund and Kurland, 1953). However, as demonstrated in
SLOS, dietary cholesterol supplementation can have a beneficial effect with respect to
remyelination (Caruso et al., 2004; Elias et al., 1997; Irons et al., 1997).
In the present study, we could neither see an anticipated beneficial effect due to the higher
circulatory availability of cholesterol, nor detect a negative impact of a high cholesterol diet
on the development of the disease. This was substantiated by clinical assessment, histology
and immunohistochemistry. The amount, onset or duration of meningitis, leukomyelitis,
demyelination or remyelination remained unchanged. The reason for the significantly
decreased density of NG2-positive cells in the Paigen diet fed mice compared with control
diet fed animals at 196dpi remains elusive. These observations cannot be explained by an
increased differentiation of OPCs to myelinating oligodendrocytes, because there was no
consecutive increase in the myelinated area. Similar to previous studies in TME, an increased
number of OPCs without maturation to myelinating oligodendrocytes was demonstrated at
earlier time points (Ulrich et al., 2008). Interestingly, a similar differentiation arrest was
observed in a cuprizone experiment in simvastatin-treated animals (Miron et al., 2009).
In the present study, the amount of sphingomyelin was increased at 98dpi in Paigen diet mice
compared to control diet mice. However, the underlying mechanism is undetermined.
Surprisingly, our results are in contrast to a similar feeding experiment using a Western-type
lipid-rich diet conducted in MOG- induced EAE in C57BL/6J mice (Timmermans et al., 2014).
It was shown that high fat diet increased the immune cell infiltration, inflammatory mediator
36
Hypercholesterolemia in TME
production and exacerbated neurologic symptoms in this EAE model (Timmermans et al.,
2014). The difference may be due to the different mouse strains used or the mode in which
myelin loss is induced. The C57BL/6J mice used by Timmermans et al. (2014) are known to
be one of the most atherosclerosis sensitive strains (Getz and Reardon, 2006), whereas the
SJL/J mouse strain develops a significant hypercholesterolemia, despite a relative resistance
to atherosclerosis, when fed with Paigen diet (Nishina et al., 1993; Paigen et al., 1990). In
contrast to the Paigen diet, the Western-type diet is known to have a higher atherogenic
potential (Getz and Reardon, 2006). Thus, the increased inflammatory reaction in
Timmermans et al. (2014) could be a secondary effect of atherosclerotic changes. Clinical
chemistry showed significantly elevated total cholesterol, HDL, LDL and free fatty acids in
the blood serum of all Paigen diet fed animals. The observed down-regulation of triglycerides
as an effect of the Paigen diet is a well-known fact in atherosclerosis research (Getz and
Reardon, 2006; Nishina et al., 1993; Paigen et al., 1990). The increased fat content of the diet
resulted in a severe hepatic lipidosis, predominantly consisting of triglycerides and cholesterol.
Based on the insights gained in this study it can be concluded that down-regulation of
cholesterol biosynthesis is a robust transcriptional marker for demyelinating conditions like
TME, EAE (Mueller et al., 2008) and MS (Lock et al., 2002). Furthermore, de- and
remyelination in the chronic progressive TME model represent two processes that develop
and precede independently from serum cholesterol levels, most likely due to the inability of
the circulatory cholesterol to enter the CNS due to a tight blood brain barrier. Moreover,
serum hypercholesterolemia and dyslipidemia exhibit no negative effect on virus- induced,
inflammatory demyelination in the CNS in this atherosclerosis-resistant mouse strain. The
reported findings could indicate that the inconclusive reports regarding dyslipidemia and MS
are maybe influenced by rather indirect pathomechanistic factors and/or the confounding
influence of the respective genetic predisposition towards atherosclerosis.
37
Hypercholesterolemia in TME
Materials and Methods:
Microarray experiment:
Microarray analysis of transcriptional changes was performed with SJL/JHanHsd mice
(Harlan Winkelmann, Borchen, Germany) intracerebrally infected with 1.63x10 6 Plaque
forming units (PFU) per mouse of the BeAn strain of Theiler´s murine encephalomyelitis
virus (TMEV) in comparison with mock- infected mice at 14, 42, 98, 196 days post infection
(dpi), as described previously (Ulrich et al., 2010). Briefly, six biological replicates were used
per group and time point, except for 5 TMEV- infected mice at 98dpi. RNA was isolated from
frozen spinal cord specimens using the RNeasy mini kit (Qiagen, Hilden, Germany),
amplified and labeled with the Message-Amp II-Biotin enhanced kit (Ambion, Austin, TX,
USA) and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays (Affymetrix,
Santa Clara, CA). Quality control and low level analysis of raw fluorescence intensities were
performed with RMAExpress (Bolstad et al., 2003).
MIAME-compliant data sets are
deposited in the ArrayExpress database (E-MEXP-1717; http://www.ebi.ac.uk/arrayexpress).
In order to focus on the transcriptional changes related to the cholesterol biosynthesis, 21
genes represented in the canonical cholesterol biosynthesis pathway of the MetacoreT M
database (version 6.5; GeneGo, St. Joseph, MO, USA) and 24 manually selected individual
genes involved in cholesterol metabolism and transport were individually analyzed using pairwise Mann-Whitney-U-tests (IBM SPSS Statistics, Version 21, IBM, Chicago, USA).
Statistical significance was generally accepted as p ≤ 0.05.
Feeding Experiment:
Three weeks old female SJL/J mice (Charles River, Sulzfeld Germany) were randomly
grouped into two feeding groups and fed conventional mouse diet (low fat control diet;
product number: S2205-E010, ssniff Spezialdiäten GmbH, Soest, Germany; Table I), or
Paigen diet (high fat Paigen 1,25% cholesterol diet; product number: S2205-E015, ssniff
Spezialdiäten GmbH, Soest, Germany; Table I; Paigen et al., 1985), beginning 13 days prior
to the infection (-13dpi) over the complete time of the experiment either ad libitum with free
access to tap water. The animals were housed in isolated ventilated cages (Tecniplast,
Hohenpeißenberg, Germany). At an age of five weeks animals from the two feeding groups
were inoculated into the right cerebral hemisphere with 1.63x10 6 PFU/mouse of the BeAn38
Hypercholesterolemia in TME
strain of TMEV as previously described (Ulrich et al., 2006; Ulrich et al., 2010; Ulrich et al.,
2008). Mock infection was performed with solvent only (Ulrich et al., 2006; Ulrich et al.,
2010; Ulrich et al., 2008). At 7, 21, 42, 98 and 196dpi, six animals per group (except 5 mice
at 42dpi in mock- infected, Paigen diet fed group) were sacrificed after general anesthesia as
described (Ulrich et al., 2006).
Immediately after death, brain, liver, spinal cord, heart and lungs were removed and weighted
(Sartorius
TE13S-DS,
Sartorius
AG,
Göttingen,
Germany).
For
histology
and
immunohistochemistry the organs was fixed in 10% formalin for 24 h and embedded in
paraffin wax (formalin- fixed and paraffin embedded, FFPE). For lipid analysis specimens
were immediately snap-frozen and stored at -80°C. For cryo-sections liver was embedded into
Optimal Cutting Temperature compound (OCT; Tissue-Tek® O.C.T.
TM
compound, Sakura,
Alphen aan den Rijn, Netherlands). Additionally, spinal cord segments were fixed with 5%
glutaraldehyde/cacodylate buffer for 24h, post fixated with 1% osmium tetroxide, dehydrated,
and embedded in epoxy resin (Baumgärtner et al., 1987; Ulrich et al., 2008). The animal
experiments were authorized by the local authorities (Regierungspräsidium Hannover,
Germany, permission number: AZ: 33.14-42502-04-11/0517).
Clinical examination and rotarod analysis
The clinical course was evaluated weekly employing weight measurements (Sartorius TE13SDS, Sartorius AG, Göttingen, Germany) and Rotarod testing (RotaRod Treadmill, TSE
Technical & Scientific Equipment, Bad Homburg, Germany). Prior to infection mice were
trained twice at –13 and –7dpi for 10 minutes at a constant speed of 5 or 10 rounds per minute
(rpm), respectively. For the measurements, the rod speed was linearly increased from 5 rpm to
55 rpm over a time period of 5 min and the attained rpm at drop was analyzed for significant
differences between the groups using three-factorial ANOVA with repeated measures and
post-hoc independent t-tests for the factors status of infection, time-point post infection and
feeding regimen using IBM SPSS Statistics (IBM SPSS Statistics, Version 21, IBM, Chicago,
USA). A mean score per mouse was calculated from three trials per day (Ulrich et al., 2010).
Clinical chemistry of blood serum
39
Hypercholesterolemia in TME
Blood was collected immediately after death from the caudal V. cava and serum stored at -80°
C. Serum concentration of total cholesterol, low density lipoprotein (LDL), high-density
lipoprotein
(HDL),
triglycerides,
alanine
transaminase
(ALT),
gamma-glutamyl
transpeptidase (GGT), glutamate dehydrogenase (GLDH), total billirubin, direct billirubin,
albumin, urea (Cobas®, Roche Diagnostics GmbH, Mannheim, Germany) and free fatty acids
(FFA; Wako chemicals GmbH, Neuss, Germany) were measured with a Hitachi Automatic
Bioanalyzer (Roche Diagnostics
GmbH,
Mannheim,
Germany) according
to
the
manufacturer’s instructions. Arithmetic means with 5-95% confidence interval of the mockinfected animals fed with normal diet were used as reference values.
Histological examination
Serial sections of FFPE liver specimens were stained with hematoxylin and eosin (HE),
Heidenhain's azan trichrome stain and Fouchet´s stain, and were semi-quantitatively scored
for lipidosis (0 = no lipidosis; 1 = single foci of lipidosis; 2 = mild lipidosis; 3 = moderate
lipidosis; 4 = marked lipidosis; 5 = severe, diffuse lipidosis), type of intracytoplasmic lipid
droplets (micro- or macrovesicular), fibrosis (0= no fibrosis; 1= mild; 2= moderate; 3=
severe), and cholestasis (0= no cholestasis; 1= mild; 2= moderate; 3= severe) respectively
(Thoolen et al., 2010). Cryo-sections of the liver were stained with Oil red O stain and the
percentage of Oil red O positive areas was morphometrically assessed using the analysis 3.1
software package (SOFT Imaging system, Münster, Germany; Haist et al., 2012).
