Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease

Abstract

The neuronal microtubule-associated protein tau plays an important role in establishing cell polarity by stabilizing axonal microtubules that serve as tracks for motor-protein-driven transport processes. To investigate the role of tau in intracellular transport, we studied the effects of tau expression in stably transfected CHO cells and differentiated neuroblastoma N2a cells. Tau causes a change in cell shape, retards cell growth, and dramatically alters the distribution of various organelles, known to be transported via microtubule-dependent motor proteins. Mitochondria fail to be transported to peripheral cell compartments and cluster in the vicinity of the microtubule-organizing center. The endoplasmic reticulum becomes less dense and no longer extends to the cell periphery. In differentiated N2a cells, the overexpression of tau leads to the disappearance of mitochondria from the neurites. These effects are caused by tau's binding to microtubules and slowing down intracellular transport by preferential impairment of plus-end-directed transport mediated by kinesin-like motor proteins. Since in Alzheimer's disease tau protein is elevated and mislocalized, these observations point to a possible cause for the gradual degeneration of neurons.

Figure 2

Mitochondria accumulate near the MTOC in CHO cells overexpressing tau. Cells stably transfected with tau40 (a–c) and mock-transfected CHO cells (d–f). Microtubules were stained with antibody DM1A (b and e) and tau with polyclonal anti–tau antibody K9JA (c and f). Mitochondria were visualized with MitoTracker™ Green (a and d). Cells were fixed in methanol 30 min after addition of MitoTracker™ Green. In tau-stable cells (a–c), there is a striking clustering of mitochondria near the MTOC (seen near the nucleus where the microtubule stain is most intense), whereas in mock-transfected cells (d–f) the mitochondria are dispersed throughout the cytoplasm. In addition, the tau-stable cells are more rounded up compared with control cells. Bar, 20 μm.

Figure 3

Effects of different tau constructs on mitochondrial distribution in CHO cells. Mitochondrial distribution in CHO cells stably transfected with different tau constructs was examined after paraformaldehyde fixation. This allows the visualization of total tau in the cells, but tends to dissociate tau from the microtubules (see Materials and Methods). Human tau23, the shortest tau-isoform (a and b), as well as K35 (c and d), a tau construct lacking nearly the entire projection domain (see Fig. 1) both led to the phenotype of mitochondrial clustering near the MTOC as seen in tau40-stable cells. On the other hand, K23 (e and f), a tau construct lacking the microtubule-binding region and K12 (g and h), consisting mainly of this MT-binding region (Fig. 1), did not alter the distribution of mitochondria. This argues for a tight binding of tau to microtubules as a prerequisite for impairment of plus-end–directed transport of these organelles and excludes the possibility that tau might inhibit kinesin by directly interacting with the motor protein. Mitochondria were stained with MitoTracker™ Red (a, c, e, and g) and transfected tau with polyclonal anti–tau antibody K9JA (b, d, f, and h). Bar, 20 μm.

Figure 11

The ER organization is altered in CHO cells overexpressing tau. Tau40-stable (a and b) and mock-transfected CHO cells (c and d) were stained with DiOC₆(3) to visualize the ER. In tau-stable cells, the ER is less branched and does not reach the cell periphery, while in control cells the ER extends almost to the plasma membrane and displays a more complex structure. Note that, although the extension of the ER of the smaller cell in Fig. 11 b seems to be less effected when compared with the larger cells, the branching and complexity of the endoplasmic reticulum is significantly reduced when compared with mock-transfected cells in c and d. The cell periphery is outlined for clarity. Bar, 10 μm.

Figure 1

Domain structure of tau isoforms and constructs. Tau40 is the longest tau isoform in the human CNS, occurring mostly in axons (Goedert et al., 1989). It consists of 441 amino acid residues and can be divided into an NH₂-terminal projection domain and a COOH-terminal microtubule binding domain containing four imperfect repeats (R1–R4) and basic proline-rich sequences on either side that act as microtubule targeting domains (shaded dark, P1, and P2). The repeats show high homology between tau, MAP2, and MAP4. The shaded NH₂-terminal inserts and repeat two can be alternatively spliced, resulting in six different tau isoforms. The predominantly embryonic isoform tau23 lacks all three inserts (352 residues). Tau constructs K12 and K10 are derived from tau23, they contain three repeats plus either a COOH-terminal flanking region (K12) or the whole COOH-terminal tail (K10). K35 contains three repeats and the basic proline-rich regions on either side, including the COOH-terminal tail. K23 contains all of tau23 except the repeats.

