Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS

Abstract

C9orf72 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) produced by unconventional translation of the C9orf72 repeat expansions cause neurodegeneration in cell culture and in animal models. We performed two unbiased screens in Saccharomyces cerevisiae and identified potent modifiers of DPR toxicity, including karyopherins and effectors of Ran-mediated nucleocytoplasmic transport, providing insight into potential disease mechanisms and therapeutic targets.

Figure 1

Yeast screens identify potent modifiers of C9orf72 DPR toxicity. a) Arginine-rich C9orf72 DPRs are toxic in yeast. Spotting assay demonstrates (GR)₅₀ and (PR)₅₀ constructs are toxic when expressed in yeast. Galactose was used to induce expression of each DPR construct and glucose was used to repress expression. Five-fold serial dilutions of yeast cells were spotted on glucose- or galactose-containing plates. b) (GR)₅₀ is less toxic than (PR)₅₀ and increasing the DPR length to 100 increased GR toxicity. c) Schematic of plasmid overexpression screen to identify genes that suppress or enhance (PR)₅₀ toxicity when overexpressed. d) Examples of overexpression suppressors of (PR)₅₀ toxicity include members of the karyopherin family of nuclear transport proteins. e) Schematic of yeast deletion screen to identify genes that suppress (PR)₅₀ toxicity when deleted. f) Examples of deletion suppressors of (PR)₅₀ toxicity, including gtr1Δ, a negative regulator of the Ran-GTPase cycle and nsr1Δ, a deletion of the yeast homolog of the human nucleolar protein nucleolin. g) A model depicting where the modifier genes from deletion and overexpression screens function. Genes colored blue suppressed toxicity when deleted. Genes colored red enhanced toxicity when overexpressed. Genes colored green suppressed toxicity when overexpressed. h) Upregulation of KPNA3 protects against (PR)₅₀ toxicity in rodent neurons. Upregulation of KPNA3 more than doubled the survival of the neurons expressing (PR)₅₀ compared to co-infection with GFP. Graph represents mean ± SEM, n = 6. ** represents p-value <0.01, by unpaired t-test. i) C9orf72-ALS patient-derived neurons show decreased nuclear localization of RCC1 (human homolog of yeast SRM1) compared to healthy control-derived neurons. j) Quantitation of nuclear vs. cytoplasmic fluorescence intensity for RCC1. Human induced neurons from 3 healthy control subjects and 2 C9orf72-ALS patients were compared. Graph represents mean ± SEM, n = 13 (healthy controls), n = 8 (C9orf72-ALS) ** represents P <0.01, by unpaired t-test.