Tocopherol derivative TFA-12 promotes myelin repair in experimental models of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is associated with demyelination and axonal loss, resulting in severe neurological handicap. Current MS therapies mostly target neuroinflammation but have only a little impact on CNS myelin repair. Progress toward treatments that enhance remyelination would therefore represent major advances in MS treatment. Here, we examined the ability of TFA-12, a new synthetic compound belonging to tocopherol long-chain fatty alcohols, to promote oligodendrocyte regeneration and remyelination in experimental models of MS. We showed that TFA-12 significantly ameliorates neurological deficit and severity of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) in mice. Histological evaluation of mouse EAE spinal cords showed that TFA-12 treatment reduces inflammation, astrogliosis, and myelin loss. Additionally, we demonstrated that TFA-12 accelerates remyelination of focal demyelinated lesions induced by lysolecithin injections. We also found that this compound induces the differentiation of oligodendrocyte precursor cells into mature oligodendrocytes through the inhibition of the Notch/Jagged1 signaling pathway. Altogether, our data provide important proof of principle indicating that TFA-12 could be a potential therapeutic compound for myelin repair in MS.

Microglia-secreted TNF-α affects differentiation efficiency and viability of pluripotent stem cell-derived human dopaminergic precursors.

Disease is a neurodegenerative disorder characterised by the progressive loss of dopaminergic cells of the substantia nigra pars compacta. Even though successful transplantation of dopamine-producing cells into the striatum exhibits favourable effects in animal models and clinical trials; transplanted cell survival is low. Since every transplant elicits an inflammatory response which can affect cell survival and differentiation, we aimed to study in vivo and in vitro the impact of the pro-inflammatory environment on human dopaminergic precursors. We first observed that transplanted human dopaminergic precursors into the striatum of immunosuppressed rats elicited an early and sustained activation of astroglial and microglial cells after 15 days' post-transplant. This long-lasting response was associated with Tumour necrosis factor alpha expression in microglial cells. In vitro, conditioned media from activated BV2 microglial cells increased cell death, decreased Tyrosine hydroxylase-positive cells and induced morphological alterations on human neural stem cells-derived dopaminergic precursors at two differentiation stages: 19 days and 28 days. Those effects were ameliorated by inhibition of Tumour necrosis factor alpha, a cytokine which was previously detected in vivo and in conditioned media from activated BV-2 cells. Our results suggest that a pro-inflammatory environment is sustained after transplantation under immunosuppression, providing a window of opportunity to modify this response to increase transplant survival and differentiation. In addition, our data show that the microglia-derived pro-inflammatory microenvironment has a negative impact on survival and differentiation of dopaminergic precursors. Finally, Tumour necrosis factor alpha plays a key role in these effects, suggesting that this cytokine could be an interesting target to increase the efficacy of human dopaminergic precursors transplantation in Parkinson's Disease.

Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMP cellular levels and controls human leukemia cell proliferation and differentiation.

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Human neural progenitors from different foetal forebrain regions remyelinate the adult mouse spinal cord.

Improving oligodendroglial differentiation from human foetal neural progenitor cells remains a primordial issue to accomplish successful cell-based therapies in myelin diseases. Here, we combined in situ, in vitro and in vivo approaches to assess the oligodendrogenic potential of different human foetal forebrain regions during the first trimester of gestation. We show for the first time that the initial wave of oligodendrocyte progenitor emergence in the ventral telencephalon onsets as early as 7.5 weeks into gestation. Interestingly, in vitro, isolation of ganglionic eminences yielded oligodendrocyte progenitor-enriched cultures, as compared with cortex and thalamus. Most importantly, single injection of human neural progenitors into rodent models of focal gliotoxic demyelination revealed the great capacity of these cells to survive, extensively migrate and successfully remyelinate the spinal cord, irrespective of their origin. Thus, our study brings novel insights into the biology of early human foetal neural progenitor cells and offers new support for the development of cellular therapeutics for myelin disorders.

[Unusual presentation of metastasis of prostate carcinoma as a mediastinal mass: A case report and a brief review of the literature.] / Presentación inusual de metástasis de carcinoma prostático como masa mediastínica: descripción de un caso y breve revisión de la literatura.

OBJECTIVE: To assess the metastasic prostatecancer in the differenctial diagnosis of mediastinal masses. METHODS: To report a case. RESULTS: We present the case of a 78-year-old male patientwith a diagnosis of prostate cancer with a mediastinalmass compatible with prostate metastasis. CONCLUSION: Mediastinum is a very rare site for prostatecancer metastasis, but it must be considered in thedifferential diagnosis of mediastinal masses. Treatment isthe usual for metastatic prostate cancer.

