doi:10.1093/brain/aws321
Brain 2013: 136; 245–258
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BRAIN
A JOURNAL OF NEUROLOGY
Chronic exposure of astrocytes to interferon-a
reveals molecular changes related to
Aicardi–Goutières syndrome
Eloy Cuadrado,1,2 Machiel H. Jansen,2 Jasper Anink,3 Lidia De Filippis,4 Angelo L. Vescovi,4,5
Colin Watts,6 Eleonora Aronica,3 Elly M. Hol1,7 and Taco W. Kuijpers2
1 Department of Astrocyte Biology and Neurodegeneration, Netherlands Institute for Neuroscience–an Institute of the Royal Netherlands Academy of
Arts and Sciences (KNAW), Amsterdam, The Netherlands
2 Department of Experimental Immunology, Academic Medical Centre, University of Amsterdam (UvA), Amsterdam, The Netherlands
3 Department of Neuropathology, Academic Medical Centre, University of Amsterdam (UvA), Amsterdam, The Netherlands
4 Department of Biotechnology and Biosciences, Università degli Studi di Milano-Bicocca, Milano, Italy
5 Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy
6 Division of Cell Biology and Immunology, College of Life Sciences, University of Dundee, Dundee, UK
7 Swammerdam Institute for Life Sciences, Centre for Neuroscience, University of Amsterdam (UvA), The Netherlands
Correspondence to: Eloy Cuadrado, PhD,
Netherlands Institute for Neuroscience,
Astrocyte Biology and Neurodegeneration Group,
Meibergdreef 47,
1105 BA Amsterdam,
The Netherlands
E-mail: [email protected]
Aicardi–Goutières syndrome is a genetically determined infantile encephalopathy, manifesting as progressive microcephaly, psychomotor retardation, and in 25% of patients, death in early childhood. Aicardi–Goutières syndrome is caused by mutations in any of
the genes encoding TREX1, RNASEH2-A, -B, -C and SAMHD1, with protein dysfunction hypothesized to result in the accumulation
of nucleic acids within the cell, thus triggering an autoinflammatory response with increased interferon-a production. Astrocytes have
been identified as a major source of interferon-a production in the brains of patients with Aicardi–Goutières syndrome. Here, we
study the effect of interferon-a treatment on astrocytes derived from immortalized human neural stem cells. Chronic interferon-a
treatment promoted astrocyte activation and a reduction in cell proliferation. Moreover, chronic exposure resulted in an alteration of
genes and proteins involved in the stability of white matter (ATF4, eIF2Ba, cathepsin D, cystatin F), an increase of antigen-presenting
genes (human leukocyte antigen class I) and downregulation of pro-angiogenic factors and other cytokines (vascular endothelial
growth factor and IL-1). Interestingly, withdrawal of interferon-a for 7 days barely reversed these cellular alterations, demonstrating
that the interferon-a mediated effects persist over time. We confirmed our in vitro findings using brain samples from patients with
Aicardi–Goutières syndrome. Our results support the idea of interferon-a as a key factor in the pathogenesis of Aicardi–Goutières
syndrome relating to the observed leukodystrophy and microangiopathy. Because of the sustained interferon-a effect, even after
withdrawal, therapeutic targets for Aicardi–Goutières syndrome, and other interferon-a-mediated encephalopathies, may include
downstream interferon-a signalling cascade effectors rather than interferon-a alone.
Keywords: interferon; astrocytes; leukodystrophy; angiopathy
Abbreviations: CST7 = cystatin F; GFAP = glial fibrillary acidic protein; IFN- = interferon-alpha; VEGF = vascular endothelial growth
factor
Received July 12, 2012. Revised October 9, 2012. Accepted October 16, 2012
ß The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
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| Brain 2013: 136; 245–258
Introduction
Aicardi–Goutières syndrome is a rare genetic disease in which
severe neurological dysfunction usually becomes apparent in
infancy. Affected children most typically present with irritability,
inconsolable crying, dystonia, hypotonia and seizures in early life,
leading to progressive microcephaly, with severe developmental
delay and death in 25% of cases (Barth et al., 1999; Crow
and Rehwinkel, 2009). Aicardi–Goutières syndrome is characterized by cerebral atrophy, white matter damage, brain calcifications
and, one of the hallmarks of the disease, the presence of high
levels of interferon-alpha (IFN-) in the CSF and serum (Lebon
et al., 1988).
Because of the clinical and biochemical features of the disease,
Aicardi–Goutières syndrome is easily confused with a congenital
infection (e.g. as a result of toxoplasmosis, HIV, rubella virus,
cytomegalovirus and/or herpes simplex virus); therefore, the diagnosis of Aicardi–Goutières syndrome is usually only considered
after exclusion of an infectious aetiology (Sanchis et al., 2005).
The observation of chilblains (skin ulcerations), hepatosplenomegaly, thrombocytopaenia and autoantibodies suggest an immune
pathogenic mechanism (Rice et al., 2007). Interestingly, IFN-
levels are higher in CSF than in the serum of these patients, indicative of an intrathecal production (Lebon et al., 1988). Indeed,
astrocytes have been identified to be a major source of IFN-,
among other cytokines, such as CXCL10, in the brain of patients
with Aicardi–Goutières syndrome (van Heteren et al., 2008).
Neuropathological data have also suggested that Aicardi–
Goutières syndrome may represent a primary microangiopathy
(Barth et al., 1999), as peri- and intravascular calcium/mineral deposits and cortical microinfarctions are occasionally found, a hypothesis supported by the pattern of changes seen on brain imaging,
and the frequently reported finding of a vasculitis on biopsy of the
chilblain lesions (Ramesh et al., 2010). These observations suggest
that an inflammatory disturbance of vascular homeostasis may be
important in the pathogenesis of Aicardi–Goutières syndrome.
