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. 2021 Oct 21;10(11):1657.
doi: 10.3390/antiox10111657.

Role of Lipocalin-2 in Amyloid-Beta Oligomer-Induced Mouse Model of Alzheimer's Disease

Heeyoung Kang  1 , Hyun Joo Shin  2 , Hyeong Seok An  2 , Zhen Jin  3 , Jong Youl Lee  2 , Jaewoong Lee  2 , Kyung Eun Kim  2 , Eun Ae Jeong  2 , Kyu Yeong Choi  4 , Catriona McLean  5 , Kun Ho Lee  4   6   7 , Soo Kyoung Kim  8 , Hae Ryong Lee  9 , Gu Seob Roh  2
Affiliations

Role of Lipocalin-2 in Amyloid-Beta Oligomer-Induced Mouse Model of Alzheimer's Disease

Heeyoung Kang et al. Antioxidants (Basel). .

Abstract

Lipocalin-2 (LCN2) is an inflammatory protein with diverse functions in the brain. Although many studies have investigated the mechanism of LCN2 in brain injuries, the effect of LCN2 on amyloid-toxicity-related memory deficits in a mouse model of Alzheimer's disease (AD) has been less studied. We investigated the role of LCN2 in human AD patients using a mouse model of AD. We created an AD mouse model by injecting amyloid-beta oligomer (AβO) into the hippocampus. In this model, animals exhibited impaired learning and memory. We found LCN2 upregulation in the human brain frontal lobe, as well as a positive correlation between white matter ischemic changes and serum LCN2. We also found increased astrocytic LCN2, microglia activation, iron accumulation, and blood-brain barrier disruption in AβO-treated hippocampi. These findings suggest that LCN2 is involved in a variety of amyloid toxicity mechanisms, especially neuroinflammation and oxidative stress.

Keywords: Alzheimer’s disease; amyloid-beta; blood–brain barrier leakage; iron accumulation; lipocalin-2; neuroinflammation; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Human LCN2 expression and white matter ischemic changes in AD patients. (A) Assessment of ischemic rating scales in control and AD patients. (B) Correlation between total ARWMC and serum LCN2 levels in AD patients. (C) Representative immunofluorescence images (×200) showing LCN2 (green), Aβ (red), and GFAP (purple) staining in frontal cortex sections from an AD patient. DAPI (blue) was used to stain nuclei. Scale bar = 10 µm. Data are shown as mean ± SEM. * p < 0.05 versus control (CTL).
Figure 2
Figure 2
Effects of AβO toxicity on memory deficits, hippocampal tau phosphorylation and microglia activation. The Morris water maze test in control and Aβ-treated mice at 34 weeks. (A) Escape latency and (B) average time spent in swimming speed over 4 days. (C) Time spent in the target quadrant, and (D) number of crossings over the platform area for the three time slices analyzed. (E) Representative swimming paths during the probe trail. (F) Western blot and quantified hippocampal phosphorylated tau (s396) expression. To normalize total protein level, β-actin was used as a loading control. (G) Representative images of Iba-1 immunohistochemistry and quantification of relative optical density (ROD) measurements (%) in hippocampal CA1 regions. Bar = 10 µm (H). The schemes graphically illustrate Sholl analysis of microglia morphology detects. (Red circles indicate Sholl intersections. Circle lines indicate Sholl sphere radius). (I) Average number of intersections at specified distances from the soma in microglia, and (J) Shoenen ramification index; 27 to 31 cells per region of n = 4 mice. Data are mean ± SEM. * p < 0.05 for control compared with Aβ-treated mice.
Figure 3
Figure 3
Effects of AβO toxicity on neuroinflammation in the hippocampus. (A) Western blot analysis of IL-6, TNF-α, HMGB1, TLR4, RAGE, and NF-κBp65 in the hippocampus. (B) Quantitation of Western blot analysis in (A). To normalize total protein level, β-actin was used as a loading control. (C) Western blot and quantified hippocampal GFAP expression. To normalize total protein level, β-actin was used. The data are presented as mean ± SEM. * p < 0.05 for control compared with Aβ-treated mice. (D) Representative immunofluorescence images (×200) of hippocampal CA1 regions showing Ly6G (green) and GFAP (red) staining. DAPI (blue) was used to stain nuclei. Scale bar = 10 µm.
Figure 4
Figure 4
Effects of AβO toxicity on BBB leakage in the hippocampus. (A) Western blot analysis of eNOS, ZO-1, VCAM-1, and albumin in the hippocampus. (B) Quantitation of Western blot analysis in (A). To normalize total protein level, β-actin was used as a loading control. The data are presented as mean ± SEM. * p < 0.05 for control compared with AβO-treated mice. (C) Representative immunofluorescence images (×200) of hippocampal CA1 regions showing ZO-1 (green). DAPI (blue) was used to stain nuclei. Scale bar = 10 µm.
Figure 5
Figure 5
Effects of AβO toxicity on LCN2, MMP9, and STAT3 expression in the hippocampus. (A) Western blot analysis of LCN2, MMP-9, p-STAT3 (Tyr705), and STAT3. (B) Quantitation of Western blot analysis in (A). To normalize total protein level, β-actin was used as a loading control. The data are presented as mean ± SEM. * p< 0.05 for control compared with AβO-treated mice. (C) Representative immunofluorescence images (×200) of hippocampal CA1 regions showing LCN2 (red) and GFAP (green). DAPI (blue) was used to stain nuclei. Scale bar = 10 µm.
Figure 6
Figure 6
Effects of AβO toxicity on iron accumulation and oxidative stress in the hippocampus. (A) Histological staining (×100) for iron with DAB-enhanced Perls’ staining in the hippocampal CA1 regions and relative optical density (ROD) measurements (%). Scale bar = 25 µm. (B) Western blots and quantified hippocampal HO-1, Ferritin, and Ceruloplasmin expressions. β-actin was used as a loading control. As shown in Figure 5A, AβO-induced LCN2 is also enhanced in same Western blot used in HO-1. The data are presented as mean ± SEM. * p < 0.05 for control compared with AβO-treated mice.
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