Cholinergic Transmission and Aβ: Boosting M1 Receptors Treats Model

An impressive data set that appeared yesterday in Neuron shows that a specific M1 muscarinic receptor agonist can reverse both cognitive deficits as well as the amyloid and tau pathology in the APP/PS1/tau triple transgenic mice mouse model of AD. The publication, from Frank LaFerla and colleagues from the University of California at Irvine and Abraham Fisher at the Israel Institute for Biological Research in Ness Ziona, advances a story that garnered significant attention when the investigators presented their preliminary results on the small molecule AF267B at the 2004 Society for Neurosciences meeting in San Diego (see ARF related news story).

The compound was licensed at that time to TorreyPines Therapeutics, Inc. (formerly Neurogenetics, Inc.) in San Diego, who made their own news this week by announcing that they have initiated a second Phase I trial of the compound in humans. The trial, which is expected to yield results as soon as next June, might give an early indication of whether the compound is capable of affecting Aβ or tau metabolism or cognitive function in humans.

For years, Fisher and his colleagues have synthesized and studied series of small molecules in an effort to prove his hypothesis that agonists that are highly selective for M1 muscarinic acetylcholine receptors could treat the symptoms of AD, as well as change its course (see, for example, Fisher et al., 2003 and Fisher et al., 1998). Muscarinic agonists have been tested before in vitro and in humans, but none have made it all the way to a useful AD drug, and early agonists failed in clinical trials. All along, Fisher has insisted that the failure was due to the compounds’ inadequate M1 selectivity and poor pharmacokinetics. AF267B provided a chance to experiment with a rigid acetylcholine mimic that is highly selective, orally available, and penetrates the blood-brain barrier.

At the 2004 SfN conference, Antonella Caccamo from the LaFerla lab presented a poster showing that daily intraperitoneal injections of AF267B in triple transgenic AD mice diminished the animals’ plaque pathology, intraneuronal pathology, and tau pathology in cortex and hippocampus. Their experiments with ELISA and Western blots indicated decreases in soluble and insoluble Aβ formation, associated with a decrease in C99 (the product of β-secretase cleavage) and an increase in C83 (the APP fragment released by α-secretase cleavage). Measuring steady-state levels, Caccamo and colleagues found a decrease in BACE, an increase in ADAM-17, and no change in ADAM-10 (see ARF related news story). AF267B also diminished phospho-reactive tau as detected with the antibody AT8. Finally, the compound reversed the memory retention deficit in the water maze.

The new paper, which features Caccamo as first author, presents all this data, and in addition shows that AF267B activates ERK and protein kinase C (PKC) in vivo, consistent with the known effects of M1 agonists to stimulate these enzymes in vitro. Other studies have shown that M1 receptor stimulation in vitro increases the production of αAPP via activation of ERK and PKC, and the new results suggest that AF267B activates the same pathway.

In a similar vein, the new work shows that amelioration of tau hyperphosphorylation is associated with a decrease in GSK3β kinase activity in brains of AF267B-treated mice. Throughout the work, the researchers compare the effects of AF267B with the M1 antagonist dicyclomine, and they consistently find that the antagonist has the expected, opposite effect on the mice.

In contrast to its effects in the cortex and hippocampus, AF267B does not reduce plaque load, Aβ, or tauopathy in the amygdala. That is consistent with its failure to correct deficits in amygdala-based fear conditioning in these mice. The authors suggest that this state of affairs may reflect the absence of ADAM-17 in the amygdala, compared to the cerebellum and hippocampus.

At TorreyPines Therapeutics, AF267B is being developed under the name of NGX267. A first Phase I trial, completed at the end of 2005, established the safety of single doses of the compound in healthy adult volunteers. Earlier this week, the company reported the initiation of a second Phase I trial that will measure the safety and tolerability of single and multiple doses in 65 healthy elderly subjects. In the study, the company will also assess several biomarkers (levels of Aβ1-42, total tau, and phospho-tau in cerebrospinal fluid) as well as cognitive function.

