Mary A. Logan, Ph.D. - US grants

DESCRIPTION (provided by applicant): Glial cells respond potently to neuronal damage, as well as neurodegenerative disease, by displaying overt changes in morphology, gene expression, migration, and phagocytic activity. Dysfunctional responses contribute to the progression of devastating neurological diseases, such as Alzheimer's disease and Parkinson's disease, and can also promote the onset of some autoimmune disorders. Despite the importance of glia in defending brain health, remarkably little is known about the molecular underpinnings of responses to neuronal damage. A vital long-term goal is to understand how basic glial immune reactions are triggered in the adult brain in response to damaged and dying neurons. The core cellular events (e.g. glial migration to injury sites and phagocytic clearance of neuronal debris) are highly conserved across species and recent work is revealing striking molecular conservation, as well. This proposal uses a well- established adult axotomy assay in Drosophila to investigate the molecular features of glial reactions; the fly offers a tractable genetic system to manipulate gene expression and function with exquisite cellular and temporal precision in vivo. Our preliminary work has identified a novel role for the evolutionarily conserved Insulin/insulin-like growth factor (IGF)-Like Signaling (ILS) pathway in orchestrating glial reactions to axon injury. Based on our findings, we hypothesize that ILS regulates glial immune responses in two fundamental ways: (1) Basal ILS activity in adult glia ensures that glia express key genes (i.e. the Draper receptor and adaptor Ced-6) required to detect and carry out responses to axon damage, and (2) Acute activation of the ILS pathway at injury sites triggers rapid responses in local glia to ensure that damaged neurons are cleared from the CNS. This proposal will employ powerful genetic-molecular tools to investigate how the ILS pathway contributes to axotomy-induced functions in glia. We will: (Aim 1) define the role of Insulin-like Receptor (InR) activity in each step of the glial response to axotomy, including altered gene expression, glial recruitment to injury sites, and glial phagocytic activity; (Aim 2) determine how insulin-like peptides (ilps), the InR ligands, influence basal expression of Draper and Ced-6, as well as each step of the injury response in local glia; and (Aim 3) define the molecular signaling cascades downstream of InR that are coupled to these important glial responses. This work will provide critical mechanistic insight into how damaged neurons communicate with glia to elicit responses and elucidate intrinsic molecular pathways that control these essential glial functions. Our findings will also offer a novel framework for exploring ILS components as therapeutic targets to treat CNS injury, as well as chronic neurodegenerative conditions. PUBLIC HEALTH RELEVANCE: Glial cells, the primary immune responders in the brain, respond swiftly and powerfully to traumatic injury and to chronic neurodegenerative disease, and abnormal glial responses contribute to the progression of devastating neurological conditions, including multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis. This proposal is relevant to public health because it will provide critically needed insight into te molecular and cellular signaling pathways by which damaged neurons elicit innate responses in glial cells. These discoveries will advance our understanding of neuron-glia communication in the brain, provide a springboard for the development of treatments to reduce the burden of neurological disorders and, thus, will be highly relevant to the NIH mission.

