The Journal of Neuroscience, April 1, 2001, 21(7):2240–2246 S100␤ Interaction with Tau Is Promoted by Zinc and Inhibited by Hyperphosphorylation in Alzheimer’s Disease W. Haung Yu,1,2 and Paul E. Fraser1,3 1Centre for Research in Neurodegenerative Diseases, 2Department of Pharmacology, and 3Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M5S 3H2 The zinc-binding protein S100␤ has been identified as an inter- sistent with an intracellular association. This was enhanced by acting partner with the microtubule-associated protein tau. the addition of zinc and eliminated by divalent metal chelators. Both proteins are individually affected in Alzheimer’s disease S100␤ uptake was also accompanied by extensive neurite (AD). S100␤, is overexpressed in the disease, whereas hyper- outgrowth that may be mediated by its interaction with tau. phosphorylated tau constitutes the primary component of neu- S100␤-tau binding may represent a key pathway for neurite rofibrillary tangles. In this study, we examine factors that mod- development, possibly through S100␤ modulation of tau phos- ulate their binding and the potential role the complex may play phorylation and/or functional stabilization of microtubules and in AD pathogenesis. Zinc was identified as a critical component process formation. S100␤–tau interaction may be disrupted by in the binding process and a primary modulator of S100␤- hyperphosphorylation and/or imbalances in zinc metabolism, associated cellular responses. Abnormally phosphorylated tau and this may contribute to the neurite dystrophy associated extracted from AD tissue displayed a dramatically reduced with AD. capacity to bind S100␤, which was restored by pretreatment with alkaline phosphatase. In differentiated SH-SY5Y cells, ex- Key words: S100␤; tau; Alzheimer’s disease; zinc; binding; ogenous S100␤ was internalized and colocalized with tau con- colocalization; neuronal development S100␤ is a small molecular weight (10 kDa) zinc–calcium binding This study examines the relationship between tau and S100␤ protein produced by astrocytes (Donato, 1991; Mrak et al., 1995). based on the observation that they are cellular binding partners In addition to metal binding, S100␤ has several functions that and each may therefore regulate specific neurite outgrowth or tau include a role in the cytokine cycle, inhibition of selected phos- hyperphosphorylation activity (Baudier and Cole, 1988; Sorci et phokinases, including phosphokinase C (PKC), and the stimula- al., 2000). Second, tau is a unique neuronal component that tion of neurite outgrowth (Kligman and Marshak, 1985; Baudier stabilizes microtubules leading to the formation of axonal pro- and Cole, 1988; Marshak and Pena 1992; Zimmer et al., 1995; cesses and, in its hyperphosphorylated state, tau is the major Griffin et al., 1998; Heizmann and Cox, 1998). S100␤ is located on component of neurofibrillary tangles (Su et al., 1994; Nagy et al., chromosome 21 and is increased in Down’s syndrome and Alz- 1995; Ikura et al., 1998; Mailliot et al., 1998). Finally, although the heimer’s disease (by as much as 20-fold) (Griffin et al., 1989, 1998; mechanism is unknown, S100␤ can induce a similar neurite Marshak et al., 1992; Castets et al., 1997). In AD, the pathology outgrowth that may be related to its association with tau. S100␤ is defined by amyloid plaques and neurofibrillary tangles (NFT) has been shown to directly affect tau, for example, by its ability to that are accompanied by neuronal loss and aberrant neuritic block PKC phosphorylation at specific sites (Ser 262 and 313) sprouting (Masilah et al., 1991). The neuritic response may be (Biernat et al., 1992; Lin et al., 1994; Singh et al., 1996a). This induced by the loss of neuronal connections or a cellular reaction activity may have a direct consequence for AD because loss of ␤ to amyloid deposition (Mrak et al., 1996). S100 overexpression PKC phosphorylation increases the susceptibility of tau to hyper- in AD has been directly correlated with plaque-associated dys- phosphorylation by GSK-3␤ (Singh et al., 1996b; Tsujo et al., trophic neurite development and the astrocyte activation, as well 2000). This AD-related phosphorylation is considered to be a ␤ as S100 overproduction, may be a direct effect of the loss of major factor in tau deposition and neurofibrillary degeneration ␤ neuronal connections and amyloid- deposition (Van Eldik and (Su et al., 1994; Friedhoff et al., 1998; Ikura et al., 1998; Mailliot ␤ Griffin, 1994; Mrak et al., 1996; Sheng et al., 2000). S100 levels et al., 1998). are elevated in brain regions with a direct relationship to the We have examined S100␤ binding proteins by affinity chroma- presence of neuritic plaques (Sheng et al., 1994). In addition, tography and immunoprecipitation to survey the potential in- ␤ astrocyte activation and S100 expression may also be correlated volvement of other AD-associated proteins. In addition to tau, with neurofibrillary tangle formation in AD (Sheng et al., 1994). S100␤ binding to the amyloid precursor protein (APP), the