Cholinergic denervation exacerbates amyloid pathology and induces hippocampal atrophy in Tg2576 mice

Neurobiology of Disease 48 (2012) 439–446

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Cholinergic denervation exacerbates amyloid pathology and induces hippocampal atrophy in Tg2576 mice Francisco J. Gil-Bea a,⁎, Gorka Gerenu b, Barbara Aisa b, Ludmil P. Kirazov c, Reinhard Schliebs d, Maria J. Ramírez a, b a

Department of Cellular and Molecular Neuropharmacology, Division of Neurosciences, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain Department of Pharmacology, University of Navarra, Pamplona, Spain Institute of Experimental Morphology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria d Paul Flechsig Institute for Brain Research, University of Leipzig, Germany b c

a r t i c l e

i n f o

Article history: Received 8 May 2012 Accepted 22 June 2012 Available online 1 July 2012 Keywords: Cholinergic lesion Basal forebrain Hippocampus Neurodegeneration APP transgenic mouse

a b s t r a c t The main pathological hallmarks of Alzheimer's disease (AD) consist of amyloid plaques and neurofibrillary tangles. Hippocampal cell loss, atrophy and cholinergic dysfunction are also features of AD. The present work is aimed at studying the interactions between cholinergic denervation, APP processing and hippocampal integrity. The cholinergic immunotoxin mu p-75-saporin was injected into the 3rd ventricle of 6‐ to 8‐ month-old Tg2576 mice to induce a cholinergic denervation. Four weeks after cholinergic immunolesion, a significant 14-fold increase of soluble Aβ1–42 was observed. Cholinergically lesioned Tg2576 mice showed hippocampal atrophy together with degenerating FluoroJade-B-stained neurons and reduction of synaptophysin expression in CA1–3 pyramidal layers. We also found that cholinergic denervation led to reduced levels of ADAM17 in hippocampus of Tg2576 mice. Inhibition of ADAM17 with TAPI-2 (5 μM) decreased viability of hippocampal primary neurons from Tg2576 brains and decreased phosphorylation of downstream effectors of trophic signalling (ERK and Akt). The cholinergic agonist carbachol (100 μM) rescued these effects, suggesting that cholinergic deficits might render hippocampus more vulnerable to neurotoxicity upon certain toxic environments. The present work proposes a novel model of AD that worsens the patent amyloid pathology of Tg2576 mice together with hippocampal synaptic pathology and neurodegeneration. Drugs aimed at favoring cholinergic transmission should still be considered as potential treatments of AD. © 2012 Elsevier Inc. All rights reserved.

Introduction Basal forebrain cholinergic neurons (BFCNs) are a subpopulation of neurons with particular vulnerability to the pathology of Alzheimer's disease (AD) (Cuello et al., 2007; Geula et al., 2008; Mufson et al., 2008). Dysfunctional atrophy of these neurons, which in turn results in a severe loss of cortical and hippocampal innervation, might be the source for the malfunction of cholinergic system in the early and late-onset AD (Mufson et al., 2007, 2008; Schliebs and Arendt, 2011). Impairments in formation and retrieval of episodic memory observed in AD are partly due to this cholinergic dysfunction (Bartus et al., 1982; Bierer et al., 1995). Little is known about the cause for the particular vulnerability of BFCNs to AD. In vivo studies have shown some deficits of cholinergic neurotransmission in mutated APP-overexpressing mice (Apelt et al., 2002). Moreover,

⁎ Corresponding author at: Center for Applied Medical Research (CIMA), University of Navarra, Dept. of Cellular and Molecular Neuropharmacology, Division of Neurosciences, Avda. Pio XII 55, 31080 Pamplona, Spain. Fax: +34 948 194715. E-mail address: [email protected] (F.J. Gil-Bea). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2012.06.020

