Naphthoquinone-Tryptophan Hybrid Inhibits Aggregation of the Tau-Derived Peptide PHF6 and Reduces Neurotoxicity

Abstract

Tauopathies, such as Alzheimer’s disease (AD), are a group of disorders characterized neuropathologically by intracellular toxic accumulations of abnormal protein aggregates formed by misfolding of the microtubule-associated protein tau. Since protein self-assembly appears to be an initial key step in the pathology of this group of diseases, intervening in this process can be both a prophylactic measure and a means for modifying the course of the disease for therapeutic purposes. We and others have shown that aromatic small molecules can be effective inhibitors of aggregation of various protein assemblies, by binding to the aromatic core in aggregation-prone motifs and preventing their self-assembly. Specifically, we have designed a series of small aromatic naphthoquinone-tryptophan hybrid molecules as candidate aggregation inhibitors of β -sheet based assembly and demonstrated their efficacy toward inhibiting aggregation of the amyloid-β peptide, another culprit of AD, as well as of various other aggregative proteins involved in other protein misfolding diseases. Here we tested whether a leading naphthoquinone-tryptophan hybrid molecule, namely NQTrp, can be repurposed as an inhibitor of the aggregation of the tau protein in vitro and in vivo. We show that the molecule inhibits the in vitro assembly of PHF6, the aggregation-prone fragment of tau protein, reduces hyperphosphorylated tau deposits and ameliorates tauopathy-related behavioral defect in an established transgenic Drosophila model expressing human tau. We suggest that NQTrp, or optimized versions of it, could act as novel disease modifying drugs for AD and other tauopathies.

Keywords

AcPHF6 peptide neurodegeneration NQTrp compound protein aggregation tau protein tauopathies

INTRODUCTION

The tau protein, a member of the microtubule-associated proteins (MAPs) family, plays a key role in stabilizing the cytoskeleton by promoting tubulin assembly into microtubules, thus defining the normal cell morphology and providing structural support [1, 2]. Under pathological conditions, intracellular aggregates of abnormal hyperphosphorylated tau protein appear in the brain, which are the neuropathological characteristic of a group of disorders termed tauopathies [3, 4]. Tauopathies belong to the family of protein misfolding disorders and include more than 20 clinicopathological conditions, such as AD, the most common tauopathy, Pick’s disease, corticobasal degeneration, frontotemporal dementia with parkinsonism linked to chromosome 17, and post-encephalitic parkinsonism. In these pathological conditions, not only does intracellular accumulations of tau protein appear, but also the abnormal phosphorylation of tau leads to microtubule disassembly due to its decreased tubulin binding capacity. Therefore, tau-mediated neurotoxicity is believed to be caused by both gain-of-function mechanism (i.e., accumulation of toxic aggregates) as well as loss-of-function mechanism (i.e., tau disassembly from tubulin). Tau aggregates are formed by the self-assembly of misfolded tau protein monomers into harmful oligomers and abnormal fibers called paired helical filaments (PHFs) that form higher-order β-sheet rich aggregates termed neurofibrillary tangles [5]. PHFs consist of two filaments twisted around one another with a width of 8–20 nm and a cross-β-sheet conformation [5].

Since protein self-assembly appears to be an initial key step in the pathology of tauopathies, intervening in this process can be both a prophylactic measure and a means for modifying the course of the disease for therapeutic purposes [6]. Based on the central role of aromatic residues in formation and stabilization of β-sheet structures, which are characteristic of various protein aggregates, we and others have shown that aromatic small molecules can be effective inhibitors of aggregation of various protein assemblies, by binding to the aromatic core in aggregation-prone motifs and preventing their self-assembly [7 –12]. Naphthoquinone-tryptophan hybrid molecules are an example of such small molecules and might serve as effective inhibitors of tau aggregation. These hybrid molecules contain: (i) Tryptophan as an aromatic core, which would target and bind the aromatic residues in the tau monomer. (ii) A quinone moiety that would also interact with the aggregative core in tau in addition to having therapeutic potential by itself, since quinones were shown to possess various therapeutic characteristics such as anti-viral, anti-cancer and anti-bacterial properties [13 –15]. Quinones by themselves were shown to inhibit the aggregation of various aggregative proteins, e.g., anthraquinones were shown to be effective inhibitors of tau aggregation [16 –19]. Indeed, a large scale in vitro screening of small molecules as inhibitors of tau assembly identified a few quinone-based molecules among the various candidates, underscoring the importance of aromatic interactions in tau aggregation and its aggregation inhibition [20]. Thus, the naphthoquinone-tryptophan hybrids are expected to prevent, or suppress, the very early steps of the molecular recognition required for the assembly of the monomers into pathogenic aggregate species.

