Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity
[['Alzheimer’s disease ( AD ) is a progressive neurodegenerative disease characterized by two pathological lesions: deposition of β-amyloid peptides ( Aβ ) as amyloid plaques and the microtubule-associated protein tau in the form of paired helical filaments in neurofibrillary tangles ( NFTs ) [1] .', 'Genetic , pathological , and biochemical evidence suggests that elevation of Aβ levels is a causal event in AD pathogenesis [2–8] that lies upstream of tau-induced neurodegeneration [3 , 5 , 9 , 10] .', 'In AD and other neurodegenerative diseases , collectively referred to as tauopathies , tau protein self-aggregates into multiple intermediate forms , including soluble oligomers and prefibrils , that may ultimately form insoluble NFTs [11] .', 'These tau aggregates all exert neurotoxicity , with some qualitative and quantitative differences; the soluble , prefibrillar aggregates are thought to cause the most damage to neurons [12–14] .', 'To prevent the cascade of events leading to neurodegeneration in AD , it is crucial to elucidate the mechanisms underlying the initial steps of abnormal metabolism of tau .', 'Tau proteins are normally enriched in neuronal axons , where they regulate microtubule stability .', 'However , in diseased brains , tau is detached from microtubules and aggregated in the cytosol .', 'The microtubule-binding domain of tau mediates interaction to proteins , including tau itself , which can cause self-aggregation into oligomers , protofibrils , and fibrils [15–20] .', 'In addition , tau is abnormally phosphorylated in diseased brains [21–24] , and tau proteins detached from microtubules are prone to be phosphorylated at disease-associated sites [19 , 25–28] .', 'Moreover , tau detached from microtubules can mislocalize to dendrites and extracellular regions , where it can disrupt neuronal functions or spread into other neurons [29–34] .', 'These observations suggest that the loss of tau binding to microtubules may be a triggering event for abnormal metabolism of tau .', 'However , the detailed molecular mechanisms underlying this event and how it relates to Aβ-mediated tau toxicity remain elusive .', 'Tau is phosphorylated at more than 40 sites in pathological lesions associated with AD [21–24] , and Aβ promotes tau phosphorylation at disease-associated sites in in vitro and in vivo models of AD [2 , 35–41] .', 'A number of kinases and phosphatases regulate the phosphorylation status of tau [42] , and the activities of two major tau kinases , GSK3 and PAR-1/microtubule affinity-regulating kinases ( MARKs ) , are often associated with tau detachment from microtubules and Aβ-induced augmentation of tau toxicity [37 , 39 , 43–45] .', 'GSK3 is a proline-directed kinase that contributes to phosphorylation of tau at serine or threonine followed by proline ( i . e . , SP/TP sites ) [46–49] , whereas PAR-1/MARKs are non-SP/TP kinases that phosphorylate tau at Ser262 and Ser356 in the repeat domains located in the microtubule-binding region [50] .', 'Tau phosphorylation at these sites decreases the protein’s affinity toward microtubules in vitro , in cultured cells , transgenic Drosophila , and transgenic mice [18 , 19 , 25–27 , 50–52] .', 'Tau phosphorylation at GSK3-target sites , as well as Ser262/356 , has prominent effects on tau toxicity [26 , 53–56] .', 'In cellular and animal models , co-expression of Aβ augments tau phosphorylation at these sites and increases the levels of active GSK3 and PAR-1/MARK [2 , 35–41 , 43 , 44] .', 'Notably , studies in cultured neurons and transgenic Drosophila showed that Aβ-induced augmentation of tau toxicity caused by Aβ depends on tau phosphorylation at these sites [39 , 45 , 56–58] .', 'These observations demonstrate the importance of tau phosphorylation by GSK3 and MARK in the abnormal metabolism and toxicity of tau .', 'However , the roles of , and interdependency between , tau phosphorylation by GSK3 and MARK/PAR-1 in an early step of tau mismetabolism , such as tau mislocalization and stabilization of mislocalized tau , are not fully understood .', 'Drosophila models expressing human tau recapitulate key features of human tauopathies , including progressive neurodegeneration and phosphorylation at AD-related sites via conserved kinases [41 , 44 , 53 , 59–63] .', 'Formation of fibrils or detergent-insoluble aggregates are not detected in the presence or absence of Aβ [58 , 59] , suggesting that the