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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 7, 2006 as doi:10.1096/fj.05-5343fje. |
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* Mayo Clinic Jacksonville, College of Medicine, Jacksonville, Florida, USA;
Stanford University, Stanford, California, USA; and
Conforma Therapeutics Corporation, San Diego, California, USA
1Correspondence: E-mail: hutton.michael{at}mayo.edu
SPECIFIC AIMS
The original aims of this study were to 1) elucidate the therapeutic relevance of HSP90 inhibitors that are able to permeate the blood brain barrier and begin to identify their mechanism of action; and 2) develop reliable cell-based assays to quantitate pathologically relevant tau species so that compound screening would be possible for treatment of tauopathies.
PRINCIPAL FINDINGS
1. Development of novel In-Cell Western assays allowed for the quantitative assessment of reductions in pathogenic tau-species elicited by a panel of low molecular weight HSP90 inhibitors
Here, standard Western blot analysis revealed that HSP90 inhibition by the classical inhibitor, geldanamycin, reduced total tau levels in primary cortical neurons. Subsequent to this initial result, a small panel of novel, low molecular weight HSP90 inhibitors was generated and using a novel 96-well microplate-based In-Cell Western assay with a near-infrared imaging platform, we analyzed the effects of these compounds on tau biology in cell culture. We demonstrated that several of these novel compounds were able to reduce endogenous total tau levels in a human neuroglioma cell line with minimal toxicity. Those compounds that elicited a >10-fold induction in HSP70 levels also produced the greatest reduction in total tau levels after drug exposure (Fig. 1
).
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2. Disease-associated phospho-tau species were preferentially reduced after HSP90 inhibition, while total tau levels were only proportionally decreased; levels of tau phosphorylated at KXGS sites within the microtubule binding domain, a substrate for normal functional phosphorylation, remained relatively despite HSP90 inhibition
To address whether chaperone activation through HSP90 inhibitor treatment was capable of selectively reducing abnormal phospho-tau species, we refined our In-Cell Western technique, changing to a CHO cell line stably transfected with 4R0N tau harboring the P301L pathogenic mutation to increase the dynamic range, and assaying for the following phospho-tau species: 1)phospho-S202/T205 tau, a reported Ser/Thr-Pro phosphorylation site for GSK3ß and MAPK (CP13 antibody); 2)phospho-S396/S404 tau, a reported Ser/Thr-Pro phosphorylation site for CdK5 (PHF1 antibody); and 3) phospho-S262/S356, a reported KXGS phosphorylation site for MARK (12E8 antibody). The CHO tau (P301L) cell line was treated with 1 µM of each HSP90 inhibitor and the levels of the three phospho-tau species (S202/T205, S96/S404, and S262/S356; Fig. 2
A) were determined by In-Cell Western analysis. Five of the compounds (EC82, 86, 102, 104, and 105) selectively reduced the proportion of total tau-phosphorylated at sites S202/T205 and S396/S404 by 50% or more (P<0.001 t test) while leaving levels of tau phosphorylated at the MARK sites S262/S356 largely unchanged (max reduction=18%) (Fig. 2A
). 17-AAG significantly reduced all three phospho-species; however the reductions in S202/T205 (74%) and S396/S404 (77%) were much greater than that of S262/S356 (30%). One of the compounds (EC107) failed to significantly (P>0.05) reduce any of the phospho-tau species, while EC108 produced only a small (18%) reduction in tau phosphorylated at S262/S356 that was significant. These results were also validated by standard Western blot analysis.
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3. We used the nondenaturing conditions of the In-Cell Western assay to measure levels of MC-1 tau, a disease-associated conformational epitope that tau presents early in the disease process
Those same compounds that produced >50% reductions in the levels of S202/T205 and S396/S404 phospho-tau species also significantly reduced the levels of conformationally altered tau with the MC-1 epitope (Fig. 2B
). Both EC104 and 17-AAG eliminated >90% MC-1 positive-tau while EC82, EC86, EC102, and EC105 reduced this species by 50–75%. Neither EC107 nor EC108 had any effect on MC-1 tau levels.
