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Purinergic implication in amyotrophic lateral sclerosis—from pathological mechanisms to therapeutic perspectives

Purinergic implication in amyotrophic lateral sclerosis—from pathological mechanisms to... Amyotrophic lateral sclerosis (ALS) is a clinically heterogeneous disorder characterized by degeneration of upper motor neurons in the brainstem and lower motor neurons in the spinal cord. Multiple mechanisms of motor neuron injury have been implicated, including more than 20 different genetic factors. The pathogenesis of ALS consists of two stages: an early neuroprotective stage and a later neurotoxic. During early phases of disease progression, the immune system through glial and T cell activities provides anti-inflammatory factors that sustain motor neuron viability. As the disease progresses and motor neuron injury accelerates, a rapidly succeeding neurotoxic phase develops. A well-orchestrated purine-mediated dialog among motor neurons, surrounding glia and immune cells control the beneficial and detrimental activities occurring in the nervous system. In general, low adenosine triphosphate (ATP) concentrations protect cells against excitotoxic stimuli through purinergic P2X4 receptor, whereas high concentrations of ATP trigger toxic P2X7 receptor activation. Finally, adenosine is also involved in ALS progression since A2A receptor antagonists prevent motor neuron death. Given the complex cellular cross-talk occurring in ALS and the recog- nized function of extracellular nucleotides and adenosine in neuroglia communication, the comprehensive understanding of purinome dynamics might provide new research perspectives to decipher ALS and help to design more efficient and targeted drugs. This review will focus on the purinergic players involved in ALS etiology and disease progression and current therapeutic strategies to enhance neuroprotection and suppress neurotoxicity. . . . . . Keywords ALS Purinergic signaling ATP Neuroinflammation Motor neuron degeneration Neuroglial activation Introduction motoneuronal cells such as microglia, astrocytes, interneu- rons, Schwann and skeletal muscle cells, oligodendrocytes, Amyotrophic lateral sclerosis (ALS), also known as Charcot’s and possibly endothelial cells and T lymphocytes [2–5]. or Lou Gehring’s disease, is the most common adult-onset Therefore, further research efforts to decipher the pathogene- motor neuron disorder (MND) characterized by degeneration sis of ALS should not only focus on potential disease triggers of motor neurons, leading to progressive paralysis and death but also on mechanisms for the propagation of pathological [1]. Due to a staggeringly complex etiology, the identification processes and signals between individual cells in a non-cell of a principal mechanism and development of an effective autonomous manner. therapeutic treatment for ALS still remains a research chal- Currently, it is postulated that an understanding of the lenge. In the light of current findings, it has become evident purinergic impact on neuroinflammation underpinning pa- that pathogenesis of ALS is not restricted to motor neurons but thology of neurological disorders is essential for the develop- attributed to the abnormal interactions of neurons and non- ment of efficacious interventions. Extracellular adenosine tri- phosphate (ATP) and adenosine (Ado) are recognized as the most powerful purinergic signaling molecules, directing inter- * M. Wujak cellular cross-talk and thereby equilibrating beneficial and mag_wuj@umk.pl detrimental activities occurring in the nervous system. In the present work, we will discuss the most relevant research ad- Neurology Clinic, Marek Cieślak, Toruń, Poland vances in deciphering a role of the purinome in mediating Department of Biochemistry, Faculty of Biology and Environmental pathological mechanisms underlying dysfunction of neuroglia Protection, Nicolaus Copernicus University in Toruń, 1 Lwowska St, and consequent motor neuron damage. By presenting the 87-100 Toruń, Poland 2 Purinergic Signalling (2019) 15:1–15 time-specific involvement of diverse purinergic receptors in cellular perturbations, including oxidative stress [10], neuroin- the disease progression, we will discuss their relevance for the flammation [11], glutamate excitotoxicity [12], mitochondrial development of new more powerful diagnostic and therapeu- dysfunction [13], RNA metabolism impairment [14, 15], pro- tic avenues for amyotrophic lateral sclerosis. tein misfolding and aggregation, and ER stress [16, 17], dysfunction of the ubiquitin–proteasome system [18], lack of trophic (growth) factors [19], aberrant axonal conduction [20], immune system deficiency [21], and blood-spinal cord barrier Basic notions of ALS impairment [22]. In 1990s, it has been discovered that some cases of fALS ALS is characterized by degeneration of motor neurons in the are associated with mutations in a gene localized on chromo- brainstem (upper motor neurons, UMN) and in the spinal cord some 21q21.1 encoding superoxide dismutase 1 (SOD1), also (lower motor neurons, LMN). This results in a progressive known as Cu/Zn superoxide dismutase [23]. Since then, over muscle denervation in upper and lower limbs, torso, and bulbar 160 mutations distributed throughout the 153-amino acid region leading ultimately to weakness and atrophy of skeletal SOD1 polypeptide have been identified in association with muscles [1]. ALS is classified as a rare adult-onset disease with ALS and till date, many cell and animal models expressing 58–60 years as average age of onset. The incidence rate of ALS exogenous mutant SOD1 have been developed in order to is around 1 to 2.6 cases per 100,000 persons annually, whereas investigate the etiology of ALS [24, 25]. Following the dis- the prevalence is approximately 6 cases per 100,000 per year covery of mutant SOD1, numerous mutations in other genes [6]. The vast majority of ALS patients die from respiratory have been identified to be associated with ALS, thereby open- failure within 3–5 years after onset of symptoms, while only ing up new avenues for the disease modeling. Figure 1 pre- 10% survive beyond 10 years [7]. Approximately 90% of ALS sents a brief overview of key cellular mechanisms and genetic cases occur randomly and are termed sporadic ALS (sALS), mutations contributing to the motor neuron damage in ALS. while the remaining 10% of cases are classified as familial ALS (fALS) having autosomal dominant pattern of inheritance, al- though some autosomal recessive pedigrees have been reported [8]. Due to a highly complex pathology and heterogeneous Purinergic players in the central nervous clinical presentations, ALS is considered as a multi-genetic, system multi-systemic, and multi-factorial disorder [9]. A composite etiology of ALS is caused by the influence of various genetic, Purinergic signaling utilizes nucleotides and nucleosides as biological, and environmental factors. The disease arises as a signaling molecules to activate two types of membrane- consequence of multiple pathophysiological mechanisms and bound receptors. The P1 receptors comprise four subtypes Fig. 1 Main key pathogenic mechanisms and cellular perturbations underlying the pathogenesis of ALS, including genetic background Purinergic Signalling (2019) 15:1–15 3 of G protein-coupled receptors (A1, A2A, A2B, A3) that dynamic changes of extracellular ATP [45]. After release, are activated exclusively by adenosine, whereas the P2 the pericellular concentration of ATP rises even up to 100– receptors are activated by nucleotides (ATP, ADP, UTP, 200 μM and is rapidly reduced to 1–100 nM by the activities UDP) and due to structural and pharmacological differ- of nucleotide-metabolizing enzymes, existing in a ences are classified into two subtypes: G protein-coupled membrane-bound or soluble form, with the consequent pro- P2Y receptors (1, 2, 4, 6, 11, 12, 13, 14) and ionotropic duction of ADP and/or AMP, including nucleotide P2X receptors (1–7) [26]. Neuroglia and neurons contain triphospho-diphosphohydrolases (NTPDases), nucleotide different combinations of purinergic receptors, which con- pyrophosphohydrolases/phosphodiesterases (NPP), and al- tributes to a versatile regulation of pathophysiological pro- kaline and acid phosphatases (Fig. 2). Finally, AMP hydro- cesses in the NCS (Table 1)[27–43]. lysis catalyzed by 5′-nucleotidase (5′-NT or CD73) results The signaling events are triggered upon the release of in production of adenosine which is further deaminated via purines and pyrimidines into the extracellular matrix by inosine into hypoxanthine by adenosine deaminase (ADA) exocytosis, facilitated diffusion, through channels, and fi- and purine nucleoside phosphorylase (PNP), respectively nally as a result of cell damage and lysis [26]. Extracellular [46, 47]. Taken together, the breakdown of ATP by ecto- ATP concentration drastically increases in response to di- nucleotidases not solely terminates its extracellular messen- verse biological, chemical, or mechanical stimuli, such as ger functions but also generates additional agonists, namely hypoxia, infection, physical trauma, neurodegeneration, ADP and adenosine. It is important to emphasize that the and neuroinflammation [44]. To measure ATP in the phosphorylation of adenosine to AMP by adenosine kinase pericellular space, several in vitro and in vivo methods are (ADK) enters the opposite metabolic pathway whereby available, including a novel pmeLUC system which is a adenosine can be converted back to its nucleotide deriva- simple and reliable in vivo tool for investigating the tives. The synthesis of ADP and ATP molecules is catalyzed Table 1 Distribution of P1 and P2 receptors in the nervous system Receptor subtype Distribution/cell type References P1 receptors A1 Widely distributed in brain (ISH); high expression levels in primary [27, 28] cultures of astrocytes from different brain regions, e.g., cerebellum, hippocampus, cortex, thalamus, spinal cord (RB, RT-PCR) A2A Brain neurons and astrocytes (RB), high expression levels in dopamine-rich [29, 30] regions (IHC), low expression levels in hippocampus (RT-PCR, IHC) A2B Widely distributed but at low expression levels; glial and neuronal cells (RT-PCR) [30, 31] A3 Neurons of cerebellum and hippocampus (RT-PCR, WB), astrocytes [32] (WB), generally low expression level P2X receptors P2X1 Cerebellum (IHC, RT-PCR), dorsal horn spinal neurons, astrocytes, microglia (IHC) [33, 34] P2X2 Widely distributed in neuronal structures, including the cortex, hippocampus, [35, 36] cerebellum, spinal cord (ISH), autonomic and sensory ganglia neurons (IHC) P2X3 Sensory neurons, nucleus tractus solarius neurons, some sympathetic [34, 35] neurons (ISH, IHC) P2X4 Widely distributed in CNS (ISH), neurons, astrocytes, activated microglia (IHC) [35, 36] P2X5 Neurons in spinal cord, astrocytes (ISH, IHC) [35] P2X6 widely distributed in CNS (ISH); motor neurons in spinal cord (IHC) [35, 36] P2X7 Ependymal cells lining the ventricles (RT-PCR), hippocampus (RT-PCR), [37] microglia, astrocytes, apoptotic cells (IHC, WB) P2Y receptors P2Y1 Widespread distribution in mammalian brain, including the cerebral cortex, [38] hippocampus and cerebellum (IHC), neurons and microglia (IHC, RT-PCR) P2Y2 Astrocytes (IHC, RT-PCR) [38] P2Y4 Brain neurons (IHC, RT-PCR) and microglia (RT-PCR) [38, 39] P2Y6 Activated microglia (ISH, IHC, RT-PCR) [39–41] P2Y11 brain neurons and oligodendrocytes of nucleus accumbens, parahippocampal [39] gyrus, putamen and striatum (RT-PCR) P2Y12 Hippocampal pyramidal neurons (RT-PCR), resting microglia (RT-PCR) [39] P2Y13 Brain (RT-PCR) neurons and oligodendrocytes (IHC), microglia (ISH, IHC) [40, 42] P2Y14 CNS astrocytes (RT-PCR), discrete brain regions (ISH) [40, 43] ISH in situ hybridization, RB radioligand binding, RT-PCR reverse transcriptase polymerase chain reaction, IHC immunohistochemistry, WB Western Blot 4 Purinergic Signalling (2019) 15:1–15 Fig. 2 Enzymes involved in the metabolism of extracellular nucleotides as CD73), adenosine