NMR Metabolomics for Stem Cell type discrimination.

Cell metabolism is a key determinant factor for the pluripotency and fate commitment of Stem Cells (SCs) during development, ageing, pathological onset and progression. We derived and cultured selected subpopulations of rodent fetal, postnatal, adult Neural SCs (NSCs) and postnatal glial progenitors, Olfactory Ensheathing Cells (OECs), respectively from the subventricular zone (SVZ) and the olfactory bulb (OB). Cell lysates were analyzed by proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy leading to metabolites identification and quantitation. Subsequent multivariate analysis of NMR data by Principal Component Analysis (PCA), and Partial Least Square Discriminant Analysis (PLS-DA) allowed data reduction and cluster analysis. This strategy ensures the definition of specific features in the metabolic content of phenotypically similar SCs sharing a common developmental origin. The metabolic fingerprints for selective metabolites or for the whole spectra demonstrated enhanced peculiarities among cell types. The key result of our work is a neat divergence between OECs and the remaining NSC cells. We also show that statistically significant differences for selective metabolites characterizes NSCs of different ages. Finally, the retrived metabolome in cell cultures correlates to the physiological SC features, thus allowing an integrated bioengineering approach for biologic fingerprints able to dissect the (neural) SC molecular specificities.

Phenotypic Modulation and Neuroprotective Effects of Olfactory Ensheathing Cells: a Promising Tool for Cell Therapy.

Olfactory Ensheathing Cells (OECs), exhibiting phenotypic characteristics of both astrocytes and Schwann Cells, show peculiar plasticity. In vitro, OECs promote axonal growth, while in vivo they promote remyelination of damaged axons. We decided to further investigate OEC potential for regeneration and functional recovery of the damaged Central Nervous System (CNS). To study OEC antigen modulation, OECs prepared from postnatal mouse olfactory bulbs were grown in different culture conditions: standard or serum-free media with/without Growth Factors (GFs) and analyzed for different neural specific markers. OEC functional characterizations were also achieved. Resistance of OECs to the neurotoxin 6-hydroxydopamine (6-OHDA) was analyzed by evaluating apoptosis and death. OEC neuroprotective properties were investigated by in vitro co-cultures or by addition of OEC conditioned medium to the neuroblastoma SH-SY5Y cells exposed to 6-OHDA. We observed: 1) modification of OEC morphology, reduced cell survival and marker expression in serum-free medium; 2) GF addition to serum-free medium condition influenced positively survival and restored basal marker expression; 3) no OEC apoptosis after a prolonged exposition to 6-OHDA; 4) a clear OEC neuroprotective tendency, albeit non statistically significant, on 6-OHDA treated SH-SY5Y cells. These peculiar properties of OECs might render them potential clinical agents able to support injured CNS.

Olfactory ensheathing cells protect cortical neuron cultures exposed to hypoxia.

A peculiar population of glial cells, Olfactory Ensheathing Cells (OECs), are able to support the continuous neuronal turn-over and sheathe olfactory axons. In vitro, they stimulate axonal growth, as produce several neurotrophic factors (GFs); in vivo they promote remyelination of damaged axons. In this in vitro study, OEC effects on survival of cortical neurons exposed to hypoxia were examined. Rat co-cultures of OECs and cortical neurons were placed both in normal and hypoxic conditions; subsequently cells were analyzed by immunocytochemistry. Furthermore, some neuronal cultures were grown with Glial cell Derived Neurotrophic Factor (GDNF) or basic Fibroblast Growth Factor (bFGF) to tentatively rescue cells from oxygen deprivation. Some cortical neurons grown in both conditions were considered as control cells. Some neuronal cultures were feed with conditioned medium from OECs. We show that both in co-cultures and with GFs-treatment there was an increase of the number of neurons in comparison with control cultures. Moreover, these neurons formed a rich axonal outgrowth. OEC-conditioned media did not affect the cell survival. In hypoxic cultures the neuron number was very low both in controls and in GFs-treated neurons, while in co-cultures and in OEC-conditioned media cultures an increased neuronal survival was observed. These data suggest that OECs promote the survival of neurons in vitro exposed to hypoxia exerting a protective influence. Since some experiments in vivo have shown that injury is often characterized by secondary insults, ischemia or hypoxia, our results suggest that OECs might be considered a possible approach for restoration in injuries.

Characterization of glial cell models and in vitro manipulation of the neuregulin1/ErbB system.

