Induction Profile of MANF/ARMET by Cerebral Ischemia and its Implication for Neuron Protection

Animals

Seventy male Sprague–Dawley rats (grade SPF, weighing 200 to 240g) were obtained from Shanghai Laboratory Animal Co. Ltd (Shanghai, China). The pregnant Sprague–Dawley rats (grade SPF) were from Anhui Experimental Animal Center (Hefei, China). The rats were kept under standard lighting conditions (12h light/dark cycle). The procedure for animal surgery was performed in accordance with the Guideline of Animal Care and Use Committee of Anhui Medical University.

Middle Cerebral Artery Occlusion

Focal cerebral ischemia was induced by using MCAO with a suture. The rats were anesthetized with 10% chloral hydrate (3 mL/kg body weight, intraperitoneally). The right middle cerebral artery (MCA) was occluded with a nylon filament (0.235 mm in diameter) with a rounded tip, which was introduced from the common carotid artery lumen into the internal carotid artery to block the origin of the right MCA. The filament was inserted at least 18 mm from the carotid bifurcation and was withdrawn at designed time points (2 or 4h after occlusion) to allow reperfusion for different time periods (2, 4, 12, 24, 36 or 48 h). For each time point, 8 to 10 animals were used. In sham operation, a right neck incision was made to expose the arteries, but the nylon thread was not inserted into internal carotid artery.

Magnetic Resonance Imaging Studies

Rats were anesthetized and placed in a general holder and positioned inside the magnet. The animal's head was held in place inside the 3 inch surface coil. All MR studies were performed using a 1.5 T superconducting magnet (General Electric Medical Systems, Waukesha, WI, USA). SE T1-weighted (TR/TE = 340/12 ms) and FSE T2-weighted images (TR/TE = 4,000/110 ms) were obtained from a 2.0 mm thick axial and coronal section without gap using a 40 mm × 40 mm field of view, and reconstructed using a 256 × 192 image matrix.

Triphenyltetrazolium Chloride Staining

Rats were killed under deep anesthesia. The brains were removed carefully and dissected into coronal sections. The fresh brain slices were immersed in a 2% solution of 2, 3, 5-triphenyltetrazolium chloride in normal saline at 37°C for 30 mins and fixed by 4% buffered formaldehyde solution for 0.5 h at 4°C. The normal area of brain was stained red whereas infarct tissue remained unstained (white). After triphenyltetrazolium chloride staining, digital photographs were taken.

Expression of ARMET and Preparation of Antibodies

Expression of ARMET and preparation of rabbit anti-ARMET polyclonal antibody have been described earlier. Monoclonal antibody was produced using standard methods. Briefly, 6-week-old female BALB/C mice were immunized three times intraperitoneally with the purified ARMET (200 μg per injection) emulsified in incomplete Freund adjuvant at 10-day intervals. Antigen-specific-antibody-secreting hybridomas were identified by ELISA assay. Monoclonal antibodies were immunotyped using isotype kit (Sigma, Saint Louis, MO, USA) to define the heavy and light chains. Specificity of monoclonal antibody was evaluated by determining the fusion protein FLAG-tagged ARMET expressed in eukaryotic cells using Western blot and double-labeled immunofluorescent staining.

Immunohistochemistry

The brain tissues were fixed with 4% paraformaldehyde. Serial coronal sections (3 μm) were hydrated. After blocking by 5% goat serum, the sections were incubated with primary antibodies overnight at 4°C. Incubation with secondary antibodies (DAKO, Glostrup, Denmark) at 37°C for 1 h, the slides were washed and incubated in 3′-3′-diaminobenzidine-tetrahydrochloride for 5 mins. Then the slides were stained with hematoxylin, dehydrated in ethanol, and mounted in neutral gum. The identical regions in different samples were selected at low magnification. For each sample, the number of ARMET-positive cells was counted in five randomly selected fields under the high-power field (x 400 magnification).

