AF710B, a concomitant activator of M1 muscarinic and sigma-1 … · 2016. 3. 14. · AF710B, a...
Transcript of AF710B, a concomitant activator of M1 muscarinic and sigma-1 … · 2016. 3. 14. · AF710B, a...
AF710B, a concomitant activator of M1 muscarinic and sigma-1 receptors:
possible disease-modifying properties in McGill-R-Thy1-APP ratsHall, Hélène1; Iulita, M. Florencia1; Ducatenzeiler, Adriana1; Fisher, Abraham2 & Cuello, A. Claudio1
1 Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada
2 Israel Institute for Biological Research, Ness Ziona, Israel
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 analysesdaily drug treatmentAF710B 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%
H2O2 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%H2O2 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 mo13 moThioflavine 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
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Iba1
/GA
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Iba1 Western blot in hippocampus
Iba1 (17 kDa)
GAPDH (35 kDa)
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*p<0.05 simple main effect following two-way ANOVA
transgenicwild type
veh
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Brown: Iba1 (microglia)Blue: NeuN (neuron)
100 µm
counting frame100x200µm
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Microglia counts in CA1
*p<0.05 simple main effect following two-way ANOVA
tg drugtg 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
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***p<0.001 simple main effectfollowing 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)
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Thioflavine S+ plaques in the subiculum
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A1
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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).