Jorge Fuentealba

Synaptic failure and adenosine triphosphate imbalance induced by amyloid-β aggregates are prevented by blueberry-enriched polyphenols extract

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 Ca2+ 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.

Blockade of ethanol-induced potentiation of glycine receptors by a peptide that interferes with Gbetagamma binding.

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 Gβγ. In the present study, we found that mutation of basic residues in the N-terminal region of the IL reduced the binding of Gβγ to 21 ± 10% of control. Two basic residues in the C-terminal region, on the other hand, contributed to a smaller extent to Gβγ binding. Using docking analysis, we found that both basic regions of the IL bind in nearby regions to the Gβγ 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 Gβγ to GlyRs to 57 ± 5% of control in glutathione S-transferase pull-down assays using purified proteins. More interestingly, when the peptide was intracellularly applied to human embryonic kidney 293 cells, it inhibited the Gβγ-mediated modulations of G protein-coupled inwardly rectifying potassium channel by baclofen (24 ± 14% of control) and attenuated the GlyR potentiation by ethanol (51 ± 10% versus 10 ± 3%).

Blueberry Extracts Protect Testis from Hypobaric Hypoxia Induced Oxidative Stress in Rats

Exposure to hypobaric hypoxia causes oxidative damage to male rat reproductive function. The aim of this study was to evaluate the protective effect of a blueberry extract (BB-4) in testis of rats exposed to hypobaric hypoxia. Morphometric analysis, cellular DNA fragmentation, glutathione reductase (GR), and superoxide dismutase (SOD) activities were evaluated. Our results showed that supplementation of BB-4 reduced lipid peroxidation, decreased apoptosis, and increased GR and SOD activities in rat testis under hypobaric hypoxia conditions (P < 0.05). Therefore, this study demonstrates that blueberry extract significantly reduced the harmful effects of oxidative stress caused by hypobaric hypoxia in rat testis by affecting glutathione reductase and superoxide dismutase activities.

Synaptic Silencing and Plasma Membrane Dyshomeostasis Induced by Amyloid-β Peptide are Prevented by Aristotelia chilensis Enriched Extract

Alzheimers disease (AD) is characterized by the presence of different types of extracellular and neurotoxic aggregates of amyloid-β (Aβ). Recently, bioactive compounds extracted from natural sources showing neuroprotective properties have become of interest in brain neurodegeneration. We have purified, characterized, and evaluated the protective potential of one extract enriched in polyphenols obtained from Aristotelia chilensis (MQ), a Chilean berry fruit, in neuronal models of AD induced by soluble oligomers of Aβ1-40. For example, using primary hippocampal cultures from rats (E18), we observed neuroprotection when the neurons were co-incubated with Aβ (0.5 μM) plus MQ for 24 h (Aβ = 23 ± 2%; Aβ + MQ = 3 ± 1%; n = 3). In parallel, co-incubation of Aβ with MQ recovered the frequency of Ca2+ transient oscillations when compared to neurons treated with Aβ alone (Aβ = 72 ± 3%; Aβ + MQ = 86 ± 2%; n = 5), correlating with the changes observed in spontaneous synaptic activity. Additionally, MAP-2 immunostaining showed a preservation of the dendritic tree, suggesting that the toxic effect of Aβ is prevented in the presence of MQ. A new complex mechanism is proposed by which MQ induces neuroprotective effects including antioxidant properties, modulation of cell survival pathways, and/or direct interaction with the Aβ aggregates. Our results suggest that MQ induces changes in the aggregation kinetics of Aβ producing variations in the nucleation phase (Aβ: k1 = 2.7 ± 0.4 × 10-3 s-1 MQ: k1 = 8.3 ± 0.6 × 10-3 s-1) and altering Thioflavin T insertion in β-sheets. In conclusion, MQ induces a potent neuroprotection by direct interaction with the Aβ aggregates, generating far less toxic species and in this way protecting the neuronal network.

Inhibition of the ethanol-induced potentiation of α1 glycine receptor by a small peptide that interferes with Gβγ binding.

