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. 2024 Jan;73(1):131-144.
doi: 10.1007/s00011-023-01824-x. Epub 2023 Dec 13.

Homer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation

Weihao Lv #  1 , Qianqian Zhang #  2 , Yuanming Li #  3 , Dan Liu  1 , Xiuquan Wu  1 , Xin He  1 , Yuanyuan Han  1 , Xiaowei Fei  4 , Lei Zhang  5 , Zhou Fei  6
Affiliations

Homer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation

Weihao Lv et al. Inflamm Res. 2024 Jan.

Abstract

Objective: Proinflammatory necroptosis is the main pathological mechanism of ischemic stroke. Homer scaffolding protein 1 (Homer1) is a postsynaptic scaffolding protein that exerts anti-inflammatory effects in most central nervous system diseases. However, the relationship between Homer1 and proinflammatory necroptosis in ischemic stroke remains unclear.

Aim: This study aimed to investigate the role of Homer1 in ischemia-induced necroptosis.

Methods: C57BL/6 mice were used to establish a model of permanent middle cerebral artery occlusion model (pMCAO). Homer1 knockdown mice were generated using adeno-associated virus (AAV) infection to explore the role of Homer1 and its impact on necroptosis in pMCAO. Finally, Homer1 protein was stereotaxically injected into the ischemic cortex of Homer1flox/flox/Nestin-Cre +/- mice, and the efficacy of Homer1 was investigated using behavioral assays and molecular biological assays to explore potential mechanisms.

Results: Homer1 expression peaked at 8 h in the ischemic penumbral cortex after pMCAO and colocalized with neurons. Homer1 knockdown promoted neuronal death by enhancing necroptotic signaling pathways and aggravating ischemic brain damage in mice. Furthermore, the knockdown of Homer1 enhanced the expression of proinflammatory cytokines. Moreover, injection of Homer1 protein reduced necroptosis-induced brain injury inhibited the expression of proinflammatory factors, and ameliorated the outcomes in the Homer1flox/flox/Nestin-Cre+/- mice after pMCAO.

Conclusions: Homer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation. These data suggested that Homer1 is a novel regulator of neuronal death and neuroinflammation.

Keywords: Homer1; Ischemic stroke; Necroptosis; Neuroinflammation.

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Conflict of interest statement

The authors have declared that no competing interest exists.

