Cab45s inhibits neuronal apoptosis following intracerebral hemorrhage in adult rats

Accepted Manuscript Title: Cab45s Inhibits Neuronal Apoptosis Following Intracerebral Hemorrhage in Adult Rats Authors: Jiabing Shen, Tingting Zhou, Haizhen Li, Wanyan Li, Shuyao Wang, Yan Song, Kaifu Ke, Maohong Cao PII: DOI: Reference:

S0361-9230(18)30356-3 https://doi.org/10.1016/j.brainresbull.2018.09.016 BRB 9521

To appear in:

Brain Research Bulletin

Received date: Revised date: Accepted date:

17-5-2018 17-9-2018 24-9-2018

Please cite this article as: Shen J, Zhou T, Li H, Li W, Wang S, Song Y, Ke K, Cao M, Cab45s Inhibits Neuronal Apoptosis Following Intracerebral Hemorrhage in Adult Rats, Brain Research Bulletin (2018), https://doi.org/10.1016/j.brainresbull.2018.09.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cab45s Inhibits Neuronal Apoptosis Following Intracerebral Hemorrhage in Adult Rats Jiabing Shen1, Tingting Zhou1, Haizhen Li1, Wanyan Li1, Shuyao Wang1, Yan Song2, Kaifu Ke1, ， Maohong Cao1, 

1. Department of Neurology, Affiliated Hospital of Nantong University, Nantong 226001,

IP T

Jiangsu Province, People's Republic of China 2. Department of Neurology, Nantong Hospital of Traditional Chinese Medicine, Nantong

Jiabing Shen, Tingting Zhou and Haizhen Li contributed equally to this work.

U

*

SC R

226006, Jiangsu Province, People’s Republic of China

corresponding authors:

N

Kaifu Ke()

M

A

E-mail: [email protected]

Maohong Cao ()

ED

E-mail: [email protected] Tel.: +86 25 5880 2883

CC E

Highlights

PT

Fax: +86 25 5850 9994

Up-regulation of Cab45s was located to neurons after ICH. Increased expression of Cab45s was relevant with neuronal apoptosis following ICH.

A

Enhanced Cab45s exerted its anti-apoptotic function against neuronal apoptosis following ICH. Abstract Recent studies have shown that Cab45s, belonging to the CREC family, can fight against apoptosis in the cancer cell lines. Here, we report that Cab45s may involve in 1

neuronal apoptosis at the early stage of intracerebral hemorrhage (ICH) in pathophysiology. We found that expression of Cab45s was enhanced in areas contiguous to hematoma following ICH in adult rats, and that so were the expressions of Glucose-regulated protein 78 (GRP78), pro-apoptotic Bcl-2-associated X protein (Bax) and active caspase-3. In vitro, coimmunoprecipitation analysis indicated the interaction between Cab45s and GRP78. Depletion of Cab45s attenuated the

IP T

expression of GRP78, but increased the expressions of Bax and caspase-3 in PC12 cells treated with hemin, which finally promoted apoptosis. Together, these results

SC R

reveal that Cab45s might exert its anti-apoptotic function against neuronal apoptosis. Thus, the study may provide evidences for regulating Cab45s as a potentially reliable treatment for the secondary damage following ICH.

N

U

Key words: ICH, Cab45s, Neuronal apoptosis, Rat

A

1. Introduction

Spontaneous intracerebral hemorrhage, as the second most common cause of stroke,

M

is defined that a cerebral artery bursts accidentally and the adjacent area is filled with

ED

blood [1]. Not only its high mortality and morbidity, but also its complex mechanism after hemorrhage, make it urgent to find effective therapeutic methods to protect the brain from the primary and secondary damage. What happens after ICH is a highly

PT

perplexed series of pathological processes [2]. First and foremost, during the formation of hematoma, mass effect and compression force contribute to the primary

CC E

damage. Second, the presence of hematoma is paralleled by many pathophysiological pathways, such as inflammatory response, neurotoxicity of excitatory amino acid, oxidative stress and apoptosis [3-6], which eventually result in the disruption of

A

blood-brain barrier, proliferation of astrocytes, activation of microglias and irreversible apoptosis of neurons [7-10]. Among them, neuronal apoptosis is regarded as the most crucial event, which is leaned upon meticulous pro-apoptotic and anti-apoptotic modulation [11]. Classical apoptosis is classified into two parts: extrinsic pathway and intrinsic pathway [12]. In the intrinsic pathway, mitochondrial outer membrane permeabilization (MOMP), which leads to the release of 2

pro-apoptotic proteins from the mitochondrial intermembrane space (IMS), is the crucial event driving initiator caspase activation and apoptosis. In the extrinsic pathway, death receptor ligation causes the recruitment of adaptor molecules, such as FAS-associated death domain protein (FADD), that bind, dimerize and activate an initiator caspase, caspase 8. Then, active caspase 8 directly cleaves and activates the executioner caspases, caspase 3 and caspase 7 [13]. Closely correlative to the two

IP T

pathways, caspase-3 is thought as one of the key executioners in the process of cell

apoptosis. Moreover, playing an essential role in the extrinsic pathway, active

SC R

caspase-3 is responsible for the cleavage of lots of proteins partially or totally,

including anti-apoptotic protein B, cell lymphoma-2 (Bcl-2) and Bax. However, the mechanism of neuronal apoptosis following ICH remains exploration, aiming to make

U

our limited therapies better and provide effective pharmacotherapy in new horizons.

