Peptides NAP and SAL attenuate human tau granular-shaped oligomers in vitro and in SH-SY5Y cells

    Peptides NAP and SAL attenuate human tau granular-shaped oligomers in vitro and in SH-SY5Y cells Farzad Mokhtari, Gholamhossein Riazi, Saeed Balalaie, Reza Khodarahmi, Saeed Karima, Azam Hemati, Bahram Bolouri, Fatemeh Hedayati Katouli, Esmat Fathi PII: DOI: Reference:

S0143-4179(16)30020-8 doi: 10.1016/j.npep.2016.06.005 YNPEP 1731

To appear in: Received date: Revised date: Accepted date:

16 March 2016 29 May 2016 26 June 2016

Please cite this article as: Mokhtari, Farzad, Riazi, Gholamhossein, Balalaie, Saeed, Khodarahmi, Reza, Karima, Saeed, Hemati, Azam, Bolouri, Bahram, Katouli, Fatemeh Hedayati, Fathi, Esmat, Peptides NAP and SAL attenuate human tau granular-shaped oligomers in vitro and in SH-SY5Y cells, (2016), doi: 10.1016/j.npep.2016.06.005

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ACCEPTED MANUSCRIPT Peptides NAP and SAL attenuate human tau granular-shaped oligomers in vitro and in SHSY5Y cells

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Farzad Mokhtari,1 Gholamhossein Riazi,1* Saeed Balalaie,2,3 Reza Khodarahmi 3, Saeed Karima4

Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran

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1

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, Azam Hemati5, Bahram Bolouri6, Fatemeh Hedayati Katouli 1, Esmat Fathi1

P.O Box 131451348

Peptide Chemistry Research Center, K. N. Toosi University of Technology, Tehran, Iran

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2

P.O. Box 158754416

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah,

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3

Iran

Clinical Biochemistry Department, faculty of medicine, Shahid Beheshti University of Medical

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4

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P.O. Box 6734667149

Sciences (SBMU), Tehran, Iran.

5

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P.O. Box 1985717434

Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran

P.O. Box 193954741 6

Department of Biophysics and Medical Physics, Iran University of Medical Sciences, Tehran,

Iran

P.O. Box 1449614525 * Corresponding author: Gholamhossein Riazi (PhD), Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran Tel: +98 21 61112473 Fax: +98 21 66404680 Email:[email protected] 1

ACCEPTED MANUSCRIPT Abbreviations 1N/4R tau: Tau protein isoform with 4R binding region in C-terminal and 1N region in N-

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terminal

ADNP: Activity-dependent neurotrophic protein

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AFM: Atomic Force Microscopy

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CD: Circular Dichroism

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DLS: Dynamic light scattering

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MT: Microtubule

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ADNF: Activity-dependent neurotrophic factor

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NAP: An octapeptide (seq: NAPVSIPQ) SAL: A nonapetide (seq: SALLRSIPA) ThT: Thioflavin T

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ACCEPTED MANUSCRIPT Abstract Accumulation of human tau protein in the central nervous system is an outstanding

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feature of Alzheimer’s Diseases and other tauopathies. Among the aggregate species, granularshaped oligomers are recently reported as toxic species and associated with neuronal loss. Many

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interests have been focused on the reducing of these neurotoxic oligomers in tauopathies. It has been approved that peptides NAP (NAPVSIPQ) and SAL (SALLRSIPA), snipped from activity-

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dependent neurotrophic protein (ADNP) and activity-dependent neurotrophic factor (ADNF), protect brain against a wide range of toxins in neurodegenerative models. The neuroprotection

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mechanisms of NAP and SAL are under investigation. We report here that the aggregation pathway of human tau protein, in the presence of NAP or SAL, could be affected and moved

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toward the less granular-shaped species. Fluorophore binding assays, kinetic parameters and circular dichroism records, showed the presence of unusual aggregated species upon treatment

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with NAP or SAL, as well as hydrophobicity studies. Also, sizing and morphological investigation of aggregates, using dynamic scattered light and atomic force microscopy,

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elucidated a significant reduction of granular-shaped oligomers under 50 nm. Furthermore,

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immunospecific detection of oligomers in SH-SY5Y cell line, using flow cytometric assessment supported our results. We conclude that one of the possible mechanisms of neuronal protection by NAP or SAL could be the attenuation of granular-shaped oligomeric tau under stress condition.

Keywords: Tauopathy, NAP, SAL, Tau protein aggregation, Neurotoxicity, Granular-shaped oligomers

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ACCEPTED MANUSCRIPT 1. Introduction Tau protein isoforms, which are mainly express in neurons, are considered as

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microtubule-associated proteins and are crucial for microtubule (MT) dynamics and integrity in vivo and in vitro (Gendron and Petrucelli, 2009). It has been revealed that axonal transport and

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synaptic viability depends on functional monomers of these isoforms (Billingsley and Kincaid, 1997; Michaelis et al., 2002). According to presence or absence of two amino terminal inserts

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(N) and microtubule binding repeats (R), tau exists in six forms (Mandelkow and Mandelkow,

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2012).

