硒酸钠与tau蛋白

Sodium selenate mitigates tau pathology, neurodegeneration, and functional deficits in Alzheimer’s disease models Janet van Eersela,b, Yazi D. Kea, Xin Liua, Fabien Deleruea, Jillian J. Krilb, Jürgen Götza,1, and Lars M. Ittnera,1 aAlzheimer’s and Parkinson’s Disease Laboratory, Brain and Mind Research Institute, University of Sydney, Camperdown, New South Wales 2050, Australia; and bDepartment of Pathology, University of Sydney, Camperdown, New South Wales 2016, Australia Communicated by Etienne-Emile Baulieu, Institut National de la Sante et de la Recherche Médicale, Le Kremlin-Bicetre, France, June 25, 2010 (received for review June 7, 2010) Alzheimer’s disease (AD) brains are characterized by amyloid- β-containing plaques and hyperphosphorylated tau-containing neurofibrillary tangles (NFTs); however, in frontotemporal demen- tia, the tau pathology manifests in the absence of overt amyloid-β plaques. Therapeutic strategies so far have primarily been target- ing amyloid-β, although those targeting tau are only slowly begin- ning to emerge. Here, we identify sodium selenate as a compound that reduces tau phosphorylation both in vitro and in vivo. Impor- tantly, chronic oral treatment of two independent tau transgenic mouse strains with NFT pathology, P301L mutant pR5 and K369I mutant K3 mice, reduces tau hyperphosphorylation and completely abrogates NFT formation. Furthermore, treatment improves contex- tual memory and motor performance, and prevents neurodegener- ation. As hyperphosphorylation of tau precedes NFT formation, the effect of selenate on tau phosphorylation was assessed in more detail, a process regulated by both kinases and phosphatases. A major phosphatase implicated in tau dephosphorylation is the ser- ine/threonine-specific protein phosphatase 2A (PP2A) that is re- duced in both levels and activity in the AD brain. We found that selenate stabilizes PP2A-tau complexes. Moreover, there was an absence of therapeutic effects in sodium selenate-treated tau trans- genic mice that coexpress a dominant-negative mutant form of PP2A, suggesting a mediating role for PP2A. Taken together, sodium selenate mitigates tau pathology in several AD models, making it a promising lead compound for tau-targeted treatments of AD and related dementias. frontotemporal lobar degeneration | protein phosphatase 2A | neurofibrillary tangle | transgenic | treatment Alzheimer’s disease (AD) is the most prevalent neurodegen-erative disorder, characterized by progressive loss of cogni- tion. Histopathologically, AD is defined by two lesions, plaques and neurofibrillary tangles (NFTs), which result from deposition of amyloid-β (Aβ) and hyperphosphorylated tau, respectively. Aβ forms upon cleavage of the amyloid precursor protein by β- and γ-secretases, and accumulates extracellularly (1). Tau accumu- lates intracellularly as it becomes increasingly phosphorylated at both physiological and pathological sites, resulting in reduced affinity to microtubules and redistribution from the axonal to the somato-dendritic compartment (2). The amyloid cascade hy- pothesis places Aβ upstream of tau, a concept supported by AD mouse models (3, 4). Interestingly, tau depletion in mice prevents Aβ pathology, suggesting that Aβ toxicity is also tau-dependent (5). This finding highlights a central pathogenic role of tau in AD. This role extends to diseases such as frontotemporal dementia, the second most common form of dementia, where tau lesions are frequent without overt Aβ pathology (2). Thus, tau can induce neurodegeneration in the absence of Aβ. Accordingly, expression of tau in transgenic mouse models recapitulates features of AD and frontotemporal lobar degeneration (FTLD) (6). Selenium is a vital trace element enriched in brain (7, 8); its levels decline with age, and particularly low levels have been linked to cognitive impairment and AD (9, 10). Short-term administra- tion of selenium improvesmemory deficits in an