ArticlePDF Available

RBM3 mediates structural plasticity and protective effects of cooling in neurodegeneration

Authors:

Abstract and Figures

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.
Lentivirally mediated overexpression of RBM3 restores structural synaptic plasticity and is neuroprotective in neurodegenerative disease. a, LV-RBM3 produces high levels of expression in hippocampus; LV-shRNA-RBM3 reduces endogenous levels. Representative western blot and bar graph, where n = 6 mice per treatment, repeated in triplicate. b, c, LV-RBM3 rescues the deficit in structural plasticity in prion-infected and 5XFAD mice at 6 w.p.i. and 3 months of age, respectively (93 images from 3 mice per time point) and produced lasting protection of synapse number in prion-infected mice at 7 and 9 w.p.i. (b), whereas LV-shRNA-RBM3 accelerated synapse loss (c) (62 images from 2 mice per time point). d, e, LV-RBM3 resulted in significant recovery in synaptic transmission (d) (left panel, inset: representative raw traces of evoked EPSCs are shown; n = 6 cells from 2 mice per time point) and protected against behavioural decline (e), whereas RNAi of RBM3 accelerated memory loss and burrowing deficits (e) (n ≥ 10 mice per group). f–h, RBM3 overexpression increases global protein synthesis rates in hippocampal slices of prion-infected mice at 9 w.p.i. (f) (n = 4–6 mice), is neuroprotective (g) (n = 3–6 mice) and significantly lengthened survival (h) (n = 20 mice), whereas knockdown accelerated disease (n = 14 mice) compared to LV-control treated mice (n = 31 mice), Mann–Whitney U-test. P < 0.05, P <0.01, P < 0.001. Student’s t-test, two tails except in e, f and g which used one-way ANOVA, Brown–Forsythe test with Tukey’s post hoc analysis for multiple comparisons. All data represent means ± s.e.m. Scale bar, 1 μm in b and c and 50 μm in g.
… 
Content may be subject to copyright.
LETTER doi:10.1038/nature14142
RBM3 mediates structural plasticity and protective
effects of cooling in neurodegeneration
Diego Peretti
1
, Amandine Bastide
1
, Helois Radford
1
, Nicholas Verity
1
, Colin Molloy
1
, Maria Guerra Martin
1
, Julie A. Moreno
1
,
Joern R. Steinert
1
, Tim Smith
1
, David Dinsdale
1
, Anne E. Willis
1
& Giovanna R. Mallucci
1,2
In the healthy adult brain synapses are continuously remodelled
through a process of elimination and formation known as struc-
tural plasticity
1
. Reduction in synapse number is a consistent early
feature of neurodegenerative diseases
2,3
, suggesting deficient com-
pensatory mechanisms. Although much is known about toxic pro-
cesses leading to synaptic dysfunction and loss in these disorders
2,3
,
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 plasticity
4,5
. 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 rela-
tionship 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) mice
7
,
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 restoredthe capacity
for synapse reassembly after cooling. RBM3overexpression, 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 partby 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 neuro-
degenerative disorders.
We used the phenomenon of physiological structural plasticity seen
in hibernating mammals to determine the capacity for synapse regen-
eration inmouse models of neurodegenerativedisease. When they enter
torpor, the neurons of hibernators undergo morphological changes in-
cluding changes in spine morphology
8,9
and/or changes in connectivity
4,5
.
These are rapidly reversedon regaining normal bodytemperature
4,5,8–10
.
We first established that the phenomenon of synapse dismantling and
reassembly (structural plasticity) on artificial cooling and rewarming
occurs in laboratory mice (Fig. 1a and Extended Data Fig. 1a). We then
explored the capacity forstructural plasticityafter cooling in two mouse
models of neurodegenerative disease: prion disease and the 5XFAD model
of Alzheimer’s disease
7
. We used tg37
1/2
mice
11
infected with Rocky
Mountain Laboratory (RML) prions used in our previous studies
12–14
.
These mice show substantial synapse loss from 7 weeks post-inoculation
1
Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
2
Department of Clinical Neurosciences, Clifford Allbutt Building, Cambridge
Biomedical Campus, University of Cambridge, Cambridge CB2 0AH, UK.
a
45 6
0
2
4
6
8***
b5XFAD
2 months
3 months
4 w.p.i.
6 w.p.i.
Prion disease
2
0
4
8
***
3
Time (months)
NS
*** 12
NS
c
Cooled Rewarmed
37°C 16-18°C 37°C
Number of synapses
per 55 μm2 area
Number of synapses
per 55 μm2 area
Control
Wild type
Number of synspses per 55 μm2 area
0
2
4
6
8***
***
2D
Number of synapses in
stratum radiatum of CA1 area (×109)
3D
0
20
40
60
80
100
**
**
Number of synapses per 100 µm3
0
0.25
0.5
0.75
1.0
** **
Time (w.p.i.)
Figure 1
|
The capacity for synaptic regeneration is lost early in
neurodegenerative disease. a, Synapse numbers decline on cooling and
recover on rewarming in wild-type mice, counted in both 3D and 2D.
Representative electron micrographs (pseudo-coloured for ease of synapse
identification: yellow, presynaptic; green, postsynaptic compartments) and bar
charts showing quantification are shown for each experiment (n54 animals at
18 uC and n52at37uC; 192 images from 2 mice per conditionfor 3D analyses;
93 images from 3 animals per condition, for 2D analyses). A typical tripartite
synapse is shown at higher magnification. b,c, The same response is seen in
prion-diseased mice (b) at 4 and 5 w.p.i. but this fails at 6 w.p.i (arrow), and in
5XFAD (c) mice, where itfails at 3 months (arrow). ***P,0.0001, **P,0.01;
NS, not significant. Student’s t-test; two tailed. All data in bar charts are
mean 6s.e.m. Scale bar, 1mm. Source Data for all figures can be found in the
Supplementary Tables.
00 MONTH 2015 | VOL 000 | NATURE | 1
Macmillan Publishers Limited. All rights reserved
©2015
(w.p.i.)
13
; 5XFAD mice have synapse loss from 4months, after which
time learning deficits emerge
7
. We tested the capacity for structural
plasticity using coolingearly in the course of disease, before the onset of
established synapse loss in both models: from 4 w.p.i. in prion-infected
animals and from 2 months of age in 5XFAD mice.
All mice were cooled to 16–18 uC for 45 min, similar to core tempera-
tures reached in small hibernators (deep hypothermia) using the bio-
molecule 59-adenosine monophosphate (59-AMP)
15
, after which they
were allowed to slowly rewarm. Animals were euthanized at each stage
of the cooling–rewarming process and synapses were counted in the
CA1 region of hippocampus. Both synapse density and total synapse
number significantly declined on cooling, but recovered on rewarming
in wild-typemice, as measured using boththree dimensional (3D)
16
and
two dimensional (2D) analyses
13
(Fig.1a).Neitherbrainvolumenorsyn-
apse size changed on cooling and rewarming, excluding the possibility
that changes in synapse density reflected changes in these parameters
(Extended Data Fig. 1a). Thus, wild-type mice showed synaptic struc-
tural plasticity with reduction in synapse number on cooling and recov-
ery on rewarming (Fig. 1a). This capacity for plasticity was also seen in
both prion-infected and 5XFADmice very early in the course of disease,
at 4 and 5 w.p.i., and at 2 months of age, respectively (Fig. 1b, c). How-
ever, this capacity was lost by 6 w.p.i. in prion-diseased mice (Fig. 1b
and Extended Data Fig. 1b) and at 3 months in 5XFADmice (Fig. 1c and
Extended Data Fig. 1c). Notably, impaired structural plasticity shortly
preceded established decline in synapse number seen in prion-infected
tg37
1/2
mice at 7 w.p.i. (ref.14), and in the 5XFAD mice from 4 months
of age (seeschematic,Extended DataFig. 1d). The lostability to reassem-
ble synapses was not due to loss of synaptic proteins at this stage (Ex-
tended Data Fig. 2) nor to increased levels of disease-specific misfolded
bPrion disease
46
Time (w.p.i.)
0
40
80
120
160
200
RBM3/GAPDH
(% relative to control mean)
**
37°C
Cooled
aWild type
0
50
100
150
200
250
RBM3/GAPDH
(% relative to control mean)
*
5XFAD
GAPDH
RBM3
months
32
35
15
23
RBM3/GAPDH
(% relative to control mean)
**
c
0
100
200
300
400
500 *
*
b
w.p.i.64
(kDa)
35
15
a
35
15
(kDa)
GAPDH
RBM3
(kDa)
35
15
Time (months)
c
Figure 2
|
Failure to induce RBM3 parallels lost capacity for synaptic
recovery in neurodegenerative disease models. a, Cooling induces increased
RBM3 levels in hippocampi of wild-typemice. b,c, The response fails at 6 w.p.i.
in prion-infected mice (b) and at 3 months in 5XFAD mice (c) (arrows).
Representative western blots are shown. Bar graphs show quantification of
RBM3 levels relative to GAPDH, (n56–11 mice per time point; all
experiments in triplicate) **P,0.01, *P,0.05, Mann–Whitney U-test in
aand c, Student’s t-test in b. All data are mean6s.e.m.
***
mEPSC frequency (s
–1
)
0
1
2
3
4
5
C7 8 9
b
d
a
*** *** ***
0
2
4
6
8
Number of synapses per 55 μm
2
area
6879
Time (w.p.i.)
