Basic Sciences

Loss of Ribosomal RACK1 (Receptor for Activated Protein

Kinase C 1) Induced by Phosphorylation at T50 Alleviates
Cerebral Ischemia-Reperfusion Injury in Rats
Xiang Li, MM*; Jinquan Li, MD*; Jinhong Qian, MM; Dongping Zhang, MM; Haitao Shen, MM;
Xiang Li, PhD; Haiying Li, PhD; Gang Chen, MD, PhD

Background and Purpose—RACK1 (receptor for activated protein kinase C 1) is an integral component of ribosomes with
neuroprotective functions. The goal of this study was to determine the role of RACK1 in cerebral ischemia-reperfusion
(I/R) injury and the underlying mechanisms.
Methods—A middle cerebral artery occlusion/reperfusion model in adult male Sprague Dawley rats (250–280 g) was
established, and cultured neurons were exposed to oxygen-glucose deprivation/reoxygenation to mimic I/R injury in
vitro. Expression vectors encoding wild-type RACK1 and RACK1 with T50A mutation (T50A) were constructed and
administered to rats by intracerebroventricular injection.
Results—The potential role of RACK1 in cerebral I/R injury was confirmed by the decreased protein levels of RACK1
within penumbra tissue, especially of neurons. Second, there was an increase in the phosphorylation ratio of RACK1
at the threonine/serine residues at 1.5 hours after middle cerebral artery occlusion onset. Third, based on site-specific
mutagenesis, we identified T50 as a key site for RACK1 phosphorylation during I/R. Fourth, wild-type RACK1
overexpression reduced infarct size, neuronal death, neuronal tissue loss, and neurobehavioral dysfunction, while RACK1
(T50A) overexpression exerted opposite effects. Finally, we found that RACK1 phosphorylation at T50 induced a loss of
ribosomal RACK1, which switched RACK1 from beclin-1 translation inhibition to autophagy induction following I/R.
Conclusions—RACK1 phosphorylation may be a potential intervention target for neurons during I/R; thus, exogenous
supplementation of RACK1 may be a novel approach for ameliorating I/R injury.
Visual Overview—An online visual overview is available for this article.   (Stroke. 2019;50:162-171. DOI: 10.1161/
STROKEAHA.118.022404.)
Downloaded from http://ahajournals.org by on January 1, 2019

Key Words: beclin-1 ◼ brain ischemia ◼ phosphorylation ◼ protein kinase C ◼ reperfusion injury

A s a main type of stroke and second most common cause

of death worldwide, acute ischemic stroke has long been
recognized as having poor outcomes despite suitable medical
care.1–4 Ischemic neurons start dying <5 minutes after the ox-
ygen supply disappears.5 The most effective measure for rescu-
ing ischemic tissue is early recanalization,6 and the penumbra
surrounding ischemia is the important target area for interven-
tion treatment.7,8 However, after recanalization, the penumbra
experiences ischemia/reperfusion (I/R)-induced injury.9–11
RACK1 (receptor for activated protein kinase C 1), as a
member of the tryptophan-aspartate (WD) family, was first
described as an intracellular receptor of PKC (protein kinase
C).12 Similar to other WD domain proteins, RACK1 works by
binding to other proteins or complexes, and many studies have
proved that RACK1 has multitudinous functions in nervous
system.13 Autophagy is an important biological mechanism for
maintaining cellular homeostasis, and under various stressors, is
immediately upregulated, leading to cell survival.14 In cerebral
ischemic stroke, autophagy is regarded as a neuroprotective first-
aid measure, as beclin-1, a key autophagy-associated protein ini-
tiates autophagy to regulate neuronal survival.15 Some reports
have shown that RACK1 promotes autophagy by enhancing
autophagy-initiation complex formation after phosphorylation
by AMPK (AMP-activated protein kinase) in the liver.16 In addi-
tion, it has been demonstrated that RACK1 is a ribosomal pro-
tein that regulates the translation process.17,18 However the role
of RACK1 in cerebral ischemic stroke is unknown.
In this study, we determined the function of RACK1 in
cerebral ischemic stroke, as well as the potential underlying
mechanisms.
Methods
Disclosure Statements
The article adheres to the American Heart Association Journals’
implementation of the Transparency and Openness Promotion

