Ralf Dahm

The GTP-Binding Protein Septin 7 Is Critical for Dendrite Branching and Dendritic-Spine Morphology

Septins, a highly conserved family of GTP-binding proteins, were originally identified in a genetic screen for S. cerevisiae mutants defective in cytokinesis [1, 2]. In yeast, septins maintain the compartmentalization of the yeast plasma membrane during cell division by forming rings at the cortex of the bud neck, and these rings establish a lateral diffusion barrier. In contrast, very little is known about the functions of septins in mammalian cells [3, 4] including postmitotic neurons [5-7]. Here, we show that Septin 7 (Sept7) localizes at the bases of filopodia and at branch points in developing hippocampal neurons. Upon downregulation of Sept7, dendritic branching is impaired. In mature neurons, Sept7 is found at the bases of dendritic spines where it associates with the plasma membrane. Mature Sept7-deficient neurons display elongated spines. Furthermore, Sept5 and Sept11 colocalize with and coimmunoprecipitate with Sept7, thereby arguing for the existence of a Septin5/7/11 complex. Taken together, our findings show an important role for Sept7 in regulating dendritic branching and dendritic-spine morphology. Our observations concur with data from yeast, in which downregulation of septins yields elongated buds, suggesting a conserved function for septins from yeast to mammals.

Dendritic Localization of the Translational Repressor Pumilio 2 and Its Contribution to Dendritic Stress Granules

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 (SGs), which are subsequently detected in the somatodendritic domain. It remains excluded from processing bodies under all conditions. When overexpressed in neurons and fibroblasts, Pum2 induces the formation of SGs that also contain T-cell intracellular antigen 1 (TIA-1)-related protein, eukaryotic initiation factor 4E, poly(A)-binding protein, TIA-1, and other RNA-binding proteins including Staufen1 and Barentsz. This induction of SGs is dependent on the RNA-binding domain and a glutamine-rich region in the N terminus of Pum2. This glutamine-rich region behaves in a similar manner as TIA-1 and prion protein, two molecules with known roles in protein aggregation. Pum2 downregulation in neurons via RNA interference (RNAi) interferes with the formation of SGs during metabolic stress. Cotransfection with an RNAi-resistant portion of the Pum2 mRNA restores SG formation. These results suggest a role for Pum2 in dendritic RNPs and SG formation in mammalian neurons.

Discovering DNA : Friedrich Miescher and the early years of nucleic acid research

In the winter of 1868/9 the young Swiss doctor Friedrich Miescher, working in the laboratory of Felix Hoppe-Seyler at the University of Tübingen, performed experiments on the chemical composition of leukocytes that lead to the discovery of DNA. In his experiments, Miescher noticed a precipitate of an unknown substance, which he characterised further. Its properties during the isolation procedure and its resistance to protease digestion indicated that the novel substance was not a protein or lipid. Analyses of its elementary composition revealed that, unlike proteins, it contained large amounts of phosphorous and, as Miescher confirmed later, lacked sulphur. Miescher recognised that he had discovered a novel molecule. Since he had isolated it from the cells’ nuclei he named it nuclein, a name preserved in today’s designation deoxyribonucleic acid. In subsequent work Miescher showed that nuclein was a characteristic component of all nuclei and hypothesised that it would prove to be inextricably linked to the function of this organelle. He suggested that its abundance in tissues might be related to their physiological status with increases in “nuclear substances” preceding cell division. Miescher even speculated that it might have a role in the transmission of hereditary traits, but subsequently rejected the idea. This article reviews the events and circumstances leading to Miescher’s discovery of DNA and places them within their historic context. It also tries to elucidate why it was Miescher who discovered DNA and why his name is not universally associated with this molecule today.

High-efficiency transfection of mammalian neurons via nucleofection

Transfection of foreign DNA is widely used to study gene function. However, despite the development of numerous methods, the transfer of DNA into postmitotic cells, such as neurons, remains unsatisfactory with regard to either transfection efficiency or cytotoxicity. Nucleofection overcomes these limitations. Direct electroporation of expression plasmids or oligonucleotides into the nucleus ensures both good cell viability and consistently high transfection rates. This allows biochemical analyses of transfected neurons, for example, western blot analyses of protein levels after RNA interference (RNAi) knockdown or microRNA transfection. We provide comprehensive protocols for performing nucleofection with high efficiency on primary neurons. The focus is on the recently developed 96-well shuttle system, which allows the simultaneous testing of up to 96 different plasmids or experimental conditions. Using this system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression. The protocol typically takes between 2 and 3 h.

