TREM2 deficiency exacerbates cognitive impairment by aggravating α-Synuclein-induced lysosomal dysfunction in Parkinson's disease

Abstract

Cognitive impairment in Parkinson's disease (PD) is a widespread and rapidly progressive feature that impacts prognosis. Although TREM2 has been implicated in neuroprotection across various neurodegenerative diseases, its specific role in PD remains to be clarified. In this study, we first detected the hippocampus of human PD specimens and of the mutant A53T α-Synuclein transgenic mice (A53T mice), and found a significant increase in the number of TREM2⁺ microglia. To evaluate the effects of TREM2 deficiency, TREM2-deficient A53T mice (TREM2^-/-/A53T mice) were generated. In these mice, exacerbated cognitive impairment, neurodegeneration, disruption of synaptic plasticity, and accumulation of pathological α-Synuclein (α-Syn) in the hippocampus were observed, without any detected motor dysfunction. Despite increased infiltration of activated microglia surrounding α-Syn aggregates, lysosomal dysfunction in microglia was aggravated in the TREM2^-/-/A53T mice. In addition, transcriptional analyses and in vitro experiments further found that TREM2 knockdown inhibited the nuclear distribution of TFEB via the ERK1/2 pathway, exacerbating α-Syn-induced lysosomal dysfunction and causing more pathological α-Syn accumulation. Finally, HT22 cells were cocultured with TREM2 knockdown of BV-2 cells pretreated with recombinant human A53T α-Syn preformed fibrils (PFFs). The coculture experiments showed that TREM2 knockdown in BV-2 cells pretreated with PFFs enhanced the phosphorylation of α-Syn and promoted apoptosis in HT22 cells via inhibiting α-Syn degradation. In conclusion, TREM2 deficiency exacerbates cognitive impairment in PD by exacerbating α-Syn-induced microglial lysosomal dysfunction, identifying TREM2 as a potential therapeutic target.

Fig. 1. TREM2 deficiency impaired cognitive function in PD mice.

A Representative image of TREM2 (red) and IBA1 (green) immunofluorescence staining in hippocampal tissue sections of the PD and control specimens. Scale bar, 50μm. B Representative image of TREM2 (red) and IBA1 (green) immunofluorescence staining in hippocampus in C57BL/6 mice and A53T mice. Scale bar, 50 μm. C Quantification of number of TREM2⁺ IBA1⁺ cells in PD specimens and control specimens (n = 3, unpaired Student’s t test) and TREM2⁺ IBA1⁺ cells in C57BL/6 mice and A53T mice (n = 3, unpaired Student’s t test). D Recognition Index was calculated in NORT (n = 9, one-way ANOVA and Šídák’s multiple comparisons test). E Representative image showing the procedure and swimming paths obtained from each group in the acquisition phase in MWM. F Time spent in the target quadrant (s) in the acquisition phase in MWM (n = 9, one-way ANOVA and Šídák’s multiple comparisons test). G Representative image showing the paths obtained from each group in the acquisition phase in Open field test. Line chart showing the average escape latencies in each group in the space exploring phase in MWM (n = 9, two-way ANOVA and Tukey’s multiple comparisons test). Column charts showing. The error bars represent the ± SDs. **p < 0.01, ***p < 0.001. NORT, novel object recognition test; MWM, Morris water maze.

Fig. 2. TREM2 deficiency exacerbated α-Syn accumulation and lysosomal dysfunction in the hippocampus.

A Representative images of western blotting analysis of Oligomer α-Syn, p-α-Syn, and α-Syn expressions in each group. B Quantification of Oligomer α-Syn, P-α-Syn, and α-Syn proteins relative expression (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). C Representative images of α-Syn (red) and IBA1 (green) in the hippocampus of each group. Scale bar, 400μm. D Quantification of mean Fluorescence intensity of α-Syn in the hippocampus of each group (n = 3, unpaired Student’s t test). E Quantification of IBA1 within 20 μm of α-Syn (%) in the hippocampus of each group (n = 3, unpaired Student’s t test). F Representative images of IBA1 (red) and CD68 (green) immunofluorescence staining in the hippocampus of each group. Scale bar, 50μm. G Quantification of IBA1 colocalized with CD68 (%) in the hippocampus of each group (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). H Representative images of western blotting analysis of CathB, CathD, and Lamp1 expressions in each group. I Quantification of CathB, CathD, and Lamp1 proteins relative expression (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). J Representative images of CathB (red) and IBA1 (green) immunofluorescence staining in the hippocampus of each group. Scale bar, 10μm. K Representative images of Lamp1 (purple) and IBA1 (green) immunofluorescence staining in the hippocampus of each group. Scale bar, 10μm. L Quantification of area CathB colocalized with IBA1 (%) in the hippocampus of each group (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). M Quantification of IBA1 colocalized with Lamp1 (%) in the hippocampus of each group (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). The error bars represent the ± SDs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3. Knockdown of TREM2 exacerbated α-Syn accumulation and lysosomal dysfunction in BV-2 cells.

