Sci. Aging Knowl. Environ., 7 September 2005
Vol. 2005, Issue 36, p. pe27
[DOI: 10.1126/sageke.2005.36.pe27]

PERSPECTIVES

Metals on the Brain

Carina Treiber

The author is at the Free University of Berlin, Thielallee 63, 14195 Berlin, Germany. E-mail: treiber{at}chemie.fu-berlin.de

http://sageke.sciencemag.org/cgi/content/full/2005/36/pe27

Key Words: zinc • metals • Alzheimer's disease • prion disease

Introduction

Metal ions play critical roles in complex signaling pathways in the central nervous system (CNS). Perhaps the best studied ion involved in signaling is calcium (1). Changes in intracellular Ca2+ concentrations trigger a cascade of cellular events mediated by the protein calmodulin (Cd) (2). In the brain, these events are key to rapid signaling and memory formation. Scientists have recently uncovered similar roles for other metal ions, in particular Zn2+ (3). Several studies implicate zinc in synaptic transmission and cell survival. Whereas normal concentrations of Zn2+ are important to the proper functioning of nerve cells, excess quantities of this metal ion cause toxicity (4). Increased concentrations of metal ions, including Zn2+, are associated with normal aging and neurodegenerative diseases, including Alzheimer's disease (AD) and transmissible spongiform encephalopathy.

The IV Metallic Network Meeting, titled "Metallothioneins and Metals: Biochemical, Anatomical, and Functional Aspects" and held in Barcelona, Spain on 16 and 17 June 2005, provided new insights into the functions of metal ions in the CNS in health and disease. The meeting, organized by Je�s P�rez-Clausell of the University of Barcelona, gave researchers an opportunity to report on recent progress in understanding the mechanisms of action of metal ions in the brain, as well as their transporters and sequestering proteins.

Zinc Signaling

Relative to other organs, the brain exhibits the highest concentration (150 µM) of Zn2+. The metal is found mainly in specific axons of the hippocampus (the mossy fibers; see figure 3 in Saito Perspective), suggesting that it plays a role in neurotransmission. Electrical stimulation of mossy fibers induces long-term potentiation (LTP), a process critical to long-term memory formation, between mossy fibers and synapses of the CA3 region of the hippocampus. Evidence suggests that after electrical stimulation, Zn2+ is released from presynaptic mossy fibers along with the neurotransmitter glutamate in a Ca2+-dependent manner.

The function of the released Zn2+ is not clear. At the meeting, M. Emilia Quinta-Ferreira of the University of Coimbra, Portugal, reported that, in rat hippocampal slices, strong electrical stimulation of mossy fiber synapses during LTP caused a depression of presynaptic Ca2+ and Zn2+ signals and synaptic responses, which could be blocked by zinc chelators (5). The work suggests that released Zn2+ has an inhibitory role on presynaptic Ca2+ mechanisms, leading to the depression of Zn2+ and of glutamate release. However, the exact role of Zn2+ in regulating presynaptic Ca2+ mechanisms needs to be further investigated.

In addition to its role in synaptic transmission, Zn2+ has been implicated in a number of cellular signaling pathways, including those involved in cell proliferation and apoptosis. Michal Hershfinkel of Ben Gurion University in Israel put forward the hypothesis that a specific zinc-sensing receptor (ZnR) regulates the signaling activities of Zn2+. Hershfinkel's group has shown that application of Zn2+ to epithelial cells in culture results in an increase in intracellular Ca2+ concentration via the inositol 1,4,5-trisphosphate (IP3) pathway--a pathway involved in cellular proliferation (6). In addition, her work suggests that ZnR is an extracellular GTP-binding protein (G protein)-coupled receptor that activates the IP3 pathway by phosphorylating the kinases ERK1/2 and Akt. At normal physiological zinc concentrations, the phosphorylation of these two proteins is accompanied by a desensitization of the ZnR (7). Functional desensitization of ZnR boosts normal cell growth, wound healing, and proliferation.

These findings have interesting implications for human cancer. Prostate cancer is characterized by decreased levels of Zn2+ in the prostate. Hershfinkel reported that, under pathogenic conditions, ZnR is no longer desensitized, because of changes in Zn concentrations. Whereas desensitized ZnR supports normal cell proliferation, activated ZnR causes aberrant cell proliferation and is a trigger for zinc-dependent survival and proliferation signals in prostate cancer cells.

Maintaining Homeostasis

Zinc is critical to cell survival and functioning, but high concentrations of this metal inside nerve cells are toxic. There are many examples of the biological consequences of Zn2+ toxicity. Injections of kainic acid, an agonist of a class of glutamate receptors, are extensively used to model temporal-lobe epilepsy, a condition that involves the degeneration of neurons in the hippocampus. Je�s P�rez-Clausell reported that after kainic acid treatment in rats, increased concentrations of Zn2+ could be detected inside degenerating neurons (8), suggesting that Zn2+ is involved in the process of neurodegeneration when concentrations are higher than normal.

