Deletion of M1 muscarinic acetylcholine receptors increases amyloid pathology in vitro and in vivo

Abstract

Alzheimer's disease (AD) is a progressive neurological disorder that causes dementia and poses a major public health crisis as the population ages. Aberrant processing of the amyloid precursor protein (APP) is strongly implicated as a proximal event in AD pathophysiology, but the neurochemical signals that regulate APP processing in the brain are not completely understood. Activation of muscarinic acetylcholine receptors (mAChRs) has been shown to affect APP processing and AD pathology, but less is known about the roles of specific mAChR subtypes. In this study, we used M(1) mAChR knock-out mice (M(1)KO) to isolate the effects of the M(1) mAChR on APP processing in primary neurons and on the development of amyloid pathology in a transgenic mouse model of AD. We demonstrate that the loss of M(1) mAChRs increases amyloidogenic APP processing in neurons, as evidenced by decreased agonist-regulated shedding of the neuroprotective APP ectodomain APPsalpha and increased production of toxic Abeta peptides. Expression of M(1) mAChRs on the M(1)KO background rescued this phenotype, indicating that M(1) mAChRs are sufficient to modulate nonamyloidogenic APP processing. In APP(Swe/Ind) transgenic mice, the loss of M(1) mAChRs resulted in increased levels of brain Abeta and greater accumulation of amyloid plaque pathology. Analysis of APP metabolites in APP(Swe/Ind) brain tissue indicates that the loss of M(1) mAChRs increases amyloidogenic APP processing. These results indicate that the M(1) mAChR is an important regulator of amyloidogenesis in the brain and provide strong support for targeting the M(1) mAChR as a therapeutic candidate in AD.

Figure 1.

M₁ mAChR regulation of APP processing in primary neurons. A, Western blot analysis of cell lysates from primary cortical neuron cultures derived from wild-type (WT) and M₁ mAChR knock-out (M₁KO) mice and transduced with a lentiviral vector encoding human sequence amyloid precursor protein (hAPP). A virus encoding green fluorescent protein was used as a control. The human-specific APP antibody 6E10 was used to detect expression of hAPP, and total APP (human plus endogenous murine) was detected using C8 (a C-terminal APP antibody). β-Actin is shown as a loading control. B, D, Representative Western blots and densitometric analysis of conditioned media and cell lysates from WT neuron cultures treated with the mAChR agonist CCh (100 μm). Quantitation of band intensity shows a 66% increase in APPsα shedding into conditioned media of WT neuron cultures treated with CCh (p < 0.05 vs vehicle), and a 68% increase in the level of CTFα (p < 0.01). Data are shown as mean ± SEM from three to five independent experiments. C, E, Western blots and densitometry from conditioned media and cell lysates from M₁KO neuron cultures. APPsα and CTFα levels were unchanged in cultures treated with CCh. In M₁KO neuron cultures transduced with an M₁ mAChR lentivirus, CCh treatment resulted in a 44% increase in APPsα shedding into conditioned media (p < 0.05), and an 88% increase in the levels of CTFα (p < 0.05). Data are shown as mean ± SEM of three independent experiments. F, G, Western blot and densitometric analysis demonstrates no change in levels of ADAM10 (p = 0.16), ADAM17 (p = 0.16), or BACE1 (p = 0.34) with CCh treatment in WT neuron cultures. Data are shown as the mean ± SD.

Figure 2.

Aβ peptide levels in conditioned media from WT and M₁KO primary neuron cultures. In WT neurons, CCh treatment (100 μm) resulted in a trend toward decreased Aβ production, although this difference did not reach statistical significance. In M₁KO neurons, CCh treatment caused an increase in Aβ production (p < 0.05 vs vehicle). Following rescue of M₁KO neuron cultures by transduction with an M₁ lentivirus, CCh treatment resulted in a significant reduction in Aβ levels compared to CCh-treated M₁KO neurons (p < 0.01). Data are shown as mean ± SEM from three independent experiments.

Figure 3.

Amyloid pathology in M₁^+/+ and M₁^−/− APP_Swe/Ind mice. A, Total number of thioflavin S-positive plaques is significantly increased at 16 months of age in M₁^−/− mice (mean ± SD, p < 0.01). B, Amyloid plaque density (mean surface area of Aβ₄₂-positive immunoreactivity (pixels) per tissue section) is significantly increased in the cortex of 16-month-old M₁^−/− mice (mean ± SD, p < 0.05). C, Light micrographs of Aβ₄₂-immunopositive plaques (brown deposits) in M₁^+/+ and M₁^−/− APP_Swe/Ind mice at 12 and 16 months of age. High-power magnification corresponding to boxed regions of cortex (a) and hippocampus (b) are shown as insets.

Figure 4.

A, B, Immunoblot analysis of APP metabolites in M₁^+/+ and M₁^−/− APP_Swe/Ind cortex. Membrane and soluble proteins from cortical homogenates of M₁^+/+ (n = 7) and M₁^−/− (n = 6) mice were fractionated by differential centrifugation and subjected to SDS-PAGE and Western blotting with antibodies to multiple APP metabolites. Representative immunoblots are shown probed with 6E10 to detect full-length APP, APPsα, and CTFβ, 192swe to detect the Swedish mutation form of APPsβ, and C8 to detect both CTFα and CTFβ. EF1α is shown as a loading control. For quantitation, APPs levels were normalized to EF1α, and CTF levels were normalized to full-length APP. Densitometric quantitation revealed a 57% increase in CTFβ in M₁^−/− mice (mean ± SD, p < 0.05). C, D, Immunoblot analysis of ADAM10 and ADAM17 in M₁^+/+ and M₁^−/− APP_Swe/Ind cortex. Levels of both proteins were unchanged in M₁^+/+ compared to M₁^−/− (data shown as mean ± SD).