凝胶

Disaggregation of Tau as a Therapeutic Approach to Tauopathies

K. Duff1,*, J. Kuret2 and E.E. Congdon1

Taub Institute / Department of Pathology, Columbia University and Dept of Integrative Neuroscience, New York State Psychiatric Institute, New York, USA; 2Ohio State University, Ohio, Cellular and Molecular Biology, Ohio, USA

Abstract: Tau aggregation is an appealing target for therapeutic intervention. However, conformational change or aggre-gation needs to be targeted without inhibiting the normal biology of tau and its role in microtubule stabilization. The num-ber of compound classes being tested at this time are very limited and include Congo red derivatives [1], anthraquinones (Pickhardt et al. 2005 [2], disputed in Crowe et al. 2007 [3]), 2,3-di(furan-2-yl)-quinoxalines , phenylthiazolyl-hydrazide (PTH) [4], polyphenols and porphyrins [5] and cyanine dyes [6-8]. Herein we have utilized a member of the cyanine dye family (C11) in an organotypic slice culture model of tangle formation. Our results demonstrate that C11 is capable of af-fecting tau polymerization in a biphasic, dose dependent manner. At submicromolar concentrations (0.001 μM) C11 re-duced levels of aggregated tau. However, higher doses resulted in an increase in tau polymerization. These effects can also be seen at the level of individual filaments with changes in filament length and number mirroring the pattern seen via immunoblotting. In addition, this effect is achieved without altering levels of phosphorylation at disease and microtubule binding relevant epitopes.

1

Keywords: Alzheimer disease, tau, tangle, cyanine, phosphorylation, polymerization. BACKGROUND

Cyanine Dyes and Tau Polymerization

The class of drugs chosen for this study is the cyanine dyes. Several members of the class have been well studied and one (indocyanine green/cardio green) has been approved for use in humans as an imaging agent [9-12]. Cyanine dye N744, an aromatic heterocycle, was shown to be a potent antagonist of tau polymerization in vitro [6-8]. While N744 appears specific for tau, the utility of the cyanine dyes for other aggregated proteins in other neurodegenerative dis-eases (󰀁-synuclein, A󰀂) is of interest as dyes with similar structure such as Congo red, have been effective in reducing filaments of huntingtin [13, 14]. The inhibitory properties of N744 against tau were discovered in a small molecule screen, under physiological conditions using a fluorescence based assay, and it was further studied in vitro using well-characterized electron microscopy methods [6-8]. The action of N744 is specific for tau with increased 󰀂-sheet and re-duced random-coil that mimics tau from brains at early stages of disease [7]. It does not bind normal tau under these conditions making it appealing from a therapeutic stand-point. To induce the aggregation-prone state in recombinant full-length tau under physiological conditions and concentra-tions, different inducers were used including fatty acid and detergent micelles and anionic microspheres that mimic fatty acids by presenting a negatively charged surface to the tau molecule [15]. Tau polymerization could be inhibited at sub-stoichiometric concentrations of N744 with an IC50 of 294 ± 23 nM [8]. An examination of tau polymerization kinetics in the presence of N744 yielded a concentration dependent de-

*Address correspondence to this author at the P&S 12th Flr Rm 12-461, Columbia University Medical Center, New York, NY 10032, USA; Tel: 212-305-8970; Fax: 212-342-0119; E-mail: ked2115@columbia.edu

crease in both total filament length and number at equilib-rium. N744 was capable of retarding filament formation in both the longest and shortest human tau isoforms, which differ in the number of microtubule binding repeats and two N terminal exons [6]. In addition, the inhibitor was effective on pseudophosporylated and glycated tau (FTD-mutant tau was not tested) [8]. Further, N744 is capable of disaggregat-ing pre-formed mature filaments via endwise depolymeriza-tion.