acute ratmodel of dementia (11) and reduces tau phosphorylation in WT rats (12). Therefore, selenium has been attributed neuroprotective proper- ties, but the underlying mechanism and its therapeutic potential remain elusive (11). To date, treatment of AD and related dementias is limited to symptomatic relief, with no cure available. Although Aβ has been the main focus of drug development until recently, tau is in- creasingly recognized as a target for the treatment of AD and FTLD (13). Here, we tested putative therapeutic effects of so- dium selenate, an oxidized form of selenium, on tau pathology, using cell culture and several transgenic mouse models. Results Sodium Selenate Reduces Tau Phosphorylation in Vitro. To test sel- enate in vitro, we first treated SH-SY5Y neuroblastoma cells that stably express human tau carrying the FTLD pathogenic mutation P301L (SH-P301L). In these cells, tau is phosphorylated at mul- tiple sites, including the pathological epitope Ser422 (pS422), a site correlating with NFT formation in vivo (3, 14) (Fig. 1 A and B). Selenate treatment reduced pS422 staining without af- fecting tau expression levels (Fig. 1A andB, and Fig. S1).Western blotting of extracts obtained from both selenate-treated and un- treated cells in the presence of the protein phosphatase 2A (PP2A) inhibitor okadaic acid (OA) revealed a dose-dependent reduction of tau phosphorylation at multiple sites, including pS422, 12E8, and PHF-1 (Fig. 1 C). Toxicity has been reported for some forms of selenium, such as sodium selenite (15), which would severely compromise a thera- peutic use. We thus assessed neurotoxicity of sodium selenate compared with selenite, a less oxidized state of selenium, by mea- suring lactate dehydrogenase (LDH) release in primary hippo- campal cultures (Fig. 1 D). Although selenite was toxic already at low concentrations, selenate showed no toxicity, even at high 100 μM concentrations. Furthermore, chronic treatment of mice with selenate over 4 mo did not cause any overt side effects or any form of neurotoxicity (see below). Selenate Improves Tau Transgenic K3 Mice. Given the profound effects on tau phosphorylation in vitro, we next tested if selenate also reduces hyperphosphorylation of tau in vivo. K3 mice express human tau carrying the pathogenic K369I mutation in neurons (16). These neurons are characterized by a particularly early-onset Author contributions: J.v.E., J.G., and L.M.I. designed research; J.v.E., Y.D.K., X.L., F.D., and L.M.I. performed research; J.v.E., Y.D.K., X.L., F.D., J.G., and L.M.I. analyzed data; and J.v.E., J.J.K., J.G., and L.M.I. wrote the paper. The authors declare no conflict of interest. 1To whom correspondence may be addressed. E-mail: jgoetz@med.usyd.edu.au or littner@med.usyd.edu.au. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1009038107/-/DCSupplemental. 13888–13893 | PNAS | August 3, 2010 | vol. 107 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1009038107 Parkinsonism associated with abundant tau phosphorylation, and axonal transport defects (16, 17). We first treated 6-wk-old K3 mice with 12 μg/mL selenate added to the drinking water ad libi- tum, with controls receiving no drug, and monitored the pro- gression of motor symptoms. Selenate did not affect total water consumption (Fig. S2). K3 mice normally exhibit motor deficits on the Rota-Rod already at 6 wk, compared with WT (Fig. 2A). However, although untreated K3 mice (further) declined dra- matically over time until they failed to stay on the rod at all, sele- nate-treated K3 mice showed a gradually improving motor performance, such that after 6 wk of treatment they were no longer distinguishable fromWT littermates. The motor improvement was associated with decreased tau phosphorylation in the motor cortex, hippocampus, and substantia nigra (Fig. 2B and Fig. S3). Staining of brain sections for tau phosphorylated at Thr231/Ser235 (AT180 epitope), a major phosphorylation site