Tauopathies such as Alzheimer’s disease (AD) feature progressive intraneuronal deposition of aggregated tau protein. The cause is unknown, but in experimental systems trans-cellular propagation of tau pathology resembles prion pathogenesis. Tau aggregate inoculation into mice produces transmissible pathology, and tau forms distinct strains, i.e. conformers that faithfully replicate and create predictable patterns of pathology in vivo. The prion model predicts that tau seed formation will anticipate neurofibrillary tau pathology. To test this idea requires simultaneous assessment of seed titer and immunohistochemistry (IHC) of brain tissue, but it is unknown whether tau seed titer can be determined in formaldehyde-fixed tissue. We have previously created a cellular biosensor system that uses flow cytometry to quantify induced tau aggregation and thus determine seed titer. In unfixed tissue from PS19 tauopathy mice that express 1 N,4R tau (P301S), we have measured tau seeding activity that precedes the first observable histopathology by many months. Additionally, in fresh frozen tissue from human AD subjects at early to mid-neurofibrillary tangle stages (NFT I-IV), we have observed tau seeding activity in cortical regions predicted to lack neurofibrillary pathology. However, we could not directly compare the same regions by IHC and seeding activity in either case. We now describe a protocol to extract and measure tau seeding activity from small volumes (.04 mm3) of formaldehyde-fixed tissue immediately adjacent to that used for IHC. We validated this method with the PS19 transgenic mouse model, and easily observed seeding well before the development of phospho-tau pathology. We also accurately isolated two tau strains, DS9 and DS10, from fixed brain tissues in mice. Finally, we have observed robust seeding activity in fixed AD brain, but not controls. The successful coupling of classical IHC with seeding and strain detection should enable detailed study of banked brain tissue in AD and other tauopathies.
Propagation of tau aggregation along neuronal networks may mediate the progressive accumulation of pathology observed in tauopathy patients. To measure tau seeding activity in well-characterized human brains, it will be necessary to analyze formaldehyde-fixed tissues. We now present a method for extracting tau seeding activity from miniscule amounts of fixed tissue (approximately .04 mm3) to permit direct comparison with tissues stained by IHC.
We first tested this method in PS19 mice that overexpress full-length human tau (1 N,4R) containing the P301S mutation. We drop-fixed brain samples that had been embedded either in paraffin or PEG and sectioned them coronally for microscopy. We analyzed adjacent 50 μm sections using standard IHC to detect phospho-tau or 1 mm circular punch biopsies of tissue for seeding assays. We homogenized punch biopsies by water-bath sonication in closed tubes, and assayed them in a cellular FRET bioassay system as described previously [10, 13].
Tau seeding activity tracked the development of pathology more efficiently than IHC, with a lower degree of inter-animal variation, and a higher dynamic range. This was perfectly comparable to previously obtained results using fresh frozen tissue . In addition, we detected seeding activity relatively early in the course of disease (1–2 months) and it steadily increased over time. Next, we tested brain tissues from animals previously inoculated with two distinct tau prion strains. We recovered these strains from fixed mouse brain tissue as accurately as we had previously from fresh frozen tissue. Finally, we tested the extraction method in fixed human brain tissue with documented AT8-positive tau pathology, including AD, and readily detected tau seeding activity in cases archived for up to 27 years in formaldehyde.
Our laboratory previously detected tau seeding activity in fresh frozen brain tissue from mouse tauopathy models and human AD cases[11, 13]. However, fresh frozen samples are much more difficult to obtain than fixed tissue sections, must be carefully stored at−80 °C, and are very challenging to dissect precisely to isolate specific brain regions. The assay described here accurately quantifies tau seeding from fixed tissue sections over three log orders of signal. Remarkably, in a mouse model from which we sampled tissue at different time points, fixed tissue seeding proved comparable to seeding activity detected in fresh frozen tissue. Thus, we expect that this assay will enable assessment of tau seeding activity in a range of fixed tissues at a similar level of sensitivity to fresh frozen samples.
Moreover, we detected seeding activity in a small sample of human tauopathy cases that were collected and stored in formalin for over 20 years prior to this study. We observed lower seeding activity in these human samples than in PS19 mice, probably because of the overexpression of an aggregation-prone form of tau in this mouse model. However, the length of fixation may affect the level of seeding observed in samples. Further, differences in seeding activity observed between patients at Braak stage III and V likely reflect differences in the level of tau aggregate burden between these patients, cell loss, or ghost-tangle formation at later disease stages. Given the early detection of seeding activity relative to AT8 staining in PS19 mice, we anticipate that this assay could represent a more sensitive metric of tau pathology. Additional studies in a large number of well-characterized human tissue samples will help address these important questions, and provide additional insight into the progression of seeding activity in human tauopathies.
Earlier work described a dose-dependent increase in tau seeding activity in the PS19 mouse tauopathy model . However, the regional specificity possible with fresh frozen tissue was limited to gross dissection. We now have reliably isolated and characterized punch biopsies as small as 1 mm diameter x 50 μm (or ~ .04 mm3). When we quantified the level of seeding activity at increasing ages vs. the tau pathology observed in adjacent tissue slices using anti-tau AT8 staining, we easily detected tau seeding activity, even in fixed tissue sections with a minimal AT8 signal. For example, when PS19 mice were inoculated with tau strains, we induced strong AT8 pathology with DS9, whereas DS10 produced a weak signal. In both cases, the pathology spread from the site of inoculation to connected regions, as described elsewhere . The fixed tissue seeding assay more readily detected the spread of tau pathology in this propagation model. Furthermore, we readily detected seeding activity in DS10 inoculated mice despite the relatively subtle AT8 staining phenotype induced by this strain (mossy fiber dots). Consequently seeding activity can serve as an important measure of tau pathology when routine AT8 staining reports otherwise minimal pathology. The combination of precise quantification of seeding activity with the ability to sample brain tissue to 1 mm resolution indicates that this method could help define the seeding activity in human brain with remarkably high accuracy.
Detection of tau strains in formaldehyde-fixed tissue
Prior experimental work indicates that distinct tau aggregate conformations may underlie different patterns of pathology, rates of progression, and disease phenotypes observed in distinct tauopathies [2, 7, 22]. Distinct tau strains are associated with different tauopathies , and inoculation of unique tau strains produces different patterns and tau pathology rates of progression . We observed that fixed tissue from mice inoculated with DS9 and DS10 produced strain phenotypes identical to the original strains upon inoculation into LM1 biosensor cells. Thus, tau strains are stable upon fixation. We anticipate that formaldehyde-fixed tissues will serve as an invaluable resource to examine the role of strain composition in tauopathies.
Studies that use traditional IHC techniques to detect tau pathology have provided important insights into the progression and anatomy of macromolecular accumulations of tau assemblies. However, these methods cannot discriminate among distinct strains, nor can they detect submicroscopic tau assemblies. The present assay measures tau pathology based on seeding activity and is also sensitive to strain composition. We anticipate that punch biopsies taken from tissue sections will be useful to measure strain identity with high anatomical precision. By carefully comparing seeding activity and strain composition with standard neuropathology, it should be possible to add new dimensions to analyses of tissue samples from a range of neurodegenerative diseases. In turn, this will facilitate more widespread testing of the putative role of tau prion activity in human tauopathies.