Notch1 deficiency in postnatal neural progenitor cells

The FASEB Journal article fj.201700216RR. Published online June 13, 2017. THE

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Notch1 deficiency in postnatal neural progenitor cells in the dentate gyrus leads to emotional and cognitive impairment Shufang Feng,*,1 Tianyao Shi,†,1 Jiangxia Qiu,* Haihong Yang,* Yan Wu,* Wenxia Zhou,† Wei Wang,‡ and Haitao Wu*,§,2

*Department of Neurobiology, Beijing Institute of Basic Medical Sciences, Beijing, China; †Department of Traditional Chinese Medicine (TCM) and Neuroimmunopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing, China; ‡Department of Orthopedics Research Institute, First Affiliated Hospital of Jinzhou Medical University, Jinzhou, China; and §Key Laboratory of Neuroregeneration, Coinnovation Center of Neuroregeneration, Nantong University, Nantong, China

It is well known that Notch1 signaling plays a crucial role in embryonic neural development and adult neurogenesis. The latest evidence shows that Notch1 also plays a critical role in synaptic plasticity in hippocampal mature neurons. So far, deeper insights into the function of Notch1 signaling during the different steps of adult neurogenesis are still lacking, and the mechanisms by which Notch1 dysfunction is associated with brain disorders are also poorly understood. In the current study, we found that Notch1 was highly expressed in the adult-born immature neurons in the hippocampal dentate gyrus. Using a genetic approach to selectively ablate Notch1 signaling in late immature precursors in the postnatal hippocampus by cross-breeding doublecortin (DCX)+ neuronspecific proopiomelanocortin (POMC)-a Cre mice with floxed Notch1 mice, we demonstrated a previously unreported pivotal role of Notch1 signaling in survival and function of adult newborn neurons in the dentate gyrus. Moreover, behavioral and functional studies demonstrated that POMC-Notch12/2 mutant mice showed anxiety and depressive-like behavior with impaired synaptic transmission properties in the dentate gyrus. Finally, our mechanistic study showed significantly compromised phosphorylation of cAMP response element-binding protein (CREB) in Notch1 mutants, suggesting that the dysfunction of Notch1 mutants is associated with the disrupted pCREB signaling in postnatally generated immature neurons in the dentate gyrus.—Feng, S., Shi, T., Qiu, J., Yang, H., Wu, Y., Zhou, W., Wang, W., Wu, H. Notch1 deficiency in postnatal neural progenitor cells in the dentate gyrus leads to emotional and cognitive impairment. FASEB J. 31, 000–000 (2017). www.fasebj.org

ABSTRACT:

KEY WORDS:

adult neurogenesis

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anxiety

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synaptic transmission

In the adult mammalian brain, the generation of new neurons in the hippocampal dentate gyrus (DG) continues postnatally and throughout life (1, 2). They functionally integrate into the local circuitry (3, 4) and can account for up ABBREVIATIONS: ACSF, artificial CSF; AP, action potential; Arc/Arg 3.1,

activity-regulated cytoskeleton-associated protein; BrdU, bromodeoxyuridine; CREB, cAMP response element-binding protein; DCX, doublecortin; DG, dentate gyrus; EPM, elevated plus maze; EPSC, excitatory postsynaptic current; FST, forced swimming test; GC, granule cell; GCL, granule cell layer; HFS, high-frequency stimulation; HRP, horseradish peroxidase; iGCL, inner GCL; KO, knockout; LTP, long-term potentiation; mEPSC, miniature EPSC; NICD, Notch intracellular domain; oGCL, outer GCL; POMC, proopiomelanocortin; PP, perforant pathway; SGZ, subgranular zone; SPE, stratum pyramidal external; SPI, stratum pyramidal internal; SVZ, subventricular zone; TOM+, tdTomato reporter-positive neurons 1 2

These authors contributed equally to this work. Correspondence: Department of Neurobiology, Beijing Institute of Basic Medical Sciences, Taiping Road 27, Haidian District, Beijing, N/A 100850, China. E-mail: [email protected].

doi: 10.1096/fj.201700216RR This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

0892-6638/17/0031-0001 © FASEB

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pCREB

to 10% of the total population of granule cells (GCs) (5). Disruption in the normal adaptive regulation of neurogenesis may occur in many pathologic contexts, resulting in cognitive and memory impairment (6, 7). Moreover, failing adult neurogenesis has been connected with the development of several neuropsychiatric disorders (8). Extrinsic and intrinsic factors have been shown to regulate adult neurogenesis, and recent studies suggest that Notch1 signaling plays an important role in the regulation of proliferation and maintenance of adult precursor cells (9, 10). Although Notch1 signaling in adult neurogenesis has been extensively investigated in recent years (11), deeper insights into its function in different phases of adult neurogenesis are still lacking. Notch1 and Notch1 intracellular domain (NICD) immunoreactivity has been found in nestin+ type 1 and 2a cells, as well as a subset of doublecortin (DCX)+ late progenitors and postmitotic GCs in the DG, whereas most type 2b and 3 cells lack Notch1 and nuclear NICD immunoreactivity (10, 11). Notch1 signaling is now believed to enhance self-renewal, while inhibiting

