Project Grant R01AG087374

LIPID IMAGING EXPANSION MICROSCOPY TO STUDY ALZHEIMER'S DISEASE - ALZHEIMER'S DISEASE (AD) IS A FATAL NEURODEGENERATIVE DISEASE CHARACTERIZED BY PROGRESSIVE COGNITIVE DECLINE AND BRAIN PATHOLOGIES INCLUDING AMYLOID-B (AB) PEPTIDE DEPOSITION, HYPERPHOSPHORYLATED TAU ACCUMULATION, INFLAMMATION, AND SYNAPTIC AND NEURONAL LOSS. IN ADDITION TO PROTEIN PATHOLOGY, THE ABNORMAL ACCUMULATION OF LIPIDS IN AD BRAINS - ORIGINALLY DESCRIBED BY ALZHEIMER HIMSELF IN HIS 1907 PUBLICATION - HAS RECENTLY BEEN REDISCOVERED AS AN IMPORTANT FACTOR IN AD. MANY RECENT DISCOVERIES IN ALZHEIMER'S DISEASE RAISE QUESTIONS OF HOW LIPID-RICH MICRODOMAINS WITHIN CELL MEMBRANES KNOWN AS LIPID RAFTS MIGHT PLAY ROLES IN ALZHEIMER'S PATHOLOGY; WHY CONTACT POINTS BETWEEN MITOCHONDRIA AND THE ENDOPLASMIC RETICULUM (ER), KNOWN AS MITOCHONDRIA-ASSOCIATED ER MEMBRANES OR MAMS, INCREASE IN AD AND ARE IMPACTED BY THE PRESENCE OF APOE4, THE STRONGEST GENETIC RISK FACTOR FOR LATE-ONSET AD; HOW INTRANEURONAL AGGREGATION OF TAU AND TOXIC AB TRIGGER ER STRESS IN EARLY STAGES OF AD; WHY CHANGES IN MITOCHONDRIAL MORPHOLOGY SUCH AS FRAGMENTATION AND ELONGATION OCCUR IN AD; AS WELL AS COUNTLESS OTHER RECENT DISCOVERIES. ONE RECENT EXAMPLE FROM OUR TEAMS WAS A DEMONSTRATION OF ACCUMULATION OF NEUTRAL LIPID DROPLETS AND CHOLESTEROL IN GLIAL CELLS HARBORING THE APOE4 ALLELE IN THE GENE CODING FOR APOLIPOPROTEIN E (APOE), ONE OF THE MOST SIGNIFICANT GENETIC RISK FACTORS FOR DEVELOPING AD. THE PRESENCE OF APOE4 LED TO LD ACCUMULATION IN ASTROCYTES, MICROGLIA, AND OLIGODENDROCYTES. WHILE THESE FINDINGS CLEARLY SUGGEST THAT SUBCELLULAR ORGANELLE ORGANIZATION AND MORPHOLOGIES PLAY IMPORTANT ROLES IN THE DEVELOPMENT OF AD, THE MECHANISMS MEDIATING THESE ROLES REMAIN UNCLEAR, LARGELY DUE TO THE LACK OF ANALYTICAL TOOLS THAT ALLOW THE UNAMBIGUOUS CHARACTERIZATION OF INTRACELLULAR STRUCTURES IN BRAIN TISSUE FROM ANIMAL MODELS OF AD AND FROM HUMAN AD PATIENTS. THE STANDARD METHOD FOR INVESTIGATING MORPHOLOGICAL CHARACTERISTICS OF ORGANELLES IS ELECTRON MICROSCOPY (EM) WHICH STRUGGLES TO IDENTIFY SPECIFIC BIOMOLECULES AMIDST ORGANELLE ARCHITECTURE. OUR RECENT INVENTION OF EXPANSION MICROSCOPY (EXM) ALLOWS NANOSCALE IMAGING OF BIOLOGICAL SPECIMENS, INCLUDING MOLECULAR CONTRAST, WITH CONVENTIONAL MICROSCOPES, AND HAS BEEN EMPLOYED IN STUDIES ON THE GOLGI APPARATUS, THE ER, MITOCHONDRIA, AND MYELIN. HERE WE PROPOSE TO EXTEND THE EXM TOOLBOX TO CONFRONT, HEAD-ON, THE KEY NEEDS OF ALZHEIMER'S LIPID RESEARCH. SPECIFICALLY, WE WILL (AIM 1) OPTIMIZE, AND VALIDATE, A FORM OF EXM THAT COMBINES LIPID PRESERVATION AND STAINING, MULTIPLEXED ANTIBODY STAINING, AND EXPANSION MICROSCOPY - WHICH WE CALL MULTIPLEXED ULTRASTRUCTURE EXPANSION MICROSCOPY (MULTIPLEX-UMEXM); (AIM 2) OPTIMIZE, AND VALIDATE, MULTIPLEX-UMEXM FOR HUMAN BRAIN TISSUE; (AIM 3) PERFORM A COMPREHENSIVE CHARACTERIZATION OF LIPID ACCUMULATION AND ORGANELLES BY BRAIN CELL TYPE AND AD RISK GENOTYPE IN MOUSE AND HUMAN TISSUE. THE NET RESULT OF THIS GRANT WILL BE A TOOLBOX THAT ANYONE IN BIOLOGY CAN USE TO CHARACTERIZE LIPID AND ORGANELLE PROPERTIES, WITH NANOSCALE PRECISION AND MOLECULAR CONTRAST, IN DISEASES SUCH AS ALZHEIMER'S DISEASE, AS WELL AS THE VALIDATION DATA TO SHOW ITS UTILITY.