Peroxiredoxin-1 is a novel danger signal involved in neurotoxic
microglial activation after experimental cardiac arrest
Mizuko Ikeda, Sarah Mader, Ines P. Koerner
Department of Anesthesiology & Perioperative Medicine, OHSU, Portland, OR
Support from American Heart Association GRNT20380839.
orally intubated
Prx1
1.0
β-actin
 cardiac arrest (CA) is induced by iv
0.8
*
0.4
0.2
temperature at 38°C
0.0
 mice are resuscitated after 10
naive 2 hours 1 day
3 days
after CA/CPR
minutes of CA (epinephrine injection
• Prx-1 expression was
normalized to β–actin;
n=4-8, *P<0.05 vs naive
Peroxiredoxin-1 is released into CSF after CA/CPR
extracted from hippocampi
 Prx-1 is detected by immunoblotting
(R&D Systems)
 cerebrospinal fluid (CSF) is
harvested from the cisterna magna
 Prx-1 is detected in CSF by ELISA
10
*
1
0.1
0.01
naive 1 day 3 days
(MyBioSource)
• While Prx-1 was barely
detectable in CSF from
naïve mice, it was rapidly
released after CA/CPR
and was easily detected
in CSF within the first
day after the insult.
n=2-5, *P<0.05
Ischemia-injured neurons release Peroxirdoxin-1
on DIV9 in the presence of absence
hours later using MTT assay
 primary microglia are harvested
from confluent mixed glial cultures
derived from postnatal (P2-4) mice
 TNF-α and IL-1β in microglial
culture medium are measured by
• Neuron-conditioned medium
(NCM) was harvested from
cultured primary neurons
injured by 90 minutes oxygenglucose deprivation (OGD).
100 kD
80 kD
60 kD
50 kD
40 kD
30 kD
20 kD
NCM conc.
 neuronal survival is measured 24
15
1.5
10
1.0
5
0.5
*
0.0
0
naive
NCM
1.0
• Prx-1 caused similar cytokine
release. n=3-6, *P<0.05 (A).
0.8
0.6
0.4
0.2
0.0
Prx1
ctrl. AB
Prx1 AB
NCM
• Precipitation of Prx1 from NCM
with Prx1 AB, but not control AB,
reduced cytokine release (B).
A
60
**
*
*
60
60
50
40
30
20
10
• Addition of untreated microglia
increased neuronal death after
OGD.
B
50
50
*
• Death increased further when
microglia where pre-treated for
24 hours with NCM (A).
40
40
30
30
20
20
• Pretreatment with Prx-1 similarly
increased microglial neurotoxicity
(B). n=6, *P<0.05
10
10
microglia -NCM
-
+
---
+
+
microglia -Prx1
-
+
---
+
+
Peroxiredoxin-1 activates microglia in vivo
E16 mice and exposed to 90
of primary mouse microglia
*
1.2
after CA/CPR
 primary neurons are cultured from
minutes oxygen-glucose deprivation
*
2.0
20
1.4
ELISA (eBioscience Ready-Set-Go)
• Proteomic analysis of NCM
identified Prx-1 as a main
protein component.
• Immunoblot confirmed
presence of Prx-1 in NCM.
 0.5 L of 5 M recombinant human
A
1 day after BSA injection
*
1 day after Prx1 injection
*
B
area fraction microglia
or 3 days after CA/CPR and protein
*
TNF-a
IL-1b
• Primary cultured microglia treated
for 24 hours with NCM released
TNF-α and IL-1β.
Peroxiredoxin-1 induces a neurotoxic microglial phenotype
and chest compressions)
 brains are harvested 2 hours, 1 day,
25
TNF-a
IL-1b
2.5
B 1.6
% neuronal death after OGD
maintained at 28°C, body
0.6
3.0
% neuronal death
 during CA, head temperature is
*
A
% neuronal
after OGD
deathdeath
% neuronal
injection of KCL and verified by ECG
*
• Prx-1 protein expression
in mouse hippocampus
increased significantly
within 2 hours of CA/CPR
and remained elevated
1 and 3 days later.
relative cytokine release
2 h expression
1 d 3 d in mouse hippocampus after CA/CPR
Prdx-1Nprotein
IL-1 [pg/100,000 cells]
are anesthetized with isoflurane and
Peroxiredoxin-1 induces pro-inflammatory cytokines
TNF- [ng/100,000 cells]
Peroxiredoxin-1 is upregulated in hippocampus after CA/CPR
NCM
While advances in cardiopulmonary
resuscitation (CPR) and critical care have
improved survival after cardiac arrest (CA) in
recent years, survivors frequently suffer
brain injury that leads to long-term cognitive
dysfunction. CA causes inflammation and
activation of microglia, the brain resident
immune cells, which precedes neuronal
death in ischemia-sensitive brain regions.
