Augmin complex activity finetunes dendrite morphology through non-centrosomal microtubule nucleation in vivo.

During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo.

Fringe-positive Golgi outposts unite temporal Furin 2 convertase activity and spatial Delta signal to promote dendritic branch retraction.

Golgi outposts (GOPs) in dendrites are known for their role in promoting branch extension, but whether GOPs have other functions is unclear. We found that terminal branches of Drosophila class IV dendritic arborization (C4da) neurons actively grow during the early third-instar (E3) larval stage but retract in the late third (L3) stage. Interestingly, the Fringe (Fng) glycosyltransferase localizes increasingly at GOPs in distal dendritic regions through the E3 to the L3 stage. Expression of the endopeptidase Furin 2 (Fur2), which proteolyzes and inactivates Fng, decreases from E3 to L3 in C4da neurons, thereby increasing Fng-positive GOPs in dendrites. The epidermal Delta ligand and neuronal Notch receptor, the substrate for Fng-mediated O-glycosylation, also negatively regulate dendrite growth. Fng inhibits actin dynamics in dendrites, linking dendritic branch retraction to suppression of the C4da-mediated thermal nociception response in late larval stages. Thus, Fng-positive GOPs function in dendrite retraction, which would add another function to the repertoire of GOPs in dendrite arborization.

An Efficient Screen for Cell-Intrinsic Factors Identifies the Chaperonin CCT and Multiple Conserved Mechanisms as Mediating Dendrite Morphogenesis.

Dendritic morphology is inextricably linked to neuronal function. Systematic large-scale screens combined with genetic mapping have uncovered several mechanisms underlying dendrite morphogenesis. However, a comprehensive overview of participating molecular mechanisms is still lacking. Here, we conducted an efficient clonal screen using a collection of mapped P-element insertions that were previously shown to cause lethality and eye defects in Drosophila melanogaster. Of 280 mutants, 52 exhibited dendritic defects. Further database analyses, complementation tests, and RNA interference validations verified 40 P-element insertion genes as being responsible for the dendritic defects. Twenty-eight mutants presented severe arbor reduction, and the remainder displayed other abnormalities. The intrinsic regulators encoded by the identified genes participate in multiple conserved mechanisms and pathways, including the protein folding machinery and the chaperonin-containing TCP-1 (CCT) complex that facilitates tubulin folding. Mutant neurons in which expression of CCT4 or CCT5 was depleted exhibited severely retarded dendrite growth. We show that CCT localizes in dendrites and is required for dendritic microtubule organization and tubulin stability, suggesting that CCT-mediated tubulin folding occurs locally within dendrites. Our study also reveals novel mechanisms underlying dendrite morphogenesis. For example, we show that Drosophila Nogo signaling is required for dendrite development and that Mummy and Wech also regulate dendrite morphogenesis, potentially via Dpp- and integrin-independent pathways. Our methodology represents an efficient strategy for identifying intrinsic dendrite regulators, and provides insights into the plethora of molecular mechanisms underlying dendrite morphogenesis.

Glia-derived exosomal miR-274 targets Sprouty in trachea and synaptic boutons to modulate growth and responses to hypoxia.

Secreted exosomal microRNAs (miRNAs) mediate interorgan/tissue communications by modulating target gene expression, thereby regulating developmental and physiological functions. However, the source, route, and function in target cells have not been formally established for specific miRNAs. Here, we show that glial miR-274 non-cell-autonomously modulates the growth of synaptic boutons and tracheal branches. Whereas the precursor form of miR-274 is expressed in glia, the mature form of miR-274 distributes broadly, including in synaptic boutons, muscle cells, and tracheal cells. Mature miR-274 is secreted from glia to the circulating hemolymph as an exosomal cargo, a process requiring ESCRT components in exosome biogenesis and Rab11 and Syx1A in exosome release. We further show that miR-274 can function in the neurons or tracheal cells to modulate the growth of synaptic boutons and tracheal branches, respectively. Also, miR-274 uptake into the target cells by AP-2-dependent mechanisms modulates target cell growth. In the target cells, miR-274 down-regulates Sprouty (Sty) through a targeting sequence at the sty 3' untranslated region, thereby enhancing MAPK signaling and promoting cell growth. miR-274 expressed in glia of an mir-274 null mutant is released as an exosomal cargo in the circulating hemolymph, and such glial-specific expression resets normal levels of Sty and MAPK signaling and modulates target cell growth. mir-274 mutant larvae are hypersensitive to hypoxia, which is suppressed by miR-274 expression in glia or by increasing tracheal branches. Thus, glia-derived miR-274 coordinates growth of synaptic boutons and tracheal branches to modulate larval hypoxia responses.