The effect of a cyanine dye like N744 is mediated by changes in the equilibrium constant, or critical concentration of the fibrillization reaction. In the presence of N744, the critical concentration, or minimal protein concentration needed for polymerization increased in a concentration de-pendent manner [8]. When examined over a large concentra-tion range, N744 produced a biphasic dose response curve that was mediated by changes in N744 aggregation state. The dimeric form however is the inhibitory species when incu-bated with tau in the presence of the inducer. Large dye polymers (J or H aggregates) of N744 relieve inhibition of tau polymerization. Thus the dose of the compound needs to be carefully monitored to avoid unexpected results. Cyanine Dye Activity is Biphasic

Previous findings demonstrate that N744 is a potent in-hibitor of tau polymerization at substoichiometric concentra-tions. However, as the concentration of N744 in solution increased above 10 μM, inhibition was relieved, and the IC50 was recalculated as ~13 μM. Further increase in N744 levels resulted in an enhancement of polymerization to levels higher than those seen with no inhibitor present ([7]data from this paper is shown in Fig. 1). This suggests a complex dose response relationship with a ratio of IC50 values of ~43 in the different phases. In parallel with changes in total fila-©2010 Bentham Science Publishers Ltd.

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236 Current Alzheimer Research, 2010, Vol. 7, No. 3 Duff et al.

Fig. (1). Preparation of organotypic slices.

A. Brains were removed from either JNPL3 or hTau pups between 9 and 11 days postnatal. Olfactory bulb and brainstem are removed and the hemispheres (cortex/hippocampus) retained. B. Each hemisphere was then sectioned into 400 μm slices. C. After sectioning, slices from the two hemispheres were plated on tissue culture inserts in separate rows of a six well plate. This method allows each animal to act as its own control.

ment length, a biphasic dose response effect was also seen in measured critical concentration. Critical concentration in-creased with N744 concentration to an optimal level at ~4 μM. At still higher dye concentrations, critical concentration began to decrease.

Properties of the Cyanine Dye Class

A structure-activity relationship (SAR) for the thiacarbo-cyanine scaffold was prepared using a filter binding assay for octadecyl sulfate induced fibrillization of human 2N4R tau. Antagonist activity depended in part on bridge length (n), meso substituent (R2), and N-substituent (R3). Molecules containing a three carbon bridge (n = 1) were the most potent cyanines (Table 1). Within this series, compound C11 re-tained the potency and efficacy of N744 but had calculated physical parameters of topological polar surface area (tPSA), octanol water partition coefficient (miLogP), and molecular weight (MW) that were more consistent with membrane and blood-brain barrier penetration. Therefore, this cyanine was selected for use in biological models. Because compound C2 had little or no tau aggregation inhibitory activity at up to 10 μM concentration, it was selected to serve as a negative con-trol for potential non-specific effects of the cyanine scaffold. METHODS

Preparation and Treatment of Organotypic Slices

Organotypic slice cultures were prepared utilizing proto-cols modified from Duff et al [16]. Brains were removed from JNPL3 mice expressing P301L mutant tau [17], at postnatal day ten or 11. Hemispheres (cortex/hippocampus) were sectioned into 40 μm thick slices and transferred to membrane inserts. Slices were allowed to incubate for 14 days in media containing 25% serum. Media was exchanged every two days. For cyanine dose response experiments, slices in one hemisphere from each brain were treated with 3,3’-Diethyl-9-methylthiacarbocyanine iodide (“C11”), a

commercially available cyanine dye similar in structure to N744, and the other with DMSO vehicle (final concentration of DMSO in media 0.008%). Slices were incubated with 1- 0.0001μm of the cyanine dye for one week. See Fig. (1) for an illustration of slice preparation. Additional slices were prepared from mice expressing all isoforms of wild-type tau on a tau null background [18] which were utilized in phos-phorylation and electron microscopy experiments. Tissue Fractionation