Immunofluorescent Staining

Brain sections were hydrated and rinsed in phosphate buffered saline (PBS). After antigen retrieval, sections were permeabilized/blocked in PBS containing 0.5% Triton X-100 and 5% goat serum. The sections were incubated with primary antibody overnight at 4°C. For dual fluorescent staining, the sections were incubated with Alexa Fluor 488-conjugated or 568-conjugated IgG (Invitrogen, Carlsbad, CA, USA) and observed under fluorescent microscopy (Olympus, Tokyo, Japan). Immunocytofluorescent staining was performed as described earlier. The nuclei of cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (5 μg/mL).

Immunoblotting or Western Blotting

The whole tissue of ispilaterally cerebral cortex was homogenized with 10 volumes of lysis buffer (50 mmol/L Tris–HCl pH 7.2, 150 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L sodium pyrophosphate, 0.5% Triton X-100, 0.5% Nonidet P-40) containing protease inhibitor cocktail. The lysate was centrifuged and processed for SDS–PAGE. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked in 5% nonfat milk. Primary and secondary antibodies were diluted in 5% milk and incubated for 1 h, respectively. Blots were developed using the enhanced chemiluminescence kit (Amersham Biosciences, NJ, USA).

Primary Neuron Culture

Pregnant Sprague–Dawley rats were killed at embryonic days 16 to 18 under anesthesia. The cortexes and hippocampi were moved into Ca²⁺- and Mg²⁺-free Hank's solution. Cells were dissociated using a fire-polished Pasteur pipette. The cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 10% horse serum and seeded onto poly-D-lysine-coated 24-well plates and incubated at 37°C and 5% CO₂ in a humidified atmosphere. After several days of culture, the cells were exposed to cytosine arabinoside for 24 h. The medium was changed into low serum (5%) when the cells were treated.

TUNEL Staining

The apoptotic cells were detected using the TUNEL kit (Promega, Madison, Wisconsin, USA) following the manufacturer's instructions. Under the high-power field (x 400 magnification) of fluorescent microscope, the number of apoptotic cells characterized by the stained nuclei was counted in five randomly selected fields. The mean number of apoptotic cells per field was calculated for further statistical analysis.

Statistical Analysis

Quantitative data were presented as the means ± s.e.m. Statistical comparisons were analyzed using a one-way ANOVA followed by Dunnett's test. P < 0.05 was considered statistically significant.

Assessment of Ischemic Lesions by Imaging and Histology

Our earlier study has shown that ARMET is an unfolded protein response-upregulated protein and inhibits ER stress-induced cell death (Apostolou et al, 2008). Focal cerebral ischemia was shown to be an in vivo ER stress model in that ischemia induces accumulation of immature proteins in the ER (DeGracia and Montie, 2004). Additionally, ARMET expression was induced in cardiac myocytes after myocardial infarction (Tadimalla et al, 2008). We wondered whether brain ischemic injury induces ARMET expression. To test this hypothesis, the focal cerebral ischemic model was successfully induced by right MCAO in adult male rats. Magnetic resonance imaging was used to estimate the lesions in vivo. Fast spin-echo T2-weighted images are shown in Figure 1A. These axial brain sections were obtained at the different levels, including caudate–putamen complex. Images were collected at different time points, including 24 and 48 h after reperfusion indicated in Figure 1A. Diffuse hyperintense signal in lesions of ispilateral brains that were subjected to ischemic injury was noted. The representative images were shown in middle column (I/R, 4/24 h) and right column (I/R, 4/48h) of Figure 1A.

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Figure 1

Assessment of ischemic lesions by imaging and histology. MCAO was performed for 4h and the images were collected at the indicated time points (24 and 48 h) after reperfusion. (A) T2-weighted images (T2WI). (B) Triphenyltetrazolium chloride staining of the brain sections. (C) HE staining of the cortexes. Scale bar = 200 μm. (D) The NeuN-positive neurons. Scale bar= 100 μm. (The color reproduction of this figure is available on the html full text (online) version of the article.)

