Targeting the Actions of Muscarinic Receptors on Dopamine Systems: New Strategies for Treating Neuropsychiatric Disorders.

Cholinergic regulation of dopamine (DA) signaling has significant implications for numerous disorders, including schizophrenia, substance use disorders, and mood-related disorders. The activity of midbrain DA neurons and DA release patterns in terminal regions are tightly regulated by cholinergic neurons found in both the striatum and the hindbrain. These cholinergic neurons can modulate DA circuitry by activating numerous receptors, including muscarinic acetylcholine receptor (mAChR) subtypes. This review specifically focuses on the complex role of M2, M4, and M5 mAChR subtypes in regulating DA neuron activity and DA release and the potential clinical implications of targeting these mAChR subtypes.

Activation of Metabotropic Glutamate Receptor 3 Modulates Thalamo-accumbal Transmission and Rescues Schizophrenia-like Physiological and Behavioral Deficits.

BACKGROUND: Polymorphisms in the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are associated with an increased likelihood of schizophrenia diagnosis and can predict improvements in negative symptoms following treatment with antipsychotics. However, the mechanisms by which mGlu3 can regulate brain circuits involved in schizophrenia pathophysiology are not clear. METHODS: We employed selective pharmacological tools and a variety of approaches including whole-cell patch-clamp electrophysiology, slice optogenetics, and fiber photometry to investigate the effects of mGlu3 activation on phencyclidine (PCP)-induced impairments in thalamo-accumbal transmission and sociability deficits. A chemogenetic approach was used to evaluate the role of thalamo-accumbal transmission in PCP-induced sociability deficits. RESULTS: We first established that PCP treatment augmented excitatory transmission onto dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) in the nucleus accumbens (NAc) and induced sociability deficits. Our studies revealed a selective increase in glutamatergic synaptic transmission from thalamic afferents to D1-MSNs in the NAc shell. Chemogenetic silencing of thalamo-accumbal inputs rescued PCP-induced sociability deficits. Pharmacological activation of mGlu3 normalized PCP-induced impairments in thalamo-accumbal transmission and sociability deficits. Mechanistic studies revealed that mGlu3 activation induced robust long-term depression at synapses from the thalamic projections onto D1-MSNs in the NAc shell. CONCLUSIONS: These data demonstrate that activation of mGlu3 decreases thalamo-accumbal transmission and thereby rescues sociability deficits in mouse modeling schizophrenia-like symptoms. These findings provide novel insights into the NAc-specific mechanisms and suggest that agents modulating glutamatergic signaling in the NAc may provide a promising approach for treating negative symptoms in schizophrenia.

Ventral Tegmental Area M5 Muscarinic Receptors Mediate Effort-Choice Responding and Nucleus Accumbens Dopamine in a Sex-Specific Manner .

Optimization of effort-related choices is impaired in depressive disorders. Acetylcholine (ACh) and dopamine (DA) are linked to depressive disorders, and modulation of ACh tone in the ventral tegmental area (VTA) affects mood-related behavioral responses in rats. However, it is unknown if VTA ACh mediates effort-choice behaviors. Using a task of effort-choice, rats can choose to lever press on a fixed-ratio 5 (FR5) schedule for a more-preferred food or consume freely available, less-preferred food. VTA administration of physostigmine (1 µg and 2 µg/side), a cholinesterase inhibitor, reduced FR5 responding for the more-preferred food while leaving consumption of the less-preferred food intact. VTA infusion of the M5 muscarinic receptor negative allosteric modulator VU6000181 (3 µM, 10 µM, 30 µM/side) did not affect lever pressing or chow consumption. However, VU6000181 (30 µM/side) coadministration with physostigmine (2 µg/side) attenuated physostigmine-induced decrease in lever pressing in female and male rats and significantly elevated lever pressing above vehicle baseline levels in male rats. In in vivo voltammetry experiments, VTA infusion of combined physostigmine and VU6000181 did not significantly alter evoked phasic DA release in the nucleus accumbens core (NAc) in female rats. In male rats, combined VTA infusion of physostigmine and VU6000181 increased phasic evoked DA release in the NAc compared with vehicle, physostigmine, or VU6000181 infusion alone. These data indicate a critical role and potential sex differences of VTA M5 receptors in mediating VTA cholinergic effects on effort choice behavior and regulation of DA release. SIGNIFICANCE STATEMENT: Effort-choice impairments are observed in depressive disorders, which are often treatment resistant to currently available thymoleptics. The role of ventral tegmental area (VTA) acetylcholine muscarinic M5 receptors, in a preclinical model of effort-choice behavior, is examined. Using the selective negative allosteric modulator of the M5 receptor VU6000181, we show the role of VTA M5 receptors on effort-choice and regulation of dopamine release in the nucleus accumbens core. This study supports M5 receptors as therapeutic targets for depression.

