PROJECT SUMMARY Neurodegenerative diseases are characterized by the progressive appearance of insoluble inclusions that arise due to protein misfolding in the brain. Much research has focused on determining mechanisms by which these protein lesions cause dysfunction and loss of neurons and glia. Accumulating evidence supports the hypothesis that protein assemblies associated with most neurodegenerative diseases propagate in the brain by templating the aggregation of properly-folded versions of the same protein, similarly to infectious prions. However, it is still not well understood how “prion-like” aggregates spread between different cell types in the brain or the relevance of this to neuropathogenesis. The long-term goal of our research is to identify mechanisms underlying protein aggregate-induced toxicity and spreading between neurons and glia in the degenerating brain. The overall objective of this project is to determine normal and pathological functions of phagocytic glia in response to formation of mutant huntingtin (mHTT) aggregates in neurons. Prior research showing that glial responses to neural injury shift from beneficial to harmful over time, and emerging evidence that phagocytic glia participate in aggregate clearance and spreading provide rationale for the current project. The central hypothesis is that incomplete clearance of engulfed neuronal mHTT aggregates by phagocytic glia promotes formation and growth of a reservoir of seeding-competent, prion-like species in the brain. This hypothesis was formulated based on findings that glia mediate prion-like transfer of mHTT, tau, and a-synuclein aggregates, and that phagocytic clearance of engulfed debris becomes less effective with age and in the disease state. Our recent work indicates that prion-like transfer of mHTT aggregates between synaptically-connected neurons requires Draper-dependent phagocytosis, an evolutionarily-conserved glial pathway for clearing debris in the brain. The central hypothesis will be tested in three Specific Aims, carried out in complementary in vivo Drosophila and in vitro mammalian cell culture models. In Aim 1, effects of neuronal mHTT aggregates on glial responses to neural injury will be measured in adult fly brains to gain insight into whether mHTT aggregate engulfment leads to glial dysfunction. In Aim 2, RNAi-based forward genetic screens will identify modifiers of prion-like spreading of engulfed neuronal mHTT aggregates to the glial cytoplasm. In Aim 3, roles of the mammalian Draper homolog MEGF10 in mHTT aggregate transfer between co-cultured neuronal and astrocytic cells will be determined. These studies will identify novel mechanisms about the basic biology underlying phagocytosis, and disease-related mechanisms that drive protein aggregate-induced neurodegeneration in multiple cell types. The proposed research is significant because it addresses critical questions of how phagocytic glia interact with neurons to drive pathogenesis in potentia...