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) 2 is one of the seven GRK family members whose primary function is to desensitize G protein-coupled receptors (GPCRs) (1, 2). We recently reported that increased soluble ␤-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) and in postmortem human AD brain samples (4). Moreover, the aged GRK5 knock-out (GRK5KO) mouse, which models this GRK5 deficiency in the absence of exogenous mutant human ␤-amyloid precursor protein (␤-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). When Swedish mutant ␤APP 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). These accumulating data strongly suggest that GRK5 deficiency significantly contributes to AD pathogenesis, although the precise molecular mechanisms remain to be delineated.Mounting evidence indicates that the substrate spectrum of broadly expressed GRKs (i.e. GRK2/3/5/6) can significantly overlap for som...

GRK5 Deficiency Accelerates β-Amyloid Accumulation in Tg2576 Mice via Impaired Cholinergic Activity

“…As we have recently demonstrated, selective impairment of presynaptic M2 receptor desensitization in young GRK5KO mice causes prolonged or persistent M2 signaling, which leads to reduced ACh release and cholinergic hypofunction without any evident structural degeneration (17). In aged GRK5KO animals, however, there are significant axonal defects and synaptic degenerative changes in cholinergic projection areas, such as hippocampus, piriform cortex, amygdaloid, and anterior olfactory nuclei.…”

“…For GRK5, studies using GRK5 knock-out (GRK5KO) mice have demonstrated that one selective substrate is the family of muscarinic acetylcholine (ACh) receptors (15,16). Moreover, the selectivity of GRK5 for muscarinic ACh receptors also appears to be subtype-specific for the G i -coupled M2/M4 subtypes, rather than the G q -coupled M1 receptors (15,17).…”

“…Once GRK5 deficiency occurs, it selectively impairs desensitization of presynaptic M2 receptors and inhibits ACh release (17). On the other hand, many studies have shown that reduced postsynaptic cholinergic activity affects APP processing in favor of the ␤-amyloidogenic pathway (6 -10).…”

Atypical Actions of G Protein-Coupled Receptor Kinases

“…The M2 muscarinic receptor is located presynapse in the hippocampus, and regulates acetylcholine release. In GRK5 knockout mice, presynaptic autoregulation by the M2 muscarinic receptor was abolished, and it caused prolonged and persistent activation (Liu et al, 2009). Furthermore, the combination of a M2 muscarinic receptor blocker and a cholinesterase inhibitor corrected β-amyloid accumulation (Cheng et al, 2010).…”

Disruption of dopamine D1 receptor phosphorylation at serine 421 attenuates cocaine-induced behaviors in mice

“…However, the in vivo role of GRKs in D1R signaling in drug addiction remains to be determined [12,19,20] . For example, although GRK6 seems to be the most prominent GRK in both the dorsal and ventral striatum, observations in mice lacking GRK6 indicate that the D2R is its physiological target [ 21,22] .…”