p70S6K1 protects against tau-mediated neurodegeneration by decreasing the level of tau phosphorylated at Ser262 in a Drosophila model of tauopathy

Neurobiology of Aging 71 (2018) 255e264

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S6K/p70S6K1 protects against tau-mediated neurodegeneration by decreasing the level of tau phosphorylated at Ser262 in a Drosophila model of tauopathy Tomoki Chiku a,1, Motoki Hayashishita a,1, Taro Saito a, b, Mikiko Oka a, Kanako Shinno a, Yosuke Ohtake c, 2, Sawako Shimizu a, Akiko Asada a, b, Shin-ichi Hisanaga a, b, Koichi M. Iijima d, e, Kanae Ando a, b, * a

Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan Department of Biological Sciences, Faculty of Science, Tokyo Metropolitan University, Tokyo, Japan c Department of Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA d Department of Alzheimer’s Disease Research, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan e Department of Experimental Gerontology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 May 2018 Received in revised form 27 July 2018 Accepted 29 July 2018 Available online 3 August 2018

Abnormal accumulation of the microtubule-associated protein tau is thought to cause neuronal cell death in a group of age-associated neurodegenerative disorders. Tau is phosphorylated at multiple sites in diseased brains, and phosphorylation of tau at Ser262 initiates tau accumulation and toxicity. In this study, we sought to identify novel factors that affect the metabolism and toxicity of tau phosphorylated at Ser262 (pSer262-tau). A biased screen using a Drosophila model of tau toxicity revealed that knockdown of S6K, the Drosophila homolog of p70S6K1, increased the level of pSer262-tau and enhanced tau toxicity. S6K can be activated by the insulin signaling, however, unlike knockdown of S6K, knockdown of insulin receptor or insulin receptor substrate nonselectively decreased total tau levels via autophagy. Importantly, activation of S6K significantly suppressed tau-mediated axon degeneration, whereas manipulation of either the insulin signaling pathway or autophagy did not. Our results suggest that activation of S6K may be an effective therapeutic strategy for selectively decreasing the levels of toxic tau species and suppressing neurodegeneration. Ó 2018 Elsevier Inc. All rights reserved.

Keywords: Tau Neurodegeneration Tau phosphorylation at Ser262 S6K/p70S6K1 Insulin signaling Autophagy

1. Introduction The microtubule-associated protein tau accumulates in the brains of patients with Alzheimer’s disease (AD) or other agedependent neurodegenerative diseases collectively called tauopathies (Holtzman et al., 2016). During disease progression, tau proteins are detached from the microtubules and subjected to a number of post-translational modifications, causing them to undergo conformational changes (Ballatore et al., 2007). The altered tau proteins aggregate into bundles of filaments, which form intracellular inclusions (Wang and Mandelkow, 2016). Tau is phosphorylated at more than 40 sites in brains of * Corresponding author at: Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan. Tel: þ81 42 677 2567; fax: þ81 42 677 2559. E-mail address: [email protected] (K. Ando). 1 Those authors contributed equally to this work. 2 Present address: Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan. 0197-4580/$ e see front matter Ó 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.neurobiolaging.2018.07.021

neurodegeneration patients, and phosphorylation at some of these sites correlates with the severity of pathology (Ballatore et al., 2007). Phosphorylation at Ser262 is an early pathological change that plays an initiating role in abnormal metabolism and accumulation of tau (Ando et al., 2016a,b; Augustinack et al., 2002; LasagnaReeves et al., 2016; Nishimura et al., 2004; Yu et al., 2012; Zempel et al., 2010). Blocking tau phosphorylation at this site effectively decreases tau levels and mitigates tau-induced neurodegeneration in cultured cells, Drosophila, and mouse models (Alonso et al., 2010; Ando et al., 2016a,b; Iijima et al., 2010; Iijima-Ando et al., 2012; Lasagna-Reeves et al., 2016; Nishimura et al., 2004). These lines of evidence suggest that decreasing the level of tau phosphorylated at Ser262 (pSer262-tau) would be an effective strategy for targeting toxic tau species, thereby suppressing abnormal tau accumulation and neuron loss. Phosphorylation of tau at Ser262 is mediated by several kinases, including MARK/Par-1 (Drewes et al., 1997; Nishimura et al., 2004), AMP-activated protein kinase (AMPK) (Mairet-Coello et al., 2013; Thornton et al., 2011), CaMKII (Sironi et al., 1998), Chk2 (Iijima-