Time (w.p.i.)Time (w.p.i.)Time (w.p.i.)
Time (w.p.i.)
Time (w.p.i.)
C
8 w.p.i. 9 w.p.i.
7 w.p.i. 7 w.p.i.
Prion infection + early cooling
Prion infection only
Uninfected controls
6
Cooling ×2
8 95 w.p.i.
4
3
RBM3/GAPDH
(% relative to control mean)
687 9C
100
200
300
400
500
10 12
***
**
*
0
*NS NS
***
*
Control
9 w.p.i
9 w.p.i
8 w.p.i
7 w.p.i
0
C7 8 9
0.5
1.0
1.5
2.0
2.5
Evoked EPSC amplitude (nA)
NS
c
f
Control
Prion sickUninfected
CA1
iii
e
Early coolingNo cooling
iii
0
20
40
60
80
100
Survival (%)
Prion
+
early
cooling
Prion
only
Prion
+
LV-shRNA-RBM3
+
early cooling
060 80 100 120
Time post-inoculation (da
y
s)
***
**
***
678910
0
20
40
60
80
100
C
Burrowed (%)
0
0.5
1.0
1.5
2.0
2.5
89
C
*
Novel object preference
*
Early cooling +
LV-shRNA-RBM3
iv
0
1
2
3
Total number of CA1 neurons (×10
5
)
**
**
1nA
50 ms
Figure 3
|
Early cooling induces RBM3 overexpression and is
neuroprotectivein prion-infected mice. a,b, Cooling at 3 and 4 w.p.i. resulted
in sustained high levels of RBM3 in hippocampus for several weeks (n53–8
mice per time point) causing marked protection (b) of synapse number at 7, 8
and 9 w.p.i. (62 images from 2 mice per time point). Scale bar, 1 mm. c,d, Early
cooling maintained synaptic transmission (n54–8 cells from 2 mice per time
point; representative raw traces of evoked EPSCs are shown) and prevented
decline in burrowing behaviour and loss of novel object recognition memory
(d), expressed as ratio of exploratory preference (n$10 mice per group) in
contrast to un-cooled mice. e, Haematoxylin and eosin stained sections show
striking reduction in hippocampal spongiosis and protection of CA1 neurons
(bar chart) in cooled mice that is abolished by RBM3 knockdown (n54–6
animals per treatment, except LV-shRNA-RBM3: n52). Scale bar, 50 mm.
One-way ANOVA, Brown–Forsythe test with Tukey’s post hoc analysis for
multiple comparisons was used in dand e.f, Early cooling significantly
prolonged survival, but this was abolished by knockdown of RBM3 (n531
cooled mice; n517 not cooled; n510 cooled 1RNAi of RBM3). Mann–
Whitney U-test. *P,0.05; **P,0.01; ***P,0.001. Two-tailed Student’s
t-test was used unless otherwise stated. All data in bar charts are mean 6s.e.m.
RESEARCH LETTER
2 | NATURE | VOL 000 | 00 MONTH 2015
Macmillan Publishers Limited. All rights reserved
©2015
prion protein(PrP
Sc
) in prion-infected mice, or of amyloid-boligomers
in 5XFAD mice induced by the cooling–rewarming process (Extended
Data Fig. 3).
In hibernation and hypothermia, global protein synthesis and cell
metabolism are downregulated, but low temperature also induces a small
subset of proteins known as cold-shock proteins that escape translational
repression
17,18
. Amongst these, RNA-binding motif protein 3 (RBM3)
and cold-inducible RNA binding protein (CIRP, also known as CIRBP)
are cold-shock proteins expressed at high levels in brain
6,19
. We found
strong induction of RBM3 by coolingin brains of wild-type mice and in
mice with priondisease at 4 w.p.i. and 5XFAD mice at 2 months (Fig. 2).
CIRP was not upregulated (Extended Data Fig. 4). However, both prion
and 5XFAD mice lost the capacity to upregulate RBM3 after cooling at
6 w.p.i. (Fig. 2b) and at 3 months of age (Fig. 2c), respectively, in parallel
with the lost ability to reassemble synapses after cooling at these time
points (Fig. 1b, c). Therefore, we asked if inductionof RBM3 expression
drives synaptic recovery.
Therapeutic hypothermia is a powerful neuroprotectant in brain injury
acting through multiple mechanisms, including enhanced gene express-
ion driving regenerative processes enhancing synapse formation (see
ref. 17 for a review). RBM3 has been implicated in protection against
cell death in various in vitro modelsof cooling and neuroprotection
20,21
,
albeit in conditions of mild hypothermia (,32 uC). It is known to in-
crease local protein synthesis at dendrites
19
and global protein synthesis
through ribosomal subunit binding and/or microRNA biogenesis
22
.The
neuroprotective effects of hypothermia on neurodegenerative disease
are unknown, however. Given that the capacity for structural plasticity
correlated with induction of RBM3, we asked if raising endogenous RBM3
levels through early therapeutic cooling would restore failed synaptic plas-
ticity. In wild-type mice, a single episode of cooling to 16–18 uC raised
RBM3 levels in brain for up to 3days (see Fig. 2a and Extended Data
Fig. 4c), suggesting the response is sustained for some time after the cold
stress. Animals were cooled twice: at 3 w.p.i. and again at 4 w.p.i., resulting
in a sustained several-fold increase in RBM3 expression up to 6 weeks
later, declining to baselinelevels at 12 w.p.i., at the terminal stage of dis-
ease (Fig. 3a and Extended Data Fig. 5). Control mice were infected with
prions but were not cooled. Early cooling and associated increased RBM3
expression protected against synapse loss in prion disease at 7, 8 and
9 w.p.i. (Fig.3b), restored synaptic transmission (Fig. 3c) and prevented
behavioural deficits, maintaining burrowing behaviours and novel object
recognition memory (Fig. 3d and Extended Data Fig. 6a). There was
also marked neuronal protectionin the hippocampus (Fig. 3e, compare
subpanels ii and iii), even in mice succumbing to prion infection, which
is ultimately overwhelming due to other toxic effects
13
. Most remark-
ably, early cooling significantly increased survival in prion-infected
mice (91 67 days in cooled mice vs 84 64 days for uncooled mice;
P50.0002). Indeed, one animal survived 117 days post-infection,
nearly a 50% increase in life expectancy (Fig. 3f). Mice cooled later
in prion disease, at 5 and 6 w.p.i., when the RBM3 induction response
is lost (see Fig. 2b), did not show increased survival (Extended Data
Fig. 7). As predicted, RBM3 knockdown by lentivirally mediated RNA
interference (RNAi) in the hippocampus abolished the protective effects
of early cooling on CA1 pyramidal neurons and spongiform change
(Fig. 3e, subpanel iv), on object recognition memory (Extended Data
Fig. 6b, c), and on survival (Fig. 3f). As before, misfolded PrP levels
were not affected by cooling (Extended Data Fig. 8). In therapeutic human
hypothermia, temperatures of ,34 uC are used, similar to those of hibern-
ating large mammals such as bears, which are known to induce similar
transcriptional changes in RBM3 (ref. 6). Therefore, these physiological
changes in small rodents at 16–18 uC may well be relevant in therapeutic
gh
LV-RBM3
LV-shRNA-RBM3
Time post-inoculation (days)
Survival (%)
075 80 85 90 95 100 105
0
20
40
60
80
100
***
***
9 w.p.i.
9 w.p.i +
LV-RBM3
0
0.5
1.0
1.5
2.0
2.5
79
C0
1
2
3
4
5
79C
mEPSC frequency (s–1)
Evoked EPSC amplitude (nA)
*
*
d
***
Number of synapses
per 55 μm2 area
976C
0
2
4
6
8***
***
c
LV-RBM3
7 w.p.i.
9 w.p.i.
LV-control
LV-control LV-shRNA-RBM3
.i.p.w 6
a
***
*
RBM3/GAPDH
(% relative to control mean)
0
100
200
300
400
b
LV-RBM3
LV-shRNA-RBM3
No virus
LV-control
Uninfected control
RBM3
GAPDH
LV-Control LV-RBM3 LV-shRNA-RBM3No virus
(kDa)
35
15
0
2
4
6
8
Number of synapses per 55 μm2 area
in cooled and rewarmed mice
Prion 5XFAD
***
***
3 months
6 w.p.i.
LV-control LV-RBM3
CA1
LV-control LV-RBM3Control
Prion sick
Uninfected
LV-shRNA-RBM3
ef
0
0.5
1.0
1.5
2.0
2.5
**
C897
Novel object preference
678910C
0
20
40
60
80
100 *
Global translation 35S-Met
incorporation (%)
0
50
100
150
*
Burrowed (%)
*
99977
Time (w.p.i.)Time (w.p.i.)
0
1
2
3
T
otal number of CA1 neurons (×105)
** ** *
i ii iii iv
Time (w.p.i.)
Time (w.p.i.)Time (w.p.i.)Time (w.p.i.)
