Identification of causal factors underlying cognitive deficits in a mouse model of childhood leukemia survival

摘要 急性淋巴细胞白血病（ALL）是最常见的儿童癌症， 接近90%，但儿童癌症的幸存者患长期认知缺陷的风险增加， 尤其影响执行功能（例如，注意力、计划、抑制控制、认知灵活性）。 化疗剂甲氨蝶呤（MTX）用于治疗大多数ALL患者，并且与化疗密切相关。 与执行功能缺陷有关。因此，迫切需要确定将 甲氨蝶呤暴露后认知功能障碍，指导制定干预策略，保护 发育大脑，减轻症状并优化所有幸存者的生活质量，在儿童时期， 青春期、青年期和成年期。 为此，我们开发了一种与白血病存活相关的小鼠模型， 将癌症暴露（小鼠白血病细胞系（L1210细胞））与当代化疗药物组合 在生命早期给予的药物（长春新碱和MTX，以及甲酰四氢叶酸补救药物）。PFC开发 这使得大脑的这一区域特别容易受到早期生活的影响 化疗我们的小鼠模型概括了所提出的实验的坚实前提， 在ALL患者中观察到执行功能缺陷。此外，为了应对早期癌症+ 化疗后，我们发现促炎分子IL-1和CCL 2增加， PFC内白色物质相关基因的减少。在Aim 1中，单细胞RNA测序将被 用于确定癌症和/或化疗对PFC转录谱的影响。 甲氨蝶呤破坏叶酸代谢，抑制细胞生长，但这种破坏也导致水平增加， 血浆和脑脊液中的促炎代谢产物同型半胱氨酸（HCY）。增加 HCY可以驱动炎症，氧化应激，并与白色和灰质损伤有关， 以及认知障碍。进一步的执行功能缺陷也与改变 突触功能，而小胶质细胞，一种脑内驻留的免疫细胞，可以通过 CD 11b（CR 3）-C3吞噬途径。因此，我们假设MTX驱动的HCY增加 水平将导致神经炎症和氧化应激，导致灰色和白色物质损伤， 前额叶皮层的突触修剪改变，这是执行功能缺陷的基础。 为了验证这一假设，降低同型半胱氨酸的策略（叶酸和B族维生素补充剂，或 抗氧化剂，N-乙酰半胱氨酸酰胺）将在目标2中进行评价。目的3检测IL-1活性的必要性 而目标4将测试小胶质细胞介导化疗相关认知功能的必要性。 缺陷，以及神经炎症和氧化应激，导致灰质和白色物质损伤，以及 前额叶皮层突触修剪的改变

Abstract Survival rates for acute lymphoblastic leukemia (ALL), the most common childhood cancer, are now close to 90%, but survivors of childhood cancer are at an increased risk for long term cognitive deficits, particularly affecting executive function (e.g., attention, planning, inhibitory control, cognitive flexibility). The chemotherapeutic agent methotrexate (MTX) is used to treat most ALL patients, and is closely associated with executive function deficits. Thus a pressing need exists to define the mechanisms that link MTX exposure to cognitive dysfunction, to guide development of intervention strategies to protect the developing brain, reduce symptoms and optimize quality of life in ALL survivors, during childhood, adolescence, young and full adulthood. To that end, we have developed a translationally relevant mouse model of leukemia survival that combines cancer exposure (mouse leukemic cell line (L1210 cells) with contemporary chemotherapeutic drugs (vincristine and MTX, with leucovorin rescue) administered during early life. PFC development extends through adolescence, which renders this area of the brain particularly vulnerable to early life chemotherapy. Providing a solid premise for the proposed experiments, our mouse model recapitulates executive function deficits observed in ALL patients. Additionally, in response to early life cancer + chemotherapy, we have found an increase in the proinflammatory molecules IL-1 and CCL2, as well as a decrease in white matter associated genes within the PFC. In Aim 1, single cell RNA sequencing will be used to define the effects of cancer and/or chemotherapy on the transcriptional profile of the PFC. MTX disrupts folate metabolism to inhibit cell growth, but this disruption also leads to increased levels of the proinflammatory metabolite, homocysteine (HCY), in both plasma and cerebrospinal fluid. Increased HCY can drive inflammation, oxidative stress and is associated with both white and gray matter damage, as well as cognitive impairment. Further executive function deficits have also been linked to altered synaptic function, and microglia, a brain resident immune cell, can contribute to synapse elimination via the CD11b(CR3)-C3 phagocytic pathway. Therefore, we hypothesize that MTX-driven increased HCY levels will lead to neuroinflammation and oxidative stress, leading to gray and white matter damage, and altered synaptic pruning in the prefrontal cortex, which underlie deficits in executive function. To test this hypothesis, HCY-lowering strategies (folate and B vitamin supplementation, or the antioxidant, N-acetylcysteine amide) will be evaluated in Aim 2. Aim 3 will test the necessity of IL-1 activity while Aim 4 will test the necessity of microglia in mediating the chemotherapy-associated cognitive deficits, as well as neuroinflammation and oxidative stress, leading to gray and white matter damage, and altered synaptic pruning in the prefrontal cortex