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The decline of motor ability is a hallmark feature of aging and is accompanied by degeneration of motor synaptic terminals. Consistent with this, Drosophila motor synapses undergo characteristic age-dependent structural fragmentation co-incident with diminishing motor ability. Here, we show that motor synapse levels of Trio, an evolutionarily conserved guanine nucleotide exchange factor (GEF), decline with age. We demonstrate that increasing Trio expression in adult Drosophila can abrogate age-dependent synaptic structural fragmentation, postpone the decline of motor ability, and maintain the capacity of motor synapses to sustain high-intensity neurotransmitter release. This preservative activity is conserved in transgenic human Trio, requires Trio Rac GEF function, and can also ameliorate synapse degeneration induced by depletion of miniature neurotransmission. Our results support a paradigm where the structural dissolution of motor synapses precedes and promotes motor behavioral diminishment and where intervening in this process can postpone the decline of motor function during aging., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT + DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1β as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health., Competing Interests: Declaration of interests The authors declare no competing interests.

Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. We present evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. We present evidence that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations., Competing Interests: Declaration of interests S.R. developed MUSE technology marketed by arcoris bio AG, has shares, and sits on the board., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Within the extracellular matrix, matricellular proteins are dynamically expressed nonstructural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The cellular communication network factors family of matricellular proteins serve regulatory roles to regulate cell function and are defined by their conserved multimodular organization. Here, we characterize the expression and neuronal requirement for the Drosophila cellular communication network factor family member. Drosophila cellular communication network factor is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. Drosophila cellular communication network factor-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of Drosophila cellular communication network factor results in reduced female fertility. In addition, Drosophila cellular communication network factor accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila cellular communication network factor family member will enhance our potential to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Alteration of the levels, localization or post-translational processing of the microtubule associated protein Tau is associated with many neurodegenerative disorders. Here we develop adult-onset models for human Tau (hTau) toxicity in Drosophila that enable age-dependent quantitative measurement of central nervous system synapse loss and axonal degeneration, in addition to effects upon lifespan, to facilitate evaluation of factors that may contribute to Tau-dependent neurodegeneration. Using these models, we interrogate the interaction of hTau with the retromer complex, an evolutionarily conserved cargo-sorting protein assembly, whose reduced activity has been associated with both Parkinson's and late onset Alzheimer's disease. We reveal that reduction of retromer activity induces a potent enhancement of hTau toxicity upon synapse loss, axon retraction and lifespan through a specific increase in the production of a C-terminal truncated isoform of hTau. Our data establish a molecular and subcellular mechanism necessary and sufficient for the depletion of retromer activity to exacerbate Tau-dependent neurodegeneration., (© 2022. The Author(s).)

Establishing with precision the quantity and identity of the cell types of the brain is a prerequisite for a detailed compendium of gene and protein expression in the central nervous system (CNS). Currently, however, strict quantitation of cell numbers has been achieved only for the nervous system of Caenorhabditis elegans . Here, we describe the development of a synergistic pipeline of molecular genetic, imaging, and computational technologies designed to allow high-throughput, precise quantitation with cellular resolution of reporters of gene expression in intact whole tissues with complex cellular constitutions such as the brain. We have deployed the approach to determine with exactitude the number of functional neurons and glia in the entire intact larval Drosophila CNS, revealing fewer neurons and more glial cells than previously predicted. We also discover an unexpected divergence between the sexes at this juvenile developmental stage, with the female CNS having significantly more neurons than that of males. Topological analysis of our data establishes that this sexual dimorphism extends to deeper features of CNS organisation. We additionally extended our analysis to quantitate the expression of voltage-gated potassium channel family genes throughout the CNS and uncover substantial differences in abundance. Our methodology enables robust and accurate quantification of the number and positioning of cells within intact organs, facilitating sophisticated analysis of cellular identity, diversity, and gene expression characteristics., Competing Interests: WJ, GS, ER, SB, SV, YS, RS, KH, BM No competing interests declared, (© 2022, Jiao et al.)

