Function and Regulation of Postsynaptic Serine Racemase

虽然精神分裂症的病因机制仍然很大程度上未知，药理学和遗传学数据的融合暗示N-甲基-D-天冬氨酸受体（NMDAR）功能失调。NMDAR是在神经发育和突触可塑性中起关键作用的配体门控离子通道，并且NMDAR功能的细微变化可以具有广泛的发育和认知影响。因此，有一个关键需要详细了解的分子机制参与的NMDAR的调节，这将产生洞察精神分裂症的病理生理学和促进新的治疗策略的发展。与所有其他神经递质受体不同，NMDAR绝对需要结合两种不同的激动剂才能被激活。除了从突触前末端释放的谷氨酸外，NMDAR还需要共激动剂，其可以是甘氨酸或D-丝氨酸。在大多数前脑突触中，D-丝氨酸是主要的NMDAR共激动剂，并且D-丝氨酸缺乏与精神分裂症的病理生理学有关。然而，我们的了解调节突触D-丝氨酸的可用性的机制仍然非常有限。甚至D-丝氨酸的细胞来源也存在激烈的争论。D-丝氨酸在脑中由丝氨酸消旋酶（SR）合成，该酶将L-丝氨酸转化为D-丝氨酸。最初的研究将SR和D-丝氨酸置于星形胶质细胞中，并且星形胶质细胞中D-丝氨酸的活性依赖性释放被尊为原型胶质递质。然而，越来越多的文献使用更具选择性的抗体和小鼠遗传学挑战了这种星形胶质细胞在D-丝氨酸调节中的作用，并指出了主要的神经元来源。在该提议中，将病毒构建体注射到条件性SR敲除小鼠的海马中，以仅在少数个别神经元中去除SR。通过比较SR缺失对相邻对照神经元的影响，我们获得了初步数据，为神经元D-丝氨酸的功能作用提供了第一个严格控制的证据。这些初步数据支持我们的中心假设，即突触NMDAR的活性和功能是由突触后SR和D-丝氨酸释放调节。在具体目标1中，将表征突触后SR缺失的细胞自主效应，并确定剩余突触共激动剂的来源。在具体目标2中，我们将详细研究突触后SR缺失对突触后和突触前NMDAR的影响。在具体目标3中，我们将确定突触后SR的功能作用，使用创新的分子替代方法，用特征性重组SR突变体替代内源性SR，以探索突触后SR的功能和调节。该研究计划的成功完成将更好地了解调节突触NMDAR共激动剂可用性的分子机制，以及这些过程如何在疾病状态下被破坏，有助于实现我们的长期目标，即开发新的疾病修饰方法来治疗精神分裂症和其他复杂的神经精神疾病。

Though etiological mechanisms underlying schizophrenia remain largely unknown, a convergence of pharmacologic and genetic data implicates a dysregulation of N-methyl-D-aspartate receptor (NMDAR) function. NMDARs are ligand-gated ion channels that play critical roles in neurodevelopment and synaptic plasticity and subtle changes in NMDAR functioning can have wide-ranging developmental and cognitive effects. Thus, there is a critical need for a detailed understanding of the molecular mechanisms involved in the regulation of NMDARs which will yield insights into the pathophysiology of schizophrenia and facilitate the development of novel therapeutic strategies. Unlike all other neurotransmitter receptors, NMDARs have an absolute requirement for the binding of two different agonists in order to be activated. In addition to glutamate released from the presynaptic terminal, NMDARs require a co-agonist, which can be either glycine or D-serine. At most forebrain synapses, D-serine is the primary NMDAR co-agonist and D-serine deficiency has been implicated in the pathophysiology of schizophrenia. However, our understanding of the mechanisms regulating the availability of synaptic D-serine remains quite limited. Even the cellular source of D-serine is fiercely debated. D-serine is synthesized in the brain by the enzyme serine racemase (SR) that converts L-serine to D-serine. Original studies placed both SR and D-serine in astrocytes, and activity-dependent release of D-serine from astrocytes was venerated as the prototypical gliotransmitter. However, a growing literature using more selective antibodies and mouse genetics has challenged this astrocytic role in D-serine regulation and pointed to a primarily neuronal source. In this proposal, conditional SR knock-out mice were injected with viral constructs into the hippocampus to remove SR in only a few individual neurons. By comparing the effects of SR deletion to adjacent control neurons, we have obtained preliminary data that provides the first rigorously- controlled evidence for a functional role of neuronal D-serine. This preliminary data supports our central hypothesis that synaptic NMDAR activity and function is regulated by postsynaptic SR and D-serine release. In Specific Aim 1, the cell-autonomous effects of postsynaptic SR deletion will be characterized and the source of the remaining synaptic co-agonist will be determined. In Specific Aim 2, we will examine in detail the effects of postsynaptic SR deletion on both postsynaptic and presynaptic NMDARs. In Specific Aim 3, we will determine the functional role of postsynaptic SR, using an innovative molecular replacement approach to replace endogenous SR with characterized recombinant SR mutants to explore the function and regulation of postsynaptic SR. Successful completion of this research program will provide a better understanding of the molecular mechanisms regulating co-agonist availability at synaptic NMDARs, and how these processes might be disrupted in disease states, helping to lead towards our long-term goal of developing novel disease-modifying approaches to treat schizophrenia and other complex neuropsychiatric disorders.