Bruno Marie - US grants

DESCRIPTION (provided by applicant): During embryonic development, transcription factors are essential for establishing a code that will determine the fate of neuronal precursors and neurons. However, most proteins that are responsible for a neuron's functional properties have a half-life ranging from minutes to several hours;they therefore must be tightly regulated long after neural development is over in order to maintain neuronal function. Surprisingly, very little is known about the role that transcription factors play in this process. We hypothesize that transcription factors that were initially characterized as early neuro-developmental genes, are required in fully developed motorneurons (MNs) to maintain neuronal function. We propose to drastically affect the levels of three transcription factors gsb, isl and eve within fully developed MNs and determine the neuronal functions they control. The long term goal of this research is to establish the molecular targets of these transcription factors. The Drosophila neuromuscular junction (NMJ) provides a model that is well-suited to the study of fully developed MNs. MNs have reached their muscle targets and are releasing neurotransmitter before the end of embryogenesis. We can test MN viability, synaptic structure and physiology 4 to 5 days after they are fully developed, near the end of the larval life, many times longer than the lifetime of the average protein. Moreover, the use of transgenic RNAi and conditional expression allows for knockout of gene expression in fully developed MNs, therefore bypassing the embryonic requirement for transcription factors. We have recently shown that gsb is ubiquitously expressed in fully developed MNs of the late larval CNS. Using immunohistochemistry, we will first show that eve and isl are expressed within a subset of fully developed MNs. In a second aim we will combine classical genetics and the use of the Gal4/UAS system in conjunction with Gal80TS to knock down or over-express transcription factors late, after development. We will then use immunohistochemistry and electron microscopy to determine whether the viability of the MN or the structure of the NMJ is affected by knocking down or over-expressing eve, isl or gsb within fully developed MNs. We will then perform intracellular electrophysiological recordings at the NMJ to determine whether eve, isl or gsb control synaptic release and homeostatic plasticity within fully developed MNs. This study will provide a conceptual template attributing a function to transcription factors within fully developed neurons;this in turn could lead to great insights into the molecular processes of neuronal aging and neuronal degeneration. PUBLIC HEALTH RELEVANCE: Very little is known about the biological mechanisms required to maintain neuronal function during life. This study proposes to show that transcription factors known to be embryonic key regulators are also required to maintain neuronal integrity and electrical properties. This will prove that transcriptional regulators have a broader role outside development and might be important in processes like aging and neurodegenerative diseases.

. PROJECT SUMMARY (See instructions): During our life time, homeostatic signaling systems secure the integrity of neuronal circuits while allowing some flexibility that will be the basis for processes of cognition, learning and memory. It is therefore not surprising that a series of neurobiological diseases (autism spectrum disorder, Alzheimer's disease and schizophrenia, among them) have been linked to alterations in homeostatic mechanisms. Despite the tremendous importance of understanding neuronal homeostasis and how homeostatic signaling systems interact with neuronal plasticity, we know close to nothing about the molecular mechanisms underlying synaptic homeostasis. We have recently discovered that the highly conserved BK-type K+ channel, slowpoke (slo) is essential to the homeostatic control of synaptic function. This study will define a new function for slo while characterizing a new molecular mechanism for the homeostatic stabilization of neuronal function. In human, mutation in slo has been implicated in generalized epilepsy and paroxysmal dyskinesia, slo is also known to control the active properties of neurons and skeletal muscles, to be essential to aspects of synaptic plasticity and alcohol addiction, and to bind to really important signaling molecules, including Ca2+ channels. This study will combine quintal analysis of different mutant backgrounds, pharmacology, molecular genetics, biochemistry and activity dependent Ca2+ imaging using two-photon microscopy to show that (1)- the modification of Ca2+ influx is the mechanism at the heart of homeostatic plasticity and that this mechanism depends on presynaptic slo. (2)- Slo conductance is not required to perform this function; the Slo C-terminus tail is sufficient to restore homeostasis, illustrating a new essential function for the regulatory Slo sequences. (3)- the Slo C-terminus tail interacts with the Ca2+ channel responsible for neurotransmitter release, CaV2.1 (Cae). This interaction is required for the upregulation of Cae during synaptic homeostasis.

From flies to humans, synapses are shaped by plastic events that promote or limit changes in synaptic strength. Indeed, changes in neuronal activity can lead to modifications in the property/strength of the synapse. This ability is called activity-dependent synaptic plasticity (ADSP). Another form of plasticity, referred to as synaptic homeostasis (SH), aims at maintaining synaptic output to ensure stable activity. These two distinct forms of synaptic plasticity are at the center of processes of cognition. Indeed, ADSP is regarded as the cellular correlate of learning and memory while perturbations of SH are linked to an array of neurological diseases. To date, most studies have considered these two forms of plasticity separately. The question remains: how does a synapse integrate the two to ensure the stability of its output while still allowing for discrete changes in synaptic strength when required? Here we propose to study, at a single synapse level, the apparent antagonism between ADSP and SH. In addition, we will highlight two opposing molecular controllers underlying the mutually exclusive choice between these two modes of plasticity. Using the Drosophila Neuromuscular junction (NMJ), we propose to show that the transcription factor gooseberry (gsb, the pax3/7 homolog) and the signaling molecule wingless (wg, the wnt homolog) have antagonistic functions which determine synaptic plasticity. Using genetics, immunohistochemistry and electrophysiology, we will first ask whether eliciting SH perturbs subsequent ADSP and vice versa. This will allow us to characterize the mutual exclusivity of the two forms of plasticity and know whether one form supersedes the other or whether the order in which they are engaged is the determining factor. We will then show that a gene characterized as essential to SH, gsb, inhibits ADSP. Similarly, we will ask whether the pro-ADSP signal Wg antagonizes SH. Finally, we will characterize genetic interactions between gsb and wg supporting the idea of antagonism between the two molecules. This work will determine whether there is a hierarchical or temporal organization that determines the predominant plasticity. It will also contribute to understanding one of the molecular systems underlying this organization. It will be a major contribution to our understanding of the integration of ADSP and SH. Furthermore, it will place us in an ideal position to dissect the function and regulation of the synaptic targets directed by Wg and Gsb during these processes of plasticity.