Different genetic mutations can converge on the same downstream pathology. The C9ORF72 repeat expansion and progranulin haploinsufficiency both trigger the RNA-binding protein TDP-43 to abandon the nucleus and settle in cytoplasm. TDP-43 deposits underlie cases of frontotemporal dementia and amyotrophic lateral sclerosis. How does each mutation promote this pathology? Quite differently, according to two new papers.
- Poly(GR) made from the C9ORF72 repeat expansion sequesters TDP-43.
- Antisense oligonucleotides suppress poly(GR) and prevent this pathology.
- Complement proteins released by progranulin-deficient microglia induce TDP-43 aggregation in neurons.
In the September 2 Science Translational Medicine, researchers led by Leonard Petrucelli and Yong-Jie Zhang at the Mayo Clinic in Jacksonville, Florida, and James Shorter at the University of Pennsylvania, Philadelphia, lay the blame on the dipeptide repeat protein poly-glycine-arginine (GR), which results from aberrant translation of C9ORF72 repeat expansion transcripts. The researchers report that poly(GR) directly interacts with TDP-43, pulling it into aggregates. In a mouse model, suppressing poly(GR) production with antisense oligonucleotides prevented TDP-43 aggregation and subsequent neurodegeneration.
“This impressive new paper from the Petrucelli group presents a unified hypothesis linking key molecular phenotypes of C9ORF72-ALS to a single mechanism,” Johnathan Cooper-Knock at the University of Sheffield, U.K., wrote to Alzforum (full comment below). “The researchers have shown that poly(GR) is sufficient to reproduce all of the important aspects of TDP-43 pathology seen in human patients.”
Coaxing TDP-43. In cells with wild-type TDP-43 (red), the protein remains nuclear (top row). When forced into cytoplasm (bottom row), TDP-43 aggregated with poly(GR) (green) and drew the stress granule protein TIA-1 (purple) into these deposits as well. [Courtesy of Cook et al., Science Translational Medicine.]
But what about cases of TDP-43 proteinopathy with other genetic causes? In the August 31 Nature, researchers led by Eric Huang at the University of California, San Francisco, focused on FTD cases caused by loss-of-function mutations in the secreted growth factor progranulin. In a mouse model, they found that a dearth of progranulin affected microglia more than any other cell in the brain. In its absence, microglia release more complement proteins. These innate immune factors in turn damage excitatory neurons, causing TDP-43 to accumulate in cytoplasm and setting the stage for its aggregation there. “Microglia are the major factor driving disease in FTD cases caused by progranulin mutation,” Huang concluded.
Rogue Polypeptides Stir Up Trouble
Aberrant translation of the C9ORF72 repeat expansion results in the production of several polydipeptide proteins, including poly-glycine-alanine (GA) and poly(GR). A previous study found that expressing these proteins in flies was sufficient to draw TDP-43 out of the nucleus and into cytoplasm, where it formed aggregates (Oct 2018 news). Petrucelli and colleagues had generated a mouse model that expressed the C9ORF72 repeat expansion and accumulated cytoplasmic TDP-43 deposits (Chew et al., 2015). However, it was unclear how the aberrant polypeptides caused this.
To investigate, co-first author Hana Odeh at UPenn combined recombinant TDP-43 with either poly(GA) or poly(GR) in a cell-free assay. Poly(GA) had no effect on TDP-43, but poly(GR) accelerated its aggregation, leading to dense deposits. Co-first authors Casey Cook and Yanwei Wu at the Mayo Clinic followed this up by transfecting a kidney cell line with each polypeptide and TDP-43. The TDP-43 construct lacked a nuclear localization signal, keeping it cytoplasmic. Again, poly(GR) aggregated with TDP-43, but poly(GA) did not (see image above). By mutating various regions of TDP-43, the authors determined that its ability to bind poly(GR) did not depend on its RNA-binding motifs, nor on its sticky C-terminal tail. Ongoing work suggests that the N-terminal domain of TDP-43 is responsible for binding poly(GR), Petrucelli told Alzforum.
The authors moved to mice, injecting a 200-repeat poly(GR) construct into the lateral ventricles of newborns. After two weeks, about 8 percent of transfected cortical cells formed polydipeptide aggregates. These almost always contained TDP-43 as well, along with stress granule markers such as ataxin-2. “Stress granules appear to be recruited to the multimeric poly(GR)-TDP-43 complex and perhaps stabilize the structure, forming an inclusion,” Petrucelli said. In addition, the aggregates sequestered nucleoporins and nucleocytoplasmic transport factors, including importin α5 and karyopherin α2. Several previous studies have found that dipeptide repeat proteins jam up nuclear traffic by depleting these factors (Jan 2018 news; Nov 2019 news). By contrast, in mice that expressed poly(GA), polydipeptide repeat inclusions formed, but did not recruit TDP-43, or other factors.
