The expression and distribution of tau protein associated with the neurofibrillary pathology in Alzheimer's disease (AD) is being studied by laser scanning confocal  microscopy.
   We perform experiments  in brain tissue slices combining immunofluorescent probes and three-dimensional reconstruction of images obtained from pathological structures.
   Confocal scanning based on the principle of rejection of out-of-focus rays of light (Fig. 1) allows collection of in-focus optical sections through several microns of the specimens without optical interference from other focal planes which normally introduce blurring in the images.
   With this x/y and z-sectioning approach stacks of single optical sections from scanned objects can be projected and analyzed as three-dimensional rotations.
 
 

Figure 1. Confocal principle. Two pinhole barriers at optically conjugated
points of the path of rays (source pinhole and detector pinhole) reject
light emitted away from the focal plane (red lines). Information only
coming from the focal plane (blue rays) is collected by the detector pinhole.
(image taken from Lance Ladic web site).
 

The neuropathological hallmarks of AD, generally referred to as neurofibrillary tangles, neuropil threads and distinct types of neuritic plaques are mainly composed by tau protein assembled in the form of abnormal paired helical filaments (PHFs) (Grundke Iqbal et al. 1986; Kosik et al. 1986; Delacourte and Defossez 1986).  Several post-translational modifications in the tau molecule like phosphorylation (Bancher et al. 1989; Alonso et al. 1996), glycosylation (Ledesma et al. 1994; Goedert et al. 1996) and truncation (Novak et al. 1993; Mena et al. 1996) have been considered  key mechanisms that promote the pathologic assembly of tau into the PHFs. Recently conformational changes in the normal tau molecule has been described as an early event in AD before the appearance of neurofibrillary changes (Jicha et al. 1997,1999: Uboga and Price 2000)
   In our laboratory we have raised the antibody Tau-66 (Ghoshal et al. 2001) that recognizes a specific conformation of the tau molecule present in various abnormal structures in the brain of AD cases.
   By using Tau-66 in combination with other antibodies, which recognize several modifications in the tau molecule, we analyze in AD brain tissue the topographical relationships between the aggregation of truncated and conformationally altered tau proteins as well as the deposition of the ßA-4 amyloid protein. For this purpose we usually process 50 µm thick brain sections for double and triple label-immunofluorescence and analysis on a Zeiss LSM510 Laser Scanning Confocal Microscope
 

EXPERIMENTS
 
 

Different populations of neurofibrillary tangles are present in the CA1 region of the hippocampus in AD.
Triple labeling with monoclonal antibodies 423 and Tau-66 combined with the fluorescent dye Thiazin red evidences a wide spectrum of neurofibrillary tangles representing specific patterns of aggregation of tau protein. The ß-pleated sheet conformation of these lesions is monitored in the red channel by using Thiazin red. Neurofibrillary tangles immunoreactive to the conformation-dependent antibody Tau-66 displays a punctate pattern of staining (channel green).  In contrast, the neurofibrillary tangle evidenced with the monoclonal antibody 423 (blue channel) is more fibrillar in appearance and is predominantly composed by truncated tau protein. The 423 antibody specifically recognizes a truncation in the tau molecule at glutamic acid-391. The smaller tangles visualized in the merge channel (asterisks) seem to represent early stages in the formation of these structures since they are strongly stained by Thiazin red and display more conformational tau epitopes recognized by Tau-66.
 
 

Colocalization of truncated and conformation altered tau protein in neurofibrillary tangles.
In some populations of  intracellular tangles, predominantly found in the subiculum/CA1 region, both truncated and conformation altered tau proteins strongly colocalize in the same structure. The triple pattern of colocalization is evidenced in white pseudocolor when the three channels are merged. The pattern of staining of 423 and Tau-66 evidences a combination between fibrillar and punctate appearance.
 
 

Different pattern of aggregation of tau protein in neurofibrillary tangles. Double labeling with monoclonal antibodies 423 and Tau-66 which  recognize truncated and conformation altered tau protein respectively evidenced different patterns of aggregation of this protein in the same structure. In the images the larger tangle is strongly and more uniformly stained with 423 than Tau-66 antibody. In the merge channel the light blue pseudocolor corresponds to the regions of strong colocalization between both markers. This pattern of staining suggests that tau protein might adopt an specific conformation state and be truncated during the assembly and maturation of neurofibrillary tangles. A different pattern of tau aggregation is seen in the smaller tangle with punctuate appearance recognized with Tau-66 (asterisks). Truncated tau protein is not evidenced in these non-fibrillar structures and represent perhaps an early event of aggregation.
movie (~2.6 MB)
 
 

Late stage extracellular neurofibrillary tangles are evidenced with monoclonal antibody 423.
Truncated tau protein recognized by 423 antibody is commonly found in neurofibrillary tangles which have undergone proteolysis in the extracellular space and lost much of the epitopes recognized by other antibodies. The image corresponds to a flame-shaped neurofibrillary tangle from the subiculum/CA1 region and represent a projection of 192 serial optical sections collected every 0.2 µm.
movie (~2.1 MB)
 
 
 

Extracellular neurofibrillary tangles in the multipolar cells layer of the entorhinal cortex in AD.
In advanced Alzheimer's disease cases the layer II of the entorhinal cortex composed predominantly by multipolar cells displays a considerable number of extracellular tangles occupying the cell body and spreading along the proximal processes. Truncated tau protein recognized by the monoclonal antibody 423 strongly stains this population of tangles.
 