4. Selective degradation of aberrant tau species by these inhibitors was completely prevented with the proteasomal inhibitor MG132, while a milder and more general overall reduction in all measured tau species was elicited by the lysosomal inhibitor chloroquine
To better elucidate the mechanism of tau degradation elicited by HSP90 inhibition, we again treated the CHO tau (P301L) cell line with the panel of HSP90 inhibitors, this time in the presence of the proteasomal inhibitor MG-132. Tau protein species were again quantified using the In-Cell Western assay. Treatment with MG-132 completely blocked all HSP90 inhibitor-mediated reduction of total tau and tau phosphorylated at proline-directed Ser/Thr sites (S202/T205 and S396/S404). Proteasomal inhibition also blocked the clearance of conformationally altered tau species (MC-1) produced by HSP90 inhibition. Exposure to MG-132 alone, however, did not significantly alter total tau levels. In contrast, cells co-treated with HSP90 inhibitors and the lysosomal inhibitor, chloroquine, for 24 h maintained the selective reductions in pS202/T205 and pS396/S404 phospho-tau species; however, the levels of tau species in cells co-treated with the HSP90 inhibitors (EC82, 86, 102, 104, 105, and 17-AAG) and with chloroquine were significantly higher (P<0.05) than in cells treated with the HSP90 inhibitors alone. This result suggests that although HSP90 inhibitor-mediated clearance of tau is proteasome-dependent, lysosomal function is likely also necessary for this process to work at maximal efficiency.
CONCLUSIONS AND SIGNIFICANCE
Here, high throughput near-infrared fluorescent assays were developed to measure the levels of different tau species within cells as a means of identifying compounds that might have therapeutic benefit in tauopathy. These In-Cell Western assays were used to examine the effects of a panel of seven novel small molecule HSP90 inhibitors on the clearance of different tau species in the CHO tau (P301L) cell line. The size of these novel compounds and their predicted pharmacokinetic properties suggests that they will cross the blood brain barrier at levels sufficient to impact chaperone levels within the brain.
These studies showed that HSP90 inhibitors producing the greatest induction of molecular chaperones were able to selectively lower levels of tau phosphorylated at the proline-directed Ser/Thr sites pS202/T205 and pS396/S404. Tau phosphorylated at these epitopes accumulates during the development of AD and other tauopathies while these species are absent or present at low levels in the normal adult human brain. Due to the nondisruptive conditions of the assay, we were also able to show selective reductions in conformationally altered tau that is detected by the MC-1 antibody. The MC-1 epitope is formed when tau takes on a folded conformation in which the N-terminus interacts with the microtubule binding region. Formation of this abnormal folded conformation is thought to be one of the earliest pathological changes observed in patients with tauopathy and is associated with the initiation of tau filament formation. This is the first time that this disease-associated tau conformational epitope has been quantitatively analyzed in vitro as part of a high-throughput assay.
One of the most striking results of these studies was the minimal effect that HSP90 inhibitor treatment had on levels of tau phosphorylated by MARK (pS262/S356 tau species), suggesting that tau phosphorylated within the microtubule binding domains by MARK is likely resistant to degradation by HSP90 inhibition. A potential reason why tau phosphorylated at S262/S356 is not reduced by HSP90 inhibition, while tau species phosphorylated at the proline-directed S202/T205 and S396/S404 sites are reduced, could be the differences in the respective protein recognition motifs. The KXGS motifs (S262/S356) that are phosphorylated by MARK/PAR-1 may not be recognized by the relevant chaperones induced by HSP90 inhibition because they lack the proline residue that is a consistent feature of the other Ser/Thr phosphorylation sites within tau.
The functional significance of the apparent resistance of tau phosphorylated at S262/S356 to degradation is an important question. Transient phosphorylation of tau by MARK, within the microtubule binding domains, causes tau release from the microtubules and consequently microtubule destabilization. MARK is thus able to regulate microtubule dynamics, which is important for axonal growth and other processes that require neuronal plasticity.
It is possible that tau phosphorylated by MARK needs to be resistant to HSP-mediated degradation so that it can provide a pool of unbound tau that is then available to rapidly bind microtubules after dephosphorylation; however this resistance of pS262/S356 tau to degradation by HSP induction may also begin to explain the reported role of tau phosphorylated by MARK in the initiation of pathogenesis in tauopathy.
These studies expand upon an increasing body of evidence implicating the chaperone cycle as a critical mechanism for the removal of abnormal protein species in neurons, and further demonstrate the potential for therapeutic development targeting this system. In addition, the ability to analyze tau species by assays such as the In-Cell Western, should allow for intensified efforts toward drug discovery targeting tau species.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5343fje;
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