Elisabetta Benedetti

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.

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.

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.

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.

Glioblastoma Stem Cells Microenvironment: The Paracrine Roles of the Niche in Drug and Radioresistance

Among all solid tumors, the high-grade glioma appears to be the most vascularized one. In fact, “microvascular hyperplasia” is a hallmark of GBM. An altered vascular network determines irregular blood flow, so that tumor cells spread rapidly beyond the diffusion distance of oxygen in the tissue, with the consequent formation of hypoxic or anoxic areas, where the bulk of glioblastoma stem cells (GSCs) reside. The response to this event is the induction of angiogenesis, a process mediated by hypoxia inducible factors. However, this new capillary network is not efficient in maintaining a proper oxygen supply to the tumor mass, thereby causing an oxygen gradient within the neoplastic zone. This microenvironment helps GSCs to remain in a “quiescent” state preserving their potential to proliferate and differentiate, thus protecting them by the effects of chemo- and radiotherapy. Recent evidences suggest that responses of glioblastoma to standard therapies are determined by the microenvironment of the niche, where the GSCs reside, allowing a variety of mechanisms that contribute to the chemo- and radioresistance, by preserving GSCs. It is, therefore, crucial to investigate the components/factors of the niche in order to formulate new adjuvant therapies rendering more efficiently the gold standard therapies for this neoplasm.

Early Biochemical and Morphological Modifications in the Brain of a Transgenic Mouse Model of Alzheimer's Disease: A Role for Peroxisomes

The central role of peroxisomes in reactive oxygen species and lipid metabolism and their importance in brain functioning are well established. The aim of this work has been to study the peroxisomal population in the Tg2576 mouse model of Alzheimers disease (AD), at the age of three months when no apparent signs of behavioral, neuroanatomical, cytological, or biochemical alterations have been so far described. The expression and localization of peroxisomal (PMP70, CAT, AOX, and THL) and peroxisome-related proteins (PEX5p, GPX1, SOD1, and SOD2) were studied in the neocortex and hippocampus of transgenic and wild-type animals. Oxidative stress markers (TBARS, acrolein, and 8-OHG) were also evaluated. Our results demonstrate that significant alterations are already detectable at this early stage of the disease and also involve peroxisomes. Their number and protein composition change concomitantly with early oxidative stress. Interestingly, the neocortex shows a compensatory response, consisting in an increase of reactive oxygen species scavenging enzymes, while the hippocampus appears more prone to the oxidative insult. This different behavior could be related to metabolic differences in the two brain areas, also involving peroxisome abundance and/or enzymatic content.

Cocoa powder triggers neuroprotective and preventive effects in a human Alzheimer's disease model by modulating BDNF signaling pathway

The molecular mechanisms linking Aβ to the onset of neurotoxicity are still largely unknown, but several lines of evidence point to reactive oxygen species, which are produced even under the effect of nanomolar concentrations of soluble Aβ‐oligomers. The consequent oxidative stress is considered as the mediator of a cascade of degenerative events in many neurological disorders. Epidemiological studies indicate that dietary habits and antioxidants from diet can influence the incidence of neurodegenerative disorders such as Alzheimers and Parkinsons diseases. In the recent years, a number of reviews have reported on neuroprotective effects of polyphenols in cell and animal models. However, the majority of these studies have focused only on the anti‐oxidant properties of these compounds and less on the mechanism/s of action at cellular level. In this work we investigated the effect of cocoa polyphenolic extract on a human AD in vitro model. The results obtained, other than confirming the anti‐oxidant properties of cocoa, demonstrate that cocoa polyphenols triggers neuroprotection by activating BDNF survival pathway, both on Aβ plaque treated cells and on Aβ oligomers treated cells, resulting in the counteraction of neurite dystrophy. On the light of the results obtained the use of cocoa powder as preventive agent for neurodegeneration is further supported. J. Cell. Biochem. 114: 2209–2220, 2013.

Hypoxia induces peroxisome proliferator-activated receptor α (PPARα) and lipid metabolism peroxisomal enzymes in human glioblastoma cells.

Glioblastoma multiforme (GBM) represents the most severe type of glioma, the most common brain tumor. Their malignancy shows a relationship with an increased proliferation and a poorly organized tumor vascularization, an event that leads to inadequate blood supply, hypoxic areas and at last to the formation of necrotic areas, a feature of glioblastoma. Hypoxic/necrotic tumors are more resistant to chemotherapy and radiation therapies, thus it is crucial to formulate new therapeutic approaches that can render these tumors more sensitive to the action of conventional therapies. It has been demonstrated that under hypoxia, gliomas accumulate lipid droplets and that this event is positively correlated with the degree of malignancy, glioblastoma being the most endowed with lipid droplets. We have previously demonstrated in ex vivo glioma specimens a grade‐dependent lipid metabolism perturbation. Here we studied the lipid pathways and the presence of stemness markers in glioma primary cultures, obtained from surgical specimens of patients affected by glioma at different grade of malignancy, GBM primary cultures cultured under both hypoxic and normoxic conditions, as well as normal human astrocytes. The results obtained demonstrate that hypoxia plays a crucial role in regulating the expression of lipid metabolism peroxisomal enzymes, the lipid droplets accumulation as well as the transcription factor PPARα. J. Cell. Biochem. 112: 3891–3901, 2011.

Glycosilated nucleolin as marker for human gliomas.

Nucleolin is a multifunctional DNA and RNA binding protein involved in regulation of gene transcription, chromatin remodeling, RNA metabolism, and ribosomal RNA synthesis. Nucleolin seems to be over‐expressed in highly proliferative cells and is involved in many aspect of gene expression: DNA recombination and replication, RNA transcription by RNA polymerase I and II, rRNA processing, mRNA stabilization, cytokinesis, and apoptosis. Although nucleolin is localized predominantly in the nucleolus, it has also been shown to be localized in a phosphorylated/glycolsilated form on the cell surface of different cells. Numerous articles dealing with surface nucleolin targeting for tumor therapy have been recently published. However, at present, no extensive informations are so far available for the presence of nucleolin in human gliomas. In the present work we investigated on the presence and localization of nucleolin in glioma on glioma specimens at different grade of malignancy and on primary glioma cell cultures derived by surgical resection, trying to correlate the presence of glycosilated membrane nucleolin with the malignancy grade. To this purpose an antibody produced by us against gp273 protein, demonstrated to recognized the glycosilated surface nucleolin, has been used. The results obtained demonstrate that surface nucleolin increase with the malignancy grade thus suggesting that it may constitute a histopathological marker for glioma grading and a possible tool for targeted therapy. J. Cell. Biochem. 113: 571–579, 2012.