Fabiana Morroni

Benzofuran-Based Hybrid Compounds for the Inhibition of Cholinesterase Activity, β Amyloid Aggregation, and Aβ Neurotoxicity

The complex etiology of Alzheimers disease (AD) prompts scientists to develop multitarget strategies to combat causes and symptoms. We therefore designed, synthesized, and tested new hybrid molecules linking a benzofuran ring to a N-methyl- N-benzylamine through a heptyloxy chain, affording a series of potential multifunctional drugs for AD. The cholinesterase inhibitory activity was extended to the inhibition of Abeta fibril formation for 1, 3, and 5. Compound 3 showed an additional neuroprotective effect.

Sulforaphane as a Potential Protective Phytochemical against Neurodegenerative Diseases

A wide variety of acute and chronic neurodegenerative diseases, including ischemic/traumatic brain injury, Alzheimers disease, and Parkinsons disease, share common characteristics such as oxidative stress, misfolded proteins, excitotoxicity, inflammation, and neuronal loss. As no drugs are available to prevent the progression of these neurological disorders, intervention strategies using phytochemicals have been proposed as an alternative form of treatment. Among phytochemicals, isothiocyanate sulforaphane, derived from the hydrolysis of the glucosinolate glucoraphanin mainly present in Brassica vegetables, has demonstrated neuroprotective effects in several in vitro and in vivo studies. In particular, evidence suggests that sulforaphane beneficial effects could be mainly ascribed to its peculiar ability to activate the Nrf2/ARE pathway. Therefore, sulforaphane appears to be a promising compound with neuroprotective properties that may play an important role in preventing neurodegeneration.

Neuroprotective effects of anthocyanins and their in vivo metabolites in SH-SY5Y cells

Recent in vivo studies have highlighted an important role for the neuroprotective actions of dietary anthocyanins. However, one consistent result of these studies is that the systemic bioavailability of anthocyanins, including cyanidin 3-O-glucopyranoside (Cy-3G), is very poor. Cy-3G has been demonstrated to be highly instable at physiological pH, so its in vivo metabolites, such as the aglycon cyanidin (Cy) and protocatechuic acid (PA), may be responsible for both the antioxidant activitiy and the neuroprotective effects observed in vivo. Therefore, we investigated the protective effects of Cy-3G, Cy and PA against H(2)O(2)-induced oxidative stress in a human neuronal cell line (SH-SY5Y). We determined their ability to counteract reactive oxygen species (ROS) formation and to inhibit apoptosis in terms of mitochondrial functioning loss and DNA fragmentation induced by H(2)O(2). We demonstrated that pretreatment of SH-SY5Y cells with Cy-3G, Cy and PA inhibits H(2)O(2)-induced ROS formation at different cellular levels: Cy-3G at membrane level, PA at cytosolic level and Cy at both membrane and cytosolic levels. In addition, Cy showed a higher antioxidant activity at membrane and cytosolic level than Cy-3G and PA, respectively. Interestingly, both Cy and PA, but not Cy-3G, could inhibit H(2)O(2)-induced apoptotic events, such as mitochondrial functioning loss and DNA fragmentation. These results suggest that Cy and PA may be considered as neuroprotective molecules and may play an important role in brain health promotion. These in vitro findings should encourage further research in animal models of neurological diseases to explore the potential neuroprotective effects of compounds generated during in vivo metabolism of anthocyanins.

Neuroprotective effect of sulforaphane in 6-hydroxydopamine-lesioned mouse model of Parkinson's disease

Parkinsons disease (PD) is characterized by the selective loss of dopaminergic nigrostriatal neurons, which leads to disabling motor disturbances. Sulforaphane (SFN), found in cruciferous vegetables, is a potent indirect antioxidant and recent advances have shown its neuroprotective activity in various experimental models of neurodegeneration. This study was undertaken to examine the effects of SFN on behavioral changes and dopaminergic neurotoxicity in mice exposed to 6-hydroxydopamine (6-OHDA). For this purpose, mice were treated with SFN (5mg/kg twice a week) for four weeks after the unilateral intrastriatal injection of 6-OHDA. The increase in 6-OHDA-induced rotations and deficits in motor coordination were ameliorated significantly by SFN treatment. In addition, SFN protected 6-OHDA-induced apoptosis via blocking DNA fragmentation and caspase-3 activation. These results were further supported by immunohistochemical findings in the substantia nigra that showed that SFN protected neurons from neurotoxic effects of 6-OHDA. The neuroprotective effect of SFN may be attributed to its ability to enhance glutathione levels and its dependent enzymes (glutathione-S-transferase and glutathione reductase) and to modulate neuronal survival pathways, such as ERK1/2, in the brain of mice. These results suggest that SFN may potentially be effective in slowing down the progression of idiopathic PD by the modulation of oxidative stress and apoptotic machinery.

