Introduction

Age-related cognitive decline is believed to be initially due to changes in synaptic function rather than loss of neurons [1], while substantial synaptic and neuronal losses are observed in the early stage of Alzheimer’s disease (AD) when hippocampus-dependent memory loss becomes detectable clinically [2, 3]. The dentate gyrus and the entorhinal cortex are vulnerable to aging and AD, respectively [4]. The perforant pathway from the entorhinal cortex innervates dentate granule cells and is one of the earliest and most severely affected pathways in AD [2, 5], suggesting that the cellular disconnection between the entorhinal cortex and the dentate gyrus is involved in the AD pathogenesis. Elderly persons with mild cognitive impairment (MCI), which is a prodromal state of AD [6], have approximately 30% fewer neurons in the entorhinal cortex, which correlate with hippocampus-dependent cognitive decline [2, 5]. Therefore, dentate granule cell degeneration is a target for defending the AD pathogenesis, and its defending strategy may be useful for progressive cognitive decline.

The basal level of extracellular Zn2+ is in the range of low nanomolar concentrations (~10 nM) in the hippocampus [7] and probably increased age-dependently [8, 9]. Extracellular Zn2+ dynamics, which is often linked with Zn2+ release from zincergic terminals, a subclass of glutamatergic neurons, plays a key role for not only synaptic plasticity, a cellular mechanism of cognition but also synaptic dysfunction associated with cognitive decline [10, 11]. The basal level of intracellular Zn2+ is estimated to be less than 1 nM (~100 pM) [12, 13], and rapid intracellular Zn2+ dysregulation is critical for not only cognitive decline but also neurodegeneration. Weakened intracellular Zn2+-buffering is linked with age-related characteristic of extracellular Zn2+ influx in the aged dentate gyrus followed by cognitive decline [14, 15] and neurodegeneration [16].

Rapid intracellular Zn2+ dysregulation is due to extracellular Zn2+ influx via excess activation of glutamate receptors, glutamate excitotoxicity, while amyloid-β (Aβ), a causative candidate for the AD pathogenesis [17, 18], also induces rapid intracellular Zn2+ dysregulation. Aβ1–42 is dynamically released from synaptic vesicles [19] and rapidly captures Zn2+ at 100–500 picomolar concentration in the hippocampus extracellular fluid. Both the levels of Zn2+ and Aβ1–42 are increased in dentate granule cells 5 min after injection of Aβ1–42 into the dentate granule cell layer of rats, followed by transient Aβ1–42-induced cognitive decline that is due to increase in intracellular Zn2+ released from Aβ1–42[20, 21]. Intracellular Zn2+ dysregulation via synaptic activity-independent mechanism occurs rapidly after injection of human Aβ1–42 into rat dentate granule cell layer [22] but not after injection of human Aβ1–40 and mouse/rat Aβ1–42 because affinity of human Aβ1–42 to Zn2+ is much higher than human Aβ1–40[21] and mouse/rat Aβ1–42[23, 24], which cannot capture extracellular Zn2+ at low nanomolar concentration.

On the basis of the idea that more Zn2+ ferried by Aβ1–42 into dentate granule cells leads to degeneration in addition to cognitive decline, here, we examined whether Aβ1–42-induced intracellular Zn2+ dysregulation causes neurodegeneration in the hippocampus and also whether blocking the intracellular Zn2+ dysregulation is a useful defense strategy against Aβ1–42-induced neurodegeneration.

Materials and Methods

Animals and Chemicals

Male ddY mice (10 weeks of age) were purchased from Japan SLC (Hamamatsu, Japan). All the experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka that refer to the American Association for Laboratory Animals Science and the guidelines laid down by the NIH (NIH Guide for the Care and Use of Laboratory Animals) in the USA. The Ethics Committee for Experimental Animals in the University of Shizuoka has approved this work.

Synthetic human Aβ1–42 was purchased from ChinaPeptides (Shanghai, China). Aβ1–42 was dissolved in saline and used immediately when the experiments were performed. SDS-PAGE showed that Aβ1–42 prepared in saline was mainly monomers with a small fraction of low order oligomers [20]. ZnAF-2DA, a membrane-permeable zinc indicator was kindly supplied from Sekisui Medical Co., LTD (Hachimantai, Japan). ZnAF-2DA is taken up into the cells through the cell membrane and is hydrolyzed by esterase in the cytosol to yield ZnAF-2 (Kd = 2.7 × 10−9 M for Zn2+), which cannot permeate the cell membrane [25, 26]. The fluorescence indicator was dissolved in dimethyl sulfoxide (DMSO) and then diluted with Ringer solution containing 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, and 11 mM D-glucose (pH 7.3).

