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We report here the first dual inhibitors of brain carbonic anhydrases (CAs) and monoamine oxidase-B (MAO-B) for the management of Alzheimer's disease. Classical CA inhibitors (CAIs) such as methazolamide prevent amyloid-β-peptide (Aβ)-induced overproduction of reactive oxygen species (ROS) and mitochondrial dysfunction. MAO-B is also implicated in ROS production, cholinergic system disruption, and amyloid plaque formation. In this work, we combined a reversible MAO-B inhibitor of the coumarin and chromone type with benzenesulfonamide fragments as highly effective CAIs. A hit-to-lead optimization led to a significant set of derivatives showing potent low nanomolar inhibition of the target brain CAs ( K I s in the range of 0.1-90.0 nM) and MAO-B (IC 50 in the range of 6.7-32.6 nM). Computational studies were conducted to elucidate the structure-activity relationship and predict ADMET properties. The most effective multitarget compounds totally prevented Aβ-related toxicity, reverted ROS formation, and restored the mitochondrial functionality in an SH-SY5Y cell model surpassing the efficacy of single-target drugs.

The term neuroinflammation defines the reactions of astrocytes and microglia to alterations in homeostasis in the diseased central nervous system (CNS), the exacerbation of which contributes to the neurodegenerative effects of Alzheimer's disease (AD). Local environmental conditions, such as the presence of proinflammatory molecules, mechanical properties of the extracellular matrix (ECM), and local cell-cell interactions, are determinants of glial cell phenotypes. In AD, the load of the cytotoxic/proinflammatory amyloid β (Aβ) peptide is a microenvironmental component increasingly growing in the CNS, imposing time-evolving challenges on resident cells. This study aimed to investigate the temporal and spatial variations of the effects produced by this process on astrocytes and microglia, either directly or by interfering in their interactions. Ex vivo confocal analyses of hippocampal sections from the mouse model TgCRND8 at different ages have shown that overproduction of Aβ peptide induced early and time-persistent disassembly of functional astroglial syncytium and promoted a senile phenotype of reactive microglia, hindering Aβ clearance. In the late stages of the disease, these patterns were altered in the presence of Aβ-plaques, surrounded by typically reactive astrocytes and microglia. Morphofunctional characterization of peri-plaque gliosis revealed a direct contribution of astrocytes in plaque buildup that might result in shielding Aβ-peptide cytotoxicity and, as a side effect, in exacerbating neuroinflammation.

(1) Background: Autophagy, the major cytoplasmic process of substrate turnover, declines with age, contributing to proteostasis decline, accumulation of harmful protein aggregates, damaged mitochondria and to ROS production. Accordingly, abnormalities in the autophagic flux may contribute to many different pathophysiological conditions associated with ageing, including neurodegeneration. Recent data have shown that extra-virgin olive oil (EVOO) polyphenols stimulate cell defenses against plaque-induced neurodegeneration, mainly, through autophagy induction. (2) Methods: We carried out a set of in vitro experiments on SH-SY5Y human neuroblastoma cells exposed to toxic Aβ 1-42 oligomers to investigate the molecular mechanisms involved in autophagy activation by two olive oil polyphenols, oleuropein aglycone (OleA), arising from the hydrolysis of oleuropein (Ole), the main polyphenol found in olive leaves and drupes and its main metabolite, hydroxytyrosol (HT). (3) Results: Our data show that the mixture of the two polyphenols activates synergistically the autophagic flux preventing cell damage by Aβ 1-42 oligomers., in terms of ROS production, and impairment of mitochondria. (4) Conclusion: Our results support the idea that EVOO polyphenols act synergistically in autophagy modulation against neurodegeneration. These data confirm and provide the rationale to consider these molecules, alone or in combination, as promising candidates to contrast ageing-associated neurodegeneration.

Polyphenolic compounds in the Mediterranean diet have received increasing attention due to their protective properties in amyloid neurodegenerative and many other diseases. Here, we have demonstrated for the first time that polyphenol oleuropein aglycone (OleA), which is the most abundant compound in olive oil, has multiple potencies for the inhibition of amyloid self-assembly of pro-inflammatory protein S100A9 and the mitigation of the damaging effect of its amyloids on neuroblastoma SH-SY5Y cells. OleA directly interacts with both native and fibrillar S100A9 as shown by intrinsic fluorescence and molecular dynamic simulation. OleA prevents S100A9 amyloid oligomerization as shown using amyloid oligomer-specific antibodies and cross-β-sheet formation detected by circular dichroism. It decreases the length of amyloid fibrils measured by atomic force microscopy (AFM) as well as reduces the effective rate of amyloid growth and the overall amyloid load as derived from the kinetic analysis of amyloid formation. OleA disintegrates already preformed fibrils of S100A9, converting them into nonfibrillar and nontoxic aggregates as revealed by amyloid thioflavin-T dye binding, AFM, and cytotoxicity assays. At the cellular level, OleA targets S100A9 amyloids already at the membranes as shown by immunofluorescence and fluorescence resonance energy transfer, significantly reducing the amyloid accumulation in GM1 ganglioside containing membrane rafts. OleA increases overall cell viability when neuroblastoma cells are subjected to the amyloid load and alleviates amyloid-induced intracellular rise of reactive oxidative species and free Ca 2+ . Since S100A9 is both a pro-inflammatory and amyloidogenic protein, OleA may effectively mitigate the pathological consequences of the S100A9-dependent amyloid-neuroinflammatory cascade as well as provide protection from neurodegeneration, if used within the Mediterranean diet as a potential preventive measure.

