Mutant superoxide dismutase 1-induced IL-1beta accelerates ALS pathogenesis.
ABSTRACT ALS is a fatal motor neuron disease of adult onset. Neuroinflammation contributes to ALS disease progression; however, the inflammatory trigger remains unclear. We report that ALS-linked mutant superoxide dismutase 1 (SOD1) activates caspase-1 and IL-1beta in microglia. Cytoplasmic accumulation of mutant SOD1 was sensed by an ASC containing inflammasome and antagonized by autophagy, limiting caspase-1-mediated inflammation. Notably, mutant SOD1 induced IL-1beta correlated with amyloid-like misfolding and was independent of dismutase activity. Deficiency in caspase-1 or IL-1beta or treatment with recombinant IL-1 receptor antagonist (IL-1RA) extended the lifespan of G93A-SOD1 transgenic mice and attenuated inflammatory pathology. These findings identify microglial IL-1beta as a causative event of neuroinflammation and suggest IL-1 as a potential therapeutic target in ALS.
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ABSTRACT: Vasoactive intestinal peptide (VIP) has potent immune modulatory actions that may influence the course of neurodegenerative disorders associated with chronic inflammation. Here, we show the therapeutic benefits of a modified peptide agonist stearyl-norleucine-VIP (SNV) in a transgenic rat model of amyotrophic lateral sclerosis (mutated superoxide dismutase 1, hSOD1(G93A)). When administered by systemic every-other-day intraperitoneal injections during a period of 80days before disease, SNV delayed the onset of motor dysfunction by no less than three weeks, while survival was extended by nearly two months. SNV-treated rats showed reduced astro- and microgliosis in the lumbar ventral spinal cord and a significant degree of motor neuron preservation. Throughout the treatment, SNV promoted the expression of the anti-inflammatory cytokine interleukin-10 as well as neurotrophic factors commonly considered as beneficial in amyotrophic lateral sclerosis management (glial derived neuroptrophic factor, insulin like growth factor, brain derived neurotrophic factor). The peptide nearly totally suppressed the expression of tumor necrosis factor-α and repressed the production of the pro-inflammatory mediators interleukin-1β, nitric oxide and of the transcription factor nuclear factor kappa B. Inhibition of tumor necrosis factor-α likely accounted for the observed down-regulation of nuclear factor kappa B that modulates the transcription of genes specifically involved in amyotrophic lateral sclerosis (sod1 and the glutamate transporter slc1a2). In line with this, levels of human superoxide dismutase 1 mRNA and protein were decreased by SNV treatment, while the expression and activity of the glutamate transporter-1 was promoted. Considering the large diversity of influences of this peptide on both clinical features of the disease and associated biochemical markers, we propose that SNV or related peptides may constitute promising candidates for amyotrophic lateral sclerosis treatment.Experimental Neurology 10/2014; · 4.62 Impact Factor
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ABSTRACT: Pathological aggregation and mutation of the 43-kDa TAR DNA binding protein (TDP-43) are strongly implicated in the pathogenesis amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 neurotoxicity has been extensively modeled in mice, zebrafish, C. elegans, and Drosophila, where selective expression of TDP-43 in motoneurons led to paralysis and premature lethality. Through a genetic screen aimed to identify genetic modifiers of TDP-43, we found that the Drosophila dual leucine kinase Wallenda (Wnd) and its downstream kinases JNK and p38 influenced TDP-43 neurotoxicity. Reducing Wnd gene dosage or overexpressing its antagonist Highwire partially rescued TDP-43 associated premature lethality. Downstream of Wnd, the JNK and p38 kinases played opposing roles in TDP-43-associated neurodegeneration. LOF alleles of the p38b gene as well as p38 inhibitors diminished TDP-43 associated premature lethality whereas p38b GOF caused phenotypic worsening. In stark contrast, disruptive alleles of Basket (Bsk), the Drosophila homologue of JNK, exacerbated longevity shortening whereas overexpression of Bsk extended lifespan. Among possible mechanisms, we found motoneuron-directed expression of TDP-43 elicited oxidative stress and innate immune gene activation that were exacerbated by p38 GOF and Bsk LOF, respectively. A key pathologic role for innate immunity in TDP-43-associated neurodegeneration was further supported by the finding that genetic suppression of the Toll/Dif and Imd/Relish inflammatory pathways dramatically extended lifespan of TDP-43 transgenic flies. We propose that oxidative stress and neuroinflammation are intrinsic components of TDP-43 associated neurodegeneration and that the balance between cytoprotective JNK and cytotoxic p38 signaling dictates phenotypic outcome to TDP-43 expression in Drosophila.Human Molecular Genetics 10/2014; 24(3). · 6.68 Impact Factor