neurodegeneration observed in these models reflects toxicity of soluble , non-aggregated forms of tau , and may recapitulate early stages of the abnormal metabolism and toxicity of tau [11–14] .', 'In this study , we investigated the roles of tau phosphorylation at Ser262/356 by PAR-1/MARK and SP/TP sites by GSK3 in an initial step of tau mismetabolism , using transgenic Drosophila co-expressing human tau and Aβ42 in neurons [59 , 64] .', 'We found that augmentation of tau toxicity by Aβ42 was concomitant with increased levels of microtubule-unbound free tau in the cytosol , regardless of its phosphorylation status .', 'Tau phosphorylation at Ser262/356 by PAR-1/MARK preferentially stabilized less phosphorylated forms of microtubule-detached tau in the cytosol , and blocking this stabilization of tau suppressed Aβ42-induced augmentation of tau toxicity and the increase in the levels of tau phosphorylated at the AD-associated residue Thr231 .', 'By contrast , blocking tau phosphorylation at GSK3-target SP/TP sites did not affect tau stability or suppress the mislocalization and toxicity of tau induced by Aβ42 .', 'These results suggest that detachment of tau from microtubules , followed by stabilization by phosphorylation at Ser262/356 via PAR-1/MARK , is a critical early step in the formation of tau species associated with neurodegeneration , and that tau mismetabolism via this pathway is facilitated by Aβ42 .'], ['To understand the mechanisms by which abnormal metabolism and toxicity of tau is triggered by Aβ42 , we analyzed changes in human tau induced by co-expression of human Aβ42 in transgenic flies [58] .', 'Because accumulation of tau is a hallmark of AD brains , and an increase in tau levels positively correlates with tau toxicity in cellular and animal models [65] , we first investigated whether tau accumulation occurred in association with Aβ42-induced augmentation of tau toxicity .', 'As reported previously , co-expression of Aβ42 in fly eyes using the pan-retinal driver gmr-Gal4 enhanced tau-mediated retinal degeneration ( [58] and S1 Fig ) .', 'In this model , the levels of tau did not differ significantly between flies expressing tau alone and those expressing tau and Aβ42 ( Fig 1A ) , suggesting that Aβ42-mediated augmentation of tau toxicity is not simply due to an overall increase in the level of tau [58] .', 'Most tau proteins are normally bound to microtubules in axons , and alterations in the distribution of tau are associated with tau toxicity [66] .', 'To determine whether mislocalization of tau occurred in our fly models , we analyzed the effect of Aβ42 expression on tau binding to microtubules using an in vivo microtubule-binding assay [25] .', 'In this assay , endogenous microtubules and microtubule-bound proteins were present in the pellet following centrifugation ( microtubule fraction ) , whereas cytosolic proteins free from microtubules were recovered in the supernatant ( cytosol fraction ) .', 'The levels of tau in each fraction were determined by western blotting with anti-tau antibody .', 'We found that the levels of tau in the cytosol fraction were significantly elevated , whereas the levels in the microtubule fraction were reduced , in the fly retina co-expressing Aβ42 and tau ( Fig 1B ) .', 'These experiments were carried out using two independent transgenic fly lines expressing Aβ42 at different expression levels .', 'Both lines yielded similar results , and the fly line with higher Aβ42 expression exhibited a larger effect ( Fig 1B , compare Aβ42#1 and Aβ42#2 ) .', 'We also investigated whether co-expression of non-toxic proteins in the secretory pathway ( CD8-GFP ) altered tau distribution non-specifically .', 'Expression of CD8-GFP did not change the levels of tau in either the cytosol fraction or the microtubule fraction ( S2 Fig ) , suggesting that alterations in tau distribution are associated with Aβ42-mediated toxicity .', 'Tau proteins expressed in the fly retina are detected as two major bands by western blotting with pan-tau antibody ( Fig 1B ) .', 'Following phosphatase treatment , these two bands merged and were detected as a single faster-migrating band ( S3 Fig ) .', 'This observation indicates that the differences between these two tau bands are related to their phosphorylation levels: tau in the slower-migrating band ( tauupper ) is more highly phosphorylated than tau in the faster-migrating band ( taulower ) .', 'To further characterize phosphorylation profiles of tauupper and