田谷 真一郎

Abstract In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

Low-grade neuroepithelial tumors are major causes of drug-resistant focal epilepsy. Clinically, these tumors are defined as low-grade epilepsy-associated neuroepithelial tumors (LEATs). The BRAF V600E mutation is frequently observed in LEAT and linked to poor seizure outcomes. However, its molecular role in epileptogenicity remains elusive. To understand the molecular mechanism underlying the epileptogenicity in LEAT with the BRAF V600E genetic mutation (BRAF V600E-LEAT), we conducted RNA sequencing (RNA-seq) analysis using surgical specimens of BRAF V600E-LEAT obtained and stored at a single institute. We obtained 21 BRAF V600E-LEAT specimens and 4 control specimens, including 24 from Japanese patients and 1 from a patient of Central Asian origin, along with comprehensive clinical data. We submitted the transcriptome dataset of 21 BRAF V600E-LEAT plus 4 controls, as well as detailed clinical information, to a public database. Preliminary bioinformatics analysis using this dataset identified 2134 differentially expressed genes between BRAF V600E-LEAT and control. Additionally, gene set enrichment analysis provided novel insights into the association between estrogen response-related pathways and the epileptogenicity of BRAF V600E-LEAT patients. Our datasets and findings will contribute toward the understanding of the pathology of epilepsy caused by LEAT and the identification of new therapeutic targets.

Abstract Granule cell progenitors (GCPs) and granule cells (GCs) in the cerebellum are excellent models for studying the differentiation of neural progenitors into neurons. Although gradual degradation of ATOH1 protein in GCPs leads to their differentiation into GCs, the underlying regulatory mechanism is unclear. We show that a homeodomain-less isoform of MEIS1 (MEIS1-HdL) regulates ATOH1 degradation and GCP differentiation in a transcriptional regulation-independent manner. BMP signaling phosphorylates Ser328 of ATOH1 via ERK. CUL3 was identified as an E3-ligase that polyubiquitinates Ser328 phosphorylated ATOH1, leading to ATOH1 degradation. MEIS1-HdL and full-length MEIS1 form a trimeric complex with CUL3 and COP9 signalosome that inhibits ATOH1 ubiquitination and degradation. MEIS1-HdL is exclusively expressed in GCPs and suppresses ATOH1 degradation and GCP differentiation into GCs, despite high BMP signaling activities in the cells. Our study provides insight into the precise regulatory machinery of the degradation of the pivotal protein ATOH1 and differentiation of neural progenitors.

<title>Abstract</title>Here we report that CyclinD1 (CCND1) directly regulates both the proliferative and immature states of cerebellar granule cell progenitors (GCPs). CCND1 not only accelerates cell cycle but also upregulates ATOH1 protein, an essential transcription factor that maintains GCPs in an immature state. In cooperation with CDK4, CCND1 directly phosphorylates Ser309 of ATOH1, which inhibits additional phosphorylation at S328, consequently preventing Ser328 phosphorylation-dependent ATOH1 degradation. PROX1 downregulates Ccnd1 expression by histone-deacetylation of Ccnd1 promoter in GCPs, leading to cell cycle exit and differentiation. WNT signaling upregulates PROX1 expression in GCPs. These findings suggest that WNT-PROX1-CCND1-ATOH1 signaling cascade cooperatively controls proliferation and immaturity of GCPs. We revealed that the expression and phosphorylation levels of these molecules dynamically change during cerebellar development, which was suggested to determine appropriate differentiation rates from GCPs to GCs at distinct developmental stages. This study contributes to understanding the regulatory mechanism of GCPs as well as neural progenitors.

Molecular groups of supratentorial ependymomas comprise tumors with ZFTA-RELA or YAP1-involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation-based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histological features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA-associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo, and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion-positive tumors.

The molecular mechanisms underlying neurodevelopmental disorders (NDDs) remain unclear. We previously identified Down syndrome cell adhesion molecule like 1 (Dscaml1) as a responsible gene for Ihara epileptic rat (IER), a rat model for human NDDs with epilepsy. However, the relationship between NDDs and DSCAML1 in humans is still elusive. In this study, we screened databases of autism spectrum disorders (ASD), intellectual disability (ID)/developmental disorders (DD) and schizophrenia for genomic mutations in human DSCAML1. We then performed in silico analyses to estimate the potential damage to the mutated DSCAML1 proteins and chose three representative mutations (DSCAML1C729R , DSCAML1R1685* and DSCAML1K2108Nfs*37 ), which lacked a cysteine residue in the seventh Ig domain, the intracellular region and the C-terminal PDZ-binding motif, respectively. In overexpression experiments in a cell line, DSCAML1C729R lost its mature N-glycosylation, whereas DSCAML1K2108Nfs*37 was abnormally degraded via proteasome-dependent protein degradation. Furthermore, in primary hippocampal neurons, the ability of the wild-type DSCAML1 to regulate the number of synapses was lost with all mutant proteins. These results provide insight into understanding the roles of the domains in the DSCAML1 protein and further suggest that these mutations cause functional changes, albeit through different mechanisms, that likely affect the pathophysiology of NDDs.

