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Aberrant amplification of A_2A receptor signaling in striatal cells expressing mutant huntingtin ¹

KATIA VARANI^*, DOROTEA RIGAMONTI, SIMONETTA SIPIONE, ALESSANDRA CAMURRI, PIER ANDREA BOREA^*, FLAMINIO CATTABENI, MARIA P. ABBRACCHIO^,23 and ELENA CATTANEO^,23

^* Dipartimento di Medicina Clinica Sperimentale, Universita’ di Ferrara, 44100 Ferrara, Italy; and
Department of Pharmacological Sciences, University of Milano, 20133 Milano, Italy

³Correspondence: Department of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy. Elena.Cattaneo@unimi.it; Mariapia.Abbracchio@unimi.it

SPECIFIC AIMS

Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of a glutamine repeat in huntingtin (Htt), a 3154 aa protein with recently described antiapoptotic functions. Degeneration involves striatal GABA/enkephalinergic neurons, key components of the ‘indirect’ efferent pathway, which express adenosine A_2A and dopamine D2 receptors. We analyzed the possibility of an involvement of the A_2A receptor and its signaling components in the pathogenesis of HD, by using striatal-derived cells and subclones engineered to express either normal or mutant Htt in its full-length or truncated forms. The rationale for the analysis of the phenotype of cells expressing truncated Htt relies on the demonstration that amino-terminal fragments resulting from proteolytic cleavage of full-length mutant Htt are more toxic than the full-length protein itself.

PRINCIPAL FINDINGS

1. Both parental striatal-derived ST14A cells and Htt-engineered subclones express the A_2A adenosine receptor
Reverse-transcriptase polymerase chain reaction was used to detect the presence of A_2A receptor mRNA levels in parental striatal-derived ST14A cells and in the various stable subclones expressing either 548 amino acids amino (N) terminal to the protein in the wild-type (N548wt) or mutant (N548mu) versions or the full-length wild-type (FLwt) and full-length mutant (FLmu) Htt proteins. All subclones expressed the 630 bp band corresponding to the A_2A receptor mRNA, although to a different extent. Despite differences in mRNA levels, comparable A_2A receptor protein densities were found in all clones, as shown by binding studies with the highly selective A_2A receptor antagonist ³H-SCH 58261. Scatchard plot analysis revealed the presence of a single class of specific, high-affinity, and saturable binding sites in all clones with comparable B_max values. K_d values were instead significantly reduced only in FLmu cells with respect to parental ST14A cells, a change that was even more significant in N548mu cells. Comparable data were obtained with the A_2A receptor ligand ³H-ZM 241385: for the ST14A, FLwt, N548wt, Flmu, and N548mu, K_d values were respectively 2.30±0.10; 2.42±0.08; 2.48±0.05; 1.61± 0.06*; 1.32±0.06** nM (*P<0.05 vs. control; **P<0.01 vs. control; no changes in B_max values). These data suggest a selective increase of binding affinity in the presence of mutant Htt.

2. Forskolin-stimulated adenylyl cyclase response is selectively altered in cells expressing mutant Htt
Since the A_2A adenosine receptor is coupled to stimulation of adenylyl cyclase via Gs stimulatory proteins leading to cAMP increases, we analyzed the sensitivity of adenylyl cyclase to agents that are known to specifically activate this effector system. Despite no change in basal enzyme activity, response of adenylyl cyclase to the direct activator forskolin (used in the presence of the cAMP-dependent phosphodiesterase inhibitor RO 201724) was notably increased in N548mu (and Flmu) cells with respect to parental or wild-type Htt cells. An amplification of cAMP formation was already demonstrable with forskolin alone in N548mu cells. To shed light on the cyclase alteration observed in cells expressing mutant Htt, we performed a Michaelis-Menten analysis of adenylyl cyclase activity in ST14A and N548mu cells by incubating cell homogenates in the presence of a fixed forskolin concentration and graded concentrations of substrate (0.001–1000 µM ATP). A statistically significant reduction of enzyme Km value was detected in N548mu cells with respect to parental cells (82±3 nM vs. 180±4 nM, respectively). No change in V_max values was detected. This alteration in cyclase activity appears to specifically involve the catalytic subunit of the enzyme, since no changes in cAMP production were observed in the presence of GTP-S, a direct G-protein activator (data not shown).

