Hippocampal CA1 Pyramidal Neurons Display Sublayer and Circuitry Dependent Degenerative Expression Profiles in Aged Female Down Syndrome Mice

People with Down syndrome (DS) have intellectual disability (ID) and develop hallmark Alzheimers disease (AD) pathology during midlife. There are several circuits underlying memory and executive function in the DS and AD brain that are particularly vulnerable and degenerate early in disease, most notably the septohippocampal circuit and the trisynaptic loop in the hippocampus. A fundamental lack of knowledge exists as to the etiology and mechanisms of disease progression within these critical circuits vulnerable to degeneration in DS, AD, and relevant models. This is compounded by new evidence that suggests spatial localization of neurons has profound effects on activity and innervation within the hippocampal CA1 region. We postulated gene expression changes in a DS mouse model, at a time that these circuits of input to the CA1 are degraded, would have a significant effect on gene expression in CA1 pyramidal neurons. Further, this dysfunction may be specific to spatial localization and innervation. Laser capture microscopy on pyramidal neurons from CA1 was performed, isolating the entire CA1 pyramidal neuron layer, which was compared to select populations of deep and superficial pyramidal neurons from CA1a (distal CA1, adjacent to the subiculum). RNA-seq and bioinformatic analysis was performed where profound differences in dysregulation in the DS mouse model based on their spatial location and postulated circuitry were examined. To determine if circuitry will effect gene expression in a mouse model of DS during degeneration, the CA1 pyramidal neuron region was analyzed in 2N (disomic control; N=11 in 2 batches) and Ts2 (N=11 in 2 batches) Female mice at 11 MO. LCM was performed on the excitatory pyramidal neuron layer from the entire rostral CA1 region and compared to the distal end of the rostral CA1 deep layer pyramidal neurons and the distal end of the rostral CA1 superficial layer pyramdial neurons. RNA was purified from the isolated neurons and RNA-seq library preparation was performed, followed by RNA-seq and bioinformatic analysis. We compared the effect of genotype on gene expression within each region of CA1 neurons collected by bioinformatic analysis to determine convergent and divergent gene expression in each region to examine changes in hippocampal CA1 gene expression in the aged DS mouse model. Circuitry effects due to innervation were taken into account for analysis of divergent gene expression between deep and superficial neurons compared to the CA1 excitatory neurons as a whole in the Ts2 mice compared to their 2N littermates.唐氏综合征（Down syndrome, DS）患者存在智力障碍（intellectual disability, ID），并在中年阶段出现典型的阿尔茨海默病（Alzheimer's disease, AD）病理特征。DS与AD大脑中支撑记忆与执行功能的多条环路极易受损，并在疾病早期发生退行性病变，其中最显著的为隔海马环路（septohippocampal circuit）以及海马内的三突触环路（trisynaptic loop）。目前学界对DS、AD及其相关模型中这些易发生退行性变的关键环路的发病病因与疾病进展机制仍存在根本性的认知空白。 新的研究证据进一步表明，神经元的空间定位对海马CA1区的神经元活动与神经支配具有显著影响。我们提出如下假设：当向CA1区提供输入的环路发生退行性病变时，DS小鼠模型中的基因表达变化会对CA1锥体神经元的基因表达产生显著影响，且这种功能障碍可能与空间定位和神经支配具有特异性。 本研究针对CA1区锥体神经元实施激光捕获显微切割（laser capture microscopy, LCM），分离完整的CA1锥体神经元层，并与来自CA1a区（远端CA1，紧邻下托subiculum）的深层、表层锥体神经元亚群进行对照。随后开展RNA测序（RNA-seq）与生物信息学分析，探究DS小鼠模型中基于空间位置与假定环路的基因表达失调的显著差异。 为明确在退行性变阶段，环路是否会影响DS小鼠模型的基因表达，我们对11月龄（11 MO）的2N（二体对照，分2批，每批n=11）与Ts2（分2批，每批n=11）雌性小鼠的CA1锥体神经元区域进行分析。对完整吻侧CA1区域的兴奋性锥体神经元层实施激光捕获显微切割，并与吻侧CA1远端的深层锥体神经元、吻侧CA1远端的表层锥体神经元亚群进行对照。 从分离得到的神经元中纯化RNA，完成RNA-seq文库构建后进行测序，随后开展生物信息学分析。我们通过生物信息学分析比较基因型对各CA1神经元区域基因表达的影响，以确定各区域的趋同与趋异基因表达模式，进而探究老年DS小鼠模型中海马CA1区的基因表达变化。在对比Ts2小鼠与其同窝2N小鼠的深层、表层锥体神经元相较于整体CA1兴奋性神经元的趋异基因表达时，本研究将神经支配带来的环路效应纳入分析考量范畴。