Astrocytes Secrete Neurocan to Control Inhibitory Synapse Formation and Function

In recent years, the role of neuron-glial cell interactions in synapse formation and maintenance of synaptic function has become a hot topic in neuroscience research. This paper, published by Dolores Irala et al. from Duke University Medical Center in the May 2024 issue of “Neuron,” reveals how astrocytes control the formation and function of specific types of inhibitory GABAergic synapses by secreting the C-terminal fragment of Neurocan.

Background

In the mammalian cerebral cortex, 80% of neurons are glutamatergic (excitatory) pyramidal neurons, and 20% are γ-aminobutyric acid (GABAergic) (inhibitory) interneurons. Interneurons can be divided into three major types based on their morphological, transcriptomic, and electrophysiological properties: somatostatin (SST), parvalbumin (PV), and 5-hydroxytryptamine receptor 3A (HTR3A). Although glial cells play an important role in synapse formation, previous studies have identified various glial-secreted proteins that promote excitatory synapse formation, such as thrombospondins, SPARCL1/Hevin, glycoproteins, etc. However, the glial signals controlling inhibitory synapse formation have remained unclear.

Research Process

To explore astrocyte-secreted proteins that control inhibitory synapse formation, the authors developed a cortical neuron culture system devoid of glial cells, including excitatory and inhibitory neurons from the neonatal rat cortex. By using immunoadsorption to isolate L1CAM-positive neurons and culture them in vitro, these neurons were allowed to form extensive neurites. Synapse formation was assessed by treating neurons cultured for 8 and 11 days with astrocyte-conditioned medium (ACM) or recombinant proteins.

The neuronal cultures contained less than 2% glial fibrillary acidic protein (GFAP)-positive glial cells, 78% excitatory neurons, and 20% inhibitory neurons. Using confocal microscopy and image analysis, the authors found that ACM significantly induced the formation of inhibitory synapses without altering the number of respective synaptic markers (Bassoon and Gephyrin). Additionally, the authors identified Neurocan (NCAN) as a significant astrocyte-secreted protein that induces inhibitory synapse formation through candidate protein screening.

Research Results

The following are the key findings of the study:

1. NCAN Fragment Localization and Synapse Formation: Using Western blot and immunofluorescence staining, the expression pattern of Neurocan and cleavage fragment locations during the development of neonatal mice were determined. It was found that the C-terminal fragment of NCAN localized to synapses and controlled the formation and function of inhibitory synapses in the mouse cortex.

2. Effects of NCAN Deletion on Synapse Formation: Using an NCAN knockout mouse model (deletion of exons 3 and 4), they found a significant reduction in the number and function of inhibitory synapses, without affecting excitatory synapse density or function. Transmission electron microscopy further confirmed that inhibitory synapse density in the ACC region of NCAN knockout mice was halved, with no significant effect on neuron survival rates, body weight, or brain density.

3. Effects of NTER and CTER Fragments on Inhibitory Synapses: In vitro culture experiments demonstrated that the C-terminal (CTER) fragment of NCAN significantly promoted inhibitory synapse formation, while the N-terminal (NTER) fragment had no significant effect. Adding the CTER fragment to ACM from NCAN knockout mice could fully rescue the reduction in inhibitory synapse numbers.

4. NCAN Interaction with Specific Synaptic Proteins: Using super-resolution microscopy and in vivo proximity labeling, the NCAN-ELS fragment was found to specifically bind and regulate the formation of SST-positive synapses while having no significant effect on PV-positive synapses.

5. Functional Studies: Electrophysiological experiments recorded miniature inhibitory postsynaptic currents (mIPSCs) in ACC layer 2-3 pyramidal neurons in mice and found a significant reduction in mIPSC frequency in NCAN knockout mice, indicating that it regulates inhibitory synaptic function, specifically affecting SST-positive synapses.

Conclusion and Practical Significance

This paper identifies the C-terminal fragment of glial-secreted Neurocan as a novel key regulatory protein for inhibitory synapse formation, revealing its role in the formation of specific types of inhibitory synapses in the mammalian cortex. This discovery not only provides new insights into neurodevelopment and neural network stability but also offers potential new targets for therapeutic intervention in neurological diseases or brain injuries.

Furthermore, the study finds that the specific regulatory role of NCAN fragments may involve different neuronal cell types and the formation and function of specific synapses, laying the foundation for future research into the more complex and precise interactions between astrocytes and neurons.