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Cold Stress Protein RBM3 Responds to Temperature Change in an Ultra-Sensitive Manner in Young Neurons

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Extremely mild hypothermia to 36.0°C is not thought to appreciably differ clinically from 37.0°C. However, it is possible that 36.0°C stimulates highly sensitive hypothermic signaling mechanism(s) and alters biochemistry. To the best of our knowledge, no such ultra-sensitive pathway/mechanisms have been described. Here we show that cold stress protein RNA Binding motif 3 (RBM3) increases in neuron and astrocyte cultures maintained at 33°C or 36°C for 24 or 48h, compared to 37°C controls. Neurons cultured at 36°C also had increased global protein synthesis (GPS). Finally, we found that melatonin or fibroblast growth factor 21 (FGF21) augmented RBM3 upregulation in young neurons cooled to 36°C. Our results show that a 1°C reduction in temperature can induce pleiotropic biochemical changes by upregulating GPS in neurons which may be mediated by RBM3 and that this process can be pharmacologically mimicked and enhanced with melatonin or FGF21. Copyright © 2015. Published by Elsevier Ltd.
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COLD STRESS PROTEIN RBM3 RESPONDS TO TEMPERATURE
CHANGE IN AN ULTRA-SENSITIVE MANNER IN YOUNG NEURONS
T. C. JACKSON,
a,b
*
M. D. MANOLE,
a,c
S. E. KOTERMANSKI,
d
E. K. JACKSON,
d
R. S. B. CLARK
a,b
AND P. M. KOCHANEK
a,b
a
University of Pittsburgh School of Medicine, Safar Center
for Resuscitation Research, 200 Hill Building, 3434 Fifth
Avenue, Pittsburgh, PA 15260, United States
b
University of Pittsburgh School of Medicine, Department of Critical
Care Medicine, 3550 Terrace Street, Pittsburgh, PA 15261,
United States
c
University of Pittsburgh School of Medicine, Department of
Pediatrics, 4401 Penn Avenue, Pittsburgh, PA 15224, United States
d
University of Pittsburgh School of Medicine, Department
of Pharmacology and Chemical Biology, Bridgeside Point Building
1, 100 Technology Drive, Pittsburgh, PA 15219, United States
Abstract—Extremely mild hypothermia to 36.0 °C is not
thought to appreciably differ clinically from 37.0 °C.
However, it is possible that 36.0 °C stimulates highly
sensitive hypothermic signaling mechanism(s) and alters
biochemistry. To the best of our knowledge, no such ultra-
sensitive pathway/mechanisms have been described. Here
we show that cold stress protein RNA binding motif 3
(RBM3) increases in neuron and astrocyte cultures main-
tained at 33 °Cor36°C for 24 or 48 h, compared to 37 °C
controls. Neurons cultured at 36 °C also had increased glo-
bal protein synthesis (GPS). Finally, we found that mela-
tonin or fibroblast growth factor 21 (FGF21) augmented
RBM3 upregulation in young neurons cooled to 36 °C. Our
results show that a 1 °C reduction in temperature can induce
pleiotropic biochemical changes by upregulating GPS in
neurons which may be mediated by RBM3 and that this pro-
cess can be pharmacologically mimicked and enhanced
with melatonin or FGF21. Ó2015 The Authors. Published
by Elsevier Ltd. on behalf of IBRO. This is an open access
article under the CC BY-NC-ND license (http://creativecom-
mons.org/licenses/by-nc-nd/4.0/).
Key words: targeted temperature management, RBM3,
CIRBP, hypothermia, global protein synthesis, FGF21.
INTRODUCTION
Hypothermia has long been known to have protective or
detrimental effects depending on depth and duration.
For CNS injury, therapeutic hypothermia (TH) is one of
few treatments proven to reduce neurologic impairment
(Dietrich, 1992; Lyden et al., 2006; Povlishock and Wei,
2009; Wei et al., 2011). Optimal target temperature
remains a matter of debate. Each 1 °C reduction in body
temperature decreases cerebral metabolism and blood
flow by 6% (Rosomoff and Holaday, 1954; Wong,
1983). Cooling at or below 35.5 °C (but above deep
hypothermia) inversely correlates with greater neuropro-
tection (Busto et al., 1987; Minamisawa et al., 1990;
Welsh et al., 1990; Weinrauch et al., 1992). In contrast,
36 °C is not thought to activate hypothermia regulated
neuroprotective mechanisms – although it does cause
thermoregulatory vasoconstriction and shivering.
Furthermore, 36 °C has been used as normothermic con-
trol in a number of brain injury studies (Busto et al., 1987,
1989; Onesti et al., 1991). A recent multicenter clinical
trial on cardiac arrest found neurological outcomes were
almost identical in patients maintained to 36 °C versus
those given classic TH to 33 °C. Fever prevention in both
groups has been suggested to explain these surprising
outcomes. Cooling to 36 °C is not a trivial intervention to
accomplish clinically (Hostler et al., 2010), and thus it is
also possible that 36 °C activates as of yet unidentified
ultra-sensitive hypothermia regulated protective
mechanisms.
Cold shock proteins are up-regulated by cold stress.
They include RNA binding motif 3 (RBM3) and cold-
inducible RNA binding protein (CIRBP) (Danno et al.,
1997; Nishiyama et al., 1997). CIRBP is a novel
damage-associated molecular pattern (DAMP) that
induces inflammation in mice (Qiang et al., 2013).
Ischemic brain injury is reduced in CIRBP KOs (Zhou
et al., 2014). In contrast, RBM3 appears to be a potent
neuroprotective protein. RBM3 mRNA/protein is
increased in hippocampal slices by mild cooling to
33.5 °C(Tong et al., 2013). Knockdown of RBM3 reduces
hypothermia mediated neuroprotection in vitro (Chip
et al., 2011). Furthermore, exposing mice to brief periods
of deep hypothermia upregulates RBM3 in the brain and
markedly reduces neurodegenerative pathologies caused
by prion or Alzheimer’s disease (Peretti et al., 2015).
Deep cooling-induced neuroprotection is blocked by
RBM3 knockdown. Non-CNS cells are also protected by
RBM3 (Ferry et al., 2011). A key function of RBM3 is to
augment global protein synthesis (GPS), which has been
http://dx.doi.org/10.1016/j.neuroscience.2015.08.012
0306-4522/Ó2015 The Authors. Published by Elsevier Ltd. on behalf of IBRO.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
*Correspondence to: T. C. Jackson, University of Pittsburgh School
of Medicine, Department of Critical Care Medicine, Safar Center for
Resuscitation Research, 3434 Fifth Avenue, Pittsburgh, PA 15260,
United States. Tel: +1-412-383-190; fax: +1-412-624-0943.
E-mail address: jacksontc@upmc.edu (T. C. Jackson).
Abbreviations: ARA-C, cytosine b-d-arabinofuranoside hydrochloride;
CIRBP, cold inducible RNA binding protein; DIV, day in vitro; EDTA,
ethylenediaminetetraacetic acid; FGF21, fibroblast growth factor 21;
GPS, global protein synthesis; HEPES, 4-(2-hydroxyethyl)-1-piperazi
neethanesulfonic acid; RBM3, RNA binding motif 3; SUnSET, surface
sensing of translation; TH, therapeutic hypothermia; UMH, ultra-mild
hypothermia.
Neuroscience 305 (2015) 268–278
268
confirmed in neurons in vitro as well as in brain in vivo
(Dresios et al., 2005; Smart et al., 2007; Peretti et al.,
2015). It also is a pleiotropic regulator of miRNA and
mRNAs (Pilotte et al., 2011; Liu et al., 2013).
Here we tested if RBM3 and/or CIRBP are increased
in primary cortical neuron and/or astrocyte cultures
cooled to 36 °C versus conventional hypothermia to
33 °C, as compared with normothermia to 37 °C.
Surprisingly, 36 °C upregulated RBM3 in young 6d
primary neuron cultures but failed to do so in mature 26-
d-old neurons. Also, 36 °C for 48 h mildly increased
RBM3 in astrocytes. RBM3 upregulation in young
neurons cooled to 36 °C was also associated with
elevated GPS. Finally, small molecule RBM3 activators
have not been reported – such drugs could theoretically
augment neurorecovery in brain-injured patients.
Fibroblast growth factor 21 (FGF21), melatonin, and
liver X receptor (LXR) agonist T0901317 increased
RBM3 levels in young neurons cooled to 36 °C but were
ineffective in mature neurons. Our findings suggest that
even a 1 °C reduction in temperature can induce a
bonafide cold shock response. Sensitivity to cold shock-
induced RBM3 may be amplified in the developing brain
and capable of being pharmacologically manipulated.
EXPERIMENTAL PROCEDURES
Reagents
Puromycin dihydrochloride was purchased from SIGMA
(Cat# P9620-10 mL; St. Louis, MO, USA). FGF21 was
purchased from R&D Systems (Minneapolis, MN, USA).
