Medicinal Chemistry

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Medicinal Chemistry

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β-Amyloid-acetylcholine molecular interaction: new role of cholinergic mediators in anti-Alzheimer therapy?

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Background: For long time Alzheimer’s disease has been attributed to a cholinergic deficit. More recently, it has been considered dependent on the accumulation of the amyloid beta peptide (Aβ), which promotes neuronal loss and impairs neuronal function. Results/methodology: In the present study, using biophysical and biochemical experiments we tested the hypothesis that in addition to its role as a neurotransmitter, acetylcholine may exert its action as an anti-Alzheimer agent through a direct interaction with Aβ. Conclusion: Our data provide evidence that acetylcholine favors the soluble peptide conformation and exerts a neuroprotective effect against the neuroinflammatory and toxic effects of Aβ. The present paper paves the way toward the development of new polyfunctional anti-Alzheimer therapeutics capable of intervening on both the cholinergic transmission and the Aβ aggregation.

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M Grimaldi1, S Di Marino1, F Florenzano2, MT Ciotti3, SL Nori4, M Rodriquez1, G Sorrentino5,6, AM D’Ursi*,1 & M Scrima*1 1 Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy 2 Confocal Microscopy Unit, EBRIEuropean Brain Research Institute, Via del Fosso di Fiorano, 64, 00143 Rome, Italy 3 Institute of Cellular Biology & Neurobiology (IBCN), CNR, IRCSS Fondazione Santa Lucia, Via del Fosso di Fiorano 64–65, 00143 Rome, Italy 4 Department of Medicine & Surgery, University of Salerno, Via Allende, 84081 Baronissi (SA), Italy 5 Università degli Studi di Napoli Parthenope, Napoli, Italy 6 Istituto di Diagnosi e Cura Hermitage Capodimonte, Napoli, Italy *Author for correspondence: Tel.: +39 089 969748 Fax: +39 089 969602 [email protected] Tel +39 089 969176 Fax: +39 089 969602 [email protected]

First date submitted: 12 January 2016; Accepted for publication: 18 April 2016; Published online: 12 July 2016 Keywords:  acetylcholine • Aβ(25–35) • Alzheimer’s disease • amyloid peptide • anti-Alzheimer agent • cholinergic trasmission

Based on the prevailing idea that many neuropsychiatric diseases are caused by dysfunctions in neurotransmission, Alzheimer’s disease (AD) was attributed to a cholinergic deficit due to degeneration of the cholinergic projections from the basal forebrain (nucleus basalis magno cellularis of Meynert) to the cortex and hippocampus until the early

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1980s [1] . Subsequently, when it became clear that the pathogenesis of the disease was more complex than a simple neurotransmitter deficit, the so-called amyloid cascade hypothesis  [2] became the main research paradigm in AD pathogenesis. In its first formulation, the amyloid cascade hypothesis claims that the formation