Triphenyltetrazolium chloride staining showed that MCAO rats developed prominently focal infarcts at 4/24 h (I/R) and 4/48 h (I/R) (Figure 1B). The core of infarction was observed as a white area and the penumbra was pink. The histologic assessment showed mild edema in cerebral cortex supplied by the MCA started from 2/2h (I/R) (Figure 4A, indicated by circle). The comparison of focal infarcts by HE staining at 4/24h (I/R) and 4/48h (I/R) was presented in Figure 1C. The number of NeuN-positive neurons was obviously reduced in ischemic cortex at 4/24 h (I/R) and 4/48 h (I/R) (Figure 1D). These results suggest that the focal ischemic model was induced successfully by MCAO.

Focal Cerebral Ischemia Induces CHOP Expression

C/EBP homologous protein is a stress-inducible nuclear protein (Zinszner et al, 1998), and was usually used as a marker of ER stress (Ohoka et al, 2005). To clarify whether focal ischemia induced by MCAO leads to ER stress, CHOP was determined by using immunofluorescent staining and immunoblotting with anti-CHOP (Santa Cruz, CA, USA) at the designed time points. It was found that CHOP-positive cells were largely dispersed in MCA-innervated cerebral cortex (Figure 2B) at the time of 2/24 h (I/R). Meanwhile, small amount of CHOP was found in ispilateral hippocampus of MCAO at that time (Figure 2D). Western blotting showed that expression of CHOP in ischemic cortexes increased beginning from the time point of 4/4 h (I/R), compared with the shams (Figures 2E–2F). More interesting, the ischemia-induced expression of CHOP was found to be localized in the different subcellular compartments, including cytosol (Figures 2G–2I), nuclei (Figures 2J–2L), and perinuclear distribution (Figures 2M–2O). Focal cerebral ischemia-induced expression of CHOP showed here further confirms the results reported before that focal cerebral ischemia induces ER stress.

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Figure 2

Focal cerebral ischemia induces CHOP expression. CHOP was detected with anti-CHOP antibody in the cortexes (A, B) and hippocampi (C, D) of sham (A, C) and MCAO rats with I/R 4/24 h (B, D). (E) CHOP expression in cortexes. The samples were collected at I/R 4/4 and 4/12h, respectively. Actin was used as a loading control. IB, immunoblot; S, sham. (F) The quantitative data of panel E. Values are expressed as mean ± s.e.m. of three independent experiments. *P < 0.05, **P < 0.01, compared with S (sham). (G–O) Subcellular distribution of CHOP. The samples were collected at I/R 4/24h. The nuclei were stained with DAPI (G, J, M). CHOP displays in cytosol (H), nuclei (K), and perinuclear distribution (N). Scale bar = 50 μm. (The color reproduction of this figure is available on the html full text (online) version of the article.)

Focal Cerebral Ischemia Upregulates ARMET Expression

We reported earlier the expression of Armet in ischemic cerebral cortex (Apostolou et al, 2008). To investigate the characteristics of ischemia-induced ARMET expression, we observed the time course of ARMET expression in different brain regions by means of immunohistochemical assay and Western blotting. It was found that ARMET was detectable in many regions of normal rat brain, including cortex, hippocampus, subcommissural organ, and substantia nigra, but the staining is weak (not shown). Compared with the shams (Figures 3A1–3D1, Figures S2 and S3), ARMET was induced in MCAO rats at the early stage of ischemia, starting from 2/2 h (I/R) and retaining in the period of observation (up to 4/48 h). The typical ARMET-positive cells were found in the regions supplied by MCA at 2/2h (I/R) (Figures 3A2–3D2, Figures S1 and S4), whereas HE staining did not show marked infarct of the brain tissue at that time. Along with the prolongation of reperfusion, not only the immunostaining with anti-ARMET antibody became darker in cytoplasm, but also the number of ARMET-positive cells was increased in the ischemic regions (Figure 3G). Western blotting shown in Figure 3H also conformed that the levels of ARMET in ispilaterally cerebral cortexes of ischemic rats were increased at 2/2h (I/R) and 4/4h (I/R). These results indicate that ARMET was induced by focal ischemia in a time-dependent manner. Furthermore, in the core of infarct tissue, ARMET expression was relatively weak, even absent. The intense ARMET immunostaining was often found in peri-infarct tissue (Figure 3I).

