Protection against β-amyloid-induced synaptic and memory impairments via altering β-amyloid assembly by bis(heptyl)-cognitin

β-amyloid (Aβ) oligomers have been closely implicated in the pathogenesis of Alzheimer’s disease (AD). We found, for the first time, that bis(heptyl)-cognitin, a novel dimeric acetylcholinesterase (AChE) inhibitor derived from tacrine, prevented Aβ oligomers-induced inhibition of long-term potentiation (LTP) at concentrations that did not interfere with normal LTP. Bis(heptyl)-cognitin also prevented Aβ oligomers-induced synaptotoxicity in primary hippocampal neurons. In contrast, tacrine and donepezil, typical AChE inhibitors, could not prevent synaptic impairments in these models, indicating that the modification of Aβ oligomers toxicity by bis(heptyl)-cognitin might be attributed to a mechanism other than AChE inhibition. Studies by using dot blotting, immunoblotting, circular dichroism spectroscopy, and transmission electron microscopy have shown that bis(heptyl)-cognitin altered Aβ assembly via directly inhibiting Aβ oligomers formation and reducing the amount of preformed Aβ oligomers. Molecular docking analysis further suggested that bis(heptyl)-cognitin presumably interacted with the hydrophobic pockets of Aβ, which confers stabilizing powers and assembly alteration effects on Aβ. Most importantly, bis(heptyl)-cognitin significantly reduced cognitive impairments induced by intra-hippocampal infusion of Aβ oligomers in mice. These results clearly demonstrated how dimeric agents prevent Aβ oligomers-induced synaptic and memory impairments, and offered a strong support for the beneficial therapeutic effects of bis(heptyl)-cognitin in the treatment of AD.


Preparation of rat hippocampal slice and electrophysiological techniques. All electrophysi-
ological experiments were carried out on transverse slices of Wistar rat hippocampus. The brains were rapidly removed after decapitation and placed in cold oxygenated media. Slices were cut at a thickness of 350 μ m using and placed in a storage container containing oxygenated medium at room temperature for 1 h. The slices were then transferred to a recording chamber for submerged slices and continuously superfused at a rate of 5-6 ml/min at 30-32 °C. The control media contained mM of: NaCl, 120; KCl, 2.5; Scientific RepoRts | 5:10256 | DOi: 10.1038/srep10256 NaH 2 P0 4 , 1.25; NaHCO 3 , 26; MgSO 4 , 2.0; CaCl 2 , 2.0; D-glucose 10. All solutions contained picrotoxin (100 μ M) to block GABA A -mediated responses.
The slices were transferred to a recording chamber, where they were held submerged between two nylon nets and maintained at 30-32 °C. The chamber consisted of a circular well of a low volume and was perfused constantly at a rate of 4-5 ml/min. All experiments were carried out in the dentate gyrus, with presynaptic stimulation applied to the medial perforant pathway of the dentate gyrus using a bipolar insulated tungsten wire electrode, and field excitatory postsynaptic potentials (fEPSPs) recorded at a control test frequency of 0.033 Hz from the middle one-third of the molecular layer of the dentate gyrus with a glass microelectrode.
In experiments involving application of Aβ 1-42 oligomers, Aβ 1-42 oligomers were perfused for 45 min prior to high frequency stimulation (HFS). In experiments involving AChE inhibitors, AChE inhibitors or vehicle were perfused over the slices for 60 min prior to HFS. Control and experimental levels of LTP were measured on slices prepared from the same hippocampus. Experiments that involved α 7 type nicotinic acetylcholine receptor (α 7nAChR) and cAMP-dependent protein kinase (PKA) inhibitors, and experiments that involved the effect of inhibitors alone and the effects of inhibitors applied together with Aβ 1-42 oligomers were carried out on slices of the same hippocampus. All values were normalized to this baseline and all values of LTP reported here were calculated as changes in fEPSP amplitude measured 60 min after tetanic stimulation.
