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综合征的鼠PEX 2无效动物模型。我们已经确定，体内过氧化物酶体功能障碍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CNS缺陷与外源性全身器官功能障碍的贡献以及过氧化物酶体脂质缺乏或特定过氧化物酶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。PEX 2-/-小鼠神经元迁移和分化的缺陷是由中枢神经系统的内在因素还是外在因素引起的？过氧化物酶体脂质缺乏或过氧化物酶体B-氧化或血浆酶原途径中的特定缺陷在引起这些异常中的作用是什么？我们将通过使用大脑和小脑切片培养物和小脑神经元-胶质细胞培养物的视频显微镜直接观察来自对照和PEX 2-/-小鼠的迁移神经元的动态。将检查对照和PEX 2突变小脑浦肯野细胞和颗粒神经元的体外分化。我们将确定纠正缩醛磷脂或二十二碳六烯酸对PEX 2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试验，比较在PEX 2-/-小鼠中观察到的CNS缺陷与在具有过氧化物酶体缺陷的小鼠中观察到的CNS缺陷，所述过氧化物酶体缺陷限于缩醛磷脂和/或B-氧化途径（MFE 2-/-、MFE 2/MFE 1-/-、PEX 7-/-）。我们将通过体外和体内转染方法评估CNS内在与全身过氧化物酶体功能障碍在CNS缺陷发病机制中的作用，以恢复选定CNS细胞类型或脑区域中的PEX 2功能。或者，我们将使用正常或过氧化物酶体缺陷的神经元祖细胞体内移植到正常或突变的发育中的大脑皮层或小脑。具体目标#2：肝脏因素是否导致过氧化物酶体缺陷小鼠的CNS缺陷？我们将评估当在PEX 2-/-小鼠体内纠正胆汁酸缺乏时，CNS发育如何改变。我们将确定胆汁酸产物是否在PEX 2/小鼠的脑中积累。我们将通过确定添加外源性胆汁酸中间体或肝细胞条件培养基对对照、PEX 2-/、缩醛磷脂或B-氧化缺陷小鼠的体外神经元缺陷的影响来检查来自PEX 2突变体肝脏的毒性物质是否引起CNS功能障碍。具体目标#3：过氧化物酶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PEX 2基因的条件性缺失将决定在没有全身器官过氧化物酶体缺乏的情况下过氧化物酶体对这些神经元发育的作用。

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.