FFPE transversal sections of the ascending aorta, heart and large pulmonary arteries were
stained with HE and examined for changes by light microscopy.
FFPE transverse sections of cervical, thoracic and lumbar spinal cord segments were stained
with HE and Luxol fast blue-cresyl violet (LFB-CV) and evaluated semi-quantitatively for
meningitis, leukomyelitis (0 = no changes; 1 = scattered infiltrates; 2 = 2-3 layers of
inflammatory cells; 3 = more than 3 layers of inflammatory cells) and demyelination (0 = no
changes; 1 = ≤25%; 2 = 25-50%; 3 = 50-100% white matter affected; Hansmann et al., 2012;
Ulrich et al., 2006; Ulrich et al., 2010). Remyelination in the spinal cord was assessed semiquantitatively in semi-thin, epoxy resin embedded, toluidine blue stained, transverse sections
of cervical, thoracic and lumbar spinal cord (0 = no changes; 1 = single focus up to 25%; 2 =
40
Hypercholesterolemia in TME
25-50%; 3 = 50-100% of white matter affected; Ulrich et al., 2008). A mean score per mouse
was calculated from cervical, thoracic and lumbal spinal cord scores.
Immunohistochemistry
Immunohistochemistry was performed on FFPE transverse serial spinal cord sections, using
the avidin-biotin-peroxidase complex (ABC) method (Vector Laboratories, Burlingame, CA,
USA) with 3,3’-diaminobezidine-tetrahydrochloride (DAB) as chromogen (Ulrich et al.,
2008). The antibodies applied were anti-CD3 (polyclonal rabbit anti- human, diluted 1:1,000,
Dako A0452, Hamburg, Germany) for T lymphocytes, anti-CD107b (monoclonal rat antimouse biotinylated, clone M3/84, diluted 1:800, Serotec MCA2293B, Oxford, UK) for
microglia/macrophages, anti-IgG (goat anti- mouse-IgG, diluted 1:200, Vetor Laboratories,
BA9200, Burlingame, CA, USA) for plasma cells, anti- glial fibrillary acidic protein (GFAP;
polyclonal rabbit anti-cow, diluted 1:1,000, Dako Z0334, Hamburg, Germany) for astrocytes,
anti- myelin
basic protein
(MBP; polyclonal rabbit
anti- human,
diluted
1:1,600,
Merck/Millipore AB980, Darmstadt, Germany) for myelin, anti-periaxin (PRX; polyclonal
rabbit anti-human; diluted 1:5,000; Sigma-Aldrich, St. Louis, USA) for peripheral myelin,
anti-neural/glial antigen 2 (NG2; polyclonal rabbit anti- rat, diluted 1:400, Merk/Millipore
AB5320, Darmstadt, Germany) for OPCs, and anti- TMEV (polyclonal rabbit anti-TMEV
capsid protein VP1, diluted 1:2,000) for virus detection. The density of GFAP, NG2, MBP,
CD3, IgG, CD107b, PRX and TMEV immunopositive cells per square millimeter was
calculated by dividing the number of immunopositive cells obtained from the complete
thoracic spinal cord section through the measured area of the spinal cord (Sun et al., 2014;
Ulrich et al., 2008). The percentage of MBP-positive white matter areas was calculated by
dividing the computationally detected immunopositive areas by the white matter area
employing the program Analysis 3.1 (Sun et al., 2014; Ulrich et al., 2010; Ulrich et al., 2008).
For clinical chemistry, histology, immunohistochemistry and lipid analysis Kruskal-WallisTest followed by pair-wise post-hoc Mann-Whitney-U-Test with Bonferroni-correction were
calculated independently for the factors infection, time-point post infection and feeding group
(IBM SPSS Statistics). Statistical significance was generally accepted as p ≤ 0.05.
High performance thin layer chromatography (HPTLC) of liver and spinal cord
41
Hypercholesterolemia in TME
HPTLC was performed to analyze liver and spinal cord cell membrane lipids. Lipids were
extracted and prepared for HPTLC with minor modifications as as previously described
(Brogden et al., 2014). Briefly, liver and spinal cord samples were homogenized in multiple
steps and dissolved in methanol and chloroform (2:1). The upper aqueous layer was removed
and the remaining fraction was vacuum-dried and stored at -20°C. Liver and spinal cord lipid
samples were dissolved in chloroform/methanol (1:1) solution, app lied on HPTLC Silica gel
60 glass plates (Merck, Darmstadt, Germany) and run with three subsequent running solutions
consisting of acetic acid ethyl ester/1-propanol/chloroform/methanol/0.25% potassium
chloride (27:27:11:10), n- hexane/diethyl ether/acetic acid (75:23:2), and 100% n- hexane. For
visualization of the lipid bands, plates were stained in phosphoric acid/copper sulphate (10:7.5)
solution. Lipid bands were identified by comparison to authentic standards (Sigma Aldrich, St.
Louis, USA) substances and analyzed using the CP ATLAS software (Lazarsoftware). The
intensity of each lipid detected by CP ATLAS software was divided by the weight of the
respective sample. An average intensity of two repeats for each sample was calculated.
Dividing the intensity/weight value by a correction factor normalized differences between the
plates. The correction factor was calculated by dividing the geometric mean of the standards
from the respective plate by the geometric mean of all standards. Statistical significance was
calculated by three-factorial ANOVA and post-hoc independent t-tests (IBM SPSS Statistics).
42
Hypercholesterolemia in TME
Acknowledgement
The authors thank Anuschka Unold, Thomas Feidl and Martin Gamber for excellent technical
support for microarray technology and Priv.-Doz. Dr. Gunter Eckert for kindly providing his
protocols for the lipid analysis. We also thank Petra Grünig, Bettina Buck, and Caro Schütz
for excellent technical support.
Author contribution:
R.U., U.D., H.Y.N. and W.B. initiated the scientific research; B.B.R., W.S, G.B., Y.S., P.K.,
F.C. performed the experiments; R.U., B.B.R., W.S., A.K., U.D., F.C. analyzed the data;
B.B.R., W.S., R.U., W.B., F.C., G.B., H.Y.N. edited the manuscript.
The authors have no conflicting financial interests.
Funding
This study was in part supported by Niedersachsen-Research Network on Neuroinfectiology
(N-RENNT) of the Ministry of Science and Culture of Lower Saxony, Germany. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript. Wenhui Sun and Yanyong Sun received a grant from the China Scholarship
Council (CSC) under the File No. 2010617013 and No. 2009617013, respectively.
43
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Figures legends:
Figure 1: Canonical cholesterol biosynthesis pathway map in Theiler’s murine
encephalomyelitis
Transcriptional changes associated with cholesterol biosynthesis pathway in Theiler´s murine
encephalomyelitis virus (TMEV)- infected mice in comparison with mock- infected control
mice, illustrated in the canonical cholesterol biosynthesis pathway map from Metacore T M
database (GeneGO, St. Joseph, USA). The bars labeled from one to four disp lay the fold
changes of significantly differentially regulated genes in TMEV- infected animals (N=6)
compared with mock-infected animals (N=6) on the four days post infection (dpi) employing
pair-wise Mann-Whitney U-tests (p ≤ 0.05) (1 = 14dpi; 2 = 42dpi; 3 = 98dpi; 4 = 196dpi).
The blue indicator scale of the bar marks down-regulation and displays a comparable
measurement of the magnitude of the negative fold change.
Green arrows = positive functional interaction; Z = catalysis; grey arrows = technical links;
orange icons = enzymes; purple hexagons = generic compound; blue rectangle = normal
process; grey rectangle = reaction; Acat1 = acetyl-Coenzyme A acetyltransferase 1; Cyp51 =
cytochrome P450, family 51; Dhcr24 = 24-dehydrocholesterol reductase; Dhcr7 = 7dehydrocholesterol reductase; Ebp = phenylalkylamine Ca2+ antagonist (emopamil) binding
protein; Fdft1 = farnesyl diphosphate farnesyl transferase 1; Fdps = farnesyl diphosphate
synthetase; Ggps1 = geranylgeranyl diphosphate synthase 1; Hmgcr = 3-hydroxy-3methylglutaryl-Coenzyme A reductase; Hmgcs1 = 3-hydroxy-3-methylglutaryl-Coenzyme A
synthase 1; Idi1 = isopentenyl-diphosphate delta isomerase; Idi2 = isopentenyl-diphosphate
delta isomerase 2; Lss = lanosterol synthase; Mvd = mevalonate (diphospho) decarboxylase;
Mvk = mevalonate kinase; Nsdhl = NAD(P) dependent steroid dehydrogenase -like; Pmvk =
phosphomevalonate kinase; Sc4mol = sterol-C4-methyl oxidase-like; Sc5d = sterol-C5desaturase (fungal ERG3, delta-5-desaturase) homolog (S. cerevisae); Sqle = squalene
epoxidase; Tm7sf2 = transmembrane 7 superfamily member 2;
Figure 2: Quantitative analysis of the lipid composition of liver, blood serum and spinal
cord in Theiler’s murine encephalomyelitis
51
Hypercholesterolemia in TME
Lipid composition of blood serum,
liver and spinal cord of Theiler´s murine
encephalomyelitis virus- infected female SJL/J mice (N=6; except 7dpi in B, C, D, E were
only 3 animals are used) compared to mock-infected female SJL/J mice (N=6) was analyzed
on five days post infection (7dpi, 21dpi, 42dpi, 98dpi, 196dpi). Displayed are serum lipid
values, as measured with Hitachi Automatic Bioanalyzer (Roche Diagnostics GmbH,
Mannheim, Germany), of (A) total Cholesterol, (B) low density lipoprotein, (C) high dens ity
lipoprotein, (D) triglycerides and (E) free fatty acids in mmol/L. Furthermore (F) liver (only
at 98dpi) and (G) spinal cord (21dpi, 42dpi, 98dpi, 196dpi) lipid intensity values per sample
weight, as measured with high performance thin layer chromatography, are shown. The data
are displayed as bars or box-and-whisker plots showing the median and 95% percentile.