Received June 2, 2018; final revision received October 31, 2018; accepted November 9, 2018.
From the Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, China.
*X. Li and Dr J. Li contributed equally to this work.
The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.118.022404.
Correspondence to Gang Chen, MD, PhD, or Haiying Li, PhD, Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated
Hospital of Soochow University. Email nju_neurosurgery@163.com
© 2018 American Heart Association, Inc.
Stroke is available at https://www.ahajournals.org/journal/str DOI: 10.1161/STROKEAHA.118.022404

162
 Li et al   RACK1 Phosphorylation in Brain I/R Injury   163
Guidelines. The data that support the findings of this study are avail-
able from the corresponding author upon reasonable request.
Animals
All experiments were approved by the Ethics Committee of the First
Affiliated Hospital of Soochow University (Jiangsu Province, China),
and were in accordance with Animal Research: Reporting of In Vivo
Experiments guidelines. Adult male Sprague Dawley rats weighing
250 to 280 g were purchased from the Animal Center of Chinese
Academy of Sciences (Shanghai, China). The rats were housed at a
constant temperature (23°C) and relative humidity (40%) under a reg-
ular light/dark schedule. Food and water were available ad libitum.
Sample sizes were determined by power analysis during the animal
ethics dossier application.
Establishing a Model of Experimental Middle
Cerebral Artery Occlusion/Reperfusion in Sprague
Dawley rats
The model of experimental middle cerebral artery occlusion/reper-
fusion (MCAO/R) experimental model was established in Sprague
Dawley rats as previously described.19 Sprague Dawley rats were
anesthetized (4% chloral hydrate, 10 mL/kg, i.p.) and fixed in a ste-
reotaxic apparatus (ZH-Lanxing B type stereotaxic frame, Anhui
Zhenghua Biological Equipment Co Ltd, Anhui, China). The rat was
placed supine on a heating pad to maintain temperature at ≈27°C to
35°C. In this study, all the MCAO/R surgeries were performed by
trained and experienced investigators. As shown in Figure IA in the
online-only Data Supplement, triphenyl tetrazolium chloride staining
of rat brain slices at acute phase postsurgery further indicated that the
establishment of MCAO/R model is mature and stable in our research
group. In addition, neurological symptoms were evaluated according
to the 4-level assessment method described by Bederson et al.20 At
0.5 hour after the rat woke up from anesthesia, postsurgery rats with
Downloaded from http://ahajournals.org by on January 1, 2019
neurological symptoms up to levels 1 to 3 were included for further
investigations in this study. In this model, there is a penumbra (peri-
infarct) region around the ischemic core that can be rescued.7,8 This
study focused on the role of RACK1 in this penumbra region.
Cell Culture
As previously reported,21 primary cerebral neurons were obtained
from 17-day-old rat embryos.
Establishing an In Vitro Oxygen-Glucose
Deprivation/Reoxygenation Model
For details, please see the online-only Data Supplement.
Experimental Groups and Study Design
Part 1: Time-course analysis of the protein and phosphorylation lev-
els of RACK1 after I/R in vivo (Figure IB in the online-only Data
Supplement).
Part 2: Roles of RACK1 in I/R injury (Figure IC in the online-
only Data Supplement).
Part 3: Mechanisms underlying RACK1 function during I/R
(Figure ID in the online-only Data Supplement).
Random Number Generator (Stat Trek) was used for selecting
random samples, which is available on the website: http://stattrek.
com/statistics/random-number-generator.aspx. All the quantification
and statistical analysis were performed by an observer who was blind
to the experimental condition.
Antibodies and Drugs
For details, please see the online-only Data Supplement.
Western Blot Analysis
For details, please see the online-only Data Supplement.
Immunoprecipitation Analysis
For details, please see the online-only Data Supplement.
Immunofluorescence Microscopy
For details, please see the online-only Data Supplement.
Ribosomal Protein Extraction
For details, please see the online-only Data Supplement.
Construction of Expression Plasmids
and Site-Directed Mutagenesis
For details, please see the online-only Data Supplement.
Transfection
For details, please see the online-only Data Supplement.
Measurements of Infarct Volume
For details, please see the online-only Data Supplement.
Brain Edema
For details, please see the online-only Data Supplement.
Terminal Deoxynucleotidyl Transferase
dUTP Nick End Labeling Staining
For details, please see the online-only Data Supplement.
Fluoro-Jade B Staining
For details, please see the online-only Data Supplement.
Lactate Dehydrogenase Assay
For details, please see the online-only Data Supplement.
Hoechst Staining
For details, please see the online-only Data Supplement.
Morris Water Maze
For details, please see the online-only Data Supplement.
Statistical Analysis
All data were statistically analyzed using GraphPad Prism 7 and pre-
sented as the mean±standard deviation. Statistical comparisons be-
tween groups were performed using 1- or 2-way ANOVA and post
hoc least significant difference tests for multiple comparisons. P
<0.05 were considered statistically significant.
Results
General Observation
The mortality rate of rats in the sham group was 0% (0/42
rats) and was 11.2% (25/223 rats) in the MCAO and MCAO/R
groups.
RACK1 Protein Level Decreases in
Penumbra Neurons After Ischemia
To evaluate the protein levels of RACK1 after MCAO and
MCAO/R, western blot analysis of protein samples from
penumbra tissue was performed. Compared with the sham
group, the protein level of RACK1 was significantly reduced
 164  Stroke  January 2019

