The first hints of a specific role for purinergic receptor signaling during development came from studies in Xenopus, where ATP degradation to ADP by E-NTPDase-2 in the anterior neural plate, with subsequent activation of the P2Y₁ receptor subtype, leads to the induction of Pax6, Rx1, and Six3 genes. These genes encode for transcription factors collectively referred to as eye field transcription factors (EFTF), and are fundamental to promote and sustain eye development (Massè et al., 2007). Altering purinergic signaling, by either overexpressing or downregulating some of the molecular components of this pathway, leads to abnormal eye development, thus confirming the crucial role of extracellular nucleotides during tissue generation and cell specification in embryos.

Data have been further confirmed and expanded to the mammalian brain, where it has been demonstrated that the expression of specific P2Y receptor subtypes is plastic and tightly controlled at different embryonic stages, and correlates with their recruitment in controlling brain development (Oliveira et al., 2016). For example, P2Y₁ receptor-mediated calcium signaling is fundamental for the specification of the cortical layers. At this stage of development, daughter cells derived from neural precursors located in the ventricular/subventricular zone start their migration toward their final localization in the developing cortex where they differentiate to either neurons or astrocytes (Pino et al., 2017). Radial glial cells, a peculiar type of glial cells, act as both neuronal progenitors in the ventricular/subventricular zone through their asymmetric division and as “guiding sign” for migrating cells. In fact, their long radial process extends up to the subpial surface, and constitute a “highway” for migrating cells to find their final localization in the correct cortical layer (Ulrich et al., 2012; Figure 1).

Extracellular nucleotides control both radial glial cell proliferation and migration of developing neuroblasts and glial cells. In fact, a paracrine ATP signaling is established through its degradation to ADP and activation of P2Y₁ receptors, leading to the generation of [Ca²⁺]_i waves that propagate through hemichannel and gap junctions, thereby synchronizing cell cycle and migration (Weissman et al., 2004; Lecca et al., 2016; Figure 1). In turn, calcium waves amplify ATP release accompanied by other neurotransmitters and growth factors, which further contribute to drive neural precursor migration toward developing cortical layers, where they terminally differentiate to neurons or glial cells (Ulrich et al., 2012).

Furthermore, extracellular ATP acts as one of the main activity-dependent axonal signal and activates P2 receptors on oligodendrocyte precursors cells (OPCs), which generate mature oligodendrocytes during early post-natal development (Fields and Stevens, 2000). ATP and uracil nucleotides interact with growth factors to trigger specific intracellular pathways that regulate OPC proliferation, migration, terminal maturation, and myelination (Fumagalli et al., 2016).

Neurotransmitters and growth factors released by migrating cells and radial glia are also fundamental for driving neuronal differentiation and maturation, and for neurite extension and myelination. As recently elegantly reviewed (Heine et al., 2016), the purinergic system is crucially involved also in these later stages of neuronal development, thanks to the recruitment of several receptor subtypes, activated by either nucleotides or adenosine. For example, both purine P2Y_{1, 13} and pyrimidine P2Y₂ receptor subtypes have been positively correlated to neurite elongation, with the latter specifically linked to the α_V integrin/Rho/ROCK pathway (del Puerto et al., 2012; Peterson et al., 2013). A significant contribution to these events is also provided by astrocytes, and the purinergic system is also directly involved in controlling their functions (Buffo et al., 2010; Heine et al., 2016).

Neurogenesis and gliogenesis initiated by purinergic signaling in the embryonic brain continue during postnatal development, and the ventricular/subventricular zone is recognized in the adult brain as neurogenic niche, together with the subgranular layer of the hippocampus (Boda et al., 2017). In vitro and in vivo studies have demonstrated that the ADP-responsive P2Y₁ receptor subtype endures in its role of modulator of precursor cell proliferation and differentiation throughout life (Suyama et al., 2012; Boccazzi et al., 2014). Several other P2Y (and also P2X) receptor subtypes have been found expressed in the adult neurogenic niches (Mishra et al., 2006; Stafford et al., 2007; Grimm et al., 2009), and could therefore contribute to modulate neural precursor cell proliferation, differentiation, and recruitment following brain injury, in an often-unsatisfactory attempt to damage repair.

