Increased expression and altered subcellular distribution of cathepsin B in microglia induce cognitive impairment through oxidative stress and inflammatory response in mice

Abstract

During normal aging, innate immunity progresses to a chronic state. However, how oxidative stress and chronic neuroinflammation arise during aging remains unclear. In this study, we found that genetic ablation of cathepsin B (CatB) in mice significantly reduced the generation of reactive oxygen species (ROS) and neuroinflammation and improved cognitive impairment during aging. In cultured microglia, pharmacological inhibition of CatB significantly reduced the generation of mitochondria-derived ROS and proinflammatory mediators induced by L-leucyl-L-leucine methyl ester (LLOMe), a lysosome-destabilizing agent. In the CatB-overexpressing microglia after treatment with LLOMe, which mimicked the aged microglia, CatB leaked in the cytosol is responsible for the degradation of the mitochondrial transcription factor A (TFAM), resulting in the increased generation of mitochondria-derived ROS and proinflammatory mediators through impaired mtDNA biosynthesis. Furthermore, intralateral ventricle injection of LLOMe-treated CatB-overexpressing microglia induced cognitive impairment in middle-aged mice. These results suggest that the increase and leakage of CatB in microglia during aging are responsible for the increased generation of mitochondria-derived ROS and proinflammatory mediators, culminating in memory impairment.

Keywords: cathepsin B; lysosomal leakage; microglia; mitochondria-derived reactive oxygen species.

Figure 1

Amelioration of age‐dependent increased oxidation and inflammation in the hippocampus of CatB ^−/− mice. (a) Immunoblotting and quantitative analyses show HNE in WT and CatB ^−/− of both young (2 months old) and aged (20 months old) groups. (b) The amount of 8‐oxo‐dG in the hippocampus of WT and CatB ^−/− of both young and aged groups. (c) CLSM images of the HNE, 8‐oxo‐dG, and merged images with microglia marker F4/80, astrocyte marker GFAP, and neuron marker Nissl in the hippocampus of aged WT mice. Scale bar, 50 μm. (d) Immunoblotting showing CatB, Iba1, iNOS, IL‐1β, and TNF‐α in WT and CatB ^−/− of both young and aged groups. (e) The quantitative analyses of Iba1, iNOS, mIL‐1β, and TNF‐α in the immunoblots of (d). The results represent the mean ± SEM of three independent experiments in a, b, and e. The asterisks indicate a statistically significant difference from the young WT group (**p < 0.01, ***p < 0.001, one‐way ANOVA test). The daggers indicate a statistically significant difference from the aged WT mice (^†† p < 0.01, ^††† p < 0.001, one‐way ANOVA test). (f) CLSM images of the IL‐1β, TNF‐α, and iNOS with microglia marker Iba1, GFAP, and Nissl in the hippocampus of aged WT mice. Scale bar, 30 μm. (g) The mean percentage of IL‐1β，TNF‐α and iNOS‐positive microglia (Iba1), neurons (Nissl) and astrocytes (GFAP) in (f). Each column and bar represents the means ± SEM (n = 6, each). Asterisks indicate a statistically significant difference from the value for astrocytes in the same group (**p < 0.01, ***p < 0.01, two‐way ANOVA test), and the daggers indicate a statistically significant difference from the value for neurons in the same group (^†† p < 0.01, ^††† p < 0.001, one‐way ANOVA test)

Figure 2

Amelioration of age‐dependent decline in learning and memory in CatB ^−/− mice. (a) Step‐through latency of WT and CatB ^−/− mice on the first trial in the acquisition trials. (b) The total number of acquisition trials. (c) Step‐through latencies in the retention trials performed 1, 3, 5, and 7 days after the acquisition trials. The columns and bars represent the mean ± SEM of young WT (2 months, n = 10), CatB ^−/− young (2 months, n = 10), aged WT (20 months, n = 14) and aged CatB ^−/− (20 months, n = 8) mice. (d) Time spent exploring the familiar and the novel object in the recognition trial. The results represent the mean ± SEM (aged WT mice, n = 6; aged CatB ^−/− mice, n = 6). The asterisks indicate a statistically significant difference from the familiar object (***p < 0.001, Student's t test). (e) Cumulative potentiation of fEPSP slope after consecutive tetanic stimulation at 25, 50, and 100 Hz in the hippocampus of aged WT and aged CatB ^−/− mice. The traces show the typical fEPSPs before and after stimulation at 100 Hz in the hippocampal slices prepared from aged CatB ^−/− and aged WT mice. The results represent the mean ± SEM of five slices from three animals of each group. (f) Representative images of CA1 dendritic branches showing spines of WT and CatB ^−/− of both young and aged groups. Scale bar, 5 μm. (g) Analysis of spine density in the apical segment of hippocampal CA1 neurons of WT and CatB ^−/− of both young and aged groups. The results represent the mean ± SEM (n = 25 dendrites in three mice). The asterisks indicate a statistically significant difference from the young WT group (*p < 0.05, **p < 0.01, and ***p < 0.001, one‐way ANOVA test). The daggers indicate a statistically significant difference from the aged WT group (^† p < 0.05 and ^†† p < 0.01, one‐way ANOVA test)

