降低組蛋白去乙醯酶活性減緩果蠅模式之Tau蛋白神經毒性之研究

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(1)國立臺灣師範大學生命科學系碩士論文. 降低組蛋白去乙醯酶活性於果蠅模式中減緩 Tau 蛋白神經毒性 Down-regulation of histone deacetylase attenuates Tau neurotoxicity in Drosophila. 研究生 : 黃昱嘉 Yu-Jia Huang. 指導教授: 蘇銘燦博士 Dr. Ming-Tsan Su. 中華民國 103 年二月.

(2) Table of content. Abstract………….…………………………………………….……. 2. 摘要……………………………………………………………….... 3. Introduction…………………………………………………………. 4. Objectives of this study……………………………………………... 7. Materials and Methods………………………………………….…..... 8. Results………………………………………………………………. 12. Discussion…………………………………………………................ 19. References………………………………………………………..…. 23. Figures…………………………………………………….……..….. 27. 1.

(3) Abstract Tau is a neuronal enriched protein whose function is to stabilize the structure of microtubules, and maintain the integrity of neuron. However, it was found that hyperphosophorylation and aberrant accumulation of Tau in the neurons can cause a group of neurodegenerations, including Alzheimer’s disease and frontotemporal dementia, which are collectively called tauopathies. Since it was reported that transcriptional dysfunction is one of the main pathomechanisms of tauopathies and inhibition of histone deactylase (HDAC) can enhance transcription and ameliorate the neuronal impairments, we would like to address the question using Drosophila as a model system. The aims of my study is to inactivate HDAC by either RNA interference (RNAi) or HDAC inhibitors (HDACIs) to test if down-regulation of HDAC is beneficial in reducing Tau toxicity in flies. We found that the notal bristle was increased in Tau-expressing flies when HDACs were knocked down by RNAi. Similarly, some novel HDAC inhibitors can exhibits same beneficial effects. We further discovered that the expression of acetylated histone H3 was increased when treated with RNAi or a dietary HDACI, Sulforaphane (SFN). The expression and phosphorylation of Tau were decrease in both treatment, and increase the solubility of Tau. Additionally, knocking down most HDAC or SFN treatment can extend the lifespan of tau expressing flies. In sum, my studies suggest that phosophorylated Tau might be the main causes of tauopathies. On the contrary, inhibition of HDAC might be beneficial to tauopathies. Key words: HDAC, Tau, neurodegeneration, Drosophila 2.

(4) 摘要 Tau 是一個被大量表現在中樞神經系統之中的蛋白，其功能為穩固神經細胞之中微管的結構，並維持神經系統的完整性。Tau 蛋白在過度磷酸化及不正常聚集會造成許多退化性神經疾病通稱為”tauopathies”。這些疾病包括了阿茲海默氏症以及額顳葉失智症。由於研究顯示基因轉錄失調為 tauopathies 的致病機轉之一，且抑制組蛋白去乙醯酶可改善上述病徵。因此本研究的目的在於利用果蠅模式，藉由組蛋白去乙醯酶抑制劑及核酸干擾檢驗上述推論。我們先前研究發現在果蠅過量表現人類的 Tau 蛋白會造成背甲上的剛毛缺少，利用此一模式以核酸干擾的方式，發現降低果蠅的組蛋白去乙醯酶的表現能有效的抑制因 Tau 蛋白表現所造成剛毛缺失的現象，同樣地，利用一些新穎的組蛋白去乙醯酶抑制劑也可以達到改善剛毛生長的情況，進一步我們發現核酸干擾及抑制劑的處理可增加組蛋白 H3 的乙醯化。而核酸干擾及組蛋白去乙醯酶抑制劑的處理會降低 Tau 及 Tau 磷酸化的表現以及提升 Tau 的溶解度。然而核酸干擾及部分組蛋白去乙醯酶抑制劑可增加 Tau 果蠅模式的壽命。綜合上述發現，我們認為降低 Tau 蛋白的表現量和磷酸化及增加組蛋白 H3 的乙醯化，可能對病徵有改善的作用。關鍵字: 組蛋白去乙醯酶，Tau，神經退化性疾病，果蠅 3.

