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ObjectiveSince the first report of CHCHD10 gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia.MethodsWhole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10. CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology.ResultsNo causal, novel or disease-associated variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease.ConclusionsGenetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.

Background: Ongoing disease gene discoveries continue to drive our understanding of the molecular and cellular mechanisms underlying ALS. Causative genes from 60% of ALS families have been identified using modern genetic techniques, but the causal gene defect is yet to be identified in the remaining 40% of families. These remaining families often do not follow true Mendelian inheritance patterns and are challenging to solve using traditional genetic analysis alone. In vitro and in vivo studies have become critical in assessing and validating these ALS candidate genes.Objectives: In this study, we aim to develop and validate the utility of an in vitro functional pipeline for the discovery and validation of novel ALS candidate genes.Methods: A panel of cell based-assays were applied to candidate genes to examine the presence/absence of known ALS pathologies in cell lines as well as human autopsy tissues. These include immunofluorescence, flow cytometry and western blotting to study toxicity, neuronal inclusion formation, interaction with TDP-43, aberrant protein degradation and accumulation in detergent-insoluble cellular fractions. Immunohistochemistry and immunofluorescence were also used to examine if candidates were present in neuronal inclusions from ALS patient spinal cord tissues.Results: The in vitro pipeline was applied to five candidate genes from an ALS family that is negative for known ALS gene mutations. Two candidates were prioritized as top candidates based on their capacity to induce known ALS cellular pathologies. In transfected cells, the variants in these two genes caused a significantly higher toxicity than wild type, formed detergent insoluble inclusions and was able to co-aggregate with TDP-43 in neuronal cells. The variants have also led to protein degradation defects. One of the candidates also co-localised with TDP-43-positive neuronal inclusions in sporadic ALS patient post-mortem tissues, a signature pathology of ALS.Discussion and conclusions: We have demonstrated the utility of a functional prioritization pipeline and successfully prioritized two novel candidate ALS genes. These genes, and its associated pathways, will be further investigated through the development of animal models to establish if there is support for its role in ALS. New ALS genes offer fresh diagnostic and therapeutic targets and tools for the generation of novel animal models to better understand disease biology and offer preclinical testing of candidate treatments for ALS in the future.

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.

BackgroundSplicing factor proline and glutamine rich (SFPQ, also known as polypyrimidine tract-binding protein-associated-splicing factor, PSF) is a RNA-DNA binding protein with roles in key cellular pathways such as DNA transcription and repair, RNA processing and paraspeckle formation. Dysregulation of SFPQ is emerging as a common pathological feature of multiple neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Increased retention of SFPQ intron nine and nuclear loss of the protein have been linked to multiple genetic subtypes of ALS. Consequently, SFPQ dysregulation has been hypothesised to be a common pathological feature of this highly heterogeneous disease.MethodsThis study provides a comprehensive assessment of SFPQ pathology in large ALS patient cohorts. SFPQ gene expression and intron nine retention were examined in multiple neuroanatomical regions and blood from ALS patients and control individuals using RNA sequencing (RNA-Seq) and quantitative PCR (RT-qPCR). SFPQ protein levels were assessed by immunoblotting of patient and control motor cortex and SFPQ expression pattern was examined by immunofluorescent staining of patient and control spinal cord sections. Finally, whole-genome sequencing data from a large cohort of sporadic ALS patients was analysed for genetic variation in SFPQ.ResultsSFPQ intron nine retention was significantly increased in ALS patient motor cortex. Total SFPQ mRNA expression was significantly downregulated in ALS patient motor cortex but not ALS patient blood, indicating tissue specific SFPQ dysregulation. At the protein level, nuclear expression of SFPQ in both control and patient spinal motor neurons was highly variable and nuclear depletion of SFPQ was not a consistent feature in our ALS cohort. However, we did observe SFPQ-positive cytoplasmic ubiquitinated protein aggregates in ALS spinal motor neurons. In addition, our genetic screen of ALS patients identified two novel, and two rare sequence variants in SFPQ not previously reported in ALS.ConclusionsThis study shows that dysregulation of SFPQ is a feature of ALS patient central nervous system tissue. These findings confirm SFPQ pathology as a feature of ALS and indicate that investigations into the functional consequences of this pathology will provide insight into the biology of ALS.