Annamaria Cimini

Expression of peroxisome proliferator-activated receptors (PPARs) and retinoic acid receptors (RXRs) in rat cortical neurons

Neuronal differentiation is a complex process involving the sequential expression of several factors. The important role of lipid molecules in brain development is well known. Many fatty acid cell signaling activities are mediated by peroxisome proliferator-activated receptors (PPARs). PPARs are ligand-activated transcription factors belonging to the steroid, thyroid and retinoid nuclear receptor superfamily. They are activated by fatty acids and their derivatives. Different isotypes of PPARs (alpha, beta/delta and gamma) have distinct physiological functions depending on their different ligand activation profiles and tissue distribution. PPARs have been involved in neural cell differentiation and death as well as in inflammation and neurodegeneration. Although PPARs have been described in neurons by in situ studies, the presence and possible modulation of these receptors during neuronal differentiation has not been explored yet. In this study we analyzed the expression of PPARs and of their heterodimeric partners, RXRs, in embryonic rat cortical neurons during their in vitro maturation. Our results demonstrate the presence of PPARs alpha, beta/delta and gamma and of RXRs beta and gamma. PPARalpha, beta/delta and gamma are differentially modulated during culture time suggesting that they may be involved in neuronal maturation. In particular, we point toward the PPARbeta/delta isotype as a key factor in neuronal differentiation.

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.

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.

Antibody-conjugated PEGylated cerium oxide nanoparticles for specific targeting of Aβ aggregates modulate neuronal survival pathways

Oxidative stress has been found to be associated with the progression of neurodegenerative diseases such as Alzheimers, Parkinsons, Lou Gehrigs, etc. In the recent years, cerium oxide nanoparticles (CNPs) have been studied as potent antioxidant agents able to exert neuroprotective effects. This work reports polyethylene glycol (PEG)-coated and antibody-conjugated CNPs for the selective delivering to Aβ aggregates, and the protective effect against oxidative stress/Aβ-mediated neurodegeneration. In this study PEG-coated and anti-Aβ antibody-conjugated antioxidant nanoparticles (Aβ-CNPs-PEG) were developed, and their effects on neuronal survival and brain-derived neurotrophic factor (BDNF) signaling pathway were examined. Aβ-CNPs-PEG specifically targets the Aβ aggregates, and concomitant rescue of neuronal survival better than Aβ-CNPs, by modulating the BDNF signaling pathway. This proof of concept work may allow in the future, once validated in vivo, for the selective delivery of CNPs only to affected brain areas.

TNFα downregulates PPARδ expression in oligodendrocyte progenitor cells: Implications for demyelinating diseases

TNFα has been implicated in several demyelinating disorders, including multiple sclerosis (MS) and X‐adrenoleukodystrophy (X‐ALD). TNFα abundance is greatly increased in the areas surrounding damaged regions of the central nervous system of patients with MS and X‐ALD, but its role in the observed demyelination remains to be elucidated. A class of nuclear receptors, the peroxisome proliferator‐activated receptors (PPARs), has been implicated in several physiological and pathological processes. In particular, PPARδ has been shown to promote oligodendrocyte (OL) survival and differentiation and PPARγ has been implicated in inflammation. In the present study, we investigate on the effects of TNFα on OLs during differentiation in vitro. The results obtained show that TNFα treatment impairs PPARδ expression with concomitant decrease of lignocerolyl‐CoA synthase and very‐long‐chain fatty acid β‐oxidation as well as plasmalogen biosynthesis. We propose a hypothetical model possibly explaining the perturbation effects of proinflammatory cytokines on myelin synthesis, maturation, and turnover. GLIA 41:3–14, 2003.

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.

Peroxisome Proliferator-Activated Receptors (PPARs) and related transcription factors in differentiating astrocyte cultures

Peroxisome proliferator-activated receptors (PPARs), retinoid X receptors (RXRs), CCAAT/enhancer binding proteins (C/EBPs) and beta-catenin are transcription factors involved in cell differentiation. The aim of this work was to investigate the occurrence and variations of these proteins during astrocyte differentiation. Primary cultures of mouse cortical astrocytes were characterized using nestin, A2B5 and glial fibrillary acidic protein (GFAP) as differentiation markers, during a period of 21 days in vitro (DIV). Glycogen and triglyceride accumulation were also studied. At 3 DIV the cultures were mainly constituted by neural progenitor cells, as assessed by their immunofluorescent pattern. At this time PPARs and beta-catenin were localized to the cytoplasm. Interestingly, some cells contained Oil Red O-positive lipid droplets. Between 7 and 21 DIV, nestin decreased, while GFAP increased, indicating ongoing astroglial differentiation. beta-catenin, predominantly nuclear at 7 DIV, later localized to membranes. Redistribution of all three PPAR isotypes from the cytoplasm to the nucleus was observed starting from 7 DIV. Between 7 and 14 DIV, C/EBPalpha, PPARalpha, RXRalpha and glycogen content increased. Between 14 and 21 DIV, PPARbeta/delta decreased, while PPARgamma, C/EBPbeta and delta and lipid droplet-containing cells increased. At 21 DIV both A2B5-/GFAP+ and A2B5+/GFAP+ cells were predominantly observed, indicating differentiation toward type-1 and type-2 astrocytes, although the presence of GFAP- cells demonstrates the persistence of neural precursors in the culture even at this time point. In conclusion, our results, reporting modifications of PPARs, RXRs, C/EBPs and beta-catenin during culture time, strongly suggest the involvement of these transcription factors in astrocyte differentiation. Specifically, beta-catenin translocation from the nucleus to plasma membrane, together with PPARbeta/delta decrease and C/EBPalpha increase, could be related to decreased proliferation at confluence, while PPARalpha and gamma and all C/EBPs could participate in differentiation processes, such as glycogenesis and lipidogenesis.

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.

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.