starting at 1.5 hours after MCAO, lasted until 4 hours after
MCAO/R, and then gradually rebounded (Figure 1A). Then,
double immunofluorescence was performed to distinguish
changes in cell type-specificity of RACK1 expression after
ischemia. The results showed that RACK1 was mainly
expressed in neurons and microglia in the sham group
(Figure 1B and 1C), whereas it was hardly detected in astro-
cytes under both sham and ischemic conditions (Figure 1D).
Compared with the sham group, the protein levels of RACK1
in neurons showed a significant decrease at 1.5 hours after
MCAO and 4 hours after MCAO/R, while both MCAO and
MCAO/R did not induce significant changes in RACK1 pro-
tein levels in microglia (Figure 1C; Figure II in the online-
only Data Supplement).
I/R Increases the Phosphorylation
of RACK1 at T50
Previous studies have demonstrated that under I/R conditions,
AMPK is activated and phosphorylates RACK1 at T50 in the
liver.16 We determined whether cerebral ischemia could affect
the phosphorylation status of RACK1. To this end, immuno-
precipitated RACK1 was subjected to western blot analysis
using an antibody against phosphorylated serine (Ser) and
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threonine (Thr) residues. The results showed an increased
phosphorylation of RACK1 at 1.5 hours after MCAO and 4
hours after MCAO/R (Figure 2A). To elucidate the primary
phosphorylation site of RACK1, rat RACK1 cDNA con-
structs with mutation at a possible key phosphorylation site
(T50) were prepared. First, GFP (green fluorescent protein)-
RACK1 and GFP-RACK1 (T50A mutation) were equally
expressed in the sham group (Figure 2B). Then, consistent
with the trend of endogenous RACK1 protein levels, the pro-
tein level of GFP-RACK1 was reduced by ischemia treat-
ment, which was almost completely abolished by the T50A
mutation (Figure 2B). In addition, wild-type GFP-RACK1
showed a significant increase in phosphorylation at Ser and
Thr residues following ischemia, while the T50A mutant sig-
nificantly abolished phosphorylation (Figure 2C). Finally,
we tested the effects of the AMPK inhibitor Dorsomorphin
dihydrochloride on the phosphorylation of RACK1 in cul-
tured neurons exposed to oxygen-glucose deprivation (OGD;
Figure 2D). Consistent with the results of in vivo experi-
ments (Figure 2A), OGD significantly induced the phos-
phorylation of RACK1 at Ser and Thr residues (Figure 2D).
Compared with the OGD group, AMPK inhibition signifi-
cantly decreased Ser and Thr phosphorylation of RACK1,

