Paolo Del Grande

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.

Impairment of neural precursor proliferation increases survival of cell progeny in the adult rat dentate gyrus.

In the present study we show that a reduction in the number of neural precursor cells enhances survival of new granule cells in the dentate gyrus allowing the recovery of the proper granule cell layer structure. To diminish the number of newborn cells methylazoxymethanol (MAM), a toxic agent for proliferating cells, was injected during neonatal life. Proliferation of precursor cells and survival of newborn cells were assessed by BrdU administration to 1-month-old rats when granule cell layer still shows a reduction in granule cell number in treated animals. Treatment with MAM reduced cell proliferation by 30% and enhanced cell progeny survival: so that the final number of newborn cells exceeded control ones by 38%. Consistently, dentate granule cell death, assessed by the TUNEL method, was significantly decreased in the MAM rats. The enhanced survival of newborn granule cells and the consistent reduced cell death suggest a link between neurogenesis and regulation of granule cell number. A comparison with previous findings shows that the recovery in the long-term of granule cell layer may be due to the re-establishing of the progenitor pool size and/or to the rescue of cell progeny.

α-Tocopherol controls cell proliferation in the adult rat dentate gyrus

Abstract The effect of α-tocopherol on cell proliferation and proliferated cell survival was investigated in the dentate gyrus of adult rats. Adult rats were supplemented with α-tocopherol, injected with 5-bromo-2′-deoxyuridine (BrdU), that is incorporated into DNA during the S-phase, and killed at different time after BrdU injection. The number of newborn cells decreased after α-tocopherol supplementation, confirming the hypothesis that α-tocopherol is able to depress cell proliferation in vivo. Most newborn cells die within few days; more newborn cells survive in α-tocopherol-treated rats, suggesting the hypothesis that α-tocopherol decreases cell death.

NADPH-consuming enzymes correlate with glucose-6-phosphate dehydrogenase in Purkinje cells: an immunohistochemical and enzyme histochemical study of the rat cerebellar cortex

In cerebellum of the adult rat, glucose-6-phosphate dehydrogenase (G6PD) activity is particularly localized in Purkinje cells, showing lower activity in the molecular and granule cell layers. G6PD is the first and rate-limiting step of the hexose monophosphate shunt (HMS), which has the physiological role of providing NADPH for reductive biosynthesis and detoxifying reactions. In this study, we searched for a possible correlation between G6PD and other NADPH-consuming enzymes, such as NADPH-cytochrome P450 reductase (P450R), glutathione reductase (GR) and NADPH-diaphorase (NADPH-d). This study was performed by means of immunohistochemistry and enzyme histochemistry followed by quantitative densitometric and confocal laser scanning microscopic analyses. Our results demonstrated that G6PD, P450R and GR have a similar distribution pattern characterized by the highest concentration of these enzymes in the somata of Purkinje cells, and by lower concentrations in the molecular and the granule cell layers. Moreover, in Purkinje cells, G6PD colocalized with both P450R and GR. NADPH-d activity showed a different distribution pattern when compared to the other enzymes, revealing the highest activity in the molecular layer and the lowest in Purkinje cells. Our results suggest a coordinated regulative mechanism of G6PD, P450R and GR based on the request of NADPH or on specific transcription factors.

Cytochemical and immunocytochemical methods for electron microscopic detection of glucose-6-phosphate dehydrogenase in brain areas.

This paper reports on protocols for the cytochemical and immunocytochemical determination of the glucose-6-phosphate dehydrogenase (G6PD) in brain areas by electron microscopy (EM). The cytochemical assay consists of a pre-embedding staining of small and flat tissue blocks, which were first mildly fixed and then floated in a staining mixture based on the reduction of tetrazolium salts by NADPH. Tissue blocks were then washed, post-fixed in OsO(4), dehydrated through graded ethanol concentrations and embedded in resin. Ultrathin sections were then obtained and observed at the EM. The immunocytochemical technique was performed on completely fixed tissues of perfused animals. After the tissue embedding in resin, ultrathin sections were obtained and treated with a primary anti-erythrocyte G6PD antibody, produced and purified in our laboratory. The immunostaining was performed with secondary gold-conjugated antibody. Gold grains were well evident by EM analysis thus revealing the G6PD protein in the subcellular compartments. These protocols are useful to detect peculiar populations of neurons which express high levels of G6PD to sustain processes of neural plasticity in some brain areas.

Glucose-6-phosphate dehydrogenase expression associated with NADPH-dependent reactions in cerebellar neurons.

This review describes the variation of glucose-6-phosphate dehydrogenase (G6PD) activity in the main neurons of the molecular and granular layers as well as in the deep nuclei of the cerebellum as observed so far by optical and electron microscopy studies. Light microscopy and semiquantitative microphotometry of histochemical staining showed that the highest G6PD activity was expressed by Purkinje cells and neurons of the deep cerebellar nuclei; the elements of the molecular layer showed a diffuse G6PD staining, while the granular layer displayed only scattered G6PD activity. Electron microscopy analysis showed that the basket and stellate cells, as well as the Golgi cells, have a remarkable G6PD activity, while in the granule cells the enzyme was barely detectable. The results show that cerebellar G6PD activity changes with different neuron types as a function of its role in sustaining NADPH dependent pathways in these cells.

Postnatal proliferation of DRG non-neuronal cells in vitamin E-deficient rats.

Changes in the number of satellite cells in neuron body sheaths in dorsal root ganglia (DRGs) were studied from 1 to 5 months of age in control and in vitamin E‐deficient rats; furthermore, the satellite cell proliferation rate was detected in the same groups of animals with immunohistochemistry for 5‐bromo‐2′‐deoxyuridine (BrdU).