Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

Cloning and expression of the A2a adenosine receptor from guinea pig brain

A full-length complementary DNA (cDNA) clone encoding the guinea pig brain A₂ adenosine receptor has been isolated by polymerase chain reaction (PCR) and low-stringency-hybridization screening of a guinea pig brain cDNA library. This cDNA contains a long open reading frame encoding a 409-amino acid-residue protein which is highly homologous to the A₂ adenosine receptors previously cloned from other species. Hydrophobicity analysis of the deduced protein sequence reveals seven hydrophobic regions, characteristic of a member of the G-protein-coupled receptor superfamily. Radioligand binding assay and functional (GTPase and cAMP) assays of the receptor, transiently expressed in mammalian cells, demonstrate typical characteristics of the A₂ type adenosine receptor. The messenger RNA (mRNA) of this A₂ receptor is found in the brain, heart, kidney and spleen. Receptor autoradiography with [³H]CGS21680, a specific A₂ agonist, and in situ hybridization with A₂ cRNA probe in guinea pig brain indicate that the receptor is expressed exclusively in the caudate nucleus. The pharmacological profile and anatomical distribution of this receptor indicate that it is of the A_2a subtype. This work represents the first cloning of an A_2a receptor in a rodent species, offers a complete pharmacological characterization of the receptor and provides an anatomical comparison between binding profile and gene expression of the receptor.Abbreviations ADAC adenosine amine congener - BA N⁶-benzyladenosine - bp nucleotide base pair - cAMP cyclic adenosine 3,5-monophosphate - CCPA 2-chloro-N⁶-cyclopentyladenosine - CGS 21680 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamido adenosine hydrochloride - CHA N⁶-cyclohexyladenosine - CNS central nervous system - CPA N⁶-cyclohexyladenosine - CNS central nervous system - CPA N⁶-cyclopentyladenosine - CPX 8-cyclopentyl-1,3-dipropylxanthine - DME Dulbecco's modified Eagle's medium - DMPX 3,7-dimethyl-1-propargylxanthine - DPMX 1,3-dipropyl-7-methylxanthine - DPX 1,3-dipropyl-8-(2-amono-4-chlorophenyl)xanthine - FCS fetal calf serum - IBMX 3-isobutyl-1-methylxanthine - KHB Kreb-HEPES buffer - MECA 5-N-methylcarboxamidoadenosine - NECA 5-N-ethylcarboxamidoadenosine - D-PBS Dulbecco's phosphate buffered saline - PCR polymerase chain reaction - R-PIA R(–)-N⁶-(2-phenylisopropyl)adenosine - SSPE sodium chloride-sodium phosphate-EDTA buffer - TM transmembrane domain - XAC xanthine amine congener Special issue dedicated to Dr. Bernard W. Agranoff     

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 A_2B receptor (A_2BR), 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_2BR 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_2BR antagonist, MRS1754 (200 nM), and was also absent in A_2BR null-mutant mice. In mouse primary astrocytic or neuronal cultures, BAY606583 similarly increased ³H-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_2BR blockade diminished newly synthesized glycogen content and at least in the striatum, increased lactate release. In conclusion, we report here that A_2BR 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.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9474-3) contains supplementary material, which is available to authorized users.     

Neurotransmitter receptor availability in the rat brain is constant in a 24 hour-period

Wakefulness and sleep are fundamental characteristics of the brain. We, therefore, hypothesized that transmitter systems contribute to their regulation and will exhibit circadian alterations.

We assessed the concentration of various neurotransmitter receptors and transporters including adenosinergic (A₁AR, A_2AAR, and ENT1), dopaminergic (D₁R, D₂R, and DAT), and serotonergic (5-HT_2AR) target proteins. Adult male Sprague Dawley rats were used and maintained in a 12 h light: 12 h dark cycle (lights on from 07:00 h to 19:00 h). We measured receptor and transporter concentrations in different brain regions, including caudate putamen, basal forebrain, and cortex in 4 hour-intervals over a 24 hour-period using quantitative in vitro autoradiography.

