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Originally published In Press as doi:10.1074/jbc.M408127200 on August 20, 2004

J. Biol. Chem., Vol. 279, Issue 43, 44795-44801, October 22, 2004
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Increased tau Phosphorylation in Apolipoprotein E4 Transgenic Mice Is Associated with Activation of Extracellular Signal-regulated Kinase

MODULATION BY ZINC*

Faith M. Harris{ddagger}§, Walter J. Brecht{ddagger}§, Qin Xu{ddagger}§, Robert W. Mahley{ddagger}§¶||, and Yadong Huang{ddagger}§**

From the Gladstone Institutes of {ddagger}Neurological Disease and §Cardiovascular Disease and the Departments of Pathology and ||Medicine, University of California, San Francisco, California 94141-9100

Received for publication, July 19, 2004 , and in revised form, August 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although apolipoprotein (apo) E4 is present in amyloid plaques and neurofibrillary tangles, its pathogenic role in Alzheimer's disease (AD) is unclear. Neuronal expression of apoE4 or apoE4 fragments in transgenic mice increases tau phosphorylation. To identify the kinase responsible for the increase, we studied transgenic mice expressing human apoE3 or apoE4 in neurons under the control of the neuron-specific enolase promoter. Brain levels of phosphorylated tau (p-tau) and phosphorylated (active) extracellular signal-regulated kinase (p-Erk) increased with age in both groups but were considerably higher in the apoE4 mice. Other candidate kinases, including glycogen synthase kinase 3{beta} and cyclin-dependent kinase-5 and its activators p25 and p35, were not significantly altered. The increases in p-Erk and p-tau were highest in the hippocampus, intermediate in the cortex, and lowest in the cerebellum. In the hippocampus, p-Erk and p-tau accumulated in the hilus and CA3 region of the dentate gyrus, where high levels of zinc are found along mossy fibers. In Neuro-2a cells stably expressing apoE3 or apoE4, treatment with ZnCl2 generated 2-fold more p-Erk and 3-fold more p-tau in the apoE4-expressing cells. Phosphorylation of Erk and tau was reduced by preincubation with the Erk pathway inhibitor U0126. Thus, increased tau phosphorylation in apoE4 transgenic mice was associated with Erk activation and could be modified by zinc, suggesting that apoE4 and zinc act in concert to contribute to the pathogenesis of AD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human apolipoprotein (apo)1 E4 has been identified as a major risk factor for Alzheimer's disease (AD) (1, 2). It increases the occurrence and lowers the age of onset of AD and is associated with ~40–65% of cases of sporadic and familial AD (1, 2). The neuropathological hallmarks of AD are neuritic amyloid plaques and neurofibrillary tangles (NFTs) in the brain (3–5). The plaques are extracellular deposits consisting primarily of amyloid-beta (A{beta}) peptides (3–5), the proteolytic products of the amyloid protein precursor. NFTs are primarily intracellular deposits composed largely of highly phosphorylated tau (p-tau), a microtubule-associated protein (4), as well as phosphorylated neurofilaments of high molecular weight (6, 7). Both plaques and NFTs contain apoE (8–10), but the role of apoE in the pathogenesis of these lesions is unclear.

Several hypotheses have been proposed to explain the association of apoE4 with AD (10–14). They include modulation of the deposition and clearance of A{beta} peptides and the formation of plaques (15–21), impairment of the antioxidative defense system (22), dysregulation of neuronal signaling pathways (23), altered phosphorylation of tau and NFT formation (24–29), depletion of cytosolic androgen receptor levels in the brain (30, 31), potentiation of A{beta}-induced lysosomal leakage and apoptosis in neuronal cells (32), and promotion of endosomal abnormalities linked to A{beta} overproduction (33–35). However, the mechanisms of these apoE4-mediated detrimental effects are largely unknown, and it is not known which are the primary effects and which are subsequent or downstream effects.

ApoE3 and apoE4 have different effects on the structure and function of the cytoskeleton (36–38). Levels of p-tau were higher in transgenic mice expressing apoE4 in neurons than in mice expressing apoE4 in astrocytes (25, 39), suggesting a neuron-specific effect of apoE4 on tau phosphorylation. We have shown that apoE4 or apoE4 fragments are much more effective than apoE3 in stimulating abnormal phosphorylation of tau in transgenic mice (28, 29, 40). Because tau normally binds to and stabilizes microtubules and stimulates their assembly by polymerizing tubulin, hyperphosphorylation may prevent tau from interacting with these structures, leading to their destabilization and NFT formation (4, 41).