Figure 1. The protein level of RACK1 (receptor for activated protein kinase C 1) in penumbra tissue. A, Western blot analysis and quantification of RACK1 lev-
els in penumbra tissue. *P<0.05, **P<0.01 vs sham group, ##P<0.01, $$P<0.01, n=6. B, Double immunofluorescence analysis was performed with antibodies
against RACK1 (green) and a neuronal marker (NeuN, red) in brain sections. Nuclei were fluorescently labeled with DAPI (blue). Scale bar =30 μm. The relative
fluorescent intensity of RACK1 in neurons is shown below. **P<0.01 vs sham group, n=6. C, Double immunofluorescence analysis was performed with anti-
bodies against RACK1 (green) and a microglia marker (CD11b, red) in brain sections. Nuclei were fluorescently labeled with DAPI (blue). Scale bar =30 μm. D,
Double immunofluorescence analysis was performed with antibodies against RACK1 (green) and an astrocyte marker (GFAP [glial fibrillary acidic protein], red)
in brain sections. Nuclei were fluorescently labeled with DAPI (blue). Scale bar =30 μm.
 Li et al   RACK1 Phosphorylation in Brain I/R Injury   165
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Figure 2. Effects of ischemia on RACK1 (receptor for activated protein kinase C 1) phosphorylation. A, Immunoprecipitation (IP) and quantification of the
phosphorylation level of RACK1 in penumbra tissue. **P<0.01 vs sham group, n=6. B, Western blots of transfection efficiency. **P<0.01, NS, no significant,
n=6. C, IP of tissue lysates with GFP (green fluorescent protein) antibodies to enrich GFP-RACK1 from total proteins. Western blots of IP showed the phos-
phorylation of wild-type GFP-RACK1 and GFP-RACK1 (T50A). **P<0.01, ##P<0.01, n=6. D, IP of tissue lysates with RACK1 antibody to enrich RACK1 from
total proteins. Western blots of IP showed RACK1 phosphorylation. **P<0.01, ##P<0.01, n=6.

suggesting that the increased RACK1 phosphorylation after
ischemia is at least partially mediated by AMPK. Together,
these results suggest that T50 is a key site for ischemia-
induced RACK1 phosphorylation in neurons, which may be
related to RACK1 degradation.
Phosphorylation at T50 Exerts a Functional
Switch of RACK1 Under I/R Stimulus
Triphenyl tetrazolium chloride staining showed that wild-
type GFP-RACK1 overexpression reduced the infarct
volume, while GFP-RACK1 (T50A) exerted opposite effects
(Figure 3A). Compared with the sham group, the brain sam-
ples in the MCAO/R group had a higher brain water content,
which was significantly attenuated by wild-type GFP-RACK1
overexpression but not GFP-RACK1 (T50A) overexpres-
sion (Figure 3B). In addition, Fluoro-Jade B and terminal
deoxynucleotidyl transferase dUTP nick end labeling stain-
ing also showed the rescue effects of wild-type GFP-RACK1
overexpression on neuronal degeneration and death, while
GFP-RACK1 (T50A) overexpression exerted the opposite
effects (Figure 3C and 3D). We further confirmed the roles of
RACK1 in cultured neurons exposed to OGD/reoxygenation
(OGD/R) stimulus.
The transfection efficiency of RACK1 overexpression
(about 40%) was showed by fluorescence assay in cultured
neurons (Figure IIIA in the online-only Data Supplement).
Hoechst staining showed that there were almost no apop-
totic cells in the normal group, while the apoptotic index was
significantly higher in the OGD/R group (Figure IIIB in the
online-only Data Supplement). Compared with the OGD/R
group, the apoptotic index was significantly attenuated by
wild-type GFP-RACK1 overexpression and exacerbated by
GFP-RACK1 (T50A) overexpression. Consistently, the ne-
crosis index tested by the lactate dehydrogenase assay showed
the same trend as that in the apoptotic index (Figure IIIC in the
online-only Data Supplement). Together, these results suggest
that phosphorylation at T50 may exert a functional switch of
RACK1 in I/R-induced neuronal injury.
 166  Stroke  January 2019
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Figure 3. Effects of RACK1 (receptor for activated protein kinase C 1) overexpression on brain injury at 24 h after MCAO/R surgery. A, Triphenyl tetrazolium
chloride (TTC) staining. ##P<0.01, $$P<0.01, @P<0.05, n=6. B, Brain water content. ***P<0.001 vs sham group, ###P<0.001, $$$P<0.001, n=6. C, Fluoro-
Jade B (FJB) staining. ***P<0.001 vs sham group, ##P<0.01, $$P<0.01, @P<0.05, n=6. Scale bar = 60 μm. D, Terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL) staining. ***P<0.001 vs sham group, ##P<0.01, $$P<0.01, n=6. Scale bar = 60 μm.

