Nicole E. Chambers,Michael Millett,Mark S. Moehle
The muscarinic M4 acetylcholine receptor exacerbates symptoms of movement disorders
acetylcholine,basal ganglia,cholinergic,motor deficits,movement disorders,muscarinic
Barbeau's seesaw hypothesis of dopamine-acetylcholine balance has predominated movement disorders literature for years. Both the simplicity of the explanation and the matching efficacy of anticholinergic treatment in movement disorders seem to support this hypothesis. However, evidence from translational and clinical studies in movement disorders indicates that many features of this simple balance are lost, broken, or absent from movement disorders models or in imaging studies of patients with these disorders. This review reappraises the dopamine-acetylcholine balance hypothesis in light of recent evidence and describes how the Gαi/o coupled muscarinic M4 receptor acts in opposition to dopamine signaling in the basal ganglia. We highlight how M4 signaling can ameliorate or exacerbate movement disorders symptoms and physiological correlates of these symptoms in specific disease states. Furthermore, we propose future directions for investigation of this mechanisms to fully understand the potential efficacy of M4 targeting therapeutics in movement disorders. Overall, initial evidence suggest that M4 is a promising pharmaceutical target to ameliorate motor symptoms of hypo- and hyper-dopaminergic disorders.
The muscarinic M4 acetylcholine receptor exacerbates symptoms of movement disorders

Barbeau's seesaw hypothesis of dopamine-acetylcholine balance has predominated movement disorders literature for years. Both the simplicity of the explanation and the matching efficacy of anticholinergic treatment in movement disorders seem to support this hypothesis. However, evidence from translational and clinical studies in movement disorders indicates that many features of this simple balance are lost, broken, or absent from movement disorders models or in imaging studies of patients with these disorders. This review reappraises the dopamine-acetylcholine balance hypothesis in light of recent evidence and describes how the Gαi/o coupled muscarinic M4 receptor acts in opposition to dopamine signaling in the basal ganglia. We highlight how M4 signaling can ameliorate or exacerbate movement disorders symptoms and physiological correlates of these symptoms in specific disease states. Furthermore, we propose future directions for investigation of this mechanisms to fully understand the potential efficacy of M4 targeting therapeutics in movement disorders. Overall, initial evidence suggest that M4 is a promising pharmaceutical target to ameliorate motor symptoms of hypo- and hyper-dopaminergic disorders.

