Ciliary Neurotrophic Factor Derived From Astrocytes Protects Retinal Ganglion Cells Through PI3K/AKT, JAK/STAT, and MAPK/ERK Pathways.

Purpose: The purpose of this study was to investigate the roles of ciliary neurotrophic factor (CNTF) on the protective effects of astrocytes on retinal ganglion cells (RGCs). Methods: Primary RGCs were isolated from neonatal rats. Oxidative stress was induced, and the effects of co-culture with astrocytes and CNTF treatment on RGCs were evaluated. The pathways commonly altered by astrocytes and CNTF were investigated. Effects of each pathway were investigated using pathway inhibitors against PI3K/AKT, JAK/STAT, and MAPK/ERK. RNA sequencing was performed to identify the genes upregulated and downregulated by CNTF treatment. Results: Astrocytes improved the viability and increased ß3-tubulin expression in RGCs. The concentration of CNTF increased in the RGC-astrocyte co-culture medium. The protective effects of astrocytes were abolished by neutralization with the anti-CNTF antibody; thus, CNTF may play an important role in the effects mediated by astrocytes. Furthermore, CNTF treatment alone enhanced the viability and ß3-tubulin expression of RGCs and increased the population of viable RGCs under oxidative stress. The PI3K/AKT pathway was associated with both RGC viability and ß3-tubulin expression. However, the JAK/STAT pathway increased the viability of RGCs, whereas the MAPK/ERK pathway was associated with ß3-tubulin expression. RNA sequencing revealed the CNTF-upregulated genes associated with response to DNA damage and downregulated genes associated with photoreceptor cell differentiation. Conclusions: Our data revealed protective effects of astrocyte-derived CNTF on RGCs. In addition, we showed that multiple pathways exert these protective effects and identified the novel genes involved. These results may be helpful in developing treatments for RGC injury.

[Delayed Post-Hypoxic Leukoencephalopathy Induced by an Overdose with Fentanyl Patches: A Case Report]. / 펜타닐 첩포 남용으로 인한 지연성 ì €ì‚°ì†Œì„± 백색질뇌증: 증례 ë³´ê³ .

Fentanyl intoxication has occasionally been reported since fentanyl patches became available for medical use. Delayed post-hypoxic leukoencephalopathy is a complication of hypoxia. However, its neuropsychiatric symptoms can be delayed, and it can progress to leukoencephalopathy even after full recovery from coma due to acute intoxication. Herein, we report a case of fentanyl intoxication in a 65-year-old man who was presented to the hospital because of unconsciousness for 13 hours after using ten fentanyl patches simultaneously. Initial brain CT findings were non-specific. Twenty days later, delayed neuropathologic symptoms manifested, and MRI showed bilaterally symmetrical, heterogeneous, confluent high signal intensities on T2-weighted/fluid attenuated inversion recovery MRI in the cerebral white matter with diffusion restriction. This was followed by rapid exacerbation of neuropathological symptoms with diffuse severe cerebral atrophy over 1 year.

[Brain MRI Findings of the Cri-Du-Chat Syndrome: A Case Report and Summary]. / 묘성증후군 환아의 뇌 ìžê¸°ê³µëª ì˜ìƒ 소견: 증례 ë³´ê³ ë° ì •ë¦¬.

Cri-du-chat syndrome is a rare genetic disorder in which the patient presents with a characteristic high-pitched monotonous cry and recurrent aspiration pneumonia, attributed to abnormalities in the larynx, epiglottis, and nervous system. The most prominent brain MRI findings are the presence of pontine and cerebellar hypoplasia, which primarily involve posterior cranial fossa structures. Although atrophy of supratentorial structures were also a common radiological finding, it was considered to be a secondary change due to pontine hypoplasia. Here, we present the case of a three-month-old patient presenting with cri-du-chat at our institution. The patient also showed the presence of prominent pontine hypoplasia similar to previously reported cases; however, contrary to other cases, there was a general delayed myelination of brain instead of decreased myelination of anterior limb of internal capsule. Since the larynx, pons, and cerebellum all originated from similar notochord level, which suggests anomaly in early stage of development, laryngeal, and brain anomaly characteristically observed in the cridu-chat syndrome.

