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O SARAIVA*,,1
* Amyloid Unit, Institute for Molecular and Cell Biology and
ICBAS, University of Porto, Portugal; and
Biotechnology Research Laboratories, University Hospital IRCCS Policlinico San Matteo and Department of Biochemistry, University of Pavia, Italy
1Correspondence: Amyloid Unit, IBMC, Rua do Campo Alegre, 823, P-4150-180 Porto, Portugal. E-mail: mjsaraiv{at}ibmc.up.pt
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: familial amyloidotic polyneuropathy • transthyretin • I-DOX • tetracyclines • amyloid disruption
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The major component of the amyloid deposits in FAP is a variant transthyretin (TTR). To date, 
80 different TTR mutations related to amyloid deposition and 10 nonpathogenic mutations have been described (2)
. Amyloid fibrils are characterized by certain tinctorial properties such as birefringence under polarized light after staining with the dye Congo red and binding to thioflavin T (Th T). The fibrils are straight or coiled, unbranched, 7.5 to 10 nm in diameter, and several microns in length. X-ray studies revealed a cross-ß pattern with the ß-sheets running perpendicular to the axis of the growing fibril.
In vitro fibril formation has been a powerful tool in the study of the mechanism that leads to amyloid formation. Theories for the events occurring in vivo have been proposed based on in vitro results. In vitro screening of drugs that reverse or avoid the amyloidogenic process is necessary to prevent or treat the disease. Therefore, it is important to elucidate the dynamics of amyloid fibril formation to prevent disease: different drugs may act on distinct species present on the amyloidogenic pathway at different stages, and this knowledge enables us to search for specific drugs for each step of the process. We recently reported on the in vitro assembly properties as observed by transmission electron microscopy (TEM) of a highly amyloidogenic TTR variant—TTR Leu55Pro: oligomers appearing as round particles of various sizes evolved into short 4–5 nm wide fibrils leading to 9–10 nm wide fibrils that elongated over time (3)
.
In this work we investigated the action on TTR fibril formation of a class of compounds known to disrupt amyloid fibrils. Drugs tested included 4'-deoxy-4'-iododoxorubicin (I-DOX) (4
5
6)
, tetracycline derivatives (7
8
9)
, and nitrophenols (10)
. Whether these drugs produce toxic intermediate species has not been addressed. We reported on the toxicity associated with the different species formed during TTR Leu55Pro fibril in vitro assembly and demonstrated that nonfibrillar initial aggregates are toxic to a Schwannoma cell line, as measured by caspase-3 activation. In contrast, soluble protein and long fibrils did not produce significant caspase-3 activation (11)
. Using the same methodology, we specifically investigated the nature and toxicity of the species generated by the action of I-DOX, tetracycline, and its derivatives on TTR amyloid.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, isolated, and purified as described previously (13)
. After growing the bacteria, the protein was isolated and purified by preparative gel electrophoresis after ion exchange chromatography. Protein concentration was determined using the Lowry method (14)
.
Preparation of amyloid fibrils
TTR Leu55Pro was dialyzed against water pH 7.0 and concentrated to 5 mg/mL. The preparation was centrifuged at 15,000 g for 30 min at 4°C, then the pellet was washed, resuspended in phosphate-buffered saline (PBS) or 0.9% NaCl at 2 mg/mL, and incubated at 37°C. When tested by Th T fluorescence, the sample was shown to be positive, presenting the characteristic novel excitation maxima at 450 nm in contrast to the initial soluble preparation.
Fibril concentrations were assessed by the Lowry method (14)
.
I-DOX effect on TTR fibrils
I-DOX (kindly provided by Pharmacia and Upjohn) was dissolved in 0.9% NaCl and filtered through a 0.2 µm filter (Schleicher and Schuell, Keene, NH, USA). I-DOX concentration was determined spectrophotometrically at 478 nm using an extinction coefficient of 13,300 M-1cm-1 based on a 690 Da molecular mass. Stock solutions were prepared at 10-3 M.
