Skip to main content
NCBI home page
Biochemistry and Biophysics Reports logoLink to Biochemistry and Biophysics Reports
. 2026 May 28;46:102650. doi: 10.1016/j.bbrep.2026.102650

Role of Dlk1–Notch signaling in impaired neurogenesis and cognitive decline in chronic cerebral hypoperfusion

Yoshiyuki Moriyama 1, Rena Hasegawa 1, Hikaru Endo 1, Sayaka Matsumura 1, Ryotaro Kaneko 1, Hideki Hayashi 1, Yui Iwatani 1, Norio Takagi 1,⁎
PMCID: PMC13235409  PMID: 42256790

Abstract

Vascular dementia (VaD) is characterized by chronic cerebral hypoperfusion, leading to cognitive decline. Impaired hippocampal neurogenesis is a crucial pathological feature of VaD. Delta-like 1 homolog (Dlk1)-Notch signaling may contribute to neural stem cell regulation, but the involvement of Dlk1-Notch signaling in impaired hippocampal neurogenesis during hypoperfusion is unclear. Here, we investigated the role of Dlk1 and Notch signaling in a mouse model of chronic cerebral hypoperfusion induced by bilateral carotid artery stenosis (BCAS). We found that BCAS increased Dlk1 levels in hippocampal tissue and cerebrospinal fluid, accompanied by elevated Notch intracellular domain (NICD) expression and reduced doublecortin (Dcx)-positive hippocampal cells. These changes were associated with impaired novel object recognition. In Neuro2a cells, hypoxia-mimetic treatment elevated NICD and extracellular Dlk1 and reduced cell viability, while pharmacological inhibition of Notch signaling partially rescued cell loss and attenuated these changes. These findings suggest that Dlk1-associated Notch activation correlates with impaired hippocampal neurogenesis and memory dysfunction under chronic hypoperfusion. Our results highlight a potential link between altered niche signaling and cognitive decline in VaD, providing a basis for further mechanistic investigation.

Keywords: Chronic cerebral hypoperfusion, Dlk1, Notch, Neurogenesis, Vascular dementia

Highlights

  • •

    Chronic hypoperfusion impaired cognitive function and reduced neurogenesis.

  • •

    Dlk1 and NICD levels were elevated in hippocampal tissue after BCAS.

  • •

    Hypoxia induced Dlk1–Notch activation in vitro, accompanied by increased apoptotic markers.

  • •

    Inhibition of γ-secretase with DAPT that blocks Notch signaling suppressed these changes and restored cell viability.

1. Introduction

Dementia is characterized by progressive cognitive dysfunction due to disrupted neural circuit networks, ultimately interfering with activities of daily living [1]. The core symptoms of dementia include memory impairment, executive dysfunction, and disorientation, and peripheral symptoms include delusions, hallucinations, delirium, and depression [1,2]. These neuropsychiatric manifestations delay the detection and treatment of comorbidities and accelerate physical decline, often leading to institutionalization and a bedridden state [3]. Dementia is classified according to its etiology. Vascular dementia (VaD) is the second most common subtype of dementia, accounting for approximately 15%–20% of dementia cases worldwide [4]. VaD is caused by cerebrovascular dysfunction, frequently associated with chronic cerebral hypoperfusion after ischemic events or age-related vascular changes [5]. No pharmacological interventions, including antiplatelet agents and drugs used in Alzheimer's disease, have shown robust clinical efficacy in patients with VaD [6,7]. Thus, novel therapeutic strategies that suppress disease progression are urgently needed.

Neural stem cells (NSCs) are multipotent cells that can self-renew and differentiate into neurons, astrocytes, and oligodendrocytes. NSCs are located in the subgranular zone of the dentate gyrus and the subventricular zone of the adult brain [8]. The continuous generation of new neurons from these regions, called neurogenesis, persists throughout life, but declines with age [9]. New neurons generated in the hippocampus contribute to synaptic plasticity and neural circuitry, which play a critical role in memory and learning [10]. Age-related cognitive decline and dementia correlate with reduced hippocampal neurogenesis [11,12]. Impaired neurogenesis contributes to the pathology of vascular dementia, but the underlying mechanisms are poorly understood.

Delta-like 1 homolog (Dlk1) is a ligand of the Notch receptor. The Dlk1-Notch signaling pathway is a key regulator of cell proliferation and differentiation. Dlk1 binding to the Notch receptor induces notch intracellular domain (NICD) translocation to the nucleus, where it binds to transcriptional repressors and converts them into transcriptional activators [13]. Dlk1-Notch signaling regulates the proliferation and differentiation of hippocampal neural stem cells in mice [14]. However, the role of Dlk1-Notch signaling in vascular dementia is unclear. The aim of this study was to determine the role of Dlk-1-Notch signaling in neurogenesis during hypoperfusion using a mouse model of VaD and Neuro2a cells exposed to hypoxia.