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ISSN 1756-8919

Research Article  Grimaldi, Di Marino, Florenzano et al. of β-amyloid plaques composed of β-sheet-rich fibrillar aggregates of Aβ(1–40) and Aβ(1–42) peptides is the cause of AD pathophysiology. Aβ peptides are produced in the form of soluble molecules; however, in response to environmental factors, they aggregate into soluble, low-molecular-weight oligomers and highermolecular-weight protofibrillar oligomers, which, in turn, give rise to the insoluble fibrils that form amyloid plaques. Insoluble fibrils have long been considered responsible for the disease; however, Aβ oligomers, which have recently been recognized as the primary toxic species responsible for the neuronal impairments and the fibril deposits in AD, are considered by different authors either protective, or pathogenic, according to different mechanisms [3–8] . Additional evidence show that both oligomeric and fibrillar aggregates are toxic but following different mechanisms: for instance Aβ aggregates induce apoptosis by caspase 8 activation, whereas oligomers induce apoptosis principally by c­aspase 9 activation [9] . Based on the amyloid cascade hypothesis, thousands of substances have been screened [10] to search for anti-Alzheimer therapeutics that are able to control the Aβ conformation. These possible drug candidates, which differ in their chemical structures and chemical properties, interact with Aβ monomers to prevent aggregation or to accelerate the formation of fibrils in order to reduce the lifetime of toxic oligomers. Alternatively, these drug candidates interact with the Aβ fibrils to exert a disaggregating effect [11,12] . Using this approach, a set of nicotine and thiazolidine derivatives that can modulate the conformation of the synthetic Aβ(25–35) fragment were previously identified in our laboratory [13,14] . Based on similar approaches, omega-3 fatty acids were investigated for their ability to affect Aβ(25–35) aggregation [15,16] . The fragment including the 25–35 residues of the Aβ(1–42), namely Aβ(25–35), is the shortest fragment of the Aβ(1–42) peptide that remains biologically active. When aggregated, it exhibits large β-sheet structures, retaining the same physical, biological and toxicological properties of the full-length peptide. Accordingly, Aβ(25–35) has been extensively investigated as a model peptide because of its short length readily allows derivatives to be synthesized [17–20] . The cholinergic hypothesis has recently been revisited [21,22] based on the clear clinical evidence that the deficits in cholinergic neurotransmission are generally correlated with impairments of neuronal homeostasis in the hippocampus of Alzheimer’s patients. Moreover increasing data show that the amyloidogenic and cholinergic hypotheses, while being apparently different, share molecular pathways that converge at several points.

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Amyloid peptide plays a central role in producing the cholinergic deficit; it reduces acetylcholine (ACh) synthesis and affects nAChR levels. In turn, the loss of ACh was reported to be associated with the production of Aβ [23–26] [27] . Relationships between cholinergic activity and Aβ aggregation and toxicity have previously been reported by several in vitro and in vivo studies [28–31] . It has been shown that the aggregation of β-amyloid peptides to form Aβ oligomers compromises ACh neurotransmission, inducing cellular dysfunction, an imbalance in neurotransmitters signaling and, ultimately, the appearance of neurological signs [32] . Many evidences show the correlation between the Aβ aggregation and the acetylcholinesterase (AChE) activity. Several cholinesterase inhibitors are used in anti-Alzheimer therapy eliciting anti-amyloid effects even by a regulation of Aβ oligomerization [33] . Development of multitarget directed ligands acting as dual inhibitors of β-amyloid aggregation and AChE is an emerging strategy for improving the quality of the treatment against AD [34,35] . In particular, several in silico techniques such as quantitative structure–activity relationship (QSAR) have been used to provide useful and rational insight to facilitate the discovery of novel compounds and biomarker or imaging agents for improving diagnosis [36] . Aβ peptides have been proved to complex with AChE suggesting a possible link between ACh and Aβ peptides [37] . By investigating the multiple roles of ACh in the pathogenesis of AD, and in the context of the screening of small molecules as preventing of aggregation or as disaggregating effect [11,12] , we explored the hypothesis that in addition to its function in cholinergic transmission, ACh may exert a direct action on the amyloid peptide, preventing the formation of the toxic oligomeric/fibrillar species. Accordingly, first we investigated by CD spectroscopy the conformational behavior of Aβ(25–35) in the presence of ACh. Then, to validate our biophysical data on a biological scale, we measured the effect of ACh in protecting neuronal cells from the toxic action of the amyloid fragment and in modulating the neuroinflammatory response caused by phospholipase A2 (PLA2) activation and reactive astrogliosis. Experimental section Chemicals & reagents

The reagents were purchased from Sigma–Aldrich (Italy). Peptide synthesis

The Aβ(25–35) peptide, GSNKGAIIGLM, was manually synthesized by conventional solid-phase chemistry using the Fmoc/tBu strategy and subsequently

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β-Amyloid-acetylcholine molecular interaction: new role of cholinergic mediators in anti-Alzheimer therapy? 

purified, as previously reported [18,52,53] . The peptide was characterized on a Finnigan LCQ Deca ion trap instrument equipped with an electrospray source (LCQ Deca Finnigan, CA, USA). The samples were directly infused into the electrospray ionization (ESI) source using a syringe pump at a flow rate of 5 μl/min. The data were analyzed with the Xcalibur software. The sample purity was >98%. To ensure sample reproducibility and the removal of aggregated states, the dry peptide was pretreated with neat trifluoroacetic acid (TFA) (from Fluka; MO, USA) for 3 h, followed by a tenfold dilution with MilliQ water and lyophilization (Millipore, MA, USA) [54] . CD measurements