(5) Introduction. Tauopathy The microtubule-associate protein Tau was identified for its activity in microtubule assemblage and the stabilization (WEINGARTEN et al. 1975). Many neurodegenerations, such as Alzheimer’s disease (AD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) and Pick disease, are collectively called tauopathies because of the abnormal deposition of Tau is a common pathology among these diseases (IQBAL et al. 2005; BRUNDEN et al. 2009). In tauopathies, Tau loses its ability to bind microtubule and aggregates into intracytoplasmic filaments known as neurofibrillary tangles (NFTs). AD, the most common and severe form of dementia, is alone an enormous burden to our society. This disease progression and severity is positive parallel with the fabrication of NFTs (ARRIAGADA et al. 1992). Because of Tau interacts with microtubules, actin and several signaling molecules, it is believed that tau-mediated neurodegeneration is cause by the aggregation of gain of tau toxic and loss the normal tau function.. Pathogenic mechanism of tauopathy Tauopathies are a class of neurodegenerative diseases associated with the pathological aggregation of tau protein and the underlying pathomechanism of neurofibrillary tangles-caused neurotoxicity is incompletely unclear. Accumulated evidence demonstrated that 4.

(6) hyperphosphorylation of tau accelerates the formation of paired helical filament (PHF) and neurodegeneration. However, some contradict results have also been obtained (YEH et al. 2010). Other posttranslational modifications, including proteolysis (KOSIK AND SHIMURA 2005), ubiquitination (BANCHER et al. 1991), nitration (GONG et al. 2005) and glycosylation (LIU et al. 2002) were also shown to be involved in the disease progression. In addition, a mutation of tau protein resistant to phosphorylation GSK3 retains substantial toxicity (CHATTERJEE et al. 2009). Moreover, transcriptional dysfunction is considered one of the main pathomechanisms of tauopathies (ERMAK AND DAVIES 2002). Drosophila models for tauopathy Tau is normally a highly soluble protein that usually does not aggregates into filaments. However, tau aggregation can be formed and the process can be accelerated by polyanionic cofactors or small molecule in vitro. In Alzheimer’s disease and other tauopathies, the neurodegeneration is involved in the abnormal hyperphosphorylation of tau protein, and this hyperphosphorylated tau is the major subunit of the paired helical filaments (PHF) and straight filaments (SF) forming neurofibrillary tangles (IQBAL et al. 2009), but hyperphosphorylated tau in Drosophila model had no affect to neurofibrillary tangle formation (COWAN et al. 2010b). Transgenic Drosophila models had been widely used to study neurodegenerative disease including AD and tauopathies (IIJIMA-ANDO AND IIJIMA 2010).. Expressing human wild-type Tau or mutant R406W. Tau in Drosophila exhibits key tauopathy features, including 5.

(7) accumulation of abnormal Tau and early deaths, but these occurred without neurofibrillary tangles (WITTMANN et al. 2001).. Histone protein in tauopathies Histones are highly alkaline proteins found in cell nuclei that package and order the DNA into structural units called nucleosomes. Histone acetylation and deacetylation are essential parts of gene regulation. These reactions are typically catalyzed by enzymes with "histone acetyltransferase" (HAT) or "histone deacetylase" (HDAC) activity. Acetylation is the process where an acetyl functional group is transferred from one molecule to another. Deacetylation is simply the reverse reaction where an acetyl group is removed from a molecule. It has been known for decade that tau induced neurotoxicity could cause the gene down-regulation (ERMAK AND DAVIES 2002). Several studies described mutation in histone deacetylase 6 (HDAC6) reduced the microtubule disorganized in both muscle and neuron cells (XIONG et al. 2013). Furthermore, down-regulation of different class of histone deacetylase displayed positive effect of tau-induced neurotoxicity in many studies (GAN 2007; AGIS-BALBOA et al. 2013; GOVINDARAJAN et al. 2013). Therefore, a decrease in the expression level of HDAC might be a therapeutic treatment for the tau-induced neurodegeneration diseases. Despite numerous reports of HDAC inhibition rescue Tau induced neurodegeneration, these studies only focused on single class of HDAC. In my study, we exam if the different classes of HDACs play similar role in modulating Tau mediated neurotoxicity. 6.

(8) Objectives of this study HDAC is an important regulator of gene expression, which could decreases the level of gene expression. The main objective of this study is to determine whether down-regulation of different classes of HDAC affect the tau induced neurotoxicity Moreover we want to know the above phenomenon in tranegenic Drosophila model. In parallel, we would also like to know HDAC inhibitors and the RNA interference of HDAC can modulate tauopathy.. 7.

