The histone H3K36 demethylase Fbxl11 plays pivotal roles in the development of retinal late-born cell types.

Histone methylation plays a vital role in retinal development. However, the role of histone H3K36 methylation in retinal development is not clear. We examined the role of H3K36 methylation by loss-of-function analysis of H3K36me1/2 demethylases, Fbxl10, and Fbxl11. We analyzed the effect of knockout of these genes in the developing and mature retina on retinal development. Knockout of Fbxl10 specifically in the developing retina did not result in gross developmental abnormalities. Although adult rod photoreceptor-specific knockout of Fbxl11 in mature retinas did not result in morphological abnormalities, Fbxl11 knockout in developing retinas increased apoptosis, suppressed the proliferation of retinal progenitor cells, and resulted in microphthalmia. Morphological analysis revealed perturbed differentiation of rod photoreceptor and bipolar cells. RNA-seq of retinas at P7 showed markedly decreased expression of genes characterizing rod photoreceptor and bipolar cells in Fbxl11-knockout retinas. In addition, perturbation of alternative splicing increased intron retention in Fbxl11-knockout retinas. Genome-wide evaluation of the H3K36 methylation status revealed that Fbxl11 knockout altered the distribution of H3K36me2/3 in genes important for rod photoreceptor development. Taken together, we show that Fbxl11 plays pivotal roles in the development of retinal late-born cell types and may contribute to tight control of H3K36 methylation during retinal development.

Clinical, radiological, and molecular analyses of neuronal intranuclear inclusion disease with polyglycine inclusions.

Neuronal intranuclear inclusion disease (NIID) is a clinically complex neurological disorder that appears sporadically or autosomally. Expansions of intronic GGC trinucleotide repeats in the NOTCH2 N-terminal-like C (NOTCH2NLC) gene cause NIID. In this study, to clarify the clinical characteristics useful for the differential diagnosis of NIID, clinical data of neurological examination, neuroimaging, and nerve conduction studies of six NIID patients diagnosed by pathological or genetic investigations were analyzed. Clinically useful characteristics for diagnosing NIID include general hyporeflexia, episodic disturbance of consciousness, sensory disturbance, miosis, and dementia. Furthermore, neuroimaging findings, such as leukoencephalopathy in T2-weighted magnetic resonance imaging and a linear high intensity of subcortical U-fibers in diffusion-weighted imaging (DWI), as well as decreased motor nerve conduction velocity, are especially important biomarkers for NIID. However, it is necessary to remember that these features may not always be present, as shown in one of the cases who did not have a DWI abnormality in this study. This study also investigated whether expanded GGC repeats were translated into polyglycine. Immunohistochemical analysis using a custom antibody raised against putative C-terminal polypeptides followed by polyglycine of uN2CpolyG revealed that polyglycines were localized in the intranuclear inclusions in skin biopsy specimens from all six patients, suggesting its involvement in the pathogenesis of NIID.

Cerebral Microbleeds, Cerebrospinal Fluid, and Neuroimaging Markers in Clinical Subtypes of Alzheimer's Disease.

Lobar cerebral microbleeds (CMBs) in Alzheimer's disease (AD) are associated with cerebral amyloid angiopathy (CAA) due to vascular amyloid beta (Aß) deposits. However, the relationship between lobar CMBs and clinical subtypes of AD remains unknown. Here, we enrolled patients with early- and late-onset amnestic dominant AD, logopenic variant of primary progressive aphasia (lvPPA) and posterior cortical atrophy (PCA) who were compatible with the AD criteria. We then examined the levels of cerebrospinal fluid (CSF) biomarkers [Aß1-42, Aß1-40, Aß1-38, phosphorylated tau 181 (P-Tau), total tau (T-Tau), neurofilament light chain (NFL), and chitinase 3-like 1 protein (YKL-40)], analyzed the number and localization of CMBs, and measured the cerebral blood flow (CBF) volume by 99mTc-ethyl cysteinate dimer single photon emission computerized tomography (99mTc ECD-SPECT), as well as the mean cortical standard uptake value ratio by 11C-labeled Pittsburgh Compound B-positron emission tomography (11C PiB-PET). Lobar CMBs in lvPPA were distributed in the temporal, frontal, and parietal lobes with the left side predominance, while the CBF volume in lvPPA significantly decreased in the left temporal area, where the number of lobar CMBs and the CBF volumes showed a significant inversely correlation. The CSF levels of NFL in lvPPA were significantly higher compared to the other AD subtypes and non-demented subjects. The numbers of lobar CMBs significantly increased the CSF levels of NFL in the total AD patients, additionally, among AD subtypes, the CSF levels of NFL in lvPPA predominantly were higher by increasing number of lobar CMBs. On the other hand, the CSF levels of Aß1-38, Aß1-40, Aß1-42, P-Tau, and T-Tau were lower by increasing number of lobar CMBs in the total AD patients. These findings may suggest that aberrant brain hypoperfusion in lvPPA was derived from the brain atrophy due to neurodegeneration, and possibly may involve the aberrant microcirculation causing by lobar CMBs and cerebrovascular injuries, with the left side dominance, consequently leading to a clinical phenotype of logopenic variant.