in K3 mice (16), revealed significantly reduced phosphorylation in selenate- compared with untreatedmice. Importantly, transgenic taumRNA levels were not altered upon treatment (Fig. S1). Hence, selenate reduces both tau phosphorylation and early-onset motor deficits in young K3 mice. Tau pathology in K3 mice is progressive. Beyond the age of 4 mo, deposition of tau results in overt Bielschowsky-silver-posi- tive NFT-like inclusions and axonal spheroids in many brain areas, as well as in neurodegeneration in the substantia nigra (16, 17). When we treated 4-mo-old K3 mice until 8 mo of age with selenate, numbers of inclusions were significantly reduced, as shown for the hippocampal subiculum and midbrain (Fig. 2 C–E). Furthermore, numbers of spheroids, a result of axonal transport defects in K3 mice (17), were significantly lower in treated mice (Fig. 2F). In fact, spheroids were completely absent from frontal cortex, suggesting that selenate reverts functional impairments leading to spheroids. K3 mice are characterized by a substantial, age-dependent degeneration of cerebellar basket neurons, re- sulting in the absence of pinceau terminals formed by clustered axons surrounding Purkinje cells (Fig. 2G). Selenate treatment fully prevented this degeneration. Taken together, these findings show that selenate reduces tau phosphorylation and deposition, mitigates pathological spheroid formation, and prevents axonal degeneration of distinct neuronal populations in K3 mice. Selenate Halts Pathology in Tau Transgenic pR5 Mice. Next, we treated 8-mo-old pR5mice that express P301Lmutant human tau in neurons (18) for 4 mo with 12 μg/mL selenate added to the drinking water. The pR5 mice present with a progressive tau pa- thology, including NFT formation initiated at around 6 mo of age (18). These mice are characterized by an amygdala-dependent impairment in the conditioned taste aversion (CTA) paradigm (19). As expected, untreated pR5 mice displayed a significant impairment, although selenate-treated pR5 mice showed no im- pairment, performing similar to wild-type littermates and hence, suggestive of improved contextual memory (Fig. 3A). Basic taste qualities were normal in untreated and treated pR5 mice (Fig. S2). Next, we determined whether the functional improvement of selenate-treated pR5 mice was associated with changes in tau pathology. Double-staining for human tau (HT7) and tau phos- phorylated at Ser422 (pS422) revealed reduced phosphorylation in CA1 neurons of selenate-treated compared with untreated pR5 mice, although staining for total tau was comparable (Fig. 3B). As for the K3 mice, transgenic tau mRNA levels were not altered by selenate (Fig. S1). Next, we determined if selenate treatment reduces levels of insoluble tau, a step critical in NFT formation. Therefore, we extracted pR5 brain tissue with either formic acid (FA) or sarkosyl to obtain insoluble proteins (16, 20). Consistent with the histopathological finding, Western blotting revealed reduced phosphorylation of tau in selenate-treated pR5 brains, although total levels of soluble tau were comparable in treated and untreated pR5 mice (Fig. 3 C and D). However, both FA and sarkosyl extraction revealed markedly less insoluble tau in selenate-treated compared with untreated pR5 mice. The amyg- dala is a major site of NFT formation in pR5mice (18). Here, both numbers of neurons stained with pS422, a marker of severe tau pathology (3), and numbers of Gallyas-positive NFTs were sig- nificantly reduced in selenate-treated compared with untreated pR5 brains (Fig. 3 E and F). Specifically, NFT numbers in 12-mo- old selenate-treated pR5 mice were similar to those in 8-mo-old untreated pR5 mice (3), suggesting that treatment had halted disease progression. Selenate-Induced Improvements Require PP2A. Reduced PP2A ac- tivity has been implicated in AD (21, 22). Because selenate antag- onized the PP2A inhibitor OA in SH-SY5Y cells (Fig. 1C), we addressed