Propagation of Tau Pathology in a Model of Early Alzheimer's Disease

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In 24-month-old rTgTauEC mice, endogenous mouse tau accumulates in the somatodendritic compartment of medial entorhinal cortex neurons and co-localizes with Alz50.

“To determine whether mouse tau is recruited by human tau to aggregate, we performed immunohistochemistry using an antibody specific for mouse tau that revealed that mouse tau indeed accumulates in the somatodendritic compartment of MEC neurons of 24 month-old rTgTauEC mice (Fig 4A).”

• Also stated in: § Human tau seeds mouse tau aggregation · plain

  “Results from double labeling using Alz50 and mouse tau antibodies shows that Alz50 and mouse tau staining co-localize in neuronal cell bodies of MEC which is further evidence for mouse tau recruitment into aggregates in the rTgTauEC mouse model (Fig 4B).”

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Human tau expression in the medial entorhinal cortex causes age-dependent accumulation of tau pathology in transgene-expressing neurons, with Alz50 axonal staining at 3 months preceding somatic Alz50 at 6 months, PHF1 at 12 months, Gallyas at 18 months, and Thioflavin S at 24 months.

“Human tau expression in the MEC results in an age-dependent accumulation of tau pathology in transgene expressing neurons.”

• Also stated in: § Restricted transgene expression and age-dependent pathology in EC of rTgTauEC mice · plain

  “The first sign of tau pathology was detected at 3 months of age with Alz50 staining, an early indicator of tau misfolding, in the projection input zone of MEC, corresponding to the middle third of the molecular layer of the dentate gyrus (Fig 1E, left most panel), while the inner and outer layers were non-reactive (for higher magnification see S2).”

• Also stated in: § Restricted transgene expression and age-dependent pathology in EC of rTgTauEC mice · plain

  “From 6 months, a slow progression of tau epitopes in the soma of MEC neurons towards later stages of pathology was observed, marked by the presence of PHF1 staining, recognizing the late phosphorylation of Ser 396/404 sites, starting at the age of 12 months (Fig 1E, middle panel and S2), Gallyas staining (Paired Helical Filament-specific silver impregnation) at the age of 18 months (Fig 1E, second”

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In rTgTauEC mice, axon terminals undergo age-dependent degeneration: Alz50 staining increases through 12 months, decreases at 18 months with more prominent somatic dentate gyrus staining, and becomes irregular patches from 21 months.

“We observed age-dependent degeneration of axon terminals in rTgTauEC mice (Fig 5A, for higher magnification images see Fig S2 and for pathology progression see ST1).”

• Also stated in: § Progressive neurodegeneration and gliosis in rTgTauEC neural circuits · hedged

  “Alz50 tau staining of misfolded tau in axon terminals increased with age through 12 months (Fig 5A, second left panel). At 18 months, the reactivity in axon terminals decreased and staining in soma of the molecular layer of the DG became more prominent, indicating the possibility that EC-II axons began to degenerate and DG granular neurons took up the misfolded protein (Fig 5A, middle panel).”

• Also stated in: § Progressive neurodegeneration and gliosis in rTgTauEC neural circuits · plain

  “From 21 months of age, the pattern of Alz50 reactivity in the middle molecular layer of the DG changed from a clear layer to irregular patches in the axon terminal zone (Fig 5A, right panels), similar to a pattern observed in AD patients (Hyman et al., 1988).”

• cites Hyman et al., 1988Not checked yet

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Mouse tau increases with age in rTgTauEC mice and is present in both sarkosyl insoluble and soluble fractions.

“Immunoblotting using the mouse tau specific antibody also revealed that mouse tau increases with age in rTgTauEC mice (Fig 4C), indicating that it may be accumulating in tangles.”

• Also stated in: § Human tau seeds mouse tau aggregation · plain

  “In confirmation of this idea, sarkosyl insoluble and soluble fractions both contain endogenous mouse tau (Fig 4D).”

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In rTgTauEC mice, human tau transgene mRNA is intensely expressed in a subset of medial entorhinal cortex and pre/parasubiculum neurons as early as 3 months, predominantly in layer II, with rare expression in other regions.

“We observed intense expression as early as 3 months of age, in a subset of neurons in the medial entorhinal cortex (MEC) and pre- and para-subiculum (Pre and Para) (Fig 1C).”

• Also stated in: § Restricted transgene expression and age-dependent pathology in EC of rTgTauEC mice · plain

  “The positive neurons in the MEC were detected prominently in layer II, although rare positive neurons were observed in layer III, especially in the area adjacent to para-subiculum. A few rare neurons in the granular layer and the hilum of the dentate gyrus (DG) showed some transgene expression in accord with observations in the parental line (Yasuda and Mayford, 2006), as well as very rare neurons”

• cites Yasuda and Mayford, 2006Not checked yet

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In rTgTauEC mice, total tau and human tau increase with age, and phospho-tau epitopes AT180, PHF1, and CP13 increase with age when normalized to total tau.

