Surfen and oxalyl surfen decrease tau hyperphosphorylation and mitigate neuron deficits in vivo in a zebrafish model of tauopathy

Tauopathies comprise a family of neurodegenerative disorders including Alzheimer’s disease for which there is an urgent and unmet need for disease-modifying treatments. Tauopathies are characterized by pathological tau hyperphosphorylation, which has been shown to correlate tightly with disease progression and memory loss in patients suffering from Alzheimer’s disease. We recently demonstrated an essential requirement for 3-O-sulfated heparan sulfate in pathological tau hyperphosphorylation in zebrafish, a prominent model organism for human drug discovery. Here, we investigated whether in vivo treatment with surfen or its derivatives oxalyl surfen and hemisurfen, small molecules with heparan sulfate antagonist properties, could mitigate tau hyperphosphorylation and neuronal deficits in a zebrafish model of tauopathies. In vivo treatment of Tg[HuC::hTauP301L; DsRed] embryos for 2 days with surfen or oxalyl surfen significantly reduced the accumulation of the pThr181 tau phospho-epitope measured by ELISA by 30% and 51%, respectively. Western blot analysis also showed a significant decrease of pThr181 and pSer396/pSer404 in embryos treated with surfen or oxalyl surfen. Immunohistochemical analysis further confirmed that treatment with surfen or oxalyl surfen significantly decreased the AT8 tau epitope in spinal motoneurons. In addition, in vivo treatment of Tg[HuC::hTauP301L; DsRed] embryos with surfen or oxalyl surfen significantly rescued spinal motoneuron axon-branching defects and, as a likely consequence, the impaired stereotypical touch-evoked escape response. Importantly, treatment with hemisurfen, a surfen derivative devoid of heparan sulfate antagonist activity, does not affect tau hyperphosphorylation, nor neuronal or behavioural deficits in Tg[HuC::hTauP301L; DsRed] embryos. Our findings demonstrate for the first time that surfen, a well-tolerated molecule in clinical settings, and its derivative, oxalyl surfen, could mitigate or delay neuronal defects in tauopathies, including Alzheimer’s disease.


Introduction
Tauopathies comprise more than 20 neurodegenerative diseases including Alzheimer's disease (AD), frontotemporal dementia (FTD), Pick's disease, progressive supranuclear palsy (PSP) and other related disorders. Tauopaties are characterized by accumulation of hyperphosphorylated isoforms of the microtubule-associated tau protein in brain forming distinct inclusions [1].
We have recently shown that in vivo depletion of Hs3st2, an enzyme involved in 3-O-sulfation of heparan sulfate chains and predominantly expressed in neuronal cells, significantly decreased tau hyperphosphorylation and partially rescued neuronal and behaviour defects in transgenic Tg[HuC::hTau P301L ; DsRed] zebrafish embryos [2]. Tg[HuC::hTau P301L ; DsRed] embryos display key features of tauopathies, such as tau hyperphosphorylation [3]. These findings suggest that inhibition of heparan sulfate-related activities could have beneficial therapeutic effects for tauopathies. Here, we have tested the hypothesis that treatment of Tg[HuC::hTau P301L ; DsRed] zebrafish embryos with small molecules displaying heparan sulfate antagonist properties could mitigate pathological tau hyperphosphorylation and rescue the induced neuronal and behavioural deficits.

Results
Surfen, oxalyl surfen and hemisurfen are well tolerated in zebrafish embryos Initially, experiments were set up to analyze potential toxicity of the surfen analogs used in this study, (Fig. 1a). One set of 24 h post-fertilization (hpf) zebrafish Tg[HuC::hTau P301L , DsRed] embryos were incubated for 2 days in E3 medium containing 1% DMSO and surfen, oxalyl surfen or hemisurfen. As negative controls, agematched wild-type and Tg[HuC::hTau P301L , DsRed] embryos were incubated for 2 days in E3 medium containing 1% DMSO. As positive control, age-matched Tg[HuC::hTau P301L , DsRed] embryos were incubated in E3 medium containing 1% DMSO and 1 lithium chloride (LiCl), a long known inhibitor of tau hyperphosphorylation [15]. For each agent, operational concentrations were defined as the highest concentration not inducing any visible morphological abnormalities, including heart rhythm and blood flow defects (Fig. 1b), nor any significant increase in embryo lethality (Additional file 1: Figure S1c). Operational concentrations (3 μM for surfen, 2 μM for oxalyl surfen, and 3 μM for hemisurfen) were then used for all subsequent experiments.

