LMP2 deficiency causes abnormal metabolism, oxidative stress, neuroinflammation, myelin loss and neurobehavioral dysfunctions

Abstract

Background: Substantial evidence suggests that immunoproteasome is implicated in the various neurological diseases such as stroke, multiple sclerosis and neurodegenerative diseases. However, whether the immunoproteasome itself deficiency causes brain disease is still unclear. Therefore, the aim of this study was to explore the contribution of the immunoproteasome subunit low molecular weight protein 2 (LMP2) in neurobehavioral functions.

Methods: Male LMP2 gene completed knockout (LMP2-KO) and littermate wild type (WT) Sprague-Dawley (SD) rats aged 12-month-old were used for neurobehavioral testing and detection of proteins expression by western blotting and immunofluorescence. A battery of neurobehavioral test tools including Morris water maze (MWM), open field maze, elevated plus maze were used to evaluate the neurobehavioral changes in rats. Evans blue (EB) assay, Luxol fast blue (LFB) and Dihydroethidium (DHE) staining were applied to explore the blood-brain barrier (BBB) integrity, brain myelin damage and brain intracellular reactive oxygen species (ROS) levels, respectively.

Results: We firstly found that LMP2 gene deletion did not cause significantly difference in rats' daily feeding activity, growth and development as well as blood routine, but it led to metabolic abnormalities including higher levels of low-density lipoprotein cholesterol, uric acid and blood glucose in the LMP2-KO rats. Compared with the WT rats, LMP2-KO rats displayed obviously cognitive impairment and decreased exploratory activities, increased anxiety-like behavior and without strong effects on gross locomotor abilities. Furthermore, multiple myelin loss, increased BBB leakage, downregulation of tight junction proteins ZO-1, claudin-5 and occluding, and enhanced amyloid-β protein deposition were observed in brain regions of LMP2-KO rats. In addition, LMP2 deficiency significantly enhanced oxidative stress with elevated levels of ROS, caused the reactivation of astrocytes and microglials and markedly upregulated protein expression levels of interleukin (IL)-1 receptor-associated kinase 1 (IRAK1), IL-6 and tumor necrosis factor-α (TNF-α) compared to the WT rats, respectively.

Conclusion: These findings highlight LMP2 gene global deletion causes significant neurobehavioral dysfunctions. All these factors including metabolic abnormalities, multiple myelin loss, elevated levels of ROS, increased BBB leakage and enhanced amyloid-β protein deposition maybe work together and eventually led to chronic oxidative stress and neuroinflammation response in the brain regions of LMP2-KO rats, which contributed to the initial and progress of cognitive impairment.

Keywords: Blood–brain barrier; Cognitive impairment; Immunoproteasome; Neuroinflammation; Oxidative stress.

Fig. 1

Comparison of basal parameters between the LMP2-KO and WT rats. A There was no significant difference in gross brain appearance between the LMP2-KO and WT rats. B There were no significant differences in heart rate and blood pressure between the two groups (^#P > 0.05). C The levels of ALT, total cholesterol, LDL-C, blood glucose, creatine and uric acid from blood samples of LMP2-KO and WT rats were comparison, and there were significant increased levels of blood LDL-C, uric and glucose in the LMP2-KO rats compared with the WT rats (^#P > 0.10,*P < 0.05). (D) Blood biochemical examination showed that the levels of low-density lipoprotein cholesterol (LDL-C), uric acid (Uric) and blood glucose in the LMP2-KO group were higher than those in the WT group, respectively (*P < 0.05)

Fig. 2

LMP2 defect increases the latency and path length to find the hidden platform in place navigation. LMP2-KO and WT rats were subjected to Morris water maze test (MWM) for place navigation for 5 consecutive days. The escape latency and the path length were analyzed using repeated measures two-way ANOVA. A Representative track plots of MWM for place navigation. B The latency significantly increased in the LMP2-KO rats as compared with the WT rats. C The path length of finding the hidden platform significantly increased in the LMP2-KO rats as compared to the WT rats. D The average swimming speed was not significantly different between the LMP2-KO and the WT rats (^#P > 0.10,*P < 0.05)

Fig. 3

LMP2 defect decreases the frequency over the target quadrant in probe test. Both LMP2-KO and WT rats were subjected to Morris water maze test (MWM) for probe tests on the 6th day followed place navigation. The data were analyzed using one-way ANOVA. A Representative track plots of MWM for probe tests. B The frequency crossing through the platform significantly lower in LMP2-KO rats than in WT rats (*P < 0.001). C The total path length and D the percentage of path length in target quardrant and E the average swimming speed was compared between LMP2-KO and WT rats, respectively (^#P > 0.10,*P < 0.05)

Fig. 4

Deletion of the LMP2 gene reduces rats’ exploratory activities. A Representative tracks for open field test. B There was no significant difference in the total distance between the WT rats and the LMP2-KO rats (^#P > 0.10). C–D Compared with the WT rats, both the distance traveled in center area and the time spent in the center were significantly lower in the LMP2-KO rats, respectively (*P < 0.001)

Fig. 5

Deletion of the LMP2 gene increased rats’ anxiety-like behavior measured by elevated plus maze. A Representative tracks for the elevated plus maze test. B–C Compared with the WT rats, both the percentages of the entries in open arms and the time spent in open arms were significantly less in the LMP2-KO rats than that in WT rats, respectively (*P < 0.05)

Fig. 6

LMP2 gene deficiency results in myelin loss and remyelination coexisted in brain of rats. A LFB staining showed myelin loss of the cortex, corpus callosum and striatum of WT, LMP2-KO rats, respectively. B–C Representation image of MBP protein expression detected by western blotting and quantification data. D–F Immunofluorescence and western blotting showed that increased protein expression of oligodendrocytes-1(Olig1) in the LMP2-KO rats compared with the WT rats. Scale bars: 500 μm and 100 μm. *P < 0.001

Fig. 7

LMP2 gene deficiency significantly induces BBB leakage and decreases tight junction proteins expression compared to the WT rats. A–B Evans blue (EB) exudation was observed under fluorescence microscope and quantificated, respectively. The level of EB exudation significantly increased in the LMP2-KO group than that in the WT group. C FITC-dextran angiographic micrographs indicated the tracer was confined to the capillaries in wild-type littermates, whereas LMP2-KO rats showed large amounts of tracer leakage in the brain parenchyma. D–E Representation images of tight junction proteinsZO-1, claudin-5 and occluding expression detected by western blotting and quantification data. Scale bars: 500 um; 100 μm.*P < 0.001

Fig. 8

LMP2 gene deficiency enhances Amyloid-β protein deposition compared to the WT rats. LMP2 deficiency significantly enhanced Aβ protein deposition. Immunofluorescence showed that there was more Aβ protein deposition in the hippocampus and cerebral cortex of LMP2 gene deficiency rats compared with the WT rats. Scale bars: 500 um; 100 μm

Fig. 9

LMP2-KO rats exhibit significantly enhanced oxidative stress, increased expression of astrocyte, microglial expression and inflammation compared to WT rats, respectively. A DHE staining showed ROS levels in the cortex and corpus callosum of WT and LMP2-KO rats, respectively. B Expression of astrocytes (GFAP), microglias (IBA1) in brain of WT and LMP2-KO rats, respectively. C–D Representation images of IRAK1, IL-6 and TNF-a proteins expression detected by western blotting and quantification data. Scale bars: 500 um; 100 μm. *P < 0.001