Figure 4
|
Lentivirally mediated overexpression
of RBM3 restores structural synaptic plasticity
and is neuroprotective in neurodegenerative
disease. a, LV-RBM3 produces high levels of
expression in hippocampus; LV-shRNA-RBM3
reduces endogenous levels. Representative western
blot and bar graph, where n56 mice per
treatment, repeated in triplicate. b,c, LV-RBM3
rescues the deficit in structural plasticity in
prion-infected and 5XFAD mice at 6 w.p.i. and
3 months of age, respectively (93 images from 3
mice per time point) and produced lasting
protection of synapse number in prion-infected
mice at 7 and 9 w.p.i. (b), whereas LV-shRNA-
RBM3 accelerated synapse loss (c) (62 images from
2 mice per time point). d,e, LV-RBM3 resulted in
significant recovery in synaptic transmission
(d) (left panel, inset: representative raw traces of
evoked EPSCs are shown; n56 cells from 2 mice
per time point) and protected against behavioural
decline (e), whereas RNAi of RBM3 accelerated
memory loss and burrowing deficits (e)(n$10
mice per group). fh, RBM3 overexpression
increases global protein synthesis rates in
hippocampal slices of prion-infected mice at
9 w.p.i. (f)(n54–6 mice), is neuroprotective
(g)(n53–6 mice) and significantly lengthened
survival (h)(n520 mice), whereas knockdown
accelerated disease (n514 mice) compared to
LV-control treated mice (n531 mice),
Mann–Whitney U-test. *P,0.05, **P,0.01,
***P,0.001. Student’s t-test, two tails except in
e,fand gwhich used one-way ANOVA, Brown–
Forsythe test with Tukey’s post hoc analysis for
multiple comparisons. All data represent
means 6s.e.m. Scale bar, 1 mminband cand
50 mming.
LETTER RESEARCH
00 MONTH 2015 | VOL 000 | NATURE | 3
Macmillan Publishers Limited. All rights reserved
©2015
human hypothermia
23
. Cooling of mice to higher core temperature of
26–28 uC, was similarly protective in prion disease, extending survival
(Extended Data Fig. 9).
We next asked if RBM3 overexpression alone, in the absence of cool-
ing, was similarly neuroprotective. We overexpressed, or knocked-down,
RBM3 in both hippocampi of mice by stereotaxic injection of lenti-
viruses LV-RBM3 and LV-shRNA-RBM3, respectively. LV-RBM3 pro-
duced a threefold increase in RBM3 levels compared to controls up
to 8 weeks post-injection; whereas knockdown by LV-shRNA-RBM3
reduced RBM3 levels to 30% of control levels(Fig. 4a). LV-RBM3 treat-
ment, but not LV-control, rescued the early deficit in synapse reassembly
in both prion-infected and 5XFAD mice at 6 w.p.i. and 3 months, re-
spectively (Fig. 4b). Furthermore, LV-RBM3 was associated with marked,
sustained neuroprotective effects in prion-infected mice: preventing
synapse loss (Fig.4c), synaptic transmissiondecline (Fig. 4d) and mem-
ory and behavioural impairments (Fig. 4e). In vitro, RBM3 has been
shown to promote translation
22
. Increased global protein synthesis
rates are profoundly neuroprotective, rescuing synapsenumber in prion
disease
13,14
. We found that RBM3 overexpression rescued levels of glo-
bal translation, whereas RBM3 knockdown further reduced them, in
prion-infected mice at 9 w.p.i. (Fig. 4f) suggesting that this action of
RBM3 along with preferential translation of specific RBM3-bound
mRNAs, contributes to the synapse regeneration process. LV-RBM3
treatment reduced prion neuropathology and prevented neuronal loss
(compare subpanels ii and iii in Fig. 4g) and significantly extended sur-
vival of prion-infected animals (Fig. 4h). This was not associated with
changes in levels of PrP
Sc
, which were not affected by overexpression of
RBM3 (Extended Data Fig. 8). Knockdown of RBM3, in contrast, ac-
celerated synapse loss and memory and behavioural deficits (Fig. 4c–e),
accelerating neuronalloss (Fig. 4g, subpanel iv) and significantly short-
ening survival (Fig. 4h). 5XFAD mice do not allowsimilar examination
of long-termeffects of RBM3 overexpression as evolutionof deficits and
neuronal loss takes many months, and life expectancy is normal. How-
ever, RNAi of RBM3 accelerated onset of synaptic loss in 5XFAD mice,
which was now seen at 3 months (Extended Data Fig. 10a), suggesting
that RBM3 has a long-term protective role in structural plasticity in these
mice also. RBM3 knockdown also reduced synapse number and novel
object memory in wild-type mice (Extended Data Fig. 10b), thus it is
likely to be involved in synaptic maintenance under normal physio-
logical conditions.
In conclusion, we have shown that early synapse loss in mouse mod-
els of neurodegenerative disease results, at least in part, from defective
synaptic repair processes associated with failure to induce the cold-
shock RNA-binding protein, RBM3. This results in impaired synaptic
reassembly after cooling, but also appears to be important in the con-
text of protecting against ongoing synaptic toxicity during disease, and
in synaptic maintenance in wild-type mice. Our data suggest that fur-
ther understanding the mechanisms of action of cold-shock proteins
such as RBM3 may yield insightsinto endogenous repair processes and
bring new therapeutic targets for neuroprotection in neurodegenera-
tive disease.
Online Content Methods, along with any additional Extended Data display items
and SourceData, are available in theonline version of the paper;references unique
to these sections appear only in the online paper.
Received 25 September 2013; accepted 5 December 2014.
Published online 14 January 2015.
1. Holtmaat, A. & Svoboda, K. Experience-dependent structural synaptic plasticity in
the mammalian brain. Nature Rev. Neurosci. 10, 647–658 (2009).
2. Selkoe, D. J. Alzheimer’s disease is a synaptic failure. Science 298, 789–791
(2002).
3. Mallucci, G. R. Prion neurodegeneration: starts and stops at the synapse. Prion 3,
195–201 (2009).
4. Magarin
˜os, A. M., McEwen, B. S., Saboureau, M. & Pevet, P. Rapid and reversible
changes in intrahippocampal connectivity during the course of hibernation in
European hamsters. Proc. Natl Acad. Sci. USA 103, 18775–18780 (2006).
5. Popov, V. I. & Bocharova, L. S. Hibernation-induced structural changes in synaptic
contacts between mossy fibres and hippocampal pyramidal neurons.
Neuroscience 48, 53–62 (1992).
6. Williams,D. R. et al. Seasonally hibernatingphenotype assessedthrough transcript
screening. Physiol Genomics 24, 13–22 (2005).
7. Oakley, H. et al. Intraneuronal b-amyloid aggregates, neurodegeneration, and
neuron loss in transgenic mice with five familial Alzheimer’s disease mutations:
potential factors in amyloid plaque formation. J. Neurosci. 26, 10129–10140
(2006).
8. Popov, V. I. et al. Reversible reduction in dendritic spines in CA1 of rat and ground
squirrel subjected to hypothermia-normothermia in vivo: A three-dimensional
electron microscope study. Neuroscience 149, 549–560 (2007).
9. Ruediger, J. et al. Dynamics in the ultrastructure of asymmetric axospinous
synapses in the frontal cortex of hibernating European ground squirrels
(Spermophilus citellus). Synapse 61, 343–352 (2007).
10. von der Ohe, C. G., Garner, C. C., Darian-Smith, C. & Heller, H. C. Synaptic protein
dynamics in hibernation. J. Neurosci. 27, 84–92 (2007).
11. Mallucci, G. R. et al. Post-natal knockout of prion protein alters hippocampal CA1
properties, but does not result in neurodegeneration. EMBO J. 21, 202–210
(2002).
12. Mallucci, G. R. et al. Targetingcellular prion proteinreverses early cognitive deficits
and neurophysiological dysfunction in prion-infected mice. Neuron 53, 325–335
(2007).
13. Moreno, J. A. et al. Sustained translational repression by eIF2a-P mediates prion
neurodegeneration. Nature 485, 507–511 (2012).
14. Moreno, J. A. et al. Oral treatmenttargeting the unfolded proteinresponse prevents
neurodegeneration and clinical disease in prion-infected mice. Sci. Transl. Med. 5,
206ra138 (2013).
15. Zhang, J., Kaasik, K., Blackburn, M. R.& Lee, C. C. Constant darknessis a circadian
metabolic signal in mammals. Nature 439, 340–343 (2006).
16. West, M. J. Counting and measuring ultrastructural features of biological samples.
Cold Spring Harb. Protoc. http://dx.doi.org/10.1101/pdb.top071886 (2013).
17. Yenari, M. A. & Han, H. S. Neuroprotective mechanisms of hypothermia in brain
ischaemia. Nature Rev. Neurosci. 13, 267–278 (2012).
18. Danno, S. et al. Increased transcript level of RBM3, a member of the glycine-rich
RNA-binding protein family, in human cells in response to cold stress. Biochem.
Biophys. Res. Commun. 236, 804–807 (1997).
19. Smart, F. et al. Two isoforms of the cold-inducible mRNA-binding protein RBM3
localize to dendrites and promote translation. J. Neurochem. 101, 1367–1379
(2007).
20. Chip, S. et al. The RNA-binding protein RBM3 is involved in hypothermia induced
neuroprotection. Neurobiol. Dis. 43, 388–396 (2011).
21. Tong, G. et al. Effects of moderate and deep hypothermia on RNA-binding proteins
RBM3 and CIRP expressions in murine hippocampal brain slices.Brain Res. 1504,
74–84 (2013).
22. Dresios, J. et al. Cold stress-induced proteinRbm3 binds 60S ribosomal subunits,
alters microRNAlevels, and enhances globalprotein synthesis.Proc. Natl Acad. Sci.
USA 102, 1865–1870 (2005) .
