Presynaptic Calcium Channel Localization and Calcium Dependent Vesicle Exocytosis Regulated by the Fuseless Protein.

Ashleigh Long, William Pak, and Kendal Broadie

A systematic forward genetic Drosophila screen for electroretinogram mutants lacking synaptic transients has identified fuseless (fusl), encoding an 8-pass transmembrane protein with homology to a family of transporters, which colocalizes with horse radish peroxidase (HRP) at the presynaptic membrane. Null fusl mutants display a >75% reduction in evoked synaptic transmission, which is rescued by presynaptic expression of a fusl transgene, whereas both the frequency and amplitude of spontaneous fusion events is increased >2-fold. Synaptic architecture is comparable to controls in fusl mutants, but presynaptic active zones are reduced ~2-fold and the density of vesicles clustered and docked at active zones is strikingly elevated. Cycling vesicle exocytosis is greatly reduced in fusl mutants, and the calcium-dependence of neurotransmitter release and calcium-dependent facilitation are both dramatically compromised, which is likely explained by the significant reduction in expression of the calcium channel pore (1 subunit) seen in fuseless mutants throughout development, and the rescue of the neurotransmitter release defects using transgenically expressed cation channels in high calcium saline. The Fusl mutation is rescued with the overexpression of the Fuseless protein using the elav-Gene Switch system during development, indicating a developmental role for fuseless. The mechanistic defect linking these mutant phenotypes is impaired calcium dynamics underlying vesicle exocytosis. These data indicate that Fusl regulates calcium channel expression and active zone domains required for efficient synaptic vesicle exocytosis mediating neurotransmission.

Drosophila Fragile X Protein Regulates Synaptic Expression of Glutamate Receptor Classes by Differential Mechanisms

Luyuan Pan and Kendal Broadie Department of Biological Science, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN 37232

Fragile X Syndrome (FXS) is the most common known cause of inherited human mental retardation and autism, resulting from loss of expression of the Fragile X Mental Retardation Protein (FMRP), a negative regulator of protein translation. A current hypothesis proposes that FMRP acts downstream of metabotropic glutamate receptor (mGluR) signaling at neuronal synapses to modulate the local translation of proteins critical for the trafficking of AMPA glutamate receptors, and consequently regulates synaptic plasticity changes in neurotransmission strength. However, direct evidence of a relationship between FMRP, mGluR and AMPA receptor synaptic expression is very limited. In this work, we have used the Drosophila FXS model established in our lab to assay the expression of different AMPA-type glutamate receptors (GluRs) at the well-characterized larval neuromuscular junction (NMJ). Two GluR classes are known at this synapse, both believed to contain common GluRIII (IIC), IID and IIE subunits, and variable GluRIIA (A-class) or GluRIIB (B-class) subunits. We found that A-class GluRs significantly accumulate in the dfmr1 null mutant, whereas B-class GluRs are significantly lost in the same synapses. This represents a change in receptor subclass as the level of all receptors combined is not changed. DmGluRA encodes the sole functional mGluR in Drosophila, which is expressed at the NMJ. Both iontotropic GluR classes increase significantly in dmGluRA null mutants. In dfmr1; dmGluRA double mutants there is a more significant increase of A-class GluRs than in either single mutant, whereas B-class GluRs tend to normal levels in the double mutants. These results show that FMRP regulates different subclasses of synaptic GluRs by independent mechanisms, and that some mechanisms overlap with mGluR-induced downstream pathways. This work is supported by the FRAXA Foundation and NIH grant HD40654 to K.B.

DROSOPHILA MODEL OF NIEMANN-PICK TYPE C (NPC) NEURODEGENERATIVE DISEASE

Scott E. Phillips, Elvin A. Woodruff III and Kendal S. Broadie Department of Biological Sciences, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN.

The mistrafficking and cytoplasmic accumulation of sterols and sphingolipids is mechanistically linked to multiple neurodegenerative diseases, suggesting a central role for the regulation of these lipids in the maintenance of neuronal function and viability. One class of these diseases, the sphingolipid storage diseases, includes Niemann-Pick disease type C (NPC), a fatal autosomal-recessive neurodegenerative condition characterized by a massive accumulation of both cholesterol and glycosphingolipids in endosomal compartments. It is totally unknown whether or how lipid mistrafficking leads to disease pathology. Drosophila models for human neurodegenerative diseases, including Parkinson’s Disease, Huntington’s Disease and Alzheimer’s Disease, have contributed vital information about the molecular foundations underlying these conditions. Therefore, we are developing a Drosophila NPC model to elucidate the causal relationship between lipid mistrafficking and neuronal dysfunction. Mutations in human NPC1 gene are known to cause 95% of disease cases: Drosophila NPC1a (dNPC1a) is a well-conserved sequence homolog expressed in the brain. Null dNPC1a mutants are lethal early in development; however, mutant animals can be rescued to adult stages simply with a diet of excess cholesterol. These ‘cholesterol rescued’ adult dNPC1a mutants mimic human NPC patients with progressive motor defects and reduced life spans. Mass spectrophotometry analysis of dNPC1a mutant adult brains shows elevated cholesterol levels compared to cholesterol fed controls. Furthermore, filipin staining of sterols in adult dNPC1a animals reveals striking punctate accumulation throughout the entire central brain and optic lobes. All of these phenotypes can be rescued by targeted neuronal expression of a wild-type NPC1a transgene, thereby demonstrating a neuronal requirement for the NPC1a protein. High resolution imaging of dNPC1a neurons by electron microscopy reveals an age-progressive accumulation of multi-vesicular bodies and immense multilamellar structures, structures detected in samples from NPC patients. Electron microscopy also reveals age-progressive neurodegeneration in dNPC1a mutant animals. These data strongly suggest a neuronal requirement for the NPC1a gene with loss of dNPC1a in neurons mimicking the human neurodegenerative condition. This work is supported by NINDS grant NS41740 to K.S.B.

