Alpha-Synuclein induces hyperphosphorylation ... - The FASEB Journal

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Alpha-Synuclein induces hyperphosphorylation of Tau in the MPTP model of Parkinsonism Tetyana Duka,* Milan Rusnak,* Robert E. Drolet,† Valeriy Duka,* Christophe Wersinger,* John L. Goudreau,† and Anita Sidhu*,1 *Department of Biochemistry, Molecular and Cellular Biology, Georgetown University, Washington, DC, USA; and †Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan, USA Many neurodegenerative diseases associated with functional Tau dysregulation, including Alzheimer’s disease (AD) and other tauopathies, also show ␣-synuclein (␣-Syn) pathology, a protein associated with Parkinson’s disease (PD) pathology. Here we show that treatment of primary mesencephalic neurons (48 h) or subchronic treatment of wild-type (WT) mice with the Parkinsonism-inducing neurotoxin MPPⴙ/MPTP, results in selective dose-dependent hyperphosphorylation of Tau at Ser396/404 (PHF-1-reactive Tau, pTau), with no changes in pSer202 but with nonspecific increases in pSer262 levels. The presence of ␣-Syn was absolutely mandatory to observe MPPⴙ/MPTP-induced increases in p-Tau levels, since no alterations in p-Tau were seen in transfected cells not expressing ␣-Syn or in ␣-Synⴚ/ⴚ mice. MPPⴙ/MPTP also induced a significant accumulation of ␣-Syn in both mesencephalic neurons and in WT mice striatum. MPTP/MPPⴙ lead to differential alterations in p-Tau and ␣-Syn levels in a cytoskeleton-bound, vs. a soluble, cytoskeleton-free fraction, inducing their coimmunoprecipitation in the cytoskeleton-free fraction and neuronal soma. Subchronic MPTP exposure increased sarkosyl-insoluble p-Tau in striatum of WT but not ␣-Synⴚ/ⴚ mice. These studies describe a novel mechanism for MPTP neurotoxicity, namely a MPTP-inducible, strictly ␣-Syn-dependent, increased formation of PHF-1-reactive Tau, suggesting convergent overlapping pathways in the genesis of clinically divergent diseases such as AD and PD. —Duka, T., Rusnak, M., Drolet, R. E., Duka, V., Wersinger, C., Goudreau, J. L., Sidhu, A. Alphasynuclein induces hyperphosphorylation of Tau in the MPTP model of Parkinsonism. FASEB J. 20, 2302–2312 (2006) ABSTRACT

Key Words: PD 䡠 Alzheimer’s disease 䡠 mesencephalic neurons 䡠 ␣-synuclein knock-out mice 䡠 neurodegeneration ␣-Synuclein (␣-syn), the missense mutants (1, 2, 3) and gene duplication/triplication of which (4, 5) cause familial Parkinson’s disease (PD), may be implicated in axonal transport (6) and dopamine homeostasis (7, 8, 9, 10). How ␣-Syn aggregates and accumulates into Lewy bodies (LBs), intracellular inclusions that contain 2302

aggregated proteins and that are symptomatic hallmarks of idiopathic and familial PD and pathological abnormalities of other synucleopathies (11, 12), is still unknown. Apart from aggregation of ␣-Syn, oxidative stress and exposure to certain neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are linked to the pathogenesis of PD. MPTP induces a selective degeneration of the nigrostriatal dopaminergic pathway in mice and primates, as seen in PD, associated with increases in ␣-Syn expression levels and aggregation, but without inducing real LBs (12), but triggering formation of nigral inclusions immunoreactive for ubiquitin and alpha-synuclein on continuous administration of MPTP (13). Another component of certain LBs is abnormally phosphorylated Tau (14), a microtubule (MT), stabilizing protein. Neuronal colocalization of Tau and ␣-Syn as aggregates or inclusions, or inside certain LBs or LBs-like inclusions, has been reported in brains of patients with familial Alzheimer’s disease (AD), Down’s syndrome, and LB disease (15, 16, 17, 18), but the significance of their codeposition in certain diseases and its underlying mechanism are poorly understood. Similarities between Tau and ␣-Syn include expression in presynaptic neurons, long half-lives in vivo, their “natively unfolded” nature allowing for their heat stability, and their propensity to fibrillize through stretches of hydrophobic residues that form the core of assembled fibrils (19, 20). Despite their codeposition in certain neurological disorders and their enormous potential to either directly or indirectly interact with one another and/or modulate the molecular properties of each other, few studies have been conducted in this regard (21, 22). Tau functional properties depend on posttranslation modifications, mostly phosphorylation, but Tau hyperphosphorylation results in loss of its biological function, with dissociation from MTs (23, 24, 25), and accumulation inside neuronal perikarya and processes, especially dendrites, as paired helical filaments PHFs (26, 27, 28), 1

Correspondence: Head, Laboratory of Molecular Neurochemistry, The Research Bldg., Rm. W222, 3970 Reservoir Rd., NW, Washington, DC. 20007. E-mail: [email protected] doi: 10.1096/fj.06-6092com 0892-6638/06/0020-2302 © FASEB

