UHRF1 Licensed Self-Renewal of Active Adult Neural Stem Cells.

Adult neurogenesis in the brain continuously seeds new neurons throughout life, but how homeostasis of adult neural stem cells (NSCs) is maintained is incompletely understood. Here, we demonstrate that the DNA methylation adapter ubiquitin-like, containing PHD and RING finger domains-1 (UHRF1) is expressed in, and regulates proliferation of, the active but not quiescent pool of adult neural progenitor cells. Mice with a neural stem cell-specific deficiency in UHRF1 exhibit a massive depletion of neurogenesis resulting in a collapse of formation of new neurons. In the absence of UHRF1, NSCs unexpectedly remain in the cell cycle but with a 17-fold increased cell cycle length due to a failure of replication phase entry caused by promoter demethylation and derepression of Cdkn1a, which encodes the cyclin-dependent kinase inhibitor p21. UHRF1 does not affect the proportion progenitor cells active within the cell cycle but among these cells, UHRF1 is critical for licensing replication re-entry. Therefore, this study shows that a UHRF1-Cdkn1a axis is essential for the control of stem cell self-renewal and neurogenesis in the adult brain. Stem Cells 2018;36:1736-1751.

Regulation of TrkB receptor translocation to lipid rafts by adenosine A(2A) receptors and its functional implications for BDNF-induced regulation of synaptic plasticity.

Brain-derived neurotrophic factor (BDNF) signalling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signalling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A(2A) receptor activation, we hypothesized that activation of A(2A) receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A(2A) receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansyl cadaverine (100 µM) did not modify the effects of the A(2A) receptor agonists, but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A(2A) receptor activation in TrkB localization was mimicked by 5 µM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors RpcAMPs and PKI-(14-22) and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF upon hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts, induced by the activation of adenosine A(2A) receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.

Modulation of brain-derived neurotrophic factor (BDNF) actions in the nervous system by adenosine A(2A) receptors and the role of lipid rafts.

In this paper we review some novel aspects related to the way adenosine A(2A) receptors (A(2A)R) modulate the action of BDNF or its high-affinity receptors, the TrkB receptors, on synaptic transmission and plasticity, as well as upon cholinergic currents and GABA transporters. Evidence has been accumulating that adenosine A(2A)Rs are required for most of the synaptic actions of BDNF. In some cases, where A(2A)Rs are constitutively activated (e.g. by endogenous extracellular adenosine), the need for A(2A)R activation for the maintenance of the synaptic influences of BDNF can be envisaged from the loss of BDNF effects upon blockade of adenosine A(2A)Rs or upon removal of extracellular adenosine with adenosine deaminase. In some other cases, it is necessary to enhance extracellular adenosine levels (e.g. depolarization) or to further activate A(2A)Rs (e.g. with selective agonists) to trigger a BDNF neuromodulatory role at the synapses. Age- and cell-dependent differences may determine the above two possibilities, but in all cases it is quite clear that there is close interplay between adenosine A(2A)Rs and BDNF TrkB receptors at synapses. The role of lipid rafts in this cross-talk will be discussed. This article is part of a Special Issue entitled: "Adenosine Receptors".

Activation of adenosine A2A receptors induces TrkB translocation and increases BDNF-mediated phospho-TrkB localization in lipid rafts: implications for neuromodulation.

Brain-derived neurotrophic factor (BDNF) signaling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signaling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A(2A) receptor activation, we hypothesized that activation of A(2A) receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A(2A) receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansylcadaverine (100 microm) did not modify the effects of the A(2A) receptor agonists but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A(2A) receptor activation in TrkB localization was mimicked by 5 microm forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors Rp-cAMPs and PKI-(14-22), and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF on hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts induced by activation of adenosine A(2A) receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.

Inhibition of renal Na+-ATPase activity by inosine is mediated by A1 receptor-induced inhibition of the cAMP signaling pathway.

We have previously demonstrated that adenosine is deaminated to inosine in the isolated basolateral membrane (BLM) of kidney proximal tubules. This work investigates the possible effect of inosine on proximal tubule Na(+)-ATPase activity. Inosine reduced Na(+)-ATPase activity by 70%. This effect of inosine was completely attenuated by 10(-8) M DPCPX, an A(1) receptor-selective antagonist, but it was not affected by either 10(-8) M DMPX or 10(-7) M MRS1523, A(2) and A(3) receptor-selective antagonists, respectively. The inhibitory effect of inosine was blocked by: (1) 10(-6) M GDPbetaS, a trimeric G protein inhibitor; (2) 1microg/ml pertussis toxin, a Gi protein inhibitor; (3) 10(-6) M forskolin, an adenylyl cyclase activator; (4) 10(-9) M cholera toxin, a Gs protein activator; (5) 10(-6)M cAMP. Our results demonstrate that the inhibitory effect of inosine on the sodium pump is mediated by the A(1) receptor/Gi/cAMP pathway.

Adenosine deamination to inosine in isolated basolateral membrane from kidney proximal tubule: implications for modulation of the membrane-associated protein kinase A.

In this work, the metabolism of adenosine by isolated BLM associated-enzymes and the implications of this process for the cAMP-signaling pathway are investigated. Inosine was identified as the major metabolic product, suggesting the presence of adenosine deaminase (ADA) activity in the BLM. This was confirmed by immunoblotting and ADA-specific enzyme assay. Implications for the enzymatic deamination of adenosine on the receptor-modulated cAMP-signaling pathway were also investigated. We observed that inosine induced a 2-fold increase in [(35)S] GTPgammaS binding to the BLM and it was inhibited by 10(-6)M DPCPX, an A(1) receptor-selective antagonist. Inosine (10(-7)M) inhibited protein kinase A activity in a DPCPX-sensitive manner. Molecular association between ADA and G(alphai-3) protein-coupled A(1) receptor was demonstrated by co-immunoprecipitation assay. These data show that adenosine is deaminated by A(1) receptor-associated ADA to inosine, which in turn modulates PKA in the BLM through A(1) receptor-mediated inhibition of adenylyl cyclase.

Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons.

Axon growth and dendrite development are key processes for the establishment of a functional neuronal network. Adenosine, which is released by neurons and glia, is a known modulator of synaptic transmission but its influence over neuronal growth has been much less investigated. We now explored the action of adenosine A2A receptors (A2AR) upon neurite outgrowth, discriminating actions over the axon or dendrites, and the mechanisms involved. Morphometric analysis of primary cultures of cortical neurons from E18 Sprague-Dawley rats demonstrated that an A2AR agonist, CGS 21680, enhances axonal elongation and dendritic branching, being the former prevented by inhibitors of phosphoinositide 3-kinase, mitogen-activated protein kinase and phospholipase C, but not of protein kinase A. By testing the influence of a scavenger of BDNF (brain-derived neurotrophic factor) over the action of the A2AR agonist and the action of a selective A2AR antagonist over the action of BDNF, we could conclude that while the action of A2ARs upon dendritic branching is dependent on the presence of endogenous BDNF, the influence of A2ARs upon axonal elongation is independent of endogenous BDNF. In consonance with the action over axonal elongation, A2AR activation promoted a decrease in microtubule stability and an increase in microtubule growth speed in axonal growth cones. In conclusion, we disclose a facilitatory action of A2ARs upon axonal elongation and microtubule dynamics, providing new insights for A2ARs regulation of neuronal differentiation and axonal regeneration.

Lipid rafts, synaptic transmission and plasticity: impact in age-related neurodegenerative diseases.

The synapse is a crowded area. In the last years, the concept that proteins can be organized in different membrane domains according to their structure has emerged. Cholesterol-rich membrane domains, or lipid rafts, form an organized portion of the membrane that is thought to concentrate signaling molecules. Accumulating evidence has shown that both the pre-synaptic and post-synaptic sites are highly enriched in lipid rafts, which are likely to organize and maintain synaptic proteins in their precise localization. Here we review recent studies highlighting the importance of lipid rafts for synaptic function and plasticity, as well as their relevance for age or disease-related cognitive impairment. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Regulation of hippocampal cannabinoid CB1 receptor actions by adenosine A1 receptors and chronic caffeine administration: implications for the effects of Δ9-tetrahydrocannabinol on spatial memory.

The cannabinoid CB(1) receptor-mediated modulation of γ-aminobutyric acid (GABA) release from inhibitory interneurons is important for the integrity of hippocampal-dependent spatial memory. Although adenosine A(1) receptors have a central role in fine-tuning excitatory transmission in the hippocampus, A(1) receptors localized in GABAergic cells do not directly influence GABA release. CB(1) and A(1) receptors are the main targets for the effects of two of the most heavily consumed psychoactive substances worldwide: Δ(9)-tetrahydrocannabinol (THC, a CB(1) receptor agonist) and caffeine (an adenosine receptor antagonist). We first tested the hypothesis that an A(1)-CB(1) interaction influences GABA and glutamate release in the hippocampus. We found that A(1) receptor activation attenuated the CB(1)-mediated inhibition of GABA and glutamate release and this interaction was manifested at the level of G-protein activation. Using in vivo and in vitro approaches, we then investigated the functional implications of the adenosine-cannabinoid interplay that may arise following chronic caffeine consumption. Chronic administration of caffeine in mice (intraperitoneally, 3 mg/kg/day, for 15 days, >12 h before trials) led to an A(1)-mediated enhancement of the CB(1)-dependent acute disruptive effects of THC on a short-term spatial memory task, despite inducing a reduction in cortical and hippocampal CB(1) receptor number and an attenuation of CB(1) coupling with G protein. A(1) receptor levels were increased following chronic caffeine administration. This study shows that A(1) receptors exert a negative modulatory effect on CB(1)-mediated inhibition of GABA and glutamate release, and provides the first evidence of chronic caffeine-induced alterations on the cannabinoid system in the cortex and hippocampus, with functional implications in spatial memory.

Influence of age on BDNF modulation of hippocampal synaptic transmission: interplay with adenosine A2A receptors.

We previously reported that adenosine, through A(2A) receptor activation, potentiates synaptic actions of brain-derived neurotrophic factor (BDNF) in the hippocampus of infant (3-4 weeks) rats. Since A(2A)-receptor-mediated actions are more evident in old than in young rats and since the therapeutic potential for BDNF-based strategies is greater in old subjects, we now evaluated synaptic actions of BDNF and the levels of TrkB receptors and of adenosine A(2A) receptors in the hippocampus of three groups of adult rats: young adults (10-16 weeks), old adults (36-38 weeks), and aged (70-80 weeks), as well as in one group of infant (3-4 weeks) rats. BDNF (20 ng/ml) enhances field excitatory postsynaptic potentials recorded from the hippocampus of young adults and aged rats, an action triggered by adenosine A(2A) receptor activation, since it was blocked by the A(2A) receptor antagonist, ZM 241385. In the other groups of animals BDNF (20 ng/ml) was virtually devoid of action on synaptic transmission. Western blot analysis of receptor density shows decreased amounts of TrkB receptors in old adults and aged rats, whereas A(2A) receptor levels assayed by ligand binding are enhanced in the hippocampus of old adults and aged rats. It is concluded that age-related changes in the density of TrkB receptors and of adenosine A(2A) receptors may be responsible for a nonmonotonous variation of BDNF actions on synaptic transmission in the hippocampus.