Research ArticleCELLULAR NEUROSCIENCE

O-GlcNAcylation ameliorates the pathological manifestations of Alzheimer’s disease by inhibiting necroptosis

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Science Advances  13 Jan 2021:
Vol. 7, no. 3, eabd3207
DOI: 10.1126/sciadv.abd3207
  • Fig. 1 Association of O-GlcNAcylation and necroptosis in AD.

    (A and B) Western blot analysis of brain tissues of patients with AD and nondemented controls (non-AD) (n = 6) (A) and 5xFAD and WT mice (n = 5; 12 months of age) (B). (C to J) Quantification of O-GlcNAcylation–related proteins (C and G), necroptosis-related proteins (D and H), phosphorylated necroptosis–related proteins (E and I), and synaptophysin (F and J) in (A) and (B). The levels of phosphorylated necroptosis–related proteins were normalized to the levels of the corresponding total proteins. Values are presented as means ± SEM. *P < 0.05 and **P < 0.01 versus non-AD (C to F) or WT (G to J); two-tailed Student’s t test.

  • Fig. 2 OGA haploinsufficiency increased global O-GlcNAc levels in the brain and decreased activation of necroptosis.

    (A) Western blot analysis of OGA and O-GlcNAcylated proteins in brain samples of indicated mouse genotypes (n = 3). (B and C) Quantification of OGA protein (B) and O-GlcNAc protein (C) level in (A). (D) Immunostaining of O-GlcNAc and P-MLKL in the cortical region of mice samples (n = 3 to 4). DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 20 μm. (E) Necroptosis-related proteins in the brain of indicated genotypes of mice (n = 3). (F to H) Quantification of P-RIPK1 (F), P-RIPK3 (G), and P-MLKL (H) in (E). The levels of phosphorylated necroptosis–related proteins were normalized to the levels of the corresponding total proteins. (I) Western blot analysis of necroptosis-related proteins in RIPK3 immunoprecipitates from mouse brain samples. The precipitated immunoglobulin G (IgG) heavy chain is marked with an asterisk. IP, immunoprecipitation. (J) Quantification of O-GlcNAc, RIPK1, P-RIPK1, and RIPK3 binding to RIPK3 in (I). (K) Necroptosis-related proteins in insoluble fractions of brain samples collected from the indicated mouse genotypes (n = 3). (L to N) Quantification of necroptosis-related proteins RIPK1 (L), RIPK3 (M), and MLKL (N) in (K). Three slices of each sample were used to normalize each sample. Values are presented as means ± SEM. #P < 0.05, ##P < 0.01, and ###P < 0.001 versus WT; *P < 0.05 and **P < 0.01 versus 5xFAD; one-way analysis of variance (ANOVA) with Tukey’s test.

  • Fig. 3 Increased O-GlcNAcylation reduced neuronal loss in the brain tissue of 5xFAD.

    (A) Nissl staining in the cortical region of indicated mouse genotypes (n = 3 to 5). Scale bars, 100 μm. (B) The number of neurons in the cortical region of mice in (A). (C) Immunostaining of NeuN and P-MLKL in the cortical region of mice samples (n = 3 to 4). Scale bar, 20 μm. (D and E) The number of NeuN-positive cells (D) and NeuN/P-MLKL double-positive cells (E) in (C). (F) Flouro-Jade C staining in the cortical region of mice samples (n = 3). Scale bar, 100 μm. (G) Quantification of Flouro-Jade C–positive area in (F). (H) Immunostaining of Aβ and synaptophysin in the cortical region of mice samples (n = 3 to 4). Scale bar, 20 μm. (I) Synaptophysin protein level in the cortical region of mice samples (n = 3). (J) Quantification of synaptophysin density in (H). (K) Quantification of synaptophysin proteins levels in (I). Three to five slices of each sample were used to normalize each sample. Values are presented as means ± SEM. #P < 0.05, ##P < 0.01, and ###P < 0.001 versus WT; *P < 0.05 and **P < 0.01 versus 5xFAD; one-way ANOVA with Tukey’s test.

  • Fig. 4 Increased O-GlcNAc levels ameliorate cognitive deficits in 5xFAD mice.

