Dion K. Dickman

The Schizophrenia Susceptibility Gene dysbindin Controls Synaptic Homeostasis

Dysbindin Function in Synaptic Homeostasis Homeostatic signaling systems are widely believed to stabilize neural function over prolonged periods of time. However, the molecular mechanisms of homeostatic signaling in the nervous system are largely unknown, and direct links between defective homeostatic signaling and disease-causing genes remain obscure. Dickman and Davis (p. 1127) performed a large-scale, electrophysiology-based genetic screen for mutations that specifically disrupt synaptic homeostasis. DTNBP1 is one of two genes that are most strongly and consistently associated with schizophrenia susceptibility in humans. The Drosophila homolog of DTNBP1 (dysbindin) was identified in the screen and was found to function during synapse development, baseline neurotransmission, and synaptic homeostasis. Dysbindin altered the calcium-dependence of vesicle release and was essential in the presynaptic neuron for both the induction and expression of synaptic homeostasis. The dysbindin protein is required for the modulation of presynaptic neurotransmitter release in Drosophila. The molecular mechanisms that achieve homeostatic stabilization of neural function remain largely unknown. To better understand how neural function is stabilized during development and throughout life, we used an electrophysiology-based forward genetic screen and assessed the function of more than 250 neuronally expressed genes for a role in the homeostatic modulation of synaptic transmission in Drosophila. This screen ruled out the involvement of numerous synaptic proteins and identified a critical function for dysbindin, a gene linked to schizophrenia in humans. We found that dysbindin is required presynaptically for the retrograde, homeostatic modulation of neurotransmission, and functions in a dose-dependent manner downstream or independently of calcium influx. Thus, dysbindin is essential for adaptive neural plasticity and may link altered homeostatic signaling with a complex neurological disease.

A Hierarchy of Cell Intrinsic and Target-Derived Homeostatic Signaling

Homeostatic control of neural function can be mediated by the regulation of ion channel expression, neurotransmitter receptor abundance, or modulation of presynaptic release. These processes can be implemented through cell autonomous or intercellular signaling. It remains unknown whether different forms of homeostatic regulation can be coordinated to achieve constant neural function. One way to approach this question is to confront a simple neural system with conflicting perturbations and determine whether the outcome reflects a coordinated, homeostatic response. Here, we demonstrate that two A-type potassium channel genes, shal and shaker, are reciprocally, transcriptionally coupled to maintain A-type channel expression. We then demonstrate that this homeostatic control of A-type channel expression prevents target-dependent, homeostatic modulation of synaptic transmission. Thus, we uncover a homeostatic mechanism that reciprocally regulates A-type potassium channels, and we define a hierarchical relationship between cell-intrinsic control of ion channel expression and target-derived homeostatic control of synaptic transmission.

Mutations in a Drosophila α2δ Voltage-Gated Calcium Channel Subunit Reveal a Crucial Synaptic Function

Voltage-dependent calcium channels regulate many aspects of neuronal biology, including synaptic transmission. In addition to their α1 subunit, which encodes the essential voltage gate and selective pore, calcium channels also contain auxiliary α2δ, β, and γ subunits. Despite progress in understanding the biophysical properties of calcium channels, the in vivo functions of these auxiliary subunits remain unclear. We have isolated mutations in the gene encoding an α2δ calcium channel subunit (dα2δ-3) using a forward genetic screen in Drosophila. Null mutations in this gene are embryonic lethal and can be rescued by expression in the nervous system, demonstrating that the essential function of this subunit is neuronal. The photoreceptor phenotype of dα2δ-3 mutants resembles that of the calcium channel α1 mutant cacophony (cac), suggesting shared functions. We have examined in detail genotypes that survive to the third-instar stage. Electrophysiological recordings demonstrate that synaptic transmission is severely impaired in these mutants. Thus the α2δ calcium channel subunit is critical for calcium-dependent synaptic function. As such, this Drosophila isoform is the likely partner to the presynaptic calcium channel α1 subunit encoded by the cac locus. Consistent with this hypothesis, cacGFP fluorescence at the neuromuscular junction is reduced in dα2δ-3 mutants. This is the first characterization of an α2δ-3 mutant in any organism and indicates a necessary role for α2δ-3 in presynaptic vesicle release and calcium channel expression at active zones.

