Preventing cognitive impairment in mouse genetic models of Down syndrome by early postnatal suppression of Kir3.2 channel signaling

标题：通过早期干预预防唐氏综合征小鼠遗传模型中的认知障碍 出生后Kir3.2通道信号抑制 摘要 导致DS认知障碍的神经机制尚不清楚，也没有治疗方法。 预防这种最常见的遗传性残疾。我们的长期目标是开发改善认知功能的治疗方法， 在DS中受损。近年来我们发现在DS模型中，突触可塑性和认知功能可以得到恢复 通过遗传去除Kcnj 6的额外拷贝，Kcnj 6是编码钾通道Kir3.2（Girk 2）亚基的基因。 这些通道，已知其在神经元超极化中的关键作用， 出生后的发育表明他们参与了神经回路的形成。我们的核心假设 在DS模型中，通过Kir3.2通道增加的信号传导减少了 新生神经元，从而影响突触发生和神经回路的形成，最终 会导致认知障碍相应地，在早期细胞周期中Kir3.2通道信号传导的抑制是可能的。 出生后的一段时间可以正常化的神经回路，从而减少神经元，突触，和认知 DS中的异常。两个特定的目标将被用来测试这个假设。目标1.为了检验 Kcnj 6基因剂量对DS小鼠遗传模型中神经回路发育和成熟的影响形成 GABA能和多巴胺能突触连接的成熟将在海马中检查。 含有2个或3个Kcnj 6拷贝的DS小鼠（Ts 65 Dn/Kcnj 6 ++- vs. Ts 65 Dn/Kcnj 6+小鼠）。 电生理学、生物化学和组织化学技术将用于评估Kcnj 6基因的作用 剂量：（i）影响新生神经元活动的神经元特性;（ii）突触的效率 在“兴奋性到抑制性GABA转换”（P5 - P12）之前去极化GABA作用;（iii） “兴奋性-抑制性GABA转换”（P14 - P22）后的GABA能抑制;（iv） 同步的新生儿神经元活动指示神经回路的形成;（v）发育变化 突触可塑性这些研究将显示Kcnj 6基因在神经回路形成中的作用， 为监测目标2中的治疗后变化提供依据。目标2.为了检验产后早期 治疗减少通过含Kir3.2亚基的钾通道对发育的信号传导 和神经回路的成熟。Kir3.2信道信令将在时间和区域上减少- 通过以下特异性方式：（i）Kcnj 6 siRNA的新生儿脑室内AAV递送;（ii）Kcnj 6 siRNA的腹膜内注射。 选择性GABAB受体拮抗剂这些治疗的后果将在新生儿和 成年老鼠将评估以下性质：Kir3.2通道信号传导的变化，新生儿的性质， 神经回路，GABA能突触传递和突触可塑性的治疗后变化。该数据 将提供Kcnj 6基因在DS的突触、结构和功能异常中的作用的信息， 提供了一个“原理证明”，即Kir3.2通道信号传导的时间限制性出生后抑制可用于 改善脑功能和认知功能。

Title: Preventing cognitive impairment in mouse genetic models of Down syndrome by early postnatal suppression of Kir3.2 channel signaling Abstract Neural mechanisms responsible for cognitive impairment in DS are still unclear, and there is no treatment to prevent this most common genetic disability. Our long-term goal is to develop treatments ameliorating cognitive impairment in DS. Recently we discovered that synaptic plasticity and cognition can be restored in DS models by genetic removal of an extra copy of Kcnj6, the gene encoding Kir3.2 (Girk2) subunits of potassium channels. These channels, known for their critical role in neuronal hyperpolarization, are highly expressed during early postnatal development suggesting their involvement in the formation of neural circuits. Our central hypothesis is that, in DS models, increased signaling through Kir3.2 channels reduces the spontaneous activity of neonatal neurons, thus affecting synaptogenesis and the formation of neural circuits, which ultimately leads to cognitive impairment. Accordingly, suppression of Kir3.2 channel signaling during the early postnatal period can normalize the neural circuits, thereby reducing neuronal, synaptic, and cognitive abnormalities in DS. Two Specific Aims will used to test this hypothesis. Aim1. To examine the effects of the Kcnj6 gene dose on the development and maturation of neural circuits in mouse genetic models of DS. Formation and maturation of GABAergic and glutamatergic synaptic connections will be examined in the hippocampus of DS mice containing either 2 or 3 copies of Kcnj6 (Ts65Dn/Kcnj6++- vs. Ts65Dn/Kcnj6+++ mice). Electrophysiological, biochemical, and histochemical techniques will be used to assess the effects of Kcnj6 gene dose on: (i) Neuronal properties affecting the activity of neonatal neurons; (ii) The efficiency of synaptic depolarizing GABA action before the ‘excitatory-to-inhibitory GABA switch’ (P5 - P12); (iii) The efficiency of GABAergic inhibition after the ‘excitatory-to-inhibitory GABA switch’ (P14 – P22); (iv) Development of synchronized neonatal neuronal activity indicative of the formation of neural circuits; (v) Developmental changes in synaptic plasticity. These studies will show the role of the Kcnj6 gene in the formation of neural circuits and give a basis for monitoring post-treatment changes in the Aim 2. Aim 2. To examine the effects of early postnatal treatments reducing signaling through the Kir3.2 subunit-containing potassium channels on the development and maturation of neural circuits in DS models. Kir3.2 channel signaling will be reduced in a time- and region- specific manner via: (i) Neonatal intraventricular AAV delivery of Kcnj6 siRNA; (ii) Intraperitoneal injections of selective GABAB receptor antagonists. Consequences of these treatments will be examined in neonatal and adult mice. Following properties will be assessed: changes in Kir3.2 channel signaling, properties of neonatal neural circuits, post-treatment changes in GABAergic synaptic transmission and synaptic plasticity. This data will provide information on the role of Kcnj6 gene in synaptic, structural, and functional abnormalities in DS, and provide a ‘proof of principle’ that time-limited postnatal suppression of Kir3.2 channel signaling can be used to improve brain functions and cognition in DS.