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Departamento de Bioquímica Médica and
Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil;
* Departamento de Química Orgânica, Instituto Militar de Engenharia, Rio de Janeiro, Brazil; and
Department of Neurobiology and Physiology, Cognitive Neurology and Alzheimer’s Disease Center, Northwestern University Institute for Neuroscience, Evanston, Illinois, USA
1Correspondence: Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21944-590, Brazil. E-mail: felice{at}bioqmed.ufrj.br; ferreira{at}bioqmed.ufrj.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: amyloid • oligomers • neuroprotection • small molecule inhibitors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 million people worldwide (1)
. The disease is characterized by extracellular accumulation of the ß-amyloid peptide (Aß) in senile plaques, the intracellular appearance of neurofibrillary tangles, and synaptic and neuronal loss (2
3
4)
.
Despite extensive data obtained from genetic and animal models supporting a central role for Aß in the pathogenesis of AD, the specific form(s) of Aß that cause(s) injury to neurons in vivo has not been clearly established. Early evidence indicated that fibrillar aggregates of Aß are neurotoxic (see, for example, refs 5
6
7
). On the other hand, recent reports suggest that the toxicity of Aß and of other amyloidogenic proteins lies not in the insoluble amyloid fibrils but rather in soluble oligomeric species (8
9
10
11
12)
. These soluble species include small globular structures 2.7 to 6.0 nm in diameter referred to as Aß-derived diffusible ligands or ADDLs (8)
and curvilinear structures called "protofibrils," which appear to represent strings of the globular structures.
Considerable effort is currently directed toward the development of anti-amyloid therapeutics as possible strategies to prevent or treat AD (13)
. Several inhibitors of the aggregation of Aß have been described. However, the vast majority of such studies have reported inhibitors of Aß fibril formation (e.g., 13
14
15
16
17
). A large body of recent evidence indicates that the main neurotoxic species involved in the pathogenesis of AD are soluble oligomeric forms of the Aß peptide (reviewed in ref 18
) rather than the amyloid fibrils originally considered to be responsible for neurotoxicity. As recently pointed out (19)
, this raises questions about the merits of fibril-disrupting compounds, as they may shift the equilibrium of Aß aggregation from fibrils to potentially more hazardous oligomeric species. Therefore, rather than simply blockers of fibril formation, a more important goal is to find compounds capable of blocking Aß oligomerization. Here we describe the characterization and neuroprotective actions of novel small molecule inhibitors of the formation of soluble Aß oligomers and fibrils. We show that such compounds effectively block Aß oligomerization and toxicity to hippocampal neurons. These results are discussed in terms of the development of novel therapeutic approaches in AD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.5% residual TFE) to a final concentration of 7 µM Aß. Aggregation was followed at 23°C by thioflavin T fluorescence (excitation at 440 nm and emission at 482 nm) on a Hitachi F-4500 fluorometer. Samples were examined by transmission electron microscopy. Aß (7 µM) was incubated in PBS in the absence or presence of different compounds, as indicated in Results. After 6 days, samples were stained with 1% uranyl acetate and examined on a Jeol 1200-EX transmission electron microscope.
Aß oligomer preparation and blocking assay
Aß was dissolved in hexafluoro-2-propanol and aliquoted. An aliquot of the Aß solution was dried and dissolved in anhydrous, cold DMSO, as described (20)
, to make a 0.5 µM stock solution. The five test compounds were dissolved individually in F-12 medium at 1 mM stock solutions, briefly heated to 60–70°C, filtered through 0.22 µM filters (Millipore, Bedford, MA, USA), then diluted with F12 to the concentrations indicated in Results. For each experiment, 2 µL of the 0.5 µM Aß-DMSO stock solution was added to a final volume of 100 µL of samples containing different concentrations of compounds (see Results) and incubated for 50 min at 4°C. Aliquots (2 µL) of each solution were then spotted on a nitrocellulose membrane. The membrane was dried and blocked with 5% non-fat milk in TBST for 1 h, probed with the M90-2 anti-Aß oligomer polyclonal antibody (1:800) (21)
, then with HRP-conjugated anti-rabbit IgG (1:60,000; Amersham). Antibody binding was visualized with the SuperSignal ECL kit (Pierce, Rockford, IL, USA) and analyzed with the 440CF imaging station (Kodak).