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and detected using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore). One of the membranes was probed with anti-actin and used as the loading control for other blots. Antitau antibody (TauC) was a kind gift from Dr A. Takashima (Gakushuin University). Anti-tau [T46, Thermo (13e6400)], Antipospho-Ser262-tau antibody [abCam (#ab92627)], anti-Myc antibody [4A6, Merck Millipore (#05e724)], anti-pospho-Thr398-S6K antibody [Cell Signaling (#9208)], anti-chico antibody [Cosmo bio (#COP-080063)], anti-ATG8 antibody [Merck Millipore (#ABC974)], anti-Ref2P antibody (abCaM #ab178440), anti-actin antibody [Sigma (#A2066)] were purchased. The chemiluminescent signal was detected by Fusion FX (Vilber) and intensity was quantified using ImageJ (NIH). Western blots were repeated a minimum of 3 times with different animals and representative blots are shown. Flies used for Western blotting were 2-day old after eclosion.

Ando et al., 2010), p70S6K1 (Pei et al., 2006), and NuaK1 (LasagnaReeves et al., 2016). This modification affects conformation (Fischer et al., 2009), aggregation propensity (Schneider et al., 1999), liquideliquid phase separation (Ambadipudi et al., 2017; Wegmann et al., 2018), interactions with microtubules (Drewes et al., 1997; Mandelkow et al., 2004) and molecular chaperones (Dickey et al., 2007; Karagoz et al., 2014), and cellular distribution (Zempel et al., 2010), suggesting that metabolism of pSer262-tau is regulated at multiple levels. The use of Ser262 tau kinase inhibitors has been proposed as a potential strategy for mitigating tau toxicity. Accordingly, elucidation of the molecular processes underlying the metabolism of pSer262-tau should yield novel therapeutic targets beyond the tau kinases. In this study, we sought to identify novel players involved in the metabolism of pSer262-tau. Drosophila models of human tau toxicity have been established by overexpressing human tau in the fly retina (Wittmann et al., 2001). These models recapitulate key features of biochemical changes observed in the diseased brain, including phosphorylation at disease-associated sites and early phases of the conformational changes with associated neurodegeneration (Wittmann et al., 2001). Using such a model, we screened for genes whose knockdown affects neurodegeneration caused by pSer262-tau. Our results revealed that knockdown of S6K, the Drosophila homolog of p70S6K1, selectively increased the level of pSer262-tau. Unlike S6K, knockdown of the components of the insulin signaling pathway, which is upstream of S6K activity, nonselectively decreased total tau levels via autophagy. Moreover, activation of S6K significantly suppressed tau-mediated axon degeneration, whereas manipulation of either the insulin signaling pathway or autophagy did not. These results suggest that activation of S6K represents an effective therapeutic strategy for selectively decreasing the levels of toxic tau species and suppressing neurodegeneration.

Statistics was done with Microsoft Excel (Microsoft). Differences were assessed using the Student’s t test. p values <0.05 were considered statistically significant.

