Giovanna Pannuzzo

Sodium L-Lactate Differently Affects Brain-Derived Neurothrophic Factor, Inducible Nitric Oxide Synthase, and Heat Shock Protein 70 kDa Production in Human Astrocytes and SH-SY5Y Cultures

The present study analyzed the in vitro effects induced by sodium L‐lactate on human astrocytes and the SH‐SY5Y cell line, when added at concentrations of 5, 10, and 25 mmol/liter. Expression of brain‐derived neurotrophic factor (BDNF), inducible nitric oxide synthase (iNOS), and heat shock protein 70 kDa (HSP70) was evaluated by Western blot analysis. Cell viability with MTT, release of nitric oxide (NO) through the Griess reaction, and production of BDNF by enzyme‐linked immunoassay was determined. Data indicate that, in SH‐SY5Y as well as in cortical astrocytes, after 4 hr sodium L‐lactate increases the expression and release of BDNF, iNOS, and NO; after 24 hr, it turns is ineffective for the production of the neurotrophin in SH‐SY5Y and not in astrocytes, but the expression of iNOS and release of NO appear to be further increased compared with those after 4 hr. Sodium L‐lactate influences differently the expression of HSP70 in SH‐SY5Y compared with astrocytes. We propose, based on these findings, that sodium L‐lactate affects the expression of BDNF in SH‐SY5Y and astrocytes in a different manner: high levels of iNOS and NO expressed in SH‐SY5Y have a profound inhibitory effect on the release of BDNF related to a more limited production of HSP70 by SH‐SY5Y. In conclusion, the results demonstrate differences in the responses of SH‐SY5Y and astrocytes to stimulation by high levels of sodium L‐lactate. Sodium L‐lactate differently and dose and time dependently influences the expression and release of BDNF, iNOS, NO, and HSP70 depending on the cell type.

A galactose-free diet enriched in soy isoflavones and antioxidants results in delayed onset of symptoms of Krabbe disease in twitcher mice.

Krabbe disease or globoid cell leukodystrophy is an autosomal recessively inherited disorder caused by the deficiency of galactocerebrosidase, the lysosomal enzyme that catalyzes the hydrolysis of galactose from galactosylceramide and galactosylsphingosine (psychosine). Psychosine accumulation results in the loss of myelin and oligodendrocytes in the brain of Krabbe patients as well as twitcher mice (natural model of human Krabbe disease). The aim of the present research was to investigate in twitcher mice the potential role of a diet deficient in galactose enriched in soy isoflavones and a pool of antioxidants molecules, such as l-glutathione, coenzyme Q10, xanthophylls, in counteracting the toxic effects derived by psychosine accumulation. A second goal of this manuscript was to demonstrate suppression of the apoptotic effects of psychosine in cultured oligodendrocyte progenitor mice cells (OLP-II) with antioxidants. The affected twitcher mice began the milk-derivatives free diet on post-natal day 15 although they also received mothers milk until post-natal day 18. Nevertheless, average life span was increased 50%, from 32+/-2 to 48+/-3 days, onset of tremor was delayed 17 days (from 21 days in the untreated twitcher mice to 38 days in the treated affected mice) and the gait in the treated mice was normal until almost a week after the untreated animals died (38+/-1 days versus 32 days at death). Weight gain in the treated animals also progressed to 38 days compared with 22 days for the untreated affected twitcher mice. Protection of the OLP-II cells against psychosine was shown using the MTT test (the ability of the tetrazolium salt MTT to form a dark blue formazan product by mitochondrial dehydrogenase in viable cells) and assay of expression of p53 and TNF-related apoptosis-inducing ligand (TRAIL). The results showed a time-dependent and concentration-dependent decrease of OLP-II viability on exposure to psychosine and dose-dependent protection with the antioxidants xanthophylls and glutathione. They also demonstrated that psychosine-induced p53 induction of apoptosis and TNF-related apoptosis-inducing ligand receptors could be decreased by l-glutathione and xanthophylls. A dietary approach may constitute a promising clinical management of the late-infantile and juvenile forms of Krabbe leukodystrophy.

