Purinergic receptors in embryonic and adult neurogenesis

Accepted Manuscript Purinergic receptors in embryonic and adult neurogenesis Ágatha Oliveira, Peter Illes, Henning Ulrich PII:

S0028-3908(15)30131-3

DOI:

10.1016/j.neuropharm.2015.10.008

Reference:

NP 6033

To appear in:

Neuropharmacology

Received Date: 28 June 2015 Revised Date:

1 October 2015

Accepted Date: 4 October 2015

Please cite this article as: Oliveira, Á., Illes, P., Ulrich, H., Purinergic receptors in embryonic and adult neurogenesis, Neuropharmacology (2015), doi: 10.1016/j.neuropharm.2015.10.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Purinergic receptors in embryonic and adult neurogenesis.

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Ágatha Oliveira1, Peter Illes2, Henning Ulrich1. 1

Departamento de Bioquímica, Instituto de Química, Universidade de São

Paulo, São Paulo, SP 05508-900, Av. Prof. Lineu Prestes, 748, São Paulo, SP, Brazil. 2

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Rudolf-Boehm-Institut für Pharmakologie und Toxikologie der Universität

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Leipzig, Haertelstrasse 16-18, 04107, Leipzig, Germany

Key words: purinergic receptors, neural embryonic development, neural

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stemcells, neurodegenerative disorders, cell therapy Send correspondence to

Prof. Henning Ulrich

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e-mail: [email protected] or to

Prof. Peter Illes

e-mail:[email protected] at their above postal address.

Abbreviations: ATP: adenosine 5′-triphosphate; ADP: adenosine 5′diphosphate; UTP: uridine-5'-triphosphate; CNS: central nervous system ; NPC: neural progenitor cell; SVZ: subventricular zone; SGZ: subgranular zone; DG: dentate gyrus; NSC: neural stem cell; EC: embryonic carcinoma cell; iPSC: induced pluripotent stem cell.

ACCEPTED MANUSCRIPT ABSTRACT ATP (adenosine 5′-triphosphate), one of the most ancient neurotransmitters, exerts essential functions in the brain, including neurotransmission and modulation of synaptic activity. Moreover, this nucleotide has been attributed with trophic properties and experimental evidence points to the participation of

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ATP-activated P2X and P2Y purinergic receptors in embryonic brain development as well as in adult neurogenesis for maintenance of normal brain functions and neuroregeneration upon brain injury. We discuss here the available data on purinergic P2 receptor expression and function during brain

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development and in the neurogenic zones of the adult brain, as well as the insights based on the use of in vitro stem cell cultures. While several P2

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receptor subtypes were shown to be expressed during in vitro and in vivo neurogenesis, specific functions have been proposed for P2Y1, P2Y2 metabotropic as well as P2X2 ionotropic receptors to promote neurogenesis. Further, the P2X7 receptor is suggested to function in the maintenance of pools of neural stem and progenitor cells through induction of proliferation or cell death, depending on the microenvironment. Pathophysiological actions have

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been proposed for this receptor in worsening damage in brain disease. The P2X7 receptor and possibly additional P2 receptor subtypes have been implicated in pathophysiology of neurological diseases including Parkinson’s disease, Alzheimer’s disease and epilepsy. New strategies in cell therapy could

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involve modulation of purinergic signaling, either in the achievement of more effective protocols to obtain viable and homogeneous cell populations or in the

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process of functional engraftment of transplanted cells into the damaged brain.

ACCEPTED MANUSCRIPT

Purinergic receptors in embryonic and adult neurogenesis.

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Ágatha Oliveira1, Peter Illes2, Henning Ulrich1. 1

Departamento de Bioquímica, Instituto de Química, Universidade de São

Paulo, São Paulo, SP 05508-900, Av. Prof. Lineu Prestes, 748, São Paulo, SP, Brazil. 2

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Rudolf-Boehm-Institut für Pharmakologie und Toxikologie der Universität

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Leipzig, Haertelstrasse 16-18, 04107, Leipzig, Germany

Key words: purinergic receptors, neural embryonic development, neural

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stemcells, neurodegenerative disorders, cell therapy Send correspondence to

Prof. Henning Ulrich

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e-mail: [email protected] or to

Prof. Peter Illes

e-mail:[email protected] at their above postal address.

Abbreviations: ATP: adenosine 5′-triphosphate; ADP: adenosine 5′diphosphate; UTP: uridine-5'-triphosphate; CNS: central nervous system ; NPC: neural progenitor cell; SVZ: subventricular zone; SGZ: subgranular zone; DG: dentate gyrus; NSC: neural stem cell; EC: embryonic carcinoma cell; iPSC: induced pluripotent stem cell.

ACCEPTED MANUSCRIPT ABSTRACT ATP (adenosine 5′-triphosphate), one of the most ancient neurotransmitters, exerts essential functions in the brain, including neurotransmission and modulation of synaptic activity. Moreover, this nucleotide has been attributed with trophic properties and experimental evidence points to the participation of

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ATP-activated P2X and P2Y purinergic receptors in embryonic brain development as well as in adult neurogenesis for maintenance of normal brain functions and neuroregeneration upon brain injury. We discuss here the available data on purinergic P2 receptor expression and function during brain

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development and in the neurogenic zones of the adult brain, as well as the insights based on the use of in vitro stem cell cultures. While several P2 receptor subtypes were shown to be expressed during in vitro and in vivo

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neurogenesis, specific functions have been proposed for P2Y1, P2Y2 metabotropic as well as P2X2 ionotropic receptors to promote neurogenesis. Further, the P2X7 receptor is suggested to function in the maintenance of pools of neural stem and progenitor cells through induction of proliferation or cell death, depending on the microenvironment. Pathophysiological actions have

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been proposed for this receptor in worsening damage in brain disease. The P2X7 receptor and possibly additionalP2 receptor subtypes have been implicated in pathophysiology of neurological diseases including Parkinson’s disease, Alzheimer’s disease and epilepsy. New strategies in cell therapy could

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involve modulation of purinergic signaling, either in the achievement of more effective protocols to obtain viable and homogeneous cell populations or in the

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process of functional engraftment of transplanted cells into the damaged brain.