taulower , we used a panel of antibodies capable of distinguishing the phosphorylation status of tau at AD-related sites .', 'Western blotting with TAU-1 antibody , which specifically recognizes tau protein without phosphorylation at several AD-related sites ( Ser194 , Ser195 , Ser198 , and Ser202 ) , detected taulower ( Fig 1C , TAU-1 ) , whereas antibodies specific to phospho-Ser202 , phospho-Thr231 , or phospho-Ser396/404 ( PHF1 ) preferentially recognized tauupper ( Fig 1C , pSer202 , pThr231 , and PHF1 , respectively ) .', 'By contrast , phospho-Ser262-specific antibody recognized both tauupper and taulower species ( Fig 1C , pSer262 ) .', 'In the Aβ42 fly , levels of both tauupper and taulower were elevated in the cytosol fraction ( Fig 1B , cytosol ) , but reduced in the microtubule fraction ( Fig 1B , microtubule ) .', 'These results suggest that , regardless of its phosphorylation status , microtubule-unbound free tau in the cytosol is more abundant in the presence of Aβ42 .', 'Cytoskeletal pathologies , including microtubule disintegrity , have been reported in AD brains and cultured cells incubated with Aβ [39 , 67–69] .', 'Under this particular experimental condition , however , co-expression of Aβ42 did not significantly change the levels of tubulin in the form of microtubules ( Fig 1B , tubulin ) or the levels of acetylated or tyrosinated tubulin ( S4 Fig ) , suggesting that the stability of microtubules was not grossly affected by Aβ42 .', 'Phosphorylation of tau negatively influences its ability to bind microtubules [25–27 , 51 , 52] .', 'In Drosophila , tau phosphorylation at proline-directed Ser and Thr ( SP/TP ) sites decreases tau binding to microtubules , whereas replacing these phosphorylation sites with unphosphorylatable Ala significantly increases binding [25 , 26] .', 'In fly neurons , many of these SP/TP sites are phosphorylated by a fly homolog of GSK3 called Sgg [26 , 53 , 61] .', 'In line with these reports , we found that RNAi-mediated knockdown of GSK3/Sgg significantly reduced tau phosphorylation at these SP/TP sites ( S5 Fig ) ; as a result , tau is detected as a single band with faster migration speed than taulower ( Fig 2A , input , asterisk ) .', 'In vivo microtubule-binding assays revealed that most tau was recovered in the microtubule fraction in the Sgg knockdown background ( Fig 2A ) .', 'These results indicate that , in this fly model , tau phosphorylation at SP/TP sites by GSK3β/Sgg plays an important role in tau binding to microtubules .', 'As we reported previously , Aβ42 increased the levels of tau phosphorylated at Ser202 ( pSer202 ) and Thr231 ( pThr231 ) ( [58] and Fig 2B ) .', 'We found that , in the retina with RNAi-mediated GSK3β/Sgg knockdown , expression of Aβ42 did not increase the levels of tau phosphorylated at either of these residues ( Fig 2C ) .', 'These results suggest that increases in the levels of phosphorylated tau mediated by GSK3β/Sgg might contribute to Aβ42-induced detachment of tau from microtubules .', 'We tested whether blocking tau phosphorylation at SP/TP sites by GSK3/Sgg was sufficient to suppress Aβ42-mediated mislocalization of tau from the microtubule fraction to the cytosol fraction .', 'In vivo microtubule-binding assays revealed that , in the GSK3/Sgg knockdown background , co-expression of Aβ42 was still capable of increasing the level of tau in the cytosolic fraction and decreasing the level in the microtubule fraction ( Fig 2D ) .', 'These results suggest that in the presence of Aβ42 , even hypophosphorylated tau species that are normally bound to microtubules ( Fig 2A ) were mislocalized to the cytosolic fraction .', 'We next investigated whether RNAi-mediated knockdown of GSK3/Sgg suppressed Aβ42-induced augmentation of tau toxicity .', 'Even in the GSK3/Sgg knockdown background , Aβ42 still promoted tau-mediated neurodegeneration , as indicated by smaller eye size in flies co-expressing tau and Aβ42 relative to those expressing tau alone ( Fig 2E , compare SggRNAi+tau and SggRNAi+tau+Aβ42 ) .', 'In addition , GSK3/Sgg knockdown did not suppress neurodegeneration caused by tau and Aβ42 ( Fig 2E , compare tau+Aβ42 and SggRNAi+tau+Aβ42 ) .', 'Under this experimental condition , expression of neither Aβ42 alone nor Aβ42 with GSK3β/Sgg RNAi caused a reduction in eye size ( S6 Fig ) , suggesting that the observed augmentation of tau toxicity was not simply due to additive effects ( Fig 2E ) .', 'Taken together , these results suggest that