the building blocks of fibrils of neurofibrillary tangles (29), the pathological hallmarks of AD (30). Tau phosphorylation at Ser202 and Ser262 is important for the regulation of MTs assembly (31). The developmentally regulated Ser262 phosphorylation strongly inhibits Tau binding to MTs (24, 32) and is likely abnormally phosphorylated in AD PHFs (33). In vitro studies suggest that Ser396/404 hyperphosphorylation [PHF-1 antibody (Ab)-reactive site, referred to as p-Tau in this study] of Tau, characteristically found in PHFs of AD brain (34), as well as of the FTDP-17 mutated Tau found in frontotemporal dementia with parkinsonism linked to chromosome 17, may promote Tau-Tau interactions and Tau oligomerization, by favoring additional phosphorylations necessary for the conformational changes needed for its oligomerization and polymerization into PHFs (28). Other in vitro data with the [396/404]S 3 E double mutant (a pseudophosphorylation construct mimicking the PHF-1 phosphorylation sites in which the two serine residues at position 396 and 404 in the longest human isoform of Tau, ht40, were replaced into glutamate residues) Tau also indicate that hyperphosphorylation at Ser396/404 may cause the C terminus of Tau to assume a more extended conformation, altering its inhibitory effect on Tau oligomerization and potentiating the rate of filament formation (35). Here we show that MPTP-induced increases in ␣-Syn expression levels in mesencephalic dopaminergic neurons promote changes in the phosphorylation patterns of Tau at the PHF-1 binding site (Ser396/404), resulting in a mislocation of both proteins and with increased coimmunoprecipitation, together with increased levels of sarkosyl-insoluble hyperphosphorylated Tau, suggesting that an initial step in MPTP-induced parkinsonism and neurotoxicity, is ␣-Syn-directed hyperphosphorylaton of Tau at Ser396/404.

MATERIALS AND METHODS Animals Mice used with the All University Committees on Animal Use and Care (AUF# 1104 –144-00) approval were bred and maintained in university approved animal facilities and handled by trained personnel, according to National Institutes of Health/PHS Animal Welfare Assurance #A3955– 01. All studies used male C57Bl6 and homozygous ␣-Syn⫺/⫺ (B6;129XSncaTMLRossl) mice aged 8 –12 wk. Mice were originally obtained in breeding pairs from Jackson Laboratories (Bar Harbor, MA) to generate a stable breeding colony as described previously (36). Generation of DNAs constructs and transfection of SHSY5Y cells Human ␣-Syn and human dopamine transporter (hDAT) cDNAs were subcloned into pcDNA3.1 as described previously (37). SH-SY5Y human neuroblastoma cells stably transfected with ␣-Syn or mock (pcDNA3.1) DNAs were generated by Lipofectamine 2000 transfection and selected over several

passages with 600 ␮g/ml gentamicin. Stably transfected cells were transiently cotransfected at 80% confluence with either human dopamine transporter (hDAT) or Mock (pcDNA3.1) DNAs by Lipofectamine 2000 and grown for a further 48 h after transfection to allow expression of the hDAT transgene. Subchronic MPTP administration Male homozygous ␣-Syn⫺/⫺ and WT littermate control mice (age 8 –12 wk, 8 per group) received subcutaneous injections of vehicle (1 ml/kg) or MPTP (20 mg/kg) once daily for 5 consecutive days. Three days after the last injection, animals were decapitated; brains were quickly removed, frozen over dry ice, and stored at ⫺80°C until analysis. Preparation of striatal samples, dopamine measurements, and neuronal cultures Coronal sections (500 ␮m) of frozen brains were prepared using a cryostat (⫺10°C). Striatum was isolated using a modification of the Palkovits method (38). Isolated striata were briefly sonicated and centrifuged in ice-cold 0.1 M phosphate-citrate buffer, and dopamine concentrations in the supernatant were assessed by HPLC with electrochemical detection as described previously (39) and normalized to protein content. Primary neuronal cultures from the ventral mesencephalon and prefrontal cortex of gestational 17- to 18-day-old rat embryos were prepared and maintained according to previously described protocols (37). Experiments were performed on 8- to 10-day-old cultures. Treatment paradigm and MTT cell viability Primary cultures were treated in the serum-free medium, whereas SH-SY5Y cells were grown and treated with Dulbecco’s modified Eagle’s medium (DMEM)/F12 ⫹ 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin, at 37°C and 5% CO2. MPP⫹ (1-methyl-4-phenylpyridinium) iodide, prepared at 5 nM–50 ␮M concentrations, was added directly to the medium in the six well-dishes. Cells were exposed to MPP⫹ for 48 h or treated with an equal volume of vehicle (0.1% DMSO) in the presence or absence of 100 nM of the dopamine transporter blocker indatraline. Cell viability was measured with the MTT test, as described by Loo and Rillema (40). Preparation of cell lysates for Western immunoblotting Two separate protocols were used to check the protein expression levels of ␣-Syn (protocol I) and the pattern of p-Tau (protocol II) by Western blot. In protocol I, tissue or cells were collected by gentle scraping, washed three times with D-PBS, and lysed in buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, and 1 mM EDTA) containing 0.1% Nonidet P-40, 0.1% Triton X-100, 1 mM PMSF, and protease inhibitor cocktail tablets (Complete Mini, EDTA-free; Roche Diagnostics, Mannheim, Germany). Lysates were left for 20 min on ice, and after centrifugation (10 min; 14,000 g), the supernatant was collected. Protein concentrations were measured using the Bradford assay (Bio-Rad, Hercules, CA). In protocol II, tissue or cells were washed twice with ice-cold PBS, homogenized in 200 ␮l 2⫻-Stop solution (500 mM Tris–Cl, pH 6.8, 10% SDS, 100 mM EDTA, and 10% glycerol) containing 1 mM Na-orthovanadate, 1 mM PMSF, and protease inhibitors, and sonicated with a Branson Sonifier 250. Protein concentrations were measured with the DC protein assay