“Western blot analysis of whole brain extracts obtained from 12, 18 and 24 month-old rTgTauEC and control mice confirm human tau protein is expressed in the brain and shows an age-dependent increase in total tau and human tau (Fig 2A–C).”

• Also stated in: § Restricted transgene expression and age-dependent pathology in EC of rTgTauEC mice · plain

  “rTgTauEC mice present an age-dependent increase in all phospho epitopes (Fig 2D–F).”

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Cortical areas with limited transgene expression outside the entorhinal cortex but without direct entorhinal input do not show tau aggregation, and the cerebellum, which expresses human P301L tau mRNA, does not develop fibrillar human tau accumulation in the soma.

“Importantly, unlike the anterior cingulate cortex, cortical areas that showed limited transgene expression outside of the EC, but do not receive direct input from the entorhinal cortex, did not show any tau aggregation. Moreover, the cerebellum, which expresses human P301L tau mRNA, did not develop any fibrillar accumulation of htau in the soma.”

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Endogenous mouse tau accumulates in the somatodendritic compartment of entorhinal cortex neurons, where it co-aggregates with human tau.

“endogenous mouse tau accumulates in the somatodendritic compartment of EC neurons where it co-aggregates with human tau”

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In rTgTauEC mice, significant neuronal loss occurs at 24 months in EC-II and parasubiculum, with a 42% decrease in neuron density in entorhinal cortex layer II, but no significant loss up to 21 months.

“In rTgTauEC mice, significant neuronal loss was detected at 24 months of age, in the area of transgene expression, EC-II and parasubiculum, compared to the average neuron number in age-matched control brains (Fig 6C).”

• Also stated in: § Progressive neurodegeneration and gliosis in rTgTauEC neural circuits · plain

  “We observed a 42% decrease in neuron density in layer II of entorhinal cortex. We did not observe significant neuronal loss up to 21 months of age.”

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Axonal degeneration in rTgTauEC mice is accompanied by microglial activation and astrogliosis, with Alz50-positive terminal patches surrounded by activated microglia at 24 months.

“Axonal degeneration was accompanied by gliosis in rTgTauEC brain. rTgTauEC mice showed evidence of microglial activation (Fig 5B–C) and astrogliosis (Fig 5D–E).”

• Also stated in: § Progressive neurodegeneration and gliosis in rTgTauEC neural circuits · hedged

  “At 24 months of age, Alz50-positive patches of axon terminals in the middle molecular layer were surrounded by activated microglia (Fig 5C) suggesting that axon terminals and their synapses were degenerating in this area.”

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In rTgTauEC mice, approximately 12% of EC-II neurons express the human tau transgene at 3 months, unchanged at 12 and 18 months, and only about 4% at 24 months.

“Stereological counts of human tau expressing neurons labeled with in situ hybridization show that approximately 12% of neurons (12.4%; sem +/− 1.69) in layer-II of entorhinal cortex expressed the transgene at 3 months of age (Fig 6E–F).”

• Also stated in: § Progressive neurodegeneration and gliosis in rTgTauEC neural circuits · plain

  “This number was unchanged at 12 months (13.21%; sem +/− 0.86; p=0.366) and 18 months of age (12.18%; sem +/− 1.96; p=0.481), showing that only a portion of neurons in the EC-II expressed the human tau transgene. At 24 months of age, only ~4% (3.71%; sem +/− 0.84) of the neurons expressed the transgene (Fig 1C and 1D) (p<0.005).”

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In rTgTauEC mice, 47% of EC-II neurons are Alz50 positive at 12 months, dropping to approximately 10% at 24 months.

“Quantification by stereological counts showed that 47% of all neurons in the EC-II were Alz50 positive at 12 months of age which dropped to approximately 10% at 24 months of age (Fig 6D) as some neurons died.”

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Parental P301L mice without the transactivator have human tau protein or mRNA below detection thresholds, consistent with a prior observation that the tau-alone parental line expresses less than 2% of rTg4510 levels and does not develop tau pathology with age.

“Moreover, the parental P301L mice without the transactivator transgene show levels of human tau protein or mRNA (supplemental figure S1) below detection thresholds, in accord with a recent observation that the “tau alone” parental line express <2% of the levels of rTg4510 mice and do not develop any tau pathological alterations with age (Barten et al. 2011).”

