The Role of Adenosine Tone and Adenosine Receptors in Huntington's Disease

Abstract

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a mutation in the IT15 gene that encodes for the huntingtin protein. Mutated hungtingtin, although widely expressed in the brain, predominantly affects striato-pallidal neurons, particularly enriched with adenosine A_2A receptors (A_2AR), suggesting a possible involvement of adenosine and A_2AR is the pathogenesis of HD. In fact, polymorphic variation in the ADORA2A gene influences the age at onset in HD, and A_2AR dynamics is altered by mutated huntingtin. Basal levels of adenosine and adenosine receptors are involved in many processes critical for neuronal function and homeostasis, including modulation of synaptic activity and excitotoxicity, the control of neurotrophin levels and functions, and the regulation of protein degradation mechanisms. In the present review, we critically analyze the current literature involving the effect of altered adenosine tone and adenosine receptors in HD and discuss why therapeutics that modulate the adenosine system may represent a novel approach for the treatment of HD.

Pathogenic Mechanisms of Huntington's Disease

Overview

Huntington's disease (HD) is an inherited neurodegenerative disorder that is caused by a single, autosomal dominant gene mutation. HD generally affects young adults and is characterized by involuntary, abnormal movements and postures (chorea, dyskinesia, dystonia), psychiatric disturbances, and cognitive alterations.^1,2 It is estimated that 1 in 10,000 people have HD and this disorder is fatal within 15–20 years after the onset of symptoms. Multiple brain regions, including several cortical and subcortical regions (cerebral cortex, layers III, V, and VI; pallidum, sub-thalamic nucleus, cerebellum), show signs of neurodegeneration, but the most pronounced neuronal loss is present in the caudate nucleus and the putamen of the striatum.³ Within the striatum, the striato-pallidal, medium spiny neurons (MSN) that express enkephalin, dopamine D₂ receptors (D₂R) and adenosine A_2A receptors (A_2AR),^4,5 appear to be the most vulnerable.^6,7

HD is caused by a mutation in the IT15 gene that encodes for the protein huntingtin (Htt).⁸ This mutation consists of a CAG trinucleotide repeat that is translated into an abnormal polyglutamine (polyQ) tract in the N-terminal region of the protein. The disease is present in all cases where the CAG repeat length is greater than 40, and longer CAG repeats are associated with an earlier onset of disease symptoms.⁹ Although onset of the disease is primarily correlated to the number of CAG repetitions, this only accounts for about 60% of the variation in the age at onset (AAO).⁹ This supports the idea that other genetic and environmental factors may be influential components that can modify the expression of the disease and potentially be used as therapeutic targets.

Htt is a large protein of about 350 kD that is expressed in various tissues in the body and is involved in many important cellular functions.^1,10–13 Htt is widely distributed in the central nervous system, particularly in cortical pyramidal neurons, striatal interneurons, Purkinje cells,^14,15 and glial cells.¹⁶ To date, the exact cellular function of Htt is still not completely understood.^1,17 Htt is involved in many physiological functions including early embryonic development,¹⁸ fate of cortical progenitors,^19,20 axonal transport^21,22 and brain-derived neurotrophic factor (BDNF) expression/transport.²³ When the Htt protein becomes mutated (mHtt), not only is normal Htt function impaired, but several mechanisms important for neuronal activity and survival become impaired, leading to a gain of function that can be toxic to the cells.¹³

It is not clear whether HD is a prion-like disorder comparable to Alzheimer's or Parkinson's diseases, but experimental evidence suggests that mHtt triggers mis-conformation of wild-type (WT) Htt and neuropathological observations in patients who received intracerebral allografts support the transfer of HD pathology from cell to cell.²⁴ This supports the idea that transcellular propagation of protein aggregation could underlie the pathological progression in HD.²⁴ In the next sections, we will discuss excitotoxicity, BDNF function, and glial cells: specifically, how mHtt impairs these pathways, and how these systems are likely to be modulated by adenosine receptors.

Excitotoxicity and mitochondrial dysfunctions

Excitotoxicity is the pathological process where nerve cells are damaged or killed due to excessive stimulation of receptors by neurotransmitters such as glutamate. There is evidence indicating that dysfunctions of the glutamatergic system in the striatum account for the toxic effect of mHtt in HD, although it is unclear whether this is mainly due to a presynaptic mechanism, with the over-release of glutamate by cortical afferents.²⁵ Neuronal impairments that lead to excitotoxicity include poor reuptake of glutamate by glial cells, the hypersensitivity of N-methyl-D-aspartate (NMDA) receptors, and the dysregulation of mitochondrial function.^26–28 mHtt has been shown to impair the expression of astrocytic glutamate transporters, thus leading to an increase in glutamate in the synaptic clef.^16,29–31

Several studies have shown that there is abnormal NMDA receptor arrangement and activity in HD. In striatal MSN, mHtt increases the activity of the NR2B subunit of the NMDA receptor, reducing its ability to interact with the docking protein PSD95.^32–34 Also, R6/2 transgenic HD mice show an enhanced response to NMDA,³⁵ and a reduced NR2A/NR2B ratio, a measurement of excitotoxic cell death vulnerability.^36,37 These abnormal interactions lead to increased NMDA firing, altered NMDA receptor trafficking,²⁸ and neuronal vulnerability to NMDA.^34,38 These data suggest that the increased expression of extra-synaptic, NR2B-contaning NMDA receptors may contribute to NMDA's deleterious effects on the neuron.³⁹