β-amyloid has been shown to mediate cholinergic neurotoxicity by binding to pan-neurotrophin receptor (p75NTR) (Sotthibundhu et al., 2008). Vice versa, it has also been reported that cholinergic neurotransmission plays a key role in the functional processes that lead to AD (Goekoop et al., 2006). In this sense, BFCNs exert a crucial role on hippocampus by modulating processes that trigger learning and memory such as long-term potentiation (LTP) or synaptic consolidation (Drever et al., 2011). Indeed, acetylcholine has not only been reported as a powerful presynaptic modulator of hippocampal excitatory transmission but exhibits an ability to promote long‐term synaptic plasticity (Fernandez de Sevilla et al., 2008), increases expression of proteins involved in synaptic consolidation, as BDNF and Arc (Gil-Bea et al., 2011), and enhances LTP in hippocampus (Auerbach and Segal, 1994; Ovsepian et al., 2004). Particularly, expression and processing of APP seems to be affected by an impaired cholinergic function (Schliebs, 2005), as forebrain cholinergic deficit is accompanied by induction of cortical APP mRNAs and increased levels of secreted APP in the CSF (Wallace and Haroutunian, 1993). Moreover, loss-of-function studies have reported that deletions of specific muscarinic or nicotinic receptors in mutated APP-overexpressing mice have worsen amyloid pathology (Davis et al., 2011; Medeiros et al., 2011).

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Dysfunction of BFCNs has also been reported in other dementing disorders, such as Parkinson's disease, progressive supranuclear palsy or traumatic brain injury (Arendt et al., 1983, 1984; Salmond et al., 2005). However, cholinergic dysfunctions observed in AD are accompanied by the occurrence of senile plaques and neurofibrillary tangles. It is still a matter of debate whether loss of cholinergic function or amyloid/tau pathology are primary events or both are two different phenomena that might interact to mediate the pathogenic development of AD. The purpose of this study was to perform a cholinergic denervation in a situation of amyloid pathology to study whether the cholinergic system might contribute to the aggravation of AD pathology. The transgenic Tg2576 mouse that over-expresses the Swedish mutation of human APP was intraventricularly injected with the selective cholinergic immunotoxin mu p75-saporin. Four weeks following the lesion, these animals demonstrated reduced hippocampal cholinergic innervation and increased amyloid pathology and hippocampal cell death and atrophy, a pathological feature that is usually not observed in a number of other transgenic animal models for AD (Gotz et al., 2004). It is suggested that amyloid pathology together with cholinergic denervation and hippocampal atrophy makes this model a better approach to AD pathology. Materials and methods Animals and surgery A total of sixty‐two 6- to-8‐month‐old mice were used. Fifteen mice were used to test the best concentration for the immunotoxin mu p75-SAP (Advanced Targeting Systems, CA, USA). Two doses of the toxin were tested, 2 and 4 μg, being the dose of 2 μg enough to manage a depletion of cholinergic markers in hippocampus (see Results) while sparing mortality. Once the dose was selected, 15 Tg2576 mice were subjected to the immunotoxin (Tg2576-SAP) and 10 sham-operated (Tg2576-Sham) with a sterile saline solution. Twelve non-transgenic littermates (wild-type) were also infused with the immunotoxin (WT-SAP), and 10 were sham-operated (WT-Sham). The total volume of immunotoxin or saline solution for the bilateral administration into the third ventricle was 2 μl. The stereotaxic injection was done at the following coordinates from bregma): AP −0.4 mm, ML ±1.2 mm, DV −2.6 mm. Four weeks after the surgical procedures mice were sacrificed by decapitation. One hemisphere was snap-frozen and further processed for histochemistry and “in situ” hybridization. The other hemisphere was dissected and stored at −80 °C until biochemical analysis. Some mice were perfused with 4% paraformaldehyde, and their brains cryoprotected in 30% sucrose, frozen, and cut into series of 20 μm for immunohistochemistry. All experiments were performed in accordance with animal care guidelines stipulated by the Animal Care and Use Committee at the University of Navarra and conformed to the European Community Council Directive (86/609/EEC). Analysis of the cholinergic marker AChE AChE activity was measured in the frontal cortex, hippocampus and striatum as previously described (Gil-Bea et al., 2005). AChE staining by histochemistry was performed following the protocol firstly described (Tago et al., 1986) in sections from hippocampus and prefrontal cortex. Assay of soluble and fibrillar β-amyloid peptide levels To differentiate between soluble and fibrillar plaque-associated Aβ, extraction was followed as previously described (Kawarabayashi et al., 2001). Cortical tissue (150 mg wet weight) was initially homogenized by sonication in 1 ml of 2% sodium dodecyl sulphate (SDS) in water containing a protease inhibitor cocktail 1 tablet in 50 ml solution (BOEHRINGER-MANNHEIM, Mannheim, Germany) and centrifuged at 100,000 ×g for 1 h at 4 °C. The supernatant containing the SDS-soluble