Aicardi–Goutières syndrome is a genetically heterogeneous
disorder. To date, mutations have been identified in five genes
accounting for 90% of Aicardi–Goutières syndrome cases. These
genes encode the DNA exonuclease 1 (TREX1; Crow et al.,
2006a), the three subunits of the endoribonuclease H2 complex
(RNASEH2B, RNASEH2C and RNASEH2A; Crow et al., 2006b),
and the deoxynucleoside triphosphate triphosphohydrolase
SAMHD1 (Rice et al., 2009; Goldstone et al., 2011; Powell
et al., 2011). All of these proteins are important in nucleic acid
metabolism, and it is now believed that the innate immune response might occur as a result of nucleic acid accumulation in cells
(Yang et al., 2007), thus mimicking a viral infection. Recently,
data have revealed that SAMHD1 might act as a restriction
factor for HIV infection (Berger et al., 2011; Hrecka et al.,
2011; Laguette et al., 2011), and that TREX1 (Yan et al., 2010)
and RNASEH2A (Genovesio et al., 2011) also have a role in the
metabolism of HIV.
Trex1 null mice exhibit a reduced survival and develop inflammatory myocarditis. A cerebral phenotype has not been observed
in these animals (Morita et al., 2004; Gall et al., 2012). The
E. Cuadrado et al.
cardiac inflammation is dependent on IFN- receptor 1 (IFNR1),
interferon responsive factor 3 (IRF3) and the ability to generate
autoantibodies by mature B cells. Abrogation of any of these three
interferon-dependent signalling cascades is sufficient to circumvent
lethality in mice (Stetson et al., 2008). Furthermore, in these animals, it has recently been demonstrated that the accumulation of
Trex1 DNA substrates leads to the activation of the
interferon-stimulatory DNA response through the adaptor protein
STING, and that type I interferons drive the autoreactive response
causing the myocarditis (Gall et al., 2012). Ablation of Rnaseh2b
in mice leads to early embryonic lethality because of elevated DNA
damage and p53-dependent growth arrest (Reijns et al., 2012).
Although not a genetic model for Aicardi–Goutières syndrome,
transgenic mice overexpressing IFN- under control of the astrocytic glial fibrillary acidic protein (GFAP) promoter (so-called GIFN
mice), develop a progressive inflammatory encephalopathy, with
calcium deposits, gliosis and neurodegeneration (Akwa et al.,
1998; Campbell et al., 1999). These latter findings are remarkably
similar to those observed in patients with Aicardi–Goutières syndrome, and thus implicate a pathogenic role for IFN- in the cerebral pathology of Aicardi–Goutières syndrome.
An additional indication of a detrimental role of IFN- in
Aicardi–Goutières syndrome was provided by microarray studies
on lymphocytes from CSF samples of patients. These studies
showed an upregulation of interferon-related genes together
with a downregulation of angiogenesis-related transcripts (Izzotti
et al., 2009). Moreover, it has been demonstrated that mutations
in TREX1 potentiate the anticancer properties of T lymphocytes to
inhibit growth of neuroblastoma cells and related angiogenesis,
and that these effects were enhanced in the presence of IFN-
(Pulliero et al., 2012).
Here, we establish an in vitro model of Aicardi–Goutières syndrome using human neural stem cell-derived astrocytes chronically
treated with IFN-. Despite the emerging evidence linking IFN-
to autoimmunity, little is known about the specific mechanisms
that lead to disease in human brain cells. We show that chronic
IFN- exposure reduces cell proliferation while promoting the
activation and reactivity of astrocytes. Although a defect in endogenous RNA/DNA metabolism in Aicardi–Goutières syndrome
may be central to disease pathogenesis (Stetson et al., 2008;
Gall et al., 2012), we demonstrate that IFN- alone may induce
changes in the expression of genes and proteins related to white
matter stability and vascular growth and development, in the absence of any mutation. Finally, we confirm our in vitro results in
astrocyte cultures using human post-mortem brain specimens from
patients with Aicardi–Goutières syndrome. Together, these findings provide support for the hypothesis that blocking interferon
signalling might be beneficial in Aicardi–Goutières syndrome and
other IFN- mediated encephalopathies.
Materials and methods
Cell culture
Immortalized human neural stem cells, derived from neural stem cells
from the diencephalic and telencephalic brain regions of one human
Effect of IFN- on astrocytes in Aicardi–Goutières syndrome
Brain 2013: 136; 245–258
foetus, were cultured and propagated as previously described (De
Filippis et al., 2007). Briefly, neurospheres were cultured in
Euromed-N medium (Euroclone) supplemented with 25 mg/ml
insulin, 100 mg/ml transferrin, 6.3 ng/ml progesterone, 9.6 mg/ml
putrescine, 520 ng/ml sodium selenite (N2 supplement, all from
Sigma), 20 ng/ml epidermal growth factor and 10 ng/ml of fibroblast
growth factor 2 (both from Tebu-Bio) in uncoated dishes.
To differentiate immortalized human neural stem cell into astrocytes,
individual spheres were mechanically dissociated and transferred at a
density of 20 000 cells/ml onto laminin-coated plates. Cells were
grown for 21 days in vitro in normal supplemented-Euromed-N
medium in the presence of 2% foetal calf serum without growth factors. During this period, cell medium was refreshed twice a week in
the presence/absence of the afterwards indicated concentrations of
human pegylated interferon alpha-2a (PegasysÕ , Roche).
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Immunocytochemistry
Immunostaining was performed according to a standard protocol.
Briefly, cells were fixed in 4% ice-cold paraformaldehyde for 15 min.
The wells were then washed with PBS and permeabilized for 10 min
with PBS containing 0.25% Triton X-100. Subsequently, cells were
washed again with PBS and blocked for 30 min in SuperMix (0.05 M
Tris, 0.9% NaCl, 0.25% gelatin and 0.5% Triton X-100, pH 7.4). After
blocking, cells were incubated with primary antibodies (see complete
list in Supplementary Table 2) diluted in SuperMix overnight at 4 C.
The following day, cells were washed three times in PBS and incubated
with secondary antibodies diluted in SuperMix for 1 h at room temperature in the dark. Finally, cells were washed three more times in
PBS, and nuclei were counterstained with 1 mg/ml Hoechst. Stainings
were analysed with a fluorescence microscope (Zeiss 200 M Axiovert,
Carl Zeiss) interfaced with an image analysis system (Image Pro Plus
6.3, Media Cybernetics Inc).
Post-mortem human brain material
Immunohistochemistry
Tissue from Aicardi–Goutières syndrome and healthy control subjects
was obtained from The Netherlands Brain Bank (Amsterdam, The
Netherlands), the Department of Neuropathology, Academic
Medical0 Centre (Amsterdam, The Netherlands) and from the
Department of Paediatrics at the Rikshospitalet (Oslo, Norway). The
brain donors gave informed consent for using the tissue and for
accessing the extensive neuropathological and clinical information
for scientiEc research, in compliance with ethical and legal guidelines. Clinicopathological information of all donors can be found in
Table 1.
