M1 muscarinic receptor activation reduces the molecular pathology and slows the progression of prion-mediated neurodegenerative disease.

Many dementias are propagated through the spread of "prion-like" misfolded proteins. This includes prion diseases themselves (such as Creutzfeldt-Jakob disease) and Alzheimer's disease (AD), for which no treatments are available to slow or stop progression. The M1 acetylcholine muscarinic receptor (M1 receptor) is abundant in the brain, and its activity promotes cognitive function in preclinical models and in patients with AD. Here, we investigated whether activation of the M1 receptor might slow the progression of neurodegeneration associated with prion-like misfolded protein in a mouse model of prion disease. Proteomic and transcriptomic analysis of the hippocampus revealed that this model had a molecular profile that was similar to that of human neurodegenerative diseases, including AD. Chronic enhancement of the activity of the M1 receptor with the positive allosteric modulator (PAM) VU0486846 reduced the abundance of prion-induced molecular markers of neuroinflammation and mitochondrial dysregulation in the hippocampus and normalized the abundance of those associated with neurotransmission, including synaptic and postsynaptic signaling components. PAM treatment of prion-infected mice prolonged survival and maintained cognitive function. Thus, allosteric activation of M1 receptors may reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein.

Long-term daily feeding of cannabidiol is well-tolerated by healthy dogs.

Cannabidiol (CBD) containing dog food and treats are widely commercially available, mirroring the growing popularity of CBD as a supplement for humans. Despite this, experimental evidence of the safety and efficacy of long-term oral exposure in dogs is lacking. The purpose of this study was to address the gap in knowledge around the longer-term suitability and tolerance of a broad-spectrum CBD (THC-free) distillate in clinically healthy dogs. The study was a randomized, placebo-controlled, and blinded study where one group of twenty dogs received daily CBD capsules at a dose of 4 mg/kg of body weight (BW) for a period of 6 months. The control group of twenty dogs received placebo capsules. A comprehensive suite of physiological health measures was performed throughout the study at baseline, and after 2, 4, 10, 18, and 26 weeks of exposure, followed by 4 weeks of washout. CBD concentrations were measured at the same cadence in plasma, feces and urine. Health measures included biochemistry, hematology, urinalysis, in addition to fortnightly veterinary examinations, twice daily well-being observations, and a daily quality-of-life survey. Biochemistry and hematology showed no clinically significant alterations apart from a transient elevation in alkaline phosphatase (ALP) in just over half of the dogs receiving CBD. This elevation was observed in the absence of concurrent elevations of other liver parameters, and without any adverse effects on health and wellbeing. Furthermore, bone alkaline phosphatase (BALP) was simultaneously elevated with a significant, strong (r > 0.9) positive correlation between the two measures, suggesting that the elevation of total ALP was at least partly due to the bone-derived isoform. This study provides evidence that a once-daily oral dose of 4 mg CBD/kg BW is well tolerated in clinically healthy dogs for a duration of 6-months.

Targeting the Type 5 Metabotropic Glutamate Receptor: A Potential Therapeutic Strategy for Neurodegenerative Diseases?

The type 5 metabotropic glutamate receptor, mGlu5, has been proposed as a potential therapeutic target for the treatment of several neurodegenerative diseases. In preclinical neurodegenerative disease models, novel allosteric modulators have been shown to improve cognitive performance and reduce disease-related pathology. A common pathological hallmark of neurodegenerative diseases is a chronic neuroinflammatory response, involving glial cells such as astrocytes and microglia. Since mGlu5 is expressed in astrocytes, targeting this receptor could provide a potential mechanism by which neuroinflammatory processes in neurodegenerative disease may be modulated. This review will discuss current evidence that highlights the potential of mGlu5 allosteric modulators to treat neurodegenerative diseases, including Alzheimer's disease, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Furthermore, this review will explore the role of mGlu5 in neuroinflammatory responses, and the potential for this G protein-coupled receptor to modulate neuroinflammation.

The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes.