Cd9 Protects Photoreceptors from Injury and Potentiates Edn2 Expression.

Purpose: Cd9 is a tetraspanin membrane protein that plays various roles in tissue development and disease pathogenesis, especially in cancer, but the expression patterns and function of Cd9 in retinal development and disease are not well understood. We asked its roles during retinal photoreceptor degeneration by using CD9-knockout mice. Methods: Cd9 knockout mice and rd1 mice were used to examine roles of Cd9 for progression of photoreceptor degeneration. Reverse transcription-polymerase chain reaction and immunohistochemistry were mainly used as analytical methods. Results: Cd9 transcripts were only weakly expressed in retina at embryonic day 14, but its expression level subsequently increased and peaked at around postnatal day 12. In 6-week-old female mice derived retina, mRNA expression decreased slightly but was maintained at a significant level. Published RNA-sequencing data and immunohistochemistry indicated that Cd9 was expressed abundantly in Müller glia and weakly in other retinal neurons. Notably, when photoreceptors were damaged, Cd9 expression was increased in rod photoreceptors and decreased in Müller glia. Cd9 knockout mice retinas developed normally; however, once the retina suffered damage, degeneration of photoreceptors was more severe in Cd9 knockout retinas than control retinas. Induction of Edn2, which is known to protect against photoreceptor damage, was severely hampered. In addition, induction of Socs3, which is downstream of gp130 (Il6st), was weaker in Cd9 knockout retinas. Conclusions: Taken together, these findings indicate that, although Cd9 was dispensable for normal gross morphological development, it protected rod photoreceptors and enhanced Edn2 expression, possibly through modulation of gp130 signaling.

MicroRNA-7a regulates Müller glia differentiation by attenuating Notch3 expression.

miRNA-7a plays critical roles in various biological aspects in health and disease. We aimed to reveal roles of miR-7a in mouse retinal development by loss- and gain-of-function analyses of miR-7a. Plasmids encoding miR-7a or miR-7a-decoy (anti-sense miR-7a) were introduced into mouse retina at P0, and the retina was cultured as explant. Then, proliferation of retinal progenitors and differentiation of retinal subtypes were examined by immunostaining. miR-7a had no apparent effect on the proliferation of retinal progenitor cells. However, the expression of Müller glia marker, cyclin D3, was reduced by miR-7a overexpression and up-regulated by miR-7a decoy, suggesting that miR-7a negatively regulates differentiation of Müller glia. Targets of miR-7a, which were predicted by using a public program miRNA.org, and Notch3 was suggested to be one of candidate genes of miR-7a target. Notch3 3' UTR appeared to contain complementary sequence to the seed sequence of miR-7a. A reporter assay in NIH3T3 cells using a plasmid containing multiple repeats of potential target sequence of 3' Notch UTR showed that miR-7a suppress expression of reporter EGFP through 3'UTR region. Expression of sh-Notch3 and over-expression of NICD3 in retina suggested that miR-7a regulates Müller glia differentiation through attenuation of Notch3 expression. Taken together, we revealed that the miR-7a regulates the differentiation of Müller glia through the suppression of Notch3 expression.

Induction of intermediate mesoderm by retinoic acid receptor signaling from differentiating mouse embryonic stem cells.