Sulforaphane as an inducer of glutathione prevents oxidative stress-induced cell death in a dopaminergic-like neuroblastoma cell line

The total GSH depletion observed in the substantia nigra (SN) appears to be responsible for subsequent oxidative stress (OS), mitochondrial dysfunction, and dopaminergic cell loss in patients with Parkinson’s disease. A strategy to prevent the OS of dopaminergic cells in the SN may be the use of chemopreventive agents as inducers of endogenous GSH, antioxidant and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic‐like neuroblastoma SH‐SY5Y cell line with sulforaphane (SF), a cruciferous vegetables inducer, resulted in significant increases of total GSH level, NAD(P)H : quinone oxidoreductase‐1, GSH‐transferase and ‐reductase, but not GSH‐peroxidase, catalase and superoxide dismutase activities. Further, the elevation of GSH levels, GSH‐transferase and NAD(P)H:quinone oxidoreductase‐1 activities was correlated to an increase of the resistance of SH‐SY5Y cells to toxicity induced by H2O2 or 6‐hydroxydopamine (6‐OHDA). The pre‐treatment of SH‐SY5Y cells with SF was also shown to prevent various apoptotic events (mitochondrial depolarization, caspase 9 and 3 activation and DNA fragmentation) and necrosis elicited by 6‐OHDA. Further, the impairment of antioxidant capacity and reactive oxygen species formation at intracellular level after exposure to 6‐OHDA was effectively counteracted by pre‐treatment with SF. Last, both the cytoprotective and antioxidant effects of SF were abolished by the addition of buthionine sulfoximine supporting the main role of GSH in the neuroprotective effects displayed by SF. These findings show that SF may play a role in preventing Parkinson’s disease.

Neuroprotective effects of cyanidin 3-O-glucopyranoside on amyloid beta (25-35) oligomer-induced toxicity.

Recent studies suggest that the oligomers of short amyloid beta (Abeta) peptides such as Abeta(25-35) as well as full-length Abeta peptides (i.e. Abeta(1-40) and Abeta(1-42) peptides) are responsible for synaptic dysfunction and/or neuronal loss in Alzheimers disease (AD). Among antioxidant phytochemicals derived from fruit and vegetables, cyanidin 3-O-glucoside (Cy-3G) has recently gained attention for its neuroprotective properties. In this in vitro study, we demonstrated that Cy-3G can inhibit Abeta(25-35) spontaneous aggregation into oligomers and their neurotoxicity in human neuronal SH-SY5Y cells. In particular, the pre- and co-treatment of SH-SY5Y cells with Cy-3G reduced the neuronal death, in terms of apoptosis and necrosis, elicited by Abeta(25-35) oligomers. Cy-3G also shows the interesting ability to prevent the early events leading to neuronal death such as the Abeta(25-35) oligomer binding to plasma membrane and the subsequent membrane integrity loss. Taken together, these findings suggest that Cy-3G may be considered a phytochemical with neuroprotective properties useful in finding potential drug or food supplements for the therapy of AD.

Multitargeted drugs discovery: Balancing anti-amyloid and anticholinesterase capacity in a single chemical entity

Memoquin (1) is a lead compound multitargeted against Alzheimers disease (AD). It is an AChE inhibitor, free-radical scavenger, and inhibitor of amyloid-β (Aβ) aggregation. A new series of 1 derivatives was designed and synthesized by linking its 2,5-diamino-benzoquinone core with motifs that are present in the structure of known amyloid binding agents like curcumin, the benzofuran derivative SKF64346, or the benzothiazole bearing compounds KHG21834 and BTA-1. The weaker AChE inhibitory potencies and the concomitant nearly equipotent anti-amyloid activities of the new compounds with respect to 1 resulted in a more balanced biological profile against both targets. Selected compounds turned out to be effective Aβ aggregation inhibitors in a cell-based assay. By properly combining two or more distinct pharmacological properties in a molecule, we can achieve greater effectiveness compared to single-targeted drugs for investigating AD.