Under chronic pathological condition(s) tau becomes abnormally accumulates and causes

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MT based axonal transport defect and neuronal loss (Himmelstein et al., 2012). Tau aggregates, commonly known as neurofibrillary tangles (NFTs), are hallmark of various neurodegenerative states such as Alzheimer’s disease (AD) (Alonso et al., 2001; Michaelis et al., 2002). Recent

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evidences indicate that tau could aggregate in different neurotoxic oligomers or filamentous

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species (Patterson et al., 2011; Ross and Poirier, 2005). Noteworthy, neurotoxicity is due to the presence of granular-shaped oligomers with low molecular weight and high -sheet content

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(Lasagna-Reeves et al., 2012b; Sahara et al., 2008; Wu et al., 2013). Extracellular release via cell membrane tunneling is proceed by elevated levels of these oligomers in prefrontal cortex of Braak stage I in AD and cerebrospinal fluid of animal models (Meraz‐Ríos et al., 2010). Granular-shaped oligomers are visible as diametrically distributed 5-50 nm particles under Atomic Force Microscopy (AFM) tip in primary stages of aggregation (Gerson and Kayed, 2013; Guzmán-Martinez et al., 2013; Lasagna-Reeves et al., 2012b; Ren and Sahara, 2013; Sahara and Avila, 2014). Accordingly, some researches have focused on the reducing of granular-shaped oligomeric population and toxicity (Ballatore et al., 2007; Maeda et al., 2006). Activity-dependent neurotrophic factor and protein (ADNF and ADNP) are proteins that co-localized with MT in the stress conditions (Gozes et al., 2003; Gozes et al., 2013; Mandel and Gozes, 2006). Neuroprotection against various toxins and MT stabilization, have evoked researchers to find agonist peptides with ADNF and ADNP mimicry properties (Bassan et al., 1999; Brenneman and Gozes, 1996; Oz et al., 2012; Smith-Swintosky et al., 2005). It has been demonstrated that peptides NAP and SAL have such characteristics (Gozes et al., 2000; 4

ACCEPTED MANUSCRIPT Matsuoka et al., 2006). Octapeptide NAP (NAPVSIPQ) and nonapetide SAL (SALLRSIPA) are snipped from ADNP and ADNF respectively. In addition to the neuroprotective impacts (Gozes et al., 2004; Greggio et al., 2011; Shiryaev et al., 2009) , NAP could interact with MT and

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protects neurons in the face of tau deficiency (Anand et al., 2014; Gozes et al., 2014a; Magen and Gozes, 2013; Oz et al., 2014). Reduction of tau phosphorylation and functional

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compensation of abnormal tau in tauopathy models are well defined by NAP (Divinski et al.,

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2006; Quraishe et al., 2013; Vulih-Shultzman et al., 2007). Several experiments supporting that SAL could keep neurons alive in response to the various insults such as amyloid beta and free

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radical toxicities (Gozes et al., 2013; Kita, 2006; Sari, 2006). It seems that SAL and NAP have similar functional outcomes via different mechanisms. Chronic administration of SAL has been

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shown to protect ADNP knocked-out mice (ADNP+/- mice) against tau hyperphosphorylation. (Shiryaev et al., 2011).

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As stated above, reducing the concentration of granular-shaped tau oligomers could decline neurotoxicity and maintain normal neuronal function (Guzmán-Martinez et al., 2013). On

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the other hand, the reports have been confirmed the important role of NAP and SAL in

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tauopathies in vivo and in vitro (Magen and Gozes, 2013; Shiryaev et al., 2009). In the current study we hypothesized that NAP and SAL could decrease the population of granular-shaped tau oligomers in the stress condition. Because among the tau isoforms 4R being overproduced in most tauopathies (Ballatore et al., 2007; Panda et al., 2003), here we studied the tau (1N/4R) aggregation kinetic parameters and structural specification of aggregates in the presence of NAP or SAL in vitro. Toxicity and population of granular-shaped species of tau was examined in SHSY5Y cell line as a neuronal model. 2. Materials and methods 2.1.Chemicals ANS (8-anilino-1-naphthalene sulfonic acid), Thioflavin T (ThT), Dithiothreitol (DTT) and Heparin (MW=6 kD), were purchased from Sigma-Aldrich (Deisenhofen, Germany). PerfectPro Ni-NTA Agarose was prepared from 5Prime (Hamburg, Germany). ABN454, AntiTau (T22) oligomeric antibody was obtained from EMD Millipore, (USA). All other chemicals including phenylmethylsulphonyl fluoride (PMSF), Imidazole, Congo Red (CR) were purchased

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ACCEPTED MANUSCRIPT as analytical grade from Merck (Darmstadt, Germany). The htau34-pET21-NHis construct, which encodes 412 amino acids, was kindly provided by Dr. MA. Nasiri (University of Tehran). 10 mM HEPES buffer, pH 7.4, containing 100 mM NaCl was used for in vitro experiments.

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Deionized nanopure water, without trace cations and contaminants, was applied for making

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solutions.

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2.2.Expression and purification of recombinant human tau (1N/4R) The pET21a(+) vector containing human tau 1N/4R (htau34) cDNA with N-terminal His-Tag

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were transformed to E. coli BL21 (DE3). Five mL of the overnight starter culture was added to 500 ml of Luria Broth (LB) culture medium and let OD600 reached 0.7-0.9. Expression was

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induced by 1 mM IPTG and grown 3 hours at 37C, 250 rpm. SDS-PAGE and immunoblotting was used for expression confirmation. Next, the remaining cells were harvested by centrifugation 4200 g, 15 min, and lysed in 100 mM PBS, 10 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, pH 8

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lysis buffer. The lysates were coupled to Ni-NTA agarose resin and eluted gradually for affinity

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precipitation in successive manner. htau34 was obtained from the beads in the presence of 80

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mM imidazole and formulated as monomer in 10 mM HEPES, 1 mM DTT, pH 7.4 buffer. Purity of htau34 was assessed by 12% SDS-PAGE following Coomassie Brilliant Blue staining. Moreover, the protein was verified by western blot using HRP conjugated rabbit polyclonal antibody (pAb) against 6XHis tagged end, 1:5000. The concentration of htau34 was measured by Lowry and Bradford assays and correlated with UV absorption at 280 nm using extinction coefficient of 7450 cm-1M-1 (Karima et al., 2012). The concentration of purified htau34 was adjusted to 20 µM and stored in -70C for and used for htau34 aggregation experiments. 2.3.Peptide synthesis, purification and mass validation Synthesis of selected peptide NAP (NAPVSIPQ) and SAL (SALLRSIPA) were carried out using 2-chlorotrityl chloride resin, 1.0 mM, following the standard Fmoc strategy. The loading capacity was determined by weight after drying the resin under vacuum and was 1.0. Fmocamino acid binding to resin, treating with 25% piperidine-DMF and coupling process was done by repetitive washing with DMF. The coupling was repeated as in the same way for all amino 6

ACCEPTED MANUSCRIPT acids of selected sequence. In all cases for the presence or absence of free primary amino groups, Kaiser Test was used. Fmoc determination was done using UV spectroscopy method. Produced peptides were cleaved from resin by treatment of TFA 1% in DCM and neutralization with

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pyridine 4% in MeOH. The solvent was removed under reduced pressure and precipitated in water, filtered and dried. Final deprotection was done using TFA 95% and reagent K

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(TFA/TES/Water 95:2.5:2.5). The desired peptides were precipitated in diisopropyl ether.