Surfen and oxalyl surfen reduce tau hyperphosphorylation in vivo
As a first attempt to determine whether treatment with surfen, oxalyl surfen or hemisurfen could decrease tau hyperphosphorylation in vivo, we quantified tau pThr181 phosphorylation by ELISA assay. Treatment of 24 hpf Tg[HuC::hTau P301L ; DsRed] embryos for 2 days with surfen or oxalyl surfen, decreased the accumulation of hyperphosphorylated tau by 30% and 51%, respectively (surfen and oxalyl surfen: P < 0.05), when compared to embryos treated with 1% DMSO (Fig. 2a). In contrast, no significant differences in pThr181 phospho-tau accumulation could be detected in embryos incubated with hemisurfen (P = 0.83, Fig. 2a). As expected, treatment with LiCl resulted in a 43% decrease in hyperphosphorylated tau when compared to embryos treated with 1% DMSO (N = 5, n = 250 (number of embryos); P < 0.05, Fig. 2a).
To further investigate the effects of surfen and its derivatives on tau hyperphosphorylation, Tg[HuC::hTau P301L ; DsRed] embryos were analysed by immunohistochemistry using the AT8 antibody directed against the pSer202/ pThr205 phospho-tau epitope and the anti-total-tau K9JA antibody (Fig. 2e). In good agreement with both the ELISA results and Western blot analysis, a significant decrease in AT8/Total tau labelling intensity ratio was observed in the spinal cord of Tg[HuC::hTau P301L ; DsRed] embryos treated with surfen, oxalyl surfen and LiCl (P < 0.05), but not hemisurfen (P = 0.3) (Fig. 2f). Interestingly, surfen or oxalyl surfen, decreases the somato-dendritic localization of hTau P301L (Fig. 2e), a feature of tau pathology in AD and FTD [16]. As tau missorting is linked to tau hyperphosphorylation, the decrease in somato-dendritic tau might be a consequence of the decrease in tau phosphorylation. In general, we observed stronger effects of oxalyl surfen than surfen (Fig. 2).
We next assessed whether treatment with surfen or oxalyl surfen could rescue the motility defects of Tg[HuC:: hTau P301L ; DsRed] zebrafish larvae in response to touch stimuli. As previously shown, Tg[HuC::hTau P301L ; DsRed] zebrafish larvae showed significantly impaired motility characterized by slower movements and reduced touchinduced escape when compared to wild-type age-matched larvae (wild-type vs. 1% DMSO Tg[HuC::hTau P301L ; DsRed] embryos: P < 0.05) (Fig. 3d). Interestingly, treatment with surfen or oxalyl surfen, fully rescued the motility deficit (surfen and oxalyl surfen vs. 1% DMSO: P < 0.05; Fig. 3d), while treatment with hemisurfen had no effect on motility defects (P = 0.27; Fig. 3d). These results provided functional evidence that surfen and oxalyl surfen not only significantly decrease tau hyperphosphorylation, but also alleviate the behavioural consequences of the neuronal deficits induced by the expression of the human mutant tau P301L protein.
Importantly, apart from rare reports of hypersensitivity reactions [25,26], surfen is well tolerated in clinical settings. Although one study linked high doses and prolonged administration of surfen to lymphosarcoma and lesions reminiscent of nutritional deficiency, surfen is well tolerated in mice [13]. In good agreement, we found surfen and oxalyl surfen to be well tolerated in zebrafish embryos at the effective concentrations of 3 μM and 2 μM, respectively. Oxalyl surfen shows a stronger effect on tau phosphorylation and behavioural rescue than surfen, suggesting the molecule to be more efficient against tau pathology. However, the derivative is toxic to zebrafish embryos at a lower concentration, pointing out a potentially less favourable safety profile than surfen. Further investigations are needed to determine whether the two molecules could also counteract neurodegenerative processes linked to tau alterations in humans.