We performed immunohistochemistry on 6-mm thick frontal sections
of paraffin-embedded brain material from two patients with Aicardi–
Goutières syndrome and two control subjects (Table 1), as described
previously (van den Berge et al., 2010). In brief, sections were
rehydrated and rinsed with Tris-buffered saline. Sections were
pre-incubated with horse serum-containing buffer and were incubated
overnight in the primary antibody solution (Supplementary Table 3).
After peroxidase blocking, sections were subsequently incubated with
a biotinylated secondary antibody, avidin–biotin complex (Vector
Laboratories) and diaminobenzidine tetrahydrochloride (Vector
Laboratories). Finally, sections were counterstained with haematoxylin,
dehydrated and mounted for microscope analysis.
Real-time quantitative polymerase chain
reaction assay
Calcium deposits staining
Õ
RNA from cell pellets was isolated using TRIzol (Invitrogen Life
Technologies Europe) and precipitated overnight in isopropanol.
Total RNA (500 ng) was DNase I treated and was used as a template
to generate complementary DNA following the manufacturer’s
instructions (QuantiTect Reverse Transcription Kit, Qiagen) with a
blend of oligo-dT and random hexamer primers. The reverse transcriptase and real-time quantitative PCR reactions were performed as
previously described (Kamphuis et al., 2012). Briefly, the reverse transcriptase reaction was incubated at 42 C for 30 min. The resulting
complementary DNA was diluted 1:20 and served as a template in
real-time quantitative PCR assays (SYBRÕ Green PCR Master Mix;
Applied Biosystems). Sequences of primers used are given in
Supplementary Table 1. GAPDH, b-actin and 18S rRNA were used
as reference genes to normalize the assessed transcript levels of the
target genes.
Cells were fixed in 4% ice-cold paraformaldehyde for 15 min at room
temperature and subsequently stained with modified alizarin red
staining (Kawazoe et al., 2008) or von Kossa staining (Bonewald
et al., 2003) by standard procedures.
Western blot
Protein was isolated from cells by homogenization with lysis buffer
(0.1 M NaCl, 0.01 M Tris–HCl pH 7.6, 1 mM EDTA pH 8.0) supplemented with protease inhibitors phenylmethylsulphonyl fluoride
(100 mg/ml) and aprotinin (0.5 mg/ml) cocktail (Roche Diagnostics).
Protein content was determined using the BCA protein assay kit
(Thermo Scientific). The same amounts of protein were dissolved in
2 loading buffer (2 : 100 mM Tris, 4% SDS, 20% glycerol,
200 mM dithiothreitol, 0.006% bromophenol blue) and boiled for
Table 1 Clinicopathological data of the brain donors
Code
Gender
Age (years)
Area
Brain weight (g)
Experiments
Diagnosis
Cause of death
T94-13424
RM991-01
T00-2033 A
S00-97
1996-115
M
F
M
M
F
18
2
9
31
92
FC,
FC,
FC,
FC,
FC
591
536
1048
1376
964
IHC
IHC
IHC
IHC
WB
Aicardi–Goutieres syndrome
Aicardi–Goutieres syndrome
Control
Control
Alzheimer’s disease
Unknown
Unknown
Pulmonary aspiration
Myocardial infarction
Dehydration and cachexia
BG
BG
BG
BG
BG = basal ganglia, F = female, FC = frontal cortex, IHC = immunohistochemistry, M = male, WB = western blot.
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| Brain 2013: 136; 245–258
5 min. Subsequently, they were run on an 8–12% SDS–PAGE gel and
blotted semi-dry on nitrocellulose. Blots were blocked in SuperMix and
incubated overnight with primary antibodies (Supplementary Table 4).
The next day, the blots were washed with Tris-buffered saline–Tween
(100 mM Tris–HCl pH 7.4, 150 mM NaCl, with 0.2% Tween-20) and
incubated with secondary antibody anti-rabbit IRDye800 or anti-mouse
Cy5 (1:5000; Rockland Immunochemicals) in SuperMix for 1 h at room
temperature. After three washes in Tris-buffered saline–Tween, bands
were visualized with the Odyssey Infrared Imaging System (LI-COR
Biosciences). Human post-mortem tissue was obtained from The
Netherlands Brain Bank (Amsterdam, The Netherlands). A human
brain homogenate from donor 1996-115 (Supplementary Table 1) was
used as a positive control sample.
Flow cytometry
Pelleted cells were resuspended in 0.1 M of phosphate buffer containing
0.1% egg albumin. Cells were then incubated with a mouse anti-human
HLA-ABC-PE antibody (BD PharmingenTM) for 30 min at 4 C in darkness.
Cells were washed and resuspended in 0.1 M of phosphate buffer containing 0.1% egg albumin, and fluorescence-activated cell sorting analysis
was immediately performed using the BD FACSCantoTM II flow cytometer (BD Biosciences). Collected data were analysed using FlowJo Software version 7.6.3 (TreeStar).
Luminex
Briefly, 25 ml of supernatants was analysed using a custom Bio-Plex
ProTM human cytokine 27-plex panel antibody kit according to the
manufacturer’s protocol (BioRad). Plates were then read on a
BioPlex Protein Array System (BioRad) using both supplied high and
low calibration curves, according to the instructions of the manufacturer. This technique allowed us to study the following cytokines:
IL-1b, IL-1r, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,
IL-13, IL-15, IL-17A, basic FGF, G-CSF, GM-CSF, IFN-, IP-10,
MCP-1 (MCAF), MIP-1, MIP-1b, PDGF-BB, CCL5, TNF- and
VEGF (vascular endothelial growth factor). Data analysis was performed with Bio-Plex Manager software (BioRad).
Statistics
All experiments were performed at least three independent times
unless otherwise specified. Graphs are presented as bar charts or
box plots to give mean standard error of mean (SEM). The variable
distribution was assessed by Kolmogorov–Smirnov test. When test distribution was normal, a Student t-test was used to determine mean
differences between two groups. When distribution was not normal, a
non-parametric Mann–Whitney test was used. Kruskal–Wallis test was
used followed by Dunn’s multiple comparison test to assess intergroup
differences. A P-value of 50.05 was considered statistically significant
at a 95% confidence level. Tests were performed as indicated using
Prism 4.0 (GraphPad Software Inc).
