Maria Paola Cerù

Immunolocalization of peroxisome proliferator-activated receptors and retinoid X receptors in the adult rat CNS.

Peroxisome proliferator-activated and retinoid X receptors (PPARs and RXRs) are transcription factors belonging to the steroid hormone receptor superfamily. Upon activation by their ligands, PPARs and RXRs bind to their target genes as heterodimers. Ligands of these receptors include lipophylic molecules, such as retinoids, fatty acids and eicosanoids, the importance of which in the metabolism and functioning of the nervous tissue is well documented. The immunohistochemical distribution of PPARs and RXRs in the CNS of the adult rat was studied by means of a sensitive biotinyl-tyramide method. All PPAR (alpha, beta/delta and gamma) and RXR (alpha, beta and gamma) isotypes were detected and found to exhibit specific patterns of localization in the different areas of the brain and spinal cord. The presence of the nuclear receptors was observed in both neuronal and glial cells. While PPAR beta/delta and RXR beta showed a widespread distribution, alpha and gamma isotypes exhibited a more restricted pattern of expression. The frontal cortex, basal ganglia, reticular formation, some cranial nerve nuclei, deep cerebellar nuclei, and cerebellar Golgi cells appeared rather rich in all studied receptors. Based on our data, we suggest that in the adult CNS, PPARs and RXRs, besides playing roles common to many other tissues, may have specific functions in regulating the expression of genes involved in neurotransmission, and therefore play roles in complex processes, such as aging, neurodegeneration, learning and memory.

Scavenging system efficiency is crucial for cell resistance to ROS-mediated methylglyoxal injury.

Methylglyoxal is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Recent research indicates that methylglyoxal is a potent growth inhibitor and genotoxic agent. The antiproliferative activity of methylglyoxal has been investigated for pharmacological application in cancer chemotherapy. However, various cells are not equally sensitive to methylglyoxal toxicity. Therefore, it would be important to establish the cellular factors responsible for the different cell-type specific response to methylglyoxal injury, in order to avoid the risk of failure of a therapy based on increasing the intracellular level of methylglyoxal. To this purpose, we comparatively evaluated the signaling transduction pathway elicited by methylglyoxal in human glioblastoma (ADF) and neuroblastoma (SH-SY 5Y) cells. Results show that methylglyoxal causes early and extensive reactive oxygen species generation in both cell lines. However, SH-SY 5Y cells show higher sensitivity to methylglyoxal challenge due to a defective antioxidant and detoxifying ability that, preventing these cells from an efficient scavenging action, elicits extensive caspase-9 dependent apoptosis. These data emphasize the pivotal role of antioxidant and detoxifying systems in determining the grade of sensitivity of cells to methylglyoxal.

An overview of the effect of linoleic and conjugated-linoleic acids on the growth of several human tumor cell lines.

Both n‐6 and n‐3 polyunsaturated fatty acids are dietary fats important for cell function, being involved in several physiologic and pathologic processes, such as tumorigenesis. Linoleic acid and conjugated linoleic acid, its geometrical and positional stereoisomer, were tested on several human tumor cell lines originating from different tissues and with different degrees of malignancy. This was to provide the widest possible view of the impact of dietary lipids on tumor development. While linoleic acid exerted different effects, ranging from inhibitory to neutral, even promoting growth, conjugated linoleic acid inhibited growth in all lines tested and was particularly effective against the more malignant cells, with the exception of mammary tumor cells, in which behavior was the opposite, the more malignant cell line being less affected. The inhibitory effect of conjugated linoleic acid on growth may be accompanied by different contributions from apoptosis and necrosis. The effects of conjugated linoleic acid on growth or death involved positive or negative variations in PPARs. The important observation is that a big increase of PPARα protein occurred in cells undergoing strong induction of apoptosis, whereas PPARβ/δ protein decreased. Although PPARα and PPARβ/δ seem to be correlated to execution of the apoptotic program, the modulation of PPARγ appears to depend on the type of tumor cell, increasing as protein content, when inhibition of cell proliferation occurred. In conclusion, CLA may be regarded as a component of the diet that exerts antineoplastic activity and its effect may be antiproliferative or pro‐apoptotic.