deaminase (ADA), and purine nucleoside and nucleosides. AMP, ADP, and ATP nucleotides breakdown to phosphorylase (PNP). ATP re-synthesis via backward phosphotransfer adenosine (Ado) and other nucleosides such as inosine (Ino) and reactions: adenylate kinases (AK), nucleoside-diphosphate kinases hypoxanthine (Hyp): nucleotide triphospho-diphosphohydrolases (NDPK), and ATP synthase. P2X7, pannexin (Panx), connexin (Cx), (NTPDases), nucleotide pyrophosphohydrolases/phosphodiesterases and ATP-binding cassette (ABC) proteins represent channel and (NPP), alkaline phosphatase (AP), 5′-nucleotidase (5′-NT, also known transport-mediated ATP release pathways by nucleotide-phosphorylating enzymes such as adenylate disproportionate neuroinflammatory responses, particular- kinases (AK) and nucleoside-diphosphate kinases (NDPK) ly when chronic, promote apoptosis and necrosis and in- and ATP synthase [47, 48]. Consequently, the interplay be- fluence the synaptic and intrinsic membrane properties of tween phosphohydrolysis and phosphotransfer reactions neurons [52]. provides a precise and dynamic control of the duration, A dominant role for neuroinflammation has been re- magnitude, and direction of purinergic and pyrimidinergic ported in the etiology of primary and secondary neurode- signals. Most importantly, the existence of many P1/P2 re- generative diseases, such as Alzheimer’s disease (AD), ceptor subtypes differing in their sensitivity to agonists and Parkinson’s disease (PD), multiple sclerosis (SM), amyo- the possibility that the same ligand activates more than one trophic lateral sclerosis, Huntington’s disease (HD), receptor subtype increase the complexity of the purinome stroke, and epilepsy [53, 54]. and generate an extensive network of overlapping cellular The main cellular effectors of neuroinflammation are astro- responses [9, 49]. cytes and microglia, as well as T lymphocytes, perivascular monocytes, and macrophages invading to the sites of insult from circulation. Microglial cells and astrocytes undergo acti- Cellular players in neuroinflammation vation in the process of microgliosis and astrogliosis, respec- tively [55]. The processes of microglia and astrocyte activa- In the central nervous system, ATP acting as a neurotrans- tion, T lymphocyte infiltration, and overproduction of a vari- mitter and neuromodulator regulates a wide array of phys- ety of inflammatory cytokines have been demonstrated in as- iological processes such as neurotransmission, cell-to-cell sociation with neuronal loss even during the pre-symptomatic communication, neurite outgrowth, as well as cell prolif- phase of ALS [56]. eration, migration, differentiation, and apoptosis [26]. Microglia are the first line of immune defense in the ATP is released extracellularly under conditions of tissue CNS. The cells have been shown to be highly plastic and stress or injury by microglia, astrocytes, and damaged could acquire distinctive phenotypes in response to vari- neurons. The increase in extracellular ATP (eATP) level ous stimuli. Under physiological conditions, microglia re- is recognized by competent cells as damage-associated tain a relatively quiescent phenotype. However, these cells molecular pattern (DAMP) signal capable of initiating constantly monitor local microenvironment and communi- and propagating neuroinflammatory response [3, 50, 51]. cate with astrocytes and neurons by secreting anti- The principle functions of neuroinflammation are to limit inflammatory molecules and neurotrophic factors like insulin-like growth factor 1 (IGF-1), transforming growth tissue damage and initiate tissue repair. However, Purinergic Signalling (2019) 15:1–15 5 factor-β (TGF-β), brain-derived neurotrophic factor enhancing the expression of neurotrophic factors, such (BDNF), and nerve growth factor (NGF) [57]. Following as IGF-1 and glial glutamate transporter-1 (GLT-1) [65]. injury or pathogen invasion and exposure to pro- At pre-symptomatic stage, treatment of ALS mice with inflammatory cytokinessuchasinterferon-γ (IFN-γ), CD4 T lymphocytes significantly delays the onset of + + IL4 and tumor necrosis factor-α (TNF-α), microglial cells symptoms (actionascribedtoCD4 CD25 regulatory T- become polarized (activated) toward a pro-inflammatory cells) and increases the latency between disease onset and phenotype. Upon activation, microglia promptly release entry into late stage (action mediated by CD4 an entirely different set of molecules, such as pro- CD25 effector cells) [66, 67]. In the recent study, the inflammatory cytokines and chemokines, reactive oxygen expansion of endogenous regulatory T-cells in a mouse species (ROS) or nitric oxide (NO), which contribute to model of ALS significantly prolonged motor neuron sur- the onset of local inflammatory response [58, 59]. vival time, suppressed glial cell immunoreactivity, and The danger signals from damaged tissues include ATP enhanced neuroprotective gene expression. Regulatory T- released by dying and abnormally functioning neurons. cells were also shown to correlate with a slower rate of The nucleotide-metabolizing enzymes are represented on disease progression in ALS patients [68]. microglial cells mainly by ecto-nucleoside triphosphate Mast cells function as environmental Bsensors^ to com- diphosphohydrolase 1 (ecto-NTPDase1, CD39) and ecto- municate with other players under physiological condi- 5′-nucleotidase (CD73) [3]. Concerted action of these en- tions or during immune responses, based on their wide- zymes restores the balance between ATP and adenosine spread tissue presence near blood vessels and surfaces concentration that will be discussed in detail in the next exposed to the environment [69]. In the nervous system, chapters. mast cells represent an important peripheral counterpart in Astrocytes are the most abundant fraction of cells with intercellular cross-communication. Upon activation, mast a glial phenotype in the brain. Despite this population of cells secrete numerous vasoactive, neurosensitizing, and cells has long been neglected or misidentified, astrocytes pro-inflammatory mediators, including histamine, seroto- represent a key component in the brain environment. They nin, cytokines, proteolytic enzymes (e.g., chymase, provide neurotrophic factors, control synaptic functions tryptase, acid hydrolases), lipid metabolites (prostaglandin and formation, regulate the concentration of neurotrans- D2, leukotriene C4, platelet-activating factor), neuropep- mitters at synapses, and are involved in a wide range of tides, growth factors (NGF and VEGF), and nitric oxide homeostatic functions [60]. In the course of neurodegen- [70]. Mast cells are also an abundant source of ATP which erative diseases, including ALS, astrocytes become reac- is stored in their granules and secreted upon activation tive by altering their morphology and molecular expres- [69]. Serum and CSF samples of ALS patients display sion patterns [55, 61]. Activation of P2X7 receptor on elevated amounts of IL-12 and IL-15 [71], the latter cyto- spinal cord astrocytes was proved to trigger cytotoxic cas- kine acting as a mast cell chemoattractant, resulting in cade and exert neurotoxic effect on motor neurons [62]. mast cells accumulation around degenerating motor neu- Accompanying processes involve induction of nuclear rons. Mast cell involvement in the neuromuscular junction factor κ light-chain enhancer of activated B cells denervation was recently investigated in an animal ALS (NF-κB) and activator protein 1 (AP-1), and stimulation model, where the authors observed a marked infiltration of cyclooxygenase-2 (COX-2) activity [3]. Another im- and degranulation of mast cells that correlated with dis- portant astrocyte dysfunction in ALS is attributed to ease progression [72]. lowered release of neurotrophic factors, such as BDNF, The cross-talk between different cell types in the ner- glia-derived neurotrophic factor (GDNF), or vascular en- vous system has a recognized role in neural information dothelial growth factor (VEGF). Neurotrophic factors are processing. Glia-neuron, mast cell-glia, and glia-glia cells known to improve the neuron survival and repair; there- communicate bi-directionally through the release of extra- fore, their deficiency significantly attenuates cellular signaling molecules. Studies from the last decades neuroregeneration [63]. have provided strong evidence for the complexity of this Lymphocyte infiltration was described in ALS patients cross-talk [50, 69, 73]. The outcome of this intercellular in the corticospinal tracts and anterior horns of the spinal communication is dependent on the local pathophysiolog- cord [64]. T-helper CD4 cells permeate at the onset of ical status, e.g., stress or injury, and on environmental fac- the disease and then accumulate during disease progres- tors, e.g., cytokine concentration. Molecules contributing sion, whereas T-cytotoxic CD8 cells are only present at to the intercommunication also include extracellular nucle- the end stage of disease. In the ALS mice model, CD4 T otides, particularly ATP. All the cellular players can both lymphocytes slowed disease progression, extended dis- release and simultaneously respond to the nucleotide signal ease duration by 50%, modified the microglial pheno- via numerous purinergic receptors. Therefore, eATP gen- erates a feedback loop that drives the persistent pro- types, and prolonged survival. This probably occurs by 6 Purinergic Signalling (2019) 15:1–15 inflammatory and detrimental response [3, 74, 75]. During ATP and P2 receptors in ALS the progression of ALS, microglia, astrocytes, and motor neurons enter in this pro-inflammatory cross-talk, followed P2X7 by exacerbation of pathological processes. More insight into the communication between different cell types should Injury to motor neurons caused by pro-inflammatory factors provide important novel therapeutic approaches to promote released by activated microglia is considered as one of the repair and reduce neuroinflammation. most critical pathogenic mechanisms in ALS. Among purinergic receptors, the P2X7 receptor was shown to be in- volved in chronic pain, neurodegeneration, and neuroinflam- mation [90]. This purinergic receptor is predominantly The involvement of purinergic signaling expressed on microglia and oligodendroglia, and at lower lev- in the ALS pathology el on astrocytes [37]. P2X7 receptor activation mediates the release of IL-1 family cytokines including IL-1α,IL-1β,and Purinergic signaling regulates glial proliferation, motility, IL-18 [91]. Release of IL-1β results in upregulation of pro- survival, and myelination, as well as facilitates interac- teins contributing to inflammatory processes, including COX- tions between neurons and vascular and immune system 2, nitric oxide synthase (NOS), TNF-α, pro-caspase 1 as well cells [3, 73]. Numerous studies indicate that a vast inter- as matrix metalloproteinase-9 (MMP-9), and cannabinoid re- play occurs also at the cell membrane among purinergic ceptor 2 (CB2). Interestingly, a significantly increased immu- receptors, ecto-nucleotidases, and transporters, resulting noreactivity of P2X7, together with COX-2 and CB2, has in the insurgence or maintenance of neuroinflammatory been shown in active microglia from post-mortem spinal cord conditions [73, 76]. In the following subsections, we will samples of sALS patients and in the mSOD1G93A transgenic present in detail the contribution of individual purinergic rodent model of fALS [81, 85, 92]. The P2X7 receptor- signaling components in the pathogenesis of ALS and mediated neurotoxicity was also confirmed in mSOD1 astro- potential therapeutic directions (Table 2). cytes [62]. P2X7 receptor alone can activate cell death via Table 2 Fundamental discoveries of purinergic contribution to ALS pathogenesis Purinergic component Contribution to ALS References Adenosine Ado Significantly increased in the cerebrospinal fluid of ALS patients [77] and A2A receptors A2A Receptors activation makes motor neurons susceptible to [78] excitotoxic challenge A2A