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

星形胶质细胞分泌的多效蛋白在唐氏综合症树突棘表型中的作用

• 批准号: 10188246
• 负责人: Ashley N Brandebura
• 金额: $ 6.64万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188246
• 关键词: Age; Architecture; Astrocytes; Autopsy; Biological; Caliber; Cells; Clinical; Coculture Techniques; Complement; Data; Dendritic Spines; Development; Down Syndrome; Down-Regulation; Environment; Exhibits; Failure; Filopodia; Functional disorder; Future; Genetic; Goals; Histologic; Human; Impairment; In Vitro; Injections; Institutes; Intellectual functioning disability; Investigation; Knockout Mice; Knowledge; Light; Link; Maintenance; Mass Spectrum Analysis; Mediating; Messenger RNA; Morphology; Mus; Neck; Neurodevelopmental Disorder; Neurons; Pathology; Pattern; Phenotype; Physiological; Process; Protein Secretion; Proteins; Pyramidal Cells; Quality of life; Rattus; Regulation; Research; Role; Seminal; Signal Transduction; Small Interfering RNA; Structure; Synapses; Testing; Thinness; Thrombospondin 1; Tissues; Transcript; Transfection; Up-Regulation; Vertebral column; Viral; Work; clinically relevant; density; dentate gyrus; equipment training; experimental study; in vivo; in vivo two-photon imaging; insight; mRNA Expression; mRNA sequencing; molecular sequence database; molecular targeted therapies; mouse Ts65Dn; mouse model; normal aging; novel; overexpression; pleiotrophin; ranpirnase; receptor; synaptogenesis; syndecan 3; therapeutic target

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.

唐氏综合征(DS)小鼠模型和DS人死后组织显示树突棘密度总体减少，但具有不成熟形态的脊椎数量增加。虽然传统上认为DS中观察到的脊椎缺陷是神经细胞内部信号转导的失败，但最近的研究表明星形胶质细胞-神经元信号转导与脊椎表型有关。一项利用人类星形胶质细胞和大鼠原代神经元共培养的开创性研究表明，与与星形胶质细胞共同培养的野生型(WT)神经元相比，在DS星形胶质细胞存在下培养的WT神经元的总脊椎密度显著降低，并倾向于更细的丝状棘突(未成熟形态)。这些结果呼应了大量研究的结果，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的突触发生、成熟和维持。为了确定DS患者星形胶质细胞分泌体的全部变化，实验室对来自三体Ts65Dn小鼠(DS的一个特征良好的小鼠模型)的培养星形胶质细胞的条件培养液进行了无偏倚的质谱仪筛选，并在相同背景下与其整倍体对照组进行了比较。Pleiotroin(PTN)是DS患者下降最多的10种蛋白之一(下降了4倍以上)。PTN是研究DS脊椎表型的一个有吸引力的候选蛋白，因为：(1)在皮质突触发生过程中，PTN在星形胶质细胞中的表达模式高度丰富；(2)最近证实，PTN在齿状回神经元的棘突发生中起作用。关于PTN参与皮质突触和脊柱形成、成熟和维持的各个方面，PTN在DS病理中的作用，以及哪个受体(S)可能在神经元突触结构中发挥转导作用，仍有许多有趣的问题。本研究的目的是研究PTN在正常皮质发育中对突触和脊柱密度、脊柱形态和脊柱稳定性的调节作用，确定在DS中恢复生理水平的PTN分泌是否可以挽救所观察到的脊柱表型，并确定PTN在树突棘上介导作用的受体(S)。主要的假说是，PTN在皮质发育中调节脊柱的密度、形态和稳定性，PTN的分泌下调是导致DS脊柱表型的关键机制。目的1利用PTN基因敲除小鼠研究PTN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利用体外和体内方法，询问上调PTN是否可以挽救DS患者的脊柱表型。目的3利用体外小干扰核糖核酸筛选，探讨S等神经受体介导甲状旁腺素的作用。这些实验将扩大目前对PTN在皮质发育中作用的认识，并对阐明PTN水平失调导致DS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。