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The large intracellular loop (IL) of the glycine receptor (GlyR) interacts with various signaling proteins and plays a fundamental role in trafficking and regulation of several receptor properties, including a direct interaction with G␤␥. In the present study, we found that mutation of basic residues in the N-terminal region of the IL reduced the binding of G␤␥ to 21 Ϯ 10% of control. Two basic residues in the C-terminal region, on the other hand, contributed to a smaller extent to G␤␥ binding. Using docking analysis, we found that both basic regions of the IL bind in nearby regions to the G␤␥ dimer, within an area of high density of amino acids having an electronegative character. Thereafter, we generated a 17-amino acid peptide with the N-terminal sequence of the wild-type IL (RQH) that was able to inhibit the in vitro binding of G␤␥ to GlyRs to 57 Ϯ 5% of control in glutathione Stransferase pull-down assays using purified proteins. More interestingly, when the peptide was intracellularly applied to human embryonic kidney 293 cells, it inhibited the G␤␥-mediated modulations of G protein-coupled inwardly rectifying potassium channel by baclofen (24 Ϯ 14% of control) and attenuated the GlyR potentiation by ethanol (51 Ϯ 10% versus 10 Ϯ 3%).

Gelsemine is one of the principal alkaloids produced by the Gelsemium genus of plants belonging to the Loganiaceae family. The extracts of these plants have been used for many years, for a variety of medicinal purposes. Coincidentally, recent studies have shown that gelsemine exerts anxiolytic and analgesic effects on behavioural models. Several lines of evidence have suggested that these beneficial actions were dependent on glycine receptors, which are inhibitory neurotransmittergated ion channels of the CNS. However, it is currently unknown whether gelsemine can directly modulate the function of glycine receptors. EXPERIMENTAL APPROACHWe examined the functional effects of gelsemine on glycine receptors expressed in transfected HEK293 cells and in cultured spinal neurons by electrophysiological techniques. KEY RESULTSGelsemine directly modulated recombinant and native glycine receptors and exerted conformation-specific and subunit-selective effects. Gelsemine modulation was voltage-independent and was associated with differential changes in the apparent affinity for glycine and in the open probability of the ion channel. In addition, the alkaloid preferentially targeted glycine receptors in spinal neurons and showed only minor effects on GABA A and AMPA receptors. Furthermore, gelsemine significantly diminished the frequency of glycinergic and glutamatergic synaptic events without altering the amplitude. CONCLUSIONS AND IMPLICATIONSOur results provide a pharmacological basis to explain, at least in part, the glycine receptor-dependent, beneficial and toxic effects of gelsemine in animals and humans. In addition, the pharmacological profile of gelsemine may open new approaches to the development of subunit-selective modulators of glycine receptors. Abbreviations

The potential neuroprotective properties of fruits have been widely recognized. In this study, we evaluated the protective properties of a blueberry extract (BB-4), rich in polyphenols, in a neurodegenerative model induced by amyloid-β peptide (Aβ). Chronic treatment with Aβ drastically reduced synaptic transmission and the extent of secretory vesicles, which were recovered partially with BB-4. Also, the extract recovered Ca(2+) transients in hippocampal neurons preincubated with Aβ (0.5 and 5 μM) by about 25% ± 3% and 30% ± 2, respectively. In this work, we demonstrate a novel effect of the BB-4 extract on Aβ-induced ATP leakage, in which this extract was able to antagonize the acute ATP leakage but not chronic ATP depletion. On the other hand, BB-4 prevented the uncoupling of mitochondrial function induced by FCCP by about 85%, but it was unable to modify the uncoupling induced by Aβ. The present results strongly indicate that BB-4 plays a role in the process of Aβ aggregation by reducing the toxic species (i.e., 40 kDa). These findings suggest that a blueberry extract can protect neuronal tissue from Aβ toxicity mainly through its antiaggregation property, and its antioxidant properties and mitochondrial membrane potential capacities are secondary mechanisms important in chronic stages. Our work suggests that BB-4 could be an important nutritional complement to neuronal health as well as a potential nutraceutical formulation useful as a dietary supplement in the elderly.

The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aβ). Because Aβ increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aβ and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca microfluorimetry to assess the effects of chronic exposure (24 h) to Aβ oligomers (0.5 μM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aβ. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aβ. Taken together, our findings suggest that Aβ disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.

Human erythropoietin is mainly recognized for its hematopoietic function; however, by binding to its receptor (EpoR), it can activate different signaling pathways as STAT, PI3K, MAPK and RAS to increase cellular differentiation or provide neuroprotective effects, among others. A recombinant human erythropoietin variant with low glycosylation and without hematopoietic effect (EpoL) was purified from skimmed goat milk. Recombinant human erythropoietin (Epo) was obtained from CHO cell line and used as control to compare EpoL effects. Neuroprotection studies were performed in PC12 cells and rat hippocampal slices. Cells were pretreated during 1 h with EpoL or Epo and exposed to oxidative agents (H2O2 or FCCP); cell viability was assayed at the end of the experiment by the MTT method. Hippocampal slices were exposed to 15 min of oxygen and glucose deprivation (OGD) and the neuroprotective drugs EpoL or Epo were incubated for 2 h post-OGD in re-oxygenated medium. Cell cultures stressed with oxidative agents, and pretreated with EpoL, showed neuroprotective effects of 30% at a concentration 10 times lower than that of Epo. Moreover, similar differences were observed in OGD ex vivo assays. Neuroprotection elicited by EpoL was lost when an antibody against EpoR was present, indicating that its effect is EpoR-dependent. In conclusion, our results suggest that EpoL has a more potent neuroprotective profile than Epo against oxidative stress, mediated by activation of EpoR, thus EpoL represents an important target to develop a potential biopharmaceutical to treat different central nervous system pathologies related to oxidative stress such as stroke or neurodegenerative diseases.