?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
Activated protein C therapy slows ALS-like
disease in mice by transcriptionally inhibiting
SOD1 in motor neurons and microglia cells
Zhihui Zhong,1 Hristelina Ilieva,2 Lee Hallagan,1 Robert Bell,1 Itender Singh,1 Nicole Paquette,1
Meenakshisundaram Thiyagarajan,1 Rashid Deane,1 Jose A. Fernandez,3 Steven Lane,1
Anna B. Zlokovic,1 Todd Liu,1 John H. Griffin,3 Nienwen Chow,4 Francis J. Castellino,5
Konstantin Stojanovic,1 Don W. Cleveland,2 and Berislav V. Zlokovic1
1Center for Neurodegenerative and Vascular Brain Disorders and Department of Neurological Surgery, University of Rochester Medical Center, Rochester,
New York, USA. 2Ludwig Institute for Cancer Research, Department of Medicine, and Department of Neuroscience, University of California, San Diego,
La Jolla, California, USA. 3Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.
4Socratech Research Laboratories, Rochester, New York, USA. 5WM Keck Center for Transgene Research,
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA.
Activated protein C (APC) is an endogenous plasma protease with
anticoagulant activity and direct cytoprotective activities (1). The
anticoagulant action of APC is mediated by irreversible proteolytic
inactivation of factors Va and VIIIa in plasma with contributions
of different cofactors. Independent of its anticoagulant action,
the protein C cellular pathway mediates cytoprotective altera-
tions in gene expression (2–4) and controls activation of several
transcription factors that regulate different antiapoptotic and
antiinflammatory pathways (2, 4–6). Most studies have indicated
that protease activated receptor–1 (PAR1) is a key receptor medi-
ating APC’s transmembrane signaling in different cell types (1).
APC protects neurons (7) and endothelial cells (8–10) from differ-
ent types of injury and limits brain damage in rodent models of
ischemia (6, 11–13) and multiple sclerosis (14). Whether APC can
influence a chronic neurodegenerative process like that in amyo-
trophic lateral sclerosis (ALS) is unknown.
Mutations in SOD1 are the most-studied forms of inherited
ALS (15). Neurodegeneration in SOD1 mutants is mediated via
mechanism(s) involving mutant damage within both motor neu-
rons and non-neuronal cells (15) such as microglia (16) and astro-
cytes (17), both of which develop mutant-mediated damage that
drives rapid disease progression. In addition, microvessels control
integrity of the blood–spinal cord barrier (BSCB) and are damaged
early in the disease process (18, 19), allowing leakage of potentially
neurotoxic blood components into the spinal cord (19). Using WT
recombinant APC, APC variants whose anticoagulant activity is
reduced (3K3A-APC; ref. 20) or minimal (5A-APC; refs. 21, 22),
or a mutant that has minimal anticoagulant activity but is pro-
teolytically inactive (S360A-APC), we tested whether peripherally
administered APC slows the course of motor neuron disease in
transgenic mice expressing ALS-linked mutant human superoxide
dismutase–1 (SOD1G93A; ref. 23). We found that APC with protease
activity initially crosses the BSCB via endothelial protein C recep-
tor (EPCR) and acts on motor neurons and their glial neighbors,
especially microglia, to directly inhibit disease progression by
reducing mutant SOD1 transcription.
APC treatment delivered after disease onset controls progression of ALS-like
disease. We randomly assigned 60 male mice expressing ALS-linked
mutant SOD1G93A into 5 groups receiving saline or each of 4 differ-
ent recombinant murine APC analogs: (a) WT-APC; (b) 3K3A-APC,
which contains 3 alanine substitutions for 3 protease domain resi-
dues (Lys191–193) and reduces factor Va binding and inactivation
Authorship?note: Zhihui Zhong and Hristelina Ilieva contributed equally to this
Conflict?of?interest: B.V. Zlokovic is a scientific founder of ZZ Biotech LLC, a startup
biotech company with a mission to develop new treatments for the aging brain,
stroke, and Alzheimer disease. B.V. Zlokovic and J.H. Griffin are inventors on issued
and pending patents related to APC.
Citation?for?this?article: J. Clin. Invest. 119:3437–3449 (2009). doi:10.1172/JCI38476.
Related Commentary, page 3205
3438?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
but does not affect those APC exosites recognizing PAR1 and EPCR,
resulting in greatly reduced anticoagulant activity (>70% reduction;
ref. 24) and normal cytoprotective activity (20); (c) 5A-APC, which
contains 5 alanine substitutions for 5 protease domain positively
charged residues (Arg229/230 and Lys191–193) and alters factor Va
binding exosites, but not exosites that recognize PAR1 and EPCR,
resulting in approximately 10% of the anticoagulant activity of WT-
APC but retaining normal cytoprotective activity (21, 22); and (d)
S360A-APC, an enzymatically inactive form of APC that lacks ser-
ine protease activity and the ability to activate PAR1 (5).
APC treatments or saline were delivered approximately 7 days after
disease onset, as determined by denervation-mediated muscle atro-
phy and accompanying weight loss (16, 25–27), which began at about
77 ± 5.1 days (Figure 1A). Treatments continued daily throughout
the symptomatic phase until death. WT-APC and 3K3A-APC were
given at a low dose of 40 μg/kg/d i.p. With this regimen, plasma APC
levels were elevated over a 3-hour period, with a peak that was about
2.3-fold higher than basal mouse endogenous plasma APC levels
(Figure 1B). The half-life of injected APC was 25 minutes. At the
higher dose of 100 μg/kg/d i.p., 5A-APC, inactive S360A-APC, and
WT-APC caused 5.8-fold higher plasma APC peak elevations (0.8–0.9
nmol/l) within 15–20 minutes of administration (Figure 1B).
Injection of enzymatically inactive S360A-APC at 100 μg/kg/d i.p.
did not have any significant effect on survival, lifespan, or duration
of the symptomatic phase (Figure 1, C–E), whereas enzymatically
active WT-APC and 3K3A-APC (both 40 μg/kg/d i.p.) yielded com-
parable increases in lifespan (by 10% and 13%, P < 0.05 to P < 0.01)
and disease duration (by 23% and 28%, P < 0.05) compared with
saline-treated controls (Figure 1, D and E). At this initial low dose,
the effects of 3K3A-APC on lifespan and duration of the symptom-
atic phase were not significantly different from those of WT-APC.