Aβ is further diluted to 1:40. The Aβ peptides present in the supernatant were analyzed using a sandwich ELISA system for either Aβ1–40 or Aβ1–42 (commercially obtained from BIOSOURCE Leuwen, Belgium) according to the manufacturer's protocol. The pellet containing non-SDS-extractable plaque-associated) β-amyloid was further extracted with 1 ml 70% formic acid (FA) in water by sonication. FA extracts were neutralized initially by 1:20 dilution into 1 M Tris-phosphate buffer, pH 11., and then diluted as necessary using the reaction buffer provided in the ELISA kit (BIOSOURCE, Leuwen, Belgium) for the determination of Aβ1–40 or Aβ1–42, according to the manufacturer's protocol. Thioflavin-S Thioflavin-S staining for amyloid plaques and neurofibrillar tangles was performed as firstly described (Schmidt et al., 1995). Western blotting Proteins (10 μg) were analyzed using standard SDS-polyacrylamide gel electrophoresis (PAGE; 8% polyacrylamide-gel minigels, Miniprotean; BIO-RAD, Munich). Nitrocellulose membranes were incubated overnight with monoclonal antibodies: ChAT (Millipore, USA), APP (22C11; Chemicon, USA), 6E10, phospho-ERK1/2, ERK1/2, phospho-Akt, Akt, β-actin (Sigma, Munich, Germany). Data were normalized to the corresponding expression level of actin in each sample. The relative abundance of protein was determined by densitometric quantification of immunoblots using Image J v. 1.43u (NIH Image) and expressed as percentage of its proper control. Fluoro-Jade B Fluoro-Jade B (Chemicon, USA) is a polyanionic fluorescein derivative that specifically binds to degenerating neurons regardless of the mechanism of cell death (Schmued and Hopkins, 2000). This procedure was performed following the manufacturer's instructions. Three sections of hippocampus were selected, were consecutively immersed in ethanol 100%, 70%, KMnO4 0,06% and destilled water. This step was followed by incubation with a solution of Fluoro-Jade B 0.0004% in acetic acid 0.1%, avoiding direct light from this step forward. After that, slides were rinsed three times in distilled water, allowed to dry overnight and cover slipped using DPX. Immunofluorescence Free floating sections were blocked for non-specific sites by addition of 10% of serum (from the species in which the secondary antibody was produced in) in PBS with 0.3% of Triton X-100 (PBS-Tx) for 30 min prior to incubations with the primary antibody. For 4G8 immunostaining, sections were incubated in 70% formic acid for 10 min to expose the epitope. Sections were incubated overnight at room temperature with primary antibodies (see Table 1 for information on primary antibodies). Sections were then incubated for 2 h with secondary antibody Alexa Fluor 594 (1:500; Invitrogen, USA) in PBS-Tx with 2% serum. Finally the sections were rinsed in PBS and mounted in fluorescence mounting medium (DAKO Cytomation, Glostrup, Denmark). For control staining the primary antibody was omitted. Nikon Eclipse E800 microscope and Nikon FDX35 camera were used to capture images. For the quantification of the area covered by the positive staining, a series of sections per mouse were processed and captured into images. Images were latter compiled, scaled and converted to 8-bit gray-scale for analyses of area in CA1–3 and DG layers by using the software Image J v. 1.43u (NIH Image). In situ hybridization The oligonucleotides probes to mouse ADAM17 mRNAs were 3'-tail labelled with αS[ 35S]dATP to a specific activity >1000 Ci/mmol

F.J. Gil-Bea et al. / Neurobiology of Disease 48 (2012) 439–446 Table 1 Primary antibodies used in the study. Antibody

Dilution

Company

4G8 6E10 22C11 ChAT GFAP ERK1/2 (phospho-T202/Y204) ERK1/2 Akt (phospho-S473) Akt Actin

1:10,000 1:1,000 1:1,000 1:200 1:500 1:1,000 1:1,000 1:1,000 1:1,000 1:1,000

Covance, USA Covance, USA Chemicon, USA Millipore, USA Sigma-Aldrich, Germany Sigma-Aldrich, Germany Sigma-Aldrich, Germany Sigma-Aldrich, Germany Sigma-Aldrich, Germany Sigma-Aldrich, Germany