Upregulated in lymphocytes from ALS patients [79, 80] A2A Upregulated in motor neurons from spinal cords of SOD1G93A [80] mice and ALS patients ATP and P2 receptors P2X7 Microglial expression increased in post-mortem spinal cord samples [81] from ALS patients P2X4, P2X7, P2Y6 Upregulated in SOD1-G93A mice microglia [75] P2X7 Activation of microglial receptor induces cell death of ALS motor neurons [75] P2X7 Activation of astrocytes receptor initiates motor neurons death in vitro [62] ATP, BzATP Small doses induce motor neuron death in vitro [82] P2X7 Activation of receptor by BzATP up-regulates the miRNAs transcriptome [15] in SOD1-G93A microglia P2X7 Ablation of receptor in SOD1-G93A mice aggravates gliosis and motor [83] neuron death P2X7 Receptor antagonist Brilliant Blue G ameliorates spinal cord pathology in [84] SOD1-G93A mice P2X4 Upregulated in degenerating motor neurons in ALS mouse and rat spinal cord [12, 85] P2Y12 Expression progressively reduced in SOD1-G93A mice microglia [86, 87] P2X4, P2X7 Upregulated in sciatic nerves of SOD1-G93A mice [9] Enzymes CD39 Gene expression downregulated in spinal cord microglia from SOD1G93A [88] mice and ALS patients CD39 Protein expression downregulated in SOD1-G93A mice microglia [9] ADK Increased activity in reactive astrocytes decreases adenosine concentration [89] and triggers neurodegeneration Purinergic Signalling (2019) 15:1–15 7 both apoptotic and necrotic mechanisms leading ultimately to delayed ALS onset, although with no effect on life span. the development of neurotoxic phenotype. For instance, a Importantly, BBG neuroprotection occurred within a specific peroxynitrite-fueled apoptotic cascade activated by P2X7 re- time frame, since BBG administration at earlier phases did not sults in motor neuron death due to trophic factor deprivation, prevent disease progression [84]. In particular, BBG treatment activation of p57 neutrophin death receptor (p75NTR), and enhanced motor neuron survival and significantly reduced expression of mutant forms of SOD1 [62, 82]. microgliosis by downregulating the expression of pro- Emerging evidence indicates that P2X7 may have a dual inflammatory proteins, NF-κB, and microglia markers of ac- function in onset and progression of ALS, either trophic and tivated phenotype (NOX2 and IL-1β), and simultaneously anti-inflammatory or toxic and pro-inflammatory. The fact that upregulating other markers (IL-10 and BDNF). The key con- ablation of P2X7 in SOD1-G93A mice exacerbates gliosis and clusion drawn from these results might be Bthe double face^ of motor neuron death at end stage of the disease supports a neuro- P2X7 receptor and the existence of a narrow therapeutic win- protective role of this receptor [83]. On the other hand, the acti- dow concerning its beneficial role in ALS. The dual action of vation of microglial P2X4, P2X6, and P2X7 receptors by 2′-3′- P2X7 during ALS progression seems to correspond to the O-(benzoyl-benzoyl) ATP (BzATP), a non-hydrolysable form of switch of microglia from protective to lethal phenotype. ATP, leads to increased content of pro-inflammatory molecules Most recently, the role of P2X7 receptor in microglia po- such as TNF-α and COX-2 with the consequent motor neuron larization has been confirmed in LPS-induced cellular model injury [75]. Moreover, the activation of P2X7 receptor by BzATP of inflammation. It was shown that P2X7 inhibition by the enhanced ROS production in SOD1-G93A mouse microglia selective antagonist A438079 suppressed microglia activation through the activation of NADPH oxidase 2 (NOX2) which is [101]. Based on these findings, P2X7 has emerged as a poten- the main ROS-producing enzyme in microglial cells and well- tial marker of activated microglia. It is postulated that the recognized player in the pathogenesis of ALS. Importantly, this microglial polarization switch is closely linked with the time microglia-mediated mechanism for motor neuron damage was when P2X7 starts to play a critical role in regulating neuroin- shown to be prevented by ablation of P2X7 and the use of P2X7 flammation in ALS [9]. This hypothesis is strongly supported specific antagonists such as A839977, A438079, and Brilliant by the fact that ALS pathogenesis consists of two distinct Blue G (BBG) [93]. Other pathological mechanism by which neuroinflammatory stages. The first stage is neuroprotective P2X7 can exacerbate a neurotoxic phenotype of ALS microglia due to the concerted action of regulatory T-cells, anti- is miRNA dysregulation. Parisi and colleagues have shown that inflammatory macrophages/microglia, and T helper cells, stimulation of P2X7 receptor by BzATP hyperactivated inflam- which sustain motor neuron viability by providing anti- matory miRNAs, such as miR-155, miR-125b, and miR-146b inflammatory agents. As the disease and motor neuron injury which are known to be upregulated in SOD1-G93A microglia. It accelerate, the second rapidly progressing stage emerges when resulted in downregulation of IL-6/STAT3 signaling and en- activation of pro-inflammatory T-cells and macrophages/ hancement of TNF-α production, switching eventually microglia microglia leads eventually to neurotoxicity [9, 59, 102]. toward a detrimental phenotype [15]. It is assumed that the use of neuroprotective agents such as P2X4 COX-2 inhibitors, P2X7 receptor antagonists, and CB2 ago- nists might exert beneficial effects in ALS pathology by A strong P2X4 immunoreactivity was shown to be associated slowing down the progressive damage to motor neurons [81, with degenerating motoneurons in spinal cord ventral horn 92]. The P2X7 antagonist BBG is considered as a promising samples from mSOD1G93A rats. In parallel, P2X4 immuno- candidate for blocking the detrimental effects of P2X7 activa- staining detected degeneration in other neuronal populations, tion due to its high selectivity, low toxicity, and ability to including noradrenergic neurons in the locus coeruleus, efficiently cross the blood–brain barrier. Other P2 antagonists Purkinje cells in the cerebellum and serotonin containing neu- such as oxATP (oxidized ATP) and PPADS (pyridoxal-phos- rons in the raphe nucleus [85]. More interestingly, the P2X4 phate-6-azophenyl-2′,4′-disulfonic acid) fail to meet all of the antibodies were able to recognize neurotoxic species of above requirements [94, 95]. Brilliant Blue G has previously misfolded SOD1G93A in motor neurons but not in glial cells. been proven to reduce neuroinflammation in traumatic brain It was suggested that neuronal P2X4-immunoreactive and spinal cord injury [95–97], cerebral ischemia reperfusion SOD1G93A conformers may have a pathogenic role in the [98], neuropathic pain [99], and in experimental autoimmune promotion of neuroinflammation since they activated microg- encephalitis [100]. The neuroprotective effects of BBG have lia and astroglia when injected intracerebrally into normal been tested in the SOD1-G93A ALS mouse model at different animals [103]. phases of ALS to elucidate the role of P2X7 receptor in Upregulation of P2X4, both at mRNA and protein level, microglial polarization and neuroinflammation. was also found in ALS microglia from SOD-G93A mice [75]. Administration of BBG, beginning at a late pre-symptomatic Most recently, Volonté and colleagues have demonstrated for phase, improved motor neuron performance and slightly the first time the increased expression of P2X4 and P2X7 8 Purinergic Signalling (2019) 15:1–15 proteins in the peripheral nervous system of SOD1-G93A authors showed that short treatment (from asymptomatic to mice, namely in sciatic nerves, and nominated these P2 recep- symptomatic phase) with antihistamine drug clemastine reduced tors as potential diagnostic biomarkers for ALS at the periph- disease progression and improved survival of SOD1G93A ALS eral level [9]. Finally, P2X4 receptor activation was shown to mice by enhancing anti-inflammatory phenotype of microglia protect motor neurons. α-Amino-3-hydroxy-5-methyl-4- via upregulation of P2Y12 together with P2Y7, arginase-1 and isoxazole propionic acid (AMPA) receptor-mediated CD1639. Because long treatment with clemastine (from asymp- excitotoxicity is considered as an important mechanism for tomatic until the end stage) failed to ameliorate ALS progres- motor neuron death in ALS. Andries and colleagues showed sion, the beneficial effects of this drug are thought to be tightly that preincubation of motor neurons with P2X4 allosteric dependent on the first phase of neuroinflammation in ALS, modulators such as ivermectin (anti-parasite medication) and characterized by neuroprotective functions of microglia [84]. Cibacron Blue 3G-A (CB) protected the cells against kainate- induced excitotoxicity in vitro. In addition, ivermectin poten- Adenosine and P1 receptors in ALS tiated the protective effect of low ATP concentrations to motor neurons, presumably by increasing the number of P2X4 mol- In 1999, Yoshida and colleagues reported a significantly in- ecules on the cell surface, and most importantly, extended creased adenosine concentration in the cerebrospinal fluid of survival of SOD1-G93A mice by almost 10% [12]. progressing ALS patients; however, neither diagnostic nor prognostic potential of these findings had been evaluated P2Y6 [77]. Adenosine, formed as the product of eATP degradation, possesses neuroregenerative potential. In ALS patients, over- P2Y6 receptor is activated mainly by UDP, partially sensitive expression of adenosine kinase (ADK) is a common patho- to UTP and ADP, and completely insensitive to ATP. P2Y6 logic hallmark, and the consequent increase in astrocyte ADK was shown to be upregulated in SOD1G93A mice microglia; activity disrupts adenosine homeostasis triggering ultimately however, the role of this receptor in ALS remains elusive [75]. neurodegeneration [89]. On the other hand, adenosine is ca- Likewise P2Y2 and P2Y4 receptors, P2Y6 regulates pable to stimulate astrogliosis by activating P1 receptors on microglial phagocytosis upon activation by UDP [104]. It astrocytes. A2A and A3 receptors are involved in the was suggested that this process may play important role in astrogliosis initiation, whereas the activation of A1 receptor facilitating the uptake of cellular debris generated after neuro- inhibits the proliferation of astrocytes [3, 108]. Moreover, nal damage. In fact, damage of hippocampal neurons by in vitro studies revealed that adenosine at physiological con- kainate in vivo and in vitro resulted in upregulation of centration of about 10 nM activates A2A receptor in astro- microglial P2Y6 expression and consequent activation of cytes, followed by affecting GLT-1 and inhibiting glutamate microglial phagocytic activity via UDP/P2Y6 signaling [105]. uptake [3]. And ALS patients and SOD1-G93A mice suffer from suppressed glutamate uptake that drives excitotoxic pro- P2Y12 cesses and motoneuron degeneration [109]. The first evidence for the involvement of A2A in ALS was The involvement of P2Y12 in microglia process dynamics is provided by A2A blockage with the selective antagonist well established, including its key role in the regulation of mi- KW6002, which protected motor neurons from toxic insult croglia activation, chemotaxis, and migration [86, 106]. This triggered by the expression of mutant versions of SOD1 and glued purinergic receptor can serve as a marker of a resting/ dynactin subunit p150 . The neuroprotective effect of A2A surveillant branched state of ALS microglia as well as a marker antagonism was attributed to attenuated tyrosine receptor ki- distinguishing CNS-resident microglia from blood-derived mac- nase B (TrkB) signaling, known to induce vulnerability of rophages infiltrating CNS upon neuronal injury [87, 107]. motor neurons to excitotoxic challenge upon activation by Moreover, a dramatic and continuous reduction of P2Y12 re- BDNF. Moreover, a physical interaction between A2A and ceptor expression was observed after microglia activation fol- TrkB within lipid rafts of motoneurons was shown to be pre- lowing brain injury [106]. Consistently, P2Y12 expression