2. Materials and methods

3. Results

2.1. Fly stocks

3.1. S6K affects the level of pSer262-tau

Flies were maintained in standard cornmeal media at 25  C under light-dark cycles of 12:12 hours, and food vials changed every 4e5 days. The transgenic fly line carrying the human 0N4R tau, which has 4 tubulin-binding domains (R) at the C-terminal region and no N-terminal insert (N), is a kind gift from Dr M. B. Feany (Harvard Medical School) (Wittmann et al., 2001). The transgenic fly line carrying UAS-S2Atau was reported previously (Ando et al., 2016a,b; Iijima-Ando et al., 2012). S6K ribonucleic acid interference (RNAi) (HMS02267), AMPK RNAi (GL00004), insulin receptor (InR) RNAi (HMS03166), chico RNAi (HMS01553), and other RNAi lines listed in Table S2 were obtained from NIG-fly stock center (National Institute of Genetics). Luciferase RNAi (BDSC#31603), UAS-S6K (BDSC#6910), UAS-S6K KQ (S6K DN) (BDSC#6911), UAS-S6K STDETE (S6K CA) (BDSC#6914), UAS-Tor (BDSC#7012) were obtained from the Bloomington Drosophila Stock Center. UAS-ATG1 (GS10797) was obtained from Kyoto Stock Center. UAS-AMPK is a gift from Dr Jay E. Brenman (University of North Carolina). UAS-chico (Naganos et al., 2012) is a gift from Dr M. Saitoe (Tokyo Metropolitan Institute of Medical Science). UAS-Par1-Myc is a gift from Dr Bingwei Lu (Stanford University). Genotypes are described in Table S1.

Forward genetic screens in the fly from several laboratories identified modifiers of tau-induced neurodegeneration (Bilen and Bonini, 2007; Blard et al., 2006; Cao et al., 2008; Fulga et al., 2007; Jackson et al., 2002; Karsten et al., 2006; Khurana et al., 2006; Shulman and Feany, 2003; Shulman et al., 2014). Among these modifiers, we screened for genes whose knockdown affects the level of pSer262-tau. For this purpose, human tau was coexpressed with RNAi targeting the candidate gene, or a control RNAi construct, under the control of the pan-retinal gmr-Gal4 driver. To compare tau protein levels, head homogenates were subjected to Western blotting with anti-tau antibody or an antibody that specifically recognizes pSer262-tau. Among 28 genes tested (Table S2), we found that RNAi-mediated knockdown of S6K significantly increased the level of total tau, as well as the level of pSer262-tau (Fig. 1A). We confirmed that expression of this RNAi effectively decreased expression of S6K (Fig. 1A). The ratio of pSer262-tau and total tau were not significantly changed (Fig. 1A). We also found that overexpression of a dominant-negative form of S6K (S6K DN) (Barcelo and Stewart, 2002; Kapahi et al., 2004; Mikeladze-Dvali et al., 2005; Shen and Ganetzky, 2009) increased the levels of total tau and pSer262-tau (Fig. 1A), suggesting that blocking S6K activity increases tau levels in this fly model. We previously reported that tau phosphorylation at Ser262 stabilized tau and enhancement of tau phosphorylation at this site increases total tau levels (Ando et al., 2016a,b). To ask whether tau phosphorylation at Ser262 is critical for the increase in total tau levels caused by S6K inhibition, we used a tau mutant harboring

2.2. Western blotting Western blotting was carried out as described previously (Ando et al., 2016b). The membranes were blotted with the antibodies described below, incubated with appropriate secondary antibody,

2.3. Histological analysis Fly heads were fixed in Bouin’s fixative for 48 hours at room temperature, incubated for 24 hours in 50 mM Tris/150 mM NaCl, and embedded in paraffin. Serial sections (7 mm thickness) through the entire heads were stained with hematoxylin and eosin and examined by bright-field microscopy. Images of the sections that include the lamina were captured with Keyence microscope BZX700 (Keyence), and vacuole area was measured using Image J (NIH). Heads from more than 3 flies (more than 5 hemispheres) were analyzed for each genotype. 2.4. Statistics