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Purification was done using preparative HPLC Knauer, 250 ml/min pump head, C18 column and water: acetonitrile 30:70 as eluents. The collected fractions were lyophilized by Christ α-2

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freeze dryer. The purification of fractions was studied by Knauer analytical HPLC. The structure of peptide was confirmed using ESI mass spectrometry (LC/Mass Agilent triple quadrupole

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2.4.Aggregation of htau34 in vitro

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6410). The m/z values range was from 100-2000.

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Monomeric htau34 was incubated triplicate with 20 μg/ml heparin into Grenier solid black 96 microwell polyester plate at 37C for 180 h without agitation (10 mM HEPES, pH 7.4, 3mM

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DTT). The plate was covered with self-adhesive foil to avoid evaporation and light exposition during the aggregation. Aggregation process was monitored using 50 μM ThT (Chirita et al., 2005; Rankin et al., 2005). The fluorescence intensity of ThT was measured every 3 hours by multimode reader Synergy H4 (Biotek Instruments, Winooski,VT). Excitation and emission wavelengths were adjusted to 440 and 495 nm respectively. NAP and SAL were pre-incubated (1, 5, 10 or 50 μg/ml) with htau34 at room temperature, 4 h and 50 rpm shaking for intervention experiments. The kinetic parameters of htau34 aggregation were studied by fitting ThT fluorescence intensity to amyloid formation sigmoidal curve which described in equation 1 by correlation coefficient of 0.97 (Bandyopadhyay et al., 2007). Eq. 1

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Where Y is the fluorescence intensity, t is time, y stands for maximum and yo for initial fluorescence intensity. Therefore, the kapp presents the apparent rate constant of aggregation growth.

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ACCEPTED MANUSCRIPT 2.5.Circular Dichroism analysis Secondary structure and conformational changes of htau34 aggregates were analyzed by

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Far-UV CD spectra in the presence or absence of NAP and SAL. The samples were diluted 1:5 in HEPES buffer pH 7.4 and data were taken in a 0.1 cm path length cuvette, using an Aviv

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model 215 Spectropolarimeter (Lakewood, NJ, USA). The spectrum was recorded in the range of 195-260 nm with a data interval of 1 nm at 25C. Each spectrum was an average of two scans,

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with a subtraction of buffer and peptide baselines. CD signals (obs millidegrees) were converted

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to delta epsilon () for secondary structure prediction using equation 2: Eq. 2 =obsMRW/10.c.l.3298

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Where MRW is the mean residual weight of the protein (MW/number of amino acids in the sequence), c and l stand for concentration (mg/ml) and path length (cm). Curves were analyzed

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using CD spectra deconvolution software (CDNN) version 2.1.

size

of

htau34

aggregate

species

was

measured

using

ZetaPlus

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The

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2.6.Dynamic light scattering (DLS) analysis

zeta potentialanalyzer (Brookhaven, USA). Mean radius (Matsuoka et al.) of diluted samples (1:10) were calculated from five assesses, following the placement of samples in optical glass fluorometer cuvettes and equilibration at 25 °C for 2 min. According the Stokes–Einstein equation (equation 3), RH was determined and categorized in four groups (0-25 nm, 25-100 nm, 100-200 nm and more than 200 nm). Eq. 3 Where k, T,

and D stand for Boltzmann constant, temperature, solvent viscosity and Brownian

motion respectively. The molecular weight of htau34 aggregate species was calculated according to the Mark– Houwink–Sakurada equation.

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ACCEPTED MANUSCRIPT 2.7.Monitoring of hydrophobicity by ANS florescence During the aggregation process in the presence or absence of NAP and SAL, samples were

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collected at different time intervals (2, 4, 24, 48 or 72 h) and hydrophobicity of htau34 aggregated species was measured by obtained emission data using multimode reader Synergy H4

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(Biotek Instruments, Winooski,VT), following incubation with 40 µM ANS for 1 h. The

2.8.Atomic Force Microscopy (AFM) imaging

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emission spectra recorded between 400 to 600 nm upon excitation at 350 nm.

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The structure and distributed population of htau34 granular-shaped oligomers were observed by quantitative Atomic Force Microscopy (AFM) by ARA-AFM, Ara-Research Company

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(Anand et al.). Imaging were conducted at room temperature in tapping mode. All samples were prepared by dropping 1 µL of aggregates, in the presence or absence of peptides, onto freshly

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cleaved highest grade V1 mica (Ted Pella Inc., Redding, CA), followed by washing with 500 L

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buffer after 10 min. HQ:NSC36/Cr-Au BS silicon probe Lever C with a typical force constant of 0.6 N/m and resonance frequency of 65 kHz from Mikromasch Company was employed. Images

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were processed using Imager version 1.01, Ara Research Company (Anand et al.) and size of each particle was measured consequently. 2.9.Induction of tau aggregates in SH-SY5Y cells SH-SY5Y neuroblastoma cells obtained from Pasteur Institute of Iran were cultured in 160 ml culture flask (Nunc™, Danmark) in DMEM+Ham's F12 medium (1:1), supplemented with 20% FBS and 1% PenStrep, all from Gibco, Canada. Cells maintained in a humidified chamber, 37C and 5% CO2. All experiments were performed at 80% confluency and on a monolayer 6 well culture plate (Corning Costar, Corning, NY, USA). Induction of tau aggregation in the SHSY5Y cells was performed as described earlier with a minor modification. (Lira-De Leon et al., 2013). Briefly, cells with density of 500,000 per dish were exposed to 100 µM Congo Red (CR) up to five days. At the third day, half of the total medium was replenished with the respective of CR concentration. Simultaneously, samples were treated with NAP and SAL (1, 5, 10 or 50 gr/ml) and incubated for 48 h.