23. Lee, C. C. Is human hibernation possible? Annu. Rev. Med. 59, 177–186 (2008).
Supplementary Information is available in the online version of the paper.
Acknowledgements We thank J. Edwards, J. McWilliam and D. Read of the MRC
Toxicology Unit; members of University of Leicester Department of Biological Services
staff; and D. Morrison, University of Cambridge, for technical assistance. We thank
J. Skepper, University of Cambridge, for advice about stereological procedures. This
work was funded by the Medical Research Council, UK.
Author Contributions D.P. performed most experimental procedures and analyses.
A.B. analysed cold-shock proteins, N.V. and M.G.M. carried out prion inoculations and
stereotaxic injections, C.M. performed behavioural tests, J.A.M. and H.R. carried out
histologicalanalyses, J.R.S. performedneurophysiological procedures, M.G.M. and T.S.
performedultramicrotomy andprocessed samples forelectron microscopy,which was
analysed by D.D. A.E.W. provided expertise on cold-shock and protein expression.
G.R.M. conceived and directed the project. D.P. and G.R.M. wrote thepaper. All authors
contributed to discussion and analysis of data and to the final draft of the paper.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on theonline version of the paper. Correspondence
and requests for materials should be addressed to G.R.M. (gm522@cam.ac.uk).
RESEARCH LETTER
4 | NATURE | VOL 000 | 00 MONTH 2015
Macmillan Publishers Limited. All rights reserved
©2015
METHODS
Animals. All animal work conformed to UK regulations and institutional guide-
lines and were performed under UK Home Office guidelines.
Prion infection of mice. The 3-week-old tg37
1/2
mice
11
were inoculated intra-
cerebrally into the right parietal lobe with 30 mlof1%brainhomogenateofChandler/
RML (Rocky Mountain Laboratories) prions under generalanaesthetic, as described
24
.
Animals were culled when they developed clinical signs of scrapie as defined in
14
.
Control mice received 1% normal brain homogenate.
5XFAD mice. Founder 5XFAD mice were obtained from the Jackson Laboratory
(Ba Harbour, ME, USA). The 5XFAD mice have the following five mutations: Swedish
(K670N and M671L), Florida(I716V) and London (V717I) in human APP695 and
human PS1 cDNA (M146L and L286V) under the transcriptional control of the
neuron-specific mouse Thy-1 promoter
7
. Colonies were maintained by crossing hemi-
zygous transgenic to wild-type littermates.
Induction of hypothermia. FVB, tg37
1/2
and 5XFAD mice weighing $20 g were
cooled using 59-AMP as described
15,25
, with slight modifications. Freshly prepared
59-AMP (Sigma) was injected intraperitoneally (0.7mg per g). Control mice were
injected with saline. Mice were maintained at room temperature until core body
temperature decreased to 25uC (approximately 60 min). Subsequently, mice were
transferred to a refrigerator(5 uC) and core body temperature lowered to 16–18 uC
for 45 min. Mice recovered normal body temperature at room temperature con-
ditions. Cooled samples were collected at the end of the 16–18 uC period and rewarmed
samples as stated elsewhere in the text.
Electron microscopy data acquisition and analysis of synapse number. Male
mice were used to avoid the effects on synapse number of theoestrus cycle. Brains
were perfusion fixed with 2% glutaraldehyde 12% paraformaldehyde in 0.1 M
sodium cacodylate buffer (final pH 7.3). Slices (300 mm) were prepared using a
vibrating blade microtome (LeicaMicrosystems, Milton Keynes, UK). These slices
were post-fixed in 1% osmiumtetroxide 11% potassium ferrocyanide,stained en-
bloc with 5% uranyl acetate and embedded in epoxy resin (TAAB Laboratories
Equipment Ltd, Aldermaston, UK), as described
26
.
For random sampling and calculation of synapse density, estimation of total
synapse number and measures of volume of tissue using the stereology disector
method, the following procedures were used, as described previously
16
. Fixed and
stained slices were flat embedded as de scribed
27
. The slices were mounted for microt-
omy, a semi-thin (1 mm) section was cut from the upper surface of each slice and
the area of thestratum radiatum (sr) measured using theCavalieri estimator (points
grid), as described
16
. These results were then used to estimate the volume of the
stratumradiatumand the systematic randomselectionof regions forelectron micro-
scopy, as described
16
. Briefly, the grid has intersections every 50 pixels (at a scale
that 1 pixel is 1 mm). Each intersection was labelled with a dot. The dots were
numbered in each of the sections generated per hippocampus. These numbers
were used for measuring the area of each section and generate the data about the
volume applying the following formula: VsrCA1 5SPsrCA1 3Ap 3T (ref. 16).
The total number of dots per hippocampus was divided by 6 (number of samples
selected to obtainrepresentative synapse variability from the whole hippocampus).
The number obtained was used for the generation of a random number for sam-
pling the first positionand defined the interval for the subsequent 5 positions to be
sampled. These regions were trimmed-down, mounted on aluminium pins and
imaged by serial block-face scanning electron microscopy in a FEI Quanta FEG
250 electron microscope (FEI Ltd, Cambridge, UK) equipped with a ‘3View2XP’
system (Gatan Ltd, Abingdon, UK). Images of 32 serial sections were generated in
each of the six points. Serial sections of areas 51.4 mm
2
were recorded at 320,000
and an accelerating voltage of 3 kV, using spot size 3.0, at a working distance of
6.1 mm, and a dwell-time of 5 ms per pixel. The mean section thickness was esti-
mated by sectioning only half of the original block face. The block was then re-
embedded,sectionedorthogonally andthe depth removed by‘3View’ was measured
by transmission electron microscopy
28
. The mean thickness of 3View sections was
86 nm. We analysed synapses in a volume stack of 88 mm
3
(with an area of 33 mm
2
,
a measured section thickness of 0.086 mm and 31 sections). Synapses that had their
first identifiableprofile below the first section in theseries were counted
16
. Synapses
were identified, within a counting frame of 5.75 mm35.75 mm, which follows the
counting frame rules to avoid edge effect for the estimation of synapse numerical
density. The total number of synapse was estimated with the synapsedensity and
the volume of each hippocampus, as described
16
.
Synapse size was calculated by measuring mean synapse length, mean synapse
area in the same serial sections used for estimations of synapse density and total
synapse number, as described
16
.
For routine 2D analyses semi-thin (1mm) sections were stained with toluidine
blue and examined to select areas for ultramicrotomy. Ultrathin sections (,70 nm)
were stained with lead citrate and examined, blind, in a Jeol 100-CXII electron
microscope (JEOL (UK)Ltd, Welwyn GardenCity, UK) equippedwith a ‘Megaview
III’ digital camera (Olympus Soft Imaging Solutions GmbH, Mu
¨nster, Germany).
A series of images were recorded from the stratum radiatum, all at a distance of
approximately 100 mm from the CA1 pyramidal layer to avoid the large dendritic
profiles in the proximal area. 31 images, each encompassing an area of 55mm
2
,
from each of two to three mice were used for scoring. For synapse quantification
the following criteria were followed: the presence of an unambiguous postsynaptic
density, a clear synaptic cleft, and three or more synaptic vesicles. An average of
300 synapses were counted per sample.
Quantifiation of numbers of neurons in CA1 region of hippocampus. For CA1
pyramidal layer volume analysis, whole hippocampus was cut and every 250mm
stained with haemotoxylin and eosin in fixed sections of 5 mm thickness. The
volume of the pyramidal layer was measured using the Cavalieri estimator (points
grid). For neuron mean density slices were stained with NeuN and calculated
within an unbiased virtual space. The total number of neurons in CA1 pyramidal
layer was estimated with the neuron density and the volume of the hippocampus.
Immunoblotting. Protein samples were isolated from hippocampi using protein
lysis buffer (50mM Tris, 150 mM NaCl,1% Triton X-100, 1% Na deoxycholate,
0.1% SDS and 125 mM sucrose) supplemented with Phos-STOP and protease inhi-
bitors (Complete, Roche), followed by centrifugation and quantification. Protein
levels were determined by resolving 20 mg of protein on SDS–polyacrylamide gel
electrophoresis gels, transferred onto either nitrocellulose or PDVF membranes
and incubated with primary antibodies. Synaptic proteins were detected using the
following antibodies: SNAP-25, (1:10,000; catalogue number: ab5666, Abcam),
VAMP2 (1:5,000; catalogue number: 104204, Synaptic Systems), NMDA-R1 (1:1,000;
cataloguenumber: G8913, Sigma) and PSD95(1:1,000; cataloguenumber: 04-1066,
Millipore). Odyssey IRDye800 secondary antibodies (1:5,000; catalogue number:
926-32210/32211 LI-COR) were applied, visualized and quantified using Odyssey
infrared imager (LI-COR; software version 3.0). Protein for PrP levels was deter-
mined using the primary antibody ICSM35 (1:10,000; catalogue number: 0130-
03501, D-GEN). PrP
Sc
was detected after Proteinase K digestion. Cold-shock protein
levels were determinedwith antibodies CIRP (1:1,000;catalogue number: 10209-2-
AP, Proteintech Group, Inc.) and RBM3 (1:500; catalogue number: 14363-1-AP,
Proteintech Group, Inc.).Amyloid-blevels in 5XFAD mice were detected by 6E10
clone antibody (1:1,000; catalogue number: SIG-39320, Covanche). Horseradish-
peroxidase-conjugated secondary antibodies (1:10,000; DAKO) were applied and
protein visualizedusing enhanced chemiluminescence(GE Healthcare) and quan-
tified using ImageJ. An antibody against GAPDH (1:5,000; catalogue number:
sc32233, Santa Cruz) was used to determine gel loading.