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neuronal differentiation and cell cycle exit of type 1 and 2a neural precursor cells (10, 11). It has also been reported that Notch1 may be responsible for the exercise-induced cell cycle exit of type 3 progenitors and initiation of further maturation (11, 12), whereas the exact role of Notch1 signaling in type 3 late precursor cells is still elusive. In general, endogenous newborn precursors can be induced to proliferate, migrate, and integrate into the local circuitry after their birth. Indeed, at least 50% of newborn neurons fail to survive longer than a couple of months after their generation (13), suggesting that the late postnatal period plays a critical role in survival and integration of newborn neurons. Moreover, survival and maturation are tightly controlled by the activity of local circuits (14, 15). A study suggested that the expression and cleavage of Notch1 increase in response to synaptic activity, which is probably regulated by the neuronal activity-related early gene activity-regulated cytoskeleton-associated protein (Arc/Arg 3.1) (16). Activation of Notch1 signaling could also induce dendritic branching and neurite remodeling and prevent apoptosis, suggesting that Notch1 controls the survival of neural progenitors in the postmitotic mature phase (9, 10, 17). So far, a direct functional relationship and genetic evidence showing that Notch1 signaling regulates the survival and function of late-stage progenitor cells in the DG are still lacking. In this study, using proopiomelanocortin (POMC)a-Cre–derived Ai9 reporter mice, we demonstrated that Cre recombinase in the POMC-Cre line is uniquely expressed in a subpopulation of neurons in the DG and can be detected from postnatal day (P)5 to P56. This agrees with a study showing that POMC-Cre+ neurons are highly expressed in DCX+ newborn neurons, but not in nestin+ cells or proliferating neural precursor cells (18). By using genetic ablation of Notch1 receptor selectively in postnatal late precursor cells in the mouse hippocampus, we demonstrated a previously unreported pivotal role of Notch1 signaling in the survival and function of type 3 progenitors in DG. Moreover, behavioral and functional studies demonstrated that POMC-Notch12/2 mutants showed anxiety- and depressive-like behavior with impaired synaptic transmission in the DG. In addition, contextual-discrimination or pattern-separation deficits in cognition were found in POMC-Notch12/2 mutants. Notch1 affects the cAMP response element-binding (CREB) protein level and phosphorylation via a noncanonical pathway (19), which supports the role of CREB phosphorylation in the differentiation, survival, and proliferation of newborn neurons in the adult hippocampus (20–22). Finally, our mechanistic study showed that the phosphorylation of CREB (pCREB) is significantly compromised in Notch1-ablated neurons in hippocampal DG, suggesting that the dysfunction of POMC-Notch12/2 mutants is associated with the inactivation of pCREB signaling in postnatal newborn neurons. MATERIALS AND METHODS

and Chinese governmental regulations. POMC-Cre transgenic mice [B6.FVB-Tg(Pomc-cre)1Stl/J] (23, 24) were crossed with Notch1 floxed mice (25) to generate POMC-Notch12/2 mutants and Notch1f/f control mice. For neural survival experiments, POMC-Notch1+/2 mice were also crossed with B6.CgGt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (also named Ai9 reporter) mice (26). The mice were housed under controlled temperature (22–25°C) and a 12-h light–dark cycle with a standard chow diet (4% fat SPF Rodent Feed; Beijing KeAoXieLi Feed Co., Beijing, China) and ad libitum drinking water. Immunofluorescent staining, imaging, and quantification Mouse brain sections were prepared by cardiac perfusion (4% paraformaldehyde). After 12 h of fixation, 40 mm brain sections were prepared on a vibratome (VT1000S; Leica Microsystems, Buffalo Grove, IL, USA). For immunofluorescence staining, sections were rinsed in 0.1 M PBS (pH 7.4) 3 times and incubated in blocking solution (0.1% Triton X-100, 1% bovine serum albumin, and 5% normal goat serum in PBS) for 1 h at room temperature, followed by overnight incubation with primary antibody at 4°C. Sections were then washed and incubated with the secondary antibody at room temperature for 1 h, then washed with 0.1% PBST 3 times. The primary antibodies used were anti-Notch1 antibody (1:50; goat; Santa Cruz Biotechnology, Dallas, TX, USA); anti-NICD antibody (1:200; rabbit), and anti-nestin antibody (1:200; mouse), anti-BrdU antibody (1:400; rat; all from Abcam, Cambridge United Kingdom); and anti-NeuN antibody (1:1000; mouse), anti-GFAP antibody (1:100; mouse), antidoublecortin antibody (1:1000; guinea pig), anti-pCREB antibody (1:200; rabbit), and anti-CREB antibody (1:200, rabbit; all from EMS Millipore, Billerica, MA, USA). Secondary antibodies from Jackson ImmunoResearch (West Grove, PA, USA) were all applied at the dilution of 1:1000. For visualization of positive expression, a confocal laser-scanning microscope (FV-1200; Olympus, Tokyo, Japan) was used to capture images and stitch them together to make large composite images. For morphometric evaluation and stereologic quantification, positive cells were counted in a series of clear 2-dimensional images and traced by using modified stereological image analysis software (ImageJ; National Institutes of Health, Bethesda, MD, USA). Images with multiple channels were separated into channels first, to use the ImageJ plug-in for tracing. Areas to be counted were traced at low power, and frames to be counted were selected at random by the image-analysis software. To trace the dendritic arbors, 3-dimensional reconstruction and analysis were used. For quantification of dendritic branching, DCX+ cell dendrites in the molecular layer were independently reconstructed to avoid sampling bias, and individual cells were reconstructed from confocal z stacks. Eight to 10 sections through the entire extent of the hippocampus were examined in POMCNotch12/2 mutants and control littermates. Nissl staining Brains from control and POMC-Notch12/2 mutant mice were immediately removed and fixed in 4% paraformaldehyde for 2 d at 4°C, followed by cryoprotection in 20 and 30% sucrose. Subsequently, 50-mm-thick brain slices were cut with a cryostat (Thermo Cryotome FSE; Thermo Fisher Scientific, Waltham, MA, USA) and stained with cresyl violet for 5–6 min.