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ACCEPTED MANUSCRIPT 2.10.

Determination of tau oligomers in SH-SY5Y cells

Induced

tau

oligomers

were

detected

by

FACS

flow

cytometry,

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Partec CyFlow space flow cytometer (Partec GmbH , Germany), using intracellular antigen staining by anti-tau oligomer antibody-T22 (EMD Millipore, USA) (Hawkins et al., 2013).

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Concisely, cells were permeabilized with 0.2% Tween-20 by three wash for 15 min, following detachment with 0.25% trypsin-EDTA, washing and re-suspension in PBA (PBS 1X, BSA 0.1%)

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and fixation with 4% paraformaldehyde for 10 min at room temperature. Next, cells were incubated in 1x PBS, 10% normal sheep serum and 0.3 M glycine to block non-specific protein-

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protein interactions for 30 min. Cells were incubated with prepared T22 antibody in PBS including 0.2% tween-20 and 1% BSA (1:500) overnight at 4C. Diluted Rabbit IgG antibody

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(1:500) was used as isotype control under the same conditions. After that, the cells were incubated with FITC-sheep anti-rabbit secondary antibody (1:500) for 1 h in the dark room.

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After washing with PBS, cells were monitored by flow cytometer blue beam. Data were analyzed

Detection of cytotoxicity of NAP and SAL treated samples

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2.11.

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with FlowJo version 7.6.1 software (TreeStar, Ashland, OR, USA).

Cell toxicity of generated species in the presence of NAP and SAL peptides was determined using the conventional MTT colorimetric assay. 96-multiwell plates were seeded at 10,000 cells per well. PC12 Cells were cultured similar to conventional method and after serum-starvation for 4 h, were incubated with the 50 µL of peptide-treated samples. After 24 hours cells were incubated with 5mg/mL MTT for 4 h at 37◦C. Then, the medium was removed and the cells were dissolved with 100 µL dimethylsulfoxide with 15 minutes of shaking. The formazan reduction was measured by reading absorbance at 570 nm.

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ACCEPTED MANUSCRIPT 2.12.

Statistical analysis

Data were adjusted to appropriate equations and graphed by GraphPad Prism Version 5

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(GraphPad Software Inc. San Diego, CA, USA). All linear and non-linear regression fits were calculated as  S.E of the estimates. The significance of assays was analyzed by t-test and P

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value < 0.005 was considered as statistically significant.

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3. Results

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3.1.Expression and purification of htau34

The purity of expressed htau34 was checked in the purification steps and finally confirmed using SDS-PAGE and western blotting methods. As shown in Fig. 1A and Fig.1B, single band at

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about 52 kD was correlated to htau34 (Fig. 1A, lane 5). Immunodetection of 6x His tagged end by HRP conjugated rabbit pAb, showed the existence of monomeric htau34 (Fig. 1B, lane 2).

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3.2.Mass validation of synthetic peptides NAP and SAL Purified peptides, by preparative HPLC, were analyzed using ESI-MS for the mass

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confirmation for NAP (Fig. 1C) and SAL (Fig. 1D) after protonation and fractionation. Deconvoluted ESI-MS spectrum demonstrated an observed m/z of 825.5 ([M+H]+) for NAP and 926.6 for SAL ([M-H]-) as main peaks. Calculated masses were matched with observed ones. Results are summarized in Table 1. As for SAL, arginine residue fragment (R 113.1) and other fragments (y3 385.1, a5-R 433.1) were nomenclature as proposed by Roepstorff and Fohlman (Roepstorff and Fohlman, 1984).

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(MW: 824.5)

[M+H]+

825.5

[M+Na]+

847.5

[2M+H]+

1650

[M-H][M-2H-R]SALLRSIPA

824.5

926.5 812.4

R (Arginine residue)

113.1

y3

385.1

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(MW: 927.5)

Calculated masses

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Observed m/z

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NAPVSIPQ

Ion

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Peptides Sequences

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Table 1 Calculated masses and observed m/z of peptides NAP and SAL fragments.

433.1

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D

a5-R

927.5

3.3.Aggregation of htau34 in the presence or absence of peptides in vitro Normally, polyanionic trigger molecules such as heparin are used for inducing the formation of tau protein fibrils in many studies. Subsequently, fluorescence emitted spectra of Thioflavin dyes (ThS or ThT) are employed for detection of amyloid contents of them (Chirita et al., 2005; Rankin et al., 2005). It has been reported that three phases involved in fibril formation including nucleation or lag phase (assembly of monomers), elongation (formation of oligomer and subsequent fibrils) and equilibrium (saturation) (Lomakin et al., 1996; Teplow, 1998). Here, ThT was used for monitoring htau34 time-dependent aggregation and an increase in the intensity of ThT emission was correlated to fibril formation in 37C without agitation. (Fig. 2A and B). As shown in Fig. 2A achievement to fibril generation without a significant lag phase was clear in NAP-treated samples after 50 hours. Fibrillization of htau34 in the presence of 1, 5, 10 or 50 g/ml of NAP decreased in an inverse concentration-dependent manner (Fig. 2A). In contrast,

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ACCEPTED MANUSCRIPT fibril formation of htau34, in the presence of SAL, did not obey the general patterns of the normal phases (see Fig. 2B).

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An increase of about 75% in the maximum ThT emission intensity was observed for the samples treated with 1g/ml NAP. Conversely, a decrease of about 30% was seen in the case of

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5 or 10 g/ml SAL (Fig 2A and B). The results proposed that aggregation of htau34 in NAP and SAL treated samples may have stemmed from the formation of fibrillary species in different

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conformations.