Indeed, we have previously demonstrated that two naphthoquinone-tryptophan hybrids, namely 1,4-naphthoquinone-2-yl-L-tryptophan (hereafter termed NQTrp, Fig. 1A) and its derivative Cl-1,4-naphthoquinone-2-yl-L-tryptophan (hereafter termed Cl-NQTrp), efficiently inhibited the assembly of synthetic amyloid-β (Aβ)_1 - 42 monomers into either toxic oligomers Aβ species (IC50 = 50 nM) or into fibrils [21, 22]. NMR and molecular modeling confirmed that the compounds bind the aromatic residues in Aβ [21]. Interestingly, the compounds were also effective in causing in disassembly of pre-formed Aβ fibrils in vitro [22].

Feeding NQTrp or Cl-NQTrp to transgenic Drosophila expressing Aβ throughout their nervous system, which serve as a canonical model for AD [21, 22], or injecting them IP, 50 mg/kg, to acute AD model mice (5×FAD) [23], every other day for 4 months resulted in remarkable amelioration of their behavioral and cognitive deficits. This was accompanied by dramatic reduction (40% ) in Aβ load in brain extracts and in brain sections of both the flies and mice [21, 22]. We have also shown that the compounds efficiently cross the blood-brain barrier (BBB) in an established ex-vivo model of BBB [22].

The rationale mentioned for the use of these molecules predicts also that they may be able to inhibit assembly of other aggregative-prone proteins. Indeed, we have recently shown that both molecules efficiently inhibit the in vitro aggregation of several tested other proteins including α-synuclein, islet amyloid polypeptide, lysozyme, and calcitonin [7].

Given these encouraging properties of naphtho-quinone-tryptophan hybrids as aggregation inhibitors of various aggregation-prone proteins we examined here whether NQTrp can be re-purposed as an inhibitor of the aggregation of the tau protein in vitro and in vivo. In the present study we employed the tau-derived VQIVYK peptide (hereafter termed PHF6), which was shown to be crucial for the aggregation of tau protein into PHFs [20, 24], and is widely used as a model system for tau aggregation [25 –29]. We found that NQTrp efficiently inhibits PHF6 aggregation in vitro. Treating flies expressing human tau, which serve as an established tauopathy model, with NQTrp, resulted in significant amelioration of tauopathy-engendered phenotypes, demonstrating that NQTrp is also an effective aggregation inhibitor of tau in vivo. Results from our study support the development of NQTrp for treating tau-mediated neurodegenerative disorders. The observation that NQTrp inhibits both Aβ and tau aggregation positions it as a superior drug candidate for the simultaneous targeting of two major culpritsof AD.

MATERIALS AND METHODS

Compounds

1,4-naphthoquinone-2-yl-L-tryptophan (NQTrp) was purchased from Topharman (Shanghai, China) (Fig. 1A). Synthetic acetylated PHF6 peptide (Ac-VQIVYK-NH2) was purchased from GL Biochem (Shanghai, China).

ThT kinetic binding fluorescence

In all of the in vitro assays described, in order to avoid pre-aggregation, synthetic lyophilized PHF6 peptide was pretreated with 100% hexafluoroisopropanol (HFIP), incubated for 10 min at 37°C and the HFIP was evaporated using a SpeedVac. Immediately prior to the experiment, PHF6 was dissolved in ultra pure water and sonicated twice for 3 min minutes to reach a 1 mM concentration (calculated according to ɛ280 of 1490 M^–1cm^–1).

NQTrp was freshly prepared by dissolving it in DMSO to a concentration of 100 mM, sonicated for 5 min and diluted to 10 mM with MOPS. A 4 mM thioflavin-T (ThT) stock solution was prepared in MOPS.

