Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Astrocytes are one of the most abundant cell types in the mammalian brain. They play essential roles in synapse formation, maturation, and elimination. However, how astrocytes migrate into the gray matter to accomplish these processes is poorly understood. Here, we show that, by combinational analyses of in vitro and in vivo time-lapse observations and lineage traces, astrocyte progenitors move rapidly and irregularly within the developing cortex, which we call erratic migration. Astrocyte progenitors also adopt blood vessel-guided migration. These highly motile progenitors are generated in the restricted prenatal stages and differentiate into protoplasmic astrocytes in the gray matter, whereas postnatally generated progenitors do not move extensively and differentiate into fibrous astrocytes in the white matter. We found Cxcr4/7, and integrin β1 regulate the blood vessel-guided migration, and their functional blocking disrupts their positioning. This study provides insight into astrocyte development and may contribute to understanding the pathogenesis caused by their defects.

DOI： 10.1038/s41467-022-34184-x

Other Link： https://www.nature.com/articles/s41467-022-34184-x

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism.

We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1.

Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

DOI： 10.1093/brain/awac106

Web of Science

Scopus

PubMed

Other Link： https://academic.oup.com/brain/article-pdf/145/9/3308/45872573/awac106.pdf

Language：English   Publishing type：Research paper (scientific journal)   Publisher：S. Karger AG  

Polo-like kinase 4 (Plk4) is a ser/thr kinase, which plays a central role in centriole duplication during the cell cycle. PLK4 gene abnormalities are responsible for autosomal recessive chorioretinopathy-microcephaly syndrome and Seckel syndrome. In this study, we performed expression analyses of Plk4 by focusing on mouse brain development. Western blotting analyses revealed that Plk4 with a molecular mass of ∼100 kDa was broadly expressed in adult mouse tissues with specific subcellular distribution. As to the central nervous system, Plk4 was expressed throughout the developmental process with drastic increase after P15, suggesting an essential role of Plk4 in differentiated neurons. In immunohistochemical analyses with mouse brain at embryonic day 14, Plk4 was detected dominantly at the cell-cell contact sites of neuronal progenitors in the ventricular zone. Plk4 was then diffusely distributed in the cell body of cortical neurons at P7, while it was enriched in the neuropil as well as soma of excitatory neurons in the cerebral cortex and hippocampus and Purkinje cells in the cerebellum at P30. Notably, biochemical fractionation analysis found an enrichment of Plk4 in the postsynaptic density fraction. Then, immunofluorescent analyses showed partial co-localization of Plk4 with excitatory synaptic markers, PSD95 and synaptophysin, in differentiated primary cultured hippocampal neurons. These results suggest that Plk4 takes part in the regulation of synaptic function in differentiated neurons.

DOI： 10.1159/000526914

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Homozygosity of the p.Arg204Trp variation in the Pleckstrin homology and RhoGEF domain containing G2 (PLEKHG2) gene, which encodes a Rho family-specific guanine nucleotide-exchange factor, is responsible for microcephaly with intellectual disability. However, the role of PLEKHG2 during neurodevelopment remains unknown. In this study, we analyzed mouse Plekhg2 function during cortical development, both in vitro and in vivo. The p.Arg200Trp variant in mouse (Plekhg2-RW), which corresponds to the p.Arg204Trp variant in humans, showed decreased guanine nucleotide-exchange activity for Rac1, Rac3, and Cdc42. Acute knockdown of Plekhg2 using in utero electroporation-mediated gene transfer did not affect the migration of excitatory neurons during corticogenesis. On the other hand, silencing Plekhg2 expression delayed dendritic arbor formation at postnatal day 7 (P7), perhaps because of impaired Rac/Cdc42 and p21-activated kinase 1 signaling pathways. This phenotype was rescued by expressing an RNAi-resistant version of wildtype Plekhg2, but not of Plekhg2-RW. Axon pathfinding was also impaired in vitro and in vivo in Plekhg2-deficient cortical neurons. At P14, knockdown of Plekhg2 was observed to cause defects in dendritic spine morphology formation. Collectively, these results strongly suggest that PLEKHG2 has essential roles in the maturation of axon, dendrites, and spines. Moreover, impairment of PLEKHG2 function is most likely to cause defects in neuronal functions that lead to neurodevelopmental disorders.

DOI： 10.3390/cells11040696

Web of Science

Scopus

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：S. Karger AG  

Rac3 is a member of Rho family small GTPases which regulate cellular signaling and cytoskeletal dynamics. The RAC3 gene abnormalities have been shown to cause neurodevelopmental disorders with structural brain anomalies, including polymicrogyria/dysgyria, callosal abnormalities, brainstem anomalies, and cerebellar dysplasia. Although this evidence indicates that Rac3 is essential in brain development, not only its molecular mechanism but also the expression profile is yet to be elucidated. In this study, we carried out expression analyses of Rac3 with mouse brain tissues. In immunoblotting, Rac3 exhibited a tissue-dependent expression profile in the young adult mouse and was expressed in a developmental stage-dependent manner in brain. In primary cultured hippocampal neurons, while Rac3 was distributed mainly in the cytoplasm, it was visualized in axon and dendrites with partial localization at synapses, in consistent with the observation in biochemical fractionation analyses. In immunofluorescence analyses with brain slices, Rac3 was distributed strongly and moderately in the axon and cytoplasm, respectively, of cerebral cortex at postnatal day (P) 2 and P18. Similar distribution profile was also observed in hippocampus. Taken together, the results obtained strongly suggest that Rac3 plays an important physiological role in neuronal tissues during corticogenesis, and defects in the Rac3 function induce structural brain anomalies leading to pathogenesis of neurodevelopmental disorders.

