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Notch1 signaling modulates neuronal progenitor activity in the subventricular zone in response to aging and focal ischemia

Authors

  • Fen Sun,

    1. Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
    2. Department of Pharmacology & Neuroscience, Institute for Aging and Alzheimer's Disease Research, University of North Texas Health Science Center, Fort Worth, TX, USA
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  • XiaoOu Mao,

    1. Buck Institute for Research on Aging, Novato, CA, USA
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  • Lin Xie,

    1. Buck Institute for Research on Aging, Novato, CA, USA
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  • Meiping Ding,

    Corresponding author
    1. Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
    • Correspondence

      Kunlin Jin, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA. Tel.: 817 7352579; fax: 817 7350408; e-mail: kunlin.jin@unthsc.edu

      and

      Meiping Ding, Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, No. 88, Jiefang Road, Hangzhou, Zhejiang 310009, China. Tel.: +86 571 87783769; fax: +86 571 87784750;

      e-mail: yichen@mail.hz.zj.cn

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  • Bei Shao,

    1. Department of Neurology, First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
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  • Kunlin Jin

    Corresponding author
    1. Department of Pharmacology & Neuroscience, Institute for Aging and Alzheimer's Disease Research, University of North Texas Health Science Center, Fort Worth, TX, USA
    2. Buck Institute for Research on Aging, Novato, CA, USA
    • Correspondence

      Kunlin Jin, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA. Tel.: 817 7352579; fax: 817 7350408; e-mail: kunlin.jin@unthsc.edu

      and

      Meiping Ding, Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, No. 88, Jiefang Road, Hangzhou, Zhejiang 310009, China. Tel.: +86 571 87783769; fax: +86 571 87784750;

      e-mail: yichen@mail.hz.zj.cn

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Summary

Neurogenesis diminishes with aging and ischemia-induced neurogenesis also occurs, but reduced in aged brain. Currently, the cellular and molecular pathways mediating these effects remain largely unknown. Our previous study has shown that Notch1 signaling regulates neurogenesis in subventricular zone (SVZ) of young adult brain after focal ischemia, but whether a similar effect occurs in aged normal and ischemic animals is unknown. Here, we used normal and ischemic aged rat brains to investigate whether Notch1 signaling was involved in the reduction of neurogenesis in response to aging and modulates neurogenesis in aged brains after focal ischemia. By Western blot, we found that Notch1 and Jagged1 expression in the SVZ of aged brain was significantly reduced compared with young adult brain. Consistently, the activated form of Notch1 (Notch intracellular domain; NICD) expression was also declined. Immunohistochemistry confirmed that expression and activation of Notch1 signaling in the SVZ of aged brain were reduced. Double or triple immunostaining showed that that Notch1 was mainly expressed in doublecortin (DCX)-positive cells, whereas Jagged1 was predominantly expressed in astroglial cells in the SVZ of normal aged rat brain. In addition, disruption or activation of Notch1 signaling altered the number of proliferating cells labeled by bromodeoxyuridine (BrdU) and DCX in the SVZ of aged brain. Moreover, ischemia-induced cell proliferation in the SVZ of aged brain was enhanced by activating the Notch1 pathway and was suppressed by inhibiting the Notch1 signaling. Reduced infarct volume and improved motor deficits were also observed in Notch1 activator–treated aged ischemic rats. Our data suggest that Notch1 signaling modulates the SVZ neurogenesis in aged brain in normal and ischemic conditions.

Introduction

The treatment for ischemic stroke remains an intimidating mission because few therapeutic strategies have proven to be effective. Systemic thrombolysis with intravenous tissue plasminogen activator (tPA) is the only proven treatment to improve clinical outcome of patients with acute ischemic stroke (Brott & Bogousslavsky, 2000; Adams et al., 2007). But because of an increased risk of hemorrhage beyond 3 h poststroke, only certain stroke patients (1–2%) can benefit from tPA. Therefore, new therapies need to be found that protect and repair the damaged brain after stroke. Regenerative cell–based therapies offer long-term hope for many patients with stroke, as neural stem/progenitor cells (NSCs) may be possible for dead or injured neural cells to be replaced after acute stroke (Kondziolka et al., 2000; Kelly et al., 2004; Jin et al., 2005). As stroke in humans usually afflicts the elderly (Arnold, 1981; Ramirez-Lassepas, 1998; Popa-Wagner et al., 2007, 2011), it is important to know whether stem cells can be a potential therapy for stroke in aged models of stroke (Popa-Wagner et al., 2010).