The neuregulin1/ErbB system plays an important role in Schwann cell behavior both in normal and pathological conditions. Upon investigation of the expression of the neuregulin1/ErbB system in vitro, we explored the possibility to manipulate the system in order to increase the migration of Schwann cells, that play a fundamental role in the peripheral nerve regeneration. Comparison of primary cells and stable cell lines shows that both primary olfactory bulb ensheathing cells and a corresponding cell line express ErbB1-ErbB2 and neuregulin1, and that both primary Schwann cells and a corresponding cell line express ErbB2-ErbB3, while only primary Schwann cells express neuregulin1. To interfere with the neuregulin1/ErbB system, the soluble extracellular domain of the neuregulin1 receptor ErbB4 (ecto-ErbB4) was expressed in vitro in the neuregulin1 expressing cell line, and an unexpected increase in cell motility was observed. In vitro experiments suggest that the back signaling mediated by the transmembrane neuregulin1 plays a role in the migratory activity induced by ecto-ErbB4. These results indicate that ecto-ErbB4 could be used in vivo as a tool to manipulate the neuregulin1/ErbB system.

Viability of olfactory ensheathing cells after hypoxia and serum deprivation: Implication for therapeutic transplantation.

Olfactory ensheathing cells (OECs) represent glial cells supporting neuronal turnover in the olfactory system. In vitro, OECs promote axonal growth as a source of neurotrophic growth factors; in vivo, they produce myelin, promoting remyelination of damaged axons. Consequently, OEC transplantation appears to be a promising treatment for spinal cord injury, although the functional recovery is limited. This might be ascribed to the microenvironment at the lesion site, lacking growth factors (GFs), nutrients, and oxygen. To mimic this condition, we used an in vitro approach by growing primary neonatal mouse OECs under hypoxic conditions and/or serum deprivation. In addition, we compared OECs survival/proliferation with that of primary cultures of Schwann cells (SCs) and astrocytes under the same experimental conditions. Cultures were analyzed by immunocytochemistry, and cell viability was evaluated by MTT assay. Different GFs, such as NGF, bFGF, and GDNF, and their combination were used to rescue cells from serum and/or oxygen deprivation. We show that the cell types were differently sensitive to the tested stress conditions and that OECs were the most sensitive among them. Moreover, OEC viability was rescued by bFGF under serum-deprived or hypoxic condition but not under conditions of drastic serum deprivation and hypoxia. bFGF was effective also for the other cell types, whereas the effect of the other GFs was negligible. This model suggests that administration of bFGF might be considered useful to sustain cell survival/proliferation after transplantation of OECs either alone or in combination with other glial cell types.

Biomarkers expression in rat olfactory ensheathing cells.

Olfactory Ensheathing Cells (OECs) ensheathe unmyelinated olfactory axons and exhibit antigenic and morphological characteristics both of astrocytes and of Schwann Cells (SCs). As a matter of fact they express an astrocyte-specific marker (GFAP) and low-affinity p75 nerve growth factor receptor (p75 NGFr), S100, as well as adhesion molecules such as laminin and N-CAM like SCs. Immunocytochemical studies reveal that OECs are able to produce different growth and survival factors. In vitro, OECs promote axonal growth, probably by secretion of neurotrophic growth factors that support axonal elongation and extension. In vivo studies have shown that OECs can form myelin promoting remyelination of damaged axons. In fact, when transplanted, they stimulate extensive sprouting and axonal regeneration of multiple axons. As OECs appear to exert a neuroprotective effect for functional restoration and for neural plasticity in neurodegenerative disorders, they might be considered a suitable approach to functional recovery. These data establish OECs as prime candidates for transplantation, showing some advantages over SC thanks to their different capacity to intermingle with astrocytes after implantation in lesion sites.

Olfactory ensheathing cells represent an optimal substrate for hippocampal neurons: an in vitro study.

Olfactory ensheathing cells (OECs) are cells that display Schwann cell or astrocyte-like properties. They are a source of growth factors and adhesion molecules which play a very important role as neuronal support enhancing cellular survival. Over the past 10 years, OECs have emerged as a leading reparative candidate, when transplanted into the injured spinal cord, having shown significant promise in the regeneration of spinal cord lesions. In this study we assessed the efficacy of OECs on the survival and neurite outgrowth of hippocampal neurons in vitro. Co-cultures of OECs and hippocampal of postnatal rats were successfully established and cells were immunocytochemically characterized. Some hippocampal cultures were added with growth factors, as bFGF, NGF and GDNF. Furthermore, conditioned medium from OECs cultures was used to feed some hippocampal neurons coverslips. Our results show that in co-cultures of hippocampal neurons and OECs the number of neurons and their neurite outgrowth were significantly increased in comparison with controls. Moreover, we showed that NGF and GDNF promoted a more positive effect in both neuronal survival and neurite outgrowth than bFGF. OEC-conditioned media stimulated both the neuronal survival and dense neurite outgrowth. These data indicate that OECs, as a source of growth factors, can promote the survival and the neurite outgrowth of hippocampal neurons in vitro and that bFGF, NGF and GDNF support them differently. Therefore, as OECs and their secreted growth factors appear to exert a neuroprotective effect for functional restoration and for neural plasticity in neurodegenerative disorders, they might be considered an approach for functional recovery.

