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AF710B, a concomitant activator of M1 muscarinic and sigma-1 receptors: possible disease-modifying properties in McGill-R-Thy1-APP rats Hall, Hélène 1 ; Iulita, M. Florencia 1 ; Ducatenzeiler, Adriana 1 ; Fisher, Abraham 2 & Cuello, A. Claudio 1 1 Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada 2 Israel Institute for Biological Research, Ness Ziona, Israel [email protected] Richard and Edith Strauss Canadian foundation - There is currently no cure for AD, the leading cause of dementia in the elderly, making it one of the largest unmet medical needs in neurology. The few therapeutic options available are only symptomatic and provide modest relief. They are mostly geared towards increasing cholinergic synaptic transmission by inhibiting the breakdown of acetylcholine. - Activation of muscarinic receptors can reduce the production of Aβ by shifting the amyloid precursor protein (APP) processing towards a non- amyloidogenic pathway (1). - Original attempts using first generation M1 muscarinic agonists were discontinued due to lack of true subtype specificity and/or peripheral effects, which prevented for a long time full exploration of the therapeutic potential of this pharmacological approach in vivo (2). Introduction AF710B: a novel drug with dual pharmacological target (3) selective allosteric modulator of M1 muscarinic receptors sigma 1 receptor agonist Fisher. 2012. J Neurochem 120 Suppl 1, 22. References: (1) R. M. Nitsch et al. 1992. Science 258, 304-307. (2) A. Fisher. 2012. J Neurochem 120 Suppl 1, 22-33. (3) A. Fisher et al. 2015. Neurodegener Dis. (4) W. Leon et al. 2010. Journal of Alzheimer's disease 20, 113-126. To investigate whether long term chronic selective stimulation of M1 and sigma 1 receptors reduces an existing severe AD-like neuropathology and reverts cognitive impairment in McGill-R-Thy1- APP transgenic rats. Aim 0 no drug Age 12-14 mo 5 months 6 months Age 18-20 mo behavior: Novel Object, Morris Water Maze post-mortem analyses daily drug treatment AF710B 10μg/kg orally Experimental design 5 week washout - McGill-R-Thy1-APP transgenic (tg) and wild type (wt) rats of 12 months of age were orally treated with AF710B (10 μg/kg; wt n=8, tg n=4) or vehicle (PBS; wt n=7, tg n=13) for 4.5 months. After a wash-out period of 5 weeks, rats were submitted to behavioral tests designed to provide measurements of short-term and long-term hippocampal and cortical learning and memory (Novel Object Recognition and Morris Water Maze tasks), following which they were sacrificed and their brain extracted. - Western blot: hippocampal samples were sonicated in RIPA buffer containing a protease inhibitor and centrifuged at 13000rpm for 45min at 4C. 20ug protein of the supernatant were loaded on 15% SDS-polyacrylamide gels and wet-transferred to nitrocellulose membranes. After blocking in 5% milk in TBS-T, membranes were incubated with rabbit Iba1 antibody (1:500; Wako) overnight at 4C. Following incubation with peroxidase- conjugated secondary antibody, immunoreactivity was visualized using enhanced chemiluminescence detection system (ECL). Following 15min film exposure, intensity of the immunoreactive bands was quantified using densitometry (TotalLab Quant). - ELISA: cortical samples were homogenized in TBS containing a protease inhibitor and ultracentrifuged at 100000g for 1h at 4C. The pellet was resuspended in 5M guanidine HCl, 50mM Tris HCl pH8.0 and further ultracentrifuged. Levels of human Aß42 in TBS- soluble and guanidine fractions were measured using an ELISA kit (Invitrogen) following manufacturer instructions. - Immunohistochemical staining was performed on 40μm-thick free-floating sections. Briefly, endogenous peroxidase activity was quenched by incubating sections in 1% H 2 O 2 in PBS. Sections were blocked in goat serum for 1h and then incubated with mouse anti-McSA1 (1:2000), anti-NeuN (1:2000, Millipore) or rabbit anti-Iba1 (1:4000; Wako) antibodies overnight at 4C. Following incubation with secondary goat anti-mouse, or biotinylated goat anti-rabbit antibody, respectively, sections were incubated with MAP- HRP or ABC kit, respectively, and the staining visualized using DAB as a chromogen and 1%H 2 O 2 or Vector SG kit (for the NeuN staining). - Thioflavine S staining: sections were rinsed in distilled water, stained in Thioflavine-S solution (0.1% in 50% ethanol) for 5 min, differentiated in 50% ethanol, rinsed in distilled water, washed in PBS and mounted with Aqua/Poly Mount medium. - Semi-quantitative assessment of microglia: images of Iba1/NeuN stained sections (3 per animal, 2 images per section) were acquired by bright-field microscopy. On