2. Materials and methods

2.1. Animals

Adult male C57BL/6J mice aged 11–13 weeks (Japan SLC, Inc., Shizuoka, Japan) were used for this study. A total of 27 mice were used in this study. In the novel object recognition test, 8 mice in the sham group and 10 mice in the BCAS group were analyzed. For immunohistochemical analysis, 7 mice in the sham group and 11 mice in the BCAS group were analyzed. For biochemical analysis, 4 mice per group (sham and BCAS) were used. The mice were 15-18 weeks old at the end of the study. Animals were housed under the following conditions: temperature: 23 ± 1 °C; humidity: 55 ± 5%; 12-h light/dark cycle. The mice had free access to food and water, and all studies complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Guidance for Experimental Animal Care issued by the Prime Minister's Office of Japan. The experimental protocol was approved by the Committee of Animal Care and Welfare of Tokyo University of Pharmacy and Life Sciences (No. P24-09, P25-40) and in compliance with the ARRIVE guidelines.

2.2. Bilateral common carotid artery stenosis model

Bilateral common carotid artery stenosis (BCAS) was induced as previously described with slight modifications [15]. Mice were anesthetized with an intraperitoneal (IP) injection of 0.3 mg/kg medetomidine hydrochloride, 4 mg/kg midazolam, and 5 mg/kg butorphanol tartrate. The right common carotid artery (CCA) was exposed and dissected from the surrounding tissue via a midline cervical incision. A microcoil (wire diameter: 0.08 mm; inner diameter: 0.18 mm; pitch: 0.5 mm; total length: 2.5 mm; Sawane Spring Co., Ltd., Shizuoka, Japan) was placed around the bifurcation of the right internal carotid artery (ICA) and external carotid artery (ECA). After 30 min, the left CCA was exposed, and a microcoil was placed around the bifurcation of the left ICA and ECA. The surgical site was sutured, and the anesthesia was reversed with atipamezole (0.3 mg/kg, IP). Sham-operated mice underwent the same surgical procedures without microcoil placement.

2.3. Novel object recognition test

The novel object recognition test was performed on day 28 after BCAS, as previously described with slight modifications [16]. After habituation in an experimental room for 30 min, mice were allowed to freely explore two identical objects for 5 min in a plastic box (W355 × D499 × H198 mm3). After familiarization, the mice were returned to their home cages. After 60 min, one of the objects was replaced by a new object and the mice were allowed to explore the box in the experimental room for another 5 min. The time spent exploring the novel object and the familiar object during the testing was recorded by an observer that was blinded to the treatment conditions. The amount of time the mouse spent exploring each object during both trials was recorded by an observer that was blinded to the treatment conditions. The discrimination index was calculated as follows: time to explore novel object − time to explore familiar object)/(total time to explore both objects). The box and the object were cleaned with 70% ethanol to eliminate the smell after each exploration.

2.4. Immunohistochemistry

On day 28 after BCAS surgery, mice were perfused with 4% paraformaldehyde in ice-cold phosphate buffered saline (PBS) via the heart. Brains were quickly removed, immersed in 30% sucrose in PBS, and sliced into 30-μm sections using a microtome (Yamoto Kohki Industrial Co., Ltd., Saitama, Japan). After washing with PBS, the sections were immersed in room temperature PBS containing 0.3% TritonX-100 for 15 min to permeabilize the cell membranes to antibodies. After incubating in Blocking One Histo (06349-64, Nacalai Tesque, Inc., Kyoto, Japan) for 1 h at room temperature, the sections were incubated overnight with goat anti-doublecortin (Dcx, sc-8066, Santacruz, Dallas, TX, USA) at 4 °C. On the following day, the sections were incubated with the Alexa Fluor 594 donkey anti-goat (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Nuclei were stained with Hoechst 33342 (346–07951, Dojindo, Kumamoto, Japan). Fluorescence was detected with a fluorescence microscope (BZ-X800; Keyence, Tokyo, Japan), and Dcx-positive cells were counted using a BZ-X Analyzer (Keyence).

2.5. In vivo sample preparation

Mice were perfused with ice-cold PBS via the heart. The hippocampi were quickly removed and homogenized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.4) with phosphatase inhibitors (Roche, Mannheim, Germany) and protease inhibitors (Roche). After sonication (Q125, QSONICA, Newtown, CT, USA), protein concentrations were measured using the BCA method. Samples were incubated in sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 5% β-mercaptoethanol) at 95 °C for 5 min. To collect cerebrospinal fluid (CSF), mice were anesthetized with an intraperitoneal (IP) injection of 0.3 mg/kg medetomidine hydrochloride, 4 mg/kg midazolam, and 5 mg/kg butorphanol tartrate and placed horizontally with their cisternae magnae surgically exposed. Ten to 20 μL of CSF were collected from each mouse 28 days after sham surgery or BCAS.