In HD, the increase in NMDA response is likely affected by environmental modulation of the NMDA receptor other than glutamate. Quinolinic acid (QA), is a downstream product of the kynurenin pathway that degrades tryptophan and acts as an NMDA agonist. QA stimulates glutamate release from cortico-striatal endings,^40,41 and is able to produce lesions that mimic those seen in HD patients.⁴² In the striatum, endogenous increases in QA have been demonstrated in the early stage of HD in both humans and animal models.^43,44

Mitochondrial dysregulation may lead to excitotoxicity. Several imaging studies have revealed an early metabolic dysfunction in the striatum of HD patients,^26,45,46 and the severity of these metabolic alterations correlate with the size of the CAG expansion.⁴⁷ Several postmortem studies point to reduced activity of complex II-III, including succinate dehydrogenase in the caudate nucleus of HD patients.^48–50 This loss of complex II activity in the striatum is due to the reduced expression of complex II subunits,⁵¹ and the presence of dopamine increases the vulnerability of these striatal neurons to mHtt toxicity.⁵² Complex II inhibition shows a specific degeneration profile in animals treated with the irreversible complex II inhibitor 3-nitropropionic acid (3-NP),²⁶ suggesting that complex II inhibition may play a significant role in the striatal degeneration that is seen in HD.

Several other works have reported mitochondrial alterations resulting from mHtt. mHtt found localized in the neuronal mitochondrial membrane⁵³ has been shown to impair mitochondrial biogenesis, fission,^54,55 axonal transport,⁵⁶ membrane potential,⁵³ ATP production,^57,58 and calcium handling.^53,59 Interestingly, a dysregulation of mitophagy in the mitochondria is also present in HD, and the prevention of mitochondrial fission and cristae remodeling has been shown to delay HD progression.⁶⁰

Since mitochondrial defects represent one condition that can contribute to neuronal excitotoxicity in HD,^27,26 the rescue of impaired mitochondria⁶¹ and correcting poor energy homeostasis might represent a valuable therapeutic approach to HD.^60,62,63

Brain-derived neurotrophic factor

BDNF is a neurotropic growth hormone, present in the mammalian brain, involved in a variety of brain processes including synaptic activity. It is also vital for neuronal development, differentiation, and plasticity.⁶⁴ BDNF is secreted and transported through neurons in the cerebral cortex and is released at nerve endings in the striatum.⁶⁵

In HD, mHtt alters BDNF transcription,⁶⁶ trafficking and axonal transport,²² and it downregulates a set of genes, including the one coding BDNF, resulting in a negative effect on the silencing activity of the RE1/NRSE protein seen in WT Htt.⁶⁷ mHtt also alters the axonal transport of BDNF vesicles,⁶⁸ and post-Golgi trafficking.⁶⁹ More recently, it has been shown that abnormal interactions between pro-BDNF and Htt-associated protein 1 can lead to the alterations in BDNF transport that is seen in HD.⁷⁰

BDNF impairment is critically involved in the early vulnerability of striato-pallidal neurons in HD, and increasing BDNF, by gene overexpression, pharmacological or environmental modulation, has been shown to be beneficial in several experimental models of HD.^71–77

Two major protein degradation systems: Proteasome and autophagy

In HD, the expansion of polyglutamine (polyQ) in the N-terminal region of Htt causes protein misfolding and aggregation.^78–80 The ubiquitin-proteasome system (UPS) degrades damaged or misfolded proteins by E3 ligase targeted polyubiquitination,^81,82 and global changes in the ubiquitin system, an indicator of the UPS function, has been found in both HD patients and in animal models of HD.^83–85 mHtt causes the suppression of UPS function in the cells and brains of HD patients and in animal models,^86–88 while the enhancement of UPS activity enables soluble mHtt to degrade and improves proteasome function and motor coordination in HD.^63,89–94

Macroautophagy, or autophagy, degrades aggregated proteins in the body by delivering them to the lysosome, and is essential for cellular function.⁹⁵ Htt has been found to function as an important regulator and substrate for selective autophagy.^96,97 In HD, it has been shown that autophagic vacuoles are unable to recognize cytosolic cargo, and this causes slower turnover and accumulation of mHtt.^98,99 Research supports the hypothesis that the clearance of mHtt is important for the treatment of HD, and that the upregulation of the autophagic process can produce beneficial effects.^91,100–103

Non-neuronal (glial) and peripheral cells

Even though neuronal cells are preferentially damaged in HD, mHtt is also expressed in glial cells.^16,104–106 The presence of mHtt in astrocytes and other glial cells is associated with age-dependent neurological symptoms, contributes to neuronal excitotoxicity, and can lead to HD pathogenesis.^{16,30,107–111} Specifically, mHtt impairs glycolysis,¹¹² increases glutamate synthesis,¹¹³ causes a reduction in GABA release,¹¹⁴ reduces the production and release of trophic factors,^107,115 decreases the expression of potassium channel that leads to neuronal excitotoxicity,¹¹⁶ and causes a dysfunction in calcium and glutamate signaling.¹¹⁷

