Cell-specific changes of laminin expression in the CNS in Alzheimer’s disease

项目总结/摘要 本申请的长期目标是：（1）确定每种单独的细胞类型如何衍生 在生理和病理条件下，基底膜（BM）中的层粘连蛋白变化， 和（2）将这些层粘连蛋白改变与AD病理学相关联以筛选独特的层粘连蛋白改变， 有助于早期AD诊断（痴呆或Aβ/tau病理学发作前）和预后 预测.该提案旨在产生一种创新的层粘连蛋白基因敲入小鼠系， 以细胞特异性和Cre依赖性方式准确评估层粘连蛋白表达;和 确定每种单个细胞类型来源的层粘连蛋白在细胞中的暂时和空间表达谱， 正常和AD脑的BM。我们已经成功地产生了Laminin-mCherry/eGFP敲入， 使用CRISPR-Cas9技术对小鼠系进行了研究，并进一步验证了这些小鼠在存在和 不存在Cre重组。在目标1中，我们将这些层粘连蛋白基因敲入小鼠与各种Cre 系以产生一系列细胞特异性层粘连蛋白报告基因（Lam-Rep）小鼠。使用这些Lam-Rep小鼠， 我们将确定脑BM中层粘连蛋白的细胞来源，估计它们的相对丰度， 表征每种单个细胞类型来源的层粘连蛋白的暂时和空间表达谱 在正常的老化过程中。在目标2中，将细胞特异性Lam-Rep小鼠与5xFAD背景杂交。 在所得到的细胞中，每个单独的细胞来源的层粘连蛋白的临时和空间表达谱是由细胞来源的层粘连蛋白的表达谱决定的。 Lam-Rep-5xFAD小鼠及其非AD Lam-Rep同窝仔将按照所述方法进行类似测定 目标1。成功完成这项研究将填补知识差距的领域，阐明 脑BM中层粘连蛋白的细胞来源和表征层粘连蛋白的暂时/空间表达谱 在正常和AD条件下每种单独的细胞类型来源的层粘连蛋白。这些发现将 能够进行特定细胞类型来源的层粘连蛋白的丢失与AD病理学之间的相关性研究;以及 可以识别独特的层粘连蛋白的变化，在早期AD诊断和预后预测有用。在 此外，该提案还将通过产生一个 创新的层粘连蛋白敲入小鼠系，其允许以细胞特异性和Cre- 依赖的方式。这种遗传工具对其他领域的研究人员也很有价值（例如体内成像 和白细胞迁移）。这些研究将导致层粘连蛋白/BM研究的突破， 大大推动了这一领域的发展。

Project Summary/Abstract The long-term objectives of this application are to: (1) determine how each individual cell type-derived laminin changes in the basement membrane (BM) under both physiological and pathological conditions, and (2) correlate these laminin alterations with AD pathology to screen for unique laminin changes that are useful in early AD diagnosis (before the onset of dementia or Aβ/tau pathology) and prognosis prediction. This proposal aims to generate an innovative laminin knock-in mouse line that enables accurate assessment of laminin expression in a cell-specific and Cre-dependent manner; and determine the temporary and spatial expression profile of each individual cell type-derived laminin in the BM in normal and AD brains. We have successfully generated the Laminin-mCherry/eGFP knock-in mouse line using CRISPR-Cas9 technique and further validated these mice in the presence and absence of Cre recombination. In Aim 1, we will cross these laminin knock-in mice with various Cre lines to generate a series of cell-specific laminin reporter (Lam-Rep) mice. Using these Lam-Rep mice, we will determine the cellular source of laminin in brain BM, estimate their relative abundance, and characterize the temporary and spatial expression profile of each individual cell type-derived laminin during normal aging. In Aim 2, cell-specific Lam-Rep mice will be crossed into the 5xFAD background. The temporary and spatial expression profile of each individual cell-derived laminin in the resulting Lam-Rep-5xFAD mice and their non-AD Lam-Rep littermates will be determined similarly as described in Aim 1. Successful completion of this study will fill the gap of knowledge in the field by elucidating the cellular source of laminin in brain BM and characterizing the temporary/spatial expression profile of each individual cell type-derived laminin under both normal and AD conditions. These findings will enable correlation studies between loss of specific cell type-derived laminin and AD pathology; and may identify unique laminin changes that are useful in early AD diagnosis and prognosis prediction. In addition, this proposal will also address a critical barrier to progress in the field by generated an innovative laminin knock-in mouse line, which allows labeling of laminin in a cell-specific and Cre- dependent manner. This genetic tool is also valuable to researchers in other fields (e.g. in vivo imaging and leukocyte transmigration). These studies will lead to a breakthrough in laminin/BM research and substantially move the field forward.