Sphingosine Kinase 2 Inhibitors: Rigid Aliphatic Tail Derivatives Deliver Potent and Selective Analogues.

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that interacts with five native G-protein coupled receptors (S1P1-5) to regulate cell growth, survival, and proliferation. S1P has been implicated in a variety of pathologies including cancer, kidney fibrosis, and multiple sclerosis. As key mediators in the synthesis of S1P, sphingosine kinase (SphK) isoforms 1 and 2 have attracted attention as viable targets for pharmacologic intervention. In this report, we describe the design, synthesis, and biological evaluation of sphingosine kinase 2 (SphK2) inhibitors with a focus on systematically introducing rigid structures in the aliphatic lipid tail present in existing SphK2 inhibitors. Experimental as well as molecular modeling studies suggest that conformationally restricted "lipophilic tail" analogues bearing a bulky terminal moiety or an internal phenyl ring are useful to complement the "J"-shaped sphingosine binding pocket of SphK2. We identified 14c (SLP9101555) as a potent SphK2 inhibitor (K i = 90 nM) with 200-fold selectivity over SphK1. Molecular docking studies indicated key interactions: the cyclohexyl ring binding in the cleft deep in the pocket, a trifluoromethyl group fitting in a small side cavity, and a hydrogen bond between the guanidino group and Asp308 (amino acid numbering refers to human SphK2 (isoform c) orthologue). In vitro studies using U937 human histiocytic lymphoma cells showed marked decreases in extracellular S1P levels in response to our SphK2 inhibitors. Administration of 14c (dose: 5 mg/kg) to mice resulted in a sustained increase of circulating S1P levels, suggesting target engagement.

Cannabinoid type-2 receptors: An emerging target for regulating schizophrenia-relevant brain circuits.

Although the cannabinoid type-2 receptor (CB2) is highly expressed in the immune system, emerging evidence points to CB2 playing a key role in regulating neuronal function in the central nervous system. Recent anatomical studies, combined with electrophysiological studies, indicate that CB2 receptors are expressed in specific dopaminergic and glutamatergic brain circuits that are hyperactive in schizophrenia patients. The ability of CB2 receptors to inhibit dopaminergic and hippocampal circuits, combined with the anti-inflammatory effects of CB2 receptor activation, make this receptor an intriguing target for treating schizophrenia, a disease where novel interventions that move beyond dopamine receptor antagonists are desperately needed. The development of new CB2-related pharmacological and genetic tools, including the first small molecule positive allosteric modulator of CB2 receptors, has greatly advanced our understanding of this receptor. While more work is needed to further elucidate the translational value of selectively targeting CB2 receptors with respect to schizophrenia, the studies discussed below could suggest that CB2 receptors are anatomically located in schizophrenia-relevant circuits, where the physiological consequence of CB2 receptor activation could correct circuit-based deficits commonly associated with positive and cognitive deficits.

mGlu1-mediated restoration of prefrontal cortex inhibitory signaling reverses social and cognitive deficits in an NMDA hypofunction model in mice.

Extensive evidence supports the hypothesis that deficits in inhibitory GABA transmission in the prefrontal cortex (PFC) may drive pathophysiological changes underlying symptoms of schizophrenia that are not currently treated by available medications, including cognitive and social impairments. Recently, the mGlu1 subtype of metabotropic glutamate (mGlu) receptor has been implicated as a novel target to restore GABAergic transmission in the PFC. A recent study reported that activation of mGlu1 increases inhibitory transmission in the PFC through excitation of somatostatin-expressing GABAergic interneurons, implicating mGlu1 PAMs as a potential treatment strategy for schizophrenia. Here, we leveraged positive allosteric modulators (PAMs) of mGlu1 to examine whether mGlu1 activation might reverse physiological effects and behavioral deficits induced by MK-801, an NMDA receptor antagonist commonly used to model cortical deficits observed in schizophrenia patients. Using ex vivo whole-cell patch-clamp electrophysiology, we found that MK-801 decreased the frequency of spontaneous inhibitory postsynaptic currents onto layer V pyramidal cells of the PFC and this cortical disinhibition was reversed by mGlu1 activation. Furthermore, acute MK-801 treatment selectively induced inhibitory deficits onto layer V pyramidal cells that project to the basolateral amygdala, but not to the nucleus accumbens, and these deficits were restored by selective mGlu1 activation. Importantly, the mGlu1 PAM VU6004909 effectively reversed deficits in sociability and social novelty preference in a three-chamber assay and improved novel objection recognition following MK-801 treatment. Together, these findings provide compelling evidence that mGlu1 PAMs could serve as a novel approach to reduce social and cognitive deficits associated with schizophrenia by enhancing inhibitory transmission in the PFC, thus providing an exciting improvement over current antipsychotic medication.

