Motor Dysfunction in Type 5 Adenylyl Cyclase-null Mice

Abstract

Various neurotransmitters, such as dopamine, stimulate adenylyl cyclase to produce cAMP, which regulates neuronal functions. Genetic disruption of the type 5 adenylyl cyclase isoform led to a major loss of adenylyl cyclase activity in a striatum-specific manner with a small increase in the expression of a few other adenylyl cyclase isoforms. D1 dopaminergic agonist-stimulated adenylyl cyclase activity was attenuated, and this was accompanied by a decrease in the expression of the D1 dopaminergic receptor and Gsα. D2 dopaminergic agonist-mediated inhibition of adenylyl cyclase activity was also blunted. Type 5 adenylyl cyclase-null mice exhibited Parkinsonian-like motor dysfunction, i.e. abnormal coordination and bradykinesia detected by Rotarod and pole test, respectively, and to a lesser extent locomotor impairment was detected by open field tests. Selective D1 or D2 dopaminergic stimulation improved some of these disorders in this mouse model, suggesting the partial compensation of each dopaminergic receptor signal through the stimulation of remnant adenylyl cyclase isoforms. These findings extend our knowledge of the role of an effector enzyme isoform in regulating receptor signaling and neuronal functions and imply that this isoform provides a site of convergence of both D1 and D2 dopaminergic signals and balances various motor functions. Various neurotransmitters, such as dopamine, stimulate adenylyl cyclase to produce cAMP, which regulates neuronal functions. Genetic disruption of the type 5 adenylyl cyclase isoform led to a major loss of adenylyl cyclase activity in a striatum-specific manner with a small increase in the expression of a few other adenylyl cyclase isoforms. D1 dopaminergic agonist-stimulated adenylyl cyclase activity was attenuated, and this was accompanied by a decrease in the expression of the D1 dopaminergic receptor and Gsα. D2 dopaminergic agonist-mediated inhibition of adenylyl cyclase activity was also blunted. Type 5 adenylyl cyclase-null mice exhibited Parkinsonian-like motor dysfunction, i.e. abnormal coordination and bradykinesia detected by Rotarod and pole test, respectively, and to a lesser extent locomotor impairment was detected by open field tests. Selective D1 or D2 dopaminergic stimulation improved some of these disorders in this mouse model, suggesting the partial compensation of each dopaminergic receptor signal through the stimulation of remnant adenylyl cyclase isoforms. These findings extend our knowledge of the role of an effector enzyme isoform in regulating receptor signaling and neuronal functions and imply that this isoform provides a site of convergence of both D1 and D2 dopaminergic signals and balances various motor functions. adenylyl cyclase wild type cAMP-dependent protein kinase The neurotransmitter dopamine acts through various dopaminergic receptor subtypes that are associated with either stimulation or inhibition of adenylyl cyclases (ACs),1 leading to the regulation of physiological functions such as the control of various motor functions or psychomotor activity (1Hardman J.G. Limbird L.E. Gilman A.G. Goodman ' Gilman's The Pharmacological Basis of Therapeutics. 10th Ed. McGraw-Hill, New York2001: 447-483Google Scholar). This dopamine-sensitive AC activity is highest in the striatum as well as in associated limbic structures of the brain where their levels of activity exceed, by orders of magnitude, those in other areas of the brain. Such differences in striatal enzymatic activity may be attributed to the amount and/or combination of the enzyme isoforms that are expressed differentially in each brain region (2Chern Y. Cell. Signal. 2000; 12: 195-204Crossref PubMed Scopus (72) Google Scholar, 3Hanoune J. Defer N. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 145-174Crossref PubMed Scopus (565) Google Scholar). The brain expresses all nine AC isoforms (AC1–AC9) that have distinct biochemical properties,i.e. regulation by Gi, Gऔγ, calcium, or various kinases (4Taussig R. Zimmermann G. Adv. Second Messenger Phosphoprot. Res. 1998; 32: 81-98Crossref PubMed Scopus (47) Google Scholar). Most, if not all, isoforms are enriched in specific brain regions (2Chern Y. Cell. Signal. 