In vitro effects of cocaine on tunneling nanotube formation and extracellular vesicle release in glioblastoma cell cultures

The effects of cocaine (150 nM, 300 nM, and 150 μM) on human glioblastoma cell cultures were studied on tunneling nanotube formation (1-h cocaine treatment) and extracellular vesicle release (1-, 3-, and 8-h cocaine treatment). Cocaine significantly increased the number of tunneling nanotubes only at the lowest concentration used. The release of extracellular vesicles (mainly exosomes) into the medium was stimulated by cocaine at each concentration used with a maximum effect at the highest concentration tested (150 μM). Moreover, cocaine (150 nM) significantly increased the number of vesicles with 61-80 nm diameter while at concentrations of 300 nM and 150 μM, and the smaller vesicles (30-40 nm diameter) were significantly increased with a reduction of the larger vesicles (41-60 nm diameter). A time dependence in the release of extracellular vesicles was observed. In view of the proposed role of these novel intercellular communication modes in the glial-neuronal plasticity, it seems possible that they can participate in the processes leading to cocaine addiction. The molecular target/s involved in these cocaine effects could be specific molecular components of plasma membrane lipid rafts and/or cocaine-induced modifications in cytoplasmic lipid composition

BV-2 Microglial Cells Respond to Rotenone Toxic Insult by Modifying Pregnenolone, 5alpha-Dihydroprogesterone and Pregnanolone Level

Neuroinflammation, whose distinctive sign is the activation of microglia, is supposed to play a key role in the development and progression of neurodegenerative diseases. The aim of this investigation was to determine levels of neurosteroids produced by resting and injured BV-2 microglial cells. BV-2 cells were exposed to increasing concentrations of rotenone to progressively reduce their viability by increasing reactive oxygen species (ROS) production. BV-2 cell viability was significantly reduced 24, 48 and 72 h after rotenone (50–1000 nM) exposure. Concomitantly, rotenone (50–100 nM) determined a dose-independent augmentation of ROS production. Then, BV-2 cells were exposed to a single, threshold dose of rotenone (75 nM) to evaluate the overtime release of neurosteroids. In particular, pregnenolone, pregnenolone sulfate, progesterone, 5alpha-dihydroprogesterone (5-DHP), allopregnanolone, and pregnanolone, were quantified in the culture medium by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. BV-2 cells synthesized all the investigated neurosteroids and, after exposure to rotenone, 5DHP and pregnanolone production was remarkably increased. In conclusion, we found that BV-2 cells not only synthesize several neurosteroids, but further increase this production following oxidative damage. Pregnanolone and 5alpha-DHP may play a role in modifying the progression of neuroinflammation in neurodegenerative diseases

Disclosing the Antioxidant and Neuroprotective Activity of an Anthocyanin-Rich Extract from Sweet Cherry (Prunus avium L.) Using In Vitro and In Vivo Models

In this study, an autochthonous variety of sweet cherry (Prunus avium L.), namely “Moretta di Vignola”, was processed to prepare extracts rich in polyphenols, which were characterized by high-performance liquid chromatography (HPLC) separation coupled to UV/DAD and ESI-MSn analysis. Then, a sweet cherry anthocyanin-rich extract (ACE) was prepared, fully characterized and tested for its activity against Parkinson’s disease (PD) in cellular (BV2 microglia and SH-SY5Y neuroblastoma) and in Drosophila melanogaster rotenone (ROT)-induced model. The extract was also evaluated for its antioxidant activity on Caenorhabditis elegans by assessing nematode resistance to thermal stress. In both cell lines, ACE reduced ROT-induced cell death and it decreased, alone, cellular reactive oxygen species (ROS) content while reinstating control-like ROS values after ROT-induced ROS rise, albeit at different concentrations of both compounds. Moreover, ACE mitigated SH-SY5Y cell cytotoxicity in a non-contact co-culture assay with cell-free supernatants from ROT-treated BV-2 cells. ACE, at 50 µg/mL, ameliorated ROT (250 μM)-provoked spontaneous (24 h duration) and induced (after 3 and 7 days) locomotor activity impairment in D. melanogaster and it also increased survival and counteracted the decrease in fly lifespan registered after exposure to the ROT. Moreover, heads from flies treated with ACE showed a non-significant decrease in ROS levels, while those exposed to ROT markedly increased ROS levels if compared to controls. ACE + ROT significantly placed the ROS content to intermediate values between those of controls and ROT alone. Finally, ACE at 25 µg/mL produced a significant increase in the survival rate of nematodes submitted to thermal stress (35 °C, 6–8 h), at the 2nd and 9th day of adulthood. All in all, ACE from Moretta cherries can be an attractive candidate to formulate a nutraceutical product to be used for the prevention of oxidative stress-induced disorders and related neurodegenerative diseases