Intracerebroventricular (ICV) Injection of Aβ

Mice (11–13 weeks of age) were anesthetized with chloral hydrate (30 mg/kg) and placed in a stereotaxic apparatus. A microinjection canula (CXG-6, Eicom Co., Kyoto) was positioned 0.5 mm posterior to the bregma, 1.0 mm lateral, 2.2–2.4 mm inferior to the dura for ICV injection. Aβ1-42 in saline (25 μM) was injected via the microinjection canula at the rate of 0.5 μl/min for 40 min (500 pmol/mouse). Ten minutes later, the microinjection canula was slowly pulled up from the brain. The mice were individual housed for the experiments□.

Object Recognition Test

Twelve days after ICV injection of Aβ1-42, mice were placed for 10 min into an open field, which was a 56 × 67.5 cm arena surrounded by 60 cm high walls, made of a black-colored plastic. Twenty-four hours after open field exploration, mice were trained and tested in a novel object recognition task. Training in the object recognition task took place in the same area used for the open field exploration. The open field exploration was thus used as a context habituation trial for the recognition memory task. The object recognition test requires that the mice recall which of two earthenware objects they had been previously familiarized with. Twenty-four hours after arena exploration, training was conducted by placing individual mice into the field, in which two identical objects (objects A1 and A2) were positioned in two adjacent corners, 13 cm from the walls. Mice were left to explore the objects for 5 min. Mice were not used for the test when the total of the object exploration time was less than 10 s. In the test given 1 h after training, the mice explored the open field for 3 min in the presence of one familiar (A) and one novel (B) object. Behavior of mice was recorded with a video camera during the training and the test, and then two persons independently measured exploratory time and the averaged time was used. All objects presented similar textures, colors, and sizes but distinctive shapes. A recognition index calculated for each mouse was expressed by the ratio TB/(TA + TB) [TA = time spent to explore the familiar object A; TB = time spent to explore the novel object B]. Between trials the objects were washed with 70% ethanol solution. Exploration was defined as sniffing or touching the object with the nose and/or forepaws. We confirmed that there was no preference for the objects used.

Propidium Iodide (PI) Staining

Neurodegeneration was determined by PI staining after the object recognition test was finished. Fourteen days after ICV injection of Aβ1-42, the brain was quickly removed from the mice under anesthesia and immersed in ice-cold choline-Ringer containing 124 mM choline chloride, 2.5 mM KCl, 2.5 mM MgCl2, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose (pH 7.3) to suppress excessive neuronal excitation. Coronal brain slices (400 μm) were prepared using a vibratome ZERO-1 (Dosaka Kyoto, Japan) in ice-cold choline-Ringer, which were continuously bubbled with 95% O2 and 5% CO2. The brain slices were bathed in PI in Ringer solution (7 μg/ml) for 30 min, bathed in Ringer solution for 30 min, and transferred to a recording chamber filled with Ringer solution. PI fluorescence (Ex/Em: 535 nm/617 nm) was captured with a confocal laser-scanning microscopic system. The region of interest was set in the dentate granule cell layer and the CA1 and CA3 pyramidal cell layers.

