Caspase-3 Modulates Regenerative Response After Stroke

Authors

  • Wenying Fan,

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
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  • Yiqin Dai,

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
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  • Haochen Xu,

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
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  • Ximin Zhu,

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
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  • Ping Cai,

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
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  • Lixiang Wang,

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
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  • Chungang Sun,

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
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  • Changlong Hu,

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
    2. School of Life Sciences, Fudan University, Shanghai, People's Republic of China
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  • Ping Zheng,

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
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  • Bing-Qiao Zhao

    1. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, People's Republic of China
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Correspondence: Bing-Qiao Zhao, M.D., Ph.D., State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, People's Republic of China. Telephone: +86-21-54237884; Fax: +86-21-64174579, e-mail: bingqiaoz@fudan.edu.cn, or Wenying Fan, M.D., Ph.D., State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, People's Republic of China. Telephone: +86-21-54237479; Fax: +86-21-64174579, e-mail: wenyingf@fudan.edu.cn

Abstract

Stroke is a leading cause of long-lasting disability in humans. However, currently there are still no effective therapies available for promoting stroke recovery. Recent studies have shown that the adult brain has the capacity to regenerate neurons after stroke. Although this neurogenic response may be functionally important for brain repair after injury, the mechanisms underlying stroke-induced neurogenesis are not known. Caspase-3 is a major executioner and has been identified as a key mediator of neuronal death in the acute stage of stroke. Recently, however, accumulating data indicate that caspase-3 also participates in various biological processes that do not cause cell death. Here, we show that cleaved caspase-3 was increased in newborn neuronal precursor cells (NPCs) in the subventricular zone (SVZ) and the dentate gyrus during the period of stroke recovery, with no evidence of apoptosis. We observed that cleaved caspase-3 was expressed by NPCs and limited its self-renewal without triggering apoptosis in cultured NPCs from the SVZ of ischemic mice. Moreover, we revealed that caspase-3 negatively regulated the proliferation of NPCs through reducing the phosphorylation of Akt. Importantly, we demonstrated that peptide inhibition of caspase-3 activity significantly promoted the proliferation and migration of SVZ NPCs and resulted in a significant increase in subsequent neuronal regeneration and functional recovery after stroke. Together, our data identify a previously unknown caspase-3-dependent mechanism that constrains stroke-induced endogenous neurogenesis and should revitalize interest in targeting caspase-3 for treatment of stroke. Stem Cells 2014;32:473–486

Introduction

Stroke is a leading cause of death and disability around the world [1, 2]. Thrombolytic therapy with tissue plasminogen activator is beneficial for acute ischemic stroke. However, many stroke patients who cannot benefit from this approach have significant physical impairment, including paralysis, sensory deficits, and memory loss [3]. Despite extensive efforts, therapeutic options for improving stroke recovery remain limited.

In mammals, neurogenesis, which is important for stroke response and recovery, occurs throughout adulthood in the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus (DG) of the hippocampus. Stroke stimulates ongoing neurogenesis in these two regions, which leads neuronal precursor cells (NPCs) to migrate to the areas adjacent to ischemic injury and to differentiate into more mature neurons [4-6]. These newly generated neurons may restore neuronal function lost by stroke. Therefore, understanding of the molecular mechanisms that control the endogenous neurogenic response after stroke may hold promise for promoting brain repair and recovery after stroke.

Caspases are a family of cysteine proteases that function as central regulators of apoptosis [7]. The important role for caspases in programmed neuronal death has been demonstrated in a wide range of central nervous system diseases [8]. In particular, caspase-3 is a major executioner caspase and has generally been viewed as a terminal step in the process of apoptotic cell death [9]. Caspase-3 cleaves a variety of cellular substrates and induces DNA fragmentation, a key feature of apoptosis [10, 11], and acute inhibition of caspase-3 has been shown to offer robust protection of neurons against ischemic and traumatic cell death [12-14]. However, recent studies have shown that activation of caspases, especially caspase-3, also participates in various other biological processes that do not cause cell death, such as dendritic pruning, synaptic depression, cell differentiation, and compensatory proliferation [15]. Interestingly, significant amounts of caspase-3 were detected in nonapoptotic astrocytes after stroke [16]. However, the exact role of caspase-3 in neurogenesis after stroke is largely unknown.

In this study, we showed that caspase-3 appears to be a critical endogenous component that negatively regulates neurogenesis after stroke. We demonstrated that inhibition of caspase-3 activity enhanced stroke-induced neurogenesis. These findings suggest that caspase-3 inhibition might make a potential approach for expanding the neurogenic capacity of the brain to repair damaged tissue after stroke.