was requisite for TrkB transactivation [110]. On the contrary, acti- found to be progressively reduced in spinal cord microglia of vation of A2A in the absence of BDNF signaling rendered SOD1-G93A mice and ALS patients during neuroinflammation these cells susceptible to excitotoxicity [78]. However, the [87, 107]. Interestingly, P2Y12 expression was significantly de- above findings are not consistent with results gained from creased at symptomatic stage, when the disease accelerates, and studies on motor neuron cultures exposed to BzATP and in completely lost at end stage of the disease when motor neuron SOD1-G93A mouse model for ALS. Namely, it was shown loss, oligodendrocyte degeneration, and microglia activation are that high doses of adenosine protect motor neurons from known to be augmented [87]. These findings indicate a neuro- death induced by BzATP, whereas low doses exert no benefi- protective action of P2Y12 at early stage of ALS that is consis- cial effect. Because adenosine production results from rapid tent with results obtained by Apolloni and colleagues. The ATP breakdown, it was concluded that ATP at high Purinergic Signalling (2019) 15:1–15 9 concentrations (1 mM), paradoxically, may be protective to NTPDases motor neurons, while at low concentrations trigger neuronal apoptosis upon P2X7 activation [82]. A protective role of CD39 (NTPDase1) is exclusively expressed on the surface of A2A has been also demonstrated in SOD1G93A mice model, microglia where it plays a predominant role in the inactivation as treatment with the selective agonist CGS21680 resulted in of P2 receptor-mediated signaling by catalyzing a rapid ATP delayed disease onset [111], whereas intake of caffeine, a non- degradationtoADP andAMP [114]. Consequently, any selective P1 receptor antagonist, significantly decreased sur- changes in CD39 expression may result in disturbed nucleo- vival time of ALS animals [112]. tide homeostasis and subsequent alterations in purinergic A possible involvement of the P1 receptors in ALS has also transmission. In fact, CD39 downregulation at mRNA level been investigated at peripheral level. Expression profiling of has been demonstrated in microglia from the spinal cord of all P1 receptor subtypes revealed a significant upregulation of SOD1-G93A mice and ALS patients [88], which was further A2A in lymphocytes from ALS patients, compared to healthy confirmed at protein level in cortical primary microglia from subjects, and a positive correlation between A2A density SOD1-G93A newborn mice [9]. CD39 downregulation values and scores of the revised ALS Functional Rating caused prolonged activation of P2 receptors as a consequence Scale, which is a useful predictor for ALS progression. of a significantly reduced hydrolysis of extracellular ATP. As Moreover, activation of A2A by the selective CGS21680 ag- three ATP-sensitive P2 receptors, namely P2X4, P2X7, and onist led to increased production of cyclic AMP in lympho- P2Y6, are found to be upregulated in ALS, the above findings cytes from ALS patients as compared with a control group. emphasize a critical role of CD39 in the regulation of The negative correlation between A2A density and the sever- neuroinflammatory events mediated by ALS microglia [75]. ity of disease symptoms, which highlights a possible role for In general, dysregulation of any purinergic element along with these receptors in immunosuppressive responses in ALS, altered concentrations of signaling agents in the extracellular might also represent a promising perspective for alternative milieu leads to the enhancement of inflammatory responses. therapeutic approaches for ALS based on modulation of CD39L1 (NTPDase2) was found to be highly expressed in A2A receptor activity [79]. In view of anti-inflammatory ef- hippocampal, cortical, and cerebellar astrocytes where it plays fects of A2A activation to peripheral immune cells, these find- a predominant role in regulation of the ATP/adenosine bal- ings support a neuroprotective role of A2A in ALS, at least at ance. Consequently, impairment of its activity can increase peripheral level. eATP concentration resulting in activation of P2X receptor The most recent discovery has provided evidence for a and initiation of inflammatory astrogliosis, leading ultimately more complex role of A2A-mediated adenosine signaling in to neuronal cell death. For this reason, NTPDase2 is consid- ALS. Firstly, it was shown that A2Awas upregulated in motor ered as another potent therapeutic target in human CNS dis- neurons from spinal cords of symptomatic SOD1G93A mice orders, but its precise role in ALS needs to be elucidated [115]. and end-stage ALS patients [80] that was in agreement with To summarize the current findings discussed in the previous studies reporting the increased expression of A2A in above chapter, in Fig. 3, we depict a network of motor neurons of end-stage SOD1G93A mice [112]. purinergic components and mechanisms recognized so Furthermore, a direct treatment of adenosine at concentration far to contribute to aberrant neuron-glia communication of 0.3 or 1.0 μM induced death of embryonic stem cell- and enhanced neuroinflammation underpinning ALS derived motor neurons (ESMN) cultured in vitro suggesting pathogenesis. that A2A blockage might be neuroprotective to motoneurons. The subsequent A2A inhibition by the selective KW6002 an- tagonist and partial genetic ablation of A2A efficiently Current and potential treatment for ALS protected ESMN from SOD1G93A+ astrocyte-induced cell death and slowed disease progression of SOD1G93A mice Despite intensive research, little in the field of new effective [80]. In light of these studies, a novel toxic effect of adenosine therapeutics for ALS has been developed so far. The most on spinal cord motor neurons has been discovered; however, frequently provided reason for explaining the difficulties in the exact mechanism of this adenosine-induced A2A activa- finding a remedy for curing or slowing the progression of tion is elusive and merits further investigation. ALS is that the disease is multi-genic, multi-factorial, and Contrary to A2A, the role of other P1 receptors in ALS still multi-systemic. Till 2016, more than 50 major phase II or III remains elusive, but evidence for a loss of A1-A2A functional clinical trials in ALS patients have been reported to fail, de- cross-talk at the neuromuscular junction in pre-symptomatic spite positive results from animal models [116]. In this con- SOD1G93A mice has been reported recently. A1-mediated text, the question arises whether the most widely used SOD1 adenosine signaling may contribute to exacerbation of the transgenic mice models properly reflect different aspects of disease during the symptomatic phase when A1 tonic activa- ALS heterogeneity in patients with both sALS and non- tion was shown to be enhanced [113]. SOD1 linked fALS. It is believed that the latest discoveries 10 Purinergic Signalling (2019) 15:1–15 Fig. 3 Purinergic dysregulation in ALS in genetics of ALS, including mutations in C9ORF72 and symptoms, arimoclomol slowed disease progression, in- TBK1 genes, will open new perspectives in the generation of creased survival, and improved muscle function. The drug is relevant animal models for ALS, with consequent potent con- currently tested in phase III clinical trial [121, 122]. tribution to the development of new biomarkers and therapeu- tic targets for the disease [117]. For the past 22 years, the only available FDA-approved Purinergic signaling as potential therapeutic intervention for ALS has been riluzole (brand names Rilutek target in ALS or Teglutik). However, this anti-excitotoxic drug extends pa- tient’s life span by only several months without improving A growing body of research provides evidence for an essential muscle strength and neurological function and is ineffective contribution of purinergic signaling to the development of many in later stages of the disease [118]. On May 5, 2017, the sec- neuroinflammatory and neurodegenerative diseases. Most im- ond drug, known as either edaravone (Radicava) or MCI-186, portantly, some results of these studies have been already imple- was finally approved by the FDA for ALS treatment [119]. mented in the treatment of Parkinson’s disease (A2A receptor Edaravone administrated intravenously once a day for 14 days, antagonist istradefylline) [123], cerebral ischemic stroke followed by a 2-week break, decreases disease progression in (P2Y12 receptor antagonists clopidogrel and ticlopidine, and early-stage ALS patients with no respiratory involvement and adenosine transporter inhibitor dipyridamole) [124, 125]. It is indications for gastrostomy [119]. This anti-oxidant and free- also believed that they might contribute to establishing new radical scavenger delays motor neuron degeneration but pro- treatment strategies for multiple sclerosis, epilepsy, migraine, vides limited survival benefit [120, 121]. Among other com- and neuropathic pain [53, 126–128]. Since the discoveries of pounds currently being tested in clinical trials, arimoclomol is microglia activation as an important mechanism of motor neu- considered as a promising therapeutic candidate. This so- ron death in ALS and extracellular ATP as a crucial neuron-to- called smart drug induces the expression of heat shock pro- glia alarm signal, the involvement of purinergic signaling in teins (HSP) exclusively under cellular stress conditions. It neuroinflammation has become evident and largely accepted. results in increased capacity of protein quality control and During the progression of ALS, microglia, astrocytes, degradation of misfolded proteins, including mutant SOD1. and motor neurons continue the pro-inflammatory cross- Administered to SOD1-G93A mice after the onset of talk, inter alia through P2X7 activation that was shown to Purinergic Signalling (2019) 15:1–15 11 be prevented by P2X7 antagonists [62, 75]. The P2X4 Compliance with ethical standards receptors, however, exert protective effects in motor neu- Conflicts of interest M. Cieślak declares that he/she has no conflict of rons. Considering these results, it can be concluded that interest. low ATP concentrations protect cells against excitotoxic K. Roszek declares that he/she has no conflict of interest. stimuli through P2X4 receptors, whereas high concentra- M. Wujak declares that he/she has no conflict of interest. tions of ATP produce toxic P2X7 activation. Finally, adenosine is also involved in ALS progression since aden- Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. osine A2A receptor antagonists prevent motor neuron death at the symptomatic phase [73, 110]. In light of cur- Open Access This article is distributed under the terms of the Creative rent findings, P2X7 and A2A are recognized as dual- Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, function purinergic receptors, which course of action distribution, and reproduction in any medium, provided you give appro- closely depends on ALS state, particularly priate credit to the original author(s) and the source, provide a link to the neuroinflammatory landscape of the disease. However, Creative Commons license, and indicate if changes were made. despite many lines of evidence for essential contribution of purinergic signaling to motor neuron damage, it is im- portant to emphasize here that there are currently no clin- ical trials targeting the purinergic players in the therapy of References ALS. In terms of the high complexity of the purinome comprising diverse nucleotide- and nucleoside- 1. 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Vande Griend JP, Saseen JJ (2008) Combination antiplatelet the pathology of amyotrophic lateral sclerosis? Curr Neurol agents for secondary prevention of ischemic stroke. Neurosci Rep 15:45 Pharmacotherapy 28:1233–1242 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Purinergic Signalling Springer Journals

Purinergic implication in amyotrophic lateral sclerosis—from pathological mechanisms to therapeutic perspectives

Cieślak, M.; Roszek, K.; Wujak, M.
