Far infrared light irradiation enhances Aβ clearance via increased exocytotic microglial ATP and ameliorates cognitive deficit in Alzheimer's disease-like mice

Abstract

Background: Exposure to sunlight may decrease the risk of developing Alzheimer's disease (AD), and visible and near infrared light have been proposed as a possible therapeutic strategy for AD. Here, we investigated the effects of the visible, near infrared and far infrared (FIR) light on the cognitive ability of AD mice, and found that FIR light also showed potential in the improvement of cognitive dysfunction in AD. However, the related mechanism remains to be elucidated.

Methods: Morris water maze was used to evaluate the cognitive ability of APPswe/PSEN1dE9 double-transgenic AD mice after light treatment. Western blot was carried out to detect the expression of protein involved in synaptic function and amyloid-β (Aβ) production. The protein amount of interleukin (IL)-1β, IL-6, Aβ_1-40 and Aβ_1-42 were determined using enzyme-linked immunosorbent assay. The mRNA level of receptors was performed using real-time quantitative polymerase chain reaction. Immunostaining was performed to characterize the Aβ burden and microglial Aβ phagocytosis in the brain of AD mice. The Aβ phagocytosis of primary cultured microglia and BV2 were assessed by flow cytometry. The energy metabolism changes were evaluated using related assay kits, including adenosine triphosphate (ATP), lactate content, mitochondrial respiratory chain complex enzymatic activity and oxidized/reduced nicotinamide adenine dinucleotide assay kits.

Results: Our results showed that FIR light reduced Aβ burden, a hallmark of AD neuropathology, alleviated neuroinflammation, restored the expression of the presynaptic protein synaptophysin, and ameliorated learning and memory impairment in the AD mice. FIR light enhanced mitochondrial oxidative phosphorylation pathway to increase ATP production. This increased intracellular ATP promoted the extracellular ATP release from microglia stimulated by Aβ, leading to the enhanced Aβ phagocytosis through phosphoinositide 3-kinase/mammalian target of rapamycin pathways for Aβ clearance.

Conclusions: Our findings have uncovered a previously unappreciated function of FIR light in inducing microglial phagocytosis to clean Aβ, which may be the mechanisms for FIR light to improve cognitive dysfunction in AD mice. These results suggest that FIR light treatment is a potential therapeutic strategy for AD.

Keywords: Alzheimer’s disease; Amyloid-β clearance; Energy mechanism; Far infrared light; Microglial phagocytosis.

Fig. 1

FIR light ameliorated learning and memory deficit of AD mice. A Timeline of treatment with visible light (VIS), near infrared light (NIR) and far infrared light (FIR) on APP/PS1 mice. B Representative tracking routes of mice in training trials and spatial probe test. C The escape latency of mice to find the platform during training trials. D The number of entries in the zones around platform. E The mean swimming speed of mice. Data were means ± SEM, n = 5–8, *p < 0.05 compared to APP/PS1 mice without light treatment. N.S., not significant

Fig. 2

FIR light treatment ameliorated Aβ deposition in the cortex and hippocampus. A Representative images of Aβ (6E10) staining in the cerebral cortex and hippocampus from WT, APP/PS1 and FIR light-treated APP/PS1 mice. B Percentage of Aβ plaque area in the cerebral cortex (n = 4). C Aβ plaque density in the cerebral cortex (n = 4). D Percentage of Aβ plaque area in the hippocampus (n = 4). E Aβ plaque density in the hippocampus (n = 4). F The levels of Aβ_1-40 and Aβ_1-42 in the PBS fractions from the cerebral cortex (n = 5). G The levels of Aβ_1-40 and Aβ_1-42 in the guanidine hydrochloride fractions from the cerebral cortex (n = 5). Data were means ± SEM, *p < 0.05, **p < 0.01. ND: not detected

Fig. 3

FIR light did not influence the Aβ production but enhanced microglial Aβ phagocytosis. A The Western blot analysis of key molecules involved in the process of Aβ production. Expression level of B APPfl, C sAPPα, D Nicastrin, E BACE1 and F PS1 (n = 5 per group). G Representative images of Aβ (6E10, red), microglia (Iba1, green) and nuclei (DAPI, blue) co-staining from APP/PS1 treated with or without FIR light. H Quantification of microglial cells within 20 μm from the Aβ plaque boundary (n = 102 to 103 plaques per group) in the cerebral cortex. I Representative images of Aβ (6E10, white), microglia (Iba1, red), phagosome (CD68, green) and nuclei (DAPI, blue) co-staining in the cerebral cortex of APP/PS1 mice treated with or without FIR light. J Quantification of the percentage of phagosome area in Iba1 positive area (n = 49–52 per group). K Quantification of the percentage of 6E10⁺/CD68⁺/Iba1⁺ co-staining area normalized to the total 6E10⁺ area (n = 49–52 per group). L Representative images of the uptake of FAM-Aβ_1-42. M Image quantification of the corresponding fluorescent intensity of FAM-Aβ_1-42 engulfed by microglia (n = 6 per group). Data were means ± SEM, ***p < 0.001. N.S., not significant. CytoD: cytochalasin D