Although for many years the expression of P2X7 receptor in adult brain has been confined to glial cells (i.e., astrocytes and microglia), a lively debate is currently open on its expression by adult neurons, with arguments both in favor and against this statement (Illes et al., 2017; Miras-Portugal et al., 2017). What is now widely accepted is that P2X7 is highly expressed by neuroblasts and neural precursors during brain development, where it inhibits cell proliferation and neurite outgrowth, but promotes neuronal differentiation (Heine et al., 2016; Oliveira et al., 2016). Interestingly, recent data point for a new role of P2X7⁺ neuroblasts which also express high levels of doublecortin, the typical marker of developing neurons (Boda et al., 2017). These cells are in fact endowed with the peculiar ability to phagocyte surrounding cells dying by programmed cell death well before brain colonization by specialized phagocytes, such as microglia/macrophages (Gu et al., 2015; Lovelace et al., 2015; Figure 2). Based on these data, since programmed cell death is a fundamental process during brain development and shaping, leading to the elimination of unnecessary, damaged, or exceeding cells, a new fundamental role for P2X7-mediated purinergic signaling during embryonal life is now emerging.

Adenosine has been shown to dramatically affect embryonic development as well. In fact, it is directly involved in the elimination of interdigital membranes, in the apoptotic death of clones of autoreactive lymphocytes in the thymus, and in the morphogenetic outgrowth of vertebrate limb buds (Jacobson et al., 1999). Moreover, the adenosine catabolizing enzyme adenosine deaminase (ADA) is expressed at high levels in the placenta, and its pharmacological inhibition disrupts fetal development (Knudsen et al., 1992), thus suggesting that developing tissues and organs are highly sensitive to increased adenosine concentrations. A direct link with the activation of specific P1 receptor subtypes has not been demonstrated; it is more likely that non-receptor mediated effects are involved in this pro-apoptotic and toxic actions of adenosine, as already demonstrated in ADA deficiency and in other disorders (see below; Jacobson et al., 1999). Additionally, in the developing brain the expression of adenosine kinase, the enzyme responsible for the rephosphorylation of adenosine to nucleotides, is switched from neurons during life in utero to exclusive astrocytic expression along with progressive brain maturation (Studer et al., 2006). It is therefore clear that a tight control of adenosine levels is fundamental for brain development and neural plasticity (Boison et al., 2012). A role for adenosine in promoting neurite outgrowth through the A_2A receptor subtype (Heine et al., 2016), and in fostering the differentiation of OPCs and their ability of myelinate axons (Butt et al., 2014) has been also highlighted.

Adenosine is involved in the regulation of several signaling pathways in CNS (Boison, 2008), acting as a neuromodulator through multiple mechanisms, including the control of neurotransmitter release, or via regulatory effects on glial cells (Boison et al., 2010, 2012). As extensively reviewed elsewhere (Fredholm et al., 2005), activation of A₁ receptors inhibits the release of neurotrasmitters, such as dopamine and glutamate, and decreases neural excitability by inducing post-synaptic hyperpolarization. Conversely, A_2A receptors promote neurotransmitter release. Based on these interactions with glutamatergic and dopaminergic neurotransmission, a role for adenosine in neurodevelopmental defects at the basis of schizophrenia has been proposed (Lara and Souza, 2000; Lewis and Levitt, 2002; see section The Adenosine Dysfunction Hypothesis in Neurodevelopment). A role for A_2B and A₃ adenosine receptors (both expressed in brain) in neural development still remains to be unveiled.

Purine de novo biosynthesis is a complex, energy-expensive process. As depicted in Figure 3, it begins with the formation of phosphoribosyl pyrophosphate (PRPP) and leads to the first fully formed nucleotide, inosine 5′-monophosphate (IMP), which can be subsequently converted into either AMP or GMP (Kelley and Andersson, 2014). PRPP is also utilized to convert the nucleobase orotic acid into uridine monophosphate (UMP), the first step of the biosynthesis of pyrimidines. Alternatively, intracellular nucleosides coming from the diet or from the dephosphorylation of endogenous nucleotides can be recycled by the salvage pathway. The catabolism of residual free bases generates uric acid in case of purines, and β-alanine and β-aminoisobutyric acid in case of pyrimidines to be excreted as end products. Both de novo and salvage pathways are feedback-regulated by their end products, whereas purine catabolism is mostly regulated by substrate availability (Micheli et al., 2011). Only 10–30% of the free purines generated by intracellular metabolism are degraded or excreted (Torres and Puig, 2007), thus highlighting the importance of nucleotide salvage, which is fundamental in brain tissues; in addition, salvaged IMP from the liver is transported by erythrocytes to the brain and other tissues, where it is released for conversion to ATP or GTP (Ipata et al., 2011).

