Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Mar 24;30(12):4190-6.
doi: 10.1523/JNEUROSCI.6393-09.2010.

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

Albert A Davis  1 , Jason J Fritz, Jürgen Wess, James J Lah, Allan I Levey
Affiliations

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

Albert A Davis et al. J Neurosci. .

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.

PubMed Disclaimer

Figures

Figure 1.
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.
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.
Figure 3.
Amyloid pathology in M1+/+ and M1−/− APPSwe/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−/− APPSwe/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.
Figure 4.
A, B, Immunoblot analysis of APP metabolites in M1+/+ and M1−/− APPSwe/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−/− APPSwe/Ind cortex. Levels of both proteins were unchanged in M1+/+ compared to M1−/− (data shown as mean ± SD).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alzheimer's Association. 2009 Alzheimer's disease facts and figures. Alzheimers Dement. 2009;5:234–270. - PubMed
    1. Anagnostaras SG, Murphy GG, Hamilton SE, Mitchell SL, Rahnama NP, Nathanson NM, Silva AJ. Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice. Nat Neurosci. 2003;6:51–58. - PubMed
    1. Beach TG, Potter PE, Kuo YM, Emmerling MR, Durham RA, Webster SD, Walker DG, Sue LI, Scott S, Layne KJ, Roher AE. Cholinergic deafferentation of the rabbit cortex: a new animal model of Abeta deposition. Neurosci Lett. 2000;283:9–12. - PubMed
    1. Beach TG, Kuo YM, Schwab C, Walker DG, Roher AE. Reduction of cortical amyloid beta levels in guinea pig brain after systemic administration of physostigmine. Neurosci Lett. 2001a;310:21–24. - PubMed
    1. Beach TG, Walker DG, Potter PE, Sue LI, Fisher A. Reduction of cerebrospinal fluid amyloid beta after systemic administration of M1 muscarinic agonists. Brain Res. 2001b;905:220–223. - PubMed

Publication types

MeSH terms

LinkOut - more resources