RACK1 Overexpression Improves marker MAP2 (microtubule-associated protein 2) to measured

Long-Term Recovery of Neurological
Function After MCAO/R
To further test the long-term effect of RACK1 overexpres-
sion on stroke outcomes, Morris water maze tests were per-
formed on rats from 22 to 26 days post-MCAO/R. As shown
in Figure 4A and 4B, Morris water maze test revealed that
RACK1 overexpression facilitated spatial learning and
memory after MCAO/R, as evidenced by less time spent find-
ing the hidden platform in the RACK1 overexpression group.
In contrast, MCAO/R + RACK1 (T50A) rats needed more
time to find the hidden platform. At 48 hours after the Morris
water maze test, rats were euthanized, and the brains were
collected for immunofluorescence staining of neuron-specific
neuronal tissue loss at 28 days after MCAO/R (Figure 4C and
4D). Over a 4-week period after MCAO/R, rats with RACK1
overexpression exhibited less neuronal tissue loss than
MCAO/R + vector group in coronal hippocampal sections,
while MCAO/R+RACK1 (T50A) rats showed significantly
increased neuronal tissue loss.
RACK1 Phosphorylation at T50 Induces Loss
of Ribosomal RACK1 After Ischemia
As shown in Figure 2B, RACK1 phosphorylation at T50 in-
duced by ischemia may be related to RACK1 degradation. It is
well known that RACK1 is an important component of the ri-
bosomal 40S subunit.22 To examine the ribosomal localization
 Li et al   RACK1 Phosphorylation in Brain I/R Injury   167

Figure 4. Effects of RACK1 (receptor for activated protein kinase C 1) overexpression on long-term recovery of neurological function after middle cerebral ar-
tery occlusion/reperfusion (MCAO/R). A, The typical swim path of rats in the Morris water maze test at 26 d after MCAO/R. B, Time to reach the submerged
platform in the water maze 22 to 26 d after MCAO/R. *P<0.05, **P<0.01, n=6. C and D, Immunofluorescence analysis was performed with antibody against
MAP2 (green) in coronal hippocampal sections 28 d after MCAO/R. ##P<0.01 vs MCAO/R group, $$P<0.01 vs MCAO/R + GFP (green fluorescent protein)-
RACK1group, @P<0.05, n=6.

of RACK1, the western blot assay was conducted using ribo-
somal protein extract. The results showed a profound decrease
in RACK1 protein level in ribosomes at 1.5 hours after OGD
and 4 hours after OGD/R (Figure 5A). Consistently, in the con-
trol group, the immunofluorescence assay revealed a clear dis-
tribution of RACK1 within ribosomes, whereas it was mainly
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ribosomes (Figure 5C). Consistent with the trend of endogenous
RACK1 (Figure 5A), the protein level of GFP-RACK1 in ribo-
somes was significantly reduced by OGD, which was almost
completely abolished by T50A mutation (Figure 5C). The im-
munofluorescence results further confirmed the OGD-induced
translocation of GFP-RACK1 from ribosomes to axons, which

located at axons at 1.5 hours after OGD and 4 hours after was inhibited by T50A mutation (Figure 5D). Together, these
OGD/R (Figure 5B). Under normal conditions, equal amounts results suggest that OGD-induced RACK1 phosphorylation at
of GFP-RACK1 and GFP-RACK1 (T50A) were located in the T50 promotes the loss of ribosomal RACK1.

Figure 5. Subcellular localization of RACK1 (receptor for activated protein kinase C 1) in cultured neurons after oxygen-glucose deprivation (OGD) or OGD/
reoxygenation (OGD/R) onset. A, Western blots of ribosomal RACK1. *P<0.05 vs control group, n=6. B, Double immunofluorescence analysis was performed
with antibody for RACK1 (green) and ribosomal marker (S6, red) in cultured neurons. Nuclei were fluorescently labeled with DAPI (blue). Scale bar = 60 μm. C,
Western blots of transfection efficiency. **P<0.01, N.S., no significant, n=6. D, Immunofluorescence analysis was performed with ribosomal marker (S6, red)
in cultured neurons. Nuclei were fluorescently labeled with DAPI (blue). Scale bar = 60 μm.
 168  Stroke  January 2019

