Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling.

Anna Konopka,Evgeni Ponimaskin,Andre Zeug,Grzegorz M Wilczynski,Franziska Mueller,Agnieszka Chwedorowicz,Joanna Dzwonek,Beata Kaza,Anna Skupien,Pontus Aspenstrom

doi:10.1371/journal.pone.0155053

Anna Konopka, Evgeni Ponimaskin + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0155053

Copy DOI

Abstract

Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of astrocytes.

Highlights

Astrocytes constitute the largest population of the glial cell type in the central nervous system (CNS) and play multiple supportive and regulatory roles in neuronal function [1]
To investigate whether HA regulates astrocyte morphology we investigated the effect of hyaluronidase treatment on astrocytes cultured in vitro
In order to evaluate the efficiency of HA digestion by hyaluronidase, astrocytes were stained with hyaluronan binding protein (HABP) (Fig 1A)

Summary

Introduction

Astrocytes constitute the largest population of the glial cell type in the central nervous system (CNS) and play multiple supportive and regulatory roles in neuronal function [1]. They represent a heterogeneous class of cells, exhibiting different morphological appearances i.e. Astrocytes contribute to this, mainly via their responses to different stimuli from extracellular space and usually this response is accompanied by morphological changes of the astrocyte [9,10] They acquire polarity to migrate [11] or play a role in glutamate clearance via invasion of thin astroglial processes into the synaptic cleft [12]. With the use of the FRET-based biosensor for Rac activity and dominant negative mutant of Rac, we investigated the function of Rac activity in regulation of astrocyte morphology

Methods

Results

Conclusion