Emerging links between homeostatic synaptic plasticity and neurological disease

Homeostatic signaling systems are ubiquitous forms of biological regulation, having been studied for hundreds of years in the context of diverse physiological processes including body temperature and osmotic balance. However, only recently has this concept been brought to the study of excitatory and inhibitory electrical activity that the nervous system uses to establish and maintain stable communication. Synapses are a primary target of neuronal regulation with a variety of studies over the past 15 years demonstrating that these cellular junctions are under bidirectional homeostatic control. Recent work from an array of diverse systems and approaches has revealed exciting new links between homeostatic synaptic plasticity and a variety of seemingly disparate neurological and psychiatric diseases. These include autism spectrum disorders, intellectual disabilities, schizophrenia, and Fragile X Syndrome. Although the molecular mechanisms through which defective homeostatic signaling may lead to disease pathogenesis remain unclear, rapid progress is likely to be made in the coming years using a powerful combination of genetic, imaging, electrophysiological, and next generation sequencing approaches. Importantly, understanding homeostatic synaptic plasticity at a cellular and molecular level may lead to developments in new therapeutic innovations to treat these diseases. In this review we will examine recent studies that demonstrate homeostatic control of postsynaptic protein translation, retrograde signaling, and presynaptic function that may contribute to the etiology of complex neurological and psychiatric diseases.

Snapin is Critical for Presynaptic Homeostatic Plasticity

The molecular mechanisms underlying the homeostatic modulation of presynaptic neurotransmitter release are largely unknown. We have previously used an electrophysiology-based forward genetic screen to assess the function of >400 neuronally expressed genes for a role in the homeostatic control of synaptic transmission at the neuromuscular junction of Drosophila melanogaster. This screen identified a critical function for dysbindin, a gene linked to schizophrenia in humans (Dickman and Davis, 2009). Biochemical studies in other systems have shown that Snapin interacts with Dysbindin, prompting us to test whether Snapin might be involved in the mechanisms of synaptic homeostasis. Here, we demonstrate that loss of snapin blocks the homeostatic modulation of presynaptic vesicle release following inhibition of postsynaptic glutamate receptors. This is true for both the rapid induction of synaptic homeostasis induced by pharmacological inhibition of postsynaptic glutamate receptors, and the long-term expression of synaptic homeostasis induced by the genetic deletion of the muscle-specific GluRIIA glutamate receptor subunit. Loss of snapin does not alter baseline synaptic transmission, synapse morphology, synapse growth, or the number or density of active zones, indicating that the block of synaptic homeostasis is not a secondary consequence of impaired synapse development. Additional genetic evidence suggests that snapin functions in concert with dysbindin to modulate vesicle release and possibly homeostatic plasticity. Finally, we provide genetic evidence that the interaction of Snapin with SNAP25, a component of the SNARE complex, is also involved in synaptic homeostasis.

Importin-β11 Regulates Synaptic Phosphorylated Mothers Against Decapentaplegic, and Thereby Influences Synaptic Development and Function at the Drosophila Neuromuscular Junction

Importin proteins act both at the nuclear pore to promote substrate entry and in the cytosol during signal trafficking. Here, we describe mutations in the Drosophila gene importin-β11, which has not previously been analyzed genetically. Mutants of importin-β11 died as late pupae from neuronal defects, and neuronal importin-β11 was present not only at nuclear pores but also in the cytosol and at synapses. Neurons lacking importin-β11 were viable and properly differentiated but exhibited discrete defects. Synaptic transmission was defective in adult photoreceptors and at larval neuromuscular junctions (NMJs). Mutant photoreceptor axons formed grossly normal projections and synaptic terminals in the brain, but synaptic arbors on larval muscles were smaller while still containing appropriate synaptic components. Bone morphogenic protein (BMP) signaling was the apparent cause of the observed NMJ defects. Importin-β11 interacted genetically with the BMP pathway, and at mutant synaptic boutons, a key component of this pathway, phosphorylated mothers against decapentaplegic (pMAD), was reduced. Neuronal expression of an importin-β11 transgene rescued this phenotype as well as the other observed neuromuscular phenotypes. Despite the loss of synaptic pMAD, pMAD persisted in motor neuron nuclei, suggesting a specific impairment in the local function of pMAD. Restoring levels of pMAD to mutant terminals via expression of constitutively active type I BMP receptors or by reducing retrograde transport in motor neurons also restored synaptic strength and morphology. Thus, importin-β11 function interacts with the BMP pathway to regulate a pool of pMAD that must be present at the presynapse for its proper development and function.

