349. Nucleoporin Pom121 Confer Passive Nuclear Translocation and Envelop Integrity Via Sigma-1 Receptor in Poly-pr-induced C9orf72 Als
Year: 2025
Type: conference-abstract
Abstract: Background Nucleocytoplasmic transport defects are associated with the progression of C9orf72- amyotrophic lateral sclerosis (ALS)1. Repeat-associated non-ATG translation of dipeptide repeat proteins (DPRs) induces abnormal translation and causes C9orf72-ALS progression2. Among these DRPs, Poly-PR (Proline-Arginine) is more toxic and affect nuclear envelope integrity and nucleocytoplasmic transport3. Aims & Objectives Poly-PR can interact with the nuclear pore complex to affect nuclear transport4. Sigma-1 receptor (Sig-1R), a multiple-functional chaperone protein, stabilizes the nucleoporin POM121 to provide neuroprotection in ALS5. Here, we test whether Sig-1R modulates Poly-PR induced C9orf72-ALS progression via nucleocytoplasmic transport. Method The NSC34 cell line was overexpressed with HA-Sig-1R, EGFP-PR42, POM121-myc/DDK, Atf3-myc/DDK or empty vectors for 24h. Transfected cells were then proceeding to immunofluorescence assays (IF), western blotting (WB) and immunoprecipitation. To examine the nucleocytoplasmic distribution, overexpression cells were collected and subjected to subcellular fractionation and proteins were analyzed by WB. For protein turnover assay, cycloheximide (100 μg/mL) was added into the transfected cells for 1 to 3h before harvested for WB. To evaluate cell viability, overexpression cells were treated with 100mM H2O2 for 3h and followed by a CCK-8 assay (ab228554, Abcam). Results 1) EGFP-PR42 overexpression caused a decrease in the Atf3 nuclear-to-cytosol ratio (N:C ratio) without Importin-b1 participation suggesting that nucleocytoplasmic transport of Atf3 is impeded by Poly-PR. EGFP-PR42 co-transfected with POM121-myc/DDK leading to increase the Atf3 N:C ratio indicating that POM121 mediates Atf3 nucleocytoplasmic transport. 2) POM121 had higher expression in cycloheximide treated EGFP-PR42/HA-Sig1R co-expression cells than EGFP-PR42 alone. In IF assay, Sig-1R also increased Atf3 N:C ratio. These data demonstrated that Sig-1R chaperones POM121 and promotes the nuclear import of Atf3. 3) In EGFP-PR42 overexpression cells, POM121 and EGFP-PR42 showed co-localization and direct interaction. 4) In IF assay, Lamin B diffusion was lower in EGFP-PR42 overexpression compared to that of co-transfected with POM121 or Sig1R. These data suggest that POM121 and Sig-1R can reduce Poly-PR induced nuclear envelop disruption. 5) In CCK-8 assay, EGFP-PR42 overexpression increased cell death after H2O2 treatment while EGFP-PR42 co-transfected with POM121 or Sig-1R significant rescued cell viability. Discussion & Conclusions In C9orf72-ALS, arginine-rich dipeptide repeats reported disrupt nucleocytoplasmic transport and lead to neuronal cell death. Here we provide novel insights into the POM121-regulated mechanism on Poly-PR toxicity and unveils the therapeutic effect of Sig-1R. POM121 has been shown to cooperate with Importin-b1 to regulate the nuclear import of transcriptional factors6. Our data show that the decrease of POM121 expression in Poly PR42-transfected NSC34 cells occurs via the proteasomal pathway. We also observed cytosolic accumulation of the neuroprotective protein Atf3 and nuclear envelope integrity disruption in Poly-PR42-transfected cells and Pom121 overexpression reverses these effects. Sigma-1R stabilizes Pom121 expression in Poly-PR42 transfected cells, maintaining nuclear envelope integrity and Atf3 levels. Overexpression of Pom121, Sigma-1R, or Atf3 in Poly-PR42-transfected cells can reverse H2O2 toxicity in Poly-PR-induced motor neuron cell death. Taken together, our data verify the role of the Sigma-1R/Pom121 axis in reducing Poly-PR toxicity through nuclear envelope integrity and can be a therapeutic target for C9orf72 ALS.
Language: English
Subfield: Neurology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,693
OpenAlex ID: W4413285011