Effect of the B-amyloid peptide on microglia activation: ATP release / Efecto del péptido B-amiloide en la activación de la microglía: liberación de ATP

Estudios previos han mostrado un papel clave de las células microgliales en los procesos neuroinflamatorios asociados con algunas enfermedades neurodegenerativas, como la enfermedad de Alzheimer (EA). La microglía detecta varios tipos de moléculas difusibles que regulan el múltiple repertorio de funciones microgliales. Entre ellos, los nucleótidos extracelulares, actuando sobre los receptores P2 microgliales, llevan a cabo un papel central. En este sentido, el receptor P2X7 ionotrópico ha sido reconocido como un regulador clave de las respuestas inflamatorias mediadas por la microglia. Se sabe que la microglía libera ATP y otros nucleótidos al medio extracelular. Aunque se han propuesto varios mecanismos, tales como la liberación a través de conexinas o panexinas, no se puede descartar un origen vesicular para estos nucleótidos liberados, basándose en la actividad del transportador vesicular de nucleótidos (VNUT).En este trabajo hemos analizado si la expresión de VNUT y el receptor P2X7, así como la liberación de ATP, podrían modificarse en la microglía reactiva. Para lograr la activación de la microglía estimulamos las células con el lipopolisacárido (LPS). Además, analizamos el efecto del péptido β1-amiloide, β1-42, que puede activar también las células microgliales, sobre la expresión de VNUT y la liberación de ATP en la microglía. (AU)

Previous studies have shown a key role of microglial cells in the neuroinflammatory processes associated with some neurodegenerative diseases, such as Alzheimer’s disease (AD). Microglia sense several types of diffusible molecules that regulate the multiple repertoire of microglial functions. Among them, extracellular nucleotides, acting on microglial P2 receptors, have central roles. In this sense, the ionotropic P2X7 receptor has gained recognition as a key regulator of microglial-mediated inflammatory responses. It is known that microglia releases ATP and other nucleotides to the extracellular medium. Although several mechanisms, such as release trough conexins or panexins, has been proposed, a vesicular origin for this released nucleotides, relying on the activity of the vesicular nucleotide transporter (VNUT), cannot be ruled out.In this work we evaluated whether the expression of VNUT and the P2X7 receptor, as well as the ATP release, could be modified in the reactive microglia. To achieve microglia activation we stimulated the cells with the lipopolysaccharide (LPS). Moreover, we analyzed the effect of the β-amyloid peptide β1-42, which is also able to activate the microglial cells, on the expression of VNUT and the ATP release in the microglia. (AU)

Drug repurposing based on a quantum-inspired method versus classical fingerprinting uncovers potential antivirals against SARS-CoV-2.

The COVID-19 pandemic has accelerated the need to identify new antiviral therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir, the first antiviral against SARS-CoV-2 approved for human use, using a quantum-inspired device. We modelled Remdesivir and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of lead compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC50) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. We also demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Lastly, we employed an in vitro polymerization assay to demonstrate that these compounds directly inhibit the RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. Together, our data reveal that our QUBO model performs accurate comparisons (BMS-986094) that differed from those predicted by Tanimoto (different forms of vitamin B12); all compounds inhibited replication of SARS-CoV-2 via direct action on RdRP, with both models being useful. While Tanimoto may be employed when performing relatively small comparisons, QUBO is also accurate and may be well suited for very complex problems where computational resources may limit the number and/or complexity of possible combinations to evaluate. Our quantum-inspired screening method can therefore be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.

Promoters of ASCL1- and NEUROD1-dependent genes are specific targets of lurbinectedin in SCLC cells.