Many kinases may be involved in tau phosphorylation in vitro and in vivo (42–53), including glycogen synthase kinase-3{beta} (GSK-3{beta}), cyclin-dependent kinase-5 (Cdk-5) and its activators, the phosphorylated (active) form of extracellular signal-regulated kinase (p-Erk), microtubule-affinity regulating kinase, and fyn kinase. Zinc, which is highly concentrated in synaptic vesicles in the mossy fiber zone of the hippocampus and may also be involved in AD pathogenesis (54), can activate some of these kinases, including Erk (55–62). How apoE4 stimulates tau phosphorylation is largely unknown.

In this study, we investigated the mechanisms by which apoE4 elicits abnormal phosphorylation of tau in transgenic mice. Here we report that apoE4 increases zinc-induced Erk activation in neuronal cells and enhances tau phosphorylation, especially in the hippocampus, in transgenic mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Minimum essential medium, N2 medium supplements, and fetal bovine serum were from Invitrogen. Polyclonal goat anti-human apoE was from Calbiochem. The phosphorylation-dependent monoclonal tau antibodies AT8 (p-Ser-202) and AT270 (p-Thr-181) were from Endogen (Woburn, MA). The phosphorylation-dependent monoclonal Erk(p-Thr-202/Tyr-204) antibody E10, the phosphorylation-independent polyclonal Erk antibody, the Erk pathway inhibitor U0126, and the phosphorylation-dependent monoclonal GSK-3(p-Tyr-279/Tyr-216) antibody 5G-2F were from Cell Signaling (Charlottesville, VA). The phosphorylation-dependent Cdk5(p-Tyr-15) monoclonal antibody and the polyclonal p35 and p25 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-rabbit, anti-mouse, and anti-goat IgGs coupled to fluorescein, rhodamine, or Cy5 were from Vector Laboratories (Burlingame, CA). Horseradish peroxidase-coupled anti-rabbit, anti-mouse, and anti-goat IgG were from Dako (Carpinteria, CA). ECL was from Amersham Biosciences.

Transgenic Mice Expressing ApoE3 or ApoE4 in Neurons—We studied transgenic mice expressing human apoE3 or apoE4 in central nervous system neurons under the control of a neuron-specific enolase (NSE) promoter on a mouse apoE-deficient background (29, 30, 63). All NSE-apoE transgenic mice had been backcrossed more than 15 times with apoE-deficient mice (C57BL/6J-ApoetmlUnc; Jackson Laboratory, Bar Harbor, ME). Because apoE4-induced behavioral deficits in NSE-apoE mice are gender dependent (30, 31), only female transgenic mice were used. Mice were kept under a 12-h light/12-h dark cycle with free access to sterile water and food (PicoLab Rodent Diet 20, No. 5053; PMI Nutrition International, St. Louis, MO).

Genotyping of Transgenic Mice—Mice were genotyped by PCR of DNA purified from tail clips (29, 30, 63). Because APOE intron 3 was included in the apoE4 but not the apoE3 construct, the amplicon generated with intron 3-spanning primers (forward primer: nucleotides 3158–3175; reverse primer: nucleotides 3815–3834; GenBankTM accession number M10065 [GenBank] ) was 670 bp in apoE4 mice and 100 bp in apoE3 mice. PCR reactions were run on a PTC-100 programmable thermal controller (MJ Research, Watertown, MA), and the products were analyzed on 1.5% agarose gels. To assess the Apoe knockout status of the mice, total plasma cholesterol levels were measured with a kit (Abbott Laboratories, Abbott Park, IL) as described (64).

Western Blotting and Quantitative Analyses of p-Erk and p-tau in Mouse Brain Lysates—Brains from hemizygous NSE-apoE3 or NSE-apoE4 transgenic mice on a mouse apoE-null background were collected after a 2-min transcardial perfusion with PBS. One hemibrain or dissected regions (cortex, hippocampus, and cerebellum) were homogenized and analyzed for apoE, p-tau, Erk, p-Erk, p25, p35, Cdk-5, and GSK-3{beta} by Western blot as described (26, 28, 29). Values are reported in arbitrary units.

