Biased M1 muscarinic receptor mutant mice show accelerated progression of prion neurodegenerative disease

Significance The M1 muscarinic acetylcholine receptor (M1-receptor) plays a crucial role in learning and memory and is a validated drug target for the treatment of Alzheimer’s disease (AD). Furthermore, M1-receptor ligands have been demonstrated to display disease-modifying effects in preclinical models of neurodegenerative disease. By employing a genetic mouse model expressing a G protein–biased M1-receptor in combination with a mouse model of terminal neurodegenerative disease, we demonstrate here that the M1-receptor exerts an inherent neuroprotective activity that is dependent on its phosphorylation status. Thus, in AD drug development programs, M1-receptor ligands that maintain the receptor phosphorylation status will be more likely to lead to beneficial neuroprotective outcomes.


Human Embryonic Kidney (HEK) cell culture
HEK293T cells were maintained in Delbecco's Modifeid Eagle Medium (DMEM) containing 10% FBS and maintained at 37°C and 5% CO2. HEK293T cells were transfected with polyethylenimine (PEI), using a 1:6 weight to weight ratio of DNA to PEI. 24 h post transfection cells were detached and seeded at either 20,000 cells/well into poly-D-lysine coated white 96-well plates for BRET assays, or 40,000 cells/well into poly-D-lysine coated clear 96-well plates for IP1 assay. Cells were then returned to the incubator and cultured for a further 24 h for BRET based assays or 48 h for IP1 accumulation assays.

IP1 Accumulation assay
HEK293T cells transiently transfected with vectors containing the M1-WT or M1-PD, or with the empty vector (pcDNA) were plated on 96-well plates at cell density of 40,000 cells/well for IP1 accumulation assays. Cells were washed and incubated in 1X stimulation buffer (Hank's Balanced Salt Solution (HBSS) w/o phenol red containing 20 mM HEPES, 1.2 mM CaCl2; 30 mM LiCl; pH7.4) for one hour at 37°C prior drug treatments. 10X concentrated test compounds were added (5 μl/well) to the 96-well plates and incubated at 37°C for one hour. Following the treatment, the stimulation buffer was removed and lysis buffer (IP-One assay kit, CisBio) was added (40 µl/well). Following 10 minute-incubation on a shaker at 600 rpm, cell suspensions (7µl/well) were added to 384-well white proxiplates (PerkinElmer).
The IP1-d2 conjugate and the anti-IP1 cryptate Tb conjugate (IP-One Tb™ assay kit, CisBio) were diluted together 1:40 in lysis buffer and 3 μl of the antibody mix were added to each well. The plate was incubated at 37°C for 1-24h and FRET between d2-conjugated IP1 (emission at 665 nm) and Lumi4™-Tb cryptate conjugated anti-IP1 antibody (emission at 620 nm) was detected using a CLARIOstar plate reader (BMG Labtech). Results were calculated from the 665/620 nm ratio and normalised to the maximum response stimulated by ACh.
Primary neuronal cells were seeded at a density of 5x10 4 cells per well onto precoated 96 well plates and maintained at 37 °C in a 5% CO2 humidified atmosphere.
On DIV7 cells were washed and incubated in stimulation buffer, and the assay was conducted as detailed above. Inhibition of agonist stimulation was measured by pre-incubating cells with 10 µM atropine for 30 min at 37 °C prior to addition of carbachol for 1 hour.

ERK 1/2 phosphorylation assay
HEK293T cells transiently transfected with vectors containing the M1-WT or M1-PD were plated on 96-well plates at cell density of 50,000 cells/well for ERK phosphorylation assay using the CisBio Phospho-ERK (Thr202/Tyr404) cellular kit (PerkinElmer). After incubation overnight at 37 °C, the culture medium was replaced with serum-free DMEM, and the cells were incubated for a further 4-5 hours prior to the assay. To inhibit Gq signalling, cells were pre-incubated with 1 μM FR900359 (1) for 1 hour. For the assay, cells were stimulated with FBS, vehicle (0.1% DMSO), or 1 μM acetylcholine for 8 different time points (0, 2.5, 5, 7.5, 10, 15, 30 and 60 minutes) at 37 °C. The reactions were terminated by the removal of the compounds and the addition of lysis buffer supplemented with blocking buffer.
The lysates were shaken (600 rpm) for 10 minutes at room temperature before being transferred to a 384-well white OptiPlate (PerkinElmer) where they were incubated with a premixed antibody solution (anti-phospho-ERK1/2 Cryptate and anti-phospho-ERK1/2 d2) for 1-24 hours at room temperature with agitation at 600 rpm. Fluorescence emission (620 nm and 665 nm) was determined using a CLARIOstar plate reader (BMG Labtech) and results normalised to the response stimulated by FBS.