Neural stem cells persist in the subventricular zone (SVZ) of the lateral ventricle and in the subgranular zone (SGZ) of dentate gyrus throughout adulthood in mammals (McDermott & Lantos, 1991; Eriksson et al., 1998; Jin et al., 2001; Yoshimura et al., 2001). There is substantial evidence for increased proliferation of NSCs in the adult brain after brain injuries. In the young adult animal, stroke induces the proliferation of endogenous NSCs located in the SVZ (focal ischemia) (Jin et al., 2001) and in the dentate SGZ (global ischemia) (Liu et al., 1998). The newborn cells can migrate into the damaged brain regions (Jin et al., 2003b), express phenotypic markers of mature neurons (NeuN, MAP-2) and region-specific mature neuronal markers like dopamine (Arvidsson et al., 2002; Parent et al., 2002), and form synapses (Yamashita et al., 2006). Evidence for functional neuronal replacement in the damaged brain regions has been reported from a model of global cerebral ischemia that affects the hippocampus predominantly. Infusion of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) led to regeneration of hippocampal CA1 neurons derived from the dentate SGZ, which integrated into the existing brain circuitry and were thought to have contributed to ameliorating neurologic deficits (Nakatomi et al., 2002). Despite an age-related reduction in basal SVZ proliferation (Tropepe et al., 1997; Jin et al., 2003a; Maslov et al., 2004; Luo et al., 2006), we found that NSCs in the SVZ of aged rats (Jin et al., 2004) and in the ischemic penumbra region of human (Jin et al., 2006) retained the capacity for proliferation and lesion-directed migration in response to cerebral ischemia, although the response was less robust than in younger animals. In addition, Yagita et al. (2001) reported increased proliferation, but reduced survival of new neurons after global ischemia in middle-aged rats in comparison with young adult rats. Darsalia et al. (2005) stated that postischemic neurogenesis in the SGZ of 15-month-old rats was reduced, but neurogenesis in the SVZ was increased. We found that conditional ablation of neurogenesis in the SVZ of young adult and middle-aged mice increased infarct size and exacerbated postischemic sensorimotor behavioral deficits (Jin et al., 2010). These findings suggest that ischemia-induced neurogenesis is critical for functional recovery after stroke and may be used as the new therapeutic strategy for stroke. However, the cellular and molecular pathways mediating these effects in aged animals remain largely unknown.

Previous studies show that Notch signaling is a fundamental pathway controlling cell fate acquisition and plays critical roles during maintenance, proliferation, and differentiation of NSCs in developing brain (Artavanis-Tsakonas et al., 1999). Recent evidence shows that Notch1 signaling is conserved in the regulation of adult neurogenesis. First, the Notch1 signaling molecules are expressed in cells of the SVZ and the SGZ of postnatal brain (Stump et al., 2002; Chojnacki et al., 2003). Second, expression of the Notch1 ligands, Jagged1 and Delta1, in neurogenic brain regions decreases with aging (Givogri et al., 2006). Third, disruption of Notch1 signaling interferes with the maintenance and proliferation of NSCs (Chojnacki et al., 2003; Wang et al., 2009a). Our previous study showed that Notch1 and its downstream targets were expressed in SVZ cells and that the number of BrdU-positive (proliferating) cells in the normal adult SVZ was significantly altered after inhibiting or activating the Notch1 pathway (Wang et al., 2009b). In addition, we found that Notch1 signaling in the SVZ was activated after focal ischemia and that ischemia-induced cell proliferation in the SVZ could be blocked by inhibiting the Notch1 pathway in young adult brain. However, whether Notch1 signaling activity contributes, directly or indirectly, to the age-dependent decline in neurogenesis, including that following stroke, remains largely unknown.

The present study was undertaken to examine the effect of Notch1 signaling on neurogenesis in the SVZ of the normal and ischemic aged rat brain in vivo. We found that the Notch1 and Jagged1 expression was decreased with aging, along with reduced activation level of Notch1 signaling. Double or triple immunostaining showed that Notch1 and activated form of Notch1 (Notch intracellular domain; NICD) were predominantly localized to doublecortin (DCX)-positive cells, whereas Jagged1 was mainly expressed in astrocytes, in the SVZ of aged rat brain. Disruption or activation of Notch1 signaling altered the number of proliferating cells in the SVZ of aged brain. In addition, ischemia-induced cell proliferation in the SVZ of aged brain was enhanced by activating the Notch1 pathway and was suppressed by inhibiting the Notch1 signaling. Reduced infarct volume and improved motor deficits were also observed in Notch1 activator–treated aged ischemic rats. Our data suggest that Notch1 signaling modulates the SVZ neurogenesis in aged brain in normal and ischemic conditions.

Materials and methods

Ethics statement

All animal procedures were approved by Institutional Animal Care and Use Committee of Buck Institute for Research on Aging and University of North Texas Health Science Center and conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize suffering and to reduce the number of animals used.

Focal cerebral ischemia

Aged (20–24 months old) male Fisher 344 rats were obtained from the National Institute of Aging rodent colony and were anesthetized with 2.0% isoflurane in 70% N2O/30% O2. Permanent distal middle cerebral artery occlusion (dMCAO) was performed as described previously (Nawashiro et al., 1997; Won et al., 2006). Briefly, a 2-cm incision was made under the surgical microscope between the left orbit and tragus, the temporal muscle was retracted laterally. A small burr hole (2 mm diameter) was made with a high-speed microdrill through the outer surface of the semitranslucent skull just over the visibly identified middle cerebral artery (MCA) at the level of the inferior cerebral vein. Saline was applied to the area throughout the procedure to prevent heat injury. The inner layer of the skull and the dura were removed with fine forceps, and left dMCAO was performed by electrocoagulation (by means of a small-vessel cauterizer) without damaging the brain surface. If the brain surface was visibly damaged or if the MCA bled owing to incomplete artery occlusion/coagulation, the animal was killed and not used. The temporal muscle was repositioned and the skin was closed. After surgery, the rats were placed in a cage under an infrared heating lamp until they recovered from anesthesia. Rectal temperature was maintained at 37.0 ± 0.5°C using a thermostat-controlled heating pad (Harvard Apparatus, Holliston, MA, USA). Sham-operated rats underwent identical surgery except that the MCA was not occluded.