(Sigma Genosis, UK). In situ hybridization for ADAM17 was performed as described (Aisa et al., 2009). The relative abundance mRNA in each region was determined by densitometric quantification of autoradiograms using an image analysis system (Scion Image v. 4.0.3.2, Scion Corporation, USA) correcting for non-specific signals. Data were reported in optical density (OD) units. MTT cell viability assay Cell viability was examined by means of the 3‐4,5dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. Hippocampal neurons from 16‐day-old Tg2576 or wild-type embryos were cultured in 48‐well plates with neurobasal medium supplemented with B27 (Invitrogen, Carlsbad, USA). At 14th day neurons were treated for three consecutive days with 5 μM of TAPI-2 (Calbiochem, Darmstadt, Germany) and/or 100 μM of carbachol (Sigma-Aldrich, Munich, Germany). After treatment, MTT (Sigma-Aldrich, Munich, Germany) was dissolved in PBS pH 7.4 at 5 mg/ml, added 10 μl per 100 μl of medium to the wells and incubated for 2 h at 37 °C in the cell incubator (5% CO2 and 37 °C). Then, the MTT solution was discarded and DMSO was added to the wells. Aliquots were transferred to a 96-well plate, and absorbance was measured at 595 nm in a plate reader. Results were expressed as percentages of non-treated control cells. Statistical analysis All data were presented as mean ± SEM. Differences between groups were tested using Student's t test when there were only two groups or two-way analysis of variance (ANOVA) when there were two grouping factors (genotype and lesion). In those cases where F of interaction was not statistically significant, the F value for the main effect was shown. In those cases where F of interaction was

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statistically significant, single effects were analyzed by a Fisher's least significant difference post hoc test when assuming homogeneity of variances or Dunnet test when assuming heterogeneity of variances. A P value b 0.05 was considered statistically significant. Statistical analyses were performed by SPSS version 15.0 software. Results Achievement of cholinergic denervation after lesion with the toxin mu p75-SAP In a pilot study, two doses of the toxin were tested in C57B6SJL mice, 2 and 4 μg. The highest dose of toxin achieved greater decreases in the activity of AChE in hippocampus when compared to sham-operated mice (24.90±3.60%, one-way ANOVA, F(2,11) =29.70, Pb 0.01) but caused 60% of mortality rate. However, the lowest dose of 2 μg managed a significant depletion of cholinergic markers in hippocampus (47.44±7.25%) while sparing death. None of the doses achieved a significant reduction of cholinergic markers in frontal cortex. Based in this preliminary data, the dose of 2 μg was selected. To fully characterize the action of the immunotoxin on BFCNs, ChAT immunostaining was performed in medial septum and AChE activity was assessed in hippocampus (Fig. 1). As observed in Fig. 1A, 2 μg of the immunotoxin induced dramatic depletion of ChAT-positive neurons in medial septum of wild-type and Tg2576 mice. Characterization of cholinergic denervation on cholinoceptive-rich regions, such as hippocampus, was performed by histochemical and biochemical analysis of AChE. This enzyme provides high reliability as indicator for loss of cholinergic innervation or cholinergic denervation (Gil-Bea et al., 2005; Lysakowski et al., 1989). Both WT-SAP and Tg2576-SAP presented reduced AChE activity in hippocampus (42.41±13.93 and 30.27±18.31, respectively; two-way ANOVA, F(1,21) =70.98, Pb 0.01; Fig. 1B) and decreased AChE-positive fibres (Fig. 1C). Effects of cholinergic immunolesion on β-amyloid levels in Tg2576 mice Cholinergic immunolesion significantly increased SDS-soluble Aβ1–40 (Student's t test, t(10) =−2.64, Pb 0.05), Aβ1–42 (Student's t test, t(10) =−3.90, Pb 0.01) and the ratio Aβ1–42/Aβ1–40 (Student's t test, t(10) =−4.19, Pb 0.01) in Tg2576 mice (Fig. 2A). However, fibrillar levels (formic acid-soluble) of both species remained unaffected (Student's t test, t(10) =−0.61, P=0.56, for Aβ1–40; t(10) =−0.86, P=0.41, for Aβ1–42; t(10) =0.29, P=0.78, for the ratio; Fig. 2B). Furthermore, thioflavin-S staining did not reveal amyloid plaques even after cholinergic immunolesion (data not shown), which is in agreement with original studies that reported plaque onset occurring at 9 months of age (Hsiao

