Targeted DNA methylation of neurodegenerative disease genes via homology directed repair.

DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5' regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.

C9orf72 intermediate repeats are associated with corticobasal degeneration, increased C9orf72 expression and disruption of autophagy.

Microsatellite repeat expansion disease loci can exhibit pleiotropic clinical and biological effects depending on repeat length. Large expansions in C9orf72 (100s-1000s of units) are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). However, whether intermediate expansions also contribute to neurodegenerative disease is not well understood. Several studies have identified intermediate repeats in Parkinson's disease patients, but the association was not found in autopsy-confirmed cases. We hypothesized that intermediate C9orf72 repeats are a genetic risk factor for corticobasal degeneration (CBD), a neurodegenerative disease that can be clinically similar to Parkinson's but has distinct tau protein pathology. Indeed, intermediate C9orf72 repeats were significantly enriched in autopsy-proven CBD (n = 354 cases, odds ratio = 3.59, p = 0.00024). While large C9orf72 repeat expansions are known to decrease C9orf72 expression, intermediate C9orf72 repeats result in increased C9orf72 expression in human brain tissue and CRISPR/cas9 knockin iPSC-derived neural progenitor cells. In contrast to cases of FTD/ALS with large C9orf72 expansions, CBD with intermediate C9orf72 repeats was not associated with pathologic RNA foci or dipeptide repeat protein aggregates. Knock-in cells with intermediate repeats exhibit numerous changes in gene expression pathways relating to vesicle trafficking and autophagy. Additionally, overexpression of C9orf72 without the repeat expansion leads to defects in autophagy under nutrient starvation conditions. These results raise the possibility that therapeutic strategies to reduce C9orf72 expression may be beneficial for the treatment of CBD.

Loss of Nuclear TDP-43 Is Associated with Decondensation of LINE Retrotransposons.

Loss of the nuclear RNA binding protein TAR DNA binding protein-43 (TDP-43) into cytoplasmic aggregates is the strongest correlate to neurodegeneration in amyotrophic lateral sclerosis and frontotemporal degeneration. The molecular changes associated with the loss of nuclear TDP-43 in human tissues are not entirely known. Using subcellular fractionation and fluorescent-activated cell sorting to enrich for diseased neuronal nuclei without TDP-43 from post-mortem frontotemporal degeneration-amyotrophic lateral sclerosis (FTD-ALS) human brain, we characterized the effects of TDP-43 loss on the transcriptome and chromatin accessibility. Nuclear TDP-43 loss is associated with gene expression changes that affect RNA processing, nucleocytoplasmic transport, histone processing, and DNA damage. Loss of nuclear TDP-43 is also associated with chromatin decondensation around long interspersed nuclear elements (LINEs) and increased LINE1 DNA content. Moreover, loss of TDP-43 leads to increased retrotransposition that can be inhibited with antiretroviral drugs, suggesting that TDP-43 neuropathology is associated with altered chromatin structure including decondensation of LINEs.

RNA metabolism in neurodegenerative disease.

Aging-related neurodegenerative diseases are progressive and fatal neurological diseases that are characterized by irreversible neuron loss and gliosis. With a growing population of aging individuals, there is a pressing need to better understand the basic biology underlying these diseases. Although diverse disease mechanisms have been implicated in neurodegeneration, a common theme of altered RNA processing has emerged as a unifying contributing factor to neurodegenerative disease. RNA processing includes a series of distinct processes, including RNA splicing, transport and stability, as well as the biogenesis of non-coding RNAs. Here, we highlight how some of these mechanisms are altered in neurodegenerative disease, including the mislocalization of RNA-binding proteins and their sequestration induced by microsatellite repeats, microRNA biogenesis alterations and defective tRNA biogenesis, as well as changes to long-intergenic non-coding RNAs. We also highlight potential therapeutic interventions for each of these mechanisms.