Administration of Notch signaling activator and inhibitor

Male Fisher 344 rats (N = 8–12 per group) were anesthetized and implanted with an osmotic minipump (Alzet 1003D; Alza Corporation, CA, USA) as described previously (Wang et al., 2009b). Briefly, the cannula was placed into the left lateral ventricle: 1.6 mm lateral to the midline, 0.8 mm posterior to the bregma, and 4.0 mm deep into the pial surface. Notch1 activator and inhibitor were prepared as described previously (Wang et al., 2009b). For Notch1 activator, Notch1-activating antibody (clone 8G10; Millipore, Temecula, CA, USA) 10 μg/ml was dissolved in the artificial cerebrospinal fluid (aCSF; Harvard apparatus, Holliston, MA, USA) at a 1:4 dilution. For Notch1 inhibitor, Jagged1–Fc (10 μg; R&D systems, Minneapolis, MN, USA) was incubated for 1 h on ice with anti-human Fc antibody (5 μg; Sigma-Aldrich, St Louis, MO, USA) at a 2:1 ratio and the final concentration of Jagged1–Fc was 50 μg mL−1. Each rat was infused for 3 days with 1 μL mL−1 of either (i) Notch1-activating antibody, (ii) the Jagged1–Fc–anti-Fc complex, or (iii) aCSF alone (vehicle). BrdU (50 mg kg−1; Sigma-Aldrich) was dissolved in saline and given intraperitoneally, twice daily for 3 days at 8 h intervals, and rats were killed on day 4.

Immunohistochemistry

Brain tissues were postfixed in paraformaldehyde overnight, embedded in paraffin, and then cut into 6-μm sections, which were deparaffinized with xylene and rehydrated with ethanol, followed by antigen retrieval using the antigen unmasking solution (Vector Laboratories, CA, USA) according to the manufacturer's instructions. Immunohistochemistry was performed as described previously (Wang et al., 2009b). In brief, endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 30 min at room temperature. After several washes with PBS, the sections were incubated in blocking solution (10% goat or rabbit serum and 0.3% Triton X-100 in PBS) for 1 h at room temperature. Primary antibodies used were (i) rabbit polyclonal anti-Notch1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:50), (ii) rabbit polyclonal anti-NICD (Abcam, Cambridge, MA, USA; 1:200), and (iii) goat polyclonal anti-Jagged1 (Santa Cruz Biotechnology; 1:50). Primary antibodies were incubated in PBS with 0.3% Triton X-100 and 1% bovine serum albumin at 4°C overnight. Sections were then washed with PBS and incubated for 1 h at room temperature with biotinylated goat anti-rabbit or rabbit anti-goat antibody (1:200). Avidin–biotin complex (Vector Elite; Vector Laboratories) and a diaminobenzidine (DAB; Vector laboratories) were used to obtain a visible reaction product. Processing was stopped with H2O and sections were dehydrated through graded alcohols, cleared in xylene, and coverslipped in permanent mounting medium (Vector laboratories). Sections were examined and photographed with a Nikon microscope and the Nikon DS-Fi1 color camera (Nikon, Melville, NY, USA). Controls included omitting the primary or secondary antibody.

Double- or triple-label immunostaining

Double or triple immunostaining was performed on brain sections as previously described (Wang et al., 2009b). The primary antibodies used, in addition to those listed above, were mouse monoclonal anti-BrdU (Sigma-Aldrich; 1:500), goat or rabbit polyclonal anti-DCX (Santa Cruz Biotechnology; 1:50), and mouse monoclonal anti-GFAP (Sigma-Aldrich; 1:500). The secondary antibodies were Alexa Fluor 488-, 594-, or 647-conjugated donkey anti-mouse, anti-goat, or anti-rabbit IgG (1:200; Invitrogen, Grand Island, NY, USA). To detect BrdU-labeled cells in brain sections, sections were incubated in 2 m of HCl at 37°C for 30 min and rinsed in 0.1 m of boric acid (pH 8.5) and PBS. Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) using the Prolong Gold antifade reagent (Invitrogen). Fluorescence signals were detected with the Nikon inverted Ti-U microscope and the Nikon DS-Qi MC camera (Nikon), and images were acquired using the Nikon Imaging System (Nikon). Selected images were recorded using an LSM 510 NLO Confocal Scanning System mounted on an Axiovert 200 inverted microscope equipped with a two-photon Chameleon laser. Controls included omitting either the primary or secondary antibody.

