Exposure to ALS-FTD-CSF generates TDP-43 aggregates in glioblastoma cells through exosomes and TNTs-like structure

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent a continuum of devastating neurodegenerative diseases, characterized by transactive response DNA-binding protein of 43 kDa (TDP-43) aggregates accumulation throughout the nervous system. Despite rapidly emerging evidence suggesting the hypothesis of ‘prion-like propagation’ of TDP-43 positive inclusion in the regional spread of ALS symptoms, whether and how TDP-43 aggregates spread between cells is not clear. Herein, we established a cerebrospinal fluid (CSF)-cultured cell model to dissect mechanisms governing TDP-43 aggregates formation and propagation. Remarkably, intracellular TDP-43 mislocalization and aggregates were induced in the human glioma U251 cells following exposure to ALS-FTD-CSF but not ALS-CSF and normal control (NC) -CSF for 21 days. The exosomes derived from ALS-FTD-CSF were enriched in TDP-43 C-terminal fragments (CTFs). Incubation of ALS-FTD-CSF induced the increase of mislocated TDP-43 positive exosomes in U251 cells. We further demonstrated that exposure to ALS-FTD-CSF induced the generations of tunneling nanotubes (TNTs)-like structure and exosomes at different stages, which mediated the propagation of TDP-43 aggregates in the cultured U251 cells. Moreover, immunoblotting analyses revealed that abnormal activations of apoptosis and autophagy were induced in U251 cells, following incubation of ALS-CSF and ALS-FTD-CSF. Taken together, our data provide direct evidence that ALS-FTD-CSF has prion-like transmissible properties. TNTs-like structure and exosomes supply the routes for the transfer of TDP-43 aggregates, and selective inhibition of their over-generations may interrupt the progression of TDP-43 proteinopathy.

In vitro prion-like behaviour of TDP-43 in ALS

“…Aside from exosome propagation of pTDP-43, other mechanisms of potential aggregate spread include free floating aggregates or seeds or nanotubule transmission as seen previously with prions (Gousset et al, 2009) and now recently observed with TDP-43 (Ding et al, 2015). Additionally, the method of seed uptake by the recipient cell has yet to be discovered.…”

“…Previous publications (Ding et al, 2015, Nonaka et al, 2013) have demonstrated that TDP-43 inclusions can spread from cell to cell in a prion-like manner although the mechanism of how this occurs remains unclear. In order to confirm these findings, we co-cultured cells containing pTDP-43 inclusions and mixed them in a 1:1 ratio with cells expressing GFP.…”

“…Indeed, other findings from cerebrospinal fluid suggest that TDP-43 aggregates may spread via exosomes and tunnelling nanotubules (Ding et al, 2015). Additionally, recent studies utilizing staging of pTDP-43 pathology in the brain (Brettschneider et al, 2013) and spinal cord (Brettschneider et al, 2014) suggest that pTDP-43 pathology may propagate transynaptically in an anterograde fashion along corticofugal axonal projections (Braak et al, 2013).…”

Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies

“…In fact, the role of TNTs in a pathological context such as inflammation has already been shown between widely spaced dendritic cells in the adult mouse cornea [28, 72]). Nevertheless, even if in different in vitro and in vivo experimental models [6, 7, 13, 14, 72], there is now a strong evidence for the role of TNTs in different stress-induced pathological processes including the transport of many pathological proteins and pathogens, it has never been addressed in vivo in the brain and remains to be investigated.…”

“…In such cells, the tip of the TNT is an active zone of actin cytoskeleton reorganization and contains ezrin, Exo70, myosin 10 and N-WASP, suggesting a regulation at the cellular level [8, 9]. Extrinsic factors such as arachidonic acid in endothelial cells [10], HIV-1 infection in macrophages [11], oxidative stress [12] and prion-like proteins (e.g., Huntingtin fibrils, TDP-43) in neuronal cells [6, 13, 14] have been shown to trigger TNT formation.…”