Research Background and Problem Statement

In social animals, competition among individuals and the establishment of hierarchical status are crucial for resource allocation, group stability, and energy conservation. However, while it is known that the medial prefrontal cortex (mPFC) plays an essential role in regulating social competition behaviors and social hierarchy, the specific molecular regulatory mechanisms are still not fully understood. This study explores the role of long non-coding RNA (lncRNA) SERA and its regulation of PKM2 in excitatory neurons of the brain during competitive behavior and social rank changes.

This research was conducted jointly by researchers from the Department of Pathophysiology at Tongji Medical College of Huazhong University of Science and Technology, the Department of Biology at Boston University, and Cincinnati Children’s Hospital. It was published in 2024 in the journal “Cell Discovery,” revealing a new mechanism involving the SERA/PKM2 pathway in the regulation of social behavior.

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Research Methods and Experimental Design

The study was divided into several stages, using various innovative experimental techniques and methods:

1. Social Competition and Rank Measurement

• Experimental Animals and Grouping: C57BL/6 mice were used, and their social competitiveness and hierarchical rank were assessed through food competition and tube tests.
• Behavioral Testing:
  • Tube Test: Two mice enter a tube from opposite directions, with the winner being the one that forces the other to retreat, establishing a rank order.
  • Warm Spot Test: Mice were observed in a cold environment to see which one occupies the only warm spot.
  • Food Competition Test: Food-deprived mice compete for a single food pellet, establishing a clear order of competitiveness.

2. Neuronal Activity Recording and Analysis

• Electrode Implantation and Recording: Microelectrode arrays were used to record the activity of excitatory neurons in the prelimbic cortex (PL) of mice during competitive behaviors.
• Neuron Classification: Neurons were classified as competition-related, rank-related, or both-related based on differing activity patterns depending on competition success or opponent rank.

3. Molecular Mechanism Analysis

• RNA Sequencing and Splicing Event Analysis: Fluorescence-activated cell sorting (FACS) was used to isolate RNA from PL excitatory neurons of mice with high and low competitiveness, followed by whole-genome RNA sequencing and alternative splicing event analysis.
• PKM Gene Splicing Pattern Detection: It was found that the splicing pattern of the PKM gene changed significantly in highly competitive mice, resulting in a decreased PKM1/PKM2 ratio.

4. Functional Validation

• PKM2 Expression Regulation: The expression of PKM2 in PL excitatory neurons was controlled using the Tet-On/Off system via adeno-associated virus (AAV)-mediated gene editing.
• SERA Functional Validation: Overexpression or knockdown experiments were performed to verify the regulatory effect of SERA on PKM splicing and competitive behavior.

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Research Results and Findings

1. PKM Splicing Pattern and Social Competition

• The use of exon 9 and exon 10 of the PKM gene changed significantly in the PL excitatory neurons of highly competitive mice, resulting in a decrease in the PKM1/PKM2 ratio.
• Modulating PKM2 expression showed an immediate effect on competition-related neuronal activity and subsequently influenced competitive behavior and social rank.

2. SERA Regulates PKM Splicing

• SERA, an lncRNA, is primarily distributed in PL excitatory neurons, showing a pattern negatively correlated with PKM1/PKM2 expression.
• Experimental validation indicated that SERA inhibits PKM2 production by blocking the binding of the splicing site after exon 10 of PKM to U1 snRNP, thereby increasing the PKM1/PKM2 ratio.

3. PKM2’s Effect on Synaptic Function

• PKM2 is localized postsynaptically and promotes synaptic transmission efficiency by phosphorylating the Ser845 site of the AMPA receptor (AMPAR) GluA1 subunit, enhancing its membrane expression.
• Blocking the phosphorylation site with a peptide showed that this process directly impacts the competitiveness and social rank of mice.

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Research Significance and Application Prospects

This study is the first to reveal the core role of the SERA/PKM2 pathway in social behavior regulation. The primary significance of this research includes:

1. Theoretical Significance:

   • Expands the understanding of the neural mechanisms of social behavior, specifically how competition behavior influences social rank through the interaction of neuronal activity and molecular mechanisms.
   • Provides a new mechanism for PKM2’s role in synaptic function, namely, its role as a protein kinase regulating AMPAR membrane expression.

2. Potential Applications:

   • Offers a new target for studying social dysfunction in mental disorders such as depression and autism.
   • PKM2 could become a critical molecular target in neural modulation and behavioral intervention to improve social behavior.

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Research Highlights and Innovations

1. Methodological Innovation: A combination of behavioral, molecular biology, and electrophysiological recording techniques provided a multi-level research perspective.
2. Discovery of New Mechanisms: The study is the first to discover that lncRNA SERA regulates competition behavior by modulating PKM splicing.
3. Application Value: The findings offer potential avenues for molecular intervention in the regulation of social behavior.

By combining multiple disciplines, this study has uncovered the complex molecular mechanisms behind social competitive behavior, providing a significant reference for understanding the formation.