Zinc (Zn) is an essential trace element for most organisms. It plays important roles in various physiological functions such as the mitotic cell division, the immune system, the synthesis of proteins and DNA as a co-factor of more than 300 enzymes or metalloproteins . Recent studies revealed that Zn signaling plays crucial roles in various biological systems of humans . Zn deficiency in human childhood is known to cause the dwarfism, the retardation of mental and physical development, the immune dysfunction, and the learning disabilities . In adults, Zn deficiency causes the taste and odor disorders.
The human body contains approximately 2 g of Zn, mostly in the testes, muscle, liver, and brain tissues. In the brain, Zn is found at the highest concentrations in the hippocampus, amygdala, cerebral cortex, thalamus, and olfactory cortex . The total Zn content of the hippocampus is estimated to be 70–90 ppm (dry weight). Although some Zn in the brain binds firmly to metalloproteins or enzymes, a substantial fraction (approximately 10% or more) either forms free Zn ions (Zn2+) or is loosely bound, and is histochemically detectable by staining using chelating reagents. This chelatable Zn is stored in presynaptic vesicles of specific excitatory glutamatergic neurons and is secreted from these vesicles into synaptic clefts along with glutamate during neuronal excitation. Recent studies have suggested that this secreted Zn2+ plays crucial roles in information processing, synaptic plasticity, learning, and memory (Fig. 1A). Indeed, Zn2+ in the hippocampus is essential for the induction of long-term potentiation (LTP), a form of synaptic information storage that has become a well-known paradigm for the mechanisms underlying memory formation .
However, despite its importance, excess Zn is neurotoxic and implicated in neurodegenerative diseases. In this chapter, we review the current understanding about the link between the disruption of Zn homeostasis and the pathogenesis of various neurodegenerative diseases including senile dementia.
2. Zinc and vascular type of dementia
2.1. Zn-induced neurodegeneration after ischemia
Senile dementia is a serious problem in a rapidly aging world. Its prevalence increases with age. Approximately 25% of elderly individuals are affected by the diseases. In Japan, 3 million people have been estimated to be affected by senile dementia by 2025, and the number continues to grow annually. Senile dementia is mainly divided to Alzheimer’s disease (AD) and vascular-type dementia (VD). VD is a degenerative cerebrovascular disease, and its risk factors include aging, sex difference (male), diabetes, and high blood pressure
Increasing evidence suggests that Zn is central to ischemia-induced neuronal death and finally the pathogenesis of VD . In ischemic conditions, a considerable amount of Zn (up to 300µM) is co-released with glutamate into synaptic clefts by membrane depolarization. Zn caused the apoptotic death of primary cultured cortical neurons. Furthermore, the chelatable Zn reportedly moved from presynaptic terminals into postsynaptic neuronal cell bodies. The increase in intracellular Zn2+ levels ([Zn2+]i), namely, “Zn translocation,” occurs in vulnerable neurons in the CA1 or CA3 regions of the hippocampus prior to the onset of the delayed neuronal death after transient global ischemia . This Zn translocation is reported to enhance the appearance of the infarct. Administration of calcium EDTA (Ca EDTA), a membrane-impermeable chelator that chelates cations except for calcium, blocked the translocation of Zn, protected the hippocampal neurons after transient global ischemia, and reduced the infarct volume . Thus, Zn translocation is recognized to be the primary event in the pathway of Zn-induced neuronal death. Sensi
In a normal condition, most hippocampal neurons express AMPA receptors with subunit GluR2, which are poorly permeable to divalent cations including Ca2+ and Zn2+(A/K-R). However, after ischemia, the acute reduction in the expression of GluR2 subunit occurs, and neurons possess specific type of AMPA receptors which channels are directly Ca2+ permeable (Ca-AMPA/kainate channels; Ca-A/K-R)) . The appearance of Ca-AMPA/kainate channels causes the increased permeability of Ca2+ and enhances the toxicity. Therefore, the expression of Zn2+-permeable Ca-AMPA/kainite channels and the entry of Ca2+ and/or Zn2+ through the channels are mediators of the delayed neuronal death after ischemia. Considering that Ca EDTA, a zinc chelator, attenuates the ischemia-induced down-regulation of GluR2 gene , Zn is also implicated in the transcriptional regulation in Ca-AMPA/kainite channels.
