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.

Huntington's disease-specific mis-splicing unveils key effector genes and altered splicing factors.

Correction of mis-splicing events is a growing therapeutic approach for neurological diseases such as spinal muscular atrophy or neuronal ceroid lipofuscinosis 7, which are caused by splicing-affecting mutations. Mis-spliced effector genes that do not harbour mutations are also good candidate therapeutic targets in diseases with more complex aetiologies such as cancer, autism, muscular dystrophies or neurodegenerative diseases. Next-generation RNA sequencing (RNA-seq) has boosted investigation of global mis-splicing in diseased tissue to identify such key pathogenic mis-spliced genes. Nevertheless, while analysis of tumour or dystrophic muscle biopsies can be informative on early stage pathogenic mis-splicing, for neurodegenerative diseases, these analyses are intrinsically hampered by neuronal loss and neuroinflammation in post-mortem brains. To infer splicing alterations relevant to Huntington's disease pathogenesis, here we performed intersect-RNA-seq analyses of human post-mortem striatal tissue and of an early symptomatic mouse model in which neuronal loss and gliosis are not yet present. Together with a human/mouse parallel motif scan analysis, this approach allowed us to identify the shared mis-splicing signature triggered by the Huntington's disease-causing mutation in both species and to infer upstream deregulated splicing factors. Moreover, we identified a plethora of downstream neurodegeneration-linked mis-spliced effector genes that-together with the deregulated splicing factors-become new possible therapeutic targets. In summary, here we report pathogenic global mis-splicing in Huntington's disease striatum captured by our new intersect-RNA-seq approach that can be readily applied to other neurodegenerative diseases for which bona fide animal models are available.

The contribution of tourism to municipal solid waste generation: A mixed demand-supply approach on the island of Tenerife.

Tourism contributes substantially to municipal solid waste generation, yet the waste from tourism systematically remains hidden behind residential waste flows. As a result, municipal fees are set without precise information about waste producers' contributions, causing budget imbalances and cross-subsidies between residential and economic activities. To estimate tourism's contribution to mixed waste generation in an island destination, socio-demographic, economic and disposal-related factors are modelled using municipal panel data from 2004 to 2015 for Tenerife (Spain). In contrast to previous studies, a mixed demand-supply approach is adopted to estimate the contribution of main tourism activities to mixed waste generation, thus, differentiating between tourists and residents' contributions. An auxiliary model is used to isolate employment levels in tourism activities attributable to residents' consumption and to capture tourists' and residents' mobility on the island. Estimates show that main tourism activities generate 0.40 kg of mixed waste per tourist daily, while residential and economic sectors account for 1.19 kg per resident daily. This tourism contribution is significantly lower compared to other studies, as it captures tourism's contribution to mixed waste generation, attributable only to tourists, following a mixed demand-supply approach. These results shift impacts from tourists to main tourism activities, which highlights the choices made by producers rather than the final customers and reinforces the producers extended responsibility principle. The implementation of a Pay-As-You-Throw tariff for mixed waste is discussed as a way of promoting waste prevention and recycling, as well as avoiding cross-subsidies among waste producers and, as a result, imbalances in municipal budgets.

The dual-function of lipoic acid groups as surface anchors and sulfhydryl reactive sites on polymer-stabilized QDs and Au nanocolloids.

Coating inorganic nanocrystals [e.g., quantum dots (QDs) and gold nanoparticles] with polymer ligands presenting multiple lipoic acid anchoring groups provides nanocolloids with remarkable long-term colloidal and photophysical stability. Here, we show that the natural swelling of macromolecules leaves a fraction of the lipoic acid groups in the surface coating free, which are targeted for activation and conjugation to target molecules, using the reliable sulfhydryl-to-maleimide reaction. This implies that simple and efficient functionalization of the nanocrystals can be achieved without introducing additional reactive groups in the coating. We apply a photomediated ligand exchange strategy to luminescent QDs and AuNPs and react the resulting nanocrystals with maleimide Cy3 dye. We then use optical absorption and resonance energy transfer measurements applied to QD-Cy3 and AuNP-Cy3 conjugates to extract estimates for the fraction of accessible lipoic acid groups per QD or AuNP. In addition, we demonstrate the potential utility of this approach by constructing a ratiometric pH sensor made of QD-SNARF conjugates. Our ligand design combined with the photoligation strategy yield colloidally stable dispersions of QDs and AuNPs that present accessible reactive thiols, without introducing new functionalities or requiring disulfide reducing reagents, making them useful for potential use in applications such as biological sensing and imaging.

Hospital productivity bias when not adjusting for cost heterogeneity: The case of Spain.