CD measurements were performed on a JASCO J-810 spectropolarimeter equipped with a temperature-controlled cell holder using a quartz cell with a 1.0 mm path length. The spectra were collected over range of wavelengths from 260–190 nm, with a bandwidth of 2.0 nm and a time constant of 8.0 s. The samples for CD analysis were prepared by dissolving the TFA-pretreated  [54] Aβ(25–35) in pH 5 water to give a final peptide concentration of 100 μM. The CD spectrum of Aβ(25–35) in the absence of ligands was recorded for comparison. The CD spectra of Aβ(25–35) were recorded in PBS and SDS micelles (8 mM c.m.c.) containing 500 μM ACh to obtain a final molar ratio of peptide:ACh of 1:5. To verify that the Ach concentration was unmodfied over the time, we recorded 1D 1H NMR spectra on Bruker 600 MHz. The CD spectra were analyzed using the CONTINN algorithm of the DICHROWEB online server to estimate the secondary structure content [39] . Preparation & treatments of the mixed NCCs

All procedures were approved by the Italian Ministry of Health (Rome, Italy) and performed in compliance with the guidelines of the NIH and the Italian Ministry of Health (D.L. 116/92). Mixed NCCs, containing both neurons and glial cells (astrocytes and microglia), were prepared from the brains of embryonic day 17–18 (E17/E18) embryos from timed pregnant Wistar rats (Charles River in 1975 from Charles River UK) as previously reported [55] . Briefly, the cortex was dissected in HEPES-buffered Hanks’ balanced salt solution and dissociated via trypsin treatment. The cells were plated on poly-L-lysine-coated 3.5-cm dishes at 1 × 106 cells/dish. After 2 days of culture in Neurobasal medium with the B-27 supplement, half of the medium was changed every 3–4 days. All experimental treatments were performed on 12-day in vitro (DIV) cultures in Neurobasal + B27 medium or in Neurobasal + ½ B27 medium. The culture cell composition

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was examined by immunocytochemical staining for neurons (NeuN antibody, Sigma, 1: 200), astrocytes (GFAP antibody, Sigma 1:400) and microglia (Iba1 antibody, Abcam 1: 200), with DAPI nuclear staining. The mixed cultures contain approximately 50% NeuN+ cells, 45% GFAP+ cells and 4% Iba1+ cells. The experimental groups included a CTRL that received only PBS; an Aβ group that received only Aβ(25–35); an ACh group (ACh) that received only ACh; and an ACh with Aβ (ACh+Aβ) group that received both ACh and Aβ. All treatments were performed for 48 h. Little amount of physostigmine were used to control the acetylcolinesterase activity. The 1 mg/ml Aβ (25–35) peptide stock solutions were prepared in PBS and stored at -20°C. Cell viability

Cell viability was assessed by counting the number of intact nuclei, according to a previously described method [55,56] . Briefly, the culture medium was removed and replaced with 0.5 ml of a detergent-containing lysing solution (0.5% ethylhexadecyldimethylammonium bromide, 0.28% acetic acid, 0.5% Triton X-100, 3 mM NaCl and 2 mM MgCl2 in PBS pH 7.4 diluted 1/10). After 2 min, the cells were collected, and the solution consisted of a uniform suspension of single, intact, viable nuclei that were then quantified by counting in a hemocytometer; the detergent-containing solution is able to dissolve the nuclei of the cells that are dying, while the healthy cells appear as phase-bright intact circles surrounded by a dark ring. Broken or damaged nuclei were not included in the count. Alternatively, the cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in 0.1 M Tris-HCl pH 7.4 for 5 min and then incubated with Hoechst 33258 (0.25 μg/ml) for 5 min at room temperature. After washing with PBS, the percentage of shrunken and condensed nuclei was assessed. Immunofluorescence

