Department of Prenatal Diagnosis, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, China.
Ministry of Education Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, Medical School, Nanjing University, Nanjing, China.
J Clin Lab Anal. 2022 Feb;36(2):e24196. doi: 10.1002/jcla.24196. Epub 2022 Jan 8.
Proline-rich transmembrane protein 2 (PRRT2) is a neuron-specific protein associated with seizures, dyskinesia, and intelligence deficit. Previous studies indicate that PRRT2 regulates neurotransmitter release from presynaptic membranes. However, PRRT2 can also bind AMPA-type glutamate receptors (AMPARs), but its postsynaptic functions remain unclear.
Whole-exome sequencing used to diagnose a patient with mental retardation identified a nonsense mutation in the PRRT2 gene (c.649C>T; p.R217X). To understand the pathology of the mutant, we cloned mouse Prrt2 cDNA and inserted a premature stop mutation at Arg223, the corresponding site of Arg217 in human PRRT2. In mouse hippocampal tissues, Prrt2 interacted with GluA1/A2 AMPAR heteromers but not GluA2/A3s, via binding to GluA1. Additionally, Prrt2 suppressed GluA1 expression and localization on cell membranes of HEK 293T cells. However, when Prrt2 was overexpressed in individual hippocampal neurons using in utero electroporation, AMPAR-mediated synaptic transmission was unaffected. Deletion of Prrt2 with the CRIPR/Cas9 technique did not affect AMPAR-mediated synaptic transmission. Furthermore, deletion or overexpression of Prrt2 did not affect GluA1 expression and distribution in primary neuronal culture.
The postsynaptic functions of Prrt2 demonstrate that Prrt2 specifically interacts with the AMPAR subunit GluA1 but does not regulate AMPAR-mediated synaptic transmission. Therefore, our study experimentally excluded a postsynaptic regulatory mechanism of Prrt2. The pathology of PRRT2 variants in humans likely originates from defects in neurotransmitter release from the presynaptic membrane as suggested by recent studies.
富含脯氨酸跨膜蛋白 2（PRRT2）是一种与癫痫、运动障碍和智力缺陷相关的神经元特异性蛋白。先前的研究表明，PRRT2 调节突触前膜神经递质的释放。然而，PRRT2 也可以结合 AMPA 型谷氨酸受体（AMPARs），但其突触后功能尚不清楚。
使用外显子组测序诊断一名智力障碍患者，发现 PRRT2 基因（c.649C>T；p.R217X）发生无义突变。为了了解突变体的病理，我们克隆了小鼠 Prrt2 cDNA，并在人 PRRT2 的精氨酸 217 对应的位置插入一个提前终止突变。在小鼠海马组织中，Prrt2 通过与 GluA1/A2 AMPAR 异聚体结合，而不是 GluA2/A3s，与 GluA1 相互作用。此外，Prrt2 抑制 GluA1 在 HEK 293T 细胞细胞膜上的表达和定位。然而，当使用子宫内电穿孔在单个海马神经元中过表达 Prrt2 时，AMPAR 介导的突触传递不受影响。使用 CRISPR/Cas9 技术删除 Prrt2 不会影响 AMPAR 介导的突触传递。此外，在原代神经元培养物中删除或过表达 Prrt2 不会影响 GluA1 的表达和分布。
Prrt2 的突触后功能表明，Prrt2 特异性地与 AMPAR 亚基 GluA1 相互作用，但不调节 AMPAR 介导的突触传递。因此，我们的研究从实验上排除了 Prrt2 的突触后调节机制。正如最近的研究表明，人类 PRRT2 变体的病理学可能源自突触前膜神经递质释放的缺陷。