Fig. 1. Characterization of cholinergic denervation. A. The cholinergic immunotoxin mu p75-SAP depletes ChAT-positive neurons in medial septum of WT and Tg2576 mice. Dashed line outlines the medial septum. Scale bar, 100 μm. B. AChE activity is decreased in hippocampus of both Tg2576 and wild-type mice by the immunotoxin (Two-way ANOVA, main effect of immunolesion, F(1,21) = 70.98, *P b 0.01). Errors bars indicate SEM. C. A detail of AChE staining in hippocampus proper shows that cholinergic immunotoxin reduced AChE-positive fibres in both Tg256 and WT mice. Hp, hippocampus; FrCx, frontal cortex. Scale bar, 100 μm.

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et al., 1996). However, immunohistochemisty with 4G8 antibody showed a higher presence of intracellular Aβ1–42 in the CA1 pyramidal cell layer of Tg2576-SAP animals (Student's t test, t(6)=1.97, Pb 0.05; Fig. 2C).

The reason for such an increase in the levels of soluble Aβ1–42 may be due to a modulation of APP processing. We then analyzed APP processing by Western blotting. Whereas full-length APP remained unchanged,

Fig. 2. Cholinergic immunolesion aggravates amyloid pathology in Tg2576 mice. A. Levels of soluble (SDS-soluble) Aβ1–40 and Aβ1–42 were found increased after cholinergic denervation. However, fibrillar (formic acid-soluble) species were not altered. Student's t test; t(10) =−2.64, *Pb 0.05 for soluble Aβ1–40; t(10) =−3.90, **Pb 0.01 for soluble Aβ1–42; t(10) =−4.19, **Pb 0.01 for the ratio Aβ1–42/Aβ1–40. B. Levels of C99 were found increased in hippocampus of Tg2576 mice after cholinergic denervation (Student's t test, t(8) =2.08, *Pb 0.05), while full-length APP and C83 remained unchanged. C. Immunohistochemical staining with 4G8 antibody showed significative augmented intracellular Aβ accumulation in Tg2576-SAP mice (Student's t test, t(6) =1.97, Pb 0.05.). Errors bars indicate SEM. Scale bar, 10 μm.

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levels of C99, but not C83, were found increased (Student's t test, t(8) = 2.08, Pb 0.05 for C99) in Tg2576-SAP when compared to Tg2576-Sham group (Fig. 2B). Effects of cholinergic immunolesion on hippocampal integrity of Tg2576 mice Gross examination revealed that cholinergic immunolesion induced severe hippocampal atrophy in Tg2576 mice (Fig. 3A). Quantification of cell layer areas in cresyl-violet-stained slices showed that CA1– 2 and CA3 pyramidal layer (two-way ANOVA followed by Dunnet test, F(1,14) =11.87, Pb 0.01 for CA1–2; F(1,14) =9.45, Pb 0.01 for CA3) were reduced in Tg2576-SAP mice when compared to Tg2576-Sham but DG granular layer remained unchanged (Fig. 3E; a detailed picture of CA1 pyramidal cell layer can be seen in Fig. 3B). Signs of neurodegeneration were observed by a high GFAP-ir astrogliosis. Tg2576-SAP showed much larger area covered by astrogliosis in the whole hippocampus proper (two-way ANOVA followed by Dunnet test, F(1,16) =78.50, Pb 0.01; Fig. 3C, F). In order to check whether the observed hippocampal atrophy in Tg2576-SAP mice accounts for cell death we used FluoroJade B staining. Notably, these animals presented some FluoroJadeB-stained

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cells specifically along the CA1–2 pyramidal layer (Fig. 3D, G). However, no FluoroJade B staining was found in either CA3 or DG (data not shown). Expression of synatophysin mRNA, a marker for synaptic density, was found significantly decreased in CA1–2 and CA3 cell layers of Tg2576 (two-way ANOVA, F(1,18) =7.24, Pb 0.05), but not in WT mice, after cholinergic lesion (Fig. 3H). Role of ADAM17 down-regulation in cholinergic immunolesion-induced hippocampal cell death in Tg2576 mice As shown by “in situ” hybridization, cholinergic immunolesion induced a decrease, almost significant, of ADAM17 mRNA levels in CA1– 2 pyramidal layers (Student's t test, t(8) = − 1.25, P = 0.08) and a significant decrease in CA3 layer (Student's t test, t(8) = − 2.02, P b 0.05) of Tg2576 mice but left unchanged the DG (Fig. 4A). Immunoblotting revealed that protein levels of ADAM17 were also decreased in the whole hippocampus proper of Tg2576-SAP (Student's t test, t(8) = − 3.52, P b 0.01; Fig. 4B). Besides APP, many other membrane-bound pro-ligands with trophic or survival properties, such as EGF, neuregulin 1β or TGFα, could be