(9) Material and Methods Fly stocks and genetics Fly stock including, Elav-Gal4, w1118 were obtained from the Bloomington Stock Center. Eq-Gal4 has been a gift from H. Sun (Tang and Sun, 2002). The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit flyis. It was developed in 1993 (BRAND AND PERRIMON 1993), and is considered a powerful technique for studying the expression of genes (DUFFY 2002). The system has two parts: the GAL4 gene, encoding the yeast transcription activator protein GAL4, and the UAS (Upstream Activation Sequence), an enhancer to which GAL4 specifically binds to activate gene transcription. UAS-Tau strains obtained by the Feany, the histone deacetylase RNAi strains were acquired from VDRC (Table below). For all the physiological and biochemical assays, Elav-Gal4 flies were crossed with UAS-Tau flies to express Tau in the central neural system (CNS). All fly stocks were maintained on standard yeast-glucose medium at 25ºC. Table1. RNAi stocks used in the study. 8.

(10) Drug treatment HDAC inhibitors (NC100, NC102, NC112, NC113, NC114, NC115) were obtained from Enamine company. These inhibitors were all dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mM (stock solution). To administer drug to the flies, compounds were diluted into yeast-glucose medium at final concentrations of 1 , 5 , or 50 Same amount of DMSO was added to medium containing as solvent control. 5 pairs of Eq>hTauWT (express Tau in fly notal) flies were collected and placed into a culture vial containing 10 ml yeast-glucose medium. First filial generation was collect for the follow-up assay.. Bristle assay Flies were collected and adhered onto the slide glass with white glue in spread-wing postures. Images of the fly nota were captured under microscope equipped with a cooled CCD camera. The notal bristles were scored. Quantification analysis were performed using Student’s t-test. p values less than 0.05 were considered to be statistically significant.. Immunoblotting To analyze phosphorylation levels of Tau proteins and acetylation levels of histone protein, adult fly heads were homogenized in the 2X Laemmli buffer (0.5M Tris-HCl (pH6.8), 10% SDS, Bremophenol blue, glycerol, β-mercaptoethanol). Extracted proteins were separated by 12% SDS-PAGE. The samples on polyvinylidene fluoride membranes were then incubated with antibodies at the following dilutions: Histone H3 9.

(11) (rabbit 1:5,000; GeneTex), Histone H3 acetyal K9 (mouse 1:1,500; GeneTex), -Tubulin 1A (rabbit 1:1,500; GeneTex), Ace-tubulin (mouse 1:2,500; Sigma), Tau (rabbit 1:50,000; Dako), PHF-1 (rabbit 1:1,000; Invitrogen), AT8 (rabbit 1:500), 12E8 (rabbit 1:1,000; Invitrogen), AT180 (rabbit 1: 1,000; Invitrogen) and β-actin (1:5,000; GeneTex). Secondary antibodies were Mouse IgG antibody (HRP) (1:10,000; GeneTex), or Goat anti-rabbit IgG HRP (1:5,000; Jackson), and signals were developed by enhanced chemiluminescence and detective by ImageQuant LAS 4000 (GE Healthcare Life Sciences). All antibodies were diluted in PBST buffer (1×PBS, 5% Tween 20 and 5% BSA). The signal was quantified using Image J.. Survivorship assay For lifespan recordings, newly eclosed adults were raised on standard yeast-glucose medium with 50 HDAC inhibitors. For the control treatment, same amount of DMSO was added to the medium. Viable flies were raised at 25ºC and scored at 4-day intervals. At least 200 flies for each treatment were used for survivorship assay. The log-rank test was used for evaluating the lifespan of flies. p values less than 0.05 were considered to be statistically significant.. Solubility assay Nonionic detergent-soluble and -insoluble protein fractions were prepared by homogenization of fly heads in TNE buffer (10 mM Tris 10.

