Role of astrocyte-secreted pleiotrophin in dendritic spine phenotypes in Down Syndrome

唐氏综合征（DS）小鼠模型和唐氏综合征人死后组织显示树突棘密度总体下降，未成熟棘数量增加。虽然在退行性椎体滑移中观察到的脊柱缺陷传统上被认为是神经元细胞内在信号传导的失败，但最近的研究表明，星形胶质细胞-神经元信号传导有助于脊柱表型。一项利用人类星形胶质细胞和大鼠原代神经元共培养的重要研究表明，与用WT星形胶质细胞培养的WT神经元相比，在DS星形胶质细胞存在下培养的野生型（WT）神经元具有显著降低的总脊柱密度，并向更高比例的细丝状样脊柱（未成熟形态）转变。这些结果与大量研究结果相呼应，这些研究表明星形胶质细胞分泌的蛋白质可以调节神经元突触的发生、成熟和维持。为了确定DS中星形胶质细胞分泌组变化的完整补体，实验室在条件培养基上对从Ts65Dn三体小鼠（一种具有良好特征的DS小鼠模型）分离的培养星形胶质细胞进行了无偏质谱筛选，并将其与相同背景下的整倍体对照进行了比较。多营养蛋白（Pleiotrophin, Ptn）在DS中下降幅度最大的10个蛋白中（下降幅度超过4倍）。Ptn是一个有吸引力的候选蛋白，用于研究在DS脊柱表型中的作用，因为(i)在皮质突触发生期间，Ptn在星形胶质细胞中高度富集的mRNA表达模式，以及（ii）最近证明Ptn在齿状回神经元脊柱发生中的作用。关于Ptn在皮质突触和脊柱形成、成熟和维持的各个方面的参与，Ptn在DS病理中的作用，以及哪些受体可能对神经元突触结构产生转导作用，还有许多有趣的问题仍然存在。本研究的目的是研究Ptn在正常皮质发育中调节突触和脊柱密度、脊柱形态和脊柱稳定性中的作用，确定恢复DS中Ptn分泌的生理水平是否可以挽救观察到的脊柱表型，并确定介导Ptn对树突棘影响的受体。主要假设是Ptn在皮质发育过程中调节脊柱密度、形态和稳定性，分泌Ptn的下调是导致退行性椎体滑移脊柱表型的关键机制。目的1将研究Ptn基因敲除小鼠是否调节皮质突触和脊柱密度、脊柱形态和脊柱稳定性。目的2询问Ptn的上调是否可以利用体外和体内方法挽救DS的脊柱表型。目的3利用体外siRNA筛选研究哪些神经元受体介导Ptn的作用。这些实验将扩大目前对Ptn在皮质发育中的作用的认识，并对阐明Ptn水平失调导致退行性椎体滑移脊柱发育不良的机制具有重要的临床意义。拟议的工作将在索尔克生物研究所进行，这是一个高水平的合作研究环境，提供所有必要的设备和培训。

Mouse models of Down Syndrome (DS) and DS human postmortem tissue display an overall decrease in dendritic spine density with an increased number of spines with immature morphology. Although the spine deficits observed in DS were traditionally viewed as a failure of neuronal cell intrinsic signaling, more recent studies suggest that astrocyte-neuron signaling contributes to the spine phenotypes. One seminal study utilizing co-cultures of human astrocytes and rat primary neurons demonstrated that wildtype (WT) neurons cultured in the presence of DS astrocytes possessed a significantly decreased total spine density with a shift towards a higher ratio of thin filopodia-like spines (immature morphology) when compared to WT neurons cultured with WT astrocytes. These results echo the findings of a multitude of studies demonstrating that proteins secreted from astrocytes can modulate neuronal synaptogenesis, maturation and maintenance. In order to identify the full complement of changes in the astrocyte secretome in DS, the lab conducted an unbiased mass spectrometry screen on the conditioned media of cultured astrocytes isolated from trisomic Ts65Dn mice (a well-characterized mouse model of DS) compared to their euploid controls on the same background. Pleiotrophin (Ptn) was identified among the top 10 proteins decreased in DS (over 4-fold decrease). Ptn is an attractive protein candidate to investigate for a role in spine phenotypes in DS due to (i) its highly enriched mRNA expression pattern in astrocytes during cortical synaptogenesis and (ii) a recently demonstrated role for Ptn in neuronal spinogenesis in dentate gyrus. There are many intriguing questions that remain regarding the involvement of Ptn in various aspects of cortical synapse and spine formation, maturation and maintenance, the role of Ptn in DS pathology, as well as which receptor(s) may be transducing effects on neuronal synaptic architecture. The goals of this proposal are to investigate the role of Ptn in regulating synapse and spine density, spine morphology and spine stability in normal cortical development, to determine if restoring physiological levels of Ptn secretion in DS can rescue the spine phenotypes observed, and to define the receptor(s) that mediate the effects of Ptn on dendritic spines. The main hypothesis is that Ptn regulates spine density, morphology and stability in cortical development and that down-regulation of secreted Ptn is a key mechanism leading to spine phenotypes in DS. Aim 1 will investigate if Ptn regulates cortical synapse and spine density, spine morphology and spine stability using Ptn knockout mice. Aim 2 asks if up-regulation of Ptn can rescue spine phenotypes in DS utilizing in vitro and in vivo approaches. Aim 3 investigates which neuronal receptor(s) mediate effects of Ptn utilizing an in vitro siRNA screen. These experiments will expand the current knowledge of the role of Ptn in cortical development and have significant clinical relevance to elucidate a mechanism by which dysregulation of Ptn levels leads to spine dysgenesis in DS. The proposed work will take place at the Salk Institute for Biological Studies, a high caliber, collaborative research environment which provides access to all necessary equipment and training.