PETISCO is a novel protein complex required for 21U RNA biogenesis and embryonic viability.

Piwi proteins are important for germ cell development in most animals. These proteins are guided to specific targets by small guide RNAs, referred to as piRNAs or 21U RNAs in Caenorhabditis elegans In this organism, even though genetic screens have uncovered 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1 (piRNA-induced silencing-defective 1), we here define a novel protein complex, PETISCO (PID-3, ERH-2, TOFU-6, and IFE-3 small RNA complex), that is required for 21U RNA biogenesis. PETISCO contains both potential 5' cap and 5' phosphate RNA-binding domains and interacts with capped 21U precursor RNA. We resolved the architecture of PETISCO and revealed a second function for PETISCO in embryonic development. This essential function of PETISCO is mediated not by PID-1 but by the novel protein TOST-1 (twenty-one U pathway antagonist). In contrast, TOST-1 is not essential for 21U RNA biogenesis. Both PID-1 and TOST-1 interact directly with ERH-2 using a conserved sequence motif. Finally, our data suggest a role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes a key complex for 21U RNA processing in C. elegans and strengthens the view that 21U RNA biogenesis is built on an snRNA-related pathway.

Adenosine A2A Receptors Contribute to the Radial Migration of Cortical Projection Neurons through the Regulation of Neuronal Polarization and Axon Formation.

Cortical interneurons born in the subpallium reach the cortex through tangential migration, whereas pyramidal cells reach their final position by radial migration. Purinergic signaling via P2Y1 receptors controls the migration of intermediate precursor cells from the ventricular zone to the subventricular zone. It was also reported that the blockade of A2A receptors (A2AR) controls the tangential migration of somatostatin+ interneurons. Here we found that A2AR control radial migration of cortical projection neurons. In A2AR-knockout (KO) mouse embryos or naïve mouse embryos exposed to an A2AR antagonist, we observed an accumulation of early-born migrating neurons in the lower intermediate zone at late embryogenesis. In utero knockdown of A2AR also caused an accumulation of neurons at the lower intermediate zone before birth. This entails the presently identified ability of A2AR to promote multipolar-bipolar transition and axon formation, critical for the transition of migrating neurons from the intermediate zone to the cortical plate. This effect seems to require extracellular ATP-derived adenosine since a similar accumulation of neurons at the lower intermediate zone was observed in mice lacking ecto-5'-nucleotidase (CD73-KO). These findings frame adenosine as a fine-tune regulator of the wiring of cortical inhibitory and excitatory networks.

PID-1 is a novel factor that operates during 21U-RNA biogenesis in Caenorhabditis elegans.

The Piwi-piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing.

Purinergic signalling and brain development.

ATP and adenosine are released from cells as a function of their metabolic activity, being important cell-to-cell communication signals. Both purines are also released from neurons in an activity-dependent manner, with several established roles to fine tune brain function in adults, as best heralded by the effects of caffeine, an antagonist of adenosine receptors. Purines are also dynamically released from early neurogenesis and different purine receptors are dynamically expressed throughout development. Accordingly, emerging evidence supports multiple roles for purinergic signalling in the control of different processes of brain development, such as embryonic neurogenesis, migration of principal neurons and interneurons, guidance for neuronal connectivity, synaptogenesis and synaptic stability/elimination. Although major efforts are still required to unravel the time and space-related engagement of the different components of the purinergic system, the relevance of purines in brain development is heralded by their association with neurodevelopmental disorders, positing novel opportunities to understand and correct brain wiring.

Symptoms awareness, emergency medical service utilization and hospital transfer delay in myocardial infarction.

BACKGROUND: The length of time between symptom onset and reperfusion therapy in patients with ST-segment elevation acute myocardial infarction (STEMI) is a key determinant of mortality. Information on this delay is scarce, particularly for developing countries. The objective of the study is to prospectively evaluate the individual components of reperfusion time (RT) in patients with STEMI treated at a University Hospital in 2012. METHODS: Medical records were reviewed to determine RT, its main (patient delay time [PDT] and system delay time [SDT]) and secondary components and hospital access variables. Cognitive responses were evaluated using a semi-structured questionnaire. RESULTS: A total of 50 patients with a mean age of 59 years (SD = 10.5) were included, 64% of whom were male. The median RT was 430 min, with an interquartile range of 315-750 min. Regarding the composition of RT in the sample, PDT corresponded to 18.9% and SDT to 81.1%. Emergency medical services were used in 23.5% of cases. Patients treated in intermediate care units showed a significant increase in SDT (p = 0.008). Regarding cognitive variables, PDT was approximately 40 min longer among those who answered "I didn't think it was serious" (p = 0.024). CONCLUSIONS: In a Brazilian tertiary public hospital, RT was higher than that recommended by international guidelines, mainly because of long SDT, which was negatively affected by time spent in intermediate care units. Emergency Medical Services underutilization was noted. A patient's low perception of severity increased PDT.

