M1 Muscarinic Receptors—Muscling APP to a Better End?

The production of toxic Aβ peptides from amyloid precursor protein (APP) proceeds via the sequential cleavage of the extracellular domain by β-secretase, followed by an intramembrane cut by γ-secretase. However, if α-secretase finds APP first, it can lop off the β-secretase cleavage site, simultaneously blocking production of Aβ and releasing a neuroprotective soluble APP fragment. Activation of muscarinic acetylcholine receptors is known to steer APP down the non-amyloidogenic, α-secretase pathway, but it has been difficult to pin the effect on a specific receptor subtype: There are five different muscarinic receptors (M1-M5) and knowing which one is involved is critical for making selective drugs and minimizing cholinergic side effects. Allan Levey and colleagues at Emory University, Atlanta, Georgia, have now used knockout mice to show that the M1 receptor is responsible for regulating APP processing in vivo. Their work, published in the March 24 Journal of Neuroscience, offers support for the ongoing development of M1-selective agonists as modulators of amyloid load.

At the same time, a second paper raises the question of whether α-secretase processing of APP is as benign as assumed. Ratnesh Lal, University of California, San Diego, and Ruth Nussinov, National Cancer Institute, Frederick, Maryland, present evidence that the small peptide derived from α- then γ-secretase cleavage forms ion channels in cells, reminiscent of pore-forming channels proposed for Aβ peptides. At high concentrations the peptide (known as P3) is toxic to neuronal cells in culture, apparently by causing calcium leakage. That work appears in this week’s edition of PNAS online.

The muscarinic acetylcholine receptor has been a target for AD therapies for decades, based first on the loss of acetylcholine in the disease and the role of M1 receptors in memory and cognition, and then on the finding that the receptors control the processing of amyloid precursor protein to Aβ peptides. Attempts to tweak muscarinic receptors to modify APP processing pointed to M1 as the main regulator, and selective agonists of the M1-type receptor have been shown to reduce amyloid and tau pathology in a mouse model of AD (see ARF related news story on Caccamo et al., 2006). However, the compound used in that study (AF267B) was subsequently shown to also activate M3 receptors (Jones et al., 2008). In the new study, first author Albert Davis and coworkers use a mouse knockout to examine regulation of APP processing by M1. They show that treating cultured mouse neurons expressing human APP with the acetylcholine receptor agonist carbachol increased the production of α-secretase cleavage products, but no such effect occurred in cells from M1 knockout mice. Expressing M1 receptor in knockout cells restored carbachol-induced α-secretase processing.

To look at the effect in vivo, the researchers knocked out M1 in a mouse that overexpresses a mutated human APP. Without M1, the mice showed a three- to fivefold increase in Aβ content in the brain at 16 months of age, and a doubling in plaque number compared to M1-expressing animals. The researchers do not report whether cognition was negatively affected in the animals, either by M1 loss or enhanced amyloid accumulation.

The results suggest that an M1-specific agonist could be doubly beneficial in AD by making up for the loss of acetylcholine and by decreasing Aβ production. Selectivity is important on both counts: Non-specific muscarinic receptor agonists can produce side effects due to peripheral cholinergic effects including gastrointestinal disturbances, changes in blood pressure, and excessive sweating. In addition, Davis and coworkers show that treating neurons from M1 knockout mice with carbachol increased Aβ production, probably via stimulation of other non-M1 muscarinic receptor subtypes. Selective agonists have been hard to come by, although pharma is hard at work on the problem, with recent progress toward promising candidates that are both selective and possess the requisite pharmacokinetic properties for testing in animal models (see ARF related news story on Jones et al., 2008 and ARF related news story on Ma et al., 2009).