Investigated receptors and transporters showed no fluctuations in any of the analyzed regions using one-way ANOVA. Only in the horizontal diagonal band of Broca, the difference of A₁AR concentration between light and dark phases (t-test) as well as the cosinor analysis of the 24 hour-course were significant, suggesting that this region underlies receptor fluctuations.

Our findings suggest that the availability of the investigated neurotransmitter receptors and transporters does not undergo changes in a 24 hour-period. While there are reports on changes in adenosine and dopamine receptors during sleep deprivation, we found no changes in the investigated adenosine, dopamine, and serotonin receptors during regular and undisturbed day-night cycles.     

Static magnetic field exposure reproduces cellular effects of the Parkinson's disease drug candidate ZM241385

Background

This study was inspired by coalescing evidence that magnetic therapy may be a viable treatment option for certain diseases. This premise is based on the ability of moderate strength fields (i.e., 0.1 to 1 Tesla) to alter the biophysical properties of lipid bilayers and in turn modulate cellular signaling pathways. In particular, previous results from our laboratory (Wang et al., BMC Genomics, 10, 356 (2009)) established that moderate strength static magnetic field (SMF) exposure altered cellular endpoints associated with neuronal function and differentiation. Building on this background, the current paper investigated SMF by focusing on the adenosine A_2A receptor (A_2AR) in the PC12 rat adrenal pheochromocytoma cell line that displays metabolic features of Parkinson''s disease (PD).

Methodology and Principal Findings

SMF reproduced several responses elicited by ZM241385, a selective A_2AR antagonist, in PC12 cells including altered calcium flux, increased ATP levels, reduced cAMP levels, reduced nitric oxide production, reduced p44/42 MAPK phosphorylation, inhibited proliferation, and reduced iron uptake. SMF also counteracted several PD-relevant endpoints exacerbated by A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 in a manner similar to ZM241385; these include reduction of increased expression of A_2AR, reversal of altered calcium efflux, dampening of increased adenosine production, reduction of enhanced proliferation and associated p44/42 MAPK phosphorylation, and inhibition of neurite outgrowth.

Conclusions and Significance

When measured against multiple endpoints, SMF elicited qualitatively similar responses as ZM241385, a PD drug candidate. Provided that the in vitro results presented in this paper apply in vivo, SMF holds promise as an intriguing non-invasive approach to treat PD and potentially other neurological disorders.     

Intravitreal Administration of HA-1077, a ROCK Inhibitor,Improves Retinal Function in a Mouse Model of Huntington Disease

Huntington disease (HD) is an inherited neurodegenerative disease that affects multiple brain regions. It is caused by an expanded polyglutamine tract in huntingtin (Htt). The development of therapies for HD and other neurodegenerative diseases has been hampered by multiple factors, including the lack of clear therapeutic targets, and the cost and complexity of testing lead compounds in vivo. The R6/2 HD mouse model is widely used for pre-clinical trials because of its progressive and robust neural dysfunction, which includes retinal degeneration. Profilin-1 is a Htt binding protein that inhibits Htt aggregation. Its binding to Htt is regulated by the rho-associated kinase (ROCK), which phosphorylates profilin at Ser-137. ROCK is thus a therapeutic target in HD. The ROCK inhibitor Y-27632 reduces Htt toxicity in fly and mouse models. Here we characterized the progressive retinopathy of R6/2 mice between 6–19 weeks of age to determine an optimal treatment window. We then tested a clinically approved ROCK inhibitor, HA-1077, administered intravitreally via liposome-mediated drug delivery. HA-1077 increased photopic and flicker ERG response amplitudes in R6/2 mice, but not in wild-type littermate controls. By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.     

Molecular basis for the dosing time-dependency of anti-allodynic effects of gabapentin in a mouse model of neuropathic pain

Background

Ecto-5'-nucleotidase (NT5E, also known as CD73) hydrolyzes extracellular adenosine 5'-monophosphate (AMP) to adenosine in nociceptive circuits. Since adenosine has antinociceptive effects in rodents and humans, we hypothesized that NT5E, an enzyme that generates adenosine, might also have antinociceptive effects in vivo.

