Identification of a primary target of thalidomide teratogenicity.

Half a century ago, thalidomide was widely prescribed to pregnant women as a sedative but was found to be teratogenic, causing multiple birth defects. Today, thalidomide is still used in the treatment of leprosy and multiple myeloma, although how it causes limb malformation and other developmental defects is unknown. Here, we identified cereblon (CRBN) as a thalidomide-binding protein. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1) and Cul4A that is important for limb outgrowth and expression of the fibroblast growth factor Fgf8 in zebrafish and chicks. Thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting the associated ubiquitin ligase activity. This study reveals a basis for thalidomide teratogenicity and may contribute to the development of new thalidomide derivatives without teratogenic activity.

Thalidomide resistance is based on the capacity of the glutathione-dependent antioxidant defense.

Thalidomide as an effective treatment for multiple myeloma and leprosy has also caused birth defects in thousands of children five decades ago particularly in Europe. Thus its use in humans remains limited. The rapid and fatal approval of thalidomide at that time ultimately was a consequence of the sole use of thalidomide-insensitive species in animal toxicity tests. Here, we aimed at elucidating the molecular basis for the resistance of mice to thalidomide teratogenicity. By using hydroethidine staining we demonstrate that thalidomide induces the formation of superoxide in embryonic fibroblasts of thalidomide-sensitive species but not in those of mice. As determined by trypan blue staining, scavenging of superoxide prevents thalidomide-induced apoptosis, a marker for thalidomide teratogenicity. Mouse embryonic fibroblasts are found to have higher glutathione levels than those of sensitive species and can be sensitized for thalidomide by glutathione depletion with diethyl maleate or diamide. Accordingly, experimental increase of glutathione levels in human embryonic fibroblasts by adding N-acetyl cysteine or glutathione ethyl ester to the culture medium counteracts thalidomide-induced apoptosis. Finally, we show that thalidomide-induced molecular pathology downstream of superoxide is essentially identical in human and sensitized mouse embryonic fibroblasts. In conclusion, thalidomide-resistance is based on the capacity of the glutathione-dependent antioxidant defense. We provide a basis to pharmacologically overcome the limitations of thalidomide use at humans and describe substantial differences between human and mouse embryonic cells regarding the protection against oxidative stress.

Early development of the optic nerve in the turtle Mauremys leprosa.

We show the distribution of the neural and non-neural elements in the early development of the optic nerve in the freshwater turtle, Mauremys leprosa, using light and electron microscopy. The first optic axons invaded the ventral periphery of the optic stalk in close relationship to the radial neuroepithelial processes. Growth cones were thus exclusively located in the ventral margin. As development progressed, growth cones were present in ventral and dorsal regions, including the dorsal periphery, where they intermingled with mature axons. However, growth cones predominated in the ventral part and axonal profiles dorsally, reflecting a dorsal to ventral gradient of maturation. The size and morphology of growth cones depended on the developmental stage and the region of the optic nerve. At early stages, most growth cones were of irregular shape, showing abundant lamellipodia. At the following stages, they tended to be larger and more complex in the ventral third than in intermediate and dorsal portions, suggesting a differential behavior of the growth cones along the ventro-dorsal axis. The arrival of optic axons at the optic stalk involved the progressive transformation of neuroepithelial cells into glial cells. Simultaneously with the fiber invasion, an important number of cells died by apoptosis in the dorsal wall of the optic nerve. These findings are discussed in relation to the results described in the developing optic nerve of other vertebrates.

Developmental changes in the fibre population of the optic nerve follow an avian/mammalian-like pattern in the turtle Mauremys leprosa.

The changes in the axon and growth cone numbers in the optic nerve of the freshwater turtle Mauremys leprosa were studied by electron microscopy from the embryonic day 14 (E14) to E80, when the animals normally hatch, and from the first postnatal day (P0) to adulthood (5 years on). At E16, the first axons appeared in the optic nerve and were added slowly until E21. From E21, the fibre number increased rapidly, peaking at E34 (570,000 fibres). Thereafter, the axon number decreased sharply, and from E47 declined steadily until reaching the mature number (about 330,000). These observations indicated that during development of the retina there was an overproduction and later elimination of retinal ganglion cells. Growth cones were first observed in the optic nerve at as early as E16. Their number increased rapidly until E21 and continued to be high through E23 and E26. After E26, the number declined steeply and by E40 the optic nerve was devoid of growth cones. These results indicated that differentiation of the retinal ganglion cells occurred during the first half of the embryonic life. To examine the correlation between the loss of the fibres from the optic nerve and loss of the parent retinal ganglion cells, retinal sections were processed with the TUNEL technique. Apoptotic nuclei were detected in the ganglion cell layer throughout the period of loss of the optic fibres. Our results showed that the time course of the numbers of the fibres in the developing turtle optic nerve was similar to those found in birds and mammals.