Statistically significant differences (p ≤ 0.05) as detected by pair-wise Mann-Whitney U-tests
variance or three- factorial ANOVA with post-hoc independent t-tests are marked with
asterisks ().
Figure 3: Body weight and quantitative analysis of the lipid composition of the blood
serum
in
Theiler’s
murine
encephalomyelitis
after
experimentally -induced
hypercholesterolemia
(A) Mean body weight in gram (g) over time (days post infection;dpi) of Theiler´s murine
encephalomyelitis virus (TMEV)- infected female SJL/J mice (N=6; except in Figure 3 D, E,
F: TMEV-infected, control diet group: 7dpi N=3) compared to mock- infected female SJL/J
mice (N=6; except mock- infected; Paigen diet group: 42dpi N=5) grouped into two feeding
groups (control diet; Paigen diet).
(B - F) Serum lipid levels at 7, 21, 42, 98 and 196dpi of the animals described in (A) as
measured with Hitachi Automatic Bioanalyzer (Roche Diagnostics GmbH, Mannheim,
Germany). Data of (B) total cholesterol, (C) low density lipoprotein, (D) high density
lipoprotein, (E) triglycerides and (D) free fatty acids are displayed in mmol/L as box-andwhisker plots showing the median and 95% percentile. Gray areas show the median and 95%
percentile of the mock- infected animals fed with control diet, used as reference values.
Statistical significant differences (p ≤ 0.05) as detected by pair-wise Mann-Whitney U-tests
are marked with asterisks () for the comparison of TMEV- infected animals with mockinfected animals of the same feeding group. Statistically significant differences (p ≤ 0.05)
52
Hypercholesterolemia in TME
between Paigen diet and control diet fed animals of the same infection group are marked with
a dot ().
Figure 4: Analysis of liver-weight, histology, quantitative lipid composition of the liver
and serum liver values in Theile r’s murine encephalomyelitis after expe rimentallyinduced hypercholesterolemia
(A) Liver weight in gram (g) on 7, 21, 42, 49, 98, and 196dpi of Theiler´s murine
encephalomyelitis virus- infected female SJL/J mice (N=6) compared to mock- infected female
SJL/J mice (N=6; except mock- infected; Paigen diet group: 42dpi N=5) grouped into two
feeding groups (control diet; Paigen diet). (B-D) Serum lipid levels of animals and timepoints described in (A) as measured with Hitachi Automatic Bioanalyzer (Roche Diagnostics
GmbH, Mannheim, Germany). Data of (B) glutamate dehydrogenase in IU/L, (C) alanine
transaminase in IU/L, and (D) albumin in g/dl, are displayed as box-and-whisker plots
showing the median and 95% percentile. Gray areas show the median and 95% percentile of
the mock- infected animals fed with control diet, used as reference values. (E) Histological
examination of the liver stained with (I) hematoxylin and eosin-stain, (II) Heidenhain's azan
trichrome stain, (III) Fouchet´s stain and (IV) oil red O stain revealed a moderate to severe,
multifocal to diffuse, centrolobularly accentuated, microvesicular hepatic lipidosis in all
Paigen diet fed mice (I). A mild, multifocal, hepatic fibrosis was associated with the fatty
degeneration in Paigen diet fed animals (II). No cholestasis was detectable in any of the
groups (III). Oil red O positive areas were significantly higher in TMEV- and mock- infected,
Paigen diet mice compared to control diet mice (IV). Exemplary displayed is the liver of a
TMEV- infected control diet fed mouse (left) and Paigen diet fed mouse (right) at 98dpi.
Pictures were taken with a 10- fold objective for (I), 20-fold objective for (II-III) and 40- fold
objective for (IV). The bar indicates 100 µm in (I), 50 µm in (II-III) and 20 µm in (IV). (F)
Lipid composition of the liver (only at 98dpi) of the animals described in (A). Displayed are
the lipid intensity values per sample weight, as measured with high performance thin layer
chromatography as bars showing the median and 95% percentile. Statistically significant
differences (p ≤ 0.05) as detected by pair-wise Mann-Whitney U-tests or three- factorial
ANOVA with post- hoc independent t-tests are marked with asterisks () for the comparison
of TMEV- infected animals with mock- infected animals of the same feeding group.
53
Hypercholesterolemia in TME
Statistically significant differences (p ≤ 0.05) between Paigen diet and control diet fed
animals of the same infection group are marked with a dot ().
Figure 5: Rotarod performance and inflammatory changes in the spinal cord in
Theiler’s murine encephalomyelitis after experimentally-induced hypercholesterolemia
(A) Rotarod performance on 0-195dpi of Theiler´s murine encephalomyelitis virus (TMEV)infected female SJL/J mice (N=6) compared to mock- infected female SJL/J mice (N=6;
except mock- infected; Paigen diet group: 42dpi N=5) grouped into two feeding groups
(control diet; Paigen diet). (B) Hematoxylin and eosin-stained thoracic spinal cord section of a
mock-infected, control diet fed mouse (I), mock- infected, Paigen diet fed mouse (II), TMEVinfected, control diet fed mouse (III), and a TMEV- infected, Paigen diet fed mouse (IV) at
196dpi. (III-IV) TMEV- infection induced a mild to moderate, mononuclear infiltration in the
meninges and the perivascular space of the white matter and a progressive demyelination. (III) Mock- infected animals show no changes. Pictures were taken with a 20-fold objective.
The bar indicates 50 µm. (C-D) Cell density of the inflammatory infiltrates characterized by
immunohistochemistry of the animals described in (A) on day 7, 21, 42, 98 and 196dpi for (C)
CD3-positive T- lymphocytes; (D) IgG-producing B- lymphocytes and (E) CD107b-positive
macrophages. The cell-density is displayed as box-and-whisker plots showing the median and
95% percentile. Statistical significant differences (p ≤ 0.05) as detected by pair-wise MannWhitney U-tests are marked with asterisks () for the comparison of TMEV- infected animals
with mock-infected animals of the same feeding group. Statistically significant differences (p
≤ 0.05) between Paigen diet and control diet fed animals of the same infection group are
marked with a dot ().
Figure 6: Semi-quantitative assessment of de- and remyelination in the spinal cord in
Theiler’s murine encephalomyelitis after experimentally-induced hypercholesterolemia
(A) Luxol fast blue-cresyl violet-stained thoracic spinal cord section of a mock- infected,
control diet fed mouse (I), a mock- infected, Paigen diet fed mouse (II), a TMEV- infected,
control diet fed mouse (III), and a TMEV- infected, Paigen diet fed mouse (IV) at 196dpi. (IIIIV) TMEV- infection induced a severe progressive demyelination. No demyelination was
observed in mock- infected animals. Pictures were taken with a 20-fold objective. The bar
54
Hypercholesterolemia in TME
indicates 50 µm. (B) Semi- thin toluidine blue-stained thoracic spinal cord section of the
groups described in (A). Remyelinated axons are characterized by thinner myelin sheaths than
normal. Myelin debris can be found intra-cytoplasmatically in macrophages. Pictures were
taken with a 60-fold immersion oil objective. The bar indicates 10 µm. (C-D) Semiquantitative scoring (0-3) of (C) demyelination, assessed in Luxol fast blue-cresyl violetstained spinal cord section and (D) remyelination, assessed in semi- thin toluidine blue-stained
spinal cord sections at 7,21, 42, 98 and 196dpi. The scores are displayed as box-and-whisker
plots showing the median and 95% percentile. Statistically significant differences (p ≤ 0.05)
as detected by pair-wise Mann-Whitney U-tests are marked with asterisks () for the
comparison of TMEV- infected animals with mock- infected animals of the same feeding
group. Statistically significant differences (p ≤ 0.05) between Paigen diet and control diet fed
animals of the same infection group are marked with a dot ().
Figure 7: Immunohistoche mical assessment of de - and re myelination and lipid
composition of the spinal cord in Theiler’s
murine
encephalomyelitis after
experimentally-induced hypercholesterolemia
(A) Myelin basic protein- immunopositive positive white matter area; (B) density of
neuron/glial-antigen 2-immunopositive cells; (C) density of periaxin- immunopositive cells,
and (D) glial fibrillary acid protein- immunopositive spinal cord area of Theiler´s murine
encephalomyelitis virus (TMEV)- infected female SJL/J mice (N=6) compared with mockinfected female SJL/J mice (N=6; except mock-infected; Paigen diet group: 42dpi N=5)
grouped into two feeding groups (control diet; Paigen diet) at 7, 21, 42, 98 and 196dpi. The
scores are displayed as box-and-whisker plots showing the median and 95% percentile. (E)
Spinal cord lipid intensity values per sample weight, as measured with high performance thin
layer chromatography, are shown for the animals described above. Statistically significant
differences (p ≤ 0.05) as detected by pair-wise Mann-Whitney U-tests or three- factorial
ANOVA with post- hoc independent t-tests are marked with asterisks () for the comparison
of TMEV- infected animals with mock- infected animals of the same feeding group.
Statistically significant differences (p ≤ 0.05) between Paigen diet and control diet fed
animals of the same infection group are marked with a dot ().
55
Hypercholesterolemia in TME
Tables:
Table I: Diet composition
Diet composition in percent dry weight of the conventional low- fat diet, used as control diet,
and the cholesterol-rich, high- fat Paigen diet, used to induce hypercholesterolemia in female
SJL/J mice applied as a maintenance diet from 14 days prior to the infection with Theiler’s
murine encephalomyelitis virus or mock substance over the complete studied period of 196
days post infection.