A principled strategy for mapping enhancers to genes.

Mapping enhancers to genes is a fundamental goal of modern biology. We have developed an innovative strategy that maps enhancers to genes in a principled manner. We illustrate its power by applying it to Myrf. Despite being a master regulator of oligodendrocytes, oligodendrocyte enhancers governing Myrf expression remain elusive. Since chromatin conformation capture studies have shown that a gene and its enhancer tend to be found in the same topologically associating domain (TAD), we started with the delineation of the Myrf TAD. A genome-wide map of putative oligodendrocyte enhancers uncovered 6 putative oligodendrocyte enhancers in the Myrf TAD, narrowing down the search space for Myrf enhancers from the entire genome to 6 loci in a principled manner. Epigenome editing experiments revealed that two of them govern Myrf expression for oligodendrocyte development. Our new method is simple, principled, and powerful, providing a systematic way to find enhancers that regulate the expression of a gene of interest. Since it can be applied to most cell types, it would greatly facilitate our effort to unravel transcriptional regulatory networks of diverse cell types.

Elucidating the transactivation domain of the pleiotropic transcription factor Myrf.

Myrf is a newly discovered membrane-bound transcription factor that plays an essential role in as diverse organisms as human, worm, and slime mold. Myrf is generated as a type-II membrane protein in the endoplasmic reticulum (ER). It forms homo-oligomers to undergo auto-cleavage that releases Myrf N-terminal fragment from the ER membrane as a homo-trimer. The homo-trimer of Myrf N-terminal fragments enters the nucleus and binds the Myrf motif to activate transcription. Despite its prominent role as a transcriptional activator, little is known about the transactivation domain of Myrf. Here, we report that the N-terminal-most (NTM) domain of Myrf is required for transcriptional activity and, when fused to a Gal4 fragment, enables it to activate transcription. The transactivation function of the NTM domain did not require homo-trimerization. We also discovered that the NTM domain can be sumoylated at three lysine residues (K123, K208, and K276), with K276 serving as the main acceptor. K276 sumoylation repressed the transactivation function of the NTM domain without affecting the stability or nuclear localization of Myrf N-terminal fragment. In sum, this study identifies the NTM domain as the transactivation domain of Myrf and the potential regulatory impact of its K276 sumoylation.

Homo-trimerization is essential for the transcription factor function of Myrf for oligodendrocyte differentiation.

Myrf is a key transcription factor for oligodendrocyte differentiation and central nervous system myelination. We and others have previously shown that Myrf is generated as a membrane protein in the endoplasmic reticulum (ER), and that it undergoes auto-processing to release its N-terminal fragment from the ER, which enters the nucleus to work as a transcription factor. These previous studies allow a glimpse into the unusual complexity behind the biogenesis and function of the transcription factor domain of Myrf. Here, we report that Myrf N-terminal fragments assemble into stable homo-trimers before ER release. Consequently, Myrf N-terminal fragments are released from the ER only as homo-trimers. Our re-analysis of a previous genetic screening result in Caenorhabditis elegans shows that homo-trimerization is essential for the biological functions of Myrf N-terminal fragment, and that the region adjacent to the DNA-binding domain is pivotal to its homo-trimerization. Further, our computational analysis uncovered a novel homo-trimeric DNA motif that mediates the homo-trimeric DNA binding of Myrf N-terminal fragments. Importantly, we found that homo-trimerization defines the DNA binding specificity of Myrf N-terminal fragments. In sum, our study elucidates the molecular mechanism governing the biogenesis and function of Myrf N-terminal fragments and its physiological significance.

Characterization of Fabry mice treated with recombinant adeno-associated virus 2/8-mediated gene transfer.

