Biology subjects

Lindsley, C. W.

Publications and source records attributed to Lindsley, C. W..

12 recordsLinked to original sources

bioRxiv2026

Targeting Protease-activated Receptor 4 (PAR4) Protects Against Acute Kidney Injury (AKI) in Ischemia Reperfusion Injury

Acute kidney injury (AKI) is a serious and common clinical syndrome that currently has no effective treatment. Emerging evidence links coagulation pathways to kidney injury, particularly through coagulation proteases. Protease-activated receptors (PARs) are a family of G-protein coupled receptors (GPCRs) that are activated by proteolytic cleavage of their N termini, exposing a tethered ligand that initiates receptor signaling. PARs have been shown to play a major role in inflammation, vascular regulation, and tissue injury. PARs play key roles in inflammation, vascular regulation, and tissue injury. Previous work from the Hamm laboratory demonstrated that PAR4 contributes to AKI progression, as PAR4 knockout mice were protected in both unilateral ureteral obstruction and ischemia-reperfusion-based models of kidney disease. In this study, we investigated the potential of a PAR4 antagonist, VU6073819, at mitigating AKI progression in an ischemia-reperfusion injury (IRI) mouse model. PAR4 antagonism not only alleviated kidney injury and inflammatory response, but it significantly improved the survival. These findings identify PAR4 as a promising therapeutic target for AKI.

pharmacology and toxicology↗

bioRxiv2026

The importance of M1 muscarinic receptor phosphorylation in learning and memory

The muscarinic acetylcholine receptor 1 (mAChR1, M1) has been identified as a primary target for Alzheimers disease (AD) and better understanding of the receptor biology, especially in regard to biased signalling of the receptor, will allow for the development of improved drugs targeting cholinergic dysfunction in AD. The aim of this study was to determine the contribution of phosphorylation of M1 to the learning and memory (LM) effects of M1 agonism. The contribution of M1 phosphorylation dependent signalling in LM was assessed using the mAChR1 positive allosteric modulator, VU0486846, in a scopolamine (1.5 mg/kg) induced LM deficit model in mice expressing HA-tagged M1 (M1-WT), phosphorylation deficient HA-tagged M1 (M1-PD), or mice deficient in M1 (M1-KO). LM was assessed using a fear conditioning (FC) testing paradigm where context and cued memory retrieval was measured 24 hrs after training and a higher level of freezing indicated intact memory. Results demonstrated that scopolamine induced a significant LM deficit in both context and cued retrieval in M1-WT mice which was partially rescued by VU0486846 confirming a contribution of M1 signalling in LM. The scopolamine induced deficit in contextual retrieval in M1-KO mice was not rescued by VU0486846, which is an M1 selective ligand, while scopolamine did not induce a deficit in cued retrieval in M1-KO mice. In M1-PD mice, scopolamine induced a LM deficit in contextual retrieval, however this was also not rescued by VU0486846 treatment. Similarly to M1-KO animals, M1-PD mice did not display a scopolamine induced deficit in cued retrieval. When freezing responses were compared across strains, M1-PD mice displayed a deficit compared to M1-WT and M1-KO mice in contextual retrieval, while both M1-PD and M1-KO mice displayed a deficit compared to M1-WT mice in cued retrieval. These results demonstrate that although M1 agonism can restore a LM deficit in both contextual and cued testing paradigms, only the cued retrieval response is dependent on the M1. Additionally, biased Gq M1 signalling is not sufficient to restore contextual memory and requires phosphorylation of the receptor. Furthermore, biased M1 signalling results in LM deficits not seen with KO of the receptor. Overall, these results reiterate the importance of considering the bias of ligands when developing M1 agonists for dementia in the future.