Small-Cell Lung Cancer (SCLC) is an aggressive neuroendocrine malignancy with a poor prognosis. Here, we focus on the neuroendocrine SCLC subtypes, SCLC-A and SCLC-N, whose transcription addiction was driven by ASCL1 and NEUROD1 transcription factors which target E-box motifs to activate up to 40% of total genes, the promoters of which are maintained in a steadily open chromatin environment according to ATAC and H3K27Ac signatures. This leverage is used by the marine agent lurbinectedin, which preferentially targets the CpG islands located downstream of the transcription start site, thus arresting elongating RNAPII and promoting its degradation. This abrogates the expression of ASCL1 and NEUROD1 and of their dependent genes, such as BCL2, INSM1, MYC, and AURKA, which are responsible for relevant SCLC tumorigenic properties such as inhibition of apoptosis and cell survival, as well as for a part of its neuroendocrine features. In summary, we show how the transcription addiction of these cells becomes their Achilles's heel, and how this is effectively exploited by lurbinectedin as a novel SCLC therapeutic endeavor.

Drug repurposing based on a Quantum-Inspired method versus classical fingerprinting uncovers potential antivirals against SARS-CoV-2 including vitamin B12.

The COVID-19 pandemic has accelerated the need to identify new therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir (RDV), the only antiviral against SARS-CoV-2 currently approved for human use, using a quantum-inspired device. We modelled RDV and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC 50 ) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. Lastly, we demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Our data reveal that BMS-986094 and different forms of vitamin B12 are effective at inhibiting replication of all these variants of SARS-CoV-2. While BMS-986094 can cause secondary effects in humans as established by phase II trials, these findings suggest that vitamin B12 deserves consideration as a SARS-CoV-2 antiviral, particularly given its extended use and lack of toxicity in humans, and its availability and affordability. Our screening method can be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.

Effect of the Beta-amyloid peptide on microglia activation: ATP release / Efecto del péptido (R)-amiloide en la activación de la microglía: liberación de ATP

Previous studies have shown a key role of microglial cells in the neuroinflammatory processes associated with some neurodegenerative diseases, such as Alzheimer’s disease (AD). Microglia sense several types of diffusible molecules that regulate the multiple repertoire of microglial functions. Among them, extracellular nucleotides, acting on microglial P2 receptors, have central roles. In this sense, the ionotropic P2X7 receptor has gained recognition as a key regulator of microglial-mediated inflammatory responses. It is known that microglia releases ATP and other nucleotides to the extracellular medium. Although several mechanisms, such as release trough conexins or panexins, has been proposed, a vesicular origin for this released nucleotides, relying on the activity of the vesicular nucleotide transporter (VNUT), cannot be ruled out. In this work we evaluated whether the expression of VNUT and the P2X7 receptor, as well as the ATP release, could be modified in the reactive microglia. To achieve microglia activation we stimulated the cells with the lipopolysaccharide (LPS). Moreover, we analyzed the effect of the b-amyloid peptide b1-42, which is also able to activate the microglial cells, on the expression of VNUT and the ATP release in the microgli

Estudios previos han mostrado un papel clave de las células microgliales en los procesos neuroinflamatorios asociados con algunas enfermedades neurodegenerativas, como la enfermedad de Alzheimer (EA). La microglía detecta varios tipos de moléculas difusibles que regulan el múltiple repertorio de funciones microgliales. Entre ellos, los nucleótidos extracelulares, actuando sobre los receptores P2 microgliales, llevan a cabo un papel central. En este sentido, el receptor P2X7 ionotrópico ha sido reconocido como un regulador clave de las respuestas inflamatorias mediadas por la microglia. Se sabe que la microglía libera ATP y otros nucleótidos al medio extracelular. Aunque se han propuesto varios mecanismos, tales como la liberación a través de conexinas o panexinas, no se puede descartar un origen vesicular para estos nucleótidos liberados, basándose en la actividad del transportador vesicular de nucleótidos (VNUT). En este trabajo hemos analizado si la expresión de VNUT y el receptor P2X7, así como la liberación de ATP, podrían modificarse en la microglía reactiva. Para lograr la activación de la microglía estimulamos las células con el lipopolisacárido (LPS). Además, analizamos el efecto del péptido (R)1-amiloide, (R)1-42, que puede activar también las células microgliales, sobre la expresión de VNUT y la liberación de ATP en la microglía

El transportador vesicular de nucleótidos (VNUT). Relevancia en tejidos neurales y neuroendocrinos. Nuevas perspectivas farmacológicas / The nucleotide vesicular transporter (VNUT). Relevance in neural and neuroendocrine tissues. New pharmacological perspectives

