Materials

High glucose Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were obtained from Invitrogen (Carlsbad, CA, USA). C57BL/6J mice were ordered from Jackson Labs (Sacramento, CA, USA) Stock 000664. The transgenic mouse line APPswe/PS1ΔE9 85 was a generous gift of Dr. J.L. Jankowsky.

The primary antibodies were used at a dilution of 1:1,000 unless otherwise stated and their sources and molecular weights were as follows: Cell Signaling Technology (Danvers, MA, USA): β-actin, monoclonal HRP conjugate, 45 kDa; CREB, monoclonal, 43 kDa. Santa Cruz Biotechnology (Santa Cruz, CA, USA): Egr-3, C-24 polyclonal, 42 kDa; BDNF, polyclonal, 16 kDa. Millipore (Temecula, CA, USA): Anti-BACE C-terminus, clone 61-3E7, 60 to 75 kDa. Novus Biologicals (Littleton, CO, USA): Homer-1, polyclonal, 47 kDa. Sigma (St Louis, MO, USA): Anti-Amyloid Precursor Protein, C-terminal, polyclonal, 95 to 100 kDa; Anti-Nerve Growth Factor 2.5S, polyclonal homodimer, 26 kDa. Covance (Princeton, NJ, USA): 6E10 monoclonal antibody.

All other materials were from Sigma (St Louis, MO, USA) unless otherwise stated.

Methods

Animal studies

All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Salk Institute for Biological Studies.

Old huAPPswe/PS1ΔE9 transgenic mice

Animals

The APPswe/PS1ΔE9 transgenic mice (line 85) have been previously characterized [10, 11]. The line 85 mice carry two transgenes, the mouse/human chimeric APP/Swe, linked to Swedish FAD and human PS1ΔE9. At 20 months of age both male and female transgenic mice were fed a high fat diet (Harlan Tekland, Madison, WI, USA) with and without J147 (200 ppm, 10 mg/kg/day). Treatment continued for three months and was followed by behavior testing and sacrifice of mice for tissue harvesting. Mouse body weights and food consumption were measured weekly, and there were no significant differences between the groups. (Data not shown).

C57BL/6J mice-scopolamine study

Animals

A total of 60 male mice aged eight weeks were housed 4 per cage and were divided into five groups with 12 mice per group. Treatments were administered in the food (TestDiet® 5015, Richmond, IN, USA) for a period of two weeks before commencement of behavioral testing. Groups included J147 at 200 ppm (10 mg/kg/day), donepezil at 20 ppm (1 mg/kg/day), a combination of J147 at 200 ppm and donepezil at 20 ppm, and two groups on the control food without any treatments. Following two weeks of treatment, memory impairment was induced by intraperitoneal (i.p.) injection of scopolamine (1 mg/kg) 30 minutes prior to each of the following behavioral tests: Y-maze, probe trial of the water maze and contextual and cued fear conditioning. Mice were allowed to rest for two days between each behavior test. All mice received scopolamine except for one of the control groups, which received saline as a control. Mice were sacrificed 24 hours after the last behavioral test for tissue harvesting.

Behavior assays

Two-day water maze

Spatial memory was determined using the two-day water maze in 23-month-old huAPPswe/PS1 transgenic mice fed J147 at 200 ppm in food for the previous three months. The protocol was adapted from a publication by Gulinello and colleagues [12]. Water temperature remained at 27°C throughout the experiment. The goal platform was positioned 45 cm from the outside wall in the north-west quadrant of the maze for all groups and all trials. Day 1 of the two-day water maze procedure involved training the mice to find the platform using cues located around the pool within a 180 s time frame. This training involved a series of visible platform trials where mice were tracked using the Noldus EthoVision software (Noldus Information Technology, Inc., Leesburg, VA, USA). There were four visible platform trials (V1 to V4) where the last visible platform trial of a mouse was considered its post-habituation baseline. If the mice failed to find the platform after 180 s, they were placed on the platform by the experimenter. All mice remained on the platform for 15 s before being placed in a heated incubator (30°C) between trials. On Day 2, 24 hours following the last visible platform trial, mice were tested in a series of three hidden platform trials (T1 to T3). Again each trial lasted for 180 s. The time it took each mouse to find the hidden platform was measured as escape latency. For the scopolamine experiment, normal mice were given an i.p. injection of saline or 1 mg/kg scopolamine 30 minutes before the first hidden platform trial on Day 2. All trials were recorded using the EthoVision Software and statistics were computed using GraphPad Instat software (GraphPad Software, San Diego, CA, USA).

