Abstract
Hyaluronan is a linear glycosaminoglycan that forms the backbone of perineuronal nets around neurons in the cerebral cortex. However, it remains controversial whether neurons are capable of independent hyaluronan synthesis. Herein, we examined the expression of hyaluronan and hyaluronan synthases (HASs) throughout cortical neuron development in vitro. Enriched cultures of cortical neurons were established from E16 rats. Neurons were collected at days in vitro (DIV) 0 (4 h), 1, 3, 7, 14, and 21 for qPCR or immunocytochemistry. In the relative absence of glia, neurons exhibited HAS1–3 mRNA at all time-points. By immunocytochemistry, puncta of HAS2–3 protein and hyaluronan were located on neuronal cell bodies, neurites, and lamellipodia/growth cones from as early as 4 h in culture. As neurons matured, hyaluronan was also detected on dendrites, filopodia, and axons, and around synapses. Percentages of hyaluronan-positive neurons increased with culture time to ~93% by DIV21, while only half of neurons at DIV21 expressed the perineuronal net marker Wisteria floribunda agglutinin. These data clearly demonstrate that neurons in vitro can independently synthesise hyaluronan throughout all maturational stages, and that hyaluronan production is not limited to neurons expressing perineuronal nets. The specific structural localisation of hyaluronan suggests potential roles in neuronal development and function.
Highlights
Hyaluronan is a linear glycosaminoglycan that forms the backbone of perineuronal nets around neurons in the cerebral cortex
Using normalised ΔCq data, HAS3 was expressed at the highest levels (~670 fold higher than HAS1), followed by HAS2 (~130 fold greater than HAS1), while HAS1 consistently had the lowest expression (Table 2)
We provide new in vitro evidence that cortical neurons express the entire family of hyaluronan synthases (HASs) enzymes, and produce hyaluronan on multiple structures important for neuronal development and synaptic function
Summary
Hyaluronan is a linear glycosaminoglycan that forms the backbone of perineuronal nets around neurons in the cerebral cortex It remains controversial whether neurons are capable of independent hyaluronan synthesis. Percentages of hyaluronan-positive neurons increased with culture time to ~93% by DIV21, while only half of neurons at DIV21 expressed the perineuronal net marker Wisteria floribunda agglutinin. As well as other grey matter regions, hyaluronan can form the backbone of specialised lattice-like extracellular matrix structures termed perineuronal nets, which surround the soma and proximal processes of mature GABAergic interneurons[19,20,21,22,23,24]. In the present study, using enriched primary cultures of cortical neurons, we assessed the specific timing and structural localisation of hyaluronan and HASs throughout neuronal development
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