Aline Araujo dos Santos

Neuronal Cell Survival

One of the central issues in neuroscience today is the study of the mechanisms of neuronal survival. Since the discovery of nerve growth factor (almost 60 years ago), many groups have clearly demonstrated the central role of neurotrophins on the regulation of neuronal cell survival during developmental stages as well as during adult life. However, neurotrophins are not alone in regulating neuronal survival, and many groups have demonstrated the effect of different cytokines on this phenomenon. In this brief review we will address the effect of interleukins (IL), particularly IL‐2, IL‐6, and IL‐4, on the survival of neuronal cells.

The effect of PKC activation on the survival of rat retinal ganglion cells in culture.

Abstract Natural cell death is a degenerative phenomenon occurring during the development of the nervous system. Approximately half the neurons initially generated during this period die. The role of trophic molecules produced by target and afferent neurons as well as by glial cells controlling this regressive event has been extensively demonstrated. The aim of this work was to study the role of activated protein kinase C (PKC), an enzyme involved in apoptosis regulation, on the survival of retinal ganglion cells kept “in vitro” for 48 h. For this purpose, we used the phorbol 12-myristate 13-acetate (PMA), a tumor promoter agent that activates PKC. Our results showed that PMA increases the survival of ganglion cells. The effect was dose-dependent and PMA concentrations of 10 or 100 ng/ml produced the maximal effect (a two-fold increase on ganglion cells survival compared with 48 h control). This effect was totally abolished by 1.25 μM chelerythrine chloride (an inhibitor of PKC) and 30 μM genistein (an inhibitor of tyrosine kinase enzymes). Otherwise, PMA was effective only when it was chronically present in the cultures. On the other hand, treatment with 20 μM 5-fluoro-2′-deoxyuridine, an inhibitor of cell proliferation, or 25 μM BAPTA-AM, an intracellular calcium chelator, did not block PMA effect. Our results suggest that the survival of retinal ganglion cells “in vitro” may be mediated by a mechanism that involves PKC activation.

Ouabain induces an increase of retinal ganglion cell survival in vitro: the involvement of protein kinase C.

Abstract Ouabain is a steroid derivative that can regulate many cellular events such as growth and proliferation. It modulates Na + ,K + -ATPase activity leading to the activation of different intracellular pathways through protein–protein interactions that have been characterized during the last few years. The aim of this work was to study the role of ouabain in rat retinal ganglion cell survival after 48 h in culture. Our results demonstrated that ouabain significantly induced an increase in retinal ganglion cell survival. The effect was dose-dependent and was maximal with 3.0 nM. The blockade of protein kinase C activity by 1.25 μM chelerythrine chloride abolished the ouabain effect, indicating an involvement of this intracellular pathway. None of the protein kinase inhibitors usually employed in the study of ouabain-driven intracellular pathways (PD98059, Ly294002, herbimycin, and genistein) was able to influence neuronal survival induced by ouabain. The data presented suggest that ouabain may be the trigger of an intracellular pathway responsible for neuronal survival.

PMA decreases the proliferation of retinal cells in vitro: the involvement of acetylcholine and BDNF.

Protein kinase C (PKC) is involved in several cell events including proliferation, survival and differentiation. The aim of this work was to investigate the role of PKC activation on retinal cells proliferation. We demonstrated that PKC activation by phorbol 12-myristate 13-acetate (PMA), a tumor promoter phorbol ester, is able to decrease retinal cells proliferation. This effect was mediated by M1 receptors and dependent on intracellular Ca(2+) increase, tyrosine kinase activity, phosphatidylinositol 3-kinase activity, polypeptide secretion and activation of TrkB receptors. The effect of PMA was not via activation of mitogen-activated protein (MAP) kinase. Carbamylcholine and brain derived neurotrophic factor were both able to decrease retinal cells proliferation to the same level as PMA did. Our results suggest that PKC activation leads to a decrease in retinal cells proliferation through the release of acetylcholine and brain derived neurotrophic factor in the culture, and activation of M1 and TrkB receptors, respectively.

PMA increases M3 muscarinic receptor levels and decreases retinal cells proliferation through a change in the levels of cell-cycle regulatory proteins.

Protein kinase C (PKC) pathway plays important roles in different phenomena in nervous system development. Our previous data demonstrated that phorbol 12-myristate 13-acetate (PMA) treatment, a PKC activator, for 48 h decreases retinal cells proliferation by a mechanism mediated by muscarinic receptor activation, involving a decrease in M1 receptors levels. The aim of this work was to analyze how PMA interferes in the levels of cell cycle control proteins p53, p21 and cyclin D1 and also to investigate its influence on M3 receptor levels. Our results show that PMA (50 ng/mL) produces a significant increase in p21 and p53 levels, decreases cyclin D1 levels, and also enhances M3 receptors levels in cell cultures. Evaluating the postnatal retinal tissue development until 30 days, we observed that tissue differentiation is accompanied by an increase in M3 and p21 levels. Based on our results we suggest that PMA treatment is promoting a change in muscarinic receptors expression mimicking the pattern observed during tissue differentiation, indicating that PMA is probably accelerating the cholinergic differentiation in rat retinal cell cultures.

