1. Gαo Signaling Pathway Is Associated with GNAO1 Encephalopathy
G-protein coupled receptors (GPCRs) are the largest class of surface receptors, constituting about 5% of the genome. To date, humans have approximately 1000 GPCRs classified, each with a unique and distinct function [
1]
. The major functions of GPCRs involve mediating most of the cellular responses to taste, olfaction, and vision [
2]
. The prominent role of GPCRs is implicated in the fact that they bind to a variety of ligands like neurotransmitters, ions, and hormones and are majorly involved in the transmission of extracellular signals into intracellular responses. The significance of GPCRs lies in their ubiquity and diversity as they are involved in a wide range of biological functions from regulation of blood pressure and heart rate to control of immune responses and release of hormones [
3]. GPCRs are coupled with G-proteins (guanosine-binding) proteins, which act as molecular switches, transmitting signals from activated cell surface receptors to intracellular proteins. The activation of G-proteins and their subsequent interaction with effector proteins mediate a wide range of cellular responses, including changes in enzyme activity, ion channel opening or closing, and modulation of intracellular signaling pathways [
4]
. In its usual conformation, the G-proteins exist as heterotrimeric complexes involving the Gα, Gβ, and Gγ subunits. Neurotransmitter receptors stimulate Gα subunits to exchange bound GDP for GTP. Gα-GTP then separates from the Gβγ, and these activated G-protein subunits can evoke responses in the cell. Signaling is terminated when the Gα proteins hydrolyze their bound GTP, thus returning to the GDP-bound state and re-associating with Gα
(Figure 1A) [
5]. G-proteins are further classified into four main families: Gα
s, Gα
i/o, Gα
q, and Gα
12, each mediating different physiological responses depending on the tissue type and signal received [
6]
. Among these, the Gα
i/o family, Gα
o (encoded by the
GNAO1 gene) is the most abundant G-protein found in brain tissue and controls both the development and adult physiology of the brain. It is particularly crucial for regulating neuronal signaling, specifically in neurodevelopment and synaptic transmission [
7]. Recently, Gα
o has been characterized as an inducer of neuronal differentiation [
8]. In
C. elegans, the homolog of the human Gα
o protein is GOA-1. This protein plays a vital role in inhibiting neurotransmitter release by negatively regulating synaptic vesicle exocytosis [
9]
. GOA-1 acts via several signaling pathways, including those involving diacylglycerol (DAG) and protein kinase C (PKC), modulating locomotion and egg-laying behaviors in
C. elegans [
10]
. Mutations in GOA-1 result in hyperactive neurotransmission that is reflected by hyperactive locomotion and increased egg-laying behavior due to dysregulated synaptic vesicle cycling [
11]
. For the other Gα subunits, the downstream effectors molecules are established using a combination of forward genetics and biochemical analysis. However, Gα
o remains the only type of Gα protein in higher eukaryotes for which an effector has not been identified
(Figure 1B). This is a remarkable gap in our knowledge since Gα
o constitutes 1% of the total membrane protein in the brain, making it orders of magnitude more abundant than other neural G-proteins [
12]
. Over the past two decades, many unsuccessful attempts have been made to classify the effector molecules (signaling proteins) for Gα
o. The challenge in identifying Gα
o effectors through forward genetics could be attributed to several factors. First, the redundancy of multiple functionally similar effectors may mask the effects of mutations in single genes, making it difficult to observe changes in Gα
o signaling [
13]. Second, if a Gα
o effector is vital for survival or reproduction, mutations could result in lethality or impair recovery of such mutants in genetic screens [
14]
.
