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, 22 (6), 959-73

CR6-interacting Factor 1 Is a Key Regulator in Aβ-induced Mitochondrial Disruption and Pathogenesis of Alzheimer's Disease

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CR6-interacting Factor 1 Is a Key Regulator in Aβ-induced Mitochondrial Disruption and Pathogenesis of Alzheimer's Disease

J Byun et al. Cell Death Differ.

Abstract

Mitochondrial dysfunction, often characterized by massive fission and other morphological abnormalities, is a well-known risk factor for Alzheimer's disease (AD). One causative mechanism underlying AD-associated mitochondrial dysfunction is thought to be amyloid-β (Aβ), yet the pathways between Aβ and mitochondrial dysfunction remain elusive. In this study, we report that CR6-interacting factor 1 (Crif1), a mitochondrial inner membrane protein, is a key player in Aβ-induced mitochondrial dysfunction. Specifically, we found that Crif1 levels were downregulated in the pathological regions of Tg6799 mice brains, wherein overexpressed Aβ undergoes self-aggregation. Downregulation of Crif1 was similarly observed in human AD brains as well as in SH-SY5Y cells treated with Aβ. In addition, knockdown of Crif1, using RNA interference, induced mitochondrial dysfunction with phenotypes similar to those observed in Aβ-treated cells. Conversely, Crif1 overexpression prevented Aβ-induced mitochondrial dysfunction and cell death. Finally, we show that Aβ-induced downregulation of Crif1 is mediated by enhanced reactive oxygen species (ROS) and ROS-dependent sumoylation of the transcription factor specificity protein 1 (Sp1). These results identify the ROS-Sp1-Crif1 pathway to be a new mechanism underlying Aβ-induced mitochondrial dysfunction and suggest that ROS-mediated downregulation of Crif1 is a crucial event in AD pathology. We propose that Crif1 may serve as a novel therapeutic target in the treatment of AD.

