. C9orf72 poly(GR) aggregation induces TDP-43 proteinopathy. Sci Transl Med. 2020 Sep 2;12(559) PubMed.


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  1. The origin and meaning of TDP-43 aggregation in neurodegenerative disorders such as ALS and FTD remain a mystery. TDP-43 undergoes physiology aggregation and disaggregation, and, as an RNA-binding protein, traverses in and out of the nucleus. Somehow in disease, the normal equilibrium of these processes is disrupted.

    This paper by Cook, Wu, Odeh, and colleagues provides a nice demonstration across a variety of experimental platforms (purified protein, cell culture, in vivo) that polyGR proteins from the C9ORF72 repeat expansion mutation can accelerate aggregation of isolated, purified TDP-43, and can drive mislocalization through altering nucleocytoplasmic transport factors. This was specific to polyGR, and was not seen with polyGA, at least for purified proteins. Other DPRs were not examined.

    Several interesting questions remain for further exploration, such as what drives TDP-43 pathology in non-C9ORF72 cases of FTD/ALS where DPRs are not present, and how this particular mechanism interacts with the many other reported toxicities of polyGR in experimental models by this group and many others. Regardless, there is a convergence of evidence from experimental systems and human pathology studies suggesting that polyGR is a key pathologic species in C9ORF72-related ALS. This study shows it could even be a more direct driver of TDP-43 pathology than previously thought.

    View all comments by Robert Baloh
  2. This impressive new paper from the Petrucelli group presents a unified hypothesis linking key molecular phenotypes of C9ORF72-ALS to a single mechanism. They present evidence that poly(GR) protein translated from the C9ORF72 expansion can accelerate the aggregation of purified TDP-43. But more than that, they show that, in vivo, poly(GR) disrupts nucleocytoplasmic transport of TDP-43 and actively recruits mislocalized TDP-43 into stress granules. Thereby, the researchers have shown that poly(GR) is sufficient to reproduce all of the important aspects of TDP-43 pathology seen in human patients.

    But a mechanism that is sufficient is not necessarily the key mechanism at work. Phrased another way, if we could prevent the formation of poly(GR) in human patients, would this prevent TDP-43 pathology and neuronal death? To answer this, the researchers moved to their previously published mouse model of C9ORF72-ALS (Chew et al., 2015), where they intervened to deplete sense transcribed GGGGCC-repeat RNA and sense dipeptide repeats (including poly-GR) using c9 antisense oligonucleotides, which are currently in clinical trial in ALS patients. As expected, depletion of G4C2-repeat RNA reduced levels of poly(GR) and other sense dipeptide repeats, reduced TDP-43 pathology, and reduced neuronal loss.

    However, there are a few outstanding concerns: The Chew et al. mouse is not a perfect model of C9ORF72-ALS in that there is no reported motor neuron loss, despite molecular pathology within motor neurons. Moreover, the phenotype in these mice is comparable to FTD but not closely comparable to ALS and there is no reduction in survival. The C9ORF72 mouse that most closely models an ALS phenotype, including TDP-43 pathology and reduced survival, is from the Ranum group (Liu et al., 2016). This model is notable for increased antisense transcription of the C9ORF72 expansion in vulnerable neuronal populations. This raises the question that perhaps antisense-specific transcripts and dipeptide repeats (poly(PR), poly(PA) are key to development of C9ORF72-ALS, which obviously excludes a poly(GR)-centric mechanism.

    We await the results of the current c9ASO clinical trial, but this paper gives reason to hope that the treatment will reverse TDP-43 pathology and neuronal death. In future, even more effective treatments may be devised to target poly(GR) directly.


    . Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. Science. 2015 Jun 5;348(6239):1151-4. Epub 2015 May 14 PubMed.

    . C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD. Neuron. 2016 May 4;90(3):521-34. Epub 2016 Apr 21 PubMed.

    View all comments by Johnathan Cooper-Knock
  3. This paper by Cook et al. describes very exciting results. The authors present strong evidence that poly(GR) promotes TDP43 pathology indirectly by inhibiting its nuclear transport and more directly through physical interaction promoting TDP43 aggregation. I would be interested to know if the interaction between TDP43 and poly(GR) could be inhibited by small molecules or peptides and if so, whether this would rescue poly(GR)-mediated toxicity.

    View all comments by Brian Freibaum
  4. This paper by Zhang et al. elegantly builds upon, and strengthens, their previous findings that progranulin is required to suppress aberrant microglial activation in the aging brain (Lui et al., 2016). For many years microglia had been overlooked as direct contributors to the pathological progression of neurodegenerative disorders, but many studies, like this one, continue to add to a growing pool of evidence that increasingly demonstrates microglia to be key drivers of neurodegeneration.

