PM2.5 exposure represents a risk factor for the public health. PM2.5 is able to cross the blood-alveolar and blood-brain barriers and reach the brain through three routes: nasal olfactory pathway, nose-brain pathway, blood-brain barrier pathway. We evaluated the effect of PM2.5 to induce cytotoxicity and reduced viability on in vitro cultures of OECs (Olfactory Ensheathing Cells) and SH-SY5Y cells. PM2.5 samples were collected in the metropolitan area of Catania, and the gravimetric determination of PM2.5, characterization of 10 trace elements and 16 polycyclic aromatic hydrocarbons (PAHs) were carried out for each sample. PM2.5 extracts were exposed to cultures of OECs and SH-SY5Y cells for 24-48-72 h, and the cell viability assay (MTT) was evaluated. Assessment of mitochondrial and cytoskeleton damage, and the assessment of apoptotic process were performed in the samples that showed lower cell viability. We have found an annual average value of PM2.5 = 16.9 ?g/m3 and a maximum value of PM2.5 = 27.6 ?g/m3 during the winter season. PM2.5 samples collected during the winter season also showed higher concentrations of PAHs and trace elements. The MTT assay showed a reduction in cell viability for both OECs (44%, 62%, 64%) and SH-SY5Y cells (16%, 17%, 28%) after 24-48-72 h of PM2.5 exposure. Furthermore, samples with lower cell viability showed a decrease in mitochondrial membrane potential, increased cytotoxicity, and also impaired cellular integrity and induction of the apoptotic process after increased expression of vimentin and caspase-3 activity, respectively. These events are involved in neurodegenerative processes and could be triggered not only by the concentration and time of exposure to PM2.5, but also by the presence of trace elements and PAHs on the PM2.5 substrate. The identification of more sensitive cell lines could be the key to understanding how exposure to PM2.5 can contribute to the onset of neurodegenerative processes.

Alzheimer's disease (AD) is characterized by agglomerated proteins constitued by amyloid-beta (A?). A? possesses neutoxic effect and is also a substrate of tissue transglutaminase (TG2), an ubiquitarian protein that plays a key role in AD. Several reports indicated that in AD is involved oxidative stress and that the treatment with antioxidants mitigates the effects of oxidative stress in the central nervous system. In particular, astaxanthin, an antioxidant with antiflammatory properties, could have an important therapeutic role in AD. Herein, we have evaluated the effect of astaxanthin pretreatment on olfactory ensheathing cells (OECs) exposed to the native peptide of A?(1-42) or its fragments A?(25-35) and A?(35-25). OECs are glial cells located in the olfactory system, the first to show a deficit in neurodegenerative diseases. Vimentin, GFAP, nestin, cyclin D1, TG2 expression and the activation of the apoptotic pathway were assessed through immunocytochemical techniques. The percentage of cell viability and ROS levels were detected. In addition, to monitor the mitochondrial status, we used delayed luminescence (DL). We found that astaxanthin was able to reduce TG2 expression up-regulated by A?, reducing also GFAP and Vimentin levels. Astaxanthin pre-treatment stimulates cellular repair increasing cyclin D1 and nestin levels and inhibing apoptotic pathway activation. In parallel, we observed a significant change of DL intensity in OECs exposed to the toxic fragment A? (25-35), that completely disappear when OECs were pre-incubated with astaxantin. Therefore, we can suggest that astaxanthin pretreatment could represent an innovative mechanism to counteract the aberrant TG2 overexpression in AD.