Elevated plus maze

The elevated plus maze analyzes the anxiety response of mice [13]. This test relies upon the tendency of mice to have a fear of heights and to navigate towards dark enclosed spaces and remain there [14]. Our maze is made of gray plastic and consists of four arms (two open without walls and two enclosed by 15.25 cm high walls) 30 cm long and 5 cm wide in the shape of a plus sign. The elevated plus maze is placed close to the center of the room, and has similar levels of illumination on both open and closed arms. A video-tracking system (Noldus EthoVision) is used to automatically collect behavioral data. The software is installed on a PC computer with a digital video camera mounted overhead on the ceiling, which automatically detects and records when mice enter the open or closed arms of the maze and the time spent in each. Mice are habituated to the room 24 hours before testing. Mice are also habituated to the maze for two minutes before testing by placing them in the center of the maze and blocking entry to the arms. Mice were then tested in the maze for a five-minute period while the software tracked and recorded the behavior of the mice. The anxiety of mice was measured by comparing the time spent in the open arms to time spent in the closed arms. Statistics were computed using GraphPad Instat software.

Fear conditioning assay

Fear conditioning to either a cue or a context represents a form of associative learning. The read-out that is measured in contextual and cued fear conditioning is a freezing response that occurs following the pairing of an unconditioned stimulus (US), such as a foot shock, with a conditioned stimulus (CS), such as a particular context or cue (tone) [15–17]. The mouse will freeze if it remembers and associates that environment with the aversive stimulus. The hippocampus and the amygdala are required for fear memory where the hippocampus is involved in the formation and retrieval of context fear associations and the amygdala is involved in conditioning and recall of associations to contextual and discrete cues [18, 19]. This assay used fear conditioning chambers from Med Associates Inc. with Video Freeze Software (Med Associates Inc, St. Albans, VT, USA). On Day 1, mice were trained by allowing them to explore the chamber for 120 sec, mice were then presented with a 30-sec tone (2 kHz with 85 dB intensity) immediately followed by a 2-sec foot shock (0.7 mA). The tone-shock pairing was repeated following a 30-sec interval and the mice were again allowed to explore for 120 sec before removing them from the chamber. On Day 2, contextual memory, which requires a functioning hippocampus, was tested by placing the mice in the chambers and allowing them to explore for the same length of time as the previous day but without the tone and the shock. On Day 3, cued or emotional memory was tested, which relies on both hippocampus and amygdala. For this, the chamber environment was altered by using plastic boards to alter the shape of the chamber and using similar plastic boards over the grid floor to alter the environment further. Vanilla essence was used to alter the smell of the environment. Testing involved placing the mice in the chambers and carrying out the same paradigm as Day 1 without the foot shock. The camera measures the amount of time the mice freeze and the software allows analysis of this freezing at any time-point of interest. On Day 2, the time spent freezing is measured over the entire time in the chamber. A mouse that remembers the chamber context and associates it with the foot shock will spend more time freezing and this response is hippocampal dependent. The percentage of time spent freezing by each mouse is averaged per group and then groups can be compared and P-values calculated to determine statistical significance. On Day 3, the percentage of time spent freezing during the two tones is averaged per group, and then groups can be compared and P-values calculated to determine statistical significance. This result relates to the recall of associations to the tone and is dependent on the amygdala and the hippocampus. For the scopolamine experiment, normal mice were administered an i.p. injection of saline or 1 mg/kg scopolamine 30 minutes before testing on Day 2 and Day 3.

Y-Maze

Spontaneous alternation, the tendency to alternate free choices in a Y-maze (three arms), is a model for studying short term working memory in mice [20, 21]. Mice were injected with 1 mg/kg of scopolamine or saline 30 minutes prior to testing. Then each mouse was placed in the center of the Y and arm entries were recorded by video camera and the order of entries were recorded for the first 15 entries. Spontaneous alternations are defined as consecutive triplets of different arm choices.

