CIB1 protects against MPTP-induced neurotoxicity through inhibiting ASK1

Abstract

Calcium and integrin binding protein 1 (CIB1) is a calcium-binding protein that was initially identified as a binding partner of platelet integrin α_IIb. Although CIB1 has been shown to interact with multiple proteins, its biological function in the brain remains unclear. Here, we show that CIB1 negatively regulates degeneration of dopaminergic neurons in a mouse model of Parkinson's disease using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Genetic deficiency of the CIB1 gene enhances MPTP-induced neurotoxicity in dopaminergic neurons in CIB1^-/- mice. Furthermore, RNAi-mediated depletion of CIB1 in primary dopaminergic neurons potentiated 1-methyl-4-phenyl pyrinidium (MPP⁺)-induced neuronal death. CIB1 physically associated with apoptosis signal-regulating kinase 1 (ASK1) and thereby inhibited the MPP⁺-induced stimulation of the ASK1-mediated signaling cascade. These findings suggest that CIB1 plays a protective role in MPTP/MPP⁺-induced neurotoxicity by blocking ASK1-mediated signaling.

Figure 1

CIB1 deficiency potentiates MPTP-induced loss of dopaminergic neurons. (A) Wild-type (WT) and CIB1^−/− mice (2~3-month-old group) were treated with MPTP (30 mg/kg every 24 h, five times) or saline by intraperitoneal injection. Immunohistochemical staining for TH was performed on coronal brain sections from WT and CIB1^−/− mice. Numbers of TH-positive neurons in the substantia nigra pars compacta were analyzed by stereological counting. Data are means ± SD (n = 5). *P < 0.05. (B,C) The representative images of TH-stained neurons in substantia nigra pars compacta (B) or striatum (C) of WT and CIB1^−/−mice were shown. Scale bars, 100 μm (B) and 250 μm (C).

Figure 2

CIB1 mitigates MPP⁺-induced neurotoxicity in dopaminergic neurons. (A) SH-SY5Y cells expressing either control (GFP) or CIB1 shRNA were incubated for 20 h in the absence or presence of 3 mM MPP⁺. Then, the cells were stained with red fluorescein (TMR red)-labeled TUNEL and analyzed by flow cytometry. The apoptosis was determined by TUNEL-positive population showing positively shifted TMR red signal. The percents of TUNEL-positive cells are indicated as apoptotic population. Representative data of apoptosis are shown in the right panel. The left graph represents mean ± SD calculated from triplicate experiments (n = 3). *P < 0.05. (B) Primary mesencephalic neurons in culture were transfected for 48 h with control (GFP) or CIB1 siRNA. The neurons were left untreated or treated with 200 μM MPP⁺ for 20 h, and then were subjected to immunocytochemistry with anti-TH antibody. The representative images of the stained cells are shown in the right panels (scale bar, 100 μm), and the viability of TH-positive neurons is shown in the left panel as a percentage of that for control siRNA-transfected untreated cells. Data are means ± SD of values from three independent experiments. *P < 0.05.

Figure 3

CIB1 inhibits the MPP⁺-induced stimulation of ASK1 in primary dopaminergic neurons. (A) Primary mesencephalic neurons were transfected for 48 h with either control (GFP) or CIB1 siRNA. The neurons were incubated in the absence or presence of 200 μM MPP⁺ for 2 h. Cell lysates were subjected to immunoprecipitation with anti-ASK1 antibody. The resulting precipitates were assayed for ASK1 activity using substrates GST-MKK6 (K82A). The lysates were also examined directly by immunoblot analysis with antibodies to CIB1 or ASK1. (B) SH-SY5Y cells were transfected for 48 h with the indicated combinations of expression vectors for HA-ASK1, Flag-CIB1, and Myc-JNK1. The cells were then lysed and subjected to immunoprecipitation with anti-Myc antibody, and the resulting precipitates were assayed for JNK1 activity.

Figure 4

CIB1 physically associates with ASK1 in mesencephalic dopaminergic neurons. (A) A schematic representation of ASK1 indicating the thioredoxin-binding region (gray box), the TRAF2-binding region (dotted box), and the kinase domain (hatched box) is shown in the upper panel. The indicated ³⁵S-labled ASK1 variants were produced by in vitro translation and incubated for 4 h at 4 °C with GST-CIB1 immobilized on glutathione-agarose beads. The bead-bound ³⁵S-labled proteins were eluted and detected by SDS-PAGE and autoradiography. The gel was also stained with Coomassie Brilliant Blue. A portion (25%) of the ³⁵S-labled protein input to the binding reaction was also directly subjected to SDS-PAGE and autoradiography. (B) Primary mesencephalic neurons in culture were incubated in the absence or presence of 200 μM MPP⁺ for 2 h, lysed, and subjected to immunoprecipitation (IP) with antibodies to ASK1 (α-ASK1) or with rabbit preimmune IgG (control). The resulting precipitates as well as cell lysates were subjected to immunoblot (IB) analysis with antibodies to CIB1 or to ASK1.

Figure 5

CIB1 inhibits the binding of TRAF2 to ASK1 and the phosphorylation of ASK1 on Thr⁸⁴⁵ in intact cells. (A) SH-SY5Y cells were transfected for 48 h with the indicated combinations of vectors encoding Flag-CIB1, HA-Trx1, and Myc-ASK1. Cell lysates were subjected to immunoprecipitation with anti-HA antibody. The resulting precipitates were subjected to immunoblot analysis with anti-Myc antibody. Cell lysates were also examined directly by immunoblot analysis with the indicated antibodies. (B,C,D) Primary mesencephalic neurons in culture were transfected with either control or CIB1 siRNA for 48 h. The neurons were left untreated or treated with 200 μM MPP⁺ for 2 h, lysed, and subjected to immunoprecipitation with antibody to ASK1 (B,C) or antibody to MKK7 (D). The resulting precipitates were immunoblotted with antibodies to ASK1. Cell lysates were also immunoblotted directly with antibodies to CIB1, to ASK1, or to MKK7. The levels of protein expression are determined by relative fold of band image intensity (the first lane, fold 1.0).

Figure 6

siRNA-mediated depletion of ASK1 abolishes the protective effect of CIB1 on MPP⁺-initiated neurotoxicity. Primary mesencephalic neurons in culture prepared from WT or CIB^−/− mice were transfected for 48 h with control (GFP) or ASK1 siRNA. The neurons were left untreated or treated with 200 μM MPP⁺ for 20 h, and then were subjected to immunostaining with anti-TH antibody. The representative images of the stained cells are shown in the bottom panels (scale bar, 100 μm), and the viability of TH-positive neurons is shown in the top panel as a percentage of that of control siRNA-transfected untreated cells. Data are means ± SD of values from three independent experiments. *P < 0.05.