Zn2+ can gain access to neurons by many mechanisms. Studies have demonstrated an influx of Zn2+ ions through glutamate receptor-associated channels [the NMDA (N-methyl-D-aspartate) receptor and the Ca2+-permeable AMPA (L-{alpha}-amino-3-hydroxy-5-methylisoxazole-4-proprionate)] and kainate receptors. In addition to these channels, specific Zn-influx proteins (ZIPs) exist in neurons. However, little is known about the mechanisms of Zn2+ uptake used by astrocytes, glial cells that also seem to participate in regulating brain Zn2+ homeostasis. Emilio Varea of the University of Valencia used fluorochromes that specifically bind to Zn2+ to demonstrate the localization of zinc ions in acidic organelles in astrocytes. His work shows that Zn2+ uptake is carried out by clathrin-mediated endocytosis and not through ZIPs. This finding distinguishes the mechanism for the uptake of zinc in astrocytes from the one used in neurons.

Because of the importance of maintaining Zn2+ homeostasis in cells, a number of different proteins are involved in transporting and sequestering this ion. Several studies have tried to elucidate the mechanism of action of members of one family of such proteins, the zinc transporters (ZnTs), which transport zinc out of cells.

The most widely expressed ZnT in mammals, ZnT1, is known to protect cells from toxicity caused by both zinc and cadmium. Zinc colocalizes with ZnT1 in neurons (9). William F. Silverman at the Ben Gurion University of Negev, Israel, discovered that free zinc regulates ZnT1 expression, which was, in turn, strongly linked to a reduction of intracellular zinc concentration. Fluorescent imaging of cells showed that ZnT1 does not, however, affect the extrusion of zinc from cells, but attenuates its permeation.

Blocking the function of the L-Type Calcium Channel (LTCC) with nifedipine was found to abolish the attenuating effect of ZnT1 on Zn2+ influx, indicating that ZnT1 is a regulator of this channel and that LTCC mediates zinc influx. On the other hand, silencing ZnT1 expression with a small interfering RNA resulted in enhanced permeation of not only zinc but also calcium and cadmium (10). Therefore, ZnT1 plays a general role in preventing heavy metal toxicity.

Israel Sekler, also at Ben Gurion University, reported that the activity of another member of the ZnT family, ZnT5, which is localized inside the cell, is regulated by intracellular pH. Although the work is still in its early stages, understanding the regulation and mechanisms of action of the ZnTs will provide important insights into how zinc exerts its effects on cells.

It is not clear why Zn2+ is toxic to neurons and other cells, but Stefano Sensi of the University of G. d'Annunzio in Chieti, Italy, suggested that, at least under pathogenic conditions, the mechanism has to do with the disruption of mitochondrial function (11). Zn2+ accumulates in mitochondria following ischemia (a low oxygen state). In these organelles, increases in Zn2+ concentrations result in the release of cytochrome c and apoptosis-inducing factor through the opening of the mitochondrial permeability transition pore. The released factors, in turn, trigger apoptosis (see also "Cell Death, Start to Finish").

Metallothioneins

Metallothioneins represent another group of proteins that maintain adequate levels of metal ions. These cysteine-rich proteins have a number of functions in the CNS, including sequestering metals. For example, the intracellular neuronal growth inhibitory factor, metallothionein-3 (MT-3), can bind seven Zn2+ ions. Interestingly, the concentrations of this protein are decreased in the brains of AD patients as compared with healthy individuals.

In contrast to MT-1 and -2, which do not show any growth inhibitory activity in primary cultures, MT-3 impairs survival and neurite formation of neurons in culture. Mutational analysis by Milan Vasak at the University of Z�rich established that the Thr(5)-Cys-Pro-CysPro-(9)-motif of MT-3 is critical to its ability to inhibit neuronal growth. This motif is essential for the extracellular biological activity and structure of MT-3 (12).

At the meeting, another function of MT-3 was discussed. In zinc-enriched neurons, Zn2+ is sequestered in presynaptic vesicles; upon stimulation by Ca2+, neurotransmitter and zinc are released in the synaptic cleft. Members of the Rab family of monomeric G proteins associate with the presynaptic vesicles to regulate the exocytosis of synaptic vesicles. The role in this process of MT-3, which colocalizes with Rab3A, is not well understood. However, Vasak has shown that MT-3 reversibly binds to the protein Rab3A-GDP, but not to Rab3-GTP, the protein conformation that regulates Ca2+-dependent exocytosis and release of neurotransmitter and Zn2+. This interaction suggests that MT-3 acts as a cellular zinc buffer and is also actively involved in synaptic vesicle trafficking upstream of vesicle fusion.

