N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X3 receptor (Asn139, Asn170, Asn194 and Asn290) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn170 is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr172 in the same glycosylation consensus. Asn194 and Asn290 are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn170 mutation or the Asn139/Asn290 double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X3 mutants where residue Asn170 is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X3 recepto

Does α-Amino-β-methylaminopropionic Acid (BMAA) Play a Role in Neurodegeneration?

The association of α-amino-β-methylaminopropionic acid (BMAA) with elevated incidence of amyotrophic lateral sclerosis/Parkinson’s disease complex (ALS/PDC) was first identified on the island of Guam. BMAA has been shown to be produced across the cyanobacterial order and its detection has been reported in a variety of aquatic and terrestrial environments worldwide, suggesting that it is ubiquitous. Various in vivo studies on rats, mice, chicks and monkeys have shown that it can cause neurodegenerative symptoms such as ataxia and convulsions. Zebrafish research has also shown disruption to neural development after BMAA exposure. In vitro studies on mice, rats and leeches have shown that BMAA acts predominantly on motor neurons. Observed increases in the generation of reactive oxygen species (ROS) and Ca2+ influx, coupled with disruption to mitochondrial activity and general neuronal death, indicate that the main mode of activity is via excitotoxic mechanisms. The current review pertaining to the neurotoxicity of BMAA clearly demonstrates its ability to adversely affect neural tissues, and implicates it as a potentially significant compound in the aetiology of neurodegenerative disease. When considering the potential adverse health effects upon exposure to this compound, further research to better understand the modes of toxicity of BMAA and the environmental exposure limits is essential

Meccanismi di vulnerabilità dei neuroni dopaminergici mesencefalici di ratto esposti a fattori neurotossici ambientali

