Introduction

Alzheimer’s disease (AD) has a preclinical phase of 20–30 years prior to clinical onset and is the most common cause of dementia1,2. Amyloid-β (Aβ) peptides are produced by processing of amyloid precursor protein (APP)3,4 and have a characteristic of self-assembly into oligomers in the extracellular compartment5. Aβ1–40 and Aβ1–42 are the two major isoforms6. Aβ1–42 more readily forms oligomers than Aβ1–40 and is more neurotoxic7. Formation and propagation of misfolded aggregates of Aβ1–42 may contribute to AD pathogenesis rather than those of Aβ1–40. C-Terminal carboxylate anion of Aβ1–42, which forms the C-terminal hydrophobic core, leads to accelerate neurotoxic oligomerization8. C-Terminal Ala42 that is absent in Aβ1–40 forms a salt bridge with Lys28 and constructs a self-recognition molecular switch. The switch is the Aβ1–42-mediated self-replicating amyloid-propagation machinery9.

In AD pathogenesis, Zn2+ has been implicated by inducing rapid Aβ oligomerization10,11,12. The aggregation property of Aβ1–42 is accelerated with Zn2+, followed by much higher affinity of Aβ1–42 (Kd = ~3–30 nM) to Zn2+ than Aβ1–40 (Kd = 0.1–60 μM), which leads to neuronal Zn-Aβ1–42 uptake and then synaptic dysfunction. In the hippocampus, Aβ1–42 released into the extracellular compartment can capture Zn2+, which is estimated to be approximately 10 nM in the extracellular fluid13, even at 500 pM Aβ1–42 in vivo. We have reported that Aβ1–42 takes Zn2+ as a cargo into dentate granule cells in the normal brain, followed by cognitive decline14,15,16. Although the mechanism of Zn-Aβ1–42 uptake remains to be solved, extracellular Zn2+ is essential for Aβ1–42 uptake into dentate granule cells15.

It had been reported that Aβ oligomers form Ca2+-permeable plasma membrane pores that form Aβ channels, leading to a disruption of neuronal Ca2+ homeostasis17,18, which may be linked with synaptic dysfunction and neurodegeneration19. Aβ-mediated Ca2+ channels interact with Zn2+ and Zn2+ blocks extracellular Ca2+ influx in the range of high micromolar concentrations (>250 μM)20, although the gating kinetics and Ca2+ permeability of Aβ pores are not well understood21.

In the present study, we postulate that Zn-Aβ1–42 oligomers formed in the extracellular compartment form Zn2+-permeable plasma membrane pores, based on the evidence of permeation of Zn2+ through Ca2+ channels22,23. Here we report the mechanism on synaptic activity-independent Aβ1–42 uptake in vivo.

Results

When Aβ1–42 is bound to extracellular Zn2+ in vivo, Aβ1–42 is rapidly taken up into dentate granule cells and affects memory via attenuated LTP14,15,16. Both uptake of Aβ1–42 and Zn2+ are observed 5 min after Aβ1–42 injection into the dentate gyrus, followed by Aβ toxicity and are blocked by coinjection of CaEDTA, an extracellular Zn2+ chelator, followed by blockade of Aβ toxicity. Thus, we need to clarify the mechanism of the rapid uptake of Aβ1–42 in vivo, which is linked with Aβ toxicity. On the other hand, endogenous Zn2+ released from the hippocampal slices promotes Aβ1–42 uptake (retention), which is determined by in vitro Aβ immunohistochemistry (monoclonal antibody 4G8), in the absence of additional extracellular Zn2+ 15.

In the present study, Aβ1–42 uptake was determined in rat hippocampal slices 15 min after incubation with Aβ1–42, Aβ1–42 uptake was preferentially high in the dentate granule cell layer in the hippocampus, compared with the CA3 and CA1 pyramidal cell layer (Fig. 1).