• cites Barten et al. 2011Not checked yet

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In rTgTauEC mice, human tau immunofluorescence in the entorhinal cortex is significantly reduced at 24 months and dentate gyrus neuron cell bodies become immunoreactive for human tau.

“There is a significant reduction in the human tau immunofluorescence staining of the EC at 24 months and the cell bodies of the DG neurons become immunoreactive for htau, suggesting the protein is being transmitted (Fig S4).”

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In Alzheimer disease, neurofibrillary tangles accumulate first in layer II of the entorhinal cortex and then spread in a hierarchical pattern to limbic and association cortices.

“Neurofibrillary tangles advance from layer II of the entorhinal cortex (EC-II) toward limbic and association cortices as Alzheimer disease (AD) evolves.”

• Also stated in: § INTRODUCTION · boosted

  “It has been known for more than 25 years that neurofibrillary tangles have a hierarchical pattern of accumulation reflecting selective vulnerability of neuronal populations in the Alzheimer brain, initially affecting the large projection neurons that connect memory related neural systems (Braak and Braak, 1991; Hyman et al., 1984).”

• Also stated in: § INTRODUCTION · hedged

  “Over years, a “march” of lesions appears to propagate across limbic and association cortices, creating a pattern so consistent as to be incorporated into criteria for the neuropathological diagnosis of the illness (Hyman and Trojanowski, 1997).”

• cites Braak and Braak, 1991 sourceNot checked yet

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• cites Hyman et al., 1984 sourceNot checked yet

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• cites Hyman and Trojanowski, 1997 sourceNot checked yet

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Human tau enters neighboring cells without detectable transgene and transmits a misfolded state that recruits endogenous mouse tau into the somatodendritic compartment, contributing to tau aggregation.

“These data suggest that human tau enters neighboring cells that do not have detectable levels of human tau transgene and transmits a misfolded state which recruits endogenous mouse tau into the somatodendritic compartment, where it becomes misfolded and contributes to tau aggregation.”

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Prior cell culture studies showed that extracellular tau aggregates can be internalized, transmit tau misfolding, seed fibril formation, and transfer between co-cultured cells.

“It has been shown previously, in cell culture experiments, that extracellular tau aggregates could be internalized transmitting tau misfolding from outside to inside of the cell where these aggregates could seed fibril formation of recombinant tau monomer. Moreover, the same study showed that tau aggregates were transferred between co-cultured cells. (Frost et al., 2009).”

• cites Frost et al., 2009Not checked yet

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The mouse tau antibody is specific: it shows reactivity in rTgTauEC and control mouse brain but not in tau knockout or human Alzheimer disease brain.

“The specificity of the mouse tau antibody was confirmed by Western blot (Fig 4E) which shows mTau reactivity in rTgTauEC and control mouse brain but not in tau knockout or human AD brain.”

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In mice, superficial entorhinal cortex layers project to the dentate gyrus via the perforant pathway, with layer II projecting to the outer two-thirds of the dentate molecular layer.

“The superficial layers provide neuronal projections to the dentate gyrus in a powerful projection referred to as the perforant pathway (Witter, 2007).”

• Also stated in: § INTRODUCTION · plain

  “In the mouse, layer II of the EC projects directly to the outer two thirds of the molecular layer of the dentate gyrus, where it connects to dendrites from the granule cells of the dentate gyrus (Hjorth-Simonsen and Jeune, 1972; Steward, 1976).”

• cites Witter, 2007Not checked yet

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• cites Hjorth-Simonsen and Jeune, 1972Not checked yet

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• cites Steward, 1976Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

A prior study reported that brain extracts from NFT-bearing mouse brain injected into wild-type tau expressing mice induce seeding of tau fibrils in neurons.

“Another recent study reported that brain extracts from NFT-bearing mouse brain injected in wild-type tau expressing mice induces seeding of tau fibrils in neurons (Clavaguera et al., 2009).”

• cites Clavaguera et al., 2009 sourceNot checked yet

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An unbiased stereologic counting method was used to determine the number of neurons per brain region.

“Unbiased stereologic counting method was used to determine the number of neurons per region of the brain (Hyman et al., 1998).”

• cites Hyman et al., 1998Not checked yet

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Reactive astrocytes in rTgTauEC mice are PHF1-positive at 24 months, and because the tau transgene is not expressed in glia and no PHF1 astrocytes appear earlier, human tau is likely released from terminals and taken up by glia as axons degenerate.

“Double labeling using PHF1, phospho tau antibody, and GFAP antibody demonstrated reactive astrocytes that were PHF1-positive at 24 months of age (Fig 5E). The tau transgene is not expressed in glia, and there were no PHF1 positive astrocytes at earlier ages, indicating human tau is likely released from terminals and taken up by glia as the axons degenerate.”

