Resumo

Oligodendrocyte is the myelinating cell of the central nervous system (CNS). Most of the cortical oligodendrocytes are generated from the subventricular zone (SVZ) during postnatal development. Once generated, oligodendrocyte progenitors (pOLGs) migrate radially out of the SVZ into the overlying cortical parenchyma differentiating into mature oligodendrocytes. Specific growth factors regulate proliferation and cell fate specification in the SVZ. Lysophosphatidic acid (LPA) is a lipid growth factor known to influence progenitor proliferation during embryogenesis. Besides, it is known to promote transient retraction of migratory postmitotic neuron processes in vitro. Here we investigated the role of LPA on proliferation, migration and differentiation of SVZ progenitors in vivo and in vitro. To assess an in vivo role for LPA, intraventricular infusion of LPA 1μM or BSA fat acid free (BSAfaf, as control) was performed in postnatal day 5 (P5) Swiss rats. Progenitors were labeled with a single injection of BrdU (60mg/Kg, i.p.) immediately or 45h after LPA infusion. Animals were sacrificed 48h later and tissue processed for immunohistochemistry. BrdU+ cell density was evaluated in the SVZ and in the overlying white and gray matter. At least three sections per animal were analyzed within the presumptive somatosensory area. LPA effect on progenitors was also studied in vitro by generating neurospheres from SVZ and cortical parenchyma at P5-7 Swiss rats. Briefly, cells were isolated and incubated in medium EGF 5ng/mL, FGF2 10ng/mL and LPA 1uM (or BSAfaf 0.04%, as control). After 5 days, cells from LPA treated and non-treated neurospheres were dissociated and plated to differentiate for 7 days on poly-L-lysine (10μg/mL) in the absence of growth factors. Neurons and oligodendrocytes were quantified after immunocytochemistry. After 48h of BrdU administration, LPA treated animals (n=3) presented an increase of 49,2±6,8% in cell labeling in the cortical layers and 53,2±9,5% in the white matter compared to control group (n=5). However, no change in cell proliferation was detected in the SVZ, white and gray matter of LPA-treated animals. These in vivo results raise the following hypotheses: LPA could (1) accelerate cell migration out of SVZ to the parenchyma; (2) stimulate radial glial transformation into astrocytes; (3) promote oligodendrogenesis and/or (4) reduce apoptosis in the SVZ. Additionally the neurosphere assay showed that LPA did not change the number of neurospheres generated from SVZ and cortical parenchyma regions studied, suggesting no effect on survival of the progenitors initially plated. LPA also reduced SVZ neurosphere size in approximately 50±9,9% and also the cell number per neurospheres in 35,8±15,3%, but no effects were observed on neurospheres generated from the cerebral parenchyma. These results suggest that LPA anticipated progenitor differentiation which suggests an effect on cell cycle duration. Furthermore, neurospheres formed in the presence of LPA gave rise to 184±9,9% more oligodendrocytes (O4+ cells) compared to control, but no changes were observed in neuron density (β-tubulin III+ cells). Taken together, these results suggest that LPA selectively increases glial cell number in the cerebral cortex by affecting postnatal SVZ progenitors directing them towards an oligodendrocyte cell fate. Future experiments will show whether LPA also affects oligodendocyte migration.