Staging of neurofibrillary degeneration caused by human tau overexpression in a unique cellular model of human tauopathy

Abstract

The hyperphosphorylation of human tau and its aggregation into neurofibrillary tangles are central pathogenic events in familial tauopathies and Alzheimer's disease. However, the cellular consequences of neurofibrillary tangle formation in vivo have not been directly studied because cellular models of human neurofibrillary degeneration have been unavailable until recently. Incorporation of human tau into filaments in vivo and the association of filamentous tau with cytodegeneration were first demonstrated experimentally with the overexpression of human tau in identified neurons (anterior bulbar cells) in the lamprey central nervous system. In this system, filamentous tau deposits are associated with the loss of dendritic microtubules and synapses, plasma membrane degeneration, and eventually the formation of extracellular tau deposits and cell death. Here we show that human tau hyperphosphorylation in anterior bulbar cells is spatiotemporally correlated with a highly stereotyped sequence of degenerative stages closely resembling those seen in human neurofibrillary degeneration. Hyperphosphorylated tau deposits first appear in the distal dendrites and somata, together with degenerative changes that begin in distal dendrites and progress proximally over time. This sequence is independent of the tau isoform used, the presence of epitope tags and the method used to overexpress tau, and thus has important implications for the cytopathogenesis of human neurofibrillary disease.

Figure 1.

Expression and immunolabeling of human tau from plasmid and SFV-derived mRNA vectors in lamprey ABCs. A: Constructs used in this study to express tau in ABCs. In all clones, human tau expression was driven by the CMV promoter. Clone 93 has EGFP fused to the htau23 N-terminal and clone 139 has GFP fused to the C terminal of htau40, whereas n123c and SFV expressed htau23 and htau40 without epitope tags. B: A schematic diagram outlining the mechanism of SFV mRNA vector amplification used for htau40 expression in ABCs. The SFV replicase consists of four subunits that combine to synthesize strands complementary (−) to the injected mRNA. Duplicate strands (+) are then synthesized from which both the replicase and the exogenous target protein (ie, htau40) are then translated, amplifying the original mRNA injected into the ABC and permitting the chronic expression of the target protein without the need for either whole virus or nuclear targeting and subsequent transcription of injected plasmids. C: Schematic diagram of the tau molecule showing the MT-binding motifs (black) and the approximate locations of the epitopes recognized by mAbs used in this study. AT8, AT100, and TG3 recognize phosphoepitopes on the N-terminal flanking region of the MT binding repeats, whereas PHF1 recognize a phosphoepitope on the C-terminal flanking region. ALZ50 recognizes the N terminal of tau in conjunction with a conformational change that occurs when human tau becomes hyperphosphorylated and dissociates from MTs.

Figure 2.

Effects of human tau overexpression on lamprey ABCs. A: Dorsal view of the hindbrain of a living lamprey showing an ABC that is expressing htau23 viewed in a combination of fluorescence and bright-field illumination under a dissecting microscope. B: A transverse section through the soma and dendritic field of an ABC (at the point indicated by the arrows in A) injected with Lucifer Yellow, showing normal ABC dendritic gross morphology in the absence of tau expression, with ABC dendrites tapering smoothly from base to tip (arrow). C: An ABC 9 days after injection with clone 93 exhibiting typical early (stage 1) dendritic changes, including a slight expansion of distal dendrites (caret) relative to proximal dendrites (arrow). PHF1 labeling is even and granular, except in some distal dendrites, where it is concentrated near the plasma membrane. D: GFP fluorescence image from two ABCs 79 days after injection with clone 93. The circle delineates the normal outline of the cell body; it is likely that this neuron is at a relatively severe stage of degeneration involving the entire somatodendritic region (ie, stage 3 or 4); note that despite this the cell retains a grossly normal axon (arrow). E: A cross-sectional view of a section from an ABC 21 days after injection with clone n123c, showing relatively severe (stage 3) degeneration. Note the marked contrast between the cells shown in C and E, in particular the roughened and distorted outline of most dendrites in E. Much of the tau in E is localized to membranous structures, as well as to filamentous deposits, and that some is outside the cell (E, large asterisk). Nuclei of ABCs showing stage 3 degeneration are typically tau-positive (E, small asterisk) unlike earlier stages (C, asterisk). F and G: Examples of dendritic ultrastructure in normal (F) and severely degenerated (G) ABCs. G is from a cell 70 days after injection of clone 93. ABC dendrites normally contain well-organized arrays of MTs (F, carets) that are disrupted by chronic tau overexpression and eventually replaced by straight filaments containing human tau (immunolabeled with anti-GFP in G) Scale bars: 50 μm (A–E), 200 nm (F), and 100 nm (G).

Figure 3.

