Drosophila Motor Neurons Research Articles

The ALS gene FUS regulates synaptic transmission at the Drosophila neuromuscular junction

Mutations in the RNA binding protein Fused in sarcoma (FUS) are estimated to account for 5-10% of all inherited cases of amyotrophic lateral sclerosis (ALS), but the function of FUS in motor neurons is poorly understood. Here, we investigate the early functional consequences of overexpressing wild-type or ALS-associated mutant FUS proteins in Drosophila motor neurons, and compare them to phenotypes arising from loss of the Drosophila homolog of FUS, Cabeza (Caz). We find that lethality and locomotor phenotypes correlate with levels of FUS transgene expression, indicating that toxicity in developing motor neurons is largely independent of ALS-linked mutations. At the neuromuscular junction (NMJ), overexpression of either wild-type or mutant FUS results in decreased number of presynaptic active zones and altered postsynaptic glutamate receptor subunit composition, coinciding with a reduction in synaptic transmission as a result of both reduced quantal size and quantal content. Interestingly, expression of human FUS downregulates endogenous Caz levels, demonstrating that FUS autoregulation occurs in motor neurons in vivo. However, loss of Caz from motor neurons increases synaptic transmission as a result of increased quantal size, suggesting that the loss of Caz in animals expressing FUS does not contribute to motor deficits. These data demonstrate that FUS/Caz regulates NMJ development and plays an evolutionarily conserved role in modulating the strength of synaptic transmission in motor neurons.

Identifying dominant modifiers of mutant FUS toxicity in vivo

FUS is a DNA/RNA binding protein found to be mutated in some cases of both sporadic and familial forms of ALS. It is still not clear how ALS-causing mutations in FUS leads to motor neuron degeneration. Here, we exploited a Drosophila model and mammalian neuronal cell lines to elucidate the role of the RNA-binding ability of FUS in regulating FUS-mediated toxicity. To determine the role of the RNA binding ability of FUS in ALS, we mutated FUS RNA binding sites (F305, F341, F359, F368) to leucines and generated RNA binding-incompetent mutants (4F-L) with and without ALS causing mutations R518K or R521C. We found that mutating 4F to L residues makes FUS RNA binding-incompetent. We observed that ectopic expression of RNA binding-incompetent FUS in fly brain, eyes and motor neurons strongly blocks neurodegenerative phenotypes as compared to RNA-binding-competent FUS carrying ALS causing mutations. Interestingly, RNA-binding deficient FUS strongly localized to the nucleus of Drosophila motor neurons and mammalian neuronal cells whereas FUS carrying ALS linked mutations was distributed to the nucleus and cytoplasm. Importantly, we found that incorporation of mutant FUS into stress granules is dependent on the RNA-binding ability of FUS. SGs are dynamic aggregates composed of proteins and RNA that are formed when cells are under a variety of stresses. We observed that normally cytoplasmic SGs rapidly disassemble when stress conditions end, whereas cytoplasmic SGs formed in ALS patient cells having a FUS mutation fail to disassemble. This suggests that mutant FUS sequesters proteins and RNAs important for cellular homeostasis and the defect in disassembly of cytoplasmic SGs contributes to ALS. Finally, following up these observations we performed an in vivo unbiased genetic screen that led to the discovery of several dominant modifiers of mutant FUS toxicity including proteins involved in regulating SG dynamics, mitochondrial functions and RNA splicing. These genetic modifiers would help in identifying potential therapeutic targets for ALS.