Treatment in vitro of retinal cells with IL-4 increases the survival of retinal ganglion cells: the involvement of BDNF.

Interleukin 4 (IL-4) is a pleiotropic cytokine involved in many functions during the development as well as in adult life. Previous work from our group demonstrated, in vitro, that this interleukin is able to prevent rat retinal ganglion cells death after axotomy. The aim of the present study was to investigate the signaling pathways involved in this trophic effect, particularly the cAMP pathway and also to demonstrate the expression of IL-4 in retinas at different stages of post natal development. Our results show that the trophic effect of IL-4 on rat retinal ganglion cells is dependent on the activation of Janus Kinase 3, Protein Kinase A, c-Jun N-terminal Kinase and Tropomyosin related Kinase receptors, on the increase in intracellular calcium levels, on polypeptide release and on the endogenous Brain Derived Neurotrophic Factor (BDNF). We also observed that treatment with IL-4 enhances c-AMP response element binding and Mitogen Activated Protein Kinase phosphorylation and increases the expression of BDNF. Concerning the IL-4 expression our data show an increase in IL-4 levels during post natal development. Taken together our results demonstrate that the trophic effect of IL-4 on retinal ganglion cells of newborn rats is mediated by cAMP pathway and BDNF release.

Aerobic exercise modulation of mental stress-induced responses in cultured endothelial progenitor cells from healthy and metabolic syndrome subjects

AIM Numerous studies have demonstrated that exercise acutely prevents the reduction in flow-mediated dilation induced by mental stress in subjects with metabolic syndrome (MetS). However, it is unknown whether a similar effect occurs in endothelial progenitors cells (EPCs). This study investigated whether exercise protects from the deleterious effect of mental stress on cultured EPCs in healthy subjects and those with MetS. MAIN METHODS Ten healthy subjects (aged 31±2) and ten subjects with MetS (aged 36±2) were enrolled. Subjects underwent a mental stress test, followed immediately by either 40 min of leg cycling or rest across two randomized sessions: mental stress+non-exercise control (MS) and mental stress+exercise (MS+EXE). The Stroop Color-Word Test was used to elicit mental stress. Blood samples were drawn at baseline and following sessions to isolate mononuclear cells. These cells were cultured in fibronectin-coated plates for seven days, and EPCs were identified by immunofluorescence (acLDL(+)/ UEA-I Lectin(+)). KEY FINDINGS All subjects presented similar increases in mean blood pressure and heart rate during the mental stress test (P<0.01) in both the MS and MS+EXE sessions. Number of EPCs was not different between groups at baseline in both sessions (P>0.05). The EPC response to MS and MS+EXE was increased in healthy subjects, whereas it was decreased in subjects with MetS (P<0.04). In healthy subjects, the EPC response to MS+EXE was greater than the response to MS alone (P=0.03). SIGNIFICANCE An exercise session increased EPCs in healthy subjects but did not prevent the EPC reduction induced by mental stress among subjects with MetS.

Impaired Circulating Angiogenic Cells Mobilization and Metalloproteinase-9 Activity after Dynamic Exercise in Early Metabolic Syndrome

Increased levels of adhesion molecules or metalloproteinases (MMPs) may indicate endothelial dysfunction. Exercise mobilizes circulating angiogenic cells (CACs) from bone marrow in healthy subjects, improving vascular function. However, it is unclear whether this mechanism is preserved in the early stages of metabolic syndrome (early MetS). We aimed to evaluate the acute effects of exercise on adhesion molecules, angiogenic factors, MMPs, and CACs in early MetS. Fifteen subjects with early MetS and nine healthy controls underwent an exercise session and a nonexercise session, randomly. Adhesion molecules, angiogenic factors, CACs, and MMPs were evaluated before and after exercise or nonexercise sessions. At baseline, levels of sE-selectin, sICAM-1, and MMP-9 were higher in early MetS than in controls (P ≤ 0.03). After exercise, sE-selectin, sICAM-1, and MMP-9 levels were still higher in early MetS (P < 0.05). Subjects with early MetS presented less CACs (P = 0.02) and higher MMP-9 activity (P ≤ 0.04), while healthy controls presented higher MMP-2 activity after exercise. There was no difference between moments in nonexercise session (P > 0.05). In conclusion, subjects with early MetS already presented impaired endothelial function at rest along with a decrease in CACs and an increase in MMP-9 activity in response to exercise.