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,1
* CNR-Istituto di Biofisica U.O. di Palermo, Palermo, Italy; and
CNR-Istituto di Biomedicina e di Immunologia Molecolare, Via Ugo La Malfa, Palermo, Italy
2Correspondence: CNR-IBF, Via Ugo La Malfa, 153, Palermo 90146, Italy. E-mail: pierluigi.sanbiagio{at}pa.ibf.cnr.it
SPECIFIC AIMS
The aims of this work are 1) the setup of a high-yield expression and purification method of production of a recombinant Aßbeta;42 peptide to obtain, by using the bacteria machinery, a product avoiding the presence of contaminant reagents as well as of residual fragments, usually poorly controlled in the synthetic methods of protein production; 2) the setup of a new toxicity assay on the morphogenic development of a model system, the Sea Urchin Paracentrotus lividus, testing in particular the effect of two different kind of Aßbeta;42 aggregates, obtained dissolving the recombinant peptide at pH 3 and pH 7.2.
PRINCIPAL FINDINGS
1. The recombinant peptide can be produced in huge quantities by using the RNA from human neuroblastoma LAN5 cells and an Escherichia coli expression vector.
2. The aggregation properties (the kinetic behavior and the ability to form fibrillar aggregates) are similar to the synthetic amyloid peptide as shown by dynamic light scattering measurements and thioflavin affinity.
3. Aggregates, with hydrodynamic diameter of 
30 nm, are present in solution when the recombinant peptide is dissolved at pH 3, whereas smaller oligomers, with hydrodynamic diameter of 5 nm, are detected when dissolved at pH 7.2.
The kinetics of aggregation is faster at low pH.
4. The toxicity assay on the morphogenic development of the Sea Urchin Paracentrotus lividus is dose dependent.
At the higher doses, the assay shows many morphological defects with respect to the control already from the first developmental stages (Fig. 1
).
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5. Small oligomers stabilized at pH 7.2 result more toxic than the aggregates detected at pH 3, as shown by the percentage of embryos that survived for both recombinant and commercial Aßbeta;42 (Fig. 2 ).
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CONCLUSIONS AND SIGNIFICANCE
In this work we have developed a procedure to improve the production and purification of a recombinant Aßbeta;42 peptide. Different species are stabilized under two conditions: small oligomers at physiological pH and larger aggregates at low pH, as shown by dynamic light scattering studies. In the framework of the relation structure-activity, the toxicity of these different kinds of Aßbeta;42 aggregates was tested on a classical powerful model system—the sea urchin—which can pass synchronously through different embryonal stages in only 48 h. Aßbeta;42 toxicity on the development of sea urchin embryos is dose dependent: By increasing the concentration, the number of embryos that survived decreased, and among those embryos that survived, the defective morphology due to difficulty in cell division was proportional to the dose. Though Alzheimer’s disease (AD) is strictly linked to Aßbeta; peptides, the relation between disease symptoms and both extracellular plaques and intracellular accumulation of small oligomers of Aßbeta; peptides is not yet clear. In neuronal cells it has been demonstrated that apoptosis is induced by the intracellular component of Aßbeta;42. We observe that, in comparison to acid solution, neutral solution significantly increased the level of toxicity on sea urchin embryos, indicating that the state of Aßbeta;42 assembly appears to influence their biological activities. The presence of small oligomers of recombinant Aßbeta;42, stabilized at pH 7.2, brings malformation and complete interruption of the embryo’s development at the pathological occluded blastula state after 48 h. At the same stage, larger aggregates of recombinant Aßbeta;42, stabilized at pH 3, allow some embryos to reach normal development or the more advanced (though pathological) occluded prism state. These results support the belief that early symptoms of AD can be the effect of cellular malfunctioning due to pathological small oligomers of Aßbeta; peptides, which, unfortunately, are hardly detectable in vivo. The mechanism responsible for the different toxicity in our system is not yet definable. However, a possibility is that the small oligomers might be more diffusible with respect to larger aggregates or fibrils and can easily be inserted into the extracellular space or in the lipid bilayer, or can be internalized within the cells of the developing embryos, altering their vital functions. In contrast, preformed larger aggregates or fibrils in sea urchin could mimic the extracellular plaques of AD in neurons and compromise cell-cell interaction and all the process related to cellular membrane. Finally, the studies described here on the sea urchin system suggest it can be used as an in vivo model to relate Aßbeta; composition with its toxicity and may likely be used as an indicative tool for pharmacological evaluation of novel therapeutic agents.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5716fje
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