A continuum approach to model neurites/dendrites with emerging subtrees

Neuronal morphology plays an important role in the processing of spatiotemporal synaptic input patterns. We formulate a new cable theoretic model to study the output in a dendritic cable with many subtrees, both analytically and computationally. The model is an extension of the continuum spine model derived by Baer and Rinzel [1] and like that model, there is no direct interaction between branches. Instead, all interactions occur through the primary dendrite. The continuum branch formulation provides a link between analytical and computational approaches and allows for detailed models of structural plasticity with the advantage that parameters like branch density can be varied dynamically. This is in contrast to multicompartment modeling approaches where different models, each having a static morphology, must be used. A multicompartment model with equally spaced discrete branches implemented in NEURON [2] is used to validate the continuum branch model. Then we demonstrate the approach using Drosophila Motoneuron 5 (MN5), a monopolar neuron, where soma and axon are connected via a long primary neurite from which dendritic subtrees emerge. Parameters for the model are based on 3D reconstructions [3] of that neuron and the subtrees are reduced to uniform cables. For a passive membrane and uniformly distributed branches, this formulation provides analytic estimates of electrical parameters, such as the input resistance. Transforming the equations to the frequency domain, we examine the steady state response properties for periodic forcing, where the impact of structural properties on transfer properties and the phase shift at different locations are calculated directly. Based on calculations of the voltage response along the primary neurite, the continuum formulation performs much better than a simple cable as an approximation of the morphologically realistic model. Simulations of the continuum model are in good agreement with the realistic model for frequencies up to 1000 Hz. In insects neurons the spike initiation zone (SIZ) is usually located far away from the recording site. We use this model to investigate the impact of the location of the SIZ and morphology on spike initiation and spike shape at the recording site as well as on activation and inactivation curves of ion channels obtained by voltage clamp recordings. Also we investigate how structural properties impact the results found in a point model of MN5, where variations of ion channel densities in physiological ranges lead to different observed firing properties [4].

Enhanced Optomotor Efficiency by Expression of the Human Gene Superoxide Dismutase Primarily in Drosophila Motorneurons

Mutation of the human gene superoxide dismutase (hSOD1) triggers the fatal neurodegenerative motorneuron disorder, familial amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease). Broad expression of this gene in Drosophila has no effect on longevity or functional senescence. We show here that restricting expression of human SOD1 primarily to motorneurons of Drosophila has significant effects on optomotor efficiency during in-flight tracking of rapidly moving visual targets. Under high-stress workloads with a recursive visual-motion stimulus cycle, young isogenic controls failed to track rapidly changing visual cues, whereas their same-aged hSOD1-activated progeny maintained coordinated in-flight tracking of the target by phase locking to the dynamic visual movement patterns. Several explanations are considered for the observed effects, including antioxidant intervention in motorneurons, changes in signal transduction pathways that regulate patterns of gene expression in other cell types, and expression of hSOD1 in a small set of neurons in the central brain. That hSOD1 overexpression improves sensorimotor coordination in young organisms may suggest possible therapeutic strategies for early-onset ALS in humans.

RNA-binding ability of FUS regulates neurodegeneration, cytoplasmic mislocalization and incorporation into stress granules associated with FUS carrying ALS-linked mutations

Amyotrophic lateral sclerosis (ALS) is an uncommon neurodegenerative disease caused by degeneration of upper and lower motor neurons. Several genes, including SOD1, TDP-43, FUS, Ubiquilin 2, C9orf72 and Profilin 1, have been linked with the sporadic and familiar forms of ALS. FUS is a DNA/RNA-binding protein (RBP) that forms cytoplasmic inclusions in ALS and frontotemporal lobular degeneration (FTLD) patients' brains and spinal cords. However, it is unknown whether the RNA-binding ability of FUS is required for causing ALS pathogenesis. Here, we exploited a Drosophila model of ALS and neuronal cell lines to elucidate the role of the RNA-binding ability of FUS in regulating FUS-mediated toxicity, cytoplasmic mislocalization and incorporation into stress granules (SGs). To determine the role of the RNA-binding ability of FUS in ALS, we mutated FUS RNA-binding sites (F305L, F341L, F359L, F368L) and generated RNA-binding-incompetent FUS mutants with and without ALS-causing mutations (R518K or R521C). We found that mutating the aforementioned four phenylalanine (F) amino acids to leucines (L) (4F-L) eliminates FUS RNA binding. We observed that these RNA-binding mutations block neurodegenerative phenotypes seen in the fly brains, eyes and motor neurons compared with the expression of RNA-binding-competent FUS carrying ALS-causing mutations. Interestingly, RNA-binding-deficient FUS strongly localized to the nucleus of Drosophila motor neurons and mammalian neuronal cells, whereas FUS carrying ALS-linked mutations was distributed to the nucleus and cytoplasm. Importantly, we determined that incorporation of mutant FUS into the SG compartment is dependent on the RNA-binding ability of FUS. In summary, we demonstrate that the RNA-binding ability of FUS is essential for the neurodegenerative phenotype in vivo of mutant FUS (either through direct contact with RNA or through interactions with other RBPs).