Cell culture, immunostaining and viability assay
Hippocampi from 18-day-old rat embryos were dissected and cultured as described (22)
with minor modifications. Cells were plated on glass coverslips coated with 1.5 µg/mL polyornithine (Sigma) in neurobasal medium supplemented with B27 (Gibco, Grand Island, NY, USA). Aß1-42 (40 µM), in the absence or presence of different compounds, was added after 72 h of culture and kept in the medium for 2 days. All Aß solutions used in cell culture experiments were prepared and kept at all times under sterile conditions. To further exclude the possibility of bacterial contamination of Aß preparations, lipopolysaccharide (LPS) levels were determined in aliquots from different preparations using the QCL 1000 kit (Biowhittaker, Walkersville, MD, USA). LPS levels in the samples were found to be below the detection limit of the method (<0.06 IU/mL or <12 pg/mL) (data not shown). Control cultures consisting of neurons cultured in growth medium alone or presence of residual TFE (0.5% v/v) were prepared.
Cell viabilities in cultures incubated with or without Aß were assessed using the LIVE/DEAD kit (Molecular Probes, Eugene, OR, USA). Randomly chosen fields were examined and counted in a Nikon Eclipse TE300 microscope. Percentages of live neurons are expressed relative to the total number of neurons observed in each field. Five independent fields were counted for each experimental condition (which were carried out in triplicate). Essentially identical results were obtained in repeat experiments using neurons from different animals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 23)
yet should be sufficiently water soluble to allow their utilization in primary neuronal culture assays. Other desirable characteristics included low molecular weight and lack of neurotoxicity at the concentrations required for effective inhibition of amyloid aggregation.
Screening for inhibitors was first carried out using the thioflavin T fluorescence assay and measuring the extent of disaggregation of preformed amyloid fibrils upon addition of a fixed concentration (200 µM) of each compound. This initial screen revealed five compounds (of a total of >50 chemically related compounds tested) that caused a significant reversal of the aggregation of Aß. These compounds were 2-amino-4-chloro-phenol; 4-amino-phenol; 3,4-dihydroxybenzoic acid; 4-amino-anisole; and 2-hydroxy-3-ethoxybenzaldehyde (Fig. 1
A). The kinetics of disaggregation of Aß1–42 fibrils by these compounds are shown in Fig. 1B
. After 6 days of incubation, 4-amino-anisole caused 50% disaggregation and all other four compounds caused between 65 and 95% disaggregation of previously formed amyloid fibrils. Three of the compounds tested (2-amino-4-chloro-phenol; 4-amino-phenol; 2-hydroxy-3-ethoxybenzaldehyde) exhibited very fast disaggregating actions, with 50% decrease in thioflavin T fluorescence after only 2 h of incubation with the drugs. Control measurements showed that those compounds exhibit no significant absorption at the wavelength of thioflavin excitation and have no direct effects on the fluorescence of this dye (data not shown). Figure 1C
shows the concentration dependence of Aß1–42 fibril disaggregation by aromatic compounds. At a fixed incubation of 24 h, an apparent IC50 value of 30 µM was found for both 2-amino-4-chloro-phenol and 2-hydroxy-3-ethoxybenzaldehyde and an IC50 value of 90 µM was found for 4-amino-phenol (Fig. 1C
). For the other two compounds, 3,4-dihydroxybenzoic acid and 4-amino-anisole, significant disaggregation of fibrillar amyloid was observed only after 6 days of incubation (Fig. 1B
).
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In addition to disrupting previously formed amyloid aggregates, the compounds identified above inhibited amyloid aggregation, as shown by transmission electron microscopy analysis (Fig. 2
). Abundant amyloid fibrils were observed in control samples of Aß1-42 aggregated in PBS (Fig. 2A
), whereas samples in which Aß was added to the medium in the presence of different compounds were devoid of fibrils and contained only occasional scattered amorphous aggregates (B, C). Careful examination of the EM grids revealed that Aß oligomers and protofibrils were not present in the samples treated with different compounds.