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To identify the changes of htau34 aggregation propensity in the presence or absence of the peptides, kinetic parameters were calculated and rate constant (kapp) has been reported in Table 2.

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Using equation 1 kapp, as the rate constants, was calculated. Data showed that the rate of htau34 filament growth drastically increased and was 3 times faster in the presence of 1g/ml NAP (kapp(control)= 0.021 h-1, kapp(1g/ml

NAP)=

0.068 h-1). In a consistent manner, other NAP-treated

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samples exhibited an increase of about 40% (kapp(5g/ml

NAP)=

0.03 h-1), (see Table 2).

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Subsequently, SAL-treated samples showed a decrease in kapp of about 20 % and 14 % in the presence of 5 and 50 g/ml (kapp(control)= 0.011, kapp(5g/ml SAL)= 0.0088 h-1, kapp(50g/ml SAL)= 0.0094 h). Therefore, the presence of NAP or SAL revealed a significant impact on htau34 fibrillization

propensity

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Table 2 First-order rate constants (h-1), kapp, of htau34 aggregations in the presence or absence of peptides NAP or SAL.

htau34+Peptides (g/ml) 0

1

5

10

50

NAP

0.021

0.068

0.031

0.020

0.011

SAL

0.011

0.010

0.0088

0.0086

0.0094

3.4.The changes of htau34 secondary structure assemblies As depicted in Fig. 2C and D, a typical CD spectra, with a minimum ellipticity degree at 200 nm and a positive value at 220 nm, was recorded for monomeric htau34 (Uversky et al., 1998), and a shift in minimum ellipticity degree at 220 was seen for aggregated htau34 under 13

ACCEPTED MANUSCRIPT mentioned conditions. This shift is commonly referred to secondary structures with rich betasheet content. The different concentrations of peptide NAP, led to a significant decrease of ellipticity degree at 220 nm besides an emergent shoulder at 200 nm, proposing a profound

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change from conventional amyloid fibrils to other polymorphic assembled structures (24.2 % beta-sheet , 38.1 % random coil), (Fig. 2C). It is noteworthy that according to the earlier

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published data, the ellipticity degree decrease at 220 nm, could pertain to the conversion of

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toxic amyloid structures to nontoxic beta-sheet rich fibrils in the presence of specific molecules (Bieschke et al., 2012). In compare to NAP, SAL-treated samples showed more decrease in

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minimum ellipticity degree at 220 nm and a disappearance of the shoulder at 200 nm has been represented in Fig. 2D. According to the relation of the ratio of random coil/beta-sheet structure

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to the ratio of ellipticity degrees of 200 nm/220 nm, we assumed that in the presence of both peptides, generated assembly species are differing from known toxic amyloid secondary

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D

structures. We summarized the deconvoluted data in Table 3.

Table 3 Secondary structure prediction by CDNN software of htau34 aggregates in the presence or absence of different concentration of peptides NAP or SAL. htau34+NAP

htau34+SAL

(g/ml)

(g/ml)

Secondary Structure

0

5

10

50

5

10

50

Helix (%)

35.01

4.48

7.67

5.65

7.83

8.68

7.92

Antiparallel beta sheet

12.38

24.29

16.68

12.28

24.93

23.60

22.12

7.90

12.56

8.90

6.55

13.62

13.52

14.28

(%) Parallel beta sheet (%)

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ACCEPTED MANUSCRIPT 3.5.Size distribution of aggregates in the presence of NAP and SAL Average hydrodynamic radius (RH) of aggregates was determined by recording dynamic scattered light intensity and sizes were categorized and are given in Table 4. Previous in vitro

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studies have indicated that the size of oligomeric, intermediate aggregates, pre-filaments and large fibrils are less than 25 nm, between 25-100 nm, between 100-200 nm and more than 200

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nm respectively (Maeda et al., 2006; Patterson et al., 2011; Ren and Sahara, 2013). Interestingly,

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as graphed in Fig. 2E and F, oligomeric species with the RH less than 25 nm were negligible in peptides treated samples. The average content of these species are 15% in non-treated ones.

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Accordingly, intermediate species are also present in low population (Table 4). Instead, the population of larger htau34 assemblies has been raised in the presence of NAP and SAL.

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However, 1 or 5 µM SAL did not induce the formation of any htau34 aggregate species with RH between100-200 nm. Calculated molecular weight of aggregate species, using Mark–Houwink– Sakurada equation, represented in Table 5.

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Table 4 Size categories (average RH) of the htau34 (20 µM) aggregates, measured by dynamic

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scattered light, in the presence or absence of peptides NAP or SAL in various concentrations.

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Data are representative of five independent experiments (P value < 0.005). htau34+NAP (g/ml)

htau34+SAL (g/ml)

RH

0

1

5

10

50

1

5

10

50

< 25 nm

15%

0

0

0

0

2%

2%

0

0

25-100 nm 15%

0

4%

0

5%

3%

3%

0

0

0

0

0

100-200 nm 10% 30% 40% 25%

10% 15%

> 200 nm 60% 70% 56% 75% 95% 95% 95% 90% 85%

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Molecular Weight (kDa 10 ) 5

htau34+NAP (g/ml)

htau34+SAL (g/ml)

Ctrl

1

10

50

1

10

50

1.62

17.3

18.6

15.1

8.19

212

37.6

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Table 5 Molecular weight of various tau aggregates in the presence or absence of 1, 10 and 50

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g/ml of NAP and SAL. Values were calculated using Mark–Houwink–Sakurada equation.