In HD, the abnormal function of microglia can cause an overactivation of the inflammatory response that is like the response seen in other neurodegenerative diseases.^109,110 A recent study indicates that mHtt in glia can create a disease phenotype in normal mice, while normal glia can abolish the disease phenotype in transgenic HD mice. This study strongly suggests a causal role for glia in HD.¹¹⁸

mHtt is also expressed in peripheral cells and can alter their normal physiology. mHtt expressed in hepatocytes can suppress the activity of the urea cycle, and can cause high blood ammonia.^119,120 mHtt expressed in the immune system leads to enhanced immune activation that can be detected in the early disease stages in both humans and mice. Thus, elevated inflammatory cytokines and chemokines levels are present in HD patients, and it has been proposed that high levels of mHtt in monocytes and T cells are significantly associated with the disease progression.¹²¹ These data suggest that the expression level of mHtt in immune cells of patients might be a useful, noninvasive disease biomarker for identifying HD.¹²²

Dysfunction of Striatal Adenosine Receptors in HD

Striatal adenosine neurotransmission

Adenosine plays a fundamental role in modulating dopaminergic and glutamatergic neurotransmission in the striatum.¹²³ The main neuronal inputs into the striatum are dopaminergic and glutamatergic in nature, and both converge in the dendritic spines of the MSN, the predominant neuronal population in the striatum.¹²⁴ Glutamate terminals make a tight, tripartite synapse with the head of the dendritic spines and with astrocytes,¹²⁵ while dopamine terminals make lose contact with the neck of the dendritic spines, allowing for dopamine to interact with dopamine receptors located around the synapses by volume transmission.^123,126

“Local module” can be defined as the minimal portion of neurons and glial cells that combine and operate as an independent and functional, integrative unit.¹²³ The “striatal spine module” is the local module centered in the dendritic spine of the MSN, and includes the glutamatergic and dopaminergic terminals and astrocytic processes.¹²³ Under normal conditions, ATP released by astrocytic vesicles is rapidly converted to adenosine by ectonucleotidases and this occurs within the striatal spine module.^127,128 The effects of extracellular adenosine are primarily mediated by adenosine A₁ receptors (A₁R) and A_2AR that are localized in the different locations of the striatal spine module. Both are G protein-coupled receptors (GPCRs), with A₁R coupling to the inhibitory Gi/o protein and A_2AR coupling to the excitatory Gs/olf protein.¹²⁸ Both are co-localized in the glutamatergic terminals and astrocytes, where they form A₁R-A_2AR heteromers.¹²⁹ These heteromers act as a concentration-dependent switch in the glutamatergic terminals, with activation of A₁R inhibiting glutamate release, while activation of A_2AR shuts down A₁R signaling and stimulates glutamate release.¹²⁹

Adenosine has higher affinity for A₁R than it has for A_2AR, and, presynaptically, it only tonically activates A₁R. Thus, gene-targeted vesicular release of ATP from the astrocytes leads to a loss of A₁R-mediated tonic inhibition of presynaptic hippocampal glutamatergic transmission.¹²⁷ Presynaptic A_2AR on the other hand are only activated by phasic increases in adenosine. This normally occurs when glutamatergic inputs are activated, leading to a release of ATP by neurons and glial cells that is later converted to adenosine by 5-nucleotidases.¹²⁸ When there is a phasic increase of adenosine, activation of A_2AR negatively modulates A₁R signaling in the heteromer resulting in an increase in glutamate release.^130–134 This same interaction is also seen in cultured cortical astrocytes, where activation of the A₁R-A_2AR heteromer has a modulatory effect on GABA uptake.¹³⁵ In dopaminergic terminals, only A₁R are expressed, resulting in the tonic inhibition of dopamine release.¹³²

Postsynaptically, A₁R and A_2AR are highly expressed in the somatodendritic region of striatal MSNs, and particularly, in the dendritic spines.¹³⁶ They are not co-localized but are segregated into two phenotypically distinct neuronal populations that connect the striatum with the output structures of the basal ganglia.¹²⁴ The striato-nigral neurons of the direct motor pathway, connect the striatum to the substantia nigra pars reticulata. These neurons selectively express A₁R and D₁R (and D₃R in the ventral striatum).^136–138 The striato-pallidal neurons of the indirect motor pathway, connects the striatum with the lateral segment of the globus pallidus and the ventral pallidum, and selectively expresses A_2AR and D₂R.^4,136 A₁R and D1R and A_2AR and D₂R form specific receptor complexes, the A₁R-D1R and A_2AR-D₂R heteromers.^{136,139–141} These heteromers act as molecular devices by which endogenous adenosine, when binding to the respective adenosine receptor, tonically inhibits the affinity and signaling of its respective dopamine receptor. Postsynaptic A_2AR, differently from presynaptic A_2AR, are therefore tonically activated, which produces significant behavioral and biochemical effects that can be disclosed when A_2AR is blocked after the administration of an antagonist (see below).¹⁴²

