Toxicity of recombinant β‐amyloid prefibrillar oligomers on the morphogenesis of the sea urchinParacentrotus lividus

Abstract

A distinctive feature of Alzheimer's disease is the deposition of amyloid beta-protein (Abeta) in senile or diffuse plaques. The 42 residue beta-peptide (Abeta42) is the predominant form found in plaques. In the present work we report a high-yield expression and purification method of production of a recombinant Abeta42. The purified recombinant peptide shows characteristics similar to the synthetic human peptide. Different size aggregates, either small oligomers or larger aggregates, were obtained upon dissolving the recombinant Abeta42 peptide under different conditions at pH 7.2 or pH 3, respectively. We report a new toxicity assay on the morphogenic development of the sea urchin Paracentrotus lividus and study the toxicity of the two kinds of aggregates. Despite the difference between the ionic strength of human extracellular fluid (0.154 mol/l) and artificial sea water (0.48 mol/l), toxicity data collected in this system have an intrinsic relevance. The different ionic strength, in fact, could change the kinetics of oligomer formation, but the effect of morphogenic development reported here is related to the final oligomer sizes. Results of the toxicity assay of Abeta42 on sea urchin development also show a dose-dependent effect. After only 4 h of embryo development, one can note morphological defects in the cell membrane. Retardation of the embryo's development, along with cellular disorders visible inside the blastocoele, can be observed after 1 day of development. Cellular degeneration in two different pathological phenotypes-the occluded blastulae and the occluded prism-is present after 48 h of development. Results show that a greater effect on cell death is induced by the small oligomers stabilized under physiological conditions than at acid pH. In this case only occluded blastulae are found after 48 h of development.

Amyloid precursor protein 96–110 and β-amyloid 1–42 elicit developmental anomalies in sea urchin embryos and larvae that are alleviated by neurotransmitter analogs for acetylcholine, serotonin and cannabinoids

“…Before or after this period, even concentrations as high as 8-10 µM had no effect. Within the vulnerable period, though, the outcomes from APP 96-110 exposure were similar to those seen with Aβ42 [13], with so-called "occluded" blastulae, gastrulae or prisms [13,20]. At the lowest effective concentration of APP 96-110 (0.5 µM), we often found cells released into the blastocoele, accumulating initially near the apex simultaneously with thinning of archenterons, and only later filling this cavity (not shown).…”

“…Aβ42 (oligomeric peptide; Chemicon International, Temecula, CA) and APP 96-110 (Ac-NWCKRGRKQCKTHPH-NH 2 , disulfide bond 3-10; AnaSpec, San Jose, CA) were used at concentrations from 0.1 to 1 µM or 0.5 to 10 µM, respectively, after preliminary studies established these ranges as appropriate to evaluations for threshold to maximal effects. These concentration ranges are similar to those shown to be required for Aβ42 toxicity in other lower organisms [20,22,30,33,51]. Lipophilic analogs of ACh (arachidonoyl dimethylaminoethanol, AA-DMAE), 5HT (arachidonoyl 5HT, AA-5-HT), and cannabinoids-vanilloids (arachidonoyl vanillylamine, AA-VAN) were evaluated at 10-40 µM, a concentration range found to be optimal in our previous experiments with a variety of neurotoxicants [12,15,19], including Aβ42 [13].…”

“…We [13] and others [20] recently showed that Aβ42, the β-amyloid fragment suspected to cause neurotoxicity in Alzheimer's Disease, elicits dose-dependent malformations in sea urchin embryos and larvae. Further, we found that membrane-permeable neurotransmitter analogs could prevent the adverse effects of Aβ42 [13]; although analogs of ACh, dopamine, serotonin (5HT) and cannabinoids all offset the toxicity, those targeting ACh systems were the most effective.…”

“…In the current study, we used the developing sea urchin embryo to evaluate the morphogenetic effects of APP 96-100 in contrast to already-identified actions of a different Aβ42 [13,20], the neurotoxic APP fragment involved in plaque formation. The sea urchin embryo, develops the ability to synthesize, store and release ACh and other neurotransmitters, and possesses the analogous receptors and downstream signaling cascades, all of which appear rapidly over a defined developmental period and act as morphogens that control cell differentiation and assembly of the embryo and larva [10,13,20,26].…”

“…The sea urchin embryo, develops the ability to synthesize, store and release ACh and other neurotransmitters, and possesses the analogous receptors and downstream signaling cascades, all of which appear rapidly over a defined developmental period and act as morphogens that control cell differentiation and assembly of the embryo and larva [10,13,20,26]. Agents that target specific neurotrophic mechanisms or signaling cascades in the mammalian brain, produce structural anomalies during sea urchin morphogenesis that are readily characterized with routine light microscopy [4,5,[9][10][11][12][17][18][19]36,41].…”