Swollen dendritic tips produced by localized aggregations of membrane-bound organelles are the first morphological changes seen as the result of human tau overexpression in ABCs. A: A swollen dendritic tip from a lightly expressing (stage 1) ABC immunostained with PHF-1. B: Electron micrograph of a similar swollen dendritic tip showing pronounced aggregations of membranous organelles. Inset region is shown at high magnification at right. C: These aggregations contain MTs (carets), tau filaments (arrows), and mitochondria (m), small vesicles (v), and smooth endoplasmic reticulum (sER) oriented in an apparently random arrangement, with some clumping of both vesicles and mitochondria. Scale bars: 10 μm (A), 5 μm (B), and 500 nm (C).

Figure 4.

Swelling and beading of dendritic tips is characteristically accompanied by tau hyperphosphorylation in ABCs showing a moderate degree of tau-induced degeneration (stage 2). Each micrograph shows immunolabel with a different mAb: PHF1 (A), ALZ50 (B), AT8 (C), TG3 (D). A and C: Adjacent sections from the same ABC (28 days after injection of pRc123c), B and D are from different cells 28 and 34 days, respectively, after the injection of this plasmid. Note that although some sections show somatic tau deposits (asterisks), and all cells show immunolabeling in distal dendrites (arrows), there is no labeling of dendritic shafts (small carets) in the proximal dendritic field by any mAb except PHF1. Note the parallel nature in the development of morphological degenerative changes (ie, distal dendritic beading, A, and the appearance of AT8, ALZ50, and TG3 immunolabel in these areas) Also note that although many of the distal dendritic deposits of hyperphosphorylated tau appear to be membranous, some are fibrillar in appearance (C, arrow). Scale bar: 25 μm.

Figure 5.

Severe degeneration of ABCs overexpressing human tau is accompanied by widespread tau hyperphosphorylation in both membrane-associated and fibrillar deposits. A, C, and E were immunolabeled with PHF1 (A and E) or anti-GFP (C) to show the distribution of total human tau within severely degenerated ABCs, whereas adjacent sections were labeled with a mAb against a hyperphosphorylation marker epitope [ALZ50 (A), TG3 (C), AT8 (E)]. A and B are from an ABC expressing htau40 28 days after injection with SFV-tau mRNA, C and D are from a cell 49 days after injection of clone 93 (EGFP-htau23) plasmid, and E and F are from a cell 34 days after injection of clone 93. Note that the somatodendritic profiles of the htau-expressing cells are highly distorted and swollen compared to normal, especially in distal dendrites (arrows). We classified these cells into stage 3 degeneration (if the nucleus was present and the somatic profile was still clearly defined as shown in A–D), or stage 4 degeneration (E–F), which represents the unequivocal breakup and death of the cell. Stage 4 degeneration was also accompanied by the loss of most dendritic immunostaining and the presence of human tau both extracellularly (asterisk) and within adjacent glial or ependymal cells. This tau was presumably scavenged from the dead ABC or the extracellular space via endocytosis, and was frequently hyperphosphorylated. Note that precisely the same pattern of pathology can be seen in ABCs expressing htau40 via the SFV mRNA vector (A and B) as by either EGFP tagged htau23 (C and D) or untagged htau23 expressed from pRc123c (Figure 2E) ▶ Scale bar: 50 μm.

Figure 6.

Stages of neurofibrillary degeneration caused by chronic human tau overexpression in ABCs. The most prominent and/or typical changes seen in degenerating ABCs that are overexpressing human tau. Stages were assigned to 67 ABCs according to morphological changes visible in immunostained sections on the basis of PHF1 and anti-GFP immunolabeling only, without reference to either the presence of tau hyperphosphorylation, the clone or technique being used to induce tau overexpression, or the length of time after injection. Although the presence and/or pattern of tau hyperphosphorylation was not an a priori criterion for assigning cells to a particular stage, the close correlation between the patterns of neurodegeneration (shown by PHF1 label at left) and hyperphosphorylated tau label (defined as immunolabeling with AT8, TG3, AT100, or ALZ50) is shown for each stage (right).

Figure 7.

Steady progression from early to late stages of NFD occurs with all human tau constructs in ABCs. A: A total of 67 ABCs examined at progressively later times after injection showed a steady progression through stages 1 to 4. Cells expressing htau40 via clone 139 and the SFV vector and htau23 via pRcCMV123c and clone 93 were combined. Each bar shows the percentage of the total number of cells examined in each time period; these were: 1 to 9 days, 9 cells; 10 to 19 days, 21 cells; 20 to 29 days, 17 cells; and 30+ days, 22 cells. There were a total of 19 stage 1 cells, 22 stage 2 cells, 19 stage 3 cells, and 8 stage 4 cells among the cells sampled. B: The stages of degeneration reached by cells sampled at early (E, 20 days or less after injection) and late (L, more than 20 days after injection) times are compared on a per clone basis. The presence of EGFP/GFP as an epitope tag, the use of the SFV expression system, and the presence of the fourth MT-binding repeat did not seem to interfere with the progression of cytopathology from mild (stages 1 to 2) to severe (stages 3 to 4) stages of degeneration (P < 0.05). Progression of degeneration within the sample as a whole was highly significant (P ≪ 0.001) Statistical comparisons between stage distributions in early and late samples were performed using the chi-square test.