M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss

The current frontline symptomatic treatment for Alzheimer’s disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR–selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.

control sample were added to a 96-well plate then mixed with 180 μl of acetonitrile/methanol (1:1, v/v) containing internal standard. After mixing, the samples were centrifuged and the resulting supernatants were diluted 12.5fold with methanol/water (1:1, v/v)  The unbound fraction of drug in brain was estimated using fast gradient elution LC-MS/MS to estimate the percent of compound bound to brain over a 4.5 hour incubation period at 37ºC, while undergoing orbital shaking. The assay was performed using a HT dialysis micro equilibrium device, using dialysis membrane strips (molecular weight cutoff 12 -14k). At time 0 sample of brain homogenate was taken and samples taken from both the protein side and buffer side of the membrane after 4.5 hours of incubation. Drug concentrations were measured as described previously. Fraction unbound was calculated by dividing the concentration of the buffer side by the concentration of the protein side.

Prion infection of mice
Tg37 hemizygous mice were inoculated by intracerebral injection into the right parietal lobe with 1% brain homogenate of Rocky Mountain Laboratory (RML) prions aged 3-4 weeks as described previously (1). Control mice received 1% normal brain homogenate (NBH).

Fear conditioning learning and memory test
For behavioural testing of C57Bl6/NTAC or M 1 -KO mice, 8 to 12 week-old male mice were used. For prion-infected mice, and the relevant control mice, behavioural experiments were conducted on male mice between 9 and 10 weeks post inoculation (w.p.i.) with prion infected or normal brain homogenate. Mice were acclimatised to the behavioural room for at least two hours prior to the test. For fear conditioning, mice were placed in the conditioning chamber (Stoelting ANY-maze fear conditioning system) and after a 2 minute adaptation period, received three tone/foot shock pairings where the foot shock (unconditioned stimulus; US; 2 seconds; 0.4 mA) always co-terminated with a tone (conditioned stimulus; CS; 2.8 kH; 85 dB; 30 seconds). The CS-US pairings were separated by 1 minute intervals. After completion of training, the mice remained in the conditioning chamber for 1 minute and were then returned to their home cages. The next day, mice were placed back in the conditioning chamber, and time spent immobile was recorded for 3 minutes to assess context-dependent learning. Data were analysed using ANY-maze software.

Pain threshold
The mice were placed on the grid floor of the conditioning chamber (described above for fear conditioning) and were given 2 second foot shocks of increasing intensity (0.10-0.4 mA) at 10 second intervals. The level of the electric current needed to elicit startle, running/jumping and vocalisation responses was determined. All animals were foot shock-naive before the experiment and were not used for any subsequent tests.

Burrowing
Assessment of burrowing was conducted on mice from 7 w.p.i. The burrowing test involved mice being placed into individual cages with a plastic cylinder filled with 140 g of food pellets. Food remaining in the cylinders after 2 hours was weighed and the amount displaced ('burrowed') was calculated. Prior to the burrowing test mice were placed in the burrowing cage for a 2 hour period.
On the following day mice received vehicle or xanomeline (5 mg/kg) via i.p.
injection 30 minutes prior to the burrowing test. This was then repeated on a weekly basis.

Open field
This test was used to analyse general locomotor activity levels. The mice were placed into a clear, Perspex square arena (50 x 50 cm) and activity was tracked for a 10 minute period using ANY-maze software.

Elevated plus maze
The elevated plus maze apparatus consisted of four non-transparent arms (50 x 10 cm); two enclosed arms (with black walls of 30 cm height) that formed a

Generation M 1 mAChR phospho-serine 228 specific antiserum
Phosphorylation specific antibody that detected phosphorylation of the M 1 mAChR at serine 228 was raised against the peptide AALQGS (P) ETPGKG corresponding to amino acid residues 223-234 of the mouse M 1 mAChR. The 87 day program which included 4 immunisations was performed by Eurogentec. The resulting antiserum was purified against the immunising peptide.

Generation of M 1 mAChR antiserum
To generate antibodies for immunoprecipitation and detection of M 1 AChR protein, rabbits and rats were immunised with the peptide RDRGGKGQKPRGKEQ that corresponds to amino acids 334-348 of the mouse M 1 mAChR. The resulting antiserum was purified against the immunising peptide.

