Activation of the immunoproteasome protects SH-SY5Y cells from the toxicity of rotenone

Abstract

This study investigated the expression and role of immunoproteasome (i-proteasome) in a cell model of Parkinson’s disease (PD). The cytotoxicity of rotenone was measured by CCK-8 assay. The i-proteasome β1i subunit PSMB9 was suppressed by a specific shRNA or transfected with an overexpression plasmid in the SH-SY5Y cells. Under the exposure to rotenone or not, the expression of constitutive proteasome β subunits, i-proteasome βi subunits, antigen presentation related proteins, α-syn and TH were detected by Western blot in PSMB9-silenced or -overexpressed cells, and the proteasomal activities were detected by fluorogenic peptide substrates. The location of i-proteasome βi subunits and α-syn were detected by immunofluorescence staining. The levels of ROS, GSH and MDA were measured by commercial kits. Cell apoptosis was detected by flow cytometry. Besides impairing the constitutive proteasomes, rotenone induced the expression of βi subunits of i-proteasome and antigen presentation related proteins such as TAP1, TAP2 and MHC-I. Silencing or overexpressing PSMB9 had no obvious effect on the levels of other subunits, but could regulate the chymotrypsin-like activity of 20S proteasome and the expression of TAP1, TAP2 and MHC-I. Three βi subunits (PSMB9, PSMB10, PSMB8) of i-proteasome were all co-localized with α-syn. PSMB9 knockdown aggravated accumulation of α-syn, degradation of TH, release of ROS, increased level of MDA, decreased level of GSH and eventually promoted apoptosis in SH-SY5Y cells after rotenone treatment, while over-expression of PSMB9 could attenuate these toxic effects of rotenone. I-proteasome is activated in SH-SY5Y cells treated with rotenone and may play a neuroprotective role.