. 2020 Sep 23;107(6):1124-1140.e11.

doi: 10.1016/j.neuron.2020.06.027. Epub 2020 Jul 15.

G₄C₂ Repeat RNA Initiates a POM121-Mediated Reduction in Specific Nucleoporins in C9orf72 ALS/FTD

Alyssa N Coyne¹, Benjamin L Zaepfel², Lindsey Hayes³, Boris Fitchman⁴, Yuval Salzberg⁴, En-Ching Luo⁵, Kelly Bowen⁶, Hannah Trost⁷, Stefan Aigner⁸, Frank Rigo⁹, Gene W Yeo¹⁰, Amnon Harel⁴, Clive N Svendsen⁷, Dhruv Sareen⁷, Jeffrey D Rothstein¹¹

Affiliations

¹ Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
² Biochemistry, Cellular, and Molecular Biology Graduate Program, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
³ Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
⁴ Azrieli Faculty of Medicine, Bar-Ilan University, Safed 1311502, Israel.
⁵ Bioengineering Graduate Program, University of California San Diego College of Engineering, La Jolla, CA 92037, USA.
⁶ Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
⁷ The Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
⁸ Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, CA 92037, USA.
⁹ Ionis Pharmaceuticals, Carlsbad, CA 92010, USA.
¹⁰ Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, CA 92037, USA; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92037, USA.
¹¹ Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: jrothstein@jhmi.edu.

PMID: 32673563
PMCID: PMC8077944
DOI: 10.1016/j.neuron.2020.06.027

G₄C₂ Repeat RNA Initiates a POM121-Mediated Reduction in Specific Nucleoporins in C9orf72 ALS/FTD

Alyssa N Coyne et al. Neuron. 2020.

. 2020 Sep 23;107(6):1124-1140.e11.

doi: 10.1016/j.neuron.2020.06.027. Epub 2020 Jul 15.

Authors

Affiliations

¹ Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
² Biochemistry, Cellular, and Molecular Biology Graduate Program, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
³ Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
⁴ Azrieli Faculty of Medicine, Bar-Ilan University, Safed 1311502, Israel.
⁵ Bioengineering Graduate Program, University of California San Diego College of Engineering, La Jolla, CA 92037, USA.
⁶ Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
⁷ The Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
⁸ Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, CA 92037, USA.
⁹ Ionis Pharmaceuticals, Carlsbad, CA 92010, USA.
¹⁰ Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, CA 92037, USA; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92037, USA.
¹¹ Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: jrothstein@jhmi.edu.

PMID: 32673563
PMCID: PMC8077944
DOI: 10.1016/j.neuron.2020.06.027

Abstract

Through mechanisms that remain poorly defined, defects in nucleocytoplasmic transport and accumulations of specific nuclear-pore-complex-associated proteins have been reported in multiple neurodegenerative diseases, including C9orf72 Amyotrophic Lateral Sclerosis and Frontotemporal Dementia (ALS/FTD). Using super-resolution structured illumination microscopy, we have explored the mechanism by which nucleoporins are altered in nuclei isolated from C9orf72 induced pluripotent stem-cell-derived neurons (iPSNs). Of the 23 nucleoporins evaluated, we observed a reduction in a subset of 8, including key components of the nuclear pore complex scaffold and the transmembrane nucleoporin POM121. Reduction in POM121 appears to initiate a decrease in the expression of seven additional nucleoporins, ultimately affecting the localization of Ran GTPase and subsequent cellular toxicity in C9orf72 iPSNs. Collectively, our data suggest that the expression of expanded C9orf72 ALS/FTD repeat RNA alone affects nuclear POM121 expression in the initiation of a pathological cascade affecting nucleoporin levels within neuronal nuclei and ultimately downstream neuronal survival.

Keywords: ALS; C9orf72; FTD; POM121; neurodegeneration; nuclear pore complex.

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Conflict of interest statement

Declarations of Interests The authors declare no competing interests.

