A significant number of genes associated with paroxysmal and non-paroxysmal movement disorders (MDs) and epilepsy have been recognized in recent years, shedding light on the biological substrates and pathways involved in these conditions.

Recently described genes in this field encode for proteins involved in postsynaptic signaling pathways downstream to G-protein-coupled receptors (GPCRs), which are ubiquitously expressed in the central nervous system (CNS) and highly enriched in striatal medium spiny neurons (MSNs) (1, 2). GPCRs control responses to a wide array of sensory stimuli, including light and odorants, and non-sensory stimuli, including neurotransmitters and hormones. Signal transduction via GPCRs relies primarily upon the activation of heterotrimeric G-proteins, which consist of an α-subunit that binds and hydrolyzes GTP and a βγ heterodimer (3). GTP binding can induce an allosteric transition leading to βγ release which, in turn, enables Gα and Gβγ signaling to their multiple downstream effectors (4). The variety and expression pattern of individual G-protein subunits define unique GPCR properties in a cell-context-specific manner.

Genes discovered in this pathway and associated with neurological disorders encode transducer molecules or components of the GPCR machinery (i.e., GNAO1, GNB1, GNAL, GPR88), or proteins controlling the synthesis and hydrolysis of the second messenger cyclic adenosine monophosphate (cAMP) (ADCY5, PDE10A, PDE2A). Although the precise functions are still largely unknown, HPCA (encoding for hippocalcin) is a calcium sensor associated with the plasma membrane that influences the activity of potassium and calcium channels and could be implied in the modulation of dopamine post signaling in striatal neurons (1, 2). Figure 1 represents the organization of this signaling pathway in MSNs (Figure 1). The exact role of these proteins in the pathophysiology of MDs and the functional impact of pathogenic variants have only begun to be explored in preclinical models. For a comprehensive review on this topic, see Golzales-Latapi et al. (2).

GNAL (OMIM 139312) and ADCY5 (OMIM 600293) genes encode for G_olf and adenylyl cyclase type 5 (AC5), the main AC isoform expressed in the striatum, respectively, which are directly involved in the GPCR–cAMP signaling cascade mediated by the activation of dopamine receptor 1 (D1R) and adenosine receptor 2A (A2AR) in MSNs (5).

G_olf is responsible for coupling D1R and A2AR stimulation to the activation of ADCY5, causing increased intracellular cAMP levels, which, in turn, enhances neuronal activity (6). Pathogenic variants in GNAL and ADCY5 may manifest through either autosomal dominant or recessive modes of inheritance. Most GNAL mutations are heterozygous missense, nonsense, or frameshift variants with a clear loss-of-function (LOF) effect (7, 8) leading to reduced cAMP levels. In contrast, most disease-causing ADCY5 variants are gain-of-function (GOF) defects causing increased cAMP production (2). These findings suggest a complex scenario in which both increased and decreased intraneuronal cAMP levels may underlie the pathogenesis of MDs.

The critical role of the GPCRs–cAMP signaling pathway in the pathophysiology of MDs has been further highlighted by the identification of disease-causing variants in PDE10A (OMIM 610652) and PDE2A (OMIM 602658). These genes encode two cyclic nucleotide phosphodiesterases (PDEs) highly expressed in MSNs and critically involved in modulating dopaminergic and adenosinergic responses through degradation of intracellular cAMP and cGMP (9–13). While both homozygous and heterozygous LOF mutations have been reported in PDE10, only biallelic changes have been identified in PDE2A so far.

GNAO1 (OMIM 139311) and GNB1 (OMIM 139380) encode proteins that are components of the GPCR machinery, respectively the alpha subunit (Gαo) and the beta-1 (Gß1) subunit of the inhibitory heterotrimeric Go-protein complex (3). GNAO1 and GNB1 are co-expressed in the cerebral cortex, hippocampus, and striatum where they are involved in transducing signals downstream to several GPCRs and in the regulation of AC activity.