Materials and Methods

Reagents and Antibodies

Bromodeoxyuridine (BrdU), paraformaldehyde, sucrose, dimethyl sulfoxide (DMSO), laminin, and LY294002 were purchased from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com). Dulbecco's modified Eagle's medium (DMEM)/F-12 medium, streptomycin, l-glutamine, B27 supplement, epidermal growth factor, and fibroblast growth factor were obtained from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). N-Benzyloxycarbonyl-Asp (OMe)-Glu(OMe)-Val-Asp-(OMe)-fluoro-methylketone (Z-DEVD-fmk), RIPA lysis buffer, chemiluminescence solution, and Fluoro-Jade B were from Merck Millipore (http://www.millipore.com). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was from BD Biosciences (San Diego, CA, http://www.bdbiosciences.com), and BCA protein assay was from Thermo Scientific. Logan, UT, http://www.thermoscientific.com, Vectashield, 4,6-diamino-2-phenylindole (DAPI), hematoxylin, avidin-biotin-peroxidase complex (Vectastain Elite ABC kit), diaminobenzidine (DAB) substrate, and mouse on mouse immunodetection kit were from Vector Laboratories (Burlingame, CA, http://www.vectorlabs.com). Protease inhibitor cocktails, in situ cell death detection kit (fluorescein), and in situ cell death detection kit (peroxidase (POD)) were from Roche Diagnostics (Basel, Switzerland, http://www.roche-applied-science.com). Primary antibodies used were: rabbit anti-cleaved caspase-3 (9661), rabbit anti-cleaved caspase-7 (8438), rabbit anti-cleaved caspase-8 (8592), rabbit anti-phospho-extracellular signal regulated kinase (ERK)1/2 (9101), rabbit anti-phospho-JNK (9251), rabbit anti-phospho-p38 (9211), rabbit anti-phospho-Akt (4060), rabbit anti-Akt (9272), rabbit anti-β-actin (4970) (all from Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), rat anti-BrdU (ab6326, Abcam, Cambridge, U.K., http://www.abcam.com), rabbit anti-cleaved caspase-3 (ab13847, Abcam), goat anti-doublecortin (DCX) (sc-8066, Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), mouse anti-neuronal nuclei (NeuN, MAB377, Merck Millipore), mouse anti-glial fibrillary acidic protein (GFAP, MAB360, Merck Millipore). Secondary antibodies used were: Alexa Fluor 594-conjugated donkey anti-rabbit IgG, donkey anti-goat IgG and donkey anti-mouse IgG, Alexa Fluor 488-conjugated donkey anti-rat IgG, and donkey anti-goat IgG (all from Invitrogen), and biotinylated donkey anti-rat secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com).

Stroke Model

All experimental procedures were approved by the Animal Care and Use Committee of Shanghai Medical College, Fudan University. Adult male C57BL/6 mice (Shanghai SLAC Laboratory Animal Co., Ltd.), 8–10-week old, were used in this study. Focal cortical strokes were produced by electrocoagulation of the distal portion of the right middle cerebral artery (MCA), as described [17, 18]. Previous studies have shown that the focal cortical strokes enhance neurogenesis in the DG [19, 20]. Mice were anesthetized with intraperitoneal injections of chloral hydrate (360 mg/kg b.wt.). The dura mater was carefully opened and the MCA was isolated, electrocauterized, and disconnected just distal to crossing the olfactory tract. Immediately following the occlusion, the right common carotid artery (CCA) was occluded with a microvascular clip (Fine Science Tools, Foster City, CA, http://www.finescience.com) for 15 minutes. To produce moderately large cortical infarcts of reproducible size, mice were subjected to transient occlusion of the ipsilateral CCA [21, 22]. Body temperature was maintained at 37 ± 0.5°C using a heating pad (World Precision Instruments, Sarasota, FL, http://www.wpiinc.com). The sham surgery was similar to the MCAO (MCA occlusion) surgery and included opening of the dura mater and exposure of CCA; however, neither the MCA nor CCA were clipped or coagulated [23, 24]. Treatment groups were assigned in a randomized and blinded manner.

For 14 days cerebral infarction, six coronal sections (20 μm) were stained with hematoxylin and eosin (H&E). Infarct volume was measured using the NIH Image J analysis system [25] and was presented as percentage of the contralateral hemisphere. Six mice were evaluated per group.

BrdU Labeling

For investigation of the kinetics of cell proliferation after stroke, intraperitoneal injections of BrdU (50 mg/kg) were given twice daily for 2 days (i.e., on day 5 and 6 or day 12 and 13) after stroke or sham surgery, and mice were killed 1 day later. Three mice were evaluated per group. For investigation of cell proliferation and differentiation after injection of caspase-3 inhibitor, intraperitoneal injections of BrdU (50 mg/kg) were given twice daily during days 5–13 after stroke [26], and mice were killed at 14 or 42 days. Six mice were evaluated per group.

Caspase-3 Inhibitor Injection

We anesthetized the mice with 360 mg/kg chloral hydrate. Using standard stereotaxic techniques, a 26-gauge stainless steel guide cannula was implanted 1 mm above the left lateral ventricle (0.2 mm caudal and 0.9 mm lateral to bregma). The cannula was secured to the skull by dental cement and screws and closed with a dummy cannula cap. Mice were allowed to recover from the surgery for 10 days before induction of stroke. The caspase-3 inhibitor, Z-DEVD-fmk was dissolved in DMSO and further diluted in phosphate-buffered saline (PBS; pH 7.4). Intraventricular injections of Z-DEVD-fmk (240 ng in 2 μL 1.8% DMSO) or vehicle (1.8% DMSO) were given twice daily, 2 days a week for 2 weeks (on day 3, 6, 9, and 12 after stroke) based on a published protocol with minor modification [27]. As expected from previous studies [28], mice treated with intraventricular injections of Z-DEVD-fmk on day 3 after stroke showed significantly reduced levels of cleaved caspase-3 in the SVZ on day 6, when compared to the vehicle-treated mice (Supporting Information Fig. 1A). All injections were performed on conscious mice. The dose used early after stroke effectively reduced infarct size in mice, according to previous reports [11]. To exclude the possibility of neuroprotective effect of caspase-3 inhibitor, we started the injection of Z-DEVD-fmk on day 3 after stroke. Treatment groups were assigned in a randomized and blinded manner.