N

Multiple proteins from the CREC family have been discovered in the last 20

A

years, including Ca2+-binding protein of 45 kDa (Cab45), reticulocalbin, ER Ca2+-binding protein of 55 kDa (ERC-55), and calumenin. Besides the original

M

secretory pathway, these proteins are also found in the cytoplasm [14]. Moreover, the dysregulation of these proteins is discovered in lots of diseases, for instance cancer,

ED

neuromuscular disease, cardiovascular disease and various treatment and ‘‘stress’’ [15-18]. With respect to Cab45, it has been reported to modulate the injury emerged

PT

by UV or ethanol in PC12 pheochromocytoma cells, which suggests that Cab45 may be related to the process of neuron-concerning disease, but the detailed molecule

CC E

mechanism is poorly understood [19]. Cab45 is consisted of three isoforms: Cab45s, Cab45G as well as Cab45C [20]. L Chen et al. has elaborated that Cab45s inhibits cell apoptosis induced by the endoplasmic reticulum (ER) stress via interplay with

A

GRP78/BiP in cancer [18]. Additionally, Cab45s is reported to promote cell proliferation and tumor progression [21]. Therefore, we speculated whether Cab45s is involved in the neuron apoptosis in molecule level. We initiated this study to find out the role of Cab45s in neuronal apoptosis following ICH. Here, we inspected the expressions of Cab45s, caspase-3, Bax, and GRP78/BiP in the PC12 cells treated with hemin and the ICH rat models. Our results 3

revealed the functions of Cab45s in the central nervous system (CNS) and the involvement of Cab45s in the molecular events following ICH.

2. Materials and Methods 2.1. Animal and the ICH Model All the animal care and experiments were performed according to the National

IP T

Institutes of Health Guidelines for the Care and Use of Laboratory Animals, which

was published by National Research Council in 1996. For the rat experiments,

SC R

220-275 g male Sprague-Dawley rats were approved by the Animal Care and Use Committee in Nantong University. With a cycle of 12-h light and 12-h darkness,

constant temperature 22-24°C, as well as relative humidity 55-60%, animals (5 rats

U

per cage) had free access to sufficient food and drinking water.

N

First of all, rats were anesthetized with mixture containing ketamine (100 mg/kg;

A

sigma) and xylazine (10 mg/kg; sigma) (2:1 v/v, intraperitoneal injection). Then, they were conducted in accordance with a previous report with minor modifications [22].

M

In short, the rats were randomly assigned into two groups: sham-operation and ICH. Next, each rat was placed in a stereotaxic frame and got the injection of saline or the

ED

autologous blood gathered from its tail tip by a microsyringe (50 μL). The 50 μL liquid was infused into right basal ganglia at coordinates 0.2 mm anterior, 5.5 mm

PT

ventral, 3.5 mm lateral to the bregma, at a rate of 5 μL/min. After injection, the needle of microsyringe was left there for 5 min. The striatum proteins encircling the

CC E

hematoma (2 mm) were extracted from the experimental animals (n=5 per time point) sacrificed at the time points of 6 h, 12 h, 1 d, 2 d, 3 d, 5 d, 7 d and 14 d following ICH.

A

Additional animals were killed for pathologic studies.

2.2. Behavioral Tests 2.2.1. Forelimb Placing Test Forelimb placing test was performed in a blind fashion as previously described [23]. Held by torsos, the rats were allowed to hang the forelimbs free. Each forelimb’s movements were induced by brushing the contralateral vibrissae at the corner of a 4

countertop. The healthy and sham-operated rats quickly placed their contralateral forelimbs onto the countertop after stimulation to the vibrissae. Due to the degree of impairment, the ICH rat may fail to place the left paralyzed forelimb which was contralateral to the injury. The test was designed as 6 units with 30 min interval. In every unit, the number of the left forelimb’s positive reaction (C) was counted out of

IP T

ten (T). In other words, the score of forelimb placing test was calculated as C/T [24].

2.2.2. Corner Turn Test

SC R

Corner turn test was also conducted blindly. The rats were allowed to proceed into a

corner with angle of 30°, then they would turn left or right to leave the corner. Only turns involving complete rearing along the wall were counted. In the light of the

U

impairment, ICH rats would trend towards turning to the ipsilateral side of injury. A

N

unit was made up of 10 times (T) and the rats would be tested for 6 units in total. The

M

2.3. Cell Cultures and Stimulation

A

percentage of right turns (I) in the corner turn test was marked as I/T [25].

ED

Rat pheochromocytoma (PC12) cells were purchased from the Institute of Biochemistry and Cell Biology in the Chinese Academy of Science (Shanghai, China). Under the environment of 37°C along with 5% CO2 humidify, PC12 cells were

PT

cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (v/v). PC12 cells were incubated in DMEM with a low

CC E

concentration of horse serum for 24 h in advance, then they were exposure to 100 μmol/L hemin for different time (1.5 h, 3 h, 6 h, 12 h and 24 h).

