Download - 1311 Poster Val Def - ANAVEX...Chronic treatment with the tetrahydrofuran derivative ANAVEX2-73, a mixed muscarinic cholinergic and sigma-1 ligand, alleviates pathology in Tg2576 mice,

Chronic treatment with the tetrahydrofuran derivative ANAVEX2-73, a mixed muscarinic cholinergic andsigma-1 ligand, alleviates pathology in Tg2576 mice, a transgenic Alzheimer's disease model

Valentine Lahmy,1,2 Vanessa Villard,2 Tangui Maurice1,2

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1 University of Montpellier 2, Inserm U. 710, 34095 Montpellier, France; 2 Amylgen, 34095 Montpellier, France.

Tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride(ANAVEX2-73, AE37) is a novel compound, binding to muscarinic cholinergicreceptors and the sigma-1 chaperone with affinities in the low micromolar range.1 Wepreviously reported that the drug showed anti-amnesic and neuroprotective potentialsin the Alzheimer's disease (AD) mouse model induced by icv injection of oligomericamyloid(25-35) peptide. In particular, the drug attenuated the oxidative stress,caspases induction, cellular loss, Aß1-42 seeding, Tau hyperphosphorylation,mitochondrial dysfunction and learning and memory deficits observed within one weekafter Aß25-35 injection.2,3 The mechanism of action appeared to involve a synergicaction at muscarinic cholinergic receptors and the sigma-1 chaperone, a potentsensor/modulator of cellular responses.2

In the present study, we treated 10-month old Tg2576 mice, overexpressing thedouble swedish mutation of hAPP, with ANAVEX2-73, 3 mg/kg/day per os, in drinkingwater, during two months. 40 male and 60 female mice were used and data wereanalyzed for each gender. Water-treated Tg2576 showed significant alterations ofspontaneous alternation in Y-maze at 11 and 12 month of age and of place learning inthe water-maze at 12 months. These deficits were prevented by the ANAVEX2-73treatment. Animals were sacrificed after the water-maze procedure and used forbiochemical analyses.

A significant increase in DCF level and a decrease in synaptophysin content weremeasured in Tg2576, showing elevated oxidative stress and synaptic alteration,respectively. Both were normalized by the ANAVEX2-73 treatment. We confirmed apositive impact of the compound on brain plasticity by measuring by qPCR increasedexpression of Arc, Egr-1, NR2A, NR2B and PSD95 proteins in treated Tg2576 mice.However, the compound only marginally decreased SDS- and formic acid-soluble Aß1-42 brain contents, and only in male mice. Morphological analyses are in progress.

These first set of data confirmed the efficacy of the mixed muscarinic cholinergic andsigma-1 receptor agonist in a chronic transgenic mouse model of AD. The compoundmay allow an effective brain protection during the most aggressive phase of thepathology.

Animals. Tg2576 mice overexpressing hAPPSwe, and C57BL/6/SJL wildtype (WT) controls werefrom Taconic (Lelystad, Netherlands). They were used at 10 month of age. They were housed in groups of 3-5individuals or isolated with free access to food and water, except during behavioral experiments. They werekept in a regulated environment (23 ± 1°C, 40-60% humidity) under a 12 h light/dark cycle (light on at 8:00A.M.). All animal procedures were conducted in adherence with the 2010/63 EU Directive.

Drug and administration procedure. Tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanaminehydrochloride (ANAVEX2-73, AE37) was synthetized by Anavex Life Science. It was administered per os, in thedrinking bottle, at a daily dose of 3 mg/kg.

Spontaneous alternation performances in the Y-maze. Each mouse was allowed to explore theY-maze during a 8-min session. The series of arm entries and alternations (entries into all three arms onconsecutive trials) was measured. The percentage of alternation was calculated as (actual alternations / totalpossible alternations) x 100.