Behavioral Measurements

A blind experimental procedure was used for all behavioral measurements. At 42 days after stroke, locomotor activity of mice (seven mice per group) was measured in a 27.3 cm × 27.3 cm × 40 cm activity chamber (Med Associates Inc., St Albans, VT, http://www.med-associates.com) [29]. The chamber contained three 16-beam infrared arrays spaced 2.9 cm apart. During the test, mice were individually placed in the center of the chamber and allowed to move freely for 30 minutes. The movements of mice were recorded by a digital video and analyzed via SOF-811 analyzer software (Med Associates Inc.). Locomotor activity was calculated using the total distance traveled and the number of infrared beams crossed by the mice. The beam-walking test was performed to assess locomotor deficit before and 1, 7, 14, 28, and 42 days after stroke as described with modifications [30, 31]. The mice (6 mice for sham, 10 mice for other groups) were placed on one end of a narrow wooden beam (12 mm in diameter, 1.2 m long and 45 cm high), and usage of each limb during beam crossing was recorded by a digital video. The total numbers of limb steps and the numbers of forelimb and hindlimb foot faults were recorded. The percentage of foot faults to total steps that occurred within 10 minutes was calculated. A foot fault was defined as any paw slips off the top surface of the beam.

Immunohistochemistry

Mice were perfused with ice-cold PBS and 4% phosphate-buffered paraformaldehyde. Brains were cryoprotected in 30% sucrose at 4°C overnight, and coronal sections (20 μm) were prepared using a cryostat (Leica Microsystems Inc., Buffalo Grove, IL, http://www.leica-microsystems.com). Immunohistochemistry was performed as described previously [32, 33]. For BrdU staining, all sections were pretreated with 2N HCl for 30 minutes to denature DNA as reported previously [26]. For immunofluorescence staining, sections were stained with Alexa Fluor 594-conjugated or Alexa Fluor 488-conjugated secondary antibodies, respectively. Nuclei were stained with DAPI. For immunoperoxidase staining, sections were treated with biotinylated secondary antibody followed by incubation with avidin-biotin-peroxidase complex. The staining was visualized with DAB substrate and briefly counterstained in hematoxylin. Images were obtained using an Olympus BX 51 microscope and an Olympus FV 1000 confocal microscope. Colocalization was verified and reconstructed using Olympus FV 10-ASW software. Further information is provided in supporting information.

TUNEL Staining

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) was evaluated using in situ cell death detection kit (fluorescein or POD) according to the manufacturer's instructions. Briefly, sections were permeabilized, and double-strand DNA breaks were labeled via a strepavidin-horseradish peroxidase and visualized with DAB or fluorescein isothiocyanate. We assessed six mice per group for each time point.

DNA Gel Electrophoresis

Mice were perfused with ice-cold PBS, and the brains were removed and frozen immediately at −80°C. DNA was isolated from brain tissue as previously described with minor modifications [34]. Briefly, brain samples were homogenized and digested in lysis buffer (10 mmol/L Tris HCl, PH 8.0, 0.1 mol/L EDTA, 0.5% sodium dodecyl sulfate, 50 μg/mL RNase A) at 37°C for 1 hour. Sample was extracted with an equal volume of phenol/chloroform/amyl (25:24:1) and treated with proteinase K (0.1 mg/mL) at 55°C for 3 hours. DNA pellet was washed with 70% ethanol and dissolved in 50 μL Tris-EDTA buffer. DNA was subjected to gel electrophoresis on a 2% agarose gel containing ethidium bromide, followed by visualization under UV illumination. The DNA isolated from the ischemic areas at 3 days after stroke exhibited clear DNA laddering and was used as a positive control. We examined three brains for each time point.

Fluoro-Jade B Staining

Mice were perfused with ice-cold PBS and 4% paraformaldehyde in PBS (pH 7.4). Brains were postfixed in 4% paraformaldehyde at 4°C, and then cryoprotected in 30% sucrose overnight at 4°C. Cerebral tissue cryosections (20 μm) were dehydrated in 100% and 70% ethanol and incubated with 0.06% potassium permanganate for 10 minutes. The sections were then rinsed in distilled water and incubated in 0.0004% Fluoro-Jade B solution in 0.1% acetic acid for 15 minutes. The sections were rinsed in distilled water, dehydrated, and cleared in xylene. Staining was visualized using an Olympus FV 1000 confocal microscope. We examined three brains for each time point.

Cell Counting

To quantify BrdU-positive, caspase-3-positive, and TUNEL-positive cells in the SVZ, DG, and the peri-infarct cortical areas, bright-field images were digitized using a ×20 objective lens. Data are expressed as the number of DAB-positive cells per section. For quantification of newly born immature neurons in the SVZ, cells double-labeled with BrdU and DCX were counted with an epifluorescence microscope using a ×40 objective lens. The dorsolateral wing of the SVZ was included for sampling. Counting of regenerating neurons in the ipsilateral cortex was performed using a ×40 objective lens. The number of BrdU and NeuN/GFAP double-positive cells in the entire cortex was examined. Data are expressed as the number of double-labeled cells per section. For each animal, three sections for SVZ (0.6 mm apart) and four sections for DG (1.0 mm apart) were analyzed. Each image was traced using ImageJ software. The traced area was measured and the total numbers of positive cells in the traced area were counted. All quantitative measurements were conducted in a blind fashion to experimental conditions. We assessed six mice per group for each time point.

