Expression of Ext1, Ext2, and heparanase genes in brain of senescence-accelerated OXYS rats in early ontogenesis and during development of neurodegenerative changes

Abstract

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) play a significant role in brain development, and their structural and quantitative changes are revealed during aging and in neurodegenerative disorders. The mechanism of these changes is not clear, but is likely to be associated with alteration in the expression and/or activity of enzymes responsible for HSPG biosynthesis and degradation. The contents of mRNAs of the genes Ext1 and Ext2 encoding polymerization enzymes and of gene Hpse of heparanase degrading HS were determined in the brain of prematurely aging OXYS rats during early postnatal development and during appearance of signs of brain accelerated aging (at age of 1, 7, 14, 30, 60, and 420 days). Wistar rats of the same age were used as controls. Expression levels of the genes Ext1, Ext2, and Hpse in the brain of rats of both strains were maximal during the two first weeks of life, and the contents of mRNAs of all genes in the brain of newborn and 7-day-old OXYS rats were significantly higher than in Wistar rats. By the 14th day of life the differences leveled, but at the age of 30 days on the background of a decrease in the contents of mRNAs of Ext1, Ext2, and Hpse in OXYS rats they became more pronounced (three-, four-, and twofold, respectively). Differences between the strains were absent at the age of 60 days and 14 months, and expression of all the genes was significantly lower than in the newborn animals. A strong positive correlation was found between contents of mRNAs of all the studied genes, and this suggested that heparanase should be involved in HSPG metabolism together with Ext1 and Ext2. Based on these and earlier findings, we conclude that development of the OXYS rat brain occurs on the background of significant alterations in HSPG metabolism that precede the development of neurodegenerative manifestations recently detected by magnetic resonance imaging.

Old gene, new phenotype: mutations in heparan sulfate synthesis enzyme, EXT2 leads to seizure and developmental disorder, no exostoses

“…Glycosylation is an essential, ubiquitous and evolutionarily conserved process required for cellular function and development 16 17. Not surprisingly, defects in genes encoding heparan sulfate biosynthesis enzymes can lead to a wide spectrum of human disorders including bone abnormalities, cancer, metabolic disorders and even late-onset neurodegeneration 16 18–21.…”

“…This is due to normal expression of the other EXT protein, which still has glycosyltransferase activity although significantly less than the amount generated by the EXT1and EXT2 complex as the enzymes cannot substitute for one another during co-polymerisation 9 38–40. Therefore, while residual levels of heparan sulfate facilitate some cellular functions, they are not sufficient for proper neural development including brain morphology and midline axon guidance—processes defective in Ext-mutants 17 18. Collectively, these studies have demonstrated the importance of sufficient and spatiotemporal expression of heparan sulfate biosynthesis enzymes in the development of brain size and structure.…”

MS2 phage ribonucleoproteins as exogenous internal control for RT-qPCR data normalization in gene expression study of developing rat brain

“…The mRNA was extracted from pre weighed homogenized whole brains using the TRI REAGENT kit (GIBCO/Life Technologies, USA) according to the manufacturer's protocol. Ribonucleoprotein particles of recombinant phage MS2 containing an artificial SNK sequence (Vector Best, Russia) were added at the stage of homoge nization in amounts proportional to the weight of the brain tissue (4·10 7 particles per gram of brain tissue) [19]. After RNA isolation, an additional deproteinization was performed using phenol-chloroform (1 : 1) extraction followed by chloroform extraction to remove the residual phenol.…”

“…The mechanism of these changes is probably associated with change in the expression and/or activity of enzymes responsible for HSPG biosynthesis and degradation (such as exostosins and heparanase). Our goal was to determine Ext1, Ext2 (genes of exostosins 1 and 2), and Hpse (gene of heparanase) mRNA levels in the brain of senescence accelerated OXYS rats during early postnatal development and during appearance of signs of accelerated brain aging (at the age of 1, 7, 14, 30, 60, and 420 days) [19].…”

“…Our previous studies of HSPG biosynthesis and degradation genes are described in detail elsewhere [19]. Initially, for normalization of the RT qPCR data we per formed a traditional search for a reference gene that would exhibit a stable level of expression from the first days of animal life and throughout maturity.…”