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
Essen, M.F. van; Schlagwein, N.; Gijlswijk-Janssen, D.J. van; Anholts, J.D.H.; Eikmans, M.; Ruben, J.M.; ... ; COMBAT Consortium 2020
Gene silencing using small interfering ribonucleic acids (siRNA) is a powerful method to interfere with gene expression, allowing for the functional exploration of specific genes. siRNA... Show moreGene silencing using small interfering ribonucleic acids (siRNA) is a powerful method to interfere with gene expression, allowing for the functional exploration of specific genes. siRNA interference can be applied in both cell lines, as well as in primary, non-dividing cell types like dendritic cells. However, the efficacy in different cell types is variable and requires optimization. Here, we showed that the type of culture medium used during lipid-based siRNA-mediated transfection acts as a critical factor, affecting dendritic cell activation. Transfection of immature monocyte-derived dendritic cells in RPMI medium, but not in IMDM, showed increased transcript levels of pro-inflammatory cytokines. Moreover, the expression of co-stimulatory molecules was enhanced, thereby increasing the T cell stimulatory capacity. Our data demonstrates that the choice of medium should be critically examined as one of the variables while optimizing cell transfection. Show less
During my research project we mainly focussed on studying the pathophysiology of venous and arterial thrombosis in mice. When we transiently lowered plasma protein levels of natural anticoagulants... Show moreDuring my research project we mainly focussed on studying the pathophysiology of venous and arterial thrombosis in mice. When we transiently lowered plasma protein levels of natural anticoagulants antithrombin and protein C using RNA interference, mice developed venous thrombosis in the head. In contrast to other mouse models for venous thrombosis where surgery is required for provoking the disease, mice injected with RNA interference against the mRNA of Serpinc1 and Proc (antithrombin and protein C, respectively) developed venous thrombosis without additional handlings. In this unique form of venous thrombosis, we studied the roles of platelets, neutrophils, and coagulation factor XII. These factors have been shown to be indispensable in experimental venous thrombosis in other mouse models, and they have been introduced as novel therapeutic targets. For the second part of my thesis we again used the RNA interference approach, to lower natural anticoagulation in atherosclerotic mice. When we lowered protein C in these mice, they developed atherothrombosis in the aortic root without any additional intervention. This unique form of atherothrombosis has been showed in multiple independent experiments, and we aimed to further characterize the process to learn more about prevention atherothrombosis in atherosclerotic mice and the role of protein C. Show less
Familial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of low density lipoprotein cholesterol (LDL-C) and increasing the risk of cardio vascular diseases. FH and many... Show moreFamilial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of low density lipoprotein cholesterol (LDL-C) and increasing the risk of cardio vascular diseases. FH and many other liver diseases can possibly be treated with RNA interference (RNAi). RNAi is a natural process of regulation of gene expression by binding of small RNA molecules to complementary sequences in the mRNA of a gene and hence inducing its degradation or translational repression. In this thesis, we aimed at developing a safe and robust RNAi-based therapy for FH by inhibiting Apolipoprotein B100 (ApoB). ApoB is a primary component of the low density lipoprotein (LDL), a lipoprotein that transports __bad__ cholesterol LDL-C. We have shown that RNAi therapy using AAV-delivered artificial miRNA is a promising approach for treatment of FH and possibly other liver diseases. Our mechanistic studies revealed differences in shRNA and miRNA processing and functioning in vivo. Finally, our findings have significant impact on understanding and overcoming toxicity and off-targeting related problems of RNAi based gene therapy in the liver using AAV vectors. Show less