A

2.4. Cell and Tissue Protein Extraction PC12 cells were lysed on ice for 30 min using RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China). Rats at different time points after operation were sacrificed by intraperitoneal injection of anesthetic containing ketamine (200 mg/kg; Sigma) and xylazine (20 mg/kg; Sigma). Then, the whole brains were quickly extracted out. Apart from the hematoma itself, the tissue of striatum encircling the 5

hematoma (2 mm) was obtained, cut into pieces, and lysed in RIPA lysis buffer. Following, the cell or tissue proteins were centrifugated at a speed of 12, 000 rpm for 15 min at 4°C. The supernatant was collected, and the protein concentration was calculated with a BCA protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). Finally, proteins added with 2× SDS loading buffer were boiled for 5 min at

IP T

100°C.

2.5. Western Blot Analysis

SC R

Each sample from cell or tissue lysates was subjected to 10% SDS-polyacrylamide gel

electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene diflouride (PVDF) filter membrane. Blocked with 5% bovine serum albumin at room

U

temperature for 2 h, the membrane was incubated overnight with primary antibodies

N

against Cab45s (anti-rabbit, 1:1000; Abcam), active caspase-3 (anti-rabbit, 1:1000; Cell Signaling Technology), GRP78 (anti-mouse, 1:500; Cell Signaling Technology),

A

Bax (anti-rabbit, 1:1000; Cell Signaling Technology), β-actin (anti-rabbit, 1:1000;

M

Sigma) at 4°C. After 3 times’ washing with PBST and incubation with secondary

ED

antibodies for 120 min at room temperature, the membrane was washed in PBST again for 3 times and then scanned with the Odyssey infrared imaging system (LI-COR Bioscience). All results shown in the test were repeated at least three times

PT

independently.

CC E

2.6. Immunoprecipitation

PC12 cell lysates were prepared in RIPA buffer and then centrifuged at 12, 000 rpm at 4°C for 15 min to collect the supernatant. Total 100 μL (1 μg/μL) cell lysates were

A

incubated with anti-Cab45s (anti-rabbit; Abcam) or anti-GRP78 (anti-mouse; Cell Signaling Technology) primary antibody on a rocker at 4°C overnight. Next, 50 μL protein G-Sepharose (Sigma, 1:1 slush in PBS) was added, then the mixture was rocked for 2 h at 4°C. The precipitate collected was washed gently for 3 times with 1 ml PBS, resuspended in 50 μL 2× SDS loading buffer and boiled for 5 min at 100°C. Finally, western blot analysis was operated as described above. 6

2.7. siRNAs and Transfection The

small

interfering

RNA

(siRNA)

target

sequence

of

Cab45s

was

5’-TCAAGTACAGCGAGTTCT-3’ (Sigma, St. Louis, MO, USA). The transfection of Cab45s siRNA vector or the nonspecific vector was performed, using lipofectamine 2000 reagent (Invitrogen) in antibiotic-free OptiMEM (Invitrogen). Transfected cells

IP T

were cultured for 48 h before the subsequent experiments.

SC R

2.8. Sections

Rats at 2 days after surgical in ICH or sham-operated group were deeply anesthetized with agents containing ketamine (200 mg/kg; Sigma) and xylazine (20 mg/kg; Sigma)

U

(2:1 v/v, intraperitoneal injection), perfused with 0.9% saline and gone on with 4%

N

paraformaldehyde (PFA) transcardially. Shortly, the whole brains were taken out,

A

immersed in PFA at room temperature for 1 day, transferred to 20% sucrose for 2-3 days and followed by 30% sucrose for another 2-3 days at 4°C. The brain sections

M

were cut coronally on a freezing microtome and kept in −20°C until use. The

ED

thickness of brain sections was 5-7 μm.

2.9. Immunofluorescent Staining

PT

The sections were baked in 37°C incubator for 1 h and rinsed in PBS for 10 min. Next, they were treated with 1% Triton-X 100 for another 10 min, washed with PBS for 3

CC E

times and confined to 10% bovine serum albumin in PBS for 2 h. Primary antibodies incubated overnight at 4°C were anti-Cab45s (anti-rabbit; 1:500; Abcam), anti-NeuN (anti-rabbit or anti-mouse; 1:300; Abcam), anti-GFAP (anti-mouse; 1:500; Sigma),

A

anti-CD11b (anti-mouse; 1:500; Sigma), and anti-active caspase-3 (anti-mouse; 1:500; Cell Signaling Technology). After 3-time washing with PBST, the sections were exposed to fluorescent secondary antibodies (1:1000; Sigma) at room temperature for 2 h in the darkness. Images around the hematoma in striatum were captured in a Leica fluorescence microscope.

7

2.10. Immunohistochemistry The sections were pretreated as previously. After 10-min exposure to 3% H2O2, the sections were incubated with blocking solution for 10 min, reacted with antibody against Cab45s for 2 h, treated with biotinylated goat anti-polyvalent for 10 min as well as subsequent streptavidin peroxidase for 10 min. Every step was operated at room temperature and followed by washing with PBS. Then, the sections were

IP T

revealed with the DAB kit (Abcam, UK). At last, the sections were air-dried,

dehydrated, covered with coverslips, and the images around the hematoma in striatum

SC R

were captured in a Leica microscope.

2.11. TUNEL Assay

U

For TUNEL assay, the apoptosis cells were stained by TUNEL kit (Roche, USA).