Neurodegenerative diseases are characterized by neuroinflammation, a symptom with growing interest directed towards the development of active drugs for the reduction or elimination of its negative effects. It is largely reported on the anti-inflammatory and their potential neuroprotective activity in some neural cells of Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to the class of ?-3 polyunsaturated fatty acids. Most of the observed biological activities of these fatty acids are maintained, and in some case enhanced, in the corresponding amide derivatives or oxygenated metabolites. While free acids EPA and DHA are commercially available, the corresponding amides were prepared by aminolysis of the ethyl esters of EPA and DHA with ethanolamine in the presence of immobilized lipase from Candida antarctica (Novozym 435) and molecular sieves in tert-butyl methyl ether. Our study aims to elucidate the protective effect of both EPA and DHA, as well as the corresponding N-ethanolamides EPA-EA and DHA-EA, on Olfactory Ensheathing Cells (OECs) exposed to lipopolysaccharide (LPS)-induced neuroinflammation for 24 h. OECs are glial cells located in the olfactory system, which is the first to show a deficit in neurodegenerative diseases. To verify the anti-inflammatory effect of these compounds on OEC cultures and on cell morphological features, the expression of some cytoskeletal proteins, such as Vimentin and Glial Fibrillary Acid Protein (GFAP), was evaluated by immunocytochemical procedures. In addition, MTT test was carried out to establish the non-toxic concentrations and the optimal time of exposure. Mitotoxicity and cytotoxicity levels in stressed were detected by using the HCS Mitochondrial Health Kit, while apoptosis was determined by staining with the TUNEL Alexa Fluor Imaging assay. Our results show a decrease of GFAP and Vimentin expression in OECs treated with EPA or DHA acids or EPA-EA or DHA-EA and stressed with LPS when compared with OECs exposed to LPS alone. While a protective role on cell morphology is predominantly observed for EPA and DHA, the amides EPA-EA and DHA-EA mainly show anti-inflammatory effects, superior to those of free acids. These results highlight that all the tested compounds have anti-inflammatory activity on LPS-exposed OECs and may provide an innovative tool to contrast neuroinflammation, which plays a key role in several neurodegenerative diseases.

Neuroinflammation is a common symptom in the onset of different neurodegenerative diseases and growing interest is directed towards the development of active drugs for the reduction or elimination of its negative effects. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to the class of ?-3 polyunsaturated fatty acids, have been largely investigated for their anti-inflammatory activity and their potential as neuroprotective agents has been evaluated on some neural cells1. Most of the observed biological activities of these fatty acids are maintained, and in some case enhanced, in the corresponding amide derivatives or oxygenated metabolites2. Our study aims to elucidate the protective effect of both EPA and DHA, as well as the corresponding N-ethanolamides EPA-EA and DHA-EA, on Olfactory Ensheathing Cells (OECs) exposed to lipopolysaccharide (LPS)-induced neuroinflammation. OECs are glial cells located in the olfactory system, which is the first to show a deficit in neurodegenerative diseases. To verify the anti-inflammatory effect of these compounds on OEC cultures and on cell morphological features, the expression of some cytoskeletal proteins, such as Vimentin and Glial Fibrillary Acid Protein (GFAP), was evaluated by immunocytochemical procedures. In addition, MTT test was carried out to establish the non-toxic concentrations and the optimal time of exposure. Our results show a decrease of GFAP and Vimentin expression in OECs treated with EPA or DHA acids or EPA-EA or DHA-EA and stressed with LPS when compared with OECs exposed to LPS alone. While a protective role on cell morphology is predominantly observed for EPA and DHA, the amides EPA-EA and DHA-EA mainly show anti-inflammatory effects, superior to those of free acids. These results highlight that all the tested compounds have anti-inflammatory activity on LPS-exposed OECs and may provide an innovative tool to contrast neuroinflammation, which plays a key role in several neurodegenerative diseases.

Nowadays, there is considerable attention toward the use of food waste from food processing as possible sources of compounds with health properties, such as anticancer activity. An example is tomato processing, which is responsible for generating a remarkable amount of waste (leaves, peel, seeds). Therefore, our goal was to evaluate the potential anticancer property of tomato extracts, in particular "Datterino" tomato (DT) and "Piccadilly" tomato (PT), and to study their phytochemical composition. Liquid chromatography with tandem mass spectrometry (LC/MS-MS) results showed that these extracts are rich in alkaloids, flavonoids, fatty acids, lipids, and terpenes. Furthermore, their potential anticancer activity was evaluated in vitro by MTT assay. In particular, the percentage of cell viability was assessed in olfactory ensheathing cells (OECs), a particular glial cell type of the olfactory system, and in SH-SY5Y, a neuroblastoma cell line. All extracts (aqueous and ethanolic) did not lead to any significant change in the percentage of cell viability on OECs when compared with the control. Instead, in SH-SY5Y we observed a significant decrease in the percentage of cell viability, confirming their potential anticancer activity; this was more evident for the ethanolic extracts. In conclusion, tomato leaves extracts could be regarded as a valuable source of bioactive compounds, suitable for various applications in the food, nutraceutical, and pharmaceutical fields.