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3.6.Hydrophobic surface detection of htau34 aggregated species by ANS Hydrophobicity level of aggregated species was determined with ANS as a fluorescent

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probe of surface-exposed hydrophobic patches (Gasymov and Glasgow, 2007). Hydrophobic content of formed species, as an index of solution availability, were declared in the time intervals, after aggregation start (detailed in materials and methods). In the absence of NAP or

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SAL, htau34 displayed a time-dependent increase in the maximum emission of ANS after 72

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hours (2 h 2709, 4 h 2770, 24 h 4004, 48 h 4208, 72 h 4456 AU), as showed in Fig. 3A. In contrast, increase of maximum emission of ANS-bound htau34 aggregates, did not follow the

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above pattern in the presence of NAP or SAL. Noticeably, the maximum emissions of ANSbound htau34 aggregates were overlapped in the various peptides concentration at 24, 48 and 72h (Fig. 3B, C, D and E). The representative data for 5, 10 or 50 g/ml NAP are 4036, 4050 and 4058 AU (Fig. 3C). As for SAL, these data are 5067, 5057 and 4993 AU (Fig. 3E).

3.7.AFM micrograph of htau34 aggregated forms AFM imaging was employed for morphological study of htau34 aggregated species (Fig. 4). Fig. 4A represents htau34 aggregate shapes in the absence of peptides. For htau34, granular oligomers and fibrillar aggregates were dominant forms, in consistent with DLS data. The diameter of granular-shaped oligomers varied in the range of 5-100 nm and 355 nm was the maximum width for a single fibril (Fig. 4A). Surprisingly, in 5 µg/ml NAP treated samples, granular shaped oligomers (<100 nm) were disappeared and thickened (>200 nm), branched and elongated species were obvious (Fig. 4B). In the presence of higher concentration of NAP (50 µg/ml), intercalated and amorphous forms were predominant (image not shown). In similar to 16

ACCEPTED MANUSCRIPT NAP treated samples, absence of granular-shaped oligomers was clear too, for SAL treated ones. Annular forms (diameter less than 1000 nm), are the main assembled forms of htau34 in the

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presence of 5 µg/ml SAL (Fig 4C).

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3.8.Tau aggregation response to the NAP or SAL in SH-SY5Y cells

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The role of peptides NAP or SAL in the formation of tau oligomers were investigated by immunoreactivity in SH-SY5Y cell line. Existence of tau oligomers was checked using anti-tau

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oligomer antibody T22 and FACS flow cytometry method in CR-treated cells (Fig. 5). The T22 antibody does not recognize the monomer or fibril form of the tau protein. As recorded in Fig.

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5B, the CR-treated SH-SY5Y cells presented 82% immunoreactivity in the absence of peptides than the isotype control (rabbit IgG antibody) (Fig. 5A). In respect to the flow cytometry records and in a similar manner, immunoreactive responses have been declined about 20% for 50 g/ml

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NAP or 5 g/ml SAL treated cells (Fig. 5C and D). As a significant immunospecific binding of

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anti-tau oligomer T22 and granular oligomers formed within the SH-SY5Y cell, the results demonstrated that the population of granular-shaped tau decreased significantly in response to 50

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g/ml NAP and 5 g/ml SAL.

3.9. Toxicity assay of aggregated species As shown in Fig. 6, when PC12 cells were exposed to NAP or SAL treated samples, generally the cell viability was increased in compare to htau34 oligomer treated cells after 24 hours. The diagrams indicate the prevention of cell growth in the treated samples with htau34 aggregates. Conversely, NAP and SAL treated htau34 aggregates, increased the viability of the cells. The observed protective effect for NAP and SAL treated samples was 20±3% and 35±2% respectively.

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ACCEPTED MANUSCRIPT 4. Discussion Intracellular deposition and chronic increase of NFTs in the specific area of brain, is a

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pathological marker of AD and other tauopathies (Goedert and Spillantini, 2006; LasagnaReeves et al., 2012a; Maeda et al., 2006). Among tau aggregated species, studies emphasized

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that appearance of granular-shaped oligomers, which precedes the accumulation of NFTs, are neurotoxic and elevates in the stages of AD (Lasagna-Reeves et al., 2010; Lasagna-Reeves et al.,

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2012b; Sahara et al., 2008), thereby implicating the importance of granular-shaped intermediates (Haroutunian et al., 2007; Polydoro et al., 2014). As a therapeutic target, growing researches

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attempt to reduce the population and eventually the neurotoxicity of these oligomers. An inverse correlation between granular-shaped oligomers and some heat shocked protein (Hsp) levels,

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suggesting a protective role of Hsp against granular-shaped oligomers toxicity (Sahara et al., 2007). Additionally, immunosuppressive treatment could also decrease soluble oligomers level. Targeting oligomeric tau with anti-tau oligomer-specific mouse monoclonal antibody (TOMA),

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has been shown to be an effective mean for prevention and reverse of tauopathy caused cognitive

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deficits, supporting the potential of oligomeric tau as a candidate therapeutic target for AD

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(Castillo-Carranza et al., 2013; Castillo-Carranza et al., 2015). In the recent years, interests have been devoted to peptides NAP and SAL, derived from ADNP and ADNF proteins, due to their neuroprotective properties against neurotoxicity associated with tauopathy. Details are reviewed by Shiryaev (Gozes et al., 2005; Shiryaev et al., 2011). The protective mechanism of NAP and SAL is unclear and under intense investigation. Previously, it has been reported that NAP reduces tau pathology via elevation of tau monomeric fraction and reduction of hyperphosphorylated tau with in vivo models (Gozes et al., 2014b; Magen et al., 2014; Matsuoka et al., 2008; Shiryaev et al., 2009). Furthermore, several efforts have been demonstrated the protection ability of NAP and SAL by decreasing the aggregation of the amyloid beta peptide species and cytotoxicity (Ashur-Fabian et al., 2003; Guo et al., 1999). The active core of SAL and NAP exhibits structural and functional similarities (Bassan et al., 1999). Here, we extended the studies and for the first time, present the finding of the NAP and SAL ability to decrease the toxic tau oligomers. Emissions obtained upon ThT binding experiments, showed a significant increase in mature fibril formation phase for NAP. In contrast, 18

ACCEPTED MANUSCRIPT in the presence of SAL, fibrillization was reduced in the factor of one-third, in compare to the control. Additionally, kapp increased up to three times for NAP and decreased 20% for SAL treated samples. According to accumulating evidences, different and distinct pre-fibrillar

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structures could generate fibrillary forms on independent routes and distinguishable kinetic parameters (Necula et al., 2007). Our results indicate a distinct aggregation pathway and kinetics,

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following NAP and SAL intervention, suggesting the generation of different htau34 aggregated

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intermediates preceding fibril formation.