Adenosine A2b receptors control A1 receptor-mediated inhibition of synaptic transmission in the mouse hippocampus.

Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A(2B)R) are G(s/q)--protein-coupled receptors with low expression in the brain. As A(2B)R function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A(2B)R-knockout (A(2B)R-KO) and C57/BL6 mice were submitted to a behavioural test battery (open field, elevated plus-maze, Y-maze). The A(2B)R agonist BAY60-6583 (300 nM) decreased the paired-pulse stimulation ratio, an effect prevented by the A(2B)R antagonist MRS 1754 (200 nM) and abrogated in A(2B)R-KO mice. Accordingly, A(2B)R immunoreactivity was present in 73 ± 5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60-6583 attenuated the A(1)R control of synaptic transmission, both the A(1)R inhibition caused by 2-chloroadenosine (0.1-1 µM) and the disinhibition caused by the A(1)R antagonist DPCPX (100 nM), both effects prevented by MRS 1754 and abrogated in A(2B)R-KO mice. BAY 60-6583 decreased glutamate release in slices and also attenuated the A(1)R inhibition (CPA 100 nM). A(2B)R-KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus-maze and the Y-maze and no alteration of locomotion, anxiety or working memory. We conclude that A(2B)R are present in hippocampal glutamatergic terminals where they counteract the predominant A(1)R-mediated inhibition of synaptic transmission, impacting on exploratory behaviour.

Adenosine A2B receptor activation stimulates glucose uptake in the mouse forebrain.

ATP consumption during intense neuronal activity leads to peaks of both extracellular adenosine levels and increased glucose uptake in the brain. Here, we investigated the hypothesis that the activation of the low-affinity adenosine receptor, the A2B receptor (A(2B)R), promotes glucose uptake in neurons and astrocytes, thereby linking brain activity with energy metabolism. To this end, we mapped the spatiotemporal accumulation of the fluorescent-labelled deoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in superfused acute hippocampal slices of C57Bl/6j mice. Bath application of the A(2B)R agonist BAY606583 (300 nM) triggered an immediate and stable (>10 min) increase of the velocity of 2-NBDG accumulation throughout hippocampal slices. This was abolished with the pretreatment with the selective A(2B)R antagonist, MRS1754 (200 nM), and was also absent in A(2B)R null-mutant mice. In mouse primary astrocytic or neuronal cultures, BAY606583 similarly increased (3)H-deoxyglucose uptake in the following 20 min incubation period, which was again abolished by a pretreatment with MRS1754. Finally, incubation of hippocampal, frontocortical, or striatal slices of C57Bl/6j mice at 37 °C, with either MRS1754 (200 nM) or adenosine deaminase (3 U/mL) significantly reduced glucose uptake. Furthermore, A(2B)R blockade diminished newly synthesized glycogen content and at least in the striatum, increased lactate release. In conclusion, we report here that A(2B)R activation is associated with an instant and tonic increase of glucose transport into neurons and astrocytes in the mouse brain. These prompt further investigations to evaluate the clinical potential of this novel glucoregulator mechanism.

CRMP2 tethers kainate receptor activity to cytoskeleton dynamics during neuronal maturation.

The CRMP2 and CRMP4 proteins are strongly expressed in the developing nervous system, mediating neurite outgrowth, neuronal polarity, and axon guidance. In the present study, we demonstrate the interaction of the CRMP2 and CRMP4 proteins with the GluK5 subunit of the kainate (KA) receptor (KAR) and investigated the role of KARs in modulating the development of cultured mouse DRG neurons. We found that KARs modulate neuronal maturation and neurite outgrowth in a bidirectional manner. Accordingly, low concentrations of KA delayed maturation and enhanced neurite outgrowth, whereas maturation was promoted by higher concentrations of KA that attenuated neuritic elongation. The effects of weak KAR activation were prevented by blocking their noncanonical signaling and involved a differential regulation of CRMP2. Whereas the delay in maturation involves PKC-mediated phosphorylation of CRMP2 at T555 leading to a downregulation of membrane Cav2.2, the promotion of neurite outgrowth is achieved by dephosphorylation at T514 at the growth cones, the latter reflecting PKC-driven enhancement of GSK3ß phosphorylation at S9. Together, these findings indicate that noncanonical KAR signaling influences neuronal development by modulating CRMP2 activity.