SPATIOTEMPORAL REQUIREMENTS OF FRAGILE X MENTAL RETARDATION PROTEIN IN SYNAPSE REGULATION

Cheryl Gatto and Kendal Broadie, Department of Biological Sciences, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN 37232 USA

Fragile X Syndrome (FraX), caused by the loss of function of a single gene (FMR1), is the most common inherited form of mental retardation and autism spectrum disorders. The FMR1 product (FMRP) is an RNA-binding translation regulator implicated in the activity-dependent control of synaptic connections. A critical question is to determine whether FraX is a ‘disease of development’ or a ‘disease of plasticity’. For defining FraX intervention strategies, we must know when and where FMRP is required for the manifestation of synaptic defects and, specifically, whether the reintroduction of FMRP at late stages can restore normal synaptic architecture and function. In our Drosophila FraX model, loss of the well-conserved dFMRP causes neuromuscular junction (NMJ) over-elaboration (overgrowth, overbranching, excess synaptic boutons), accumulation of developmentally arrested mini/satellite boutons, and altered functional neurotransmission properties due to both pre- and postsynaptic defects. We are using the GeneSwitch system to drive conditional dFMRP expression to define the spatiotemporal requirements for dFMRP function in these synaptic phenotypes. Constitutive induction of neuronal FMRP expression at levels comparable to wild-type is sufficient to rescue all the synaptic architectural defects in dfmr1 null mutants, indicating a presynaptic dFMRP requirement for synaptic structuring. In contrast, presynaptic dFMRP expression does not ameliorate the neurotransmission defect in the null mutant, suggesting a postsynaptic dFMRP requirement for synaptic function. Most importantly, acute (hours) dFMRP expression at larval maturity significantly alleviates dfmr1 null synaptic defects. Although rescue of mutant phenotypes is not as complete as with constitutive dFMRP expression, these findings minimally indicate that 1) synaptic properties are subject to plastic modulation, and 2) dFMRP provided late can quickly restore synaptic defects caused by genetic loss of dFMRP. These ongoing studies will inform future pharmacological treatments about developmental windows that are accessible for therapeutic intervention in FraX patients.

FRAGILE X MENTAL RETARDATION PROTEIN IS DEVELOPMENTALLY REGULATED DURING EARLY STAGE ACTIVITY-DEPENDENT PRUNING OF MUSHROOM BODY NEURONS

Charles R. Tessier and Kendal Broadie, Department of Biological Sciences, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville TN 37232 USA

Fragile X Syndrome (FraX) is the most common genetic form of mental retardation, affecting 1:4000 males and 1:6000 females. This broad spectrum disorder results from loss of the RNA-binding protein FMRP and manifests symptoms including severely reduced I.Q., hyperactivity, susceptibility to seizures, and autistic behaviors. FMRP function is activity-regulated to mediate the translational repression of mRNAs important for controlling synaptic connections. In the absence of FMRP, an increase in protein translation leads to changes in synapse structure (excessive length/number of dendritic and axonal branches), and function (altered neurotransmission and plasticity). In mouse mutants, hippocampal neurons display increased long term depression (LTD) and neocortical neurons display decreased long term potentiation (LTP). What remains totally unclear is whether developmental defects underlie the mature plasticity aberrations. To address this vital issue, we have used the Drosophila FraX model to characterize the developmental profile of dFMRP. We have identified a developmental peak of dFMRP expression during the early initial use period of brain circuits which correlates with structural changes in brain mushroom body neurons. We further show that mushroom body neurons undergo activity-dependent pruning during the window of high dFMRP expression. This use-dependent pruning is slowed in the absence of dFMRP, indicating a role for dFMRP in mediating activity driven remodeling of synaptic connections. Together, these results suggest dFMRP regulates activity-dependent protein synthesis during a late developmental stage of circuit formation, resulting in transient developmental defects, but also perhaps constitutively interfering with normal manifestation of synaptic plasticity.