(12) pH7.5, 5 mM EDTA, 150 mM NaCl and 0.5% NP40). The concentration of protein was quantified and adjusted to the same protein concentration. The homogenate was centrifuged at 100,000 × g for 5 min). The resulting supernatant fractions and pellet fractions were collected. The pellet fraction was resuspended in TNE buffer, and centrifuged at 100,000 × g for 5 min again, the resulting pellet (nonionic detergent-insoluble) was solubilized in TNE buffer containing 1% SDS. The protein were homogenized in Laemmli buffer and heat denatured for 5 min at 95°C, and resolved by standard 8~16% SDS-PAGE gels. Proteins were transferred to PVDF membranes and blocked in 4% nonfat dry milk dissolved in PBST overnight, and then incubated with primary antibody Tau (DAKO) at 1:50,000 dilutions overnight. Membranes were washed three times in PBST, and then incubated with HRP-linked secondary antibody for 1 h in room temperature. The membrane was washed three times, and then developed with the ECL. Chemiluminescence was recorded on an image scanner (GE LAS4000).. 11.

(13) Result. Genetic inhibition of HDAC suppresses Tau toxicity in flies To investigate possible connections between HDAC inhibition and tauopathy, we use the Drosophila notal bristle as an assessment tool to evaluate the toxicity of tau. The assay system is based on the fact that overexpression of human tau cause the loss of notal bristle (YEH et al. 2010). As Shown in Fig. 1 overexpression of human Tau driven by Eq-gal4 (Eq>tau) in the notum of Drosophila result in bristle lost in transgenic flies. To test if the downregulation of HDAC suppresses tau induced bristle loss phenotype in Eq>tau flies, RNAi approach was conducted. We out-crossed virgin of transgenic flies carrying various HDAC RNAi constructs (HDAC 1 (v30599, v46929, 46930), HDAC 3 (v107073), HDAC 4 (v20522), HDAC 6 (v108831) and HDAC 11 (v108098)) with Eq>hTauWT male flies. We found that knocking down the expression of all classes of HDAC suppress the bristle loss phenotype in Eq>tau flies (Eq>Tau; HDAC-RNAi, Figure 1).. Genetic inhibition of HDAC extends the lifespan of tauopathy flies To further demonstrate that the genetic inhibition of HDAC is amenable to the manifestation of tau neurotoxicity in flies, we have overexpressed tau in neuronal tissues of flies using the Elav-gal4 driver (Elav>tau). Similar to previous study, neuronal overexpression of tau reduced the survivorship of flies. In the survivorship assay, we found that the lifespan of Elav>Tau; HDACi flies is significantly longer than that of 12.

(14) Elav>Tau flies. This data suggest that HDAC downregulation significantly extended the lifespan of Elav-hTau flies (figure 2).. Knocking down the expression of HDAC increases acetylation of Histone H3, but not -tubulin Having established that genetic modification of HDAC expression could suppress the defect of Tau transgenic flies, we explored how knocking down of HDAC affect cell acetylation. Because tubulin has been shown to be a substrate of HDAC. Microtubules organized by tubulin protein, we determined that down-regulation of HDAC influence the acetylation level of tubulin and histone. Western blotting experiment revealed that down-regulation of HDAC significantly increased the expression levels of acetylated histone H3, but the expression levels of histone H3 were not changed. Additionally, silencing the expression of HDAC has no effect on the expression of acetylased tubulin in Tau flies (Figure 3).. HDAC silencing decreases total Tau and phosphorylated Tau The expression levels of tau and phosphorylated tau have been shown to be the pathological hallmark of tauopathies. In AD and other tauopathies, accumulation of abnormally phosphorylated Tau is a hallmark of these diseases and is involved in fibril formation and neuron loss (ALONSO et al. 1996; NECULA AND KURET 2004; NISHIMURA et al. 2004; IQBAL et al. 2005; HERNANDEZ AND AVILA 2007). Hyperphosphorylation of tau at serine and threonine sites have been 13.