ACCEPTED MANUSCRIPT 1. Introduction Purinergic signaling, based on the activation of signaling cues by ATP is one of the most ancient signaling throughout evolution, expanding the longknown function of ATP as store for chemical energy within cells for metabolism. First major attention was drawn on ATP as a molecule with neurotransmitter

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properties based on a pioneering study of Geoffrey Burnstock (Burnstock, 1970), which two years later led to the emergence of the purinergic system hypothesis (Burnstock, 1972).

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Extracellular ATP is physiologically released from cells by exocytosis or through membrane channels. The levels of the bioavailable extracellular ATP are controlled by enzymes that catalyze its degradation, the ectonucleotidases.

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They act by hydrolyzing ATP, adenosine 5′-diphosphate (ADP) and uridine-5'triphosphate (UTP), whose sub-products can interact with the P2 subtype of purinergic receptors. The enzymes involved in this process include the ectonucleoside triphosphate diphosphohydrolases (E-NTPDases, NTPDase1– 8) and the ectonucleotide pyrophosphatase / phosphodiesterases (E-NPPs). These reactions can result also in monophosphate nucleotides degraded into

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adenosine by ecto-alkaline phosphatase and ecto-5′-nucleotidase/CD73 that are able to bind to P1 receptors (Zimmerman, 2001; 2006). Subsequent studies contributed to the elucidation of the purinergic system. Purinergic receptors are

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classified into two different subtypes, P1 and P2 (Burnstock, 1978). P1 receptors are activated by adenosine and can be subdivided into four types: A1, A2A, A2B and A3, coupled to different G proteins that are associated with

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various intracellular transduction cascades (Ciruela et al., 2010). P2 receptors are subdivided into two classes based on their structural

properties: (1) P2X ionotropic receptors assemble as functional homo- or heteromeric ligand-gated receptors from three subunits of the P2X1-7 subtypes, and (2) P2Y metabotropic receptors occur in mammals as eight subtypes, namely P2Y1-2,4,6,11-14 (Burnstock, 2007). P2X receptors are activated by ATP and its analogues such as α,β-methylene ATP and dibenzoyl ATP (BzATP), binding to the extracellular ligand-binding site and leading to a conformational change that opens a channel permeable to Na+, K+, and

ACCEPTED MANUSCRIPT Ca2+(Khakh et al., 2001). The ensuing depolarization and the opening of voltage-sensitive Ca2+ channels induces intracellular signaling, and triggers different cellular responses. G protein-coupledP2Y receptor ligands include ATP, ADP, UTP, UDP or UDP-glucose (Burnstock, 1997). While ATP is an agonist for all P2 receptors,

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the further mentioned nucleotides are agonists for only some of the P2Y subtypes: ADP for P2Y1, P2Y12 and P2Y13 receptors; UTP for P2Y2, P2Y4 and Y6 receptors; UDP for P2Y6 and P2Y14 receptors and UDP-glucose for

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P2Y14 receptors (Burnstock, 2014).

Depending on the activated P2Y receptor subtype, the induced signal transduction

pathway

may

vary.

P2Y1,2,4,6,11

receptors

activate

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phospholipase C-β, promoting subsequent cleavage of phosphatidylinositol 4,5bisphosphate into inositol 1,4,5-trisphosphate (InsP3) and releasing Ca2+ from endoplasmic reticulum (Dubyak and el-Moatassim, 1993). P2Y12-14 subtypes act by inhibiting adenylyl cyclase activity, reducing intracellular cAMP levels and decreasing Ca2+ cellular concentration (Abbracchio et al., 2006). These alterations in calcium levels modulate several secondary messengers involved

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in many physiological processes, like cell death, proliferation and stemcell differentiation. However, Gprotein-mediated pathways may vary in different cellular contexts.

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Purinergic receptor activation may be of para- or autocrine nature. Besides of classic neurotransmitter signaling within the synaptic cleft, it is

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known that ATP through P2 receptor activation functions in modulating synaptic activity, such as i.e. through presynaptic receptors. Paracrine signaling, including ATP release is also characteristic for astrocytes, which by this mechanism regulate neuronal activity (Pascual et al. 2005). Furthermore, as explained in the following, astrocytic signaling through purinergic receptors also promotes ATP release and a gradient of this trophic compound which regulates the migration of differentiating neuroblasts (Ulrich et al. 2012). Overall, various P2 receptor subtypes are expressed by almost every cell type, presumably participating in all cellular processes, including proliferation, differentiation and cell fate determination as well as in the elimination of undesired cell 2

ACCEPTED MANUSCRIPT populations. Importantly, aberrant purinergic receptor signaling may be the cause or result of various disease states, including inflammation, tissue degeneration and tumor formation.

