PICK1 knockout mice: role for D-serine in neonatal forebrains

描述（由申请人提供）：与C-激酶相互作用的蛋白质（PICK 1）是一种多功能支架蛋白，与神经元和神经胶质细胞中的许多蛋白质相互作用。我们最近报道了PICK 1直接与D-丝氨酸合成酶丝氨酸消旋酶（SR）结合。敲低PICK 1的表达降低了细胞培养物中SR的D-丝氨酸水平。我们发表了在PICK 1敲除（PICK 1 KO）的新生儿前脑中观察到D-丝氨酸水平降低，这与细胞生物学的观察结果一致。最近的几项研究表明，D-丝氨酸在与皮质回路相关的精神障碍中发挥作用，包括精神分裂症。然而，据我们所知，PICK 1的作用主要是在突触可塑性的背景下研究的，主要是通过使用小脑和海马的切片。因此，在这项拟议的研究中，我们将在行为测定和电生理方法中检查PICK 1敲除小鼠，并关注蛋白质对D-丝氨酸处置和脑功能的影响，特别是与皮质电路相关的影响。在我们的初步研究中，我们获得了（1）在新生期前脑中D-丝氨酸水平降低，但在成年期没有;（2）成年期的几种行为缺陷，包括空间工作记忆和前脉冲抑制;（3）成年期皮质锥体神经元对NMDA的反应降低。基于初步数据，我们的总体假设是，新生儿D-丝氨酸缺陷，这可能与异常谷氨酸功能，影响前额叶皮层回路的成熟，并导致在成年期锥体神经元和中间神经元的突触和NMDA依赖性反应的赤字，而在这个时候的D-丝氨酸水平是正常的。在目的1中，我们将检查在发育过程中，特别是在PICK 1 KO小鼠的新生儿阶段，前脑中D-丝氨酸的水平。在目标2中，我们将通过一组行为测定来表征成年PICK 1 KO小鼠，特别是用于测量皮质功能的那些，并且还通过电生理学方法。在目标3中，我们将通过在新生阶段给予D-丝氨酸（如有必要）与DAAO抑制剂组合，使PICK 1 KO小鼠中的D-丝氨酸水平正常化。然后，我们将研究在新生期的D-丝氨酸治疗是否会影响成年PICK 1 KO小鼠可能的异常行为和生理表型。通过这些实验，我们将研究PICK 1与D-丝氨酸在新生儿前脑中的作用，希望从这项研究中获得的信息将成为理解精神障碍和大脑发育的重要基础。 公共卫生相关性：PICK 1基因敲除小鼠最近的证据表明，PICK 1可能通过调节新生儿前脑中的D-丝氨酸代谢来调节谷氨酸神经传递。新生儿阶段神经元回路的形成对于成年大脑的正常发育和功能至关重要。我们将通过对PICK 1基因敲除小鼠的特征分析，研究PICK 1在新生儿前脑中与D-丝氨酸结合的作用，希望从这项研究中获得的信息将成为理解精神障碍和脑发育的重要基础。

DESCRIPTION (provided by applicant): Protein Interacting with C-Kinase (PICK1) is a multifunctional scaffold protein that interacts with many proteins in neurons and glial cells. We recently reported that PICK1 binds directly to the D-serine synthesizing enzyme, serine racemase (SR). Knockdown expression of PICK1 decreased levels of D-serine from SR in cell cultures. We published that decreased levels of D-serine were observed in neonatal forebrains in PICK1 knockout (PICK1 KO), consistent with the observation from cell biology. Several recent studies have suggested roles for D-serine in mental disturbances associated with cortical circuitry, including schizophrenia. To our knowledge, however, roles for PICK1 have largely been studied in the context of synaptic plasticity, mainly by using slices from the cerebellum and hippocampus. Therefore, in this proposed study, we will examine PICK1 knockout mice in behavioral assays and electrophysiological approaches and focus on the impact of the protein on D- serine disposition and brain functions, especially those associated with cortical circuitry. In our preliminary studies, we obtained (1) decreased levels of D-serine in the forebrain during the neonatal period, but not in the adulthood; (2) several behavioral deficits in adulthood, which includes those in spatial working memory and prepulse inhibition; and (3) decreased response of the cortical pyramidal neurons to NMDA in adulthood, in PICK1 KO mice. Based on preliminary data, our overall hypothesis is that neonatal deficits in D-serine, which is likely to associate with abnormal glutamate function, affect the maturation of prefrontal cortical circuits and result in deficits in synaptic and NMDA-dependent responses in pyramidal neurons and interneurons in adulthood, whereas at this time the levels of D-serine are normal. In Aim 1, we will examine levels of D-serine in the forebrain during development, especially in neonatal stages in PICK1 KO mice. In Aim 2, we will characterize adult PICK1 KO mice by a set of behavioral assays, in particular those for measuring cortical functions, and also by electrophysiological approaches. In Aim 3, we will normalize the levels of D-serine in PICK1 KO mice by administering D-serine, if necessary, combined with a DAAO inhibitor during the neonatal stage. We will then examine whether the D-serine treatment during neonatal periods influences possible abnormal behaviors and physiological phenotypes in adult PICK1 KO mice. Through these experiments, we will study a role for PICK1 in the neonatal forebrain in conjunction with D-serine, hoping that the information obtained from this study will be an important basis in understanding mental disorders and brain development. PUBLIC HEALTH RELEVANCE: PICK1 knockout mice Recent evidence has suggested that PICK1 may regulate glutamate neurotransmission via regulating D-serine metabolism in the neonatal forebrain. Formation of neuronal circuitry during neonatal stages is crucial for proper brain development and functions of adult brain. We will study role for PICK1 in the neonatal forebrain in conjunction with D- serine by characterizing PICK1 knockout mice, hoping that the information obtained from this study will be an important basis in understanding mental disorders and brain development.