Cell biology: Silenced RNA on the move

Proteins are often produced at their site of action, but the RNAs from which they are made must be kept inactive until they reach the right spot. It seems this ‘silencing’ of RNA is linked to its transport around the cell.

Visualizing mRNA Localization and Local Protein Translation in Neurons

Fluorescent proteins (FPs) have been successfully used to study the localization and interactions of proteins in living cells. They have also been instrumental in analyzing the proteins involved in the localization of RNAs in different cell types, including neurons. With the development of methods that also tag RNAs via fluorescent proteins, researchers now have a powerful tool to covisualize RNAs and associated proteins in living neurons. Here, we review the current status of the use of FPs in the study of transport and localization of ribonucleoprotein particles (RNPs) in neurons and provide key protocols used to introduce transgenes into cultured neurons, including calcium-phosphate-based transfection and nucleofection. These methods allow the fast and efficient expression of fluorescently tagged fusion proteins in neurons at different stages of differentiation and form the basis for fluorescent protein-based live cell imaging in neuronal cultures. Additional protocols are given that allow the simultaneous visualization of RNP proteins and cargo RNAs in living neurons and aspects of the visualization of fluorescently tagged proteins in neurons, such as colocalization studies, are discussed. Finally, we review approaches to visualize the local synthesis of proteins in distal dendrites and axons.

Alzheimer's discovery

On November 3rd 1906, the young doctor Alois Alzheimer delivers a talk in which he describes the psychiatric symptoms and changed brain histology of his late patient Auguste D. This moment marks the first publication of what was to become one of the most infamous afflictions, the disease that today bears his name: Alzheimers disease.

Transfection of Cultured Primary Neurons via Nucleofection

Despite the development of various transfection methods, the transfection of post‐mitotic cells, including neurons, poses a challenging task. Nucleofection, a specialized form of electroporation described in this unit, achieves high transfection efficiencies in primary mammalian neurons, such as hippocampal neurons, while simultaneously maintaining high cell viability. Therefore, it allows for biochemical analyses that rely on large numbers of transfected cells. The recently developed 96‐well shuttle system described in this unit further permits the transfection of up to 96 different constructs in a single experiment. This opens up the possibility for large‐scale experiments in primary neurons, such as shRNA‐mediated knock‐down of a wide range of target genes. Curr. Protoc. Neurosci. 47:4.32.1‐4.32.21.

Human pathologies associated with defective RNA transport and localization in the nervous system.

RNA localization is emerging as an important process to restrict certain proteins to specific subcellular domains and thus spatially control the expression of genes within cells. It is used, for instance, to compartmentalize the developing embryo during early embryogenesis. The localization of RNA also plays important roles later during development, such as in asymmetric cell divisions, cell migration and the outgrowth and pathfinding of axons and dendrites. In differentiated cells, it serves to subdivide the cell into functionally distinct compartments. For example, in mature neurons it is believed to contribute to the plastic changes of individual synapses underlying learning and memory. In this review, we highlight the importance of subcellular RNA localization for the function of the nervous system and neurological diseases associated with defective RNA localization and translation. These diseases include fragile X mental retardation syndrome, spinocerebellar ataxia and spinal muscular atrophy.

High-Efficiency Transfection of Short Hairpin RNAs-Encoding Plasmids Into Primary Hippocampal Neurons

The transfection of expression constructs encoding a variety of transgenes is a widely used method to study gene function in cultured cells. Especially when the efficiency of the knock‐down of target proteins via small interfering RNAs (siRNAs) is to be determined by quantitative Western blotting, large proportions of untransfected cells compromise the analysis. Achieving high transfection efficiencies in postmitotic cells, such as neurons, poses a particular problem in that these cells cannot be selected for the expression of the transgene following transfection. It is therefore important to develop protocols that allow for the highly efficient transfection of these cells. In the present study, we identify three important parameters that prove especially useful for chronically difficult to transfect short hairpin RNA (shRNA)‐encoding plasmids: the amount and quality of the plasmid DNA used and the use of new nucleofection programs. Combining those changes increases the rate of transfected cells from less than 5% to up to ∼80%. Importantly, these high transfection efficiencies can be obtained while maintaining good cell viability and normal cellular development. Taken together, these improvements allow for a detailed biochemical and phenotypical analysis of neurons that have been nucleoporated with a wide variety of shRNAs.