N

Make it easy, cells were confined to 4% PFA for 1 h and treated with permeabilisation

A

solution for 2 min on ice. After washing, 100 μL DNase 1 (1500 U/ml) was added to the positive control group and placed in a wet box for 20 min. Reaction mixture

M

solution was made up of 50 μL enzyme solution and 450 μL label solution. While 50 μL reaction mixture solution was added to the positive control group and each

ED

experimental group, 50 μL label solution was added to the negative control group. The sections were incubated in the wet box for 60 min darkly. The apoptosis cells

PT

were labeled with green fluorescence, and all cells were stained with Hoechst 33342 (Beyotime Biotechnology, China). The samples can be analyzed directly under a

CC E

fluorescent microscopy.

2.12. Quantitative Analysis

A

For cell quantification in immunohistochemistry and immunofluorescent staining, the number of cells 2 mm around the hematoma in the basal ganglia was counted using a Leica microscope. To avoid counting the same cell in more than one section, we counted every fifth section. For each section, the number of cells of three separate regions (per square millimeter) was calculated. The final statistical values of per square millimeter were the representative of six sections. 8

2.13. Statistical Analysis All data was analyzed with Stata 8.0 statistical software (Systat Software Inc., San Jose, CA, USA) and expressed as mean ± standard error of the mean (SEM). The statistical analysis was implemented by One-way ANOVA and following Tukey’s multiple comparison tests. Only P < 0.05 was considered statistically significant. Each

IP T

experiment was repeated in triplicate.

SC R

3. Result

3.1. ICH induces the changes of neurological deficits in rat models

To evaluate the acute and chronic impacts in sensory and motor function in rat models

U

of unilateral brain injury, a range of behavior tests were performed [23]. At different

N

time points after ICH, we applied forelimb placing test and corner turn test to appraise

A

the neurological deficits of rats (Supplementary Figure A, B). The lower score means the worse motor function of limbs contralateral to the injury. When compared to the

M

sham-operated group, the rats in ICH group presented the neurological deficits at the

ED

first 7 days and a gradual recovery in a time-dependent manner. Till 14 days, the neurological deficits of ICH rats were almost restored.

ICH

PT

3.2. The expression of Cab45s distributed in the perihematoma is increased after

CC E

To determine the trend of Cab45s’ expression, western blot was employed. The protein level of Cab45s in ICH group increased at 6 h, peaked at day 2, and then gradually decreased in the ipsilateral region, while it was low in sham-operated group

A

(Fig. 1A, B). The data indicated that the expression of Cab45s had a transient change following ICH. To further confirm the expression and distribution of Cab45s, we conducted immunohistochemistry staining with anti-Cab45s antibody on coronal section of brain. As shown in Fig. 2, the positive signals of Cab45s were present in the cytoplasm and nucleus of neuron-like cells. At day 2 after ICH, the damage in the ipsilateral induced a marked increase of Cab45s-positive cells (Fig. 2E, F). 9

Comparing to the low level of Cab45s in the sham-operated group (Fig. 2A, B), the expression of Cab45s in the contralateral showed no significance (Fig. 2C, D). Moreover, nothing could be seen from the negative control (Fig. 2G). Corresponding with western blot, the data demonstrated that the expression of Cab45s was visibly

3.3. Cab45s is significantly up-regulated in neurons following ICH

IP T

higher in the ipsilateral region surrounding the hematoma at day 2 following ICH.

To find out which cell phenotype expressing Cab45s after ICH, we chose the double

SC R

immunofluorescent staining by co-labeling Cab45s with different cell makers, such as

NeuN, GFAP and CD11b, which are makers of neuron, astrocyte and microglia respectively. Our immunostaining showed that Cab45s was especially observed in the

U

cytoplasm and nucleus of neurons (Fig. 3A-D), whereas there was no presence of

N

Cab45s in astrocytes or microglias (Fig. 3E-L). Additionally, at day 2 following

A

operation, the number of Cab45s-positive neurons in ICH group was more than that of the sham-operated group. The proportion of Cab45s in neurons was counted in the

M

experimental group and the sham-operated group (Fig. 3O). As expected, Cab45s’

ED

expression was markedly increased in neurons following ICH, when compared with the sham group (Fig. 3C, D). Then, we speculated that Cab45s might be related to

PT

neuronal function following ICH.

3.4. Cab45s is associated with apoptosis in the model of ICH rats

CC E

Neuronal apoptosis has been regarded as one of the major mechanisms following ICH [26]. As shown in Fig. 4A, the expressions of active caspase-3 and Bax were up-regulated in the boundary of hematoma and peaked at day 3 after ICH. To further

A

ascertain the phenomena, we used double immunofluorescent staining to label active caspase-3 and NeuN (Fig. 4C-E), as well as active caspase-3 and Cab45s (Fig. 4F-H). The images showed that active caspase-3 appeared in most neurons, and that most active caspase-3 was co-located with Cab45s. Thus, we suggested that elevated Cab45s protein might participate in ICH-induced neuronal apoptosis. What is worth mentioning is that Cab45s can suppress cancer cell apoptosis by increasing GRP78 10

[18]. We wanted to explore the potent ability of GRP78 in Cab45s-related neuronal apoptosis, so we measured the expression of GRP78 adjacent to the hematoma by western blot. Like Cab45s, the protein level of GRP78 increased, peaked at day 3 and decreased (Fig. 4B). All results implied that Cab45s might participate in neuronal apoptosis via caspase-3 dependent pathway after ICH, which might be regulated by

IP T

GRP78.