Adenosine receptor single nucleotide polymorphisms and caffeine intake

In early pathological stages of HD and even in symptomatic patients with a grade of 0 on Vonsattel’s neuropathological severity in HD scale, both D₂R and A_2AR are significantly and differentially downregulated when compared with D₁R.⁶ These data suggest a selective functional alteration in this MSN subpopulation, and this is not surprising considering the preferential vulnerability of the striato-pallidal neurons in HD.^6,7 Using different HD mouse models, downregulation of A_2AR has also been reported, and several studies imply that the mHtt in these HD mice causes abnormal A_2AR signaling and amplification.⁶¹

In HD, questions remain whether the changes seen in A_2AR expression and function are markers of selective degeneration of indirect MSNs, or if they are involved in the pathological process. Genetic studies suggest that changes in A_2AR are involved in the pathogenesis of the disease. Three single nucleotide polymorphisms in the ADORA2A gene that have nearly complete linkage disequilibrium and can potentially modify A_2AR transcription are rs5751876 (C>T substitution in exon 5), rs35320474 (T deletion in the 3′ untranslated region that includes U-rich motifs, which provide active sites of interaction with RNA-binding proteins), and rs2298383 (C>T substitution in a potential promoter region with a regulatory element predicted from alignment of human and other mammalian genes).^143–146 Rs5751876, a synonymous mutation (normally encoded amino acid), has been associated to an earlier AAO of HD,^147,148 and a recent study demonstrated a significant increase in the expression of A_2AR in the brain of HD subjects homozygous for a rs5751876 polymorphic block (including rs35320474 and rs2298383).¹⁴⁶ These results suggest that transcriptional dysregulation of A_2AR is associated with HD. How these data relate to the previous binding and expression studies in postmortem human brain in HD patients and in HD mouse models remains to be investigated.

Another link between the adenosine receptors and HD is the epidemiological evidence that associates the habitual consumption of caffeine with an earlier AAO of HD.¹⁴⁹ Caffeine is a nonselective A₁R and A_2AR antagonist, and it is proposed that the blockade of these receptors by caffeine may result in an increased acceleration of neurodegeneration. This could possibly be related to the fact that chronic caffeine exposure is associated with tolerance to the A₁R but not to the A_2AR.¹⁵⁰ Studies show that high doses of A_2AR antagonists or global genetic A_2AR blockade worsen disease progression in HD models,^151,152 whereas A_2AR agonists as well as A₁R agonists have been shown to protect against neurodegeneration.^151,153

In these animal models of HD, recently described alterations in adenosine metabolism leads to a hypotonic adenosine state (see below), and this condition could be mimicked by chronic caffeine exposure. Interestingly, Cornelis et al. described an association between the ADORA2A rs5751876 polymorphism and caffeine intake, which may link them to the HD progression.¹⁵⁴ However, this association was not confirmed in a recent genome-wide meta-analysis of polymorphisms and habitual coffee intake.¹⁵⁵

Alterations of A₁R function during HD progression

Data suggest that A₁R stimulation has clear neuroprotective effects in animal and human models of HD.^156,157 A₁R activation attenuated limb dystonia and striatal degeneration in the 3-NP model of HD,¹⁵¹ and prevented 3-NP-induced seizures in mice.¹⁵⁸ Also, A₁R blockade caused deleterious effects in malonate-induced metabolic models of HD.¹⁵⁹

Differing results were found when investigating A₁R binding in various transgenic rodent models of HD. In the Tg51 HD rat model, no changes in A₁R density were observed.¹⁶⁰ However, when the widely used symptomatic R6/2 HD mouse model was used, a decrease in cortical and striatal A₁R antagonist binding density was observed.¹⁶¹ Interestingly, when comparing R6/2 mice to WT mice, despite the reduced A₁R density, the A₁R agonist N6-Cyclopentyl-adenosine was able to further reduce synaptic transmission and glutamate release. This decrease in A₁R density and its apparently increased functionality was further confirmed using mHtt expressing striatal cells.¹⁶¹ Translating these preclinical results to human HD patients, a noninvasive positron emission tomography (PET) imaging study revealed that, in symptomatic HD patients, A₁R were significantly reduced compared with healthy non-HD subjects (see below).¹⁶²

Alterations of A_2AR during HD progression

The first evidence of A_2AR downregulation in HD was found in tissue sections of the human brain using autoradiography,¹⁶³ and was later confirmed in the basal ganglia of HD patients in early, intermediate, and late stages of the disease.⁶ Downregulation of A_2AR has been reported in HD patients that have not yet displayed any motor dysfunction,⁶ and in animal models before any significant neuronal loss has occurred.^{152,160,164–166}

Animal and cell models of HD have been fundamental in identifying the molecular mechanisms by which mHtt causes a reduction in the expression of A_2AR. Most of these models clearly show A_2AR downregulation at the protein and transcription level, but there are conflicting results when tested in the H46, YAC72, and Tg51 transgenic models.^{153,164,167–171}

