Stability of small non-coding RNA reference gene expression in the rat retina during exposure to cyclic hyperoxia

Purpose: Oxygen-induced retinopathy (OIR) is a robust animal model of human retinopathy of prematurity that readily allows changes in retinal gene and microRNA (miRNA) expression in response to fluctuations in oxygen levels to be studied. We sought to identify small non-coding RNA (ncRNA) genes that showed stable expression upon exposure to varying levels of oxygen, with different developmental stages and in different rat strains, to act as reference genes for normalizing miRNA expression in a rat model of OIR. Methods: Expression of five small ncRNAs (U6 snRNA, miR-16, U87, 4.5S RNA (H) “Variant 1”, and 5S ribosomal RNA [rRNA]) were tested on a standard RNA pool and representative retinal samples from P5, P6, P9, and P14 from room air– and cyclic hyperoxia–exposed rats using reverse transcription (RT)-qPCR, to assess the effect of developmental stage and exposure to fluctuations in oxygen levels, respectively. Two strains of inbred albino rats, Fischer 344 (F344, resistant to OIR) and Sprague-Dawley rats (SD, susceptible to OIR), were used to assess the effect of rat strain on the stability of the small ncRNAs. Results: In this rat model of OIR, 5S rRNA expression was variable with strain, fluctuations in oxygen levels, and developmental stage. U6 snRNA was stably expressed with changes in oxygen levels, and minimal variation was observed with strain and developmental stage. MiR-16 showed less stable expression with changes in oxygen levels and between strains compared to U6 snRNA. Some variation in expression in response to developmental stage was also observed. The PCR amplification efficiencies of the U6 snRNA and miR-16 TaqMan assays were 56% and 78%, respectively. U87 and 4.5S RNA (H) “Variant 1” expression varied with strain, exposure to cyclic hyperoxia, and in particular developmental stage, and was at low levels in the neonatal rat retina. Conclusions: We conclude that U6 snRNA and miR-16 are the most suitable reference RNAs for normalizing miRNA expression, as they are relatively stable with strain, exposure to cyclic hyperoxia, and developmental stage in a rat model of OIR.The work presented was supported by the National Health and Medical Research Council (NHMRC) of Australia and the Ophthalmic Research Institute of Australia (ORIA)

Gene expression microarray analysis of early oxygen-induced retinopathy in the rat

Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague–Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague–Dawley list returned the term “response to hypoxia,” absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1α were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat

Targeting the Sphingolipid System as a Therapeutic Direction for Glioblastoma

Glioblastoma (GBM) is the most commonly diagnosed malignant brain tumor in adults. The prognosis for patients with GBM remains poor and largely unchanged over the last 30 years, due to the limitations of existing therapies. Thus, new therapeutic approaches are desperately required. Sphingolipids are highly enriched in the brain, forming the structural components of cell membranes, and are major lipid constituents of the myelin sheaths of nerve axons, as well as playing critical roles in cell signaling. Indeed, a number of sphingolipids elicit a variety of cellular responses involved in the development and progression of GBM. Here, we discuss the role of sphingolipids in the pathobiology of GBM, and how targeting sphingolipid metabolism has emerged as a promising approach for the treatment of GBM

A drug screening pipeline using 2D and 3D patient-derived in vitro models for pre-clinical analysis of therapy response in glioblastoma

Glioblastoma is one of the most common and lethal types of primary brain tumor. Despite aggressive treatment with chemotherapy and radiotherapy, tumor recurrence within 6–9 months is common. To overcome this, more effective therapies targeting cancer cell stemness, invasion, metabolism, cell death resistance and the interactions of tumor cells with their surrounding microen-vironment are required. In this study, we performed a systematic review of the molecular mechanisms that drive glioblastoma progression, which led to the identification of 65 drugs/inhibitors that we screened for their efficacy to kill patient-derived glioma stem cells in two dimensional (2D) cul-tures and patient-derived three dimensional (3D) glioblastoma explant organoids (GBOs). From the screening, we found a group of drugs that presented different selectivity on different patient-derived in vitro models. Moreover, we found that Costunolide, a TERT inhibitor, was effective in reducing the cell viability in vitro of both primary tumor models as well as tumor models pre-treated with chemotherapy and radiotherapy. These results present a novel workflow for screening a relatively large groups of drugs, whose results could lead to the identification of more personalized and effective treatment for recurrent glioblastoma.</p

A Neurogenic Perspective of Sarcopenia: Time Course Study of Sciatic Nerves From Aging Mice

To elucidate the neural basis for age-related sarcopenia, we quantified morphologic and molecular changes within sciatic nerves of aging male and female C57BL/6J mice aged between 3 and 27 months using immunoblotting, immunohistochemistry, and electron microscopy. Protein analyses by immunoblotting of nerves of male mice aged 4, 15, 18, 22, and 24 months showed increased levels of heavy chain SMI-32-positive neurofilaments, vimentin, tau5, choline acetyltransferase (ChAT), and p62 by 18–22 months. Similar protein increases were seen in 26-month-old compared with 3-month-old female mice. Immunostaining of longitudinal sections of old (27-month-old) male sciatic nerves revealed intense staining for tau5 and p62 that was increased compared with that at 3 months, but there were decreased numbers of axon profiles stained for ChAT or isolectin B4 (motor and sensory axons, respectively). Ultrastructural analysis revealed electron-dense aggregates within axons in peripheral nerves of old male mice; the proportion of axons that contained aggregates more than doubled between 15 and 27 months. Overall, the observed age-related accumulation of many proteins from about 18 months of age onward suggests impaired mechanisms for axonal transport and protein turnover. These peripheral nerve changes may contribute to the morphological and functional muscle deficits associated with sarcopenia