The human
GNAO1 gene encodes Gα
o, the α subunit of heterotrimeric G-proteins that play important neuro-modulatory functions by coupling with various GPCRs, including dopamine, serotonin, and opioid receptors. [
15]
. Although various signaling events in which Gα
o is involved have been described, systematic actions of Gα
o in the nervous system have not been fully understood. Numerous
de-novo GNAO1 mutations are associated with neurodevelopmental disorders, collectively termed
GNAO1 encephalopathy-related disorders or GNAO1 encephalopathy, which mainly include developmental and epileptic encephalopathy 17 (also called early infantile epileptic encephalopathy) (DEE) and neurodevelopmental disorder with involuntary movements (NEDIM) [
16]. GNAO1 encephalopathy has a broad, emerging phenotypic spectrum. One core phenotype is weakened movement which can be a characteristic of chorea, dystonia, and dyskinesia, and other altered phenotypes can feature epilepsy and developmental delay. [
17]. Evaluation of GNAO1 disorder-associated mutations in mice has reiterated some of the phenotypes of GNAO1 encephalopathy, including impaired movement and seizure susceptibility. Testing pathological GNAO1 mutations in other model organisms will be valuable in assessing the conserved functional effects of these genetic perturbations, and their influence on movement. Gα
o is highly conserved in invertebrates including the nematode
C. elegans where its ortholog, G-protein o-alpha subunit (GOA)-1, regulates locomotion and egg-laying circuits [
14,
18]
. The extremely well-defined genetics of GOA-1 in
C. elegans make this an ideal
in-vivo system for evaluating the functional impacts of pathological GNAO1 mutations. To date, efforts to characterize pathological mutations of
GNAO1 at the molecular level have yielded promising, but conflicting results in mammalian and rodent systems. An early study evaluated a pertussis toxin-insensitive version of Gα
o using a heterologous cell-based assay and placed these pathological mutations in three categories: loss of function, gain of function, and normal function [
14]
. Recent evaluation of rescued Gα
o indicated that
GNAO1 mutations result in loss of function with several mutations reported to antagonize transduction of GPCR signals by acting as dominant negatives [
15]
. Particularly notable are differing in vitro results with G203R, R209C, and the less well-characterized G42R mutation. These were initially described as gain of function or normal function and were subsequently found to be loss of function and dominant negative. As a result of these differing conclusions, the functional effects, and mechanisms of GNAO1 pathological mutations remain unresolved. Intense interest has emerged in understanding Gα
o function in the nervous system and developing intervention strategies for GNAO1 encephalopathy. Thus, there is a persuasive need to use
in-vivo models to study the behavioral impact of GNAO1 disorder-associated mutations. Here, we highlight the importance of studying
GNAO1 mutations and effectors of Gα
o, in the context of GNAO1 encephalopathy using
C. elegans as the model system.
3. C. elegans as an Advanced Model System to Study GNAO1 Mutations
GNAO1 mutations remain highly understudied because of the non-availability of human subjects due to the extremely low number of reported cases and technical difficulties, permissions, and ethical concerns associated with obtaining human samples. To mitigate the mutations responsible for GNAO1 encephalopathy, researchers have traditionally been using rodent models, particularly mice [
30]. There were reported studies of unsuccessful attempts in mice models to study GNAO1-related disorders [
35]. Further, in the quest to find a simpler model system, an attempt was made to replace
Drosophila melanogaster’s Gα
o with human
GNAO1 which led to the production of an inaccurate nucleotide sequence of the gene. While approximately 75% of human genes associated with diseases have counterparts in
Drosophila, the proteins they produce differ, which may limit the translation of findings from the
Drosophila model to human conditions, particularly in drug discovery [
36]. These reports establish the need for the use of the alternative model system with the ease of genetic manipulation and evident phenotypic defects.
C. elegans has been established as a valuable experimental model for understanding GNAO1-related disorders and exploring potential treatments [
37]. The researchers created two genetically modified strains with mutations at key positions Glu246 and Arg209, known to be crucial in Gα
o. These loss-of-function (LOF) mutations caused varied reductions in Gα
o-mediated signaling, resulting in excessive neurotransmitter release from different neuron types. This led to hyperactive behaviors like increased egg laying and movement [
38]. Notably, single-copy mutations demonstrated cell-specific dominant-negative effects, determined by the specific altered residue. Similar to earlier mutants S47G and A221D, caffeine effectively mitigated the excessive movement in animals with the R209H and E246K mutations, implying caffeine’s independent action from the mutation type. In contrast, the adenosine receptor antagonist Istradefylline worked in R209H animals but not in E246K worms, suggesting caffeine’s multifaceted mode of action [
37]. Overall, these findings deepen our understanding of the disease mechanisms and provide more evidence for caffeine’s potential efficacy in managing dyskinesia linked to
GNAO1 loss of function mutations. In contrast, models involving the gain-of-function (GOF) mutation of Gα
o, [G203R] or Gα
o [R209C] in striatal neurons led to impaired locomotor behavior in mice which provides insights into G protein functioning and its mutants [
39]
, but its relevance as a disease model is still not clear. Interestingly, hyperactivity seen in C215Y/+ mice is associated with the motor cortex rather than anatomical defects in the striatum [
30]
. These findings underscore the complexity of modeling GNAO1 encephalopathy and the importance of accurately replicating the disease phenotype in animal models for meaningful research and therapeutic development.
