Etifoxine improves peripheral nerve regeneration and functional recovery.

Peripheral nerves show spontaneous regenerative responses, but recovery after injury or peripheral neuropathies (toxic, diabetic, or chronic inflammatory demyelinating polyneuropathy syndromes) is slow and often incomplete, and at present no efficient treatment is available. Using well-defined peripheral nerve lesion paradigms, we assessed the therapeutic usefulness of etifoxine, recently identified as a ligand of the translocator protein (18 kDa) (TSPO), to promote axonal regeneration, modulate inflammatory responses, and improve functional recovery. We found by histologic analysis that etifoxine therapy promoted the regeneration of axons in and downstream of the lesion after freeze injury and increased axonal growth into a silicone guide tube by a factor of 2 after nerve transection. Etifoxine also stimulated neurite outgrowth in PC12 cells, and the effect was even stronger than for specific TSPO ligands. Etifoxine treatment caused a marked reduction in the number of macrophages after cryolesion within the nerve stumps, which was rapid in the proximal and delayed in the distal nerve stumps. Functional tests revealed accelerated and improved recovery of locomotion, motor coordination, and sensory functions in response to etifoxine. This work demonstrates that etifoxine, a clinically approved drug already used for the treatment of anxiety disorders, is remarkably efficient in promoting acceleration of peripheral nerve regeneration and functional recovery. Its possible mechanism of action is discussed, with reference to the neurosteroid concept. This molecule, which easily enters nerve tissues and regulates multiple functions in a concerted manner, offers promise for the treatment of peripheral nerve injuries and axonal neuropathies.

Translocator protein (18 kDa) is involved in primitive erythropoiesis in zebrafish.

The translocator protein (18 kDa) (TSPO), also known as peripheral-type benzodiazepine receptor, is directly or indirectly associated with many biological processes. Although extensively characterized, the specific function of TSPO during development remains unclear. It has been reported that TSPO is involved in a variety of mechanisms, including cell proliferation, apoptosis, regulation of mitochondrial functions, cholesterol transport and steroidogenesis, and porphyrin transport and heme synthesis. Although the literature has reported a murine knockout model, the experiment did not generate information because of early lethality. We then used the zebrafish model to address the function of tspo during development. Information about spatiotemporal expression showed that tspo has a maternal and a zygotic contribution which, during somatogenesis, seems to be erythroid restricted to the intermediate cell mass. Genetic and pharmacological approaches used to invalidate Tspo function resulted in embryos with specific erythropoietic cell depletion. Although unexpected, this lack of blood cells is independent of the Tspo cholesterol binding site and reveals a new in vivo key role for Tspo during erythropoiesis.

Grafts of brain-derived neurotrophic factor and neurotrophin 3-transduced primate Schwann cells lead to functional recovery of the demyelinated mouse spinal cord.

Experimental studies provided overwhelming proof that transplants of myelin-forming cells achieve efficient remyelination in the CNS. Among cellular candidates, Schwann cells can be used for autologous transplantation to ensure robust remyelination of lesions and to deliver therapeutic factors in the CNS. In the present study, macaque Schwann cells expressing green fluorescent protein (GFP) were infected with human immunodeficiency virus-derived vectors overexpressing brain-derived neurotrophic factor (BDNF) or Neurotrophin 3 (NT-3), two neurotrophins that also modulate glial cell biology. The ability of transgenic Schwann cells to secrete growth factors was assessed by ELISA and showed 35- and 62-fold increases in BDNF and NT-3, respectively, in transduced macaque Schwann cell supernatants. Conditioned media of BDNF- and NT-3-transduced Schwann cells reduced Schwann cell proliferation and favored their differentiation in vitro. Transgenic cells were grafted in demyelinated spinal cords of adult nude mice. Two behavioral assays showed that NT-3- and BDNF-transduced Schwann cells promoted faster and stronger functional recovery than GFP-transduced Schwann cells. Morphological analysis indicated that functional recovery correlated with enhanced proliferation and differentiation of resident oligodendrocyte progenitors and enhanced oligodendrocyte and Schwann cell differentiation. Moreover, NT-3-transduced Schwann cells provided neuroprotection and reduced astrogliosis. These results underline the potential therapeutic benefit of combining neuroprotection and activation of myelin-forming cells to restore altered functions in demyelinating diseases of the CNS.

Expression and functional state of the corticosteroid receptors and 11 beta-hydroxysteroid dehydrogenase type 2 in Schwann cells.

To investigate the role of steroid receptors in mediating the reported effects of steroids on Schwann cell (SC) myelination and growth, we determined mRNA contents and transcriptional activities of the corticosteroid (glucocorticosteroid and mineralocorticosteroid) receptors (GR and MR) and sex steroid (progesterone, androgen, and estrogen alpha and beta) receptors in rat SC cultured under proliferative (in the presence of insulin and forskolin, which induces a high intracellular cAMP content) and quiescent conditions. We found no or very low expression and activity of the sex steroid receptors, as shown by mRNA concentrations determined with real-time PCR and transcriptional activities using transient expression of reporter plasmids in SC. These data and binding studies in SC lines demonstrated that the levels of the sex steroid receptors were the limiting factors. GR was clearly expressed (approximately 8000 sequences/ng total RNA) and functional. No significant modification in GR mRNA levels was observed, but an increase in transcriptional efficiency was recorded in proliferating cells compared with quiescent cells. MR was also significantly expressed at the mRNA level (approximately 450 sequences/ng total RNA) under the two culture conditions. No MR transcriptional activity was observed in SC, but a low specific binding of aldosterone was detected in SC lines. 11 beta-Hydroxysteroid-dehydrogenase type 2 (HSD2), an enzyme that inactivates glucocorticoids, was strongly expressed and active in quiescent SC, although in proliferating cells, HSD2 exhibited a strong decrease in activity and mRNA concentration. These data support a physiological role for HSD2 regulation of glucocorticosteroid concentrations in nerve SC.

