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amaxa eNews #4
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Down-regulation of Staufen2, a Key Component of the RNA Localization Machinery, in Hippocampal Neurons
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Manuel Zeitelhofer and Ralf Dahm
Division of Neuronal Cell Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria
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Abstract
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The localization of mRNAs is an efficient way to target proteins to specific regions in a cell. In mammalian neurons, RNA localization is involved in compartmentalizing the cell during differentiation and is assumed to play an important role in synaptic plasticity. RNAs are packaged together with RNA-binding proteins into transport-competent ribonucleoprotein particles (RNPs) and transported into dendrites. The RNA-binding protein Staufen2 is a key component of RNPs and implicated in dendritic RNA transport. In this study, Staufen2 was successfully down-regulated by RNAi. The knock-down efficiency of Staufen2 expression was assessed after nucleofection of shRNA constructs targeting Staufen2 mRNA. This approach was used to explore the function of Staufen2 in mammalian neurons.
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Introduction
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The localization of mRNAs is an efficient way to target gene products to specific regions in a cell. It occurs in a wide range of organisms and cell types (Kloc & Zearfoss, 2002; Jansen, 2001). During Drosophila development, for example, localized mRNAs act as cell fate determinants and thereby specify the body axes (St Johnston, 2005). In addition to its role in development, RNA localization also occurs in differentiated cells. In migrating cells, for instance, RNAs are localized to the leading edges, thereby enabling directed movement. This plays a role in wound closure when fibroblasts migrate into the lesion or when cancer cells metastasize.
Polarized cells, such as neurons, display a functional compartmentalization of the cytoplasm. The cell body is molecularly distinct from the axon and the dendrites. RNA localization is involved in compartmentalizing neurons during differentiation and it is assumed to also play an important role in synaptic plasticity, the experience-dependent remodeling of synapses that forms the basis of learning and memory (Sutton and Schuman, 2006).
How is RNA localization achieved? Localized RNAs contain cis-acting sequence elements, often within the 3’-untranslated region (3’-UTR), which are recognized by proteins (trans-acting factors). The mRNAs and trans-acting factors are packaged into ribonucleoprotein particles (RNPs). These RNPs are then transported to and retained by sites of local synthesis (Martin and Zukin, 2006). During transport, the RNAs must be kept translationally silenced to prevent the ectopic expression of the corresponding protein. At their destinations, this translational block can be lifted by signals inducing translation (Hüttelmaier et al., 2005). A central hypothesis assumes that translation is only activated at synapses where synaptic plasticity has been induced.
In this study, the nucleofection technique was used to assess the efficiency of down-regulation of Staufen2 (Stau2) in hippocampal neurons. Stau2 is one of the key components implicated in dendritic RNA localization. In Drosophila, Staufen is required for bicoid and oscar mRNA localization in the oocyte (St Johnston, 2005) and for the localization of prospero mRNA in embryonic neuroblasts (Broadus et al., 1998). In mammals, it is hypothesized that Stau2 is important in the transport of RNAs into dendrites of mature hippocampal neurons. To assess the consequences of a loss of Stau2 on dendritic RNA localization as well as the morphology of dendrites and dendritic spines, Stau2 was down-regulated by RNAi in primary cultures of hippocampal neurons (Goetze et al., 2006).
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Material and Methods
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Primary hippocampal neuron culture
Neurons were isolated from the hippocampi of 17 day-old embryonic (E17) rat brains and cultured as described (Zeitelhofer et. al., 2007). For nucleofection experiments, neurons were immediately used after dissociation. For the assessment of dendritic spine phenotypes and for fluorescence in situ hybridization (FISH) experiments, cells were used after 15 days in vitro (DIV).
Nucleofection and western blot analysis
After dissociation, neurons were co-nucleofected with an expression vector encoding citrine and shRNA constructs (pSUPER, Oligoengine; Brummelkamp et al., 2002) against Staufen2 (Satu2), mismatch Staufen2 (misStau2), and Septin7 (Sept7; CDC10). Cells were plated onto 6 cm cell culture dishes and lysed after 3 days of expression. The knock-down efficiency was assessed by western blot analysis.