DOI： 10.1159/000521168

Web of Science

Scopus

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbc.2022.101579

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Rho family guanosine triphosphatases (GTPases) regulate cellular signaling and cytoskeletal dynamics, playing a pivotal role in cell adhesion, migration, and cell cycle progression. The Rac subfamily of Rho GTPases consists of three highly homologous proteins, Rac 1–3. The proper function of Rac1 and Rac3, and their correct interaction with guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) are crucial for neural development. Pathogenic variants affecting these delicate biological processes are implicated in different medical conditions in humans, primarily neurodevelopmental disorders (NDDs). In addition to a direct deleterious effect produced by genetic variants in the RAC genes, a dysregulated GTPase activity resulting from an abnormal function of GEFs and GAPs has been involved in the pathogenesis of distinctive emerging conditions. In this study, we reviewed the current pertinent literature on Rac-related disorders with a primary neurological involvement, providing an overview of the current knowledge on the pathophysiological mechanisms involved in the neuro-RACopathies.

DOI： 10.3390/cells10123395

PubMed

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbc.2021.101427

PubMed

Publishing type：Research paper (scientific journal)   Publisher：American Society for Clinical Investigation  

DOI： 10.1172/jci139933

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abnormalities of PLEKHG2 gene, encoding a Rho family-specific guanine nucleotide exchange factor, are involved in microcephaly with intellectual disability. However, not only the role of PLEKHG2 in the developmental process but also its expression profile is unknown. In this study, we prepared a specific antibody against PLEKHG2 and carried out expression analyses with mouse tissues. In western blotting, PLEKHG2 exhibited a tissue-dependent expression profile in adult mouse and was expressed in a developmental stage-dependent manner in brain. Then, in immunohistochemical analyses, while PLEKHG2 was observed in the cortical plate and ventricular zone surface of the cerebral cortex at embryonic day 14, it came to be distributed throughout the cerebral cortex in layer II/III and V during corticogenesis. PLEKHG2 was also detected mainly in the nucleus of neurons in the hippocampal CA regions and dentate gyrus at P7. Notably, the nuclear accumulation disappeared at P30 and PLEKHG2 came to be located at the axons and/or dendrites at this time point. Moreover, in vitro immunofluorescence revealed that PLEKHG2 was at least partially localized at both excitatory and inhibitory synapses in primary cultured hippocampal neurons. These results suggest roles of PLEKHG2 in the development of the central nervous tissue and synaptic function.

DOI： 10.1007/s00795-020-00275-1

PubMed

Other Link： https://link.springer.com/article/10.1007/s00795-020-00275-1/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：S. Karger AG  

MED13L (mediator complex subunit 13-like) is a component of the mediator complex, which functions as a regulator for gene transcription. Since gene abnormalities in MED13L are responsible for neurodevelopmental disorders, MED13L is presumed to play an essential role in brain development. In this study, we prepared a specific antibody against MED13L, anti-MED13L, and analyzed its expression profile in mouse tissues with focusing on the central nervous system. In Western blotting, MED13L exhibited a tissue-dependent expression profile in the adult mouse and was expressed in a developmental stage-dependent manner in brain. In immunofluorescence analyses, MED13L was at least partially colocalized with pre- and post-synaptic markers, synaptophysin, and PSD95, in primary cultured hippocampal neurons. Immunohistochemical analyses revealed that MED13L was relatively highly expressed in ventricular zone surface of cerebral cortex, and was also located both in the cytoplasm and nucleus of neurons in the cortical plate at embryonic day 14. Then, MED13L showed diffuse cytoplasmic distribution throughout the cerebral cortex at the postnatal day (P) 30. In addition, MED13L appeared to be localized in cell type- and developmental stage-specific manners in the hippocampus and cerebellum. These results suggest that MED13L is involved in the development of the central nervous system and synaptic function.

DOI： 10.1159/000515188

PubMed

Event date： 2022.6 - 2022.7

Presentation type：Poster presentation  

Event date： 2022.6 - 2022.7

Event date： 2022.6 - 2022.7

Presentation type：Poster presentation  

Event date： 2021.10

Event date： 2021.10

Event date： 2021.9 - 2021.10

Presentation type：Oral presentation (general)  

Presentation type：Oral presentation (general)  

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Presentation type：Oral presentation (general)