Neuroprotective Effect of Chlorogenic Acid on Mitochondrial Dysfunction-Mediated Apoptotic Death of DA Neurons in a Parkinsonian Mouse Model

Abstract

Mitochondrial dysfunction and oxidative stress characterize major factors involved in the activation of complex processes corresponding to apoptosis-mediated neuronal senescence of dopaminergic neurons (DA) in Parkinson's disease (PD). Here, we evaluated the molecular mechanisms participating in the treatment of a 1-methyl-4-phenyl-1,2,3,6-tetrahydopyridine- (MPTP-) intoxicated PD mouse model in response to chlorogenic acid (CGA). The results indicate that CGA treatment significantly improved the motor coordination of the MPTP-intoxicated mice. CGA also alleviated the fall in activity of mitochondrial complexes I, IV, and V in accordance with ameliorating the level of superoxide dismutase and mitochondrial glutathione in the midbrain of MPTP-induced mice. CGA inhibited the activation of proapoptotic proteins including Bax and caspase-3, while elevating the expression of antiapoptotic protein like Bcl-2 consequently preventing the MPTP-mediated apoptotic cascade. The study also revealed the improved phosphorylation state of Akt, ERK1/2, and GSK3β which was downregulated as an effect of MPTP toxicity. Our findings signify that CGA may possess pharmacological properties and contribute to neuroprotection against MPTP induced toxicity in a PD mouse model associated with phosphorylation of GSK3β via activating Akt/ERK signalling in the mitochondrial intrinsic apoptotic pathway. Thus, CGA treatment may arise as a potential therapeutic candidate for mitochondrial-mediated apoptotic senescence of DA neurons in PD.

Figure 1

Mouse behavioral analysis. CGA mediated alteration in the neurobehavior of MPTP-injected mice. (a) Time on the rotarod test. (b) T-turn time in the pole test. (c) Traction score in the traction test. (d) Cataleptic test. Values are represented in the form of mean ± SEM (n = 10). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Results were studied using the one-way ANOVA and further using the Newman-Keuls test.

Figure 2

CGA mediated alterations on mitochondrial complexes I-III (a), IV (b), and V (c) of the electron transport chain in the midbrain of mice. Values are represented in the form of mean ± SEM (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Results were studied using the one-way ANOVA and further using the Newman-Keuls test.

Figure 3

Alterations in mitochondrial antioxidant defence in respective treatment groups. (a) Mitochondrial reduced glutathione level. (b) Mitochondrial superoxide dismutase level. Values are represented in the form of mean ± SEM (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Results were studied using the one-way ANOVA and further using the Newman-Keuls test.

Figure 4

(a–f) Relative expression of Bax, Cas-3, Bcl-2, p-Akt, p-ERK1/2, and p-GSK3β in SN of mice was studied using the Western blot technique and densitometry analysis of proteins. CGA suppressed MPTP-induced apoptosis via Akt, GSK3β, and ERK1/2 signalling pathways in parkinsonian mice. It has normalized the deregulated expression of Bax, Bcl-2, and Cas-3 in MPTP-injected mice (a, b). β-Actin protein served as an internal control. Values are represented in the form of mean ± SEM (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Results were studied using the one-way ANOVA and further using the Newman-Keuls test. Abbreviations: CGA: chlorogenic acid; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SEM: standard error of the mean.

Figure 5

Immunohistochemical staining to analyse the expression of p-Akt in SN of different experimental groups. CGA administration enhanced the expression of p-Akt in SN of parkinsonian mice (20x magnification after staining). Values are expressed as mean ± SEM (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Results were studied using the one-way ANOVA and further using the Newman-Keuls test.

Figure 6

Immunohistochemical staining to analyse the expression of p-GSK3β in SN of different experimental groups (a, b). Profound expression of p-GSK3β in the CGA-administered group compared to the MPTP-intoxicated PD mice (20x). Values are represented as mean ± SEM (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Results were studied using the one-way ANOVA and further by the Newman-Keuls test.

Figure 7

Immunohistochemical staining to analyse the expression of p-ERK1/2 in SN of different experimental groups. Upregulated expression of p-ERK1/2 due to CGA administration in parkinsonian mice (20x). Values are represented as mean ± SEM (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Results were studied using the one-way ANOVA and further by the Newman-Keuls test.

Figure 8

(a, b) Immunohistochemical staining to analyse the expression of TH-positive DA neurons in SN of different experimental groups. CGA protected the TH-positive DA neurons in MPTP-induced mice. (a) 10x and (b) 20x. TH immunoreactivity in SN of mice. Values are represented as mean ± SEM (n = 5). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Results were studied using the one-way ANOVA and further by the Newman-Keuls test.