. Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer's disease. Cell. 2012 Mar 30;149(1):232-44. PubMed.

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  1. This intriguing study claims that a localized conformational switch, in the form of the cis/trans conformation of the prolyl bond separating the phosphorylated Threonine 231 (pThr231) and Proline 232 (Pro232) of Tau, carries toxic properties. In order to make this claim, the authors present several polyclonal antibodies that specifically recognize cis or trans forms of this prolyl bond in tau.

    In a tau peptide phosphorylated at the Thr231 site, the authors find the prolyl bond equilibrium yields 91 percent in the trans conformation, and 9 percent in cis. Earlier studies (Daly et al., Biochemistry 2000; Smet et al., Febs Lett. 2005) mentioned even lesser amounts of the cis conformer form in slightly longer peptides. Our NMR results on full-length tau point in the same direction, with less than 5 percent of the cis form for this particular prolyl bond (I. Landrieu and GL, unpublished results). An, at first, amazing finding is that the cis antibody in an Elisa test recognizes the natural (majorly trans) pThr-Pro as strongly as the pThr-dimethyl-proline containing one, although the presence of two methyl groups on the proline ring forces the prolyl bond to adopt almost exclusively the cis conformation. The authors’ argument that “cis and trans isomers in the phosphopeptide can be interchanged relatively easily” implies that the cis-specific antibody would basically displace the equilibrium towards the all-cis form by continuously sequestering the soluble cis-form. The same argument can evidently be applied to full-length phospho-Tau, thereby rendering a quantification of the initial cis- content at least hazardous. Equally intriguing is the overlap of the described cis-specific polyclonal antibody epitope with the epitope of the AT180/TG3 antibodies. AT180 was raised against purified human paired helical fragments (Goedert et al., 1994), and would thus be expected to recognize the cis isoform if that is the dominant form in PHFs, as the present study suggests. However, we showed earlier that AT180 recognizes, with nanomolar affinity, tau phosphorylated by CDK2/CycA3 (Amniai et al., BBRC 2011) at the pThr231-Pro232 epitope, where it is almost exclusively in the trans form. An experiment whereby the cis peptide (with the dimethyl-proline) would be probed with the AT180 antibody may bring clarity to this apparent contradiction (M. Mercken, personal communication).

    The authors use the modified peptides to confirm the specificity of protein phosphatase 2A (PP2A) for the trans conformation of pThr231-Pro232. Whereas we agree on the trans specificity of PP2A, we did unambiguously show by NMR spectroscopy that PP2A containing the B55 regulatory subunit (which is the one the authors used - see Zhou et al., Mol Cell 2000) does not dephosphorylate the pThr231 position (Landrieu et al., Plos One 2011) but rather the pSer205. Phosphorylation of the Thr231 thereby interferes with the catalytic efficiency of the phosphatase. Because dephosphorylation by PP2A was B55 dependent, we assume that the anchoring of the phospho-Tau substrate to the phosphatase via the regulatory B55 unit is regulated by the phospho-status of Thr231. Pin1 has an effect on the PP2A catalyzed dephosphorylation of phospho-Tau, as it seems to counter-act the negative effect of phospho-Thr231, and thereby stimulates the dephosphorylation of the pSer205 despite the presence of a phospho-Thr231. Whereas it is not clear what exact phosphorylation pattern is generated by the cdc2/CycB kinase used by the authors, disruption of the microtubule (MT) assembly capacity of tau (but not binding to MTs) requires at least three phosphates in the pS202/pS205/pT231/pS235 cluster (Amniai et al., Faseb J 2009). If Pin1 stimulates the dephosphorylation of pSer205, and thereby reduces the phospho content, tau, while still containing the phospho-Thr231, might indeed regain its MT assembly capacity after PP2A treatment (Figure 3G).

    In conclusion, the role of Pin1 in changing the phospho-tau conformation remains of considerable interest, but more work is required to evaluate the importance of its isomerase activity on the cis form of the pT231-P232.

  2. The development of cis- and trans-specific anti-tau antibodies by these authors has added very significant tools to the armory of research into AD (and the FTLD tauopathies). They have used these well to demonstrate that, while the trans isoform of tau promotes microtubule assembly, the phosphorylated cis form accumulates in the frontal cortex and hippocampus of patients with MCI and AD. The authors discuss the beneficial effects of cis->trans isomerization catalyzed by Pin1. These demonstrated accumulations of cis-p-tau are as expected, perhaps, but have never been demonstrated before, to our knowledge. We say "expected" because others’ work has shown that Pin1 function is compromised in MCI hippocampus, with the authors concluding that the oxidative inactivation of Pin1 could be involved in the progression from MCI to AD (Butterfield et al., 2006); thus, if Pin1 is the prime mediator of trans-specific tau dephosphorylation, increases in the cis form of tau would be expected in Pin1-deficient MCI or AD brain regions. Also, our own previous work has relevance here, as we showed apparent deficits of Pin1 in FTD brain and also aging-related deficits of the protein in normal brain (Thorpe et al., 2004; Hashemzadeh-Bonehi et al., 2006). This creates a good case for using targeted antibodies against the cis form of p-tau in immunotherapy to prevent AD progression. A caveat to this is that proline isomerization occurs spontaneously in solution from cis to trans and vice versa, and prolyl isomerases such as Pin1 accelerate this isomerization to equilibrium. A drug or antibody that targets and removes cis-p-tau from the environment may thus result in depletion of trans-tau as well as cis-p-tau. Thus, given the beneficial role of trans-tau in microtubule assembly, there may be adverse side effects to this kind of immunotherapy.

    References:

    . Redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of Alzheimer's disease. Neurobiol Dis. 2006 May;22(2):223-32. PubMed.

    . Shortfalls in the peptidyl-prolyl cis-trans isomerase protein Pin1 in neurons are associated with frontotemporal dementias. Neurobiol Dis. 2004 Nov;17(2):237-49. PubMed.

    . Pin1 protein associates with neuronal lipofuscin: potential consequences in age-related neurodegeneration. Exp Neurol. 2006 Jun;199(2):328-38. Epub 2006 Feb 14 PubMed.

    View all comments by Julian Thorpe
  3. The cis/trans configuration of tau adds another layer of complexity to the changes tau undergoes in diseases such as AD, but first of all, this study underlines that it is the serine/threonine-specific hyperphosphorylation of tau rather than any other modification (such as truncation, glycation, or nitration) that causes, at a very early stage, a gain of toxic function of tau and a loss of physiological functions. It will be interesting to see whether the findings for a role of the cis phospho-Thr231 epitope in pathogenesis can be extended to tauopathies other than AD. Furthermore, it will be crucial to determine (as the pThr231-proline motif seems to be the only tau epitope recognized by the isomerase Pin1) whether other phospho-epitopes of tau are also predominantly in the cis configuration in AD, and which enzymes regulate their cis/trans isomerization. Overall, this is an exciting study with interesting antibody tools to be exploited in a wider context.

    View all comments by Jürgen Götz

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  1. Pinning Down Role for Tau Proline Isomers in Alzheimer’s