Tomato by-products represent a good source of phytochemical compounds with health properties, such as the steroidal glycoalkaloid ?-tomatine (?-TM) and its aglycone tomatidine (TD). Both molecules have numerous beneficial properties, such as potential anticancer activity. Unfortunately, their therapeutic application is limited due to stability and bioavailability issues. Therefore, a valid strategy seems to be their encapsulation into Solid Lipid Nanoparticles (SLN). The nanoformulations containing ?-TM (?-TM-SLN) and TD (TD-SLN) were prepared by solventdiffusion technique and subsequently characterized in terms of technological parameters (particle size, polydispersity index, zeta potential, microscopy, and calorimetric studies). To assess the effect of ?-TM and TD on the percentage of cellular viability in Olfactory Ensheathing Cells (OECs), a peculiar glial cell type of the olfactory system used as normal cells, and in SH-SY5Y, a neuroblastoma cancer cell line, an MTT test was performed. In addition, the effects of empty, ?-TMSLN, and TD-SLN were tested. Our results show that the treatment of OECs with blank-SLN, free ?-TM (0.25 µg/mL), and TD (0.50 µg/mL) did not induce any significant change in the percentage of cell viability when compared with the control. In contrast, in SH-SY5Y-treated cells, a significant decrease in the percentage of cell viability when compared with the control was found. In particular, the effect appeared more evident when SH-SY5Y cells were exposed to ?-TM-SLN and TD-SLN. No significant effect in blank-SLN-treated SH-SY5T cells was observed. Therefore, SLN is a promising approach for the delivery of ?-TM and TD.

Astaxanthin, a natural compound of Haematococcus pluvialis, possesses antioxidant, antiinflammatory, anti-tumor and immunomodulatory activities. It also represents a potential therapeutic in Alzheimer's disease (AD), that is related to oxidative stress and agglomeration of proteins such as amyloid-beta (A?). A? is a neurotoxic protein and a substrate of tissue transglutaminase (TG2), an ubiquitary protein involved in AD. Herein, the effect of astaxanthin pretreatment on olfactory ensheathing cells (OECs) exposed to A?(1-42) or by A?(25-35) or A?(35-25), and on TG2 expression were assessed. Vimentin, GFAP, nestin, cyclin D1 and caspase-3 were evaluated. ROS levels and the percentage of cell viability were also detected. In parallel, delayed luminescence (DL) was used to monitor mitochondrial status. ASTA reduced TG2, GFAP and vimentin overexpression, inhibiting cyclin D1 levels and apoptotic pathway activation which induced an increase in the nestin levels. In addition, significant changes in DL intensities were particularly observed in OECs exposed to A? toxic fragment (25-35), that completely disappear when OECs were pre-incubated in astaxantin. Therefore, we suggest that ASTA pre-treatment might represent an innovative mechanism to contrast TG2 overexpression in AD.

Epilepsy is one of the most common neurological disorders in the world. The therapeutic treatment is challenging since conventional drugs have limited efficacy and several side effects that impair patient management. Efforts are being made to find innovative strategies to control epileptic seizures. Intranasal administration provides a convenient route to deliver the drug to the brain. Carbamazepine (CBZ) is an anticonvulsant characterized by poor water solubility, nanonization can improve its bioavailability. Therefore, the design of CBZ nanocrystals (NCs) was assessed to obtain a formulation suitable for nose-to-brain delivery. CBZ NCs were prepared by sonoprecipitation following the Quality by Design approach identifying the impact of process and formulation variables on the critical quality attributes of the final product. The formulation was characterized by a technological point of view (thermotropic behavior, crystallinity, morphology, mucoadhesive strength). Response surface methodology was a reliable tool (error % 2.6) to optimize CBZ NCs with size <=300 nm. Incubation of CBZ NCs in artificial cerebrospinal fluid at 37 °C did not promote aggregation and degradation phenomena. Preliminary biological studies revealed the biocompatibility of CBZ NCs towards Olfactory Ensheating Cells. The suspension was successfully converted into a powder. The highly concentrated formulation can be obtained, providing the possibility to administer the maximum dose of the drug in the lowest volume.