Alzheimer's disease (AD) and similar dementias present substantive challenges to patients and families, including medical, emotional, and fiscal hardships. Understanding the basic molecular underpinnings associated with AD progression is imperative to develop targeted strategies to intervene before notable cognitive decline occurs in patients. Glial cells, the first immune responders in the brain, are believed to influence AD pathology, although the molecular and cellular details are still unclear. Healthy glial cells can efficiently engulf amyloid-beta (A?), one of the major neurotoxic proteins that forms aggregates in the AD brain, and it has been proposed that defects in glial clearance of A? may contribute to the onset or advancement of AD. How do glia clear A? in the brain? What are the molecules and signaling pathways that govern glial recognition and engulfment of A?? Finally, what is the fate of A? once it has been internalized by glial cells? Using a well-established AD model in Drosophila melanogaster, we have identified the Draper receptor as a novel neuroprotective molecule against A?-induced toxicity. Draper is a highly conserved glial engulfment receptor required for glial phagocytic clearance of apoptotic neurons and degenerating axons. Notably, the role of Draper or the mammalian orthologs (MEGF10/Jedi) in glial clearance of A? function have never been explored in vivo. Here, we show that loss of glial Draper results in greater A? accumulation, exacerbates locomotor defects, and further reduces lifespan, while activation of glial Draper reverses these molecular and behavioral phenotypes. Our preliminary work also suggests that Draper-dependent activation of autophagy pathways may influence the progression of A?-induced CNS dysfunction. Thus, we hypothesize that glial cells utilize the Draper receptor to internalize and/or degrade neurotoxic A? peptides in the adult brain and that Draper activity attenuates A?-induced phenotypes. Draper activates several downstream signaling pathways, including altered cytoskeletal remodeling, autophagy, and transcription (specifically, STAT92E and AP-1). In Aim 1, we will use genetic and microscopy methods, as well as behavioral assays, to interrogate known downstream signaling effectors of Draper to determine which pathways protect against A? accumulation, motor defects, and reduced longevity. In Aim 2, we will investigate the possibility that Draper influences A? propagation throughout the CNS. More specifically, we propose that glial Draper/autophagy promotes A? destruction, thereby inhibiting A? peptide spreading. Using in vivo genetic manipulations and super resolution microscopy, we will inhibit glial Draper and autophagy pathways to determine if A? propagates more readily in the adult brain. This work will rapidly offer new molecular insight into how Draper/MEGF10 is coupled to AD progression and, more broadly, will provide a significant advancement in our understanding of how glial immunity is linked AD, as well as other proteinopathies.

SUMMARY Glial cells play an essential role in defending brain health and managing neuronal stress and damage. Neurodegeneration triggers robust glial immune responses, including changes in cytoskeletal dynamics, glial cell migration, and increased phagocytic activity. Timely removal and degradation of degenerating axons and neuronal debris by glia confers neuroprotection in the brain. Despite the importance of glial responses to axon injury, we still know surprisingly little about how damaged neurons invoke immune reactions in glial cells. What signals are released from degenerating neurons? What prompts the release of these injury cues? Finally, how are these signals translated by glia to carry out efficient immune responses to damage? We are using the fruit fly Drosophila melanogaster as a tractable model to investigate the immune communication relays that exist between neurons and glial cells in vivo. The fly nervous system contains distinct glial subtypes that are molecularly and functionally similar to vertebrate glia. Moreover, well-established axotomy assays in the adult olfactory system and the adult wing reveal that Drosophila axons undergo a classic Wallerian degeneration (WD) program, which includes increased intra-axonal calcium waves, axon fragmentation, and subsequent clearance by phagocytic glia. Notably, our lab has recently shown that axon degeneration triggers activation of the insulin-like signaling (ILS) pathway in reactive ensheathing glia, which, in turn, elicits essential glial immune responses, including transcriptional upregulation of immune genes (e.g. the engulfment receptor Draper) and phagocytic activity. We hypothesize that neuropeptide-containing dense core vesicles (DCVs) are broadly released from severed axons to trigger immune responses in local glial cells. Here, we propose to use static and live confocal imaging, transcriptional profiling, and newly developed in vivo reporters to investigate how neuropeptide signaling between neurons and glia informs glial immune responses to nerve injury. Specifically, we will 1) monitor DCV dynamics and release in adult severed nerves, 2) utilize novel single transcript labeling methods to visualize local translation of immune mRNA transcripts in glial extensions at sites of injury, and 3) determine how neuropeptide signaling between discrete glial subtypes ensures that glial responses to degenerating axons are properly carried out. Together, these findings will offer exciting molecular and cellular insight into how neuropeptide signaling between neurons and glia govern immune responses in both acute and chronic degenerative conditions.