RACK1 Phosphorylation at T50 Switches
RACK1 Function From Inhibiting Beclin-1
Translation in Ribosomes to Promoting
Beclin-1 Binding in Axons After Ischemia
Previous reports have shown that RACK1 can work as a
signal-transduction platform in the translation process to
regulate protein translation in various cell types,23,24 and its
depletion in the ribosome may induce autophagy.25 In addi-
tion, induction of RACK1-mediated autophagy is corre-
lated with the upregulation of beclin-1 in RACK1-deficient
ribosomes.26 Here, we found that, OGD/R induced an in-
crease in the protein level of beclin-1 in cultured neurons
(Figure 6A). Under normal or OGD/R conditions, both GFP-
RACK1 and GFP-RACK1 (T50A) overexpression induced
a significant decrease in beclin-1 protein level in cultured
neurons. However, under OGD/R conditions, GFP-RACK1
(T50A) overexpression exerted more significant effects than
GFP-RACK1 overexpression in decreasing beclin-1 protein
levels, whereas there was only a slight difference between
the levels under normal conditions. These results suggest
that RACK1 inhibits beclin1 translation in neurons, which
is reversed by its phosphorylation at T50, especially under
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OGD/R conditions. It has also been reported that the phos-
phorylation of RACK1 promotes autophagy by enhanc-
ing formation of the autophagy-initiation complex through
interactions with beclin-1-Atg14L-Vps34-Vps15.16 Thus, we
performed immunofluorescence to verify the interaction be-
tween RACK1 and beclin-1 under I/R injury. At 1.5 hours
after OGD and 4 hours after OGD/R, RACK1 was abundant
in axons and showed more RACK1/beclin-1 colocalization
than the control group (Figure 6B; Figure IV in the online-
only Data Supplement). These results suggest that in neurons,
OGD-induced RACK1 phosphorylation at T50 switches the
function of RACK1 from inhibiting beclin-1 translation in
ribosomes to promoting beclin-1 binding in axons.
RACK1 Phosphorylation at T50 Promotes
Autophagy Induction After Ischemia
When neurons suffer from ischemia, the activation of autoph-
agy is an important defensive strategy for alleviating I/R
injury.27 Moreover, beclin-1 plays an important role in autoph-
agy initiation. Therefore, we tested the ratio of LC3II/I to eval-
uate the role of RACK1 in autophagy under I/R conditions.
As shown in Figure 6C, the ratio of LC3II/I was significantly

