Abstract
We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.
Highlights
In the adult brain, ECM2 is mainly present in the intercellular spaces between neurons and glial cells
We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs)
To extract and analyze CSPGs loosely associated in the extracellular matrix (ECM), those associated with membranes and those bound in stable ternary complexes in PNNs, adult rat brain was sequentially extracted with four different buffers as described under “Experimental Procedures.”
Summary
CSPGs in the CNS can interact with various growth factors and cell adhesion molecules, playing a significant role in development [18, 20] They mostly have an inhibitory effect toward neurite outgrowth and regeneration, either via their CS chains or core proteins [29]. PNNs form late in development, surrounding particular classes of neurons Their time of appearance corresponds with the termination of plasticity at the end of critical periods in many parts of the CNS, and their appearance in the visual cortex can be delayed by dark rearing, which prolongs plasticity. Chondroitinase digestion of CSPGs in the PNNs in the adult visual cortex reactivates plasticity after the critical period [11] This and other evidence has led to the hypothesis that PNNs. Extraction of Perineuronal Net Proteoglycans are involved in the control of plasticity in the CNS. This enabled us to examine the composition of PNNs and changes in development; to analyze the GAG chain composition of the different extracts in the adult brain; to study how the various components are retained within the net structure and to compare the properties of the rather different PNNs from brain and spinal cord
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