Zn-specific membrane transporter proteins (Zn transporters) also control Zn homeostasis; they facilitate zinc influx in deficiency and efflux during zinc excess. Recent genetic and molecular approaches revealed the implications of abnormalities in Zn transporters in various human diseases . Zn transporter 1 (ZnT-1), a membrane protein with six transmembrane domains, is widely distributed in mammalian cells, and is co-localized with chelatable Zn in the brain. ZnT-1 is activated by excess Zn and the expression of ZnT-1 is induced after transient global ischemia. On the contrary, dietary Zn deficiency decreases expression of ZnT-1. Consequently, it is provable that ZnT-1 plays a pivotal role in efflux of Zn and in protection from Zn toxicity. Another important Zn transporter in the brain is ZnT-3, which localizes in the membranes of presynaptic vesicles, transports Zn into synaptic vesicles, and maintains high Zn concentrations in the vesicles. Although the physiological role of ZnT-3 and vesicular zinc remain elusive, recent studies have suggested the implication of ZnT-3 or other Zn transporters in the pathogenesis of AD and other neurodegenerative diseases .
2.2. Molecular mechanism of Zn-induced neurotoxicity: GT1-7 cells as an
in vitromodel system
Understanding the molecular mechanism of Zn-induced neuronal death is of great importance for the treatment of VD. Numerous studies have been undertaken to elucidate the mechanism of Zn-induced neuronal death. To this end, many researchers have investigated Zn neurotoxicity
We found that GT1-7 cells, immortalized hypothalamic neurons, are much more sensitive to Zn than other neuronal cells are [17,18] (Fig. 2A). Zn caused the apoptotic death of GT1-7 cells in a dose-dependent and time-dependent manner. The degenerated GT1-7 cells were terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL) positive and exhibited the DNA fragmentation.
The GT1-7 cells were originally developed by Mellon
We investigated the detailed characteristics of Zn-induced death in GT1-7 cells and its mechanisms. First, we tested the effects of various pharmacological agents prior to Zn treatment of GT1-7 cells. Neither antagonists nor agonists of excitatory neurotransmitters (D-APV, glutamate, and CNQX), or those of inhibitory neurotransmitters (bicuculline, muscimol, baclofen, and GABA) attenuated the viability of GT1-7 cells after Zn exposure. Our findings in GT1-7 cells, which lack such glutamate receptors, are inconsistent with previous studies that agonists of glutamate receptors, such as NMDA or AMPA, enhance Zn-induced neurotoxicity in cultured cortical neurons .
To evaluate the involvement of other metal ions in Zn neurotoxicity, we investigated the viability of GT1-7 cells with or without various metal ions after exposure to Zn . The equimolar addition of Al3+ and Gd3+ significantly inhibited Zn-induced neurotoxicity. Moreover, overloading of Ca2+ and Mg2+ inhibited the Zn-induced death of GT1-7 cells; Zn protected GT1-7 cells from neurotoxicity induced by Ca2+ overload, and
2.3. Implication of Ca dyshomeostasis in Zn-induced neuronal death
To address this issue, we employed a high-resolution multi-site video imaging system with fura-2 as the cytosolic free calcium reporter fluorescent probe for the observation of temporal changes in [Ca2+]i after exposure to Zn (Fig. 4). This multisite fluorometry system enables the simultaneous long-term observation of temporal changes in [Ca2+]i of more than 50 neurons. The elevations in [Ca2+]i were observed among GT1-7 cells after 3-30 min of the exposure to Zn . Detailed analysis of Zn-induced [Ca2+]i revealed that pretreatment of Al3+ significantly blocked the Zn-induced [Ca2+]i elevations. Thus, it is possible that Al3+, a known blocker of various types of Ca2+ channels, attenuate Zn-induced neurotoxicity by blocking Zn-induced elevations in [Ca2+]i.