Purinergic Signalling , Volume 15 (1) – Nov 14, 2018
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Springer Journals
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Copyright © 2018 by The Author(s)
Subject
Biomedicine; Biomedicine, general; Pharmacology/Toxicology; Human Physiology; Neurosciences; Cancer Research
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1573-9538
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1573-9546
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10.1007/s11302-018-9633-4
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Abstract

Amyotrophic lateral sclerosis (ALS) is a clinically heterogeneous disorder characterized by degeneration of upper motor neurons in the brainstem and lower motor neurons in the spinal cord. Multiple mechanisms of motor neuron injury have been implicated, including more than 20 different genetic factors. The pathogenesis of ALS consists of two stages: an early neuroprotective stage and a later neurotoxic. During early phases of disease progression, the immune system through glial and T cell activities provides anti-inflammatory factors that sustain motor neuron viability. As the disease progresses and motor neuron injury accelerates, a rapidly succeeding neurotoxic phase develops. A well-orchestrated purine-mediated dialog among motor neurons, surrounding glia and immune cells control the beneficial and detrimental activities occurring in the nervous system. In general, low adenosine triphosphate (ATP) concentrations protect cells against excitotoxic stimuli through purinergic P2X4 receptor, whereas high concentrations of ATP trigger toxic P2X7 receptor activation. Finally, adenosine is also involved in ALS progression since A2A receptor antagonists prevent motor neuron death. Given the complex cellular cross-talk occurring in ALS and the recog- nized function of extracellular nucleotides and adenosine in neuroglia communication, the comprehensive understanding of purinome dynamics might provide new research perspectives to decipher ALS and help to design more efficient and targeted drugs. This review will focus on the purinergic players involved in ALS etiology and disease progression and current therapeutic strategies to enhance neuroprotection and suppress neurotoxicity. . . . . . Keywords ALS Purinergic signaling ATP Neuroinflammation Motor neuron degeneration Neuroglial activation Introduction motoneuronal cells such as microglia, astrocytes, interneu- rons, Schwann and skeletal muscle cells, oligodendrocytes, Amyotrophic lateral sclerosis (ALS), also known as Charcot’s and possibly endothelial cells and T lymphocytes [2–5]. or Lou Gehring’s disease, is the most common adult-onset Therefore, further research efforts to decipher the pathogene- motor neuron disorder (MND) characterized by degeneration sis of ALS should not only focus on potential disease triggers of motor neurons, leading to progressive paralysis and death but also on mechanisms for the propagation of pathological [1]. Due to a staggeringly complex etiology, the identification processes and signals between individual cells in a non-cell of a principal mechanism and development of an effective autonomous manner. therapeutic treatment for ALS still remains a research chal- Currently, it is postulated that an understanding of the lenge. In the light of current findings, it has become evident purinergic impact on neuroinflammation underpinning pa- that pathogenesis of ALS is not restricted to motor neurons but thology of neurological disorders is essential for the develop- attributed to the abnormal interactions of neurons and non- ment of efficacious interventions. Extracellular adenosine tri- phosphate (ATP) and adenosine (Ado) are recognized as the most powerful purinergic signaling molecules, directing inter- * M. Wujak cellular cross-talk and thereby equilibrating beneficial and mag_wuj@umk.pl detrimental activities occurring in the nervous system. In the present work, we will discuss the most relevant research ad- Neurology Clinic, Marek Cieślak, Toruń, Poland vances in deciphering a role of the purinome in mediating Department of Biochemistry, Faculty of Biology and Environmental pathological mechanisms underlying dysfunction of neuroglia Protection, Nicolaus Copernicus University in Toruń, 1 Lwowska St, and consequent motor neuron damage. By presenting the 87-100 Toruń, Poland 2 Purinergic Signalling (2019) 15:1–15 time-specific involvement of diverse purinergic receptors in cellular perturbations, including oxidative stress [10], neuroin- the disease progression, we will discuss their relevance for the flammation [11], glutamate excitotoxicity [12], mitochondrial development of new more powerful diagnostic and therapeu- dysfunction [13], RNA metabolism impairment [14, 15], pro- tic avenues for amyotrophic lateral sclerosis. tein misfolding and aggregation, and ER stress [16, 17], dysfunction of the ubiquitin–proteasome system [18], lack of trophic (growth) factors [19], aberrant axonal conduction [20], immune system deficiency [21], and blood-spinal cord barrier Basic notions of ALS impairment [22]. In 1990s, it has been discovered that some cases of fALS ALS is characterized by degeneration of motor neurons in the are associated with mutations in a gene localized on chromo- brainstem (upper motor neurons, UMN) and in the spinal cord some 21q21.1 encoding superoxide dismutase 1 (SOD1), also (lower motor neurons, LMN). This results in a progressive known as Cu/Zn superoxide dismutase [23]. Since then, over muscle denervation in upper and lower limbs, torso, and bulbar 160 mutations distributed throughout the 153-amino acid region leading ultimately to weakness and atrophy of skeletal SOD1 polypeptide have been identified in association with muscles [1]. ALS is classified as a rare adult-onset disease with ALS and till date, many cell and animal models expressing 58–60 years as average age of onset. The incidence rate of ALS exogenous mutant SOD1 have been developed in order to is around 1 to 2.6 cases per 100,000 persons annually, whereas investigate the etiology of ALS [24, 25]. Following the dis- the prevalence is approximately 6 cases per 100,000 per year covery of mutant SOD1, numerous mutations in other genes [6]. The vast majority of ALS patients die from respiratory have been identified to be associated with ALS, thereby open- failure within 3–5 years after onset of symptoms, while only ing up new avenues for the disease modeling. Figure 1 pre- 10% survive beyond 10 years [7]. Approximately 90% of ALS sents a brief overview of key cellular mechanisms and genetic cases occur randomly and are termed sporadic ALS (sALS), mutations contributing to the motor neuron damage in ALS. while the remaining 10% of cases are classified as familial ALS (fALS) having autosomal dominant pattern of inheritance, al- though some autosomal recessive pedigrees have been reported [8]. Due to a highly complex pathology and heterogeneous Purinergic players in the central nervous clinical presentations, ALS is considered as a multi-genetic, system multi-systemic, and multi-factorial disorder [9]. A composite etiology of ALS is caused by the influence of various genetic, Purinergic signaling utilizes nucleotides and nucleosides as biological, and environmental factors. The disease arises as a signaling molecules to activate two types of membrane- consequence of multiple pathophysiological mechanisms and bound receptors. The P1 receptors comprise four subtypes Fig. 1 Main key pathogenic mechanisms and cellular perturbations underlying the pathogenesis of ALS, including genetic background Purinergic Signalling (2019) 15:1–15 3 of G protein-coupled receptors (A1, A2A, A2B, A3) that dynamic changes of extracellular ATP [45]. After release, are activated exclusively by adenosine, whereas the P2 the pericellular concentration of ATP rises even up to 100– receptors are activated by nucleotides (ATP, ADP, UTP, 200 μM and is rapidly reduced to 1–100 nM by the activities UDP) and due to structural and pharmacological differ- of nucleotide-metabolizing enzymes, existing in a ences are classified into two subtypes: G protein-coupled membrane-bound or soluble form, with the consequent pro- P2Y receptors (1, 2, 4, 6, 11, 12, 13, 14) and ionotropic duction of ADP and/or AMP, including nucleotide P2X receptors (1–7) [26]. Neuroglia and neurons contain triphospho-diphosphohydrolases (NTPDases), nucleotide different combinations of purinergic receptors, which con- pyrophosphohydrolases/phosphodiesterases (NPP), and al- tributes to a versatile regulation of pathophysiological pro- kaline and acid phosphatases (Fig. 2). Finally, AMP hydro- cesses in the NCS (Table 1)[27–43]. lysis catalyzed by 5′-nucleotidase (5′-NT or CD73) results The signaling events are triggered upon the release of in production of adenosine which is further deaminated via purines and pyrimidines into the extracellular matrix by inosine into hypoxanthine by adenosine deaminase (ADA) exocytosis, facilitated diffusion, through channels, and fi- and purine nucleoside phosphorylase (PNP), respectively nally as a result of cell damage and lysis [26]. Extracellular [46, 47]. Taken together, the breakdown of ATP by ecto- ATP concentration drastically increases in response to di- nucleotidases not solely terminates its extracellular messen- verse biological, chemical, or mechanical stimuli, such as ger functions but also generates additional agonists, namely hypoxia, infection, physical trauma, neurodegeneration, ADP and adenosine. It is important to emphasize that the and neuroinflammation [44]. To measure ATP in the phosphorylation of adenosine to AMP by adenosine kinase pericellular space, several in vitro and in vivo methods are (ADK) enters the opposite metabolic pathway whereby available, including a novel pmeLUC system which is a adenosine can be converted back to its nucleotide deriva- simple and reliable in vivo tool for investigating the tives. The synthesis of ADP and ATP molecules is catalyzed Table 1 Distribution of P1 and P2 receptors in the nervous system Receptor subtype Distribution/cell type References P1 receptors A1 Widely distributed in brain (ISH); high expression levels in primary [27, 28] cultures of astrocytes from different brain regions, e.g., cerebellum, hippocampus, cortex, thalamus, spinal cord (RB, RT-PCR) A2A Brain neurons and astrocytes (RB), high expression levels in dopamine-rich [29, 30] regions (IHC), low expression levels in hippocampus (RT-PCR, IHC) A2B Widely distributed but at low expression levels; glial and neuronal cells (RT-PCR) [30, 31] A3 Neurons of cerebellum and hippocampus (RT-PCR, WB), astrocytes [32] (WB), generally low expression level P2X receptors P2X1 Cerebellum (IHC, RT-PCR), dorsal horn spinal neurons, astrocytes, microglia (IHC) [33, 34] P2X2 Widely distributed in neuronal structures, including the cortex, hippocampus, [35, 36] cerebellum, spinal cord (ISH), autonomic and sensory ganglia neurons (IHC) P2X3 Sensory neurons, nucleus tractus solarius neurons, some sympathetic [34, 35] neurons (ISH, IHC) P2X4 Widely distributed in CNS (ISH), neurons, astrocytes, activated microglia (IHC) [35, 36] P2X5 Neurons in spinal cord, astrocytes (ISH, IHC) [35] P2X6 widely distributed in CNS (ISH); motor neurons in spinal cord (IHC) [35, 36] P2X7 Ependymal cells lining the ventricles (RT-PCR), hippocampus (RT-PCR), [37] microglia, astrocytes, apoptotic cells (IHC, WB) P2Y receptors P2Y1 Widespread distribution in mammalian brain, including the cerebral cortex, [38] hippocampus and cerebellum (IHC), neurons and microglia (IHC, RT-PCR) P2Y2 Astrocytes (IHC, RT-PCR) [38] P2Y4 Brain neurons (IHC, RT-PCR) and microglia (RT-PCR) [38, 39] P2Y6 Activated microglia (ISH, IHC, RT-PCR) [39–41] P2Y11 brain neurons and oligodendrocytes of nucleus accumbens, parahippocampal [39] gyrus, putamen and striatum (RT-PCR) P2Y12 Hippocampal pyramidal neurons (RT-PCR), resting microglia (RT-PCR) [39] P2Y13 Brain (RT-PCR) neurons and oligodendrocytes (IHC), microglia (ISH, IHC) [40, 42] P2Y14 CNS astrocytes (RT-PCR), discrete brain regions (ISH) [40, 43] ISH in situ hybridization, RB radioligand binding, RT-PCR reverse transcriptase polymerase chain reaction, IHC immunohistochemistry, WB Western Blot 4 Purinergic Signalling (2019) 15:1–15 Fig. 2 Enzymes involved in the metabolism of extracellular nucleotides as CD73), adenosine deaminase (ADA), and purine nucleoside and nucleosides. AMP, ADP, and ATP nucleotides breakdown to phosphorylase (PNP). ATP re-synthesis via backward phosphotransfer adenosine (Ado) and other nucleosides such as inosine (Ino) and reactions: adenylate kinases (AK), nucleoside-diphosphate kinases hypoxanthine (Hyp): nucleotide triphospho-diphosphohydrolases (NDPK), and ATP synthase. P2X7, pannexin (Panx), connexin (Cx), (NTPDases), nucleotide pyrophosphohydrolases/phosphodiesterases and ATP-binding cassette (ABC) proteins represent channel and (NPP), alkaline phosphatase (AP), 5′-nucleotidase (5′-NT, also known transport-mediated ATP release pathways by nucleotide-phosphorylating enzymes such as adenylate disproportionate neuroinflammatory responses, particular- kinases (AK) and nucleoside-diphosphate kinases (NDPK) ly when chronic, promote apoptosis and necrosis and in- and ATP synthase [47, 48]. Consequently, the interplay be- fluence the synaptic and intrinsic membrane properties of tween phosphohydrolysis and phosphotransfer reactions neurons [52]. provides a precise and dynamic control of the duration, A dominant role for neuroinflammation has been re- magnitude, and direction of purinergic and pyrimidinergic ported in the etiology of primary and secondary neurode- signals. Most importantly, the existence of many P1/P2 re- generative diseases, such as Alzheimer’s disease (AD), ceptor subtypes differing in their sensitivity to agonists and Parkinson’s disease (PD), multiple sclerosis (SM), amyo- the possibility that the same ligand activates more than one trophic lateral sclerosis, Huntington’s disease (HD), receptor subtype increase the complexity of the purinome stroke, and epilepsy [53, 54]. and generate an extensive network of overlapping cellular The main cellular effectors of neuroinflammation are astro- responses [9, 49]. cytes and microglia, as well as T lymphocytes, perivascular monocytes, and macrophages invading to the sites of insult from circulation. Microglial cells and astrocytes undergo acti- Cellular players in neuroinflammation vation in the process of microgliosis and astrogliosis, respec- tively [55]. The processes of microglia and astrocyte activa- In the central nervous system, ATP acting as a neurotrans- tion, T lymphocyte infiltration, and overproduction of a vari- mitter and neuromodulator regulates a wide array of phys- ety of inflammatory cytokines have been demonstrated in as- iological processes such as neurotransmission, cell-to-cell sociation with neuronal loss even during the pre-symptomatic communication, neurite outgrowth, as well as cell prolif- phase of ALS [56]. eration, migration, differentiation, and apoptosis [26]. Microglia are the first line of immune defense in the ATP is released extracellularly under conditions of tissue CNS. The cells have been shown to be highly plastic and stress or injury by microglia, astrocytes, and damaged could acquire distinctive phenotypes in response to vari- neurons. The increase in extracellular ATP (eATP) level ous stimuli. Under physiological conditions, microglia re- is recognized by competent cells as damage-associated tain a relatively quiescent phenotype. However, these cells molecular pattern (DAMP) signal capable of initiating constantly monitor local microenvironment and communi- and propagating neuroinflammatory response [3, 50, 51]. cate with astrocytes and neurons by secreting anti- The principle functions of neuroinflammation are to limit inflammatory molecules and neurotrophic factors like insulin-like growth factor 1 (IGF-1), transforming growth tissue damage and initiate tissue repair. However, Purinergic Signalling (2019) 15:1–15 5 factor-β (TGF-β), brain-derived neurotrophic factor enhancing the expression of neurotrophic factors, such (BDNF), and nerve growth factor (NGF) [57]. Following as IGF-1 and glial glutamate transporter-1 (GLT-1) [65]. injury or pathogen invasion and exposure to pro- At pre-symptomatic stage, treatment of ALS mice with inflammatory cytokinessuchasinterferon-γ (IFN-γ), CD4 T lymphocytes significantly delays the onset of + + IL4 and tumor necrosis factor-α (TNF-α), microglial cells symptoms (actionascribedtoCD4 CD25 regulatory T- become polarized (activated) toward a pro-inflammatory cells) and increases the latency between disease onset and phenotype. Upon activation, microglia promptly release entry into late stage (action mediated by CD4 an entirely different set of molecules, such as pro- CD25 effector cells) [66, 67]. In the recent study, the inflammatory cytokines and chemokines, reactive oxygen expansion of endogenous regulatory T-cells in a mouse species (ROS) or nitric oxide (NO), which contribute to model of ALS significantly prolonged motor neuron sur- the onset of local inflammatory response [58, 59]. vival time, suppressed glial cell immunoreactivity, and The danger signals from damaged tissues include ATP enhanced neuroprotective gene expression. Regulatory T- released by dying and abnormally functioning neurons. cells were also shown to correlate with a slower rate of The nucleotide-metabolizing enzymes are represented on disease progression in ALS patients [68]. microglial cells mainly by ecto-nucleoside triphosphate Mast cells function as environmental Bsensors^ to com- diphosphohydrolase 1 (ecto-NTPDase1, CD39) and ecto- municate with other players under physiological condi- 5′-nucleotidase (CD73) [3]. Concerted action of these en- tions or during immune responses, based on their wide- zymes restores the balance between ATP and adenosine spread tissue presence near blood vessels and surfaces concentration that will be discussed in detail in the next exposed to the environment [69]. In the nervous system, chapters. mast cells represent an important peripheral counterpart in Astrocytes are the most abundant fraction of cells with intercellular cross-communication. Upon activation, mast a glial phenotype in the brain. Despite this population of cells secrete numerous vasoactive, neurosensitizing, and cells has long been neglected or misidentified, astrocytes pro-inflammatory mediators, including histamine, seroto- represent a key component in the brain environment. They nin, cytokines, proteolytic enzymes (e.g., chymase, provide neurotrophic factors, control synaptic functions tryptase, acid hydrolases), lipid metabolites (prostaglandin and formation, regulate the concentration of neurotrans- D2, leukotriene C4, platelet-activating factor), neuropep- mitters at synapses, and are involved in a wide range of tides, growth factors (NGF and VEGF), and nitric oxide homeostatic functions [60]. In the course of neurodegen- [70]. Mast cells are also an abundant source of ATP which erative diseases, including ALS, astrocytes become reac- is stored in their granules and secreted upon activation tive by altering their morphology and molecular expres- [69]. Serum and CSF samples of ALS patients display sion patterns [55, 61]. Activation of P2X7 receptor on elevated amounts of IL-12 and IL-15 [71], the latter cyto- spinal cord astrocytes was proved to trigger cytotoxic cas- kine acting as a mast cell chemoattractant, resulting in cade and exert neurotoxic effect on motor neurons [62]. mast cells accumulation around degenerating motor neu- Accompanying processes involve induction of nuclear rons. Mast cell involvement in the neuromuscular junction factor κ light-chain enhancer of activated B cells denervation was recently investigated in an animal ALS (NF-κB) and activator protein 1 (AP-1), and stimulation model, where the authors observed a marked infiltration of cyclooxygenase-2 (COX-2) activity [3]. Another im- and degranulation of mast cells that correlated with dis- portant astrocyte dysfunction in ALS is attributed to ease progression [72]. lowered release of neurotrophic factors, such as BDNF, The cross-talk between different cell types in the ner- glia-derived neurotrophic factor (GDNF), or vascular en- vous system has a recognized role in neural information dothelial growth factor (VEGF). Neurotrophic factors are processing. Glia-neuron, mast cell-glia, and glia-glia cells known to improve the neuron survival and repair; there- communicate bi-directionally through the release of extra- fore, their deficiency significantly attenuates cellular signaling molecules. Studies from the last decades neuroregeneration [63]. have provided strong evidence for the complexity of this Lymphocyte infiltration was described in ALS patients cross-talk [50, 69, 73]. The outcome of this intercellular in the corticospinal tracts and anterior horns of the spinal communication is dependent on the local pathophysiolog- cord [64]. T-helper CD4 cells permeate at the onset of ical status, e.g., stress or injury, and on environmental fac- the disease and then accumulate during disease progres- tors, e.g., cytokine concentration. Molecules contributing sion, whereas T-cytotoxic CD8 cells are only present at to the intercommunication also include extracellular nucle- the end stage of disease. In the ALS mice model, CD4 T otides, particularly ATP. All the cellular players can both lymphocytes slowed disease progression, extended dis- release and simultaneously respond to the nucleotide signal ease duration by 50%, modified the microglial pheno- via numerous purinergic receptors. Therefore, eATP gen- erates a feedback loop that drives the persistent pro- types, and prolonged survival. This probably occurs by 6 Purinergic Signalling (2019) 15:1–15 inflammatory and detrimental response [3, 74, 75]. During ATP and P2 receptors in ALS the progression of ALS, microglia, astrocytes, and motor neurons enter in this pro-inflammatory cross-talk, followed P2X7 by exacerbation of pathological processes. More insight into the communication between different cell types should Injury to motor neurons caused by pro-inflammatory factors provide important novel therapeutic approaches to promote released by activated microglia is considered as one of the repair and reduce neuroinflammation. most critical pathogenic mechanisms in ALS. Among purinergic receptors, the P2X7 receptor was shown to be in- volved in chronic pain, neurodegeneration, and neuroinflam- mation [90]. This purinergic receptor is predominantly The involvement of purinergic signaling expressed on microglia and oligodendroglia, and at lower lev- in the ALS pathology el on astrocytes [37]. P2X7 receptor activation mediates the release of IL-1 family cytokines including IL-1α,IL-1β,and Purinergic signaling regulates glial proliferation, motility, IL-18 [91]. Release of IL-1β results in upregulation of pro- survival, and myelination, as well as facilitates interac- teins contributing to inflammatory processes, including COX- tions between neurons and vascular and immune system 2, nitric oxide synthase (NOS), TNF-α, pro-caspase 1 as well cells [3, 73]. Numerous studies indicate that a vast inter- as matrix metalloproteinase-9 (MMP-9), and cannabinoid re- play occurs also at the cell membrane among purinergic ceptor 2 (CB2). Interestingly, a significantly increased immu- receptors, ecto-nucleotidases, and transporters, resulting noreactivity of P2X7, together with COX-2 and CB2, has in the insurgence or maintenance of neuroinflammatory been shown in active microglia from post-mortem spinal cord conditions [73, 76]. In the following subsections, we will samples of sALS patients and in the mSOD1G93A transgenic present in detail the contribution of individual purinergic rodent model of fALS [81, 85, 92]. The P2X7 receptor- signaling components in the pathogenesis of ALS and mediated neurotoxicity was also confirmed in mSOD1 astro- potential therapeutic directions (Table 2). cytes [62]. P2X7 receptor alone can activate cell death via Table 2 Fundamental discoveries of purinergic contribution to ALS pathogenesis Purinergic component Contribution to ALS References Adenosine Ado Significantly increased in the cerebrospinal fluid of ALS patients [77] and A2A receptors A2A Receptors activation makes motor neurons susceptible to [78] excitotoxic challenge A2A Upregulated in lymphocytes from ALS patients [79, 80] A2A Upregulated in motor neurons from spinal cords of SOD1G93A [80] mice and ALS patients ATP and P2 receptors P2X7 Microglial expression increased in post-mortem spinal cord samples [81] from ALS patients P2X4, P2X7, P2Y6 Upregulated in SOD1-G93A mice microglia [75] P2X7 Activation of microglial receptor induces cell death of ALS motor neurons [75] P2X7 Activation of astrocytes receptor initiates motor neurons death in vitro [62] ATP, BzATP Small doses induce motor neuron death in vitro [82] P2X7 Activation of receptor by BzATP up-regulates the miRNAs transcriptome [15] in SOD1-G93A microglia P2X7 Ablation of receptor in SOD1-G93A mice aggravates gliosis and motor [83] neuron death P2X7 Receptor antagonist Brilliant Blue G ameliorates spinal cord pathology in [84] SOD1-G93A mice P2X4 Upregulated in degenerating motor neurons in ALS mouse and rat spinal cord [12, 85] P2Y12 Expression progressively reduced in SOD1-G93A mice microglia [86, 87] P2X4, P2X7 Upregulated in sciatic nerves of SOD1-G93A mice [9] Enzymes CD39 Gene expression downregulated in spinal cord microglia from SOD1G93A [88] mice and ALS patients CD39 Protein expression downregulated in SOD1-G93A mice microglia [9] ADK Increased activity in reactive astrocytes decreases adenosine concentration [89] and triggers neurodegeneration Purinergic Signalling (2019) 15:1–15 7 both apoptotic and necrotic mechanisms leading ultimately to delayed ALS onset, although with no effect on life span. the development of neurotoxic phenotype. For instance, a Importantly, BBG neuroprotection occurred within a specific peroxynitrite-fueled apoptotic cascade activated by P2X7 re- time frame, since BBG administration at earlier phases did not sults in motor neuron death due to trophic factor deprivation, prevent disease progression [84]. In particular, BBG treatment activation of p57 neutrophin death receptor (p75NTR), and enhanced motor neuron survival and significantly reduced expression of mutant forms of SOD1 [62, 82]. microgliosis by downregulating the expression of pro- Emerging evidence indicates that P2X7 may have a dual inflammatory proteins, NF-κB, and microglia markers of ac- function in onset and progression of ALS, either trophic and tivated phenotype (NOX2 and IL-1β), and simultaneously anti-inflammatory or toxic and pro-inflammatory. The fact that upregulating other markers (IL-10 and BDNF). The key con- ablation of P2X7 in SOD1-G93A mice exacerbates gliosis and clusion drawn from these results might be Bthe double face^ of motor neuron death at end stage of the disease supports a neuro- P2X7 receptor and the existence of a narrow therapeutic win- protective role of this receptor [83]. On the other hand, the acti- dow concerning its beneficial role in ALS. The dual action of vation of microglial P2X4, P2X6, and P2X7 receptors by 2′-3′- P2X7 during ALS progression seems to correspond to the O-(benzoyl-benzoyl) ATP (BzATP), a non-hydrolysable form of switch of microglia from protective to lethal phenotype. ATP, leads to increased content of pro-inflammatory molecules Most recently, the role of P2X7 receptor in microglia po- such as TNF-α and COX-2 with the consequent motor neuron larization has been confirmed in LPS-induced cellular model injury [75]. Moreover, the activation of P2X7 receptor by BzATP of inflammation. It was shown that P2X7 inhibition by the enhanced ROS production in SOD1-G93A mouse microglia selective antagonist A438079 suppressed microglia activation through the activation of NADPH oxidase 2 (NOX2) which is [101]. Based on these findings, P2X7 has emerged as a poten- the main ROS-producing enzyme in microglial cells and well- tial marker of activated microglia. It is postulated that the recognized player in the pathogenesis of ALS. Importantly, this microglial polarization switch is closely linked with the time microglia-mediated mechanism for motor neuron damage was when P2X7 starts to play a critical role in regulating neuroin- shown to be prevented by ablation of P2X7 and the use of P2X7 flammation in ALS [9]. This hypothesis is strongly supported specific antagonists such as A839977, A438079, and Brilliant by the fact that ALS pathogenesis consists of two distinct Blue G (BBG) [93]. Other pathological mechanism by which neuroinflammatory stages. The first stage is neuroprotective P2X7 can exacerbate a neurotoxic phenotype of ALS microglia due to the concerted action of regulatory T-cells, anti- is miRNA dysregulation. Parisi and colleagues have shown that inflammatory macrophages/microglia, and T helper cells, stimulation of P2X7 receptor by BzATP hyperactivated inflam- which sustain motor neuron viability by providing anti- matory miRNAs, such as miR-155, miR-125b, and miR-146b inflammatory agents. As the disease and motor neuron injury which are known to be upregulated in SOD1-G93A microglia. It accelerate, the second rapidly progressing stage emerges when resulted in downregulation of IL-6/STAT3 signaling and en- activation of pro-inflammatory T-cells and macrophages/ hancement of TNF-α production, switching eventually microglia microglia leads eventually to neurotoxicity [9, 59, 102]. toward a detrimental phenotype [15]. It is assumed that the use of neuroprotective agents such as P2X4 COX-2 inhibitors, P2X7 receptor antagonists, and CB2 ago- nists might exert beneficial effects in ALS pathology by A strong P2X4 immunoreactivity was shown to be associated slowing down the progressive damage to motor neurons [81, with degenerating motoneurons in spinal cord ventral horn 92]. The P2X7 antagonist BBG is considered as a promising samples from mSOD1G93A rats. In parallel, P2X4 immuno- candidate for blocking the detrimental effects of P2X7 activa- staining detected degeneration in other neuronal populations, tion due to its high selectivity, low toxicity, and ability to including noradrenergic neurons in the locus coeruleus, efficiently cross the blood–brain barrier. Other P2 antagonists Purkinje cells in the cerebellum and serotonin containing neu- such as oxATP (oxidized ATP) and PPADS (pyridoxal-phos- rons in the raphe nucleus [85]. More interestingly, the P2X4 phate-6-azophenyl-2′,4′-disulfonic acid) fail to meet all of the antibodies were able to recognize neurotoxic species of above requirements [94, 95]. Brilliant Blue G has previously misfolded SOD1G93A in motor neurons but not in glial cells. been proven to reduce neuroinflammation in traumatic brain It was suggested that neuronal P2X4-immunoreactive and spinal cord injury [95–97], cerebral ischemia reperfusion SOD1G93A conformers may have a pathogenic role in the [98], neuropathic pain [99], and in experimental autoimmune promotion of neuroinflammation since they activated microg- encephalitis [100]. The neuroprotective effects of BBG have lia and astroglia when injected intracerebrally into normal been tested in the SOD1-G93A ALS mouse model at different animals [103]. phases of ALS to elucidate the role of P2X7 receptor in Upregulation of P2X4, both at mRNA and protein level, microglial polarization and neuroinflammation. was also found in ALS microglia from SOD-G93A mice [75]. Administration of BBG, beginning at a late pre-symptomatic Most recently, Volonté and colleagues have demonstrated for phase, improved motor neuron performance and slightly the first time the increased expression of P2X4 and P2X7 8 Purinergic Signalling (2019) 15:1–15 proteins in the peripheral nervous system of SOD1-G93A authors showed that short treatment (from asymptomatic to mice, namely in sciatic nerves, and nominated these P2 recep- symptomatic phase) with antihistamine drug clemastine reduced tors as potential diagnostic biomarkers for ALS at the periph- disease progression and improved survival of SOD1G93A ALS eral level [9]. Finally, P2X4 receptor activation was shown to mice by enhancing anti-inflammatory phenotype of microglia protect motor neurons. α-Amino-3-hydroxy-5-methyl-4- via upregulation of P2Y12 together with P2Y7, arginase-1 and isoxazole propionic acid (AMPA) receptor-mediated CD1639. Because long treatment with clemastine (from asymp- excitotoxicity is considered as an important mechanism for tomatic until the end stage) failed to ameliorate ALS progres- motor neuron death in ALS. Andries and colleagues showed sion, the beneficial effects of this drug are thought to be tightly that preincubation of motor neurons with P2X4 allosteric dependent on the first phase of neuroinflammation in ALS, modulators such as ivermectin (anti-parasite medication) and characterized by neuroprotective functions of microglia [84]. Cibacron Blue 3G-A (CB) protected the cells against kainate- induced excitotoxicity in vitro. In addition, ivermectin poten- Adenosine and P1 receptors in ALS tiated the protective effect of low ATP concentrations to motor neurons, presumably by increasing the number of P2X4 mol- In 1999, Yoshida and colleagues reported a significantly in- ecules on the cell surface, and most importantly, extended creased adenosine concentration in the cerebrospinal fluid of survival of SOD1-G93A mice by almost 10% [12]. progressing ALS patients; however, neither diagnostic nor prognostic potential of these findings had been evaluated P2Y6 [77]. Adenosine, formed as the product of eATP degradation, possesses neuroregenerative potential. In ALS patients, over- P2Y6 receptor is activated mainly by UDP, partially sensitive expression of adenosine kinase (ADK) is a common patho- to UTP and ADP, and completely insensitive to ATP. P2Y6 logic hallmark, and the consequent increase in astrocyte ADK was shown to be upregulated in SOD1G93A mice microglia; activity disrupts adenosine homeostasis triggering ultimately however, the role of this receptor in ALS remains elusive [75]. neurodegeneration [89]. On the other hand, adenosine is ca- Likewise P2Y2 and P2Y4 receptors, P2Y6 regulates pable to stimulate astrogliosis by activating P1 receptors on microglial phagocytosis upon activation by UDP [104]. It astrocytes. A2A and A3 receptors are involved in the was suggested that this process may play important role in astrogliosis initiation, whereas the activation of A1 receptor facilitating the uptake of cellular debris generated after neuro- inhibits the proliferation of astrocytes [3, 108]. Moreover, nal damage. In fact, damage of hippocampal neurons by in vitro studies revealed that adenosine at physiological con- kainate in vivo and in vitro resulted in upregulation of centration of about 10 nM activates A2A receptor in astro- microglial P2Y6 expression and consequent activation of cytes, followed by affecting GLT-1 and inhibiting glutamate microglial phagocytic activity via UDP/P2Y6 signaling [105]. uptake [3]. And ALS patients and SOD1-G93A mice suffer from suppressed glutamate uptake that drives excitotoxic pro- P2Y12 cesses and motoneuron degeneration [109]. The first evidence for the involvement of A2A in ALS was The involvement of P2Y12 in microglia process dynamics is provided by A2A blockage with the selective antagonist well established, including its key role in the regulation of mi- KW6002, which protected motor neurons from toxic insult croglia activation, chemotaxis, and migration [86, 106]. This triggered by the expression of mutant versions of SOD1 and glued purinergic receptor can serve as a marker of a resting/ dynactin subunit p150 . The neuroprotective effect of A2A surveillant branched state of ALS microglia as well as a marker antagonism was attributed to attenuated tyrosine receptor ki- distinguishing CNS-resident microglia from blood-derived mac- nase B (TrkB) signaling, known to induce