Here we show that glioblastoma express high levels of branched-chain amino acid transaminase 1 (BCAT1), the enzyme that initiates the catabolism of branched-chain amino acids (BCAAs). Expression of BCAT1 was exclusive to tumors carrying wild-type isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and was highly correlated with methylation patterns in the BCAT1 promoter region. BCAT1 expression was dependent on the concentration of α-ketoglutarate substrate in glioma cell lines and could be suppressed by ectopic overexpression of mutant IDH1 in immortalized human astrocytes, providing a link between IDH1 function and BCAT1 expression. Suppression of BCAT1 in glioma cell lines blocked the excretion of glutamate and led to reduced proliferation and invasiveness in vitro, as well as significant decreases in tumor growth in a glioblastoma xenograft model. These findings suggest a central role for BCAT1 in glioma pathogenesis, making BCAT1 and BCAA metabolism attractive targets for the development of targeted therapeutic approaches to treat patients with glioblastoma.

Pumilio (Pum) protein acts as a translational inhibitor in several organisms including yeast, Drosophila, Xenopus, and mammals. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals, but their function in neurons has not been identified. In this study, we found that Pum2 mRNA is expressed during neuronal development and that the protein is found in discrete particles in both the cell body and the dendritic compartment of fully polarized neurons. This finding indicates that Pum2 is a novel candidate of dendritically localized ribonucleoparticles (RNPs). During metabolic stress, Pum2 is present in stress granules (

Editor's Note: Toolboxes are intended to describe and evaluate methods that are becoming widely relevant to the neuroscience community or to provide a critical analysis of established techniques. For more information, see http://www.jneurosci.org/misc/ ifa_minireviews.dtl. Transfection Techniques for Neuronal CellsDaniela Karra 1 and Ralf Dahm 2,3 1 Institute for Neuropathology, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany, 2 Department of Biology, University of Padua, I-35121 Padua, Italy, and 3 Spanish National Cancer Research Centre (CNIO), E-28029 Madrid, Spain IntroductionThe transfection of nucleic acids into cells is crucial for the study of many aspects of neuronal cell biology. These include investigating gene and protein function by knocking down target proteins via RNA interference (RNAi) or microRNAs, expressing tagged proteins to track their subcellular localization, behavior, and turnover; and expressing mutant versions of proteins to study the functions of specific domains or mimic disease conditions. Moreover, reporter proteins can be used to detect intracellular ion concentrations or levels of gene expression.Despite efforts to optimize transfection techniques and protocols for neurons, no method has yet been developed that is suitable for all applications. Instead, the various established methods have their own advantages and drawbacks concerning transfection efficiency, expression levels, cell survival, and viability. Other considerations are the ease of use, reproducibility, cost, and applicability to a given experiment. Researchers therefore often face a bewildering roster of possibilities, making it difficult to decide which approach to take.In this review we provide a brief overview of methods used to transfect mam-

The dendritic localization of mRNAs and their subsequent translation at stimulated synapses contributes to the experience-dependent remodeling of synapses and thereby to the establishment of long-term memory. Localized mRNAs are transported in a translationally silent manner to distal dendrites in specific ribonucleoprotein particles (RNPs), termed transport RNPs. A recent study suggested that processing bodies (P-bodies), which have recently been identified as sites of RNA degradation and translational control in eukaryotic cells, may participate in the translational control of dendritically localized mRNAs in Drosophila neurons. This study raised the interesting question of whether dendritic transport RNPs are distinct from P-bodies or whether those structures share significant overlap in their molecular composition in mammalian neurons. Here, we show that P-body and transport RNP markers do not colocalize and are not transported together in the same particles in dendrites of mammalian neurons. Detailed time-lapse videomicroscopy analyses reveal, however, that both P-bodies and transport RNPs can interact in a dynamic manner via docking. Docking is a frequent event involving as much as 50% of all dendritic P-bodies. Chemically induced neuronal activity results in a 60% decrease in the number of P-bodies in dendrites, suggesting that P-bodies disassemble after synaptic stimulation. Our data lend support to the exciting hypothesis that dendritically localized mRNAs might be stored in P-bodies and be released and possibly translated when synapses become activated.