Figure 3

Involvement of leaked CatB in mitochondria‐derived ROS generation and inflammatory response by cultured microglia. (a) Representative FACS histograms of H₂DCFDA analyses of ROS production in microglia when treated with 100 μM LLOMe or pre‐treated with 50 μM CA‐074Me. (b) The quantitative analyses of the ROS level in the FACS histogram shown in (a). (c) Detection of mitochondria‐derived ROS generated in the cultured microglia using MitoSOX 48 hr after treatment with 100 μM LLOMe or pre‐treatment with 50 μM CA‐074Me. Scale bar, 10 μm. (d) Mean relative intensity of MitoSOX oxidation in the CLSM images of (c). (e) Detection of ROS generated in the microglia using CM‐H₂DCFDA 48 hr after treatment with 100 μM LLOMe, 100 nM rotenone, or pre‐treatment with 50 μM CA‐074Me. The mitochondria were visualized using MitoTracker. Nuclei were stained by Hoechst. Scale bar, 30 μm. (f) The mitochondrial membrane potential (MMP, Δψm) in microglia was determined by a FACS analysis with JC‐1 staining which reversibly change color from green to red as membrane potential increase. The negative control means cultured cells without JC‐1 staining. The none group means cultured cells without stimulation stained with JC‐1 dye. (g) The quantitative analyses of MG6 cells exhibiting mitochondria‐derived ROS production. The results in b, d, and g represent the mean ± SEM of three independent experiments. The asterisks indicate a statistically significant difference from the non‐treated group (***p < 0.001, one‐way ANOVA test). The daggers indicate a statistically significant difference from the LLOMe‐treated group (^†† p < 0.01 and ^††† p < 0.001, one‐way ANOVA test)

Figure 4

Inflammatory responses after treatment with rotenone in cultured microglia. (a) Detection of mitochondria‐derived ROS generated in the cultured microglia using MitoSOX 48 hr after treatment with 100 nM rotenone or pre‐treatment with 50 µM CA‐074Me. Scale bar, 10 µm. Histograms show the mean relative intensity of MitoSOX oxidation in the CLSM images. (b) Immunoblots showing IκBα, p‐IκBα, iNOS, TNF‐α, and pro‐IL‐1β in the cultured microglia after 48 hr after treatment with 0.1, 1, 10, and 100 nM rotenone or pre‐treatment with 50 µM CA‐074Me. (c, d) The quantitative analyses of IκBα (c) and p‐IκBα (d) in the immunoblots of (b). (e–g) The quantitative analyses of iNOS (e), TNF‐α (f), and mIL‐1β (g) in the Immunoblotting of (b). (h) Detection of the mitochondrial complex I activity in the cultured microglia 48 hr after treatment with 1, 10, and 100 nM rotenone or pre‐treatment with 50 µM CA‐074Me. The results in (a) and (c–h) represent the mean ± SEM of three independent experiments. The asterisks indicate a statistically significant difference from the untreated group (**p < 0.01 and ***p < 0.001, one‐way ANOVA test). The daggers indicate a statistically significant difference from the 100 nM rotenone‐treated group (^††† p < 0.001, one‐way ANOVA test)