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 (27b) was identified as a novel M5 orthosteric antagonist with high potency (human M5 IC50 = 36 nM), M5 subtype selectivity (>100-fold selectivity against human M1-4) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

A retrospective observational study of patient analgesia outcomes when regional anaesthesia procedures are performed by consultants versus supervised trainees.

At teaching hospitals, consultants must provide effective supervision, including appropriate selection of teaching cases, such that the outcomes achieved by trainees are similar to that of consultants. Numerous studies in the surgical literature have compared patient outcomes when surgery is performed by consultant surgeons or surgical trainees but, to our knowledge, none exist in the field of anaesthesia. We aimed to compare analgesia outcomes of regional anaesthesia when performed by supervised trainees versus consultants. We designed a retrospective observational study using registry data. The primary outcome was inadequate analgesia, defined as a numerical rating scale (NRS) for pain >5 reported at any time in the post-anaesthesia care unit (PACU). Secondary outcomes included the maximum pain NRS, pain experienced in the PACU, and the requirement for systemic opioid analgesia in the PACU. Of the 1814 patients analysed, the primary proceduralist was a consultant for 514 (28.3%) patients and a trainee for 1300 (71.7%) patients. All trainees were supervised by an on-site consultant. There were no statistically significant differences between consultants and supervised trainees in terms of the primary outcome (NRS >5 in 24.9% and 24.5% of patients, respectively; P = 0.84) and secondary outcomes. Compared to trainees, consultants had a slightly higher rate of patients with a body mass index >30 kg/m2, an American Society of Anesthesiologists Physical Status Classification of 3 or 4, nerve blocks performed under general anaesthesia, paravertebral/neuraxial blocks and blocks with perineural catheter placement. Regional anaesthesia performed by supervised trainees can achieve similar analgesia outcomes to consultant-performed procedures.

Muscarinic receptors: from clinic to bench to clinic.

Efforts to target muscarinic acetylcholine receptors in the brain have been hampered by dose-limiting side effects. In a tour de force of team science, Brown and colleagues have designed a muscarinic agonist that has been optimized to possess properties that could position it to succeed where other agonists have failed.

Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency.

Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.

Discovery of the First Selective M4 Muscarinic Acetylcholine Receptor Antagonists with in Vivo Antiparkinsonian and Antidystonic Efficacy.

Nonselective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson's disease and dystonia. Despite their efficacy in these and other central nervous system disorders, antimuscarinic 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 antiparkinsonian and antidystonic efficacy observed with the use of nonselective antimuscarinic 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 nonselective antimuscarinic 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 nonselective mAChR antagonists to confirm that the M4 receptor activation is required for the locomotor-stimulating and antiparkinsonian 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 antiparkinsonian and antidystonic efficacy in pharmacological and genetic models of movement disorders.

Targeting muscarinic receptors to treat schizophrenia.

Schizophrenia is a severe neuropsychiatric disorder characterized by a diverse range of symptoms that can have profound impacts on the lives of patients. Currently available antipsychotics target dopamine receptors, and while they are useful for ameliorating the positive symptoms of the disorder, this approach often does not significantly improve negative and cognitive symptoms. Excitingly, preclinical and clinical research suggests that targeting specific muscarinic acetylcholine receptor subtypes could provide more comprehensive symptomatic relief with the potential to ameliorate numerous symptom domains. Mechanistic studies reveal that M1, M4, and M5 receptor subtypes can modulate the specific brain circuits and physiology that are disrupted in schizophrenia and are thought to underlie positive, negative, and cognitive symptoms. Novel therapeutic strategies for targeting these receptors are now advancing in clinical and preclinical development and expand upon the promise of these new treatment strategies to potentially provide more comprehensive relief than currently available antipsychotics.

TRIM71 binds to IMP1 and is capable of positive and negative regulation of target RNAs.