Molecular Docking and Fluorescence Characterization of Benzothieno[3,2-d]pyrimidin-4-one Sulphonamide Thio-Derivatives, a Novel Class of Selective Cyclooxygenase-2 Inhibitors

The aims of this study were: (i) to explore the structure-activity relationship of some new anti-inflammatory benzothieno[3,2-d]pyrimidin-4-one sulphonamide thio-derivatives 1–11; and (ii) to evaluate the possibility of using the most active compounds as fluorescent probes to determine tumours or their progression. Therefore, to know the precise mechanism by which these compounds interact with cyclooxygenase (COX)-2 enzyme, a molecular docking study was carried out; to assess spectroscopic characteristics, their absorption and emission properties were determined. The results demonstrated that some derivatives of benzothieno[3,2-d] pyrimidine exhibit interesting anti-inflammatory properties related to interactions with active sites of COX-2 and are fluorescent. The antipyrine-bearing compound 4 displayed high COX-2 affinity (ΔG = −9.4) and good fluorescent properties (Φfl = 0.032). Thus, some members of this new class of anti-inflammatory may be promising for fluorescence imaging of cancer cells that express the COX-2 enzyme. Further in vitro and in vivo studies are needed to confirm this hypothesis.

New insights on Parkinson's disease from differentiation of SH-SY5Y into dopaminergic neurons: An involvement of aquaporin4 and 9

&NA; The purpose of this research was to explore the behavior of aquaporins (AQPs) in an in vitro model of Parkinsons disease that is a recurrent neurodegenerative disorder caused by the gradual, progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Because of postmortem studies have provided evidences for oxidative damage and alteration of water flow and energy metabolism, we carried out an investigation about AQP4 and 9, demonstrated in the brain to maintain water and energy homeostasis. As an appropriate in vitro cell model, we used SH‐SY5Y cultures and induced their differentiation into a mature dopaminergic neuron phenotype with retinoic acid (RA) alone or in association with phorbol‐12‐myristate‐13‐acetate (MPA). The association RA plus MPA provided the most complete and mature neuron phenotype, as demonstrated by high levels of &bgr;‐Tubulin III, MAP‐2, and tyrosine hydroxylase. After validation of cell differentiation, the neurotoxin 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) and H2O2 were applied to reproduce a Parkinsons‐like stress. The results confirmed RA/MPA differentiated SH‐SY5Y as a useful in vitro system for studying neurotoxicity and for using in a MPTP and H2O2‐induced Parkinsons disease cell model. Moreover, the data demonstrated that neuronal differentiation, neurotoxicity, neuroinflammation, and oxidative stress are strongly correlated with dynamic changes of AQP4 and 9 transcription and transduction. New in vitro and in vivo experiments are needed to confirm these innovative outcomes. HighlightsWe used an in vitro cellular model as controlled system of Parkinsons disease.We induced differentiation of SH‐SY5Y into a mature neuronal phenotype.We evaluated the effect of neurotoxin MPTP and H2O2 on differentiated SH‐SY5Y.We hypothesized an involvement of aquaporin4 and 9 in MPTP and H2O2‐induced damage.

Chaperones as potential therapeutics for Krabbe disease.

Krabbes disease (KD) is an autosomal recessive, neurodegenerative disorder. It is classified among the lysosomal storage diseases (LSDs). It was first described in , but the genetic defect for the galactocerebrosidase (GALC) gene was not discovered until the beginning of the 1970s, 20 years before the GALC cloning. Recently, in 2011, the crystal structures of the GALC enzyme and the GALC‐product complex were obtained. For this, compared with other LSDs, the research on possible therapeutic interventions is much more recent. Thus, it is not surprising that some treatment options are still under preclinical investigation, whereas their relevance for other pathologies of the same group has already been tested in clinical studies. This is specifically the case for pharmacological chaperone therapy (PCT), a promising strategy for selectively correcting defective protein folding and trafficking and for enhancing enzyme activity by small molecules. These compounds bind directly to a partially folded biosynthetic intermediate, stabilize the protein, and allow completion of the folding process to yield a functional protein. Here, we review the chaperones that have demonstrated potential therapeutics during preclinical studies for KD, underscoring the requirement to invigorate research for KD‐addressed PCT that will benefit from recent insights into the molecular understanding of GALC structure, drug design, and development in cellular models.