Fig. 4

Increased ATP production was involved in the FIR light-enhanced microglial Aβ phagocytosis. A Intracellular ATP of microglia under different conditions. B Intracellular ATP of microglia exposed to FIR light. Upon the FIR light treatment, C before Aβ phagocytosis, intracellular ATP of microglia treated with antimycin A or not, D Aβ phagocytosis of microglia treated with antimycin A or not, E after Aβ phagocytosis, intracellular ATP of microglia treated with antimycin A or not. Upon the treatment with antimycin A or not, F before Aβ phagocytosis, intracellular ATP of microglia cultured in medium containing glucose of 25 mM and 0.1 mM, respectively, G Aβ phagocytosis of microglia cultured in medium containing glucose of 25 mM and 0.1 mM, respectively, H after Aβ phagocytosis, intracellular ATP of microglia cultured in medium containing glucose of 25 mM and 0.1 mM, respectively. Data were means ± SEM, n = 6, *p < 0.05, **p < 0.01, ***p < 0.001. AA: antimycin A

Fig. 5

FIR light enhanced microglial Aβ phagocytosis through increased microglial extracellular ATP release. A Extracellular ATP of microglia under different stimulation conditions. Without Aβ stimulation, extracellular ATP of microglia cultured in B medium containing glucose of 25 mM and 0.1 mM, respectively (n = 12), or C medium containing glucose of 25 mM and additionally adding AA or not (n = 12). With Aβ stimulation, extracellular ATP of microglia cultured in D medium containing glucose of 25 mM and 0.1 mM, respectively (n = 12), or E medium containing glucose of 25 mM and additionally adding AA or not (n = 12). F Upon the FIR light treatment, extracellular ATP of microglia treated with antimycin A or not (n = 6). G Extracellular ATP of microglia treated with FIR light or not (n = 6). H Upon the FIR light treatment, Aβ phagocytosis of microglia pretreated with suramin or not (n = 6). I Upon the suramin pretreatment, Aβ phagocytosis of microglia treated with FIR light or not (n = 6). J Upon the FIR light treatment, Aβ phagocytosis of microglia pretreated with apyrase or not (n = 6). K Upon the apyrase pretreatment, Aβ phagocytosis of microglia treated with FIR light or not (n = 6). L Upon the FIR light treatment, Aβ phagocytosis of microglia pretreated with wortmannin or not (n = 6). M Upon the FIR light treatment, Aβ phagocytosis of microglia pretreated with rapamycin or not (n = 6). N Upon the wortmannin pretreatment, Aβ phagocytosis of microglia treated with FIR light or not (n = 6). O Upon the rapamycin pretreatment, Aβ phagocytosis of microglia treated with FIR light or not (n = 6). Data were means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. N.S.: not significant. AA: antimycin A. Su: suramin. Ap: apyrase. Wo: wortmannin. Ra: rapamycin

Fig. 6

FIR light promoted microglial ATP production mainly through mitochondrial OXPHOS pathway. A Upon the 2-DG pretreatment, Aβ phagocytosis of microglia treated with FIR light or not (n = 6). B Upon the antimycin A pretreatment, Aβ phagocytosis of microglia treated with FIR light or not (n = 6). C Upon the 2-DG plus antimycin A pretreatment, Aβ phagocytosis of microglia treated with FIR light or not (n = 6). D The lactate level of microglia treated with FIR light or not (n = 6). The enzymatic activity of E complex I, F complex II, G complex III, H complex IV, and I ATP synthase in the mitochondrial respiratory chain in BV2 microglia (n = 6). J The NAD⁺ level and K the ratio of NAD⁺/NADH of BV2 microglia treated with FIR light or not (n = 9). Data were means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. N.S.: not significant. AA: antimycin A

Fig. 7

Schematic presentation showing the pathways that mediate the effects of FIR light on microglial Aβ clearance