We hypothesized that injured neurons
release danger signals after CA, which
activate microglia to a neurotoxic phenotype
that exacerbates neuronal death. We tested
whether the antioxidant Peroxiredoxin (Prx)-1
acts as a danger signal after CA/CPR.
 adult male C57Bl/6 mice (20-25g)
marker
Background
Results
Prx-1/b-actin
Background: Cardiac arrest (CA) is a common manifestation of ischemic heart
disease. While advances in cardiopulmonary resuscitation (CPR) and critical
care have improved survival after cardiac arrest, survivors frequently suffer
brain injury that leads to long-term cognitive dysfunction. Cardiac arrest
causes inflammation and activation of microglia, the brain resident immune
cells, which precedes neuronal death in ischemia-sensitive brain regions [1].
We hypothesized that microglia are activated to a neurotoxic phenotype after
cardiac arrest and exacerbate neuronal death, and that a danger signal
released by injured neurons drives this activation.
Methods: In vivo: CA was induced in anesthetized and intubated male adult
C57BL/6 mice by injection of potassium chloride. CPR was begun after 10 min
of CA. Hippocampal tissue was harvested and cerebrospinal fluid (CSF)
collected 1 or 3 days after CA/CPR for quantification of antioxidant protein
Peroxiredoxin-1 (Prx1) by immunoblot (tissue) or ELISA (CSF). Recombinant
Prx1 was injected into the hippocampus of additional mice and microglial
activation assessed by immunohistochemistry using Iba1 antibody 1 day later.
In vitro: Primary cultured mouse neurons were exposed to oxygen-glucose
deprivation (OGD) to simulate ischemia, and cell death assessed 1 day later.
Neurotoxicity of primary mouse microglia was assessed by measuring
neuronal death in microglia-neuronal co-cultures after OGD. Microglial release
of cytokines was quantified by ELISA. Group differences were evaluated using
ANOVA or Student’s t-test, as appropriate. Results are mean±SEM.
Results: Prx1 protein was upregulated in mouse hippocampus within 2 hrs
after CA/CPR (Fig. 1A) and released into the CSF, where it became detectable
within a day after CA/CPR (Fig. 1B). Similarly, cultured neurons released Prx1
into the medium after OGD. This neuron-conditioned medium (NCM) induced
a pro-inflammatory phenotype in cultured microglia, characterized by release
of TNF-α (104±31 pg/105 cells vs 0.8±0.4 untreated) and IL-β (11.4±6.5 vs
0±0 untreated). Similar inflammation was induced when microglia were treated
with recombinant Prx1 (TNFα 1482±798, IL-1β 8.0±5), while depletion of Prx1
from neuron-conditioned medium by immunoprecipitation abolished the
cytokine release. Microglia activated by NCM or recombinant Prx1 significantly
increased neuronal cell death after OGD, compared to untreated microglia
(NCM 37±6% death, Prx1 34±3%, untreated 25±5%, P<0.05). Finally, injection
of recombinant Prx1 into the hippocampus caused morphologic activation of
microglia that mimicked activation after cardiac arrest.
Conclusions: We identified Prx1 as a novel danger molecule that is released
by ischemia-injured neurons and activates microglia to a pro-inflammatory and
neurotoxic phenotype, which exacerbates neuronal death. As Prx1 release
after CA/CPR precedes microglial activation, it provides a promising new
target for interventions aimed at reducing brain injury and dysfunction after
CA/CPR by blocking inflammation and microglial neurotoxicity.
Reference: [1] Wang J J Cereb Blood Flow Metab. 2013 Oct;33(10):1574-81
Methods
Prx1 conc. CSF [ng/ml]
Abstract
*
6
4
2
0
BSA
Prx1
• Injection of Prx-1 into the hippocampus (right panel) induced
widespread activation of microglia (retracted processes,
enlarged cell bodies, and increased expression of Iba1, yellow).
• Microglial activation was limited to the injection site (white
star) after injection of BSA (left panel).
Prx-1 (ProSpec) or BSA control is
injected stereotactially into the
hippocampal CA1 region
 brains are harvested 24 hours later
and microglial activation assessed
by immunohistochemistry for Iba1
Conclusion
We conclude that Prx-1 is a novel danger molecule that is released from injured neurons after ischemic stress in vitro and after
CA/CPR in vivo. We found that Prx-1 is necessary and sufficient in our model to activate microglia to a pro-inflammatory and neurotoxic phenotype in
response to neuronal injury. These novel observations improve our understanding of intercellular communication after ischemic brain injury. Future
work will determine how Prx-1 transforms microglia to the neurotoxic phenotype. Blocking the detrimental microglial signaling induced by neuronal
Prx-1 release after injury may provide a promising new target for neuroprotective intervention after CA/CPR. Support from AHA GRNT20380839.
Nashville, TN, 2015
korneri@ohsu.edu