Neurosphere Cultures and Immunocytochemistry

Ipsilateral SVZ cells were dissociated from ischemic hemispheric brains at 7 days after stroke as reported previously [35]. After digestion, the cells were cultured in DMEM/F-12 medium supplemented with 1% penicillin plus streptomycin (10,000 U/mL), 2 mM l-glutamine, 2% B27 supplement, 20 ng/mL epidermal growth factor, and 20 ng/mL fibroblast growth factor at a concentration of 10,000 cells per milliliter. Cells were maintained at 37°C with 5% CO2 in an incubator with high humidity. Primary neurospheres were mechanically dissociated and reseeded into 96-well plates (1,000 cells per well) in DMEM/F-12 medium including growth factors. Z-DEVD-fmk (25 μM) or LY294002 (25 μM) was added to the cultures at the time of seeding. The expression of cleaved caspase-3 was measured by Western blot after 3 days of Z-DEVD-fmk treatment. The neurosphere number was counted on day 7. Drug treatments were performed by an investigator blinded to the experimental protocol.

For imunostaining of cleaved caspase-3, neurospheres were transferred to glass coverslips precoated with poly-d-lysine and laminin and fixed in 4% paraformaldehyde in PBS. Cells were blocked in 10% donkey serum and incubated with rabbit anti-cleaved caspase-3 antibody overnight at 4°C. After three washes, Alexa Fluor 594 donkey anti-rabbit IgG was added for 30 minutes at room temperature. Nuclei were stained with DAPI. Slides were mounted in Vectashield. Apoptosis was detected by TUNEL using in situ cell death detection kit (Fluorescein).

Flow Cytometry

Neurospheres were gently collected, and 100,000 cells were labeled with Annexin V-FITC and propidium iodide (Annexin V-FITC apoptosis detection kit) according to the manufacturer's protocol. The cells were measured by an Epics Altra FACScan Flow Cytometer (Beckman Coulter, Fullerton, CA, https://www.beckmancoulter.com). Fluorescence activated cell sorting data were analyzed by EXPO32 V1.2 analysis software (Beckman Coulter).

Western Blot Analysis

SVZ from ischemic hemispheric brains at 12 days after stroke (n = 6 per group) were dissected. SVZ or neurosphere protein was extracted with RIPA lysis buffer including protease inhibitor cocktails [36]. After centrifugation, protein concentration was determined with the BCA protein assay. Equal amounts of protein samples were loaded on 8%, 10%, and 12% Tris-glycine gel, electrophoresed, and transferred onto PVDF membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 and probed with primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Signals were detected with an enhanced chemiluminescence solution and quantified by scanning densitometry using a Bio-Image Analysis System (Bio-Rad, Hercules, CA, http://www.bio-rad.com). For the loading control, the same membrane was blotted with anti-β-actin antibody.

Statistical Analysis

Values are represented as mean ± SD. Multiple comparisons were analyzed using one-way ANOVA followed by Bonferroni's multiple comparison test. When two groups were compared, unpaired two-tailed Student's t test or Mann Whitney U test was used. p < .05 was considered statistically significant.

Results

Kinetics of Neurogenic Responses and Cell Death After Stroke

Stroke stimulates neurogenesis in the SVZ and the DG [5, 6]. The peri-infarct cortical areas are critical for functional recovery after stroke [37, 38]. We therefore analyzed occurrence of neurogenic responses and cell death in the neurogenic niches and the peri-infarct cortical areas in mice subjected to stroke. BrdU uptake was analyzed to assess cell neurogenic responses. After stroke, dramatic increase of BrdU immunoreactivity was observed at 3 days, reached maximal expression at 7 days, and persists to at least 14 days in the SVZ, DG, and the peri-infarct cortical areas (Fig. 1A, 1B), similar to results reported previously [39, 40]. Surprisingly, only a few TUNEL-stained cells were detected in the SVZ and DG during the 14-day period after stroke (Fig. 1C, 1D). In the peri-infarct cortex areas, the number of TUNEL-labeled cells was significantly increased at 1–3 days after stroke but returned to basal levels at 7–14 days (Fig. 1C, 1D). These data suggest differential spatio-temporal patterns of neurogenic responses and cell death in the ischemic brain, and that the window of neurogenic responses is longer than that of cell death in the lesioned cortex.

Figure 1.

Kinetics of neurogenic responses and cell death after stroke. (A): Representative photomicrographs of BrdU immunostaining in the SVZ, DG, and the peri-infarct cortex from sham-operated mice and ischemic mice at 1, 3, 7, and 14 days after stroke. Scale bar = 100 μm (upper and middle panels) and 25 μm (bottom panel). (B): Time course quantification of BrdU-positive cells after stroke. Values are mean ± SD (n = 3 per group). p < .05 versus sham-operated group. (C): Representative photomicrographs of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) staining in the SVZ, DG, and the peri-infarct cortex from sham-operated mice and ischemic mice at 1, 3, 7, and 14 days. Arrows show TUNEL-positive cells. Scale bar = 5 μm (upper and middle panels) and 20 μm (bottom panel). (D): Time course quantification of TUNEL-positive cells after stroke. Values are mean ± SD (n = 3 per group). p < .05 versus sham-operated group. Abbreviations: BrdU, bromodeoxyuridine; DG, dendate gyrus; GCL, granule cell layer; HIL, hilus; LV, lateral ventricle; S, sham; SVZ, subventricular zone.