Modeling transport of a pulse of radiolabeled organelles in a Drosophila unipolar motor neuron

Based on published experimental evidence, this paper develops a model for the transport of a pulse of radiolabeled organelles in a unipolar Drosophila motor neuron. In particular, since published data indicate that no microtubules (MTs) travel from the primary neurite into the dendrite, it is investigated how organelles are transported into the dendrite. Analytical solutions describing concentrations of kinesin- and dynein-driven organelles in the primary neurite, axon, and dendrite are obtained. The effects of increasing the width of the pulse and increasing the rate of organelle transition rate from the kinesin-driven to the dynein-driven state are investigated.

Ras‐dependent and Ras‐independent effects of PI3K in Drosophila motor neurons

The lipid kinase PI3K plays key roles in cellular responses to activation of receptor tyrosine kinases or G protein coupled receptors such as the metabotropic glutamate receptor (mGluR). Activation of the PI3K catalytic subunit p110 occurs when the PI3K regulatory subunit p85 binds to phosphotyrosine residues present in upstream activating proteins. In addition, Ras is uniquely capable of activating PI3K in a p85-independent manner by binding to p110 at amino acids distinct from those recognized by p85. Because Ras, like p85, is activated by phosphotyrosines in upstream activators, it can be difficult to determine if particular PI3K-dependent processes require p85 or Ras. Here, we ask if PI3K requires Ras activity for either of two different PI3K-regulated processes within Drosophila larval motor neurons. To address this question, we determined the effects on each process of transgenes and chromosomal mutations that decrease Ras activity, or mutations that eliminate the ability of PI3K to respond to activated Ras. We found that PI3K requires Ras activity to decrease motor neuron excitability, an effect mediated by ligand activation of the single Drosophila mGluR DmGluRA. In contrast, the ability of PI3K to increase nerve terminal growth is Ras-independent. These results suggest that distinct regulatory mechanisms underlie the effects of PI3K on distinct phenotypic outputs.

The LIM-Homeodomain Protein Islet Dictates Motor Neuron Electrical Properties by Regulating K+ Channel Expression

SummaryNeuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K+ currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K+ current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes.

Drosophila Motor Neuron Retraction during Metamorphosis Is Mediated by Inputs from TGF-β/BMP Signaling and Orphan Nuclear Receptors

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. In this study, we describe the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, we show that phagocytes have a key role in this process. Importantly, we show the presence of peripheral glial cells close to the neuro-muscular junction that retracts before the motor neuron. We show also that in muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. We propose that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation.

Bone morphogenetic protein 2 inhibits neurite outgrowth of motor neuron‐like NSC‐34 cells and up‐regulates its type II receptor

Bone morphogenetic proteins (BMPs) regulate several aspects of neuronal behavior. For instance, BMP-2 has the ability to modulate, either positively or negatively, the outgrowth of neuronal processes in diverse cell types. In Drosophila motor neurons, the BMP type II receptor (BMPRII) homolog wishful thinking plays crucial roles on neuromuscular synaptogenesis signaling through Smad-dependent and Smad-independent pathways. However, a role for BMP signaling at the vertebrate neuromuscular junction has not been addressed. Herein, we have analyzed the expression of BMPRII and the effect of BMP-2 during the morphological differentiation of motor neuron-like NSC-34 cells. Our data indicate that BMPRII is up-regulated and becomes accumulated in somas and growth cones upon motor neuronal differentiation. BMP-2 inhibits the differentiation of NSC-34 cells, an effect that correlates with activation of a Smad-dependent pathway, induction of the inhibitory Id1 transcription factor, and down-regulation of the neurogenic factor Mash1. BMP-2 also activates effectors of Smad-independent pathways. Remarkably, BMP-2 treatment significantly increases the expression of BMPRII. Our findings provide the first evidence to suggest a role for BMP pathways on the differentiation of motor neurons leading to successful assembly and/or regeneration of the vertebrate neuromuscular synapse.

