RESUMO
Astrocytes react to brain injury in a heterogeneous manner with only a subset resuming proliferation and acquiring stem cell properties in vitro. In order to identify novel regulators of this subset, we performed genomewide expression analysis of reactive astrocytes isolated 5 days after stab wound injury from the gray matter of adult mouse cerebral cortex. The expression pattern was compared with astrocytes from intact cortex and adult neural stem cells (NSCs) isolated from the subependymal zone (SEZ). These comparisons revealed a set of genes expressed at higher levels in both endogenous NSCs and reactive astrocytes, including two lectins-Galectins 1 and 3. These results and the pattern of Galectin expression in the lesioned brain led us to examine the functional significance of these lectins in brains of mice lacking Galectins 1 and 3. Following stab wound injury, astrocyte reactivity including glial fibrillary acidic protein expression, proliferation and neurosphere-forming capacity were found significantly reduced in mutant animals. This phenotype could be recapitulated in vitro and was fully rescued by addition of Galectin 3, but not of Galectin 1. Thus, Galectins 1 and 3 play key roles in regulating the proliferative and NSC potential of a subset of reactive astrocytes.
Assuntos
Astrócitos/metabolismo , Galectina 1/metabolismo , Galectina 3/metabolismo , Córtex Somatossensorial/lesões , Córtex Somatossensorial/metabolismo , Animais , Astrócitos/patologia , Proliferação de Células/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Galectina 1/genética , Galectina 3/genética , Perfilação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Substância Cinzenta/lesões , Substância Cinzenta/metabolismo , Substância Cinzenta/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Córtex Somatossensorial/patologia , Nicho de Células-Tronco/fisiologiaRESUMO
A cell membrane is an essential cellular component providing protection against the outer environment. It is also a host for proteins and carbohydrates responsible for, e.g. transporter, receptor, or enzymatic functions. In parallel, the membrane may also be implicated in pathological processes leading, e.g. to the oligomerization of amyloid-forming proteins, a hallmark of i.a. Alzheimer's disease. The increasing need for detailed information on mechanisms driving the amyloid formation and the potential role of cell membranes in the process proves the research on protein-membrane interactions biologically relevant. Considering the potential and limitations of the relatively well established and newly developed methods, this study focused on selecting methods that allow a broad and comprehensive description of interactions between amyloidogenic protein human cystatin C and lipid bilayers. In the first step, dot-blot and ELISA tests were selected as techniques allowing fast screening for protein-ligand interactions. Next, surface plasmon resonance, spectral shift, biolayer interferometry, and switchSENSE® technology were used to determine kinetic parameters and binding constants for interactions between human cystatin C and the selected lipid bilayers. Based on the obtained results we have proposed the most promising candidates for monitoring of interactions and determining affinity between amyloidogenic proteins and membrane mimetics.