Pharmacological manipulation of brain galaninergic system and sexual behavior in male mice

Available data suggest a complex role for the brain galaninergic system in male sexual behavior; however, the results so far obtained in animals with either galanin or galanin antagonists are conflicting. Objective: To define the better influence of galanin on male sexual behavior by studying, in mice, (i) the effect of galanin and of the chimeric galanin peptide M40 on the copulatory performance, and (ii) galanin mRNA levels in hypothalamic arcuate and dorso-medial nuclei. Methods: For the behavioral testing, only sexually sluggish male mice were used. Galanin mRNA levels were studied in both sexually potent and impotent mice by means of in situ hybridization. Standard behavioral parameters for sexual behavior were recorded or calculated. Synthetic galanin (0.05, 0.1 or 1 mug/mouse) and M40 (5 or 20 mug/mouse) were intracerebroventricularly (ICV) injected, 15 min before the copulatory test. Galanin mRNA levels were evaluated. Results: In sexually sluggish male mice, both galanin (0.1 and 1 mug/mouse ICV) and M40 (20 mug/mouse ICV), significantly increased intromission frequency and ejaculation latency; M40 also improved copulatory efficacy. On the other hand, in the hypothalamic arcuate and dorso-medial nuclei, the levels of galanin mRNA were not significantly different in sexually potent and impotent male mice. Conclusions.-These results show that in sexually sluggish male mice the ICV injection of either galanin or the chimeric analogue M40 greatly prolongs the duration of the copulation; without a reduction of the sexual drive or of the copulatory performance. On the other hand, the hybridization experiments seem to rule out an important physiological role of the brain galaninergic system in the regulation of male sexual behavior, at least in mice

Differential Sensitivity of A(2A) and Especially D (2) Receptor Trafficking to Cocaine Compared with Lipid Rafts in Cotransfected CHO Cell Lines. Novel Actions of Cocaine Independent of the DA Transporter.

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A2A and D2 immunoreactivities in previously characterized A2A-D2 CHO cell lines. Receptor double immunofluorescence staining was performed with D2 and A2A antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D2 immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A2A immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 µM at which concentration the rise of D2 immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D2 receptors may primarily increase the insertion of D2 monomers, homomers and also of a subpopulation of A2A-D2 heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D2 receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D2 and A2A-D2 trafficking induced by allosteric actions of cocaine at D2 receptors may contribute to the alterations of D2 signaling found in cocaine abusers

The antinociceptive effect of acetylsalicylic acid is differently affected by a CB1 agonist or antagonist and involves the serotonergic system in rats

Combinations of non steroidal anti-inflammatory drugs (NSAIDs) and cannabinoids are promising because of their potential synergistic effects in analgesia, resulting in a reduction in dosage and minimizing adverse reactions. The analgesic effect of Acetylsalicylic acid (ASA), probably due to a central mechanism, also implicates changes in the central monoaminergic system. Therefore, we decided to evaluate the antinociceptive interaction between the CB1 receptor agonist, HU210, and ASA in tests involving central pain in rats as well as the implication of the central serotonergic system thereon

Influence of f-MLP, ACTH(1-24) and CRH on in vitro chemotaxis of monocytes from centenarians.

OBJECTIVE: The lifelong exposure to a variety of stressors activates a plethora of defense mechanisms, including the hypothalamic-pituitary-adrenal axis which releases neuropeptides affecting the immune responses. Here, we report data on the capability of monocytes from young subjects and centenarians to migrate towards chemotactic stimuli (formyl-methionyl-leucyl-phenylalanine, f-MLP; adrenocorticotropic hormone, ACTH, and corticotrophin-releasing hormone, CRH). Plasma levels of ACTH, CRH and cortisol were measured as an index of ongoing stress response. METHODS: Monocyte chemotaxis towards f-MLP (10(-8)M), ACTH(1-24) (10(-14) and 10(-8)M) and CRH (10(-14) and 10(-8)M) was evaluated in vitro in young subjects (n = 8, age range 25-35 years) and centenarians (n = 9, age >100 years) and expressed as chemotactic index. In 9 young subjects and 6 centenarians, plasma levels of cortisol, ACTH and CRH were measured. RESULTS: Monocyte chemotaxis towards f-MLP, ACTH(1-24) and CRH (10(-8)M) was well preserved in centenarians, except when the lowest concentration of CRH was used. CRH, ACTH and cortisol plasma levels were significantly higher in centenarians than in young subjects. CONCLUSIONS: The capability of monocytes from centenarians to respond to chemotactic neuropeptides is well preserved. The decreased responsiveness to the lowest concentration of CRH might be due to downregulation of CRH receptors or to defects in the intracellular signal transduction pathway. The high plasma levels of cortisol, CRH and ACTH in centenarians indicate an activation of the entire stress axis, likely counteracting the systemic inflammatory process occurring with age. This activation fits with the hypothesis that lifelong low-intensity stressors activate ancient, hormetic defense mechanisms, favoring healthy aging and longevity

