Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction.

Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.

Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons.

Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes, respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS), and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.

A Transgenic Pig Model With Human Mutant SOD1 Exhibits the Early Pathology of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) causes progressive degeneration of the motor neurons. In this study, we delivered the genetic construct including the whole locus of human mutant superoxide dismutase 1 (SOD1) with the promoter region of human SOD1 into porcine zygotes using intracytoplasmic sperm injection-mediated gene transfer, and we thereby generated a pig model of human mutant SOD1-mediated familial ALS. The established ALS pig model exhibited an initial abnormality of motor neurons with accumulated misfolded SOD1. The ALS pig model, with a body size similar to that of human beings, will provide opportunities for cell and gene therapy platforms in preclinical translational research.

Induction of inverted morphology in brain organoids by vertical-mixing bioreactors.

Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization. Here we examined the vertical-mixing effect on brain organoid structures using bioreactors and established inverted brain organoids. The organoids generated by vertical mixing showed neurons that migrated from the outer periphery to the inner core of organoids, in contrast to orbital mixing. Computational analysis of flow dynamics clarified that, by comparison with orbital mixing, vertical mixing maintained the high turbulent energy around organoids, and continuously kept inter-organoid distances by dispersing and adding uniform rheological force on organoids. To uncover the mechanisms of the inverted structure, we investigated the direction of primary cilia, a cellular mechanosensor. Primary cilia of neural progenitors by vertical mixing were aligned in a multidirectional manner, and those by orbital mixing in a bidirectional manner. Single-cell RNA sequencing revealed that neurons of inverted brain organoids presented a GABAergic character of the ventral forebrain. These results suggest that controlling fluid dynamics by biomechanical engineering can direct stem cell differentiation of brain organoids, and that inverted brain organoids will be applicable for studying human brain development and disorders in the future.

Latency-associated Peptide Degradation Fragments Produced in Stellate Cells and Phagocytosed by Macrophages in Bile Duct-ligated Mouse Liver.

Transforming growth factor-ß (TGF-ß) activation is involved in various pathogeneses, such as fibrosis and malignancy. We previously showed that TGF-ß was activated by serine protease plasma kallikrein-dependent digestion of latency-associated peptides (LAPs) and developed a method to detect LAP degradation products (LAP-DPs) in the liver and blood using specific monoclonal antibodies. Clinical studies have revealed that blood LAP-DPs are formed in the early stages of liver fibrosis. This study aimed to identify the cell source of LAP-DP formation during liver fibrosis. The N-terminals of LAP-DPs ending at residue Arg58 (R58) were stained in liver sections of a bile duct-ligated liver fibrosis model at 3 and 13 days. R58 LAP-DPs were detected in quiescent hepatic stellate cells at day 3 and in macrophages on day 13 after ligation of the bile duct. We then performed a detailed analysis of the axial localization of R58 signals in a single macrophage, visualized the cell membrane with the anti-CLEC4F antibody, and found R58 LAP-DPs surrounded by the membrane in phagocytosed debris that appeared to be dead cells. These findings suggest that in the early stages of liver fibrosis, TGF-ß is activated on the membrane of stellate cells, and then the cells are phagocytosed after cell death.

Simple derivation of skeletal muscle from human pluripotent stem cells using temperature-sensitive Sendai virus vector.

Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature-sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV-Myod1), a myogenic master transcription factor. SeV-Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38℃, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre-like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell-targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human-induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future.

Epilepsy protein Efhc1/myoclonin1 is expressed in cells with motile cilia but not in neurons or mitotic apparatuses in brain.

