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 (TDP-43) 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 able to interfere with the underlying pathological process and early detection of TDP-43 pathology would facilitate the development of disease modifying drugs. The Real Time Quaking Induced Conversion reaction (RT-QuIC) showed the ability to detect prions in several peripheral tissues of patients with different forms of prion and prion-like diseases. Despite TDP-43 displays prion-like properties, to date the RT-QuIC technology has not yet been adapted to this protein. The aim of this study was to adapt the RT-QuIC technique for the TDP-43 substrate and to exploit the intrinsic ability of this technology to amplify minutes amount of misfolded proteins for the detection of pathological TDP-43 species in the CSF of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. We first optimized the technique with synthetic TDP-43 preformed aggregates and with autopsy-verified brain homogenate samples and subsequently analyzed CSF samples from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients and controls. TDP-43 RT-QuIC was able to detect as little as 15 picograms of TDP-43 aggregates, discriminating between a cohort of subjects 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 TDP-43 is a suitable substrate for the RT-QuIC. TDP-43 RT-QuIC could be an innovative and useful tool for diagnosis and drug development in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. CSF detection of TDP-43 pathological aggregates may be exploited as a disease biomarker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.

The Cellular Prion Protein Increases the Uptake and Toxicity of TDP-43 Fibrils

“…A brain sample (0.5 g) from post-mortem frozen frontal cortex of a patient with a confirmed neuropathological diagnosis of FTLD-TDP associated with C9orf72 expansion was homogenized as previously described [ 39 ]. Briefly, the sample was homogenized in 2.5 mL of homogenization buffer (HB: 10 mM Tris-HCl, 0.8 M NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1X protease and phosphatase inhibitor cocktail tablets (Roche, Basel Switzerland), pH 7.5).…”

“…Here, capitalizing on our recently adapted TDP-43 real-time quaking induced reaction (RT-QuIC) [ 39 ] we aimed at investigating the involvement of PrP C in the uptake of TDP-43 fibrils and its potential role in mediating at least part of their detrimental effects. We devised an in vitro assay to verify the interaction between TDP-43 LCD fibrils and recombinant PrP C and subsequently tested TDP-43 fibril toxicity in two different cell lines, human SH-SY5Y and mouse neuroblastoma (N2a) cells, engineered to express low or high levels of PrP C .…”

Protein Aggregation Landscape in Neurodegenerative Diseases: Clinical Relevance and Future Applications

“…Particularly, the advent of dyes capable of specifically labelling aggregating structures [101,230], together with the exploitation of the prion-like seeded conversion mechanism, produced outstanding results in the early and differential diagnosis of neurodegenerative diseases. Protein misfolding cyclic amplification and real-time quaking-induced conversion assays have been applied to an increasing number of pathologies, starting with prion-related pathologies [231,232] and expanding to synucleinopathies [233,234], tauopathies and AD [235][236][237] and TDP-43-related pathologies [238], yielding a very a high diagnostic accuracy from ex vivo samples. Moreover, evidence of strain typing, as in the case of prion pathologies and synucleinopathies, has been documented with these technologies [239][240][241].…”

State-of-the-Art Methods and Emerging Fluid Biomarkers in the Diagnostics of Dementia—A Short Review and Diagnostic Algorithm

“…TDP-43 is a major neuropathological protein accumulating in familial FTD (linked to mutations in GRN and C9orf72 genes) as well as in amyotrophic lateral sclerosis (ALS), and it normally contains various physiological functions such as RNA translation, autophagy, and synaptic plasticity [ 103 ]. Recently, also this protein was shown to be highly detectable in the CSF via an RT-QuIC method [ 104 ]. The study by Scialò et al reports that RT-QuIC is the first diagnostic tool, which can be used to accurately detect antemortem neuropathological changes in TDP-43-associated brains.…”