Early life stress (ELS) refers to harmful environmental events (i.e., poor maternal health, metabolic restraint, childhood trauma) occurring during the prenatal and/or postnatal period, which may cause the 'epigenetic corruption' of cellular and molecular signaling of mental and physical development. While the impact of ELS in a wide range of human diseases has been confirmed, the ELS susceptibility to bone diseases has been poorly explored. In this review, to understand the potential mediating pathways of ELS in bone diseases, PRISMA criteria were used to analyze different stress protocols in mammal models and the effects elicited in dams and their progeny. Data collected, despite the methodological heterogeneity, show that ELS interferes with fetal bone formation, also revealing that the stress type and affected developmental phase may influence the variety and severity of bone anomalies. Interestingly, these findings highlight the maternal and fetal ability to buffer stress, establishing a new role for the placenta in minimizing ELS perturbations. The functional link between ELS and bone impairments will boost future investigations on maternal stress transmission to the fetus and, parallelly, help the assessment of catch-up mechanisms of skeleton adaptations from the cascading ELS effects.

Alzhèimer Disease (AD), one of the most common neurodegenerative diseases, is characterized by progressive neuronal loss and accumulation of neurotoxic proteins, including Amyloid-beta (A?). It is known that tissue transglutaminase (TG2), an ubiquitary calcium-dependent protein, is involved in protein aggregation in AD. Previous our studies showed that A?(1-42) and its fragments A?(25-35) and A?(35-25) induced an overexpression of TG2 on Olfactory Ensheathing Cells (OECs), a glial population of the olfactory system that express neural stem cell markers, including Nestin. In the last years, growing attention rose on neuronutraceutics and their effect on mental health. Among these molecules, we focused our research on indicaxanthin and astaxanthin, natural compounds that are able to cross the blood-brain barrier. In this study, the effect of indicaxanthin or astaxanthin pre-treatment on TG2 expression in OECs exposed to A? was immunocytochemically evaluated by Vimentin and Glial Fibrillary Acid Protein (GFAP) expression. The percentage of cell viability by MTT test and the apoptotic pathway activation were also evaluated. Since Nestin, a marker of neural precursors, is co-expressed in pluripotent stem cells with cyclin D1, a marker of cellular proliferation, the effect of indicaxanthin and astaxanthin pre-treatment on their expression was also tested. We found that indicaxanthin or astaxanthin pre-treatment was able to reduce TG2 overexpression, decreasing GFAP and Vimentin levels. In addition, both compounds inhibited apoptotic pathway activation and induced an increase in the Nestin and cyclin D1 expression. These results show that indicaxanthin and astaxanthin are able to prevent the TG2 change conformation induced by A?. Our data demonstrate that indicaxanthin or astaxanthin pre-treatment stimulated OECs self-renewal through the reparative activity played by TG2. Therefore, indicaxanthin and astaxanthin might represent an innovative mechanism to contrast TG2 overexpression in AD

Alzhèimer Disease (AD), one of the most common neurodegenerative diseases, is characterized by progressive neuronal loss and accumulation of proteins, including Amyloid-beta (A?), a neurotoxic protein. It is known that tissue transglutaminase (TG2), an ubiquitary calcium-dependent protein, is involved in protein aggregation in AD. Previous our studies showed that A?(1-42) and its fragments A?(25-35) and A?(35-25) induced an overexpression of TG2 and its isoforms on Olfactory Ensheathing Cells (OECs), a glial population of the olfactory system that express neural stem cell markers, including Nestin. In the last years growing attention rose on neuronutraceutics and their effect on mental health. Among these molecules, we focused our research on indicaxanthin, and astaxanthin, natural compounds that were able to cross the blood-brain barrier. In this study, the effect of indicaxanthin or astaxanthin pre-treatment on TG2 and its isoform expression levels exposed to A?(1-42) or by A?(25-35) or A?(35-25) on OECs was assessed. Furthermore, we evaluated thei effect on the expression levels of Vimentin and Glial Fibrillary Acid Protein (GFAP). The percentage of cell viability and the apoptotic pathway activation were also evaluated. Since Nestin, a marker of neural precursors, is co-expressed in pluripotent stem cells with cyclin D1, a marker of cellular proliferation, the effect of indicaxanthin and astaxanthin pre-treatment on their expression levels was also tested. In addition, the production of total reactive oxygen species (ROS) and superoxide anion (O2-.), were assessed. In parallel, docking studies were performed to obtain informations relative to the interaction between the indicaxanthin or astaxanthin and TG2. We found that indicaxanthin or astaxanthin pre-treatment was able to reduce TG2 overexpression and its isoforms, decreasing total ROS and O2-.production and GFAP and Vimentin expression levels. In addition, they inhibited apoptotic pathway activation and induced an increase in the Nestin and cyclin D1 expression levels. Docking results showing that indicaxanthin and astaxanthin were able to prevent the TG2 change conformation induced by A?. Our data demonstrated that indicaxanthin or astaxanthin pre-treatment stimulated OECs self-renewal through the reparative activity played by TG2. Therefore, indicaxanthin and astaxanthin might represent an innovative mechanism to contrast TG2 overexpression in AD.