E’ sempre più accettata l’ipotesi secondo cui le malattie neurodegenerative come il morbo di Parkinson siano di origine multifattoriale (“multiple hit hypothesis”), cioè siano causate dalla concomitante o ripetitiva presenza di diversi fattori che cooperano alla morte cellulare. Tra questi quelli ambientali occupano un posto rilevante. Sebbene una grande varietà di processi neurologici potrebbero essere influenzati da neurotossine ambientali, il sistema dopaminergico sembra essere quello più colpito. Negli ultimi 10 anni si è rivolta sempre maggiore attenzione alla L-BMAA (L-β-N-methylamino-L-alanine), un aminoacido non proteico trovato nei semi della Cycas micronesica, che sembra essere alla base dell’ “ALS-PDC complex”, una sindrome complessa caratterizzata da sintomatologie cliniche tipiche della sclerosi laterale amiotrofica (SLA), del morbo di Parkinson e dell’Alzheimer. Recenti scoperte hanno dimostrato che questa tossina viene prodotta da una grande varietà di cianobatteri del genere Nostoc presenti in tutto il mondo. Pertanto, potenzialmente, tutta la popolazione umana potrebbe essere esposta a tale sostanza. Ciò potrebbe determinare il suo accumulo nell’organismo, sia in forma libera che legata alle proteine, da cui verrebbe poi rilasciata lentamente durante il catabolismo proteico. Sulla base di queste considerazioni abbiamo voluto analizzare gli effetti di questo aminoacido sulle cellule dopaminergiche della SNc da un punto di vista elettrofisiologico, farmacologico, morfologico e tossicologico. La BMAA (3 mM, 10 psi 1.0 s) causa una depolarizzazione dei neuroni dopaminergici della SNc inducendo una corrente entrante (media = 454.48 ± 34.65, n = 73) e aumenti transienti della concentrazione di calcio intracellulare (R medio = 0.368 ± 0.062, n = 13). Questi effetti sono mediati prevalentemente dall’attivazione dei recettori metabotropici del glutammato di gruppo I (mGluR1), in quanto vengono ridotti reversibilmente dall’antagonista selettivo, CPCCOEt (100 μM) (corrente: 41.56 ± 3.61 % del controllo, n = 24; calcio: 28.43 ± 5.96 % del controllo, n = 7). La corrente, ma non il calcio, indotta dalla BMAA è ridotta in piccola parte dal CNQX (10 μM) (corrente: 93,09 ± 1,97 % del controllo, n = 24; calcio: 100.17 ± 9.93 % del controllo, n=6), antagonista competitivo dei recettori AMPA. Nelle cellule dopaminergiche della SNc gli aumenti di calcio indotti dall’attivazione dei recettori mGluR1 sono mediati dai canali SOCs/TRPC. Infatti, sia le correnti entranti che le variazioni di calcio intracellulare indotte dalla BMAA sono ridotte dagli antagonisti di tali canali, SKF 96365 (100 μM) (corrente: 42.125 ± 4.35 % del controllo, n = 8; calcio: 43.57 ± 7.9 % del controllo, n = 7) e Ruthenium Red (20 μM) (corrente: 27.05 ± 8.3 % del controllo, n = 6). Inoltre, nonostante la BMAA in presenza di carbonato presenti una struttura chimica simile a quella dell’acido glutammico, essa non viene ricaptata dalla cellule dopaminergiche attraverso il trasportatore degli aminoacidi eccitatori EAAT. E’ interessante notare che negli interneuroni GABAergici della SNc la BMAA attiva i recettori AMPA, ma non quelli metabotropici, senza indurre variazioni della concentrazione del calcio intracellulare. In queste cellule, tuttavia, l’agonista selettivo degli mGluR1, il DHPG (30 μM), evoca correnti entranti, a dimostrazione della presenza degli mGluR1 sugli interneuroni GABAergici. A conferma della sua potenziale tossicità, esposizioni prolungate di fettine mesencefaliche (12, 20 e 30 minuti) alla BMAA inducono cambiamenti irreversibili su numerose proprietà cellulari. Tali modificazioni sono accompagnate dal rilascio massivo del citocromo C (Cyt C) nel citoplasma delle cellule dopaminergiche che viene completamente bloccato dall’aggiunta, nel mezzo di perfusione, di antagonisti dei recettori mGluR1 e AMPA. I dati che ho riportato in questa tesi forniscono una chiara e plausibile dimostrazione di come la BMAA può essere tossica verso le cellule dopaminergiche della SNc, causando i sintomi neurologici della malattia di Parkinson.It is well known that several neurodegenerative diseases, such as Parkinson disease, have a multifactorial origin (multiple hit hypothesis), which suggests that neuronal loss is a result of multiple factors. Among them, environmental factors are the most important. Although a variety of neurological processes can be adversely affected, the dopaminergic system appears to be a major target for environmental neurotoxins. The hypothesis that L-BMAA (L-β-methylamino-L-alanine), a nonprotein amino acid found in the Cycas micronesica seeds in western pacific islands, is involved in the development of amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS-PDC complex) has risen and fallen since its initial proposal in 1987. In the last ten years the interest for this toxin has grown due to the discovery that it can be produced by many strains of Nostoc cyanobacterias, present throughout the world. Moreover L-BMAA can bind proteins. This bound form may function as an endogenous neurotoxic reservoir, accumulating and being released during protein catabolism. In order to analyze the effects of this amino acid, we have performed electrophysiological, pharmacological, morphological and toxicological studies on dopaminergic neurons of SNc. In these neurons puff-application of L-BMAA (3 mM, 10 psi 1.0 s) causes an inward current (mean = 454.48  34.65, n = 73) and a transient increase of intracellular calcium (R mean = 0.368 ± 0.062, n = 13). These effects are mediated by the activation of group I metabotropic glutamate receptors (mGluR1) and they are reversibly blocked by the application of the antagonist CPCCOEt (100 μM) (current: 41.56 ± 3.61 % of control, n = 24; calcium: 28.43 ± 5.96 % of control, n = 7). Bath application of CNQX (10 μM), a competitive antagonist of AMPA receptors, partially inhibits the L-BMAA-induced current (current: 93,09 ± 1,97 % of control, n = 24) but it has no effect on the calcium concentration (100.17 ± 9.93 % of control, n = 6). SOCs/TRPC channels are present in the dopaminergic cells of SNc and they mediate the intracellular calcium increase due to the activation of mGluR1. Indeed SKF 96365 (100 μM) and Ruthenium Red (20 μM), two antagonists of TRPC channels, are able to reduce the L-BMAA-induced inward current (42.125 ± 4.35 % of control, n = 8 and 27.05 ± 8.3 % of control, n = 6 respectively). Moreover SKF 96365 (100 μM) reduces the intracellular calcium increase induced by L-BMAA (43.57 ± 7.9 % of control). It is known that L-BMAA, in the presence of carbonate, has a chemical structure similar to glutamic acid, however it is not re-uptaken by EAATs, the excitatory amino acid transporters. Interestingly, in GABAergic interneurons, L-BMAA activates AMPA receptors but not mGluR1, and this activation causes inward current without any change in intracellular calcium concentration. However mGluR1 are present in these neurons because application of DHPG (30 μM), the selective agonist, produces inward currents. In order to confirm the toxic effects of this amino acid we have treated midbrain slices with L-BMAA for 12, 20 and 30 minutes and we have seen irreversible modification of cellular properties (decrease in membrane resistance, inability to evoke firing, elevated intracellular calcium). As a consequence of the treatments, cytocrome C is released in the cytoplasm, but in the presence of AMPA and mGluR1 antagonists, this effect is blocked. In conclusion this study demonstrates that L-BMAA could be considered a possible toxic agent for the dopaminergic neurons and provides new insights into the role of this amino acid in the aetiology of Parkinson disease

Substantia nigra control of basal ganglia nuclei

Abstract. The substantia nigra, located in the ventral mesencephalon, is one of the five nuclei that constitute the basal ganglia circuit, which controls voluntary movements. It is divided into the pars compacta and the pars reticulata, which mainly contain dopaminergic and GABAergic cells respectively. Here we overview the electrophysiological properties of these substantia nigra neurons in the pars compacta and reticulata, together with their synaptic connections, and discuss the functional effects of dopaminergic and GABAergic inputs within the basal ganglia. We also examine the phenomenon that when a deficiency of dopamine (DA) occurs (e.g. in Parkinson’s disease), there is an aberrant synaptic plasticity in the basal ganglia. Moreover, we point out that the appearance of an altered pattern of neuronal firing (beta-oscillations) and synchrony among neurons in the subthalamic nucleus, the internal globus pallidus, and the substantia nigra pars reticulata has been related to motor symptoms and possibly, persistent degeneration of DA-containing neurons. Finally, we believe that, based on pathophysiological data, new and significant targets for therapeutic intervention can be identified and tested

The midbrain slice preparation. An in vitro model to select potential anti-parkinsonian drugs?