Following treatment, the neuronal cultures were washed twice with PBS and fixed in PBS containing 4% (w/v) paraformaldehyde for 15 min at room temperature. The cells were washed three times with PBS and incubated overnight at 4°C with a mixture of the following primary antibodies: mouse anti-NeuN (dilution 1:500; Millipore) or mouse anti-MAP2 (dilution 1:600; Cell Signaling) and rabbit anti-GFAP (dilution 1:500, Sigma). The unbound antibody was removed by three washes with 1X PBS for 5 min at room temperature. The bound antibody was detected by incubation with the Alexa Fluor 488-conjugated donkey anti-rabbit (1:500) and Alexa Fluor 555-conjugated donkey antimouse secondary antibodies (1:500) (Invitrogen) at 4°C

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Research Article  Grimaldi, Di Marino, Florenzano et al. for 2 h. The nuclei were counterstained with a 1:1000 dilution of 4′,6-diamidino-2-phenylindole dihydrochloride, DAPI dihydrochloride (DAPI; Sigma, MO, USA) in PBS for 10 min. After that, the cells were coverslipped using gel mount (Biomeda Corp., CA, USA). The controls included cells treated with secondary antibody alone, and no specific staining was observed. Confocal microscopy

The slides were examined under a confocal laser scanning microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany) equipped with 4 laser lines: violet diode emitting at 405 nm, argon emitting at 488 nm, helium/neon emitting at 543 nm and helium/neon emitting at 633 nm. In addition, a transmitted light detector for differential interference contrast (DIC; Nomarski) imaging was available. The confocal acquisition settings were identical between the control and experimental cases. The brightness and contrast of the images were adjusted to produce the figures, and the final figures were assembled using Adobe Photoshop 6 and Adobe Illustrator 10. Image analysis was performed using Imaris Suite 7.4® (Bitplane A.G., Zurich, Switzerland) or Image J 1.4 software on five different images derived from each experimental group. Image analysis was performed under visual control to determine the thresholds that subtract the background noise and take into account the neuronal and mitochondrial structures. During image processing, the images were compared with the original raw data to make sure that no structures were introduced that were not observed in the original data series and that the structures present in the original data series were not removed. Statistical analysis

The experiments were performed in triplicate and repeated at least three times. The data were expressed as the means ± SD, and n was the number of independent experiments. The statistical analyses were performed using SPSS 11.0.0 for Windows (SPSS Inc., USA). The significance of the effect was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s test for multiple comparisons. The significance level was set at p < 0.05 (*) and p < 0.01 (**). The differences in cell viability were expressed as the means ± SEM. The differences between treatments were examined using Student’s T-test and statistical significance was set at p < 0.05. LA-N-2 neuroblastoma cells and Aβ(25–35) treatment

The cells were maintained in Leibovitz’s L-15 medium with 15% FBS. The cells were grown in an incubator

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with 5% CO2 at 37°C. They were split at 95% confluence before every experimental procedure. The LA-N-2 cells were treated for 1 h with 5 μM Aβ(25–35) with and without 100 μM ACh at day 0. Each experiment was repeated three times. Protein extraction & western blotting

The cells were lysed with buffer containing 25 mM HEPES, 150 mM NaCl, 1% Triton and 5 mM EDTA. Ten μg of the total protein were separated on a 4–12% SDS-polyacrylamide gel and electrotransferred (iBlot system) onto nitrocellulose membranes. The nonspecific binding of proteins was prevented by treating the membranes with 5% nonfat dry milk in PBS for 1 h at room temperature. Western blot experiments were performed using two different types of primary antibodies: the first one was directed against the phosphorylated form of cytosolic phospholipase A2 (anti-pPLA2, dilution 1:500, LSBio), while the second one was used against the ribosomal protein RPL7 (anti-RPL7, dilution 1:1000, LSBio) which was used as loading control. After washing the membrane three times in 1× PBS, a horseradish peroxidase-conjugated anti-rabbit IgG was used (1:1000, R&D Systems) as secondary antibody. Subsequently, the nitrocellulose membrane was washed again and incubated with an enhanced chemiluminescence (ECL) solution (Bio-Rad), the signals were revealed using Alliance Mini (UVITEC Cambridge) system, and then quantified and analyzed using the UVITEC software. This program is able to quantify the intensity of each band, counting the number of pixels in the band and considering the background as zero. The background is the region of the membrane where bands are not presents. The intensity of the pPLA2 bands was normalized to the intensity of the loading control RPL7; and the levels of phosphorylated cPLA2 were expressed as percentages by comparing the relative intensity of the bands in the treated samples with those of the untreated samples (control), which were present on the same nitrocellulose membrane. Each experiment was repeated three times. Results & discussion Circular dichroism analysis