Would suppressing poly(GR) production prevent TDP-43 aggregation? The authors turned to a mouse model that expresses a C9ORF72 expansion with 149 repeats. These animals make all the polypeptides and develop TDP-43 aggregates and neurodegeneration as they age. The researchers injected C9 antisense oligonucleotides (ASOs) into the brains of 3-month-old mice, which have abundant polydipeptide repeat deposits but no TDP-43 mislocalization yet. Three months later, treated animals had far fewer deposits containing TDP-43 than did untreated littermates. Untreated mice lost neurons over this time period, and the neurodegeneration marker neurofilament light shot up in the plasma. ASO treatment maintained neurons and plasma NfL at wild-type levels.
That antisense oligonucleotide treatment can ameliorate TDP-43 pathology is new, Petrucelli noted. “That’s a message of optimism for trials targeting C9ORF72 RNA,” he said. He believes the finding will translate to the clinic, because he found poly(GR) and TDP-43 sequestering together in postmortem tissue samples from people with the C9ORF72 expansion. ASOs directed against C9ORF72 are in clinical trials (May 2018 conference news).
“This paper describes very exciting results,” Brian Freibaum at St. Jude Children’s Research Hospital in Memphis, Tennessee, wrote to Alzforum (full comment below). “I would be interested to know if the interaction between TDP-43 and poly(GR) could be inhibited by small molecules or peptides and if so, whether this would rescue poly(GR)-mediated toxicity.”
Robert Baloh at Cedars-Sinai Medical Center, New York, agreed that the evidence suggests poly(GR) drives TDP-43 pathology. “Several interesting questions remain for further exploration, such as what drives TDP-43 pathology in non-C9ORF72 cases of FTD/ALS where dipeptide repeat proteins are not present,” he wrote to Alzforum (full comment below).
Complement Lures TDP-43 Away. In neurons from wild-type mice (left), TDP-43 (green) is nuclear, and nucleoporin Nup98 (red) stays in the nuclear envelope. In GRN knockouts (center), both escape into cytoplasm, but when the knockouts also lack C1q and C3 (right), the proteins stay put. [Courtesy of Zhang et al., Nature.]
Microglia Play the Heavy in Progranulin Disease
A case in point are FTD cases caused by progranulin mutations. Progranulin participates in lysosomal processing, the stress response, and innate immunity (for review see Kao et al., 2017). Huang and colleagues previously found that low levels of progranulin cause microglia to supercharge production of complement proteins, leading to synapse elimination (May 2016 news).
To look more closely at how loss of progranulin affects microglia and other cell types, joint first authors Jiasheng Zhang, Dmitry Velmeshev, Kei Hashimoto, and Yu-Hsin Huang used RNA-Seq to compare gene expression in the thalami of wild-type and GRN knockout mice at 2, 4, 7, 12, and 19 months of age. The thalamus typically atrophies in FTD (Bocchetta et al., 2018). In wild-type animals, microglia express much more progranulin than do other cell types. Perhaps not surprisingly, then, thalamic microglia were the first cell type to show effects in the knockouts. At 7 months of age, expression of 45 microglial genes either rose or fell, and this went up to 65 genes at 12 months and 265 at 19 months. In general, microglia turned down homeostatic genes and boosted inflammatory and lysosomal genes. The gene expression profile partially overlapped with microglial changes seen in Alzheimer’s disease and ALS, the authors noted (Jun 2017 news; Sep 2017 news).
The microglial changes had their greatest effect on excitatory neurons in the thalamus. At 12 months, these neurons accumulated cytoplasmic TDP-43. By 19 months, some of the neurons had died off, while gene expression in the survivors had gone haywire.
To parse what was going on between microglia and neurons, the authors established primary cell cultures of each from wild-type and knockout mice. Cultured microglia are notorious for changing their phenotype, but by using serum-free culture conditions, Huang and colleagues were able to maintain these cells in something close to their native state, with a similar gene expression profile to microglia in mouse brain (Jun 2017 news). After 14 days in vitro, the authors collected media from the microglial cultures and added them to the excitatory neuron cultures. They found that conditioned media from knockout microglia was far more toxic than that from wild-types. It killed 30 percent of wild-type excitatory neurons, and more than half of GRN knockout neurons. Curiously, inhibitory neurons were fairly resistant to these toxic effects.
How did conditioned media kill? When added to GRN-/- neurons, it depleted nuclear pore proteins and caused TDP-43 to bolt into the cytoplasm. Cytoplasmic TDP-43 partially localized with lysosomes but, unlike in Petrucelli’s study, attracted no stress granules. Because the authors previously found that progranulin knockout microglia make more C1q and C3b complement protein than do wild-types, they wondered if these inflammatory factors might mediate the harmful effects. Indeed, treating cultured wild-type and GRN-/- neurons with C1q, with or without C3b, mimicked the effects of conditioned media and triggered cytoplasmic TDP-43 granules. However, it did not kill as many neurons as did conditioned media, suggesting there might be other toxic factors involved.