Melatonin, T0901317, and SRT1720 were purchased
from Tocris (Bristol, UK). AZD1080 was purchased from
Cayman Chemical (Ann Arbor, MI, USA). Primary
antibodies: Anti-RBM3 was purchased from Proteintech
Group and used at 1:1000 (Cat#14363-1-AP; Chicago,
IL, USA). Anti-CIRBP, anti-a-Tubulin, anti-
pAKT(Ser473), anti-AKT total, anti-pERK, anti-ERK
total, anti-eIF2a(Ser51), anti-eIF2atotal were purchased
from Cell Signaling Technology(Danvers, MA, USA).
Anti-puromycin (12D10) was purchased from EMD
Millipore and used at 1:15,000 (Cat#MABE343; Billerica,
MA, USA). Goat anti-rabbit secondary antibodies were
purchased from Life Technologies (Grand Island, NY,
USA).
Animals
Animal studies were approved by the Institutional Animal
Care and Use Committee of the University of Pittsburgh.
Euthanasia protocols follow recommendations
established by the American Medical Veterinary
Association Guideline for Euthanasia. Female timed
pregnant Sprague–Dawley rats were purchased from
Charles River (Wilmington, MA, USA) and granted ab
libitum accesses to food and water, and maintained on
a 12-h light/dark cycle prior to euthanasia to harvest
embryos.
Primary cortical neuron culture
Embryos (mixed gender) were isolated from timed
pregnant (E16-17) Sprague–Dawley rats. Neuron
cultures were performed as previously described by our
group (Jackson et al., 2013). Cortices were dissected in
ice-cold Hanks balanced salt solution (HBSS; Life
Technologies, Grand Island, NY, USA) containing sodium
bicarbonate (SIGMA), penicillin–streptomycin (Life tech-
nologies), and 4-(2-hydroxyethyl)-1-piperazineethanesul
fonic acid (HEPES; Life Technologies). Brain tissues
were dissected under a Leica M651light microscope
(Buffalo Grove, IL, USA), and placed in a 1.5 mL tube
containing 1 mL prepared HBSS. Tissues were minced
and transferred to a 15 mL conical tube. Cells were spun
5 min/200 g/4 °C. Supernatant was aspirated, cells resus-
pended in 2 mL trypsin solution, and incubated 8 min at
37 °C with gentle mixing. Trypsin activity was quenched
in 10 mL Neurobasal/B27 (Life Technologies) containing
10% fetal bovine serum (FBS; Thermo Fisher Scientific,
Pittsburgh, PA, USA). Cells were transferred into a new
15 mL conical tube and spun 5 min/200 g/4 °C.
Supernatant was replaced with 1.5 mL trituration media.
Cells were dissociated by 10 passages through a fire-
polished glass Pasteur pipette. The cell pellet was resus-
pended in plating media warmed to 37 °C (Neurobasal
Media/B27 supplement prepared + 25 lM
L
-Glutamic
Acid + Pen-Strep). Cell number was quantified by auto-
matic counting on a Cellometer (Nexcelom Bioscience,
Lawrence, MA, USA). Cells were seeded onto 6-well
culture plates coated with poly-
D
-lysine (density 1.2–
1.5 10
6
/well). Cultures were maintained by half media
replacement every 3–4 days in vitro (DIV). The mitotic
inhibitor cytosine b-
D
-arabinofuranoside hydrochloride
(ARA-C; 4 lM final concentration) was added in study 1
experiment (e.g. DIV10–11 cultures) to ensure purity of
neuronal biochemistry for initial assessment of tempera-
ture treatments. Mitotic inhibitors at low levels are non-
toxic but still potentially represent a mild stressor (though
necessary to guarantee neuron purity) (Ahlemeyer et al.,
2003). Therefore we also confirmed results of tempera-
ture treatments in younger DIV6 neurons in the absence
of ARA-C (neuron enriched cultures; >90% neurons).
Astrocytes are allowed to proliferate in aged cultures,
which are not treated with ARA-C. We observe that
astrocytes are necessary for long-term survival of neuron
cultures (i.e. DIV26 mature neuron cultures).
Primary astrocyte culture
Pure rat astrocyte cultures were prepared as previously
reported by our group (Jackson et al., 2013). In brief,
brains were collected from postnatal day 2 SD rat pups.
Mixed brain cells were seeded onto T75 culture flasks
and maintained in DMEM/F12/10%FBS/Pen-Strep culture
media. Astrocytes were repeatedly split and propagated
in new T75 flasks until pure. At propagation #5, astrocytes
were seeded onto poly-
D
-lysine coated 6-well plates
(7.5 10
4
/well) and maintained for an additional 3d.
At 60–70% confluency in 6-well plates, astrocytes were
given fresh media exchange then temperature treatments
were initiated for 24 h and 48 h.
T. C. Jackson et al. / Neuroscience 305 (2015) 268–278 269
Hypothermia protocols
Neurons were maintained in a 37 °C (normothermic)
incubator. Before start of hypothermia, two identical
incubators of the same make/model with humidity of
95% (all in the same room) were programed to 36 °C
and 33 °C. Incubators were allowed a minimum of 48 h
equilibration time prior to starting hypothermic
experiments. In some experiments, as an additional
control, a thermometer was placed inside hypothermic
incubators as a secondary gauge of temperature.
Temperature as assessed by that approach was
independently confirmed to never be under target
temperature and fluctuate by no more than 0.5 °C. On
DIV of interest, culture plates were assigned to one of
the three experimental temperatures for 24 or 48 h.
Drug treatments: Drugs were prepared in DMSO or PBS
(i.e. for FGF21). All drugs were diluted in conditioned
culture media and applied for 24 h. Controls received
equal amounts of DMSO/PBS without drugs. Primary
neurons were harvested for biochemistry at key time
points. RBM3 and CIRBP levels were measured by
Western blot. Investigators were not blinded to
temperature treatment groups.
Analysis of de novo protein synthesis
Surface sensing of translation (SUnSET) is a recently
developed non-radioactive method using puromycin
incorporation to accurately measure rate of new protein
synthesis in cells (Schmidt et al., 2009). We adapted that
protocol with a slight modification. Briefly, at the end of
temperature treatments (on DIV6), conditioned media
were collected by removing 1 mL from each well of six-
well plate, for all plates, and set aside. Conditioned media
were pooled but separated by the temperature group to
control for potential secreted proteins during 48-h treat-
ment. Pooled conditioned media (separated by tempera-
ture group) and fresh neurobasal/B27 media (stored at
4°C) were pre-warmed to 37 °C. Remaining culture
media from six-well plates were then aspirated off and
replaced with either 1 mL of conditioned media (specific
for each temperature) or 1 mL fresh media; fresh
neurobasal/B27 media were applied in some neurons to
stimulate global protein synthesis. Neurons were incu-
bated for 1 h, media aspirated off, and replaced with
1.5 mL 37 °C warmed PBS containing 10 ng/mL puro-
mycin. Neurons were incubated for 30 min, washed
once with ice cold PBS (without puromycin), and har-
vested for biochemistry. Investigators were not blinded
to temperature treatment groups.
Western blot
Cells were quickly washed with ice cold PBS and
harvested in RIPA buffer containing EDTA, protease
inhibitors, and phosphatase inhibitors (Thermo
Scientific-PIERCE). Samples were sonicated 20 s in
0.6 mL conical tubes. Homogenized material was spun
10 min/16,000 g/4 °C, and supernatant was used for
downstream SDS–PAGE (whole cell extracts). Protein
concentration was determined using the BCA assay
(Thermo Scientific-PIERCE). 15–30 lg of protein was
mixed with Laemmli loading buffer (BioRad, Hercules,
CA, USA). Samples were heated 95 °C/5 min, and
loaded on pre-cast TGX 4–15% gradient gels (BioRad).
Gels were run at 150 V and proteins were transferred
onto polyvinylidene fluoride (PVDF) membrane (GE
Healthcare Bio-Sciences, Pittsburgh, PA, USA).
Transfers were run at 100V for 1 h 15 min. During the
course of investigations GE Healthcare discontinued our
preferred PVDF membrane Hybond-P (Cat#RPN2020F).
While testing alternative PVDFs, prior to consuming
current stock, we inadvertently discovered that RBM3 is
difficult to detect using some brands of membrane
(Fig. 1). This suggests PVDF selection may be an
important variable to study RBM3. All RBM3/CIRBP
Western blots in this report used (now discontinued)
Hybond-P membrane; we found it displays optimal
RBM3 binding/detection characteristics. However,
results suggest new Hybond P 0.2 membrane is also a
good alternative to detect RBM3 with lower non-specific
background. Precision Plus Kaleidoscope protein
standards (BioRad) were used for MW estimation.
Membranes were washed using 1X Tris-Buffered-Saline
(TBS; BioRad) and blocked in TBS-Tween-20 (TBST)
containing 7.5% blotting grade milk (TBS-T/milk) for 1 h.
Primary antibodies were prepared in TBS-T/7.5%milk
and incubated overnight in a 4 °C cooler. Blots were
washed with TBS and incubated with secondary
antibodies for 2 h. Blots were given final TBS washes,
incubated with ECL-2 HRP-detection reagent (Thermo
Fisher-PIERECE), and imaged in a dark room. Films
were scanned and compiled in Photoshop. Densitometry
analyzed by UN-SCAN-IT software (Silk Scientific,
Orem, UT, USA).