Aquaporin1 and 3 modification as a result of chondrogenic differentiation of human mesenchymal stem cell

Chondrocytes are cells of articular cartilage particularly sensitive to water transport and ionic and osmotic changes from extracellular environment and responsible for the production of the synovial fluid. Aquaporins (AQPs) are a family of water and small solute transport channel proteins identified in several tissues, involved in physiological pathways and in manifold human diseases. In a recent period, AQP1 and 3 seem to have a role in metabolic water regulation in articular cartilage of load bearing joints. The aim of this study was to examine the levels of AQP1 and 3 during the chondrogenic differentiation of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT). For the determination of chondrogenic markers and AQPs levels, glycosaminoglycans (GAGs) quantification, immunocytochemistry, RT‐PCR, and Western blot were used after 0, 7, 14, 21, and 28 days from the start of differentiation. At 21 days, chondrocytes derived from AT‐MSCs were able to produce augmented content of GAGs and significant quantity of SOX‐9, lubricin, aggrecan, and collagen type II, suggesting hyaline cartilage formation, in combination with an increase of AQP3 and AQP1. However, while AQP1 level decreased after 21 days; AQP3 reached higher values at 28 days. The expression of AQP1 and 3 is a manifestation of physiological adaptation of functionally mature chondrocytes able to respond to the change of their internal environment influenced by extracellular matrix. The alteration or loss of expression of AQP1 and 3 could contribute to destruction of chondrocytes and to development of cartilage damage.

Krabbe's leukodystrophy: Approaches and models in vitro: Cell Models and Krabbe's Disease

This Review describes some in vitro approaches used to investigate the mechanisms involved in Krabbes disease, with particular regard to the cellular systems employed to study processes of inflammation, apoptosis, and angiogenesis. The aim was to update the knowledge on the results obtained from in vitro models of this neurodegenerative disorder and provide stimuli for future research. For a long time, the nonavailability of established neural cells has limited the understanding of neuropathogenic mechanisms in Krabbes leukodystrophy. More recently, the development of new Krabbes disease cell models has allowed the identification of neurologically relevant pathogenic cascades, including the major role of elevated psychosine levels. Thus, direct and/or indirect roles of psychosine in the release of cytokines, reactive oxygen species, and nitric oxide and in the activation of kinases, caspases, and angiogenic factors results should be clearer. In parallel, it is now understood that the presence of globoid cells precedes oligodendrocyte apoptosis and demyelination. The information described here will help to continue the research on Krabbes leukodystrophy and on potential new therapeutic approaches for this disease that even today, despite numerous attempts, is without cure.

Zoledronate derivatives as potential inhibitors of uridine diphosphate-galactose ceramide galactosyltransferase 8: A combined molecular docking and dynamic study

Krabbes disease is a neurodegenerative disorder caused by deficiency of galactocerebrosidase activity that affects the myelin sheath of the nervous system, involving dysfunctional metabolism of sphingolipids. It has no cure. Because substrate inhibition therapy has been shown to be effective in some human lysosomal storage diseases, we hypothesize that a substrate inhibition therapeutic approach might be appropriate to allow correction of the imbalance between formation and breakdown of glycosphingolipids and to prevent pathological storage of psychosine. The enzyme responsible for the biosynthesis of galactosylceramide and psychosine is uridine diphosphate‐galactose ceramide galactosyltransferase (2‐hydroxyacylsphingosine 1‐β‐galactosyltransferase; UGT8; EC 2.4.1.45), which catalyzes the transferring of galactose from uridine diphosphate‐galactose to ceramide or sphingosine, an important step of the biosynthesis of galactosphingolipids. Because some bisphosphonates have been identified as selective galactosyltransferase inhibitors, we verify the binding affinity to a generated model of the enzyme UGT8 and investigate the molecular mechanisms of UGT8–ligand interactions of the bisphosphonate zoledronate by a multistep framework combining homology modeling, molecular docking, and molecular dynamics simulations. From structural information on UGTs’ active site stereochemistry, charge density, and access through the hydrophobic environment, the molecular docking procedure allowed us to identify zoledronate as a potential inhibitor of human ceramide galactosyltransferase. More importantly, zoledronate derivates were designed through computational modeling as putative new inhibitors. Experiments in vivo and in vitro have been planned to verify the possibility of using zoledronate and/or the newly identified inhibitors of UGT8 for a substrate inhibition therapy useful for treatment of Krabbes disease and/or other lysosomal disorders.