Food ingredients
Low fat Control diet
High fat Paigen diet
S2205-E010
S2205-E015
[% dry weight]
[% dry weight]
Casein
20.00
20.00
Corn starch
30.00
-
Maltodextrin
20.39
0.94
Dextrose
11.00
-
Sucrose
-
50.00
Cellulose
5.00
5.00
DL-Methionine
0.20
0.20
L-Cystine
0.10
0.10
Vitamin premixture
1.00
1.00
Mineral
5.00
5.00
Choline Cl
0.30
0.30
Butylated hydroxytoluene
0.01
0.01
premixture,
AIN93G
56
Hypercholesterolemia in TME
Continuation of Table I
Food ingredients
Low fat Control diet
High fat Paigen diet
S2205-E010
S2205-E015
[% dry weight]
[% dry weight]
Cholesterol
-
1.25
Corn oil
1.00
1.00
Soybean oil
6.00
-
Cocoa butter
-
15.20
Protein
17.8
17.8
Fat
7.1
17.6
Fiber
5.0
5.0
Ash
4.3
4.3
Starch
29.4
-
Sugar
12.1
50.9
NfE (carbohydrates)a
62.9
53.0
MEb (Atwater) [MJ/kg]
16.2
18.5
MEb (Atwater) [kcal/g]
3.9
4.4
kcal% Protein
18
16
kcal% Fat
17
36
kcal% Carbohydrates
65
48
a
NfE = Nitrogen-free extract; bME= metabolized energy;
57
Hypercholesterolemia in TME
Supplemental Material:
Suppleme ntal Table 1: Fold changes and p-values of genes associated with cholesterol
synthesis, metabolism and transport
The table displays the 48 probe sets (21 genes) represented in the canonical cholesterol
biosynthesis pathway map in the MetacoreT M database (version 6.5; GeneGo, St. Joseph, MO,
USA) and manually selected 43 probe sets (24 genes) used for the analysis of the cholesterol
biosynthesis, metabolism and transport in TME. Shown are the fold changes (FC) and pvalues. Fold changes are calculated as the ratio of the inverse-transformed arithmetic means
of the expression values of Theiler’s murine encephalomyelitis virus (TMEV)- infected
animals versus mock- infected mice animals on 14, 42, 98, and 196dpi. Down-regulation is
shown as negative reciprocal values (Ulrich et al., 2010). P-values were calculated by
performing pair-wise Mann-Whitney-U-tests in IBM SPSS Statistics (Version 21, IBM,
Chicago, USA). MIAME-compliant row data sets are deposited in the ArrayExpress database
(E-MEXP-1717; http://www. ebi.ac.uk/arrayexpress).
58
Hypercholesterolemia in TME
Figure 1:
59
Hypercholesterolemia in TME
Figure 2A-F:
60
Hypercholesterolemia in TME
Figure 2G:
61
Hypercholesterolemia in TME
Figure 3A-B:
62
Hypercholesterolemia in TME
Figure 3C-F:
63
Hypercholesterolemia in TME
Figure 4A-D:
64
Hypercholesterolemia in TME
Figure 4E:
E
65
Hypercholesterolemia in TME
Figure 4F:
66
Hypercholesterolemia in TME
Figure 5A-B:
67
Hypercholesterolemia in TME
Figure 5C-E:
68
Hypercholesterolemia in TME
Figure 6A-B:
69
Hypercholesterolemia in TME
Figure 6C-D:
70
Hypercholesterolemia in TME
Figure 7A-D:
71
Hypercholesterolemia in TME
Figure 7E:
72
Hypercholesterolemia in TME
Supplemental Table 1: Cholesterol Biosynthesis Pathway
Probe set ID
Gene Symbol
Gene Name
FC14
p14
FC42
p42
FC98
p98
FC196
p196
1424182_at
1451271_a_at
1424183_at
1422533_at
1450646_at
1422534_at
1451895_a_at
1418129_at
1418130_at
1448619_at
1416667_at
Acat1
Acat1
Acat1
Cyp51
Cyp51
Cyp51
Dhcr24
Dhcr24
Dhcr24
Dhcr7
Ebp
-1.10
-1.11
-1.08
-1.17
-1.11
1.03
-1.06
-1.23
1.07
-1.07
-1.10
0.31
0.13
0.13
0.13
0.13
0.94
0.59
0.02
0.82
0.48
0.09
-1.17
-1.14
-1.10
-1.27
-1.23
-1.11
-1.10
-1.23
1.04
-1.12
-1.00
0.09
0.00
0.01
0.01
0.00
0.24
0.04
0.00
0.82
0.03
0.82
-1.02
-1.12
-1.10
-1.34
-1.39
-1.32
-1.51
-1.53
1.07
-1.34
-1.08
0.79
0.01
0.03
0.01
0.00
0.00
0.00
0.05
0.05
0.00
0.33
-1.14
-1.11
-1.05
-1.57
-1.55
-1.40
-1.55
-1.55
-1.05
-1.32
-1.06
0.09
0.00
0.06
0.00
0.00
0.00
0.00
0.04
0.59
0.00
0.09
1438322_ x_at
1448130_at
1438919_ x_at
Fdft1
Fdft1
Fdft1 ///
Gm6781
Fdft1 ///
Gm6781
Fdps
Fdps ///
LOC674674
Ggps1
Ggps1 ///
Gm5630 ///
LOC100045315
Ggps1 ///
LOC100045315
Ggps1 ///
LOC100045315
Hmgcr
acetyl-Coenzyme A acetyltransferase 1
acetyl-Coenzyme A acetyltransferase 1
acetyl-Coenzyme A acetyltransferase 1
cytochrome P450, family 51
cytochrome P450, family 51
cytochrome P450, family 51
24-dehydrocholesterol reductase
24-dehydrocholesterol reductase
24-dehydrocholesterol reductase
7-dehydrocholesterol reductase
phenylalkylamine Ca 2+ antagonist (emopamil)
binding protein
farnesyl diphosphate farnesyl transferase 1
farnesyl diphosphate farnesyl transferase 1
farnesyl diphosphate farnesyl transferase 1 ///
predicted gene 6781
farnesyl diphosphate farnesyl transferase 1 ///
predicted gene 6781
farnesyl diphosphate synthetase
farnesyl diphosphate synthetase /// similar to
farnesyl diphosphate synthetase
geranylgeranyl diphosphate synthase 1
geranylgeranyl diphosphate synthase 1 ///
geranylgeranyl diphosphate synthase 1
pseudogene /// similar to Ggps1 protein
geranylgeranyl diphosphate synthase 1 ///
similar to Ggps1 protein
geranylgeranyl diphosphate synthase 1 ///
similar to Ggps1 protein
3-hydroxy-3-methylglutaryl-Coenzy me A
reductase
-1.09
-1.11
-1.07
0.13
0.13
0.70
-1.15
-1.22
1.01
0.01
0.00
0.82
-1.40
-1.43
-1.07
0.00
0.00
0.18
-1.46
-1.46
-1.02
0.00
0.00
0.94
-1.17
0.39
1.11
0.13
-1.02
0.79
1.10
0.31
-1.13
-1.02
0.13
0.94
-1.26
1.08
0.01
0.48
-1.54
-1.00
0.00
1.00
-1.38
1.19
0.02
0.03
-1.14
-1.12
0.04
0.18
-1.08
-1.05
0.18
0.59
-1.15
-1.05
0.18
0.43
-1.18
-1.08
0.06
0.09
-1.01
1.00
-1.03
0.94
-1.17
0.00
-1.15
0.09
-1.14
0.18
-1.03
0.94
-1.07
0.66
-1.05
0.48
-1.18
0.09
-1.26
0.03
-1.39
0.01
-1.36
0.03
1438918_at
1423418_at
1438150_at
1419506_at
1419805_s_at
1429356_s_at
1419505_a_at
1427229_at
73
Hypercholesterolemia in TME
Continuation of supplemental Table 1:
74
Probe set ID
Gene Symbol
Gene Name
FC14
p14
FC42
p42
FC98
p98
FC196
p196
1451766_at
Hmgcr
1.06
0.70
-1.07
0.70
-1.13
0.13
-1.07
0.31
1433446_at
Hmgcs1
-1.13
0.02
-1.32
0.00
-1.47
0.02
-1.54
0.00
1433443_a_at
Hmgcs1
-1.07
0.18
-1.16
0.03
-1.35
0.02
-1.46
0.00
1433444_at
Hmgcs1
-1.12
0.03
-1.16
0.01
-1.20
0.02
-1.39
0.02
1433445_ x_at
Hmgcs1
-1.11
0.02
-1.15
0.00
-1.26
0.00
-1.37
0.02
1441536_at
Hmgcs1
1.14
0.48
1.00
0.82
-1.08
0.54
-1.30
0.00
1423804_a_at
1451122_at
1440852_at
Idi1
Idi1
Idi2
-1.30
-1.16
1.03
0.06
0.04
0.94
-1.38
-1.26
-1.05
0.02
0.02
0.48
-1.51
-1.58
-1.13
0.00
0.00
0.02
-1.92
-1.66
1.06
0.00
0.00
0.59
1430619_a_at
-1.14
0.06
-1.09
0.04
-1.23
0.02
-1.20
0.03
1426913_at
1420013_s_at
1420014_at
1448663_s_at
1417303_at
1418052_at
1416222_at
LOC637711 ///
Mvk
Lss
Lss
Lss
Mvd
Mvd
Mvk
Nsdhl
-1.12
-1.09
-1.05
-1.13
-1.14
-1.10
-1.08
0.04
0.39
0.94
0.18
0.09
0.39
0.09
-1.22
-1.16
-1.11
-1.22
-1.17
-1.17
-1.15
0.00
0.13
0.39
0.04
0.00
0.04
0.00
-1.31
-1.17
-1.13
-1.47
-1.42
-1.25
-1.25
0.00
0.02
0.01
0.00
0.00
0.00
0.05
-1.42
-1.20
-1.15
-1.42
-1.23
-1.26
-1.40
0.00
0.01
0.01
0.00
0.02
0.00
0.00
1458831_at
Nsdhl
-1.03
0.48
1.04
0.70
1.03
0.54
-1.05
0.31
1427893_a_at
1423078_a_at
Pmvk
Sc4mol
3-hydroxy-3-methylglutaryl-Coenzy me
A reductase
3-hydroxy-3-methylglutaryl-Coenzy me
A synthase 1
3-hydroxy-3-methylglutaryl-Coenzy me
A synthase 1
3-hydroxy-3-methylglutaryl-Coenzy me
A synthase 1
3-hydroxy-3-methylglutaryl-Coenzy me
A synthase 1
3-hydroxy-3-methylglutaryl-Coenzy me
A synthase 1
isopentenyl-diphosphate delta isomerase
isopentenyl-diphosphate delta isomerase
isopentenyl-diphosphate delta isomerase
2
similar to mevalonate kinase ///
mevalonate kinase
lanosterol synthase
lanosterol synthase
Lanosterol synthase
mevalonate (diphospho) decarboxylase
mevalonate (diphospho) decarboxylase
mevalonate kinase
NAD(P) dependent steroid
dehydrogenase-like
NAD(P) dependent steroid
dehydrogenase-like
phosphomevalonate kinase
sterol-C4-methyl oxidase-like
-1.08
-1.16
0.09
0.00
-1.09
-1.24
0.13
0.00
-1.22
-1.53
0.00
0.00
-1.20
-1.64
0.00
0.00
Hypercholesterolemia in TME
Continuation of supplemental Table 1:
Probe set ID
Gene Symbol
Gene Name
FC14
p14
FC42
p42
FC98
p98
FC196
p196
1459627_at
1451457_at
Sc4mol
Sc5d
-1.02
-1.11
0.70
0.03
-1.18
-1.21
0.24
0.00
-1.00
-1.42
0.79
0.02
-1.11
-1.41
0.31
0.00
1434520_at
Sc5d
-1.09
0.06
-1.21
0.01
-1.52
0.01
-1.38
0.00
1424709_at
Sc5d
-1.14
0.18
-1.09
0.39
-1.18
0.13
-1.33
0.01
1415993_at
1460684_at
Sqle
Tm7sf2
Sterol-C4-methyl oxidase-like
sterol-C5-desaturase (fungal ERG3,
delta-5-desaturase) homolog (S.
