A newly designed molecule J2326 for Alzheimer's disease disaggregates amyloid fibrils and induces neurite outgrowth.

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Neuropharmacology xxx (2015) 1e12

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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

A newly designed molecule J2326 for Alzheimer's disease disaggregates amyloid fibrils and induces neurite outgrowth Q5

Pei-Teh Chang a, b, Rahul Subhash Talekar a, Fan-Lu Kung a, Ting-Rong Chern c, Chen-Wei Huang a, Qing-qing Ye a, b, Min-Yan Yang a, b, Chao-Wu Yu a, b, Shin-Yu Lai a, b, Ravindra Ramesh Deore a, Jung-Hsin Lin a, c, d, Chien-Shu Chen e, Grace Shiahuy Chen f, **, Ji-Wang Chern a, b, g, * a

School of Pharmacy, National Taiwan University, No. 33, Lin-Sen South Road, Taipei, 10051, Taiwan, ROC Center for Innovative Therapeutics Discovery, National Taiwan University, No. 33, Lin-Ssen South Road, Taipei, 10051, Taiwan, ROC c Division of Mechanics, Research Center for Applied Sciences, Academia Sinica, No. 128, Section 2, Academia Road, Taipei, 11529, Taiwan, ROC d Institute of Biomedical Sciences, Academia Sinica, No. 128, Section 2, Academia Road, Taipei, 11529, Taiwan, ROC e School of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung, 40402, Taiwan, ROC f Department of Applied Chemistry, Providence University, No. 200, Section 7, Taiwan Boulevard, Taichung, 43301, Taiwan, ROC g Department of Life Science, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei, 10617, Taiwan, ROC b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 August 2014 Received in revised form 25 December 2014 Accepted 7 January 2015 Available online xxx

Alzheimer's disease is a neurodegenerative disorder characterized by deposition of b-amyloid (Ab) fibrils accompanied with progressive neurite loss. None of the clinically approved anti-Alzheimer's agents target both pathological processes. We hypothesized that conjugation of a metal chelator to destabilize Ab fibrils (fAbs) and a long-chain fatty alcohol to induce neurite outgrowth may generate a novel molecular scaffold that targets both pathologies. The hydroxyalkylquinoline J2326 was designed and synthesized by joining an 11-carbon alcohol to 5-chloro-8-methoxyquinoline at the 2-position and its antineurodegenerative potentials in vitro and in vivo were characterized. It attenuated fAb formation and disaggregated the existing fAb zinc-dependently as well as zinc-independently. It also triggered extracellular signal-regulated kinase-dependent neurite outgrowth and increased synaptic activity in neuronal cells. In fAb-driven neurodegeneration in vitro, J2326 reversed neurite collapse and neurotoxicity. These roles of J2326 were also demonstrated in vivo and were pivotal to the observed improvement in memory of mice with hippocampal fAb lesions. These results show that the effectiveness of J2326 on fAb-driven neurodegeneration is ascribed to its novel scaffold. This might give clues to evolving attractive therapy for future clinical trials. © 2015 Published by Elsevier Ltd.

Keywords: Alzheimer's disease b-amyloid Disaggregation Neurite outgrowth and neurodegeneration

1. Introduction Alzheimer's disease (AD) continues to be an incurable neurodegenerative disorder and a leading cause of dementia that affects

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Abbreviations: AD, Alzheimer's disease; Ab, b-amyloid; fAb, Ab fibrils; NGF, nerve growth factor; AChE, acetylcholinesterase; GAP43, growth-associated protein 43; MAPK, mitogen-activated protein kinase; ERK, extracellular signaleregulated kinase; BBB, bloodebrain barrier; Pe, effective permeability; SPR, surface plasmon resonance; CD, circular dichroism. * Corresponding author. School of Pharmacy, National Taiwan University, No. 33, Lin-Sen South Road, Taipei, 10051, Taiwan, ROC. ** Corresponding author. E-mail address: [email protected] (J.-W. Chern).

an increasing number of individuals (Wimo et al., 2006). The extracellular deposition of b-sheet-rich b-amyloid fibrils (fAbs) in the brain is believed to be one of the primary mechanisms of the pathogenesis of AD (Iwatsubo et al., 1994; Selkoe, 1991). The accumulation of fAb is facilitated by zinc (Chen et al., 2006; Bush et al., 1994a,b Sep), which is significantly increased in AD patients compared to age-matched subjects (Danscher et al., 1997). The fAb elicits oxidative stress (Matsuoka et al., 2001) and tau hyperphosphorylation (Zheng et al., 2002), leading to neurite dystrophy, synaptic failure and neuronal death (Puttfarcken et al., 1996). The fAb-driven neurodegeneration proceeds, even if the fAb load has been reduced (Tohda et al., 2003, 2004), and gradually becomes fAb-independent and eventually results in memory loss (Holmes et al., 2008; Hyman, 2011; Karran et al., 2011; Sperling et al., 2011).

http://dx.doi.org/10.1016/j.neuropharm.2015.01.004 0028-3908/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Chang, P.-T., et al., A newly designed molecule J2326 for Alzheimer's disease disaggregates amyloid fibrils and induces neurite outgrowth, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.01.004

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The fAb-driven neurodegeneration cannot be reversed by currently available therapies, which include acetylcholinesterase (AChE) inhibitors and NMDA antagonists, but these can only control the symptoms by affecting the function of brain neurotransmitters and slowing cognitive decline. In recent years, amyloid based strategies are likely to show potency against AD; however, numerous clinical trials of these types of agents, for example AN1792 (Ab-mediated immunotherapy reported by Sperling et al., 2011), tramiprosate (anti-Ab aggregating agent reported by SantaMaria et al., 2007), tarenflurbil (Ab-lowering agent reported by Wan et al., 2009), clioquinol and PBT2 and PA1637 (chelating agents reported by Cherny et al., 1999; Barnham et al., 2003, 2014, and Ceccom et al., 2012) and semagacestat (secretase inhibitor reported by Extance, 2010), have been discontinued because of either safety reasons or the absence of clinically significant benefits. Another strategy with a long history being a potential therapy for AD is neurotrophic supplementation by giving especially nerve growth factor (NGF). NGF not only contains neurotrophic and neuroprotective activities but also specifically modulate the activities of adult cholinergic neurons (Seiler and Schwab, 1984). Additionally, it was reported to be able to maintain neurogenesis and cell survival in neurodegenerative disorders (Hefti and Will, 1987; Koliatsos et al., 1991). NGF has been widely tested as an anti-AD agent targeting neurite dystrophy to restore neuronal function; however, it triggers severe back pain and weight reduction (Eriksdotter€nhagen et al., 1998; Williams et al., 2006) and is also easily Jo degraded and allergenic. The effectiveness of the single target strategy for AD is limited; thus, a great need exists for novel therapeutic agents against AD having a favorable risk-benefit ratio. In view of the complex mechanisms in AD processes (Youdim and Buccafusco, 2005), the development of multifunctional strategy to combat fAb-driven neurodegeneration has been inspired. Compounds with dual or multiple targets for AD are in development to offer both symptomatic and disease-modifying efficiencies; for instance, anti-AChE activity combine with either secretase inhibitory activity (Bolognesi et al., 2009; Kupershmidt et al., 2012), antioxidant activity (Bajda et al., 2011; Luo et al., 2013) or monoamine oxidase inhibition (Weinstock et al., 2000). None of these multifunctional anti-AD agents target both fAb deposition and neurite dystrophy. Inducing fAb disaggregation and stimulating neurite outgrowth simultaneously may be a promising therapeutic strategy for fAbdriven AD patients. The present study aimed to design a multifunctional ligand strategy that could both induce fAb disaggregation and stimulate neurite outgrowth in fAb-driven neurodegeneration, and then constructed a concept molecule, verified the molecule's mechanistic actions and to characterize its anti-Alzheimer effects in vitro and in vivo. 2. Materials and methods 2.1. Determinations of zinc-binding activity J2326 (5 mM) was incubated with ZnCl2 (0e50 equivalents) or with 50 equivalents of metal ions (CuCl2, MgCl2, CaCl2, MgSO4, FeSO4, or ZnSO4) in methanol at room temperature for 24 h, and then the UVevisible absorption and fluorescence excitation spectra were recorded with a UVevisible spectrophotometer (DU730, Beckman) and a fluorescence spectrophotometer (F4500, Hitachi), respectively. Representative spectra are shown from three independent experiments. The zinc-binding activities of J2326, clioquinol, compound 4, and undecanol were also quantified using the zinc-specific fluorescent indicator ZnAF2 (Merck). The indicator (0.5 mM) and ZnCl2 (10 mM) in 25 mM sodium acetate buffer (pH 6.0, final volume 500 ml) were incubated at room temperature for 30 min in the dark before additional incubation with the compound for 30 min. The fluorescence intensity of ZnAF-2 was measured with excitation at 498 nm and emission at 518 nm, at which J2326 showed no emission. The data are shown as fold change compared with the fluorescence of ZnAF-2/ZnCl2. Then, the Kd of J2326 for Zn2þ was calculated from data acquired with a MicroCal™ isothermal titration calorimetry system. Briefly, titrations of 16 mM ZnCl2 into 0.4 mM J2326