neuroscience↗

bioRxiv2025

Cryo-EM reveals a new allosteric binding site at the M5 mAChR

The M5 muscarinic acetylcholine receptor (M5 mAChR) represents a promising therapeutic target for neurological disorders. However, the high conservation of its orthosteric binding site has posed significant challenges for drug development. While selective positive allosteric modulators (PAMs) offer a potential solution, a structural understanding of the M5 mAChR and its allosteric binding sites has remained limited. Here, we present a 2.8 [A] cryo-electron microscopy structure of the M5 mAChR complexed with heterotrimeric Gq protein and the agonist iperoxo, completing the active-state structural characterization of the mAChR family. To identify the binding site of M5-selective PAMs, we implemented an integrated approach combining mutagenesis, pharmacological assays, structural biology, and molecular dynamics simulations. Our mutagenesis studies revealed that selective M5 PAMs bind outside previously characterized M5 mAChR allosteric sites. Subsequently, we obtained a 2.1 [A] structure of M5 mAChR co-bound with acetylcholine and the selective PAM VU6007678, revealing a novel allosteric pocket at the extrahelical interface between transmembrane domains 3 and 4 that was confirmed through mutagenesis and simulations. These findings demonstrate the diverse mechanisms of allosteric regulation in mAChRs and highlight the value of integrating pharmacological and structural approaches to identify novel allosteric binding sites.

pharmacology and toxicology↗

bioRxiv2025

Identification of M4 muscarinic acetylcholine receptor expressing astrocytes that regulate locomotion and survival in murine prion disease

Whilst there has been much focus on neuronally expressed muscarinic acetylcholine receptors (mAChRs), less attention has been paid to their expression and role in non-neuronal brain cells. Using genetically engineered mice, we identify a previously unappreciated subpopulation of astrocytes expressing the M4 mAChR subtype dispersed across various regions that include the brainstem, hypothalamus and, most abundantly, the cerebellum. Signalling and proteomic analysis of M4 mAChR positive astrocytes from the cerebellum confirmed functional receptor expression and its role in regulating protein expression. Genetic ablation of these astrocytes in mice revealed a specific role in locomotion. Importantly, in the context of murine prion disease, we observed a significant expansion of M4 mAChR positive astrocytes, and ablation experiments established that this astrocyte subpopulation has a detrimental effect on late-stage disease. Together we identify M4 mAChR expressing astrocyte subpopulation that plays a specific role in normal neurophysiology and the progression of inflammatory neurodegenerative disease.

neuroscience↗

bioRxiv2024

Rostral Pedunculopontine Nucleus Infusion of M4 Positive Allosteric Modulator VU0467154 Augments L-DOPA Effects in Hemiparkinsonian Rats

Standard treatment for Parkinsons disease (PD) is dopamine replacement therapy with L-DOPA. However, chronic treatment often results in abnormal involuntary movements called L-DOPA-induced dyskinesia (LID). Prior evidence indicates that heightened striatal cholinergic tone may contribute to LID. Restoring cholinergic inhibition by targeting the inhibitory M4 muscarinic acetylcholine (ACh) receptor (M4) reduces LID in preclinical models. Although intrinsic striatal sources of ACh have been considered for their role in LID, extrinsic sources of ACh such as the pedunculopontine nucleus (PPN) have not been well investigated for their role in LID. Therefore, the current study employed hemiparkinsonian Long-Evans rats with a PPN-targeted cannula ipsilateral to 6-OHDA lesion. Following chronic treatment with L-DOPA, we examined the effect of local unilateral PPN infusion of M4 PAM VU0467154 on LID, motor performance, and c-fos expression within the PPN. It was expected that PPN infusion of VU0467154 would reduce LID, reduce L-DOPAs motor benefit, and globally reduce c-fos expression in the PPN. Contrary to our expectations, PPN infusion of M4 PAM did not significantly affect LID severity. Furthermore, the group receiving M4 PAM showed slightly elevated motor improvement compared to L-DOPA, and decreased c-fos expression specifically in PPN cholinergic neurons. These results suggest that local PPN ACh dynamics differ from those of the striatum. Specifically, our results suggest that PPN cholinergic neurons may be a promising therapeutic target for augmenting L-DOPA-mediated motor benefit without increasing LID.

neuroscience↗

bioRxiv2022

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular level understanding of the general principals that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a well-validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. Here, we present high-resolution cryo-electron microscopy structures of the M4 mAChR bound to a cognate Gi1 protein and the high affinity agonist, iperoxo, in the absence and presence of two different positive allosteric modulators, LY2033298 or VU0467154. We have also determined the structure of the M4 mAChR-Gi1 complex bound to its endogenous agonist, acetylcholine (ACh). Structural comparisons, together with molecular dynamics, mutagenesis, and pharmacological validations, have provided in-depth insights into the role of structure and dynamics in orthosteric and allosteric ligand binding, global mechanisms of receptor activation, cooperativity, probe-dependence, and species variability; all key hallmarks underpinning contemporary GPCR drug discovery.