GNAO1 modulates inhibitory signaling from several GPCRs, including GABA-B, dopamine D2, α2A adrenergic, and adenosine A1, regulating neuronal excitability and neurotransmission (3), and controls neurodevelopment (14). In the brain, Go controls the synthesis of cAMP, directly prevents neurotransmitter release, inhibits N-type and P/Q-type calcium channels, and activates G-protein-coupled inward rectifying potassium (GIRK) channels. Regarding striatal pathways, Gαo activity influences the excitability of neurons of the indirect (inhibitory MSNs, iMSNs) and direct pathways (dMSNs) by tuning inputs from dopamine D2 and adenosine A2A receptors, with crucial effects on movement control (15). In dMSNs, Gao affects both the efficacy (defined as the power to produce an effect) and potency (defined as the capacity to produce strong physiological or chemical effects) of responses to dopamine while only modulating adenosine efficacy. Instead, in iMSNs, Gao affects both efficacy and potency of responses to adenosine while only modulating dopamine efficacy. Taken together, these data indicate that Gao plays a pivotal role in controlling the potency and efficacy of stimulatory neuromodulation while only affecting the efficacy of inhibitory inputs in both populations of striatal neurons (15). A preclinical model of Gao defect revealed a different motor impairment as a result of knocking out Gao in dMSN or iMSN. In the first case, the acquisition and retention of motor skills were mainly affected. In the second one, dystonia and profound coordination deficits were observed (15).

Aberrant cAMP synthesis was originally proposed as the main pathogenic mechanism of the disease in GNAO1 disorders, with LOF and GOF alleles that appeared to be primarily associated with epilepsy and MD, respectively (16). These data apparently contradict the original findings from Nakamura et al. (17) suggesting a LOF behavior of GNAO1 variants on Gα_o-mediated signaling, regardless of the associated clinical presentation. In a recently published and elegant study performed by Muntean and coworkers, GNAO1 variants were shown to disturb Gαo function in a cell-type-specific manner via a combination of LOF and dominant-negative mechanisms that are not mutually exclusive (15). More recently, a strong hypomorphic effect or a complete LOF behavior has been confirmed in genetically modified nematodes harboring GNAO1 pathogenic variants, leading to excessive neurotransmitter release by different classes of motor neurons (18, 19). Of note, the observed phenotype was shown to be ameliorated by caffeine via adenosine receptor antagonism (18).

GNB1 encodes Gβ1, the third component of the heterotrimeric G-protein complex. Upon receptor activation, Gβ1 dissociates from the Gα subunit and, together with the Gγ subunit, activates downstream signaling pathways, leading to inhibition of presynaptic voltage-gated calcium (i.e., Cav2.1 and Cav2.2) and potassium channels, with effects on neurotransmitter release (20, 21). Gβ1 interacts also with Gα_olf in striatal neurons, and Lohmann et al. showed that pathogenic GNB1 variants might reduce association with Gα_olf thus reducing coupling to D1R (22). The functional impact of dominant GNB1 mutations is still debated.

The clinical spectrum associated to this group of conditions ranges from developmental epileptic encephalopathy with severe early-onset movement disorder to isolated paroxysmal and/or non-paroxysmal movement disorders.

Clinical studies, preclinical models, and systems biology analysis helped to understand the relevance of these genes encoding postsynaptic signaling proteins in different subtypes of striatal cells to the pathogenesis of hyperkinetic MDs (2). The clinical features other than MD and the pathophysiology of epilepsy and developmental issues in these disorders have been less investigated and remain poorly understood.

Here, we reviewed the clinical phenotypes and mutational spectrum associated with GPCRs signaling disorders focusing on the conditions of this group presenting with epilepsy, movement disorders, and neurodevelopmental disorders. We aimed to verify the presence of common clinical features and characterize the core clinical phenotype of this group of severe early-onset genetic neurological disorders. Considering their clinical severity and susceptibility and precipitation with specific triggers, this in-depth clinical characterization has implications for timely diagnosis, management, and therapeutic strategies.

A comprehensive search of the medical literature (PubMed, Medline, Cochrane CENTRAL, Google Scholar) was conducted to identify papers reporting patients with pathogenic or likely pathogenic variants in GNAO1, GNB1, ADCY5, GNAL PDE2A, PDE10A, HPCA. “GNAO1,” “GNB1,” ”ADCY5,” ”GNAL,” “PDE2A,” “PDE10A,” and “HPCA” were used as search terms. As possible limitations of our search, we declare to have selected only English-written articles. It is possible that by doing so, some information included in non-English-written papers and useful to further delineate the clinical phenotype of these rare disorders could have been missed. Furthermore, variants were not independently re-evaluated as they were already judged as pathogenic or likely pathogenic according to ACMG criteria or reported in multiple patients with similar clinical presentations. We selected and reviewed cases with pathogenic or likely pathogenic variants according to ACMG criteria for which clinical, neuroradiological, neurophysiological, and genetic data were available. We focused on disorders with MDs, epilepsy, and neurodevelopmental disorders in their clinical spectrum.