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ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a clinically heterogeneous disorder characterized by loss of motor neurons, resulting in paralysis and death. Multiple mechanisms of motor neuron injury have been implicated based upon the more than 20 different genetic causes of familial ALS. These inherited mutations compromise diverse motor neuron pathways leading to cell-autonomous injury. In the ALS transgenic mouse models, however, motor neurons do not die alone. Cell death is noncell-autonomous dependent upon a well orchestrated dialogue between motor neurons and surrounding glia and adaptive immune cells. The pathogenesis of ALS consists of 2 stages: an early neuroprotective stage and a later neurotoxic stage. During early phases of disease progression, the immune system is protective with glia and T cells, especially M2 macrophages/microglia, and T helper 2 cells and regulatory T cells, providing anti-inflammatory factors that sustain motor neuron viability. As the disease progresses and motor neuron injury accelerates, a second rapidly progressing phase develops, characterized by M1 macrophages/microglia, and proinflammatory T cells. In rapidly progressing ALS patients, as in transgenic mice, neuroprotective regulatory T cells are significantly decreased and neurotoxicity predominates. Our own therapeutic efforts are focused on modulating these neuroinflammatory pathways. This review will focus on the cellular players involved in neuroinflammation in ALS and current therapeutic strategies to enhance neuroprotection and suppress neurotoxicity with the goal of arresting the progressive and devastating nature of ALS.Journal of the American Society for Experimental NeuroTherapeutics 01/2015; · 3.88 Impact Factor
Mutant superoxide dismutase 1-induced IL-1β
accelerates ALS pathogenesis
Felix Meissner1, Kaaweh Molawi1, and Arturo Zychlinsky2
Department of Cellular Microbiology, Max Planck Institute for Infection Biology, 10117 Berlin, Germany
Edited* by Charles A. Dinarello, University of Colorado, Aurora, CO, and approved June 2, 2010 (received for review February 26, 2010)
ALS is a fatal motor neuron disease of adult onset. Neuroinflam-
mation contributes to ALS disease progression; however, the
inflammatory trigger remains unclear. We report that ALS–linked
mutant superoxide dismutase 1 (SOD1) activates caspase-1 and
IL-1β in microglia. Cytoplasmic accumulation of mutant SOD1 was
sensed by an ASC containing inflammasome and antagonized by
autophagy, limiting caspase-1–mediated inflammation. Notably,
mutantSOD1 induced IL-1β correlatedwith amyloid-likemisfolding
and was independent of dismutase activity. Deficiency in caspase-1
or IL-1β or treatment with recombinant IL-1 receptor antagonist
(IL-1RA) extended the lifespan of G93A-SOD1 transgenic mice and
attenuated inflammatory pathology. Thesefindingsidentifymicro-
glial IL-1β as a causative event of neuroinflammation and suggest
IL-1 as a potential therapeutic target in ALS.
caspase-1|inflammasome|interleukin 1|Lou Gehrig’s disease|
finally death within 1–5 years after diagnosis. Dominant gain of
function mutations of SOD1 are the most common genetic cause
of ALS and also lead to motor neuron disease in mutant SOD1
transgenic mice (1). Mutations induce toxic misfolding and ag-
gregation of SOD1 and interfere with cellular homeostasis in
neurons and glia cells (2, 3). Neurodegeneration in ALS is ac-
companied by glia mediated neuroinflammation, which accel-
erates disease progression non–cell-autonomously (2, 4). One of
the inflammatory markers found in the CNS of ALS mice is active
caspase-1, which is also implicated in amyloid-β–mediated in-
flammation (5–7). Caspase-1 is activated in response to danger
signals by cytosolic protein complexes called inflammasomes and
proteolytically matures IL-1β and IL-18 (8). Accordingly, IL-1β
levels are elevated in the CNS of mutant SOD1 transgenic mice
to induce an inflammatory reaction by microglia (9, 10); however,
the underlying mechanisms are poorly understood.