But is there be a twist to the story? The peptide products of sequential α- and γ-secretase processing (known as P3, spanning residues 17-40/42) are widely viewed as harmless. However, the Lal study suggests that P3 peptides share properties with their amyloidogenic relatives, including the ability to form channels in the cell membrane that allow calcium to leak into the cell. In fact, calling P3 “non-amyloidogenic” is a misnomer, Lal told ARF. The P3 peptide includes the transmembrane region of Aβ and can form amyloid fibrils (Pike et al., 1995). The new work, led by first authors Hyunbum Jang, Fernando Teran Arce, and Srinivasan Ramachandran, shows that the peptides form channel-like structures in vitro similar to those described for the Aβ peptides (Quist et al., 2005). When reconstituted into lipid bilayers, the peptides assemble into cation-selective channels, and in cells, they appear to mediate calcium influx. The peptides were toxic for neurons, but only at very high concentrations (100 μM). The physiological significance of the findings remains to be seen. While these peptides do appear as a component of amyloid plaques in AD and Down syndrome, it is not clear what concentrations they achieve and whether channel formation plays a role in amyloid toxicity in vitro. One recent report suggests the P3 peptides do not form synaptotoxic oligomers in the way that Aβ does (Dulin et al., 2008). Based on their results, however, Lal and colleagues caution that efforts to detoxify APP by steering processing toward these peptides may be a futile effort.—Pat McCaffrey

Comments

  1. The paper by Davis et al. adds further support to the major and pivotal role of the M1 muscarinic receptor (M1 AChR) in AD pathology and possible treatment with selective M1 agonists. It adds indirect support to our earlier findings that M1 agonists such as AF267B could be a highly promising and causal treatment in AD (Caccamo et al., 2006; Fisher, 2008). While this paper represents a major step towards our understanding in M1 AChR-mediated APP processing, I would like to address the following points:

    1. Carbachol in the M1 knockout mice increased Aβ production, probably via stimulation of other non-M1 muscarinic receptor subtypes. It can be speculated that this is not due to its agonistic activity on M3 AChR or some nicotinic receptor subtypes. Furthermore, since it appears that mainly the activation of the M1 AChR is responsible for the decrease in Aβ production, while the M2 and M4 AChR may have an opposite effect, this may explain why treatment with cholinesterase inhibitors lack an effect on Aβ production in AD patients. Notably, such treatments increase synaptic ACh that will interact with all cholinergic receptor subtypes.

    2. The role of M1 AChR on cognitive functions or effects on tau hyperphosphorylation were not reported in this study in spite of the major role of this receptor in modulating these two major hallmarks of AD (Caccamo et al., 2006).

    3. AF267B (originated from our lab, >99.9 percent chemical and enantiomeric purity) was shown in our laboratory to be a selective functional M1 agonist, both in vitro and in vivo (see my comments to Alzforum). Furthermore, AF267B is also specific for muscarinic receptors, unlike several selective allosteric M1 agonists reported recently that interact with some other G protein-coupled receptors. However, the authors refer to Jones and colleagues’ claim of similar activities on M1 and M3 AChRs using AF267B (though source and purity were not reported; see Jones et al., 2008).

    4. M1 selective agonists represent a rational treatment for AD both in treating cognitive deficits and as potential disease modifiers on AD hallmarks (Fisher, 2008). The previous clinical experience in AD with muscarinic agonists was disappointing. However, it is important to emphasize that the compounds that failed in clinical trials lacked selectivity for the M1 AChR, had major side effects, and some had poor pharmacokinetics. Thus, the question whether M1 agonists are useful treatments in AD was not really addressed so far in AD patients. Based on a plethora of recent studies, the M1 AChR is a promising target for future drug development in AD and was prematurely abandoned by major pharma. Unfortunately, recently major pharma preferred other strategies rather than the M1 therapeutic strategy in their drug development programs to treat AD.

    5. Given some major recent disappointments in Phase 2 and 3 studies with compounds that target at best one hallmark but ignore the others in AD, it is imperative and timely "to go back to the drawing board." Due to an elusive etiology, it is imperative to treat comprehensively all major hallmarks, regardless of the real culprit. In this context, M1 selective agonists may provide such a therapy. Notably, AF267B, tested successfully in three Phase 1 and Phase 2a studies (not AD), is the first low-molecular-weight monotherapy that targets comprehensively the major hallmarks of AD (cognitive dysfunctions, cholinergic hypofunction, Aβ and tau pathologies). Clinical studies in AD patients are timely and justified to explore the role of AF267B and some other M1 selective agonists in AD treatment.