2000; 12: 195-204Crossref PubMed Scopus (72) Google Scholar, 5Cooper D.M. Mons N. Karpen J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (557) Google Scholar) rather than diffusely distributed throughout the brain. AC5, for example, is the dominant isoform in the striatum as well as in the heart (6Premont R.T. Chen J. Ma H.W. Ponnapalli M. Iyengar R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9809-9813Crossref PubMed Scopus (170) Google Scholar, 7Mons N. Decorte L. Jaffard R. Cooper D.M. Life Sci. 1998; 62: 1647-1652Crossref PubMed Scopus (76) Google Scholar, 8Ishikawa Y. Katsushika S. Chen L. Halnon N.J. Kawabe J. Homcy C.J. J. Biol. Chem. 1992; 267: 13553-13557Abstract Full Text PDF PubMed Google Scholar). However, the coupling of each enzyme isoform to a specific neuronal function or functions and a receptor signal remains unknown as does whether the function of an AC isoform, unlike that of the receptors, can be substituted by another isoform. The striatum is considered to be the center of sensorimotor integration within the basal ganglia (9Smith Y. Kieval J.Z. Trends Neurosci. 2000; 23: S28-S33Abstract Full Text PDF PubMed Scopus (251) Google Scholar) and receives widespread excitatory input from all regions of the cortex that converge with extensive dopaminergic signals, both D1 and D2, afferent from the midbrain. Concerted and balanced activity of these two dopaminergic signals is believed to play a key role in regulating striatal motor functions. In this study, we examined the role of their potential target enzyme isoform, AC5, by the use of knockout mice in which the AC5 gene was disrupted. We disrupted the AC5 gene by the homologous recombination technique at the exon with the first translation initiation site (Fig. 1A). The type 5 AC gene has another translation initiation site with a reasonable Kozak consensus sequence within the same exon (8Ishikawa Y. Katsushika S. Chen L. Halnon N.J. Kawabe J. Homcy C.J. J. Biol. Chem. 1992; 267: 13553-13557Abstract Full Text PDF PubMed Google Scholar) that was excised in the final targeting vector. The integration of the knockout transgene was confirmed by genomic Southern analysis (Fig. 1B). All mice were 129/SvJ-C57BL/6 mixed background littermates from F1 heterozygote crosses. All experiments were performed in 8–12-week-old homozygous (AC5−/−) and wild-type (WT) littermates. This study was approved by the Animal Care and Use Committee at the Yokohama City University School of Medicine and New Jersey Medical School. Partial fragments of mouse AC cDNA clones for each isoform (types 1–9) and neuropeptides,i.e. enkephalin, substance P, and dynorphin, were obtained by PCR. A human 28 S ribosomal RNA probe was used as an internal control. RNase protection assay was performed using the RPA III kit (Ambion, Austin, TX). Striatal tissues were dissected from mice, and membrane preparations were prepared for AC assays as described previously (10Hess E.J. Battaglia G. Norman A.B. Creese I. Mol. Pharmacol. 1987; 31: 50-57PubMed Google Scholar, 11Ishikawa Y. Sorota S. Kiuchi K. Shannon R.P. Komamura K. Katsushika S. Vatner D.E. Vatner S.F. Homcy C.J. J. Clin. Investig. 1994; 93: 2224-2229Crossref PubMed Scopus (121) Google Scholar). D1 and D2 dopaminergic receptor binding assays were performed using [3H]SCH23390 and [3H]spiperone, respectively, as described previously (12Hess E.J. Battaglia G. Norman A.B. Iorio L.C. Creese I. Eur. J. Pharmacol. 1986; 121: 31-38Crossref PubMed Scopus (114) Google Scholar,13Baik J.H. Picetti R. Saiardi A. Thiriet G. Dierich A. Depaulis A. Le Meur M. Borrelli E. Nature. 1995; 377: 424-428Crossref PubMed Scopus (591) Google Scholar). Preliminary experiments demonstrated that theKd and Bmax values for D1 and D2 dopaminergic receptors were similar to those reported previously (12Hess E.J. Battaglia G. Norman A.B. Iorio L.C. Creese I. Eur. J. Pharmacol. 1986; 121: 31-38Crossref PubMed Scopus (114) Google Scholar, 13Baik J.H. Picetti R. Saiardi A. Thiriet G. Dierich A. Depaulis A. Le Meur M. Borrelli E. Nature. 1995; 377: 424-428Crossref PubMed Scopus (591) Google Scholar). Motor functions of mice were assessed by Rotarod test (14Brandon E.P. Logue S.F. Adams M.R. Qi M. Sullivan S.P. Matsumoto A.M. Dorsa D.M. Wehner J.M. McKnight G.S. Idzerda R.L. J. Neurosci. 1998; 18: 3639-3649Crossref PubMed Google Scholar), locomotor activity tests (14Brandon E.P. Logue S.F. Adams M.R. Qi M. Sullivan S.P. Matsumoto A.M. Dorsa D.M. Wehner J.M. McKnight G.S. Idzerda R.L. J. Neurosci. 