Canonical literature in movement disorders and basal ganglia physiology suggests a fine-tuned balance between levels of the neurotransmitters dopamine (DA) and acetylcholine (ACh; Barbeau's seesaw hypothesis; [1,2]). This viewpoint has been largely supported by observed changes in cholinergic transmission in disorders affecting DA such as Parkinson's disease (PD) and schizophrenia [3,4]. Additional clinical evidence points to this imbalance: hypo-DAergic movement disorders can be treated with anticholinergic therapies, whereas hyper-DAergic disorders can be treated with pro-cholinergic therapies [5,6]. The seesaw view of ACh and DA is an oversimplification which largely hinges on research describing levels of neurotransmitters. However, neurotransmitter levels alone do not directly indicate or correlate to neural activity or plasticity in the basal ganglia circuit. Furthermore, the relationship between DA and ACh is more nuanced than simply two neurotransmitters suppressing one another, as there are complex signaling mechanisms among a number or receptors that bind these neurotransmitters. DA acts in opposition to acetylcholine release in the striatum, via Gαi/o-coupled DA D2 receptors (D2) expressed on striatal cholinergic interneurons (ChI; [7,8]). However, there is considerable overlap in expression of DA receptors and ACh receptors in spiny projection neurons (SPNs), corticostriatal terminals, and even in ChI, which suggests a more complicated interplay between ACh and DA receptor signaling. Conversely, ACh exerts bidirectional effects on DA release. Specifically, ACh can both increase nigrostriatal DA release through activation of nicotinic receptors [9] and can also both oppose or facilitate the effects of DA release and signaling depending on which muscarinic ACh receptors it activates on spiny projection neurons (SPNs; [10,11]),corticostriatal glutamate terminals [12], or DA terminals themselves. In particular, recent research has highlighted a unique role for the M4 muscarinic ACh receptor (M4) specifically in regulating DA signaling and direct pathway plasticity. M4 are metabotropic ACh receptors which are coupled to Gαi/o. ACh binding to M4 leads to the inhibition of adenylyl cyclase and subsequently decreases cyclic adenosine monophosphate (cAMP). Of note, M2 muscarinic ACh receptors show significant functional overlap with M4; however, unlike M4, their location in peripheral heart and lung tissue precludes them from being a safe and effective therapeutic target for central nervous system disorders [13]. M4 are most abundantly expressed in the striatum, and mechanistic electrophysiological and behavioral studies using newly developed pharmacological tool compounds have indicated several key sites of M4 regulation of the basal ganglia motor circuit (See Figure 1). First, at corticostriatal terminals, M4 inhibits glutamate release [12]. Second, M4 is thought to inhibit ACh release from striatal ChIs [14,15]. Third, M4 acts on cell bodies and terminals of direct pathway spiny projection neurons (dSPNs; [10,11,16]), where they directly oppose Gαolf-coupled DA D1 receptor (D1) signaling and promote dSPN long-term depression (LTD). When ACh binds to M4 on dSPN cell bodies, it also reduces the probability of DA release through autacoid signaling, specifically the release of the endocannabinoid 2-arachidonoylglycerol (2-AG) which then binds to Gɑi/o-coupled CB2 cannabinoid receptors (CB2) located on nigrostriatal terminals [16]. Historically, selectively targeting M4 with pharmaceutical compounds has been challenging due to a close sequence homology between the orthosteric site of muscarinic receptors.There are compounds under development that target the orthosteric site; however these efforts have yet to produce a truly selective M4 compound agonist, but success has very recently been reported with orthosteric antagonists. This has led to an abundance of behavioral pharmacology research in which the interpretation of results are confounded by the use of non-selective muscarinic compounds, namely tropicamide. For more selective interrogation of M4’s role in the brain and behavior, the field has relied upon genetic global or conditional M4 knockout (KO) mice. More recently, the discovery of compounds which target M4 through allosteric sites has allowed for pharmacological agents to potentiate M4 signaling that are truly selective and specific for M4 [10–12,17,18]. These positive allosteric modulators and recently developed selective M4 orthosteric antagonists have permitted the direct testing of the utility of M4 inhibition and potentiation in preclinical models of movement disorders. This is especially exciting as M4 modulation may be better tolerated by patients than general cholinergic therapies due to higher M4 expression in the central nervous system and limited presence in the periphery. Of note, an M4 allosteric modulator emraclidine, was recently tested in a double blind, placebo-controlled Phase 1B clinical trial in schizophrenia patients [19]. It is promising that the only peripheral side effect noted was a small and transient increase in heart rate and systolic and diastolic blood pressure that went away after 6 weeks of treatment. Below, we discuss the function of M4 in the normal brain, how M4 signaling may be altered in different movement disorders, and how this may be targeted for therapeutic benefit.

Under normal physiological conditions, evidence indicates that the net consequence of M4 activation on movement is the suppression of locomotion [20]. M4 also plays a role in behaviors and functions outside of the scope of this review, such as social behavior, and reward learning, (see [21,22]). This is supported by the fact that M4 KO animals show increases in basal locomotion and in novelty-induced locomotion [20,22]. Additionally, M4 KO mice exhibit augmented locomotor responses to D1 agonists [20]. M4 also modify effects of indirect DA agonists such as amphetamine. For instance, in wild-type animals M4 positive allosteric modulators (PAM) decrease amphetamine-induced hyperlocomotion and striatal and accumbal DA release [23,24]. In sum, this work implies that M4 deletion or blockade increases movement and DA signaling, whereas augmenting ACh binding to M4 decreases movement and DA signaling, especially in the direct pathway. Recent work suggests that M4 opposes D1 signaling specifically on dSPN terminals in the substantia nigra pars reticulata (SNr), and not on dendrites or cell bodies in the striatum, implicating brainstem ACh transmission in the regulation of the direct pathway [11]. Brainstem cholinergic terminals also likely express M4 as autoreceptors, and some evidence suggests that they also project to the striatum [25], and the substantia nigra pars compacta [26]. It is possible also that M4 are expressed on targets of brainstem cholinergic transmission; however, the exact role of brainstem ACh remains undetermined. In addition to opposing D1 signaling, M4 also regulate dSPN plasticity in the striatum. Long-term potentiation (LTP) in the striatum occurs as a result of coactivation of N-Methyl-D-Aspartate (NMDA) glutamate receptors by corticostriatal glutamate, tropomyosin-related kinase B receptors which bind brain-derived neurotrophic factor, and cAMP signaling [27]. M4 likely affects LTP in two ways: first through the inhibition of cAMP signaling, and second through interactions with the NMDA receptor. Prior research shows that M4 PAM suppresses NMDA-evoked postsynaptic current influx and calcium transients in dSPNs [10] and promotes depotentiation in spike-time-dependent plasticity protocols. Furthermore, M4 signaling promotes LTD in dSPNs. Interestingly, a system of plasticity checks and balances is built into dSPNs, as calmodulin-dependent protein kinase II, which is active in LTP, binds to the second intracellular loop of M4 and can potentiate its activity through threonine phosphorylation [28]. Prepulse inhibition of startle is also decreased by M4 KO. Critically, PPI is reduced in animals with increases or decreases in DA tone, indicating that M4 loss likely leads to enhanced DAergic signaling [22]. Finally, in contrast with general anticholinergic therapies, targeting M4 likely would not produce severe adverse effects on memory, as M4 KO did not alter Morris Water Maze performance compared with controls [22]. Additionally, targeting M4 has been shown to be efficacious for cognition in recent studies [29,30]. Overall, these studies provided critical evidence that M4 regulates basal ganglia nuclei linked to several diseases and implicates that M4 should be further studied for their role in modulating the symptoms of movement disorders.