Circular dichroism (CD) spectra of Aβ(25–35) were recorded in phosphate buffered saline (PBS) solution in the presence and absence of ACh. The CD spectra were recorded immediately after the defibrillating treatment, according to the Zagorski procedure [38] , which was considered to be the starting time (t = 0). We acquired the CD spectra immediately after the treatment, 4 h after the treatment and then every 24 h, for 14 days. Figure 1A shows representative Aβ(25–35)

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β-Amyloid-acetylcholine molecular interaction: new role of cholinergic mediators in anti-Alzheimer therapy? 

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like environments [16,41] . Accordingly, we recorded the CD spectra of Aβ(25–35) in a sodium dodecyl sulfate (SDS) micelle solution chosen to mimic the membrane environment in solution (Figure 1B) . The quantification of the Aβ secondary structure in this medium confirms the previously described helix-inducing effect of micelle solutions [42] . Moreover, even in these conditions, ACh was effective in increasing and stabilizing the soluble helical content of the Aβ(25–35) secondary structures over time. Therefore, our CD data in PBS and in micelle solution provide clear evidence that ACh stabilizes the Aβ(25–35) random coil and helical structures. The β-strand structures are the initial seeds of insoluble β-fibrils, whereas the random coil and α-helix struc-

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Figure 1. Effect of Ach on Aβ(25–35) conformation. (A & B) CD spectra of Aβ(25–35) (100 μM) in PBS (A) or in sodium dodecyl sulfate micelle solution (B) (8 mM c.m.c.) in the presence and absence of ACh at times t = 0, t = 7 days and t = 14 days. Peptide/ACh final molar ratio 1:5. (C & D) Variations of the Aβ(25–35) secondary structure calculated according to evaluation of the CD curves using DICHROWEB in response to the addition of ACh in PBS (C) and SDS micelle solution (D). The quantitative estimation of Aβ(25–35) secondary structures based on CD curves was done using DICHROWEB [39] online web server. Aβ: Amyloid beta peptide; ACh: Acetylcholine; CD: Circular dichroism; PBS: Phosphate-buffered saline.

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Research Article  Grimaldi, Di Marino, Florenzano et al. tures represent the soluble forms of Aβ(25–35). Our data strongly suggest that ACh is effective in preserving the soluble form of Aβ(25–35) and therefore to reduce the initiation of the Aβ aggregation phenomena. Effects of ACh on Aβ-induced toxicity

We investigated the possible neuroprotective activity of ACh against Aβ(25–35) toxicity on mixed neural cell cultures (NCCs). Preliminary experiments on cultures treated with the reverse-sequence peptide Aβ(25–35) alone did not show an increase in cell death compared with the cultures treated with saline (data not shown). The NCCs incubated with 20 μM Aβ(25–35) for 48 h exhibited dramatically reduced cellular survival rates at approximately 40% of the CTRL values (Figure 2) . This Aβ(25–35) concentration was previously reported to be effective in provoking extensive cell death in NCCs [43] . To test the neuroprotective actions of ACh, we incubated the NCCs with different concentrations of ACh (10 mM–1 nM) and 20 μM Aβ(25–35) for 48 h. ACh was able to significantly reverse the Aβ(25–35) toxicity over a wide range of

concentrations from 10 mM to 1 μM. Among these concentrations, 1 μM ACh achieved the maximum protection against Aβ(25–35)-induced cell death, accounting for an 80% cell survival with respect to the untreated CTRL values. The administration of ACh alone did not induce toxic effect. At concentrations below 1 μM (0.1–0.001 μM), ACh did not show any neuroprotective effects against Aβ(25–35) toxicity. Effects of ACh on Aβ-induced neuronal death & astrogliosis

