Kir2.1

A traumatic brain injury (TBI) can quadruple your risk for developing dementia and increase your chances of developing neurodegenerative diseases such as ALS. In a new study scientists use lab-grown human brain structures known as organoids to offer insights into why this is the case and how to mitigate the risk. A traumatic brain injury (TBI) can quadruple your risk for developing dementia and increase your chances of developing neurodegenerative diseases such as ALS. In a new study published in Cell Stem Cell, USC scientists use lab-grown human brain structures known as organoids to offer insights into why this is the case and how to mitigate the risk. In the study, former postdoc Jesse Lai and PhD student Joshua Berlind from the USC Stem Cell laboratory of Justin Ichida used human patient-derived stem cells to grow rudimentary brain structures known as organoids in the lab. They then injured these organoids with high-intensity ultrasound waves. The injured organoids showed some of the same features seen in TBI patients, including nerve cell death and pathological changes in tau proteins, as well as in a protein called TDP-43. The scientists found that the pathological changes in TDP-43 were more prevalent in organoids derived from patients with ALS or frontotemporal dementia, making their nerve cells more suspectable to dysfunction and death following injury. This suggests that TBI might increase the risk of developing these diseases even more for patients with a genetic predisposition. The worst injuries were sustained by nerve cells that share information -- called excitatory neurons -- located in the deep layers of the organoids. In their search for ways to protect these neurons against the effects of TBI, the scientists identified a gene called KCNJ2, which contains instructions for making channels that selectively allow potassium to pass through the cell membrane, helping to enable muscle contraction and relaxation. Inhibiting this gene had a protective effect on organoids derived from patients with and without ALS, as well as on mice, following a TBI. "Targeting KCNJ2 may reduce the death of nerve cells after TBI," said Ichida, who is the John Douglas French Alzheimer's Foundation Associate Professor of Stem Cell Biology and Regenerative Medicine at USC, and a principal investigator at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC. "This could have potential as either a post-injury treatment or as a prophylactic for athletes and others at high risk for TBI." About the authors and the study Co-corresponding author Ichida is also a co-founder of AcuraStem and Modulo Bio, a Scientific Advisory Board (SAB) member at Spinogenix and Vesalius Therapeutics, and an employee in the Research and Early Development group at BioMarin Pharmaceutical. Co-corresponding author Lai and co-author Violeta Yu were both employees of Amgen during the study, and currently work at Dewpoint Therapeutics. Named companies were not involved in this research project. First author Berlind is a PhD student in the Ichida Lab. Additional co-authors are Gabriella Fricklas, Cecilia Lie, Jean-Paul Urenda, Kelsey Lam, Naomi Sta Maria, Russell Jacobs, and Zhen Zhao from USC. Fifty percent of the work was supported by federal funding from the National Institute of Neurological Disorders and Stroke (NINDS) and the National Institute on Aging (grant F31NS117075), NINDS (grant R01 1R01NS097850-01), and the Department of Defense (grant 12907280). The project was also privately funded by an Amgen postdoctoral fellowship, the New York Stem Cell Foundation, the Tau Consortium, the Harrington Discovery Institute, the Alzheimer's Drug Discovery Foundation, the Association for Frontotemporal Dementia, and the John Douglas French Alzheimer's Foundation.

