Abstract
Aberrant nucleocytoplasmic localization of proteins has been implicated in many neurodegenerative diseases. Evidence suggests that cytoplasmic mislocalization of nuclear proteins such as transactive response DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS) may be associated with neurotoxicity in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. This study investigated the changes in nucleocytoplasmic distributions of the proteome and transcriptome in an in vitro model of ALS. After subcellular fractionation of motor neuron-like cell lines expressing wild-type or G93A mutant hSOD1, quantitative mass spectrometry and next-generation RNA sequencing (RNA-seq) were performed for the nuclear and cytoplasmic compartments. A subset of the results was validated via immunoblotting. A total of 1,925 proteins were identified in either the nuclear or cytoplasmic fractions, and 32% of these proteins were quantified in both fractions. The nucleocytoplasmic distribution of 37 proteins was significantly changed in mutant cells with nuclear and cytoplasmic shifts in 13 and 24 proteins, respectively (p<0.05). The proteins shifted towards the nucleus were enriched regarding pathways of RNA transport and processing (Dhx9, Fmr1, Srsf3, Srsf6, Tra2b), whereas protein folding (Cct5, Cct7, Cct8), aminoacyl-tRNA biosynthesis (Farsb, Nars, Txnrd1), synaptic vesicle cycle (Cltc, Nsf), Wnt signalling (Cltc, Plcb3, Plec, Psmd3, Ruvbl1) and Hippo signalling (Camk2d, Plcb3, Ruvbl1) pathways were over-represented in the proteins shifted to the cytoplasm. A weak correlation between the changes in protein and mRNA levels was found only in the nucleus, where mRNA was relatively abundant in mutant cells. This study provides a comprehensive dataset of the nucleocytoplasmic distribution of the proteome and transcriptome in an in vitro model of ALS. An integrated analysis of the nucleocytoplasmic distribution of the proteome and transcriptome demonstrated multiple candidate pathways including RNA processing/transport and protein synthesis and folding that may be relevant to the pathomechanism of ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive fatal neurodegenerative disease that affects motor neurons [1]
Based on quantitative mass spectrometry and RNA sequencing (RNA-seq), this study provides a comprehensive unbiased dataset of the nucleocytoplasmic distribution of the proteome and transcriptome in an in vitro model of ALS
The pathway analysis revealed that the proteins shifted towards the nucleus were associated with RNA transport and processing (Dhx9, Fmr1, Srsf3, Srsf6, Tra2b) and that the proteins shifted towards the cytoplasm were associated with protein folding (Cct5, Cct7, Cct8), aminoacyl-tRNA biosynthesis (Farsb, Nars, Txnrd1), synaptic vesicle cycle (Cltc, Nsf), Wnt signalling (Cltc, Plcb3, Plec, Psmd3, Ruvbl1) and Hippo signalling (Camk2d, Plcb3, Ruvbl1) pathways
Summary
Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive fatal neurodegenerative disease that affects motor neurons [1]. It has been increasingly recognized that protein mislocalization may play an important role in ALS/FTLD pathology. Recent studies have suggested that the C9orf hexanucleotide repeat expansion (HRE), the most common causative mutation in familial ALS, exerts toxicity by disrupting nucleocytoplasmic transport [6,7,8]. Several of the latest studies using different HRE disease models have identified the disease-modifying genes that encode elements of nuclear pore complex and nuclear RNA export/nuclear protein import machinery [6,7,8]. Proteins associated with nucleocytoplasmic transport such as RanGAP1 were found to be mislocalized in autopsied brain tissues and in induced pluripotent stem cells derived from ALS patients with the C9orf mutation [7]
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