The administration of Aβ(25–35) has been widely reported to induce neuronal death and neuroinflammation in several experimental models. Depending on physical and morphological characteristics of oligomers, in neuroblastoma LAN cell cultures fibrillar Aβ aggregates induce apoptosis by caspase 8 activation, whereas oligomers induce apoptosis principally by caspase 9 activation [9] . In our experiments, we tested the ability of ACh to protect NCCs from Aβ(25–35)-induced neuronal damage and inflammation. Based on the findings from the

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Figure 2. Protective effect of acetylcholine against Aβ(25–35) neurotoxicity in mixed neural cell cultures. The histogram shows the quantification of the percentage of surviving cells compared with the control cells (white bar). The 12 cultures were treated with 20 μM Aβ(25–35) alone (black bar), both 20 μM Aβ(25–35) and decreasing concentrations of ACh (10 mM–1 nM; red bar), or acetylcholine alone (gray bar) for 48 h. Fourteen fields were selected for each treatment from each individual experiment and each experiment was performed in triplicate. The data represent the mean percentages (±SEM). t-Test p-value:***p < 0.0005 indicates a significant difference compared with the Aβ group. Aβ: Amyloid beta peptide; ACh: Acetylcholine.

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β-Amyloid-acetylcholine molecular interaction: new role of cholinergic mediators in anti-Alzheimer therapy? 

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Figure 3. Confocal microscopy images of mixed neural cell cultures treated for 48 h with 20 μM Aβ peptide (25–35) + 100 μM acetylcholine, 100 μM acetylcholine or phosphate-buffered saline (CTRL) and double immunostained for neuronal (NeuN) and astrocytic (GFAP) markers; the nuclei were counterstained (DAPI). The Aβ group (A–D) exhibited many condensed nuclei (A), indicating the presence of massive cell death. Several cells (B) exhibited displaced NeuN immunoreactivity in the cytoplasm, suggesting the loss of the nuclear integrity. Reactive astrocytes were present and displayed hypertrophic phenotypes, and, in some cases, they surrounded and contacted the NeuN-positive neurons. In the Aβ + ACh group (E–H), the ACh treatment was able to partially rescue the cell death and decrease astrocyte hypertrophy. In the ACh group (I–L), the administration of ACh alone was able to induce a low level of reactive astrocytosis and low-to-moderate cell death. In the CTRL group (M-P), the saline buffer administration did not trigger reactive astrogliosis, and low-to-moderate cell death was detected. Scale bar: 30 μm.Aβ: Amyloid beta peptide; ACh: Acetylcholine; CD: Circular dichroism; PBS: Phosphatebuffered saline.

cell viability assay showing a substantial protection over a wide range of concentrations (from 10 mM to 1 μM), we decided to infuse an ACh concentration of 100 μM, which is in the middle of the above-mentioned range of concentrations. The NCCs treated with 20 μM Aβ(25–35) for 48 h exhibited substantial neuronal death, as shown by immunostaining for the neuronal nuclei marker NeuN and the blue fluorescent dye 4´,6-diamidino-2-phenylindole dihydrochloride (DAPI) dihydrochloride used to stain the DNA (Figure 3A–D). The figure shows the presence of a dense cluster of fragmented and pyknotic DAPI-positive nuclei (Figure 3A), together with several NeuN-positive cells showing dislocation of the NeuN immunoreactivity from the nucleus to the cytoplasm. These data indicate the occurrence of extensive neuronal death at different degenerative stages in this experimental group. After the administration of 20 μM Aβ(25–35) and ACh 100 μM, neuronal death was substantially reversed (Figure 3E–H), as can be observed by the lack of either degenerating nuclei clusters

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and dislocated NeuN immunoreactivity in the neurons. The DAPI- and NeuN-positive nuclei in the ACh and CTRL groups exhibited the absence of dense clusters of degenerated cells and a preserved NeuN morphology. Effects of ACh on Aβ-induced astrogliosis

Neuroinflammation is a prominent feature of the AD brain, and considerable evidence indicates that glial inflammation plays a significant role in the progression of AD [44] . In response to injury, astrocytes become reactive (reactive astrogliosis) and attain functions different from their natural physiological response. Reactive astrocytes can be recognized by their morphological hypertrophy and by increased GFAP synthesis. It is generally believed that Aβ accumulates in the extracellular space, triggering reactive astrogliosis [45] . Cultured astrocytes have been shown to phagocytize and degrade Aβ. In vivo, reactive astrogliosis first becomes apparent in the vicinity of nascent Aβ plaques and progresses as the plaque load increases.