Caspase-3 Expression was Upregulated in the Neurogenic Niches During the Period of Stroke Recovery

We found that the expression of cleaved caspase-3 was increased within ischemic areas at 24 hours after stroke (Supporting Information Fig. 1B). These areas were extensively labeled with TUNEL-positive dead cells (Supporting Information Fig. 1C). Immunohistochemical staining showed that cleaved caspase-3 was presented both in the cell nucleus and in the cytoplasm (Supporting Information Fig. 1D). In contrast, we observed a marked increase in cleaved caspase-3 in the neurogenic niches SVZ (Fig. 2A) and DG (Fig. 2B) at 7–14 days after stroke compared to sham controls. Quantitative analysis showed that the cleaved caspase-3 enhanced roughly in parallel with the stroke-induced increase in proliferating NPCs (Figs. 1B, 2C) but not related to an increase in cell death (Figs. 1D, 2C). These data suggest that caspase-3 may have a different role during the delayed stages after stroke. To test our hypothesis, we first identified the cellular localization of cleaved caspase-3. Double immunostaining for cleaved caspase-3 with BrdU or DCX demonstrated that caspase-3 was expressed in both BrdU-positive cells and DCX-positive NPCs in the SVZ (Fig. 3A, 3B) and the DG (Fig. 3C, 3D), indicating that these cleaved caspase-3-positive cells were proliferating NPCs. In addition, we failed to detect cleaved caspase-7 and cleaved caspase-8 in these regions (data not shown).

Figure 2.

Increased expression of caspase-3 in the SVZ and the DG during the period of stroke recovery. (A, B): Representative photomicrographs of caspase-3 immunostaining in the SVZ and the DG from sham-operated mice and ischemic mice at 1, 3, 7, and 14 days after stroke. Scale bar = 50 μm (A) and 25 μm (B). (C): Time course quantification of caspase-3-positive cells in the SVZ and the DG after stroke. Values are mean ± SD (n = 3 per group). *, p < .05 versus sham-operated mice. Abbreviations: Cas-3, caspase-3; DG, dentate gyrus; DAPI, 4,6-diamino-2-phenylindole; GCL, granule cell layer; HIL, hilus; LV, lateral ventricle; S, sham; SVZ, subventricular zone.

Figure 3.

Caspase-3 expression was increased in proliferating neuronal precursor cell (NPCs) in the neurogenic niches during the period of stroke recovery. (A, B): Cleaved caspase-3 was extensively expressed in both BrdU-positive cells and DCX-positive cells in the subventricular zone (SVZ) at 14 days. Scale bar = 20 μm. (C, D): Cleaved caspase-3 was extensively expressed in both BrdU-positive cells and DCX-positive cells in the DG at 14 days. Scale bar = 20 μm. (E, F): No colocalization of cleaved caspase-3 and TUNEL-positive cells in the SVZ and the dentate gyrus (DG) at 14 days. Arrows show TUNEL-positive and cleaved caspase-3-negative cells. Scale bar = 20 μm (inset, 2 μm). (G, H): None of the cleaved caspase-3-positive cells appeared to have pyknotic nuclei (arrows) in the SVZ and the DG at 14 days. Scale bar = 20 μm. (I): DNA laddering was not detected by agarose gel electrophoresis in samples taken from the ipsilateral SVZ and DG at 14 days (lane 3 and lane 4). DNA markers are shown in lane 1. Lane 2 is a positive control prepared from the ischemic areas at 3 days after stroke. (J): No neuronal injury was detected by cresyl violet staining in the ipsilateral SVZ and DG at 14 days. Bottom panel is a positive control from the ischemic areas at 1 day after stroke. Scale bars = 20 μm. (K): No degenerating neurons were detected by Fluoro-Jade B staining in the ipsilateral SVZ and DG at 14 days. Bottom panel is a positive control from the ischemic areas at 1 day after stroke. Scale bars = 20 μm. Each observation was repeated three times. Abbreviations: BrdU, bromodeoxyuridine; Cas-3, caspase 3; DAPI, 4,6-diamino-2-phenylindole; DCX, doublecortin; GCL, granule cell layer; HIL, hilus; LV, lateral ventricle; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

To examine whether these cleaved caspase-3-positive cells in the SVZ and the DG were dying, we performed double-labeling with antibody against cleaved caspase-3 and DNA fragmentation (TUNEL). We found that none of the caspase-3-positive cells was TUNEL-positive (Fig. 3E, 3F). We also observed that the caspase-3-positive nucleus were nonpyknotic cells (Fig. 3G, 3H). Furthermore, we demonstrated that neither SVZ nor DG showed DNA damage when detected with DNA gel electrophoresis (Fig. 3I) or histological features of neuronal injury when examined with cresyl violet (Fig. 3J) and Fluoro-Jade B staining (Fig. 3K). These data indicate that proliferating NPCs activate caspase-3 in the neurogenic niches without undergoing apoptosis during stroke recovery.

