GRK5 Deficiency Leads to Reduced Hippocampal Acetylcholine Level via Impaired Presynaptic M2/M4 Autoreceptor Desensitization

Abstract

G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive. Here we report that overexpression of dominant negative mutant of GRK5 (dnGRK5) in a cholinergic neuronal cell line led to decreased acetylcholine (ACh) release. This reduction was fully corrected by pertussis toxin, atropine (a nonselective muscarinic antagonist), or methoctramine (a selective M2/M4 muscarinic receptor antagonist). Consistent with results in cultured cells, high potassium-evoked ACh release in hippocampal slices from young GRK5 knock-out mice was significantly reduced compared with wild type littermates, and this reduced ACh release was also fully corrected by methoctramine. In addition, following treatment with the nonselective muscarinic agonist oxotremorine-M, M2, and M4 receptors underwent significantly reduced internalization in GRK5KO slices compared with wild type slices, as assessed by plasma membrane retention of receptor immunoreactivity, whereas M1 receptor internalization was not affected by loss of GRK5 expression. Moreover, Western blotting revealed no synaptic or cholinergic degenerative changes in young GRK5 knock-out mice. Altogether, these results suggest that GRK5 deficiency leads to a reduced hippocampal ACh release and cholinergic hypofunction by selective impairment of desensitization of presynaptic M2/M4 autoreceptors. Because this nonstructural cholinergic hypofunction precedes the hippocampal cholinergic hypofunction associated with structural cholinergic degeneration and cognitive decline in aged GRK5 knock-out mice, this nonstructural alteration may be an early event contributing to cholinergic degeneration in AD. G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive. Here we report that overexpression of dominant negative mutant of GRK5 (dnGRK5) in a cholinergic neuronal cell line led to decreased acetylcholine (ACh) release. This reduction was fully corrected by pertussis toxin, atropine (a nonselective muscarinic antagonist), or methoctramine (a selective M2/M4 muscarinic receptor antagonist). Consistent with results in cultured cells, high potassium-evoked ACh release in hippocampal slices from young GRK5 knock-out mice was significantly reduced compared with wild type littermates, and this reduced ACh release was also fully corrected by methoctramine. In addition, following treatment with the nonselective muscarinic agonist oxotremorine-M, M2, and M4 receptors underwent significantly reduced internalization in GRK5KO slices compared with wild type slices, as assessed by plasma membrane retention of receptor immunoreactivity, whereas M1 receptor internalization was not affected by loss of GRK5 expression. Moreover, Western blotting revealed no synaptic or cholinergic degenerative changes in young GRK5 knock-out mice. Altogether, these results suggest that GRK5 deficiency leads to a reduced hippocampal ACh release and cholinergic hypofunction by selective impairment of desensitization of presynaptic M2/M4 autoreceptors. Because this nonstructural cholinergic hypofunction precedes the hippocampal cholinergic hypofunction associated with structural cholinergic degeneration and cognitive decline in aged GRK5 knock-out mice, this nonstructural alteration may be an early event contributing to cholinergic degeneration in AD. G protein-coupled receptor kinase-5 (GRK5) 2The abbreviations used are: GRKG protein-coupled receptor kinaseADAlzheimer diseaseAChacetylcholinemAChRmuscarinic acetylcholine receptorWTwild typePTXpertussis toxinoxo-Moxotremorine-Mdndominant negativeGFPgreen fluorescent proteinhrhumanized RenillaMCSmultiple cloning siteRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenaseHACThigh affinity choline transporterChATcholine acetyltransferaseVAChTvesicular acetylcholine transporterICCimmunocytochemistryCIcholinesterase inhibitor. is one of the seven GRK family members whose primary function is to desensitize G protein-coupled receptors (GPCRs) (1.Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (359) Google Scholar, 2.Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1068) Google Scholar). We recently reported that increased soluble β-amyloid decreases membrane (functional) levels of GRK5 in vitro, and this membrane GRK5 deficiency occurs in vivo as well in an Alzheimer disease (AD) transgenic model (3.Suo Z. Wu M. Citron B.A. Wong G.T. Festoff B.W. J. Neurosci. 2004; 24: 3444-3452Crossref PubMed Scopus (65) Google Scholar) and in postmortem human AD brain samples (4.Suo Z. Wu M. Citron B.A. Wong G.T. Festoff B.W. Neurobiol. Aging. 2005; 25: S2-291Google Scholar). Moreover, the aged GRK5 knock-out (GRK5KO) mouse, which models this GRK5 deficiency in the absence of exogenous mutant human β-amyloid precursor protein (β-APP) or any other known AD-related genes (i.e. presenilins or tau), develops axonal defects and mild cholinergic degeneration with associated amnestic mild cognitive impairment (5.Suo Z. Cox A.A. Bartelli N. Rasul I. Festoff B.W. Premont R.T. Arendash G.W. Neurobiol. Aging. 2007; 28: 1873-1888Crossref PubMed Scopus (56) Google Scholar). When Swedish mutant βAPP is overexpressed in the GRK5KO mice by cross-breeding with Swedish APP transgenic mice, the aged double mutant mice display significantly exaggerated brain inflammation (6.Li L. Liu J. Suo W.Z. J. Neuroinflammation. 2008; 5: 24Crossref PubMed Scopus (20) Google Scholar). These accumulating data strongly suggest that GRK5 deficiency significantly contributes to AD pathogenesis, although the precise molecular mechanisms remain to be delineated. G protein-coupled receptor kinase Alzheimer disease acetylcholine muscarinic acetylcholine receptor wild type pertussis toxin oxotremorine-M dominant negative green fluorescent protein humanized Renilla multiple cloning site reverse transcription glyceraldehyde-3-phosphate dehydrogenase high affinity choline transporter choline acetyltransferase vesicular acetylcholine transporter immunocytochemistry cholinesterase inhibitor. Mounting evidence indicates that the substrate spectrum of broadly expressed GRKs (i.e. GRK2/3/5/6) can significantly overlap for some receptors, suggesting that a lack of one of these members may have only a limited impact on GPCR regulation (2.Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1068) Google Scholar). On the other hand, compensation for loss of a particular GRK member by others in vivo can be incomplete or selective for other receptor types. For example, GRK2KO and GRK6KO mice have been shown to display selective impairments of adrenergic and dopaminergic receptor desensitization, respectively (7.Jaber M. Koch W.J. Rockman H. Smith B. Bond R.A. Sulik K.K. Ross Jr., J. Lefkowitz R.J. Caron M.G. Giros B. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12974-12979Crossref PubMed Scopus (285) Google Scholar, 8.Gainetdinov R.R. Bohn L.M. Sotnikova T.D. Cyr M. Laakso A. Macrae A.D. Torres G.E. Kim K.M. Lefkowitz R.J. Caron M.G. Premont R.T. Neuron. 2003; 38: 291-303Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Findings from different GRK isoform-targeted animals strongly support the conclusion that although redundancy exists between GRK isoforms, each isoform has its own selective substrates; should one GRK be deficient or inactivated, desensitization of its selective substrates will be impaired (1.Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (359) Google Scholar). For GRK5 in particular, previous studies have demonstrated that GRK5KO mice display selectively impaired desensitization of muscarinic acetylcholine receptors (mAChRs) (9.Gainetdinov R.R. Bohn L.M. Walker J.K. Laporte S.A. Macrae A.D. Caron M.G. Lefkowitz R.J. Premont R.T. Neuron. 1999; 24: 1029-1036Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 10.Walker J.K. Gainetdinov R.R. Feldman D.S. McFawn P.K. Caron M.G. Lefkowitz R.J. Premont R.T. Fisher J.T. Am. J. Physiol. Lung Cell Mol. Physiol. 2004; 286: L312-319Crossref PubMed Scopus (48) Google Scholar). To date, five mAChR subtypes have been identified, with M1, M3, and M5 receptors being Gq/11-coupled, and M2 and M4 receptors being Gi/o-coupled (11.Matsui M. Yamada S. Oki T. Manabe T. Taketo M.M. Ehlert F.J. Life Sci. 2004; 75: 2971-2981Crossref PubMed Scopus (71) Google Scholar). In hippocampal memory circuits, M2 receptor (M2R) is primarily a presynaptic autoreceptor that inhibits ACh release (12.Levey A.I. