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Yetman MJ, Lillehaug S, Bjaalie JG, Leergaard TB, Jankowsky JL. Transgene expression in the Nop-tTA driver line is not inherently restricted to the entorhinal cortex. Brain Struct Funct. 2015 Apr 14; PubMed.
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University of Florida
I think the data nicely show that is possible for expression of tTA in the Nop-tTA mice to extend beyond what has been reported for these animals. As pointed out by Yetman et al., it is quite possible that the strain background of the mice modifies expression patterns for this tTA transgene. Thus, it is difficult to know whether the publications that used these mice to conclude that Aβ and tau spread trans-synaptically are flawed. Still, the Yetman study really sets a standard for how one goes about defining the pattern of transgene expression, and the data cast a considerable shadow on the published Aβ/tau work. It now becomes incumbent on the authors of these studies to more carefully prove an absence of APP or tau transgene expression in the regions of the brain that were said to have acquired pathology by trans-synaptic spreading.
Emory University
The study by Yetman and colleagues is nicely done, and the resolution of the images is superb. The authors convincingly show that the “leakage” of transgene expression in the Nop-tTA driver line is much more extensive than was previously thought. The results undoubtedly give pause, but they don’t necessarily negate earlier conclusions that a pathogenic agent (such as Aβ or tau seeds) is being transported by neurons (indeed, the authors concur with Harris et al., 2010, that Aβ appears to be transported to regions distant from the primary sites of transgene expression); whether specific agents are conveyed trans-synaptically to connected neurons in vivo remains to be determined. The conclusion “that leak rather than (or in addition to) trans-synaptic spread could have contributed” to the anatomical distribution of lesions in the AD mouse models seems reasonable in light of present knowledge. Numerous capable researchers are addressing the problem of cellular trafficking of seeds in vitro; I am especially looking forward to studies that nail down the mechanism of lesion spread within the living brain.
References:
Harris JA, Devidze N, Verret L, Ho K, Halabisky B, Thwin MT, Kim D, Hamto P, Lo I, Yu GQ, Palop JJ, Masliah E, Mucke L. Transsynaptic progression of amyloid-β-induced neuronal dysfunction within the entorhinal-hippocampal network. Neuron. 2010 Nov 4;68(3):428-41. PubMed.
The Jackson Lab
The Jankowsky group should be commended for doing this careful and important work; too often, this type of careful characterization is not done, which can lead to ambiguous or even misleading results.
Anyone selecting a mouse for a specific application should take advantage of the existing information resources. In addition to those mentioned in the paper, the Mouse Genome Informatics creportal presents annotated expression data on more than 2,200 distinct cre-expressing strains, representing more than 600 different genetic transgenic drivers and more than 400 knock-in drivers. The Allen Brain Atlas data portal presents brain serial sections of in situ analysis of cre reporters for more than 100 different cre lines, all of which are available from a mouse repository. We at JAX are also developing data on off-target expression in the developing embryo and in adult organs other than the brain (all information is available in the creportal).
While tet-expressing strains have not had the same level of analysis, there is a similar Klk8-tTA strain (same construct, different founder line from the Mayford lab) to that used in this paper with expression analysis presented in the Allen Brain Atlas data portal. This mouse is available from the MMRRC repository.
Ideally, other journals and funding agencies will take note of this type of work and encourage some common-sense practices to promote experimental reproducibility. It is very inefficient for each lab to have to do this type of analysis, so having central resources such as the Allen Brain Atlas and the Mouse Genome Informatics data are essential. The use of congenic, or at least standard, genetic backgrounds should be encouraged. Preferably, each project would assay multiple transgenic lines, to minimize the chance that the result is an artifact of aberrant expression in a single founder line. Where possible, use a mouse from a repository or donate your mouse to a repository, so that other labs can easily obtain the mouse and reproduce/extend your work. The simplest and most effective change would be to require that publications unambiguously identify the strain(s) that were used, ideally with a unique ID; very often it is not clear from a publication what experimental tools were used. PubMed has the “LinkOut” feature that can provide a quick link to the resources (e.g. mouse models). These practices don’t have to be costly, but can make results significantly more meaningful.
