Mouse Astrocytes Cerebellar

S-equol Increased the Proliferation, Invasion, Lamellipodia Formation, and Rearrangement of Cortical F-actin Activity in Mouse Cerebellar Astrocytes

Soy isoflavone metabolite S-equol is a phytoestrogen receptor binder for estrogen receptors, which influences many brain areas including the cerebellum. Although widely known, it has not been fully investigated for its impact on cerebellar development and function. Ariyani's team used mouse primary cerebellar cultures, Neuro-2A cells and astrocyte-enriched cultures to explore how S-equol affected cerebellar development by targeting estrogen receptors and G-protein coupled receptors in neurons and astrocytes.

Studies in the primary cerebellar culture demonstrated that S-equol increased the dendrite arborisation of purkinje cells and the neurite outgrowth of neuro-2A cells. To investigate whether S-equol promoted astrocyte proliferation, they performed a BrdU incorporation assay, MTS cell proliferation assay, and immunocytochemistry for pERK1/2. S-equol dose-dependently increased proliferation (1-100 nM) in a BrdU assay (Fig. 1A). An MTS assay showed that S-equol enhanced astrocyte proliferation at 24, 48, and 72 hours. Proliferation was inhibited by G15 (10 nM), but ICI (10 nM) had a minor effect only after 72 hours (Fig. 1B). S-equol increased pERK1/2 positive cells within 30 minutes, and this was inhibited by G15 (Fig. 3C), indicating proliferation via the GPR30 pathway. For S-equol-induced astrocyte migration, invasion assays and F-actin immunocytochemistry were performed. S-equol (10 nM) boosted astrocyte invasion, inhibited by G15 (Fig. 2A). Stress fiber formation and the cortical F-actin score (CFS) increased with S-equol and were suppressed by G15, not ICI (Fig. 2B). This suggests GPR30's key role in astrocyte migration.

Fig. 1. Effects of S-equol on mouse cerebellar astrocyte proliferation (Ariyani W, Miyazaki W, et al., 2019).

Fig. 2. Effects of S-equol on mouse cerebellar astrocyte invasion and F-actin rearrangement (Ariyani W, Miyazaki W, et al., 2019).

Neuron-glia Interaction Enhanced TH-induced Neuritogenesis

Membrane-associated receptors are vital in brain development, with thyroid hormones (TH) affecting both nuclear and membrane-associated receptors, particularly integrin αvβ3 in neurons and glia. Integrin αvβ3 influences cellular events like morphogenesis and synaptogenesis. However, its role in brain development remains unclear.

Ariyani et al. investigated TH's effects via integrin αvβ3 on neurons and astrocytes using primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture of neurons and astrocytes. The study confirmed that TH promotes dendrite growth, which is hindered by inhibiting integrin αvβ3 or TRα. Additionally, TH impacts protein phosphorylation through TRα-dependent pathways, vital for neuritogenesis, indicating a role for neuron-astrocyte interaction. To examine the effects of TH exposure on neuron-glia interactions, they performed a co-culture study. Neuro-2A cells cultured with cerebellar astrocytes enriched culture for 3-5 days were stained with β-tubulin III and anti-doublecortin (DCX). In co-cultures, DCX stained Neuro-2A and astrocyte soma, so β-tubulin III and F-actin were used to study cell interactions. Photographs of Neuro-2A and co-cultures are shown (Fig. 3A). Neurite growth was measured using Sholl analysis, showing enhanced TH-induced neurite branching and elongation in co-cultures (Fig. 3B-D). Inhibiting integrin αvβ3 reduced these effects (Fig. 4), indicating its importance in neuron-glia interaction for neuritogenesis.

Fig. 3. Co-culture with astrocytes enhanced TH-augmented neurite length in Neuro-2A cells (Ariyani W, Miyazaki W, et al., 2022).

Fig. 4. Integrin αvβ3 affect neurite outgrowth induced by TH in co-culture of astrocytes and Neuro-2A cells (Ariyani W, Miyazaki W, et al., 2022).