 
 

Perivascular glial elements recognized by the monoclonal antibody Tau-66. Tau protein aggregates within glial cells in several tauopathies and the picture corresponds to these elements immunostained with the tau conformation-dependent antibody Tau-66. Besides the staining of cell bodies and thin cellular processes several wide and large astrocytic feet are clearly evidenced.
 
 
 

In AD a population of diffuse early "ghosty" plaques is evidenced with Tau-66 antibody. Conformationally altered tau protein recognized by Tau-66 was identified in a population of plaques displaying a punctate, diffuse staining. These plaques were also immunoreactive to a rabbit anti-human ß-amyloid antibody.
Also, some colocalization is evidenced in the plaque (in yellow) however, the side view demonstrates that most of tau (small arrow) and the amyloid peptide (large arrow) are distributed in different sides of the structure (for a full text go to Ghoshal et al. 2001)
movie (~6.4 MB)
 
 

Neuritic plaques are double immunolabeled with Tau-66 and Tau-5. The conformation-dependent antibody Tau-66 recognizes two discontinuous regions in the tau molecule. One of these sites is also recognized by the non conformation dependent antibody Tau-5. This immunochemical feature is also reflected in the pattern of neurofibrillary staining of neuritic plaques in AD. In the pictures, while both Tau-66 and Tau5 stain the main body of the plaque, the neuritic component is predominantly stained by Tau- 5 (for a full text go to Ghoshal et al. 2001).
movie (~6.4 MB)
 
 

Tau-66 antibody stains some reticulated plaques in AD.  In the pictures a cored and neuritic plaque visualized with Thiazin red is also stained with Tau-66.  The core of the plaque consisting perhaps of amyloid and strongly stained with Thiazin red is surrounded by more diffuse components colocalizing with Tau-66 staining (yellow pseudocolor in the merge channel). Elements in the periphery of the plaque, perhaps consisting of dystrophic neurites are predominantly stained with Tau-66.
movie (~5.2 MB)
 
 

References

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Bancher C, Brunner C, Lassmann H, Budka H, Jellinger K, Wiche G, Seitelberger F, Grundke-Iqbal I, Iqbal K & Wisniewski HM (1989). Accumulation of abnormaly phosphorylated tau precedes the formation of neurofibrillary tangles in Alzheimer’s disease. Brain Res 477: 90-99.

Delacourte A, Defossez A (1986). Alzheimer’s disease: tau proteins, the promoting factors of microtubule assembly, a major component  of paired helical filaments. J Neurol Sci 76: 173-186.

Goedert, M., Jakes, R., Spillantini, M. G., Hasegawa, M., Smith, M. J., and Crowther, R. A. (1996). Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans. Nature 383, 550-3

Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y. C., Zaidi, M. S. and Wisniewski, H. M. (1986a) Microtubule-associated protein tau. A component of Alzheimer paired helical filaments. J Biol Chem 261, 6084-6089.

Jicha GA, Bowser R, Kazam IG, Davies P. (1997). Alz-50 and MC1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau. J Neurosci Res 48: 128-32.

Jicha GA, Berenfeld B, Davies P (1999). Sequence requirements for formation of conformational variants of tau similar for those found in Alzheimer's disease. J Neurosci res 55: 713-23

Kosik KS, Joachim CL, Selkoe DJ (1986). Microtubule-associated protein tau. A component of Alzheimer paired helical filaments. Proc Natl Acad Sci USA 83: 4044-4048.

Ledesma, M. D., Bonay, P., Colaco, C. and Avila, J. (1994). Analysis of microtubule-associated protein tau glycation in paired helical filaments. J Biol Chem 269, 21614-21619.

Mena R, Edwards P, Harrington CR, Mukaetova-Ladinska EB, Wischik CM. (1996). Staging the pathological assembly of truncated tau protein into paired helical filaments in Alzheimer’s disease. Acta Neuropathol 91: 633-641.

Novak M, Kabat J, Wischik CM (1993). Molecular characterization of the minimal protease resistant tau unit of the Alzheimer’s disease paired helical filament. EMBO J 12: 365-370.

Uboga NV, Price JL (2000). Formation of diffuse and fibrillar tangles in aging and early Alzheimer’s disease. Neurobiology of Aging 21: 1-10.