<i>GNAO1</i>organizes the cytoskeletal remodeling and firing of developing neurons

Akamine, Satoshi; Okuzono, Sayaka; Yamamoto, Haruyuki; Setoyama, Daiki; Sagata, Noriyuki; Ohgidani, Masahiro; Kato, Takahiro A.; Ishitani, Tohru; Kato, Hiromichi; Masuda, Keiji; Matsushita, Yuki; Ono, Hiroaki; Ishizaki, Yoshito; Sanefuji, Masafumi; Saitsu, Hirotomo; Matsumoto, Naomichi; Kang, Dongchon; Kanba, Shigenobu; Nakabeppu, Yusaku; Sakai, Yasunari; Ohga, Shouichi

doi:10.1096/fj.202001113r

Cited by 23 publications

(19 citation statements)

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“…This discovery was followed by an avalanche of clinical analyses that cumulatively led to the recognition of GNAO1 encephalopathy as a spectrum of neurodevelopmental disorders manifesting as motor dysfunction, epileptic seizures, and developmental delay first appearing mostly in infancy (6)(7)(8). Although G o , in addition to neurons, is also strongly expressed in glial cells (www.proteinatlas.org), disease modeling in mice (9) and brain organoids (10) demonstrated strong effects of GNAO1 mutations on neuronal rather than glial differentiation, highlighting neurons as the main target of G o malfunctioning in the disease. G o couples to many neuronal GPCRs, such as D2 dopamine, -opioid, M2 muscarinic, 2-adrenergic, and more.…”

Section: Introductionmentioning

confidence: 99%

Restoration of the GTPase activity and cellular interactions of Gα _o mutants by Zn ²⁺ in GNAO1 encephalopathy models

et al. 2022

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De novo point mutations in GNAO1 , gene encoding the major neuronal G protein Gα o , have recently emerged in patients with pediatric encephalopathy having motor, developmental, and epileptic dysfunctions. Half of clinical cases affect codons Gly 203 , Arg 209 , or Glu 246 ; we show that these mutations accelerate GTP uptake and inactivate GTP hydrolysis through displacement Gln 205 critical for GTP hydrolysis, resulting in constitutive GTP binding by Gα o . However, the mutants fail to adopt the activated conformation and display aberrant interactions with signaling partners. Through high-throughput screening of approved drugs, we identify zinc pyrithione and Zn 2+ as agents restoring active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with negligible effects on wild-type Gα o . We describe a Drosophila model of GNAO1 encephalopathy where dietary zinc restores the motor function and longevity of the mutant flies. Zinc supplements are approved for diverse human neurological conditions. Our work provides insights into the molecular etiology of GNAO1 encephalopathy and defines a potential therapy for the patients.

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Section: Introductionmentioning

confidence: 99%

Restoration of the GTPase activity and cellular interactions of Gα _o mutants by Zn ²⁺ in GNAO1 encephalopathy models

et al. 2022

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“…Neurons were reported in cerebral organoids as early as seven days of culture [ 95 ], and mature neurons were reported at 28 days. In dorsal forebrain organoids, neurons were reported from 14 days onwards, although one article described the presence of mature neurons after twelve days [ 96 ]. In ventral forebrain organoids, neurons were reported after 25 days.…”

Section: Resultsmentioning

confidence: 99%

A beginner’s guide on the use of brain organoids for neuroscientists: a systematic review

et al. 2023

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Background The first human brain organoid protocol was presented in the beginning of the previous decade, and since then, the field witnessed the development of many new brain region-specific models, and subsequent protocol adaptations and modifications. The vast amount of data available on brain organoid technology may be overwhelming for scientists new to the field and consequently decrease its accessibility. Here, we aimed at providing a practical guide for new researchers in the field by systematically reviewing human brain organoid publications. Methods Articles published between 2010 and 2020 were selected and categorised for brain organoid applications. Those describing neurodevelopmental studies or protocols for novel organoid models were further analysed for culture duration of the brain organoids, protocol comparisons of key aspects of organoid generation, and performed functional characterisation assays. We then summarised the approaches taken for different models and analysed the application of small molecules and growth factors used to achieve organoid regionalisation. Finally, we analysed articles for organoid cell type compositions, the reported time points per cell type, and for immunofluorescence markers used to characterise different cell types. Results Calcium imaging and patch clamp analysis were the most frequently used neuronal activity assays in brain organoids. Neural activity was shown in all analysed models, yet network activity was age, model, and assay dependent. Induction of dorsal forebrain organoids was primarily achieved through combined (dual) SMAD and Wnt signalling inhibition. Ventral forebrain organoid induction was performed with dual SMAD and Wnt signalling inhibition, together with additional activation of the Shh pathway. Cerebral organoids and dorsal forebrain model presented the most cell types between days 35 and 60. At 84 days, dorsal forebrain organoids contain astrocytes and potentially oligodendrocytes. Immunofluorescence analysis showed cell type-specific application of non-exclusive markers for multiple cell types. Conclusions We provide an easily accessible overview of human brain organoid cultures, which may help those working with brain organoids to define their choice of model, culture time, functional assay, differentiation, and characterisation strategies.

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“…Available models for in vitro proof-of-concept studies of RNA therapeutics for GNAO1 encephalopathy are patient-specific neurons and organoids derived from induced pluripotent stem cells ( Akamine et al, 2020 ). To evaluate drug efficacy in vivo , a mouse model with a heterozygous c.607 G>A mutation in murine Gnao1 is required.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

et al. 2023

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The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the “humanized” fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts.

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GNAO1organizes the cytoskeletal remodeling and firing of developing neurons

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Cited by 23 publications

References 68 publications

Restoration of the GTPase activity and cellular interactions of Gα _o mutants by Zn ²⁺ in GNAO1 encephalopathy models

Restoration of the GTPase activity and cellular interactions of Gα _o mutants by Zn ²⁺ in GNAO1 encephalopathy models

A beginner’s guide on the use of brain organoids for neuroscientists: a systematic review

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

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GNAO1organizes the cytoskeletal remodeling and firing of developing neurons

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Cited by 23 publications

References 68 publications

Restoration of the GTPase activity and cellular interactions of Gα o mutants by Zn 2+ in GNAO1 encephalopathy models

Restoration of the GTPase activity and cellular interactions of Gα o mutants by Zn 2+ in GNAO1 encephalopathy models

A beginner’s guide on the use of brain organoids for neuroscientists: a systematic review

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

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Restoration of the GTPase activity and cellular interactions of Gα _o mutants by Zn ²⁺ in GNAO1 encephalopathy models

Restoration of the GTPase activity and cellular interactions of Gα _o mutants by Zn ²⁺ in GNAO1 encephalopathy models