Glial response to hypoxia in mutants of NPAS1/3 homolog Trachealess through Wg signaling to modulate synaptic bouton organization.

Synaptic structure and activity are sensitive to environmental alterations. Modulation of synaptic morphology and function is often induced by signals from glia. However, the process by which glia mediate synaptic responses to environmental perturbations such as hypoxia remains unknown. Here, we report that, in the mutant for Trachealess (Trh), the Drosophila homolog for NPAS1 and NPAS3, smaller synaptic boutons form clusters named satellite boutons appear at larval neuromuscular junctions (NMJs), which is induced by the reduction of internal oxygen levels due to defective tracheal branches. Thus, the satellite bouton phenotype in the trh mutant is suppressed by hyperoxia, and recapitulated in wild-type larvae raised under hypoxia. We further show that hypoxia-inducible factor (HIF)-1α/Similar (Sima) is critical in mediating hypoxia-induced satellite bouton formation. Sima upregulates the level of the Wnt/Wingless (Wg) signal in glia, leading to reorganized microtubule structures within presynaptic sites. Finally, hypoxia-induced satellite boutons maintain normal synaptic transmission at the NMJs, which is crucial for coordinated larval locomotion.

Epidermis-Derived L1CAM Homolog Neuroglian Mediates Dendrite Enclosure and Blocks Heteroneuronal Dendrite Bundling.

Building sensory dendritic arbors requires branching, growth, spacing, and substrate support. The conserved L1CAM family of cell-adhesion molecules generates neuronal isoforms to regulate neurite development in various aspects. However, whether non-neuronal isoforms participate in any of these aspects is unclear. In Drosophila, the L1CAM homolog Neuroglian (Nrg) is expressed as two isoforms: the neuronal isoform Nrg180 on dendritic surfaces of dendritic arborization (da) neurons and the non-neuronal isoform Nrg167 in epidermis innervated by dendrites. We found that epidermal Nrg167 encircles dendrites by interactions with dendritic Nrg180 to support dendrite growth, stabilization, and enclosure inside epidermis. Interestingly, whereas Nrg180 forms homophilic interactions to facilitate axonal bundling, heteroneuronal dendrites in the same innervating field avoid bundling through unknown mechanisms to maintain individual dendritic patterns. Here, we show that both epidermal Nrg167 depletion and neuronal Nrg180 overexpression can cause dendrite bundling, with genetic analyses suggesting that Nrg167-Nrg180 interactions antagonize Nrg180-Nrg180 homophilic interaction to prevent dendrite bundling. Furthermore, internalization of Nrg180 also participates in resolving dendrite bundling, as overexpression of endocytosis-defective Nrg180 and compromising endocytosis in neurons both exacerbated dendrite-bundling defects. Together, our study highlights the functional significance of substrate-derived Nrg167 in conferring dendrite stability, positioning, and avoidance.

USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin.

Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. Here, we show that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth.

Dbo/Henji Modulates Synaptic dPAK to Gate Glutamate Receptor Abundance and Postsynaptic Response.

In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. Here, we analyzed mutant synapses of henji, also known as dbo, at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but that of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. We propose that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK.

Lrrk regulates the dynamic profile of dendritic Golgi outposts through the golgin Lava lamp.