We next characterized the secondary structure status of htau34 aggregates. The ratio of

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random coil to beta sheet conformations, in the presence of peptides, demonstrated that classical conformation of beta-sheet rich content of htau34 aggregates moved toward the transition of

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beta-sheets to random coils. It has been confirmed that this transition is a common phenomenon for beta-sheet disruption in the presence of specific peptides (Soto et al., 1996). Then, we can confirm that assembled structures, in the presence of NAP and SAL, don not have the usual

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amyloid forms.

We found near identical hydrophobic surface properties between NAP and SAL treated

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htau34 in 24,48 or 72 hours after initiation process. However, as described in the results section, the hydrophobicity of htau34 aggregated species, was distinct and showed a time-dependent increase. Accordingly, it could be suggested that after 24 hours, generated species in response to NAP or SAL are mostly uniform.

Using Dynamic light scattering we showed a significant disappearance of the particles under 100 nm in peptide-treated samples which presumably correspond to the oligomeric species. Considering all RH values, given in table 4, one would expect an increase in the amount of larger aggregated species in the presence of peptides. To learn the impact of NAP and SAL on htau34 aggregates structure and morphology, we conducted AFM imaging analysis. Performing AFM, granular-shaped oligomers and fibril structure could be observed (Ren and Sahara, 2013). Obviously, thickened and branched species with negligible number of granular-shaped ones, were detected in response to the presence of NAP, but in response to the presence of SAL, main assembled forms were annular. We would suggest a clear reduction of granular-shaped aggregated species in response to the both peptides. Seemingly, NAP and SAL 19

ACCEPTED MANUSCRIPT can introduce a change to the aggregation pathway toward the generation of special and unusual forms rather than granular-shaped oligomers.

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In the subsequent step we aimed to check whether NAP or SAL would change the population of oligomeric tau in SH-SY5Y cell line. Using anti-tau T22 sensitive

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immunodetection against tau oligomers in peptides treated cells, followed by flow cytometric assays, elucidated a 20% decrease of tau oligomers population than untreated cells. Moreover,

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dead cells region was excluded from NAP and SAL treated cells. Based on cellular dot plots, and in consistent with our previous in vitro results, we also suggest that NAP and SAL may be able

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to decline the tau oligomers within cellular model as well as cell toxicity (Fig. 5 and 6).

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In conclusion, the presented data could support the hypothesis that peptide NAP attenuate granular-shaped tau oligomers in vitro and in the neural cells. Cause of intrinsic hydrophobic nature, NAP may interact with hydrophobic patches of small aggregates and push them toward

confer

beta

sheet

breaking

characteristics

to

NAP.

Then

neither normal

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which

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the formation of larger assemblies. As shown in NAP sequence, it contains proline residues

fibrillar nor oligomeric species is stable in the presence of NAP. Figure 7 represents a schematic

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pathway of this formation. As for the peptide SAL, the homology between its sequence (SALLRSIPA) and a chaperonin-like motif of Hsp60 (CALLRCIPA), (Brenneman and Gozes, 1996), suggesting a chaperon like activity for SAL and subsequent decrease of granular-shaped oligomers.

5. Conclusions

In summary, we conclude that peptides NAP and SAL can attenuate granular-shaped tau oligomer in vitro and in cellular model and our finding support the role of these peptides in neural protection and pave the way for exploring the NAP and SAL molecular mechanism of neuroprotection action in deeper way.

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ACCEPTED MANUSCRIPT 6. Conflict of interest The authors declare no conflict of interest

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7. Acknowledgments

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This work was supported by research grants from the University of Tehran, Institute of Biochemistry and Biophysics (IBB). We are grateful to Dr. Jesus Avila (Universidad Autonoma

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de Madrid, Spain) for tau (1N/4R) construct. The authors would like to thank Dr. Lornejad,

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Dr.Shahmoradi and Mr. Akbari for their precious help in AFM imaging.

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8. References

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Sahara, N., Maeda, S., Yoshiike, Y., Mizoroki, T., Yamashita, S., Murayama, M., Park, J.M., Saito, Y., Murayama, S., Takashima, A., 2007. Molecular chaperone mediated tau protein metabolism counteracts the formation of granular tau oligomers in human brain. Journal of neuroscience research 85, 3098-3108. Sari, Y., 2006. Peptides derived from activity-dependent neurotrophic factor and activitydependent neuroprotective protein protect the developing brain against the insult of prenatal alcohol exposure in C57 mice. Neuropeptides 40, 432. Shiryaev, N., Jouroukhin, Y., Giladi, E., Polyzoidou, E., Grigoriadis, N.C., Rosenmann, H., Gozes, I., 2009. NAP protects memory, increases soluble tau and reduces tau hyperphosphorylation in a tauopathy model. Neurobiology of disease 34, 381-388. Shiryaev, N., Pikman, R., Giladi, E., Gozes, I., 2011. Protection against tauopathy by the drug candidates NAP (davunetide) and D-SAL: biochemical, cellular and behavioral aspects. Current pharmaceutical design 17, 2603-2612. Smith-Swintosky, V.L., Gozes, I., Brenneman, D.E., D’Andrea, M.R., Plata-Salaman, C.R., 2005. Activity-dependent neurotrophic factor-9 and NAP promote neurite outgrowth in rat hippocampal and cortical cultures. Journal of molecular neuroscience 25, 225-238. Soto, C., Kindy, M.S., Baumann, M., Frangione, B., 1996. Inhibition of Alzheimer's amyloidosis by peptides that prevent β-sheet conformation. Biochemical and biophysical research communications 226, 672-680. Teplow, D.B., 1998. Structural and kinetic features of amyloid -protein fibrillogenesis: Review. Amyloid 5, 121-142. Uversky, V.N., Winter, S., Galzitskaya, O.V., Kittler, L., Lober, G., 1998. Hyperphosphorylation induces structural modification of tau-protein. FEBS letters 439, 21-25. Vulih-Shultzman, I., Pinhasov, A., Mandel, S., Grigoriadis, N., Touloumi, O., Pittel, Z., Gozes, I., 2007. Activity-dependent neuroprotective protein snippet NAP reduces tau hyperphosphorylation and enhances learning in a novel transgenic mouse model. Journal of Pharmacology and Experimental Therapeutics 323, 438-449. Wu, J.W., Herman, M., Liu, L., Simoes, S., Acker, C.M., Figueroa, H., Steinberg, J.I., Margittai, M., Kayed, R., Zurzolo, C., 2013. Small misfolded Tau species are internalized via bulk endocytosis and anterogradely and retrogradely transported in neurons. Journal of Biological Chemistry 288, 1856-1870.