HEK cells
To assess arrestin recruitment to M1-WT and M1-PD, a bystander BRET assay was employed (2). HEK293T cells were co-transfected with plasmids encoding: 1) human β-Arrestin-2 fused to Nanoluciferase (Nluc) at its N-terminus; 2) the mNeonGreen (mNG) fluorescent protein fused with the prenylation CAAX sequence of KRas at its C-terminus; and 3) Either an empty pcDNA3 plasmid, a plasmid encoding wild-type mouse M1, or a plasmid encoding mouse M1-PD. For these transfections a DNA weight ratio of 1:25:5 was used for the Nluc-β-Arrestin-2 : mNG-CAAX : pcDNA3/M1-WT/M1-PD plasmids. After transfection, cells were cultured 24h, transferred to white 96-well plates and cultured a further 24h. For the assay, transfected cells in white 96-well plates were first washed twice with HBSS supplemented with 20mM HEPES (HBSS-H) and incubated for 30 min at 37°C. Nluc substrate, Coelenterazine 400a, was then added to a final concentration of 5 µM and incubated for 10 min. Dual 535 and 475 nm luminescent emission measurements were then taken at one minute intervals using a PherStar FS plate reader (BMG labtech) for 5 min prior to and 30 min following the addition of the indicated test compounds. Net BRET responses were calculated as the 535/475 ratio after correcting for both the well baseline and test compound vehicle response. BRET data was then reported as the area under the Net BRET curve for the 30 min following test compound addition.

Primary neurons
Primary cortico-hippocampal neuron cultures from wild type or M1-PD mouse strains were cultured for 7 days in white half area 96-well plates. After 7 days in culture neurons were transfected using lipofectamine MessengerMAX transfection reagent (ThermoFisher) according to the manufacturer instructions with mRNA coding for Nluc-β-arrestin-2 and mNG-CAAX in a 1:5 ratio. For positive control experiments, neurons were also transfected with mRNA coding for the human free fatty acid 4 receptor, which we have previously shown to interact strongly with barrestin-2 (3). After transfection, Neurons were cultures for an additional 24h before using in arrestin recruitment assays. Neurons were washed twice with HBSS-H then incubated HBSS-H for 30 min at 37°C. NanoGlow substrate (Promega) was added to a final 1:800 dilution before plates were incubated a further 10 minutes. BRET measurements were then taken using a PheraStar FS plate reader (BMG Labtech), recording the donor emission at 475 nm and acceptor 6 excitation at 535 nm at one minute intervals. After five minutes, either vehicle or test compounds were added (100μM Ach, or 10μM TUG-891 for positive controls) before continuing to take measurements for an additional 30 min. BRET ratios were calculated as the emission at 535/475, corrected for basal BRET prior to compound addition and expressed as the net BRET above vehicle treatment. The area under the curve (AUC) was then calculated over the full 30 min of compound addition.

BRET early endosome association assay
Internalization of M1-WT and M1-PD was assessed using a bystander BRET assay designed to measure the translocation of M1 receptor to early endosomes (2). HEK293T cells were co-transfected with: 1) a plasmid encoding wild-type or PD mouse M1 fused at its C terminal to Nluc; and 2) a plasmid encoding mNG fused at its C terminal to the FYVE domain of endofin. A DNA weight transfection ratio of 1:2 was used for Nluc-tagged M1 receptor to mNG-FYVE. 24 h after transfection cells were transferred to white 96-well plates and then plates were cultured a further 24 h prior to the assay. For the assay, plates were washed twice with HBSS-H, before incubating in HBSS-H for 30 min at 37 °C. Coelenterazine 400a was added to a final concentration of 5 µM, plates were then incubated a further 10 min. BRET measurements were taken using a PherStar FS plate reader, measuring luminescent emission at 535 and 475 nm at 2 min intervals for 6 min prior to addition of test compounds, and a further 1 h after addition of test compounds. Net BRET responses were calculated as the 535/475 ratio after 7 correcting for both the well baseline and test compound vehicle response. BRET data was then reported as the area under the Net BRET curve for the 60 min following test compound addition.

Bias Factor calculations
Bias factors were calculated by first fitting an operation model (5) to concentration response data in the arrestin or Gq activation assays to obtain τ/ΚΑ values for ACh at both M1-WT or M1-PD. Δτ/ΚΑPD values were obtained by subtracting the τ/ΚΑ obtained for the M1-WT from the value obtain for M1-PD from the same assay.
ΔΔτ/ΚΑ values were then calculated as the difference between the Δτ/ΚΑPD the two assays. The antilog(10) of this value was then reported as the 'bias factor'.