OBJETIVO: Considerar el cáncer de próstatametastásico en el diagnóstico diferencial de masas mediastínicas. MÉTODOS: Presentación de un caso clínico. RESULTADOS: Se presenta el caso de un paciente varónde 78 años con diagnóstico de cáncer de próstata en elque se objetiva una masa mediastínica compatible conmetástasis prostática. CONCLUSIÓN: El mediastino es un lugar muy infrecuentede metástasis de cáncer de próstata, pero hay que considerarloen el diagnóstico diferencial de masas mediastínicas.El tratamiento es el estándar del cáncer de próstatametastásico.

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.

A familiar study on self-limited childhood epilepsy patients using hIPSC-derived neurons shows a bias towards immaturity at the morphological, electrophysiological and gene expression levels.

BACKGROUND: Self-limited Childhood Epilepsies are the most prevalent epileptic syndrome in children. Its pathogenesis is unknown. In this disease, symptoms resolve spontaneously in approximately 50% of patients when maturity is reached, prompting to a maturation problem. The purpose of this study was to understand the molecular bases of this disease by generating and analyzing induced pluripotent stem cell-derived neurons from a family with 7 siblings, among whom 4 suffer from this disease. METHODS: Two affected siblings and, as controls, a healthy sister and the unaffected mother of the family were studied. Using exome sequencing, a homozygous variant in the FYVE, RhoGEF and PH Domain Containing 6 gene was identified in the patients as a putative genetic factor that could contribute to the development of this familial disorder. After informed consent was signed, skin biopsies from the 4 individuals were collected, fibroblasts were derived and reprogrammed and neurons were generated and characterized by markers and electrophysiology. Morphological, electrophysiological and gene expression analyses were performed on these neurons. RESULTS: Bona fide induced pluripotent stem cells and derived neurons could be generated in all cases. Overall, there were no major shifts in neuronal marker expression among patient and control-derived neurons. Compared to two familial controls, neurons from patients showed shorter axonal length, a dramatic reduction in synapsin-1 levels and cytoskeleton disorganization. In addition, neurons from patients developed a lower action potential threshold with time of in vitro differentiation and the amount of current needed to elicit an action potential (rheobase) was smaller in cells recorded from NE derived from patients at 12 weeks of differentiation when compared with shorter times in culture. These results indicate an increased excitability in patient cells that emerges with the time in culture. Finally, functional genomic analysis showed a biased towards immaturity in patient-derived neurons. CONCLUSIONS: We are reporting the first in vitro model of self-limited childhood epilepsy, providing the cellular bases for future in-depth studies to understand its pathogenesis. Our results show patient-specific neuronal features reflecting immaturity, in resonance with the course of the disease and previous imaging studies.

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.

[IgG4-Related Retroperitoneal fibrosis]. / FIBROSIS RETROPERITONEAL RELACIONADA IgG4.

OBJECTIVE: To report two new cases of IgG4-related retroperitoneal fibrosis, a recently described pathology. METHODS: We analyze two cases diagnosed in our center and performed a literature review. RESULT: IgG4 related disease is a recently described entity that includes previously not related pathologies. The clinical manifestations are highly variable and its presentation is usually subacute. The treatment of choice is glucocorticoids. In our first case the outcome was favorable with corticosteroids and azathioprine. However, the second case required surgery on 2 occasions with radical nephrectomy. The diagnosis of the latter was made nine years after the onset of symptoms when the biopsy was reviewed; at that moment immunosuppressive therapy was not started. CONCLUSIONS: It is very important to know and diagnose this disease because of the good response to treatment that prevents complications.

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.

Number of oligodendrocyte progenitors recruited to the lesioned spinal cord is modulated by the levels of the cell cycle regulatory protein p27Kip-1.

Remyelination is a critical step for recovery of function after demyelination and defines the ability to generate new myelin. This repair process is dependent on the presence of resident oligodendrocyte progenitors (OLPs) that have been shown to remyelinate axons after demyelination. We have previously shown that the levels of the cell cycle inhibitor p27Kip-1 modulate the number of neonatal cortical OLPs. We now asked whether this cell cycle molecule plays also a role in regulating the number of adult OLP in the spinal cord after demyelination induced by lysolecithin injection. The proliferative response of OLP in the spinal cord of injected wild-type (wt) and p27Kip-1 null mice was evaluated 3 days after lesion. In vivo labeling with bromodeoxyuridine (BrdU) was used to identify cells in S phase. Double immunofluorescence for the OLP marker NG2, and for BrdU was used to count the number of proliferating progenitors. Consistent with a role of p27Kip-1 in regulating the number of adult OLP in the injured spinal cord, a larger number of proliferating OLPs was observed in p27Kip-1null mice compared with wild-type controls. These cells were able to differentiate as assessed by the presence of MBP+ cells in the spinal cord 14 days after injury. We conclude that the cellular levels of the cell cycle inhibitor p27Kip-1 modulate the repair response of OLPs to injury in the adult spinal cord.

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.