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Chaperones increase association of tau protein with microtubules - PubMed

  • ️Wed Jan 01 2003

Chaperones increase association of tau protein with microtubules

Fei Dou et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Molecular chaperones and their functions in protein folding have been implicated in several neurodegenerative diseases, including Parkinson's disease and Huntington's disease, which are characterized by accumulation of protein aggregates (e.g., alpha-synuclein and huntingtin, respectively). These aggregates have been shown in various experimental systems to respond to changes in levels of molecular chaperones suggesting the possibility of therapeutic intervention and a role for chaperones in disease pathogenesis. It remains unclear whether chaperones also play a role in Alzheimer's disease, a neurodegenerative disorder characterized by beta-amyloid and tau protein aggregates. Here, we report an inverse relationship between aggregated tau and the levels of heat shock protein (Hsp)7090 in tau transgenic mouse and Alzheimer's disease brains. In various cellular models, increased levels of Hsp70 and Hsp90 promote tau solubility and tau binding to microtubules, reduce insoluble tau and cause reduced tau phosphorylation. Conversely, lowered levels of Hsp70 and Hsp90 result in the opposite effects. We have also demonstrated a direct association of the chaperones with tau proteins. Our results suggest that up-regulation of molecular chaperones may suppress formation of neurofibrillary tangles by partitioning tau into a productive folding pathway and thereby preventing tau aggregation.

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Figures

Figure 1
Figure 1

The tau and Hsp70/90 levels in hippocampus of mutant tau Tg mice and in AD brain. (a) Hippocampal sections of control and mutant tau (4Rm V337M) Tg mice were analyzed for intraneuronally accumulated tau and Hsp90 by immunofluorescence confocal microscopy. Images were overlapped with nuclear counterstaining (Nuc). White arrowheads indicate those neurons with intensive immunoreactivity for accumulated tau but little signal for Hsp90. Black arrowheads indicate neurons with strong immunostaining for Hsp90 but lacking immunoreactivity for accumulated tau. (Bar = 10 μm.) (b) Hippocampal tissues were homogenized and Hsp90 was assayed by Western blotting. Levels of β-tubulin were similar between control and Tg mice. Quantitative data represent averages of three experiments (mean ± SE); *, P < 0.01, with respect to control. (c) Intraneuronal immunostaining for NFT tau, Hsp70, and Hsp90 in hippocampal sections of an AD patient. Adjacent sections were doubly immunostained for hyperphosphorylated (NFT) tau (red, arrows) and for either Hsp70 or Hsp90 (blue, arrowheads). (Bar = 40 μm.)

Figure 2
Figure 2

GA induction of molecular chaperones regulates partitioning, aggregation, and phosphorylation of tau. (a) GA induces expression of Hsp70 and Hsp90 in N2a cells, primary neurons, and tau-transfected COS-1. A dose-dependent increase of both Hsp70 and Hsp90 was observed after cells were treated with indicated concentrations of GA for 48 h. Data represent mean ± SE, n = 4, P < 0.01 at all concentrations of GA. (b) The partitioning of the smallest tau isoform (arrow) into nonionic detergent insoluble (Pellet, Left) and soluble (Supernatant, Right) fractions in transfected COS-1 cells shows a concentration-dependent shift from insoluble to soluble fraction after GA treatment. Data represent mean ± SE, n = 4. (c) Effect of GA on insoluble, aggregated tau in transfected COS-1 cells. A dose-dependent decrease of insoluble tau was evident after GA treatment by using a filter-trap/immunoblotting assay. Data represent mean ± SE, n = 4. (d) GA reduces basal and okadaic acid-induced (5 μM) tau phosphorylation. COS-1 cells expressing human tau (T44) were pretreated with GA for 24 h, and okaidic acid was added for another 6 h. Cell lysates (2% SDS extraction) were analyzed by Western blotting by using phosphorylated tau-specific Ab (AT270, Innogenetics). Quantitative data represent an average of three experiments (mean ± SE).

Figure 3
Figure 3

COS cells transfected with different tau isoforms were subjected to multiple, serial extractions to corroborate aggregation of transfected tau and attenuation of aggregation by GA. (Upper) Serial extractions of GA-treated and -untreated COS cells expressing WT tau isoforms WT/T44 and WT/T40, and tau mutants P301L/T40 and R406W/T40. (Lower) Histogram of high salt extraction (RAB), RIPA extraction, and formic acid (FA) extraction, respectively. Note: the amount of material loaded for the high-salt (RAB), ionic detergents, and formic acid extraction represented 0.2%, 1%, and 10% of total material extracted, respectively. Data represent mean ± SE, n = 3.

Figure 4
Figure 4

Hsp70 and Hsp90 induce partitioning of tau. (a) Fractionation scheme used to resolve different cellular pools of tau. Total membrane and cytosolic fractions were prepared. The tau fraction sedimenting with total membranes was treated with nocodazole to solubilize microtubule-associated tau (Supernatant 1). The remaining membrane-bound/aggregated tau (Pellet 1) was extracted with Na2CO3 to solubilize membrane-associated tau (Supernatant 2), thus separating it from aggregated tau (Pellet 2). (b) Western blot analysis of GA effects on tau pools generated from fractionation scheme of a. (c) siRNA-induced reduction in Hsp70 and Hsp90 levels. Data represent mean ± SE, n = 3. (d) Western blot analysis of effects of reduction of Hsp70 and Hsp90 by siRNA on tau partitioning into subcellular fractions generated from fractionation scheme of a.

Figure 5
Figure 5

Colocalization of molecular chaperones, tau, and microtubules. (a) Coimmunoprecipitation of tau with Hsp90 (Left) and Hsp70 (Right) from COS-1 cells, (+) anti-tau Ab, (−) control Ab. (b) Colocalization by triple immunofluorescence microscopy of Hsp70, tau, and microtubules. (Upper) Immunofluorescence staining of COS-1 cells expressing the shortest tau isoform (T44). (Lower) Immunofluorescence staining of COS-1 cells expressing the longest tau isoform (T40). (Bar = 10 μm.)

Figure 6
Figure 6

Hsp70 and Hsp90 mediate tau microtubule partitioning/aggregation in a cell-free system. Permeabilized reconstituted cells were incubated for 90 min (Left) or 180 min (Right) and then fractionated as described in Fig. 4a. Membrane-bound tau refers to supernatant 2. “+Hsp70” and “+Hsp90” refer to added recombinant chaperones; “−Hsp70/90” refers to cytosol prepared from siRNA-transfected cells. Arrowheads indicate the absence of phosphorylated tau.

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