Background: Gβγ interaction with GlyR is an important determinant in ethanol potentiation of this channel. Results: A small peptide, RQHC7, can inhibit ethanol potentiation of GlyR currents. Conclusion: Results with RQHC7 indicate that ethanol mediated potentiation of GlyR is in part by Gβγ activation. Significance: Molecular interaction between Gβγ and GlyR could be used as a target for pharmacological modification of ethanol effects. Previous studies indicate that ethanol can modulate glycine receptors (GlyR), in part, through Gβγ interaction with basic residues in the intracellular loop. In this study, we show that a seven-amino acid peptide (RQHC7), which has the primary structure of a motif in the large intracellular loop of GlyR (GlyR-IL), was able to inhibit the ethanol-elicited potentiation of this channel from 47 ± 2 to 16 ± 4%, without interfering with the effect of Gβγ on GIRK (G protein activated inwardly rectifying potassium channel) activation. RQHC7 displayed a concentration-dependent effect on ethanol action in evoked and synaptic currents. A fragment of GlyR-IL without the basic amino acids did not interact with Gβγ or inhibit ethanol potentiation of GlyR. In silico analysis using docking and molecular dynamics allowed to identify a region of ∼350Å2 involving aspartic acids 186, 228, and 246 in Gβγ where we propose that RQHC7 binds and exerts its blocking action on the effect of ethanol in GlyR.

Functional modulation of glycine receptors by the alkaloid gelsemine

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 neurotransmitter‐gated ion channels of the CNS. However, it is currently unknown whether gelsemine can directly modulate the function of glycine receptors.

Copper-uptake is critical for the down regulation of synapsin and dynamin induced by neocuproine: Modulation of synaptic activity in hippocampal neurons

Extracellular and intracellular copper and zinc regulate synaptic activity and plasticity, which may impact brain functionality and human behavior. We have found that a metal coordinating molecule, Neocuproine, transiently increases free intracellular copper and zinc levels (i.e., min) in hippocampal neurons as monitored by Phen Green and FluoZin-3 fluorescence, respectively. The changes in free intracellular zinc induced by Neocuproine were abolished by the presence of a non-permeant copper chelator, Bathocuproine (BC), indicating that copper influx is needed for the action of Neocuproine on intracellular Zn levels. Moreover, Neocuproine decreased the mRNA levels of Synapsin and Dynamin, and did not affect the expression of Bassoon, tubulin or superoxide dismutase (SOD). Western blot analysis showed that protein levels of synapsin and dynamin were also down regulated in the presence of Neocuproine and that these changes were accompanied by a decrease in calcium transients and neuronal activity. Furthermore, Neocuproine decreased the number of active neurons, effect that was blocked by the presence of BC, indicating that copper influx is needed for the action of Neocuproine. We finally show that Neocuproine blocks the epileptiform-like activity induced by bicuculline in hippocampal neurons. Collectively, our data indicates that presynaptic protein configuration and function of primary hippocampal neurons is sensitive to transient changes in transition metal homeostasis. Therefore, small molecules able to coordinate transition metals and penetrate the blood-brain barrier might modify neurotransmission at the Central Nervous System (CNS). This might be useful to establish therapeutic approaches to control the neuronal hyperexcitabiltity observed in brain conditions that are associated to copper dyshomeotasis such as Alzheimer’s and Menkes diseases. Our work also opens a new avenue to find novel and effective antiepilepsy drugs based in metal coordinating molecules.

Modulation of Neuronal Nicotinic Receptor by Quinolizidine Alkaloids Causes Neuroprotection on a Cellular Alzheimer Model

