PROJECT SUMMARY/ABSTRACT Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are now thought to represent a spectrum of rapidly progressing neurodegenerative diseases. There are currently no cures or effective treatments for them. Though separate entities, these diseases share common genetic and pathological hallmarks and can have overlapping clinical presentations resulting in FTD-ALS. The underlying genetics linked to FTD, ALS, and FTD-ALS span a broad range of functions, making it difficult to interpret their contribution to disease pathophysiology. Moreover, most cases (70% FTD, 95% ALS) are sporadic with unknown etiology. Therefore, new techniques are needed to evaluate cellular pathomechanisms in the FTD-ALS spectrum of diseases. Because many causative genes are integral to organelle functions or contacts, we propose to use Multispectral Imaging (MSI) as a novel approach to accomplish this goal. MSI is a cutting-edge confocal microscopy technique used to simultaneously visualize up to seven fluorescent labels in living cells. In the proposed work, we will label major membrane-bound organelles (endoplasmic reticulum, peroxisomes, Golgi apparatus, mitochondria, lysosomes, lipid droplets) to quantify organelle morphology, interactions, and dynamics that are integral to cell function and/or perturbed in disease. The resulting organelle metrics will be combined to produce an organelle signature for neurons and astrocytes, cell types relevant to FTD-ALS. To our knowledge, this is the first study to utilize MSI to assess cellular mechanisms of the brain. Therefore, Aim 1 will establish organelle signatures for healthy neurons and astrocytes, examine how the signatures change upon pharmacological perturbations, and create a platform to automate the MSI analysis process. We hypothesize that neurons and astrocytes will have disparate organelle signatures related to their differing physiology. Aim 2 will evaluate FTD-ALS phenotypes induced by a familial mutation in the TARDBP gene. TARDBP encodes the TDP-43 protein, which has been identified to aggregate in the majority of FTD and ALS cases (45% and 97%, respectively). We hypothesize that this neurodegenerative perturbation will result in aberrant organelle morphology and dynamics in both neurons and astrocytes. Our preliminary findings demonstrate the feasibility of our approach and a likelihood of success, as we have optimized MSI labels in both cell types and began to examine pharmacological and genetic perturbations that result in altered organelle morphology and interactions. To substantiate our findings, we will confirm and expand on the MSI data in human induce pluripotent stem cell-derived neurons and astrocytes and validate significant phenotypes in FTD-ALS brain tissue. Our study will establish MSI as an innovative approach to evaluate neurodegeneration and could reveal cellular phenotypes integral to FTD-ALS onset or progression that traditional techniques have thus far m...