Fig. 3. Effects of cholinergic immunolesion on hippocampal integrity. A, B. Cresyl-violet staining showed hippocampal atrophy in Tg2576 mice after cholinergic denervation. C, D. Cholinergic immunolesion induced an increase of GFAP-immunoreactive astrogliosis in the whole hippocampus proper and degenerating FluoroJade B-stained neurons in the CA1– 2 pyramidal cell layer, but not in pyramidal CA3 or granular DG layers. E. Quantification of cresyl-violet stained area covered by the pyramidal CA1–2 (two-way ANOVA followed by Dunnet test, F(1,14) = 11.87, *P b 0.01 vs. Tg2576-Sham) and CA3 layer (F(1,14) = 9.45, *P b 0.01 vs. Tg2576-Sham), as well as the DG granular layer. F. Quantification of GFAP-ir area in the whole hippocampus proper (F(1,16) = 78.50, *P b 0.01 vs. Tg2576-Sham). G. Quantification of FluoroJade B-stained area in the CA1–2 subfield. H. In situ hybridization revealed that cholinergic immunolesion decreased Synaptophysin mRNA expression in CA1–2 and CA3 cell layers (F(1,18) = 7.24, *P b 0.05 vs. Tg2576-Sham) of Tg2576 mice. Histograms show quantification of optical density (OD units) in H and stained area (arbitrary units) in E, F and G. Error bars indicate SEM. Scale bar, 200 μm in A, 50 μm in B, C and D, and 150 μm in H.

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substrate of ADAM17 (Dang et al., 2011). Thus, the possibility that a deficit of ADAM17 activity might be limiting the release of these neurotrophic factors and, therefore, might be rendering the hippocampus more vulnerable to toxic insults should not be ruled out. We treated hippocampal primary neurons with the inhibitor of ADAM17, TAPI-2, and performed MTT assay for the analysis of survival. Cultured neurons from Tg2576 mice reduced viability after treatment with TAPI-2 (71.1 ± 4.2% vs. saline; two-way ANOVA followed by Fisher's least significant difference test, F(1,23) = 4.77, P b 0.05; Fig. 5A), which was reversed by carbachol (93.9 ± 4.4% vs. saline; Fig. 5A). In contrast, cultured neurons from WT mice did not show any survival compromise after the same exposure to TAPI-2. In parallel, TAPI-2 reduced the active state of two key players involved in survival pathways, as phospho-ERK1/2 and phospho-Akt in Tg2576 primary neurons (46.8±4.2% and 68.3±3.5% vs. saline; two-way ANOVA followed by Fisher's least significant difference test, F(1,23) =5.01 and 4.82, respectively, Pb 0.05; Fig. 5B). Decrease in phospho-Atk was partially recovered by carbachol (84.5±2.0% vs. saline, respectively; Fig. 5B). Discussion Our data show that a cholinergic dysfunction, which was achieved by surgical immunolesion of BFCNs, aggravates the amyloid pathology and affects the integrity of hippocampus in the transgenic mouse known as Tg2576. The immunolesion was performed by the intracerebroventricular injection of the immunotoxin mu p75-SAP, an anti-p75 NTR antibody conjugated to saporin that was first developed in the last decade (Berger-Sweeney et al., 2001) to specifically kill BFCNs in mice. The assessment of the immunolesion-induced cholinergic denervation was performed by the analysis of the marker AChE. This enzyme provides high reliability as indicator for loss of cholinergic innervation or cholinergic denervation (Gil-Bea et al., 2005; Lysakowski et al., 1989). The injection of 2 μg was sufficient to produce a significant and selective cholinergic dysfunction in hippocampus and, as previously shown, must be devoid of collateral effects towards other neurotransmission systems (Berger-Sweeney et al., 2001; Hunter et al., 2004). Cholinergic immunolesion exacerbated amyloid pathology in 6‐ to 8‐month-old Tg2576 mice, in terms of enhanced levels of soluble Aβ1–42 and a seemingly increased intracellular accumulation. Whether cholinergic denervation could affect amyloid pathology at an older age is not assessed in the present study. Consistent with this result, genetic