Place learning in the water-maze. The water-maze was a circular pool (140 x 40 cm high) withopacified water at 23°C. A transparent Plexiglas platform (10 cm) was immersed during acquisition. Swimmingcould be videotracked (Viewpoint), with trajectories being analyzed as latencies and distances. The softwaredivides the pool into four quadrants. Acquisition: between days 7 to 11 after peptide injection, training consistedin 3 swims per day for 5 days, with 20 min ITI, randomized start positions, 90 s swim duration and 20 s on thePF. The median latency was calculated each day and expressed as mean ± S.E.M. Retention: a probe test wasperformed on day 12. The PF was removed and each animal swum during 60 s. The swimming wasvideotracked and the time spent in the training (T) quadrant analyzed.

Tissue preparation. Mice were killed by decapitation and their brain rapidly removed on ice. Onehemibrain was fixed in a 4% paraformaldehyde solution for histological analysis and cortex and hippocampus ofthe other hemibrain were microdissected and rapidly frozen in liquid nitrogen and stored at -80°C. Cortex werehomogenized in a 1M Tris solution pH 7.6, containing 2% SDS and protease and phosphatase inhibitors(Roche). After sonication and centrifugation (13,600 g for 30 min), the supernatant was collected (SDS-solublefraction). The pellet was solubilized with 70% formic acid (FA) under agitation for 2 h. The resulting solution wasneutralized (pH 7.6) with a 1.5 M Tris solution pH 11 (SDS-insoluble fraction).

ELISA assays. Aß1-42 content was measured in both SDS-soluble and SDS-insolublefractions in the mouse cortex, using a kit (Invitrogen). The SDS-soluble fraction was diluted 1/25e and FA-soluble fraction was diluted 1/150e or 1/300e. Synaptophysin content was determined in the SDS-solublefraction according to manufacturer’s instructions (USCN Life Science inc.).

DCF fluorescence assay. 2’,7’-dichlorofluorescein diacetate (DCF-DA, 0.5µM) was applied to thesoluble hippocampus extract. DCF fluoresence was quantified (exc = 485 nm, em = 530 nm) using afluorescence multiwell reader.

qRT-PCR. RNA was extracted NucleoSpin RNA II kit (Marchery Nagel). RNA concentration wasmesured using a spectrophotometer at 260/280 nm. Total RNA was reverse transcribed with dNTPs, randomprimers and reverse transcriptase (Applied Biosystems). Incubations time were 10 min at 25°C, 120 min at 37°Cand 5 min at 85°C. Quantitative real-time PCR amplification reactions were performed using SYBR-Green(Roche) and a Light-Cycler (Roche). Appropriate primers were used for Synaptophysin, PSD-95, NR2A, NR2B,Arc, Egr-1 and ß-actin genes.

Histochemistry. Hemibrains were sliced in 30 µm coronal sections with a microtome (Thermo) and sliceswere stored at -20°C until used. Sections were incubated overnight with an anti-GFAP antibody (Sigma Aldrich).Sections when the incubated 2 hours with a biotinylated secondary antibody and 1 h with the avidin-biotincomplex (ABC Kit, Vector). The signal was detected with a diaminobenzidine kit according to the manufacturer’sinstructions.

Performances of Tg2576 mice in the Y-maze testafter 1 month of p.o. treatment with

ANAVEX2-73 (3 mg/kg/day)

Performances of Tg2576 mice in the Y-maze testafter 2 months of p.o. treatment with


10 month-old Tg2576 and WT mice were administered p.o. with tap water or ANAVEX2-73 (3 mg/kg/day) in drinking bottle. After 1 month, they were tested for spontaneousalternation in the Y-maze. Alternation performance (a, c) and number of arm entries (b, d)are shown for male (a, b) and female (c, d) mice. N = 5-14 for males and 8-16 forfemales. Two-way ANOVA: F(1,33) = 7.23, p < 0.05 for the genotype, F < 1 for thetreatment, F(1,33) = 2.61, p > 0.05 for the interaction, in (a); F < 1 for the genotype, F < 1for the treatment, F < 1 for the interaction, in (b); F(1,49) = 1.72, p > 0.05 for the genotype,F(1,49) = 4.29, p < 0.05 for the treatment, F < 1 for the interaction, in (c); F(1,49) = 2.66, p >0.05 for the genotype, F < 1 for the treatment, F(1,49) = 1.41, p > 0.05 for the interaction, in(d). * p < 0.05, ** p < 0.01 vs. V-treated WT mice; Student’s t-test.

