. 3D Visualization of the Temporal and Spatial Spread of Tau Pathology Reveals Extensive Sites of Tau Accumulation Associated with Neuronal Loss and Recognition Memory Deficit in Aged Tau Transgenic Mice. PLoS One. 2016;11(7):e0159463. Epub 2016 Jul 28 PubMed.


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  1. A major unresolved question in the pathogenesis of Alzheimer's disease (AD) is the mechanism by which Aβ aggregates induce neurofibrillary degeneration in nerve cells and their processes. Microscopic studies reveal that the Aβ plaque, one of the signature lesions of AD, the other being the neurofibrillary tangle (NFT), is a complex extracellular structure composed of filamentous Aβ, abnormal nerve cell processes called dystrophic neurites, reactive astrocytes, and activated microglial cells. The glial cells may have both a beneficial effect by removing “bad proteins,” e.g., Aβ filaments, and a detrimental effect by releasing neuroinflammatory chemicals that cause neuronal stress and abnormal cell function. Visualizing these components of the Aβ plaque in three dimensions may help to elucidate the temporal contribution of each to the evolution of the plaque and subsequent neurodegeneration. Thus, this paper by Liebmann and colleagues at Rockefeller University will be of great interest to students of AD.

    To facilitate visualization of the intrinsic structure of the mouse brain and of small volumes of human postmortem brain, Liebmann and colleagues have applied an established method to make the brain more translucent called immunolabelling-enabled three-dimensional imaging of solvent cleared organs (iDISCO). The advantages of this method are several and include: rapid staining of tissue, automated whole (mouse) brain imaging, atlas alignment (e.g., Allen Brain Atlas), confocal microscopy, and automated object quantification (e.g., number, volume, inter-object distance). These methods are likely to engender more complex, but more realistic, modeling of plaque formation in both animal models of AD and postmortem brain.

    The authors show what may be achieved using iDISCO in the study of AD. The spatial organization of plaques is better appreciated in three dimensions than in the conventional two-dimensional histological section. The relationship between one molecular pathology, the Aβ plaque, and the local neuroanatomy can be readily appreciated using antibodies to markers of blood vessels and glial cells, for example. Similarly, other molecular pathologies, such as tauopathy, the other molecular pathology of AD, can be visualized in a similar manner, which will allow for more sophisticated spatial studies that are likely to throw light on the pathogenesis of AD.

    As with any new technology, there are potential pitfalls. iDISCO is most suitable for small molecular probes, such as the dye Congo red, and less suitable for large ones such as antibodies, which have a harder time penetrating the matrix supporting the tissue. This is a concern because antibodies are typically more sensitive than histological stains such as Congo red. In the Liebmann study, of 11 anti-Aβ antibodies, only one was sufficiently sensitive and penetrated the whole mouse brain and only 1 cm in human postmortem brain. As few well-characterized antibodies are used currently in the routine histology laboratory, new antibodies may need to be developed that are compatible with iDISCO. The software used to visualize microstructures could be improved. For example, the segmentation of the amyloid plaques generated globular structures where both immunohistochemistry and fine structural studies reveal that the Aβ plaque contains filamentous structures. 

    Since the original descriptions of senile plaques, pathologists have known that this is a three-dimensional structure and has different morphologies depending on the plane of section. The introduction of a new acronym, the three-dimensional amyloid pathology (TAP), to describe this well-characterized morphology is not illuminating, whereas iDISCO has every possibility of generating new insights into plaque formation. In summary, iDISCO has both advantages and some pitfalls in characterizing the neuroanatomy of the signature lesions of AD. Neuroscientists and neuropathologists will need to balance these competing factors in determining how best their research goals may be met with iDISCO.    

    View all comments by Nigel Cairns
  2. A third dimension of brain research?

    Liebmann et al. used the iDISCO clearing methods to stain amyloid plaques immunohistochemically as well as with staining dyes (Congo red, thioflavin s) to assess their three-dimensional structure. Compared to the classical approach of three-dimensional reconstruction using serial sections, the iDISCO method is less laborious and, thus, permits the analysis of three-dimensional relationships in more detail and with higher numbers of cases.

    In their published study, Liebmann et al. tested several staining methods for their feasibility in cleared tissue and could show that immunohistochemistry works well in this approach. The use of immunohistochemistry makes this clearing technique attractive for all kind of morphological studies aiming at the description of three-dimensional relations, especially for studying not only plaques but also neuronal connectivity, neuron-glia relations, etc. Such studies will produce novel insights into brain structure and function or disease pathogenesis constituting a novel level (“the third dimension”) of neuroscience.

    When comparing plaques in the 2xtg AD-transgenic mouse brain with those in the human brain they could show that human plaques are more complex and larger than those in 2xtg AD-transgenic mice. This finding fits well with the well-known complex architectures of diffuse plaques that, for example, result in lake-like presubicular amyloid or in fleecy amyloid in the entorhinal cortex.

    Thus, Liebmann et al. gave us a small but very promising look at what will be possible with the novel tissue-clearing methods.

    However, when deciding between the use of classical immunohistochemistry and three-dimensional clearing approaches to address a research question, it is essential to keep in mind that classical immunohistochemistry will address more than 90 percent of the research questions, whereas three-dimensional clearing methods like iDISCO or CLARITY should be reserved for those questions that require the third dimension. Otherwise, normal immunohistochemistry provides a better cellular/subcellular resolution and may be more convincing, especially for double-labelling studies.

    View all comments by Dietmar Thal
  3. This is an elegant study by Drs. Duff and Hyman’s groups on the longitudinal characterization of the EC-tau mouse model. iDisco three-dimensional imaging of the whole translucent hemibrain is impressive and certainly quantitative at the mesoscale. Significant neuroinflammation is observed in the amygdala, which is an important region for aversive learning and can be tested by fear conditioning.

    The authors distinguish cell autonomous/non-autonomous human tau (htau) accumulation based on in situ hybridization (ISH) of human tau mRNA. However, they observe hardly any htau mRNA signal in the MEC region (Fig. 5), which is where the htau transgene was originally designed to be expressed. If it is almost undetectable in the MEC, it will not be detectable anywhere else. Therefore, control ISH for mouse tau mRNA in the MEC region would be desirable to verify the ISH technique.

    Because the authors found little evidence for tau accumulation in astrocytes and microglia in the mouse brains, they suggested these cells play no major role in the propagation of tau. However, the lack of accumulation of tau in microglia does not disprove their role in phagocytosis of neurons or pathogenic proteins. Immune cells, and microglia in particular, are not designed to store proteins intracellularly. After phagocytosis of exogenous materials, they digest them, then secrete for antigen presentation, or die if neither digestion nor secretion is effective. In addition, while the authors use IBA1 (or Cx3cr1-GFP) staining to detect microglia, P2ry12 is microglia-specific and is expressed on their extracellular regions, which extend much further in space than the cytoplasmic regions. Therefore, P2ry12 staining is better for visualizing extended and more-branched microglial terminals that might envelope cytopathic neurons accumulating tau or other toxic molecules. 

    View all comments by Tsuneya Ikezu

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