Animals
Zebrafish were maintained at 28°C in our zebrafish facility under standard conditions as described by Westerfield (1995) [27]. Developmental stages were determined as hours post-fertilization (hpf) as described by Kimmel et al. [28]. AB strain was used as wild-type fish. The zebrafish transgenic line stably expressing the human mutant Tau P301L protein that is associated with frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) (the Tg[HuC::hTau P301L ; DsRed]), has been previously described [3], and was kindly provided by Christian Haass, Bettina Schmid, and Dominik Paquet (Deutsches Zentrum für Neurodegenerative Erkrankungen or DZNE, Munich, Germany).

Treatments
As surfen and oxalyl surfen bind avidly to plastic, we either pre-coated all plasticware with serum containing medium or used glass vessels. Stock solutions (surfen and hemisurfen, 30 mM; oxalyl surfen, 21.7 mM) were prepared in DMSO. Working solutions were prepared as needed by diluting stock solutions in E3 medium and adjusting the DMSO concentration to 1% (vol/vol). Final concentrations for treatments were determined as 3 μM for surfen, 2 μM for oxalyl surfen, 3 μM for hemisurfen and 80 mM for lithium chloride (LiCl) based on maximal non-toxic concentrations for zebrafish embryos (Additional file 1). Embryos (24 hpf) were manually dechorionated and incubated for 2 days in 1-2 ml of either control medium (E3 medium containing 1% DMSO) or E3 medium containing 1% DMSO and the surfen derivatives in BSA-coated 6-well microtiter plates. All solutions were changed daily.

Western blot
Zebrafish larvae were collected, anaesthetized in MS-222, and lysed on ice with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 10 mM NaF, 1 mM Na 3 VO 4 , pH 8.0) supplemented with protease and phosphatase inhibitors (Pierce). Lysates were homogenized by sonication and centrifuged at 12000 g for 15 min. The protein content in the supernatants was quantified using a Bradford protein assay (Bio-Rad).
Samples containing 10 μg proteins were subjected to SDS-PAGE in 10% acrylamide gel. Primary antibodies against phosphorylated tau, AT270 and PHF1 (Pierce, Thermo Scientific), anti-human total tau antibody K9JA (Rabbit Polyclonal Antibody, Dako Cytomation), and anti-GADPH (Abcam) were used. Blots were subsequently incubated for 1 h at room temperature with the corresponding secondary antibodies diluted in phosphate-buffered saline containing 5% milk and revealed using ECL RevelBlOt® Plus (Ozyme) following manufacturer's instructions.

Image analysis
Bright field images of embryos were captured using a stereomicroscope (SteREO Lumar. V12, Zeiss) equipped with a digital camera (DXM 1200F, Nikon) controlled by the ACT-1 software (Version 2.63 Nikon). Fluorescently labelled embryos were imaged using a microscope equipped with an ApoTome system (Zeiss) fitted with an AxioCam MRm camera (Zeiss) controlled by the Axiovision or ZEN software. All images were processed with Adobe Photoshop 7.0 (Adobe System, San Jose, CA). When necessary, brightness, contrast, and colour balance, were uniformly optimized. Fluorescence intensities and densimetric quantification of protein immunoblots were performed using ImageJ/Fiji (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2012) on grayscale images. For each value, quantifications were performed using images from three independent experiments.

Behavioural analysis
Larvae behaviour was analysed at 48 hpf after 1 day treatments with the different compounds. The larval escape response reflex was assessed by gently touching the tip of the tail with a fine plastic rod. Embryos were classified as responders or non-responders, with nonresponders failing to respond by swimming at least three times their own body length.