Yetman et al. report a straightforward experiment crossing Mayford’s Nop line S (which they obtained five years ago) with a reporter line. The initial line was selected from multiple lines because of its relative restriction to neurons of interest, predominantly but not exclusively in the medial entorhinal cortex. Yetman et al. show data from a handful of animals – using semiquantitative scoring – to illustrate the extent to which the promoter drives a reporter mostly but not exclusively in the entorhinal cortex. We (and I imagine all groups that have used this line) are in broad agreement with this observation, although in our hands the extent to which the reporter is expressed is not as widespread as they report. Still, our line has also been separately bred from the parent strain for ~five years, and some drift could easily occur.
Where we differ is the implication that the idea of tau propagation, put forward by our lab and by Karen Duff’s lab in 2012 (de Calignon et al., 2012; Liu et al., 2012) based in part on results from this line, should be questioned based on Yetman et al.’s observations that some neurons outside of the entorhinal cortex express the promoter. The critical observation by our lab and Duff’s is in the dentate gyrus, the neurons that receive projections from the entorhinal cortex become immunopositive for human tau protein as the mice age. We did not simply assume that entorhinal cortex was the only place that expressed human tau in the transgene. We did extensive control experiments to conclusively demonstrate that the dentate gyrus cells that contained human tau protein did not express human tau mRNA.
Five lines of evidence support this critical point, using a variety of techniques, have been presented in DeCalignon et al. and subsequently (Polydoro et al., 2014):
1. A fluorescent protein reporter line demonstrates absence of expression of a reporter protein in the dentate gyrus.
2. Double in-situ hybridization/immunofluorescence demonstrates absence of human tau mRNA in the exact neurons that are immunopositive for human tau protein
3. Laser capture microdissection of neurons that contain human tau mRNA versus those that did not, with or without being immunopositive for human tau protein, showed by a sensitive qPCR specific for human tau mRNA that none could be detected in the neurons that were in situ negative, tau protein positive.
4. The number of neurons that become immunopositive for tau increases with age and corresponds to the pace at which the dentate gyrus terminal zone degenerates. This observation would be difficult to explain if it were due to mistaken expression. These experiments directly ask if tau mRNA is expressed in the relevant cells, and the answer is unequivocally no.
5. To this list we can now add a fifth line of evidence: Yetman et al.’s table 1 shows a score of zero for the dentate gyrus, suggesting that with yet another and potentially more sensitive reporter, there is no misexpression in dentate gyrus.
It is worth noting that tau propagation has been seen in vitro and in vivo by multiple labs using multiple techniques, including direct injection of human tau protein and viral-based human tau expression (Dujardin et al., 2014; Wu et al., 2013).
Yetman et al. make an interesting point with regard to the possibility that strain or colony drift have changed the properties of the animals they examine. In our hands there are scattered neurons in other fields, although by far the predominant expression is in the entorhinal cortex and adjacent pre- and parasubiculum. In other transgenic lines we have observed phenotype drift over time and breeding, and we remain committed to carefully documenting expression patterns in the exact animals we use for experiments on an ongoing basis. Yetman et al. make the excellent point that this is important in any experimental paradigm where anatomical patterns of expression matter, and we definitely agree.
References:
Yetman MJ, Lillehaug S, Bjaalie JG, Leergaard TB, Jankowsky JL. Transgene expression in the Nop-tTA driver line is not inherently restricted to the entorhinal cortex. Brain Struct Funct. 2015 Apr 14; PubMed.
de Calignon A, Polydoro M, Suárez-Calvet M, William C, Adamowicz DH, Kopeikina KJ, Pitstick R, Sahara N, Ashe KH, Carlson GA, Spires-Jones TL, Hyman BT. Propagation of tau pathology in a model of early Alzheimer's disease. Neuron. 2012 Feb 23;73(4):685-97. PubMed.
Liu L, Drouet V, Wu JW, Witter MP, Small SA, Clelland C, Duff K. Trans-synaptic spread of tau pathology in vivo. PLoS One. 2012;7(2):e31302. PubMed.
Polydoro M, Dzhala VI, Pooler AM, Nicholls SB, McKinney AP, Sanchez L, Pitstick R, Carlson GA, Staley KJ, Spires-Jones TL, Hyman BT. Soluble pathological tau in the entorhinal cortex leads to presynaptic deficits in an early Alzheimer's disease model. Acta Neuropathol. 2014 Feb;127(2):257-70. Epub 2013 Nov 24 PubMed.