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Figure 3

Focal cerebral ischemia induces ARMET expression. ARMET was detected with anti-ARMET antibody in the cortexes (A1, A2), substantia nigra (B1, B2), subcommissural organs (C1, C2), and hypothalamuses (D1, D2) of sham (A1–D1) and MCAO rats (A2–D2) with I/R 2/2h. (E1–E3) Double labeling of ARMET (red) and DAPI (blue). (F1–F3) Double labeling of ARMET (red) and NeuN (green). Scale bar= 50 μm. (G) The time course of ARMET expression in different brain regions. Values are expressed as mean ± s.e.m. n = 4. *P < 0.05, **P < 0.01, compared with S (sham). CA1, CA1 subfield of hippocampus; Hyp, hypothalamus; SN, substantia nigra; AM, amygdaloid nucleus. (H) ARMET was determined using immunoblot (IB). The samples were collected at I/R 2/2 and 2/4h. Actin was used as a loading control. S, sham; N, normal. (I) ARMET was detected in infarct tissue of cerebral cortex using immunohistochemistry (I1) and immunofluorescent staining (I2). The tissues were collected at 48h of reperfusion after 4h ischemia. (The color reproduction of this figure is available on the html full text (online) version of the article.)

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Figure 4

Susceptibility of ARMET to ischemia. (A) The cerebral cortexes of sham control (A1) and MCAO rat with I/R 2/2h (A2). Scale bar= 100 μm. (B–D) ARMET and CHOP were double labeled with antibodies against ARMET (red) and CHOP (green), respectively, in sham (B1–B3) and MCAO rats with I/R 2/2h (C1–C3) and 4/24h (D1–D3). Scale bar = 50 μm.

ARMET is a secreting protein. It is synthesized in the ER and transferred to the Golgi for secreting. To observe the subcellular localization of ARMET induced by the stimulation of cerebral ischemia, DAPI was used to counterstain the nuclei. It was found that ARMET had no overlap with DAPI (Figures 3E1–3E3), suggesting that ARMET localizes in cytosol and focal ischemia did not induce its relocalization. To investigate the subpopulations of ARMET-positive cells, we first performed the doubly immunofluorescent staining with antibodies against ARMET and NeuN, a neuron-specific nuclear protein associated with withdrawal from the cell cycle and terminal differentiation (Mullen et al, 1992). We showed that ARMET was expressed in neurons (Figures 3F1–3F3). Meanwhile, we found that some NeuN-negative cells presented ARMET-positive immunoreactions. Further identification with glial markers showed that cerebral ischemia also induced ARMET expression in various glial cell types, including astrocytes, microglial cells, and oligodendrocytes (data not shown).

Susceptibility of ARMET to Ischemia

We also found that ARMET expression was susceptible to focal cerebral ischemia. For example, the early ischemia-induced dissociation of meninx from cortex at 2/2h (I/R) stimulated ARMET expression (Figure 4A2, region III) and tissue edema (indicated by circle), which was not observed in the sham controls (Figure 4A1). This result suggests that ARMET is sensitive to ischemic simulation. Additionally, double-labeled immunofluorescence showed that there were many ARMET-positive cells in ischemic regions at 2/2 h (I/R) (Figure 4C2), whereas few CHOP-positive cells was found at that time (Figure 4C1). CHOP was remarkably induced at 4/48 h (I/R) (Figure 4D1). These results suggest that ARMET was induced earlier than CHOP.