Protein levels of glycogen synthase 3 kinase are normal in progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by pure neurofibrillary tau pathology involving mainly basal ganglia and brain stem nuclei. One of the kinases involved in tau phosphorylation is glycogen synthase 3 kinase (GSK3). In mammals GSK3 is present in two isoforms, alpha and beta regulated by phosphorylation: phosphorylation of Ser9 in GSK3beta or Ser21 in GSK3alpha leads to inactivation while phosphorylation of Tyr216 in GSK3beta or Tyr279 in GSK3alpha leads to activation. We analyzed the protein levels of GSK3alpha/beta and of the phosphorylated forms GSK3beta S(9), GSK3beta Y(216), GSK3alpha Y(279) in brain tissues of subjects with PSP. The analysis failed to show significant differences of all GSK3 isoforms in PSP in comparison to age-matched control cases. This negative result argues against the role of GSK3 in the pathogenesis of PSP.

Growth factor-estradiol interaction on DNA labeling and cytoskeletal protein expression in cultured rat astrocytes.

Growth factors are major signaling agents regulating neuron-glia dialogue. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin growth factor-I (IGF-I) and insulin (INS) induce neuronal and astroglial cell proliferation and differentiation. This is true also for estrogens that influence astrocytes and exert neuroprotectant activity. In this study interactions between growth factors and estradiol on DNA labeling and glial fibrillary acidic protein (GFAP) and vimentin expression in cultured astrocytes were investigated. DNA labeling was significantly stimulated by bFGF pretreatment followed by 24 h estradiol and EGF or IGF-I or INS added in the last 12 h. Western blotting showed also a modulation of GFAP and vimentin expression in treated astrocytes. This suggests the occurrence of a crucial growth factor-estradiol interaction on DNA labeling and cytoskeletal protein expression during astrocyte proliferation and differentiation in culture.

Effect of growth factors on DNA labeling and cytoskeletal protein expression in 17-beta-estradiol and basic fibroblast growth factor pre-treated astrocyte cultures.

Astrocytes react to all noxae which damage neurons. Their reactions include degeneration, hypertrophy, hyperplasia and fibre formation. Growth factors inducing proliferation and differentiation of both neurons and astrocytes in culture play a pivotal role in the dynamic flow of signaling molecules between neurons and astroglia. Estrogens as well influence astroglia and are neuroprotectants. This study has investigated the interactions between growth factors and estrogens on DNA labeling and cytoskeletal protein [glial fibrillary acidic protein (GFAP) and vimentin] expression in 22 DIV astrocyte cultures treated for 24 or 36 h under different experimental conditions. Contemporary addition of 17-beta-estradiol (E2) with two or three growth factors for 24 h, significantly stimulated methyl-[3H]thymidine incorporation into DNA from 22 days in vitro (DIV) astrocyte cultures. This effect reached a peak when E2 was co-added with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and insulin. In astrocyte cultures treated for 36 h with E2 and EGF + insulin or bFGF + insulin added in the last 12 h, DNA labeling was remarkably increased. The parallel cyclin Dl expression positively correlated with ERK2 activation. Western blot analysis for cytoskeletal proteins showed also changes of both GFAP and vimentin expression. The above data suggest the occurrence of a scheduled interaction between "competence" or "progression" growth factors and estrogens on DNA labeling and cytoskeletal protein expression during astroglial cell proliferation and differentiation in culture. A better understanding of the mechanisms of these interactions may contribute to develop strategies for controlling astroglial reaction in cerebrovascular disease including stroke and hypertensive brain damage.

Neuronal apoptosis is accompanied by amyloid beta-protein accumulation in the endoplasmic reticulum.