El almacenamiento vesicular de los neurotransmisores, que permite su subsecuente liberación exocitótica, es un proceso esencial para la transmisión química en neuronas y células endocrinas. La acumulación de los neurotransmisores en vesículas de secreción se lleva a cabo por medio de transportadores vesiculares, que utilizan el gradiente electroquímico de protones generado por una ATPasa vacuolar como fuerza impulsora del transporte. El ATP, así como otros nucleótidos y dinucleótidos, son importantes moléculas señalizadoras que intervienen en una gran variedad de procesos biológicos. Aunque el transporte activo de nucleótidos se ha caracterizado desde el punto de vista bioquímico y farmacológico en una variedad de vesículas de secreción, la proteína responsable de esta acumulación vesicular permaneció durante mucho tiempo desconocida. En 2008, se demostró que SLC17A9, el último miembro identificado de la familia de transportadores SLC17, codifica el transportador vesicular de nucleótidos (VNUT). VNUT se expresa en una variedad de células que liberan ATP y ha mostrado ser capaz de transportar varios nucleótidos de manera dependiente del potencial de membrana vesicular. Ratones deficientes en VNUT pierden la capacidad de almacenar y liberar ATP de neuronas y células neuroendocrinas, lo que resulta en un bloqueo de la transmisión química purinérgica. En esta revisión se pretende resumir los estudios llevados a cabo hasta la fecha sobre VNUT y analizar la relevancia del transporte vesicular de nucleótidos en distintos tipos celulares y tejidos. Asimismo, se discute el posible uso de inhibidores de VNUT, así como de ARNs de interferencia que reduzcan su expresión, con fines terapéuticos

Vesicular storage of neurotransmitters, which allows their subsequent exocytotic release, is essential for chemical transmission in neurons and endocrine cells. Neurotransmitter uptake to secretory vesicles is carried out by vesicular transporters, which use the electrochemical gradient of protons generated by a vacuolar proton-ATPase as transport driving force. ATP and other nucleotides and dinucleotides are relevant signaling molecules that participate in a variety of biological process. Although the active transport of nucleotides has been pharmacologically and biochemically characterized in a diversity of secretory vesicles, the protein responsible for such vesicular accumulation remained unidentified for some time. In 2008, SLC17A9, the last identified member of the SLC17 transporter family, was found to encode the vesicular nucleotide transporter (VNUT). VNUT is expressed in various ATP-secreting cells and is able to transport several nucleotides in a vesicular membrane potential- dependent fashion. VNUT knockout mice lack vesicular storage and release of ATP from neurons and neuroendocrine cells, resulting in blockage of the purinergic chemical transmission. This review summarizes the current studies on VNUT and analyzes the relevance of vesicular nucleotide transport in different cells types and tissues. The possible use of VNUT inhibitors and interference RNA to reduce VNUT gene expression for therapeutic purposes is also discussed

TNAP stimulates vascular smooth muscle cell trans-differentiation into chondrocytes through calcium deposition and BMP-2 activation: Possible implication in atherosclerotic plaque stability.

Atherosclerotic plaque calcification varies from early, diffuse microcalcifications to a bone-like tissue formed by endochondral ossification. Recently, a paradigm has emerged suggesting that if the bone metaplasia stabilizes the plaques, microcalcifications are harmful. Tissue-nonspecific alkaline phosphatase (TNAP), an ectoenzyme necessary for mineralization by its ability to hydrolyze inorganic pyrophosphate (PPi), is stimulated by inflammation in vascular smooth muscle cells (VSMCs). Our objective was to determine the role of TNAP in trans-differentiation of VSMCs and calcification. In rodent MOVAS and A7R5 VSMCs, addition of exogenous alkaline phosphatase (AP) or TNAP overexpression was sufficient to stimulate the expression of several chondrocyte markers and induce mineralization. Addition of exogenous AP to human mesenchymal stem cells cultured in pellets also stimulated chondrogenesis. Moreover, TNAP inhibition with levamisole in mouse primary chondrocytes dropped mineralization as well as the expression of chondrocyte markers. VSMCs trans-differentiated into chondrocyte-like cells, as well as primary chondrocytes, used TNAP to hydrolyze PPi, and PPi provoked the same effects as TNAP inhibition in primary chondrocytes. Interestingly, apatite crystals, associated or not to collagen, mimicked the effects of TNAP on VSMC trans-differentiation. AP and apatite crystals increased the expression of BMP-2 in VSMCs, and TNAP inhibition reduced BMP-2 levels in chondrocytes. Finally, the BMP-2 inhibitor noggin blocked the rise in aggrecan induced by AP in VSMCs, suggesting that TNAP induction in VSMCs triggers calcification, which stimulates chondrogenesis through BMP-2. Endochondral ossification in atherosclerotic plaques may therefore be induced by crystals, probably to confer stability to plaques with microcalcifications.