Dujardin S, Lécolle K, Caillierez R, Bégard S, Zommer N, Lachaud C, Carrier S, Dufour N, Aurégan G, Winderickx J, Hantraye P, Déglon N, Colin M, Buée L. Neuron-to-neuron wild-type Tau protein transfer through a trans-synaptic mechanism: relevance to sporadic tauopathies. Acta Neuropathol Commun. 2014 Jan 30;2(1):14. PubMed.
Wu JW, Herman M, Liu L, Simoes S, Acker CM, Figueroa H, Steinberg JI, Margittai M, Kayed R, Zurzolo C, Di Paolo G, Duff KE. Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons. J Biol Chem. 2013 Jan 18;288(3):1856-70. PubMed.
View all comments by Bradley HymanAllen Institute for Brain Science
Gladstone Institutes/UCSF
The Gladstone Institutes
The paper by Yetman et al. provides a valuable resource for investigators planning experiments with this neuropsin-tTA driver line, and the availability of this data in an open-access resource should be lauded. While bringing important attention to the fact that this neuropsin-tTA mouse line is not perfect in its restriction to the EC, the findings of Yetman et al. should not be seen in any way as “casting a shadow” on the results or interpretation of our APP/Aβ and hTau studies using the Nop-tTA line (Harris et al., 2010; Harris et al., 2012).
In fact, we systematically assessed expression of APP and hTau protein, and APP mRNA, in both the neuropsin-tTA/TetO-APP or TetO-hTau double-transgenic mice as well as in the singly transgenic TetO-APP and TetO-hTau across the cortex and hippocampus. We used this information to interpret our results with care (see, e.g., Figure 1 and Supplemental Figures 2-4 in Harris et al., 2010). Our analysis revealed essentially similar results as in the current paper. Thus, we very purposefully referred to expression in this mouse line as “predominantly” in the EC, a decision recognized by Yetman and co-authors.
The paper by Yetman et al. does bring to light a very important caveat on the use of transgenic mouse lines. The point should be made that not only do driver lines need to be carefully analyzed, as was done nicely in this paper, but reporter lines must be as well, as they carry their own intrinsic expression patterns (TetO lines can be leaky). We found this to be an issue with both the TetO-APP and TetO-hTau lines; there was true leaky expression of APP in the absence of tTA in CA1 of the TetO-APP line and of hTau in the dentate gyrus granule cells of the TetO-hTau mice. Leak of hTau in TetO-hTau singly transgenic mice was age-dependent. At young ages, some singly transgenic mice did not express any hTau, but at older ages, all mice did (Harris et al., 2012). This leakiness could be avoided by using the second generation tetO promoter (TRE-tight) in our hands (unpublished).
However, in agreement with our paper, Yetman et al. confirmed that the Nop-tTA/TetO-APP mice do not show detectable expression of APP in granule cells of the dentate gyrus, providing at least one pathway with which to test the transmission hypothesis of Aβ from point A (entorhinal cortex) to point B (granule cells of the dentate gyrus). In fact, the authors of this study agreed with our conclusion that the spread of Aβ pathology from the EC to the molecular layer of the DG likely arose through release from perforant path terminals. We also found alterations in calbindin and fos expression within DG granule cells and physiological changes specifically at EC to DG synapses which suggest a network-based transmission of pathology (here meaning molecular and functional changes) from one region to another without a clear understanding yet as to the exact mechanism of this transfer.
Unlike with the APP mice, we did detect hTau in DG granule cells of singly transgenic TetO-hTau mice. In light of this result, our opinion, as also stated in Harris et al., 2012, was that it was impossible to definitively conclude that hTau was transferred from the EC to the DG. We did, however, observe pathological forms of Tau in these same cells only in the Nop-tTA/TetO-hTau animals.
Thus, the main findings and conclusions of our APP/Aβ and hTau papers remain unchanged in light of this new paper: APP overexpression predominantly in the EC-hippocampal network is sufficient to drive age-dependent molecular and functional pathologies and cognitive impairments similar to those seen with brain-wide overexpression of APP/Aβ. On the other hand, although overexpression of hTau in a similar spatial pattern causes pathologies within this network, it is not sufficient to induce age-dependent cognitive decline, at least up to 16 months of age.
References:
Harris JA, Koyama A, Maeda S, Ho K, Devidze N, Dubal DB, Yu GQ, Masliah E, Mucke L. Human P301L-mutant tau expression in mouse entorhinal-hippocampal network causes tau aggregation and presynaptic pathology but no cognitive deficits. PLoS One. 2012;7(9):e45881. PubMed.
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