The nuclear protein FUS, which accumulates in the cytoplasm in some forms of frontotemporal dementia and amyotrophic lateral sclerosis (ALS), gets its marching orders from the DNA repair machinery, according to a paper in the June 4 Journal of Neuroscience. Senior author Thomas Kukar and colleagues at Emory University School of Medicine in Atlanta show that breaks in the genetic material lead to phosphorylation of FUS, which causes the DNA- and RNA-binding protein to leave the nucleus. This work was done in cultured cells. DNA damage might contribute to pathology in frontotemporal lobar degeneration linked to FUS (FTLD-FUS), the authors suggest, and they add some evidence of DNA damage in postmortem samples from several human cases. Therapeutics that protect DNA might help treat the disease, they propose.
First author Qiudong Deng was interested in how FUS distributes in cells. He noticed that TDP-43, another nuclear protein that moves into the cytoplasm in neurodegenerative disorders, can be coaxed into doing so when cells are exposed to the kinase inhibitor staurosporine (Zhang et al., 2007). Given the similarities between TDP-43 and FUS (see Feb 2009 news story), Deng decided to examine FUS localization after treating human H4 neuroglioma cultures with the inhibitor. Sure enough, a fraction of nuclear FUS made off to the cytoplasm after the treatment, though plenty of FUS stayed back in the nucleus as well. Moreover, Deng used antibodies to phosphorylated serine and threonine to show that FUS had picked up phosphate groups on its way to the cytoplasm.
By deleting different parts of the FUS gene, Deng was able to pinpoint the phosphorylation to the amino terminus of the protein. Clipping off this end abolished the cytoplasmic FUS after staurosporine treatment. Because staurosporine inhibits several kinases, Deng used online prediction algorithms, specific inhibitors, and in-vitro phosphorylation assays to determine that a kinase called DNA-PK is the one that modifies FUS. Phosphomimetic FUS mutants, with aspartate at all of the possible DNA-PK phosporylation sites, also localized to the cytoplasm (see image below). “We think [the phosphorylation] is driving localization,” Kukar said.
Because DNA-PK participates in DNA repair pathways, the researchers speculated that FUS might be phosphorylated in response to chromosome damage. They used a chemical called calicheamicin g1 to cause double-stranded DNA breaks in H4 cells and, sure enough, this led to FUS phosphorylation.
Other scientists have also linked FUS to DNA repair, reporting that the protein finds double-stranded DNA breaks in the nucleus within seconds (see Sep 2013 news story).
How to reconcile this with Deng and Kukar’s new finding that FUS takes off for the cytoplasm? The two findings do not conflict, said Kukar, because they occur on different timescales. Deng and colleagues observed FUS in the cytoplasm starting one hour after the damage, and peaking after two hours. FUS could go straight to DNA after chromosome damage, then become phosphorylated and head for the cytoplasm, suggest the authors. The phosphorylation event might exist to remove FUS from the damage site, Kukar speculated.
Li-Huei Tsai from the Massachusetts Institute of Technology in Cambridge, who conducted some of the work linking FUS to immediate DNA repair (Wang et al., 2013), told Alzforum in an email that she liked this idea. Randal Tibbetts of the University of Wisconsin in Madison, who was not involved in the study, called the idea “provocative” and “plausible.” He speculated that FUS might guide certain RNAs out of the nucleus in response to DNA damage.
Deng’s data convinced Tibbetts and others who commented on the work for Alzforum that DNA damage leads to FUS phosphorylation, which then promotes the protein’s cytoplasmic localization. “The bigger issue now is how this relates to neurodegenerative disease,” Tibbetts said. To begin to address this, Deng analyzed the frontal cortices from 11 FTLD-FUS cases for phosphorylated H2AX, a marker for DNA double-stranded breaks, via Western blots. The samples contained more phosphorylated H2AX than tissue from seven normal controls. “I think it is pretty compelling that there are profound DNA damage markers in these cases,” Kukar said. The story has a gap, however, as the authors found no evidence for FUS phosphorylation in these cases. The phosphorylation effect might be subtler in human tissue than in cell culture, Kukar said, particularly since the FUS in human tissue might have degraded during storage.
Others wondered why DNA damage would cause this specific form of neurodegeneration. “What is the initial cause and nature of the DNA damage?” asked Ian Mackenzie of the University of British Columbia in Vancouver, in an email to Alzforum. He suggested it must be something fairly unique to cause such a rare disease.
In fact, DNA damage occurs in many neurodegenerative diseases (see Feb 2013 conference story). “What is missing is an analysis of other neurodegenerative disease brains,” commented Dorothee Dormann of Ludwig-Maximilians-University in Munich. However, Dormann noted that human tissues suitable for Western blotting are hard to come by. Without those controls, there is no way to know if this pathway occurs specifically in FTLD-FUS, she said. Indeed, if Kukar is right that DNA damage triggers FUS mislocalization, then cytoplasmic FUS might show up in other neurological diseases linked to DNA damage, such as ataxia telangiectasia, Tibbetts suggested.
It will be difficult to prove that DNA damage and FUS phosphorylation directly cause disease, he added. “Whether or not this [pathway] is relevant to the ALS/FTLD-associated functions of FUS, we just do not know yet,” Tibbetts said.—Amber Dance
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