Toxic PR _n poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

Abstract

The toxic proline:arginine (PR n ) poly-dipeptide encoded by the (GGGGCC) n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PR n poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-β polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PR n poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PR n -mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PR n poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PR n poly-dipeptide toxicity in the context of a prominent form of ALS.C9orf72 repeat expansion | PR n poly-dipeptide | nuclear pore | FG domain | labile cross-β polymers

Lost in Transportation: Nucleocytoplasmic Transport Defects in ALS and Other Neurodegenerative Diseases

“…Insight into a potential common pathway has recently been highlighted by a series of studies that have implicated defects in nucleocytoplasmic transport as a shared consequence downstream of a variety of ALS-initiating mutations. These include the identification of ALS-causing mutations in the nuclear localization signals (NLSs) of FUS and hnRNPA1 (Dormann et al, 2010; Gal et al, 2011; Liu et al, 2016), evidence of impaired nucleocytoplasmic transport downstream of C9ORF72 -related ALS-FTD (Boeynaems et al, 2016; Freibaum et al, 2015; Jovičić et al, 2015; Shi et al, 2017; Zhang et al, 2015), and recognition that cytoplasmic protein aggregates of TDP-43, and cytoplasmic deposition of amyloids more generally, interfere with nucleocytoplasmic transport of protein and RNA (Woerner et al, 2016). In this review, we provide an overview of the current understanding of nucleocytoplasmic transport through the nuclear pore complex (NPC) in mammalian cells.…”

“…Nuclear transport receptors, which enhance the transport efficiency of cargos through the NPC, directly interact with the FG repeats of FG-Nups and dissolve into and through the FG-hydrogel network (Figure 2). Importantly, FG-Nups emerged as genetic modifiers in a Drosophila model of C9ORF72 -related neurodegeneration and were identified as direct binding targets of toxic poly-dipeptides produced from mutant C9ORF72 (Freibaum et al, 2015; Lee et al, 2016; Lin et al, 2016; Shi et al, 2017). …”

Mechanisms of protein toxicity in neurodegenerative diseases

“…The mechanisms by which nucleocytoplasmic transport becomes disrupted range from sequestration of nuclear pore complex (NPC) molecules by toxic RNA or proteins [19, 51–56] to direct blockage of nuclear pores by toxic disease proteins [57]. Some excellent reviews on this topic have recently been published, which we recommend for detailed discussion [19, 50].…”

A PR plug for the nuclear pore in amyotrophic lateral sclerosis

“…The study by Shi et al (2) not only provides a satisfying explanation for the nucleocytoplasmic transport defect detected in model systems of C9-ALS/FTD but may also illuminate the molecular basis of ALS-FTD pathogenesis more broadly. Two recent studies used proteomic approaches to identify the cellular protein targets of the toxic, arginine-containing poly-dipeptides GR and PR (16,21).…”

“…Mounting evidence suggests that impairment of nucleocytoplasmic transport contributes to the most common genetic form of ALS and FTD, although the underlying mechanism has remained obscure. In PNAS, a study by Shi et al (2) provides evidence that toxic disease-related peptides directly obstruct the central channel of the nuclear pore complex. Fittingly, this study appears on the 50th anniversary of Gall's seminal electron microscopy study (3) that revealed the architecture of the nuclear pore.…”