Age-Dependent Modification of Intracellular Zn²⁺ Buffering in the Hippocampus and Its Impact

Abstract

The basal concentrations of extracellular Zn²⁺ and intracellular Zn²⁺, which are approximately 10 nM and 100 pM, respectively, in the brain, are markedly lower than those of extracellular Ca²⁺ (1.3 mM) and intracellular Ca²⁺ (100 nM), respectively, resulting in much less attention paid to Zn²⁺ than to Ca²⁺. However, intracellular Zn²⁺ dysregulation, which is closely linked with glutamate- and amyloid β-mediated extracellular Zn²⁺ influx, is more critical for cognitive decline and neurodegeneration than intracellular Ca²⁺ dysregulation. It is estimated that the age-dependent increase in the basal concentration of extracellular Zn²⁺ in the hippocampus plays a key role in cognitive decline and neurodegeneration. The characteristics of extracellular Zn²⁺ influx in the hippocampus may be modified age-dependently, probably followed by modification of intracellular Zn²⁺ buffering that is closely linked with age-related cognitive decline and neurodegeneration. Reduction of intracellular Zn²⁺-buffering capacity may be linked with the pathophysiology of progressive neurodegeneration such as Alzheimer’s disease. This paper deals with age-dependent modification of intracellular Zn²⁺ buffering in the hippocampus and its impact. On the basis of the estimated impact, we propose a potential defense strategy against Zn²⁺-mediated neurodegeneration, i.e., metallothionein induction in the hippocampus.

1. INTRODUCTION

Zrt-Irt-like proteins (ZIPs), which are involved in the transport of Zn²⁺ into the cytoplasm, and the zinc transporter (ZnT) family, which is involved in the transport of Zn²⁺ out of the cytoplasm, serve to maintain Zn²⁺ homeostasis in the living body including the brain.^1–3) The zinc concentration in the brain increases in the process of development (infant brain, 8.2 ± 0.8 µg/g wet weight) and reaches 13.3 ± 0.3 µg/g wet weight in the human adult brain.⁴⁾ Zinc homeostasis in the brain is strictly regulated through the brain barrier system, i.e., the blood–brain and blood–cerebrospinal fluid (CSF) barriers.^5,6) The cells constructing the brain barrier system, which express ZIPs and ZnT, regulate Zn²⁺ transport into the brain parenchyma, i.e., neurons and glial cells.⁷⁾ Zn²⁺ is very slowly transported through the brain barrier system, especially the blood–CSF barrier.^8,9)

Approximately 80% of brain zinc is zinc metalloproteins, while approximately 20%, which is histochemically reactive as determined by Timm’s sulfide-silver staining,^10,11) serves as a signal factor, free Zn²⁺, in both the intracellular and extracellular compartments. Synaptic Zn²⁺ dynamically functions in conjunction with synaptic activity, especially glutamatergic synapse activity, and is involved in synaptic function.¹²⁾ Synaptic Zn²⁺ plays a key role in cognitive function, while it can also lead to cognitive decline.

Brain aging is characterized by cognitive decline and neuronal loss, while it elevates susceptibility to neurological disorders.¹³⁾ The changes emerging in the process of brain aging are often linked with the modification of synaptic Zn²⁺ dynamics. Zinc transporter-3 (ZnT-3) protein, which is responsible for Zn²⁺ transport into presynaptic vesicles,¹⁴⁾ regulates the availability of Zn²⁺ at zincergic synapses that concentrate zinc in presynaptic vesicles and activity-dependently releases Zn²⁺ with glutamate. ZnT-3 is involved in hippocampus-dependent memory and its expression decreases with aging.¹⁵⁾ The decrease in ZnT-3 protein may be involved in age-related cognitive decline. Adlard et al. reported that clioquinol, a copper/zinc ionophore, and PBT2, a second-generation 8-hydroxyquinoline analogue, which serve as metal chaperones in the brain, increase the availability of Zn²⁺ and prevent normal age-related cognitive decline.^16,17) It is estimated that the decrease in the availability of Zn²⁺, which originates in presynaptic vesicles, is a cause of normal age-related cognitive decline. On the other hand, intracellular Zn²⁺ dysregulation, which is linked with extracellular Zn²⁺ influx mediated by glutamate and amyloid β (Aβ), a key pathogenic peptide of Alzheimer’s disease (AD), may readily emerge with aging and is also a cause of normal age-related cognitive decline, as described below.^18,19) Therefore, the regulation of intracellular Zn²⁺ dynamics is critical for cognitive activity under physiological and pathological conditions.

