NPC1 regulates the distribution of phosphatidylinositol 4-kinases at Golgi and lysosomal membranes.

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann-Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4-phosphate (PtdIns4P) countertransport cycle between Golgi-endoplasmic reticulum (ER), as well as lysosome-ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4-kinases-PI4KIIα and PI4KIIIß-which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.

Methods for high throughput discovery of fluoroprobes that recognize tau fibril polymorphs.

Aggregation of microtubule-associated protein tau (MAPT/tau) into conformationally distinct fibrils underpins neurodegenerative tauopathies. Fluorescent probes (fluoroprobes), such as thioflavin T (ThT), have been essential tools for studying tau aggregation; however, most of them do not discriminate between amyloid fibril conformations (polymorphs). This gap is due, in part, to a lack of high-throughput methods for screening large, diverse chemical collections. Here, we leverage advances in protein adaptive differential scanning fluorimetry (paDSF) to screen the Aurora collection of 300+ fluorescent dyes against multiple synthetic tau fibril polymorphs. This screen, coupled with orthogonal secondary assays, revealed pan-fibril binding chemotypes, as well as fluoroprobes selective for subsets of fibrils. One fluoroprobe recognized tau pathology in ex vivo brain slices from Alzheimer's disease patients. We propose that these scaffolds represent entry points for development of selective fibril ligands and, more broadly, that high throughput, fluorescence-based dye screening is a platform for their discovery.