This research quantifies the bias caused in hospital productivity measurements when cost heterogeneity is not considered. A multi-output stochastic cost frontier under a normalised translog specification is used to approximate the structure of technology of a sample of public general hospitals in Spain during the period 2002-2009. To control for observable heterogeneity in costs, a set of variables related to hospital characteristics are included in the cost frontier specification (i.e., hospital complexity, degree of specialisation, availability of outpatient clinics, variety of high-technology equipment available, teaching activity and quality of care), whereas unobservable heterogeneity is accounted for by means of individual dummy variables. A measure of hospitals' cost efficiency is first obtained, and the analysis is then completed by measuring and decomposing the total factor productivity index (TFP-I) change. Findings reveal that controlling for heterogeneity decreases total productivity from an annual average rate of 0.028% to 1.330%, mainly driven by the negative contribution of the cost efficiency change component. Hence, a bias of 1.303 percentage points in the overall TFP-I is found as consequence of not controlling for heterogeneity. In addition to this, if heterogeneity factors are not accounted for, the mean cost efficiency index during the period analysed is 0.730, figure that increases up to 0.974 if heterogeneity is considered. Hence, the omission of heterogeneity leads to a bias of 24.4 percentage points in the mean cost efficiency. Therefore, not adjusting for heterogeneity in costs gives rise to distorted measurements of hospital productivity, as well as distortions in the contribution of each of its components, which may lead to the adoption of inadequate policies and decisions on resource allocation.

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

Autism-like phenotype and risk gene mRNA deadenylation by CPEB4 mis-splicing.

Common genetic contributions to autism spectrum disorder (ASD) reside in risk gene variants that individually have minimal effect sizes. As environmental factors that perturb neurodevelopment also underlie idiopathic ASD, it is crucial to identify altered regulators that can orchestrate multiple ASD risk genes during neurodevelopment. Cytoplasmic polyadenylation element binding proteins 1-4 (CPEB1-4) regulate the translation of specific mRNAs by modulating their poly(A)-tails and thereby participate in embryonic development and synaptic plasticity. Here we find that CPEB4 binds transcripts of most high-confidence ASD risk genes. The brains of individuals with idiopathic ASD show imbalances in CPEB4 transcript isoforms that result from decreased inclusion of a neuron-specific microexon. In addition, 9% of the transcriptome shows reduced poly(A)-tail length. Notably, this percentage is much higher for high-confidence ASD risk genes, correlating with reduced expression of the protein products of ASD risk genes. An equivalent imbalance in CPEB4 transcript isoforms in mice mimics the changes in mRNA polyadenylation and protein expression of ASD risk genes and induces ASD-like neuroanatomical, electrophysiological and behavioural phenotypes. Together, these data identify CPEB4 as a regulator of ASD risk genes.

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

[Biomechanical analysis of cervical spine movement on removal of motorcycle helmets]. / Análisis biomecánico del movimiento cervical en la extracción del casco en motoristas.

OBJECTIVES: To measure cervical spine movement during removal of a motorcycle helmet by health care professionals. MATERIAL AND METHODS: Observational study using biomechanical inertial sensors to detect movement in the spinal column during removal of helmets. RESULTS: Thirty-four emergency medicine specialists and nurses participated. The mean (SD) rotation was 1.14° (0.82°) to the left and 3.30° (1.69°) to the right (P<.001). Mean flexion was 9.82° (7.46°) and mean extension was 6.23° (6.86°) (P<.001). Mean lateral displacement was 5.73° (2.97°) to the left and 5.62° (8.22°) to the right (P=.678). The removal maneuvers took a mean of 70 seconds (4 seconds). CONCLUSION: Helmet removal was completed in an average of 70 seconds with flexion and rotation mainly toward the side where the professional supporting the head was positioned.

OBJETIVO: Determinar el movimiento cervical durante la extracción de un casco realizada por profesionales sanitarios. METODO: Estudio observacional mediante análisis biomecánico con sensores inerciales de los movimientos producidos en la columna durante la extracción de un casco. RESULTADOS: La muestra final la componen 34 profesionales de servicios de urgencias y emergencias. La rotación fue de 1,14 (DE 0,82)° hacia el lado izquierdo y de 3,30 (1,69)° hacia el lado derecho (p < 0,001). La flexoextensión fue de 9,82 (7,46)° para la flexión y de 6,23 (6,86)° para la extensión (p < 0,001). La lateralización fue de 5,73 (2,97)° para el lado izquierdo y de 5,62 (8,22)° para el lado derecho (p = 0,678). El tiempo medio de realización de la extracción fue 70 (4) seg. CONCLUSIONES: La extracción del casco se realizó en 70 segundos con flexión y rotación hacia el lado donde se encuentra colocado el profesional que sujeta la cabeza.