    References:

    Fisher A. M1 muscarinic agonists target major hallmarks of Alzheimer's disease--the pivotal role of brain M1 receptors. Neurodegener Dis. 2008;5(3-4):237-40. PubMed.

    Fisher A. Cholinergic treatments with emphasis on m1 muscarinic agonists as potential disease-modifying agents for Alzheimer's disease. Neurotherapeutics. 2008 Jul;5(3):433-42. PubMed.

    Caccamo A, Oddo S, Billings LM, Green KN, Martinez-Coria H, Fisher A, Laferla FM. M1 receptors play a central role in modulating AD-like pathology in transgenic mice. Neuron. 2006 Mar 2;49(5):671-82. PubMed.

    Jones CK, Brady AE, Davis AA, Xiang Z, Bubser M, Tantawy MN, Kane AS, Bridges TM, Kennedy JP, Bradley SR, Peterson TE, Ansari MS, Baldwin RM, Kessler RM, Deutch AY, Lah JJ, Levey AI, Lindsley CW, Conn PJ. Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats. J Neurosci. 2008 Oct 8;28(41):10422-33. PubMed.

    View all comments by Abraham Fisher

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References

News Citations

  1. Cholinergic Transmission and Aβ: Boosting M1 Receptors Treats Model
  2. TBPB or Not to Be—The Latest on Muscarinic Receptor Agonists
  3. Targeting M1—The Agony of Agonists, the Power of Potentiators

Paper Citations

  1. Caccamo A, Oddo S, Billings LM, Green KN, Martinez-Coria H, Fisher A, Laferla FM. M1 receptors play a central role in modulating AD-like pathology in transgenic mice. Neuron. 2006 Mar 2;49(5):671-82. PubMed.
  2. Jones CK, Brady AE, Davis AA, Xiang Z, Bubser M, Tantawy MN, Kane AS, Bridges TM, Kennedy JP, Bradley SR, Peterson TE, Ansari MS, Baldwin RM, Kessler RM, Deutch AY, Lah JJ, Levey AI, Lindsley CW, Conn PJ. Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats. J Neurosci. 2008 Oct 8;28(41):10422-33. PubMed.
  3. Ma L, Seager MA, Seager M, Wittmann M, Jacobson M, Bickel D, Burno M, Jones K, Graufelds VK, Xu G, Pearson M, McCampbell A, Gaspar R, Shughrue P, Danziger A, Regan C, Flick R, Pascarella D, Garson S, Doran S, Kreatsoulas C, Veng L, Lindsley CW, Shipe W, Kuduk S, Sur C, Kinney G, Seabrook GR, Ray WJ. Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation. Proc Natl Acad Sci U S A. 2009 Sep 15;106(37):15950-5. PubMed.
  4. Pike CJ, Overman MJ, Cotman CW. Amino-terminal deletions enhance aggregation of beta-amyloid peptides in vitro. J Biol Chem. 1995 Oct 13;270(41):23895-8. PubMed.
  5. Quist A, Doudevski I, Lin H, Azimova R, Ng D, Frangione B, Kagan B, Ghiso J, Lal R. Amyloid ion channels: a common structural link for protein-misfolding disease. Proc Natl Acad Sci U S A. 2005 Jul 26;102(30):10427-32. PubMed.
  6. Dulin F, Léveillé F, Ortega JB, Mornon JP, Buisson A, Callebaut I, Colloc'h N. P3 peptide, a truncated form of A beta devoid of synaptotoxic effect, does not assemble into soluble oligomers. FEBS Lett. 2008 Jun 11;582(13):1865-70. PubMed.

Further Reading

Primary Papers

  1. Davis AA, Fritz JJ, Wess J, Lah JJ, Levey AI. Deletion of M1 muscarinic acetylcholine receptors increases amyloid pathology in vitro and in vivo. J Neurosci. 2010 Mar 24;30(12):4190-6. PubMed.
  2. Jang H, Arce FT, Ramachandran S, Capone R, Azimova R, Kagan BL, Nussinov R, Lal R. Truncated beta-amyloid peptide channels provide an alternative mechanism for Alzheimer's Disease and Down syndrome. Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6538-43. PubMed.

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