Aislamiento y caracterizacion de un nuevo oxidante de origen marino con propiedades neuroprotectoras y antiamiloidogenicas

Resumen

Multiple lines of evidence implicates oxidative stress in the cellular mechanisms leading to neuronal cell death associated to certain neurodegenerative conditions such as Alzheimer¿s disease, Parkinson¿s disease or Amyotrophic Lateral Sclerosis. Thus, neuroprotection constitute a promising approach to slow down the progression of some neurodegenerative processes. It has been also described that some antioxidants are able to reduce the levels of amyloid-¿ peptide (A¿¿, the main constituent of amyloid plaques in the brains of Alzheimer¿s disease patients. The marine environment provides us with an enormous resource for the discovery of potential chemotherapeutic agents. Primary screening to look for new antioxidants was performed on SH-SY5Y neuroblastoma cell line. NPM01, 02 and 03, described previously as antibiotic and antitumoral compounds, were isolated from Streptomyces spp. having very strong antioxidant activity. Another neuroprotection assays, with widely used toxins as cellular models for Parkinson¿s or Alzheimer¿s disease, and different in vitro antioxidant enzymatic assays were performed in order to evaluate different biological activities and to gain insight into the molecular mechanisms of action underlying the neuroprotective effect of these molecules. The compounds showed potent neuroprotective activity against 6-hydroxydopamine (6-OHDA) and A¿25-35 toxicity and exhibited Fe2+ chelating effect. We have evaluated antiamyloidogenic properties using an amyloid precursor protein (APP) transfected cell line. Carried out experiments to quantitatively determine A¿1-40 and A¿1-42 production of NPM01 treated cells showed a time-dependent, dose-dependent reduction in A¿ secretion, at all different times studied as compared to the untreated cells. Interestingly, the finding that the extracellular APP levels were increased after NPM01 treatment, due to an increase in the secreted sAPP¿¿form, suggested that this effect was mediated by the activation of the non-amyloidogenic ¿-secretase pathway. Western-blot analysis and real-time quantitative PCR indicated that this effect could be mediated through the enzyme ADAM-10. TAPI-2, an ¿-secretase inhibitor, abolished the sAPP¿ increased levels but not the reduction on A¿ levels after NPM01 treatment, demonstrating another independent mechanism of the compound for A¿ decrease. Real-time quantitative PCR and zymography showed an increase in the metalloproteinase (MMP) MMP-2 and MMP-9 expression and activity, respectively. Moreover, MMP inhibitor GM6001 treatment prevented the A¿ reduction levels induced by NPM01. Finally, we have studied different cellular signalling pathways demonstrating that the increase in sAPP¿ levels could be due to an increase on the PI3K/Akt and ERK phosphorilation and, subsequently, activation of these signalling pathways. In conclusion, NPM01 is a new marine neuroprotective compound against oxidative stress and other insults such as 6-OHDA and A¿25-35. Additionally, NPM01 has Fe2+ chelating activity. Furthermore, NPM01 is able of promote A¿ degradation probably by MMP activation and enhances the release of the neurotrophic and neuroprotective sAPP¿ fragment most likely due to an activation of the ADAM-9 and ADAM-10 ¿-secretases. All these effects are maybe mediated by the activated PI3K/Akt and ERK signalling pathways. Taking all the results together, NPM01 may serve as an ideal drug for neurodegenerative diseases, especially Alzheimer¿s disease.