BACKGROUND: Enzyme replacement therapy (ERT) with alpha-galactosidase A (alpha-Gal A) is currently the most effective therapeutic strategy for patients with Fabry disease, a lysosomal storage disease. However, ERT has limitations of a short half-life, requirement for frequent administration, and limited efficacy for patients with renal failure. Therefore, we investigated the efficacy of recombinant adeno-associated virus (rAAV) vector-mediated gene therapy for a Fabry disease mouse model and compared it with that of ERT. METHODS: A pseudotyped rAAV2/8 vector encoding alpha-Gal A cDNA (rAAV2/8-hAGA) was prepared and injected into 18-week-old male Fabry mice through the tail vein. The alpha-Gal A expression level and globotriaosylceramide (Gb3) levels in the Fabry mice were examined and compared with Fabry mice with ERT. Immunohistochemical and ultrastructural studies were conducted. RESULTS: Treatment of Fabry mice with rAAV2/8-hAGA resulted in the clearance of accumulated Gb3 in tissues such as liver, spleen, kidney, heart, and brain with concomitant elevation of alpha-Gal A enzyme activity. Enzyme activity was elevated for up to 60 weeks. In addition, expression of the alpha-Gal A protein was identified in the presence of rAAV2/8-hAGA at 6, 12, and 24 weeks after treatment. alpha-Gal A activity was significantly higher in the mice treated with rAAV2/8-hAGA than in Fabry mice that received ERT. Along with higher alpha-Gal A activity in the kidney of the Fabry mice treated with gene therapy, immunohistochemical studies showed more alpha-Gal A expression in the proximal tubules and glomerulus, and less Gb3 deposition in Fabry mice treated with this gene therapy than in mice given ERT. The alpha-gal A gene transfer significantly reduced the accumulation of Gb3 in the tubules and podocytes of the kidney. Electron microscopic analysis of the kidneys of Fabry mice also showed that gene therapy was more effective than ERT. CONCLUSIONS: The rAAV2/8-hAGA mediated alpha-Gal A gene therapy provided improved efficiency over ERT in the Fabry disease mouse model. Furthermore, rAAV2/8-hAGA-mediated expression showed a greater effect in the kidney than ERT.

Tissue-specific activation of mitogen-activated protein kinases for expression of transthyretin by phenylalanine and its metabolite, phenylpyruvic acid.

Phenylketonuria is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase. Transthyretin has been implicated as an indicator of nutritional status in phenylketonuria patients. In this study, we report that phenylalanine and its metabolite, phenylpyruvic acid, affect MAPK, changing transthyretin expression in a cell- and tissue-specific manner. Treatment of HepG2 cells with phenylalanine or phenylpyruvic acid decreased transcription of the TTR gene and decreased the transcriptional activity of the TTR promoter site, which was partly mediated through HNF4alpha. Decreased levels of p38 MAPK were detected in the liver of phenylketonuria-affected mice compared with wild-type mice. In contrast, treatment with phenylalanine increased transthyretin expression and induced ERK1/2 activation in PC-12 cells; ERK1/2 activation was also elevated in the brainstem of phenylketonuria-affected mice. These findings may explain between-tissue differences in gene expression, including Ttr gene expression, in the phenylketonuria mouse model.

Expression of genes and their responses to enzyme replacement therapy in a Fabry disease mouse model.

Fabry disease is a lysosomal storage disease caused by a deficiency of alpha-galactosidase A, which results in aberrant glycosphingolipid metabolism and accumulation of globotriaosylceramide (Gb3). Since a correlation between the level of Gb3 and clinical manifestations of Fabry disease has not been observed, we investigated potential diagnostic biomarkers. Hepatic and renal gene expression of male alpha-galactosidase A-deficient mice (Fabry mice) was compared with that of wild-type mice. Microarray analyses were performed using samples taken before and after intravenous infusion of alpha-galactosidase A. The identified genes were validated using quantitative real-time PCR and Western blot assay. Expression of hepatic Serum Amyloid A1 (Saa1), S100 Calcium-binding protein A8 and A9 (S100a8 and a9), and Lipocalin 2 (Lcn2) and renal Neuropeptide Y (Npy), Thrombospondin 2 and 4 (Tsp-2 and -4) was significantly upregulated in Fabry mice compared with wild-type mice and normalized by enzyme replacement therapy. Plasma concentrations of Lcn2 and Npy were also greater in Fabry mice and reduced to wild-type levels after enzyme replacement therapy, although the plasma concentrations of these proteins show heterogeneity. Upregulation of Saa1, S100a8, S100a9 and Lcn2 may modulate inflammation and Lcn2, Npy and Tsp may be associated with vascular and renal involvement in Fabry disease. Furthermore, these genes are promising targets for developing biomarkers for monitoring disease progression and therapeutic efficacy in patients with Fabry disease.