Cell counting

BrdU- and DCX-positive and double-labeled cells in the SVZ, along the lateral walls of the lateral ventricles (beginning at 1.18 mm anterior to bregma), were counted in five to seven 6-μm paraffin coronal sections per animal (n = 3–4 per group), spaced 120 μm apart, by an observer blind to experimental condition using the Nikon inverted Ti-U microscope in fluorescein isothiocyanate (FITC) and Texas (Tx) Red channels with a 40 × objective. An LSM 510 NLO Confocal Scanning System was also used to record the z-stack images to confirm colocalization of DCX and BrdU-labelled cells in the SVZ. Results were expressed as the average number of BrdU- and DCX-positive cells in SVZ.

Western blotting

Western blotting was conducted as previously described (Wang et al., 2009b). The SVZ was dissected from both young adult (3 months old) and aged (24 months old) Fisher 344 rat brains as previously described (Ortega et al., 2011). Rats were transcardially perfused with PBS. The brains were removed, placed in a coronal brain matrix, and cut into 1-mm sequential sections throughout the site of the SVZ. The specimens were collected in PBS, and the SVZs were dissected from the surrounding tissue with a Zeiss V8 dissecting microscope. Cell lysates were extracted in PBS containing 0.05% Nonidet P-40 (Roche, South San Francisco, CA, USA), 0.25% sodium deoxycholate, 50 mm Tris–HCl (pH 8.5), 100 mm NaCl, 1 mm ethylenediaminetetraacetic acid (EDTA, pH 8.0), 1 mg mL−1 aprotinin, and 100 mg mL−1 phenylmethylsulfonyl fluoride. Protein (50 μg) was boiled at 100°C in the SDS sample buffer for 5 min, electrophoresed on SDS/12% PAGE gels, and transferred into polyvinyldifluoridine membranes, which were incubated overnight at 4°C using one of the following primary antibodies: (i) rabbit polyclonal anti-Notch1 (Santa Cruz Biotechnology; 1:50), (ii) rabbit polyclonal anti-NICD (Abcam; 1:500), (iii) goat polyclonal anti-Jagged1 (Santa Cruz Biotechnology; 1:500), or (iv) mouse monoclonal anti-actin (Oncogene Science, Cambridge, MA, USA; 1:20 000). Membranes were washed with PBS/0.1% Tween 20, incubated at room temperature for 60 min with horseradish peroxidase conjugated anti-mouse, anti-rabbit, or anti-goat secondary antibody (Santa Cruz Biotechnology; 1:3000), and thereafter washed three times for 15 min with PBS/Tween 20. Peroxidase activity was visualized by chemiluminescence (NEN Life Science Products, Boston, MA, USA). Antibodies were removed with stripping buffer (100 mm 2-mercaptoethanol/2% SDS/62.5 mm Tris–HCl, pH 6.7) at 50°C for 30 min, followed by washing with PBS/Tween 20, and membranes were reprobed. Densitometry measurements were normalized to actin.

Neurobehavioral testing

Rats (N = 8 per group) underwent neurobehavioral tests to evaluate functional outcome. Animals were trained prior to dMCAO and deficits were assessed 72 h thereafter. The investigator performing the tests was blinded to the experimental condition.

Ladder rung walking test

The ladder rung walking test is sensitive for quantifying skilled locomotion. The degree of motor dysfunction after MCAO was measured by counting the number of foot-faults of the impaired limbs per round, as described previously (Sun et al., 2012). Baseline and postoperative testing sessions consisted of three traverses across the ladder. An error was scored for any foot slip or misstep. The number of errors of the affected forelimb and hindlimb in each trial was counted. The mean number of errors in three traverses was calculated.

Limb placing test

The limb placing test was used to assess sensorimotor dysfunction (De Ryck et al., 1989). Rats were evaluated for the ability to place the limbs on a tabletop surface in response to visual, tactile, or proprioceptive input, as described previously (Sun et al., 2012). Each of seven tasks was scored by a blinded observer as 0, no placing; 1, incomplete or delayed placing; or 2, complete, immediate placing. Forelimb and hindlimb scores were averaged for each animal (normal score, 14).

Elevated body swing test

The elevated body swing test (EBST) was conducted to evaluate asymmetric motor behavior. Rats were held by the tail and raised ~10 cm above the testing surface (Borlongan & Sanberg, 1995). The initial direction of body swing which was turning of the upper body >10° to either side was recorded in 10 trials, observed over 5 min. The number of turns in each (left or right) direction was recorded, and the percentage of left turns was calculated.

Cylinder test

Forelimb use bias was analyzed by observing the rat's movements over 3-min intervals in a transparent, 18-cm-wide, 30-cm-high poly (methyl methacrylate) cylinder. A mirror behind the cylinder made it possible to observe and record forelimb movements when the rat was facing away from the examiner. After an episode of rearing and wall exploration, a landing was scored for the first limb to contact the wall or for both limbs if they made simultaneous contact. Percentage use of the impaired limb was calculated.

Infarct volume

Rats were deeply anesthetized and decapitated 72 h after dMCAO. Brains were removed and 50-μm coronal sections (400-μm apart) were prepared and stained with crysel violet. Stained sections (seven per rat, four rats per group) were scanned on a desktop scanner, and infarct area was measured by a blinded observer using Image J software, and areas were multiplied by the distance between sections to obtain the respective volumes. Infarct volumes were expressed as a percentage of the volume of the structures in the control hemispheres, as described previously (Swanson et al., 1990).

