Proteasome activation to protect the white matter in neonatal hypoxic-ischemic encephalopathy.

新生儿窒息所致的新生儿缺氧缺血性脑病(HIE)可导致持续严重的神经系统疾病 残疾，即使在接受治疗性低温的患者中也是如此。我们在临床研究中发现，脑白质损伤 低温治疗后MRI上仍有异常改变。因此，体温过低并不能完全起到保护作用。脑白质损伤是一个突出的 然而，在接受HIE低温治疗的新生儿中观察到的神经功能障碍的部分尚未得到充分研究。 保护脑白质的治疗辅助手段可能会降低HIE患者永久性神经损伤的风险。 新生猪的缺氧缺血(HI)具有类似人类的白质束，产生类似于 包括临床研究中观察到的脑白质损伤在内的足月儿缺氧缺血性脑病。我们的模特 包括临床相关的全身降温、0.5C/h复温、镇静、持续血流动力学 监测、呼吸机支持和纠正血气和电解质异常以模拟临床新生儿 重症监护室。初步数据表明，蛋白酶体功能不足介导了持续性脑白质损伤 低氧和低体温后。我们推测白质蛋白酶体不足导致氧化清除失败。 受损的蛋白质，导致少突胶质细胞凋亡，可能破坏少突胶质细胞前体成熟， 缺氧缺血和亚低温后髓鞘和轴突损伤，脑白质体积丢失。 我们将阐明蛋白酶体在全身缺氧和过夜低温后脑白质损伤中的作用。 新生的猪。白质损伤和少突胶质细胞生物学将通过神经病理学(包括 少突胶质前体成熟、体视学、细胞死亡和电子显微镜)和生物化学(包括 蛋白质翻译后修饰和蛋白酶体组成和活性)在HI后1个月恢复。T- 迷宫神经认知行为测试与神经病理学的相关性将提供一个功能结果。我们开发了 基因调控新生猪白质不同靶区蛋白酶体活性的新方法 前脑使用病毒介导的蛋白酶体激活亚基的强制表达或蛋白酶体的抑制 短发夹状小干扰RNA。我们还将使用一种小分子蛋白酶体抑制剂来确定 蛋白酶体抑制会加重脑白质损伤。此外，我们将测试药物oluropein的潜在保护作用 白色物质。油菜素是一种易于生物利用的化合物，具有蛋白酶体激活特性，临床副作用少。 效果。将使用一种静脉给药方案来保护少突胶质细胞和髓鞘，增加 蛋白酶体表达，并促进缺氧和低温后氧化蛋白的清除。我们将确定是否 油菜素作用于标准蛋白酶体或免疫蛋白酶体。培养人少突胶质细胞的实验研究 将验证蛋白酶体作为治疗靶点，以及在缺氧糖剥夺后oluropein的作用。这 该项目将通过研究蛋白酶体的新机制来推动新生儿缺氧缺血性脑病和细胞生物学领域的发展 功能不全导致脑白质耐低温性损伤。我们将发现蛋白酶体激活是否是一种 相关的亚低温辅助治疗，以保护脑白质和改善HIE的神经预后。

Neonatal hypoxic-ischemic encephalopathy (HIE) from birth asphyxia causes persistent and severe neurologic disabilities, even in patients who receive therapeutic hypothermia. We found in clinical studies that white matter injury on MRI persists after hypothermic treatment. Thus, hypothermia is not fully protective. White matter injury is a prominent yet understudied component of the neurologic disabilities observed in neonates who receive hypothermia for HIE. Therapeutic adjuncts that protect the white matter might reduce the risk of permanent neurologic injury in HIE. Hypoxia-ischemia (HI) in neonatal pig, which has human-like white matter tracts, produces brain damage similar to that of full-term human newborns with HIE, including the white matter injuries observed in clinical studies. Our model includes clinically relevant whole-body hypothermia, rewarming at 0.5°C/h, sedation, continuous hemodynamic monitoring, ventilator support, and correction of blood gas and electrolyte abnormalities to mimic clinical neonatal intensive care. Preliminary data suggest that insufficient proteasome function mediates persistent white matter injury after HI and hypothermia. We postulate that white matter proteasome insufficiency causes a failure to clear oxidatively damaged proteins, causing oligodendrocyte apoptosis, potential disruption of oligodendrocyte precursor maturation, myelin and axonal injury, and white matter volume loss after HI and hypothermia. We will elucidate the proteasome’s role in white matter injury after whole-body HI and overnight hypothermia in neonatal swine. White matter injury and oligodendrocyte biology will be studied with neuropathology (including oligodendrocyte precursor maturation, stereology, cell death, and electron microscopy) and biochemistry (including protein post-translational modification and proteasome composition and activity) through 1 month recovery after HI. T- maze neurocognitive behavior testing with neuropathology correlation will provide a functional outcome. We developed new methods to genetically modulate proteasome activity in distinct, targeted regions of white matter in neonatal pig forebrain using virus-mediated enforced expression of a proteasome activator subunit or proteasome inhibition with short hairpin small interfering RNA. We will also use a small molecule proteasome inhibitor to determine whether proteasome inhibition aggravates white matter injury. Moreover, we will test the potential of the drug oleuropein to protect white matter. Oleuropein is a readily bioavailable compound with proteasome activating properties and few clinical side effects. An intravenous oleuropein dosing regimen will be used that protects oligodendrocytes and myelin, increases proteasome expression, and promotes clearance of oxidized proteins after HI and hypothermia. We will identify whether oleuropein acts on the standard proteasome or the immunoproteasome. Cultured human oligodendrocyte experiments will validate the proteasome as a therapeutic target and oleuropein’s actions after oxygen glucose deprivation. This project will advance the neonatal HI and cell biology fields by investigating novel mechanisms by which proteasome insufficiency mediates hypothermia-resistant injury in white matter. We will discover whether proteasome activation is a relevant therapeutic adjunct to hypothermia to protect white matter and improve neurologic outcomes in HIE.