Análisis biomecánico del movimiento cervical en la extracción del casco en motoristas / Biomechanical analysis of cervical spine movement on removal of motorcycle helmets

Objetivo. Determinar el movimiento cervical durante la extracción de un casco realizada por profesionales sanitarios. Métodos. Estudio observacional mediante análisis biomecánico con sensores inerciales de los movimientos producidos en la columna durante la extracción de un casco. Resultados. La muestra final la componen 34 profesionales de servicios de urgencias y emergencias. La rotación fue de 1,14 (DE 0,82)° hacia el lado izquierdo y de 3,30 (1,69)° hacia el lado derecho (p < 0,001). La flexoextensión fue de 9,82 (7,46)° para la flexión y de 6,23 (6,86)° para la extensión (p < 0,001). La lateralización fue de 5,73 (2,97)° para el lado izquierdo y de 5,62 (8,22)° para el lado derecho (p = 0,678). El tiempo medio de realización de la extracción fue 70 (4) seg. Conclusión. La extracción del casco se realizó en 70 segundos con flexión y rotación hacia el lado donde se encuentra colocado el profesional que sujeta la cabeza (AU)

Objective. To measure cervical spine movement during removal of a motorcycle helmet by health care professionals. Methods. Observational study using biomechanical inertial sensors to detect movement in the spinal column during removal of helmets. Results. Thirty-four emergency medicine specialists and nurses participated. The mean (SD) rotation was 1.14° (0.82°) to the left and 3.30° (1.69°) to the right (P<.001). Mean flexion was 9.82° (7.46°) and mean extension was 6.23° (6.86°) (P<.001). Mean lateral displacement was 5.73° (2.97°) to the left and 5.62° (8.22°) to the right (P=.678). The removal maneuvers took a mean of 70 seconds (4 seconds). Conclusion. Helmet removal was completed in an average of 70 seconds with flexion and rotation mainly toward the side where the professional supporting the head was positioned (AU)

Specific Temporal Distribution and Subcellular Localization of a Functional Vesicular Nucleotide Transporter (VNUT) in Cerebellar Granule Neurons.

Adenosine triphosphate (ATP) is an important extracellular neurotransmitter that participates in several critical processes like cell differentiation, neuroprotection or axon guidance. Prior to its exocytosis, ATP must be stored in secretory vesicles, a process that is mediated by the Vesicular Nucleotide Transporter (VNUT). This transporter has been identified as the product of the SLC17A9 gene and it is prominently expressed in discrete brain areas, including the cerebellum. The main population of cerebellar neurons, the glutamatergic granule neurons, depends on purinergic signaling to trigger neuroprotective responses. However, while nucleotide receptors like P2X7 and P2Y13 are known to be involved in neuroprotection, the mechanisms that regulate ATP release in relation to such events are less clearly understood. In this work, we demonstrate that cerebellar granule cells express a functional VNUT that is involved in the regulation of ATP exocytosis. Numerous vesicles loaded with this nucleotide can be detected in these granule cells and are staining by the fluorescent ATP-marker, quinacrine. High potassium stimulation reduces quinacrine fluorescence in granule cells, indicating they release ATP via calcium dependent exocytosis. Specific subcellular markers were used to assess the localization of VNUT in granule cells, and the transporter was detected in both the axonal and somatodendritic compartments, most predominantly in the latter. However, co-localization with the specific lysosomal marker LAMP-1 indicated that VNUT can also be found in non-synaptic vesicles, such as lysosomes. Interestingly, the weak co-localization between VNUT and VGLUT1 suggests that the ATP and glutamate vesicle pools are segregated, as also observed in the cerebellar cortex. During post-natal cerebellar development, VNUT is found in granule cell precursors, co-localizing with markers of immature cells like doublecortin, suggesting that this transporter may be implicated in the initial stages of granule cell development.

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.

Role of P2X7 and P2Y2 receptors on α-secretase-dependent APP processing: Control of amyloid plaques formation "in vivo" by P2X7 receptor.

Amyloid precursor protein (APP) is expressed in a large variety of neural and non-neural cells. The balance between non-pathogenic and pathologic forms of APP processing, mediated by α-secretase and ß-secretase respectively, remains a crucial step to understand ß-amyloid, Aß42 peptide, formation and aggregation that are at the origin of the senile plaques in the brain, a characteristic hallmark of Alzheimer's disease (AD). In Neuro-2a, a neuroblastoma cell line that constitutively expresses APP, activation of the P2X7 receptor leads to reduction of α-secretase activity, the opposite effect being obtained by P2Y2 receptor activation. The in vivo approach was made possible by the use of J20 mice, a transgenic mouse model of familial Alzheimer's disease (FAD) expressing human APP mutant protein. This animal exhibits prominent amyloid plaques by six months of age. In vivo inhibition of the P2X7 receptor induced a significant decrease in the number and size of hippocampal amyloid plaques. This reduction is mediated by an increase in the proteolytic processing of APP through α-secretase activity, which correlates with an increase in the phosphorylated form of GSK-3, a less active form of this enzyme. The in vivo findings corroborate the therapeutic potential of P2X7 antagonists in the treatment of FAD.