ECM-integrin signaling drives glutamatergic synapse differentiation

Jeff Rohrbough, Emma Rushton, Elvin Woodruff III & Kendal Broadie Vanderbilt Kennedy Center for Research on Human Development, Department of Biological Sciences, Vanderbilt University, Nashville TN

Glutamate is the primary excitatory neurotransmitter in the nervous system. The formation of glutamatergic synapses is therefore essential for sculpting neural circuits. A key goal is to identify signal(s) that induce/organize the postsynaptic glutamate receptor (GluR) domain. Using systematic Drosophila mutant screens, we identified mind the gap (mtg) mutants based on severely impaired neurotransmission and severely disrupted localization of postsynaptic signaling/scaffold proteins, including Pix, Pak, Dock, DLG/PSD-95, and GluRs at the Drosophila glutamatergic neuromuscular junction (NMJ). Null mtg NMJs lack the distinctive electron-dense ECM found only in the synaptic cleft. MTG has a signal peptide and C-terminus GlcNAc-binding domain characteristic of ECM family proteins. MTG is expressed neuronally, localized in synaptic terminals and synaptic cleft ECM, and presynaptic knockdown of mtg impairs differentiation of the postsynaptic GluR domain, suggesting that MTG is secreted from the terminal to induce receptor field formation. Synaptic integrins (PSPS1, PS2 orPS23) localize to the NMJ, complexed with the postsynaptic DLG scaffold. In vertebrate synapses, integrins also act upstream of the Pix/Pak pathway. Presynaptic mtg knockdown reduces the postsynaptic accumulation of integrin receptors, suggesting that MTG operates upstream of integrins. PS mutants appear to exhibit PSD phenotypes resembling those of mtg mutants. We are currently investigating the proposed MTG-integrin interaction during synaptogenesis. Taken together, these studies suggest that MTG is a novel ECM molecule, required to establish a specialized synaptic ECM, acting in part via integrin receptors to induce postsynaptic differentiation.

THE DROSOPHILA MODEL OF FRAGILE X SYNDROME: TRANSLATIONAL REGULATION OF SYNAPTOGENESIS

Charles R. Tessier, Luyuan Pan, Elvin Woodruff III and Kendal Broadie Department of Biological Sciences, Kennedy Center for Research on Human Development, Program in Developmental Biology, Vanderbilt University

Fragile X Syndrome (FXS), a developmental disease of aberrant neural circuit formation, is the most common form of inherited mental retardation, conservatively affecting 1:4000 males and 1:6000 females. FXS is caused by transcriptional silencing of the FMR1 gene, which encodes a multi-domain RNA-binding protein that mediates translational repression. FXS neurons display abnormal structural development, characterized by supernumerary, anatomically immature synaptic spines. We developed a Drosophila FXS model that similarly displays excess dendritic/axonal elaboration, synaptogenic defects and cognitive/behavioral impairments. We used confocal and electron microscopy, in addition to biochemical assays, to identify and analyze molecular alterations occurring in the developing and mature mutant nervous system. We find that FMRP is developmentally regulated, with peak expression correlating with late stages of synaptogenesis and neural circuit refinement. Consistent with FMRP being a translational repressor, we identified upregulated expression of several cytoskeletal and synaptic proteins in mutants, including microtubule-associated Futsch/MAP1b, post-translationally modified tubulin isoforms, postsynaptic density protein DLG/PSD-95 and the presynaptic vesicle regulator UNC-13. Interestingly, all protein upregulation is exacerbated in adults, following the peak of FMRP expression in late developmental stages, consistent with the aberrant maintenance of an immature developmental state into the adult brain. Our working hypothesis is that FMRP negatively regulates translation of a small set of proteins to coordinate cytoskeletal dynamics and synaptic differentiation, likely in an activity-dependent mechanism. Future work will aim to systematically identify RNA targets of FMRP, and characterize the relationship between molecular alterations and defects in neuronal/synaptic architecture that result from this altered developmental program.

ECM-integrin signaling directs synaptic differentiation

Jeffrey Rohrbough, Emma Rushton, Elvin Woodruff & Kendal Broadie Department of Biological Sciences, Vanderbilt University

The formation and modification of excitatory glutamatergic synapses is essential for shaping neural circuits. A key goal is to identify molecular signal(s) that induce/organize the postsynaptic glutamate receptor (GluR) field. We identified mind the gap (mtg) in systematic screens for Drosophila mutants with impaired neurotransmission. Mtg null mutants exhibit severe mislocalization of postsynaptic density (PSD) signaling/scaffold proteins at the glutamatergic neuromuscular junction (NMJ), including Pix, Pak, Dock, DLG/PSD-95, and GluRs, and severely reduced postsynaptic GluR response. 70% of mtg mutant NMJs lack the distinctive electron-dense ECM present at normal NMJs. MTG has predicted signal peptide and C-terminus GlcNAc-binding domains characteristic of ECM family proteins. MTG is neuronally expressed and detected within presynaptic terminals and the synaptic cleft ECM, and presynaptic mtg knockdown likewise impairs PSD differentiation, suggesting that MTG is presynaptically secreted to induce GluR field formation. At vertebrate synapses, integrins act upstream of Pix/Pak pathway signaling. The Drosophila PSandPS1-3 integrins are concentrated postsynaptically, and complexed with DLG/PSD-95. Presynaptic mtg knockdown reduces NMJ integrin expression, and Drosophila PS mutants appear to exhibit PSD phenotypes resembling those of mtg mutants, suggesting that MTG acts upstream of integrins. We are currently investigating the proposed MTG-integrin synaptic interaction. Current results suggest that MTG may be a novel ECM signaling molecule, acting in part via integrins to regulate postsynaptic development.

