Zinc and Zinc Transport in AD 675 cysteines. Metallothioneins display high Zn binding affinity (K Zn = 3.2 × 10 –13 M –1 at pH 7.4), can bind 7 atoms of Zn per molecule, and function to sequester Zn immediately after uptake by cells to prevent toxicity (Palmiter, 1998). These pro- teins are ubiquitous and expression can be induced by metals including mercury and cadmium, glucocorticoids, proinflammatory cytokines, oxidative stress, elec- trophilic compounds, and xenobiotics (Kagi and Schaffer, 1988; Palmiter, 1998; Vallee, 1995). In mammals, four subfamilies of MT exist (MT-I, MT-II, MT-III, and MT-IV) with three functional isoforms expressed in brain including MT-I and II, which are expressed in astrocytes, the perivascular space, and pia mater (Penkowa et al., 1999) in a Zn-dependent manner (Atar et al., 1995; Durnam and Palmiter, 1981) and MT-III, which is most abundant in neurons that sequester Zn in synaptic vesicles (Bush et al., 1994). In general MT are thought to be largely intracellular with localization in the cytoplasm, lysosomes, and mitochondria (Penkowa, 2006) and are regularly translocated to the nucleus during cell division and under oxidative stress (Cherian and Apostolova, 2000; Klaassen et al., 1999; Maret, 2002; Trayhurn et al., 2000). Because of their small size MT-I and MT-II are able to diffuse into the nucleus through nuclear pore complexes where they are retained by nuclear factors (Penkowa, 2006). Metallothioneins have a low redox potential (–366 mV) that allows mild oxidation to decrease Zn binding and facilitates release of Zn for binding to Zn finger and other transcription factors that modulate DNA binding efficiency and expression of antioxidant genes during periods of oxidative stress (Mocchegiani et al., 2005). 3.2 ZIP Proteins Although the mechanism of transport of Zn from brain extracellular environments to intracellular compartments in neurons and glia is not completely understood, it is thought to involve members of the ZIP family of proteins (Chromy et al., 2003). The ZIP family of proteins was initially identified based on their functional and structural similarity to the ZRT yeast family (Eide, 1998) and the IRT transporters of Arabidopsis thaliana (Grotz et al., 1998). ZIP proteins are predicted to have 8 transmembrane domains with a histidine-rich intracellular loop between domains 3 and 4 (Huang et al., 2005) and are part of the plasma membrane or membranes of intracellular organelles. Using mouse and human sequence analysis 14 mammalian ZIP proteins that elevate intracellular Zn by increasing Zn uptake (ZIP 1–5; 7–15) or by releasing Zn from intracellular stores when Zn is deficient (ZIP 6 and 7) have been identified. ZIPs have no ATP binding sites or ATPase domains and function in an energy-independent manner (Gaither and Eide, 2000, 2001). ZIP-1 mRNA is expressed ubiquitously (Gaither and Eide, 2001) whereas ZIP-2 is specific to spleen, small intestine, and bone marrow (Gaither and Eide, 2000). Similarly, ZIP-3 expres- sion is high in bone marrow and spleen (Gaither and Eide, 2000). ZIP-4 expression is associated primarily with small intestine, and kidney (Wang et al., 2002b) and is increased during periods of Zn deficiency (Cousins et al., 2003; Dufner-Beattie 676 M.A. Lovell et al., 2003). Human ZIP-5 is highest in intestine, liver, kidney, and pancreas (Wang et al., 2004) whereas ZIP-6 is associated with prostate and placenta (Taylor et al., 2003). In humans ZIP-7 is ubiquitously expressed and is subcellularly localized to the Golgi apparatus where it functions to release Zn to the cytoplasm during periods of low intracellular Zn (Huang et al., 2005). The remaining ZIP proteins (8–15) have been identified by database searches but are yet to be localized. In addition to ZIP proteins, neuronal Zn uptake may also be mediated by a variety of Zn-permeable membrane spanning channels including Ca 2+ permeable AMPA/kainate channels (Jia et al., 2002), voltage-gated L-type Ca 2+ (Colvin et al., 2003), N-methyl-D aspartate (NMDA) receptor gated (Koh and Choi, 1994), and Na + /Zn 2+ exchangers (Cheng and Reynolds, 1998). 