1998; 18: 3639-3649Crossref PubMed Google Scholar, 15Krezel W. Ghyselinck N. Samad T.A. Dupe V. Kastner P. Borrelli E. Chambon P. Science. 1998; 279: 863-867Crossref PubMed Scopus (301) Google Scholar), pole test (16Matsuura K. Kabuto H. Makino H. Ogawa N. J. Neurosci. Methods. 1997; 73: 45-48Crossref PubMed Scopus (282) Google Scholar), and tail suspension test (17Yamamoto A. Lucas J.J. Hen R. Cell. 2000; 101: 57-66Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar). Given that AC is the major effector enzyme of dopaminergic signals in the striatum, we conducted various motor function tests to evaluate striatal function in an animal model in which the AC5 gene was disrupted (AC5−/−). The most dramatic change was found in coordinated movement, which was evaluated by Rotarod performance. In this test, we measured the time that mice could stay on an accelerating Rotarod without falling. In general, there was a major impairment in AC5−/− and to a lesser degree in the heterozygous mice relative to WT (Fig.2A). AC5−/−could spend significantly less time on the Rotarod, and heterozygous mice could spend slightly less time than WT. When tests were repeated, their performance improved significantly after a few trials. However, AC5−/− performed very poorly even after several trials. When the test was repeated on the following day, the results were similar, disputing the possibility that AC5−/− required a longer period to learn the performance (data not shown). Spontaneous activity was determined both horizontally (locomotion) and vertically (rearings). Mice were placed in a cage, and their movements were videotaped for analysis. WT and heterozygous mice revealed a similar performance in open field locomotor activity, while AC5−/− showed a small but significant degree of reduction (Fig. 2, B and C). To evaluate bradykinesia, pole test was performed. The time until they turned downward (Tturn) and the time until they descended to the floor were measured (TLA). We found that AC5−/− had marked deficits in this test; they showed an over 3-fold prolongation of both recording time indexes (Fig.2D). It was also possible that striatal dysfunction led to choleric or dystonic movements. Such abnormal movements may be demonstrated most readily in mice as a clasping of the limbs that is triggered by tail suspension test (17Yamamoto A. Lucas J.J. Hen R. Cell. 2000; 101: 57-66Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar). However, we found no such abnormal movements in both AC5−/− and WT (data not shown). These results indicated impairments of striatal functions in AC5−/− presumably induced by the loss of AC5. While AC5 may be striatum-specific with regard to its distribution (18Mons N. Cooper D.M.F. Mol. Brain Res. 1994; 22: 236-244Crossref PubMed Scopus (95) Google Scholar), it remained unknown whether it was dominant for cAMP production in the striatum. AC5 mRNA was expressed at least 10–20-fold more abundantly in the striatum than in the other brain regions, such as the cortex and the cerebellum, in WT (Fig.3A); this was in agreement with previous findings (18Mons N. Cooper D.M.F. Mol. Brain Res. 1994; 22: 236-244Crossref PubMed Scopus (95) Google Scholar). In AC5−/−, AC5 mRNA expression was negated, but histological examinations revealed no changes such as neuronal loss and/or reactive gliosis at 8–12 weeks old (data not shown). We found, however, that AC activity was greatly decreased in striatal membrane preparations in AC5−/−(Fig. 3B). In contrast, AC activity was significantly, but only to a small degree, decreased in the cortex where AC5 could be detected in WT and showed no difference in the cerebellum where AC5 was scarcely detected in WT. For comparison, AC activity in the heart, another tissue in which AC5 is dominantly expressed (8Ishikawa Y. Katsushika S. Chen L. Halnon N.J. Kawabe J. Homcy C.J. J. Biol. Chem. 1992; 267: 13553-13557Abstract Full Text PDF PubMed Google Scholar), was decreased by only 307 (data not shown), indicating that the contribution of AC5 to cAMP production is greater in the striatum than in the heart. We also examined receptor agonist-stimulated AC activity (Fig.3C). In general, in many tissues including the heart, marked stimulation of AC is readily attainable with Gs-coupled receptor agonists, although the inhibition of AC with Gi-coupled receptor agonists may not always be easy. In the striatum, however, SKF38393, a D1 dopaminergic receptor agonist, modestly stimulated AC activity in