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(Bio-Rad) for determination of protein after detergent solubilizations. Samples obtained from both protocols were analyzed by Western blots with 12% SDS-PAGE. Primary antibodies used were TAU-5 (phosphorylation-independent Ab; 1/1000; Chemicon, Temecula, CA), PHF-1 (pSer396/404; 1/500; kindly provided by P. Davies, Albert Einstein College of Medicine, Bronx, NY), CP13 (pSer202; 1/500; kindly provided by P. Davies), and pSer262 (1/1,000; Biosource, Camarillo, CA); and ␣-Syn [mouse monoclonal antibody (mAb); 1/1,000; BD Transduction Laboratories, San Diego, CA, USA]; DAT (1/1,000, Chemicon). Equal protein loading was confirmed with anti-␤-actin Ab (1/500; Santa Cruz Biotechnology, Santa Cruz, CA, sc-1616).

rabbit anti-␣ -Syn polyclonal antibody (pAb; 1:300; C-20, Santa Cruz Biotechnology) and then for 2 h with Alexa-488-conjugated goat-anti-rabbit antibodies (1:750; Invitrogen). After 3 ⫻ 5 min washes in PBS, sections were blocked (1 h; RT) in blocking solution of Mouse on Mouse kit (Vector Laboratories, Burlingame, CA), and all following procedures were performed according to the protocol of the kit, with incubation in Mouse on Mouse diluent followed first by incubation with PHF-1 Ab (30 min; 1:100 dilutions), and then by subsequent incubations with first biotinylated anti-Mouse IgG reagent and secondly streptavidin-conjugated- and Alexa-594-conjugated-secondary antibodies (Invitrogen). After 3 ⫻ 5 min washes in PBS, sections were rinsed in distilled water, mounted on glass slides with Prolong Gold antifade reagent, and visualized under a Nikon Eclipse E800 fluorescent microscope.

Immunoprecipitation Statistical analysis Cells were lysed in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1 mM NaVO4, and 0.5% Nonidet P-40) supplemented with protease inhibitor cocktail tablets. Solubilized proteins (300 ␮g) were precleared for 3 h at 4°C with protein A-Sepharose beads (50% slurry, CL-4B; Pfizer, Inc., Pharmacia, Piscataway, NJ, USA). After immunoprecipitation overnight at 4°C with ␣-Syn polyclonal Abs (1/100, Chemicon), or nonimmune sera, immune complexes were recovered with protein A-Sepharose beads added for 5 h at 4°C. After three washes with lysis buffer, samples were boiled for 10 min and analyzed by Western blots. Fractionation and sarkosyl extractions Cells were rinsed once with warm PBS and once with warm extraction buffer [80 mM PIPES (pH 6.8), 1 mM MgCl2, 2 mM EGTA, 0.1 mM EDTA, 0.1% Triton X-100, and 30% glycerol] containing protease inhibitor cocktail tablets and a phosphatase inhibitor [0.5 ␮M okadaic acid (OA)]. Lysate was incubated at 37°C for 10 min before centrifugation (20 min; 14,000 g; 25°C). The supernatant (soluble fraction) was removed, whereas the pellet (cytoskeletal-associated fraction) was resuspended in 100 ␮l 2⫻-stop solution without DTT or dye and sonicated with a Branson Sonifier 250. Samples were analyzed by Western blots. For analysis of aggregated tau, sarkosyl extractions were performed on striatal tissue samples as described elsewhere (41, 42). Immunocytochemistry on mesencephalic neurons After fixation with 4% (w/v) paraformaldehyde and nonspecific sites blockage and cell permeabilization in D-PBS ⫹ 2% BSA ⫹ 0.3% Triton X-100 for 2 h, neurons grown on coverslips were incubated (16 h; 4°C) with PHF-1 monoclonal (1:200) and anti-␣-Syn rabbit polyclonal (1:300; Santa Cruz Biotechnology) antibodies. After incubation in the dark for 2 h at room temperature (RT) with Alexa 594- or Alexa 488conjugated secondary antibodies (1:2,000 in PBS; Molecular Probes, Eugene, OR), coverslips were mounted on glass slides with Prolong Gold antifade reagent and visualized under a Nikon Eclipse E800 fluorescent microscope equipped with a Nikon DXM1200 digital camera. Immunohistochemistry Mouse brains (age 8 –12 wk) were perfused with 4% (w/v in PBS) paraformaldehyde, and 50 ␮M coronal sections were cut on a vibratome and stored in cryoprotectant at 4°C until use. After being blocked with 1% (w/v in PBS) BSA for 1 h at RT, brain floating sections were incubated overnight at 4°C first with 2304

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Results are mean ⫾ sd and statistically analyzed by the t test between two groups and ANOVA among multiple groups. Statistical significance was accepted at the P ⬍ 0.05 level.

RESULTS Accumulation of ␣-Syn and hyperphosphorylated Tau in rat primary mesencephalic neurons on MPPⴙ treatment Mesencephalic neurons (8 –10 days in culture) were treated with increasing amounts of the active metabolite of MPTP, MPP⫹, for 48 h, and ␣-Syn levels were examined by Western blots and compared with cortical neurons used as controls (Fig. 1A). In cortical neurons, which do not express the dopamine transporter (DAT) and consequently do not take up MPP⫹, there was no change in the pattern of expression of ␣-Syn. In mesencephalic neurons, however, a significant (P⫽0.019) dose-dependent increase (148⫾12%, compared with vehicle-treated neurons, at 50 ␮M of MPP⫹, n⫽5) in ␣-Syn levels was seen on MPP⫹ treatment (Fig. 1A). These findings are consistent with those of others who also showed that MPP⫹ can increase ␣-Syn levels in SH-SY5Y neuroblastoma cells (43). Parallel studies to assess the levels of Tau phosphorylation at several amino acid residues, which are heavily phosphorylated in AD brain (44, 45), were performed in primary neuronal cultures after MPP⫹ treatment. In these studies, the following monoclonal antibodies from Peter Davies (Albert Einstein University, NY) were used: CP13 (pSer202) and PHF-1 (pSer396/404); Western blot analysis was also performed using a commercially available Ab against the pSer262 epitope of phosphorylated Tau. In mesencephalic neurons, there was a significant (P⫽0.032) dose-dependent increase (76⫾9% compared with vehicle-treated neurons, at 50 ␮M of MPP⫹, n⫽5) in PHF-1-immunoreactive Tau levels (Fig. 1B), which was completely blocked by the DAT inhibitor indatraline (data not shown). In cortical neurons, however, MPP⫹ failed to alter PHF-1 immunoreactivity, consistent with the requirement for DAT in the uptake of MPP⫹. We also probed for two other

