Human P301L-mutant tau expression in mouse entorhinal-hippocampal network causes tau aggregation and presynaptic pathology but no cognitive deficits

Abstract

Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer's disease (AD). It can occur before significant Aβ deposition and appears to "spread" into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here.

Figure 1. Spatially restricted expression of P301L-mutant hTau in EC-hTau mice.

Representative images of sagittal (A, B, E, F) and horizontal (C, D, G, H) brain sections from 4-, 8-, 12-, and 16-month-old EC-hTau mice (top) and NTG controls (bottom) immunostained for hTau with the anti-hTau antibody HT7 (A–D). In EC-hTau mice, hTau expression was observed primarily in cell bodies and neuropil of the EC and in PP terminals in the hippocampus (arrows). Mossy fiber axons of DG GC also stained for hTau (arrowheads). From 8 months onward, there was labeling of scattered cells in the DG, CA3 and CA1 regions in the hippocampus. (E–H) Only nonspecific background staining was observed in NTG mice. Scale bar (H) = 500 µm.

Figure 2. EC-hTau mice do not have deficits in the Morris water maze (MWM).

(A–H) Mice from each of the four genotypes were tested at 4, 8, 12 and 16 months of age in the MWM. (A–D) Learning curves. (E–H) Twenty-four hours after the last training session, spatial memory was tested in a probe trial. (I, J) An independent cohort of 8-month-old behaviorally naïve EC-hTau mice also showed normal learning (I) and memory retention (J). (K–P) EC-hTau mice also displayed normal reversal-learning in the MWM. (K–M) After the initial training to find a hidden platform, the platform was moved to a new location in the opposite pool quadrant. Mice were then trained to navigate to this new location for 3 days. (N–P) Twenty-four hours after the last reversal training session, spatial memory was tested in a probe trial. *p<0.05, **p<0.005, ***p<0.0005 vs. the average percent time spent in non-target quadrants (Tukey test). Values are mean ± SEM.

Figure 3. EC-hTau mice do not have deficits in other tests of learning and memory.

(A–C) Mice of the four genotypes were tested in a novel object recognition task at 4 (A), 12 (B), and 16 (C) months of age. At all ages, all groups of mice spent significantly more time with the novel object, indicating memory of the familiar object. (D) At 12 months, mice were tested in a fear-conditioning task. Mice from all genotypes spent significantly more time freezing in the test session 24 hours after training. (E) At 16 months, mice were tested in a novel place recognition task. Again, all genotypes spent significantly more time with the object in the novel location, indicating memory for the familiar place. ***p<0.0005 vs. empty bars (Tukey test). Values are mean ± SEM.

Figure 4. Expression of pathological tau in superficial EC layers of EC-hTau mice.

(A–D) No obvious change in expression of the hTau transgene was observed between 4 and 16 months of age. (E–H) Abnormal conformation of tau, detected with the MC-1 antibody, was observed in EC neuropil at 4 months of age. At 12 and 16 months, cell bodies were also positively stained by MC-1 (G,H). (I–L) Phosphorylation of tau at serine 202 was detected with the CP-13 antibody. Cell bodies were positively stained for CP-13 across all ages, but some more intensely labeled neurons were observed at the older ages. CP-13 positive neuropil labeling also increased by 12 months (K). (M–P) Phosphorylations of tau at serine residues 199, 202, and 205 were detected with the AT8 antibody. Cell bodies were positively stained for AT8 across all ages. AT8 staining of the neuropil was maximal at 12 months (O). (Q–T) Phosphorylations of tau at serine 396 and 404 were detected with the PHF1 antibody. Scattered PHF1-positive cell bodies were seen at all ages. The neuropil and neurons were stained more intensely at 12 and 16 months (S,T). Scale bar (T) = 50 µm.

Figure 5. Pathological tau in the DG of EC-hTau mice.

(A–D) hTau was detected in PP terminals in the outer molecular layer of the DG with the HT7 antibody at all ages examined. However, darkly stained cells in the granule cell layer (GCL) were seen primarily at 12 and 16 months (C, D). (E–H) PP terminals stained positive for MC-1 at all ages. By 12 and 16 months, GC also stained with MC-1 (G,H). (I–L) Faint CP13-positive PP terminals (arrows in K,L) and many more GC were observed at 12 and 16 months of age. (M–P) AT8 staining of the PP and GC increased markedly at 12 months (O) and many more positive GC were seen at 16 months (P). (Q–T) PHF1 staining of GC and the neuropil was also increased at 12 and 16 months (S,T). Scale bar (T) = 50 µm.

Figure 6. DG GC of tet-hTau singly transgenic mice express hTau but not abnormal tau.

