Prionic protein gene dosage is critical for maintenance of neuronal homeostasis of hippocampal circuits

We demonstrate that Prnp dosage is critical for the maintenance of neuronal homeostasis since both its absence and, more relevantly, its overexpression induce higher sensitivity to kainate (KA) damage. These data correlate with electrophysiological results in freely behaving mutant mice showing an imbalance in activity-dependent synaptic processes, as determined from input/output curves, paired-pulse facilitation, and LTP studies. Gene expression profiling showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission among others were co-regulated in knockout and PrPc overexpressing mice. RT-qPCR analysis of neurotransmission-related genes confirmed GABA-A and AMPA-Kainate receptor subunit transcriptional co-regulation in both Prnp -/- and Tg20 mice. Our results demonstrate that PrPc is necessary for the proper homeostatic functioning of hippocampal circuits, because of its interactions with GABAA and AMPA-Kainate receptors. Keywords: steady state expression; adult brain tissue; hippocampus; genetic modification; transgenic gain of function mutation; targetted deletion loss of function mutation We performed a global transcriptome analysis of three strains that differ in their Prnp gene dose using Illumina Sentrix 6 mouse v1.1 beadarrays. We analyzed mRNA expression in the hippocampi of four individual male mice from each of three strains: 1) Tg20, with a 30 exogenous copies of the Prnp gene within a Prnp -/- background that overexpresses PrPc about 6-7 times the normal wild type levels; 2) littermates lacking the transgene and therefore carrying only the homozygous knock out Prnp -/- strain which lacks expression of PrPc; and 3) their wild type counterparts carrying two copies of the intact Prnp gene and expressing normal levels of PrPc本研究证实，朊蛋白基因（Prnp）的剂量对于维持神经元稳态至关重要：无论是其缺失，还是更值得关注的过表达，均会提升神经元对红藻氨酸（kainate, KA）损伤的敏感性。上述结果与自由活动突变小鼠的电生理实验结果相契合——通过输入-输出曲线、配对脉冲易化及长时程增强（LTP）实验，可观察到活动依赖性突触过程存在失衡。基因表达谱分析显示，在Prnp纯合敲除小鼠与朊蛋白C（PrPc）过表达小鼠中，共有129个参与泛素化、神经传递等经典通路的基因呈现共调控特征。实时定量聚合酶链反应（reverse transcription quantitative polymerase chain reaction, RT-qPCR）对神经传递相关基因的分析进一步证实，在Prnp -/-与Tg20两种小鼠中，γ-氨基丁酸A型（GABA-A）受体及AMPA-红藻氨酸受体亚基均存在转录共调控现象。本研究结果表明，PrPc可通过与GABA-A及AMPA-红藻氨酸受体相互作用，保障海马环路的稳态正常运作。 关键词：稳态表达；成年脑组织；海马体；基因修饰；转基因功能获得性突变；靶向敲除功能丧失性突变 本研究使用Illumina Sentrix 6小鼠基因芯片v1.1（Illumina Sentrix 6 mouse v1.1 beadarrays），对3个Prnp基因剂量存在差异的小鼠品系开展了全转录组分析。我们对3个品系各4只雄性小鼠的海马mRNA表达水平进行了检测：1）Tg20品系：在Prnp -/-遗传背景中整合了30个外源性Prnp基因拷贝，其PrPc表达量约为正常野生型水平的6~7倍；2）同窝敲除对照品系：不携带转基因，仅为纯合Prnp敲除（Prnp -/-）品系，无PrPc表达；3）野生型对照品系：携带2个完整的Prnp基因拷贝，PrPc表达水平处于正常范围。