3.5. Cab45s correlates with GRP78 in hemin-induced PC12 cells apoptosis

SC R

From all results in vivo, we have speculated that Cab45s might take part in neuronal

apoptosis after ICH through caspase-3 dependent pathway and be regulated by GRP78. However, further experiments in vitro were needed to confirm the speculation.

U

In PC12 cells, cell apoptosis was induced by exposure to 100 μmol/L hemin, as

N

evidenced by increased protein level of active caspase-3 in western blot (Fig. 5A, B).

A

After simulation, Cab45s and GRP78 reached their peak at hour 24 while active caspase-3 peaked at hour 12. So we surmised that GRP78 might be a crucial member

M

in Cab45s-related apoptosis in PC12 cells. To this aim, we explored the direct

ED

connection between Cab45s and GRP78 by co-immunoprecipitation (Fig. 5C, D). Then, we found that Cab45s interacted with GRP78 in a time-dependent manner in which the association between Cab45s and GRP78 was significantly enhanced at hour

PT

12 after stimulation. Based on these results, we suggested that Cab45s might join in

CC E

caspase-3 dependent cell apoptosis via interacting with GRP78 after ICH.

3.6. Cab45s protects PC12 cells from apoptosis induced by hemin To figure out the exact role of Cab45s in neuronal apoptosis, hemin-induced PC12

A

cell apoptosis model was used to simulate neuronal apoptosis following ICH. Cells transfected with Cab45s-siRNA, non-specific siRNA or vehicle were tested 48 h later. Cab45s-siRNA dramatically knocked down the protein expression of Cab45s in PC12 cells (Fig. 6A, B). In the presence of hemin for 12 h, levels of GRP78, Bax and active caspase-3 were significantly up-regulated in the non-specific siRNA group. However, the Cab45s silencing group showed lower level of GRP78 but higher levels of Bax 11

and active caspase-3 after simulation (Fig. 6C, D). The phenomenon indicated that with attenuate of Cab45s, the cell apoptosis was increased after exposure to hemin for 12 h, which was also confirmed by immunofluorescence staining (Fig. 6E). Next, we used the TUNEL assay to further determine whether Cab45s protects cells from apoptosis. Briefly, for this assay, TUNEL staining recognizes the damaged DNA including double- and single-stranded DNA breaks. As predicted, compared to the

IP T

hemin+ group, low-expressing Cab45s increased the cell apoptosis induced by hemin, while the apoptosis in the hemin+/non-specific siRNA group showed no statistical

SC R

significance (Fig. 6F, G). In conclusion, these data showed the potent anti-apoptotic abilities of Cab45s in ICH.

U

4. Discussion

N

In this paper, ICH model was induced by autologous blood injection into right basal

A

ganglia of rats, and cultured PC12 cells were exposed to hemin to imitate neuronal apoptosis in vitro. We provided evidence that Cab45s was rapidly up-regulated in the

M

perihematomal brain tissues following ICH and mainly co-located with neurons.

ED

Likewise, the protein levels of active caspase-3, Bax and GRP78 were increased in vivo and in vitro. We also found the interaction between Cab45s and GRP78 in hemin-treated PC12 cells. Depletion of Cab45s by its special siRNA up-regulated the

PT

expressions of active caspase-3 and Bax, but down-regulated the expression of GRP78. These data indicates that Cab45s has a potent neuroprotective role in

CC E

neuronal apoptosis and might provide a new intervention target for ICH. Neuronal apoptosis, the ultimate consequence of ICH, contains complicated pro-

and anti-apoptotic regulations. Bax and active caspase-3 were increased in our ICH

A

model, which are members of pro-apoptotic agents [27]. Bax, in charge of the permeabilization of mitochondrial outer membrane (MOM), is a pivotal effector of apoptosis [28]. Furthermore, the formation of apoptosome brings the initiator molecules of apoptotic pathways together. As the significant factor in the execution phase of apoptosis, caspase-3 is always activated via both intrinsic and extrinsic pathways. Our findings above indicated that Cab45s was increased and mainly 12

distributed in neurons after ICH. Moreover, some researches have shown that Cab45s has an important role in inhibiting cell apoptosis and promoting cell proliferation [18, 21]. In this study, active caspase-3 appeared in most neurons and most active caspase-3 was co-located with Cab45s. Thus, we suggested that elevated Cab45s protein might participate in ICH-induced neuronal apoptosis. How does Cab45s exert itself to fight against apoptosis?

IP T

The endoplasmic reticulum is an essential organelle for diverse cellular functions, such as apoptosis [29]. GRP78, also known as BiP, is a key mediator of the ER stress

SC R

[30-32]. Under conditions of chronic or irreversible ER stress, GRP78 is dissociated from ER-located transmembrane proteins, which leads to the activation of these

proteins. The activated proteins can induce apoptosis, including the up-regulation of

U

the transcription factor C/EBP-homologous protein (CHOP), the increase of

N

pro-apoptotic components in the Bcl-2 protein family and the activation of the caspase

A

cascade, like caspase-3 [33]. These studies indicate that GRP78 play a crucial role in modulating apoptosis induced by ER stress. Still, researches show that overexpression

