To understand the sequence of events from expression of pathological tau in the entorhinal cortex to the development of widespread cortical involvement, we recreated an early stage of AD neurofibrillary pathology in transgenic mice to investigate how, starting in the entorhinal area, tau pathology leads to neural system dysfunction. We observed two important consequences of the formation of tangles in the entorhinal cortex: (1) spreading of the pathology to downstream connected neurons despite regional and cellular restriction of transgene expression, and (2) evidence favoring very slow synaptic, then axonal, then somatic degeneration associated with accumulation of misfolded tau.
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. This transmission has first been described to occur inside cells, since incorrectly folded tau proteins convert to an aggregate-prone state acting as a nucleus that recruits additional tau monomers (de Calignon et al., 2010
; Iliev et al., 2006
; Mocanu et al., 2008
). In cell culture experiments (Frost et al., 2009
), extracellular tau aggregates could enter cells and trigger tau fibrillization. In living mouse brain, intracortical injections of tau aggregates seed tau fibrillization in neurons carrying the human transgene (Clavaguera et al., 2009
). Here, we found that in aged rTgTauEC mice, human tau protein is present in neurons that do not do not have detectable levels of human tau mRNA, suggesting that transneuronal propagation of tau occurs. This idea is also supported by our data showing that (1) in EC-II, the number of transgene-expressing neurons decreases in older age – correlating with neuronal loss - while (2) the proportion of transgene-negative Alz50-positive neurons robustly increases with age, suggesting that the remaining Alz50-positive neurons were secondarily affected by transneuronal transfer. It has been reported that glial tau pathology occurs in tauopathies (Ballatore et al., 2007
; Chin and Goldman, 1996
) and in Alzheimer’s disease (Nakano et al., 1992
; Nishimura et al., 1995
; Papasozomenos, 1989a
) where tau inclusions can be found in astrocytes and oligodendrocytes. 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.
The specificity of the neurospsin driven transactivator for EC and related structures is demonstrated by in situ
hybridization, qPCR, immunostaining, and western blot analysis of rTgTauEC mice. 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
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. In the dentate gyrus, CA regions, and the cingulate cortex, we found neurons that do not have detectable human tau mRNA at any of the ages examined, which accumulated tau immunoreactive species (recognized by multiple antibodies) at advanced ages (21 and 24 months). In parallel to our study, a recent report described a mouse model of mutant APP expressed predominantly in the entorhinal cortex which used the same promoter as rTgTauEC mice. Progression of Aβ deposition to the hippocampus and cingulate cortex was also reported (Harris et al., 2010
). These data suggest that misfolded tau and Aβ share properties that allow propagation through the extracellular space to disrupt neuronal systems.
Our data support the idea that tau, when accumulated in the terminal zones, induces synaptic destruction. We cannot distinguish between the possibilities that misfolded axonal tau induces dying back terminal degeneration, or that release of tau is synaptotoxic. It is not clear how, or if, misfolded tau gets released and/or taken up by neurons, but the presence of increased tau CSF levels after injury is consistent with the possibility that injury induces release (Blennow et al., 1995
). In rTgTauEC mice, propagation seems more tightly linked to the time frame when axons are dying back (21–24 months) than when Alz50-positive tau can be detected in axon terminals (3 months), 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.
Interestingly, by using a new specific mouse tau antibody, we also show evidence that endogenous mouse tau accumulates in the somatodendritic compartment of EC neurons where it co-aggregates with human tau. Furthermore, we also report that mouse tau can be detected in both the sarkosyl soluble and insoluble fractions, suggesting that misfolded human tau can recruit endogenous mouse tau to aggregate.
If the propagation of AD tangle pathology from Braak stage I to VI entails, to some extent, the type of neuronal system propagation events described here, several critical questions remain. 1) Why are some neuronal populations protected from developing tangles despite being anatomically strongly connected to neuronal populations that do develop tangles? 2) Does tau need to emerge into the extracellular space where it is potentially available to therapies such as immunotherapy or might it be released from the cytoplasm, yet remain sequestered within membrane bound compartments such as exosomes? 3) Which species of tau are responsible for transferring the aggregated state to non-expressing cells? Is the tau that is capable of initiating misfolded aggregates in downstream cells an aggregated form or a consequence of a unique post-translational modification such as phosphorylation, acetylation, glycosylation, or truncation? Understanding these issues may help inform therapeutic approaches that have promise to slow the progression of AD.