A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons

Benedikt V Hölbling; Yashica Gupta; Paolo M Marchi; Magda L Atilano; Michael Flower; Enric Ureña; Rajkumar A Goulden; Hannah K Dobbs; Eszter Katona; Alla Mikheenko; Ashling Giblin; Ali Raza Awan; Chloe L Fisher-Ward; Niamh O'Brien; Deniz Vaizoglu; Liam Kempthorne; Katherine M Wilson; Lauren M Gittings; Mireia Carcolé; Marc-David Ruepp; Sarah Mizielinska; Linda Partridge; Pietro Fratta; Sarah J Tabrizi; Bhuvaneish T Selvaraj; Siddharthan Chandran; Emma Armstrong; Paul Whiting; Adrian M Isaacs

doi:10.1016/j.celrep.2025.115695

A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons

Benedikt V Hölbling, Yashica Gupta, Paolo M Marchi, Magda L Atilano, Michael Flower, Enric Ureña, Rajkumar A Goulden, Hannah K Dobbs, Eszter Katona, Alla Mikheenko, Ashling Giblin, Ali Raza Awan, Chloe L Fisher-Ward, Niamh O'Brien, Deniz Vaizoglu, Liam Kempthorne, Katherine M Wilson, Lauren M Gittings, Mireia Carcolé, Marc-David RueppSarah Mizielinska, Linda Partridge, Pietro Fratta, Sarah J Tabrizi, Bhuvaneish T Selvaraj, Siddharthan Chandran, Emma Armstrong, Paul Whiting, Adrian M Isaacs

Research output: Contribution to journal › Article › peer-review

Abstract

Repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation generates neurotoxic dipeptide repeat proteins (DPRs). To study endogenous DPRs, we inserted the minimal HiBiT luciferase reporter downstream of sense repeat derived DPRs polyGA or polyGP in C9orf72 patient iPSCs. We show these "DPReporter" lines sensitively and rapidly report DPR levels in lysed and live cells and optimize screening in iPSC neurons. Small-molecule screening showed the ERK1/2 activator periplocin dose dependently increases DPR levels. Consistent with this, ERK1/2 inhibition reduced DPR levels and prolonged survival in C9orf72 repeat expansion flies. CRISPR knockout screening of all human helicases revealed telomere-associated helicases modulate DPR expression, suggesting common regulation of telomeric and C9orf72 repeats. These DPReporter lines allow investigation of DPRs in their endogenous context and provide a template for studying endogenous RAN-translated proteins, at scale, in other repeat expansion disorders.

Original language	English
Article number	115695
Pages (from-to)	115695
Journal	Cell Reports
Volume	44
Issue number	5
Early online date	10 May 2025
DOIs	https://doi.org/10.1016/j.celrep.2025.115695
Publication status	Published - 27 May 2025

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Hölbling, B. V., Gupta, Y., Marchi, P. M., Atilano, M. L., Flower, M., Ureña, E., Goulden, R. A., Dobbs, H. K., Katona, E., Mikheenko, A., Giblin, A., Awan, A. R., Fisher-Ward, C. L., O'Brien, N., Vaizoglu, D., Kempthorne, L., Wilson, K. M., Gittings, L. M., Carcolé, M., ... Isaacs, A. M. (2025). A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons. Cell Reports, 44(5), 115695. Article 115695. https://doi.org/10.1016/j.celrep.2025.115695

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title = "A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons",

abstract = "Repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation generates neurotoxic dipeptide repeat proteins (DPRs). To study endogenous DPRs, we inserted the minimal HiBiT luciferase reporter downstream of sense repeat derived DPRs polyGA or polyGP in C9orf72 patient iPSCs. We show these {"}DPReporter{"} lines sensitively and rapidly report DPR levels in lysed and live cells and optimize screening in iPSC neurons. Small-molecule screening showed the ERK1/2 activator periplocin dose dependently increases DPR levels. Consistent with this, ERK1/2 inhibition reduced DPR levels and prolonged survival in C9orf72 repeat expansion flies. CRISPR knockout screening of all human helicases revealed telomere-associated helicases modulate DPR expression, suggesting common regulation of telomeric and C9orf72 repeats. These DPReporter lines allow investigation of DPRs in their endogenous context and provide a template for studying endogenous RAN-translated proteins, at scale, in other repeat expansion disorders.",

