Genetic and Pathological Assessment of hnRNPA1, hnRNPA2/B1, and hnRNPA3 in Familial and Sporadic Amyotrophic Lateral Sclerosis

Abstract

Background: Mutations in the genes encoding the heterogeneous nuclear ribonucleoproteins hnRNPA1 and hnRNPA2/B1 have been reported in a multisystem proteinopathy that includes amyotrophic lateral sclerosis (ALS) and inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia. Mutations were also described in the prion-like domain of hnRNPA1 in patients with classic ALS. Another hnRNP protein, hnRNPA3, has been found to be associated with the ALS/frontotemporal dementia protein C9orf72. Objective: To further assess their role in ALS, we examined these hnRNPs in spinal cord tissue from sporadic (SALS) and familial ALS (FALS) patients, including C9orf72 repeat expansion-positive patients, and controls. We also sought to determine the prevalence of HNRNPA1, HNRNPA2B1, and HNRNPA3 mutations in Australian ALS patients. Methods: Immunostaining was used to assess hnRNPs in ALS patient spinal cords. Mutation analysis of the HNRNPA1, HNRNPA2B1, and HNRNPA3 genes was performed in FALS and of their prion-like domains in SALS patients. Results: Immunostaining of spinal motor neurons of ALS patients with the C9orf72 repeat expansion showed significant mislocalisation of hnRNPA3, and no differences in hnRNPA1 or A2/B1 localisation, compared to controls. No novel or known mutations were identified in HNRNPA1, HNRNPA2B1, or HNRNPA3 in Australian ALS patients. Conclusions: hnRNPA3 pathology was identified in motor neurons of ALS patients with C9orf72 repeat expansions, implicating hnRNPA3 in the pathogenesis of C9orf72-linked ALS. hnRNPA3 warrants further investigation into the pathogenesis of ALS linked to C9orf72. This study also determined that HNRNP mutations are not a common cause of FALS and SALS in Australia.

Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3

“…We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA. We and others also found that hnRNPA3 is mislocalized from the nucleus to the cytoplasm specifically in hippocampal, cerebellar, and spinal motor neurons of C9orf72 patients [17,41]. Moreover, mislocalized hnRNPA3 colocalizes with poly-glycine-alanine (poly-GA) deposits [42].…”

Role of RNA Binding Proteins with prion-like domains in muscle and neuromuscular diseases

“…First, hnRNPA3 was found to bind to mutant C9ORF72 RNA in ALS and could mediate some of its toxic effects [146,147]. Furthermore, hnRNPA3 was also reported to be present in TDP43, p62 immunoreactive dipeptide repeat (DPR) inclusions in C9orf72 cases [148,149] further linking hnRNPA3 to C9orf72 ALS/FTD. Second, mutations in hnRNPA1 and hnRNPA2B1 have been identified in multisystem proteinopathy, a disorder combining IBM, FTD, ALS or Paget's disease of the bone (PDB) [20].…”

Genetic and immunopathological analysis of CHCHD10 in Australian amyotrophic lateral sclerosis and frontotemporal dementia and transgenic TDP-43 mice

“…Immunohistochemistry (IHC) analysis of CHCHD10 for visualisation of staining patterns and semi-quantification of protein levels in motor cortex and frontal cortex tissues was performed on spinal cord, motor cortex and frontal cortex tissue sections as previously described using rabbit polyclonal anti-CHCHD10 (1:400; Sigma-Aldrich, Missouri, USA) 38. To assess colocalisation between CHCHD10 and phosphorylated TDP-43 or a mitochondrial marker VDAC1, dual immunofluorescence (IF) was performed on spinal cord, motor cortex and frontal cortex tissues using primary anti-CHCHD10 and either mouse monoclonal anti-VDAC1 (1:500, BioLegend, California, USA), or mouse monoclonal anti-TDP-43 phosphorylated Ser409/410 (1:5000; Cosmo Bio, Japan) antibodies, followed by relevant Alexa Fluor conjugated secondary antibodies.…”

“…To quantify CHCHD10 protein expression in spinal cord tissues, the mean CHCHD10 fluorescence intensity from at least four motor neurons were obtained using methods described by Fifita et al 38. For frontal cortex tissues, the semi-quantification was performed by a researcher and each section was categorised into strong, moderate or weak based on staining level blindly.…”