EFHC1 gene encodes the myoclonin1 protein, also known as Rib72-1. Pathogenic variants in EFHC1 have been reported in patients with juvenile myoclonic epilepsy (JME). Although several studies of immunohistological investigations reproducibly showed that the myoclonin1 is expressed in cells with flagella and motile cilia such as sperm, trachea and ependymal cells lining the brain ventricles, whether myoclonin1 is also expressed in neurons still remains controversial. Here we investigated myoclonin1 expression using widely-used polyclonal (mRib72-pAb) and self-made monoclonal (6A3-mAb) anti-myoclonin1 antibodies together with Efhc1 homozygous knock-out (Efhc1-/-) mice. All of the western blot, immunocytochemical, and immunohistochemical analyses showed that mRib72-pAb crossreacts with several mouse proteins besides myoclonin1, while 6A3-mAb specifically recognized myoclonin1 and detected it only in cells with motile cilia but not in neurons. In dividing cells, mRib72-pAb signals were observed at the midbody (intercellular bridge) and mitotic spindle, but 6A3-mAb did not show any signals at these apparatuses. We further found that the complete elimination of myoclonin1 in Efhc1-/- mouse did not critically affect cell division and migration of neurons in cerebral cortex. These results indicate that myoclonin1 is not expressed in neurons, not a regulator of cell division or neuronal migration during cortical development, but expressed in choroid plexus and ependymal cells and suggest that EFHC1 mutation-dependent JME is a motile ciliopathy.

Histological and biochemical evaluation of transforming growth factor-ß activation and its clinical significance in patients with chronic liver disease.

Transforming growth factor-ß (TGF-ß) is a key driver for liver fibrogenesis. TGF-ß must be activated in order to function. Plasma kallikrein (PLK) is a TGF-ß activator that cleaves the latency-associated protein (LAP) between arginine58 and lysine59 residues and releases active TGF-ß from the latent TGF-ß-LAP complex. Thus, the generation of two LAP degradation products, ending at arginine58 (R58/LAP-DPs) and beginning from lysine59 (L59/LAP-DPs), reflects PLK-dependent TGF-ß activation. However, the significance and details of TGF-ß activation in patients with chronic liver disease (CLD) remain uncertain. We herein examined the PLK-dependent TGF-ß activation in patients by detecting R58 and L59/LAP-DPs. A total of 234 patients with CLD were included in this study. Liver biopsy specimens were used for immunostaining to detect R58/LAP-DPs, while plasma samples were subjected to an enzyme-linked immunosorbent assay to measure the L59/LAP-DP concentration. R58/LAP-DP was robustly expressed in and around the sinusoidal cells before the development of the fibrous regions. The R58/LAP-DP expression at fibrosis stage 1 was higher than at any other stages, and the relationship between the plasma L59/LAP-DP level and the stage of fibrosis also showed a similar trend. The abundance of plasma L59/LAP-DP showed no correlation with the levels of direct serum biomarkers of liver fibrosis; however, its changes during interferon-based therapy for chronic hepatitis C were significantly associated with virological responses. Our results suggest that PLK-dependent TGF-ß activation occurs in the early stages of fibrosis and that its unique surrogate markers, R58 and L59/LAP-DPs, are useful for monitoring the clinical course of CLD.

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

Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor ß (TGF-ß) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-ß 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-ß activation. This study aimed to explore the potential roles of PLK-dependent TGF-ß 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-ß1 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-ß activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-ß 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-ß activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-ß activation has potential as a therapeutic strategy for ALI.

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

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/ß-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.

Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes.

The acute phase protein orosomucoid-1 (Orm1) is mainly expressed by hepatocytes (HPCs) under stress conditions. However, its specific function is not fully understood. Here, we report a role of Orm1 as an executer of HPC proliferation. Increases in serum levels of Orm1 were observed in patients after surgical resection for liver cancer and in mice undergone partial hepatectomy (PH). Transcriptome study showed that Orm1 became the most abundant in HPCs isolated from regenerating mouse liver tissues after PH. Both in vitro and in vivo siRNA-induced knockdown of Orm1 suppressed proliferation of mouse regenerating HPCs and human hepatic cells. Microarray analysis in regenerating mouse livers revealed that the signaling pathways controlling chromatin replication, especially the minichromosome maintenance protein complex genes were uniformly down-regulated following Orm1 knockdown. These data suggest that Orm1 is induced in response to hepatic injury and executes liver regeneration by activating cell cycle progression in HPCs.

L(59) TGF-ß LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice.