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tures, as indexed by MTT assays, and found decreased cell viability at all concentrations of MPP⫹ used (Fig. 1C). By contrast, cortical neurons were relatively resistant to the effects of MPP⫹, except at high toxin concentration (50 ␮M), where modest cell death (30%) was observed, which was nonetheless lower than that seen in mesencephalic neurons (65%). Treatment of transfected SH-SY5Y neuroblastoma cells with MPPⴙ increases p-Tau levels in an ␣-Synand DAT-dependent manner To further assess the MPTP-induced increases in p-Tau levels, additional studies were conducted in SH-SY5Y neuroblastoma cells stably transfected with ␣-Syn (SH␣Syn) or mock-transfected with vector (SH-SY5Y); both groups of cells lack DAT. In other studies, both groups of cells were also transiently cotransfected with either human DAT (hDAT) or vector DNA. Cells were treated with increasing amounts of MPP⫹ for 48 h and ␣-Syn or phosphorylated Tau was examined by Western blots (Fig. 2A) and quantified (Fig. 2B). MPP⫹ (50 ␮M) increased ␣-Syn levels (by 124%⫾8%, P⫽0.002, n⫽4) and Tau phosphorylation at Ser396/404 (52⫾4%,

Figure 1. MPP⫹ causes overexpression of ␣-Syn (A), increases in p-Tau (pSer396/404; B), and reduces cell viability (C) in mesencephalic neurons. Increasing concentrations of MPP⫹ (0.5–50 ␮M) were added for 48 h to 8- to 10-day-old, primary mesencephalic, and cortical neuronal cultures. Blots were probed with antibodies to ␣-Syn (A); total Tau; p-Tau epitopes: pSer396/404 (PHF-1), pSer202 (CP13), and pSer262 (B). To confirm equal protein loading, blots were reprobed with ␤-actin antibodies. Multiple immunoblots were subjected to quantitative analysis, and percentage increase (mean⫾sd) of relative levels of ␣-Syn (␣-Syn to ␤-actin) and p-Tau (tau phosphorylated at serine 396/404, 262, or 202 to total Tau) associated with MPP⫹ treatment was plotted in right panels. C) Mesencephalic and cortical neurons were treated with increasing concentrations of MPP⫹ for 48 h and cell viability assessed by MTT assay. Data from 4 independent cultures in triplicate are expressed as mean absorbance at 564 nm ⫾ sd *P ⬍ 0.05, compared with respective vehicle-treated controls in A–C; t test.

epitopes of phosphorylated Tau (pSer202 and pSer262), as well as total Tau (Clone TAU 5 Ab, to detect all phosphorylated and nonphosphorylated forms of Tau, respectively), but did not observe any other significant MPP⫹-induced changes that were specific to mesencephalic neuron compared with those seen with the PHF-1 Ab. Thus, the increased immunoreactivity in pSer262 induced by MPP⫹ in mesencephalic neurons was also observed in cortical neurons treated with MPP⫹ (Fig. 1B). This is the first demonstration that MPP⫹ is able to induce increases in PHF-1 immunoreactive Tau in mesencephalic neurons. For the sake of clarity, PHF-1 immunoreactive Tau is referred to as p-Tau in this study. We next measured cell death in mesencephalic cul-

Figure 2. MPP⫹ effects on p-Tau require the presence of both ␣-Syn and DAT. A) Parental SH-SY5Ycells, or SH-SY5Y cells stably transfected with human ␣-Syn (SH␣-Syn), cotransfected with either hDAT or vector DNA, were exposed to increasing concentrations of MPP⫹ for 48 h or treated with an equal volume of solvent (0.1% DMSO). Western blots were probed for ␣-Syn and hyperphosphorylated Tau antibodies. B) Mean ⫾ sd of densitometries of ␣-Syn and hyperphosphorylated Tau relative to the respective ␤-actin and total Tau densities. *P ⬍ 0.05; t test; n⫽4, significantly different from vehicle-treated controls.


