Drosophila highwire gene modulates acute ethanol sensitivity in the nervous system

Animals exhibit behavioral differences in their sensitivity to ethanol, a trait that is at least in part due to genetic predispositions. This study has implicated a large neuronal protein involving Highwire, a Drosophila E3 ubiquitin ligase (Hiw, a homolog of Pam, a protein associated with Myc found in humans) in acute sensitivity to ethanol sedation. Flies lacking Hiw were hypersensitive to the sedating effect of ethanol whereas those overexpressing Hiw showed decreased sensitivity to ethanol. Furthermore, RNAi functional knockdown of Hiw in adult neurons or ellipsoid body neurons showed increased sensitivity to ethanol sedation. None of these manipulations of the hiw gene caused changes in the rate of ethanol absorption and/or metabolism. These results suggest a previously unknown role for this highly conserved gene in regulating the behavioral responses to an addictive drug.     

Epsin 1 Promotes Synaptic Growth by Enhancing BMP Signal Levels in Motoneuron Nuclei

Bone morphogenetic protein (BMP) retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad) is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid facets (Lqf) positively regulates synaptic growth through post-endocytotic processing of pMad signaling complex. Lqf and the BMP receptor Wishful thinking (Wit) interact genetically and biochemically. lqf loss of function (LOF) reduces bouton number whereas overexpression of lqf stimulates bouton growth. Lqf-stimulated synaptic overgrowth is suppressed by genetic reduction of wit. Further, synaptic pMad fails to accumulate inside the motoneuron nuclei in lqf mutants and lqf suppresses synaptic overgrowth in spinster (spin) mutants with enhanced BMP signaling by reducing accumulation of nuclear pMad. Interestingly, lqf mutations reduce nuclear pMad levels without causing an apparent blockage of axonal transport itself. Finally, overexpression of Lqf significantly increases the number of multivesicular bodies (MVBs) in the synapse whereas lqf LOF reduces MVB formation, indicating that Lqf may function in signaling endosome recycling or maturation. Based on these observations, we propose that Lqf plays a novel endosomal role to ensure efficient retrograde transport of BMP signaling endosomes into motoneuron nuclei.     

Fat facets and Liquid facets promote Delta endocytosis and Delta signaling in the signaling cells

Endocytosis modulates the Notch signaling pathway in both the signaling and receiving cells. One recent hypothesis is that endocytosis of the ligand Delta by the signaling cells is essential for Notch activation in the receiving cells. Here, we present evidence in strong support of this model. We show that in the developing Drosophila eye Fat facets (Faf), a deubiquitinating enzyme, and its substrate Liquid facets (Lqf), an endocytic epsin, promote Delta internalization and Delta signaling in the signaling cells. We demonstrate that while Lqf is necessary for three different Notch/Delta signaling events at the morphogenetic furrow, Faf is essential only for one: Delta signaling by photoreceptor precluster cells, which prevents recruitment of ectopic neurons. In addition, we show that the ubiquitin-ligase Neuralized (Neur), which ubiquitinates Delta, functions in the signaling cells with Faf and Lqf. The results presented bolster one model for Neur function in which Neur enhances Delta signaling by stimulating Delta internalization in the signaling cells. We propose that Faf plays a role similar to that of Neur in the Delta signaling cells. By deubiquitinating Lqf, which enhances the efficiency of Delta internalization, Faf stimulates Delta signaling.     

Highwire regulates synaptic growth in Drosophila

The formation, stabilization, and growth of synaptic connections are dynamic and highly regulated processes. The glutamatergic neuromuscular junction (NMJ) in Drosophila grows new boutons and branches throughout larval development. A primary walking behavior screen followed by a secondary anatomical screen led to the identification of the highwire (hiw) gene. In hiw mutants, the specificity of motor axon pathfinding and synapse formation appears normal. However, NMJ synapses grow exuberantly and are greatly expanded in both the number of boutons and the extent and length of branches. These synapses appear normal ultrastructurally but have reduced quantal content physiologically. hiw encodes a large protein found at presynaptic terminals. Within presynaptic terminals, HIW is localized to the periactive zone surrounding active zones; Fasciclin II (Fas II), which also controls synaptic growth, is found at the same location.     