3. A_2A receptor-stimulated adenylyl cyclase is aberrantly increased in cells expressing mutant Htt
When the adenosine analog 5'-N-ethylcarboxamidoadenosine (NECA) was applied to cells, an aberrant amplification of adenylyl cyclase response was detected selectively in cells expressing mutant Htt. In all clones, NECA (0.1 nM-10 µM) significantly increased cAMP production in a concentration-dependent manner. As shown in Fig. 1, however, which reports the log dose-response curve for NECA in cells expressing either N548wt and FLwt (Fig. 1A ) or N548mu and FLmu (Fig. 1B ), cAMP formation was significantly higher in the latter two at almost all agonist concentrations with respect to parental ST14A cells (EC₅₀ values: 198±15*; 93±9** nM in FLmu and N548mu, respectively, vs. 270±10 in ST14A cells; *P<0.05; **P<0.01 vs. control). No differences in NECA responses were detected in FLwt and N548wt cells with respect to ST14A cells at any of the agonist concentrations (EC₅₀ values: 253±11 and 236±13, respectively). These results suggest a selective increase in responsivity of the adenosine A_2A receptor/adenylyl cyclase system in the presence of mutant Htt. This change is unrelated to modifications of endogenously produced adenosine since adenosine deaminase was present during the adenylyl cyclase assay.

View larger version (18K): [in this window] [in a new window]   Figure 1. Stimulation of cyclic AMP levels by NECA (0.1 nM-10 µM) in ST14A (•), FL wt (), N548 wt () (A) (EC50=270±10; 253±11; 236±13 nM, respectively), and ST14A(•), in FL mu (), in N548 mu () (B) (EC50=270±10; 198±15*; 93±9** nM, respectively; *P<0.05; **P<0.01 vs. control). Curves represent the mean of four independent experiments performed in triplicate. Bars represent the standard error of the mean.

4. Selective A_2A receptor antagonists retain their ability to revert A_2A-stimulated cAMP formation in both parental and Htt-expressing cells
We tested the ability of adenosine receptor antagonists characterized by various degrees of A_2A receptor selectivity to prevent NECA stimulation of adenylyl cyclase. As shown in Fig. 2, in ST14A cells the relatively selective antagonists CGS 15943 and ZM 241385 and the highly selective antagonist SCH 58261 completely prevented increases in cAMP induced by NECA at an agonist concentration (100 nM), which selectively stimulates adenylyl cyclase via the A_2A receptor. All the antagonists tested also retained the ability to counteract NECA-induced cAMP formation in cells expressing mutant Htt, where adenylyl cyclase activity is aberrantly increased.

View larger version (47K): [in this window] [in a new window]   Figure 2. Schematic diagram showing the potential interaction of Htt with A2A receptor signaling in striatal cells. In parental ST14A cells (not shown) and subclones expressing wtHtt (A), stimulation of the A2A receptor by adenosine analogs results, as expected, in adenylyl cyclase activation, which can be prevented by the concomitant exposure to A2A receptor antagonists (B). In cells expressing mutant Htt, A2A antagonists still retain their ability to revert this response (D) despite increased A2A binding affinity and aberrantly increased stimulation of cAMP production (thick arrow in panel C). Question marks indicate possible direct or indirect interaction between mutant Htt and components of the A2A receptor signaling pathway.