(15) found in disease cases (YEN et al. 1995), and several studies have described possible connections between HDAC and Tau hyperphosphorylation (BOOM et al. 2009; SUN et al. 2012). To investigate the beneficial effects of HDAC downregulation on tauopathy, we analyzed Tau phosphorylation with a set of phosphor-specific antibodies, including PHF-1, 12E8, AT180, and AT8, which can recognize different phosphor-epitopes of Tau (WITTMANN et al. 2001). Western blotting results show that the expressed level of phosphorylated Tau were decreased obviously when HDACs were silenced (Figure 4), these phosphorylation sites that have been previously demonstrated as important contributors of Tau toxicity. These results showed the total Tau decreased, therefore down-regulation of HDAC could reduce the expression of total tau and phosphorylated tau in tauopathy fly models.. Reduced HDAC increases Tau solubility The pathogenesis of tau protein have not been full revealed. In one way, it is generally believed that hyperphosphorylation of tau causes the Tau to form insoluble aggregated, such as neurofibrillary tangle, which exert its neurotoxicity (CASTELLANI et al. 2008). Nevertheless, in another way, the hyperphosphorylated tau reduced its affinity toward microtubule, thereby leading to microtubule breakdown which impair neuronal function (COWAN et al. 2010a). For this reason we suspect that in our model knocking down HDAC, other than reduce Tau expression and phosphorylation, could also affect to Tau solubility. Proteins extracted form 10-day old adult flies were subjected for differential centrifugation 14.

(16) to separate the soluble and insoluble fractions. Both protein fractions were resolved using SDS-PAGE and the soluble and detergent soluble tau were revealed by immunoblotting. As shown in Fig. 5 we demonstrate that knocking down the expression of HDAC increase the solubility of tau because the amount of insoluble Tau is significantly reduced when compared with the control Elav>tau flies (Figure 5). Similar results were also observed in 5 day old Elav>tau flies when HDACs were knocked down.. HDAC inhibitors mitigate Tau toxicity in flies Since downregulation tau exhibit beneficial affect on tauopthy, suggesting that HDAC is a potential therapeutic target for tauopathies. With this in mind we felt that HDAC inhibitors could reduce the toxicity of tau. We have conducted a drug screening. Eggs of transgenic flies (Eq>hTauWT) were collected and placed into cultured vials containing different concentrations of HDAC inhibitors (i.e. 1, 5, 50 ). Notal bristles were used as an assessing tool. In our primary screen, we have tested 15 novel HDAC inhibitors. We found that 6 inhibitors, including SAHA, NC102, NC112, NC113, NC114 and NC115, can increase the bristle number in Eq>tau flies. As can be seen, these compounds restoring bristle phenotype significantly (Figure S1). The above compounds were subjected for second round of drug screening to determine the dosage effect on bristles recovery (Figure 6). Since the inhibition of HDAC shown to promote gene transcription, our data suggested that HDAC inhibitors is a potential treatment for tauopathies. Our data also suggest 15.

(17) that Tau-induced neurotoxicity might correlate with transcriptional dysregulation.. HDAC inhibitors increase the longevity of tauopathy flies With the success of HDACi in alleviating Tau toxicity in notal bristle, we would like to test whether the above 6 HDAC inhibitors could ameliorate tau toxicity in neuronal tissues. We oral administration of the compound to transgenic fly overexpression Tau driven by Elav-Gal4 driver. As shown in Figure 7, only NC113 can significantly extended the lifespan of tauopathy fly model. Both NC112 and NC114 have no effect on extending the lifespan of tauopathy transgeneic flies. To our surprised HDAC inhibitors, including NC100, NC102 and NC115, even reduced the survivorship of tauopathy flies (Table 1).. HDAC inhibitors increase acetylation of Histone 3 and -tubulin To further investigate how HDAC inhibitors exhibit beneficial effect on tauopathy, we determined the protein expression levels in Elav>Tau flies treated with HDAC inhibitors. The Tau transgenic flies (Elav-hTauWT) were raised in standard yeast-glucose medium containing with 50 M HDACi. Heads of 5-day old adult flies were collected and proteins were extracted. Immunoblottings were conducted to determine the expression levels of the following proteins, including histone H3, acetylated histone H3, -tubulin and acetylation -tubulin. In our immunoblotting results, we could observe that expression of acetylation tubulin increased significantly when tauopathy flies were treated with 16.