cerevisae)
sterol-C5-desaturase (fungal ERG3,
delta-5-desaturase) homolog (S.
cerevisae)
sterol-C5-desaturase (fungal ERG3,
delta-5-desaturase) homolog (S.
cerevisae)
squalene epoxidase
transmembrane 7 superfamily member 2
-1.03
-1.21
0.70
0.00
-1.15
-1.19
0.00
0.00
-1.42
-1.53
0.02
0.00
-1.36
-1.32
0.00
0.00
75
Hypercholesterolemia in TME
Continuation of supplemental Table 1: Manually selected genes
76
Probe set ID
Gene Symbol
Gene Name
FC14
p14
FC42
p42
FC98
p98
FC196
p196
1425771_at
Akr1d1
-1.12
0.59
-1.08
0.48
1.00
0.93
-1.11
0.18
1455100_at
Akr1d1
-1.05
0.39
-1.11
0.13
1.02
0.79
-1.05
0.06
1417257_at
1417590_at
Cel
Cyp27a1
-1.04
1.11
0.48
0.24
-1.01
-1.08
0.94
0.24
1.06
-1.24
0.25
0.05
1.05
-1.23
0.59
0.00
1457665_x_at
Cyp27a1
-1.06
0.09
-1.03
0.18
1.01
1.00
-1.07
0.59
1418780_at
Cyp39a1
-1.12
0.31
-1.18
0.02
-1.32
0.01
-1.28
0.09
1422100_at
Cyp7a1
-1.17
0.04
-1.22
0.24
-1.09
0.43
-1.16
0.18
1438743_at
Cyp7a1
1.01
0.59
-1.03
0.82
-1.01
0.93
1.01
0.94
1421074_at
Cyp7b1
-1.07
0.13
1.04
0.39
1.14
0.18
1.23
0.00
1421075_s_at
Cyp7b1
-1.03
0.70
1.04
0.39
1.04
0.66
1.13
0.01
1446995_at
Cyp7b1
-1.03
0.94
-1.06
0.48
1.03
0.54
-1.08
0.24
1449038_at
1448453_at
Hsd11b1
Hsd3b1
-1.28
1.01
0.02
1.00
-1.25
-1.07
0.09
0.39
-1.59
-1.08
0.00
0.08
-1.60
-1.01
0.00
1.00
1460232_s_at
Hsd3b2 /// Hsd3b3
/// Hsd3b6
-1.01
0.94
1.11
0.18
-1.11
0.25
-1.09
0.18
1423140_at
1423141_at
Lipa
Lipa
aldo-keto reductase family 1, member
D1
aldo-keto reductase family 1, member
D1
carboxyl ester lipase
cytochrome P450, family 27, subfamily
a, polypeptide 1
cytochrome P450, family 27, subfamily
a, polypeptide 1
cytochrome P450, family 39, subfamily
a, polypeptide 1
cytochrome P450, family 7, subfamily a,
polypeptide 1
cytochrome P450, family 7, subfamily a,
polypeptide 1
cytochrome P450, family 7, subfamily b,
polypeptide 1
cytochrome P450, family 7, subfamily b,
polypeptide 1
Cytochrome P450, family 7, subfamily
b, polypeptide 1
hydroxysteroid 11-beta dehydrogenase 1
hydroxy-delta-5-steroid dehydrogenase,
3 beta- and steroid delta-isomerase 1
hydroxy-delta-5-steroid dehydrogenase,
3 beta- and steroid delta-isomerase 2 ///
hydroxy-delta-5-steroid dehydrogenase,
3 beta- and steroid delta-isomerase 3 ///
hydroxy-delta-5-steroid dehydrogenase,
3 beta- and steroid delta-isomerase 6
lysosomal acid lipase A
lysosomal acid lipase A
1.06
1.10
0.24
0.31
1.15
1.21
0.13
0.18
1.20
1.67
0.18
0.01
1.26
1.90
0.04
0.00
Hypercholesterolemia in TME
Continuation of supplemental Table 1:
Probe set ID
Gene Symbol
Gene Name
FC14
p14
FC42
p42
FC98
p98
FC196
p196
1450872_s_at
1449112_at
Lipa
Slc27a5
1.02
1.00
0.59
0.70
1.16
-1.06
0.00
0.48
1.34
-1.08
0.01
0.33
1.51
-1.17
0.00
0.02
1417695_a_at
1417696_at
1417697_at
1448068_at
1460722_at
1417709_at
Soat1
Soat1
Soat1
Soat1
Soat2
Cyp46a1
1.12
1.00
1.04
-1.15
-1.01
1.04
0.48
0.82
0.39
0.31
0.82
0.52
1.68
1.40
1.49
1.23
1.11
-1.05
0.00
0.01
0.00
0.00
0.13
0.20
2.75
2.08
1.31
-1.12
1.20
-1.14
0.00
0.00
0.03
0.05
0.18
0.01
2.53
1.98
1.35
-1.03
1.06
-1.19
0.00
0.00
0.01
0.82
0.48
0.01
1432466_a_at
Apoe
lysosomal acid lipase A
solute carrier family 27 (fatty acid
transporter), member 5
sterol O-acyltransferase 1
sterol O-acyltransferase 1
sterol O-acyltransferase 1
sterol O-acyltransferase 1
sterol O-acyltransferase 2
cytochrome P450, family 46, subfamily
a, polypeptide 1
apolipoprotein E
1.03
0.63
1.15
0.00
1.24
0.01
1.53
0.00
1417561_at
Apoc1
apolipoprotein C-I
-1.02
0.75
1.28
0.20
1.71
0.02
2.52
0.00
1450883_a_at
Cd36
CD36 antigen
1.02
0.75
1.71
0.01
2.92
0.01
2.29
0.00
1423166_at
Cd36
CD36 antigen
1.11
0.26
1.36
0.05
3.95
0.01
3.20
0.00
1450884_at
Cd36
CD36 antigen
1.08
0.38
-1.06
0.34
-1.01
1.00
1.05
0.52
1438322_ x_at
Fdft1
-1.09
0.11
-1.15
0.01
-1.40
0.01
-1.46
0.00
1438918_at
Fdft1 /// Gm6781
-1.17
0.34
1.11
0.11
-1.02
0.78
1.10
0.26
1438919_ x_at
Fdft1 /// Gm6781
-1.07
0.63
1.01
0.75
-1.07
0.14
-1.02
0.87
1448130_at
Fdft1
-1.11
0.11
-1.22
0.00
-1.43
0.01
-1.46
0.00
1426744_at
Srebf2
1.07
0.34
1.08
0.04
-1.14
0.27
-1.14
0.01
1452174_at
Srebf2
1.05
0.63
-1.09
0.02
-1.19
0.01
-1.08
0.87
1421839_at
Abca1
farnesyl diphosphate farnesyl transferase
1
farnesyl diphosphate farnesyl transferase
1 /// predicted gene 6781
farnesyl diphosphate farnesyl transferase
1 /// predicted gene 6781
farnesyl diphosphate farnesyl transferase
1
sterol regulatory element binding factor
2
sterol regulatory element binding factor
2
ATP-binding cassette, sub-family A
(ABC1), member 1
1.07
0.63
1.12
0.15
-1.04
0.72
1.04
0.26
77
Hypercholesterolemia in TME
Continuation of supplemental Table 1:
78
Probe set ID
Gene Symbol
Gene Name
FC14
p14
FC42
p42
FC98
p98
FC196
p196
1421840_at
Abca1
1.23
0.02
1.52
0.00
2.01
0.01
2.63
0.00
1450392_at
Abca1
-1.07
0.34
1.05
0.20
-1.09
0.14
-1.04
0.94
1424437_s_at
Abcg4
1.08
0.42
-1.09
0.04
-1.16
0.02
-1.13
0.01
1427732_s_at
Abcg4
-1.00
0.87
1.04
0.52
-1.03
0.58
-1.09
0.01
1455221_at
Abcg1
1.11
0.05
-1.02
0.87
-1.03
0.47
1.00
0.75
1423570_at
Abcg1
-1.00
0.87
1.06
0.15
1.13
0.10
1.11
0.02
1426690_a_at
Srebf1
ATP-binding cassette, sub-family A
(ABC1), member 1
ATP-binding cassette, sub-family A
(ABC1), member 1
ATP-binding cassette, sub-family G
(WHITE), member 4
ATP-binding cassette, sub-family G
(WHITE), member 4
ATP-binding cassette, sub-family G
(WHITE), member 1
ATP-binding cassette, sub-family G
(WHITE), member 1
sterol regulatory element binding
transcription factor 1
1.12
0.00
1.09
0.11
1.13
0.27
1.30
0.01
Discussion
5. Discussion
5.1 Role of JAK-STAT signalling in TME24
Gene Set Enrichment Analysis was performed to combine the data of gene expression
microarray studies with the morphometrically assessed density of OPCs within the spinal
cords from the same animals in order to search for transcriptional changes intimately
associated with the OPCs at the pathway level (Subramanian et al. 2005; Ulrich et al. 2010).