TRIM71 is an important RNA-binding protein in development and disease, yet its direct targets have not been investigated globally. Here we describe a number of disease and developmentally-relevant TRIM71 RNA targets such as the MBNL family, LIN28B, MDM2, and TCF7L2. We describe a new role for TRIM71 as capable of positive or negative RNA regulation depending on the RNA target. We found that TRIM71 co-precipitated with IMP1 which could explain its multiple mechanisms of RNA regulation, as IMP1 is typically thought to stabilize RNAs. Deletion of the NHL domain of TRIM71 impacted its ability to bind to RNA and RNAs bound by congenital hydrocephalus-associated point mutations in the RNA-binding NHL domain of TRIM71 clustered closely with RNAs bound by the NHL deletion mutant. Our work expands the possible mechanisms by which TRIM71 may regulate RNAs and elucidates further potential RNA targets.

Examining the role of muscarinic M5 receptors in VTA cholinergic modulation of depressive-like and anxiety-related behaviors in rats.

Acetylcholine is implicated in mood disorders including depression and anxiety. Increased cholinergic tone in humans and rodents produces pro-depressive and anxiogenic-like effects. Cholinergic receptors in the ventral tegmental area (VTA) are known to mediate these responses in male rats, as measured by the sucrose preference test (SPT), elevated plus maze (EPM), and the forced swim test (FST). However, these effects have not been examined in females, and the VTA muscarinic receptor subtype(s) mediating the pro-depressive and anxiogenic-like behavioral effects of increased cholinergic tone are unknown. We first examined the behavioral effects of increased VTA cholinergic tone in male and female rats, and then determined whether VTA muscarinic M5 receptors were mediating these effects. VTA infusion of the acetylcholinesterase inhibitor physostigmine (0.5 µg, 1 µg and 2 µg/side) in males and females produced anhedonic-like, anxiogenic, pro-depressive-like responses on the SPT, EPM, and FST. In females, VTA administration of the muscarinic M5 selective negative allosteric modulator VU6000181 (0.68 ng, 2.3 ng, 6.8 ng/side for a 3 µM, 10 µM, 30 µM/side infusion) did not alter SPT, EPM nor FST behavior. However, in males intra-VTA infusion of VU6000181 alone reduced time spent immobile on the FST. Furthermore, co-infusion of VU6000181 with physostigmine, in male and female rats, attenuated the pro-depressive and anxiogenic-like behavioral responses induced by VTA physostigmine alone, in the SPT, EPM, and FST. Together, these data reveal a critical role of VTA M5 receptors in mediating the anhedonic, anxiogenic, and depressive-like behavioral effects of increased cholinergic tone in the VTA.

Activation of the mGlu1 metabotropic glutamate receptor has antipsychotic-like effects and is required for efficacy of M4 muscarinic receptor allosteric modulators.

Recent clinical and preclinical studies suggest that selective activators of the M4 muscarinic acetylcholine receptor have potential as a novel treatment for schizophrenia. M4 activation inhibits striatal dopamine release by mobilizing endocannabinoids, providing a mechanism for local effects on dopamine signaling in the striatum but not in extrastriatal areas. G protein-coupled receptors (GPCRs) typically induce endocannabinoid release through activation of Gαq/11-type G proteins whereas M4 transduction occurs through Gαi/o-type G proteins. We now report that the ability of M4 to inhibit dopamine release and induce antipsychotic-like effects in animal models is dependent on co-activation of the Gαq/11-coupled mGlu1 subtype of metabotropic glutamate (mGlu) receptor. This is especially interesting in light of recent findings that multiple loss of function single nucleotide polymorphisms (SNPs) in the human gene encoding mGlu1 (GRM1) are associated with schizophrenia, and points to GRM1/mGlu1 as a gene within the "druggable genome" that could be targeted for the treatment of schizophrenia. Herein, we report that potentiation of mGlu1 signaling following thalamo-striatal stimulation is sufficient to inhibit striatal dopamine release, and that a novel mGlu1 positive allosteric modulator (PAM) exerts robust antipsychotic-like effects through an endocannabinoid-dependent mechanism. However, unlike M4, mGlu1 does not directly inhibit dopamine D1 receptor signaling and does not reduce motivational responding. Taken together, these findings highlight a novel mechanism of cross talk between mGlu1 and M4 and demonstrate that highly selective mGlu1 PAMs may provide a novel strategy for the treatment of positive symptoms associated with schizophrenia.

High density is a property of slow-cycling and treatment-resistant human glioblastoma cells.