Animals

BrdU labeling and quantification of neurogenesis

Animal care and use conformed to institutional guidelines of the Beijing Institute of Basic Medical Sciences (Beijing, China)

The control and POMC-Notch12/2 mutant mice (6 for each group) were given bromodeoxyuridine (BrdU) injections 3 times

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per day (50 mg/kg in 0.9% saline, i.p.; Sigma-Aldrich, St. Louis, MO, USA), 4 h apart, at P21. Twenty-four hours and 4 wk after injection, rodent brains were fixed as previously described (3 animals for each time point). A series of every sixth section (240 mm apart) through each hippocampus was processed. Freefloating sections were washed twice in PBS, incubated in 2 N HCl (30 min at 37°C), and rinsed in 0.1 M borate buffer (pH 8.4; 10 min). Immunostaining was then performed. In each section, every BrdU+ cell within the DG, including the GC layer and adjacent subgranular zone (SGZ), were counted with a fluorescence microscope through a 310 objective for the whole series of sections. The absolute number of BrdU+ cells was obtained by multiplying BrdU cell density by the reference volume (27). Data are presented as average number of BrdU+ cells per cubic millimeter. Results are expressed as the mean of 3 mice per group. High-frequency stimulation in the brain Mice were anesthetized intraperitoneally with urethane (1.2 g/kg) and placed in a stereotaxic frame fitted with ear cuffs (SR-6N; Narishige, East Meadow, NY, USA) to restrain head movement. The stimuli electrodes were made from stainless steel needles (0.25 mm diameter), coated with insulated lacquer except at the tips, and were placed 3.80 mm posterior to the bregma, 3.00 mm lateral to the midline, and 1.50 mm ventral to the surface of the skull located at the perforant pathway (PP) to the DG (PP-DG). Longterm potentiation (LTP) was induced with high-frequency stimulation (HFS) by 6 series of 6 trains of 6 pulses at 400 Hz (100 ms between trains, 20 s between series) (28). Three hours after the stimulation, the animals were euthanized for immunostaining. Brain slice preparation and electrophysiology The mice were anesthetized with 2% isoflurane, and transverse hippocampal slices (300 mm) were cut at 4°C with a vibratome in oxygenated ACSF containing: 124 mM NaCl, 2 mM KCl, 26 mM NaHCO3, 2 mM CaCl2, 2 mM MgSO4, 1 mM NaH2PO4, and 10 mM D-glucose (pH 7.4). Brain slices were transferred to a submerged recovery chamber with oxygenated ACSF at room temperature. Experiments were performed in a recording chamber on the stage of a BX51W1 microscope (Olympus) equipped with infrared differential interference contrast optics. For whole-cell patch-clamp recordings, the recording pipettes (2–5 MV) were filled with a solution containing the following: 145 mM K-gluconate, 5 mM NaCl, 1 mM MgCl2, 0.2 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, 0.1 mM Na3-GTP, and 10 mM phosphocreatine disodium (pH 7.2). Neurons were voltage clamped at 270 mV. Spontaneous excitatory postsynaptic current (EPSC) and action potentials (APs) were recorded from GCs in the DG neurons with an Axon 700B amplifier (Molecular Devices, Sunnyvale, CA, USA). The access resistance of 15–30 MV was monitored throughout the experiment. Data were discarded if the access resistance changed by 15% during an experiment. For field recordings, a bipolar stimulating electrode was placed in the medial (m)PP, and evoked field potentials were recorded in the molecular layer with a glass capillary microelectrode (1–3 MV) filled with ACSF. Isolation of the mPP was confirmed by assessing paired pulse depression of the mPP-DG synaptic connection at 50 ms, which generated the highest level of depression (29). Input–output curves were obtained after 10 min of stable recordings. The stimulation intensity that produced onethird of the maximum response was used for the test pulses and tetanus. After 15 min of stable baseline (once every 20 s), LTP was induced with 4 trains of 1 s each, 100 Hz within the train, repeated every 15 s. Responses were recorded every 20 s for 60 min after LTP induction. All experiments were conducted in the presence of picrotoxin (100 mM) to block GABAA receptor-mediated inhibitory synaptic currents. NOTCH1 DEFICIENCY IN DG LEADS TO BRAIN DYSFUNCTION

Measurement of behavioral test All behaviors were performed in a double-blinded manner. Body weight was evaluated every week after P7. Behavioral tests described below were performed at P56 in the following order: handling, sucrose preference, open-field, elevated plus maze (EPM), and forced swimming test (FST) and contextualdiscrimination/pattern separation. Intervals between each behavioral test were 1–3 d. Sucrose preference test The mice were placed in separate cages. Two preweighted bottles, one containing 0% (tap water) and the other containing 1% sucrose solution, were placed on each cage. Bottle order (left– right placement of water vs. sucrose bottles) was counterbalanced among mice in each group. The preference for sucrose was calculated as a percentage of consumed solution of the total amount of liquid drunk and tested at 1 h. Open-field test The apparatus of this test was as described in Feng et al. (30). Mouse movements were recorded with a video camera for 15 min. The locomotor activity level of each mouse was quantified by measuring total distance traveled in each 5 min for 15 min. EPM and FST FST and EPM were conducted according to published protocols (30). Contextual-discrimination/pattern-separation test This procedure was based on the method described by McHugh et al. (31), with slight modifications. One conditioning chamber (30 3 25 3 25 cm; Med-Associates, St. Albans, VT, USA) was used for both context A (the context paired with a shock) and context B (never paired with a shock), with a stainless grid floor with 60 dB background noise. Each chamber was wiped down with 70% ethanol before conditioning and between animals. Contexts A and B were different in wallpaper, bottom color, and lighting. The experiments were performed in the same conditions of time, temperature, and humidity: 9:00 AM–4:00 PM, 20–22°C, and 45%, respectively. For the first 3 d, mice were placed in one of the contexts for 3 min and given a single foot shock of 0.65 mA for 2 s. On d 4 and 5 (for generalization), mice of each group were divided into 2 groups, one visiting chamber A, the other visiting chamber B (1 trial/d) on d 4 and visiting the unvisited chamber on d 5. No foot shock was delivered in A or B during this generalization period, and freezing behavior was assessed. During d 6–11 (for discrimination training), mice were placed in both chambers every day for 6 d and always received a foot shock 3 min after being placed in chamber A but not when placed in chamber B. Freezing during the 3 min preceding the shock was measured in each chamber to calculate a daily discrimination ratio. The order of training followed a double alternation schedule: d 6, B/A; d 7, A/B; d 8, A/B; d 9, B/A, and so on. Western blot The ventral hippocampus from different genotypes was dissected at P56 on ice and lysed with ice-cold RIPA lysis buffer. Subsequently, protein concentration measurement and