Statistical analysis
Data were analyzed and graphed using GraphPad PRISM
software (GraphPad Software Inc., La Jolla, CA, USA).
Multiple comparisons were analyzed by ANOVA and
Newman–Keuls Multiple Comparison post hoc analysis.
Data were considered significant at p< .05 using two-
tailed tests. All graphs show mean + SEM. Intrablot
group comparisons of densitometry, collected as relative
pixel intensity, were standardized for graphing by
expressing group differences as values between 0 and
1 on the yaxis. In Fig. 4D, a-tubulin normalized
densitometry values were transformed to give log(Y)
values and used for statistical analysis to correct for
non-normality in distribution.
RESULTS
Experimental hypothermic temperatures were tested in
pure neuron cultures as outlined (Fig. 2A). The
predicted molecular weight of RBM3 and CIRBP is
17 kDa and 18 kDa, respectively. Antibodies
detected RBM3 and CIRBP proteins in DIV11 neurons
cooled to 33 °C for 48 h. Western blots show a
270 T. C. Jackson et al. / Neuroscience 305 (2015) 268–278
dominant 17 kDa RBM3 band, and a fainter 18 kDa
CIRBP band (Fig. 2B, C). We next compared 24-h
exposures to either 36 °C or conventional mild
hypothermia (33 °C) to induce RBM3/CIRBP in neurons
(Fig. 2D). Neuronal RBM3 protein levels were twofold
higher after 24 h 36 °C, compared to 37 °C
normothermic controls (Fig. 2F; n= 5/group; ANOVA,
p< 0.0001). RBM3 levels were highest in the 33 °C
group at 24 h (n= 5) although statistically significant
this represented only a minor increase above 36 °C
RBM3 levels at 24 h (Fig. 2F). By 48-h RBM3 levels in
36 °C and 33 °C groups were twofold higher than
37 °C normothermic controls (Fig. 2E, G; n= 5/group;
ANOVA, p= 0.0169). RBM3 levels were not
significantly different comparing 36 °C versus
conventional 33 °C groups at 48 h (Fig. 2G). CIRBP was
barely detectable in primary rat neurons and did not
increase with cooling which is consistent with prior
studies by others (Fig. 2D, E).
We next tested if temperature treatments alter global
protein translation in young DIV6 neurons. Experiments
were performed as outlined (Fig. 3A). RBM3 levels
increased with 48-h cooling in both 36 °C and 33 °C
groups (Fig. 3B–E). In the 36 °C group, RBM3 levels
were augmented more if re-warmed for 1 h in fresh
neurobasal/B27 media compared to neurons re-warmed
in conditioned media (Fig. 3D, E). In young DIV6
neurons, RBM3 levels were highest in the 33 °C group -
regardless of 1-h treatment with fresh or conditioned
media. Fresh media exchange on cells is assumed to
stimulate protein translation by providing renewed
nutrients and growth factors but other factors such as
altered pH may also be involved. To validate that
assumption, de novo GPS was measured by SUnSET
in 37 °C treated control neurons given conditioned or 1-
h fresh media. As expected, neurons given fresh media
had higher levels of puromycin incorporation (i.e. higher
GPS). Also, anti-puromycin antibody failed to detect
signal in DIV6 neurons that were not treated with
puromycin (Fig. 3F). Next we compared if conditioned
media versus fresh media affected or unmasked,
respectively, the ability of hypothermia treatments to
alter protein translation. 36 °C increased protein
synthesis in neurons given fresh media but not to the
same extent in those given conditioned media (Fig. 3G,
H). GPS was reduced in neurons given 33 °C for 48 h.
Consistent with that observation, eIF2a(a master
regulator of global cap-dependent protein translation)
Fig. 1. Comparison of PVDF Membranes Used to Detect RBM3 by Western Blot. (A) Methods used to equalize factors for comparison of PVDF
membranes. Neuron homogenates (i.e. samples 1and 2) were loaded onto a 15-well 4–15% SDS gradient gel in five replicates. PVDF membranes
were precisely cut to span the width of two lanes and match gel length. Proteins were transferred to all five membranes at the same time (in the
same tank/cassette). Membranes were processed using the same volume/time incubation in blocking solution, TBS washes, primary antibody,
secondary antibody, and ECL detection reagent. Membranes were put inside a single film holder for equivalent film exposures in a dark room. (B)
Western blots show comparison of PVDF membranes to capture and/or detect RBM3.
T. C. Jackson et al. / Neuroscience 305 (2015) 268–278 271
was inhibited most in 33 °C cooled neurons compared to
36 °Cor37°C groups (i.e. increased Ser51
phosphorylation; Fig. 3I, J).
We next explored if 36 °C and/or 33 °C induces RBM3
in rat brain astrocytes. Astrocytes were propagated to
purity. Approximately 3d after seeding onto 6-well
plates, astrocytes were given fresh media exchange and
temperature treatments immediately initiated (Fig. 4A).
24-h exposure to hypothermia temperatures did not
appear to alter RBM3 levels compared to 37 °C controls
(Fig. 4B). RBM3 was significantly increased after 48-h
exposure to 36 °C(Fig. 4C, D). However, 33 °C induced
much higher levels of RBM3 compared to 36 °C
(Fig. 4C, D). Phosphorylation of eIF2ain astrocytes was
unaffected by temperature treatments at either time
point (Fig. 4B, C).
We next screened several agents for potential RBM3
augmenting effects in young neurons. DMSO (drug
dissolving vehicle) did not alter baseline RBM3 levels
(data not shown). In young DIV6 neurons, cooling to
36 °C for 24 h was insufficient duration to induce
RBM3 (Fig. 5A) (i.e. in contrast RBM3 was mildly
increased in DIV6 neurons by 36 °C for 48 h; Fig. 3B).
However, addition of FGF21 or melatonin (Mel-T)
during the shorter 24-h cooling period augmented
RBM3 levels at 36 °C(Fig. 5A). The LXR agonist T090
mildly increased RBM3 levels at both 37 °C and 36 °C.
Neither SRT1720 (Sirtuin antagonist) nor AZD1080
(GSK-3bantagonist) increased RBM3 levels at 37 °C
and 36 °C. Similarly, 24-h treatment with increasing
dose of FGF21, Mel-T, and T090 had minor effects on
RBM3 protein expression in DIV7 neurons cultured at
37 °C(Fig. 5B). In contrast, FGF21 increased RBM3
above baseline in a bi-phasic manner when cultured at
36 °C(Fig. 5C). As a positive control we also tested if
FGF21 activated AKT/ERK survival signaling at 37 °C
as previously reported in neurons (Leng et al., 2015).
Paradoxically, AKT/ERK phosphorylation was decreased
24 h later potentially suggesting feedback inhibition
(Fig. 5D). Combination therapy of drugs increased
Fig. 2. Mild Hypothermia Increases RBM3 in DIV10–11 Neurons. (A) Timeline of experimental procedures. (B) Western blot showing specificity of
antibodies to detect RBM3 and (C) CIRBP in neurons given 33 °C hypothermia for 48 h. (D) Western blot show increased RBM3 but not CIRBP in
primary cortical neurons treated 24 h or (E) 48 h with hypothermia. (F) Densitometry of protein changes for RBM3 (n= 5/group) at 24 h and (G) 48 h
(n= 5/group). Multiple comparisons were analyzed by a one-way-ANOVA and Newman–Keuls post hoc. Data were significant at p< .05.
*
Indicates post hoc significant difference compared to 37 °C normothermia.
#
Indicates post hoc significant difference comparing UMH to mild
hypothermia. Graphs show mean + SEM.
272 T. C. Jackson et al. / Neuroscience 305 (2015) 268–278
RBM3 levels above baseline in DIV7 neurons cooled to
36 °C. CIRBP levels were unaffected by treatments
(Fig. 5E). Finally, 48 h to 36 °C alone or with drug
combinations failed in induce RBM3 in mature DIV26
neurons. RBM3 was marginally increased in control neu-
rons cooled to 33 °C for 48 h (Fig. 5F).
Fig. 3. Mild Hypothermia Increases RBM3 in DIV6 Neurons and Increases Protein Synthesis. (A) Timeline of experimental procedures. (B) 20-ug
protein was loaded onto gels. Western blot show increased RBM3 in primary cortical neurons treated 48 h with hypothermia and subjected to 1 h re-
warm in conditioned media (C.M.) or (C) fresh neurobasal B27 supplement (NBM/B27). (D) Densitometry of RBM3 protein changes in neurons
treated with hypothermia for 48 h (n= 3/group) and then re-warmed in C.M. or (E) Fresh NBM/B27. Multiple comparisons were analyzed by a one-
way-ANOVA and Newman–Keuls post hoc. (F) Western blot shows validation of SUnSET and effect of fresh media exchange on protein translation
at 37 °C. Negative control (no puromycin; NP) are ARA-C treated DIV6 neuron homogenates. Neuron homogenates incubated with puromycin for
30 min stain positive with anti-puromycin antibody indicating level of de novo protein synthesis. (G) Level of puromycin staining in neurons treated
with hypothermia and rewarmed 1 h in C.M. or (H) NBM/B27. (I) Western blot show increased phosphorylation of eIF2ain neurons treated with
hypothermia. (J) Densitometry of phosphorylated eIF2alevels, normalized to eIF2atotal, in hypothermia treated neurons (n = 3/group). Data were
significant at p< .05.