LS is a fatal neurodegenerative disorder characterized by
progressive loss of motor neurons causing paralysis and
Mutant SOD1 Activates Caspase-1 in Microglia. To test whether mu-
tant SOD1 can act as a danger signal that activates caspase-1 in
resident microglia, we stimulated these cells with purified WT or
mutant G93A-SOD1. G93A-SOD1, but not the WT protein, acti-
vatedcaspase-1inmicroglia andmacrophages ina dose-dependent
manner (Fig. 1 A and B). Consistently, time- and dose-dependent
secretion of mature IL-1β was observed specifically upon G93A-
SOD1 stimulation of microglia or macrophages (Figs. 1C and S1).
caspase-1–mediated IL-1β release in response to G93A-SOD1 was
independent of nonproteinaceous contaminants, LPS priming or
transgenic mutant SOD1 expression in microglia or macrophages
(Figs. S2 and S3). The inflammasome adaptor protein apoptosis-
associated speck-like protein containing a caspase recruitment do-
main (ASC) was essential for IL-1β release in response to G93A-
SOD1, whereas the Nod-like receptors (NLRs) NALP3 and IPAF
were not required (Fig. S4). Collectively, these data demonstrate
that mutant G93A-SOD1 activates caspase-1 and induces IL-1β
Autophagy Counteracts Cytoplasmic SOD1 Accumulation and Cas-
pase-1 Activation. Further analysis of the mechanism of caspase-1
activation by mutant SOD1 revealed rapid endocytosis of fluo-
rescently labeled SOD1 (Fig. 2 A and B). This could be blocked by
cytochalasin D, an inhibitor of actin polymerization, which also
inhibited mutant SOD1–induced IL-1β release, indicating that up-
take ofmutantSOD1isrequired forcaspase-1activation (Fig.2C).
Because several caspase-1 activators require endocytosis and
translocate into the cytoplasm, we next analyzed the subcellular
distribution of SOD1 by differential subcellular extraction (5,
11). Biochemical analysis of the cytosol and the organelle
fraction of SOD1 stimulated macrophages showed that SOD1
partially relocated from the organelle fraction into the cyto-
plasm within 5 min where it accumulated at later time points
(Fig. 2D). Confocal microscopy of macrophages coincubated
with fluorophore-labeled mutant SOD1 and fluorescent dextran,
which traffics into the endo-lysosomal pathway, support these
results (Fig. 2E). Notably, mutant SOD1 is taken up more ef-
ficiently than WT SOD1, resulting in increased cytoplasmic
accumulation of the mutant protein (Fig. S5).
We speculated that cytoplasmic accumulation of SOD1 induces
autophagy, a homeostatic cellular process evolved to degrade long-
lived cytosolic proteins and macromolecules (12). Pharmacological
inhibition of autophagy increased the amount of SOD1 in the cy-
tosolic fraction and strongly increased IL-1β release upon mutant
SOD1 stimulation (Fig. 3 A and B). Notably, WT SOD1-induced
IL-1β release was comparable to cells primed only with LPS (13).
Furthermore, deficiency in autophagy-related 5 (ATG5), a protein
required for autophagosome formation (14), increased caspase-1
activation in response to G93A-SOD1, indicating that autophago-
somes were formed as a cellular stress response to remove cyto-
plasmic SOD1 and to reduce inflammation (Fig. 3C).