Preparation of primary rat hippocampal neurons. Primary cultured hippocampal neurons were obtained from 18-day-old Sprague-Dawley (SD) rat embryos as previously described by us with minor modification 12 . Briefly, the hippocampus was dissected and incubated with 0.25% trypsin at 37 °C. Cells were then mechanically dissociated by using a Pasteur pipette with a fire-narrowed tip in culture medium and plated at a density of 2 × 10 5 cells/ml on 35-mm culture dishes pre-coated with poly-L-lysine (10 μ g/ ml).Cells were maintained in neurobasal/B27 medium containing 0.5 mM glutamine, 100 units/ml penicillin, 100 μ g/ml streptomycin at 37 °C. Half-changes of medium were done twice weekly.
Immunocytochemistry and image acquisition. All steps were performed at room temperature as described previously 13 unless otherwise stated. For synapse quantification, primary hippocampal neurons were fixed for 20 min in 4% paraformaldehyde, 10% sucrose, 15 μ g/ml Hoechst-33342 in PBS, then washed twice with PBS. Cultures were incubated in blocking buffer (10% goat serum, 1% BSA, 0.1% Triton X-100 in PBS) for 1 h, followed by addition of rabbit anti-synapsin-1 antibody (Calbiochem, Darmstadt, Germany), in blocking buffer for 1 h. Cells were subsequently washed three times with washing buffer (1% BSA, 0.1% Triton X-100 in PBS) before addition of mouse anti-β III-tubulin antibody (Cell signaling, Beverly, MA) in blocking buffer for 1 h. Following three additional washes, Alexafluor-488 anti-mouse and Alexafluor-568 anti-rabbit secondary antibodies, diluted 1:300 in blocking buffer, were added simultaneously for 1 h. Cells were finally washed three times by PBS before analysis.
Image acquisition and analysis. Image acquisition was carried out according to previous publications with modification 13,14 . Briefly, images were acquired by a Nikon ECLIPSE Ti-U microscope (Nikon Instruments Inc, Melville, NY) at × 400 magnifications. All images in this study were acquired as 12-bit TIFF images. Optimal settings were achieved empirically as we had changed those parameters until images with clear and distinctive signals were obtained. Image analysis was carried out using ImageJ Software according to previous publications with modification 13,14 . Briefly, ultraviolet excitation and emission wavelengths were used to obtain images of nuclei labeled with Hoechst-33342, which allowed identification of the correct focal plane for further image acquisition. Second and third excitation wavelengths were used to illuminate neurites and synapses labeled with Alexa-568 and -488 secondary antibodies, respectively. Neuronal cell bodies were identified as β III-tubulin positive objects. Neurites were identified as β III-tubulin positive structures, and analyzed by a NeuriteTracer program 15 . Briefly, image pairs were opened as neuronal and nuclear stacks. Then, images were pre-processed to equalize the illumination with the stack and reduce imaging artifacts. Following pre-processing, images were thresholded and speckles were moved. The thresholded neuronal marker images were skeletonized and measured. Synapsin I integrated immunofluorescence intensity at each pixel across the images by using ImageJ 16 . Cell bodies were digitally removed from the images so that synapsin I immunostaining on β III-tubulin positive neurites was quantified. All of the above selection criteria were user-defined and all subsequent image analysis used the same criteria.
Immunoblotting analysis. The inhibition of Aβ 1-42 oligomers formation was performed as previously described with modification 1 . Briefly, synthetic Aβ 1-42 was dissolved in HFIP to 1 mg/ml, evaporated under vacuum, and reconstituted in DMSO to 5 mM. Aβ 1-42 was diluted to 50 μ M by using phenol red-free DMEM medium and incubated with various agents for 24 h at 4 °C. The Aβ 1-42 solution was centrifuged at 14000 g for 10 min, and the soluble oligomeric Aβ 1-42 in the supernatant was mixed with an equal part of Tricine sample buffer without reducing agents. Samples were electrophoresed and transferred. Membranes were boiled for 10 min, and blocked for 1 hour at room temperature, probed with rabbit anti-Aβ 1-17 antibody 6E10 (Sigma, 1:1000) followed by goat anti-rabbit horseradish peroxidase, and developed with an enhanced chemiluminescence plus kit (Amersham Bioscience, Aylesbury, UK).