Dystonia is a set of sporadic and genetic movement disorders associated with involuntary, repetitive muscle contractions and abnormal postures (See Figure 2 for circuit changes in dystonia). Disrupted striatal DA neurotransmission may be a unifying theme which is perpetuated by various mechanisms in different types of dystonia, as DA synthesis is impaired in DOPA-responsive dystonia (DRD), DA release is reduced in Tor1A-associated dystonia [31], and loss-of-function mutations in GNAL (which encodes Gαolf, and couples to D1 in dSPNs) prevent efficient signal transduction of DA (Fuchs et al. [32]). Some studies have also found a reduction in D2 protein or receptor availability in animal models of DYT1 [33–36] and in humans dystonia patients [37,38]. Furthermore, other genetic dystonias which do not have direct links to DA synthesis or signaling appear to have altered DA homeostasis, suggesting that disturbed DA may be critical to dystonia pathogenesis. One possible mechanism to ameliorate this disturbed DA signaling is through targeted cholinergic modulation. Changes in DA may alter cholinergic release or signaling, which provides interesting therapeutic targets for dystonia. Currently, non-selective anti-muscarinic antagonists remain the only orally bioavailable small molecule therapeutics for dystonia. In mouse models of dystonia, increased striatal cholinergic tone may result in increased M4 signaling at corticostriatal terminals, as well as dSPNs [24,25]. This is supported by initial studies with pharmacological inhibitors of M4 which relieve motor dysfunction in preclinical dystonia models [18]. The increase in cholinergic tone may also suggest that M4 autoreceptors are dysfunctional in dystonia. Death of ChIs within the first 1–2 months in certain mouse models of DYT1 linked dystonia, occurs possibly due to excitotoxicity. Death of ChIs is accompanied by reductions in choline acetyltransferase and vesicular ACh transporter [39,40]. Dendritic trees of striatal ChIs in DRD dystonia mice also show marked decreases in complexity [40,41]. These cholinergic modifications lead to changes in DA signaling in ChIs via M4 saturation of Gαi/o signaling where increased cholinergic tone elicits paradoxical excitation of ChIs by D2 receptors [33 42,43]. However, whether normalizing these cholinergic mediated deficits is therapeutically relevant remains untested. Recent evidence from dystonia patients suggests that there is disrupted functional connectivity in the motor cortex which may be a compensatory mechanism to counter reduced DA signaling [44–46]. This is in accordance with observed alterations in LTD multiple dystonia models [47]. One factor that may contribute to this abnormal activity is the blunting of M4 signaling in dystonia, as M2/M4 knockout mice show similar abnormalities in synaptic plasticity [48]. Perhaps, in response to the reduction or lack of DA signaling in the direct pathway, DA receptors sensitize whereas M4 desensitize to allow for more movement. This is broadly supported by evidence that multiple types of dystonia are responsive to anticholinergic therapy, which could increase the likelihood of M4 inhibition of LTP [49–51]. Relatedly, recent evidence shows that M4 antagonism reverses dystonic movements in DRD mouse models and normalizes DA release TOR1A dystonia models [18,31]. Furthermore, M4 are required for the effects of Trihexyphenidyl on dystonia [31]. However, the extent of efficacy of M4 antagonism in different types of dystonia both genetic and sporadic has largely not been examined.

