Tau-containing neurofibrillary tangles that mark Alzheimer disease represent the end stage of a long pathway that starts with the production of normal tau protein, progresses through deregulated phosphorylation and ends at aggregation. Just where in this process tau deals cells their death blow is a topic of much debate and research these days, and two papers this week take a closer look at that question: one, from Roland Brandt and colleagues at the University of Osnabruck in Germany, examines the effects of a pseudohyperphosphorylated tau mutant in hippocampal slice cultures as a model of tauopathy in the brain. They show that even as neurons start to die, their mutant tau does not appear to change dendritic spine number or morphology, in contrast to the well-known pathologic effects of its co-perpetrator in Alzheimer disease, Aβ.

A second report, from the lab of Khalid Iqbal at the New York State Institute for Basic Research in Developmental Disabilities in Staten Island, New York, focuses on the question of exactly which forms of tau are toxic. Their work demonstrates that soluble hyperphosphorylated tau, but not aggregated filaments, binds to normal tau and inhibits microtubule assembly in vitro. These results fit with the growing body of evidence suggesting that aberrantly phosphorylated soluble tau, and not filaments, produces pathology, and that neurofibrillary tangles may actually help protect cells from the wrath of tau.

In Alzheimer disease, the accumulation of tau, and also of Aβ, accompanies early loss of synapses and later, neuronal death. The complex web of cause and effect for both these proteins has yet to be fully unraveled. Brandt and colleagues turned to a hippocampal slice culture system to get a fresh look at tau toxicity. First author Neelam Shahini used Sindbis virus to introduce green fluorescent protein (GFP)-labeled tau expression constructs, and then followed the fate of infected neurons over several days. They chose to study a pseudohyperphosphorylated form of tau (PHP tau), which had nine serine and one threonine residues mutated to glutamic acid, to mimic pathologic hyperphosphorylation. Previously, they had shown that, like bona fide phospho-tau, this mutant was cytotoxic in cultured neurons and failed to stabilize microtubules Fath et al., 2002). In the hippocampal slice system, they saw that expression of PHP tau was associated with enhanced apoptotic cell death compared to wild-type tau, as indicated by lactate dehyrogenase (LDH) release, caspase3 activation, and DNA laddering. But PHP tau also caused non-apoptotic cell death, as indicated by a ballooned phenotype in many cells. Analysis of different hippocampal subfields revealed a region-specific effect of PHP tau, with significant neuron loss in the CA3 and dentate gyrus, but not CA1 subregions.

In contrast to Aβ, PHP tau did not seem to affect dendritic health. Measurements of dendritic morphology and spine density revealed no difference between cells expressing wild-type tau and PHP tau. While the authors did not show comparison to control cells expressing just GFP, the implication is that PHP tau does not induce synaptic loss on these neurons even in regions of the hippocampus where other cells are dying.

Does this mean that Aβ might be solely to blame for early synaptic loss, as the authors suggest? It’s difficult to draw that conclusion, given the caveats of the glutamic acid substitution approach to studying protein phosphorylation. For one thing, such substitutions do not entirely recapitulate the extent or the dynamics of tau phosphorylation. The researchers do present evidence that PHP tau can undergo additional phosphorylation and adopt a pathologic conformation, based on its reactivity with the Alz50 and MC1 antibodies, and the appearance of insoluble forms of the mutant. However, the subcellular distribution of the expressed PHP tau does not match the natural hyperphosphorylated protein in these cells. Effects of GFP fusion on protein function could also complicate the experiments (see Van Leuven comment below). The hippocampus slice system and Sindbis virus mediated protein expression offers an opportunity to sort out these issues, and also to look at the interactions of Aβ and tau in future experiments.