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Inhibition of the formation of soluble Aß oligomers
Direct demonstration that the present compounds block the oligomerization of Aß was obtained using a dot blot immunoassay that discriminates between soluble Aß oligomers and monomers (Fig. 3
). Monomeric Aß1–42 was incubated in F12 medium in the absence or presence of test compounds under conditions that lead to oligomerization. Under such conditions, Aß oligomers are formed but fibrils are not present in the samples, as shown by atomic force microscopy (21)
. Samples were then examined by dot blot using an oligomer-specific antibody (21)
. As shown in Fig. 3A
, the four compounds tested blocked the formation of Aß oligomers in a concentration-dependent manner. The most effective compound was 2-hydroxy-3-ethoxybenzaldehyde, which exhibited an apparent IC50 of 3 µM for the inhibition of oligomer formation. The IC50 values for the other compounds ranged from 10 µM to 20 µM (B). DNP, which we earlier described as an amyloid fibril disrupter (22)
, also blocked the formation of Aß oligomers (Fig. 3)
.
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Blockade of the neurotoxicity of Aß
The possible protective effects of the inhibitors described above against Aß-induced neurotoxicity were investigated. To this end, 72 h primary cultures of E18 rat hippocampal neurons were used, as described (22)
. Cultures were maintained in the absence or presence of Aß plus different compounds for 48 h and neuronal viability was determined using the LIVE/DEAD assay (Fig. 4
A–F). Control hippocampal neurons exhibited large cell bodies and long, branched neurites (not shown), with 82% neuronal survival (Fig. 4A
). On the other hand, significant neuronal degeneration and death was observed in the presence of Aß1–42, with only 10% neuronal survival (Fig. 4B
). The four compounds tested afforded almost complete blockade of the neurotoxicity of Aß (Fig. 4C-F
). Indeed, in the presence of such compounds, neurons treated with Aß showed large cell bodies and long neurites with good adhesion properties, and morphological aspects of the cultures were similar to control 5 day hippocampal cultures (data not shown). The compounds by themselves had no effects on the survival of neurons in culture (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, accumulated data indicate that Aß1–42 is the strongest candidate for being the neurotoxic agent. The aggregation states of these peptides are controversial and the molecular identity of the specific neurotoxic species of Aß remains unclear. Considerable early evidence indicated that amyloid fibrils are toxic (for examples, see refs 5
6
7
), but recent studies support the notion that Aß oligomers are the main neurotoxins (reviewed in ref 18
). Correlations between fibrillar plaque density and severity of dementia are not always found in Alzheimer’s diseased brains (25, 26), whereas correlations between soluble Aß levels and the extent of synaptic loss and cognitive impairment are stronger (27
, 28)
. SDS-stable Aß oligomers have been detected in the buffer-soluble fraction of Alzheimer’s diseased cortex (28
, 29)
. Oligomer levels are specifically augmented by the expression of AD-related mutations in the amyloid precursor protein (APP) or in presenilin, which increase Aß142 production (30)
.
Evidence that soluble oligomeric forms of Aß are the main neurotoxins has come from a large number of in vitro studies as well as from studies using various animal models of AD. For example, it was recently shown that naturally secreted Aß oligomers potently inhibit hippocampal long-term potentiation in vivo (9
, 31)
, supporting their pathological relevance. In addition, mice overexpressing APP751, which exhibit diffuse cerebral amyloid deposits but not plaques, exhibited age-dependent spatial learning deficits, suggesting that accumulation of soluble Aß species was responsible for the impairment (32)
. In another transgenic mouse model that does exhibit plaques, memory impairment correlated negatively with Aß-insoluble levels, suggesting that a soluble neurologically active species was associated with memory loss (33)
. That study demonstrated the presence of soluble Aß oligomers in older mice and a correlation between oligomer levels and memory impairment. Active or passive vaccination studies have provided compelling evidence that memory deficits are associated with the presence of soluble oligomeric Aß species in the brain (e.g., 34
35
36
37
38
). In fact, it has been pointed out that use of antibodies that specifically target soluble Aß assemblies may provide the memory benefits seen in transgenic mouse studies without the CNS inflammation observed in recent human clinical vaccination trials (39)
. A recent study showed that vaccination against soluble Aß oligomers leads to the development of toxicity-neutralizing antibodies (40)
. Therefore, identifying efficient ways to block the formation and neurotoxicity of Aß oligomers remains an important goal in the AD field.
