Deconstructing the perineuronal net: Cellular contributions and molecular composition of the neuronal extracellular matrix

Abstract

Perineuronal nets (PNNs) are lattice-like substructures of the neural extracellular matrix that enwrap particular populations of neurons throughout the central nervous system. Previous work suggests that this structure plays a major role in modulating developmental neural plasticity and brain maturation. Understanding the precise role of these structures has been hampered by incomplete comprehension of their molecular composition and cellular contributions to their formation, which is studied herein using primary cortical cell cultures. By defining culture conditions to reduce (cytosine-β-d-arabinofuranoside/AraC addition) or virtually eliminate (elevated potassium chloride (KCl) and AraC application) glia, PNN components impacted by this cell type were identified. Effects of depolarizing KCl concentrations alone were also assessed. Our work identified aggrecan as the primary neuronal component of the PNN and its expression was dramatically up-regulated by both depolarization and glial cell inhibition and additionally, the development of aggrecan-positive PNNs was accelerated. Surprisingly, most of the other PNN components tested were made in a glial-dependent manner in our culture system. Interestingly, in the absence of these glial-derived components, an aggrecan- and hyaluronan-reactive PNN developed, demonstrating that these two components are sufficient for base PNN assembly. Other components were expressed in a glial-dependent manner. Overall, this work provides deeper insight into the complex interplay between neurons and glia in the formation of the PNN and improves our understanding of the molecular composition of these structures.

Introduction

Within the central nervous system, enigmatic structures known as perineuronal nets (PNNs) enwrap the cell soma and proximal neurites of particular populations of neurons in a lattice-like fashion (Hockfield and McKay, 1983, Zaremba et al., 1989, Guimaraes et al., 1990, Hockfield et al., 1990, Brückner et al., 1993, Celio and Blumcke, 1994, Celio et al., 1998). PNNs are specialized substructures of the neural extracellular matrix (ECM), which is molecularly distinct from other classical matrices, in that it contains low amounts of several traditional ECM components, such as: fibronectin, collagen and laminin (Miner and Sanes, 1994) and is uniquely enriched with chondroitin sulfate proteoglycans (CSPGs) and hyaluronan (for review, see Bandtlow and Zimmermann, 2000). Based on the coincident temporal appearance of PNNs with the maturation and stabilization of synapses (Zaremba et al., 1989, Guimaraes et al., 1990, Hockfield et al., 1990), they are postulated to impact these important developmental processes (Hockfield et al., 1990; for review, see Wang and Fawcett, in press). However, despite the known existence of PNNs for more than a century, our understanding of their assembly and function has been elusive, due in part to an incomplete understanding of their molecular components and how they interact.

Previous work has identified members of the lectican family of CSPGs, hyaluronan (for review, see Yamaguchi, 2000), tenascins (Celio and Chiquet-Ehrismann, 1993, Wintergerst et al., 1996, Hagihara et al., 1999, Galtrey et al., 2008) and hyaluronan and proteoglycan link proteins (HAPLNs) (Hirakawa et al., 2000, Bekku et al., 2003, Carulli et al., 2006, Carulli et al., 2007, Carulli et al., 2010, Galtrey et al., 2008, Kwok et al., 2010) all to comprise the PNN. There is also some evidence for both neuronal and glial contributions to PNN formation (Miyata et al., 2005, Carulli et al., 2006), but their distinct roles remain poorly understood. To understand the function of PNNs it is critical to dissect their molecular composition, which will then enable us to specifically disrupt these structures in a precise manner.

The goal of the present study was to unravel the molecular composition of PNNs to determine the cellular contributions to these structures, gain insights into the role of extrinsic factors such as neural activity and establish the minimal components required to form PNNs. Here we utilized dissociated cortical cultures to determine how chemical glial inhibition and chronic depolarization impact the expression pattern of PNN components as well as the overall structure of PNNs. To achieve this, we performed immunocytochemistry, Western blotting and real-time PCR analyses. Taken together, our results reveal specific contributions by neurons and glia to the production of individual PNN constituents and provide a more complete understanding of PNN structure.