Antonios G, Borgers H, Richard BC, Brauß A, Meißner J, Weggen S, Pena V, Pillot T, Davies SL, Bakrania P, Matthews D, Brownlees J, Bouter Y, Bayer TA. Alzheimer therapy with an antibody against N-terminal Abeta 4-X and pyroglutamate Abeta 3-X. Sci Rep. 2015 Dec 2;5:17338. PubMed.

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  1. This study by Gregory Antonios and the group of Thomas Bayer—and their previous study in 2013—pertains to actions of their antibody, NT4X, in a broad array of assays and models in vitro, ex vivo, and in vivo.

    The data mainly confirm the seminal work of Schenk et al., 15 years ago: preclinical validation of immunotherapy as potential cure of AD, and by extension other neurological diseases that have neurotoxic protein-aggregates as cause or correlate—notwithstanding the fact that we fail to understand the underlying molecular mechanisms.

    When immunotherapy proves effective—and it does so in mice—the medical benefits might even be overshadowed by what we learn about neuroscience. I advocated on many an occasion that when immunotherapy does make it into the clinic, as everybody hopes, it will help us understand some of the most fundamental biochemical and cellular processes in our brain—something you would normally try to do before developing a treatment. Among the many mysteries that are still unsolved, despite being chased for decades by so many great minds, are: the origin and spreading of pathology, the identity of neurotoxic protein species, the physiological function (if any) of amyloid peptides, network-functional interrelations of brain regions, the ApoE conundrum, immune surveillance, the role of the blood brain barrier, and many more …  

    The current study offers NT4X as one more candidate antibody that looks convincing in mice. However, we must await more studies to include it eventually into the ranks of those already in human trials: bapineuzumab, solanezumab, gantenerumab, crenezumab, aducanumab. 

    At face value, and given our own efforts in this sub-field, one is no longer surprised that immunotherapy works in mice, although with respect to the actual models used here, several caveats are in order: 

    • Despite their convincing reports and data, provided here and previously, it is still early days for the hypothesis that the truncated Aβ4-4X, the Tg4-42 mice, and the NTX4 antibody are important in/for AD patients—they all need wider and deeper studies, not just in mice but also in human neurons and brains.
    • The NT4X antibody was tested here mainly in “purpose-built” mice that express truncated Aβ4-42 from a Thy1-TRH gene promoter/processing construct, obviating the complex routes of cellular trafficking and proteolyting processing of the parent APP. Tg4-42 mice show cell-associated amyloid pathology at very young ages, with severe hippocampal neuron death from age five to eight months onwards, translating not surprisingly into severe defects in learning and memory (Bouter et al., 2013). 
    • Model and data stemming from them that depends on intracerebroventricular injection of, in this study Aβ4-42, but in general of any synthetic peptide, must be taken with caution.
    • The inclusion or add-on of the 5XFAD model is, compared to the Tg4-42 mice, rather brief in terms of assays and data, while the authors go to great lengths explaining the less-than-impressive outcome. This reminded us of the genetically complex construction of these mice that can model all but sporadic AD.
    • Lastly, a rather strange counterpoint surfaced of the current study in comparison with their first NT4X report (Antonios et al., 2013), which stated in the abstract: “While NT4X-167 significantly rescued Aβ4-42 toxicity in vitro no beneficial effect was observed against Aβ1-42 or AβpE3-42 toxicity”—as opposed to the current claim that the antibody acted effectively not only against the 4-42 but also against pE3-42 amyloid peptides. This raises interesting questions concerning the methodology used in both studies, and even more so about the actual epitope that is defined or recognized by the NT4X Mab, which they claimed to be totally dependent on the F4 residue.

    As usual, interesting aspects addressed by fine research generate even more interesting questions … 

    References:

    Bouter Y, Dietrich K, Wittnam JL, Rezaei-Ghaleh N, Pillot T, Papot-Couturier S, Lefebvre T, Sprenger F, Wirths O, Zweckstetter M, Bayer TA. N-truncated amyloid β (Aβ) 4-42 forms stable aggregates and induces acute and long-lasting behavioral deficits. Acta Neuropathol. 2013 Aug;126(2):189-205. PubMed.

    Antonios G, Saiepour N, Bouter Y, Richard BC, Paetau A, Verkkoniemi-Ahola A, Lannfelt L, Ingelsson M, Kovacs GG, Pillot T, Wirths O, Bayer TA. N-truncated Abeta starting with position four: early intraneuronal accumulation and rescue of toxicity using NT4X-167, a novel monoclonal antibody. Acta Neuropathol Commun. 2013 Sep 6;1(1):56. PubMed.

