Cell Death Differ︱ Song Xiaoyuan’s team reported on the molecular mechanism of long non-coding RNA Synage regulating synaptic structure and neuronal function in the cerebellum

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Written by Wang Fei, edited by Wang Sizhen.
Synapse is the basic structure of nerve cell interconnection and the key part of information transmission.

Due to the typical "sandwich" layered structure and relatively simple cell types of the cerebellar cortex, the cerebellum has become an ideal model for studying synaptic development and function [1].

The formation and maintenance of synapses depend on the participation of a variety of proteins and RNA molecules [2-3].

 Long non-coding RNAs (lncRNAs) are an important class of molecules that regulate neurodevelopment and synaptic function [4].
Therefore, it is necessary to understand the exact biological role and molecular mechanism of lncRNA in synaptic structure and function.

However, the mechanism by which most lncRNAs regulate synapses is not clear.

 On March 24, 2021, Professor Song Xiaoyuan’s research team from the School of Life Sciences of the University of Science and Technology of China published a research paper entitled The long noncoding RNA Synage regulates synapse stability and neuronal function in the cerebellum online in Cell Death & Differentiation, reporting lncRNA Synage regulates the molecular mechanism of synaptic structure and neuronal function in the cerebellum.

 LncRNA Synage is transcribed from the Gm2694 gene and is mainly expressed in the cerebellar cortex in the nervous system.

Studies have shown that Synage is related to neuroectodermal differentiation [5], regulating FGF/ERK signal transduction and embryonic stem cell self-renewal [6].

However, whether and how Synage functions in the synapses of the cerebellar cortex is unclear.

 In this work, the researchers found that lncRNA Synage is mainly distributed in the cytoplasm and synapses of neurons in the cerebellar cortex.

The distribution of Synage in the genome of mice, rhesus monkeys and humans and the specific distribution of expression in the cerebellar cortex are very conservative.

Loss of Synage can lead to loss of cerebellar neurons, decreased number of synapses, impaired synaptic function, and motor dysfunction.
These phenotypic defects can be significantly improved by AAV-mediated overexpression of Synage.

These results suggest that Synage regulates neuron development and synaptic function.

 Cbln1 is the upstream neighboring gene of Synage.
CBLN1 protein is secreted by cerebellar granule cells and is an important organizer of parallel fiber-Purkinje cell (PF-PC) synapses [7].

In order to explore the molecular mechanism of lncRNA Synage regulating cerebellar development and synaptic function, the researchers first analyzed whether Synage regulates the expression of the neighboring gene Cbln1.

The expression of Cbln1 was significantly down-regulated after knocking down or knocking down Synage in vivo and in vitro.

Through the analysis of the two databases of StarBase v3 and DIANA-LNCBase v2, it is predicted that microRNA mmu-miR-325-3p is the shared miRNA target of Synage and Cbln1 mRNA.

Through experiments such as dual luciferase reporter system, mmu-miR-325-3p pull-down, mmu-miR-325-3p overexpression and inhibition, etc.
, the molecular mechanism of Synage's regulation of Cbln1 mRNA level through mmu-miR-325-3p was confirmed .

 Because the neuron and synaptic damage phenotype of Synage knockout mice is more severe than that of Cbln1 knockout mice [7-9].

Therefore, the researchers speculate that in addition to regulating the expression of Cbln1, Synage may also regulate cerebellar development and synaptic function through other molecular mechanisms.

In vivo RNA pull-down-mass spectrometry (mass spectrometry) experiments in adult mouse cerebellum found that Synage binds to HSP90AA1 and LRP1 proteins.

Studies have shown that LRP1 protein and HSP90AA1 protein regulate synaptic structure and function, and LRP1 interacts with postsynaptic density protein PSD-95 to participate in transmitter-dependent postsynaptic response and synaptic transmission [2, 10-11].

LRP1 is a multi-ligand receptor expressed abundantly in neurons [2].

Loss of LRP1 can cause severe damage to neuron and synaptic function [2, 12].

HSP90AA1 is also involved in regulating neuron development and synapse formation [13].

The researchers further confirmed the formation of a complex between Synage and HSP90AA1-LRP1-PSD-95 synaptic protein through RNA immunoprecipitation and electrophoretic mobility detection.

RNA fluorescence in situ hybridization and protein-specific immunofluorescence experiments also found that they co-localized in the cerebellar cortex, and protein co-localization was significantly reduced after Synage was deleted.

Co-immunoprecipitation experiments between LRP1 and HSP90AA1 and PSD-95 proteins confirmed that the interaction of the three synaptic proteins was significantly weakened after the loss of Synage.

Overexpression of Synage in the HT-22 cell line and overexpression of Synage after LRP1 knockdown significantly increased the interaction between LRP1 and HSP90AA1 protein.

 This work found that lncRNA Synage regulates synaptic structure and function through at least two molecular mechanisms during cerebellar development.

 One is that Synage acts as a sponge for adsorbing microRNA mmu-miR-325-3p, which regulates the mRNA and protein levels of cerebellar synaptic protein CBLN1; the other is that Synage acts as a scaffold to regulate LRP1 in PF-PC synapses.
-Assembly of HSP90AA1-PSD-95 synapse protein complex.  A summary diagram of the molecular mechanism of Synage regulating the structure and function of cerebellar synapses (picture quoted from: Wang, F.
, et al.
Cell Death & Differentiation, 2021: 1-17) Wang Fei (second row, third from left), Wang Qianqian (first row) Third from left), Liu Baowei (third row, first from left), Song Xiaoyuan (first row, fourth from left) (picture source: provided by Song Xiaoyuan's laboratory) Dr.
Wang Fei, Wang Qianqian (undergraduate PhD student), School of Life Sciences, University of Science and Technology of China, and Liu Baowei with a master's degree The first author of the paper, Professor Song Xiaoyuan is the corresponding author of the paper.

This research was supported by the National Natural Science Foundation of China (91540107), the State Key Laboratory of Neuroscience (SKLN-201805), the Natural Science Foundation of Anhui Province (2008085QC160), and the Anhui Science and Technology Research Project (1604a0802069).

 Original link: https://doi.
org/10.
1038/s41418-021-00774-3 Recommended high-quality scientific research training courses [1] Medicine plus patch clamp and optogenetic and calcium imaging technology seminar (April 24-25, 2 days and 1 night) [2] Online ︱Single Cell Sequencing Data Analysis and Research Thinking Seminar (January 16-17, 21) (courses can be booked from April to May 2021) [3] Multimodal Brain Image data processing analysis/machine learning application online training brain image: 17-18 Machine learning: 23-24 reference (slide up and down to view) [1] Yang H, Yang C, Zhu Q, Wei M, Li Y, Cheng J, et al.
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