Treatment with a Ginkgo biloba extract, EGb 761, inhibits excitotoxicity in an animal model of spinocerebellar ataxia type 17

Figures & data

Figure 1 Protective effects of EGb 761 in glutamate-induced excitotoxicity of SH-SY5Y cells.

Notes: (A) Viability of SH-SY5Y neuroblastoma cells is measured after 24 hours of glutamate treatment (Glu, 100 mM). EGb 761 (5–20 μg/mL) is added in the presence of glutamate. MK-801 (10 μM) is an NMDA receptor antagonist, which is used as positive control for the glutamate-induced cell death. (B) Representative images of Fluo-4 AM (FL1) labeling of SH-SY5Y cells analyzed by flow cytometry. (C) Cells are harvested after glutamate treatment for 24 hours and immunoblotted with the anti-calpain 2 and anti-α-spectrin antibodies (left panel). Expression of calpain 2 is normalized for the internal control actin, and data (mean ± SEM, n=3) are presented as the relative intensity of calpain 2 (right panel). *P<0.05; **P<0.01.
Abbreviations: NMDA, N-methyl-d-aspartate; SBDPs, spectrin breakdown products; SEM, standard error of the mean; Ctrl, control.

Figure 2 Treatment with EGb 761 downregulates apoptosis markers induced by glutamate.

Notes: Western blot for cleaved caspase 3 (A), and cleaved PARP (B) after glutamate treatment of 24 hours (left panel). Relative intensities of cleaved caspase 3 and cleaved PARP are presented in the right panel (mean ± SEM, n=3). (C) Protein expression of Bcl-2 and Bax is detected after a treatment with glutamate for 12 hours. The relative ratio of Bcl-2 and Bax is normalized to the actin control. (D) Immunoblotting is performed on cytoplasmic extracts using an anti-cytochrome C antibody after a glutamate treatment for 12 hours. Actin is used as an internal control. *P<0.05; **P<0.01.
Abbreviations: PARP, poly(adenosine diphosphate-ribose) polymerase; SEM, standard error of the mean; Ctrl, control.

Figure 3 Inhibitory effects of EGb 761 on apoptosis in TBP/79Q-EGFP cells.

Notes: TBP expression is induced for 5 days using doxycycline, and lysates are analyzed for the expression of cleaved caspase 3 (A) and cleaved PARP (B) (top panels). Relative intensities of cleaved caspase 3 and cleaved PARP are presented in the lower panel (mean ± SEM, n=3). *P<0.05; **P<0.01.
Abbreviations: PARP, poly(adenosine diphosphate-ribose) polymerase; SEM, standard error of the mean; TBP, TATA box-binding protein; Ctrl, control; Dox, doxycycline; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4 Suppression of protein aggregation by EGb 761 in the TBP/79Q-EGFP cells.

Notes: (A) Representative images of protein aggregation (arrows) in TBP-expressing cells. Cells are induced for 5 days using doxycycline to express TBP/36Q-EGFP or TBP/79Q-EGFP. (B) After TBP expression, insoluble materials are resuspended in an SDS-containing buffer. Cell lysates (20 μg/μL) are bound to a cellulose acetate membrane and are subjected to anti-TFIID dot blot immunoassay. *P<0.05; **P<0.01.
Abbreviations: EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; TBP, TATA box-binding protein; TFIID, transcription factor II D; Dox, doxycycline.

Figure 5 Protective effects of EGb 761 in the SCA 17 transgenic mice (TG).

Notes: (A) Calbindin immunostaining reaction developed with DAB in the cerebellar vermis of WT and SCA 17 transgenic mice. (B) IHC staining with 1TBP18 in vermis of WT (arrows) and SCA 17 transgenic mice. The images in (B) represent the area shown by the red rectangles in (A). (C) The latency to fall is recorded every week (9–24 weeks) in the accelerating rotarod test. (D) The cerebellar extracts of mice treated in vivo are immunostained against SBDPs (left panel). Relative intensity of calpain-specific SBDPs is presented in the right panel (mean ± SEM, n=3). *P<0.05; **P<0.01.
Abbreviations: DAB, 3,3′-diaminobenzidine; IHC, immunohistochemistry; SBDPs, spectrin breakdown products; SCA 17, spinocerebellar ataxia type 17; SEM, standard error of the mean; TBP, TATA box-binding protein; WT, wild type.