Stock solution was then diluted in 100 μL wells of a 96-well black plate such that the final concentrations were 100 μM PHF6 peptide, 100 μM ThT solution, NQTrp at varying concentrations (500 μM, 100 μM, and 20 μm, to obtain 5:1, 1:1, and 1:5 molar ratios [NQTrp:PHF6], respectively), in 20 mM MOPS pH 7.2. As commonly used, 1 μM heparin was added immediately prior to experiment in order to initiate PHF6 aggregation at 25°C [24 , 30–32]. Fluorescence data was collected in triplicates in kinetic mode, using a TECAN Infinite F-200 microplate fluorescence reader, with measurements taken at 1 min intervals (the excitation and emission wavelengths were 450 nm and 480 nm, respectively).

Transmission electron microscopy (TEM)

The PHF6 peptide (100 μM), incubated with increasing concentrations of NQTrp (molar ratio of 5:1, 1:1, and 1:5, NQTrp:PHF6), was aggregated for 20 min at 25°C after aggregation has been initiated by the addition of heparin (1:100 molar ratio, in favor of PHF6). 10 μL samples were placed on 400-mesh copper grids covered with carbon-stabilized Formvar film (Electron Microscopy Sciences (EMS), Hatfield, PA). After 2 min, excess fluid was removed, and the grids were negatively stained with 10 μL of 2% uranyl acetate solution for 2 min. Finally, excess fluid was removed and the samples were viewed by a Tecnai 12 TWIN TEM (FEI) operating at 80 kV.

CD analysis

To analyze the secondary structure of PHF6 aggregates, the PHF6 peptide (100 μM), incubated with increasing concentrations of NQTrp (molar ratio of 5:1, 1:1, and 1:5, NQTrp:PHF6), was aggregated for 3 h at 25°C after aggregation has been initiated by the addition of heparin (1:100 molar ratio, in favor of PHF6). Samples were diluted to 40 μM PHF6 peptide and placed in a 0.1 mm cuvette and CD spectra in the range of 185–260 nm were recorded on a Chirascan spectrometer. Background was subtracted from the CD spectra.

Fly keeping

Flies were reared on standard cornmeal-molasses medium and were kept at 25°C. Crosses were performed at 25°C. Adult offspring (F1) from the crosses were collected up to two days after the beginning of their eclosion at 25°C.

Genetics

The transgenic strain overexpressing the longest isoform of human Tau^WT (hTau), comprising 441 amino acids [33, 34], under the upstream activating sequence (UAS) has been described previously [35] and was a generous gift from Dr. George Jackson (University of Texas Medical Branch, Galveston, TX). The GMR-Gal4 strain used to drive the expression of hTau in the eyes or fly CNS was a kind gift from Dr. Hermann Steller (The Rockefeller University, New York, NY). Transgenic elav ^c155-Gal4 flies were obtained from BDRC (Bloomington Drosophila Research Center).

Fly crossing

Virgin females, carrying either the eye GMR-Gal4 driver or the pan-neuronal driver elav^c155 -Gal4 on chromosome X, were collected and crossed with males carrying UAS-hTau on the 2nd chromosome or with wild type Oregon-R (OR) males as a control. This resulted in F1 female and male offspring whose eyes or brain express hTau.

Fly feeding

NQTrp was added to standard molasses medium at a concentration of 0.75 mg/mL, as previously described [21, 22], and the mixture was aliquoted into rearing vials. The vials were kept at 4°C until use. Crosses were performed either on standard Drosophila medium (control) or on medium supplemented with NQTrp. Animals fed on the appropriate medium from the beginning of the larval stage onwards throughout adult life. After eclosion, adult offspring were transferred into a fresh vial containing standard Drosophila medium on top of which a solution of 0.75 mg/mL NQTrp was dripped every other day [21, 22].

Scanning electron microscopy (SEM)

Twelve-day-old female flies from each of the resultant F1 groups, crossed and reared at 25°C, were fixed in 4% paraformaldehyde (PFA, #28908, ThermoScientific) for 1 h at room temperature. After repeated washes with PBS, the flies were dehydrated with increasing concentrations of ethanol, critical-point dried and coated with gold. The samples were analyzed with a Jeol SEM JSM 840A at a magnification of 250X.

