Elvin Woodruff

AP-3 Directs the Intracellular Trafficking of HIV-1 Gag and Plays a Key Role in Particle Assembly

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.

The Drosophila Fragile X Gene Negatively Regulates Neuronal Elaboration and Synaptic Differentiation

Fragile X Syndrome (FraX) is the most common form of inherited mental retardation. The disease is caused by the silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the RNA binding translational regulator FMRP . In FraX patients and fmr1 knockout mice, loss of FMRP causes denser and morphologically altered postsynaptic dendritic spines . Previously, we established a Drosophila FraX model and showed that dFMRP acts as a negative translational regulator of Futsch/MAP1B and negatively regulates synaptic branching and structural elaboration in the peripheral neuromuscular junction (NMJ) . Here, we investigate the role of dFMRP in the central brain, focusing on the mushroom body (MB), the learning and memory center . In MB neurons, dFMRP bidirectionally regulates multiple levels of structural architecture, including process formation from the soma, dendritic elaboration, axonal branching, and synaptogenesis. Drosophila fmr1 (dfmr) null mutant neurons display more complex architecture, including overgrowth, overbranching, and abnormal synapse formation. In contrast, dFMRP overexpression simplifies neuronal structure, causing undergrowth, underbranching, and loss of synapse differentiation. Studies of ultrastructural dfmr mutant neurons reveal enlarged and irregular synaptic boutons with dense accumulation of synaptic vesicles. Taken together, these data show that dFMRP is a potent negative regulator of neuronal architecture and synaptic differentiation in both peripheral and central nervous systems.

CNS-derived glia ensheath peripheral nerves and mediate motor root development

Motor function requires that motor axons extend from the spinal cord at regular intervals and that they are myelinated by Schwann cells. Little attention has been given to another cellular structure, the perineurium, which ensheaths the motor nerve, forming a flexible, protective barrier. Consequently, the origin of perineurial cells and their roles in motor nerve formation are poorly understood. Using time-lapse imaging in zebrafish, we show that perineurial cells are born in the CNS, arising as ventral spinal-cord glia before migrating into the periphery. In embryos lacking perineurial glia, motor neurons inappropriately migrated outside of the spinal cord and had aberrant axonal projections, indicating that perineurial glia carry out barrier and guidance functions at motor axon exit points. Additionally, reciprocal signaling between perineurial glia and Schwann cells was necessary for motor nerve ensheathment by both cell types. These insights reveal a new class of CNS-born glia that critically contributes to motor nerve development.

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. C5-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.

Neuronal Loss of Drosophila NPC1a Causes Cholesterol Aggregation and Age-Progressive Neurodegeneration

The mistrafficking and consequent cytoplasmic accumulation of cholesterol and sphingolipids is linked to multiple neurodegenerative diseases. One class of disease, the sphingolipid storage diseases, includes Niemann-Pick disease type C (NPC), caused predominantly (95%) by mutation of the NPC1 gene. A disease model has been established through mutation of Drosophila NPC1a (dnpc1a). Null mutants display early lethality attributable to loss of cholesterol-dependent ecdysone steroid hormone production. Null mutants rescued to adults by restoring ecdysone production mimic human NPC patients with progressive motor defects and reduced life spans. Analysis of dnpc1a null brains shows elevated overall cholesterol levels and progressive accumulation of filipin-positive cholesterol aggregates within brain and retina, as well as isolated cultured brain neurons. Ultrastructural imaging of dnpc1a mutant brains reveals age-progressive accumulation of striking multilamellar and multivesicular organelles, preceding the onset of neurodegeneration. Consistently, electroretinogram recordings show age-progressive loss of phototransduction and photoreceptor synaptic transmission. Early lethality, movement impairments, neuronal cholesterol deposits, accumulation of multilamellar bodies, and age-dependent neurodegeneration are all rescued by targeted neuronal expression of a wild-type dnpc1a transgene. Interestingly, targeted expression of dnpc1a in glia also provides limited rescue of adult lethality. Generation of dnpc1a null mutant neuron clones in the brain reveals cell-autonomous requirements for dNPC1a in cholesterol and membrane trafficking. These data demonstrate a requirement for dNPC1a in the maintenance of neuronal function and viability and show that loss of dNPC1a in neurons mimics the human neurodegenerative condition.

Mechanistic relationships between Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A signaling

Fragile X syndrome is caused by loss of the FMRP translational regulator. A current hypothesis proposes that FMRP functions downstream of mGluR signaling to regulate synaptic connections. Using the Drosophila disease model, we test relationships between dFMRP and the sole Drosophila mGluR (DmGluRA) by assaying protein expression, behavior and neuron structure in brain and NMJ; in single mutants, double mutants and with an mGluR antagonist. At the protein level, dFMRP is upregulated in dmGluRA mutants, and DmGluRA is upregulated in dfmr1 mutants, demonstrating mutual negative feedback. Null dmGluRA mutants display defects in coordinated movement behavior, which are rescued by removing dFMRP expression. Null dfmr1 mutants display increased NMJ presynaptic structural complexity and elevated presynaptic vesicle pools, which are rescued by blocking mGluR signaling. Null dfmr1 brain neurons similarly display increased presynaptic architectural complexity, which is rescued by blocking mGluR signaling. These data show that DmGluRA and dFMRP convergently regulate presynaptic properties.

