Protective effects of a natural product, curcumin, against amyloid β induced mitochondrial and synaptic toxicities in Alzheimer's disease

Abstract

The purpose of our study was to investigate the protective effects of a natural product-'curcumin'- in Alzheimer's disease (AD)-like neurons. Although much research has been done in AD, very little has been reported on the effects of curcumin on mitochondrial biogenesis, dynamics, function and synaptic activities. Therefore, the present study investigated the protective effects against amyloid β (Aβ) induced mitochondrial and synaptic toxicities. Using human neuroblastoma (SHSY5Y) cells, curcumin and Aβ, we studied the protective effects of curcumin against Aβ. Further, we also studied preventive (curcumin+Aβ) and intervention (Aβ+curcumin) effects of curcumin against Aβ in SHSY5Y cells. Using real time RT-PCR, immunoblotting and immunofluorescence analysis, we measured mRNA and protein levels of mitochondrial dynamics, mitochondrial biogenesis and synaptic genes. We also assessed mitochondrial function by measuring hydrogen peroxide, lipid peroxidation, cytochrome oxidase activity and mitochondrial ATP. Cell viability was studied using the MTT assay. Aβ was found to impair mitochondrial dynamics, reduce mitochondrial biogenesis and decrease synaptic activity and mitochondrial function. In contrast, curcumin enhanced mitochondrial fusion activity and reduced fission machinery, and increased biogenesis and synaptic proteins. Mitochondrial function and cell viability were elevated in curcumin treated cells. Interestingly, curcumin pre- and post-treated cells incubated with Aβ showed reduced mitochondrial dysfunction, and maintained cell viability and mitochondrial dynamics, mitochondrial biogenesis and synaptic activity. Further, the protective effects of curcumin were stronger in pretreated SHSY5Y cells than in post-treated cells, indicating that curcumin works better in prevention than treatment in AD-like neurons. Our findings suggest that curcumin is a promising drug molecule to treat AD patients.

Keywords: Aging; Alzheimer Disease; Antioxidants.

Figure 1

Experimental strategy of amyloid β (Aβ), curcumin, Aβ+curcumin and curcumin+Aβ treatments in human neuroblastoma (SHSY5Y) cells.

Figure 2

Immunoblotting analysis of human neuroblastoma (SHSY5Y) cells treated with amyloid β (Aβ), curcumin, Aβ+curcumin and curcumin+Aβ relative to untreated cells. (A) Representative immunoblot. (B–D) Quantitative densitometry analysis of mitochondrial dynamics, mitochondrial biogenesis and synaptic proteins. Cur, curcumin; Unt, untreated.

Figure 2

Immunoblotting analysis of human neuroblastoma (SHSY5Y) cells treated with amyloid β (Aβ), curcumin, Aβ+curcumin and curcumin+Aβ relative to untreated cells. (A) Representative immunoblot. (B–D) Quantitative densitometry analysis of mitochondrial dynamics, mitochondrial biogenesis and synaptic proteins. Cur, curcumin; Unt, untreated.

Figure 2

Immunoblotting analysis of human neuroblastoma (SHSY5Y) cells treated with amyloid β (Aβ), curcumin, Aβ+curcumin and curcumin+Aβ relative to untreated cells. (A) Representative immunoblot. (B–D) Quantitative densitometry analysis of mitochondrial dynamics, mitochondrial biogenesis and synaptic proteins. Cur, curcumin; Unt, untreated.

Figure 3

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

Figure 3

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

Figure 3

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

Figure 4

Mitochondrial functional parameters in control human neuroblastoma (SHSY5Y) cells, in amyloid β (Aβ) incubated SHSY5Y cells, in SHSY5Y cells treated with curcumin and in SHSY5Y cells incubated with Aβ and then treated with Aβ and in SHSY5Y cells treated with curcumin 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, curcumin, Aβ+cucumin and curcumin+Aβ and (2) Aβ incubated SHSY5Y cells were compared with Aβ+curcumin SHSY5Y cells and curcumin+Aβ treated cells. We performed statistical analysis using ANOVA following the Dunnett correction, for: (a) H₂O₂ production, (b) lipid peroxidation, (c) ATP levels, (d) cytochrome oxidase activity and (e) cell viability.