These data collectively show that endocytosed mutant SOD1
enters the cytoplasm and activates caspase-1, whereas autophagy
counteracts cytoplasmic SOD1 accumulation to dampen the
Amyloid Conformation Correlates with IL-1β Maturation. Mutations
vivo (3). To test whether mutation-induced structural changes are
involved in caspase-1 activation by mutant SOD1, we analyzed
WT-SOD1 and three well-studied SOD1 mutants (G93A, G85R,
Author contributions: F.M., K.M., and A.Z. designed research; F.M. and K.M. performed
research; F.M., K.M., and A.Z. analyzed data; and F.M., K.M., and A.Z. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
See Commentary on page 12741.
1F.M. and K.M. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 20, 2010
| vol. 107
| no. 29www.pnas.org/cgi/doi/10.1073/pnas.1002396107
and G37R) for their propensity to form amyloids using Thioflavin-
T (Th-T) fluorescence. All three mutant SOD1 proteins showed
protein (Fig. S6A).G93A-SOD1,which exhibited the highest Th-T
fluorescence, potently activated caspase-1 and IL-1β secretion;
G37R- and G85R-SOD1 mutants, with intermediate amyloid
characteristics, were weaker caspase-1 and IL-1β activators than
G93A-SOD1 (Fig. S6 B and C), suggesting a connection between
these two parameters. Interestingly, when we tested nine different
SOD1 mutants and the WT protein, we observed a significant,
directcorrelationbetweenthe Th-Tfluorescence andthesecretion
of mature IL-1β (Fig. 4 A–C).
Furthermore, caspase-1 activation was not dependent on the
superoxide degrading activityofSOD1.Themetalloprotein SOD1
was enzymatically inactive when purified and tested in the absence
of copper and zinc (Fig. S6D). Metalation restored SOD1 activity,
which was equally high in WT and G93A-SOD1, intermediate in
G37R-SOD1, and poor in G85R-SOD1. However, metalation did
not affect caspase-1 activation nor influence Th-T binding in any
of the SOD1 proteins tested (Fig. S6 E and F). Together, these
results suggest that mutant SOD1 induces caspase-1 activation
independently of its enzymatic activity through a gain of amyloid
IL-1β Promotes Disease Progression in G93A-SOD1 Mice. To de-
termine the contribution of caspase-1 and caspase-1–dependent
transgenic mice (15) with caspase-1–, IL-1β– or IL-18–deficient
mice and monitored survival. Deficiency in caspase-1 or IL-1β
equally extended the lifespan of G93A-SOD1 transgenic animals,
whereas deficiency in IL-18 did not affect survival (Fig. 5A). Dis-
ease onset remained unchanged in IL-1β–deficient G93A-SOD1
transgenic mice (Fig. S7). Immunohistochemical analysis revealed
reduced microgliosis and astrogliosis coinciding with an increased
number of motor neurons in the spinal cord of caspase-1– and IL-
1β–deficient G93A-SOD1 transgenic mice in comparison with
controls (Fig. 5 B–D). These findings suggested IL-1β as a thera-
peutic target to slow down disease progression in G93A-SOD1
transgenic mice. IL-1 bioactivity is controlled by the highly specific
endogenous interleukin-1 receptor antagonist (IL-1RA) (16).
Treatment of transgenic mice with recombinant human IL-1RA
(Anakinra) significantly extended survival and improved motor
function during the symptomatic phase in comparison with pla-
cebo-treated controls (Fig. 5 E and F).
These data demonstrate that IL-1β contributes significantly to
disease progression in G93A-SOD1 transgenic animals by pro-
treatmentrepresents a potentialmedicalinterventioninALS,asit
prolonged the lifespan of G93A-SOD1 transgenic animals.
Motor neuron degeneration in ALS is accompanied by in-
flammation, which involves microgliosis and astrogliosis as well as
the up-regulation of proinflammatory molecules in the CNS (4,
17, 18). Here we identified misfolded mutant SOD1 as a disease-
associated endogenous danger signal that is sensed by microglia
and initiates inflammation through caspase-1–dependent IL-1β
maturation. Mutant SOD1 has been described to form amyloid-
like oligomers and aggregates in vitro and in vivo (3, 19–21). We
demonstrate that the degree of amyloid-like misfolding of mutant
SOD1 correlated with IL-1β maturation, supporting the recent
finding that SOD1 aggregate formation is linked to accelerated
disease progression in ALS patients (22). Notably, mutant SOD1-
induced inflammasome activation was independent of LPS
priming and the NLRs NALP3 and IPAF. These observations
innate immune receptors that also account for the priming,
analogously to the recognition of amyloid-β by an inflammasome
and TLRs (5, 23, 24).