Lentiviruses. GenTarget (SanDiego, CA, USA) generated lentiviral plasmids. The
neuron-specificpromoter CAMKII was used to drive RBM3; the H1 promoter was
used for shRNA-RBM3 expression and scrambled sequence-shRNA. Viruses were
injected stereotaxically into the CA1 region of the hippocampus as described
29
.
Mouse RBM3 isoform 2 (NM_001166410.1) overexpression was induced using
the pLentiCAMKII (RBM3)Rsv (GFPBsd) plasmid. pLentiCAMKII (empty)Rsv
(GFPBsd) was used as control. RBM3 down regulation was achieved by using
pLentiH1shRNA(m RBM3) sequence number 2Rsv(GFPBsd). This plasmid con-
tains the following shRNA-RBM3 sense, anti-sense and loop sequences (sequence
number 2: 59-GTTGATCATGCAGGAAAGTCTcgagAGACTTTCCTGCATGA
TCAAC-39).
pLentyH1shRNA (negative control)Rsv (GFPBsd) containing the sequence 59-
GTCTCCACGCGCAGTACATTT-39was used as control. Lentiviral sequences and
viral stocks were generated by GenTarget (San Diego, CA, USA). Virus titre was
determined using FACS(BD FACS Calibur). Viruses were used witha final titre of
0.6 310
8
to 1.5 310
8
transducing units.
Stereotaxic injection. Under general anaesthetic, mice were injected with 5mlof
lentivirus per site into the CA1 region of the hippocampus. Mice were injectedat 2
locations per hemisphere; at 22mm and 22.7 mm posterior, 62 mm lateral and
22.2 mm ventral relative to bregma, using a 26 s-gauge needle and Hamilton
syringe as described
29
.
Burrowing assay. This was performed as described
14,30
. Briefly, mice were placed
in individual large plasticcages containing a clearPerspex tube,20 cm long 36.8 cm
diameter, filled with 140g of normal food pellets. The weight of pellets remaining
in the tube was measured after 2 h and the percentage burrowed calculated. Behavioural
data were analysedusing one-way ANOVA with Brown–Forsythetest and Tukey’s
post hoc test. For behaviouraltesting no formal randomizationwas needed or used.
Experimenter was blind to group allocation during all experiments and when
assessing outcome.
Novel object recognition memory. This was performed as described
12
. Briefly,
mice were tested in a blackcylindrical arena (69 cm diameter) mounted with a 100
LED strip infrared light source and a high resolution day/night video camera(Sony).
Mice wereacclimatizedto the arena 5 days before testing. During the learning phase,
two identical objects were placed 15 cm from the sides of the arena. Each mouse
was placed in the arena by an operator blind to the experimental group for two
blocks of 10 min for explorationof the objects with an inter-trial interval of 10 min.
LETTER RESEARCH
Macmillan Publishers Limited. All rights reserved
©2015
Two hours later, one of the objects was exchanged for a novel one, and the mouse
was replaced in the arena for 5 min (test phase). The amount of time spent explor-
ing all objects was trackedand measured for each animal using Ethovisionsoftware
(Tracksys). All objects and the arena were cleansed thoroughly between trials to
ensure the absence ofolfactory cues. The amount of time spent exploring the novel
object over the familiarobject is expressed as a ratio, where a ratio of 1 reflects ran-
dom exploration, and .1 reflects memory. Behavioural data were analysed using
one-way ANOVA with Brown–Forsythe test and Tukey’s post hoc test. For beha-
vioural testing no formal randomization was needed or used. Experimenter was
blind to group allocation during all experiments and when assessing outcome.
Electrophysiology. Whole-cell recordings were made in acute hippocampal slices
to measure synaptic transmission from identified CA1 neurons and recording per-
formed as described
31
. In brief, neurons were voltage clamped using a Multiclamp
700B amplifier and pClamp 10.3 software (Molecular Devices) and EPSCs were
evoked by stimulation with bipolar platinum electrode at 37 uC. Pipettes (2.5–3.5 MV)
were filled with a solution containing (in mM): KCl 110, HEPES 40, EGTA 0.2,
MgCl
2
1, CaCl
2
0.1; pH was adjusted to 7.2 with KOH. Neurons were visualized
with 360 objective lenses on a Nikon FS600 microscope fitted with differential
interference contrastoptics. Four to eight cells were measured permouse in at least
two animals per experiment. Male mice were used to avoid effects of the oestrus
cycle.
Hippocampal slice preparation and
35
S-methionine labelling. Slices were dis-
sected in an oxygenated cold(2–5 uC) sucrose artificial cerebrospinal fluid(ACSF)
containing (mm): 26mM NaHCO
3
, 2.5mM KCl, 4 mM MgCl
2
, 0.1 mM CaCl
2
, and
250mM sucrose. Hippocampal slices were prepared using a tissue chopper (McIlwain).
Slices were allowed to recover in normal ACSF buffer while being oxygenated at
37 uC for 1 h, then incubated with [
35
S]-methionine label for 1h, then homoge-
nized
14
. Proteins were TCA precipitated and incorporation of radiolabel was mea-
sured by scintillation counting (Winspectal, Wallac).
Statistics. Statistical analyses were performed using Prism v5 software, using Student’s
t-test for data sets with normal distribution and a single intervention; when the
F-test to comparevariances was significant, Mann–WhitneyU-test was performed
instead.
Behavioural data, neuronal counts and
35
S-met were analysed using one-way
ANOVA and Tukey’s post hoc test for multiple variables.
For behavioural testing no formal randomization was needed or used. Experimenter
was blind to group allocationduring the experiments and when assessing outcome.
Statistical analyses for
in vivo
experiments. Sample size estimation for induction
of hypothermia for volume, synaptic density and estimation of total number of synapses
was based in effectsize of the preliminary experiment on dissector method (5.9511)
and obtained with the free software G*Power version 3.1.9.2.
The software prediction shows that with a sample size of 6 animals for 2 con-
ditions (control and cooled or cooled and rewarmed), the experiment has a 99.8%
of chance of detecting a differenceand avoid a type II error (b-error), with a 0.05%
chance of a type I (a-error). Sample size estimation for novel object recognition
experiment was established based on the effect size of 1.6161 from control and
prion mice at 8 w.p.i. This parameter was applied in the following F tests calcula-
tion of power analysis with G*Power version 3.1.9.2.
Similar analyses were performed for burrowing tests.
24. Mallucci, G. et al. Depletingneuronal PrP in prion infection prevents disease and
reverses spongiosis. Science 302, 871–874 (2003).
25. Daniels, I. S. et al. A role of erythrocytes in adenosine monophosphate initiationof
hypometabolism in mammals. J. Biol. Chem. 285, 20716–20723 (2010).
26. Deerinck, T. J. Enhancing serial block-facing scanning electron microscopy to
enable high resolution 3-D nanohistology of cells and tissues. Microsc. Microanal.
16, 1138–1139 (2010).
27. Nguyen, K. B. & Pender, M. P. A simple technique for flat osmicating and flat
embedding of immunolabelled vibratome sections of the ratspinal cord for light
and electron microscopy. J. Neurosci. Methods 65, 51–54 (1996).
28. Yang, G. C. H.& Shea, S. M. Precisemeasurementof thicknessof ultrathinsections
by a ‘re-sectioned-section’ technique. J. Microscopy 103, 385–392 (1975).
29. White, M. D. et al. Single treatment with RNAi against prion protein rescuesearly
neuronal dysfunction and prolongs survival in micewith prion disease. Proc. Natl
Acad. Sci. USA 105, 10238–10243 (2008).
30. Cunningham, C. et al. Synaptic changes characterize early behavioural signs in
the ME7 modelof murine prion disease. Eur.J. Neurosci. 17, 2147–215 5 (2003).
31. Haustein, M. D. et al. Acute hyperbilirubinaemia induces presynaptic
neurodegeneration at a central glutamatergic synapse. J. Physiol. 588,
4683–4693 (2011).
RESEARCH LETTER
Macmillan Publishers Limited. All rights reserved
©2015
a
bc
4 w.p.i.6 w.p.i.
5xFAD
2 months 3 months
Prion disease
0
5.0 x10
8
1.0 x10
9
1.5 x10
9
Synapse mean length (nm)
CA1 volume (mm3)
0
0.01
0.05
0.08
Synapse mean area (mm2)
37 °C 16-18 °C 37 °C
0
0
150
300
450
400
350
250
200
100
50
0.07
0.06
0.04
0.03
0.02
7
synapse
loss memory
loss
8 9 10 11 126
neuronal
loss terminally
sick w.p.i.
0
Prion
infection
4
synapse
loss
56
3months
memory
loss
0
5xFAD lost synaptic
recovery
2
54
9
neuronal
loss
d
Extended Data Figure 1
|
Stereological assessment of volume and synapse
size to validate 2D assumption-based approaches for counting synapse
density. a, CA1 volume and synapse mean length and area in the stratum
radiatum remain essentially unchanged on cooling and rewarming in wild-type
mice. Volume was measured using disector principle and synapse mean length
and area determined in the same sections, as described
1
,nvalues as reported
for Fig. 1a. b,c, Representative electron micrographs (pseudo-coloured for
ease of synapse identification) for data not shown in Fig. 1b, c, from prion-
infected mice at 4 and 6 w.p.i. (b) and for 5XFAD mice at 2 and 3 months
(c) before cooling (black framed images) and cooled (blue framed images).
d, Schematic showing lost capacity for structural plasticity precedes synapse
loss and neuronal loss in both mouse models. Scale bar, 1 mm. All data in bar
charts are mean 6s.e.m. Student’s t-test, two tailed. Non-significant Pvalues.
