Bilateral Frontoparietal Polymicrogyria Research Articles

Re-routing GPR56 signalling using Gα12/13 G protein chimeras.

Adhesion G protein-coupled receptors (aGPCRs) constitute the second largest subclass of the GPCR superfamily. Although canonical GPCRs are explored pharmacologically as drug targets, no clinically approved drugs target the aGPCR family so far. The aGPCR GPR56/ADGRG1 stands out as an especially promising target, given its direct link to the monogenetic disease bilateral frontoparietal polymicrogyria and implications in cancers. Key to understanding GPCR pharmacology has been mapping out intracellular signalling activity. Detection of GPCR signalling in the Gαs /Gαi /Gαq G protein pathways is feasible with second messenger detection systems. However, in the case of Gα12/13 -coupled receptors, like GPR56, signalling detection is more challenging due to the lack of direct second messenger generation. To overcome this challenge, we engineered a Gαq chimera to translate Gα12/13 signalling. We show the ability of the chimeric GαΔ6q12myr and GαΔ6q13myr to translate basal Gα12/13 signalling of GPR56 to a Gαq readout in transcription factor luciferase reporter systems and show that the established peptide ligands (P7 and P19) function to enhance this signal. We further demonstrate the ability to directly influence the generation of second messengers in inositol-3-phosphate assays. In the future, these chimeric G proteins could facilitate basic functional studies, drug screenings and deorphanization of other aGPCRs.

Downstream signalling of the disease-associated mutations on GPR56/ADGRG1.

GPR56/ADGRG1 is an adhesion G protein-coupled receptor (GPCR) and mutations on this receptor cause cortical malformation due to the over-migration of neural progenitor cells on brain surface. At pial surface, GPR56 interacts with collagen III, induces Rho-dependent activation through Gα12/13 and inhibits the neuronal migration. In human glioma cells, GPR56 inhibits cell migration through Gαq/11 -dependent Rho pathway. GPR56-tetraspanin complex is known to couple Gαq/11 . GPR56 is an aGPCR that couples with various G proteins and signals through different downstream pathways. In this study, bilateral frontoparietal polymicrogyria (BFPP) mutants disrupting GPR56 function but remaining to be expressed on plasma membrane were used to study receptor signalling through Gα12 , Gα13 and Gα11 with BRET biosensors. GPR56 showed coupling with all three G proteins and activated heterotrimeric G protein signalling upon stimulation with Stachel peptide. However, BFPP mutants showed different signalling defects for each G protein indicative of distinct activation and signalling properties of GPR56 for Gα12 , Gα13 or Gα11 . β-arrestin recruitment was also investigated following the activation of GPR56 with Stachel peptide using BRET biosensors. N-terminally truncated GPR56 showed enhanced β-arrestin recruitment; however, neither wild-type receptor nor BFPP mutants gave any measurable recruitment upon Stachel stimulation, pointing different activation mechanisms for β-arrestin involvement.

Case Report: Diffuse Polymicrogyria Associated With a Novel ADGRG1 Variant

Pathogenic variants of the ADGRG1 gene are associated with bilateral frontoparietal polymicrogyria, defined radiologically by polymicrogyria with an anterior-posterior gradient, pontine and cerebellar hypoplasia and patchy white matter abnormalities. We report a novel homozygous ADGRG1 variant with atypical features. The patient presented at 8 months of age with motor delay, esotropia, hypotonia with hyporeflexia and subsequently developed refractory epilepsy. At the last assessment, aged 12 years, head control, sitting and language were not acquired. Magnetic resonance imaging revealed diffuse polymicrogyria with relative sparing of the anterior temporal lobes, without an anterior-posterior gradient, diffuse hypomyelination and pontine and cerebellar hypoplasia. A panel targeting brain morphogenesis defects yielded an unreported homozygous ADGRG1 nonsense variant (dbSNP rs746634404), present in the heterozygous state in both parents. We report a novel ADGRG1 variant associated with diffuse polymicrogyria without an identifiable anterior-posterior gradient, diffuse hypomyelination and a severe motor and cognitive phenotype. Our case highlights the phenotypic diversity of ADGRG1 pathogenic variants and the clinico-anatomical overlap between recognized polymicrogyria syndromes.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα12/13-mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gαq/11-mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways.

