Abstract
Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and >95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded ‘prion-like’ protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation in vitro directly from both ALS brain and spinal cord. We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.
Highlights
Amyotrophic lateral sclerosis (ALS) is a progressive, age-related, fatal neurodegenerative disorder that predominantly causes the death of upper and lower motor neurons
Co-transfection of Human embryonic kidney (HEK) cells with the full length Full length wild type TDP-43 plasmid (WT) construct (FL WT) and ALS brain or spinal cord samples, revealed pathological phosphorylated TDP-43 (pTDP-43) bands on Western blotting (WB) (Fig. 1B) after 3 days. The quantification of these bands demonstrates a significant increase in the 46 kDa band of pTDP-43 at 3 days post incubation with ALS temporal cortex (TCX) and motor cortex (MC) samples (***p b 0.001, n = 3) compared to the cells expressing the FL WT and FL WT seeded with control inocula
Whilst the signal from the ALS spinal cord (SC) demonstrates an increase, this does not reach significance here (Fig. 1C). In this experimental condition alone, we observed the formation of numerous pTDP-43 positive cytoplasmic aggregates, which co-localised with the FL WT TDP-43, and depleted the nucleus of FL WT TDP-43 (Fig. 1E)
Summary
Amyotrophic lateral sclerosis (ALS) is a progressive, age-related, fatal neurodegenerative disorder that predominantly causes the death of upper and lower motor neurons. This results in muscle spasticity, weakness, atrophy and eventual paralysis, where death is usually caused by degeneration of motor neurons innervating respiratory musculature (Wijesekera and Leigh, 2009). ALS demonstrates similarities to the prion diseases with a characteristic spreading of degeneration within the CNS. In ALS, this is often well recognised by a clear focal onset of clinical features which spreads to neighbouring. Pathological examination corroborates clinical symptom spread, with a site of severe degeneration and cell loss from a focal onset in the neuraxis that diminishes further from the onset site (Ravits et al, 2007a, 2007b)
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