The Sci Adv Shengneng Yin/Mao Bingyu/Ding Yuqiang team collaborated to discover a new mechanism of AMPA receptor ubiquitination in the regulation of excitatory synaptic function

The human brain contains about 10 billion neurons, which are connected to each other through synaptic structures to form neural networks for information exchange and integration, which is the material basis for
performing various physiological functions of the brain.
Its functional regulation disorder is one of the important causes of the occurrence of neuropsychiatric diseases, and it is also an important target for the intervention and treatment of related diseases
.
Glutamic acid is the main excitatory neurotransmitter in the brain, and the expression level of the corresponding glutamate receptor at the synaptic site of neurons is an important determinant
of the efficiency of synaptic information transmission and the activity of neural networks.

The expression localization and functional regulation of AMPA glutamate receptors in the postsynaptic membrane determine the intensity of excitatory synaptic transport activity, a process that neuronal activity depends
.
The transport regulation process of AMPA receptors in neurons determines their dynamic changes in the postsynaptic membrane and is also a key prerequisite for the production of synaptic plasticity (long-term enhanced LTP and long-term inhibition of LTD) [1
].
AMPA receptors are tetrameric complexes composed of different combinations of GluA1/2/3/4 subunits, and various protein post-translational modifications (phosphorylation, glycosylation, and palmitoylation) of these subunits are known to be involved in regulating the dynamic transport process of postsynaptic membrane AMPA receptors [2
].
Although ubiquitinization-proteasome system-mediated regulation of protein stability plays an important role in synaptic transport and plasticity[3], little
is known about the molecular mechanisms of AMPA receptor ubiquitination regulation and how excitatory synaptic transport activity is affected.
At present, the AMPA receptor E3 ubiquitin ligases are Nedd4, Nedd4L, APC^Cdh1 and RNF167, which are also involved in the regulation of excitatory synaptic transport activity [4, 5], and their mediated AMPA receptor protein stability regulation disorders may also be closely related to the development of related neurological diseases, such as epilepsy-related Nedd4L gene point mutations affect the ubiquitination of GluA1 receptors and lead to neuron overexcitement [5].

。 However, the role and molecular mechanism of AMPA receptor ubiquitination regulation in the regulation of excitatory synaptic function, and what kind of internal relationship between this molecular regulation and neurological diseases still need to be further studied
.

The team has long been concerned about the mechanism of the E3 ubiquitin ligase RNF220 in the development of the central nervous system, and in recent years, systematic research has found that it plays an important role in the development process of nerve cell fate determination and neural tube patterning [6-11].

A recent genetic study of the disease reported that RNF220 homozygous mutations (R363Q and R365Q) cause abnormal brain function, and a variety of clinical symptoms are strongly associated with synaptic regulatory disorders, such as intellectual disability and epilepsy [12].

Although our previous findings also suggest that this gene is also highly expressed in the adult cortex and hippocampus of mice, we still do not know anything about the ubiquitinated substrate and biological function of RNF220 in mature neurons, especially whether the molecule is involved in synaptic regulation and brain physiological functions and pathological processes
。 In view of this scientific problem, Professor Sheng Nengyin and Professor Mao Bingyu of Kunming Institute of Zoology, Chinese Academy of Sciences and Professor Ding Yuqiang of Fudan University collaborated to reveal the function and molecular mechanism of RNF220 as a new ubiquitinated ligase of AMPA receptor in the regulation of synaptic transport activity, and the relevant results were based on RNF220 is an E3 ubiquitin ligase for AMPA receptors to regulate synaptic Transmission Title, published online in Science Advances on September 30, 2022
.


In order to resolve the functional mechanism of RNF220 in mature neurons, in view of its main expression in the cortex and hippocampus in the adult brain, the researchers crossed RNF220 fl^/fl conditional knockout mice with Emx1-Cre tool mice to achieve specific knockout of RNF220 molecules in the forebrain region, and found that:
(1) In this mouse model, there were no abnormalities in the development and morphological structure of the cerebral cortex and hippocampus, but the amplitude and frequency of the tiny excitatory synaptic current (mEPSC) mediated by the AMPA receptor, and the stimulatory excitatory synaptic current (eEPSC) were significantly enhanced, while the LTP ability of long-term enhancement was significantly weakened, and the expression level of AMPA receptors in the postsynaptic dense body (PSD) increased significantly, while the expression of NMDAR and other key proteins of the excitatory synapse was significantly increased, while the expression of NMDAR and other key proteins of the excitatory synapse was significantly increased
。 This finding suggests that the E3 ligase RNF220 is involved in regulating excitatory synaptic transport and synaptic plasticity, and suggests that it may function by modulating protein stability in AMPA receptors
.

(2) To test this hypothesis, the researchers further combined with in vitro cells and cultured hippocampal brain tablets and in vivo mouse models, and found that GluA1 and GluA2 receptors are direct substrates of RNF220 ubiquitin ligase, and that the enhancement of RNF220 to AMPA receptor-mediated eEPSCs depends on its ubiquitinated ligase activity, and when the potential RNF220 ubiquitinization site at the intracellular end of GluA1 and GluA2 receptors is mutated, RNF220 also does not regulate the expression localization
of these two receptors in the excitatory postsynaptic membrane.
In addition, RNF220 is also involved in the transport expression of
AMPA receptors in neuronal cell membranes for protein stability regulation mechanisms.

(3) Neurobehavioral analysis of the above mouse models found that RNF220 molecules significantly reduced the short-term memory ability of mice, such as fear memory, new object recognition and social memory, after the absence of the forebrain, while its long-term memory level, such as water maze and fear memory, did not change
significantly.

(4) Molecular biochemical analysis found that the two mutations R363Q and R365Q found in the genetic discovery of the disease significantly weakened the interaction between RNF220 and the AMPA receptor, as well as the ubiquitination regulation process of the latter, consistent with this, the electrophysiological results are also these two mutations that make RNF220 lose the ability to
regulate the activity of excitatory synapse.

In summary, the study found that RNF220 is a new AMPA receptor E3 ubiquitin ligase, which elucidates its mechanism in excitatory synapse transmission and plasticity regulation and learning and memory function, and is conducive to further in-depth study of the mechanism
of synapsin stability regulation in the physiological function and pathological process of the brain.

Ma Pengcheng and Li Yuwei, associate professors and doctoral students at the Kunming Institute of Zoology, Chinese Academy of Sciences, and He Chunhui, doctoral student of Tongji University, are the co-first authors of the paper; Sheng Nengyin, researcher of Kunming Institute of Zoology, Chinese Academy of Sciences, and Professor Mao Bingyu and Professor Ding Yuqiang of Fudan University are co-corresponding authors
of the paper.

RNF220 as a novel AMPA receptor ubiquitinated ligase for regulatory synaptic activity and neurobehavioral schematic

Article link: http://doi.
org/10.
1126/sciadv.
abq4736

Plate Maker: Eleven

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