LETTER RESEARCH
Macmillan Publishers Limited. All rights reserved
©2015
kDa
kDa
0
50
100
150
200
4 6 w.p.i.6
0
50
100
150
4 w.p.i.
0
40
80
120
160
4 6 w.p.i. 0
20
40
60
80
100
120
140
4 6 w.p.i.
GAPDH
VAMP2
GAPDH
NR1
w.p.i.
PSD 95
SNAP25
GAPDH
46
25
95
35
19
105
35
35
n.s.
PSD95/GAPDH
(% relative to mean control)
VAMP2/GAPDH
(% relative to control mean)
SNAP25/GAPDH
(% relative to mean control)
NR1/GAPDH
(% relative to control mean)
0
20
40
60
80
100
120
140
0
20
40
60
80
100
120
140
months 0
20
40
60
80
100
120
140
160
0
20
40
60
80
100
120
140
GAPDH
VAMP2
PSD 95
SNAP25
GAPDH
23
NR1
months
23
25
95
35
19
105
35
PSD95/GAPDH
(% relative to mean control)
SNAP25/GAPDH
(% relative to mean control)
VAMP2/GAPDH
(% relative to control mean)
NR1/GAPDH
(% relative to control mean)
months2 3 months23 months23
a
b 5xFAD
Prion disease
Extended Data Figure 2
|
Synaptic protein levels during cooling–
rewarming in prion and 5XFAD mice. a, Levels of presynaptic (SNAP25,
VAMP2) and postsynaptic (PSD95, NR1) proteins do not change before
(black bars) and after cooling to 16–18 uC (bluebars) in prion-infected mice at
4 and 6 w.p.i., b, 5XFAD mice at 2 and 3 months. Representative western blots
are shown for 3 mice per temperature and time point. Bar graphs show
quantification of synaptic protein levels relative to GAPDH. All data represent
means 6s.e.m. (n53–11 mice per time point). Student’s t-test, two tailed.
n.s. 5non-significant Pvalues.
RESEARCH LETTER
Macmillan Publishers Limited. All rights reserved
©2015
b
GAPDH
5x FAD 3 months
Ab
10
15
kDa
kDa
35
25 PrP
25
15
PrPSc
prion terminal
a
Prion disease 6 w.p.i.
uninfected control
A oligomers
C+
Extended Data Figure 3
|
Cooling does not induce changes in PrP
Sc
or
amyloid-blevels. a, Levels of total PrP (upper blot) and PrP
Sc
(lower blot) do
not change notably before (white line), during (blue line) or after (red line)
cooling to 16–18 uC in prion-infected mice. PrP
Sc
is detected after digestion
with proteinase K. Levels are undetectable by western bloting at 6 w.p.i., as
expected. b, Cooling does not change levels of amyloid-boligomers in 5XFAD
mice, arrow indicates amyloid-bmonomers (lane 1, synthetic amyloid-b
oligomers; last lane, one-year-old 5XFAD control (C1)). Representative
western blots are shown for 3 mice per temperature and time point.
Non-significant Pvalues.
LETTER RESEARCH
Macmillan Publishers Limited. All rights reserved
©2015
37°C
cooled
wpi
46
GAPDH
CIRP
35
18
kDa
150
0
25
50
75
100
125
46
w.p.i.
CIRP/GAPDH
(% relative to control mean)
months
23
GAPDH
CIRP
35
18
kDa
0
50
100
150
200
months23
n.s.
CIRP/GAPDH
(% relative to control mean)
0
50
100
150
200
250
12 48 72 hrs
RBM3/GAPDH
(% relative to control mean)
*
wild type
rewarmed
a
b
5xFAD
Prion disease
c
Extended Data Figure 4
|
Cooling induces sustained increase in RBM3
levels but not in CIRP. a,b, Levels of CIRP do not change after cooling in
prion-infected mice at 4 and 6 w.p.i. (a) or in 5XFAD mice at 2 and 3 months
(b). Representative western blots are shown for 3 mice per temperature and
time point. Bar graphs show quantification of CIRP levels relative to GAPDH.
All data represent means 6s.e.m. (n56–9 mice per time point). Student’s
t-test, two tailed. n.s. 5non-significant Pvalues. c, Increasedlevels of RBM3 are
sustained for at least 72h after cooling in wild-type mice. Bar graph shows
quantification of RMB3 against GAPDH in control (white bar), cooled (blue
bar), and 12, 48 and 72 h recovery after cooling (red bars). All data represent
means 6s.e.m., (n53–6 mice per time points, *P,0.05, Mann–Whitney
U-test, two tailed).
RESEARCH LETTER
Macmillan Publishers Limited. All rights reserved
©2015
wpi10
GAPDH
RBM3
12
67
uninfected
control 89
kDa
35
15
Extended Data Figure 5
|
Early cooling induces sustained elevation of
RBM3 levels. RBM3 levels remain high after cooling to 16–18 uC in prion-
infected mice (magentaboxes) compared to control prion-infected mice.These
levels remained high up to 6 weekslater and declined at 12 w.p.i. Representative
western blots are shown for 3 mice per time point.
LETTER RESEARCH
Macmillan Publishers Limited. All rights reserved
©2015
Prion infection + LV-shRNA-control
+ early cooling
Prion infection + LV-shRNA-RBM3
+ early cooling
89C w.p.i.
0
100
200
300
a
0
100
200
300
9w.p.i.C
c
Prion infection + early cooling
0
0.5
1.0
1.5
2.0
2.5
Novel object preference
9 w.p.i.C
** **
b
Prion infection only
Uninfected controls
Total exploration time (s)
Total exploration time (s)
Extended Data Figure 6
|
Exploration time in exposure phase of novel
object testing is normal in all groups and RBM3 knockdown abolishes
improved memory after cooling. a, Exploratory behaviour measured in
seconds is not different in mice with early cooling from prion-diseased mice
and is not affected by the duration of disease (nas reported in Fig. 3d).
b,c, Lentivirally mediated RNAi of RBM3 eliminates the protective effect of
cooling on novel object memory impairment in prion disease (b) (dark green
bar); but does not affect exploratory behaviour in training phase (c). All data
represent means 6s.e.m. Data analysed using one way ANOVA, Brown–
Forsythe test with Tukey’s post hoc analysis for multiple comparisons
(n511–16 mice per time point, **P,0.01).
RESEARCH LETTER
Macmillan Publishers Limited. All rights reserved
©2015
75 80 85 90 95 100
0
20
40
60
80
100
Survival (%)
Days post inoculation
Prion infection
+
5 and 6 wpi cooling
Prion
infection
only
Extended Data Figure 7
|
Induction of hypothermia at time point when
RBM3 induction fails is not neuroprotective. Cooling at 5 and 6 w.p.i., when
synaptic plasticity andRBM3 induction fails (see Fig. 1 and 2, main text), does
not increase survival in prion-infected mice. Kaplan–Meier survival plots for
prion-infected mice (black line, no cooling; n510; orange line, mice cooled at
5 and 6 w.p.i., n516). Student’s t-test, two tailed. Non-significant Pvalues.
LETTER RESEARCH
Macmillan Publishers Limited. All rights reserved
©2015
a
Prion disease terminal
kDa LV-
Control LV-
RBM3 LV-
shRNA-RBM3
35
25
35
PrP
25
15 PrPSc
GAPDH
C
b
kDa
35
25 PrP
25
15
PrPSc
CPrion disease terminal
35 GAPDH
kDa
35
25
CPrion disease 9 w.p.i.
PrP
25
15
PrPSc
35 GAPDH
Extended Data Figure 8
|
PrP
Sc
levels remain unchanged in prion with
overexpression of RMB3. a,b, In prion-infected mice total PrP and PrP
Sc
levels do not alter after early cooling to 16–18 uC(a) (magenta boxes) or
following treatment with LV-RBM3 (dark green) and LV-shRNA-RBM3
(pale green) (b). PrP and PrP
Sc
levels tested in 9 w.p.i. and terminal mice. PrP
Sc
is detected after digestion with proteinase K. Representative western blots are
shown for 3 mice per temperature and time point, the lane marked C shows
uninfected control mouse.
RESEARCH LETTER
Macmillan Publishers Limited. All rights reserved
©2015
**
0Days post inoculation
Prion
infection
only
Prion infection
+
mild cooling
75 80 85 90 95 100
0
20
40
60
80
100
Survival (%)
Extended Data Figure 9
|
Mild hypothermia also extends survival in prion-infected mice. Kaplan–Meier plot showing that cooling to 26uC at an early stage
also significantly lengthens survival (n527 cooled vs n516 non-cooled mice); **P,0.01, Student’s t-test, two tailed.
