Sandra H. Vaz

A1R–A2AR heteromers coupled to Gs and Gi/0 proteins modulate GABA transport into astrocytes

Astrocytes play a key role in modulating synaptic transmission by controlling extracellular gamma-aminobutyric acid (GABA) levels via GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that a further level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A1 (A1R) and A2A (A2AR) receptors. This regulation occurs through A1R–A2AR heteromers that signal via two different G proteins, Gs and Gi/0, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how GPCR heteromers signal. Furthermore, we uncover a previously unknown mechanism where adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron–glia–neuron) synapse.

Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1

Neurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G‐protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT‐1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2‐(2‐furanyl)‐7‐(2‐phenylethyl)‐7H‐pyrazolo[4,3‐e][1,2,4]triazolo[1,5‐c]pyrimidin‐5‐amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4‐[2‐[[6‐amino‐9‐(N‐ethyl‐β‐d‐ribofuranuronamidosyl)‐9H‐purin‐yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3–100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of KM. This was mimicked by adenylate cyclase activation (forskolin, 10 μM) and prevented by protein kinase A (PKA) inhibition (N‐[2‐(p‐bromocinnamylamino) ethyl]‐5‐isoquinolinesulfonamide dihydrochloride, 1 μM), which per se did not influence GABA transport. Blockade of protein kinase C (PKC) (2‐[1‐(3‐dimethylaminopropyl)indol‐3‐yl]‐3‐(indol‐3‐yl) maleimide, 1 μM) facilitated GABA transport whereas PKC activation (4‐β‐phorbol‐didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT‐1 mediated GABA transport into nerve endings by restraining tonic PKC‐mediated inhibition.

Brain-derived neurotrophic factor (BDNF) enhances GABA transport by modulating the trafficking of GABA transporter-1 (GAT-1) from the plasma membrane of rat cortical astrocytes

Background: Transport of GABA into astrocytes is crucial for excitability control. Results: The neurotrophin BDNF, through TrkB-t receptor activation, enhances GABA transport into astrocytes, which requires adenosine A2A receptor signaling. Conclusion: BDNF plays an active role in the synaptic clearance of GABA. Significance: This new regulatory role for TrkB-t receptors discloses their relevance for excitability control at the tripartite synapse. The γ-aminobutyric acid (GABA) transporters (GATs) are located in the plasma membrane of neurons and astrocytes and are responsible for termination of GABAergic transmission. It has previously been shown that brain derived neurotrophic factor (BDNF) modulates GAT-1-mediated GABA transport in nerve terminals and neuronal cultures. We now report that BDNF enhances GAT-1-mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in the Vmax kinetic constant. This action involves the truncated form of the TrkB receptor (TrkB-t) coupled to a non-classic PLC-γ/PKC-δ and ERK/MAPK pathway and requires active adenosine A2A receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope was incorporated into the second extracellular loop. An increase in plasma membrane of HA-rGAT-1 as well as of rGAT-1 was observed when both HA-GAT-1-transduced astrocytes and rGAT-1-overexpressing astrocytes were treated with BDNF. The effect of BDNF results from inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1 rather than from facilitation of the monensin-sensitive recycling of GAT-1 molecules back to the plasma membrane. We therefore conclude that BDNF enhances the time span of GAT-1 molecules at the plasma membrane of astrocytes. BDNF may thus play an active role in the clearance of GABA from synaptic and extrasynaptic sites and in this way influence neuronal excitability.

Glial cell line-derived neurotrophic factor (GDNF) enhances dopamine release from striatal nerve endings in an adenosine A2A receptor-dependent manner

Both glial cell line-derived neurotrophic factor (GDNF) and adenosine influence dopaminergic function in the striatum. We now evaluated the GDNF effect on dopamine release from rat striatal nerve endings and if this effect of GDNF is modulated by adenosine A(2A) receptors. Dopamine release was evoked twice (S(1) and S(2)); GDNF was added before S(2) and drugs used to modify GDNF actions were present during both stimulation periods. The effect of GDNF was taken as the change in the S(2)/S(1) ratio in the absence and in the presence of GDNF in the same experimental conditions. GDNF (3-30 ng/ml) increased dopamine release from K(+) (20 mM, 2 min) stimulated synaptosomes and electrically (2 Hz, 2 min) stimulated striatal slices, an effect dependent upon tonic adenosine A(2A) receptor activation, since it was blocked by the A(2A) receptor antagonist, SCH 58261 (50 nM). Activation of A(2A) receptors with CGS 21680 (10 nM) potentiated the effect of GDNF in synaptosomes. CGS 21680 also potentiated the effect of GDNF in striatal slices, providing that GABAergic transmission was inhibited; if not, the action of GDNF was attenuated by CGS 21680. Blockade of GABAergic transmission per se increased dopamine release, but attenuated the effect of GDNF upon dopamine release in slices. The results suggest that GDNF enhances dopamine release by acting presynaptically at the striatum, an action that requires adenosine A(2A) receptor activity. Furthermore, in striatal slices, the action of GDNF as well as its modulation by adenosine A(2A) receptor activation appears to be also under control of GABAergic transmission.

