Pharmacological inhibition of OTULIN deubiquitinase or deletion of OTULIN gene decreases tau levels in human neuroblastoma cell line, SH-SY5Y. (IMAGE)
Caption
Pharmacological inhibition of OTULIN deubiquitinase or deletion of OTULIN gene decreases tau levels in human neuroblastoma cell line, SH-SY5Y. (A,B) Pharmacological inhibition of OTULIN with UC495 compound significantly decreases the levels of hyperphosphorylated and total tau, and OTULIN in SH-SY5Y
cells. Western blot quantifications show significantly reduced AT8, AT180 and PHF-1 positive tau as well as total tau upon inhibition of OTULIN deubiquitinase with UC495 compound in SH-SY5Y cells as compared to DMSO-treated control. Inhibiting the deubiquitinase activity of OTULIN with UC495 also decreases the OTULIN levels. (C–E) CRISPR-Cas9–mediated OTULIN KO in SH-SY5Y causes a complete loss of tau and an accumulation of polyubiquitinated proteins. To study the dynamics of protein degradation in SH-SY5Y WT (C) and OTULIN KO (D) cells, protein translation was inhibited with 50 μM cycloheximide (CHX) 1 h prior adding the proteostasis inhibitors, 10 or 20 μM MG132 (26S proteasome), 10 μM Lactacystin (20S proteasome), 100 nM Bafilomycin A1 (Baf A1, autophagy), 100 μM PD 150606 (calpain 1/2) either individually or all four inhibitors together for 4 h. None of the inhibitors stabilizes tau in the absence of OTULIN (D) or change the OTULIN and Tau levels in wild-type cells except with 20 μM MG132, which decreases tau levels (C, E). (F) OTULIN deficiency in SH-SY5Y downregulates the transcription of MAPT gene. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis shows the absence of MAPT mRNA in OTULIN KO compared to wild type SH-SY5Y. 18S ribosomal RNA (18s rRNA) was used as a housekeeping reference gene for normalizing qRT-PCR data (F).
Credit
Kiran Bhaskar
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