Figures

**Figure 1:. Specific nucleoporins are altered in nuclei from *C9orf72* iPSNs.**
(**A-C**) Maximum intensity projections from SIM imaging of Nups in nuclei isolated from control and *C9orf72* iPSNs. Genotype and time point as indicated on left, antibodies as indicated on top. (**D-E**) Quantification of Nup spots and volume. Time point as indicated on top. N = 8 control and 8 *C9orf72* iPSC lines (including 1 isogenic pair), 50 NeuN+ nuclei per line/time point. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. Scale bar = 5 μm. * indicates significantly altered Nups.

**Figure 2.. Specific nucleoporins are altered in nuclei from *C9orf72* patient motor cortex.**
(**A-B**) Maximum intensity projections from SIM imaging of Nups in nuclei isolated from postmortem human brain tissue. Genotype and brain region as indicated on left, antibodies as indicated on top. (**C-D**) Quantification of Nup spots and volume. Brain region as indicated on top. N = 3 control and 3 *C9orf72* cases, 50 NeuN+ nuclei per case. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. Scale bar = 5 μm. * indicates significantly altered Nups.

**Figure 3:. Sense repeat RNA targeting ASOs mitigate alterations in specific Nups, restore the localization of Ran GTPase, and protect against cellular toxicity in *C9orf72* iPSNs.**
(**A-E**) Maximum intensity projections from SIM imaging of Nups in nuclei isolated from 5 μM sense strand targeting or scrambled ASO treated control and *C9orf72* iPSNs. Treatment as indicated on left, genotype and antibodies as indicated on top. (**F-T**) Quantification of Nup spots and volume. N = 5 control and 5 *C9orf72* iPSC lines, 50 NeuN+ nuclei per line/treatment. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. (U) Confocal imaging of 5 μM sense strand targeting or scrambled ASO treated control and *C9orf72* iPSNs immunostained for Ran. Treatment as indicated on left, genotype and antibodies as indicated on top. (V) Quantification of nuclear to cytoplasmic ratio of Ran. N = 3 control and 3 *C9orf72* iPSC lines, 30 cells per line. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. (W) Quantification of percent cell death following exposure to glutamate. N = 4 control and 4 *C9orf72* iPSC lines, 10 frames per well. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. Scale bar = 5 μm (**A-E**), 10 μm (U). * indicates significantly altered Nups, ** indicates significantly restored Nups.

**Figure 4:. POM121 overexpression restores the nuclear expression of specific Nups, localization of Ran GTPase and mitigates cellular toxicity.**
(**A-E**) Maximum intensity projections from SIM imaging of Nups in nuclei isolated from control and *C9orf72* iPSNs overexpressing GFP tagged Nup98, Nup133, or POM121. Overexpression as indicated on left, genotype and antibodies as indicated on top. (**F-T**) Quantification of Nup spots and volume. N = 4 control and 4 *C9orf72* iPSC lines, 50 GFP+ nuclei per line/overexpression. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. (U) Confocal imaging of control and *C9orf72* iPSNs overexpressing GFP tagged Nup98, Nup133, or POM121 immunostained for Ran. Overexpression as indicated on left, genotype and antibodies as indicated on top. (V) Quantification of nuclear to cytoplasmic ratio of Ran. N = 4 control and 4 *C9orf72* iPSC lines, at least 50 cells per line/overexpression. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. ** p < 0.01, **** p < 0.0001. (W) Quantification of percent cell death following exposure to glutamate. N = 4 control and 4 *C9orf72* iPSC lines, 10 frames per well. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. Scale bar = 5 μm (**A-E**), 10 μm (U). * indicates significantly altered Nups, ** indicates significantly restored Nups.

