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.

FIGURE 1.

Construction and expression of dnGRK5GFP and M2 in HT22 cells. A, schematic illustration of the plasmids that contain hrGFP, bovine dnGRK5GFP, or wtGRK5GFP cDNA inserts at MCS1 and M2 cDNA at MCS2 of pVITRO1. B, semi-quantitative RT-PCR and Western blot characterization of dnGRK5GFP and wtGRK5GFP expression in the HT22 cells stably transfected with pVITRO1-GFP-M2, pVITRO1-dnGRK5GFP-M2, and pVITRO1-wtGRK5GFP-M2 (abbreviated as GFP, DN, and WT, respectively). bGRK5 refers to exogenous bovine GRK5; mGRK5 refers to intrinsic murine GRK5. GAPDH was used as an internal reference for RT-PCR. C, Western blot characterization of cholinergic markers (ChAT, HACT, M1, M2, and M4), in the GFP, dnGRK5, and wtGRK5 transfected cells. Actin was used as an internal control.

FIGURE 2.

Membrane localization of GRK5GFP fusion proteins and their colocalization with M2 at synaptic terminals. A, D, and G, representative images of the expression and subcellular distribution of GFP, dnGRK5GFP, and wtGRK5GFP fusions in the GFP, dnGRK5, and wtGRK5 cell lines, respectively. B, E, and H, representative images of ICC staining with antibody to M2 (red) in the GFP, dnGRK5, and wtGRK5 cell lines, respectively. C, F, and I, merged panels for GFP imaging and M2 ICC staining in the GFP, dnGRK5, and wtGRK5 cell lines, respectively. The insets in C, F, and I show the high power views of the overlap of GFP imaging and M2 ICC staining at synaptic terminals as indicated by the arrowheads. 4′,6-Diamidino-2-phenylindole (blue) stains all nuclei. The scale bar in A is for all panels (30 μm).

FIGURE 3.

Effects of PTX and muscarinic antagonists on high potassium-evoked [³H]ACh release from the GRK5 deficient HT22 cells. A, high K⁺-evoked [³H]ACh release in the GFP, dnGRK5, and wtGRK5 cells. A single optimal concentration of potassium (50 mm) was used to evoke the [³H]ACh release (n ≥ 3). *, p < 0.05, as compared with the GFP cells. B–D, show the effects of PTX (100 ng/ml), atropine (1 μm), and MT (100 nm) on the ACh release in the GFP, dnGRK5, and wtGRK5 cells, respectively. **, p < 0.001, as compared with the potassium-alone treated vehicles.

FIGURE 4.

Membrane retention of M1, M2, and M4 receptors in the GRK5 deficient HT22 cells. The dnGRK5 cells, along with the GFP and wtGRK5 control cells, were treated with a saturation concentration of oxo-M (5 mm) before the membrane proteins were separated for Western blotting analysis. A, representative Western blots for M1, M2, and M4 receptors in the membrane fraction in the presence and absence of the saturated oxo-M challenge. B, the semi-quantification of the Western blotting results. The data were expressed as the percentage of the receptors remained in the membrane after the oxo-M treatment (the treated divided by the untreated) (n ≥ 3). Separate one-way analysis of variance for M1, M2, and M4, respectively, revealed significant membrane retention of M2 and M4 (p < 0.01 for both), but not M1, in the GRK5-deficient dnGRK5 cells. In the contrast, the wtGRK5-overexpressing cells displayed significant less membrane retention of M2 (p < 0.001) and M4 (p < 0.05), but not M1, as compared with the GFP control cells. *, p < 0.05; **, p < 0.01, and p < 0.001, as compared with the corresponding GFP controls.

FIGURE 5.

Expression of synaptic and cholinergic markers in the young adult GRK5KO mice. Levels of synaptic and cholinergic markers in the 3-month-old GRK5KO mouse brains were analyzed by Western blotting using antibodies against synaptophysin, synapsin II, synaptotagmin, SNAP-25, GAP-43, M1, M2, M4, acetylcholine esterase, ChAT, HACT, and VAChT. The representative blots were shown on the left, and the corresponding semi-quantitative results (n = 4) are plotted on the right. The results revealed no significant changes for any of the examined molecules, suggesting that the cholinergic system in these young animals is normal at the structural level.

FIGURE 6.

Reduced ACh release in the hippocampal slices of the young adult GRK5KO mice. The high K⁺-evoked [³H]ACh release from the hippocampal slices of 3-month-old GRK5^−/− (KO, n = 3) and GRK5^+/+ (WT, n = 5) mice were measured as described under “Experimental Procedures.” A, ACh release from the hippocampal slices of the young adult GRK5KO mice. The slices were separated into two parallel groups, with one group pretreated with a saturated concentration of carbachol (1 mm) to desensitize the muscarinic receptors, and with the other group as nontreated control. The ACh release was stimulated with high potassium (24 mm) in the presence and absence of 20 nm MT. We found that the GRK5KO mice displayed ∼40% (p < 0.05) and 65% (p < 0.01) less ACh release than the WT mice in the cases of without and with the predesensitization, respectively. In either case, however, coperfusion of MT eliminated the differences between the GRK5KO and WT. *, p < 0.05; **, p < 0.01, as compared with the corresponding K⁺-alone treated WT controls. B, the total HACT activity was estimated by measuring the total uptake of [³H] into the slices. The results revealed no difference between the GRK5KO and WT mice.

FIGURE 7.

Membrane retention of M1, M2, and M4 receptors in the young adult GRK5KO mice. Cortical slices from the 3-month-old GRK5 KO mice were treated with the saturated carbachol (Carb, 1 mm) in the oxygenated Kreb's buffer at 37 °C for 20 min and then subjected to multi-step homogenization and centrifugations to isolate subcellular fractions, including pellets for nuclear (P1), mitochondria/microsome (P2), and crude membrane/synaptosome (P3). Equal amount (50 μg/lane) of proteins were analyzed by SDS-PAGE, followed by Western blotting. A, representative Western blots for M1, M2 and M4. B–D, semi-quantification (n = 3) for M1, M2, and M4, respectively. All of the data were standardized against the mean of the untreated membrane fraction, and the percentages over the P3 controls (Cont) were plotted as shown. *, p < 0.05; **, p < 0.001, as compared with the same fraction of the WT mice.