Defects in mitochondrial function and subcellular trafficking along neuronal subcompartments are hallmarks of genetic and idiopathic Alzheimer’s disease (AD). Neurons rely heavily on proper mitochondrial function and localization, as mitochondria play a dual role: buffering calcium and also providing the energy required for synaptic transmission and local translation. Mitochondria maintain their own extrachromosomal genome and must coordinate gene expression programs with the nuclear genome (mitonuclear coordination) to correctly assemble oxidative phosphorylation (OXPHOS) complexes that make up the electron transport chain. This coordination is vital for proper cellular function, as mitonuclear imbalance leads to orphan OXPHOS subunits that can overwhelm proteostasis pathways. How mitonuclear balance is affected in AD and the collective effects of AD pathology in subcellular transcriptome dynamics —from transcription to degradation and localization—, for both nuclear- and mitochondrial-encoded transcripts are unknown. We hypothesize that neuronal architecture predisposes mitochondria located distal to the soma to mitonuclear imbalance, making them prone to malfunction in neurodegenerative disease states. Recently, we developed a technique (subcellular TimeLapse-seq: STLseq) that simultaneously quantifies the kinetic rates of transcription, nuclear export, ribosome association, and degradation for each transcript, a process we refer to as RNA-flow. This method combines RNA metabolic labeling and subcellular fractionation with Bayesian modeling and has been adapted to capture mitochondrial-encoded genes, enabling quantitative analysis of mitochondrial transcriptome dynamics and of mitonuclear co-regulation. Here, we will analyze RNA-flow dynamics in AD by adapting STL-seq to neurons harboring AD-related mutations, combined with the isolation of soma and neuronal projections. The overall goal of this proposal is to determine the effects of AD in subcellular transcriptome dynamics and in mitonuclear coordination in the soma and neuronal projections. In Specific Aim 1, we will use STL-seq to analyze the subcellular transcriptome dynamics in AD and identify key regulatory factors. In Specific Aim 2, we will determine the soma and neurites translatome, the mitonuclear coordination differences in soma and neuronal projections, and how AD influences mitonuclear balance. The expected outcome from this grant will be an understanding of AD pathology at the transcriptome, the translatome, and the mitonuclear coordination levels in the soma and neuronal projections. These results will positively impact our understanding of mitochondrial gene regulation in healthy neurons and in AD, exposing putative new areas for therapeutic intervention.