DUSP1/MKP-1 represents another piece in the P2X7R intracellular signaling puzzle in cerebellar cells: our last journey with Mª Teresa along the purinergic pathways of Eden.

The P2X7 receptor (P2X7R) stands out within the purinergic family as it has exclusive pharmacological and regulatory features, and it fulfills distinct roles depending on the type of stimulation and cellular environment. Tonic activation of P2X7R promotes cell proliferation, whereas sustained activation is associated with cell death. Yet strikingly, prolonged P2X7R activation in rat cerebellar granule neurons and astrocytes does not affect cell survival.

BCI, an inhibitor of the DUSP1 and DUSP6 dual specificity phosphatases, enhances P2X7 receptor expression in neuroblastoma cells.

P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where high levels of P2RX7 are correlated with a poor prognosis for patients. Tonic activation of P2X7 receptor favors cell metabolism and angiogenesis, thereby promoting cancer cell proliferation, immunosuppression, and metastasis. Although understanding the mechanisms that control P2X7 receptor levels in neuroblastoma cells could be biologically and clinically relevant, the intracellular signaling pathways involved in this regulation remain poorly understood.

Combining low-density cell culture, single-cell tracking, and patch-clamp to monitor the behavior of postnatal murine cerebellar neural stem cells.

Low-density cell culture of the postnatal cerebellum, combined with live imaging and single-cell tracking, allows the behavior of postnatal cerebellar neural stem cells (NSCs) and their progeny to be monitored. Cultured cerebellar NSCs maintain their neurogenic nature giving rise, in the same relative proportions that exist to the neuronal progeny generated by the three postnatal cerebellar neurogenic niches. This protocol describes the identification of the nature of the progeny through both post-imaging immunocytochemistry and patch-clamp recordings.

P2X7 receptors in the central nervous system.

For the past three decades, our laboratory has conducted pioneering research to elucidate the complexity of purinergic signaling in the CNS, alone and in collaboration with other groups, inspired by the ground-breaking efforts of Geoffrey Burnstock. This review summarizes our contribution to understand the nucleotide receptor signaling in the CNS with a special focus on the P2X7 receptor.

Salient brain entities labelled in P2rx7-EGFP reporter mouse embryos include the septum, roof plate glial specializations and circumventricular ependymal organs.

The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits relevant functions in the regulation of the central nervous system (CNS) development. Amongst the components of the purinergic system, the ionotropic P2X7 receptor (P2X7R) stands out as a potential regulator of brain pathology and physiology. Thus, P2X7R is known to regulate crucial aspects of neuronal cell biology, including axonal elongation, path-finding, synapse formation and neuroprotection.

Live Imaging Reveals Cerebellar Neural Stem Cell Dynamics and the Role of VNUT in Lineage Progression.

Little is known about the intrinsic specification of postnatal cerebellar neural stem cells (NSCs) and to what extent they depend on information from their local niche. Here, we have used an adapted cell preparation of isolated postnatal NSCs and live imaging to demonstrate that cerebellar progenitors maintain their neurogenic nature by displaying hallmarks of NSCs. Furthermore, by using this preparation, all the cell types produced postnatally in the cerebellum, in similar relative proportions to those observed in vivo, can be monitored.

Changes in melatonin receptor expression in a murine model of glaucoma.

Purpose: The objective of this study was to evaluate the changes in the melatoninergic receptors of DBA/2J and C57BL/6J mice with the development of glaucoma. DBA/2J mice are widely used to study the physiopathology of glaucoma due to the similarities of their eyes to human eyes and the resulting similarity in the development of their pathology. In addition, melatoninergic receptors are known for their control of intraocular pressure (IOP), reducing the production of aqueous humor; however, little is known about their relationship with the development of this pathology.

Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment.

Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca binding. One of the most common calcium indicators is the ratiometric dye Fura-2.

Physiopathological Role of the Vesicular Nucleotide Transporter (VNUT) in the Central Nervous System: Relevance of the Vesicular Nucleotide Release as a Potential Therapeutic Target.