Immunostaining
The following antibodies were used: an affinity-purified rabbit anti-Stau1 antibody (1µg/ml) and an affinity-purified rabbit anti-Stau2 antibody (1µg/ml). Fluorophore-coupled phalloidin was used to label F-actin. Immunostaining experiments were performed as described (Goetze et al., 2004).
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Results
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Staufen2 was down-regulated in hippocampal neurons by nucleofection of shRNA constructs to assess its function during neuronal differentiation and in mature neurons. To evaluate the success of the down-regulation, western blot analysis of the nucleofected neurons was performed. As neurons are post-mitotic cells, they are difficult to transfect. Conventional transfection methods like the calcium phosphate (CaPi) method have very poor transfection efficiencies for subsequent biochemical analyses and virus-based methods are too time-consuming and costly (Dahm et al., 2008). The nucleofection technique overcomes these limitations.
Primary hippocampal neurons were co-transfected with an expression vector encoding citrine and shRNA constructs (pSUPER, Oligoengine) against Staufen2, mismatch Staufen2 (misStau2), and Septin7. First, the transfection efficiency was evaluated. To this aim, nucleofected neurons were fixed 3 days post-transfection and the percentage of transfected cells quantified (Figure 1a, b). We reached transfection efficiencies of approximately 60 percent that allowed for the performing of biochemical experiments. Importantly the nucleofected neurons developed normally (Figure 1c, d). This allowed us to quantitatively down-regulate Staufen2 in neurons via RNAi. The levels and the specificity of Stau2 down-regulation were assessed via western blot analysis (Figure 2a). The protein levels of Stau2 were significantly down-regulated in shRNA nucleofected cells whereas the levels of the Stau2 paralogue Stau1 as well as that of the unrelated protein Septin7 remained unchanged. Moreover, transfection with misStau2 did not affect Stau2 protein levels, indicating that the down-regulation of Stau2 is specific.
The extent of down-regulation in mature neurons was further controlled by immunocytochemistry (Figure 2b). 15 DIV neurons were co-transfected using the CaPi method with an expression vector encoding ECFP together with pSUPER vectors against Stau2, misStau2 and RFP, respectively, and immunostained with Stau2 and Stau1 antibodies. Stau2 staining was substantially reduced in neurons transfected with the shStau2 plasmid but was abundant in neurons transfected with misStau2 or RFP plasmid. The down-regulation of Stau2 did not affect Stau1 staining.
Interestingly, down-regulation of Stau2 caused a rearrangement of the actin cytoskeleton, which plays an important role in the maintenance and plasticity of dendritic spines. This effect was mirrored by changes in the morphology of dendritic spines from their characteristic mushroom-like shape to filopodia. In addition, it could be observed that the levels of PSD95, a key component of the postsynaptic density, were reduced in Stau2 down-regulated neurons, indicating that there are fewer functional synapses than in normal cells. This hypothesis was borne out by electrophysiological recordings that indicate a reduction in synaptic transmission in Stau2 knock-down neurons (Goetze et al., 2006).
As Stau2 has been implicated in dendritic mRNA transport and since the actin cytoskeleton is rearranged upon Stau2 down-regulation, it was further tested whether the levels of ß-actin mRNA are changed in Stau2 down-regulated neurons. It was indeed observed that the ß-actin mRNA levels were reduced by approximately 37%, indicating that Stau2 is important for the transport of this mRNA into dendrites (Goetze et al., 2006).
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Figure 1: Hippocampal neurons are transfected efficiently via nucleofection. Hippocampal neurons were nucleofected with an shRNA construct against Stau2 and cultured in 6 cm cell culture dishes. After 3 DIV, neurons were fixed and analyzed to examine transfection efficiencies (B). The transfection efficiency reached up to 60 percent. Neurons displayed normal morphology upon nucleofection (A). Higher magnification phase contrast (C) and fluorescence images (D) show the integrity of the nucleofected neurons. The arrow shows an outgrowing axon and the arrowheads indicate extending neurites with the typical growth cones at their tips. The inset in C shows the corresponding DAPI stained nucleus of the nucleofected neuron.