**Figure 5:. Reduction in POM121 in wildtype iPSNs recapitulates *C9orf72* mediated alterations in specific Nups and Ran GTPase and increases susceptibility to glutamate induced excitotoxicity.**
(**A-P**) Maximum intensity projections from SIM imaging (A) and quantification (**B-P**) of Nup spots and volume in nuclei isolated from wildtype iPSNs following knockdown of Nup133, POM121, NDC1, or GAPDH. Antibody used for Trim21 GFP mediated knockdown as indicated on left, antibodies as indicated on top. N = 3 wildtype iPSC lines, 50 GFP+ nuclei per line/knockdown. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (Q) Confocal imaging of control iPSNs following Trim21 GFP knockdown of Nup133, POM121, or GAPDH immunostained for Ran. Antibody used for Trim21 knockdown as indicated on left, antibodies as indicated on top. (R) Quantification of nuclear to cytoplasmic ratio of Ran. N = 3 wildtype iPSC lines, at least 50 cells per line/knockdown. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. (S) Quantification of percent cell death following exposure to glutamate. N = 3 control iPSC lines, 10 frames per well. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. (T) Confocal imaging of cell death in control and C9orf72 iPSNs as measured by propidium iodide (PI) incorporation. Antibody used for Trim Away as indicated on left, glutamate concentration and stain as indicated on top. Scale bar = 5 μm (A), 10 μm (Q), 100 μm (T). * indicates significantly altered Nups.

**Figure 6:. Loss of C9ORF72 protein or DPRs do not alter the nuclear expression of POM121**
(A) Maximum intensity projections from SIM imaging of Nups in nuclei isolated from control and *C9orf72*^−/− iPSNs. Genotype as indicated on left, antibodies as indicated on top. (B) Quantification of Nup spots and volume. N = 1 control and 1 *C9orf72* null line, differentiations conducted in triplicate, 50 NeuN+ nuclei per line/differentiation. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. (C) SIM imaging of POM121 in nuclei isolated from wildtype iPSNs overexpressing GFP tagged Poly(GA), Poly(GR), and Poly(PR) DPRs. Overexpression as indicated on left, antibodies and time points as indicated on top. (**D-E**) Quantification of percent total nuclear volume occupied by POM121 2 days (D) and 7 days (E) after overexpression of DPRs. N = 3 wildtype iPSC lines, 50 GFP+ nuclei per line/overexpression. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. Scale bar = 5 μm.

**Figure 7.. Expression of pathologic G₄C₂ repeat RNA initiates the nuclear reduction in POM121.**
(**A-B**) RT-qPCR for G₄C₂ repeat RNA (A) and MSD Elisa for Poly(GP) DPR levels (B) in *C9orf72* iPSNs following 5 days of treatment with G₄C₂ targeting ASO. (C) SIM imaging for POM121 in nuclei isolated from control and *C9orf72* iPSNs. Genotype as indicated on left, treatment as indicated on top. (D) Quantification of percent total nuclear volume occupied by POM121 following 5 days of treatment with G₄C₂ targeting ASO. N = 4 control and 4 *C9orf72* IPSC lines, 50 NeuN+ nuclei per line/treatment. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. (E) Maximum intensity projections from SIM imaging of POM121 in nuclei isolated from wildtype iPSNs overexpressing 36, 106, or 288 G₄C₂ RNA repeats. Time point as indicated on left, overexpression as indicated on top. (**F-I**) Quantification and histogram distributions of POM121 spots 2 days (**F-G**) and 7 days (**H-I**) after overexpression of G₄C₂ repeat RNA. N = 4 wildtype iPSC lines, 100 NeuN+ nuclei per line/overexpression. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. (J) Quantification of percent cell death following exposure to glutamate in iPSNs treated with ASOs for 5 days. N = 4 control and 4 *C9orf72* iPSC lines, 10 frames per well. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. (K) Quantification of percent cell death following exposure to glutamate in wildtype iPSNs overexpressing G₄C₂ repeat RNA. N = 4 control iPSC lines, 10 frames per well. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. Scale bar = 5 μm.

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G₄C₂ Repeat RNA Initiates a POM121-Mediated Reduction in Specific Nucleoporins in C9orf72 ALS/FTD

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G₄C₂ Repeat RNA Initiates a POM121-Mediated Reduction in Specific Nucleoporins in C9orf72 ALS/FTD

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