Treatment with enzymatically active 5A-APC at 100 μg/kg/d
i.p., administered 1 week after disease onset, increased lifespan
from 122 ± 4 to 150 ± 12 days (a 25% extension; P < 0.001) and
the symptomatic phase from 44 ± 4 to 72 ± 12 days (64% longer;
P < 0.05) compared with saline-treated mice (Figure 1, C–E). Clot-
ting time of both the ineffective SA360A-APC and the disease-
slowing 5A-APC was reduced similarly (Figure 1G), comparable
to values previously reported for human S360A-APC (28), whereas
5A-APC retained fully amidolytic activity and S360A-APC was
inactive (Figure 1F). Thus, the serine protease activity of APC, but
not its anticoagulant activity, is critical for the observed beneficial
effects in slowing disease in SOD1 mutant–mediated ALS, which
suggests that PARs are likely involved (1).
APC crosses the blood–spinal cord barrier via EPCR. APC, 3K3A-APC,
and 5A-APC cross the blood-brain barrier (BBB) in different brain
regions via EPCR-dependent receptor-mediated transport (29). To
determine whether slowing disease progression in SOD1G93A mice
correlates with 5A-APC action within the spinal cord, 125I-radio-
labeled 5A-APC (125I–5A-APC) was used to determine the con-
centrations of 5A-APC in the spinal cord interstitial fluid (ISF) in
nontransgenic and SOD1G93A mice treated with 5A-APC or saline.
125I–5A-APC levels in the lumbar ISF — determined 15 minutes after
i.p. injection of 100 μg/kg 125I–5A-APC, a time point within the ini-
tial linear phase of 125I–5A-APC accumulation in the arterial plasma
(Figure 2A) — was identical to that of unlabeled 5A-APC (Figure 1B).
After correcting for the residual vascular radioactivity (see Methods),
substantial 5A-APC accumulation (to about 3 nmol/l) was observed
in the spinal cord ISF in normal mice (Figure 2B). 5A-APC uptake
was reduced by greater than 80% in severely depleted EPCR mice
(Figure 2C), but not in PAR1-null mice (Figure 2B), consistent with
a requirement for EPCR for APC transport across the BSCB. There
was no change in entry of 99mTc-albumin into the spinal cord in
comparing EPCR-depleted and control mice (data not shown).
5A-APC accumulation in the lumbar ISF doubled in naive
SOD1G93A mice compared with nontransgenic mice (Figure 2D),
as expected from the reported BSCB disruption in SOD1G93A
mutants (18, 19). Increased 5A-APC entry was not caused by an
increase in EPCR (Figure 2, E and F), but was eliminated after
treatment of comparably aged SOD1G93A mice with 5A-APC (Fig-
ure 2D), consistent with the observed stabilization of the BSCB
permeability in response to APC therapy (see below).
APC lowers SOD1 expression in motor neurons of SOD1G93A mice. APC
modulates expression of several genes, including nuclearly encoded
mitochondrial SOD2 (2–4), and suppresses activity of transcription
factors such as NF-κB (2, 6) and p53 (4, 5). To examine whether APC
therapy affects SOD1 expression in motor neurons in vivo, mRNA
levels were determined in laser-captured spinal cord motor neurons
from treated and untreated SOD1G93A mice 4 weeks after disease
onset. Levels of transgene encoded SOD1G93A mRNA (transcribed
from the authentic human SOD1 promoter), as well as levels of
endogenous mouse SOD1 mRNA, were approximately 40% lower
in motor neurons isolated from 5A-APC–treated mice compared
with saline-treated controls (Figure 3A). Treatment with S360A-
APC did not affect SOD1 expression. Immunoblotting of extracts
of cell populations enriched in motor neurons (greater than 85%
motor neurons) from lumbar spinal cords of 5A-APC–treated and
control mice confirmed a significant 50% reduction in SOD1G93A
and endogenous mouse SOD1 protein levels (P < 0.05; Figure 3,
B and C). Treatment of differentiated N2a neuroblastoma cells
expressing an ALS-linked mutant SOD1G85R (N2a-SOD1G85R cells)
confirmed that APC can act directly on neuronal cells to selectively
downregulate SOD1G85R and endogenous mouse SOD1 mRNA
(Figure 4A) and protein levels (Figure 4, B and C) to about 40%–60%
their initial levels. Similar EC50 values were found for reduction
in SOD1 mRNA levels by WT-APC and 5A-APC (0.56 ± 0.09 and
0.51 ± 0.09 nM, respectively; Figure 4D), consistent with their com-
parable cytoprotective effects in endothelial cells (21).
Neuroprotection by APC correlates with PAR1- and PAR3-dependent
decrease in SOD1. PAR1 is a principal signaling receptor for APC in
different cell types (1, 3–7, 13), and PAR3 is apparently required
for APC action on neuronal cells (7, 30). As in non-neuronal cells
(31), pulldown experiments confirmed that PAR1 and PAR3
coexist in neuronal cells, at least in part, as heterodimers (Sup-
plemental Figure 1; supplemental material available online with
this article; doi:10.1172/JCI38476DS1). Antibody inhibition was
used to determine whether downregulation of mutant SOD1 pro-
vided by APC was mediated through any of the 4 PAR receptors,
all of which were determined by immunoblotting to be expressed
in differentiated neuroblastoma N2a cells (see Supplemental
Methods for PAR-specific blocking antibodies). Blockage of PAR1
and PAR3 activation, but not that of PAR2 and PAR4, abolished
5A-APC–mediated downregulation of SOD1 (Figure 4E), similar
to what has previously been reported in murine cortical neurons
(7, 30). WT-APC and 5A-APC protected N2a-SOD1G85R cells from
xanthine/xanthine oxidase–induced stress in a dose-dependent
manner (Figure 4G), with comparable EC50 values of 0.70 ± 0.13
nM and 0.63 ± 0.17 nM, respectively. APC-mediated protection
was again dependent on PAR1 and PAR3 levels, as it was blocked
by antibody addition (Figure 4F) and by small interfering RNA
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
APC analogs delivered after disease onset control disease progression in SOD1G93A mice. (A) Weight curves of SOD1G93A mice treated with
saline (n = 19) or 5A-APC (100 μg/kg/d i.p.; n = 10) after disease onset (84 days; red arrow) and in nontransgenic littermate controls (n = 12).