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electrophoresis were performed (32). The transferred membrane was incubated with primary antibody CREB or pCREB (1:500; EMS-Millipore). After they were washed, the membranes were incubated with the appropriate horseradish peroxidase–coupled secondary antibody diluted 1:3000 for 1 h, followed by ECL detection of the proteins with Western Lightening Chemiluminescence Reagent Plus according to the instructions of the manufacturer (Perkin-Elmer, Waltham, MA, USA). To verify equal loading, we also probed the membranes with an antibody against glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology). The density of the immunoblots was measured with ImageJ.

activated in the early developmental stage, but also can be reactivated in a neuronal activity-dependent manner in the hippocampal CA1 and CA3 regions (16). To investigate whether Notch1 signaling could be activated by neuronal activity in the adult hippocampal DG, we used HFS to induce the depolarization of neurons in vivo. Based on Notch1-specific immunofluorescent staining in brain sections, we demonstrated that Notch1 signaling was robustly activated in mature GCs of the DG after HFSinduced depolarization (Fig. 1B).

Data analysis and statistics

Notch1 deficiency in postmitotic GCs affects hippocampal CA3 lamination

Statistical comparisons were made by ANOVA to assess the interaction between genotype and time. Significant major effects were analyzed further by post hoc Student’s t test with Bonferroni adjustment (Statview 5.0 software; SAS Institute, Cary, NC, USA). Values of P , 0.05 were considered significant. All data are expressed as means 6 SEM.

RESULTS HFS activates Notch1 signaling in DG We first demonstrated that Notch1 is highly expressed in a subset of lineage-restricted DCX+ immature neurons in DG at P30 (Fig. 1A). This result was consistent with previous reports which showed high, specific Notch1 immunoreactivity in nestin+ type 1 and 2a cells, as well as DCX+ late progenitor and postmitotic GCs, but lack of expression in type 2b progenitor cells in the DG (10). Recent studies have shown that Notch1 signaling is not just

The subventricular zone (SVZ) and the SGZ represent the preservation of neurogenic niches throughout the adult brain, where Notch pathway components are highly expressed (10, 33, 34). To investigate the role of Notch1 signaling in DCX+ late progenitor and postmitotic GCs in adult hippocampal DG, we used a Cre-loxP strategy to specifically ablate Notch1 gene expression in the celltype–specific pro-POMC Cre transgenic line. Based on the Ai9 reporter line, we found the activity of POMC-Cre was significantly high in newborn immature neurons in hippocampal DG from P5, as well as in the arcuate nucleus of the hypothalamus (Supplemental Fig. 1), consistent with a previous report (35). Therefore , this strategy allowed us to study the role of Notch1 signaling selectively in postnatal newborn immature neurons and reduce or bypass its embryonic developmental effects. When POMC-Cre transgenic mice were bred with Notch1 floxed allele, Notch1 signaling was specifically

Figure 1. Expression of Notch1 in the adult-born late progenitor neurons in the DG. A) Immunofluorescent staining of Notch1 in green and DCX in red in DG at P30. Blue, nuclei counterstained with DAPI. White arrowheads, a small number of DCX+ cells with morphologic characteristics of type 2b and 3 cells lacked Notch1 expression. Yellow arrowheads, Notch1+ mature DCX+ cells with characteristic dendritic branches. Scale bars: 100 mm (top), 20 mm (bottom). B) Three hours after HFS-LTP induction in DG from P56 mice, Notch1 expression was significantly upregulated in GCs within the SGZ and GZ layer.

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disrupted in newborn neurons in the DG, whereas the Notch1 gene in the proliferating neural progenitor cells was not affected. As shown in Fig. 2A, no significant volume and gross anatomic abnormalities were found in the DG in POMC-Notch12/2 mutants compared to control littermates by Nissl staining (n = 5; P , 0.05). However, an obvious lamination deficit was found in the CA3 region in the POMC-Notch12/2 mutant hippocampus, showing dispersed pyramidal neurons with double separated layers (Fig. 2A, B). As described (36, 37), our morphologic and quantitative analysis demonstrated that the significant disorganized hippocampal CA3 region in POMCNotch12/2 mutants could be divided into 2 distinct layers, showing double peaks termed stratum pyramidale

Figure 3. The numbers and migration pattern of POMC-Cre+ progenitor cells in POMC-Cre and POMC-Notch12/2 mice. A) Ai9 reporter mice were bred with POMC-Notch1+/2 mice to get POMC-Cre control and POMC-Notch12/2 mutants to illustrate the morphology of POMC-Cre+ neurons. Scale bars: 100 mm (top), 20 mm (bottom). B) Quantitative analysis of total number of TOM+ neurons. C ) Distribution of TOM+ cells in the GCL was divided into oGCL, iGCL, and SGZ. TOM+ neurons migrate to a significantly decreased proportion in oGCL and iGCL layers in POMC-Notch12/2 mutants vs. controls. *P , 0.01 (Student’s t test).

internal (SPI) and stratum pyramidale external (SPE) (Fig. 2C), which were similar to what has been observed in doublecortin knockout (Dcx-KO) mice (38). Impaired survival and migration of hippocampal adult newborn neurons in POMC-Notch12/2 mutants

Figure 2. Altered morphologic characteristics of hippocampus in POMC-Notch12/2 mutants. A) Arrowhead, cresyl violet staining shows abnormally organized CA3 pyramidal cells in POMC-Notch12/2 mutants. B) Immunofluorescent staining with NeuN to label pyramidal neurons within the hippocampus. The disorganized CA3 region was characterized by 2 distinct layers termed SPI and SPE. C ) Histogram of the distribution of pixel intensities along the red lines as indicated in B. The distribution of gray values showed a double peak in POMC-Notch12/2 mutants with SPI and SPE respectively, but only a single peak (stratum pyramidale; SP) in Notch1f/f control mice. Scale bars, 500 mm. NOTCH1 DEFICIENCY IN DG LEADS TO BRAIN DYSFUNCTION