Results
Immortalized human neural stem cells
give rise to mature functional astrocytes
in vitro
We dissociated neurospheres into single cell suspension and cultured them as indicated above for 21 days in vitro (Fig. 1A and B).
E. Cuadrado et al.
After this period, we stained the cells for different cell markers to
assess the purity of the cultures. Cells showed positive staining of
the astrocyte marker GFAP (Fig. 1C) and the glia-specific
calcium-binding protein S100b (Fig. 1D). Cells were slightly positive for bIII-tubulin (Fig. 1E) and negative for mature neuronal
marker NeuN (Fig. 1F). Cells showed no staining for both oligodendrocytic CNPase (Fig. 1G) and microglial CD11b (Fig. 1H)
markers. Cells were harvested, and lysates were analysed by western blot against different commonly used astrocyte markers, which
showed that after 21 days in vitro, they were positive for
pan-GFAP, S100b, glutamine synthetase and vimentin (Fig. 1I).
The differences in GFAP and S100b pattern observed in the
brain homogenate can be attributed to a different GFAP degradation and its different isoforms, and the increase of S100b that
occurs in the aged and Alzheimer brain as previously described
(DeArmond et al., 1983; Sheng et al., 1996; Hol et al., 2003).
To assess the capacity of these astrocytes to react to an innate
immune system stimulus, we treated the astrocytes for 48 h with
10 mg/ml of polyinosinic:polycytidylic acid. Gene expression analysis (Fig. 1J) clearly showed that polyinosinic:polycytidylic acid
promoted a robust increase in the expression of different IFN-
subtypes, representative of the different anti-viral activity categories (Moll et al., 2011): low activity IFNA1 (P = 0.034), high
activity IFNA2 (P = 0.0079) and intermediate activity IFNA4
(P = 0.034). The cytokine CXCL10 (P = 0.015) and the intermediate filament protein GFAP (P = 0.015), which is a marker for
reactive gliosis, were also highly upregulated in treated cells
compared with vehicle-treated control cells. Furthermore, we
also analysed the release to the supernatant of cytokines in
these cultures (Fig. 1K). After polyinosinic:polycytidylic acid treatment, the astrocytes produced and released significant amounts of
IL-6 (P = 0.021), IL-8 (P = 0.046), CXCL10 (P = 0.015), CCL5
(P = 0.028) and TNF- (P = 0.047).
Chronic IFN-a exposure reduces
astrocyte proliferation while
promoting reactive gliosis
In Trex1-deficient mice IRF3-dependent type I interferon response
is identifiable in utero and is reliably detectable in specific tissues
within a day after birth (Gall et al., 2012). In humans, the moment
at which the interferon response is initiated remains unknown and
may vary according to genotype and/or stochastic factors.
To assess whether IFN- has an effect in the early stages of
neurodevelopment, we plated the single cell dissociatedneurospheres in complete medium containing increasing doses of
IFN-. After 5 days in vitro, the number of newly formed neurospheres and their diameters were measured (Fig. 2A). Analysis of
these data revealed no differences when compared with
vehicle-treated control cells (Fig. 2B and C).
We next examined the effect of IFN- in the differentiation and
growth of the neural stem cells into astrocytes. Cultures were
treated during the 21 days in vitro of differentiation process
with increasing doses of IFN-. After this period, cells were stained
against proliferation marker Ki67 (Fig. 2D). We found that cells
treated with 20 pg/ml of IFN- already showed a reduction in the
Effect of IFN- on astrocytes in Aicardi–Goutières syndrome
Brain 2013: 136; 245–258
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Figure 1 Differentiation of immortalized human neural stem cell (ihNSC) into mature astrocytes. (A) Phase contrast image of a free
floating neurospheres culture. Scale bar = 500 mm. (B) Phase images of immortalized human neural stem cell during the differentiation
process that takes 21 days in vitro. Scale bars = 100 mm. Immunofluorescence images of mature cultures positively stained against
astrocyte GFAP (C) and S100b (D) and negatively stained against neuronal bIII-tubulin (E), NeuN (F), oligodendrocytic CNPase (G) and
microglial CD11b (H). Scale bars = 100 mm, scale bars in inserts = 25 mm. (I) Immunoblots from cell lysates prove that cells are positive
against astrocyte markers pan-GFAP, S100b, glutamine synthetase (GS) and vimentin. A human brain homogenate was loaded as a
positive control. (J) Real-time quantitative PCR gene expression shows an increase in IFNA1,-2,-4, CXCL10 and GFAP in astrocytes
treated with 10 mg/ml of polyinosinic:polycytidylic acid (poly I:C) after 48 h compared with vehicle-treated control cells. Results are
presented in arbitrary units (AU). Bars on graphs represent the mean SEM of three different experiments. (K) Secreted cytokines levels
were evaluated by Luminex from the supernatant of wells treated with 10 mg/ml of polyinosinic:polycytidylic acid for 48 h compared with
vehicle-treated control cells. Results are expressed in pg/ml (mean SEM, n = 5). Statistically significant differences are expressed as
*P 5 0.05 and **P 5 0.01.
proliferation (percentage of Ki67-positive nuclei: 53.35% 5.38,
P = 0.039, Fig. 2E) compared with vehicle-treated control cells
(69.89% 1.16). This reduction in the proliferation was even
more pronounced when cells were treated with 200 pg/ml of
IFN- (45.68% 1.58, P = 0.0002).
We observed that after IFN- treatment, immortalized human
neural stem cells gave rise to mature astrocytes, without affecting
their viability (Supplementary Fig. 1), but those astrocytes
underwent morphological changes compared with control cells.
Treated cells presented a more pronounced star-like cellular shape
and a more bundled intermediate filament network (Fig. 2F), which
is a typical sign of astrogliosis (Stichel and Müller, 1998; Sofroniew
and Vinters, 2010). We, therefore, studied the expression of
intermediate filaments by real-time quantitative PCR (Fig. 2G).
We demonstrated that IFN--treated cells indeed showed
increased expression of GFAP (P 5 0.05), S100B (P 5 0.05) and
vimentin (VIM, P 5 0.05). To corroborate these results, we performed western blot analysis to confirm the increase in protein
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E. Cuadrado et al.