Ion channels play a crucial role in many cellular processes, including neuronal communication, muscle contraction or cell proliferation. Most multi subunit ion channels exist in two functional states, either closed or open. During gating, one should expect that all subunits undergo conformational changes. The absence of intermediate conduction levels is surprising and asks for an explanation. A team of researchers has created a smart model system to answer this important question. Ion channels play a crucial role in many cellular processes, including neuronal communication, muscle contraction or cell proliferation. Most multi subunit ion channels exist in two functional states, either closed or open. During gating, one should expect that all subunits undergo conformational changes. The absence of intermediate conduction levels is surprising and asks for an explanation. A team of researchers from the University of Vienna and the Washington University in St. Louis created a smart model system to answer this important question. The study is currently published in Nature Communications. Ion channels are membrane proteins that regulate the electrical activity of cells. In this study the scientific team investigated the inwardly rectifying potassium channel Kir2. This channel is crucial for maintaining a negative membrane potential in many cells. These channels are promising drug targets for treatment of cardiovascular diseases. To foster drug development, a detailed understanding of the gating mechanism is important. Intelligent model system & innovative methods "We designed a model system that allowed us to visualize the gating of individual subunits and track conductance changes," explains Grigory Maksaev from the Washington University in St. Louis. As a model system, the inwardly rectifying potassium channel Kir2 was used. This channel is crucial for maintaining a negative membrane potential in many cells. "We introduced an acidic residue near the channel gate. This led to novel states, so-called sub-conductance states" explains Eva Plessl from the Department of Pharmaceutical Sciences, University of Vienna. The life times of these sub-states were long enough to resolve them experimentally. Each of the observed sub-states represents a distinct subunit conformation. Interestingly, the sub-state occupancy is titratable by pH. "This suggests that protonation or deprotonation of individual acidic residues causes this phenomenon," explains Sun-Joo Lee from the Washington University in St. Louis. Sour is…less conductive "Molecular dynamics simulations with different protonation states of the acidic residue support this finding," explains Anna Weinzinger from the Department of Pharmaceutical Sciences, University of Vienna. The study reveals that each subunit gating transition leads to conductance level changes. This suggests that for a fully open channel, all subunits must move together. "By designing a smart model system, wehave answered a long-standing question about ion channel gating," explains Colin Nichols from the Washington University in St. Louis.

