Dion K. Dickman, PhD - US grants

DESCRIPTION (provided by applicant): Dysbindin and the Mechanisms Controlling the Homeostatic Modulation of Presynaptic Neurotransmitter Release Nervous system function remains remarkably stable despite the many changes that occur during the development, maturation, and aging of the brain. There is increasing evidence that neurons are endowed with potent mechanisms that compensate for perturbations to their activity and maintain the stability of neural function within proper physiological ranges. Although these homeostatic properties have been demonstrated in a variety of systems from invertebrates to humans, the mechanisms that mediate these fundamental and complex processes are poorly understood. Using Drosophila as a model for homeostasis at the level of the synapse, we have recently demonstrated that the gene dysbindin is required for synaptic homeostasis. Interestingly, the human homolog of dysbindin (DTNBP1) has emerged as a primary susceptibility gene for schizophrenia. The overall objective of this proposal is to define the mechanisms through which Dysbindin modulates neural function and achieves the homeostatic control of synaptic stability. The initial aim will be to define the role of Snapin in synaptic function and homeostasis. Snapin has been shown to bind Dysbindin and separately to modulate the synaptic fusion machinery. Next, biochemical and live imaging approaches will be used to monitor and test the importance of the Snapin-Dysbindin interaction for the homeostatic modulation of presynaptic release. Finally, I will explore the role of other proteins that interact with Dysbindin and go on to search for new genes that are required for synaptic homeostasis. The training phase of this research will be performed at the University of California, San Francisco in the laboratory of Dr. Graeme Davis. In this environment at UCSF, I will enhance both my experimental skills as well as the skills necessary to become a successful independent researcher. My long term goal is to understand the molecular mechanisms that govern the homeostatic control of neural function and how dysfunction in this process may contribute to complex neurological and psychiatric disease. I am committed to researching these areas at an academic institution. Public Health Relevance: Dysbindin has emerged as a primary susceptibility gene for schizophrenia in humans. This proposal seeks to elucidate the role of Dysbindin in the homeostatic control of neural function and to search for new genes involved in this process. Together, these efforts have to potential to implicate synaptic homeostasis in the etiology of schizophrenia and other complex psychiatric diseases. PUBLIC HEALTH RELEVANCE: Dysbindin has emerged as a primary susceptibility gene for schizophrenia in humans. This proposal seeks to elucidate the role of Dysbindin in the homeostatic control of neural function and to search for new genes involved in this process. Together, these efforts have the potential to implicate defects in synaptic homeostasis as a plausible contributing factor in the etiology of schizophrenia and perhaps other complex psychiatric diseases.

? DESCRIPTION (provided by applicant): Nervous systems from invertebrates to humans have shown remarkably resilient and adaptive abilities to maintain stable functionality despite challenges that may otherwise lead to suboptimal or uncontrolled activity. In each of these systems, perturbations to synaptic activity initially lead to corresponding alterations in synaptic strength. However, given sufficient time, nervous systems in these organisms adapt by modulating presynaptic release or postsynaptic neurotransmitter receptors to re-target previous levels of synaptic strength. This process, termed homeostatic synaptic plasticity, is thought to enable stable, yet flexible, synaptic activity and to play key roles in tuning neural function in health and disease. Yet there is a major gap in our knowledge of the molecular and cellular mechanisms that endow synapses with these extraordinary abilities. The long term goal of this proposal is to identify the genes and elucidate the mechanisms that achieve and maintain the homeostatic control of synaptic strength. To understand the principles governing homeostatic synaptic signaling, we will utilize the Drosophila neuromuscular junction, which has been established as a powerful genetic system to study this process. This proposal will use a combination of genetic analysis, electrophysiology, and imaging approaches to investigate the homeostatic mechanisms that enhance presynaptic release in response to a perturbation to postsynaptic neurotransmitter receptor function. In particular, three genes encoding neuronal transmembrane proteins have been identified that appear to function together in the presynaptic terminal to promote the calcium-dependent, homeostatic potentiation of synaptic transmission. Interestingly, these genes have been associated with epilepsy, schizophrenia, and bipolar disorder. The proposed experiments will first characterize these molecules in synaptic function and homeostatic plasticity. Confocal and super-resolution microscopy will then be utilized to reveal the subsynaptic localization and cellular activities of these proteins. Finally, complementary forward genetic screens are proposed to identify new genes that orchestrate homeostatic synaptic plasticity. Together, this work is expected to reveal new homeostatic genes and mechanisms that control the adaptive modulation of synaptic strength and provide a foundation from which to understand how transcellular homeostatic signaling systems more generally are established in the nervous system.

This BRAIN EAGER will support an interdisciplinary team of investigators to jointly develop and deploy optimized light sheet microscopy and novel genetically encoded probes to image synaptic function in the intact nervous system. This team will optimize a novel 3D imaging microscope, in which multi-photon light sheet illumination is combined with light field microscopy to permit a single snapshot to capture the full 3D image, enabling the team to visualize these probes with unprecedented speed and coverage. They will also develop new genetically encoded glutamate and calcium probes, targeted to defined synaptic compartments, to optimize signal magnitude and report synaptic activity with high sensitivity and fidelity. These innovations will be exploited to synergistically visualize synaptic structure and monitor glutamate and calcium dynamics in the intact Drosophila central nervous system.