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The data do not rule out the possibility that some tau is released and taken up at earlier ages or under normal physiological conditions.

“but this does not preclude the possibility that that some tau is released and taken up at earlier ages, or even under normal physiological circumstances.”

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cDNA samples were run in triplicate in a 25 μl reaction with 12.5 μl SYBR green Mastermix.

“Triplicates of cDNA samples were added to a 25 μl reaction containing 12.5 μl SYBR green Mastermix (Applied Biotechnology).”

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Band density values were normalized to β-actin or total tau when analyzing tau phosphorylation.

“Band density values were normalized to β-actin or total tau levels when tau phosphorylation levels were analyzed.”

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Human tau protein is present in GFAP-positive astrocytes in rTgTauEC mice, suggesting that release of tau from neurons and uptake by glia also takes place in this model.

“The presence of human tau protein in GFAP positive astrocytes in rTgTauEC mice suggests that release of tau from neurons and uptake by glia also takes place in this model.”

Classified as probably not a substantial claim.

Prior data suggest that intracellular tau aggregates can seed aggregation of native tau proteins and propagate their misfolded state in a prion-like manner.

“Recent data suggest that intracellular protein aggregates of tau have the capacity to seed aggregation of native tau proteins and might propagate their misfolded state in a prion-like manner.”

• cites de Calignon et al., 2010 sourceNot checked yet

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• cites Iliev et al., 2006Not checked yet

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• cites Mocanu et al., 2008Not checked yet

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cDNA clones were verified by sequencing for specificity before being used to generate standard curves.

“After verifying the respective specificities of the cDNA clones by sequencing, these were used to generate individual standard curves”

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In rTgTauEC mice, tau is transferred to neighboring cells and to synaptically connected neurons, suggesting that tau or a particular tau species may be released at the synapse.

“Taken together, data presented in this study indicates that, in the rTgTauEC mice, tau was not only transferred to neighboring cells, but also to synaptically-connected neurons, which suggested that tau, or a particular species of tau such as hyperphosphorylated tau, misfolded tau, or a fragment of tau, may have been released at the synapse.”

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The mouse tau-specific antibody mTau was characterized by western blot and immunohistochemistry for sensitivity and specificity and did not recognize unspecific proteins in tau knockout mice or human brain.

“The antibody was characterized by western blot analysis and immunohistochemistry for sensitivity and specificity and did not recognize any unspecific proteins in tau knockout mice or in human brain.”

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The levels of tau mRNA were normalized against mouse tau mRNA.

“Finally, the respective tau mRNA levels were normalized to murine tau mRNA levels.”

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Only F1 offspring were used, ensuring a uniform 50:50 mix of FVB and C57BL/6 genetic backgrounds.

“Only F1 offspring were used as experimental animals ensuring a uniform 50:50 mix of FVB and C57BL/6 genetic background.”

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Increased tau levels in cerebrospinal fluid after injury are consistent with injury causing tau release.

“the presence of increased tau CSF levels after injury is consistent with the possibility that injury induces release (Blennow et al., 1995).”

• cites Blennow et al., 1995 sourceNot checked yet

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Classified as probably not a substantial claim.

Age-matched littermates carrying only the activator and responder transgenes served as controls.

“Age matched littermates expressing only the activator transgene and the responder transgene were used as controls.”

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Mean band densities from rTgTauEC samples were normalized to corresponding control samples.

“Mean band densities for samples from rTgTauEC mice were normalized to corresponding samples from control mice.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Counting frames of 21.8 μm × 21.8 μm and step lengths of 100-200 μm were chosen to count more than 200 neurons per animal.

“Counting frames of size 21.8 μm x 21.8 μm, and step length from 100 μm to 200 μm were selected to count >200 neurons per animal.”

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Cell loss at 24 months exceeds the number of transgene-expressing neurons, suggesting that neurons developing tau pathology without expressing the transgene might also be lost.

“Further, the cell loss observed at 24 months exceeds the number of neurons that were expressing the transgene, suggesting that neurons that develop tau pathology without expressing the transgene might also be lost.”

Classified as probably not a substantial claim.

Each of the eight age groups studied (3, 6, 9, 12, 15, 18, 21, and 24 months) included both transgenic and control animals.

“Each of the 8 age groups studied (3, 6, 9, 12, 15, 18, 21, 24 months) contained transgenic and control animals.”

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RNA samples were assessed for quality using an Agilent 6000 Bioanalyzer and a Nanodrop spectrophotometer.

“RNA samples were assayed for quality with an Agilent 6000 Bioanalyzer and a Nanodrop spectrophotometer.”