Ikuyo Inoue

Nav1.1 Localizes to Axons of Parvalbumin-Positive Inhibitory Interneurons: A Circuit Basis for Epileptic Seizures in Mice Carrying an Scn1a Gene Mutation

Loss-of-function mutations in human SCN1A gene encoding Nav1.1 are associated with a severe epileptic disorder known as severe myoclonic epilepsy in infancy. Here, we generated and characterized a knock-in mouse line with a loss-of-function nonsense mutation in the Scn1a gene. Both homozygous and heterozygous knock-in mice developed epileptic seizures within the first postnatal month. Immunohistochemical analyses revealed that, in the developing neocortex, Nav1.1 was clustered predominantly at the axon initial segments of parvalbumin-positive (PV) interneurons. In heterozygous knock-in mice, trains of evoked action potentials in these fast-spiking, inhibitory cells exhibited pronounced spike amplitude decrement late in the burst. Our data indicate that Nav1.1 plays critical roles in the spike output from PV interneurons and, furthermore, that the specifically altered function of these inhibitory circuits may contribute to epileptic seizures in the mice.

SCN1A Mutation Mosaicism in a Family with Severe Myoclonic Epilepsy in Infancy

Summary:  Purpose: To investigate the genetic background of familial severe myoclonic epilepsy in infancy (SMEI) cases.

Efhc1 deficiency causes spontaneous myoclonus and increased seizure susceptibility

Mutations in EFHC1 gene have been previously reported in patients with epilepsies, including those with juvenile myoclonic epilepsy. Myoclonin1, also known as mRib72-1, is encoded by the mouse Efhc1 gene. Myoclonin1 is dominantly expressed in embryonic choroid plexus, post-natal ependymal cilia, tracheal cilia and sperm flagella. In this study, we generated viable Efhc1-deficient mice. Most of the mice were normal in outward appearance, and both sexes were found to be fertile. However, the ventricles of the brains were significantly enlarged in the null mutants, but not in the heterozygotes. Although the ciliary structure was found intact, the ciliary beating frequency was significantly reduced in null mutants. In adult stages, both the heterozygous and null mutants developed frequent spontaneous myoclonus. Furthermore, the threshold of seizures induced by pentylenetetrazol was significantly reduced in both heterozygous and null mutants. These observations seem to further suggest that decrease or loss of function of myoclonin1 may be the molecular basis for epilepsies caused by EFHC1 mutations.

Increased lipid peroxidation in Down’s syndrome mouse models

Elevated oxidative stress has been suggested to be associated with the features of Down’s syndrome (DS). We previously reported increased oxidative stress in cultured cells from the embryonic brain of Ts1Cje, a mouse genetic DS model. However, since in vivo evidence for increased oxidative stress is lacking, we here examined lipid peroxidation, a typical marker of oxidative stress, in the brains of Ts1Cje and another DS mouse model Ts2Cje with an overlapping but larger trisomic segment. Accumulations of proteins modified with the lipid peroxidation‐derived products, 13‐hydroperoxy‐9Z,11E‐octadecadienoic acid and 4‐hydroxy‐2‐nonenal were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation‐sensitive fluorescent probe also showed that reactive oxygen species themselves were increased in Ts1Cje brain. However, electron spin resonance analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that the reactive oxygen species production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13‐hydroperoxy‐9Z,11E‐octadecadienoic acid and/or 4‐hydroxy‐2‐nonenal to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.

Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid

Significance Hepatocellular carcinoma (HCC) is a highly lethal cancer, partly because of its high rate of recurrence, which is caused by the presence of liver cancer stem cells (CSCs). Here, using a selective chemopreventive agent, acyclic retinoid (ACR), as a bioprobe, we identified MYCN, which is mostly recognized as an oncogene in neuroblastoma, as a therapeutic target of ACR for HCC through a selective deletion of MYCN+ liver CSCs. We also demonstrated that the expression of MYCN in HCC served as a prognostic biomarker and positively correlated with recurrence of de novo HCC after curative treatment. Our study highlighted MYCN as a biomarker and therapeutic target in drug discovery for screening chemopreventive agents against the recurrence of HCC. Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix–loop–helix–zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/β-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM− cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

TDP-43 accelerates age-dependent degeneration of interneurons

TDP-43 is an RNA-binding protein important for many aspects of RNA metabolism. Abnormal accumulation of TDP-43 in the cytoplasm of affected neurons is a pathological hallmark of the neurodegenerative diseases frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Several transgenic mouse models have been generated that recapitulate defects in TDP-43 accumulation, thus causing neurodegeneration and behavioural impairments. While aging is the key risk factor for neurodegenerative diseases, the specific effect of aging on phenotypes in TDP-43 transgenic mice has not been investigated. Here, we analyse age-dependent changes in TDP-43 transgenic mice that displayed impaired memory. We found the accumulation of abundant poly-ubiquitinated protein aggregates in the hippocampus of aged TDP-43 transgenic mice. Intriguingly, the aggregates contained some interneuron-specific proteins such as parvalbumin and calretinin, suggesting that GABAergic interneurons were degenerated in these mice. The abundance of aggregates significantly increased with age and with the overexpression of TDP-43. Gene array analyses in the hippocampus and other brain areas revealed dysregulation in genes linked to oxidative stress and neuronal function in TDP-43 transgenic mice. Our results indicate that the interneuron degeneration occurs upon aging, and TDP-43 accelerates age-dependent neuronal degeneration, which may be related to the impaired memory of TDP-43 transgenic mice.