Constructing the dendritic arbor of neurons requires dynamic movements of Golgi outposts (GOPs), the prominent component in the dendritic secretory pathway. GOPs move toward dendritic ends (anterograde) or cell bodies (retrograde), whereas most of them remain stationary. Here, we show that Leucine-rich repeat kinase (Lrrk), the Drosophila melanogaster homologue of Parkinson's disease-associated Lrrk2, regulates GOP dynamics in dendrites. Lrrk localized at stationary GOPs in dendrites and suppressed GOP movement. In Lrrk loss-of-function mutants, anterograde movement of GOPs was enhanced, whereas Lrrk overexpression increased the pool size of stationary GOPs. Lrrk interacted with the golgin Lava lamp and inhibited the interaction between Lva and dynein heavy chain, thus disrupting the recruitment of dynein to Golgi membranes. Whereas overexpression of kinase-dead Lrrk caused dominant-negative effects on GOP dynamics, overexpression of the human LRRK2 mutant G2019S with augmented kinase activity promoted retrograde movement. Our study reveals a pathogenic pathway for LRRK2 mutations causing dendrite degeneration.

Neurofibromin mediates FAK signaling in confining synapse growth at Drosophila neuromuscular junctions.

Neurofibromatosis type I (NF1), caused by the mutation in the NF1 gene, is characterized by multiple pathological symptoms. Importantly, ~50% of NF1 patients also suffer learning difficulty. Although downstream pathways are well studied, regulation of the NF1-encoded neurofibromin protein is less clear. Here, we focused on the pathophysiology of Drosophila NF1 mutants in synaptic growth at neuromuscular junctions. Our analysis suggests that the Drosophila neurofibromin protein NF1 is required to constrain synaptic growth and transmission. NF1 functions downstream of the Drosophila focal adhesion kinase (FAK) Fak56 and physically interacts with Fak56. The N-terminal region of NF1 mediates the interaction with Fak56 and is required for the signaling activity and presynaptic localization of NF1. In presynapses, NF1 acts via the cAMP pathway, but independent of its GAP activity, to restrain synaptic growth. Thus, presynaptic FAK signaling may be disrupted, causing abnormal synaptic growth and transmission in the NF1 genetic disorder.

Activity-dependent retrograde laminin A signaling regulates synapse growth at Drosophila neuromuscular junctions.

Retrograde signals induced by synaptic activities are derived from postsynaptic cells to potentiate presynaptic properties, such as cytoskeletal dynamics, gene expression, and synaptic growth. However, it is not known whether activity-dependent retrograde signals can also depotentiate synaptic properties. Here we report that laminin A (LanA) functions as a retrograde signal to suppress synapse growth at Drosophila neuromuscular junctions (NMJs). The presynaptic integrin pathway consists of the integrin subunit ßν and focal adhesion kinase 56 (Fak56), both of which are required to suppress crawling activity-dependent NMJ growth. LanA protein is localized in the synaptic cleft and only muscle-derived LanA is functional in modulating NMJ growth. The LanA level at NMJs is inversely correlated with NMJ size and regulated by larval crawling activity, synapse excitability, postsynaptic response, and anterograde Wnt/Wingless signaling, all of which modulate NMJ growth through LanA and ßν. Our data indicate that synaptic activities down-regulate levels of the retrograde signal LanA to promote NMJ growth.

Liquid chromatography-tandem mass spectrometry determination of the toxicity and identification of fish species in a suspected tetrodotoxin fish poisoning.

Suspected tetrodotoxin (TTX) poisoning was associated with eating unknown fish in April 2009 in Taiwan. After ingestion of the fish, symptoms of the victim included perioral paresthesia, nausea, vomiting, ataxia, weakness of all limbs, respiration failure, and death within several hours. The toxicity in the remaining fish was determined, with the mice exhibiting symptoms of neurotoxin poisoning. The implicated fish and deceased victim tissues were analyzed for TTX by liquid chromatography-tandem mass spectrometry. The urine, bile, cerebrospinal fluid (spinal cord), pleural effusion, and pericardial effusion of the victim contained TTX. In addition, the partial cytochrome b gene of the implicated fish was determined by PCR. The DNA sequence in the partial 465-bp cytochrome b gene identified the implicated fish as Chelonodon patoca (puffer fish). These results indicate that people should avoid eating unknown fish species from fish markets where harvested fish may include toxic species.