Glial cell phagocytosis of pH-dependent amyloid-β, AβpH, in live and fixed cultures, brain tissue sections, retina, cortex and in live animals useful for studying function in health and disease.

Several microglia-expressed genes have emerged as top risk variants for Alzheimer′s disease (AD). Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD-risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. Here we show that microglia form lipid droplets (LDs) upon exposure to amyloid-beta (Aβ), and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. LD formation is dependent upon age and disease progression and is more prominent in the hippocampus in mice and humans. Despite variability in LD load between microglia from male versus female animals and between cells from different brain regions, LD-laden microglia exhibited a deficit in Aβ phagocytosis. Unbiased lipidomic analysis identified a substantial decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TAGs) as the key metabolic transition underlying LD formation. We demonstrate that DGAT2, a key enzyme for the conversion of FFAs to TAGs, promotes microglial LD formation, is increased in microglia from 5xFAD and human AD brains, and that inhibiting DGAT2 improved microglial uptake of Aβ. These findings identify a new lipid-mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.

Targeting live cell organelles is essential for imaging, understanding, and controlling specific biochemical processes. Typically, fluorescent probes with distinct structural scaffolds are used to target specific cell organelles. Here, we have designed a modular one‐step synthetic strategy using a common reaction intermediate to develop new lysosomal, mitochondrial, and nucleus‐targeting pH‐activable fluorescent probes that are all based on a single boron dipyrromethane scaffold. The divergent cell organelle targeting was achieved by synthesizing probes with specific functional group changes to the central scaffold resulting in differential fluorescence and pKa. Specifically, we show that the functional group transformation of the same scaffold influences cellular localization and specificity of pH‐activable fluorescent probes in live primary microglial cells with pKa values ranging from ∼3.2–6.0. We introduce a structure‐organelle‐relationship (SOR) framework to target nuclei (NucShine), lysosomes (LysoShine), and mitochondria (MitoShine) in live microglia. This work will result in future applications of SOR beyond imaging to target and control organelle‐specific biochemical processes in disease‐specific models.

One scaffold – different organelle sensors: The structure‐organelle relationship (SOR) of an important synthetic intermediate targeting the nucleus and its transformation into other pH‐activable fluorescent probes is demonstrated. The fluorescent probes were synthesized using a single synthetic route. As shown in the cover picture, the assembly line illustrates that a common synthetic intermediate (rectangle shape) was transformed into another molecule (triangle or hexagonal shape) in a one‐step reaction to target specific cell organelles in live microglial cells. The common intermediate targets the cell nucleus, the cationic probe targets mitochondria, whereas the amine‐containing probe with pH‐activable property targets acidic lysosomes. The graphic was created by Ryann Davis. More information can be found in the Communication by G. Chopra et al.

Targeting live cell organelles is important for imaging and to understand and control specific biochemical processes. Typically, fluorescent probes with distinct structural scaffolds have been used for targeting specific cell organelle. Herein, we aimed to design modular one-step synthetic strategies using a common reaction intermediate to develop new lysosomal, mitochondrial and nucleus targeting pH-activable fluorescent probes that are all based on a single boron dipyrromethane analogs. The divergent cell organelle targeting was achieved by synthesizing pH-activable fluorescent probes with specific functional groups changes to the main scaffold resulting in differential fluorescence and pKa. Specifically, we show that the functional group transformation of the same scaffold influences cellular localization and specificity of pH-activable fluorescent probes in live primary microglial cells with pKa’s ranging from ~4.5-6.0. We introduce a structure-organelle-relationship (SOR) framework targeting the nucleus (NucShine), lysosomes (LysoShine) and mitochondria (MitoShine) in primary mouse microglial cells. This work will result in future applications of SOR beyond imaging to target and control organelle-specific biochemical processes in disease-specific models.