Erythropoietin regulates developmental myelination in the brain stimulating postnatal oligodendrocyte maturation.

Myelination is a process tightly regulated by a variety of neurotrophic factors. Here, we show-by analyzing two transgenic mouse lines, one overexpressing EPO selectively in the brain Tg21(PDGFB-rhEPO) and another with targeted removal of EPO receptors (EPORs) from oligodendrocyte progenitor cells (OPC)s (Sox10-cre;EpoRfx/fx mice)-a key function for EPO in regulating developmental brain myelination. Overexpression of EPO resulted in faster postnatal brain growth and myelination, an increased number of myelinating oligodendrocytes, faster axonal myelin ensheathment, and improved motor coordination. Conversely, targeted ablation of EPORs from OPCs reduced the number of mature oligodendrocytes and impaired motor coordination during the second postnatal week. Furthermore, we found that EPORs are transiently expressed in the subventricular zone (SVZ) during the second postnatal week and EPO increases the postnatal expression of essential oligodendrocyte pro-differentiation and pro-maturation (Nkx6.2 and Myrf) transcripts, and the Nfatc2/calcineurin pathway. In contrast, ablation of EPORs from OPCs inactivated the Erk1/2 pathway and reduced the postnatal expression of the transcripts. Our results reveal developmental time windows in which EPO therapies could be highly effective for stimulating oligodendrocyte maturation and myelination.

Dissecting the role of diazepam-sensitive γ-aminobutyric acid type A receptors in defensive behavioral reactivity to mild threat.

Moderate reductions in synaptic γ-aminobutyric acid(A) receptors (GABA(A)Rs) have been associated with an enhanced defensive behavioral reactivity to mild threat, sensitive to diazepam. We here tested whether a deficit in α2 subunit-containing GABAergic synapses is sufficient to cause this anxiety-related phenotype and to prevent its attenuation by the benzodiazepine. Wild type (α2+/+), heterozygous (α2+/-) and homozygous (α2-/-) knock-out littermates were tested in the free-choice exploratory (FCE) and the light/dark choice (LDC) paradigms. α2-/- mice, double mutant α1H101Rα2-/- and α3H126Rα2-/- mice, which combine a lack of α2-GABA(A)Rs with point-mutated diazepam-insensitive either α1H101R or α3H126R-GABA(A)Rs, and double point-mutated α1H101Rα2H101R and α1H101Rα3H126R mice were used to uncover the GABA(A)R subtype(s) mediating the drug effects. Data show that in the FCE, α2-/- mice exhibited more retractions (i.e. risk assessment) and longer latencies to first occurrence into the novel compartment and less transitions and time spent inside it in comparison to α2+/- and α2+/+ mice. In the LDC, α2-/- mice visited and spent less time in the lit box and stayed longer in the tunnel than the other two groups. Minor differences were found between α2+/- and α2+/+ mice in the two paradigms. Diazepam (1.5mg/kg per os) normalized retractions and latencies in the FCE in α2-/- and α3H126Rα2-/- mice, but not in α1H101Rα2-/- mice. The same drug treatment failed to attenuate behavioral aversion in both paradigms in all mutants with impaired α2-GABA(A)R function. These results reveal α2-containing GABA(A)Rs as key molecular determinants in the regulation of anxiety-related responses elicited by exposure to relative novelty and mild threat. In the absence of these receptors, diazepam through activation of α1-GABA(A)Rs remains effective in reducing risk assessment, but not behavioral aversion.

Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABAA receptor alpha3 subunit-null mice.

Multiple GABAA-receptor subtypes are assembled from alpha, beta and gamma subunit variants. GABAA receptors containing the alpha3 subunit represent a minor population with a restricted distribution in the CNS. In addition, they predominate in monoaminergic neurons and in the nucleus reticularis thalami (nRT), suggesting a role in the regulation of cortical function and sleep. Mice with a targeted deletion of the alpha3 subunit gene (alpha3(0/0)) are viable and exhibit a subtle behavioural phenotype possibly related to dopaminergic hyperfunction. Here, we investigated immunohistochemically the consequences of the loss of alpha3 subunit for maturation of GABAA receptors and formation of GABAergic synapses in the nRT. Throughout postnatal development, the regional distribution of the alpha1, alpha2, or alpha5 subunit was unaltered in alpha3(0/0) mice and the prominent alpha3 subunit staining of nRT neurons in wildtype mice was not replaced. Subcellularly, as seen by double immunofluorescence, the alpha3 and gamma2 subunit were clustered at postsynaptic sites in the nRT of adult wildtype mice along with the scaffolding protein gephyrin. In alpha3(0/0) mice, gamma2 subunit clustering was disrupted and gephyrin formed large aggregates localized at the cell surface, but unrelated to postsynaptic sites, indicating that nRT neurons lack postsynaptic GABAA receptors in mutant mice. Furthermore, GABAergic terminals were enlarged and reduced in number, suggesting a partial deficit of GABAergic synapses. Therefore, GABAA receptors are required for gephyrin clustering and long-term synapse maintenance. The absence of GABAA-mediated transmission in the nRT may have a significant impact on the function of the thalamo-cortical loop of alpha3(0/0) mice.

Molecular heterogeneity of the dystrophin-associated protein complex in the mouse kidney nephron: differential alterations in the absence of utrophin and dystrophin.

The dystrophin-associated protein complex (DPC) consisting of syntrophin, dystrobrevin, and dystroglycan isoforms is associated either with dystrophin or its homolog utrophin. It is present not only in muscle cells, but also in numerous tissues, including kidney, liver, and brain. Using high-resolution immunofluorescence imaging and Western blotting, we have investigated the effects of utrophin and dystrophin gene deletion on the formation and membrane anchoring of the DPC in kidney epithelial cells, which co-express utrophin and low levels of the C-terminal dystrophin isoform Dp71. We show that multiple, molecularly distinct DPCs co-exist in the nephron; these DPCs have a segment-specific distribution and are only partially associated with utrophin in the basal membrane of tubular epithelial cells. In utrophin-deficient mice, a selective reduction of beta2-syntrophin has been observed in medullary tubular segments, whereas alpha1-syntrophin and beta1-syntrophin are retained, concomintant with an upregulation of beta-dystroglycan, beta-dystrobrevin, and Dp71. These findings suggest that beta2-syntrophin is dependent on utrophin for association with the DPC, and that loss of utrophin is partially compensated by Dp71, allowing the preservation of the DPC in kidney epithelial cells. This hypothesis is confirmed by the almost complete loss of all DPC proteins examined in mice lacking full-length utrophin and all C-terminal dystrophin isoforms (utrophin(0/0)/mdx(3Cv)). The DPC thus critically depends on these proteins for assembly and/or membrane localization in kidney epithelial cells.