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Fig. 1. Blocking S6K activity increases the levels of pSer262-tau. (A) S6K knockdown increases tau protein levels. (Top) Expression of S6K RNAi reduces the levels of active S6K protein. Western blot analysis of fly heads expressing control RNAi (control) or S6K RNAi (S6K RNAi) driven by gmr-Gal4. Blots were performed with anti-pThr398 S6K antibody. Representative blots (left) and quantitation (right) are shown. Means SD; n ¼ 4. ***, p < 0.005 (Student’s t-test). (Middle) Western blot analysis of fly heads coexpressing tau and either control RNAi (Tau þ control RNAi) or S6K RNAi (Tau þ S6K RNAi) driven by the pan-retinal gmr-Gal4 driver. (Bottom) Western blot of fly heads expressing tau (Tau) or coexpressing tau and a dominant-negative form of S6K (Tau þ S6K DN). (B) The increase in tau levels caused by S6K knockdown depends on Ser262. Western blots of heads of flies coexpressing tau with nonphosphorylatable Ala substitution at Ser262 and Ser356 (S2A-Tau) with control RNAi (S2A-Tau þ control RNAi), S6K RNAi (S2A-Tau þ S6K RNAi), or alone (S2A-Tau) or with S6K DN (S2A-Tau þ S6K DN). (C) Overexpression of a constitutively active form of S6K reduced the level of pSer262-tau. Western blot of fly heads expressing tau (Tau) or coexpressing tau and a constitutively active form of S6K (Tau þ S6K CA). Blots were performed with anti-tau antibody (total-Tau) or anti-phospho Ser262 tau antibody (pSer262-Tau). Actin was used as a loading control. Representative blots (left) and quantitation (right) are shown. Means SD; n ¼ 4. N.S., p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.005 (Student’s t-test).

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nonphosphorylatable alanine in place of Ser262 and Ser356 (S2A) (Iijima-Ando et al., 2012). Neither expression of S6K DN nor RNAimediated knockdown of S6K increased the level of S2A-tau (Fig. 1B). These results indicate that blocking S6K activity increases tau levels via the mechanisms that depends on S262, such as increasing tau phosphorylation at Ser262 or boosting protein degradation mechanisms that target pSer262-tau. This result also indicated that the increase in tau levels caused by blocking S6K is mediated post-translationally.

We further asked whether activation of S6K was capable of decreasing the levels of pSer262-tau. Indeed, expression of a constitutively active form of S6K (Barcelo and Stewart, 2002; Kapahi et al., 2004; Lee et al., 2010; Liu and Lu, 2010; Shen and Ganetzky, 2009) significantly decreased the level of total tau and pSer262-tau (Fig. 1C). Taken together, these results suggest that S6K activity selectively affects tau levels in a manner that depends on Ser262 phosphorylation.

Fig. 2. InR, chico, and TOR negatively regulate tau levels in a Ser262/356-independent manner. (A) InR and chico knockdown decreases tau protein levels. Western blot analysis of fly heads expressing tau and control RNAi (Tauþcontrol RNAi) or coexpressing tau and InR RNAi (TauþInR RNAi), or fly heads expressing tau or coexpressing tau and chico RNAi (Tauþchico RNAi). (B) Tor overexpression increases tau protein levels. Western blot analysis of fly heads expressing tau (Tau) or coexpressing tau and Tor (Tau þ Tor OE). (C) Effects of chico knockdown or Tor overexpression on tau levels are independent of Ser262. Western blots of fly heads coexpressing tau with S2ATau, either with control RNAi (S2ATau þ control RNAi) or with a chico RNAi (S2A-Tau þ chico RNAi), or alone (S2A-Tau) or with Tor overexpression (S2A-Tau þ Tor OE). Blots were performed with anti-tau antibody (totalTau) or anti-phospho Ser262 tau antibody (pSer262-Tau). Actin was used as a loading control. Representative blots (left, middle) and quantitation (right) are shown. Means SD; n ¼ 4. N.S., p > 0.05; *, p < 0.05; ***, p < 0.005 (Student’s t-test). Abbreviation: InR, insulin receptor.

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3.2. The insulin signaling pathway and autophagy nonselectively affect tau levels in a pSer262-independent manner S6K is a major downstream target of Tor in the insulin signaling pathway (Wullschleger et al., 2006). The InR and insulin receptor substrate (IRS) trigger a cascade of phosphorylation events leading to Tor, which phosphorylates S6K at Thr398 and positively regulates S6K activity (Lizcano et al., 2003) (Fig. 2A). Hence, we asked

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whether InR or IRS regulates the level of pSer262-tau in the same way that S6K does. Initially, we hypothesized that blocking insulin signaling would increase the pSer262-tau level. However, we observed that RNAi-mediated knockdown of InR or the Drosophila homolog of IRS, chico (Fig. S1), significantly decreased the levels of total tau and pSer262-tau (Fig. 2A). Similarly, overexpression of Tor increased the levels of both phosphorylated and total tau (Fig. 2B).