Scientific RepoRts | 5:10256 | DOi: 10.1038/srep10256 Dot blotting analysis. Dot blotting analysis was performed as previously described 17 . Briefly, nitrocellulose membrane was divided into 1.5 cm grid lines and 2 μ l of each sample was spotted onto the membrane with air-dry. The membrane was blocked in 10% milk TBST solution overnight. Then the membrane was incubated with anti-oligomer antibody A11 (Invitrogen, 1:1000) or anti-Aβ 1-17 antibody 6E10 (Sigma, 1:1000) with gentle shaking for 1 h. After three washes with TBST, the membrane was incubated with secondary antibodies for 1 h, developed with an enhanced chemiluminescence plus kit and quantified by using ImageJ. Circular dichroism spectroscopy. Circular dichroism (CD) spectroscopy was performed as previously described 18 . Briefly, synthetic Aβ 1-42 was dissolved in HFIP to 1 mg/ml, evaporated under vacuum, and reconstituted in DMSO to 5 mM. Then Aβ 1-42 was diluted to 20 μ M by using 10 mM sodium phosphate (pH = 7.4), and incubated with or without 1 μ M bis(heptyl)-cognitin. CD spectra were acquired at 15 min, 6 h, 1d and 2d after incubation at 37 °C. CD measurements were made by removing a 200 μ l aliquot from the reaction mixture, adding the aliquot to a 1 mM path length CD cuvette and acquiring spectra in a J-805spectropolarimeter (JASCO, Tokyo, Japan). Following temperature equilibration, spectra were recorded from ~195-240 nm at 0.2 nm resolution with a scan rate of 100 nm/min. Raw data were manipulated by smoothing and subtraction of buffer spectra according to the manufacturer's instructions.
Transmission electron microscopy. Assay of Aβ 1-42 oligomers formation using Transmission electron microscopy (TEM) images was performed as previously described 5 . Briefly, synthetic Aβ 1-42 was dissolved in HFIP to 1 mg/ml, evaporated under vacuum, and reconstituted in DMSO to 5 mM. Then Aβ 1-42 was diluted to 20 μ M by using 10 mM sodium phosphate (pH = 7.4), and incubated with 1 μ M bis(heptyl)-cognitin. The TEM samples were prepared by placing 2 μ l of the pre-incubated solution on a carbon-coated grid. The samples were stained with 1% uranylacetate and then placed on a clean paper for removing excess staining solution. The grids were thoroughly examined using JEM-2011transmission electron microscope (JEOL, Tokyo, Japan).

Molecular docking analysis.
The solution NMR structure of Aβ 1-42 assemblies (PDB: 2BEG) 19 was taken from PDB and prepared for molecular modeling using the Internal Coordinate Mechanics method (ICM-pro 3.6-1d, MolSoft L.L.C., La Jolla, CA) 20 . According to the ICM method, the molecular system was described by using internal coordinates as variables. Energy calculations were based on the ECEPP/3 force field with a distance-dependent dielectric constant. The biased probability Monte Carlo minimization procedure was used for global energy optimization.
ICM docking was performed to find out the most favourable orientation. The resulting trajectories of the complex between the small molecules and Aβ 1-42 were energy minimized, and the interaction energies, which are expressed in kJ/mol, were computed. The compound was docked three times and the minimum of the three scores was used. Aβ 1-42 oligomers administration and drug treatment. Aβ 1-42 oligomers administration was performed as previously described with modification 21 . Briefly, male ICR mice (20-26 g) anesthetized with chloral hydrate were positioned in a Narishige stereotaxic instrument, and 1 μ l Aβ 1-42 oligomers (1 μ g/μ l) were injected into the bilateral hippocampi. Sham control mice were infused with the vehicle. Both bis(heptyl)-cognitin and tacrine were dissolved in saline.