Figure 6. Effects of RACK1 (receptor for activated protein kinase C 1) overexpression on beclin-1 and autophagy induction. A, Western blots of beclin-1.
*P<0.05, ##P<0.01, $P<0.05, $$P<0.01, @P<0.05, n=6. B, Double immunofluorescence analysis was performed with antibodies for RACK1 (green) and
beclin-1 (red) in cultured neurons. Nuclei were fluorescently labeled with DAPI (blue). Scale bar = 60 μm. C, Western blots of LC3. *P<0.05, #P<0.05,
##P<0.01, n=6. D, Schematic representations of potential mechanisms of RACK1 actions in ischemia-reperfusion (I/R) injury.
 Li et al   RACK1 Phosphorylation in Brain I/R Injury   169
increased after OGD/R. Under normal conditions, both GFP-
RACK1 and GFP-RACK1 (T50A) overexpression induced a
significant decrease in the ratio of LC3II/I in cultured neu-
rons. However, under OGD/R conditions, wild-type RACK1
overexpression induced a significant increase in the ratio of
LC3II/I, while RACK1 (T50A) overexpression exerted op-
posite effects. These results suggest that, in neuron exposed
to OGD/R conditions, RACK1 initiates autophagy in its T50
phosphorylation-dependent manner.
Discussion
Cerebral ischemic stroke as a main cerebrovascular disease
has occupied a large amount of medical resources for a long
time. However, its clinical prognosis remains unsatisfactory
because of inevitable I/R injury. This study showed the role
of RACK1 in the pathogenesis of cerebral I/R injury in a rat
MCAO/R model for the first time. We found decreased RACK1
protein expression and increased RACK1 phosphorylation in
neurons in a MCAO/R model. Through site-directed mutagen-
esis of T50 to alanine in RACK1, we found that wild-type
RACK1 overexpression rescued I/R-induced brain injury,
while RACK1 (T50A) overexpression exerted adverse effects.
As previously reported, RACK1 is a ribosome scaffold pro-
tein.23 We found that I/R conditions induced a loss of RACK1
in the ribosomes, which was controlled by AMPK-mediated
RACK1 phosphorylation at T50. In addition, phosphoryl-
ation at T50 after ischemia switched RACK1 function from
inhibition of beclin-1 translation in ribosomes to promotion
of beclin-1 binding in axons, which subsequently promoted
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autophagy induction and improved I/R injury (Figure 6D). A
complete understanding of RACK1-mediated physiological
changes because of different phosphorylation states could
lead to new models and therapeutic approaches for the treat-
ment of cerebrovascular disease.
In this study, we focused on the phosphorylation status of
RACK1 at T50 and its function in regulating autophagy after
I/R. To this end, GFP was fused with the exogenous RACK1
plasmid to differentiate between endogenous and exogenous
RACK1. As shown in Figures 2 and 5, exogenous GFP-
RACK1 showed similar changes as endogenous RACK1, sug-
gesting that the GFP tag did not induce unwanted side effects
in the study.
In addition to phosphorylation at T50, there are other
posttranslational modifications of RACK1. For example, ty-
rosine phosphorylation and protein sumoylation of RACK1
have been implicated in mediating its protein-protein inter-
actions.28,29 In this study, phosphorylation at T50 played an
important role under I/R conditions. However, whether other
sites of RACK1 phosphorylation or sumoylation participate
in I/R injury needs further investigation. We also observed
that the protein level of RACK1 in penumbra was clearly
decreased after MCAO (Figure 1). Furthermore, the protein
level of wild-type exogenous GFP-RACK1 was also signifi-
cantly reduced, while mutant exogenous GFP-RACK1 T50A
was unchanged (Figure 2B). These results suggest that the
decrease of RACK1 expression under ischemia conditions is
regulated by the increased degradation level of the protein,
which is possibly caused by its phosphorylation at T50.
In this study, we investigated the function of RACK1
under I/R conditions. As an intracellular adaptor protein,
RACK1 is a member of the WD repeat family, which has var-
ious roles in regulating several major nervous system path-
ways. RACK1 can regulate ion channel fluxes and receptor
desensitization by interacting with protein kinase C, which
may be related to memory phenomena involved in neurode-
generative disorders.30,31 In addition, RACK1 can also partic-
ipate in neurotransmitter neurotransmission.32 As a core 40S
ribosomal protein, an electron microscopy study showed that
RACK1 is localized next to the mRNA exit channel of the
ribosome,23 and exposes the WD-repeats as a platform for
interactions with manifold kinases and receptors.33 RACK1
interaction with the translation factor eIF6 and loading ribo-
somal 60S subunit with eIF6 may cause a translational block
and impairment of 80S formation, whereas this inhibitory
effect can be reversed by RACK1 expression.34 In this study,
we found that, under normal conditions, RACK1 localized in
ribosomes functions as a translational inhibitor of beclin-1.
However, when I/R occurs, RACK1 is phosphorylated and
moves from the ribosome to axon, accompanied by the release
of translation inhibition.
Autophagy is a double-edged sword, in that extreme loss
or gain of autophagy may be detrimental to stroke progres-
sion. As shown in Figure 1A, the fluctuation in the protein
level of RACK1 after MCAO suggests that there is a flex-