*P < 0.05, 5A-APC versus saline treatment, repeated-measures ANOVA. (B) APC arterial plasma profiles after i.p. administration of WT-APC
(40 μg/kg/d, filled circles; 100 μg/kg/d, open circles) or 5A-APC (100 μg/kg/d, triangles) determined by ELISA. n = 3. (C) Cumulative probability
of survival in SOD1G93A mice treated with saline (n = 19), S360A-APC (100 μg/kg/d i.p.; n = 10) or 5A-APC (100 μg/kg/d; n = 10). (D and E)
Lifespan (D) and duration of symptomatic phase (E) in SOD1G93A mice treated i.p. with saline (n = 19), WT-APC (40 μg/kg/d; n = 10), 3K3A-APC
(40 μg/kg/d; n = 11), or S360A-APC or 5A-APC (as in C). Differences were calculated from the survival curves by the Cox proportional hazard
method (D) and by 1-way ANOVA followed by Tukey post-hoc test (E). *P < 0.05 versus saline; #P < 0.05 versus S360A-APC. S360A-APC
was not significantly different from saline. (F and G) Amidolytic activity of (F) and clotting time for (G) WT-APC (filled circles), 3K3A-APC (filled
squares), 5A-APC (open circles), and S360A-APC (triangles).
3440? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
reduction of either receptor, to about 20% of the initial level.
Finally, although another receptor, EPCR, is required for APC
effects on endothelial cells (present study and refs. 3, 4, 20, 32,
33), EPCR cannot mediate direct effects of APC on SOD1 levels in
cortical neurons, spinal cord motor neurons, or N2a cells: immu-
noblotting and RT-PCR revealed that these cells did not express
it (Supplemental Figure 2 and Figure 2, C and E).
Neuroprotection by APC reduces hemoglobin and ROS toxicity. In
SOD1 mutant mice, microhemorrhages of red blood cells release
hemoglobin (Hb) and free iron that
potentially produce oxidant stress
within mutant spinal cords (19). Con-
sistent with this, added Hb was toxic to
N2a cells expressing either dismutase-
active SOD1G37R or dismutase-inactive
SOD1G85R mutants (Figure 4H), while
similar cells expressing SOD1WT were
resistant, as previously reported (19).
Addition of 5A-APC at concentrations
comparable to therapeutically effective
levels in SOD1G93A mice (Figure 2B)
abolished Hb-induced toxicity (Fig-
ure 4H) and lowered detectable levels
of ROS (Figure 4I). S360A-APC was
ineffective in improving cell survival
(Figure 4H) or preventing ROS gen-
eration (data not shown). Reduction
in mutant SOD1 by half (through use
of shRNA species) blocked Hb toxicity
(Figure 4, J and K), just as did the simi-
lar mutant SOD1 reduction (Figure 4,
A–C) after 5A-APC treatment (Figure
4H). Control shRNA had no effect
on SOD1G85R protein levels and did
not protect against Hb toxicity (Fig-
ure 4, J and K). 5A-APC also protected
N2a-SOD1G85R cells from excitotoxic
overstimulation of NMDA glutamate
receptors (Figure 4L), consistent with
the previously reported action of
APC in models of glutamate-induced
neuronal excitotoxicity (7, 30, 34).
The APC–PAR1/PAR3 pathway reduces nuclear levels of Sp1. APC’s
activation of PAR1 and PAR3 can suppress activity of transcription
factors, including Sp1, Egr-1/WT, NF-κB, and PPAR (35), and/or
can activate negative regulators of transcription, such as Yin-Yang 1
or MyoD (36). Because inspection of the human SOD1 transgene
construct revealed the absence of NF-κB and PPAR binding sites
(23), and MyoD is not expressed in neuronal cells (37), we tested
whether APC affects activation of Sp1, whose DNA-binding activ-
ity has previously been shown to be blocked by phosphorylation
Uptake of radiolabeled 5A-APC by the spinal cord. (A) Arterial plasma profile of 125I-5A-APC (TCA-pre-
cipitable 125I-radioactivity) after i.p. injection at 100 μg/kg. n = 3. (B) Transport of circulating 125I-5A-APC
into the lumbar cord ISF in severely depleted EPCR mice (EPCRδ/δ), PAR1–/– mice, and their matching
littermate controls (all on C57BL/6 background) after i.p. injection of 125I-5A-APC (100 μg/kg). Concen-
tration of 125I-5A-APC was calculated from TCA-precipitable 125I-radioactivity corrected for the residual
vascular radioactivity (see Methods). n = 3–5. (C) Immunoblot of EPCR in spinal cord microvessels
isolated from severely depleted EPCR mice and littermate controls. (D) Transport of circulating 125I-5A-
APC into the lumbar cord ISF in nontransgenic B6SJL controls compared with SOD1G93A mice treated
with 5A-APC (100 μg/kg/d) or saline for 4 weeks after disease onset. n = 5. (E) Immunoblot analysis of
EPCR in spinal cord microvessels isolated from B6SJL or SOD1G93A mice treated with saline or 5A-APC
(100 μg/kg/d) for 4 weeks after disease onset. β-Actin was used as a loading control. (F) Densitometry
of EPCR signal intensity from experiments in E. n = 3–5.
5A-APC transcriptionally downregulates SOD1 expression in spinal cord motor neurons in SOD1G93A mice. (A) Human SOD1G93A and murine
mSOD1 mRNA levels were determined by QPCR analysis in laser-captured motor neurons. SOD1G93A mice were treated with 5A-APC (100
μg/kg/d i.p.) or saline beginning at 84 days of age, after disease onset. Mice were sacrificed 4 weeks after disease onset. n = 5. (B) SOD1G93A
and mSOD1 protein levels, determined by immunoblot analysis of motor neuron cell lysates from SOD1G93A mice treated as in A. Enriched motor
neuron cell populations were isolated from the spinal cord as described in Methods. n = 5. (C) Scanning densitometry of SOD1G93A and mSOD1
bands in B, relative to β-actin. n = 3–5.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
5A-APC–PAR1/PAR3–mediated SOD1 suppression and neuroprotection in N2a cells. (A) SOD1G93A and mSOD1 mRNA levels, determined by
QPCR in N2a-SOD1G85R cells treated with 5A-APC (5 nM) or saline for 48 h. (B) SOD1G93A and mSOD1 protein levels, determined by immunoblot
analysis of N2a-SOD1G85R cell lysates in A. (C) Scanning densitometry of SOD1G93A and mSOD1 bands in B, relative to β-actin. (D and E)
SOD1G93A mRNA levels in N2a-SOD1G85R cells treated with 0–10 nM WT-APC or 5A-APC for 48 h (D) or 5 nM 5A-APC with and without 20 μg/ml
of cleavage site–blocking PAR1, PAR2, PAR3, and PAR4 antibodies (E). (F and G) N2a-SOD1G85R cell viability 16 h after incubation with xan-
thine/xanthine oxidase (X/XO) with and without 5 nM 5A-APC and 20 μg/ml PAR-blocking antibodies (F) and 0–10 nM WT-APC or 5A-APC (G).