PP2A function in more detail. Regulation of tau phos- phorylation by PP2A involves direct binding (23). Interestingly, coimmunoprecipitation of PP2A from SH-SY5Y cells with a tau- specific antibody revealed a markedly increased tau-PP2A inter- action in the presence of increasing doses of selenate (Fig. 4A and Fig. S4), suggesting that selenate enhances tau binding of PP2A. To determine the role of PP2A in mediating the effects of sel- enate on tau pathology in vivo, we crossed pR5 with Dom5 mice (pR5.Dom5). Dom5 mice express a substrate-specific dominant- negative mutant form of PP2A, L309A (24). The two transgenes show an overlapping expression pattern in pR5.Dom5 mice, in- cluding the hippocampus (25). Although selenate treatment of pR5 mice reduced levels of tau phosphorylation significantly, treatment had no effect in pR5.Dom5 mice, as determined by Western blot analysis of hippocampal extracts (Figs. 2C and 4B D controlA B 224 Sp 7T H egre m selenate HT7 * † pS422 Gapdh C 20nM OA † - - 1.0 1 01 001 0001 selenate: (µM) 12E8 † PHF-1 † selenate selenite * * * 4 3 2 1 0 yti ci xot ev ita ler )rt Cf o d lof ( conc (mM) 0000.0 1 000.0 1 00.0 1 0.0 1 .0 1 10 50 100) %( sllec # HT7pS422 I selenate control I Fig. 1. Sodium selenate mitigates aberrant tau phosphorylation in SH-SY5Y neuroblastoma cells. (A) Immunocytochemistry of human P301L mutant tau expressing SH-SY5Y cells treated with selenate. Phosphorylation of tau at the pathological epitope Ser422 (pS422) is markedly reduced in selenate- treated cells, although total tau (HT7) is comparable to untreated controls. (B) Flow cytometry confirms reduced pS422 phosphorylation, but similar HT7 staining upon selenate treatment, compared with control. I, fluorescence intensity. (C) OA induces phosphorylation of tau at multiple epitopes (pS422, 12E8, PHF-1), resulting in a molecular weight shift (†) in SH-SY5Y cells. Cotreatment with increasing doses of selenate shows a dose-dependent reduction of tau phosphorylation, but levels of nonphosphorylated tau (asterisk) remain unaltered. (D) Dose-dependent increase in toxicity of sel- enite, but not selenate in primary hippocampal neurons, as determined by LDH release (*P < 0.001). van Eersel et al. PNAS | August 3, 2010 | vol. 107 | no. 31 | 13889 N EU RO SC IE N CE and Fig. S4). Furthermore, extraction of insoluble proteins revealed high levels of insoluble tau in selenate-treated pR5. Dom5, but not pR5 mice. Consistent with these findings, the his- tological analysis revealed that selenate had no effect on pre- venting tau phosphorylation and NFT formation in pR5.Dom5 mice (Fig. 4 C and D). Therefore, the absence of therapeutic effects in pR5.Dom5mice suggests a role for PP2A in the selenate- induced reduction of tau phosphorylation. Increased kinase activities have been implicated in AD (26). Although selenate may alter kinase function, Western blot analysis of wild-type and K3 brains with phosphorylation (ac- tivity)- and total kinase-specific antibodies (GSK3β, ERK1/2, p38 Mapk, and cdk5) revealed no alterations upon chronic selenate treatment (Fig. S5). Therefore, altered kinase activity seems not to contribute to the effects of selenate on tau phosphorylation. Discussion In the present study, we show that chronic low doses of sodium selenate reduce tau phosphorylation in both cell culture and mouse models of disease. Treatment prevents memory and motor deficits, NFT formation, and degeneration in two tau transgenic mouse lines with robust pathologies. These effects are, at least in part, mediated by PP2A, because selenate does not reduce tau phosphorylation in mice coexpressing a defective PP2A subunit (which causes reduced activity for substrates such as tau). PP2A is a heterotrimeric complex composed of a structural A, a catalytic C, and a variable regulatory B subunit (27). Four classes of regulatory subunits, B (PR55), B’ (B56 or PR61), B’’ (PR72), and B’’’ (PR93/PR110), with several members in each