Abstract. Most anti-parkinsonian drugs produce a dopamine-like effect on single dopaminergic neurons. Using electrophysiological recordings, this effect is observed as an inhibition of the spontaneous firing activity. The firing activity of dopaminergic cells throughout dopamine-mediated mechanisms is inhibited not only by dopamine direct agonists but also by substances that increase the release or synthesis, or block the uptake or reduce degradation of dopamine in the brain. Here we propose the electrophysiological approach on dopaminergic neurons in in vitro slice preparation as a preliminary tool for selecting new potential anti-parkinsonian agents, before they are tested in more complex animal models

Memantine inhibits ATP-dependent K+ conductances in dopamine neurons of the rat substantia nigra pars compacta

1-Amino-3,5-dimethyl-adamantane (memantine) is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist used in clinical practice to treat neurodegenerative disorders that could be associated with excitotoxic cell death. Because memantine reduces the loss of dopamine neurons of the substantia nigra pars compacta (SNc) in animal models of Parkinson’s disease, we examined the effects of this drug on dopamine cells of the SNc. Besides inhibition of NMDA receptor-mediated currents, memantine (30 and 100 M) increased the spontaneous firing rate of whole-cell recorded dopamine neurons in a midbrain slice preparation. Occasionally, a bursting activity was observed. These effects were independent from the block of NMDA receptors and were prevented in neurons dialyzed with a high concentration of ATP (10 mM). An increase in firing rate was also induced by the ATP-sensitive potassium (KATP) channel antagonist tolbutamide (300 M), and this increase occluded further effects of memantine. In addition, KATP channel-mediated outward currents, induced by hypoxia, were inhibited by memantine (30 and 100 M) in the presence of the NMDA receptor antagonist (5S,10R)-()-5-methyl-10,11-dihydro- 5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (10 M). An increase in the spontaneous firing rate by memantine was observed in dopamine neurons recorded with extracellular planar 8 8 multielectrodes in conditions of hypoglycemia. These results highlight KATP channels as possible relevant targets of memantine effects in the brain. Moreover, in view of a proposed role of KATP conductances in dopamine neuron degeneration, they suggest another mechanism of action underlying the protective role of memantine in Parkinson’s disease

Metabotropic glutamate receptor 1 mediates the electrophysiological and toxic actions of the cycad derivative beta-N-Methylamino-L-alanine on substantia nigra pars compacta DAergic neurons

Amyotrophic lateral sclerosis–Parkinson dementia complex (ALS-PDC) is a neurodegenerative disease with ALS, parkinsonism, and Alzheimer’s symptoms that is prevalent in theGuampopulation.-N-Methylamino alanine(BMAA)has been proposed as the toxic agent damaging several neuronal types in ALS-PDC, including substantia nigra pars compacta dopaminergic (SNpc DAergic) neurons. BMAA is a mixed glutamate receptor agonist, but the specific pathways activated in DAergic neurons are not yet known. We combined electrophysiology, microfluorometry, and confocal microscopy analysis to monitor membrane potential/current, cytosolic calcium concentration ([Ca2]i ) changes, cytochrome-c (cyt-c) immunoreactivity, and reactive oxygen species (ROS) production induced by BMAA. Rapid toxin applications caused reversible membrane depolarization/inward current and increase of firing rate and [Ca2]i in DAergic neurons. The inward current (IBMAA) was mainly mediated by activation of metabotropic glutamate receptor 1 (mGluR1), coupled to transient receptor potential (TRP) channels, and to a lesser extent,AMPAreceptors. Indeed, mGluR1 (CPCCOEt) and TRP channels (SKF 96365; Ruthenium Red) antagonists reduced IBMAA, and a small component of IBMAA was reduced by the AMPA receptor antagonist CNQX. Calcium accumulation was mediated by mGluR1 but not by AMPA receptors. Application of a low concentration of NMDA potentiated the BMAA-mediated calcium increase. Prolonged exposure to BMAA caused significant modifications of membrane properties, calcium overload, cell shrinkage, massive cyt-c release into the cytosol and ROS production. In SNpc GABAergic neurons, BMAA activated only AMPA receptors. Our study identifies the mGluR1-activated mechanism induced by BMAA that may cause the neuronal degeneration and parkinsonian symptoms seen in ALS-PDC. Moreover, environmental exposure toBMAAmight possibly also contribute to idiopathic PD