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Herein, we assessed the effect of Ferulic Acid (FA), a natural antioxidant with anti-cancer effect, on the human glioblastoma cells through molecular and Delayed Luminescence (DL) studies. DL, a phenomenon of ultra-week emission of optical photons, was used to monitor mitochondrial assessment. The effect of FA loaded in nanostructured lipid carriers (NLCs) was also assessed. To validate NLCs as a drug delivery system for glioblastoma treatment, particular attention was focused on their effect. We found that free FA induced a significant decrease in c-Myc and Bcl-2 expression levels accompanied by the apoptotic pathway activation. Blank NLCs, even if they did not induce cytotoxicity and caspase-3 cleavage, decreased Bcl-2, ERK1/2, c-Myc expression levels activating PARP-1 cleavage. The changes in DL intensity and kinetics highlighted a possible effect of nanoparticle matrix on mitochondria, through the involvement of the NADH pool and ROS production that, in turn, activates ERK1/2 pathways. All the effects on protein expression levels and on the activation of apoptotic pathway appeared more evident when the cells were exposed to FA loaded in NLCs. We demonstrated that the observed effects are due to a synergic pro-apoptotic influence exerted by FA, whose bio-availability increases in the glioblastoma cells, and NLCs formulation.

Metabotropic glutamate receptor subtype 5 (mGluR5) is crucially implicated in the pathophysiology of Fragile X Syndrome (FXS); however, its dysfunction at the sub-cellular level, and related synaptic and cognitive phenotypes are unexplored. Here, we probed the consequences of mGluR5/Homer scaffold disruption for mGluR5 cell-surface mobility, synaptic N-methyl-D-Aspartate receptor (NMDAR) function, and behavioral phenotypes in the second-generation Fmr1 knockout (KO) mouse. Using single-molecule tracking, we found that mGluR5 was significantly more mobile at synapses in hippocampal Fmr1 KO neurons, causing an increased synaptic surface co-clustering of mGluR5 and NMDAR. This correlated with a reduced amplitude of synaptic NMDAR currents, a lack of their mGluR5-Activated long-Term depression, and NMDAR/hippocampus dependent cognitive deficits. These synaptic and behavioral phenomena were reversed by knocking down Homer1a in Fmr1 KO mice. Our study provides a mechanistic link between changes of mGluR5 dynamics and pathological phenotypes of FXS, unveiling novel targets for mGluR5-based therapeutics.

Introduction. Olfactory mucosa is considered a potential source of neural stem cells, since supported by peculiar glial cells, Olfactory Ensheathing Cells (OECs). Studies have shown that OECs share some common features with astrocytes and Schwann Cells (Ramon-Cueto and Avila 1998). The finding that OECs are a source of multiple Growth Factors (GFs) is very important as they play a decisive role in regeneration, promoting functional restoration in lesioned area (Raisman and Li 2007). Immunocytochemical investigations have reported that OECs express different markers and adhesion molecules (Pellitteri et. al., 2010). Therefore, OECs have attracted attention because of their remarkable neural plasticity. Materials and Methods. Primary OECs were prepared from neonatal rat olfactory bulbs. In some experiments were used GFs. Cells were identified by immunocytochemical procedures, using different antibodies and the results were highlighted by fluorescent microscopy. Results. Our in vitro studies have reported the ability of OECs to stimulate neuronal survival and neurite sprouting mediated through secretion of GFs; moreover, OECs have shown the capability to exert a protective effect on neurons exposed to stressed condition, such as hypoxia, simulating secondary events during spinal cord injury. Conclusion. These data suggest that OECs might be a suitable tool in cellular therapy for their exceptional ability to promote axonal regeneration and functional restoration making them attractive candidates for neurogenesis, in different neurological disorders or injured CNS.

In the U87-MG glioma cell line we assessed the effect of unloaded/NLC-loaded FA on the TG2-mediated apoptotic pathway activation, and cell cycle progression.