Aliquots of TTR Leu55Pro (100 µg) prepared as described above and resuspended in 0.9% NaCl or PBS were incubated at 37°C in the dark alone or in the presence I-DOX 10-5 M. The same amount of I-DOX was added every day for 15 days. In one aliquot I-DOX was added only once in the first day. At given times, the samples were visualized by TEM for the presence of amyloid fibrils.
In a different set of experiments, TTR Leu55Pro prepared as above and resuspended in 0.9% NaCl was allowed to grow into fibrils for 10 days at 37°C, then I-DOX was added in concentrations ranging from 10-4 to 10-7 M. After 5 more days in the dark at 37°C, the samples were analyzed by TEM.
Transmission electron microscopy
For visualization by TEM, samples aliquots were vortexed, diluted (1:50) with NaCl 0.15 M, and immediately adsorbed to glow-discharged carbon-coated collodion film supported on 200-mesh copper grids. Just before adding the drop to the EM grid, the sample was pipetted up and down. For negative staining, the grids were washed with deionized water and stained with 1% uranyl acetate. The grids were visualized with a Zeiss microscope operated at 60 kV and exhaustively examined. Measurements of the fibril widths and lengths were made from enlarged prints of the electron micrographs using a magnifying glass with a built-in calibrated ruler.
Screening for TTR fibril disrupters
A series of compounds was tested for their ability to interfere with TTR amyloid fibril formation or to disrupt mature fibrils. These included 1) minacycline, tetracycline (Sigma, St. Louis, MO, USA) and its derivatives rolitetracycline and doxycycline, a kind gift from the Department of Neuroscience, SmithKline Beecham Pharmaceuticals, UK; 2) 2,4-dinitrophenol (DNP) and 3-nitrophenol (NP) from Sigma.
Tetracycline, rolitetracycline, doxycycline, minocycline, DNP, and NP were prepared in dimethyl sulfoxide (DMSO) at 10 mg/mL; 100 µg of fibrillar TTR Leu55Pro prepared as described (2 mg/mL, PBS) was incubated with a single dose of each of the compounds at 360 µM.
Control (TTR Leu55Pro alone and TTR Leu55Pro with an equal amount of DMSO) and compound-added aliquots were analyzed by TEM at different times.
Caspase-3 assay
RN22 cells (rat Schwannoma cell line) were propagated in 25 cm2 flasks and maintained at 37°C in a humidified atmosphere of 95% and 5% CO2. Cells were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Gaithersburg, MD, USA).
TTR Leu55Pro fibrillar material (500 µg) was treated with I-DOX, tetracycline, rolitetracycline, or doxycycline added fresh every day for 3 or 15 days (10-5 M drug/100 µg fibrils). The sample was then centrifuged at 14,000 rpm, washed three times with 0.9% NaCl to remove the drug, and resuspended in 0.9% NaCl. Toxicity of the treated fibrils was evaluated by activation of caspase-3, measured using the CaspACE colorimetric 96-well plate assay system (Promega, Madison, WI, USA), following the manufacturer’s instructions. Briefly, 80% confluent RN22 cells in DMEM in 1% FBS were exposed for 48 h to 2 µM TTR (either soluble, fibrillar TTR Leu55Pro alone, or I-DOX treated). Subsequently, each well was trypsinized and the cell pellet was lysed in 100 µL hypotonic lysis buffer (Promega) by five cycles of freeze/thawing; 40 µL of each cell lysate was used in duplicate to determine caspase-3 activation. The remaining cell lysate was used to measure total cellular protein concentration using the BIO-RAD protein assay kit (Bio-Rad, Hercules, CA, USA), using BSA as standard. Values shown are the mean of duplicates and the experiment was performed twice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The continuous addition of the drug to the preparation up to 15 days led to disruption of the fibrillar material into round particles 5-6 nm wide, which resembled soluble protein. Therefore, the drug does not act on initial oligomers but on structured fibrils of a certain length.