Fig. 3. The reduction in tau levels caused by inhibition of insulin signaling may be due to disruption of autophagy. (A) ATG1 overexpression decreased the levels of total tau and pSer262-tau. Western blot analysis of fly heads expressing tau alone (Tau) or with overexpression of ATG1 (Tau þ ATG1 OE). Blots were performed with anti-tau antibody (total-Tau) or anti-phospho Ser262 tau antibody (pSer262-Tau). (B) Reduction in tau levels caused by induction of autophagy does not depend on Ser262/356. Western blots of fly heads expressing S2A-Tau alone (S2A-Tau) or with ATG1 overexpression (S2A-Tau þ ATG1 OE). (C) Chico knockdown promotes autophagy. Western blotting of fly heads expressing control RNAi or chico RNAi. Blots were performed with anti-LC3 antibody (Top) or anti-Ref2P, the Drosophila melanogaster homologue of mammalian p62 (Bottom). (D) S6K inhibition does not enhance autophagy. Western blot of fly heads without (control) or with expression of S6K DN in the retina (S6K DN). Representative blots (left) and quantitation (right) are shown. Means SD; n ¼ 4. N.S., p > 0.05; *, p < 0.05; ***, p < 0.005 (Student’s t-test). Actin was used as a loading control.

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We also asked whether knockdown of IRS/chico or overexpression of Tor would affect the level of S2A-tau. Knockdown of chico significantly decreased the level of S2A-tau, whereas overexpression of Tor increased the levels of the mutant protein (Fig. 2C), indicating that the insulin signaling pathway affects tau levels independently of phosphorylation at Ser262. These results suggest that insulin signaling and S6K regulate tau levels in opposite directions via different underlying mechanisms. 3.3. S6K inhibition does not affect autophagy induction Because insulin signaling and Tor negatively regulate autophagy (Kaur and Debnath, 2015), and tau is degraded by autophagy (Menzies et al., 2017), we postulated that the observed effects of InR, Chico, and Tor on tau levels were mediated by autophagy. Indeed, we found that overexpression of ATG1, which induces autophagy in Drosophila (Scott et al., 2007), caused a significant reduction in tau levels (Fig. 3A) independent of tau phosphorylation at Ser262 (Fig. 3B). To detect the induction of autophagy by chico knockdown in this background, the levels of the markers of autophagic vesicles, the LC3-II/LC3-I ratio, and the autophagic substrate p62 (Nagy et al., 2015), were analyzed. Knockdown of chico caused reductions in the LC3-II/I ratio and the levels of the autophagic substrate p62, suggesting that blocking insulin signaling enhances autophagy (Fig. 3C). Together, these results suggest that insulin signaling affects tau levels via autophagy. S6K can regulate autophagy either positively or negatively, depending on the cellular conditions (Scott et al., 2004). Hence, we examined the effects of blocking S6K on induction of autophagy in our fly model. In contrast to inhibition of insulin signaling, expression of S6K DN did not affect either the LC3-II/LC3-I ratio or the p62 level (Fig. 3D), suggesting that the observed effects of S6K inhibition on tau levels in this model were independent of autophagy. 3.4. S6K inhibition does not increase activity of either MARK/PAR-1 or AMPK Because the effect of S6K depends on tau phosphorylation at Ser262, we hypothesized that S6K affects kinases that phosphorylate tau at this site. A major kinase regulating tau phosphorylation at Ser262 in this fly model is PAR-1, the Drosophila homolog of MARK (Nishimura et al., 2004). PAR-1 activity is regulated by phosphorylation at T408, which stabilizes the protein (Yu et al., 2012). Hence, we asked whether S6K inhibition would increase PAR-1 levels in the fly retina. However, we found that RNAimediated knockdown of S6K did not increase, but rather decreased, the level of PAR-1 (Fig. 4A). AMPK is also known to phosphorylate tau at Ser262 in mammalian neurons (Mairet-Coello et al., 2013) and also in this model (Fig. S2). Hence, we investigated whether blocking S6K would increase AMPK activity in the fly retina and found that S6K knockdown did not affect the levels of the active form of AMPK (Fig. 4B). Taken together, these results suggest that the effect of inhibition of S6K on tau levels is not mediated by upregulation of tau phosphorylation at Ser262 by PAR-1 or AMPK. 3.5. Knockdown of InR or chico does not rescue tau-mediated axon degeneration Because tau levels were significantly decreased by knockdown of InR or chico, as well as by ATG1 overexpression, we asked whether neurodegeneration was also suppressed in these flies. However, neither knockdown of InR or chico significantly suppressed tauinduced neurodegeneration in the lamina (Fig. 5A and B). Because

