Regulation and role of Acyl-CoA synthetase 4 in glial cells.

Acyl-CoA synthetase 4 (Acsl4), an enzyme involved in arachidonic acid (AA) metabolism, participates in physiological and pathological processes such as steroidogenesis and cancer. The role of Acsl4 in neurons and in nervous system development has also been documented but little is known regarding its functionality in glial cells. In turn, several processes in glial cells, including neurosteroidogenesis, stellation and AA uptake, are regulated by cyclic adenosine monophosphate (cAMP) signal. In this context, the aim of this work was to analyze the expression and functional role of Acsl4 in primary rat astrocyte cultures and in the C6 glioma cell line by chemical inhibition and stable silencing, respectively. Results show that Acsl4 expression was regulated by cAMP in both models and that cAMP stimulation of steroidogenic acute regulatory protein mRNA levels was reduced by Acsl4 inhibition or silencing. Also, Acsl4 inhibition reduced progesterone synthesis stimulated by cAMP and also affected cAMP-induced astrocyte stellation, decreasing process elongation and increasing branching complexity. Similar effects were observed for Acsl4 silencing on cAMP-induced C6 cell morphological shift. Moreover, Acsl4 inhibition and silencing reduced proliferation and migration of both cell types. Acsl4 silencing in C6 cells reduced the capacity for colony proliferation and neurosphere formation, the latter ability also being abolished by Acsl4 inhibition. In sum, this work presents novel evidence of Acsl4 involvement in neurosteroidogenesis and the morphological changes of glial cells promoted by cAMP. Furthermore, Acsl4 participates in migration and proliferation, also affecting cell self-renewal. Altogether, these findings provide insights into Acsl4 functions in glial cells.

Sputnik V vaccine elicits seroconversion and neutralizing capacity to SARS-CoV-2 after a single dose.

Massive vaccination offers great promise for halting the global COVID-19 pandemic. However, the limited supply and uneven vaccine distribution create an urgent need to optimize vaccination strategies. We evaluate SARS-CoV-2-specific antibody responses after Sputnik V vaccination of healthcare workers in Argentina, measuring IgG anti-spike titers and neutralizing capacity after one and two doses in a cohort of naive or previously infected volunteers. By 21 days after receiving the first dose of the vaccine, 94% of naive participants develop spike-specific IgG antibodies. A single Sputnik V dose elicits higher antibody levels and virus-neutralizing capacity in previously infected individuals than in naive ones receiving the full two-dose schedule. The high seroconversion rate after a single dose in naive participants suggests a benefit of delaying administration of the second dose to increase the number of people vaccinated. The data presented provide information for guiding public health decisions in light of the current global health emergency.

Oligodendrocytes and myelination: the role of iron.

Iron is an essential trophic factor that is required for oxygen consumption and ATP production. Thus it plays a key role in vital cell functions. Although the brain has a relatively high rate of oxygen consumption compared to other organs, oligodendrocytes are the principal cells in the CNS that stain for iron under normal conditions. The importance of iron in myelin production has been demonstrated by studies showing that decreased availability of iron in the diet is associated with hypomyelination. The timing of iron delivery to oligodendrocytes during development is also important because hypomyelination and the associated neurological sequelae persist long after the systemic iron deficiency has been corrected. Therefore, identifying the molecular roles of iron in oligodendrocyte development and myelin production, and the mechanisms and timing of iron acquisitions are important prerequisites to developing effective therapies for dysmyelinating disorders. It is the purpose of this review to give a comprehensive overview of the existing literature on role of iron in oligodendrocytes and the mechanisms of iron acquisition and intracellular handling.

Differential gene expression during development in two oligodendroglial cell lines overexpressing transferrin: a cDNA array analysis.

In the central nervous system, transferrin (Tf) is produced by oligodendroglial cells (OLGcs) and is essential for their development. Recently, using the complete cDNA of the human Tf gene, we obtained clones overexpressing Tf in two OLGc lines, N19 and N20.1, which represent different stages of differentiation. We showed that the overexpression of this glycoprotein promotes the maturation and myelinogenic capacity of both cell lines. In this work, using cDNA array technology, we examined changes induced by Tf in 1,176 genes. We found 41 genes differentially expressed in both cell lines, all of them involved in OLGc development. In the less mature cells (N19) overexpressing Tf, there was a significant increase in key enzymes of neurosteroid metabolism, such as cholesterol side chain cleavage cytochrome P450, 3beta-hydroxysteroid dehydrogenase and 5alpha-reductase type 1. In the more mature cell line (N20.1), Tf overexpression produced an induction of several mRNAs of the GABA(A) receptor subunits, of thyroid hormone receptors and of proteins involved in axon-glia interactions such as F3/contactin. In addition, in both cell lines, Tf overexpression induced an increase in the expression of different isoforms of transforming growth factor beta receptors and in several genes related to mitochondrial function and to complex lipid metabolism, crucial steps in myelin synthesis. Differentiation produced by Tf in both cell lines seems to occur by modulation of different genes depending on the maturational stage of the cells. Our findings provide new insights into the molecular basis of OLGc differentiation and on the role played by Tf in this process.