Previous work has found increased cell cycle exit of early precursor cell types and impaired expansion of the progenitor pool in the SGZ of adult inducible Notch1-KO mice, with eventual depletion of the stem population (39, 40). To examine the effect of Notch1 signaling on adult born neurons in the DG, Ai9 (also called Rosa26tdTomato) reporter mice were bred with POMCNotch12/2 mutants to specifically label the morphology of Cre+ neurons. We found that adult-born neurons in the DG contribute almost exclusively to the limited two-thirds of the inner GC layers including SGZ and inner granule cell layer (iGCL) (Fig. 3A) at P56. The number of tdTomato reporter-positive neurons (TOM+) in the granule cell layer (GCL) and molecular cell layer were significantly decreased in POMC-Notch12/2 mutants (Fig. 3B). Most of the TOM+ neurons in POMC-Cre/Ai9 mice migrated into the iGCL (Notch1f/f: 55 6 3.10%; POMC-Notch12/2: 18 6 1.05%) and outer GCL (oGCL) (Notch1f/f: 10 6 2.75%;


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POMC-Notch12/2: 4 6 0.1.62%). In contrast, at P56, most of the TOM+ neurons in POMC-Notch12/2 mutants were still localized in the inner layer of the SGZ (Notch1f/f: 35 6 3.84%; POMC-Notch12/2: 81 6 5.38%) (Fig. 3C). This result was further confirmed by immunostaining and quantitative analysis of DCX+ neurons in the DG. The average number of dendritic branches in DCX+ neurons was 1.41 6 0.28 per neuron in POMC-Notch12/2 mutants, significantly less than that in Notch1f/f control littermates (2.31 6 0.54/neuron) at P29 (Fig. 4D, E). No significant difference was found between POMC-Notch12/2 mutants and control at P56. Next, we investigated whether Notch1 signaling is necessary for the proliferation and survival of type 3 newborn GCs in the DG. A BrdU labeling experiment demonstrated that there was no significant difference in BrdU+ cells between POMC-Notch12/2 mutants and control littermates 1 d after BrdU injection (Fig. 4A–C). However, the survival of newborn neurons in DG was significantly decreased in POMC-Notch12/2 mutants (131 6 25/mm3) compared to control littermates (1109 6 52/mm3; P , 0.05), which suggests that Notch1 signaling is essential for the survival, but not for the proliferation, of type 3 newborn cells in the DG. Compromised neural plasticity of postmitotic GCs in POMC-Notch12/2 mutants To determine whether loss of Notch1 signaling in postmitotic GCs affects the synaptic release probability, miniature EPSCs (mEPSCs) were recorded in TOM+ neurons in

the DG in POMC-Notch12/2 mutants and control to reflect quantal neurotransmitter release (Supplemental Fig. S2A). Slight increases in mEPSC frequency (Notch1f/f: 1.6 6 0.2 Hz, n = 14; POMC-Notch12/2: 1.8 6 0.1 Hz, n = 12; P . 0.05) and amplitude (Notch1f/f: 30.1 6 1.8 pA, n = 12; POMC-Notch12/2: 35.4 6 1.2pA, n = 11; P . 0.05) were detected in POMC-Notch1 2/2 mutants compared to Notch1f/f controls, but no significant difference was found (Supplemental Fig. S2C, D). Analysis of the mEPSC kinetics showed no differences between these 2 groups (increase time, Notch1f/f: 2.1 6 0.2 ms, n = 13; POMC-Notch12/2: 2.2 6 0.3 ms, n = 11; decay time, Notch1f/f: 11.7 6 2.1 ms, n = 10; POMC-Notch12/2: 12.8 6 3.2 ms, n = 10) (Supplemental Fig. S2B). Because TOM+ neurons were distributed in the GCL, we first divided those cells in the GL into 3 layers: L1–3 (Fig. 5A). Three major firing patterns were recorded by whole-cell patch recording. In L1, the majority of TOM+ neurons (32/40) fired with or without mini-spike APs in response to depolarizing-current injection, and some of these cells were converted to firing a single AP when they were hyperpolarized by current injection (Fig. 5B). In L2 and L3, hyperpolarizing TOM+ neurons with current injection induced most of those cells to fire longer trains of APs, although most of them were unable to maintain a sustainable train (Fig. 5B). The remaining TOM+ neurons were capable of firing a sustainable train of APs, similar to that observed in mature GCs in the DG. Therefore, we defined the first 2 firing types of neurons as immature neurons. In L1, there was no significant difference between

Figure 4. Decreased progenitor survival and dendritic branches in DG of POMC-Notch12/2 mutants. A) Experimental protocol for generation of BrdU-labeled proliferating neurons in DG. Animals were injected with BrdU at P28 and were collected at P29 and 56 for immunofluorescent staining. B) Immunofluorescent staining of BrdU in DG of Notch1f/f control and POMC-Notch12/2 mutants at the indicated time points. Scale bars, 100 mm. C ) Stereologic quantification of BrdU+ neurons in Notch1f/f control and POMC-Notch12/2 mutants. The number of BrdU cells was significantly decreased at P56 in POMC-Notch12/2 mutants compared to controls, but no significant difference was found at P29. D) Immunofluorescent staining of DCX+ neurons in the DG of POMC-Notch12/2 mutants and Notch1f/f control at P29 and 56. E ) Quantification of dendritic branches of DCX+ immature neurons in POMC-Notch12/2 mutant and Notch1f/f control at P29 and 56 showed a significantly decreased number of dendritic branches in POMC-Notch12/2 mutant compared to controls at P29, but no difference at P56. Means 6 SEM. *P , 0.01 (Student’s t test). Scale bars, 100 mm.