ible and sensitive regulation of RACK1. It is well known
that 1 protein level is regulated by both the expression and
the degradation of the protein. We also observed that, at 1.5
hours after MCAO, the protein level of wild-type exogenous
GFP-RACK1 was significantly reduced, while mutant ex-
ogenous GFP-RACK1 T50A was unchanged (Figure 2B).
These results suggested that the overexpression of RACK1
induced by plasmid transfection is transient and the decrease
of RACK1 under ischemia conditions may be because of
increased degradation, which is possibly caused by its phos-
phorylation at T50. The loss of ribosomal RACK1 switched
RACK1’s function from beclin-1 translation inhibition to
autophagy, which may eventually implement RACK1 degra-
dation and exert as a negative regulation of RACK1. These
results mean such turned on switched for autophagic activity
mediated by RACK1 is transient and moderate.
As shown in Figure 1, under ischemia conditions, RACK1
expression is predominantly altered in neurons. Although neu-
rons have always been the focus for almost all of neurosci-
ence research, the concept of neurovascular unit has gradually
become a new paradigm for both physiology and pathology
studies in recent years.35–38 Neurons together with astro-
cytes, microglia, brain microvascular endothelial cell, peri-
cytes, and the extracellular matrix, constitute a neurovascular
unit, which is essential for the function and health of central
nervous system.36 A growing body of evidence indicates that,
after acute central nervous system injury, neurovascular unit
components undergo a program of pathophysiology changes
include tissue hypoxia, inflammatory and angiogenic activa-
tion, which jointly manifest as cerebral edema and neuronal
damage.39–41 As shown in Figure 3, RACK1 overexpression
significantly ameliorated MCAO/R-induced cerebral edema
and neuronal damage. Consistently, it was reported recently
 170  Stroke  January 2019
that, RACK1 upregulation significantly inhibited neuronal
apoptosis and brain edema at 48 hours after traumatic brain
injury.42 All these results suggest that RACK1 may be able
to promote neurovascular unit homeostasis after acute central
nervous system injury. Moreover, more comprehensive and
deeper researches are needed to draw a map of the role of
RACK in neurovascular unit.
This study also had some limitations. First, we chose
healthy adult male Sprague Dawley rats as experimental ani-
mals. However, ischemia stroke mainly occurs in humans ≈50
to 60 years of age, especially in high-risk populations with
arteriosclerosis, hypertension, hyperlipidemia, and diabetes
mellitus.43 Our experimental animals did not mimic human
high-risk populations. In addition, the roles of RACK1 in dif-
ferent sex animals should be tested in further studies. Second,
in clinical treatment, the most appropriate method for drug de-
livery is oral or intravenous infusion. However, in our study,
we used exogenous plasmid transfection through intracerebro-
ventricular injection to enhance the function of RACK1. Third,
we only researched autophagy through the interaction between
RACK1 and beclin-1 under I/R conditions. In fact, RACK1
also participates in many other signaling pathways such as
the IRE1 (inositol-requiring enzyme 1)-RACK1 axis, which
orchestrates endoplasmic reticulum stress.44 The RACK1-
ATG5 (autophagy-related 5) interaction regulates autophagy45
and suppression of RACK1-regulated apoptosis through
increasing Bcl-2 (B-cell lymphoma 2).46 Therefore, when I/R
occurs, the underlying mechanism by which RACK1 partici-
pates may exist multifariously. Future studies are needed on
Downloaded from http://ahajournals.org by on January 1, 2019
whether other signaling pathways play important roles in I/R
injury. In conclusion, the results of our experiments provide in-
sight into the rescue effects of RACK1 overexpression on I/R
injury. However, there is still a long way to go before RACK1
can be used in clinical application.
Conclusions
Ischemic stroke is becoming the leading cause of mortality
and morbidity worldwide with early recanalization as the
preferred treatment strategy. However, I/R often results in
unavoidable damage and leads to unsatisfactory clinical out-
comes. At present, penumbra rescue by attenuating I/R injury
is a treatment strategy, but there is an urgent need for more
efficacious therapies. Using a clinically relevant rat MCAO/R
model, we explored a novel neuroprotective strategy target-
ing RACK1. Phosphorylation at T50 after ischemia switched
RACK1 function from inhibition of beclin-1 translation in
ribosomes to promotion of beclin-1 binding in axons, which
subsequently promoted autophagy induction and improved
I/R injury. By promoting autophagy induction, exogenous
RACK1 and promotion of RACK1 phosphorylation at T50
may limit I/R complications and lead to beneficial neurolog-
ical outcomes.
Sources of Funding
This work was supported by the Project of Jiangsu Provincial Medical
Innovation Team (No. CXTDA2017003), Jiangsu Provincial Medical
Youth Talent (No. QNRC2016728), Suzhou Key Medical Centre
(No. Szzx201501), Scientific Department of Jiangsu Province (No.
BE2017656), and Suzhou Government (LCZX201601).
Disclosures
None.
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