# Memo: C9orf72 Repeat Expansion and Nucleocytoplasmic Transport Defects in ALS **Date:** 2026-05-04 **Subject:** Current evidence linking C9orf72 HRE to NPC/NCT dysfunction—recent mechanistic findings, downstream consequences, and contested open questions --- ## 1. Most Recent Mechanistic Findings (2023–2025) ### Direct molecular binding of arginine-rich DPRs to transport machinery The strongest recent mechanistic advance comes from **coarse-grained molecular dynamics simulations** showing that polyPR (an arginine-rich dipeptide repeat, R-DPR) binds directly and promiscuously to multiple classes of nuclear transport components. **Jafarinia et al. (eLife, 2024)** demonstrated that polyPR binds the Kapβ family of importins/exportins, FG-nucleoporins, Ran, and CRM1—driven largely by electrostatic interactions that are **length-dependent and salt-sensitive** (longer polyPR chains bind more avidly) [1]. This builds on earlier structural work (Hayes et al., eLife 2020; PMC 2020) showing R-DPRs disrupt **karyopherin-mediated cargo loading** rather than physically occluding the NPC channel or sequestering transport proteins [2,3]. The emerging consensus is that polyPR acts as a **competitive cargo mimic** that saturates importin-β binding surfaces, slowing nuclear import of authentic NLS-bearing cargoes. ### NPC subunit disassembly in patient-derived neurons A landmark series of studies identified **loss of the transmembrane nucleoporin POM121** as an early, initiating event in C9orf72 iPSC-derived neurons (iPSNs) and postmortem tissue (Coyne et al., bioRxiv 2020 / later published work) [4,5]. Super-resolution imaging of 23 nucleoporins revealed a selective subset of ~8 nucleoporins depleted from neuronal nuclei in C9-ALS/FTD, with POM121 loss preceding broader NPC injury. In parallel, **Bilican et al. (FEBS Letters, 2025)** showed that the scaffold nucleoporin **Nup107 aberrantly interacts with G3BP1** and aggregates into stress granules (SGs) specifically in **C9orf72 patient iPSC motor neurons**, suggesting a physical sequestration mechanism that removes NPC components from the nuclear envelope [6]. ### Nuclear envelope / LINC-complex homeostasis as an upstream driver A 2024 preprint/later publication (Sirtori et al., *Acta Neuropathologica Communications*, 2024; bioRxiv 2024) demonstrated that **altered nuclear envelope tensional homeostasis**—mediated by LINC complex dysfunction—is a hallmark of both sporadic and familial ALS/FTD [7,8]. In C9ORF72 mutant neurons, loss of NE integrity was shown to **precede and drive NPC injury**, and disconnecting the nucleus from the cytoskeleton (rescuing NE tension) could **rescue NPC injury and reduce DNA damage** [8]. This reframes NPC disruption not merely as a passive consequence of DPR toxicity, but as part of a broader biomechanical cascade. --- ## 2. Downstream Consequences for Motor Neuron Survival ### TDP-43 mislocalization and aggregation NCT defects are tightly linked to the **cytoplasmic mislocalization and aggregation of TDP-43**, the near-universal pathological hallmark of ALS. In C9-ALS models, impaired nuclear import/export permits TDP-43 egress from the nucleus, and its subsequent cytoplasmic aggregation can **feed forward** to further disrupt NPC components (Grima et al., Nature Neuroscience 2018; Zhang et al., Nature 2015; reviewed in PMC/Frontiers 2023) [9,10,11]. Recent work (Scientific Reports 2022) explicitly shows C9orf72 dipeptides disrupt NCT machinery and **cause TDP-43 mislocalization** in a mechanistic cascade [12]. ### DNA damage and impaired TFEB-mediated autophagy Impaired nuclear import of **TFEB**—a master transcriptional regulator of autophagy-lysosomal biogenesis—has been documented in C9-ALS/FTD models and human motor cortex (Cunningham et al., 2020; Frontiers 2023 review). The sigma-1 receptor (Sig-1R) normally chaperones **POM121 and importin-β1** to enable TFEB nuclear entry; this axis is compromised in C9-ALS, linking NPC injury to **autophagy failure** [11,13]. Separately, NCT disruption is associated with **DNA damage accumulation** in patient-derived motor neurons, and small-molecule epigenetic modulators (e.g., HDAC inhibitors) that rescue NCT can concurrently reduce DNA damage markers and improve viability (Cell Reports 2021) [14]. ### Stress granule–NPC cross-talk The Bilican 2025 finding that Nup107 is sequestered into SGs in C9 motor neurons suggests a **bidirectional pathology**: SG dynamics perturb the NPC, and NPC dysfunction may alter SG composition, creating a vicious cycle that overwhelms proteostasis in motor neurons [6]. --- ## 3. Strongest Open Questions and Contested Evidence ### Is the NCT defect direct or indirect? This remains the **central mechanistic controversy**: - **Evidence for direct interference:** PolyPR binds transport components directly (Jafarinia 2024), disrupts karyopherin loading (Hayes 2020), and locks FG-repeats in a polymerized state (Shi et al., PNAS 2017) [1,2,15]. - **Evidence against direct blockade:** **Vanneste et al. (Scientific Reports, 2019)** reported that poly-GR and poly-PR **do not directly impede active