Activity-dependent facilitation of Synaptojanin and synaptic vesicle recycling by the Minibrain kinase

Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signaling required for proper synaptic development, function, and plasticity, particularly during changes in neuronal activity. Here we present evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). We find that Mnb-dependent phosphorylation of synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. Our data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity.

Gene Dosage in the Dysbindin Schizophrenia Susceptibility Network Differentially Affect Synaptic Function and Plasticity

Neurodevelopmental disorders arise from single or multiple gene defects. However, the way multiple loci interact to modify phenotypic outcomes remains poorly understood. Here, we studied phenotypes associated with mutations in the schizophrenia susceptibility gene dysbindin (dysb), in isolation or in combination with null alleles in the dysb network component Blos1. In humans, the Blos1 ortholog Bloc1s1 encodes a polypeptide that assembles, with dysbindin, into the octameric BLOC-1 complex. We biochemically confirmed BLOC-1 presence in Drosophila neurons, and measured synaptic output and complex adaptive behavior in response to BLOC-1 perturbation. Homozygous loss-of-function alleles of dysb, Blos1, or compound heterozygotes of these alleles impaired neurotransmitter release, synapse morphology, and homeostatic plasticity at the larval neuromuscular junction, and impaired olfactory habituation. This multiparameter assessment indicated that phenotypes were differentially sensitive to genetic dosages of loss-of-function BLOC-1 alleles. Our findings suggest that modification of a second genetic locus in a defined neurodevelopmental regulatory network does not follow a strict additive genetic inheritance, but rather, precise stoichiometry within the network determines phenotypic outcomes.

The Innate Immune Receptor PGRP-LC Controls Presynaptic Homeostatic Plasticity

It is now appreciated that the brain is immunologically active. Highly conserved innate immune signaling responds to pathogen invasion and injury and promotes structural refinement of neural circuitry. However, it remains generally unknown whether innate immune signaling has a function during the day-to-day regulation of neural function in the absence of pathogens and irrespective of cellular damage or developmental change. Here we show that an innate immune receptor, a member of the peptidoglycan pattern recognition receptor family (PGRP-LC), is required for the induction and sustained expression of homeostatic synaptic plasticity. This receptor functions presynaptically, controlling the homeostatic modulation of the readily releasable pool of synaptic vesicles following inhibition of postsynaptic glutamate receptor function. Thus, PGRP-LC is a candidate receptor for retrograde, trans-synaptic signaling, a novel activity for innate immune signaling and the first known function of a PGRP-type receptor in the nervous system of any organism.

New Approaches for Studying Synaptic Development, Function, and Plasticity Using Drosophila as a Model System

The fruit fly Drosophila melanogaster has been established as a premier experimental model system for neuroscience research. These organisms are genetically tractable, yet their nervous systems are sufficiently complex to study diverse processes that are conserved across metazoans, including neural cell fate determination and migration, axon guidance, synaptogenesis and function, behavioral neurogenetics, and responses to neuronal injury. For several decades, Drosophila neuroscientists have taken advantage of a vast toolkit of genetic and molecular techniques to reveal fundamental principles of neuroscience illuminating to all systems, including the first behavioral mutants from Seymour Benzers pioneering work in the 1960s and 1970s, the cloning of the first potassium channel in the 1980s, and the identification of the core genes that orchestrate axon guidance and circadian rhythms in the 1990s. Over the past decade, new tools and innovations in genetic, imaging, and electrophysiological technologies have enabled the visualization, in vivo, of dynamic processes in synapses with unprecedented resolution. We will review some of the fresh insights into synaptic development, function, and plasticity that have recently emerged in Drosophila with an emphasis on the unique advantages of this model system.