Figure 1
figure 1

In vitro Aβ1–42 uptake in the hippocampus. Aβ immunostaining was determined in the dentate gyrus (DG), the CA3, and the CA1 15 min after incubation with 50 μM Aβ1–42 in ACSF. GCL, dentate granule cell layer; PCL, pyramidal cell layer. Aβ uptake in the GCL (n = 32), CA3 PCL (n = 22), and CA1 PCL (n = 22) was determined with Alexa 633 intensity, which was represented by the ratio to Alexa 633 intensity in the control GCL without 50 μM Aβ1–42 in ACSF expressed as 100% (lower-right). Alexa 633 intensity in the control CA3 PCL and the control CA1 PCL was also represented by the ratio to Alexa 633 intensity in the control GCL. **p < 0.01, ***p < 0.001, vs. each control with vehicle (DG, n = 29; CA3, n = 20; CA1, n = 20), ###p < 0.001, vs. DG with Aβ1–42. Bar, 50 μm.

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To examine the involvement of neuronal activity in Aβ1–42 uptake, Aβ1–42 and tetrodotoxin, a Na+ channel blocker, was co-injected into the dentate gyrus of rats. The present dose (15 μM) of tetrodotoxin was determined based on the in vivo action as a Na+ channel blocker (10 μM)24. Aβ1–42 uptake was determined by ex vivo Aβ immunohistochemistry 5 min after the co-injection. The rapid uptake of Aβ1–42 was not modified in the dentate granule cell layer even by co-injection of tetrodotoxin (Fig. 2).

Figure 2
figure 2

In vivo uptake of Aβ1–42 in the dentate gyrus in the presence of tetrodotoxin. Aβ1–42 immunostaining was determined in the dentate gyrus 5 min after injection of 50 μM Aβ1–42 (n = 6), and 50 μM Aβ1–42 + 15 μM tetrodotoxin (n = 8) in ACSF into the dentate gyrus of anesthetized rats (left). GCL, dentate granule cell layer. Bar, 50 μm. Aβ1–42 uptake in the dentate granule cell layer determined with Alexa 633 intensity, which is represented by the ratio to the control (n = 12) without 50 μM Aβ1–42 in ACSF expressed as 100%. *p < 0.05, vs. control.

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AMPA receptor activation induces extracellular Zn2+ influx in vivo, which is blocked in the presence of 2 mM CNQX, and the excess influx leads to cognitive decline via attenuated LTP25,26. The present dose of CNQX (2 mM) was used according to the previous papers. Both the rapid uptake of Aβ1–42 and Zn2+ into the dentate granule cell layer was not modified by co-injection of CNQX (Fig. 3), suggesting Aβ1–42-mediated extracellular Zn2+ influx.

Figure 3
figure 3

In vivo uptake of Aβ1–42 and Zn2+ in the dentate gyrus in the presence of CNQX. Aβ1–42 immunostaining (upper-left) and Zn2+ imaging (upper-right) were determined in the dentate gyrus 5 min after injection of 50 μM Aβ1–42, and 50 μM Aβ1–42 + 2 mM CNQX in ACSF into the dentate gyrus of anesthetized rats. GCL, dentate granule cell layer. Bar, 50 μm. Aβ1–42 uptake in the dentate granule cell layer determined with Alexa 633 intensity, which is represented by the ratio to the control (control, n = 6; Aβ, n = 5; Aβ/CNQX, n = 6) without 50 μM Aβ1–42 in ACSF expressed as 100% (lower-left). Zn2+ uptake in the dentate granule cell layer determined with ZnAF-2 intensity, which is represented by the ratio to the control (control, n = 11; Aβ, n = 10; Aβ/CNQX, n = 6) without 50 μM Aβ1–42 in ACSF expressed as 100% (lower-right). *p < 0.05, **p < 0.01, vs. control.

