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.

Degradome products of the matricellular protein CCN1 as modulators of pathological angiogenesis in the retina.

CCN1 is a matricellular protein involved in normal vascular development and tissue repair. CCN1 exhibits cell- and context-dependent activities that are reflective of its tetramodular structure phylogenetically linked to four domains found in various matrix proteins. Here, we show that vitreal fluids from patients with proliferative diabetic retinopathy (PDR) were enriched with a two-module form of CCN1 comprising completely or partially the insulin-like growth factor-binding protein (IGFBP) and von Willebrand factor type C (vWC) domains. The two- and three-module forms comprising, in addition to IGFBP and vWC, the thrombospondin type 1 (TSP1) repeats are CCN1 degradome products by matrix metalloproteinase-2 and -14. The functional significance of CCN1 and its truncated variants was determined in the mouse model of oxygen-induced retinopathy, which simulates neovascular growth associated with PDR and assesses treatment outcomes. In this model, lentivirus-mediated expression of either CCN1 or the IGFBP-vWC-TSP1 form reduced ischemia-induced neovascularization, whereas ectopic expression of the IGFBP-vWC variant exacerbated pathological angiogenesis. The IGFBP-vWC form has potent proangiogenic properties promoting retinal endothelial cell growth, migration, and three-dimensional tubular structure formation, whereas the IGFBP-vWC-TSP1 variant suppressed cell growth and angiogenic gene expression. Both IGFBP-vWC and IGFBP-vWC-TSP1 forms exhibited predictable variations of their domain folding that enhanced their functional potential. These data provide new insights into the formation and activities of CCN1-truncated variants and raise the predictive value of the form containing completely or partially the IGFBP and vWC domains as a surrogate marker of CCN1 activity in PDR distinguishing pathological from physiological angiogenesis.

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.

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.

Leigh Syndrome Mimicking Wernicke's Encephalopathy: A Case Report.

Leigh syndrome or subacute necrotizing encephalomyelopathy is a rare, rapidly progressive neurodegenerative disorder. In general, symptoms such as shortness of breath and decreased cardiac function usually occur within 1 year of life. It is a serious disease with a mortality rate of 75% in 2-3 years. The cause of Leigh syndrome is DNA mutation. Approximately 75% of patients have nuclear DNA mutations while 25% have mitochondrial DNA mutations. Clinical symptoms vary depending on the affected brain area. Neuroimaging plays an important role in diagnosing patients with Leigh syndrome. Late-onset Leigh syndrome is rarer and progresses more slowly compared to the classic form. Here, we report a case of late-onset Leigh's syndrome mimicking Wernicke's encephalopathy.

Intradural-extramedullary solitary fibrous tumor of the thoracic spine: A case report.

Spinal solitary fibrous tumors are extremely rare neoplasms and of those, intradural extramedullary location is even rarer. A 64-year-old male presented to the emergency department with worsening right leg pain over 1 month. Whole spine magnetic resonance imaging revealed a well-circumscribed mass with low T1 and markedly low T2 signal intensity at the level of T1-2. Spine computed tomography showed no evidence of calcification or acute hemorrhage. Surgical removal was performed and the final diagnosis was intradural extramedullary solitary fibrous tumor.

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.

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.

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.