Background: Throughout the animal kingdom, GABA is the principal inhibitory neurotransmitter of the nervous system. It is essential for maintaining the homeostatic balance between excitation and inhibition required for the brain to operate normally. Identification of GABAergic neurons and their GABA release sites are thus essential for understanding how the brain regulates the excitability of neurons and the activity of neural circuits responsible for numerous aspects of brain function including information processing, locomotion, learning, memory, and synaptic plasticity, among others., New Method: Since the structure and features of GABA synapses are critical to understanding their function within specific neural circuits of interest, here we developed and characterized a conditional marker of GABAergic synaptic vesicles for Drosophila, 9XV5-vGAT., Results: 9XV5-vGAT is validated for conditionality of expression, specificity for localization to synaptic vesicles, specificity for expression in GABAergic neurons, and functionality. Its utility for GABAergic neurotransmitter phenotyping and identification of GABA release sites was verified for ellipsoid body neurons of the central complex. In combination with previously reported conditional SV markers for acetylcholine and glutamate, 9XV5-vGAT was used to demonstrate fast neurotransmitter phenotyping of subesophageal ganglion neurons., Comparison With Existing Methods: This method is an alternative to single cell transcriptomics for neurotransmitter phenotyping and can be applied to any neurons of interest represented by a binary transcription system driver., Conclusion: A conditional GABAergic synaptic vesicle marker has been developed and validated for GABA neurotransmitter phenotyping and subcellular localization of GABAergic synaptic vesicles., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. Here, we have investigated Drosophila motor neuron synaptic terminals during ageing. We observed cumulative fragmentation of presynaptic structures accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability. Through discrete manipulation of each neurotransmission modality, we find that miniature but not evoked neurotransmission is required to maintain presynaptic architecture and that increasing miniature events can both preserve synaptic structures and prolong motor ability during ageing. Our results establish that miniature neurotransmission, formerly viewed as an epiphenomenon, is necessary for the long-term stability of synaptic connections., (© 2021. The Author(s).)

Age is the greatest risk factor for Parkinson's disease (PD) which causes progressive loss of dopamine (DA) neurons, with males at greater risk than females. Intriguingly, some DA neurons are more resilient to degeneration than others. Increasing evidence suggests that vesicular glutamate transporter (VGLUT) expression in DA neurons plays a role in this selective vulnerability. We investigated the role of DA neuron VGLUT in sex- and age-related differences in DA neuron vulnerability using the genetically tractable Drosophila model. We found sex differences in age-related DA neurodegeneration and its associated locomotor behavior, where males exhibit significantly greater decreases in both DA neuron number and locomotion during aging compared with females. We discovered that dynamic changes in DA neuron VGLUT expression mediate these age- and sex-related differences, as a potential compensatory mechanism for diminished DA neurotransmission during aging. Importantly, female Drosophila possess higher levels of VGLUT expression in DA neurons compared with males, and this finding is conserved across flies, rodents, and humans. Moreover, we showed that diminishing VGLUT expression in DA neurons eliminates females' greater resilience to DA neuron loss across aging. This offers a new mechanism for sex differences in selective DA neuron vulnerability to age-related DA neurodegeneration. Finally, in mice, we showed that the ability of DA neurons to achieve optimal control over VGLUT expression is essential for DA neuron survival. These findings lay the groundwork for the manipulation of DA neuron VGLUT expression as a novel therapeutic strategy to boost DA neuron resilience to age- and PD-related neurodegeneration., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease wherein motor neuron degeneration leads to muscle weakness, progressive paralysis, and death within 3-5 years of diagnosis. Currently, the cause of ALS is unknown but, as with several neurodegenerative diseases, the potential role of neuroinflammation has become an increasingly popular hypothesis in ALS research. Indeed, upregulation of neuroinflammatory factors have been observed in both ALS patients and animal models. One such factor is the inflammatory inducer NF-κB. Besides its connection to inflammation, NF-κB activity can be linked to several genes associated to familial forms of ALS, and many of the environmental risk factors of the disease stimulate NF-κB activation. Collectively, this has led many to hypothesize that NF-κB proteins may play a role in ALS pathogenesis. In this review, we discuss the genetic and environmental connections between NF-κB and ALS, as well as how this pathway may affect different CNS cell types, and finally how this may lead to motor neuron degeneration.

Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both "classical" and "modulatory" signals that could produce diverse and/or complementary effects in associated circuits. Here, we establish that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identify the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. Our findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network., Competing Interests: The authors have declared that no competing interests exist.

Metastasis-the disseminated growth of tumours in distant organs-underlies cancer mortality. Breast-to-brain metastasis (B2BM) is a common and disruptive form of cancer and is prevalent in the aggressive basal-like subtype, but is also found at varying frequencies in all cancer subtypes. Previous studies revealed parameters of breast cancer metastasis to the brain, but its preference for this site remains an enigma. Here we show that B2BM cells co-opt a neuronal signalling pathway that was recently implicated in invasive tumour growth, involving activation by glutamate ligands of N-methyl-D-aspartate receptors (NMDARs), which is key in model systems for metastatic colonization of the brain and is associated with poor prognosis. Whereas NMDAR activation is autocrine in some primary tumour types, human and mouse B2BM cells express receptors but secrete insufficient glutamate to induce signalling, which is instead achieved by the formation of pseudo-tripartite synapses between cancer cells and glutamatergic neurons, presenting a rationale for brain metastasis.

Essential Tremor (ET) is one of the most common neurological diseases, with an estimated 7 million affected individuals in the US; the pathophysiology of the disorder is poorly understood. Recently, we identified a mutation (KCNS2 (Kv9.2), c.1137 T > A, p.(D379E) in an electrically silent voltage-gated K + channel α-subunit, Kv9.2, in a family with ET, that modulates the activity of Kv2 channels. We have produced transgenic Drosophila lines that express either the human wild type Kv9.2 (hKv9.2) or the ET causing mutant Kv9.2 (hKv9.2-D379E) subunit in all neurons. We show that the hKv9.2 subunit modulates activity of endogenous Drosophila K + channel Shab. The mutant hKv9.2-D379E subunit showed significantly higher levels of Shab inactivation and a higher frequency of spontaneous firing rate consistent with neuronal hyperexcitibility. We also observed behavioral manifestations of nervous system dysfunction including effects on night time activity and sleep. This functional data further supports the pathogenicity of the KCNS2 (p.D379E) mutation, consistent with our prior observations including co-segregation with ET in a family, a likely pathogenic change in the channel pore domain and absence from population databases. The Drosophila hKv9.2 transgenic model recapitulates several features of ET and may be employed to advance our understanding of ET disease pathogenesis.

SMN (Survival Motor Neuron) deficiency is the predominant cause of spinal muscular atrophy (SMA), a severe neurodegenerative disorder that can lead to progressive paralysis and death. Although SMN is required in every cell for proper RNA metabolism, the reason why its loss is especially critical in the motor system is still unclear. SMA genetic models have been employed to identify several modifiers that can ameliorate the deficits induced by SMN depletion. Here we focus on WDR79/TCAB1, a protein important for the biogenesis of several RNA species that has been shown to physically interact with SMN in human cells. We show that WDR79 depletion results in locomotion defects in both Drosophila and Caenorhabditis elegans similar to those elicited by SMN depletion. Consistent with this observation, we find that SMN overexpression rescues the WDR79 loss-of-function phenotype in flies. Most importantly, we also found that WDR79 overexpression ameliorates the locomotion defects induced by SMN depletion in both flies and worms. Our results collectively suggest that WDR79 and SMN play evolutionarily conserved cooperative functions in the nervous system and suggest that WDR79/TCAB1 may have the potential to modify SMA pathogenesis., (Copyright © 2017. Published by Elsevier Inc.)