*
Indicates post hoc significant difference compared to 37 °C normothermia.
#
Indicates post hoc significant difference
comparing UMH to mild hypothermia. Graphs show mean + SEM.
T. C. Jackson et al. / Neuroscience 305 (2015) 268–278 273
DISCUSSION
Young neurons respond to cold stress in an ultra-
sensitive manner: implications for temperature
management in developmental brain injury
To the best of our knowledge only a single study has
tested if extremely mild hypothermia (a temperature
reduction of only 2 °C) upregulates cold-stress proteins.
In that study, long-term maintenance of embryonic stem
cells to 35 °C did not increase mRNA levels of
RBM3/CIRBP (Belinsky and Antic, 2013). Here we report
in pure primary cortical neurons and astrocytes that either
ultra-mild hypothermia (UMH) to 36 °C or traditional clini-
cally used mild hypothermia levels to 33 °C increase pro-
tein levels of RBM3. Notably, potentially harmful CIRBP
did not increase in neurons.
RBM3 is developmentally regulated. It is abundant in
the postnatal brain but low in adults (Pilotte et al.,
2009). We anticipated cold stress to induce RBM3 with
greater amplitude in young neurons. Consistent with that
prediction 36 °C robustly increased RBM3 in young DIV6–
DIV11 neurons but not in mature DIV26 cultures. The
mechanism(s) regulating inhibition of RBM3 during CNS
development are unclear. Neurons in culture sponta-
neously fire excitatory potentials beginning around
DIV7–10 followed by other progressive biochemical
changes including alterations in expression of glutamater-
gic AMPA versus NMDA receptors DIV14–21 (Zona et al.,
1994; Lin et al., 2002). Neurons adopt a more stable adult
phenotype by DIV25 (Lesuisse and Martin, 2002). We
speculate that synaptic activity and other coordinated
developmental processes regulate epigenetic changes
suppressing protective RBM3 in the adult brain
(Martinowich et al., 2003; Fagiolini et al., 2009). Future
studies need to test that hypothesis.
Clinically conventional levels of mild TH have greater
efficacy in newborns than adults with hypoxic-ischemic
brain injury (Shankaran et al., 2005; Nielsen et al.,
2013). RBM3 is more easily induced by hypothermia in
developing neurons and thus may be an underappreci-
ated component of endogenous neuroprotective
responses activated by TH in the young. The American
Academy of Pediatrics recently published 2014 recom-
mendations to enhance/extend TH in community hospi-
tals for treatment of neurological injury in neonates. It
will be important to understand if RBM3 has a role in brain
recovery in that population.
Pharmacological strategies to augment RBM3
upregulation in young and mature neurons
RBM3 signaling is downregulated in the adult brain. To
the best of our knowledge only a single study
demonstrated methodology to upregulate RBM3 in the
adult brain of non-hibernating animals. In that report,
deep hypothermia to 16 °C was used in combination
with 5-AMP injection to induce a hypometabolic state
which mimics conditions in hibernating animals (Peretti
et al., 2015). Induction of RBM3 by deep cooling/5-
AMP injection is ill-advised in the setting of acute brain
Fig. 4. Mild Hypothermia Increases RBM3 in Rat Astrocytes. (A) Timeline of experimental procedures. Western blots (20 lg/well) showing eIF2a
and RBM3 changes in astrocytes given 37 °C, 36 °C, or 33 °C for hypothermia for (B) 24 h and (C) 48 h. (D) Densitometry of protein changes for
RBM3 (n= 3/group) at 48 h. Data were transformed to log(Y) for analysis. Multiple comparisons were analyzed by a one-way-ANOVA and
Newman–Keuls post hoc. Data were significant at p< .05.
*
Indicates post hoc significant difference compared to 37 °C normothermia.
#
Indicates
post hoc significant difference comparing UMH to mild hypothermia. Graphs show mean + SEM.
274 T. C. Jackson et al. / Neuroscience 305 (2015) 268–278
injury. 5-AMP is well known to chemically induce
hypothermia. Unfortunately, its obvious benefits as an
easy strategy to lower core body temperature are
eclipsed by serious side effects including profound
hypotension and hyperglycemia, which together aggra-
vate ischemic brain injury (Zhang et al., 2009).
Furthermore, it is well known that deep hypothermia
can exacerbate ischemic brain damage whereas mild
cooling is protective (Weinrauch et al., 1992). Given
those limitations, we attempted to identify drugs which
might safely augment RBM3 signaling under mild
hypothermic conditions. We tested several agents
including the hormones FGF21 and melatonin. Both
molecules cause slight body temperature reduction in
mammals (Dawson et al., 1996; Inagaki et al., 2007).
The LXR agonist T0901317 is a potential caloric restric-
tion (CR) mimetic known to upregulate RBM3 mRNA in
mouse liver as measured by microarray (Corton et al.,
2004). We also tested Sirtuin agonist SRT1720 – which
belongs to a different class of potential CR mimetic
drugs (Smith et al., 2009). Finally, we tested the GSK-
3binhibitor AZD1080 (Georgievska et al., 2013). GSK-
3binhibitors activate the developmentally essential
Wnt/b-catenin pathway (Castelo-Branco et al., 2004).
Fig. 5. FGF21 and Melatonin augment RBM3 induction in young neurons. (A) Western blots (n= 2) show effect of 24-h treatment of DIV6 neurons
with 5 nM FGF21, 100 lM melatonin, 1 lM T090, 5 lM SRT1720, or 1 lM AZD1080. (B) Western blots show dose effect of 24 h FGF21, melatonin,
or T090 at 37 °C on RBM3 levels in DIV7 neurons. (C) Western blot shows dose effect of 24 h FGF21 at 36 °C on RBM3 levels in DIV7 neurons. (D)
Western blots show the effect of FGF21 on AKT and ERK activation. (E)Western blots show effect of 24-h treatment combinations (n= 3/group) to
induce RBM3 in DIV7 neurons cooled to 36 °C. FGF21 was applied at 50 nM, melatonin 100 lM, and T090 at 1 lM. (F) Western blots show effect of
48 h treatment combinations to induce RBM3 in DIV26 neurons cooled to 36 or 33 °C. FGF21 was applied at 50 nM, melatonin 100 lM, and T090 at
1lM(n= 3/group).
T. C. Jackson et al. / Neuroscience 305 (2015) 268–278 275
FGF21 and melatonin increased hypothermia-induced
RBM3 in young neurons cooled to 36 °C but failed to do
so in mature neurons or young neurons incubated at
37 °C. T0901317 mildly induced RBM3 in young
neurons at both temperatures but failed to do so in
mature neurons. SRT1720 and AZD1080 did not affect
RBM3 levels in young neurons. Our results suggest that
FGF21 and/or melatonin merit pre-clinical evaluation
in vivo as promoters of RBM3 in the immature injured
brain. Of note, melatonin reportedly augments
hypothermia (33.5 °C)-induced neuroprotection in a
piglet model of perinatal ischemic brain injury
(Robertson et al., 2013). It remains to be determined if
RBM3 could have contributed to improved outcomes in
that study. More work is needed to investigate the mech-
anism(s) underlying RBM3 induction by those compounds
in pre-clinical models of developmental brain injury.
Global protein translation is a target for treatment of
brain injury
Though not addressed by this study, we speculate RBM3
is neuroprotective in acute brain injury such as in stroke,
cardiac arrest, or traumatic brain injury. A major function
of RBM3 is to stimulate GPS (Dresios et al., 2005; Liu
et al., 2013)(Smart et al., 2007). Loss of GPS persists
in vulnerable CA1 neurons after brain ischemia, for exam-
ple, and coincides with delayed cell death (Vosler et al.,
2012). Interestingly, TH has been shown to reverse aber-
rant GPS in injured CA1 although to the best of our knowl-
edge the underlying mechanism(s) have not been
elucidated (Yamashita et al., 1991; Widmann et al.,
1993). As RBM3 is a cold shock-induced protein logic
suggests that it is in some way involved in TH-mediated
alterations in GPS. Furthermore, increasing RBM3 in
the diseased hippocampus is thought to augment GPS
which improves synaptic plasticity/sparing in vivo
(Peretti et al., 2015). Here we show for the first time that
cooling neurons to 36 °C is sufficient to increase RBM3
and is associated with enhanced GPS.
Unexpectedly, GPS was not increased by exposure to
33 °C despite higher RBM3 levels. That observation can
be explained by overall greater inhibition of GPS in the
33 °C group. Hypothermia is well known to temporally
decrease rates of GPS but at the same time upregulate
cold stress proteins prior to rewarming. In our
experiments, before harvesting neurons, all groups
received a 1-h re-warming period to 37 °C in media
following 48 h of experimental temperature exposure.