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We examined whether endocytosis is involved in the rapid uptake of Aβ1–42 and Zn2+. Chlorpromazine (500 μM), a clathrin/caveolae-mediated endocytosis blocker, was used based on the in vivo action as a blocker (200 μM)27. Genistein (250 μM), a caveolae/raft-mediated endocytosis blocker, was used based on the blocking effect on the primary entry pathway for aquareovirus (<200 μM)28. Both the rapid uptake of Aβ1–42 and Zn2+ into the dentate granule cell layer was not also modified by either co-injection of chlorpromazine (Fig. 4) or genistein (Fig. 5).

Figure 4
figure 4

In vivo uptake of Aβ1–42 and Zn2+ in the dentate gyrus in the presence of chlorpromazine (CPZ). Aβ1–42 immunostaining (upper-left) and Zn2+ imaging (upper-right) were determined in the dentate gyrus 5 min after injection of 50 μM Aβ1–42, and 50 μM Aβ1–42 + 500 μM chlorpromazine in ACSF into the dentate gyrus of anesthetized rats. GCL, dentate granule cell layer. Bar, 50 μm. Aβ1–42 uptake in the dentate granule cell layer determined with Alexa 633 intensity, which is represented by the ratio to the control (control, n = 11; Aβ, n = 10; Aβ/CPZ, n = 5) without 50 μM Aβ1–42 in ACSF expressed as 100% (lower-left). Zn2+ uptake in the dentate granule cell layer determined with ZnAF-2 intensity, which is represented by the ratio to the control (control, n = 10; Aβ, n = 9; Aβ/CNQX, n = 11) without 50 μM Aβ1–42 in ACSF expressed as 100% (lower-right). *p < 0.05, **p < 0.01, ***p < 0.001, vs. control.

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Figure 5
figure 5

In vivo uptake of Aβ1–42 and Zn2+ in the dentate gyrus in the presence of genistein. Aβ1–42 immunostaining (upper-left) and Zn2+ imaging (upper-right) were determined in the dentate gyrus 5 min after injection of 50 μM Aβ1–42, and 50 μM Aβ1–42 + 250 μM genistein in ACSF into the dentate gyrus of anesthetized rats. GCL, dentate granule cell layer. Bar, 50 μm. Aβ1–42 uptake in the dentate granule cell layer determined with Alexa 633 intensity, which is represented by the ratio to the control (control, n = 5; Aβ, n = 4; Aβ/genistein, n = 6) without 50 μM Aβ1–42 in ACSF expressed as 100% (lower-left). Zn2+ uptake in the dentate granule cell layer determined with ZnAF-2 intensity, which is represented by the ratio to the control (control, n = 8; Aβ, n = 5; Aβ/genistein, n = 5) without 50 μM Aβ1–42 in ACSF expressed as 100% (lower-right). *p < 0.05, vs. control.

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Discussion

In vivo LTP at medial perforant pathway-dentate granule cell synapses, which is closely linked to object recognition memory29, is not affected even under perfusion with 1,000 nM Aβ1–42 in ACSF without Zn2+, but attenuated under pre-perfusion with 500 pM Aβ1–42 in ACSF containing 10 nM Zn2+. The attenuation is rescued by extracellular Zn2+-chelation with CaEDTA. These data indicate that high picomolar Aβ1–42 captures extracellular Zn2+ and subsequently attenuates LTP15. The evidence is consistent with Aβ1–42-induced object recognition memory deficit, which is also rescued by CaEDTA and ZnAF-2DA, an intracellular Zn2+ chelator13. Thus, extracellular Zn2+ is essential for Aβ1–42-induced cognitive decline via attenuated LTP in the normal brain, consistent with in vivo complete blocking rapid uptake of Aβ1–42 and Zn2+ into dentate granule cells after co-injection of Aβ1–42 and CaEDTA into the dentate gyrus15. In the present study, we tested the uptake mechanism of Aβ1–42 oligomers in vitro and in vivo.

