One Scaffold, Different Organelle Sensors: pH-Activable Fluorescent Probes for Targeting Live Microglial Cell Organelles.

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.

Comprehensive Lipidomic Automation Workflow using Large Language Models.

Lipidomics generates large data that makes manual annotation and interpretation challenging. Lipid chemical and structural diversity with structural isomers further complicates annotation. Although, several commercial and open-source software for targeted lipid identification exists, it lacks automated method generation workflows and integration with statistical and bioinformatics tools. We have developed the Comprehensive Lipidomic Automated Workflow (CLAW) platform with integrated workflow for parsing, detailed statistical analysis and lipid annotations based on custom multiple reaction monitoring (MRM) precursor and product ion pair transitions. CLAW contains several modules including identification of carbon-carbon double bond position(s) in unsaturated lipids when combined with ozone electrospray ionization (OzESI)-MRM methodology. To demonstrate the utility of the automated workflow in CLAW, large-scale lipidomics data was collected with traditional and OzESI-MRM profiling on biological and non-biological samples. Specifically, a total of 1497 transitions organized into 10 MRM-based mass spectrometry methods were used to profile lipid droplets isolated from different brain regions of 18-24 month-old Alzheimer's disease mice and age-matched wild-type controls. Additionally, triacyclglycerols (TGs) profiles with carbon-carbon double bond specificity were generated from canola oil samples using OzESI-MRM profiling. We also developed an integrated language user interface with large language models using artificially intelligent (AI) agents that permits users to interact with the CLAW platform using a chatbot terminal to perform statistical and bioinformatic analyses. We envision CLAW pipeline to be used in high-throughput lipid structural identification tasks aiding users to generate automated lipidomics workflows ranging from data acquisition to AI agent-based bioinformatic analysis.

Amyloid ß Induces Lipid Droplet-Mediated Microglial Dysfunction in Alzheimer's 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 (Aß), 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 Aß 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 Aß. These findings identify a new lipid-mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.

Monitoring phagocytic uptake of amyloid ß into glial cell lysosomes in real time.

Phagocytosis by glial cells is essential to regulate brain function during health and disease. Therapies for Alzheimer's disease (AD) have primarily focused on targeting antibodies to amyloid ß (Aß) or inhibitng enzymes that make it, and while removal of Aß by phagocytosis is protective early in AD it remains poorly understood. Impaired phagocytic function of glial cells during later stages of AD likely contributes to worsened disease outcome, but the underlying mechanisms of how this occurs remain unknown. We have developed a human Aß1-42 analogue (AßpH) that exhibits green fluorescence upon internalization into the acidic organelles of cells but is non-fluorescent at physiological pH. This allowed us to image, for the first time, glial uptake of AßpH in real time in live animals. We find that microglia phagocytose more AßpH than astrocytes in culture, in brain slices and in vivo. AßpH can be used to investigate the phagocytic mechanisms responsible for removing Aß from the extracellular space, and thus could become a useful tool to study Aß clearance at different stages of AD.