Hydroxytyrosol from olive fruits prevents blue-light-induced damage in human keratinocytes and fibroblasts: AVOLA et al.

Skin aging is a complex biological process influenced by a combination of endogenous or intrinsic and exogenous or extrinsic factors due to environmental damage. The primary environmental factor that causes human skin aging is the ultraviolet irradiation from the sun. Recently, it was established that the long‐term exposure to light‐emitting‐diode‐generated blue light (LED‐BL) from electronic devices seems to have a relevant implication in the molecular mechanisms of premature photoaging. BL irradiation induces changes in the synthesis of various skin structures through DNA damage and overproduction of reactive oxygen species (ROS), matrix metalloproteinase‐1 and ‐12, which are responsible for the loss of the main components of the extracellular matrix of skin like collagen type I and elastin. In the current study, using human keratinocytes and fibroblasts exposed to specific LED‐BL radiation doses (45 and 15 J/cm 2), we produced an in vitro model of skin photoaging. We verified that, compared with untreated controls, the treatment with LED‐BL irradiation results in the alteration of metalloprotease‐1 (collagenase), metalloprotease‐12 (elastase), 8‐dihydroxy‐2′‐deoxyguanosine, proliferating cell nuclear antigen, and collagen type I. Moreover, we showed that the photoaging prevention is possible via the use of hydroxytyrosol extracted from olive fruits, well known for antioxidant properties. Our results demonstrated that hydroxytyrosol protects keratinocytes and fibroblasts from LED‐BL‐induced damage. Thus, hydroxytyrosol might be proposed as an encouraging candidate for the prevention of BL‐induced premature photoaging.

Synthesis, characterization, molecular modelling and biological evaluation of thieno-pyrimidinone methanesulphonamide thio-derivatives as non-steroidal anti-inflammatory agents

This study reports the synthesis, molecular docking and biological evaluation of eight (5‐8 and 5a‐8a) newly synthesized thieno‐pyrimidinone methanesulphonamide thio‐derivatives. The synthetic route used to prepare the new isomers thioaryl and thio‐cycloesyl derivatives of the heterocyclic system 6‐phenylthieno[3,2]pyrimidinone was economically and environmentally very advantageous and characterized by the simplicity of procedure, reduction in isolation steps, purification phases, time, costs and waste production. The study in silico for the evaluation of cyclooxygenase (COX)‐1 and COX‐2 selective inhibition was carried out by AutoDock Vina, an open‐source program for doing molecular docking which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. The research in vitro for the biological evaluation was performed by using human cartilage and chondrocytes cultures treated with 10 ng/mL of interleukin‐1beta as inflammation models. The anti‐inflammatory activity of each new compound at the concentration of 10 μmol/L was determined by assaying COX‐2, inducible nitric oxide synthetase (iNOS) and intercellular adhesion molecule 1 (ICAM 1) through Western blot. The examined derivatives showed interesting pharmacological activity, and the compound N‐[2‐[2,4‐difluorophenyl)thio]‐4‐oxo‐6‐phenylthieno[3,2‐d]pyridine‐34H‐yl]methanesulphonamide (7) was excellent COX‐2 inhibitor. In agreement with the biological data, compound 7 was able to fit into the active site of COX‐2 with highest interaction energy. These results can support the design of novel specific inhibitors of COX‐2 by the comparative modelling of COX‐1 and COX‐2 enzymes with the available pharmacophore.