The cAMP-responsive unit of the human insulin-like growth factor-binding protein-1 coinstitutes a functional insulin-response element.

Insulin-like growth factor-binding protein-1 (IGFBP-1) is one of the genes involved in glucose homeostasis. In vivo, its level is increased by counter-regulatory hormones (glucocorticoids and glucagon via its second messenger cAMP) and decreased by insulin, these variations being primarily correlated with IGFBP-1 gene transcription. Previous reports described a functional insulin response element (IRE), immediately 5'- to the glucocorticoid response element (GRE). This IRE has been shown to mediate partial inhibition (1) of basal IGFBP-1 promoter activity and (2) of glucocorticoid-induced stimulation of gene transcription by insulin. In this work, using human HepG2 hepatoma cells as a model system, we showed: (1) that insulin inhibited both basal and cAMP-induced hIGFBP-1 promoter (nt-1 to -341) activity; (2) that in the absence of insulin, forkhead box class O (FOXO) transcription factors enhance constitutive hIGFBP-1 promoter activity without interfering with the stimulatory effect of cAMP; (3) that PI-3' kinase signaling is involved in the inhibition of constitutive and cAMP-induced promoter activities by insulin; (4) that wild-type FOXO-1 mediates the inhibitory effect of insulin on the promoter, although FOXO-1(Ala3), a nonphosphorylatable mutant of FOXO-1, does not; (5) that the cAMP-responsive unit (CRU), that includes a putative IRE (nt-265 to -282) and a cAMP responsive element (CRE; nt-258 to -263), is sufficient per se to mediate both cAMP stimulation of a heterologous promoter, and inhibition of both basal and cAMP-induced promoter activities by insulin; and (6) that the inhibitory effects of insulin on the isolated CRU are mediated by the FOXOs. This study is the first evidence for the occurrence of a second IRE within hIGFBP-1 promoter sequences, IRE(CRU), located 5'- to the CRE.

Efficient myelin repair in the macaque spinal cord by autologous grafts of Schwann cells.

Experimental transplantation in rodent models of CNS demyelination has led to the idea that Schwann cells may be candidates for cell therapy in human myelin diseases. Here we investigated the ability of Schwann cells autografts to generate myelin in the demyelinated monkey spinal cord. We report that monkey Schwann cells derived from adult peripheral nerve biopsies retain, after growth factor expansion and transduction with a lentiviral vector encoding green fluorescent protein, the ability to differentiate in vitro into promyelinating cells. When transplanted in the demyelinated nude mouse spinal cord, they promoted functional and anatomical repair of the lesions (n = 12). Furthermore, we obtained evidence by immunohistochemistry (n = 2) and electron microscopy (n = 4) that autologous transplantation of expanded monkey Schwann cells in acute lesions of the monkey spinal cord results in the repair of large areas of demyelination; up to 55% of the axons were remyelinated by donor Schwann cells, the remaining ones being remyelinated by oligodendrocytes. Autologous grafts of Schwann cells may thus be of therapeutic value for myelin repair in the adult CNS.

Efficiency of adeno-associated virus type-2 vectors in non-human primate Schwann cells.

Adult macaque Schwann cells were infected using adeno-associated virus type-2-derived vectors expressing the green fluorescent protein reporter gene under the control of the cytomegalovirus, the hybrid cytomegalovirus-betaactin, the myelin basic protein or the tetracycline-inducible promoters. On the basis of green fluorescent protein expression, gene transfer efficiency was compared in resting and dividing conditions following or not following hydroxyurea or etoposide treatment. Hydroxyurea allowed promoter-dependent expression of green fluorescent protein in infected Schwann cells. Etoposide treatment led to a high percentage of green fluorescent protein expressing cells (over 50%) with all promoters tested. When infected cells were grafted into demyelinated nude mice spinal cord, green fluorescent protein expression was only observed with the cytomegalovirus-betaactin and tetracycline-inducible promoters. In addition, adeno-associated virus type-2 infection reduced the grafted cell survival but increased their differentiation.

Mineralocorticoid and glucocorticoid receptors in sciatic nerve function and regeneration.

The contribution of the two corticosteroid (mineralocorticoid and glucocorticoid) receptor (MR and GR) pathways to the function and regeneration of the sciatic nerve was investigated. We found that the corticosterone-inactivating enzyme 11ß-hydroxysteroid dehydrogenase type 2 (HSD2) was up-regulated 7 days after lesion in freeze-injured nerve. The maintenance of a low intracellular level of corticosterone by HSD2 activity in the regenerating nerve is concordant with the improvement of nervous function in injured animals (as measured by walking ability) after treatment by the GR antagonist mifepristone and with the reduction in GR participation in accumulation of the mRNA for numerous endogenous genes (from the renin-angiotensin system and other classical mineralocorticoid-responsive genes), in the same animals. Furthermore, using the MR antagonist spironolactone, we demonstrated that MR plays an active role in the function of the intact sciatic nerve: MR is required for walking ability and participates in the control of the accumulation of the mRNA for several endogenous genes. However, after injury, changes in gene expression cannot be fully explained by changes in MR/GR activity, due to an HSD2 effect, and other signalling pathway(s) induced by the lesion likely combine with the effect of the corticosteroid receptors.