Regulation of proteasome activity by P2Y2 receptor underlies the neuroprotective effects of extracellular nucleotides.

The Ubiquitin-Proteasome System (UPS) is essential for the regulation of the cellular proteostasis. Indeed, it has been postulated that an UPS dysregulation is the common mechanism that underlies several neurological disorders. Considering that extracellular nucleotides, through their selective P2Y2 receptor (P2Y2R), play a neuroprotective role in various neurological disorders that course with an UPS impairment, we wonder if this neuroprotective capacity resulted from their ability to modulate the UPS. Using a cellular model expressing two different UPS reporters, we found that the stimulation of P2Y2R by its selective agonist Up4U induced a significant reduction of UPS reporter levels. This reduction was due to an increase in two of the three peptidase proteasome activities, chymotrypsin and postglutamyl, caused by an increased expression of proteasome constitutive catalytic subunits ß1 and ß5. The intracellular signaling pathway involved required the activation of IP3/MEK1/2/ERK but was independent of PKC or PKA. Interestingly, the P2Y2R activation was able to revert both UPS-reporter accumulation and the cell death induced by a prolonged inhibition of UPS. Finally, we also observed that intracerebroventricular administration of Up4U induced a significant increase both of chymotrypsin and postglutamyl activities as well as an increased expression of proteasome subunits ß1 and ß5 in the hippocampus of wild-type mice, but not in P2Y2R KO mice. All these results strongly suggest that the capacity to modulate the UPS activity via P2Y2R is the molecular mechanism which is how the nucleotides play a neuroprotective role in neurological disorders.

Nucleotides in neuroregeneration and neuroprotection.

Brain injury generates the release of a multitude of factors including extracellular nucleotides, which exhibit bi-functional properties and contribute to both detrimental actions in the acute phase and also protective and reparative actions in the later recovery phase to allow neuroregeneration. A promising strategy toward restoration of neuronal function is based on activation of endogenous adult neural stem/progenitor cells. The implication of purinergic signaling in stem cell biology, including regulation of proliferation, differentiation, and cell death has become evident in the last decade. In this regard, current strategies of acute transplantation of ependymal stem/progenitor cells after spinal cord injury restore altered expression of P2X4 and P2X7 receptors and improve functional locomotor recovery. The expression of both receptors is transcriptionally regulated by Sp1 factor, which plays a key role in the startup of the transcription machinery to induce regeneration-associated genes expression. Finally, general signaling pathways triggered by nucleotide receptors in neuronal populations converge on several intracellular kinases, such as PI3K/Akt, GSK3 and ERK1,2, as well as the Nrf-2/heme oxigenase-1 axis, which specifically link them to neuroprotection. In this regard, regulation of dual specificity protein phosphatases can become novel mechanism of actions for nucleotide receptors that associate them to cell homeostasis regulation. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions.

The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome.

Increased levels of extracellular ATP in glaucomatous retinas: Possible role of the vesicular nucleotide transporter during the development of the pathology.