Drosophila Model of Fragile X Syndrome

Kendal Broadie, Vanderbilt University, USA Kendal.Broadie@Vanderbilt.edu

A compromised ability to learn and remember may be due defects in the developmental program of neural circuit formation and/or a later loss of neural plasticity. Fragile X Syndrome (FXS) is the primary inherited form of cognitive impairment. This disease is caused by the loss of a single RNA-binding protein (FMRP). FMRP is known to function as a negative regulator of translation; only a handful of targets have been verified, although long lists of putative targets have been proposed. FMRP may function both during synaptic development and synaptic plasticity: FMRP levels spike greatly during late stages of synaptogenesis and FMRP levels also are regulated in an activity-dependent manner in mature neurons. A popular hypothesis is that FMRP is regulated downstream of glutamatergic synaptic signaling via metabotropic glutamate receptors (mGluRs). Drosophila has a single highly conserved FMRP (dFMRP) and a single functional mGluR (dmGluRA), making this system particularly tractable for the study of mechanisms involved in neuronal developmental and maintained plasticity. dFMRP is under strong developmental regulation and null dfmr1 mutants show altered expression of a select number of proteins with key functions in synaptogenesis. We are combining studies at the well-characterized neuromuscular junction (NMJ) synapse, the Mushroom Body (MB) learning/memory brain center and in primary cultured neurons to determine the molecular and temporal mechanisms of dFMRP and dmGluRA is the regulation of neuronal architecture and synaptic function. We have shown that dFMRP is a negative regulator of neuronal structural complexity within NMJ synaptic terminals, and throughout the entirety of central neurons, including branching from the soma, within the dendritic arbor and in axonal/synaptic outputs. We have shown that dFMRP acts as a negative regulator of synaptic function and regulates the ultrastructural differentiation of synaptic contacts. We have shown that dFMRP acts independently in the regulation of both pre- and postsynaptic differentiation. We have shown that one primary target of dFMRP regulation during these events is control of the stability/dynamics of microtubule/microfilament cytoskeletons. We have shown that dmGluRA mutants display antagonistic phenotypes for many of these parameters. With pharmacological and genetic approaches, we have found that dmGluRA interacts with dFMRP in some, but not all, of these mechanisms. We are using a combination of microarrays, proteomics and genetic screens to determine the full scope of biological function of dFMRP and dmGluRA in neuronal mechanisms. This work is supported by NIH grant HD40654 and a grant from the Fragile X Association.

Characterization of Eight Novel Synaptic Loci in Drosophila

Ashleigh Long, Bill Pak and Kendal Broadie Human Genetics Graduate Program, Vanderbilt University, Nashville, TN

A decades-long genetics screen has identified mutants with defective visual processing based on electroretinogram (ERG) recordings from the adult eye. A subset of these mutants lack the ERG lights on/off transients that represent synaptic transmission between photoreceptors and their downstream targets. We have identified eight mutant complementation groups that specifically lack synaptic transients but appear to display normal axonal projections and morphological synapse formation. Thus, these “transientless” mutants are presumed to be defective in synaptic transmission. Our aim is to map, clone, and characterize the function of these eight synaptic transmission genes. We have shown that all eight mutants display major defects in synaptic transmission at the larval neuromuscular junction (NMJ), indicating defects in a common set of gene products that mediate synaptic transmission in the visual system and NMJ. These mutants display severely reduced amplitudes during evoked transmission and significant defects in synaptic vesicle endocytosis and/or exocytosis as assayed by FM1-43 imaging. We are currently characterizing synaptic defects in detail. To identify the genes, we are using sequence polymorphisms in the genome to map mutations. We will provide an update on the identity of the highest priority genes.

ROLLING BLACKOUT IS REQUIRED FOR A LATE STEP IN SYNAPTIC VESICLE EXOCYTOSIS IN DROSOPHILA

Fu-De Huang, Elvin Woodruff and Kendal Broadie Department of Biological Sciences, Vanderbilt Kennedy Center & Brain Institute, Vanderbilt University, Nashville, TN 37235-1634

Rolling Blackout (RBO) is a novel transmembrane lipase identified in our lab (Huang et al., 2004), for which there is an activity-dependent requirement in phototransduction. In photoreceptors, RBO regulates PLC-dependent PIP2-DAG signaling required for TRP channel opening. Here, we show that conditional rbo mutants display complete, reversible temperature-sensitive (ts) paralysis with similar kinetics to conditional shibire mutants. RBO is subcellularly restricted to synapses, and rbo ts mutants display a complete, reversible block of both central and peripheral synaptic transmission. Electron microscopy reveals an increase in the total synaptic vesicles (SVs) and an accumulation of docked SVs at the active zone in rbo ts mutants, within minutes at restrictive temperature. These results demonstrate a block of SV exocytosis downstream of SV docking. Conditional rbo mutants show a strong synergistic genetic interaction with syntaxin1A ts mutants, indicating a defect near SNARE-mediated SV fusion. Analysis of the SNARE complex in rbo; syx ts double mutants indicates that RBO functions downstream of SNARE complex assembly, or in a parallel pathway. We conclude that RBO plays a critical role in downstream of SV docking, mediating SV fusion.

Drosophila Model of Human Spastic Paraplegia (HSP) Disease.