When Aβ1–42 uptake was determined in hippocampal slices after incubation with Aβ1–42, Aβ1–42 uptake was preferentially high in the dentate granule cell layer in the hippocampus. Aβ1–42 uptake was also considerably high in the CA3 pyramidal cell layer, while Aβ1–42 uptake was not observed in the CA1 pyramidal cell layer. Extracellular Zn2+ interaction with Aβ1–42 is essential for Aβ1–42 uptake into dentate granule cells in vivo and its essentiality is the same even under in vitro hippocampal slice condition15. Endogenous Zn2+ release from the hippocampal slices is required for the Aβ1–42 uptake. In hippocampal slices bathed in ACSF without Zn2+, extracellular Zn2+ level determined with ZnAF-2 is the highest in the hilus close to the dentate granule cell layer and the second highest in the stratum lucidum close to the CA3 pyramidal cell layer30. Endogenous Zn2+, which is released from mossy fibers, may be closely linked with the Aβ1–42 uptake. Although the Schaffer collaterals also release Zn2+, it is estimated that its release is not enough for the Aβ1–42 uptake into CA1 pyramidal cells in vitro. In contrast, it is estimated that Aβ1–42 captures extracellular Zn2+ and is taken up into CA1 pyramidal cell in vivo. In vivo CA1 LTP is impaired after intracerebroventricular injections of Aβ peptide fragments31, implying that extracellular Zn2+ is potentially involved in the impairment. Intracellular infusion of oligomerised Aβ1–42 via passive diffusion from the patch pipette induces the rapid synaptic insertion of Ca2+-permeable AMPA receptors in CA1 pyramidal cells32. When oligomeric Aβ1–42 captures extracellular Zn2+ in vivo, it is also possible that intracellular oligomeric Aβ1–42 affect neuronal functions. Postmortem studies suggest that the hippocampus and entorhinal cortex are the first brain regions to be affected in Alzheimer’s disease1,33,34. The perforant pathway-dentate granule cell synapses are vulnerable to Aβ synapse toxicity35, which may be linked with extracellular Zn2+-mediated Aβ1–42 uptake into dentate granule cells. This uptake may be associated with the high level of extracellular Zn2+ in the hilus that may be due to Zn2+ release form mossy fibers.

The mechanism of Aβ1–42 uptake was tested in the dentate granule cell layer in vivo. When Aβ1–42 was co-injected with tetrodotoxin, a Na+ channel blocker, into the dentate gyrus, the rapid uptake of Aβ1–42 was not modified in the dentate granule cell layer. AMPA receptor activation induces extracellular Zn2+ influx and the excess influx leads to cognitive decline via attenuated LTP25,26. In contrast, both the rapid uptake of Aβ1–42 and Zn2+ into the dentate granule cell layer was not modified after co-injection of CNQX, an AMPA receptor antagonist, suggesting that Zn-Aβ1–42 oligomers are ionotropic glutamate receptor activation-independently taken up into dentate granule cells. Both the rapid uptake of Aβ1–42 and Zn2+ into the dentate granule cell layer was not also modified after either co-injection of chlorpromazine or genistein, an endocytic repressor. It is likely that Zn-Aβ1–42 oligomers are rapidly taken up into dentate granule cells without interaction with plasma membrane receptor proteins.

In human SH-SY5Y neuroblastoma, monomeric Aβ1–42 is selectively internalized via clathrin- and dynamin-independent endocytosis compared to monomeric Aβ1–40 36. Genistein, a major phytoestrogen in soybean, may reduce the Aβ1–42-induced cell toxicity by suppressing the formation of toxic, cell membrane penetrating Aβ1–42 oligomers in human SH-SY5Y neuroblastoma37. Zn2+ interaction with Aβ1–42 has not been taken into account in these in vitro cell culture systems, suggesting that Zn-Aβ1–42 oligomers formed in vivo are taken up into dentate granule cells via a novel mechanism and causes cytotoxicity. On the other hand, Aβ production occurs in acidic vesicular organelles and contributes both to Aβ secretion and to the direct accumulation of Aβ within neurons37. Intracellular Aβ produced by the direct accumulation can exist as a monomeric form that further aggregates into oligomers and it may mediate pathological events38, although it is unknown whether Zn2+ is involved in the pathological effects.

