February 2023
Jian Zhang,  Yuan Zhao, Yidan Zhang, Ya Gao, Shuyue Li, Cui Chang, Xuan Gao, Jingru Zhao, Guofeng Yang

 

Highlights

• ALA improves cognitive function in ageing mice.

• TMT based LC-MS/MS is executed to identify the protein change of ALA in brain ageing.

• ALA upregulates the terminal bouton and lipid transporter activity in ageing mice.

• ALA downregulates apoptosis, and neuroinflammatory associated proteins in ageing mice.


Abstract

Alpha lipoic acid (ALA), a powerful antioxidant, has the potential to relieve age-related cognitive impairment and neurodegenerative disease. Clinical randomized controlled studies have demonstrated the cognitive improvement effects of lipoic acid in Alzheimer's disease (AD).

In the present study, we examined the effects of ALA on cognitive function in ageing mice and its protective mechanisms. Eighteen-month-old male C57BL6/J mice received ALA or normal saline for 2 months. The Morris water maze test revealed improved cognitive function in animals that received ALA. Furthermore, tandem Mass Tags (TMT) based liquid chromotography with mass spectrometry/mass spectrometry (LC-MS/MS) was established to identify the target proteins.

The results showed that 10 proteins were changed significantly. Gene Ontology (GO) analysis indicated that the upregulated proteins were enriched in terminal bouton, synaptic transmission and lipid transporter activity while the down-regulated proteins were involved in nuclear transcription factor-κB binding, apoptosis and mitogen-activated protein kinase binding. Based on the GO results, two upregulated proteins oxysterol-binding protein-related protein 10 (OSBPL10) and oligophrenin 1 (OPHN1), and one downregulated protein, CDK5 regulatory subunit-associated protein 3 (CDK5rap3), were validated through Western blotting. The results were consistent with the proteomic results. Modulation of synaptic transmission, lipid transporter activity and neuroinflammation appears to be the mechanisms of ALA in the aged brain.

 

Introduction

Brain ageing is a complex multifactorial process characterized by continuous and gradual loss of neuronal functions, the decline of which is a major feature of many neurological disorders such as Alzheimer's disease (AD) [1], [7]. The hippocampus, a brain region that processes spatial and episodic memory and learning, is sensitive to the deleterious effects of ageing at both morphological and molecular levels. With advancing age, synapses in different subfields of the hippocampus exhibit impaired long-term potentiation, an electrophysiological function associated with learning and memory [1], [3].

Alpha lipoic acid (ALA) is a sulfur-containing and both water-soluble and lipid-soluble coenzyme involved in the energy metabolism of carbohydrates, proteins and lipids [4], [13]. The neuroprotective action of alpha lipoic acid has been demonstrated in a number of cellular or animal models of Parkinson's disease (PD), AD and amyotrophic lateral sclerosis (ALS) due to its antioxidative and anti-inflammatory properties [8], [21]. To date, we have not been able to retrieve any randomized controlled trials or other types of alpha-lipoic acid in PD. With regard to AD, a recent pilot study reported that combination therapy with omega-3 fatty acids and ALA may delay functional and cognitive decline in mild to moderate AD patients [16]. Another open-label study by Fava et al. [5] evaluated the effect of alpha-lipoic acid on cognitive function in AD patients with and without diabetes. At the end of the study, MMSE scores showed significant improvement in patients with diabetes compared to patients without diabetes.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a mature protein recognition method that is widely used in proteomic mapping. To improve the sensitivity and specificity of the assay, tandem mass tags (TMT) labelling was performed before quantification of the proteome without bias [2]. The advantages of TMT labelling include high penetrability and robustness to changes in chromatographic reproducibility and sensitivity, which are essential for accurate proteome quantification.

In this study, we found that ALA treatment improved the cognition of ageing mice during the Morris water maze (MWM) test. Furthermore, TMT-based LC-MS/MS was established to identify the potential protective targets of ALA in the hippocampus during brain ageing.

Section snippets

Animals and experimental design
Ageing male C57BL/6 mice (17–18 months, 30–35 g) and young male C57BL/6 mice (2.5–3 months, 25–30 g) were purchased from Vital River Laboratory Animal Technology Co., ltd. China. All mice were provided a diet and water ad libitum under a 12 h light/dark cycle with humidity of 60 % ± 5 % and 22 ± 3 °C. Experiments were performed according to the regulations of laboratory animal management of the Ministry of Science and Technology of the People's Republic of China and were approved by the Ethics

MWM test

In the acquisition trial, significant differences in the path length and escape latency were found between the ageing vehicle mice and ALA-treated mice in the fourth and fifth trials (P < 0.05, Fig. 1A, B), and the swimming routes of the mice in the two groups are shown in Fig. 1C. In the probe trial, when the platform was removed, the number of times the mice in the ALA-treated ageing group crossed the platform site significantly increased compared with that in the ageing vehicle group (P

Discussion

The study demonstrated that 2 months of ALA treatment commencing in late middle age leads to better cognitive function in old age during the MWM tests. To identify the target protein of ALA, we established a quantitative protein expression profile in the ageing hippocampus after treatment with ALA using the TMT-labelled LC-MS/MS technique. Quantitative proteomic analysis showed that 5 proteins were upregulated and 5 proteins were downregulated. GO analysis indicated that the DEPs were

Credit authorship contribution statement

Jian Zhang: Writing – original draft, Investigation, Writing – review & editing. Yuan Zhao: Investigation. Yidan Zhang: Investigation. Ya Gao: Validation. Shuyue Li: Data curation. Cui Chang: Data curation. Xuan Gao: Supervision. Jingru Zhao: Writing – review & editing. Guofeng Yang: Conceptualization, Methodology, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by Hebei Natural Science Foundation (H2022206294), Department of Health of Hebei Province (Grant No. 20221058) and the National Natural Science Foundation of China (Grant No. 82001229).

Data availability statement

All data and materials that support the results or analyses presented are freely available upon request...