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13541-13546Crossref PubMed Scopus (303) Google Scholar, 13.Zhang W. Basile A.S. Gomeza J. Volpicelli L.A. Levey A.I. Wess J. J. Neurosci. 2002; 22: 1709-1717Crossref PubMed Google Scholar), whereas M1R is postsynaptic and is believed to be critical in memory processes involving an interaction between the cerebral cortex and hippocampus (11.Matsui M. Yamada S. Oki T. Manabe T. Taketo M.M. Ehlert F.J. Life Sci. 2004; 75: 2971-2981Crossref PubMed Scopus (71) Google Scholar). In AD, there is a selective loss of cholinergic neurons that leads to a cholinergic hypofunction, primarily a hypoactivity of postsynaptic nicotinic and M1 muscarinic receptors (14.Terry Jr., A.V. Buccafusco J.J. J. Pharmacol. Exp. Ther. 2003; 306: 821-827Crossref PubMed Scopus (872) Google Scholar). GRK5KO mice, when challenged with nonselective muscarinic agonists, display augmented hypothermia, hypoactivity, tremor, and salivation, as well as antinociceptive changes (9.Gainetdinov R.R. Bohn L.M. Walker J.K. Laporte S.A. Macrae A.D. Caron M.G. Lefkowitz R.J. Premont R.T. Neuron. 1999; 24: 1029-1036Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). These behavioral changes are typical M2 and/or M4 receptor-mediated functions, according to the findings from muscarinic receptor subtype knock-out mice (11.Matsui M. Yamada S. Oki T. Manabe T. Taketo M.M. Ehlert F.J. Life Sci. 2004; 75: 2971-2981Crossref PubMed Scopus (71) Google Scholar, 15.Wess J. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 423-450Crossref PubMed Scopus (324) Google Scholar). Therefore, GRK5 deficiency in vivo may selectively impair M2/M4R desensitization. If so, the resulting presynaptic M2/M4R hyperactivity would overly inhibit ACh release from cholinergic neurons and eventually compromise the learning and memory function. This study was undertaken to investigate the impact of GRK5 deficiency on ACh release and desensitization of mAChR subtypes using GRK5-deficient models both in vitro and in hippocampal slices from the GRK5KO mice. [Methyl-3H]Choline chloride was from Amersham Biosciences. Fetal bovine serum was from Atlanta Biologicals (Norcross, GA). Pertussis toxin (PTX), methoctramine tetrahydrochloride (MT), atropine, Eserine, and oxotremorine-M (oxo-M) were purchased from Sigma. Other routine biochemical and cell culture reagents and supplies were purchased from Sigma, Invitrogen, or Fisher. The coding sequence of K215R dominant negative (dn) mutant bovine GRK5 cDNA in the previously constructed pRK5-GRK5K215R plasmid (16.Pitcher J.A. Fredericks Z.L. Stone W.C. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus Google Scholar) was used as a for with and a GRK5 reverse that the of the GRK5 and The the and were with and to with the humanized Renilla green fluorescent protein of the The of the was corrected by to the wild type The of both the and was to any Because of levels of in the cells, this study used to the with M2 The from and and to human both display that levels of expression. The to in both and Therefore, and of genes of high levels in For the human cDNA of cDNA was with and multiple cloning site of the and from the were with and the of For the the was with and the of The of the was are a from Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U.S.A. 1998; PubMed Scopus Google Scholar). The were cultured in with bovine and as previously Z. Wu M. Citron B.A. Festoff B.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). was using of plasmid and with reagents according to the was to the for of the were and with of cloning The were in the culture to the of and that of the were used for this For the were as previously Z. Wu M. Citron B.A. Festoff B.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), any were The levels of were by was from the and using and by was used to cDNA using of bovine or was for of for for and for by a The were on and the were using an one The were shown in of used for sequence sequence of bovine of of bovine of in a This was in both cultured and hippocampal a of 5 in were for as previously Z. Wu M. Citron B.A. Festoff B.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The were with chloride a of for This of the selective of choline high affinity choline transporter Z. S. J. PubMed Scopus Google Scholar). the the were by high in the of of the ACh M. A. J. Pharmacol. Exp. Ther. 1999; Google for with as for of release. The was for previously the in W. Basile A.S. Gomeza J. Volpicelli L.A. Levey A.I. Wess J. J. Neurosci. 