Frontotemporal dementia (FTD) is an age-related non-Alzheimer dementia characterized by progressive neuronal loss in the frontotemporal lobes. A subset of FTD is defined by the pathology of protein inclusions positive for Fused in Sarcoma (FUS), thus named FUS-FTD. FTD and amyotrophic lateral sclerosis (ALS) share a wide spectrum of clinical, pathological and genetic features. Pathogenic mutations of FUS cause both ALS and FTD, and FUS proteinopathy is also detected in sporadic diseases. Thus, it is crucial to examine relevant types of neurons in affected brain regions in different disorders. This project focuses on studying pathogenic FUS in forebrain cortical neurons in FTD. FUS is primarily in the nucleus, but the protein with a disease-causing mutation mis-localizes to the cytoplasm and form dynamic membraneless granules, which can compromise cellular functions and impair neurons. We recently showed that FTD/ALS mutations of FUS suppressed protein translation and hyper-activated the nonsense-mediated decay (NMD) of mRNAs. Thus, we hypothesize that the dysregulation of protein translation and mRNA surveillance contributes to cortical neuron loss in FUS-FTD.
Three specific aims are designed to test the hypothesis using in vitro and animal models as well as FTD patient tissues.
Aim 1 is to determine how mRNA NMD and protein translation are perturbed by pathogenic FUS in FTD mice and patient tissues. We will determine whether NMD factors and translation-related proteins are sequestered in FUS inclusions in forebrain neurons in R521G FUS transgenic mice at different ages. mRNA turnover rates and protein translation efficiency in mouse forebrains will be measured and correlated with neuronal dysfunction and FTD disease progression. Moreover, perturbations in NMD factors and protein translation will be examined in FTD patient tissues.
Aim 2 is to identify specific proteins and mRNAs affected by pathogenic FUS in FTD mice. We will apply proteomic approaches to identify changes in protein translation impacted by mutant FUS in forebrain neurons. Actively translated mRNAs will be identified and quantified in polysomes using RNA-Seq. The -omics data will be integrated for pathway analysis to reveal which specific pathways are impaired by mutant FUS. Functional studies of affected pathways will be carry out to determine their mechanistic relevance in the FTD etiology.
Aim 3 is to elucidate the significance of RNA binding and post-translational modifications in the dysfunction of pathogenic FUS. We will use a cohort of RNA binding-deficient mutations in an optogenetic Cry2olig-FUS-mCherry system to examine the significance of RNA binding in FUS inclusion formation and dysfunction in cortical neurons. We will evaluate the status of FUS acetylation in FTD mice and patient tissues using a new acetylation-specific antibody we generated. We will also test how acetylation-null and -mimicking mutations affect FUS inclusions, NMD, protein translation and neuronal toxicity. The proposed experiments will thoroughly examine a novel disease mechanism using innovative approaches. Completion of our proposed work will help elucidate molecular mechanisms underlying FUS FTD.
Frontotemporal dementia (FTD, also known as frontotemporal lobar dementia or FTLD) is an age-related non-Alzheimer dementia characterized by progressive degeneration of cortical neurons in the frontal and temporal lobes, which share a wide spectrum of clinical, pathological and genetic features with amyotrophic lateral sclerosis (ALS). A subset of FTD is defined by the pathology of protein inclusions containing Fused in Sarcoma (FUS) and is caused by FUS mutations (FUS-FTD). This project is to determine the mechanisms how pathogenic FUS interferes with RNA surveillance and turnover, suppresses protein translation, and disrupts cellular pathways in cortical neurons in FTD using cellular and animal models as well as FTD patient tissues.