Performances of male Tg2576 mice in thewater-maze test after 2 months of p.o. treatment

with ANAVEX2-73 (3 mg/kg/day)

1. Vamvakides A (2002). Ann Pharm Fr 60: 88-92.2. Villard V, Espallergues J, Keller E, et al. (2009) Neuropsychopharmacology 34: 1552-66.3. Villard V, Espallergues J, Keller E, et al. (2011) J Psychopharmacol 25: 1101-17.4. Lahmy V, Meunier J, Malmström S, et al. (2013) Neuropsychopharmacology 38: 1706-23.

ABSTRACT

MATERIALS AND METHODS

REFERENCES

Performances of female Tg2576 mice in thewater-maze test after 2 months of p.o. treatment


10 month-old Tg2576 and WT female mice were administered p.o. with tap water orANAVEX2-73 (3 mg/kg/day) in drinking bottle. After 2 months, they were tested for placelearning in the water-maze. Acquisition profiles (a) and time in T quadrant (b) andswimming speed during the probe test. N = 10-15 per group. Friedman repeated-measureANOVA: Fr = 21.8, p < 0.001 for WT/V, Fr = 19.0, p < 0.001 for WT/AN2-73, Fr = 10.6, p <0.05 for Tg2576/V, Fr = 23.4, p < 0.0001 for Tg2576/AN2-73. Two-way ANOVA: F(1,41) =2.65, p > 0.05 for the genotype, F < 1 for the treatment, F(1,41) = 5.13, p < 0.05 for theinteraction, in (b); F < 1 for the genotype, F(1,41) = 2.01, p > 0.05 for the treatment, F(1,41) =1.65, p > 0.05 for the interaction, in (c). * p < 0.05 vs. V-treated WT mice, # p < 0.05 vs. V-treated Tg2576 mice; Student’s t-test.

10 month-old Tg2576 and WT mice were administered p.o. during 2 months with tapwater or ANAVEX2-73 (3 mg/kg/day). After 2 month, synaptic markers were assayed inthe hippocampus using qPCR. N = 3-6 per group. * p < 0.05, ** p < 0.01 vs. V-treatedWT mice, # p < 0.05, ## p < 0.01 vs. V-treated Tg2576 mice; Student’s t-test.

Synaptic markers in the hippocampus ofTg2576 mice after 2 months of p.o. treatment


10 month-old Tg2576 and WT mice were administered p.o. during 2 months with tap wateror ANAVEX2-73 (3 mg/kg/day) in drinking bottle. After 2 months, Amyloid load was assayedin the hippocampus by measuring SDS-soluble and insoluble Aß1-42 contents: (a) male and(b) female mice. N = 6-12 per group. Kruskal-Wallis ANOVA: H = 2.25, p > 0.05, in (a); H =1.89, p > 0.05, in (b). In (c,d), pictures show typical congophilic deposits in the prefrontalcortex (1,4), the entorhinal-piriform cortex (2,5) and hippocampus (3).

10 month-old Tg2576 and WT mice were administered p.o. with tap water or ANAVEX2-73 (3 mg/kg/day) in drinking bottle. After 2 months, they were tested for spontaneousalternation in the Y-maze. Alternation performance (a, c) and number of arm entries (b, d)are shown for male (a, b) and female (c, d) mice. N = 6-12 for males and 10-17 forfemales. Two-way ANOVA: F(1,33) = 4.18, p < 0.05 for the genotype, F(1,33) = 2.57, p > 0.05for the treatment, F(1,33) = 1.65, p > 0.05 for the interaction, in (a); F(1,33) = 6.76, p < 0.05for the genotype, F < 1 for the treatment, F < 1 for the interaction, in (b); F(1,48) = 4.47, p <0.05 for the genotype, F(1,48) = 1.22, p > 0.05 for the treatment, F(1,48) = 1.41, p > 0.05 forthe interaction, in (c); F(1,48) = 11.98 p < 0.01 for the genotype, F < 1 for the treatment, F <1 for the interaction, in (d). * p < 0.05, ** p < 0.01 vs. V-treated WT mice; Student’s t-test.