Figures

Figure 1
Figure 1
Crif1 expression was decreased in the brains of mouse models of AD and AD patients. (a and b) WB analysis showed that Crif1 was decreased in the frontal cortex (not in the cerebellum) of 6-month-old Tg6799 mice (n=5 per group). Data are presented as mean percentage±S.E.M. **P<0.01, NS indicates no significant difference. (ce) Representative micrographs showing immunohistochemistry in the frontal cortex of 6-month-old Tg6799 mice (n=6 per group). (d and e) Both the intensity (d) and area fraction (e) of the Crif1 signaling were significantly reduced. The fluorescent signals were captured by fluorescence microscopy and analyzed by Image J software. Data are presented as mean percentage±S.E.M. **P<0.01. Scale bar represents 20 μm. (f and g) qPCR analysis (f) and WB analysis (g) of brains from AD patients (AD) and controls (normal) revealed downregulation of Crif1 mRNA and protein expression in AD patients (n=4 per group), *P<0.05; **P<0.01. (h) Nickel-enhanced diaminobenzidine/peroxidase reaction showed decreased Crif1 signal in AD patients. Scale bars represent 200 μm (hippocampus) and 20 μm (CA1 and CA3), respectively
Figure 2
Figure 2
Aβ reduced Crif1 levels in SH-SY5Y cells through transcriptional regulation. (a) Crif1 levels were significantly reduced by Aβ (5 μM) in a time-dependent manner. Data were obtained from at least four replicates for each group (N=4 experiments). Data are presented as mean±S.E.M. *P<0.05; **P<0.01 versus vehicle (dimethyl sulfoxide (DMSO))-treated cells. (b) Crif1 levels were significantly decreased by Aβ1-42 (24 h), but not the reversed form of Aβ1-42, in a dose-dependent manner. Data were obtained from at least four replicates for each group (N=4 experiments). Data are presented as mean±S.E.M. *P<0.05, **P<0.01. β-Actin is used as a loading control in a and b. (c) Confocal images showing reduced Crif1 signal in mitochondria of SH-SY5Y cells treated with 2.5 μM Aβ for 24 h. Scale bar represents 10 μm. Quantification of Crif1 signal was analyzed using the Image J program. Data are represented as mean±S.E.M. from three independent experiments. ***P<0.001. (d) Aβ-induced Crif1 reduction was not mediated by protein degradation pathway. MG132 (10 μM), 3-MA (2 mM), and bafilomycin (5 nM) were administered 1 h before Aβ treatment, and 24 h after incubation, WB was performed to measure Crif1 levels. β-Actin serves as a loading control. Representative images are shown. Data are represented as mean±S.E.M. from three independent experiments. **P<0.01 versus vehicle-treated cells; NS versus Aβ-treated cells. (e) qRT-PCR analysis showed that Aβ decreased Crif1 mRNA levels in SH-SY5Y cells without affecting mRNA levels of other mitochondrial proteins such as TOM20 and TIM50. Reduced levels of Crif1 after siRNA transfection were detected by qRT-PCR. Data are represented as the mean±S.E.M. from three independent experiments. *P<0.05, **P<0.01, versus vehicle-treated cells
Figure 3
Figure 3
Aβ-induced ROS decreased the transcription of Crif1 gene by facilitating sumoylation of Sp1. (a) Treatment with H2O2-reduced Crif1 levels. 10–750 μM of H2O2 were administered for 6 h into SH-SY5Y cells, and then WB was performed to measure Crif1 levels. GAPDH is used as a loading control. Representative images are shown. (b) Treatment with a ROS scavenger (NAC; 1 mM) rescued Aβ-induced Crif1 downregulation. β-Actin is used as a loading control. Representative images are shown. Data are represented as mean±S.E.M. from three independent experiments. **P<0.01 versus vehicle-treated cells; #P<0.05 versus Aβ-treated cells. (c) Inhibition of NADPH oxidase rescued Aβ-induced Crif1 reduction. Apo (apocynin; 10 μM) or DPI (10 μM) was administered to vehicle- or Aβ-treated cells, and then WB was performed. Representative images are shown. Data are represented as mean±S.E.M. from three independent experiments (N=3 experiments). **P<0.01 versus vehicle-treated cells. #P< 0.05, ##P<0.01 versus Aβ-treated cells. (d) EMSA analysis showed that Aβ (5 μM, 24 h treatment) decreased the binding of Sp1 on the promoter region of Crif1. Asterisk (*) represents the nonspecific band, whereas the black arrowhead (protein-DNA probe complex) represents specific bands. NE indicates ‘nuclear extracts', Comp indicates ‘competition probe', and free probe indicates ‘non-binding probe'. The intensity of the protein-probe complex was analyzed using Image J software. *P<0.05. Representative images are shown (N=2 experiments). (e, f) Co-immunoprecipitation (Co-IP) analysis showed that sumoylation of Sp1 by SUMO-1 was enhanced after treatment with Aβ. The left panel (e) shows the increased Sp1-Sumo1 interaction in Aβ-treated cells, and the right panel (f) shows the increased Sp1 sumoylation in Aβ-treated cells using an anti-Sumo1 antibody. The lower panel (‘Input') shows a WB of Sp1 and Sumo1, with β-actin as a loading control. Representative images are shown (N=3 experiments). (g) Sumoylation-deficient mutant form of Sp1 (Sp1 K16A) shows less reduction in Crif1 levels by Aβ compared with Sp1 wild-type (wt)-transfected cells. β-Actin as a loading control. Representative images are shown. Data are represented as mean±S.E.M. from five independent experiments (N=5 experiments). **P< 0.01.versus vehicle-treated Sp1 wt-transfected cells
Figure 4
Figure 4
