Figure 1.
Model of tau protein with potential phosphosites.
The analysed tau phosphosites are indicated on the right designating the applied antibodies.
Figure 2.
Immunohistological analysis of tau phosphorylation in the neocortex of Syrian hamsters (Mesocricetus auratus).
The monoclonal antibody AT8 was used to determine the extent of tau phosphorylation in different states of hibernation. A–G: low magnification images showing the reversible phosphorylation pattern. Note that after the entrance into torpor the labelling in the apical dendrites and the band of Bechterew appears a few hours later than in the cell bodies of the pyramidal cells. H–L and M–Q: high magnification images corresponding to the insets in the images B–F and show the staining of pyramidal cells in detail. The labelling of apical dendrites is present very early after the entrance into torpor (arrow in H and M) and disappears at last during arousal (arrow in L). A few basal dendrites were labelled in late torpor (arrow in J). Scale bars: A–G, 200 µm; H–Q, 20 µm.
Figure 3.
Brain region specific, hibernation-state dependent tau phosphorylation in arctic ground squirrels (Spermophilus parryii).
Using various phospho site-specific antibodies we analysed the degree of tau phosphorylation by Western blot in brain extracts of neocortex, hippocampus, cerebellum, brainstem and the midbrain including the hypothalamus. The diagrams show the alteration in tau phosphorylation (mean ± SE) in animals of five different hibernation-states; euthermic animals (EU; n = 9), animals in early torpor (TE; n = 5), animals in late torpor (TL; n = 5), animals sampled shortly after spontaneous arousal (AE; n = 7) and animals sampled later after spontaneous arousal (AL; n = 5). The corresponding Western blots are displayed on the right showing representatively the immunoreactivity of the applied antibodies in the investigated brain regions (from top to bottom: neocortex, hippocampus, cerebellum, brainstem, midbrain. The approximate molecular weights (in kDa) are indicated on the left of the panel.
Figure 4.
Brain region specific, hibernation-state dependent tau phosphorylation in Syrian hamsters (Mesocricetus auratus).
Using various phospho site-specific antibodies we analysed the degree of tau phosphorylation by Western blot in brain extracts of neocortex, hippocampus, cerebellum, brainstem and the midbrain including the hypothalamus. The diagrams show the alteration in tau phosphorylation (mean ± SE) in animals of five different hibernation-states; euthermic animals (EU; n = 3), animals in early torpor (TE; n = 5), animals in late torpor (TL; n = 5), animals sampled shortly after induced arousal (AE; n = 3) and animals sampled later after induced arousal (AL; n = 4). The corresponding Western blots are displayed on the right showing representatively the immunoreactivity of the applied antibodies in the investigated brain regions (from top to bottom: neocortex, hippocampus, cerebellum, brainstem, midbrain. The approximate molecular weights (in kDa) are indicated on the left of the panel.
Figure 5.
Characterisation of hibernation-state dependent tau phosphorylation in American black bears (Ursus americanus).
We analysed tau status in the frontal cortex and hippocampus of summer active and hibernating black bears by Western blot using phospho-site-specific and conformation-specific antibodies. The histograms show the relative change in tau phosphorylation of hibernating animals in the frontal cortex (euthermic animals n = 6, hibernating animals n = 5) and hippocampus (euthermic animals n = 6, hibernating animals n = 4). The corresponding Western blots are displayed below, showing the immunoreactivity in frontal cortex (upper panel) and hippocampus (lower panel) (E – euthermic animal; H – hibernating animal). X indicates empty lanes caused by an unavailable hippocampal sample. The approximate molecular weights (in kDa) are indicated on the left. The application of the conformation dependent antibody Alz-50 revealed a significant alteration in tau conformation in the frontal cortex of hibernating animals. Significant alterations are indicated as follows: * p≤0.05; ** p≤0.01; *** p≤0.001.
Table 1.
Synopsis of hibernation dependent tau phosphorylation in arctic ground squirrels (Spermophilus parryii) and Syrian hamsters (Mesocricetus auratus).
Table 2.
Brain region specific comparison of hibernation related tau phosphorylation.
Figure 6.