BACKGROUND: Hepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. There are no validated biomarkers that can non-invasively monitor excessive production of ECM (i.e., fibrogenesis). Transforming growth factor (TGF)-ß, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-ß is enveloped by its pro-peptide, the latency-associated protein (LAP). Thus, active TGF-ß must be released from the complex for binding to its receptor and inducing ECM synthesis. We recently reported that during the pathogenesis of liver fibrosis, plasma kallikrein (PLK) activates TGF-ß by cleavage between R(58) and L(59) residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R(58) (R(58) LAP-DPs), can be detected mainly around activated HSCs by specific antibodies against R(58) cleavage edges and functions as a footprint of PLK-dependent TGF-ß activation. Here, we describe a sandwich enzyme-linked immunosorbent assay (ELISA) that detects the other by-products, the C-terminal side LAP-DPs starting from residue L(59) (L(59) LAP-DPs). We demonstrated that the L(59) LAP-DPs are a potentially novel blood biomarker reflecting hepatic fibrogenesis. RESULTS: We established a specific sandwich ELISA to quantify L(59) LAP-DPs as low as 2 pM and measured L(59) LAP-DP levels in the culture media of a human activated HSC line, TWNT-4 cells. L(59) LAP-DPs could be detected in their media, and after treatment of TWNT-4 cells with a TGF-ß receptor kinase inhibitor, SB431542, a simultaneous reduction was observed in both L(59) LAP-DP levels in the culture media and the mRNA expression levels of collagen type (I) α1. In carbon tetrachloride- and bile duct ligation-induced liver fibrosis models in mice, plasma L(59) LAP-DP levels increased prior to increase of hepatic hydroxyproline (HDP) contents and well correlated with α-smooth muscle actin (αSMA) expression in liver tissues. At this time, αSMA-positive cells as well as R(58) LAP-DPs were seen in their liver tissues. CONCLUSIONS: L(59) LAP-DPs reflect PLK-dependent TGF-ß activation and the increase in αSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.

Surmounting chemoresistance by targeting the Y-box binding protein-1.

The Y-box binding protein 1 (YB-1), characterized by the presence of the cold shock domain, has been reported to induce chemoresistance in cancer therapy. Chemotherapy is one of the main therapeutic strategies in the treatment of cancer, in addition to surgery, radiation therapy, and hormonal therapy. However, chemoresistance remains a key obstacle to successful cancer management. In this review, we will focus on the role of YB-1, an important regulator of gene transcription, in cancer therapy and chemoresistance.

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.

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.

Sequential expression of Efhc1/myoclonin1 in choroid plexus and ependymal cell cilia.

EFHC1 is a gene mutated in patients with idiopathic epilepsies, and encodes the myoclonin1 protein. We here report the distribution of myoclonin1 in mouse. Immunohistochemical analyses revealed that the myoclonin1 first appeared at the roof of hindbrain at embryonic day 10 (E10), and moved on to choroid plexus at E14. At E18, it moved to ventricle walls and disappeared from choroid plexus. From neonatal to adult stages, myoclonin1 was concentrated in the cilia of ependymal cells at ventricle walls. At adult stages, myoclonin1 expression was also observed at tracheal epithelial cilia in lung and at sperm flagella in testis. Specificities of these immunohistochemical signals were verified by using Efhc1-deficient mice as negative controls. Results of Efhc1 mRNA in situ hybridization were also consistent with the immunohistochemical observations. Our findings raise "choroid plexusopathy" or "ciliopathy" as intriguing candidate cascades for the molecular pathology of epilepsies caused by the EFHC1 mutations.

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.

PURPOSE: To investigate the genetic background of familial severe myoclonic epilepsy in infancy (SMEI) cases. METHODS: We performed mutation analyses of the sodium-channel gene SCN1A in two Japanese brothers with clinical features of SMEI and their parents, who had no history of febrile and epileptic seizures. RESULTS: Each patient showed nucleotide changes (c.[730G>T; 735G>T; 736A>T]) in the coding exon 6 of SCN1A that led to a truncation of the channel protein. Their father showed no mutations, but their mother showed the same mutation in a subpopulation of lymphocytes. CONCLUSIONS: The maternal mosaicism explains the identical SCN1A mutations in the two brothers. This highlights the importance of investigating parental mosaicism even in sporadic SMEI cases.