Glucocorticoids (GCs) exert profound effects on a variety of physiological processes, including adaptation to stress, metabolism, immunity, and neuronal development. Cellular responsiveness to GCs depends on numerous factors, including the amount of the glucocorticoid receptor (GR) protein. We tested the hypothesis that micro-RNAs (miRs), a recently discovered group of noncoding RNAs involved in mRNA translation, might control GR activity by reducing GR protein levels in neuronal tissues. We tested a panel of five miRs consisting of 124aa, 328, 524, 22, and 18. We found that miRs 18 and 124a reduced GR-mediated events in addition to decreasing GR protein levels. miR reporter assays revealed binding of miR-124a to the 3' untranslated region of GR. In correspondence, the activation of the GR-responsive gene glucocorticoid-induced leucine zipper was strongly impaired by miR-124a and -18 overexpression. Although miR-18 is expressed widely throughout the body, expression of miR-124a is restricted to the brain. Endogenous miR-124a up-regulation during neuronal differentiation of P19 cells was associated with a decreasing amount of GR protein levels and reduced activity of luciferase reporter constructs bearing GR 3' untranslated regions. Furthermore, we show that miR-124a expression varies over time during the stress hyporesponsive period, a neonatal period when GC signaling is modulated. Our findings demonstrate a potential role for miRs in the regulation of cell type-specific responsiveness to GCs, as may occur during critical periods of neuronal development. Ultimately, our results may provide a better understanding of the etiology of stress-related diseases as well as the efficacy of GC therapy.

Glucocorticoids (GCs) secreted after stress reduce adult hippocampal neurogenesis, a process that has been implicated in cognitive aspects of psychopathology, amongst others. Yet, the exact role of the GC receptor (GR), a key mediator of GC action, in regulating adult neurogenesis is largely unknown. Here, we show that GR knockdown, selectively in newborn cells of the hippocampal neurogenic niche, accelerates their neuronal differentiation and migration. Strikingly, GR knockdown induced ectopic positioning of a subset of the new granule cells, altered their dendritic complexity and increased their number of mature dendritic spines and mossy fiber boutons. Consistent with the increase in synaptic contacts, cells with GR knockdown exhibit increased basal excitability parallel to impaired contextual freezing during fear conditioning. Together, our data demonstrate a key role for the GR in newborn hippocampal cells in mediating their synaptic connectivity and structural as well as functional integration into mature hippocampal circuits involved in fear memory consolidation.

During corticogenesis, progenitors divide within the ventricular zone where they rely on radial process extensions, formed by radial glial cell (RG) scaffolds, along which they migrate to the proper layers of the cerebral cortex. Although the microtubule-associated proteins doublecortin (DCX) and doublecortin-like kinase (DCLK) are critically involved in dynamic rearrangement of the cytoskeletal machinery that allow migration, little is known about their role in early corticogenesis. Here we have functionally characterized a mouse splice-variant of DCLK, doublecortin-like (DCL), exhibiting 73% amino acid sequence identity with DCX over its entire length. Unlike DCX, DCL is expressed from embryonic day 8 onwards throughout the early neuroepithelium. It is localized in mitotic cells, RGs and radial processes. DCL knockdown using siRNA in vitro induces spindle collapse in dividing neuroblastoma cells, whereas overexpression results in elongated and asymmetrical mitotic spindles. In vivo knockdown of the DCLK gene by in utero electroporation significantly reduced cell numbers in the inner proliferative zones and dramatically disrupted most radial processes. Our data emphasize the unique role of the DCLK gene in mitotic spindle integrity during early neurogenesis. In addition, they indicate crucial involvement of DCLK in RG proliferation and their radial process stability, a finding that has thus far not been attributed to DCX or DCLK.

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In neuronal cells, activated glucocorticoid receptor (GR) translocates to the nucleus guided by the cytoskeleton. However, the detailed mechanisms underlying GR translocation remain unclear. Using gain and loss of function studies, we report here for the first time that the microtubule-associated protein doublecortin-like (DCL) controls GR translocation to the nucleus. DCL overexpression in COS-1 cells, neuroblastoma cells, and rat hippocampus organotypic slice cultures impaired GR translocation and decreased GR-dependent transcriptional activity, measured by a specific reporter gene assay, in COS-1 cells. Moreover, DCL and GR directly interact on microtubule bundles formed by DCL overexpression. A C-terminal truncated DCL with conserved microtubule-bundling activity did not influence GR translocation. In N1E-115 mouse neuroblastoma cells and neuronal progenitor cells in rat hippocampus organotypic slice cultures, laser-scanning confocal microscopy showed colabeling of endogenously expressed DCL and GR. In these systems, RNA-interference-mediated DCL knockdown hampered GR translocation. Thus, we conclude that DCL expression is tightly regulated to adequately control GR transport. Because DCL is primarily expressed in neuronal progenitor cells, our results introduce this microtubule-associated protein as a new modulator of GR signaling in this cell type and suggest the existence of cell-specific mechanisms regulating GR translocation to the nucleus.