were performed in 10 mM MES, pH 5.8, containing 2% DMSO (v:v) at 25 C with an injection volume of 39 ml for 13 injections and a stirring speed of ~500 rpm and ~400 s between injections. The resulting thermogram was used for Kd value determination, which is calculated from the best fit obtained using a model of a single set of sites using Origin supplied by MicroCal. Experiments were performed independently at least three times. 2.2. Effects on aggregation kinetic and neurotoxic of fAb Stock Ab (Bachem) was prepared as described (Chang et al., 2009). Ab1e42 was incubated with or without 10 mM ZnCl2 at 37 C for 24 h in 50 mM MES (pH 5.8) to promote zinc-induced or self-aggregated (zinc-independent) Ab polymerization, respectively. Five microgram Ab in 15 ml MES for western blotting (detected with anti-fAb antibody from Biosource), 50 ml of 10 mM Ab for Congo red staining (Chang et al., 2009) or 10 mM fAb for neurotoxic evaluation (10 ml of 100 mM fAb was added into 90 ml of neuronal culture in 96-well plate simultaneously with compounds to test the neurotoxicity) were used. The anti-aggregation and antifAb neurotoxicity studies were performed in the presence of J2326, clioquinol, or compound 4. For dissociation of pre-existing aggregates, the zinc-induced and self-aggregated fAbs were first harvested with centrifugation at 13,000 g for 30 min in, 4 C (Su and Chang, 2001). The fAb pellets were further incubated with or without J2326, clioquinol, or compound 4 for 6 h (thioflavin T assay) or 24 h (western blot and Congo red assays) in the absence of Zn2þ. Then, the kinetics of zinc-induced aggregation and self-aggregation of fAbs in the presence or absence of J2326 or clioquinol were examined using the surface plasmon resonance (SPR) system BIAcore1000 (GE Healthcare Life Sciences). Briefly, 5 mM Ab1e42 in 0.01 M sodium acetate, pH 4.0, was immobilized on a CM3 chip via amine coupling. The level of immobilized Ab1e42 reached 450e500 response units, which was sufficient for investigating Ab association. In the absence or presence of 10 mM Zn2þ with or without 10 mM clioquinol or J2326, 5 mM Ab1e42 in PBS containing 0.5% DMSO was injected onto an immobilized surface at a flow rate of 5 ml/min for 10 min. PBS containing 0.5% DMSO was used as a buffer control. The inhibitory effects of clioquinol or J2326 on Ab aggregation were recorded. Next, disaggregation of existing fAb by various compounds was also measured. After the polymerization of self-aggregated fAb (or zinc-induced fAb) on the CM3 surface reached a level of 1800e2000 (or 5500) response units, the non-associated Ab was removed, and dissociation of surface-coupled fAbs was monitored by injecting 10 mM clioquinol or J2326 for 7 min at the same flow rate in the absence of zinc. The changes in the amplitudes of the reflected beam were acquired and recorded as sensorgrams. Experiments were performed in triplicate and generated similar responses. 2.3. Ab interaction 2.3.1. SPR analysis SPR-based ProteOn XPR36 (Bio-Rad) was used to determine the Ab binding motif of J2326. Peptides Ab1e42 (pH 4.0), Ab42e1 (pH 4.0), Ab1e11 (pH 3.0), Ab35e42 (pH 3.0), and Ab12e24 (pH 5.0) in 10 mM sodium acetate buffer and Ab25e35 (pH 7.0) in 100 mM sodium phosphate buffer were individually immobilized on a GLM chip at a flow rate of 30 ml/min at 25 C, resulting in immobilization of 1700e2100 response units for each fAb peptide. Binding sensorgrams were obtained by injecting 200 mM J2326 or compound 4 (in 0.5% DMSO in PBS) onto chips containing immobilized fAb at a flow rate of 10 ml/min at 25 C for 9 min. Then, to explore the conformational transitions of Ab fragments during interactions with J2326, each Ab fragment at 10 mM was incubated in 100 ml PBS (pH 7.4) for 5 min, incubated with 10 mM J2326 for 9 min, and finally mixed with 900 ml of 30 mM thioflavin T (in 50 mM sodium phosphate, pH 6.0). Experiments were performed independently at least three times and generated similar responses. 2.3.2. Thioflavin T analysis The thioflavin T fluorescence intensity (representing the b-sheet content in fAbs) was immediately measured with a fluorescence spectrophotometer (excitation at 450 nm, emission at 460e520 nm). Sequential incubation was used to investigate the change in the Ab secondary structure during J2326-Ab interactions. The Abs used to study this interaction were demonstrated to be initially b-sheet rich but acquired a lower b-sheet load after J2326 interaction. Experiments were carried out in three times independently. 2.3.3. Circular dichroism analysis Circular dichroism (CD; J-720, Jasco) was used to monitor the interaction of J2326 with fAbs by incubating with 50 mM Ab1e42 or Ab42e1 (in PBS, incubation at 37 C for 6 h) with or without 10 mM J2326 at 25 C for 1 h. Finally, to obtain the Kd value of J2326 with Ab1e42, the biolayer interferometry system ForteBio Octet (Menlo Park, CA) was used. Biotin-Ab1e42 (Anaspec) was conjugated to Super Streptavidin sensor tips, which were then transferred into wells containing J2326 at varying concentrations. The binding curve was plotted based on data for the deflection in the wavelength of light passing through the sensor, and the Kd value (shown as the mean ± SEM from at least three independent experiments) of J2326 on Ab was calculated using Data Analysis version 7.0.


