S100B-RAGE induces changes in astrocyte shape and proliferation towards a reactive state in vivo and in vitro.

VILLARREAL A, SEOANE R, ANGELO MF, BARKER P, RAMOS AJ

Barcelona

Congreso; FENS Forum; 2012

The understanding of autocrine glial crosstalk after brain injury has become an important milestone to comprehend the mechanisms that propagate damage in the injured brain. After ischemic brain injury a plethora of neuronal and glial molecules are released, they are collectively known as DAMP (Damage Associated Molecular Pattern) proteins. One of these molecules is glial derived S100B that is also released by reactive astrocytes surrounding brain lesions and signals in a paracrine (to neurons) and autocrine way (to astroglia and microglia). Here we show that S100B signals through RAGE (Receptor for Advanced Glycosilated End products) to activate NFkB in astrocytes and promotes morphological features resembling reactive gliosis in vivo and in vitro. Our in vitro studies using primary cultures showed that S100B exposure induces astrocyte proliferation (evidenced by BrdU incorporation) and increases astrocytic branching. The increased branching effect is RAGE dependent being blocked by RAGE neutralizing antibody or by the transfection of a RAGE-cyto (dominant negative) and exacerbated by RAGEwt overexpression. Downstream signaling seems to involve Cdc42 but not RhoA since the transfection of a DN-Cdc42 but not the overexpression of active RhoA blocks the S100B-induced branching. Indeed, protrusion of astrocytic branch into an artificial wound in vitro (wound healing assay) is promoted by S100B and reduced when astrocytes were transfected with the DN-Cdc42. S100B exposure also induced a dose-dependent activation of NFkB in astrocytes as shown by the activation of NFkB reporter plasmid and nuclear p65 localization. Expression of RAGE was also promoted by S100B, and effect partially abolished by chemical NFkB blockage. In vivo, intracortical release of S100B by direct infusion of S100B using 50um diameter glass pipettes in adult rats (Wistar) showed that increased focal release of S100B reproduces many features of ischemia-induced reactive gliosis with the extension of glial projections to the site of injection, increased proliferation being significantly different from effects observed with the vehicle (PBS) administration. Together these results suggest molecular mechanisms by that S100B>RAGE pathway is able to induce many morphological features of reactive gliosis and activates an autocrine loop that may sustain NFkB activity after brain injury.