Morris water maze task. The water maze apparatus consisted of a circular pool 110 cm in diameter, filled with water at 23 ± 2 °C to cover a platform. The platform always resided in the center of southwest quadrant except on the last day. Each mouse's swimming was monitored by a video camera linked to a computer-based image analyzer. Learning performance was tested for 5 consecutive days beginning at 12th day after the infusion of Aβ 1-42 oligomers. Each mouse was trained to find the platform, with two trials a day. In each trial, the time required to escape onto the hidden platform was recorded. On day 5 of training, a probe trial was made by removing the platform and allowing the mice to swim for 90 s in search of the platform. Swimming time and distance in each of the four quadrants in the pool were calculated as percentages of the totals. A persistent preference for the quadrant previously occupied by the platform was taken to indicate that the mice had acquired and remembered the spatial task.
Data analysis and statistics. The data are expressed as the means ± SEM. Statistical significance was determined by ANOVA with Dunnett's test in the case of multiple comparisons with control or Tukey's test. Differences were accepted as significant at p < 0.05.
To further examine the threshold concentration of bis(heptyl)-cognitin for the prevention of

Blockage of α7nAChR could not abolish the prevention of Aβ 1-42 oligomers-induced inhibition of LTP caused by bis(heptyl)-cognitin. AChE inhibitors could enhance HFS-induced LTP
and prevent Aβ -mediated LTP inhibition via potentiating α 7nAChR and activating downstream PKA pathway [26][27][28] . In our study, MLA, a specific α 7nAChR inhibitor, and KT5720, a specific inhibitor of PKA, could block the enhancement of HFS-induced LTP and the prevention of Aβ 1-42 oligomers-induced inhibition of LTP by tacrine (Fig. 2E,F). Interestingly, although MLA and KT5720 reduced the increase of HFS-induced LTP caused by 3 μ M bis(heptyl)-cognitin, neither of these agents affected the protection of bis(heptyl)-cognitin on Aβ 1-42 oligomers-induced inhibition of LTP (Fig. 2E,F). Moreover, MG624 (a specific α 7nAChR inhibitor) and H89 (a specific PKA inhibitor) could not abolish the protection of bis(heptyl)-cognitin on Aβ 1-42 oligomers-induced inhibition of LTP (Fig. 2F). To date, none of these inhibitors altered baseline neurotransmission or the induction of LTP by HFS in our experiments.
Bis(heptyl)-cognitin, but not tacrine, prevents Aβ 1-42 oligomers-induced synaptotoxicity in primary hippocampal neurons. It is well established that Aβ 1-42 oligomers not only inhibit synaptic plasticity, but also induce synaptotoxicity in hippocampal neurons 13 . To further study the effects of bis(heptyl)-cognitin on the prevention of synaptic impairments, an immunostaining-based quantitative study was used. At 14 day in vitro (DIV), primary mature hippocampal neurons were exposed to Aβ 1-42 oligomers for 4d. Aβ 1-42 oligomers (0.1-1.5 μ M) caused a concentration-dependent reduction in the length of β III-tubulin positive neurites with a threshold concentration of 1 μ M (Fig. 3A).