We also showed that the administration of sodium pyruvate, an energy substrate, significantly inhibited the Zn-induced death of GT1-7 cells . The results are consistent with findings of other studies using primary cultured cortical neurons, oligodendrocyte progenitor cells, or retinal cells. Furthermore, the administration of pyruvate attenuated the neuronal death after ischemia
2.4. Carnosine as an endogenous protective substance against Zn neurotoxicity
Considering the implication of Zn in transient global ischemia, substances that protect against Zn-induced neuronal death could be potential candidates for the prevention or treatment of neurodegeneration following ischemia, and ultimately provide a lead to treatments for VD. With the aim of exploring this idea, we developed a rapid, sensitive, and convenient assay system for the mass-screening of such substances by using GT1-7 cells. We examined the potential inhibitory effects of various agricultural products such as vegetable extracts, fruits extracts, and fish extracts, and found that extracts from eel muscles significantly protected against Zn-induced neurotoxicity . Finally, we demonstrated that carnosine (ß-alanyl histidine), a small hydrophilic peptide abundant in eel muscles, protected GT1-7 cells from Zn-induced neurotoxicity in a dose-dependent manner. Therefore, we applied for the patent on carnosine as a drug for the treatment of VD or for slowing the progress of cognitive decline after ischemia (the application No. 2006-145857; the publication No. 2007-314467 in Japan) . Carnosine is a naturally occurring dipeptide and is commonly present in vertebrate tissues, particularly within the skeletal muscles and nervous tissues . It is found at high concentrations in the muscles of animals or fish which exhibit high levels of exercise, such as horses, chickens, and whales. The concentration of carnosine in the muscles of such animals is estimated to be 50–200 mM, and carnosine is believed to play important roles in the buffering capacities of muscle tissue. During high-intensity anaerobic exercise, proton accumulation causes a decrease in intracellular pH, which influences various metabolic functions. The p
Therefore, carnosine contributes to physicochemical non-bicarbonate buffering in skeletal muscles, and the administration of carnosine has been reported to induce hyperactivity in animals.
Carnosine reportedly has various functions including anti oxidant, anti glycation, anti crosslink, and considered to be an endogenous neuroprotective, anti-aging substances. Considering the advantegeous properties of carnosine Considering the advantegeous properties of carnosine (relatively non-toxic, heat-stable, and water-soluble), the dietary supplementation of carnosine might be an effective strategy for the prevention or treatment of neurodegenerative diseases such as ischemia, VD, AD, and prion diseases. Corona et al. reported that supplementation of carnosine improved learning abilities of Alzheimer’s model mice . We demonstrated that neurotoxicity of prion protein fragment was attenuated by Zn and carnosine .
3. Zn and Alzheimer’s disease diseases
3.1. Amyloid cascade hypothesis and Zn
AD is a severe senile type of dementia first reported in 1906. The pathological hallmarks of AD are the deposition of extracellular senile plaques, intracellular neurofibrillary tangles (NFTs), and the selective loss of synapses and neurons in the hippocampal and cerebral cortical regions. The major component of NFTs is the phosphorylated tau protein. Senile plaques are largely comprised of ß-amyloid protein (AßP) . Numerous biochemical, toxicological, cell biological, and genetic studies have supported the idea termed “amyloid cascade hypothesis” which suggests that the neurotoxicity caused by AßP play a central role in AD [32,33]. AβP is a small peptide with 39–43 amino acid residues. It is derived from the proteolytic cleavage of a large precursor protein (amyloid precursor protein; APP). AβP has an intrinsic tendency to self-assemble to form sodium dodecyl sulfate (SDS)-stable oligomers. Moreover, oligomerization and conformational changes in AßP are important for its neurodegeneration process. In an aqueous solution, freshly prepared and dissolved AβP exists as a monomeric protein with a random coil structure. However, following incubation at 37°C for several days (
Interestingly, rodent AßP exhibits less tendency to oligomerization than human AßP
We have investigated the metal-induced oligomerization of AßP and found that the metals including Al, Zn, Fe, Cu, and Cd enhanced the oligomerization. However, the oligomerization induced by Al is more marked than that induced by other metals [36,37]. Furthermore, while Zn-aggregated AßPs are rarely observed on the surface of cultured neurons several days after its exposure, Al-aggregated AßPs bind tightly to the surface of cultured neurons and form fibrillar deposits. Bush et al. reported the Zn- or Cu- induced oligomerization of AßP [38,39], and have developed the chelation therapy for AD treatment . Clioquinol (quinoform), a chelator of Cu2+ or Zn2+, inhibits oligomerization of AßP and attenuates the accumulation of amyloid in the brains of experimental animals. Clinical trials using its analogue PBT2 are under investigation. However, considering that the morphology of AßP oligomers treated with metals including Al, Cu, Fe, Zn are quite different  and that recent approaches using size-exclusion chromatography, gel electrophoresis, and atomic force microscopy have demonstrated that identified soluble oligomers are neurotoxic, further studies about metal-induced oligomerization are necessary.