Moving a little bit more to the in vitro side, the work from the Iqbal lab and first author Alejandra del C. Alonso aims to pin down just what form of tau is responsible for interfering with microtubule formation, a process that can trigger apoptosis in neurons. Their previous work shows that hyperphosphorylated tau sequesters normal tau (N-tau) and the microtubule-associated proteins MAP1 and MAP2, resulting in disassembly of microtubules (Alonso et al., 1997). Their new work carries this further, by showing that phospho-tau, but not single or paired helical filaments (all isolated from postmortem AD brain), binds N-tau and inhibits microtubule assembly in vitro. Likewise, addition of the AD phospho-tau, but not the filaments, inhibited microtubule bundle formation in cell-free extracts. Finally, they showed that when they phosphorylated and polymerized recombinant human brain tau in vitro, it lost its ability to bind N-tau, but the binding was restored by breaking up the filaments by sonication. They got similar results starting with soluble phospho-tau isolated from AD brain and aggregated in vitro. From these results, they postulate that abnormal hyperphosphorylation of tau, and not the formation of neurofibrillary tangles, is the key element in neurodegeneration. This is an increasingly accepted idea, as it fits well with growing evidence from animal models of tauopathies (Spires et al., 2006; also see ARF related news story and the recent ARF Eibsee coverage).

In the model that Iqbal and coauthors propose, after dysregulation of tau phosphorylation, either by Aβ-related processes in AD or by mutation in other tauopathies, the first accumulation of phospho-tau disrupts microtubule networks by binding N-tau. (According to their data, phospho-tau can tie up super-stoichiometric amounts of N-tau, so this disruption might occur at very low phospho-tau concentrations). With time and increasing phospho-tau, the protein starts to self-associate. This scenario suggests that rather than trying to reverse tau aggregation, therapeutic approaches aimed at blocking tau phosphorylation, or preventing hyperphosphorylated tau from interacting with normal microtubule-associated proteins, will be most effective, Iqbal and colleagues write.—Pat McCaffrey.

Reference:
Shahani N, Subramaniam S, Wolf T, Tackenberg C, Brandt R. Tau aggregation and progressive neuronal degeneration in the absence of changes in spine density and morphology after targeted expression of Alzheimer’s disease-relevant tau constructs in organotypic hippocampal slices. J. Neuroscience. 31 May 2006; 26:6103-6114. Abstract

Alonso AC, Li B, Grundke-Iqbal I, Iqbal K. Polymerization of hyperphosphorylated tau into filaments eliminates its inhibitory activity. PNAS. 6 June 2006; 103:8864-8869. Abstract

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  1. While this study helps us to understand some of the actions of tau, several questions come to mind.

    • It is surprising that no effects are noted on spines or dendrites, while synapses are not really analysed here. The evident neurotoxicity is remarkable in its regional difference since CA1 is the most vulnerable hippocampal region, as the authors properly discuss.

    • Do the recombinant “ten E-tau” molecules still bind to microtubules (MT), and is displacement of normal tau from MT part of the problem?
    • The S/T to E mutation is a fairly easy method to assess effects of phosphorylation, but it remains “pseudo-phosphorylation” and therefore not relevant to the real thing, as opposed to the S/T to A mutations to prevent phosphorylation.
    • In our hands, in cellular transfections, when EGFP is fused to tau at either the N- or C-terminus, it interferes with its "normal" behavior, including formation of the MC1 epitope. Moreover, this epitope is special and should in principle not be evident by Western blotting after SDS PAGE.
  2. Alzheimer disease (AD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Guam parkinsonism dementia, cortical basal degeneration, dementia pugilistica, Pick’s disease, progressive supranuclear palsy, and tangle-only dementia, among other tauopathies, are linked to the progressive accumulation of filamentous hyperphosphorylated tau inclusions. Besides the accumulation of tau, another distinctive feature is the loss of neuritic arborization and the disruption of microtubules and synaptic terminals. This neurodegeneration is also present in FTDP-17, diseases linked to tau mutations, pointing to tau and tau function as the origin of dementia. Shahani et al. recently described a very interesting model to test the role of hyperphosphorylated tau in neurodegeneration; they infected neurons in mouse brain slices with pseudo-hyperphosphorylated tau using recombinant Sindbis virus. This method allows the authors to study behavior of hyperphosphorylated tau in living neurons. The expressed tau filled up the neurons highlighting all the processes. In this paradigm, spines and dendrites were not altered, but neurons underwent apoptosis—an acute cell death. This scenario is markedly different from AD where neurofibrillary degeneration is a slow, chronic, progressive cell death associated with a retrograde degeneration, that is, loss of spines and dendrites.