2. ZN²⁺ PHYSIOLOGY IN THE BRAIN

Divalent cations such as Ca²⁺ and Mg²⁺ are required for neuronal activity, i.e., synaptic neurotransmission.²⁰⁾ The Ca²⁺ concentration is the highest among divalent cations in the brain and is approximately 1.3 mM in the CSF and brain extracellular fluid in adult rats.²¹⁾ Artificial cerebrospinal fluid (ACSF) used for in vivo and in vitro experiments contains approximately 2 mM Ca²⁺, based on the essentiality of intracellular Ca²⁺ signaling for neurons and glial cells.^22,23) However, excess influx of extracellular Ca²⁺ into neurons, which is induced by glutamate excitotoxicity, is involved in the pathophysiological process of neuronal death.^24–26)

The basal concentrations of extracellular Zn²⁺ and intracellular Zn²⁺, which are approximately 10 nM²⁷⁾ and 100 pM,^28,29) respectively, in the brain, are markedly lower than those of extracellular Ca²⁺ (1.3 mM) and intracellular Ca²⁺ (100 nM), respectively, resulting in much less attention paid to Zn²⁺ than to Ca²⁺. For studying synaptic function, no attention has been paid to Zn²⁺; Zn²⁺ is not included in ACSF, i.e., the brain extracellular medium widely used for in vitro and in vivo experiments. Spontaneous presynaptic activity in the stratum lucidum where mossy fibers are contained, which is determined with FM4-64, an indicator of exocytosis (presynaptic activity), is suppressed in brain slices from young rats bathed in ACSF containing 10 nM Zn²⁺, indicating that glutamatergic presynaptic activity is enhanced in brain slices bathed in ACSF without Zn²⁺.³⁰⁾ We reported that not only neuronal excitation but also synaptic plasticity such as long-term potentiation (LTP) are modified in brain slices bathed in ACSF without Zn²⁺ where the original neurophysiology may be modified.^30,31)

The action of extracellular Zn²⁺ at physiological concentrations is important to understand synaptic function precisely as well as to understand bidirectional Zn²⁺ actions under physiological and pathological conditions. It has been recognized that low nanomolar concentrations of Zn²⁺ are more physiologically relevant than its micromolar concentrations, which have been widely used and are often neurotoxic. On the other hand, the role of endogenous Zn²⁺ released from zincergic neurons has been studied in acute brain slice preparations using ACSF without Zn²⁺. Because zinc concentrations in the presynaptic vesicles are reduced in the process of slice preparation, it is estimated that in vitro Zn²⁺ release is reduced to approximately 25% as compared with in vivo Zn²⁺ release.³²⁾ Interestingly, the extracellular zinc concentration, which was determined by in vivo microdialysis, increases age-dependently in the hippocampus, suggesting that the extracellular Zn²⁺ concentration is also physiologically increased in the hippocampus with aging.³³⁾

3. MODIFICATION OF INTRACELLULAR ZN²⁺ BUFFERING WITH AGING

It was reported that vulnerability to Ca²⁺ dysregulation is facilitated in the process of brain aging.^34–36) Ca²⁺ dysregulation is not ubiquitous in the brain but has been observed in specific cell populations and areas. For example, the expression of L-type Ca²⁺ channels is age-dependently elevated in hippocampal pyramidal cells.³⁷⁾ N-Methyl-D-aspartate (NMDA) receptor function is age-dependently reduced in the frontal cortex and the hippocampus,³⁸⁾ suggesting that a compensatory mechanism is induced in the process of brain aging to maintain the availability of intracellular Ca²⁺ signaling. On the other hand, intracellular Ca²⁺ buffering, which is involved not only in cognitive function but also in neurodegeneration, is weakened during brain aging.³⁵⁾