Statistical analysis

Quantitative results were expressed as the mean ± SEM. The statistical significance of difference between means was evaluated using Student's t-test or one-way analysis of variance (anova), followed by Newman–Keuls post hoc multiple comparison tests. < 0.05 was regarded as statistically significant.

Results

Our previous study has shown that Notch1 signaling regulates neural progenitor fate in the SVZ of young adult brain. However, whether a similar effect occurs in aged brain is unknown. Therefore, we first determined the expression profile of Notch signaling in the SVZ of aged rat brain. Rats were trained for 3 days prior to dMACO, and behavioral tests were performed 72 h after ischemia. Notch1 inhibitor, activator, or vehicle was injected into the lateral ventricle immediately after dMCAO for three consecutive days, and rats were then euthanized on fourth day (Fig. 1A). By Western blot analyses, we found that Notch1 expression in the SVZ of aged brain was significantly decreased compared with young adult brain (Fig. 1B,C), which was confirmed by immunocytochemistry (Fig. 1D). Confocal images showed that Notch1 protein was predominantly in the DCX-positive cells, but not GFAP-positive cells (Fig. 1E,F). As shown in Fig. 2A and C, expression of Jagged1, a Notch1 ligand, was also declined in response to aging. However, Jagged1 was expressed in GFAP-positive cells, but not DCX-positive cells (Fig. 2D,E).

Figure 1.

Expression of Notch1 in the subventricular zone (SVZ) of young adult and aged rat brain. (A) Rats were trained for 3 days prior to distal middle cerebral artery occlusion (dMCAO), and motor deficits were assessed 72 h after ischemia. Notch1 inhibitor, activator, or vehicle was injected into the left lateral ventricle immediately after dMCAO for three consecutive days. BrdU was injected twice per day for 3 days. Rats were euthanized after behavioral tests for immunocytochemistry and histology analysis. (B) The SVZ was dissected from normal young adult (Yang rats) and aged rat brains, and Western blots were performed using anti-Notch1 antibody (top). The blot was reprobed using anti-actin antibody to control for protein loading (bottom). (C) The optical density of the respective band in panel B, which was normalized according the corresponding actin band. Data are means ± SEM; N = 3 to 4 per group; *P < 0.05, analyzed by Student's t-test. (D) Notch1 immunoreactivity (brown) in the SVZ of young adult (top) and aged (bottom) rat brain. Inset: high magnification. (E) Confocal image showed that Notch1 (green) was mainly expressed in doublecortin (DCX)-positive cells (red), but not GFPA-positive cells (purple), in the SVZ of aged brain. DAPI (blue) was used to counterstain nuclei. (F) Maximum z-projections (main panels) and selected orthogonal yz (right) and xz (above) sections (with the plane of yz and xz sections indicated by the vertical red and horizontal green lines, respectively) of confocal image stacks. SVZ cells were stained with anti-Notch 1 (green), anti-DCX (red), and anti-GFAP antibodies (purple), and DAPI (blue) was used to counterstain nuclei.

Figure 2.

Expression of Jagged1 in the subventricular zone (SVZ) of young adult and aged rat brain. (A) The SVZ was dissected from normal young adult (Yang rats) and aged rat brains, and Western blots were performed using anti-Jagged1 antibody (top). The blot was reprobed using anti-actin antibody to control for protein loading (bottom). (B) The optical density of the respective band in panel A, which was normalized according the corresponding actin band. Data are means ± SEM; N = 3 to 4 per group; * P < 0.05, analyzed by Student's t-test. (C) Jagged1 immunoreactivity (brown) in the SVZ of young adult (top) and aged (bottom) rat brain. Insets: higher magnification. (D) Triple-label immunofluorescence staining recorded by a two-photon confocal microscopy showed that Jagged1 (green) was mainly expressed in GFAP-positive cells (purple), but not doublecortin (DCX)-positive cells (red). DAPI (blue) was used to counterstain nuclei. (E) Confocal image stacks show that SVZ cells were stained with anti-Jagged1 (green), anti-DCX (red), and anti-GFAP antibodies (purple), and DAPI (blue) was used to counterstain nuclei.

Next, we asked whether Notch1 signaling activation was also declined in the SVZ with aging. Notch signaling is triggered upon interaction of the receptor with its transmembrane ligand. Upon this ligand–receptor interaction, Notch receptor undergoes to proteolytic cleavage at the membrane by presenilins. This cleavage releases NICD, which is then translocated to the nucleus where it can interact with a transcriptional activator, resulting in subsequent events. Therefore, NICD is the activated form of Notch1 signaling. As shown in Fig. 3 A and B, NICD protein in the SVZ of aged brain was significantly reduced compared with young adult brain. Consistently, immunohistochemistry showed that NICD-positive cells were found in the SVZ of both young adult and aged brain, but expression level was reduced in aged brain compared with young adult brain. High-magnification views showed that NICD was located in the nuclei of SVZ cells (insets in Fig. 3C). In addition, we also found that Hes1, a downstream element of Notch1 signaling, was reduced in the aged brain (data not shown). To determine the phenotype of NICD-expressing cells, triple immunostaining was performed using anti-GFAP, anti-DCX, and anti-NICD. Consistent with our findings in young adult rat (Wang et al., 2009b), triple-label immunofluorescence recorded by a two-photon confocal microscopy showed that NICD was expressed in the nuclei of DCX-positive cells in the SVZ of aged brain (Fig. 3D,E).