Expression of myelin basic protein in two oligodendroglial cell lines is modulated by apotransferrin through different transcription factors.

We have shown that apotransferrin (aTf) promotes the differentiation of two oligodendroglial cell (OLGc) lines, N19 and N20.1, representing different stages of OLGc maturation. Although in both cell lines aTf promoted myelin basic protein (MBP) expression, an increase in cAMP levels and CREB phosphorylation was observed only in the less mature cells (N19), suggesting that the maturation induced by aTf is achieved probably through different signaling pathways. We transfected both cell lines with the proximal region of the human MBP promoter fused to the lacZ reporter gene. In both transfected cell lines, addition of aTf produced an activation of the promoter. To elucidate the mechanisms involved in this action, Western blot analysis, EMSAs, and RT-PCR were performed for different transcription factors involved in mbp regulation. In the N20.1 line, treatment with aTf increased the expression and the DNA-binding capacity of thyroid hormone (TH) receptors, Sp1, and nuclear factor-kappaB (NFkappaB). For these cells we found that an inductor of NFkappaB (tumor necrosis factor-alpha) promoted MBP messenger synthesis, whereas mithramycin, a specific inibitor of Sp1, and a cAMP analog (db-cAMP) inhibited its transcription. In the N19 cell line, aTf stimulated NF-I and NFkappaB activation, but, aside from aTf, only db-cAMP induced mbp transcription. These data suggest that, depending on the OLGc maturational stage, aTf modulates MBP expression and OLGc differentiation through different signaling pathways and different transcription factors.

Overexpression of human transferrin in two oligodendroglial cell lines enhances their differentiation.

We have previously demonstrated that the addition of apotransferrin (aTf) to oligodendroglial cell (OLGc) primary cultures accelerates their maturation. Cells treated with aTf developed a multipolar morphology and displayed increased expression of mature OLGc markers. In this work, we studied the effect of Tf overexpression in two OLGc lines, N19 and N20.1. The former cells exhibit characteristics of OLGc precursors (O2A), while N20.1 cells express markers of more mature OLGcs. Using the complete cDNA of the human Tf gene, we obtained clones overexpressing Tf in both cell lines. These clones were evaluated for the expression of OLGc differentiation markers. In agreement with our previous results, we found that in the cells overexpressing Tf, there was an increased O(4), GC, and MBP immunoreactivity. To study the myelinogenic potential of these cells, we co-cultured N19 and N20.1 Tf-transfected cells together with cortical neurons. There was a dramatic increase in the morphological differentiation of the OLGcs accompanied by enhanced GC and MBP expression. The OLGcs appeared to establish contact with neurites and extend their processes along them. Only two MBP isoforms were detected in Tf-overexpressing clones, while all the isoforms were present in the co-cultures, suggesting that there was a modulation of MBP expression by neurons. Concomitantly, we found an increase in several proteins involved in axon-glia interaction, such as MAG, N-CAM, and F3/Contactin. This co-culture system represents a potentially powerful tool to study neuron-glia interactions that occur during myelinogenesis and the role of Tf in this process.

Apotransferrin promotes the differentiation of two oligodendroglial cell lines.

We have previously shown that addition of apotransferrin (aTf) accelerates maturation of oligodendroglial cells (OLGcs) in primary cultures. In this work, we examined the effect of aTf on two conditionally immortalized cell lines: N19 and N20.1. These cells proliferate at 34 degrees C and differentiate into mature OLGcs at 39 degrees C. In vitro addition of aTf to both cell lines at the differentiation temperature for 7 days showed increased expression of galactocerebroside, O4, and myelin basic protein (MBP) and a drop in the percentage of BrdU+ cells. The effect on MBP expression was particularly interesting in the less mature N19 cells. These cells do not express either MBP mRNAs or proteins, so aTf induced, rather than modulated, MBP expression in this cell line. In addition, even though MBP mRNAs for all four isoforms were induced, only the 17 and 21.5 kDa appeared to be translated. OLGc differentiation has been shown to be stimulated by the cAMP-CREB pathway. In N19 cells, following a pulse of aTf, there was a 10-fold increase in cAMP levels accompanied by elevated levels of pCREB. In the more mature N20.1 cells, there were no changes in cAMP levels. We conclude that addition of aTf to immature OLGc lines can enhance their expression of differentiated markers, such as MBP. The action of aTf on MBP gene expression in the least mature line is likely to be mediated by the cAMP pathway. In the N20.1 cells, it appears that different signals and/or mechanisms are involved in modulating myelin lipid and MBP expression. The results suggest that aTf can influence OLGc gene expression and differentiation through multiple mechanisms depending on the maturation of the cell.