Beneficial effects of increased lysozyme levels in Alzheimer's disease modelled in Drosophila melanogaster

Abstract

Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer's disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome-wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aβ_1-42 or AβPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aβ_1-42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aβ_1-42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aβ_1-42 and caused a beneficial effect by binding of lysozyme to toxic species of Aβ_1-42 , which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.

Keywords: Drosophila; Alzheimer's disease; amyloid-β; lysozyme.

Figure 1

Lysozyme mRNA expression is increased in brains of transgenic AD mice. (A–C) Lysozyme mRNA expression (LYZ) in the cortex, hippocampus and cerebellum of wild‐type (WT) mice and amyloid transgenic mice expressing human AβPP or human PSEN1 or both genes homo‐ or heterozygously (HOM_AβPP–PSEN1 and HET_AβPP–PSEN1) at 2, 4, 8 and 18 months of age. Significant increases of lysozyme in HOM_AβPP–PSEN1 and HET_AβPP–PSEN1 compared with WT mice are denoted with asterix (*). Significant differences were determined by two‐way ANOVA with Tukey's post hoc test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Mean and SD are presented at the different ages, n = 4. (D–F) Lysozyme mRNA expression in the cortex, hippocampus and cerebellum in tau transgenic mice at 2, 4, 8 and 18 months of age. Significant increases of lysozyme in TAU mice compared with WT mice are denoted with asterix (*). Significant differences were determined by two‐way ANOVA with Tukey's post hoc test. Mean and SD are presented at the different ages, n = 4. (G,H) Correlation analysis of lysozyme mRNA expression and Aβ pathology in the cortex and hippocampus of homozygous or heterozygous AβPP–PSEN1 mice and AβPP mice using the Pearson correlation coefficient. (I) Correlation analysis of lysozyme mRNA expression and tau pathology in cortex and hippocampus of tau transgenic mice using nonlinear regression.

Figure 2

Lysozyme protein expression is increased in brains of transgenic AD mice. (A) Representative western blot of brain homogenates from 12‐month‐old WT and AβPP transgenic mice analysed for lysozyme, Aβ and β‐actin. (B) Densitometric quantification of the western blot. Significant differences were determined by Student's t‐test. Error bars represent mean ± SD, n = 4. (C) Immunohistochemistry of brain tissue prepared from 15‐month‐old transgenic AβPP_{S we} mice, stained with 6E10 antibody (brown) and anti‐lysozyme (green). The white box shows one single amyloid plaque in magnification. Lysozyme is present inside the plaque (white arrow). Green circular structures (black arrow) are cells stained with intracellular lysozyme.

Figure 3

Lysozyme is increased in brains from AD patients. (A) Lysozyme mRNA expression (LYZ; log‐values of the mean intensities, normalized to the average intensities of all samples) in the visual cortex, prefrontal cortex and cerebellum of healthy controls (C) (n = 173) and AD patients (n = 376). Significant differences were determined by Student's t‐test; ***P ≤ 0.001. Bars represent mean ± SEM. (B) Representative western blot of human temporal cortex tissue from healthy controls (Braak 0‐IV, n = 24) and AD patients (Braak V‐VI, n = 10). Shown are densitometric quantifications of the western blots, normalized to GAPDH levels and to a standard sample loaded on each gel. Significant differences were determined by the nonparametric Mann–Whitney U test. Bars represent the mean ± SD. (C) Lysozyme concentrations in CSF from controls (n = 25) and biochemical and clinical diagnosed AD patients (n = 25) measured using the Meso Scale Discovery technique. Significant differences was determined by Student's t‐test, *P ≤ 0.05. Bars represent the mean ± SD.

Figure 4

Lysozyme protects transgenic AD flies from Aβ‐induced toxicity. (A) Images of rough eye phenotype obtained by scanning electron microscopy of two AD Drosophila models expressing Aβ_1‐42 or AβPP–BACE1 with or without lysozyme (Lys) and controls; only expressing Gal4, AβPP and Lys at the day of eclosion (150× magnification). Scale bars = 50 μm. (B) Quantification of the abnormal ommatidia seen in A (n ≥ 4 of each genotype). All samples are related to their respective control. Significant differences were determined by one‐way ANOVA followed by Tukey's test, **P ≤ 0.01, ***P ≤ 0.001. Meso Scale Discovery analyses performed on fly heads from flies at the day of eclosion to quantify the levels of (D) soluble Aβ_1‐42 (n = 4) and (E) insoluble Aβ_1‐42 (n = 4). All samples are compared to their respective control. Significant differences were determined by one‐way ANOVA followed by Tukey's test, **P ≤ 0.01, ***P ≤ 0.001. (C) Immunoprecipitation assay of extract from AβPP–BACE1 flies with and without coexpression of lysozyme. Brain homogenates were immunoprecipitated with lysozyme antibodies bound to resin and the urea‐eluted samples were analysed for Aβ_1‐42 using the Meso Scale Discovery technique. The assay was performed on 100 flies of each genotype. Significant differences were determined by Student's t‐test, *P ≤ 0.05.

Figure 5

Longevity and behavioural analyses show beneficial effects of lysozyme on Aβ_Arc flies. (A) Lifespan trajectories for Drosophila flies expressing Aβ with the Arctic mutation (Aβ_Arc) in the CNS, in the absence or presence of lysozyme expressed as one copy or as two copies compared to control flies (n = 100). Kaplan–Meier graph shows per cent survival against age of flies in days after eclosion. Significant differences were determined by log‐rank analysis, *P ≤ 0.05 and ***P ≤ 0.001. The fly behaviour was analysed for control flies and Aβ_Arc flies with or without coexpression of one or two copies of lysozyme by (B) velocity measurement, (C) velocity measurement at day 9, (D) angle of movement analysis and (E) angle of movement analysis at day 6. Significant differences were determined by one‐way ANOVA followed by Tukey's test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Error bars represent mean ± SD, n = 30. (F) Meso Scale Discovery analysis performed on the fly heads of control, Aβ_Arc, Aβ_Arc‐Lys and Aβ_Arc‐Lys‐Lys at day 10 after eclosion to quantify the levels of soluble and insoluble Aβ_Arc. Significant differences were determined by one‐way ANOVA followed by Tukey's test, ***P ≤ 0.001. Error bars represent mean ± SD, n = 4.