Vesicular storage of neurotransmitters, which allows their subsequent exocytotic release, is essential for chemical transmission in the central nervous system. Neurotransmitter uptake into secretory vesicles is carried out by vesicular transporters, which use the electrochemical proton gradient generated by a vacuolar H-ATPase to drive neurotransmitter vesicular accumulation. ATP and other nucleotides are relevant extracellular signaling molecules that participate in a variety of biological processes.

Dual-Specificity Phosphatase Regulation in Neurons and Glial Cells.

Dual-specificity protein phosphatases comprise a protein phosphatase subfamily with selectivity towards mitogen-activated protein (MAP) kinases, also named MKPs, or mitogen-activated protein kinase (MAPK) phosphatases. As powerful regulators of the intensity and duration of MAPK signaling, a relevant role is envisioned for dual-specificity protein phosphatases (DUSPs) in the regulation of biological processes in the nervous system, such as differentiation, synaptic plasticity, and survival. Important neural mediators include nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) that contribute to transcriptional induction and post-translational mechanisms of DUSP protein stabilization to maintain neuronal survival and differentiation.

P2 receptor interaction and signalling cascades in neuroprotection.

Nucleotides can contribute to the survival of different glial and neuronal models at the nervous system via activation of purinergic P2X and P2Y receptors. Their activation counteracts different proapoptotic events, such as excitotoxicity, mitochondrial impairment, oxidative stress and DNA damage, which concur to elicit cell loss in different processes of neurodegeneration and brain injury. Thus, it is frequent to find that different neuroprotective mediators converge in the activation of the same intracellular survival pathways to protect cells from death.

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.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results.

Changes in P2Y Purinergic Receptor Expression in the Ciliary Body in a Murine Model of Glaucoma.

Glaucoma is a neuropathology, often accompanied by an elevated intraocular pressure (IOP), which can lead to blindness. Since DBA/2J mice develop glaucoma, several studies of the physiopathology of glaucoma have been reported in this animal model. It is also known that purinergic receptors are involved in the pathology of glaucoma by controlling aqueous humor production and drainage and therefore controlling IOP.

An Update on P2Y Receptor Signalling and Function.

The distribution of nucleotide P2Y receptors across different tissues suggests that they fulfil key roles in a number of physiological and pathological conditions. P2Y is one of the latest P2Y receptors identified, a novel member of the Gi-coupled P2Y receptor subfamily that responds to ADP, together with P2Y and P2Y. Pharmacological studies drew attention to this new ADP receptor, with a pharmacology that overlaps that of P2Y receptors but with unique features and roles.

Expression and function of the metabotropic purinergic P2Y receptor family in experimental seizure models and patients with drug-refractory epilepsy.

Objective: ATP is released into the extracellular space during pathologic processes including increased neuronal firing. Once released, ATP acts on P2 receptors including ionotropic P2X and metabotropic P2Y receptors, resulting in changes to glial function and neuronal network excitability. Evidence suggests an involvement of P2Y receptors in the pathogenesis of epilepsy, but there has been no systematic effort to characterize the expression and function of the P2Y receptor family during seizures and in experimental and human epilepsy.

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.

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.

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.

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 β-secretase respectively, remains a crucial step to understand β-amyloid, Aβ42 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.

Purinergic Receptors in Spinal Cord-Derived Ependymal Stem/Progenitor Cells and Their Potential Role in Cell-Based Therapy for Spinal Cord Injury.

Spinal cord injury (SCI) is a major cause of paralysis with no current therapies. Following SCI, large amounts of ATP and other nucleotides are released by the traumatized tissue leading to the activation of purinergic receptors that, in coordination with growth factors, induce lesion remodeling and repair. We found that adult mammalian ependymal spinal cord-derived stem/progenitor cells (epSPCs) are capable of responding to ATP and other nucleotidic compounds, mainly through the activation of the ionotropic P2X4, P2X7, and the metabotropic P2Y1 and P2Y4 purinergic receptors.