deletion of either the acetylcholine M1-muscarinic (mAChR) or α7nicotinic receptor (nAChR) (Davis et al., 2010; Hernandez et al., 2010) resulted in higher amyloid plaque pathology. This data is further supported by a recent study demonstrating that deletion of M1-mAChR in 3xTgAD or Tg-SwDI mice increased number of plaques and elevated cerebrovascular deposition of fibrillar Aβ, which led to tau hyperphosphorylation (Medeiros et al., 2011). The administration of the antagonist for M1 receptor, dicyclomine, was also reported to exacerbate amyloid pathology, while its agonist, AF267B, rescued amyloid pathology in the 3xTg-AD model (Caccamo et al., 2006). Enhanced levels of amyloid in cholinergically lesioned Tg2576 mice may be due to either impaired Aβ clearance or altered APP processing. Cholinergic system has been mostly reported to affect the latter. The election of the age to perform the cholinergic immunolesion was based in this assumption, since amyloid pathology starts developing at 6–8 months (Hsiao et al., 1996) and therefore at this age cholinergic deficit, by affecting APP processing, could have greater impact. Previous reports have shown that selective activation of M1/M3- but not M2/M4-mAChR increased sAPPα secretion and decreased total Aβ formation both in vitro (Ensinger et al., 1993) and in vivo in AD patients (Hock et al., 2003). Facilitation of cholinergic neurotransmission by AChE inhibitors increased the non-amyloidogenic processing in animals (Ratia et al., 2010) and in neuroblastoma cells (Zimmermann et al., 2004). As well, scopolamine treatment in Tg2576 mice resulted in increased levels of fibrillar Aβ and decreased α-secretase activity (Liskowsky and Schliebs, 2006). Moreover, nAChR stimulation has also been observed to modulate APP processing (Seo et al., 2001). The fact that there is much evidence in literature about the regulation of the α-processing of APP by cholinergic system led us to study the effects of cholinergic immunolesion on the levels of α-secretase. As it will be further discussed, we particularly focused on ADAM17 for its broad spectrum of action to different substrates. Although some studies did not find any effects of muscarinic manipulation on levels of ADAM17 (Davis et al., 2010), others have reported strong increases of ADAM17 activity state or protein levels after muscarinic activation (Alfa Cisse et al., 2007). We found decreases on the expression levels of ADAM17 in hippocampus proper. Howerer, this reduction did not affect the α-processing of APP since levels of αsecretase-derived APP peptide C83 remained unaltered. As ADAM17 is not the constitutive α-secretase of APP, changes in its levels do not necessarily have to led to altered APP processing. ADAM10, which is

Fig. 4. Effect of cholinergic immunolesion on ADAM17 hippocampal levels in Tg2576 mice. A. “In situ” hybridization revealed that cholinergic lesion induced a slight decrease, although non-significant, of ADAM17 mRNA levels in CA1–2 pyramidal layers, and a significant decrease in CA3 layer (Student's t test, t(8) =−2.02, Pb 0.05). B. Immunoblotting shows decreased proteins levels of ADAM17 in hippocampal homogenates after cholinergic denervation (Student's t test, t(8) =−3.52, Pb 0.01). Histograms show quantification of optical density (OD). Error bars indicate SEM. Scale bar, 150 μm.

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Fig. 5. Inhibition of ADAM17 compromises viability of Tg2576 primary hippocampal neurons. A. Three‐day exposure to the inhibitor of ADAM17, TAPI-2 (5 μM), reduced the viability on MTT assay of hippocampal primary neurons from Tg2576 mice (two-way ANOVA followed by Fisher's least significant difference test, F(1,23) =4.77, *Pb 0.05 vs. saline), which was rescued by carbachol at 100 mM (#Pb 0.05 vs. TAPI-2). TAPI-2 did not alter the viability of WT primary neurons. B. TAPI-2 decreased the phosphorylation of ERK1/2 and Akt in primary neurons from Tg2576 mice (two-way ANOVA followed by Fisher's least significant difference test, F(1,23) =5.01 and 4.82, respectively, *Pb 0.05 vs. saline). Carbachol partially recovered the decrease in phospho-Akt (#Pb 0.05 vs. TAPI-2). Error bars indicate SEM. Dashed line indicates the level of 100% given to saline treatment.