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Figure 5. Impaired firing properties and HFSinduced LTP in POMC-Notch12/2 mutants. A) Schematic and confocal imaging of POMC-Cre and Ai9 reporter double-positive (TOM+) neurons distributed at the indicated layers in DG. The GL was divided into 3 subregions: L13. ML, molecular layer. B) The firing properties of TOM+ immature GCs in Notch1f/f control and POMC-Notch12/2 mutants were recorded by patch clamp recording. C ) Percentage of immature neurons in different layers in the GL region between Notch1f/f control and POMCNotch12/2 mutants. D) Normal basal synaptic transmission properties in POMC-Notch12/2 mutants compared with Notch1f/f control. E) The paired-pulse facilitation curve was similar between POMC-Notch12/2 mutants and Notch1f/f control. F) Compromised LTP induced by HFS in brain slices of POMC-Notch12/2 mutants compared with Notch1f/f control. *P , 0.05, **P , 0.01 (Student’s t test).

control and POMC-Notch12/2 mutants (Notch1f/f: 81.6 6 5.6, n = 25; POMC-Notch12/2: 85.4 6 6.9, n = 30; P . 0.05). In L2, there were many more immature cells in the POMCNotch12/2 mutants vs. the controls (Notch1f/f: 48.1 6 6.7, n = 20; POMC-Notch12/2: 68.2 6 3.2, n = 20; P , 0.05). There were ;3-fold more immature neurons recorded in POMC-Notch12/2 mutants than in controls in L3 (Notch1f/f: 17.5 6 4.1, n = 18; POMC-Notch12/2: 45.7 6 6.2, n = 16; P , 0.01) (Fig. 5C). For field recordings, basal synaptic transmission was similar between POMC-Notch12/2 mutants and the control littermates (10–11 slices) (Fig. 5D), and the pairedpulse facilitation protocol revealed that POMC-Notch12/2 slices had presynaptic strength comparable to that of controls (Fig. 5E). However, when we induced the LTP in the PP-DG synapses in vivo, the magnitude of LTP in the DG region was uniformly higher in controls (166.5 6 23.1, n = 6) than in POMC-Notch12/2 mutant slices (114.9 6 20.6, n = 5; P , 0.05) (Fig. 5F), suggesting that Notch1 signaling is essential for the maintenance of synaptic efficacy and potentiation in the DG and most likely serves as a postsynaptic component. Impaired emotional and cognitive capacity in POMC-Notch12/2 mutants To investigate whether the deletion of the Notch1 gene in postmitotic GCs affects the behavior and cognition of mutant animals, we first checked the body weight and metabolism of POMC-Notch12/2 mutants to rule out the possibility of side effects of POMC-Cre leaking in the arcuate nucleus of the hypothalamus. We did not found any significant body weight changes from P1 to wk 8 (F1,15 = 3.73; P . 0.05) (Fig. 6A), in contrast to another study that shows altered body size with loss-of-function of Notch signaling in the ventral hypothalamus (35), probably because of the different line of Cre transgenic mice. For a behavioral assay, we first studied the open-field test and found no significant differences in either total distance traveled (F1,15 = 3.41; P . 0.05) (Fig. 6B) or average speed NOTCH1 DEFICIENCY IN DG LEADS TO BRAIN DYSFUNCTION

(data not shown) between POMC-Notch12/2 mutants and Notch1f/f controls, suggesting no motor activity deficits in the mutants. Moreover, the time in the center area in the first 5 min was calculated, and no difference was found between the 2 genotypes (F1,15 = 3.88, P . 0.05; Fig. 6C). In the EPM test, we found that POMC-Notch12/2 mutants spent much less time in the open-field arms than did the Notch1f/f controls (F1,15 = 7.73; P , 0.05; Fig. 6E). During the FST, depletion of Notch1 in immature neurons in the DG resulted in significantly increased immobility time compared to that in the controls (F1,15 = 6.95; P , 0.05; Fig. 6F). The results of these assays suggest that the deletion of the Notch1 gene in type 3 postmitotic GCs leads to anxietyand depressive-like behaviors in POMC-Notch12/2 mutants. It should also be noted that we did not find significant differences between POMC-Notch12/2 mutants and controls in the sucrose solution preference and consumption test (F1,15 = 2.55; P . 0.05; Fig. 6D). Because adult neurogenesis in the DG is crucial for pattern separation, to differentiate similar context in cognition, we next subjected POMC-Notch12/2 mutants and controls to the following 2 different pattern-separation assays. In the first assay, mice that had been fear conditioned in context A (d 1–3) were exposed to the same context A or similar context B (d 4–5), followed by exposure to context A combined with foot shock or context B without foot shock for 6 d (d 6–11), to associate context A with foot shock (Fig. 7A). We found that POMC-Notch12/2 mutants showed decreased levels of freezing in the context A relative to context B during d 6–11, but not d 1–5, compared with Notch1f/f controls (n = 8; P , 0.05; Fig. 7B–D), suggesting significant deficits in learning and acquisition of associative fear memory. Decreased phosphorylation of CREB in DG immature neurons in POMC-Notch12/2 mutants It has been reported that the phosphorylation of the CREB protein transcription factor was present in most immature


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Figure 6. Impaired emotional and cognitive behaviors in POMC-Notch12/2 mutants. A) Similar body weight between Notch1f/f control and POMC-Notch12/2 mutants from 1 to 8 wk after birth. B) Measurement of the total distance traveled in an open-field test. No significant differences were found between POMC-Notch12/2 mutants and Notch1f/f controls in 15 min. C ) Measurement of the time traveled in center area in open-field test. No significant differences were found between POMC-Notch12/2 mutants and Notch1f/f controls in the first 5 min. D) Measurement of sucrose preference. No significant differences were found between POMC-Notch12/2 mutants and Notch1f/f controls. E ) Measurement of EPM between POMC-Notch12/2 mutants and Notch1f/f controls. POMC-Notch12/2 mutants spent much less time in open arms than Notch1f/f controls (n = 8). F ) Measurement of FST between POMC-Notch12/2 mutants and Notch1f/f controls. POMC-Notch12/2 mutants exhibited significantly increased immobility time compared to Notch1f/f controls (n = 8). N.s., not significant. *P , 0.05 (Student’s t test).

neurons in the adult DG to regulate the maturation and survival of newly generated neurons (20, 21). We found that there are significant deficits in maturation and survival of immature neurons in the adult DG of POMC-Notch12/2 mutants. To further investigate whether CREB phosphorylation (pCREB) is involved in the regulation of Notch1 function in the adult mouse DG, we compared the expression of pCREB between POMC-Notch12/2 mutants and controls at P56. Both immunofluorescent staining (Fig. 8A) and

immunoblot (Fig. 8B) results demonstrated that the pCREB level was significantly decreased in the adult hippocampal DG in POMC-Notch12/2 mutants (relative value: 36.75 6 4.21 in POMC-Notch12/2 mutants vs. 72.50 6 5.46 in controls; n = 4; P , 0.05) (Fig. 8A–C). These data suggested that one of the underlying molecular mechanisms of Notch1 signaling is dependent on the pCREB activity in the regulation of the survival and function of adult immature neurons in the DG.