Figure 7

The ratio between plus- and minus-end–directed transport of mitochondria is altered by tau overexpression. Tau-stable and control cells were treated with nocodazole to disrupt the microtubule cytoskeleton and to achieve an even distribution of mitochondria in both cell lines. The drug was washed out after 2 h of incubation and mitochondria were stained with MitoTracker™ Red. Translocations of the organelles were then recorded for 2 min with a time interval of 1 s. The direction of movement was regarded as plus-end directed when the organelles were transported to the periphery (+), minus-end directed when transport was towards the nucleus (−), and neutral (−/+) when movement was vertical to the axis between nucleus and periphery of the cells. Note that the relative number of transported mitochondria is significantly lower for all three groups in the tau-stable cell line (P < 0.0001, two tailed Student's t test). However, in tau-stable cells, the plus-end– directed movement is significantly more affected (∼10-fold) than the reverse direction (2-fold). This leads to the phenotype of mitochondrial clustering. At least 40 cells were analyzed for quantitation, the total number of mitochondria ranged between 100 and 700 in both cell lines.

Figure 10

Quantitation of mitochondrial clustering after overexpression of tau, drug treatment, and inhibition of dynein. The distribution of mitochondria was quantified as described in Materials and Methods. A minimum of 30 cells were analyzed per experiment (n). In mock-transfected cells, mitochondria occupy up to 65% of the total cell area. The same is true in cells stably transfected with K23, a tau construct lacking the microtubule-binding repeats (CHO-K23). This value drops to ∼30% in tau40-stable cells (CHO-tau). Treatment of the stably transfected cells with either nocodazole or taxotere restores the wild-type distribution of mitochondria (CHO-tau nocodazole or CHO-tau taxotere, respectively). The same effect can be seen after transfection with dynamitin (CHO-tau transfection dynamitin), but not after transfection of EGFP as a control protein (CHO-tau transfection EGFP; P < 0.0001, two-tailed Student's t test). Removal of nocodazole again leads to clustering of mitochondria at the nucleus (CHO-tau noc. removal; 4 h).

Figure 4

Microinjection of recombinant tau protein leads to mitochondrial clustering near the MTOC. Wild-type CHO cells were microinjected with 1.3 mg/ml (∼1.2 μM) of purified recombinant tau protein and incubated for 4 h. After addition of MitoTracker™ Red (a and b), cells were fixed with paraformaldehyde and stained with antibodies against tubulin (c and d) and tau (e and f). Mitochondrial clustering is visible even within 4 h after microinjection. Note that the microtubule network retains its normal appearance and that mitochondria in microinjected cells similar as in the tau-stable CHO and N2a cells tend to cluster at one side of the nucleus in the vicinity of the MTOC. The cell periphery of microinjected cells is outlined for clarity. Bar, 10 μm.

Figure 5

Disruption of microtubules by nocodazole leads to a random distribution of mitochondria in tau-stable CHO cells. Tau-stable cells (a and b) and mock-transfected CHO cells (c and d). Cells were treated with nocodazole to disrupt the MT cytoskeleton, fixed with methanol, and immunolabeled with antibodies against tubulin (b and d). In both cell lines, mitochondria, visualized by addition of MitoTracker™ Green before fixation (a and c), are now visible throughout the entire cytoplasm even in the cell periphery, suggesting the existence of an intact MT network as a prerequisite for mitochondrial clustering. Bar, 20 μm.

Figure 6

Reassembly of microtubules after removal of nocodazole restores the clustering of mitochondria. The microtubule cytoskeleton of tau-transfected CHO cells was disrupted by addition of 5 μM nocodazole for 2 h. Pictures of living cells were then taken after removal of nocodazole and reassembly of microtubules at the indicated time points. Mitochondria were stained with MitoTracker™ Green. Note that, concomitant with microtubule reassembly, the phenotype of mitochondrial clustering at the MTOC near the nucleus begins to reappear after ∼1 h. The cell periphery is outlined for clarity. Bar, 10 μm.

