The novel aminoglycoside, ELX-02, permits CTNSW138X translational read-through and restores lysosomal cystine efflux in cystinosis.

BACKGROUND: Cystinosis is a rare disorder caused by recessive mutations of the CTNS gene. Current therapy decreases cystine accumulation, thus slowing organ deterioration without reversing renal Fanconi syndrome or preventing eventual need for a kidney transplant.15-20% of cystinosis patients harbour at least one nonsense mutation in CTNS, leading to premature end of translation of the transcript. Aminoglycosides have been shown to permit translational read-through but have high toxicity level, especially in the kidney and inner ear. ELX-02, a modified aminoglycoside, retains it read-through ability without the toxicity. METHODS AND FINDINGS: We ascertained the toxicity of ELX-02 in cells and in mice as well as the effect of ELX-02 on translational read-through of nonsense mutations in cystinotic mice and human cells. ELX-02 was not toxic in vitro or in vivo, and permitted read-through of nonsense mutations in cystinotic mice and human cells. CONCLUSIONS: ELX-02 has translational read-through activity and produces a functional CTNS protein, as evidenced by reduced cystine accumulation. This reduction is comparable to cysteamine treatment. ELX-02 accumulates in the kidney but neither cytotoxicity nor nephrotoxicity was observed.

Vascular patterning regulates interdigital cell death by a ROS-mediated mechanism.

Blood vessels serve as key regulators of organogenesis by providing oxygen, nutrients and molecular signals. During limb development, programmed cell death (PCD) contributes to separation of the digits. Interestingly, prior to the onset of PCD, the autopod vasculature undergoes extensive patterning that results in high interdigital vascularity. Here, we show that in mice, the limb vasculature positively regulates interdigital PCD. In vivo, reduction in interdigital vessel number inhibited PCD, resulting in syndactyly, whereas an increment in vessel number and distribution resulted in elevation and expansion of PCD. Production of reactive oxygen species (ROS), toxic compounds that have been implicated in PCD, also depended on interdigital vascular patterning. Finally, ex vivo incubation of limbs in gradually decreasing oxygen levels led to a correlated reduction in both ROS production and interdigital PCD. The results support a role for oxygen in these processes and provide a mechanistic explanation for the counterintuitive positive role of the vasculature in PCD. In conclusion, we suggest a new role for vascular patterning during limb development in regulating interdigital PCD by ROS production. More broadly, we propose a double safety mechanism that restricts PCD to interdigital areas, as the genetic program of PCD provides the first layer and vascular patterning serves as the second.

The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf.

Limb development constitutes a central model for the study of tissue and organ patterning; yet, the mechanisms that regulate the patterning of limb vasculature have been left understudied. Vascular patterning in the forming limb is tightly regulated in order to ensure sufficient gas exchange and nutrient supply to the developing organ. Once skeletogenesis is initiated, limb vasculature undergoes two seemingly opposing processes: vessel regression from regions that undergo mesenchymal condensation; and vessel morphogenesis. During the latter, vessels that surround the condensations undergo an extensive rearrangement, forming a stereotypical enriched network that is segregated from the skeleton. In this study, we provide evidence for the centrality of the condensing mesenchyme of the forming skeleton in regulating limb vascular patterning. Both Vegf loss- and gain-of-function experiments in limb bud mesenchyme firmly established VEGF as the signal by which the condensing mesenchyme regulates the vasculature. Normal vasculature observed in limbs where VEGF receptors Flt1, Flk1, Nrp1 and Nrp2 were blocked in limb bud mesenchyme suggested that VEGF, which is secreted by the condensing mesenchyme, regulates limb vasculature via a direct long-range mechanism. Finally, we provide evidence for the involvement of SOX9 in the regulation of Vegf expression in the condensing mesenchyme. This study establishes Vegf expression in the condensing mesenchyme as the mechanism by which the skeleton patterns limb vasculature.

HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis.

During early stages of limb development, the vasculature is subjected to extensive remodeling that leaves the prechondrogenic condensation avascular and, as we demonstrate hereafter, hypoxic. Numerous studies on a variety of cell types have reported that hypoxia has an inhibitory effect on cell differentiation. In order to investigate the mechanism that supports chondrocyte differentiation under hypoxic conditions, we inactivated the transcription factor hypoxia-inducible factor 1alpha (HIF1alpha) in mouse limb bud mesenchyme. Developmental analysis of Hif1alpha-depleted limbs revealed abnormal cartilage and joint formation in the autopod, suggesting that HIF1alpha is part of a mechanism that regulates the differentiation of hypoxic prechondrogenic cells. Dramatically reduced cartilage formation in Hif1alpha-depleted micromass culture cells under hypoxia provided further support for the regulatory role of HIF1alpha in chondrogenesis. Reduced expression of Sox9, a key regulator of chondrocyte differentiation, followed by reduction of Sox6, collagen type II and aggrecan in Hif1alpha-depleted limbs raised the possibility that HIF1alpha regulation of Sox9 is necessary under hypoxic conditions for differentiation of prechondrogenic cells to chondrocytes. To study this possibility, we targeted Hif1alpha expression in micromass cultures. Under hypoxic conditions, Sox9 expression was increased twofold relative to its expression in normoxic condition; this increment was lost in the Hif1alpha-depleted cells. Chromatin immunoprecipitation demonstrated direct binding of HIF1alpha to the Sox9 promoter, thus supporting direct regulation of HIF1alpha on Sox9 expression. This work establishes for the first time HIF1alpha as a key component in the genetic program that regulates chondrogenesis by regulating Sox9 expression in hypoxic prechondrogenic cells.

Free diffusion of steroid hormones across biomembranes: a simplex search with implicit solvent model calculations.

Steroid hormones such as progesterone, testosterone, and estradiol are derived from cholesterol, a major constituent of biomembranes. Although the hormones might be expected to associate with the bilayer in a fashion similar to that of cholesterol, their biological action in regulating transcription of target genes involves transbilayer transfer by free diffusion, which is not observed for cholesterol. We used a novel combination of a continuum-solvent model and the downhill simplex search method for the calculation of the free energy of interaction of these hormones with lipid membranes, and compared these values to that of cholesterol-membrane interaction. The hormones were represented in atomic detail and the membrane as a structureless hydrophobic slab embedded in implicit water. A deep free-energy minimum of approximately -15 kcal/mol was obtained for cholesterol at its most favorable location in the membrane, whereas the most favorable locations for the hormones were associated with shallower minima of -5.0 kcal/mol or higher. The free-energy difference, which is predominantly due to the substitution of cholesterol's hydrophobic tail with polar groups, explains the different manner in which cholesterol and the hormones interact with the membrane. Further calculations were conducted to estimate the rate of transfer of the hormones from the aqueous phase into hexane, and from hexane back into the aqueous phase. The calculated rates agreed reasonably well with measurements in closely related systems. Based on these calculations, we suggest putative pathways for the free diffusion of the hormones across biomembranes. Overall, the calculations imply that the hormones may rapidly cross biomembrane barriers. Implications for gastrointestinal absorption and transfer across the blood-brain barrier and for therapeutic uses are discussed.