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)-linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M1 and anti-green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M1-mEGFP was expressed at levels equal to the M1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M1-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M1-mEGFP-expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M1-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

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

There are currently no treatments that can slow the progression of neurodegenerative diseases, such as Alzheimer's disease (AD). There is, however, a growing body of evidence that activation of the M1 muscarinic acetylcholine receptor (M1-receptor) can not only restore memory loss in AD patients but in preclinical animal models can also slow neurodegenerative disease progression. The generation of an effective medicine targeting the M1-receptor has however been severely hampered by associated cholinergic adverse responses. By using genetically engineered mouse models that express a G protein-biased M1-receptor, we recently established that M1-receptor mediated adverse responses can be minimized by ensuring activating ligands maintain receptor phosphorylation/arrestin-dependent signaling. Here, we use these same genetic models in concert with murine prion disease, a terminal neurodegenerative disease showing key hallmarks of AD, to establish that phosphorylation/arrestin-dependent signaling delivers neuroprotection that both extends normal animal behavior and prolongs the life span of prion-diseased mice. Our data point to an important neuroprotective property inherent to the M1-receptor and indicate that next generation M1-receptor ligands designed to drive receptor phosphorylation/arrestin-dependent signaling would potentially show low adverse responses while delivering neuroprotection that will slow disease progression.

From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Energy Requirements for Growth in the Norfolk Terrier.

An appropriate energy intake for healthy growth can reduce the risk of obesity and co-morbidities, such as orthopaedic diseases. The 2006 National Research Council (NRC) universal equation calculates the energy requirement of growing dogs based on predicted adult body weight, but evidence suggests a revision may be required. This study investigates the energy requirements of seventeen Norfolk terrier puppies over their first year (10 to 52 weeks). Puppies were individually fed complete and balanced diets in amounts to maintain an optimal body condition score (BCS), recording intake daily and body weight and BCS weekly. To monitor health a veterinary examination, haematology and plasma biochemistry and serum measures of bone turnover were undertaken every 12 weeks. Skeletal development was assessed using dual-energy X-ray absorptiometry (26 and 52 weeks). Puppies were clinically healthy with normal skeletal development and healthy growth throughout. The energy intake to achieve this was significantly lower than that predicted by the NRC (2006) equation at all time points, with largest mean difference of 285 kJ/kg0.75 per day at 10 weeks. If fed according to the NRC 2006 equation, dogs would have been in positive energy balance, possibly leading to obesity. These data support a revision to the NRC (2006) equation.

Deep Learning Classification of Canine Behavior Using a Single Collar-Mounted Accelerometer: Real-World Validation.

Collar-mounted canine activity monitors can use accelerometer data to estimate dog activity levels, step counts, and distance traveled. With recent advances in machine learning and embedded computing, much more nuanced and accurate behavior classification has become possible, giving these affordable consumer devices the potential to improve the efficiency and effectiveness of pet healthcare. Here, we describe a novel deep learning algorithm that classifies dog behavior at sub-second resolution using commercial pet activity monitors. We built machine learning training databases from more than 5000 videos of more than 2500 dogs and ran the algorithms in production on more than 11 million days of device data. We then surveyed project participants representing 10,550 dogs, which provided 163,110 event responses to validate real-world detection of eating and drinking behavior. The resultant algorithm displayed a sensitivity and specificity for detecting drinking behavior (0.949 and 0.999, respectively) and eating behavior (0.988, 0.983). We also demonstrated detection of licking (0.772, 0.990), petting (0.305, 0.991), rubbing (0.729, 0.996), scratching (0.870, 0.997), and sniffing (0.610, 0.968). We show that the devices' position on the collar had no measurable impact on performance. In production, users reported a true positive rate of 95.3% for eating (among 1514 users), and of 94.9% for drinking (among 1491 users). The study demonstrates the accurate detection of important health-related canine behaviors using a collar-mounted accelerometer. We trained and validated our algorithms on a large and realistic training dataset, and we assessed and confirmed accuracy in production via user validation.

Inhibition of neuroinflammatory nitric oxide signaling suppresses glycation and prevents neuronal dysfunction in mouse prion disease.