Membrane preparation
All of the following procedures were performed at 4°C. For prion mouse hippocampus preparations (NBH, prion 9wpi or 10wpi) 8-10 mice were pooled. For human control or AD frontal cortex ~1.5g of tissue from each patient was used (Oregon Alzheimer's Disease Center). Frozen tissue was homogenised in ice-cold HEPES buffer (10 mM HEPES, 1 mM EGTA, 1 mM dithiothreitol) plus 10% (w/v) sucrose and 1 x protease inhibitor cocktail by 20 strokes with a hand held glass/Teflon tissue grinder. The homogenate was centrifuged at 1000 xg for 10 minutes and the supernatant collected. The resulting pellet was rehomogenised and centrifuged again at 1000 xg for 10 minutes. The combined supernatant was centrifuged at 11,000 xg for 20 minutes and the resulting pellet resuspended in final storage buffer (HEPEs buffer 2 plus 1 mM MgCl 2 ) before a final centrifugation at 27,000 xg for 20 minutes. The final pellet was resuspended in final storage buffer before performing protein estimation by the Bradford method using the BioRad Protein assay kit. The homogenate was then further diluted in final storage buffer to produce a concentration of 3 mg/ml.  terminated by the addition of 1 mL ice-cold assay buffer and immediate transfer to an ice bath. Samples were centrifuged (20,000 xg, 6 min, 4°C) and membrane pellets solubilised by the addition of 50 µL ice-cold solubilisation buffer (100 mM Tris HCI, 200 mM NaCI, 1 mM EDTA, 1.25% Igepal and 0.2% SDS, pH 7.4) and incubation for 1 h at 4°C on a shaking platform. Following complete protein re-solubilisation, 50 µL of solubilisation buffer without SDS was added. Solubilised protein was pre-cleared using normal rabbit serum at a dilution of 1:100 and 3% (w/v) protein A-sepharose beads in TE buffer (10 mM Tris HCI, 10 mM EDTA, pH 8.0) added for 60 min at 4°C. Protein Asepharose beads and insoluble material were collected by centrifugation (20,000 xg, 6 min, 4°C) and 100 µL of the supernatant was transferred to fresh tubes containing G q -specific anti-serum (Santa Cruz; sc393) and incubated overnight at 4°C. Protein A-sepharose beads were added to samples, vortex mixed and rotated at 4°C for 90 min before being centrifuged (10,000 xg, 1 min, 4°C). Supernatants were aspirated and the protein Asepharose beads washed three times with ice-cold solubilisation buffer (without SDS). Recovered beads were then mixed with 1 mL FloScint-IV scintillation cocktail and counted by liquid scintillation spectrometry.

Electrophysiology
Brain slices were prepared from animals killed by decapitation (age < P20) or by cervical dislocation. Whole-cell patch recordings were made from visually identified mouse CA1 neurons in acute brain slices (250 μm thick) of the hippocampus (2-3 cells were measured per mouse in a total of 7 animals) essentially as described previously (2). Experiments were performed at 32 ± 1°C (LinLab software, Scientifica). Synaptic stimulation (using a DS2A Xanomeline stock (1 mM) was prepared in 5% glucose and was diluted to 100 nM in aCSF. Following baseline recordings, muscarinic receptor ligands were perfused for at least 6 minutes before starting the paired recording. In this way the impact of mAChRs on AMPA currents in hippocampal CA1 pyramidal neurons was determined by calculating the area under the current curve (AUC, time-integral; reflecting synaptic strength also known as current density analysis) of the EPSC compared to control.

Immunohistochemistry
For drug administration, mice were injected (i.p.) with muscarinic receptor compounds 30 minutes prior to anaesthesia and perfusion fixation.
For fear conditioning, mice were placed into the fear conditioning chamber and underwent the training protocol as described above. Control mice were placed into the chamber and received an immediate foot shock (2 sec; 0.4 mA). Approximately 30 seconds after training or the immediate foot shock, mice were returned to their home cage.
Following fixation, brains were immediately removed, and further fixed overnight in 4% PFA at 4°C. Brains were processed in paraffin wax and sliced at 5 µM using a microtome. Slices were either stained with haematoxylin and Eosin (H&E) or underwent antigen retrieval. Following antigen retrieval, sections were washed in TBS + 0.1% triton x-100 and blocked for 2 h at RT in TBS, 0.1% triton X-100, 10% goat serum and 5% BSA.
For assessing PrPsc levels, hippocampi and cortex were homogenised in lysis buffer (as above) and then treated with proteinase K (100 μg/ml) for 1 hour at 37°C. Samples were then resuspended into sample buffer and treated as above.
Multiple concentrations of donepezil, xanomeline, BQCA or BQZ-12 were administered via intraperitoneal injection (in 5% glucose) to wild-type mice and were observed for a range of side-effects. Shown are the proportion of mice displaying each side-effect following administration of drug (n=3-5).