C. elegans provides an excellent opportunity to define the genetic mechanisms by which
GNAO1 variants affect locomotor behavior and could be key for resolving outstanding mechanistic issues about the molecular pathology of GNAO1 encephalopathy. Moreover,
C. elegans has the potential to be developed as an
in-vivo platform capable of evaluating large numbers of
GNAO1 variants and could be used for genetic and small molecule screens targeting
GNAO1. In
C. elegans mutations associated with disorders, tested by CRISPR editing of the native GOA-1/Gα
o locus, lead to abnormal locomotor behavior also same result was found in previous research in rodents and suggests emerging common principles governing how
GNAO1 pathological variants impact Gα
o, function in live organisms. The similarities between locomotor behaviors in
C. elegans and rodent models make this correspondence quite reasonable. Both are sensitive to dopaminergic modulation, the
C. elegans motor circuit comprising excitatory cholinergic and inhibitory GABAergic motor neurons, and GABAergic striatal neurons (dMSNs and iMSNs) responsible for motor coordination in mice [
40,
41,
42,
43]. In both cases, loss of Gα
o function results in hyperactive locomotion. Furthermore, Gα
o, signaling serves to inhibit neuronal activity in both
C. elegans and mammals [
44,
45]. These conserved features have proven advantageous in the profiling of
GNAO1 variants.
The spectrum of GNAO1 encephalopathy is wide and continually evolving. As mentioned previously, the central aspect of this condition is compromised motor function, along with epilepsy and developmental delay which are prevalent features of this phenotype. Gα
o being a major G-protein, is expressed across many brain regions including the cerebellum wherein it is cellularly localized within the Purkinje fibers, basket, and stellate cells which inhibit GABAergic interneurons and Golgi neuronal cells. It forms connections with various crucial G protein-coupled receptors (GPCRs) such as GABA-β, α2 adrenergic, adenosine A1 (A1R), and dopamine D2 (D2R) receptors. These receptors are essential for controlling neurotransmitter release, movement, and neural development functions. There are numerous postulated downstream targets in the signaling pathway of Gα
o, as well as the other members of the Gα family. Many of the targets affected by Gα
o signaling are also implicated in disorders related to movement. Mutations in other signaling molecules remain understudies but are of great importance. For example, genetic mutations in ADCY5, the gene responsible for encoding adenylyl cyclase type 5, have been identified in patients with dyskinesia and dystonia [
46,
47]. Acknowledging the significant diversity in clinical manifestations is crucial to comprehend the molecular mechanisms behind GNAO1-related disorders. This diversity encompasses early-onset epileptic encephalopathy, as indicated by [
17], as well as patients with intricate movement disorders, some accompanied by epilepsy [
22]
. The prevailing characteristics among patients harboring
GNAO1 mutations are hypotonia and developmental delay irrespective of their clinical presentation or biochemical traits. After these, choreoathetosis and dystonia are the subsequent most frequent observations [
17,
48,
49]. While a notable proportion of individuals exhibit atypical EEG or MRI results, fewer than fifty percent of those with
GNAO1 mutations displayed distinctly aberrant EEG patterns, primarily among patients with epilepsy and loss-of-function (LOF) mutations. This diversity, encompassing variations in clinical presentation and impact on brain structure/function, suggests an involvement of both neurodevelopmental changes and disruptions in functional signaling. The latter factor seems to be more prominent in patients with gain-of-function (GOF) mutations, who exhibit fewer indications of structural brain abnormalities and display partial positive responses to drug interventions. The potentially uncertain underlying factors contributing to GNAO1-related movement disorders can be elucidated by examining GNAO1 signaling. One potential pathway involves the canonical inhibition of cAMP by Gα
o, which can be facilitated by Gα
o or the liberated Gβγ [
50,
51]
. Notably, mutations in ADCY5, responsible for encoding an AC protein that generates cAMP, also lead to movement abnormalities in human patients. The disruption of cAMP signaling has been linked to impaired brain function [
52]. Consequently, disturbances in cAMP levels could perturb the delicately balanced neurodevelopmental system suggesting the operation of Gα
o via the cAMP pathway might be important in movement disorders, yet the downstream effectors are not studied [
48].
