Histopathology studies demonstrate progressive tauopathy radiating from the EC

A detailed histopathological analysis was performed on relatively young (10–11 mon) and aged (∼22 mon) neuropsin-tTA-tau (NT) transgenic mice using three different antibodies, MC1, CP27 and AT8. The MC1 monoclonal antibody detects human tau in an abnormal conformation [6] that is associated with early stage NFT–tau in human AD patients [7]. In young NT mice (Figs. 2A, D, G), abnormal human tau recognized by MC1 was most abundant in the medial EC (MEC). Relatively dense staining was also seen in the lateral EC (LEC) and the para-(PaS) subiculum while the presubiculum (PrS) was less intensely stained. Dense staining was seen in superficial layers II and III of the EC, whereas deeper layers showed considerably less staining. Human tau was present in some cell bodies, but mostly in neurites within the superficial layers of the MEC and LEC (Fig. 2G). Dense tau staining was seen in the middle third of the molecular layer of the DG and CA3 (Fig. 2D) but not the outer layer indicating tau in axon terminals of the perforant pathway (pp) that originate from layer II of the MEC [8]. In the CA1 and subiculum, the outer molecular layer was labeled extensively (Fig. 2D), indicating tau in perforant path terminals from layer III cells in both LEC and MEC [8]. Mice expressing only the uninduced tau transgene (control) showed negligible (Fig. 2C), or very limited immunoreactivity with the antibodies used, and it was usually restricted to the mossy fibers (for example, antibody CP27, Fig. 3C). Some non-specific staining in the fornix was seen in all mice, with all antibodies. By 22 months of age, the distribution of human tau in old NT mice had changed dramatically to resemble that seen in more affected AD brain tissue (Braak stages II–III). Intense MC1 immunoreactivity was readily detected not only in neurons in the superficial layers of the EC and throughout the subiculum (Figs. 2B, E, H), but in pyramidal neurons in the hippocampus, especially in CA1, and also in dentate gyrus granule cells (DG GCs) (Fig. 2E). Somatodendritic staining with MC1 was intense for cells in the MEC (Fig. 2F). Scattered MC1 positive neuronal cell bodies could also be seen in the perirhinal and the parietal cortices (Fig. 2I), and more extensively in the deeper layers of the EC.

The pattern of staining was reproduced in young and old NT mice using a human specific tau antibody (CP27) that recognizes all human tau, regardless of phosphorylation or conformation status (Fig. 3). Subtle differences in the relative intensity of staining in different areas were observed for different antibodies, especially in the DG GC layer where CP27 staining was more intense and extensive than MC1 (Figs. 3G and 4B vs. Fig. 2E). This could either indicate differential sensitivity of the antibodies, differential synthesis or clearance of tau forms recognized by the two antibodies, or retarded development of the conformational change in tau recognized by MC1.

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larger image TIFF original image Download: Figure 4. Progressive spread of tauopathy to monosynaptically connected regions of the hippocampus. Young NT mice (Fig. 4A) show accumulation of human tau immunolabeled with CP27 predominately in the endzones of the perforant pathway that terminate in the middle third of the molecular layer of the DG (area 3). Terminals from neurons in the LEC terminating in the outer third of the molecular layer are shown in area 4. Human tau was also seen in cells in the hilus (area 1). Granule cell layers of the DG (area 2) did not accumulate human tau at this age. Old NT mice (Fig. 4B) show accumulation of human tau in cell bodies in the granule cells of the DG (area 2). Increased accumulation of human tau is seen in layers 1, 2 and 4 but the perforant pathway endzone in layer 3 was significantly depleted of tau. Magnification = 20×. https://doi.org/10.1371/journal.pone.0031302.g004

