Reversible paired helical filament-like phosphorylation of tau is an adaptive process associated with neuronal plasticity in hibernating animals

Abstract

Neurofibrillary pathology [paired helical filaments (PHFs)] formed by the microtubule-associated protein tau in a hyperphosphorylated form is a major hallmark of Alzheimer's disease and related disorders. The process of tau phosphorylation, thought to be of critical importance for PHF formation, and its potential link to neurodegeneration, however, is not understood very well, mostly because of the lack of a physiological in vivo model of PHF-like tau phosphorylation. Here we describe the formation of highly phosphorylated tau, containing a number of PHF-like epitopes in torpor during hibernation. PHF-like phosphorylation of tau was not associated with fibril formation and was fully reversible after arousal. Distribution of PHF-like tau followed a consistent pattern, being most intense in the entorhinal cortex, hippocampus, and isocortical areas. Within the hippocampus, a particularly high labeling was seen in CA3 pyramidal cells. Somewhat lesser reactivity was present in CA1 neurons while dentate gyrus granule cells were not reactive. Formation of PHF-like tau in CA3 neurons was paralleled by the regression of synaptic contacts of the mossy fiber system terminating on CA3 apical dendrites. Mossy fiber afferentation was re-established during arousal, concomitantly with the decrease of PHF-like tau in CA3 neurons. These findings implicate an essential link between neuronal plasticity and PHF-like phosphorylation of tau. The repeated formation and degradation of PHF-like tau might, thus, represent a physiological mechanism not necessarily associated with pathological effects. Hibernation will, therefore, be a valuable model to study the regulation of PHF-like tau-phosphorylation and its cell biological sequelae under physiological in vivo conditions.

Figure 1.

Phosphorylation of tau protein in the neocortex and hippocampus in an euthermic animal (EU), in long torpor (TL), and after long arousal (AL). Immunoblots were reacted for phosphorylation-independent detection of tau (BR134), specific PHF-like tau-phospho-epitopes (AT8, AT100, AT180, AT270, PHF-1, 12E8) and tau, unphosphorylated at Ser198, Ser199, and Ser202 (Tau-1).

Figure 2.

Tau protein isoform expression in hibernating and euthermic animals. Heatstable brain extracts from euthermic (EU), torpor (TL), and aroused (AL) animals were dephosphorylated with alkaline phosphatase and probed with anti-tau C-terminal antibody BR134. For comparison of the relative electrophoretic mobility, a mixture of the six recombinant human (τ) isoforms was run in parallel. No obvious change in isoform composition is apparent during the hibernation cycle.

Figure 3.

Relative expression levels of tau isoforms containing exon 10 and isoforms lacking exon 10. RT-PCR of a tau fragment comprising exon 10. Brains of ground squirrel contain a higher relative proportion of exon 10 containing tau mRNAs compared with human brain.

Figure 4.

Sequenz homology of tau in ground squirrel, rat, and human. Top, Alignment of amino acid sequence deduced from the cDNA sequence of the longest, most abundant tau isoform of ground squirrel with rat and human tau amino acid sequence (ClustalW program). Bottom, The cladogram, obtained by multiple sequence alignments using ClustalW, places ground squirrel tau in the group of rodent where it is most closely related to human.

Figure 5.

Immunohistochemical detection of PHF-like phosphorylated tau by the monoclonal antibody AT8 in long torpor (middle) as compared with a non-hibernating euthermic animal (left) and an animal after long arousal (right). Note the strong reactivity in the entorhinal cortex (arrowheads), hippocampus, cortex, as well as hypothalamic and epithalamic nuclei in torpor and its complete reversal within hours after arousal. Scale bar, 1 mm.

Figure 6.

Distribution of immunohistochemically detectable PHF-like phosphorylated tau (AT8) in the hippocampus during hibernation. A, Euthermic animal; insets are shown at higher magnification in B-E; A′, Animal in long torpor (TL); insets are shown at higher magnification in B′-E′. F-H, Neocortex (corresponding to the area shown in B, B′) in animals shortly after beginning of torpor (TS; F, F′), shortly after arousal (AS; G, G′), and after longer arousal period (AL; H, H′). F′-H′ correspond to insets in F-H. Scale bars: A, A′, 500 μm; B-E, B′-H′, 30 μm; F-H, 200 μm.

Figure 7.

Electron micrographs demonstrating longitudinally sectioned dendritic profiles (D) in the neocortex of an euthermic animal (A) and during long torpor (B). Arrows indicate microtubules. There are no indications for the formation of neurofibrillary aggregations. Scale bar, 0.5 μm.

Figure 8.

Cyclic changes in the hippocampal mossy fiber system during hibernation. A1-A4, Mossy fibers labeled by PSA-NCAM (arrowheads). Note the disappearance of staining during torpor and its progressive reappearance during arousal. B1-B5, Immunohistochemical reaction for synaptophysin in the stratum lucidum (arrowheads). Reactivity decreases and increases again with a similar time course as the PSA-NCAM labeling of mossy fibers. C1-C5, Double immunofluorescence for MAP2 (green) and AT8 (red), coexpression in yellow. Although MAP2 reactivity in the stratum lucidum (arrowheads) disappears in torpor and reappears during arousal with a similar time course as PSA-NCAM (A) and synaptophysin (B), an inverse pattern is observed for AT8 reactivity in corresponding pyramidal cell bodies. Scale bars: A, 300 μm; B, C, 50 μm.

Figure 9.

Immunoblots for MAP2 and synaptophysin from extracts of the neocortex and hippocampus in an euthermic animal (EU), in long torpor (TL), and after long arousal (AL).

Figure 10.

Synopsis of cyclic changes in the hippocampal mossy fiber system and CA3 target neurons during hibernation obtained by densitometrical analysis on immunohistochemical preparations, processed in parallel. An inverse cyclic relationship is observed between presynaptic markers (PSA-NCAM, synaptophysin) and postsynaptic markers (MAP2) in the stratum lucidum on one side and PHF-like tau (AT8) in somata of CA3 pyramidal neurons on the other side.