Accordingly, a cluster of 6 GO terms related to tyrosine phosphorylation and the JAK -STAT
cascade represented the most interesting finding from the list of 26 GO terms significantly
positively correlated to the density of OPCs. Notably, a recent meta-analysis identified the
JAK-STAT cascade to be one out of 15 GO terms significantly positively correlated with
demyelination in MS, TME, EAE and a transgenic TNF-overexpressing mouse model of
demyelination (Raddatz et al. 2014), highlighting that JAK-STAT signalling is of general
importance in demyelinating conditions. The JAK-STAT cascade is involved in a plethora of
physiological and pathological conditions including inflammatory and immune responses
(Rodriguez et al. 2006; Darnell et al. 1994), and tumorigenesis (Brantley and Benveniste 2008;
Hansmann et al. 2012). Binding of many extracellular ligands such as cytokines, growth
factors and hormones to their cell surface receptors can lead to a recruitment of JAKs
followed by activation of a context dependent set out of the seven different STATs by
tyrosine phosphorylation. The activated STATs homo- or heterodimerize, translocate to the
nucleus, bind to specific DNA motifs and induce the expression of their target genes
(Kisseleva et al. 2002; Rawlings et al. 2004; Harrison 2012).
With the JAK-STAT cascade being an interesting candidate pathway, we focused the analysis
on a manually curated list of genes involved in JAK-STAT signalling. The demonstration of
up-regulated expression of Stat1, -2, -3, -4, and -6 as well as several upstream activators and
downstream targets in TME did not unequivocally indicate which of the multiple STAT
cascades is most important in the setting of OPC dysregulation. Furthermore, the
transcriptional profiles give no information concerning the phosphorylation dependent
activation status of the various JAK-STAT pathways (Harrison 2012). However, the different
STAT proteins are activated by various upstream events with considerable specificity and are
known to be involved in different processes (Akira 1999). STAT1 and -2 are both
participating in interferon signalling and the activation of the inflammatory response (Darnell
et al. 1994; Liu et al. 1998). STAT4 and -6 are essential for the differentiation of T cells into
79
Discussion
the Th1- or Th2-phenotype, respectively, and thus the activation of the adaptive immune
response (Liu et al. 1998; Rodriguez et al. 2006). Remarkably, the STAT3 pathway is known
to be a key regulator that promotes astrocytic differentiation in neuroglial progenitor cells
during embryogenesis (Bonni et al. 1997; Rajan and McKay 1998; He et al. 2005; Cao et al.
2010), and represents a molecular hub for signalling pathways in glial tumors such as
glioblastoma (Brantley and Benveniste 2008; Hansmann et al. 2012). Furthermore, multiple
genome-wide association studies implicate the STAT3 gene as a MS susceptibility locus
(Jakkula et al. 2010; Lill et al. 2012). Based on this knowledge, STAT3 was selected for
further experimental analysis.
The identification of intralesional spindloid cells co-expressing p-STAT3 and NG2 supports
the GSEA results, suggesting that the JAK-STAT cascade is an important signalling pathway
within intralesional OPCs. It seems reasonable that this active intranuclear p-STAT3 shifts the
differentiation of the intralesional OPCs towards an astrocytic fate, since STAT3 is known to
induce astrocytic differentiation in neuroglial progenitor cells during embryogenesis (Bonni et
al. 1997; Rajan and McKay 1998; He et al. 2005; Cao et al. 2010). In agreement with this, a
minor amount of intralesional cells exhibiting NG2 and GFAP colocalization has been
described in a previous TME study (Ulrich et al. 2008), MHV- induced demyelination
(Redwine and Armstrong 1998), as well as traumatic CNS injury (Alonso 2005). Furthermore,
a genetic fate- mapping study employing a freeze- injury of the brain displayed a high amount
of OPCs differentiating into astrocytes (Tatsumi et al. 2008). Additionally, a low but distinct
percentage of OPCs is known to differentiate into astrocytes in demyelinating lesions induced
by ethidium bromide and in experimental autoimmune encephalomyelitis (Zawadzka et al.
2010; Tripathi et al. 2010). In contrast to these demyelinating conditions, there is progressive
demyelination and astrogliosis with only delayed and insufficient remyelination in TME
(Ulrich et al. 2008), which is suggested to result from a different micromilieu within the
lesions reducing the capability of the OPCs to differentiate into oligodendrocytes. Obviously,
not all astrocytes within the sclerotic lesions of chronic progressive TME are derived from
OPCs, since in analogy to other pathological conditions the majority of astrocytes is generated
by parenchymal astrocytes that re-enter the cell cycle (Young el al. 2010; Zawadzka et al.
2010; Richardson et al. 2011).
In summary,
TMEV- induced chronic progressive demyelinating leukomyelitis was
accompanied by an increased population of intralesional NG2-positive OPCs and astrogliosis.
GSEA suggested an association between OPCs and the JAK-STAT signalling cascade.
80
Discussion
Immunohistochemistry revealed an increased intralesional number of p-STAT3-positive cells
co-expressing either GFAP, NG2, or CD107b. Meteorin- induced activation of STAT3signalling in BO-1 cells and primary rat OPCs resulted in an enhanced GFAP- and reduced
CNPase-expression in some of the tested media. In contrast, an oppositional result was
observed in BO-1 cells treated with STAT3 inhibitor VII. These results suggest that STAT3
signalling represents an important factor shifting the differentiation of OPCs from an
oligodendrocytic to an astrocytic fate, thereby simultaneously inducing remyelination failure
and glial scarring in chronic progressive demyelinating TME.
5.2 Influence of dietary cholesterol supplementation in TME
On the transcriptional level we observed an overall down-regulation of genes associated with
cholesterol biosynthesis, comparable to observations in MOG- induced experimental
autoimmune encephalomyelitis (EAE) in rats (Mueller et al. 2008) and in MS patients (Lock
et al. 2002). This down-regulation was suggested as a transcriptional representation of a
reduced capacity for myelin repair (Lock et al. 2002). The majority of the examined genes
showed progressive down-regulation, indicating a continuous decline in the ability to
synthesize cholesterol. This correlates with the chronic progressive clinical course of TME.
Seven genes of the cholesterol biosynthesis pathway were down-regulated at all examined
time-points. The rather early decrease of their expression, already at 14dpi, indicates their
regulatory importance or a special vulnerability of oligodendrocytes triggered by the virus
infection or the inflammatory changes in the environment. Based on these observations, it can
be concluded that the observed failure of sufficient remyelination might be caused by a
change in the above-described genes causing a severe dysregulation during myelination.
Observations during developmental myelination, suggested the presence of specific
checkpoints to ensure sufficient production cholesterol to proceed brain myelination
(Fünfschilling et al. 2012; Herz and Farese 1999). Considering the concept of an orchestrated
remyelination process, it seems plausible that the suggested dysregulation of OPCs in MS and
its animal models could be caused by insufficient cholesterol biosynthesis. However, a downregulation of the genes associated with cholesterol biosynthesis secondary to demyelination or
loss of oligodendrocytes cannot be excluded. To further elucidate the underlying processes
the lipid composition of the main cholesterol repositories in the body was analyzed in the
second part of the study.
81
Discussion
Recent studies indicate that serum dyslipidemia could be a comorbidity of MS (Marrie and
Horwitz 2010). Different MRI studies in MS patients showed an associatio n with an adverse
lipid profile and disability progression (Giubilei et al. 2002; Tettey et al. 2014a; Tettey et al.
2014b; Weinstock-Guttman et al. 2011; Weinstock-Guttman et al. 2013), an effect we could
not reproduce in the present study. It can eventua lly be related to the fact that mice have
relative high HDL and low LDL levels under physiological conditions (Getz and Reardon
2006). Therefore the mouse is generally not as susceptible as humans to a disruption of this
lipoprotein balance (Getz and Reardon 2006). In the spinal cord, galactocerebroside and
sphingomyelin levels were significantly decreased in TMEV- infected animals compared to
mock-infected animals at 196 dpi. Galactocerebroside (galactosylceramide; GalC) is the most
typical myelin lipid and is used as a marker for mature oligodendrocytes (Baumann and
Pham-Dinh 2001). In addition GalC is proportional to the amount of myelin during
development (Norton and Poduslo 1973). GalC-deficient mice show impaired insulator
function of the myelin sheath (Baumann and Pham- Dinh 2001). Sphingomyelin is involved in
cell adhesion and forms lipid rafts with cholesterol for signal transduction (Podbielska et al.
2012). Interestingly cholesterol levels are unchanged between TMEV- and mock infected
animals, although a decrease in cholesterol is described in lesions and normal appearing white
matter in MS patients (Cumings 1955; Gerstl et al. 1961; Wender et al. 1974). However, our
findings are in line with a study in EAE. In the latter transcriptional changes in cholesterol
transporters, but no alterations in spinal cord cholesterol levels were detected (Mueller et al.