Tissue preparation and immunoblotting

Hippocampal and entorhinal cortex tissue samples were homogenized in 10 volumes of RIPA lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% sodium dodecyl sulfate and 0.5% deoxycholate, and 1% NP40) containing a cocktail of protease and phosphatase inhibitors (20 mg/ml each of pepstatin A, aprotinin, phosphoramidon, and leupeptin; 0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; 1 mM EGTA; 5 mM fenvalerate; and 5 mM cantharidin). Samples were sonicated (2 × 10 s) and centrifuged at 100,000 × g for 60 minutes at 4°C. Protein concentrations in the cell extracts were determined using the BCA protein assay (Pierce supplied by Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of protein were solubilized in 2.5x SDS-sample buffer, separated on 12% SDS-polyacrylamide gels, transferred to Immobulin P and immunoblotted with the antibodies indicated in the Materials section. For Western blots, protein levels were normalized to actin levels. An unpaired t test was performed to compare between two groups at a single time point. When comparing multiple groups, one-way ANOVA followed by a Tukey's post hoc test was used. All statistical analysis was conducted using GraphPad Instat software.

Immunohistochemistry

Brains were fixed with 4% paraformaldehyde in 100 mM sodium tetraborate, pH 9.5, for 3 h, cryoprotected with 20% sucrose-potassium-PBS (KPBS), and sectioned into coronal (30 μm) sections using a sliding microtome (Leica Microsystems Inc, Buffalo Grove, IL, USA). Sections were submerged in 0.3% H 2 O 2 for 10 minutes to eliminate endogenous peroxidase activity and treated with 1% borate to eliminate free paraformaldehyde. Sections were incubated with primary antibody in 0.3% Triton X-100 in KPBS plus 2% filtered serum or BSA overnight at 4°C, and with primary antibodies (1:1,000) in 0.3% Triton X-100 for 1 hr at room temperature. After incubation with secondary antibody and ABC reagent (Vector Laboratories Inc, Burlingame, CA, USA), sections were developed using metal-enhanced DAB solution. Sections were mounted to slides, dried, dehydrolyzed, treated with xylene, and covered using permount (Fisher Scientific, Pittsburgh, Pennsylvania, USA). Images were captured by a Zeiss digital camera connected to a Zeiss VivaTome microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA), and image analysis on sections was performed using Axiovision software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).

Quantification of amyloid plaque burden was based on the image captured by immunohistochemical staining with antibody 6E10. Sections of each mouse cortex and hippocampus were imaged together and the areas and densities of the plaques in the hippocampus only were measured by the Image J software (NIH). The total counts of Aβ plaques in sections per six mouse brains of each group were determined in an unbiased fashion.

Aβ ELISA

Aβ 1 to 40 and 1 to 42 levels in hippocampal lysate were analyzed using the Aβ 1-40 and Aβ 1-42 ELISA kits from Invitrogen (# KHB3481 and # KHB3442, respectively). All kit reagents were brought to room temperature before use. Standards were prepared according to the manufacturer's guidelines and samples were diluted as follows; RIPA fractions were diluted 1:10 for both Aβ 1-40 and Aβ 1-42 ; and RIPA insoluble fractions were diluted 1:2,000 for Aβ 1-40 and 1:5,000 for Aβ 1-42 . A total of 50 μl of Aβ peptide standards and samples were added in duplicate to 96-well plates pre-coated with mAb to the NH 2 terminus region of Aβ. Plates were incubated at 4°C overnight and then 50 μl of Hu Aβ40 or Aβ42 detection antibody was added to each well except the chromogen blanks. Plates were incubated at room temperature with gentle shaking for three hours and then washed four times with the provided wash buffer. At this time, 100 μl of anti rabbit IgG HRP working solution was added to each well except the chromogen blanks for 30 minutes at room temperature. Wells were then washed as before four times and incubated with 100 μl of stabilized chromogen for 25 minutes at room temperature in the dark. Stop solution was then added at 100 μl to each well followed by reading the absorbance of each well at 450 nm. Curve fitting software was used to generate the standard curve where a four-parameter algorithm provided the best standard curve fit. The concentrations of the samples were calculated from the standard curve and multiplied by the dilution factor.