A second theoretical foundation for GNAO1-associated movement disorders pertains to Gα
o’s involvement in governing neurotransmitter release. The deficiency of crucial neurotransmitters like catecholamines (such as dopamine, epinephrine, and norepinephrine) and serotonin has been extensively studied in the context of movement disorders or seizures [
53]. Gα
o’s presynaptic role in regulating neurotransmitter release presents another potential avenue in the understanding of movement disorder etiology.
A third conceivable perspective, centered on developmental considerations, involves potential alterations in the maturation of neurons, a process vital during appropriate stages of neurological development. Consequently, children with developmental abnormalities might display irregular behaviors. Notably, most individuals with GNAO1-linked movement disorders also suffer from significant developmental delays. Morphologically, MRI scans may reveal widespread atrophy and delayed myelination. In general, genetic factors account for around 40% of cases involving developmental delay, including intellectual disability [
54]
. Control over cAMP levels and neurotransmitter release can influence ongoing neural functions as well as neurological development. Within this framework, GNAO1-associated movement disorders could arise from disruptions in either or both processes. The former scenario would likely be more amenable to therapeutic interventions compared to the latter.
The conserved nature of G-protein signaling across species enables us to use the
C. elegans model system, while also highlighting the specific downstream effectors unique to each organism.
GNAO1 mutations significantly impair normal biological functions, manifesting as distinct phenotypes—locomotory defects in
C. elegans and defective neuronal development in humans. Understanding these pathways can provide critical insights into the molecular mechanisms underlying GNAO1-related diseases and may offer therapeutic targets for treating associated disorders. In
C. elegans, the Gα
o protein is involved in several signaling cascades. Upon activation, Gα dissociates from the Gβγ subunits, initiating a downstream signaling cascade that regulates key biological processes. These include serotonin release via
tph-1, muscle contraction through the hermaphrodite-specific motor neuron (HSN) [
55], and GPCR-independent signaling through Ric8 [
56], which mediates Gα signaling even in the absence of receptor activation. The regulator of G-protein signaling (RGS) protein, egl-10, modulates Gα activity by accelerating GTP hydrolysis [
57], returning Gα to its inactive state bound to GDP. Mutations in
GNAO1 disrupt these pathways, resulting in egg-laying and locomotory defects in
C. elegans, highlighting the critical role of G-protein signaling in neural and muscular functions. In
Homo sapiens, mutations in
GNAO1 are linked to neurological disorders, including epilepsy and movement disorders. Similar to
C. elegans, Gα dissociates from Gβγ upon activation by GPCRs. However, in humans,
GNAO1 mutations impact neuronal signaling pathways involved in neurite outgrowth. Specifically, these mutations influence Rho-GTPase signaling, which is modulated by GPRIN (G-protein-regulated inducer of neurite outgrowth) [
58]. These proteins possibly interact with GNAO1 and Rho-GTPase via the CDC-42 pathway [
59], which regulates cytoskeletal dynamics essential for neurite outgrowth and neuronal morphology. Additionally, the interaction between GNAO1 and Necdin-E2F1 modulates cell cycle regulation, further affecting neuronal differentiation [
60]. Defects in these pathways due to
GNAO1 mutations result in impaired neurite development, contributing to the observed neurological phenotypes in patients
. (Figure 3). A clear picture of all the events is still unknown, opening a plethora of opportunities in the field of GNAO1 biology.
4. Conclusion and Perspective
The study of GNAO1 encephalopathy, a genetic disorder caused by mutations in the
GNAO1 gene, which encodes for the Gα
o subunit of G-proteins, presents significant challenges due to the complexity of G-protein-coupled receptor (GPCR) signaling pathways in the nervous system [
61]. Gα
o, one of the most abundant membrane proteins in the brain, plays a crucial role in neuro-modulation by interacting with various GPCRs, such as dopamine, serotonin, and opioid receptors [
39]. This genetic disorder manifests in patients as a broad spectrum of symptoms, including developmental delays, epilepsy, involuntary movements, and other motor dysfunctions [
62]. The exact mechanisms underlying these phenotypes remain poorly understood, and while mammalian models have provided some insight into the effects of
GNAO1 mutations, they often present conflicting results, making it difficult to derive conclusive findings. In this context,
C. elegans a nematode with a well-conserved G-protein signaling pathway similar to that in humans, has emerged as an ideal
in-vivo model for studying the functional impacts of
GNAO1 mutations.