Qualitative classification of eye phenotype

Both eyes of each two day old female flies from the resultant F1 groups, were examined under a stereomicroscope. The eye defects observed were classified into three classes according to their severity (see Fig. 4B): Class 2 had mucosal eyes, but had otherwise normal morphology; class 3 exhibited a collapse of the eye tissue; and class 4 had black spots of apoptotic tissue on their eyes. Chi-squared test was performed in order to examine whether the distribution of eye classes in each experimental group was significantly different from the untreated flies overexpressing hTau (p-value <0.05 was considered to be significantly different). The validity of this categorization was verified beforehand by finding that the classification results of independent researchers do not differ significantly.

Quantification of eye pigment

Seven-day-old female flies from each of the F1 groups were stored for one day at –80°C, decapitated and their heads were distributed into 3–5 tubes containing 15 heads each. Next, 50 μL of AEA buffer (50% ethanol, pH = 2) were added to each tube and the tubes were stored for three days at room temperature in the dark. Then, each sample was diluted with equivalent volume of AEA buffer. The absorbance of the samples was measured using a Thermo Fisher Scientific NanoDrop at 480 nm and was normalized according to the formula:

$\frac{\begin{matrix} Average absorbance (GMR - Gal 4; USA - hTau, treaded with \\ NQTrp) \times Average absorbance (GMR - Gal 4, untreated) \times 100 \end{matrix}}{Average absorbance (GMR - Gal 4, treated with NQTrp)}$

T-test was performed for evaluating statistical significance of the observed differences (p-value <0.05 was considered to be statistically different).

For calibration and for examining a possible correlation between qualitatively-classified eye phenotypes and pigment level, virgin females carrying the GMR-Gal4 driver on chromosome×were crossed to either UAS-hTau or OR males. Three repeats of six fly heads from the resultant offspring genotypes (GMR-Gal4;OR and GMR-Gal4; UAS-hTau, the latter classified qualitatively into class ‘2’ or classes ‘3’–‘4’), were used for pigment measurement using the above mentioned protocol.

Quantification of tau levels in larval eye discs

Eye discs of third instar female larvae, from each of the resultant F1 groups, were fixed in 4% PFA for 20 min, washed with PBS-TX (0.3% Triton in PBS) and blocked with 0.1% BSA in PBS-TX. The discs were incubated overnight at 4°C with a monoclonal 5A6 anti tau antibody (Developmental Studies Hybridoma Bank, Iowa City, USA), in a 1:50 ratio, washed with PBS-TX, incubated with 0.1% BSA in PBS-TX for 20 min and incubated with Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch), in a 1:200 ratio, for 2 h. After thorough washes with PBS-TX, the eye discs were mounted on slides using VECTASHILED (#H-1200, Vector Laboratories, Burlingame, CA). Images were taken with LSM510 confocal microscope (Zeiss).

Quantification of the mean staining intensity of the discs (calculated by integrated density divided by the area defined by the threshold) was done using the ImageJ program. T-test was performed for evaluating statistical significance of the observed differences (p-value <0.05 was considered to be statistically different).

Immunoprecipitation and western-blot of fly head extracts

Immunoprecipitation

Two weeks old female and male flies (n = 40), at a 1:1 ratio, from each of the resultant F1 groups, were decapitated. The heads were washed with PBS and homogenized in ice using 40 μl lysis buffer consisting of 1% SDS in PBS, supplemented with cOmplete Protease Inhibitor Cocktail (Roche Molecular Biochemicals GmbH, Mannheim, Germany). The homogenates were centrifuged twice at 13,000 RPM for 15 s to obtain a clear lysate, and the resulting supernatants were used for immunoprecipitation. Monoclonal antibody 5A6 anti human tau (Developmental Studies Hybridoma Bank, Iowa City, USA), in a 1:10 ratio, was added to the supernatant together with protein G-sepharose beads (P3296, Sigma–Aldrich, Schnelldorf, Germany). After overnight incubation at 4°C, the samples were centrifuged at 10000 RPM for 30 s and the beads were washed several times with PBS. Next, sample buffer was added to the beads and the samples were boiled for 5 minutes to elute the tau proteins from the beads.

