Adenylate Kinase 2 Deficiency and the Failure of Myelopoiesis

腺苷酸激酶 2 缺乏和骨髓生成失败

基本信息

批准号：
10446518
负责人：
Katja Gabriele Weinacht
金额：
$ 59.91万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10446518
关键词：
ATP Synthesis Pathway Adenine Nucleotides Adult Affect Aspartate B-Lymphocytes Blood Carbon Carnitine Catabolism Cell Cycle Cells Citric Acid Cycle Clustered Regularly Interspaced Short Palindromic Repeats Cytoplasm Deamination Defect Development Electrons Energy Metabolism Enzymes Erythroid Excretory function Exhibits FRAP1 gene Failure Generations Genetic Transcription Hematological Disease Hematopoietic Hematopoietic stem cells Homeostasis Human Image Immunosuppression Impairment In Vitro Inosine Monophosphate Isotopes Knock-out Life Link Lipids Lymphopenia Malignant Neoplasms Mass Spectrum Analysis Mediating Megakaryocytes Metabolic Metabolic Diseases Mitochondria Mitochondrial Diseases Modeling Mus Myelogenous Myeloid Cells Myelopoiesis NADH NADH oxidase Natural regeneration Neutropenia Nucleoside Hydrolases Nucleotides Oxidation-Reduction Pathology Pathway interactions Patients Phenotype Phosphorylation Purines Pyruvate Recycling Ribosomal RNA Ribosomes Severe Combined Immunodeficiency Signal Transduction Stress System T-Lymphocyte Testing Therapeutic immunosuppression Translations Transplantation Xenograft procedure adenylate kinase cancer therapy cell type improved in vitro Model in vivo in vivo Model insight loss of function mutation metabolomics mitochondrial metabolism novel novel strategies nucleotide metabolism overexpression progenitor purine metabolism reticular dysgenesis stem stress reduction

项目摘要

PROJECT SUMMARY/ABSTRACT Cellular ATP demand and mitochondrial ATP synthesis are tightly linked. ATP synthesis, in turn, depends on (1) NADH generation in the TCA cycle and (2) recycling of the adenine nucleotide pool. When ATP is depleted and AMP rises, the cell increases energy generation and curtails energy expenditure until homeostasis is restored. In the context of mitochondrial pathology, the system becomes unhinged… Reticular Dysgenesis (RD) is a rare hematologic disease, caused by biallelic loss-of-function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the phosphorylation of AMP to ADP in the inter-membrane space to generate substrate for ATP synthesis1. RD patients suffer from severe congenital neutropenia, lymphopenia, and die early in life unless cured by hematopoietic stem cell transplantation5. We have developed a novel biallelic CRISPR-knockout model of AK2 in primary human hematopoietic stem and progenitor cells to precisely mimic the failure of human myelopoiesis in culture and after transplantation into mice. Using broad metabolomic profiling, our preliminary studies revealed that AK2-deficient myeloid progenitors exhibit a high NADH/NAD+ ratio and NAD+ depletion, consistent with reductive stress. In addition, AK2-deficient myeloid progenitors displayed a decrease in mitochondrial metabolites, including TCA cycle intermediaries and aspartate, while lipid carnitines were increased, and lipid droplets were found in the cytoplasm. We also detected highly elevated levels of the purine intermediate inosine monophosphate (IMP) and a decrease in rRNA and ribosome subunits. Interestingly, our studies suggest the high IMP stems from deamination of AMP, rather than a block in purine de novo synthesis. Taken together, these observations raise the possibility that AK2 deficiency causes mitochondrial reductive stress, curtailing TCA cycle activity and diverting carbon and electron pools into lipid synthesis while counteracting the accumulation of AMP. These findings led us to hypothesize that AK2 deficiency causes two interconnected but distinct pathologies: I. Reductive stress redirecting energy metabolism into lipid storage rather than OXPHOS; II. Accumulation of AMP and IMP, leading to defects in nucleotide metabolism. Our proposed studies will test if failure of myelopoiesis is primarily a result of reductive stress and impaired energy utilization, versus impaired purine metabolism, or both. We will determine if myelopoiesis can be rescued by correcting the NADH/NAD+ ratio or nucleotide pools. Lastly, we will validate our findings in an in vivo model of RD and investigate if different compensatory mechanisms in different blood lineages result in the RD phenotype. We use RD as a model to dissect escape mechanisms at the juncture of energy metabolism, redox stress, and nucleotide homeostasis. These insights will advance therapies for mitigating reductive stress and using cell type-specific manipulation of purine metabolism as a strategy for immunosuppression and cancer therapy.

项目摘要/摘要细胞ATP需求和线粒体ATP合成密切相关。 ATP合成又取决于（1）NADH在TCA循环中产生，（2）腺嘌呤核苷酸池的回收。当ATP是耗尽并放大了，细胞增加了能量的产生，并减少了能量消耗直到稳态已恢复。在线粒体病理的背景下，系统变得无关… 网状失调（RD）是一种罕见的血液学疾病，是由双重功能丧失突变引起的线粒体酶腺苷酸激酶2（AK2）。 AK2催化AMP在ADP中的磷酸化内膜间空间生成ATP合成1的底物1。 RD患者患有严重的先天性除非造血干细胞移植5固化，否则中性粒细胞减少症，淋巴细胞减少症和生命早期死亡。我们已经开发了一种新型的Biallec CRISPR-KNOCKOUT AK2的AK2中的原代人造血茎和祖细胞精确模仿人髓磷脂在培养和移植后的失败使用广泛的代谢组分析，我们的初步研究表明AK2缺乏髓样祖细胞暴露了高NADH/NAD+比率和NAD+部署，这与减轻的压力一致。在此外，AK2缺乏髓样祖细胞显示出包括TCA在内的线粒体代谢物的降低循环中间体和天冬氨酸，同时增加脂质肉碱，并在脂质液滴中发现细胞质。我们还检测到了高度升高的嘌呤中间肌苷一磷酸（IMP）和rRNA和核糖体亚基的减少。有趣的是，我们的研究表明了高IMP植物来自AMP的脱氨，而不是从头合成中的块。两者一起，这些观察提高AK2缺乏会导致线粒体减轻压力，减少TCA周期活动和将碳和电子池转移到脂质合成中，同时抵消AMP的积累。这些发现使我们假设AK2缺乏会导致两种相互联系但不同的病理： I.还原应力将能量代谢重定向到脂质储存而不是Oxphos； ii。 AMP和IMP的积累，导致核苷酸代谢缺陷。我们提出的研究将测试骨髓虫的失败是否是压力减轻和能量利用受损的结果，而不是纯代谢受损或两者兼而有之。我们将确定是否可以通过校正骨髓性检索 NADH/NAD+比率或核苷酸池。最后，我们将在RD的体内模型中验证我们的发现和研究不同血统中的不同补偿机制是否导致RD表型。我们使用RD作为模型，以剖析能量代谢，氧化还原应激，氧化还原的连接处的逃生机制和核苷酸稳态。这些见解将推进减轻压力并使用的疗法细胞类型的购买代谢作为免疫抑制和癌症治疗的策略。