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P.-T. Chang et al. / Neuropharmacology xxx (2015) 1e12 2.4. Docking study Molecular docking was performed using PLANTS 1.2 software (Korb et al., 2009). The PLANTSCHEMPLP scoring function (suitable for 11e15 rotatable bonds) and speed 1 were used. To assess fAb protein dynamics, 10 hairpin-like NMR models for fAb1e42 (PDB ID: 2BEG) were used. Because the N-terminal residues of Ab are disordered and lack suitable binding pockets (Bertini et al., 2011), N-terminal residues 1e17 were omitted. The search space was set as a sphere of radius 7.5 nm at the geometric center of each conformation. Twenty clusters were maintained for the binding pose analysis. 2.5. Assessments of neurotrophic effects PC12 and SH-SY5Y cells, which were purchased from Bioresource Collection and Research Center (HsinChu, Taiwan), were cultured in RPMI1640 medium supplemented with 25 mM HEPES, 10% horse serum, and 5% fetal bovine serum or in MEM/ F12 (1:1) supplemented with 2 mM glutamine, 1% non-essential amino acids, and 15% fetal bovine serum, respectively. 2.5.1. Neuritogenic activity The undifferentiated neuronal cells were grown on poly-D-lysine/laminin/ fibronectin pre-coated glass coverslips in six-well plates (1 105 cells/well). The cells were treated with 0, 1 or 10 mM J2326 with or without the extracellular signaleregulated kinase (ERK) inhibitor U0126 (1 mM) or the p38 inhibitor SB203580 (1 mM) for 3 days, with daily medium replenishment. The cells were photographed under a light microscope (Nikon T100), and six fields were randomly chosen after cytochemical Liu's staining (Tonyar Biotech) or colorimetric immunostaining (diaminobenzidine, Vector Labs) with anti-growth-associated protein 43 (GAP43) (Sigma, 1:500), anti-b-tubulin (Santa Cruz Biotechnology, 1:1000), or anti-synapsin (Biovision, 1:250). Neurite length and neuron numbers were analyzed using ImageProPlus based on morphological results. At least three independent experiments were performed. 2.5.2. Synaptic activity Synaptic activity was measured using the action potentialedependent synaptic fluorescent indicator FM1-43 (Molecular Probes). After treatment with tested compounds, cells were washed twice for 30 s each with 100 ml prewarmed KrebseRinger buffer (115 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 1.2 mM Na2SO4, 2.5 mM CaCl2, 25 mM NaHCO3, and 10 mM glucose, pH 7.4). Subsequently, 100 ml KrebseRinger buffer containing 100 mM potassium ions and 2 mM FM1-43 was added, and the cells were incubated at 37 C for 5 min and washed twice with 100 ml KrebseRinger buffer for 30 s. Finally, 100 ml KrebseRinger buffer was added, and fluorescence was measured using a fluorescence microplate reader (excitation at 470 nm, emission at 540 nm). Experiments were done at least three times, independently. 2.5.3. Mitogenic signalings and mitogenic effect The levels of phospho-ERK/p38 and ERK/p38 in 25 mg protein harvested from neuronal cells, which were treated with J2326 with/without a mitogen-activated protein kinase (MAPK) inhibitor for 12 h, were detected with western blotting using antibodies from Santa Cruz Biotechnology. Immunoblot data were quantified using Un-Scan-It version 5.1. Each neurotrophic response (neurite length, neuron number, synaptic activity, or activation of MAPK) compared to that induced by 50 ng/ml NGF was considered as 100%. Neuronal survival was measured using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 570 nm) assay and cells that were only treated with vehicle were taken as control (100%). Data were calculated from at least three independent experiments and represent the mean ± SEM. 2.6. In vitro fAb-driven neurodegeneration models NGF-differentiated neurite-abundant neurons (undifferentiated PC12 cells treated with 50 ng/ml NGF for 3 days) and dystrophic neurons (NGF-differentiated PC12 cells treated with 10 mM zinc-induced fAb for 2 days) were used to assess the effects of J2326 on different stages of fAb-induced neurodegeneration. Neuriteabundant neurons and dystrophic neurons were treated with 0, 1, or 10 mM J2326 for 72 h in the presence or absence of 10 mM zinc-induced fAb to mimic fAbdependent, fAb-independent, or chronic degeneration stages. The morphologies of these neurons were analyzed as described above. Neurotrophic effects of J2326 in different conditions were quantified and expressed as percentages (mean ± SEM) by taking the effects of 50 ng/ml NGF for 3 days as the control. The changes in neurite, synaptic activity, and neuronal number between vehicle-treated and J2326-treated cells were evaluated by the analysis of variance with triplicated measurements followed by paired Student's test (*P < 0.05). 2.7. Blood-brain barrier (BBB) permeability assay in vitro The parallel artificial membrane permeation assay was carried out in a sandwich-like 96-well plate formed with a top filter plate containing acceptor wells (200 ml PBS containing 5% DMSO) and a bottom plate containing donor wells (300 ml J2326 or low/high permeability standards) separated with a 0.45-mm polyvinylidene

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fluoride membrane coated with 5 ml BBB-1 Lipid (pION). Tested compounds were prepared in DMSO and diluted 20-fold in PBS to yield final concentrations of 100 mg/ ml working stock solutions. The final DMSO concentration in the working stock solution was 5%. The sandwich plate was then incubated at room temperature for approximately 4 h. After reaching diffusion equilibrium, the sandwich plate was disassembled, and the concentration of compounds in the acceptor, donor, and reference wells was determined using a microplate reader. Effective permeability (Pe, 106 cm/s) of the compounds was calculated, and the effective permeability of a BBB þ compound (high BBB permeability predicted) was greater than 4.0 106 cm/ s. Experiments were repeated at least three times independently. 2.8. AD mice model All procedures were carried out in accordance with the Institutional Animal Care and Use Committee of National Taiwan University, and based on the animal welfare aspects of minimizing pain and suffering in an experiment and of reducing the number of animals used. Prior to the induction of neurodegeneration, Balb/c mice that demonstrated a poor learning ability were excluded from the rotarod and water maze pre-training that was performed three times a week. Hippocampal lesions were produced with stereotaxic injection of zinc-induced fAb. The fAb (0.5 ml of 20 mM fAb aggregated for 24 h in the presence of 10 mM ZnCl2 in 50 mM MES buffer at 37 C) was injected bilaterally into the hippocampal CA3 region (AP, 1.9 mm from bregma; L, ±2.2 mm from midline; V, þ2.0 mm ventral to dura). Mice without a learning disability 1 week after surgery were excluded. Subsequently, non-lesioned (sham) and fAb-lesioned mice were divided into different groups that received 0, 1, or 10 mg/kg/day J2326, clioquinol, undecanol, or a combination of clioquinol and undecanol (10 mg/kg/day each) by i.p. injection (100 ml in 10:10:80 [v:v:v] DMSO:Cremophor:saline) daily for 30 days. Behavioral training was performed three times a week and started at 4 h after the drug injection. The performance on the last trail day was analyzed. The mice were randomly assigned to each groups (n ¼ 6 per group for rotarod test and n S 4 per group for water maze test). 2.8.1. Rotarod and water maze tests For the rotarod test (IITC Life Science), training comprised three linear accelerations (5e30 rotations within 5 min), and the latency to fall was scored. In the water maze test, cued navigation directed the mice from the starting quadrant toward a transparent plexiglass platform (15 cm high, 10 10 cm). The platform was placed in the quadrant located diagonally across from the starting quadrant and was immersed 0.5e1 cm under the water surface. Four trials were conducted each time, and each trial lasted approximately 5 min with an interval of 5 min. The swim paths of all trials during training were monitored using tracking (Noldus Ethovision) and videotaping systems. The swimming paths at day 30 are shown in Supplemental material. 2.8.2. Immunohistochemistry and analyses After 30 days, mice were anaesthetized with sodium pentobarbital 100 mg/kg (i.p.), and intracardial perfusion was performed with 4% paraformaldehyde in PBS at pH 7.4. Brains were fixed in the same solution and coronal sections (10 mm) containing the hippocampus were prepared. Diaminobenzidine-based colorimetric immunostaining was performed using anti-fAb (MyBiosource, 1:250) and antiGAP43 (Abcam, 1:250) followed by Nissl and methylgreen counterstaining, and sections were examined under light microscopy (T100, Nikon). The swimming paths analyses and histological hippocampus profile quantifications of vehicletreated and J2326-treated fAb-lesioned mice were compared using one-way ANOVA (*P < 0.05). 2.9. Statistical analysis of data All experiment data were expressed as mean value ± standard error mean (SEM). In vitro experiment data were analyzed using paired Student t-test. The differences between groups in vivo experiments were assessed by one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. Data were considered statistically significant if P values were less than 0.05.