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2. Purinergic System in the Developing Nervous System Mammalian embryo formation starts from a single totipotent cell, the zygote, which undergoes consecutive morphological and physiological changes to form the blastocyst. In this stage, cells belonging to blastocyst inner cell mass

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of embryo development lose their ability to form placental structure and can only produce embryonic tissue cells, i.e., they become pluripotent (Mitalipov and

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Wolf, 2009). This cell mass rearranges and forms a discoid structure giving rise to the three germ layers and the neural tube. Transcriptional and epigenetic signals triggered by cell-to-cell communication and the activation of signaling cascades induce the migration of cells to diverse locations in the embryo (Savagner, 2010). Mesenchymal transitory cells migrate from the dorsal neural tube to produce most of the peripheral nervous system cells, including sensory,

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sympathetic and enteric neurons (Bronner, 2012). Regional changes in gene expression patterns of the neural tube cells determine the progenitors of different types of neuronal cells that form the central nervous system (CNS) (Lang et al., 2004). When the organism has been completely formed and

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progenitor differentiation has been terminated, these cells lose their multipotency and are no longer able to change their cell commitment. Several

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lines of evidence support the importance of extrinsic signals, activating cell surface receptors and specific intracellular pathways to define these events and the phenotypes of differentiating cells. Ligand-induced signaling, activating tyrosine kinases, G protein-coupled receptors and ion channels are known to participate in the events of embryonic differentiation (Trujillo et al. 2009; Oliveira et al. 2013). ATP is considered to be the phylogenetically most ancient epigenetic factor, exerting short-time actions, as modulating excitability of neurons, and long-term trophic effects necessary for embryonic development. In accordance with such functions, ATP-induced signaling provides a principal sperm-to-egg 3

ACCEPTED MANUSCRIPT cue for fertilization (Foresta et al. 1992; Ishikawa and Seguchi, 1985). In brain development, ATP increases intracellular calcium release in radial glial cells the embryonic neural stem cells (NSCs) from which all neural cell types originate - culminating in increased proliferation and consequently contributing to neocortex development (Weissman et al., 2004). Embryonic NSCs have

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been characterized as multipotent stem cells derived from the CNS with the capacity to give rise to cells belonging to all three major lineages of the nervous system: neurons, oligodendrocytes and astrocytes. More recently, this increased proliferation caused by ATP was hypothesized to take place via

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recruiting quiescent cells into the cell cycle by cycling glial cells and in consequence enhancing the number of radial glial cells (Barrack et al., 2014). Moreover, inhibition of ATP production in zebrafish development decreased

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motoneuron axon arborization and prevented formation of retina, showing that not only mammalian neural development is modulated by ATP (Bestman et al., 2015).

The expression pattern of purinergic receptors throughout embryonic development suggests the importance of these receptors. In situ hybridization

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revealed P2X3 receptor subunit gene expression patterns in neural crestderived trigeminal cells during early zebrafish embryo development (Norton et al., 2000), that will give rise to the facial sensory neurons. Subsequently, Cheung and Burnstock (2002) showed that this receptor subunit is expressed in

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the developing rat nervous system from embryonic day 11 (E11) onwards, when gangliogenesis starts. Intense staining for P2X3 receptor subunit expression

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appears in the hindbrain neural tube, that gives rise to the medulla oblongata involved in the autonomic nervous system, with subsequent staining in brain and spinal cord as development proceeds, showing a prominent involvement of this receptor in the generation of sensory nerves and craniofacial motoneurons (Massé et al. 2007; Massé and Dale, 2012). Corroborating these data, translation disruption of the mammalian P2X3 receptor paralog p2xr3.1 of zebrafish led to defects in craniofacial and sensory circuit formation (Kucenas et al., 2009). Expression of P2X3 receptor subunits, together with P2X4 and P2Y1 receptors, was observed during mouse embryo neurulation. The transient

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ACCEPTED MANUSCRIPT expression of P2X3 receptors during development enforces the role of this receptor subtype in orchestrating embryonic neurogenesis. In rat E11 to 18, the period in which organs begin to be formed, P2Y1 and P2Y4 receptors are strongly expressed and P2Y2 and P2Y6 receptor subtypes gradually appear. P2Y4 and P2Y1 receptors were detected in different

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periods in the developing brain. However, P2Y4 receptor expression becomes down-regulated in the brainstem after birth, suggesting its specific role in prenatal brain development. On E12, P2Y1 and P2Y4 receptor expression becomes prominent in the neural tube and P2Y2 receptors appear specifically

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in the spinal motor nerves. In the progress of development, while P2Y1 receptor expression occurs in the spinal cord, the P2Y2 and P2Y4 receptor subtypes are

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expressed in the ventral horns, indicating a participation of purinergic signaling in the motor neuron development. P2Y2 receptors are also expressed in the dorsal root ganglia onE18 (Cheung et al., 2003), reinforcing the importance of the purinergic system in the development of the sensory system. Moreover, coordinated spontaneous intracellular calcium waves mediated by ATP, as verified by the use of non-specific purinergic receptor and P2Y1 subtype-

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specific antagonists, is suggested to induce proliferation of subventricular zone radial glial cells, such as shown in E16-17 rat embryo brain slices (Weissman et al., 2004), connecting P2Y1 receptor-induced intracellular calcium signaling with the organized development of cortical layers. Further work revealed deficient

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migration capabilities of NSCs from embryonic connexin-43 knock-out mouse brain, which also show diminished P2Y1 receptor expression levels (Scemes et

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al. 2003). Interestingly, expression of recombinant P2Y1 receptors by connexin43 null mice restored migration capability. This was again inhibited in the presence of the P2Y1 receptor-selective antagonist MRS2179, confirming the importance of this P2 receptor subtype in NSC migration. P2X5 receptor expression was also studied in developing mouse nervous system. The mouse brain exhibits a gradually increasing expression of P2X5 receptors, which reaches its maximum on E9 to E13 and then continuously decreases until E17, when the formation of most organs takes place. Immunolabeling

showed

that

P2X5

receptors

are

expressed

by

neuroectodermal cells and, as development proceeds to E11, their expression 5

ACCEPTED MANUSCRIPT appears in the cortical plate, where neurons start to differentiate, in accordance with purinergic signaling involvement in prenatal neurogenesis. P2X5 receptor expression occurs in the whole spinal cord on E9 and by E11 only in the ventral horn (Guo et al., 2013), giving rise to motor fibers. In summary, P2X5 receptors appear to be involved in the formation of mouse brain as a whole, but also in