To maintain the availability of intracellular Zn²⁺ signaling, it is likely that a compensatory mechanism is also induced in the process of brain aging. The ZnT-3-dependent zinc concentration in presynaptic vesicles is decreased with aging,^15,39) while the extracellular zinc concentration is age-dependently increased in the hippocampus.³³⁾ Intracellular Zn²⁺ buffering is critical not only for cognitive function but also for neurodegeneration via regulating the availability of intracellular Zn²⁺ signaling. However, the Zn²⁺-buffering system is more poorly understood than the Ca²⁺-buffering system. It was reported that weakened intracellular Ca²⁺ buffering, with a net decrease in the Ca²⁺-buffering capacity, is linked with both normal aging³⁵⁾ and neurological disorders such as AD.⁴⁰⁾

The Zn²⁺-buffering system is composed of Zn²⁺ transporters (ZIPs and ZnT), Zn²⁺-binding proteins such as metallothioneins (MTs), internal stores containing Zn²⁺, and Ca²⁺-permeable channels, which is dynamically linked with synaptic activity. Judging from the increased extracellular Zn²⁺ concentration and extracellular Zn²⁺ influx in the aged rat hippocampus,³³⁾ it is estimated that intracellular Zn²⁺ buffering is modified in the dentate gyrus of aged rats.⁴¹⁾ ZnAF-2DA, a membrane-permeable Zn²⁺ indicator, is taken up into the cells through the plasma membrane and is hydrolyzed by esterase in the cytosol, resulting in the production of ZnAF-2 (dissociation constant [K_d] = 2.7 × 10^–9 M for Zn²⁺). Intracellular ZnAF-2 cannot permeate the plasma membrane.^42,43) Thus we used ZnAF-2DA to determine the changes in the Zn²⁺-binding capacity, which was assessed by the changes in intracellular ZnAF-2 fluorescence (Fig. 1). The in vivo increase in intracellular ZnAF-2 fluorescence is more rapidly lost in the aged dentate molecular layer where medial perforant pathway-dentate granule cell synapses are present than in the young dentate molecular layer, suggesting that intracellular Zn²⁺-buffering capacity is reduced in the aged dentate gyrus.⁴¹⁾ The characteristics (easiness) of extracellular Zn²⁺ influx may lead to reduced intracellular Zn²⁺-buffering capacity in the aged dentate gyrus, which represents weakened intracellular Zn²⁺ buffering. It was reported that the dentate gyrus is vulnerable to aging and AD in the hippocampal formation.^44,45) The vulnerability seems to be linked with age-related modification of extracellular Zn²⁺ influx, which is induced by glutamate and Aβ_1–42 in the extracellular compartment.^33,41,46,47)

Fig. 1. Intracellular Zn²⁺-Buffering Capacity as Assessed with Intracellular ZnAF-2

MTs function in intracellular Zn²⁺ buffering and are mainly in the form of Zn₅MT under basal (static) conditions. Extracellular glutamate accumulation induces Zn²⁺ influx through Ca²⁺-permeable AMPA receptors (Ca²⁺-AMPAR). Extracellular Aβ_1–42 also induces Zn²⁺ influx via the formation of Zn-Aβ_1–42 complexes, which is independent of AMPA receptor activation.⁶²⁾ Increased Zn²⁺ is captured with MTs. When MTs are saturated with Zn²⁺ via the formation of Zn₇MT, intracellular free Zn²⁺ is neurotoxic. Induced MTs increase the intracellular Zn²⁺-buffering capacity, as assessed based on intracellular ZnAF-2 fluorescence. [Zn²⁺]_i, basal concentration of intracellular Zn²⁺; [Zn²⁺]_o, basal concentration of extracellular Zn²⁺. (Color figure can be accessed in the online version.)

4. IMPACT OF MODIFIED INTRACELLULAR ZN²⁺ BUFFERING ON NEURODEGENERATION

Glutamate excitotoxicity is a common pathway to neurodegeneration, and extracellular glutamate accumulation causes neurodegeneration via Ca²⁺ influx through calcium channels such as NMDA receptors. On the other hand, Zn²⁺ can pass through calcium channels and preferentially passes through GluR2-lacking Ca²⁺-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors.^48,49) It was reported that glutamate excitotoxicity, which is involved in the pathology of most neurodegenerative disorders including ischemia, epilepsy, and AD, is due to Zn²⁺ neurotoxicity rather than to Ca²⁺ neurotoxicity.^50–52) This may be due to the extremely low concentration (approx. 100 pM) of basal intracellular Zn²⁺,^28,29) which reflects the necessity of strict Zn²⁺ regulation in the intracellular (cytosolic) compartment to maintain the intracellular environment.