Heparin interacts with the adhesion GPCR GPR56, reduces receptor shedding, and promotes cell adhesion and motility.

GPR56 is an adhesion-class G-protein-coupled receptor responsible for bilateral frontoparietal polymicrogyria (BFPP), a severe disorder of cortical formation. Additionally, GPR56 is involved in biological processes as diverse as hematopoietic stem cell generation and maintenance, myoblast fusion, muscle hypertrophy, immunoregulation and tumorigenesis. Collagen III and tissue transglutaminase 2 (TG2) have been revealed as the matricellular ligands of GPR56 involved in BFPP and melanoma development, respectively. In this study, we identify heparin as a glycosaminoglycan interacting partner of GPR56. Analyses of truncated and mutant GPR56 proteins reveal two basic-residue-rich clusters, R(26)GHREDFRFC(35) and L(190)KHPQKASRRP(200), as the major heparin-interacting motifs that overlap partially with the collagen III- and TG2-binding sites. Interestingly, the GPR56-heparin interaction is modulated by collagen III but not TG2, even though both ligands are also heparin-binding proteins. Finally, we show that the interaction with heparin reduces GPR56 receptor shedding, and enhances cell adhesion and motility. These results provide novel insights into the interaction of GPR56 with its multiple endogenous ligands and have functional implications in diseases such as BFPP and cancer.

Bilateral fronto-parietal polymicrogyria in an Indian infant

A 1-year-old boy who presented with global developmental delay, spastic quadriparesis, seizures and squint with similar history in an elder sibling has been described. Magnetic resonance imaging of brain revealed features consistent with bilateral fronto-parietal polymicrogyria, which is a cerebral cortical malformation with characteristic clinical and neuroimaging findings.

P43 – 2841: Development coordination disorder (DCD) and attention deficit hyperactivity disorder (ADHD) as the initial presentation of bilateral fronto-parietal polymicrogyria type 2 (BFPP2) secondary to GPR56 mutation

Introduction Bilateral Fronto-Parietal Polymicrogyria type 1 (BFPP1; OMIM 606854) is an autosomic recessive affection usually linked to GPR56. Clinical (mental retardation, cerebellar signs, severe psychomotor delay and symptomatic generalized epilepsy) and radiological (bilateral fronto-parietal polymicrogyria with an anterior to posterior gradient with brainstem and cerebellar hypoplasia) signs are specific. Patients with BFPP without cerebellar or brainstem abnormalities (BFPP2) have a milder phenotype and are not linked to GPR56 mutation (Piao et al., 2005; Bahi-Buisson et al., 2010). We report here a patient with specific learning disorder (Development Coordination Disorder-DCD and attention deficit hyperactivity disorder-ADHD) that presented the radiological characteristics of BFPP2 and harbor a GPR56 mutation. Case report 4-year-old boy with a normal psychomotor and speech development, presenting awkwardness, learning difficulties and ADHD. Familial and personal history was unremarkable. Neurological examination was normal. Neuropsychological tests performed at 5.5 years showed DCD (Mouvement assessment Battery for Children-M-ABC scale –2SD and WPPSI-III: ICV=100, IRP=71, IVT=71 with severe dysgraphia) and ADHD. EEG was normal. He received a multidisciplinary rehabilitation and methylphenidate with an improvement of his learning disabilities even though he needed special school when he left kindergarten. When he was 7, he presented shock-like episodes with absences. Prolonged video-EEG showed 2Hz generalized spikes and waves, atypical absences and tonic seizures. Brain MRI revealed BFPP2. DNA analysis revealed compound heterozygoty in GPR56, parents where heterozygous for GPR56. Conclusion This case is, to our knowledge, the first case of BFPP2 phenotype presenting initially as a specific learning disorder and harbouring compound heterozigoty in GPR56.