The nasal route has received a great deal of attention as a convenient and reliable method for the brain target on administration of drugs. When drugs are loaded into nanoparticles (NPs) the interaction with mucosa transports directly into the brain, skipping the blood- brain barrier and achieving rapid cerebrospinal fluid levels. Poly-lactic acid (PLA), poly-lactic-co-glycolic acid (PLGA), and chitosan (CS) were chosen to prepare NPs. After optimization of CS nanocarriers, our goal was to evaluate the different type of NPs uptake into olfactory ensheathing cells (OECs). We then correlated obtained biological data to zeta potential measurements of cells treated with NPs. Rodhamine-loaded NPs were used to study the uptake of OECs carried out by confocal microscopy at different times (1, 2, and 4 h). Our results showed that uptake of rodhamine-NPs by OECs was time dependent and it was influenced by the carrier charge. Confocal imaging of OECs demonstrated that NPPLGA showed a higher increase in uptake compared with NPPLA and NPCS after 1 h and it increased at 2-4 h. Zeta potential values of treated cells were more amplified with respect to untreated cells. The highest values were showed by unloaded NPPLGA, confirming microscopy data.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and autism and is caused by the lack of Fragile X Mental Retardation Protein (FMRP), an RNA binding protein involved in RNA metabolism and protein synthesis. Several studies report that FMRP is associated with the pool of mRNAs that go into stress granules (SGs) upon cellular stress. Stress granules are cytoplasmic aggregates that are formed under stress conditions such as exposure to heat, oxidative agents, UV irradiation; these foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SGs contain translation initiation components and many additional proteins affecting mRNA function. After environmental stress, mRNAs stored in this abortive translation initiation complex can be routed to either translation initiation or degradation. The link between SG formation and cell survival after stress is poorly understood. In this study we tested the appearance of SGs and the cell survival in wild type (WT) and Fmr1 knockout (KO) cultured astrocytes after exposure to sodium arsenite (200 uM for 1 hour or 500 µM for 30 minutes) and hydrogen peroxide (500 uM for 1 hour), by immunocytochemistry using an anti-TIA antibody and MTT assay, respectively. We also tested the effect of (S)-3,5-Dihydroxyphenylglycine (DHPG, 100 uM for 5 minutes), a selective agonist of group-I metabotropic glutamate (mGlu) receptor subtype1/5 on both SG formation and oxidative stress induced damage in both WT and Fmr1 KO astrocytes. We observed an increased number of cells with granules after exposure to oxidative stress, as expected; however, we detected a lower number of cells with granules in Fmr1 KO than WT astrocytes both under basal condition and after exposure to oxidative stress. Interestingly, under the same oxidative stress conditions cell survival was lower in Fmr1 KO than WT astrocytes. The activation of mGlu receptors before oxidative stress reduced both SG formation and cell survival in WT but had no effect in Fmr1 KO astrocytes. These results indicate that the lack of FMRP impairs the formation of SGs and sensitize to oxidative stress-induced damage. Furthermore, the observed differential effect of group-I mGlu receptors activation on both SG formation and cell survival in the presence and absence of FMRP suggests an interesting signalling interplay among SGs and mGlu receptors which may be related to their involvement in FMRP-mediated RNA transport and protein synthesis.

Fragile X Syndrome (FXS) is a leading genetic cause of intellectual disability. It is caused by the lack of expression of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in mRNA transport, stability and translation. A growing number of studies supports a role of group-I mGlu receptors (mGlu1 and mGlu5) in the pathophysiology of FXS. However, the expression of mGlu5 receptors have not been systematically investigated in FXS mouse model. In FXS a reduced number of mGlu5 receptors are associated to Homer proteins, a class of scaffolding proteins which anchor mGlu receptors to post-synaptic density (Giuffrida et al., J Neurosci., 2005). Considering that mGlu5-Homer interaction regulates the surface expression and axonal/dendritic distribution of mGlu5 receptors (Ango et al., J Neurosci., 2000; Mol Cell Neurosci 2002), we hypothesised that mGlu5 receptors might be differently targeted to cell surface of Fmr1 knock out (KO) neurons. We studied the expression of mGlu5 receptors in hippocampal synaptic proteins prepared from mice of different ages by Western blot analysis and the surface expression of mGlu5 receptors in hippocampal cultured neurons by confocal analysis of surface fluorescence-labeled receptors (kind gift of Prof. Shigemoto). Hippocampal cell cultures were prepared from wild type (WT) and Fmr1 KO newborn mice (P0-P1) and immunocytochemistry was performed at different days in vitro (3, 7, 12 DIV). Double- or triple-labelling experiments with anti-MAP2 and/or anti-tau1 antibodies were carried out to visualize the presence of mGlu5 receptors in dendrites and axons, respectively. Our results show that: a) mGlu5 receptors are more expressed in hippocampal synaptic proteins at postnatal day (P) 21 and 45, but not at 15 month of age; b) mGlu5 receptors are more expressed on the cell surface in Fmr1 KO than in WT cultured neurons. Interestingly, mGlu5 receptors were clearly detectable in the cellular body and dendrites in WT neurons, whereas in Fmr1 KO neurons a strong staining was also observed in the axons. Our data suggest that mGlu5 receptors are differently targeted to the cell surface and differently distributed in dendrites and axons in WT and Fmr1 KO mice.

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 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 cells 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. 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.