Synapsins I and II Are Not Required for β-Cell Insulin Secretion: Granules Must Pool Their Own Weight

Synapsins I and II Are Not Required for β-Cell Insulin Secretion: Granules Must Pool Their Own Weight

Drosophila as a model for MECP2 gain of function in neurons.

Methyl-CpG-binding protein 2 (MECP2) is a multi-functional regulator of gene expression. In humans loss of MECP2 function causes classic Rett syndrome, but gain of MECP2 function also causes mental retardation. Although mouse models provide valuable insight into Mecp2 gain and loss of function, the identification of MECP2 genetic targets and interactors remains time intensive and complicated. This study takes a step toward utilizing Drosophila as a model to identify genetic targets and cellular consequences of MECP2 gain-of function mutations in neurons, the principle cell type affected in patients with Rett-related mental retardation. We show that heterologous expression of human MECP2 in Drosophila motoneurons causes distinct defects in dendritic structure and motor behavior, as reported with MECP2 gain of function in humans and mice. Multiple lines of evidence suggest that these defects arise from specific MECP2 function. First, neurons with MECP2-induced dendrite loss show normal membrane currents. Second, dendritic phenotypes require an intact methyl-CpG-binding domain. Third, dendritic defects are amended by reducing the dose of the chromatin remodeling protein, osa, indicating that MECP2 may act via chromatin remodeling in Drosophila. MECP2-induced motoneuron dendritic defects cause specific motor behavior defects that are easy to score in genetic screening. In sum, our data show that some aspects of MECP2 function can be studied in the Drosophila model, thus expanding the repertoire of genetic reagents that can be used to unravel specific neural functions of MECP2. However, additional genes and signaling pathways identified through such approaches in Drosophila will require careful validation in the mouse model.

Toxic and non-toxic aggregates from the SBMA and normal forms of androgen receptor have distinct oligomeric structures

Hormone-dependent aggregation of the androgen receptor (AR) with a polyglutamine (polyQ) stretch amplification (>38) is considered to be the causative agent of the neurodegenerative disorder spinal and bulbar muscular atrophy (SBMA), consistent with related neurodegenerative diseases involving polyQ-extended proteins. In spite of the widespread acceptance of this common causal hypothesis, little attention has been paid to its apparent incompatibility with the observation of AR aggregation in healthy individuals with no polyQ stretch amplification. Here we used atomic force microscopy (AFM) to characterize sub-micrometer scale aggregates of the wild-type (22 glutamines) and the SBMA form (65 glutamines), as well as a polyQ deletion mutant (1 glutamine) and a variant with a normal length polyQ stretch but with a serine to alanine double mutation elsewhere in the protein. We used a baculovirus-insect cell expression system to produce full-length proteins for these structural analyses. We related the AFM findings to cytotoxicity as measured by expression of the receptors in Drosophila motoneurons or in neuronal cells in culture. We found that the pathogenic AR mutants formed oligomeric fibrils up to 300–600nm in length. These were clearly different from annular oligomers 120–180nm in diameter formed by the nonpathogenic receptors. We could also show that melatonin, which is known to ameliorate the pathological phenotype in the fly model, caused polyQ-extended AR to form annular oligomers. Further comparative investigation of these reproducibly distinct toxic and non-toxic oligomers could advance our understanding of the molecular basis of the polyQ pathologies.