(A–G) n = 5. (H) Viability of N2a-SOD1WT, N2a-SOD1G37R, and N2a-SOD1G85R cells 16 h after incubation with Hb with and without 5 nM 5A-APC
or S360A-APC. (I) Double staining for ROS and Hoechst in N2a-SOD1G85R cells 16 h after incubation with Hb with and without 5 nM 5A-APC or
S360A-APC. Scale bars: 20 μm. (J) SOD1 reduction in hSOD1 shRNA–transfected N2a-SOD1G85R cells by immunoblot analysis. (K) Viability
of N2a-SOD1G85R cells transfected with hSOD1 shRNA or control shRNA and incubated with Hb as in H. (H–K) n = 3–5. (L) N2a-SOD1G85R cell
viability 24 hours after 10 minutes of exposure to 300 μM NMDA with or without 5 nM 5A-APC. n = 5.
3442? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
after PAR1 activation (38). We used 3-color confocal microscopy to
simultaneously localize Sp1 and the neuronal microtubule–associ-
ated protein MAP2 in N2a-SOD1G85R cells, and found that almost
all of these cells normally expressed nuclear Sp1 (Figure 5A). Treat-
ment with 1 nM 5A-APC significantly reduced nuclear Sp1 by 60%
compared with untreated controls (P < 0.01; Figure 5, A and B).
Reductions in nuclear Sp1 were also observed in motor neurons
in spinal cords of SOD1G93A mice treated with 5A-APC, but not
S360A-APC or saline (data not shown). Antibody inhibition in
vitro of PAR1 (Figure 5A) or PAR3, but not PAR2 or PAR4 (Figure
5B), abolished 5A-APC suppression of nuclear Sp1. Quantitative
immunoblotting of 5A-APC–treated cells confirmed a 60% reduc-
tion in nuclear Sp1 (P < 0.05) that was blocked by antibodies to
PAR1 and PAR3, but not PAR2 and PAR4 (Figure 5, C and D).
Levels of phosphorylated Sp1 in cytoplasmic cell lysates were
elevated after 5A-APC treatment of N2a-SOD1G85R cells (Figure 5,
E and F), consistent with an earlier report suggesting that PAR1
signaling results in SP1 phosphorylation, preventing its nuclear
translocation or retention (38). Increased Sp1 phosphorylation was
inhibited both by the PI3K inhibitor LY294002 and by PAR1 and
PAR3 antibodies. Parallel immunostaining and immunoblotting
for 2 other transcription factors whose cis elements are also present
in SOD1 transgene, Egr-1 and Yin-Yang, revealed no APC-depen-
dent change in their respective nuclear levels (data not shown).
The 5A-APC–PAR1/PAR3 pathway blocks nuclear translocation of Sp1 in N2a-SOD1G85R–expressing cells. (A and B) Confocal microscopy
images (A) and signal intensity (B) of Map2 (red), Sp1 (green), and Hoechst nuclear staining (blue) in N2a-SOD1G85R cells treated with 1 nM
5A-APC for 48 hours with and without PAR1, PAR2, PAR3, and PAR4 cleavage site–blocking antibodies. Scale bar: 20 μm. n = 5. (C and D)
Immunoblot analysis (C) and densitometry relative to histone 1 (D) of Sp1 in nuclear lysates of N2a-SOD1G85R cells treated as in B. n = 3–5. (E
and F) Immunoblot analysis (E) and scanning densitometry relative to β-actin (F) of phosphorylated Sp1 in cytoplasmic lysates of N2a-SOD1G85R
cells treated as in B or with 50 μM of the PI3K inhibitor LY294002. n = 3–5.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
APC downregulates SOD1 in spinal cord microvessels and maintains
the BSCB. A significant (50%; P < 0.01) APC-dependent reduction
was seen after 4 weeks of APC treatment initiated at disease onset
in both SOD1G93A and endogenous mouse SOD1 proteins and
mRNAs, determined by immunoblotting of extracts from lumbar
spinal cord capillaries (Figure 6, A and B) and in the correspond-
ing mRNAs isolated from laser-captured microvessels (Figure 6C).
APC treatment after disease onset was also accompanied by greatly
reduced damage to the BSCB, as evidenced by blockade of serum
protein IgG leakage (Figure 6, D and E) and elimination of micro-
hemorrhages (detected by Prussian blue staining for hemosiderin;
Figure 6, F and G). Similarly, a low dose of WT-APC, but not a high
dose of S360A-APC, effectively controlled BSCB disruption (Figure
6E) and microhemorrhages (Figure 6G). Reduced accumulation
of endothelial tight junction proteins ZO-1 and occludin, which
function to maintain BSCB integrity (19), was found in SOD1G93A
APC downregulates SOD1 in spinal cord microvessels and blocks BSCB disruption in SOD1G93A mice. (A and B) Immunoblotting (A) and
densitometry (B) of human SOD1G93A and mouse mSOD1 in spinal cord microvessels isolated from SOD1G93A mice treated with 5A-APC or S360A-
APC at 100 μg/kg/d i.p. for 4 weeks after disease onset. Relative band density was normalized to β-actin. n = 5 per group. (C) SOD1G93A and
mSOD1 mRNA levels determined by QPCR in laser-captured microvessels from mice as in B. n = 5 per group. (D) Immunostaining for IgG (green)
and endothelium (CD31, red) in the lumbar spinal cords of SOD1G93A mice treated with S360A-APC or 5A-APC as in B. Scale bar: 50 μm. (E) IgG
signal intensity in the lumbar spinal cords of SOD1G93A mice treated as in B or with 40 μg/kg/d WT-APC. n = 5–8. (F) Hemosiderin deposits in the
lumbar spinal cord of SOD1G93A mice treated as in B. Scale bar: 20 μm. (G) Quantification of lumbar spinal cord hemosiderin deposits in SOD1G93A
mice from D and F. n = 5–8. (H) Relative abundance of ZO-1 and occludin, normalized to β-actin, from immunoblots of spinal cord microvessels
isolated from SOD1G93A mice treated with 5A-APC as in B or with saline. n = 3–5. (E, G, and H) B6SJL denotes littermate controls.