Renal lineages including kidney are derived from intermediate mesoderm, which are differentiated from a subset of caudal undifferentiated mesoderm. The inductive mechanisms of mammalian intermediate mesoderm and renal lineages are still poorly understood. Mouse embryonic stem cells (mESCs) can be a good in vitro model to reconstitute the developmental pathway of renal lineages and to analyze the mechanisms of the sequential differentiation. We examined the effects of Activin A and retinoic acid (RA) on the induction of intermediate mesoderm from mESCs under defined, serum-free, adherent, monolayer culture conditions. We measured the expression level of intermediate mesodermal marker genes and examined the developmental potential of the differentiated cells into kidney using an ex vivo transplantation assay. Adding Activin A followed by RA to mESC cultures induced the expression of marker genes and proteins for intermediate mesoderm, odd-skipped related 1 (Osr1) and Wilm’s Tumor 1 (Wt1). These differentiated cells integrated into laminin-positive tubular cells and Pax2-positive renal cells in cultured embryonic kidney explants. We demonstrated that intermediate mesodermal marker expression was also induced by RA receptor (RAR) agonist, but not by retinoid X receptor (RXR) agonists. Furthermore, the expression of these markers was decreased by RAR antagonists. We directed the differentiation of mESCs into intermediate mesoderm using Activin A and RA and revealed the role of RAR signaling in this differentiation. These methods and findings will improve our understanding of renal lineage development and could contribute to the regenerative medicine of kidney.

[Neuromelanin-related contrast in the substantia nigra semiquantitatively evaluated by magnetic resonance imaging at 3T: comparison between normal aging and Parkinson disease].

Fast spin-echo (FSE) T1-weighted magnetic resonance imaging (MRI) at 3T, which was optimized to detect neuromelanin-related contrast (NRC), was applied to quantitative estimation of signal alterations in the substantia nigra pars compacta (SNc) of 72 normal volunteers and 59 patients with Parkinson disease (PD). We examined relationship between NRC in SNc and clinical parameters. The NRC showed significant positive correlation with normal aging and was slightly but significantly higher in women than in men. Significant reduction in the NRC was found in PD as compared with 59 age- and sex-matched normal volunteers. The NRC in PD was negatively and significantly correlated with duration of illness and disease severity assessed by UPDRS and Hoehn & Yahr stage. Significant reduction of the NRC was demonstrated in patients with visual hallucinations as compare with patients without the symptoms. REM sleep behavior disorder also contributed reduction of NRC although it did more mildly than visual hallucination. Anosmia or hyposmia had no statistical relationship with the amount of NRC in PD. The overall visual inspection indicated that the reduction of the NRC in PD should start at the ventrolateral portion of SNc and advance medially. Additionally, we studied dementia with Lewy body disease (DLB). The NRC was reduced more significantly in DLB patients with PD symptoms than in those without them who also showed a significant reduction compared with normal controls. Quantification and distribution of the NRC obtained by 3T MRI was well correlated with pathological findings reported previously and clinical parameters in this study. Visualization and quantification of the NRC provide some parts of clinical and diagnostic information about pathologic condition of SNc.

Induction of neural crest cells from mouse embryonic stem cells in a serum-free monolayer culture.

The neural crest (NC) is a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells,including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early development remain poorly understood. We previously established a defined serum-free culture condition for mouse embryonic stem (mES) cells without feeders. Here, using this defined condition, we have developed a protocol to promote mES cell differentiation into NC cells in an adherent monolayer culture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblast growth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we have established a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocol expressed EGFP. This protocol could be used to help clarify the mechanisms by which cells differentiate into the NC lineage and to assist the development of applications for clinical therapy.

BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin.

Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast.

The Xenopus Bowline/Ripply family proteins negatively regulate the transcriptional activity of T-box transcription factors.

Bowline, which is a member of the Xenopus Bowline/Ripply family of proteins, represses the transcription of somitogenesis-related genes before somite segmentation, which makes Bowline indispensable for somitogenesis. Although there are three bowline/Ripply family genes in each vertebrate species, it is not known whether the Bowline/Ripply family proteins share a common role in development. To elucidate their developmental roles, we examined the expression patterns and functions of the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline, and a novel member of this protein family, xRipply3. We found that the expression patterns of bowline and ledgerline overlapped in the presomitic mesoderm (PSM), whereas ledgerline was additionally expressed in the newly formed somites. In addition, we isolated xRipply3, which is expressed in the pharyngeal region. Co-immunoprecipitation assays revealed that Ledgerline and xRipply3 interacted with T-box proteins and the transcriptional co-repressor Groucho/TLE. In luciferase assays, xRipply3 weakly suppressed the transcriptional activity of Tbx1, while Ledgerline strongly suppressed that of Tbx6. In line with the repressive role of Ledgerline, knockdown of Ledgerline resulted in enlargement of expression regions of the somitogenesis-related-genes mespb and Tbx6. Inhibition of histone deacetylase activity increased the expression of mespb, as seen in the Bowline and Ledgerline knockdown experiments. These results suggest that the Groucho-HDAC complex is required for the repressive activity of Bowline/Ripply family proteins during Xenopus somitogenesis. We conclude that although the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline and xRipply3 are expressed differentially, they all act as negative regulators of T-box proteins.

