C9ORF72 hexanucleotide repeat expansions are a major cause of Frontotemporal Dementia and underlie 10% of all Motor Neuron Disease cases. Important pathophysiology delineating the toxic effect of the repeat expansion was published in Nature Neuroscience this week. This paper is the result of a cross-faculty effort between SITraN and the Department of Molecular Biology and Biotechnology, jointly supervised by Professors Mimoun Azzouz and Sherif-El-Khamisy.
Video animation credit: Nora El-Khamisy
The C9orf72 gene contains an intronic repeating region of guanine and cytosine nucleotides that is expanded in C9orf72 linked FTD and MND. GC-RNA repeats are prone to hybridize with the DNA template strand during transcription producing a stable 3-stranded nucleic acid structure called an R-loop that predisposes DNA breakage.
Enzymatic DNA repair is part of routine maintenance for cells when structural damage like single or double stranded breaks occur. Double stranded breaks (DSBs) can lead to dangerous genome rearrangements and trigger apoptosis at high levels. In post-mitotic cells like neurons, low levels of unrepaired DNA damage can accumulate over-time aging the cell. A master kinase called ATM responds to DSBs as part of the DNA repair response.
Immunohistochemistry on post-mortem tissues from patients with C9ORF72-MND confirmed an increased presence of R-loops and possible marks of an impaired ATM repair system. To model these findings in vitro, the authors used AAV viral vectors to deliver the toxic products of the C9ORF72 gene to cells. Increased R-loops and DSBs were subsequently observed. The master kinase ATM was not activated in response to the DSBs and was below activation levels found in control cells. This led to the hypothesis that the C9ORF72 repeat expansion impaired ATM-mediated DNA repair.
To test the hypothesis in vivo, mice were transfected with normal and repeat expansion versions of C9ORF72 products. Mice with the repeat expansions developed an MND phenotype and the hallmarks of R-loops, DSBs and an impaired ATM response. The next question was, what is it about the repeat expansion that causes the ATM response to be impaired?
Autophagy; the garbage disposal system of the cell, whereby defective or otherwise unwanted proteins and other constituents are recycled into their reusable parts, was found to be initiated by the normal function of the C9ORF72 gene by Kurt de Vos’ team published in EMBOJ last year. A protein called p62 accumulates in cells with a defective autophagy system and p62 aggregates are a characteristic feature of C9ORF72 pathology. Furthermore, deficient autophagy was reported to impair DNA repair leading to genome instability elsewhere in the literature. A p62 complex directly binds to components of the DNA repair response preventing DNA repair proteins from being recruited to the site of DSBs. In the current paper, a gene therapy technique using a complementary RNA sequence to silence p62 mRNA transcripts depleted the level of p62, restored the ATM repair process and reduced the level of DSBs in cells transfected with C9ORF72 repeat expansion products.
Elucidating the cell pathways that cause motor neurons to die in MND and FTD paves the way for new therapies to be developed to tackle these targets.
Find out more:
Read the full paper:C9orf72 expansion disrupts ATM-mediated chromosomal break repair
University of Sheffield press release: https://www.sheffield.ac.uk/news/nr/new-discovery-in-motor-neurone-disease-and-dementia-1.716976
This work was funded by the European Research Council and Wellcome Trust.