Brain-derived neurotrophic factor inhibits GABA uptake by the rat hippocampal nerve terminals.

The lifespan of the predominant inhibitory neurotransmitter in the central nervous system, gamma-aminobutyric acid (GABA), is determined by its uptake into neurons and glia, through high-affinity Na(+)/Cl(-) dependent transporters (GATs). We now evaluated how the uptake of GABA by nerve endings, which is mostly mediated by the GAT-1 subtype, is modulated by brain-derived neurotrophic factor (BDNF). BDNF (10-200 ng/ml) decreased GAT-1-mediated GABA uptake by isolated hippocampal rat nerve terminals (synaptosomes), an effect that occurred within 1 min of incubation with BDNF and blocked by the tyrosine kinase inhibitor K252a (100 nM) as well as by the PLC inhibitor, U73122 (3 microM). Maximum inhibition was attained with 100 ng/ml BDNF. In contrast with what has been observed for other synaptic actions of BDNF, the inhibition of GABA transport by BDNF does not require tonic activation of adenosine A(2A) receptors since it was not blocked by the A(2A) receptor antagonist SCH 58261 (50 nM). However, in synaptosomes previously depleted of extracellular endogenous adenosine by incubation with adenosine deaminase (1 U/ml), activation of A(2A) receptors with the A(2A) receptor agonist, CGS 21680 (30 nM), enhanced the inhibitory effect of BDNF upon GABA transport, an action prevented by the A(2A) receptor antagonist, SCH 58261 (50 nM). It is concluded that BDNF, through TrkB and PLCgamma signalling inhibits GAT-1-mediated GABA transport by nerve endings and that this action is not dependent on, but can be enhanced by, TrkB/A(2A) receptor cross talk.

Dopamine-galanin receptor heteromers modulate cholinergic neurotransmission in the rat ventral hippocampus

Previous studies have shown that dopamine and galanin modulate cholinergic transmission in the hippocampus, but little is known about the mechanisms involved and their possible interactions. By using resonance energy transfer techniques in transfected mammalian cells, we demonstrated the existence of heteromers between the dopamine D1-like receptors (D1 and D5) and galanin Gal1, but not Gal2 receptors. Within the D1–Gal1 and D5–Gal1 receptor heteromers, dopamine receptor activation potentiated and dopamine receptor blockade counteracted MAPK activation induced by stimulation of Gal1 receptors, whereas Gal1 receptor activation or blockade did not modify D1-like receptor-mediated MAPK activation. Ability of a D1-like receptor antagonist to block galanin-induced MAPK activation (cross-antagonism) was used as a “biochemical fingerprint” of D1-like–Gal1 receptor heteromers, allowing their identification in the rat ventral hippocampus. The functional role of D1-like–Gal receptor heteromers was demonstrated in synaptosomes from rat ventral hippocampus, where galanin facilitated acetylcholine release, but only with costimulation of D1-like receptors. Electrophysiological experiments in rat ventral hippocampal slices showed that these receptor interactions modulate hippocampal synaptic transmission. Thus, a D1-like receptor agonist that was ineffective when administered alone turned an inhibitory effect of galanin into an excitatory effect, an interaction that required cholinergic neurotransmission. Altogether, our results strongly suggest that D1-like–Gal1 receptor heteromers act as processors that integrate signals of two different neurotransmitters, dopamine and galanin, to modulate hippocampal cholinergic neurotransmission.