blocking tau phosphorylation at GSK3/Sgg sites is not sufficient to suppress either mislocalization of tau to the cytosol or Aβ42-induced augmentation of tau toxicity .', 'Previous studies from several groups , including ours , showed that substitution of two Ser residues at 262 and 356 in tau to unphosphorylatable Ala ( S2A ) suppressed Aβ42-induced augmentation of tau toxicity ( [58] and Fig 3A ) .', 'These mutations abolished tau phosphorylation at Ser262 ( Fig 3B , pSer262 ) .', 'Moreover , the SDS-PAGE migration pattern of S2A tau differed from that of wild-type tau .', 'As described above ( Fig 1 and S3 Fig ) , wild-type tau proteins expressed in fly eyes are detected as two major bands reflecting different levels of phosphorylation ( Fig 3B , Aβ42+tau in tauC ) .', 'By contrast , S2A tau exhibited only a single major band corresponding to tauupper ( Fig 3B , Aβ42+S2A in tauC ) .', 'Western blotting with the pan-tau antibodies tauC and tau46 , which were raised against different epitopes and detect taulower to tauupper with slightly different sensitivities , yielded similar results .', 'To quantify this difference , we calculated the ratio of signal intensities of taulower to tauupper and found that S2A tau had a lower ratio of taulower to tauupper than wild-type tau ( Fig 3C ) .', 'In addition , western blotting with TAU-1 tau antibody , which specifically recognizes tau protein without phosphorylation at Ser194 , Ser195 , Ser198 , and Ser202 , and only detected taulower ( Fig 1C ) , revealed that the level of TAU-1-positive tau was significantly reduced in S2A tau relative to wild-type tau ( Fig 3B and 3D ) .', 'These results suggest that less phosphorylated forms of tau ( taulower ) were preferentially reduced in S2A tau .', 'Because taulower represents less phosphorylated forms of tau , S2A mutation might reduce the relative ratio of taulower to tauupper by promoting tau phosphorylation .', 'However , this was not the case: when the phosphorylation levels of S2A tau at Ser202 were normalized against total levels of S2A tau , they were not elevated; instead , they were reduced relative to those of wild-type tau ( Fig 3B and 3E ) .', 'These results suggest that tau phosphorylation at Ser262 and Ser356 stabilizes less phosphorylated forms of tau ( taulower ) but does not promote tau phosphorylation at SP/TP sites ( see also Fig 4 ) .', 'Next , we analyzed the effects of S2A mutation on Aβ42-mediated mislocalization of tau to the cytoplasm using an in vivo microtubule-binding assay .', 'As described above , the levels of taulower were diminished in both the cytosolic and microtubule fractions .', 'Moreover , although Aβ42 still increased the level of S2A tauupper in the cytosol and decreased that in the microtubule fraction ( Fig 3F ) , the effects were smaller than those in wild-type tau: Aβ42 caused a 3-fold increase in the level of wild-type tauupper in the cytosol ( Fig 1B ) , but only a 1 . 6-fold increase in the level of S2A tauupper ( Fig 3F , right ) .', 'Taken together , these data demonstrate that S2A mutation preferentially reduces the levels of taulower , and partially suppresses the Aβ42-mediated increase in the levels of tau mislocalized from the microtubule to the cytosol .', 'Drosophila PAR-1 kinase , a functional homolog of MARKs , is the major kinase of human tau at Ser262 and Ser356 expressed in fly eye and brain [53 , 63] , and RNAi-mediated knockdown of PAR-1 effectively decreases tau phosphorylation at Ser262 and Ser356 [63 , 70] .', 'To further elucidate the role of tau phosphorylation at Ser262 and Ser356 in the stabilization of tau , we asked whether knockdown of PAR-1 would also decrease the levels of less phosphorylated forms of tau ( taulower ) , as the S2A mutation did .', 'Knockdown of PAR-1 significantly reduced the levels of taulower ( Fig 4A ) , whereas overexpression of PAR-1 preferentially increased the levels of taulower ( Fig 4B ) .', 'The effects on the levels of tauupper were less prominent .', 'Importantly , PAR-1-mediated increases in tau levels required the presence of the Ser262 and Ser356 phosphorylation sites in tau .', 'Knockdown of PAR-1 did not decrease the levels of S2A tau ( Fig 4C ) , whereas co-expression of PAR-1 did not increase the levels of S2A tau ( Fig 4D ) .', 'Expression of tau in the brain neurons has been reported to cause structural and functional abnormalities in Drosophila [53 , 58 , 71 , 72] .', 'Pan-neuronal expression of tau in the central nervous