PURPOSE: To study retinal extracellular ATP levels and to assess the changes in the vesicular nucleotide transporter (VNUT) expression in a murine model of glaucoma during the development of the disease. METHODS: Retinas were obtained from glaucomatous DBA/2J mice at 3, 9, 15, and 22 months together with C57BL/6J mice used as age-matched controls. To study retinal nucleotide release, the retinas were dissected and prepared as flattened whole mounts and stimulated in Ringer buffer with or without 59 mM KCl. To investigate VNUT expression, sections of the mouse retinas were evaluated with immunohistochemistry and western blot analysis using newly developed antibodies against VNUT. All images were examined and photographed under confocal microscopy. Electroretinogram (ERG) recordings were performed on the C57BL/6J and DBA/2J mice to analyze the changes in the electrophysiological response; a decrease in the scotopic threshold response was observed in the 15-month-old DBA/2J mice. RESULTS: In the 15-month-old control and glaucomatous mice, electrophysiological changes of 42% were observed. In addition, 50% increases in the intraocular pressure (IOP) were observed when the pathology was fully established. The responses in the retinal ATP net release as the pathology progressed varied from 0.32±0.04 pmol/retina (3 months) to 1.10±0.06 pmol/retina (15 months; threefold increase). Concomitantly, VNUT expression was significantly increased during glaucoma progression in the DBA/2J mice (58%) according to the immunohistochemical and western blot analysis. CONCLUSIONS: These results may indicate a possible correlation between retinal dysfunction and increased levels of extracellular ATP and nucleotide transporter. These data support an excitotoxicity role for ATP via P2X7R in glaucoma. This modified cellular environment could contribute to explaining the functional and biochemical alterations observed during the development of the pathology.

Age-related nuclear translocation of P2X6 subunit modifies splicing activity interacting with splicing factor 3A1.

P2X receptors are ligand-gated ion channels sensitive to extracellular nucleotides formed by the assembling of three equal or different P2X subunits. In this work we report, for the first time, the accumulation of the P2X6 subunit inside the nucleus of hippocampal neurons in an age-dependent way. This location is favored by its anchorage to endoplasmic reticulum through its N-terminal domain. The extracellular domain of P2X6 subunit is the key to reach the nucleus, where it presents a speckled distribution pattern and is retained by interaction with the nuclear envelope protein spectrin α2. The in vivo results showed that, once inside the nucleus, P2X6 subunit interacts with the splicing factor 3A1, which ultimately results in a reduction of the mRNA splicing activity. Our data provide new insights into post-transcriptional regulation of mRNA splicing, describing a novel mechanism that could explain why this process is sensitive to changes that occur with age.

The vesicular nucleotide transporter (VNUT) is involved in the extracellular ATP effect on neuronal differentiation.

Before being released, nucleotides are stored in secretory vesicles through the vesicular nucleotide transporter (VNUT). Once released, extracellular ATP participates in neuronal differentiation processes. Thus, the expression of a functional VNUT could be an additional component of the purinergic system which regulates neuronal differentiation and axonal elongation. In vitro expression of VNUT decreases neuritogenesis in N2a cells differentiated by retinoic acid treatment, whereas silencing of VNUT expression increases the number and length of neurites in these cells. These results highlight the role of VNUT in the neuritogenic process because this transporter regulates the ATP content in neurosecretory vesicles.

Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

In adult brains, ionotropic or metabotropic purinergic receptors are widely expressed in neurons and glial cells. They play an essential role in inflammation and neurotransmission in response to purines secreted to the extracellular medium. Recent studies have demonstrated a role for purinergic receptors in proliferation and differentiation of neural stem cells although little is known about their role in regulating the initial neuronal development and axon elongation. The objective of our study was to investigate the role of some different types of purinergic receptors, P2Y1, P2Y13 and P2X7, which are activated by ADP or ATP. To study the role and crosstalk of P2Y1, P2Y13 and P2X7 purinergic receptors in axonal elongation, we treated neurons with specific agonists and antagonists, and we nucleofected neurons with expression or shRNA plasmids. ADP and P2Y1-GFP expression improved axonal elongation; conversely, P2Y13 and ATP-gated P2X7 receptors halted axonal elongation. Signaling through each of these receptor types was coordinated by adenylate cyclase 5. In neurons nucleofected with a cAMP FRET biosensor (ICUE3), addition of ADP or Blue Brilliant G, a P2X7 antagonist, increased cAMP levels in the distal region of the axon. Adenylate cyclase 5 inhibition or suppression impaired these cAMP increments. In conclusion, our results demonstrate a crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K-Akt-GSK3 pathway.