WT (40.7 ± 2.67 increase over that with 10 ॖm GTP). Quinpirole, a D2 agonist, inhibited SKF38393-stimulated AC activity; the inhibition was significant but small (13.5 ± 1.17 decrease). In AC5−/−, the responses to D1 and D2 dopaminergic receptor agonists were markedly diminished; the D1 dopaminergic agonist-mediated stimulation was very small, and the D2 dopaminergic agonist-mediated inhibition in AC5−/− was hardly detectable. It is tentative to speculate that the loss of D2 agonist-mediated inhibition was due to the loss of AC5, which is Gi-inhibitable, as proposed recently in a similar model (19Lee K.W. Hong J.H. Choi I.Y. Che Y. Lee J.K. Yang S.D. Song C.W. Kang H.S. Lee J.H. Noh J.S. Shin H.S. Han P.L. J. Neurosci. 2002; 22: 7931-7940Crossref PubMed Google Scholar), while it is also possible that the AC catalytic activity was too low to demonstrate inhibition by AC assays with membrane preparations. Thus, in vitro AC assays may not be sufficient in terms of sensitivity to study changes in selective dopaminergic signal in AC5−/−. We did not understand, however, why the response to D1 agonist stimulation as shown by percent increase was also attenuated in AC5−/− because other remnant AC isoforms must be able to respond to Gs, if not Gi, stimulation. We also examined cAMP accumulation in intact striatal neuronal cells from the fetus; however, the difference in cAMP production was not as great as in the above AC assays using membrane preparations from adults (data not shown). The disruption of the major striatal AC isoform may change the expression of other molecules involved in dopaminergic signaling. D1 dopaminergic receptor binding sites were modestly decreased in AC5−/−, while D2 receptor binding sites were similar to those in WT (Fig.4A). The short, but not the long, form of Gs protein expression was decreased in AC5−/− (Fig. 4B) presumably due to the loss of positive feed forward regulatory loop. Western blotting for various molecules, using either the membrane preparation or whole tissue homogenates, revealed that the protein expression of Golf, Gi, Gq, Gऔ, and PKA (the α catalytic subunit) were not changed (data not shown). Changes in neurotransmitters, such as dynorphin, substance P, and enkephalin, were examined by RNase protection assays that may be linked to the activity of D1 and D2 dopaminergic receptors. The expression of dynorphin, which acts on presynaptic κ-receptors to inhibit AC (20Xie G.X. Meng F. Mansour A. Thompson R.C. Hoversten M.T. Goldstein A. Watson S.J. Akil H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3779-3783Crossref PubMed Scopus (70) Google Scholar), was modestly increased (Fig. 4C). In contrast, the expressions of enkephalin and substance P were unchanged (Fig. 4C). The expressions of glutamic acid decarboxylase and tyrosine hydroxylase, which are involved in the synthesis of γ-aminobutyric acid and dopamine, respectively, were also unchanged as determined by immunoblotting (data not shown). The above findings suggested that the expression of some molecules, i.e. D1 dopaminergic receptors, Gs, and dynorphin, was changed in such a way to suppress the D1 dopaminergic pathway despite the disruption of the major AC isoform. We then examined whether there was any increase in the expression of other AC isoforms in AC5−/−. Since AC isoform antibodies that can convincingly determine the level of protein expression are not available, we quantitated the mRNA expression of the AC isoforms by RNase protection assays (Fig. 4D). All AC isoforms except AC4 and AC8 were detected. Among these isoforms, we found a modest increase of AC6, the most relevant isoform to AC5, as well as AC2, AC7, and AC9 but not AC1 and AC3 in AC5−/−. We thought, however, that such small increases in the expression of AC isoforms were not sufficient to explain decreases in the expression of Gs and D1 dopaminergic receptors, which occurred as if to inhibit the D1 dopaminergic pathway. If either or both D1 and D2 dopaminergic signals were attenuated in AC5−/− leading to motor dysfunction in vivo, then stimulation of dopaminergic receptors, D1 and/or D2, with specific agonists may restore the function. Administration of SKF38393 (25 or 50 mg/kg), a D1 dopaminergic agonist, increased locomotor activity in both WT and AC5−/−. In particular, locomotor activity response appeared pronounced, and might be supersensitive, in AC5−/− relative to WT (Fig.5A). This finding was reminiscent of the supersensitive response of the direct pathway neurons observed in dopamine depletion of Parkinson's disease in which the D1 dopaminergic function becomes supersensitive but is accompanied by an actual reduction of D1 dopamine receptor levels (21Marshall J.F. Navarrete R. Joyce J.N. Brain Res. 1989; 493: 247-257Crossref PubMed Scopus (107) Google Scholar, 22Gerfen C.R. Engber T.M. Mahan L.C. Susel Z. Chase T.N. Monsma Jr., F.J. Sibley D.R. Science. 