PMesogenin1 and 2 function directly downstream of Xtbx6 in Xenopus somitogenesis and myogenesis.

T-box transcription factor tbx6 and basic-helix-loop-helix transcription factor pMesogenin1 are reported to be involved in paraxial mesodermal differentiation. To clarify the relationship between these genes in Xenopus laevis, we isolated pMesogenin2, which showed high homology with pMesogenin1. Both pMesogenin1 and 2 appeared to be transcriptional activators and were induced by a hormone-inducible version of Xtbx6 without secondary protein synthesis in animal cap assays. The pMesogenin2 promoter contained three potential T-box binding sites with which Xtbx6 protein was shown to interact, and a reporter gene construct containing these sites was activated by Xtbx6. Xtbx6 knockdown reduced pMesogenin1 and 2 expressions, but not vice versa. Xtbx6 and pMesogenin1 and 2 knockdowns caused similar phenotypic abnormalities including somite malformation and ventral body wall muscle hypoplasia, suggesting that Xtbx6 is a direct regulator of pMesogenin1 and 2, which are both involved in somitogenesis and myogenesis including that of body wall muscle in Xenopus laevis.

[Clinical features and laboratory findings in children with both anti-dsDNA and anti-U1-RNP antibody].

Mixed connective tissue disease (MCTD) includes clinical features of systemic lupus erythematosus (SLE), dermatomyositis/polymyositis (DM/PM), and systemic sclerosis (SSc) occurring in conjunction with a high anti-U1-RNP antibody titer. Childhood MCTD rarely manifests the symptoms and signs of DM/PM and SSc, and mostly does those of SLE. Thus, the diagnosis of childhood MCTD is inevitably based on the two major findings, Raynaud's phenomenon and a high titer of anti-U1-RNP antibody. However, in clinical setting there exist patients who have both anti-dsDNA antibody, a SLE disease-marker, and anti-U1-RNP antibody, a MCTD disease-marker, and thus it is hard to differentiate MCTD patients from SLE. Eighty children were enrolled in this study, and divided into 3 groups ; group A, those who are positive for anti-dsDNA antibody/negative for anti-U1-RNP antibody (48 cases, 60.0%), group B : those who are positive for both anti-dsDNA and anti-U1-RNP antibody (22 cases, 27.5%), group C; those who are negative for anti-dsDNA antibody/positive for anti-U1-RNP antibody (10 cases, 12.5%), and each of the clinical characteristics among these 3 groups was mutually examined. The results indicated that the frequency of hypocomplementemia in group B was close to group A rather than group C, and the frequencies of both hyper-gamma-globulinemia and Raynaud's phenomenon were very close to group C, but not to group A. On the contrary, the findings which seemed to be specific to MCTD, high titers of speckled type anti-nuclear antibody and rheumatoid factor, located at the middle between group A and group C. Thus, children in group B essentially carried characteristic symptoms and signs of both SLE and MCTD, and it will be difficult to differentiate these two diseases at the onset of the disease. Taken together, children with high titers of both anti-dsDNA antibody and anti-U1-RNP antibody as well as clinical symptoms and signs such as hyper-gamma-globulinemia, Raynaud's phenomenon, membranous nephritis, positive speckled type anti-nuclear antibody and rheumatoid factor should be followed and treated as children with MCTD along with SLE.

[A case of aortitis syndrome diagnosed with 18F-fluorodeoxyglucose-positron tomography (18F-FDG-PET) in the early pre-pulseless phase].

A 14-year-old girl with aortitis syndrome in the early pre-pulseless phase was admitted to our hospital because of slight fever, neck bruit, asymmetrical blood pressure, stenosis or dilatation of the main branch arteries in aorta on chest computed tomography. Laboratory examination revealed a high level of C-reactive protein and an elevated erythrocyte sedimentation rate, as well as hypergammaglobulinemia, and 18F-FDG-PET revealed an accumulation of 18 fluorodeoxyglucose in the great vessels. She was first given pulse therapy with a combination of methylprednisolone and intravenous cyclophosphamide, and then maintenance therapy with oral prednisolone and azathioprine. All the abnormal laboratory parameters improved to normal levels within a month. We suggest that early diagnosis of aortitis syndrome may permit early treatment in the early pre-pulseless phase and could possibly prevent progression to the occlusive phase.