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Figure 2. Assessment of the extent of down-regulation of Stau2. (A) Western blot analysis of nucleofected hippocampal neurons. Neurons were co-nucleofected with a plasmid expressing citrine fluorescent protein (lane 1, mock) and shRNA-expressing plasmids against Septin7 (lane 2, labelled unr.), mismatch Stau2 (lane 3, mis) and Stau2 (lane 4, si2-2). Cells were lysed after 3 days of expression and processed for western blot analysis. The levels of the three Stau2 isoforms (lines indicate 62, 59 and 52 kD) were significantly down-regulated only in cells nucleofected with the shRNA plasmid si2-2 (lane 4). Calnexin served as internal loading control. The levels of Stau1 did not change upon down-regulation of Stau2. (B) Down-regulation of Stau2 in mature neurons. 15 DIV neurons were co-transfected with the following constructs: siStaufen2-2 (si2-2), si-RFP or mismatch Staufen2 (mis) pSUPER vectors together with ECFP (green). Neurons were stained 3 days after transfection with anti-Stau2 or anti-Stau1 antibodies (red). The Stau2 signal was strongly reduced in both the cell body (asterisk) and dendrites of transfected neurons (green) compared with untransfected neurons. By contrast, expression of misStau2 or si-RFP did not alter the Stau2 levels. Down-regulation of Stau2 also did not affect Stau1 expression. Scale bar: 10 µm. Figure 2a reproduced from Journal of Cell Biology, 2006, 172:221-231. Copyright 2006 Rockefeller University Press.
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Discussion
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The nucleofection technology is ideally suited to performing RNAi studies in post-mitotic cells, such as hippocampal neurons. In contrast to other transfection methods, the high transfection efficiencies attainable with the nucleofection technique allow a proper analysis of the levels of Stau2 protein down-regulation by quantitative western blot analysis. In summary this study showed that the down-regulation of Stau2 causes changes in dendrite morphology and suggests a role of Stau2 in the transport of ß-actin mRNA into dendrites of hippocampal neurons.
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References
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[1] Broadus, J., S. Fuerstenberg, et al. (1998). "Staufen-dependent localization of prospero mRNA contributes to neuroblast daughter-cell fate." Nature 391(6669): 792-5.
[2] Brummelkamp, T. R., R. Bernards, et al. (2002). "A system for stable expression of short interfering RNAs in mammalian cells." Science 296(5567): 550-3.
[3] Dahm R, Zeitelhofer M, Götze B, Kiebler M and Macchi P (2008): Visualising mRNA localisation and local protein translation in neurons. Methods in Cell Biology (in press).
[4] Goetze, B., B. Grunewald, et al. (2004). "Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons." J Neurobiol 60(4): 517-25.
[5] Goetze, B., F. Tuebing, et al. (2006). "The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis." J Cell Biol 172(2): 221-31.
[6] Jansen, R. P. (2001). "mRNA localization: message on the move." Nat Rev Mol Cell Biol 2(4): 247-56.
[7] Kloc, M., N. R. Zearfoss, et al. (2002). "Mechanisms of subcellular mRNA localization." Cell 108(4): 533-44.
[8] Martin, K. C. and R. S. Zukin (2006). "RNA trafficking and local protein synthesis in dendrites: an overview." J Neurosci 26(27): 7131-4.
[9] St Johnston, D. (2005). "Moving messages: the intracellular localization of mRNAs." Nat Rev Mol Cell Biol 6(5): 363-75.
[10] Sutton, M. A. and E. M. Schuman (2006). "Dendritic protein synthesis, synaptic plasticity, and memory." Cell 127(1): 49-58.
[11] Zeitelhofer, M., J. P. Vessey, et al. (2007). "High-efficiency transfection of mammalian neurons via nucleofection." Nat Protoc 2(7): 1692-704.
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