Figures

Fig. 1
Fig. 1
Spatiotemporal expression pattern of Homer1 after pMCAO. A Homer1 protein expression of ischemic penumbra cortex after pMCAO 4 h, 8 h, and 12 h. The blots are representative of other replicates in those groups. B Quantification of result in A. C Representative images of Homer1 expression in ischemic penumbra cortex. D Immunofluorescence was used to detect the co-localization of Homer1 with neurons, microglia, and astrocytes. E Optical microscopic identification images of primary neurons. F Immunofluorescence identification of primary neurons. G Representative immunoblot of Homer1 in cultured primary neurons after OGD 1 h, 2 h, 4 h, and 8 h. H Quantification of result in G. For B, H: ***P < 0.001, and ****P < 0.0001 by one-way-ANOVA analysis. Data are presented as the mean ± SD; n = 6/group. All data are representative of three independent experiments
Fig. 2
Fig. 2
Homer1-KD promoted post-ischemic neuronal necroptosis. A, B Representative images A and quantification B of neuronal death based on TUNEL assay in the ischemic penumbra cortex of each group of mice at 8 h after pMCAO. C Effects of Homer1-KD on the expression level of p-RIPK1, p-RIPK3, and p-MLKL in the ischemic penumbra cortex of each group at 8 h after pMCAO. D–G Quantification of result in C. H Representative photographs of p-MLKL-positive neurons of brain tissue in each group at 8 h after pMCAO. I Quantification of result in H. J, K Representative images J and quantification K of primary neuronal death based on TUNEL assay in vitro of different groups at 4 h after OGD. L Effects of Homer1-KD on the expression level of p-RIPK1, p-RIPK3, and p-MLKL of different groups in vitro experiment at 4 h after OGD. M–P Quantification of result in L. For B, D–G, I, K–N: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P  < 0.0001 by one-way-ANOVA analysis. Data are presented as the mean ± SD; n = 6/group. All data are representative of three independent experiments
Fig. 3
Fig. 3
Homer1-KD aggravated proinflammatory cytokine production. A Representative experimental diagram of chip slide carrier from ischemic penumbra cortex tissue at 8 h after pMCAO. B Heat map of protein chip results. C ELISA of TNFα in supernatants of primary neurons in different groups. D ELISA of TNFR I in supernatants of primary neurons in different groups. E ELISA of IL-1β in supernatants of primary neurons in different groups. F ELISA of Fas Ligand in supernatants of primary neurons in different groups. For in vitro cell experiments C–F, detection time is at 4 h after OGD. For C–F: **P < 0.01, ****P  < 0.0001 by one-way ANOVA analysis. Data are presented as the mean ± SD; n = 3/group. All data are representative of three independent experiments
Fig. 4
Fig. 4
Homer1-KD exacerbated ischemic brain injury. A Longa scores of different groups. B Corner test of different groups. C Rota-rod test of different groups. D Detection of brain water content. E TTC-stained sections of different groups of mice. F Quantification of the infarct volume. G Representative photographs of Nissl staining of brain tissue in each group. H Apoptosis rate of different modified primary neuron cells by flow cytometry. I Quantification of result in H. J Cell viability test in different groups. K LDH release detection in different groups. Cell viability J or LDH release K was assessed and quantified for transfected neurons or without treatment (Con). The primary cultured neurons were transfected with plasmid for 2 days before cell viability or LDH release assay. For in vivo experiment A–G, detection time is at 8 h after pMCAO. For in vitro experiment H–K, detection time is at 4 h after OGD. For A–D, F, I, J, K: **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA analysis. Data are presented as the mean ± SD; n = 6/each group and neurological scores n = 10/each group. All data are representative of three independent experiments
Fig. 5
Fig. 5
Homer1 protein ameliorated the pathological indexes of pMCAO. A Genotype identification of Homer1flox/flox/ Nestin-Cre+/− mice. B Representative photographs of Tunel staining of brain tissue in each group. C Quantification of result in B. D Representative photographs of p-MLKL-positive neurons of brain tissue in each group. E Quantification of result in D. F Representative experimental diagram of chip slide carrier. G IgG staining for blood–brain barrier damage. H ELISA of TNFα of ischemic penumbra cortex in different groups. I ELISA of TNFR I of ischemic penumbra cortex in different groups. J ELISA of IL-1β of ischemic penumbra cortex in different groups. K ELISA of Fas ligand of ischemic penumbra cortex in different groups. All brain tissues in the experiment were taken from ischemic penumbra cortex tissue at 8 h after pMCAO. For C, E, H–K: *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test. Data are presented as the mean ± SD; n = 6/group. All data are representative of three independent experiments
Fig. 6
Fig. 6
Homer1 protein ameliorated neurological deficits and outcomes of pMCAO. A Representative photograph of TTC-stained sections of different groups of mice at 8 h after pMCAO. B Quantification of result in A. C Representative photographs of Nissl staining of ischemic penumbra cortex in each group. D Longa scores of different groups. E Rota-rod test of different groups. F Corner test of different groups. G Survival curve of mice in each group (n = 15) after pMCAO. For in vivo experiment (A–F), testing time is at 8 h after pMCAO. For B, D–G: **P < 0.01, and ***P < 0.001 by one-way-ANOVA analysis. Data are presented as the mean ± SD; TTC stain and Nissl stain n = 6/each group, neurological scores n = 10/each group, and survival time n = 15/each group. All data are representative of three independent experiments
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