Aging is a major risk factor for the onset of AD. The first step in pathological alteration in the AD brain is the abnormal processing of Aβ peptides and their accumulation, which is initiated more than two decades before the onset of AD.^53,54) Aβ is secreted into the extracellular space after sequential cleavage of the amyloid precursor protein (APP).⁵⁵⁾ Aβ has self-aggregation ability and Aβ fibrils are the main components of Aβ plaques, a pathological hallmark of AD. Aβ is bound to Zn²⁺ via histidine residues, and the K_d values of Zn²⁺ to Aβ_1–40 are in the range of 0.1–60 µM.⁵⁶⁾ On the other hand, it was reported that the formation and propagation of misfolded aggregates of Aβ_1–42 rather than of Aβ_1–40 contribute to AD pathogenesis. However, the structural details of misfolded Aβ_1–42 are poorly understood.⁵⁷⁾ The C-terminal carboxylate anion of Aβ_1–42 constructs the C-terminal hydrophobic core, which accelerates neurotoxic oligomerization.⁵⁸⁾ C-terminal Ala42 that is absent in Aβ_1–40 constructs a salt bridge with Lys28 to create a self-recognition molecular switch, which is the Aβ_1–42-selective self-replicating amyloid-propagation machinery.⁵⁹⁾ The aggregating property of Aβ_1–42 is rapidly promoted with Zn²⁺, resulting in much higher affinity of Aβ_1–42 to Zn²⁺ than that of Aβ_1–40, and the K_d values of Zn²⁺ to Aβ_1–42 are in the range of 3–30 nM.⁴⁶⁾ Aβ_1–42, unlike Aβ_1–40, captures extracellular Zn²⁺ at high picomolar levels, and the formation of Zn-Aβ_1–42 in the extracellular compartment is essential for Aβ_1–42 uptake into dentate granule cells, followed by cognitive decline (Fig. 2). Soluble Aβ_1–42 oligomers that are strong synaptotoxic molecules in AD induce synaptic dysfunction and cognitive decline.^60,61)

Fig. 2. Proposed Defense Strategy against Neurotoxic Zn²⁺ Released from Zn-Aβ_1–42 Complexes

When Zn-Aβ_1–42 complexes are taken up into dentate granule cells, neurotoxic Zn²⁺ is released from the complexes. Neurotoxic Zn²⁺ can be captured with MTs. Thus, the induction of MTs, which increases the intracellular Zn²⁺-buffering capacity, is a promising defense strategy against neurotoxic Zn²⁺. (Color figure can be accessed in the online version.)

The cytosolic Zn²⁺ concentration in dentate granule cells is much lower than the extracellular Zn²⁺ concentration. When Zn-Aβ_1–42 formed is taken up into dentate granule cells, Zn²⁺ can be released from Aβ_1–42, resulting in an increase in neurotoxic Zn²⁺ (Fig. 2), which is assessed based on the increase in intracellular ZnAF-2 fluorescence^46,62) (Fig. 1). Zn-Aβ_1–42 uptake is increased in the aged dentate gyrus compared with the young dentate gyrus⁴⁷⁾, consistent with the estimated increase in extracellular Zn²⁺ in the hippocampus with aging.³³⁾ It is likely that Zn²⁺ neurotoxicity, which originates in Aβ_1–42, is readily induced with aging. On the other hand, weakened intracellular Zn²⁺ buffering is observed in the aged dentate gyrus.⁴¹⁾ Although aging is a major risk factor for AD, the characteristic age-related Zn-Aβ_1–42 formation in the extracellular compartment and weakened intracellular Zn²⁺ buffering may be linked with the major risk.