Figure 3.

Expression of Notch intracellular domain (NICD) in the subventricular zone (SVZ) of young adult and aged rat brain. (A) The SVZ were dissected from normal young adult (Yang rats) and aged rat brains, and Western blots were performed using anti-NICD antibody. The blot was reprobed using anti-actin antibody to control for protein loading (bottom). (B) The optical density of the respective band in panel A, which was normalized according the corresponding actin band. Data are means ± SEM; N = 3 to 4 per group; *P < 0.05, analyzed by Student's t-test. (C) NICD immunoreactivity (brown) in the SVZ of young adult (top) and aged (bottom) rat brain. Insets: high magnification. (D) Triple-label immunofluorescence staining recorded by a two-photon confocal microscopy shows that NICD (green) was largely expressed in doublecortin (DCX)- (red), but not GFAP-positive cells (purple). DAPI (blue) was used to counterstain nuclei. (E) Confocal image stacks confirm that NICD (green) was expressed in DCX- (red), but not GFAP-positive cells. DAPI (blue) was used to counterstain nuclei.

To further test the significance of Notch1 signaling in the proliferation of SVZ cells in the aged brain, we activated Notch1 signaling by administration of Notch1 extracellular domain antibody (Conboy et al., 2003; Wang et al., 2009b) and inhibited Notch1 signaling by injection of a soluble Jagged1–Fc complex protein into the lateral ventricle of aged brain and BrdU- and DCX-positive cells in the SVZ were assessed. We found that the numbers of both BrdU-positive cells (Fig. 4A) and DCX-positive cells (Fig. 4B) in the SVZ were significantly increased after the Notch1 signaling activator injection and decreased after infusion of Notch1 signaling inhibitor, which is consistent with the findings in young adult rat brain (Wang et al., 2009b). Double-label immunohistochemistry showed that ~50% of BrdU-positive cells expressed DCX, suggesting that these cells were proliferative neuronal progenitors (Fig. 4C,D). The confocal images through the series of z-stacks indicated that BrdU-positive cells expressed DCX (Fig. 4E).

Figure 4.

Modulation of Notch signaling significantly alters cell proliferation in the subventricular zone (SVZ) of young adult and aged rat brain. (A) Quantification of BrdU-positive cells in the SVZ of aged brain after administration of Notch1 activator, inhibitor, or vehicle. (B) Quantification of doublecortin (DCX)-positive cells in the SVZ of aged brain after administration of Notch1 activator, inhibitor, or vehicle. (C) Quantification of BrdU/DCX-positive cells in the SVZ of aged brain after administration of Notch1 activator, inhibitor, or vehicle. (D) Representative confocal images of BrdU/DCX-immunoreactive cells in the SVZ of aged brain after administration of Notch1 activator. (E) Confocal images through the series of z-stacks confirmed that BrdU-positive cells (red) expressed DCX (green). Data are means ± SEM; N = 3 to 4 per group.

Then, we asked whether Notch1 signaling modulated ischemia-induced neurogenesis in aged brain. Focal ischemia was induced in aged rats, and Notch1 activator and inhibitor were administrated by intracerebroventricular route. As shown in Fig. 5A–D, the numbers of both BrdU- and DCX-positive cells in the SVZ were significantly increased 3 days after administration of the Notch1 signaling activator and dramatically reduced 3 days after infusion of the Notch1 signaling inhibitor compared with vehicle-treated group. Similarly, double-label immunohistochemistry also showed that ~50% of BrdU-positive cells in the SVZ expressed DCX after ischemic stroke (Fig. 5E–F), which were recorded by two-photon confocal microscopy.

Figure 5.

Modulation of Notch signaling pathway significantly alters cell proliferation in aged rat brain subventricular zone (SVZ) after distal middle cerebral artery occlusion (dMCAO). (A) Representative images of BrdU-positive cells (red) in the SVZ of aged brain after focal ischemia followed administration of Notch1 activator, inhibitor, or vehicle. (B) Quantification of BrdU-positive cells in the SVZ of aged brain after focal ischemia followed administration of Notch1 activator, inhibitor, or vehicle. (C) Representative images of doublecortin (DCX)-positive cells (green) in the SVZ of aged brain after focal ischemia followed administration of Notch1 activator, inhibitor, or vehicle. (D) Quantification of DCX-positive cells in the SVZ of aged brain after focal ischemia followed administration of Notch1 activator, inhibitor, or vehicle. (E) Representative images of BrdU/DCX-positive cells (red/green) in the SVZ of aged brain after focal ischemia followed administration of Notch1 activator. Left panel: a confocal 3D image of BrdU/DCX-positive cells in the SVZ; right panel: confocal image z-stacks of BrdU/DCX-positive cells in the SVZ. (F) Quantification of BrdU/DCX-positive cells in the SVZ of aged brain after focal ischemia followed administration of Notch1 activator, inhibitor, or vehicle (control). Data are means ± SEM; N = 3 to 4 per group.