We showed that mutant SOD1 was efficiently endocytosed and
leaked into the cytoplasm to activate caspase-1. Future studies
should address whether the increased uptake of mutant SOD1 is
a result of a specific cell surface receptor interaction, which
might contribute to the innate immune sensing of misfolded
proteins such as mutant SOD1. Autophagy antagonized cyto-
plasmic accumulation of misfolded SOD1 and therefore limited
or G93A-SOD1 for 20 h or as indicated. (A) Flow cytometry analysis of caspase-1 activation in primary microglia by a fluorescent cell-permeable inhibitor that
binds to active caspase-1 (FLICA). Numbers above bracketed lines indicate percentage of cells with active caspase-1. Unstimulated and ATP-stimulated cells
served as controls. (B) Immunoblot analysis of cell lysates and supernatants of stimulated bone marrow–derived macrophages (BMMs) with antibodies to the
p10 subunit of caspase-1 and to IL-1β, respectively. Actin shows equal loading of lanes. (C–E) ELISA analysis of secreted, mature IL-1β in the cell supernatant of
stimulated primary microglia (C), caspase-1–deficient BMMs (D), or primary astrocytes (E). Data are representative of at least three independent experiments;
error bars represent SEM of triplicate wells.
Mutant SOD1 activates caspase-1 in microglia and macrophages. (A–E) Primed cells were stimulated with 10 μM or the depicted concentrations of WT
Meissner et al.PNAS
| July 20, 2010
| vol. 107
| no. 29
caspase-1–mediated inflammation, confirming a beneficial func-
tion of autophagy in ALS (13).
In G93A-SOD1 ALS mice, caspase-1 acts through IL-1β, as
caspase-1– and IL-1β–deficient transgenic mice displayed equal
survival extensions. Treatment of ALS mice with IL-1RA further
confirmed the relevance of IL-1–mediated inflammation, in-
dicating that caspase-1–induced neuronal cell death described
elsewhere seems to play a minor role in this disease model (7).
Previous results suggesting that IL-1β does not affect ALS
pathogenesis in G37R transgenic ALS mice might be due to the
different aggregation and inflammatory properties of the SOD1
mutation (G93A vs. G37R) or to the different mouse strains
used (congenic C57BL/6 vs. mixed background B10RIII ×
C57BL/6) (17). In our experiments, IL-1β deficiency slowed
disease progression but did not affect disease onset, arguing for
a non–cell-autonomous, microglia-mediated acceleration of
neurodegeneration (25). IL-1 is a pleiotropic cytokine involved in
the induction of inflammatory and neurotoxic molecules such as
COX2, inducible NOS, NO, or IL-6, which are implicated in ALS
(4, 16, 18, 26). Therefore our data and the previous finding of
signal of neuroinflammation in ALS (6, 27). It is tempting to
speculate that the results presented here not only identified IL-1
asapotential therapeutictargetin ALSbut alsohaveimplications
on other conformational diseases involving inflammation.
Mice. CASP1−/−, IL1β−/−, IL18−/−, IL18/IL1β−/−, and TLR4−/−mice have been
described previously (4, 16). TgSOD1-G93A mice on a C57BL/6 background
were kindly provided by Albert Ludolph (University of Ulm, Ulm, Germany)
(15). TgSOD1-G93A/CASP1−/−, TgSOD1-G93A/IL1β−/−, TgSOD1-G93A/IL18−/−,
and TgSOD1-G93A/IL18/IL1β−/−mice were generated as F2 litters from in-
terbreeding of TgSOD1-G93A and the corresponding knockout mice. The
number of SOD1 transgenes was controlled semiquantitatively by real-time
PCR as described elsewhere (28). Mice were tested weekly for neuromuscular
dysfunction with the hanging-wire test. Latency to fall (maximum 3 min) was
measured after a mouse was placed on the bars and turned upside down
(height 20 cm). Mice that fell in <10 s were given a second trial. For IL-1RA
treatment, SOD1 transgenic animals were injected every 24 h in-
traperitoneally with either 150 mg/kg or 75 mg/kg IL-1RA in 200 μL NaCl
BMMs were stimulated with WT or G93A-SOD1 as indicated. (A and B) Up-
take of fluorescently labeled G93A-SOD1 (5 μM; red) in the presence of
Cytochalasin D analyzed by confocal microscopy (A) or flow cytometry (B).