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I-DOX stability and optimal concentration
When a single dose of I-DOX 10-5 M was added on day 1 to TTR Leu55Pro initial aggregates, with further incubation at 37°C for 14 days, TEM revealed the presence of fibrillar structures (not shown); these fibrils appeared shorter when compared with the untreated sample (roughly, the fibrils appeared to be one-fourth the length of fibrils grown in the absence of the drug); a mixture of round particles and short prefibrillar elements was also visible. In contrast, when the drug was added daily, round particles were observed, as documented in Fig. 1
; these findings suggest that the drug is inactive after some time, probably due to a destabilization process, and loses its activity as a fibril disrupter.
I-DOX ability to interact with amyloid fibrils has been preferentially studied in AL-related amyloidosis and used in concentrations ranging from 10-6 to 10-5 M (5
). To determine the optimal concentration at which I-DOX acts on TTR Leu55Pro fibrils, TTR Leu55Pro initial aggregates were incubated at 37°C and allowed to grow into fibrils for 10 days. After this period, aliquots of 100 µg were incubated with a single dose of I-DOX at 10-4, 10-5, 10-6, or 10-7 M. The samples were further incubated at 37°C for 5 more days in the dark, then analyzed by TEM. I-DOX at 10-7 and 10-6 M concentrations did not disrupt completely the TTR Leu55Pro fibrils formed at 10 days (not shown). In contrast, concentrations of 10-4 or 10-5 M completely disrupted the fibrils previously produced
Study of TTR fibril disrupters
Tetracyclines
It has been reported that tetracyclines exhibit anti-amyloidogenic properties toward Aß-amyloid not only by inhibiting ß-amyloid aggregates formation, but also by disaggregating preformed fibrils (8)
. This class of antibiotics presents a safe toxicological profile and thus is a good candidate for treatment of amyloid-related diseases.
We tested tetracycline and three tetracycline derivatives—rolitetracycline, doxycycline, and minocycline—for their ability to interfere with TTR amyloid formation and assessed their capacity to inhibit fibril formation and/or disrupt TTR fibrils. For up to 17 days this capacity was determined by exposing TTR Leu55Pro initial aggregates to the drugs, followed by TEM analysis.
We found that the antibiotics tested act primarily as fibril disrupters since fibrils were observed in intermediate times (data not shown) before 17 days. After 17 days of incubation at 37°C with tetracycline, the sample clearly showed small round particles abundantly, when compared with the control situation; a small portion of fibrils with apparent lengths similar to the control were also observed (Fig. 2
, upper panels), implying that although tetracycline behaves as a disrupter, it is not completely effective in disaggregating TTR Leu55Pro fibrils in vitro under the concentrations tested (360 µM/100 µg fibrils). Minocycline revealed significant disrupter activity, apparently higher than tetracycline, since only a few fibrils were present after incubation with this compound (Fig. 2
, lower panel); rolitetracycline showed lower disrupting activity than tetracycline and minocycline (since a large amount of fibrils were still visible after 17 days of incubation at 37°C as documented in Fig. 2
, left middle panel). Finally, doxycycline was revealed as the most effective of the three compounds of this class tested: no fibrils were visible after incubation (Fig. 2
, right middle panel). Doxycycline is therefore a good candidate in therapeutic approaches in TTR-related amyloidosis. DMSO had no effect on fibril formation (not shown).
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Nitrophenols
Another group of compounds tested for their capacity to obstruct TTR fibril formation included DNP and NP. NP resulted in partial disruption of the fibrils whereas DNP activity resulted in complete disruption (not shown) at 360 µM/100 µg fibrils.
Cytotoxicity of the species resulting from the action of I-DOX and tetracyclines
The toxicity of the species generated upon treatment of TTR amyloid fibrils with I-DOX was next investigated and compared with different species present during the TTR Leu55Pro fibril assembly process, including the initial aggregates and fibrils assembled for 15 days, using the caspase-3 activation assay on an Schwannoma cell line. Figure 3
A depicts results obtained showing that I-DOX treatment for 15 days did not produce toxic species as compared with untreated fibrils or soluble TTR, contrary to the toxic initial aggregates that we had earlier demonstrated to lead to cell death (11)
.
