The interplay between the biochemical and biomechanical properties to the establishment of perineuronal nets

Perineuronal nets (PNNs) are an important pericellular matrix structure that form around the soma and dendrites of sub-populations of neurons in the mature central nervous system (CNS). They have been implicated in regulating synaptic plasticity, increasing synaptic stability, neuroprotection and memory formation. They are composed of hyaluronan (HA), chondroitin sulfate proteoglycans (CSPGs), Haplns and tenascin-R, that can assemble in many different potential configurations to form morphologically distinct PNNs. Currently there is a proposed model for the assembly of this extracellular matrix structure, but the detailed mechanism for the stabilisation of the morphologies of PNNs is not yet known. The aim of this thesis project is to characterise the biochemical heterogeneity of PNNs and understand how PNN molecules assemble to form a pericellular coat with distinct morphologies, while ascertaining the biomechanical contribution of each molecule to the pericellular coat.

Using Western blotting, regional variations in the molecular composition of PNN-associated molecules were observed that may present an opportunity to modulate PNNs region specifically in the future. Different splice variants and levels of glycosylation of CSPGs were profiled in different CNS regions. To analyse the assembly of PNNs in real-time and measure the biomechanical properties of the film, an in vitro methodology was established using quartz crystal microbalance with dissipation monitoring (QCM-D). The PNN film was modelled using a tethered film of HA, to which HA and proteoglycan link protein 1 (Hapln1) and aggrecan (Acan) were sequentially added. Hapln1 bound to HA films stably, while binding of Acan was reversible, though could be stabilised by sequentially adding Acan to Hapln1-presenting HA films. While the density of HA films influenced the interaction of Acan, increasing the density of pre-bound Hapln1 did not. Furthermore, Hapln1 addition caused a rigidification of HA films, while Acan addition caused a softening effect, highlighting the important role of Hapln1 in stabilising the PNN matrix and a potential mechanism for regulating the biomechanical properties of PNNs. As cells in the CNS can mechanosense, understanding these mechanisms may offer new therapeutic avenues for targeting neurological diseases correlated with changes to PNNs.

In addition, a new methodology for sizing surface-anchored GAG chains was established using QCM-D by utilising the relationship between the ratio of ∆D/-∆f and GAG size. This methodology highlights the effect of HA size on the biomechanical properties of the PNN film and presents a potential future methodology for the characterisation of PNN HA size. Mechanisms for biomechanically manipulating PNN films by increasing HA size and varying the density of Hapln1 and HA suggest PNNs may partially function via a biomechanical signalling mechanism in addition to already established mechanisms.