    We have shown that hyperphosphorylated tau from AD brains inhibits and disrupts microtubules probably by sequestering normal tau, MAP1, and MAP2 (Alonso et al., 1994, 1996, 1997, 2001), and that on aggregation into filaments this pathological tau loses its inhibitory activity (Alonso et al., 2006). Shahani et al. pointed out that synaptic loss might require Aβ pathology, a second hallmark of AD, which is lacking in the mouse brain slice model. Though one cannot rule out the possible role of Aβ in AD-type neurofibrillary degeneration, there are several other more likely explanations for the lack of synaptic loss in the mouse slice model which ought to be considered.

    For example, the pseudophosphorylated tau is not AD-like abnormally hyperphosphorylated tau. One should at least verify whether, like the AD p-tau, the pseudophosphorylated protein sequesters normal MAPs and inhibits and disrupts microtubule assembly (see Alonso, et al. 1994, 1996, 1997). The AD p-tau has been shown to self-assemble in vitro into bundles of paired helical filaments (Alonso, et al., 2001). The dynamics of the self-assembly of the pseudophosphorylated tau may be compared with those of AD p-tau.

    The level of tau expression in neurons in the model was several-fold higher than the endogenous tau expression. It is possible that the presence of such a high level of protein increases the chances of misfolding. This could induce stress in the neurons, triggering apoptosis independent of tau physiological function. The expression of pseudophosphorylated tau, even with the limitation of the mutation to glutamic acid to mimic phosphorylation, means the appearance in the cytosol of a molecule of tau phosphorylated at 10 sites. It is unlikely that tau gets phosphorylated at all the sites simultaneously in vivo. In fact, our in vitro studies suggest that tau phosphorylation is a stepwise process where the ability to sequester normal tau is acquired at ~4-6 moles of phosphate per mole of protein, and tau is able to self-assemble at ~10 moles of phosphate per mole of protein. This suggests a sequential change of conformation (Alonso et al., 2004). In this context, the nature of the sites selected for mutation is very important. One significant absence in this selection in the mouse slice model by Shahani et al. was Ser262, one of the tau phosphorylation sites implicated in tau-tubulin binding and a site that has been found to be phosphorylated in most of the tauopathies.

    In short, the approach presented by Shahani et al. appears to be quite attractive to study tau phosphorylation. However, a number of refinements might be required for such a model to reproduce the scenario found in Alzheimer disease brains.

    References:

    . Role of abnormally phosphorylated tau in the breakdown of microtubules in Alzheimer disease. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5562-6. PubMed.

    . Alzheimer's disease hyperphosphorylated tau sequesters normal tau into tangles of filaments and disassembles microtubules. Nat Med. 1996 Jul;2(7):783-7. PubMed.

    . Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: sequestration of microtubule-associated proteins 1 and 2 and the disassembly of microtubules by the abnormal tau. Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):298-303. PubMed.

    . Hyperphosphorylation induces self-assembly of tau into tangles of paired helical filaments/straight filaments. Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6923-8. PubMed.

    . Interaction of tau isoforms with Alzheimer's disease abnormally hyperphosphorylated tau and in vitro phosphorylation into the disease-like protein. J Biol Chem. 2001 Oct 12;276(41):37967-73. PubMed.

    . Promotion of hyperphosphorylation by frontotemporal dementia tau mutations. J Biol Chem. 2004 Aug 13;279(33):34873-81. PubMed.

    . Polymerization of hyperphosphorylated tau into filaments eliminates its inhibitory activity. Proc Natl Acad Sci U S A. 2006 Jun 6;103(23):8864-9. PubMed.