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neurogenesis and in motor neuron development. On E15.5 of rat brain development, P2X7 receptors are expressed by subventricular (SVZ) and subgranular zones (SGZ) NSCs. NSCs have been isolated from many regions of the CNS, indicating their ubiquity. However,

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following embryonic mammalian development, NSCs begin to concentrate in specific neurogenic niches: the SVZ of the lateral ventricle and the SGZ of the

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dentate gyrus (DG) of the hippocampus, where NSCs survive for the duration of the whole adult life (Lledo et al., 2006; Mu et al., 2010). These cells differentiate while migrating to the cortical plate area, where a large amount of glial cells express this subtype two embryonic days later. Simultaneously, immature neurons of SGZ exhibit P2X7 receptor gene expression until adulthood, suggesting that this purinergic receptor is involved in alteration of cellular

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functions during brain development (Tsao et al., 2013). Furthermore, P2X7 receptor expression in NSCs could mean that these cells will be sent to an apoptosis program in order to control unbalanced proliferation, preserving the embryonic development process, as further discussed in the sections of our

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review on in vitro studies and cell therapy. In agreement with the mRNA and immunohistochemistry data, P2Y1

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receptors have been shown to be involved in regulating numerous functions of embryonic and adult NSCs. The first step after receptor activation appeared to be an increase in intracellular [Ca2+]i caused by the release of Ca2+ from its storage sites by the Gq,11 protein/phospholipase C/InsP3 pathway (Rubini et al., 2009; Illes et al., 2013). Furthermore, MAPK (mitogen-activated protein kinase) / ERK1-2 / CREB (cAMP response element-binding) signaling (Milosevic et al., 2006; Grimm et al., 2009) or phosphoinositide 3-kinase/P70 S6-kinase signaling (Ryu et al., 2003) also participates in the transduction mechanism of P2Y receptors. Increases of [Ca2+]i in embryonic and adult NSCs have been

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ACCEPTED MANUSCRIPT repeatedly demonstrated, and attributed to P2Y1 receptor stimulation (Rubini et al., 2009; Mishra et al., 2006; Stafford et al., 2007) Reinforcing the importance of the purinergic system in the developing brain, a study conducted in Xenopus laevis showed that damaged neural cells release ATP, activating P2X1 or P2X3 receptors with participation of P2Y

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receptors that induce calcium waves in NSCs leading to the expelling of these damaged cells from the brain ventricle and preventing the spread of injury (Herrgen et al., 2014). In view of the above-discussed P2X7 receptor functions

pool of these cells remains available.

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in selecting NSCs for the cell-death program, thereby a limited physiological

Purinergic signaling has also been shown to participate in embryonic eye

gastrulation.

Retinal pigment

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development by acting on expression of the eye field gene network following epithelium

releases

ATP

and

increases

proliferation of the early developmental neural retina in chick. This effect is mediated by P2Y2 receptor activation, and it is highly suggested that this purinergic subtype participates in generation of retinal neurons (Pearson et al, 2005). Moreover, P2Y1 receptor activity was observed in cells of the

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neuroblastic layer of retinas from chick embryos on E8, demonstrating that purinergic receptor expression pattern changes along the embryonic process, modulating different stages of retinal development (Nunes et al., 2007).

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Corroborating with purinergic function in retina development, the P2X5 receptor is heavily expressed on E11 in the neural layer of the retina and in the optic nerve (Guo et al., 2013). Additionally, P2X3 receptor expression is present in

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the whole optic tract of rat E16.5, from the neural layer of the retina through the optic chiasm to the lateral geniculate of the diencephalon (Cheung et al., 2002). In newborn mice, late developing progenitors in retinal explants showed increased proliferation, when challenged with ATP, possibly via P2Y1 receptor activation (Sholl-Franco et al., 2010). Altogether, these data suggest a constant modulation of P2 receptor expression levels during nervous system development with major implications for P2Y1, P2X3 and P2X7 receptor subtypes, as summarized in Figure 1. Several aspects of purinergic system functioning in embryonic development of 7

ACCEPTED MANUSCRIPT different species and other tissues have been discussed in a recent review by

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Burnstock and Ulrich (2011).

Figure 1. Purinergic receptor expression patterns during nervous system

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development. P2X and P2Y receptor subtype expression levels change together with the phenotypic transitions and morphological changes occurring during development of the brain, spinal cord, peripheral nervous system and retina. N.S.: Nervous System. [2-column fitting image]

3. Purinergic receptors in adult neural stem cells A historic landmark was the quantitative determination of the selfrenewing ability of adult tissues (Becker et al., 1963), identifying the so far

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ACCEPTED MANUSCRIPT unknown adult pluripotent stem cells. These cells are capable of generating tissue-specific cells, such as neurons, astrocytes and oligodendrocytes in adulthood, by taking over the function of NSCs (Sousa et al., 2014). In the adult brain, neurogenesis occurs preferentially in the previously mentioned two neurogenic niches (see also Chapter 2). In the SVZ, three closely adjacent cell

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types are situated below the ependyma. Type B cells represent NSCs that give rise to the highly proliferating type C cells corresponding to neural progenitor cells (NPC), which in turn generate glioblasts and migrating neuroblasts termed type A cells (Suh et al., 2009; Neary and Zimmermann, 2009). These neuroblasts move via the rostral migratory stream to the olfactory bulb, where

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they form mature interneurons, using γ-aminobutyric acid (GABA) and dopamine as neurotransmitters. A similar migration of neuroblasts takes place

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from the subgranular zone of the DG over a short distance into the DG itself, where they differentiate to glutamatergic granule cells and become integrated into the local neuronal circuits (Taupin and Gage, 2002). A number of signals direct the proliferation and fate commitment of the transit amplifying cells in the SVZ and the stem cells in the DG (Urbán and Guillemot, 2014). Different from

and Gage, 2002).