A series of evidences suggests that enhanced susceptibility to programmed cell death (PCD) is a major pathogenetic factor in Alzheimer's disease (AD). We investigated this issue, analyzing amyloid beta-protein (A beta) production in a model of neuronal PCD, induced by staurosporine in a murine neuroblastoma cell line. When PCD was induced, a 280-290% secreted A beta occurred, in spite of a 20% metabolism and an unchanged A betaPP expression. The increased intracellular A beta reactivity largely colocalized with a marker of ER. Inhibition of caspases blocked the cleavage at the C-terminus of beta PP, but only partially rescued A beta overproduction caused by staurosporine treatment. Our findings suggest that PCD fosters the physiological pathways of A beta production characteristic of neuronal cells, and they confirm the theory that unbalance of PCD is a central event in AD pathogenesis. Moreover, our data indicate that still unidentified cellular mechanisms, other than caspases activation, are responsible of the specific alteration of A betaPP processing during PCD.

Oxidative stress increases expression and activity of BACE in NT2 neurons.

Recently an aspartyl protease with beta-secretase activity called BACE was identified. In the present paper we showed that BACE is modulated by the oxidative stress product 4-hydroxynonenal (HNE). Exposure of NT(2) neurons to the two classical pro-oxidant stimuli ascorbate/FeSO(4) and H(2)O(2)/FeSO(4) resulted in a significant generation of HNE, which is temporally followed by an increased production of BACE protein levels. HNE mediated BACE induction is accompanied by a proportional elevation of carboxy-terminal fragments of amyloid precursor protein. Moreover, the direct relationship between BACE induction and lipid peroxidation products was strongly confirmed by the protection exerted by a short pretreatment with alpha-tocopherol, the most important antioxidant known to prevent the formation of aldehydic end-products of lipid peroxidation, including HNE. Our results support the hypothesis that oxidative stress and A beta production are strictly interrelated events and suggest that inhibition of BACE may have a therapeutic effect synergic with antioxidant compounds.

Postnatal development of dopamine receptor expression in rat peripheral blood lymphocytes.

Postnatal development in the expression of dopamine D1-like and D2-like receptors was investigated in peripheral blood lymphocytes of male Wistar rats aged 1, 3, 4, 8, 12 and 16 weeks of age by radioligand binding assay techniques. Sample of frontal cortex, striatum and hippocampus were also investigated as reference tissues. The dopamine D1-like receptor antagonist [3H]SCH 23390 and the dopamine D2-like receptor agonist [3H]7-OH-DPAT were used as radioligands. The affinity (K(d)) of [3H]SCH 23390 or of [3H]7-OH-DPAT binding was unchanged in lymphocytes of rats of different age groups. The density (B(max)) of [3H]SCH 23390 binding sites increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 8th week of age, and then increased slightly at 12 and 16 weeks of age. The B(max) value of [3H]7-OH-DPAT binding to lymphocytes increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 4th week, increased again until the 12th week and then plateaued. Dopamine D1-like and D2-like receptor maturation in frontal cortex, hippocampus and striatum revealed an increased receptor density until the 4th week of age and a relative stabilization of receptor density values between the 4th to the 12th week depending on the area considered. Comparatively postnatal maturation of lymphocyte dopamine D1-like receptors displayed a pattern different from that of brain areas investigated, whereas maturation of D2-like receptors displayed a pattern similar to that of striatum. The quantitative and/or qualitative dissimilarities between development of lymphocyte and brain dopamine receptors suggest that from a developmental point of view lymphocyte dopamine receptors probably cannot be considered as a marker of homologous brain receptors.

Muscarinic cholinergic receptor subtypes in the hippocampus of aged rats.

In spite of the suggestion of impaired muscarinic function in adult-onset cognitive disorders, data on the expression of muscarinic receptors in the hippocampus as a function of age are inconsistent. One reason may be that the majority of investigations were unable to differentiate the five brain muscarinic receptors subtypes. In this study, using a protocol based on a combination of both kinetic and equilibrium binding approaches, we have assessed the expression and the density of M1-M5 muscarinic cholinergic receptors in the hippocampus of Fisher 344 rats aged 6, 15 and 22 months. An age-related decrease of the density of M1 receptor was found in pyramidal neurons of the CA1 subfield. In this area, other subtypes of muscarinic receptors were unchanged with the exception of a loss of M2 receptor in the radial layer. In the CA3 subfield, receptor changes involved M2, M3 and M5 subtypes, whereas in the dentate gyrus, the main changes affected M1 and M2 receptors of the granular layer and M2 and M3 receptors of the molecular layer. The above findings indicate that analysis of age-related changes of different muscarinic cholinergic receptors might represent a useful contribution to identifying the basis of cholinergic neurotransmission impairment in adult-onset cognitive dysfunction.