Nick Trotta and Kendal Broadie, Department of Biological Sciences, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville TN 37232 USA

Human spastic paraplegia (HSP) diseases are a group of related neurological disorders that cause progress loss of lower limb coordination and eventual axonal degeneration. Over 20 genes have been linked to HSP in humans, however mutations in one gene, spastin (SPG4), are the cause of over 40% of all disease cases. Spastin is a member of the ATPases Associated with diverse cellular Activities (AAA) protein family, and contains a Microtubule Interacting and organelle Transport (MIT) domain. Previous work has suggested a role for Spastin in microtubule dynamic instability. Employing transgenic methods for overexpression and RNA interference, we have examined the neural consequences of altering the homologous Drosophila spastin (Dspastin) gene dosage. We find that Dspastin is highly concentrated in the nervous system. Subcellularly the protein is particularly abundant at the sites of synaptic contact. Altering Dspastin dosage, in either direction, has dramatic consequences on the motor coordination and viability of mutant animals. At the neuromuscular synapse, Dspastin dosage regulates synaptic size, with animals expressing Dspastin RNAi exhibiting reduced synaptic area and loss of structural elaboration. Dspastin dosage directly impacts the efficacy of synaptic transmission. RNAi-mediated knockdown of Dspastin increases evoked synaptic currents (~60%), whereas neurotransmission is significantly reduced in animals overexpressing Dspastin. These alterations in synaptic structure and function are causally linked to local changes in microtubule stability at the synapse. Dspastin RNAi causes enhanced microtubule stability and Dspastin over-expression reduces microtubule stability. Pharmacological agents that counter these effects on microtubule stability rescues synaptic defects associated with both Dspastin over-expression and knockdown. Thus, Dspastin acts as a negative regulator for microtubule stability at the synapse, underlying alterations in synaptic structure and function. These data predict a specific molecular mechanism regarding microtubule cytoskeleton stability which may be causal in spastin-mediated HSP.

Fragile X, mental retardation and synaptic defects

Yong Q. Zhang1, Adina M. Bailey2, Gerry M. Rubin2 and Kendal Broadie
1. Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, USA. 2. Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.

Fragile X syndrome (FRAXA) is the most common inheritable disease which causes mental retardation in humans. It is caused by a triplet nucleotide CGG expansion in the 5Õ regulatory region of the gene FMR1, resulting in the "shut-down" of the production of the gene product, FMRP. FMRP is a selective RNA binding protein with three RNA binding domains, two KH domains and one RGG box, although its RNA targets remain unknown. Immuno-electronmicroscopy has shown that FMRP is localized at synapses in the developing rat brain. Furthermore, examination of cerebral cortical in fragile X patients and knockout mice has shown structurally abnormal synapses. These data suggest that the underlying mechanism of mental retardation in FRAXA patients is most probably the result of defects in FMRP function at the synapse.
To elucidate the molecular mechanisms by which the loss of FMRP leads to mental retardation, we have developed a genetic model for FRAXA. The homologue of FMR1 in Drosophila, named dFXR (drosophila fragile X related gene), shows 35%/56% overall identity/similarity to FMRP and contains all the functional domains, including the RNA binding domains. It is mapped at 85F9-12 on polytene chromosome. dFXRP is a cytoplasmic protein which is highly enriched in muscle and subsets of neurons, including motor neurons in the larval CNS. Two P element insertions and four dFXR deletions derived from one of the P element insertions were identified. Behavioral test demonstrated that dFXR mutants have profound locomotory defects. Assays of the neuromuscular junction model synapse have shown that levels of dFXRP regulate synaptic structure, as in humans and mice: dFXR mutants display an expanded terminal with more numerous but smaller "mini-boutons", whereas dFXR over-expression caused a reduced terminal with fewer but larger boutons. We therefore propose that dFXR regulates synaptic growth and maturation. On-going experiments are examining the role of dFXR in dendritic structural modification within the CNS and alteration in synaptic function in this promising FRAXA model.

PS integrins regulate stabilization of the synaptic field by mediating activity-dependent CaMKII function at the neuromuscular synapse.

K. J. Beumer and K. Broadie. Biology Dept. University of Utah, Salt Lake City, UT, 84112-0840.

Integrins play multiple roles in nervous system development and plasticity. Recently, genetic work in Drosophila and pharmacological work in rodents has shown that integrins are required for associative short-term memory and both structural and functional synaptic plasticity. Drosophila is an ideal system to study integrin function since only a single b integrin family is present at the synapse. Mutations in myospheroid (bPS integrin) that alter integrin expression at the neuromuscular junction (NMJ) modulate structural architecture in ways reminiscent of mutations of the Ig-domain NCAM Fasciclin II (FASII), and calcium/calmodulin dependent kinase II (CaMKII). We now demonstrate that integrins play a critical role in regulating a common pathway with these molecules at the NMJ. Alleles of myospheroid that decrease integrin levels at the NMJ also reduce CaMKII expression. This reduction of CaMKII activity causes a concomitant increase in the synaptic expression/localization of FASII and Discs-large (DLG), a PDZ-domain protein responsible for the localization of FASII and Shaker K+ ion channels at the NMJ. Integrin mutant phenotypes can be partially rescued by transgenically restoring FASII or CaMKII protein to their normal levels, indicating that the observed alterations in structural morphology are due to the changes in FASII expression mediated by the loss of integrin function. We present a model for a mechanism by which integrins stabilize NMJ growth during development and plasticity by regulating CaMKII translation at the NMJ.