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P⫽0.004, n⫽4) only in cells cotransfected with hDAT. In cells not transfected with any hDAT, MPP⫹ failed to alter either ␣-Syn or Tau Ser396/404 phosphorylation levels, consistent with the requirement for DAT in the cellular uptake of MPP⫹. In controls using the SH-SY5Y parental cell line lacking both ␣-Syn and hDAT, ␣-Syn was not detected. Elevated Tau Ser262 phosphorylation levels were found both in SH␣-Syn/DAT cells (22⫾3%, P⫽0.019, n⫽4) and in parental SH-SY5Y cells lacking DAT, attesting to a MPP⫹-induced, non-DAT- and non-␣-Syn-dependent, increase in phosphorylation at this Tau epitope. In SH-SY5Y cells expressing DAT and no ␣-Syn, MPP⫹ failed to alter PHF-1 reactivity, indicating the mandatory requirement for ␣-Syn in this process. MPP⫹ did not significantly alter Tau Ser202 phosphorylation in any cell line tested. These data confirm the mandatory requirement of both ␣-Syn and DAT in mediating MPTP-induced specific increases of Tau phosphorylation at Ser396/404. Treatment of rat mesencephalic primary neurons with MPPⴙ causes dissociation of both p-Tau and ␣-Syn from the cytoskeleton-bound fraction Since Tau is a microtubule-binding protein stabilizing the MTs, cell fractionation studies were conducted using mesencephalic neurons treated for 48 h with varying concentrations of MPP⫹. Cell lysates were separated into the cytoskeleton-bound and cytoskeletonfree fractions, and p-Tau levels were examined in both fractions. MPP⫹ treatment caused a significant (P⫽0.019, n⫽5) increase in p-Tau levels (35⫾9% at 50 ␮M MPP⫹) in soluble fractions. Such changes were not observed in cortical neurons similarly treated with MPP⫹ (data not shown). The increase in p-Tau in soluble cytoskeleton-free fractions was simultaneously accompanied by a progressive decrease in the levels of p-Tau associated with the cytoskeletal fraction (39⫾0.5%, at 50 ␮M MPP⫹, Fig. 3A). These results suggest a cellular redistribution of p-Tau due to its dissociation from the cytoskeletal network. Since ␣-Syn is known to bind to tubulin and/or microtubules (46, 47, 48), parallel studies were conducted to examine the cellular distribution of ␣-Syn in these neurons and found a redistribution of ␣-Syn from the cytoskeleton-bound to the soluble cytoskeleton-free fraction. Thus, in mesencephalic neurons (Fig. 3B), much larger increases (74⫾9%) in soluble ␣-Syn levels were seen compared with p-Tau (⬇2-fold), at 50 ␮M of MPP⫹. This was accompanied by a virtual total loss (97⫾3% at 50 ␮M MPP⫹) of ␣-Syn associated with the cytoskeleton-bound fraction, suggesting that MPP⫹ completely abolished the binding of ␣-Syn to microtubules. p-Tau and ␣-Syn colocalize and interact to form protein:protein heteromers in MPPⴙ treated mesencephalic neurons Neuronal colocalization of Tau and ␣-Syn has been reported in brains of patients with familial AD, Down’s 2306

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Figure 3. Exposure of mesencephalic neurons to MPP⫹ (48 h) decreases levels of p-Tau and ␣-Syn associated with cytoskeleton. Mesencephalic neurons (8- to 10-day-old) were treated with increasing concentrations of MPP⫹ for 48 h, and cell lysates were fractionated into cytoskeleton-bound and soluble, cytoskeleton-free, fractions. These fractions were immunoblotted with antibodies to (A) Tau and (B) ␣ -Syn and ␤-actin. Densitometric scans of p-Tau relative to total Tau (A), and of ␣-Syn relative to ␤-actin (B), were measured (right panels), and are the mean ⫾ sd of 5 and 3 different experiments, respectively (*P⬍0.05, compared with respective vehicle-treated controls; t test). Western blots represent experiments. Densitometric data are expressed as percentage of vehicle-treated control cultures, which were set as 100%.

syndrome, and LB disease (15–18). To gain insights to the relationship between p-Tau and ␣-Syn in MPP⫹ treated mesencephalic neurons, double immunostaining was performed (Fig. 4A). Immunocytochemical analysis of vehicle-treated mesencephalic neurons showed both diffuse and punctate ␣-Syn staining (Fig. 4A,a,c). Vehicle-treated neurons also showed modest PHF-1 immunoreactivity, which is coexpressed with ␣-Syn in these neurons (Fig. 4A,b,c). However, a substantial increase in ␣-Syn and PHF-1 immunoreactivities, as well as a shift of PHF-1 immunoreactivity from neuronal processes to the cell body, was seen in the mesencephalic neurons treated with MPP⫹ (Fig. 4A,d,e). A partial colocalization between ␣-Syn and PHF-1 immunoreactivities was also seen in the neurons exposed to MPP⫹ (Fig. 4A,f ). These results suggest that MPP⫹-treated neurons show increased p-Tau, concomitant with increased ␣-Syn immunoreactivities, and both proteins colocalize in the neuronal soma only after MPP⫹ treatment. Although ␣-Syn can interact with Tau forming protein:protein complexes (49, 50), its ability to interact with hyperphosphorylated Tau is not documented. We therefore ascertained if p-Tau and ␣-Syn were able to directly interact with one another through reciprocal coimmunoprecipitation (co-IP) assays. In solubilized lysates from MPP⫹-treated neurons, enhanced co-IP between ␣-Syn and p-Tau was seen in an indatraline (INDT)-sensitive manner (Fig. 4B, upper panel). Thus, using PHF-1 antibodies to pull-down p-Tau, elevated

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and total Tau antibodies, as controls for lysate inputs. Modest reductions in both ␣-Syn and p-Tau levels, without changes in ␤-actin, were observed in the post-IP lysates of MPP⫹-treated neurons, suggesting these proteins were lost due to their presence in the co-IPs. These combined co-IPs data suggest that ␣-Syn and p-Tau are able to interact with one another. Increased p-Tau formation and ␣-Syn levels observed in vivo in the subchronic MPTP mice model of Parkinsonism

Figure 4. Increased colocalization and interactions between p-Tau and ␣-Syn after MPP⫹ treatment. A) Double-immunofluorescent labeling of ␣-Syn and p-Tau in MPP⫹-treated (50 ␮M; 48 h) mesencephalic neurons was conducted as described in Materials and Methods (a–f ). Control neurons were treated with vehicle alone. Bar ⫽ 100 ␮m. B) Mesencephalic neurons were treated for 48 h with either MPP⫹ (50 ␮M) or vehicle (0.1% DMSO), in the presence or absence of the dopamine transporter blocker, indatraline (INDT). Treatment was followed by coimmunoprecipitations (co-IPs) on solubilized protein fractions, using ␣-Syn and PHF-1 antibodies, as described in Materials and Methods. NI, IPs with respective nonimmune sera. Levels of residual, unbound proteins were checked in postimmunoprecipitation lysates (post-IP lysate), to demonstrate equal protein amounts between samples during IPs, by immunobloting with antibodies to ␤-actin and total Tau. Blots shown represent 2 experiments yielding identical results.