10 month-old Tg2576 and WT male mice were administered p.o. with tap water orANAVEX2-73 (3 mg/kg/day) in drinking bottle. After 2 months, they were tested for placelearning in the water-maze. Acquisition profiles (a) and time in T quadrant (b) andswimming speed during the probe test. N = 6-12 per group. Friedman repeated-measureANOVA: Fr = 25.0, p < 0.0001 for WT/V, Fr = 15.6, p < 0.01 for WT/AN2-73, Fr = 3.44, p> 0.05 for Tg2576/V, Fr = 7.09, p > 0.05 for Tg2576/AN2-73. Two-way ANOVA: F(1,26) =5.17, p < 0.05 for the genotype, F < 1 for the treatment, F(1,26) = 5.62, p < 0.05 for theinteraction, in (b); F < 1 for the genotype, F(1,26) = 5.85, p < 0.05 for the treatment, F(1,26) =6.77, p < 0.05 for the interaction, in (c). * p < 0.05 vs. V-treated WT mice, # p < 0.05 vs.V-treated Tg2576 mice; Student’s t-test.

Amyloid load in the cortex of Tg2576 mice after2 months of p.o. treatment with ANAVEX2-73 (3

mg/kg/day)

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Oxidative stress in the hippocampus of Tg2576mice after 2 months of p.o. treatment with


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10 month-old Tg2576 and WT mice were administered p.o. during 2 months with tapwater or ANAVEX2-73 (3 mg/kg/day) in drinking bottle. After 2 month, oxidative stresswas assayed in the hippocampus using the DCF fluorescence assay: (a) male and (b)female mice. N = 5-14 for males and 10-16 for females. Two-way ANOVA: F(1,32) = 4.98, p< 0.05 for the genotype, F < 1 for the treatment, F(1,32) = 6.09, p < 0.05 for the interaction,in (a); F(1,47) = 4.76, p < 0.05 for the genotype, F < 1 for the treatment, F < 1 for theinteraction, in (b). * p < 0.05, ** p < 0.01 vs. V-treated WT mice; Student’s t-test.

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DISCUSSION

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Y-maze test

water-maze test

Sacrifice and biochemical analyses

AN2-73/water p.o.Month of age

…

ANAVEX2-73 has been shown to alleviate the toxicity and functional deficits inducedby icv injection of Aß25-35 peptide in mice. We reported that the learning and memoryimpairments, the oxicdative stress, neuroinflammation and cholinergic cell loss can beblocked by ANAVEX2-73 treatment. 2,3 We also showed that icv injection of Aß25-35

resulted in the mouse hippocampus in increased APP and Aß1-42 contents, andactivation of GSK-3ß leading to hyper- and abberrant phosphorylation of Tau protein.Thus the acute AD model reconcile the amyloido- and Tauopathies aspects of the ADpathology.4 The compound protected against both Aß seeding and GSK-3ß activation,suggesting that it may be effective in counteracting the progression of the diseaseunder chronic treatment.4

In the present study, we started to address this question by administering chronicallythe compound per os at a daily dose of 3 mg/kg in Tg2576 mice, a referencetransgenic model of AD. The results showed that the treatment alleviated significantlythe learning and memory deficits developing with time in the animals, at least in termsof spatial working memory (spontaneous alternation in the Y-maze) and long-termspatial reference memory (place learning in the water-maze). The treatment alsoprotected against the oxidative stress developing in the mouse brain and significantlyincreased the expression of functional markers, such as Arc or Egr1, or synapticplasticity markers or protein, such as NR2A or NR2B, the NMDA receptor subunits orsynaptophysin. A marginal effect was however measured on PSD95 mRNA.

Although the treatment appeared effective on funtional responses and biochemicalmarkers of the toxicity developing in Tg2576 mice, a first attempt to measure its impacton Aß load was negative. No significant change in soluble or insoluble Aß contents inthe mouse cortex preparations. Further investigations are therefore needed toestablish whether such drug treatment may indeed lead to disease-modifying effects,since its strong neuroprotective effect may be mitigated by a lower impact on Aß load.

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Probe test typical trajectories

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##