system affects development of the mushroom body structure in the brain [53 , 58 , 71–73] , and tau expression in otherwise structurally intact adult neurons causes learning deficits [73] .', 'Blocking tau phosphorylation at Ser262 and Ser356 suppresses these tau-induced defects [58 , 71 , 73] , indicating that tau phosphorylation at Ser262 is critical for tau toxicity also in the brain neurons .', 'To ask whether blocking tau phosphorylation at Ser262 and Ser356 also reduces tau levels in the brain neurons , we expressed tau by using the pan-neuronal driver elav-Gal4 and tested the effect of PAR-1 knockdown on tau levels .', 'PAR-1 knockdown reduced the levels of tau , ( S7 Fig ) , indicating that reduction in tau toxicity caused by blocking tau phosphorylation at Ser262 and Ser356 is concomitant with reductions in tau levels in the brain neurons as well .', 'Taken together , these results indicate that PAR-1 stabilizes less phosphorylated forms of tau ( taulower ) through phosphorylation at Ser262 and Ser356 .', 'The level of tau phosphorylated at Ser262 is elevated in the Aβ42 fly retina [58] ( Fig 5A ) , and this increase was suppressed in the PAR-1 knockdown background ( Fig 5B ) .', 'These results suggest that tau is stabilized via phosphorylation at Ser262 by PAR-1 in the Aβ42 fly retina .', 'We next tested whether knockdown of PAR-1 would reduce the levels of less phosphorylated forms of tau ( taulower ) in the presence of Aβ42 .', 'Western blot analyses using the pan-tau antibodies tauC and Tau46 revealed that PAR-1 knockdown reduced the levels of tau ( Fig 5C , tauC/Tau46 ) , and the reductions in the levels of taulower were more prominent than those of tauupper ( Fig 5C , tauC/Tau46 , ) .', 'The levels of co-expressed Aβ42 were not influenced by PAR-1 knockdown ( Fig 5D ) , suggesting that PAR-1 knockdown did not simply increase protein degradation in a non-specific manner .', 'To further characterize the reduction of tau phosphorylation in the PAR-1 knockdown background , we used a panel of antibodies ( Fig 1C ) that could distinguish the phosphorylation status of tau at AD-associated sites .', 'Western blot analyses using these antibodies confirmed that PAR-1 knockdown significantly decreased the levels of tau phosphorylated at Ser262 .', 'PAR-1 knockdown also caused a prominent reduction in the levels of TAU-1-positive , taulower ( Fig 5D ) .', 'PAR-1 knockdown also reduced the levels of pThr231-positive tau , whereas the PAR-1 knockdown-mediated reductions in the levels of pSer202- and PHF-1-positive tau were less prominent ( Fig 5D ) .', 'A previous study reported that blocking tau phosphorylation at Ser262/356 decreased the levels of tau phosphorylation at SP/TP sites including Ser202 [53] .', 'In that study , it has been suggested that tau phosphorylation at Ser262/356 primes the protein for subsequent phosphorylation events .', 'In this model , blocking tau phosphorylation at Ser262/356 sites should abolish the downstream phosphorylation events and a larger proportion of tau protein should be non-phosphorylated at SP/TP sites , including Ser202 .', 'However , western blot analysis using the TAU-1 antibody , which specifically recognizes tau lacking phosphorylation at Ser202 , revealed that blocking tau phosphorylation at Ser262/356 by either by PAR-1 knockdown ( Fig 5D ) or introduction of the S2A mutation ( Fig 3B ) , did not increase , but rather significantly decreased , the levels of tau species that are not phosphorylated at Ser202 .', 'These data suggest that tau phosphorylation at Ser262/356 rather regulates the stability of taulower species .', 'Taken together , these results suggest that blocking tau phosphorylation at Ser262/356 by PAR-1 knockdown preferentially reduces the levels of taulower also in the presence of Aβ42 .', 'Hyperphosphorylation of tau at SP/TP sites is thought to promote aggregation and accumulation of tau [61 , 74 , 75] .', 'A previous report showed that tau phosphorylation at Ser262 and Ser356 by PAR-1 plays a priming role in subsequent tau phosphorylation at SP/TP sites , and that knockdown of PAR-1 reduces the level of tau phosphorylated at these sites [53] ( also see Fig 5D ) .', 'Moreover , a mammalian homolog of PAR-1 , MARK , positively regulates the activity of GSK3β and may promote tau phosphorylation at SP/TP sites [26 , 53 , 61] .', 'Consistent with this , phosphorylation levels of GSK3/Sgg at Ser9 , which negatively regulates kinase activity [76] , were increased