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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 4, 2003 as doi:10.1096/fj.03-0079fje. |
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,2,3
Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, Italy;
* Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, Italy;
Neurogenetics Unit, IRCCS Neuromed, Pozzilli (IS), Italy;
Section of Neurology, "Fornaroli" Hospital, Magenta (MI), Italy; and
Neurogenetics Section, IRCCS "C. Besta," Milan, Italy
3Correspondence: F.S., Neurogenetics Unit, IRCCS Neuromed, Località Camerelle, 86077 Pozzilli (IS), Italy; E-mail: neurogen{at}neuromed.it; or E.C., Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milano, Via Balzaretti 9, 20133 Milano, Italy; E-mail: elena.cattaneo{at}unimi.it
SPECIFIC AIMS
Huntington’s disease (HD) is a progressively disabling neurodegenerative disease caused by an expanded CAG mutation in the gene encoding huntingtin (Htt). There is increasing interest in the identification of peripheral biomarkers of the disease that could be used to predict its progression and/or to monitor the efficacy of upcoming therapeutic treatments. A2A adenosine receptors are specifically found on striatal medium spiny neurons undergoing death in HD, suggesting they may play a role in HD-associated neurodegeneration. In a previous study we demonstrated the presence of an aberrantly increased A2A adenosine receptor function in immortalized striatal cells engineered to express mutant Htt. The present study represents the follow-up of that original observation and was aimed at testing whether a similar dysfunction is present in the peripheral blood cells (platelets, lymphocytes, and neutrophils) of human subjects carrying the mutant HD gene.
PRINCIPAL FINDINGS
1. Increased A2A adenosine receptor density in the peripheral circulating cells of subjects carrying the HD gene
The study involved 48 heterozygous, 3 homozygous HD patients, and 7 asymptomatic at-risk subjects compared to 58 healthy subjects. Sex and age of all groups of subjects are reported in Table 1
. We have determined the kinetic parameters of A2A adenosine receptors in membranes prepared from platelets, lymphocytes, and neutrophils of all subjects by the selective radioligand [3H]-ZM 241385. In all cell preparations from control subjects, A2A receptor binding was saturable and indicated the presence of a single receptor population characterized by affinity (expressed as ligand dissociation constant, KD) and density (expressed as fmol of bound ligand/mg protein, Bmax) values that agree with those already reported in the literature for these cells (Table 1)
. Analysis of the same parameters in subjects carrying the HD gene revealed a highly significant increase of the density (Bmax) of A2A receptors in all cell populations with respect to normal controls (P=0.0001, Table 1
). Such significance was statistically obtained by considering HD subjects as a whole or each cohort of mutation carriers compared with the healthy controls (heterozygotes P=0.0001, asymptomatic at risk P=0.0001, Table 1
). Increased A2A receptor density in HD was not due to medication of these patients with either benzodiazepines or neuroleptics (the typical drugs administered in HD), since in a small cohort of five HD patients who had never been treated with such drugs a comparable increase of the peripheral A2A receptor was found. Given that the mean age of asymptomatic mutation carriers was remarkably lower than that of symptomatic patients (as expected), these subjects were compared with another group of age-matched control subjects (Table 1)
, revealing a highly significant increase of A2A receptor density in the at risk mutation carriers. The increase in Bmax was also accompanied by an increase in KD values (P=0.0001, Table 1
). In three subjects homozygous for the HD mutation, a marked increase of A2A receptor parameters in all three blood cell types was detected (Table 1)
. Neutrophil cells from these subjects showed an even significantly higher alteration of A2A receptor binding parameters with respect to heterozygotes (Table 1
; see % increase of Bmax in right-hand column). The A2A receptor changes detected in HD are highly specific for this receptor subtype, since a parallel analysis of other adenosine receptors (the A1 and A3 receptor subtypes) and another unrelated G-protein-coupled receptor (i.e., the 
2-adrenergic receptor) did not reveal any differences in HD subjects with respect to healthy controls.