Results

To test this hypothesis, we purified a soluble version of mouse NT5E (mNT5E) using the baculovirus expression system. Recombinant mNT5E hydrolyzed AMP in biochemical assays and was inhibited by α,β-methylene-adenosine 5'-diphosphate (α,β-me-ADP; IC₅₀ = 0.43 μM), a selective inhibitor of NT5E. mNT5E exhibited a dose-dependent thermal antinociceptive effect that lasted for two days when injected intrathecally in wild-type mice. In addition, mNT5E had thermal antihyperalgesic and mechanical antiallodynic effects that lasted for two days in the complete Freund's adjuvant (CFA) model of inflammatory pain and the spared nerve injury (SNI) model of neuropathic pain. In contrast, mNT5E had no antinociceptive effects when injected intrathecally into adenosine A₁ receptor (A ₁ R, Adora1) knockout mice.

Conclusion

Our data indicate that the long lasting antinociceptive effects of mNT5E are due to hydrolysis of AMP followed by activation of A₁R. Moreover, our data suggest recombinant NT5E could be used to treat chronic pain and to study many other physiological processes that are regulated by NT5E.     

Adenosine A<Subscript>1</Subscript> receptors contribute to immune regulation after neonatal hypoxic ischemic brain injury

Neonatal brain hypoxic ischemia (HI) often results in long-term motor and cognitive impairments. Post-ischemic inflammation greatly effects outcome and adenosine receptor signaling modulates both HI and immune cell function. Here, we investigated the influence of adenosine A₁ receptor deficiency (A₁R^?/?) on key immune cell populations in a neonatal brain HI model. Ten-day-old mice were subjected to HI. Functional outcome was assessed by open locomotion and beam walking test and infarction size evaluated. Flow cytometry was performed on brain-infiltrating cells, and semi-automated analysis of flow cytometric data was applied. A₁R^?/? mice displayed larger infarctions (+33 %, p?<?0.05) and performed worse in beam walking tests (44 % more mistakes, p?<?0.05) than wild-type (WT) mice. Myeloid cell activation after injury was enhanced in A₁R^?/? versus WT brains. Activated B lymphocytes expressing IL-10 infiltrated the brain after HI in WT, but were less activated and did not increase in relative frequency in A₁R^?/?. Also, A₁R^?/? B lymphocytes expressed less IL-10 than their WT counterparts, the A₁R antagonist DPCPX decreased IL-10 expression whereas the A₁R agonist CPA increased it. CD4⁺ T lymphocytes including FoxP3⁺ T regulatory cells, were unaffected by genotype, whereas CD8⁺ T lymphocyte responses were smaller in A₁R^?/? mice. Using PCA to characterize the immune profile, we could discriminate the A₁R^?/? and WT genotypes as well as sham operated from HI-subjected animals. We conclude that A₁R signaling modulates IL-10 expression by immune cells, influences the activation of these cells in vivo, and affects outcome after HI.     

BDNF prevents NMDA‐induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved

NMDA receptor‐mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain‐derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A_2ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA‐induced effects in HD models, and the possible involvement of A_2ARs. In corticostriatal slices from wild‐type mice and age‐matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild‐type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A_2AR blockade. The protective effect of BDNF against NMDA‐induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A_2AR ligands in HD.     

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Adenosine A1 and A3 receptor agonists reduce hypoxic injury through the involvement of P38 MAPK

