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Alzheimer's could be stopped from progressing after scientists find disease 'spreads like an infection'
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By Reuters Reporter
Alzheimer's disease spreads in a predictable pattern like an infection, going from one brain cell to another along linked circuits known as synapses, researchers say.
The findings, published in the online journal PloS One, suggest that blocking the process early on may keep the disease from spreading.
Brain effect: This computer graphic shows a slice of the brain of an Alzheimer patient (left) compared with a normal brain (right). The Alzheimer's brain is considerably shrunken
'This is a phenomenon that is increasingly recognised and potentially very important,' said Dr Samuel Gandy, of the Mount Sinai Alzheimer's Disease Research Center in New York.
'If we understood this process, we could potentially arrest progression at an early stage.'
Imaging studies in people have suggested that Alzheimer's spreads from region to region in the brain rather than popping up spontaneously in different areas, but the evidence was not strong enough to say for sure.
'Everyone talks about Alzheimer's 'spreading', but there really has not been a standard theory,' study authors Dr Karen Duff and Dr Scott Small from the Columbia University Medical Center in New York, said.
'In the past, we have asked many of our colleagues in the field of Alzheimer's research what they mean when they say 'spread'. Most think that the disease just pops up in different areas of the brain over time, not that the disease actively jumps from one area to the next,' they said.
'Our findings show for the first time that the latter might be true.'
More than five million Americans and 465,000 people in the UK suffer from Alzheimer's, a brain disease that causes dementia.
Despite costly efforts, no drug has been found that can keep the disease from progressing.
There is currently no cure for Alzheimer's, which is a progressive condition and most common in people over 65
For their study, the team used mice that were genetically engineered to accumulate deposits of tau in a key memory center of the brain known as the entorhinal cortex, which is where that toxic protein starts to deposit in people.
Their aim was to map the progression of tau, an abnormal protein that forms tangles of protein fibers in the brains of people with Alzheimer's disease.
The team analysed the brains of the mice periodically over a period of 22 months to see how the disease progressed.
They found that as the mice aged, the abnormal human tau spread along a linked pathway, traveling from the entorhinal cortex to the hippocampus to the neocortex, areas of the brain needed to form and store memories.
That pattern closely follows the progression of Alzheimer's as it passes through various stages in people, Dr Duff said.
The team also saw signs that tau moved from brain cell to brain cell across synapses, connection points that allow nerve cells to communicate.
The researchers think those findings suggest new strategies for diagnosing and treating Alzheimer's disease.
'First, it would suggest that imaging tools that can detect entorhinal cortex dysfunction will be particularly helpful in diagnosing the earliest stages of the disease,' they said.
'More importantly, it might suggest ways of improving treatment.
'The implication of our study is that if it were possible to 'treat' Alzheimer's when it was first detected in the entorhinal cortex, this would prevent spread,' they said.
They likened the approach to treating cancer early, when it is still in one spot, and not waiting until it has spread.
The study may bring a new focus to diagnostics and treatments that focus on tau, rather than amyloid, the protein that causes plaques to form in the brain.
Current imaging agents used with PET scanners can identify amyloid deposits in the brain, but not tau.
Most late-stage Alzheimer's drugs, including Eli Lilly and Co's solanezumab, and Johnson & Johnson and Pfizer's bapineuzumab, take aim at amyloid, which accumulates silently 15 to 20 years before signs of dementia appear.
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Earlier treatment might be key in Alzheimer's
Feb. 3, 2012
Courtesy of Columbia University Medical Center
and World Science staff
Sci-en-tists have long de-bat-ed wheth-er Alzheimer's dis-ease starts in-de-pend-ently at dif-fer-ent times in dif-fer-ent brain ar-eas-or if it be-gins in one, and then spreads to con-nect-ed re-gions.
Re-search-ers have now an-nounced new find-ings that they say strongly sup-port the lat-ter the-o-ry about the dev-as-tat-ing, mem-o-ry-erasing ill-ness. They say an ab-nor-mal mol-e-cule known as tau-a key fea-ture of struc-tures known as "tan-gles" seen in Alzheimer's pa-tients' brains-seems to prop-a-gate along brain cir-cuits, "jump-ing" from cell to cell.