We collected and reviewed 74 articles (17 for GNB1, 46 for GNAO1, 6 for PDE10A, 2 for PDE2A, and 3 for HCPCA) describing motor, epileptic, and developmental phenotype of patients with genetic disorders of GPCRs–cAMP signaling pathway. Six additional papers on GNAO1 were excluded (four because of insufficient information and two because of the presence of concomitant mutations in other genes). Finally, to the scope of this review 203 cases from 72 papers published up to May 2022 were selected. Figure 2 represents the main clinical features associated with the above-mentioned genes. Characteristics of GNAO1 and GNB1 deficiency were further evaluated to assess for a genotype–phenotype correlation (Figures 3, 4).

Here, we reviewed the clinical phenotypes and mutational spectrum associated with genetic disorders affecting the GPCRs–cAMP signaling pathway and having epilepsy and movement disorder in their clinical spectrum. Common pathophysiology has been proposed but not fully investigated, as well as the degree of clinical overlap, which is the object of this work.

A complex hyperkinetic MD with or without paroxysmal exacerbations seems to be the clinical signature of the whole group. Dominant mutations in GNAO1, GNB1, and PDE2A have been associated to a complex early-onset neurological disorder characterized by a variable association of hyperkinetic MD, epilepsy, and developmental delay generally evolving into intellectual disability (23, 42, 56). GNAL and ADCY5 genes have not been associated with epilepsy so far. A homozygous non-sense variant affecting the GPR88 gene has been reported in four sisters of a single family with childhood-onset chorea and psychomotor retardation. Since then, no additional patients carrying LOF variants in this gene have been identified, raising concerns on the effective relevance of GPR88 as a disease-causing gene. Dyskinetic storms or minor paroxysmal choreo-dystonic spells, baseline dystonia and/or chorea, prominent cranial involvement leading to dysarthria and dysphagia, orofacial dyskinesia, axial hypotonia, and severe impairment of postural development characterize the MD phenotype of these conditions. Susceptibility to a wide range of triggers and severe paroxysmal exacerbations evolving into status dystonicus are typical (ADCY5, GNAO1, GNB1, PDE2A). Febrile and upon awakening exacerbations of movement disorder have been described for GNAO1, ADCY5, PDE2A, and GNB1 (12, 42, 48, 57). Other typical triggers are emotional stress, sudden movements, and sudden sensorial stimuli. Benzodiazepines (clonazepam, lorazepam, midazolam) are useful for controlling or aborting paroxysmal episodes (GNAO1, GNB1), while tetrabenazine can partially control the baseline hyperkinetic MD and prevent MD exacerbations (GNAO1, ADCY5). Neuromodulation (GPi-DBS or pallidotomy) has been successful in controlling paroxysmal exacerbations and evolution into status dystonicus in GNAO1, GNB1, and PDE2A-related MDs.

Epilepsy can be prominent in GNB1 and GNAO1 encephalopathy, while it is anecdotical in individuals with HPCA, PDE2A, and PDE10A pathogenic variants.

DEE, described in the literature as Ohtahara syndrome, infantile spasms, or EIFMS can be the epileptic presentation of GNAO1, GNB1, and PDE2A. Onset with tonic seizures or infantile spasms with EEG patterns of burst suppression or hypsarrhythmia has been described for GNAO1 and GNB1.

These infantile forms are usually drug-resistant. Childhood-onset presentations with focal (motor and non-motor) and/or generalized seizures are usually milder. Febrile status epilepticus and febrile seizures are typical of GNB1 and GNAO1.

Global developmental delay and subsequent moderate to severe intellectual disability are observed in almost all patients with GNB1, GNAO1, PDE2A, and HPCA variants. Severe motor impairment with limited postural control, absence of independent walking, and absent speech are more frequent in GNB1 and GNAO1 encephalopathy. Patients with PDE10A and ADCY5 disorders can be cognitively normal.

