Li Fang, Li Zhen, Li Shuying, Zhou Hong, Guo Yunhui, Wang Yongchen, Lei Bo, Miao Yanying, Wang Zhongfeng
State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institute of Brain Science, Fudan University, Shanghai, China.
Institute of Neuroscience and Third Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan Province, China.
Neural Regen Res. 2026 Apr 1;21(4):1628-1640. doi: 10.4103/NRR.NRR-D-24-00461. Epub 2024 Nov 13.
JOURNAL/nrgr/04.03/01300535-202604000-00043/figure1/v/2025-06-30T060627Z/r/image-tiff Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells, which is involved in retinal ganglion cell apoptosis in glaucoma. Modulation of Kir4.1 expression in Müller cells may therefore be a potential strategy for attenuating retinal ganglion cell damage in glaucoma. In this study, we identified seven predicted phosphorylation sites in Kir4.1 and constructed lentiviral expression systems expressing Kir4.1 mutated at each site to prevent phosphorylation. Following this, we treated Müller glial cells in vitro and in vivo with the mGluR I agonist DHPG to induce Kir4.1 or Kir4.1 Tyr 9 Asp overexpression. We found that both Kir4.1 and Kir4.1 Tyr 9 Asp overexpression inhibited activation of Müller glial cells. Subsequently, we established a rat model of chronic ocular hypertension by injecting microbeads into the anterior chamber and overexpressed Kir4.1 or Kir4.1 Tyr 9 Asp in the eye, and observed similar results in Müller cells in vivo as those seen in vitro . Both Kir4.1 and Kir4.1 Tyr 9 Asp overexpression inhibited Müller cell activation, regulated the balance of Bax/Bcl-2, and reduced the mRNA and protein levels of pro-inflammatory factors, including interleukin-1β and tumor necrosis factor-α. Furthermore, we investigated the regulatory effects of Kir4.1 and Kir4.1 Tyr 9 Asp overexpression on the release of pro-inflammatory factors in a co-culture system of Müller glial cells and microglia. In this co-culture system, we observed elevated adenosine triphosphate concentrations in activated Müller cells, increased levels of translocator protein (a marker of microglial activation), and elevated interleukin-1β mRNA and protein levels in microglia induced by activated Müller cells. These changes could be reversed by Kir4.1 and Kir4.1 Tyr 9 Asp overexpression in Müller cells. Kir4.1 overexpression, but not Kir4.1 Tyr 9 Asp overexpression, reduced the number of proliferative and migratory microglia induced by activated Müller cells. Collectively, these results suggest that the tyrosine residue at position nine in Kir4.1 may serve as a functional modulation site in the retina in an experimental model of glaucoma. Kir4.1 and Kir4.1 Tyr 9 Asp overexpression attenuated Müller cell activation, reduced ATP/P2X receptor-mediated interactions between glial cells, inhibited microglial activation, and decreased the synthesis and release of pro-inflammatory factors, consequently ameliorating retinal ganglion cell apoptosis in glaucoma.
内向整流钾通道Kir4.1的下调是诱导视网膜Müller细胞活化并与其他神经胶质细胞相互作用的关键步骤，这一过程参与青光眼视网膜神经节细胞的凋亡。因此，调节Müller细胞中Kir4.1的表达可能是减轻青光眼视网膜神经节细胞损伤的潜在策略。在本研究中，我们鉴定了Kir4.1中的七个预测磷酸化位点，并构建了在每个位点突变以防止磷酸化的Kir4.1慢病毒表达系统。在此之后，我们在体外和体内用代谢型谷氨酸受体I激动剂DHPG处理Müller神经胶质细胞，以诱导Kir4.1或Kir4.1 Tyr 9 Asp过表达。我们发现Kir4.1和Kir4.1 Tyr 9 Asp过表达均抑制Müller神经胶质细胞的活化。随后，我们通过向前房注射微珠建立了慢性高眼压大鼠模型，并在眼中过表达Kir4.1或Kir4.1 Tyr 9 Asp，在体内Müller细胞中观察到与体外相似的结果。Kir4.1和Kir4.1 Tyr 9 Asp过表达均抑制Müller细胞活化，调节Bax/Bcl-2平衡，并降低包括白细胞介素-1β和肿瘤坏死因子-α在内的促炎因子的mRNA和蛋白质水平。此外，我们研究了Kir4.1和Kir4.1 Tyr 9 Asp过表达对Müller神经胶质细胞和小胶质细胞共培养系统中促炎因子释放的调节作用。在这个共培养系统中，我们观察到活化的Müller细胞中三磷酸腺苷浓度升高，转运体蛋白（小胶质细胞活化的标志物）水平增加，以及活化的Müller细胞诱导的小胶质细胞中白细胞介素-1β mRNA和蛋白质水平升高。这些变化可通过Müller细胞中Kir4.1和Kir4.1 Tyr 9 Asp过表达来逆转。Kir4.1过表达，但不是Kir4.1 Tyr 9 Asp过表达，减少了活化的Müller细胞诱导的增殖和迁移小胶质细胞的数量。总的来说，这些结果表明，在青光眼实验模型中，Kir4.1第9位的酪氨酸残基可能作为视网膜中的功能调节位点。Kir4.1和Kir4.1 Tyr 9 Asp过表达减弱了Müller细胞活化，减少了ATP/P2X受体介导的神经胶质细胞间相互作用，抑制了小胶质细胞活化，并减少了促炎因子的合成和释放，从而改善了青光眼视网膜神经节细胞的凋亡。