2.6. Western blotting

Western blotting was performed according to standard protocols with the following primary antibodies: mouse anti-β-actin (A5441, Sigma-Aldrich Corp., St. Louis, MO, USA), rabbit anti-NICD (ab52301, Abcam, Cambridge, UK), mouse anti-Dlk-1 (B1315, Santa Cruz), rabbit anti-Hif1α (bs-0737R, Thermo Fisher Scientific), rabbit anti-cleaved caspase3 (9661S, Cell Signaling Technology, MA, USA), rabbit anti-LC3 (12741, Cell Signaling Technology, MA, USA), and horseradish peroxidase-conjugated secondary antibodies. Immunoreactive proteins were detected using ImmunoStar zeta (FUJIFILM Wako, Osaka, Japan), Immobilon ECL Ultra Western HRP Substrate (WBULS0100, Millipore, Burlington, MA, USA), or Immobilon Crescendo Western HRP Substrate (WBLUR0100, Millipore). Quantification of antibody binding was performed using computerized densitometry (LuminoGraph II, ATTO Co., Tokyo, Japan) and an image analyzer (CS Analyzer, ATTO Co.).

2.7. Cell culture

Mouse neuroblastoma Neuro2a cells were obtained from ATCC (VA, USA) and grown in Dulbecco's Modified Eagle Medium (FUJIFILM Wako) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin–streptomycin (Thermo Fisher Scientific). Cells were cultured in 6-well plates (8.0 × 105 cells/well) in 5% CO2 at 37 °C. The medium was replaced with serum-free medium supplemented with cobalt chloride and γ-secretase inhibitor (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester; DAPT, MERCKMillipore, EMD_BIO-565770) for 24 h.

2.8. Cell viability

The viability of Neuro2a cells was determined using a WST-1 assay. Cells were incubated in media containing 0.5 mM 2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1; 346–06454, Dojindo, Kumamoto, Japan) and 20 μM 1-methoxy-5-methylphenazinium methylsulfate (345–04001, Wako) for 1 h at 37 °C. Absorbance at 450 nm was measured using a plate reader (EMax Plus Microplate Reader, Molecular Devices, Sunnyvale, CA, USA). The relative cell viability was expressed as the ratio of the treatment group and the corresponding nontreated group.

2.9. In vitro sample preparation

To analyze changes in protein levels in Neuro2a cells exposed to cobalt chloride to induce hypoxia, cells were rinsed with ice-cold PBS, collected in RIPA buffer with phosphatase inhibitors (Roche) and protease inhibitors (Roche), and sonicated. Protein concentrations were determined using the BCA method. Samples were incubated in sample buffer at 95 °C for 5 min.

2.10. Statistical analyses

Statistical analyses were performed using GraphPad Prism (version 10.5.0, GraphPad Software, CA, USA). When the data were normally distributed, parametric tests (unpaired t-tests or one-way ANOVA followed by appropriate post hoc tests) were used. Parametric analyses of multiple groups were performed using one-way analyses of variance followed by a Tukey post hoc test. P values of <0.05 were considered statistically significant. When the data were not normally distributed, non-parametric tests (Mann-Whitney U test or Kruskal-Wallis test) were applied. All experimental results are shown as means ± standard deviations and individual values.

3. Results

3.1. Mice exhibit cognitive dysfunction and reduced hippocampal neurogenesis 28 Days after BCAS

The novel object recognition test was conducted 28 days after BCAS to assess cognitive performance. The discrimination index was significantly reduced in the BCAS mice compared to the sham mice on day 28 after surgery (Fig. 1A). No significant difference in total exploration time was detected between the BCAS and sham groups (Fig. 1B). These results demonstrate that chronic cerebral hypoperfusion-induced cognitive dysfunction occurs 28 days after BCAS.

Fig. 1.

Fig. 1

Open in a new tab

Cognitive dysfunction in the novel object recognition test and impaired neurogenesis on day 28 after BCAS.

Changes in the discrimination index (A) and total exploration time (B) in the novel object recognition test on day 28 after BCAS. The discrimination index was significantly lower in the BCAS-(red closed triangle) mice compared with the sham-operated (open circle) mice on day 28 after surgery. All results are presented as the means ± SD and individual values (n = 8 in the sham group, n = 10 in the BCAS group). Groups were compared using unpaired two-tailed t-tests. A significant difference was observed between the groups (t(16) = 4.129, ∗, P = 0.001). Representative immunofluorescence images of doublecortin (Dcx) positive cells in the hippocampal dentate gyrus of sham- (opened circle) and BCAS-operated (red closed triangle) mice 28 days after surgery (C). Scale bar shows 100 μm. Dcx-positive cells were counted in the hippocampal dentate gyrus of sham- and BCAS-operated mice 28 days after surgery (D). Results are presented as means ± SD and individual values (n = 7 in the sham group, n = 11 in the BCAS group). The number of Dcx-positive cells per hippocampal dentate gyrus region was quantified in each group. Groups were compared with unpaired two-tailed t-tests. A significant decrease in Dcx-positive cells was observed in the BCAS-operated mice group compared to the Sham-operated group (t(16) = 2.132, ∗P = 0.049).