Immunohistochemistry—The other hemibrain was fixed in 3% paraformaldehyde, sectioned, and stained with anti-apoE, anti-p-tau, and anti-p-Erk (26, 28, 29, 63). Vibratome sections were incubated in 0.3% H2O2 in PBS for 20 min to quench endogenous peroxidase activity. To facilitate penetration of antibodies, sections used for immunoperoxidase staining were preincubated for 4 min in 1 µg/ml proteinase K in a buffer containing 250 mM NaCl, 25 mM EDTA, 50 mM Tris-HCl, pH 8. To block nonspecific reactions, all sections were incubated for 1 h in 15% normal serum from the same species that produced the secondary antibodies (Jackson ImmunoResearch, West Grove, PA) in PBS or for 7 min in Superblock (Scytec, Logan, UT), followed by a 1-h incubation in PBS with primary antibody (anti-apoE, anti-p-tau, anti-p-Erk, or anti-Erk). Sections were washed three times in PBS and incubated for 1 h with the corresponding secondary antibodies labeled with fluorescein (Jackson ImmunoResearch) or biotin (Vector). After three washes in PBS, the sections were mounted in VectaShield (Vector) and viewed with a Radiance 2000 laser-scanning confocal system (Bio-Rad) mounted on an Optiphot-2 microscope (Nikon, Tokyo, Japan). For immunoperoxidase staining, secondary antibody binding was detected with the ABC-Elite kit (Vector). Six sections of each brain were stained for each antibody.

Generation of Neuro-2a Cells Stably Expressing Human ApoE—Neuro-2a cells (American Type Culture Collection, Manassas, VA) were transfected with 17 kb of human apoE genomic DNA encoding apoE3 or apoE4 and a plasmid containing the neomycin resistance gene by the LipofectAMINE method (40). The apoE genomic DNA construct consists of 5 kb of 5'-flanking region, four exons, three introns, and 8 kb of 3'-flanking region. Stably transfected cells were selected by growth in minimal essential medium containing 10% fetal bovine serum and 400 µg/ml G418 for more than 2 weeks and cloned with a cell sorter (40). Cell lines stably expressing similar levels of apoE3 or apoE4, as determined by reverse transcriptase-PCR and anti-apoE Western blotting, were used in this study. Neo-transfected Neuro-2a cells were used as controls.

Western Blot Analysis of Cell Lysates—Stably transfected Neuro-2a cells expressing Neo, apoE3, or apoE4 were grown to 80% confluence in 6-well plates, incubated with ZnCl2 (50–200 µM) in the presence or absence of the Erk pathway inhibitor U0126 (10 µM) for 24 h, harvested, and lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and a mixture of protease and phosphatase inhibitors) for 30 min. After centrifugation at 35,000 rpm for 30 min, proteins (100 µg) in the supernatant were subjected to SDS-PAGE and detected by anti-apoE, anti-Erk, anti-p-Erk, or anti-p-tau Western blotting as described above.

Statistical Analysis—Results are reported as mean ± S.D. Differences were evaluated by t test or analysis of variance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Age-dependent Accumulation of p-tau and p-Erk Is Increased in NSE-ApoE4 Mice—Brain homogenates of 3- to 21-month-old NSE-apoE3 or NSE-apoE4 mice, which expressed similar levels of human apoE in brains (apoE3, 331 ± 52 ng/mg protein; apoE4, 316 ± 58 ng/mg protein) were analyzed by Western blotting with monoclonal antibody AT8, which recognizes p-tau isoforms with molecular masses of 55–60 kDa. The levels of p-tau were much higher in NSE-apoE4 mice (Fig. 1A). Semi-quantitative analysis of 3- to 21-month-old mice revealed considerably greater age-dependent accumulation of p-tau (all isoforms together) in NSE-apoE4 mice, especially aged mice (Fig. 1B).



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  		FIG. 1.
Age-dependent accumulation of p-tau in the brains of NSE-apoE4 mice. A, p-tau in brain homogenates (150 µg of total protein) of three NSE-apoE3 or NSE-apoE4 mice at 9 months of age was detected by Western blotting with monoclonal antibody AT8. B, quantitative analysis of p-tau (all isoforms) in brain homogenates of NSE-apoE3 and NSE-apoE4 mice at 3–21 months of age (n = 4–6 per genotype and age; p <0.01, apoE3 versus apoE4 mice at 7–21 months).

 
Immunoblotting of brain sections from 5- to 18-month-old mice revealed age-dependent accumulation of p-Erk, especially p-Erk2, that was significantly greater in NSE-apoE4 mice, although total Erk levels were similar (Fig. 2A). The ratios of p-Erk to Erk were also significantly higher in NSE-apoE4 mice at all ages (Fig. 2B). No age- or isoform-specific differences were seen in other candidate tau kinases or their activators, including active GSK-3{alpha}/{beta} (Fig. 2C), active Cdk-5 (data not shown), p35 (Fig. 2D), and p25 (data not shown).