Fluorescent cholera toxin B (CTB; green) stains cell membranes. (C) ELISA of
SOD1 (10 μM) induced mature IL-1β in the presence of Cytochalasin D. (D)
Immunoblot analysis of cytoplasmic and organelle fraction of BMMs stimu-
lated with G93A-SOD1 (2 μM) for indicated times using antibodies against
human SOD1, GAPDH (cytoplasmic marker protein), and LAMP1 (organelle
marker protein). (E) Confocal microscopy of BMMs stimulated for the in-
dicated time points with labeled G93A-SOD1 (5 mM; green) and Dextran
(red) or Dextran only. (Scale bars, 4 μm.) Data are representative of at least
three independent experiments; error bars represent SEM of triplicate wells.
IL-1β maturation requires endocytosis of mutant SOD1. (A–D) Primed
autophagy. (A–C) Primed cells were stimulated with WT or G93A-SOD1 as in-
dicated. (A) Immunoblot analysis of cytoplasmic and organelle fraction
of BMMs stimulated with G93A-SOD1 (2 μM) for 6 h in the presence of
3-methyladenine (3MA) using antibodies against human SOD1, GAPDH (cyto-
plasmic marker protein), and LAMP1 (organelle marker protein). (B) ELISA of
or 3-methyladenine. (C) Caspase-1 activation determined by flow cytometry
analysis with caspase-1 FLICA in autophagy-related 5–deficient (ATG5−/−, ○)
and nondeficient ATG5+/+(•) mouse embryonic fibroblasts (MEFs) transfected
with the indicated concentrations of SOD1. Data are representative of three
independent experiments; error bars represent SEM of triplicate wells.
Mutant SOD1–induced caspase-1 activation is counteracted by
| www.pnas.org/cgi/doi/10.1073/pnas.1002396107 Meissner et al.
solution starting at the age of 70 d. For the placebo group, 200 μL NaCl
solution was used for injection. All mice were housed in the Institute’s
pathogen-free facility. Animal studies were approved by the Landesamt für
Gesundheit und Soziales Berlin.
Tissue Culture. Murine microglia and astrocytes were isolated from neonatal
brains. Neonatal mouse brains were dissected free of meninges and blood
vessels. Minced brains were dissociated with trypsin in HBSS and filtered with
200-μm pore size. A mixed glial culture was prepared by maintaining cells in
complete medium containing penicillin/streptomycin in poly-D-lysine–coated
flasks for 2 wk. A microglial culture was obtained from the mixed glial culture
by shaking flasks for 1 h at 200 rpm in an orbital shaker and allowing the
transferreddislodgedcellsto adhere tonew tissueculture dishes. An astrocyte
culture was obtained from a trypsinized and detached mixed glial culture by
depletion of CD11b–positive cells using MACS (130-093-634, Miltenyi) and
replating on new tissue culture dishes. Purity of the microglia and astrocyte
culture was analyzed by FACS using antibodies against CD11b and GFAP re-
spectively. Bone marrow–derived macrophages (BMMs) were collected from
the femur and tibia of mice. Bone marrow cells were plated on sterile Petri
dishes and incubated in DMEM containing 10%FCS, 5% equine serum, 10 mM
Hepes, 1 mM pyruvate, 10 mM L-glutamine, and 20% M-CSF–conditioned
medium. M-CSF–conditioned medium was collected from an L929 M-CSF cell
were harvested after 6 d. Bone marrow deficient in NALP3, ASC, and IPAF was
kindly provided by Eicke Latz (University of Massachusetts Medical School,
Worcester, MA) and Vishva Dixit (Genentech, San Francisco). ATG5−/−and
ATG5+/+mouse embryonic fibroblasts (MEF) were kindly provided by Noboru
Mizushima(Tokyo Medical andDentalUniversity, Tokyo)andgrown inDMEM
medium supplemented with 10% FCS at 37 °C in 5% CO2.