The Highwire Ubiquitin Ligase Promotes Axonal Degeneration by Tuning Levels of Nmnat Protein

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury.     

Nibbling away at synaptic development

Beyond its role as a response to starvation, autophagy has been increasingly implicated as part of the normal mechanisms regulating growth and remodeling of various cells and tissues during development. In recently published work we demonstrate that autophagy promotes synaptic development of the Drosophila larval neuromuscular junction (NMJ). We find that autophagy acts by downregulating an E3 ubiquitin ligase, Highwire (Hiw), which limits NMJ growth via a MAPKKK pathway. A similar role for autophagy in the synaptic remodeling that occurs during learning and memory remains an intriguing possibility.     

The neural cell adhesion molecule and synaptic plasticity

Highly stereotyped patterns of neuronal connections are laid down during the development of the nervous system via a range of activity independent and activity dependent mechanisms. Whereas the coarse hard-wiring of the nervous system appears to rely on molecular recognition events between the neuron, its pathway, and its target, the establishment of precisely patterned functional circuits is thought to be driven by neuronal activity. In this review we discuss the role that the neuronal cell adhesion molecule (NCAM) plays in morphological plasticity. Recent studies on NCAM and its probable species homologue in Aplysia (apCAM) suggests that an individual CAM can function to both promote synaptic plasticity and maintain the structure of the synapse. In the adult brain, changes between stability and plasticity are likely to underlie dynamic morphological changes in synaptic structures associated with learning and memory. In this review we use NCAM as an example to illustrate mechanisms that can change the function of an individual CAM from a molecule that promotes plasticity to one that does not. We also discuss evidence that NCAM promotes plasticity by activating a conventional signal transduction cascade, rather than by modulating adhesion perse. Finally, we consider the evidence that supports a role for NCAM in learning and memory. © 1995 John Wiley & Sons, Inc.     

Either part of a Drosophila epsin protein,divided after the ENTH domain,functions in endocytosis of delta in the developing eye

Epsin is part of a protein complex that performs endocytosis in eukaryotes. Drosophila epsin, Liquid facets (Lqf), was identified because it is essential for patterning the eye and other imaginal disc derivatives [2]. Previous work has provided only indirect evidence that Lqf is required for endocytosis in Drosophila [2, 3]. Epsins are modular and have an N-terminal ENTH (epsin N-terminal homology) domain that binds PIP(2) at the cell membrane and four different classes of protein-protein interaction motifs. The current model for epsin function in higher eukaryotes is that epsin bridges the cell membrane, a transmembrane protein to be internalized, and the core endocytic complex. Here, we show directly that Drosophila epsin (Lqf) is required for endocytosis. Specifically, we find that Lqf is essential for internalization of the Delta (Dl) transmembrane ligand in the developing eye. Using this endocytic defect in lqf mutants, we develop a transgene rescue assay and perform a structure/function analysis of Lqf. We find that when we divide Lqf into two pieces, an ENTH domain and an ENTH-less protein, each part retains significant ability to function in Dl internalization and eye patterning. These results challenge the model for epsin function that requires an intact protein.     