Figure 2 IFN- reduces the proliferation and activates the astrocytes in vitro. (A) Contrast phase microscopy images of free floating
neurospheres cultures treated with vehicle, 20 or 200 pg/ml of IFN-. Scale bar = 500 mm. (B) Box plot graph represents the mean number
of neurospheres per field. (C) Box plot graph represents the mean diameter of the treated neurospheres in millimetres. Both graphs
represent the mean SEM (n = 4). (D) Fully differentiated astrocytes were stained against proliferation marker Ki67 and GFAP (D.1).
Representative fluorescence images of astrocytes treated with vehicle (D.2), IFN- 20 pg/ml (D.3) and IFN- 200 pg/ml (D.4) are shown.
(E) Graph shows average percentage of Ki67 positive cells SEM (n = 4). (F) Images depict the morphological changes that follow IFN-
treatment where cells show stronger staining against GFAP and S100b, and extended cellular processes. (G) Real-time quantitative PCR
gene expression showing an increase in treated astrocytes of the transcript levels of GFAP, S100B and VIM. Normalized values are
presented as ratio compared with vehicle-treated control cells (mean SEM, n = 3). (H) Immunoblots from cell lysates showing an
increase in pan-GFAP, S100b and vimentin protein after treatment with IFN-. Actin was used as loading control. Scale bars: D and
F = 100 mm. Statistically significant differences are expressed as *P 5 0.05 and ***P 5 0.001.
Effect of IFN- on astrocytes in Aicardi–Goutières syndrome
amount of these components after chronic IFN- treatment
(Fig. 2H).
Astrocytes chronically exposed to IFN-a
exhibit an alteration of expression of
genes and proteins related to white
matter stability and antigen
presentation
We investigated whether IFN- alters the expression of ATF4 and
EIF2B1, both crucial factors for another leukodystrophy known as
vanishing white matter disease (Bugiani et al., 2010). In lymphocytes of patients with Aicardi–Goutières syndrome, an alteration in
the expression of genes, such as the myelin-degrading protease
cathepsin D (CTSD) and its inhibitor cystatin F (CST7) (Izzotti
et al., 2009), has been shown previously. It is assumed that the
cathepsin/cystatin ratio is crucial for the induction of white matter
damage. Although suggested, a direct relationship between IFN-
levels and changes in gene expression has not been proven.
Moreover, these changes have been described in lymphocytes,
whereas a pathological role is more likely relevant to brain cells
(Bugiani et al., 2010).
To confirm these assumptions, we examined the effect of acute
and chronic IFN- exposure to our astrocytes in vitro. When fully
differentiated mature astrocytes were treated with IFN- acutely for
24 h, no significant differences in gene expression variation were
detected (Fig. 3A). However, we observed an induction in expression of the antigen presenting HLA-C gene. On the other hand,
when astrocytes were differentiated in the continuous presence of
IFN-, we found a significant reduction of ATF4 (P 5 0.05, Fig. 3B),
EIF2B1 (P 5 0.05) and CTSD (P 5 0.05). Although not significant,
we also observed an increase in CST7 and a marked induction of
the HLA-C gene (P 5 0.001). Consistent with these data, western
blot analysis confirmed a reduction of ATF4 and CTSD levels, together with an increase in CST7 (Fig. 3C). Furthermore, analysis of
the cells by flow cytometry (Fig. 3D) revealed a significant increase
in overall surface presentation of human leukocyte antigen class I
compared with vehicle-treated control cells (P 5 0.01, Fig. 3E).
As in humans, the mutations in Aicardi–Goutières syndromerelated genes drive a type I interferon response, we also explore
the possibility of a cross-talk between these genes and IFN-
(Fig. 3F). We analysed the expression of the five Aicardi–Goutières
syndrome causative genes, but neither acute nor chronic IFN- treatment produced an alteration in TREX1 or RNASEH2 A/B/C expression. In contrast, SAMHD1 expression was enhanced after an acute
exposure of IFN- (P 5 0.05), although this effect disappeared after
chronic treatment.
Astrocytes reduce the production and
release of angiogenic factors, but do not
produce calcium deposits after chronic
IFN-a exposure in vitro
Neuropathological data have suggested that Aicardi–Goutières syndrome may represent a primary angiopathy (Barth et al., 1999)
Brain 2013: 136; 245–258
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affecting not only small blood vessels (Richards et al., 2007) but
also, at least in the case of SAMHD1-related disease, large arteries
(Ramesh et al., 2010). In our autopsy specimens from patients with
Aicardi–Goutières syndrome, we also observed a proliferation of
small vessels and arterioles and many foci of juxtaposed blood vessels (Fig. 4A). We, therefore, explored whether IFN- directly influences the vascular homeostasis by studying the production of
angiogenic/vascular factors by astrocytes in vitro.
The expression levels of pro-angiogenic factors IL-1 and VEGF in
chronically IFN--treated astrocytes were dramatically reduced (Fig.
4B). Transcript levels of VEGFB (P 5 0.05), IL1A (P 5 0.01) and
IL1B (P 5 0.01) were significantly lower than vehicle-treated control
cells. Transcript levels of VEGFA were also lower in treated astrocytes, but those differences were not significant. Moreover, western
blots clearly demonstrated an intracellular reduction of VEGF-A,
VEGF-B and IL-1b, especially after high dose IFN- treatment
(Fig. 4C). We subsequently measured the levels of cytokines in
the supernatants. There was a trend towards a reduction in the
level of pro-angiogenic cytokines VEGF-A (P = 0.09) and IL-1b
(P = 0.22) in supernatants of astrocytes treated chronically with
IFN- (Fig. 4D). The levels of TNF- (P = 0.035) were significantly
reduced. The rest of the analysed cytokines did not show any significant changes (Supplementary Fig. 2).
