Hexanucleotide expansions in the C9ORF72 gene—the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia—mount a multipronged attack on the DNA repair system, according to a July 17 study in Nature Neuroscience. Researchers led by Mimoun Azzouz and Sherif El-Khamisy at the University of Sheffield in England reported that the repeat expansions trigger the formation of DNA-RNA hybrids, called R-loops, that break DNA. At the same time, the unusual dipeptide repeats (DPRs) translated from these expansions derail efforts to mend the damage. The researchers found evidence of broken DNA and a subpar repair response in mice expressing the expansions, and also in postmortem tissue from C9-ALS patients. They proposed that the onslaught of DNA damage in neurons ultimately leads to their demise, and that targeting the pathway could become a therapeutic strategy.
DNA damage is a common hazard inside cells, and an extensive repair system exists to lessen its toll. Neurons are acutely dependent on this repair machinery, as they cannot easily wipe the slate clean through replication (see Pan et al., 2014). Making matters worse, oxidative DNA damage increases in the brain, and repair mechanisms start to falter with age and in the context of neurodegenerative disease (see Feb 2013 conference news; Borgesius et al., 2011).
Against this backdrop, El-Khamisy and colleagues wondered if an additional stressor—C9ORF72 hexanucleotide expansions—might add fuel to the fire. These expansions of the GGGGCC sequence exist in hundreds to thousands of copies in people with ALS/FTD. Both RNA foci formed from their transcription, and the DPRs generated by their translation, reportedly inflict damage on neurons. The researchers hypothesized that due to the expansions’ repetitive nature, and the abundance of GC repeats within them, the C9ORF72 expansions could be extremely prone to folding into R-loops, a type of DNA-RNA hybrid structure that can form during transcription (Aguilera and Garcia-Muse, 2012). R-loops are known triggers of double-stranded DNA breaks (Hamperl and Cimprich, 2014).
To learn if the expansions caused R-loops, first author Callum Walker and colleagues transfected the expansions into human fetal lung fibroblasts. Then they probed with antibodies specific to R-loops and phosphorylated histone γH2AX, an established indicator of double-stranded breakages. Indeed, they found that cells expressing 102 repeats that could not be translated—and thus only formed RNA foci, not DPRs—harbored elevated numbers of R-loops and breaks. This was also the case in cells transfected with constructs that did result in the translation of 34 or 69 DPRs. Notably, overexpression of senataxin, an RNA helicase known to resolve R-loops, reduced the number of breakages and even normalized the uptick in cell death the breakages triggered. Together, the findings suggested that the repeat expansions caused R-loops, which snapped DNA and harmed cells.
The researchers further wondered whether the repeat expansions would affect DNA repair. In both human fibroblasts and primary rat cortical neurons expressing the expansions, the researchers found the repair machinery to be profoundly hobbled. For starters, ataxia telangiectasia (ATM), the master DNA repair kinase, was hypophosphorylated and failed to activate when the researchers treated cells with DNA-damaging toxins. This led to a dismal nuclear recruitment of 53BP1, a factor that rejoins broken DNA, as well as subpar phosphorylation of another key ATM target, p53.
Through an extensive battery of biochemical and immunostaining experiments, the researchers zeroed in on the mechanisms that derailed the DNA repair machinery. The E3 ubiquitin ligase RN168 normally ubiquitylates histone H2A, an adornment that is needed to recruit 53BP1 to damaged DNA. However, in cells expressing the expansions, the researchers found RN168 tied up in p62 inclusions instead. This led to a reduction in ubiquitylated H2A and stymied 53BP1 recruitment. Interestingly, previous studies have reported that successful recruitment of 53BP1 to DNA helps sustain further ATM signaling (Lee et al., 2010). Therefore, RN168’s entrapment in p62 inclusions could potentially derail the entire DNA repair process. In support of this idea, overexpression of RN168, or depletion of p62, restored 53BP1 recruitment and reduced the number of DNA breaks in cells expressing the repeat expansions.
Strikingly, the researchers also observed R-loops, double-stranded breaks, and signs of weak ATM signaling in neurons from mice injected with viral vectors harboring the repeat expansions. These animals suffered a 20 percent loss in brainstem neurons, as well as motor deficits. The researchers proposed that DNA damage was the primary cause of this neurodegeneration, a hypothesis they will test by overexpressing senataxin and/or RN168 in the animals, El-Khamisy told Alzforum.
The researchers also found evidence of DNA in disrepair in postmortem spinal cord tissue from ALS patients, which were wrought with R-loops, double-stranded DNA breaks, and signs of ATM signaling defects.
The researchers proposed that C9ORF72 hexanucleotide expansions attacked DNA via two distinct, yet intertwined, pathways: through directly causing damage via R-loops, and by dismantling ATM-mediated DNA repair. El-Khamisy proposed that the repeat-laden RNA causes R-loops, while the DPRs manifest the p62 inclusions that sequester RN168 and disrupt repair. Interestingly, the latter pathway meshes with other recent findings implicating RN168 sequestration in p62 inclusions in the disruption of DNA repair (Wang et al., 2017).
Walker’s findings dovetail with a previous study led by Li-Huei Tsai of MIT, which reported that the ALS gene FUS is recruited to DNA breaks and helps orchestrate repair (see September 2013 news). “The findings of the current paper … are very consistent with ours, and together make a strong argument for the role of unrepaired DNA breaks in ALS,” Tsai commented.
“This paper is particularly well done,” commented Ray Truant of McMaster University in Hamilton, Canada. “It really establishes ATM-mediated DNA repair as a common ‘node’ in neurodegenerative disease,” he said.
Truant recently reported that mutated huntingtin protein disrupted DNA repair (Maiuri et al., 2017). He added that as reactive oxidation builds in the brain with age, the efficiency of the DNA repair response could strongly influence the onset of neurodegenerative disease, a hypothesis supported by recent genome wide association studies (Bettencourt et al., 2016; Jones et al., 2017).
The study also provides researchers with a number of therapeutic targets, some of which may prove useful across neurodegenerative diseases, Truant added.
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