Nav1.1 predominantly localizes to axons of parvalbumin-positive inhibitory interneurons: a circuit basis for epileptic seizures in Nav1.1-deficient mice

1P-EO2 Nav1.1 predominantly localizes to axons of arvalbumin-positive inhibitory interneurons: a circuit basis for pileptic seizures in Nav1.1-deficient mice . Ogiwara1, H. Miyamoto2, N. Morita3, N. Atapour2, . Mazaki1, I. Inoue1, Y. Yanagawa5, K. Obata4, T. Furuichi3, .K. Hensch2, K. Yamakawa1 Laboratory of Neurogenetics, RIKEN-BSI, Japan; 2 Laboratory of euronal Circuit Development, RIKEN-BSI, Japan; 3 Laboratory for olecular Neurogenesis, RIKEN-BSI, Japan; 4 Neuronal Circuit echanisms Research Group, RIKEN-BSI, Japan; 5 Department f Genetics and Behavioural Neuroscience, Gunma University and ORST, JST, Japan

Prevention of acute liver injury by suppressing plasma kallikrein-dependent activation of latent TGF-β

Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor β (TGF-β) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-β is activated partly through proteolytic cleavage by a serine protease plasma kallikrein (PLK) between the R58 and L59 residues of its propeptide region. Recently, we developed a specific monoclonal antibody to detect the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs) that reflect PLK-dependent TGF-β activation. This study aimed to explore the potential roles of PLK-dependent TGF-β activation in the pathogenesis of ALI. We established a mouse ALI model via the injection of anti-Fas antibodies (Jo2) and observed increases in the TGF-β1 mRNA level, Smad3 phosphorylation, TUNEL-positive apoptotic hepatocytes and R58-positive cells in the liver tissues of Jo2-treated mice. The R58 LAP-DPs were observed in/around F4/80-positive macrophages, while macrophage depletion with clodronate liposomes partly alleviated the Jo2-induced liver injury. Blocking PLK-dependent TGF-β activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-β receptor-mediated signaling pathway using SB431542 significantly prevented Jo2-induced hepatic apoptosis and mortality. Furthermore, similar phenomena were observed in the mouse model of ALI with the administration of acetaminophen (APAP). In summary, R58 LAP-DPs reflecting PLK-dependent TGF-β activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-β activation has potential as a therapeutic strategy for ALI.

No visible abnormality of migrating neurons in Efhc1-deficient mouse, a model for juvenile myoclonic epilespy

Juvenile myoclonic epilepsy (JME) accounts for 10 to 12% of all epilepsies. By genetic linkage analysis, we mapped the responsible gene on chromosome 6p12-p11 and we found disease-associated missense mutations of EFHC1 (EF-Hand Containing-1) gene in the region in JME patients (Suzuki et al., 2004. Nat. Genet. 36: 842-9). Subsequently other groups reported additional disease-associated mutations not only in JME but also in other types of idiopathic epilepsies. Whereas most of the genes for idiopathic epilepsies encode ion-channels, EFHC1 encodes non-ion channel protein myoclonin1 that is composed of three consecutive DM10 domains, a motif with unknown function, and an EF-hand calcium-binding motif at the C terminus. In mouse brain, myoclonin1 is prominently expressed in choroid plexus during embryonic stages and in cilia of ependymal cells lining the ventricles at postnatal stages (Suzuki et al., Biochem. Biophys. Res. Commun. 367: 226-33, 2008). We generated Efhc1-deficient mice and found that both heterozygous and null mutants showed frequent spontaneous myoclonus and increased seizure susceptibility for chemoconvulsant (Suzuki et al., Hum. Mol. Genet. 18: 1099-109, 2009). Recently, one group in Belgium reported that myoclonin1 is expressed at mitotic spindle and RNAi suppression of myoclonin1 lead to marked disruption of migration of neurons (de Nijs, L. et al. Nat. Neurosci. 12, 1266-1274, 2009). We re-investigated their results with the same myoclonin1 antibody and found that it surely developed signals at mitotic spindles but those signals did not disappear in our Efhc1 null mouse, suggesting that the signals were nonspecific. In addition, immuno-histological investigations of Efhc1 null mouse revealed no visible abnormality in migrating neurons and/or SOX2-, PH3-, or TUNEL-positive cells including their locations and cell numbers. These results require further re-investigations on the proposed role of myoclonin1 in cell division and neuronal migration.

Analysis of voltage-gated sodium channel α1 expression using BAC transgenic mice

in the thalamocortical loops. Involvement of basal ganglia in absence seizures was not shown in mice models, and roles of basal ganglia in the SWD generation were not known in any animal models. To address these issues, we performed in vivo and in vitro experiments using tottering (tg) mice, a well established model of absence epilepsy. In vivo experiments showed the involvement of basal ganglia in the SWD generation. In vitro experiments in the subthalamic nucleus (STN) neurons showed the enhanced membrane excitability in tg mice. This enhancement seemed to result from the decrement of the HCN channel activity. Unilateral blockades of STN HCN channels of tg mice extended the mean duration of SWDs. The results suggested that the basal ganglia play a positive role in the SWD generation through the enhanced membrane excitability caused by decreased HCN channel activity in the STN.