the constitutive α-secretase of APP (Kuhn et al., 2010), might be compensating the lack of ADAM17. In contrast, it is important to note that β-secretase-derived APP peptide C99 was found increased by cholinergic immunolesion, which might potentially account for the increased Aβ1–42 production observed in the present study. We found that Tg2576 mice subjected to immunotoxic cholinergic dysfunction of BFCNs led to a reduction of hippocampal volume that was patent by gross examination. In particular, CA1–3 pyramidal layers were affected, showing reduced volumes and FluoroJade B-stained degenerative cells. As most of pyramidal cells within hippocampus formation express p75 NTR, degeneration of these cells and the subsequent hippocampal atrophy could be attributed to an unspecific toxicity brought about by the diffused action of the toxin. However, by the fact that FluoroJade B staining do not label degenerative neurons after 3 weeks from the toxic insult (Poirier et al., 2000), it is reasonable to argue that, 4 weeks after the lesion was performed, the observed hippocampal degeneration is an event secondary to the action of the toxin. In keeping with this finding, a previous study found some hippocampal atrophy, although to a minor extent and restricted to the CA3 pyramidal cell layer, in a Tg2576 mouse with deletion of the α7-nAChR

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(Hernandez et al., 2010). In parallel, it is important to note that expression of synaptophysin, which is as a marker for synaptic density and has been used to estimate synaptic pathology (Aisa et al., 2009; Eastwood et al., 1995), was also decreased in CA1–3 pyramidal layers. The reason by which cholinergic denervation induces hippocampal atrophy in Tg2576 but not in WT animals is hard to decipher. Several cellular and molecular mechanisms must participate, but undoubtedly, amyloid pathology must be actively contributing to this event. We hypothesized that cholinergic dennervation might be rendering hippocampus more vulnerable to certain toxic insults, as β-amyloid, by inducing a withdrawal of trophic input. ADAM17, which besides being an α-secretase is further known for its wide sheddase activity towards different types of substrates, mostly trophic factors among them (Dang et al., 2011; Gooz et al., 2006), could be a potential link between cholinergic dennervation and augmented vulnerability of hippocampus to amyloid toxicity. We tested this hypothesis ex vivo in primary neurons. Inhibition of ADAM17 resulted in reduced cell viability on hippocampal primary neurons from Tg2576 primary but not from wild-type mice, and that was notably reversed by the muscarinic agonist carbachol. Moreover, main downstream effectors of survival signalling cascade, such as phospho-ERK and phospho-Akt, were found decreased after ADAM17 inhibition and, as well, partially reversed by carbachol in Tg2576 neurons. It is still inappropriate to drag any conclusion from this experiment. First, the inhibitor TAPI-2 is not fully specific for ADAM17, so that other metalloproteases might be mediating in this process, and second, further in vivo studies are needed to validate the finding. Anyway, we have shed some light on a potential mechanism of cholinergic-induced neurodegeneration under pathological circumstances. The majority of AD research is carried out using APP transgenic mice that have increased Aβ levels in comparison to wild-type. Although these animal represent good models to mimic amyloid deposition and behavioral symptoms of AD, they are devoided of significant neuronal cell loss (Irizarry et al., 1997; Morgan, 2006; Takeuchi et al., 2000). Here we present a mouse model that, besides exacerbating the classical AD feature mimicked by Tg2576 mouse, resembles other important AD pathological traits as hippocampal atrophy and synaptic pathology. Furthermore, our data provide further support to encourage the research on cholinomimetic-based pharmacological approaches to treat AD.

Acknowledgments The expert technical assistance of Mrs. Renate Jendrek is gratefully acknowledged. The authors like to express their gratitude to Dr. Karen Hsiao Ashe, Department of Neurology, University of Minnesota, USA, for kindly providing three Tg2576 founder mice. L.K. and F.G.B. acknowledge the receipt of travel grants by the German Academic Exchange Foundation (DAAD) for a 3-month research stay at the Paul Flechsig Institute for Brain Research, Leipzig. The present study was supported by the Instituto de Salud Carlos III FIS project PI10/01748 (to M.J.R.) and the Interdisziplinäre Zentrum für Klinische Forschung (IZKF) Leipzig at the Faculty of Medicine of the University of Leipzig projects C18 and C29 to R.S). None of the authors have conflicts of interest.

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