LETTER RESEARCH
Macmillan Publishers Limited. All rights reserved
©2015
0
1
2
3
4
5
6
7
***
LV-Control
3 months
LV-shRNA-RBM3
5xFAD
0
0.5
1.0
1.5
2.0
Novel object preference
*
0
2
4
6
8***
Number of synapses per 55µm2 area
wild type
a
b
LV-Control LV-shRNA-RBM3
Number of synapses per 55µm2 area
Extended Data Figure 10
|
RNAi of RBM3 downregulation accelerates
impaired structural synaptic plasticity in the 5XFAD mouse model, and also
reduced synapse number and function in wild-type mice. a, Impaired
structural synaptic plasticity after cooling occurs in shRNA-RBM3 treated
5XFAD mice at 3 months. Representative electron micrographs areshown and
are pseudo-coloured as in main text figures. Quantification shows significant
reduction in synapse number by RNAi of RBM3 (n582–93 images from 3
mice per time point, Student’s t-test, two tailed). b, RBM3 knockdown reduces
synapse number and novel object memory in wild-type mice (n593 images
from 3 mice per time point,Student’s t-test, two tailed ***P,0.0001; for novel
object recognition task n511 mice, LV-shRNA-control and 10 mice,
LV-shRNA-RBM3, Mann–Whitney U-test, *P,0.05). Scale bar, 1 mm.
RESEARCH LETTER
Macmillan Publishers Limited. All rights reserved
©2015
... [18] RBM3 has been observed to function in neuroprotection during hypothermic conditions by preventing neural cell death and improving synaptic plasticity. [19] This protein does so by increasing local protein synthesis at dendrites [20] and global protein synthesis through binding ribosomal subunits and/or by micro RNA biogenesis. [21] Specifically, RBM3 is shown to bind the messenger RNA of the cold-shock protein reticulon 3 (RTN3) and upregulate its expression. ...
... [24] Peretti et al. performed an experiment to determine whether increasing RBM3 expression would restore the failed synaptic plasticity in mice with Alzheimer's disease or prion disease. [19] The utilization of hypothermia to increase RBM3 expression in mice with prion disease was shown to prevent loss of synapses, improve synaptic communication, and thwart behavior deficits. [19] In addition, prion-infected mice that were exposed to hypothermic conditions were observed to have a prolonged lifespan compared to the ones that were not. ...
... [19] The utilization of hypothermia to increase RBM3 expression in mice with prion disease was shown to prevent loss of synapses, improve synaptic communication, and thwart behavior deficits. [19] In addition, prion-infected mice that were exposed to hypothermic conditions were observed to have a prolonged lifespan compared to the ones that were not. [19] RBM3 knockdown through lentiviral-mediated RNAi eliminated the neuroprotective effects of hypothermia on prion disease in mice. ...
... This study found that the same happens in cooled laboratory rodents. [7] RNA-binding motif protein 3 (RMB3) is an RNA-binding protein that was evaluated for its role in structural plasticity and the role it plays on neurodegeneration during cooling. 5XFAD mice were the AD-type mice used in this experiment. ...
... This is a hopeful therapeutic target for future AD treatment. [7] A newer cold-shock protein, reticulon 3 (RTN3), was evaluated in another study by Bastide et al. Since RBM3 has been already by proven by these authors to play a critical role in mediated synaptic repair processes, but the mechanisms by which micro ribonucleic acid (mRNA) encoding cold-shock proteins escape repression is unknown, they wanted to focus on another cold-shock protein. ...
Article
Alzheimer's disease is a neurological condition that causes the disruption of neuronal connections in the human brain. It is progressive and targets about 10% of the United States population over the age of 65.3 to date, there is no cure to the disease. Physicians can treat symptoms but lack the ability to stop the progression of the disease. However, promising research has come to the surface in recent years. A collection of these therapeutic targets, which have yielded positive results in mice models, are presented in this article. They include targets such as meningeal lymphatics, mitochondrial homeostasis, genomic instability, calcium homeostasis, and cold-shock proteins such as RNA-binding motif protein 3 and reticulon-3, high-density lipoprotein, and antibodies.
... On the other spectrum, it is interesting to note that hypothermia has been discussed as a potential neuroprotective therapy against dementia. 36,37 Although the exact mechanisms are still unknown, the overexpression of a cold-shock protein, RNA binding motif 3 (RBM3), results in reduced synaptic and neuronal loss in mouse models of neurodegeneration. ...
Article
Full-text available
The aggregation of Aβ42 is a hallmark of Alzheimer's disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to Aβ42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers to show that Aβ42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of Aβ42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model Aβ peptides to examine the factors that hinder thermal dissipation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides, and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by Aβ peptides are favored under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.
... On the other spectrum, it is interesting to note that hypothermia has been discussed as a potential neuroprotective therapy against dementia. 31,32 Although the exact mechanism are still unknown, the overexpression of a cold-shock protein, RNA binding motif 3 (RBM3), results in reduced synaptic and neuronal loss in mouse models of neurodegeneration. ...
Preprint
Full-text available
The aggregation of Aβ42 is a hallmark of Alzheimer′s disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to Aβ42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers (FPTs) to show that Aβ42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of Aβ42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model Aβ peptides to examine the factors that hinder thermal dissipation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by Aβ peptides are favoured under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.
... Cold-shock proteins such as RNA-binding motif protein 3, RBM3, are essential for maintaining synapses in laboratory mouse models of neurodegenerative diseases. These results suggest a potential role for therapeutic human hypothermia in achieving neuroprotective effects (Peretti et al., 2015). In-depth analyses in higher-order animals may help to exploit the benefits of temperature in improving organismal health. ...
Article
Full-text available
Temperature is an important environmental condition that determines the physiology and behavior of all organisms. Animals use different response strategies to adapt and survive fluctuations in ambient temperature. The hermaphrodite Caenorhabditis elegans has a well-studied neuronal network consisting of 302 neurons. The bilateral AFD neurons are the primary thermosensory neurons in the nematode. In addition to regulating thermosensitivity, AFD neurons also coordinate cellular stress responses through systemic mechanisms involving neuroendocrine signaling. Recent studies have examined the effects of temperature on altering various signaling pathways through specific gene expression programs that promote stress resistance and longevity. These studies challenge the proposed theories of temperature-dependent regulation of aging as a passive thermodynamic process. Instead, they provide evidence that aging is a well-defined genetic program. Loss of protein homeostasis (proteostasis) is one of the key hallmarks of aging. Indeed, proteostasis pathways, such as the heat shock response and aggregation of metastable proteins, are also controlled by thermosensory neurons in C. elegans. Prolonged heat stress is thought to play a critical role in the development of neurodegenerative protein misfolding diseases in humans. This review presents the latest evidence on how temperature coordinates proteostasis and aging. It also discusses how studies of poikilothermic organisms can be applied to vertebrates and provides new therapeutic strategies for human disease.
... In fact, hippocampal synaptic dysfunction is an early pathological hallmark in AD and is the underlying reason for memory impairment [105]. Hippocampal synaptic dysfunction associated with AD has been reported as decreased synaptic number [106][107][108], impaired synapse regeneration/synaptogenesis [109,110], loss of synaptic proteins (i.e., synaptophysin, synaptogyrin, synaptotagmin, syntaxin 1, PSD95, and Homer-1) [103,109,[111][112][113][114][115], impaired Figure 1. Schematic flow diagram of the correlation between etiopathogenesis and hippocampal synaptic dysfunction in neurodegenerative diseases. ...
Article
Full-text available
Neuroplasticity is the capacity of neural networks in the brain to alter through development and rearrangement. It can be classified as structural and functional plasticity. The hippocampus is more susceptible to neuroplasticity as compared to other brain regions. Structural modifications in the hippocampus underpin several neurodegenerative diseases that exhibit cognitive and emotional dysregulation. This article reviews the findings of several preclinical and clinical studies about the role of structural plasticity in the hippocampus in neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and multiple sclerosis. In this study, literature was surveyed using Google Scholar, PubMed, Web of Science, and Scopus, to review the mechanisms that underlie the alterations in the structural plasticity of the hippocampus in neurodegenerative diseases. This review summarizes the role of structural plasticity in the hippocampus for the etiopathogenesis of neurodegenerative diseases and identifies the current focus and gaps in knowledge about hippocampal dysfunctions. Ultimately, this information will be useful to propel future mechanistic and therapeutic research in neurodegenerative diseases.
... During torpor, the cold-shock protein Rbm3 was upregulated. Studies on natural and enforced hypothermia in the hibernating 13-line ground squirrels and in cooled mice suggest that Rbm3 and other cold-inducible RNA-binding proteins within the brain might enable RNA stability and protein synthesis at low body temperatures, which is important for synaptic regeneration and therefore structural plasticity [67][68][69]. ...
Article
Full-text available
The energy‐saving strategy of Djungarian hamsters (Phodopus sungorus, Cricetidae) to overcome harsh environmental conditions comprises of behavioral, morphological, and physiological adjustments, including spontaneous daily torpor, a metabolic downstate. These acclimatizations are triggered by short photoperiod and orchestrated by the hypothalamus. Key mechanisms of long‐term photoperiodic acclimatizations have partly been described, but specific mechanisms that acutely control torpor remain incomplete. Here we performed comparative transcriptome analysis on hypothalamus of normometabolic hamsters in their summer‐ and winter‐like state to enable us to identify changes in gene expression during photoperiodic acclimations. Comparing non‐torpid and torpid hamsters may also be able to pin down mechanisms relevant for torpor control. A de novo assembled transcriptome of the hypothalamus was generated from hamsters acclimated to long photoperiod or to short photoperiod. The hamsters were sampled either during long photoperiod normothermia, short photoperiod normothermia, or short photoperiod‐induced spontaneous torpor with a body temperature of 24.6 ± 1.0 °C, or. The mRNA‐seq analysis revealed that 32 and 759 genes were differentially expressed during photoperiod or torpor, respectively. Biological processes were not enriched during photoperiodic acclimatization but were during torpor, where transcriptional and metabolic processes were reinforced. Most extremely regulated genes (those genes with |log2(FC)| > 2.0 and padj <0.05 of a pairwise group comparison) underpinned the role of known key players in photoperiodic comparison, but these genes exhibit adaptive and protective adjustments during torpor. Targeted analyses of genes from potentially involved hypothalamic systems identified gene regulation of previously described torpor‐relevant systems and a potential involvement of glucose transport.
