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

<div><p>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.</p></div

Deactivation of Rac1 activity rescues CD44 knockdown and hyaluronidase-induced morphological changes of astrocytes.

<p>A: Representative images of astrocytes transfected with CD44shRNA/pSuper or co-transfected with pcDNA3-EGFP-Rac1-T17N (Rac1-DN) constructs. The β-actin-RFP construct was used for cell visualization. Scale: 20 μm. B: Morphometric analysis of shape-describing parameters of cells treated as in A. One way ANOVA, area: F(3.112) = 2.456, p>0,05, solidity: F(3.114) = 30.173, p<0.001 Sidak post hoc test, circularity: F(3.114) = 13.834, p<0.001, branching: F(3.114) = 51, 825, p<0,001. Dunnett’s C post hoc test. C: Representative images of astrocytes transfected with pcDNA3-EGFP-Rac1-T17N (Rac1-DN) and β-actin-RFP constructs and treated with hyaluronidase. Scale: 20 μm. D: Morphometric analysis of shape-describing parameters of cells treated as in C. One way ANOVA, area: F(2.147) = 1.520, p>0.05, solidity: F(2.147) = 106.292, p<0.001, circularity: F(2.147) = 96.843, p<0.001, branching: F(2.147) = 135.932; p<0,001, Dunnett’s C post hoc tests.</p

Hyaluronidase treatment and CD44-knockdown leads to enhanced Rac1 activity.

<p>A: Cells were transfected with FRET based biosensor pRaichu-Rac1/1011X and then treated or not with hyaluronidase for 24h. YFP-CFP ratio was calculated as a readout of Rac1 activity. T-Student test, t(589) = 3.212; p<0,001. B: Cells were co-transfected with pRaichu-Rac1/1011X and pSuper/CD44shRNA/CD44-RFP constructs. YFP-CFP ratio was calculated as a readout of Rac1 activity. One way ANOVA, F(2.527) = 39.998; p<0.001, Sidak post hoc test.</p

Astrocytes treated with hyaluronidase acquire the stellate-like morphology.

<p>A. Hyaluronan digestion by hyaluronidase was evaluated by staining with hyaluronan binding protein (HABP) (red). Scale: 30 μm. B. Measurement of fluorescence intensity. One way ANOVA test was performed, F(2.57) = 53.169; p<0.001, Dunnett’s C post hoc. C. Representative images of astrocytes transfected with β-actin GFP and either untreated (control) or treated with hyaluronidase or heat inactivated hyaluronidase for 48h. Cell nuclei were visualized with DAPI staining. Scale: 30 μm.D. Morphometric analysis of shape-describing parameters of cells treated as described in C. One way ANOVA test was performed, area: F(2.57) = 2.658; p>0.05, solidity: F(2.57) = 16.814; p<0.001, circularity: F(2.57) = 13.799; p<0.001, branching: F(2.57) = 16.774; p<0,001 Dunnett’s C post hoc test.</p

CD44 regulates astrocyte morphology.

<p>A: Validation of CD44shRNA and CD44-GFP constructs. Astrocytes were transfected with pSuper, CD44shRNA or CD44-GFP constructs (together with β-actin-GFP plasmid) and then immunostained with anti-CD44 antibody (red). The level of CD44 expression was evaluated by measuring CD44 immunofluorescence (IF) signal intensity with the use of ImageJ program. One way ANOVA, F(2.71) = 71.187, p<0.001, Dunnett C post hoc tests. Scale: 30 μm. B: Morphological analysis of shape-describing parameters of astrocytes in 2D cultures co-transfected with pSuper, CD44shRNA or CD44shRNA/CD44Rescue constructs together with β-actin-GFP plasmid. One way ANOVA test was performed, area: F(3.150) = 8.169; p<0.001, solidity: F(3.153) = 21.454; p<0.001, circularity: F(3.153) = 18.873; p<0.001, Dunnett’s C post hoc tests, branching: F(3.151) = 33,478; p<0.001, Sidak post hoc test. Scale: 30 μm. C: The morphological analysis of shape-describing parameters of astrocytes in 3D cultures transfected with pSuper or CD44shRNA constructs (together with β-actin-GFP plasmid) or CD44-GFP. One way ANOVA test was performed, area: F(2.92) = 12.311; p<0.001, Sidak post hoc test; solidity: F(2.95) = 42.208; p<0.001, Dunnett’s C post hoc test, circularity: F(2.94) = 20.609; p<0.001, Dunnett’s C post hoc test, branching: F(2.95) = 17.703; p<0.001, Sidak post hoc test. Scale: 30 μm.</p