1990; 250: 1429-1432Crossref PubMed Scopus (2477) Google Scholar) (Fig.4A). SKF38393 did not improve Rotarod performance in both WT and AC5−/− (Fig. 5B). We then examined the effect of cabergoline (0.2 or 1.0 mg/kg), a D2 dopaminergic agonist that has been used in the treatment of Parkinson's disease. Cabergoline had no significant effect on locomotor activity in both WT and AC5−/−, although both showed a tendency of small increases (Fig. 5D). In contrast, cabergoline improved Rotarod performance selectively in AC5−/−; their performance reached an equivalent level to that of WT (Fig.5E), while cabergoline essentially had no effect on WT, suggesting that coordination in AC5−/− was restored by D2 dopaminergic stimulation. We also examined the effect of these agonists on pole test performance (Fig. 5, C and F). Both SKF38393 and cabergoline improved pole test performance in AC5−/−, the of which induced a dramatic even with a (0.2 We have demonstrated that the disruption of the AC5 gene led to a major in AC activity in a striatal specific manner and an abnormal coordination by Rotarod performance as well as other motor disorders that Parkinson's disease. Selective stimulation of D2 dopaminergic receptors by cabergoline restored suggesting that the of D2 dopaminergic signal abnormal coordination in AC5−/− and that D2 dopaminergic signal AC5 as a major effector isoform. activity was also attenuated and restored by selective D1 dopaminergic suggesting that this dopaminergic signal also AC5. In contrast, both dopaminergic signals may be able to to other AC isoforms as well because D1 or D2 dopaminergic stimulation could restore specific motor i.e. coordination or such selective dopaminergic agonist stimulation could not restore all of the motor indicating that AC5 is in and both coordination and and may the site of convergence of both D1 and D2 dopaminergic D1 and D2 are the most dopaminergic receptors expressed in the and both are involved in the two major striatal the and the which are dominantly by D1 and D2 receptors, it is unknown whether these receptor subtypes are expressed in the distinct of striatal neurons C.J. E. H. J. Neurosci. 1995; PubMed Google Scholar) or within the same Z. Song Adv. Pharmacol. 1998; PubMed Scopus Google Scholar), it has been believed that the and balanced of these two and their control striatal motor functions. findings that the and balanced is by the of AC5 that is to both dopaminergic In the of AC5, and AC1 are but for the function of AC5. The supersensitive response to D1 dopaminergic stimulation the in Parkinson's disease. in Parkinson's AC5−/− also had decreased D1 dopaminergic receptor expression (21Marshall J.F. Navarrete R. Joyce J.N. Brain Res. 1989; 493: 247-257Crossref PubMed Scopus (107) Google Scholar, 22Gerfen C.R. Engber T.M. Mahan L.C. Susel Z. Chase T.N. Monsma Jr., F.J. Sibley D.R. Science. 1990; 250: 1429-1432Crossref PubMed Scopus (2477) Google Scholar). there was no of protein or PKA changes for this must be of although we not that compensation in the AC pathway could increased translation and/or of AC isoforms or other pathway The for this have also remained in Parkinson's but a very study suggested that a in the regulation of protein kinase signal may be involved C.R. S. R. P. J. Neurosci. 2002; 22: PubMed Google Scholar). The dopamine depletion in Parkinson's disease and the of its major effector enzyme isoform may be similar in many and AC5−/− may be to the for in The of our however, that the neurotransmitter signal at the level of an effector the integration of receptor signals on effector isoform, may be as as the neurotransmitter signal at the level of their receptor subtypes in regulating neuronal functions that has been more findings also that targeting an AC such as AC5, in may be an way to motor dysfunction in human Y. M. H. S. H. Y. I. Homcy C.J. K. Y. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We of for and City for