Glial-Derived Prodegenerative Signaling in the Drosophila Neuromuscular System

We provide evidence for a prodegenerative, glial-derived signaling framework in the Drosophila neuromuscular system that includes caspase and mitochondria-dependent signaling. We demonstrate that Drosophila TNF-α (eiger) is expressed in a subset of peripheral glia, and the TNF-α receptor (TNFR), Wengen, is expressed in motoneurons. NMJ degeneration caused by disruption of the spectrin/ankyrin skeleton is suppressed by an eiger mutation or by eiger knockdown within a subset of peripheral glia. Loss of wengen in motoneurons causes a similar suppression providing evidence for glial-derived prodegenerative TNF-α signaling. Neither JNK nor NFκβ is required for prodegenerative signaling. However, we provide evidence for the involvement of both an initiator and effector caspase, Dronc and Dcp-1, and mitochondrial-dependent signaling. Mutations that deplete the axon and nerve terminal of mitochondria suppress degeneration as do mutations in Drosophila Bcl-2 (debcl), a mitochondria-associated protein, and Apaf-1 (dark), which links mitochondrial signaling with caspase activity in other systems.

Slamdance: seizing a fly model for epilepsy

brain seizures, as in epilepsy, are characterized by massively synchronous firing of neurons. Given the extraordinary complexity of the human brain, it is surprising that epilepsy is not a more common complaint. Episodic brain seizures currently affect ∼1% of the human population, and causes for

RNA Polymerase 1-driven Transcription as a Mediator of BDNF-induced Neurite Outgrowth

Neurite outgrowth is essential for development of the nervous system. Neurotrophins including BDNF are among extracellular signals that regulate neurite outgrowth. The ERK1/2 pathway contributes to intracellular signaling networks transducing the pro-neuritic effects of BDNF. In the nucleolus, RNA polymerase-1 (Pol1)-mediated transcription regulates ribosomal biogenesis, enabling cellular protein synthesis and growth. Hence, we tested the possibility that Pol1 is an effector for pro-neuritic signals such as BDNF. We report that Pol1-mediated nucleolar transcription was increased by BDNF in an ERK1/2-dependent manner in rat forebrain neurons. Conversely, in cultured hippocampal neurons, knockdown of a Pol1 coactivator, transcription initiation factor 1A (TIF1A), attenuated BDNF- or ERK1/2-induced neurite outgrowth. Also, upon overexpression, a constitutively active mutant of TIF1A strongly promoted neurite outgrowth, including increases in total neurite length and branching. Finally, overexpression of wild-type TIF1A enhanced the pro-neuritic effects of ERK1/2 activation. These observations indicate that the Pol1-mediated nucleolar transcription regulates neurite outgrowth and serves as a major pro-neuritic effector of the BDNF-activated ERK1/2 pathway. Thus, development of the nervous system appears critically dependent on the nucleolus.

Structural insights into membrane fusion at the endoplasmic reticulum

The endoplasmic reticulum (ER) in eukaryotes is composed of an interconnected network of membrane tubules that undergoes permanent membrane fusion and fission. A hallmark of this membrane system is the presence of so-called three-way junctions (Fig. 1). These connections between adjacent membrane tubules are essential for the continuity of the ER luminal space. In 2009, two groups identified the ER resident atlastin GTPases as key players involved in the maintenance of normal ER morphology (1, 2). Whereas down-regulation of atlastin function in Drosophila motor neurons resulted in shorter and unconnected ER tubules (1), interference with atlastin function in humans and yeast resulted in long unbranched ER tubules (2). Furthermore, purified atlastin was shown to catalyze membrane fusion in a GTPase-dependent fashion (1) and interact with proteins from the reticulon and deleted in polyposis 1 (DP1) families (2), which are also involved in maintaining ER shape (3). The work by Byrnes and Sondermann (4) in PNAS now sheds light on the structural basis of how atlastins perform this function.

