Hyaluronan synthesis by developing cortical neurons in vitro

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.

Figure 1. Hyaluronan synthase (HAS)1–3 mRNA expression during cortical neuron development in vitro.

Gene expression was normalised to days in vitro (DIV)0 to provide relative quantification values (RQ). N = 3–5 samples from 3–4 independent cultures for each developmental stage. Error bars are minimum and maximum RQ; *P < 0.05, **P < 0.01, ***P < 0.001 relative to DIV0.

Figure 2. HAS2 protein expression on cortical neurons in vitro.

HAS2 (left: grey, right: red) and actin (green) at DIV3 (A), DIV7 (B), and DIV14 (C). A high magnification example of a neuronal process expressing HAS2 is shown (B, inset). Scale bar: 5 μm (inset), 30 μm.

Figure 3. HAS3 protein expression on cortical neurons in vitro.

HAS3 (left: grey, right: red) and actin (green) at DIV3 (A), DIV7 (B), and DIV14 (C). A high magnification example of a growth cone expressing HAS3 is shown (A, inset). Scale bar: 5 μm (inset), 30 μm.

Figure 4. Hyaluronan expression on cortical neurons in vitro.

Neurons at DIV0, 1, 3, 7, 14, and 21 (A–F) were stained with biotinylated hyaluronic acid binding protein (bHABP) (left: grey, right: red), MAP2 (green), and Hoechst 33258 (blue). Scale bar: 30 μm.

Figure 5. Structural localisation of hyaluronan on cortical neurons in vitro.

Subcellular structures were visualised under differential interference contrast (DIC) optics. bHABP localisation (red) was observed on neuronal lamellipodia (arrowheads; A), immature neurites (between arrowheads; B), growth cones (arrowheads; C), filopodia (arrows; B,C), dendrites (arrows; D), and axons (defined as a thin, MAP2-negative process, arrowheads; D). MAP2, green. Scale bars: 10 μm (D), 30 μm (A–C).

Figure 6. Spatial relationship between hyaluronan and synaptic markers on cortical neurons in vitro.

Representative examples of neurons labelled with hyaluronan (red) and synaptophysin (A, green) or PSD-95 (B, green) at DIV21 are shown. High magnification images indicate small spatial separation, but no colocalisation, between hyaluronan (C,D) and synaptophysin (SYN; E,G) or PSD-95 (F,H) immunoreactive puncta on neuronal processes. Scale bars: 5 μm (A,B), 20 μm (C–H).

Figure 7. Perineuronal net expression on cortical neurons in vitro.

Neurons at DIV7, 14, and 21 (A–C) were stained with biotinylated WFA (left: grey, right: red), MAP2 (green), and Hoechst 33258 (blue). Scale bar: 30 μm.