Altered neuronal excitability is emerging as an important feature in Alzheimer's disease (AD). Kv2.1 potassium channels are important modulators of neuronal excitability and synaptic activity. We investigated Kv2.1 currents and its relation to the intrinsic synaptic activity of hippocampal neurons from 3xTg-AD (triple transgenic mouse model of Alzheimer's disease) mice, a widely employed preclinical AD model. Synaptic activity was also investigated by analyzing spontaneous [Ca2+]i spikes. Compared with wild-type (Non-Tg (non-transgenic mouse model)) cultures, 3xTg-AD neurons showed enhanced spike frequency and decreased intensity. Compared with Non-Tg cultures, 3xTg-AD hippocampal neurons revealed reduced Kv2.1-dependent Ik current densities as well as normalized conductances. 3xTg-AD cultures also exhibited an overall decrease in the number of functional Kv2.1 channels. Immunofluorescence assay revealed an increase in Kv2.1 channel oligomerization, a condition associated with blockade of channel function. In Non-Tg neurons, pharmacological blockade of Kv2.1 channels reproduced the altered pattern found in the 3xTg-AD cultures. Moreover, compared with untreated sister cultures, pharmacological inhibition of Kv2.1 in 3xTg-AD neurons did not produce any significant modification in Ik current densities. Reactive oxygen species (ROS) promote Kv2.1 oligomerization, thereby acting as negative modulator of the channel activity. Glutamate receptor activation produced higher ROS levels in hippocampal 3xTg-AD cultures compared with Non-Tg neurons. Antioxidant treatment with N-Acetyl-Cysteine was found to rescue Kv2.1-dependent currents and decreased spontaneous hyperexcitability in 3xTg-AD neurons. Analogous results regarding spontaneous synaptic activity were observed in neuronal cultures treated with the antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Our study indicates that AD-related mutations may promote enhanced ROS generation, oxidative-dependent oligomerization, and loss of function of Kv2.1 channels. These processes can be part on the increased neuronal excitability of these neurons. These steps may set a deleterious vicious circle that eventually helps to promote excitotoxic damage found in the AD brain.

The effects of microgravity on functions of the human body are well described, including alterations in the male and female reproductive systems. In the present study, TCam-2 cells, which are considered a good model of mitotically active male germ cells, were used to investigate intracellular signalling and cell metabolism during exposure to simulated microgravity, a condition that affects cell shape and cytoskeletal architecture. After a 24 hour exposure to simulated microgravity, TCam-2 cells showed 1) a decreased proliferation rate and a delay in cell cycle progression, 2) increased anaerobic metabolism accompanied by increased levels of intracellular Ca2+, reactive oxygen species and superoxide anion and modifications in mitochondrial morphology. Interestingly, all these events were transient and were no longer evident after 48 hours of exposure. The presence of antioxidants prevented not only the effects described above but also the modifications in cytoskeletal architecture and the activation of the autophagy process induced by simulated microgravity. In conclusion, in the TCam-2 cell model, simulated microgravity activated the oxidative machinery, triggering transient macroscopic cell events, such as a reduction in the proliferation rate, changes in cytoskeleton-driven shape and autophagy activation.

The ionotropic P2X7 receptor (P2X7R) is involved in bone homeostasis but its role in osteogenesis is controversial. Thus, we investigated the expression of P2X7R and the effects exerted by its modulation in mesenchymal stromal cells from human subcutaneous adipose tissue (S-ASCs), which have potential therapeutic application in bone regenerative medicine. We found that undifferentiated S-ASCs expressed P2X7R and its functional splice variants P2X7AR and P2X7BR. Cell stimulation by P2X7R agonist BzATP (100 μM) neither modified proliferation nor caused membrane pore opening while increasing intracellular Ca 2+ levels and migration. The P2X7R antagonist A438079 reversed these effects. However, 25-100 μM BzATP, administered to S-ASCs undergoing osteogenic differentiation, dosedependently decreased extracellular matrix mineralization and expression of osteogenic transcription factors Runx2, alkaline phosphatase and osteopontin. These effects were not coupled to cell proliferation reduction or to cell death increase, but were associated to decrease in P2X7AR and P2X7BR expression. In contrast, expression of P2X7R, especially P2X7BR isoform, significantly increased during the osteogenic process. Noteworthy, the antagonist A438079, administered alone, at first restrained cell differentiation, enhancing it later. Accordingly, A438079 reversed BzATP effects only in the second phase of S-ASCs osteogenic differentiation. Apyrase, a diphosphohydrolase converting ATP/ADP into AMP, showed a similar behavior. Altogether, findings related to A438079 or apyrase effects suggest an earlier and prevailing pro-osteogenic activity by endogenous ATP and a later one by adenosine derived from endogenous ATP metabolism. Conversely, P2X7R pharmacological stimulation by BzATP, mimicking the effects of high ATP levels occurring during tissue injuries, depressed receptor expression/activity impairing MSC osteogenic differentiation.

Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca 2þ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca 2þ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca 2þ influx. ROS and Ca 2þ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca 2þ increase, eventually leading to apoptosis in cancer cells.

The effects induced by microgravity on human body functions have been widely described, in particular those on skeletal muscle and bone tissues. This study aims to implement information on the possible countermeasures necessary to neutralize the oxidative imbalance induced by microgravity on osteoblastic cells. Using the model of murine MC3T3-E1 osteoblast cells, cellular morphology, proliferation, and metabolism were investigated during exposure to simulated microgravity on a random positioning machine in the absence or presence of an antioxidant—the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Our results confirm that simulated microgravity-induced morphological and metabolic alterations characterized by increased levels of reactive oxygen species and a slowdown of the proliferative rate. Interestingly, the use of Trolox inhibited the simulated microgravity-induced effects. Indeed, the antioxidant-neutralizing oxidants preserved cell cytoskeletal architecture and restored cell proliferation rate and metabolism. The use of appropriate antioxidant countermeasures could prevent the modifications and damage induced by microgravity on osteoblastic cells and consequently on bone homeostasis.