Before FUS clumps up in the cytoplasm and motor neurons degenerate, an early event, in at least some forms of ALS, may be DNA damage. These DNA breaks could feed into a vicious cycle of motor neuron toxicity by promoting mislocalizaton of FUS, a key repair protein, leading to ALS, according to a study published January 23 in Nature Communications.
The results suggest that blocking key enzymes in the DNA damage response may give FUS a better chance at reaching broken DNA in the nucleus, keeping motor neurons healthier.
Scientists are currently targeting these enzymes to sensitize cancer to chemotherapy, and some of these inhibitors are already undergoing clinical testing. “One should directly try to look whether these can be used for ALS,” said Technische Universität Dresden’s Andreas Hermann in Germany, leader of the study.
The concept of DNA damage in ALS has a long history (reviewed in Coppedè, 2011). Reactive oxidative species (ROS), for one, can batter the genetic molecule, and people with the disease show signs of fractured DNA (Aguirre et al., 2005; Bogdanov et al., 2000; Fitzmaurice et al., 1996). And, to make matters worse, DNA damage repair processes may become ineffective (Bradley et al., 1987; Tandan et al., 1987; Kisby et al., 1997).
More recently, scientists discovered that key proteins implicated in ALS, including FUS, help fix the genome, explaining why this damage may go unrepaired in diseased motor neurons (Sep 2013 news, Wang et al., 2013; Qiu et al, 2014; Zhou et al., 2014). FUS is known for its propensity to decamp the nucleus and accumulate in the cytosol in ALS– where it’s barred from the genome.
But precisely how FUS contributes to the repair of DNA damage remains uncertain. However, it is becoming increasingly clear that a key DNA-repair enzyme called poly(ADP-ribose) polymerase (PARP) localizes to these genetic blemishes, and recruits FUS to these sites (Rulten et al., 2014). “[This] is a very upstream event in the DNA damage response, most likely important for identifying DNA double strand breaks,” said Hermann. “It is thought [FUS] is important to initiate a proper DNA damage response.”
A Vicious Cycle
Hermann’s group aimed to study the role of FUS in ALS. They developed patient-derived induced pluripotent stem cells (iPSCs) from people with FUS mutations, and induced these cells to develop into spinal motor neurons, watching for signs of ALS-like pathology as the cultures matured.
By 14 days, the cells had accumulated double-stranded DNA breaks. However, mutant FUS (mtFUS) did not localize to damage sites as readily as wild-type FUS, the authors found, when they made their own lesions with lasers.
After 21 days, the mtFUS cells began to develop key pathologies that resembled ALS. Mitochondria in distal axons shrank and lost their membrane potential. By day 35, axons swelled. mtFUS moved into the cytoplasm and aggregated. Axons began to wither. Finally, after about 60 days, the neurons began to degenerate – just like in the disease.
Since DNA damage built up first, the authors hypothesized that genomic breaks may cause these tell-tale signs of ALS. To test this idea, they used etoposide to fragment the genome of healthy control motor neurons. The result: FUS accumulated in the cytoplasm, the membrane potential of mitochondria dropped, and organelle transport stalled. “You get exactly the phenotype the FUS mutants have,” said Hermann.
How could damaged DNA cause ALS? Hermann’s team suspects that this build-up of DNA breaks promotes the mislocalization of FUS, contributing to a vicious cycle of motor neuron toxicity. That’s because DNA-PK, a key DNA damage sensor, is activated, and it phosphorylates FUS still remaining in the nucleus (see Deng et al., 2014). That phosphorylated FUS is then exported from the nucleus to the cytoplasm where it can aggregate, potentially contributing to ALS.
The idea of DNA damage as a key early event in ALS doesn’t come as a complete surprise; scientists suspected this based on previous work, notes Massachusetts Institute of Technology’s Li-Huei Tsai in Cambridge (September 2013 news). “Here, they actually show functional data to bring home this message,” said Tsai, who was not involved in the new paper. “If one can figure out how to target this DNA response signaling pathway or to enhance DNA repair, then I think this could be a very novel way to think about therapeutic interventions.”
Putting FUS in Its Place
Perhaps, a therapy could be developed that gives FUS a better chance at reaching the DNA damage. Hermann’s team tried two different strategies: one that buys FUS more time to get to the DNA breaks, and another that prevents FUS from leaving the nucleus.
Approach #1. Buy time. During the DNA repair process, PARP recruits FUS to the DNA damage. Another enzyme, poly(ADP-ribose) glycohydrolase (PARG) degrades PARP, once the DNA break has been fixed. Hermann and colleagues reasoned that if they blocked PARG, PARP would remain at these DNA breaks longer. That might give FUS more time to find the damage, and perform its (as-yet-uncertain) role in the repair.
To test this idea, the researchers treated the cells with gallotannin to inhibit PARG. The result: mtFUS localized to the DNA damage sites in the nucleus. Moreover, the treatment kept mtFUS from aggregating in the cytoplasm, and restored trafficking to distal axons.
Approach #2 Retain FUS in the nucleus. Next, Hermann’s team tried to keep FUS on the job. To prevent any nuclear FUS from escaping, the researchers targeted DNA-PK, which normally phosphorylates FUS, sending it into the cytosol. The researchers treated the cells with the DNA-PK inhibitor NU7441. Again, FUS localized to sites of DNA damage, and axonal transport was restored. “You can completely [rescue] the FUS mutant phenotype,” said Hermann.
In fact, Merck is currently testing a DNA-PK inhibitor in a phase 1 trial for people with certain types of cancer. It’s worth testing for ALS, Hermann said, but he worried there could be side effects, because DNA-PK itself is also necessary to repair the genome. Inhibiting PARG would likely be safer, Hermann thought. Though no such compound has reached the clinical stage, scientists are developing PARG inhibitors for potential therapeutic use (James et al., 2016; Finch et al., 2012). At least one of these teams is setting their sights on neurodegenerative disease.
Nicolas Fawzi, of Brown University in Providence, Rhode Island, said it would be a “good move” to target FUS in neurons as early as possible, ideally to avoid creating aggregates in the cytoplasm. He is developing a different approach, hoping to prevent inclusions. His group reported in 2017 that phosphorylation by DNA-PK makes FUS proteins repel each other, inhibiting aggregation (Aug 2017 news, Monahan et al., 2017).
He doesn’t want to develop a potential ALS therapy that promotes DNA-PK activity, though. Rather, he hopes to identify small-molecular activators of other kinases that would phosphorylate any FUS that does reach the cytoplasm, to prevent aggregation–potentially reducing motor neuron toxicity in the disease.
Hermann’s work raises plenty of questions. For one, how general is the role of the DNA damage pathway in ALS—does it extend beyond those few people with FUS mutations? That’s plausible, Tsai said. In fact, TDP-43, which is mislocalized to the cytoplasm in most cases of ALS, has also been implicated in the DNA repair process (Hill et al., 2016).
Repeat expansions in the ALS-linked gene C9orf72 may create havoc in the genome, too. RNA-DNA complexes called R-loops, formed during the transcription of these sequences, can lead to DNA breaks (Wang et al., 2015). At the same time, the dipeptide repeat proteins encoded by these repetitive sequences may promote R-loop formation and interfere with repairing the resulting damage (July 2017 news, Walker et al., 2017).
Understanding how DNA repair falls short in ALS may help researchers develop strategies that target many forms of ALS. In the meantime, Hermann’s group plans to focus on how FUS helps fix DNA damage. He’s also investigating why people with FUS ALS don’t have a high rate of cancer.
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