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Hölbling, BV, Gupta, Y, Marchi, PM, Atilano, ML, Flower, M, Ureña, E, Goulden, RA, Dobbs, HK, Katona, E, Mikheenko, A, Giblin, A, Awan, AR, Fisher-Ward, CL, O'Brien, N, Vaizoglu, D, Kempthorne, L, Wilson, KM, Gittings, LM, Carcolé, M, Ruepp, M-D , Mizielinska, S, Partridge, L, Fratta, P, Tabrizi, SJ, Selvaraj, BT, Chandran, S, Armstrong, E, Whiting, P & Isaacs, AM 2025, 'A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons', Cell Reports, vol. 44, no. 5, 115695, pp. 115695. https://doi.org/10.1016/j.celrep.2025.115695

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T1 - A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons

AU - Hölbling, Benedikt V

AU - Gupta, Yashica

AU - Marchi, Paolo M

AU - Atilano, Magda L

AU - Flower, Michael

AU - Ureña, Enric

AU - Goulden, Rajkumar A

AU - Dobbs, Hannah K

AU - Katona, Eszter

AU - Mikheenko, Alla

AU - Giblin, Ashling

AU - Awan, Ali Raza

AU - Fisher-Ward, Chloe L

AU - O'Brien, Niamh

AU - Vaizoglu, Deniz

AU - Kempthorne, Liam

AU - Wilson, Katherine M

AU - Gittings, Lauren M

AU - Carcolé, Mireia

AU - Ruepp, Marc-David

AU - Mizielinska, Sarah

AU - Partridge, Linda

AU - Fratta, Pietro

AU - Tabrizi, Sarah J

AU - Selvaraj, Bhuvaneish T

AU - Chandran, Siddharthan

AU - Armstrong, Emma

AU - Whiting, Paul

AU - Isaacs, Adrian M

PY - 2025/5/27

Y1 - 2025/5/27

N2 - Repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation generates neurotoxic dipeptide repeat proteins (DPRs). To study endogenous DPRs, we inserted the minimal HiBiT luciferase reporter downstream of sense repeat derived DPRs polyGA or polyGP in C9orf72 patient iPSCs. We show these "DPReporter" lines sensitively and rapidly report DPR levels in lysed and live cells and optimize screening in iPSC neurons. Small-molecule screening showed the ERK1/2 activator periplocin dose dependently increases DPR levels. Consistent with this, ERK1/2 inhibition reduced DPR levels and prolonged survival in C9orf72 repeat expansion flies. CRISPR knockout screening of all human helicases revealed telomere-associated helicases modulate DPR expression, suggesting common regulation of telomeric and C9orf72 repeats. These DPReporter lines allow investigation of DPRs in their endogenous context and provide a template for studying endogenous RAN-translated proteins, at scale, in other repeat expansion disorders.

AB - Repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation generates neurotoxic dipeptide repeat proteins (DPRs). To study endogenous DPRs, we inserted the minimal HiBiT luciferase reporter downstream of sense repeat derived DPRs polyGA or polyGP in C9orf72 patient iPSCs. We show these "DPReporter" lines sensitively and rapidly report DPR levels in lysed and live cells and optimize screening in iPSC neurons. Small-molecule screening showed the ERK1/2 activator periplocin dose dependently increases DPR levels. Consistent with this, ERK1/2 inhibition reduced DPR levels and prolonged survival in C9orf72 repeat expansion flies. CRISPR knockout screening of all human helicases revealed telomere-associated helicases modulate DPR expression, suggesting common regulation of telomeric and C9orf72 repeats. These DPReporter lines allow investigation of DPRs in their endogenous context and provide a template for studying endogenous RAN-translated proteins, at scale, in other repeat expansion disorders.

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U2 - 10.1016/j.celrep.2025.115695

DO - 10.1016/j.celrep.2025.115695

M3 - Article

C2 - 40349338

SN - 2211-1247

VL - 44

SP - 115695

JO - Cell Reports

JF - Cell Reports

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ER -

A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons

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