Cysteinyl Leukotrienes in HIV Brain Injury

HIV-1感染可诱发痴呆，目前尚无治疗方法。我们和其他实验室的实验证据强烈表明，HIV-1感染和大脑中小胶质细胞和巨噬细胞(M)的神经毒性刺激会引发神经元损伤和神经发生障碍。我们最近观察到，有丝分裂原活化蛋白激酶（MAPK） p38在M - /小胶质细胞中诱导HIV/gp120毒性和在M -毒素启动细胞凋亡的神经元中都是必需的。在后续研究中，我们发现p38？MAPK通过特异性sirna下调半胱氨酸白三烯合成酶（LTC4S）。我们还发现，阻断半胱氨酸-白三烯受体1 （CysLTR1）可以保护脑皮质神经元免受gp120刺激或hiv感染的毒性。因此，我们拟在体内研究LTC4S基因缺失或CysLTR1阻断如何影响HIV-1或其包膜gp120引起的脑损伤。我们假设消除CysLT的产生或抑制CysLTR1可以防止HIV和gp120诱导的神经元损伤和行为障碍。长期目标是找到新的保护策略，防止艾滋病毒感染造成脑损伤。具体目的（SA）是：(1)在体内确定半胱氨酸-白三烯合成酶（LTC4S）基因缺失是否能预防HIV/gp120转基因小鼠模型的神经元损伤和行为障碍。(2)在体内研究药物抑制CysLTR1是否能改善HIV/gp120转基因小鼠模型中的神经元损伤。(3)体外评估阻断CysLTR1或敲除LTC4S （KO）如何在hiv诱导的巨噬细胞毒素存在下促进神经元存活。表达HIV/gp120的转基因小鼠将与LTC4SKO动物（SA1）杂交，孟鲁司特将用于阻断CysLTR1 （SA2）。将分别比较LTC4SKO与野生型、孟鲁司特与载药治疗的HIV/gp120转基因小鼠SA1和sa2的记忆和认知行为表现、神经元损伤和胶质细胞活化。对于SA3，我们将使用除LTC4SKO外的CysLTR1以及暴露于hiv感染和未感染的原代人M的条件培养基中的野生型脑皮质神经元和星形胶质细胞的药理抑制。这三种sa都将采用基于多维质谱的散弹枪脂质组学来分析cyslt与大脑不同部位（SA1和sa2）以及神经元、星形胶质细胞和M - (SA3)中的其他细胞脂质和介质的比较。对所有三种sa进行神经细胞和突触标记免疫标记，并用Hoechst染料染色核DNA后，用反褶积显微镜分析神经元损伤和死亡。所有三个Specific Aims都将验证一个前提，即删除CysLTR1的产生或阻断CysLTR1可以阻止炎症和损伤过程，从而有利于神经保护机制，如p38 MAPK和Caspase 3活性的降低，Akt活性的增加。我们还将在体内评估LTC4SKO和CysLTR1阻断是否可以在HIV/gp120存在的情况下保持记忆和认知并改善胶质瘤。

DESCRIPTION: Infection with HIV-1 can induce dementia for which a treatment is currently not available. Experimental evidence from our and other laboratories strongly suggests that HIV-1 infection and neurotoxic stimulation of microglia and macrophages (M�) in the brain trigger neuronal damage and impairment of neurogenesis. We recently observed that mitogen-activated protein kinase (MAPK) p38 was required in both M�/microglia for induction of HIV/gp120 toxicity and in neurons for initiation of apoptosis by M� toxins. In follow-up studies we found that knockdown of p38? MAPK by specific siRNAs down-regulated cysteinyl leukotriene synthase (LTC4S) in M�. We also discovered that blockade of the cysteinyl-leukotriene receptor 1 (CysLTR1) protected cerebrocortical neurons against toxicity of gp120-stimulated or HIV-infected M�. Therefore, we propose to study in vivo how genetic deletion of LTC4S or blockade of CysLTR1 affects brain injury caused by HIV-1 or its envelope gp120. We hypothesize that ablation of CysLT production or CysLTR1 inhibition can prevent HIV and gp120 from inducing neuronal injury and behavioral impairment. The long-term objectives are to find new protective strategies against brain injury by HIV infection. The specific aims (SA) are: (1) To determine in vivo whether genetic deletion of cysteinyl-leukotriene synthase (LTC4S) prevents neuronal injury and behavioral impairment in a HIV/gp120 transgenic mouse model. (2) To investigate in vivo whether pharmacological inhibition of CysLTR1 ameliorates neuronal damage in a HIV/gp120 transgenic mouse model. (3) To assess in vitro how the blockade of CysLTR1 or knockout (KO) of LTC4S enables neuronal survival in the presence of HIV-induced macrophage toxins. Transgenic mice expressing HIV/gp120 will be cross-bred with LTC4SKO animals (SA1) and Montelukast will be used to block CysLTR1 (SA2). Memory and cognition-based behavioral performance, neuronal injury and glial cell activation will be compared in LTC4SKO versus wild-type and Montelukast- versus vehicle-treated HIV/gp120-transgenic mice for SA1 and 2, respectively. For SA3, we will use pharmacological inhibition of CysLTR1 besides LTC4SKO and wild-type cerebrocortical neurons and astrocytes exposed to conditioned media of HIV-infected and un-infected, primary human M�. All three SAs will employ multi-dimensional mass spectrometry-based shotgun lipidomics to profile CysLTs in comparison to other cellular lipids and mediators in different parts of the brain (SA1 and 2) and neurons, astrocytes and M� (SA3). For all three SAs neuronal injury and death will be analyzed by deconvolution microscopy after immunolabeling for neuronal cellular and synaptic markers and staining of nuclear DNA by Hoechst dye. All three Specific Aims will test the premise that deletion of CysLT production or blockade of CysLTR1 prevents inflammatory and injurious processes in favor of neuroprotective mechanisms, such as reduced activity of p38 MAPK and Caspase 3, and increased activity of Akt,. We will also assess in vivo whether LTC4SKO and CysLTR1 blockade can preserve memory and cognition and ameliorate gliosis in the presence of HIV/gp120.