Aggregated mHtt causes changes in gene expression profiles leading to specific protein–protein interactions with several transcription factors,^172–175 while mHtt does not change gene expression relating to cytoskeleton proteins, enzymes of metabolism, mitochondrial proteins, and caspases.^164,176 Chiang et al. found that mHtt expression significantly reduced the transcript levels of endogenous A_2AR in PC12 cells and striatal neurons in culture.¹⁶⁸ They identified that cells expressing mHtt have an atypical C-AMP response element-binding protein (CREB) responsive element site located in the core promoter of the A_2AR gene that prevents the binding of CREB, and thus suppresses the core promoter activity of the A_2AR gene. Interestingly, stimulation of the A_2AR restored normal CREB binding and reduced the aggregation caused by the mHtt protein.¹⁶⁸

The length of poly(Q)-expanded Htt appears to be critical for the downregulation of the A_2AR transcript, and it has been hypothesized that transcriptional dysfunctions only occur in the presence of a short N-terminal fragment (<171 amino acids) of mHtt. No reduction in the expression of A_2AR and other mHtt sensitive genes were seen in either a model that expresses an extended N-terminal fragment (HD46), or in a model that expresses a full-length mHtt (YAC72).¹⁶⁷ DNA methylation (5-methylcytosine, 5mC, and 5-hydroxymethylcytosine, 5hmC), an important mechanism for epigenetic silencing that regulates basal A_2AR level in the human brain,¹⁷⁷ has been proposed to be a key mechanism for the reduced striatal A_2AR levels observed in the brains of HD patients and in R6/1 and R6/2 mice.^170,178,179 In their study, Villar-Menéndez et al. found an increase in 5mC levels and a reduction in 5hmC levels in the 5′ untranslated region of the A_2AR gene, and these findings were closely associated with the downregulation of the A_2AR transcript seen in R6/2 mice and in the putamen of HD patients.¹⁷⁰ These results suggest that using strategies that modulate A_2AR expression could be a new approach to treating HD.

In addition to studies showing A_2AR reduction in HD, others have reported that as HD progresses the presence of mHtt can abnormally amplify signaling of the A_2AR. Varani et al. reported an amplification of A_2AR-mediated adenylyl cyclase stimulation in striatal-derived cells engineered to express mHtt,¹⁸⁰ a result confirmed in the striatum of R6/2 mice.^153,169 This amplification of A_2AR signaling was also found in peripheral blood cells in HD subjects, where overstimulation of A_2AR-mediated cAMP production was associated with an aberrant increase in A_2AR function and density.¹⁸¹

A_2AR density in blood platelets has been shown to correlate with the age of onset and CAG repeat expansion in HD patients.¹⁸² These data suggest that further studies investigating abnormal A_2AR signaling in peripheral blood cells are needed to validate using this receptor target as a potential biomarker for disease prognosis and drug efficacy.

PET imaging for adenosine receptor occupancy in HD

PET is a functional imaging technique that is used to observe receptor binding capacity in the body and can aid in the diagnosis of many neurological diseases, including HD.¹⁸³ Different radiotracers have been used with PET to measure brain metabolism, dopaminergic function, neuro-inflammation, phosphodiesterases, and other targets in HD.¹⁸³ However, few adenosine analog radiotracers have been developed and employed to noninvasively image A₁R and A_2AR occupancy.

One such radio-ligand that can be used to image the A₁R in vivo is [¹⁸F]CPFPX.^184–186 A cross-sectional study using [¹⁸F]CPFPX and MRI was performed to assess differences in A₁R density between controls and HD patients at different stages of the disease progression (“premanifest patients far from the onset of predicted symptoms”, “premanifest patients near the onset of predicted symptoms”, and “manifest patients”).¹⁶² In this study, [¹⁸F]CPFPX binding in the caudate of “manifest HD patients” was reduced by 25%, and [¹⁸F]CPFPX binding in the thalamus of “premanifest patients far from the onset of predicted symptoms” was 31% higher than health controls. Interestingly, thalamic [¹⁸F]CPFPX binding between “premanifest patients near the onset of predicted symptoms” and healthy controls was similar. These data suggest that A₁R switch from upregulation to downregulation as HD progresses. This study reveals the importance of A₁R in the pathology of HD, and shows that [¹⁸F]CPFPX and PET can be a useful tool to explore A₁R interactions in preclinical and clinical trials.¹⁶²

Radio-ligands developed and tested to image A_2AR antagonists, in particular, the xanthine ligands [¹¹C]TMSX, [¹¹C]KF17837, [¹¹C]TMSX, [¹¹C]KF21213, [¹¹C]KF19631, and [¹¹C]KW6002, have not been particularly useful for molecular imaging due to their low signal to noise ratio and high degree of nonspecific binding.¹⁸⁷ In general, radio-ligands that lack the xanthine structure appear to have better specificity for the A_2AR subtype and allow quantitative imaging of A_2AR in the mammalian striatum, but even these are less effective at imaging other areas of the brain.¹⁸⁸ [¹¹C]SCH442416 was the first non-xanthine ligand that was suitable for mapping of A_2AR using PET.¹⁸⁹ Recently, [¹¹C]preladenant has been used to quantify A_2AR binding sites in the rat brain. It displayed high uptake in the striatum, and low, homogenous uptake in all extra-striatal regions, a binding profile that corresponds to the known A_2AR expression in the rat brain.¹⁹⁰ Although [¹¹C]preladenant is tolerated, and has been used in both humans and monkeys to study A_2AR expression in the brain,^191,192 very few PET imagining studies investigating the role of A_2AR in HD pathology have been performed.

