Differential regulation of microtubule dynamics by three- and four-repeat tau: implications for the onset of neurodegenerative disease

Abstract

The microtubule (MT)-associated protein tau is important in neuronal development and in Alzheimer's and other neurodegenerative diseases. Genetic analyses have established a cause-and-effect relationship between tau dysfunction/misregulation and neuronal cell death and dementia in frontotemporal dementia and parkinsonism associated with chromosome 17; several mutations causing this dementia lead to increased ratios of four-repeat (4R) to three-repeat (3R) wild-type tau, and an attractive hypothesis is that the abnormally high ratio of 4R to 3R tau might lead to neuronal cell death by altering normal tau functions in adult neurons. Thus, we tested whether 3R and 4R tau might differentially modulate the dynamic instability of MTs in vitro using video microscopy. Although both isoforms promoted MT polymerization and decreased the tubulin critical subunit concentration to approximately similar extents, 4R tau stabilized MTs significantly more strongly that 3R tau. For example, 4R tau suppressed the shortening rate, whereas 3R tau had little or no detectable effect. Similarly, 3R tau had no effect on the length shortened during a shortening event, whereas 4R tau strongly reduced this parameter. Further, when MTs were diluted into buffer containing 4R tau, the MTs were stabilized and shortened slowly. In contrast, when diluted into 3R tau, the MTs were unstable and shortened rapidly. Thus, 4R tau stabilizes MTs differently and significantly more strongly than 3R tau. We suggest a "dosage effect" or haploinsufficiency model in which both tau alleles must be active and properly regulated to produce appropriate amounts of each tau isoform to maintain MT dynamics within a tolerable window of activity.

Fig. 1.

Schematic of 4R and 3R tau. Boxes above each line correspond to the 18-aa imperfect repeats. Dark boxes below each line correspond to regions encoded by alternatively spliced exons. The inclusion or excision of sequences encoded by the exon in the repeat region leads to the synthesis of 4R or 3R tau, respectively. Arrows above the line mark the positions of amino acid substitutions that lead to FTDP-17.

Fig. 2.

The effects of 3R (circles) and 4R tau (squares) on the tubulin critical subunit concentration. MTs were assembled at 37°C in the presence of 0.09 μM tau isoforms (A) or 0.74 μM isoforms (B). After 30 min, the MTs were sedimented at 150,000 × g in a Beckman TL100 centrifuge (Beckman Coulter). The quantity of polymer was determined after dissolving the MT pellets in 100 μl of 100 mM Pipes/1 mM MgS0₄/1 mM EGTA, pH 6.8, at 0°C for 1 h.

Fig. 3.

Life history traces; the effects of 0.74 μM 3R and 4R tau on the dynamics of individual MTs at 37°C near steady state. The growing and shortening dynamic of individual MTs was determined by video microscopy (see Materials and Methods). (A) Control (no added tau). (B) 4R tau. (C) 3R tau. Each trace represents a single MT.

Fig. 4.

The effects of 3R and 4R tau on the stability of individual MTs after dilution-induced disassembly. MTs were polymerized in PMME buffer at 37°C (12 μM tubulin) and diluted 4-fold into PMME buffer containing 0.74 μM 4R (A)or3Rtau(B). The diluted MT suspensions were analyzed between 2 and 10 min after dilution. Each trace represents a single MT.

Fig. 5.

Dosage effect model for normal and pathogenic tau action. Cell function and viability require properly regulated MT dynamics. Overly suppressed dynamics, such as those that occur in the presence of taxol and, as we propose, might occur in the FTDP-17 RNA splicing mutations, result in cell death. Similarly, overly dynamic MTs, such as those that occur in taxol-resistant and -dependent cells in the absence of taxol (16) and those that might occur in the FTDP-17 missense mutations and in cells expressing hyperphosphorylated tau, also result in cell death.