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We also studied the toxicity of the species resulting from the action of tetracycline, rolitetracycline, and doxycycline. The results are shown in Fig. 3B
and reveal the absence of toxicity after treatment with these compounds when compared with the untreated initial aggregates.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and although the amyloidogenic precursor protein is eliminated from blood, clinical improvement is not observed in all patients. Alternative therapies should therefore be considered. One possible route for treatment is to inhibit amyloid deposition and/or to disrupt already formed fibrils. In the present work we tested different classes of compounds for their ability to inhibit fibril elongation and/or disrupt TTR fibrils.
The data presented in this study clearly showed that I-DOX disrupts in vitro preformed amyloid fibrils assembled for 15 days but does not inhibit fibril formation until a certain fibril length. TTR Leu55Pro fibril elongation pattern was ascertained previously (3)
: the preparations start with abundant oligomers with diameters of 4–5 nm or 8 nm wide and short fibrils that elongate over time; the most prominent species is an 8 nm wide fibril, but thinner fibrils of 4 nm wide and thicker ones 10 nm in diameter are also found. Fibril growth was shown to be sigmoidal in character; the fastest rate of fibril growth occurred between days 11 and 18, corresponding to an elongation rate of 9.2 nm/day. The expected average fibril length after 15 days of incubation at 37°C is 89 nm (3)
. The observation that I-DOX exerts its effect within this time interval suggests that the drug has an important action on fibril elongation and raises the question for the need of a structural motif that allows the drug to bind to fibrils and disrupt them. In fact, I-DOX was shown to rapidly destroy TTR Leu55Pro crystals presenting an oligomeric-like structure composed by the association of monomeric units; TTR WT crystals were quite stable under the same conditions (16)
. Molecular modeling studies were performed and the results suggest that the I-DOX binding site to TTR Leu55Pro crystals corresponds to a region of interaction between monomers. In this variant structure, the main crystal-packing interactions are loops CE, loop AB-
-helix, and loops FG, which are disturbed by I-DOX, leading to the disruption of the amyloid-like oligomer. Although the structures of TTR Leu55Pro oligomer and fibrils are different, it is conceivable that in TTR amyloid fibrils the interactions described above also occur and that I-DOX acts in a similar mode. The lack of activity of I-DOX on TTR Leu55Pro oligomers and short fibrils (up to 10 days) may underlie the lack of binding between the two (I-DOX and oligomers/short fibrils) or a nonactive binding. The latter possibility implies that I-DOX binds in a nonperturbing way, still facilitating fibril growth. However, this hypothesis was not explored in the current work and should be addressed in the future by other molecular methodologies.
The concentration study revealed that I-DOX holds its maximum activity toward fibril disruption at 10–5 M/100 µg fibrils, which is within the range of concentrations regularly used in amyloid-related studies. Anthracyclines are a well-known category of compounds used in clinical practice and constitute a promising group of drugs to be used in the treatment of amyloidosis. The most promising therapeutic results were obtained in AL patients treated with I-DOX at dosages ranging from 100 to 30 mg/m2 (4
, 17)
, with response rates ranging from 5/8 to 6/13. A recent paper reports the results of a multicenter trial in patients with AL amyloidosis (18)
. Patients received I-DOX at a dose of 15 mg per m2 once a week for 4 consecutive weeks, repeated every 3 months for up to four times. The study showed improvement in 15% of patients but concludes that it failed regarding the decision of the effective dosage. Nevertheless, the toxicity was minimal and future investigations concerning pharmacokinetic studies are under way. The results obtained infer the need for optimization of the conditions in each particular situation.
