Hexanucleotide expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, but how the dipeptide repeats (DPRs) translated from the expansions incite disease still puzzles researchers. A study published March 16 in the EMBO Journal proposes a mechanistic link between the dipeptides and cytoplasmic aggregates of TDP-43—the pathology thought to trigger neurodegeneration.
- Poly-GA dipeptide repeats travel between neurons, at least in culture.
- The peptides derail the work of the proteasome.
- Proteasome deficits trigger TDP-43 mislocalization, aggregation.
Researchers led by Dieter Edbauer at the German Center for Neurodegenerative Diseases in Munich reported that poly-GA peptides travel between neurons in cell culture. In both donor and recipient neurons, the dipeptides disrupted proteasomal function. This, in turn, derailed the normal trafficking of TDP-43 into the nucleus, rendering this protein prone to aggregation in the cytoplasm. The findings connect DPRs and mislocalized TDP-43, two pathologies rarely spotted cohabitating in the same cell.
Using a noncanonical translation mechanism called repeat associated non-ATG (RAN) translation, DPRs are translated from C9ORF72 hexanucleotide transcripts in six reading frames, producing five unique DPRs. Poly-GA is the most abundant. Previously, Edbauer and colleagues reported that poly-GA dipeptides triggered mislocalization of TDP-43, preventing its entry into the nucleus (Khosravi et al., 2017). While this offered a potential causal link between DPRs and TDP-43, it did not explain the observation that in patient tissue, cytoplasmic aggregates of TDP-43 are usually spotted in different cells than are DPR inclusions. Might DPRs somehow bungle TDP-43’s nuclear transport from afar?
To address this question, first author Bahram Khosravi and colleagues set up a neuronal co-culture. They transduced rat primary hippocampal neurons with a gene encoding 175 poly-GA dipeptide repeats linked to green fluorescent protein. After growing the “donor” cells on coverslips for four days, they washed them and positioned them next to a coverslip of untreated, “recipient” neurons that did not themselves express the poly-GA dipeptide. Four days later, the researchers observed poly-GA aggregates in both the donor and recipient cells. What’s more, they found cytoplasmic TDP-43 in both donor and recipient cells, even in cells in which the fluorescent poly-GA aggregates were undetectable. Addition of a poly-GA antibody to the culture system blocked transmission of poly-GA aggregates as well as TDP-43 mislocalization, suggesting that the dipeptide repeats were directly responsible for TDP-43 pathology, even at exceedingly low levels.
Traveling Trouble. Compared with “donor” cells transduced with GFP alone (top left panel), donor cells transduced with Poly-GA-GFP (bottom left) had GA aggregates (green) and some TDP-43 excluded from the nucleus (red). The same effects occurred in non-transduced receiver cells (right panels). [Courtesy of Khosravi et al., EMBO Journal, 2020.]
Poly-GA pumped from neighboring cells also coaxed the formation of cytoplasmic aggregates of TDP-43 in cells expressing a version of the protein lacking a nuclear localization sequence (NLS). Again, poly-GA antibodies blocked this effect. Together, the findings suggested that poly-GA dipeptides instigate the mislocalization and aggregation of TDP-43 in the cytoplasm, in the cells expressing the dipeptides as well as in cells stationed nearby.
How could poly-GA be doing this? In a series of experiments, the researchers zeroed in on the proteasome. Previous structural studies had spotted poly-GA dipeptides gumming up proteasomes (Feb 2018 news). Using a reporter of proteasome function, the researchers confirmed that indeed, poly-GA dipeptides not only derailed proteasome function in the cells expressing the dipeptides, but also in receiver cells nearby in the co-culture. This proteasomal shutdown created a backlog of proteasome substrates—in particular, ubiquitinated TDP-43—in the cytoplasm. Interestingly, the researchers also found that poly-GA triggered a buildup of TDP-43 ubiquitinated on lysine-95—a residue smack dab in the middle of the nuclear localization sequence (NLS). Ubiquitination on this residue directly blocked TDP-43 from interacting with nuclear import machinery. Treating cells with the proteasome activator rolipram countered poly-GA’s promotion of TDP-43 mislocalization and aggregation.
In all, the findings suggested that poly-GA dipeptides can clog the proteasome in nearby neurons. This would lead to a buildup of ubiquitinated TDP-43 in the cytoplasm. TDP-43 is not degraded by the weakened proteasome, and is also is blocked from entering the nucleus because of ubiquitination within its NLS.
The researchers speculate that in the human brain, some neurons, such as those in the cerebellum, are more adept at the noncanonical translation required to generate poly-GA peptides, but less susceptible to their toxicity. Others, including motor neurons, are less efficient at noncanonical translation and more susceptible to proteasomal inhibition triggered by even small amounts of traveling poly-GA. This could explain the different neuronal populations affected by DPR inclusions and TDP-43 pathology in patients, the researchers proposed.
The researchers found that only poly-GA, not other dipeptide repeats translated from different reading frames of C9ORF72, inhibited the proteasome. However, they speculate that RNA foci formed by C9ORF72 transcripts, as well as other DPRs translated from them, likely exert additional toxic effects, such as inhibiting nucleocytoplasmic transport.
The findings cast proteasomal activators, as well as anti-poly-GA antibodies, as potential therapeutic candidates.—Jessica Shugart
- Khosravi B, Hartmann H, May S, Möhl C, Ederle H, Michaelsen M, Schludi MH, Dormann D, Edbauer D. Cytoplasmic poly-GA aggregates impair nuclear import of TDP-43 in C9orf72 ALS/FTLD. Hum Mol Genet. 2017 Feb 15;26(4):790-800. PubMed.
- Khosravi B, LaClair KD, Riemenschneider H, Zhou Q, Frottin F, Mareljic N, Czuppa M, Farny D, Hartmann H, Michaelsen M, Arzberger T, Hartl FU, Hipp MS, Edbauer D. Cell-to-cell transmission of C9orf72 poly-(Gly-Ala) triggers key features of ALS/FTD. EMBO J. 2020 Mar 16;:e102811. PubMed.