subfamily, define substrate specificity and subcellular localization of the holoenzyme. This result is also true for the PP2A substrate tau BA ED C control selenate controlWT ** * * * * *** * * ** * * * ** * * * * * * * * * * selenate K3 selenate K3 control WT control WT selenate 0 0 1 2 3 4 5 6 7 8 9 101112 60 120 180 )ces( do R-ato R no e mit duration of treatment (wks) F G cortex CA1 SN yks wohcslei B e tan eles l ortnoc yks wohcslei B F N / AP lortnoc etaneles 60 40 20 0 noitc es/ sn ois el * lortnoc etaneles 150 100 50 0 noi tces / s diorehps * lortnoc etaneles 12 8 4 0 noitc es/ s nois el * # * Fig. 2. Chronic selenate treatment improves pathology in both young and aged K369I mutant tau transgenic K3 mice. (A) Rota-Rod testing in acceleration mode of selenate-treated and untreated (control) WT and K3 mice. Despite pronounced deficits in K3 compared with WT mice at the onset of treatment (6 wk of age), after 5 wk of chronic selenate treatment motor performance of K3 mice is improved such that performance does not differ significantly from WT (#n.s.), although untreated K3 control mice continue to deteriorate, staying only for a minimal time on the rod (*P < 0.05, treated vs. untreated K3 mice). (B) Selenate treatment for 12 wk significantly reduces staining of K3 brains with the phospho-tau antibody AT180 (Thr231/Ser235), a dominant phospho-epitope in K3 mice, in neurons of cortex, hippocampal CA1, and substantia nigra (SN), compared with untreated K3 mice (for quantification, see Fig. S3). (Scale bars, 50 μm.) (C) Bielschowsky-silver positive NFT-like lesions (arrows) are numerous in the superior colliculus of the midbrain of 8-mo-old untreated K3 mice (control), but are significantly reduced after 4 mo of treatment with selenate. (Scale bar, 50 μm.) (D) Quantification reveals that numbers of lesions are 4.4-fold lower in the superior colliculus of selenate-treated compared with untreated (control) K3 mice (*P < 0.0001). (E) Numbers of NFT-like lesions in the subiculum are 2.87-fold lower in selenate-treated compared with untreated (control) K3 mice (*P < 0.0001). (F) Axonal spheroids are 3.68-fold less frequent in the cortex of selenate-treated compared with untreated (control) K3 mice (*P < 0.0001). (G) Bundled axons of cerebellar basket cell form Pinceau terminals (arrows) around the initial axon segment of the Purkinje cell (PC, asterisks), as visualized by Bielschowsky-silver impregnation and neurofilament (NF) staining (red) in WT brains. Eight-month-old untreated K3 mice (control) show a pronounced degeneration of Pinceau terminals, resulting in the absence of NF- and Bielschowsky-positive axons around PCs (open arrows). Treatment with selenate fully prevents this degeneration. PCs are stained with parvalbumin (PA, green). (Scale bar, 50 μm.) 13890 | www.pnas.org/cgi/doi/10.1073/pnas.1009038107 van Eersel et al. that is dephosphorylated, upon binding, by distinct isoforms of PP2A (23, 28). Interestingly, we found that selenate increases binding of PP2A and tau in cell culture, which may explain its effects on tau phosphorylation. Other mechanisms of regulating PP2A activity may include alterations in the methylation of the core enzyme or dislodging of catalytic metal atoms (29, 30). Here, the exact molecular effects of selenate on PP2A and other likely, yet unidentified cellular targets remain to be elucidated. How- ever, given the role proposed for PP2A in tau pathology in AD (21, 22, 28, 31), activating PP2A represents an attractive thera- peutic approach (26). It should be considered, however, that in- terfering with the activity of PP2A may cause side effects, as the holoenzyme participates in several signaling cascades in many tissues other than brain. Hence, a substrate-specific activation of PP2A rather than a broad inhibition needs to be achieved. Al- though in K3 and pR5 mice, tau phosphorylation was reduced in the absence of overt central or peripheral side effects, it cannot fo