by PAR-1 knockdown ( S8 Fig ) , suggesting that PAR-1 may positively regulate GSK3/Sgg activity in the fly retina as well .', 'Because tau phosphorylation at SP/TP sites is resistant to proteolytic degradation [77] , these observations raise the possibility that knockdown of PAR-1 might reduce tau phosphorylation at SP/TP sites , thereby promoting the degradation of less phosphorylated forms of tau .', 'To investigate this possibility , we next asked whether blocking tau phosphorylation at SP/TP sites by knockdown of GSK3/Sgg was sufficient to reduce tau levels .', 'Western blot analyses using a panel of anti-pan-tau , anti-non-phospho-tau , and anti-phospho-tau antibodies confirmed that knockdown of GSK3β/Sgg significantly decreased tau phosphorylation levels at SP/TP sites ( Fig 6 ) .', 'Migration speed of tau ( Fig 6 , tau46/tauC ) and the level of non-phosphorylated tau were elevated ( Fig 6 , TAU-1 ) , whereas the level of PHF1-positive tau was reduced ( Fig 6 , PHF1 ) .', 'These results suggest that knockdown of GSK3β/Sgg significantly decreased tau phosphorylation levels at SP/TP sites .', 'Under this condition , the levels of tau detected with pan-tau antibodies tau46 and tauC , TAU-1-positive tau , and tau phosphorylated at Ser262 were not reduced , but were instead slightly elevated ( Fig 6 , tau46 , tauC , TAU-1 , and pSer262 ) .', 'These results suggest that tau phosphorylation at SP/TP sites by GSK3/Sgg does not play a major role in stabilization of tau in the fly retina .', 'Because tau phosphorylation at Ser262 was increased by Aβ42 [58] ( Fig 5A ) , we asked whether stabilization of tau through tau phosphorylation at Ser262 and Ser356 contributed to the Aβ42-mediated increase in the level of tau phosphorylated at AD-related SP/TP sites , Ser202 and Thr231 [58] ( Fig 2B ) .', 'Introduction of unphosphorylatable Ala at the Ser262 and Ser356 sites in tau ( S2A ) blocked Aβ42-mediated increases in the levels of tau phosphorylated at Thr231 ( Fig 7A ) .', 'Similar effects were also observed upon knockdown of PAR-1 ( Fig 7B ) .', 'By contrast , Aβ42 increased the level of S2A tau phosphorylation at Ser202 ( Fig 7A: pS202 level was elevated , whereas TAU-1 level was reduced ) .', 'To further validate the dependence of tau phosphorylation at Thr231 on Ser262/356 phosphorylation , we investigated whether upregulation of tau phosphorylation at Ser262/356 by PAR-1 was sufficient to increase the levels of tau phosphorylated at Thr231 .', 'Overexpression of PAR-1 significantly increased the levels of tau phosphorylation levels at Ser262 as expected ( Fig 7C , pSer262 ) .', 'In addition , the levels of TAU-1-positive tau were markedly increased ( Fig 7C , TAU-1 ) , consistent with its stabilizing effect on taulower ( Fig 4B ) .', 'Under these conditions , the level of tau phosphorylated at Thr231 was significantly increased ( Fig 7C , pThr231 ) .', 'Because GSK3/Sgg is required for the Aβ42-mediated increase in the levels of tau phosphorylated at Ser202 and Thr231 ( Fig 2C ) , we also investigated whether Aβ42 increased GSK3/Sgg activity in this model .', 'Phosphorylation levels of GSK3/Sgg at Ser9 sites , which negatively correlate with GSK3/Sgg activity , were not significantly altered by Aβ42 ( S9 Fig ) , suggesting that Aβ42-mediated increases in the levels of tau phosphorylated at Ser202 and Thr231 were not due to global activation of GSK3/Sgg .', 'Indeed , the levels of tau phosphorylated at other GSK3/Sgg-target sites were not altered by Aβ42 in this model [58] .', 'Taken together , these results suggest that tau phosphorylation at Thr231 depends strongly on tau phosphorylation at Ser262 and Ser356 by PAR-1/MARK , and stabilization of tau may contribute to the increase in the levels of tau phosphorylated at an AD-related SP/TP site , Thr231 , caused by Aβ42 .', 'These data also suggest that increases in the levels of tau phosphorylated at other AD-related SP/TP sites , such as Ser202 , might be mediated by mechanisms distinct from tau species phosphorylated at Thr231 ( model in Fig 8 ) .'], ['Tau phosphorylation at Ser262/356 is detected in the pre-tangle stage of neurons in AD brains [78] .', 'Several reports suggest that tau phosphorylation at these sites initiates mismetabolism of tau by increasing the levels of microtubule-unbound free tau [66] .', 'Ser262 and Ser356 are located in the microtubule-binding domain , and phosphorylation at these sites decreases tau binding to microtubules [18 , 19 , 25 , 26 , 50 , 51] .', 'In addition , tau phosphorylation at Ser262/356 is reported to promote tau phosphorylation at other SP/TP sites [53 , 55] , which may also decrease tau binding to microtubules and increases the levels of microtubule-unbound tau .', 'This study demonstrates a novel mechanism by which tau phosphorylation at Ser262/356 via PAR-1/MARK regulates the levels of microtubule-unbound tau in the cytosol: tau phosphorylation at Ser262/356 sites preferentially stabilizes less phosphorylated forms of tau ( taulower ) .', 'Importantly , blocking this stabilization of tau suppressed tau-mediated neurodegeneration and reduced the level of tau phosphorylated at the AD-associated residue Thr231 ( Fig 7 ) .', 'Furthermore , these processes are involved in Aβ42-induced augmentation of tau toxicity ( Fig 3 ) , suggesting the relevance of this step to AD pathogenesis .', 'Collectively , our data reveal a novel role for tau phosphorylation by PAR1/MARK at Ser262 and Ser356 in the stabilization of tau at an early step of mismetabolism , ultimately leading to tau toxicity .', 'We demonstrate that blocking tau phosphorylation at AD-related Ser262/356 sites by introduction of the S2A mutation preferentially decreases the levels of taulower species and suppresses Aβ-mediated augmentation of tau toxicity ( Fig 3 ) .', 'These results suggest that taulower species contribute to neurotoxicity in this experimental model .', 'By contrast , blocking tau phosphorylation at SP/TP sites by RNAi-mediated knockdown of GSK3β/Sgg did not suppress Aβ42-mediated enhancement of tau toxicity ( Fig 2 ) .', 'This observation suggests that tau phosphorylation by GSK3/Sgg is not required for promotion of tau toxicity by Aβ in this model .', 'In addition , knockdown of Sgg significantly decreased phosphorylation levels of tau at SP/TP sites and shifted all tau species to lower–molecular weight regions ( Fig 2 ) ; therefore , Aβ is capable of promoting tau toxicity under conditions in which the levels of tauupper species are diminished .', 'It has been shown that both introduction of S2A mutation and Sgg knockdown reduce tau toxicity [26 , 53] , suggesting that multiple tau species mediate neurotoxicity in a context-dependent manner .', 'From the results of this study , we cannot draw an explicit conclusion regarding which tau species , tauupper or taulower , is more toxic .', 'In our model system , however , microtubule-detached taulower species contributed to the augmentation of tau toxicity caused by Aβ42 , and tau phosphorylation at Ser262/356 played a critical role in the stabilization of taulower in this process .', 'Our results also suggest that the difference between taulower and tauupper species is not simply due to different levels of phosphorylation at SP/TP sites mediated by GSK/Sgg .', 'If it were the case , Sgg RNAi would increase the abundance of tau species qualitatively similar to the original taulower species and greatly promote neurodegeneration .', 'However , we found that Sgg RNAi did not enhance the neurodegeneration phenotype ( Fig 2E ) , suggesting that the original taulower species may be qualitatively different from tau species that are less phosphorylated at SP/TP sites as a consequence of Sgg knockdown .', 'These results raise the possibility that the difference between the original taulower and tauupper species includes not only phosphorylation status at GSK3-target sites but also additional post-transcriptional modifications and/or differences in cellular localization .', 'Our results show that blocking tau phosphorylation at Ser262/356 via PAR-1/MARK reduces tau levels and suppresses augmentation of tau-mediated neurodegeneration caused by Aβ42 ( Fig 3 ) , suggesting that elevated levels of tau phosphorylated at Ser262/356 promote neurodegeneration .', 'Although the mechanisms underlying tau-mediated neurodegeneration remain unclear , one potential mechanism might involve disruption of protein degradation pathways .', 'The majority of tau species ( including tau lacking phosphorylation at Ser262/356 ) are degraded by the proteasome [20] , while aggregated tau or tau phosphorylated at Ser262/356 is degraded by the autophagy-lysosome system [79–84] .', 'This suggests that abnormal increases in the level of tau phosphorylated at Ser262/356 may interfere with or overload the autophagy-lysosome pathway .', 'Consistent with this , the autophagic system and endosome/lysosome pathways are compromised in AD brains , and chronic disturbance