Western blotting

Samples were subjected to SDS-PAGE using 4–20% (w/v) polyacrylamide GeBaGels (GeBa, Interchim, Montluçon, France) under reducing conditions and transferred into PVDF membrane using a dry blot technique (iBlot^®, Life Technologies, Grand Island, NY). Western blot analysis was performed in order to detect the presence of phosphorylated tau using AT180 antibody which detects pThr231 (Thermo Fisher Scientific Inc., Rockford, IL) or total tau levels using ab64193 antibody (Abcam, Cambridge, United Kingdom). Briefly, the membrane was blocked for one hour using 5% milk diluted in TBS. AT180 or ab64193 antibodies, diluted 1:1,000 in 5% milk, were added to the membrane for an overnight incubation at 4°C, followed by several washes with TBST (0.1% Tween). Next, the membrane was incubated with appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature while shaking. Blots were developed after thorough TBST washes, using an Enhanced Chemiluminescence System (EZ-ECL, Biological Industries, Kibbutz Beit Haemek, Israel) according to the manufacturer’s manual. Densitometric analysis of phosphorylated and total tau levels was performed using GE Healthcare Densitometer (Pharmacia Biotech Inc., Uppsala, Sweden).

Locomotion (climbing) assay

Vials of each of the following four F1 classes: 1) Females expressing hTau reared on regular medium; 2) Females expressing hTau reared on medium supplemented with NQTrp; 3) Control females carrying only the elav ^c155-Gal4 driver, reared on regular medium; 4) Control females carrying only the elav ^c155-Gal4 driver, reared on medium supplemented with NQTrp, each containing 10 flies, were tapped gently on the table and were let to climb for 18 s. The percent of flies which climbed higher than 1 cm from the bottom of the test tube during that time period was then calculated, as we have previously reported [21, 36]. Each class had at least five independent vial repeats. The climbing ability of the offspring flies was monitored for up to eight days.

RESULTS

ThT analysis reveals inhibition of fibril formation by NQTrp

Two hexapeptide motifs within tau protein, VQIVYK and VQIINK, were shown to be critical for tau aggregation into oligomers and neurofibrillary tangles [20, 24]. To determine and quantify the ability of NQTrp (Fig. 1A) to interfere with tau aggregate formation, we used the PHF6 (VQIVYK) peptide as a model system for in vitro aggregation of tau. The ³⁰⁶VQIVYK³¹¹ peptide is a short segment found in the third repeat of the microtubule-binding region of the tau protein [24, 30], where the residue numbers correspond to the longest tau isoform. This peptide was shown to be critical for neurotoxic tau oligomerization and aggregation and is widely used as a model system for studying tau aggregation and examining candidate inhibitors [25 –29]. Notably, PHF6 contains an aromatic Tyrosine residue, which could be targeted by the Tryptophan moiety of NQTrp. First we employed the Thioflavin-T (ThT) binding assay to assess inhibition of aggregation and induced PHF6 aggregation with heparin, as previously described [24]. PHF6 peptide (100 μM) was allowed to aggregate at 25°C in the absence or presence of NQTrp at 5:1, 1:1, and 1:5 molar ratio (NQTrp:PHF6). The kinetic and end point results (Fig. 1B and C, respectively) indicate that NQTrp inhibited PHF6 aggregation in a dose-dependent manner.

TEM analysis of PHF6 aggregation supports the effective inhibition of PHF6 aggregation by NQTrp

Transmission electron microscopy (TEM) analysis was performed on the samples of PHF6 incubated in the presence or absence of NQTrp at different molar ratio concentrations (5:1, 1:1 and 1:5, NQTrp:PHF6). Images were taken after PHF6 was allowed to aggregate in the presence of heparin at 25°C for 20 min. Representative TEM images of PHF6 aggregates are shown in Fig. 2A In agreement with the ThT analysis, while the fibrils formed by PHF6 alone were large and mature, only few fibrils, most of them short, were detected in the presence of 5:1 and 1:1 molar ratio concentrations of NQTrp. In the presence of the lowest concentration of NQTrp (1:5 in favor of PHF6), less, albeit still detectable, inhibition of aggregation was evident.

NQTrp affect the secondary structure of PHF6

Samples containing PHF6 aggregates formed in the presence or absence of NQTrp were analyzed using circular dichroism (CD), to gain information about the secondary structural changes that occur during the aggregation process (Fig. 2B). Naive PHF6 monomers exhibit a large negative peak near 195 nm, indicating a random coil conformation (Fig. 2B). Following three hours of incubation after initiation of aggregation by heparin, PHF6 adopts a strong positive peak around 195 nm and a negative peak around 220 nm, indicating a β-sheet conformation. Consistent with previous findings, when assembled in the presence of NQTrp at the different concentrations, a decrease in the intensity of these peaks was evident (Fig. 2B), indicating fewer β-sheet-rich structures in the presence of the compound.