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ACCEPTED MANUSCRIPT Fig. 1 Confirmation of htau34 expression and purification using (A) 12% SDS-acrylamide gel electrophoresis in the steps of purification, stained with Coomassie Brilliant Blue G-250, (lane 1 and 2) bacterial lysate before and after induction with 1 µM IPTG, (lane 3 and 4) the first and the

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second washing cycle with 15 mM imidazole, (lane 5) eluted fraction with 80 mM imidazole and (B) immunoblotting of 6x-His tagged with HRP conjugated rabbit pAb, (lane 1) recombinant 6x-

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His tagged Fc receptor-like protein as control+, (lane 2) purified recombinant htau34, (lane 3)

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non-transformed control-. Peptides NAP and SAL mass validation, (C) NAP mas spectrum in

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ESI+1 charge state, (D) SAL mass spectrum in ESI-1 charge state.

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Fig. 2 Changes in htau34 aggregation kinetics, secondary structures and size distribution in the

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presence or absence of peptides NAP or SAL in vitro. (A, B) Time-dependent, non-agitated, htau34 (20 M) aggregation through recording of fluorescence intensity of ThT, induced by

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heparin (20 g/ml), in the absence (__) or presence of 1 (), 5 (▲), 10 () or 50 g/ml (▼) of peptides NAP or SAL (10 mM HEPES, pH 7.4, 3 mM DTT, 37°C, 180 h). (C, D) Far-UV circular dichroism spectra of htau34 (20 M) in the monomeric and aggregated states in the absence (○) or presence of 5 (▲), 10 () or 50 g/ml (▼) of peptides NAP or SAL (wavelength 190-260 nm, 25°C), control baseline has been subtracted from samples. (E, F) Size distribution of htau34 (20 M) aggregated species, determined by dynamic scattered light, in the absence ( ) or presence of 1 (

), 5 (

), 10 (

) or 50 g/ml (

sizes are categorized in Table 3.

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) of peptides NAP or SAL (25°C),

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Fig. 3 Fluorescence emission spectra of ANS-bound htau34 (20 µM) aggregated species in the (A) absence or presence of (B) 1 µg/ml NAP, (C) 5, 10, 50 µg/ml NAP, (D) 1 µg/ml SAL and

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(E) 5, 10, 50 µg/ml SAL. Resulting spectra were drawn from treated or untreated samples in the

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several intervals (__) time zero (monomeric htau34), () 2 h, (▲) 4 h, () 24 h, () 48 h or (▼)

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72 hours.

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Fig. 4 AFM micrographs of htau34 aggregates in the presence or absence of peptides NAP or

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SAL. The samples prepared as described in materials and methods and 1 µL dropped on mica surface. AFM equipment was set to tapping mode and images are demonstrated as (A) untreated

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htau34 aggregate species, boxes correspond to granular-shaped oligomers with the size less than 100 nm and arrow indicates fibrils, (B) htau34 aggregate species in the presence of 5 µg/ml NAP, arrow shows branched and elongated aggregates with width more than 200 nm, (C) htau34 aggregate species in the presence of 5 µg/ml SAL, arrows imply the annular forms with diameter less than 1000 nm. Each images are presented in three scanned size and scale bars are 1000, 300 and 100 nm.

Fig. 5 Light scatter cytogram of immunospecific interaction of anti-tau oligomer antibody T22 and oligomer species in SH-SY5Y cells. (A) Isotype control (rabbit IgG antibody), 1:500. (B) Immunoresponse of CR induced cells against T22 in the absence and (C) presence of 50 g/ml NAP or (D) 5 g/ml SAL. In contrast to the significant density of dead cells in the absence of NAP or SAL (B), in peptides treated samples dead population were not seen (C and D). T22 does not recognize oligomers from other amyloidogenic proteins like A or -synuclein

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Fig. 6 Cell viability assay. PC12 cells were treated with equal doses of aggregated species of htau34 in presence or absence of NAP and SAL peptides for 24 h. Cell viability was measured

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by the MTT assay, both NAP and SAL treated samples increased the cell viability.

Fig. 7 A schematic representation of proposed granular-shaped oligomer attenuation by peptides

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NAP or SAL

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Graphical abstract

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Highlights Changes in human tau fibrillization kinetics induced by neuropeptides NAP and SAL,

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

suggesting formation of diverse aggregated species

Conformational shift from beta-sheet rich content structures toward random-coil,

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confirming unusual amyloid forms in the presence of NAP and SAL Disappearance of aggregated particles under 100 nm in peptides NAP and SAL treated

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samples which corresponded to the attenuation of oligomeric tau forms. Significant reduction of granular-shaped tau forms was observed using atomic

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microscopy technique

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Peptide-treated SH-SY5Y cells, elucidates a twenty percent decrease of oligomeric tau

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species

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

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