Figure 7. Impaired contextual fear discrimination in POMC-Notch12/2 mutants. A) Experimental procedures for contextual fear discrimination with 2 similar contexts. B) Averaged percentage of freezing levels recorded during the stage of fear conditioning in context A in d 1–3. No significant differences were found between POMC-Notch12/2 mutants and controls (n = 8). P . 0.05 (Student’s t test). C ) Averaged percentage of freezing levels recorded during introduction of a similar context B in d 4–5. No significant differences were found between POMC-Notch12/2 mutants and controls (n = 8). N.s, not significant. P . 0.05 (Student’s t test and repeated-measures ANOVA). D) Averaged percentage of freezing levels recorded during the stage of contextual fear discrimination in context A/B on d 6–11. POMC-Notch12/2 mutant exhibited deficits of fear discrimination in 2 similar contexts (context A-solid line, context B-dashed line) compared to Notch1f/f controls (n = 8). *P , 0.05 (context A vs. context B for Notch1f/f control); #P , 0.05 (context A vs. context B for POMC-Notch12/2 mutants; Student’s t test and repeated-measures ANOVA). 8

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Figure 8. Decreased phosphorylation of CREB in DG in POMC-Notch12/2 mutant. A) Immunofluorescent staining of pCREB in Notch1f/f controls and POMC-Notch12/2 mutants at P56. Scale bars, 100 mm. B) Western blot analysis of total lysate from ventral hippocampus of Notch1f/f controls and POMC-Notch12/2 mutants at P56. Representative results showing levels of total CREB protein and pCREB from 2 different genotypes. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal loading control for protein normalization. C ) Quantitative analysis of the gray value of the Western blot results showing significantly decreased expression of pCREB in POMC-Notch12/2 mutants compared to controls. N.s., not significant. *P , 0.05 (Student’s t test).

DISCUSSION Adult neurogenesis in the hippocampus has gained increasing attention over the past decades (41, 42), and interest has intensified with the discovery of differences in adult neurogenesis in normal brain function and disorders (43–45). The SGZ, one of the major adult neurogenesis niches in the brain, differs from another niche, the SVZ, providing a constant connection and supply to the olfactory bulb that gives rise to inhibitory olfactory bulb GCs (42). Note that a massive number of adult-born neurons in the DG undergo programmed cell death and differentiation within a month after birth (46). During mammalian neural development, the role of Notch1 signaling in the maintenance of neural progenitor cells has been supported by both loss-of-function and gain-of-function studies showing that Notch1 activation prevents neuronal differentiation (47–49). In addition, Notch1 signaling in adult neurogenesis has also been intensively investigated in recent years, but whether and how Notch1 signaling regulates the survival and function of late progenitor cells in the DG is still elusive. Previous studies have demonstrated that NICD, the active cleavage form of Notch1, is expressed in nestin+ type 1 and 2a cells as well as DCX+ late progenitor and postmitotic GCs, whereas most type 2b and 3 cells lack NICD expression (10, 11). Similarly, we found that the Notch1 receptor is highly expressed in DCX+ immature neurons in the DG at P30 (Fig. 1A). Moreover, the expression of Notch1 in DG GCs is neuronal activity dependent, which we induced by electrical HFS in vivo (Fig. 1B). Notch1 has been shown to be cleaved and activated in mature pyramidal neurons in the hippocampal CA1-CA3 region in response to synaptic activity, which requires the NOTCH1 DEFICIENCY IN DG LEADS TO BRAIN DYSFUNCTION

neuronal activity-related gene Arc/Arg3.1 to modulate LTP and LTD in hippocampal networks (16). Our results further demonstrate that the activation of Notch1 signaling is not only restricted in the CA1-CA3 region in adult hippocampus, but also in GCs in the hippocampal DG, suggesting a conserved role in the regulation of synaptic plasticity of adult mature neurons. In addition, one of the most striking results of this study is that deletion of Notch1 in late progenitors in the DG caused the dispersed lamination of CA3 (Fig. 2). The lamination deficit of hippocampal CA3 in POMCNotch12/2 mice phenocopied the defects in doublecortin knockout (Dcx-KO) mice, which exhibit anatomic and physiologic defects in CA3 showing abnormal double layers of pyramidal cells with reduced dendritic arbors (38, 50). Indeed, both POMC-Cre and Notch1 floxed mice have a B6 background; however, we never observed a dispersed lamination of CA3 on the POMC-Cre, Notch1f/f, or F1 hybrid background. Thus, the lamination deficits of CA3 in POMC-Notch12/2 mice were bona fide, not related to the genetic background. The hippocampal CA3 region could be the convergence point of inputs from the entorhinal cortex via the PP, the DG via mossy fibers, and its own inputs via the recurrent collaterals, which may play critical roles in many specific cognitive processes (51–53). As DG and mossy fiber could send input into CA3, the abnormal lamination of CA3 may be related to the dissociation between the DG and CA3 subregion of the hippocampus. Because CA3 is involved in the regulation of temporal pattern separation and completion for spatial locations (54–56), the pattern separation defects in POMC-Notch12/2 mutants may be mostly related to the disorganized CA3 lamination in addition to the dysfunction of type 3 cells in the DG.