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H(+) antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects.

Tau is a highly abundant and multifunctional brain protein that accumulates in neurofibrillary tangles (NFTs), most commonly in Alzheimer's disease (AD) and primary age-related tauopathy. Recently, microRNAs (miRNAs) have been linked to neurodegeneration; however, it is not clear whether miRNA dysregulation contributes to tau neurotoxicity. Here, we determined that the highly conserved brain miRNA miR-219 is downregulated in brain tissue taken at autopsy from patients with AD and from those with severe primary age-related tauopathy. In a Drosophila model that produces human tau, reduction of miR-219 exacerbated tau toxicity, while overexpression of miR-219 partially abrogated toxic effects. Moreover, we observed a bidirectional modulation of tau levels in the Drosophila model that was dependent on miR-219 expression or neutralization, demonstrating that miR-219 regulates tau in vivo. In mammalian cellular models, we found that miR-219 binds directly to the 3'-UTR of the tau mRNA and represses tau synthesis at the post-transcriptional level. Together, our data indicate that silencing of tau by miR-219 is an ancient regulatory mechanism that may become perturbed during neurofibrillary degeneration and suggest that this regulatory pathway may be useful for developing therapeutics for tauopathies.

Miniature neurotransmission is the transsynaptic process where single synaptic vesicles spontaneously released from presynaptic neurons induce miniature postsynaptic potentials. Since their discovery over 60 years ago, miniature events have been found at every chemical synapse studied. However, the in vivo necessity for these small-amplitude events has remained enigmatic. Here, we show that miniature neurotransmission is required for the normal structural maturation of Drosophila glutamatergic synapses in a developmental role that is not shared by evoked neurotransmission. Conversely, we find that increasing miniature events is sufficient to induce synaptic terminal growth. We show that miniature neurotransmission acts locally at terminals to regulate synapse maturation via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Our results establish that miniature neurotransmission, a universal but often-overlooked feature of synapses, has unique and essential functions in vivo., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The dopamine transporter (DAT) is the primary molecular target responsible for the rewarding properties of the psychostimulants amphetamine (AMPH) and cocaine. AMPH increases extracellular dopamine (DA) by promoting its nonexocytotic release via DAT-mediated efflux. Previous studies in heterologous cells have shown that phosphorylation of the amino terminus of DAT is required for AMPH-induced DA efflux but not for DA uptake. However, the identity of many of the modulatory proteins and the molecular mechanisms that coordinate efflux and the ensuing behavioral effects remain poorly defined. Here, we establish a robust assay for AMPH-induced hyperlocomotion in Drosophila melanogaster larvae. Using a variety of genetic and pharmacological approaches, we demonstrate that this behavioral response is dependent on DA and on DAT and its phosphorylation. We also show that methylphenidate (MPH), which competitively inhibits DA uptake but does not induce DAT-mediated DA efflux, also leads to DAT-dependent hyperlocomotion, but this response is independent of DAT phosphorylation. Moreover, we demonstrate that the membrane raft protein Flotillin-1 is required for AMPH-induced, but not MPH-induced, hyperlocomotion. These results are the first evidence of a role for a raft protein in an AMPH-mediated behavior. Thus, using our assay we are able to translate molecular and cellular findings to a behavioral level and to differentiate in vivo the distinct mechanisms of two psychostimulants.