Fluoro-Jade B (FJB) Staining

Neurodegeneration was also determined by FJB staining after the object recognition test was finished. Fourteen days after ICV injection of Aβ1-42, the mice were anesthetized with chloral hydrate and perfused with ice-cold 4% paraformaldehyde in PBS, followed by removal of the brain and overnight fixation in 4% paraformaldehyde in PBS at 4 °C. Fixed brains were cryopreserved in 30% sucrose in PBS for 2 days and frozen in Tissue-Tek Optimal Cutting Temperature embedding medium. Coronal brain slices (30 μm) were prepared at − 20 °C in a cryostat, picked up on slides, adhered at 50 °C for 60 min, and stored at − 20 °C. The slides were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol (20 ml of 5% NaOH added to 80 ml ethanol) for 5 min. This was followed by 2 min in 70% ethanol and 2 min in distilled water. The slides were then transferred to a solution of 0.06% potassium permanganate for 15 min, preferably on a shaker table to insure consistent background suppression between slices. The slides were then rinsed in distilled water for 2 min. The staining solution was prepared from a 0.01% stock solution of FJB that was made by adding 10 mg of the dye powder to 100 ml of distilled water. The stock solution and 0.1% 4′,6-Diamidino-2-phenylindole (DAPI) in distilled water were diluted with 0.1% acetic acid vehicle, resulting in a final dye concentration of 0.0004% FJB and 0.0001% DAPI in the staining solution. The staining solution was prepared within 10 min of use. The slides were bathed in the staining solution for 30 min and were rinsed for 2 min in each of three distilled water washes. Excess water was briefly removed by using a paper towel. The slides were placed at 50 °C for drying. The dry slides were twice immersed in xylene for 2 min twice before coverslipping with DPX, a non-aqueous, non-fluorescent plastic mounting media. FJB-positive cells in the unit area were measured with a confocal laser-scanning microscopic system (Ex/Em: 480 nm/525 nm). The region of interest was set in the dentate granule cell layer and the CA1 and CA3 pyramidal cell layers.

In Vivo Aβ1-42-Mediated Zn2+Dynamics

Aβ1–42 (25 μM) in saline containing 100 μM ZnAF-2DA was intracerebroventricularly injected via a microinjection canula at the rate of 0.5 μL/min for 40 min (500 pmol/mouse) into anesthetized mice as described above. One hour after the start of injection, coronal brain slices (400 μm) were prepared in ice-cold choline-Ringer solution in the same manner and transferred to a recording chamber filled with Ringer solution. The fluorescence of ZnAF-2 (laser, 488 nm; emission, 505–530 nm) was captured with a confocal laser-scanning microscopic system.

In another experiment, saline and dexamethasone sodium phosphate (10 mg/kg) in saline were intraperitoneally (i.p.) injected into mice once a day for two days. Twenty-four hours later, Aβ1–42 (25 μM) in saline containing 100 μM ZnAF-2DA was intracerebroventricularly injected, and the fluorescence of ZnAF-2 was captured in the same manner.

Data Analysis

Student’s paired t test was used for comparison of the means of paired data. For multiple comparisons, differences between treatments were assessed by one-way ANOVA followed by post hoc testing using the Tukey’s test (the statistical software, GraphPad Prism 5). A value of p < 0.05 was considered significant. Data were expressed as means ± standard error. The results of statistical analysis are described in each figure legend.

Results

Preferential Neurodegeneration in the Dentate Gyrus by Aβ1–42

To assess hippocampal neurodegeneration, we used PI, a fluorescent intercalating agent, which binds to DNA by intercalating between the bases with little or no sequence preference in dead cells, and FJB, an anionic fluorescein derivative, which is used for the histological staining of neurons undergoing degeneration [27, 28].

PI fluorescence was enhanced in the dentate granule cell layer 14 days after ICV injection of Aβ1-42 but not in the CA1 and CA3 pyramidal cell layers (Fig. 1). FJB-positive cells were also increased in the dentate granule cell layer but not in the CA1 and CA3 pyramidal cell layers (Fig. 2). On the other hand, intracellular Zn2+ level, which was determined with intracellular ZnAF-2 fluorescence, was extensively increased in the hippocampal CA1 and CA3, in addition to the dentate gyrus (Fig. 3).

Fig. 1
figure 1

A single ICV injection of Aβ1–42 induces neurodegeneration in the dentate gyrus. a Fourteen days after ICV injection of Aβ1-42, PI fluorescence was measured in the dentate granule cell layer (DGCL), the CA1 pyramidal cell layer (CA1 PCL), and the CA3 pyramidal cell layer (CA3 PCL) surrounded by the dotted line (upper). Bar, 50 μm. b Each bar and line (mean ± SEM) represent the rate (%) of PI fluorescence after Aβ1–42 injection to that after saline (vehicle) injection, which was represented as 100% (lower). ***p < 0.001 vs. saline (t test). DGCL; saline, n = 17, Aβ1–42 = 23; CA1, saline, n = 21, Aβ1–42 = 30; CA3, saline, n = 13, Aβ1–42 = 28

