Paola Ferri

Thyroid hormones affect neurogenesis in the dentate gyrus of adult rat.

Thyroid hormones play a crucial role in new neuron production and maturation during brain development. However, the knowledge about the involvement of these hormones on adult neurogenesis is still incomplete. Hippocampus is an anatomical region where neurogenesis occurs throughout adulthood and where high levels of thyroid hormone receptors have been found. In this work the possible involvement of thyroid hormones in the regulation of adult neurogenesis in the granule cell layer of rat hippocampus dentate gyrus was investigated using an experimental model of adult-onset pharmacologically-induced hypothyroidism. Neurogenesis was assessed by means of the thymidine analogue 5-bromo-2′-deoxyuridine 24 h and 30 days after its last administration in order to study neural precursor proliferation and newborn cell survival, respectively. Mitotic activity of the neural precursors was not affected by thyroid hormone deficiency; on the contrary, newborn cell survival dramatically decreased under these conditions when compared with controls, leading to a lower number of immature neurons being added to the granule cell layer. Moreover, in conditions of hypothyroidism, new neurons exhibit a delay in neuronal differentiation showing a prolonged expression of the neuritogenesis-associated immature neuron marker TUC-4 and a very immature morphology. Finally, the total number and size of granule cells, and granule cell layer volume decreased in hypothyroid rats. These results suggest that thyroid hormones play a role in regulating new neuron production during adult life in dentate gyrus of rat hippocampus.

ERK MAPK activation mediates the antiapoptotic signaling of melatonin in UVB-stressed U937 cells

The pineal gland hormone melatonin has been recently described to downregulate the intrinsic (or damage-induced) pathway of apoptosis in human leukocytes. These properties appear to depend on a specific mitochondrial signaling of melatonin which is associated with a lower generation of reactive oxygen species and a better control of redox-sensitive components such as the antiapoptotic protein Bcl-2. Other elements upstream in this signaling are expected to contribute regulatory roles that remain unexplored. The aim of this study was to investigate whether the extracellular signal-regulated kinase (ERK), which controls the balance between survival and death-promoting genes throughout the MAPK pathway, is involved in the antiapoptotic signaling of melatonin. Human monocytic U937 cells irradiated with UVB light were used as a model of stress-induced apoptosis. In this model we found that pharmacological concentrations of melatonin (1 mM) were able to decrease superoxide anion production, mitochondrial damage, and caspase-dependent apoptosis by improved Bcl-2 levels and decreased Cyt c release in the cytoplasm. Moreover, melatonin increased the phosphorylative activation of ERK 1/2 independently from the presence of UVB stress, and decreased the UVB-mediated activation of the stress kinases p38 MAPK and JNK. The ERK 1/2 inhibitor PD98059, but not the p38 MAPK inhibitor SB203580, abolished to different extents the effects that melatonin had on the UVB-induced ROS generation, mitochondrial dysfunction, and apoptosis. Using these inhibitors, a cross-talk effect between stress and survival-promoting kinases was tentatively identified, and confirmed the hierarchical role of ERK MAPK phosphorylation in the signaling of melatonin. In conclusion, melatonin sustains the activation of the survival-promoting pathway ERK MAPK which is required to antagonize UVB-induced apoptosis of U937 cells. This kinase mediates also the antioxidant and mitochondrial protection effects of this hormonal substance that may find therapeutic applications in inflammatory and immune diseases associated with leukocyte oxidative stress and accelerated apoptosis.

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

BACKGROUND Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

Persistently High Corticosterone Levels but Not Normal Circadian Fluctuations of the Hormone Affect Cell Proliferation in the Adult Rat Dentate Gyrus

The dentate gyrus is one of the few brain structures where new neurons are added throughout adulthood in several mammalian species, including humans. Production of new neurons can be regulated by factors which influence cell proliferation or newborn cell survival. Supplementation or deprivation of glucocorticoids, adrenal hormones involved in the response to stress, affect cell proliferation, leading to a decrease or an increase, respectively, in the number of newborn cells. Glucocorticoid secretion under physiological conditions follows a circadian pattern. We thus investigated a possible relationship between cell proliferation and circadian oscillations of corticosterone secretion in the adult rat dentate gyrus. Corticosterone is the species-specific glucocorticoid hormone of the rat. 5-Bromo-2′-deoxyuridine was used to evaluate cell proliferation at 4 different time points in the light-dark cycle. No correlation was found between corticosterone circadian oscillations and cell proliferation in the adult dentate gyrus. In contrast, constantly high corticosterone levels, obtained by implanting corticosterone pellets, decreased cell proliferation in particular zones of the dentate gyrus, i.e. the hilus and the superior blade of the granule cell layer. These findings show that a short, physiologically occurring exposure to high corticosterone levels does not influence cell proliferation, whereas a lengthy exposure to this hormone does induce anatomically localized proliferative changes.

Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts

It is known that the MyoD family members (MyoD, Myf5, myogenin, and MRF4) play a pivotal role in the complex mechanism of skeletal muscle cell differentiation. However, fragmentary information on transcription factor‐specific regulation is available and data on their post‐transcriptional and post‐translational behavior are still missing. In this work, we combined mRNA and protein expression analysis with their subcellular localization. Each myogenic regulator factor (MRF) revealed a specific mRNA trend and a protein quantitative analysis not overlapping, suggesting the presence of post‐transcriptional mechanisms. In addition, each MRF showed a specific behavior in situ, characterized by a differentiation stage‐dependent localization suggestive of a post‐translational regulation also. Consistently with their transcriptional activity, immunogold electron microscopy data revealed MRFs distribution in interchromatin domains. Our results showed a MyoD and Myf5 contrasting expression profile in proliferating myoblasts, as well as myogenin and MRF4 opposite distribution in the terminally differentiated myotubes. Interestingly, MRFs expression and subcellular localization analysis during C2C12 cell differentiation stages showed two main MRFs regulation mechanisms: (i) the protein half‐life regulation to modulate the differentiation stage‐dependent transcriptional activity and (ii) the cytoplasmic retention, as a translocation process, to inhibit the transcriptional activity. Therefore, our results exhibit that MRFs nucleo‐cytoplasmic trafficking is involved in muscle differentiation and suggest that, besides the MRFs expression level, also MRFs subcellular localization, related to their functional activity, plays a key role as a regulatory step in transcriptional control mechanisms. J. Cell. Biochem. 108: 1302–1317, 2009.

α-Tocopherol, an exogenous factor of adult hippocampal neurogenesis regulation

In previous work, we found that adult hippocampal neurogenesis in rat is affected by vitamin E deficiency. Because vitamin E deficiency is a complex condition involving numerous biological systems, it is possible that its effect on postnatal new neuron production could be mediated by unknown changes in different factors that in turn play a role in this process. To clarify if vitamin E plays a direct role in regulating hippocampal neurogenesis, we studied the neurogenesis in adult control rats and in adult rats under supplementation with α‐tocopherol, the most important compound of vitamin E. The α‐tocopherol level in control and supplemented rats was monitored. Qualitative and quantitative analysis of cell proliferation and death was carried out and expression of immature neuron markers PSA‐NCAM, TUC 4, and DCX was investigated in hippocampus dentate gyrus. α‐Tocopherol levels increased significantly in both plasma and brain after supplementation. Cell proliferation was inhibited in α‐tocopherol‐supplemented rats, the number of dying cells was reduced, and the number of cells expressing the immature neuron markers was increased. The results obtained confirm and extend the idea that vitamin E is an exogenous factor playing a direct role in regulation of different steps of adult hippocampal neurogenesis. Some hypotheses about the possible mechanisms underlying the complex action of α‐tocopherol, related to its antioxidant and molecule‐specific non‐antioxidant properties, are proposed and discussed.

Neurogenesis in the adult rat dentate gyrus is enhanced by vitamin E deficiency

Neurogenesis occurs throughout adult life in rat dentate gyrus. Factors and mechanisms of adult neurogenesis regulation are not well known. Vitamin E deficiency has been found to deliver a neurogenetic potential in rat dorsal root ganglia. To determine whether the role of tocopherols in adult neurogenesis may be generalized to the central nervous system, changes in adult rat dentate gyrus neurogenesis were investigated in vitamin E deficiency. Neurogenesis was quantitatively studied by determination of the density of 5‐bromo‐2′‐deoxyuridine (BrdU)‐labeled cells and by determination of the total number of cells in the granule cell layer. The BrdU‐labeled cells were immunocytochemically characterized by demonstration of neuronal marker calbindin D28K. The following results were found: (1) the volume of the granule layer increased in controls from 1 to 5 months of age, mainly due to cell density decrease; (2) the volume increased by a similar amount in vitamin E–deficient rats, mainly because of an increase in cell number; (3) BrdU‐positive cells were more numerous in vitamin E–deficient rats in comparison to age‐matched controls; (4) the increase in proliferated cells was located in the hilus and in the plexiform layer. This study confirms that neurogenesis occurs within adult dentate gyrus and demonstrates that this process is enhanced in vitamin E deficiency. This finding indicates that vitamin E may be an exogenous factor regulating adult neurogenesis. J. Comp. Neurol. 411:495–502, 1999.

Are there proliferating neuronal precursors in adult rat dorsal root ganglia

The origin of new neurons in dorsal root ganglia of adult rat was investigated using an experimental model in which postnatal neurogenesis naturally occurring is enhanced and restricted in a brief period of life. Possible mitotic origin of new neurons was investigated by means of 5-bromo-2-deoxyuridine, anti-NF 200 antibody was used to detect if proliferated cells showed a neuronal phenotype. The results suggest that postnatal neurogenesis in dorsal root ganglia could depend only in part on precursor proliferation and that normally new neurons derive from the late differentiation of postmitotic cells.

Actin involvement in apoptotic chromatin changes of hemopoietic cells undergoing hyperthermia

During apoptosis, cell chromatin undergoes characteristic morphological changes, which have been long described in a variety of experimental models but are mostly not yet understood. The aim of the present study was to investigate the mechanisms underlying this phenomenon and the possible role of cytoskeleton, in particular actin. The chosen apoptotic model were HL60 hemopoietic cells undergoing hyperthermia and the starting point was the observation of thin filament bundles in decondensed chromatin of their early apoptotic nuclei. The characterization of these structures was undertaken by cytochemical, fluorescent and immunogold techniques, directed to actin identification. Taken together, our results suggest, in apoptotic cells, a deep actin rearrangement. Moreover, this cytoskeletal component, never present in normal nucleus, appears in the early apoptotic one, where it can be found in polymerized form, promptly recognizable both by conventional and immunogold electron microscopy. We suggest that, similarly to the role played by nuclear matrix in interphase and mitotic nucleus, actin could be directly involved in chromatin rearrangement occurring in apoptosis.

In vitro apoptotic cell death during erythroid differentiation

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development.Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches.Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3–4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Glybright) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Glybright) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation.These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.