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Research Article  Grimaldi, Di Marino, Florenzano et al.

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PLA2 activity is relevant in the inflammatory responses associated with neurological disorders. Activated PLA2 catalyzes the cleavage of arachidonic acid from the sn-2 position of phospholipids, generating inflammatory mediators, such as prostaglandins, leukotrienes and

Conclusion In the present study, we tested the hypothesis that in addition to its role as a neurotransmitter, ACh may exert its action as an anti-Alzheimer agent through a direct interaction with the amyloid peptide, affecting the formation of the toxic oligomeric/fibrillar species. Using CD experiments, we studied the biophysical effect of ACh on the conformation of Aβ(25–35). Using biochemical experiments, we investigated the ability of ACh to protect the neuronal cells from the toxic action of the amyloid peptide and to modulate the neuroinflammatory response, which occurs via PLA2 activation and reactive astrogliosis. The CD experiments showed that the variations in the Aβ(25–35) secondary structure over time are consistent with a linearly increasing content of β-strand Aβ + ACh

Effects of ACh on Aβ-induced PLA2 activation

thromboxanes, which seem to be altered in the brains of patients with dementia [47] . We have previously showed that Aβ(25–35) treatment activates PLA2 in a human cholinergic neuroblastoma cell line (LA-N-2) [48] . Cytosolic PLA2 mediates neuronal apoptosis induced by soluble Aβ oligomers. More recently, D­esbene et al. confirmed our previous observations [49] . The cholinergic LA-N-2 neuroblastoma cells were treated with 5 μM Aβ(25–35) in PBS in the presence or absence of ACh. The cells were stimulated immediately after the defibrillating treatment according to the Zagorski procedure [38] . The levels of active PLA2 (phosphorylated at position 505) were measured. Figure 4 shows a western blot of the relative levels of phosphorylated PLA2 in the presence of Aβ(25–35). The experiments showed a significant 2.5-fold increase in the active form of PLA2 compared with the untreated cells. Analogous experiments performed in the presence of 100 μM ACh showed that ACh was able to blunt the activation of PLA2 induced by Aβ(25–35), and its activity was comparable to that measured in the untreated cells.

Aβ + ACh

In addition, reactive astrocytes seem to accumulate large amounts of the neuronal subtype of the nicotinic choline receptor (a7nAChR), which is known to have an exceptional high affinity for the Aβ peptide [46] . Based on these findings, we tested the protective effect of ACh on astrocyte-mediated inflammation, which is known to be induced by Aβ administration. The NCCs were challenged with 20 μM Aβ(25–35) for 48 h and were immunostained for GFAP and neuronal (NeuN) markers. A qualitative inspection of the morphological features by confocal microscopy revealed the presence of intense astrogliosis characterized by clusters of highly hypertrophic and ramified astrocytes in the Aβ group (Figure 3A–D), which, in some cases, were in contact with the surviving neurons. Some astrocytes displayed a globose cell body endowed with thick and shorter processes or, in other cases, with well-developed ramifications of different calibers, ranging from thick to thin. In the Aβ + ACh 100 μM group (Figure 3E–H), the astrocytes still appeared to be hypertrophic and ramified, although to a lesser degree when compared with the Aβ group. Globose astrocyte phenotypes were not encountered, while the stellate morphology endowed by the different caliber ramifications was common. The astroglial distribution in the clusters appeared less prominent. In the ACh 100 μM group (Figure 3I–L), the astrocytes were slightly reactive and endowed with a well-defined cell body and longer processes. In the CTRL group, the astrocytes displayed slender, sheet-like or filopodial processes that are morphologically typical of resting astroglia.