3444?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
capillaries (Figure 6H and ref. 19). 5A-APC treatment in SOD1G93A
mice restored normal ZO-1 and occludin expression (Figure 6H).
Selective gene excision of mutant SOD1G37R from endothelial cells does
not affect disease. Quantitative RT-PCR (QPCR) was used to con-
firm that endothelial cells synthesize high levels of SOD1: mutant
SOD1G93A mRNA levels were 40% higher in laser-captured spinal
cord capillaries than in motor neurons (Figure 7A). Combined
with presymptomatic damage to spinal cord capillaries previously
observed in SOD1 mutant mice (19), this finding suggested that
slowing of disease by APC might be caused by transcriptional
reduction of mutant SOD1 within endothelial cells. Prior efforts
using cell type–selective expression of the Cre recombinase and
a transgenic line carrying a SOD1 mutant gene excisable by Cre
action (loxSOD1G37R mice) have proven that mutant damage with-
in motor neurons (16, 17) drives disease onset, while mutant dam-
age within astrocytes (17) and microglia (16) drives rapid disease
progression. As expected, the loxSOD1G37R mice exhibited a leaky
BSCB phenotype at disease onset (Figure 7F), consistent with pre-
vious findings in other mutant SOD1 mouse lines (19).
To selectively silence mutant SOD1 expression within endothelia,
the loxSOD1G37R mice were mated to a line of mice carrying a
Ve-cadherin–promoted Cre transgene previously shown to be selec-
tively expressed in endothelial cells (39), generating Ve-Cre/SOD1G37R
mice. Gene excision within endothelia was efficient (75%; Figure 7B)
and attenuated BSCB disruption by 70%, relative to comparably aged
SOD1G37R mice, although the Ve-Cre/SOD1G37R double transgenic
mice still had about 50% greater IgG leakage than did normal mice
(Figure 7F). Unlike slowed disease and extended survival from APC
treatment, however, highly diminished mutant synthesis within
endothelia did not affect the timing of disease onset (Figure 7C), pro-
gression to early disease (Figure 7D), survival (Figure 7E), or disease
duration (Table 1). Thus, although the timing of BSCB disruption
and how different degrees of BSCB disruption affect disease in SOD1
mutants remain to be established, our present results demonstrate
that mutant SOD1 synthesized by the capillary endothelia is not an
important contributor to pathogenesis and that the benefit in slow-
ing disease progression and extending survival by APC therapy can-
not be attributed to mutant SOD1 downregulation in endothelia.
APC downregulates mutant SOD1 in microglia and delays an inflam-
matory response. Because SOD1 mutant expression within microglia
drives rapid disease progression (16, 40), we tested whether APC
treatment after disease onset affected the levels of SOD1 in
microglia or microglial activation. Accompanying 5A-APC treat-
ment was reduction by about 40% in microglial SOD1G93A expres-
sion 7 weeks after disease onset (Figure 8A). Administration of
low-dose WT-APC or of 5A-APC delayed microglia activation and
Disease onset, early disease, end stage, and duration in SOD1G37R
and Ve-Cre/SOD1G37R mice
Age at onset (d)
Age at early disease (d)
Age at end stage (d)
Disease duration (d)
240 ± 37
313 ± 44
377 ± 29
137 ± 15
240 ± 39
301 ± 47
368 ± 21
128 ± 21
Data are mean ± SD.
Selective reduction of mutant SOD1 expression within
endothelial cells by Cre-mediated gene excision does not
affect ALS-like disease onset, progression, or survival.
(A) Relative mRNA abundance of SOD1G93A in laser-
captured microvessels and motor neurons in SOD1G93A
mice. n = 5. (B) Excision of the SOD1G37R transgene in
microvessels purified from SOD1G37R mice or Ve-Cre/
SOD1G37R mice promoted Cre recombinase transgene.
DNA copy number for the mutant SOD1 gene was
normalized to mouse apoB gene. From 2–3 mice per
extraction, 3 or 4 preparations of vessel-extracted DNA
were analyzed. Excision frequency was corrected for the
known 4:1 ratio of endothelial cells/pericytes. Data are
mean ± SD. n = 3–4. (C–E) Age of disease onset (C),
progression to an early phenotypic stage (D), and surviv-
al (E) in SOD1G37R and Ve-Cre/SOD1G37R mice. (F) IgG
signal intensity in the lumbar spinal cords in SOD1G37R
mice (n = 4) and Ve-Cre/SOD1G37R mice (n = 4)
at disease onset at 7 months of age compared with age-
matched nontransgenic littermate controls (n = 3).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
proliferation 4 weeks after onset (Figure 8, B–D), while spinal cord
levels of microglia in saline-treated SOD1G93A mice showed increases
of 12- and 20-fold compared with nontransgenic littermate con-
trols at 4 and 7 weeks after onset, respectively (Figure 8, C and D).
In contrast to the highly elevated levels in saline-treated SOD1G93A
mice, as reported previously (19), 5A-APC treatment effectively
reduced the expression of inflammatory markers, including mono-
cyte chemoattractant protein 1 (MCP-1, also known as Ccl-2; ref. 40)
and Icam-1, a neuroinflammatory marker of activated endothelium
(Figure 8, E and F, and ref. 19). 5A-APC treatment also reduced the
expression of hemoxygenase-1 in the spinal cord (Supplemen-
tal Figure 3). Moreover, treatment with both WT-APC (data not
shown) and 5A-APC (Figure 8, G and H), but not with S360A-APC,
substantially preserved innervation of the neuromuscular junctions
4 weeks after onset, an age at which untreated and saline-treated
animals had developed significant muscle weakness.
APC-EPCR interactions at the spinal cord endothelium. EPCRδ/δ hypo-
morphs exhibited severely depleted EPCR levels in the spinal cord
endothelium (Figure 2C) and were therefore unable to transport
WT-APC, 3K3A-APC, or 5A-APC from the circulation across the BBB
(29) and into the spinal cord (Figure 2B). Indeed, in EPCRδ/δ hypo-
morphs, systemic administration of 5A-APC did not reduce SOD1
in motor neurons or spinal cord endothelium in vivo, although, as
above, it lowered SOD1 by half in EPCR+/+ animals (Figure 9, A–F).