Recent genome-wide association studies have linked common variants in the human genome to Parkinson's disease (PD) risk. Here we show that the consequences of variants at 2 such loci, PARK16 and LRRK2, are highly interrelated, both in terms of their broad impacts on human brain transcriptomes of unaffected carriers, and in terms of their associations with PD risk. Deficiency of the PARK16 locus gene RAB7L1 in primary rodent neurons, or of a RAB7L1 ortholog in Drosophila dopamine neurons, recapitulated degeneration observed with expression of a familial PD mutant form of LRRK2, whereas RAB7L1 overexpression rescued the LRRK2 mutant phenotypes. PD-associated defects in RAB7L1 or LRRK2 led to endolysosomal and Golgi apparatus sorting defects and deficiency of the VPS35 component of the retromer complex. Expression of wild-type VPS35, but not a familial PD-associated mutant form, rescued these defects. Taken together, these studies implicate retromer and lysosomal pathway alterations in PD risk., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Defects in endosomal sorting have been implicated in Alzheimer's disease. Endosomal traffic is largely controlled by phosphatidylinositol-3-phosphate, a phosphoinositide synthesized primarily by lipid kinase Vps34. Here we show that phosphatidylinositol-3-phosphate is selectively deficient in brain tissue from humans with Alzheimer's disease and Alzheimer's disease mouse models. Silencing Vps34 causes an enlargement of neuronal endosomes, enhances the amyloidogenic processing of amyloid precursor protein in these organelles and reduces amyloid precursor protein sorting to intraluminal vesicles. This trafficking phenotype is recapitulated by silencing components of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway, including the phosphatidylinositol-3-phosphate effector Hrs and Tsg101. Amyloid precursor protein is ubiquitinated, and interfering with this process by targeted mutagenesis alters sorting of amyloid precursor protein to the intraluminal vesicles of endosomes and enhances amyloid-beta peptide generation. In addition to establishing phosphatidylinositol-3-phosphate deficiency as a contributing factor in Alzheimer's disease, these results clarify the mechanisms of amyloid precursor protein trafficking through the endosomal system in normal and pathological states.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, we investigated the role of SMN-dependent U12 splicing events in the regulation of motor circuit activity. We show that SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Spinal muscular atrophy (SMA) is a lethal human disease characterized by motor neuron dysfunction and muscle deterioration due to depletion of the ubiquitous survival motor neuron (SMN) protein. Drosophila SMN mutants have reduced muscle size and defective locomotion, motor rhythm, and motor neuron neurotransmission. Unexpectedly, restoration of SMN in either muscles or motor neurons did not alter these phenotypes. Instead, SMN must be expressed in proprioceptive neurons and interneurons in the motor circuit to nonautonomously correct defects in motor neurons and muscles. SMN depletion disrupts the motor system subsequent to circuit development and can be mimicked by the inhibition of motor network function. Furthermore, increasing motor circuit excitability by genetic or pharmacological inhibition of K(+) channels can correct SMN-dependent phenotypes. These results establish sensory-motor circuit dysfunction as the origin of motor system deficits in this SMA model and suggest that enhancement of motor neural network activity could ameliorate the disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pre-mRNA alternative splicing is an important mechanism for the generation of synaptic protein diversity, but few factors governing this process have been identified. From a screen for Drosophila mutants with aberrant synaptic development, we identified beag, a mutant with fewer synaptic boutons and decreased neurotransmitter release. Beag encodes a spliceosomal protein similar to splicing factors in humans and Caenorhabditis elegans. We find that both beag mutants and mutants of an interacting gene dsmu1 have changes in the synaptic levels of specific splice isoforms of Fasciclin II (FasII), the Drosophila ortholog of neural cell adhesion molecule. We show that restoration of one splice isoform of FasII can rescue synaptic morphology in beag mutants while expression of other isoforms cannot. We further demonstrate that this FasII isoform has unique functions in synaptic development independent of transsynaptic adhesion. beag and dsmu1 mutants demonstrate an essential role for these previously uncharacterized splicing factors in the regulation of synapse development and function.