PP01.4 – 2720: Novel mutation in GRIN1 gene expressed as neonatal-onset drug-resistant epilepsy associated with microcephaly and polymicrogyria

Objective A male infant with severe psychomotor retardation, congenital progressive microcephaly who presented in the first week of life with intractable neonatal seizures underwent genetic analysis. Brain MRI showed migration and sulcation disorder of the gray matter associated with bilateral fronto-parietal and temporal polymicrogyria. Genes mutations associated with abnormal cortical migration (such as CDX, LIS1, ARX, TUBBB1A, TUBB2A, TUBB2B, WDR62 and GPR56) were not detected. Methods Whole exome sequencing was employed and revealed 20,149 changes. Analysis of these mutations, filtration of nonspecific mutations and separation of alterations that were not present in the DNA samples of his parents provided us with one heterozygous mutation. Results We detected a novel mutation (c.2365 G> A p. D789N) in the GRIN1 gene. No single nucleotide polymorphisms were found in this region and its probability score was 1.0. The GRIN1 gene encodes the subunit 1 of the N-methyl D-aspartate (NMDA) ionotropic glutamate receptor which has an excitatory effect that may control synaptic plasticity in the central nervous system. Conclusion GRIN1 mutations were previously reported in children with “idiopathic” epilepsy, non-syndromic intellectual disabilities and recently with epileptic encephalopathy (such as infantile spasms) but not with malformations of cortical migration. The present report expands the clinical spectrum of GRIN1-associated neurological disorders, and suggests that early fetal NMDAR-induced exitotoxicity (during the late neuronal migration period) underlies neuronal damage which lead to microcephaly, polymicrogyria and intractable epilepsy.

Bilateral Frontoparietal Polymicrogyria: A Novel GPR56 Mutation and an Unusual Phenotype

Loss of function of GPR56 causes a specific brain malformation called the bilateral frontoparietal polymicrogyria (BFPP), which has typical clinical and neuroradiological findings. So far, 35 families and 26 independent mutations have been described.We present a Portuguese 5-year-old boy, born from nonconsanguineous parents, with BFPP. This patient has a novel GPR56 mutation (R271X) and an unusual phenotype, because he presents hot water epilepsy.To the best of our knowledge, this is the first reported case of BFPP evolving hot water epilepsy.

The adhesion G protein-coupled receptor GPR56 is a cell-autonomous regulator of oligodendrocyte development

Mutations in GPR56, a member of the adhesion G protein-coupled receptor family, cause a human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Magnetic resonance imaging (MRI) of BFPP brains reveals myelination defects in addition to brain malformation. However, the cellular role of GPR56 in oligodendrocyte development remains unknown. Here, we demonstrate that loss of Gpr56 leads to hypomyelination of the central nervous system in mice. GPR56 levels are abundant throughout early stages of oligodendrocyte development, but are downregulated in myelinating oligodendrocytes. Gpr56-knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation and diminished levels of active RhoA, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum and optic nerves. Conditional ablation of Gpr56 in OPCs leads to a reduced number of mature oligodendrocytes as seen in constitutive knockout of Gpr56. Together, our data define GPR56 as a cell-autonomous regulator of oligodendrocyte development.

Mechanism for adhesion G protein-coupled receptor GPR56-mediated RhoA activation induced by collagen III stimulation.

GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Despite the importance of GPR56 in brain development, where mutations cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP), the signaling mechanism(s) remain largely unknown. Like many other adhesion GPCRs, GPR56 is cleaved via a GPCR autoproteolysis-inducing (GAIN) domain into N- and C-terminal fragments (GPR56N and GPR56C); however, the biological significance of this cleavage is elusive. Taking advantage of the recent identification of a GPR56 ligand and the presence of BFPP-associated mutations, we investigated the molecular mechanism of GPR56 signaling. We demonstrate that ligand binding releases GPR56N from the membrane-bound GPR56C and triggers the association of GPR56C with lipid rafts and RhoA activation. Furthermore, one of the BFPP-associated mutations, L640R, does not affect collagen III-induced lipid raft association of GPR56. Instead, it specifically abolishes collagen III-mediated RhoA activation. Together, these findings reveal a novel signaling mechanism that may apply to other members of the adhesion GPCR family.

