| Name | Luca |
| Surname | Colucci-D'Amato |
| Institution | Università degli Studi della Campania Luigi Vanvitelli |
| luca.colucci@unicampania.it | |
| Address | Department of Environmental, Biological, Pharmaceutical Science and Technology, "Luigi Vanvitelli", Caserta, Italy |
The proteomic profiling, by means of label-free qualitative and quantitative LC-MS analysis of proliferating/undifferentiated vs nonproliferating/differentiated mes-c-myc A1 cell line (A1), has been performed. A1 cells were generated from mouse embryonic central nervous system. The study was aimed at surveying the molecular changes following neural differentiation. The results provide a list of candidate proteins with potential relevance for the transition of A1 cells from the proliferative to the differentiated status.
The discovery of neurogenesis and neural stem cells (NSC) in the adult CNS has overturned a long-standing and deep-routed "dogma" in neuroscience, established at the beginning of the 20(th) century. This dogma lasted for almost 90 years and died hard when NSC were finally isolated from the adult mouse brain. The scepticism in accepting adult neurogenesis has now turned into a rush to find applications to alleviate or cure the devastating diseases that affect the CNS. Here we highlight a number of methodological, technical and conceptual drawbacks responsible for the historical denial of adult neurogenesis. Furthermore, we discuss old and new issues that need to be faced before NSC or endogenous neurogenesis can safely enter into the doctor's bag for therapies.
The glial cell line-derived neurotrophic factor (GDNF) exerts trophic actions on a number of cell types, including mesencephalic dopaminergic (mDA) neurons. Using rat mesencephalic primary cultures enriched in mDA neurons, we show that protracted GDNF stimulation increases their survival and neurite outgrowth. It modulates the expression of genes essential for DA function (tyrosine hydroxylase, TH and dopamine transporter, dat) and of DA high affinity uptake. To identify genes involved in GDNF signaling pathways, we have used DNA microarray on mDA cultures stimulated with GDNF for 3 h. Here we show that GDNF signaling sequentially activates the genes encoding for the transcription factors EGR1 and TIEG. In addition, it increases the expression of cav1, which encodes for the major component of caveolae. GDNF also modulates the expression of the genes encoding for the Calcineurin subunits ppp3R1 and ppp3CB, and inhibits calcium-calmodulin-dependent protein kinase II beta isoform (CaMKIIbeta) gene expression. These proteins are involved in neuronal differentiation and synaptic plasticity. Moreover, GDNF stimulation down regulates the expression of the glycogen synthase kinase 3beta (gsk3beta) gene, involved in neuronal apoptosis. Using inhibitors of specific intracellular signal transduction pathways we show that changes of egr1, tieg, cav1, CaMkIIbeta and gsk3beta genes expression are extracellular-signal regulated kinases 1/2 (ERK)-dependent, while the cAMP-dependent protein kinase (PKA) pathway influences the up-regulation of ppp3R1 and ppp3CB gene expression. These results demonstrate that GDNF stimulation results in the transcriptional modulation of genes involved in neuronal plasticity and survival and in mDA function, mediated in part by ERK and PKA signaling.
The transcription factor Nurr1 is essential for the generation of midbrain dopaminergic neurons (mDA). Only a few Nurr1-regulated genes have so far been identified and it remains unclear how Nurr1 influences the development and function of dopaminergic neurons. To identify novel Nurr1 target genes we have used genome-wide expression profiling in rat midbrain primary cultures, enriched in dopaminergic neurons, following up-regulation of Nurr1 expression by depolarization. In this study we demonstrate that following depolarization the hyperexpression of Nurr1 and the brain derived neurotrophic factor (BDNF) are phospholipase C- and protein kinase C-dependent. We show that Bdnf, which encodes a neurotrophin involved also in the phenotypic maturation of mDA neurons, is a novel Nurr1 target gene. By RNA interference experiments we show that a decreased Nurr1 expression is followed by tyrosine hydroxylase and BDNF mRNA and protein down-regulation. Reporter gene assay experiments performed on midbrain primary cultures using four Bdnf promoter constructs show that Bdnf is a direct target gene of Nurr1. Taken together, our findings suggest that Nurr1 might also influence the development and the function of midbrain dopaminergic neurons via direct regulation of Bdnf expression.
Serotonin (5-HT) is a neurotransmitter involved in a variety of CNS functions during development and in adulthood. 5-HT neurons are also involved in the pathogenesis of a number of psychiatric disorders. FLUOXETINE (FLX), a prototypic antidepressant, is a selective 5-HT uptake inhibitor (SSRI) with a demonstrated clinical efficacy in these disorders. SSRI, in a short-term period, binds 5-HT transporter (SERT) raising 5-HT levels at the synapse. Nevertheless, clinical improvement is observed only after 3-4 weeks of treatment. Recently, it has been shown that antidepressants, besides interfering with neurotransmission, can also display an effect on neural cells' proliferation and differentiation. Therefore it has been proposed that antidepressant may exert their clinical effects also acting on cellular functions other then neurotransmission. Here we show that a mesencephalic neural cell line, mes-c-myc A1 (A1) produces 5-HT and expresses SERT and both peripheral (TPH1) and CNS-specific (TPH2) form of tryptophan hydroxylase, the limiting enzyme in 5-HT biosynthesis. Cyclic AMP-dependent neuronal differentiation of A1 cells modulates the expression of TPHs. FLX, as well as citalopram (CIT), another SSRI inhibitor, modulates expression of serotonergic markers depending on the differentiation status of the cells. Interestingly, long-term but not short-term FLX treatment selectively modulates mRNA levels of TPH2, only in differentiated A1 cells. Finally, FLX and citalopram selectively decrease the proliferation rate of undifferentiated A1 cells, whereas have no effects on NIH-3T3 fibroblasts proliferation. In conclusion, neuronal differentiation of A1 cells not only modulates the expression of serotonergic markers, but appears to affect the response to FLX.
