Glycosphingolipids are linked to elevated neurotransmission and neurodegeneration in a Drosophila model of Niemann Pick type C.

The lipid storage disease Niemann Pick type C (NPC) causes neurodegeneration owing primarily to loss of NPC1. Here, we employed a Drosophila model to test links between glycosphingolipids, neurotransmission and neurodegeneration. We found that Npc1a nulls had elevated neurotransmission at the glutamatergic neuromuscular junction (NMJ), which was phenocopied in brainiac (brn) mutants, impairing mannosyl glucosylceramide (MacCer) glycosylation. Npc1a; brn double mutants had the same elevated synaptic transmission, suggesting that Npc1a and brn function within the same pathway. Glucosylceramide (GlcCer) synthase inhibition with miglustat prevented elevated neurotransmission in Npc1a and brn mutants, further suggesting epistasis. Synaptic MacCer did not accumulate in the NPC model, but GlcCer levels were increased, suggesting that GlcCer is responsible for the elevated synaptic transmission. Null Npc1a mutants had heightened neurodegeneration, but no significant motor neuron or glial cell death, indicating that dying cells are interneurons and that elevated neurotransmission precedes neurodegeneration. Glycosphingolipid synthesis mutants also had greatly heightened neurodegeneration, with similar neurodegeneration in Npc1a; brn double mutants, again suggesting that Npc1a and brn function in the same pathway. These findings indicate causal links between glycosphingolipid-dependent neurotransmission and neurodegeneration in this NPC disease model.

Multiple system atrophy.

This is a practical guide to diagnosing and managing multiple system atrophy (MSA). We explain the newly published Movement Disorders Society Consensus Diagnostic Criteria, which include new 'Clinically Established MSA' and 'Possible Prodromal MSA' categories, hopefully reducing time to diagnosis. We then highlight the key clinical features of MSA to aid diagnosis. We include a list of MSA mimics with suggested methods of differentiation from MSA. Lastly, we discuss practical symptom management in people living with MSA, including balancing side effects, with the ultimate aim of improving quality of life.

RNA-binding FMRP and Staufen sequentially regulate the Coracle scaffold to control synaptic glutamate receptor and bouton development.

Both mRNA-binding Fragile X mental retardation protein (FMRP; Fmr1) and mRNA-binding Staufen regulate synaptic bouton formation and glutamate receptor (GluR) levels at the Drosophila neuromuscular junction (NMJ) glutamatergic synapse. Here, we tested whether these RNA-binding proteins act jointly in a common mechanism. We found that both dfmr1 and staufen mutants, and trans-heterozygous double mutants, displayed increased synaptic bouton formation and GluRIIA accumulation. With cell-targeted RNA interference, we showed a downstream Staufen role within postsynaptic muscle. With immunoprecipitation, we showed that FMRP binds staufen mRNA to stabilize postsynaptic transcripts. Staufen is known to target actin-binding, GluRIIA anchor Coracle, and we confirmed that Staufen binds to coracle mRNA. We found that FMRP and Staufen act sequentially to co-regulate postsynaptic Coracle expression, and showed that Coracle, in turn, controls GluRIIA levels and synaptic bouton development. Consistently, we found that dfmr1, staufen and coracle mutants elevate neurotransmission strength. We also identified that FMRP, Staufen and Coracle all suppress pMad activation, providing a trans-synaptic signaling linkage between postsynaptic GluRIIA levels and presynaptic bouton development. This work supports an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulating structural and functional synapse development.

Secreted C-type lectin regulation of neuromuscular junction synaptic vesicle dynamics modulates coordinated movement.

