FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations

Abstract

Accumulation of the DNA/RNA binding protein fused in sarcoma as cytoplasmic inclusions in neurons and glial cells is the pathological hallmark of all patients with amyotrophic lateral sclerosis with mutations in FUS as well as in several subtypes of frontotemporal lobar degeneration, which are not associated with FUS mutations. The mechanisms leading to inclusion formation and fused in sarcoma-associated neurodegeneration are only poorly understood. Because fused in sarcoma belongs to a family of proteins known as FET, which also includes Ewing's sarcoma and TATA-binding protein-associated factor 15, we investigated the potential involvement of these other FET protein family members in the pathogenesis of fused in sarcoma proteinopathies. Immunohistochemical analysis of FET proteins revealed a striking difference among the various conditions, with pathology in amyotrophic lateral sclerosis with FUS mutations being labelled exclusively for fused in sarcoma, whereas fused in sarcoma-positive inclusions in subtypes of frontotemporal lobar degeneration also consistently immunostained for TATA-binding protein-associated factor 15 and variably for Ewing's sarcoma. Immunoblot analysis of proteins extracted from post-mortem tissue of frontotemporal lobar degeneration with fused in sarcoma pathology demonstrated a relative shift of all FET proteins towards insoluble protein fractions, while genetic analysis of the TATA-binding protein-associated factor 15 and Ewing's sarcoma gene did not identify any pathogenic variants. Cell culture experiments replicated the findings of amyotrophic lateral sclerosis with FUS mutations by confirming the absence of TATA-binding protein-associated factor 15 and Ewing's sarcoma alterations upon expression of mutant fused in sarcoma. In contrast, all endogenous FET proteins were recruited into cytoplasmic stress granules upon general inhibition of Transportin-mediated nuclear import, mimicking the findings in frontotemporal lobar degeneration with fused in sarcoma pathology. These results allow a separation of fused in sarcoma proteinopathies caused by FUS mutations from those without a known genetic cause based on neuropathological features. More importantly, our data imply different pathological processes underlying inclusion formation and cell death between both conditions; the pathogenesis in amyotrophic lateral sclerosis with FUS mutations appears to be more restricted to dysfunction of fused in sarcoma, while a more global and complex dysregulation of all FET proteins is involved in the subtypes of frontotemporal lobar degeneration with fused in sarcoma pathology.

Structural and energetic basis of ALS-causing mutations in the atypical proline–tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

“…The few disease mutation sites found thus far are located in the RGG regions that are N-terminal of the PY-NLSs. TAF15 and EWS have not been found to be mutated in FTLD but significant cytoplasmic mislocalization of both proteins was found in FTLD-FUS cases, suggesting impaired Kapβ2-mediated nuclear import even in these wild-type situations (28,29). We can now explain how all three FET proteins are recognized and imported into the nucleus by Kapβ2.…”

FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns

“…Mutations in the genes coding for the other two FET proteins, TAF15 and EWS, are also found in familial and sporadic ALS cases (Couthouis et al 2011(Couthouis et al , 2012Ticozzi et al 2011). Furthermore, prominent cytoplasmic accumulation and aggregation of FUS, TAF15, and EWS are found in a subset of FTLD cases, further supporting the central role of FUS and of other FET proteins in neurodegeneration (Mackenzie and Neumann 2011;Neumann et al 2011).…”

Phosphorylation of C-terminal tyrosine 526 in FUS impairs its nuclear import

“…In contrast to ALS-FUS, FTLD-FUS individuals do not have mutations in the gene encoding FUS (Neumann et al, 2009a,b;Urwin et al, 2010;Lashley et al, 2011;Snowden et al, 2011). Another difference is that cytoplasmic inclusions in FTLD-FUS cases also include the FUS homologues EWS RNA-binding protein 1 (EWSR1) and TATA box binding protein associated factor (TAF15), and nuclear import factor TNPO1, which are absent in ALS-FUS inclusions, implying that there are key differences in the mechanisms underlying inclusion formation in the two disease groups Neumann et al, 2011Neumann et al, , 2012Troakes et al, 2013). FUS, TAF15 and EWSR1 form the FET-family of proteins, which share a homologous C-terminal NLS, characterised by an overall basic charge, a central hydrophobic or basic motif, followed by a C-terminal R/H/Kx (2)(3)(4)(5) PY consensus sequence [where x (2)(3)(4)(5) is any sequence of 2-5 residues] (Lee et al, 2006;Zakaryan and Gehring, 2006;Marko et al, 2012).…”