p150(Glued) is the major subunit of dynactin, a complex that functions with dynein in minus-end-directed microtubule transport. Mutations within the p150(Glued) CAP-Gly microtubule-binding domain cause neurodegenerative diseases through an unclear mechanism. A p150(Glued) motor neuron degenerative disease-associated mutation introduced into the Drosophila Glued locus generates a partial loss-of-function allele (Gl(G38S)) with impaired neurotransmitter release and adult-onset locomotor dysfunction. Disruption of the p150(Glued) CAP-Gly domain in neurons causes a specific disruption of vesicle trafficking at terminal boutons (TBs), the distal-most ends of synapses. Gl(G38S) larvae accumulate endosomes along with dynein and kinesin motor proteins within swollen TBs, and genetic analyses show that kinesin and p150(Glued) function cooperatively at TBs to coordinate transport. Therefore, the p150(Glued) CAP-Gly domain regulates dynein-mediated retrograde transport at synaptic termini, and this function of dynactin is disrupted by a mutation that causes motor neuron disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transgenic Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues.

The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination(1). Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP's) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP's and minature excitatory junction potentials (mEJP's) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution(2) (HL3) that contains 5 mM Mg(2+) and 1.5 mM Ca(2+). Changes in the amplitude of evoked EJP's can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

A molecular understanding of synaptogenesis is a critical step toward the goal of understanding how brains "wire themselves up," and then "rewire" during development and experience. Recent genomic and molecular advances have made it possible to study synaptogenesis on a genomic scale. Here, we describe the results of a screen for genes involved in formation and development of the glutamatergic Drosophila neuromuscular junction (NMJ). We screened 2185 P-element transposon mutants representing insertions in approximately 16% of the entire Drosophila genome. We first identified recessive lethal mutants, based on the hypothesis that mutations causing severe disruptions in synaptogenesis are likely to be lethal. Two hundred twenty (10%) of all insertions were homozygous lethal. Two hundred five (93%) of these lethal mutants developed at least through late embryogenesis and formed neuromusculature. We examined embryonic/larval NMJs in 202 of these homozygous mutants using immunocytochemistry and confocal microscopy. We identified and classified 88 mutants with altered NMJ morphology. Insertion loci in these mutants encode several different types of proteins, including ATP- and GTPases, cytoskeletal regulators, cell adhesion molecules, kinases, phosphatases, RNA regulators, regulators of protein formation, transcription factors, and transporters. Thirteen percent of insertions are in genes that encode proteins of novel or unknown function. Complementation tests and RT-PCR assays suggest that approximately 51% of the insertion lines carry background mutations. Our results reveal that synaptogenesis requires the coordinated action of many different types of proteins--perhaps as much as 44% of the entire genome--and that transposon mutageneses carry important caveats that must be respected when interpreting results generated using this method., (Copyright 2006 Wiley Periodicals, Inc.)

Autosomal-dominant pure hereditary spastic paraplegia (AD-HSP) is characterized by the degeneration of long axons in corticospinal tracts and dorsal columns, resulting in spasticity and difficulty walking. Mutations in the SPG4 gene product spastin are the predominant genetic lesions associated with this inherited disease. In this issue, Orso et al. examine and reconcile existing Drosophila mutants of spastin and generate a new model for HSP by overexpression of a fly spastin transgene that carries a mutation prevalent in human AD-HSP (see the related article beginning on page 3026). Expression of this mutant spastin protein produces pathology in flies reminiscent of the human disease, including adult locomotion defects, in addition to causing aberrant synaptic morphology and altered microtubule stability. Both movement and synaptic defects in fly mutants were ameliorated by treatment with the microtubule-modifying agent vinblastine. The results are consistent with disease-causing mutations in human spastin producing dominant-negative proteins and confirm the usefulness of Drosophila genetic techniques to understand HSP and other neurodegenerative diseases.

