Biology subjects

Gao, F.-B.

Publications and source records attributed to Gao, F.-B..

4 recordsLinked to original sources

bioRxiv2022

Translation of dipeptide repeat proteins in C9ORF72-ALS/FTD through unique and redundant AUG initiation codons

A hexanucleotide repeat expansion in the first intron of C9ORF72 is the most common monogenic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A hallmark of ALS/FTD pathology is the presence of dipeptide repeat (DPR) proteins, produced from both sense GGGGCC (poly-GA, poly-GP, poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) transcripts. Although initiation codons and regulatory factors have been identified for sense DPR translation, they remain mostly unknown for antisense DPRs. Here, we show that an AUG initiation codon is necessary for poly-PR synthesis, suggesting canonical AUG dependent translation. Remarkably, although an AUG located 194 base pairs (bp) upstream of the repeat is the main start codon for poly-PG synthesis, two other AUG codons (-212 bp, -113 bp) can also initiate translation, demonstrating a striking redundancy in start codon usage. eIF2D is required for CUG start codon-dependent poly-GA translation from the sense transcript in human motor neurons derived from induced pluripotent stem cells of C9ORF72 ALS/FTD patients, but AUG-dependent poly-PG or poly-PR synthesis does not require eIF2D, indicating that distinct translation initiation factors control DPR synthesis from sense and antisense transcripts. Our findings provide key molecular insights into DPR synthesis from the C9ORF72 locus, which may be broadly applicable to many other nucleotide-repeat expansion disorders.

molecular biology↗

bioRxiv2022

CRISPR/Cas9-Mediated Excision of ALS/FTD-Causing Hexanucleotide Repeat Expansion in C9ORF72 rescues major disease mechanisms in vivo and in vitro

A hexanucleotide repeat expansion (HRE) consisting of GGGGCC24+ in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are fatal neurodegenerative diseases with no current approved treatments that significantly slow disease progression or extend life expectancy. Several hypotheses have emerged to explain how this HRE causes neuronal death, including C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. In the present study we used a CRISPR/Cas9 gene-editing approach to remove the HRE from the C9ORF72 genomic locus, designing guide RNAs (gRNAs) flanking the HRE, and delivered Cas9 and gRNAs using adeno-associated virus serotype 9 (AAV9) vectors. Here, we demonstrate successful excision of the HRE in C9ORF72 in primary cortical neurons and brains of three mouse models containing the C9ORF72 expanded HRE (ranging from 500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, the major hallmarks of C9-ALS/FTD, making this an extremely attractive therapeutic approach to these diseases.

molecular biology↗

bioRxiv2020

Activated iPSC-microglia from C9orf72 ALS/FTD patients exhibit endosomal-lysosomal dysfunction

While motor and cortical neurons are affected in C9orf72 ALS/FTD, it remains still largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We generated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells differentiated into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G4C2 repeat RNA foci, exhibit reduced C9orf72 protein levels and generate dipeptide repeat proteins. Healthy control and C9orf72 iPSC-MG equivalently express microglial specific genes and display microglial functions including inflammatory cytokine release and phagocytosis of extracellular toxic cargos such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. Select C9orf72 iPSC-MG patient lines show inability to efficiently remove phagocytosed contents, suggesting dysfunction of the endosomal-lysosomal pathways. Finally, RNA sequencing revealed overall transcriptional changes in diseased microglia yet no significant differentially expressed microglial-enriched genes. These minimal differences in cellular, molecular and functional characteristics of microglial mono-cultures suggest that a diseased microenvironment is associated with microglial activation and subsequent regulation of neuronal dysfunction.

neuroscience↗

bioRxiv2020

Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

Toxic dipeptide repeat (DPR) proteins are produced from expanded G4C2 hexanucleotide repeats in the C9ORF72 gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of [≥] 20 repeats inhibit the ribosomes peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with the DPR proteins and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

biochemistry↗