To assess whether tauopathy could spread across a synapse, we examined cells in the DG that are monosynaptically connected with cells in the EC (Fig. 4). Young NT mice (Fig. 4A) showed robust accumulation of CP27 immunoreactive human tau in the endzones of the perforant pathway that originate from neurons in the MEC and terminate in the middle third of the molecular layer of the DG (area 3). Low, but detectable levels of immunoreactivity were seen in the outer third of the molecular layer (area 4) which represents terminals from neurons originating in the LEC. Some human tau was seen in cells in the hilus (area 1), most likely in mossy cells. Notably, human tau did not accumulate in DG GCs (area 2) in young NT mice. Old NT mice however showed a very different distribution of human tau (Fig. 4B). Robust accumulation of human tau was now seen in DG GCs (area 2) and increased accumulation of human tau was seen in layers 1, 2 and 4. The appearance of tau in DGGCs strongly supports the idea that tauopathy initiated in the EC can spread between cells that are connected, but physically separated by a synapse. Interestingly, the perforant pathway endzone in layer 3 was significantly depleted of tau which coincided with accumulation in originating cell bodies in the MEC (Fig. 2H, 2F). This apparent relocalization of tau from axons to somatodendritic compartments is one of the earliest events in the pathological cascade of early Alzheimer's disease [1].

Tauopathy in AD is usually staged using the antibody AT8 [9]. This antibody recognizes phosphorylated epitopes S202/205 (in both mouse and human tau) that are abundant in tau from AD brain, but not normal brain [10]. In young NT mice, (Figs. 5A, D) AT8 immunoreactive tau was mainly concentrated in the EC with no staining visible in the hippocampal subfields. Cell body staining was predominant with relatively less staining seen in neurites. In old NT mice (Figs. 5B, E, F, G), there was considerably more neurite staining throughout the EC (Fig. 5B, E), and in all fields of the hippocampus (Fig. 5F), with cell body immunoreactivity being seen in scattered neurons that were most prominent in pyramidal cells in the CA1 and in DG GCs. As for MC1, in the old mice, additional cell body staining was apparent in the deeper layers of the EC, and in cells in the perirhinal and parietal cortices (Fig. 5G). The control mouse (Fig. 5C) was essentially negative for this antibody. Overall, the pattern of staining, including extensive staining of cell bodies and neurites throughout the EC and hippocampus was reminiscent of that described for early Braak stages of AD [9].

Although the exact type of tau associated with functional impairment and degeneration is not known [11], the accumulation of insoluble, conformationally abnormal, hyperphosphorylated tau into mature neurofibrillary tangles in the somatodendritic cell compartment is generally associated with more severe pathology, degeneration and cell death. To test whether mature tangles had formed in the NT mice, we examined tissue sections stained with thioflavin S (thioS), a dye that binds to proteins in a β- sheet conformation, indicative of tau in mature tangles (Fig. 6). Special care was taken to mask lipofuscin fluorescence which is significant in old mice. A small number of neurons restricted to the MEC were positive for thioS in old NT mice (Figs. 6B, E). Young NT (Fig. 6D) and old control mice (Figs. 6A, C) were essentially negative. Not all of the tau immunoreactive neurons in the MEC of old NT mice were thioS positive, and cells in the LEC, CA1 and DG GC layer were thioS negative, as were neurites and axonal terminals in the perforant pathway. As cells with the highest level of human tau occur in the MEC, the lack of staining in other areas is most likely explained by the lower tau levels rather than by regional sensitivity to tangle formation, but the latter interpretation cannot be ruled out in these studies.

Altered conformation of proteins can also be visualized by silver staining using one of several methods [12]. Argyophilic plaques, tangles and neurites are abundant in the human AD brain. Abundant, argyrophilic cell body and neurite staining was also seen in the old (Figs. 7B, D, F), but not the young NT mice (Fig. 7A), and it was related to tauopathy development rather than aging as parallel-processed, old littermate control mice were negative (Figs. 7C, E, G). The distribution of histopathology in the old NT mouse was extensive, with robust staining being seen in cells in the EC, as well as in the subiculum (Fig. 7D). Staining was also extensive in the CA1, but to a lesser extent in the DG GC layer (Fig. 7F). In general, the distribution of silver-staining matched that seen with the MC1 antibody more closely than that seen with the CP27 or AT8 antibodies, suggesting that it is the conformational change in tau recognized by MC1 that is recognized by the silver stain. Interestingly, MC1 immunoreactivity was robust in neurites in the young NT mice but these mice were negative for silver staining. Therefore the silver stain recognizes a more advanced conformational abnormality that lies between pre-tangle MC1 immunoreactivity seen in the young mice, and the overt conformational change recognized by thioS, which in the old mice, is restricted to cells in the MEC.