The Aβ ion channel hypothesis has been reported in AD pathogenesis. These authors have reported that Aβ peptides disrupt Ca2+ homeostasis in neurons and increase intracellular Ca2+ level, resulting in synaptic dysfunction and neurodegeneration. The hypothesis is based on the results from in vitro experimental systems such as artificial membranes and neuronal culture18,39. Judging from capturing extracellular Zn2+ with high picomolar (100–500 pM) Aβ1–42 in the hippocampal extracellular compartment, which disrupts Zn2+ homeostasis in neurons and increases intracellular Zn2+ level14, the Aβ-mediated Ca2+ channel hypothesis might be changed into the Aβ1–42-mediated Zn2+ channel hypothesis in AD pathogenesis. Because Aβ stain is observed around the nuclei in dentate granule cells15, a portion of Aβ1–42 is co-taken up with Zn2+ into dentate granule cells even if Aβ1–42-mediated Zn2+ channels (pores) is formed in the plasma membrane.

In conclusion, the present study suggests that amyloid β1–42 and Zn2+ are synaptic activity-independently co-taken up into dentate granule cells in the normal brain and the co-uptake is preferential in dentate granule cells. We propose a hypothesis that Zn-Aβ oligomers formed in the extracellular compartment are directly incorporated into neuronal membranes and form Zn2+-permeable ion channels. Because extracellular Zn2+ is age-relatedly increased in the rat hippocampus40, Zn-Aβ1–42 oligomers are more readily produced in the extracellular compartment of the aged hippocampus, followed by vulnerability to Zn-Aβ1–42 oligomers in aging16,41. Therefore, controlling intracellular Zn2+ dysregulation may be an effective strategy for overcoming AD pathogenesis.

Experimental Procedures

Animals and chemicals

Wistar rats (male, 7–9 weeks of age) were obtained from Japan SLC (Hamamatsu, Japan). Rats were housed under the standard laboratory conditions (23 ± 1 °C, 55 ± 5% humidity) and had access to water and food ad libitum. Rats were used for experiments approximately 1 week after housing. All the experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals in the University of Shizuoka, which 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 present study has been approved by the Ethics Committee for Experimental Animals in the University of Shizuoka.

Synthetic human Aβ1–42 was purchased from ChinaPeptides (Shanghai, China). Aβ was dissolved in saline at the time of need. Aβ1–42 prepared in saline was mainly monomers with a small fraction of low order oligomers in SDS-PAGE14. ZnAF-2DA (Kd = 2.7 × 10−9 M for Zn2+) is a membrane-permeable Zn2+ indicator and was kindly supplied from Sekisui Medical Co., LTD (Hachimantai, Japan). This indicator is taken up into the cells through the plasma membrane and is hydrolyzed by esterase in the cytosol to produce ZnAF-2, which does not permeate the plasma membrane42,43. ZnAF-2 is selectively bound to Zn2+, but not bound to other divalent cations such as Ca2+, Mg2+, and Cu2+ 42. Calcium Orange AM is a membrane-permeable Ca2+ indicator and was purchased from Molecular Probes, Inc. (Eugene, OR). The fluorescence indicators were dissolved in dimethyl sulfoxide and diluted to artificial cerebrospinal fluid (ACSF) 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).

Hippocampal slice preparation

Rats were anesthetized with ether and decapitated under anesthesia. The brain was quickly removed from rats and bathed in ice-cold choline-ACSF, which consists of 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 block excessive neuronal excitation. Horizontal hippocampal slices (400 μm) were prepared in ice-cold choline-ACSF by using a vibratome ZERO-1 (Dosaka Kyoto, Japan). Hippocampal slices were placed in ACSF at 25 °C for at least 30 minutes. All solutions were continuously bubbled with 95% O2 and 5% CO2.

