Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

Abstract

Perineuronal nets (PNNs) are implicated in closure of critical periods of synaptic plasticity in the brain, but the molecular mechanisms by which PNNs regulate synapse development are obscure. A receptor complex of NCAM and EphA3 mediates postnatal remodeling of inhibitory perisomatic synapses of GABAergic interneurons onto pyramidal cells in the mouse frontal cortex necessary for excitatory/inhibitory balance. Here it is shown that enzymatic removal of PNN glycosaminoglycan chains decreased the density of GABAergic perisomatic synapses in mouse organotypic cortical slice cultures. Neurocan, a key component of PNNs, was expressed in postnatal frontal cortex in apposition to perisomatic synapses of parvalbumin-positive interneurons. Polysialylated NCAM (PSA-NCAM), which is required for ephrin-dependent synapse remodeling, bound less efficiently to neurocan than mature, non-PSA-NCAM. Neurocan bound the non-polysialylated form of NCAM at the EphA3 binding site within the immunoglobulin-2 domain. Neurocan inhibited NCAM/EphA3 association, membrane clustering of NCAM/EphA3 in cortical interneuron axons, EphA3 kinase activation, and ephrin-A5-induced growth cone collapse. These studies delineate a novel mechanism wherein neurocan inhibits NCAM/EphA3 signaling and axonal repulsion, which may terminate postnatal remodeling of interneuron axons to stabilize perisomatic synapses in vivo.

Decreased Density of Perineuronal Net in Prelimbic Cortex Is Linked to Depressive-Like Behavior in Young-Aged Rats

“…Genetic studies in humans revealed that the variation of NCAN, a gene encoding PNN component protein neurocan, is a common risk factor for schizophrenia and bipolar disorder (Mühleisen et al, 2012;Oruč et al, 2012;Schultz et al, 2014). Neurocan has also been shown to regulate brain development, dendritic spine remodeling and the function of GABAergic interneurons (Zhou et al, 2001;Mohan et al, 2018;Sullivan et al, 2018). The assembly and function of PNNs are disrupted when these component proteins are hydrolyzed or the structures are destroyed, which could ultimately lead to negative consequences and pathophysiology of neuropsychiatric disorders (Wen et al, 2018;Testa et al, 2019).…”

Post‐mortem multiple sclerosis lesion pathology is influenced by single nucleotide polymorphisms

“…How this SNP influences NCAN RNA and Neurocan protein expression or function and how it relates to neuronal functions remains to be experimentally determined. The Neurocan protein is involved in the regulation of peri‐neuronal nets around (parvalbumin) neurons and it is shown that peri‐neuronal nets are decreased in MS cortical gray matter lesions compared to normal appearing gray matter without a reduction in the number of neurons . This suggests that Neurocan and the stability of peri‐neuronal nets could be associated with cortical demyelination in MS, and could possibly be influenced by the genotype at rs1064395.…”

Neurocan Inhibits Semaphorin 3F Induced Dendritic Spine Remodeling Through NrCAM in Cortical Neurons

“…Cells were pre-treated with or without Neurocan for 30 min, then stimulated with 3 nM Sema3F-Fc or Fc protein for 30 min. We used a concentration of Neurocan (20 nM) that exceeded the Kd for Neurocan binding to L1-CAM (∼1 nM) ( Friedlander et al, 1994 ; Milev et al, 1998 ), effectively inhibited ephrinA5-induced axon terminal retraction in cortical neuron cultures ( Sullivan et al, 2018 ), and was within the estimated physiological range in rodent brain, which varies with age and region ( Rauch et al, 1992 ; Bhattacharyya et al, 2015 ). Analysis of spine density on apical dendrites of EGFP-labeled neurons showed that Neurocan (20 nM) blocked Sema3F-induced spine retraction, but had no effect on spine density of Fc-treated neurons ( Figures 3A,B ).…”

“…NrCAM-Fc protein was adsorbed to ELISA plates that were pre-coated with Protein A, then incubated with Neurocan-AP or AP control protein for 1.5 h. Binding was quantified by colorimetric detection of bound AP using p-nitrophenylphosphate. As a positive control, Neurocan-AP was also assayed for binding to NCAM, a different cell adhesion molecule known to engage Neurocan at its extracellular Ig2 domain ( Sullivan et al, 2018 ). Neurocan-AP bound to NrCAM-Fc to a significantly greater extent than control AP ( Figure 4F ).…”

“…C57BL/6 WT mice (P18 and P80) were anesthetized and perfused transcardially with phosphate buffer (0.15 M sodium phosphate, pH 7.4) and postfixed in 4% PFA, 0.1% glutaraldehyde in PBS. Coronal vibratome sections (50 μm) were subjected to pre-embedding immunogold labeling with silver enhancement using Neurocan antibodies and streptavidin-nanogold (Nanoprobes 2016), as we previously described ( Sullivan et al, 2018 ). Sections were silver-enhanced using HQ silver enhancement kit (Nanoprobes 2012), and postfixed in osmium tetroxide.…”