Considering the crucial roles of purines, pyrimidines, and their derivatives in brain development (see section Contribution of Purinergic Transmission to the Development of the Central Nervous System), it is evident that alterations in their synthesis, catabolism, and concentrations may lead to significant functional consequences. Inborn errors in purine metabolism are usually rare, and characterized by the absence or abnormal concentrations of purine nucleotides in cells, or by the presence of toxic intermediates in body fluids. At present, more than 35 enzyme defects of nucleotide synthesis, salvage, and catabolism of both purines and pyrimidines have been identified, some of which are associated with serious clinical consequences during development (Table 1; for review see Micheli et al., 2011). These disorders, previously considered as pediatric diseases, are now increasingly recognized in adults with milder phenotypes. Although purines and pyrimidines are essential in all tissues, the clinical outcomes of these disorders often suggest that the CNS is more seriously affected than other organs.

In the following paragraphs, we will introduce some of these disorders, focusing on the possible links between enzyme alterations during development and their neurological outcomes.

Down Syndrome (DS) represents the most typical example of genetic disorder associated to mental retardation. Trisomy of chromosome 21 leads to the pathological overexpression of various gene products and miRNAs, but also to additional alterations spanning from the deregulation of non-coding DNA, the abnormal expression of non-HSA21 (non-Homo sapiens autosome 21) genes and epigenetic modifications (for review see Dierssen, 2012). These genetic abnormalities lead to a consequent dysregulation of related biochemical pathways in all tissues (Table 1; Spellman et al., 2013), but the most relevant alterations are localized in the brain, with consequent moderate to severe mental retardation (Dierssen, 2012). The most evident anatomical correlates of mental retardation in DS children and fetuses are brain hypoplasia and hypocellularity, leading to a decreased brain size (Rachidi and Lopes, 2008). Consistent with this observation, many groups have detected an impaired proliferation of neural precursors and reduction in neurogenesis in the developing neocortex, hippocampus, and cerebellum of mouse models of DS and of DS fetuses (Contestabile et al., 2007, 2009; Guidi et al., 2008). In the adult brain the above-mentioned modifications also translate in reduced spine density and impaired synaptic plasticity, paralleled by profound alterations in several neurotransmitter pathways (i.e., GABA, glutamate, serotonin etc.), with a consequent imbalance between excitatory and inhibitory neurotransmission mostly in the hippocampus (Dierssen, 2012). Additionally, amyloid precursor protein (APP) is triplicated in DS and it is believed to contribute to neurodevelopmental alterations and to trigger the development of Alzheimer-like pathology in DS adults (Stagni et al., 2017). As extensively reviewed elsewhere (Stagni et al., 2017), various biochemical pathways have been causally associated to the impairment of neurogenesis and the consequent shift in cell destiny leading to reduced neurons and increased percentage of astrocytes.

