Glial swip-10 controls systemic mitochondrial function, oxidative stress, and neuronal viability via copper ion homeostasis.

Cuprous copper [Cu(I)] is an essential cofactor for enzymes that support many fundamental cellular functions including mitochondrial respiration and suppression of oxidative stress. Neurons are particularly reliant on mitochondrial production of ATP, with many neurodegenerative diseases, including Parkinson's disease, associated with diminished mitochondrial function. The gene MBLAC1 encodes a ribonuclease that targets pre-mRNA of replication-dependent histones, proteins recently found in yeast to reduce Cu(II) to Cu(I), and when mutated disrupt ATP production, elevates oxidative stress, and severely impacts cell growth. Whether this process supports neuronal and/or systemic physiology in higher eukaryotes is unknown. Previously, we identified swip-10, the putative Caenorhabditis elegans ortholog of MBLAC1, establishing a role for glial swip-10 in limiting dopamine (DA) neuron excitability and sustaining DA neuron viability. Here, we provide evidence from computational modeling that SWIP-10 protein structure mirrors that of MBLAC1 and locates a loss of function coding mutation at a site expected to disrupt histone RNA hydrolysis. Moreover, we find through genetic, biochemical, and pharmacological studies that deletion of swip-10 in worms negatively impacts systemic Cu(I) levels, leading to deficits in mitochondrial respiration and ATP production, increased oxidative stress, and neurodegeneration. These phenotypes can be offset in swip-10 mutants by the Cu(I) enhancing molecule elesclomol and through glial expression of wildtype swip-10. Together, these studies reveal a glial-expressed pathway that supports systemic mitochondrial function and neuronal health via regulation of Cu(I) homeostasis, a mechanism that may lend itself to therapeutic strategies to treat devastating neurodegenerative diseases.

An ankyrin-binding motif regulates nuclear levels of L1-type neuroglian and expression of the oncogene Myc in Drosophila neurons.

L1 cell adhesion molecule (L1CAM) is well-known for its importance in nervous system development and cancer progression. In addition to its role as a plasma membrane protein in cytoskeletal organization, recent in vitro studies have revealed that both transmembrane and cytosolic fragments of proteolytically cleaved vertebrate L1CAM translocate to the nucleus. In vitro studies indicate that nuclear L1CAM affects genes with functions in DNA post-replication repair, cell cycle control, and cell migration and differentiation, but its in vivo role and how its nuclear levels are regulated is less well-understood. Here, we report that mutations in the conserved ankyrin-binding domain affect nuclear levels of the sole Drosophila homolog neuroglian (Nrg) and that it also has a noncanonical role in regulating transcript levels of the oncogene Myc in the adult nervous system. We further show that altered nuclear levels of Nrg correlate with altered transcript levels of Myc in neurons, similar to what has been reported for human glioblastoma stem cells. However, whereas previous in vitro studies suggest that increased nuclear levels of L1CAM promote tumor cell survival, we found here that elevated levels of nuclear Nrg in neurons are associated with increased sensitivity to oxidative stress and reduced life span of adult animals. We therefore conclude that these findings are of potential relevance to the management of neurodegenerative diseases associated with oxidative stress and cancer.

Transsynaptic coordination of synaptic growth, function, and stability by the L1-type CAM Neuroglian.

The precise control of synaptic connectivity is essential for the development and function of neuronal circuits. While there have been significant advances in our understanding how cell adhesion molecules mediate axon guidance and synapse formation, the mechanisms controlling synapse maintenance or plasticity in vivo remain largely uncharacterized. In an unbiased RNAi screen we identified the Drosophila L1-type CAM Neuroglian (Nrg) as a central coordinator of synapse growth, function, and stability. We demonstrate that the extracellular Ig-domains and the intracellular Ankyrin-interaction motif are essential for synapse development and stability. Nrg binds to Ankyrin2 in vivo and mutations reducing the binding affinities to Ankyrin2 cause an increase in Nrg mobility in motoneurons. We then demonstrate that the Nrg-Ank2 interaction controls the balance of synapse growth and stability at the neuromuscular junction. In contrast, at a central synapse, transsynaptic interactions of pre- and postsynaptic Nrg require a dynamic, temporal and spatial, regulation of the intracellular Ankyrin-binding motif to coordinate pre- and postsynaptic development. Our study at two complementary model synapses identifies the regulation of the interaction between the L1-type CAM and Ankyrin as an important novel module enabling local control of synaptic connectivity and function while maintaining general neuronal circuit architecture.

Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition.

The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3rd instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.

Application for the Drosophila ventral nerve cord standard in neuronal circuit reconstruction and in-depth analysis of mutant morphology.