Figure 5

Possible involvement of CatB leaked in the cytosol of microglia in the degradation of TFAM. (a, b) CLSM images of the CatB (green) merged images with LysoTracker (a) and MitoTracker (b) in MG6 cells 48 hr after CatB‐overexpressing plasmid transfection. Scale bar, 5 µm. (c) CLSM images of acridine orange and Z‐Arg‐Arg‐cresyl violet in non‐treated and LLOMe‐treated CatB/MG6 cells. LLOMe quenched fluorescent emission of acridine orange, but not of Z‐Arg‐Arg‐cresyl violet. Scale bar, 5 µm. (d) Immunoblots showing a decrease in TFAM in CatB/MG6 cells pre‐treated with LLOMe. (e) The quantitative analyses of immunoblots in (d). The results represent the mean ± SEM of three independent experiments. The asterisks indicate a statistically significant difference from the control (**p < 0.01, one‐way ANOVA test). (f) The immunoblots show degradation of mouse recombinant TFAM by human recombinant CatB at 37°C, pH = 7. (g) The immunoblots show degradation of mouse TFAM 24 hr after incubated with and without CatB at 37°C or 0°C, PH = 7. (h) The quantitative analyses of TFAM in the immunoblotting shown in (f). The results represent the mean ± SEM of three independent experiments. The asterisks indicate a statistically significant difference from the control (**p < 0.01, one‐way ANOVA test). (i) The quantitative analyses of TFAM in the immunoblotting shown in (g). (j) Mitochondrial complex I activity in MG6 cells 24 hr after treatment with LLOMe alone or combination of LLOMe with CA‐074Me. The results represent the mean ± SEM of three independent experiments. The asterisks indicate a statistically significant difference from the controls (***p < 0.001, one‐way ANOVA test). The daggers indicate a statistically significant difference from the LLOMe alone (^†† p < 0.001, one‐way ANOVA test)

Figure 6

Impairment of learning and memory in middle‐aged mice by intralateral ventricle injection of CatB‐overexpressing microglia. (a) Immunoblots showing HNE and IL‐1β 48 hr after CatB‐overexpressing plasmid transfection, LLOMe treatment in MG6 cells, and LLOMe treatment in CatB/MG6 cells. (b, c) The quantitative analyses of HNE and IL‐1β in the immunoblotting shown in (a). The results represent the mean ± SEM of three independent experiments. The asterisks indicate a statistically significant difference from the controls (*p < 0.05, ***p < 0.001, one‐way ANOVA test). (d) Experimental timeline. (e) Evens blue was injected into the right lateral ventricle, and the dye was distributed throughout the mouse ventricular system. Scale bar, 2 mm. (f) CLSM images of MG6 cells labeling by CFDA SE cell tracer. Scale bar, 50 µm. (g) CLSM image of transplanted MG6 cells in lateral ventricle 3 days after transplantation. Scale bar, 2 mm. (h) CLSM images of Hoechst and mIL‐1β in the periventricular area and hippocampal CA3 subfield of middle‐aged mice after intralateral ventricular injection of MG6 and LLOMe‐treated CatB/MG6. Scale bar, 100 µm. (i) CLSM images of Hoechst, mIL‐1β, and Iba1 in the periventricular area and hippocampal CA3 subfield after intralateral ventricular injection of LLOMe‐treated CatB/MG6. Scale bar, 100 µm. (j, k) The recognition memory was evaluated by the novel object recognition test in 10‐month‐old mice 5 days after intralateral ventricle injection of cultured medium (control), MG6 cells, and LLOMe‐treated CatB/MG6 cells. (j) Time spent exploring the familiar and the novel object in the recognition trial. The results represent the mean ± SEM (cultured medium injection [control], n = 15; MG6 cell transplantation [MG6], n = 14; LLOMe‐treated CatB/MG6 cell transplantation [CatB/MG6 + LLOMe], n = 12). The asterisks indicate a statistically significant difference from the vehicle group (***p < 0.001, Student's t test). (k) Discrimination index (exploration of novel object minus exploration of familiar object/total exploration time). The results represent the mean ± SEM (cultured medium injection [control], n = 15; MG6 cells transplantation [MG6], n = 14; LLOMe‐treated CatB/MG6 cells transplantation [CatB/MG6 + LLOMe], n = 12). The asterisks indicate a statistically significant difference from 0 (**p < 0.01 and ***p < 0.001, Student's t test). The daggers indicate a statistically significant difference from the MG6 transplantation group (^††† p < 0.001, one‐way ANOVA test). (l) A schematic illustration representing the CatB leaked in the cytosol during normal aging play a critical role in the mitochondria‐derived ROS generation and inflammatory response through proteolytic degradation of TFAM, resulting in impairment of learning and memory