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Proc Natl Acad Sci U S A 91:7787–7791 Zinc and Zinc Transport and Sequestration Proteins in the Brain in the Progression of Alzheimer’s Disease Mark A. Lovell Abstract Multiple studies over the past 25 years have demonstrated alterations of zinc (Zn) in the brain in Alzheimer’s disease (AD), although the potential fole of these alterations in the pathogenesis of AD remains unclear. The following examines normal and abnormal roles of Zn and Zn transport (ZIP and ZnT) proteins in brain and the potential effects of t heir alterations in the pathogenesis of AD. Keywords Zinc · Early Alzheimer’s disease · Mild cognitive impairment · Zinc transporter proteins · Amyloid beta peptide · Neurodegeneration Contents 1 Introduction 670 1.1 Clinical Parameters of Mild Cognitive Impairment (MCI), Early AD (EAD), and Late Stage AD (LAD) 670 1.2 Pathological Characterization of MCI, EAD, and LAD 671 2 Zinc and Zinc Homeostasis 672 2.1 Zinc Transport and Sequestration 673 3 Maintenance of Zinc Homeostasis 674 3.1 Metallothioneins 674 3.2 ZIP Proteins 675 3.3 ZnT Proteins 676 4 Zinc, Zinc Transport, Alzheimer’s Disease, and Mouse Models of AD 677 5 Zinc and Amyloid Beta (Aβ) Peptide Processing and Aggregation 680 6 Zinc as a Therapeutic Target in AD 681 7 Conclusions and Future Directions 682 References 682 M.A. Lovell (B) Department of Chemistry, Sanders-Brown Center on Aging and Alzheimer’s Disease Center, University of Kentucky, Lexington, KY 40536, USA e-mail: malove2@uky.edu 669 J.P. Blass (ed.), Neurochemical Mechanisms in Disease, Advances in Neurobiology 1, DOI 10.1007/978-1-4419-7104-3_20, C Springer Science+Business Media, LLC 2011 670 M.A. Lovell 1 Introduction 1.1 Clinical Parameters of Mild Cognitive Impairment (MCI), Early AD (EAD), and Late Stage AD (LAD) Alzheimer’s disease (AD), the fourth leading cause of death in the United States, affected 4.5 million Americans in 2000 and may affect as many as 14 million by 2040 (Hebert et al., 2003). Current estimates suggest ∼3% of Americans between ages 65 and 74, 19% ages 75–84, and 47% over age 85 are victims of the disease (Evans et al., 1989) with ∼60% of nursing home patients over age 65 suffering from AD. Alzheimer’s disease is characterized clinically by a progressive decline in multiple cognitive functions and is thought to begin with amnestic mild cognitive impairment (MCI), widely considered to be a transition between normal aging and dementia. Recent studies suggest conversion from MCI to dementia occurs at a rate of 10–15% per year (Petersen and Morris, 2003) with a conversion rate of ∼80% by the sixth year of followup. Of the remain- ing MCI subjects ∼5% remain stable or convert back to normal (Bennett et al., 2002; DeCarli, 2003). Clinically, MCI is diagnosed based on the Petersen et al. criteria and is characterized by: (a) memory complaints, (b) objective memory impairment for age and education, (c) intact general cognitive function, (4) intact activities of daily living (ADLs), and (5) the subject is not demented (Petersen et al., 1999). Objective memory test impairment is based on a score of ≤1.5 stan- dard deviations from the mean of controls on the CERAD Word List Learning Task (Morris et al., 1989) and corroborated in some cases with the Free and Cued Selective Reminding Test. As the disease progresses patients are classified as early AD (EAD), patients and are clinically characterized by (a) a decline in cognitive function from a previous higher level, (b) decline in one or more areas of cognition in addition to memory, (c) a clinical dementia rating scale score of 0.5–1, (d) impaired ADLs, and (e) a clinical evaluation that excludes other causes of dementia. Disease progression ultimately leads to late stage AD (LAD) which is characterized clinically by impairment of recent memory, language distur- bances, and alterations of abstract reasoning, concentration, and thought sequencing (executive function) ( American Psychiatric Association, 2000). Diagnosis of prob- able AD is based on criteria from the National Institute of Neurological and Communicative Diseases and Stroke/Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) and is made when patients demonstrate (a) demen- tia established by clinical examination and documented by mental status tests, (b) deficits in two or more areas of cognition, (c) progressive worsening, (d) no dis- turbance in consciousness, (e) onset between age 40 and 90, and (f) no systemic or other brain diseases that could account for