A cell-penetrant peptide blocking C9ORF72 -repeat RNA nuclear export reduces the neurotoxic effects of dipeptide repeat proteins

Abstract

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72 -repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72 -linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.

Atypical Neuropsychiatric Presentation of FTD-ALS Caused by a Pathogenic Repeat Expansion in C9orf72: A Case Report

“… 34 In addition, molecules have been studied to reduce the neurotoxic effects of DPRs by blocking their export from the nucleus. 35 Loss of function effects have been less frequently studied, but new knockdown mouse models are now allowing researchers to further assess these effects. 36 In this model, C9orf72 loss of function has been shown to be associated with the development of FTD-like symptoms, 36 paving the way for deeper understanding of the effect of C9orf72 expansions in FTD-ALS and ultimately help develop new therapeutic approaches.…”

Activation of the Keap1/Nrf2 pathway suppresses mitochondrial dysfunction, oxidative stress, and motor phenotypes inC9orf72ALS/FTD models

“…Similarly, the progressive loss of motor function in pan-neuronal GR1000 flies was DMF treatment is beneficial in C9orf72-ALS/FTD patient-derived neurons Since we had observed elevated ROS in C9orf72 Drosophila models and found that DMF treatment provided phenotypic benefit, we wanted to test the translatable potential of DMF treatment on patient-relevant material. Fibroblasts from ALS patients carrying C9orf72 mutations and healthy age-matched controls (Table 2), previously reported and successfully differentiated (42), were reprogrammed into induced neural progenitor cells (iNPCs), then differentiated into induced neurons (iNeurons), as assessed by the expression of neuronal markers βIII-tubulin, MAP2, and NeuN (Fig S3A and B). Although these cultures did not show overt signs of excess toxicity or cell death (Fig S3C ), assessing the level of oxidative stress using MitoSOX, we found a modest increase in mitochondrial ROS in C9orf72 iNeurons (Fig 7A and B), in line with that observed in Drosophila.…”

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

“…A recent study has demonstrated that this is a viable route for C9orf72 -ALS therapy, using a cell-penetrant peptide that competes with the interaction between SRSF1 and NXF1, thereby blocking nuclear export of DPR-encoding transcripts and reducing DPR production (Castelli et al, 2023 ). This study demonstrated that the cell-permeable peptides used decreased DPR production in BAC transgenic mice expressing (G4C2)500, in addition to decreasing DPR production and rescuing toxicity in a C9orf72 -ALS patient-derived neuronal cell model and (G4C2)36-expressing Drosophila (Castelli et al, 2023 ). Thus, mRNA export factors can modulate DPR toxicity by disrupting nuclear export of (G4C2) containing mRNA, thus lowering DPR expression.…”