I-DOX belongs to a class of chemotherapeutic agents presenting cardiotoxicity as the most important side effect. Nevertheless, this compound is clearly less cardiotoxic than the parental drug, doxorubicin (DOX), both in animal models (19
20
21)
and humans (22
, 23)
. Due to its cardiotoxicity, however, use of I-DOX has been limited to AL-related amyloidosis caused by a neoplastic plasma cell population in the bone marrow. Efforts should be made to find other I-DOX-related compounds with anti-amyloidogenic activity but producing fewer side effects.
In screening for compounds capable of inhibiting and/or disrupting amyloid fibrils, great attention must be paid not only to the compound toxicity, but also to the toxicity of the structures resulting from the action of these drugs in tissues. From the present report we have no evidence for toxicity of the species after I-DOX treatment. It has been shown that the receptor for advanced glycation end products (RAGE) interacts with TTR fibrils, triggering the activation of nuclear transcription factor 
B (24)
. Using the RN22 cell line, anti-RAGE abolished caspase-3 activation induced by TTR fibrils, suggesting toxicity of TTR fibrils to be a RAGE-mediated process (25)
. In vivo it was observed that RAGE expression is increased in FAP tissues overlapping with TTR deposition and increased with the progression of the disease. Therefore, future in vivo studies in FAP animal models using selected drugs with in vitro activity need to quantify RAGE, inflammatory, and oxidative stress gene expression in tissues.
A group of tetracyclines with structural analogies with Congo red and I-DOX were tested and shown not only to inhibit Aß self-aggregation, but also to disrupt preformed Aß-amyloid aggregates (8)
. Tetracycline also affects the properties of synthetic prion protein (PrP) aggregation by disrupting the fibrils and reverts the protease resistance of PrP Sc from Creutzfeldt-Jakob disease patients (9)
. Our results suggested tetracyclines as TTR fibril disrupters but not as inhibitors of fibril formation, in contrast to the inhibition provoked in Aß aggregation, probably reflecting structural differences between TTR and Aß unfolded intermediates. The disruption ability of this class of antibiotics toward both amyloid fibrils implies the recognition of a common motif present in amyloid fibrils. It has been hypothesized that hydrophobic interactions mediated by the C-terminal portion of Aß are important for fibril stability (10)
, leading to an investigation of hydrophobic compounds effective in destabilizing and disaggregating amyloid fibrils. The results showed that DNP and NP, the two compounds tested, possessed both inhibitory and disrupter activities. Our data support only the latter, and DNP was shown to be the most active. Thus, DNP might additionally recognize a common motif shared by amyloid fibrils.
Tetracyclines are pluripotent drugs that affect many mammalian cell functions including proliferation, migration, apoptosis, and matrix remodeling (26)
. Here we showed that tetracycline, rolitetracycline, and doxycycline treatment generates TTR Leu55Pro species without toxicity, as measured by caspase-3 activation. In our studies, doxycycline presented the highest disrupter activity, making it a good candidate for FAP treatment. This derivative is readily accessible to the central nervous system (27)
, making it especially interesting for amyloid diseases with involvement of the nervous system. It has recently been shown that minocycline, another derivative of tetracycline able to cross the blood-brain barrier (28)
, exerts a neuroprotective effect in a transgenic model of Huntington disease (29)
and in mice with amyotrophic lateral sclerosis (ALS) (28)
. In this process, inhibition of caspase-1 and -3 has been proposed as a potential mechanism responsible for this drug effect. Further testing of tetracycline derivatives for their ability to prevent amyloid deposition (either by inhibiting or disrupting fibrils) and for neuroprotection through caspase inhibition merits investigation. Compounds showing both effects would constitute an added value in treating amyloidosis.
The work described here provides a strategy for studying compounds affecting the amyloidogenic cascade, allowing assessment of the stage affected and the relevance of the results by determining the toxicity of the generated species.
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ACKNOWLEDGMENTS |
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Received for publication September 26, 2002. Accepted for publication January 7, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (NF-B) activation. Lab. Invest. 80,1101-1110[Medline]
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