Protective effect of recombinant adeno-associated virus 2/8-mediated gene therapy from the maternal hyperphenylalaninemia in offsprings of a mouse model of phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pahenu2, by the AAV serotype 2-mediated gene delivery of a human PAH transgene. However, the therapeutic efficacy had been limited to only male PKU mice. In this study, we generated a pseudotyped recombinant AAV2/8-hPAH vector and infused it into female PKU mice through the hepatic portal vein or tail vein. Two weeks after injection, complete fur color change to black was observed in female PKU, as in males. The PAH activity in the liver increased to 65-70% of the wild-type activity in female PKU mice and to 90% in male PKU mice. Plasma phenylalanine concentration in female PKU mice decreased to the normal value. In addition, the offsprings of the treated female PKU mice can rescue from the harmful effect of maternal hyperphenylalaninemia. These results indicate that recombinant AAV2/8-mediated gene therapy is a potential therapeutic strategy for PKU.

Migration of styrene monomer, dimers and trimers from polystyrene to food simulants.

Migration experiments with polystyrene were performed in two-sided contact with n-heptane and distilled water as the food simulants at temperatures of 10, 24 and 40, and 40, 60 and 90 degrees C, respectively. The surface/volume ratios in the migration cell were set at 8.04 and 10.05 dm2 l(-1) for n-heptane and distilled water, respectively. Styrene monomer, styrene dimers and styrene trimers migrating to the food simulants were determined by GC-FID analysis. Heptane fully extracted the styrene monomer and the oligomers from the polystyrene sheet, whereas in the distilled water only the migrated styrene trimers could be detected. To determine the apparent diffusion coefficient, the migration process was analysed based on Fick's law. The higher the molecular weight of the oligomers, the more significant the reduction in the diffusion coefficient. Higher molecular weight oligomers also had lower activation energy of diffusion when the temperature dependence of diffusivity was analysed by the Arrhenius equation. The diffusion coefficient of the trimers was much higher for heptane contact than for water. The activation energy of the diffusion of trimers for water contact was higher than that for heptane.

Migration of surrogate contaminants in paper and paperboard into water through polyethylene coating layer.

The migration of five surrogate contaminants, anthracene, benzophenone, dimethyl phthalate, methyl stearate and pentachlorophenol, from paper and paperboard into water through a polyethylene (PE) coating layer was investigated. Virgin paper and paperboard coated with PE films of 0.012 and 0.030 mm thickness were spiked evenly with standard 1-ml solutions containing 5mg of each surrogate. The spiked papers were placed in contact with the PE coating layer at 10 and 24 degrees C for 21 days. The resulting surrogate migration through the PE layer into 100ml water was measured by an analytical method developed here that used gas chromatography equipped with a flame ionization detector. Non-polar surrogates of anthracene and methyl stearate did not show any significant migration. In the case of the thin layer coating of 0.012 mm, polar water-soluble contaminants of benzophenone, dimethyl phthalate and pentachlorophenol showed an equilibrated or maximized migration after 1 day, even at a refrigerated temperature of 10 degrees C. A thick PE coating of 0.030 mm thickness delayed the progress of contaminant migration, which was also slower at lower temperature. Our results indicate that polyethylene coatings should not be seen as a complete barrier against possible contaminants in paper packaging materials under chilled or ambient conditions. Several variables such as coating thickness, temperature and suspected contaminants need to be considered to control the possible contamination risk from recycled or printed paper.