Introduzione L'esposizione al PM2,5 rappresenta un importante fattore di rischio per la salute pubblica. Oltre ad effetti respiratori e cardiovascolari, è stato evidenziato come l'esposizione al PM2,5 possa essere un fattore di rischio ambientale coinvolto nei processi neurodegenerativi. Tuttavia, i meccanismi attraverso cui il PM2.5 esercita tali effetti non sono completamente compresi. In questo studio riportiamo i primi dati di un progetto che mira a valutare la capacità del PM2.5 di indurre citotossicità e ridotta vitalità in colture in vitro di cellule neuronali SH-SY5Y. Materiali e metodi I campioni di PM2,5 sono stati raccolti in aree ad alta densità di traffico veicolare della città di Catania. È stata effettuata la caratterizzazione chimica quali-quantitativa dei campioni, mediante determinazione in ICP-MS di As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Se, V, e dei 16 IPA classificati prioritari dalla US EPA mediante determinazione in HPLC UV-FL. Successivamente, è stata validata una metodologia adeguata a garantire la sterilità del terreno di coltura (DMEM-F12) per le prove di esposizione in vitro del PM2,5 alle SH-SY5Y, così da valutare il potenziale effetto citotossico mediante saggio MTT. Risultati Le analisi sono state effettuate su un totale di 40 campioni. É stato rilevato un valore medio annuo di PM2,5 = 17,1 ?g/m3, e un valore massimo di PM2,5 = 27,6 ?g/m3 durante la stagione invernale. I metalli pesanti e gli IPA hanno mostrato i valori più elevati durante la stagione invernale, sebbene al di sotto dei limiti riportati dal D.Lgs. 152/2007 per As (6 ng/m3), Cd (5 ng/m3) e Ni (20 ng/m3), dal DM 60/2002 per il Pb (0,5 ?g/ m3) e dal D.Lgs 155/10 per il Benzo(a)pirene (1 ng/m3). I successivi test in vitro sulle cellule SH-SY5Y hanno evidenziato una discreta riduzione della vitalità cellulare dopo esposizione agli estratti del PM2.5. In particolare, i campioni della stagione invernale, con maggior quantità di PM2,5 e più elevati valori di Cr, Cu, Mn, Ni, Pb, V, naftalene, pyrene, dibenzo(a,h)antracene, hanno mostrato una riduzione della vitalità cellulare del 16,3%, 16,6% e 28,1% dopo 24, 48 e 72 ore di esposizione, rispettivamente. Conclusioni I risultati ottenuti forniscono una prima indicazione sui possibili effetti del PM2,5, e delle molecole ad esso correlate, nei confronti delle SH-SY5Y. Nei prossimi step studieremo la risposta delle cellule al PM2,5 analizzando il potenziale danno genomico, l'attività mitocondriale, lo stress ossidativo indotto e l'interferenza dei livelli di espressione proteica, al fine di indicare linee di intervento preventivo per le patologie neurodegenerative.

Neuroinflammation induces neurometabolic alterations and increases in energy consumption. Recent research has attempted to clarify the role of Ghrelin (Ghre) signaling in microglia on the regulation of energy balance, obesity, neuroinflammation and the occurrence of neurodegenerative diseases. This orexigenic hormone not only regulates food intake, energy content and glucose homeostasis, but also modulates plasticity and cognition in the central nervous system (CNS). Additionally, microglia may constitute an important therapeutic target in neuroinflammation. In fact, microglial cells are able of producing a wide range of chemokines to promote inflammatory processes being the protective cells of the CNS. These cells have also been identified as specialized macrophages sharing many phenotypical and functional characteristics. Ghre and microglia are involved in the pathophysiology of neurodegenerative diseases characterized by neuronal damage such as Alzheimer's disease and Parkinson's disease. The reported evidences show that Ghre modulates microglia activity and thus affecting pathophysiology of neurodegenerative diseases interferes in the control of neurometabolism. The aim was to evaluate the underline mechanisms by which Ghre modulates microglia activity during obesity-induced neuroinflammation by emphasizing the effects of Ghre in inducing these cells towards an anti-inflammatory phenotype. The understanding of this peptide's functions will allow for the development and implementation of new therapeutic and neurological diagnostic strategies.