Feeding tauopathy model flies with NQTrp alleviates eye neurodegeneration

The fruit fly Drosophila melanogaster has proven to be a useful model for various neurodegenerative diseases [18 , 37–39]. Specifically, Drosophila overexpressing the human tau protein is a widely used transgenic model for studying tauopathies [38 , 41]. We used a fly UAS-hTau strain that allows overexpression of the largest 441aa-long isoform of wild type human tau (hTau) [35] in tissues of interest and targeted it, using the Gal4-UAS system [42], to the fly eyes or brain. When overexpressed in the fly central nervous system (CNS), tau causes defects that resemble symptoms in AD patients, which deteriorate with age [43]. These include memory and learning deficits, reduced mobility, reduced lifespan, and region-specific neurodegeneration in the adult brain, accompanied by accumulation of tau protein aggregates. Neurodegeneration can also be easily monitored when tau is overexpressed and aggregates in the fly eyes, where the retina is part of the CNS, resulting in a ‘rough eye’ phenotype with disordered ommatidia and bristle abnormalities [35, 38].

Based on our encouraging in vitro results that collectively demonstrated inhibition of aggregation of the tau-derived peptide by NQTrp, we were prompted to test whether the compound will also have an in vivo ameliorative effect toward tau-engendered defects. In order to evaluate the efficacy of NQTrp as neuroprotective agents in vivo, we first examined by scanning electron microscopy (SEM) eyes of flies that overexpress hTau in their eyes (using GMR-Gal4), treated or untreated with the compound (Fig. 3A). It appeared that treatment with NQTrp dramatically ameliorated the degenerative phenotype of the eye. Importantly, feeding control flies, that do not express hTau, with NQTrp, had no detectable effect on their lattice-like eye morphology.

We classified qualitatively, via a stereo-microscope, the eye defects of flies that overexpress hTau in their eyes (Fig. 3B). We defined four severity classes (class 1 = normal, class 2 = moderate, through class 4 = most severe, See Materials and Methods). Using this classification we observed a remarkable amelioration of eye neurodegeneration when these animals were fed with NQTrp (Fig. 3C). This was evident by a clear shift in the distribution of severity classes in the population, from the more severe to the milder. Treatment with NQTrp resulted in 95% class ‘2’ eyes, compared to only 61% class ‘2’ eyes in untreated hTau expressing flies.

We sought to corroborate the qualitative results by an independent quantitative assay. We quantified pigment level in the eyes of flies, belonging to different qualitatively classified groups, using a colorimetric measurable readout [44] (Fig. 4A). When flies, expressing only hTau in their eyes (GMR-Gal4; UAS-hTau), were distributed qualitatively according to the severity of the eye phenotypes, we found a correlation between the qualitatively classified eye phenotypes and the amount of pigment in them. Specifically, there were significant differences between pigment levels in mildly as compared to severely neurodegenerative eyes (class ‘2’ versus combined groups of classes ‘3’–‘4’). Importantly, the pigment levels of class ‘2’ eyes were less than 40% of the pigment levels found in the control flies (GMR-Gal4 flies, class ‘1’), and over 1.5 times higher than the pigment level of a combined group of classes ‘3’ and ‘4’. Hence, the results justify the use of the qualitative phenotypic classification described above. Moreover, these results suggest that quantification of pigment levels could serve as a quantitative assay for evaluating amelioration or exacerbation of eye degeneration. We speculated that following treatment with NQTrp, the observed shift in the phenotypic distribution in the qualitative assay will be reflected in differences in pigment levels in the fly eyes.

The results were normalized compared to the pigment levels of untreated flies that overexpress hTau as well as to the change in the level of pigment that resulted from treatment of control flies with the compound (See formula in Materials and Methods). When treated with NQTrp, a marked increase in the amount of eye pigment of 40% was observed, compared to untreated flies (Fig. 4B), indicating less eye degeneration. Notably and as expected, the pigment levels of untreated flies that overexpress hTau were lower (by 35% ) than the pigment levels found in the control flies that do not express hTau (Fig. 4B). Importantly, there is correlation between the qualitative morphological classification and the pigment assays, indicating that treatment with NQTrp clearly led to less eye degeneration.

