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ABSTRACT: RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of RNAi in mice, short hairpin (sh) RNAs expressed stably from the genome are a fast alternative to conventional knockout approaches. We developed a strategy for complete or conditional gene knockdown in mice, where the Cre/loxP system is used to activate RNAi in a time and tissue dependent manner. Alternatively doxycycline controlled shRNA expression vectors can be used for conditional gene silencing. Single copy RNAi constructs are placed into the Rosa26 locus of ES cells by recombinase mediated cassette exchange and transmitted through the germline of chimeric mice. The shRNA transgenic offspring can be either directly used for phenotypic analysis or are further crossed to a Cre transgenic strain to activate conditional shRNA vectors. The site specific insertion of single copy shRNA vectors allows the expedite and reproducible production of knockdown mice and provides an easy and fast approach to assess gene function in vivo.
Methods 02/2011; 53(2):142-50. · 4.01 Impact Factor
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ABSTRACT: Soluble amyloid beta (Aβ) oligomers are widely accepted to be neurotoxic and lead to the memory loss and neuronal death observed in Alzheimer's disease (AD). Ample evidence suggests that impairment in glutamatergic signalling is associated with AD pathology. In particular, Aβ(1-42) is thought to affect N-methyl-d-aspartate (NMDA) receptor function and abolish the induction of long-term potentiation (LTP), which is regarded to be a phenomenon relevant to memory formation. The involvement of glutamatergic signalling in the pathology of AD is underscored by the therapeutic success of memantine, an uncompetitive NMDA receptor antagonist, used to treat patients with moderate to severe AD. In this study we show that Aβ(1-42) oligomers applied to acute murine hippocampal slices prevented, in a concentration-dependent manner, the development of CA1-LTP after tetanic stimulation of the Schaffer collaterals with a half maximal inhibitory concentration of around 2 nM (before oligomerization). The highest concentration of Aβ(1-42) oligomers (50 nM before oligomerization) completely blocked LTP (105 ± 1% potentiation versus 141 ± 3% in control) whereas scrambled Aβ(1-42) (50 nM) was without effect (144 ± 10% potentiation). Pre-incubation with memantine (1 μM) restored LTP in the presence of Aβ(1-42) (50 nM; 135 ± 5% potentiation). NMDA receptors containing the NR2B subunit have been proposed to play a particularly important role in excitotoxicity, functioning as extracellular "death receptors". The metabotropic glutamate receptor 5 (mGluR5) is mechanistically coupled to postsynaptic NMDA receptors. As such, allosteric sites on both receptors offer alternative means to modulate NMDA receptor function. We therefore tested low concentrations (each 300 nM) of allosteric antagonists of NR2B (Ro 25-6981, [R-(R∗,S∗)]-α-(4-Hydroxyphenyl)-β-methyl-4(phenylmethyl)-1-piperidine propanol hydrochloride) and mGluR5 receptors (MPEP, 2-methyl-6-(phenylethynyl)-pyridine). Both compounds restored LTP in the presence of Aβ(1-42) oligomers (50 nM, fEPSPs were potentiated to 129 ± 13% and 133 ± 7% respectively). Finally, we demonstrated that slices from mice heterozygous for NR2B receptor) in the forebrain are not susceptible to the toxic effects of Aβ(1-42) oligomers but express normal LTP (138 ± 6%). These experiments demonstrate that glutamate receptor antagonists delivered at concentrations which still allow physiological activities in vitro, are able to prevent Aβ(1-42) oligomer-induced synaptic toxicity and further support the glutamatergic system as a target for the development of improved symptomatic/neuroprotective treatments for AD.
Neuropharmacology 02/2011; 60(6):982-90. · 4.81 Impact Factor
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ABSTRACT: In spite of growing evidence linking vitamin D(3) levels to mental health disorders, little is known about its direct targets in the brain. This study set out to investigate targets of vitamin D(3) in a human brain stem cell line. We employed arrays with antibodies directed against more than 600 structural and signalling proteins, including phospho-variants. Over 180 proteins responded to vitamin D(3), such as cyclin-dependent protein-serine kinase 1/2, epidermal growth factor receptor-tyrosine kinase, protein kinase A, protein-serine kinase Bgamma and protein-serine kinase Calpha. PEA-15 (phosphoprotein enriched in astrocytes-15 kD, also known as PED), known to be involved in various anti-proliferative and anti-apoptotic effects, was strongly up-regulated. In silico promoter analysis revealed conserved binding sites for vitamin D(3) receptor, suggesting a strong vitamin D(3) dependency of the PEA-15 promoter. PEA-15 up-regulation by vitamin D(3) could be confirmed by Western blot in two different cell lines. Analysis of mRNA and protein phosphorylation status of PEA-15 suggests that increased PEA-15 promoter activity and increased protein stabilization contribute to the overall rise of PEA-15 protein. In a functional test of this novel pathway, we demonstrated that vitamin D(3) was able to rescue cells from TRAIL-induced apoptosis through regulation of the PEA-15 expression and function. Summarized, our study presents novel targets of vitamin D(3) relevant for apoptosis and cell proliferation, and thus strongly supports a function of vitamin D(3) in the brain that impacts on processes highly relevant for major neurological disorders.
Journal of Cellular and Molecular Medicine 04/2009; 13(9B):3315-28. · 4.13 Impact Factor
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ABSTRACT: CRF receptor type (CRHR) 1 exerts neuroregulatory control on associative learning processes such as fear and anxiety like behaviour. Using hippocampal slices, we investigated the neuronal excitability in mice lacking CRHR1 (Crhr1(-/-)). Compared to wild-type mice, long-term potentiation (LTP) elicited by 100 pulses at 100Hz was not different. Unexpectedly, at lower frequencies (1, 5 or 10Hz), the resulting synaptic changes in CA1 neurons of Crhr1(-/-) were systematically shifted towards long-term depression (LTD). Furthermore, testing paired-pulse paradigm revealed a GABA receptor-dependent decrease of paired-pulse ratio in Crhr1(-/-). It might be assumed that a lack of CRHR1 induce developmental changes which resulted in altered GABAergic activity, producing attenuated synaptic potentiation after repetitive stimulation and thus favouring LTD in principal neurons. Since CRHR1 are located in GABAergic somata, axons and boutons the activity of these receptor types rather might contribute to the development of the neuronal ability for plasticity like processes on the level of NMDAR subunit composition and GABAergic activity.
Neuroscience Letters 05/2007; 416(1):82-6. · 2.11 Impact Factor