nucleocytoplasmic transport** in several cell models, arguing the effect must be indirect (e.g., via sequestration or upstream biomechanical stress) [16]. The reconciliation may lie in **context-dependence**: in reconstituted in vitro systems and overexpression models, direct binding is observable, but in intact neurons the dominant pathology may require **POM121 loss, NE disruption, or SG sequestration** as preceding or parallel events. ### Which toxic species drives NPC injury—RNA foci or DPRs? C9orf72 pathogenesis involves (1) **loss of C9orf72 protein function**, (2) **G4C2 repeat RNA foci**, and (3) **RAN-translated DPRs**. The relative contribution of each to NPC/NCT dysfunction is unresolved. The 2020 bioRxiv study emphasized **G4C2 repeat RNA–mediated disassembly** of the NPC, independent of DPR translation [4]. Meanwhile, the 2024 biophysical and 2025 Nup107 studies implicate **DPRs (polyPR)** as the proximal insult [1,6]. These mechanisms are not mutually exclusive but complicate therapeutic targeting. ### Is NCT disruption the primary cause of motor neuron death or a convergent downstream pathway? NCT impairment has been proposed as a **unifying mechanism** across genetic and sporadic ALS (including TDP-43 and FUS mutations). However, it remains debated whether restoring NCT alone is sufficient to rescue motor neuron survival in vivo, or whether it is one of several parallel degenerative cascades (membraneless organelle dysfunction, DNA damage, ER stress, ion channel hyperexcitability). Small-molecule rescues in cellular models are promising but have not yet translated to robust in vivo efficacy [14]. ### Cell-type specificity Why C9orf72-related NPC injury preferentially affects motor neurons—and why the cerebellum can harbor heavy DPR loads without equivalent NPC disruption or neurodegeneration—remains unexplained. Recent iPSC work suggests **motor neuron–specific stress granule composition** (e.g., G3BP1–Nup107 interaction) may confer selective vulnerability [6]. --- ## Summary The evidence now supports a **multi-hit model**: C9orf72 repeat expansion causes nucleocytoplasmic transport dysfunction through at least three interlocking routes—(i) **direct electrostatic binding of polyPR to importins/exportins and FG-nucleoporins**, (ii) **nucleoporin loss/sequestration (POM121, Nup107) altering NPC composition**, and (iii) **nuclear envelope/LINC-complex biomechanical failure** upstream of NPC integrity. Downstream, these defects converge on **TDP-43 mislocalization, TFEB/autophagy impairment, DNA damage, and SG-mediated proteostasis collapse**. The major unresolved questions center on **which toxic species is dominant**, whether NCT defects are **primary or secondary** to neuronal death, and how to reconcile **apparently contradictory findings** of direct vs. indirect polyPR toxicity across model systems. --- ## References (Inline Citations) [1] Jafarinia H, van der Giessen E, Onck PR. *C9orf72 polyPR directly binds to various nuclear transport components.* eLife. 2024;12:RP89694. [2] Hayes LR, Duan L, Bowen K, Kalab P, Rothstein JD. *C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import.* eLife. 2020;9:e51685. [3] PMC/eLife (Hayes et al., 2020) — transport disruption occurs via cargo mimicry, not sequestration or FG-blockade. [4] Coyne AN, et al. *G4C2 repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD.* bioRxiv. 2020.02.13.947721. [5] Frontiers in Cell and Developmental Biology (2025) — POM121 depletion and RanGAP mislocalization in C9-ALS. [6] Bilican et al. *C9orf72 ALS-causing mutations lead to mislocalization and aggregation of nucleoporin Nup107 into stress granules.* FEBS Letters. 2025;599(17). [7] Sirtori R, et al. *LINC complex alterations are a hallmark of sporadic and familial ALS/FTD.* Acta Neuropathologica Communications. 2024;12:69. [8] Sirtori R, et al. *Altered nuclear envelope homeostasis is a key pathogenic event in C9ORF72-linked ALS/FTD.* bioRxiv. 2024.02.01.578318. [9] Grima JC, et al. *Mutant huntingtin impedes axonal BDNF transport by suppressing huntingtin/HAP1 interactions.* (and related TDP-43/NPC work) — see Grima et al. / Nature Neuroscience NPC/TDP-43 literature. [10] Zhang K, et al. *The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.* Nature. 2015;525(7567):56–61. [11] PMC/Frontiers in Cellular Neuroscience (2023) — comprehensive review of disrupted NCT in C9-ALS and TFEB/autophagy deficits. [12] *C9orf72 dipeptides disrupt the nucleocytoplasmic transport machinery and cause TDP-43 mislocalisation to the cytoplasm.* Scientific Reports. 2022;12:4927. [13] Wang SM, et al. *Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine.* Autophagy. 2022/2023. [14] *Epigenetic Small Molecules Rescue Nucleocytoplasmic Transport and DNA Damage Phenotypes in C9ORF72 ALS/FTD.* Cell Reports. 2021;37(9):110036. [15] Shi KY, et al. *Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export.* PNAS. 2017;114(7):E1111–E1117. [16] Vanneste J, et al. *C9orf72-generated poly-GR and poly-PR do not directly interfere with nucleocytoplasmic transport.* Scientific Reports. 2019;9:15292. --- *End of memo*