Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model

Abstract

Immune responses in the brain are thought to play a role in disorders of the central nervous system, but an understanding of the process underlying how immune cells get into the brain and their fate there remains unclear. In this study, we used a 2-photon microscopy to reveal that neutrophils infiltrate brain and migrate toward amyloid plaques in a mouse model of Alzheimer's disease. These findings suggest a new molecular process underlying the pathophysiology of Alzheimer's disease.

Keywords: Alzheimer's disease; Amyloid plaques; Immune cells; Neutrophils.

Fig. 1

The transendothelial migration of neutrophils from blood vessels into the brain parenchyma occurs in 5XFAD mice. The brain of a 5XFAD mouse, which exhibited massive Aβ plaque burden and neuronal cell death, was imaged with a 2-photon microscope after craniotomy surgery, and the dynamic extravasation of neutrophils into the brain parenchyma was detected. Intravenously (i.v.) injected Texas red-conjugated 70-kDa Dextran (red) labels brain blood vessels, and the Ly6C/G (Gr-1) Alexa Fluor 488-conjugated antibody (green) labels neutrophils. (A) Left: neutrophils in the blood vessels exited the brain parenchyma (transendothelial migration of the neutrophils, asterisk). Right: the different side-view images of the transendothelial migration of the same neutrophils (white arrow) from Fig. 1A, left, were taken at intervals of 30 seconds; 1 unit, 10.96 µm. The in vivo imaging of the brain of wild-type ([WT]; B) and 5XFAD (C) mice with Texas red-conjugated 70-kDa Dextran (red) and Gr-1 Alexa Fluor 488-conjugated antibody (green). The neutrophils that migrated into the brain parenchyma were found in 5XFAD mice but not in WT mice. Scale bar, 40 µm. (D) A fluorescence-activated cell sorting analysis also shows that Gr-1-positive neutrophils were more abundant in the brain of 5XFAD mice (middle) than in WT mice (left). N = 2 per group; the experiments were repeated 4 times; * p < 0.05. (E) The populations of tracked neutrophils from the 5XFAD mice (Video 2) and WT mice (Video 8) were quantified with respect to their average velocity, displacement rate, and meandering index; *** p < 0.0001. (F) In addition, the populations of tracked neutrophils from 5XFAD mice (Video 2) and WT mice (Video 8) were also examined with respect to their XY track plot, which was relative to their starting position. These quantitative data and plots suggest that neutrophils migrated into the brain parenchyma and that they have vigorous behavior and movement in 5XFAD mice compared with WT mice. Abbreviation: Aβ, beta-amyloid.

Fig. 2

Neutrophils migrate toward β-amyloid plaques in the 5XFAD mouse brain. (A) The 2-photon in vivo time series imaging of the 5XFAD mouse brain reveals the interaction between transmigrated neutrophils, which seem to be activated, and some spots in the brain parenchyma. Neutrophils (green), which moved randomly outside the vessels (red), were suddenly and massively recruited to the specific spot (white circle). T1: 10 minutes, 0 seconds; T2: 28 minutes, 0 seconds; T3: 32 minutes, 30 seconds; T4: 39 minutes, 0 seconds; T5: 46 minutes, 0 seconds; and T6: 48 minutes, 30 seconds from Video 3. Scale bar, 20 µm. (B) The traced neutrophils from Video 3 were shown as green color at each time point. This tracking data also indicated recruitment of neutrophils in the 5XFAD mouse brain. The Volocity software program (PerkinElmer Inc, Waltham, MA, USA) was used. (C) Chemotactic effects are expressed by the color index. The blue area indicates less chemotactic, while more chemotactic movement is indicated by the red color. The center of the circle is represented by a red dot in Fig. 3B at each time point, and the chemotactic neutrophils were counted at intervals of 5 µm in the vector states. The Volocity software program was used. Neutrophils were recruited to β-amyloid plaques in the cortex of 5XFAD mice. To visualize the plaques, methoxy-X04 dye, which can cross the blood-brain barrier well, was intraperitoneally (i.p.) injected 24 hours before the imaging. (D) The in vivo time series imaging in the brain of a 5XFAD mouse. Gr-1-positive neutrophils are recruited near βamyloid plaques (white circle, red), and they stick. T1: 0 minutes, T2: 10 minutes, T3: 20 minutes, T4: 30 minutes, T5: 40 minutes, and T6: 50 minutes from Video 4; 1 unit, 13.87 µm. (E) The direct binding of neutrophils to β-amyloid plaques. A neutrophil (arrow) is directly bound to the small plaque (red) that is already covered with a few neutrophils. T1: 45 minutes, 30 seconds; T2: 46 minutes, 0 seconds; T3: 46 minutes, 30 seconds; T4: 47 minutes, 0 seconds; T5: 47 minutes, 30 seconds; T6: 48 minutes, 0 seconds; T7: 48 minutes, 30 seconds; and T8: 49 minutes, 0 seconds from Video 5; 1 unit, 4.3 µm.

Fig. 3

Quantification of neutrophil chemotaxis by using a microfluidic platform. The platform is composed of a central compartment for cells, both side compartments for 2 different conditions, and migration channels connecting the compartments. The cell compartment is separated from migration channels by a central valve (default-open) and the migration channels are separated from chemokine compartment by 2 side valves (default-closed). Neutrophils were stained in a red fluorescence for easy tracking and white migration channels are overlaid for visualization after imaging. (A) The photo shows the strong chemotactic activity of neutrophils by fMLP 100 nM, which is taken at 28 minutes after starting the experiment. (B) On the other hand, Aβ at 10 nM does not induce any chemotatic activity on the neutrophils from the observation for 2 hours. Scale bar, 200 µm. Abbreviations: Aβ, beta-amyloid; fMLP, N-formyl-methyl-leucyl-phenylalanine.