The synaptic cleft manifests enriched glycosylation, with structured glycans coordinating signaling between presynaptic and postsynaptic cells. Glycosylated signaling ligands orchestrating communication are tightly regulated by secreted glycan-binding lectins. Using the Drosophila neuromuscular junction (NMJ) as a model glutamatergic synapse, we identify a new Ca2+-binding (C-type) lectin, Lectin-galC1 (LGC1), which modulates presynaptic function and neurotransmission strength. We find that LGC1 is enriched in motoneuron presynaptic boutons and secreted into the NMJ extracellular synaptomatrix. We show that LGC1 limits locomotor peristalsis and coordinated movement speed, with a specific requirement for synaptic function, but not NMJ architecture. LGC1 controls neurotransmission strength by limiting presynaptic active zone (AZ) and postsynaptic glutamate receptor (GluR) aligned synapse number, reducing both spontaneous and stimulation-evoked synaptic vesicle (SV) release, and capping SV cycling rate. During high-frequency stimulation (HFS), mutants have faster synaptic depression and impaired recovery while replenishing depleted SV pools. Although LGC1 removal increases the number of glutamatergic synapses, we find that LGC1-null mutants exhibit decreased SV density within presynaptic boutons, particularly SV pools at presynaptic active zones. Thus, LGC1 regulates NMJ neurotransmission to modulate coordinated movement.

Extracellular heparan sulfate proteoglycans and glycan-binding lectins orchestrate trans-synaptic signaling.

The exceedingly narrow synaptic cleft (<20 nm) and adjacent perisynaptic extracellular space contain an astonishing array of secreted and membrane-anchored glycoproteins. A number of these extracellular molecules regulate intercellular trans-synaptic signaling by binding to ligands, acting as co-receptors or modulating ligand-receptor interactions. Recent work has greatly expanded our understanding of extracellular proteoglycan and glycan-binding lectin families as key regulators of intercellular signaling at the synapse. These secreted proteins act to regulate the compartmentalization of glycoprotein ligands and receptors, crosslink dynamic extracellular and cell surface lattices, modulate both exocytosis and endocytosis vesicle cycling, and control postsynaptic receptor trafficking. Here, we focus closely on the Drosophila glutamatergic neuromuscular junction (NMJ) as a model synapse for understanding extracellular roles of the many heparan sulfate proteoglycan (HSPG) and lectin proteins that help determine synaptic architecture and neurotransmission strength. We particularly concentrate on the roles of extracellular HSPGs and lectins in controlling trans-synaptic signaling, especially that mediated by the Wnt and BMP pathways. These signaling mechanisms are causally linked to a wide spectrum of neurological disease states that impair coordinated movement and cognitive functions.

N-glycosylation requirements in neuromuscular synaptogenesis.

Neural development requires N-glycosylation regulation of intercellular signaling, but the requirements in synaptogenesis have not been well tested. All complex and hybrid N-glycosylation requires MGAT1 (UDP-GlcNAc:α-3-D-mannoside-ß1,2-N-acetylglucosaminyl-transferase I) function, and Mgat1 nulls are the most compromised N-glycosylation condition that survive long enough to permit synaptogenesis studies. At the Drosophila neuromuscular junction (NMJ), Mgat1 mutants display selective loss of lectin-defined carbohydrates in the extracellular synaptomatrix, and an accompanying accumulation of the secreted endogenous Mind the gap (MTG) lectin, a key synaptogenesis regulator. Null Mgat1 mutants exhibit strongly overelaborated synaptic structural development, consistent with inhibitory roles for complex/hybrid N-glycans in morphological synaptogenesis, and strengthened functional synapse differentiation, consistent with synaptogenic MTG functions. Synapse molecular composition is surprisingly selectively altered, with decreases in presynaptic active zone Bruchpilot (BRP) and postsynaptic Glutamate receptor subtype B (GLURIIB), but no detectable change in a wide range of other synaptic components. Synaptogenesis is driven by bidirectional trans-synaptic signals that traverse the glycan-rich synaptomatrix, and Mgat1 mutation disrupts both anterograde and retrograde signals, consistent with MTG regulation of trans-synaptic signaling. Downstream of intercellular signaling, pre- and postsynaptic scaffolds are recruited to drive synaptogenesis, and Mgat1 mutants exhibit loss of both classic Discs large 1 (DLG1) and newly defined Lethal (2) giant larvae [L(2)GL] scaffolds. We conclude that MGAT1-dependent N-glycosylation shapes the synaptomatrix carbohydrate environment and endogenous lectin localization within this domain, to modulate retention of trans-synaptic signaling ligands driving synaptic scaffold recruitment during synaptogenesis.

Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila.

Fragile X syndrome (FXS), the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1) gene product (FMRP), an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1) null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs): GPI-anchored glypican Dally-like protein (Dlp) and transmembrane Syndecan (Sdc). Our recent work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg) ligand abundance and downstream Frizzled-2 (Fz2) receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb), and downstream ERK phosphorylation (dpERK) are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb) and downstream signaling via phosphorylation of the transcription factor MAD (pMAD) seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1) Wg and Jeb trans-synaptic signaling, and (2) synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state.

Structure-function analysis of endogenous lectin mind-the-gap in synaptogenesis.

Mind-the-Gap (MTG) is required for neuronal induction of Drosophila neuromuscular junction (NMJ) postsynaptic domains, including glutamate receptor (GluR) localization. We have previously hypothesized that MTG is secreted from the presynaptic terminal to reside in the synaptic cleft, where it binds glycans to organize the heavily glycosylated, extracellular synaptomatrix required for transsynaptic signaling between neuron and muscle. In this study, we test this hypothesis with MTG structure-function analyses of predicted signal peptide (SP) and carbohydrate-binding domain (CBD), by introducing deletion and point-mutant transgenic constructs into mtg null mutants. We show that the SP is required for MTG secretion and localization to synapses in vivo. We further show that the CBD is required to restrict MTG diffusion in the extracellular synaptomatrix and for postembryonic viability. However, CBD mutation results in elevation of postsynaptic GluR localization during synaptogenesis, not the mtg null mutant phenotype of reduced GluRs as predicted by our hypothesis, suggesting that proper synaptic localization of MTG limits GluR recruitment. In further testing CBD requirements, we show that MTG binds N-acetylglucosamine (GlcNAc) in a Ca(2+)-dependent manner, and thereby binds HRP-epitope glycans, but that these carbohydrate interactions do not require the CBD. We conclude that the MTG lectin has both positive and negative binding interactions with glycans in the extracellular synaptic domain, which both facilitate and limit GluR localization during NMJ embryonic synaptogenesis.

Presynaptic secretion of mind-the-gap organizes the synaptic extracellular matrix-integrin interface and postsynaptic environments.

Mind-the-Gap (MTG) is required during synaptogenesis of the Drosophila glutamatergic neuromuscular junction (NMJ) to organize the postsynaptic domain. Here, we generate MTG::GFP transgenic animals to demonstrate MTG is synaptically targeted, secreted, and localized to punctate domains in the synaptic extracellular matrix (ECM). Drosophila NMJs form specialized ECM carbohydrate domains, with carbohydrate moieties and integrin ECM receptors occupying overlapping territories. Presynaptically secreted MTG recruits and reorganizes secreted carbohydrates, and acts to recruit synaptic integrins and ECM glycans. Transgenic MTG::GFP expression rescues hatching, movement, and synaptogenic defects in embryonic-lethal mtg null mutants. Targeted neuronal MTG expression rescues mutant synaptogenesis defects, and increases rescue of adult viability, supporting an essential neuronal function. These results indicate that presynaptically secreted MTG regulates the ECM-integrin interface, and drives an inductive mechanism for the functional differentiation of the postsynaptic domain of glutamatergic synapses. We suggest that MTG pioneers a novel protein family involved in ECM-dependent synaptic differentiation.

Presynaptic establishment of the synaptic cleft extracellular matrix is required for post-synaptic differentiation.

Formation and regulation of excitatory glutamatergic synapses is essential for shaping neural circuits throughout development. In a Drosophila genetic screen for synaptogenesis mutants, we identified mind the gap (mtg), which encodes a secreted, extracellular N-glycosaminoglycan-binding protein. MTG is expressed neuronally and detected in the synaptic cleft, and is required to form the specialized transsynaptic matrix that links the presynaptic active zone with the post-synaptic glutamate receptor (GluR) domain. Null mtg embryonic mutant synapses exhibit greatly reduced GluR function, and a corresponding loss of localized GluR domains. All known post-synaptic signaling/scaffold proteins functioning upstream of GluR localization are also grossly reduced or mislocalized in mtg mutants, including the dPix-dPak-Dock cascade and the Dlg/PSD-95 scaffold. Ubiquitous or neuronally targeted mtg RNA interference (RNAi) similarly reduce post-synaptic assembly, whereas post-synaptically targeted RNAi has no effect, indicating that presynaptic MTG induces and maintains the post-synaptic pathways driving GluR domain formation. These findings suggest that MTG is secreted from the presynaptic terminal to shape the extracellular synaptic cleft domain, and that the cleft domain functions to mediate transsynaptic signals required for post-synaptic development.