Cyanidin 3-O-glucopyranoside protects and rescues SH-SY5Y cells against amyloid-beta peptide-induced toxicity.

The amyloid-&bgr; (A&bgr;) peptide (1–42) aggregation into oligomeric and fibrillar species affects neuronal viability, having a causal role in the development of Alzheimers disease. Among dietary anthocyanins, cyanidin 3-O-glucoside (Cy-3G) and its metabolites, such as protocatechuic acid (PA), have gained attention as potential neuroprotective agents. In this in-vitro study, we demonstrated that Cy-3G, but not PA, can inhibit A&bgr;1–42 spontaneous aggregation using thioflavin T fluorescence assay and transmission electron microscopy. Furthermore, treatment of human neuronal SH-SY5Y cells with Cy-3G during oligomeric and fibrillar A&bgr;1−42 treatment prevents neuronal viability loss. These protective effects were still evident when Cy-3G treatment was initiated after the appearance of oligomeric A&bgr;1−42 neurotoxicity. Taken together, these results suggest that Cy-3G may protect and rescue the neuronal cells from toxicity induced by A&bgr;1−42.

Influence of Cultivation System on Bioactive Molecules Synthesis in Strawberries: Spin‐off on Antioxidant and Antiproliferative Activity

Strawberries (Fragaria ananassa L., cv. favette) were studied to investigate the influence of cultivation practices (biodynamic, conventional) on the synthesis of bioactive molecules (ascorbic acid, ellagic acid, anthocyanins, flavonols) and to evaluate their antioxidant activity. Additionally, the in vitro bioactivity, in terms of antioxidant and antiproliferative activity, of the same strawberry samples in human colon carcinoma (Caco-2) cells was also studied. Compared to conventional strawberries, biodynamic fruits had a significantly higher content of ascorbic acid (P < 0.01), pelargonidin-3-glucoside (P < 0.05), cyanidin-3-glucoside (P < 0.01), ellagic acid (P < 0.01), quercetin, and kaempferol (both P < 0.01). Antioxidant activity of biodynamic strawberry crude extract was significantly higher than that of the conventional one (P < 0.05); in addition, while the antioxidant activity of water-soluble fraction was very similar in both biodynamic and conventional strawberries, that of water-insoluble fraction of biodynamic fruits was significantly higher (P < 0.05). The same crude extract of biodynamic strawberry samples effectively corresponded to an increase of bioactivity, in terms of both cellular antioxidant activity and antiproliferative activity, in Caco-2 cells differentiated to normal intestinal epithelia and in undifferentiated Caco-2, respectively. Further studies are needed to confirm whether the practice of biodynamic agriculture is likely to increase the bioactivity of other varieties of fruits and vegetables.

Neuroprotective Effects of Erucin against 6-Hydroxydopamine-Induced Oxidative Damage in a Dopaminergic-like Neuroblastoma Cell Line

Oxidative stress (OS) contributes to the cascade leading to the dysfunction or death of dopaminergic neurons during Parkinson’s disease (PD). A strategy to prevent the OS of dopaminergic neurons may be the use of phytochemicals as inducers of endogenous antioxidants and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic-like neuroblastoma SH-SY5Y cell line with isothiocyanate erucin (ER), a compound of cruciferous vegetables, resulted in significant increases of both total glutathione (GSH) levels and total antioxidant capacity at the cytosolic level. The increase of GSH levels was associated with an increase in the resistance of SH-SY5Y cells to neuronal death, in terms of apoptosis, induced by 6-hydroxydopamine (6-OHDA). The pretreatment of SH-SY5Y cells with ER was also shown to prevent the redox status impairment, in terms of intracellular ROS and O2•− formation, and loss of mitochondrial membrane potential, early events that are initiators of the apoptotic process, induced by 6-OHDA. Last, the antiapoptotic and antioxidant effects of ER were abolished by buthionine sulfoximine, supporting the main role of GSH in the neuroprotective effects recorded by ER. These results suggest that ER may prevent the oxidative damage induced by 6-OHDA.