of this system is thought to contribute to protein accumulation and neurodegeneration [85–87] .', 'Alternatively , sustained activation of the autophagy pathway might shift the balance of proteostasis and disturb normal cellular functions , potentially causing neuronal dysfunction and degeneration .', 'Supporting this hypothesis , the lysosomal system is upregulated in vulnerable cell populations in AD brains [88] .', 'Further studies regarding the fate of tau phosphorylated at Ser262/356 may reveal the mechanisms underlying the compromised autophagy-lysosome system caused by tau and tau-mediated neurodegeneration in AD and other tauopathies .', 'We found that the Aβ-induced increase in tau phosphorylation at Thr231 depends on phosphorylation at Ser262/356 ( Fig 7 ) .', 'Interestingly , similar to tau phosphorylated at Ser262/356 , tau phosphorylated at Thr231 is detected in the pre-tangle stage in AD brains [89] , and is often found detached from microtubules in vitro and in cultured cells [51 , 90] .', 'These observations suggest that toxic tau species generated in the early stage of mismetabolism are phosphorylated at both Ser262/356 and Thr231 .', 'There are ample data showing the reduction and modification in tau phosphorylated at Thr231 , which have been linked to s
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer’s disease ( AD ) .', 'β-amyloid ( Aβ ) lies upstream of abnormal tau behavior , including detachment from microtubules , phosphorylation at several disease-specific sites , and self-aggregation into toxic tau species in AD brains .', 'To prevent the cascade of events leading to neurodegeneration in AD , it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism .', 'Currently , however , these mechanisms remain unclear .', 'In this study , using transgenic Drosophila co-expressing human tau and Aβ , we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism , leading to neurodegeneration .', 'Aβ increased the level of tau detached from microtubules , independent of the phosphorylation status at GSK3-targeted SP/TP sites .', 'Such mislocalized tau proteins , especially the less phosphorylated species , were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK .', 'Levels of Ser262 phosphorylation were increased by Aβ42 , and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231 , suggesting that this process may be involved in AD pathogenesis .', 'In contrast to PAR-1/MARK , blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels , suppress tau mislocalization to the cytosol , or diminish Aβ-mediated augmentation of tau toxicity .', 'These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis , and that such tau species may represent a potential therapeutic target for AD .']
['Alzheimer’s disease ( AD ) is the most common cause of dementia resulting from progressive neuron loss .', 'Two proteins , β-amyloid ( Aβ ) and tau , accumulate in AD brains and are involved in AD pathogenesis .', 'In healthy neurons , tau binds to microtubules to regulate its stability; in AD brains , however , tau is detached from microtubules and phosphorylated at multiple sites .', 'Such abnormal tau behavior , which is likely to be triggered by Aβ , results in generation of pathological tau species that mediate neuron loss .', 'However , the detailed mechanisms underlying this event remain incompletely understood .', 'Using transgenic flies expressing human tau and Aβ as a model system , we found that tau phosphorylation at specific AD-related sites stabilized microtubule-unbound tau in the early phase of tau mismetabolism to generate toxic tau species .', 'Moreover , this process is critical for Aβ to promote subsequent tau phosphorylation and neurodegeneration .', 'Our results reveal a critical step in the initiation of tau mismetabolism , and this process may represent a potential therapeutic target for AD .']
['phosphorylation', 'medicine and health sciences', 'rna interference', 'microtubules', 'pathology and laboratory medicine', 'neurodegenerative diseases', 'toxicology', 'toxicity', 'epigenetics', 'tubulins', 'alzheimer disease', 'cellular structures and organelles', 'cytoskeleton', 'genetic interference', 'proteins', 'animal cells', 'gene expression', 'dementia', 'mental health and psychiatry', 'biochemistry', 'rna', 'cytoskeletal proteins', 'cell biology', 'post-translational modification', 'nucleic acids', 'neurology', 'neurons', 'genetics', 'biology and life sciences', 'cellular types', 'cytosol']