In Aicardi–Goutières syndrome brain, lamellar calcium deposits
are widespread in the cerebral and cerebellar white matter as well
as around small blood vessels (Barth et al., 1999). Thus, we tested
whether astrocytes were responsible for producing these calcium
inclusions after constant IFN- exposure. We assessed the expression of genes related to intracranial calcifications, such as SLC20A2
(Wang et al., 2012), COL4A1 (Livingston et al., 2011) and osteopontin (now known as SSP1) (Shin et al., 2012). We could not
find any significant differences in SLC20A2 and COL4A1 expression between cell cultures treated with IFN- or vehicle, and we
were not able to detect any osteopontin transcripts in our astrocyte cultures (Fig. 4E; data not shown). We also stained the astrocyte cultures for calcium deposits by using alizarin red and von
Kossa techniques (Fig. 4F). The dyed wells revealed no positive
staining compared with the CaCO3 control wells. These results
indicate that astrocytes are not responsible for producing the
extracellular calcium inclusions after IFN- treatment in vitro.
CST7 is overexpressed and VEGF and
IL-1 are downregulated in
Aicardi–Goutières syndrome brains
To study whether the observed in vitro changes also occur within
the brain of patients with Aicardi–Goutières syndrome, we performed immunohistochemical stainings on autopsy specimens
from two patients with Aicardi–Goutières syndrome (Barth et al.,
1999; Rasmussen et al., 2005; van Heteren et al., 2008).
As previously described, Aicardi–Goutières syndrome samples
showed an intense immunoreactivity against IFN- in cells identified
as astrocytes (van Heteren et al., 2008), in both the grey matter (Fig.
5A) and even more so in the white matter (Fig. 5B) compared with
control white matter (Fig. 5C). Moreover, in some areas, some blood
vessels also appeared positive for IFN- (Fig. 5A). We performed
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E. Cuadrado et al.
Figure 3 Continuous IFN- exposure alters the expression of white matter-stability related genes and increases HLA expression.
(A) Quantitative PCR analysis shows no gene expression changes compared with vehicle-treated control cells (dashed line) when cells are
treated with IFN- for 24 h. (B) Bar graph shows a reduction on the expression of EIF2B1, ATF4 and myelin-degrading protease
CTSD, together with an increase of CST7 and HLA-C after 21 days in vitro (21d) chronic IFN- exposure. Results are presented as percentage
of control (Cntrl) values (mean SEM, n = 5). (C) Protein expression of ATF4, CTSD and CST7 was investigated by immunoblotting of
whole-cell extracts. Results parallel to those found by quantitative PCR. Actin was used as loading control. (D) Histograms corresponding to
flow cytometry analysis of human leukocyte antigen expression in IFN--treated astrocytes (dark grey) compared with control cells (light
grey). (E) Quantification of fluorescence data represent the geometric mean SEM (n = 3) and is presented as percentage of vehicle-treated
control cells. (F) Quantitative PCR analysis of Aicardi–Goutières syndrome-causative genes after acute (light grey) or chronic (dark grey) IFN-
treatment (20 or 200 pg/ml). Results are presented in arbitrary units (AU) and represent mean SEM (n = 5). Statistically significant differences are expressed as *P 5 0.05, **P 5 0.01 and ***P 5 0.001. PE = phycoerithryn.
similar staining against CST7. Those stainings revealed a modest increase for CST7 in cortical neurons in patients with Aicardi–Goutières
syndrome (Fig. 5E) compared with control subjects (Fig. 5D). The
increase, however, was more pronounced in the white matter of patients with Aicardi–Goutières syndrome (Fig. 5G and H) compared
with healthy control white matter (Fig. 5F). Double staining with
GFAP confirmed astrocytes as the major source of CST7 (Fig. 5I),
and these cells typically appeared to surround blood vessels.
We also evaluated the expression of pro-angiogenic factors
IL-1b and VEGF-B. Immunostaining of IL-1b was strong in both
grey matter and white matter of control specimens (Fig. 5J and L)
compared with an almost absent reactivity in Aicardi–Goutières
syndrome specimens (Fig. 5K and M). For VEGF-B, a strong expression was found confined to the white matter of control cases
(Fig. 5O and Q), and almost no expression was observed in the
white matter of Aicardi–Goutières syndrome cases (Fig. 5R). On
the other hand, some neurons in patients with Aicardi–Goutières
syndrome showed stronger staining for VEGF-B than in control
brains (Fig. 5P). Taken together, these results validated our previous in vitro findings.
Effect of IFN- on astrocytes in Aicardi–Goutières syndrome
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Figure 4 IFN- treatment reduces angiogenic factor production/release but does not affect calcium deposition. (A) Brightfield micrographs of haematoxylin-stained blood vessels in control tissue and Aicardi–Goutières syndrome (AGS) brain samples. Scale bars = 500 mm.
(B) Bar graph shows a decrease in transcript levels in treated astrocytes compared with control cells (dashed line). Normalized values are
presented as percentage of vehicle-treated control cells (mean SEM, n = 5). (C) Representative immunoblots show a reduction of
angiogenic factors in cell lysates. Actin was used as loading control. (D) Secreted cytokines levels were measured in the supernatant of
wells treated with IFN- or vehicle. Results are expressed in pg/ml (mean SEM, n = 8). (E) Bar graph shows quantitative PCR values of
SLC20A2 and COL4A. Results are presented as percentage (mean SEM, n = 5) of control values (dashed line). (F) Images of wells
stained with alizarin red (top) and von Kossa staining (bottom). Wells coated with gelatin and CaCO3 were used as positive control.
Statistically significant differences are expressed as *P 5 0.05 and **P 5 0.01.
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E. Cuadrado et al.
Figure 5 Overexpression of CST7 and downregulation of VEGF-B and IL-1b in patient-derived brain tissue. (A) Panels showing IFN-
immunoreactivity of astrocytes in Aicardi–Goutières syndrome (AGS) grey-(GM) and white-matter (WM) (B). Control white matter shows
no staining for IFN- (C). Immunostainings for CST7 in the grey matter of control (D) and Aicardi–Goutières syndrome specimens (E). (F)
Control white matter shows no reactivity for CST7 compared with Aicardi–Goutières syndrome white matter panels (G and H). (I)
Representative double immunofluorescence image of GFAP (white) and CST7 (red). Blood vessel (asterisk). Panels show robust staining
against IL-1b in control grey matter (J) and white matter (l) compared with Aicardi–Goutières syndrome grey matter (K) and white matter
(M). Images show immunoreactivity for VEGF-B in control tissue (O, Q) compared with Aicardi–Goutières syndrome grey matter (P) and
white matter (R) specimens. Scale bars: A–H, J, K, O–R = 100 mm; I, L and M = 50 mm.