Activation of either the A₁ adenosine receptor (A₁R) or the A₃ adenosine receptor (A₃R), by their specific agonists CCPA and Cl-IB-MECA, respectively, protects cardiac cells in culture against ischemic injury. Yet the full protective mechanism remains unclear. In this study, we therefore examined the involvement of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) phosphorylation in this protective intracellular signaling mechanism. Furthermore, we investigated whether p38 MAPK phosphorylation occurs upstream or downstream from the opening of mitochondrial ATP-sensitive potassium (K_ATP) channels. The role of p38 MAPK activation in the intracellular signaling process was studied in cultured cardiomyocytes subjected to hypoxia, that were pretreated with CCPA or Cl-IB-MECA or diazoxide (a mitochondrial K_ATP channel opener) with and without SB203580 (a specific inhibitor of phosphorylated p38 MAPK). Cardiomyocytes were also pretreated with anisomycin (p38 MAPK activator) with and without 5-hydroxy decanoic acid (5HD) (a mitochondrial K_ATP channel blocker). SB203580 together with the CCPA, Cl-IB-MECA or diazoxide abrogated the protection against hypoxia as shown by the level of ATP, lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Anisomycin protected the cardiomyocytes against ischemic injury and this protection was abrogated by SB203580 but not by 5HD. Conclusions Activation of A₁R or A₃R by CCPA or Cl-IB-MECA, respectively, protects cardiomyocytes from hypoxia via phosphorylation of p38 MAPK, which is located downstream from the mitochondrial K_ATP channel opening. Elucidating the signaling pathway by which adenosine receptor agonists protect cardiomyocytes from hypoxic damage, will facilitate the development of anti ischemic drugs.     

Nucleosides Present on Phlebotomine Saliva Induce Immunossuppression and Promote the Infection Establishment

Background

Sand fly saliva plays a crucial role in establishing Leishmania infection. We identified adenosine (ADO) and adenosine monophosphate (AMP) as active pharmacologic compounds present in Phlebotomus papatasi saliva that inhibit dendritic cell (DC) functions through a PGE₂/IL 10-dependent mechanism.

Methodology/Principal Findings

Herein, we prepared a mixture of ADO and AMP in equimolar amounts similar to those present in the salivary-gland extract (SGE) form one pair of salivary glands of P. papatasi and co-injected it with Leishmania amazonensis or L. major into mouse ears. ADO+AMP mimicked exacerbative effects of P. papatasi saliva in leishmaniasis, increasing parasite burden and cutaneous lesions. Enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect associated with IL-10 enhancement. Immunosuppressive factors COX2 and IL-10 were upregulated and failed to enhance ear lesion and parasite burden in IL 10^-/- infected mice. Furthermore, nucleosides increased regulatory T cell (Treg) marker expression on CD4⁺CD25^- cells, suggesting induction of Tregs on effector T cells (T eff). Treg induction (iTreg) was associated with nucleoside-induced tolerogenic dendritic cells (tDCs) expressing higher levels of COX₂ and IL-10. In vitro generation of Tregs was more efficient in DCs treated with nucleosides. Suppressive effects of nucleosides during cutaneous leishmaniasis were mediated through an A2_AR-dependent mechanism. Using BALB/c mice deficient in A2A ADO receptor (A2_AR^–/–), we showed that co-inoculated mice controlled infection, displaying lower parasite numbers at infection sites and reduced iTreg generation.

Conclusion/Significance

We have demonstrated that ADO and AMP in P. papatasi saliva mediate exacerbative effects of Leishmania infection by acting preferentially on DCs promoting a tolerogenic profile in DCs and by generating iTregs in inflammatory foci through an A2_AR mechanism.     

Inhibition of Platelet Activation and Thrombus Formation by Adenosine and Inosine: Studies on Their Relative Contribution and Molecular Modeling

Background

The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.

Objectives

The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.

Results

Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A_2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A_2A is the adenosine receptor present in platelets; it is known that inosine is not an A_2A ligand. Docking of adenosine and inosine inside A_2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH₂ of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A_2A receptor.

Conclusion

Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.     

Alterations in Adenosine Metabolism and Signaling in Patients with Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis

Background

Adenosine is generated in response to cellular stress and damage and is elevated in the lungs of patients with chronic lung disease. Adenosine signaling through its cell surface receptors serves as an amplifier of chronic lung disorders, suggesting adenosine-based therapeutics may be beneficial in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Previous studies in mouse models of chronic lung disease demonstrate that the key components of adenosine metabolism and signaling are altered. Changes include an up-regulation of CD73, the major enzyme of adenosine production and down-regulation of adenosine deaminase (ADA), the major enzyme for adenosine metabolism. In addition, adenosine receptors are elevated.