To determine the role of Notch1 signaling in histological outcome after focal ischemia, we induced focal ischemia and then administered Notch1 inhibitor, activator or vehicle into the brains for 3 days. As shown in Fig. 6, infarct volume was significantly decreased in rats treated with Notch1 activator, compared with vehicle-treated or Notch1 inhibitor–treated rats after dMCAO. Although the infarct volume was increased in the ischemic rats after treatment of Notch1 inhibitor, no significant difference was reached.

Figure 6.

Infarct volume in aged ischemic rats after administration of Notch1 inhibitor or activator. (A) Rats were treated with vehicle, Notch1 inhibitor, or activator for three consecutive days after distal middle cerebral artery occlusion (dMCAO). The brain sections were stained with crystal violent. (B) Infarct volume was expressed as percentage of hemispheric volume. The significant difference was found between the vehicle- and Notch1 activator–treated dMCAO groups by anova analysis, followed by Newman–Keuls post hoc multiple comparison tests.

We also measured neurologic deficits in ischemic rats after Notch1 inhibitor, activator, or vehicle treatment. We found that motor deficits in the ladder running walking tests (Fig. 7A), EBST (Fig. 7B), cylinder test (Fig. 7C), and limp placing test (Fig. 7D) were improved in the Notch1 activator–treated rats tested at 3 days after dMCAO, compared with vehicle- or Notch1 inhibitor–treated rats, suggesting involvement of Notch1 signaling in functional outcome in aged rats after focal ischemia.

Figure 7.

Neurobehavioral outcome in ischemic aged rats after administration of Notch1 inhibitor or activator. Neurobehavioral function was assessed by the ladder running walking test (forelimb slip steps: left panel; hindlimb slip steps: right panel) (A), EBST (B), cylinder test (C), and limp placing test (D) at 3 days after distal middle cerebral artery occlusion (dMCAO), followed administration of vehicle, Notch1 inhibitor, or activator. The significant differences were found between the vehicle-treated and Notch1 activator–treated dMCAO groups and between the Notch inhibitor–treated and Notch1 activator–treated dMCAO groups by anova analysis, followed by Newman–Keuls post hoc multiple comparison tests.

Discussion

In the present study, we found that the expression of Notch1 and Notch1 ligand (Jagged1) in the SVZ decreased with aging, in parallel with reduced activation of Notch1 signaling. In addition, we also found that Notch1 and NICD were predominantly expressed in neural precursor cells, but Jagged1 mainly in astrocytes. Enhancing activation of Notch1 signaling promoted neurogenesis, while inhibiting Notch1 signaling decreased neurogenesis of the SVZ in aged brain. Moreover, ischemia-induced cell proliferation in the SVZ of aged brain was also increased by activating the Notch1 pathway and reduced by inhibiting the Notch1 signaling. Moreover, we also found that the Notch1 activator administration reduced infarct volumes and improved neurologic deficits in aged rats after focal cerebral ischemia. Our data suggest that Notch1 signaling plays a critical role in diminished SVZ neurogenesis with aging and modulates the SVZ neurogenesis as well as functional outcome after focal ischemia.

The Notch1 signaling pathway is regarded as one of the main regulators of neural development, of which signaling components have been found to express in neural germinal zone of both embryonic and adult brains (Stump et al., 2002; Irvin et al., 2004; Givogri et al., 2006; Wang et al., 2009b). Activation of Notch1 signaling contributes to the maintenance and proliferation of NSCs (Chojnacki et al., 2003; Givogri et al., 2006; Ehm et al., 2010; Gaiano, 2011). Studies have shown that Notch1 expression decreases significantly with aging in the neural germinal zone, including SVZ and SGZ (Givogri et al., 2006; Crews et al., 2008), which is consistent with our data. Notch1 is a transmembrane protein that is activated by binding the ligands Delta1 or Jagged1. Studies report that the cells expressing the ligands typically need to be adjacent to the Notch1 expressing cell for signaling to occur. Consistent with our previous findings (Wang et al., 2009b) and other studies (Givogri et al., 2006; Ables et al., 2011) in young adult brain, we also found that Notch1 and NICD were expressed in DCX-positive cells (Type A cells), while Jagged1 was found in neighboring cells of Notch1 expressing cells such as GFAP-positive cells (Type B cells) in the SVZ of aged brain. Such a unique expression pattern may form a certain microenvironments or niches that instruct the fate of NSCs in the SVZ.

With age, the capacity for neurogenesis in adult brain diminishes. We found that the forced Notch1 signaling activation restores the reduced proliferation rate of NSCs in aged brain, which is evidenced by increase in the number of BrdU-positive cells in the SVZ. Conversely, after inhibiting Notch1 signaling, the proliferation level of NSCs in the SVZ of the aged brain is significantly reduced or abolished. In our previous study (Wang et al., 2009b), we applied intracerebroventricular administration of Notch1 extracellular domain antibody to activate Notch1 signaling (Conboy et al., 2003; Wang et al., 2009b) and of a soluble Jagged1–Fc complex protein to inhibit Notch1 signaling (Conboy et al., 2003; Wang et al., 2009b) and found that manipulating Notch1 signaling could alter the neurogenesis levels in the SVZ of normal young adult brain. In addition, studies also show that NICD overexpression recues proliferation and self-renewal of NSCs in the SVZ (Aguirre et al., 2010) and inducible knockout Notch1 fails to self-renew and expand of NSCs (Ables et al., 2010). These data suggest that Notch1 signaling plays a critical role in the proliferation of NSCs in the SVZ of young adult and aged brain.