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.
Article
Background: Neonates have high levels of cold-shock proteins (CSPs) in the normothermic brain for a limited period following birth. Hypoxic-ischemic (HI) insults in term infants produce neonatal encephalopathy (NE), and it remains unclear whether HI-induced pathology alters baseline CSP expression in the normothermic brain. Methods: Here we established a version of the Rice-Vannucci model in PND 10 mice that incorporates rigorous temperature control. Results: Common carotid artery (CCA)-ligation plus 25 min hypoxia (8% O2) in pups with targeted normothermia resulted in classic histopathological changes including increased hippocampal degeneration, astrogliosis, microgliosis, white matter changes, and cell signaling perturbations. Serial assessment of cortical, thalamic, and hippocampal RNA-binding motif 3 (RBM3), cold-inducible RNA binding protein (CIRBP), and reticulon-3 (RTN3) revealed a rapid age-dependent decrease in levels in sham and injured pups. CSPs were minimally affected by HI and the age point of lowest expression (PND 18) coincided with the timing at which heat-generating mechanisms mature in mice. Conclusions: The findings suggest the need to determine whether optimized therapeutic hypothermia (depth and duration) can prevent the age-related decline in neuroprotective CSPs like RBM3 in the brain, and improve outcomes during critical phases of secondary injury and recovery after NE. Impact: The rapid decrease in endogenous neuroprotective cold-shock proteins (CSPs) in the normothermic cortex, thalamus, and hippocampus from postnatal day (PND) 11-18, coincides with the timing of thermogenesis maturation in neonatal mice. Hypoxia-ischemia (HI) has a minor impact on the normal age-dependent decline in brain CSP levels in neonates maintained normothermic post-injury. HI robustly disrupts the expected correlation in RNA-binding motif 3 (RBM3) and reticulon-3 (RTN3). The potent neuroprotectant RBM3 is not increased 1-4 days after HI in a mouse model of neonatal encephalopathy (NE) in the term newborn and in which rigorous temperature control prevents the manifestation of endogenous post-insult hypothermia.
Article
Full-text available
During prion disease, an increase in misfolded prion protein (PrP) generated by prion replication leads to sustained overactivation of the branch of the unfolded protein response (UPR) that controls the initiation of protein synthesis. This results in persistent repression of translation, resulting in the loss of critical proteins that leads to synaptic failure and neuronal death. We have previously reported that localized genetic manipulation of this pathway rescues shutdown of translation and prevents neurodegeneration in a mouse model of prion disease, suggesting that pharmacological inhibition of this pathway might be of therapeutic benefit. We show that oral treatment with a specific inhibitor of the kinase PERK (protein kinase RNA–like endoplasmic reticulum kinase), a key mediator of this UPR pathway, prevented UPR-mediated translational repression and abrogated development of clinical prion disease in mice, with neuroprotection observed throughout the mouse brain. This was the case for animals treated both at the preclinical stage and also later in disease when behavioral signs had emerged. Critically, the compound acts downstream and independently of the primary pathogenic process of prion replication and is effective despite continuing accumulation of misfolded PrP. These data suggest that PERK, and other members of this pathway, may be new therapeutic targets for developing drugs against prion disease or other neurodegenerative diseases where the UPR has been implicated.
Article
Full-text available
Therapeutic hypothermia has emerged as an effective neuroprotective therapy for cardiac arrest survivors. There are a number of purported mechanisms for therapeutic hypothermia, but the exact mechanism still remains to be elucidated. Although hypothermia generally down-regulates protein synthesis and metabolism in mammalian cells, a small subset of homologous (>70%) cold-shock proteins (RNA-binding motif protein 3, RBM3 and cold-inducible RNA-binding protein, CIRP) are induced under these conditions. In addition, RBM3 up-regulation in neuronal cells has recently been implicated in hypothermia-induced neuroprotection. Therefore, we compared the effects of moderate (33.5 °C) and deep (17 °C) hypothermia with normothermia (37 °C) on the regulation of RBM3 and CIRP expressions in murine organotypic hippocampal slice cultures (OHSC), hippocampal neuronal cells (HT-22), and microglia cells (BV-2).
Article
Full-text available
The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer's, Parkinson's and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the α-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2α-P levels are seen in patients with Alzheimer's, Parkinson's and prion diseases, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2α-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2α-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2α-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation, increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion-diseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.
Article
Full-text available
of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.
Article
Full-text available
Cooling can reduce primary injury and prevent secondary injury to the brain after insults in certain clinical settings and in animal models of brain insult. The mechanisms that underlie the protective effects of cooling - also known as therapeutic hypothermia - are slowly beginning to be understood. Hypothermia influences multiple aspects of brain physiology in the acute, subacute and chronic stages of ischaemia. It affects pathways leading to excitotoxicity, apoptosis, inflammation and free radical production, as well as blood flow, metabolism and blood-brain barrier integrity. Hypothermia may also influence neurogenesis, gliogenesis and angiogenesis after injury. It is likely that no single factor can explain the neuroprotection provided by hypothermia, but understanding its myriad effects may shed light on important neuroprotective mechanisms.
Article
Ultrastructural features of cells can be fractions of a micrometer in diameter, and electron microscopy is needed to resolve them to a degree that is compatible with stereological techniques. Because the focal depth of transmission electron microscopy (TEM) images is thousands of times greater than the thickness of the sections used with TEM, virtual sectioning of sections suitable for TEM is not possible, as it is with light microscopy and the optical disector probe. With features the size of neuronal synapses, for example, this necessitates the use of physical sections and physical disectors. Regardless of how the imaging is performed, the design of stereological studies for quantifying ultrastructural features will be essentially the same as that used in the example described here, which uses physically separated ultrathin sections viewed with conventional TEM to estimate the number and size of synapses in a particular brain region.
Article
The thickness of ultrathin tissue sections embedded in Epon-Araldite and cut with a diamond knife was measured by re-sectioning and electron microscopic examination of the section profiles. A secondary section mounted on a Formvar-coated slot grid provided enough normally cut segments (seven to seventeen) for measurements giving a precise estimate of mean thickness, comparable to that obtainable by interference microscopy (±2.3% or less for grey to dark gold sections). The standard deviation of section thickness within sections was never more than 5 nm, corresponding to a coefficient of variation of 6.5% or less for sections more than 48 nm thick. This suggests that variation in section thickness, within sections, may be less than has been supposed, so that quantitative work may be based on thickness measurements made over a limited representative area. A silver interference colour was associated with sections 49–60 nm thick.
Article
Although the cold-shock responses of microorganisms have been extensively investigated, those of mammalian cells are just beginning to be understood. Recently, CIRP, a member of the glycine-rich RNA-binding protein (GRP) family, has been identified as the first cold-shock protein in mammalian cells. Here, we report that RBM3, another member of the GRP family, is induced in human cells in response to cold stress (32°C).RBM3transcripts were constitutively expressed in all cell lines examined including K562, HepG2, NC65, HeLa, and T24 cells. In all of them, the transcript levels ofRBM3were increased at 24 h after the 37 to 32°C temperature down-shift. In NC65 cells, the kinetics ofRBM3induction was different from that ofCIRP.Protein synthesis inhibitors cycloheximide and puromycin inducedRBM3transcripts, but cadmium chloride, H2O2, ethanol, and osmotic shock had no effect. Combined with the different tissue distribution of expression, these results suggest that RBM3 and CIRP play distinct roles in cold responses of human cells.
Article
A diverse set of mRNA-binding proteins (BPs) regulate local translation in neurons. However, little is known about the role(s) played by a family of cold-inducible, glycine-rich mRNA-BPs. Unlike neuronal mRNA-BPs characterized thus far, these proteins are induced by hypothermia and are comprised of one RNA recognition motif and an adjacent arginine- and glycine-rich domain. We studied the expression and function of the RNA-binding motif protein 3 (RBM3), a member of this family, in neurons. RBM3 was expressed in multiple brain regions, with the highest levels in cerebellum and olfactory bulb. In dissociated neurons, RBM3 was observed in nuclei and in a heterogeneous population of granules within dendrites. In sucrose gradient assays, RBM3 cofractionated with heavy mRNA granules and multiple components of the translation machinery. Two alternatively spliced RBM3 isoforms that differed by a single arginine residue were identified in neurons; both were post-translationally modified. The variant lacking the spliced arginine exhibited a higher dendritic localization and was the only isoform present in astrocytes. When overexpressed in neuronal cell lines, RBM3 isoforms-enhanced global translation, the formation of active polysomes, and the activation of initiation factors. These data suggest that RBM3 plays a distinctive role in enhancing translation in neurons.