Cross-regulation between an alternative splicing activator and a transcription repressor controls neurogenesis.

Neurogenesis requires the concerted action of numerous genes that are regulated at multiple levels. However, how different layers of gene regulation are coordinated to promote neurogenesis is not well understood. We show that the neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4) negatively regulates REST (NRSF), a transcriptional repressor of genes required for neurogenesis. nSR100 directly promotes alternative splicing of REST transcripts to produce a REST isoform (REST4) with greatly reduced repressive activity, thereby activating expression of REST targets in neural cells. Conversely, REST directly represses nSR100 in nonneural cells to prevent the activation of neural-specific splicing events. Consistent with a critical role for nSR100 in the inhibition of REST activity, blocking nSR100 expression in the developing mouse brain impairs neurogenesis. Our results thus reveal a fundamental role for direct regulatory interactions between a splicing activator and transcription repressor in the control of the multilayered regulatory programs required for neurogenesis.

A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis.

Here, we have asked about post-transcriptional mechanisms regulating murine developmental neurogenesis, focusing upon the RNA-binding proteins Smaug2 and Nanos1. We identify, in embryonic neural precursors of the murine cortex, a Smaug2 protein/nanos1 mRNA complex that is present in cytoplasmic granules with the translational repression proteins Dcp1 and 4E-T. We show that Smaug2 inhibits and Nanos1 promotes neurogenesis, with Smaug2 knockdown enhancing neurogenesis and depleting precursors, and Nanos1 knockdown inhibiting neurogenesis and maintaining precursors. Moreover, we show that Smaug2 likely regulates neurogenesis by silencing nanos1 mRNA. Specifically, Smaug2 knockdown inappropriately increases Nanos1 protein, and the Smaug2 knockdown-mediated neurogenesis is rescued by preventing this increase. Thus, Smaug2 and Nanos1 function as a bimodal translational repression switch to control neurogenesis, with Smaug2 acting in transcriptionally primed precursors to silence mRNAs important for neurogenesis, including nanos1 mRNA, and Nanos1 acting during the transition to neurons to repress the precursor state. SIGNIFICANCE STATEMENT: The mechanisms instructing neural stem cells to generate the appropriate progeny are still poorly understood. Here, we show that the RNA-binding proteins Smaug2 and Nanos1 are critical regulators of this balance and provide evidence supporting the idea that neural precursors are transcriptionally primed to generate neurons but translational regulation maintains these precursors in a stem cell state until the appropriate developmental time.

FoxP2 regulates neurogenesis during embryonic cortical development.

The transcription factor FoxP2 has been associated with the development of human speech but the underlying cellular function of FoxP2 is still unclear. Here we provide evidence that FoxP2 regulates genesis of some intermediate progenitors and neurons in the mammalian cortex, one of the key centers for human speech. Specifically, knockdown of FoxP2 in embryonic cortical precursors inhibits neurogenesis, at least in part by inhibiting the transition from radial glial precursors to neurogenic intermediate progenitors. Moreover, overexpression of human, but not mouse, FoxP2 enhances the genesis of intermediate progenitors and neurons. In contrast, expression of a human FoxP2 mutant that causes vocalization deficits decreases neurogenesis, suggesting that in the murine system human FoxP2 acts as a gain-of-function protein, while a human FoxP2 mutant acts as a dominant-inhibitory protein. These results support the idea that FoxP2 regulates the transition from neural precursors to transit-amplifying progenitors and ultimately neurons, and shed light upon the molecular changes that might contribute to evolution of the mammalian cortex.

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.

A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis.