Immunocytochemical localization of d-amino acid oxidase in rat brain

Abstractd-amino acid oxidase (d-AAO) is a peroxisomal flavoenzyme, the physiological substrate and the precise function of which are still unclear. We have investigated D-AAO distribution in rat brain, by immunocytochemistry, with an affinity-purified polyclonal antibody. Immunoreactivity occurred in both neuronal and glial cells, albeit at different densities. Glial immunostaning was strongest in the caudal brainstem and cerebellar cortex, particularly in astrocytes, Golgi-Bergmann glia, and tanycytes. Hindbrain neurons were generally more immunoreactive than those in the forebrain. Immunopositive forebrain cell populations included mitral cells in the olfactory bulb, cortical and hippocampal neurons, ventral pallidum, and septal, reticular thalamic, and paraventricular hypothalamic nuclei. Within the positive regions, not all the neuronal populations were equally immunoreactive; for example, in the thalamus, only the reticular and anterodorsal nuclei showed intense labelling. In the hindbrain, immunopositivity was virtually ubiquitous, and was especially strong in the reticular formation, pontine, ventral and dorsal cochlear, vestibular, cranial motor nuclei, deep cerebellar nuclei, and the cerebellar cortex, especially in Golgi and Purkinje cells.

Peroxisome proliferator-activated receptors (PPARs) and peroxisomes in rat cortical and cerebellar astrocytes

Astrocytes are the most versatile cells of the neural tissue. Numerous astrocytic functions—such as protection from oxidative damage, catabolism of neuroactive D-amino acids acting as neuromodulators, synthesis and catabolism of some lipid molecules, and, possibly, gluconeogenesis—reside in peroxisomes. The expression of several peroxisomal enzymes, particularly those of the acyl-CoA β-oxidation pathway, is regulated by a class of ligand-activated transcription factors, known as peroxisome proliferator-activated receptors (PPARs), acting on their target genes as heterodimers with the retinoid X receptors (RXRs). In this work, primary and secondary cultures of astrocytes from the cerebral cortices and cerebella of neonatal rats (2 and 7 days of postnatal age) were utilized to investigate the expression of peroxisomal enzymes, PPAR and RXR isotypes (α, β and γ), by both biochemical and immunological methods. The results obtained demonstrate that astrocytes in vitro express peroxisomal enzymes, PPARs, and RXRs and that differences dependent on brain area, animal age, and culture time are reminiscent of the in vivo situation. Therefore, primary cultures of astrocytes and, particularly, high purified subcultures may constitute a useful model for further studies aimed to gain further insights into the roles of peroxisomes and PPARs related to lipid and glucose metabolism in these cells.

Cerium Oxide Nanoparticles Trigger Neuronal Survival in a Human Alzheimer Disease Model By Modulating BDNF Pathway

In engineering and materials science, nanotechnology has made significant advances in the reduction of free radical damage. Despite such advances, there has been little application to biomedical problems. Cross-disciplinary interactions and the application of this technology to biological systems has led to the elucidation of novel nanoparticle antioxidants. Oxidative stress and free radical produc- tion are associated with neurodegenerative conditions, including aging, trauma, Alzheimers and Parkinsons diseases, etc. The antioxi- dant properties of cerium oxide nanoparticles show promise in the treatment of such diseases. Recent reports suggest that CeO2 and other nanoparticles are potent, and probably regenerative, free radical scavengers in vitro and in vivo. In this work, the effects of CeO2 nanopar- ticles on an in vitro human AD model are investigated. The validation of new therapeutic agents implies the understanding of their mechanisms of action, therefore the following parameters were investigated under nanoparticles treatment: cell viability, cell death, neu- rite atrophy, neuronal marker localization and the expression of factors, i.e. PPARs, BDNF, TrkB, involved in the signal transduction pathways of neuronal survival. The data obtained, demonstrate that CeO2 nanoparticles do not act as mere anti-oxidant agents, but they seems to affect, directly or indirectly, signal transduction pathways involved in neuronal death and neuroprotection, raising the possibility of their use as therapeutic tools for neurodegenerative diseases.

PPARβ agonists trigger neuronal differentiation in the human neuroblastoma cell line SH-SY5Y

Neuroblastomas are pediatric tumors originating from immature neuroblasts in the developing peripheral nervous system. Differentiation therapies could help lowering the high mortality due to rapid tumor progression to advanced stages. Oleic acid has been demonstrated to promote neuronal differentiation in neuronal cultures. Herein we report on the effects of oleic acid and of a specific synthetic PPARβ agonist on cell growth, expression of differentiation markers and on parameters responsible for the malignancy such as adhesion, migration, invasiveness, BDNF, and TrkB expression of SH‐SY5Y neuroblastoma cells. The results obtained demonstrate that many, but not all, oleic acid effects are mediated by PPARβ and support a role for PPARβ in neuronal differentiation strongly pointing towards PPAR ligands as new therapeutic strategies against progression and recurrences of neuroblastoma. J. Cell. Physiol. 211: 837–847, 2007.