Crif1 knockdown (KD) disrupted mitochondrial morphology and impaired mitochondrial function. (a) Massive mitochondrial fission and loss (indicated by arrows) occurred in the frontal cortex of 6-month-old Tg6799 mice, whereas healthy filamentous mitochondria (indicated by arrowheads) were observed in age-matched littermate controls. The right panels show figures under higher magnification. Scale bar represents 2 μm (left panels) and 1 μm (right panels). (b and c) Crif1 KD induced increased mitochondrial fission (b). Mito-DsRed-expressing SH-SY5Y cells were transfected with control siRNA or Crif1 siRNA, and then treated with vehicle or Aβ for 12 h. The images were obtained by a confocal microscope, and the right panels show figures under higher magnification. (c) Mitochondrial morphology was analyzed using the Image J program (200 cells for each group). Mitochondria in Crif1 KD cells were shorter (represented by the aspect ratio) and had a more circular shape (represented by the form factor) in comparison to control siRNA-transfected cells. Data are represented as the mean±S.E.M. from three independent experiments. *P<0.05, **P <0.01, ***P<0.001 versus vehicle-treated control siRNA-transfected cells. Scale bars represent 10 μm. (d) EM analysis revealed disrupted mitochondrial morphology in Crif1 KD cells similar to Aβ-treated cells. Arrowheads indicate intact and healthy mitochondria, whereas arrows point to disrupted mitochondria. Scale bar represents 1 μm. (e) The MMP was measured by JC-1 assay. Data presented as mean±S.E.M. of three experiments (N=3 experiments). *P<0.05, **P<0.01 versus vehicle-treated control siRNA-transfected cells. (f) ATP generation in Crif1 KD cells was determined by ATP-luciferase assay. Crif1 KD cells show impaired ATP production. Data presented as mean±S.E.M. from three experiments (N=3 experiments). *P<0.05, **P<0.01 versus vehicle-treated control siRNA-transfected cells. (g and h) ROS generation in Crif1 KD cells was determined by MitoSOX staining. (g) Left panel shows phase contrast imaging and right panel shows the fluorescence of MitoSOX dye representing production of mitochondrial superoxide. (h) Quantification data of g are presented here. Crif1 KD cells show massive ROS generation in mitochondria. Scale bar represents 50 μm. Data are represented as mean±S.E.M. from three to five independent experiments. *P<0.05, ***P<0.001 versus vehicle-treated control siRNA-transfected cells
Figure 5
Figure 5
Crif1 overexpression restored Aβ-induced mitochondrial dysfunction in SH-SY5Y cells. (a and b) Mock vector or Crif1 cDNA-transfected (Crif1 o/e) Mito-DsRed-expressed cells were treated with vehicle or 5 μM Aβ for 12 h. In small boxes, enlarged images were converted to 8-bit format to analyze mitochondrial morphology using the Image J program. Crif1 o/e recovered the increased mitochondrial fission by Aβ in terms of shape (form factor (b)) and length (aspect ratio (b)). Scale bar represents 10 μm. Data presented as mean±S.E.M. from three experiments (200 cells for each group). *P<0.05, ***P<0.001 versus vehicle-treated mock-transfected cells. #P<0.05, ##P<0.01 versus Aβ-treated mock-transfected cells. (c) The MMP was measured by JC-1 assay. Data presented as mean±S.E.M. of four experiments. ***P<0.001 versus Aβ-treated mock vector-transfected cells. (d) ATP generation in Crif1 o/e cells was determined by ATP-luciferase assay. Data presented as mean±S.E.M. from three experiments. ***P<0.001 versus Aβ-treated mock vector-transfected cells. (e and f) ROS generation in Crif1 o/e cells was determined by dichloro-dihydrofluoresceindiacetate (DCFDA; e) and MitoSOX (f) staining. Scale bar represents 10 μm (e) or 20 μm (f). Data are represented as the mean ±S.E.M. of five independent experiments. **P<0.01, ***P<0.001 versus vehicle-treated mock vector-transfected cells; ###P<0.001 versus Aβ-treated mock vector-transfected cells
Figure 6
Figure 6
Crif1 levels are important for Aβ-induced cell death in SH-SY5Y cells. (a and b) Crif1 KD induced cell death by MTT assay (a) and Calcein-AM assay (b). Cell viability assays showed that Crif1 KD enhanced cell death as much as Aβ-treated condition (5 μM, 24 h). *P<0.05, **P<0.01 versus vehicle-treated control siRNA-transfected cells (c and d) Increased Crif1 expression blocked Aβ-induced cell death. After cDNA transfection for 24 h, 5 μM Aβ was administered for 24 h, and then MTT (c) and Calcein-AM assay (d) were performed. *P<0.05 versus vehicle-treated mock vector-transfected cells. Data were obtained from at least five replicates per group (N=5 experiments). (e) To confirm cell death in Crif1 KD or Crif1 o/e cells, TUNEL assay was performed. Scale bar represents 20 μm. Data are shown as mean±S.E.M. of three independent experiments (n=200 each groups, N=3 experiments). **P<0.01 versus vehicle-treated mock vector-transfected cells; ##P<0.01 versus Aβ-treated mock vector-transfected cells; NS indicates no significant difference
Figure 7
Figure 7
Schematic diagram for Aβ-induced mitochondrial dysfunction via ROS-Sp1-Crif1 pathway. Based on our experimental data, Aβ-induced ROS facilitated the reduction of Crif1 mRNA levels by increasing the sumoylation of Sp1. Reduced Crif1 expression resulted in disruption of mitochondrial morphology and mitochondrial functions, which leads to neuronal cell death shown in AD brains

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