Cell-type-specific and spatial proteogenomics analysis of aberrant RNA splicing and misfolded proteins in Alzheimer's related tauopathies - Project Summary / Abstract The molecular mechanisms of mis-folded protein accumulation in Alzheimer’s related tauopathies have been difficult to determine. In response to RFA-AG-25-017, we have assembled a strong inter-institutional multi- investigator team with complementary expertise in spatial transcriptomics and proteogenomics platforms for the proposed project. We hypothesize that vulnerable cell types in AD-impacted brain regions have detectable maladaptive gene expression profiles and epigenetic states that cause aberrant splicing and subsequent aberrant protein accumulation during AD/ADRD pathogenesis. To test this hypothesis, we will comparatively study new tau-directed AD/ADRD mouse models and human AD/ADRD brain tissues. We will use the “humanized” Tau mouse models that replace the endogenous mouse Mapt gene with either a normal or pathogenic variant of the entire human MAPT gene (MAPT gene replacement, MAPT-GR), which express isoforms of human tau at physiologic levels and ratios. In Specific Aim 1, we will generate single-cell spatial proteo-transcriptomics maps for molecular characterization of aberrant RNA splicing and abnormal protein products in specific cell types in the brain regions developing Alzheimer’s related tauopathy in mouse and human brains. Specifically, we will combine multiple protein immunofluorescent staining with RNA MERFISH (multiplexed error-robust fluorescence in situ hybridization) on the same mouse brain sections to map aberrant mRNA splicing, spliceosome factors and Alzheimer’s hallmark proteins at different ages (3 months, 12 months; 18 months). The mouse model studies will be complemented by the studies of misprocessed and aberrant RNAs and proteins in human postmortem brain tissues from early- and late-stage AD. In Specific Aim 2, we will determine the impact of aberrant splicing on proteome dynamics and the development of Alzheimer’s related proteinopathies in mouse and human brains. Considering that dysregulated RNA splicing disrupts proteome homeostasis in specific cell types and may cause the development of proteinopathies in AD/ADRD brains, we will generate a new MetRS*; MAPT-GR mouse line by crossing the MetRS* mouse with MAPT-GR mice to perform bio-orthogonal, non-cannonical amino acid tagging (BONCAT) to incorporate the non-canonical amino acid azidonorleucine into newly synthesized proteins in four specific brain cell types: excitatory neurons, inhibitory neurons, microglia and astrocytes. We will use a mass spectrometry-based proteomic Hyperion system to examine aberrantly spliced protein products and will use BONCAT to examine pulse labeled steady- state proteomics to demonstrate the effects of alternative/aberrant splicing on proteomics of specific cell types. We will further test and validate the effects of alternative/aberrant splicing in human postmortem brain tissues from early- and late-stage AD subjects. Together our proposed research will create cell-type-specific atlases of misprocessed and aberrant RNAs and proteins in mouse model and human brains, and will identify new molecular pathways and novel misprocessed protein interaction networks in Alzheimer’s related tauopathies.