Neurotrophins improve synaptic transmission in the adult rodent diaphragm

Neurotrophins are usually viewed as secreted proteins that control long-term survival and differentiation of neurons. However, recent studies have established that among the most important functions of neurotrophins is their capacity to regulate synaptic functions and plasticity. When altering synaptic function, neurotrophins are able to produce two types of outcomes, an immediate effect on synaptic transmission and long-term control of synaptic structure and function. The first effect occurs within seconds or minutes after the neurotrophic factor has been applied and usually involves acute modification of synaptic transmission. The second effect takes hours and days, as protein synthesis is required to complete the structural changes. Neurotrophins and their receptors are expressed within the neuromuscular system, making these agents ideal candidates for the short-and long-term regulation of skeletal muscle function. For instance, neurotrophins can alter neuromuscular function acutely, by modulating the amount of neurotransmitter released with each nerve impulse, or chronically, by changing postsynaptic properties or the content and size of synaptic vesicles. It is obvious that the effects of neurotrophins depend on the specific neurotrophin involved (four neurotrophins have been found in mammals; these are nerve growth factor, brain-derived neurotrophic factor, and neurotrophins-3 and-4) and on the specific synapse being studied. Growing evidence highlights the role of neurotrophins in the development and function of neuromuscular synapses. This review will examine the role of neurotrophins in the regulation of neuromuscular transmission. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 327–337, July–October, 2007.     

Distinct molecular mechanisms control levels of synaptic F‐actin

Polymerization of filamentous (F)‐actin at the neuronal synapse plays an important role in neuronal function. However, the regulatory mechanisms controlling the levels of synaptic actin remain incompletely understood. Here, I used established pharmacological blockers to acutely disrupt the function of actin polymerization machinery, then quantified their effect on synaptic F‐actin levels. Synaptic F‐actin was modestly decreased by inhibition of Arp2/3‐dependent actin branching. Blockade of formin‐dependent actin elongation resulted in an Arp2/3‐dependent increase in synaptic actin that could be mimicked by limited actin depolymerization. Limited actin depolymerization was also sufficient to reverse a decrease in synaptic F‐actin caused by prolonged blockade of synaptic NMDA‐type glutamate receptors. These results suggest that interplay between different actin polymerization pathways may regulate synaptic actin dynamics.     

The role of microglia in synaptic stripping and synaptic degeneration: a revised perspective

Chronic neurodegenerative diseases of the CNS (central nervous system) are characterized by the loss of neurons. There is, however, growing evidence to show that an early stage of this process involves degeneration of presynaptic terminals prior to the loss of the cell body. Synaptic plasticity in CNS pathology has been associated with microglia and the phenomenon of synaptic stripping. We review here the evidence for the involvement of microglia in synaptic stripping and synapse degeneration and we conclude that this is a case of guilt by association. In disease models of chronic neurodegeneration, there is no evidence that microglia play an active role in either synaptic stripping or synapse degeneration, but the degeneration of the synapse and the envelopment of a degenerating terminal appears to be a neuron autonomous event. We highlight here some of the gaps in our understanding of synapse degeneration in chronic neurodegenerative disease.     

LAR-RPTPs: synaptic adhesion molecules that shape synapse development

The synapse is the most elementary operating unit in neurons, creating neural circuits that underlie all brain functions. Synaptic adhesion molecules initiate neuronal synapse connections, promote their stabilization and refinement, and control long-term synaptic plasticity. Leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) have previously been implicated as essential elements in central nervous system (CNS) development. Recent studies have demonstrated that LAR-RPTP family members are also involved in diverse synaptic functions, playing a role in synaptic adhesion pathways together with a host of distinct transmembrane proteins and serving as major synaptic adhesion molecules in governing pre- and postsynaptic development, dysfunctions of which may underlie various disorders. This review highlights the emerging role of LAR-RPTPs as synapse organizers in orchestrating synapse development.     

Long-term synaptic changes induced in the cerebellar cortex by fear conditioning

To better understand learning mechanisms, one needs to study synaptic plasticity induced by behavioral training. Recently, it has been demonstrated that the cerebellum is involved in the consolidation of fear memory. Nevertheless, how the cerebellum contributes to emotional behavior is far from known. In cerebellar slices at 10 min and 24 hr following fear conditioning, we found a long-lasting potentiation of the synapse between parallel fibers and Purkinje cells in vermal lobules V-VI, but not in the climbing fiber synapses. The mechanism is postsynaptic, due to an increased AMPA response. In addition, in hotfoot mice with a primary deficiency of the parallel fiber to Purkinje cell synapse, cued (but not contextual) fear conditioning is affected. We propose that this synapse plays an important role in the learned fear and that its long-term potentiation may represent a contribution to the neural substrate of fear memory.     

