Susan Tsunoda - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] An important issue in the field of signal transduction is how signaling molecules are organized into different pathways within the same cell. Our goal is to understand how the signal transduction cascade that underlies the phototransduction process is organized, and how individual components interact and participate in normal visual function. The importance of assembling signaling molecules into architecturally defined complexes has emerged as an essential cellular strategy to ensure speed and specificity of signaling. Results in Drosophila photoreceptors as well as in other systems and organisms have further demonstrated that the subcellular localization of these signaling complexes is essential for effective signaling. Mislocalization of signaling components is often the equivalent of their absence, and consequences can be severe. Critical questions that arise then are: how are signaling complexes targeted to the right subcellular domain? How and where are they assembled? How is this assembly regulated? How are they anchored or stabilized in the proper locale? This grant proposal focuses on a molecular-genetic dissection of the assembly and localization of signaling complexes in Drosophila photoreceptors. We will 1) perform a comprehensive genetic screen to isolate mutations affecting the proper localization of signaling complexes in photoreceptors, 2) characterize the mutants genetically, cell biologically, and physiologically, 3) isolate the defective genes, compare the homologous sequence from wild-type flies, identify the nature of the change, and introduce the wild-type gene back into flies and test for rescue of the phenotype, 4) for those genes that warrant further investigation, we will study how the proteins they encode function in assembly and localization processes. Components and strategies used in Drosophila are likely to be conserved in mammals. We expect to identify and characterize key components in the assembly, regulation of assembly, targeting, and anchoring of signaling complexes. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our long-term interest is to understand how ion channels are localized to particular sub-cellular sites and regulated by specific protein interactors. In this grant proposal, we focus on the voltage-gated K+ channel, Shal (Kv4), which has been implicated in setting the rhythmic firing of central pattern generators, learning and memory, and shaping the cardiac action potential. Therefore, understanding the mechanisms of Shal K+ channel localization and regulation has important implications for vital processes in the heart and central nervous system. We focus our initial studies on two newly identified interactors, K30 and K29, of Drosophila Shal channels. Both interactors are expressed primarily in the nervous system, co-localize with Shal channels, and exhibit strong and specific binding to the C-terminus of Shal channels. Interestingly, K29 binds to a highly conserved motif required for dendritic targeting of mammalian Shal channels, implicating K29 as a key regulator of Shal channel localization. We will characterize Shal-K30 and Shal-K29 interactions and examine the function of K30 and K29 in the subcellular localization and regulation of Shal channels in vivo. Using Drosophila as a model system will allow us to combine genetic, electrophysiological, cell and molecular biological approaches to study how all identified interactors function in the regulation and subcellular localization of Shal channels. Since strategies and proteins identified are likely to be conserved in mammals, our findings are expected to be significant not only for understanding Drosophila ion channels, but also for mammalian systems. Relevance to public health: Ion channels are the basic components that shape electrical and chemical communication in the nervous system, and the function of ion channels is highly dependent on their subcellular localization and regulation. When ion channels are mis-localized or mis-regulated, consequences are often severe, resulting in conditions such as epilepsy, episodic ataxia, periodic paralysis, myotonia, and Long QT syndrome. Therefore, understanding how ion channels are regulated and localized to subcellular compartments is likely to give important insights into the prevention and treatment of these conditions.

DESCRIPTION (provided by applicant): Ion channels and receptors are the basic components that shape electrical and chemical communication in the nervous system. Our long-term interest is to understand how ion channels are trafficked and regulated to perform their physiological roles. In this proposal, we focus on the highly conserved, voltage-gated Shal/Kv4 channel. Across species, these channels are localized to somato-dendritic sites, where they regulate dendritic excitability, the integration of synaptic inputs, the shape of mEPSCs, backpropagating action potentials, and long-term potentiation. Because of these important functions, animal models with decreased/mutant Shal/Kv4 channels display spatial learning defects, seizure behavior, as well as temporal lobe epilepsy. Shal/Kv4 channels have also been shown to underlie the Ito current, which is responsible for initial repolarization of the cardiac action potential. Therefore, understanding the mechanisms of Shal/Kv4 channel localization and regulation have important implications for the health and functioning of vital processes in the nervous system and heart. Using Drosophila as our model system, we propose studies to examine mechanisms underlying the somato-dendritic localization of Shal/Kv4 channels, and investigate a new role Shal/Kv4 channels play at postsynaptic sites. In Specific Aim #1, we explore two mechanisms that underlie the polarized distribution of Shal/Kv4 channels. One mechanism depends on a highly conserved di-leucine motif (LL-motif) on the C-terminus of Shal/Kv4 channels. We propose to generate a mutant of a recently identified protein, Shal Interactor of Di-Leucine Motif (SIDL) that interacts with this LL-motif, and examine how GFP-Shal/Kv4 localization is affected in vivo. This SIDL mutant will also be engineered to allow us to knock-in mutant SIDL constructs, and perform structure-function studies. We will also test whether the second mechanism involves a cytoskeletal barrier at the axon initial segment (AIS) by perturbing the AIS cytoskeleton, tracking single Shal/Kv4 channels, and analyzing how localization and mobility are affected. Specific Aim #2 is based on strong preliminary studies showing that Shal/Kv4 channels are up-regulated in response to synaptic inactivity. We will test the model that it is the homeostatic up-regulation of specific postsynaptic receptors that triggers an increase in Shal/Kv4 channel expression, for the purpose of modulating postsynaptic potentials and their homeostatic regulation. This novel regulation will add a new dimension to the role Shal/Kv4 channels play in synaptic plasticity. Using Drosophila as our model system, we combine genetic, biochemical, electrophysiological, cell and molecular biological approaches to gain unique insight into these questions that would be more difficult to address in mammalian systems. 1