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  		FIG. 2.
Age-dependent accumulation of p-Erk in the brains of NSE-apoE4 mice. A, total Erk and p-Erk levels in brain homogenates (150 µg of total protein) of NSE-apoE3 or NSE-apoE4 mice at 5–18 months of age were detected by Western blotting with polyclonal anti-Erk or monoclonal anti-p-Erk. B, quantitative analysis of the ratio of p-Erk (p-Erk1 + p-Erk2) to Erk (Erk1 + Erk2) in brain homogenates (n = 4–5 mice per genotype and age; p <0.01, apoE3 versus apoE4 mice at any age). C, phosphorylated (active) GSK-3{alpha} and GSK-3{beta} in brain homogenates (150 µg of total protein) were detected by Western blotting with phosphorylation-dependent GSK-3{alpha}(Tyr-279)/GSK-3{beta}(Tyr-216) monoclonal antibody. D, p35 in brain homogenates (200 µg of total protein) was detected by Western blotting with polyclonal anti-p35.

 
Accumulation of p-tau and p-Erk Is Brain Region- and ApoE Isoform-dependent—Next, we assessed the brain region specificity of the increases in p-tau and p-Erk in 9-month-old mice. Homogenates of neocortex, hippocampus, and cerebellum were subjected to SDS-PAGE and analyzed by anti-p-tau and anti-p-Erk Western blotting. The p-tau and p-Erk levels were highest in the hippocampus, intermediate in the neocortex, and lowest in the cerebellum and were ~2-fold higher in NSE-apoE4 mice in all three regions (Fig. 3, A and B).



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  		FIG. 3.
Brain region- and apoE isoform-dependent accumulation of p-tau and p-Erk. Quantitative analyses of p-tau (A) and the ratio of p-Erk (p-Erk1 + p-Erk2) to Erk (Erk1 + Erk2) (B) in homogenates (150 µg of total protein) of different regions of brains of NSE-apoE3 or NSE-apoE4 mice at 9 month of age as determined by anti-p-tau and anti-p-Erk or anti-Erk, (n = 3–5 per genotype; p <0.01, apoE3 versus apoE4 mice for the neocortex and hippocampus; p <0.05, apoE3 versus apoE4 mice for the cerebellum).

 
p-Erk- and p-tau-positive Neurons Are Prominent in the CA3 Region and the Dentate Gyrus of the Hippocampus in NSE-ApoE4 Mice—Immunostaining of brain sections from 18 to 21-month-old mice with anti-p-Erk antibodies revealed p-Erk-positive neurons predominantly in the CA3 region (Fig. 4A) and hilus of the dentate gyrus (Fig. 4B) in NSE-apoE4 mice. Such neurons were detected much less frequently in NSE-apoE3 mice (Fig. 4C). Immunostaining with monoclonal antibody AT8 revealed p-tau-positive neurons, some containing p-tau-positive inclusions in the CA3 region (Fig. 4D, arrowheads) and hilus (Fig. 4E) in the same NSE-apoE4 mice. Again, such p-tau-positive neurons were detected much less frequently in age-matched NSE-apoE3 mice (Fig. 4F). These results suggest that Erk activation is associated with increased phosphorylation and aggregation of tau.



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  		FIG. 4.
Detection of p-Erk- and p-tau-positive neurons in the CA3 region and hilus of the dentate gyrus of the hippocampus. Brain sections from 18 to 21-month-old NSE-apoE4 mice were immunostained with monoclonal anti-p-Erk (A and B, immunofluorescence staining) or anti-p-tau (AT8) (D and E, immunoperoxidase staining; arrowheads indicate p-tau-containing inclusions). Quantitative analyses of neurons positive for p-Erk (C) or p-tau (F) in 18 to 21-month-old NSE-apoE4 (E4) and NSE-apoE3 (E3) mice (n = 4–5 per genotype; p <0.01, apoE3 versus apoE4 mice).