Highwire (Hiw), a putative RING finger E3 ubiquitin ligase, negatively regulates synaptic growth at the neuromuscular junction (NMJ) in Drosophila. hiw mutants have dramatically larger synaptic size and increased numbers of synaptic boutons. Here we show that Hiw binds to the Smad protein Medea (Med). Med is part of a presynaptic bone morphogenetic protein (BMP) signaling cascade consisting of three receptor subunits, Wit, Tkv, and Sax, in addition to the Smad transcription factor Mad. When compared to wild-type, mutants of BMP signaling components have smaller NMJ size, reduced neurotransmitter release, and aberrant synaptic ultrastructure. BMP signaling mutants suppress the excessive synaptic growth in hiw mutants. Activation of BMP signaling, which in wild-type does not cause additional growth, in hiw mutants does lead to further synaptic expansion. These results reveal a balance between positive BMP signaling and negative regulation by Highwire, governing the growth of neuromuscular synapses.

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. Here, we show that this retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, we show that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, we demonstrate that presynaptic BMP receptor wishful thinking is required for the retrograde signal to function. Our results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila.

We show that the BMP ortholog Gbb can signal by a retrograde mechanism to regulate synapse growth of the Drosophila neuromuscular junction (NMJ). gbb mutants have a reduced NMJ synapse size, decreased neurotransmitter release, and aberrant presynaptic ultrastructure. These defects are similar to those we observe in mutants of BMP receptors and Smad transcription factors. However, whereas these BMP receptors and signaling components are required in the presynaptic motoneuron, Gbb expression is required in large part in postsynaptic muscles; gbb expression in muscle rescues key aspects of the gbb mutant phenotype. Consistent with this notion, we find that blocking retrograde axonal transport by overexpression of dominant-negative p150/Glued in neurons inhibits BMP signaling in motoneurons. These experiments reveal that a muscle-derived BMP retrograde signal participates in coordinating neuromuscular synapse development and growth.

Synaptobrevins or VAMPs are vesicle-associated membrane proteins, often called v-SNARES, that are important for vesicle transport and fusion at the plasma membrane. Drosophila has two characterized members of this gene family: synaptobrevin (syb) and neuronal synaptobrevin (n-syb). Mutant phenotypes and gene-expression patterns indicate that n-Syb is exclusively neuronal and required only for synaptic vesicle secretion, whereas Syb is ubiquitous and, as shown here, essential for cell viability. When the eye precursor cells were made homozygous for syb(-), the eye failed to develop. In contrast, n-syb(-) eye clones developed appropriately but failed to activate downstream neurons. To determine whether the two proteins are structurally specialized to accomplish these distinct in vivo functions, we have driven the expression of each gene in the absence of the other to look for phenotypic rescue. We find that expression of n-syb during eye development can rescue the cell lethality of the syb mutations, as can rat VAMP2 and cellubrevin. Expression of syb can restore synaptic transmission to n-syb mutants as assayed both by electroretinogram and recordings of excitatory junctional currents at the neuromuscular junction. Therefore, we find that Syb, which usually is not involved in synaptic function, can mediate Ca(2+)-triggered synaptic activity and that no particular specialization of the v-SNARE is required to differentiate synaptic exocytosis from other forms.

We conducted a large-scale screen for Drosophila mutants that have structural abnormalities of the larval neuromuscular junction (NMJ). We recovered mutations in wishful thinking (wit), a gene that positively regulates synaptic growth. wit encodes a BMP type II receptor. In wit mutant larvae, the size of the NMJs is greatly reduced relative to the size of the muscles. wit NMJs have reduced evoked excitatory junctional potentials, decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The NMJ phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ.

S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) is an important enzyme in the trans-sulphuration pathway, mediating the conversion of S-adenosyl-L-homocysteine to adenosine and L-homocysteine. We have identified a cDNA clone from Xenopus laevis, encoding a protein of 433 aa, which is highly conserved with S-Adenosyl-L-homocysteine hydrolases (Adohcyases) from other species. Expression of Adohcyase mRNA in X.laevis tadpoles is detectable from developmental Stage 27 onwards. Phylogenetic analysis of available Adohcyase sequences indicates that species cluster essentially as predicted from morphological data. Furthermore, we estimate that S-adenosyl-L-homocysteine hydrolase is evolving very slowly, almost 10 times slower than the average rate.