P2Y1 receptor inhibits GABA transport through a calcium signalling-dependent mechanism in rat cortical astrocytes

Astrocytes express a variety of purinergic (P2) receptors, involved in astrocytic communication through fast increases in [Ca2+]i. Of these, the metabotropic ATP receptors (P2Y) regulate cytoplasmic Ca2+ levels through the PLC‐PKC pathway. GABA transporters are a substrate for a number of Ca2+‐related kinases, raising the possibility that calcium signalling in astrocytes impact the control of extracellular levels of the major inhibitory transmitter in the brain. To access this possibility we tested the influence of P2Y receptors upon GABA transport into astrocytes. Mature primary cortical astroglial‐enriched cultures expressed functional P2Y receptors, as evaluated through Ca2+ imaging, being P2Y1 the predominant P2Y receptor subtype. ATP (100 μM, for 1 min) caused an inhibition of GABA transport through either GAT‐1 or GAT‐3 transporters, decreasing the Vmax kinetic constant. ATP‐induced inhibition of GATs activity was still evident in the presence of adenosine deaminase, precluding an adenosine‐mediated effect. This, was mimicked by a specific agonist for the P2Y1,12,13 receptor (2‐MeSADP). The effect of 2‐MeSADP on GABA transport was blocked by the P2 (PPADS) and P2Y1 selective (MRS2179) receptor antagonists, as well as by the PLC inhibitor (U73122). 2‐MeSADP failed to inhibit GABA transport in astrocytes where intracellular calcium had been chelated (BAPTA‐AM) or where calcium stores were depleted (α‐cyclopiazonic acid, CPA). In conclusion, P2Y1 receptors in astrocytes inhibit GABA transport through a mechanism dependent of P2Y1‐mediated calcium signalling, suggesting that astrocytic calcium signalling, which occurs as a consequence of neuronal firing, may operate a negative feedback loop to enhance extracellular levels of GABA. GLIA 2014;62:1211–1226

Differential Role of the Proteasome in the Early and Late Phases of BDNF-Induced Facilitation of LTP

The neurotrophin brain-derived neurotrophic factor (BDNF) mediates activity-dependent long-term changes of synaptic strength in the CNS. The effects of BDNF are partly mediated by stimulation of local translation, with consequent alterations in the synaptic proteome. The ubiquitin-proteasome system (UPS) also plays an important role in protein homeostasis at the synapse by regulating synaptic activity. However, whether BDNF acts on the UPS to mediate the effects on long-term synaptic potentiation (LTP) has not been investigated. In the present study, we show similar and nonadditive effects of BDNF and proteasome inhibition on the early phase of synaptic potentiation (E-LTP) induced by theta-burst stimulation of rat hippocampal CA1 synapses. The effects of BDNF were blocked by the proteasome activator IU1, suggesting that the neurotrophin acts by decreasing proteasome activity. Accordingly, BDNF downregulated the proteasome activity in cultured hippocampal neurons and in hippocampal synaptoneurosomes. Furthermore, BDNF increased the activity of the deubiquitinating enzyme UchL1 in synaptoneurosomes and upregulated free ubiquitin. In contrast to the effects on posttetanic potentiation, proteasome activity was required for BDNF-mediated LTP. These results show a novel role for BDNF in UPS regulation at the synapse, which is likely to act together with the increased translation activity in the regulation of the synaptic proteome during E-LTP.

Modulation of GABA Transport by Adenosine A1R–A2AR Heteromers, Which Are Coupled to Both Gs- and Gi/o-Proteins

Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A1 (A1R) and A2A (A2AR) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A1R–A2AR that signal via two different G-proteins, Gs and Gi/o, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron–glia–neuron) synapse.

Modeling the functional network of primary intercellular Ca2+ wave propagation in astrocytes and its application to study drug effects.

We introduce a simple procedure of multivariate signal analysis to uncover the functional connectivity among cells composing a living tissue and describe how to apply it for extracting insight on the effect of drugs in the tissue. The procedure is based on the covariance matrix of time resolved activity signals. By determining the time-lag that maximizes covariance, one derives the weight of the corresponding connection between cells. Introducing simple constraints, it is possible to conclude whether pairs of cells are functionally connected and in which direction. After testing the method against synthetic data we apply it to study intercellular propagation of Ca(2+) waves in astrocytes following an external stimulus, with the aim of uncovering the functional cellular connectivity network. Our method proves to be particularly suited for this type of networking signal propagation where signals are pulse-like and have short time-delays, and is shown to be superior to standard methods, namely a multivariate Granger algorithm. Finally, based on the statistical analysis of the connection weight distribution, we propose simple measures for assessing the impact of drugs on the functional connectivity between cells.