vulnerability of rophages infiltrating CNS upon neuronal injury [87, 107]. motor neurons to excitotoxic challenge upon activation by Moreover, a dramatic and continuous reduction of P2Y12 re- BDNF. Moreover, a physical interaction between A2A and ceptor expression was observed after microglia activation fol- TrkB within lipid rafts of motoneurons was shown to be pre- lowing brain injury [106]. Consistently, P2Y12 expression was requisite for TrkB transactivation [110]. On the contrary, acti- found to be progressively reduced in spinal cord microglia of vation of A2A in the absence of BDNF signaling rendered SOD1-G93A mice and ALS patients during neuroinflammation these cells susceptible to excitotoxicity [78]. However, the [87, 107]. Interestingly, P2Y12 expression was significantly de- above findings are not consistent with results gained from creased at symptomatic stage, when the disease accelerates, and studies on motor neuron cultures exposed to BzATP and in completely lost at end stage of the disease when motor neuron SOD1-G93A mouse model for ALS. Namely, it was shown loss, oligodendrocyte degeneration, and microglia activation are that high doses of adenosine protect motor neurons from known to be augmented [87]. These findings indicate a neuro- death induced by BzATP, whereas low doses exert no benefi- protective action of P2Y12 at early stage of ALS that is consis- cial effect. Because adenosine production results from rapid tent with results obtained by Apolloni and colleagues. The ATP breakdown, it was concluded that ATP at high Purinergic Signalling (2019) 15:1–15 9 concentrations (1 mM), paradoxically, may be protective to NTPDases motor neurons, while at low concentrations trigger neuronal apoptosis upon P2X7 activation [82]. A protective role of CD39 (NTPDase1) is exclusively expressed on the surface of A2A has been also demonstrated in SOD1G93A mice model, microglia where it plays a predominant role in the inactivation as treatment with the selective agonist CGS21680 resulted in of P2 receptor-mediated signaling by catalyzing a rapid ATP delayed disease onset [111], whereas intake of caffeine, a non- degradationtoADP andAMP [114]. Consequently, any selective P1 receptor antagonist, significantly decreased sur- changes in CD39 expression may result in disturbed nucleo- vival time of ALS animals [112]. tide homeostasis and subsequent alterations in purinergic A possible involvement of the P1 receptors in ALS has also transmission. In fact, CD39 downregulation at mRNA level been investigated at peripheral level. Expression profiling of has been demonstrated in microglia from the spinal cord of all P1 receptor subtypes revealed a significant upregulation of SOD1-G93A mice and ALS patients [88], which was further A2A in lymphocytes from ALS patients, compared to healthy confirmed at protein level in cortical primary microglia from subjects, and a positive correlation between A2A density SOD1-G93A newborn mice [9]. CD39 downregulation values and scores of the revised ALS Functional Rating caused prolonged activation of P2 receptors as a consequence Scale, which is a useful predictor for ALS progression. of a significantly reduced hydrolysis of extracellular ATP. As Moreover, activation of A2A by the selective CGS21680 ag- three ATP-sensitive P2 receptors, namely P2X4, P2X7, and onist led to increased production of cyclic AMP in lympho- P2Y6, are found to be upregulated in ALS, the above findings cytes from ALS patients as compared with a control group. emphasize a critical role of CD39 in the regulation of The negative correlation between A2A density and the sever- neuroinflammatory events mediated by ALS microglia [75]. ity of disease symptoms, which highlights a possible role for In general, dysregulation of any purinergic element along with these receptors in immunosuppressive responses in ALS, altered concentrations of signaling agents in the extracellular might also represent a promising perspective for alternative milieu leads to the enhancement of inflammatory responses. therapeutic approaches for ALS based on modulation of CD39L1 (NTPDase2) was found to be highly expressed in A2A receptor activity [79]. In view of anti-inflammatory ef- hippocampal, cortical, and cerebellar astrocytes where it plays fects of A2A activation to peripheral immune cells, these find- a predominant role in regulation of the ATP/adenosine bal- ings support a neuroprotective role of A2A in ALS, at least at ance. Consequently, impairment of its activity can increase peripheral level. eATP concentration resulting in activation of P2X receptor The most recent discovery has provided evidence for a and initiation of inflammatory astrogliosis, leading ultimately more complex role of A2A-mediated adenosine signaling in to neuronal cell death. For this reason, NTPDase2 is consid- ALS. Firstly, it was shown that A2Awas upregulated in motor ered as another potent therapeutic target in human CNS dis- neurons from spinal cords of symptomatic SOD1G93A mice orders, but its precise role in ALS needs to be elucidated [115]. and end-stage ALS patients [80] that was in agreement with To summarize the current findings discussed in the previous studies reporting the increased expression of A2A in above chapter, in Fig. 3, we depict a network of motor neurons of end-stage SOD1G93A mice [112]. purinergic components and mechanisms recognized so Furthermore, a direct treatment of adenosine at concentration far to contribute to aberrant neuron-glia communication of 0.3 or 1.0 μM induced death of embryonic stem cell- and enhanced neuroinflammation underpinning ALS derived motor neurons (ESMN) cultured in vitro suggesting pathogenesis. that A2A blockage might be neuroprotective to motoneurons. The subsequent A2A inhibition by the selective KW6002 an- tagonist and partial genetic ablation of A2A efficiently Current and potential treatment for ALS protected ESMN from SOD1G93A+ astrocyte-induced cell death and slowed disease progression of SOD1G93A mice Despite intensive research, little in the field of new effective [80]. In light of these studies, a novel toxic effect of adenosine therapeutics for ALS has been developed so far. The most on spinal cord motor neurons has been discovered; however, frequently provided reason for explaining the difficulties in the exact mechanism of this adenosine-induced A2A activa- finding a remedy for curing or slowing the progression of tion is elusive and merits further investigation. ALS is that the disease is multi-genic, multi-factorial, and Contrary to A2A, the role of other P1 receptors in ALS still multi-systemic. Till 2016, more than 50 major phase II or III remains elusive, but evidence for a loss of A1-A2A functional clinical trials in ALS patients have been reported to fail, de- cross-talk at the neuromuscular junction in pre-symptomatic spite positive results from animal models [116]. In this con- SOD1G93A mice has been reported recently. A1-mediated text, the question arises whether the most widely used SOD1 adenosine signaling may contribute to exacerbation of the transgenic mice models properly reflect different aspects of disease during the symptomatic phase when A1 tonic activa- ALS heterogeneity in patients with both sALS and non- tion was shown to be enhanced [113]. SOD1 linked fALS. It is believed that the latest discoveries 10 Purinergic Signalling (2019) 15:1–15 Fig. 3 Purinergic dysregulation in ALS in genetics of ALS, including mutations in C9ORF72 and symptoms, arimoclomol slowed disease progression, in- TBK1 genes, will open new perspectives in the generation of creased survival, and improved muscle function. The drug is relevant animal models for ALS, with consequent potent con- currently tested in phase III clinical trial [121, 122]. tribution to the development of new biomarkers and therapeu- tic targets for the disease [117]. For the past 22 years, the only available FDA-approved Purinergic signaling as potential therapeutic intervention for ALS has been riluzole (brand names Rilutek target in ALS or Teglutik). However, this anti-excitotoxic drug extends pa- tient’s life span by only several months without improving A growing body of research provides evidence for an essential muscle strength and neurological function and is ineffective contribution of purinergic signaling to the development of many in later stages of the disease [118]. On May 5, 2017, the sec- neuroinflammatory and neurodegenerative diseases. Most im- ond drug, known as either edaravone (Radicava) or MCI-186, portantly, some results of these studies have been already imple- was finally approved by the FDA for ALS treatment [119]. mented in the treatment of Parkinson’s disease (A2A receptor Edaravone administrated intravenously once a day for 14 days, antagonist istradefylline) [123], cerebral ischemic stroke followed by a 2-week break, decreases disease progression in (P2Y12 receptor antagonists clopidogrel and ticlopidine, and early-stage ALS patients with no respiratory involvement and adenosine transporter inhibitor dipyridamole) [124, 125]. It is indications for gastrostomy [119]. This anti-oxidant and free- also believed that they might contribute to establishing new radical scavenger delays motor neuron degeneration but pro- treatment strategies for multiple sclerosis, epilepsy, migraine, vides limited survival benefit [120, 121]. Among other com- and neuropathic pain [53, 126–128]. Since the discoveries of pounds currently being tested in clinical trials, arimoclomol is microglia activation as an important mechanism of motor neu- considered as a promising therapeutic candidate. This so- ron death in ALS and extracellular ATP as a crucial neuron-to- called smart drug induces the expression of heat shock pro- glia alarm signal, the involvement of purinergic signaling in teins (HSP) exclusively under cellular stress conditions. It neuroinflammation has become evident and largely accepted. results in increased capacity of protein quality control and During the progression of ALS, microglia, astrocytes, degradation of misfolded proteins, including mutant SOD1. and motor neurons continue the pro-inflammatory cross- Administered to SOD1-G93A mice after the onset of talk, inter alia through P2X7 activation that was shown to Purinergic Signalling (2019) 15:1–15 11 be prevented by P2X7 antagonists [62, 75]. The P2X4 Compliance with ethical standards receptors, however, exert protective effects in motor neu- Conflicts of interest M. Cieślak declares that he/she has no conflict of rons. Considering these results, it can be concluded that interest. low ATP concentrations protect cells against excitotoxic K. Roszek declares that he/she has no conflict of interest. stimuli through P2X4 receptors, whereas high concentra- M. Wujak declares that he/she has no conflict of interest. tions of ATP produce toxic P2X7 activation. Finally, adenosine is also involved in ALS progression since aden- Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. osine A2A receptor antagonists prevent motor neuron death at the symptomatic phase [73, 110]. In light of cur- Open Access This article is distributed under the terms of the Creative rent findings, P2X7 and A2A are recognized as dual- Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, function purinergic receptors, which course of action distribution, and reproduction in any medium, provided you give appro- closely depends on ALS state, particularly priate credit to the original author(s) and the source, provide a link to the neuroinflammatory landscape of the disease. However, Creative Commons license, and indicate if changes were made. despite many lines of evidence for essential contribution of purinergic signaling to motor neuron damage, it is im- portant to emphasize here that there are currently no clin- ical trials targeting the purinergic players in the therapy of References ALS. In terms of the high complexity of the purinome comprising diverse nucleotide- and nucleoside- 1. 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