3.3 ZnT Proteins Zinc transport (ZnT) proteins serve as a counterpoint to ZIP proteins and function in the export of cytoplasmic Zn to the extracellular space or the sequestration of Zn in intracellular organelles. ZnT proteins are members of the cation diffusion facilitator family of proteins and are predicted to have 6 transmembrane domains with a histidine-rich loop between transmembrane domains 4 and 5. Presently, eight ZnT proteins have been described (reviewed in Eide, 2006) with two additional ZnT genes (ZNT-9 and ZNT-10) predicted based on analysis of the mouse and human genome (Seve et al., 2004; Sim and Chow, 1999). ZnT-1 is located at the plasma membrane, whereas the other ZnT proteins are expressed at the membrane of intra- cellular organelles. ZnT-1 is present in multiple organs including brain (Palmiter, 1995) and is induced in the presence of elevated cytoplasmic Zn through direct bind- ing of Zn to the Zn-finger domain of metal response element-binding transcription factor-1 (MTF-1; reviewed in Andrews, 2001). After binding Zn, MTF-1 translo- cates to the nucleus where it binds the metal response element (MRE) in genes for ZnT-1, MT, and gamma glutamylcysteine synthetase heavy chain which con- trols the rate-limiting step in glutathione synthesis (reviewed in Andrews, 2001). Initial in vitro studies of ZnT-1 showed overexpression in baby hamster kidney cells conferred resistance to increased Zn with the rate of Zn efflux increasing as extracellular Zn concentrations increased suggesting Zn efflux mediated by ZnT-1 is an energy-dependent process and argues against ZnT-1 being a channel or facil- itated transporter (Palmiter, 1995). Later studies demonstrated that ZnT-1 reduces Zn influx through the L-type calcium channels (LTCC) without increasing Zn efflux (Nolte et al., 2004; Ohana et al., 2006; Segal et al., 2004). In addition, in vivo studies (Chowanadisai et al., 2005) showed rats provided a Zn-deficient diet demonstrated decreased brain ZnT-1, suggesting low systemic Zn could decrease ZnT-1 to main- tain or increase brain Zn stores which is consistent with studies of Takeda et al. (2001) who found rats on a Zn-deficient diet showed increased brain Zn. Studies from our laboratory show ZnT-1 protein expression and function can be inactivated by HNE (2006aSmith et al., ), a neurotoxic aldehydic marker of lipid peroxidation present in MCI and LAD brain (Lovell et al., 1997; Williams et al., 2005). Zinc and Zinc Transport in AD 677 ZnT-2, a component of vesicular acid intracellular compartments, is predomi- nantly expressed in intestine, kidney, and testis and is scarcely detected in brain in mice (Palmiter et al., 1996). Overexpression of ZnT-2 in baby hamster kidney cells conferred resistance to elevated Zn with sequestration into acidic compart- ments at higher concentrations (Palmiter et al., 1996). In contrast, coexpression of ZnT-1 suppressed ZnT-2 mediated transport into acidic vesicles suggesting ZnT-2 has a relatively low affinity for Zn and functions only under excessive elevations of Zn as a second line of defense when other ZnTs fail to function properly (Palmiter et al., 1996). ZnT-3 sequesters Zn in vesicles and has expression limited to brain and testis (Palmiter et al., 1996). In mouse brain, ZnT-3 is associated with hippocampal dentate granule cells, pyramidal, and intraneurons as evidenced by levels of mRNA (Palmiter et al., 1996). ZnT-4 exhibits considerable homology with ZnT-2 and 3 and has expression in mammary gland and brain (Huang and Gitschier, 1997). Functionally, ZnT-4 sequesters Zn in acidic vesicles and is involved in the transport of Zn 2+ into milk during lactation (Kelleher and Lonnerdal, 2002). In contrast to other ZnT proteins, ZnT-5 is predicted to have 15 membrane spanning domains and is less than twice the size of other ZnT proteins (Colvin et al., 2003). In mice ZnT-5 mRNA is found in most organs although the highest protein expression is in the pancreas where is it associated with Zn-enriched secretory gran- ules in insulin containing β cells. ZnT-5 is scarcely detected in brain (Kambe et al., 2004) in