Endoplasmic Reticulum Stress Induces ARMET Expression in Cultured Neural Cells

To further confirm that ARMET expression can be induced by ischemia, we first use SH-SY5Y cells. It was found that the expression of ARMET (Figure 5B2), but not CHOP (Figure 5B3), was moderately increased after deprivation of serum for 24h. However, both CHOP and ARMET were remarkably increased after exposure of cells to ER stress inducer tunicamycin (2.5 μg/mL) for 4 h (Figures 5C1–5C4). Furthermore, we isolated and cultured the neurons from embryonic day 18 rats and identified with neuron marker NeuN. We found that NeuN was dispersed in cytoplasm during neuron differentiation and it concentrates to nuclei after the neurons get mature (Figure 5D3). The number of neurons was reduced and the nuclei became small and condensed (Figure 5F1) after treatment with tunicamycin for 4 h. ARMET and CHOP strongly expressed in the primary cultured neurons after treatment with tunicamycin (Figure 5F2–5F3), suggesting that ER stress induces ARMET expression in primary cultured neurons.

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Figure 5

ER stress-induced ARMET expressions in neural cells. (A–C) SH-SY5Y cells were cultured with normal medium (A1–A4), serum-free medium (B1–B4) for 24 h, or treated with tunicamycin (2.5 μg/mL) for 4h before staining (C1–C4). ARMET and CHOP were detected with antibodies of anti-ARMET (red) and anti-CHOP (green), respectively. The nuclei were stained with DAPI (blue). (D1–D4) The primary cultured neurons were double labeled with anti-NeuN (green) and anti-GFAP (red), respectively. (E–F) The neurons were treated with vehicle (E1–E4) or tunicamycin (2.5 μg/mL) (F1–F4) for 4h before staining. ARMET and CHOP were detected with anti-ARMET (red) and anti-CHOP (green), respectively. Scale bar= 50 μm.

Recombinant Human ARMET Promotes Neuronal Survival

To investigate the physiological significance of ARMET upregulation during ER stress in neurons either in vivo or in vitro, we expressed recombinant human ARMET in bacteria and purified it. After treatment with ARMET (0.5 μg/mL) for 2 weeks, the primary cultured neurons formed proliferative clones (Figure 6B, indicated by arrows). As a control, the recombinant human full-length tau protein inhibited the neuronal proliferation (Figure 6C). To test whether ARMET prevents ER stress-induced apoptosis, TUNEL staining was used. The neurons were pretreated with ARMET (0.5 μg/mL) for 2 weeks, and then tunicamycin (2.5 μg/mL) was added to medium for 4h. We found that there were fewer TUNEL-positive cells in ARMET-treated neurons (Figure 6E4), compared with those treated with bovine serum albumin (Figure 6D4) or tau protein (Figure 6F4). These results indicate that ARMET is protective to neurons during ER stress.

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Figure 6

Protective effects of ARMET on primary cultured neurons. (A–F) The primary cultured neurons were treated with bovine serum albumin (BSA, 0.5 μg/mL) (A, D1–D4), ARMET (0.5 μg/mL) (B, E1–E4), or tau (0.5 μg/mL) (C, F1–F4) for 2 weeks. The cells were treated with vehicle (D1–D2, E1–E2, F1–F2) or tunicamycin (10 μg/mL) (D3, D4; E3, E4; F3, F4) for 4h before staining. The cells were stained with TUNEL and the images were collected under fluorescent microscope (D2, D4, E2, E4, F2, and F4). The identical fields were taken under bright field (D1, D3, E1, E3, F1, and F3). Scale bar = 100 μm in panel A, B and 50 μm in panel D–F. (G) The quantitative data of TUNEL-positive cells. Values are expressed as mean ± s.e.m. of three independent experiments. **P < 0.01, compared with BSA.