(A–P) Brain sections from tet-hTau singly transgenic mice of different ages were immunostained with the hTau-specific antibody HT7 (A–H) or with antibodies that recognize misfolded (MC1; I–L) or abnormally phosphorylated (CP-13; M–P) tau. (A–D) Low power images show prominent expression of hTau in GC axons (mossy fibers, arrowheads) at all ages. (E–H) Cell bodies of GC were also variably immunoreactive for hTau at all ages examined. (I–P) GC cell bodies of tet-hTau singly transgenic mice did not stain with MC-1 (I–L) or CP-13 (M–P). However, we did observe MC-1 positive MFs in tet-hTau singly transgenic mice at all ages (data not shown). Scale bars: 500 µm (A–D) and 50 µm (E–P).

Figure 7. Tau aggregates in the DG of 16-month-old EC-hTau mice.

(A–E) Gallyas silver staining revealed no abnormalities in NTG (A) and tet-hTau singly transgenic (B) controls. In contrast, EC-hTau mice had neuropil threads in the outer molecular layer of the DG (C, box 1 enlarged in panel D) and tangle-like inclusions in GC of the DG (box 2 enlarged in panel E). (F) Low magnification (5,000X) view of a GC. (G–H) Higher magnification (30,000X) view of intracellular filamentous aggregates in GC. (I) Immuno-EM analysis of the packed intracellular filaments with the PHF1 antibody. Gold particles decorate straight filaments; gold particles enhanced with silver solution are 15–20 nm. (J) Negative control (no primary antibody) shows the specificity of the immunogold labeling. Scale bars: 30 µm (A, B), 10 µm (D, E), 2 µm (F), 0.5 µm (G, H), 0.1 µm (I) and 0.05 µm (J).

Figure 8. Biochemical detection of abnormal tau aggregation in EC-hTau mice.

(A, B) Levels of tau dimers (d) in the EC (A) and DG (B) of EC-hTau mice, NTG mice (negative control) and rTg4510 mice (positive control) were detected by western blot analysis with antibodies against total tau (Tau5). The middle panels show shorter exposures of the tau monomer (m) band, used for optical density quantification. GAPDH was used as a loading control. (C–D) Ratios of tau dimers to monomers as determined by densitometric quantitation of western blot signals obtained from EC (C) and DG (D) homogenates. ***p<0.0005 vs. all EC-hTau groups or *p<0.05 as indicated by brackets (Tukey test). Values are mean ± SEM. (E) Sarcosyl-insoluble tau was extracted from the EC and DG of EC-hTau and rTg4510 mice and detected by western blot analysis with the Tau5 antibody.

Figure 9. Synaptic alterations in the DG of 16-month-old EC-hTau mice.

(A–F) Sagittal brain sections of NTG and EC-hTau mice were immunostained for synaptophysin (A–C) or synapsin (D–F). Levels of immunoreactive terminals were quantitated in the DG molecular layer (C, F). n = 8 mice per group. *p<0.05 (Student’s t test). Values are mean ± SEM. (G–P) Ultrastructural analysis of synaptic alterations in EC-hTau mice. Electron micrographs were obtained at 25,000X from the molecular layer of the DG. Representative images from NTG controls (G–K) show presynaptic terminals (PST), spines (Sp) and dendrites (Den) with normal characteristics, including abundant round, clear synaptic vesicles and dense postsynaptic apparatus. In EC-hTau mice (L–P), presynaptic terminals were enlarged and irregular with laminated electrondense bodies (LEB), vesicular-tubular structures (arrows in M), enlarged vesicles (arrows in N), a paucity of small synaptic vesicles (SV), and a diffuse appearance of postsynaptic sites (encircled). Dendrites of EC-hTau mice also contained multivesicular bodies (MVB in O). Scale bars = 20 µm (in E for A, B, D and E) and 0.5 µm (in P for G–P).

Figure 10. Enrichment of PHF1-tau in PP to GC synapses of 16-month-old EC-hTau mice.

(A–F) Images from the outer molecular layer of the DG from sections of EC-hTau and NTG mice colabeled for pTau (PHF1, red) and synaptophysin (SY38, green). (G) Quantification of the number of synaptophysin-positive punctae that were also positive for pTau. (H–M) Images from the outer molecular layer of the DG from sections of EC-hTau and NTG mice colabeled for pTau (PHF1, red) and MAP2 (green). (N) Quantification of the number of MAP2-positive structures that were also positive for pTau. (O–R) Immuno-EM images of PP to GC synapses in the molecular layer of the DG from immunogold (PHF1)-labeled sections of NTG and EC-hTau mice. Arrows in (P) indicate gold particles. (S, T) Quantification of gold particles in presynaptic (S) and postsynaptic (T) structures. n = 8 mice per group, *p<0.05 (Student’s t test). Values are mean ± SEM.