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NSCs, NPCs revealed limited capacity to undergo replication cycles (Taupin

Mobilization of mouse SVZ NPCs and NSCsis likely to be mediated by ATP; its injection into the lateral ventricle increased the number of both cell

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types. Additionally, infusion of a selective P2Y1 receptor antagonist prevented neurogenic proliferation (Suyama et al., 2012). Corroborating these data,

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knockout mice for NTPDase2 expression, one of the enzymes responsible for nucleoside triphosphate degradation, showed increased proliferation of NPCs in these zones, when compared with wild-type animals (Gampe et al., 2015). Crucial functions have been suggested for the P2Y1 receptor, since ADPβS injection into mouse cerebral ventricles resulted in increased number of astrocytes in the brain parenchyma and proliferation of NsCs in neurogenic areas of the SVZ (Bocazzi et al., 2014). The purinergic system does not only interfere with proliferation and differentiation of NSCs but also with the migration of these cells (Agresti et al., 2005; Scemes et al., 2003). More recently, NSCs obtained from adult mice and kept in culture after withdrawing epidermal growth 9

ACCEPTED MANUSCRIPT factor (EGF) and fibroblast growth factor-2 (FGF-2), responded with increased migration rates when treated with purinergic agonists ADPβS, ATP and UDP, possibly via P2Y1, P2Y2 and P2Y13 receptor activation (Grimm et al., 2010). Thereby, stimulation of the purinergic receptors mentioned above appears to induce cellular migration, an important process to direct newborn neurons to

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their sites of synapse and network formation. Under pathologic events, NSCs can undergo uncontrolled proliferation resulting in a detrimental increase in the number of these cells (Urbán and Guillemot, 2014). Therefore, as already emphasized above in the section on

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brain development, a balance between proliferation and cell death is also crucial in the adult brain for the proper maintenance of neuroplasticity and for

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the prevention of possible pathophysiological conditions resulting from uncontrolled proliferation of these cells. Furthermore, just as for brain development, cell surface receptor activation patterns, including those of purinergic receptors, are key events in maintaining defined NSC and NPC pools. Studies conducted in mouse brain slices showed not only that SVZ NSCs express functional P2X7 receptors (Messemer et al., 2013a), but also that

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ependymal cells located along the lateral cerebral ventricle in contact with NSCs from the SVZ express functional P2X7 receptors (Genzen et al., 2009). Since this purinergic receptor subtype regulates apoptosis and necrosis of NSCs (Delarasse et al., 2009), it has been suggested that P2X7 receptors

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exert- as described for various cell types (Sperlagh et al., 2006; Sperlagh and

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Illes, 2014) – also in SVZ NSCs’ cytotoxic properties (Ulrich and Illes, 2014).

4. Purinergic signaling during in vitro neural differentiation In vitro cultures of NSCs allow studying basic mechanisms under simplified

conditions,

deepening

the

understanding

of

CNS

development

and

maintenance. This holds true also for neurological and neurodegenerative diseases and possible repair mechanisms. In vitro models allow to reduce the number of interference variables, focusing on stem cell biology and its extrinsic and intrinsic regulation by interaction of neurotransmitters, growth factors, and other biologically active molecules with cell surface and intracellular receptors, 10

ACCEPTED MANUSCRIPT their signaling cascades and subsequent effects on gene and protein expression patterns. Furthermore, stem cells have turned into promising tools for transplantation therapy, and, therefore, directed differentiation protocols into specific neuronal phenotypes. For instance, transplantation of NPCs expanded in vitro have shown promising results in treatment of spinal cord injury (Yan et

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al., 2007), Parkinson’s Disease (Anderson and Caldwell, 2007), Down Syndrome (Rachubinski et al., 2012), ischemic brain (Hermann et al., 2014), traumatic brain injury (Shear et al., 2004) and other neurological damage situations in animal models. Alternatively, in vitro differentiation of NSCs in

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desired neuron types is performed prior to transplantation. NSCs are obtained as primary cultures from embryonic brain as well as from SVZ and SGZ of the adult brain. Furthermore, NSCs yield also from in vitro differentiation of

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pluripotent cells: embryonic stem cells (ESCs), embryonic carcinoma cells (ECs) and induced pluripotent stem cells (iPSCs). The latter ones can be used for disease modelling and the development of new treatments for enabling the establishment of in vitro models.