Structure-based drug design approaches to develop inhibitors of Aß aggregation and neurotoxicity are hampered by the lack of detailed structural information on soluble or aggregated Aß. Given this limitation, we have approached the development of anti-amyloidogenic compounds by targeting molecular interactions that are important for amyloid stability. Earlier work has proposed that hydrophobic interactions between Aß side chains are determinants of amyloid aggregation (see below). We previously showed that 2,4-dinitrophenol and 3-nitrophenol, two moderately hydrophobic aromatic compounds, cause the disassembly of preaggregated Aß fibrils (22)
. DNP has also been found to inhibit the amyloid aggregation of transthyretin implicated in familial amyloidotic polyneuropathy and senile systemic amyloidosis (41
, 42)
. In the present study, we have identified novel small molecule anti-amyloidogenic compounds that inhibit the formation of soluble Aß oligomers, as well as amyloid fibrils, and disrupt already formed aggregates. Of greater interest, these compounds completely block the neurotoxicity of Aß to rat hippocampal neurons in primary culture.
It is interesting to consider the possible mechanisms of action of the present compounds in blocking Aß oligomerization and aggregation. Different lines of evidence support the notion that hydrophobic interactions play important roles in Aß aggregation. Disaggregation of previously formed amyloid fibrils by guanidine hydrochloride shows that fibrils obtained using a truncated version of the Aß peptide (Aß1-28) are considerably less stable than fibrils obtained from Aß1-42 (22)
. Inspection of the amino acid sequence of Aß reveals that the portion of the molecule spanning residues 29-42 (which is buried in the plasma membrane before cleavage of Aß from the amyloid precursor protein) contains a large proportion of nonpolar amino acids. The stability of Aß fibrils is markedly decreased at low temperatures (22)
, a characteristic behavior of hydrophobic protein interactions. It has been proposed that pi-stacking interactions between aromatic side chains of Aß may be directly involved in amyloid aggregation (23)
. Thus, it is conceivable that the compounds identified in the present study interfere with hydrophobic and/or pi-pi interactions and thus prevent, or severely impair, the self-association of Aß. A role for hydrophobic interactions has been proposed in the aggregation of other amyloidogenic proteins or peptides such as the islet amyloid polypeptide and calcitonin (23)
. The formation of amyloid aggregates of lysozyme is blocked by compounds similar to those used in the present study (M. N. N. Vieira et al., unpublished results), suggesting that the approach of blocking amyloid aggregation via the use of small aromatic compounds may be applicable to other amyloidosis.
Considering the importance of soluble oligomeric forms of Aß in AD pathogenesis, it is now clear that drug development in this area should focus on inhibitors of the oligomerization of Aß rather than simply inhibitors of fibril formation. A literature search of inhibitors of the oligomerization of Aß yields remarkably few results. A recent study, for example, reported that neuroprotective extracts from Gingko biloba retard oligomer assembly (43). It was recently reported that certain functionalized cyclodextrins effectively block oligomer assembly and neurotoxicity (44)
. With our present results, these earlier findings suggest that blockade of Aß oligomerization by small molecules could potentially lead to effective AD drugs.
The neuroprotective efficiencies of the inhibitors identified in the present study appeared to correlate better with the extent of inhibition of Aß oligomerization than with the disruption of fibrils. Figures 3
and 4
show that full neuroprotection was obtained with inhibitor concentrations that completely blocked the oligomerization of Aß, whereas Aß fibrils were only partially disrupted under such conditions (Fig. 1)
. The apparent IC50 values obtained for blockade of Aß oligomerization (Fig. 3)
were lower than those obtained for disruption of previously formed fibrils (Fig. 1)
, showing that, although able to disrupt preformed fibrils, our compounds more efficiently target Aß oligomerization. These results provide important proof-of-concept to the notion that blocking Aß oligomerization is a valid means to develop neuroprotective agents.
In conclusion, the present findings suggest that the small molecule anti-amyloidogenic compounds described here should be considered as lead compounds for the development of drugs to inhibit oligomerization and prevent the neurotoxicity of Aß in Alzheimer’s disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication March 2, 2004. Accepted for publication May 3, 2004.
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REFERENCES |
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