M

of GRP78 reprograms ER stress signaling and blocks apoptosis in neurons. For

ED

example, mesencephalic astrocyte-derived neurotrophic factor reduces cell apoptosis via up-regulating GRP78 in SH-SY5Y cells [34]. The loss of GRP78 augments human alpha-synuclein (α-syn) toxicity to rat nigral neurons during normal aging or from

PT

siRNA knockdown [35]. In addition, it is reported that Cab45s can increase GRP78 protein level to inhibit cell apoptosis under stress conditions [18]. During ER stress,

CC E

decreasing GRP78 expression and increasing Bax level can be detected in Cab45s-depleted cells. It is proposed that Cab45s could regulate apoptosis through modulating the level of intracellular GRP78. In our vitro experiments, we found that

A

the loss of Cab45s leaded to the lower expression of GRP78 and more apoptosis in PC12 cells exposure to hemin. L Chen et al. has elaborated that: (1) during ER stress condition, Cab45s increases the GRP78 protein level and prevents GRP78 to disassociate from ER-located transmembrane proteins; (2) Cab45s interacts with GRP78. So we conjectured that whether Cab45s plays the analogous role in neuronal apoptosis. We found that Cab45s interacted with GRP78 in PC12 cells. What’s more, 13

another study also elaborates that Cab45s binds to and inhibits SERCA2b to promote cell proliferation [21]. Thereby, whether this effect of inhibiting apoptosis is mainly due to the interaction between Cab45s and GRP78 or requires other proteins for assistance remains further exploration. Besides, we found that the neurological deficits of ICH rats were almost restored till 14 days after ICH (Supplementary Figure A, B). In this study, we make a

IP T

conclusion that Cab45s has a potent neuroprotective role in neuronal apoptosis after intracerebral hemorrhage. However, the absorption of hematoma and the regression of

SC R

edema also make a certain recovery of neurobehavioral in ICH rats. The significance of Cab45s’ anti-apoptotic ability in ICH remains further study.

Another issue should be raised is about the cell-type of TUNEL-positive cells.

U

Cells with any kinds of double-strand and single-stranded DNA damage will be

N

TUNEL-positive, so TUNEL-positive cells are not always indications of apoptotic

A

cells and the reduction of TUNEL-positive cells cannot be illustrated only by the reduced apoptosis. In previous studies, the nature of TUNEL-positive cells encircling

M

the hematoma has been already studied and clarified. Most TUNEL-positive cells

ED

were neurons and astrocytes [36, 37]. As mentioned above, the prominent form of cell death associated with ICH around the hematoma is neuronal apoptosis. Moreover, the active caspase-3 precedes the appearance of TUNEL-positive cells [37]. Accordingly,

PT

the decrease of TUNEL-positive cells observed in our paper is likely to be explained by the decreased apoptosis.

CC E

Collectively, we conclude that Cab45s attenuates apoptosis in ICH rats and

hemin-treated PC12 cells. However, we can not entirely exclude the possibility of other signaling pathways besides GPR78 involving in Cab45s-related cell apoptosis.

A

It is interesting to further investigate the underlying mechanisms of Cab45s in neurons, which may provide novel clinical therapies for ICH treatment.

Acknowledgments This work was supported by the National Natural Science Foundation of China 14

(Nos.81471188, 81671135); Postgraduate Resarch & Practice Innovation Program of Jiangsu Province (KYZZ16_0356); Technology Innovation Programme of Nantong University (YKC16058); Science and Technology Planning Project of Nantong (YYZ16016).

Conflict of Interests

IP T

The authors have declared that no conflict of interests exists.

References

Roquer, J., et al., Sex-related differences in primary intracerebral hemorrhage. Neurology,

SC R

1.

2016. 2.

Karuppagounder, S.S., et al., Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models. Sci Transl Med, 2016. 8(328): p. 328ra29.

Sharp, F., et al., Intracerebral hemorrhage injury mechanisms: glutamate neurotoxicity,

U

3.

thrombin, and Src. Acta Neurochir Suppl, 2008. 105: p. 43-6.

Wang, J., et al., Raf-kinase inhibitor protein attenuates microglia inflammation in an in vitro

N

4.

model of intracerebral hemorrhage. Cell Mol Biol (Noisy-le-grand), 2016. 62(6): p. 86-91. Duan, X., et al., Intracerebral Hemorrhage, Oxidative Stress, and Antioxidant Therapy. Oxid

A

5. 6.

M

Med Cell Longev, 2016. 2016: p. 1203285.

Zhai, W., et al., A1 adenosine receptor attenuates intracerebral hemorrhage-induced secondary brain injury in rats by activating the P38-MAPKAP2-Hsp27 pathway. Mol Brain,

7.

ED

2016. 9(1): p. 66.

He, X.F., et al., Deferoxamine inhibits microglial activation, attenuates blood-brain barrier disruption, rescues dendritic damage and improves spatial memory in a mouse model of

PT

microhemorrhages. J Neurochem, 2016. 8.

Sukumari-Ramesh, S., C.H. Alleyne, Jr., and K.M. Dhandapani, Astrocyte-specific expression of survivin after intracerebral hemorrhage in mice: a possible role in reactive gliosis? J

CC E

Neurotrauma, 2012. 29(18): p. 2798-804. 9.

Wan, S., et al., Microglia Activation and Polarization After Intracerebral Hemorrhage in Mice: the Role of Protease-Activated Receptor-1. Transl Stroke Res, 2016.

