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
- Fath T, Eidenmüller J, Brandt R. Tau-mediated cytotoxicity in a pseudohyperphosphorylation model of Alzheimer's disease. J Neurosci. 2002 Nov 15;22(22):9733-41. PubMed.
- Alonso AD, Grundke-Iqbal I, Barra HS, Iqbal K. 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.
- Spires TL, Orne JD, Santacruz K, Pitstick R, Carlson GA, Ashe KH, Hyman BT. 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.
No Available Further Reading
- 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 Neurosci. 2006 May 31;26(22):6103-14. PubMed.
- Alonso Ad, Li B, Grundke-Iqbal I, Iqbal K. 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.