Feeding tauopathy model flies with NQTrp reduces the level of total and hyperphosphorylated hTau in their eyes

In AD both total tau protein levels as well as hyperphosphorylated tau levels are increased as the disease progresses [45, 46]. In order to evaluate neurotoxicity in hTau-expressing flies following treatment with NQTrp, we measured levels of total hTau in larval eye discs (using 5A6 antibody) and in extracts of eyes from adult flies (using ab64913). Levels of hTau, phosphorylated at Thr231, were measured in extracts of eyes from adult flies (using AT180 antibody). In eye discs, total hTau levels were normalized according to hTau signal in discs of untreated control larvae overexpressing hTau. Importantly, eye tissue of control larvae that do not express hTau (GMR-Gal4 flies) was not immune-reactive with the antibody used for staining total hTau (Fig. 5A). This most likely indicates that the signal measured reflects only the transgenic hTau protein and not the endogenous fly tau protein. When treated with NQTrp, a significant 25% decrease in total hTau levels was evident (Fig. 5A, B).

Hyperphosphorylation of tau is considered as the main cause of pathological aggregation of tau in AD [47, 48]. Therefore, estimation of the level of phosphorylated tau in the eyes of the flies overexpressing tau represents a direct measure of the severity of the neurodegenerative effect provoked by it. To evaluate levels of hyperphosphorylated tau following treatment with the compound, we immunoprecipitated total hTau from the eyes of flies, treated or untreated with NQTrp. Immunoprecipitated hTau was evaluated after subjecting it to SDS-PAGE and western blotting against both total and phosphorylated tau (See Materials and Methods). The ratio between phosphorylated tau and total tau levels was normalized according to the ratio found in untreated control larvae overexpressing hTau. It was evident that treated flies had less phosphorylated tau relative to the total level of tau compared to untreated flies (Fig. 5C, D). Importantly, no tau was detected in control flies that do not express hTau (GMR-Gal4 flies) (Fig. 5C).

Notably, levels of total as well as phosphorylated hTau in treated flies negatively correlated with pigment levels, i.e., while treatment with NQTrp led to an increase in pigment levels, it caused reduction of both total and phosphorylated tau levels. This is also in agreement with the amelioration in the eyes of treated flies, monitored by SEM and the qualitative classification. Taken together, these in vivo results indicate that NQTrp reduced tau accumulation and its toxic hyperphosphorylation, and by that possibly ameliorated the eye degeneration in these tauopathy model flies.

Treatment with NQTrp ameliorate neurotoxicity caused by overexpression of hTau in the fly CNS

Locomotion (climbing) is a behavioral readout commonly used for assessing neurotoxic effects in Drosophila flies expressing aggregative proteins in their brain [21 , 50]. We employed this assay to evaluate the behavioral effect of NQTrp on the tauopathy Drosophila model in which overexpression of hTau was targeted to the nervous system via elav-Gal4. While normal flies tend to climb up the side of a tube, hTau-expressing flies remain at the bottom (Fig. 6) [51, 52]. Wild type flies (Oregon-R, OR), serving as a control and hTau-expressing flies, were grown on regular Drosophila medium or on medium supplemented with NQTrp. Climbing ability of the flies was monitored for up to 8 days (Fig. 6). Control flies retained a 90% –99% of their climbing ability at day 8 (Fig. 6). In contrast, only 20% of hTau-expressing flies climbed along the tube at day 8, displaying a severe motor dysfunction (Fig. 6), as reported. When fed on a medium supplemented with NQTrp, hTau-expressing flies exhibited a remarkable phenotypic recovery, with 66% climbing ability at day 8 (Fig. 6). NQTrp had no significant effect on locomotion of the control (elav-Gal4) flies, as previously reported (Fig. 6) [21].