The Drosophila standard brain has been a useful tool that provides information about position and size of different brain structures within a wild-type brain and allows the comparison of imaging data that were collected from individual preparations. Therefore the standard can be used to reveal and visualize differences of brain regions between wild-type and mutant brains and can provide spatial description of single neurons within the nervous system. Recently the standard brain was complemented by the generation of a ventral nerve cord (VNC) standard. Here the authors have registered the major components of a simple neuronal circuit, the Giant Fiber System (GFS), into this standard. The authors show that they can also virtually reconstruct the well-characterized synaptic contact of the Giant Fiber with its motorneuronal target when they register the individual neurons from different preparations into the VNC standard. In addition to the potential application for the standard thorax in neuronal circuit reconstruction, the authors show that it is a useful tool for in-depth analysis of mutant morphology of single neurons. The authors find quantitative and qualitative differences when they compared the Giant Fibers of two different neuroglian alleles, nrg(849) and nrg(G00305), using the averaged wild-type GFS in the standard VNC as a reference.

Axon Termination, Pruning, and Synaptogenesis in the Giant Fiber System of Drosophila melanogaster Is Promoted by Highwire.

The ubiquitin ligase Highwire has a conserved role in synapse formation. Here, we show that Highwire coordinates several facets of central synapse formation in the Drosophila melanogaster giant fiber system, including axon termination, axon pruning, and synaptic function. Despite the similarities to the fly neuromuscular junction, the role of Highwire and the underlying signaling pathways are distinct in the fly's giant fiber system. During development, branching of the giant fiber presynaptic terminal occurs and, normally, the transient branches are pruned away. However, in highwire mutants these ectopic branches persist, indicating that Highwire promotes axon pruning. highwire mutants also exhibit defects in synaptic function. Highwire promotes axon pruning and synaptic function cell-autonomously by attenuating a mitogen-activated protein kinase pathway including Wallenda, c-Jun N-terminal kinase/Basket, and the transcription factor Jun. We also show a novel role for Highwire in non-cell autonomous promotion of synaptic function from the midline glia. Highwire also regulates axon termination in the giant fibers, as highwire mutant axons exhibit severe overgrowth beyond the pruning defect. This excessive axon growth is increased by manipulating Fos expression in the cells surrounding the giant fiber terminal, suggesting that Fos regulates a trans-synaptic signal that promotes giant fiber axon growth.

Whole mount preparation of the adult Drosophila ventral nerve cord for giant fiber dye injection.

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies. In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective (CvC) containing the big axons of the giant fibers (GFs) are exposed, while the brain containing the GF cell body and dendrites remains in the intact head. In this preparation most nerves of the VNC should remain attached to their muscles. Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures suitable for high resolution confocal microscopy.

Average shape standard atlas for the adult Drosophila ventral nerve cord.

Neuroanatomy benefits from quantification of neural structures, i.e., neurons, circuits, and brain parts, within a common reference system. Recent improvements in imaging techniques and increased computational power have made the creation of Web-based databases possible, which serve as common platforms for incorporating anatomical data. This study establishes a standard average shape atlas for the ventral nerve cord (VNC) of Drosophila melanogaster. This atlas allows for the registration of morphological, developmental, and genetic data into one quantitative 3D reference system. The standard is based on an average adult Drosophila VNC neuropil as labeled in 24 whole-mount preparations with the commercially available antibody (nc82) recognizing the Drosophila Bruchpilot protein (Brp). For the standardization procedure no expert knowledge of brain anatomy is required and global thresholding as well as straightforward affine and elastic registration procedures minimize user interactions. Successful registration is demonstrated for tracts and commissures, gene expression patterns, and geometric reconstructions of individual neurons. Any structure that is counterstained with anti-Brp can be registered into the standard, allowing for fast comparison of data from different experiments and different laboratories. In addition, standard transformations can be applied to gray scale image data, so that any confocal image stack that is colabeled with anti-Brp can be analyzed within standardized 3D reference coordinates. This allows for the creation of putative neural connectivity maps and the comparison of expression patterns derived from different preparations. The standard and protocols for histological methods, segmentation, and registration procedures will be made available on the Web.

Crystallization on confined engineered surfaces: a method to control crystal size and generate different polymorphs.

Patterned glycine crystals nucleated on functionalized metallic square islands. This approach can be used to fabricate particles with micron dimensions and screen solid forms under different conditions. The size of the glycine crystals is controlled by the dimensions of the islands. High energy metastable beta-glycine crystallizes on small metallic islands, whereas for large islands, the polymorphic outcome becomes biased toward the alpha-form.