Caspase-1 Activity Assays. Cells were primed for 2 h with LPS (100 ng/mL) if
indicated. Primary microglia or macrophages were stimulated with WT or
mutant SOD1 protein. Unstimulated, ATP stimulated or Shigella flexneri
(M90T)–infected cells served as controls. MEFs were transfected with purified
SOD1 using DOTAP (Roche) according to the manufacturer’s instructions.
Caspase-1 activation was analyzed in cell lysates by immunoblot or with
a fluorescent inhibitor of active caspase-1 (FAM-YVAD-FMK, Immunochem-
istry Technologies) by FACS (exitation at 488 nm, emission at 515–545 nm).
by ELISA from BD or by immunoblot.
Immunoblot. Cells were lysed in buffer containing 1% Nonidet-P40 supple-
mented with complete protease inhibitor “mixture” (Roche). The protein
concentration was measured with BCA Protein Assay Reagent (Pierce), and
lysates were adjusted accordingly. For cell fractionation experiments, cellular
subfractions were obtained using ProteoExtract Subcellular Proteome Ex-
traction Kit (Calbiochem) according to the manufacturer’s instructions.
Lysates were boiled 5 min with SDS sample buffer under reducing con-
ditions, resolved by SDS/PAGE, and transferred to nitrocellulose membranes
by electroblotting. Samples were incubated for 4 h at 4 °C with the appro-
priate antibody and analyzed by immunoblot.
Immunohistochemistry. Tissue from 120-d-old mice was fixed by transcardiac
perfusion with 4% paraformaldehyde in phosphate buffer. Coronal cryo-
sectioning of the cervical spinal cord was performed at a thickness of 35 μm
per section. Every 12th section was stained for microglia or astrocytes using
misfolding. (A) Th-T fluorescence of the indicated SOD1 proteins. (B) IL-1β
maturation induced by the indicated SOD1 proteins in primed BMMs
determined by ELISA. (C) Correlation of IL-1β maturation and its corre-
sponding Th-T fluorescence at 490 nm. P = 0.0052; Pearson´s correlation
coefficient r = 0.8027. AU, arbituary units. Data are representative of two
SOD1-induced IL-1β maturation correlates with its amyloid-like
mice(152.2 ±1.2 d;n =25), caspase-1–deficient(162.0±1.8 d;n =24), IL-1β–deficient(159.6±1.7d;n= 21), and IL-18–deficient(152.2±1.4ds;n =24)G93A-SOD1
transgenic mice. Caspase-1– and IL-1β–deficient mice lived significantly longer than G93A-SOD1 transgenic mice (P < 0.001). (B–D) Immunohistochemical analysis
of cervical spinal cord sections of 120-d-old G93A-SOD1 transgenic mice, caspase-1–deficient G93A-SOD1 transgenic mice, and IL-1β–deficient G93A-SOD1
transgenic mice using antibodies against the microglial marker protein ionized calcium binding adaptor molecule 1 (IBA1; B), the astrocytic marker glial fibrillary
acidic protein (GFAP; C), or the motor neuron marker protein choline acetyltransferase (ChAT; D). Representative images of the ventral horn area of mice with
treated with indicated dosages of IL-1RA (75 mg/kg: 157.8 ± 1.8 d, n = 23; 150 mg/kg: 159.4 ± 1.8 d; n = 21) or placebo (153.1 ± 1.3 d, n = 19). IL-1RA–treated mice
lived significantly longer than placebo-injected mice (75 mg/kg, P = 0.0145; 150 mg/kg, P < 0.005). (F) Motor performance of IL-1RA (150 mg/kg) and placebo-
treated G93A-SOD1 transgenic mice determined by the hanging-wire test. (Scale bar, 200 μm.) *P < 0.05. n.s, Not significant. Values are mean ± SEM.