An essential Drosophila glutamate receptor subunit that functions in both central neuropil and neuromuscular junction.

A Drosophila forward genetic screen for mutants with defective synaptic development identified bad reception (brec). Homozygous brec mutants are embryonic lethal, paralyzed, and show no detectable synaptic transmission at the glutamatergic neuromuscular junction (NMJ). Genetic mapping, complementation tests, and genomic sequencing show that brec mutations disrupt a previously uncharacterized ionotropic glutamate receptor subunit, named here "GluRIID." GluRIID is expressed in the postsynaptic domain of the NMJ, as well as widely throughout the synaptic neuropil of the CNS. In the NMJ of null brec mutants, all known glutamate receptor subunits are undetectable by immunocytochemistry, and all functional glutamate receptors are eliminated. Thus, we conclude that GluRIID is essential for the assembly and/or stabilization of glutamate receptors in the NMJ. In null brec mutant embryos, the frequency of periodic excitatory currents in motor neurons is significantly reduced, demonstrating that CNS motor pattern activity is regulated by GluRIID. Although synaptic development and molecular differentiation appear otherwise unperturbed in null mutants, viable hypomorphic brec mutants display dramatically undergrown NMJs by the end of larval development, suggesting that GluRIID-dependent central pattern activity regulates peripheral synaptic growth. These studies reveal GluRIID as a newly identified glutamate receptor subunit that is essential for glutamate receptor assembly/stabilization in the peripheral NMJ and required for properly patterned motor output in the CNS.

Ceramidase regulates synaptic vesicle exocytosis and trafficking.

A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C(5)-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50-70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50-70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission.

Developmental regulation of glutamate receptor field size by nonvesicular glutamate release.

We hypothesized that presynaptic glutamate regulates postsynaptic ionotropic glutamate receptor number during synaptogenesis. To test this idea, we genetically manipulated presynaptic glutamate levels at the glutamatergic Drosophila neuromuscular junction (NMJ), then microscopically and electrophysiologically measured postsynaptic glutamate receptor field size and function. Our data show that presynaptic glutamate is a strong negative regulator of postsynaptic receptor field size and function during development. Glutamate-triggered receptor downregulation was not affected by block of synaptic vesicle fusion, demonstrating that receptors are regulated by nonvesicular glutamate release. Our results reveal an elegant mechanism for receptor field regulation during synaptogenesis and reveal a nonpathological role for nonvesicular glutamate release at the synapse.

Genes required for embryonic muscle development in Drosophila melanogaster A survey of the X chromosome.

We have begun a genetic analysis to dissect the process of myogenesis by surveying the X chromosome of Drosophila melanogaster for mutations that affect embryonic muscle development. Using polarised light microscopy and antibody staining techniques we analysed embryos hemizygous for a series of 67 deletion mutations that together cover an estimated 85% of the X chromosome, or 16.5% of the genome. Whereas the mature wild type embryo has a regular array of contractile muscles that insert into the epidermis, 31 of the deletion mutants have defects in muscle pattern, contractility or both, that cannot be attributed simply to epidermal defects and identify functions required for wild type muscle development. We have defined mutant pattern phenotypes that can be described in terms of muscle absences, incomplete myoblast fusion, failure of attachment of the muscle to the epidermis or mispositioning of attachment sites. Thus muscle development can be mutationally disrupted in characteristic and interpretable ways. The areas of overlap of the 31 deletions define 19 regions of the X chromosome that include genes whose products are essential for various aspects of myogenesis. We conclude that our screen can usefully identify loci coding for gene products essential in muscle development.