Many approaches to brain delivery of bioactive compounds have emerged in recent years. Intranasal (IN) route is one of the newly focused delivery options for central nervous system (CNS) targeting, since this is the only way through which the brain is in connection with the outside environment. Nose-to-brain (N2B) drug delivery can be achieved by different mechanisms that are not yet fully understood. Drugs can be transported directly from the nasal cavity to the CNS by the trigeminal and the olfactory nerves pathway by passing the blood brain barrier. In order to overcome some of the limitations associated with the administration of free drugs (i.e. rapid elimination, poor water solubility, stability etc.), the use of nanomedicine could be considered a promising approach and is under intense investigation. Nanoplatforms of various nature [polymeric nanoparticles (PNP), lipid nanoparticles (SLN, NLC) nanocrystals (NCs)], characterized by different physico-chemical and technological properties have been designed to improve drug transfer after N2B delivery. Fluorescent dye (Rhodamine B) loaded PNP have been detected after IN administration in healthy rats to evaluate their distribution in brain areas. Once confirmed PNP transport to hippocampus, assessment of drug efficacy was performed after IN administration of oxcarbazepine loaded PNP (OXC_PNP) in an in vivo model of epilepsy. Our results demonstrated significant protective effects against seizures with a reduced dose and number administrations of OXC_PNP compared to the neat drug. Recently, nanocrystalline drug technology approach was also investigated to overcome one of the main limitations of colloidal systems, namely the difficulty to obtain an highly concentrated drug formulation in the small volume necessary for IN administration. NCs were formulated starting from natural (curcumin, resveratrol) and synthetic molecules (carbamazepine) obtaining concentrated formulations with high drug loading, particularly useful for for the treatment of neurodegenerative diseases by the N2B strategy. NCs have been evaluated in vitro on Olfactory Ensheating Cells, a unique class of glia that envelopes bundles of olfactory axons, that project to the olfactory bulb which we consider to play a key role in the direct pathway of drug transport through the N2B route. Overall, nanomedicine combined with IN administration is a relevant research topic that can rise as a hope for a new era of brain diseases treatment.

Alzheimer's disease (AD) is a neurodegenerative disease representing the most prevalent cause of dementia. It is also related to the aberrant amyloid-beta (A) protein deposition in the brain. Since oxidative stress is involved in AD, there is a possible role of antioxidants present in the effected person's diet. Thus, we assessed the effect of the systemic administration of solid lipid nanoparticles (SLNs) to facilitate curcumin (CUR) delivery on TG2 isoform expression levels in Wild Type (WT) and in TgCRND8 (Tg) mice. An experimental model of AD, which expresses two mutated human amyloid precursor protein (APP) genes, was used. Behavioral studies were also performed to evaluate the improvement of cognitive performance and memory function induced by all treatments. The expression levels of Bcl-2, Cyclin-D1, and caspase-3 cleavage were evaluated as well. In this research, for the first time, we demonstrated that the systemic administration of SLNs-CUR, both in WT and in Tg mice, allows one to differently modulate TG2 isoforms, which act either on apoptotic pathway activation or on the ability of the protein to repair cellular damage in the brains of Tg mice. In this study, we also suggest that SLNs-CUR could be an innovative tool for the treatment of AD.