Control and C11 treated slices were homogenized in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1μg/ml protease inhibi-tors, 1μg/ml phosphatase inhibitors) and centrifuged at 20,000 G at 4oC for 20 minutes. The pellet fraction was dis-carded and the resulting supernatant assayed for total protein concentration. The heat stable (S) fraction was generated using an aliquot of the low speed supernatant which was retained and boiled for 10 minutes. The samples were then centrifuged at 20,000 x G for 20 minutes at 4o C and the su-pernatant was retained and diluted in O+ buffer (62.5 mM Tris-HCl pH 6.8, glycerol 5%, 2-mercaptoehtanol, 2.3% SDS, 1mM EGTA, 1mM PMSF, 1mM NaVO4, 1 μg/ml pro-tease inhibitors, 1 μg/ml phosphatase inhibitors). An addi-tional fraction of low speed supernatant was incubated in 1% sarkosyl for 30 minutes at room temperature and then centri-fuged for 1 hour at 100,000 x G at 20oC. Supernatant was discarded and the sarkosyl insoluble (SI) pellet was resus-pended in 20 μL O+ buffer. This sarkosyl insoluble fraction represents the polymerized tau. Immunoblotting

Total, heat stable, and sarkosyl in soluble fractions were assayed using a variety of antibodies including Tau C, S202/205 (AT8), S396/404 (PHF-1), S262, T212, and S396 at appropriate dilutions. Bands were visualized with en-hanced chemiluminescence reagent using a Fujifilm

Disaggregation of Tau as a Therapeutic Approach to Tauopathies Current Alzheimer Research, 2010, Vol. 7, No. 3 237

Table 1.

Summary of Molecular Properties of Cyanine Derivatives Used in SAR Studies

SR1NR3nR2SR1NR3

Cpd n N744 1 C2 0 C3 1 C5 2 C7 3 C11 1 C3-B 1 C3-D 1 C3-O 1 ab

R1 R2 R3 tPSAa miLogPa MWb IC50 (μM)

67.7 8.8 8.8 8.8 8.8 8.8

1.38 2.52 3.04 3.56 3.07 6.67

485.6 365.5 391.6 417.6 379.6 477.7

0.29 ± 0.02 0.96 ± 0.25 2.15 ± 0.47 5.00 ± 3.30c 0.28 ± 0.04 3.29 ± 2.07c >>10c

5-OCH3 -CH2CH3 -(CH2)2OH -H -H -H -H -H -H -H -H

N/A -H -H -H -H -H

-CH2CH3 -CH2CH3 -CH2CH3 -(CH2)5CH3

-CH2CH3 8.8 1.94 339.5 ~10c

-CH3 -CH2CH3

-CH3 -(CH2)3SO3- 123.2 -2.22 565.7 -CH2CH3 35.1 1.24 333.4 >>10c

tPSA and miLogP values were calculated from website http://www.molinspiration.com/

MW for organic component of the salt. c

Accurate estimates of SE could not be determined owing to poor inhibition.

A structure-activity relationship (SAR) for the thiacarbocyanine scaffold was prepared using a filter binding assay for octadecyl sulfate induced fibrillization of human 2N4R tau. Antagonist activity depended in part on bridge length (n), meso substituent (R2), and N-substituent (R3). Molecules containing a three carbon bridge (n = 1) were the most potent cyanines. Within this series, compound C11retained the potency and efficacy of N744 but had calculated physical parameters of topological polar surface area (tPSA), octanol water partition coefficient (miLogP), and molecular weight (MW) that were more consistent with membrane and blood-brain barrier penetration. Therefore, this cyanine was selected for use in biological models. Because compounds C2 had little or no tau aggregation inhibitory activity at up to 10 μM concentration, it was selected to serve as negative controls for potential non-specific effects of the cyanine scaffold.