The duration of time necessary for GPS to return to
normal after cooling in primary neurons has not been
reported to the best of our knowledge. It might be that
1-h rewarming was insufficient to allow return of normal
translational mechanisms in the 33 °C group, delaying
RBM3 from maximally augmenting GPS. In contrast
36 °C, being closer to normothermia, might not alter
baseline GPS to the same extent thus facilitate the
ability of RBM3 to augment protein translation at that
temperature. This interpretation is supported by results
on eIF2a. eIF2ais a master regulation of cap-
dependent protein synthesis. Phosphorylation at Ser51
is a potent mechanism cells use to shutdown global
protein translation (Clemens, 2001). We found eIF2a
phosphorylation was greatest in the 33 °C group.
Phosphorylation of eIF2adid not increase in
astrocytes cooled to 36 °Cor33°C for 48 h. Thus,
in vitro, astrocytes appear more resistant than neurons
to translational shutdown by eIF2ainhibition after mild
hypothermia. The cause of that resistance is unclear but
may relate to differences in biochemical mechanisms
regulating energy and metabolism; for instance access
to glycogen stores in astrocytes may alter protein
translation homeostasis. Alternatively, differences might
relate to relative cell culture conditions. Astrocytes were
maintained, and temperature treatments initiated, under
high serum conditions (i.e. 10% FBS). In contrast,
neurons were maintained, and temperature treatments
initiated, with serum free neurobasal/B27 supplement.
High growth factor support in astrocytes might make
them resistant to mild translational inhibition by
hypothermia. Nevertheless, RBM3 was potently induced
by 33 °C and mildly increased by 36 °C in astrocytes
after 48 h. Thus our work confirms 36 °C can increase
RBM3 in multiple CNS cell types. Future studies also
need to test if RBM3 is differentially induced by cooling
in type-1 versus type-2 astrocytes – the former being
the majority of cells in monolayer cultures in our studies.
Besides its effect on GPS, RBM3 has other beneficial
actions which have been noted. RBM3 knockdown blocks
mild TH-induced neuroprotection in vitro (Chip et al.,
2011). In contrast, RBM3 overexpression prevents death
in serum starved cells (Wellmann et al., 2010).
Small temperature shifts may alter disease outcomes
Tiny temperature shifts are increasingly being recognized
to influence outcomes in critically ill patients. Recent trials
suggest that targeted temperature management (TTM) to
36 °C is equally effective on neurologic outcome and
mortality in brain-injured patients compared to
conventional TH (Nielsen et al., 2013; Moler et al.,
2015). Hyperthermia (such as by fever) exacerbates neu-
ronal death after brain ischemia (Noor et al., 2003).
Clinically, it has been shown that increases in 1 °C body
temperature in the initial 72 h post hypoxic ischemic per-
iod is associated with an over threefold increase in the
odds of poor outcome in newborns (Laptook et al.,
2008). Prevention of fever is likely a key mechanism of
benefit with TTM. Nevertheless it is tempting to speculate
that temperatures representing UMH might also increase
protective cold shock proteins.
Ultra-mild hypothermia: semantics or biologically
relevant?
Ironically, 36 °C has been used as the temperature in the
control group in many studies testing traditional levels of
TH but our data suggest it may not represent a
normothermic control –at least in young (Onesti et al.,
1991; Clark et al., 1996). Contrary to concern, classic
studies by Busto et al. in adult rodents show that mild
hypothermia to 33 °Cor34°C dramatically improves his-
tological outcomes after global forebrain ischemia com-
pared to 36 °C controls (Busto et al., 1987). Our findings
276 T. C. Jackson et al. / Neuroscience 305 (2015) 268–278
do not prove RBM3 activation or 36 °C is protective.
Rather, results suggest that it may be important to recog-
nize that extremely mild levels of hypothermia may have
biological effects in some circumstances. Temperatures
slightly below normothermia may activate molecular cold
shock mechanisms in the very young – until now thought
to be only induced by traditional levels of TH. We believe
it is appropriate to consider these extremely mild temper-
ature reductions to represent UMH (i.e. 36–35.6 °C).
UMH may have important distinctions from ‘‘mild
hypothermia” which technically includes 36 °C but more
colloquially implies temperatures proven to induce neuro-
protection (35.5–33 °C). Notwithstanding the latter defini-
tion, UMH might still be neuroprotective over and above
benefits gained by preventing fever after brain injury -
depending on the myriad circumstances of the model
(such as in neonates) or clinical condition. In summary,
here we show for the first time that UMH at 36 °C induces
a bonafide cold-stress response in young neurons and
astrocytes in vitro.
AUTHOR CONTRIBUTIONS
TCJ designed experiments. TCJ and SEK performed
experiments. TCJ, MDM, EKJ, RSBC, and PMK
contributed to data analysis and interpretation. TCJ,
MDM, EKJ, RSBC, and PMK contributed to writing the
manuscript.
CONFLICT OF INTEREST/DISCLOSURE
Travis C. Jackson and Patrick M. Kochanek are co-
innovators on a submitted provisional patent application
titled ‘‘Method to Improve Neurologic Outcomes in
Temperature Managed Patients.” (Application
#62/164,205).
Acknowledgments—This work was supported by NIH/NINDS
grant 1R21NS088145 to Travis C. Jackson, and U.S. Army grant
W81XWH-10-1-0623 to Patrick M. Kochanek.
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(Accepted 5 August 2015)
(Available online 8 August 2015)
278 T. C. Jackson et al. / Neuroscience 305 (2015) 268–278
... In line with previous in vivo experimental studies [9][10][11][12], we have recently reported that the RNA-binding motif protein 3 (RBM3), a member of CSPs, mediates the good prognosis of ischemic stroke patients with mild body temperatures [13]. Pharmacological induction of RBM3 represents a potential means of neuroprotection for stroke in the absence of hypothermia [14]; however, a drug or agonist that directly targets RBM3 expression or activity has not been developed [14]. ...
... In line with previous in vivo experimental studies [9][10][11][12], we have recently reported that the RNA-binding motif protein 3 (RBM3), a member of CSPs, mediates the good prognosis of ischemic stroke patients with mild body temperatures [13]. Pharmacological induction of RBM3 represents a potential means of neuroprotection for stroke in the absence of hypothermia [14]; however, a drug or agonist that directly targets RBM3 expression or activity has not been developed [14]. ...
... Fibroblast growth factor 21 (FGF21) is an integral hormone with critical regulatory functions for glycemia, lipid profile, body weight, and normothermia, among others [15]. The recombinant form (rFGF21) of FGF21 has been proposed as a novel inductor of RBM3, via downstream signaling mechanisms in cells that express the transmembrane receptor β-klotho, which is restricted to a few sites in the body, such as liver, adipose tissue, and brain [9,14]. FGF21, just like RBM3, is sensitive to cold stress and readily crosses the bloodbrain barrier, and participates in the protective effect of hypothermia treatment [16][17][18][19]. ...
Article
Full-text available
Background: RNA-binding motif protein 3 (RBM3) is a cold-induced marker of good functional outcome of ischemic stroke that is promising as a protective target. Fibroblast growth factor 21 (FGF21) is an obesity- and temperature-related hormone that upregulates the expression of RBM3, which is beneficial as a recombinant treatment and has been tested under different experimental pathological conditions, including stroke. However, the interaction between RBM3 and FGF21 has not yet been tested for clinical stroke conditions. Methods: In a sample of 66 stroke patients, we analyzed the associations between the FGF21 and RBM3 serum concentrations on admission and at 72 h, body weight, maximum temperature during the first 24 h, and the outcome of patients at 3 months. We also analyzed their association with biomarkers of obesity (adiponectin and leptin) and inflammation (interleukin-6 (IL-6) and interleukin (IL-10)). Results: Higher concentrations of FGF21 on admission and RBM3 at 72 h were associated with good outcomes. Serum FGF21 and RBM3 were directly related to body mass index and inversely related to the maximum temperature during the first 24 h. We found a positive association between the FGF21 concentrations in obese patients with leptin and a negative correlation with adiponectin in non-obese participants. Conclusions: This clinical study demonstrates the association between RBM3 and FGF21 levels and the outcome of stroke patients. Although further investigations are required, these data support the pharmacological induction of RBM3 as a promising protective therapy.
... Elle diminue également la production de l'enzyme iNOS (oxyde nitrique synthase inductible), qui catalyse la production d'oxyde nitrique (Frink et al., 2012;Lee et al., 2016b;Schmitt et al., 2007;Seo et al., 2012;Tong et al., 2017). (Jackson et al., 2015). Ces protéines sont principalement localisées dans le noyau où elles régulent la transcription des gènes ou se lient à l'ARN messager pour la régulation posttranscriptionnelle. ...