Genetic Dissection of Glutamatergic Synapse Formation

Kendal Broadie, Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840 USA. 28th Gottingen Neuroscience Conference (2000):

The Drosophila NMJ is a leading model for the genetic investigation of mechanisms underlying glutamatergic synapse formation, function and plasticity. This system has been used extensively in reverse genetic approaches to characterize the in vivo function of previously identified synaptic proteins. It has been even more important in forward genetic screens to identify novel synaptic molecules including ion channels and their regulators, proteins implicated in synaptic vesicle endocytosis and diverse classes of proteins involved in both functional and structural plasticity. In numerous cases, genes identified in these screens have revolutionized the field.
For the past five years, my lab has been conducting a forward genetic screen to identify proteins essential for synaptogenesis in the Drosophila system. We have been using both chemical mutagens and transposable DNA elements to mutate the fully sequenced Drosophila genome, focussing primarily on the 3rd chromosome which contains ~40% of the genome. The final stage of the screen identifies mutants with grossly normal synaptic morphology but synaptic function reduced by >50% as judged by whole-cell patch-clamp recordings of synaptic currents. Hence, we are targeting aspects of functional physiological development rather than morphological synaptogenesis. This screen identifies genes required both for synaptic "development" and "function", since these two groups are impossible to distinguish until a late stage in the analyses.
To date, we have screened ~8000 mutant chromosomes to identify >40 genetic complementation groups with severe dysfunction phenotypes. The mutant phenotypes range from a complete loss of neurotransmission, down to the level of single SV transmission events, through milder phenotypes including a loss of transmission amplitude, fidelity and fatigue-resistance. We have identified mutants that disrupt both presynaptic and postsynaptic development. A subset of these mutant classes map to known genes, revealing both known and previously unsuspected roles for their gene products in synaptic development. However, the majority of complementation groups map to chromosomal regions that do not contain previously identified genes. We have used positional mapping and deficiency mapping techniques to locate small chromosomal sites for our highest priority lines. We are currently cloning and characterizing a number of these genes.
I will present the current results of this on-going screen. The presentation will survey the number of phenotypic classes of mutant complementation groups that have been identified and discuss the molecular nature of all genes and gene products that have been characterized. Detailed descriptions of the most fascinating mutant phenotypes will be provided. Mutant characterization includes whole-cell patch clamp recordings of transmission, confocal imaging of synaptic architecture, optical imaging of presynaptic function and ultrastructural studies with electron microscopy. To my knowledge, this is the first attempt to use forward genetics to systematically identify genes required for synaptogenesis at a glutamatergic synapse. The products of this screen are likely to make an enormous contribution to our understanding of the molecular and genetic pathways underlying synapse formation. This work is supported by grants from the National Institutes of Health.

Targeted Conditional Gene Disruption in Drosophila via a Heat Inducible N-Degron

S.Speese, C. Rodesch, M.M. Proschel, K. Broadie.
Neuroscience Meeting Abstract (2000):

Our lab utilizes Drosophila melanogaster as a model system to study the function of genes involved in synaptogenesis, synaptic transmission, and synaptic plasticity. However, probing the function of such genes is made difficult by the fact that many have separate roles in neural development and later in the function of the mature nervous system. Coincident with these genes are those that have essential roles outside of the nervous system in addition to their neural function. As techniques for circumventing these problems have yet to achieve a superior ability to dissect neural function, the field of neurogenetics requires new methods to look at the function of such genes.
For these reasons, an acute gene disruption technique that has temporal and spatial control is desired. We are establishing a conditional gene knockout system in Drosophila that was first developed in yeast to rapidly destroy tagged proteins. The basis of this targeted protein destruction is an interchangeable, temperature induced N-degron (td). An N-degron is a signal on a protein that marks it for destruction by stimulating ubiquination of the protein, thus causing it to be destroyed by the proteasome. We have taken the yeast td, dihydrofolate reductase (DHFRtd) and fused it to the green fluorescent protein (GFP) and expressed it in different tissues. We then utilized time lapse fluorescent imaging to quantitate destruction of the DHFRtd-GFP fusion protein. Initial tests of the system indicate that the DHFRtd-GFP fusion is rapidly degraded in some tissues. We are currently testing this system with known synaptic genes to assay the kinetics of degradation of DHFRtd tagged proteins.

CAPS is Essential for Regulated Neuromodulator Release

R.B. Renden, B. Berwin, C.-T. Chin, K. Ann, R. Kreber, B. Ganetzky, T.F.J. Martin, K. Broadie
Neuroscience Meeting Abstract (2000):

Calcium-Activated Protein For Secretion (CAPS) has been proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here, we report the cloning, mutation and characterization of the Drosophila melanogaster ortholog (dCAPS) which is 59% identical to the mammalian protein. dCAPS expression is restricted to the nervous system and the dCAPS protein is highly concentrated in the CNS neuropil and presynaptic terminals of peripheral neuromuscular junctions (NMJ). The protein is pan-synaptic and is not restricted to neural/synapse classes specialized for peptidergic transmission. Null dCAPS mutants show severe locomotory deficits and embryonic lethality. Presynaptic terminals display highly significant morphological overgrowth and differentiate 80% more synaptic boutons than normal. Electrophysiological recordings at the mutant embryonic NMJ reveal a 50% loss in evoked glutamatergic transmission although spontaneous events are unaffected. The dCAPS mutant defects are rescued by transgenic rat CAPS demonstrating conserved CAPS function across species. We conclude that dCAPS is an essential modulator of neurotransmitter release present in all presynaptic terminals.