Increased ATP Release and Higher Impact of Adenosine A2A Receptors on Corticostriatal Plasticity in a Rat Model of Presymptomatic Parkinson's Disease.

Extracellular ATP can be a danger signal, but its role in striatal circuits afflicted in Parkinson's disease (PD) is unclear and was now investigated. ATP was particularly released at high stimulation intensities from purified striatal nerve terminals of mice, which were endowed with different ATP-P2 receptors (P2R), although P2R antagonists did not alter corticostriatal transmission or plasticity. Instead, ATP was extracellularly catabolized into adenosine through CD73 to activate adenosine A2A receptors (A2AR) modulating corticostriatal long-term potentiation (LTP) in mice. In the presymptomatic phase of a 6-hydroxydopamine rat model of PD, ATP release from striatal nerve terminals was increased and was responsible for a greater impact of CD73 and A2AR on corticostriatal LTP. These observations identify increased ATP release and ATP-derived formation of extracellular adenosine bolstering A2AR activation as a key pathway responsible for abnormal synaptic plasticity in circuits involved in the onset of PD motor symptoms. The translation of these findings to humans prompts extending the use of A2AR antagonists from only co-adjuvants of motor control in Parkinsonian patients to neuroprotective drugs delaying the onset of motor symptoms.

Extracellular vesicles improve GABAergic transmission in Huntington's disease iPSC-derived neurons.

Background: Extracellular vesicles (EVs) carry bioactive molecules associated with various biological processes, including miRNAs. In both Huntington's disease (HD) models and human samples, altered expression of miRNAs involved in synapse regulation was reported. Recently, the use of EV cargo to reverse phenotypic alterations in disease models with synaptopathy as the end result of the pathophysiological cascade has become an interesting possibility. Methods: Here, we assessed the contribution of EVs to GABAergic synaptic alterations using a human HD model and studied the miRNA content of isolated EVs. Results: After differentiating human induced pluripotent stem cells into electrophysiologically active striatal-like GABAergic neurons, we found that HD-derived neurons displayed reduced density of inhibitory synapse markers and GABA receptor-mediated ionotropic signaling. Treatment with EVs secreted by control (CTR) fibroblasts reversed the deficits in GABAergic synaptic transmission and increased the density of inhibitory synapses in HD-derived neuron cultures, while EVs from HD-derived fibroblasts had the opposite effects on CTR-derived neurons. Moreover, analysis of miRNAs from purified EVs identified a set of differentially expressed miRNAs between manifest HD, premanifest, and CTR lines with predicted synaptic targets. Conclusion: The EV-mediated reversal of the abnormal GABAergic phenotype in HD-derived neurons reinforces the potential role of EV-miRNAs on synapse regulation.

Resistance Training for Older Women: Do Adaptive Responses Support the ACSM and NSCA Position Stands?

PURPOSE: The optimal intensity of resistance training (RT) to improve muscular, physical performance, and metabolic adaptations still needs to be well established for older adults. Based on current position statements, we compared the effects of two different RT loads on muscular strength, functional performance, skeletal muscle mass, hydration status, and metabolic biomarkers in older women. METHODS: One hundred one older women were randomly allocated to perform a 12-wk whole-body RT program (eight exercises, three sets, three nonconsecutive days a week) into two groups: 8-12 repetitions maximum (RM) and 10-15RM. Muscular strength (1RM tests), physical performance (motor tests), skeletal muscle mass (dual-energy X-ray absorptiometry), hydration status (bioelectrical impedance), and metabolic biomarkers (glucose, total cholesterol, HDL-c, HDL-c, triglycerides, and C-reactive protein) were measured at baseline and posttraining. RESULTS: Regarding muscular strength, 8-12RM promoted higher 1RM increases in chest press (+23.2% vs +10.7%, P < 0.01) and preacher curl (+15.7% vs +7.4%, P < 0.01), but not in leg extension (+14.9% vs +12.3%, P > 0.05). Both groups improved functional performance ( P < 0.05) in gait speed (4.6%-5.6%), 30 s chair stand (4.6%-5.9%), and 6 min walking (6.7%-7.0%) tests, with no between-group differences ( P > 0.05). The 10-15RM group elicited superior improves in the hydration status (total body water, intracellular and extracellular water; P < 0.01), and higher gains of skeletal muscle mass (2.5% vs 6.3%, P < 0.01), upper (3.9% vs 9.0%, P < 0.01) and lower limbs lean soft tissue (2.1% vs 5.4%, P < 0.01). Both groups improved their metabolic profile. However, 10-15RM elicited greater glucose reductions (-0.2% vs -4.9%, P < 0.05) and greater HDL-c increases (-0.2% vs +4.7%, P < 0.01), with no between-group differences for the other metabolic biomarkers ( P > 0.05). CONCLUSIONS: Our results suggest that 8-2RM seems more effective than 10-15RM for increasing upper limbs' muscular strength, whereas the adaptative responses for lower limbs and functional performance appear similar in older women. In contrast, 10-15RM seems more effective for skeletal muscle mass gains, and increased intracellular hydration and improvements in metabolic profile may accompany this adaptation.