[A case of autoimmune hepatitis needed to be differentiated from EBV hepatitis, in that the histology of liver biopsy specimen was useful for diagnosis].

A 10-year-old girl with autoimmune hepatitis (AIH) was reported. She was admitted to our hospital because of cholestasis and elevation of liver enzymes for 2 months. Laboratory examination revealed that EBV-DNA copy number in the PBMNC (peripheral mononuclear cells) was 1.2 x 10(3) copies/microg of DNA, hypergammaglobulinemia, and positive antinuclear antibody, positive anti-smooth muscle antibody. The histology of her liver biopsy specimen revealed interface hepatitis, dense mononuclear cell infiltrates, mild fibrosis, and negative for EBV in situ hybridization assay indicating AIH and not EBV-associated hepatitis. She was treated firstly with methylprednisolone pulses, then will prednisolone p.o.+azathioprine p.o.. Intravenous cyclophosphamide pulse therapy was introduced because of her abnormal immune pathology. All abnormal laboratory parameters improved to normal levels within 2 months, and EBV-DNA copy number in the PBMNC became negative after 4 months. The histology of liver biopsy specimen was useful for the diagnosis of AIH in such a difficult case needed to be differentiated from EBV hepatitis.

[A case of classical polyarteritis nodosa diagnosed by myocardial biopsy].

We experienced a girl with polyarteritis nodosa (PN) diagnosed by myocardial biopsy. The symptoms began with high fever and skin rash. These symptoms and laboratory findings temporarily improved by oral prednisolone, however, she flared up with chest pain about 40 days after onset of the disease. Electrocardiogram indicated the elevation of ST-T levels and low voltage, and blood examination showed remarkable elevation of creatine phosphokinase (CK), white blood cell count (WBC), aspartate aminotransferase (AST) and lactic dehydrogenase (LDH) levels. We suspected systemic vasculitis and damage of coronary artery or/and heart muscle. Finally, she was diagnosed with classical polyarteritis nodosa by myocardial biopsy. Coronary angiography revealed no abnormalities. The combination therapy of cyclophosphamide pulses and plasma-exchange was very effective to suppress the disease activity.

[Intravenous cyclophosphamide pulse therapy for refractory juvenile dermatomyositis].

We described three children with juvenile dermatomyositis (JDM) refractory to the conventional therapy. They were successfully treated with intravenous cyclophosphamide (IVCY) pulses, and two of them were administered plasma exchange (PE) before IVCY. Case 1. A 17-year-old girl with JDM was previously treated for 2 years with the combination of prednisolone, intravenous gamma-globulin, methotrexate, and azathioprine. However, muscle weakness gradually progressed. She failed to hold her sitting position and to rise her arms, but both serum CK and aldolase were stable. After the episode of aspiration pneumonia the follow-up muscle biopsy was performed, which revealed muscle degeneration and massive mononuclear cell infiltration in perivascular area. The erythrocyte sedimentation rate (ESR) and fibrin degradation product E (FDP-E) levels were gradually increased. Because the active inflammation of muscle and muscle vasculature was suspected, the PE and IVCY combination therapy was administered. During the 6 courses of the therapy, muscle weakness was markedly improved so that she could hold herself at the sitting position and could have meals by herself. Case 2. A 5-year-old boy with JDM was treated for 8 months with prednisolone p.o., but his muscle strength became worse. The muscle enzyme levels, such as serum CK and aldolase, were not reflecting his status of the disease, but FDP-E levels were increased. Muscle MRI and biopsy revealed the inflammatory changes of perivascular area of muscle. The PE and IVCY combination therapy was effective, and he became able to walk and run by himself. Case 3. A 14-year-old boy was diagnosed as having JDM when he was 10 years of age, and treated with prednisolone p.o., and subsequently with intravenous methylprednisolone pulses and azathioprine. Three years later the flares were observed accompanied with the elevations of serum CK and FDP-E. The administration of IVCY improved muscle strength as well as serum muscle enzyme and FDP-E levels. These findings indicated that the clinical manifestations of JDM should be closely monitored, that the serum levels of muscle enzymes including CK and aldolase were sometimes not indicative for the flares of JDM, and that muscle MRI and re-biopsy examination were needed for the children with progressive muscle weakness. In addition, determination of ESR and FDP-E was not specific but helpful to detect flares of the disease in some cases.