(18) HDAC inhibitors, but not with NC102 treatment. HDAC inhibitors have no effect on the expression of tubulin (Figure 8). The expression of acetylated histone H3 was also increased with the treatment of HDAC inhibitors. We concluded that HDAC inhibitors, except NC102, increases acetylated H3 and tubulin in tauopathy flies.. HDAC inhibitors decreases total Tau and phosphorylated Tau As shown above, HDACi treatment could increase the acetylation level of both histone H3 and tubulin. We next investigated how HDACi affect Tau expression and phosphorylation level. Heads of 5-day old adult flies treated with HDACi were collected and subjected to western blotting analysiss. We aim to determine Tau phosphorylation with the following antibodies. Including phosphor-specific antibodies PHF-1 and AT8, which could recognize different Tau phosphor-epitopes of Tau. Our results suggest that total Tau expression decrease when Elav>tau flies were treated with HDACi. However, NC102 treatment did not confer the same effect (Figure 9). Similarly, HDACi treatment also decrease the expression levels of phosphorylated Tau (PHF-1 and AT8).Taken together, our data indicates that HDACi exerts its beneficial effect through reducing the phosphorylation of Tau in Elav>tau flies.. Reduced HDAC increases Tau solubility As described above, HDACi could reduce the phosphorylation of Tau in transgene fly model, we would like to determine if insoluble tau is also reduced in Elav>tau flies treated with HDACi. Protein was extracted 17.

(19) from heads of 10-day old adult flies, and was subjected to the solubility assay as described above. As shown in Fig. 10, HDACi treatment increases soluble Tau (Figure 10). The increasing in soluble tau was not observed in Elav>tau flies when treated with NC102.. 18.

(20) Discussion Tau is a microtubule association protein which binds onto microtubule and stabilizes the cytoskeleton structure in neurons. Tau contains two major etiology of neurotoxicity. First, hyper-phosphorylation of Tau cause the protein departed from microtubule and induced MT defect. This has counted for the hallmark of tauopathies and contributed directly to neurodegeneration (SPITTAELS et al. 1999; STOKIN et al. 2005; BALLATORE et al. 2007). Second, departed Tau aggregation to forming neurofibrillary tangles which would pile in neuron cells and leading to cell death (BANCHER et al. 1989; GOEDERT et al. 1989; ARRIAGADA et al. 1992). Microtubule defect and neurofibrillary tangles are two main pathologies of neurodegeneration had been established. Over the past decade, substantial progress has been made in studying human tauopathies in the fruit fly Drosophila melanogaster. These models mimic, at least in some extent, the human disease state, including age-dependent neuronal degeneration, adult-onset, and shortened lifespan (WITTMANN et al. 2001; NISHIMURA et al. 2004; KHURANA 2008). However to date, the Drosophila model rely on overexpression of human wild type or mutant forms including R406W, V337M, and P301L (BRANDT et al. 2005; RAMSDEN et al. 2005; TERWEL et al. 2005).. Several screens based on Tau neurotoxicity have been performed in Drosophila by using different morphological phenotypes as readout. From these studies, phosphatases, and cytoskeletal proteins have been 19.

(21) shown to modulate tau induced neurotoxicity (SHULMAN AND FEANY 2003; BLARD et al. 2007). In the present studies we demonstrate that inhibition of histone deacetylation decrease Tau toxicity (GOVINDARAJAN et al. 2013; XIONG et al. 2013). By knocking down of each class of HDAC, we would like to test which class of HDAC can be a potential target for treating tauopathies. We downregulated HDAC using either genetic and HDAC inhibitor. In the first part of our study, we used notal bristles loss which developed in our lab, as assessment tool to evaluate different classes of HDAC systematically on the tau toxicity. We demonstrate that knocking down the expression of HDAC with RANi significantly suppress the bristle loss phenotype in Eq>tau flies. Similar effectiveness also observed when tauopathy flies were treated with HDAC inhibitors.. We further demonstrated that HDAC inhibition extend the lifespan of Elav>tau flies. It had been proved that overexpression of human Tau in fly central neuron system decrease the longevity. Despite knocking down the expression of HDAC all increase the longevity of tauopathy flies, NC113 is the only HDACi that exhibits the same beneficial effect on extending the lifespan of Tauopathy flies. Other HDAC inhibitors, such as NC100, NC102, and NC115, gave the oppositely effect (Figure 2, 7). We speculation that NC113 inhibits HDAC1 specifically, while NC100, NC102, and NC115 that decrease lifespans of tauopathy flies, inhibit both HDAC 1 and HDAC 3. Because different HDACs play important roles in animal development, inhibition of both HDAC 1 and HDAC 3 could 20.