Cell culture with growth conditioned medium

The HT22 cell line was used to make growth conditioned medium. HT22 is a nerve cell line derived from mouse brain and is widely used to study nerve cell physiology [22, 23]. To make HT22 growth conditioned medium, cells were grown in DMEM with 10% fetal calf serum. Then, semiconfluent cultures were washed three times with serum-free DMEM and cultured overnight in a reduced volume of DMEM in the presence or absence of 100 nM J147. The following day, the growth conditioned medium was collected and centrifuged at 10,000 × g to remove detached cells. To determine the effect of the conditioned medium on NGF-induced neurite outgrowth, PC12 cells were dissociated and plated on polyornithine-coated tissue culture dishes in the following conditions: 1) HT22 conditioned medium, 2) J147 treated HT22 conditioned medium, 3) DMEM alone plus J147, 4) DMEM plus NGF at 50 nanograms per ml, 5) J147 treated HT22 conditioned medium pre-incubated for one hour with 10 μg/ml anti-NGF and N2 supplement (Invitrogen). N2 supplement, which contains transferrin, was used in the presence of antibody to minimize the possibility that antibody protein non-specifically modified cell-substrate from adhesion and therefore neurite outgrowth. Phase contrast photographs were taken 24 hrs later.

GeneChip

HT22 cells

HT22 cells were plated in DMEM plus 10% FCS. The next day, the cells were treated with 10 μM J147 for 1 hr before RNA isolation.

RNA isolation

RNA was isolated with the use of RNeasy Mini kit (Qiagen, #74104; Valencia, CA, USA) according to the manufacturer's instructions. Total RNA was quantified using the ND-1000 Nanodrop and assessed for quality using the ratios: A260/280 (range: 1.9 to 2.1) and A260/230 (range: 2.0 to 2.2, if <2.0, contamination), in addition to the Bioanalyzer (Agilent Technologies, Cedar Creek, TX, USA) if further quality assessment was required.

RNA isolation and microarray hybridization experiments

After RNA isolation for each sample, double stranded cDNA was synthesized from 500 ng total RNA and biotin-labeled using the GeneChip 3' IVT Express Kit (Affymetrix, Santa Clara, CA, USA, #901228-A) per the manufacturer's instructions and protocol. RNA was purified, quantified, fragmented randomly to an average size of 50 to 200 bases and hybridized to GeneChip® Mouse Genome 430 2.0 Arrays (Affymetrix,) consisting of over 45,000 probe sets representing more than 34,000 named mouse genes. The hybridization and processing of the GeneChips were conducted by Salk Institute's Functional Genomics Core Facility using the following systems from Affymetrix (Santa Clara, CA, USA): GeneChip® Hybridization Oven 640, GeneChip® Fluidics Station 450 to the wash and stain operation of Affymetrix GeneChip® arrays, and the GeneChip® Scanner 3000 7G.

GeneChip quantification and normalization

Affymetrix Expression Console Software (version 1.0) was used to perform quality assessment of the microarray scan/experiments. Array data were normalized via scaling to adjust the average intensity of each array to be similar. GeneChips were analyzed by the GeneChip Operating Software (Affymetrix) with the default settings except that the target signal was set to 200 for GeneChip quality control. Raw data were analyzed through the gcRMA-algorithm using the Affymetrix package in R software for statistical computing and graphics [24]. The median microarray intensity for all microarrays was normalized to 100, and probe sets with median intensities >100 were scored. Fold changes were calculated in Microsoft Excel Microsoft, Redmond, Washington, USA). Genes of interest and genes with the highest fold-changes were validated using Real-Time Quantitative PCR. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [25] and are accessible through GEO Series accession number GSE45534 [26].

Commercial screening for molecular targets and "off target" effects

All screening was done at 10 μM J147 by various contract research organizations (CROs), including MDS Pharma Services (King of Prussia, PA, USA), Ricerca, now Panlabs (Concord, OH, USA), Ambit (La Jolla, CA, USA), Caliper (Hopkinton, MA, USA) and NovaScreen Biosciences (Hanover, MD, USA) by standard protocols described in their catalogs. The only two assays that yielded greater than 60% inhibition at 10 μM J147 were then re-assayed to determine EC 50 values: the dopamine transporter (EC 50 = 0.649 μM) and monoamine oxidase B (EC 50 = 1.88 μM) assays both carried out by MDS Pharma Services.