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most prevalent form of dementia in the elderly population. Most AD research has focused on understanding how beta-amyloid (Abeta) peptide accumulation, and neurofibrillary tangles (NFT), contribute to the cause of AD. There is, however, less known about how these events lead to the later manifestations of AD, such as progressive neurodegeneration and a decline in cognitive and motor function. With no current cure for AD (prevention of the primary events), understanding the subsequent cellular events that underlie disease progression may give important insight into potential treatments that could halt or slow the devastating effects of the disease. Recently, over-production of Abeta has been shown to result in hyperexcitability and Ca2+ overload in hippocampal and cortical neurons. Increased excitability is also consistent with behavioral studies which have shown enhanced seizure activity in mouse models with increased Abeta expression, and increased risk of epilepsy in AD patients. The goal of this proposal is to determine whether a transgenic Drosophila model that expresses the secreted human Abeta42, which exhibits many of the hallmarks of AD (e.g. Abeta deposits, age-dependent learning/memory and locomotor deficits, neurodegeneration), also displays neuronal hyperexcitability. We will identify how intrinsic electrical properties of neurons are altered, and how these changes affect neuronal excitability. These studies are essential for establishing the Abeta42-Drosophila transgenic line as an effective model for investigations into how Abeta42-induced intrinsic changes, and hyperexcitability, contribute to downstream cellular and behavioral deficits seen AD. Since ion channels are so highly conserved, cellular strategies are likely to be shared across species and findings are expected to be significant for mammalian systems.

DESCRIPTION (provided by applicant): Synaptic homeostasis is a protective mechanism employed by neurons to counterbalance changes in global neural activity. The last decade has seen intensive study in this field for glutamatergic synapses, however, almost nothing is known about whether synaptic homeostasis is also mediated by other excitatory receptors, such as nicotinic acetylcholine receptors (nAChRs). Indeed, cholinergic synaptic homeostasis has been implicated in important pathological conditions, such as Alzheimer's disease (AD) and nicotine dependence. In this R21 application, our goal is to validate a new system for studying cholinergic synaptic homeostasis and break new ground in an important area that has not yet been explored. The Drosophila CNS provides an ideal model for studying cholinergic synaptic homeostasis since nAChRs are the major excitatory receptor in Drosophila central neurons. Since mammalian ?7 nAChRs are among the most abundant and widespread of nAChRs, and have been implicated in Alzheimer's disease, nicotine addiction, nicotine-induced seizures, and schizophrenia, we focus on the Drosophila ?7 like receptor (D?7) to validate our model. Using primary neuronal cultures in parallel with a cultured brain preparation, we propose to manipulate neural activity, and assay for changes in synaptic strength and changes in nAChR localization and distribution. We will also test the physiological relevance of cholinergic synaptic homeostasis by examining effects on a known behavioral output mediated by D?7 nAChRs, the giant fiber mediated escape behavior. The development of this novel system, which involves the combination of primary cultures, an ex-vivo brain preparation, and a quantifiable behavioral output, will be unique to the field and allow us to answer long-awaited questions about cholinergic synaptic homeostasis.

MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that are increasingly being shown to play important roles in regulating gene expression in both plants and animals. Recent reports have shown that small deletions affecting the brain-enriched miRNA, miR-137, have been identified in unrelated families with intellectual disability (ID), and SNPs in miR-137 have been linked to schizophrenia; subsequent studies have shown that reduced and over-expression of miR-137 result in defects in long-term depression (LTD), long-term potentiation (LTP), as well as learning and memory function. Since miRNAs usually have hundreds of predicted targets, it will be essential to not only identify miRNAs significant to human health, but also to identify relevant targets, and the specifics of their regulation. Although there are interesting targets of miR-137 already reported, most in vivo tests performed affect miR-137, and not the endogenous mRNA target sites; thus, it is not clear whether physiological defects resulting from loss/gain of miR-137 are due to other/additional targets. Interestingly, predicted targets of miR-137 across fly, mouse and human genomes, include multiple voltage-dependent ion channels, suggesting that miR-137 contributes to shaping the intrinsic electrical properties of neurons; these channel targets include K+ channels, Kv4/Shal (KCND1, KCND2), Shaw/Kv3 (KCNC1, KCNC3), and eag-like channels (KCNH1, KCNH7), as well as Cav3 T-type calcium channels (CACNAg, CACNAh, CACNAi). Our long-term goal is to understand if and how miR-137 regulates the intrinsic excitability of neurons, and whether mis-regulation contributes to ID and/or schizophrenia. In this R21 application, we propose to validate a system in which we can characterize how miR-137 regulates neuronal excitability, identify the affected ion channel targets, and test endogenous target site(s) in vivo. We use Drosophila as a model since available genetic tools in this system enable rapid generation of null mutants of miR-137, targeted loss/over-expression of miR-137 in subsets of neurons, as well as generation of mutations that affect endogenous target sites in the 3? UTR of individual genes. We will evaluate effects on mRNA, protein, as well as on electrical/synaptic signaling in identified neurons and eventually, on whole animal behavior. We expect molecular and cellular mechanisms to be conserved, and that our findings will provide the rationale to form and test hypotheses more efficiently in mammalian systems.

Kv4 channels have been shown to play important roles in modulating neural activity: regulating the integration of high-frequency trains of synaptic input, regulating backpropagating action potentials, and contributing to long-term potentiation. Consequently, mutations that affect Kv4 function/availability have been shown to result in spatial learning defects, seizure behavior, as well as temporal lobe epilepsy. In the last funding period, we showed that expression and turnover of Kv4 channels are affected in three new contexts: in modulating cholinergic synaptic homeostasis, in response to over-expression of human A?42, and during normal aging. In the proposed studies, we investigate the mechanisms underlying Kv4 expression during cholinergic synaptic homeostasis. Synaptic homeostasis/scaling is a form of plasticity that has been heavily studied in the last decade as a protective mechanism that counterbalances changes in global neural activity; this likely occurs during physiological processes, such as learning/memory and development, as well as during pathological conditions. We used Drosophila central neurons as a model, and showed that Drosophila ?7 (D?7) nAChRs are up-regulated after cholinergic blockade, thereby enhancing synaptic currents and providing a homeostatic response. We found that this homeostatic response triggered a novel regulatory mechanism ? the up-regulation of Kv4 channels, which we showed prevents an ?overshoot? of the homeostatic response. We further showed that the up-regulation of Kv4 channels is blocked by transcriptional inhibitors, and is dependent on D?7 nAChRs and Ca2+ influx. Drosophila continues to be an ideal model system for these studies because of its cholinergic CNS, the genetic tools it offers, its less redundant genome (eg. there is only a single Drosophila NFAT and Kv4 gene, each of which represents a multi-gene family in mammals), and the ability to go from mechanisms of gene regulation to physiological relevance in the intact brain, and eventually, whole animal behavior. The proposed studies will apply new optogenetic approaches to elicit cholinergic synaptic homeostasis in vivo (Aim-1) ?something that has not been explored in any system, and which would currently not be feasible in mammalian systems. We will examine underlying molecular mechanisms, including a novel relationship between ?7 nAChRs and Kv4 channels (Aim-2), and inactivity-induced transcription of Kv4 (Aim-3) that is mediated by NFAT (Aim-4). We will also test all molecular mechanisms for their physiological relevance in identified neurons in the intact brain. Our studies are likely to reveal important insights into the underlying mechanisms of cholinergic synaptic homeostasis.