Finally, in the QA model of HD, that results in a loss of striato-pallidal GABAergic enkephalin neurons, the binding potential of [¹¹C] KF18446 in the striatum and globus pallidus was reduced by 25%.¹⁶⁵ This amount of loss is similar to that seen when [¹¹C]raclopride is used to measure the binding potential of D₂R.¹⁶⁵ As new and more suitable A_2AR radiotracers are developed, more detailed PET studies investigating the role of A_2AR in HD can be performed.

Alterations in striatal adenosine tone in HD

In the Tg51 rat model of HD there is a slower disease progression as compared to other animal models, and this allows for evaluating possible biomarkers that might be present during the initial phases of HD.¹⁹³ Using this model, we found that there were alterations in the adenosine system that were independent of alterations in A_2AR expression.¹⁷¹ Analyzing the function of pre- and postsynaptic A_2AR in the striatum, it was originally believed that during the initial stages of HD, the postsynaptic striatal A_2AR were selectively and functional impaired.¹⁶⁶ This was based on the observation that A_2AR antagonists were unable to produce locomotor activity in this model, while their ability to modulate cortical-striatal neurotransmission remained unchanged.¹⁹⁴ It was assumed that this difference was due to the downregulation of A_2AR that is seen in both human and animal models of HD. A pharmacological characterization of the Tg51 however, revealed that postsynaptic A_2AR function was not altered at all. After the administration of an A_2AR agonist, no difference in postsynaptic-dependent locomotor depression was seen when Tg51 rats were compared to their WT littermates.¹⁷¹ More convincingly, no difference in the number of striatal A_2AR sites or affinity was seen when WT rats were compared with either Tg51 homozygous or heterozygous rats.¹⁷¹ Together, the agonist effect, and the lack of antagonist effect suggested a low adenosinergic tone, resulting in a decreased ability of endogenous adenosine to activate the postsynaptic A_2AR. This would explain the preserved ability of A_2AR antagonists to block cortical-striatal transmission, because presynaptic activation of A_2AR is dependent on a phasic increase of extracellular adenosine. In fact, we showed a reduction in extracellular adenosine levels in the striatum of Tg51 rats and also of zQ175 knock-in mice,¹⁷¹ a recently developed transgenic animal model of HD.¹⁹⁵ Unlike the Tg51 rats, we observed a downregulation of A_2AR in the zQ175 mice.¹⁷¹

It is well accepted that under physiological conditions, the main source of extracellular adenosine production is the release of ATP by astroglial vesicles.¹²⁷ Extracellular ATP is rapidly converted to adenosine by a series of ectonucleotidases and extracellular levels of adenosine (adenosinergic tone) is maintained by the equilibrative transporter's and the astrocytic adenosine kinase's (ADK) ability to uptake and metabolize adenosine, respectively.^128,196 In mammals, there are two types of nucleoside transporters: bidirectional, equilibrative transporters that are driven by chemical gradients, and unidirectional, concentration transporters that are driven by sodium electrochemical gradients.¹⁹⁷ Adenosine uptake in the brain is mostly facilitated by the equilibrative nucleoside transporters, and when these transporters are blocked pharmacologically, adenosine accumulates in the extracellular space^128,197,198 Of the four equilibrative transporters identified (ENT1, ENT2, ENT3, and ENT4), ENT1 and ENT2 are the ones mostly present in the brain, expressed by both neurons and astrocytes.¹⁹⁷ Studies suggest that because of its dependence on glutamate receptor activation, the ENT1 plays a more prominent role in determining the concentration of extracellular adenosine in the brain.^199,200

Using the ENT1 selective inhibitor [³H]-S-(4-Nitrobenzyl)-6-thioinosine ([³H]NBTI), we found an upregulation of the ENT1 transporter in zQ175 mice.¹⁷¹ Also, the ENT1 gene transcript (SLC29A1) was upregulated in HD patients during early stages of neuropathological severity, but not in those with higher stages of severity, relative to controls.¹⁷¹ In a more recent study, we found that cerebral spinal fluid (CSF) adenosine levels were significantly lower in HD patients and CSF concentrations of ATP were inversely correlated with the number of CAG repeats.²⁰¹ Also, the disease duration of these HD patients was negatively correlated to the adenosine/ATP ratio.²⁰¹ These data suggest that adenosine levels may constitute another biomarker that can be used for diagnosing and treating HD.