Fig. 4. Reduction in pSer262-tau levels caused by blocking S6K is not mediated by enhancement of activity of MARK/PAR-1 or AMPK. (A) S6K knockdown does not increase Par-1 levels. Western blot of fly heads expressing myc-tagged Par-1 (Par-1 þ control RNAi) or coexpressing Par-1 and S6K RNAi (Par-1þS6K RNAi). Blots were performed with anti-myc antibody (Par-1). (B) S6K knockdown does not increase the level of the active form of AMPK. Western blot of fly heads expressing control RNAi (control RNAi) or S6K RNAi (S6K RNAi). Blots were performed with anti-phosphoeAMPK antibody (p-AMPK). Actin was used as a loading control. Representative blots (left) and quantitation (right) are shown. Means SD; n ¼ 4. N.S., p > 0.05; *, p < 0.05 (Student’s t-test).

blocking insulin signaling promotes autophagy, we hypothesized that sustained activation of autophagy could itself cause neurodegeneration. Indeed, fly eyes overexpressing ATG1 exhibited degeneration in the lamina (Fig. 5B), suggesting that chronic inhibition of insulin signaling has detrimental effects on neural integrity. Expression of human tau in Drosophila eyes also decreases the external eye size due to apoptosis during the larval stage (Jackson et al., 2002; Wittmann et al., 2001). Accordingly, we asked whether rough-eye phenotypes would be ameliorated by knockdown of InR or chico. Consistent with the results described above, the reduced eye size of tau flies was not recovered by RNAimediated knockdown of InR or chico (Fig. S3). Thus, although knockdown of InR or chico or overexpression of ATG1 decreased tau levels, they failed to rescue tau-induced neurodegeneration in this model. 3.6. Activation of S6K suppresses, while inhibition of S6K promotes, tau-mediated axon degeneration Finally, we investigated whether activation of S6K would suppress tau toxicity. Overexpression of a constitutively active form of S6K (Barcelo and Stewart, 2002) significantly mitigated tauinduced neurodegeneration (Fig. 6A). By contrast, RNAi-mediated knockdown of S6K RNAi (Fig. 6B) and expression of S6K DN both significantly promoted tau-mediated neurodegeneration (Fig. 6C). Expression of S6K RNAi in the retina without tau did not cause neurodegeneration (Fig. 6B and C). These results suggest that enhancement of S6K activity represents an effective strategy for selectively decreasing the levels of toxic tau species and suppressing neurodegeneration.

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Fig. 5. Neither knockdown of InR or chico nor overexpression of ATG1 rescues tau-mediated axon degeneration. (A) (Left) Lamina of flies expressing the driver gmr-GAL4 alone (control), expressing tau alone (Tau), coexpressing tau and InR RNAi (TauþInR RNAi), or coexpressing tau and chico RNAi (Tauþchico RNAi). Neurodegeneration is observed as vacuoles (arrows). (Right) Quantification of vacuole area. Means SEM; n ¼ 6e10. N.S., p > 0.05, *, p < 0.05 (Student’s t-test). (B) (Left) Lamina of flies expressing tau (Tau), tau with overexpression of ATG1 (TauþATG1 OE), overexpression of ATG1 without tau (ATG1 OE). (Right) Quantification of vacuole area. Means SEM; n ¼ 6e10. N.S., p > 0.05, *, p < 0.05; ***, p < 0.005 (Student’s t-test). Flies used for experiments were 10-day old after eclosion. Abbreviation: InR, insulin receptor.

