CNS defects in a murine Zellweger syndrome model

描述(由申请人提供)：我们的实验室一直在研究人类过氧化物酶体生物发生障碍Zellweger综合征的小鼠PEX2缺失动物模型。我们已经确定体内的过氧化体功能障碍会影响中枢神经系统发育过程中神经元的增殖、迁移、存活和分化。然而，内源性中枢神经系统缺陷对外源性全身器官功能障碍的作用以及过氧素体脂质缺陷或特定的过氧素体途径缺陷的作用尚未确定。我们提出了一系列综合的体外和体内方法来解决以下问题：具体目标1.PEX2-/-小鼠神经元迁移和分化的缺陷是由中枢神经系统固有的还是外部的因素引起的？在导致这些异常的过程中，过氧物体性脂质缺陷或特定的过氧物性B氧化途径缺陷或纤溶酶原途径的作用是什么？我们将使用大脑和小脑切片培养以及小脑神经元-神经胶质培养，通过视频显微镜直接显示对照组和PEX2-/-小鼠神经元迁移的动力学。将检测对照和PEX2突变的小脑浦肯野细胞和颗粒神经元的体外分化情况。我们将确定纠正血浆原或二十二碳六烯酸缺陷对PEX2神经元细胞缺陷的影响。我们将使用这些神经元迁移和分化检测方法，将在PEX2-/-小鼠中观察到的中枢神经系统缺陷与仅限于血浆蛋白原和/或B-氧化途径(MFE2-/-、MFE2/MFE1-/-、PEX7-/-)的过氧化体缺陷小鼠中观察到的中枢神经系统缺陷进行比较。我们将通过体外和体内转染法在选定的CNS细胞类型或脑区恢复PEX2功能的方法来评估CNS固有的和系统性的过氧化体功能障碍在CNS缺陷发病机制中的作用。或者，我们将使用体内移植方法，将正常或过氧化物酶体缺陷的神经前体细胞移植到发育正常或突变的大脑皮层或小脑中。具体目标#2：肝脏因素是否与过氧化物酶缺陷症小鼠的中枢神经系统缺陷有关？我们将评估当体内胆汁酸缺乏被纠正时，PEX2-/-小鼠的中枢神经系统发育是如何改变的。我们将确定胆汁酸产物是否在PEX2/小鼠的大脑中积聚。我们将通过测定添加外源性胆汁酸中间体或肝细胞条件培养液对对照、PEX2-/、血浆丙二醛或B-氧化缺陷小鼠体外神经元缺陷的影响，来检验来自PEX2突变肝脏的有毒物质是否导致中枢神经系统功能障碍。具体目标#3：过氧化物体在小脑浦肯野细胞和/或颗粒神经元发育中的作用是什么？小脑浦肯野细胞和/或颗粒神经元中PEX2基因的条件性缺失将决定在没有系统性器官过氧化酶体缺乏症的情况下，过氧酶体在这些神经元的发育中所起的作用。

DESCRIPTION (provided by applicant): Our laboratory has been studying a murine PEX2 null animal model for the human peroxisomal biogenesis disorder Zellweger syndrome. We have determined that peroxisomal dysfunction in vivo affects neuronal proliferation, migration, survival and differentiation during CNS development. However, the contributions from intrinsic CNS defects versus extrinsic systemic organ dysfunctions and the role of peroxisomal lipid deficiencies or specific peroxisomal pathway defects have not been determined. We propose an integrated series of in vitro and in vivo approaches to address the following questions: Specific Aim #1. Are the defects in neuronal migration and differentiation in PEX2 -/- mice caused by factors intrinsic or extrinsic to the CNS? What is the role of peroxisomal lipid deficiencies or specific defects in peroxisomal B-oxidation or plasmalogen pathways in causing these abnormalities? We will directly visualize the dynamics of migrating neurons from control and PEX2 -/-mice by videomicroscopy using cerebral and cerebellar slice cultures and cerebellar neuron-glial cultures. The in vitro differentiation of control and PEX2 mutant cerebellar Purkinje cells and granule neurons will be examined. We will determine the effect of correcting deficiencies for plasmalogens or docosahexaenolc acid on the cellular defects in PEX2 neurons. We will compare the CNS defects observed in PEX2 -/- mice with those seen in mice with peroxisomal defects limited to plasmalogen and/or B-oxidation pathways (MFE2 -/-, MFE2/MFE1 -/-, PEX7 -/-) using these assays for neuronal migration and differentiation. We will evaluate the role of CNS intrinsic versus systemic peroxisomal dysfunction in the pathogenesis of CNS defects by both in vitro and in vivo transfection methods to restore PEX2 function in selected CNS cell types or brain regions. Alternatively, we will use in vivo transplantation of normal or peroxisome-defective neuronal progenitors into normal or mutant developing cerebral cortex or cerebellum. Specific Aim #2: Do hepatic factors contribute to the CNS defects in peroxisome defective mice? We will evaluate how CNS development is altered when bile acid deficiency is corrected in vivo in PEX2 -/-mice. We will determine whether bile acid products accumulate in the brain of PEX2 / mice. We will examine whether toxic substances from the PEX2 mutant liver cause CNS dysfunction by determining the effect of adding exogenous bile acid intermediates or hepatocyte conditioned medium on the in vitro neuronal defects in control, PEX2 -/, plasmalogen or B-oxidation defective mice. Specific Aim #3: What is the role for peroxisomes in developing cerebellar Purkinje cells and/or granule neurons? Conditional deletion of the PEX2 gene in cerebellar Purkinje cells and/or granule neurons will determine the role of peroxisomes for the development of these neurons in the absence of systemic organ peroxisome deficiency.