2008). A plausible explanation could be the different degradation capacities for various lipid
components in the myelin sheath and the sustained duration of the disease in the human
patients. Under physiological circumstances cholesterol has by far the longest half- life of
about 300 days. In contrast cerebrosides have a half- life of about 20 days (Ando et al. 2003).
Furthermore hyperactivities of degradation enzymes as described for the hydrolysis of
spingomyelin may play a central role (Wheeler et al. 2008). However, an impaired response
or clearing activity of macrophages might represent a possible cause for the poor clearance of
myelin debris leading to dysregulation of OPC differentiation (Kotter et al. 2006; Robinson
and Miller 1999).
In the present study we could not see an anticipated beneficial effect due to the higher
circulatory availability of cholesterol, neither could we detect a negative impact of a high
cholesterol diet on the disease course. This was substantiated by clinical assessment, histology,
and immunohistochemistry. The amount, onset or duration of meningitis, leukomyelitis,
82
Discussion
demyelination or remyelination remained unchanged. The reason for the significantly
decreased density of NG2-positive cells in the Paigen diet fed mice compared with control
diet fed animals at 196dpi remains elusive. The observation cannot be explained by an
increased differentiation of OPCs to myelinating oligodendrocytes, because there was no
consecutive increase in myelinated area. Similar to previous studies in TME an increased
number of OPC without maturation to myelinating oligodendrocytes was observable at earlier
time points (Ulrich et al. 2008). Interestingly, a similar differentiation arrest was observed in a
cuprizone experiment in simvastatin-treated animals (Miron et al. 2009).
Surprisingly our results are in contrast to a similar, feeding experiment using a Western-type
lipid rich diet conducted in MOG- induced EAE in C57BL/6J mice (Timmermans et al. 2014).
It was shown that high fat diet increased the immune cell infiltration, inflammatory mediator
production and exacerbated neurologic symptoms in this EAE model ( Timmermans et al.
2014). The difference may be due to the different mouse strains used or the mode myelin loss
is induced. The C57BL/6J used by Timmermans et al. (2014) are known to be one of the most
atherosclerosis sensitive strains (Getz and Reardon 2006), whereas our SJL/J mouse strain
develops a significant hypercholesterolemia, despite a relative resistance to atherosclerosis,
when fed with Paigen diet (Nishina et al. 1993; Paigen et al. 1990). In contrast to the Paigen
diet, the Western-type diet is known to have a higher atherogenic potential (Getz and Reardon
2006). Thus, the increased inflammatory reaction in Timmermans et al. (2014) could be a
secondary effect of atherosclerotic changes. Clinical chemistry showed significantly elevated
total cholesterol, HDL, LDL and free fatty acids in the blood serum of all Paigen diet fed
animals. The observed down-regulation of triglycerides as an effect of Paigen diet is a wellknown fact in atherosclerosis research (Getz and Reardon 2006; Nishina et al. 1993; Paigen et
al. 1990). The increased fat content of the diet resulted in a severe hepatic lipidosis,
predominantly consisting of triglycerides and cholesterol.
Based on the insights gained in this study it can be concluded, that down-regulation of
cholesterol biosynthesis is a robust transcriptional marker for demyelinating conditions like
TME, EAE (Mueller et al. 2008) and MS (Lock et al. 2002). Furthermore, de- and
remyelination in the chronic progressive TME model represent two processes that develop
and precede independent from serum cholesterol levels, most likely due to the inability of the
circulatory cholesterol to enter the CNS due to a tight blood brain barrier. Moreover, serum
hypercholesterolemia and dyslipidemia exhibit no negative effect on virus- induced,
inflammatory demyelination in the CNS in this atherosclerosis-resistant mouse strain. The
83
Discussion
reported findings could indicate. that the inconclusive reports regarding dyslipidemia and MS
maybe influenced by rather indirect pathomechanistic factors and/or the confounding
influence of the respective genetic predisposition towards atherosclerosis.
5.3 Interaction between STAT3 and cholesterol biosynthesis in TME
The basis for the progressive nature of neurological disabilities in MS is ongoing myelin
destruction and a failure of remyelination, resulting in a progressive axonal damage with
limited remyelination (Ferguson et al. 1997; Trapp et al. 1999; Trapp and Nave 2008;
Podbielska et al. 2012). Observations in MS and its animal models suggest a block of
oligodendroglial differentiation as a possible cause for the insufficient remyelination in over
80% of MS patients (Patrikios et al. 2006; Franklin and Ffrench-Constant 2008; Kuhlmann et
al. 2008; Ulrich et al. 2008). Despite the presence of multiple oligodendrocyte precursor cells
in the demyelinated lesion, these cells fail to differentiate into myelinating oligodendrocytes
(Chang et al. 2000; Kuhlmann et al. 2008; Ulrich et al. 2008). Transcriptional analysis and
preliminary observations in TME directed in two directions (Ulrich et al. 2008; Ulrich et al.
2010). On the one hand the observation, that some OPCs underwent astrocytic differentiation
(Ulrich et al. 2008) is an indication for an abnormal lineage decision of OPCs. On the other
hand transcriptional changes in the cholesterol biosynthesis (Ulrich et al. 2010), interpreted
together with similar observations in MS and its animals models (Lock et al. 2002; Mueller et
al. 2008; Ulrich et al. 2010), the unsatisfying effects of statin treatment (Miron et al. 2007;
Klopfleisch et al. 2008; Miron et al. 2009), and the decreased cholesterol level in normal
appearing white and grey matter in MS patients (Cumings 1955; Gerstl et al. 1961; Wender et
al. 1974) pointed to an involvement of the cholesterol biosynthesis in the insufficient
differentiation process in OPCs.
Both reasonable implications were further characterized in two independent studies. The first
study revealed a correlation of JAK-STAT signaling with the amount of NG2-positive OPCs
on the transcriptional level. In subsequent experiments STAT3 was identified as a key
regulator of OPC- differentiation and suggested to induce astrogliosis due to a shift in OPCs
towards astrocytic fate. The second study displayed an over-all down-regulation of genes
associated with cholesterol biosynthesis on the transcriptional level. In the following feeding
experiment, no substantial influence of cholesterol supplementation on the inflammatory
response, astrogliosis, lipid composition, demyelination and remyelination was observed.
84
Discussion
The involvement of astrocytes in the pathophysiology of MS in general and their interaction
with OPCs in particular is still not fully understood. Astrocytes are assumed to be beneficial
in initial stages of MS, but in chronic stages a switch towards negative effects was supposed
(Kipp et al. 2011). On the one hand a contribution of astrocytes to various degenerative and
inflammatory reactions was described, but on the other hand they are suggested to create a
permissive environment for remyelination (Kipp et al. 2011). The glial scar formation in MS
is thought to serve as a barrier between the damaged and the surrounding tissue but also may
secret factors to inhibit OPC migration and interact with cholesterol biosynthesis (Miron et al.
2011).
Interestingly, in an in vitro model of the blood brain-barrier co-culture of brain capillary
endothelial cells with astrocytes lead to an increased number of LDL-receptors and a threefold enhanced capacity to bind LDL (Dehouck et al. 1994). Due to this observation a
modulatory function of astrocytes on the lipid up-take was suggested (Dehouck et al. 1994).
Further studies indicated, that astrocytes immediately incorporate and process these LDL
particles from the circulatory and are therefore suggested as key regulators for the lipid
homeostasis in the brain (Ferretti and Bacchetti 2011). Moreover, it was demonstrated, that
the interaction of apolipoproteins with ATP-binding cassette transporter ABCA1 (member 1
of human transporter sub-family ABCA) (ABCA1) activated the JAK2/STAT3 signalling
pathway. JAK2 in turn causes two independent effects, lipid export from the cell and STAT3mediated anti- inflammatory activity (Liu and Tang 2012). However, no differences in the
lipid levels of the spinal cord were detectable in the present study.
Summarizing the above a possible explanation for the lack of myelinating oligodendrocyte in
TME lesions is a STAT3- mediated dysregulation of OPCs, an involvement of the cholesterol
metabolismus in this process seems likely, but could not be verified. Dietary cholesterol
supplementation had no influence on these processes in TME.
85
Summary
6. Summary
Pathogenetic role of cholesterol biosynthesis and STAT3 signaling in chronic
demyelinating diseases
Wenhui Sun
Multiple sclerosis is an immune- mediated demyelinating disease of the central nervous
system with still unknown etiology. The basis for the progressive nature of neurological
disabilities in MS is ongoing myelin destruction and a failure of remyelination. This is
resulting in a progressive axonal damage with limited remyelination. Transcriptional analysis
and observations from previous studies in murine Theiler’s virus encephalomyelitis (TME),
an experimental, virus- induced animal model of MS lead to different underlying mechanisms.
On the one hand oligodendrocyte precursor cells (OPC) are present in the lesion but do not
differentiate into myelinating oligodendrocytes and on the other hand, substantial,
transcriptional changes are present concerning the cholesterol biosynthesis.
Therefore, the objectives of this thesis were first to identify the underlying mechanisms
leading to the dysregulation of OPC differentiation and second to analyze the changes in
cholesterol metabolism and study the possible influence on de- and remyelination by dietary
cholesterol supplementation.
Therefore five weeks old, female SJL/J mice were infected intracerebrally with the low
virulent BeAn strain of TME virus. Mock- infected animals served as controls. The motoric
performance of the animals was evaluated weekly by Rotarod test over the studied period
from 7-196 days post infection (dpi). At different stages of the disease 6 animals per group
was necropsied and evaluated by histology, immunohistochemistry and oligonucleotide
microarrays. In addition, the effect of activation and inhibition of the STAT3 signalling
pathway on glial precursor cells was examined in vitro on BO-1 cells and primary rat OPC
cultures for in the first part of the study.
In the second part an additional feeding experiment was performed to clarify the influence of
hypercholesterolemia on the disease course in TME. For this purpose one group of animals
was fed a conventional mouse diet, the other group received a high- fat and high-cholesterol
Paigen-diet throughout the complete period of the experiment. Subsequently, the lipid
composition of liver, blood and spinal cord was assessed by high performance liquid
chromatography.