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Fig. 2
figure 2

A single ICV injection of Aβ1–42 induces neurodegeneration in the dentate gyrus. a Fourteen days after ICV injection of Aβ1-42, FJB fluorescence was measured in the dentate granule cell layer (DGCL), the CA1 pyramidal cell layer (CA1 PCL), and the CA3 pyramidal cell layer (CA3 PCL) surrounded by the dotted line. Bar, 50 μm. b The area surrounded by the white line, which was shown in a, was magnified. c Each bar and line (mean ± SEM) represent FJB-positive cells in the unit area after injection of vehicle and Aβ1-42. ***p < 0.001 vs. saline (t test). DGCL; saline, n = 16, Aβ1–42 = 16; CA1, saline, n = 16, Aβ1–42 = 16; CA3, saline, n = 16, Aβ1–42 = 16

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Fig. 3
figure 3

A single ICV injection of Aβ1–42 increases intracellular Zn2+ level in the hippocampus. a Fourteen days after ICV injection of Aβ1-42, ZnAF-2 fluorescence was measured in the dentate granule cell layer (DGCL), the CA1 pyramidal cell layer (CA1 PCL), and the CA3 pyramidal cell layer (CA3 PCL) surrounded by the dotted line (upper). Bar, 50 μm. b Each bar and line (mean ± SEM) represent the rate (%) of ZnAF-2 fluorescence after Aβ1–42 injection to that in the DGCL after saline (vehicle) injection, which was represented as 100% (lower). *p < 0.05; **p < 0.001 vs. saline (t test). DGCL; saline, n = 7, Aβ1–42 = 6; CA1, saline, n = 7, Aβ1–42 = 6; CA3, saline, n = 6, Aβ1–42 = 6

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Rescue of Neurodegeneration by Reducing Aβ1–42-Induced Increase in Intracellular Zn2+ in the Dentate Gyrus

The enhanced PI fluorescence and FJB-positive cells were reduced in the dentate granule cell layer after co-injection of extracellular and intracellular Zn2+ chelators, i.e., CaEDTA and ZnAF-2DA, respectively (Figs. 4 and 5). CaEDTA competes with Aβ1–42 for binding to Zn2+ and reduces the formation of Zn-Aβ1–42 complexes, resulting in reducing both the uptake of Zn2+ and Aβ1–42[21]. Extracellular ZnAF-2DA also competes with Aβ1–42 for binding to Zn2+ and reduces the formation of Zn-Aβ1–42 complexes, while intracellular ZnAF-2 taken up into cells chelates Zn2+ released from Aβ1–42[20, 21].

Fig. 4
figure 4

Zn2+ chelators rescue Aβ1–42-induced neurodegeneration. Fourteen days after ICV injection of Aβ1-42 and Zn2+ chelators, PI fluorescence was measured in the dentate granule cell layer (DGCL) surrounded by the dotted line (upper). Bar, 50 μm. Each bar and line (mean ± SEM) represent the rate (%) of PI fluorescence after Aβ1–42 injection to that after saline (vehicle) injection, which was represented as 100% (lower). ***p < 0.001 vs. saline; #p < 0.05, ##p < 0.01 vs. Aβ1–42 (Tukey’s test). Saline, n = 17, Aβ1–42, n = 23; Aβ1–42 + CaEDTA, n = 16, Aβ1–42 + ZnAF-2DA, n = 18

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Fig. 5
figure 5

Zn2+ chelators rescue Aβ1–42-induced neurodegeneration. Fourteen days after ICV injection of Aβ1-42 and Zn2+ chelators, FJB fluorescence was measured in the dentate granule cell layer (DGCL) surrounded by the dotted line (upper). Bar, 50 μm. Each bar and line (mean ± SEM) represent FJB-positive cells in the unit area after injection of vehicle and Aβ1-42 (lower). ***p < 0.001 vs. saline; ###p < 0.001 vs. Aβ1–42 (Tukey’s test). Saline, n = 16, Aβ1–42, n = 19; Aβ1–42 + CaEDTA, n = 16, Aβ1–42 + ZnAF-2DA, n = 16

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Newly synthesized metalothioneins (MTs), Zn2+-binding proteins can capture Zn2+ released from intracellular Aβ1–42 without modifying intracellular Aβ1–42 level followed by rescuing Aβ1–42 neurotoxicity [29], indicating that Zn2+ released from Aβ1–42 is more neurotoxic than Aβ1–42 itself. Pretreatment with dexamethasone, an inducer of MTs reduced not only the enhanced PI fluorescence and FJB-positive cells in the dentate granule cell layer (Figs. 6 and 7) but also Aβ1–42-induced increase in intracellular Zn2+ in the dentate gyrus (Fig. 8).

