Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration

Background

Nucleocytoplasmic transport is crucial for cellular homeostasis and occurs across the nuclear membrane via large multi-protein complexes that form aqueous channels, called nucleopore complexes (NPCs) (Stoffler et al., 1999; Ryan and Wente, 2000). Although these channels allow small proteins to passively equilibrate across the nuclear membrane (passive transport), most proteins appear to be actively transported by karyopherins, namely importins or exportins (active transport). Cargo proteins that contain a nuclear localization signal (NLS) are targeted by importins. Various receptor-mediated import pathways have been identified, but the best characterized pathway involves importin-β1/importin-α (KPNB1/KPNAx) of which the cargos contain a classical NLS (cNLS), exemplified by the SV40 large T antigen NLS (Lange et al., 2007). Protein export from the nucleus into the cytoplasm is mediated by exportins. Several pathways of nuclear export have been identified. The most common type of nuclear export signal (NES) is a leucine-rich sequence motif recognized by exportin1 (XPO1), of which the NES from protein kinase inhibitor (PKI) is the most often used prototype (Ossareh-Nazari et al., 2001).

Altered subcellular distribution of proteins is a common characteristic among neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS) (Fahrenkrog and Harel, 2018). Increasing evidence indicates impaired nucleocytoplasmic transport as a possible mechanism explaining this aberrant protein localization in the degenerating neurons. For example, in the majority of ALS patients the RNA binding protein TDP-43 (Arai et al., 2006; Neumann et al., 2006) or, to a lesser extent, FUS (Kwiatkowski et al., 2009; Vance et al., 2009; Tyzack et al., 2019) becomes partially depleted from the nucleus and aggregates in the cytoplasm. Interestingly, the most common genetic cause associated with ALS, a G₄C₂-repeat expansion in the C9orf72 gene (Majounie et al., 2012), has been suggested to affect nucleocytoplasmic transport in various ways (Boeynaems et al., 2016; Yuva-Aydemir et al., 2018). One possible toxic pathway is the bidirectional translation of the repeat expansion by repeat-associated non-ATG (RAN) translation into dipeptide repeat proteins (DPRs), namely poly-GP, poly-GA, poly-GR, poly-PA and poly-PR (Ash et al., 2013; Zu et al., 2010; Mori et al., 2013a and 2013b). Here, we expressed the DPR poly-PR using a lentiviral vector transduced into our reporter cells, to subsequently measure nucleocytoplasmic transport.

Although there is growing evidence indicating that impaired nucleocytoplasmic transport is a component of neurodegenerative diseases, it is still under large debate whether nucleocytoplasmic transport deficits are a consequence of neurodegeneration or an instigator (Hutten and Dormann, 2019). As nucleocytoplasmic transport is a complex and stress-sensitive process, reliable assays with a minimal interference are essential to answer this question, as we did before (Vanneste et al., 2019). We developed several reporter cell lines stably expressing a shuttling-fluorophore allowing us to consistently measure nucleocytoplasmic import as a function of time and in intact cells without the need for transfection. Moreover, this method is cheap and can easily be up scaled for high throughput screening purposes. By fine-tuning the NLS and NES fused to the reporter, the subcellular localization of the reporter can be modified and different transport pathways of interest can be analyzed. While the experiments here are performed with Hela Kyoto cells, the protocol can easily be adapted for other cell types.