3. Results 3.1. Design and synthesis of J2326 The design concept for a newly molecular scaffold was built on two approaches. First, the long-chain fatty alcohol hexacosanol extracted from Hygrophila erecta Hochr. (Acanthaceae), a traditional Chinese medicine, promotes neuronal survival and neurite outgrowth, especially on peripheral cholinergic neurons in nerveinjured animals (Azzouz et al., 1996; Borg et al., 1987, 1990; Borg, 1991). Even some shorter-chain fatty alcohols (Girlanda-Junges et al., 1988; Gonzalez et al., 2001; Hanbali et al., 2004; Luu et al., 2000; Natarajan and Schmid, 1977) have the same neuritogenic


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effects. Second, the 8-hydroxyquinoline scaffold based metal chelators clioquinol and PBT2 (5,7-Dichloro-2-dimethylaminomethylquinolin-8-ol) had been used to reduce zinc-triggered fAb deposition and memory impairment in patients with AD (Cherny et al., 1999; Barnham et al., 2003; Faux et al., 2010). Clioquinol causes neurite retraction in vitro (Asakura et al., 2009) and its clinical trial was discontinued owing to safety concerns (Tateishi et al., 1973); and PBT2 had improved solubility, BBB penetrability and greater preclinical efficacy (Adlard et al., 2008; Barnham and Bush, 2014; Maria and Chris, 2013). All these results with the 8hydroxyquinoline scaffold agents suggest that disrupting zinctriggered fAb accumulation may be a useful approach for treatment of AD. Hence, a hybrid compound called J2326, which combines the properties of clioquinol and fatty alcohol, was designed (Fig. 1A). To our knowledge, this type of multifunctional agent has not been reported. Through knowledge-based molecular engineering (Fig. 1A), the molecule J2326 was formulated by attaching hexacosanol to the C2 position of clioquinol, followed by additional modifications to optimize its characteristics. First, the 7-iodo group from clioquinol was removed because of its neuropathy (Fisher et al., 1993). The 8hydroxyl group was replaced with an 8-methoxy group since 8hydroxyquinoline is neurotoxic and diabetogenic (Foye, 1961; Oakley, 1973; Root and Chen, 1952) and non-selective for biologically vital metal ions, whereas 8-alkoxyquinolines are selective for specific metals (Xue et al., 2011). Lastly, based on the optimal chain lengths of fatty alcohols for neurite outgrowth (Coowar et al., 2004; Girlanda-Junges et al., 1988) and an adequate logP for penetration of the BBB, the C26 alcohol was replaced with a C11 alcohol. The concept molecule was then synthesized as following. Methylation of commercially available 2-methylquinolin-8-ol (1) generated 8-methoxy-2-methylquinoline (2), which was further alkylated to produce 11-(8-methoxyquinolin-2-yl)undecan-1-ol (3). Subsequently, compounds 2 and 3 were chlorinated to produce 5-chloro-2-methylquinolin-8-ol (4) and 11-(5-chloro-8methoxyquinolin-2-yl)undecan-1-ol (5, J2326), respectively. These synthetic steps are illustrated in Fig. 1B, and the detailed

synthesis and identification of compounds are described in Supplemental material. 3.2. Interaction with Zn2þ The UVevis ans fluorescence spectrometries were performed to assess the metal binding ability of J2326. The absorption spectra of J2326 did not differ substantially in the absence or presence of Zn2þ; however, Zn2þ did cause an impressive decrease in J2326's intrinsic fluorescence (Fig. 2A) that was more prominent than that observed with other metal ions such as Fe2þ, Cu2þ, Mg2þ, and Ca2þ (Fig. 2B). Next, zinc-binding activity was elucidated using the fluorescent Zn2þ sensor, ZnAF-2. J2326 concentration-dependently reduced the fluorescence of ZnAF-2 (0.5 mM) in the presence of Zn2þ (Fig. 2C). It also indicates, on the zinc binding activity, that J2326 and compound 4 are equal and undecanol doesn't have and clioquinol is superior among all. The affinity of J2326 for Zn2þ was then measured using the isothermal titration calorimetry system. The titration profile of J2326 was exothermic and non-typically sigmoidal (Fig. 2D), and the apparent Kd value of J2326 to Zn2þ was estimated to be 23.1 ± 0.5 mM. 3.3. Effects of J2326 on Ab aggregation kinetics and its neurotoxicity 3.3.1. Zinc-mediated anti-fAb aggregation Since J2326 could bind to Zn2þ, the effect of J2326 on the formation and stability of zinc-induced fAbs was examined next by determining the extent, morphology and aggregation kinetics of the zinc-induced fAb in the presence of J2326. Western blotting revealed that zinc-induced formations of various fAb forms (dimeric, oligomeric, and polymeric) were significantly reduced in the presence of 10 mM J2326 (Fig. 3A). Furthermore, light microscopy using Congo red staining showed that the formation of polygonal fAb was appeared after aggregation and was disappeared by J2326 (Fig. 3B, left panels in the presence of zinc). The aggregation rate of zinc-induced fAb was measured by SPR and was

Fig. 1. Design and synthesis of J2326. (A) Knowledge-based molecular engineering for new scaffold. (B) Synthesis of J2326. Reagents and conditions: (a) K2CO3, CH3I, acetone, room temperature, 14 h; (b) (1) Lithium diisopropylamide, tetrahydrofuran, 0 C, 1 h; (2) 10-bromo-1-decanol, 0 C to room temperature, 2 h; (c) Iodine trichloride, 12 M HCl, 98% glacial acetic acid, room temperature, 6 h. LogP refers to partition coefficient for n-octanol/water and is used to describe lipophilicity.