RCR-252, an anti-human EPCR antibody directed to an APC
binding site on EPCR (41), has been reported to block murine
APC downregulates SOD1 in microglia and controls neuroinflammatory response in SOD1G93A mice. (A) QPCR analysis of SOD1G93A and mSOD1
mRNA levels in laser-captured microglia in SOD1G93A mice treated with 5A-APC or S360A-APC at 100 μg/kg/d for 7 weeks after disease onset.
n = 5. (B) Immunostaining of activated microglia (CD11b, red) in SOD1G93A mice treated with saline, 40 μg/kg/d WT-APC, or 100 μg/kg/d 5A-APC
for 4 weeks after disease onset. Nuclei were stained with DAPI (white). Scale bar: 20 μm. (C) Microglia numbers, in SOD1G93A mice treated as in
B, relative to control B6SJL mice (arbitrarily taken as 1). n = 5–6 per group. (D) CD11b-positive microglia numbers in SOD1G93A mice treated with
100 μg/kg/d 5A-APC or S360A-APC for 7 weeks. n = 5 per group. (E and F) QPCR analysis of mRNA transcripts for Ccl-2 (E) and Icam-1 (F) in
the lumbar spinal cords of mice treated with saline or 100 μg/kg/d 5A-APC for 4 weeks. n = 3–5 per group. (G) Double immunostaining of skeletal
muscle sections with α-bungarotoxin and VAChT, to label endplates and axon terminals, respectively, in SOD1G93A mice treated with 100 μg/kg/d
5A-APC or S360A-APC for 4 weeks after disease onset. Denervated endplates were labeled only with bungarotoxin (green; left). Innervated end-
plates were labeled with both bungarotoxin and VAChT (right). Scale bar: 25 μm. (H) Percentage of innervated plates in G. n = 4 per group.
3446?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
EPCR activity in vivo, thereby inhibiting APC transport across
the BBB (29). We determined that this antibody also recognized
murine EPCR in spinal cord endothelium (Supplemental Figure 2).
Systemic administration of this antibody, but not nonimmune
IgG used at a comparable concentration, blocked 5A-APC–medi-
ated mutant SOD1 downregulation in SOD1G93A mice at both the
mRNA and the protein levels in motor neurons (Figure 9, G–I) and
capillary endothelia (Figure 9, J–L).
We have determined that treatment of mutant SOD1–expressing
mice with enzymatically active APC analogs delivered after disease
onset retarded progression of ALS-like disease, increased lifespan,
and, more importantly, increased duration of the symptomatic
phase. The enzymatic activity of APC, but not its anticoagulant
activity, was critical for the beneficial effects. A primary mechanism
for APC’s action is through passage across the BSCB in an EPCR-
5A-APC–mediated SOD1 downregulation in motor neurons and spinal cord endothelium requires endothelial EPCR. (A–C) mSOD1 mRNA lev-
els, determined by QPCR (A), and immunoblotting (B) and densitometry analysis (C) of mSOD1 protein, in laser-captured motor neurons from
EPCRδ/δ and EPCR+/+ mice treated with saline or 100 μg/kg/d 5A-APC i.p. for 7 days. (D–F) mSOD1 mRNA levels (D), and immunoblotting (E)
and densitometry (F) analysis of mSOD1 protein levels, in spinal cord microvessels isolated from EPCRδ/δ and EPCR+/+ mice treated as in A–C.
(A–F) n = 3–4 per group. (G–I) SOD1G93A and mSOD1 mRNA levels (G), and immunoblotting (H) and densitometry (I) analysis of SOD1G93A and
mSOD1 protein levels, in laser-captured spinal cord motor neurons of SOD1G93A mice treated with saline or 100 μg/kg/d 5A-APC i.p. for 7 days in
the absence or presence of an EPCR blocking antibody (RCR-252) or nonimmune IgG infused through the femoral vein (40 μg/mouse) at day 1
and 3. (J–L) SOD1G93A and mSOD1 mRNA levels (J), and immunoblotting (K) and densitometry (L) analysis of SOD1G93A and mSOD1 protein
levels, in spinal cord microvessels isolated from SOD1G93A mice treated as in G–I. (G–L) n = 3–5.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
dependent manner, followed by transcriptional downregulation of
mutant SOD1 in motor neurons and their non-neuronal neigh-
bors, including microglia and cells comprising microvessels.
BSCB disruption (18, 19), swelling (18, 19), and ischemic changes
(42) are consistent features in spinal cord pathology of ALS mice
(18, 19). Ischemia worsens motor neuron degeneration and func-
tional outcome, as shown in mice with a mutation that eliminates
hypoxia-responsive induction of the vascular endothelial growth
factor A gene (42), which develop late-onset motor neuron degen-
eration (43). Endothelial cells of the microvasculature, along with
pericytes and astrocytes, were essential to stabilize the BSCB, but
we have also shown that APC’s slowing of disease was not medi-
ated by reduction of mutant SOD1 within the endothelial cells,
the first cells to encounter plasma-delivered APC. Indeed, mutant
SOD1 damage directly within the endothelial cells was shown by
selective gene excision to play little, if any, role in disease patho-
genesis. Activation of microglia and astrocytes and the accompa-
nying inflammatory response, on the other hand, play a major role
in progressive BSCB opening in SOD1 mutants after disease onset
(18, 19). Thus, we conclude that BSCB stabilization by APCs given
postsymptomatically — as in the present study — will critically
depend on mutant SOD1 reduction in microglia and astrocytes
after APC transport across the capillary wall. Additional transgenic
mouse models will be needed to resolve whether the well-described
protective effects of APC on endothelium (8, 9, 44) can improve
capillary integrity in SOD1 mutants independent of the observed
SOD1 blockade in nonendothelial cells and/or endothelia.
