C2-lacking isoform of Nedd4-2 regulates excitatory synaptic strength through GluA1 ubiquitination-independent mechanisms.
C2-lacking isoform of Nedd4-2 regulates excitatory synaptic strength through GluA1 ubiquitination-independent mechanisms.
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Nedd4-2 缺乏 C2 亚型通过 GluA1 泛素化独立机制调节兴奋性突触强度。
DOI：
10.1111/jnc.14840
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发表时间：
2019
影响因子：
4.7
通讯作者：
Tsai,Nien-Pei
中科院分区：
文献类型：
--
作者：
Zhu,Jiuhe;Lee,KwanYoung;Jong,TiffanyT;Tsai,Nien-Pei
神经前体细胞表达的发育下调基因4样（Nedd 4 - 2）是一种癫痫相关基因，编码一种在大脑中高度表达的泛素E3连接酶。Nedd 4 - 2的底物包括许多离子通道和受体，因为其N末端C2结构域将Nedd 4 - 2引导到细胞膜。我们之前发现Nedd 4 - 2泛素化α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体的谷氨酸受体亚基1（GluA 1）亚基，导致小鼠神经元兴奋性降低和癫痫发作。然而，尽管意识到Nedd 4 - 2亚型没有C2结构域，但这种亚型的功能仍然难以捉摸。在这项研究中，我们发现缺乏C2的Nedd 4 - 2减少了膜分布，并对泛素化GluA 1表现出降低的亲和力。然而，当在原代皮层神经元中表达时，我们发现缺乏C2的Nedd 4 - 2表现出与含有C2的Nedd 4 - 2相似的降低兴奋性突触强度的活性。为了鉴定可以介导兴奋性突触强度的新型Nedd 4 - 2底物，我们使用无偏蛋白质组学筛选，发现了在条件性Nedd 4 - 2敲除小鼠大脑中上调的多种突触调节因子，包括蛋白磷酸酶3催化亚基-α（PPP 3CA;也称为钙调磷酸酶A-α）。我们证实PPP 3CA是C2缺失的Nedd 4 - 2的底物，并表明Nedd 4 - 2的所有三种癫痫相关错义突变都破坏了PPP 3CA泛素化。总而言之，我们的研究结果揭示了新的潜在Nedd 4 - 2底物，并表明缺乏C2的Nedd 4 - 2最有可能通过GluA 1泛素化非依赖性机制抑制兴奋性突触强度。这些发现提供了新的信息，以进一步了解Nedd 4 - 2依赖的神经元兴奋性稳态和病理性过度兴奋时，Nedd 4 - 2受损。
Neural precursor cell expressed developmentally downregulated gene 4‐like (Nedd4‐2) is an epilepsy‐associated gene, which encodes a ubiquitin E3 ligase that is highly expressed in the brain. Nedd4‐2’s substrates include many ion channels and receptors because its N‐terminal C2 domain guides Nedd4‐2 to the cell membrane. We previously found that Nedd4‐2 ubiquitinates the glutamate receptor subunit 1 (GluA1) subunit of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor, which leads to reduction of neuronal excitability and seizures in mice. However, despite awareness of a Nedd4‐2 isoform with no C2 domain, the functions of this isoform remain elusive. In this study, we showed that the C2‐lacking Nedd4‐2 has reduced membrane distribution and exhibits reduced affinity toward ubiquitinating GluA1. However, when expressed in primary cortical neurons, we found that the C2‐lacking Nedd4‐2 exhibits a similar activity toward reducing excitatory synaptic strength as does the C2‐containing Nedd4‐2. In an attempt to identify novel Nedd4‐2 substrates that could mediate excitatory synaptic strength, we used unbiased proteomic screening and found multiple synaptic regulators that were up‐regulated in the brain of conditional Nedd4‐2 knockout mice, including protein phosphatase 3 catalytic subunit‐α (PPP3CA; alternately called calcineurin A‐α). We confirmed PPP3CA as a substrate of the C2‐lacking Nedd4‐2 and showed that all three epilepsy‐associated missense mutations of Nedd4‐2 disrupted PPP3CA ubiquitination. Altogether, our results revealed novel potential Nedd4‐2 substrates and suggest that the C2‐lacking Nedd4‐2 represses excitatory synaptic strength most likely through GluA1 ubiquitination‐independent mechanisms. These findings provide novel information to further our knowledge about Nedd4‐2‐dependent neuronal excitability homeostasis and pathological hyperexcitability when Nedd4‐2 is compromised.