mice although more recent studies observed ZnT-5 immunostaining in SP of AD brain (Zhang et al., 2008a). In mice ZnT-6 mRNA is present in multiple organs including brain and sequesters cytoplasmic Zn in the trans-Golgi network (TGN) and vesicular compartments (Huang et al., 2002). ZnT-6 mRNA is present in multiple organs including brain. Similarly, ZnT-7 sequesters Zn in the TGN but has expression limited to lung and small intestine (Kirschke and Huang, 2003). ZnT-8 has been characterized and is primarily associated with secretory granules of pancreatic β cells (Kleineke and Brand, 1997; Rivlin et al., 1999) where it likely plays a role in insulin transport. In mice ZnT-8 has limited expression in brain. 4 Zinc, Zinc Transport, Alzheimer’s Disease, and Mouse Models of AD The potential role of Zn in the pathogenesis of AD has been of interest since 1981 when Burnet (Burnet, 1981) proposed that Zn deficiencies led to dementia. Initial studies of AD and control brain showed significantly decreased Zn in the hippocam- pus, inferior parietal lobule, and occipital cortex of LAD subjects (Andrasi et al., 1990, 1993; Corrigan et al., 1993; Deng et al., 1994). In contrast, later studies using short postmortem interval tissue specimens from well-characterized LAD and con- trol subjects showed significant elevations of Zn in LAD hippocampus, amygdala, and multiple neocortical areas (Cornett et al., 1998; Danscher et al., 1997; Deibel 678 M.A. Lovell et al., 1996; Ehmann et al., 1986; Samudralwar et al., 1995; Wenstrup et al., 1990). The use of formalin-fixed tissues in some of the earlier studies, which could have led to mobilization and loss of Zn, has been suggested to account for the observed differences in these studies. In addition, earlier studies may have also included con- trol subjects that were not prospectively evaluated. Although multiple studies show alterations of Zn in LAD, there are few reports of Zn concentrations in brain in earlier stages of the disease. Although several studies have quantified changes in Zn at the bulk level, changes in the cellular localization of Zn in the progression of AD remains unclear. Studies of Zn at the microprobe level have primarily focused on the association of Zn with SP. Initial studies using microparticle induced X-ray emission (micro-PIXE), showed increased Zn in SP compared to adjacent neuropil and an elevation of Zn in LAD neuropil compared to age-matched normal control (NC) subjects (Lovell et al., 1998). Subsequent studies confirmed t hose findings in AD (Cherny et al., 1999; Frederickson et al., 2000; Miller et al., 2006; Stoltenberg et al., 2005) and in amyloid plaques of Tg2576 transgenic mice expressing mutant APP (Friedlich et al., 2004; Lee et al., 1999). Using Raman microscopy to evaluate the structure and com- position of isolated senile plaques Dong et al. (2003)showedZn 2+ and Cu 2+ were specifically coordinated with histidine residues in Aβ. Despite considerable study of Zn in SP, there have been relatively few studies that measure Zn in individual neurons in AD. Although the subject of extensive study over the past 25 years, the reasons for elevated brain Zn in AD are unclear. Several studies have attempted to relate changes in peripheral Zn to elevated brain levels, although results have been contradictory. Haines et al. (1991), Molina et al. (1998), and Shore et al. (1984) showed no signif- icant differences between AD and control serum Zn, whereas Jeandel et al. (1989) showed a significant decrease in Zn and other nutrients and antioxidant properties in AD serum, although the AD group may have contained malnourished subjects. The study of Haines et al. (1991) may also be questioned because it included control subjects whose Mini Mental Status Examination scores were considered cognitively impaired. In contrast, Rulon et al. (2000) and Gonzales et al. (1999) showed signifi- cant elevations of Zn in AD serum. Additionally, Gonzales et al. (1999) showed that serum Zn correlated with the presence of APOE4 alleles and concluded that of the indices analyzed in their study, only serum Zn appeared to be