In vitro immunostaining

Hippocampal slices were bathed in 50 μM Aβ1–42 in ACSF for 15 min. Slices were then rinsed twice with ACSF for 5 min to remove extracellular agents, and fixed with paraformaldehyde (4% in 0.01 M PBS) for 15 min. Slices were rinsed in 0.01 M PBS three times. Slice tissues were blocked in 10% normal goat serum for 30 min, followed by rinse in 0.01 M PBS three times, incubated with 70% formic acid for 5 min, rinsed with 0.01 M PBS three times, and bathed at 4 °C in Aβ monoclonal antibody, 4G8 (COVANCE, 1:500 dilution in 0.01 M PBS) for 48 h. Slices were then rinsed with 0.01 M PBS three times, bathed in Alexa Fluor 633 goat anti-mouse IgG secondary antibody (1: 200 dilution in 0.01 M PBS) for 1 h, rinsed with 0.01 M PBS three times, bathed in 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, rinsed again with 0.01 M PBS three times, and mounted on glass slides. Immunostaining images were obtained by using a confocal laser-scanning microscopic system (Nikon A1 confocal microscopes, Nikon Corp.) through a 10× objective. Florescence intensity was analyzed by the NIH Image J.

Ex vivo immunostaining

Rats anesthetized with chloral hydrate (400 mg/kg) were placed in a stereotaxic apparatus. The skull was exposed and two burr holes were drilled. Injection cannulae (internal diameter, 0.15 mm; outer diameter, 0.35 mm) were bilaterally inserted into the dentate granule cell layer (4.0 mm posterior to the bregma, 2.3 mm lateral, 2.9 mm inferior to the dura) of the anesthetized rats. Thirty minutes later following recovery from the insertion damage, 50 μM Aβ1–42 in ACSF, or 50 μM Aβ1–42 with either 15 μM tetrodotoxin, a Na2+ channel blocker, 2 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, 500 μM chlorpromazine, a clathrin/caveolae-mediated endocytosis blocker, or 250 μM genistein, a caveolae/raft-mediated endocytosis blocker, in ACSF were bilaterally injected into the dentate granule cell layer at the rate of 0.25 μl/min for 8 min via the injection cannulae. Five minutes later, the brain was quickly removed from the rats and immunostaining images in hippocampal slices were obtained in the same manner except for changing the 10% goat serum with 5% goat serum. The positions of the injection cannulae were confirmed in the slice preparation.

In vivo intracellular Zn2+ imaging

Fifty μM Aβ1–42 in ACSF containing 100 μM ZnAF-2DA, or 50 μM Aβ1–42 with either 15 μM tetrodotoxin, 2 mM CNQX, 500 μM chlorpromazine, or 250 μM genistein in ACSF containing 100 μM ZnAF-2DA was bilaterally injected via injection cannulae into the dentate granule cell layer of anesthetized rats at the rate of 0.25 μl/min for 8 min. Five minutes later, the hippocampal slices were prepared in the same manner. Slices were transferred to a chamber filled with ACSF, loaded with 2 μM Calcium Orange AM in ACSF for 30 min, and then rinsed in ACSF for 30 min. The hippocampal slices were transferred to a recording chamber filled with ACSF. The fluorescence of ZnAF-2 (laser, 488.4 nm; emission, 500–550 nm), and calcium orange (laser, 561.4 nm; emission, 570–620 nm) was measured with a confocal laser-scanning microscopic system (Nikon A1 confocal microscopes, Nikon Corp.). Calcium Orange AM was used to identify hippocampal regions in slices. The positions of the injection cannulae were confirmed in the slice preparation.

Data analysis

For multiple comparisons, differences between treated groups were analyzed by one-way ANOVA followed by post hoc testing using the Tukey’s test. A value of p < 0.05 was considered significant (the statistical software, GraphPad Prism 5). Data were expressed as means ± standard error. The control group with vehicle was compared with the treated groups in all figures for the statistical analysis. In Fig. 1, Aβ group in the DG was also compared with those in the CA3 and the CA1.

Ethics statement

All experiments were done according to the Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka, which refer to 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. All experimental protocols were approved by the ethics committee of the University of Shizuoka