Ankyrin-G induces nucleoporin Nup358 to associate with the axon initial segment of neurons.

Nup358 (also known as RanBP2) is a member of the large nucleoporin family that constitutes the nuclear pore complex. Depending on the cell type and the physiological state, Nup358 interacts with specific partner proteins and influences distinct mechanisms independent of its role in nucleocytoplasmic transport. Here, we provide evidence that Nup358 associates selectively with the axon initial segment (AIS) of mature neurons, mediated by the AIS scaffold protein ankyrin-G (AnkG, also known as Ank3). The N-terminus of Nup358 is demonstrated to be sufficient for its localization at the AIS. Further, we show that Nup358 is expressed as two isoforms, one full-length and another shorter form of Nup358. These isoforms differ in their subcellular distribution in neurons and expression level during neuronal development. Overall, the present study highlights an unprecedented localization of Nup358 within the AIS and suggests its involvement in neuronal function.This article has an associated First Person interview with the first author of the paper.

The cell adhesion molecule neuroplastin-65 is a novel interaction partner of γ-aminobutyric acid type A receptors.

γ-Aminobutyric acid type A (GABA(A)) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABA(A) receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABA(A) receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABA(A) receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABA(A) receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABA(A) receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABA(A) receptor subtypes and might contribute to anchoring and/or confining GABA(A) receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength.

Mammalian Pumilio 2 regulates dendrite morphogenesis and synaptic function.

In Drosophila, Pumilio (Pum) is important for neuronal homeostasis as well as learning and memory. We have recently characterized a mammalian homolog of Pum, Pum2, which is found in discrete RNA-containing particles in the somatodendritic compartment of polarized neurons. In this study, we investigated the role of Pum2 in developing and mature neurons by RNA interference. In immature neurons, loss of Pum2 led to enhanced dendritic outgrowth and arborization. In mature neurons, Pum2 down-regulation resulted in a significant reduction in dendritic spines and an increase in elongated dendritic filopodia. Furthermore, we observed an increase in excitatory synapse markers along dendritic shafts. Electrophysiological analysis of synaptic function of neurons lacking Pum2 revealed an increased miniature excitatory postsynaptic current frequency. We then identified two specific mRNAs coding for a known translational regulator, eIF4E, and for a voltage-gated sodium channel, Scn1a, which interacts with Pum2 in immunoprecipitations from brain lysates. Finally, we show that Pum2 regulates translation of the eIF4E mRNA. Taken together, our data reveal a previously undescribed role for Pum2 in dendrite morphogenesis, synapse function, and translational control.

Dendritically localized transcripts are sorted into distinct ribonucleoprotein particles that display fast directional motility along dendrites of hippocampal neurons.

Localization of mRNAs to postsynaptic sites and their subsequent translation is thought to contribute to synapse-specific plasticity. However, the direct visualization of dendritic RNA transport in living neurons remains a major challenge. Here, we analyze the transport of Alexa-labeled RNAs microinjected into mature hippocampal neurons. We show that microinjected MAP2 and CaMKIIalpha RNAs form particles that localize into dendrites as their endogenous counterparts. In contrast, nonlocalizing RNAs or truncated CaMKIIalpha, lacking the dendritic targeting element, remain in the cell body. Furthermore, our microinjection approach allowed us to identify a novel dendritically localized RNA, Septin7. Time-lapse videomicroscopy of neurons injected with CaMKIIalpha and Septin7 RNAs demonstrates fast directional movement along the dendrites of hippocampal neurons, with similar kinetics to Staufen1 ribonucleoprotein particles (RNPs). Coinjection and simultaneous visualization of two RNAs, as well as double detection of the corresponding endogenous RNAs, reveal that neuronal transcripts are differentially sorted in dendritic RNPs.

The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis.

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.

Dynamic interaction between P-bodies and transport ribonucleoprotein particles in dendrites of mature hippocampal neurons.

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.

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 approximately 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.

Staufen2 regulates neuronal target RNAs.

RNA-binding proteins play crucial roles in directing RNA translation to neuronal synapses. Staufen2 (Stau2) has been implicated in both dendritic RNA localization and synaptic plasticity in mammalian neurons. Here, we report the identification of functionally relevant Stau2 target mRNAs in neurons. The majority of Stau2-copurifying mRNAs expressed in the hippocampus are present in neuronal processes, further implicating Stau2 in dendritic mRNA regulation. Stau2 targets are enriched for secondary structures similar to those identified in the 3' UTRs of Drosophila Staufen targets. Next, we show that Stau2 regulates steady-state levels of many neuronal RNAs and that its targets are predominantly downregulated in Stau2-deficient neurons. Detailed analysis confirms that Stau2 stabilizes the expression of one synaptic signaling component, the regulator of G protein signaling 4 (Rgs4) mRNA, via its 3' UTR. This study defines the global impact of Stau2 on mRNAs in neurons, revealing a role in stabilization of the levels of synaptic targets.

Interactome of two diverse RNA granules links mRNA localization to translational repression in neurons.

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.

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.

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.