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2. Up-regulation of A2A receptors in the peripheral cells of HD subjects is associated with an aberrantly increased adenylyl cyclase (AC) stimulation
In the blood peripheral cells of a selected number of heterozygous subjects (see below), the coupling of A2A receptors to AC stimulation and cAMP formation (the typical transduction system used by this receptor) was compared with control subjects. Figure 1
shows typical concentration-response curves to the A2A receptor agonist N-ethyl-carboxamido-adenosine (NECA) in platelets, lymphocytes, and neutrophils obtained from control and HD heterozygous subjects. Concentration-dependent stimulation of cAMP formation was obtained in the presence of NECA in all cell preparations. In all peripheral cells from HD subjects, NECA EC50 values (agonist concentrations eliciting 50% of maximal cAMP formation) were significantly lower with respect to controls, indicating an increased potency of NECA in stimulating cAMP accumulation.
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CONCLUSIONS AND SIGNIFICANCE
In this study we show for the first time that peripheral blood cells from HD subjects exhibit an aberrantly increased A2A receptor number, as demonstrated by Bmax values significantly higher compared to healthy controls. In line with the currently accepted receptor theory, this increase of Bmax was associated with an overstimulation of A2A receptor-mediated cAMP production. In the three homozygous patients available for the study, a more severe alteration of A2A receptor phenotype with respect to heterozygous patients was detected. As these three homozygotes showed a particularly accelerated disease progression toward disability and no difference in age at onset with respect to the heterozygotes, this biochemical finding may reflect clinical and genetic differences between these two groups of patients. The aberrant A2A receptor phenotype was also found in the circulating cells of a small cohort of presymptomatic individuals carrying the CAG mutation. This suggests that the aberrant A2A receptor behavior may accumulate in the presymptomatic years and is likely to contribute to disease progression. This conclusion is consistent with reports highlighting neuronal dysfunctions in the presymptomatic life stages in HD animal models and humans.
In the brain, A2A receptor stimulatory on adenylyl cyclase activity has long been known to be almost exclusively expressed by striatal medium spiny neurons, where it regulates motor behavior in cooperation with D2 dopamine receptors. More recently, activation of this receptor under specific pathological conditions has been associated with the induction of cell death in experimental models of cerebral damage (Fig. 2
). Our previous findings showing that this receptor is aberrantly amplified in a cellular model of HD combined with the findings reported here on the peripheral cells of HD subjects suggest that the changes detected in A2A receptor activity may reproduce in the periphery a molecular dysfunction present in HD brain. The fact that this dysfunction is easily accessible and measurable and likely correlates with the underlying disease mechanisms would make it an ideal peripheral biomarker of the disease.
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Consistent with the possible involvement of the A2A receptor in HD pathogenesis, selective A2A receptor antagonists were able to reduce the loss of striatal neurons in an animal model of HD and could also revert the aberrantly increased A2A receptor stimulation of adenylyl cyclase in striatal cells expressing mutant Htt.
Altogether, the evidence presented here points to the A2A receptor as a potential target to be exploited for HD therapy and as a tool to monitor disease evolution.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0079fje; doi: 10.1096/fj.03-0079fje 
2 These authors contributed equally to this work.
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  M. Valenza, D. Rigamonti, D. Goffredo, C. Zuccato, S. Fenu, L. Jamot, A. Strand, A. Tarditi, B. Woodman, M. Racchi, et al. Dysfunction of the Cholesterol Biosynthetic Pathway in Huntington's Disease J. Neurosci., October 26, 2005; 25(43): 9932 - 9939. [Abstract] [Full Text] [PDF]
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) (platelets: EC50=308±7 nM, lymphocytes: EC50=199±4 nM, neutrophils: EC50=168±5 nM, n=10), and HD heterozygous subjects (•) (platelets: EC50=119±13* nM, lymphocytes: EC50=79±10* nM, neutrophils: EC50=85±9* nM, n=10, *P=0.0001).