PD is neurodegenerative disorder which is characterized by motor symptoms including akinesia, bradykinesia, gait difficulties, and in some patients, development of cognitive impairment or dementia (See Figure 3 for circuit changes in PD). Death of nigrostriatal DA neurons leads to low DAergic tone; whereas striatal cholinergic tone is augmented [3,52]. This increase in cholinergic tone appears to be clinically relevant, as non-selective cholinergic antagonists were originally used to treat PD and remain an additive therapy. The increase in cholinergic tone may be a result of M4 autoreceptors on striatal ChIs becoming desensitized in PD after an increase in regulator of G-protein signaling 4 (RGS4; [53]). RGS4 is a guanosine triphosphatase-accelerating protein associated with autoreceptors, and it reduces G-protein coupling between receptors and effectors, in this case, specifically Cav2 calcium channels and potassium channels, which regulate ChI activity and ACh release. As a result of augmented cholinergic tone, striatal M4 signaling may be increased on dSPNs which likely contributes to LTD and motor symptoms such as bradykinesia and akinesia. Furthermore, M4 signaling on dSPN terminals in the SNr is also likely increased in PD. In multiple preclinical lesion models, pedunculopontine nucleus cholinergic neurons become hyperactive [54–56], which could even further inhibit dSPN firing. Corticostriatal glutamate release would also be reduced by M4 signaling. Within the cortex, there are two types of neurons that project to the striatum: the pyramidal tract (PT) neurons which innervate the ipsilateral striatum and encode information about ongoing motor activity and intratelencephalic tract (IT) neurons which innervate the striatum bilaterally and carry information related to motor planning and the environment [57–59]. IT and PT neurons synapse onto both dSPNs and iSPNs [57]. Recent studies in PD models demonstrate inhibition of PT but not IT neurons [60,61], which may contribute to the lateralized symptom profile in PD patients, where pathology is more severe on one side of the body. M4 signaling on corticostriatal terminals of PT neurons may further exacerbate this effect as observed in preclinical models of parkinsonian motor deficits and blocking this may normalize corticostriatal transmission and alleviate some PD motor symptoms. Interestingly, in 6-OHDA lesion models there is a loss of synaptic plasticity in SPNs at 3–4 weeks post-lesion [62], which could be due to a marked decrease in glutamate and DA signaling onto SPNs [63,64]. By 2 months post-6-OHDA-lesion, dSPNs undergo spine pruning, which is consistent with a state of LTD, which could be perpetuated by increased M4 signaling. Finally, by 3 months post-lesion, dSPNs undergo substantial spine loss [62]. Later in disease progression, compensatory changes occur whereby dSPNs show increases in intrinsic excitability. This change may be due, in part, to simultaneous increase in D1 sensitization paired with M4 desensitization, which may be mediated by the progressive inactivity of calmodulin-dependent protein kinase II in the absence of LTP [28]. Despite the contribution of M4 to directly regulating neurons and circuits shown to be altered in preclinical models of parkinsonian motor deficits as discussed above, the ability for M4 to directly modulate the symptoms of PD remains largely unexplored with only one study directly showing that M4 antagonism relieves bradykinesia and forelimb asymmetry in models of parkinsonian motor deficits [18].

Huntington's disease (HD) is an autosomal dominant disorder caused by an expansion of the CAG triplet repeat in the huntington gene (htt) which leads to a progressive degeneration of SPNs and cortical cells (See Figure 4 for circuit changes in HD). Early in disease progression there are heightened levels of nigrostriatal DA release; whereas later in disease progression there are reduced levels of DA release [65]. There are marked cholinergic abnormalities in HD including a reduction in ChAT, and likely ACh synthesis (See Figure 4). Additionally, striatal ACh release is reduced, and ChI function is impaired. Although mixed results have occurred in human HD patients, some studies suggest that inhibiting the breakdown of ACh via inhibitors of acetylcholinesterase may rescue deficits in some patients. Corticostriatal drive from the intratelencephalic tract (IT) neurons to SPNs is increased, promoting dSPN LTP [66]. Intriguingly, deleting mutant htt from ChIs but not from the cortex normalized cholinergic tone and restore IT neurons to their normal state of connectivity with SPNs, suggesting that cholinergic signaling is regulating corticostriatal drive in early HD. Underlying these changes in corticostriatal signaling may be altered M4 activity. There is aberrant M4-dependent plasticity in HD. In the R6/2 preclinical mouse model of HD, M4 expression is reduced in the cortex and likely at corticostriatal synapses [67]. This reduction in expression of M4 enhances corticostriatal glutamate release, and likely contributes to dSPN LTP and chorea. Directly targeting M4 through selective potentiation of M4 in presymptomatic periods normalized corticostriatal glutamate release, normalized nigrostriatal DA release, and prevented the onset of motor symptoms [68]. Thus, targeting M4 may be beneficial early in HD progression through countering the effects of over-active corticostriatal drive through depotentiation, and possibly represents a disease modifying strategy in HD.