...  Condition hypothermique RBM3 est très sensible aux variations de température, puisqu'une diminution de seulement un degré Celsius suffit à doubler son expression après 24 heures ( Figure 51, (Jackson et al., 2015)). Son expression est rapide après le choc froid, avec une apparition dès 3 heures à 32°C et une expression maximale à partir de 13 heures, maintenue jusqu'à plus de 24 heures (Figure 52, (Ushio and Eto, 2018)). ...
Thesis
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Glioblastoma is a particularly aggressive malignant brain tumor, currently treated by surgical removal followed by the combined used of radiotherapy and chemotherapy. Despite all treatments, patients have a median survival of only 16 months after diagnosis. Indeed, the tumor recurs locally in the resection margins in the months following surgery, due to the activation of residual tumor cells that were previously dormant. In this thesis, we suggest the use of therapeutic hypothermia as an adjuvant therapy to maintain residual tumor cells in a dormant state and thus prevent glioblastoma recurrence.First, we focused our work on evaluating the effects of hypothermia on four human glioblastoma cell lines (A172, U251, U87 and T98G). We demonstrated that hypothermia inhibited glioblastoma cell proliferation and migration, induced cycle arrest in the G2/M phase and altered cell morphology. In addition, we showed that its effects lasted beyond hypothermic conditioning, with a significant inhibition of proliferation despite rewarming to 37°C. We also showed that hypothermia had more consistent effects than chemotherapy, and that a combination of the two treatments further inhibited tumor cell proliferation. Then, we studied the effects of hypothermia on healthy tissue, by using human primary astrocytes and performing mouse primary neuronal cultures. We demonstrated similar effects than those observed on tumor cells, with an inhibition of astrocyte proliferation and migration, as well as good tolerance of neurons to hypothermia. Then, we performed an ex vivo proof of concept by culturing mouse embryonic brains, in order to study the effects of hypothermia on tumor growth in a complex environment. We were able to maintain the brains in culture, but the histological analyses did not show a significant therapeutic effect of hypothermia, and have to be completed by further studies. Finally, we suggested a technological solution, with the development of an implantable cooling device.Ultimately, our work showed that hypothermia keeps tumor cells dormant in a durable manner, arresting their proliferation without inducing cell death. This thesis work confirms the therapeutic interest of hypothermia in the treatment of glioblastoma, and constitutes a solid basis for future preclinical in vivo studies.
... The temperature level in the intervention group showed the strongest impact of all effect modifiers on neurologic function. Our results reflect in vitro pathophysiologic studies, reporting that neuronscan sense and have an adaptive response to even a single degree reduction in temperature from 37 to 36 C, 31 and reflect experimental cardiac arrest studies, showing a Random effects meta-regression of potential effect modifiers. SE: standard error; CI: confidence interval; TTM: targeted temperature management; aetiology: ventricular fibrillation in comparison to asphyxia; noflow: duration from cardiac arrest to resuscitation (min); lowflow: duration from start of cardiopulmonary resuscitation to return of spontaneous circulation (ROSC) (dichotomous 0 À15 min and 30À60 min); ROSC to start: duration from ROSC to start of cooling (min); cooling rate: temperature difference between intervention and control temperature divided by hours to reach target temperature (dichotomous 0.9À9.8 ...
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Aim Animal studies are an important knowledge base when information from clinical trials is missing or conflicting. The goal of this systematic review and meta-analysis was to investigate the effect of conventional targeted temperature management (TTM) between 32-36 °C in animal cardiac arrest models, and to estimate the influence of effect modifiers on the pooled effect of TTM. Data sources We searched Medline and Scopus from inception to May 2020 for randomised controlled animal trials assessing the effect of conventional TTM versus normothermia on neurologic outcome after cardiac arrest. We extracted data on study characteristics, study quality data, neurologic outcome, mortality, and potential effect modifiers. Results We retrieved 1635 studies, 45 studies comprising data of 981 animals met the inclusion criteria. Risk of bias was high in 17 studies and moderate in 28 studies. We undertook random-effects meta-analyses and meta-regression analyses to calculate the pooled effect and the influence of effect modifiers. There was a strong beneficial effect of TTM as compared to normothermia on neurologic outcome (standardised mean difference of 1.4 [95% CI -1.7 to -1.1; I² = 75%]). Faster cooling rates, lower target temperature of TTM within the range of 32-36 °C, and shorter duration of cooling were independently associated with an increasing effect size of TTM. Conclusions This systematic review of animal cardiac arrest studies showed a consistent favorable effect of postresuscitation TTM as compared to normothermia on neurologic outcome that increased with lower target temperatures.
... The downstream RNA targets of RBM3, other than RTN3, also remain unknown, as does the precise mechanism by which synapses are disassembled and regenerated also and are all questions for future studies. Possible mechanisms contributing to synapse regeneration relate to the dendritic localization of the specific RBM3 isoform that rescues synapse loss in disease (Peretti et al, 2015), which is associated with increased global protein synthesis rates (Smart et al, 2007;Jackson et al, 2015), but also, most recently has been shown to control neuronal endogenous activity through local translation at synapses (Sertel et al, 2020). Furthermore, high expression of RBM3 induces the formation of long protrusions with F-actin component in several cell types (Pilotte et al, 2018), and its reduction compromises neuronal differentiation and cell polarity and migration (Pilotte et al, 2018;Xia et al, 2018). ...
Article
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Increasing levels of the cold-shock protein, RNA-binding motif 3 (RBM3), either through cooling or by ectopic over-expression, prevents synapse and neuronal loss in mouse models of neurodegeneration. To exploit this process therapeutically requires an understanding of mechanisms controlling cold-induced RBM3 expression. Here, we show that cooling increases RBM3 through activation of TrkB via PLCγ1 and pCREB signaling. RBM3, in turn, has a hitherto unrecognized negative feedback on TrkB-induced ERK activation through induction of its specific phosphatase, DUSP6. Thus, RBM3 mediates structural plasticity through a distinct, non-canonical activation of TrkB signaling, which is abolished in RBM3-null neurons. Both genetic reduction and pharmacological antagonism of TrkB and its downstream mediators abrogate cooling-induced RBM3 induction and prevent structural plasticity, whereas TrkB inhibition similarly prevents RBM3 induction and the neuroprotective effects of cooling in prion-diseased mice. Conversely, TrkB agonism induces RBM3 without cooling, preventing synapse loss and neurodegeneration. TrkB signaling is, therefore, necessary for the induction of RBM3 and related neuroprotective effects and provides a target by which RBM3-mediated synapse-regenerative therapies in neurodegenerative disorders can be used therapeutically without the need for inducing hypothermia.
... Chemicals and Reagents mdi Membrane. We previously demonstrated that polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membranes robustly alter the fidelity of antibodies to detect cold-shock proteins (CSPs) by Western blot analysis [35]. Furthermore, we showed that the mdi brand of membrane has optimal detection properties among the currently available options, germane to the investigation of CSPs/ CSHs [3]. ...
Article
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Cold-stress hormones (CSHs) stimulate thermogenesis and have direct neuroprotective effects on the brain. The obligatory receptor components of two new CSHs (irisin and growth differentiation factor-15 [GDF15]) were recently discovered. Irisin binds integrin-αV/β5 heterodimers while GDF-15 binds to the orphan receptor glial cell-derived neurotrophic factor (GDNF) family receptor α-like (GFRAL). In addition, integrin-αV/β5 was just identified as the key receptor mediating Zika virus infection in the CNS. We measured integrin-αV, integrin-β5, and GFRAL protein levels across 78 high-quality human male/female brain tissues in infants, toddlers, preschoolers, adolescent, and adults—providing the most robust analysis to date on their levels in the human cortex and hippocampus. We report that integrin-αV was detected at all ages in the prefrontal cortex with levels greatest in adults. Integrin-αV was also detected in the hippocampus in all age groups. In contrast, integrin-β5 was detected in cortex and hippocampus largely restricted to infants. Co-expression of integrin-αV/β5 in the human infant hippocampus and cortex suggests the possibility that irisin has a more robust effect on the developing vs. the adult brain and may have implications for Zika virus infection in infants and young children.
... RNA binding motif protein 3 (RBM3) is one of the first discovered cold shock protein and belongs to RBMs' family. Based on previous studies, RBM3 is an RNAbinding protein that associates with the various types of mRNAs, microRNAs, and RBPs (RNA binding protein) through the AUrich element (ARE) in the 3 -UTR, which has a neuroprotective function in the condition of brain injury (Lunde et al., 2007;Hogan et al., 2008;Jackson et al., 2015;Zhu et al., 2016;Xia et al., 2018). Previous studies have shown that RBM3 knockdown can eliminate the neuroprotective effect induced by deep freezing, while RBM3 overexpression prevents cell death in serum starvation (Chip et al., 2011;Tong et al., 2013;Peretti et al., 2015). ...