A Representative images of α-Syn (red) and Lamp1 (green) immunofluorescence staining in TREM2^KD BV-2 cells and NC^KD BV-2 cells. Scale bar, 5μm. B Quantification of mean Fluorescence intensity of α-Syn in TREM2^KD BV-2 cells and NC^KD BV-2 cells (n = 3, unpaired Student’s t test). C Quantification of MCC of α-Syn and Lamp1 in TREM2^KD BV-2 cells and NC^KD BV-2 cells (n = 3, unpaired Student’s t test). D Quantification of mean Fluorescence intensity of Lamp1 in TREM2^KD BV-2 cells and NC^KD BV-2 cells (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). E Representative images of DQ-BSA (red), Lysotracker (red), and CathB (red) in TREM2^KD BV-2 cells and NC^KD BV-2 cells. Scale bar, 10μm. F Quantification of integrated density of DQ-BSA, Lysotracker, and CathB in TREM2^KD BV-2 cells and NC^KD BV-2 cells (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). The error bars represent the ± SDs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PFFs, recombinant human A53T α-Syn pre-formed fibrils; MCC, Manders’ Colocalization Coefficients; TREM2^KD BV-2, BV-2 transfected with TREM2-shRNA, NC^KD BV-2, BV-2 transfected with LV-NC; MCC, Manders’ Colocalization Coefficients.

Fig. 4. Transcriptome analysis.

A Volcano plot for differentially expressed genes. B GO database on biological process of differentially expressed genes. C Identification key pathway by GO of differentially expressed genes. D KEGG pathway classification of differentially expressed genes during environment information processing. GO Gene Ontology, KEGG Kyoto Encyclopedia of Genes and Genomes.

Fig. 5. Knockdown of TREM2 aggravated α-Syn-induced lysosomal dysfunction in BV-2 cells via the ERK1/2/TFEB pathway.

A Representative images of western blotting analysis of TREM2, ERK1/2, and p-ERK1/2 expressions in TREM2^KD BV-2 cells and NC^KD BV-2 cells. B Representative images of western blotting analysis of Total-TFEB, Nuclear-TFEB, and Cytoplasm-TFEB expressions in TREM2^KD BV-2 cells and NC^KD BV-2 cells. C Quantification of TREM2, ERK1/2, p-ERK1/2, Total-TFEB, Nuclear-TFEB, and Cytoplasm-TFEB expressions proteins relative expression (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). D Representative images of TFEB (red) immunofluorescence staining in TREM2^KD BV-2 cells and NC^KD BV-2 cells. Scale bar, 10 μm. E Quantification of Nuclear- Cytoplasm TFEB Fluorescence ratio in TREM2^KD BV-2 cells and NC^KD BV-2 cells (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). F Representative images of DQ-BSA (red), Lysotracker (red), and CathB (red) in TREM2^KD BV-2 cells and NC^KD BV-2 cells. Scale bar, 10μm. G Representative images of α-Syn (red) and Lamp1 (green) immunofluorescence staining in TREM2^KD BV-2 cells and NC^KD BV-2 cells. Scale bar, 5 μm. H Quantification of integrated density of DQ-BSA, Lysotracker, and CathB in TREM2^KD BV-2 cells and NC^KD BV-2 cells (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). I Quantification of MCC of α-Syn and Lamp1 in TREM2^KD BV-2 cells and NC^KD BV-2 cells (n = 3, unpaired Student’s t test) and mean Fluorescence intensity of α-Syn in TREM2^KD BV-2 cells and NC^KD BV-2 cells (n = 3, unpaired Student’s t test). The error bars represent ± SDs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PFFs, recombinant human A53T α-Syn pre-formed fibrils; MCC, Manders’ Colocalization Coefficients; TREM2^KD BV-2, BV-2 transfected with TREM2-shRNA, NC^KD BV-2, BV-2 transfected with LV-NC.

Fig. 6. Knockdown of TREM2 aggravated α-Syn phosphorylation and apoptosis in HT22 cells.

A Coculture of BV-2 cells and HT22 cells. B Representative images of flow cytometry of HT22 cells in each group. C Representative images of TEM of apoptotic morphology of HT22 cells in each group. D Quantification of apoptosis of HT22 cells in each group (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). E Representative images of p-α-Syn (red) immunofluorescence staining in HT22 cells in each group. Scale bar, 10μm. F Quantification of mean Fluorescence intensity of p-α-Syn in HT22 cells in each group (n = 3, one-way ANOVA and Šídák’s multiple comparisons test). The error bars represent the ± SDs. ***p < 0.001, ****p < 0.0001. PFFs, recombinant human A53T α-Syn pre-formed fibrils; TEM, transmission electron microscopy.