Synthesis of J147 and donepezil

Materials

Compounds J147 and donepezil were synthesized in our laboratory at the Salk Institute. All starting materials, chemicals and reagents were obtained from Sigma Aldrich, (Milwaukee, WI, USA), and were used as received. Solvents used for synthesis and chromatographic analysis were HPLC or ACS reagent grade and were purchased from Fisher Scientific Co (Pittsburg, PA, USA). Thin layer chromatography (TLC) used EMD silica gel F-254 plates (thickness of 0.25 mm). Flash chromatography used EMD silica gel 60, 230 to 400 mesh and were purchased from EMD Chemicals (San Diego, CA, USA).

Analytical methods

1H NMR recorded at 500, on a Varian VNMRS-500 spectrometer at the Salk Institute (La Jolla, CA, USA) using the indicated solvents. Chemical shift (δ) is given in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Coupling constants (J) are expressed in hertz (Hz), and conventional abbreviations used for signal shape are as follows: s = singlet; d = doublet; t = triplet; m = multiplet; dd = doublet of doublets; brs = broad singlet. Liquid chromatography mass spectrometry (LCMS) was carried out using a Shimadzu LC-20AD spectrometer at The Scripps Research Institute (La Jolla, CA, USA), and electrospray ionization (ESI) mass analysis with a Thermo Scientific LTQ Orbitrap-XL spectrometer at the Salk Institute. Melting points were determined with a Thomas-Hoover capillary melting point apparatus at the Salk Institute, and are uncorrected. All final compounds were characterized by LCMS and 1H NMR and gave satisfactory results in agreement with the proposed structure. J147 and donepezil have purity of 98% and 99%, respectively, which was determined by analysis on a C18 reverse phase HPLC column (Phenomenex Luna (50 mm × 4.60 mm, 3 μm)) at The Scripps Research Institute, using 10 to 90% CH 3 CN/H 2 O containing a 0.02% AcOH with a flow rate of 1 mL/min (5-minute gradient) and monitoring by a UV detector operating at 254 nm.

Chemical synthesis of compounds

The synthesis of J147 has been carried out using simple chemistry as described in our previous paper by condensation of 3-methoxybenzaldehyde and (2, 4-dimethylphenyl) hydrazine hydrochloride in EtOH at room temperature, followed by acetylation using trifluoroacetic anhydride and triethylamine in CH 2 Cl 2 gave J147 (Scheme 1). Donepezil has been synthesized with 99% purity according to the literature procedure published in Organic Process Research & Development 2008, 12:731-735 (Scheme 2).

Synthesis of (E)-N-(2,4-dimethylphenyl)-2,2,2-trifluoro-N'-(3methoxybenzyli-dene) acetohydrazide (J147)

A mixture of 3-methoxybenzaldehyde (50 g, 367.64 mmol) and (2, 4-dimethylphenyl) hydrazine hydrochloride (63.23 g, 367.64 mmol) in EtOH (50 mL) was stirred at room temperature for 1 h, the obtained solid was filtered off, washed with ethanol and dried under vacuum to afford hydrazone hydrochloride 1 (95.94 g) in 90% yield as a light brown solid. This unstable hydrazone (50 g, 172.41 mmol) was dissolved in CH2Cl2 (50 ml), Et3N (57.56 mL, 413.79 mmol) followed by (CF3CO)2O (28.77 mL, 206.89 mmol), was added at 0°C and the mixture was stirred at room temperature for 1 h. Reaction mixture was diluted with aq. sat. NaHCO3 solution (500 mL), extracted with CH2Cl2 (2 × 500 mL), dried (Na2SO4) and evaporated, resulting solid was recrystalized from ethanol to give J147 (49.11 g, 81%) as a white solid: mp 70 to 72°C; LCMS purity 98%; 1H NMR (CDCl3, 500 MHz) δ ppm 2.10 (s, 3H), 2.42 (s, 3H), 3.82 (s, 3H), 6.98 (dd, J = 8.5, 2.0 Hz, 1H), 7.07 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.28 (m, 3H). MS (ESI): m/z calcd for C18H17F3N2O2 ((M + H) +) 351.1314; found 351.1366 ((M + H) +).