Tsunoda, Susan PROJECT SUMMARY The ability of animals to detect sensory stimulation, generate appropriate motor responses, and adapt detection mechanisms to changes in stimuli is crucial for survival. All of this signaling depends on the coordinated action of ion channels in the membranes of neurons. For example, there are Na+ channels specially evolved to carry out the fast depolarization of the neuronal membrane, as well as a wide range of K+ channels that subsequently repolarize the membrane and shape the action potential (AP) output of a neuron. In the proposed studies, the PI will test the role of KNa/slo2 channels, whose molecular identity has, relatively speaking, only recently been described. KNa/slo2 channels are Na+-activated K+ channels, which may have evolved to provide a protective ?brake? on membrane depolarization when neurons are over-stimulated. Although a neuroprotective role against over-excitation and a physiological role in sensory transduction/adaptation have been suggested, these role(s) of KNa/slo2 channels remain controversial. The PI proposes to use Drosophila as an in vivo model to address questions about how Na+ activates KNa/slo2 channels to affect neuronal signaling and behavior. The studies will look directly at AP firing and neuronal excitability, and explore behavioral effects on responsiveness to touch stimulation. Finally, the studies will use hyper-excitable genetic mutants with enhanced Na+ currents to test whether KNa/dslo2 channels are indeed able to counter over-excitation. 1

Tsunoda, Susan Project Summary Age is perhaps the most significant contributing factor to multiple neurological diseases, including Alzheimer?s Disease (AD). Our overarching hypothesis is that protein targets affected during normal aging may be especially affected in age-related disease conditions. In this proposal, we focus on the voltage-dependent K+ channel, Kv4, as such a target. Multiple studies have found that A?42 induces a decline in Kv4 channels that contributes to downstream cognitive and motor pathologies. Here, we will examine whether there is a decline in Kv4 channels with normal aging, whether reactive oxygen species (ROS) that arise with both normal aging and A?42 accumulation lead to this progressive loss of Kv4, and whether loss of Kv4 leads to signs of early aging and a shortened lifespan. Drosophila offers an ideal model for combining its powerful molecular-genetic toolkit with a short lifespan to study how aging/A?42 accumulation affects neuronal signaling. We propose: 1) to test the hypothesis that Kv4 channels are progressively lost with age by examining Kv4 mRNA, protein level and localization, as well as current, 2) to test the hypothesis that the age/A?42-dependent accumulation of ROS affects Kv4 channels, and 3) to test if normal age-related decline in motor activity and lifespan are improved when levels of Kv4 are genetically restored, and exacerbated when Kv4 is absent.

Kv4 channels have been shown to play important roles in modulating neural activity: regulating the integration of high-frequency trains of synaptic input, regulating backpropagating action potentials, and contributing to long-term potentiation. Consequently, mutations that affect Kv4 function/availability have been shown to result in spatial learning defects, seizure behavior, as well as temporal lobe epilepsy. We have recently shown that expression and turnover of Kv4 channels are also affected in three new contexts: in modulating cholinergic synaptic homeostasis, in response to over-expression of human A?42, and during normal aging. In the proposed studies, we investigate the mechanisms underlying Kv4 expression during cholinergic synaptic homeostasis (also referred to as synaptic scaling). Synaptic homeostasis is a form of plasticity that has been heavily studied in the last decade as a protective mechanism that counterbalances changes in global neural activity; this likely occurs during physiological processes, such as learning/memory and development, as well as during pathological conditions. We used Drosophila central neurons as a model, and showed that Drosophila ?7 (D?7) nAChRs are up-regulated after cholinergic blockade, thereby enhancing synaptic currents and providing a homeostatic response. We found that this homeostatic response triggered a novel regulatory mechanism ?the up-regulation of Kv4 channels, which we showed prevents an ?overshoot? of the homeostatic response. We further showed that the up-regulation of Kv4 channels is blocked by transcriptional inhibitors, and is dependent on D?7 nAChRs and Ca2+ influx. Drosophila continues to be an ideal model system for these studies because of its cholinergic CNS, the genetic tools it offers, its less redundant genome (eg. there is only a single Drosophila NFAT and Kv4 gene, each of which represents a multi-gene family in mammals), and the ability to go from mechanisms of gene regulation to physiological relevance in the intact brain, and whole animal behavior. The proposed studies will apply new optogenetic approaches to elicit cholinergic synaptic homeostasis in vivo (Aim-1) ?something that has not been explored in any system, and which would currently not be feasible in mammalian systems. We will examine underlying molecular mechanisms, including a novel relationship between ?7 nAChRs and Kv4 channels (Aim-2), and inactivity-induced transcription of Kv4 (Aim-3) that is mediated by NFAT (Aim-4). We will also test all molecular mechanisms for their physiological relevance in identified neurons in the intact brain and behaving animal (Aims 4-5). Our studies are likely to reveal novel insights into the underlying mechanisms of cholinergic synaptic homeostasis.