Heather T. Broihier - US grants

The goal of this proposal is to study the genetic and molecular mechanisms behind the differentiation and proliferation of neural precursors. Since the cells in the developing CNS are characterized by very different mitotic potentials (i.e. NBs are stem cells and GMCs each divide once), a study of genes involved in determining the unique fates of neural precursors may lead to insights into how their cell divisions are controlled. In particular, extra-extra, a gene whose mutant phenotype is defined by the presence of ectopic neurons will be cloned and characterized. Additionally, genes required for sibling neuron fates will be identified by screening for deficiencies which dominantly modify the CNS phenotype produced by loss of zygotic numb. This work may clarify general principles of asymmetric cell division as well as determine the nature of the cell-intrinsic factors or signaling pathways required for the unique identity of individual neurons. With a general understanding of how normal cell proliferation and differentiation are controlled, we should be able to improve the rationale behind cancer treatments.

[unreadable] DESCRIPTION (provided by applicant): Our long term aim is to use a genetic approach in Drosophila to understand molecular mechanisms of motorneuron fate specification and differentiation. In this proposal, we focus on mechanisms linking motorneuron fate to axon guidance. In a large-scale screen for mutants affecting motorneuron development, we have identified foxO and Mmp2, both of which are expressed in motorneuron subsets and necessary for proper motor axon guidance. We focus on these two evolutionarily-conserved proteins as they provide us with novel entry points for elucidating how distinct behaviors of motorneurons are developmentally regulated. Our goals here are to complete molecular, genetic, and phenotypic analyses of these genes in order to understand the observed mutant phenotypes. FoxO is a transcription factor best known for its role in the insulin signaling pathway where it is regulated by extracellular signals. Our preliminary data suggest that FoxO is expressed specifically in clusters of motorneurons in response to a target-derived signal. Detailed expression and phenotypic analyses will elucidate the role of FoxO in motorneuron development. Furthermore, we will identify the signaling pathway(s) regulating FoxO expression in motorneurons through molecular and genetic epistasis experiments. Matrix metalloproteinases (Mmps) comprise a large family of. transmembrane and secreted proteases that together cleave nearly every component of the ECM. Mmp2 is expressed in stereotyped populations of motorneurons and is necessary for proper motor axon guidance. We will characterize the expression pattern of Mmp2 in post-mitotic neurons and elucidate its role in motor axon guidance by analyzing motor axon outgrowth in Mmp2 mutant embryos. Additionally, we will establish whether Mmp2 is necessary for motor axon defasciculation by analyzing genetic interactions between Mmp2 and guidance molecules known to regulate this key pathfinding step. These studies will advance our understanding of how molecules acting in distinct motorneuron populations coordinate to establish proper patterns of neuromuscular connectivity. Motorneuron differentiation is an essential event in neuronal development and can be disrupted in human development and disease. Furthermore, since both foxO and Mmp2 are evolutionarily-conserved proteins acting in pathways of intense clinical interest, these studies should have broad biological and medical significance. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We found that the transcription factor FoxO controls neuronal microtubule organization and neuronal morphogenesis in Drosophila. Arguing for evolutionary conservation, FoxO homologs have also recently been implicated in neuronal morphogenesis in worms and mice. In these systems, FoxOs regulate polarization, outgrowth, and morphology-all processes demanding appropriate microtubule organization. Our work demonstrated that Drosophila FoxO limits microtubule stability (or alternatively, promotes microtubule destabilization) at the NMJ. We also found that FoxO protein levels are strikingly reduced in several cytoskeletal stress paradigms, arguing that FoxO dynamically regulates the microtubule network. We propose that FoxO levels are reduced as part of a cellular strategy to counteract microtubule perturbation. This hypothesis is based on FoxO's microtubule- destabilizing function as revealed by phenotypic analysis of foxO mutants. Our identification of FoxO as both a regulator of the neuronal microtubule network, and a neuronal stress-regulated transcription factor, argues that FoxO contributes to the neuronal response to cytoskeletal disruption. In this proposal we will define FoxO signaling systems to gain insight into mechanisms that regulate stability and plasticity of neuronal morphology. Moreover, neuronal microtubule dysfunction is linked to debilitating neuronal pathologies, including motorneuron and neurodegenerative diseases, as well as age and disease-related neuropathies. Thus, analyzing the dynamic regulation of microtubule organization will shed light on mechanisms important to human health and aging. We present a systematic set of experiments to establish FoxO-dependent pathways. We will determine the role of the lipid phosphatase PTEN in promoting FoxO activity at the NMJ, and the role of the E3 ubiquitin ligase Nedd4 in FoxO degradation following cytoskeletal perturbation. Furthermore, we present phenotypic analysis and expression data arguing that the microtubule - associated protein CRMP (Collapsin Response Mediator Protein) is repressed by FoxO. Thus, we will establish the role of CRMP in regulating microtubule stability in motorneurons, and define its relationship with FoxO. Finally, we will establish the FoxO's function in dendritogenesis and in aging neurons, to define the scope of its neuronal phenotypes, and to illuminate FoxO function in these diverse neuronal contexts.

