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.

Membrane G protein-coupled receptor kinase 5 (GRK5) deficiency is linked to Alzheimer disease, yet its precise roles in the disease pathogenesis remain to be delineated. We have previously demonstrated that GRK5 deficiency selectively impairs desensitization of presynaptic M2 autoreceptors, which causes presynaptic M2 hyperactivity and inhibits acetylcholine release. Here we report that inactivation of one copy of Grk5 gene in transgenic mice overexpressing β-amyloid precursor protein (APP) carrying Swedish mutations (Tg2576 or APPsw) resulted in significantly increased β-amyloid (Aβ) accumulation, including increased Aβ⁺ plaque burdens and soluble Aβ in brain lysates and interstitial fluid (ISF). In addition, secreted β-APP fragment (sAPPβ) also increased, whereas full-length APP level did not change, suggesting an alteration in favor of β-amyloidogenic APP processing in these animals. Reversely, perfusion of methoctramine, a selective M2 antagonist, fully corrected the difference between the control and GRK5-deficient APPsw mice for ISF Aβ. In contrast, a cholinesterase inhibitor, eserine, although significantly decreasing the ISF Aβ in both control and GRK5-deficient APPsw mice, failed to correct the difference between them. However, combining eserine with methoctramine additively reduced the ISF Aβ further in both animals. Altogether, these findings indicate that GRK5 deficiency accelerates β-amyloidogenic APP processing and Aβ accumulation in APPsw mice via impaired cholinergic activity and that presynaptic M2 hyperactivity is the specific target for eliminating the pathologic impact of GRK5 deficiency. Moreover, a combination of an M2 antagonist and a cholinesterase inhibitor may reach the maximal disease-modifying effect for both amyloid pathology and cholinergic dysfunction.

Although mutations and variations of several genes have been identified to be involved in Alzheimer's disease (AD), the efforts towards understanding the pathogenic mechanisms of the disease still have a long journey to go. One such effort is to identify the signal transduction deficits, for which previous studies have suggested that the central problems appear to be at the interface between G proteins and their coupled receptors. G protein-coupled receptor kinases (GRKs) are a small family of serine/threonine protein kinases primarily acting at the “receptor-G protein interface””. Recent studies have indicated the possible involvement of GRK, primarily GRK2 and GRK5, dysfunction in the pathogenesis of AD. It seems that mild, soluble, β-amyloid accumulation can lead to a reduced membrane (functional) and an elevated cytosolic GRK2/5. The increased cytosolic GRK2 appears to be colocalized with damaged mitochondria and neurofibrillary tangles. Moreover, the total levels of GRK2, not only in the brain, but also in peripheral blood samples, are increased in a manner inversely correlated with the patient's cognitive levels. The deficiency of GRK5, on the other hand, impairs presynaptic M2 autoreceptor desensitization, which leads to a reduced acetylcholine release, axonal/synaptic degenerative changes, and associated amnestic, mild cognitive impairment. It also promotes an evil cycle to further increase Beta-amyloid accumulation and exaggerates brain inflammation, possibly even the basal forebrain cholinergic degeneration. Therefore, continuous efforts in this direction are necessary before translating the knowledge to any therapeutic strategies.

Why certain diseases primarily affect one specific neuronal subtype rather than another is a puzzle whose solution underlies the development of specific therapies. Selective basal forebrain cholinergic (BFC) neurodegeneration participates in cognitive impairment in Alzheimer’s disease (AD), yet the underlying mechanism remains elusive. Here, we report the first recapitulation of the selective BFC neuronal loss that is typical of human AD in a mouse model termed GAP. We created GAP mice by crossing Tg2576 mice that over-express the Swedish mutant human β-amyloid precursor protein gene with G protein-coupled receptor kinase-5 (GRK5) knockout mice. This doubly defective mouse displayed significant BFC neuronal loss at 18 months of age, which was not observed in either of the singly defective parent strains or in the wild type. Along with other supporting evidence, we propose that GRK5 deficiency selectively renders BFC neurons more vulnerable to degeneration.

Prevention of Alzheimer’s disease (AD) is a high priority mission while searching for a disease modifying therapy for AD, a devastating major public health crisis. Clinical observations have identified a prodromal stage of AD for which the patients have mild cognitive impairment (MCI) though do not yet meet AD diagnostic criteria. As an identifiable transitional stage before the onset of AD, MCI should become the high priority target for AD prevention, assuming successful prevention of MCI and/or its conversion to AD also prevents the subsequent AD. By pulling this string, one demonstrated cause of amnestic MCI appears to be the deficiency of G protein-coupled receptor-5 (GRK5). The most compelling evidence is that GRK5 knockout (GRK5KO) mice naturally develop into aMCI during aging. Moreover, GRK5 deficiency was reported to occur during prodromal stage of AD in CRND8 transgenic mice. When a GRK5KO mouse was crossbred with Tg2576 Swedish amyloid precursor protein transgenic mouse, the resulted double transgenic GAP mice displayed exaggerated behavioral and pathological changes across the spectrum of AD pathogenesis. Therefore, the GRK5 deficiency possesses unique features and advantage to serve as a prophylactic therapeutic target for MCI due to AD.