We have recently identified a novel plasticity protein, doublecortin-like (DCL), that is specifically expressed in the shell of the mouse suprachiasmatic nucleus (SCN). DCL is implicated in... Show moreWe have recently identified a novel plasticity protein, doublecortin-like (DCL), that is specifically expressed in the shell of the mouse suprachiasmatic nucleus (SCN). DCL is implicated in neuroplastic events, such as neurogenesis, that require structural rearrangements of the microtubule cytoskeleton, enabling dynamic movements of cell bodies and dendrites. We have inspected DCL expression in the SCN by confocal microscopy and found that DCL is expressed in GABA transporter-3 (GAT3)-positive astrocytes that envelope arginine vasopressin (AVP)-expressing cells. To investigate the role of these DCL-positive astrocytes in circadian rhythmicity, we have used transgenic mice expressing doxycycline-induced short-hairpin (sh) RNA's targeting DCL mRNA (DCL knockdown mice). Compared with littermate wild type (WT) controls, DCL-knockdown mice exhibit significant shorter circadian rest-activity periods in constant darkness and adjusted significantly faster to a jet-lag protocol. As DCL-positive astrocytes are closely associated with AVP-positive cells, we analyzed AVP expression in DCL-knockdown mice and in their WT littermates by 3D reconstructions and transmission electron microscopy (TEM). We found significantly higher numbers of AVP-positive cells with increased volume and more intensity in DCL-knockdown mice. We found alterations in the numbers of dense core vesicle-containing neurons at ZT8 and ZT20 suggesting that the peak and trough of neuropeptide biosynthesis is dampened in DCL-knockdown mice compared to WT littermates. Together, our data suggest an important role for the astrocytic plasticity in the regulation of circadian rhythms and point to the existence of a specific DCL+ astrocyte-AVP(+) neuronal network located in the dorsal SCN implicated in AVP biosynthesis. Show less
Saaltink, D.J.; Zwet, E.W. van; Vreugdenhil, E. 2020
Doublecortin (DCX)-like (DCL) is a microtubule (MT)-associated protein (MAP) that is highly homologous to DCX and is crucially involved in embryonic neurogenesis. Here, we have investigated the in... Show moreDoublecortin (DCX)-like (DCL) is a microtubule (MT)-associated protein (MAP) that is highly homologous to DCX and is crucially involved in embryonic neurogenesis. Here, we have investigated the in vivo role of DCL in adult hippocampal neurogenesis by generating transgenic mice producing inducible shRNA molecules that specifically target DCL but no other splice variants produced by the DCLK gene. DCL knock-down (DCL-KD) resulted in a significant increase in the number of proliferating BrdU+ cells in the subgranular zone (SGZ) 1 d after BrdU administration. However, the number of surviving newborn adult NeuN+/BrdU+ neurons are significantly decreased when inspected four weeks after BrdU administration suggesting a blockade of neuronal differentiation after DCL-KD. In line with this, we observed an increase in the number of proliferating cells, but a significant decrease in postmitotic DCX+ cells that are characterized by long dendrites spanning all dentate gyrus layers. Behavioral analysis showed that DCL-KD strongly extended the escape latency of mice on the circular hole board (CHB) but did not affect other aspects of this behavioral task. Together, our results indicate a function for DCL in adult neurogenesis and in the motivation to escape from an aversive environment. In contrast to DCX, its pivotal role in the maturation of postmitotic neuronal progenitor cells (NPCs) marks DCL as a genuine adult neurogenesis indicator in the hippocampus. Show less
The results in this thesis showed for the first time doublecortin-like (DCL)-specific expression in the adult mouse brain. Besides the expected regions with the capacity to generate new neurons ... Show moreThe results in this thesis showed for the first time doublecortin-like (DCL)-specific expression in the adult mouse brain. Besides the expected regions with the capacity to generate new neurons (hippocampus and olfactory forebrain), DCL expression was found in three novel brain areas namely hypothalamic tanycytes, suprachiasmatic nucleus and Islands of Calleja. A state of the art conditional shRNA expressing mouse model was used to target DCL mRNA. The analysis of these DCL knockdown animals using qPCR and Western blot revealed strong reduction of DCL protein expression. Subsequent stereological analysis using BrdU and several stem cell and neuronal markers revealed increased progenitor proliferation, but impaired neurogenesis in the hippocampus. This impaired neurogenesis was associated, however, with an apparent normal spatial and contextual fear memory formation in circular hole board and in a contextual fear conditioning paradigm. Therefore, DCL-regulated adult neurogenesis seems not crucial for hippocampus-dependent learning. However, more subtle functions like pattern separation and context distinction might be regulated by DCL. DCL knockdown also increased D2 activity within the hypothalamus. Altogether, the DCL-KD mouse seems a good working model to study adult neurogenesis and the role of DCL in this process. Show less
Doublecortin-like (DCL) is a microtubule-binding protein crucial for neuroblastoma (NB) cell proliferation. We have investigated whether the anti-proliferative effect of DCL knockdown is linked to... Show moreDoublecortin-like (DCL) is a microtubule-binding protein crucial for neuroblastoma (NB) cell proliferation. We have investigated whether the anti-proliferative effect of DCL knockdown is linked to reduced mitochondrial activity. We found a delay in tumor development after DCL knockdown in vivo in doxycycline-inducible NB tumor xenografts. To understand the mechanisms underlying this tumor growth retardation we performed a series of in vitro experiments in NB cell lines. DCL colocalizes with mitochondria, interacts with the mitochondrial outer membrane protein OMP25/ SYNJ2BP and DCL knockdown results in decreased expression of genes involved in oxidative phosphorylation. Moreover, DCL knockdown decreases cytochrome c oxidase activity and ATP synthesis. We identified the C-terminal Serine/Proline-rich domain and the second microtubule-binding area as crucial DCL domains for the regulation of cytochrome c oxidase activity and ATP synthesis. Furthermore, DCL knockdown causes a significant reduction in the proliferation rate of NB cells under an energetic challenge induced by low glucose availability. Together with our previous studies, our results corroborate DCL as a key player in NB tumor growth in which DCL controls not only mitotic spindle formation and the stabilization of the microtubule cytoskeleton, but also regulates mitochondrial activity and energy availability, which makes DCL a promising molecular target for NB therapy. Show less
Fitzsimons, C.P.; Hooijdonk, L.W.A. van; Schouten, M.; Zalachoras, I.; Brinks, V.; Zheng, T.; ... ; Vreugdenhil, E. 2013
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... Show moreGlucocorticoids (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. Show less