The find-ings, pub-lished Feb. 1 in the on-line jour-nal PLoS One, open new av-enues to bet-ter un-der-stand and help cure Alzheimer's and re-lat-ed dis-eases, said the stu-dy's sen-ior au-thor, Ka-ren Duff of Co-lum-bia Uni-vers-ity Med-i-cal Cen-ter and the New York State Psy-chi-at-ric In-sti-tute.
The re-sults sug-gest "the most ef-fec-tive ap-proach may be to treat Alzheimer's the way we treat can-cer-through early de-tec-tion and treat-ment, be-fore it has a chance to spread," said Co-lum-bia re-search-er Scott Small, a co-au-thor of the stu-dy. "That is the ex-cit-ing clin-i-cal prom-ise down the road."
Alzheimer's, the most com-mon form of de-men-tia, is char-ac-ter-ized by the ac-cu-mula-t-ion of ab-nor-mal struc-tures in brain cells called neu-rons. Scientists describe these ab-norm-alities as as plaques and fi-brous tan-gles, both of which are made up of pro-tein mol-e-cules. Stud-ies have sug-gested the dis-ease, es-pe-cially the tan-gles, be-gins in the en-torhi-nal cor-tex, a part of the brain that plays a key role in mem-o-ry. Then the ab-nor-mal-i-ties ap-pear in an-a-tom-ic-ally linked, high-er brain re-gions.
"But these var-i-ous find-ings do not de-fin-i-tively show that Alzheimer's spreads di-rectly from one brain re-gion to an-oth-er," said Small.
The re-search-ers de-vel-oped a ge-net-ic-ally en-gi-neered mouse in which the ab-nor-mal, hu-man ver-sion of tau is pro-duced mainly in the en-to-rhi-nal cor-tex. As the mice aged, the de-formed pro-tein was seen to spread along a linked ana-tom-i-cal path-way. "This pat-tern very much fol-lows [what] we see at the ear-li-est stages of hu-man Alzheimer's dis-ease," said Duff.
The group al-so found ev-i-dence that the pro-tein was mov-ing from neu-ron to neu-ron across synapses, the junc-tions that these cells use to com-mu-ni-cate with each oth-er. So treat-ments could con-ceivably tar-get tau while it's be-tween cells, added Duff. "If we can find the mech-an-ism by which tau spreads from one cell to anoth-er, we could po-ten-tially stop it from jump-ing across the syn-apses - per-haps us-ing some type of im-mu-noth-erapy."
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Alzheimer's Disease: Tau Spreads in the Brain, Not Between People
Released: 2/3/2012
Source: Alzheimer Research Forum Foundation
Newswise - Tau tangles are one half of the twin hallmark pathologies of Alzheimer's disease-the half that is most closely tied to the death of neurons. A study in the February 1 PLoS One offers evidence that the toxic forms of tau that cause these tangles spread throughout the brain by moving from one neuron to the next in a pattern that tracks anatomical synaptic connections of the earliest-hit nerve cells in the brain. This spread, the scientists found, recreates the typical disease progression usually observed in Alzheimer's, starting in a part of the brain called the entorhinal cortex.
This work made the front page of yesterday's New York Times. The Times article suggests that "Alzheimer's disease seems to spread like an infection from brain cell to brain cell...." Such an analogy may be misleading to the public, experts caution, implying falsely that Alzheimer's itself is contagious and can be passed between people.
In an Alzforum article posted today, reporter Tom Fagan takes apart this important scientific finding. Fagan puts it into the broader context of an emerging research trend, explains what it really means, and gets input from top experts in the field about why Alzheimer's cannot be caught like a cold.
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Trans-Synaptic Spread of Tau Pathology In Vivo
PLos one Jan 31 2012
PLoS ONE: Trans-Synaptic Spread of Tau Pathology In Vivo
www.plosone.org/article/info:doi/10.1371/journal.pone.0031302
Trans-Synaptic Spread of Tau Pathology In Vivo. Tauopathy in the brain of patients with Alzheimer's disease starts in the entorhinal cortex (EC) and spreads
"So now that you know that the disease is spreading by this protein, I imagine that's exciting, because there might be someway - to find someway to sop up the protein from spreading around.