APP also possesses copper/zinc binding sites in its amino-terminal domain and in the AßP domain and may be involved in homeostasis of these metals . Duce
3.2. AßP -induced neuronal death and Zn
Zn is involved in the mechanism of AßP-induced neurotoxicity. There is considerable interest regarding the mechanism by which AßPs cause neuronal death. In 1993, Arispe
Inorganic cations such as Al3+ or Zn2+ inhibit current induced by amyloid channels [44,45]. Zn reportedly inhibited AßP-induced Ca2+ increase. We have revealed that the amyloid channel activity formed on membranes of GT1-7 cells was inhibited by addition of Zn2+, and recovered by Zn chelator,
Based on our and other findings about the link between Zn and the pathogenesis of AD, we made a hypothetical scheme about the link between AD pathogenesis and Zn (Fig. 6). AßPs are normally secreted from APP, which exists in the synapse. Secreted AßPs are usually degraded proteolytically by proteases within a short period. However, Zn or other metals enhance the oligomerization and accumulation of AßP. After incorporation into the membrane, the conformation of AßPs change and the accumulated AßPs aggregate on the membranes.. Finally, aggregated AßP oligomers form ion channels leading to the various neurodegenerative processes.Unlike endogenous Ca2+ channels, these AßP channels are not regulated by usual blockers. Thus, once formed on membranes, a continuous flow of [Ca2+]i is initiated. Disruption of calcium homeostasis triggers several apoptotic pathways and promotes numerous degenerative processes, including free radical formation and tau phosphorylation, thereby accelerating neuronal death. Meanwhile, Zn2+, which are secreted into synaptic clefts in a neuronal activity-dependent manner, inhibit AßP-induced Ca2+ entry, and thus have a protective function in AD.
Based on results of our own and other numerous studies, the disruption of Zn homeostasis, namely both zinc depletion and excess zinc, cause severe damage to neurons and linked with various neurodegenerative diseases including VD and AD. Increasing evidence suggests the implications of Zn in the pathogenesis of other neurodegenerative disease including prion diseases, Parkinson disease, ALS etc. Zn acts as a contributor of the disease in one part, and as a protector in another part. Thus, Zn might play a role like that of Janus, an ancient Roman god of doorways with two different faces, in the brain (Fig. 7).
Our new approach to ischemia-induced neurodegeneration from the perspective of the Zn hypothesis will lead to new therapeutic tools for the treatment and/or prevention of VD. Further research about the role of Zn in neuronal injury and the significance of Zn homeostasis might give rise to the development of new treatments for neurodegenerative diseases. In this context, the advantegeous properties of carnosine (relatively non-toxic, heat-stable, and water-soluble) as a possible candidate for the prevention or treatment of neurodegenerative diseases such as ischemia, VD, AD, and prion diseases are important.
As described here, Zn plays important roles in memory formation, and protects neurons from various neurodegenerative diseases. Meanwhile, excess Zn is neurotoxic and may enhance the pathogenesis of the diseases.