A convergence of evidence has led to a consensus that SOD1
mutations cause disease by acquisition of 1 or more toxic proper-
ties, rather than by loss of dismutase activity, including mutant
damage to mitochondria, damage from aberrant mutant SOD1
secretion (45), endoplasmic reticulum stress from blockage of
ejection of misfolded proteins from it (46), and hyperactivation of
extracellular superoxide production by microglia (47), as reviewed
recently (48). We would emphasize that whatever the most rel-
evant toxicities, APC-mediated mutant SOD1 downregulation
within motor neurons and microglia represents a therapeutic
approach that is directly linked to disease mechanism. APC-medi-
ated diminution of mutant SOD1 synthesis in motor neurons may
contribute to the delay in disease initiation, whereas — perhaps
more importantly — lowered SOD1 mutant levels within astro-
cytes, microglia, or peripheral macrophages are highly likely to be
responsible for slowed disease progression. This conclusion would
be in accordance with prior findings that selective mutant SOD1
gene excision from astrocytes (17), from microglia and peripheral
macrophage lineages (16, 49), or by bone marrow replacement of
mutant myeloid cells with normal ones (50) can strikingly slow
disease progression despite no effect on disease onset.
The only other SOD1 gene–silencing approach previously prov-
en to slow disease progression is antisense DNA oligonucleotide
infusion (51), but these oligonucleotides are not BBB or BSCB
permeant, requiring delivery by direct CNS infusion following
invasive surgery. Methods of gene silencing through retroviral
delivery of transcription-mediated shRNAs have been shown to
dramatically slow disease onset, but only when administered to
very young SOD1G93A animals. Even with delivery at a juvenile
age, this approach was of no benefit in slowing the rate of disease
progression (52). Modest survival benefits from slowing disease
onset, but without benefit in slowing progression, have also been
seen with viral delivery prior to disease onset by direct injection
into the spinal cord to very focally silence SOD1 (53). Thus, the
ability of APC to slow disease progression, combined with simple
peripheral administration, represent unique therapeutic advan-
tages compared with other SOD1-silencing strategies. Moreover,
APC is already approved for use in adult patients. Indications
include severe sepsis (54), and a clinical trial is currently underway
to assess the benefit of APC in acute ischemic stroke (http://www.
clinicaltrials.gov/ct2/show/NCT00533546). What is clear from
our efforts and the prior ones (5, 6, 13, 14, 55) is that transient
exposure, without continuous infusion, of APC can produce long-
lasting neuroprotective effects. With the recognition that accumu-
lation of aberrant SOD1 species has been linked to most cases of
sporadic ALS (56), strategies based on activation of the protein C
cellular pathway are promising directions for treating patients
with familial, and possibly sporadic, ALS.
Mouse recombinant APC variants. WT-APC, S360A-APC, 3K3A-APC, and
5A-APC were prepared as described previously (5, 21).
APC treatment of SOD1G93A mice. Studies were performed in male SOD1G93A
mice (The Jackson Laboratory) according to NIH guidelines, using a pro-
tocol approved by the University of Rochester. Mice were injected i.p. daily
with saline or APC analogs after disease onset and continued throughout
the symptomatic stage until death. Disease onset was determined from the
weight curves (16, 25–27). Mice were randomized into 5 groups receiving
saline (n = 19), WT-APC (40 μg/kg/d; n = 10), 3K3A-APC (40 μg/kg/d; n = 11),
S360A-APC (100 μg/kg/d; n = 10), and 5A-APC (100 μg/kg/d; n = 10). Mor-
tality was defined when the mouse could not right itself within 30 seconds.
APC arterial plasma profiles. Mice (n = 3 per group) were anesthetized i.p.
with 100 mg/kg ketamine and 10 mg/kg xylazine. WT-APC (40 or 100 μg/
kg) and 5A-APC (100 μg/kg) were administered i.p., and blood samples
were collected from the femoral artery.
Amyloidotic assay, coagulation assay, and APC ELISA. Assays were performed
as reported previously (30, 57). See Supplemental Methods.
125I–5A-APC uptake into the lumbar spinal cord. Studies were performed
according to the NIH guidelines using a protocol approved by the University
of Rochester. 5A-APC was radioiodinated as reported previously (29). Male
C57BL/6 mice, severely depleted EPCR mice (58), PAR1-null mice, SOD1G93A
mice, and nontransgenic littermate controls weighing about 23–24 g were
anesthetized i.p. with ketamine (100 mg/kg) and xylazine (10 mg/kg) and
injected with 125I–5A-APC (MW, 56 kDa; 100 μg/kg i.p. or 1.25 μCi per
mouse) simultaneously with 99mTc-albumin (MW, 67 kDa; 10 μCi, Cardinal
Health), a reference vascular space marker (59). Plasma samples were collected
from the femoral artery. Mice were sacrificed, and the lumbar region was
carefully dissected and prepared for counting. See Supplemental Methods
for details regarding iodination, counting, and calculations (19, 59).
Treatment of EPCRδ/δ hypomorphs with 5A-APC. Severely depleted male
EPCR mice (58) and their littermate controls (2–3 months old) were
treated with 100 μg/kg/d 5A-APC i.p. or saline for 7 days. Animals were
sacrificed at day 7. SOD1 mRNA levels were determined in laser-captured
motor neurons and microvessels by QPCR analysis. SOD1 protein levels
were analyzed by immunoblotting of isolated motor neurons and spinal
cord microvessels lysates (see below). Blockade of EPCR in SOD1G93A mice
is described in Supplemental Methods.
Laser capture microdissection. The lumbar (L2–L5) OCT-embedded 10-μm
tick spinal cord sections were mounted on RNase-free PALM membrane
slides (Carl Zeiss Microimaging). Motor neurons were stained using cresyl
violet (26). Microvessels were stained with a rabbit polyclonal anti-mouse
laminin antibody (diluted 1:500; Sigma-Aldrich). HRP-conjugated goat
anti-rabbit antibody was used as a secondary antibody (diluted 1:200;
3448? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 11 November 2009
Dako North America). Microglia were stained with a rat monoclonal anti-
mouse CD11b (diluted 1:100; BD Biosciences — Pharmingen). Contami-
nation-free laser capture microdissection was performed on dry, stained
sections at ×400 magnification with a Zeiss Axiovert 200 inverted micro-
scope equipped with PALM LCM system including a 337-nm laser and a
robotic microscope table operated by PalmRobo software. Motor neurons,
microvessels, and microglia were captured from 10–12 randomly chosen
sections of the ventral horns of SOD1G93A mice treated with saline, S360A-
APC, 5A-APC, or WT-APC (n = 5 per group).
Motor neuron–enriched cell suspension. Motor neurons were isolated as
reported previously (60). See Supplemental Methods.
Isolation of microvessels by dextran density centrifugation gradient. The spi-
nal cord microvessels were isolated as described previously (19). See
N2a cultures. N2a-SOD1WT, N2a-SOD1G37R, and N2a-SOD1G85R cells were
cultured as described previously (61).