(⬇3-fold) ␣-Syn levels were found to be present in the immunoprecipitates. In reciprocal co-IP assays using ␣-Syn antibodies (Fig. 4B, upper panel) increased (⬎2fold) p-Tau was seen in the immunoprecipitates only in extracts from neurons treated with MPP⫹. In control studies using nonimmune sera (NI), neither p-Tau nor ␣-Syn was found to be coimmunoprecipitated. Moreover, in vehicle-treated neurons, only negligible levels of p-Tau or ␣-Syn were found in the coimmunoprecipitates. The postimmunoprecipitation fractions (post-IP lysates) were analyzed to determine the immunoprecipitations efficiencies and relative amounts of each protein that can coimmunoprecipitate (Fig. 4B, lower panels) and were also immunoblotted with anti-␤-actin

Earlier studies by Drolet et al. (36) and others (13, 51, 52) previously observed a partial resistance of ␣-Syn⫺/⫺ mice to chronic MPTP-induced neurotoxicity, but the underlying mechanisms were unknown. To investigate this and to test the validity of our in vitro findings in vivo, WT or a-Syn⫺/⫺ (KO) mice were subchronically treated with MPTP; WT mice showed an 80% depletion (P⬍0.01) of striatal dopamine (Fig. 5A), compared with saline-treated WT mice, as measured by HPLC. Striatal dopamine levels were also decreased (P⬍0.01) in MPTP-treated ␣-Syn⫺/⫺ mice, as compared with control saline-treated WT mice, but this dopamine depletion was significantly (P⬍0.01, n⫽4) attenuated, compared with MPTP-treated WT mice, consistent with previous findings that ␣ -Syn⫺/⫺ mice are relatively more resistant to MPTP neurotoxicity (36). In striatum from MPTP-treated WT mice, there was a significant increase in p-Tau levels (30⫾2%, P⫽0.015, n⫽4); a slight rise in Tau phosphorylation at Ser262 (14⫾1.5%, P⫽0.124, n⫽4), which was similar in WT and ␣-Syn⫺/⫺ mice (therefore, MPTP dependent but ␣-Syn independent); and no difference in CP13 immunoreactivity (4⫾0.3%, P⫽0.314, n⫽4; Fig. 5B). These patterns of Tau phosphorylation are similar to the alterations seen in mesencephalic neurons. By contrast, no statistically significant changes in p-Tau levels were observed in MPTP-treated KO mice, indicating that ␣ -Syn is mandatory for the MPTP-induced increase in p-Tau. Reduced PHF-1 labeling in ␣-Syn KO mice relative to WT mice after subchronic MPTP exposure was confirmed by immunohistochemistry on brain striatal sections (Fig. 5C). By contrast, striatal ␣-Syn labeling was increased (39⫾3%, P⫽0.018, n⫽4) in sections from MPTP-treated WT mice (Fig. 5D) in agreement with other studies (53, 54) Redistribution of ␣-Syn and p-Tau levels in the striatal soluble and cytoskeletal fractions of mice subchronically treated with MPTP To assess whether MPTP causes a redistribution and subcellular shift of ␣-Syn (Fig. 6A) or p-Tau (Fig. 6B) in vivo, striatal lysates from mice were fractionated into a cytoskeleton-bound and a cytoskeleton-free fraction and analyzed by Western blots. In WT mice, MPTP increased ␣-Syn levels (43⫾3%, P⫽0.003, n⫽4) in the


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soluble fraction and decreased ␣-Syn levels (43⫾2%, P⫽0.003, n⫽4) in the cytoskeletal-bound fraction. In WT mice, MPTP increased p-Tau levels (41⫾4%,

Figure 5. Increases in p-Tau occur in striatum of WT, but not in ␣-Syn KO, mice after subchronic treatment with MPTP. A) Dopamine levels in the striatum of WT and ␣ -Syn KO mice after subchronic MPTP treatment. Results are the mean ⫾ se. of four to five determinations; *P ⬍ 0.05, significantly different from saline-treated (–) groups; #P ⬍ 0.05, significantly different from MPTP-treated (⫹) WT. B, D) Representative immunoblots and right-hand panels show densitometry of p-Tau (relative to total Tau) and ␣ -Syn (relative to ␤-actin) expression as percent of relative levels found in saline-treated control animals, which were set as 100%. *P ⬍ 0.05; t test, n ⫽ 4, significantly different compared with respective salinetreated controls. C) p-Tau and ␣-Syn immunoreactivities in striatal sections of either ␣-Syn KO or their WT counterparts after a subchronic MPTP regimen. st, striatum; cc, corpus callosum. Bar ⫽ 100 ␮m. 2308

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P⫽0.001, n⫽4) in the soluble fraction, with a decrease (34⫾2%, P⫽0.034, n⫽4) in the cytoskeleton-bound fraction, relative to saline-treated animals. No changes in p-Tau were found in KO mice, attesting to the requirement for ␣-Syn in the MPTP-induced dynamic modulation of cellular redistribution in p-Tau levels in vivo. Abnormally hyperphosphorylated Tau in diseased brain often shifts to an insoluble pool on Tau fibrillization. To determine whether MPTP generated such a shift in mice striatum, homogenized striatal tissue was extracted with the detergent sarkosyl and fractionated into sarkosyl-soluble and sarkosyl-insoluble fractions, as described previously (41, 42). A representative immunoblot showed a substantial increase (42⫾5%, P⫽0.0299, n⫽3) in p-Tau levels both in the sarkosylinsoluble and sarkosyl-soluble fractions of MPTPtreated WT mice striatum (Fig. 6C). No increases in sarkosyl-insoluble p-Tau were observed in striata of vehicle-treated WT mice or in any of the KO groups.