Inhibition of Caspase-3 Promoted the Proliferation of NPCs After Stroke

We next tested whether the delayed upregulation of caspase-3 participate in neurogenic responses after stroke. For this purpose, we infused the caspase-3 inhibitor Z-DEVD-fmk or vehicle into the lateral ventricle twice a week for 2 weeks starting from 3 days after stroke and examined mice at 14 days. The pharmacological approach was used because studies have shown that genetic ablation of caspase-3 protected mice from stroke [41]. To determine the effects of caspase-3 on long-term behavioral and histological outcomes, we induced stroke by permanent distal MCAO in mice. This cortical stroke model was used in this study because it generally produces a lower stroke mortality [42]. The overall mortality in our model was less than 10%. Our data showed that there was no significant difference in ischemic damage between Z-DEVD-fmk-treated and vehicle-treated mice at 14 days (Supporting Information Fig. 1E, 1F). Thus, this approach allowed us to provide evidence that effects of caspase-3 inhibition on neurogenesis are not secondary to the reduced infarct volume. Compared with vehicle controls, mice treated with Z-DEVD-fmk showed significantly increased BrdU-positive cells and BrdU-positive NPCs in the SVZ by 45.8% (Fig. 4A, 4C) and 34.3% (Fig. 4B, 4D), respectively. To test if caspase-3 inhibition could affect the survival of the NPCs indirectly, we assessed cell death in the SVZ from mice treated with Z-DEVD-fmk or vehicle at 14 days after stroke. We observed that Z-DEVD-fmk did not affect the number of TUNEL-positive cells (Z-DEVD-fmk-treated mice, 2.00 ± 0.89; vehicle-treated mice, 1.50 ± 1.22; p = .34, n = 6) at 14 days. Together, these data indicate the importance of caspase-3 in NPCs proliferation after stroke.

Figure 4.

Caspase-3 negatively regulated subventricular zone (SVZ) neuronal precursor cells (NPCs) proliferation during stroke recovery. (A): Representative photomicrographs of BrdU immunostaining in the SVZ from sham-operated mice and mice treated with vehicle or caspase-3 inhibitor Z-DEVD-fmk at 14 days after stroke. Scale bar = 50 μm. (B): Representative images of cells double-labeled for BrdU and DCX in the SVZ from sham-operated mice and mice treated with vehicle or caspase-3 inhibitor Z-DEVD-fmk at 14 days after stroke. Scale bar = 50 μm (left panel) and 10 μm (right panel). (C): Quantitative determination of BrdU-positive cells for each group. Values are mean ± SD (n = 6 per group). *, p < .05. (D): Quantitative determination of BrdU and DCX double-labeled cells for each group. Values are mean ± SD (n = 6 per group). *, p < .05. Abbreviations: BrdU, bromodeoxyuridine; DCX, doublecortin; DEVD, Z-DEVD-fmk; LV, left ventricle.

Caspase-3 Limited NPCs Self-Renewal in Culture

To further investigate the function of caspase-3 in NPCs, we prepared neurosphere cultures from the SVZ of ischemic mice at 7 days after stroke (Fig. 5A). During neurosphere formation, cleaved caspase-3 was consistently detected (Fig. 5B). This observation was further confirmed through measurement of cleaved caspase-3 by immunocytochemistry (Fig. 5C). Importantly, 87.3% of the cleaved caspase-3-positive cells were TUNEL-negative cells (Fig. 5D, 5E). Thus, the expression of caspase-3 in neurospheres may not be involved in cell death. To directly test the effects of caspase-3 on apoptosis, we treated neurosphere cultures with the caspase-3 inhibitor Z-DEVD-fmk. Despite this treatment clearly inhibited the expression of caspase-3 (Fig. 5B), we did not observe substantial differences in percentage of apoptosis between Z-DEVD-fmk-treated and vehicle-treated neurospheres (Fig. 5F, 5G). In addition, counts of TUNEL-positive NPCs showed cell death rate in cultures was not changed by the treatment (percentage of TUNEL-positive cells in Z-DEVD-fmk-treated vs. vehicle-treated neurospheres: 1.92 ± 0.04 vs. 1.86 ± 0.03; n = 15). We next dissociated primary neurospheres and examined the putative nonapoptotic effects of caspase-3 on the formation of secondary neurospheres. Whereas treatment of cultures with Z-DEVD-fmk did not affect neurosphere diameter and BrdU incorporation (data not shown), Z-DEVD-fmk-treated cultures generated more secondary neurospheres than vehicle-treated cultures (Fig. 5H, 5I). These results demonstrated that caspase-3 is not a cell-death executing factor for NPCs but instead negatively regulates NPCs self-renewal.

Figure 5.

Caspase-3 regulated self-renewal of neuronal precursor cells (NPCs) but not cell death in cultured subventricular zone (SVZ) NPCs. (A): Schematic drawing showing the isolation of SVZ NPCs from ischemic brains at 7 days after stroke. (B): Western blot analysis of cleaved caspase-3 in neurospheres under proliferative condition. Neurospheres were incubated in proliferative media for the indicated time points. DEVD reduced the expression of cleaved caspase-3 after 3 days of treatment. (C): Immunocytochemical staining of cleaved caspase-3 in neurospheres. Nuclei were visualized with DAPI. Scale bar = 50 μm (inset, 5 μm). (D): Double staining of cleaved caspase-3 with TUNEL in neurospheres. Arrowhead shows a TUNEL-positive and cleaved caspase-3-positive cell. Scale bar = 15 μm (inset, 2.5 μm). (E): Percentage of TUNEL-positive and -negative cells among the cleaved caspase-3-positive cells in neurospheres. Values are mean ± SD (n = 15 per group). Data are representative of three independent experiments. (F): Representative flow cytometry analysis of Annexin V/PI staining in neurospheres treated with vehicle or DEVD for 7 days. FITC, Fluorescein isothiocyanate. (G): Flow cytometry quantitation showing no substantial difference in the frequency of apoptotic events between vehicle and DEVD-treated neurospheres. Values are mean ± SD (n = 10 per group). Data are representative of three independent experiments. (H): Representative images of neurospheres treated with vehicle or DEVD for 7 days. Scale bar = 100 μm. (I): Caspase-3 inhibition with DEVD increased the number of secondary neurospheres at 7 days after treatment. Values are mean ± SD (n = 10 per group). Data are representative of three independent experiments. *, p < .05. Abbreviations: Cas-3, cleaved caspase-3; DAPI, 4,6-diamino-2-phenylindole; DEVD, Z-DEVD-fmk; FITC, Fluorescein isothiocyanate; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Akt Phosphorylation Was Involved in the Effects of Caspase-3