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Fig. 2. J2326 selectively binds Zn2þ. (A) Absorption (solid lines) and excitation (250 nm, dotted lines) spectra of 5 mM J2326 upon the addition of ZnCl2. a.u.: atomic unit. (B) Excitation (250 nm) spectra of J2326 in the presence of 50 equivalents of Zn2þ, Ca2þ, Mg2þ, Fe2þ, or Cu2þ. (C) The fluorescence (mean ± SEM) of the ZnAF-2/zinc complex was reduced by J2326, clioquinol, and compound 4. Statistical analysis was performed using paired Student t-test. *P < 0.05, **P < 0.005 compared with vehicle control (0 mM J2326). (D) Representative binding affinity profile for J2326 titrated with ZnCl2. The results of one of three independent experiments are shown in (A), (B) and (D).

found to be reduced by J2326 (Fig. 3E, left panel). Altogether, these results indicated that J2326 inhibited the formation of zinc-induced fAb. 3.3.2. Disaggregation of fAb The results of the above analyses also indicated that J2326 caused more pronounced disaggregation of pre-existing zincinduced fAb (Fig. 3A, C and E). Although the inhibitory effects of J2326 and clioquinol on the formation of zinc-induced fAbs were comparable (Fig. 3A and E), the destabilizing effect of J2326 on existing zinc-induced fAbs was much greater than that of clioquinol (Fig. 3A and E). This notable finding was additionally confirmed using a solid-phase aggregation platform (Table S1). Interestingly, J2326 was found to block fAb aggregation in the absence of Zn2þ as well as to destabilized the self-aggregated fAbs (formed in the absence of zinc), whereas clioquinol was of no effect on self-

aggregated fAb. The distinctively zinc-independent effects of J2326 on the formation and dissociation of self-aggregated fAbs were testified by Congo red staining (right panels in Fig. 3B and C), western blotting (Fig. 3D), and SPR (Fig. 3E) and were consistent with results from the solid-phase platform (Table S1). 3.3.3. Anti-fAb neurotoxicity The neuroprotective effects of compounds on undifferentiated neuronal cells treating with zinc-induced or self-aggregated fAbs were further elucidated by using MTT cell viability assay. J2326 reduced the neuronal death caused by either zinc-induced or selfaggregated fAbs in PC12 cells as well as in SH-SY5Y cells (Table S1). In contrast, clioquinol was only effective in protecting cells from zinc-induced fAbs. These results agreed with the difference between the effects of J2326 and clioquinol on the zinc-independent fAb formation and stability.

Fig. 3. J2326 inhibits fAb formation and dissociates preformed fAbs. (A) Western blots showing that J2326 inhibited the formation of polymeric fAbs in the presence of Zn2þ and dissociated all zinc-induced fAb deposits. Clioquinol only inhibited the formation of fAbs. (B) Congo red staining showing polygonal fAb deposits and that 10 mM J2326 inhibited both zinc-dependent and -independent formation of polygonal fAbs. (C) J2326 (10 mM) dissociated both zinc-induced and self-aggregated polygonal fAb deposits. (D) J2326 dissociated self-aggregated fAbs. (E) SPR sensorgrams showing the inhibition of fAb formation and the dissociation of fAbs by vehicle (black line) or 10 mM J2326 (blue line) or clioquinol (red line). The results shown are representative of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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3.4. Direct interactions with fAbs 3.4.1. SPR analysis J2326 was identified to induced fAb disaggregation zincindependently; thus, the interaction between J2326 and selfaggregated fAb was investigated by using SPR. The sensorgrams showed that J2326 bound to fAb1e42 (Fig. 4A), and this interaction was verified using biolayer interferometry, which yielded an apparent Kd of 1.84 ± 0.45 mM. Clioquinol, compound 4, and the C11 alcohol undecanol did not bind to Ab1e42 in SPR study (sensorgrams not shown). Further insights into aspects of the J2326-interacting motif on amyloid, the fragments Ab1e11, Ab25e35, Ab12e24, Ab35e42, Ab1e42, and the reverse sequence Ab42e1 were used. The sensorgrams showed that J2326 did not interact with Ab1e11, Ab25e35, Ab35e42, or the reverse sequence Ab42e1. Importantly, in addition to Ab1e42, J2326 also bound to the Ab12e24 fragment (Fig. 4A).

3.4.2. Thioflavin T ana CD analyses Considering the zinc-independent anti-fAb activity and fAbinteracting ability of J2326, the correlation between them was further explored with the thioflavin T fluorescence assay and CD. Afterward CD spectra showed that the addition of J2326 to selfaggregated fAb1e42 resulted in an inverted signal compared with the b-sheeterich fAbs alone (Fig. 4B); this inverted signal was similar to that generated by Ab42e1, which has a reverse sequence of Ab and lacks a b-sheet structure. The zinc-independent action of J2326 was consistent with the loss of b-sheet conformation in the existing fAb peptides that only contain the Ab12e24 motif, as evidenced by the drastic reductions in b-sheetedependent thioflavin T fluorescence of pre-existing fAb1e42 or fAb12e24 after J2326 addition (Fig. 4C). As a result, two assays validated that J2326 lowered the bsheet content of self-aggregated fAbs. 3.4.3. Docking study To gain further insight into the mode of J2326:self-aggregated fAb interaction, the molecular docking was performed by using PLANTS 1.2 software. The self-aggregated fAb1e42 pentamer (PDB ID 2BEG) (Lührs et al., 2005) was chosen for docking study, and 11 possible J2326-binding sites within 10 conformational variants were recognized (Table S2). The major two favorable binding sites for J2326 on fAb were identified (Fig. 4D). First, the C11 side chain of J2326 was anchored in the open hydrophobic tunnel comprised of residues Ala21/Asp23/Leu34/Val36 (models 01, 05, 07, 08, 09, and 10) from either fibril axis with hydrogen bonding between the terminal hydroxyl group of the C11 side chain and backbone amide group of Ala21 (models 01, 07, 09, and 10). In the other four fAb models (models 02, 03, 04, and 06), however, the tunnels were too narrow to accommodate the C11 side chain. Second, the 5-chloro8-methoxyquinoline core of J2326 played a part in hydrophobic interactions along the side clefts of fAb pentamer, indicating this modified quinoline core also contributes to fAb binding. 3.5. Stimulation of neurite outgrowth in vitro To analyze the effects of J2326, as well as clioquinol and compounds 3 and 4, on neurite outgrowth, the undifferentiated PC12 and SH-SY5Y cells were used for study. J2326 led to neurite outgrowth (Fig. 5A). These newly sprouted neurites were identified to be b-tubulin positive (Fig. 5A), and considered to be mature and functional on account of the localizations of GAP43 and/or synapsin (Fig. 6A) within neurite, the increased expressions of GAP43 and neuronespecific enolase (Fig. S1C) and the augmented synaptic activity (Fig. 5C). These effects of J2326 showed concentrationdependence (Fig. 5). The neuritogenic effect of J2326 (Fig. 5B and Fig. S1A) was comparable to those of NGF and hexacosanol (Fig. S1B). The intermediate of J2326, compound 3, also promoted neurite outgrowth but its effect was not remarkable in compared with that of J2326 (Fig. S1A). As regards clioquinol and compound 4, they did not (Fig. S1A). 3.6. MAPK-dependent neurotrophic effects

