Moreover, we performed additional validation with western blots which quantitatively confirm a decrease in total POM121, Nup133, and Nup50 protein in isolated nuclei from iPSNs (Fig

Moreover, we performed additional validation with western blots which quantitatively confirm a decrease in total POM121, Nup133, and Nup50 protein in isolated nuclei from iPSNs (Fig. ultimately downstream neuronal survival. eTOC Blurb Coyne et al. demonstrate that G4C2 repeat RNA initiates a reduction of POM121 expression within neuronal nuclear pore complexes. Decreased nuclear POM121 impacts the expression of 7 additional nucleoporins resulting in altered nuclear pore composition. This combined nucleoporin reduction impacts the localization of nucleocytoplasmic transport proteins and neuronal survival. Introduction A GGGGCC GADD45gamma (G4C2) hexanucleotide repeat expansion (HRE) Ixazomib citrate in intron 1 of the gene is causative of familial and sporadic forms of the motor neuron disease Amyotrophic Lateral Sclerosis (ALS) and the second most common form of dementia, Frontotemporal dementia (FTD) (DeJesus-Hernandez et al., 2011; Renton et al., 2011). The HRE is bidirectionally transcribed to form sense (G4C2) and antisense (G2C4) RNA species that pathologically accumulate into nuclear RNA foci. Repeat associated non-ATG translation (RANT) of G4C2 and G2C4 RNA produces five dipeptide repeat (DPR) proteins [poly(GA), poly(GP), poly(GR), poly(PR), poly(PA)]. Together, these RNA species and DPR proteins are thought to Ixazomib citrate contribute to disease Ixazomib citrate through gain of toxicity mechanisms. In addition, the HRE may also lead to haploinsufficiency of C9ORF72 protein resulting in a mild cellular, but not clinical, loss of function phenotype (Gitler and Tsuiji, 2016; Taylor et al., 2016). Recently, alterations in an essential cellular process, nucleocytoplasmic transport (NCT), have Ixazomib citrate been identified in models of ALS/FTD (Freibaum et al., 2015; Jovicic et al., 2015; Zhang et al., 2015). However, the molecular mechanisms underlying these disruptions and the normal biology governing NCT in human neurons remain largely unknown. The mammalian nuclear pore complex (NPC) is a ~120 MDa protein complex consisting of multiple copies of approximately 30 nucleoporins (Nups) which collectively function to maintain NCT, genome organization, and gene expression. These Nups are found within modular subcomplexes that form the major architectural units of the NPC including the outer and inner ring complexes which form the core NPC scaffold. This scaffold is anchored to the nuclear envelope via transmembrane Nups. Ultimately, this structure anchors the cytoplasmic filaments, nuclear basket, and central transport channel. The central channel is comprised of intrinsically disordered proteins containing multiple FG-repeat motifs. These establish a size-selective diffusion barrier and transport channel for nuclear transport receptors (NTRs) and their bound cargo (Beck and Hurt, 2017; Li et al., 2016; Lin and Hoelz, 2019; Raices and DAngelo, 2012). Additionally, a subset of Nups can also function outside of the NPC in regulating gene transcription (Pascual-Garcia and Capelson, 2019). While small ( 5 nm) macromolecules can passively diffuse through the NPC (Popken et al., 2015; Timney et al., 2016), the nuclear transport of signal-bearing macromolecules is mediated by NTRs (also called karyopherings, importins, and exportins). NTRs recognize import and export signals and transport cargo through the NPC by binding to FG-repeat sequences. The energy to fuel multiple transport cycles is provided by RanGTPase which binds to NTRs in its GTP-bound form to destablize import complexes and stablize export complexes. As a result, in order for bidirectional nuclear transport to occur, a chromatin-bound RanGEF (RCC1) ensures that RanGTP is maintained at high levels within the nucleus and RanGAP drives GTP hydrolysis in the cytoplasm (Melchior, 2001; Raices Ixazomib citrate and DAngelo, 2012). The relative steady-state localization of Ran between the nucleus and cytoplasm has previously been used as a metric for global alterations to NCT.