Yeah. That's exactly what we're hoping to be able to do. We are a long way from any sort of development of therapy that could do this. But the idea here is that, yes, you can develop specific biological reagents, such as an antibody against the tau protein that's in these tangles. And as it leaves the cell, an antibody could perhaps intervene, you know, and prevent it from actually being picked up by the neighboring cell and suck it out, and prevent this, what we're calling spread, between brain regions."
"We need first a biomarkers. You know, we've been trying to develop biomarkers for - that we can test in the CSF of patients and the stage their disease and also imaging biomarkers where we can actually use something like PET imaging to identify these abnormal forms of the protein and where they are. We've been very successful for imaging biomarkers for amyloids, and it's used quite extensively, now. But we really haven't got anything for tau. So, yes, we need these tools. They'll help us with diagnosis and with estimating progression and staging of the disease, and then, you know, efficacy of treatments, if we can actually monitor how well the treatments have attacked these proteins."
"In general, our NT mouse model replicates the spatial and temporal aspects of the earliest stages (I-III) of Braak staging of tauopathy in Alzheimer's disease. We have demonstrated that tau pathology initiating in the EC can spread to other synaptically connected brain areas as the mice age, supporting the idea that AD progresses via an anatomical cascade as opposed to individual events occurring in differentially vulnerable regions. Thus, our NT transgenic mouse provides a model in which the spatial and temporal propagation of the disease can be predicted, and correlative functional outcomes can now be tested. Given that the earliest Braak stages are not associated with cognitive decline, identifying an EC based "biomarker" for pathology or dysfunction and developing therapeutic strategies to prevent propagation are likely to be both possible, and beneficial."
Li Liu1, Valerie Drouet1, Jessica W. Wu1, Menno P. Witter2, Scott A. Small3, Catherine Clelland1, Karen Duff1,4*
1 Department of Pathology and Cell Biology, Taub Institute for Alzheimer's Disease Research, Columbia University, New York, New York, United States of America, 2 Kavli Institute for Systems Neuroscience and Centre for the Biology of Memory, Norwegian University of Science and Technology, Trondheim, Norway, 3 Department of Neurology, Taub Institute for Alzheimer's Disease Research, Columbia University, New York, New York, United States of America, 4 Department of Psychiatry, New York State Psychiatric Institute, New York, New York, United States of America
Abstract
Tauopathy in the brain of patients with Alzheimer's disease starts in the entorhinal cortex (EC) and spreads anatomically in a defined pattern. To test whether pathology initiating in the EC spreads through the brain along synaptically connected circuits, we have generated a transgenic mouse model that differentially expresses pathological human tau in the EC and we have examined the distribution of tau pathology at different timepoints. In relatively young mice (10-11 months old), human tau was present in some cell bodies, but it was mostly observed in axons within the superficial layers of the medial and lateral EC, and at the terminal zones of the perforant pathway. In old mice (>22 months old), intense human tau immunoreactivity was readily detected not only in neurons in the superficial layers of the EC, but also in the subiculum, a substantial number of hippocampal pyramidal neurons especially in CA1, and in dentate gyrus granule cells. Scattered immunoreactive neurons were also seen in the deeper layers of the EC and in perirhinal and secondary somatosensory cortex. Immunoreactivity with the conformation-specific tau antibody MC1 correlated with the accumulation of argyrophilic material seen in old, but not young mice. In old mice, axonal human tau immunoreactivity, especially at the endzones of the perforant pathway, was greatly reduced. Relocalization of tau from axons to somatodendritic compartments and propagation of tauopathy to regions outside of the EC correlated with mature tangle formation in neurons in the EC as revealed by thioflavin-S staining. Our data demonstrate propagation of pathology from the EC and support a trans-synaptic mechanism of spread along anatomically connected networks, between connected and vulnerable neurons. In general, the mouse recapitulates the tauopathy that defines the early stages of AD and provides a model for testing mechanisms and functional outcomes associated with disease progression.