Oxidant stress. Cells were challenged with 10 μM mouse Hb (19) or with
xanthine/xanthine oxidase (100 μM to 10 mU/ml; refs. 42, 61) in the pres-
ence and absence of 5 nM 5A-APC or S360A-APC. Cell viability was deter-
mined with a water-soluble tetrazolium-8 assay kit (CCK-8 kit; Dojindo
NMDA-induced apoptosis in N2a-SOD1G85R cells. This was performed as
described previously (7, 30). See Supplemental Methods.
Inhibition of PARs. Antibodies against PARs were from Santa Cruz Bio-
technology Inc. The following cleavage site–blocking PAR antibodies were
used, each highly specific for blocking its respective PAR: polyclonal rabbit
against human PAR1 (H-111), monoclonal mouse against human PAR2
(SAM-11), polyclonal rabbit against human PAR3 (H-103), and polyclonal
goat against mouse PAR4 (S-20). See Supplemental Methods.
Silencing through RNA interference. See Supplemental Methods.
Immunostaining and immunoblot analysis for Sp1. See Supplemental Methods.
Immunoblot analysis. Microvessels, motor neurons, and N2a cell lysates were
prepared in the Cell Lysis Buffer (Cell Signaling Technology). We analyzed
20–40 μg lysate proteins using 10% SDS-PAGE. Reactivity was detected using
an enhanced chemiluminescence detection system (Amersham). The density
of bands was quantified by scanning densitometry relative to β-actin signal
(Alpha Imager; Alpha Innotech). For each studied protein, the signal was
within the linear range. See Supplemental Methods for antibodies.
Real-time QPCR. Total RNA was isolated from laser-captured motor neu-
rons, microvessels and microglia, and N2a cells using RNeasy Mini kit
(Qiagen Inc.) and reverse transcripted to cDNA using the iScript cDNA
Synthesis kit (Bio-Rad Laboratories). The cDNA products of the RT reac-
tion were stored at –80°C or used immediately for QPCR. QPCR, using
iQ SYBR Green Supermix (Bio-Rad Laboratories) as the fluorescent DNA
intercalating agent, was analyzed using a IQ4 multicolor detection QPCR
system (Bio-Rad Laboratories). The relative abundance of target mRNA
was normalized to β-actin. See Supplemental Methods for primers.
Endothelial-specific mutant SOD1G37R deletion. The deletion of mutant
SOD1G37R gene from endothelial cells was accomplished using Ve-cad-
herin-Cre mice (39) with selective expression of the Cre recombinase in
endothelial cells and a transgenic line carrying SOD1G37R mutant (16).
This approach has been successfully used to delete SOD1 mutant gene in
microglia (16), motor neurons (16, 17), and astrocytes (17). The gene exci-
sion efficiency was determined by QPCR of SOD1G37R DNA in microves-
sels from brain and spinal cord (see below). See Supplemental Methods for
measurements of BSCB disruption.
DNA extraction from microvessels in SOD1G37R mice and QPCR. Vessels from
brain and spinal cords were isolated as described above. DNA was extracted as
described previously (62). For QPCR, 30 ng DNA was amplified as described
previously (63). SOD1 and apoB primer/probe sets were run in the same reac-
tion, with every reaction run in triplicates and all samples run in parallel. The
entire experiment was repeated twice, and results were averaged.
Histology and immunohistochemistry in SOD1G93A mice. Mice were anesthetized
i.p. with 100 mg/kg ketamine and 10 mg/kg xylazine and perfused trans-
cardially with heparinized PBS. Lumbar spinal cords (L2–L5 region) were
embedded in OCT medium (Sakura) and cut in the coronal plane at 14 μm.
Serum protein leakage. Sections were fixed in acetone for 5 minutes,
blocked by 5% swine serum for 1 hour at room temperature, and incubated
with fluorescein-conjugated affinity-purified goat anti-mouse IgG (fluo-
rescein-labeled primary antibody, diluted 1:200; Jackson ImmunoResearch
Laboratories) and rat monoclonal anti-mouse CD31 (endothelial marker,
diluted 1:100; BD Biosciences — Pharmingen). Alexa Fluor 594 donkey
anti-rat IgG (diluted 1:200; Invitrogen) was used as a secondary antibody
to CD31. Images were taken using a Zeiss 510 meta confocal microscope.
In each mouse, 12 nonadjacent sections (greater than 250 μm apart in the
L2–L5 region) were examined. The signal intensity of mouse IgG extravas-
cular deposits was analyzed using NIH Image J. Sections of all control and
experimental mice were processed in parallel.
Prussian blue staining. Staining was performed as described previously
(19). See Supplemental Methods.
Immunofluorescent staining of microglia. We used rat monoclonal anti-
mouse CD11b (diluted 1:100; BD Biosciences — Pharmingen), and nuclei
were stained with DAPI. Secondary antibody was Alexa Fluor 594 donkey
anti-rat IgG (diluted 1:200; Invitrogen). All sections from control and
experimental animals were processed in parallel.
Statistics. We used the Cox proportional hazards model (64), with the
treatment group as the unique covariate, to study the effect of treatment
on the survival distributions and lifespan of mice from different treatment
groups. Symptomatic phase duration was calculated in days as the differ-
ence between lifespan and disease onset. We used 1-way ANOVA followed by
Tukey post-hoc test to calculate differences in symptomatic phase as well as
to compare the treatment effects of ordinal data between groups. S-plus 7.0
was used for statistical calculations. All data are mean ± SEM unless other-
wise indicated. A P value less than 0.05 was considered significant.
This work was supported by NIH grants HL63290 and HL81528
(to B.V. Zlokovic), HL73750 (to F.J. Castellino), HL52246 (to J.H.
Griffin), and NS27036 (to D.W. Cleveland), as well as by Socrat-
ech LLC. D.W. Cleveland receives salary support from the Ludwig
Institute for Cancer Research. H. I receives salary support from a
Career Development Award from the Muscular Dystrophy Associ-
ation. The authors thank Xin Tu for help with statistical analysis.
Received for publication January 5, 2009, and accepted in revised
form September 9, 2009.
Address correspondence to: Berislav V. Zlokovic, Arthur Korn-
berg Medical Research Building, University of Rochester Medi-
cal Center, 601 Elmwood Avenue, Box 645, Rochester, New York
14642, USA. Phone: (585) 273-3132; Fax: (585) 273-3133; E-mail:
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