Restored Fyn Levels in Huntington's Disease Contributes to Enhanced Synaptic GluN2B-Composed NMDA Receptors and CREB Activity.

N-methyl-D-aspartate receptors (NMDARs) are important postsynaptic receptors that contribute to normal synaptic function and cell survival; however, when overactivated, as in Huntington's disease (HD), NMDARs cause excitotoxicity. HD-affected striatal neurons show altered NMDAR currents and augmented ratio of surface to internal GluN2B-containing NMDARs, with augmented accumulation at extrasynaptic sites. Fyn protein is a member of the Src kinase family (SKF) with an important role in NMDARs phosphorylation and synaptic localization and function; recently, we demonstrated that Fyn is reduced in several HD models. Thus, in this study, we aimed to explore the impact of HD-mediated altered Fyn levels at post-synaptic density (PSD), and their role in distorted NMDARs function and localization, and intracellular neuroprotective pathways in YAC128 mouse primary striatal neurons. We show that reduced synaptic Fyn levels and activity in HD mouse striatal neurons is related to decreased phosphorylation of synaptic GluN2B-composed NMDARs; this occurs concomitantly with augmented extrasynaptic NMDARs activity and currents and reduced cAMP response element-binding protein (CREB) activation, along with induction of cell death pathways. Importantly, expression of a constitutive active form of SKF reestablishes NMDARs localization, phosphorylation, and function at PSD in YAC128 mouse neurons. Enhanced SKF levels and activity also promotes CREB activation and reduces caspase-3 activation in YAC128 mouse striatal neurons. This work supports, for the first time, a relevant role for Fyn protein in PSD modulation, controlling NMDARs synaptic function in HD, and favoring neuroprotective pathways and cell survival. In this respect, Fyn Tyr kinase constitutes an important potential HD therapeutic target directly acting at PSD.

Adenosine A2A receptors control synaptic remodeling in the adult brain.

The molecular mechanisms underlying circuit re-wiring in the mature brain remains ill-defined. An eloquent example of adult circuit remodelling is the hippocampal mossy fiber (MF) sprouting found in diseases such as temporal lobe epilepsy. The molecular determinants underlying this retrograde re-wiring remain unclear. This may involve signaling system(s) controlling axon specification/growth during neurodevelopment reactivated during epileptogenesis. Since adenosine A2A receptors (A2AR) control axon formation/outgrowth and synapse stabilization during development, we now examined the contribution of A2AR to MF sprouting. A2AR blockade significantly attenuated status epilepticus(SE)-induced MF sprouting in a rat pilocarpine model. This involves A2AR located in dentate granule cells since their knockdown selectively in dentate granule cells reduced MF sprouting, most likely through the ability of A2AR to induce the formation/outgrowth of abnormal secondary axons found in rat hippocampal neurons. These A2AR should be activated by extracellular ATP-derived adenosine since a similar prevention/attenuation of SE-induced hippocampal MF sprouting was observed in CD73 knockout mice. These findings demonstrate that A2AR contribute to epilepsy-related MF sprouting, most likely through the reactivation of the ability of A2AR to control axon formation/outgrowth observed during neurodevelopment. These results frame the CD73-A2AR axis as a regulator of circuit remodeling in the mature brain.

Enhanced role of adenosine A(2A) receptors in the modulation of LTP in the rat hippocampus upon ageing.