Methodology/Principal Findings

The focus of this study was to utilize tissues from patients with COPD or IPF to examine whether changes in purinergic metabolism and signaling occur in human disease. Results demonstrate that the levels of CD73 and A_2BR are elevated in surgical lung biopsies from severe COPD and IPF patients. Immunolocalization assays revealed abundant expression of CD73 and the A_2BR in alternatively activated macrophages in both COPD and IPF samples. In addition, mediators that are regulated by the A_2BR, such as IL-6, IL-8 and osteopontin were elevated in these samples and activation of the A_2BR on cells isolated from the airways of COPD and IPF patients was shown to directly induce the production of these mediators.

Conclusions/Significance

These findings suggest that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of patients with COPD and IPF, and provide proof of concept information that these disorders may benefit from adenosine-based therapeutics. Furthermore, this study provides the first evidence that A_2BR signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders.     

Fall in oxygen tension of culture medium stimulates the adenosinergic signalling of a human T cell line

We examined the short-course expression of various parameters involved in the adenosinergic signalling of a human T cell line during in vitro decrease of the medium culture oxygen tension mimicking in vivo hypoxia. Fall of 92 mmHg in oxygen tension of culture medium induced in CEM, a CD4+ human T cell line, a continuous production of hypoxia-inducing factor-1α with a plateau value at 9 h, a rapid increase in adenosine production peaking at 3 h and a decrease in adenosine deaminase peaking at 6 h. The adenosine A_2A receptor (A_2AR) protein level of CEM cells was enhanced with a peak at 6 h. Intracellular 3′,5′-cyclic adenosine monophosphate accumulated in CEM cells with a maximal level at 9 h. These results show that a human-cultured T cells line can upregulate its own adenosine production and A_2AR expression during exposure to acute hypoxia. Hypoxia-increased stimulation of the adenosinergic signalling of T cells may have immunosuppressive properties and, consequently, A_2AR agonists may have therapeutic relevance.     

Phosphorylation of Threonine 3: IMPLICATIONS FOR HUNTINGTIN AGGREGATION AND NEUROTOXICITY*

Huntingtin (Htt) is a widely expressed protein that causes tissue-specific degeneration when mutated to contain an expanded polyglutamine (poly(Q)) domain. Although Htt is large, 350 kDa, the appearance of amino-terminal fragments of Htt in extracts of postmortem brain tissue from patients with Huntington disease (HD), and the fact that an amino-terminal fragment, Htt exon 1 protein (Httex1p), is sufficient to cause disease in models of HD, points to the importance of the amino-terminal region of Htt in the disease process. The first exon of Htt encodes 17 amino acids followed by a poly(Q) repeat of variable length and culminating with a proline-rich domain of 50 amino acids. Because modifications to this fragment have the potential to directly affect pathogenesis in several ways, we have surveyed this fragment for potential post-translational modifications that might affect Htt behavior and detected several modifications of Httex1p. Here we report that the most prevalent modifications of Httex1p are NH₂-terminal acetylation and phosphorylation of threonine 3 (pThr-3). We demonstrate that pThr-3 occurs on full-length Htt in vivo, and that this modification affects the aggregation and pathogenic properties of Htt. Thus, therapeutic strategies that modulate these events could in turn affect Htt pathogenesis.Aberrant behavior of mutant Huntingtin protein (Htt),² caused by an expansion of the CAG triplet repeat sequence within the first exon of the huntingtin (IT15) gene, results in neurodegeneration and leads to Huntington disease (HD) (1). Full-length Htt protein is 350 kDa in size, but a truncated form of Htt (Httex1p), which includes the expanded polyglutamine region, is sufficient to cause pathology in animal models (2–4). Moreover, an amino-terminal fragment of Htt is detected in nuclear extracts from patient brain and is not detected in control cortex samples (5). In fact, recent studies suggest that production of truncated fragments is essential for disease (6, 7).The first 17 amino acids of Htt, MATLEKLMKAFESLKSF, are highly conserved throughout mammalian evolution (8, 9), suggesting an important function for these residues. It is well established that post-translational modifications of a protein can affect activity state, intracellular localization, turnover rate, and protein-protein interactions. Several modifications of Htt, without the addition of exogenous modifiers, have been identified (10–18) and implicated in HD (18, 19), but to date, none of these occur within the pathogenic Httex1p fragment. Given that this domain is sufficient to cause HD-like phenotypes, modifications that occur within this pathologic fragment may directly affect either its biophysical properties or its interaction with cellular components that affect pathology. Within the first 17 amino acids of Httex1p, there are several candidate amino acids for post-translational modification. Whereas genetic mutation of the lysines in this region alters HD pathology (20, 21), direct evidence for modifications of the amino-terminal fragment, e.g. by mass spectrometry, and identification of the modified residues, remains undocumented.In addition to affecting interactions with cellular components, recent reports indicate that mutations in the first 17 amino acids can alter the intrinsic structure of the peptide and modulate the propensity of Htt to aggregate (8, 22). The role of Htt-containing aggregates in HD remains unclear, with recent studies suggesting that visible aggregates may be protective and function as a coping response to toxic mutant Htt (22, 23). An increasingly popular notion is that oligomer/protofibrillar soluble intermediates formed during the aggregation process are the pathogenic structures (24). Post-translational modification of the first 17 amino acids could influence Httex1p aggregation behavior by changing the properties of the modified residue much like the amino acid substitutions reported (8, 22).In this study, we use mass spectrometry to present the first direct physical evidence for post-translational modification of the pathogenic exon 1 fragment of Htt without overexpressing modifying moieties or enzymes. We find that Htt is modified by the native cellular machinery and that the most common modifications of Httex1p are amino (NH₂)-terminal acetylation and phosphorylation of threonine 3 (Thr³). Furthermore, we show that Thr-3 phosphorylation occurs in vivo on full-length, endogenous Htt, that the length of the poly(Q) tract affects the relative abundance of this modification, and that Thr-3 phosphorylation affects HD pathology and the propensity for Htt aggregation in vitro and in vivo.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

