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Background/Aims: Runt-related transcription factor 2 (Runx2) is a master regulator of osteogenic differentiation, but most of the direct downstream targets of RUNX2 during osteogenesis are unknown. Likewise, High-temperature requirement factor A1 (HTRA1) is a serine protease expressed in bone, yet the role of Htra1 during osteoblast differentiation remains elusive. We investigated the role of Htra1 in osteogenic differentiation and the transcriptional regulation of Htra1 by RUNX2 in primary mouse mesenchymal progenitor cells. Methods: Overexpression of Htra1 was carried out in primary mouse mesenchymal progenitor cells to evaluate the extent of osteoblast differentiation. Streptavidin agarose pulldown assay, chromatin immunoprecipitation assay, and dual luciferase assay were carried out to investigate the interaction of RUNX2 protein at the Htra1 promoter during osteoblast differentiation. Results: Overexpression of Htra1 increased the production of mineralized bone matrix, upregulating several osteoblast genes, such as Sp7 transcription factor (Sp7) and Alkaline phosphatase, liver/bone/kidney (Alpl). In addition, Htra1 upregulated osteogenesis-related signalling genes, such as Fibroblast growth factor 9 (Fgf9) and Vascular endothelial growth factor A (Vegfa). A series of experiments confirmed Htra1 as a direct RUNX2 transcriptional target. Overexpression of Runx2 resulted in the upregulation of Htra1 mRNA and protein. Chromatin immunoprecipitation and streptavidin agarose pull-down assays showed that RUNX2 binds a proximal -400 bp region of the Htra1 promoter during osteogenic differentiation. Dual luciferase assays confirmed that RUNX2 activates the proximal Htra1 promoter during osteogenic differentiation. Mutation of putative RUNX2 binding sites revealed that RUNX2 interacts with the Htra1 promoter at -252 bp and -84 bp to induce Htra1 expression. Conclusion: We demonstrate that Htra1 is a positive regulator of osteogenic differentiation, showing for the first time that Htra1 is a direct downstream target of RUNX2. [ABSTRACT FROM AUTHOR]

Sirtuin2 (SIRT2) is a deacetylase enzyme predominantly expressed in myelinating glia of the central nervous system (CNS). We have previously demonstrated that Sirt2 expression enhances oligodendrocyte (OL) differentiation and arborization in vitro, but the molecular targets of SIRT2 in OLs remain speculative. SIRT2 has been implicated in cholesterol biosynthesis by promoting the nuclear translocation of sterol regulatory element binding protein (SREBP)-2. We investigated this further in CNS myelination by examining the role of Sirt2 in cholesterol biosynthesis in vivo and in vitro employing Sirt2 mice, primary OL cells and CG4-OL cells. Our results demonstrate that expression of cholesterol biosynthetic genes in the CNS white matter or cholesterol content in purified myelin fractions did not differ between Sirt2 and age-matched wild-type mice. Cholesterol biosynthetic gene expression profiles and total cholesterol content were not altered in primary OLs from Sirt2 mice and in CG4-OLs when Sirt2 was either down-regulated with RNAi or overexpressed. In addition, Sirt2 knockdown or overexpression in CG4-OLs had no effect on SREBP-2 nuclear translocation. Our results indicate that Sirt2 does not impact the expression of genes encoding enzymes involved in cholesterol biosynthesis, total cholesterol content, or nuclear translocation of SREBP-2 during OL differentiation and myelination. [ABSTRACT FROM AUTHOR]

Experimental models of multiple sclerosis (MS) have significantly advanced our understanding of pathophysiology and therapeutic interventions. Although in vivo rodent models are considered to most closely represent the complex cellular and molecular disease states of the human central nervous system (CNS), these can be costly to maintain and require long timelines. Organotypic slice cultures maintain the cytotypic organization observed in the intact CNS, yet provide many of the experimental advantages of in vitro cell culture models. Cerebellar organotypic cultures have proven useful for studying myelination and remyelination, but this model has only been established using early postnatal tissue. This young brain tissue allows for neuro development ex vivo to mimic the 'mature' CNS; however, there are many differences between postnatal and adult organotypic cultures. This may be particularly relevant to MS, as a major barrier to myelin regeneration is age. This paper describes a modified protocol to study demyelination and remyelination in adult cerebellar tissue, which has been used to demonstrate neuroprotection with omega-3 fatty acids. Thus, adult cerebellar organotypic cultures provide a novel ex vivo platform for screening potential therapies in myelin degeneration and repair. [ABSTRACT FROM AUTHOR]