Splicing factor proline and glutamine rich intron retention, reduced expression and aggregate formation are pathological features of amyotrophic lateral sclerosis

Abstract

Aim Splicing factor proline and glutamine rich (SFPQ) is an RNA–DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case–control cohorts. Methods We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA‐sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. Results At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ‐positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. Conclusions Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.

Prematurely terminated intron-retaining mRNAs invade axons in SFPQ null-driven neurodegeneration and are a hallmark of ALS

“…In light of our findings, we propose that perturbed control of intron retention in ALS includes the formation of pathogenic PreT-IR transcripts induced by loss of SFPQ function in both the nucleus and neurites, and that this misregulation impacts patients’ neurites most severely. Supporting this model, we found that the intron retention signature shared across ALS studies is also present in zebrafish null neurons 18 , 19 , 33 , 34 . Loss of SFPQ function likely perturbs the splicing of SFPQ-binding introns.…”

“…An emerging hallmark of ALS/FTLD, increased retention of specific introns in spliced mRNAs, has been observed across a range of genetic backgrounds, in cellular and animal models and patient tissue. Key affected introns are common across multiple datasets 18 , 19 , 33 , 34 . A functional connection between SFPQ misregulation and aberrant intron retention in ALS/FTLD has not yet been explored.…”

“…Depletion of SFPQ protein has been shown to cause mis-splicing, accompanied by tauopathy and an FTLD-like phenotype 6 , 7 , 21 . Missense mutations within sfpq found in a small number of ALS patients have also been shown to cause neurodegeneration ALS 4 , 18 . Studies across sporadic and familial cases have established SFPQ misregulation as a hallmark of ALS/FTLD 5 .…”

Familial ALS-associated SFPQ variants promote the formation of SFPQ cytoplasmic aggregates in primary neurons

“…Mislocalization and cytoplasmic aggregation of nuclear SFPQ are associated with the pathogenesis of ALS [4][5][6]. We have previously demonstrated that the application of ZnCl 2 enhances the cytoplasmic accumulation and aggregation of SFPQ when overexpressed in cultured neurons [12].…”

“…These RBPs are best exemplified by trans-activation response element DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS), both of which predominantly function in the nucleus under normal conditions but are mislocalized and aggregated in the disease state [2,3]. Recent evidence has also demonstrated abnormal cytoplasmic accumulation and loss of the nuclear pool of an RBP, splicing factor prolineand glutamine-rich (SFPQ) protein, in ALS [4][5][6]. However, the precise molecular mechanisms that underpin these pathological changes, and their effects on neuronal functions are not well understood.…”