RNA extraction
RNA extraction was performed immediately following tissue lysis using the Qiagen lipid tissue RNeasy Plus Mini kit as per manufacturer instructions. Briefly, following homogenisation in RLT Buffer containing 10% β-mercaptoethanol, the homogenate was centrifuged at 10,000 x g for 30 sec in a gDNA eliminator column.
The aqueous phase containing RNA was collected and mixed with 70% ethanol, then applied to an RNeasy Mini spin column and subjected to centrifugation at 8000 x g for 15 sec at room temperature. The flow-through was discarded and the column was washed with guanidine containing stringent wash buffer, then washed with mild wash buffer, centrifuging for 2 min after the final wash to remove residual ethanol. RNA was eluted in 40 μl nuclease-free water.

Determination of RNA concentration
RNA concentration was determined by measuring absorbance at 260 nm using a NanoDrop spectrophotometer. RNA purity was assessed using the A230/A260 and A260/A280 ratios, with ratios of 2-2.2 considered pure. RNA was stored at -80°C until use.

qRT-PCR
For cDNA synthesis, 1 μg total RNA template per reaction was used using High Capacity cDNA Reverse Transcription Kit (Agilent). RNAase-free water (total 4.2 μl), 2 μl 10x RT buffer, 1 μl RT enzyme, 0.8 µl 25x dNTP Mix (100 mM) and 2µl 10x RT random primers were mixed together and incubated for 10 min at 25°C, followed by 120 min at 37°C, followed by 5 min at 85°C. Samples were then chilled at 4°C. Each reaction included control reactions in the absence of RT enzyme (-RT control). cDNA samples were stored at -20°C until qRT-PCR was performed.
QuantiTect Primer Assays (Qiagen) were used for all the genes analysed.

Sample preparation for Western blotting
Animal tissues were snap frozen in liquid nitrogen at sacrifice and stored at −80°C. For the preparation of membrane extracts, frozen hippocampi and cortices were homogenised by sonication at 3-5 Hz amplitude in 500 µl of T/E buffer (10 mM Tris, 1 mM EDTA, pH 8.0) containing phosphatase and protease inhibitors.
Samples were then centrifuged at 10,000 x g for 10 min at 4 °C. The supernatants were mixed with additional 500 µl T/E buffer, and then centrifuged at 15,000 x g for 1 hour at 4 °C. The pellets were then solubilised in 400 µL of RIPA buffer including phosphatase and protease inhibitors and incubated for at least 2 hours at 4°C with end over end rotation. After centrifugation of samples at 14,000 x g for 10 min at 4°C, the supernatants (membrane extracts) were transferred to fresh microcentrifuge tubes and stored at -80°C until use.
Protein concentrations were determined by using the Micro BCA protein assay reagent kit according to the manufacturer's instructions.
Nitrocellulose membranes were equilibrated in transfer buffer, before assembly the blot sandwich. The transfer was performed for 2 hours at constant 60V. Then, membranes were blocked for 30-45 min in 5% (w/v) fat-free milk ( Biotechnologies, #926-32219). Proteins were visualised using LICOR Odyssey SA scanner using the appropriate lasers.

Immunohistochemistry
Following heat-induced epitope retrieval, sections were washed in TBS + 0.1% triton x-100, and blocked overnight at 4°C in TBS, 0.1% triton X-100, 10% goat serum and 1% BSA. Incubation with primary antibodies (anti-GFAP, Sigma-Aldrich, P/N: G3893; anti-Iba1, Thermo Fisher, P/N: PA5-27436) was conducted in blocking buffer overnight at 4°C or for two hours at room temperature. Following three washes, slides were incubated with Alexa Fluor fluorescent secondary antibodies for 2 hours at room temperature in blocking buffer. Following three washes, slices were mounted on glass slides using VECTASHIELD HardSet Antifade Mounting Medium with DAPI, let dry overnight at 4°C and sealed using nail varnish. All images were taken using LSM 880 confocal laser scanning microscope (Zeiss).

Data analysis
Data statistical analyses were carried out using GraphPad Prism 9 software.
Functional concentration-response curves were fitted according to a fourparameter logistic equation to determine basal and maximal responses, log half maximal effective concentration (EC50) and slope. Burrowing data were compared using mixed-effects model with uncorrected Fisher's LSD test. Symptom onset and survival curves were analysed with a Gehan-Breslow-Wilcoxon test.