Fig. 4. J2326 binds to fAb1e42 and induces secondary structure transition of fAbs. (A) SPR analyses show significant interaction between J2326 and peptides Ab1e42 and Ab12e24, as demonstrated by an increase in resonance units, but not with Ab42e1, Ab1e11, Ab25e35, or Ab35e42. (B) CD spectra showing the inverted signal of fAb1e42 induced by the addition of J2326 at approximately 195 nm, which is similar to the signal of the reverse sequenced Ab peptide Ab42e1. (C) The fluorescence spectra of thioflavin T mixed with each preformed fAb peptide, which was incubated with (red line) or without (black line) J2326. (D) Two of the 10 conformational models (01 and 08) of J2326 interacting with the fAb1e42 pentamer, demonstrating insertion of the C11 side chain of J2326 into the hydrophobic tunnel from either side of the pentamer. The results shown in (A, B and C) are typical of three independent experiments with similar results. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

There are two MAPKs, ERK and p38, reported to be critical for neurite outgrowth (Lambeng et al., 2003) and to be required for the neurotrophic actions of some long-chain fatty alcohols (Hanbali et al., 2004). Hence, the roles of both MAPKs on J2326etriggered neurite outgrowth were investigated. Quantification of western blots proved that J2326 induced phosphorylations on ERK and p38 for the period of neurite induction by J2326 in PC12 (Fig. 6EeH) and SH-SY5Y (Fig. S1D) neuronal cells. Addition of an ERK inhibitor could completely abolish the J2326etriggered neurite outgrowth, but addition of p38 inhibitor did not affect (Fig. 6A and B).


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Fig. 5. The neurotrophic effects of J2326 on undifferentiated PC12 cells. (A) J2326 (10 mM) induces neurite outgrowth as observed with b-tubulin immunostaining. The morphology of non-differentiated PC12 cells was shown in inset with same scale. A result typical of three independent experiments is shown in (A). The neurite length (B, using image-based quantification), synaptic activity (C, using FM1-43, a fluorescent dye taken up at synaptic terminals in an activity-dependent manner), and neuron number (D, using image-based quantification) are expressed as the percentage relative to treatment with NGF. All data are presented as mean ± SEM. Statistical analysis was performed using paired Student t-test. *P < 0.05, **P < 0.01 compared with vehicle group (0 mM J2326).

Regarding outgrowth and function, the neuritogenic activities of J2326 seemed to be dependent on the activation of ERK but not of p38 (Fig. 6BeD). In addition, J2326 did not show any neurotoxicity in vitro, even at a concentration of up to 100 mM (Fig. 5D). At last, as revealed by the increases in the number of neurons (Fig. 5D), the expression of proliferating cell nuclear antigen (Fig. S1C) and the activations of ERK/p38 kinases in neuronal growth (Fig. 6EeH), J2326 encouraged neuronal proliferation in an MAPK-dependent manner.

peripheral injection of J2326. J2326 had a high BBB permeability (Pe of J2326 was 12.74 ± 5.65 106 cm/s), indicating J2326 is BBBpermeable. Evidences for crossing of the BBB by J2326 was also found in CSF (Supplemental Fig. S3AeS3C) from normal wild-type mice that were i.v. injected with J2326 (10 mg/kg). The concentration of J2326 in CSF was identified to be 0.35 ± 0.09 nM, and its CSF/plasma concentration ratio was 0.144 ± 0.034.

3.7. fAb-derived neurodegeneration in vitro

To explore the effects of J2326 on learning and memory in mice with neurodegeneration, the rotarod test and water maze test were used after mice were subjected to fAb-induced hippocampal lesioning and following i.p. administration of J2326 once daily for 1 month. In the rotarod test (Fig. 8A), the learning-based motor functions in fAb-lesioned mice were severely impaired and became progressively worse during the observation period. Administration of J2326 markedly ameliorated the fAb-mediated deficit, whereas undecanol treatment resulted in only a slight improvement. Treatment with clioquinol prevented further deterioration but did not reduce impairment. The effect of co-treatment with undecanol and clioquinol was not as high as that with J2326. In the Morris water maze test, the non-lesioned mice receiving vehicle or J2326 revealed their quick recognitions to hidden platform founded on swimming fast around platform and on reaching the platform rapidly (Fig. 8BeE and Fig. S3D). The fAb-lesioned mice demonstrated poor performance and failed to reach the platform. Administration of J2326 improved the performance of fAb-lesioned mice in a dose-dependent manner; however, lesioned mice given a lower dose (1 mg/kg) of J2326 did not climb the platform finally (Fig. 8). After treatment with 10 mg/kg J2326, all the fAb-lesioned mice swam to the hidden platform in a shorter time and distance and spent more time in the target zone (Fig. 8CeE) and five of them successfully climbed the platform. Additionally, in terms of the relationship between total movement time and distance swam, the lesioned and non-lesioned mice with/without J2326 treatment demonstrated similar swimming speeds. The observations that J2326 considerably reduced the in vivo effects of fAb suggested a correlation between the restoration of learning-based behavior owing to the recovery of neurites/neurons and the elimination of fAbs in from hippocampus of fAblesioned mice. As shown in Fig. 8B, F and H, the observed increase in fAb, decrease in GAP43, and neuronal survival in the hippocampus of fAb-lesioned mice were significant observed. When J2326 was administered daily, these patterns in fAb-lesioned mice tended to change toward levels observed in sham (nonlesioned) groups (Fig. 8FeH). These quantitative results showed that J2326 improved the memories of the mice both by eliminating fAbs and restoring the neurons and neurites. All mice

NGFedifferentiated PC12 neuronal cells were used to evaluate the antieneurodegeneration effects of J2326 on neurodegeneration from fAbedependent to fAbeindependent stage, and then to chronic stage in vitro (Fig. 7). First, to establish NGFedifferentiated neuronal cells, undifferentiated PC12 cells were treated with NGF, and substantial neurite sprouting was observed. Adding J2326 into neurite-rich containing NGFedifferentiated PC12 cells expanded the neurite growth, synaptic activity, and neuronal proliferation. Next, fAb-dependent neurodegeneration was initiated by addition of zinc-induced fAb to the NGF-differentiated neurons, resulting in neurite retraction, synaptic loss, and neuronal death. The concomitant addition of J2326 prevented these neurodegenerative events caused by fAbs. The retracted neurites did not spontaneously regrow even after the removal of exogenous fAbs, indicating that neurodegeneration moved forward into fAb-independent stage. Compound 3 exhibited weak effects on fAbedriven neurodegeneration, whereas clioquinol and undecanol showed no effects (Fig. S2A). Throughout the fAbeindependent phase, addition of J2326 improved but not totally restored the growth and function of neurites and survival. The chronic fAb neurodegeneration model was created by continuous exposure of the injured neurons to fAb. Each neurodegenerative phenotype became extremely severe after chronic exposure to fAb. In spite of such an irreparable condition, J2326 still have the ability to battle with chronic fAb neurodegeneration. These phenomena of J2326 (Fig. 7A) in different stages of fAbedriven neurodegeneration are qualitatively and quantitatively shown in Fig. 7AeD, respectively, and were consistent in both NGF-differentiated PC12 and SH-SY5Y cell models (Fig. 7A and S2A and S2B). 3.8. BBB permeability Before testing the efficacy of J2326 on fAb-induced memoryimpaired mice, the BBB permeability of J2326 was evaluated by using the in vitro parallel artificial membrane permeation assay and also by measuring the level of J2326 in CSF from rodents receiving