Disease progression in G93A-SOD1 transgenic mice is accelerated by IL-1β and can be delayed by IL-1RA treatment. (A) Survival of G93A-SOD1 transgenic
Meissner et al.PNAS
| July 20, 2010
| vol. 107
| no. 29
antibodies against IBA1 (1:15,000; WAKO), GFAP (1:20,000; DAKO) or ChAT
(1:1,000; Chemicon) respectively. Immunoreactivity was visualized with dia-
minobenzidine (DAB; Vectastain elite ABC kit, Vectorlabs). Anterior horn
area of every 12th section was photographed (Zeiss Axioplan microscope),
and DAB-positive area was quantified using ImageJ software (National
Institutes of Health). A minimum of eight sections per mouse were analyzed.
Each group contained at least four mice.
Immunofluorescence. For immunofluorescence, cells were seeded on cover-
slips and stimulated with WT or mutant SOD1 conjugated with ATTO-488 or
Cy-5. To follow endocytosis and subcellular distribution upon SOD1 uptake,
cells were coincubated with Alexa Fluor 647–conjugated dextran as described
elsewhere (11). At defined time points, cells were rinsed with PBS, fixed with
PFA, and permeabilized with 0.1% Triton X-100. All antibodies were diluted
in PBS + 0.5% BSA according to the manufacturer’s instructions. Coverslips
were mounted on microscope slides and analyzed using a confocal fluores-
cence microscope (TCS SP, Leica).
Flow Cytometry. At least 5,000 cells were acquired and analyzed by flow
cytometry using a FACSCalibur flow cytometer (BD Biosciences). Data were
analyzed and processed by FCS Express version 3 (De Novo Software).
Thioflavin-T Fluorescence. Measurements were taken in a LS55 fluorescence
quantity of SOD1 was incubated in 1 mL of 5 μM of Th-T in 50 mM glycine/
NaOH, pH 8.2. Excitation was at 446 nm and emission was recorded from 460
to 600 nm at 1-nm intervals. Scans were done in triplicate per sample. Ap-
propriate blank spectra were recorded on buffer components and sub-
tracted from spectra obtained on protein samples.
Expression and Purification of SOD1. Human SOD1 cDNA sequences (WT, A4V,
E21G, E21K, G37R, H43R, H46R, G85R, G93A, and E100K) were cloned into
pGEX-6P-1 Escherichia coli expression vector (GE Healthcare). After induction
by 0.5 mM isopropyl-1-thio-s-D-galactopyranoside (IPTG) for 1 h at 30 °C in E.
coli (BL21 RIL), cells were lysed and centrifuged at 10,000 × g. GST-SOD1 was
cleared from the supernatant with GSH-Sepharose (Sigma), and SOD1 was
released from Sepharose by cleavage with PreScission Protease (GE Health-
care). SOD1 was subjected to gel filtration chromatography on a Superdex
75 column (GE Healthcare) and was stored in 20 mM Hepes (pH 7.4), 50 mM
NaCl, and 1 mM DTT. EndoTrap Red endotoxin removal system (Profos) was
used in case further purification was necessary.
Statistical Analysis. Data were analyzed with the two-tailed Student’s t test.
Survival rates were analyzed by the Kaplan–Meier method and were com-
pared by the log-rank test. Correlation was analyzed using linear regression
and Pearson’s correlation coefficient.
ACKNOWLEDGMENTS. We thank Eicke Latz (University of Massachusetts),
Vishva Dixit (Genentech), and Noboru Mizushima (Tokyo Medical and Dental
University) for generously providing knockout cells; the members of the
Zychlinsky laboratory for advice and discussion, especially Juana de Diego,
Anna Brotcke, and Venizelos Papayannopoulos; and Steven Dewitz, Robert
Hurwitz, Britta Laube, Jutta Lambers, Yvonne Uhlemann, Jens Otto, and the
animal caretakers for technical assistance.
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| www.pnas.org/cgi/doi/10.1073/pnas.1002396107 Meissner et al.