4. Discussion p70S6K1 plays evolutionarily conserved roles in cell growth, cell differentiation, cell cycle control, and lifespan (Selman et al., 2009; Um et al., 2006; Wullschleger et al., 2006). Activation of p70S6K1 is observed in AD brains and is correlated with deposition of neurofibrillary tangles (An et al., 2003). Because p70S6K1 is essential for cell survival, the observed activation of p70S6K1 in AD may represent a protective response against stress (Pei et al., 2008). In this study, we demonstrated that S6K, the Drosophila homolog of p70S6K1, protects against tau toxicity by selectively decreasing the level of pSer262-tau. This effect was not associated with changes in the activities of the major tau Ser262 kinases, MARK/PAR-1, and AMPK, and furthermore was independent of autophagy induction. Together, these results suggest that activation of S6K represents a novel strategy for decreasing tau accumulation and toxicity. Several previous studies suggested that p70S6K1 may augment tau toxicity, either directly or indirectly, and promote AD pathogenesis. p70S6K1 phosphorylates tau at the diseaserelated residues T212, S214, and S262 in vitro (Pei et al., 2006), and positively regulates the translation of tau mRNA by phosphorylating the 40S ribosomal protein S6 (Pei et al., 2006). Loss of p70S6K1 protects against neurodegeneration in 3xTg-AD mice by decreasing translation of b-site amyloid precursor protein cleaving enzyme 1 and tau (Caccamo et al., 2015). By contrast, our

results suggest a novel role of S6K activity in tau metabolism and toxicity; S6K activity selectively reduces the levels of pSer262tau at the post-translational level (Fig. 1) to suppress neurodegeneration (Fig. 6). Notably in this regard, in contrast to the 3xTg-AD mouse, which shows massive Ab accumulation in addition to tau pathology, the fly model used here does not coexpress Ab, suggesting that the observed effects of S6K on tau metabolism and toxicity do not involve pathogenic interactions between Ab and tau. The toxicity of human tau expressed in the fly retina can be observed as a “rough-eye” phenotype, that is, eyes having a smaller size and rough surface due to apoptosis during development (Jackson et al., 2002; Wittmann et al., 2001). Loss of 1 copy of S6K suppresses the rough-eye phenotype caused by tau (Khurana et al., 2006). Hence, we examined the effects of S6K knockdown on rough-eye phenotype induced by tau. Consistent with the results of the previous study, we also observed that S6K knockdown significantly increased eye size in tau flies (Fig. S4). Thus, even in the same fly model system, S6K can modify tau-induced phenotypes in the opposite direction. A possible explanation for this observation is that, because the rough-eye phenotype occurs during development, it might be due to alterations in the cell cycle regulation of S6K (Khurana et al., 2006). By contrast, in this study, we focused on adult-onset, age-dependent degeneration of photoreceptor axons, which does not involve cell cycle regulation.

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Fig. 6. Tau toxicity is mitigated by elevation of S6K activity. (A) Tau toxicity is mitigated by expression of a constitutively active form of S6K. (Left) Lamina of flies expressing tau alone (Tau) or coexpressing tau and a constitutively active form of S6K (TauþS6K CA). Neurodegeneration is observed as vacuoles, indicated by arrows. (Right) Quantification of vacuole area in Tau and TauþS6K CA. Means SEM; n ¼ 8e10. ***, p < 0.005 (Student’s t-test). (B) Tau toxicity is increased by S6K knockdown. (Left) lamina of flies expressing tau and control RNAi (Tauþcontrol RNAi), coexpressing tau and S6K RNAi (TauþS6K RNAi), or expressing S6K RNAi without tau (S6K RNAi). (Right) Quantification of vacuole area in Tau, TauþS6K RNAi, and S6K RNAi. Means SEM; n ¼ 6e10. **, p < 0.01 between Tauþcontrol RNAi and TauþS6KRNAi (Student’s t-test). (C) Tau toxicity is increased by expression of a dominant-negative form of S6K (S6K DN). (Left) Lamina of flies expressing tau (Tau), coexpressing tau and S6K DN (TauþS6K DN), or expressing S6K DN without tau (S6K DN). (Right) Quantification of vacuole area. Means SEM; n ¼ 6e10. *, p < 0.05 between Tau and TauþS6KRNAi (Student’s t-test). Flies used for experiments were 10-day old after eclosion.