2002; 22: 1709-1717Crossref PubMed Google Scholar, J. L. Suo W.Z. Life Sci. PubMed Scopus Google Scholar). Therefore, the the release was used to the release in to a For the impact of the were with the in the absence or of high other as muscarinic were with the high For the brain slices, from and mice were and slices with a with the hippocampal slices were to with and for with The was by of the slices for in the chloride with the the slices were to and with the for For the of the muscarinic receptors, one of the slices was with a for by a with the For high the was In addition, was used with high and M2 to of the ACh M. A. J. Pharmacol. Exp. Ther. 1999; Google Scholar). samples were with treatment for line and the of the the hippocampal slices were with an for protein and the for The for of the samples was by For cultured cells, the membrane and were as previously L. Rasul I. Liu J. B. Premont R.T. Suo W.Z. PubMed Scopus Google Scholar). the were in and for the was as the The was in and for the was as the membrane The of brain membrane was according to a previously M. S. J. Neurosci. PubMed Google Scholar), with In the brain slices were in of 5 a of and by for 5 to the The was for to the The was with of by for to the The were with with and for routine of Western For the cells, or were with the For the mice, were to for the which is other protein protein Western and were as previously (5.Suo Z. Cox A.A. Bartelli N. Rasul I. Festoff B.W. Premont R.T. Arendash G.W. Neurobiol. Aging. 2007; 28: 1873-1888Crossref PubMed Scopus (56) Google Scholar, Z. Wu M. Citron B.A. Festoff B.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Z. Wu M. Citron B.A. Festoff B.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The only was the for the different primary which neuronal associated M1 and M4 M2 acetylcholine choline acetyltransferase vesicular acetylcholine transporter and The a of were with in for by and with as previously Z. Wu M. Citron B.A. Festoff B.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Z. Wu M. S. Citron B.A. Festoff B.W. J. 2002; PubMed Scopus Google Scholar). data are expressed as the and by of using of were using or are hippocampal neuronal precursor We have recently that the neurons cholinergic neuronal J. L. Suo W.Z. Life Sci. PubMed Scopus Google Scholar). To investigate impact of GRK5 deficiency on desensitization of muscarinic receptors, we an in vitro model in by of K215R mutant of the has been previously used to inhibit GRK5 as to GRK5 deficiency in vitro (16.Pitcher J.A. Fredericks Z.L. Stone W.C. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). of in cells, we human cDNA the of and bovine cDNA on its with of the In the or were to the of and were used as and these cell will be as and were using Western and The revealed that the levels of exogenous bovine and were whereas the GRK5 in the was that in the a the levels of and were and GRK5 in cell Western data that the exogenous and as well as were expressed in levels in cell of other cholinergic of as and were also to be the cell the primary cell membrane of both and GRK5 primarily (1.Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (359) Google Scholar), which is in to the of the and In addition, with an revealed a of from the the to the and which is with the known and of of axonal B.A. Neurosci. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. Neurosci. PubMed Scopus Google Scholar). In particular, axonal the was to be with or and These results suggest that although the and cell have different and GRK5 cholinergic are and are in vitro models for impact of GRK5 hypofunction on muscarinic the in vitro models were we high ACh release from these cell We that and on release with the significantly decreased and the significantly increased compared with the When these were with a cell in ACh release for as compared with but the the cell In to treatment with a nonselective muscarinic atropine and a selective M2/M4R methoctramine each in significantly increased ACh release and also the the cell and These results suggest that the of GRK5 in these cell leads to ACh which