Early lethality and neuronal proteinopathy in mice expressing cytoplasm-targeted FUS that lacks the RNA recognition motif

Abstract

Mutations to the RNA binding protein, fused in sarcoma (FUS) occur in ~5% of familial ALS and FUS-positive cytoplasmic inclusions are commonly observed in these patients. Altered RNA metabolism is increasingly implicated in ALS, yet it is not understood how the specificity with which FUS interacts with RNA in the cytoplasm can affect its aggregation in vivo. To further understand this, we expressed, in mice, a form of FUS (FUS ΔRRMcyt) that lacked the RNA recognition motif (RRM), thought to impart specificity to FUS-RNA interactions, and carried an ALS-associated point mutation, R522G, retaining the protein in the cytoplasm. Here we report the phenotype and results of histological assessment of the brain of transgenic mice expressing this isoform of FUS. Results demonstrated that neuronal expression of FUS ΔRRMcyt caused early lethality often preceded by severe tremor. Large FUS-positive cytoplasmic inclusions were found in many brain neurons; however, neither neuronal loss nor neuroinflammatory response was observed. In conclusion, the extensive FUS proteinopathy and severe phenotype of these mice suggests that affecting the interactions of FUS with RNA in vivo may augment its aggregation in the neuronal cytoplasm and the severity of disease processes.

Behavioural impairments in mice of a novel FUS transgenic line recapitulate features of frontotemporal lobar degeneration

“…Total protein samples were prepared by homogenisation of dissected mouse tissues in 2× Laemmly loading buffer followed by incubation at 100°C for 5 minutes. Separation of proteins in sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and consequent semidry transfer on polyvinylidene difluoride membrane (GE Healthcare) were performed as described elsewhere . Human FUS protein was detected using rabbit polyclonal antibody 14080 (a kind gift from Don Cleveland) specific to the N‐terminal epitope of human FUS protein, secondary anti‐rabbit horseradish peroxidase‐conjugated antibodies (GE Healthcare) and WesternBright TM Sirius chemiluminescent detection system (Advansta).…”

“…Separation of proteins in sodium dodecyl sulphatepolyacrylamide gel electrophoresis and consequent semidry transfer on polyvinylidene difluoride membrane (GE Healthcare) were performed as described elsewhere. 10,11 Human FUS protein was detected using rabbit polyclonal antibody 14080 (a kind gift from Don Cleveland) specific to the N-terminal epitope of human FUS protein, secondary anti-rabbit horseradish peroxidase-conjugated antibodies (GE Healthcare) and…”

TDP-43/FUS in motor neuron disease: Complexity and challenges

“…Mice expressing FUS that lack RRM domain and carry a R522G mutation showed significant neuronal proteinopathy but no apparent neurodegeneration in brain or brainstem region. However, both mouse models displayed shorter lifespans (Robinson et al, 2015; Shelkovnikova et al, 2013a). …”

“…A number of transgenic FUS models have been established by independent research teams (Robinson et al, 2015; Shelkovnikova et al, 2013a). Human FUS mutants, such as R495X, H517Q, R521G, and R521C, were ectopically expressed into D. melanogaster , zebrafish or mouse models (Lanson et al, 2011).…”

Pathogenesis of FUS-associated ALS and FTD: insights from rodent models

“…No reduction in motor activity or observation of ALS phenotypic features.Non-progressive vacuolation of CA3 region at 8-10 weeks. No evidence of neurodegenerationNo significant enrichment of specific profiles or changes in expression of other ALS-FTD related genesUnderexpression of FUS mRNARobinson et al [67]MouseTransgenic, FUS gene including R522G mutation and lacking RNA recognition motifB6CBAF1/JLowered body weight, early lethality, pronounced tremor around two days before deathLarge cytoplasmic FUS-positive inclusions in cortex and brainstem. No evidence of neurodegenerationNot studied.Significant FUS overexpressionShelkovnikova et al [73]MouseTransgenic, using human aggregate prone FUS-variant lacking Nuclear localization signal and RNA binding motif (expressed at lower levels than endogenous FUS)Mixed C57BL/6-CBASevere motor dysfunction at ~3 months, death within 2 weeks of symptom onsetFUS-positive inclusions in lower motor-neuron cell bodies, some ubiquinated inclusions.…”