Loss of MeCP2 function is associated with distinct gene expression changes in the striatum

Rett syndrome (RTT) is a neurodevelopmental disorder characterized by developmental regression around 6-18 months after birth, followed by a lifetime of intellectual disability, stereotyped behaviors, and motor deficits. RTT is caused by mutations in MeCP2, a methyl-CpG binding protein that was traditionally believed to repress gene expression. Gene expression studies of individual brain regions, however, have revealed that MeCP2 loss-of-function leads to the subtle activation and repression of its gene targets. However, these results may be confounded by the extensive neuronal cell heterogeneity inherent in these brain structures. To minimalize this issue of heterogeneity, we assessed whether Mecp2-null mice exhibited alterations in gene expression patterns in the striatum, a brain nucleus with relatively homogenous neuronal types and is highly relevant to the motor deficits observed in RTT. Despite the homogeneity of the tissue, the fold-change of the 127 differentially expressed genes we identified remained low with a mean change consistent with other studies. However, many of those genes differentially expressed in the striatum have not been previously identified in gene expression analyses of other brain regions. This suggests therefore that the differential expression of genes following loss of MeCP2 occurs in a tissue, or cell-type specific manner and thus MeCP2 function should be understood in a cellular context. In initiating this study, we reasoned that reducing the number of cell types in a microarray experiment may reveal transcriptional changes that are masked in a whole tissue analysis. We therefore focused on tissues more homogeneous in regards to the diversity of neuronal cell types they contain in order to discern gene expression changes in the absence of MeCP2. We chose to isolate the striatum, a tissue composed predominantly of GABAergic medium spiny neurons (MSNs). The striatum was resected from five symptomatic Mecp2-null (KO) male mice bearing the Bird allele and five wild-type (WT) littermates in a C57BL/6 background. We also isolated liver from the same individuals to serve as a non-neuronal control. RNA was isolated from these tissues, converted to cDNA, and hybridized to a single-channel Affymetrix GeneChip Mouse Exon 1.0 ST array for a total of 20 individual arrays.雷特综合征（Rett syndrome, RTT）是一种神经发育障碍，以出生后6~18个月左右出现发育倒退为核心特征，继而伴随终身的智力障碍、刻板行为与运动功能缺损。该病由MeCP2基因突变所致，MeCP2是一种甲基CpG结合蛋白，既往认为其可抑制基因表达。然而，针对单个脑区的基因表达研究显示，MeCP2功能缺失会导致其靶基因出现轻微的激活与抑制，但这类研究结果可能受到脑组织结构固有的广泛神经元细胞异质性的干扰。为尽可能降低异质性带来的实验偏差，本研究探究了Mecp2敲除（Mecp2-null）小鼠纹状体（striatum）中的基因表达模式变化——纹状体是一种神经元类型相对均一的脑核团，且与雷特综合征患者出现的运动功能缺损高度相关。尽管该组织的细胞均一性较好，但我们鉴定出的127个差异表达基因的表达倍数变化仍较低，平均变化幅度与其他研究结果一致。然而，纹状体中出现差异表达的诸多基因，此前在其他脑区的基因表达分析中均未被发现。这表明，MeCP2缺失后基因的差异表达具有组织或细胞类型特异性，因此对MeCP2功能的阐释需结合细胞环境进行。本研究启动之初，我们便推测：在微阵列实验中减少细胞类型的数量，可揭示出全组织分析中被掩盖的转录组变化。因此，我们选取了神经元类型多样性更低、细胞均一性更高的组织，以探究MeCP2缺失时的基因表达变化。最终我们选择分离纹状体——该组织主要由γ-氨基丁酸能中型多棘神经元（GABAergic medium spiny neurons, MSNs）构成。我们从5只携带Bird等位基因的有症状Mecp2敲除（KO）雄性小鼠，以及5只C57BL/6背景的野生型（WT）同窝小鼠中分离纹状体；同时从同一批个体中分离肝脏作为非神经元对照组织。从上述组织中分离RNA并反转录为cDNA，随后与单通道Affymetrix GeneChip Mouse Exon 1.0 ST阵列进行杂交，共完成20张独立芯片的检测。