Molecular Analysis Of Amyotrophic Lateral Sclerosis And Frontotemporal Degeneration Brain Tissue Identifies Disease Mechanisms Associated With Repetitive Dna Elements

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Doctor of Philosophy (PhD)
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RNA binding proteins
Molecular Biology
Neuroscience and Neurobiology
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Aging-related neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) are two fatal progressive neurodegenerative diseases that carry genetic and pathologic overlap: a hexanucleotide G4C2 repeat expansion in C9orf72 and loss of a nuclear RNA binding protein, TAR DNA binding protein-43 (TDP-43), into cytoplasmic aggregates. The C9orf72 expansion is the most common genetic cause of ALS/FTD and is associated with reduced C9orf72 expression and accumulation of toxic RNA and protein aggregates. In Chapter 2 of my thesis, using molecular analyses from human post-mortem ALS/FTD brain, I show that the C9orf72 promoter is hypermethylated within a subset of expansion carriers. C9orf72 promoter hypermethylation is associated with reduced C9orf72 pathology and may be protective in these patients. Another key feature in ALS/FTD is the characteristic pathology of nuclear TDP-43 loss in degenerating neurons. TDP-43 is a ubiquitous nuclear RNA binding protein and is heavily involved in RNA processing. TDP-43 has been shown to bind genic elements and repetitive transposable elements such as long interspersed nuclear elements (LINE). Considering that TDP-43 is a ubiquitous RNA binding protein, I hypothesize that nuclear TDP-43 loss can lead to large transcriptomic changes and may contribute to alterations in LINE elements. For Chapters 3 and 4 of my thesis, I use a novel method of subcellular fractionation and fluorescent activated cell sorting (FACS) from post-mortem ALS/FTD human brain to perform high-throughput sequencing analyses to study neuronal molecular changes. In Chapter 3 of my thesis, I use FACS coupled with RNA-seq on neuronal nuclei with and without TDP-43 to show that loss of nuclear TDP-43 is associated with large transcriptome changes and increased LINE accessibility. Furthermore, loss of nuclear TDP-43 leads to increased retrotransposition. I also extend this subcellular fractionation-FACS method to study the effects of the C9orf72 expansion in neuronal nuclei. In Chapter 4, I demonstrate that the C9orf72 expansion is linked to mild gene expression changes that reflect C9orf72 protein loss and not gain of toxic C9orf72 RNA. Through my work, I have shown disease mechanisms linked to repetitive DNA elements, in that I propose (1) the C9orf72 repeat expansion may contribute to disease primarily via a gain of toxic C9orf72 pathology (2) loss of neuronal nuclear TDP-43 may be associated with increase retrotransposon activity which may contribute to disease. Overall, my work has broadened the field of neurodegeneration in my implementation of cell-type specific molecular analyses on post-mortem brain of ALS/FTD patients to identify disease mechanisms with the intent of discovering new therapeutics and biomarkers that can be extended into the clinic.

Edward B. Lee
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