Identification of the endogenous ligand of SAP97 PDZ3

描述(由申请人提供)：成年神经元复杂的形态和电生理特性在出生前和出生后早期的较长时间内出现。新生儿与周围环境的相互作用激发了中枢神经系统内有模式的突触活动，这种活动在塑造神经元的最终形态和功能方面起着至关重要的作用。这个 活性依赖可塑性的分子机制包括激活NMDA受体和钙触发的信号事件。在之前使用脊髓神经元的工作中，我们发现了使用由GluA1组装的AMPA-R的额外分子机制的证据。GluA1通过其细胞内与SAP97(分子量为97 kDa的突触相关蛋白)的结合伙伴将活性转化为树突生长。GluA1/SAP97形式的可塑性是NMDA-R独立的，我们认为与NMDA-R介导的事件平行操作。为了使GluA1促进树突生长，SAP97必须共定位在质膜上，推测是在突触后密度。我们假设GluA1/SAP97协调组装了一个多蛋白复合体，将AMPA-R的活性转化为树突生长。我们的初步数据表明，SAP97的PDZ3的结合伙伴在这一过程中起着至关重要的作用。为了确定内源性PDZ3结合伙伴，我们将对六个最佳候选分子进行越来越严格的验证测试。首先，候选人必须绑定到WT SAP97，但不能绑定在PDZ3中突变的SAP97版本。内源蛋白质必须相互作用。第二，候选蛋白在WT神经元中的表达，而不是GluA1 7神经元，必须促进树突的生长。GluA1 7神经元具有正常的突触AMPA受体，但SAP97不进入质膜。第三，候选蛋白的敲除必须抑制正常的树突生长。除了这项定向研究外，还将部署一种使用质谱学的发现方法。总体而言，这些研究将提供一个基本的神经生物学过程的洞察，并可能对正常的大脑发育以及成人受伤后功能的恢复具有翻译意义。 神经系统。

DESCRIPTION (provided by applicant): The complex morphological and electrophysiological properties of adult neurons emerge over an extended period of prenatal and early postnatal life. The interaction of neonates with their environment evokes patterned synaptic activity within the CNS and this activity plays a crucial role in sculpting the final form and function of neurons. The best characterized molecular mechanism for activity-dependent plasticity involves activation of NMDA receptors and calcium triggered signaling events. In prior work using spinal cord neurons we have found evidence for an additional molecular mechanism that employs AMPA-R assembled from GluA1. GluA1 translates activity into dendrite growth via its intracellular binding partner with SAP97 (synapse-associated protein of 97 kDa molecular weight). The GluA1/SAP97 form of plasticity is NMDA-R independent and we think operates in parallel with NMDA-R mediated events. For GluA1 to promote dendrite growth, SAP97 must be co-localized at the plasma membrane, presumably at the postsynaptic density. We hypothesize that GluA1/SAP97 coordinately assemble a multiprotein complex that translates activity of AMPA-R into dendrite growth. Our preliminary data indicate that the binding partner of PDZ3 of SAP97 plays an essential role in this process. To identify the endogenous PDZ3 binding partner we will subject the six best candidate molecules to increasingly stringent validation tests. First, the candidate must bind to WT SAP97 but not a version of SAP97 that is mutant in PDZ3. The endogenous proteins must interact. Second, expression of the candidate protein in WT, but not GluA1 7 neurons, must promote dendrite growth. GluA1 7 neurons have normal synaptic AMPA-Rs but SAP97 does not traffic into the plasma membrane. Third, knockdown of the candidate protein must inhibit normal dendrite growth. In addition to this directed study, a discovery approach using mass spectrometry will be deployed. Overall these studies will provide insight into a fundamental neurobiological process and may have translational implications for normal brain development as well as recovery of function after injury to the adult nervous system.