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Figure 4. Western blot of the relative levels of phosphorylated phospholipase A2 in the presence of 5 μM amyloid-β peptide (25–35) and 5 μM amyloid-β peptide (25–35) +100 μM acetylcholine. Western blot experiments were performed using the first antibody directed against the phosphorylated form of cytosolic phospholipase A2 (anti-pPLA2, dilution 1:500, LSBio), and the second one against the ribosomal protein RPL7 (anti-RPL7, dilution 1:1000, LSBio) which was used as loading control. The signals were revealed using Alliance Mini (UVITEC Cambridge) system, and then quantified and analyzed using the UVITEC software. Aβ: Amyloid-β; ACh: Acetylcholine; PBS: Phosphate-buffered saline.

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β-Amyloid-acetylcholine molecular interaction: new role of cholinergic mediators in anti-Alzheimer therapy? 

structures in the absence of ACh, which is opposite to the stabilized random coil and β-strand structures in the presence of ACh. In an SDS micelle solution, which was chosen to mimic membrane-like environment, ACh was shown to be effective in increasing and stabilizing the soluble helical content of the Aβ(25–35) secondary structures over time. CD spectroscopy experiments provide low-resolution data to comprehend the molecular mechanism by which Ach stabilizes the soluble form of Aβ(25–35). However considering the chemical nature of Ach, it is reasonable to hypothesize that, similarly to other amphipatic molecules such as hexadecyl-N-methylpiperidinium bromide [50] and ‘lipid-like’ mimetics [51] Ach may bind the surface of Aβ(25–35) precluding the formation of amyloid fibrils. We demonstrated that the ACh is able to significantly reverse the Aβ-induced astrocyte inflammation in NCCs; indeed, the ACh treatment partially rescued the cell death phenomena and decreased the astrocyte hypertrophy associated with NCC toxicity, with the maximum protection evident at 1 μM ACh. Under these conditions, we observed 80% of cell survival with respect to the untreated CTRL values achieved during Aβ(25–35)-induced cell death. Finally, ACh blunted the Aβ(25–35)-induced PLA2 activation in the cholinergic LA-N-2 neuroblastoma cells. Future perspective Taken together, our data support the hypothesis that in addition to its role in cholinergic transmission, ACh

Research Article

may control Alzheimer disease by directly interacting with the Aβ peptide. Due to its chemical nature, Ach has unfavourable pharmacokinetic properties and lability to esterase degradation. However, in a future medicinal chemistry view it may be considered the lead compound for the synthesis of new molecules characterized by polypharmacological profile, able to modulate cholinergic transmission and to control β-amyloid conformational state. Based on the ACh model, this result provides a new perspective that AD may be managed by designing poly-functional compounds that are capable of modulating cholinergic transmission and the Aβ aggregation state. Supporting Information CD spectra of Aβ(25–35) in PBS solution in absence and in presence of Ach. CD spectra of Aβ(25–35) in micelle solution in absence and in presence of Ach. Quantitative evaluation of CD curves.

Financial & competing interests disclosure This work was supported by a grant from MIUR (FIRB-MERIT RBNE08LN4P:006) to G Sorrentino. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary • Cholinergic and amyloid hypothesis converge at several points for the understanding and the management of Alzheimer disease. • Circular dichroism data indicate that acetylcholine (ACh) is effective in preserving the soluble form of Aβ(25–35) and to reduce the initiation of the amyloid beta peptide (Aβ) aggregation phenomena. • On mixed neural cell cultures treated with toxic Aβ(25–35), ACh induced 80% cell survival and reversed neuronal death. • Astrocytes treated with toxic Aβ(25–35), in the presence of ACh 100 μM were endowed with a well-defined cell body as compared with CTRL group, displaying slender, sheet-like or filopodial processes that are morphologically typical of resting astroglia. • ACh was able to blunt the activation of phospholipase A2 induced by Aβ(25–35), and its activity was comparable to that measured in the untreated cells. • Ach may be considered the lead compound for the synthesis of new molecules able to modulate cholinergic transmission and β amyloid conformational state.

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