Carboxypeptidase E is required for normal synaptic transmission from photoreceptors to the inner retina

Defects in the gene encoding carboxypeptidase E (CPE) in either mouse or human lead to multiple endocrine disorders, including obesity and diabetes. Recent studies on Cpe-/- mice indicated neurological deficits in these animals. As a model system to study the potential role of CPE in neurophysiology, we carried out electroretinography (ERG) and retinal morphological studies on Cpe-/- and Cpe fat/fat mutant mice. Normal retinal morphology was observed by light microscopy in both Cpe-/- and Cpe(fat/fat) mice. However, with increasing age, abnormal retinal function was revealed by ERG. Both Cpe-/- and Cpe fat/fat animals had progressively reduced ERG response sensitivity, decreased b-wave amplitude and delayed implicit time with age, while maintaining a normal a-wave amplitude. Immunohistochemical staining showed specific localization of CPE in photoreceptor synaptic terminals in wild-type (WT) mice, but in both Cpe-/- and Cpe fat/fat mice, CPE was absent in this layer. Bipolar cell morphology and distribution were normal in these mutant mice. Electron microscopy of retinas from Cpe fat/fat mice revealed significantly reduced spherule size, but normal synaptic ribbons and synaptic vesicle density, implicating a reduction in total number of vesicles per synapse in the photoreceptors of these animals. These results suggest that CPE is required for normal-sized photoreceptor synaptic terminal and normal signal transmission to the inner retina.     

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.     

Roles of ubiquitination at the synapse

The ubiquitin proteasome system (UPS) was first described as a mechanism for protein degradation more than three decades ago, but the critical roles of the UPS in regulating neuronal synapses have only recently begun to be revealed. Targeted ubiquitination of synaptic proteins affects multiple facets of the synapse throughout its life cycle; from synaptogenesis and synapse elimination to activity-dependent synaptic plasticity and remodeling. The recent identification of specific UPS molecular pathways that act locally at the synapse illustrates the exquisite specificity of ubiquitination in regulating synaptic protein trafficking and degradation events. Synaptic activity has also been shown to determine the subcellular distribution and composition of the proteasome, providing additional mechanisms for locally regulating synaptic protein degradation. Together these advances reveal that tight control of protein turnover plays a conserved, central role in establishing and modulating synapses in neural circuits.     

Protein homeostasis and synaptic plasticity

It is clear that de novo protein synthesis has an important function in synaptic transmission and plasticity. A substantial amount of work has shown that mRNA translation in the hippocampus is spatially controlled and that dendritic protein synthesis is required for different forms of long‐term synaptic plasticity. More recently, several studies have highlighted a function for protein degradation by the ubiquitin proteasome system in synaptic plasticity. These observations suggest that changes in synaptic transmission involve extensive regulation of the synaptic proteome. Here, we review experimental data supporting the idea that protein homeostasis is a regulatory motif for synaptic plasticity.     

Dopamine-mediated calcium channel regulation in synaptic suppression in L. stagnalis interneurons

D2 dopamine receptor-mediated suppression of synaptic transmission from interneurons plays a key role in neurobiological functions across species, ranging from respiration to memory formation. In this study, we investigated the mechanisms of D2 receptor-dependent suppression using soma-soma synapse between respiratory interneuron VD4 and LPeD1 in the mollusk Lymnaea stagnalis (L. stagnalis). We studied the effects of dopamine on voltage-dependent Ca²⁺ current and synaptic vesicle release from the VD4. We report that dopamine inhibits voltage-dependent Ca²⁺ current in the VD4 by both voltage-dependent and -independent mechanisms. Dopamine also suppresses synaptic vesicle release downstream of activity-dependent Ca²⁺ influx. Our study demonstrated that dopamine acts through D2 receptors to inhibit interneuron synaptic transmission through both voltage-dependent Ca²⁺ channel-dependent and -independent pathways. Taken together, these findings expand our understanding of dopamine function and fundamental mechanisms that shape the dynamics of neural circuit.