CONCLUSIONS AND SIGNIFICANCE

Besides its involvement in motor function, the A_2A adenosine receptor may also regulate cell survival. The role of this receptor in the etiopathology of HD is still unclear. In R6/2 transgenic mice (an animal model of HD expressing a fragment of the disease-causing variant of human Htt), selective reductions of dopamine D₁ and D₂ receptors, of A_2A receptor mRNA and A_2A receptor binding, and of the expression of enkephalin (but not substance P) have been found. Yet such changes may still be subsequent to or independent of earlier aberrant receptor(s) behavior.

The present study describes for the first time an alteration of A_2A adenosine receptor signaling specifically associated with the expression of mutant Htt. We have used a striatal-derived cell line engineered to express either wild-type or mutant Htt. In this cellular system, Htt had previously been found to act as an apoptotic protein, whereas expression of an expanded CAG variant of Htt led to caspases activation and apoptosis.

Both parental striatal cells and the Htt-engineered derivatives express the A_2A receptor, hence validating this model as suitable to study possible changes induced by mutant Htt on this receptor system. Consistent with the reduction of A_2A receptor mRNA in R6/2 transgenic mice, we also found decreased levels of A_2A receptor mRNAs in mutant Htt cells. However, the density of the receptor protein was comparable in all clones as detected by binding studies. Despite no change in B_max values, we found a statistically significant increase of ligand binding affinity in cells expressing mutant Htt, suggesting that mutant but not wild-type Htt may interfere with the kinetics of A_2A receptor binding by endogenous adenosine. Our results also highlight profound changes of adenylyl cyclase response selectively in mutant Htt cells. Response to forskolin was significantly increased in these cells and, more remarkably, an even higher amplification of A_2A receptor-mediated cAMP formation was observed. Direct activation of the G-protein -subunit with GTPS did not result in higher cyclase stimulation in mutant Htt cells, suggesting that this subunit apparently is not affected. This may imply a specific effect of mutant Htt on G-protein ß-subunits, which play key roles in both cyclase activity and in receptor desensitization. A dysfunction of G-protein ß-subunits leading to reduced receptor desensitization hence may contribute to the observed aberrant amplification of A_2A receptor response. Changes of A_2A receptor signaling are much more evident in cells expressing truncated mutant Htt with respect to full-length mutant Htt. This finding may have intriguing implications, since the N548 fragment mimicks one of the potential fragments suggested to be produced by proteolytic cleavage of Htt and that may be required for expression of mutant Htt citotoxicity. Recent data demonstrate that the full-length mutant protein is gradually processed over time in vivo to generate amino-terminal fragments that progressively accumulate into the nucleus and in axons and synaptic terminals. Truncated mutant Htt has been also demonstrated to carry significantly higher toxicity with respect to the full-length protein. One could speculate that N548mu cells may reflect a more advanced stage of the disease, where the aberrant behavior of the A_2A receptor is maximally expressed.

Our data are at variance with a recent study where dopamine signaling was evaluated in R6/2 transgenic mice. At a presymptomatic stage of the pathology that manifests mainly neurological changes, the response of striatal adenylyl cyclase to either a D₁ receptor agonist or forskolin was greatly attenuated with respect to control mice. Such differences (aberrant adenylyl cyclase stimulation in our study vs. reduced cyclase responsivity in R6/2 transgenic mice) may represent changes in striatal signaling at different stages of the pathology, differences in the coupling between different receptors (D₁ or A_2A) and adenylyl cyclase, or it may depend on the fact that D1 and A_2A receptors are expressed by different sets of neurons in the basal ganglia.

A possible role for this receptor in the etiopathology of HD is also consistent with published data suggesting that the A_2A receptor may contribute to cell death in ischemia-associated neurodegeneration. In this respect, our demonstration that selective A_2A receptor antagonists retain their ability to fully block NECA-stimulated adenylyl cyclase may disclose new avenues in the pharmacological manipulation of Huntington’s disease.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0730fje ; to cite this article, use FASEB J. (March 5, 2001) 10.1096/fj.00-0730fje

² These authors contributed equally to this paper.

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