Protein Expression Profiling of the Drosophila Fragile X Mutant Brain Reveals Up-regulation of Monoamine Synthesis

Fragile X syndrome is the most common form of inherited mental retardation, associated with both cognitive and behavioral anomalies. The disease is caused by silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the mRNA-binding, translational regulator FMRP. Previously we established a disease model through mutation of Drosophila fmr1 (dfmr1) and showed that loss of dFMRP causes defects in neuronal structure, function, and behavioral output similar to the human disease state. To uncover molecular targets of dFMRP in the brain, we use here a proteomic approach involving two-dimensional difference gel electrophoresis analyses followed by mass spectrometry identification of proteins with significantly altered expression in dfmr1 null mutants. We then focus on two misregulated enzymes, phenylalanine hydroxylase (Henna) and GTP cyclohydrolase (Punch), both of which mediate in concert the synthetic pathways of two key monoamine neuromodulators, dopamine and serotonin. Brain enzymatic assays show a nearly 2-fold elevation of Punch activity in dfmr1 null mutants. Consistently brain neurochemical assays show that both dopamine and serotonin are significantly increased in dfmr1 null mutants. At a cellular level, dfmr1 null mutant neurons display a highly significant elevation of the dense core vesicles that package these monoamine neuromodulators for secretion. Taken together, these data indicate that dFMRP normally down-regulates the monoamine pathway, which is consequently up-regulated in the mutant condition. Elevated brain levels of dopamine and serotonin provide a plausible mechanistic explanation for aspects of cognitive and behavioral deficits in human patients.

Fragile X mental retardation protein has a unique, evolutionarily conserved neuronal function not shared with FXR1P or FXR2P

SUMMARY Fragile X syndrome (FXS), resulting solely from the loss of function of the human fragile X mental retardation 1 (hFMR1) gene, is the most common heritable cause of mental retardation and autism disorders, with syndromic defects also in non-neuronal tissues. In addition, the human genome encodes two closely related hFMR1 paralogs: hFXR1 and hFXR2. The Drosophila genome, by contrast, encodes a single dFMR1 gene with close sequence homology to all three human genes. Drosophila that lack the dFMR1 gene (dfmr1 null mutants) recapitulate FXS-associated molecular, cellular and behavioral phenotypes, suggesting that FMR1 function has been conserved, albeit with specific functions possibly sub-served by the expanded human gene family. To test evolutionary conservation, we used tissue-targeted transgenic expression of all three human genes in the Drosophila disease model to investigate function at (1) molecular, (2) neuronal and (3) non-neuronal levels. In neurons, dfmr1 null mutants exhibit elevated protein levels that alter the central brain and neuromuscular junction (NMJ) synaptic architecture, including an increase in synapse area, branching and bouton numbers. Importantly, hFMR1 can, comparably to dFMR1, fully rescue both the molecular and cellular defects in neurons, whereas hFXR1 and hFXR2 provide absolutely no rescue. For non-neuronal requirements, we assayed male fecundity and testes function. dfmr1 null mutants are effectively sterile owing to disruption of the 9+2 microtubule organization in the sperm tail. Importantly, all three human genes fully and equally rescue mutant fecundity and spermatogenesis defects. These results indicate that FMR1 gene function is evolutionarily conserved in neural mechanisms and cannot be compensated by either FXR1 or FXR2, but that all three proteins can substitute for each other in non-neuronal requirements. We conclude that FMR1 has a neural-specific function that is distinct from its paralogs, and that the unique FMR1 function is responsible for regulating neuronal protein expression and synaptic connectivity.

Filamin A protein interacts with human immunodeficiency virus type 1 Gag protein and contributes to productive particle assembly.

HIV-1 Gag precursor directs virus particle assembly and release. In a search for Gag-interacting proteins that are involved in late stages of the HIV-1 replication cycle, we performed yeast two-hybrid screening against a human cDNA library and identified the non-muscle actin filament cross-linking protein filamin A as a novel Gag binding partner. The 280-kDa filamin A regulates cortical actin network dynamics and participates in the anchoring of membrane proteins to the actin cytoskeleton. Recent studies have shown that filamin A facilitates HIV-1 cell-to-cell transmission by binding to HIV receptors and coreceptors and regulating their clustering on the target cell surface. Here we report a novel role for filamin A in HIV-1 Gag intracellular trafficking. We demonstrate that filamin A interacts with the capsid domain of HIV-1 Gag and that this interaction is involved in particle release in a productive manner. Disruption of this interaction eliminated Gag localization at the plasma membrane and induced Gag accumulation within internal compartments. Moreover, blocking clathrin-dependent endocytic pathways did not relieve the restriction to particle release induced by filamin A depletion. These results suggest that filamin A is involved in the distinct step of the Gag trafficking pathway. The discovery of the Gag-filamin A interaction may provide a new therapeutic target for the treatment of HIV infection.

Rolling Blackout Is Required for Synaptic Vesicle Exocytosis

Rolling blackout (RBO) is a putative transmembrane lipase required for phospholipase C-dependent phosphatidylinositol 4,5-bisphosphate–diacylglycerol signaling in Drosophila neurons. Conditional temperature-sensitive (TS) rbo mutants display complete, reversible paralysis within minutes, demonstrating that RBO is acutely required for movement. RBO protein is localized predominantly in presynaptic boutons at neuromuscular junction (NMJ) synapses and throughout central synaptic neuropil, and rbo TS mutants display a complete, reversible block of both central and peripheral synaptic transmission within minutes. This phenotype appears limited to adults, because larval NMJs do not manifest the acute blockade. Electron microscopy of adult rbo TS mutant boutons reveals an increase in total synaptic vesicle (SV) content, with a concomitant shrinkage of presynaptic bouton size and an accumulation of docked SVs at presynaptic active zones within minutes. Genetic tests reveal a synergistic interaction between rbo and syntaxin1A TS mutants, suggesting that RBO is required in the mechanism of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated SV exocytosis, or in a parallel pathway necessary for SV fusion. The rbo TS mutation does not detectably alter SNARE complex assembly, suggesting a downstream requirement in SV fusion. We conclude that RBO plays an essential role in neurotransmitter release, downstream of SV docking, likely mediating SV fusion.