Epigenetic Small Molecules Rescue Nucleocytoplasmic Transport and DNA Damage Phenotypes in C9ORF72 ALS/FTD

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport “biosensors”, composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype—persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects—resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.

Unraveling the impact of disrupted nucleocytoplasmic transport systems in C9orf72-associated ALS

“…Although most evidence points toward R-DPRs having the greatest effect on NTFs, there is also evidence for GA-induced NTF dysfunction. Similarly to R-DPRs, in cell models, GA50 can inhibit the nuclear import of multiple types of NLSs, which can be rescued by compounds that are epigenetic modifiers (Ramic et al, 2021 ), as well as inhibit XPO1-mediated nuclear export (Frottin et al, 2021 ; Ramic et al, 2021 ). GA-induced inhibition of NCT was linked to the cytoplasmic localization of GA aggregates (Khosravi et al, 2017 ; Frottin et al, 2021 ).…”

Regulating Phase Transition in Neurodegenerative Diseases by Nuclear Import Receptors

“…Phase 1 trials of another CRM1 inhibitor were recently initiated to explore the safety and positive benefits of CRM1 inhibition vs. the off-target effects in ALS patients to determine whether inhibiting nucleocytoplasmic export is adequate to prevent abnormal neuronal death in humans [ 269 ]. DPRs can disrupt multiple nucleocytoplasmic transport pathways [ 270 ]. In a new study using a cell-based phenotypic screen, several commercially available compound libraries containing 2714 compounds were evaluated to counteract the toxicity caused by PR-50 and its effect in disrupting the nucleocytoplasmic transport pathways [ 270 ].…”

“…DPRs can disrupt multiple nucleocytoplasmic transport pathways [ 270 ]. In a new study using a cell-based phenotypic screen, several commercially available compound libraries containing 2714 compounds were evaluated to counteract the toxicity caused by PR-50 and its effect in disrupting the nucleocytoplasmic transport pathways [ 270 ]. Several epigenetic protein inhibitors (such as HDAC inhibitors, EZH1/EZH2 inhibitors, HAT activators, and SIRT1 activators) were discovered as possible hits, and they improved cell viability and restored nucleocytoplasmic transport in PR-50-expressing cells in addition to a compound that is already in clinical trials for the treatment of ALS (Na-4-phenylbutyrate) [ 270 ].…”

Myospreader improves gene editing in skeletal muscle by myonuclear propagation

“…1B). Using GFP as a reporter of protein localization, we screened multiple NLS/NES combinations (Table S1)( 27 ) by establishing stable C2C12 myoblasts expressing each reporter and fusing them to naïve C2C12 myoblasts on micropatterned gelatin ( 28 ). Chimeric myotubes were then imaged to assess GFP signal in myonuclei (Fig.…”