an independent risk factor associated with the development of AD. In a subsequent study of serum Zn in the progression of AD, we showed a statistically significant decrease of serum Zn in men with MCI compared to women with MCI or age-matched normal con- trol men (Dong et al., 2008). In contrast, there were no significant differences in serum Zn between well-characterized LAD subjects and cognitively normal control subjects. The observation of decreased serum Zn in MCI is of interest in light of previous in vivo rat studies that showed systemic Zn deficiencies led to diminished ZnT-1 levels and increased brain Zn (Chowanadisai et al., 2005; Nunomura et al., 2001; Takeda et al., 2001). These data support the hypothesis that elevated brain Zn in AD may be due to increased Zn uptake by brain under conditions of diminished extraparenchymal Zn in MCI. Zinc and Zinc Transport in AD 679 Similar to serum studies, measures of CSF Zn levels have also been inconsistent. Molina et al. (1998) showed decreased Zn in AD CSF compared to age-matched control subjects whereas Basun et al. (1991) showed no significant changes. In addi- tion, recent studies (Gerhardsson et al., 2008; Strozyk et al., 2007) showed there is an inverse relationship between Zn and copper concentrations and levels of Aβ 1-42 in CSF of LAD subjects and that degradation of soluble Aβ is normally promoted by physiological concentrations of both Cu and Zn (Strozyk et al., 2007). Although the potential variation of Zn through the progression of AD is of interest there have been no published studies of CSF levels of Zn in MCI subjects. Despite considerable interest in the mechanism by which Zn accumulates in the brain in AD, there has been relatively little study of proteins responsible for Zn influx and efflux. In the first study of ZnT-1 in AD, we used Western blot analyses to show significantly decreased ZnT-1 levels in the hippocampus/parahippocampal gyri (HPG) of MCI, but significant elevations in EAD and LAD (Lovell and Markesbery, 2005). In studies of multiple ZnT proteins in SP in AD brain, Zhang et al. (2008a) used confocal microscopy and double immunolabeling to show colo- calization of ZnT-1, ZnT-3, ZnT-4, ZnT-5, ZnT-6, and ZnT-7 with amyloid in SP in AD. Although all six ZnT proteins were present to varying degrees in SP, ZnT-5 demonstrated the most pronounced immunostaining in SP whereas ZnT-3 immunos- taining was more pronounced in amyloid angiopathic vessels. These data are similar to those observed in our studies of ZnT-4 and ZnT-6 in the progression of AD which showed significantly elevated ZnT-4 in the HPG and superior and middle temporal gyri (SMTG) of EAD and LAD subjects compared to age-matched controls (Smith et al., 2006b) and significantly increased ZnT-6 in the HPG of EAD and LAD sub- jects compared to normal control subjects and a trend toward a significant elevation in MCI (Smith et al., 2006b). We also observed a striking association of ZnT-6 with NFT-bearing neurons identified using the modified Bielschowsky stain in LAD and in neurons positive for MC-1, a marker of early NFT formation in MCI (Lovell et al., 2006). In studies of transgenic mouse models of amyloid deposition, Zhang et al. (2008b) used Western blot analysis to show significant elevations of ZnT-1, ZnT- 3, ZnT-4, ZnT-6, and ZnT-7 in the hippocampus and neocortex of mice expressing mutant APP and PS1 (APPSwePS1dE9). Immunolocalization showed that most amyloid plaques of APP/PS1 mice were immunopositive for ZnT-1 and ZnT-4 whereas ZnT-3, ZnT-5, and ZnT-6 were mainly associated with degenerating neu- rites at the plaque periphery. Levels of ZnT-1 were increased 300% in hippocampus and 200% in neocortex of APP/PS1 mice compared to wild-type (WT) mice of the same age. Levels of ZnT-6 and ZnT-7 showed the smallest increase in APP/PS1 hippocampus and neocortex with levels ∼150% those of WT mice. Levels of ZnT-5 were also elevated in APP/PS1 mice but did not reach statistical signif- icance. Of the proteins studied, ZnT-3 showed the most pronounced changes in hippocampus and neocortex of APP/PS1 mice (400 and 200%) compared