Fig. 6
figure 6

Dexamethasone rescues Aβ1–42-induced neurodegeneration. Saline and dexamethasone (DEX, 10 mg/kg) in saline were i.p. injected into mice once a day for two days. Twenty-four hours later, Aβ1–42 (25 μM) in saline was intracerebroventricularly injected and PI fluorescence was measured in the dentate granule cell layer (DGCL) surrounded by the dotted line after 14 days after ICV injection (upper). Bar, 50 μm. Each bar and line (mean ± SEM) represent the rate (%) of PI fluorescence after Aβ1–42 injection to that after saline (vehicle) injection, which was represented as 100% (lower). **p < 0.01 vs. saline/saline; #p < 0.05, ##p < 0.01 vs. saline/Aβ1–42 (Tukey’s test). Saline/saline, n = 12, saline/Aβ1–42, n = 9; DEX/saline, n = 15, DEX/Aβ1–42, n = 16

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Fig. 7
figure 7

Dexamethasone rescues Aβ1–42-induced neurodegeneration. Saline and dexamethasone (DEX, 10 mg/kg) in saline were i.p. injected into mice once a day for two days. Twenty-four hours later, Aβ1–42 (25 μM) in saline was intracerebroventricularly injected and FJB fluorescence was measured in the dentate granule cell layer (DGCL) surrounded by the dotted line 14 days after ICV injection (upper). Bar, 50 μm. Each bar and line (mean ± SEM) represent FJB-positive cells in the unit area after injection of vehicle and Aβ1-42 (lower). **p < 0.01 vs. saline/saline; #p < 0.05 vs. saline/Aβ1–42 (Tukey’s test). Saline/saline, n = 20, saline/Aβ1–42, n = 23, DEX/saline, n = 12, DEX/Aβ1–42, n = 14

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Fig. 8
figure 8

Dexamethasone reduces Aβ1–42-induced increase in intracellular Zn2+. Saline and dexamethasone (DEX, 10 mg/kg) in saline were i.p. injected into mice once a day for two days. Twenty-four hours later, Aβ1–42 (25 μM) in saline was intracerebroventricularly injected and ZnAF-2 fluorescence was measured in the dentate granule cell layer (DGCL) surrounded by the dotted line 1 h after the start of ICV injection (upper). Bar, 50 μm. Each bar and line (mean ± SEM) represent the rate (%) of ZnAF-2 fluorescence after Aβ1–42 injection to that in the DGCL after saline (vehicle) injection, which was represented as 100% (lower). ***p < 0.001 vs. saline/saline; ###p < 0.001 vs. saline/Aβ1–42 (Tukey’s test). Saline/saline, n = 7, saline/Aβ1–42, n = 6; DEX/saline, n = 8, DEX/Aβ1–42, n = 7

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Rescue of Cognitive Decline by Zn2+Chelators and Dexamethasone

Object recognition memory was impaired after ICV injection of Aβ1-42, while the impairment was rescued by co-injection of CaEDTA and ZnAF-2DA (Recognition index: saline/training, 49.1 ± 1.5%, saline/test, 64.3 ± 2.9, p < 0.01; Aβ1–42/training, 49.6 ± 3.6%, Aβ1–42/test, 48.6 ± 2.2%; Aβ1–42 + CaEDTA/training, 49.6 ± 2.4%, Aβ1–42 + CaEDTA/test, 68.3 ± 3.5%, p < 0.01; Aβ1–42 + ZnAF-2DA/training, 50.0 ± 2.5%, Aβ1–42 + ZnAF-2DA/test, 67.6 ± 3.1%, p < 0.01) (Fig. 9a). The impairment was also rescued by pretreatment with dexamethasone (Recognition index: saline/saline/training, 49.1 ± 1.3%, saline/saline/test, 64.1 ± 2.8, p < 0.01; saline/Aβ1–42/training, 49.6 ± 2.6%, saline/Aβ1–42/test, 49.6 ± 1.8%; dexamethasone (DEX)/saline/training, 45.3 ± 3.7%, DEX/saline/test, 63.9 ± 3.5%, p < 0.01; DEX/Aβ1–42/training, 50.2 ± 1.2%, DEX/Aβ1–42/test, 62.0 ± 2.2%, p < 0.05) (Fig. 9b).