Dcx is a marker of immature neurons. Fig. 1C and D show the number of Dcx-positive cells in the dentate gyrus of the hippocampus on day 28 after BCAS. On day 28 after surgery, significantly fewer Dcx-positive cells were detected in the dentate gyrus of hippocampi from BCAS mice compared to the hippocampi from sham mice. These results indicate that the cognitive impairment observed in BCAS mice on day 28 after surgery was likely due to reduced neurogenesis.

3.2. Dlk1–notch signaling is induced in the mouse brain after BCAS

Dlk1 is an atypical ligand of the Notch signaling pathway. Western blot analysis revealed that Dlk1 protein levels were significantly higher in the hippocampal tissue (Fig. 2A) and cerebrospinal fluid (Fig. 2B) of BCAS mice compared to the sham mice on day 28 after surgery. NICD is a marker of the active form of Notch receptor signaling. NICD protein levels were significantly elevated in the BCAS mice compared with sham mice (Fig. 2C). These results indicate that the Dlk1–Notch1 signaling pathway was activated and may contribute to reduced neurogenesis after BCAS.

Fig. 2.

Fig. 2

Open in a new tab

Dlk1-Notch signaling pathway in the hippocampus 28 days after BCAS or sham surgery.

Representative western blots showing Dlk1 and Notch intracellular domain (NICD) levels in the hippocampus and cerebrospinal fluid (CSF) in mice 28 days after sham surgery or BCAS (A-C). Dlk1 and NICD protein levels in the hippocampus were normalized to β-actin (A and C). Dlk1 protein levels in CSF were normalized to protein concentration (B). Data are presented as the means ± SD and individual values (n = 4 each for hippocampal samples; n = 3 each for CSF samples). An unpaired two-tailed t-test revealed a significant increase in Dlk1 levels in the hippocampus after BCAS compared to sham controls (t(6) = 4.024, ∗P = 0.007), NICD levels in BCAS mice compared to sham controls (t(6) = 4.113, ∗P = 0.006), and Dlk1 levels in CSF in BCAS mice compared to sham controls (t(4) = 3.745, ∗P = 0.02). ∗P < 0.05 was considered statistically significant.

3.3. Effects of DAPT on the viability of Neuro2a cells treated with cobalt chloride

In vitro experiments were conducted using mouse neuroblastoma-derived Neuro2a cells to investigate the role of Dlk1–Notch1 signaling in neurogenesis under hypoperfusion. Neuro2a cells exhibit the self-renewal and neuronal differentiation potential characteristic of neural progenitor cells. Thus, Neuro2a cells are commonly used as an in vitro model of neurogenesis [17]. To mimic the cerebral hypoperfusion condition in the BCAS model, intracellular hypoxia was induced using cobalt chloride. Cell viability significantly decreased after 24 h of cobalt chloride exposure (Fig. 3A–C, F). The reduced cell viability was dose-dependently attenuated by DAPT, a γ-secretase inhibitor that prevents Notch signaling activation by inhibiting Notch receptor cleavage (Fig. 3D–F).

Fig. 3.

Fig. 3

Open in a new tab

Effects of DAPT on viability in Neuro2a cells after CoCl2-induced hypoxia.

Representative images after no treatment (A), DMSO (Control; B), CoCl2 100 μM (C), DAPT 10 μM (D), and DAPT 20 μM (E) in Neuro2a cells exposed to CoCl2-induced hypoxia. The viability of Neuro2a cells was determined using the WST-1 assay. Absorbance at 450 nm was measured, and values are expressed as the ratio of the absorbance at 450 nm of the nontreated group (F). Data are presented as the means ± SD and individual values (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by the Tukey's multiple comparison test to compare groups (F(3, 20) = 11.09, ∗P < 0.05, ∗∗∗∗P < 0.0001).

3.4. Effects of DAPT on Neuro2a cells exposed to cobalt chloride-induced hypoxia

The effects of DAPT on Neuro2a cells exposed to cobalt chloride-induced hypoxia were examined. After 24 h of cobalt chloride treatment, HIF1α protein levels were increased compared to the control group, but DAPT treatment did not attenuate the elevated HIF1α protein levels (Fig. 4A). NICD protein levels increased after cobalt chloride treatment, and DAPT treatment attenuated the increased NICD levels (Fig. 4B). Cobalt chloride or DAPT treatment did not affect Dlk1 protein levels in Neuro2a cells (Fig. 4C). In contrast, Dlk1 protein levels in the conditioned medium (extracellular fraction) was significantly elevated 24 h after cobalt chloride treatment compared to the control, and DAPT significantly attenuated this elevation (Fig. 4D).

Fig. 4.

Fig. 4

Open in a new tab

Effects of DAPT on HIF1α, NICD, and Dlk-1 protein levels in Neuro2a cells and extracellular Dlk-1 protein levels in the culture medium after exposure to CoCl2-induced hypoxia.

