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.

Phenylbutyrate up-regulates the adrenoleukodystrophy-related gene as a nonclassical peroxisome proliferator.

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease due to mutations in the ABCD1 (ALD) gene, encoding a peroxisomal ATP-binding cassette transporter (ALDP). Overexpression of adrenoleukodystrophy-related protein, an ALDP homologue encoded by the ABCD2 (adrenoleukodystrophy-related) gene, can compensate for ALDP deficiency. 4-Phenylbutyrate (PBA) has been shown to induce both ABCD2 expression and peroxisome proliferation in human fibroblasts. We show that peroxisome proliferation with unusual shapes and clusters occurred in liver of PBA-treated rodents in a PPARalpha-independent way. PBA activated Abcd2 in cultured glial cells, making PBA a candidate drug for therapy of X-ALD. The Abcd2 induction observed was partially PPARalpha independent in hepatocytes and totally independent in fibroblasts. We demonstrate that a GC box and a CCAAT box of the Abcd2 promoter are the key elements of the PBA-dependent Abcd2 induction, histone deacetylase (HDAC)1 being recruited by the GC box. Thus, PBA is a nonclassical peroxisome proliferator inducing pleiotropic effects, including effects at the peroxisomal level mainly through HDAC inhibition.

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.

Deterministic dynamics control oscillations of bone marrow cell proliferation.

OBJECTIVE: The production of blood cells in vivo, both normal and tumoral, displays oscillatory dynamics. Many cells in long-term cultures also show large amplitude oscillations of proliferative rate. Therefore we examined the proliferation dynamics of mouse bone marrow cells (MBM) and their clonogenic progenitor production (BMP), in order to characterize these dynamics. METHODS: Five Dexter-type cultures of MBM cells and their clonogenic BMP production were examined for up to seven-months periods of time. The recorded time series exhibited a complex pattern of oscillations with variable amplitudes. We previously reported a method that allowed analysis of such nonlinear dynamics of hepatoma cell proliferation. We applied this method, based on the two-dimensional recurrent representation of data, to analyze the fluctuations of bone marrow cells proliferation. RESULTS: The proliferation rate of mouse bone marrow cells shows large amplitude oscillations every 2 to 3 weeks. Mathematical analysis revealed a deterministic mechanism that controls all proliferation local maxima of MBM cells. Dynamics for progenitor production resembled that of parental cells. This reflects a predominant negative feedback on bone marrow cell proliferation. CONCLUSION: These dynamics were opposite of that previously described for hepatoma cells where the dominant control is applied to the local minima (troughs of proliferation). Therefore, the complex system of cell proliferation is controlled by a bipolar mechanism, with a predominant dampening command depending on the cell type. We propose that the dominant dampening control of local maxima in bone marrow cells protects the stock of stem cells.

Steroid receptors in various glial cell lines expression and functional studies.

In glial cell lines from the central (CG4, C6) and peripheral (MSC80, CR3a1, and CR1b4) nervous systems, glucocorticoid receptor (GR) messenger RNA was clearly detected and was consistent with significant GR binding and transcriptional activity. Mineralocorticosteroid receptor mRNA was less abundant, with no corresponding binding and lack of transcriptional activity. Other steroid receptors were not significantly detected.

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.

Downregulation of steroidogenic acute regulatory protein (StAR) gene expression by cyclic AMP in cultured Schwann cells.

Steroidogenic acute regulatory protein (StAR) plays a key role in the availability of cholesterol to the inner mitochondrial membrane, where the first step of steroidogenesis, its conversion to pregnenolone, takes place. Here, we demonstrate for the first time that the StAR gene is also expressed in the rat sciatic nerve and in cultured Schwann cells. The addition to the culture medium of the cAMP-elevating agent forskolin or of the cAMP analogue 8Br-cAMP produced a time-course extinction of StAR gene expression. An inverse relationship was demonstrated between StAR gene expression and the intracellular cAMP content. Accordingly, pharmacological inhibition of the activities of Schwann cell adenylyl cyclase or of phosphodiesterase IV resulted in modifications of StAR gene expression. Since StAR gene expression is stimulated by cAMP in classical steroidogenic cells, our work is the first demonstration of a negative regulation of StAR gene by cAMP.

3alpha,5alpha-Tetrahydroprogesterone (allopregnanolone) and gamma-aminobutyric acid: autocrine/paracrine interactions in the control of neonatal PSA-NCAM+ progenitor proliferation.

The earliest identified neonatal neural progenitors are cells that express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). One of these progenitors is the early PSA-NCAM+ progenitor (ePSA-NCAM+ progenitor; Gago et al. [2003] Mol Cell Neurosci 22:162-178), which corresponds to a multipotential cell with a default differentiation through glial lineages. The ePSA-NCAM+ progenitor can synthesize the neurosteroid progesterone (PROG) and its reduced metabolite 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP, or allopregnanolone; Gago et al. [ 2001] Glia 36:295-308). The latter is a potent positive allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors. In the present work, we demonstrate that PROG and 3alpha,5alpha-THP both stimulate ePSA-NCAM+ progenitor proliferation. PROG exerted its mitogenic effect indirectly, through its conversion to 3alpha,5alpha-THP, since it could be abolished by an inhibitor of the 5alpha-reductase (L685-273) and mimicked by 3alpha,5alpha-THP. A dose-response curve revealed a bell-shaped effect of 3alpha,5alpha-THP on ePSA-NCAM+ progenitor proliferation, with greatest stimulation at nanomolar concentrations. The mitogenic effect of 3 alpha,5 alpha-THP was mediated by GABAA receptors, insofar as it could be blocked by the selective antagonist bicuculline. ePSA-NCAM+ progenitors indeed expressed mRNAs for GABAA receptor subunits, and GABA enhanced cell proliferation, an effect that was also bicuculline sensitive. Moreover, these cells synthesized GABA, which was involved in a tonic stimulation of their proliferation. These results reveal complex autocrine/paracrine loops in the control of ePSA-NCAM+ progenitor proliferation, involving both neurosteroid and GABA signaling, and suggest a novel key role for 3alpha,5alpha-THP in the development of the nervous system.

Thyroid hormone induction of the adrenoleukodystrophy-related gene (ABCD2).

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disorder associated with impaired very-long-chain fatty-acid (VLCFA) beta-oxidation caused by mutations in the ABCD1 (ALD) gene that encodes a peroxisomal membrane ABC transporter. ABCD2 (ALDR) displays partial functional redundancy because when overexpressed, it is able to correct the X-ALD biochemical phenotype. The ABCD2 promoter contains a putative thyroid hormone-response element conserved in rodents and humans. In this report, we demonstrate that the element is capable of binding retinoid X receptor and 3,5,3'-tri-iodothyronine (T3) receptor (TRbeta) as a heterodimer and mediating T3 responsiveness of ABCD2 in its promoter context. After a T3 treatment, an induction of the ABCD2 gene was observed in the liver of normal rats but not that of TRbeta-/- mice. ABCD2 was not induced in the brain of the T3-treated rats. However, we report for the first time that induction of the ABCD2 redundant gene is feasible in myelin-producing cells (differentiated CG4 oligodendrocytes). The induction was specific for this cell type because it did not occur in astrocytes. Furthermore, we observed T3 induction of ABCD2 in human and mouse ABCD1-deficient fibroblasts, which was correlated with normalization of the VLCFA beta-oxidation. Finally, ABCD3 (PMP70), a close homolog of ABCD2, was also induced by T3 in the liver of control rats, but not that of TRbeta-/- mice, and in CG4 oligodendrocytes.