Noncanonical notch function in motor axon guidance is mediated by Rac GTPase and the GEF1 domain of trio

The receptor Notch interacts with the Abl tyrosine kinase signaling pathway to control axon growth and guidance in Drosophila motor neurons. In part, this is mediated by binding to Trio, a guanine nucleotide exchange factor (GEF) for Rho GTPases. We show here that one of the two GEF domains of Trio, the Rac-specific GEF1, is essential for Trio-dependent motor axon guidance and for the genetic suppression of Notch function in motor axon patterning, but the Rho-specific GEF2 domain is not. Consistent with this, we show that Rac, and not Rho1 or Cdc42, interacts genetically with Notch in a manner indistinguishable from that of bona fide Abl signaling components. We infer, therefore, that Rac is a key component of Abl signaling in Drosophila motor axons, and specifically that it is the crucial Rho GTPase in "noncanonical" Notch/Abl signaling.

Steroid-triggered, cell-autonomous death of a Drosophila motoneuron during metamorphosis

BackgroundThe metamorphosis of Drosophila melanogaster is accompanied by elimination of obsolete neurons via programmed cell death (PCD). Metamorphosis is regulated by ecdysteroids, including 20-hydroxyecdysone (20E), but the roles and modes of action of hormones in regulating neuronal PCD are incompletely understood.ResultsWe used targeted expression of GFP to track the fate of a larval motoneuron, RP2, in ventral ganglia. RP2s in abdominal neuromeres two through seven (A2 to A7) exhibited fragmented DNA by 15 hours after puparium formation (h-APF) and were missing by 20 h-APF. RP2 death began shortly after the 'prepupal pulse' of ecdysteroids, during which time RP2s expressed ecdysteroid receptors (EcRs). Genetic manipulations showed that RP2 death required the function of EcR-B isoforms, the death-activating gene, reaper (but not hid), and the apoptosome component, Dark. PCD was blocked by expression of the caspase inhibitor p35 but unaffected by manipulating Diap1. In contrast, aCC motoneurons in neuromeres A2 to A7, and RP2s in neuromere A1, expressed EcRs during the prepupal pulse but survived into the pupal stage under all conditions tested. To test the hypothesis that ecdysteroids trigger RP2's death directly, we placed abdominal GFP-expressing neurons in cell culture immediately prior to the prepupal pulse, with or without 20E. 20E induced significant PCD in putative RP2s, but not in control neurons, as assessed by morphological criteria and propidium iodide staining.ConclusionsThese findings suggest that the rise of ecdysteroids during the prepupal pulse acts directly, via EcR-B isoforms, to activate PCD in RP2 motoneurons in abdominal neuromeres A2 to A7, while sparing RP2s in A1. Genetic manipulations suggest that RP2's death requires Reaper function, apoptosome assembly and Diap1-independent caspase activation. RP2s offer a valuable 'single cell' approach to the molecular understanding of neuronal death during insect metamorphosis and, potentially, of neurodegeneration in other contexts.

Presynaptic Activity and CaMKII Modulate Retrograde Semaphorin Signaling and Synaptic Refinement

Establishing synaptic connections often involves the activity-dependent withdrawal of off-target contacts. We describe an in vivo role for temporally patterned electrical activity, voltage-gated calcium channels, and CaMKII in modulating the response of Drosophila motoneurons to the chemorepellent Sema-2a during synaptic refinement. Mutations affecting the Sema-2a ligand, the plexin B receptor (plexB), the voltage-gated Ca(v)2.1 calcium channel (cac), or the voltage-gated Na(v)1 sodium channel (mle(nap-ts);tipE) each result in ectopic neuromuscular contacts. Sema-2a interacts genetically with both of the channel mutations. The cac phenotype is enhanced by the Sema-2a mutation and is suppressed by either plexB overexpression or patterned, low-frequency (0.01 Hz) bouts of electrical activity in the embryo. The calcium-dependent suppression of ectopic contacts also depends on the downstream activation of CaMKII. These results indicate a role for patterned electrical activity and presynaptic calcium signaling, acting through CaMKII, in modulating a retrograde signal during the refinement of synaptic connections.