Until the 1990s, neurologists were practising their profession under the doctrine established in the late 19th to early 20th century by the prominent histologist Ramon y Cajal: "Once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably. In the adult centers, the nerve paths are something fixed, ended, and immutable. Everything may die, nothing may be regenerated. It is for the science of the future to change, if possible, this harsh decree." Similarly, Giulio Bizzozero, the most prominent Italian histologist and mentor of Camillo Golgi, classified the tissues of the human body into "labile, stable and perennial". Among the latter were the nerve cells, believed to be unable to proliferate in the postnatal brain. This classification was taught until a few years ago to generations of medical students and biologists all over the world. We have investigated the historical, methodological and technical reasons why this "central dogma of neurology", so influential in clinical and experimental neurology, has lasted so long. We examined how this dogma was broken and who contributed, and the difficulties encountered by the "heretical" researchers who contributed to this goal, especially between the 1960s and the early 1990s, when at last neurogenesis in the adult brain could no longer be denied. Finally, we propose that the understanding of the mechanisms underlying various neurological diseases and the interpretations of clinical syndromes, as well as the design of new therapies, are being revolutionised by the breaking of this dogma and the discovery of the presence of neural stem cells in the adult brain.
The growth cycle in nutrient-rich, aquatic environments starts with a diatom bloom that ends in mass sinking of ungrazed cells and phytodetritus. The low grazing pressure on these blooms has been attributed to the inability of overwintering copepod populations to track them temporally. We tested an alternative explanation: that dominant diatom species impair the reproductive success of their grazers. We compared larval development of a common overwintering copepod fed on a ubiquitous, early-blooming diatom species with its development when fed on a typical post-bloom dinoflagellate. Development was arrested in all larvae in which both mothers and their larvae were fed the diatom diet. Mortality remained high even if larvae were switched to the dinoflagellate diet. Aldehydes, cleaved from a fatty acid precursor by enzymes activated within seconds after crushing of the cell, elicit the teratogenic effect. This insidious mechanism, which does not deter the herbivore from feeding but impairs its recruitment, will restrain the cohort size of the next generation of early-rising overwinterers. Such a transgenerational plant-herbivore interaction could explain the recurringly inefficient use of a predictable, potentially valuable food resource--the spring diatom bloom--by marine zooplankton.
The transcription factor/nuclear receptor Nurr1 is essential for the differentiation of midbrain dopaminergic neurones. Here we demonstrate that, during the ontogeny of rat ventral mesencephalon, nurr1 gene expression is developmentally regulated and its levels show a sharp peak between embryonic day E13 and E15, when most dopaminergic neurones differentiate. In addition, in primary cultures from embryonic rat mesencephalon, nurr1 gene follows a temporal pattern of expression comparable to that observed in vivo. We also report that exposure of embryonic mesencephalic cultures to depolarizing stimuli leads to a robust increase in nurr1 mRNA and protein. The depolarizing effect is also detected in mesencephalic cultures enriched in dopaminergic neurones by using a combination of bFGF and Sonic hedgehog. The latter further increases the number of dopaminergic neurones in these 'expanded' cultures, an effect abolished in the presence of anti-Sonic hedgehog antibodies. Our data show that nurr1 gene is highly expressed in midbrain dopaminergic neurones in a sharp temporal window and that its expression is plastic, both in vivo and in vitro. In addition we show that Sonic hedgehog can direct dopaminergic differentiation in proliferating dopaminergic neuroblasts in vitro.
We analyzed the molecular mechanisms involved in the acquisition and maturation of dopaminergic (DA) neurons generated in vitro from rat ventral mesencephalon (MES) cells in the presence of mitogens or specific signaling molecules. The addition of basic fibroblast growth factor (bFGF) to MES cells in serum-free medium stimulates the proliferation of neuroblasts but delays DA differentiation. Recombinant Sonic hedgehog (SHH) protein increases up to three fold the number of tyrosine hydroxylase (TH)-positive cells and their differentiation, an effect abolished by anti-SHH antibodies. The expanded cultures are rich in nestin-positive neurons, glial cells are rare, all TH+ neurons are DA, and all DA and GABAergic markers analyzed are expressed. Adding ascorbic acid to bFGF/SHH-treated cultures resulted in a further five- to seven-fold enhancement of viable DA neurons. This experimental system also provides a powerful tool to generate DA neurons from single embryos. Our strategy provides an enriched source of MES DA neurons that are useful for analyzing molecular mechanisms controlling their function and for experimental regenerative approaches in DA dysfunction.
The extracellular-signal regulated kinases 1/2 (ERK or ERKs) are involved in the regulation of important neuronal functions, including neuronal plasticity in normal and pathological conditions. We present findings that support the notion that the kinetics and localization of ERK are intrinsically linked, in that the duration of ERK activation dictates its subcellular compartmentalization and/or trafficking. The latter, in turn, dictates whether ERK-expressing cells would enter a program of cell death, survival or differentiation. We summarize experimental data showing that chronic activation of ERK plays a role in the mechanisms that trigger neurodegeneration. We also discuss how MKPs, members of the subclass of dual specificity phosphatases, might be the link between ERK kinetics and its subcellular localization.