    View all comments by Fred Van Leuven

  2. What evidence is there for N-truncated pyroglutamate Aβ3-42 and Aβ4-42 as therapeutic targets of Alzheimer’s disease?

    As early as 1985, truncated Aβ peptides were described as precipitating in AD plaques, including a major species beginning with phenylalanine at position 4 ( Aβ4-x) (Masters et al., 1985). A majority of 64 percent of the peptides in amyloid plaques of two sporadic AD cases and 45 percent of the peptides in the patients with Down’s syndrome started with a Phe-4 residue. At the same time, Glenner and Wong demonstrated that full-length Aβ beginning with Asp-1 was the main species detected in cerebrovascular deposits (Glenner and Wong, 1984). There is common agreement that plaque-associated Aβ peptides mainly terminate at Ala-42. Analyzing familial AD patients, Ancolio and colleagues (Ancolio et al., 1999) first showed a selective and drastic increase of N-truncated Aβx–42 species triggered by the mutation APP V715M, postulating that all Aβx–42 variants are main factors driving AD pathology. Miller et al. compared the peptide compositions of the cerebrovascular and senile plaque core amyloid deposits in AD. Matrix-assisted, laser-desorption-time-of-flight (MALDI-TOF) mass spectrometry of plaque-Aβ revealed an array of peptides ending with Ala-42, while cerebrovascular Aβ began with Arg-1 ending at Val-40. They verified that Phe-4 is the main component in plaques, but cautioned that their MALDI-TOF spectral data suggest the presence of two pyroglutamyl amino termini (pyroGlu-3 and pyroGlu-11) that might escape detection by other methods. Using immunoprecipitation in combination with mass spectrometric analysis, the dominating Aβ isoforms in the three different brain regions analyzed from control, sporadic, and familial AD were described as Aβ1-42, AβpE3-42, Aβ4-42, and Aβ1-40, with Aβ1-42 and Aβ4-42 being the dominant isoforms in the hippocampi and cortices in all groups analyzed  (Portelius et al., 2010). The question whether N-truncations of Aβ are a postmortem artifact or might even precede the symptomatology of AD was addressed by Sergeant and co-workers (Sergeant et al., 2003). They adapted a proteomic method in combination with western blotting and mass spectrometry for the characterization of insoluble Aβ extracted in formic acid. Full-length Aβ peptides represented 37 percent of all Aβ species, while 17 percent corresponded to N-truncated species starting at residues Phe-4 and Arg-5, and 20 percent started with Ser-8, Gly-9, and Tyr-10. They also demonstrated that at the first stage of amyloid deposition in non-demented individuals, plaques comprised N-terminal truncated variants starting at positions 4-, 5-, 8- and 9–42, or with a pyroglutamyl residue at position 3. At this stage, Aβ oligomers were exclusively made of Aβx-42 species. 

    In a recent review (Bayer and Wirths, 2014), we summarized these arguments and discussed the validity of available mouse models focusing on Aβ3-42 and Aβ4-42 as Alzheimer’s targets.

    • N-truncated Aβ variants, predominantly with pyroGlu-3 and Phe-4, correlate well with presymptomatic AD.
    • N-truncated variants pyroGlu-3 and Phe-4 Aβ peptides are more abundant than full-length Aβ in the brains of patients diagnosed with sporadic or familial AD.
    • APP transgenic mouse models generate N-truncated Aβ, albeit at quite low levels not reflecting the situation in AD brains. The 5XFAD model, for example, produces mostly Aβ1-42 and much lower levels of pyroglutamate Aβ3-42 and Aβ4-42 (Oakley et al., 2006; Wittnam et al., 2012). 
    • Transgenic mouse models that express solely AβpE3-42 (Glu-3 mutated to Gln-3 in order to facilitate pyroGlu-3 formation) consistenly develop neuron loss and associated neurological deficts. Plaque load is low.
    • The transgenic mouse model Tg4-42 expressing Aβ4-42 (Bouter et al., 2013) develops age-dependent hippocampus-related reference memory deficits in the Morris water maze due to the drastic CA1 neuron loss. No plaque pathology is observed.
    •  AβpE3-42 and Aβ4-42 rapidly form soluble toxic aggregates in vitro having different biochemical properties than full-length Aβ1-42 (Bouter et al., 2013). 

    The comments by Fred van Leuven are well accepted. Of course, the Tg4-42 animal model was designed to test Aβ4-42-induced pathology (Antonios et al., 2015). We wanted to ask whether full-length and Fab fragement of NT4X administered peripherally can act in the central nervous system, even reaching aggregates within neurons. The neuron loss is fast in Tg4-42, reaching 50 percent at six months of age. It was significantly slowed down after 12-week-long NT4X treatment. As a consequence, the mice performed better in the memory test with both the full-length and Fab antibodies. Seemingly, there is agreement that NT4X can act in the CNS, even protecting against neuronal degeneration, a therapeutic feature not reported by other antibody treatments (so far). Moreover, we think that the therapeutic mechanism does not involve the Fc-part of the antibody (i.e., microglia clearence), as the Fab worked as well as the full antibody. As a matter of fact, NT4X treatment of 5XFAD mice lowered the plaque load of N-truncated peptides, but not of full-length Aβ1-42, as expected, because the 5XFAD model produces only low levels of AβpE3-42 and Aβ4-42. This raises a question about the face validity of animal models for human disorders in general. 