The dsRNA-binding protein Staufen was the first RNA-binding protein proven to play a role in RNA localization in Drosophila. A mammalian homolog, Staufen1 (Stau1), has been implicated in dendritic RNA localization in neurons, translational control, and mRNA decay. However, the precise mechanisms by which it fulfills these specific roles are only partially understood. To determine its physiological functions, the murine Stau1 gene was disrupted by homologous recombination. Homozygous stau1(tm1Apa) mutant mice express a truncated Stau1 protein lacking the functional RNA-binding domain 3. The level of the truncated protein is significantly reduced. Cultured hippocampal neurons derived from stau1(tm1Apa) homozygous mice display deficits in dendritic delivery of Stau1-EYFP and beta-actin mRNA-containing ribonucleoprotein particles (RNPs). Furthermore, these neurons have a significantly reduced dendritic tree and develop fewer synapses. Homozygous stau1(tm1Apa) mutant mice are viable and show no obvious deficits in development, fertility, health, overall brain morphology, and a variety of behavioral assays, e.g., hippocampus-dependent learning. However, we did detect deficits in locomotor activity. Our data suggest that Stau1 is crucial for synapse development in vitro but not critical for normal behavioral function.

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.

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.

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.

Proton-mediated feedback inhibition of presynaptic calcium channels at the cone photoreceptor synapse.

Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals. Both conventional and unconventional neurotransmitters, as well as an ephaptic effect, have been proposed, but the intercellular messaging that mediates the inhibitory feedback signal from postsynaptic horizontal cells (HCs) to cones remains unknown. We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones. In isolated, dark-adapted goldfish retina, we assessed feedback in the responses of HCs to light and found that strengthened pH buffering reduced both rollback and the depolarization to red light. In zebrafish retinal slices loaded with Fluo-4, depolarization with elevated K(+) increased Ca signals in the synaptic terminals of cone photoreceptors. Kainic acid, which depolarizes HCs but has no direct effect on cones, depressed the K(+)-induced Ca signal, whereas CNQX, which hyperpolarizes HCs, increased the Ca signals, suggesting that polarization of HCs alters inhibitory feedback to cones. We found that these feedback signals were blocked by elevated extracellular pH buffering, as well as amiloride and divalent cations. Voltage clamp of isolated HCs revealed an amiloride-sensitive conductance that could mediate modulation of cleft pH dependent on the membrane potential of these postsynaptic cells.

Transient maternal IL-6 mediates long-lasting changes in neural stem cell pools by deregulating an endogenous self-renewal pathway.

The mechanisms that regulate the establishment of adult stem cell pools during normal and perturbed mammalian development are still largely unknown. Here, we asked whether a maternal cytokine surge, which occurs during human maternal infections and has been implicated in cognitive disorders, might have long-lasting consequences for neural stem cell pools in adult progeny. We show that transient, maternally administered interleukin-6 (IL-6) resulted in an expanded adult forebrain neural precursor pool and perturbed olfactory neurogenesis in offspring months after fetal exposure. This increase is likely the long-term consequence of acute hyperactivation of an endogenous autocrine/paracrine IL-6-dependent self-renewal pathway that normally regulates the number of forebrain neural precursors. These studies therefore identify an IL-6-dependent neural stem cell self-renewal pathway in vivo, and support a model in which transiently increased maternal cytokines can act through this pathway in offspring to deregulate neural precursor biology from embryogenesis throughout life.

An asymmetrically localized Staufen2-dependent RNA complex regulates maintenance of mammalian neural stem cells.

The cellular mechanisms that regulate self-renewal versus differentiation of mammalian somatic tissue stem cells are still largely unknown. Here, we asked whether an RNA complex regulates this process in mammalian neural stem cells. We show that the RNA-binding protein Staufen2 (Stau2) is apically localized in radial glial precursors of the embryonic cortex, where it forms a complex with other RNA granule proteins including Pumilio2 (Pum2) and DDX1, and the mRNAs for ß-actin and mammalian prospero, prox1. Perturbation of this complex by functional knockdown of Stau2, Pum2, or DDX1 causes premature differentiation of radial glial precursors into neurons and mislocalization and misexpression of prox1 mRNA. Thus, a Stau2- and Pum2-dependent RNA complex directly regulates localization and, potentially, expression of target mRNAs like prox1 in mammalian neural stem cells, and in so doing regulates the balance of stem cell maintenance versus differentiation.

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.

More than just synaptic building blocks: scaffolding proteins of the post-synaptic density regulate dendritic patterning.