Although these tools could be used in a variety of settings in the vertebrate or the invertebrate nervous system, these will be first applied to address the fundamental relationship between sleep and synaptic plasticity. Although sleep is ancient, the essential biological function of this behavior remains a great mystery of science. This project will explore the exciting possibility that a fundamental function of sleep, operating at the level of individual neurons and synapses, is the homeostatic modulation of synaptic strength. Addressing this hypothesis has been beyond our capabilities because visualizing neural activity in the central nervous system during sleep-wake behavior has been limited in both speed and resolution. Through a combination of new genetically encoded probes reporting synaptic structure and activity and cutting-edge imaging approaches, this project will permit the imaging of synapses over time without perturbing the nervous system or the sleep-wake cycle. These test experiments will advance our knowledge of the complex, fundamental, and poorly understood signaling systems that orchestrate the homeostatic control of synaptic strength, and their modulation during sleep behavior. The education and outreach activities of the research team will be intimately linked with their research programs, and will include a research project with local inner-city Los Angeles high school students investigating sleep and circadian behavior. In addition, a new undergraduate course will be developed exploring the biological functions of sleep.

Homeostatic signaling systems are crucial forms of biological regulation that permit flexible yet stable information transfer in the nervous system. These fundamental mechanisms operate to maintain such properties as synaptic strength and glutamate levels within stable physiological ranges. Although intensive research has been focused on understanding how excitatory synapses are homeostatically modulated to stabilize synaptic strength, far less is known about how these synapses adjust to control glutamate release itself. Excess glutamate release can lead to a variety of diseases and dysfunctions in the nervous system, contributing to seizures, excitotoxity, and neurodegeneration. Here, we propose to characterize a glutamate homeostat that controls presynaptic function using the Drosophila neuromuscular junction as a unique and powerful model system. At this glutamatergic synapse, excess presynaptic glutamate secretion induces a homeostatic inhibition of neurotransmitter release, an adaptation referred to as presynaptic homeostatic depression (PHD). This process parallels a similar phenomenon observed in a variety of other organisms, including mammalian central synapses. We hypothesize that excess glutamate is sensed by a presynaptic glutamate receptor and activates an autocrine signaling system to homeostatically depress synaptic vesicle release. To test this model, we will use a systematic electrophysiology screen to test glutamate receptors in Drosophila for roles in PHD. Next, we will leverage a combination of cell biology, heterologous expression, pharmacology, and innovative functional imaging techniques to determine the mechanisms through which excess glutamate signals a precise reduction in presynaptic vesicle release. Finally, we will assess how synapses, neurons, and glia adapt to chronic glutamate imbalance using several approaches, including a cell-specific translational profiling technology we have developed as well as a new generation of glutamate indicators. Together, these experiments will advance our understanding of the mechanisms that endow synapses with the ability homeostatically tune glutamate release, and will identify maladaptive responses to glutamate imbalance in the nervous system. Ultimately, this knowledge will inform therapeutic strategies towards counteracting diseases associated with glutamate imbalance, including epilepsy, fragile X syndrome and neurodegeneration.

PROJECT SUMMARY Stable functionality in the nervous system is maintained despite the challenges associated with development, growth, experience, aging, and disease. This remarkable stability is controlled by potent and adaptive homeostatic signaling systems that sustain robust and reliable information transfer across synapses. Synapses are therefore critical substrates to achieve and maintain the homeostatic control of neural activity. Indeed, homeostatic synaptic plasticity is fundamental form of plasticity endowed at synapses in the central and peripheral nervous systems of invertebrates, rodents, and humans. Defects in homeostatic signaling contribute to the etiology of a variety of neurological diseases including epilepsy, Fragile X Syndrome, and neurodegeneration. However, the molecular mechanisms that induce and sustain homeostatic plasticity remain enigmatic. To understand synaptic dysfunction during disease states, we must first decipher the intercellular dialogue that controls homeostatic signaling. Our long-term goals are to define the mechanisms that achieve and maintain the homeostatic control of synaptic function in health and disease. Towards this end, we have pioneered forward genetic screens using electrophysiology that have discovered new genes and revealed fundamental mechanisms mediating homeostatic signaling at the Drosophila neuromuscular junction. At this model glutamatergic synapse, acute pharmacological inhibition or chronic genetic elimination of postsynaptic glutamate receptors (GluRs) triggers a retrograde signaling system in the postsynaptic compartment that precisely increases presynaptic neurotransmitter release to maintain stable synaptic strength. This process is referred to as Presynaptic Homeostatic Potentiation (PHP). Work from the Dickman lab and others have uncovered many presynaptic genes that converge on two key mechanisms that serve to increase neurotransmitter release and enable the expression of PHP. In addition, candidate retrograde signals have also been identified. In contrast to the emerging framework from which we now understand how enhanced presynaptic neurotransmitter release is controlled following PHP expression, almost nothing is known about the signaling system in the postsynaptic compartment that senses diminished GluR function and transforms this into tunable information transmitted to specific presynaptic compartments. Therefore, illuminating the nature of the postsynaptic induction mechanisms that initiate and maintain PHP will be the primary goal of this proposal. In this supplement, we propose to investigate the function of two newly identified postsynaptic genes, peflin and ALG2, in retrograde homeostatic signaling.