Representative western blots showing HIF1α (A), NICD (B), and Dlk-1 (C) protein levels in Neuro2a cells and extracellular Dlk-1 protein levels (D) in conditioned medium after exposure to CoCl2-induced hypoxia. HIF1α, NICD, and Dlk-1 protein levels in Neuro2a cells were normalized to β-actin (A-C). Extracellular Dlk-1 protein levels in conditioned medium normalized to protein concentration (D). Data are presented as means ± SD and individual values (n = 4–5). Groups were compared using one-way ANOVA followed by Tukey's multiple comparison tests to evaluate differences between the control group and each treatment group (F(3, 16) = 5.164 in A, F(3, 12) = 7.728 in B, F(3, 12) = 0.446 in C, and F(3, 12) = 15.11 in D). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

3.5. Effects of DAPT on apoptotic protein levels in Neuro2a cells exposed to cobalt chloride-induced hypoxia

We examined the effects of DAPT on apoptotic protein levels, including cleaved caspase-3 and LC3, in Neuro2a cells exposed to cobalt chloride-induced hypoxia. After 24 h of cobalt chloride treatment, cleaved caspase-3 protein levels were significantly increased compared to the control group, and this increase was significantly attenuated by DAPT treatment (Fig. 5A). Similarly, LC3 protein levels were significantly elevated after cobalt chloride treatment, and the increased LC3 levels were significantly attenuated by DAPT (Fig. 5B).

Fig. 5.

Fig. 5

Open in a new tab

Effects of DAPT on cleaved caspase 3 and LC-3 protein levels in Neuro2a cells after exposure to CoCl2-induced hypoxia.

Representative Western blot images showing cleaved caspase 3 (A) and LC-3 (B) protein levels in Neuro2a cells after exposure to CoCl2-induced hypoxia. Cleaved caspase 3 and LC-3 protein levels in Neuro2a cells were normalized to β-actin. Data are presented as the means ± SD and individual values (n = 4–6). Groups were compared using one-way ANOVA followed by Tukey's multiple comparison tests to evaluate differences between the control and treatment groups (F(3, 12) = 9.959 in A, F(3, 20) = 9.559 in B, ∗P < 0.05, ∗∗∗P < 0.001).

4. Discussion

This study aimed to elucidate the pathophysiological changes in Dlk1-Notch signaling associated with reduced neurogenesis in cerebral hypoperfusion-induced vascular dementia. In vitro experiments using Neuro2a cells were conducted to clarify the role of Dlk1-Notch signaling. The discrimination index of the novel object recognition test was reduced 28 days after BCAS compared with the sham mice. Thus, chronic cerebral hypoperfusion led to impaired recognition memory. The number of Dcx-positive cells, a marker for immature neurons, decreased significantly in the dentate gyrus of the hippocampus in the BCAS mice, suggesting reduced neurogenesis. Previous studies showed that neurogenesis is reduced in the adult hippocampus with aging and in Alzheimer's disease [12,18]. In agreement with these previous reports, our results suggest that reduced hippocampal neurogenesis contributes to cognitive decline in vascular dementia.

To investigate the mechanism of impaired neurogenesis after BCAS, we examined the Dlk1-Notch signaling pathway [14]. The Notch signaling pathway is a central regulator of neural stem/progenitor cell maintenance and differentiation [19,20]. Activation of Notch preserves stemness and inhibits premature neuronal differentiation, and downregulation of Notch promotes neuronal lineage commitment [13]. Dlk1, a preadipocyte factor-1 acting as a non-canonical Notch ligand, may regulate stem cell regulation and metabolic homeostasis [21]. However, the precise role of Dlk1 in the adult hippocampus is unclear. Biochemical analyses of hippocampal tissue 28 days after BCAS revealed increased protein levels of Dlk1 and NICD compared to the sham group. These results suggest that activation of Dlk1-Notch signaling after BCAS is involved in the reduced neurogenesis.

In vitro experiments were conducted in Neuro2a cells to explore the role of Dlk1-Notch signaling in neurogenesis in response to cerebral hypoperfusion. Neuro2a cells exhibit the self-renewal and differentiation potential of neural progenitor cells and are commonly used as an in vitro model of neurogenesis [17]. To mimic the cerebral hypoperfusion in the BCAS model in vitro, the Neuro2a cells were treated with cobalt chloride to induce intracellular hypoxia. Cobalt chloride inhibits prolyl hydroxylase and stabilizes HIF-1α to simulate hypoxia [22]. Neuro2a cell death and increased HIF-1α protein levels after treatment with cobalt chloride were confirmed. These results indicate the induction of intracellular hypoxia. Thus, the cobalt chloride-treated Neuro2a cell model was used as an in vitro model of cerebral hypoperfusion. Intracellular and extracellular Dlk1 protein levels were measured in Neuro2a cells. Intracellular Dlk1 levels were not affected by cobalt chloride treatment, but the Dlk1 concentration in the culture supernatant was significantly increased. Notch signaling has been reported to regulate the expression of multiple differentiation-related genes, suggesting that Dlk1 expression or secretion may also be influenced by Notch activity depending on the cellular context [23,24]. Therefore, it is possible that a feedback regulatory loop exists between Dlk1 and Notch signaling, rather than a simple linear upstream-downstream relationship. NICD protein levels also increased after cobalt chloride treatment compared to controls. Notably, these results were consistent with increased Dlk1 and NICD protein levels in hippocampal tissue and cerebrospinal fluid after BCAS. Taken together, these results suggest that hypoperfusion-induced hypoxia leads to neural stem/progenitor cell death, and activation of Dlk1-Notch signaling after BCAS may originate from neural stem/progenitor cells.