Although the basal rate of neurogenesis declines with age (Kuhn et al., 1996; Kempermann et al., 1998; Cameron & McKay, 1999), declined hippocampal neurogenesis in aged mice living in an enriched environment was fivefold higher than in controls, along with significant improvements in learning parameters, exploratory behavior, and locomotor activity. Our recent study also showed that FGF-2 and HB-EGF restore neurogenesis in aged brain and that stroke-induced neurogenesis is present, but reduced in aged brain, suggesting that the aged brain retains the ability to react to functional challenges with a neurogenic response (Kempermann et al., 2002). We and others show that the Notch1 signaling responds to ischemic stroke in young adult brain. After focal ischemia in aged rat, we infused the Notch1 signaling inhibitor into lateral ventricular for 3 days and found that ischemia-induced neurogenesis is significantly reduced after inhibiting Notch1 signaling (Wang et al., 2009b). Consistently, transient administration of Notch ligand, deltalike protein 4 (DLL4) to activate Notch1 increased the numbers of newly generated precursor cells in the SVZ and improves motor skills after ischemic injury (Androutsellis-Theotokis et al., 2006). Wang et al. (2009a) found that expression of Notch, NICD, and Hes1 mRNA in neural progenitor cells is significantly increased in vitro. siRNA against Notch or a γ-secretase inhibitor significantly reduced Notch, NICD, and Hes1 expression and cell proliferation after experimental stroke. As stroke generally occurs in aged populations (Jin, 2010), experimental studies employing aged animals may be more clinically relevant than that in young adult animals. Our findings in aged rat brain are in agreement with and extend these previous studies in young adult animals. Study shows that hypoxia enhances Notch1 signaling. Hypoxia inducible factor (HIF)-1α binds to NICD and stabilizes it (Gustafsson et al., 2005). NICD carries HIF-1α to Notch1-responsive promoters, and the complex enhances transcription, which further support the notion that Notch1 signaling may play a role in stroke-induced neurogenesis. However, a recent study shows that blocking Notch1 signaling by γ-secretase inhibitors exerts a neuroprotective and anti-inflammatory effect in focal cerebral ischemia, suggesting that Notch1 signaling may contribute to ischemic damage (Arumugam et al., 2006). In contrast, our study showed that administration of Notch1 inhibitor did not significantly cause larger infarct volume, which may be due to the fact that the maximized infarct sizes have been reached. However, we found that administration of Notch1 activator not only reduced infarct volumes, but also improved neurologic deficits in aged rats after focal cerebral ischemia. Consistently, a more delayed activation of Notch1 signaling through infusion of the Notch1 ligand Dll4 into the lateral ventricle of adult rats had no effect on the infarct size but improved motor skills over a period of 45 days (Androutsellis-Theotokis et al., 2006). In addition, a recent study also shows that Notch 3−/− mice developed ischemic lesions approximately twice as large as those seen in WT or heterozygous (Notch 3 +/−) male mice (Arboleda-Velasquez et al., 2008). Very recent studies show that the activation of Notch signaling is involved in the ischemic tolerance (Yang et al., 2012; Zhao et al., 2012). The reason for the discrepancy between these studies is unknown, but a possible explanation for these apparently contradicting results may be due to different animal strains and animal models of stroke, and the different Notch1 signaling inhibitor used in these studies, as γ-secretase inhibitors may not only involve in Notch1 signaling, but also other signaling transduction pathways, which may play a role in neuroprotection in the ischemic brain.

Notch signaling integrates with a number of other pathways, thereby tuning downstream expression to regulate the maintenance of NSCs in both the embryonic brain (Takizawa et al., 2003; Shimizu et al., 2008) and adult brain (Andreu-Agullo et al., 2009). It is likely that Notch signaling controls NSC maintenance, proliferation, and survival in the adult brain in cooperation with other signals. One study suggested that modulation of the PI3 kinase pathway and of the Wnt/β-catenin pathway potentiate Notch-induced promoting effect on adult NSCs (Ehm et al., 2010), indicating that the fine-tuning neurogenesis role of Notch1 signaling occurs in cooperation with cross-talking pathways. However, much of this is speculations and future studies will have to make a clearer understanding of the Notch signaling network. Notch may be a dominant regulator of cell fate, but it does not work alone.

Taken together, our findings in the present study indicate the beneficial effect of Notch1 signaling on neurogenesis in the aged brain under both physiologic and ischemic conditions. Understanding how Notch1 signaling can modulate neurogenesis has broad implications for the development of clinical treatments aimed at combating injury and neurodegeneration in aged brain.

Acknowledgments

This work was supported by National Institute of Health (NIH) grants AG21980 and NS057186 (K Jin) and by a grant from Wenzhou technology division (H20090067) (B Shao).

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