TDP-43 Joins Cell-To-Cell Propagation Gang

New data suggest that TDP-43 from diseased human brains propagates along neuronal connections in mice. According to a report in the October 11 Nature Communications, extracts containing pathological TDP-43 from postmortem brains of patients with frontotemporal lobar degeneration, when injected into transgenic mouse brains, seeded new pathology, corrupting human TDP-43 protein that was expressed in the cytoplasm along its path.

  • Injected human TDP-43 spreads pathology though transgenic mouse connectome.
  • Most potent seeding seen with extract from people with progranulin mutations.
  • Data raise question if similar protein propagation occurs in human brains, causing disease progression.

The result places TDP-43 in the same vein as misfolded Aβ, tau, and α-synuclein, which also propagate from neuron to neuron in this type of mouse paradigm, said senior author Virginia Lee, Perelman School of Medicine, University of Pennsylvania, Philadelphia. “TDP-43 had never been shown to spread cell-to-cell from one brain region to another in animal models,” she told Alzforum. “We now show all neurodegenerative disease proteins spread this way, which allows us to generalize that cell-to-cell transmission is a common mechanism for the progression of all neurodegenerative diseases.” TDP-43 pathology afflicts motor neurons of patients with amyotrophic lateral sclerosis (ALS), meaning the results may have implications for that disease as well.

TDP-43 was previously reported to travel from cell to cell in culture (Feiler et al., 2015; Nonaka et al., 2013). However, this propagation had yet to be demonstrated in an animal model.

Slow Migration. Heat maps depict the march of TDP-43 pathology from the ipsilateral side of the brain one month post-injection (mpi) to more distal brain areas, including in the contralateral hemisphere, at nine months. Red areas lie closest to the injection site. [Image courtesy of Porta et al., 2018. Nat Commun.]

To look for spreading of TDP-43 in mouse brain, first author Sílvia Porta used the CamKIIa-hTDP43ΔNLSm model that the Lee group had generated to express human TDP-43 in forebrain neurons inducible by removing doxycycline from their chow. TDP-43 is a nuclear protein, but this version had a mutation in its nuclear localization signal, meaning the protein wandered out into the cytoplasm.

Into this model, Porta and colleagues planned to inject brain extracts from the frontal cortices of patients who had died with FTLD. Those patients had either had sporadic disease, a causative repeat expansion in C9ORF72, or progranulin (GRN) mutation. Porta first evaluated the extracts’ seeding capacity in cultured QBI-293 cells that express human TDP-43 either in the nucleus or cytoplasm. Extracts seeded pathology in both cell types, but led to at least three times as much if TDP-43 appeared in the cytoplasm, suggesting mislocalization spurred seeding.

Extracts from patients with progranulin mutations seeded pathology faster in cultured cells than extracts from sporadic FTD. Extracts from patients with C9ORF72 expansions fell in between the two. Lee did not know why that is. It may hint that different mutations lead to different strains of misfolded TDP-43 that seed at different efficiencies, or it may be that progranulin mutations lead to more abundant pathology that seeds more effectively, Lee said, noting that her team tried to control for TDP-43 levels between samples.

Where did the researchers inject the human extract? Into the neocortices, hippocampi, and thalami of the mice. They then analyzed these brains immunohistochemically one to nine months later for phosphorylated TDP-43. After a month, pTDP-43 pathology turned up in the deep layers of the neocortex, hippocampus, subiculum, and cortex (see image below); it was most obvious on the side of the injection, but faint inclusions appeared on the contralateral side as well, indicating that TDP-43 pathology had begun to cross into the opposite hemisphere.

Outward-bound. Immunohistochemistry reveals pTDP-43 in deep layers of the ipsilateral cortex one month after injection. [Image courtesy of Porta et al., 2018. Nat Commun.]

In mice sacrificed at later time points, inclusions appeared in more superficial layers of the neocortex, contralateral cortex, and hippocampus, as well as more distant brain regions such as the nucleus accumbens and lateral septum (see image above). These regions are all anatomically connected to the injection sites. Both gray and white matter became affected over time. As aggregates matured, they became more compact. Like inclusions in the brains of people with ALS or FTLD with TDP-43 pathology, the aggregates stained positive for markers of autophagy and proteolysis, suggesting activation of similar protein-degrading pathways between mice and humans.

Pathology was mostly neuronal in this model, with little to no accumulation in astrocytes, microglia, or oligodendrocytes. That is to be expected in a host that expresses cytoplasmic TDP-43 only in forebrain neurons, the authors wrote. Altogether, the results reveal that TDP-43 spread along the connectome of the brain, with inclusions maturing and condensing over time.

Propagation seemed to require the full-length protein. If extracts from patients were injected into the CamKIIa-208 mouse, which expresses only a C-terminal fragment of TDP-43, a bit of pathology did appear, but it was but a fraction of the inclusions found in mice expressing full-length cytoplasmic TDP-43.

Human brain extracts seeded much less pathology in non-transgenic mice that expressed only mouse TDP-43 in the nucleus, though it was detectable. One month post-injection, rare inclusions popped up in the cortex and hippocampus; they became more visible after nine months.

This study used only extracts from FTLD patients. TDP-43 pathology marks spinal cord neurons from most ALS patients, as well. Asked about ALS, Lee told Alzforum she assumes that the mechanism of spread is the same.