Adenosine neuromodulation depends on a balanced activation of inhibitory A1 (A1R) and facilitatory A(2A) receptors (A(2A) R). Both A1 R and A(2A) R modulate hippocampal glutamate release and NMDA-dependent long-term potentiation (LTP) but ageing affects the density of both A1 R and A(2A) R. We tested the effects of selective A1 R and A(2A) R antagonists in the modulation of synaptic transmission and plasticity in rat hippocampal slices from three age groups (young adults, 2-3 month; middle-aged adults, 6-8 months; aged, 18-20 months). The selective A(2A) R antagonist SCH58261 (50 nm) attenuated LTP in all age groups, with a larger effect in aged (-63 ± 7%) than in middle-aged adults (-36 ± 9%) or young adult rats (-36 ± 9%). In contrast, the selective A1 R antagonist DPCPX (50 nm) increased LTP magnitude in young adult rats (+42 ± 6%), but failed to affect LTP magnitude in the other age groups. Finally, in the continuous presence of DPCPX, SCH58261 caused a significantly larger inhibition of LTP amplitude in aged (-71 ± 45%) than middle-aged (-28 ±9%) or young rats (-11 ± 2%). Accordingly, aged rats displayed an increased expression of A(2A) R mRNA in the hippocampus and a higher number of glutamatergic nerve terminals equipped with A(2A) R in aged (67 ± 6%) compared with middle-aged (34 ± 7%) and young rats (25 ± 5%). The results show an enhanced A(2A) R-mediated modulation of LTP in aged rats, in accordance with the age-associated increased expression and density of A(2A) R in glutamatergic terminals. This age-associated gain of function of A(2A) R modulating synaptic plasticity may underlie the ability of A(2A) R antagonists to prevent memory dysfunction in aged animals.

Convergence of adenosine and GABA signaling for synapse stabilization during development.

During development, neural circuit formation requires the stabilization of active γ-aminobutyric acid­mediated (GABAergic) synapses and the elimination of inactive ones. Here, we demonstrate that, although the activation of postsynaptic GABA type A receptors (GABAARs) and adenosine A2A receptors (A2ARs) stabilizes GABAergic synapses, only A2AR activation is sufficient. Both GABAAR- and A2AR-dependent signaling pathways act synergistically to produce adenosine 3',5'-monophosphate through the recruitment of the calcium­calmodulin­adenylyl cyclase pathway. Protein kinase A, thus activated, phosphorylates gephyrin on serine residue 303, which is required for GABAAR stabilization. Finally, the stabilization of pre- and postsynaptic GABAergic elements involves the interaction between gephyrin and the synaptogenic membrane protein Slitrk3. We propose that A2ARs act as detectors of active GABAergic synapses releasing GABA, adenosine triphosphate, and adenosine to regulate their fate toward stabilization or elimination.

A tudor domain protein, SIMR-1, promotes siRNA production at piRNA-targeted mRNAs in C. elegans.

piRNAs play a critical role in the regulation of transposons and other germline genes. In Caenorhabditis elegans, regulation of piRNA target genes is mediated by the mutator complex, which synthesizes high levels of siRNAs through the activity of an RNA-dependent RNA polymerase. However, the steps between mRNA recognition by the piRNA pathway and siRNA amplification by the mutator complex are unknown. Here, we identify the Tudor domain protein, SIMR-1, as acting downstream of piRNA production and upstream of mutator complex-dependent siRNA biogenesis. Interestingly, SIMR-1 also localizes to distinct subcellular foci adjacent to P granules and Mutator foci, two phase-separated condensates that are the sites of piRNA-dependent mRNA recognition and mutator complex-dependent siRNA amplification, respectively. Thus, our data suggests a role for multiple perinuclear condensates in organizing the piRNA pathway and promoting mRNA regulation by the mutator complex.

In the biological world, a process known as RNA interference helps cells to switch genes on and off and to defend themselves against harmful genetic material. This mechanism works by deactivating RNA sequences, the molecular templates cells can use to create proteins. Overall, RNA interference relies on the cell creating small RNA molecules that can target and inhibit the harmful RNA sequences that need to be silenced. More precisely, in round worms such as Caenorhabditis elegans, RNA interference happens in two steps. First, primary small RNAs identify the target sequences, which are then combatted by newly synthetised, secondary small RNAs. A number of proteins are also involved in both steps of the process. RNA interference is particularly important to preserve fertility, guarding sex cells against 'rogue' segments of genetic information that could be passed on to the next generation. In future sex cells, the proteins involved in RNA interference cluster together, forming a structure called a germ granule. Yet, little is known about the roles and identity of these proteins. To fill this knowledge gap, Manage et al. focused on the second stage of the RNA interference pathway in the germ granules of C. elegans, examining the molecules that physically interact with a key protein. This work revealed a new protein called SIMR-1. Looking into the role of SIMR-1 showed that the protein is required to amplify secondary small RNAs, but not to identify target sequences. However, it only promotes the creation of secondary small RNAs if a specific subtype of primary small RNAs have recognized the target RNAs for silencing. Further experiments also showed that within the germ granule, SIMR-1 is present in a separate substructure different from any compartment previously identified. This suggests that each substep of the RNA interference process takes place at a different location in the granule. In both C. elegans and humans, disruptions in the RNA interference pathway can lead to conditions such as cancer or infertility. Dissecting the roles of the proteins involved in this process in roundworms may help to better grasp how this process unfolds in mammals, and how it could be corrected in the case of disease.

Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation.

Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

Presynaptic control of striatal glutamatergic neurotransmission by adenosine A1-A2A receptor heteromers.

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R-A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R-A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R-A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R-A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

Involvement of cannabinoid receptors in the regulation of neurotransmitter release in the rodent striatum: a combined immunochemical and pharmacological analysis.

Despite the profound effect of cannabinoids on motor function, and their therapeutic potential in Parkinson's and Huntington's diseases, the cellular and subcellular distributions of striatal CB1 receptors are not well defined. Here, we show that CB1 receptors are primarily located on GABAergic (vesicular GABA transporter-positive) and glutamatergic [vesicular glutamate transporter-1 (VGLUT-1)- and VGLUT-2-positive] striatal nerve terminals and are present in the presynaptic active zone, in the postsynaptic density, as well as in the extrasynaptic membrane. Both the nonselective agonist WIN552122 [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate salt] (EC50, 32 nM) and the CB1-selective agonist ACEA [N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide] inhibited [3H]GABA release from rat striatal slices. The effect of these agonists was prevented by the CB1-selective antagonists SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (1 microM) and AM251 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide trifluoroacetate salt] (1 microM), indicating that cannabinoids inhibit the release of GABA via activation of presynaptic CB1 receptors. Cannabinoids modulated glutamate release via both CB1 and non-CB1 mechanisms. Cannabinoid agonists and antagonists inhibited 25 mM K+-evoked [3H]glutamate release and sodium-dependent [3H]glutamate uptake. Partial involvement of CB1 receptors is suggested because low concentrations of SR141716A partly and AM251 fully prevented the effect of WIN552122 and CP55940 [5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol]. However, the effect of CB1 agonists and antagonists persisted in CB1 knock-out mice, indicating the involvement of non-CB1,CB1-like receptors. In contrast, cannabinoids did not modulate [3H]dopamine release or [3H]dopamine and [3H]GABA uptake. Our results indicate distinct modulation of striatal GABAergic and glutamatergic transmission by cannabinoids and will facilitate the understanding of the role and importance of the cannabinoid system in normal and pathological motor function.

Dual presynaptic control by ATP of glutamate release via facilitatory P2X1, P2X2/3, and P2X3 and inhibitory P2Y1, P2Y2, and/or P2Y4 receptors in the rat hippocampus.

ATP is released in a vesicular manner from nerve terminals mainly at higher stimulation frequencies. There is a robust expression of ATP (P2) receptors in the brain, but their role is primarily unknown. We report that ATP analogs biphasically modulate the evoked release of glutamate from purified nerve terminals of the rat hippocampus, the facilitation being mediated by P2X1, P2X2/3, and P2X3 [antagonized by 8-(benzamido)naphthalene-1,3,5-trisulfonate and 2',3'-O-(2,4,6-trinitrophenyl)-ATP] and the inhibition by P2Y1, P2Y2, and/or P2Y4 [antagonized by reactive blue 2 and 2'deoxy-N6-methyladenosine-3',5'-bisphosphate and mimicked by P1-(urinine 5'-),P4-(inosine 5'-) tetraphosphate and 2-methylthio-ADP] receptors. The combination of single-cell PCR analysis of rat hippocampal pyramidal neurons, Western blot analysis of purified presynaptic active zone fraction, and immunocytochemical analysis of hippocampal glutamatergic terminals revealed that the P2 receptors expressed in glutamatergic neurons, located in the active zone and in glutamatergic terminals, were precisely P2X1, P2X2, and P2X3 subunits and P2Y1, P2Y2, and P2Y4 receptors. This provides coincident functional and molecular evidence that P2 receptors are present and act presynaptically as a modulatory system controlling hippocampal glutamate release.