In vivo P2X7 inhibition reduces amyloid plaques in Alzheimer's disease through GSK3ß and secretases.

ß-Amyloid (Aß) peptide production from amyloid precursor protein (APP) is essential in the formation of the ß-amyloid plaques characteristic of Alzheimer's disease. However, the extracellular signals that maintain the balance between nonpathogenic and pathologic forms of APP processing, mediated by α-secretase and ß-secretase respectively, remain poorly understood. In the present work, we describe regulation of the processing of APP via the adenosine triphosphate (ATP) receptor P2X7R. In 2 different cellular lines, the inhibition of either native or overexpressed P2X7R increased α-secretase activity through inhibition of glycogen synthase kinase 3 (GSK-3). In vivo inhibition of the P2X7R in J20 mice, transgenic for mutant human APP, induced a significant decrease in the number of hippocampal amyloid plaques. This reduction correlated with a decrease in glycogen synthase kinase 3 activity in J20 mice, increasing the proteolytic processing of APP through an increase in α-secretase activity. The in vivo findings presented here demonstrate for the first time the therapeutic potential of P2X7R antagonism in the treatment of familiar Alzheimer's disease (FAD).

Opposite effects of P2X7 and P2Y2 nucleotide receptors on α-secretase-dependent APP processing in Neuro-2a cells.

The amyloid precursor protein (APP) is proteolytically processed by ß- and γ-secretases to release amyloid-ß peptide (Aß), the main component found in senile plaques of Alzheimer's disease (AD) patient brains. Alternatively, APP can be cleaved within the Aß sequence by α-secretase, thus precluding the generation of Aß. We have demonstrated that activation of the P2X7 receptor leads to a reduction of α-secretase activity in Neuro-2a cells. Moreover, the P2X7 ligand 2'(3')-O-(4-benzoylbenzoyl) ATP (BzATP) can also activate a different P2 receptor in these cells. This receptor, whose pharmacology resembles that of the P2Y(2) receptor, has an opposite effect, leading to increases in α-secretase activity. Our study suggests that P2X7R and P2Y(2)R could be novel therapeutic targets in AD.

Characteristics and consequences of muscarinic receptor activation by tau protein.

It was recently suggested that tau protein released as a result of neuronal death is toxic to neighbouring cells, an effect that is mediated through the activation of muscarinic M1 and/or M3 receptors. Nevertheless, why tau protein and not other native muscarinic agonists, like ACh, can induce this neurotoxicity remains unknown. To clarify this issue, we analysed the different responses and properties of muscarinic receptors in response to stimulation by tau or ACh. The results revealed that the tau protein has an affinity for muscarinic receptors of around one order of magnitude higher than that of ACh. Furthermore, while the repeated stimulation with ACh induces desensitization of the muscarinic receptors, reiterate stimulation with tau failed to produce this phenomenon. Finally, we found the tau protein to be very stable in the extracellular milieu. These studies provide valuable information to help understand tau toxicity on neural cells bearing M1 or M3 muscarinic receptors and its contribution to neurodegenerative progression in tauopathies.

Inhibition of the ATP-gated P2X7 receptor promotes axonal growth and branching in cultured hippocampal neurons.

During the establishment of neural circuits, the axons of neurons grow towards their target regions in response to both positive and negative stimuli. Because recent reports show that Ca2+ transients in growth cones negatively regulate axonal growth, we studied how ionotropic ATP receptors (P2X) might participate in this process. Our results show that exposing cultured hippocampal neurons to ATP induces Ca2+ transients in the distal domain of the axon and the concomitant inhibition of axonal growth. This effect is mediated by the P2X7 receptor, which is present in the growth cone of the axon. Pharmacological inhibition of P2X7 or its silencing by shRNA interference induces longer and more-branched axons, coupled with morphological changes to the growth cone. Our data suggest that these morphological changes are induced by a signalling cascade in which CaMKII and FAK activity activates PI3-kinase and modifies the activity of its downstream targets. Thus, in the absence or inactivation of P2X7 receptor, axons grow more rapidly and form more branches in cultured hippocampal neurons, indicative that ATP exerts a negative influence on axonal growth. These data suggest that P2X7 antagonists have therapeutic potential to promote axonal regeneration.