To explore the potential mechanism of caspase-3 on NPCs proliferation after stroke, we compared the protein levels of known substrates of caspase-3 [43, 44] in the SVZ from mice treated with Z-DEVD-fmk or vehicle at 12 days after stroke. Z-DEVD-fmk did not alter the levels of phospho-ERK, phospho-JNK, and phospho-p38, but it significantly increased phospho-Akt levels (Fig. 6A, 6B). These data imply that in our stroke recovery model, inhibition of caspase-3 activity may contribute to NPCs proliferation by modulating phosphorylation of Akt. Furthermore, we found a significant decrease of Z-DEVD-fmk-induced increase in NPCs self-renewal in neurosphere cultures after being challenged with LY294002 (Fig. 6C, 6D). Taken together, our experiments suggest that the caspase-3-dependent regulation of NPCs proliferation after stroke is likely mediated through phosphorylation of Akt.

Figure 6.

Akt phosphorylation mediated the effects of caspase-3 on neuronal precursor cell (NPCs) proliferation. (A): Caspase-3 inhibition with DEVD induced an increase in pAkt in the subventricular zone at 12 days after stroke, while the pERK, pJNK, and p-p38 signals remained unperturbed. (B): Quantitative determinations of the levels of pAkt, pERK, pJNK, and p-p38. Values are mean ± SD (n = 6 per group). *, p < .05. (C, D): LY abolished the effects of DEVD on NPCs self-renewal. Scale bar = 100 μm. Values are mean ± SD (n = 8 per group). Data are representative of three independent experiments. *, p < .05. Abbreviations: DEVD, Z-DEVD-fmk; LY, LY294002; pAkt, phospho-Akt; pERK, phospho-ERK; pJNK, phospho-JNK; p-p38, phospho-p-38.

Inhibition of Caspase-3 Activity Promoted Neuronal Regeneration and Functional Recovery After Stroke

To elucidate whether inhibition of caspase-3 affects migration and neuronal differentiation of the proliferated NPCs, we treated mice with either vehicle or Z-DEVD-fmk and examined brains at 14 and 42 days after stroke (Fig. 7A). Our data showed that the number of BrdU-positive cells in the SVZ was reduced in both vehicle-treated and Z-DEVD-fmk-treated mice when examined at 42 days rather than 14 days (Figs. 4C, 7D), showing that NPCs had migrated from the SVZ into the lesioned tissue in the ischemic hemisphere. Treatment with Z-DEVD-fmk significantly increased the number of BrdU-positive cells in the lesioned cortex at 42 days (Fig. 7B, 7D), suggesting that caspase-3 inhibition promoted newborn cells migration induced by stroke. Z-DEVD-fmk did not significantly alter BrdU and GFAP double-positive cells (BrdU and GFAP double-positive cells in Z-DEVD-fmk-treated vs. vehicle-treated mice: 104.61 ± 38.71 vs. 85.56 ± 15.28; p = .29, n = 6), but it significantly increased BrdU and NeuN double-positive cells in the lesioned cortex (Fig. 7C, 7E). These data suggest that caspase-3 inhibition promoted regeneration of neuronal cells. In parallel with these effects, caspase-3 inhibition improved motor activity in mice after stroke, as demonstrated by increase in total distance traveled and number of movements at 42 days (Fig. 7F, 7G). To confirm these results, we assessed locomotor deficit using the beam walking test. This analysis showed that all mice exhibited the same levels of functional deficits at 1-day after stroke and there were no significant differences between vehicle- and Z-DEVD-fmk-treated groups (Fig. 7H, 7I). Compared with vehicle-treated controls, Z-DEVD-fmk-treated mice showed a significant decrease in the number of forelimb foot faults after 14 days and a significant reduction in the number of hindlimb foot faults after 28 days, respectively.

Figure 7.

Caspase-3 inhibition promoted neuronal regeneration in the cortex and improved functional recovery after stroke. (A): Schematic representation of the experimental design. Cannulation was performed 10 days before induction of stroke. Intracerebroventricular (i.c.v.) injections of DEVD were given twice daily on day 3, 6, 9, and 12 after stroke. Intraperitoneal injections (i.p.) of BrdU were given twice daily during days 5–13 after stroke, and mice were killed on day 14 or 42. (B): Representative photomicrographs of BrdU immunostaining in the lesioned cortex at 42 days. Scale bar = 50 μm. (C): Representative images of BrdU-positive neurons (NeuN) in the lesioned cortex at 42 days. Scale bar = 20 μm (left panel) and 10 μm (right panel). (D, E): Few BrdU-labeled cells were found in the SVZ on day 42. Inhibition of caspase-3 with DEVD increased the number of BrdU-positive cells (D) and BrdU-positive neurons (E) in the lesioned cortex at 42 days. Values are mean ± SD (n = 6 per group). *, p < .05. (F, G): Effects of Z-DEVD-fmk on locomotor activity at 42 days. Values are mean ± SD (n = 7 per group). *, p < .05. (H, I): Effects of DEVD on locomotor deficit. Analysis of forelimb (H) and hindlimb (I) footfaults was performed at the indicated times after stroke. Values are mean ± SD. n = 6 for sham-operated group, n = 10 for other groups. *, p < .05. (J): Schematic drawing of caspase-3-mediated regulation of Akt phosphorylation and its connection to NPC-mediated neurogenesis and functional recovery after stroke. The “↓”and “⊥” symbols indicate excitatory and inhibitory connections, respectively. Abbreviations: BrdU, bromodeoxyuridine; DEVD, Z-DEVD-fmk; dMCAO, distal middle cerebral artery occlusion; NeuN, neuronal nuclei; NPCs, neuronal precursor cells; SVZ, subventricular zone.

