Critical tools enabling analysis of biomolecular condensates in microglial signaling and function in aging and Alzheimer Disease

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Project Summary: Genetic studies have identified sequence variants in several genes that are predominantly expressed in microglia and are associated with either enhanced risk or resilience to Alzheimer Disease (AD). Our preliminary experiments reveal that the proteins encoded by some of these genes (e.g. TREM2, PLCG2, ABI3) likely function as components of an intracellular signaling pathway downstream of the TREM2 receptor. This cascade regulates microglial function in response to TREM2 activation. Crucially, several of these proteins (e.g. ABI3) or their interacting proteins (e.g. BLNK, an interactor with PLCG2) contain low complexity and intrinsically disordered motifs. These motifs are characteristic of proteins that phase separate to form biomolecular condensates. Our preliminary experiments confirm that these proteins do indeed phase separate to form two biomolecular condensates. One contains PLCG2. The other contains ABI3. Both are regulated by posttranslational modifications (PTMs). Crucially, AD-associated mutations alter these condensate-regulating PTMs and alter microglial migration and phagocytosis. Our observations suggest that both condensates play a central role in regulating microglial functions relevant to AD and aging. Consequently, this signaling pathway, and the biomolecular condensates within it, are likely to contain unrecognised molecular targets for biomarkers and therapeutics to manage microglial dysfunction in aging and AD. This proposal will develop two critical enabling resources that will underpin future work. First, we will create human iPSC-derived microglial expressing mEmerald + SPOT and mScarlet + HA tags CRISPR engineered into the endogenous ABI3 and PLCG2 genes. This tool will allow simultaneous investigation of the biophysics and cell biology of both condensates in living cells. The pluripotent nature of iPSCs will allow future analysis of these condensates in other cell types without additional resources. Second, we will develop novel protein purification and mass spectrometry workflows to obtain a more complete inventory of the proteins within these condensates. We will initially focus on simple immunopurification protocols to identify stable interacting proteins. We will exploit the HA/SPOT tags and robust, well- characterized antibodies to these tags to coIP PLCG2 and ABI3 with their interacting partners, which will be identified by LC-MS/MS. As proof of principle, ~10 binders will be authenticated as real condensate components using reciprocal coIP and colocalisation studies in human cultured microglia and brain sections. The authenticated condensate partners will be intrinsically useful. However, the validated workflows developed here will support future, large-scale studies in human iPSC derived microglia under various conditions (TREM2 activation, aging, AD-associated sequence variants). These resources will underpin future work by the field to understand how the condensates regulate microglial function, and uncover molecular targets for precision biomarkers and therapies to manage microglial dysfunction in aging and in AD.