Abstract
SummaryThrough the generation of humanized FUS mice expressing full-length human FUS, we identify that when expressed at near endogenous murine FUS levels, both wild-type and ALS-causing and frontotemporal dementia (FTD)-causing mutations complement the essential function(s) of murine FUS. Replacement of murine FUS with mutant, but not wild-type, human FUS causes stress-mediated induction of chaperones, decreased expression of ion channels and transporters essential for synaptic function, and reduced synaptic activity without loss of nuclear FUS or its cytoplasmic aggregation. Most strikingly, accumulation of mutant human FUS is shown to activate an integrated stress response and to inhibit local, intra-axonal protein synthesis in hippocampal neurons and sciatic nerves. Collectively, our evidence demonstrates that human ALS/FTD-linked mutations in FUS induce a gain of toxicity that includes stress-mediated suppression in intra-axonal translation, synaptic dysfunction, and progressive age-dependent motor and cognitive disease without cytoplasmic aggregation, altered nuclear localization, or aberrant splicing of FUS-bound pre-mRNAs.Video
Highlights
The 526-amino-acid FUS protein includes a glycine-rich, lowcomplexity, prion-like domain and a C-terminal, non-classical PY nuclear localization signal (PY-NLS) in which most of the Amyotrophic Lateral Sclerosis (ALS)/frontotemporal lobar degeneration (FTLD)-linked mutations are clustered (Da Cruz and Cleveland, 2011)
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder leading to paralysis from death of motor neurons
FUS is present in the pathological inclusions of patients with FUS-mediated ALS and most of frontotemporal dementia (FTD) instances without TDP-43 or Tau-containing aggregates, accounting for about 10% of the frontotemporal lobar degeneration (FTLD) cases, known as FTLD-FUS (Mackenzie et al, 2010)
Summary
METHOD DETAILSImmunofluorescence Mice were perfused intracardially and fixed with 4% paraformaldehyde in 0.1 M Sorenson’s phosphate buffer, pH 7.2, the entire spinal cord was dissected, post-fixed for 2 hours in the same fixative and transferred in a 30% sucrose phosphate buffer for at least 2 days. The lumbar spinal cord or brain was embedded in OCT compound (Sakura) and snap frozen in isopentane (2-methylbutane) cooled at À40C on dry ice. Floating lumbar spinal cord or brain cryosections (30mm or 35mm, respectively) were incubated in a blocking solution containing PBS1x, 0.5% Tween-20, 1.5% BSA for 1.5 hours at room temperature and in PBS1x, 0.3% Triton X-100 overnight at room temperature with the primary antibodies (listed in antibodies section). Primary antibodies were washed with PBS1x and detected using donkey anti-rabbit or anti-mouse FITC or Cy3 (1:500) coupled secondary antibodies (Jackson ImmunoResearch). Analysis was performed on a Nikon Eclipse laser scanning confocal microscope. Fluorescence intensity from unsaturated images captured with identical confocal settings (minimum of four spinal sections were imaged per animal) was quantified using NIS elements software (Nikon)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