这项研究证实Kir2.1是TAMs代谢编程和功能极化的重要调控因子，系统地描述了TME中离子紊乱对免疫细胞和其他成分的影响，揭示了代谢模式和表型状态之间的内部联系，为阐明TAMs功能极化以适应TME的 要说肿瘤组织中的大冤种，非巨噬细胞莫属！ 肿瘤微环境（TME）作为一个稳定的微型生态体系，是肿瘤细胞快乐繁殖的“伊甸园”，但对于巨噬细胞来说，无疑是块连呼吸都费劲的“盐碱地”。巨噬细胞表示很委屈：“我不是真正的妥协，墙头草只是我求生存的保护色。” 物竞天择，适者生存。从肿瘤-免疫协同进化的角度来看，免疫细胞的确需要采取一些灵活走位来对抗严酷的TME，同时可能也会被赋予免疫抑制特性，例如瘤内高钾（K+）对肿瘤浸润T细胞的免疫抑制作用[1-2]。 受限于营养缺乏的TME，不同的细胞群会根据自身需求优先选择特定的代谢物维系生存，这种细胞特异性的营养偏好在驱动免疫细胞特定表型方面起着关键作用[3-4]。 尽管研究已经证实，在营养缺乏的TME中，肿瘤相关巨噬细胞（TAMs）的功能状态受到了极大影响[5-6]，然而，TAMs代谢重编程与其对外源营养物质偏好之间的内在联系尚未阐明。 针对此问题，浙江大学医学院丁克峰和王迪团队进行了系统研究，并作为共同通讯在《细胞·代谢》杂志上发表了研究成果。他们发现，瘤内高K+可通过内向整流的K+通道（Kir2.1）塑造TAMs表型，调控其功能极化，并显著抑制其抗肿瘤能力（M2型）[7]。 论文首页截图 该研究指出，Kir2.1是肿瘤调控TAMs功能极化的重要枢纽，对TAMs电化学依赖的营养摄取至关重要。基因敲除或药理阻断Kir2.1会干扰TAMs对谷氨酰胺（GLN）的摄取，导致TAMs代谢重编程，由氧化磷酸化为优势的代谢特征转化为糖酵解模式，使TAMs复极化至抗肿瘤状态（M1型），进而增强局部抗肿瘤免疫反应，显著抑制肿瘤进展。 瘤内高K+通过Kir2.1塑造TAMs代谢表型和功能状态的模式图 研究人员分析人结直肠癌（CRC）的肿瘤间质液（TIF）和TAMs表型发现，瘤内间质K+水平与M1型TAMs显著减少相关。体外TAMs诱导实验也证实，细胞外高K+会促进骨髓源性巨噬细胞（BMDMs）向促肿瘤状态的M2型TAMs表型极化。 为进一步探究高K+诱导TAMs功能极化的机制，研究人员对其进行了基因富集分析，结果显示，高K+抑制了一系列炎症相关基因集，而提高了氧化磷酸化、血管生成和免疫抑制细胞因子产生的基因集，这表明瘤内高K+对TAMs抗肿瘤表型的抑制，可能与其代谢特征改变相关。 经常规培养液和高K+培养液处理后巨噬细胞转录组的基因富集分析 没有艰难的生存环境，只有勇敢的巨噬细胞！在瘤内高K+的大环境下，巨噬细胞只能改造自身代谢来适应TME。但是，对于巨噬细胞上感应高K+并驱动TAMs功能极化的“哨兵”，我们依然无从得知。 于是，研究人员利用RNA高通量测序和膜片钳技术，最终锁定了内向整流K+通道Kir2.1。电生理数据显示，巨噬细胞上存在功能性Kir2.1，可被高K+电流激活，促进巨噬细胞向M2型TAMs极化。 基因敲除编码Kir2.1的基因Kcnj2后，M2型TAMs水平降低，且抗肿瘤细胞因子和生长因子分泌增加。这些数据表明，Kir2.1是高K+条件下TAMs极化的重要调节因子，基因敲除或药理阻断Kir2.1会导致TAMs复极化趋向抗肿瘤状态分化。 野生型（Kcnj2f/f）与Kcnj2敲除（Kcnj2CKO）TAMs分泌细胞因子的热图 接下来，研究人员建立了多种移植瘤模型，以探究TAMs表达Kir2.1在抗肿瘤免疫中的潜在作用。结果显示，敲除Kcnj2显著抑制了移植瘤的生长，解除了高K+对TAMs抗肿瘤作用的抑制，促使TAMs复极化为抗肿瘤的M1型，并明显增强了局部抗肿瘤免疫效应（TAMs吞噬凋亡肿瘤细胞能力下降；瘤内cGAMP、IFNγ和TNF-a水平增加；CD4+Treg细胞减少）。 以上结果表明，在离子紊乱的TME内，Kir2.1作为TAMs上感应高K+的“哨兵”，在TAMs促肿瘤表型极化中发挥了重要作用，而特异性阻断Kir2.1不仅使TAMs复极化恢复到抗肿瘤状态，而且增强了局部抗肿瘤免疫效应。 绕了一大圈，终于弄清楚了感知高K+并驱动TAMs极化的重要调控因子，那么问题来了，TAMs是如何一步步调整代谢模式来适应高K+环境的？ 研究人员进一步分析发现，敲除Kcnj2的TAMs，其优势代谢模式从氧化磷酸化转向了糖酵解。此外，Kcnj2CKO TAMs比Kcnj2f/f TAMs具有更高的膜电位，但GLN和脂肪酸摄取却明显下降，这表明Kir2.1调节的膜电位对TAMs摄取GLN至关重要，可能决定了TAMs水解GLN的代谢偏好，进而影响TAMs极化状态。 为进一步探索Kir2.1与肿瘤预后的关联性，研究人员对70例CRC患者进行了单中心回顾性队列研究。他们发现早期CRC患者TAMs上Kir2.1表达强度低于晚期患者，并且高表达Kir2.1的TAMs浸润与患者预后不良有关，这表明Kir2.1在TAMs上的表达强度与人类癌症进展和临床预后存在关联性。 单中心回顾性队列建立示意图及患者的生存曲线 最后，研究人员利用Kir2.1的选择性抑制剂ML133，对靶向Kir2.1的抗肿瘤治疗潜力进行了评估。 在CCR患者来源的类器官（PDO）中，ML133治疗极大增加了TAMs向M1型复极化的比例，而且，在PDO建立的异种移植瘤模型中，ML133与抗PD-1单抗联合可显著抑制肿瘤生长，具有更好的治疗效果，证实ML133靶向Kir2.1可能是一种调节TAMs复极化恢复抗肿瘤作用的潜在策略。 总的来说，这项研究证实Kir2.1是TAMs代谢编程和功能极化的重要调控因子，系统地描述了TME中离子紊乱对免疫细胞和其他成分的影响，揭示了代谢模式和表型状态之间的内部联系，为阐明TAMs功能极化以适应TME的基本机制，以及开发TAMs靶向免疫疗法提供新的见解。