DESCRIPTION (provided by applicant): Mechanistic analysis of activity-dependent BMP/TGF¿ release at a model synapse How do cells diversify signaling outputs of widely used intercellular signaling pathways? A handful of conserved growth factor pathways mediate communication between cells throughout life. Yet remarkably, the signaling outcomes of these pathways are finely tuned to developmental and cellular context. We will investigate signaling specificity of the BMP/TGF¿ pathway. This pathway serves a number of independent functions at the Drosophila NMJ. In particular, it regulates both NMJ morphological growth and neurotransmitter release. We present evidence that the cellular source of the BMP ligand Gbb discriminates between these two pathways. We propose a model in which the single-pass transmembrane protein Crimpy is a sorting receptor for the BMP for dense core vesicles in the regulated secretory pathway. We argue that (1) activity-dependent re- lease of the BMP promotes neurotransmission and (2) Crimpy defines the neuron-derived ligand pool. BMP/TGF¿ family members are synaptically localized and subject to activity-dependent release in mammalian neurons. However, the cellular mechanisms responsible for their localization to dense core vesicles are opaque. Crimpy would represent the first BMP/TGF¿ dense core vesicle sorting receptor identified in any system. Given clinical interest in identifying tools to selectively target synaptic functions of growth factor signaling pathways, a mechanistic understanding of Crimpy and its mammalian homologs will be of significant interest.

? DESCRIPTION (provided by applicant): Establishing a transcriptional pathway for cell-fate and synaptic plasticity We hypothesize that the FOXO transcription factor controls motoneuron plasticity across lifespan in Drosophila. FOXOs are evolutionarily conserved proteins that coordinate cellular responses to developmental and environmental stimuli. Well known for their central position in molecular circuits regulating healthy aging and stress responses, their developmental functions have recently come into focus. In particular, FOXOs have emerged as important regulators of brain development. Neuronal functions of FOXOs have been investigated in mice, C. elegans, and Drosophila. To date, these functions include neuronal polarity, morphology, synaptic function, and memory consolidation. Though FOXO proteins are key regulators of multiple aspects of neuronal development and physiology, the neuronal-specific pathways in which they act are as yet undefined. Here we propose to analyze components of a novel neuronal FOXO pathway using combined molecular, genetic, and genome-wide approaches. We will test the hypothesis that FOXO activity is stimulated by Toll-6 signaling to inhibit apoptosis during embryogenesis and promote synaptic organization and plasticity during larval development. Mechanistic under- standing of FOXO's role in these processes requires the identification of its transcriptional targets. To this end, we propose an unbiased large-scale RNA-seq approach to identify the FOXO-dependent transcriptome. Thus, we propose an initial characterization of an entirely novel pathway, as well as a genome-wide screen for effector molecules. Together, these studies aim to define a novel neurotrophic pathway from cell surface to nuclear response in a powerful genetic model system. There is significant interest in modulating both the survival and synaptic functions of neurotrophic pathways in contexts as varied as neurodegenerative diseases, normal aging, and injury. The proposed genome- wide screens for effectors may suggest unexpected and novel players in these critical signaling pathways.