pharmacology and toxicology↗

bioRxiv2021

Effects of acute and repeated administration of the selective M4 PAM VU0152099 on cocaine vs. food choice in male rats

AO_SCPLOWBSTRACTC_SCPLOWLigands that stimulate muscarinic acetylcholine receptors 1 and 4 (M1, M4) have shown promising effects as putative pharmacotherapy for cocaine use disorder in rodent assays. We have previously shown reductions in cocaine effects with acute M4 stimulation, as well as long-lasting, delayed, reductions in cocaine taking and cocaine seeking with combined M1/M4 receptor stimulation or with M1 stimulation alone. M4 stimulation opposes dopaminergic signaling acutely, but direct dopamine receptor antagonists have proved unhelpful in managing cocaine use disorder because they lose efficacy with long-term administration. It is therefore critical to determine whether M4 approaches themselves can remain effective with repeated or chronic dosing. We assessed the effects of repeated administration of the M4 positive allosteric modulator (PAM) VU0152099 in rats trained to choose between intravenous cocaine and a liquid food reinforcer, to obtain quantitative measurement of whether M4 stimulation could produce delayed and lasting reduction in cocaine taking. VU0152099 produced progressively augmenting suppression of cocaine choice and cocaine intake, but produced neither rebound nor lasting effects after treatment ended. To compare and contrast effects of M1 vs. M4 stimulation, we tested whether the M4 PAM VU0152100 suppressed cocaine self-administration in mice lacking CalDAG-GEFI signaling factor, required for M1-mediated suppression of cocaine self-administration. CalDAG-GEFI ablation had no effect on M4-mediated suppression of cocaine self-administration. These findings support the potential usefulness of M4 PAMs as pharmacotherapy to manage cocaine use disorder, alone or in combination with M1-selective ligands, and show that M1 and M4 stimulation modulate cocaine-taking behavior by distinct mechanisms.

neuroscience↗

bioRxiv2021

Activation of M1 muscarinic receptors reduce pathology and slow progression of neurodegenerative disease.

The most prevalent types of dementias, including Alzheimers disease, are those that are propagated via the spread of "prion-like" misfolded proteins. Despite considerable effort no treatments are available to slow or stop the progression of these dementias. Here we investigate the possibility that activation of the M1-muscarinic receptor (M1-receptor), which is highly expressed in the brain and that shows pro-cognitive properties, might present a novel disease modifying target. We demonstrate that the progression of murine prion disease, which we show here displays many of the pathological, behavioural and biochemical hallmarks of human neurodegenerative disease, is slowed and normal behaviour maintained by the activation of the M1-receptor with a highly tolerated positive allosteric modulator (VU846). This correlates with a reduction in both neuroinflammation and indicators of mitochondrial dysregulation, as well as a normalisation in the expression of markers associated with neurodegeneration and Alzheimers disease. Furthermore, VU846 preserves expression of synaptic proteins and post-synaptic signalling components that are altered in disease. We conclude that allosteric regulation of M1-receptors has the potential to reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein in a manner that extends life span and maintains normal behaviour.

neuroscience↗

bioRxiv2020

Activating mGlu3 metabotropic glutamate receptors rescues schizophrenia-like cognitive deficits through metaplastic adaptations within the hippocampus.