DISCUSSION

Currently there are no disease-modifying treatments for tauopathies. In the present study we demonstrated that a naphthoquinone-tryptophan hybrid, NQTrp, remarkably inhibits the mis-assembly of the aggregative core of the tau protein, PHF6, in vitro. We have previously shown by NMR analysis and molecular modeling that the mode of inhibition of Aβ aggregation by NQTrp is via binding of the inhibitory molecule to the aromatic residues in Aβ [21], though other mechanisms of action of NQTrp should be considered [53]. Thus, it is possible that the effect of NQTrp on PHF6 is mediated through binding of their Tryptophan moiety to the aromatic Tyrosine residue in PHF6. Along these lines, a large scale in vitro screening of small molecules as inhibitors of tau assembly identified a few quinone-based molecules among the various candidates, underscoring the importance of aromatic interactions in tau aggregation and its aggregation inhibition [20]. It was proposed in agreement with our findings that these molecules intercalate between the hydrophobic residues around the hexapeptide motifs of tau and disrupt the β-structure interface between two neighboring sheets.

Moreover, we found that feeding flies expressing human tau with NQTrp, which serve as an established tauopathy model, resulted in significant amelioration of tauopathy-engendered phenotypes, demonstrating that NQTrp is also effective aggregation inhibitors of tau in vivo. Specifically, NQTrp reduced tau accumulation and its toxic hyperphosphorylation in treated flies, and this in turn probably induced the resultant observed amelioration of eye and brain degeneration in these tauopathy model flies.

It is worth noting that various tauopathy model flies exhibit tau toxicity without overt tau aggregation [41 , 54–58]. This implies that in these models of tauopathy, tau-induced toxicity may be caused by soluble hyperphosphorylated tau [41 , 54–58]. Since the mode of action of NQTrp is believed to be via inhibition of the toxic aggregation process, we deliberately employed a tauopathy Drosophila model that in addition to hyperphosphorylated tau also exhibits tau accumulation, as shown by the presence of insoluble tau in immunochemistry and visible by EM [35]. It is likely that this model exhibits tau accumulations due to its relatively robust expression of tau, compared to other tauopathy model flies [41 , 54–58]. This is evident by the severe rough eye morphology and also by the fast deterioration of climbing ability [35]. Given this degree of severity of degeneration, the remarkable ameliorating effect seen by treating these tauopathy model flies with NQTrp suggests that this molecule has a very potent therapeutic potential.

Our results confirm the generic mode of inhibition of protein aggregation assembly by naphthoquinone-tryptophan hybrid molecules, which were shown to be efficient inhibitors of in vitro aggregation of several aggregative proteins including Aβ, α-synuclein, islet amyloid polypeptide (IAPP), and lysozyme [7, 21], and extend it to the tau protein in vitro as well as in vivo. The NQTrp concentrations tested in this study resemble those used in our work involving α-synuclein and lysozyme, but higher than those reported in the case of Aβ and islet amyloid polypeptide [7, 21]. However, the molar ratio concentrations in all of these studies including the present one, being more important than the absolute concentrations, are very similar. This is a result of the aggregation-specific concentration of each of these protein/peptides.

It remains to be seen whether our results can be replicated in a rodent tauopathy model. If so, NQTrp, or optimized versions of it, could act as novel disease modifying drugs for tauopathies such as AD. Importantly, the compound appears to cross the BBB efficiently [22], a main advantage when targeting brain-related disorders, and a close derivative molecule, Cl-1,4-naphthoquinone-2-yl-l-Tryptophan (Cl-NQTrp), was shown to be non-toxic when administered to wild type mice, with no apparent adverse effects on either weight gain, mobility (Rotarod test) or viability [22].

AD typically involves formation and deposition of both Aβ peptide and tau protein aggregates. Therefore, we suggest that the combinatorial mechanism of action of NQTrp, targeting both the toxic aggregation of Aβ peptide, as previously reported by us, as well as the tau protein mis-assembly, reported here, may offer disease modifying treatment of AD and other tauopathies.

Footnotes

ACKNOWLEDGMENTS

This work was supported in part by the Israel Science Foundation, the Alliance Family Foundation and the Rosetrees Trust (to DS). Moran Frenkel-Pinter gratefully acknowledges the Eshkol fellowship by the Israeli Ministry of Science and Technology. Malak Abu-Hussien gratefully acknowledges a fellowship from the Israel Council for Higher Education.

We are grateful to the Segal and Gazit research groups for fruitful discussions.

Authors’ disclosures available online ().

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