Several neurodegenerative diseases associated with protein misfolding (Alzheimer's and Parkinson's disease) exhibit oxidative and nitrergic stress following initiation of neuroinflammatory pathways. Associated nitric oxide (NO)-mediated posttranslational modifications impact upon protein functions that can exacerbate pathology. Nonenzymatic and irreversible glycation signaling has been implicated as an underlying pathway that promotes protein misfolding, but the direct interactions between both pathways are poorly understood. Here we investigated the therapeutic potential of pharmacologically suppressing neuroinflammatory NO signaling during early disease progression of prion-infected mice. Mice were injected daily with an NO synthase (NOS) inhibitor at early disease stages, hippocampal gene and protein expression levels of oxidative and nitrergic stress markers were analyzed, and electrophysiological characterization of pyramidal CA1 neurons was performed. Increased neuroinflammatory signaling was observed in mice between 6 and 10 wk postinoculation (w.p.i.) with scrapie prion protein. Their hippocampi were characterized by enhanced nitrergic stress associated with a decline in neuronal function by 9 w.p.i. Daily in vivo administration of the NOS inhibitor L-NAME between 6 and 9 w.p.i. at 20 mg/kg prevented the functional degeneration of hippocampal neurons in prion-diseased mice. We further found that this intervention in diseased mice reduced 3-nitrotyrosination of triose-phosphate isomerase, an enzyme involved in the formation of disease-associated glycation. Furthermore, L-NAME application led to a reduced expression of the receptor for advanced glycation end-products and the diminished accumulation of hippocampal prion misfolding. Our data suggest that suppressing neuroinflammatory NO signaling slows functional neurodegeneration and reduces nitrergic and glycation-associated cellular stress.

Restoring Agonist Function at a Chemogenetically Modified M1 Muscarinic Acetylcholine Receptor.

Designer receptors exclusively activated by designer drugs (DREADDs) have been successfully employed to activate signaling pathways associated with specific muscarinic acetylcholine receptor (mAChR) subtypes. The M1 DREADD mAChR displays minimal responsiveness to the endogenous agonist acetylcholine (ACh) but responds to clozapine-N-oxide (CNO), an otherwise pharmacologically inert ligand. We have previously shown that benzyl quinolone carboxylic acid (BQCA), an M1 mAChR positive allosteric modulator (PAM), can rescue ACh responsiveness at these receptors. However, whether this effect is chemotype specific or applies to next-generation M1 PAMs with distinct scaffolds is unknown. Here, we reveal that new M1 PAMs restore ACh function at the M1 DREADD while modulating ACh binding at the M1 wild-type mAChR. Importantly, we demonstrate that the modulation of ACh function by M1 PAMs is translated in vivo using transgenic M1 DREADD mice. Our data provide important insights into mechanisms that define allosteric ligand modulation of agonist affinity vs efficacy and how these effects play out in the regulation of in vivo responses.

M1 muscarinic acetylcholine receptors: A therapeutic strategy for symptomatic and disease-modifying effects in Alzheimer's disease?

The M1 muscarinic acetylcholine receptor (mAChR) plays a crucial role in learning and memory processes and has long been identified as a promising therapeutic target for the improvement of cognitive decline in Alzheimer's disease (AD). As such, clinical trials with xanomeline, a mAChR orthosteric agonist, showed an improvement in cognitive and behavioral symptoms associated with AD. Despite this, the clinical utility of xanomeline was hampered by a lack of M1 receptor selectivity and adverse cholinergic responses attributed to activation of peripheral M2 and M3 mAChRs. More recently, efforts have focused on developing more selective M1 compounds via targeting the less-conserved allosteric binding pockets. As such, positive allosteric modulators (PAMs) have emerged as an exciting strategy to achieve exquisite selectivity for the M1 mAChR in order to deliver improvements in cognitive function in AD. Furthermore, over recent years it has become increasingly apparent that M1 therapeutics may also offer disease-modifying effects in AD, due to the modulation of pathogenic amyloid processing. This article will review the progress made in the development of M1 selective ligands for the treatment of cognitive decline in AD, and will discuss the current evidence that selective targeting of the M1 mAChR could also have the potential to modify AD progression.

Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs.

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including 'epileptic-like' seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD.

Fine Tuning Muscarinic Acetylcholine Receptor Signaling Through Allostery and Bias.