to WT mice providing further support for the studies of Gosavi et al. (2002) who showed that crossing mice expressing mutant APP with ZnT-3-null mice led to diminished Aβ deposition. More recently, Friedlich et al. (2004) showed that these mice also 680 M.A. Lovell demonstrate reduced cerebral amyloid angiopathy that is hypothesized to be due to diminished Zn concentrations in the perivascular space of ZnT-3-null mice. In additional studies, Stoltenberg et al. (2007) showed that providing APP/PS1 mice a Zn-deficient diet from 9 to 12 months of age led to increased Aβ deposition but no significant changes in autometallographic staining of Zn or ZnT immunostain- ing. Although the mechanism by which Zn deficiencies would lead to increased Aβ deposition, but not alterations in ZnT proteins, is unclear, the data do sup- port the hypothesis that alterations of Zn may contribute to the pathogenic changes in AD. 5 Zinc and Amyloid Beta (Aβ) Peptide Processing and Aggregation Although considerable evidence suggests there are alterations of Zn homeostasis in the AD brain, direct evidence for its role in the pathogenesis of AD has been lacking. Although Zn may play a role in multiple pathways relevant to AD, to date the most widely studied has been the possible role of Zn in processing of APP and aggrega- tion of Aβ. APP synthesis is regulated by Zn-containing transcription factors, NF-κβ and sp1, and although Zn is essential for their activity (Yang et al., 1995; Zabel et al., 1991; Zeng et al., 1991), it is unclear whether the activity in vivo is regulated by Zn availability. In addition to the potential influence of Zn on APP expression, it may also affect proteolytic processing of the protein. Normal (nonamyloidogenic) pro- cessing of APP by α-secretase cleavage in the Golgi complex leads to formation of sAPP, a neurotrophic factor (Wilquet and De Strooper, 2004). In contrast, proteolytic processing of APP by β-secretase (BACE) at the β-cleavage site (Andrasi et al., 2000; Calingasan et al., 1999; Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999) occurs in endosomes (Kinoshita et al., 2003; Koo and Squazzo, 1994), where acidic pH necessary for β-secretase activity is possible (Wilquet and De Strooper, 2004) and coupled with further processing by the γ-secretase complex at the plasma membrane (reviewed in Sisodia and St. George-Hyslop, 2002) leads to formation of Aβ, a 40 or 42 amino acid peptide that is the major component of SP in AD (Selkoe, 1999) (amyloidogenic pathway). Additionally, APP contains a ligand-binding site for Zn spanning the α-secretase position (Bush et al., 1993, 1994). Zn concentra- tions less than 50 μM inhibit α-secretase-mediated sAPP formation and increase generation of Aβ (Bush et al., 1994) perhaps through altered protein conformation. In addition, high Zn concentrations can inhibit matrix metalloproteinase-2 (MMP-2) (Backstrom et al., 1992) an enzyme that partially degrades soluble Aβ 1-42 in vitro (Bergeron et al., 1996) which could lead to increased amyloidogenic Aβ levels. Most APP molecules are transported through the TGN where α-secretase cleavage likely occurs leading to formation of secreted APP (Wilquet and De Strooper, 2004). Because ZnT-6-mediated accumulation of Zn in the TGN could initially diminish α- secretase cleavage of APP, Zn could significantly modulate APP processing leading to increased Aβ production. In addition, the presence of elevated Zn in endosomes Zinc and Zinc Transport in AD 681 mediated by ZnT-2 or ZnT-4 or both could further enhance β-secretase activity through modulation of pH. Once generated, several reports indicate that Zn at low physiological concen- trations induces Aβ aggregation (Bush et al., 1996, 1994; Bush et al., 1995; Mantyh et al., 1993), although later studies indicate that higher Zn concentrations are required (Clements et al., 1996; Esler et al., 1996) for significant aggrega- tion (fibril formation). A subsequent study using atomic force and transmission electron microscopy and Aβ 13-21 shows Zn 2+ specifically controls the rate of fib- ril assembly and regulates fibril morphology