(22) leads to lethality in Drosophila.. To investigate the underlying pathomechanisms by which HDAC downregulation affects the pathogenesis of tauopathy, we determined the change in the expression of various proteins when HDAC was downregulated in taupathy flies. Acetylated histone H3 was significantly increased when HDAC3, HDAC 4, or HDAC 6 was downregulated by RNAi. Additionally, acetylated tubulin acetylation was elevated in HDACi treatment (Figure 8).The expression of acetylated -tubulin was not affected when HDAC was downregulated (Figure 3). These results also demonstrated that the different HDAC may have different specific targets. All the tested HDAC inhibitors, except NC102, have similar effect on acetylated Histone H3.. The expression levels of total Tau were decreased when HDAC was silenced with RNAi or treated with HDACi. Tau expression were down-regulated when HDAC 4, HDAC 6, or HDAC 11 was inhibited. HDAC 1 or HDAC 3 downregulation has non-effect on Tau level. Phosphorylated Tau was also decreased when HDAC was downregulated in tauopathy flies. NC102 might increase the Tau toxicity in fly models.. Many studies have great debated with regard to the pathological role of soluble tau over insoluble tau. In our study we show that HDAC downregulation extends the lifespan of tau and increases tau solubility. Our result suggest that insoluble tau is likely to be pathogenic. 21.

(23) Although many lines of evidence demonstrate that Tau plays an important role in neurodegeneration, but this view need to refine because many molecular and environmental factors that contribute to the pathogenesis has not been solved. In our study, we have determined the links between HDAC inhibition and Tau toxicity. We manifested down-regulation of HDAC could reduce Tau expression and phosphorylation. Importantly, HDAC downregulation also extentends the lifespan of Tau overexpressing flies. Additionally, we demonstrate that inhibitions of different HDAC exhibits different consequence, suggesting that HDAC has multiple targets.. 22.

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(27) Yen, S. H., W. K. Liu, F. L. Hall, S. D. Yan, D. Stern et al., 1995 Alzheimer neurofibrillary lesions: molecular nature and potential roles of different components. Neurobiol Aging 16: 381-387.. 26.

(28) Figures Figure 1.. 27.

(29) Fig. 1. Genetic inhibition of HDAC suppresses Tau toxicity in flies. (A-I) Representative images of Drosophila notal overexpression of human Tau (hTauWT) driven by Eq-Gal 4 line cross to HDAC RNAi lines. Overexpression of human Tau caused losing of notum bristle phenotypes. Control fly Eq-Gal4 showed normal bristle pattern. (B) Quantification of the bristle number of trancgenic flies expression Tau and cross with HDAC RNAi. Counting of bristles were sent to T-test analyze. Asterisks indicate significant in bristle counts (***, P<0.05).. 28.

(30) Figure 2. 100. Perscent survial. 80. 60 Elav>hTauWT (39.64 d) hTauWT + v30599 (54.67 d). 40. hTauWT + v107073 (56.04 d). hTauWT + v20522 (60.55 d) 20. hTauWT + v108831 (52.88 d) hTauWT + v108098 (62.01 d). 0. 1. 8. 16. 24. 32. 40 48 56 64 72 Age (days after eclosion). 80. 88. 96. Fig 2. Genetic inhibition of HDAC extends the lifespan of tauopathy flies. Neuronal overexpression of human TauWT by Elav-Gal4 driver caused premature death. Cross with HDAC RNAi extent the lifespan of transgenic flies. (all P<0.05). 29. 104. 112.

(31) Figure 3.. Fig 3. Knock down the expression of HDAC increases acetylation of Histone 3, but not -tubulin. (A) Expression of different types of HDAC 30.

(32) RNAi in Drosophila brains. Protein extracts from transgenic Tau crossing with different types of HDAC RNAi fly heads at 5 day after eclosion was prepared to western blotting. Elav-Gal4 flies were used as control. Ac-Histone H3, Histone, Ac--Tubulin, and -Tubulin antibodies used to recognize the protein expression level. -Actin was used as an additional loading control. (B) Quantification of acetylation histone, cross with HDAC RNAi in creased the acetylation of histone, but no affect to histone expression. (C) Quantification of acetylation tubulin, HDAC RNAi expression did not alter the acetylation level of tubulin. (*, P<0.05, **, P<0.01). 31.

(33) Figure 4.. 32.