P459: Electro-clinical features of focal epilepsy patients with postictal generalized EEG suppression

P459: Electro-clinical features of focal epilepsy patients with postictal generalized EEG suppression

G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities.

Compound heterozygosity in GPR56 with bilateral frontoparietal polymicrogyria

Polymicrogyria is caused by a diverse etiology, one of which is gene mutation. At present, only one gene (GPR56) is known to cause polymicrogyria, which leads to a distinctive phenotype termed bilateral frontoparietal polymicrogyria (BFPP). BFPP is an autosomal recessive inherited human brain malformation with abnormal cortical lamination. Here, we identified compound heterozygous GPR56 mutations in a patient with BFPP. The proband was a Japanese female born from non-consanguineous parents. She presented with mental retardation, developmental motor delay, epilepsy exhibiting the feature of Lennox–Gastaut syndrome, exotropia, bilateral polymicrogyria with a relatively spared perisylvian region, bilateral patchy-white-matter MRI signal changes, and hypoplastic pontine basis. GPR56 sequence analysis revealed a c.107G>A substitution leading to a p.S36N, and a c.113G>A leading to a p.R38Q. Although affected individuals with compound heterozygosity in GPR56 have not been previously described, we presume that compound heterozygosity of these two mutations in a ligand binding domain within the extracellular N-terminus of protein could result in BFPP. In addition, we observed unusually less involvement of perisylvian cortex for polymicrogyria, and Lennox–Gastaut syndrome for epilepsy, which are likely common features in patients with BFPP caused by GPR56 mutations.

GPR56 Functions Together with α3β1 Integrin in Regulating Cerebral Cortical Development

Loss of function mutations in GPR56, which encodes a G protein-coupled receptor, cause a specific human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Studies from BFPP postmortem brain tissue and Gpr56 knockout mice have previously showed that GPR56 deletion leads to breaches in the pial basement membrane (BM) and neuronal ectopias during cerebral cortical development. Since α3β1 integrin also plays a role in pial BM assembly and maintenance, we evaluated whether it functions together with GPR56 in regulating the same developmental process. We reveal that loss of α3 integrin enhances the cortical phenotype associated with Gpr56 deletion, and that neuronal overmigration through a breached pial BM occurs earlier in double knockout than in Gpr56 single knockout mice. These observations provide compelling evidence of the synergism of GPR56 and α3β1 integrin in regulating the development of cerebral cortex.

Conventional magnetic resonance imaging and diffusion tensor imaging studies in children with novel GPR56 mutations: further delineation of a cobblestone-like phenotype

GPR56-related bilateral frontoparietal polymicrogyria (BFPP) is a rare recessively inherited disorder of neuronal migration caused by mutations of GPR56. To better delineate the clinical, molecular, and neuroradiological phenotypes associated with BFPP, we performed conventional magnetic resonance imaging and diffusion tensor imaging studies in a series of prospectively enrolled patients carrying novel GPR56 mutations. All subjects with GPR56-related BFPP showed a characteristic morphological pattern, including abnormalities of the cerebellar cortex with cerebellar cysts located at the periphery, a mildly thick corpus callosum, and a flat pons. Significant alterations of myelination and white matter tract abnormalities were documented. The present study confirms the phenotypic overlap between GPR56-related brain dysgenesis and other cobblestone-like syndromes and illustrates the contribution of 3D neuroimaging in the characterization of malformations of cortical development.