In vitro characterization of the cytokine Drosophila Helical factor

Drosophila Helical factor (Hf) is a protein discovered through the QT method, an algorithm specifically designed for finding helical cytokines. Since in vivo experiments suggested the involvement of Hf in Drosophila melanogaster immunity, we have proceeded with the characterization of Hf functions in the macrophage-like Drosophila embryonic hemocytes, SL2 cell line. qPCR results demonstrated that Hf gene is induced in the SL2 cell line, after either 6 or 24 h incubation with Escherichia coli-purified peptidoglycan. The silencing of Hf expression through RNAi resulted in the reduced capability of synthesizing antimicrobial peptides (AMP) after exposure to heat-inactivated E. coli. The effects of the recombinant peptide rHf have also been tested in the SL2 cell line. rHf promotes the expression and triggers the release of Hf from the hemocytes, and stimulates the synthesis of the antimicrobial peptides (AMP) Defensin and Drosomycin, without any further immune stimulation. Consistent with the output of the QT method, which predicts Hf as a secreted protein, chromatin immune-precipitation experiments confirmed that Hf does not bind DNA, excluding that it acts as an immune-regulated transcription factor. Finally, rHf neither exerts chemotactic action nor triggers bacterial phagocytosis in SL2 cells.Altogether, our data supports the prediction that Hf is an helical cytokine produced and secreted by the hemocytes and it is mainly involved in the regulation of the humoral component of the immune response of D. melanogaster

Human Microglia Synthesize Neurosteroids to Cope with Rotenone-Induced Oxidative Stress

We obtained evidence that mouse BV2 microglia synthesize neurosteroids dynamically to modify neurosteroid levels in response to oxidative damage caused by rotenone. Here, we evaluated whether neurosteroids could be produced and altered in response to rotenone by the human microglial clone 3 (HMC3) cell line. To this aim, HMC3 cultures were exposed to rotenone (100 nM) and neurosteroids were measured in the culture medium by liquid chromatography with tandem mass spectrometry. Microglia reactivity was evaluated by measuring interleukin 6 (IL-6) levels, whereas cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. After 24 h (h), rotenone increased IL-6 and reactive oxygen species levels by approximately +37% over the baseline, without affecting cell viability; however, microglia viability was significantly reduced at 48 h (p p < 0.05). Interestingly, treatment with exogenous allopregnanolone (1 nM) efficiently prevented the reduction in HMC3 cell viability. In conclusion, this is the first evidence that human microglia can produce allopregnanolone and that this neurosteroid is increasingly released in response to oxidative stress, to tentatively support the microglia’s survival

A beta peptides as one of the crucial volume transmission signals in the trophic units and their interactions with homocysteine. Physiological implications and relevance for Alzheimer's disease

Amyloid peptides (A\u3b2) can operate as volume transmission (VT) signals since they are continuously released from cells of the central nervous system and diffuse in the extra-cellular space of the brain. They have both regulatory and trophic functions on cellular networks. In agreement with A\u3b2 regulatory actions on glial-neuronal networks, the present paper reports new findings demonstrating that intrastriatal injections of A\u3b2 peptides reduce striatal tyrosine hydroxylase, increase striatal GFAP immunoreactivities and lower pain threshold in experimental rats. Furthermore, it has been demonstrated that exogenous homocysteine (Hcy) binds A\u3b2(1-40) favouring its \u3b2-sheet conformation both in vitro and in vivo and hence the formation of \u3b2-fibrils and development of neurotoxicity.Thus, the hypothesis is discussed that A\u3b2 peptides represent crucial VT-signals in the brain and their action is altered by dysmetabolic signals such as high Hcy extra-cellular levels, known to be an important risk factor for Alzheimer\u2019s disease