10.

Grossetete, M. and G.A. Rosenberg, Matrix metalloproteinase inhibition facilitates cell death in intracerebral hemorrhage in mouse. J Cereb Blood Flow Metab, 2008. 28(4): p. 752-63. Jellinger, K.A., Challenges in neuronal apoptosis. Curr Alzheimer Res, 2006. 3(4): p. 377-91.

12.

Gallotta, I., et al., Neuron-specific knock-down of SMN1 causes neuron degeneration and

A

11.

13.

death through an apoptotic mechanism. Hum Mol Genet, 2016. 25(12): p. 2564-2577. Tait, S.W. and D.R. Green, Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol, 2010. 11(9): p. 621-32.

14.

Honore, B., The rapidly expanding CREC protein family: members, localization, function, and role in disease. Bioessays, 2009. 31(3): p. 262-77.

15.

Luo, J., et al., A Novel Role of Cab45-G in Mediating Cell Migration in Cancer Cells. Int J Biol Sci,

15

2016. 12(6): p. 677-87. 16.

Herweh, C., et al., Frequency and temporal profile of recanalization after cerebral vein and sinus thrombosis. Eur J Neurol, 2016. 23(4): p. 681-7.

17.

Lee, J.H., E.J. Kwon, and H. Kim do, Calumenin has a role in the alleviation of ER stress in neonatal rat cardiomyocytes. Biochem Biophys Res Commun, 2013. 439(3): p. 327-32.

18.

Chen, L., et al., Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP. Cell Death Dis, 2014. 5: p. e1219.

19.

Zhu, Y., et al., The ethanol response gene Cab45 can modulate the impairment elicited by ethanol and ultraviolet in PC12 cells. J Genet Genomics, 2008. 35(3): p. 153-61. Scherer, P.E., et al., Cab45, a novel (Ca2+)-binding protein localized to the Golgi lumen. J Cell

IP T

20.

Biol, 1996. 133(2): p. 257-68. 21.

Chen, L., et al., Cab45S promotes cell proliferation through SERCA2b inhibition and Ca2+

22.

SC R

signaling. Oncogene, 2016. 35(1): p. 35-46.

Ke, K., et al., Increased expression of small heat shock protein alphaB-crystallin after intracerebral hemorrhage in adult rats. J Mol Neurosci, 2013. 51(1): p. 159-69.

23.

Karabiyikoglu, M., et al., Intracerebral hirudin injection attenuates ischemic damage and neurologic deficits without altering local cerebral blood flow. J Cereb Blood Flow Metab, 2004.

24.

U

24(2): p. 159-66.

Ke, K., et al., CDK5 contributes to neuronal apoptosis via promoting MEF2D phosphorylation

25.

N

in rat model of intracerebral hemorrhage. J Mol Neurosci, 2015. 56(1): p. 48-59. Hua, Y., et al., Behavioral tests after intracerebral hemorrhage in the rat. Stroke, 2002. 33(10): Wu, J., et al., Microglial activation and brain injury after intracerebral hemorrhage. Acta

M

26.

A

p. 2478-84. Neurochir Suppl, 2008. 105: p. 59-65. 27.

Liu, C., et al., Increased Expression of Ubiquitin-Specific Protease 4 Participates in Neuronal

28.

ED

Apoptosis After Intracerebral Hemorrhage in Adult Rats. Cell Mol Neurobiol, 2016. Goyal, S., et al., ROS mediated crosstalk between endoplasmic reticulum and mitochondria by Phloxine B under environmental UV irradiation. J Photochem Photobiol B, 2016. 161: p.

PT

284-294. 29.

Anelli, T. and R. Sitia, Protein quality control in the early secretory pathway. EMBO J, 2008. 27(2): p. 315-27.

Chowdhury, S., et al., Taurine protects cisplatin induced cardiotoxicity by modulating

CC E

30.

inflammatory and endoplasmic reticulum stress responses. Biofactors, 2016.

31.

Gu, X.W., et al., Endoplasmic reticulum stress in mouse decidua during early pregnancy. Mol Cell Endocrinol, 2016.

A

32. 33. 34.

Cerezo, M., et al., Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance. Cancer Cell, 2016. 29(6): p. 805-19. Scorrano, L., et al., BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science, 2003. 300(5616): p. 135-9. Huang, J., et al., Mesencephalic astrocyte-derived neurotrophic factor reduces cell apoptosis via upregulating GRP78 in SH-SY5Y cells. Cell Biol Int, 2016. 40(7): p. 803-11.

35.

Salganik, M., et al., The loss of glucose-regulated protein 78 (GRP78) during normal aging or from siRNA knockdown augments human alpha-synuclein (alpha-syn) toxicity to rat nigral neurons. Neurobiol Aging, 2015. 36(6): p. 2213-23.

16

36.

Matsushita, K., et al., Evidence for apoptosis after intercerebral hemorrhage in rat striatum. J Cereb Blood Flow Metab, 2000. 20(2): p. 396-404.

37.

Gong, C., et al., Intracerebral hemorrhage-induced neuronal death. Neurosurgery, 2001. 48(4): p. 875-82; discussion 882-3.