Yang et al. demonstrated that Notch activation induces p53-dependent apoptosis in neural stem/progenitor cells [25]. Furthermore, Cheng et al. demonstrated that the expression levels of Notch-1, phosphorylated-JNK, and active caspase-3 were increased in the intracerebral hemorrhage model and that inhibition of Notch-1 suppressed the phosphorylation of JNK and the expression of active caspase-3 [26]. Our results are consistent with these previous studies, although the activation of JNK/c-Jun should be determined in future studies. To elucidate the mechanism of cell death in response to hypoxia, we conducted inhibition experiments using DAPT, a γ-secretase inhibitor that blocks Notch signaling. DAPT treatment attenuated the cobalt chloride-induced reduction in cell viability in a concentration-dependent manner. Furthermore, DAPT treatment attenuated cobalt chloride-induced increases in protein levels of cleaved caspase-3 and LC3, as an indicator of autophagy-related cellular stress under hypoxic conditions, in a dose-dependent manner and the increases in extracellular Dlk1 protein levels induced by cobalt chloride. These results suggest that Dlk1-Notch signaling is involved in apoptotic neural stem/progenitor cell death in response to cobalt chloride-induced hypoxia.

In interpreting our results, several limitations should be considered. First, although CoCl2 treatment is widely used to mimic hypoxic stress, it does not fully reproduce the complex metabolic and vascular alterations of chronic cerebral hypoperfusion. This paradigm primarily activates hypoxia-inducible pathways and should therefore be regarded as a reductionist model that requires complementary validation in more physiological settings. Second, we employed Neuro2a cells as a convenient system for mechanistic studies; however, these neuroblastoma-derived cells do not fully recapitulate the heterogeneity and differentiation potential of primary hippocampal neural progenitors. Accordingly, the findings obtained in Neuro2a cultures should be viewed as preliminary and warrant confirmation in primary neural progenitor cells or in vivo models. Despite these limitations, our study highlights a potential link between Dlk1/Notch signaling and impaired neurogenesis under chronic hypoperfusion, providing a basis for future investigations.

Cerebrovascular dysfunction is widely recognized as a central component in the pathogenesis of vascular cognitive impairment and dementia [27]. In addition to direct neuronal injury, chronic cerebral hypoperfusion can disrupt the neurovascular unit, impair cerebral blood flow regulation, and compromise BBB integrity, thereby contributing to cognitive decline [28], [29]. Previous studies have highlighted that dysfunction of the neurovascular unit and dysregulation of cerebral blood flow are key mechanisms underlying vascular cognitive impairment, while chronic hypoperfusion has been associated with endothelial damage, BBB breakdown, and impaired neurovascular homeostasis [30]. Therefore, although the present study primarily focused on Dlk1-Notch signaling and neurogenesis-related changes, further investigation of cerebrovascular impairment represents an important future direction. In particular, direct assessment of cerebral blood flow dynamics, BBB integrity, and other neurovascular parameters may provide deeper insight into how vascular pathology interacts with Dlk1-Notch signaling in the development of BCAS-induced cognitive dysfunction.

In conclusion, our study identifies an association between elevated Dlk1/Notch signaling and impaired neurogenesis with memory deficits under chronic cerebral hypoperfusion. While causality remains to be fully established, these findings support the hypothesis that aberrant niche signaling contributes to the pathophysiology of vascular dementia and warrant further mechanistic studies.

Ethics approval

All animal care and experimental procedures were conducted in accordance with the Tokyo University of Pharmaceutical Sciences Animal Experiment Regulations and Guidelines “Laboratory Animals in Universities.” The experimental protocol was reviewed and approved by the Tokyo University of Pharmaceutical Sciences Animal Care and Use Committee (No. P24-09, P25-40).

Consent for publication

All authors concur with the submission.

Significance statement

Our study suggests an association between elevated Dlk1/Notch signaling and impaired hippocampal neurogenesis with memory dysfunction under chronic cerebral hypoperfusion.

Funding

Not applicable.