BackgroundPolymorphisms in GRM3, the gene encoding the mGlu3 metabotropic glutamate receptor, are associated with impaired cognition and neuropsychiatric disorders such as schizophrenia. Limited availability of selective genetic and molecular tools has hindered progress in developing a clear understanding of the mechanisms through which mGlu3 receptors regulate synaptic plasticity and cognition. MethodsWe examined associative learning in mice with trace fear conditioning, a hippocampal-dependent learning task disrupted in patients with schizophrenia. Underlying cellular mechanisms were assessed using ex vivo hippocampal slice preparations with selective pharmacological tools and selective genetic deletion of mGlu3 receptor expression in specific neuronal subpopulations. ResultsmGlu3 receptor activation enhanced trace fear conditioning and reversed deficits induced by subchronic phencyclidine. Mechanistic studies revealed that mGlu3 receptor activation induced metaplastic changes, biasing afferent stimulation to induce long-term potentiation through a mGlu5 receptor-dependent, endocannabinoid-mediated, disinhibitory mechanism. Selective genetic deletion of either mGlu3 or mGlu5 from hippocampal pyramidal cells eliminated effects of mGlu3 activation, revealing a novel mechanism by which mGlu3 and mGlu5 interact to enhance cognitive function. ConclusionsThese data demonstrate that activation of mGlu3 receptors in hippocampal pyramidal cells enhances hippocampal-dependent cognition in control and impaired mice by inducing a novel form of metaplasticity to regulate circuit function - providing a clear mechanism through which genetic variation in GRM3 can contribute to cognitive deficits. Developing approaches to positively modulate mGlu3 receptor function represents an encouraging new avenue for treating cognitive disruption in schizophrenia and other psychiatric diseases.

pharmacology and toxicology↗

bioRxiv2020

Discovery of the first selective M4 muscarinic acetylcholine receptor antagonists with in vivo anti-parkinsonian and anti-dystonic efficacy

Non-selective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson disease and dystonia. Despite their efficacy in these and other central nervous system disorders, anti-muscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the anti-parkinsonian and anti-dystonic efficacy observed with the use of non-selective anti-muscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of non-selective anti-muscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here we utilize genetic mAChR knockout animals in combination with non-selective mAChR antagonists to confirm that the M4 receptor underlies the locomotor-stimulating and anti-parkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have anti-parkinsonian and anti-dystonic efficacy in pharmacological and genetic models of movement disorders.

pharmacology and toxicology↗

bioRxiv2019

Biased M1 positive allosteric modulators reveal novel role of phospholipase D in M1-dependent rodent cortical plasticity

Highly selective positive allosteric modulators (PAMs) of the M1 subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach for the potential improvement of cognitive function in patients suffering from Alzheimers disease and schizophrenia. M1 PAM discovery programs have produced a structurally diverse range of M1 PAMs with distinct pharmacological properties, including different levels of agonist activity and differences in signal bias. This includes the recent discovery of novel biased M1 PAMs that can potentiate coupling of M1 to activation of phospholipase C but not phospholipase D (PLD). However, little is known about the role of PLD in M1 signaling in native systems and it is not clear whether biased M1 PAMs will display differences in modulating M1-mediated responses in native tissue. We now report a series of studies using novel PLD inhibitors and PLD knockout mice to show that PLD is necessary for the induction of M1-dependent long-term depression (LTD) in the prefrontal cortex (PFC). Importantly, biased M1 PAMs that do not couple to PLD not only fail to potentiate orthosteric agonist-induced LTD but also block M1-dependent LTD in the PFC. In contrast, biased and non-biased M1 PAMs act similarly in potentiating M1-dependent electrophysiological responses that are PLD-independent. These findings demonstrate that PLD plays a critical role in the ability of M1 PAMs to modulate certain CNS functions and that biased M1 PAMs function differently in brain regions implicated in cognition.\n\nSummaryWe demonstrate a novel role of phospholipase D in M1-dependent rodent cortical plasticity and M1 PAMs that do not couple to phospholipase D have functionally distinct effects on cortical plasticity than non-biased M1 PAMs.

neuroscience↗

bioRxiv2019

Crystal structure of the M5 muscarinic acetylcholine receptor

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all five mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies together with chimeric swaps between the extracellular regions of the M2 and M5 mAChR further revealed the structural insight into \"kinetic-selectivity\", where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective ligands.\n\nSignificance StatementThe five subtypes of the muscarinic acetylcholine receptors (mAChRs) are expressed throughout the central and peripheral nervous system where they play a vital role in physiology and pathologies. Recently, the M5 mAChR subtype has emerged as an exciting drug target for the treatment of drug addiction. We have determined the atomic structure of the M5 mAChR bound to the clinically used inverse agonist tiotropium. The M5 mAChR structure now allows for a full comparison of all five mAChR subtypes and reveals subtle differences in the extracellular loop (ECL) regions of the receptor that mediate orthosteric and allosteric ligand selectivity. Together these findings open the door for future structure-based design of selective drugs that target this therapeutically important class of receptors.

pharmacology and toxicology↗