Figure 6. MPTP causes cellular redistribution of ␣-Syn and p-Tau in vivo. Striata from subchronically treated MPTP or vehicle-treated WT and ␣ Syn⫺/⫺ animals were fractionated into a cytoskeletal-containing and a soluble fraction, as described in Materials and Methods. Both fractions were probed with antibodies to ␣-Syn (A) and PHF-1 (B). Mice striata were also extracted with sarkosyl and probed for p-Tau (C). Densitometric data are percentage (mean⫾sd) of saline-treated control (set as 100%). *P ⬍ 0.05; t test, significantly different compared with saline-treated control; #P ⬍ 0.05; t test, significant difference between MPTP-treated groups.

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DISCUSSION Our findings describe a novel linkage between MPTP neurotoxicity, ␣-Syn accumulation, and increases in p-Tau levels, providing unique insights into the roles of these proteins in the genesis of not only MPTP-induced Parkinsonian syndromes but also of other neurodegenerative diseases associated with dysregulation of Tau function (42, 55, 56). Our results show that, in addition to its known ability to inhibit Complex I of the mitochondrial respiratory chain, MPTP also increases p-Tau formation, an abnormally phosphorylated form of Tau classically linked to AD, with ␣-Syn facilitating this process. Another very recent study (42) found that overexpression of the A30P mutant of ␣-Syn in mice spontaneously leads to hyperphosphorylation of Tau in the brain stem. However, this was seen only in symptomatic mice containing aggregates of the A30P ␣-Syn mutant and was not observed in mice overexpressing WT ␣-Syn (42), and this study was moreover done in the brain stem and not in striatum, and in the absence of MPTP. Interestingly, our Western blots and immunohistochemistry results show that MPTP/MPP⫹ treatment induced ␣-Syn-dependent increases in p-Tau in striatum and neurons, which express DAT, attesting to the dopaminergic specificity of this reaction. Moreover, indatraline blocked this effect in mesencephalic neurons and MPP⫹ effects were not observed in cortical neurons. Although MPTP/MPP⫹ exposure did not alter pSer202 (CP13) levels, it induced a nonspecific (as observed in KO mice and in both cortical and mesencephalic neurons), non-dose-dependent increase in pSer262 levels that was ␣-Syn-independent. These latter findings suggest that in the MPTP/MPP⫹ model of PD there are no specific alterations in the phosphorylation of the Ser202 and Ser262 sites of Tau, sites that govern MTs assembly or polymerization. Although p-Tau is occasionally found in the periphery of certain LBs in some neurodegenerative conditions, its mechanism of formation and subsequent accumulation into these LBs is not known. Our data suggest that an initial step in p-Tau alterations is strictly through an ␣-Syn-directed event and not as a direct consequence of neurotoxicity and/or oxidative stress, since MPTP/MPP⫹ treatment failed to cause any increases in p-Tau in the absence of ␣-Syn, as seen in ␣-Syn⫺/⫺ mice and in DAT-transfected cells lacking ␣-Syn. The contributory effect of the neurotoxin, however, may be to cause an intial increase in ␣-Syn protein levels, subsequently leading to increased p-Tau formation. Several reports have previously shown that MPTP induces ␣-Syn accumulation in mice through up-regulation of its gene (57, 58), while other studies suggest that both chronic (59) and acute (60) MPTP treatments cause decreases in ␣-Syn gene expression. Moreover, it was suggested that defective lysosomal protein degradation, rather than altered gene expression, was responsible for ␣-Syn accumulation after chronic MPTP treatment (59). Since we observed an increase in ␣-Syn protein levels also in cotransfected cells, in absence of

the ␣-Syn gene promoter, it is unlikely that changes in gene expression are responsible for its apparent accumulation after MPTP exposure. Rather, it is probable that alterations in protein degradation are induced by the neurotoxin, leading to an accumulation of ␣-Syn; such accumulations are known to lead to a toxic gain of function of this protein, resulting in its aggregation through its non-A␤-amyloid component domain, leading to its eventual deposition as insoluble inclusions (61, 9, 10). Earlier studies by Drolet et al. (36) and others (13, 51, 52) have demonstrated that ␣-Syn⫺/⫺ mice were partially resistant to MPTP neurotoxicity, but the mechanism(s) underlying such resistance were unknown. These in vivo findings are substantiated by our results in vitro, where accelerated cell death was observed in mesencephalic neurons but not in cortical neurons. Moreover, transfected SH-SY5Y cells lacking ␣-Syn were more resistant to MPP⫹ compared with cells that expressed the protein (data not shown). We propose that at least part of the increased susceptibility of WT mice to MPTP observed previously (13, 36, 52, 52) is due to the accumulation of ␣-Syn and the subsequent increased formation of p-Tau. Our findings also provide evidence that free radical generation associated with MPTP-induced mitochondrial Complex I inhibition is not the mode by which p-Tau formation is increased, since in SH-SY5Y cells lacking ␣-Syn and in ␣-Syn⫺/⫺ mice, both of which express DAT and thus take up the neurotoxin, increases in p-Tau levels were not observed. A striking possibility for the mode of ␣-Syn action is that this protein, like presenilin 1 in AD, may act as a chaperone protein to facilitate Tau hyperphosphorylation at the PHF-1 binding site, by altering the presentation of these serines to Ser396/404-reactive kinases, such as PKA, Cdk5, Cdk2, Cdc2, MAPK, GSK3␤, Ca2⫹calmodulin-dependent protein kinase II, caseine kinase I and II, and microtubule affinity-regulating kinase. In this regard, an earlier study (49) demonstrated that Tau and ␣-Syn can interact with one another in vitro to form protein:protein complexes and that ␣-Syn stimulates Tau phosphorylation by PKA. In addition, a very recent study showed that binding between ␣-Syn and Tau was abolished by the most common Tau mutation (P301L) associated with frontotemporal dementia (50). Our results show that MPTP/MPP⫹-induced increases in ␣-Syn levels are associated with a shift from the cytoskeletal fraction to the cytoskeleton-free, soluble fraction. MPTP/MPP⫹ treatment increased p-Tau levels differentially in the soluble- vs. cytoskeletal-associated fraction. Our co-IP results show a direct binding between ␣-Syn and MT-unbound p-Tau in the cytoskeletal-free cellular fraction after MPP⫹ treatment. Interestingly, coincubation of Tau and ␣-Syn synergistically promoted the fibrilization of both recombinant proteins in vitro (21). In addition, A53T ␣-Syn and Tau synergistically promoted the fibrilization of each other in transgenic mice models of ␣-synucleinopathy generated by overexpression of human A53T ␣-Syn and in