CRediT authorship contribution statement

Yoshiyuki Moriyama: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. Rena Hasegawa: Formal analysis, Investigation, Methodology, Visualization. Hikaru Endo: Formal analysis, Investigation, Methodology. Sayaka Matsumura: Investigation, Methodology. Ryotaro Kaneko: Conceptualization, Methodology. Hideki Hayashi: Supervision, Validation, Writing – review & editing. Yui Iwatani: Supervision, Validation, Writing – review & editing. Norio Takagi: Conceptualization, Formal analysis, Investigation, Validation, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Misaki Hashimoto (Tokyo University of Pharmacy and Life Sciences) for technical support. We also thank Enago (www.enago.com) for English language editing.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrep.2026.102650.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.pdf (3.1MB, pdf)

Data availability

Data will be made available on request.

References

  • 1.Livingston G., Huntley J., Sommerlad A., Ames D., Ballard C., Banerjee S., Brayne C., Burns A., Cohen-Mansfield J., Cooper C., Costafreda S.G., Dias A., Fox N., Gitlin L.N., Howard R., Kales H.C., Kivimaki M., Larson E.B., Ogunniyi A., Orgeta V., Ritchie K., Rockwood K., Sampson E.L., Samus Q., Schneider L.S., Selbaek G., Teri L., Mukadam N. Dementia prevention, intervention, and care: 2020 report of the Lancet Commission. Lancet. 2020;396:413–446. doi: 10.1016/S0140-6736(20)30367-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.O'Brien J.T., Thomas A. Vascular dementia. Lancet. 2015;386:1698–1706. doi: 10.1016/S0140-6736(15)00463-8. [DOI] [PubMed] [Google Scholar]
  • 3.Cerejeira J., Lagarto L., Mukaetova-Ladinska E.B. Behavioral and psychological symptoms of dementia. Front. Neurol. 2012;3:73. doi: 10.3389/fneur.2012.00073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Wolters F.J., Ikram M.A. Epidemiology of vascular dementia. Arterioscler. Thromb. Vasc. Biol. 2019;39:1542–1549. doi: 10.1161/ATVBAHA.119.311908. [DOI] [PubMed] [Google Scholar]
  • 5.Iadecola C. The pathobiology of vascular dementia. Neuron. 2013;80:844–866. doi: 10.1016/j.neuron.2013.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Dichgans M., Leys D. Vascular cognitive impairment. Circ. Res. 2017;120:573–591. doi: 10.1161/CIRCRESAHA.116.308426. [DOI] [PubMed] [Google Scholar]
  • 7.Malouf R., Birks J. Donepezil for vascular cognitive impairment. Cochrane Database Syst. Rev. 2004;1 doi: 10.1002/14651858.CD004395.pub2. CD004395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ming G.L., Song H. Adult neurogenesis in the mammalian brain: significant answers and significant questions. Neuron. 2011;70:687–702. doi: 10.1016/j.neuron.2011.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Encinas J.M., Michurina T.V., Peunova N., Park J.H., Tordo J., Peterson D.A., Fishell G., Koulakov A., Enikolopov G. Division-coupled astrocytic differentiation and age-related depletion of neural stem cells in the adult hippocampus. Cell Stem Cell. 2011;8:566–579. doi: 10.1016/j.stem.2011.03.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Toni N., Schinder A.F. Maturation and functional integration of new granule cells into the adult Hippocampus. Cold Spring Harb Perspect Biol. 2015;8 doi: 10.1101/cshperspect.a018903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Babcock K.R., Page J.S., Fallon J.R., Webb A.E. Adult hippocampal neurogenesis in aging and alzheimer's disease. Stem Cell Rep. 2021;16:681–693. doi: 10.1016/j.stemcr.2021.01.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Moreno-Jimenez E.P., Flor-Garcia M., Terreros-Roncal J., Rabano A., Cafini F., Pallas-Bazarra N., Avila J., Llorens-Martin M. Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer's disease. Nat. Med. 2019;25:554–560. doi: 10.1038/s41591-019-0375-9. [DOI] [PubMed] [Google Scholar]
  • 13.Imayoshi I., Sakamoto M., Yamaguchi M., Mori K., Kageyama R. Essential roles of notch signaling in maintenance of neural stem cells in developing and adult brains. J. Neurosci. 2010;30:3489–3498. doi: 10.1523/JNEUROSCI.4987-09.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Surmacz B., Noisa P., Risner-Janiczek J.R., Hui K., Ungless M., Cui W., Li M. DLK1 promotes neurogenesis of human and mouse pluripotent stem cell-derived neural progenitors via modulating Notch and BMP signalling. Stem Cell Rev. Rep. 2012;8:459–471. doi: 10.1007/s12015-011-9298-7. [DOI] [PubMed] [Google Scholar]
  • 15.Shibata M., Ohtani R., Ihara M., Tomimoto H. White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke. 2004;35:2598–2603. doi: 10.1161/01.STR.0000143725.19053.60. [DOI] [PubMed] [Google Scholar]