is by an function of G protein-coupled muscarinic M2 and/or M4 To the impact of GRK5 function on internalization of we the to a of (a nonselective muscarinic 5 for and membrane retention of M1, M2 and M4 receptors by Western We that M1 receptor on the membrane in the and was the membrane M2 and M4 receptors were significantly in the for both M2 and but in the for M2 and for as compared with in the In addition, we also that following M2 and but not M1, were in the and in the as compared with in the not which is with the membrane retention of M2 and M4 in the and the membrane M2 and M4 in the these results suggest that overexpression of impaired the internalization of M2 and M4 but not M1 receptors, whereas overexpression of the of in vitro and in vivo that of GRK regulation of GPCR substrates is a function of R.T. Gainetdinov R.R. Annu. Rev. Physiol. 2007; PubMed Scopus Google Scholar). Therefore, to the of in vitro we also in an vivo using hippocampal from young GRK5KO mice. hippocampal from GRK5KO and were used for Western blotting to levels of cholinergic and synaptic in these animals are The acetylcholine and the decreased levels of some of these that we have reported in aged GRK5KO mice (5.Suo Z. Cox A.A. Bartelli N. Rasul I. Festoff B.W. Premont R.T. Arendash G.W. Neurobiol. Aging. 2007; 28: 1873-1888Crossref PubMed Scopus (56) Google Scholar), young GRK5KO mice levels of both cholinergic and synaptic as compared with These data suggest that the hippocampal cholinergic in GRK5KO mice is the structural and can be to in cholinergic function of structural To cholinergic we used high release from hippocampal We that the ACh release from GRK5KO hippocampal slices was from slices and that the M2/M4R methoctramine not only increased ACh release from both the and GRK5KO but also fully corrected the between GRK5KO and Moreover, when the hippocampal slices were with a of the nonselective muscarinic agonist we that the in high release between the and GRK5KO of increased ACh release from the but not from the GRK5 knock-out When with high ACh release from the slices not compared with but the ACh release from GRK5KO increased significantly compared with to the to that in the In to ACh we also the by the and GRK5KO hippocampal slices, and we no between suggesting that the in ACh release between the and GRK5KO be to an alteration of choline Because hippocampal M2/M4 receptors are primarily presynaptic that inhibit ACh release (12.Levey A.I. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13541-13546Crossref PubMed Scopus (303) Google Scholar, 13.Zhang W. Basile A.S. Gomeza J. Volpicelli L.A. Levey A.I. Wess J. J. Neurosci. 2002; 22: 1709-1717Crossref PubMed Google Scholar), these results suggest that the GRK5 deficiency leads to a reduction of hippocampal ACh release an impaired desensitization of M2 and/or M4 To investigate the of GRK5 on in we also membrane retention of M1, M2, and M4 receptors treatment with a of in hippocampal treatment with for the slices were to and and Western revealed that there were no for and between the and GRK5KO in any but that the membrane significantly retention in the GRK5KO for as compared with the and as compared with the but not for M1R These results suggest that the GRK5 deficiency in vivo the internalization of and to a but has no on M1 Because of a receptor leads both to receptor desensitization from G protein and to receptor from the cell the mechanism receptor with (1.Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (359) Google Scholar, R.T. Gainetdinov R.R. Annu. Rev. Physiol. 2007; PubMed Scopus Google Scholar), we used receptor internalization as a for receptor desensitization. This study an in vitro cholinergic neurons that and an vivo hippocampal slices from GRK5KO mice, to GRK5 deficiency and of the GRK5 deficiency on ACh release and desensitization of M1, M2, and M4 muscarinic In the in vitro the K215R was overexpressed to with the to a GRK5 deficiency (16.Pitcher J.A. Fredericks Z.L. Stone W.C. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). a this GRK5 deficiency led to reduced ACh which be corrected by muscarinic receptor of and by muscarinic receptors or M2/M4 receptors Moreover, the GRK5 deficiency also in an impaired internalization of M2 and M4 but not M1 receptors, in to treatment with the nonselective agonist is known that and function as presynaptic in hippocampal cholinergic neurons to inhibit ACh release (12.Levey A.I. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13541-13546Crossref PubMed Scopus (303) Google Scholar, 13.Zhang W. Basile A.S. Gomeza J. Volpicelli L.A. Levey A.I. Wess J. J. Neurosci. 2002; 22: 1709-1717Crossref PubMed Google Scholar). In addition, of these receptors leads to the receptor desensitization and internalization by GRKs or other (1.Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (359) Google Scholar, 2.Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1068) Google Scholar). Therefore, the impaired internalization of M2/M4R in the indicates that a of fully GRK5 is for the desensitization and internalization of M2/M4 receptors in these these in vitro results suggest that the GRK5 deficiency in the hippocampal cholinergic neuronal model leads to decreased ACh release a selective impairment of M2/M4 receptor on the in vitro we to of the GRK5 deficiency on ACh release and desensitization of M1, M2, and M4 receptors in an vivo hippocampal slices from GRK5KO mice. GRK5KO mice mild cholinergic and synaptic degenerative changes (5.Suo Z. Cox A.A. Bartelli N. Rasul I. Festoff B.W. Premont R.T. Arendash G.W. Neurobiol. Aging. 2007; 28: 1873-1888Crossref PubMed Scopus (56) Google Scholar). Therefore, of structural is to the cholinergic between aged and GRK5KO mice. In the GRK5KO mice, indicates that these young levels of cholinergic and synaptic as compared with and these mice are for of cholinergic the hippocampal slices from these young we a reduction in ACh release in to high in the GRK5KO mice. Consistent with the in vitro the reduced ACh release was fully corrected by the M2/M4 receptor methoctramine. In we the muscarinic receptors with a of high and that slices significantly ACh of reduced autoreceptor of but that the ACh release in the GRK5KO mice with the between the and GRK5KO was corrected by methoctramine when the slices were we retention of membrane and membrane in the GRK5KO samples compared with with a to receptor These results strongly suggest that GRK5 deficiency selectively M2 and M4 receptor both in vitro and in and the of this impaired desensitization of presynaptic M2/M4 receptors is M2/M4 receptor which leads to reduced ACh release and an hippocampal cholinergic hypofunction is one of the changes in AD. is that the to reduced cholinergic associated with cholinergic neuronal degeneration or postsynaptic M1 and nicotinic degenerative changes (14.Terry Jr., A.V. Buccafusco J.J. J. Pharmacol. Exp. Ther. 2003; 306: 821-827Crossref PubMed Scopus (872) Google Scholar). Here we a presynaptic cholinergic hypofunction in the absence of degenerative We have previously that the aged GRK5KO mice mild cholinergic and synaptic degenerative changes (5.Suo Z. Cox A.A. Bartelli N. Rasul I. Festoff B.W. Premont R.T. Arendash G.W. Neurobiol. Aging. 2007; 28: 1873-1888Crossref PubMed Scopus (56) Google Scholar). In this we that the hippocampal cholinergic hypofunction can structural degenerative and this hippocampal cholinergic hypofunction structural degeneration in young mice precedes hypofunction with structural degeneration in aged GRK5KO mice. Therefore, the of the is a of the We have previously shown that GRK5 deficiency is associated with soluble β-amyloid in AD (4.Suo Z. Wu M. Citron B.A. Wong G.T. Festoff B.W. Neurobiol. Aging. 2005; 25: S2-291Google Scholar). we that the hippocampal cholinergic hypofunction results from GRK5 Moreover, the hippocampal cholinergic hypofunction in the GRK5KO mice can be corrected by a selective M2/M4 that M2/M4 may be in AD, in the of the cholinergic hypofunction in AD. the AD are cholinesterase that the ACh the synaptic the are to the ACh synaptic and the postsynaptic cholinergic the increased ACh should also on presynaptic to inhibit ACh release. The as demonstrated in this is in the of GRK5 as in AD. The presynaptic may to an to the were for and have on in cholinergic deficiency can to cholinergic Therefore, studies are to presynaptic M2/M4 hyperactivity and to a of ACh release and the of an M2/M4 receptor in to the an for the cholinergic hypofunction in AD. We J. Lefkowitz the GRK5KO and of this