Masato Hasegawa, Tokyo Metropolitan Institute of Medical Science, told Alzforum the paper represents major progress (see full comment below).—Gwyneth Dickey Zakaib

Comments

  1. In this paper, Porta et al. report that pathological TDP-43 derived from FTLD-TDP brains induced formation of de novo TDP-43 pathology, with subsequent spreading throughout the central nervous system in a regional- and time-dependent manner, in experimental animal models. This is big progress.

    It has been already confirmed that α-synuclein and tau protein pathologies are propagated in cellular and animal models. The propagation of TDP-43 pathology was observed in a cellular model (Nonaka et al., 2013), but so far the animal model was not successful.

    This paper indicates that cytoplasmic localization of TDP-43 is important for seeding and propagation of TDP-43 pathology. Unlike α-synuclein and tau, TDP-43 pathology propagation does not easily progress in non-transgenic mice because TDP-43 is localized in the nucleus. Therefore, overexpression and mislocalization of TDP-43 in the cytoplasm may be very important. In addition, Porta et al. used CamKIIa-208 tg-mice, which produced C-terminal fragment TDP-43, for their propagation study, and showed that in these mice propagation efficiency is lower as compared to CamKIIa-hTDP-43NLSm mice; this suggests that the N-terminal region of TDP-43 is important for fibril formation and propagation.

    TDP-43 proteinopathy has been classified into four types based on the predominant TDP-43-positive structures: type A mainly includes FTLD-TDP with GRN mutations, type B contains ALS and FTLD- MND, type C is representative of sporadic FTLD-TDP showing impairment of semantic memory, and type D refers to the pathology associated with IBM and VCP mutations (Mackenzie et al., 2011). Each type is also characterized biochemically by the patterns of insoluble TDP-43 CTFs and protease-resistant CTFs detected with anti-pTDP (Hasegawa et al., 2008; Tsuji et al., 2012). Porta et al. here describe that no specific subtypes explained the differences found in vivo seeding activity. Hence it is necessary to further analyze the relationship between seeding activity or strain-like properties of pathological TDP-43 in patients and neuropathological phenotypes of the mice injected.

    References:

    Nonaka T, Masuda-Suzukake M, Arai T, Hasegawa Y, Akatsu H, Obi T, Yoshida M, Murayama S, Mann DM, Akiyama H, Hasegawa M. Prion-like properties of pathological TDP-43 aggregates from diseased brains. Cell Rep. 2013 Jul 11;4(1):124-34. PubMed.

    Mackenzie IR, Munoz DG, Kusaka H, Yokota O, Ishihara K, Roeber S, Kretzschmar HA, Cairns NJ, Neumann M. Distinct pathological subtypes of FTLD-FUS. Acta Neuropathol. 2011 Feb;121(2):207-18. PubMed.

    Hasegawa M, Arai T, Nonaka T, Kametani F, Yoshida M, Hashizume Y, Beach TG, Buratti E, Baralle F, Morita M, Nakano I, Oda T, Tsuchiya K, Akiyama H. Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Ann Neurol. 2008 Jul;64(1):60-70. PubMed.

    Tsuji H, Arai T, Kametani F, Nonaka T, Yamashita M, Suzukake M, Hosokawa M, Yoshida M, Hatsuta H, Takao M, Saito Y, Murayama S, Akiyama H, Hasegawa M, Mann DM, Tamaoka A. Molecular analysis and biochemical classification of TDP-43 proteinopathy. Brain. 2012 Nov;135(Pt 11):3380-91. Epub 2012 Oct 3 PubMed.

    View all comments by Fuyuki Kametani View all comments by Masato Hasegawa

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References

Research Models Citations

  1. hTDP-43ΔNLS

Paper Citations

  1. Feiler MS, Strobel B, Freischmidt A, Helferich AM, Kappel J, Brewer BM, Li D, Thal DR, Walther P, Ludolph AC, Danzer KM, Weishaupt JH. TDP-43 is intercellularly transmitted across axon terminals. J Cell Biol. 2015 Nov 23;211(4):897-911. PubMed.
  2. Nonaka T, Masuda-Suzukake M, Arai T, Hasegawa Y, Akatsu H, Obi T, Yoshida M, Murayama S, Mann DM, Akiyama H, Hasegawa M. Prion-like properties of pathological TDP-43 aggregates from diseased brains. Cell Rep. 2013 Jul 11;4(1):124-34. PubMed.

Further Reading

Papers

  1. Smethurst P, Newcombe J, Troakes C, Simone R, Chen YR, Patani R, Sidle K. In vitro prion-like behaviour of TDP-43 in ALS. Neurobiol Dis. 2016 Dec;96:236-247. Epub 2016 Aug 30 PubMed.
  2. Hock EM, Polymenidou M. Prion-like propagation as a pathogenic principle in frontotemporal dementia. J Neurochem. 2016 Aug;138 Suppl 1:163-83. PubMed.
  3. Shimonaka S, Nonaka T, Suzuki G, Hisanaga S, Hasegawa M. Templated Aggregation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Seeding with TDP-43 Peptide Fibrils. J Biol Chem. 2016 Apr 22;291(17):8896-907. Epub 2016 Feb 17 PubMed.

Primary Papers

  1. Porta S, Xu Y, Restrepo CR, Kwong LK, Zhang B, Brown HJ, Lee EB, Trojanowski JQ, Lee VM. Patient-derived frontotemporal lobar degeneration brain extracts induce formation and spreading of TDP-43 pathology in vivo. Nat Commun. 2018 Oct 11;9(1):4220. PubMed.

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