Adenosine Neurotransmission as a Therapeutic Target in HD

Targeting A_2AR in chemical- and lesion-induced HD models

Effective tools to study the neurodegenerative effects seen in HD are chemical- and lesion-induced rodent models. Injecting the NMDA receptor agonist QA into the striatum or systemically administering the mitochondrial toxin 3-NP can mimic the anatomical and behavioral deficits seen in HD. These manipulations can also produce direct and indirect excitotoxicity and trigger the selective loss of MSNs in the striatum similar to that seen in HD.^26,202–205 Likewise, intra-striatal injections of malonate, a competitive inhibitor of succinate dehydrogenase, can produce significant lesions in the striatum like those present in HD patients.^206–208

Using these models, it has been shown that administering various A_2AR antagonists (DMPX, SCH58261, ZM241385, ST1535, MSX-3, and CSC) can have multiple beneficial effects. A_2AR antagonism can reduce striatal degeneration (atrophy), normalize EEG patterns, and normalize motor activity. It can also improve the loss of GABA content and lower the outflow of glutamate, all of which can increase the life span in HD patients.^{40,41,159,209–213} In contrast, the A_2AR agonist (CGS21680) was shown to increase the 3-NP-induced striatal lesion size.⁴⁰

Opposing results were found when 3-NP-evoked striatal damage was assessed in A_2AR-null mice. Global A_2AR knock-out mice showed opposite effects on the 3-NP-induced neurological deficit behaviors and striatal damage at different dosages,^40,209,214 and the selective depletion of A_2AR in forebrain neurons did not contribute to the 3-NP-evoked striatal damage.²¹⁴ However, the selective removal of A_2AR in bone marrow-derived cells caused the enhanced 3-NP-induced striatal damage in global A_2AR knock-out mice to return. These finding argue against the importance of A_2AR-mediated glutamate release in 3-NP-induced striatal damage,²¹⁴ suggesting the potential involvement of diverse cell types. The possible role of A_2AR in controlling non-neuronal cells (e.g., glia) might also contribute to the function of A_2AR in the brain. Inactivation of A_2AR appears to be beneficial in the chemical- and lesion-induced HD models, but further evaluation is required.

Targeting A₁R in phenotypic HD models

Targeting the A₁R for the treatment of HD has been mostly under evaluated. Using the 3-NP mouse model of HD, we observed that acute administration of the A₁R agonist adenosine amine congener reduced the size of 3-NP-induced striatal lesions, limited striatal degeneration, and prevented the development of the hind limb dystonia that is present after 3-NP treatment.¹⁵¹ Other studies also show evidence that A₁R activation has protective effects. The administration of an A₁R agonist was able to prevent 3-NP-induced seizures in mice,¹⁵⁸ and A₁R blockade potentiated the damage to dopaminergic neurons caused by an intra-striatal infusion of malonate.¹⁵⁹ The protective effects of A₁R agonists can be attributed to their ability to reduce glutamate release by activating presynaptic receptors in the striatum.¹⁵¹ The potential cardiovascular side effects of A₁R activation has limited its investigation as a potential therapeutic for treating HD,²¹⁵ but new studies targeting basal levels of adenosine (see below) might lead researchers to reconsider the A₁R as a potential direct or indirect target in HD treatment.

Targeting A_2AR in phenotypic HD models

The first genetic mouse model of HD was developed and characterized two decades ago.¹⁷⁸ Since then, more than 30 genetic mouse models of HD are now available from various sources.²¹⁶ Multiple genetic models of HD including transgenic, conditional transgenic, and knock-in mice have been created and are being used to study various stages of HD progression.^217–220

One such model that has been useful in studying the involvement of A_2AR in the pathogenesis of HD is the R6/2 mouse model. These mice express the exon 1 of the human huntingtin gene,¹⁷⁸ and quickly show a progression of many HD symptoms, including motor impairment, aggregate formation, and body weight loss.^176,221 Pharmacological treatments with A_2AR agonists have beneficial effects in R6/2 mice. Mice chronically treated with CGS21680 had reduced mHtt aggregate accumulation and had lowered enhancing proteasome activity, while the administration of T1-11 in R6/2 mice enhanced the performance on the rota-rod test, and enhanced proteasome activity.^{36,120,153,63,222–225}

Interestingly, the blockade of A_2AR also has beneficial effects in R6/2 mice. Studies showed that the administration of the A_2AR antagonist SCH58261 in R6/2 mice reduced glutamate and adenosine outflow, normalized the alteration in emotional response, and reduced NMDA-induced toxicity,^226,227 but had no effect on locomotor capability.^226,228 Genetic and pharmacological inactivation of A_2AR reduced working memory deficits in R6/2 mice,²²⁹ and the combined blockade of D₁R and A_2AR improved cognitive dysfunction in a different transgenic HD mouse model (R6/1).²³⁰ Taken together, these results suggest that A_2AR blockade may be able to reverse the cognitive impairment that develops in these HD mice.

While A_2AR blockade appears to aid cognitive function in HD, its effects on motor dysfunction is less clear. In the N171-82Q HD model, mHtt is expressed only in the neurons. These mice develop behavioral abnormalities including loss of coordination, tremors, hypokinesis, and abnormal gait, before dying prematurely. When the A_2AR is genetically deleted, by crossing A_2AR knockout mice with the N171-82Q, the life expectancy of these mice decreases, and their motor impairments are more severe when compared to the N171-82Q mice.¹⁵² These data are in agreement with the earlier studies showing that activation of A_2AR improved motor function in the R6/2 HD mice,¹⁵³ and might raise concerns about investigating A_2AR antagonists as a treatment in human HD patients. Given that activation of A_2AR has a beneficial effect on the motor impairment seen in HD mouse models, and inactivation of A_2AR in some models improves cognitive function, the symptom-specific effects of A_2AR treatments needs to be further investigated.