To determine the morphology of Aβ 1-42 assemblies in the samples, TEM was used. Globular Aβ 1-42 oligomers with diameters of about 10-20 nm were detected predominantly in the untreated Aβ 1-42 samples (Fig. 6). In contrast, mostly large chain-like assemblies were observed in the bis(heptyl)-cognitin-treated samples (Fig. 6). These chain-like assemblies were detected even at 15 min after bis(heptyl)-cognitin and Aβ 1-42 co-incubation. Globular oligomers were mainly observed in the tacrine-treated samples (Fig. 6). Bis(heptyl)-cognitin binds favorably to the hydrophobic clefts of Aβ assemblies. To elucidate the mechanism underlying the alteration of Aβ assembly by bis(heptyl)-cognitin, we performed molecular docking analysis by using ICM-pro 3.6-1d molecular docking algorithm. Since no co-crystal structure of inhibitor with Aβ was available, the solution NMR structure of Aβ assemblies (PDB: 2BEG) was taken from PDB and used for docking analysis. In our model, no detectable hydrogen bonds between small molecules (bis(heptyl)-cognitin and tacrine) and Aβ assemblies were recorded. The interactions between small molecules and Aβ assemblies were mainly governed by hydrophobic interactions. Bis(heptyl)-cognitin was predicted to bind favorably to the hydrophobic clefts formed by Gly33-Met35 and Met35-Gly37 on the surface of Aβ 1-42 (Fig. 7). Surprisingly, bis(heptyl)-cognitin situated longitudinally to Aβ assemblies where its two tacrine subunits tied up four to five units of Aβ 1-42 molecules that are linked by the heptyl linkers, while the related monomer tacrine was only predicted to interact with two Aβ molecules (Fig. 7). Collectively, our results suggested that bis(heptyl)-cognitin presumably interacted with the hydrophobic pockets (Gly33-Met35 and Met35-Gly37) through multiple hydrophobic interactions which confers stabilizing powers and assembly alteration effects on Aβ .

Bis(heptyl)-cognitin reduces cognitive impairments after infusion of Aβ oligomers in mice.
Previous studies have shown that Aβ 1-42 oligomers lead to the learning and memory impairments in mice 33,34 . In our study, Morris water maze was used to evaluate spatial memory of mice with administration of Aβ 1-42 oligomers by intra-hippocampal infusion (Fig. 8A). In the acquisition trials, typical swimming paths on the fourth training day and quantitative escape latencies indicated that mice treated with Aβ 1-42 oligomers took longer time to find the platform than did the vehicle-treated mice (p < 0.01, Fig. 8D). This prolongation of latency was significantly shortened by bis(heptyl)-cognitin at the concentrations of 0.1 and 0.2 mg/kg, and tacrine at the concentrations of 2 mg/kg (p < 0.01, Fig. 8D). In the probe trials, the swimming distance in the quadrant that had held the hidden platform was used to estimate performance. The swimming distance in the probe quadrant is longer in groups of mice treated with Aβ 1-42 oligomers plus bis(heptyl)-cognitin (0.1, 0.2 mg/kg) and Aβ 1-42 oligomers plus tacrine (2 mg/kg) than the group of mice treated with Aβ 1-42 oligomers alone (p < 0.01, Fig. 8E). Neither Aβ 1-42 oligomers nor drugs altered swimming speed (Fig. 8F).

Discussion
Bis(heptyl)-cognitin was first reported as a potent AChE inhibitor 8 . Previous studies have reported that synaptic transmission in the hippocampus is enhanced by the activation of α 7nAChR and its downstream PKA pathway 35 . Inhibition of AChE could lead to the accumulation of acetylcholine in the synaptic cleft, and result in the activation of α 7nAChR and the enhancement of normal LTP. We have shown that 1) bis(heptyl)-cognitin and other AChE inhibitors increase HFS-induced LTP in hippocampal slice; and 2) the enhancement of LTP by bis(heptyl)-cognitin and other AChE inhibitors could be abolished by α 7nAChR antagonists and PKA inhibitors. These results suggested that bis(heptyl)-cognitin, as well as other AChE inhibitors, increased HFS-induced LTP via activating α 7nAChR/PKA pathway. We have further compared the prevention of bis(heptyl)-cognitin on Aβ oligomers-induced inhibition of LTP with tacrine and donepezil. All these AChE inhibitors prevented Aβ oligomers-induced inhibition of LTP but with different threshold concentrations. It is published that α 7nAChR agonists could prevent the inhibition of LTP by Aβ 1-42 oligomers, which could be abolished by α 7nAChR antagonists, suggesting that the activation of α 7nAChR prevents Aβ 1-42 oligomers-mediated inhibition of LTP 36 . In our study, both α 7nAChR antagonists and PKA inhibitors abolished the prevention of Aβ oligomers-induced inhibition of LTP by tacrine, suggesting that these AChE inhibitors prevent Aβ oligomers-induced inhibition of LTP via the activation of α 7nAChR/PKA pathway. These results are in accordance with previous studies showing that typical AChE inhibitors prevent Aβ -impaired LTP via potentiating α 7nAChR/PKA pathway 26,36 .