Article
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As a sensitive cold-shock protein, RNA binding protein motif 3 (RBM3) exhibits a neuroprotective function in the condition of brain injury. However, how RBM3 is involved in acute ischemic stroke by affecting stress granules (SGs) remains unclear. Here, we established an oxygen-glucose deprivation/reperfusion (OGD/R) model in rat primary cortical neurons and PC12 cells to explore the potential mechanism between RBM3 and SG formation in acute ischemic/reperfusion (I/R) condition. The immunofluorescence results showed that the SG formation significantly decreased in rat primary cortical neurons and PC12 cells during the reperfusion period after 6 h of OGD stimulation. The western blot results, flow cytometry analysis, and cell viability assessment showed that the RBM3 expression and ratio of cell viability significantly decreased, while the rate of apoptosis increased in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Co-immunoprecipitation (Co-IP) and immunofluorescence indicated that RBM3 and GTPase-activating protein-binding protein 1 (G3BP1) colocalized cytoplasm of PC12 cells after 6 h of OGD stimulation when the SGs formation reached the highest level. Besides, overexpression and knockdown of the RBM3 were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. The results of overexpression and knockdown of RBM3 gene illustrated the pivotal role of RBM3 in affecting SG formation and apoptosis level in OGD-treated PC12 cells. In conclusion, RBM3 could combine with G3BP1 resulted in increasing stress granules generation in rat primary cortical neurons and PC12 cells after 6 h of oxygen-glucose deprivation (OGD) injury, which ultimately reduced the apoptosis in OGD-induced cells. Our study may enable a new promising target for alleviating ischemia-reperfusion injury in cells.
... Late temperature fluctuations after the end of registration are possible confounders in many experiments [75] and subsequently in this meta-analysis. There is also an ongoing discussion whether a controlled temperature of 36°C is to be regarded as a very mild form of hypothermia rather than normothermia, which if true, would change the interpretation of a majority of animal research [76]. ...
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Background: Targeted temperature management (TTM) of 32 to 34 °C has been the standard treatment for out-of-hospital cardiac arrest since clinical trials in 2002 indicated benefit on survival and neurological outcome. In 2013, a clinical trial showed no difference in outcome between TTM of 33 °C and TTM of 36 °C. In this meta-analysis, we investigate the evidence for TTM in animal models of cardiac arrest. Methods: We searched PubMed and EMBASE for adult animal studies using TTM as a treatment in different models of cardiac arrest or global brain ischemia which reported neurobehavioural outcome, brain histology or mortality. We used a random effects model to calculate estimates of efficacy and assessed risk of bias using an adapted eight-item version of the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) quality checklist. We also used a scoring system based on the recommendations of the Stroke Treatment Academic Industry Roundtable (STAIR), to assess the scope of testing in the field. Included studies which investigated a post-ischemic induction of TTM had their treatment regimens characterized with regard to depth, duration and time to treatment and scored against the modified STAIR criteria. Results: The initial and updated search generated 17809 studies after duplicate removal. One hundred eighty-one studies met the inclusion criteria, including data from 1,787, 6,495 and 2,945 animals for neurobehavioural, histological and mortality outcomes, respectively. TTM was favoured compared to control for all outcomes. TTM was beneficial using short and prolonged cooling, deep and moderate temperature reduction, and early and delayed time to treatment. Median [IQR] study quality was 4 [3 to 6]. Eighteen studies checked seven or more of the eight CAMARADES quality items. There was no clear correlation between study quality and efficacy for any outcome. STAIR analysis identified 102 studies investigating post-ischemic induction of TTM, comprising 147 different treatment regimens of TTM. Only 2 and 8 out of 147 regimens investigated comorbid and gyrencephalic animals, respectively. Conclusions: TTM is beneficial under most experimental conditions in animal models of cardiac arrest or global brain ischemia. However, research on gyrencephalic species and especially comorbid animals is uncommon and a possible translational gap. Also, low study quality suggests risk of bias within studies. Future animal research should focus on mimicking the clinical scenario and employ similar rigour in trial design to that of modern clinical trials.
Article
RNA‐binding motif protein 3 (RBM3), an outstanding cold shock protein, is rapidly upregulated to ensure homeostasis and survival in a cold environment, which is an important physiological mechanism in response to cold stress. Meanwhile, RBM3 has multiple physiological functions and participates in the regulation of various cellular physiological processes, such as antiapoptosis, circadian rhythm, cell cycle, reproduction, and tumogenesis. The structure, conservation, and tissue distribution of RBM3 in human are demonstrated in this review. Herein, the multiple physiological functions of RBM3 were summarized based on recent research advances. Meanwhile, the cytoprotective mechanism of RBM3 during stress under various adverse conditions and its regulation of transcription were discussed. In addition, the neuroprotection of RBM3 and its oncogenic role and controversy in various cancers were investigated in our review.
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The history of cardiopulmonary resuscitation and the Society of Critical Care Medicine have much in common, as many of the founders of the Society of Critical Care Medicine focused on understanding and improving outcomes from cardiac arrest. We review the history, the current, and future state of cardiopulmonary resuscitation.
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In the healthy adult brain synapses are continuously remodelled through a process of elimination and formation known as structural plasticity1. Reduction in synapse number is a consistent early feature of neurodegenerative diseases2, 3, suggesting deficient compensatory mechanisms. Although much is known about toxic processes leading to synaptic dysfunction and loss in these disorders2, 3, how synaptic regeneration is affected is unknown. In hibernating mammals, cooling induces loss of synaptic contacts, which are reformed on rewarming, a form of structural plasticity4, 5. We have found that similar changes occur in artificially cooled laboratory rodents. Cooling and hibernation also induce a number of cold-shock proteins in the brain, including the RNA binding protein, RBM3 (ref. 6). The relationship of such proteins to structural plasticity is unknown. Here we show that synapse regeneration is impaired in mouse models of neurodegenerative disease, in association with the failure to induce RBM3. In both prion-infected and 5XFAD (Alzheimer-type) mice7, the capacity to regenerate synapses after cooling declined in parallel with the loss of induction of RBM3. Enhanced expression of RBM3 in the hippocampus prevented this deficit and restored the capacity for synapse reassembly after cooling. RBM3 overexpression, achieved either by boosting endogenous levels through hypothermia before the loss of the RBM3 response or by lentiviral delivery, resulted in sustained synaptic protection in 5XFAD mice and throughout the course of prion disease, preventing behavioural deficits and neuronal loss and significantly prolonging survival. In contrast, knockdown of RBM3 exacerbated synapse loss in both models and accelerated disease and prevented the neuroprotective effects of cooling. Thus, deficient synapse regeneration, mediated at least in part by failure of the RBM3 stress response, contributes to synapse loss throughout the course of neurodegenerative disease. The data support enhancing cold-shock pathways as potential protective therapies in neurodegenerative disorders.
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Background: Unconscious survivors of out-of-hospital cardiac arrest have a high risk of death or poor neurologic function. Therapeutic hypothermia is recommended by international guidelines, but the supporting evidence is limited, and the target temperature associated with the best outcome is unknown. Our objective was to compare two target temperatures, both intended to prevent fever. Methods: In an international trial, we randomly assigned 950 unconscious adults after out-of-hospital cardiac arrest of presumed cardiac cause to targeted temperature management at either 33°C or 36°C. The primary outcome was all-cause mortality through the end of the trial. Secondary outcomes included a composite of poor neurologic function or death at 180 days, as evaluated with the Cerebral Performance Category (CPC) scale and the modified Rankin scale. Results: In total, 939 patients were included in the primary analysis. At the end of the trial, 50% of the patients in the 33°C group (235 of 473 patients) had died, as compared with 48% of the patients in the 36°C group (225 of 466 patients) (hazard ratio with a temperature of 33°C, 1.06; 95% confidence interval [CI], 0.89 to 1.28; P=0.51). At the 180-day follow-up, 54% of the patients in the 33°C group had died or had poor neurologic function according to the CPC, as compared with 52% of patients in the 36°C group (risk ratio, 1.02; 95% CI, 0.88 to 1.16; P=0.78). In the analysis using the modified Rankin scale, the comparable rate was 52% in both groups (risk ratio, 1.01; 95% CI, 0.89 to 1.14; P=0.87). The results of analyses adjusted for known prognostic factors were similar. Conclusions: In unconscious survivors of out-of-hospital cardiac arrest of presumed cardiac cause, hypothermia at a targeted temperature of 33°C did not confer a benefit as compared with a targeted temperature of 36°C. (Funded by the Swedish Heart-Lung Foundation and others; TTM ClinicalTrials.gov number, NCT01020916.).
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A systemic inflammatory response is observed in patients undergoing hemorrhagic shock and sepsis. Here we report increased levels of cold-inducible RNA-binding protein (CIRP) in the blood of individuals admitted to the surgical intensive care unit with hemorrhagic shock. In animal models of hemorrhage and sepsis, CIRP is upregulated in the heart and liver and released into the circulation. In macrophages under hypoxic stress, CIRP translocates from the nucleus to the cytosol and is released. Recombinant CIRP stimulates the release of tumor necrosis factor-α (TNF-α) and HMGB1 from macrophages and induces inflammatory responses and causes tissue injury when injected in vivo. Hemorrhage-induced TNF-α and HMGB1 release and lethality were reduced in CIRP-deficient mice. Blockade of CIRP using antisera to CIRP attenuated inflammatory cytokine release and mortality after hemorrhage and sepsis. The activity of extracellular CIRP is mediated through the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex. Surface plasmon resonance analysis indicated that CIRP binds to the TLR4-MD2 complex, as well as to TLR4 and MD2 individually. In particular, human CIRP amino acid residues 106-125 bind to MD2 with high affinity. Thus, CIRP is a damage-associated molecular pattern molecule that promotes inflammatory responses in shock and sepsis.