    (A) Schematic drawing of the schedule of cognitive tests in indicated mouse genotypes (n = 9 to 14). (B) The number of entries in the Y-maze. (C) Time spent to the exploration of indicated mouse genotypes in novel object test. (D) Swimming speed of indicated mouse genotypes in the Morris water maze. (E) The Y-maze spontaneous alteration. (F) Preference for familiar and novel objects in the novel object recognition test. F, familiar object; N, novel object; N.S., not significant. (G) Duration of memory retention in the passive avoidance test. (H) The escape latency measured during the Morris water maze training trials. (I) Swimming traces obtained during training trials. (J) Time spent in the target quadrant where the hidden platform was previously placed during probe trial. (K) The number of times mice passed through the area in which the hidden platform was previously located during probe trial. (L) Swimming traces obtained during probe test. Values are presented as means ± SEM. #P < 0.05, ##P < 0.01, and ###P < 0.001 versus WT; *P < 0.05 and **P < 0.01 versus 5xFAD except familiar object (F); one-way ANOVA with Tukey’s test.

  • Fig. 5 Increased levels of O-GlcNAc reduce Aβ accumulation.

    (A) Immunostaining of Aβ plaques in the brain of 5xFAD and 5xFAD;OGA+/− mice (n = 4). Scale bars, 1 mm. (B) Quantification of Aβ plaque loads in (A). (C and D) Images of Aβ immunoreactivity with DAB staining in the cortex (C) and hippocampus (D) tissue of mice samples (n = 3 to 5). Scale bars, 100 μm. (E and F) Quantification of Aβ immunoreactivity detected with DAB staining in the cortex (E) and hippocampus (F) tissue of mice samples. (G to J) The levels of soluble (G and H) and insoluble (I and J) Aβ40 and Aβ42 measured by ELISA in mouse cerebral cortex samples (n = 4 to 6). (K) Staining of Aβ plaques with thioflavin S in the hippocampus of mice samples (n = 3 to 5). Scale bar, 150 μm. (L) The number of thioflavin S–positive dots in (K). Three slices of each sample were used to normalize each sample. Values are presented as means ± SEM. *P < 0.05 and **P < 0.01 versus 5xFAD; one-way ANOVA with Tukey’s test (G to J) and two-tailed Student’s t test (B, E, F, and L). n.d., not detectable.

  • Fig. 6 Increasing O-GlcNAcylation alters the inflammatory response in the brain of AD mice.

    (A) Aβ plaques and GFAP in the cortical region of 5xFAD and 5xFAD;OGA+/− mice (n = 4). Scale bar, 20 μm. (B) Aβ plaques and Iba1 in the cortical region of 5xFAD and 5xFAD;OGA+/− mice (n = 4). Scale bar, 20 μm. (C) Quantification of the GFAP area in (A). (D) Quantification of the Iba1 area in (B). (E and F) Western blot analysis of GFAP (E) and Iba1 (F) in mice brain samples (n = 3). (G and H) Quantification of GFAP (G) and Iba1 (H) proteins in mice samples. (I) Nitrite concentrations in conditioned media of 5xFAD and 5xFAD;OGA+/− primary microglia stimulated by LPS (n = 4). (J) Aβ-associated microglia in cerebral cortex of 5xFAD (n = 4) and 5xFAD;OGA+/− mice (n = 5). Scale bar, 20 μm. (K) The number of Aβ-associated microglia in (J). Three slices of each sample were used to normalize each sample. Values are presented as means ± SEM. ##P < 0.01 versus WT; *P < 0.05 and **P < 0.01 versus 5xFAD; except for 5xFAD microglia stimulated by LPS (I); two-tailed Student’s t test.

  • Fig. 7 Diagram showing the mechanism of how O-GlcNAcylation ameliorates AD pathology.

    RIPK3 is directly O-GlcNAcylated, which suppresses the phosphorylation of RIPK3 and interaction between RIPK1 and RIPK3. O-GlcNAcylation reduces the activation of necroptosis factors, formation of necrosome, and necroptotic cell death. In addition, O-GlcNAcylation restores phagocytic activity of microglia and mitochondria function. Therefore, O-GlcNAcylation ameliorates AD pathologies including accumulated Aβ, neuroinflammation, and neuronal loss.

Supplementary Materials

  • Supplementary Materials

    O-GlcNAcylation ameliorates the pathological manifestations of Alzheimer’s disease by inhibiting necroptosis

    Jinsu Park, Hee-Jin Ha, Eun Seon Chung, Seung Hyun Baek, Yoonsuk Cho, Hark Kyun Kim, Jihoon Han, Jae Hoon Sul, Jeongmi Lee, Eunae Kim, Junsik Kim, Yong Ryoul Yang, Mikyoung Park, Sung Hyun Kim, Thiruma V. Arumugam, Hyemin Jang, Sang Won Seo, Pann-Ghill Suh, Dong-Gyu Jo

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