the progressive deficits (National Institute on Aging and Reagan Institute Working Group, 1997). The mean length of life following diagnosis is 8.5 years with a range of 1–25 years (Jost and Grossberg, 1995). Zinc and Zinc Transport in AD 671 1.2 Pathological Characterization of MCI, EAD, and LAD Pathological examination of the AD brain shows an abundance of neurofibrillary tangles (NFT), senile plaques (SP), increased neuropil thread formation, increased neuron and synapse loss and proliferation of reactive astrocytes, primarily in the hippocampus, amygdala, entorhinal cortex, and neocortex. Neurofibrillary tangles are intracellular lesions consisting of paired helical filaments composed primarily of hyperphosphorylated tau. Senile plaques are extracellular lesions and are present in two forms: (a) diffuse plaques (DP) composed of amorphous extracellular deposits of Aβ lacking neurites, and (b) neuritic plaques (NP) composed of extracellular deposits of insoluble Aβ surrounded by dystrophic neurites, reactive astrocytes, and activated microglia. In addition to insoluble Aβ present in SP, recent studies suggest soluble Aβ oligomers are present in the AD brain and may represent the main toxic form of Aβ, thus implicating them in the disease process (Glabe, 2006; Klein, 2002; Walsh et al., 2002). Senile plaques and NFT are the hallmark pathological lesions employed for the histopathologic diagnosis of AD based on the National Institute on Aging-Reagan Institute (NIA-RI) criteria (The National Institute on Aging, 1997). The NIA-RI criteria combine NP scores used by the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) with Braak staging scores to provide classifications of low, intermediate, and high likelihood for the diagnosis of AD. The CERAD cri- teria use NP densities in three neocortical regions (frontal, temporal, and parietal) to provide an age-related NP score that is used in conjunction with the clinical his- tory to reach a diagnosis of possible, probable, or definite AD. Braak staging scores (Braak and Braak, 1994) are based on the observation that NFT pathology pro- gresses in a topographically predictable manner from the transentorhinal (stages I and II) to entorhinal, hippocampus, amygdala, and adjacent temporal cortex (limbic stages III and IV) and then to the isocortex (stages V and VI). Pathologically, MCI subjects show significant increases in neocortical NP and NFT densities in entorhinal cortex, hippocampus, and amygdala compared to nor- mal control subjects (Markesbery et al., 2006) with Braak staging scores ranging from III to IV. Subjects with EAD generally meet NIA-RI high likelihood criteria for the histopathological diagnosis of AD with Braak staging scores of V but have less severe overall NFT and NP formation than observed in LAD. Multiple risk factors have been identified for AD and include age (Evans et al., 1989), a variety of genetic factors including mutations of presenilin 1 (PS1) and 2 (PS2), and the amyloid precursor protein (APP) (Levy-Lahad et al., 1995; St. George-Hyslop, 1994). In addition, single nucleotide polymorphisms in ubiquilin-1 (Bertram et al., 2005), a genetic locus on chromosome 10 that includes the insulin-degrading enzyme (Bertram et al., 2000; Ertekin-Taner et al., 2000; Myers et al., 2000) that may interact with and degrade Aβ, inherited variants in SORL1 (Rogaeva et al., 2007), and the presence of apolipoprotein E4 alleles (Corder et al., 1993) are associated with the risk of AD. Additional risk factors for AD 672 M.A. Lovell include head injury (Mortimer et al., 1991), diabetes (Chan et al., 1999; Leibson et al., 1997; Peila et al., 2002), hyperlipidemia (Jick et al., 2000), hypertension (Skoog et al., 1996), heart disease (Kleineke and Brand, 1997), smoking (McMahon and Cousins, 1998; Merchant et al., 1999), elevated plasma homocysteine (Seshadri et al., 2002), obesity (Gustafson et al., 2003), and low educational attainment and low linguistic ability early in life (Snowdon et al., 2000, 1996). Despite considerable research, the major barrier to treating and eventually pre- venting AD is a lack of understanding of the cause and mechanisms of neuron degeneration and loss. Because of the complexity of the disease, AD is likely a heterogeneous disease of multiple, probably interrelated, etiologic/pathogenic fac- tors. Numerous etiologic/pathogenic mechanisms have been suggested for the cause of AD including genetic defects (St. George-Hyslop et al., 1987; St. George-Hyslop, 1994), the amyloid cascade hypothesis (reviewed in Sommer, 2002), the oxidative stress hypothesis (Coyle and Puttfarcken, 1993), mitochondrial defects ( Wallace, 1992), trace element (including Zn) toxicity (reviewed in Markesbery and Ehmann, 1994), or a combination of the above. One hypothesis receiving renewed interest is the potential role of alterations of Zn homeostasis in the pathogenesis of AD. 2 Zinc and Zinc Homeostasis Zinc is an essential trace (μg/g) element (Prasad et al., 1963) in human health and biology. Although Zn is present in all organs ∼90% of total body zinc (1–2 g) is associated with bones and skeletal muscle (Sturniolo et al., 2000). Most dietary Zn is absorbed from the jejunum through passive diffusion and specific transporter proteins (Sturniolo et al., 2000). Once absorbed, Zn is transported in the plasma bound largely to albumin (Smith et al., 1979). Circulating Zn is trans- ported into the brain via the blood/brain and blood/cerebrospinal fluid (CSF) barriers (Nunomura et al., 2001) where brain capillary endothelial cells respond to changes in Zn status by increasing or decreasing Zn uptake (Lehmann et al., 2002). Once transferred to the CSF, Zn is quite mobile and is taken up by the brain in pro- cesses that are not completely understood but likely involve transporters of the Zrt-Irt (ZIP) family, zinc transporter (ZnT) family, or through a variety of other specific gated Zn permeable channels. At the cellular level Zn is redox inert and has structural, catalytic, and regulatory roles (Bettger and O’Dell, 1981; Golden, 1989; Vallee and Falchuk, 1993). Zinc is a crucial component in over 300 enzymes and transcription factors where it serves as an essential cofactor for catalytic activity (Frederickson, 1989) or by conferring structural stability to Zn finger domains of DNA binding proteins (Colvin et al., 2003) including stimulating protein-1 (sp-1), a transcription factor responsible for ∼30% of APP transcription (Bittel et al., 1998; Dalton et al., 1997, 1996). Additionally, recent studies suggest free Zn may possess important signaling functions including modulation of protein kinase C (PKC) sig- naling pathways (Korichneva et al., 2002), modulation of p53 mediated DNA repair through stabilization of p53/genomic DNA interactions (Mocchegiani et al., 2005), Zinc and Zinc Transport in AD 673 inhibition of gamma aminobutyric acid (GABA-ergic) neurotransmission (Haase and Beyersmann, 2002), and modulation of glycogen synthase kinase 3β (An et al., 2005; Ilouz et al., 2002). 2.1 Zinc Transport and Sequestration In the brain, Zn is distributed in discrete pools: (a) a membrane-bound met- alloprotein, or protein–metal complex pool involved in metabolic reactions and nonmetabolic functions such as biomembrane structure and support; (b) a vesicular pool present in nerve terminal synaptic vesicles; and (c) a cytoplasmic pool of free or loosely bound ions (Frederickson, 1989). The easily chelated vesicular pool may be the most important (Danscher et al., 1985; Frederickson et al., 1983; Haug, 1967; Perez-Clausell and Danscher, 1986) because it is released during neurotransmission and may reach neurotoxic levels of 300 μM in the synapse. Without immediate uptake and sequestration these Zn gradients could potentially induce neurodegener- ation. Mean brain Zn concentrations are highest in the hippocampus, amygdala, and neocortex and are relatively low in cerebellum (Danscher et al., 1997; Frederickson et al., 2005), a pattern that mirrors the distribution of pathological features in AD. These Zn concentrations range between 150 and 200 μM (Ebadi et al., 1995;Price and Joshi, 1982) and are ∼10 times serum Zn levels (Takeda, 2000). At the cellular level Zn concentrations range from nanomolar levels in the cytoplasm of most neu- rons to millimolar concentrations in vesicles of mossy fiber terminals (Frederickson et al., 1983; Williams, 1989). Although Zn is critical for normal brain function, in vitro and in vivo studies show high concentrations of Zn are toxic to neurons (Choi et al., 1988; Duncan et al., 1992; Yokoyama et al., 1986; Chuah et al., 1995; Cuajungco and Lees, 1996; Koh et al., 1996) resulting in increased oxidative stress, and necrotic and apoptotic cell death occurring in as little as 30 min (Choi et al., 1988; Gaskin and Kress, 1977; Manev et al., 1997; Kim et al., 1999). Although elevated Zn can be neurotoxic, the exact mechanism of Zn-induced cell death remains unclear. One possible mecha- nism by which Zn mediates neurotoxicity is through the potentiation of glutamate (Beaulieu et