Overexpression of HTRA1 Leads to Ultrastructural Changes in the Elastic Layer of Bruch's Membrane via Cleavage of Extracellular Matrix Components

Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruch's membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3

Increased BMI is Associated with School Absenteeism Among US School Aged Children and Adolescents

Objective: Severe school absence may be one underlying cause of poor school performance among overweight and obese children. We examined the associations between school absenteeism and body mass index (BMI) in a nationally representative sample of children. Methods and Procedures: We analyzed the data of 1,387 children (6-11 y) and 2,185 adolescents (12-19 y), who completed an interview and anthropometric measurement as a part of the National Health and Nutrition Examination Survey, 2005-2008. CDC 2000 growth chart was used to categorize BMI status, and school days missed during the past 12 months was assessed by asking the proxies or interviewees. Results: The prevalence of obese and overweight were 18.96 (SE=1.44), and 16.41(0.78)% respectively among study populations. The means of school days missed in the last 12 months were not statistically different between normal weight, overweight and obese groups, 3.79 (SE=0.56), 3.86 (0.38) and 4.31 (0.01) days respectively. However, when more than 2 days missed per school month was defined as severe absenteeism, the prevalence of severe absenteeism were 1.57%, 2.99% and 4.94% respectively among 6- 11 years old children with normal, overweight and obese. The adjusted odds of being severe absentee were 2.18 (95% CI = 0.61-7.73) and 3.79 (1.45-9.91) respectively among overweight and obese children compared to normal weight peers (p for trend test \u3c 0.01). No significant association was found among adolescents. Conclusion: Increased body weight is independently associated with severe absenteeism

Synonymous variants in HTRA1 implicated in AMD susceptibility impair its capacity to regulate TGF-β signaling

High-temperature requirement A1 (HTRA1) is a secreted serine protease reported to play a role in the development of several cancers and neurodegenerative diseases. Still, the mechanism underlying the disease processes largely remains undetermined. In age-related macular degeneration (AMD), a common cause of vision impairment and blindness in industrialized societies, two synonymous polymorphisms (rs1049331:C>T, and rs2293870:G>T) in exon 1 of the HTRA1 gene were associated with a high risk to develop disease. Here, we show that the two polymorphisms result in a protein with altered thermophoretic properties upon heat-induced unfolding, trypsin accessibility and secretion behavior, suggesting unique structural features of the AMD-risk-associated HTRA1 protein. Applying MicroScale Thermophoresis and protease digestion analysis, we demonstrate direct binding and proteolysis of transforming growth factor β1 (TGF-β1) by normal HTRA1 but not the AMD-risk-associated isoform. As a consequence, both HTRA1 isoforms strongly differed in their ability to control TGF-β mediated signaling, as revealed by reporter assays targeting the TGF-β1-induced serpin peptidase inhibitor (SERPINE1, alias PAI-1) promoter. In addition, structurally altered HTRA1 led to an impaired autocrine TGF-β signaling in microglia, as measured by a strong down-regulation of downstream effectors of the TGF-β cascade such as phosphorylated SMAD2 and PAI-1 expression. Taken together, our findings demonstrate the effects of two synonymous HTRA1 variants on protein structure and protein interaction with TGF-β1. As a consequence, this leads to an impairment of TGF-β signaling and microglial regulation. Functional implications of the altered properties on AMD pathogenesis remain to be clarified