Interestingly, bis(heptyl)-cognitin, but not other AChE inhibitors, prevented Aβ oligomers-induced inhibition of LTP at concentrations that did not interfere with normal LTP. The protection of bis(heptyl)-cognitin on Aβ oligomers-induced inhibition of LTP could not be inhibited by α 7nAChR antagonists or PKA inhibitors. These bis(heptyl)-cognitin may act on target(s) other than α 7nAChR. The modification of Aβ oligomers-induced inhibition of LTP by bis(heptyl)-cognitin might be attributed to a mechanism different from that of typical AChE inhibitors.
Several studies have shown that agents which alter Aβ assembly could prevent Aβ -mediated LTP inhibition in hippocampal slices 5,37 . Moreover, curcumin (1 μ M), a known blocker of Aβ oligomerization, significantly prevented Aβ oligomers-induced inhibition LTP in our model (data not shown), suggesting that bis(heptyl)-cognitin might prevent Aβ oligomers-induced inhibition of LTP via altering Aβ assembly.
Apart from the inhibition of LTP, other aspects of synaptic impairments including the decrease in neurite length and the loss of synapses, were also reported to be highly correlated with cognitive impairments in AD 38 . By using a quantitative assay based on synapsin I and β III-tubulin co-immunostaining, we investigated the effects of Aβ oligomers on synaptotoxicity in primary mature hippocampal neurons 13,39 . Treatments of Aβ oligomers for four days displayed a concentration-dependent decrease in neurite length in hippocampal neurons with a minimal concentration of 1 μ M. However, shorter exposure (less than 2d) of 1 μ M Aβ oligomers did not exhibit any significant alternation in our study (data not shown). In our study, acute application of Aβ oligomers resulted in LTP inhibition in the hippocampal slice but not detectable synaptotoxicity in our primary culture system. Our previous studies have shown that acute Aβ oligomers-induced LTP impairment was mediated by the activation of MAPK pathways, and the stimulation of inducible nitric oxide synthase and superoxide 40,41 . However, these acute effects produced by Aβ oligomers may not be sufficient to cause detectable synaptotoxicity in our primary culture system. Chronic treatment of Aβ oligomers could induce long-term oxidative stress, tau protein hyper-phosphorylation, insulin signaling impairments etc, leading to detectable synaptotoxicity [42][43][44] . It has been reported that agents that alter Aβ assembly can reduce Aβ oligomers-induced synaptotoxicity 45 . We found that curcumin at 10 μ M almost completely prevented Aβ oligomers-induced synaptotoxicity. Using fluorescent microscopy and a powerful image analysis software, it was determined that bis(heptyl)-cognitin significantly prevented Aβ oligomers-induced synaptotoxicity. However, tacrine did not produce such effects. These results also match their effects on the prevention of Aβ oligomers-induced inhibition of LTP.
How could bis(heptyl)-cognitin prevent synaptic impairments induced by Aβ oligomers? We have shown that bis(heptyl)-cognitin significantly reduced the formation of Aβ oligomers and decreased the amount of pre-formed Aβ oligomers. Western blotting analysis further demonstrated that bis(heptyl)-cognitin preferably reduced the formation of Aβ oligomers. Previous studies have shown that Aβ assembly is a highly complex process that involves the sequential formation of different forms of amyloid assemblies, such as monomers, oligomers, protofibrils and mature fibrils 46 . Aβ monomers could assemble into unstructured oligomers, which convert over time into protofibrils and fibrils with a β -sheet-rich structure 47 . Our CD study showed that bis(heptyl)-cognitin froze secondary structure transitions during Aβ assembly. Our TEM data further suggested that Aβ monomers formed large chain-like assemblies but not small globular oligomers in the presence of bis(heptyl)-cognitin.
