TDP-43 loss of cellular function through aggregation requires additional structural determinants beyond its C-terminal Q/N prion-like domain

Abstract

TDP-43 aggregates are the neurohistological landmark of diseases like amyotrophic lateral sclerosis and frontotemporal dementia. Their role in the pathogenesis of these conditions is not yet clear mainly due to the lack of proper models of aggregation that may allow the study of the mechanism of formation, their interactions with other cellular components and their effect on the cell metabolism. In this work, we have used tandem repeats of the prion like Q/N-rich region of TAR DNA-binding protein (TDP-43) fused to additional TDP-43 protein sequences to trigger aggregate formation in neuronal and non-neuronal cell lines. At the functional level, these aggregates are able to sequester endogenous TDP-43 depleting its nuclear levels and inducing loss of function at the pre-mRNA splicing level. No apparent direct cellular toxicity of the aggregates seems to be present beyond the lack of functional TDP-43. To our knowledge, this is the only system that achieves full functional TDP 43 depletion with effects similar to RNAi depletion or gene deletion. As a result, this model will prove useful to investigate the loss-of-function effects mediated by TDP-43 aggregation within cells without affecting the expression of the endogenous gene. We have identified the N-terminus sequence of TDP-43 as the domain that enhances its interaction with the aggregates and its insolubilization. These data show for the first time that cellular TDP-43 aggregation can lead to total loss of function and to defective splicing of TDP-43-dependent splicing events in endogenous genes.

Structural insights into the multi-determinant aggregation of TDP-43 in motor neuron-like cells

“…This may be relevant since oligomers may contribute to the propagation of the disease as recently suggested (Feiler et al, 2015). This is in line both with our in silico data, indicating that oligomerization and fibril formation may take place through hydrophobic interactions, and with recent reports that, in cellular models overexpressing tandem repeats of the TDP-43 prion like Q/N region fused to additional TDP-43 domains, residues 1-75 of the N-terminal domain may be involved not so much in the aggregation process itself but rather in increasing the efficiency of aggregates to sequester endogenous TDP-43 (Budini et al, 2015;Romano et al, 2015).…”

“…However, in a study using an artificial Flag-TDP-12X-Q/N fragment of TDP-43 fused to additional TDP-43 protein sequences, Baralle et al have identified the N-terminal sequence of TDP-43 as the domain that enhances its interaction with the aggregates and its insolubilization in neuronal and non-neuronal cell lines (Budini et al, 2015). In a recent study, using the same constructs with tandem repeats of the prion like Q/N-rich region, Buratti et al reported that the N-terminal domain structure needs to be intact in order to bind and sequester endogenous TDP-43 within inclusions.…”

Neuronal cytoplasmic inclusions in tau, TDP-43, and FUS molecular subtypes of frontotemporal lobar degeneration share similar spatial patterns

“…Fourth, TDP-43 is a dimeric nuclear protein in which the C-terminal region exhibits 'prion-like' behaviour [49], the majority of gene mutations associated with frontotemporal dementia (FTD) and motor neuron disease (MND) being located in this region [49]. Moreover, tandem repeats of the 'prion-like' Q/N region of TDP-43, when fused to additional TDP-43, can cause aggregate formation in neuronal and non-neuronal cell lines [24]. Fifth, aggregates of phosphorylated TDP-43 (pTDP-43) are frequently present in axons of hypoglossal and facial nerves and in spinal cord anterior cells in MND, consistent with propagation of the protein [44], while FUS activity is found in granules in gray matter of the brain stem and spinal cord, which co-localise with synaptophysin [1], consistent with transport of the protein and synaptic disconnection.…”

Old versus New Mechanisms in the Pathogenesis of ALS

“…as well as to a glycine-rich sequence containing a prion-like domain (105,122,136) or to the N-terminal domain (11,114,146).…”