via specific coordination s ites (Dong et al., 2006). Multiple studies show that treatment of cortical neuron cultures with Aβ leads to increased levels of reactive oxygen species, increased lipid peroxidation, pro- tein oxidation, mitochondrial dysfunction, caspase activation, and neuron death (Butterfield, 2003; Canzoniero et al., 1999; Keller et al., 2005; Yatin et al., 1999). In addition, several transgenic models of AD including those with mutant APP, mutant APP/PS1, or mutant APP/PS1 and tau show increased Aβ deposition (Gotz et al., 2001; Lewis et al., 2001; Oddo et al., 2004). Although Aβ deposits are associated with AD, the specific Aβ species responsible for neurodegeneration are unclear. Fibrillar Aβ, the predominant component of insoluble amyloid plaques, is neuro- toxic (Lorenzo and Yankner, 1994; Pike et al., 1993). However, in vivo, insoluble Aβ deposits do not accurately predict the severity of dementia in AD subjects (Cherny et al., 1999). In addition, studies of transgenic mice including those with APP mutations show cognitive dysfunction and synaptic damage that precede amy- loid plaque deposition and neuron loss (Irizarry et al., 1997; Kumar-Singh et al., 2000; Moechars et al., 1996; Mucke et al., 2000; Westerman et al., 2002), leading to the suggestion that soluble oligomeric or protofibril Aβ species may the most toxic. In vitro studies of synthetic Aβ show monomeric Aβ aggregates in a time- dependent manner that may be accelerated by Zn leading to oligomeric species, which may eventually form fibrils (Chromy et al., 2003; Pike et al., 1991;Walsh et al., 1997). Increasing evidence suggests that these soluble oligomeric species are the predominant neurotoxic species for neurons (Demuro et al., 2005; Klein, 2002), leading to inhibition of long-term potentiation in synaptic hippocampal slices (Lambert et al., 1998; Wang et al., 2002a), calcium dysregulation, and membrane dysfunction (Demuro et al., 2005; Kayed et al., 2004). Although the exact Aβ species responsible for mediating neurodegeneration in AD is unclear, several lines of evidence support a role for Zn in their formation. 6 Zinc as a Therapeutic Target in AD Because of the potential role of Zn and Cu in the deposition of Aβ in AD brain, there has been considerable interest in the use of metal chelation to decrease amyloid pathology (Bush, 2003). In vitro studies show clioquinol (CQ), an 8-OH quinoline 682 M.A. Lovell inhibits Aβ aggregation mediated by Cu and Zn (Cherny et al., 2001). In vivo studies show transgenic mouse models of amyloid deposition (Tg 2576) treated with CQ for 9 weeks showed significantly reduced amyloid plaque burdens (Cherny et al., 2001). In initial, phase-2 double-blind placebo-controlled clinical trials, CQ significantly slowed cognitive decline in AD patients compared to placebo controls (Ritchie et al., 2003). More recently, PBT2, an 8-hydroxy quinoline with increased blood–brain barrier permeability has been developed (Adlard et al., 2008) and in a 12-week phase-IIa clinical trial of AD subjects reversed frontal lobe functional deficits and significantly decreased Aβ 1-42 levels in CSF (Lannfelt et al., 2008). Together, these data suggest modulation of Zn may be an effective potential therapeutic target in AD. 7 Conclusions and Future Directions Although considerable evidence suggests a link between alterations in Zn and Zn transport and sequestration proteins in the progression of AD, further in-depth study is needed particularly early in the progression of AD (MCI) when therapeutic inter- ventions would have greater efficacy. In particular Zn levels in CSF of subjects with MCI and EAD need to be quantified and correlated with brain ZnT, ZIP, and Zn lev- els. Based on in vivo studies, it is tempting to hypothesize that low extraparenchymal Zn early in disease progression may lead to decreased ZnT-1 levels and a concomi- tant elevation of intracellular Zn that leads to increased levels of ZnT-2, ZnT-4, and ZnT-6 and increased localization of Zn in subcellular organelles in which Aβ processing occurs. 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