Tacrine, the monomeric form of bis(heptyl)-cognitin, even at a very high concentration, hardly altered Aβ assembly, suggesting that the dimeric form of bis(heptyl)-cognitin might be critical for altering Aβ assembly. A previous study has revealed a high free energy barrier between Aβ oligomers and large Aβ assemblies 48 . Although large Aβ assemblies have much lower free energy than Aβ oligomers, the  assembly from Aβ oligomers into large Aβ assemblies involves crossing over this high free energy barrier, implying that Aβ oligomers are quite stable and could not be easily assembled into large Aβ assemblies 48 .
Our molecular docking results suggested that tacrine might bind to Aβ molecule in large Aβ assemblies. Bis(heptyl)-cognitin situated longitudinally to large Aβ assemblies where its two tacrine subunits tied up four to five units of Aβ molecules. Therefore, the interaction between bis(heptyl)-cognitin and large Aβ assemblies might be stronger than that between tacrine and large Aβ assemblies, indicating that bis(heptyl)-cognitin might be more potent than tacrine to stabilize large Aβ assemblies. These results also suggested that bis(heptyl)-cognitin might decrease the high free energy barrier between other Aβ forms and large Aβ assemblies, and facilitate assembly from Aβ oligomers into large Aβ assemblies. Interestingly, although the concentrations of Aβ and bis(heptyl)-cognitin used in various in vitro experiments are different, the molecular ratios of bis(heptyl)-cognitin to Aβ in these studies are similar, being close to 1:5, which further support our molecular docking prediction that bis(heptyl)-cognitin might promote Aβ assembly from Aβ oligomers into large Aβ assemblies with one bis(heptyl)-cognitin molecule tied up four to five Aβ molecules.
Previously, many these compounds with symmetric structures are known to inhibit Aβ oligomerization. We conjured that the symmetric structure of these compounds, including bis(heptyl)-cognitin in particular, might facilitate their binding to the regular Aβ assemblies, leading to the inhibition of Aβ oligomers. However, the detailed mechanisms are under investigated in our laboratories.
Finally, we have investigated the effects of bis(heptyl)-cognitin in mice with intra-hippocampal infusion of Aβ 1-42 oligomers. Evidenced by the decrease in escape latency during the hidden platform sessions and the increase in swimming distance in the target quadrant as compared with the Aβ 1-42 oligomers-treated group, bis(heptyl)-cognitin could significantly ameliorate the impairments of learning and memory following Aβ 1-42 oligomers administration. Although tacrine could also reduce learning and memory impairments in this model, bis(heptyl)-cognitin has shown much higher efficacy (0.1 mg/kg vs. 2 mg/kg) and higher potency than tacrine in reducing these impairments.
In conclusion, we have demonstrated that bis(heptyl)-cognitin attenuated Aβ oligomers-induced synaptic and memory impairments. Our previous study has shown that bis(heptyl)-cognitin is a potent AChE inhibitor 8 . AD is a multifaceted neurodegenerative disorder. Therapeutic pharmacological approaches with one-drug-one-target are limited in their abilities to treat such a complex disease. One-molecule-multi-target drugs might provide greater therapeutic efficacy by concurrently targeting different sites in the brain of AD patients. AChE inhibitors have been proven to be effective to stabilize the symptoms of AD. Aβ oligomers are considered the main neurotoxin for synaptic dysfunctions and memory impairment in the AD progress. Therefore, it is likely that multi-functional molecules, such as bis(heptyl)-cognitin, which target AChE and Aβ oligomers simultaneously might not only minimize cholinergic dysfunction but also reverse the cognitive impairments in AD. In our study, we have demonstrated that bis(heptyl)-cognitin, a novel AChE inhibitor derived from tacrine, could reduce the neurotoxicity of Aβ oligomers, possibly via altering Aβ assembly, suggesting that bis(heptyl)-cognitin might be effective in the treatment of AD.