al., 1992; Bramham et al., 1990; Danscher et al., 1985; Frederickson et al., 1983; Kesslak et al., 1987; Stengaard-Pedersen et al., 1983), AMPA (Buschke et al., 1999; Choi et al., 1988; Freund and Reddig, 1994; Koh and Choi, 1987), or kainic acid (Choi et al., 1988; Shore et al., 1984; Yin and Weiss, 1995) toxic- ity. In addition, Zn has been shown to play a role in mitochondrial dysfunction by inhibiting the transfer of an electron between coenzyme Q and cytochrome b of the bc 1 complex (Blennow et al., 1995; Hunter and Ford, 1955; Kleiner and von Jagow, 1972), thus inhibiting the initial step of respiration. At high Zn concentrations levels of complex I and II and cytochrome oxidase are inhibited (Skhulachev et al., 1967), although Yamaguchi et al. (1982) demonstrated increased mitochondrial function in rat liver after a single low dose of Zn. Later studies (Canzoniero et al., 1999; Ho et al., 2000; Krotkiewska and Banas, 1992)showednM–μM concentrations of Zn can inhibit a number of enzymes required for mitochondrial respiration and 674 M.A. Lovell glycolysis. Zinc-mediated dysfunction in oxidative phosphorylation and the resul- tant increase of free radical generation could in turn lead to release of Zn from MT and further increased intracellular concentrations of Zn (Fliss and Menard, 1992). Zinc at relatively low concentrations can inhibit sodium/potassium ATPase (Na + K + ATPase) activity in isolated protein, inhibit glutamate and GABA uptake in mice synaptosomes (Gabrielsson et al., 1986), and glutamate transport by human excita- tory amino acid transporter (EAAT) 1 in Xenopus laevis oocytes (Vandenberg et al., 1998). Zinc is hypothesized to influence assembly and disassembly of tubulin (Eagle et al., 1983; Gaskin and Kress, 1977; Gaskin et al., 1978) and several microtubule associated proteins in vitro ( Backstrom et al., 1992; Gaskin and Kress, 1977; Gaskin et al., 1978; Kress et al., 1981) contributing to structural abnormalities. In addition, Zn may mediate tau phosphorylation through modulation of P13/AKT, ERK1/2, and p38/MAPK signaling cascades (An et al., 2005). Influx of Zn through NMDA receptor channels may lead to neuronal depolarization and an increase of intracellular calcium (Ca) that could further activate second messenger systems via PKC-mediated phosphorylation of receptor ion channels or voltage-dependent gene expression (Atar et al., 1995; Murakami et al., 1987; Rubin and Koide, 1973). Calcium homeostasis may be further disrupted by Zn binding to calmod- ulin (Baudier et al., 1983) and the inhibition of calmodulin-complexed Ca ATPase (Brewer et al., 1979). Chelatable Zn has been shown to accumulate in the cell perikarya of apoptotic neurons before and during degeneration following ischemia insult (Kress et al., 1981; Tonder et al., 1990) or seizure activity (Frederickson, 1989), and is suggested to play a pathological role in neuron death. More recent studies demonstrated increased intracellular Zn as an early event in the apoptotic pathway that occurs in the absence of exogenous Zn and is consistent with a release of Zn from intracellular stores (Zalewski et al., 1994). Because of the essential but potentially toxic qualities of Zn it is imperative that cells regulate Zn levels through control of influx and efflux and through chelation to Zn sequestering proteins. 3 Maintenance of Zinc Homeostasis In general, Zn homeostasis is maintained by three families of proteins: (a) met- allothioneins (MT) that quickly bind, sequester, and hold Zn after influx into the cytoplasm, (b) Zrt–Irt-like (ZIP) proteins that likely mediate Zn influx into the cell, and (c) zinc transporter (ZnT) proteins that mediate efflux of cytoplasmic Zn to the extracellular space or sequestration in intracellular organelles. 3.1 Metallothioneins Metallothioneins are a superfamily of nonenzymatic low molecular weight (6–7 kDa) single polypeptide chains of 61–68 amino acids, 25–30% of which are . activity (Frederickson, 1989) or by conferring structural stability to Zn finger domains of DNA binding proteins (Colvin et al., 2003) including stimulating protein-1 (sp-1), a transcription factor responsible. to Zn sequestering proteins. 3 Maintenance of Zinc Homeostasis In general, Zn homeostasis is maintained by three families of proteins: (a) met- allothioneins (MT) that quickly bind, sequester,. alterations of zinc (Zn) in the brain in Alzheimer’s disease (AD), although the potential fole of these alterations in the pathogenesis of AD remains unclear. The following examines normal and