Continuous IFN-a exposure changes
are persistent, and astrocytes do not
recover pretreatment status after
7 days withdrawal of IFN-a
Given the effects of IFN- in the astrocyte cultures, and
considering that an anti-interferon might potentially be beneficial
for patients with Aicardi–Goutières syndrome, we decided to
explore the effect of withdrawing the IFN- in our in vitro
system. For this purpose, cells were grown in the presence of
IFN- as previously described but, after 14 days in vitro, IFN-
treatment was discontinued, and cells were washed and cultured
for an additional 7 days (Fig. 6A). After this period, we measured
the messenger RNA expression of the genes that we have previously shown to be affected by IFN- continuous exposure. We
found that analysed transcripts did not recover pre-treatment
levels after 7 days of IFN- withdrawal (Fig. 6B). Only HLA-C
showed a significantly reduced expression after IFN- removal
(P 5 0.001).
Effect of IFN- on astrocytes in Aicardi–Goutières syndrome
We also measured cytokines in the supernatant from these cell
cultures. IFN- withdrawal did not significantly produce a recovery
of the cytokine levels of IL-1b, VEGF or TNF-, although a trend
was observed in the latter two (Fig. 6C). The levels of IL-6
(P = 0.021) and G-CSF (P = 0.0092) did, however, show a recovery after IFN- treatment discontinuation. The rest of the analysed
cytokines did not show any significant changes (Supplementary
Fig. 3). Altogether, these results demonstrate that the changes
that follow IFN- chronic treatment are persistent over time.
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Discussion
IFN- is associated with a number of diseases affecting the CNS
including, among others, Aicardi–Goutières syndrome, systemic
lupus erythematosus, TORCH congenital infections and
HIV-associated dementia. With the in vitro model presented
here, we confirm that IFN-, even in the absence of an underlying
Aicardi–Goutières syndrome mutation, can induce many of the
characteristic changes that occur in the context of
Figure 6 Withdrawal of IFN- for 7 days does not recover astrocytes pretreatment status. (A) Images show that IFN--treated cells, even
after withdrawal, still display a strong staining against GFAP (green) and S100b (red), and extended cellular processes. Scale bar = 100 mm.
(B) Bar graph shows no changes in transcript levels in astrocytes after withdrawal but in HLA-C messenger RNA. Normalized values are
presented as percentage of vehicle-treated control cells (dashed line). Bars on graphs represent the mean SEM of four different
experiments. (C) Bars represent the levels of cytokines measured in the supernatant of cell cultures treated with vehicle and 200 pg/ml of
IFN- for 21 days (21d) or 200 pg/ml of IFN- for 14 days (14d) and IFN- withdrawal for 7 days. Results are expressed in pg/ml
(mean SEM, n = 6). Statistically significant differences are expressed as *P 5 0.05, **P 5 0.01 and ***P 5 0.001.
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Aicardi–Goutières syndrome-related pathophysiology. We also
confirm that astrocytes constitute a major source of CXCL10 in
CNS tissue when activated, as previously described in IFN-overexpressing (GIFN) mice (Akwa et al., 1998; Campbell et al.,
1999) and in Aicardi–Goutières syndrome brain samples (van
Heteren et al., 2008).
One of the questions relating to Aicardi–Goutières syndrome
pathogenesis is the stage at which the disease process begins. In
Trex1-deficient mice, the interferon response develops in utero
and precedes lymphocyte-dependent inflammation and autoimmune tissue damage (Gall et al., 2012). In humans, it remains
unknown when exactly the interferon-stimulatory DNA response
becomes activated. Depending on the genotype, patients might
present with signs and symptoms after several months of normal
development (Aicardi and Goutières, 1984; McEntagart et al.,
1998; Rice et al., 2007). Our observations confirm that exposure
of neural stem cells to IFN- neither affects their growth nor their
differentiation into mature astrocytes, thus supporting the idea
that brain development during the embryonic stage may well be
normal, at least for patients with a milder phenotype.
Moreover, our results also demonstrate that astrocytes on
chronic exposure to IFN-, reduced their proliferation and
became reactive by increasing astrocyte-specific proteins and by
changing their morphology with extended membrane processes.
Similar findings were observed in murine astrocytes treated with
IFN-/b together, with a disorganization of intermediate GFAP
filaments (Tedeschi et al., 1986).
Our data may also provide a link between IFN- and the white
matter pathology observed in post-mortem Aicardi–Goutières syndrome brains. In the histology of these brains, subcortical white
matter showed dense anisomorphic fibrous astrocytosis and in the
cerebellum, excessive Bergmann glial fibres were also found, in
addition to demyelination (Barth et al., 1999).
Abnormal white matter on brain imaging is observed in almost
all patients with Aicardi–Goutières syndrome during the first year
of life (Rice et al., 2007). Additionally, in GIFN mice, a degeneration of myelinated axons in the cerebellar white matter is seen
(Campbell et al., 1999). Although the exact mechanism by which
IFN- might lead to a disturbance of myelin is unknown, a microarray study analysing Aicardi–Goutières syndrome lymphocytes
from CSF showed an upregulation in the proteolytic enzyme
CTSD and its inhibitor CST7 (Izzotti et al., 2008, 2009). This
study also showed an age-dependent increase in the expression
of DNAJ (heat shock protein 40), an important component of a
negative feedback loop involved in inhibiting IFN- signalling (van
Huizen et al., 2003; Izzotti et al., 2009). Our data confirm that
chronic IFN- produced a downregulation of CTSD together with
an upregulation of CST7 as a counteracting mechanism to attenuate IFN- signalling, as was previously suggested (Izzotti et al.,
2009; Pulliero et al., 2011). The fact that we found a downregulation of CTSD in our cultures differs from what has been
described in Aicardi–Goutières syndrome lymphocytes, but this
might be attributed either to the lack of Aicardi–Goutières syndrome mutations in our cells or differences in tissue specificity.
Indeed, a recent study demonstrates that only in TREX1-deficient
lymphocytes is a remarkable upregulation of CTSD after IFN-
exposure observed, in comparison with the moderate increase
E. Cuadrado et al.
described in healthy control cells (Pulliero et al., 2012).