Fig. 9
figure 9

Zn2+ chelators and dexamethasone rescue Aβ1–42-induced cognitive decline. a Thirteen days after ICV injection of Aβ1-42, object recognition test was performed. Each bar and line (mean ± SEM) represent the recognition index (%) in the test 1 h after training. *p < 0.05 vs. saline, ##p < 0.01 vs. Aβ1–42 (Tukey’s test). Saline, n = 6, Aβ1–42, n = 6; Aβ1–42 + CaEDTA, n = 7, Aβ1–42 + ZnAF-2DA, n = 7. b Saline and dexamethasone (DEX, 10 mg/kg) in saline were i.p. injected into mice once a day for two days. Twenty-four hours later, Aβ1–42 (25 μM) in saline was intracerebroventricularly injected and object recognition test was performed 13 days after ICV injection. **p < 0.01 vs. saline/saline; #p < 0.05, ##p < 0.01 vs. saline/Aβ1–42(Tukey’s test). Saline/saline, n = 9, saline/Aβ1–42, n = 11; DEX/saline, n = 6, DEX/Aβ1–42, n = 7

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Discussion

On the basis of the rapid oligomerization of Aβ, the toxic action of soluble low-molecular-weight Aβ oligomers has been emphasized in the AD pathogenesis. Aβ1–40 and Aβ1–42 are the two most abundant isoforms, and the former is more than 10 times as abundant as the latter in the cerebrospinal fluid [30]. Importantly, Aβ1–42 far more rapidly forms aggregates and is more neurotoxic than Aβ1–40[31,32,33,34]. The neurotoxic action of low-molecular-weight Aβ oligomers has been tested in not only primary neuronal cultures or brain slices but also animal models. To determine the action of Aβ1–42 in cognitive decline and neurodegeneration in vivo, Aβ1–42 oligomers have been exclusively prepared in vitro [35, 36] and injected into experimental animals to test the neurotoxicity in vivo [37,38,39,40].

On the other hand, Zn2+ has been implicated in the AD pathogenesis because Zn2+ readily accelerates Aβ oligomerization [41, 42]. The trial for drug therapy targeting metal ions, e.g., Zn2+ has been reported for AD [43]. As a matter of fact, Zn2+ has remarkable impacts on Aβ1–42 aggregation in the range of physiological pH [44]. Zn2+ shifts the self-association of Aβ1–42 toward a non-fibrillary pathway by interfering with the aggregation process at multiple levels [45]. Judging from these findings, it is important to evaluate neurotoxicity of soluble Aβ1–42 oligomers, which is rapidly formed in the presence of extracellular Zn2+ in vivo. It is estimated that chemical structures of soluble Aβ1–42 oligomers with Zn2+ are different from those of soluble Aβ1–42 oligomers prepared without Zn2+ in vitro, which might lead to misunderstanding of Aβ1–42 neurotoxicity in vivo. On the basis of rapid intracellular Zn2+ dysregulation in dentate granule cells induced by Aβ1–42 in vivo [21], we tested the idea that rapid intracellular Zn2+ dysregulation induced by Aβ1–42 causes neurodegeneration in the hippocampus after a single injection of Aβ1–42 into the lateral ventricle.

Rapid increase in intracellular Zn2+ induced by Aβ1–42 was extensively observed in the hippocampus 20 min after finishing a single Aβ1–42 injection into the lateral ventricle. In contrast, neurodegeneration was preferentially observed in the dentate gryrus 14 days after ICV injection but not in the CA1 and CA3. When Aβ1–42 concentration reaches 100–500 pM, Aβ1–42 is taken up into dentate granule cells extracellular Zn2+-dependently. The formation of Zn-Aβ1–42 complexes in the extracellular compartment is critical for Aβ1–42 uptake [21], which is the highest in dentate gyrus neurons in hippocampal slices in vitro [22], consistent with the highest level of intracellular Zn2+ in the dentate gyrus in the present study. These data suggest that the preferential uptake of Aβ1–42 into dentate gyrus neurons causes the preferential neurodegeneration. The perforant pathway from the entorhinal cortex innervates dentate granule cells and is one of the earliest and most severely affected pathways in AD [2, 5]. Therefore, vulnerability of dentate granule cells in the AD pathogenesis is probably due to the preferential uptake of Aβ1–42, which might be linked with Aβ1–42 degradation and its elimination from the dentate gyrus [46]. However, the relationship between the preferential Aβ1–42 uptake and Aβ1–42 metabolism in the dentate gyrus remains to be clarified.