 
ApoE4 Potentiates Zinc-induced Erk Activation in Neuro-2a Cells—The CA3 region and the hilus contain the mossy fiber system of the hippocampus, which contains high levels of zinc (54). To determine whether zinc participates in apoE4-associated Erk activation and tau phosphorylation, we treated stably transfected Neuro-2a cells expressing similar levels of apoE3 and apoE4 (40) (Fig. 5A) with zinc. Neo-transfected Neuro-2a cells were used as controls. At a concentration of 200 µM, ZnCl2 activated Erk, especially p-Erk2, to a significantly greater extent in apoE4-expressing cells than in apoE3-expressing or Neo-expressing cells but did not alter the total (unphosphorylated) Erk level (Fig. 5B). Thus, the ratio of p-Erk to Erk was much higher in ZnCl2-treated than control cells and was 2-fold higher in cells expressing apoE4 (Fig. 5C).



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  		FIG. 5.
ApoE4 potentiates zinc-induced Erk activation in stably transfected Neuro-2a (N2a) cells. A, anti-apoE Western blotting showed similar levels of expression of apoE3 and apoE4 in stably transfected Neuro-2a cells (73 ± 11 and 80 ± 9 ng apoE/mg cell protein/24 h). B, lysates of Neuro-2a cells treated with different concentrations of ZnCl2 for 24 h were analyzed by anti-Erk and anti-p-Erk Western blotting. C, quantitative analysis of the ratio of p-Erk (p-Erk1 + p-Erk2) to Erk (Erk1 + Erk2) in lysates of Neuro-2a cells treated with ZnCl2 (n = 6 per genotype and treatment; p <0.001, Neo or apoE3 versus apoE4 at 200 µM ZnCl2).

 
Potentiation of Zinc-induced Erk Activation by ApoE4 Is Associated with a More Pronounced Increase in Phosphorylation and Aggregation of tau in Neuro-2a Cells—We next assessed the effect of zinc-induced Erk activation on p-tau levels in Neuro-2a cells expressing apoE3 or apoE4. As shown by Western blotting, the levels of monomeric p-tau isoforms (molecular mass 60–80 kDa) and SDS-resistant p-tau aggregates (molecular mass >100 kDa) after treatment with zinc were higher in apoE4-expressing cells than in apoE3-expressing or Neo-expressing cells (Fig. 6A). Densitometric analysis showed 3-fold more p-tau in the apoE4 cells (Fig. 6B).



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  		FIG. 6.
Potentiation of zinc-induced activation of Erk by apoE4 is associated with a more pronounced increase in phosphorylation and aggregation of tau in Neuro-2a (N2a) cells. A, p-tau in lysates of Neuro-2a cells stably expressing Neo, apoE3, or apoE4 treated with ZnCl2 for 24 h were analyzed by anti-p-tau (AT8) Western blotting. B, quantitative analysis of p-tau in lysates of Neuro-2a cells stably expressing Neo, apoE3, or apoE4 treated with ZnCl2 (n = 6 per genotype and treatment; p <0.05, Neo or apoE3 versus apoE4 for control; p <0.001, Neo or apoE3 versus apoE4 at 200 µM ZnCl2; p <0.01, Neo or apoE3 versus apoE4 at 200 µM ZnCl2 + 10 µM U0126). C, Erk and p-Erk in lysates of Neuro-2a cells stably expressing Neo, apoE3, or apoE4 treated with ZnCl2 for 24 h were analyzed by Western blotting. D, quantitative analysis of the ratio of p-Erk (p-Erk1 + p-Erk2) to Erk (Erk1 + Erk2) in lysates of Neuro-2a cells stably expressing Neo, apoE3, or apoE4 treated with ZnCl2 (n = 6 per genotype and treatment; p <0.001, Neo or apoE3 versus apoE4 at 200 µM ZnCl2; p <0.05, Neo or apoE3 versus apoE4 at 200 µM ZnCl2 + 10 µM U0126).

 
Finally, we measured tau phosphorylation after inhibition of zinc-induced Erk activation in apoE-expressing Neuro-2a cells with U0126, a specific inhibitor of the upstream Erk kinase (MEK). Zinc-induced activation of Erk was blocked almost completely (Fig. 6, C and D), and p-tau levels were reduced nearly to baseline levels (Fig. 6, A and B) in Neo-, apoE3-, and apoE4-expressing cells. Thus, Erk appears to be the major (or only) kinase responsible for increased phosphorylation of tau by apoE4 and zinc in Neuro-2a cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study shows that apoE4 stimulates tau phosphorylation by activating the Erk pathway in transgenic mice and in cultured neuronal cells. Erk activation increased with age in the brains of mice expressing apoE3 or apoE4 in neurons. The increases were most pronounced in the hippocampus, particularly in the CA3 region and the hilus of the dentate gyrus, and were significantly greater in the apoE4-expressing mice, resulting in increased phosphorylation and aggregation of tau. ApoE4 also increased zinc-induced Erk activation in neuronal cells, leading to enhanced tau phosphorylation that was significantly reduced by a specific Erk pathway inhibitor (U0126). These findings suggest that apoE4 and zinc act in concert to activate Erk and subsequently cause tau phosphorylation in transgenic mice and may be relevant to the pathogenesis of AD.