Alzheimer Disease (AD), one of the most common neurodegenerative diseases, is characterized by progressive neuronal loss and accumulation of proteins, including Amyloid-beta (A?), a neurotoxic protein. It is known that tissue transglutaminase (TG2), an ubiquitary calcium-dependent protein, is involved in protein aggregation in AD. Previous our studies showed that A?(1-42) and its fragments A?(25-35) and A?(35-25) induced an overexpression of TG2 and its isoforms on Olfactory Ensheathing Cells (OECs), a glial population of the olfactory system expressing neural stem cell markers, including Nestin. In the last years growing attention rose on neuronutraceutics and their effect on mental health, as many compounds are characterized by antioxidant and anti-inflammatory activities, these properties could have beneficial effects in counteracting the multifactorial onset and progression of AD. Among these molecules, we focus on Astaxanthin, a natural compound predominantly found in marine organisms, the green microalgae Haematococcus pluvialis and Chlorella zofingiensis; it can be transported into the brain through the blood-brain barrier and exhibits powerful anti-oxidant activity. In addition, Astaxanthin can effectively scavenge intracellular free radicals and destroy peroxide chain reactions, protecting cell and biological membranes from oxidative damage. Therefore, it exhibits numerous health benefits, such as anti-inflammatory actions, anti-tumor effects, hepatoprotective effects, and immunomodulatory activity. In this study, the effect of Astaxanthin pretreatment on TG2 and its isoform expression exposed to A?(1-42) or by A?(25-35) or A?(35-25) on OECs was assessed. Moreover, we evaluated Astaxanthin effect on the expression of some cytoskeletal proteins, Vimentin, Glial Fibrillary Acid Protein (GFAP), Nestin and Caspase-3. Astaxanthin pre-treatment reduced TG2 overexpression, modulating the level of TG2 isoforms and reduced ROS production, GFAP and Vimentin expression, inhibiting apoptotic pathway activation and induced an increase in the Nestin levels. Our data demonstrated that Astaxanthin pre-treatment stimulated OECs self-renewal through the reparative activity played by TG2. Therefore, pre-treatment Astaxanthin might represent an innovative mechanism to contrast TG2 overexpression in AD.

Nowdays, neurodegenerative diseases represent a great challenge from both the therapeutic and diagnostic points of view. Indeed, several physiological barriers of the body, including the blood brain barrier (BBB), nasal, dermal, and intestinal barriers, interpose between the development of new drugs and their effective administration to reach the target organ or target cells at therapeutic concentrations. Currently, the nose-to-brain delivery with nanoformulations specifically designed for intranasal administration is a strategy widely investigated with the goal to reach the brain while bypassing the BBB. To produce nanosystems suitable to study both in vitro and/or in vivo cells trafficking for potential nose-to-brain delivery route, we prepared and characterized two types of fluorescent poly(ethylene glycol)-methyl-ether-block-poly(lactide-coglycolide) (PLGA-PEG) nanoparticles (PNPs), i.e., Rhodamine B (RhB) dye loaded- and grafted- PNPs, respectively. The latter were produced by blending into the PLGA-PEG matrix a RhB-labeled polyaspartamide/polylactide graft copolymer to ensure a stable fluorescence during the time of analysis. Photon correlation spectroscopy (PCS), UV-visible (UV-vis) spectroscopies, differential scanning calorimetry (DSC), atomic force microscopy (AFM) were used to characterize the RhBloaded and RhB-grafted PNPs. To assess their potential use for brain targeting, cytotoxicity tests were carried out on olfactory ensheathing cells (OECs) and neuron-like differentiated PC12 cells. Both PNP types showed mean sizes suitable for nose-to-brain delivery (<200 nm, PDI < 0.3) and were not cytotoxic toward OECs in the concentration range tested, while a reduction in the viability on PC12 cells was found when higher concentrations of nanomedicines were used. Both the RhBlabelled NPs are suitable drug carrier models for exploring cellular trafficking in nose-to-brain delivery for short-time or long-term studies.

Both dopamine (DA) loaded Solid Lipid Nanoparticles (SLN) and liposomes (Lip), designed for intranasal administration of the neurotransmitter as an innovative Parkinson disease treatment, were already characterized in vitro in some extent by us (Trapani et al., 2018a and Cometa et al., 2020, respectively). Herein, to gain insight into the structure of SLN, X-ray Photoelectron Spectroscopy Analysis was carried out and DA-SLN (SLN 1) were found to exhibit high amounts of the neurotransmitter on the surface, whereas the external side of Glycol Chitosan (GCS) containing SLN (SLN 2) possessed only few amounts. However, SLN 2 were characterized by the highest encapsulation DA efficiency (i.e., 81%). Furthermore, in view of intranasal administration, mucoadhesion tests in vitro were also conducted for SLN and Lip formulations, evidencing high muchoadesive effect exerted by SLN 2. Concerning ex-vivo studies, SLN and Lip were found to be safe for Olfactory Ensheathing Cells and fluorescent SLN 2 were taken up in a dose-dependent manner reaching the 100% of positive cells, while Lip 2 (chitosan-glutathione-coated) were internalised by 70% OECs with six-times more lipid concentration. Hence, SLN 2 formulation containing DA and GCS may constitute interesting formulations for further studies and promising dosage form for non-invasive nose-to-brain neurotransmitter delivery