The first, and up to now the only available, hint of altered purinergic signaling in DS came indirectly from the observation that lack of sortin nexin 27 (SNX27), a PDZ-containing protein of the endosome-associated retromer complex controlling the trafficking of several proteins (Carlton et al., 2005), leads to severe impairment of brain functions, including cognitive manifestations mimicking those observed in DS (Wang et al., 2013, 2014). Additionally, SNX27 is down-regulated in Ts65Dn mice, the most common mouse model of DS, as a consequence of the hyperexpression of miR-155 which is physiologically responsible for its degradation (Wang et al., 2013, 2014). Our research group got interested in SNX27 while searching for the biochemical pathways responsible for the membrane-to-cytosol recycling or intracellular degradation of the G protein-coupled P2Y-like receptor GPR17. GPR17 responds to both extracellular uracil nucleotides (UDP, UDP-glucose) and cysteinyl-leukotrienes (Ciana et al., 2006). GPR17 is highly expressed by cells of the oligodendrocyte lineage, specifically during the transition from OPCs to immature oligodendrocytes. After this stage, GPR17 expression must be down-regulated to promote the generation of fully myelinating mature cells (Lecca et al., 2008; Chen et al., 2009; Boda et al., 2011; Ceruti et al., 2011; Fumagalli et al., 2011, 2015). In agreement with these findings, forced overexpression of GPR17 during late stages of differentiation leads to defective myelination in vitro and in vivo, whereas in vivo GPR17 knock-out accelerates oligodendrocyte myelination (Chen et al., 2009). Based on these data, we evaluated the possible involvement of SNX27 in the fine-tuned regulation of GPR17 membrane expression, and demonstrated that the endocytic trafficking of the receptor is mediated by the interaction of a type I PDZ-binding motif located at its C-terminus with SNX27. Additionally, SNX27 knock-down in vitro reduced GPR17 plasma membrane recycling in differentiating oligodendrocytes while fostering their terminal maturation (Meraviglia et al., 2016). When analyzing the brains of Ts65Dn mice, we observed that trisomy-linked down-regulation of SNX27 was paralleled by decreased GPR17 expression and increased number of mature oligodendrocytes, which, however, fail in reaching full maturation, eventually leading to brain hypomyelination (Meraviglia et al., 2016; Figure 4).

We therefore speculate that altered GPR17 signaling and oligodendrocyte maturation, with consequent defective axon myelination, could contribute to the overall brain pathological phenotype of DS, both in terms of brain structure and functions.

The availability of innovative and automated strategies to evaluate the presence of gene variants and mutations is accelerating our comprehension of the genetic alterations at the basis of rare or sporadic pathological phenotypes, such as some forms of mental retardation. The discovery of the first patient bearing a homozygous Pannexin 1 (Panx1) loss-of-function gene variant associated with multisystem dysfunction and intellectual disability has been recently published (Shao et al., 2016). Panx1 is a membrane channel allowing the passage of ions and small molecules, among which ATP is one of the most common (Boyce and Swayne, 2017). In the CNS, Panx1 is highly expressed by developing and mature neurons, and contributes to brain development, maturation, and to synaptic plasticity (Wicki-Stordeur et al., 2016). Thus, reduced ATP release in the presence of mutated Panx1 could lead to defective purinergic transmission which in turn could be responsible for alterations in brain structures and mental retardation. It is worth mentioning that a close relationship has been demonstrated between Panx1 and the P2X7 receptor subtype, which is crucially involved in brain development (see Contribution of Purinergic Transmission to the Development of the Central Nervous System). Data demonstrate that the two proteins can physically interact (Boyce and Swayne, 2017) and that ATP released through Panx1 in the microenvironment of P2X7 receptor ultimately leads to its activation. Defects in this signaling pathway can therefore have profound impact on brain development during embryonic and fetal life and on intellectual functions after birth.

It is now increasingly recognized that inflammation is a crucial contributor to an altered fetal brain development (Hagberg et al., 2015). In particular, epidemiological studies have shown that maternal infection is an important environmental risk factor for neurodevelopmental disorders (Boksa, 2010). In preclinical animal models, perinatal infection has been indeed reported to cause maternal immune activation (also called mIA) that is associated with later appearance of autism spectrum disorder, and schizophrenia by altering fetal brain development at critical periods of pregnancy (Knuesel et al., 2014; Estes and McAllister, 2016; Table 1).