? DESCRIPTION (provided by applicant): This proposal examines how a growth factor signaling pathway is linked to synapse structure and function. Growth factors are potent neuromodulators and play diverse roles at synapses. They regulate fundamental features of synaptic biology including baseline transmitter release, synapse morphology, and plasticity. Given their importance directing synapse organization, it is important to define the molecular and cellular mechanisms coupling synaptic growth factor signaling to synapses and neuronal activity. BMP/TGF? family members are evolutionarily conserved regulators of synapse structure and function. In particular, a BMP/TGF? pathway has neurotrophic pathway activity at the Drosophila NMJ. BMP pathway mutants display striking defects in NMJ morphology and function. Remarkably, distinct ligand pools independently regulate these synaptic features. We demonstrate that the presynaptic pool regulates synaptic structure and function, while the postsynaptic pool directs overall growth of the NMJ terminal. Understanding how these information channels are separated at an endogenous synapse is essential to understand how distinct synaptic features are independently controlled. Our entry point to this work is the novel neuronal transmembrane protein Crimpy. We have demonstrated that Crimpy enables discrimination between pre- and postsynaptic BMP pools. Crimpy binds a BMP homolog called Gbb and traffics it to presynaptic dense core vesicles (DCVs). Without Crimpy, Gbb is no longer found in DCVs and is not released by presynaptic activity. In the absence of Crimpy, pre- and postsynaptic ligand pools cannot be distinguished, and the NMJs are characterized by aberrant trophic signaling at the expense of the presynaptic synapse-organizing cue. In this proposal, we build on our novel preliminary findings to define the role of activity-induced presynaptic BMP signaling in synapse structure and function. First, we define the molecular identity of the pre- and postsynaptic signals. Because Crimpy is key for marking the presynaptic pool, we will define biochemically the role of Crimpy in BMP signal transduction. Second, we will elucidate how activity-dependent BMP signals direct synapse organization. We test the hypothesis that the Crimpy-mediated presynaptic Gbb signal is a local and acute cue instructive cue driving synapse organization. And third, we will define the downstream signaling cascade. We have exciting preliminary evidence that a novel BMP receptor transduces the activity-dependent signal. We will establish the receptor's role in the pathway and characterize novel downstream components of a non-canonical activity-dependent BMP cascade.

Glia continuously survey neuronal health during development, providing trophic support to healthy neurons while rapidly engulfing dying ones. These diametrically-opposed functions necessitate a foolproof mechanism enabling glia to unambiguously identify those neurons to support versus those to engulf. To ensure specificity, glia are proposed to interact with dying neurons via a series of carefully choreographed steps. However, these crucial interactions are largely obscure. We present novel preliminary data that dying neurons and glia communicate via Toll receptor-regulated innate immune signaling. Neuronal apoptosis drives processing and activation of the Toll-6 ligand, Spätzle5 (Spz5). This cue activates a dSARM-mediated Toll-6 transcriptional pathway in glia, which controls expression of the Draper engulfment receptor. Our results identify an upstream priming signal that prepares glia for phagocytosis. Thus, a core innate immune pathway plays an unprecedented role setting the valence of neuron-glia interactions during development. The identification of this non-canonical and previously unexplored pathway raises fundamental questions about its signaling mechanism, which we address in this proposal. First, we will define the mechanism of action of dSARM by rigorously testing its biochemical requirements in the pathway. Second, we will establish the regulation and function of Spz5 in dying neurons. Together, these experiments will define an entirely new molecular mechanism regulating interactions between dying neurons and glial phagocytes. By defining this cascade, we open the door to a systematic understanding of this innate immune pathway in neuron-glia interactions in development.

This proposal examines how a novel TLR glial signaling pathway drives phagocytic competence in glia, and defines its function in pruning neuronal number and connectivity across lifespan. Glia provide an extensive support system for healthy neurons by promoting their survival, connectivity, and synaptic function. Remarkably, glia can rapidly switch roles to precisely eliminate dying neurons or unwanted neurites/synapses by phagocytosis. These diametrically opposed functions necessitate fail-safe signaling mechanisms between neurons and glia; yet hese crucial regulatory mechanisms have remained largely obscure. Toll-like receptor (TLR) pathways were first identified for their roles in embryonic patterning and have since been defined as a conserved centerpiece of innate immunity. Our lab made the unexpected discovery that one of the most pronounced phenotypes associated with loss of a Drosophila TLR, a dramatic increase in the number of apoptotic neurons during development, is caused by selective loss of the TLR in glia. We demonstrated that release of the TLR ligand from dying neurons activates a novel TLR pathway in glia to drive phagocytic competence. In this proposal we build on our novel preliminary findings to establish how this pathway regulates the speed and specificity of debris clearance, and define its roles in neuron-glia interactions in synapse, neurite, and neuron removal across lifespan. Our unifying hypothesis is that non-canonical TLR signaling underlies the speed and specificity of debris clearance critical for proper CNS development and function. In the first aim, we focus on elucidating how glia are transformed into phagocytes during development by defining how information is relayed through the TLR pathway to elucidate how glia are primed to become phagocytic. In the second aim, we seek to extend our published work to investigate whether TLR signaling is a widespread early detection system to alert glia to the presence of neuronal debris. And in the third aim, we examine the function of TLR signaling in sculpting circuits in the olfactory system based on our preliminary findings that glial TLR signaling constrains synapse number in this well defined circuit. Here we propose to leverage the fly olfactory circuit as a model for defining glial phagocytic function in synapse maintenance. Together, these studies will shed critical light on the early signaling interactions between glia and their phagocytic substrates essential for brain health across lifespan.