PT

ED

M

A

N

U

SC R

IP T

Figure captions:

CC E

Fig. 1 Alterations of Cab45s protein level after ICH in western blot. (A) Western blot was adopted to detect the protein level of Cab45s around the hematoma at different survival periods. (B) The relative optical density of Cab45s versus β-actin at each

A

time point was shown in the quantification graph. β-actin was used as a loading control. Error bars are ± SEM. n = 3. * P < 0.05 vs the sham-operated group.

17

IP T SC R U N A M ED PT CC E A

Fig. 2 Immunohistochemical analysis of Cab45s in the rats’ basal ganglia adjacent to the hematoma. (A, B) In the sham-operated group (right basal ganglia), the level of Cab45s was detected. At day 2 after ICH, the contralateral group (contralateral basal ganglia of the ICH rats) (C, D) and the ipsilateral group (basal ganglia adjacent to the hematoma) (E, F) showed changes in the expression of Cab45s. (G) No positive 18

signal was found in the negative-control group. (H) Positive cells shown in a-f were counted. n = 3. * P < 0.05 vs the sham-operated group. Scale bar: left column 200 μm,

CC E

PT

ED

M

A

N

U

SC R

IP T

right column 100 μm.

Fig. 3 Double-immunofluorescence staining of Cab45s and unequal cell markers in ICH rats. The cryosections of the ICH rats at day 2 were labeled with Cab45s (green,

A

A, E, I) and different cell markers (red, B, F, J) as follows: neuronal marker (NeuN), astrocyte marker (GFAP), and microglia maker (CD11b). (C, G, K) In the merged images, the yellow color represented the colocalization of Cab45s with different specific phenotype markers after ICH. (D, H, L) So was it in the merged images of sham-operated group. (M, N) No positive signal was found in the negative control. (O) The percentage of cells expressing Cab45s in NeuN-positive cells was shown in the 19

CC E

PT

ED

M

A

N

U

SC R

IP T

graph. n =3. * P < 0.05 vs the sham-operated group. Scale bars 100 μm.

Fig. 4 Association of Cab45s with cell apoptosis following ICH. (A, B) Western blot was applied to measure the level of active caspase-3, Bax and GRP78 surrounding the

A

hematoma at different time points. The relative optical density of these proteins versus β-actin at each time point was shown in the quantification graph (bottom). β-actin was used as a loading control. Error bars are ± SEM. n = 3.

*, #

P < 0.05 vs the

sham-operated group. In double-immunofluorescent staining, the cryosections of the ICH rats at day 2 were labeled with active caspase-3 (red, C, F) and NeuN (green, D), as well as Cab45s (green, G). (E, H) In the merged images, the yellow color signified 20

the colocalization of active caspase-3 with NeuN and Cab45s, respectively. n = 3.

ED

M

A

N

U

SC R

IP T

Scale bars 50 μm.

Fig. 5 Relation between Cab45s and GRP78 in hemin-induced PC12 cell apoptosis.

PT

(A) Western blot showed the expressions of Cab45s, active caspase-3 and GRP78 in PC12 cells treated with hemin for different time. (B) The relative density versus

CC E

β-actin at each time point was shown in the bar graph. β-actin was used as a loading control. Error bars are ± SEM. n = 3. *, #, ^ P < 0.05 vs the control group. (C) The PC12 cells exposed to hemin for 12 h were immunoprecipitated with Cab45s and

A

immunoblotted with GRP78. (D) The PC12 cells exposed to hemin for 12 h were immunoprecipitated with GRP78 and immunoblotted with Cab45s. IgG was used to exclude specific binding. The relative density versus β-actin was shown in the quantification graph (bottom). β-actin was used as a loading control. Error bars are ± SEM. n = 3. * P < 0.05 vs the control group. (‘‘IP’’ means samples subjected to the IP procedure; ‘‘Input’’ means samples before the IP procedure; ‘‘WB’’ is abbreviated 21

A

CC E

PT

ED

M

A

N

U

SC R

IP T

form of western blot)

22

IP T SC R U N A M ED PT CC E A

Fig. 6 Regulation of Cab45s in PC12 cell apoptosis induced by hemin. (A, B) Western blot was employed to assess the protein level of Cab45s in PC12 cells after transfection of Cab45s siRNA or non-specific siRNA. The relative density of Cab45s versus β-actin was shown in the quantification graph. β-actin was used as a loading 23

control. Error bars are ± SEM. n = 3. * P < 0.05 vs the control group. (C, D) In the presence or absence of hemin, the expressions of GRP78, Bax and active-caspase-3 in Cab45s siRNA or non-specific siRNA group were examined. The bar chart indicated the density of these proteins versus β-actin. β-actin was used as a loading control. Error bars are ± SEM. n = 3.

*, #, ^

P < 0.05 vs the hemin+/non-specific siRNA group.

(E) Immunofluorescence showed the co-localization of Cab45s and active caspase-3

IP T

in control, hemin+/non-specific siRNA and hemin+/Cab45s siRNA groups. (F) After exposure to hemin for 12 h, PC12 cells were analyzed by DAPI (blue), NeuN (red)

SC R

and TUNEL (green) staining to determine the proportion of apoptotic cells. (G) The

radio of TUNEL-positive cells in each group was displayed in the graph. n =3. * P <

A

CC E

PT

ED

M

A

N

U

0.05 vs the control group, # P < 0.05 vs the hemin+ group. Scale bars 50 μm.

24