DNA-damage-induced transcription errors provide a constant stream of amyloid and prion-like proteins in human cells

University Of Southern California, Los Angeles CA

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Abstract

ABSTRACT Aging is characterized by the accumulation of amyloid and prion-like proteins. However, the molecular mechanisms by which these proteins arise remain unclear. The overarching hypothesis of this proposal is that the amyloid and prion-like proteins that accumulate in aging cells are generated by mistakes that occur during transcription. To test this hypothesis, we will screen a database of 260,000 transcription errors identified in human stem cells, brain organoids and fully differentiated neurons for candidates that are likely to create amyloid peptides. We will then use a variety of cellular, biochemical and biophysical tools, including cryo-EM, TEM and fluorescent imaging to test the amyloid behavior of these proteins. Our preliminary data indicates that these experiments will create a powerful proof of principle of our hypothesis. Next, we will test how these transcription errors are generated. Several studies suggest that DNA damage plays an important role in this process, indicating that age-related DNA damage could drive the accumulation of amyloid and prion-like proteins. To test this hypothesis, we will use single-cell-sequencing coupled to a new, custom-made bio-informatic pipeline to determine if human neurons exposed to DNA damage display error prone transcription, and if so, how many mutant proteins are generated as a result. Our preliminary data indicates that O6-methyl guanine lesions, a common form of DNA damage in the brain, creates vast amounts of mutant proteins in single cells, strongly supporting our hypothesis. Next, we will determine whether these errors are sufficient to induce protein aggregation in human neurons using a plasmid-based system that contains a carefully placed O6-methyl guanine lesion. Finally, we will test if O6-methyl guanine -induced errors can affect the pathology of Alzheimer’s disease by removing the DNA repair gene MGMT in the neurons of a mouse model that carries a humanized version of the APP protein. MGMT repairs O6-methyl guanine lesions, which will allow these lesions to accumulate over time, increasing transcriptional mutagenesis. Moreover, it was recently found that MGMT is down-regulated in female patients with non-familial cases of AD, so that this experiment closely mimics the conditions of human patients. Accordingly, we think that in addition to testing our hypothesis, these experiments could also lead to the first mouse model that fully mimics late onset, non-familial cases of AD. The most exciting aspect of our work though, is the challenge we pose to the DNA-centric way of thinking in modern medicine. Our work shows that in some cases, the mutations that give rise to disease do not have to occur in the genome, they can also occur in the transcriptome. In doing so, our research could establish a new paradigm in modern medicine and open up a new field of aging research to widespread experimentation.