Ameliorating Alzheimer's-like Pathology by Minocycline via Inhibiting Cdk5/p25 Signaling

Abstract

Background: Minocycline has multiple neuroprotective roles in abundant brain diseases, including the prevention and treatment of Alzheimer's disease (AD). Cdk5/p25 signaling plays an important role in the onset and development of Alzheimer's-like pathology. The aim of the present work was to further explore the underlying mechanism which minocycline effects on Cdk5/p25 signaling related to Alzheimer's-like pathology.

Methods: The cognitive function of animals was measured by the Morris water maze test. The levels of Aβ were determined by an enzyme-linked immunosorbent assay. The levels of APP, β- and γ- secretases, and the biomarkers of tau (total tau and hyperphosphorylated tau), inflammatory cytokine and matrix metalloproteinases (MMP-2 and MMP-9), and biomarkers of synapse and Cdk5/p25 signaling, were detected by the Western blotting. The biomarkers of the synapse, inflammatory cytokine, and matrix metalloproteinases (MMP-2 and MMP-9) were also determined by immunofluorescence.

Results: Minocycline improved learning and memory in APP/PS1 mice. It limited the production of Aβ and hyperphosphorylation of tau in the hippocampus and ameliorated synaptic deficit. Moreover, it also inhibited the activation of Cdk5/p25 signaling, inflammation, and matrix metalloproteinases.

Conclusion: Minocycline mitigates Alzheimer's-like pathology via limiting the activation of Cdk5/p25 signaling pathway and improves cognitive deficits.

Keywords: Alzheimer’s disease; Minocycline; anti-inflammatory drugs; cyclin-dependent kinase 5; pathology; synapse.

Fig. (1)

The experimental process and schematic schedule. i.g.: intragastric administration; MWM, Morris water maze.

Fig. (2)

Minocycline alleviates spatial learning and memory of AD mice. After treatment with minocycline (50 mg/kg/d, i.g.) for 30 consecutive days, mice were examined using the Morris water maze (MWM) task. The latency to find the platform during 7 days in the acquisition trials was recorded in the navigation training. The minocycline-treated mice exhibited lower escape latency on days 3, 4, 5, 6, and 7 during training trials compared to vehicle-treated APP/PS1 mice (**P<0.01) (A). The number of times that mice cross the target quadrant (removed platform) (B) and the time that mice stay in the target quadrant (C) were calculated. A significant increase in the number of times mice cross the target quadrant and the time of mice staying in the target quadrant (**P<0.01) was observed in the minocycline-treated mice compared to APP/PS1 transgenic mice. No difference was observed between the two groups (D) regarding swimming speed. Values were represented as mean ± SD (n=10). AD: Alzheimer’s disease, MINO: minocycline.

Fig. (3)

Down-regulation of Aβ in the brain of AD mice by minocycline. Both Aβ42 and Aβ40 levels significantly decreased in the hippocampal and cortex tissues after minocycline treatment (*P<0.01). Values were represented as mean ± SD (n=10). Aβ: beta-amyloid peptides, AD: Alzheimer’s disease, MINO: minocycline.

Fig. (4)

The effects of minocycline on β- and γ-secretases. Relative amounts of sAPP-β, BACE, and BACE1 were expressed as the densitometry OD ratio to β-actin (mean ± SED) for Western blotting. The sAPP-β, BACE, and BACE1 levels in the hippocampus of minocycline-treated mice were found to be lower than the model control ones (P<0.05) (A). Minocycline treatment also markedly decreased the expression of PS1, NCT, Aph-1α, and Pen-2 in the hippocampus of AD mice (B). AD: Alzheimer’s disease, Aph-1α: anterior pharynx-defective 1alpha, BACE1: the beta-amyloid precursor protein cleavage enzyme 1, MINO: minocycline, NCT: nicastrin, OD: optical density, Pen-2: presenilin enhancer 2, PS1: presenilin 1.

Fig. (5)

Minocycline inhibits phosphorylation of tau. Minocycline down-regulated the levels of both total tau (HT7) and phosphorylated tau proteins, including pre-tangle marker phospho-tau antibody TG3 (pThr205/pThr231), intraneuronal tangle marker phospho-tau protein (pSer199/pSer202), and extracellular tangle marker PHD finger gerprotein-1 ([PHF-1] pSer396/pSer404) in the hippocampus of AD mice. AD: Alzheimer’s disease, MINO: minocycline; OD: optical density.

Fig. (6)

Minocycline treatment reduces the synaptic deficit. Western blot (A) and immunofluorescence staining (B) analysis showed that minocycline increased the levels of the three synaptic proteins (microtubule-associated protein 1, synaptophysin, and post-synaptic density protein 95) in the hippocampus compared to the model control mice (A and B). Quantitative results were found to be normalized against the levels of β-actin. Values were presented as the group mean ± SEM (n = 6). *p< 0.001 vs. the AD control group. The scale bar was 50 µm. AD: Alzheimer’s disease, MAP1: microtubule-associated protein 1, MINO: minocycline; OD: optical density, PSD95: post-synaptic density protein 95, SYP: synaptophysin.

Fig. (7)

Minocycline inhibits the activity of Cdk5/p25 signaling. Minocycline significantly decreased the levels of p25 and Cdk5 while the levels of p35 were markedly increased after minocycline administration compared to the control ones (*P<0.001 vs. the control group). AD: Alzheimer’s disease, Cdk5: cyclin-dependent kinase 5, MINO: minocycline; OD: optical density.

Fig. (8)

Minocycline limits inflammation and activation of matrix metalloproteinases. Western blot (A) and immunofluorescence staining (B) analysis showed that minocycline reduced the levels of IL-1β and matrix metalloproteinases (MMP-2 and MMP-9) proteins in the cerebral cortex compared to the model control mice (A and B). Quantitative results were found to be normalized against the levels of β-actin. Values were presented as the group mean ± SEM (n = 6). *p< 0.001 vs. the AD control group. The scale bar was 20µm. AD: Alzheimer’s disease, IL-1β: Interleukin-1beta, MINO: minocycline; MMP-2:matrix metalloproteinase 2, MMP-9:matrix metalloproteinase 9, OD: optical density.

Fig. (9)

The possible mechanisms of anti-pathology in AD by minocycline. Minocycline inhibits the activation of Cdk5/p25 signaling and limits the development of Alzheimer’s-like pathology, including production of anti-Aβ, anti-hyperphosphorylation of tau, and improvement of synaptic dysfunction. Aβ: beta-amyloid peptides; AD: Alzheimer’s disease; Cdk5: cyclin-dependent kinase 5; MINO: minocycline.