amyloid-␤ peptide, and the presenilins (PS1 and PS2) were also Received Nov. 15, 2000; revised Jan. 11, 2001; accepted Jan. 18, 2001. assessed. Among the proteins we evaluated, tau was the only This work was supported by the Medical Research Council of Canada, Ontario Mental Health Foundation, and the Alzheimer Society of Ontario. W.H.Y. is significant binding protein and furthermore, based on immuno- supported by an Alzheimer’s Society of Canada Doctoral Award. fluorescence studies, colocalized with S100␤ after internalization Correspondence should be addressed to Haung Yu, Centre for Research in by neuronal cells. Zinc has also been implicated in some aspects Neurodegenerative Diseases, 6 Queen’s Park Crescent West, University of Toronto, Toronto, Ontario, Canada M5S 3H2. E-mail: [email protected]. of AD pathology, such as promotion of amyloid fibril formation Copyright © 2001 Society for Neuroscience 0270-6474/01/212240-07$15.00/0 (Bush et al., 1994) and, when examined in the current system, it Yu and Fraser • S100␤ Binding to Tau J. Neurosci., April 1, 2001, 21(7):2240–2246 2241 significantly affected the relationship between S100␤ and tau. This may be attributable to zinc-induced conformational changes that result in the exposure of a hydrophobic domain and could represent a key site for tau binding (Fujii et al., 1986; Baudier and Cole, 1988; Baudier et al., 1992). In addition, changes to tau also regulated this interaction, as shown by the altered binding of S100␤ to the AD-related hyperphosphorylated NFT-tau. Based on our observations, S100␤-tau binding, overexpression of S100␤, and tau hyperphosphorylation in Alzheimer’s disease pathology suggest that S100␤–tau interactions may contribute to neuronal development as well as neuronal dysfunction. Figure 1. Affinity chromatography using immobilized S100␤ for identi- fication of binding proteins (A). Immunoblotting of zinc (lanes 1, 3, 5, 7)- and EDTA (lanes 2, 4, 6, 8)-eluted fractions indicated a significant amount MATERIALS AND METHODS of S100␤-associated tau in control samples from both frontal (lanes 1, 2) Purification of S100␤. Extracts containing S100␤ were prepared from and temporal cortices (lanes 3, 4). Comparable affinity analysis with fresh bovine brains using the method described by Isobe et al. (1977). A AD-extracted proteins from frontal (lanes 5, 6) or temporal (lanes 7, 8) cortex indicated only weak tau immunoreactivity consistent with a re- 20% homogenate was made in a potassium phosphate buffer (0.1 M duced interaction with S100␤. Zinc-treated samples did not elute any KPO ,pH7.1,1mM EDTA, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 4 proteins with tau immunoreactivity. Immunoblotting of total brain ho- 1mM polymethonyl sulfate) with 2.66 M (or 50%) ammonium sulfate ␤ (AmSO ). Cell debris was removed by centrifugation at 10,000 ϫ g, and mogenates from AD and control indicating the elevated levels of S100 , 4 as has been previously demonstrated by Griffin et al. (1989) (B). the supernatant was adjusted to 85% AmSO4 at pH 4.2 and incubated at 4°C for 2 hr. Precipitated proteins were recovered by centrifugation, dialyzed against phosphate buffer, and stored at Ϫ20°C in lyophilized form. From this crude material, S100␤ was purified using a modified eluted with 500 mM NaCl with 1 mM EDTA. Samples were collected, method as described by Baudier et al. (1982). Crude extracts were dissolved dialyzed, and examined by Western blotting using tau antibodies. ␤ ␤ in the elution buffer (50 mM Tris-Base, pH 7.4) with 1 mM ZnSO and S100 internalization and subcellular distribution. Bovine S100 (final 4 ␮ applied to a Phenyl Sepharose 650 M column (ToyoPearl, Montgomer- concentration, 5 g/ml) was added to culture and incubated for pulse of yville, PA). S100␤ was eluted using a step gradient containing 300 mM 4 or 24 hr. Cells were washed with fresh medium and harvested at 0, 15, NaCl, 0.25 mM ZnSO ,or2mM EDTA. Protein purity was assessed by 30, or 60 min and 4, 24, or 48 hr. Cells lysates were examined by 4 ␤ SDS-PAGE with Coomassie staining and by Western blotting with an immunoblotting to determine cellular uptake of S100 . SH-SY5Y cells S100␤ monoclonal antibody (clone SH-B1; Sigma, St. Louis, MO). were grown in 10% fetal bovine serum/DMEM (Life Technologies, Electrophoresis and Western blotting. S100␤ (1 ␮g) was dissolved in Burlingame, CA) at 37°C under 5% CO2. Cells were placed on poly-L- ␮ Laemmli buffer and separated on a 10–20% Tricine gel (Novex, Carls- lysine-coated coverslips and differentiated using 10 M trans-retinoic ␤ bad, CA). Gels were either stained with 0.2% Coomassie blue reagent in acid. To examine colocalization with tau, S100 was preincubated with ␮ 5% acetic acid, or transferred to a polyvinylidene difluoride membrane. the cells for 4, 12, and 24 hr under control conditions or with 50 M ␮ ␤ ␮ The membrane was washed in Tris-buffered saline (200 mM Tris-base, EDTA or 5 M EGTA for 1 hr before addition of S100 or with 10 g/ml pH 7.4, 150 mM NaCl), blocked with skim milk and incubated overnight ZnSO4.