Introduction
AD is characterized neuropathologically by the presence of amyloid-ß containing plaques and neurofibrillary tangles (NFTs) composed of aggregated, fibrillar, hyperphosphorylated forms of the microtubule-associated protein tau. The earliest stages of the disease show accumulation of abnormal tau in the entorhinal cortex (EC) whereas later stages show accumulation in the hippocampus followed by neocortical areas [1]. As shown in Fig. 1, the EC is monosynaptically connected to other hippocampal subregions and it is trans-synaptically connected with affected regions in the temporal and parietal lobes [2], [3]. One of the most intriguing and poorly explored questions in the field is whether pathology, and/or dysfunction of the EC initiates anatomical progression of the disease, or whether pathology and/or dysfunction in extrahippocampal areas develops independently, and is unrelated to events occurring in the EC. There are a number of interesting, albeit circumstantial observations that support the trans-synaptic spread hypothesis for AD both in terms of pathology development and functional outcome. First, by simply charting the anatomical distribution of the pathology in human post-mortem tissue, the affected areas appear to be trans-synaptically linked [4]. Second, functional imaging studies in non-human primates have shown that lesioning the rhinal cortex (perirhinal and entorhinal) causes secondary dysfunction in the temporal and parietal lobes [5]. Currently available AD transgenic mouse models do not allow for studies of disease circuitry and progression as they generally overexpress APP or tau in inappropriate areas, or at high levels throughout the brain making it hard to identify temporal and spatial progression between vulnerable areas. To address this shortcoming, we have generated a transgenic mouse model with restricted expression of pathological human tau that predominates in the entorhinal cortex. We have performed a detailed histopathological analysis of the mice to map the change in distribution of tauopathy as the mice age. Our data support a temporal and spatially defined mechanism of trans-synaptic spread along anatomically connected networks, between connected and vulnerable neurons that replicates the early stages of AD.
Results and Discussion
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.
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 ß- 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.
Trans-synaptic spread of pathology identified, but mechanism unknown
One of the most intriguing observations from our studies is the appearance of tauopathy in cells outside of the entorhinal cortex. As shown in Fig. 4B, granule cells in the DG of old NT mice accumulate human tau protein, but it is unknown whether the human tau protein accumulated in the DG GCs derives solely from uptake or transfer of human tau from neurons in the EC, or if the human tau protein could be generated from endogenously produced human tau mRNA in the DG GCs resulting from "leaky" expression of the transgene. To test whether endogenously produced human tau in the DG GCs could be contributing to the tauopathy seen there, we collected approximately 1000 individual neurons by laser-capture microdissection (LCM) from the DG GC layer from old (Fig. 8A) and young (Fig. 8B) mice and assessed whether human tau mRNA was expressed in them. For this experiment, young mice at ~4 months of age were sampled to reduce the likelihood that incipient pathology had developed. Old NT, young NT and non transgenic mouse tissue sections were double immunolabeled with both CP27 and NeuN to ensure that neurons were isolated. Fig. 8C shows total RNA extracted from LCM isolated cell populations from one mouse from each type. A gel image from the 2100 Bioanalyzer, which employs capillary gel electrophoretic methodology to measure RNA integrity (RIN) and abundance, demonstrated the quality of sample from the LCM isolated cells in young mice - Non Tg (RIN 6.3; 418 pg/μl; total yield 4.18 ng), tau protein negative (Tau-) GCs (RIN 8.1; 443 pg/μl; total yield 4.43 ng), and old mice - tau protein positive (Tau+) GCs (RIN 8.6; 907 pg/μl; total yield 9.07 ng) and Tau- GCs (RIN 7.8; 351 pg/μl; total yield 3.51 ng). The level of human tau mRNA was then measured by quantitative (q) RT-PCR. To confirm the specificity of our human tau primers, LCM was used to isolate DG GCs from a non Tg young mouse that had been processed in the same way as the young NT mouse. QRT-PCR identified no amplification from the non Tg sample (CT>40) demonstrating that the primers were completely human specific (Fig. 8D). To assess whether amplification could result from residual DNA contamination even after DNAase treatment, RNA