It is possible that the Aβ_1–42-mediated increase in intracellular Zn²⁺ facilitates hyperphosphorylation of the microtubule-associated protein tau, which is a main pathological hallmark as well as Aβ deposits and affects axonal transport, subsequently leading to neurodegeneration. Neuronal loss, tau hyperphosphorylation, and hippocampus-dependent cognitive decline are observed in mice subjected to repeated injection of Aβ_1–42 oligomers into the hippocampus.⁶³⁾ Tau hyperphosphorylation in AD is linked with a reduction in protein phosphatase 2A (PP2A) activity.^64,65) PP2A regulates tau phosphorylation at multiple sites in the normal human brain. Zn²⁺ promotes tau hyperphosphorylation by inactivating PP2A.^66,67) Zn²⁺ binds PP2A and directly inhibits its activity at a low micromolar concentration of Zn²⁺ (10 µM) in vitro.⁶⁸⁾ Furthermore, Zn²⁺ 0.25 µM changes the conformation of tau, and Zn²⁺ 5 µM promotes tau aggregation in vitro.⁶⁹⁾ Although the Zn²⁺ concentrations used in in vitro experiments is extremely high compared with physiologically estimated intracellular Zn²⁺ in the in vivo picomolar range, it is possible that a local increase in intracellular Zn²⁺ induced by Aβ_1–42 is linked with tau hyperphosphorylation via PP2A inactivation and tau aggregation (Fig. 2).

5. PERSPECTIVE ON A DEFENSE STRATEGY AGAINST NEURODEGENERATION INDUCED BY ZN-Aβ_1–42

The blockage of Aβ_1–42 binding to extracellular Zn²⁺ and capturing Zn²⁺ from Zn-Aβ_1–42 in the intracellular compartment, which are performed using extracellular Zn²⁺ and intracellular Zn²⁺ chelators, respectively, may be an effective strategy for preventing Aβ_1–42-mediated cognitive decline and neurodegeneration⁶²⁾ (Fig. 2). In vivo local co-injection of Aβ_1–42 and Ca ethylenediaminetetraacetic acid (EDTA), an extracellular Zn²⁺ chelator, or CdCl₂, a zinc-replacing metal, into the rat dentate gyrus prevents Aβ_1–42-mediated impairments of LTP induced at perforant path-dentate granule cell synapses and of recognition memory.⁴⁶⁾ However, CaEDTA and CdCl₂ are not adaptable for clinical use because they cannot permeate the blood–brain barrier.

On the other hand, increasing the intracellular Zn²⁺-buffering capacity may be also an effective strategy for preventing intracellular Zn²⁺ toxicity induced by Aβ_1–42 and glutamate⁴⁷⁾ (Fig. 1). MTs are major Zn²⁺-binding proteins in the cytosol and play a critical role in intracellular Zn²⁺ buffering. Thus, the induction of MT synthesis can increase the capacity for intracellular Zn²⁺ buffering. Our data show that the systemic administration of dexamethasone, an inducer of MT synthesis, prevents the Aβ_1–42-mediated increase in intracellular Zn²⁺ and Aβ_1–42-mediated impairments of LTP and recognition memory in young rats⁴⁷⁾ (Fig. 2). The basal level of MTs in the hippocampus is approximately 65 nM in young rats, as determined in the silver-binding assay.⁴⁷⁾ MTs can bind seven Zn²⁺ in the form of Zn₇MT with varying affinity (K_d: approx. 10^–8 to 10^–12 M), and cytosolic MTs exist mostly in the form of Zn₅MT under physiological (static) conditions, which has the capacity to capture two more free Zn²⁺.^70,71) Thus, it is estimated that hippocampal MTs can buffer 130 nM of free Zn²⁺ in the cytosol of young rats. Even if the cytosolic Zn²⁺ concentration reaches the extracellular concentration (approx. 10 nM) via Aβ_1–42-mediated Zn²⁺ influx, induced MTs capture Zn²⁺ released from Zn-Aβ_1–42. On the other hand, the basal level of MTs in the hippocampus is slightly higher in aged than in young rats. When the increase in intracellular Zn²⁺-buffering capacity is compared between the young and aged hippocampus, as determined based on the reduction in intracellular ZnAF-2 fluorescence, after the systemic administration of dexamethasone, there is no appreciable different between them.⁴⁷⁾ Therefore, a blood–brain barrier-permeable MT-inducing agent may be promising for preventing neurodegenerative disorders such as AD linked with intracellular Zn²⁺ toxicity⁷²⁾ (Fig. 2).

Conflict of Interest

The authors declare no conflict of interest.

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