Over-expression of δC-DCLK-short in mouse brain results in a more anxious behavioral phenotype.

Products of the Doublecortin-Like Kinase (DCLK) gene are associated with cortical migration and hippocampal maturation during embryogenesis. However, the functions of those DCLK gene transcripts that encode kinases and are expressed during adulthood are incompletely understood. To elucidate potential functions of these DCLK gene splice variants we have generated and analyzed transgenic mice with neuronal over-expression of a truncated, constitutively active form of DCLK-short, designated δC-DCLK-short. Previously, we have performed an extensive molecular characterization of these transgenic δC-DCLK-short mice and established that a specific subunit of the GABA(A) receptor, which is involved in anxiety-related GABAergic neurotransmission, is down-regulated in the hippocampus. Here we show that δC-DCLK-short mRNA is highly expressed in the hippocampus, cortex and amygdala of transgenic mice. We provide evidence that the δC-DCLK-short protein is expressed and functional. In addition, we examined anxiety-related behavior in δC-DCLK-short mice in the elevated plus maze. Interestingly, δC-DCLK-short mice spend less time, move less in the open arms of the maze and show a reduction in the number of rim dips. These behaviors indicate that δC-DCLK-short mice display a more anxious behavioral phenotype.

Lentivirus-mediated transgene delivery to the hippocampus reveals sub-field specific differences in expression.

BACKGROUND: In the adult hippocampus, the granule cell layer of the dentate gyrus is a heterogeneous structure formed by neurons of different ages, morphologies and electrophysiological properties. Retroviral vectors have been extensively used to transduce cells of the granule cell layer and study their inherent properties in an intact brain environment. In addition, lentivirus-based vectors have been used to deliver transgenes to replicative and non-replicative cells as well, such as post mitotic neurons of the CNS. However, only few studies have been dedicated to address the applicability of these widespread used vectors to hippocampal cells in vivo. Therefore, the aim of this study was to extensively characterize the cell types that are effectively transduced in vivo by VSVg-pseudotyped lentivirus-based vectors in the hippocampus dentate gyrus. RESULTS: In the present study we used Vesicular Stomatitis Virus G glycoprotein-pseudotyped lentivirual vectors to express EGFP from three different promoters in the mouse hippocampus. In contrast to lentiviral transduction of pyramidal cells in CA1, we identified sub-region specific differences in transgene expression in the granule cell layer of the dentate gyrus. Furthermore, we characterized the cell types transduced by these lentiviral vectors, showing that they target primarily neuronal progenitor cells and immature neurons present in the sub-granular zone and more immature layers of the granule cell layer. CONCLUSION: Our observations suggest the existence of intrinsic differences in the permissiveness to lentiviral transduction among various hippocampal cell types. In particular, we show for the first time that mature neurons of the granule cell layer do not express lentivirus-delivered transgenes, despite successful expression in other hippocampal cell types. Therefore, amongst hippocampal granule cells, only adult-generated neurons are target for lentivirus-mediated transgene delivery. These properties make lentiviral vectors excellent systems for overexpression or knockdown of genes in neuronal progenitor cells, immature neurons and adult-generated neurons of the mouse hippocampus in vivo.

Temporal and functional dynamics of the transcriptome during nerve growth factor-induced differentiation.

The rat pheochromocytoma cell line (PC12) is an extensively used model to study neuronal differentiation. The initial signaling cascades triggered by nerve growth factor (NGF) stimulation have been subject to thorough investigation and are well characterized. However, knowledge of temporal transcriptomal regulation during NGF-induced differentiation of PC12 cells remains far from complete. We performed a microarray study that characterized temporal and functional changes of the transcriptome during 4 subsequent days of differentiation of Neuroscreen-1 PC12 cells. By analyzing the transcription profiles of 1595 NGF-regulated genes, we show a large diversity of transcriptional regulation in time. Also, we quantitatively identified 26 out of 243 predefined biological process and 30 out of 255 predefined molecular function classes that are specifically regulated by NGF. Combining the temporal and functional transcriptomal data revealed that NGF selectively exerts a temporally coordinated regulation of genes implicated in protein biosynthesis, intracellular signaling, cell structure, chromatin packaging and remodeling, intracellular protein traffic, mRNA transcription, and cell cycle. We will discuss how NGF-induced changes may modulate the transcriptional response to NGF itself during differentiation.

The microtubule-associated protein doublecortin-like regulates the transport of the glucocorticoid receptor in neuronal progenitor cells.

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.

Functional differences between two DCLK splice variants.

Recently, we have cloned two splice variants of the doublecortin-like kinase (DCLK) gene, called DCLK-short-A and -B, both of which encode calcium/calmodulin-dependent protein kinase (CaMK)-like proteins with different C-terminal ends. Using in situ hybridization, we have found that both are highly expressed in limbic structures of the brain and that their expression differs in a number of brain areas. DCLK-short-A is relatively more strongly expressed than DCLK-short-B in the subependymal zone. The DCLK-short-B variant shows stronger expression in the cortex, the ventromedial and dorsomedial hypothalamic nuclei, the arcuate nucleus, the zona incerta and the subincertal nucleus. Also, within the hippocampus, the relative distribution of these two splice variants differs. DCLK-short-B expression compared to DCLK-short-A is highest in the CA1 area. The expression of the A variant is highest in the CA3/CA4 area. Additionally, DCLK-short-B is expressed at a higher level than DCLK-short-A in the substantia nigra and the mammillary nucleus. Both DCLK-short-A and -B were located in the cytoplasm, however DCLK-short-B was also found specifically in growth cone like structures and near the nucleus. Both DCLK-short proteins phosphorylate autocamtide and syntide, two highly specific CaMK substrates. Finally, removal of the C-terminal end of DCLK-short leads to a 10-fold increase of kinase activity, indicating that the different C-termini represent auto-inhibitory domains. Our results indicate that DCLK-short-A and -B control different neuronal processes that overlap with those controlled by CaMKs.