Figure 8

Randomization of microtubule polarity by taxotere allows clustered mitochondria to disperse again. Tau-stable cells were treated with 10 μM taxotere for 6 h, which leads to an altered microtubule organization. Note that the MTOC in taxotere-treated cells is barely visible in the tubulin stain (b), in contrast to nontreated cells (compare Fig. 2). Microtubules are nucleated randomly throughout the cytoplasm, thus abolishing the role of the MTOC as an organizing element and therefore abolishing the polarity of microtubules. Minus-end–directed motors are now able to transport mitochondria back to peripheral regions in the tau-stable cells. Cells were fixed in methanol and mitochondria were stained with MitoTracker™ Green (a), tubulin was immunostained with DM1A (b). Bar, 10 μm.

Figure 9

Dynamitin overexpression restores the wild-type distribution of mitochondria. Tau-stable cells were transfected with dynamitin (p50), a subunit of the dynactin complex. 6 h after transfection, the mitochondria began to disperse again, visualized with MitoTracker™ Red after paraformaldehyde fixation (a). Note that the cell transfected with tau and dynamitin (arrowheads) shows dispersed mitochondria, whereas the neighboring tau-stable cells show the typical clustering near the MTOC. Dynamitin was stained with antidynamitin 50-1 antibody (b) and tubulin with YL1/2 antibody (c). Bar, 20 μm.

Figure 12

Transferrin recycling is retarded by tau overexpression. Cells were seeded on cover slips and preincubated with TMR-labeled transferrin in serum-free medium for 2 h. Excess transferrin was removed and tau-stable and mock-transfected cells were fixed in methanol. Residual transferrin fluorescence was then quantitated and plotted (a). Note that, at early time points (7 min), the transferrin fluorescence in tau-stable cells is decreased by ∼10%, whereas in control cells 50% of transferrin is exocytosed in the same time interval. 300 cells were quantitated per time point and cell line. (b) Time course of the exocytosis of transferrin after equilibration of tau-stable and mock-transfected cells with fluorescently labeled transferrin. A significant decrease of exocytosed transferrin can be seen 5–10 min after start of the experiment in tau-stable compared with control cells. Fluorescent signals were normalized to total cell numbers in each experiment. Data represent averages of four independent experiments.

Figure 13

Transport of mitochondria into neurites of differentiated neuroblastoma cells is impaired by overexpression of tau. Tau40-stable (a–c) and mock-transfected N2a cells (d–f) were differentiated for 2 d by addition of 1 μM retinoic acid into medium containing 0.1% fetal calf serum and fixed in methanol. Mitochondria were visualized with MitoTracker™ Green (a and d), immunostaining was done with antibodies against tubulin (b and e) and tau (c and f). It can be clearly seen that significantly fewer mitochondria are present in the neurites of tau-stable cells in contrast to control cells (compare a and d, and Table I). In addition, mitochondria in tau-stable cells appear to be more clustered in the vicinity of the MTOC (a), similar to tau-stable CHO cells. The comparable staining intensity of the cell bodies indicates that the clustering effect is not due to a lower number of mitochondria in the tau-transfected cell. Bar, 10 μm.

Figure 14

The expansion of the endoplasmic reticulum in differentiated neuroblastoma cells is significantly reduced by overexpression of tau. Tau-stable N2a cells (a–c) or mock-transfected cells (d–f) were induced to differentiate, and then fixed in paraformaldehyde and triple-labeled with antibodies against the ER (a and d), tubulin (b and e), and tau (c and f). In contrast to control cells, ∼82% of tau-stable neuroblastoma cells show the ER clustered at one side of the nucleus (a), whereas in mock-transfected cells the ER surrounds the entire nucleus (d; compare Table I). Furthermore, the expansion of the ER into the neurites in control cells (d) can clearly be seen. In tau-stable cells, only a faint signal of ER-immunoreactivity is visible in neuritic processes. The total intensity of the ER stain is the same in both cells. Arrowheads in a and d are indicating the position of the neurite relative to the cell body. Bar, 10 μm.