    . Tau aggregation and progressive neuronal degeneration in the absence of changes in spine density and morphology after targeted expression of Alzheimer's disease-relevant tau constructs in organotypic hippocampal slices. J Neurosci. 2006 May 31;26(22):6103-14. PubMed.

  3. Microscopic observation of many regions of the brain in advanced AD reveals, in addition to ghost tangles, a very high density of presumably still living neurons exhibiting an intact nucleus and also with a dense accumulation of neurofibrillary tangles (NFTs). Logic suggests that if a neuronal state is fleeting, it should be observed only rarely in the slice of time seen in fixed tissue under a microscope. On the other hand, if a neuronal state is long-lasting, one can expect to see it represented in many neurons in a slice of time—as is the case of apparently living neurons containing NFTs in AD brains. It is possible to derive counts in the hippocampus of these living neurons containing NFTs and combine these data with data, for example, of Mark West on numbers of neurons in hippocampus and rate of neuron loss in AD, to derive an estimate of how long a neuron may live, on average, after it has developed frank NFTs. The answer is clear that such neurons live for decades, suggesting that NFTs may not be the cause of their ultimate death (Morsch, Simon, and Coleman). This conclusion is consistent with earlier data of Gomez-Isla et al. (1997) that NFTs cannot fully account for the neuronal loss seen in AD. There are ample other factors that have been implicated in neuron death in AD.

    References:

    . Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer's disease. Ann Neurol. 1997 Jan;41(1):17-24. PubMed.

    . Neurons may live for decades with neurofibrillary tangles. J Neuropathol Exp Neurol. 1999 Feb;58(2):188-97. PubMed.

    . Differences in the pattern of hippocampal neuronal loss in normal ageing and Alzheimer's disease. Lancet. 1994 Sep 17;344(8925):769-72. PubMed.

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References

News Citations

  1. No Toxicity in Tau’s Tangles?
  2. News on Tau: More Mice Enter the Picture, Structure Takes Shape

Paper Citations

  1. . Tau-mediated cytotoxicity in a pseudohyperphosphorylation model of Alzheimer's disease. J Neurosci. 2002 Nov 15;22(22):9733-41. PubMed.
  2. . Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: sequestration of microtubule-associated proteins 1 and 2 and the disassembly of microtubules by the abnormal tau. Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):298-303. PubMed.
  3. . Region-specific dissociation of neuronal loss and neurofibrillary pathology in a mouse model of tauopathy. Am J Pathol. 2006 May;168(5):1598-607. PubMed.
  4. . Tau aggregation and progressive neuronal degeneration in the absence of changes in spine density and morphology after targeted expression of Alzheimer's disease-relevant tau constructs in organotypic hippocampal slices. J Neurosci. 2006 May 31;26(22):6103-14. PubMed.
  5. . Polymerization of hyperphosphorylated tau into filaments eliminates its inhibitory activity. Proc Natl Acad Sci U S A. 2006 Jun 6;103(23):8864-9. PubMed.

Further Reading

Papers

  1. . Tau aggregation and progressive neuronal degeneration in the absence of changes in spine density and morphology after targeted expression of Alzheimer's disease-relevant tau constructs in organotypic hippocampal slices. J Neurosci. 2006 May 31;26(22):6103-14. PubMed.
  2. . Polymerization of hyperphosphorylated tau into filaments eliminates its inhibitory activity. Proc Natl Acad Sci U S A. 2006 Jun 6;103(23):8864-9. PubMed.

Primary Papers

  1. . Tau aggregation and progressive neuronal degeneration in the absence of changes in spine density and morphology after targeted expression of Alzheimer's disease-relevant tau constructs in organotypic hippocampal slices. J Neurosci. 2006 May 31;26(22):6103-14. PubMed.
  2. . Polymerization of hyperphosphorylated tau into filaments eliminates its inhibitory activity. Proc Natl Acad Sci U S A. 2006 Jun 6;103(23):8864-9. PubMed.