Aqua-soluble DDQ reduces the levels of Drp1 and Aβ and inhibits abnormal interactions between Aβ and Drp1 and protects Alzheimer's disease neurons from Aβ- and Drp1-induced mitochondrial and synaptic toxicities

Abstract

The purpose of our study was to develop a therapeutic target that can reduce Aβ and Drp1 levels, and also can inhibit abnormal interactions between Aβ and Drp1 in AD neurons. To achieve this objective, we designed various compounds and their 3-dimensional molecular structures were introduced into Aβ and Drp1 complex and identified their inhibitory properties against Aβ-Drp1 interaction. Among all, DDQ was selected for further investigation because of 1) its best docking score and 2) its binding capability at interacting sites of Drp1 and Aβ complex. We synthesized DDQ using retro-synthesis and analyzed its structure spectrally. Using biochemical, molecular biology, immunostaining and transmission electron microscopy (TEM) methods, we studied DDQ's beneficial effects in AD neurons. We measured the levels of Aβ and Drp1, Aβ and Drp1 interaction, mRNA and protein levels of mitochondrial dynamics, biogenesis and synaptic genes, mitochondrial function and cell viability and mitochondrial number in DDQ-treated and untreated AD neurons. Our qRT-PCR and immunoblotting analysis revealed that reduced levels of mitochondrial fission and increased fusion, biogenesis and synaptic genes in DDQ-treated AD neurons. Our immunoblotting and immunostaining analyses revealed that Aβ and Drp1 levels were reduced in DDQ-treated AD neurons. Interaction between Aβ and Drp1 is reduced in DDQ-treated AD neurons. Aβ42 levels were significantly reduced in DDQ-treated mutant APPSwe/Ind cells. Mitochondrial number is significantly reduced and mitochondrial length is significantly increased. Mitochondrial function and cell viability were maintained in AD neurons treated with DDQ. These observations indicate that DDQ reduces excessive mitochondrial fragmentation, enhances fusion, biogenesis and synaptic activity and reduces Aβ42 levels and protects AD neurons against Aβ-induced mitochondrial and synaptic toxicities.

Figure 1.

Interaction of designed drug molecules against Aβ and Drp1 Complex. (A) Interaction of Drp1 (PDB ID: 4h1u) and Aβ1–40 (PDBID: 1ba4) proteins. (B) Inhibition of Drp1 and Aβ40 peptide Interaction by DDQ. (C) Interaction of Drp1 and designed drug molecules. We designed 82 molecules based on existing mitochondrial division inhibiting drug molecules in AD and subjected to molecular docking studies. We prepared Aβ and Drp1 complex and introduced these molecular structures into this complex. Few of these molecule showed better docking score compared to existed drug molecules. Amongst DDQ is only involved in the prevention of Aβ-Drp1 interaction by interacting at the active site such as ser8 and Leu34 of Aβ and ASN16 Glu16 of Drp1. And also DDQ showed better binding score (-10.8462) than existed drug molecules. DDQ is readily stopping the Drp1 before forming the complex. DDQ exhibited binding interaction at Arg225 (C = O→O-P) and phenyl part of DDQ is also showing one arene cationic interaction at Arg225. Hence, among all designed molecules we selected only DDQ for our further steps.

Figure 2.

Schematic of the synthetic procedure used to develop DDQ.

Figure 3.

Overview strategy of DDQ treatments in cell cultures. As shown in Figure, we studied five different groups of cells: (1) untreated SHSY5Y cells; (2) SHSY5Y cells treated (incubated) with DDQ (250 nM) for 24 h; (3) SHSY5Y cells incubated with the Aβ1–42 peptide (20 µM final concentration) for 6 h; (4) SHSY5Y cells incubated with Aβ1–42 for 6 h, followed by DDQ treatment for 24 h, and (5) SHSY5Y cells treated with DDQ for 24 h, followed by Aβ1–42 incubation for 6 h.

Figure 4.

Immunoblotting analysis mitochondrial dynamics, biogenesis and synaptic proteins. (A) Representative Immunoblotting images (mitochondrial fission, fusion proteins and synaptic proteins) of DDQ, Aβ, Aβ+DDQ and DDQ + Aβ-treated and untreated SHSY-5Y cells. (B) Representative Immunoblotting images (mitochondrial biogenesis proteins) of DDQ, Aβ, Aβ+DDQ and DDQ + Aβ-treated and untreated SHSY-5Y cells. (C) Quantitative densitometry analysis of mitochondrial dynamics and synaptic proteins. (D) Quantitative densitometry analysis of mitochondrial biogenesis. Fission proteins levels were increased in cells treated with Aβ; and reduced in cells treated with DDQ, Aβ+DDQ and DDQ + Aβ-treated cells. Whereas mitochondrial fusion proteins Mfn1 and Mfn2 and synaptic proteins, synaptophysin and PSD95 were decreased in cells treated with Aβ, and enhanced in cells treated with DDQ, Aβ+DDQ and DDQ + Aβ-treated cells.

Figure 5.

Co-immunoprecipitation analysis of Drp1 and Aβ in SHSY5Y cells. (A) represents immunoprecipitation with the 6E10 antibody and immunoblotting with the 6E10 antibody, indicating that the specificity of 6E10 in our Co-IP analysis. In cells treated with DDQ + Aβ and Aβ+DDQ, 4 kDa Aβ levels were reduced relative cells treated Aβ alone. (B) represents Co-IP with Aβ antibody 6E10 and western blotting with Drp1 antibody, indicating that Drp1 interacts with 4 kDa Aβ. Reduced interaction between Aβ and Drp1 was found in cells pretreated with DDQ and then Aβ added. Reduced interaction was strong in DDQ + Aβ cells compared to cells treated with Aβ alone.