    Their single nuclear RNA sequencing results demonstrate that progranulin-deficient microglia undergo a shift from a homeostatic to a disease-specific transcriptional state—akin to, yet distinct from, the disease-associated microglia (DAM) populations that have been observed in AD and ALS. Using a combination of in vitro techniques and mouse genetics, the authors further demonstrate that Grn-/- microglia promote TDP-43 proteinopathy, nuclear pore defects, and cell death in Grn-/- excitatory neurons, all of which could be mitigated by blocking complement activation.

    These findings further elevate the complement system as a potentially viable therapeutic target for PGRN-mediated FTD, though there is still much to untangle in the relationship between the complement cascade and neurotoxicity. For example, the finding that Grn-/-;C1qa-/-;C3-/- have near-complete rescue of microgliosis may be attributable to impaired formation of the C5 convertase and its subsequent pro-inflammatory activity via C5a (An et al., 2018)—conceivably linking C1qa/C3 deletion indirectly to neuroprotection.

    The authors also point out that, while their results support that blocking complement activation can mitigate Grn­-/- microglia toxicity, other cell-intrinsic defects such as lysosomal dysfunction may also propagate neurodegeneration. Indeed, their data demonstrating that TDP-43 granules preferentially attach to lysosomes in Grn-/- neurons (Extended Data figure 8a), as well as their data showing that Grn-/- microglial conditioned media did not further exacerbate Grn-/- cortical neuron apoptosis in vitro (Extended data Figure 7b and 7c) each demonstrate neuron-intrinsic deficits. Additionally, TDP-43 cytoplasmic localization and nuclear pore dysfunction were each much more pronounced in vitro when both microglia and neurons were Grn-deficient.

    Although progranulin haploinsufficiency is known to cause FTD, and heterozygous mice were not examined in detail, this study provides key insights into the way progranulin-mediated maintenance of microglial homeostasis may be contributing to FTD progression. Moving forward it will be important to continue to dissect the respective contributions of various CNS cell types in the pathogenesis of FTD and related degenerative disorders.


    . Progranulin Deficiency Promotes Circuit-Specific Synaptic Pruning by Microglia via Complement Activation. Cell. 2016 May 5;165(4):921-35. Epub 2016 Apr 21 PubMed.

    . Complement protein C5a enhances the β-amyloid-induced neuro-inflammatory response in microglia in Alzheimer's disease. Med Sci (Paris). 2018 Oct;34 Focus issue F1:116-120. Epub 2018 Nov 7 PubMed.

    View all comments by Luke Daly
  5. This study by Cook et al. nicely builds on many years of work documenting the effects of the C9ORF72 G4C2 expansion and dipeptide repeat (DPR) proteins in vivo.

    Previously, it was shown that expressing the G4C2 repeat leads to RNA foci, DPRs and TDP-43 pathology. Amongst the various DPRs, this paper indicates that poly(GR) protein is likely driving TDP-43 pathology in vivo. They also found that poly(GR) and TDP-43 protein aggregates can co-localize. In human tissues, most DPR aggregates do not co-localize with TDP-43 aggregates, although we and others have documented that some DPR aggregates do co-localize with TDP-43 inclusions where the DPR forms a central core which is coated with TDP-43 protein. Importantly, antisense oligonucleotides (ASOs) were able to reduce levels of poly(GR) and TDP-43 inclusions.

    A robust literature suggests that proteins, including RNA-binding proteins such as TDP-43, can undergo liquid phase separation, which is mediated through low complexity domains and modulated by RNA binding. Intriguingly, the authors find that TDP-43 co-aggregation with poly(GR) is not dependent on the low-complexity domain or RNA binding. This highlights that there may be diverse mechanisms that lead to TDP-43 inclusion formation.

    A longstanding argument against the hypothesis that DPRs drive toxicity is human neuropathology data, which shows that some regions, such as the cerebellum, do not degenerate but exhibit abundant DPR aggregates. There is an overall poor correlation between DPR inclusion burden and neurodegeneration. I think this study suggests that there may be an interaction between DPRs and TDP-43. So perhaps DPR accumulation promotes TDP-43 inclusion formation in cell types that are predisposed to TDP-43 dysfunction. In contrast, perhaps DPRs may be relatively innocuous in regions and neurons that are resistant to TDP-43, such as the cerebellum.

    This interaction between DPRs and TDP-43 could be one reason why the DPR burden in postmortem human tissues does not correlate with neurodegeneration.

    View all comments by Edward B. Lee

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  1. In ALS and FTD, Two Different Routes to TDP-43 Aggregation