During neuronal development, NSCs change their surface antigens as

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differentiate, e.g. from nestin to Musashi-1in NPC stage, and finally Tuj-1 (βIIItubulin), the latter as a marker of mature neurons. Nestin is a NSC and NPC marker, and GFAP stains both NPCs and astrocytes (the embryonic transiently occurring radial glia is a precursor of neurons, astrocytes, and oligodendrocytes

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and the putative origin of the neurogenic zones; Kriegstein et al., 2009). Further stem cell markers at different stages of development include neural transcription

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factors such as Sox-1, Ptx-1, NeuroD1, and neurogenin-2, demonstrating their neuroectodermal origin (Hermann et al., 2004). At the same time, the passive membrane properties and ion channel

endowment of NSCs also change (Yasuda et al., 2010). Adult NSCs presented usually no or only aborted sodium action potentials during current injection (Feldman et al., 1996; Wang et al., 2003a); in some cases, they were capable of eliciting action potentials, but not more than a single one (Liu et al., 1999). After differentiation, NSC-derived neurons became electrically excitable, expressing voltage-dependent, tetrodotoxin-sensitive Na+ channels (Hogg et al., 2004). 11

ACCEPTED MANUSCRIPT The purinergic system is involved in the maintenance, proliferation and differentiation of pluripotent stem cells in vitro (Burnstock and Ulrich, 2011). Purinergic receptor expression can vary between different stem cell models and mammalian species (Figure 2), suggesting that its modulation could direct and increase specificity of stem cell commitment to a given neural type. Human

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ESCs differentiated into NPCs; subsequent neuronal differentiation shows divergent patterns of purinergic receptor expression between cell types. While P2X5 receptor expression increased during phenotype transitions both in ESCs and NPCs, (i.e., increased cell specificity required increased P2X5 receptor

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activation), P2X2 receptors underwent a significant decrease in comparison with NPCs, when differentiated into neurons, suggesting that,

its decrease

influences the NPC transition to neurons. Moreover, P2X4 receptor expression

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decreased when ESCs differentiated into NPCs, showing that this receptor is not required for this latter stage, but that it recovers when NPCs differentiate into neurons, demonstrating a possible role of P2X4 receptor in increasing neuronal fate (Young et al., 2011). Functional co-expression of P2X4 and P2X7 receptors was observed in adult NPCs of the mouse SVZ; ivermectin, a positive allosteric modulator of P2X4 receptors, potentiated the current responses

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induced by the ATP structural analogue Bz-ATP and this effect was abolished by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5BDBD), a negative allosteric modulator of this receptor type (Messemer et al.,

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2013b).

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Figure 2. Profiles of P2 receptor expression during in vitro differentiation of stem cells from different species. Rat-derived embryonic NPCs display

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increased P2X2 and P2X6 receptor expression during in vitro development into neurons, while decreased expression of P2X7, P2Y2, P2Y4 and P2Y6

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receptors was observed (Schwindt et al., 2011; Oliveira et al., 2015). Variable expression of P2X3, P2X5 and P2Y12-14 receptors was also observed in rat NPCs (Trujillo et al., 2012; Oliveira et al., 2015). During differentiation of mouse embryonic stem cells into neurons there is a gradually increased expression of P2X7 receptors (black). Pluripotent mouse P19 embryonic carcinoma stem cells (brown) express functional P2Y1, P2Y2 as well as P2X4 and P2X7 receptors. Differentiation induction into neurons goes along with down-regulation of P2X3, P2Y1 and P2Y4 receptor expression, together with up-regulation P2X2, P2X6, P2X7 and P2Y2 and P2Y6 receptor subtype expression (Resende et al. 2007). Human stem cells exhibit decreased expression of P2X4 receptors when 13

ACCEPTED MANUSCRIPT induced differentiation to NPCs is initiated; afterwards P2X7 receptors are again up-regulated when differentiation proceeds to form neurons. P2X2 receptor expression decreases in NPCs when neuronal differentiation is induced. During the process of human stem cell to neuron differentiation, P2X5 receptor expression is increased. SC: stem cell; NPC: neural progenitor cell.[1-column

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fitting image] Using pluripotent mouse P19 EC cells as a model for early neuroectodermal differentiation, expression and activity patterns of P2X and P2Y receptors were tracked throughout the differentiation of these cells into neurons.

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Activity screening using purinergic subtype-selective agonists and antagonists revealed that P2Y1 and P2Y2 metabotropic receptors and possibly P2X4

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ionotropic receptors were involved in initial proliferation and embryonic body formation, a prerequisite for further neuronal differentiation (Resende et al. 2008). Additional differentiation of NPCs into neurons expressing functional cholinergic and glutamatergic receptors relied on expression and activity of P2Y2, P2X2 and P2X6 receptors (Resende et al. 2007), while for gliogenic differentiation P2X7 receptor expression was essential (Yuahasi et al. 2012).

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Besides, maintaining mouse ESC proliferation in vitro, P2X7 receptor suppression is needed for inducing neuronal differentiation (Glaser et al., 2014). Gene expression of P2X1-7 receptors was present in NPC cultures

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obtained from adult mice brain. In these cells, the P2X7 receptor was functional (Messemer et al., 2013), suggesting the involvement of this receptor in in vivo maintenance of NPCs, and probably also in in vitro differentiation. In a NPC derived

from

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culture

rat

embryonic

telencephalon

forced

to

neural

differentiation, P2X2-7 receptors as well as P2Y1,2,4,6,12 and 14 receptors were expressed (Trujillo et al. 2012). Maintenance of cells in culture medium favoring neurogenesis led to augmented expression of P2X2 and P2X6 receptors, suggesting an important role of these receptors in this process. Further ionotropic P2 receptors, such as P2X3, P2X4 and P2X5 receptor subtypes, did not undergo significant alterations in their expression levels (Schwindt et al., 2011).