Alzheimers disease (AD) is a progressive and neurodegenerative disorder and one of the current therapies involves strengthening the cholinergic tone in central synapses. Neuroprotective properties for nicotine have been described in AD, through its actions on nicotinic receptors and the further activation of the PI3K/Akt/Bcl-2 survival pathway. We have tested a quinolizidine alkaloid extract (TM0112) obtained from Teline monspessulanna (L.) K. Koch seeds to evaluate its action on nicotinic acetylcholine receptor (nAChR) in a neuronal AD model. Our data show that PC-12 cells pretreated with amyloid-β (Aβ) peptide for 24 h in presence of TM0112 modified Aβ-reduction on cellular viability (Aβ = 80 ± 3%; +TM0112 = 113 ± 4%, n = 15). In addition, this effect was blocked with atropine, MLA, and α-BTX (+TM0112+atropine = 87 ± 4%; +TM0112+MLA = 86 ± 4%; +TM0112+α-BTX = 92 ± 3%). Furthermore, similar protective effects were observed in rat cortical neurons (Aβ = 63 ± 6%; +TM0112 = 114 ± 8%), but not in HEK293T cells (Aβ = 61.4 ± 6.1%; +TM0112 = 62.8 ± 5.2) that do not express α7 nAChR. Moreover, the frequency of synaptic activity in the neuronal network (Aβ = 51.6 ± 16.9%; +TM0112 = 210.8 ± 47.9%, n > 10), as well as the intracellular Ca2+ transients were recovered by TM0112 (Aβ = 61.4 ± 6.9%; +TM0112 = 112.0 ± 5.7%; n = 3) in rat hippocampal neurons. TM0112 increased P-Akt, up to 250% with respect to control, and elevated Bcl-2/Bax percentage (Aβ = 61.0 ± 8.2%; +TM0112 = 105.4 ± 19.5%, n = 4), suggesting a coupling between nAChR activation and an intracellular neuroprotective pathway. Our results suggest that TM0112 could be a new potential source for anti-AD drugs.

Ibuprofen inhibits the synaptic failure induced by the amyloid-β peptide in hippocampal neurons.

Epidemiological studies have reported a decrease in the prevalence of Alzheimers disease in individuals who chronically use non-steroidal anti-inflammatory drugs (NSAIDs). Clinical trials, on the other hand, have been less positive. Nevertheless, it has been proposed that NSAIDs exert part of their effects by reducing long-term cerebral neuroinflammation, although this mechanism has not been proven. In this study, we report that ibuprofen, one of the more widely used non-steroidal anti-inflammatory drugs, was able to alter the ultrastructure of amyloid-β peptide (Aβ) and significantly decrease its association to neuronal membranes, and consequently, its synaptotoxic effect in rat primary hippocampal and cortical cultures at 24 h incubation. In agreement with these results, we found that the decrease in the frequency of calcium transients with Aβ was partly recovered by addition of ibuprofen (8.0 × 10-2 Hz in control; 3.4 × 10-2 Hz in 5 μM Aβ, and 5.9 × 10-2 Hz in the presence of Aβ and 200 μM ibuprofen). Additionally, this effect correlated well with the increment and recovery of miniature spontaneous currents (47 ± 5% of control in 1 μM Aβ alone and 104 ± 14% in the presence of Aβ and ibuprofen). Our results suggest that ibuprofen could be exerting its neuroprotective effect by directly interacting with Aβ and altering its toxic aggregated forms. We postulate that other ibuprofen analogs with better pharmacological properties might have a higher efficacy in AD.

Modulation of the neuronal network activity by P2X receptors and their involvement in neurological disorders.

ATP is a key energetic molecule, fundamental to cell function, which also has an important role in the extracellular milieu as a signaling molecule, acting as a chemoattractant for immune cells and as a neuro- and gliotransmitter. The ionotropic P2X receptors are members of an ATP-gated ion channels family. These ionotropic receptors are widely expressed through the body, with 7 subunits described in mammals, which are arranged in a trimeric configuration with a central pore permeable mainly to Ca(2+) and Na(+). All 7 subunits are expressed in different brain areas, being present in neurons and glia. ATP, through these ionotropic receptors, can act as a neuromodulator, facilitating the Ca(2+)-dependent release of neurotransmitters, inducing the cross-inhibition between P2XR and GABA receptors, and exercising by this way a modulation of synaptic plasticity. Growing evidence shows that P2XR play an important role in neuronal disorders and neurodegenerative diseases, like Parkinsons and Alzheimers disease; this role involves changes on P2XR expression levels, activation of key pathways like GSK3β, APP processing, oxidative stress and inflammatory response. This review is focused on the neuromodulatory function of P2XR on pathophysiological conditions of the brain; the recent evidence could open a window to a new therapeutic target.