Intracellular Zn2+ level was also considerably high in CA3 and CA1 neurons, while neurodegeneration was scarcely observed in the CA3 and CA1. The high Zn2+ levels might be linked with less Aβ1–42 uptake into CA3 and CA1 neurons [22] followed by less vulnerability of CA3 and CA1 neurons to neurodegeneration. On the other hand, it is estimated that Zn2+ released from intracellular Zn-Aβ1–42 complexes taken up into dentate gyrus neurons causes neurodegeneration. This estimation was confirmed using Zn2+ chelators (Figs. 4 and 5). Extracellular Zn2+ chelators reduce the formation of Zn-Aβ1–42 complexes in the extracellular compartment and both the uptake of Aβ1–42 and Zn2+ followed by reducing Aβ1–42 toxicity[20]. Intracellular Zn2+ chelators do not reduce the formation of Zn-Aβ1–42 complexes in the extracellular compartment, while they capture Zn2+ released from intracellular Zn-Aβ1–42 complexes followed by reducing Aβ1–42 toxicity[20]. In the present study, neurodegeneration in the dentate gyrus was rescued after co-injection of extracellular and intracellular Zn2+ chelators, i.e., CaEDTA and ZnAF-2DA, respectively. Aβ1–42-induced cognitive impairment was also rescued by co-injection of CaEDTA and ZnAF-2DA. Intracellular Zn-Aβ1–42 complexes taken up into dentate gyrus neurons release toxic Zn2+, while Aβ1–42 may capture functional Zn2+ after the release, followed by neurodegeneration via dysfunction of Zn2+-requiring proteins such as a metabotropic Gq-coupled Zn2+-sensing receptor, mZnR/GPR39[47].

Intracellular MTs buffer free Zn2+ based on its varying affinities for Zn2+[48,49,50]. If intracellular Zn2+ concentration reaches approximately 10 nM, which is the estimated concentration of extracellular Zn2+[7], MTs bind up to 7 equivalents of Zn2+ and become the holo-MTs, Zn7MTs [51]. When the binding capacity of MTs is saturated by the rapid increase in intracellular Zn2+, which is induced by extracellular Aβ1–42 influx, intracellular MTs lose the Zn2+-buffering ability. Therefore, it is thought that an increase in intracellular MTs is effective for buffering the rapid increase in intracellular Zn2+ by Aβ1–42. In vivo Kd value of Zn2+ to Aβ1–42 may be in the range of ~3–30 nM [21], which is higher than that of MTs (~1 pM) [52]. Pretreatment with dexamethasone, an inducer of MT-I and MT-II, reduced neurodegeneration in the dentate granule cell layer and rescued cognitive impairment via blocking the rapid increase in intracellular Zn2+ induced by Aβ1–42. Because intracellular MTs induced by dexamethasone does not reduce the uptake of Zn-Aβ1–42, the induced MTs, which capture Zn2+ released from Aβ1–42, reduce intracellular Zn2+ levels but not modify intracellular Aβ1–42 levels [29]. In contrast, dexamethasone may reduce the increase in intracellular Zn2+ induced by Aβ1–42 via another mechanism because dexamethasone has a great many effects. In addition to the effect of Zn2+ chelators, the effect of dexamethasone indicates that neurodegeneration and cognitive decline induced by Aβ1–42 are due to intracellular Zn2+ toxicity, which is caused by release from Aβ1–42.

The present study indicates that rapid intracellular Zn2+ dysregulation induced by Aβ1–42 preferentially causes neurodegeneration in the dentate gryrus, resulting in hippocampus-dependent cognitive decline. It is likely that controlling intracellular Zn2+ dysregulation, which is induced by the rapid uptake of Zn-Aβ1–42 complexes, is a defense strategy for the AD pathogenesis.