87
Summary
In the first part of the study, a high correlation between the immunohistochemically evaluated
OPC-density and the transcriptional change in the JAK-STAT signaling pathway was
observed by using microarray technology. Immunohistologically an increased amount of
STAT3-expressing astrocytes, OPCs and microglia / macrophages was observed in TME
infected animals. In subsequent cell culture experiment in both, BO-1 cells and primary OPC
cultures, an increased expression of GFAP and a reduced expression of CNPase was detect,
after activating the STAT3 signaling pathway by Meteorin. Inhibiting the STAT3 signaling
pathway by STAT3 inhibitor VII initiated the opposite effect in BO-1 cells.
In the second part of the present study, a progressive, transcriptional down-regulation of
genes involved in cholesterol biosynthesis was detected. The content of galactocerebrosides
and sphingomyelins in the spinal cord was reduced in the advanced stage of TME. The die tary
cholesterol supplementation had no impact on the degree and quality of inflammatory changes,
de- and remyelination in the TME model. Interestingly, no negative effect of serum
hypercholesterolemia was recognizable in atherosclerosis-resistant SJL/J mice.
In summary, the study shows that STAT3 signaling pathway plays a key role in the regulation
of OPC differentiation. Due to the changes observed in vitro, it is likely that STAT3 promotes
the differentiation of OPCs to astrocytes and is thus leading to a persistent astrogliosis.
Moreover, serum hypercholesterolemia exhibits no negative effect on virus- induced,
inflammatory demyelination in the CNS in this atherosclerosis-resistant mouse strain. The
reported findings could indicate, that the inconclusive reports regarding hypercholesterolemia
and MS maybe influenced by rather indirect pathomechanistic factors or the confounding
influence of the respective genetic predisposition towards atherosclerosis.
88
Zusammenfasung
7. Zusammenfassung
Pathogenetische Rolle der Cholesterol-Biosynthese und des STAT3-Signalweges in
chronisch-demyelinisierenden Erkrankungen
Wenhui Sun
Die Multiple Sklerose (MS) des Menschen stellt eine immunmediierte, demyelinisierende
Erkrankung des zentralen Nervensystems mit bislang ungeklärter Ursache dar. Die
fortschreitende Zerstörung der Myelinscheide und eine bislang ungeklärte Behinderung der
Remyelinisierung sind verantwortlich für eine progressive Schädigung der Axone. Dies stellt
die Basis der progredienten Verschlechterung des klinischen Bildes dar. Transkriptionale
Analysen und Beobachtungen aus vorangegangenen Untersuchungen der TheilervirusEnzephalomyelitis (TME) der Maus, einem experimentell, virus-induziertem Tiermodel der
MS deuten daraufhin, dass einerseits die in MS-Läsionen vorliegenden Vorläuferzellen der
Oligodendrozyten
nicht
zu
myelinisierenden
andererseits substanzielle, transkriptionale
Oligodendrozyten
differenzieren
und
Veränderungen die Cholesterol-Biosynthese
vorliegen.
Die Ziele der vorliegenden These bestanden deshalb darin, die zugrundeliegenden
Mechanismen der dysregulierten Vorläuferzell-Differenzierung zu identifizieren und die
Veränderungen im Cholesterol-Stoffwechsel sowie die mögliche Beeinflussung der De- und
Remyelinisierung durch dietätische Cholesterol-Supplementierung zu analysieren.
Hierfür wurden fünf Wochen alte, weibliche SJL/J-Mäuse mit dem schwach virulenten BeAnStamm des TME-Virus intrazerebral infiziert. Mock- infizierte Tiere dienten als Kontrolltiere.
Die motorische Leistung der Tiere wurde wöchentlich mittels Rotarod-Test über den
Untersuchungszeitraum von 7-196 Tage nach der Infektion evaluiert. In verschiedenen
Stadien der Erkrankung wurde jeweils eine Gruppe von 6 Tieren obduziert und mittels
Histologie, Immunhistologie und Oligonukleotid-Mikroarrays ausgewertet. Zusätzlich wurde
für den ersten Teil der These die Auswirkung der Aktivierung und Inhibierung des STAT3Signalweges auf gliale Vorläuferzellen zellkulturell an BO-1 und primären OligodendrozytenVorläuferzellen der Ratte untersucht. Im zweiten Teil der Studie wurde außerdem, zur
Klärung des Einflusses einer Hypercholesterolämie auf den Krankheitsverlauf der TME, ein
Fütterungsversuch im oben beschriebenen Tierexperiment durchgeführt. Hierfür wurde eine
Gruppe mit normaler handelsüblicher Mäusediät, eine andere mit einer hochgradig Fett- und
89
Zusammenfassung
Cholesterol- haltiger Paigen-diät über den gesamten Versuchszeitraum gefüttert. Nachfolgend
wurde
die
Lipid-Zusammensetzung
von
Leber,
Blut
und
Rückenmark
mittels
Hochleistungsflüssigkeitschromatographie beurteilt.
Mittels Mikroarray-Technologie konnte im ersten Teil der Studie eine hohe Korrelation der
immunhistologisch
transkriptionalen
ausgewerteten
Veränderung
Oligodendrozyten-Vorläuferzelldichte
des
JAK-STAT
Signalweges
dargestellt
und
der
werden.
Immunhistologisch wurde ein erhöhter Gehalt an STAT3 expremierenden Astrozyten,
Oligodendrozyten Vorläuferzellen und
nachfolgenden
Mikroglia/Makrophagen
zellkulturellen Untersuchungen war
festgestellt.
Im den
nach Aktivierung des STAT3-
Signalweges durch Meteorin, sowohl in BO-1 Zellen als auch in primären OligodendrozytenVorläuferzellkulturen eine vermehrte Expression von GFAP und eine verminderte Expression
von CNPase zu detektieren. Durch Inhibierung des STAT3-Signalwegs mittels STAT3
Inhibitor VII war in BO-1 Zellen der gegensätzliche Effekt zu erzielen.
Im zweiten Teil der vorliegenden Studie wurde eine progressive, transkriptionale
Herunterregulierung von Genen der Cholesterol-Biosynthese erkannt. Der Gehalt an
Galactocerebrosiden und Sphingomyelinen im Rückenmark war im fortgeschrittenen
Krankheitsstadium durch die Infektion mit TME-Virus vermindert. Die dietätische
Cholesterol-Supplementierung hatte weder einen Einfluss auf den Grad und Charakter der
entzündlichen Veränderung, noch auf die De- und Remyelinisierung im TME-Model.
Interessanterweise war kein negativer Effekt der Serum-Hypercholesterolämie auf die im
Allgemeinen als atherosklerose-resistent geltende SJL/J Mäuse zu erkennen.
Zusammenfassend
zeigten
die
Untersuchungen,
dass
der
STAT3-Signalweg
eine
Schlüsselrolle in der Regulation der Differenzierung oligodendroglialer Vorläuferzellen
einnimmt. Durch die in vitro gezeigten Veränderungen ist es wahrscheinlich, dass STAT3
eine Differenzierung von Oligodendrozyten Vorläuferzellen zum Astrozyten begünstigt und
somit die für MS kennzeichnende Astrogliose hervorruft.
Des Weiteren, stellt die Beobachtung, dass der Serum-Cholesterolspiegel keinen Einfluss auf
den Krankheitsverlauf der TME hat, eine mögliche Erklärung für die teils widersprüchlichen
Aussagen
bezüglich
eines
pathogenetischen
Zusammenhangs
zwischen
der
Hypercholesterolämie und der MS dar. Die Ergebnisse dieses Fütterungsversuchs deuten
90
Zusammenfasung
darauf hin, dass keine primäre Beeinflussung des Krankheitsbilds durch fettreiche Diät
besteht. Der Eindruck eines pathogenetischen Zusammenhangs entstand wahrscheinlich durch
die hohe Sensibilität der Menschen zur Atherosklerose.
91
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Acknowledgements
9. Acknowledgements
It has been a long journey to complete my PhD study with the support of several people. I
would like to express my sincere gratitude to all of them.
Firstly, I will forever be thankful to my supervisor Prof. Dr. Baumgärtner for his constantly
supportive and encouraging supervision to me during these past four years. He is the funniest
supervisor and one of the smartest people I know. I am so lucky to meet him and to be guided
by him. He is always patient to try his best to help me whenever I encounter difficulty in my
study and daily life.
Secondly, I am also very grateful to my co-supervisors Prof. Stangel and Prof. GerardySchahn for always critically and patiently guiding and reviewing my work during my study.
Thanks for the comment for all my meetings, presentations and manuscripts.
I would also like to show my gratitude to Dr. Reiner Ulrich for his professional help and
advice during my study. I would not have made it this far without his friendly help. He has
always been helpful with new ideas and comments on my work. I did learn a lot from him.
I really appreciate the help from Prof. Dr. Hassan Naim and his team for supporting my work
in the Department of Physiological Chemistry. I was so lucky that Prof. Naim always made
his time for me to discuss my work and helped me to solve the problems of my experiment. I
especially thank Dr. Graham Brogden, Ariana Neumann and Heike Kanapin for giving me
lots of helpful advice for my work and built a warm friendship with me.
I thank the ZSN for the good organization and providing many study chances to me by
holding conferences and the Graduate school day.
I thank to Dannuta, Petra, Bettina, Claudia, Kerstin R., Kerstin S., and Caro for their excellent
help in the lab.
I thank Kathrin and Ann-Kathrin, who built a nice working atmosphere in our office where I
can feel peaceful and happy to work there.
I thank Vanessa, Florian, Barbara, Angelika and Kerstin S. for their help on my work and
daily life. Leila, I will miss our coffee break.
I would also like to thank all my colleagues in my department. It is my pleasure to work with
them. I could always get support from them whenever I lost my motivation and faith to finish
my work.
I also thank the China Scholarship Council (CSC) for financial support by providing the
scholarship.
I also thank all my friends I meet in Germany. They made my life more colorful and funny.
Lastly, I deeply appreciate my family for all their love, encouragement and faithful company.
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