The results, expected in June 2006 will determine how development will proceed, said Neil Kurtz, the president and CEO of TorreyPines Therapeutics. "So far the drug is demonstrating good pharmaceutical properties. Based on the data from our second Phase I trial, we will decide whether to move forward with NGX267 for symptomatic treatment, or for disease modification or progression. If we get the expected changes in the cerebrospinal fluid biomarkers, it will provide a strong rationale to move forward in disease modification or progression trials,” Kurtz told ARF.—Pat McCaffrey

Comments

  1. M1 receptors play a central role in modulating AD-like pathology in transgenic mice
    The activation of M1 and M3 muscarinic receptors have long been regarded as a promising approach for AD therapy, because it was shown that they activate the non-amyloidogenic α-secretase pathway. However, the clinical trials did not support their application in clinical practice. The development of new M1 agonists with higher specificity, which can cross the blood-brain barrier, has been the main aim of the research group of Abraham Fisher for several years. The results with such a newly developed M1 agonist obtained in a triple-transgenic mouse model now provide new hope that these compounds will be more successful in clinical trials.

    In the paper by Caccamo et al., the M1 agonist AF267B is shown to reduce both the Aβ and tau pathology in the hippocampus and cortex, and to reverse cognitive deficits. Recently my group, in collaboration with the group of Fred van Leuven (Belgium) has shown that overexpression of the α-secretase ADAM-10 prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model, and in this way a proof of concept has been provided that up-regulation of the α-secretase activity in vivo could preclude the formation of Aβ and alleviate cognitive deficits (Postina et al., 2004). The paper by the LaFerla group and Fisher now moves an important step further by showing convincingly that small compounds, like the newly developed M1 agonist, can reverse AD-like pathology in transgenic mice.

    Concerning the up-regulation of the ADAM-17, I would like to respectfully question the results the authors provided. From our own experiments, we know that in brain homogenates, due to the low abundance of ADAMs, it is very difficult to quantitate their amount without enrichment of these glycoproteins by lectin chromatography. As the authors had not enriched these enzymes and do not show the molecular weights of the ADAM enzymes and also of BACE, these experiments concerning the expression of α- and β-secretase are not definitive just yet. One also has to take into account that activation of the α-secretase may be achieved without increasing its protein level. This has just been described in a paper by Kojro et al., 2006. From my point of view, it would also have been interesting to see whether the muscarinic agonist also increased the amount of secreted APP (sAPPα).

    In spite of some open questions concerning the mechanism, the authors now have made an important step forward to causal therapy of AD and have provided evidence that compounds activating the non-amyloidogenic α-secretase pathway might have clinical relevance. This work opens a way to new clinical trials with a new generation of M1 agonists.

    References:

    Postina R, Schroeder A, Dewachter I, Bohl J, Schmitt U, Kojro E, Prinzen C, Endres K, Hiemke C, Blessing M, Flamez P, Dequenne A, Godaux E, Van Leuven F, Fahrenholz F. A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest. 2004 May;113(10):1456-64. PubMed.

    Kojro E, Postina R, Buro C, Meiringer C, Gehrig-Burger K, Fahrenholz F. The neuropeptide PACAP promotes the alpha-secretase pathway for processing the Alzheimer amyloid precursor protein. FASEB J. 2006 Mar;20(3):512-4. PubMed.

    View all comments by Falk Fahrenholz

  2. Caccamo and colleagues did a great job in demonstrating the potentially important role of M1 receptors in treatment of AD.

    One question I have based on my limited knowledge is: Does the receptor agonist have to be specific to the M1 receptor? Would agonist(s) with broader specificity have any side effect(s)? Would M1-specific agonist(s) be more beneficial than donepezil?"

    In this respect, I would have used a control group treated with donepezil to demonstrate that M1 agonist(s) would be better medications than aceetylcholine esterase inhibitors. Such a control group would also help to clarify the pathophysiological relevance of the 3XTg mice as an AD model.

    View all comments by Takaomi Saido

  3. The news that a specific M1 muscarinic receptor agonist can reverse both cognitive deficits as well as the amyloid and tau pathology in this mouse model is most interesting.

    I wonder whether the study by Ganzinelli and colleagues (1) explains why you don't see AD in people with schizophrenia. They report that "circulating antibodies from schizophrenic patients interacting with cerebral M1 muscarinic acetylcholine receptors can act as an inducer of m(1) mAChR-mRNA, and neuronal nitric oxide synthase (nNOS) mRNA gene expression of rat frontal cortex." Might these antibodies be expected to reverse the amyloid pathology in AD?

    Martin et al. (2) report lower protein levels of nNOS in the Tg2576 transgenic mouse model. Has anyone used nitroglycerine as a nitric oxide donor in AD and, if so, what have been the results?

    References:

    Ganzinelli S, Borda T, Sterin-Borda L. Regulation of m1 muscarinic receptors and nNOS mRNA levels by autoantibodies from schizophrenic patients. Neuropharmacology. 2006 Mar;50(3):362-71. PubMed.