14

ACCEPTED MANUSCRIPT Induced

pluripotent

stem

cells

obtained

through

reprogramming

specialized somatic cells may be promising for therapy as well as for modeling diseases, which might be caused by dysfunctions during development. However, the knowledge on purinergic receptor expression and activity patterns in iPSCs is scarce. A study conducted with iPSCs from human fibroblasts was

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used for modeling Lesch–Nyhan disease, characterized by mutations of the hypoxanthine guanine phosphoribosyltransferase purine biosynthesis (HPRT) gene. Knock-down of HPRT gene expression resulted in markedly diminished P2Y1 receptor expression, providing an interrelationship between the purine-

SC

synthesizing enzyme and purinergic signaling (Mastrangelo et al. 2012). Further studies, recapitulating aberrant purinergic signaling in iPSCs from patients with

targets. In

human

embryonic

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neurodegenerative diseases will provide new mechanisms and therapeutic

midbrain-derived

NPCs,

doublecortin-

immunoreactivity was used as a marker indicating incipient neuronal differentiation (Rubini et al., 2009). While ATP depressed (through P2Y1 receptors), UTP increased (probably through P2Y4 receptors) the generation of

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the neuronal-committed NPCs. The promotion of neuronal differentiation by UTP in general was paralleled by an increase in the number of tyrosine hydroxylase-positive cells and tyrosine hydroxylase protein expression, indicating a selective boosting of dopaminergic neurogenesis (Milosevic et al.,

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2006). ATP was found to be a negative regulator of terminal neuronal differentiation in embryonic mouse NPCs as well (Lin et al., 2007). However, in

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contrast to this finding, ATP appeared to accelerate neuronal differentiation, determined by the analysis of the nestin and neuron-specific enolase gene and protein expression, via the activation of P2Y1 and P2Y2 receptors; this discrepancy may be due to the use of an embryonic carcinoma cell line as a model for NPCs (Resende et al., 2008).

5. Purinergic signaling in neurological diseases and cell therapy Stem cell transplantation has created promises for therapy of several diseases and turned into an emerging field of investigations, also relating to 15

ACCEPTED MANUSCRIPT neurological diseases such as detailed in Table 1. As covered by this review, the purinergic system plays an important role in proliferation, migration and differentiation of ESCs and NPCs. Moreover, purinergic receptors are involved in the pathophysiology of neurological diseases, including Parkinson’s disease, Alzheimer’s disease and epilepsy. We postulate here that cell therapy involves

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the modulation of purinergic signaling, either in the achievement of more effective protocols to obtain viable and homogeneous cell populations orto foster the process of incorporation of transplanted cells into the individual (Figure 3).

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The activation or inhibition of purinergic receptors in the transplanted cells may affect their potential in neurological diseases. For instance,P2Y2 and

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P2X2 receptor activities have been shown to be important for differentiation of pluripotent cells into neurons expressing cholinergic and glutamatergic receptors (Resende et al., 2007), and P2Y1 receptors have been attributed with functions in NSC mobilization and migration. P2X7 receptor inhibition should limit cell death of transplanted NPCs or neurons, besides of favoring neurogenesis in stem cells (Glaser et al. 2014). On the other hand, due to their

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capacity of self-renewal, cell therapy with ESCs could lead to uncontrolled proliferation and tumor formation. In this case, P2X7 receptor activation should have beneficial effects by limiting cell proliferation through death of transplanted ESCs, demonstrating that both activation and inhibition of P2X7 receptors have

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promising effects in transplanted cells.

16

ACCEPTED MANUSCRIPT Table 1.Transplantation of different cell types as a therapy of neurological disorders Source

Reference

Parkinson’s dopaminergic

induced pluripotent stem

Hargus et al.,

disease

cells obtained from

2010

neurons

Parkinsonian patients

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Cell type

dopaminergic

human embryonic stem

neuron-derived

cells

neural stem

-

SC

Ziavra et al., 2012

neural stem cell-

human embryonic stem

derived

cells

dopaminergic

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cells

neurons

Kriks et al., 2011

Daadi et al., 2012

Human embryonic stem

Sundberg et al.,

cells, human induced

2013

pluripotent stem cells

and non-human primate induced pluripotent stem

neural

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cells

human embryonic stem

Ambasudhan et

progenitor cells

cells

al., 2014

embryonic

-

Cordeiro et al., 2014

EP

ventral

mesencephalon

AC C

cells

Epilepsy

neural

embryonic stem cells

Carpentino et al.,

progenitor cells

2008

striatal precursor -

Hattiangady et al.,

cells

2008

cortical

-

GABAergic

Baraban et al., 2009

interneuron precursors neural

mouse embryonic stem

Shindo et al., 17

ACCEPTED MANUSCRIPT stem/progenitor

cells

2010

neuronal

medial ganglionic

Calcagnotto et al.,

precursor cells

eminence cells

2010; Henderson

cells

et al, 2014 embryonic stem cells

Maisano et al.,

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GABAergic

2012

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SC

interneurons

Figure 3. Suggested roles for P2X7 receptors in neurodegenerative diseases and neuroregeneration. Following stem cell transplantation, P2X7 receptors are suggested to have several functions. In the injury site, P2X7 receptor activation in adjacent cells leads to cell death and expands injury. In cell transplantation, without cell death promotion by P2X7 receptor-activation, ESC injected into the 18

ACCEPTED MANUSCRIPT lesion site of neurological diseases could undergo uncontrolled and damaging proliferation with possible tumor formation. P2X7 receptor could restrain the excessive proliferation of engrafted cells. On the other hand, P2X7 receptor activity could inhibit neuronal differentiation of engrafted cells and may be responsible for the poor survival rate of these cells. Thus, P2X7 receptor

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inhibition may be beneficial for functional engraftment of transplanted cells. In addition to promoting survival and engraftment of transplanted cells, purinergic receptors are supposed to act on neuroprotection and mobilization/migration of neural stem cells from neurogenic zones to the lesion sites. [2-column fitting