The M1 and M4 muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, in particular for Alzheimer's disease and schizophrenia. Due to high sequence homology, selective targeting of any of the M1-M5 mAChRs through the endogenous ligand binding site has been notoriously difficult to achieve. With the discovery of highly subtype selective mAChR positive allosteric modulators in the new millennium, selectivity through targeting an allosteric binding site has opened new avenues for drug discovery programs. However, some hurdles remain to be overcome for these promising new drug candidates to progress into the clinic. One challenge is the potential for on-target side effects, such as for the M1 mAChR where over-activation of the receptor by orthosteric or allosteric ligands can be detrimental. Therefore, in addition to receptor subtype selectivity, a drug candidate may need to exhibit a biased signaling profile to avoid such on-target adverse effects. Indeed, recent studies in mice suggest that allosteric modulators for the M1 mAChR that bias signaling toward specific pathways may be therapeutically important. This review brings together details on the signaling pathways activated by the M1 and M4 mAChRs, evidence of biased agonism at these receptors, and highlights pathways that may be important for developing new subtype selective allosteric ligands to achieve therapeutic benefit.

Chemogenetics defines receptor-mediated functions of short chain free fatty acids.

Differentiating actions of short chain fatty acids (SCFAs) at free fatty acid receptor 2 (FFA2) from other free fatty acid-responsive receptors and from non-receptor-mediated effects has been challenging. Using a novel chemogenetic and knock-in strategy, whereby an engineered variant of FFA2 (FFA2-DREADD) that is unresponsive to natural SCFAs but is instead activated by sorbic acid replaced the wild-type receptor, we determined that activation of FFA2 in differentiated adipocytes and colonic crypt enteroendocrine cells of mouse accounts fully for SCFA-regulated lipolysis and release of the incretin glucagon-like peptide-1 (GLP-1), respectively. In vivo studies confirmed the specific role of FFA2 in GLP-1 release and also demonstrated a direct role for FFA2 in accelerating gut transit. Thereby, we establish the general principle that such a chemogenetic knock-in strategy can successfully define novel G-protein-coupled receptor (GPCR) biology and provide both target validation and establish therapeutic potential of a 'hard to target' GPCR.

Alterations in neuronal metabolism contribute to the pathogenesis of prion disease.

Neurodegenerative conditions are characterised by a progressive loss of neurons, which is believed to be initiated by misfolded protein aggregations. During this time period, many physiological and metabolomic alterations and changes in gene expression contribute to the decline in neuronal function. However, these pathological effects have not been fully characterised. In this study, we utilised a metabolomic approach to investigate the metabolic changes occurring in the hippocampus and cortex of mice infected with misfolded prion protein. In order to identify these changes, the samples were analysed by ultrahigh-performance liquid chromatography-tandem mass spectroscopy. The present dataset comprises a total of 498 compounds of known identity, named biochemicals, which have undergone principal component analysis and supervised machine learning. The results generated are consistent with the prion-inoculated mice having significantly altered metabolic profiles. In particular, we highlight the alterations associated with the metabolism of glucose, neuropeptides, fatty acids, L-arginine/nitric oxide and prostaglandins, all of which undergo significant changes during the disease. These data provide possibilities for future studies targeting and investigating specific pathways to better understand the processes involved in neuronal dysfunction in neurodegenerative diseases.

Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes.

The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.

DREADD Agonist 21 Is an Effective Agonist for Muscarinic-Based DREADDs in Vitro and in Vivo.

Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine-N-oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di in vivo can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for in vivo studies where metabolic conversion of CNO to clozapine is a concern.

The use of chemogenetic approaches to study the physiological roles of muscarinic acetylcholine receptors in the central nervous system.

Chemical genetic has played an important role in linking specific G protein-coupled receptor (GPCR) signalling to cellular processes involved in central nervous system (CNS) functions. Key to this approach has been the modification of receptor properties such that receptors no longer respond to endogenous ligands but rather can be activated selectively by synthetic ligands. Such modified receptors have been called Receptors Activated Solely by Synthetic Ligands (RASSLs) or Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Unlike knock-out animal models which allow detection of phenotypic changes caused by loss of receptor functions, RASSL and DREADD receptors offer the possibility of rescuing "knock-out" phenotypic deficits by administration of the synthetic ligands. Here we describe the use of these modified receptors in defining the physiological role of GPCRs and validation of receptors as drug targets. This article is part of the Special Issue entitled 'Neuropharmacology on Muscarinic Receptors'.