Exploring the Impact of BDNF Val66Met Genotype on Serotonin Transporter and Serotonin-1A Receptor Binding

Background

The brain-derived neurotrophic factor (BDNF) Val66Met polymorphism (rs6265) may impact on the in-vivo binding of important serotonergic structures such as the serotonin transporter (5-HTT) and the serotonin-1A (5-HT_1A) receptor. Previous positron emission tomography (PET) studies on the association between Val66Met and 5-HTT and 5-HT_1A binding potential (BP_ND) have demonstrated equivocal results.

Methods

We conducted an imaging genetics study investigating the effect of Val66Met genotype on 5-HTT or 5-HT_1A BP_ND in 92 subjects. Forty-one subjects (25 healthy subjects and 16 depressive patients) underwent genotyping for Val66Met and PET imaging with the 5-HTT specific radioligand [¹¹C]DASB. Additionally, in 51 healthy subjects Val66Met genotypes and 5-HT_1A binding with the radioligand [carbonyl-¹¹C]WAY-100635 were ascertained. Voxel-wise and region of interest-based analyses of variance were used to examine the influence of Val66Met on 5-HTT and 5-HT_1A BP_ND.

Results

No significant differences of 5-HTT nor 5-HT_1A BP_ND between BDNF Val66Met genotype groups (val/val vs. met-carrier) were detected. There was no interaction between depression and Val66Met genotype status.

Conclusion

In line with previous data, our work confirms an absent effect of BDNF Val66Met on two major serotonergic structures. These results could suggest that altered protein expression associated with genetic variants, might be compensated in vivo by several levels of unknown feedback mechanisms. In conclusion, Val66Met genotype status is not associated with changes of in-vivo binding of 5-HTT and 5-HT_1A receptors in human subjects.     

腺苷和睡眠觉醒调节

腺苷作为神经调质,调节多种神经生物学功能.随觉醒时间延长,动物脑内腺苷水平逐渐增高,在睡眠期显著降低.因此,腺苷被认为是调节睡眠的内稳态因子之一.腺苷受体(receptor,R)有A1R、A2AR、A2BR和A3R四种亚型,其中A1R和A2AR与诱导睡眠相关.激活A1R可抑制促觉醒神经元诱导睡眠,也可抑制促眠神经元导致...