Flow cytometric measurement of the cellular propagation of TDP-43 aggregation

Zeineddine, Rafaa; Whiten, Daniel R.; Farrawell, Natalie E.; McAlary, Luke; Hanspal, Maya A.; Kumita, Janet R.; Wilson, Mark R.; Yerbury, Justin J.

doi:10.1080/19336896.2017.1314426

Cited by 23 publications

(28 citation statements)

References 26 publications

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“…We used a recently developed flow cytometry method, which counts fluorescent particles in cell lysates and normalizes this count against the number of separately enumerated nuclei (FloIT; [ 25 , 36 , 37 ]) to quantify the numbers of TDP-43 M337V -GFP inclusions in differently treated N2a cells. The results indicated that (i) in unstressed cells, overexpression of CLU or mCherry had no significant effect on the numbers of TDP-43 M337V -GFP inclusions, (ii) the numbers of inclusions were increased in cells treated with either thapsigargin (Tg) alone (to induce ER stress), or with Tg and MG132 (the latter to also inhibit the proteasome), and (iii) overexpression of CLU, but not mCherry, reduced the numbers of TDP-43 M337V -GFP inclusions in ER-stressed cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Clusterin protects neurons against intracellular proteotoxicity

Gregory

Whiten

Brown

et al. 2017

acta neuropathol commun

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It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease.Electronic supplementary materialThe online version of this article (10.1186/s40478-017-0481-1) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Clusterin protects neurons against intracellular proteotoxicity

Gregory

Whiten

Brown

et al. 2017

acta neuropathol commun

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“…In vitro, the intercellular transfer of TDP-43 has been demonstrated [21, 47, 57, 67]. However, whether this also occurs in vivo remains an open question.…”

Section: Discussionmentioning

confidence: 99%

“…Significant recent interest in the field has focussed on the potential cell-to-cell transfer of TDP-43 and/or associated proteins, and the possibility that this might facilitate propagation of the disease process through the motor system. In vitro, the intercellular transfer of TDP-43 has been demonstrated [ 21 , 47 , 57 , 67 ]. However, whether this also occurs in vivo remains an open question.…”

Section: Discussionmentioning

confidence: 99%

Nucleo-cytoplasmic transport of TDP-43 studied in real time: impaired microglia function leads to axonal spreading of TDP-43 in degenerating motor neurons

et al. 2018

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Transactivating DNA-binding protein-43 (TDP-43) deposits represent a typical finding in almost all ALS patients, more than half of FTLD patients and patients with several other neurodegenerative disorders. It appears that perturbation of nucleo-cytoplasmic transport is an important event in these conditions but the mechanistic role and the fate of TDP-43 during neuronal degeneration remain elusive. We have developed an experimental system for visualising the perturbed nucleocytoplasmic transport of neuronal TDP-43 at the single-cell level in vivo using zebrafish spinal cord. This approach enabled us to image TDP-43-expressing motor neurons before and after experimental initiation of cell death. We report the formation of mobile TDP-43 deposits within degenerating motor neurons, which are normally phagocytosed by microglia. However, when microglial cells were depleted, injury-induced motor neuron degeneration follows a characteristic process that includes TDP-43 redistribution into the cytoplasm, axon and extracellular space. This is the first demonstration of perturbed TDP-43 nucleocytoplasmic transport in vivo, and suggests that impairment in microglial phagocytosis of dying neurons may contribute towards the formation of pathological TDP-43 presentations in ALS and FTLD.Electronic supplementary materialThe online version of this article (10.1007/s00401-018-1875-2) contains supplementary material, which is available to authorized users.

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“…In cell culture, “prion-like” seeding and propagation of aggregation have been observed for proteins that are heavily involved in neurodegenerative diseases, such as Parkinson’s, Huntington’s and ALS [ 6 , 7 , 8 ]. More specifically, researchers have observed this “prion-like” behavior in TDP-43 extracted from diseased brains of ALS and frontotemporal lobar dementia (FTLD) patients [ 9 , 10 , 11 ], as well as from overexpressed TDP-43 in co-culture [ 12 ] and in conditioned medium [ 13 ]. This propagation has been associated with cytotoxicity.…”

Section: Introductionmentioning

confidence: 99%

Conditioned Medium from Cells Overexpressing TDP-43 Alters the Metabolome of Recipient Cells

Hergesheimer

Lanznaster

Bourgeais

et al. 2020

Cells

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive death of both upper and lower motor neurons. The disease presents a poor prognosis, and patients usually die 2–5 years after the onset of symptoms. The hallmark of this disease is the presence of phosphorylated and ubiquitinated aggregates containing trans-active response DNA-binding protein-43 (TDP-43) in the cytoplasm of motor neurons. TDP-43 pathology has been associated with multiple pathways in ALS, such as metabolic dysfunction found in patients and in in vivo models. Recently, it has been described as a “prion-like” protein, as studies have shown its propagation in cell culture from ALS brain extract or overexpressed TDP-43 in co-culture and conditioned medium, resulting in cytotoxicity. However, the cellular alterations that are associated with this cytotoxicity require further investigation. Here, we investigated the effects of conditioned medium from HEK293T (Human Embryonic Kidney 293T) cells overexpressing TDP-43 on cellular morphology, proliferation, death, and metabolism. Although we did not find evidence of TDP-43 propagation, we observed a toxicity of TDP-43-conditioned medium and altered metabolism. These results, therefore, suggest (1) that cells overexpressing TDP-43 produce an extracellular environment that can perturb other cells and (2) that TDP-43 propagation alone may not be the only potentially cytotoxic cell-to-cell mechanism.

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Flow cytometric measurement of the cellular propagation of TDP-43 aggregation

Cited by 23 publications

References 26 publications

Clusterin protects neurons against intracellular proteotoxicity

Clusterin protects neurons against intracellular proteotoxicity

Nucleo-cytoplasmic transport of TDP-43 studied in real time: impaired microglia function leads to axonal spreading of TDP-43 in degenerating motor neurons

Conditioned Medium from Cells Overexpressing TDP-43 Alters the Metabolome of Recipient Cells

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