    Martin BL, Tokheim AM, McCarthy PT, Doms BS, Davis AA, Armitage IM. Metallothionein-3 and neuronal nitric oxide synthase levels in brains from the Tg2576 mouse model of Alzheimer's disease. Mol Cell Biochem. 2006 Feb;283(1-2):129-37. PubMed.

    View all comments by Mary Reid

  4. Reply by Abraham Fisher to Takaomi Saido
    In reply to Takaomi Saido’s questions: The progression of Alzheimer disease (AD), associated with loss of the cholinergic neurons and decreases in acetylcholine (ACh), limit the therapeutic potential of the FDA-approved acetylcholinesterase inhibitors (AChEIs) such as donepezil, galantamine, rivastigmine, or tacrine. Postsynaptic M1 muscarinic receptors (M1 mAChR) are predominant in cerebral cortex and hippocampus and have a major role in hippocampal-based learning and memory, particularly for short-term memory, which is impaired in AD. As M1 mAChR are relatively preserved in AD, use of M1 muscarinic agonists in AD treatment is rational.

    Unlike AChEIs, M1 muscarinic agonists in theory are independent of ACh levels in the brain, and thus less affected by the extent of degeneration of presynaptic cholinergic terminals. Whilst activation of M1 mAChR is advantageous, stimulation of the other mAChR subtypes leads to side effects. Therefore, the ideal M1 muscarinic agonist should be devoid of M2, M3, and M5 agonistic effects. Some muscarinic agonists improved cognition and reduced psychotic episodes in AD patients. However, a scarcity of selective M1 muscarinic agonists has limited the clinical use of muscarinic agonists in AD due to side effects observed at higher doses. The failure of most of the tested agonists was due to the compounds’ inadequate M1 selectivity and poor pharmacokinetics, narrow safety margin, and side effects. AF267B is an M1 selective agonist, is orally available, penetrates the blood-brain barrier, and has a high bioavailability and wide safety margin.

    M1 mAChR-mediated activation of α-secretase can increase α-APPs, preventing the formation of Aβ. Activation of M3 mAChR also elevates α-APPs, yet selective M1 agonists are preferable to prevent peripheral M3 mediated-side effects (e.g., gastrointestinal). M2 mAChR and M4 mAChR are ineffective in activating α-secretase as tested by α-APPs release, and the M2 mAChR may even have an inhibitory effect.

    The multitude of animal models available complicates comparison among studies from various labs. In this context, the 3xTg-AD model offers the possibility to answer fundamental questions in AD pathology and therapeutic strategies. How validated are the animal models? One validation should include studies of AChEIs, even if such compounds are not expected to cause a disease modification (e.g., reduction in Aβ levels and/or tau hyperphosphorylation). Whether AF267B is more beneficial than donepezil cannot be addressed properly since donepezil was not yet tested in the 3xTg-AD mice. Yet, our study and previous findings indicate that AF267B, unlike donepezil, in addition to treatment of cognitive deficits, can be also regarded as a disease modifier. While few studies reported some beneficial effects of donepezil on behavioral deficits (memory and learning) in Tg mice such as Tg2576, APP23, and AD11, none of these studies reported a decrease in Aβ levels and/or tau hyperphosphorylation.

    References:

    Dong H, Csernansky CA, Martin MV, Bertchume A, Vallera D, Csernansky JG. Acetylcholinesterase inhibitors ameliorate behavioral deficits in the Tg2576 mouse model of Alzheimer's disease. Psychopharmacology (Berl). 2005 Aug;181(1):145-52. PubMed.

    Van Dam D, Abramowski D, Staufenbiel M, De Deyn PP. Symptomatic effect of donepezil, rivastigmine, galantamine and memantine on cognitive deficits in the APP23 model. Psychopharmacology (Berl). 2005 Jun;180(1):177-90. PubMed.

    Capsoni S, Giannotta S, Stebel M, Garcia AA, De Rosa R, Villetti G, Imbimbo BP, Pietra C, Cattaneo A. Ganstigmine and donepezil improve neurodegeneration in AD11 antinerve growth factor transgenic mice. Am J Alzheimers Dis Other Demen. 2004 May-Jun;19(3):153-60. PubMed.

    View all comments by Abraham Fisher
    • User Profile Image Thomas Beach
      Banner Sun Health Research Institute
    • Posted:

    The paper by LaFerla and colleagues adds to considerable evidence suggesting that M1 receptor activation leads to decreased amyloidogenic processing of APP while reduced M1 receptor activation, by means of either pharmacological agents or cholinergic lesion, results in increased Aβ production.