3.9. Long-term effect of J2326 on AD mice


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Fig. 6. J2326 induces neurite outgrowth in an ERK-dependent manner. (A) Immunocytochemical staining showing neurite-specific markers (GAP43and synapsin) in 10 mM J2326induced newly sprouted neurites on PC12 cells. The ERK inhibitor U0126 prevented J2326-triggered neurite extension, whereas the p38 inhibitor SB203580 did not. Insets show synapsin- or GAP43-positive nerve terminals. The effects of U0126 (red line), SB203580 (blue line) and vehicle (black line) on J2326-induced neurite extension (B), synaptic activity (C), and neuron number (D) were quantified and expressed as a percentage relative to 50 ng/ml NGF. Phosphorylation of ERK (E, F) and p38 (G, H) was increased by J2326. The increased levels of p-ERK and p-p38 were abolished by U0126 and SB203580. The open (closed) circles represent with (without) MAPK inhibitor treatment. The results shown in (A), (E) and (G) are representative of three independent experiments. All data are presented as mean ± SEM. Statistical analysis was performed using paired Student t-test. *P < 0.05, **P < 0.01 compared with vehicle group (0 mM J2326). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

treated with J2326 survived, and none of them exhibited any significant deterioration in their general health during the experimental period. 4. Discussion Our results indicate that J2326 can remarkably attenuate the pathological changes and improve neuronal function after neurodegeneration triggered by fAb. The characteristics of hybrid scaffold might contribute to the molecular mechanisms of J2326's antidegeneration effects after fAb challenge.

4.1. J2326 displays both zinc-mediated and zinc-independent antifAb effects The 5-chloro-8-methoxyquinoline core was designed with intent to be a moderate metal interacting moiety for specifically removing Zn2þ, which could facilitate the aggregation rate (Fig. 3E) and increase the level of fAb (Fig 3A and B), in order to dissolve the fAb. The atypical sigmoid exothermic curve (Fig. 2D) and the reduction on the ZnAF-2 fluorescence in the presence of Zn2þ (Fig. 2C) exhibit that J2326 binds Zn2þ ion in a coordinated and competitive manner. J2326 bound to Zn2þ more weakly


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(23 106 M) than clioquinol (kd for zinc 1.4 109 M reported by Ferrada et al., 2007) or amyloid (kd for zinc 1 107 M nM and 5.2 106 M for high and low affinity binding, respectively, reported by Bush et al., 1994a,b Apr and Bush et al., 1994a,b Sep). Although lacking higher zinc binding activity, J2326 has metal selectivity toward to zinc. This make J2326 different from clioquinol and PBT2, of which both liberate either copper or zinc ions trapped by amyloid (Ferrada et al., 2007; Barnham and Bush, 2014), and also unlike PA1637, which is copper-specific (Ceccom et al., 2012; Nguyen et al., 2014); and lead J2326 to display an effective zinc-dependent fAb disaggregation. Apart from zincdependent effect, J2326 also showed zinc-independent anti-fAb effect (Figs. 3 and 4 and Table. S1) that was also different from the zinc-dominant anti-fAb effect of clioquinol, results also reported by Mancino et al., 2009. The utilization of 8-methoxy quinolone scaffold in replacing 8-hydroxy ones lowered molecule's zinc binding activity but given J2326 superior efficacy in against either zinc-induced or self-aggregated fAbs, whereas 8hydroxyquinolone scaffold agent clioquinol reveals its consistence in affecting only zinc-induced fAb (Fig. 3A, E and Table. S1; Mancino et al., 2009). 4.2. J2326 disaggregates the self-aggregated fAb after wedging to the hydrophobic center interface of fAb

Fig. 7. J2326 rescues neurons from neurodegeneration caused by zinc-induced fAbs and triggers neurite regrowth at different stages in vitro. (A) GAP43 immunostaining showing that, in NGF-differentiated PC12 cells, 10 mM J2326 promoted the extension of mature neurites and prevented fAb-triggered neurite retraction. Dystrophic neurites did not spontaneously regrow even after the removal of fAbs, and the re-extension of dystrophic neurites was triggered by J2326 in the absence or presence of fAbs. The results typical of three independent experiments are shown in (A). Neurite length (B), neuron number (C), and synaptic activity (D) for PC12 cells (closed bars) and SH-SY5Y (open bars) are expressed as the percentage relative to 50 ng/ml NGF. All data are presented as mean ± SEM. Statistical analysis was performed using paired Student ttests. *P < 0.05, **P < 0.01 compared with each own vehicle group (0 mM J2326) in each model.

The zinc-independent effect of J2326 involves a direct fAb interaction, which was demonstrated by SPR analysis. The interaction only occurred after the addition of J2326 but not the additions of clioquinol or C11 alcohol undecanol or even its intermediate compound 4, indicating that the hybrid scaffold of J2326 is indispensably responsible for fAb interaction. Together, the C11 fatty alcohol and chloro substituents on 8-methoxyquinoline unindividually took part in the fAb interaction and the subsequently Ab conformational change. An extensive SPR work (Fig. 4A) further identified that only the Ab12e24 was key motif for the binding of J2326 in the absence of zinc, suggesting that the recognition of J2326 by fAb is dependent of specific Ab amino-acid sequence. Subsequent docking study indicated the consistency that the flexibly hydrophobic C11 alcohol side chain of J2326 wedged in the b-sheet-rich center interface of fAb and then spawned intermolecular interactions between the amino acids at the Ab12e24 range and J2326 (Fig. 4D and Table S2). From Lys16 through Ala21 within Ab12e24 segment, the KLVFFA sequence is important for antiparallel b-sheet structure formation and constitutes the hydrophobic spine in fAb (Tjernberg et al., 1996). These amino acids within Ab12e24 motif, especially for Ala21, are also necessary for fibrillogenesis and stabilization of fAb1e42 (Baumketner et al., 2006; Petkova et al., 2002; Yu and Zheng, 2011), which are both connected to fAbmediated neurotoxicity. Therefore, the binding of J2326 onto Ab12e24 of existing fAb presumably would allow Ab to assume a more a-helixelike structure and finally lead to disaggregation of fAb and reduce its neurotoxicity. Clioquinol did not show these characteristic, consistent with a previous report (Mancino et al., 2009). These results agreed with the difference between the effects of J2326 and clioquinol on the zinc-independent fAb formation and stability. The zinc-independent action is a unique feature of J2326 itself and is not limited to specific type of Ab-self aggregates. 4.3. J2326 displays a MAPK-mediated neurotrophic activity The C11 alcohol side chain of J2326 was used for providing neuritogenic activity. As expected, neurite outgrowth was triggered owing to the presence of a long-chain fatty alcohol.