GPR56 and the Developing Cerebral Cortex: Cells, Matrix, and Neuronal Migration

GPR56, a member of the adhesion G protein-coupled receptor (GPCR) family, is integral to the development of the cortex, as mutations in GPR56 cause bilateral frontoparietal polymicrogyria (BFPP). BFPP is a cobblestone-like cortical malformation, characterized by overmigrating neurons and the formation of neuronal ectopias on the surface of the brain. Since its original cloning a decade ago, GPR56 has emerged from an orphaned and uncharacterized protein to an increasingly well-understood receptor, both in terms of its signaling and function. Collagen III is the ligand of GPR56 in the developing brain. Upon binding to collagen III, GPR56 activates RhoA via coupling to Gα(12/13). This pathway appears to be particularly critical in the preplate neurons, which are the earliest born neurons in the cortex, as the expression pattern of GPR56 in these neurons mimics the anterior to posterior gradient of malformation associated with loss of GPR56 in both humans and mice. Further characterizing the role of GPR56 in the preplate will shed light on the mechanism of cortical development and patterning.

Characterization of G protein‐coupled receptor 56 protein expression in the mouse developing neocortex

GPR56, one of the adhesion G-protein-coupled receptors (GPCRs), plays an important role in the development of the cerebral cortex. Mutations in GPR56 cause a severe human cortical malformation called bilateral frontoparietal polymicrogyria (BFPP), characterized by a global malformation of the cerebral cortex that most severely affects the frontal and parietal regions. To characterize the expression pattern of GPR56 in the developing cerebral cortex, we developed a mouse monoclonal antibody against mouse GPR56. We revealed that GPR56 is expressed in multiple cell types in the preplate, marginal zone, subventricular zone (SVZ), and ventricular zone (VZ). Most interestingly, the expression of GPR56 in preplate neurons showed an anterior-to-posterior gradient at embryonic day (E) 10.5-11.5. In contrast, the expression pattern of the GPR56 ligand, collagen III, revealed no visible gradient pattern. With the widespread expression of GPR56 in the developing cortex, it is difficult to draw a specific conclusion as to which of the GPR56-expressing cells are critical for human brain development. However, the correlation between GPR56 expression in neurons at E10.5-E11.5 and the anatomic distribution of the cortical malformation in both humans and mice suggests that its function in preplate neurons is indispensible.

Disease-Associated Mutations Prevent GPR56-Collagen III Interaction

GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Mutations in GPR56 cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Using the N-terminal fragment of GPR56 (GPR56N) as a probe, we have recently demonstrated that collagen III is the ligand of GPR56 in the developing brain. In this report, we discover a new functional domain in GPR56N, the ligand binding domain. This domain contains four disease-associated mutations and two N-glycosylation sites. Our study reveals that although glycosylation is not required for ligand binding, each of the four disease-associated mutations completely abolish the ligand binding ability of GPR56. Our data indicates that these four single missense mutations cause BFPP mostly by abolishing the ability of GPR56 to bind to its ligand, collagen III, in addition to affecting GPR56 protein surface expression as previously shown.

Loss of Col3a1, the gene for Ehlers-Danlos syndrome type IV, results in neocortical dyslamination.

It has recently been discovered that Collagen III, the encoded protein of the type IV Ehlers-Danlos Syndrome (EDS) gene, is one of the major constituents of the pial basement membrane (BM) and serves as the ligand for GPR56. Mutations in GPR56 cause a severe human brain malformation called bilateral frontoparietal polymicrogyria, in which neurons transmigrate through the BM causing severe mental retardation and frequent seizures. To further characterize the brain phenotype of Col3a1 knockout mice, we performed a detailed histological analysis. We observed a cobblestone-like cortical malformation, with BM breakdown and marginal zone heterotopias in Col3a1 −/− mouse brains. Surprisingly, the pial BM appeared intact at early stages of development but starting as early as embryonic day (E) 11.5, prominent BM defects were observed and accompanied by neuronal overmigration. Although collagen III is expressed in meningeal fibroblasts (MFs), Col3a1 −/− MFs present no obvious defects. Furthermore, the expression and posttranslational modification of α-dystroglycan was undisturbed in Col3a1 −/− mice. Based on the previous finding that mutations in COL3A1 cause type IV EDS, our study indicates a possible common pathological pathway linking connective tissue diseases and brain malformations.