A model of neuronal cell development and differentiation: Mouse rod photoreceptor

描述（由申请人提供）：光感受器是高度特化的纤毛上皮细胞，很容易接近并在出生后进行细胞分化。我们研究项目的长期目标是了解脊椎动物光感受器发育和细胞分化的分子信号通路，特别关注这些通路的破坏如何导致早发性视力丧失。该提案的目的是确定蛋白PRA1在视杆光感受器细胞分化中的作用。在发育过程中，前体光感受器细胞退出细胞周期，并经历转化，产生具有独特感觉特化的高度极化细胞。虽然光感受器的形态分化被很好地表征，但对其潜在的分子机制知之甚少。rd1小鼠视网膜以杆状光感受器的早发性、快速变性为特征，是第一个被发现的视网膜色素变性动物模型。有趣的是，在退行性改变出现之前，rd1视杆细胞会表现出发育缺陷，包括不能进行正常的突触发生和外节分化，这为光感受器发育和先天性神经元疾病的研究提供了一个模型。在初步研究中，我们对野生型小鼠视网膜进行了微阵列分析，并将其与rd1突变体视网膜在P2-8时进行了比较，之后发生了光感受器凋亡。在rd1小鼠中，只有两个基因在所有时间点持续且显著下调：突变基因PDE6b和戊烯酰化兔受体1 （PRA1）。已知Rab家族中的小gtpase对于参与维持成熟光感受器外节和突触的囊泡运输至关重要。PRA1调节真核细胞中Rab gtpase向细胞膜的传递，尽管其确切的功能和作用机制尚不清楚。我们假设PRA1对水泡运输的调节对于杆状光感受器的突触和外节分化是必要和充分的。我们提出通过以下具体目的来验证我们的假设：(1)使用Western blot和免疫组织化学确定PRA1在成年和发育中的野生型和rd1小鼠视网膜中的表达模式；(2)使用基于RNA的功能丧失和功能获得技术确定PRA1在视杆光感受器突触和外节分化中是否必要和充分。这项工作的创新之处在于，它首次确定了PRA1在神经元发育和疾病的动物模型中的重要性。在完成这项工作后，我们期望确定PRA1表达是否可以恢复rd1小鼠视网膜中正常的视杆光感受器细胞分化。这些结果最终有望对发育神经生物学领域产生重大影响，并为异常分化的潜在机制提供见解，从而改善儿童视觉健康。

DESCRIPTION (provided by applicant): Photoreceptors are highly specialized ciliated epithelial cells that are easily accessible and undergo cell differentiation postnatally. The long-term goal of our research program is to understand the molecular signaling pathways underlying vertebrate photoreceptor development and cell differentiation, with particular focus on how disruption of these pathways leads to early onset vision loss. The objective of this proposal is to determine the role of the protein PRA1 in rod photoreceptor cell differentiation. During development, precursor photoreceptor cells exit the cell cycle and undergo a transformation to produce highly polarized cells with unique sensory specializations. Although morphological differentiation of photoreceptors is well characterized, less is known about the underlying molecular mechanisms. The rd1 mouse retina, characterized by early-onset, rapid degeneration of rod photoreceptors, was the first identified animal model of retinitis pigmentosa. Interestingly, rd1 rods show developmental defects, including failure to undergo normal synaptogenesis and outer segment differentiation, significantly before degenerative changes appear, presenting a model for investigations of photoreceptor development as well as a congenital neuronal disease. In preliminary studies, we have performed microarray analysis of wild type mouse retina compared to rd1 mutant retina at P2-8, prior to onset of photoreceptor apoptosis. Only two genes were consistently and significantly downregulated in the rd1 mouse at all time points: PDE6b, the mutant gene, and Prenylated rab acceptor 1 (PRA1). Small GTPases in the Rab family are known to be critical for vesicular trafficking involved in maintenance of both the outer segment and the synapse in the mature photoreceptor. PRA1 regulates delivery of Rab GTPases to membranes in eukaryotic cells, although its precise function and mechanism of action are poorly understood. We hypothesize that PRA1 regulation of vesicular trafficking is necessary and sufficient for synaptic and outer segment differentiation in rod photoreceptors. We propose to test our hypothesis with the following specific aims: (1) Determine the expression pattern of PRA1 in the adult and developing wild type and rd1 mouse retinas using Western blot and immunohistochemistry, and (2) determine whether PRA1 is necessary and sufficient for synaptic and outer segment differentiation in rod photoreceptors using RNA based loss-of-function and gain-of-function techniques. The proposed work is innovative in being the first to identify the significance of PRA1 in an animal model of neuronal development and disease. Upon completion of this work, we expect to have determined whether PRA1 expression can restore normal rod photoreceptor cell differentiation in the rd1 mouse retina. These results are ultimately expected to have a significant impact on the field of developmental neurobiology and to provide insights into mechanisms underlying aberrant differentiation, leading to improvement in childhood visual health. PUBLIC HEALTH RELEVANCE: The proposed experiments will elucidate mechanisms involved in development and differentiation of a highly specialized neuronal epithelial cell type: the rod photoreceptor cell. The rd1 mouse model of juvenile retinitis pigmentosa will be used to gain insights into the source of developmental aberrations. Greater understanding of these mechanisms could ultimately lead to targeted therapeutic interventions for juvenile retinitis pigmentosa and improved childhood visual health.