L-DOPA-induced dyskinesia (LID) is a side effect of the standard treatment for PD that involves the development of abnormal involuntary movements that can be extremely debilitating (See Figure 5 for circuit changes). L-DOPA improves parkinsonian motor deficits and re-establishes dSPN LTP in animal models of disease. In the parkinsonian state, raphestriatal serotonin neurons convert exogenous L-DOPA into DA [69], but unlike nigrostriatal DA neurons, serotonin neurons lack D2 and DA transporter, resulting in uncontrolled DA release and pulsatile DA stimulation in the striatum. Coupled with changes in dSPNs such as D1 sensitization and increased arborization, this leads to an inability for dSPNs to be depotentiated. Additionally, striatal cholinergic tone remains elevated [3,52], suggesting that M4 autoreceptors on ChIs are likely still dysfunctional due to increases in RGS4 [53]. Because of elevated cholinergic tone, excessive M4 signaling may also contribute to the decoupling of striatal signaling from behavioral outcomes. Interestingly, selectively killing ChIs can attenuate LID [52]. Motor cortex activity is reduced by both acute and chronic L-DOPA treatment in both humans [70,71] and preclinical models [72], and existing data suggest that motor cortex activity is positively correlated with LID severity, and that pharmacologically inhibiting motor cortex neurons may decrease LID [72]. M4 provide another more specific avenue by which cholinergic transmission can be normalized, corticostriatal glutamate release can be reduced, and the direct pathway can be depotentiated. Indeed, M4 PAM VU0467154 has been shown to reduce LID in preclinical rodent and non-human primate models [10]. This is corroborated by new evidence that chemogenetic modulation of dSPNs with modified human M4 receptors that respond to designer drugs (hm4di) delays onset of dyskinesia, reduces the duration of dyskinesia, and attenuates total dyskinetic behavior [73]. However, follow-up studies are needed, as potentiating M4 effects may worsen PD motor symptoms, especially in the time window of the off-period before the next dose of L-DOPA, where synaptic availability of DA is low.

Taken together, experimental evidence suggests that the overall role of M4 is to modulate DA release and signaling which opposes locomotion in normal physiological states. Furthermore, M4 show great promise as a pharmacological target for treating movement disorders symptoms through normalization of aberrant synaptic plasticity underlying movement disorders symptoms. Specifically, an M4 PAM has already shown efficacy in lessening the severity of LID in preclinical models [10] and for normalizing DA release and preventing onset of motor symptoms in an HD mouse model [68]. M4 antagonists reverse dystonic movements in DRD mouse models and normalize DA release in TOR1A dystonia models. Finally, M4 antagonism relieves bradykinesia and forelimb asymmetry in preclinical mouse models of PD [18]. However, the efficacy of M4 modulating agents to remove symptoms of movement disorders remains largely clinically untested. In particular, M4 sensitization and desensitization, trafficking, and protein production may be altered as a compensatory mechanism in movement disorders, leading to homeostatic changes in neurons, and these changes may occur with both region and cell-specificity. Furthermore, the potential disease modifying properties of brainstem and basal forebrain cholinergic projections activating M4 need further investigation not only for symptomatic relief, but also as disease modifying strategies as recent data from HD and Alzheimer's models suggest that cholinergic manipulations may be disease modifying. Finally, while great progress has been made in identifying the behaviors and synaptic processes of M4 with cell type specificity in regulating basal ganglia motor circuits, this has not translated to understanding of specific behaviors that can be altered by targeting M4 on specific neurons in animal models of disease. This will be critical to understanding the mechanism of action and the extent of efficacy of targeting M4. Overall, M4 represents an exciting pharmaceutical target for improvement of movement disorders symptoms, and possibly disease modification.

The development of M4 selective compounds is an exciting and important development for the investigation and treatment of movement disorders. M4 both directly and indirectly counters DA transmission in the basal ganglia, and are present at multiple loci that have potential disease-modifying effects. Potentiation of M4 reduces symptoms hyper-dopaminergic diseases such as HD and LID, and M4 antagonism reduces symptoms of hypo-dopaminergic diseases such as dystonia and PD. Future investigations into M4 plasticity in movement disorders are necessary. The disease-modifying effects of M4 need to be further investigated, and specific symptoms and aberrant neuroplasticity that are improved by M4 modulation should be defined.