Quiverin, a novel member of the immunoglobulin superfamily plays a role in bilateral motor control in Drosophila

KD Bodily, CM Morrison, KS Broadie
Neuroscience Meeting Abstract (2000):

Several members of the immunoglobulin superfamily of proteins (e.g. robo, Neogenin/DCC/Unc-40, Axo, and FasII) function in axon pathfinding by serving in ligand-receptor or cell adhesion pathways. We have identified a novel member of this superfamily in Drosophila, which we have named quiverin, that is expressed exclusively in the nervous system and plays an essential role in neuronal functional development. Alternative splicing of quiverin produces several protein isoforms and data suggests that both transmembrane and secreted forms of the protein are expressed. The largest protein isoform appears to be an integral membrane protein that includes 5 Ig domains and 2 FnIII repeats in the extracellular region and an extensive cytoplasmic region. The primary structures of the quiverin isoforms are most similar to Neo/DCC/Unc-40 protein family from several phyla (29% identity, 42% similarity).
Preliminary analyses of mutations in the quiverin gene show that the protein plays a critical role in the development of bilateral motor control. Null quiverin mutants do not survive past the 3rd instar larval stage and exhibit significant movement defects relative to wild-type larval behavior. Null mutants react abnormally to cephalic tactile stimulation by contracting and quivering rather than the reverse-and-turn response characteristic in wild-type larvae. The null mutants display drastically reduced ability to manifest coordinated bilateral movement as displayed in numerous assays of motor coordination. Hypomorphic quiverin mutants survive into adulthood but also exhibit compromised bilateral motor control. Homozygous hypomorph adults are unable to display any flight and exhibit varying degrees of compromised coordination in terrestrial movement.
Currently, we are conducting experiments to determine the role of quiverin in neural development and/or function. Preliminary studies have not detected obvious defects in synaptic transmission. Null quiverin mutants appear to have morphologically normal nervous systems. Specifically, we have yet to detect any defects in the development and maintenance of axonal tracts in the CNS or PNS using a variety of molecular markers. However, it is well known that functional redundancy exists in many neural development pathways so experiments are underway to uncover genetic interactions between quiverin and other known genes in developmental axon pathfinding pathways.

STONED AND SYNAPTOTAGMIN INTERACT DURING SYNAPTIC VESICLE RECYCLING

T. Fergestad, W. Davis and K. Broadie
Neuroscience Meeting Abstract (2000):

Presynaptic function demands the rapid and precise recycling of synaptic vesicles (SVs). We are taking a molecular genetic approach to analyze this mechanism at the Drosophila NMJ. We have previously shown that Stoned, a dicistronic locus encoding a novel protein (StnA) and a protein with domain homology to AP50 (StnB), plays an essential role in SV recycling and the synaptic recruitment/maintenance of Synaptotagmin (Fergestad et al., 1999). The physiological and ultrastructural phenotypes of stoned mutants are strikingly reminiscent of synaptotamin mutants (Broadie et al., 1994; Reist et al., 1998), raising the possibility that Stoned mediates its essential function via the recruitment of Synaptotagmin to SVs. We have recently shown that overexpression of Synaptotagmin I rescues the embryonic lethality of stoned lethal alleles and the behavioral defects of stoned viable alleles. We are currently using a combination of physiology and optical assays to determine the extent of the phenotypic rescue at the synapse. We are also characterizing stoned in double mutant combinations with synaptotagmin and shibire to assay possible synergistic interactions. Electrophysiological, ultrastructural and fluorescent optical imaging studies are being performed to establish the precise location and function of the Stoned proteins in SV recycling. Our working hypothesis is that the Stoned proteins bind directly to Synaptotagmin to regulate a choice point between degradation and recycling during the SV cycle. Thus, Stoned may be a crucial regulator of Ca2+-dependent transmission amplitude and plasticity.

Genetic Investigation of the Role of Integrins in Synaptic Plasticity

Searle Meeting Abstract (1999):