Physiological role of extracellular nucleotides at the central nervous system: signalling through P2X and P2Y receptors

En los últimos años los receptores de nucleótidos, receptores ionotrópicosP2X1-7 y metabotrópicos P2Y1, 2, 4, 6, 11, 12, 13, 14, han adquirido una importancia excepcionaldebido a su localización estratégica en órganos y tejidos, a su gran variedadjunto con la complejidad de vías de señalización a las que están asociados y a lasprimeras evidencias de importantes alteraciones debidas a su mal funcionamiento.Nuestro grupo ha sido pionero en la caracterización estos receptores en el sistemanervioso, donde definimos su localización y su funcionalidad. La abundante presencia,a nivel presináptico, de las subunidades P2X3 y P2X7 debe ser resaltada,donde gracias a la entrada de calcio inducen la exocitosis de varios neurotransmisores,como glutamato, GABA, catecolaminas y acetilcolina entre otros, como hasido descrito por nuestro grupo en trabajos previos. Además, estos receptores inducenuna profunda remodelación del citoesqueleto de las terminales nerviosas yde los mecanismos exocitóticos a través de la CaMKII y pueden interactuar conotros receptores ionotrópicos y metabotrópicos co-existentes en sus cercanías. Lamayoría de los receptores P2Y también están presentes en las células nerviosas,activando vías de señalización a través de una gran variedad de cascadas intracelulares.Recientemente hemos demostrado que los receptores metabotrópicos P2Ypertenecientes a la sub-familia de receptores activados por ADP, especialmente elP2Y13, están conectados con la señalización hacia GSK3 y β-catenina, lo que abrenuevas vías para la comprensión de la función de los nucleótidos en la supervivenciay el mantenimiento de las células nerviosas. Además, tanto los receptores P2Xcomo los P2Y juegan un papel en los estadíos iniciales del desarrollo y en lamaduración neuronal donde su función aún ha de ser plenamente comprendida.Los receptores de nucleótidos son también muy abundantes en las células gliales, y nuestro grupo ha demostrado que la mayoría de los receptores P2Y están presentesy son plenamente funcionales en astrocitos en cultivo, donde, dependiendo delsubtipo de receptor, activan una gran variedad de cascadas de señalización

In the last few years nucleotide receptors, the ionotropic P2X1-7 subunits andthe metabotropic P2Y1, 2, 4, 6, 11, 12, 13, 14, have acquired an excepcional importance dueto their strategic location in organs and tissues, their great variety along with thecomplexity of the associated signalling pathways and the first evidence of theserious alterations entailed in their dysfunctions. Our group has been pioneer inthe characterization of these receptors in the nervous system, where we definedtheir location and functionality. The abundant presence, at a presynaptic level, ofP2X3 and P2X7 should be emphasized, where by means of calcium intake theyinduce neurotransmitter exocytosis, such as glutamate, GABA, catecholamines andacetylcholine among others, as described in previous works by our group. In addition, they induce an extensive remodeling of the terminal’s cytoskeleton and exocytoticmechanisms through CaMKII and they can interact widely with other ionotropicand metabotropic receptors co-existing in nearby areas. Neural cells alsoexhibit the presence of most P2Y receptors signalling through a large variety ofintracellular cascades. Recently we have demostrated that P2Y metabotropic receptorsof the sub-family activated by ADP, especially P2Y13, are connected withthe signalling towards GSK3 and Beta-catenin, opening new ways of understading thenucleotide function in survival and maintenance of neural cells. In addition bothP2X and P2Y receptors play a role in early developmental stages and neural maturationwhere their function has to be fully understanded. Nucleotide receptorsare also very abundant in glial cells, and our group has shown that most P2Yreceptors are present and fully functional in cultured astrocytes, where, dependingon the subtype receptor they activate a large variety of signalling cascades