    We completely agree that wider and deeper studies ultimately will be needed in AD patients, but also in mice, human neurons, and human brains. We have in fact compared the immunostaining profile of biosimilars of bapineuzumab, solanezumab, and crenezumab with NT4X in different mouse models and brains from patients with sporadic AD (Bouter et al., 2015) and found that NT4X has low plaque-binding potential in comparison with the other antibodies. We believe that this feature is therapeutically beneficial because it improves the antibody bioavailability for soluble Aβ in the CNS.  

    References:

    Ancolio K, Dumanchin C, Barelli H, Warter JM, Brice A, Campion D, Frébourg T, Checler F. Unusual phenotypic alteration of beta amyloid precursor protein (betaAPP) maturation by a new Val-715 --> Met betaAPP-770 mutation responsible for probable early-onset Alzheimer's disease. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):4119-24. PubMed.

    Antonios G, Borgers H, Richard BC, Brauß A, Meißner J, Weggen S, Pena V, Pillot T, Davies SL, Bakrania P, Matthews D, Brownlees J, Bouter Y, Bayer TA. Alzheimer therapy with an antibody against N-terminal Abeta 4-X and pyroglutamate Abeta 3-X. Sci Rep. 2015 Dec 2;5:17338. PubMed.

    Bayer TA, Wirths O. Focusing the amyloid cascade hypothesis on N-truncated Abeta peptides as drug targets against Alzheimer's disease. Acta Neuropathol. 2014 Jun;127(6):787-801. Epub 2014 May 7 PubMed.

    Bouter Y, Dietrich K, Wittnam JL, Rezaei-Ghaleh N, Pillot T, Papot-Couturier S, Lefebvre T, Sprenger F, Wirths O, Zweckstetter M, Bayer TA. N-truncated amyloid β (Aβ) 4-42 forms stable aggregates and induces acute and long-lasting behavioral deficits. Acta Neuropathol. 2013 Aug;126(2):189-205. PubMed.

    Bouter Y, Lopez Noguerola JS, Tucholla P, Crespi GA, Parker MW, Wiltfang J, Miles LA, Bayer TA. Abeta targets of the biosimilar antibodies of Bapineuzumab, Crenezumab, Solanezumab in comparison to an antibody against N‑truncated Abeta in sporadic Alzheimer disease cases and mouse models. Acta Neuropathol. 2015 Nov;130(5):713-29. PubMed. Correction.

    Glenner GG, Wong CW. Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun. 1984 May 16;120(3):885-90. PubMed.

    Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4245-9. PubMed.

    Miller DL, Papayannopoulos IA, Styles J, Bobin SA, Lin YY, Biemann K, Iqbal K. Peptide compositions of the cerebrovascular and senile plaque core amyloid deposits of Alzheimer's disease. Arch Biochem Biophys. 1993 Feb 15;301(1):41-52. PubMed.

    Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, Guillozet-Bongaarts A, Ohno M, Disterhoft J, Van Eldik L, Berry R, Vassar R. Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation. J Neurosci. 2006 Oct 4;26(40):10129-40. PubMed.

    Portelius E, Bogdanovic N, Gustavsson MK, Volkmann I, Brinkmalm G, Zetterberg H, Winblad B, Blennow K. Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer's disease. Acta Neuropathol. 2010 Aug;120(2):185-93. PubMed.

    Sergeant N, Bombois S, Ghestem A, Drobecq H, Kostanjevecki V, Missiaen C, Wattez A, David JP, Vanmechelen E, Sergheraert C, Delacourte A. Truncated beta-amyloid peptide species in pre-clinical Alzheimer's disease as new targets for the vaccination approach. J Neurochem. 2003 Jun;85(6):1581-91. PubMed.

    Wittnam JL, Portelius E, Zetterberg H, Gustavsson MK, Schilling S, Koch B, Demuth HU, Blennow K, Wirths O, Bayer TA. Pyroglutamate amyloid β (Aβ) aggravates behavioral deficits in transgenic amyloid mouse model for Alzheimer disease. J Biol Chem. 2012 Mar 9;287(11):8154-62. PubMed.

    View all comments by Thomas Bayer View all comments by David Matthews View all comments by Janet Brownlees

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