C. elegans Gα
o (
goa-1) shares approximately 80% sequence similarity with the human
GNAO1 gene [
63], making it a valuable tool for assessing the consequences of mutations on locomotion, neurotransmitter release, and other nervous system functions.
Several advantages contribute to
C. elegans being the preferred model system for GNAO1 research. The simplicity of its neural circuit, coupled with its highly conserved genetic pathways, allows researchers to effectively replicate the mutations observed in human GNAO1 encephalopathy. These mutations can be introduced into
C. elegans using CRISPR editing, enabling precise study of their effects on behavior, such as locomotion and egg-laying, which mirror the impaired movement and motor dysfunctions seen in human patients [
38,
64]. Additionally,
C. elegans’ rapid life cycle and ease of genetic manipulation facilitate large-scale screenings of
GNAO1 variants, providing a high-throughput platform for studying the functional impact of these mutations. Previous studies in rodent models have confirmed the involvement of Gα
o in movement disorders, but
C. elegans offers the added benefit of being able to dissect the molecular mechanisms of these mutations with greater precision and at a faster pace. For example, recent research using
C. elegans has demonstrated that mutations in key residues, such as Glu246 and Arg209, lead to hyperactive behaviors like excessive movement and egg-laying, reflecting the overactive neurotransmitter release seen in patients with
GNAO1 mutations [
38]. The ability to observe these phenotypic changes in real time makes
C. elegans an invaluable model for understanding how specific mutations disrupt Gα
o-mediated signaling pathways.
In terms of therapeutic research,
C. elegans has proven instrumental in identifying potential treatments for GNAO1 encephalopathy. For instance, studies have shown that caffeine can mitigate the hyperactive phenotypes caused by certain
GNAO1 mutations, offering a potential avenue for pharmacological intervention. This finding highlights the multifaceted nature of caffeine’s action, as it was effective across different mutations, suggesting that it may act on multiple pathways involved in Gα
o signaling [
21]. Similarly, drugs like oxcarbazepine, traditionally used to treat epilepsy, have shown promise in managing movement disorders associated with
GNAO1 mutations, further emphasizing the utility of
C. elegans in pre-clinical drug screening [
20]. These findings underscore the potential of
C. elegans not only as a model for understanding the genetic basis of GNAO1 encephalopathy but also as a platform for discovering novel therapeutic approaches. Beyond its use in studying movement disorders,
C. elegans offers a unique opportunity to investigate the role of
GNAO1 mutations in epilepsy, a common feature of GNAO1 encephalopathy. Although much of the research has focused on the motor dysfunctions caused by
GNAO1 mutations, seizures are a significant and often life-threatening symptom of this disorder [
65].
C. elegans’ well-characterized nervous system and responsiveness to various neurotransmitters, including GABA and dopamine, make it an ideal system for studying the mechanisms of seizure induction and progression. This model has already been used to study seizure-like activity in other genetic disorders, and its application to GNAO1 research could provide new insights into how these mutations increase seizure susceptibility. While
C. elegans provides a powerful model for studying GNAO1 encephalopathy, there remain significant challenges in fully elucidating the downstream pathways through which Gα
o mutations exert their effects. The pathways involving GNAO1 are intricate, with numerous interactions between GPCRs, G proteins, and their downstream effectors, many of which remain undefined. Moreover, the clinical diagnosis of GNAO1 encephalopathy is often delayed due to the overlap of symptoms with other neurological disorders, further complicating efforts to understand the full spectrum of this disease. Future research in
C. elegans will need to focus on mapping these pathways and identifying specific molecular targets that could be used for early diagnosis and intervention. In conclusion,
C. elegans serves as an invaluable model for studying the molecular and phenotypic consequences of
GNAO1 mutations. Its conservation of Gα
o signaling pathways, combined with its genetic tractability, provides a unique platform for both basic research and therapeutic development. By utilizing
C. elegans researchers can gain deeper insights into the mechanisms underlying GNAO1 encephalopathy and develop targeted treatments that address the complex interplay of motor dysfunctions, seizures, and developmental delays characteristic of this disorder. As research continues to advance,
C. elegans will undoubtedly play a crucial role in unraveling the mysteries of GNAO1-related neurological disorders and improving outcomes for affected individuals.