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Fig. 8. (A) The learning-based rotarod test (n ¼ 6 per group) showing the effects of J2326, clioquinol, undecanol, or a combination of clioquinol with undecanol on the time fAblesioned mice remained on the rotating rod. The values of latency to fall were the mean ± SEM. On days 35, the lesioned mice receiving 10 mg/kg J2326 showed significant improvement, measured as seconds of latency to fall, compared to fAb-lesioned mice receiving 0 mg/kg J2326 (one-way ANOVA followed by Tukey multiple comparison test, n ¼ 6, *P < 0.05) (B) Morris water maze test (nS4 per group) showing that J2326 improved the learning-based memory of fAb-lesioned mice in a dose-dependent manner. The hippocampal region of fAb-lesioned mice tested in the water maze, showing the differing immunoreactivity for fAbs and GAP43 between mice receiving vehicle and J2326, as well as the neuronal densities observed by methylgreen counterstaining (green) and Nissl staining (purple). The typical images are shown. Quantitative profiles for the behavior performance (C, total swimming time; D, total swimming distance; E, time in target zone) and for data obtained from tissue sections (F, fAb level; G, surviving neurons; H, GAP43 level) are shown as the percentage relative to those from the sham group (non-fAb lesion control) that received the vehicle (0 mg/kg J2326). Data for the Morris water maze test in the hidden platform trial are presented as mean ± SEM and assessed by one-way ANOVA followed by Tukey multiple comparison test. *P < 0.05, **P < 0.01 for comparing with the fAblesioned group receiving 0 mg/kg J2326. The red dotted lines indicate the average level of the sham group in each assessment, in which the variation was less than 10%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Compound 3 revealed itself superior effect than undecanol (Fig. S1A); showing that 8-methoxyquinoline core enhanced the neurite outgrowth effect of C11 alcohol. At equal concentration, J2326 owns the highest neurite outgrowth effect as hexacosanol does; evidencing that the addition of 5-chloro substitution on 8methoxyquinoline of compound 3 further enhanced the effect on neurite growth and arborization. The neurite outgrowth driven by J2326 might be mediated through the similar signaling pathways of some long-chain fatty alcohols, as reported as to be cyclic nucleotides (Girlanda-Junges et al., 1988), calcium (Jover et al., 2005), Notch (Gonzalez et al., 2001) or Sema3Acounteracting (Coowar et al., 2004) signaling; all favoring neurite outgrowth. The knowledge-based molecular engineering also let to circumvent the neurotoxic properties associated with clioquinol or the reported fatty alcohols (Gonzalez et al., 2001; Koliatsos et al., 1991; Luu et al., 2000). This makes J2326 more advantageous than some fatty alcohols and clioquinol when treating neurons. Additionally, ERK activation was necessary for the neurotrophic and proliferative effects of J2326 but not p38 activation. This ERK-dependent and p38-independent result of J2326 is distinct from that of other cyclohexenoic alkyl alcohols, the neuritogenic activities of which are dependent on activation of both ERK and p38 (Coowar et al., 2004). This prototype molecule, which generated by conjugating 5-chloro-8methoxyquinoline and undecanol, was validated to be sufficiently enough to encourage neuronal differentiation and proliferation.

4.4. J2326 improves the memory in the AD mice via altering the accumulation of fAb and neurite dystrophy Our in vitro and in vivo neurodegeneration models (Figs. 7 and 8) then indicate that the mechanism underlying the effect of J2326 in fAb-induced neurite degeneration encompasses the simultaneous blockage of advanced dystrophic processes via targeting fAbs and the restoration of synaptic activity via neurite outgrowth (Fig. 7). The outcomes from in vitro neurodegeneration model suggest that J2326 could serve as an effectual treatment in fAb-driven neurodegeneration from acute to chronic phases. These observed performance improvements reflected memory restoration in vivo but not alterations in motor ability (Fig. 8CeE). Long-term treatment with J2326 will be essential to reveal the concerted actions for these anti-degeneration events that protect neurons from death, which is another key process during the development of AD. 4.5. Concluding remarks Based on these biological results, J2326 appears to break down fAb, and its consequences and to evoke neurite outgrowth. The integration of a C11 alkyl alcohol onto 5-chloro-8methoxyquinoline successfully generated a multifunctional agent, which might prevent and reverse the deleterious effects of fAbs both in vitro and in vivo. Of note, these actions of J2326 differ from those of clinically used acetylcholinesterase inhibitors and NMDA antagonists, which only provide transient effects, and neither stimulates


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the regrowth of dystrophic neurites or disaggregates fAbs. J2326 may not only provide therapeutic benefits but also improve the pharmacological efficacy of current drug therapies that alleviate the cholinergic deficiency and restore cholinergic neuronal activity. Our results represent an important step toward developing a more useful treatment that counters the progression of the pathological processes themselves, such as fAb accumulation and neurite collapse, and promotes neurotrophic activities. The conceptual design of J2326 provides an alternatively disease-modifying strategy for the development of other anti-neurodegenerative agents by targeting fAb-like misfolded proteins and neurite collapse. The future optimizations on the neurotrophic and fAb-disaggregating scaffold of J2326 will be assessed and further investigated experimentally, and will improve the outlook. Acknowledgments The authors thank Tsai YF, Tai MY and Wu SC as well as the NTU Animal Center and Department of Medical Research of NTUH for providing experimental facilities. This work was supported by the National Science Council in Taiwan NSC99-2320-B-002-008 and NSC100-2320-B-002-008 (to J.-W.C.) and in part by NTU 99R51101 (to J.-W.C.). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuropharm.2015.01.004. Q4

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Synergic interaction between amyloid precursor protein and neural cell adhesion molecule promotes neurite outgrowth.

The amyloid protein precursor of Alzheimer's disease is a mediator of the effects of nerve growth factor on neurite outgrowth.

A soluble form of the F3 neuronal cell adhesion molecule promotes neurite outgrowth.

HMGB1, a pathogenic molecule that induces neurite degeneration via TLR4-MARCKS, is a potential therapeutic target for Alzheimer's disease.

Mechanosensitive channels in neurite outgrowth.

The changing role of NCAM as a neurite outgrowth-promoting molecule during development.

Ganglioside modulation of neural cell adhesion molecule and N-cadherin-dependent neurite outgrowth.

Amyloid Fibrils from Hemoglobin.

ROCK signaling.

Direct activation of second messenger pathways mimics cell adhesion molecule-dependent neurite outgrowth.

Menthol blocks dihydropyridine-insensitive Ca2+ channels and induces neurite outgrowth in human neuroblastoma cells.

Amyloid fibrils in urine.

Sur8 protein regulates neurite outgrowth.

Enhanced Neurite Outgrowth by Intracellular Stimulation.

Competitive dynamics during resource-driven neurite outgrowth.

Nucleation of polymorphic amyloid fibrils.

Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction.

A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides.

Protein chemistry: catalytic amyloid fibrils.

Intralysosomal formation of amyloid fibrils.

Neurite outgrowth in peripherin-depleted PC12 cells.

Newly designed occluder pin for presacral hemorrhage.

Chirality and chiroptical properties of amyloid fibrils.

Repeated ER-endosome contacts promote endosome translocation and neurite outgrowth.