Moreover, we found an upregulation of CST7 in astrocytes in
the white matter of Aicardi–Goutières syndrome brain specimens
and demonstrated that ATF4 and eIF2B (a DNAJ downstream
transcription and translation factor, respectively) were both downregulated in astrocytes on chronic exposure to IFN-. These findings are remarkable, as mutations in EIF2B subunit genes cause
vanishing white matter disease with a severe loss of oligodendrocytes and astrocytes early in life (Pronk et al., 2006; Carter, 2007).
Indeed, defects in EIF2B in vanishing white matter disease lead to
an increased unfolded protein response (ATF4 induction) in fibroblasts as a consequence of endoplasmic reticulum stress (Kantor
et al., 2005). Even though more research is needed, eIF2B might
be a common factor in the pathogenesis of both leukodystrophies.
Having said that, these two pathologies have different disease
mechanisms. Although treatment of astrocyte cultures with
IFN- dramatically increased the expression of human leukocyte
antigens, as was reported previously (Tedeschi et al., 1986), it is
important to emphasize that neither neurons nor oligodendrocytes
express human leukocyte antigen molecules when exposed to
IFN- in vitro. Our findings agree with those of others (Akwa
et al., 1998; Campbell et al., 1999; van Heteren et al., 2008) in
pointing to astrocytes as the main players in the autoinflammation
of the brain in Aicardi–Goutières syndrome.
Our finding that chronic exposure to IFN- reduced the production of pro-angiogenic factors by astrocytes may offer a partial
explanation for the cerebrovascular problems reported in patients
with Aicardi–Goutières syndrome (Barth et al., 1999; Rasmussen
et al., 2005; Ramesh et al., 2010). IFN- is known to reduce
angiogenesis and is actually used as adjuvant therapy to treat
certain types of cancer (Kirkwood, 2002). For the first time, we
now report IFN- to inhibit the production of VEGF by astrocytes
as well as the production of some cytokines, including IL-1 and
TNF-. Downregulation of VEGF and IL-1 was confirmed in
Aicardi–Goutières syndrome brain specimens. The potential importance of this finding lies in the fact that VEGF is expressed
exclusively by astrocytes in the adult brain (Yang et al., 2003;
Bernal and Peterson, 2011). We speculate that the impaired availability of these angiogenic factors in the brains of infants might
lead to an abnormal vessel formation and proliferation. Indeed, we
observed an aberrant vasculature with excessive number of
capillary-like blood vessels, especially in cortical areas in the
Aicardi–Goutières syndrome specimens. Moreover, degeneration
of neurons is found in subcortical areas (mammillary bodies, thalamus, striatum and globus pallidus) of Aicardi–Goutières syndrome
brains (Barth et al., 1999; Rasmussen et al., 2005). Besides the
primary role of VEGF in vascular development, it is also known to
induce neurogenesis (Jin et al., 2002) and to be neuroprotective
under stress conditions (Falk et al., 2011; Ma et al., 2011).
Although vascular insufficiency might be crucial for the degenerative process, the reduced amount of trophic factors may worsen
the neurodegeneration in these cerebral areas.
Cerebral calcifications are one of the most common findings in
Aicardi–Goutières syndrome (Uggetti et al., 2009). However, brain
calcifications are non-specific and are found in many pathological
conditions. Given the anatomical distribution of microcalcifications
in the white matter and around blood vessels, we hypothesized
Effect of IFN- on astrocytes in Aicardi–Goutières syndrome
that astrocytes were good candidates for the production of calcium deposits. However, we did not find supportive evidence for
such. Also, we observed no change in transcriptional activity of
SLC20A2 and COL4A1, both genes being related to intracranial
calcifications on loss of function mutations (Livingston et al., 2011;
Wang et al., 2012). Similarly, osteopontin has also been described
to be involved in brain calcification and localizes in degenerating
neurites (Shin et al., 2012). However, we were not able to detect
any osteopontin messenger RNA in our astrocyte cultures. Hence,
it remains to be elucidated which cell type is responsible for the
cerebral calcifications observed in Aicardi–Goutières syndrome.
In summary, we describe a new in vitro model of Aicardi–
Goutières syndrome that provides insight into the dysregulated
expression of genes and proteins affecting the stability of white
matter and the cerebral vasculature in the postnatal manifestations
of this brain disease. Noteworthy is the fact that IFN- withdrawal
did not produce a clear recovery to pre-treatment gene/protein
expression levels in vitro. Instead, the IFN--induced gene expression signature seems to be persistent over time. Several authors
have suggested that anti-IFN- therapy might be beneficial in the
treatment of Aicardi–Goutières syndrome. Based on our data, we
postulate that therapeutic targets for Aicardi–Goutières syndrome,
and other IFN--related encephalopathies, might include downstream IFN- signalling effectors besides IFN- itself, as the
effect of IFN- may be long-lasting and cannot always be reversed
instantly on simple withdrawal or neutralization.
Acknowledgements
Post-mortem human brain material was obtained from The
Netherlands Brain Bank (http://www.brainbank.nl/) and the
Department of Neuropathology from the Academic Medical
Centre in Amsterdam. We thank Jacqueline Sluijs for cell culture
technical assistance. We are grateful for insight and comments
from all the NIMBL Consortium members.
The NIMBL Consortium is composed of David Bonthron,
Genetics Section, Leeds Institute of Molecular Medicine (LIMM),
St James’s University Hospital, Leeds, UK; Antonio Celada,
Institute for Research in Biomedicine (IRB) Barcelona, Spain;
Yanick Crow, Genetic Medicine, Manchester Academic Health
Science Centre, Manchester, UK; Taco Kuijpers, Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands; Arn
van den Maagdenberg, Departments of Human Genetics and
Neurology, Leiden University Medical Centre, Leiden, The
Netherlands; Simona Orcesi, Department of Child Neurology and
Psychiatry, IRCCS C. Mondino Institute of Neurology Foundation,
Pavia, Italy; Dan Stetson, Department of Immunology, University of
Washington, Seattle, WA, USA; Adeline Vanderver, Children
Research Institute, Washington DC, USA.
Funding
European Union (EU) Seventh Framework Programme (241779)
(Nuclease Immune Mediated Brain and Lupus-like conditions
Brain 2013: 136; 245–258
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(NIMBL): natural history, pathophysiology, diagnostic and therapeutic
modalities with application to other disorders of autoimmunity).
Supplementary material
Supplementary material is available at Brain online.
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