Allium roseum is an important medicinal and aromatic plant, specific to the North African flora and a rich source of important nutrients and bioactive molecules including flavonoids and organosulfur compounds whose biological activities and pharmacological properties are well known. In the present study, the inhibition of amyloid beta protein toxicity by the ethanolic extract of this plant is investigated for the first time. Preliminary biochemical analyses identified kæmpferol and luteolin-7-o-glucoside as the more abundant phenolic compounds. The effects of A. roseum extract (ARE) on aggregation and aggregate cytotoxicity of amyloid beta-42 (Aβ42), whose brain aggregates are a hallmark of Alzheimer's disease, were investigated by biophysical (ThT assay, Dynamic light scattering and transmission electron microscopy) and cellular assays (cytotoxicity, aggregate immunolocalization, ROS measurement and intracellular Ca2+ imaging). The biophysical data suggest that ARE affects the structure of the Aβ42 peptide, inhibits its polymerization, and interferes with the path of fibrillogenesis. The data with cultured cells shows that ARE reduces Aß42 aggregate toxicity by inhibiting aggregate binding to the cell membrane and by decreasing both oxidative stress and intracellular Ca2+. Accordingly, ARE could act as a neuroprotective factor against Aβ aggregate toxicity in Alzheimer's disease., Competing Interests: The authors have declared that no competing interests exist.

The increasing extension in life expectancy of human beings in developed countries is accompanied by a progressively greater rate of degenerative diseases associated with lifestyle and aging, most of which are still waiting for effective, not merely symptomatic, therapies. Accordingly, at present, the recommendations aimed at reducing the prevalence of these conditions in the population are limited to a safer lifestyle including physical/mental exercise, a reduced caloric intake, and a proper diet in a convivial environment. The claimed health benefits of the Mediterranean and Asian diets have been confirmed in many clinical trials and epidemiological surveys. These diets are characterized by several features, including low meat consumption, the intake of oils instead of fats as lipid sources, moderate amounts of red wine, and significant amounts of fresh fruit and vegetables. In particular, the latter have attracted popular and scientific attention for their content, though in reduced amounts, of a number of molecules increasingly investigated for their healthy properties. Among the latter, plant polyphenols have raised remarkable interest in the scientific community; in fact, several clinical trials have confirmed that many health benefits of the Mediterranean/Asian diets can be traced back to the presence of significant amounts of these molecules, even though, in some cases, contradictory results have been reported, which highlights the need for further investigation. In light of the results of these trials, recent research has sought to provide information on the biochemical, molecular, epigenetic, and cell biology modifications by plant polyphenols in cell, organismal, animal, and human models of cancer, metabolic, and neurodegenerative pathologies, notably Alzheimer's and Parkinson disease. The findings reported in the last decade are starting to help to decipher the complex relations between plant polyphenols and cell homeostatic systems including metabolic and redox equilibrium, proteostasis, and the inflammatory response, establishing an increasingly solid molecular basis for the healthy effects of these molecules. Taken together, the data currently available, though still incomplete, are providing a rationale for the possible use of natural polyphenols, or their molecular scaffolds, as nutraceuticals to contrast aging and to combat many associated pathologies., Competing Interests: The authors declare no conflict of interest

Amyloid aggregation starts with the initial misfolding of peptide/protein precursors, with subsequent structural rearrangement into oligomers and protofibrils; the latter eventually organize into fibrils with shared basic structural features, found deposited in amyloid diseases. Mounting evidence indicates early oligomers as the most toxic amyloid species; accordingly, the search of inhibitors of their growth is considered a promising target to prevent amyloid toxicity. We recently showed that oleuropein aglycon, a polyphenol abundant in the extra virgin olive oil, interferes with the aggregation of amylin (involved in type-2 diabetes), eliminating its cytotoxicity. Here we report that oleuropein aglycon also hinders amyloid aggregation of Aβ(1-42) and its cytotoxicity, suggesting a general effect of such polyphenol. In particular, by using a wide panel of different spectroscopic, immunologic, cell viability and imaging techniques we provide a more detailed description of Aβ(1-42) structural modifications arising in the presence of the inhibitor and the resulting cytotoxicity. We here report that the polyphenol eliminates the appearance of early toxic oligomers favouring the formation of stable harmless protofibrils, structurally different from the typical Aβ(1-42) fibrils. We also show that oleuropein aglycon is maximally effective when is present at the beginning of the aggregation process; furthermore, when added to preformed fibrils, it does not induce the release of toxic oligomers but, rather, neutralizes any residual toxicity possibly arising from the residual presence of traces of soluble oligomers and other toxic aggregates. The possible use of this polyphenol as anti-aggregation molecule is discussed in the light of these data.