Analysis of hibernation-state dependent alternative splicing of tau exon 10 in arctic ground squirrels (Spermophilus parryii) (euthermic n = 4; torpor long n = 4, arousal long n = 4).
Species-specific primers were used to determine the expression of tau exon 10 in different states of hibernation. A: Two distinct PCR products with a size of about 390 bp and 290 bp were obtained corresponding to the predicted PCR product size depending on the presence or absence of exon 10 (representative set). B: The ratio of expressed tau isoforms including and lacking exon 10, respectively, was determined by analysing the optical density of both PCR products. The expression was calculated as percentage related to the summative intensity of all bands (set to 100 %). The tau isoform expression pattern is significantly altered during hibernation (p = 0.0043; ANOVA) and the expression of tau isoforms including exon 10 is significantly decreased in the state of torpor (p = 0.0081; t-Test) and after arousal (p = 0.002; t-Test).
Figure 7.
Hibernation-state dependent formation of phospho-proteins in arctic ground squirrels (Spermophilus parryii) (euthermic n = 4; torpor long n = 4, arousal long n = 4).
We found no evidence for a state-dependent alteration of phospho-protein content (p = 0.618; ANOVA).
Figure 8.
Temperature dependent phosphate net turnover of tau protein in different hibernation-states.
The summative effect of kinase and phosphatase activities was assessed in cortical brain samples of arctic ground squirrels (Spermophilus parryii) (euthermic n = 4; torpor long n = 4, arousal long n = 4). Samples were incubated at various temperatures and times (37°C, 5 min; 33°C, 7 min; 30°C, 8.5 min; 25°C, 11 min; 15°C, 16 min; 5°C, 21 min; 0°C, 23.5 min). ANOVA was used to perform the statistical analysis and significant alterations are highlighted (** p≤0.01).
Figure 9.
Hibernation-state related tau phosphate net turnover in a decreasing (I-A, I-B) and increasing (II-A, II-B) temperature gradient.
The effect of enzyme activities was analysed in cortical brain samples of arctic ground squirrels (Spermophilus parryii) in different hibernation-states (euthermic n = 4; torpor long n = 4, arousal long n = 4). I-A and I-B: reactions were started at 37°C and aliquots were taken successively from the samples after incubation at different temperatures. II-A and II-B: Samples were pre-incubated 15 minutes at 33°C and instantly chilled on ice. Record of reaction was started at 5°C and aliquots were taken successively from the samples after incubation at different temperatures. Progress of reactions is shown in figures I-A and II-A while the differences in tau phosphate net turnover between two reading points (Δ) is shown in diagram I-B and II-B. Data indicating hibernation-state dependent differences in enzyme kinetics, consequently resulting in altered degree of tau phosphorylation after termination of reaction. Furthermore, these results demonstrate that enzyme kinetics are differentially regulated dependent on hibernation-state and temperature. Statistical analyses were performed with Student's t-Test and significant alterations are highlighted (** p≤0.01, * p≤0.05).
Figure 10.
Activity state profile of tau kinases in hibernating animals.
Using phospho-specific antibodies we analysed the activation status in neocortical brain extracts of Spermophilus parryii (I) and Mesocricetus auratus (II) respectively. The diagrams show the alteration in kinase phosphorylation (mean ± SE) in euthermic animals (EU), animals in late torpor (TL), and animals sampled after arousal (AL). The lanes of the corresponding Western blot panels are labelled with 1 (euthermic animals), 2 (torpid animals) and 3 (aroused animals) and show the detection of the phosphorylated enzyme (upper panel) as well as its expression level (lower panel) using phospho-independent antibodies. The approximate molecular weights (in kDa) are indicated on the left of the panel. Statistical analyses were performed using ANOVA and significant alterations are indicated as follows: * p≤0.05; ** p≤0.01; *** p≤0.001.
Figure 11.
Schematic illustration comparing the cellular consequences of physiological (hibernation) and pathological (AD) tau phosphorylation.
Table 3.
List of applied antibodies.
Table 4.
List of applied primers for cloning and isoforms expression analysis of tau in arctic ground squirrels.