Primary fibroblasts cultures reveal TDP-43 abnormalities in amyotrophic lateral sclerosis patients with and without SOD1 mutations

Abstract

TAR DNA-binding protein 43 (TDP-43) is a major component of the pathologic inclusions observed in the motor neurons of amyotrophic lateral sclerosis (ALS) patients. We examined TDP-43 expression in primary fibroblasts cultures from 22 ALS patients, including cases with SOD1 (n = 4), TARDBP (n = 4), FUS (n = 2), and C9ORF72 (n = 3) mutations and 9 patients without genetic defect. By using a phosphorylation-independent antibody, 15 patients showed notable alterations of TDP-43 level in the nuclear or cytoplasmic compartments. In particular, a marked accumulation of TDP-43 was observed in the cytoplasm of all cases with C9ORF72 and TARDBP mutations, 1 patient with FUS mutation and 3 patients without genetic defect. Patients with SOD1 mutations revealed a significant reduction of TDP-43 in the nuclei without cytoplasmic mislocalization. These changes were associated with the presence of truncated and phosphorylated TDP-43 species. Our results show that fibroblasts recapitulate some of hallmark TDP-43 abnormalities observed in neuronal cells. The reduction of full-length TDP-43 level in mutant SOD1 cells indicates that at least some SOD1 mutations alter TDP-43 metabolism.

Introduction

A landmark in amyotrophic lateral sclerosis (ALS) research was the discovery of TAR DNA-binding protein 43 (TDP-43) as a major component of the abnormal protein aggregates that form skein-like and spherical inclusions or appear as diffuse granular material in neuronal and glial cells (Neumann et al., 2006). Since then, additional studies have confirmed this finding and have shown that TDP-43 plays a central role in the pathogenesis of ALS and related neurodegenerative conditions, including frontotemporal lobar degeneration with ubiquitin-positive aggregates (Arai et al., 2006, Davidson et al., 2007, Mackenzie et al., 2007, Tan et al., 2007). The description of TARDBP mutations in a proportion of ALS cases confirmed that TDP-43 dysfunction is mechanistic in causing disease (Kabashi et al., 2008, Sreedharan et al., 2008). TDP-43 is normally localized to the nucleus, but in neuronal cells from ALS patients, it is redistributed from the nucleus to the cytoplasm (Neumann et al., 2006). It is unclear whether motor neuron degeneration in ALS occurs because of the gain of a toxic function of cytoplasmic TDP-43, the depletion of nuclear TDP-43, or a combination of these processes (Lagier-Tourenne and Cleveland, 2009). TDP-43 inclusions have been detected in the great majority of ALS patients, including sporadic ALS with unknown etiology and genetic cases with mutations in the genes VCP, TARDBP, SQSTM1, C9ORF72, OPTN, and UBQLN2 (Arai et al., 2006, Blokhuis et al., 2013, Davidson et al., 2007, Johnson et al., 2010, Mackenzie et al., 2014, Neumann et al., 2006, Ravits et al., 2013, Tan et al., 2007, Teyssou et al., 2013, Van Deerlin et al., 2008, Yokoseki et al., 2008). The distribution of TDP-43 pathology in patients with TDP-43 mutations has been suggested to be more widespread than in nonmutant TDP-43 ALS patients (Van Deerlin et al., 2008, Yokoseki et al., 2008), but this finding was not confirmed in another work (Pamphlett et al., 2009). A notable exception to this rule are cases with mutations in the SOD1 gene, which are consistently reported to be devoid of TDP-43 pathology, suggesting that mechanistically ALS is heterogeneous (Mackenzie et al., 2007, Tan et al., 2007).

TDP-43 is a 414-amino-acid protein with 2 RNA recognition motifs and a carboxy-terminal glycine-rich domain. Nearly all mutations reported in ALS patients are localized in the glycine-rich domain, suggesting that alterations in this region have a critical role in neurodegeneration (Kabashi et al., 2008, Sreedharan et al., 2008). This region contains a glutamine/asparagine prion-like domain that participates in protein-protein interactions and in the TDP-43 aggregation process (Budini et al., 2012, D'Ambrogio et al., 2009). To explore whether TDP-43 abnormalities affect primary fibroblasts cultures from ALS patients, we performed a series of immunoblotting and immunostaining experiments in a cohort of patients with different forms of ALS.