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Pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) inhibits protein kinase B (AKT) survival signaling in neurons. Small molecule pan-PHLPP inhibitors (selective for PHLPP1 and PHLPP2) may offer a translatable method to induce AKT neuroprotection. We tested several recently discovered PHLPP inhibitors (NSC117079 and NSC45586) in rat cortical neurons and astrocytes, and compared the biochemical response of these agents with shRNA mediated PHLPP1 Knockdown (KD). In neurons both PHLPP1 KD and experimental PHLPP inhibitors activated AKT and ameliorated staurosporine (STS) induced cell death. Unexpectedly, in astrocytes, both inhibitors blocked AKT activation, and NSC117079 reduced viability. Only PHLPP2 KD mimicked PHLPP inhibitors on astrocyte biochemistry. This suggests that these inhibitors could have possible detrimental effects on astrocytes by blocking novel PHLPP2 mediated pro-survival signaling mechanisms. Finally, because PHLPP1 levels are reportedly high in the hippocampus (a region prone to ischemic death), we characterized hippocampal changes in PHLPP and several AKT targeting pro-death phosphatases after cardiac arrest (CA) induced brain injury. PHLPP1 levels increased in rat brains subjected to CA. None of the other AKT inhibitory phosphatases increased after global ischemia (i.e. PHLPP2, PTEN, PP2A, and PP1). Selective PHLPP1 inhibition (such as by shRNA KD) activates AKT survival signaling in neurons and astrocytes. Non-specific PHLPP inhibition (by NSC117079 and NSC45586) only activates AKT in neurons. Taken together these results suggest that selective PHLPP1 inhibitors should be developed, and may yield optimal strategies to protect injured hippocampal neurons and astrocytes - namely from global brain ischemia.
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In response to low ambient temperature, mammalian cells as well as microorganisms change various physiological functions, but the molecular mechanisms underlying these adaptations are just beginning to be understood. We report here the isolation of a mouse cold-inducible RNA-binding protein (cirp) cDNA and investigation of its role in cold-stress response of mammalian cells. The cirp cDNA encoded an 18-kD protein consisting of an amino-terminal RNAbinding domain and a carboxyl-terminal glycine-rich domain and exhibited structural similarity to a class of stress-induced RNA-binding proteins found in plants. Immunofluorescence microscopy showed that CIRP was localized in the nucleoplasm of BALB/3T3 mouse fibroblasts. When the culture temperature was lowered from 37 to 32°C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37°C. By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37°C with prolongation of G1 phase of the cell cycle. These results indicate that CIRP plays an essential role in cold-induced growth suppression of mouse fibroblasts. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in mammalian cells.
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Background: Therapeutic hypothermia is recommended for comatose adults after witnessed out-of-hospital cardiac arrest, but data about this intervention in children are limited. Methods: We conducted this trial of two targeted temperature interventions at 38 children's hospitals involving children who remained unconscious after out-of-hospital cardiac arrest. Within 6 hours after the return of circulation, comatose patients who were older than 2 days and younger than 18 years of age were randomly assigned to therapeutic hypothermia (target temperature, 33.0°C) or therapeutic normothermia (target temperature, 36.8°C). The primary efficacy outcome, survival at 12 months after cardiac arrest with a Vineland Adaptive Behavior Scales, second edition (VABS-II), score of 70 or higher (on a scale from 20 to 160, with higher scores indicating better function), was evaluated among patients with a VABS-II score of at least 70 before cardiac arrest. Results: A total of 295 patients underwent randomization. Among the 260 patients with data that could be evaluated and who had a VABS-II score of at least 70 before cardiac arrest, there was no significant difference in the primary outcome between the hypothermia group and the normothermia group (20% vs. 12%; relative likelihood, 1.54; 95% confidence interval [CI], 0.86 to 2.76; P=0.14). Among all the patients with data that could be evaluated, the change in the VABS-II score from baseline to 12 months was not significantly different (P=0.13) and 1-year survival was similar (38% in the hypothermia group vs. 29% in the normothermia group; relative likelihood, 1.29; 95% CI, 0.93 to 1.79; P=0.13). The groups had similar incidences of infection and serious arrhythmias, as well as similar use of blood products and 28-day mortality. Conclusions: In comatose children who survived out-of-hospital cardiac arrest, therapeutic hypothermia, as compared with therapeutic normothermia, did not confer a significant benefit in survival with a good functional outcome at 1 year. (Funded by the National Heart, Lung, and Blood Institute and others; THAPCA-OH ClinicalTrials.gov number, NCT00878644.).
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A two-dimensional side discharge into a cross channel flow was simulated by solving the 2-D depth averaged governing equations with a modified k-ε model on a non-uniform rectangular grid. By comparisons with experimental results and the standard k-ε model, the recirculation zones calculated using the modified k-ε model generally agree well with the experimental results, while those calculated using the standard k-ε model are smaller than the experimental results. The main reason for the accurate simulation by the modified k-ε model is due to the accurate computation of eddy viscosity. The influences of the velocity distribution on the recirculation zone are also investigated in detail. The numerical results show that proposed model can be widely used in predicting flows with streamline curvature in hydraulic and environmental engineerings.
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Induced hypothermia has been reported to provide neuroprotection against traumatic brain injury. We recently developed a novel method of selective brain cooling (SBC) and demonstrated its safety and neuroprotection efficacy in a rat model of ischemic brain injury. The primary focus of the current study was to evaluate the potential neuroprotective efficacy of SBC in a rat model of penetrating ballistic-like brain injury (PBBI) with a particular focus on the acute cerebral pathophysiology, neurofunction, and cognition. SBC (34°C) was induced immediately after PBBI, and maintained for 2 hours, followed by a spontaneous re-warming. Intracranial pressure (ICP) and regional cerebral blood flow were monitored continuously for 3 hours, and the ICP was measured again at 24 hours postinjury. Brain swelling, blood-brain barrier permeability, intracerebral hemorrhage, lesion size, and neurological status were assessed at 24 hours postinjury. Cognitive abilities were evaluated in a Morris water maze task at 12-16 days postinjury. Results showed that SBC significantly attenuated PBBI-induced elevation of ICP (PBBI = 33.2 ± 10.4; PBBI + SBC = 18.8 ± 6.7 mmHg) and reduced brain swelling, blood-brain barrier leakage, intracerebral hemorrhage, and lesion volume by 40%-45% for each matrix, and significantly improved neurologic function. However, these acute neuroprotective benefits of SBC did not translate into improved cognitive performance in the Morris water maze task. These results indicate that 34°C SBC is effective in protecting against acute brain damage and related neurological dysfunction. Further studies are required to establish the optimal treatment conditions (i.e., duration of cooling and/or combined therapeutic approaches) needed to achieve significant neurocognitive benefits.
Article
Fibroblast growth factor-21 (FGF-21) is a new member of the FGF super-family and an important endogenous regulator of glucose and lipid metabolism. It has been proposed as a therapeutic target for diabetes and obesity. Its function in the central nervous system (CNS) remains unknown. Previous studies from our laboratory demonstrated that aging primary neurons are more vulnerable to glutamate-induced excitotoxicity, and that co-treatment with the mood stabilizers lithium and valproic acid (VPA) induces synergistic neuroprotective effects. This study sought to identify molecule(s) involved in these synergistic effects. We found that FGF-21 mRNA was selectively and markedly elevated by co-treatment with lithium and VPA in primary rat brain neurons. FGF-21 protein levels were also robustly increased in neuronal lysates and culture medium following lithium-VPA co-treatment. Combining glycogen synthase kinase-3 (GSK-3) inhibitors with VPA or histone deacetylase (HDAC) inhibitors with lithium synergistically increased FGF-21 mRNA levels, supporting that synergistic effects of lithium and VPA are mediated via GSK-3 and HDAC inhibition, respectively. Exogenous FGF-21 protein completely protected aging neurons from glutamate challenge. This neuroprotection was associated with enhanced Akt-1 activation and GSK-3 inhibition. Lithium-VPA co-treatment markedly prolonged lithium-induced Akt-1 activation and augmented GSK-3 inhibition. Akt-1 knockdown markedly decreased FGF-21 mRNA levels and reduced the neuroprotection induced by FGF-21 or lithium-VPA co-treatment. In addition, FGF-21 knockdown reduced lithium-VPA co-treatment-induced Akt-1 activation and neuroprotection against excitotoxicity. Together, our novel results suggest that FGF-21 is a key mediator of the effects of these mood stabilizers and a potential new therapeutic target for CNS disorders.Molecular Psychiatry advance online publication, 28 January 2014; doi:10.1038/mp.2013.192.