each image, two counting frames (CF; 100x200μm) were centered on the CA1 neuronal layer and the number of microglia within each CF counted. - Thioflavine-S stained sections (3 per animal) were visualized under an epi-fluorescence microscope. Acquired images were analyzed using ImageJ (Bethesda, USA). The subiculum was manually delineated on each image. Following subtraction of the level background staining using a median filter of 40 pixel, the mean gray value within the subiculum was obtained through the “analyse particles” function. Methods McGill-R-Thy1-APP rats (4) Hanzel et al, Neurobol Aging 2014 an ideal model for preclinical testing > Cognitive impairment as early as at 3 months of age > Early intraneuronal accumulation of Aβ progressing to full amyloid plaques > One single transgene: Swedish and Indiana mutations Leon et al, JAD 2010 6 mo 20 mo 13 mo Thioflavine S + 3 mo 20 mo > Pro-inflammatory process at the pre-plaque stage (6 months) COX2 Il1β - AF710B rescued the cognitive deficits present at the later stages of the AD-like amyloid neuropathology in McGill-R-Thy1-APP rats. - Importantly, the robust beneficial effects of AF710B were maintained following a 5- week interruption in the treatment, suggesting disease-modifying properties. - AF710B may act in part by modulating the processing of the APP metabolism via a reduction in toxic amyloid species. - AF710B shows anti-inflammatory properties. - Ongoing biochemical analyses will help determine additional molecular targets involved. Conclusion AF710B has anti-inflammatory properties 0 1 2 3 4 5 wt tg Iba1/GAPDH OD vehicle drug Iba1 Western blot in hippocampus Iba1 (17 kDa) GAPDH (35 kDa) * * *p<0.05 simple main effect following two-way ANOVA transgenic wild type vehicle drug Brown: Iba1 (microglia) Blue: NeuN (neuron) 100 μm counting frame 100x200μm 0 5 10 15 wt tg average number of Iba1+ cells/Counting frame vehicle drug * ** Microglia counts in CA1 *p<0.05 simple main effect following two-way ANOVA tg drug tg vehicle wt vehicle wt drug Heatmaps representing swim path on trial 4 of day 5 AF710B reverts the cognitive deficit in aged McGill rats Novel Object Recognition 0 20 40 60 80 100 120 0 1 2 3 4 5 latency to reach platform (s) days wt vehicle wt drug tg vehicle tg drug Average latency across days Morris Water Maze: learning phase 0.00 0.25 0.50 0.75 1.00 wt discrimination index vehicle drug tg *** ** **p<0.01; ***p<0.001 simple main effect following two-way ANOVA Latency on trial 4 of day 5 0 50 100 150 wt latency to reach platform (s) vehicle drug tg *** *** ***p<0.001 simple main effect following two-way ANOVA Figure 1. Discrimination index in the Novel Object Recognition task. After being exposed and familiarized to five objects on day 1, one of the familiar object is replaced on day 2 and time spent exploring the new object is recorded. Figure 2. Average latency in the learning phase of the Morris Water Maze task. In the Morris Water Maze, rats rely on distal cues to navigate in an open swimming arena to locate a submerged escape platform. Rats were trained for 5 consecutive days (4 trials per day) to locate the platform. The latency to locate the platform is recorded. AF710B reduces AD-like amyloid pathology in McGill rats Cortical Aβ42 (ELISA) 0 100 200 300 400 500 . concentration (μg/mg protein) vehicle drug Insoluble fraction *p<0.05 one tailed t-test 0 100 200 300 400 500 . concentration (ng/mlg protein) vehicle drug Soluble fraction * tg vehicle CA1 DG subiculum Thioflavine S+ plaques in the subiculum 0.00 0.25 0.50 0.75 1.00 mean gray value vehicle drug * *p<0.05 two tailed t-test tg drug 500 μm cortex hippocampus 1 cm tg vehicle tg drug McSA1 immunostaining Figure 4. Quantification of insoluble (guanidine fraction) and soluble (TBS fraction) Aβ42 by ELISA performed on cortical homogenates. Figure 3. Representative photomicrographs of brain sections from a vehicle (top) and drug-treated (bottom) tg animal. The Aβ immuno-reactivity detected with McSA1 antibody in cortex and hippocampus is cleared following treatment with AF710B. Figure 5. Thioflavine S positive plaques are reduced in AF710B- treated tg rats, as shown by reduction in mean fluorescent intensity (analysed with ImageJ). Figure 6. Left: representative photomicrographs of Iba1 (brown) and NeuN (blue) stained hippocampal sections. Right: semi-quantitative assessment of Iba1+ cells revealed that the number of microglial cells is increased in the CA1 area of tg rats, as compared to wt rats. AF710B reduces the number of microglia to the level of wt rats. Figure 7. Top: Iba1 representative Western blot in hippocampus homogenates. Bottom: results are expressed as relative optical density (OD).