REGIONAL AND ULTRASTRUCTURAL IMMUNOLOCALIZATION OF COPPER-ZINC SUPEROXIDE DISMUTASE IN RAT CENTRAL NERVOUS SYSTEM

We examined the distribution of copper-zinc superoxide dismutase (CuZn-SOD) in adult rat central nervous system by light and electron microscopic immunocytochemistry, using an affinity-purified polyclonal antibody. The enzyme appeared to be exclusively localized in neurons. No immunoreactivity was seen in non-neuronal cells. The staining intensity was variable, depending on the brain region and, within the same region, on the neuron type. Highly immunoreactive elements included cortical neurons evenly distributed in the different layers, hippocampal interneurons, neurons of the reticular thalamic nucleus, and Golgi, stellate, and basket cells of the cerebellar cortex. Other neurons, i.e., pyramidal cells of the neocortex and hippocampus, Purkinje and granule cells of the cerebellar cortex, and the majority of thalamic neurons, showed much weaker staining. In the spinal cord, intense CuZnSOD immunoreactivity was present in many neurons, including motor neurons. Pre-embedding immunoelectron microscopy of the neocortex, hippocampus, reticular thalamic nucleus, and cerebellar cortex showed cytosolic and nucleoplasmic labeling. Moreover, single membrane-limited immunoreactive organelles identified as peroxisomes were often found, even in neurons that appeared weakly stained at the light microscopic level. In double immunogold labeling experiments, particulate CuZn-SOD immunoreactivity co-localized with catalase, a marker enzyme for peroxisomes, thus demonstrating that in neural tissue CuZnSOD is also present in peroxisomes.

Emerging Roles of Peroxisome Proliferator-Activated Receptors (PPARs) in the Regulation of Neural Stem Cells Proliferation and Differentiation

The molecular mechanisms controlling the specification of neural cell fates have been the focus of intense research in recent years. Neural precursor cells (NPCs) sequentially undergo expansion, neurogenic and gliogenic fates during development, but the underlying mechanisms are poorly understood. Recent studies have identified a number of extrinsic factors that regulate the fate of NPCs. Wnt signaling induces neuronal differentiation of NPCs in an instructive manner. Wnt plays this role in the neurogenic phase of NPCs but not in the early expansion phase, when this pathway promotes proliferation. Likewise, STAT3-activating ligands induce astrocytic differentiation in late gliogenic phase of NPCs but not in the early expansion and neurogenic phases. The mechanisms underlying these remarkable changes in progenitor behaviour and fate during development are not understood, but are thought to include changes in the intrinsic properties of neural progenitors, as well as changes in their signalling environment. PPARs are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily, which activate the transcription of their target genes as heterodimers with retinoid X receptors (RXR). PPARs have been recently involved in NSC acquisition of a specific fate. They have been described to be involved in pathways present also in the control of the proliferation, migration and differentiation of NSC, i.e. Wnt signalling pathway, STAT3 and NFkB pathways. In this review the findings related to PPARs and NSC are reported as well as their possible linkage to other signal transduction pathways involved in NSC specification.

PPARγ-dependent effects of conjugated linoleic acid on the human glioblastoma cell line (ADF)

Conjugated linoleic acid (CLA) has been shown to exert beneficial effects against carcinogenesis, atherosclerosis and diabetes. It has been demonstrated that CLA modulates lipid metabolism through the activation of peroxisome proliferator‐activated receptors (PPARs). The PPAR family comprises 3 closely related gene products, PPAR α, β/δ and γ, differing for tissue distribution, developmental expression and ligand specificity. It has also been demonstrated that activated PPARγ results in growth inhibition and differentiation of transformed cells. These observations stimulated a great interest toward PPARγ ligands as potential anticancer drugs to be used in a differentiation therapy. Glioblastomas are the most commonly diagnosed primary tumors of the brain in humans. The prognosis of patients with high‐grade gliomas is poor and only marginally improved by chemotherapy. The aim of this work was to study the effects of CLA and of a specific synthetic PPARγ ligand on cell growth, differentiation and death of a human glioblastoma cell line as well as on parameters responsible for the metastatic behavior of this tumor. We demonstrate here that CLA and PPARγ agonist strongly inhibit cell growth and proliferation rate and induce apoptosis. Moreover, both treatments decrease cell migration and invasiveness. The results obtained show that CLA acts, directly or indirectly, as a PPARγ activator, strongly suggesting that this naturally occurring fatty acid may be used as brain antitumor drug and as a chemopreventive agent. Moreover, the γ‐agonist, once experimented and validated on man, may represent a useful coadjuvant in glioblastoma therapy and in the prevention of recurrences.