    We have done considerable work in this field that readers may find interesting (see attached citations for examples). In particular, we have evaluated three of Abraham Fisher’s M1-selective compounds, including AF267B, and found that all three reduced CSF and cortical levels of Aβ in rabbits.

    The present conventional wisdom that cholinergic agents are only palliative and do not affect disease progression is challenged by the collective contrary data that has accumulated over the last 10 years. It is likely that cholinergic therapy would have its greatest potential effects on Aβ deposition and disease progression if it were given as primary prevention, since Aβ deposition has already reached a plateau by the time the clinical diagnosis of Alzheimer disease is made. (1,2)

    References:

    Beach TG, Walker DG, Roher AE, Potter PE. Anti-Amyloidogenic Activity of Cholinergic Agents. Drug Dev Res. 2002;56 (2):242-47.

    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. 2001 Jun 29;905(1-2):220-3. PubMed.

    View all comments by Thomas Beach
    • User Profile Image Nunzio Pomara
      Nathan S Kline Institute- NYU School of Medicine
    • Posted:

    The results reported by LaFerla and colleagues are very promising. However, before embarking on large-scale clinical studies with selective M1 agonists, certain points, which might have important implications both with respect to the efficacy and safety of these agents, deserve consideration.

    While brain muscarinic (M1) receptor density has generally been found to be preserved across all stages of AD, numerous observations from in-vitro studies indicate a loss of the coupling of cortical M1 receptors to G-proteins, which could limit the efficacy of these agents.

    Additionally, the precise origin of the M1 receptor-mediated reductions in CSF Aβ levels, which have been reported in pilot clinical studies with this class of compounds in AD, is not known, and this isolated finding may not necessarily reflect brain M1 receptor activation.

    Paradoxically, there is also in-vivo evidence that certain central and peripheral responses to nonspecific M1 agonists may be increased in AD patients (1,2) which could potentially influence the safety of these drugs in this population.

    References:

    Pomara N, Sitaram N. Detecting Alzheimer's disease. Science. 1995 Mar 17;267(5204):1579-80; author reply 1580-1. PubMed.

    Pomara N, Stanley M, Lewitt PA, Galloway M, Singh R, Deptula D. Increased CSF HVA response to arecoline challenge in Alzheimer's disease. J Neural Transm Gen Sect. 1992;90(1):53-65. PubMed.

    View all comments by Nunzio Pomara

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References

News Citations

  1. San Diego: Treating Forgetfulness—Triple Transgenics Provoke
  2. α-Secretase Returns to Center Stage

Paper Citations

  1. Fisher A, Pittel Z, Haring R, Bar-Ner N, Kliger-Spatz M, Natan N, Egozi I, Sonego H, Marcovitch I, Brandeis R. M1 muscarinic agonists can modulate some of the hallmarks in Alzheimer's disease: implications in future therapy. J Mol Neurosci. 2003;20(3):349-56. PubMed.
  2. Fisher A, Brandeis R, Chapman S, Pittel Z, Michaelson DM. M1 muscarinic agonist treatment reverses cognitive and cholinergic impairments of apolipoprotein E-deficient mice. J Neurochem. 1998 May;70(5):1991-7. PubMed.

Further Reading

Papers

  1. Liskowsky W, Schliebs R. Muscarinic acetylcholine receptor inhibition in transgenic Alzheimer-like Tg2576 mice by scopolamine favours the amyloidogenic route of processing of amyloid precursor protein. Int J Dev Neurosci. 2006 Apr-May;24(2-3):149-56. PubMed.
  2. Fisher A, Pittel Z, Haring R, Bar-Ner N, Kliger-Spatz M, Natan N, Egozi I, Sonego H, Marcovitch I, Brandeis R. M1 muscarinic agonists can modulate some of the hallmarks in Alzheimer's disease: implications in future therapy. J Mol Neurosci. 2003;20(3):349-56. PubMed.
  3. Hock C, Maddalena A, Raschig A, Müller-Spahn F, Eschweiler G, Hager K, Heuser I, Hampel H, Müller-Thomsen T, Oertel W, Wienrich M, Signorell A, Gonzalez-Agosti C, Nitsch RM. Treatment with the selective muscarinic m1 agonist talsaclidine decreases cerebrospinal fluid levels of A beta 42 in patients with Alzheimer's disease. Amyloid. 2003 Mar;10(1):1-6. PubMed.

Primary Papers

  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.

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