Because of their protective effects against high concentrations of metal ions, metallothioneins are being investigated as potential drug targets. Brain injury induces the expression of metallothionein isoforms MT-1 and MT-2, which, in turn, induce the expression of other neurotrophins, neuroplastins, and growth factors essential to neuronal survival, plasticity, and brain tissue repair. Milena Penkowa of the University of Copenhagen reported that a synthetic peptide resembling a binding site (designated FGL) on the neural-cell adhesion molecule (NCAM) has neuroprotective effects in a rat model of brain injury (13). Studies using MT-1 and MT-2 knockout mice and transgenic mice overexpressing MT-1 revealed that the therapeutic action of the NCAM peptide is dependent on the expression of these two metallothioneins. The study suggests that peptides that stimulate MT-1 and MT-2 may provide candidate drugs for treating brain injury.

Neurodegenerative Diseases

There is a large body of evidence implicating Zn2+, Cu2+, and Fe2+ in the etiology of AD (see "Detangling Alzheimer's Disease" and "Mindful of Metal"). These metals are found in amyloid plaques at millimolar concentrations, and in vitro studies reveal that they promote the aggregation of {beta} amyloid (A{beta})--a protein fragment derived from the amyloid precursor protein (APP) and the major constituent of neuritic plaques (14). Moreover, binding sites for Zn2+ and Cu2+ have been found in the N-terminal domain of APP.

Peter Faller of the University of Toulouse, France, has studied the interaction of Zn2+ with synthetic peptides corresponding to portions of A{beta} and APP. He reported that a single Zn2+ is coordinated by residues His6, His13, His14, and Asp1 in a synthetic peptide representing the first 16 residues of A{beta} (15). In addition, a single Zn2+ ion is coordinated by two copies of APP170-188, a synthetic peptide representing an N-terminal region of APP (16). These observations suggest that Zn2+ is able to induce dimerization of APP. Under oxidative pathologic conditions, this could result in enhanced A{beta} formation and aggregation. The finding gives more support to the growing body of evidence that zinc is involved in AD.

A major impediment to studying the role of Zn2+ in AD is the lack of powerful visualization methods. Histochemical staining techniques to visualize Zn2+-containing plaques include the fluorescent probes TSQ (17) and zinquin (18). Meredin Stoltenberg of the University of Aarhus, Denmark, described a more sensitive method called immersion autometallography (19). This method relies on the silver enhancement of zinc-sulfur nanocrystals, which are created when 1- to 2-mm thick brain slices are immersed in a 0.1% sodium sulfide and 3% glutaraldehyde phosphate-buffered NeoTimm solution for 3 days. Using this method, the researchers were able to detect for the first time Zn2+ ions in plaques of the cerebellum of Tg2576 mice, a transgenic mouse model of AD.

Oxidative stress, an important feature of aging (see "The Two Faces of Oxygen"), is increased in several age-related neurodegenerative diseases, including AD, Huntington's disease, Parkinson's disease (see Andersen Review and Giasson Perspective), and prion diseases (20). The latter occur exclusively in elderly patients, with the exception of the new variant Creutzfeldt-Jakob disease caused by contaminated beef. It has been suggested that an imbalance in metal ions, such as depletion of Cu2+ and accumulation of Mn2+, can result in conditions that lead to the formation of the pathogenic, protease-resistant prion protein (PrP) from its normal, protease-sensitive counterpart (21). The work I presented at the meeting showed that protease K-resistant PrP molecules could be generated recombinantly in yeast cells in vivo by increasing extracellular Cu2+ and Mn2+ concentrations. In addition, analysis of yeast cells grown in medium supplemented with these ions further supported an active role for PrP23-230 (a form of the prion protein that lacks the N-terminal signal peptide of 22 amino acids and the C-terminal signal peptide of 23 amino acids required for the attachment of the glycosyl phosphatidyl inositol membrane anchor) in metal-ion homeostasis by decreasing intracellular Cu2+ concentrations.

In contrast to protease-resistant conformations of yeast PrP (PrPes) generated in vitro, the new protease-resistant PrPes molecules generated in vivo were not structurally interconvertible by the chelating agent ethylenediaminetetraacetic acid. Thus, PrPres molecules generated in vivo may more closely resemble the prion proteins deposited in the brains of patients with neurodegenerative diseases, which share this characteristic.

Conclusion

A disturbance of brain metal-ion homeostasis during aging could be one of the major risk factors for AD and prion disorders. Iron and copper concentrations increase with normal aging, whereas zinc concentrations either remain unchanged or are slightly decreased or elevated in AD. The research highlighted in this Perspective is important not only to understand the mechanisms of normal metal-ion homeostasis in more detail but also to elucidate the role of metal ions in these diseases. Whereas the research is still in its early stages, future experiments should provide a much greater understanding of these issues and assist in the development of novel therapeutic strategies.


September 7, 2005
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Citation: C. Treiber, Metals on the Brain. Sci. Aging Knowl. Environ. 2005 (36), pe27 (2005).








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