Sonic hedgehog signalling mediates astrocyte crosstalk with neurons to confer neuroprotection.

Christopher I Ugbode,Warren D Hirst,Benjamin J Whalley,Imogen Smith,Marcus Rattray

doi:10.1111/jnc.14064

Abstract

Sonic hedgehog (SHH) is a glycoprotein associated with development that is also expressed in the adult CNS and released after brain injury. Since the SHH receptors patched homolog‐1 and Smoothened are highly expressed on astrocytes, we hypothesized that SHH regulates astrocyte function. Primary mouse cortical astrocytes derived from embryonic Swiss mouse cortices, were treated with two chemically distinct agonists of the SHH pathway, which caused astrocytes to elongate and proliferate. These changes are accompanied by decreases in the major astrocyte glutamate transporter‐1 and the astrocyte intermediate filament protein glial fibrillary acidic protein. Multisite electrophysiological recordings revealed that the SHH agonist, smoothened agonist suppressed neuronal firing in astrocyte‐neuron co‐cultures and this was abolished by the astrocyte metabolic inhibitor ethylfluoroacetate, revealing that SHH stimulation of metabolically active astrocytes influences neuronal firing. Using three‐dimensional co‐culture, MAP2 western blotting and immunohistochemistry, we show that SHH‐stimulated astrocytes protect neurons from kainate‐induced cell death. Altogether the results show that SHH regulation of astrocyte function represents an endogenous neuroprotective mechanism.

Highlights

Primary neuron cultures contained 90% neurons to 10% astrocytes. This is based on staining using CD68/ionized calcium-binding adapter molecule 1 for Sonic hedgehog (SHH), astrocytes and neuroprotection 431 microglia, glial fibrillary acidic protein (GFAP)/S100b for astrocytes, b-3 tubulin/Tau/microtubule-associated protein 2 (MAP2)/NeuN/Doublecortin staining for neurons (Data not shown)
Smoothened agonist signals via astrocytes to decrease neuronal firing Having shown that activation of SHH signalling pathways caused changes in levels of key astrocyte proteins which accompanied the morphological changes, we addressed the functional consequences of SHH pathway activation
Comparing neurons conditioned with astrocytes on transwells to neurons which had been cultured with transwells and treated with kainic acid (100 lM), using western blotting, we found that kainic acid significantly reduced MAP2 protein levels of neurons (84.4 Æ 4.6% decrease, p = 0.0003, DF = 4, n = 3)

Summary

Introduction

Astrocytes change their morphology contributing to neurodegeneration through increasing the levels of proinflammatory cytokines and decreasing the levels of glutathione (John et al 2004). This change in functional characteristics is described as a ‘reactive’ phenotype. A2 astrocytes, were thought to be protective as a result of their up-regulation of neurotrophic factors after intraperitoneal lipopolysaccharide injection This may help to disseminate the functional changes between normal and reactive astrocytes identifying whether reactive astrocytes gain ‘function’ or gain ‘detrimental effects’ dependant on the type of pathology (Sofroniew and Vinters 2010).

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