Figure 6.

Co-immunoprecipitation analysis of mutant APP_Swe/Ind cells treated with DDQ. We transfected mutant APP_Swe cDNA construct into mouse neuroblastoma (N2a) cells. After 24 h of transfection, cells were treated with DDQ (250nM) for 24 h. Harvested mutant APP_Swe/Ind cells treated and untreated with DDQ and prepared protein lysates and performed immunoprecipitation with Aβ (6E10) antibody and conducted immunoblotting analysis with 6E10 and Drp1 antibodies. Lanes 1 and 2 represents IP with 6E10 and western blot with 6E10 and lanes 3 and 4 represents IP with 6E10 and western blot with Drp1 antibody respectively. As shown in Figure, reduced levels of full-length APP and 4 kDa Aβ were found in lane 2 mutant APP_Swe/Ind cells treated with DDQ compared to lane 1 of mutant APP_Swe/Ind cells untreated with DDQ. Reduced levels of Drp1 were found in lane 4 of mutant APP_Swe cells treated with DDQ compared to lane 3 of mutant APP_Swe/Ind cells untreated with DDQ.

Figure 7.

Immunofluorescence analysis. Immunofluorescence analysis of human neuroblastoma (SHSY5Y) cells treated with Aβ, DDQ, Aβ+DDQ and DDQ + Aβ relative to untreated cells. (A) Representative immunofluorescence images of mitochondrial dynamic proteins and synaptic proteins. (B) Quantitative immunofluorescence analysis of mitochondrial dynamics and synaptic proteins.

Figure 8.

Double labeling Immunofluorescence analysis of Drp1 and Aβ. Double-labeling immunofluorescence analysis of Aβ (6E10 antibody) and Drp1 in SHSY5Y cells. The localization of Drp1 (green) and Aβ (red) and the colocalization of Drp1 and Aβ (yellow, merged) at 60× the original magnification. Top panel, represents Aβ-treated cells, middle panel shows Aβ+DDQ-treated cells and the bottom panel shows DDQ+Aβ-treated cells. As shown in Figure 7, increased levels of Drp1 and intra-neuronal Aβ (full-length APP) and colocalization of Drp1 and Aβ in top panel, where as in the middle panel Aβ+DDQ cells, reduced Drp1 and Aβ and also reduced colocalization and in the bottom panel Drp1 and Aβ levels markedly reduced compared to top panel and also colocalization of Drp1 and Aβ. These findings strongly suggest that DDQ 1) reduces Drp1 and Aβ levels and also 2) inhibit the interaction of Drp1 and Aβ in SHSY5Y cells. These findings agree with our Co-IP findings.

Figure 9.

Sandwich ELISA analysis of Aβ40 and 42 in mutant APP_Swe/Ind cells treated and untreated with DDQ. We performed sandwich ELISA using protein lysates mutant APP cells treated and untreated with DDQ. (A) represents Aβ42 and (B) represents Aβ40. Significantly reduced levels of Aβ42 in mutant APP_Swe/Ind cells treated with DDQ compared to mutant APP_Swe/Ind cells untreated with DDQ. On the contrary, Aβ40 levels were significantly increased in mutant APP_Swe/Ind cells treated with DDQ relative to mutant APP_Swe/Ind cells untreated with DDQ.

Figure 10.

Electron microscopy of SH-SY5Y cells. We quantified mitochondrial architectures within the cell in all 5 groups to identify mitochondrial number and morphology. Average number of mitochondria per cell is shown in graphs. Error bars indicate the standard deviation. Mitochondrial number is significantly decreased in DDQ-treated SH-SY5Y cells, relative to untreated cells. On the contrary, mitochondrial number is significantly increased Aβ-treated SH-SY5Y cells. DDQ-pre and post-treated cells in the presence of Aβ showed reduced mitochondrial number compared to cells treated with Aβ alone. Mitochondrial length was measured for all groups of cells. Mitochondrial length was significantly reduced Aβ-treated SH-SY5Y cells. DDQ-pre and post-treated cells in the presence of Aβ showed increased mitochondrial length compared to cells treated with Aβ alone.

Figure 11.

Mitochondrial function. Mitochondrial functional parameters in control human neuroblastoma (SHSY5Y) cells, in amyloid β (Aβ) incubated SHSY5Y cells, in SHSY5Y cells treated with DDQ and in SHSY5Y cells incubated with Aβ and then treated with DDQ and in SHSY5Y cells treated with DDQ and then incubated with Aβ (n = 4). We analyzed mitochondrial functional data in two ways: (1) the control SHSY5Y cells were compared with the SHSY5Y cells treated with Aβ, DDQ, Aβ+DDQ and DDQ + Aβ and (2) Aβ-incubated SHSY5Y cells were compared with Aβ+DDQ SHSY5Y cells and DDQ + Aβ-treated cells. We performed statistical analysis using ANOVA following the Dunnett correction, for: (a) H₂O₂ production, (b) lipid peroxidation, (c) cytochrome oxidase activity, (d) ATP levels, and (e) GTPase-Drp1 activity.

Figure 12.

Cell viability analysis. Cell viability of human neuroblastoma (SHSY5Y) cells, while treated with DDQ, Aβ, Aβ+DDQ and DDQ + Aβ relative to untreated cells. Cell viability of pre-treated DDQ and post treated DDQ in Aβ-incubated SHSY5Y cells relative to Aβ-treated cells.