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Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: sequestration of microtubule-associated proteins 1 and 2 and the disassembly of microtubules by the abnormal tau - PubMed

  • ️Wed Jan 01 1997

Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: sequestration of microtubule-associated proteins 1 and 2 and the disassembly of microtubules by the abnormal tau

A D Alonso et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The microtubule-associated protein (MAP) tau is abnormally hyperphosphorylated in Alzheimer disease and accumulates in neurons undergoing neurofibrillary degeneration. In the present study, the associations of the Alzheimer-hyperphosphorylated tau (AD P-tau) with the high molecular weight MAPs (HMW-MAPs) MAP1 and MAP2 were investigated. The AD P-tau was found to aggregate with MAP1 and MAP2 in solution. The association of AD P-tau to the MAPs resulted in inhibition of MAP-promoted microtubule assembly. However, unlike the coaggregation of AD P-tau and normal tau, the association between AD P-tau and the HMW-MAPs did not result in the formation of filaments/tangles. The affinity of the tau-AD P-tau association was higher than that of HMW-MAPs-AD P-tau because normal tau inhibited the latter binding. The association between AD P-tau and the HMW-MAPs also appeared to occur in situ because these proteins cosedimented from the Alzheimer brain extracts, and, in the sediment, the levels of the HMW-MAPs correlated with the levels of AD P-tau. These studies suggested that the abnormally phosphorylated tau can sequester both normal tau and HMW-MAPs and disassemble microtubules but, under physiological conditions, can form tangles of filaments only from tau.

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Figures

Figure 1
Figure 1

Binding of MAP1 and MAP2 to AD P-tau. The binding of MAP1 and MAP2 to AD P-tau was determined as described using a fixed amount of AD P-tau or normal tau (8 μg in 100 μl) and MAP1 and MAP2 (70 μg/100 μl). The proteins were mixed in 100 mM Mes buffer (pH 6.7) containing 2 mM EGTA, 0.5 mM MgCl2, 2% BSA, 1 μM aprotinin, and 20 μM leupeptin (binding buffer) and were incubated for 30 min at 37°C. Similar incubations were carried out using rat brain cytosol (100 μg protein/100 μl) as the source of tau, MAP1, and MAP2. The incubated samples were overlaid a 100-μl cushion of 8% sucrose in the binding buffer and centrifuged at 100,000 × g for 60 min. As a control, MAP1, MAP2, and brain cytosol mixed with normal tau (8 μg) were processed identically to the mixtures of these proteins with AD P-tau. The amounts of tau, MAP1, MAP2, and tubulin (in the case of cytosolic extract) in the pellet and the supernatant fractions were assayed by radioimmuno-slot blot. AD P-tau bound to all of the MAPs when using both purified proteins and proteins in brain extract. The levels of binding of AD P-tau were within tau > MAP2 > MAP1.

Figure 2
Figure 2

Electron micrographs showing the products of association of AD P-tau with tau, MAP1, and MAP2 negatively stained with phosphotungstic acid. MAP1– and MAP2–AD P-tau aggregates were induced as described in Fig. 1. Under identical conditions, 150 μg of normal tau and 8 μg of AD P-tau/100 μl also were incubated. Aliquots of the incubated mixture of MAP1 and AD P-tau (a), MAP1 alone (b), MAP2 and AD P-tau (c), or tau and AD P-tau (d and e) were taken after centrifugation and were examined by negative stain electron microscopy. Association of AD P-tau with only tau and not MAP1 or MAP2 resulted in the formation of filaments/tangles. (Bars = 1 μm for a and d; 0.5 μm for b, c, and e.)

Figure 3
Figure 3

Relationship of the ratio of HMW-MAPs in the pellet/supernatant to the levels of AD P-tau in the pellet. The levels of MAP1 or MAP2 were determined in the 200,000 × g supernatant (Supt) and the 27,000 to 200,000 × g pellet (Pellet) from brain homogenates of 12 (MAP2) to 13 (MAP1) AD cases (•) and four Huntington disease cases (○). The levels of AD P-tau also were determined in the 27,000 × g to 200,000 × g fraction from the same brains by radioimmuno-slot blot using Tau-1 as the primary antibody (18). The AD P-tau values are expressed as cpm of the 125I secondary antibody used. The Pellet/Supt ratios of the HMW-MAPs were obtained from the means of triplicate assays determined at three different concentrations. The levels of the MAPs correlate directly with the levels of AD P-tau in the 27,000 × g to 200,000 × g pellet, and levels of the HMW-MAPs in the 200,000 × g supernatant correlate inversely with the AD P-tau in the pellet. The Pellet/Supt ratios of the HMW-MAPs show a significant direct correlation with the AD P-tau levels (p < 0.05).

Figure 4
Figure 4

Inhibition of the MAP1- and MAP2-promoted microtubule assembly by AD P-tau. Polymerization of tubulin was determined as described. The assembly reaction was carried out using (i) 1.0 mg/ml MAP1 or a mixture of MAP1 and 2 mg/ml AD P-tau (a) or (ii) 1.0 mg/ml MAP2 or MAP2 plus 2 mg/ml AD P-tau (b). For comparison, the inhibition of tau-promoted microtubule assembly is reproduced (c) from our previous report (5).

Figure 5
Figure 5

Electron micrographs showing the products of microtubule assembly with MAP1 and MAP2 and the effect of AD P-tau on the assembly. Microtubule assembly was carried out from rat brain tubulin by the addition of MAP1 (a), MAP2 (b), or MAP2 and AD P-tau (c and d, respectively) as in Fig. 4. Aliquots of the reaction mixture were negatively stained with phosphotungstic acid after 4 (c) or 30 (a, b, and d) min of incubation. Only an occasional microtubule was seen with tubulin alone (data not shown) and with AD P-tau after 30 min of incubation (d), and a large number of microtubules was observed in the assembly with MAP1 (a), MAP2 (b), and MAP2 with AD P-tau after 4 min of incubation (c). (Bars = 1 μm for a-c and 0.5 μm for d.)

Figure 6
Figure 6

Disruption of the MAP2-promoted microtubules by AD P-tau. The assembly of microtubules was determined turbidimetrically as described. The arrow indicates the time of addition of the AD P-tau (2 mg/ml final concentration) or the same volume of buffer to MAP2-preassembled microtubules. Samples of the reaction mixture were examined by negatively stained electron microscopy. Only an occasional microtubule was seen after 20 min of AD P-tau addition (not shown).

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