(34) Fig. 4. HDAC silencing decreases total Tau and phosphorylated Tau. (A) Tau and phosphorylated Tau expression level altered under HDAC RNAi crossing. Antibodies AT8, AT180, 12E8, and PHF-1, were used to detect different phosphor-epitopes of Tau. Tau was used to indicate the total Tau protein level, and -Actin was used as an additional loading control. (B) Quantification of AT8, decrease in all types of RNAi crossing. (C) Quantification of PHF-1, HDAC RNAi reduced the phosphorylation level except for v30599. (D) Quantification of total Tau expression v20522, v108831, and v108098 reduced the level of Tau, others showed non-effective in Tau expression. (*, P<0.05, **, P<0.01). 33.

(35) Figure 5.. Fig 5. Reduced HDAC increases Tau solubility. Lanes labeled “p” denote insoluble Tau, “s” indicates the corresponding soluble Proteins. Protein extract from Tau transgenic fly heads by Elav-Gal4 driver cross with HDAC RNAi flies. We used total Tau antibody to recognize the signal. Insoluble form of Tau decrease in the HDAC RNAi crossing lines compare with the positive control lanes.. 34.

(36) Figure 6.. Fig 6. HDAC inhibitors mitigate Tau toxicity in flies. (A) Representative images of Drosophila notal overexpression of human Tau (hTauWT) drove by Eq-Gal 4 lines. The HDAC inhibitors treatments contain different dosage of drugs in medium, DMSO used as control. (B) Quantification of the bristle number of fly was treating by HDAC inhibitors. Counting of bristles were sent to T-test analyze. Asterisks indicate significant in bristle counts (*, P<0.05).. 35.

(37) Figure 7.. 100 DMSO (33.0 d, n = 288) NC100 (30.1 d, p = 0.007, n = 300). 80. Perscent survial. NC114 (35.8 d, p = 0.347, n = 387). NC112(35.3 d, p = 0.830, n = 335) 60. NC113(37.5 d, p = 0.007, n = 408) NC115(25.2 d, p = 0.000, n = 398) NC102(17.7 d, p = 0.000, n = 534). 40. 20. 0. 4. 8. 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 Age (days after eclosion). Fig 7. HDAC inhibitors increase the longevity of tauopathy flies. Neuronal overexpression of hTauWT by Elav-Gal4 driver caused premature death. Treat with HDAC inhibitor NC113 extent the lifespan of transgenic flies. NC100, NC102, and NC115 showed negative effect. (P value as showed.). 36.

(38) Figure 8.. Fig. 8. HDAC inhibitors increase acetylation of Histone 3 and -tubulin. (A) Treating different types of HDAC inhibitors in transgenic Tau flies. Protein extracts from transgenic Tau fly heads at 5 day after eclosion was prepared to western blotting. DMSO treatment flies were used as control. Ac-Histone H3, Histone, Ac--Tubulin, and -Tubulin antibodies used to recognize the protein expression level. -Actin was used as an additional 37.

(39) loading control. (B) Quantification of acetylation histone, HDACi treatment increased the acetylation of histone expect for NC102 (C) Quantification of acetylation tubulin, HDACi treatment increased the acetylation level of tubulin expect for NC102. (*, P<0.05, **, P<0.01). 38.

(40) Figure 9.. Fig. 9. HDAC inhibitors decreases total Tau and phosphorylated Tau (A) Tau and phosphorylated Tau expression level altered under HDACi treatment. Antibodies AT8, and PHF-1, were used to detect different phosphor-epitopes of Tau. Tau was used to indicate the total Tau protein 39.

(41) level, and -Actin was used as an additional loading control. (B-C) Quantification of AT8, and PHF-1, the phosphorylation level decreases in all types of HDACi expect for NC102. (D) Quantification of total Tau, only NC102 showed non-affect. (P<0.05, **, P<0.01, ***, P<0.001). 40.

(42) Figure 10.. Fig. 10. Reduced HDAC increases Tau solubility. We used total Tau antibody to recognize the signal. Insoluble form of Tau decrease in the HDAC inhibitors treated lanes compare with the DMSO control lanes.. 41.

(43) Figure S1.. Fig. S1. HDAC inhibitors screening in bristle loss assay. 15 inhibitors used three different concentrations to the assay to select which has better recovery effect in Tau flies. The red marked were using to follow-up test.. 42.

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