These results suggest that the roles of S6K in the disease progression are complex and affected by the disease stages or other pathological factors. S6K acts as a downstream target of the insulin signaling pathway, disruption of which has been linked to neurodegenerative diseases (Um et al., 2006). We found that knockdown of insulin signaling and S6K affects tau levels differently: knockdown of InR

and chico decreased tau levels presumably by inducing autophagy (Figs. 2 and 3), whereas blocking S6K selectively increased the level of pSer262-tau (Fig. 1). Furthermore, although knockdown of InR and chico decreased tau levels, it failed to suppress tau-induced neurodegeneration (Fig. 5). This was presumably due to chronic induction of autophagy (Fig. 3) because although autophagy protects neurons against an accumulation of aggregated proteins,

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chronic activation of autophagy causes neurodegeneration (Mizushima et al., 2008). The mechanism by which S6K regulates pSer262-tau levels remains to be elucidated. The substrates of S6K include a group of proteins involved in RNA metabolism, protein synthesis, and metabolic reprogramming, as well as oncogenic proteins and tumor suppressors (Um et al., 2006). AMPK is one of the kinases that phosphorylates tau at Ser262 (Mairet-Coello et al., 2013), and in the mouse hypothalamus, S6K phosphorylates AMPK-a at Ser491 in response to leptin, inhibiting phosphorylation at Thr172 and decreasing its activity (Dagon et al., 2012). However, the phosphorylation site corresponding to AMPK-a Ser491 in Drosophila AMPK has not been identified, and blocking S6K activity did not significantly increase Thr172 phosphorylation (Fig. 4). Because S6K is involved in physiological changes associated with aging, including senescence and alterations in energy metabolism, future studies aimed at identifying the S6K substrates that regulate tau levels may reveal molecular links between aging and neurodegenerative diseases. 5. Conclusions These results demonstrate that S6K protects against tau toxicity by selectively decreasing the level of pSer262-tau. This effect was not associated with changes in the activities of the major tau Ser262 kinases, MARK/PAR-1, and AMPK, and furthermore was independent of autophagy induction. Together, these results reveal a novel mechanism that regulate metabolism and toxicity of tau, and suggest that activation of S6K represents a novel strategy for decreasing tau accumulation and toxicity. Disclosure statement The authors have no actual or potential conflicts of interest. Acknowledgements For fly stocks, we thank Drs. Jay Brenman, Mel B. Feany, Bingwei Lu, and Minoru Saitoe; TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947); the Bloomington Stock Center; and the Vienna Drosophila RNAi center. We thank Dr. Akihiko Takashima for tau antibody. We thank T. Oba and A. Ishio for technical assistance. We thank Drs. K. Kawahara and N. Takatori for critical comments. Funding: This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control) [Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number 15H01564 and 17H05703] (to KA), Grantsin-Aid for Scientific Research [Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number 15K06712] (to KA), a research award from the Japan Hoan-sha Foundation (to KA), the Takeda Science Foundation (to KA), Research Funding for Longevity Sciences (28e26) from the National Center for Geriatrics and Gerontology (NCGG), Japan (to KMI), Grants-in-Aid for Scientific Research [JSPS KAKENHI Grant number 16K08637] (to KMI), and the Takeda Science Foundation, Japan (to KMI). Authors’ contributions: KA and KMI were responsible for the design of the study. TC, MH, MO, TS, KS, YO, SS, AA, and KA performed experiments and analyzed data. TC, MH, SH, KMI and KA interpret the data. KA, MH and KMI drafted the article. All authors read and approved the final article. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.neurobiolaging.2018. 07.021.

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