Several lines of evidence suggest that Erk is involved in tau phosphorylation. Erk is associated with neuronal microtubules in rodent and bovine brains (65–67), and p-Erk is increased in a subpopulation of neurons containing p-tau and NFTs and in dystrophic neurites in senile plaques in AD brains (46, 68–71). In addition, stress-induced tau phosphorylation is associated with increased p-Erk immunoreactivity in neurons (72). Finally, p-Erk phosphorylates serines in Lys-Ser-Pro repeats in tau in vitro (73–75). In human and rhesus monkey brains, anti-Erk immunostaining showed that Erk levels are highest in the CA3 region, the mossy fiber zone, and the granule cell layer of the dentate gyrus (68, 76). These are the areas where we observed the greatest increases in p-Erk (the current study) and p-tau (Ref. 29 and the current study) in NSE-apoE4 mice, supporting anatomically the relationship between Erk activation and tau phosphorylation.

The possibility that apoE4 and zinc act in concert to activate Erk and cause tau phosphorylation is intriguing. Zinc appears to participate in the regulation of N-methyl-D-aspartate receptor activity (55, 77–81). Zinc also activates many kinases (including protein kinases A and C, calmodulin-dependent kinase, ribosomal S6 protein kinase, and Erk (55–62)) and may contribute to neuronal death due to transient global ischemia or traumatic brain injury (82–86). Increased zinc concentrations (>300 µM) have been found in AD brains (87–89) and in the serum of AD patients with an APOE {epsilon}4 allele (90). These findings are consistent with a synergistic role of zinc and apoE4 in AD pathogenesis.

Zinc also stimulates A{beta} aggregation in vitro (91, 92) and accumulates in amyloid plaques in the brains of AD patients and amyloid protein precursor transgenic mice (89, 93, 94). Moreover, in amyloid protein precursor transgenic mice, reducing the zinc concentration in the brain by treatment with a zinc chelator or by disrupting the zinc transporter protein dramatically inhibits A{beta} accumulation and plaque formation (93, 94). Zinc is highly concentrated in synaptic vesicles at the mossy fiber zone of the hippocampus (54) and is released into the synaptic cleft during synaptic activity, reaching concentrations that routinely exceed 10–20 µM (81) and may approach 300 µM under pathophysiological conditions (95, 96).

Because the increases in p-Erk (the current study) and p-tau (Ref. 29 and the current study) were maximal in the CA3 region and the mossy fiber zone of the hippocampus in NSE-apoE4 mouse brains, we speculate that neuronal deficits induced by apoE4 or apoE4 fragments, as previously shown in transgenic mice (28, 63, 97, 98), cause zinc release from synaptic termini, leading to Erk activation and abnormal phosphorylation of tau. Alternatively, apoE4 or its fragments could increase tau phosphorylation by potentiating zinc-induced Erk activation in neurons, as demonstrated in vitro in the current study. These possibilities are not mutually exclusive. Consequently, decreasing the concentration of zinc in the brain might help prevent AD in carriers of the APOE {epsilon}4 allele.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Heath Grants P01 AG022074, R01 HL37063, and R21 NS046465. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Gladstone Institute of Neurological Disease, P. O. Box 419100, San Francisco, CA 94141–9100. Tel.: 415-826-7500; Fax: 415-285-5632; E-mail: yhuang{at}gladstone.ucsf.edu.

1 The abbreviations used are: apo, apolipoprotein; AD, Alzheimer's disease; Cdk-5, cyclin-dependent kinase-5; Erk, extracellular signal-regulated kinase; GSK-3, glycogen synthase kinase-3; NFT, neurofibrillary tangle; NSE, neuron-specific enolase; PBS, phosphate-buffered saline; p-tau, phosphorylated tau; A{beta}, amyloid-beta. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Lennart Mucke and Karl Weisgraber for critical reading of the manuscript, Jennifer Polizzotto and Sylvia Richmond for manuscript preparation, Stephen Ordway and Gary Howard for editorial assistance, John C. W. Carroll and John Hull for graphics, and Stephen Gonzales and Chris Goodfellow for photography.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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