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TDP-43 real-time quaking induced conversion reaction optimization and detection of seeding activity in CSF of amyotrophic lateral sclerosis and frontotemporal dementia patients.

ABSTRACT: The pathological deposition of the transactive response DNA-binding protein of 43?kDa occurs in the majority (?97%) of amyotrophic lateral sclerosis and in around 45% of frontotemporal lobar degeneration cases. Amyotrophic lateral sclerosis and frontotemporal lobar degeneration clinically overlap, presenting a continuum of phenotypes. Both amyotrophic lateral sclerosis and frontotemporal lobar degeneration lack treatments capable of interfering with the underlying pathological process and early detection of transactive response DNA-binding protein of 43?kDa pathology would facilitate the development of disease-modifying drugs. The real-time quaking-induced conversion reaction showed the ability to detect prions in several peripheral tissues of patients with different forms of prion and prion-like diseases. Despite transactive response DNA-binding protein of 43?kDa displays prion-like properties, to date the real-time quaking-induced conversion reaction technology has not yet been adapted to this protein. The aim of this study was to adapt the real-time quaking-induced conversion reaction technique for the transactive response DNA-binding protein of 43?kDa substrate and to exploit the intrinsic ability of this technology to amplify minute amount of mis-folded proteins for the detection of pathological transactive response DNA-binding protein of 43?kDa species in the cerebrospinal fluid of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. We first optimized the technique with synthetic transactive response DNA-binding protein of 43?kDa-pre-formed aggregates and with autopsy-verified brain homogenate samples and subsequently analysed CSF samples from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients and controls. Transactive response DNA-binding protein of 43?kDa real-time quaking-induced conversion reaction was able to detect as little as 15?pg of transactive response DNA-binding protein of 43?kDa aggregates, discriminating between a cohort of patients affected by amyotrophic lateral sclerosis and frontotemporal lobar degeneration and age-matched controls with a total sensitivity of 94% and a specificity of 85%. Our data give a proof-of-concept that transactive response DNA-binding protein of 43?kDa is a suitable substrate for the real-time quaking-induced conversion reaction. Transactive response DNA-binding protein of 43?kDa real-time quaking-induced conversion reaction could be an innovative and useful tool for diagnosis and drug development in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The cerebrospinal fluid detection of transactive response DNA-binding protein of 43?kDa pathological aggregates may be exploited as a disease biomarker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.

SUBMITTER: Scialo C

PROVIDER: S-EPMC7566418 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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TDP-43 real-time quaking induced conversion reaction optimization and detection of seeding activity in CSF of amyotrophic lateral sclerosis and frontotemporal dementia patients.

Scialò Carlo C Tran Thanh Hoa TH Salzano Giulia G Novi Giovanni G Caponnetto Claudia C Chiò Adriano A Calvo Andrea A Canosa Antonio A Moda Fabio F Caroppo Paola P Silani Vincenzo V Ticozzi Nicola N Ratti Antonia A Borroni Barbara B Benussi Luisa L Ghidoni Roberta R Furlanis Giovanni G Manganotti Paolo P Senigagliesi Beatrice B Parisse Pietro P Brasselet Romain R Buratti Emanuele E Legname Giuseppe G

Brain communications 20200914 2

The pathological deposition of the transactive response DNA-binding protein of 43 kDa occurs in the majority (∼97%) of amyotrophic lateral sclerosis and in around 45% of frontotemporal lobar degeneration cases. Amyotrophic lateral sclerosis and frontotemporal lobar degeneration clinically overlap, presenting a continuum of phenotypes. Both amyotrophic lateral sclerosis and frontotemporal lobar degeneration lack treatments capable of interfering with the underlying pathological process and early ...[more]

PMID: 33094285

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Project description:The G4C2 hexanucleotide repeat expansion mutation in the C9orf72 gene is the most common genetic cause underlying both amyotrophic lateral sclerosis and frontotemporal dementia. Pathologically, these two neurodegenerative disorders are linked by the common presence of abnormal phosphorylated TDP-43 neuronal cytoplasmic inclusions. We compared the number and size of phosphorylated TDP-43 inclusions and their morphology in hippocampi from patients dying with sporadic versus C9orf72-related amyotrophic lateral sclerosis with pathologically defined frontotemporal lobar degeneration with phosphorylated TDP-43 inclusions, the pathological substrate of clinical frontotemporal dementia in patients with amyotrophic lateral sclerosis. In sporadic cases, there were numerous consolidated phosphorylated TDP-43 inclusions that were variable in size, whereas inclusions in C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration were quantitatively smaller than those in sporadic cases. Also, C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration homogenized brain contained soluble cytoplasmic TDP-43 that was largely absent in sporadic cases. To better understand these pathological differences, we modelled TDP-43 inclusion formation in fibroblasts derived from sporadic or C9orf72-related amyotrophic lateral sclerosis/frontotemporal dementia patients. We found that both sporadic and C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia patient fibroblasts showed impairment in TDP-43 degradation by the proteasome, which may explain increased TDP-43 protein levels found in both sporadic and C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration frontal cortex and hippocampus. Fibroblasts derived from sporadic patients, but not C9orf72 patients, demonstrated the ability to sequester cytoplasmic TDP-43 into aggresomes via microtubule-dependent mechanisms. TDP-43 aggresomes in vitro and TDP-43 neuronal inclusions in vivo were both tightly localized with autophagy markers and, therefore, were likely to function similarly as sites for autophagic degradation. The inability for C9orf72 fibroblasts to form TDP-43 aggresomes, together with the observations that TDP-43 protein was soluble in the cytoplasm and formed smaller inclusions in the C9orf72 brain compared with sporadic disease, suggests a loss of protein quality control response to sequester and degrade TDP-43 in C9orf72-related diseases.

Dataset Information

TDP-43 real-time quaking induced conversion reaction optimization and detection of seeding activity in CSF of amyotrophic lateral sclerosis and frontotemporal dementia patients.

Publications

TDP-43 real-time quaking induced conversion reaction optimization and detection of seeding activity in CSF of amyotrophic lateral sclerosis and frontotemporal dementia patients.

Similar Datasets

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