A proper balance between proliferation and differentiation of cerebellar granule cell precursors (GCPs) is required for appropriate cerebellar morphogenesis. The Skp1-Cullin1-F-box (SCF) complex, an E3 ubiquitin ligase complex, is involved in polyubiquitination and subsequent degradation of various cell cycle regulators and transcription factors. However, it remains unknown how the SCF complex affects proliferation and differentiation of GCPs. In this study, we found that the scaffold protein Cullin1, and F-box proteins Skp2, β-TrCP1 and β-TrCP2 are expressed in the external granule layer (EGL). Knockdown of these molecules in the EGL showed that Cullin1, Skp2 and β-TrCP2 enhanced differentiation of GCPs. We also observed accumulation of cyclin-dependent kinase inhibitor p27 in GCPs when treated with a Cullin1 inhibitor or proteasome inhibitor. Furthermore, knockdown of p27 rescued enhancement of differentiation by Cullin1 knockdown. These results suggest that the SCF complex is involved in the maintenance of the proliferative state of GCPs through p27 degradation. In addition, inhibition of Cullin1 activity also prevented cell proliferation and enhanced accumulation of p27 in Daoy cells, a cell line derived from the sonic hedgehog subtype of medulloblastoma. This suggested that excess degradation of p27 through the SCF complex causes overproliferation of medulloblastoma cells.

The Ihara epileptic rat (IER) is a mutant model with limbic-like seizures whose pathology and causative gene remain elusive. In this report, via linkage analysis, we identified Down syndrome cell adhesion molecule-like 1(Dscaml1) as the responsible gene for IER. A single base mutation in Dscaml1 causes abnormal splicing, leading to lack of DSCAML1. IERs have enhanced seizure susceptibility and accelerated kindling establishment. Furthermore, GABAergic neurons are severely reduced in the entorhinal cortex (ECx) of these animals. Voltage-sensitive dye imaging that directly presents the excitation status of brain slices revealed abnormally persistent excitability in IER ECx. This suggests that reduced GABAergic neurons may cause weak sustained entorhinal cortex activations, leading to natural kindling via the perforant path that could cause dentate gyrus hypertrophy and epileptogenesis. Furthermore, we identified a single nucleotide substitution in a human epilepsy that would result in one amino acid change in DSCAML1 (A2105T mutation). The mutant DSCAML1A2105T protein is not presented on the cell surface, losing its homophilic cell adhesion ability. We generated knock-in mice (Dscaml1A2105T) carrying the corresponding mutation and observed reduced GABAergic neurons in the ECx as well as spike-and-wave electrocorticogram. We conclude that DSCAML1 is required for GABAergic neuron placement in the ECx and suppression of seizure susceptibility in rodents. Our findings suggest that mutations in DSCAML1 may affect seizure susceptibility in humans.

For normal neurogenesis and circuit formation, delamination of differentiating neurons from the proliferative zone must be precisely controlled; however, the regulatory mechanisms underlying cell attachment are poorly understood. Here, we show that Down syndrome cell adhesion molecule (DSCAM) controls neuronal delamination by local suppression of the RapGEF2-Rap1-N-cadherin cascade at the apical endfeet in the dorsal midbrain. Dscam transcripts were expressed in differentiating neurons, and DSCAM protein accumulated at the distal part of the apical endfeet. Cre-loxP-based neuronal labeling revealed that Dscam knockdown impaired endfeet detachment from ventricles. DSCAM associated with RapGEF2 to inactivate Rap1, whose activity is required for membrane localization of N-cadherin. Correspondingly, Dscam knockdown increased N-cadherin localization and ventricular attachment area at the endfeet. Furthermore, excessive endfeet attachment by Dscam knockdown was restored by co-knockdown of RapGEF2 or N-cadherin Our findings shed light on the molecular mechanism that regulates a critical step in early neuronal development.