  • 16.Oules B., Del Prete D., Greco B., Zhang X., Lauritzen I., Sevalle J., Moreno S., Paterlini-Brechot P., Trebak M., Checler F., Benfenati F., Chami M. Ryanodine receptor blockade reduces amyloid-beta load and memory impairments in Tg2576 mouse model of Alzheimer disease. J. Neurosci. 2012;32:11820–11834. doi: 10.1523/JNEUROSCI.0875-12.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Sugahara M., Nakaoki Y., Yamaguchi A., Hashimoto K., Miyamoto Y. Vitronectin is involved in the morphological transition of neurites in retinoic acid-induced neurogenesis of neuroblastoma cell line Neuro2a. Neurochem. Res. 2019;44:1621–1635. doi: 10.1007/s11064-019-02787-4. [DOI] [PubMed] [Google Scholar]
  • 18.Spalding K.L., Bergmann O., Alkass K., Bernard S., Salehpour M., Huttner H.B., Bostrom E., Westerlund I., Vial C., Buchholz B.A., Possnert G., Mash D.C., Druid H., Frisen J. Dynamics of hippocampal neurogenesis in adult humans. Cell. 2013;153:1219–1227. doi: 10.1016/j.cell.2013.05.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ables J.L., Breunig J.J., Eisch A.J., Rakic P. Not(ch) just development: notch signalling in the adult brain. Nat. Rev. Neurosci. 2011;12:269–283. doi: 10.1038/nrn3024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Louvi A., Artavanis-Tsakonas S. Notch signalling in vertebrate neural development. Nat. Rev. Neurosci. 2006;7:93–102. doi: 10.1038/nrn1847. [DOI] [PubMed] [Google Scholar]
  • 21.Wang Y., Sul H.S. Ectodomain shedding of preadipocyte factor 1 (Pref-1) by tumor necrosis factor alpha converting enzyme (TACE) and inhibition of adipocyte differentiation. Mol. Cell Biol. 2006;26:5421–5435. doi: 10.1128/MCB.02437-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Yang G., Xu S., Peng L., Li H., Zhao Y., Hu Y. The hypoxia-mimetic agent CoCl(2) induces chemotherapy resistance in LOVO colorectal cancer cells. Mol. Med. Rep. 2016;13:2583–2589. doi: 10.3892/mmr.2016.4836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Bray S.J. Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 2006;7:678–689. doi: 10.1038/nrm2009. [DOI] [PubMed] [Google Scholar]
  • 24.Kopan R., Ilagan M.X. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell. 2009;137:216–233. doi: 10.1016/j.cell.2009.03.045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Yang X., Klein R., Tian X., Cheng H.T., Kopan R., Shen J. Notch activation induces apoptosis in neural progenitor cells through a p53-dependent pathway. Dev. Biol. 2004;269:81–94. doi: 10.1016/j.ydbio.2004.01.014. [DOI] [PubMed] [Google Scholar]
  • 26.Chen M., Sun J., Lu C., Chen X., Ba H., Lin Q., Cai J., Dai J. The impact of neuronal Notch-1/JNK pathway on intracerebral hemorrhage-induced neuronal injury of rat model. Oncotarget. 2016;7:73903–73911. doi: 10.18632/oncotarget.12094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Gorelick P.B., Scuteri A., Black S.E., Decarli C., Greenberg S.M., Iadecola C., Launer L.J., Laurent S., Lopez O.L., Nyenhuis D., Petersen R.C., Schneider J.A., Tzourio C., Arnett D.K., Bennett D.A., Chui H.C., Higashida R.T., Lindquist R., Nilsson P.M., Roman G.C., Sellke F.W., Seshadri S., American Heart Association Stroke Council, Council on Epidemiology and Prevention, Council on Cardiovascular Nursing, Council on Cardiovascular Radiology and Intervention, and Council on Cardiovascular Surgery and Anesthesia Vascular contributions to cognitive impairment and dementia: a statement for healthcare professionals from the american heart association/american stroke association. Stroke. 2011;42:2672–2713. doi: 10.1161/STR.0b013e3182299496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Liu H., Zhang J. Cerebral hypoperfusion and cognitive impairment: the pathogenic role of vascular oxidative stress. Int. J. Neurosci. 2012;122:494–499. doi: 10.3109/00207454.2012.686543. [DOI] [PubMed] [Google Scholar]
  • 29.Markus H.S., de Leeuw F.E. Cerebral small vessel disease: recent advances and future directions. Int. J. Stroke. 2023;18:4–14. doi: 10.1177/17474930221144911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Di Marco L.Y., Venneri A., Farkas E., Evans P.C., Marzo A., Frangi A.F. Vascular dysfunction in the pathogenesis of Alzheimer's disease--A review of endothelium-mediated mechanisms and ensuing vicious circles. Neurobiol. Dis. 2015;82:593–606. doi: 10.1016/j.nbd.2015.08.014. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
mmc1.pdf (3.1MB, pdf)

Data Availability Statement

Data will be made available on request.

Articles from Biochemistry and Biophysics Reports are provided here courtesy of Elsevier

RESOURCES