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Adult-born neurons display distinct electrophysiological properties from that of mature neurons in the DG, including firing properties and number of excitatory synapses (57). In this study, we specifically knocked out Notch1 expression in type 3 late progenitor cells by using a postmitotic neuron-specific POMC-Cre line in the postnatal brain without affecting the development of neural stem cells (24). We found significant deficiency in neural migration and dendritic arborization in POMC-Notch12/2 mutants (Fig. 3), indicating that Notch1 signaling plays a pivotal role in the regulation of adult-born neuron migration and dendritic development, consistent with previous findings that Notch1-mediated intermittent hypoxia induces newborn neuron survival, migration, and spinal morphogenesis in the DG (58). We also found that deletion of Notch1 in type 3 late progenitor cells in the DG caused irregular and immature firing patterns (Fig. 5B, C). Another study has shown that Notch1 colocalizes with PSD95 in cultured neurons, and the activated form of the receptor, NICD1, is present at the synapse (16). In addition, we have shown that Notch1 signaling is upregulated in response to neuronal activity via stimulation of neurons in vivo by HFS (Fig. 1B). Together, our morphologic and LTP results demonstrate that ablation of Notch1 receptor in adult-born neurons in DG affects both dendritic morphology and the synaptic potentiation in mutants (Figs. 4D, E, and 5F). These results are consistent with the reduced potentiation observed in antisense Notch1 transgenic or g-secretasedisrupted mice (11, 59, 60), suggesting that Notch1 signaling is essential for the maintenance of synaptic efficacy and potentiation in the adult hippocampal DG. To assess the effect of Notch1 signaling disruption on emotional and cognitive competence in the hippocampal DG, we tested POMC-Notch12/2 mutant mice with numerous behavioral paradigms. DG is critical for the formation of new episodic memories and for transformation of similar experiences into discrete, nonoverlapping representation, which is known as pattern separation. Disruption of adult hippocampal neurogenesis impairs pattern separation (61, 62). In this study, we observed anxiety- and depressive-like behaviors in POMCNotch12/2 mutant mice (Fig. 6). Although the role of adult hippocampal neurogenesis in affective behaviors is still under debate (62–64), our results suggest that Notch1 signaling is involved in the regulation of affective behaviors, probably by regulating the survival and synaptic transmission of adult-born neurons in the hippocampal DG. Moreover, we noted that conditional fear discrimination was significantly impaired in POMC-Notch12/2 mutants (Fig. 7). Impaired pattern separation may lead to excessive generalization, which cannot discriminate similar events if the discrepancy is minimal. This increased generalization of new innocuous experiences with previously encountered aversive events frequently happens in patients with panic disorder and posttraumatic stress disorder. Thus, our results provided novel evidence to better understand the role of Notch1 signaling in the regulation of emotional, cognitive, and psychiatric disorders such as posttraumatic stress disorder. Finally, we found compromised phosphorylation of CREB in DG in 10

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POMC-Notch12/2 mutants (Fig. 8). According to previous results, phosphorylation of CREB probably plays a crucial role in the formation of long-term memory and regulation of hippocampal plasticity (22, 29). Thus, our study suggests that the function of Notch1 receptor in type 3 late progenitor cells in the DG is dependent on the activation of CREB signaling. In summary, this study demonstrated that ablation of Notch1 signaling in postnatal newborn late progenitor cells in DG could cause abnormal hippocampal CA3 lamination, reduced survival of adult-born neurons without affecting the proliferation of newborn neurons. In addition, impaired functional excitatory postsynaptic potential and LTP of GCs were found by ablation of Notch1 expression in late progenitor cells in the DG. Moreover, disruption of Notch1 signaling in postnatal newborn type3 cells in the DG caused emotional and cognitive impairment, including anxiety and depressive behaviors, and impaired contextual fear discrimination, as well. Finally, mechanistic experiments found that Notch1 may regulate the function of postnatal newborn cells by phosphorylation of the CREB protein transcription factor. ACKNOWLEDGMENTS The authors thank all of the members of the H.W. laboratory for their encouragement and discussions. This work was supported, in part, by Program 973, Grant 2014CB542203, from the State Key Development Program for Basic Research of China (to H.W.), National Natural Science Foundation of China Grants 31371149 and 31522029 (to H.W.), and Grant Z161100000216154 Beijing Municipal Science and Technology Commission (to H.W.) The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS H. Wu and S. Feng designed the research; H. Wu, S. Feng, T. Shi, and J. Qiu analyzed the data; S. Feng, T. Shi, J. Qiu, H. Yang, and Y. Wu performed the research; S. Feng, T. Shi, and H. Wu wrote the paper; and W. Wang and W. Zhou contributed new reagents and analytic tools. REFERENCES 1. Altman, J., and Das, G. D. (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neurol. 124, 319–335 2. Ming, G. L., and Song, H. (2011) Adult neurogenesis in the mammalian brain: significant answers and significant questions. Neuron 70, 687–702 3. Toni, N., Laplagne, D. A., Zhao, C., Lombardi, G., Ribak, C. E., Gage, F. H., and Schinder, A. F. (2008) Neurons born in the adult dentate gyrus form functional synapses with target cells. Nat. Neurosci. 11, 901–907 4. Zhao, C., Deng, W., and Gage, F. H. (2008) Mechanisms and functional implications of adult neurogenesis. Cell 132, 645–660 5. Imayoshi, I., Sakamoto, M., Ohtsuka, T., Takao, K., Miyakawa, T., Yamaguchi, M., Mori, K., Ikeda, T., Itohara, S., and Kageyama, R. (2008) Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain. Nat. Neurosci. 11, 1153–1161 6. Sahay, A., Wilson, D. A., and Hen, R. (2011) Pattern separation: a common function for new neurons in hippocampus and olfactory bulb. Neuron 70, 582–588



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Received for publication March 16, 2017. Accepted for publication May 30, 2017.


FENG ET AL.

Notch1 deficiency in postnatal neural progenitor cells in the dentate gyrus leads to emotional and cognitive impairment Shufang Feng, Tianyao Shi, Jiangxia Qiu, et al. FASEB J published online June 13, 2017 Access the most recent version at doi:10.1096/fj.201700216RR

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