Synapses mediate communication between electrically-excitable cells in the nervous system and neuromusculature. Precise determination of synaptic partners during development specifies the functional circuitry of the nervous system, which then directs the flow and storage of neuronal information. Such synapses are not static, but rather exist in a constant state of adaptive plasticity. The information flowing through synapses mediates profound changes in synaptic architecture and function. This synaptic plasticity is believed to be the mechanistic basis of higher brain functions including the ability to learn and remember.
One fruitful avenue to investigate synaptic plasticity mechanisms is to isolate genetic mutants defective in learning and memory tests. Such mutants allow the characterization of genes which mediate distinct aspects of functional and anatomical synaptic plasticity. We employ this strategy using the fruitfly, Drosophila melanogaster. Drosophila shares the advantages of 100 years of intensive genetic investigation. It is also a system in which robust genetic behavioral screens can be coupled to detailed morphological and alectrophysiological assays of synaptic plasticity.
Mutations of the Drosophila Volado gene dominantly interfere with short-term memory retention. Volado encodes an alpha-integrin subunit, thus suggesting that integrins are involved in memory formation. Recent results in rodents have also suggested that integrins may play an essential role is mammalian learning and memory formation. Therefore, we set out to determine whether integrins, and the Volado integrin in particular, are expressed at synaptic connections and may play functions in synaptic adaptive plasticity.
Integrins function as transmembrane heterodimers composed of one alpha and one beta subunit. These dimers function as cell-surface receptors with known function in intercellular adhesion and signal transduction in a variety of cellular contexts. Drosophila contains five known integrin subunits, two betas and three alphas. We have shown that all five subunits are expressed at synapses, specifically in postembryonic stages in which these connections are subject to active plastic modifications. Integrin subunits are found both in the pre- and postsynaptic membranes.
Genetic manipulation of a beta subunit (myospheroid) and an alpha subunit (volado) have idendependently shown that integrins regulate both mophological and functional synaptic plasticity. Alteration in integrin expression levels results in either severe reduction or elaboration of the synaptic terminal arbor. Removal of volado results in an increase in basal transmission amplitude and severe impair of both short-term and long-term functional plasticity properties. These results suggest that the integrins represent a class of proteins integral to the regulation of multiple forms of synaptic plasticity and suggest that these molecules may play key roles in learning and memory processes.

PS integrins regulate morphological plasticity at the neuromuscular synapse.

K. Beumer, K. Broadie J. Rohrbough, Department of Biology, University of Utah, 257 S. 1400 E. Salt Lake City, UT 84112 801-585-9425

Several adhesion molecules have been shown to play a role in synaptic plasticity in vertebrates and Drosophila. Mutations in adhesion molecules can affect both morphological and functional plasticity and the processes of learning and memory. Mutations in one Drosophila integrin, the alpha subunit encoded by the Volado gene, have been shown to affect both learning and function at the neuromuscular junction (NMJ). Integrin agonists have also been shown to disrupt the stabilization of LTP in rat hippocampal slice preparations. The Drosophila NMJ is a model system for incorporating genetic, molecular and cellular techniques in the study of the role of integrins at the synapse, and thereby in synaptic plasticity and behavior modulation.
We have shown that two Drosophila alpha integrin subunits appear to be localized to the postsynapticly at the NMJ. A beta integrin subunit, betaPS, is also localized postsynaptically and may be present presynapticly. We have tested several viable mutants in myospheroid, the gene encoding betaPS, for alterations in protein localization, function and morphology. Two mutants, mysb9 and mysts1, show reduced levels of protein at the NMJ in third instar larvae. Morphological alterations include deformation of bouton shape, alteration in arborization and change in bouton number. Surprisingly, one mutant, mysb9, reduces branching and bouton number, while the other, mysts1, increases these parameters. We are determining the effect of these mutations on function and attempting to determine why the two mutations differ in their effect on morphology.

THE VOLADO a-INTEGRIN REGULATES SYNAPTIC MORPHOLOGY, TRANSMISSION, AND PLASTICITY.

Jeffrey Rohrbough1, M. Grotewiel2, R.L. Davis3 & K. Broadie1. University of Utah Department of Biology, Salt Lake City, UT (1); Michigan State University Department of Zoology, East Lansing, MI (2); Baylor College of Medicine, Houston, TX (3).

Volado, a gene encoding a new Drosophila a-integrin, was identified in a recent screen for new learning and memory mutants (Grotewiel et al, 1998, Nature 391). Volado has enriched expression in the mushroom bodies, and viable adult Volado mutants have impaired olfactory short-term memory (STM). Mutant STM defects are reversibly rescued by conditional Volado expression 3 hrs prior to training (Grotewiel et al, 1998). Integrins have also been implicated in rapid (minutes) consolidation of mammalian LTP (Staubli et al, 1998). These findings suggest a novel, dynamic role for integrin-mediated adhesion and signalling functions in modulating synaptic efficacy as well as behavioral learning and memory.
Volado's requirement in memory predicts a synaptic role for the Volado (VOL) protein, particularly in forms of functional modulation likely to be relevant for memory at central synapses. To test this hypothesis, we have assayed VOL expression in the nervous system, and examined synaptic morphology and functional synaptic transmission in Volado mutants. Surprisingly, VOL is present only at low or undetectable levels at most wild-type larval synaptic terminals. However, VOL is strongly localized in a limited, variable subset of synaptic boutons in the central neuropil and at the neuromuscular junction (NMJ). These results suggest VOL is present in both central and peripheral synapses, but is dynamically and transiently concentrated in individual synaptic boutons.
Null Volado (Vol-) mutants die during late larval stages. Vol- mutant NMJs exhibit mild (30-50%) morphological overgrowth, suggesting VOL has a role in limiting developmental synaptic growth. However, VOL clearly has an additional role regulating functional synaptic transmission and plasticity. Both viable and null Vol mutant larval NMJs have reduced Ca2+-dependence of evoked synaptic transmission, and abnormally large evoked synaptic currents. Additionally, both short- and longer-term forms of Ca2+- and activity-dependent synaptic plasticity are strongly reduced or absent in mutants. At normal NMJs, disrupting integrin-ligand interactions with an Arg-Gly-Asp inhibitory peptide phenocopies Vol- mutant transmission features within 30-60 min. Our results provide evidence that integrins may dynamically regulate synaptic efficacy and functional plasticity processes utilized in memory formation.