As the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), repeat expansions in the C9ORF72 gene spew a poisonous brew of abnormal RNAs and small dipeptide repeat (DPR) proteins. In a new mouse model, researchers led by Leonard Petrucelli at the Mayo Clinic in Jacksonville, Florida, reveal that one of those proteins, the poly glycine-arginine (GR) dipeptide, kills neurons by targeting ribosomes and quenching new protein synthesis. The investigators also spotted poly(GR) and ribosomal proteins together in brain tissue from people with C9-ALS/FTD, suggesting that finding ways to break up the toxic union could offer potential therapies for the disease. In cells and in the mice, though not in human samples, poly(GR) also localized to stress granules. The study appeared June 25 in Nature Medicine.
“This new paper reports the first mouse model expressing specifically poly(GR), which is an exciting step forward for the field,” said Adrian Isaacs, University College, London (full comment below). Paul Taylor of St. Jude Children’s Research Hospital in Memphis, Tennessee, agreed. “Arginine-containing poly-dipeptides (PR and GR) produced in C9-ALS/FTD are exquisitely toxic in a wide variety of model systems. … This paper adds important new insight to this emerging story by showing that, in transgenic mice, expressing poly-GR at levels comparable to, or lower than, levels observed in human C9-ALS/FTD is sufficient to drive neurodegeneration,” Taylor wrote to Alzforum.
The study extends recent findings from Dieter Edbauer at Ludwig-Maximilians University, Munich (see Jun 2018 news). Edbauer’s group discovered that poly(GR) mainly targets ribosomes in primary neurons and in brain samples from C9-FTD patients. “It will be interesting to determine whether boosting translation would prevent neuronal death, or even worsen toxicity through enhanced DPR production,” Edbauer wrote in an email to Alzforum.
Expansion of the six-nucleotide repeat GGGGCC in C9ORF72 gives rise to two abnormal RNAs, which encode five DPR proteins: GR, proline-arginine (PR), glycine-alanine (GA), proline-alanine, and glycine-proline. All accumulate in ALS/FTD. Previously, Petrucelli’s group had generated a mouse model expressing 66 of the GGGGCC repeats from an adenovirus vector they injected into the newborn mouse brain (see May 2015 news). Those mice recapitulated key features of ALS pathology, including DPR-containing aggregates, cytoplasmic inclusions harboring phosphorylated TDP-43, neurodegeneration, and behavioral changes.
In the new study, first author Yong-Jie Zhang used a similar approach to express just the poly(GR) DPR. Based on studies in yeast, flies, and cells, arginine-containing peptides are the most toxic in the C9ORF72 mix, and that held true in the mice. Injection of a virus encoding green fluorescent protein tagged with 100 GR repeats resulted in diffuse cytoplasmic poly(GR) expression. The mice expressed levels of poly(GR) equivalent to some patient samples and the GGGGCC mice, but had almost none of the compact cytoplasmic inclusions seen in those instances. This is consistent with previous work indicating that poly(GR) needs other DPR proteins to promote its own aggregation (Yang et al., 2015).
But even without aggregating, poly(GR) produced severe neurodegeneration. Already at six weeks of age, neuroinflammation markers in the cortices and hippocampi of GFP-(GR)100 mice were elevated. By six months of age, the brains of GR mice weighed just over half those of GFP-expressing controls. The GR mice had but one-third as many cortical neurons, and one-quarter as many CA1-CA3 hippocampal neurons as did controls. They developed significant motor and cognitive deficits: They travelled less in an open field test, were clumsier when walking on a rod or hanging from a wire, and were less able to associate a noise cue with imminent foot shock in a fear conditioning test.
Consistent with the prevailing view that GR repeat peptides are more toxic than GA, the new GR mice had earlier onset and more severe neurodegeneration than mice expressing poly(GA) (see Mar 2016 news).
But how does it happen? In cells, poly(GR) DPRs bind ribosome proteins and disrupt translation (Kanekura et al., 2016; Oct 2016 news; Lopez-Gonzalez et al., 2016). The researchers observed the same in mice. Using immunofluorescence, they saw the distribution of poly(GR) overlap with S6, L21, and S25 ribosomal proteins, as well as the translation initiation factor eIF3η. The effect was selective for GR, as the same pairing was absent in mice expressing poly(GA). Importantly, both diffuse and aggregated poly(GR) co-localized with S6, L21, and eIF3η in postmortem brain tissue from C9-FTD/ALS patients, as well as in mice expressing the GGGGCC repeat.
A transcriptome analysis of brain tissue from the mice revealed expression changes in two main gene clusters: One involved the immune response, and the other spanned the activity and constituents of ribosomes and protein translation. Expression of ribosomal proteins was up in GR mice, presumably to compensate for low ribosomal activity. Indeed, GR dipeptide repeat protein inhibited new protein synthesis in HEK293T cells, as measured by incorporation of puromycin. The investigators also detected this in the mice, where cells expressing GR incorporated less puromycin than cells without (see image). Poly(GR) even shut down the special translational pathway cells use to produce DPRs from the C9ORF72 repeat RNA (see Jan 2018 news), suggesting that GR might negatively regulate its own production.
Another likely poly(GR) target is the stress granule, a dynamic collection of RNA and protein that forms in cells in distress. In cells, poly(GR) overexpression promotes stress granule formation and impedes their dissolution (see Oct 2016 news; Tao et al., 2015). In mice, diffuse poly(GR) did not increase formation of stress granules like the GR-containing aggregates could, but when stress granules were induced by way of heat shock, diffuse poly(GR) was recruited to them and impaired their disassembly. Besides inhibiting protein synthesis, promoting the persistence of SGs could be another way in which poly(GR) induces a state of chronic stress in cells, the authors speculated. They did not find evidence of poly(GR) in stress granules in human ALS/FTD brain. The reasons are unclear, Petrucelli told Alzforum.
The mice are notable for the pathology they do not show, Petrucelli said. For example, neurons degenerated even when there was no TDP-43 accumulation. It’s unclear how C9ORF72 expansions are linked to cytosolic accumulation of phosphorylated TDP-43, and Petrucelli said his group intends to use mouse models to systematically query each of the DPRs to see which elicit neurotoxicity, and how. The story will likely be complicated, as RNA appears to foment TDP-43 aggregation and TDP-43 itself was recently linked to disrupted protein synthesis (see May 2015 news; Feb 2017 news). Petrucelli wants to explore drivers of poly(GR) aggregation in vivo, and whether poly(GA) or other DPRs cooperate in that process.
The investigators also found no evidence for nuclear dysfunction, which surprised Ben Wolozin of Boston University (see April 2018 news). “Since poly(GGGGCC) pathology is associated with dysfunction of nuclear and nuclear pore biology in other animal models, the absence of such dysfunction in the poly(GR) models once again points to the mechanistic specificity by which poly(GR) causes damage, and suggests that nuclear pore pathology does not inevitably occur during neurodegeneration,” he wrote to Alzforum.
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