LAS3000 imaging system. Signal was quantified using Multigauge version 3.0. Electron Microscopy

Aliquots from the sarkosyl insoluble fractions were fixed with glutaraldehyde, adsorbed onto formvar coated copper grids and negatively stained with 2% uranyl acetate. For each condition, a total of at least five images were collected using a JEOL 1200 transmission electron microscope operated at 80 kV at 12,000x magnification. Individual filament lengths were measured using the Image J program. For each image, total filament length, number, and length-distribution was. These data provide information regarding the relative abundance of different tau species in the tissue as well as the mechanism of action for C11. RESULTS

Treatment with C11 Modulates Tau Polymerization Organotypic slices from JNPL3 mice were treated with a range of C11 concentrations. Both treated and control hemi-spheres were pooled and then assayed for levels of sarkosyl insoluble tau. A biphasic dose response effect was observed in which treatment with 0.001 μM C11 resulted in a signifi-cant reduction (p<0.05) in the levels of polymerized tau. In contrast, as the dosage was increased inhibition was lost and at the highest concentrations (0.3-1 μM) produced a signifi-cant increase in aggregation [19]. (Fig. (2)) No change in polymerization was seen with the inactive cyanine C2.

Fig. (2). C11 has a biphasic dose response curve.

Slices from JNPL3 pups were incubated with varying doses of cyanine dye C11. A. Treatment with 0.001μM C11 results in a sig-nificant decrease in sarkosyl insoluble tau in treated slices (T) rela-tive to control (C). In contrast, incubation with 1 μM C11 produced a significant increase. B. Levels of sarkosyl insoluble tau relative to control were determined over a range of C11 concentrations.

238 Current Alzheimer Research, 2010, Vol. 7, No. 3 Duff et al.

Fig. (3). C11 does not affect phosphorylation.

A. Aliquots from the heat stable fraction were used to determine the relative levels of phosphorylation at disease and polymerization relevant epitopes for. B-F. Immunoblots were quantified and the levels of phosphorylation relative to control were determined for each treatment group. No changes were seen in band intensity or mobility relative to control at either dose of C11 or with C2.

C11 Does Not Affect Phosphorylation

Slices treated with 1 μM C11, 0.001 μM C11, or 0.001 μM C2 were assayed for levels of phosphorylation at disease and microtubule biding relevant epitopes. In all cases no significant changes in occupancy were detected [19]. These data indicate that tau polymerization can be targeted inde-pendently of phosphorylation. (Fig. (3)) Because tau kinases have multiple targets within the cell, directly targeting ab-normal tau may provide a way to reduce potential side ef-fects.

C11 Treatment Alters Filament Length and Number but not Length Distribution

Treatment with 0.001 μM C11 resulted in a significant decrease (p=0.01) in filament number relative to control. In contrast, following incubation with 1 μM C11 average fila-ment number significantly increased (p=0.01). (Fig. (4)). Analysis of total filament mass revealed a similar pattern. In all cases the filament length distribution was exponential and no significant differences were observed between control and treated samples [20]. This is not insignificant because these data suggest that changes in filament mass and number are not the result of random breakage. DISCUSSION

Our data demonstrates the utility of the cyanine dye class for tau dissagregation in a physiologically relevant model system. The lack of effect on phospho tau suggests that the drug’s primary mode of action is on tau disassociation. At low doses the drug appears to be non-toxic and efficacious at dissociating tau in an ex vivo brain, but its effects in vivo are as yet unknown. Further testing of tau disaggregation as a therapeutic target are warranted.

Disaggregation of Tau as a Therapeutic Approach to Tauopathies Current Alzheimer Research, 2010, Vol. 7, No. 3 239

Fig. (4). C11 modulates filament length and number.

Aliquots from the sarkosyl insoluble fraction were adsorbed onto formvar coated copper grids and negatively stained with uranyl acetate. Individual filament lengths were measured using Image J and then quantified. A. A significant increase in total filament number was observed at 1 󰀁M produced a significant decrease. B. Similarly a significantly higher total filament mass was observed at 1μM while treatment with 0.001 󰀁M treated samples showed a significant reduction. C,D. The number of filaments falling into each 10 nm length range was determined and expressed as a percentage of total filament number. No significant difference was seen between control (black bars) and treated (grey bars) samples. E, F. Electron microscopy images obtained from samples incubated with 1 and 0.001μM C11 relative to control. In contrast 0.001 󰀁M C11 respectively.

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Received: November 19, 2009

Revised: November 24, 2009

Accepted: December 04, 2009

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