However, deleting C1q or C3 from knockout microglia rendered their conditioned media nontoxic, suggesting these factors are essential for toxicity. Vitronectin, which prevents complement proteins from forming a membrane attack complex, likewise protected neurons from the toxic effects of microglial conditioned media. Progranulin knockout mice lacking C1q and C3 did not accumulate TDP-43, and their neurons stayed healthy (see image above).
Huang is now analyzing gene expression in postmortem human brain from progranulin carriers to see if similar changes occur as in mice. If so, it would suggest that microglia are the cell type to target in FTD caused by progranulin mutations, Huang said. Such therapies should aim to cool microglial activation, he suggested.
Luke Daly at the University of Massachusetts Medical School, Worcester, agreed. “These findings further elevate the complement system as a potentially viable therapeutic target for PGRN-mediated FTD,” he wrote to Alzforum (full comment below). Shorter was intrigued by the fact that the data appear to describe a path to cytoplasmic TDP-43 aggregation that is independent of stress granules.
These findings further imply that complement proteins could become a biomarker of disease progression in progranulin mutation carriers. On this, Huang’s preliminary data show that complement in cerebrospinal fluid rises as progranulin carriers become impaired. “Perhaps CSF complement level could be used as an indicator for treatment efficacy,” he speculated.—Madolyn Bowman Rogers
- Dipeptide Repeat Proteins Trigger TDP-43 Pathology, Faulty Nuclear Import
- TDP-43 Snarls Nuclear Traffic
- Dipeptide Repeat Proteins Don’t Directly Block Nuclear Transport
- At AAN, Sights Set on Antisense Therapies for Diseases of the Brain
- Microglia Prune Synapses in a Subtype of Frontotemporal Dementia
- Hot DAM: Specific Microglia Engulf Plaques
- ApoE and Trem2 Flip a Microglial Switch in Neurodegenerative Disease
- What Makes a Microglia? Tales from the Transcriptome
- Chew J, Gendron TF, Prudencio M, Sasaguri H, Zhang YJ, Castanedes-Casey M, Lee CW, Jansen-West K, Kurti A, Murray ME, Bieniek KF, Bauer PO, Whitelaw EC, Rousseau L, Stankowski JN, Stetler C, Daughrity LM, Perkerson EA, Desaro P, Johnston A, Overstreet K, Edbauer D, Rademakers R, Boylan KB, Dickson DW, Fryer JD, Petrucelli L. 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.
- Kao AW, McKay A, Singh PP, Brunet A, Huang EJ. Progranulin, lysosomal regulation and neurodegenerative disease. Nat Rev Neurosci. 2017 Jun;18(6):325-333. Epub 2017 Apr 24 PubMed.
- Bocchetta M, Gordon E, Cardoso MJ, Modat M, Ourselin S, Warren JD, Rohrer JD. Thalamic atrophy in frontotemporal dementia - Not just a C9orf72 problem. Neuroimage Clin. 2018;18:675-681. Epub 2018 Feb 23 PubMed.
- C9ORF72 Toxicity Tied to Mitochondria, Transcriptional Machinery
- Beyond the Nucleus: TDP-43 Sticks Together, For Better or Worse
- It’s ‘And,’ Not ‘Either-Or’: C9ORF72 Mechanisms of Action are Linked
- In New ALS/FTD Mouse Model, Poly(GR) Peptides Poison Ribosomes
- Dipeptide Repeats May Hobble Ribosomes in C9ORF72-FTD Patient Brain
- No, TDP-43 and FUS Are Not Actively Exported From the Nucleus
- Lack of C9ORF72 Protein Renders Neurons More Vulnerable to Degeneration
- Stressed-Out Cells Translate C9ORF72 Repeats, Unleash Toxic Peptides
- Stabilizing C9ORF72 RNA Lowers Toxicity
- Granulins: The Missing Link between Progranulin and Lysosome Function?
- Cook CN, Wu Y, Odeh HM, Gendron TF, Jansen-West K, Del Rosso G, Yue M, Jiang P, Gomes E, Tong J, Daughrity LM, Avendano NM, Castanedes-Casey M, Shao W, Oskarsson B, Tomassy GS, McCampbell A, Rigo F, Dickson DW, Shorter J, Zhang YJ, Petrucelli L. C9orf72 poly(GR) aggregation induces TDP-43 proteinopathy. Sci Transl Med. 2020 Sep 2;12(559) PubMed.
- Zhang J, Velmeshev D, Hashimoto K, Huang YH, Hofmann JW, Shi X, Chen J, Leidal AM, Dishart JG, Cahill MK, Kelley KW, Liddelow SA, Seeley WW, Miller BL, Walther TC, Farese RV Jr, Taylor JP, Ullian EM, Huang B, Debnath J, Wittmann T, Kriegstein AR, Huang EJ. Neurotoxic microglia promote TDP-43 proteinopathy in progranulin deficiency. Nature. 2020 Dec;588(7838):459-465. Epub 2020 Aug 31 PubMed.