Slow-cycling and treatment-resistant cancer cells escape therapy, providing a rationale for regrowth and recurrence in patients. Much interest has focused on identifying the properties of slow-cycling tumor cells in glioblastoma (GBM), the most common and lethal primary brain tumor. Despite aggressive ionizing radiation (IR) and treatment with the alkylating agent temozolomide (TMZ), GBM patients invariably relapse and ultimately succumb to the disease. In patient biopsies, we demonstrated that GBM cells expressing the proliferation markers Ki67 and MCM2 displayed a larger cell volume compared to rare slow-cycling tumor cells. In optimized density gradients, we isolated a minor fraction of slow-cycling GBM cells in patient biopsies and tumorsphere cultures. Transcriptional profiling, self-renewal, and tumorigenicity assays reflected the slow-cycling state of high-density GBM cells (HDGCs) compared to the tumor bulk of low-density GBM cells (LDGCs). Slow-cycling HDGCs enriched for stem cell antigens proliferated a few days after isolation to generate LDGCs. Both in vitro and in vivo, we demonstrated that HDGCs show increased treatment-resistance to IR and TMZ treatment compared to LDGCs. In conclusion, density gradients represent a non-marker based approach to isolate slow-cycling and treatment-resistant GBM cells across GBM subgroups.

Bile diversion, a bariatric surgery, and bile acid signaling reduce central cocaine reward.

The gut-to-brain axis exhibits significant control over motivated behavior. However, mechanisms supporting this communication are poorly understood. We reveal that a gut-based bariatric surgery chronically elevates systemic bile acids and attenuates cocaine-induced elevations in accumbal dopamine. Notably, this surgery reduces reward-related behavior and psychomotor sensitization to cocaine. Utilizing a knockout mouse model, we have determined that a main mediator of these post-operative effects is the Takeda G protein-coupled bile acid receptor (TGR5). Viral restoration of TGR5 in the nucleus accumbens of TGR5 knockout animals is sufficient to restore cocaine reward, centrally localizing this TGR5-mediated modulation. These findings define TGR5 and bile acid signaling as pharmacological targets for the treatment of cocaine abuse and reveal a novel mechanism of gut-to-brain communication.

lin-4 and the NRDE pathway are required to activate a transgenic lin-4 reporter but not the endogenous lin-4 locus in C. elegans.

As the founding member of the microRNA (miRNA) gene family, insights into lin-4 regulation and function have laid a conceptual foundation for countless miRNA-related studies that followed. We previously showed that a transcriptional lin-4 reporter in C. elegans was positively regulated by a lin-4-complementary element (LCE), and by lin-4 itself. In this study, we sought to (1) identify additional factors required for lin-4 reporter expression, and (2) validate the endogenous relevance of a potential positive autoregulatory mechanism of lin-4 expression. We report that all four core nuclear RNAi factors (nrde-1, nrde-2, nrde-3 and nrde-4), positively regulate lin-4 reporter expression. In contrast, endogenous lin-4 levels were largely unaffected in nrde-2;nrde-3 mutants. Further, an endogenous LCE deletion generated by CRISPR-Cas9 revealed that the LCE was also not necessary for the activity of the endogenous lin-4 promoter. Finally, mutations in mature lin-4 did not reduce primary lin-4 transcript levels. Taken together, these data indicate that under growth conditions that reveal effects at the transgenic locus, a direct, positive autoregulatory mechanism of lin-4 expression does not occur in the context of the endogenous lin-4 locus.

Activity in LIP, But not V4, Matches Performance When Attention is Spread.

The enhancement of neuronal responses in many visual areas while animals perform spatial attention tasks has widely been thought to be the neural correlate of visual attention, but it is unclear whether the presence or absence of this modulation contributes to our striking inability to notice changes in change blindness examples. We asked whether neuronal responses in visual area V4 and the lateral intraparietal area (LIP) in posterior parietal cortex could explain the limited ability of subjects to attend multiple items in a display. We trained animals to perform a change detection task in which they had to compare 2 arrays of stimuli separated briefly in time and found that each animal's performance decreased as function of set-size. Neuronal discriminability in V4 was consistent across set-sizes, but decreased for higher set-sizes in LIP. The introduction of a reward bias produced attentional enhancement in V4, but this could not explain the vast improvement in performance, whereas the enhancement in LIP responses could. We suggest that behavioral set-size effects and the marked improvement in performance with focused attention may not be related to response enhancement in V4 but, instead, may occur in or on the way to LIP.