Targeting ENT1 in phenotypic HD models

Because ENT1 is upregulated while adenosine tone is reduced in both animal models and HD patients, it is proposed that ENT1 could constitute a new therapeutic target that may delay the progression of HD. Pharmacological blockade with the low affinity ENT1 inhibitor JMF1907²³¹ or genetic blockade of ENT (global ENT1 knockout) led to a significant increase in the mean survival time in the R6/2 mouse model of HD.²⁰¹ In the same study, there was also an increase in the expression and activity of both ENT1 and ENT2.²⁰¹ More support for this hypothesis comes from the fact that there is a decrease in tonic striatal adenosine levels in both R6/2 mice and in the knock-in Hdh(CAG)150 mouse.²³²

In two genetic animal models, the expression of ectonucleotidases and ADK were also analyzed. ADK transcript was upregulated only in R6/2 mice,²⁰¹ indicating that equilibrative transporters are more likely to represent a key pathogenetic mechanism in HD. This suggests that ENT1 and less selective ENT1/ENT2 inhibitors should be considered as new therapeutic drugs that may decrease the progression of HD.

JMF 1907 belongs to a group of multifunctional adenosine compounds that are both ENT1 inhibitors and A_2AR agonists, and although it readily crosses the blood-brain barrier, it is still under preclinical evaluation.^201,231 Given that inhibitors of ENT1 such as dipyridamole, ticagrelor, or dilazep are already being used to treat different pathological conditions related to vascular relaxation and platelet aggregation, and that the ENT1 inhibiting nonsteroidal anti-inflammatory drug sulindac sulfide is being used as an anti-inflammatory, it has been suggested that these compounds should be clinically studied in HD patients to evaluate their ability to delay the progression or the AAO of HD, even though some of these drugs have low brain penetrability.²³³

The classical reserpinized mice model is a method that can be used to study specific antagonistic interactions between adenosine and dopamine receptor ligands and it led to the discovery of the A_2AR-D₂R and A₁R-D₁R heteromers.^234,235 Using this method, we evaluated dipyridamole's ability to decrease the locomotor activation induced by dopamine receptor agonists. At a minimal dose of 30 mg/kg administered systemically, dipyridamole significantly decreased the locomotor-activating effect of equipotent doses of selective D₁R and D₂R agonists and this depressant effect was completely counteracted by caffeine.²³⁶ These results suggest that basal extracellular levels of adenosine in the striatum were increased following the systemic administration of dipyridamole and this increase in adenosine levels could explain the observed ability of dipyridamole to depress both D₁R and D₂R agonist-mediated locomotor activation in the reserpinized mice. The fact that this depressant effect was counteracted by caffeine, a non-selective adenosine antagonist, suggests that an increase in basal levels of adenosine may exert influence on both the tonic activation of pre- and postsynaptic A₁R and postsynaptic A_2AR. Since the activation of presynaptic A₁R leads to a decrease in glutamate release, therapeutics that can increase tonic levels of adenosine may be a beneficial treatment in patients with HD

Concluding Remarks

Many genetic, epidemiological, and experimental studies suggest that adenosine receptors, both A₁R and A_2AR are linked to HD pathophysiology, although their exact involvement remains unclear. Additional investigations using specific ligands and genetic murine tools are needed to dissect the pre- and postsynaptic aspects of A_2AR, and the relationship between A_2AR and mHtt induced glial dysfunction, which has been largely underestimated. Also, since HD is a chronically progressive disease, there are multiple mechanisms along the degenerative process that may be affected by their interactions with A_2AR. Since A_2AR heteromerize with several other GPCR that are involved in HD-related striatal dysfunction and degradation including D₂R and A₁R, A_2AR-containing heteromers should also be considered targets for HD-related drug development. Finally, the role of A₁R in HD pathogenesis needs to be reconsidered. The role of low adenosine tone in HD and recent studies indicating that ENT1 is upregulated in HD suggest that in addition to A_2AR, A₁R may also be a possible target for developing drugs that maybe beneficial in treating HD.

Acknowledgments

D.B. and L.B. are supported by grants from “France Alzheimer/Fondation de France,” FHU VasCog Research Network and “Program d'Investissements d'Avenir” LabEx (excellence laboratory), DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer's disease), ANR, “Fondation pour la Recherche Médicale,” Vaincre Alzheimer, “Fondation Plan Alzheimer,” Inserm, CNRS, Université Lille, Lille Métropole Communauté Urbaine, Région Hauts de France, and DN2M. Y.C. and C.Y.L. are supported by the Institute of Biomedical Sciences of Academia Sinica (103-Academia Sinica Investigation Award-06). M.R.D. and P.P. are supported by grants from the Italian Ministry of Health. W.R. and S.F. are supported by the intramural funds of the National Institute on Drug Abuse.

Author Disclosure Statement

No competing financial interests exist.