SC

image]

Parkinson’s disease (PD) is a neurodegenerative disorder characterized

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by a deficit of dopaminergic neurons in the nigrostriatal pathway. Until today, there is no known causal therapyfor this disease and great efforts have been made to elucidate the mechanisms underlying its pathophysiology and to find new treatment options. In the injury site of PD, there is an extensive release of ATP that activates cell death pathways in adjacent cells, expanding the lesion and worsening the pathological picture. It is known that P2X7 receptor

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activation by ATP leads to these effects and, as a support for this hypothesis, chronic P2X7 receptor blockade by different P2X7 antagonists prevented synaptotoxicity, neurotoxicity and gliosis in an animal model of PD (Marcellino et al. 2010; Carmo et al. 2014). Further relevance of the P2X7 receptor in this

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disease emerged fromthe demonstrated coincidence of P2X7 receptor polymorphism with PD in a Han Chinese population (Liu et al. 2013).

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The massive release of ATP in, or its injection to the injury site could

activate purinergic receptors other than P2X7 subtype. ATP has been reported to induce increased release of dopamine in the substantia nigra (Burnstock, 2008). In fact, P2Y1 and P2X1,2,3,4,6 subtypes are expressed on dopaminergic neurons in organotypic cultures of brain slices prepared from the substantia nigra / ventral tegmentum, with co-expression of P2X1 with D1 dopaminergic receptors (Heine et al., 2007). In vivo, stimulation of P2 receptors by ATP in the ventral tegmental area, striatum and nucleus accumbens - brain regions also involved in the etiopathology of PD - increased the release of dopamine (Zhang et al., 1995; Krugel et al., 2001a, 2001b). Moreover, rats submitted to an animal 19

ACCEPTED MANUSCRIPT model of PD treated with diadenosine tetraphosphate presented a reversal of dopaminergic loss in the nigrostriatal pathway possibly by P2Y1 and P2Y4 receptor activation (Wang et al., 2003b). These data show that modulation of purinergic receptors in the lesioned brain of patients with PD must be treated with highly specific purinergic ligands, since a non-selective agonist that would

death and neuroprotective signaling cascades.

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cause activation of purinergic receptors in general could concurrently trigger cell

In epilepsy, among other factors, seizures are caused by a misbalance between excitatory and inhibitory transmission, involving GABA and glutamate

SC

neurotransmissions(reviewed by Dale and Frenguelli, 2009).Experimental evidence suggested that aberrant purinergic signaling is operating in epilepsy,

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since ATP and its derivatives play an important role in excitatory and inhibitory signal transduction. For instance, rodents susceptible to seizures revealed increased extracellular ATP levels upon electrical stimulation in vivo and in hippocampal slices, suggesting an involvement of purinergic signaling in the event of a seizure, possibly due to a reduced Ca2+-ATPase activity (Wieraszko and Seyfried., 1989). epilepsy

(TLE),

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Temporal-lobe

characterized

by

expansion

of

spontaneous recurrent motor seizures originating in the limbic system region (French et al., 1993), has been widely studied in animal models of epilepsy.

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Expression of P2X7 receptors was increased in both glial and glutamatergic neurons during acute and chronic phases of TLE (Doná et al., 2009). As previously stated, the sustained activation of P2X7 receptors by ATP triggers

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pro-apoptotic and pro-inflammatory pathways, supporting the conclusion that this receptor aggravates status epilepticus. In chronic phase of TLE, P2X4 receptor expression decreased; this was attributed to malfunctioning of remaining neurons (Doná et al., 2009) which could aggravate the misbalance between excitatory and inhibitory transmission during subsequent seizures. More recently, a study showed that injection of Bz-ATP into the piriform cortex or in the cerebral ventricle of mice induced seizure activity, suggesting that the activation of P2X7 receptors is involved in the pathophysiology of epilepsy (Engel et al., 2012) In fact, the administration of a P2X7 receptor antagonist

20

ACCEPTED MANUSCRIPT decreased seizure severity in immature rats (Mesuret et al., 2014) and suppressed seizure in mice (Engel et al., 2012). The here described involvement of the P2X7 receptor in neurological diseases supports the hypothesis that hyperactivity of this receptor due to the availability of excessive extracellular concentrations of ATP due to previous

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brain injury aggravates already existing lesions. Enhanced P2X7 receptor levels may be the cause or consequence of disease development. This point needs to be clarified in more detail. Recently, P2X7 receptor functions have been proposed to participate both in proliferation stimulation and elimination of

SC

superfluous stem cells and NPCs. The decision, which of these contradictory actions will occur, depends on the cellular context and the involved micro-

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niches. Fine tuning of this receptor activity is needed to control the balance between cell proliferation and death, which disproportion may culminate in the development of diseases including tumors and neuronal necrosis / apoptosis (Figure 3). 6. Acknowledgements

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This work of HU was supported by research grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESPProject No. 2012/508804) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Project No. 486294/2012-9 and 467465/2014-2), Brazil. AO is grateful for a

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doctorate fellowship granted by CNPq. The work of PI was supported by the German Research Council (DFG, IL-20/19; IL-20/21-1) and the Sino-German

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Centre for the Promotion of Science (GZ 919). 7. References

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ACCEPTED MANUSCRIPT Highlights Purinergic signaling is present throughout nervous system development. P2Y1 receptors in neural progenitor migration are important in cortical layer

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development. P2X7 receptors act as dual sword, promoting proliferation or inducing cell death.

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P2X7 receptors are overexpressed in neurodegenerative diseases.

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P2X7 receptor inhibition is neuroprotective and enhances stem cell

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engraftment.