α-Lipoic acid improves abnormal behavior by mitigation of oxidative stress, inflammation, ferroptosis, and tauopathy in P301S Tau transgenic mice

Abstract

Alzheimer's disease (AD) is the most common neurodegenerative disease and is characterized by neurofibrillary tangles (NFTs) composed of Tau protein. α-Lipoic acid (LA) has been found to stabilize the cognitive function of AD patients, and animal study findings have confirmed its anti-amyloidogenic properties. However, the underlying mechanisms remain unclear, especially with respect to the ability of LA to control Tau pathology and neuronal damage. Here, we found that LA supplementation effectively inhibited the hyperphosphorylation of Tau at several AD-related sites, accompanied by reduced cognitive decline in P301S Tau transgenic mice. Furthermore, we found that LA not only inhibited the activity of calpain1, which has been associated with tauopathy development and neurodegeneration via modulating the activity of several kinases, but also significantly decreased the calcium content of brain tissue in LA-treated mice. Next, we screened for various modes of neural cell death in the brain tissue of LA-treated mice. We found that caspase-dependent apoptosis was potently inhibited, whereas autophagy did not show significant changes after LA supplementation. Interestingly, Tau-induced iron overload, lipid peroxidation, and inflammation, which are involved in ferroptosis, were significantly blocked by LA administration. These results provide compelling evidence that LA plays a role in inhibiting Tau hyperphosphorylation and neuronal loss, including ferroptosis, through several pathways, suggesting that LA may be a potential therapy for tauopathies.

Keywords: Alzheimer's disease; Ferroptosis; Oxidative stress; Tau; α-Lipoic acid.

Fig. 1

LA mediated the distribution of iron in the brains of P301S mice. (A) Iron staining and the quantitative analysis of 9-month-old P301S mouse brains. The iron-staining results were processed by ImageJ to allow iron deposits to be easily observed as red granules. (B, C) Detection of iron content in the brain and serum by atomic absorption spectrometry. (D-G) Western blot analysis and quantification of Fpn1, TFR, and DMT1 with β-actin as an internal control. All results are presented as the mean ± SEM (n = 7). *p < 0.05, **p < 0.01.

Fig. 2

Effect of LA on neuroinflammation in P301S mice. (A) GFAP-stained sections through the hippocampus or cortex in vehicle-, low-dose LA- and high-dose LA-treated mice. (B) Western blot analysis of GFAP, Iba1, IL-1β, and TNFα. (C-F) Quantitative analyses of Western blot for GFAP, Iba1, IL-1β and TNFα. β-actin was used as an internal control. All results are presented as the mean ± SEM (n = 7). *p < 0.05, **p < 0.01.

Fig. 3

Effect of LA on oxidative stress in the brains of P301S mice. (A) ROS production was detected in the brains of each group by the DCF-DA method. (B) SOD activity levels in the brains of each group. (C-F) Western blot and quantification analysis of SOD1, GPx4, and xCT with β-actin as a loading control. (G) Detection of calcium content by atomic absorption spectrometry. (H) Immunofluorescence with anti-GPx4 antibody and MitoTracker Green in the hippocampus and cortex. All results are presented as the mean ± SEM (n = 7). *p < 0.05, **p < 0.01.

Fig. 4

LA inhibited Tau phosphorylation at multiple AD-related sites. (A) Detection of Tau phosphorylation in the brain by immunohistochemistry using anti-phospho-Tau (Ser416). (B) Western blot analysis of phosphorylated Tau at residues Ser-202, Ser396, Ser404, Ser-416, Thr181, and Thr231 in the brain. β-actin served as a loading control. (C-I) Quantitative analysis of Tau phosphorylation levels at Ser-202, Ser396, Ser404, Ser-416, Thr181, and Thr231 sites. All results are presented as the mean ± SEM (n = 7). *p < 0.05, **p < 0.01.

Fig. 5

LA inhibited P38 MAPK, calpain1/CDK5 and GSK3β pathways but promoted PP2A. (A) Western blotting analysis of p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-P38, and P38. β-actin served as the loading control. (B-D) Quantitative analysis of p-ERK1/2, p-JNK1/2 and p-P38. (E) Western blot detection of calpain1, p-CDK5, CDK5 and P35/25 in the brains. (F-H) Quantification of calpain1, p-CDK5 and the P25/P35 ratio. (I) Western blot for p-GSK3α/β, GSK3α/β and PP2A expression. (J-L) Quantitative analysis of p-GSK3α, p-GSK3β and PP2A. All results are presented as the mean ± SEM (n = 7). *p < 0.05, **p < 0.01.

Fig. 6

LA rescued synaptic loss and inhibited apoptosis. (A-D) Western blot analysis and quantification of NeuN, SYP, and PSD95. β-actin is shown as a loading control. (E) Western blot analysis for AIF, Bcl2, Bax, caspase3, Beclin1, and LC3A/B. (F-J) Quantitative analysis of AIF, the Bcl2/Bax ratio, the cleaved caspase3/caspase3 ratio, Beclin1, and the LC3B/LC3A ratio. All results are presented as the mean ± SEM (n = 7). *p < 0.05, **p < 0.01.

Fig. 7

LA-treated mice exhibited improved learning and spatial memory in behavioral tests. (A) Escape latency in the visible and hidden platform tests of the Morris water maze. (B) The passing times of the three groups in the probe trial of the Morris water maze test. (C) Graph of a typical path in the hidden platform trial of the Morris water maze test. (D) Total distance traveled during 5 min of open field exploration. (E) Time spent in the center of the open field during 5 min of open field exploration. (F) The movement tracks showing 5 min of open field exploration by the mice. (G) Location preference index was determined by the ratio of the time spent with the left object and the total time. (H) Recognition index was determined by the ratio of the time spent with the novel object and the total time. (I) Object placement in the training and test periods of the novel object recognition test. Two identical objects were presented in the training phase, and one of the familiar objects was replaced by a novel object in the test phase. All results are presented as the mean ± SEM (n = 7). *p < 0.05, **p < 0.01.

Fig. 8

Schematic diagram of the role of LA in inhibiting Tau phosphorylation and neuronal loss including ferroptosis. Under Tau overexpression and hyperphosphorylated conditions, P301S mice developed iron overload. After LA administration, TFR expression level was downregulated while Fpn1 level was upregulated, thereby reducing the iron overload. Thus, iron overload-induced mitochondrial dysfunction and Ca²⁺ overload were not sufficient to induce calpain overactivation. p-P38, P25, and p-CDK5 levels were decreased and p-GSK3β level was increased, thereby inhibiting the hyperphosphorylation of Tau and Tau-induced iron overload. In addition, LA inhibited ferroptosis not only by reducing iron overload but also via the upregulation of xCT and GPx4. Moreover, the inhibition of calpain decreased the level of cleaved caspase3, which is involved in apoptosis, thus neuronal loss was rescued through the inhibition of apoptosis and ferroptosis.