Discussion

In this study, we showed a nonapoptotic role of caspase-3 during stroke recovery. We provided evidence that cleaved caspase-3 that was expressed by SVZ NPCs cultured from ischemic brain constrained its self-renewal. Using a mouse stroke model, we found that local upregulation of cleaved caspase-3 in the neurogenic niches was important for the proliferation, migration, and maturation of NPCs. Furthermore, we demonstrated that inhibition of caspase-3 activity improved functional recovery after stroke (Fig. 7J). These findings suggest that the apoptosis-associated factor caspase-3 is a critical inhibitory element for stroke-induced neurogenesis.

Caspase-3 is thought to function in neuronal apoptosis during development and has been identified as a key mediator of cell death following cerebral ischemia, traumatic brain injury, spinal cord injury, and cerebral hemorrhage [41, 45-47]. However, caspase-3 was also shown to play nonapoptotic roles in cell proliferation and differentiation [48, 49]. Several lines of evidence indicate that caspase-3 mediates diverse nonapoptotic functions in the central nervous system. Caspase-3 is expressed in mitotic and postmitotic cells of the murine brain, and there is evidence that caspase-3-activated cells can divide in the neurogenic niches and migrate to the olfactory bulb [50]. Caspase-3 may contribute to synaptic plasticity and axon guidance [51-53]. It has been recently reported that caspase-3 promotes regeneration of injured axons in Caenorhabditis elegans [54]. Overall, the majority of studies suggest that caspase-3 has nonapoptotic beneficial functions during development and in the adult nervous system. However, at least one study has demonstrated that caspase-3 can drive synaptic failure and contribute to cognitive dysfunction in Alzheimer's disease [55]. Our data here showed that caspase-3 enhanced roughly in parallel with the stroke-induced increase in proliferating NPCs. We observed that the expression of caspase-3 in proliferative NPCs was not associated with increased apoptosis during the delayed stages after stroke. Furthermore, we demonstrated that inhibition of caspase-3 activity not only enhanced NPCs proliferation but also enhanced regenerated neurons. This result here is also consistent with the finding that deficiency of caspase-3 increases the density of neurons in the cortex [41]. We did not detect effects of the caspase-3 inhibitor Z-DEVD-fmk on infarct volume but observed the effects of Z-DEVD-fmk infusion on functional recovery. We thus conceive that increase in endogenous neuronal regeneration may contribute to functional recovery seen after inhibition of caspase-3.

The serine/threonine protein kinase Akt is a cell survival signaling and a known target of caspase-3 during apoptosis [56-58]. Akt was shown to play a critical role in stroke-induced NPCs proliferation [59, 60]. Depletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative regulator of the phosphoinositide 3 kinase (PI3K)-AKT signaling pathway, leaded to persistently enhanced NPCs self-renewal [61]. Our data revealed that inactivation of Akt is indeed required for the effects of caspase-3 on NPCs proliferation (Fig. 7J). Subsequently, we showed that the effects of caspase-3 appear not to be associated with the ERK, JNK, or p38 pathways as these were not different between Z-DEVD-fmk- and vehicle-treated mice. However, in addition to Akt, multiple other caspase-3 substrates may also contribute to the caspase-3 effects. Future studies are needed to investigate how caspase-3 signal may modulate neurogenesis after stroke.

Conclusions

Neurogenesis holds promise for brain repair and stroke recovery. Optimization of neurogenesis after stroke requires identification of the molecular pathways that regulate endogenous neurogenic capacity of the brain. Our data here identified caspase-3 signaling as a novel mechanism that limits the neurogenic response after stroke. Considering that caspase-3 also mediates neuronal death during acute stroke, our data should revitalize interest in targeting caspase-3 for treatment of stroke.

Acknowledgments

We thank Dr. Eng H Lo (Department of Radiology, MA General Hospital, Harvard Medical School) and Dr. Mingming Ning (Cardio-Neurology Clinic, Clinical Proteomics Research Center and Department of Neurology, MA General Hospital, Harvard Medical School) for carefully reading the manuscript and many helpful discussions. This work was supported by grants from the National Natural Science Foundation of China (30971014, 81071062, and 81271457) and National Program of Basic Research, sponsored by the Ministry of Science and Technology of China (2011CB503700-G).

Author Contributions

W.F.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; Y.D. and H.X: collection and assembly of data, data analysis and interpretation, and manuscript writing; X.Z.: collection and assembly of data and data analysis and interpretation, P.C., L.W., C.S., C.L., and P.Z.: data analysis and interpretation; B.Q.Z.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript.

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflict of interest.

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