Although the initial prenatal insult alone may not be enough for clinical manifestation in human, many studies have focused on animal models of maternal infection to investigate the causal chain of events linking immune exposure and genetic predisposition, to neurodevelopmental abnormalities, as extensively reviewed elsewhere (Knuesel et al., 2014). Maternal infection is mainly mimicked by exposing pregnant rats to either LPS, or the inflammatory viral mimetic polyinosinic:polycytidylic acid (poly I:C) in the gestational period or by direct intrauterin administration of LPS (Cai et al., 2000; Bell and Hallenbeck, 2002). Immune mediators (cytokines and chemokines), reactive oxygen or nitrogen species, excitotoxicity, mitochondrial impairment, and altered vascular integrity have been described to be critical contributors to abnormal brain development in the offspring after maternal immune activation (Hagberg et al., 2015). In particular, microglial priming and elevation of maternal pro-inflammatory cytokines (i.e., TNF-α, IL-6, IL1-β) have been casually linked to dysregulation of fundamental neurodevelopment programs (Vargas et al., 2005; Morgan et al., 2010; Upthegrove et al., 2014). In this respect, members of the nucleotide-binding domain-like receptor (NLR) protein family act as inflammasome sensors, and mediate the translation of maternal immune activation to pathologically relevant neurodevelopmental abnormalities. Inflammasomes critically control different aspects of innate immunity, including the release of pro-inflammatory cytokines that induce and sustain the inflammatory response (Latz et al., 2013). The NLRP3 inflammasome is one of the major signaling routes that mediates the shift from innate immune activation to inflammation in response to different pathogenic or endogenous danger signals, including ATP (de Torre-Minguela et al., 2017). ATP-dependent stimulation of P2X7 receptors indeed promotes inflammasome activation, leading to caspase-1 cleavage and release of mature IL1-β (Ferrari et al., 2006; Di Virgilio et al., 2017). In line with this, recent data have demonstrated that both genetic deletion and the pharmacological inhibition of P2X7 receptors mitigate schizophrenia-like behavioral changes in a phencyclidine-induced rodent model of schizophrenia (Koványi et al., 2016).

Another study has revealed the critical role of P2X7 receptor in affecting astrocyte-neuron cross-talk after LPS prenatal exposure. The infection during gestation triggers the release of ATP from astrocytes via Cx43 and Panx1 unopposed channel, resulting in increased neuronal death mediated by P2X7 receptors and Panx1 channels (Avendano et al., 2015).

Maternal inflammation induced by perinatal infections can also result in preterm infants (Hagberg et al., 2012). Diffuse perinatal white matter injuries, including periventricular leukomalacia caused by ischemic damage, are the most common type of brain injury in preterm infants. Pathological demyelination and impaired oligodendrocyte maturation are known to cause cerebral palsy, and cognitive, behavioral, and sensory deficits as well as psychological problems later in life (van Tilborg et al., 2016). As detailed in section Toward a Purinergic-Based Therapy for Congenital Neurodevelopmental Disorders?, administration of UDP-glucose, acting on different purinergic receptors, has proven successful in improving the survival of newly generated oligodendrocytes in neonatal rats with ischemic periventricular leukomalacia (Mao et al., 2012; Li et al., 2015; Table 2).

Interestingly, another approach targeting purinergic receptors with suramin has been reported to correct the autism spectrum disorder-like phenotype and to restore normal social behavior in a maternal immune activation murine model of autism-like behaviors using polyI:C exposure, and in the Fragile X (Fmr1 knockout) model (Naviaux et al., 2013, 2015; Table 2). These effects have been ascribed to the capability of the anti-purinergic treatment to normalize the expression of two purinergic receptors (P2Y₂ and P2X7) and the phosphorylation of ERK1, ERK2, and CAMKII, all of which modulate purinergic signaling. In addition, suramin has been reported to correct the concentrations of some altered purine metabolites (9 out of 11, including ATP and allantoin) in the plasma of mice subjected to maternal immune activation (Naviaux et al., 2013, 2014, 2015). Metabolomic studies have also revealed similar alterations in the purine and pyrimidine metabolite profile in human biofluids (urines and plasma) of children suffering from autism spectrum disorder (Gevi et al., 2016). Moreover, purinergic signaling in the brain has been identified as one of the top-regulated gene expression pathways correlated with abnormal behaviors in children with autism spectrum disorder suggesting a key role of purines in driving a cell danger response in these disorders (Ginsberg et al., 2012).

Although the molecular basis of the altered purine metabolism in murine models of autism spectrum disorder still remains to be elucidated, these data suggest that metabolic pathways that synthesize and catabolize purines are critical regulatory elements in autism spectrum disorder, in accordance with their increasingly recognized role in promoting other neurodevelopmental and behavioral abnormalities (Micheli et al., 2011, see sections Defects in Nucleotide Metabolism and Toward a Purinergic-Based Therapy for Congenital Neurodevelopmental Disorders?).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.