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mice expressing both WT human ␣-Syn and TauP301L (22). It is likely that such cofibrilization events are enhanced after increased formation of p-Tau by MPP⫹ in an ␣-Syn-dependent manner, and indeed our data show for the first time that ␣-Syn is able to form heteromeric protein:protein complexes with a hyperphosphorylated form of Tau. In this regard, our results show an increase in the sarkosyl-insoluble levels of p-Tau in WT mice but not in ␣-Syn⫺/⫺ mice, suggesting that, indeed, the increased ␣-Syn levels induced by MPTP may promote the fibrilization of p-Tau or at least increase the levels of either insoluble Tau or of a less soluble form of Tau. This observation is in accordance with our immunocytochemistry data showing a shift of the PHF-1 immunoreactivity from neuronal processes to the cell bodies, and the increased colocalization of p-Tau and ␣-Syn, on MPP⫹ treatment of primary rat mesencephalic neurons. Together, our present results provide evidence in favor of a novel and emerging hypothesis that direct interactions between ␣-Syn and p-Tau accelerate a series of pathogenic events, eventually leading to their coaccumulation, cofibrilization, and possible codeposition. Thus far, at least two triggers for the in vivo increased formation of pathological and neurotoxic p-Tau have been identified: 1) the overexpression and aggregation of the A30P mutant in symptomatic transgenic mice, and 2) exposure of normal mice to very low levels of mitochondrial complex I inhibitors. Our findings that MPP⫹/MPTP causes increased dissociation of ␣-Syn from MTs, together with decreases in p-Tau levels associated with the cytoskeletal bound fraction, may also be of relevance to the mechanism(s) underlying the neurodegenerative process. Hyperphosphorylation of Tau greatly reduces the affinity of Tau for MTs, causing their destabilization (23, 24, 25). In addition, p-Tau is also known to bind to and deplete other MT binding proteins, such as MAP1 and MAP2, from MTs (62). Moreover, since ␣-Syn is known to bind to MTs (48) with a possible role in axonal transport (9), it is likely that the dissociation of this protein from MTs further aggravates the instability of MTs, disrupting the cytoskeletal network and cellular homeostasis. Thus, it is possible that the dissociation of ␣-Syn from MTs and abnormalities in the properties of Tau bound to MTs, comprise another link in the chain of events leading to the neurodegenerative processes associated with inclusion formation. Our studies show that MPP⫹/MPTP-induced abnormalities in ␣-Syn levels modulate p-Tau formation, and the PHF-1 form of Tau, in particular, may provide insights in the development of the early phases of both PHF formation and associated loss of vital neuronal function and suggest that MPTP-induced parkinsonian syndromes or neurotoxicity may be a tauopathy with concomitant alterations in ␣-Syn in a manner reminiscent of synucleopathies. In our study, it is clear that abnormalities of a protein (Tau) known to be mobilized during the pathogenesis of AD, may also be mobilized in parkinsonism but in a region of the brain 2310

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not associated with AD, thereby suggesting considerable overlap in the genesis of certain neurodegenerative diseases. A most important aspect of our studies is the notion that neurodegenerative diseases that seem unrelated may actually have common triggering events and subsequent pathologies, which sets in motion neuronal degeneration. We are grateful to Dr. P. Davies for providing the antibodies PHF-1 and CP13, against Tau phosphorylated at Ser-396/ 404 and Ser-202. This work was supported in part by National Institute of Health Grants NS-45326, NS-34914, and NS-41555 to Prof. A. Sidhu.

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accompanies alpha-synuclein aggregation in a progressive mouse model of Parkinson’s disease. Brain Res. 956, 156 –165 60. Xu, Z., Cawthon, D., McCastlain, K. A., Slikker, W., Jr., and Ali, S. F. (2005) Selective alterations of gene expression in mice induced by MPTP. Synapse 55, 45–51 61. Du, H., Tang, L., Luo, X., Li, H.., Hu, J., Zhou, J., and Hu, H. Y. (2003) A peptide motif consisting of glycine, alanine, and valine is required for the fibrillization and cytotoxicity of human alpha-synuclein. Biochemistry 29, 8870 – 8878 62. Iqbal, K., and Grundke-Iqbal, I. (2005) Metabolic/signal transduction hypothesis of Alzheimer’s disease and other tauopathies. Acta Neuropathol. (Berl.) 109, 25–31

The FASEB Journal

Received for publication April 28, 2006. Accepted for publication June 12, 2006.

DUKA ET AL.