FUS/TLS undergoes calcium-mediated nuclear egress during excitotoxic stress and is required for Gria2 mRNA processing

Abstract

27Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to 28 amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence 29 indicates a role for neurodegenerative disease linked RNA-binding proteins (RBPs) in the cellular 30 stress response. However, the relationships between excitotoxicity, RBP function and pathology 31 have not been explored. Here, we found that excitotoxicity induced the translocation of select 32 ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by 33 excitotoxicity include TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in 34 sarcoma/translocated in liposarcoma (FUS/TLS). FUS translocation occurs through a calcium-35 dependent mechanism and coincides with striking alterations in nucleocytoplasmic transport.36 Further, glutamate-induced upregulation of Gria2 in neurons was dependent on FUS expression, 37 consistent with a functional role for FUS under excitotoxic stress. These findings reveal a link 38 between prominent factors in neurodegenerative disease, namely excitotoxicity, disease-39 associated RBPs and nucleocytoplasmic transport.40 41 on poly-ornithine (final concentration of 1.5 µg/mL; MilliporeSigma P4957) coated plates or 104 coverslips. Neuronal cultures were grown under standard culture conditions (37°C, 5% CO2/95% 105 air) fed every 3-4 days by adding half volumes of supplemented neurobasal media to each 106 well/dish, with additional half changes of media occurring every other feeding. Unless indicated, 107 during the first feeding (DIV 2 or 3) neuron cultures were also treated with a final concentration of 108 0.5-1µM Cytosine β-D-arabinofuranoside hydrochloride (MilliporeSigma C6645) to inhibit non-109 neuronal cell growth. Experiments were performed on day in vitro (DIV) 14-16. 110Primary motor neurons were isolated from embryonic day 12.5 murine spinal cords as 111 described 68 . Briefly, after dissociation in 0.1% trypsin (Worthington LS003707, Columbus, OH, 112 USA) at 37°C for 12 minutes, primary motor neurons were purified using a 6% Optiprep 113 (MilliporeSigma D1556) density gradient and plated on glass coverslips coated with 0.5g/L poly-

Nucleo–cytoplasmic transport defects and protein aggregates in neurodegeneration

“…At their C-terminus, FET proteins have a homologous PY-NLS motif interacting with β-Kaps TNPO1 or TNPO2, which are involved in nuclear import [119,120]. FUS was seen to translocate through a calcium-dependent mechanism, and NCT alterations result from its cytosolic translocation [121]. The majority of ALS-associated FUS mutations affect the C-terminal NLS, interfering with TNPO1 binding and consequently leading to FUS cytoplasmic accumulation [122].…”