from DG GCs was used for qPCR with, or without the reverse-transcription step required to synthesize cDNA. Human tau amplicons in the sample that had not been reverse-transcribed were found to be at least twenty five fold (25X) lower than in the transcribed samples, suggesting that genomic DNA transgene priming resulted in only very low levels of PCR amplification compared to mRNA priming. Fig. 8D shows human tau expression in the human tau protein negative DG GCs (Tau- GCs) from a young NT mouse (left side of panel), and in human tau protein positive (Tau+ GCs) and negative (Tau- GCs) DG GCs from an old NT mice (right side of panel). Surprisingly, in the young NT mouse where all of the DG GCs were tau protein negative, the Tau- GCs had low but detectable levels of human tau mRNA after normalization to ß-actin. In old mice, human tau mRNA was again detected in DG GCs from the NT mouse, both in cells that were positive or negative for tau protein. Relative expression changes (2-∇∇CT) between the two populations of DG GCs in the old NT mouse showed a 27% increase in expression levels between Tau- and Tau+ DG GCs. A second, old NT mouse also had quantifiable levels of human tau mRNA in all three cell populations, with mRNA levels in Tau+ GCs>Tau- GCs (data not shown). The reason for this apparent increase is unknown but if it is significant in a larger sample group, it could reflect a feedback mechanism whereby tau transcription is upregulated if tau is losing its ability to perform its' normal function as it accumulates in the cytosol in the Tau+ DG GCs. The apparent increase in tau levels in old compared to young samples could reflect an age-dependent increase in transcription, or it could simply represent inter-animal variability.
Despite the absence of detectable human tau protein immunoreactivity in the tau negative DG GCs from young or old NT mice; human tau mRNA was identified in cells from three separate experiments suggesting that transgene expression was slightly leaky. To further examine whether ectopic expression in the DG GCs could explain the accumulation of protein at old age, we examined mice from two other crosses to the neuropsin tTA activator mouse. The first cross was to a reporter gene expressing LacZ with a nuclear localization signal to restrict the cellular distribution of the reporter to the nucleus of the cell in which it was produced [13]. As shown in Fig. 8E, positive staining for LacZ was restricted to very few cells in the DG GC layer. The second cross was to a mutant APP responder line. When crossed to the neuropsin-tTA activator, this mouse expresses APP predominantly in the EC as expected (data not shown), but due to secretion of Aß, plaques accumulate in regions outside of the EC, including layers of the DG. Of note, the cells of the DG GC layer did not accumulate APP/Aß supporting our observations that ectopic expression of responder genes in this cell layer is negligible. Therefore, despite our findings of some human tau mRNA in DG GCs, ectopic expression in these cells is very limited and unlikely to account for the extensive immunolabeling with human tau specific antibodies seen in the old mice. As we pooled many DG GCs and analysis of mRNA levels by qPCR is extremely sensitive, the actual number of cells contributing the human tau mRNA could be a very low percentage of the total that were human tau protein positive. How human tau protein accumulating in DG GCs that were likely to be human tau mRNA negative was derived is unknown, but it is possible that tau was released from cells originating in the EC, and internalized by DG GCs synapsing on to them. In support of this mechanism, a recent study has shown that tau can be released from cells via exosomes and tau positive exosomes have been identified in human CSF from AD patients [14].
In general, our NT mouse model replicates the spatial and temporal aspects of the earliest stages (I-III) of Braak staging of tauopathy in Alzheimer's disease. We have demonstrated that tau pathology initiating in the EC can spread to other synaptically connected brain areas as the mice age, supporting the idea that AD progresses via an anatomical cascade as opposed to individual events occurring in differentially vulnerable regions. Thus, our NT transgenic mouse provides a model in which the spatial and temporal propagation of the disease can be predicted, and correlative functional outcomes can now be tested. Given that the earliest Braak stages are not associated with cognitive decline, identifying an EC based "biomarker" for pathology or dysfunction and developing therapeutic strategies to prevent propagation are likely to be both possible, and beneficial.
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