Language impairment can be profound, especially in GNAO1 and GNB1 encephalopathy, and it is not clear if it is related to the prominent oromandibular distribution of movement disorders, or if it depends on cognitive impairment with or without oral dyspraxia. Unfortunately, this differentiation is not detailed in the actual literature where the language impairment is reported early in life, and in most cases, it is not clear if MD with oromandibular involvement coexisted at that time. MRI findings are usually nonspecific and include delayed or abnormal myelination (GNB1, GNAO1), cortical atrophy (GNAO1, GNB1), increased ventricular spaces (GNB1), and abnormalities of basal ganglia (GNAO1, PDE10A).

The presence of a core of very distinctive and shared features suggests that genetic disorders affecting the GPCRs–cAMP pathway can recognize, at least in part, the same pathophysiology.

Conversely, the different distribution and differential expression of clinical manifestations among the different disorders suggest that differentiated biological substrates, neuronal populations, or brain areas can be involved in pathophysiology and phenomenology.

GNB1 mutations have been found in different hematological neoplasm cell lines and are thought to increase the activation of AKT/ERK/mTOR signaling (58). This finding may account for the presence of cutaneous mastocytosis and acute lymphoblastic leukemia in GNB1 patients and, possibly, even for the presence of polymicrogyria (23).

Interestingly, a recent study in human blood neoplasms has also found GNAO1 mutations to activate AKT/ERK/mTOR signaling (59). It is thought that the hyperactivation of mTOR pathway, as seen in other disorders such as sclerosis tuberous, PTEN, and GATOR1 complex deficiency (including DEPDC, NPRL2, and NPRL3 deficiencies), may cause epilepsy through the alteration of normal neural networks (60). The GNAO1 variant associated to mTOR hyperactivation is the R209C variant (58). Interestingly, we found the same variant to be associated with epilepsy in 50% of the cases. Preclinical studies are needed to understand the pathophysiology of epilepsy in GNAO1 and GNB1 deficiency and to clarify the role of the mTOR signaling pathway activation.

Coherently with previous observations, a different brain tissue expression of these proteins has been reported. GNAO1, GNB1, and PDE2A are ubiquitously expressed in the brain, with high levels in cortical areas, while the highest HPCA, ADCY5, and PDE10A expressions are within putamen, caudate, and nucleus accumbens [data from the Genotype-Tissue Expression (GTEx) project (https://gtexportal.org/home/)] (61) (Figure 5).

Transduction of dopaminergic and adenosine inputs from heterotrimeric GPCRs activates a cascade contributing, together with other molecular actors (e.g., PDEs), to cAMP production. The generated cAMP propagates downstream signaling via specific cAMP-binding proteins (e.g., cAMP-dependent kinases, transcription factors, or ion transporters). This pathway, ubiquitously expressed in the central nervous system, seems to be particularly relevant for the proper functioning of MSNs of the direct and indirect pathways, and therefore for postural control, initiation of voluntary movements, prevention of unwanted movements, and motor learning (2, 5, 15).

Specifically, cAMP levels finely regulate the activity of protein such as the cAMP-regulated phosphoprotein molecular mass 32 (DARPP-32) and the cAMP-response element-binding protein (CREB). These proteins are thought to play an important role by mediating the dopaminergic neuromodulatory effects on GABAergic transmission and regulating the long-term synaptic plasticity and neuronal growth at MSNs level (62). Thus, through an altered basal ganglia activity, altered cAMP levels may underpin movement disorders such as dystonia, chorea, and parkinsonism.

The contribution of this pathway to neurodevelopment has been less explored. The possible individuation of common mechanisms rather than mechanisms specific to certain disorders deserves further studies.

Here, we reviewed the motor, epileptic, and neurodevelopmental phenotype of genetic neurological disorders affecting GPCRs–cAMP signaling pathway. This group of disorders presents with a highly recognizable clinical phenotype with distinctive movement disorder, epileptic, and neurodevelopmental features. While no biomarker is available for this group of potentially life-threatening disorders, the existence of distinctive clinical features prompting diagnostic suspicion and early detection has relevant implications for patient management.

SG and LP: data collection, first draft writing. VL and SM: critical review. MD, KB, and MN: data collection. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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