The dendritic arbor is responsible for receiving and consolidating neuronal input. Outgrowth and morphogenesis of the arbor are complex stages of development that are poorly understood. However, recent findings have identified synaptic scaffolding proteins as novel regulators of these important events. Scaffolding proteins are enriched in the post-synaptic density where they bind and bring into close proximity neurotransmitter receptors, signaling molecules, and regulators of the actin cytoskeleton. This property is important for dendritic spine morphogenesis and maintenance in the mature neuron. Scaffolding proteins are now being described as key regulators of neurite outgrowth, dendritic development, and pattern formation in immature neurons. These proteins, which include post-synaptic-95, Shank and Densin-180, as well as many of their interacting partners, appear to regulate both the microtubule and actin cytoskeleton to influence dendrite morphology. Through a large array of protein-protein interaction domains, scaffolding proteins are able to form large macromolecular complexes that include cytoskeletal motor proteins as well as microtubule and actin regulatory molecules. Together, the new findings form a persuasive argument that scaffolding proteins deliver critical regulatory elements to sites of dendritic outgrowth and branching to modulate the formation and maintenance of the dendritic arbor.

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.

Mutation of the calcium channel gene Cacna1f disrupts calcium signaling, synaptic transmission and cellular organization in mouse retina.

Retinal neural transmission represents a key function of the eye. Identifying the molecular components of this vital process is helped by studies of selected human genetic eye disorders. For example, mutations in the calcium channel subunit gene CACNA1F cause incomplete X-linked congenital stationary night blindness (CSNB2 or iCSNB), a human retinal disorder with abnormal electrophysiological response and visual impairments consistent with a retinal neurotransmission defect. To understand the subcellular basis of this retinal disorder, we generated a mouse with a loss-of-function mutation by inserting a self-excising Cre-lox-neo cassette into exon 7 of the murine orthologue, Cacna1f. Electroretinography of the mutant mouse revealed a scotopic a-wave of marginally reduced amplitude compared with the wild-type mouse and absence of the post-receptoral b-wave and oscillatory potentials. Cone ERG responses together with visual evoked potentials and multi-unit activity in the superior colliculus were also absent. Calcium imaging in Fluo-4 loaded retinal slices depolarized with KCl showed 90% less peak signal in the photoreceptor synapses of the Cacna1f mutant than in wild-type mice. The absence of post-receptoral ERG responses and the diminished photoreceptor calcium signals are consistent with a loss of Ca((2+)) channel function in photoreceptors. Immunocytochemistry showed no detectable Ca(v)1.4 protein in the outer plexiform layer of Cacna1f-mutant mice, profound loss of photoreceptor synapses, and abnormal dendritic sprouting of second-order neurons in the photoreceptor layer. Together, these findings in the Cacna1f-mutant mouse reveal that the Ca(v)1.4 calcium channel is vital for the functional assembly and/or maintenance and synaptic functions of photoreceptor ribbon synapses. Moreover, the outcome of this study provides critical clues to the pathophysiology of the human retinal channelopathy of X-linked incomplete CSNB.

Carbenoxolone inhibition of voltage-gated Ca channels and synaptic transmission in the retina.

We show that carbenoxolone, a drug used to block hemichannels in the retina to test the ephaptic model of horizontal cell inhibitory feedback, has strong inhibitory effects on voltage-gated Ca channels. Carbenoxolone (100 microM) reduced photoreceptor-to-horizontal cell synaptic transmission by 92%. Applied to patch-clamped, isolated cone photoreceptors, carbenoxolone inhibited Ca channels with an EC(50) of 48 microM. At 100 microM, it reduced cone Ca channel current by 37%, reduced depolarization-evoked [Ca(2+)] signals in fluo-4 loaded retinal slices by 57% and inhibited Ca channels in Müller cells by 52%. A synaptic transfer model suggests that the degree of block of Ca channels accounts for the reduction in synaptic transmission. These results suggest broad inhibitory actions for carbenoxolone in the retina that must be considered when interpreting its effects on inhibitory feedback.

Hyposmotic activation of ICl,swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane.

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl(-) conductance (I(Cl,swell)), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the I(Cl,swell) following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl(-) current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as I(Cl,swell). Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of I(Cl,swell). Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to I(Cl,swell) following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.