Marta Di Carlo

Aβ Oligomers and Fibrillar Aggregates Induce Different Apoptotic Pathways in LAN5 Neuroblastoma Cell Cultures

Fibril deposit formation of amyloid beta-protein (Abeta) in the brain is a hallmark of Alzheimers disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Abeta oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Abeta small oligomers and fibrillar aggregates to simulate the action of diffusible Abeta oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Abeta. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Abeta peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.

Mitochondrial Dysfunction: Different Routes to Alzheimer’s Disease Therapy

Mitochondria are dynamic ATP-generating organelle which contribute to many cellular functions including bioenergetics processes, intracellular calcium regulation, alteration of reduction-oxidation potential of cells, free radical scavenging, and activation of caspase mediated cell death. Mitochondrial functions can be negatively affected by amyloid β peptide (Aβ), an important component in Alzheimers disease (AD) pathogenesis, and Aβ can interact with mitochondria and cause mitochondrial dysfunction. One of the most accepted hypotheses for AD onset implicates that mitochondrial dysfunction and oxidative stress are one of the primary events in the insurgence of the pathology. Here, we examine structural and functional mitochondrial changes in presence of Aβ. In particular we review data concerning Aβ import into mitochondrion and its involvement in mitochondrial oxidative stress, bioenergetics, biogenesis, trafficking, mitochondrial permeability transition pore (mPTP) formation, and mitochondrial protein interaction. Moreover, the development of AD therapy targeting mitochondria is also discussed.

Ferulic acid inhibits oxidative stress and cell death induced by Ab oligomers: Improved delivery by solid lipid nanoparticles

Oxidative stress and dysfunctional mitochondria are among the earliest events in AD, triggering neurodegeneration. The use of natural antioxidants could be a neuroprotective strategy for blocking cell death. Here, the antioxidant action of ferulic acid (FA) on different paths leading to degeneration of recombinant β-amyloid peptide (rAβ42) treated cells was investigated. Further, to improve its delivery, a novel drug delivery system (DDS) was used. Solid lipid nanoparticles (SLNs), empty or containing ferulic acid (FA-SNL), were developed as DDS. The resulting particles had small colloidal size and highly negative surface charge in water. Using neuroblastoma cells and rAβ42 oligomers, it was demonstrated that free and SLNs-loaded FA recover cell viability. FA treatment, in particular if loaded into SLNs, decreased ROS generation, restored mitochondrial membrane potential (Δψm) and reduced cytochrome c release and intrinsic pathway apoptosis activation. Further, FA modulated the expression of Peroxiredoxin, an anti-oxidative protein, and attenuated phosphorylation of ERK1/2 activated by Aβ oligomers.

Ferulic Acid: A Hope for Alzheimer’s Disease Therapy from Plants

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the deposition of extracellular amyloid-beta peptide (Aβ) and intracellular neurofibrillar tangles, associated with loss of neurons in the brain and consequent learning and memory deficits. Aβ is the major component of the senile plaques and is believed to play a central role in the development and progress of AD both in oligomer and fibril forms. Inhibition of the formation of Aβ fibrils as well as the destabilization of preformed Aβ in the Central Nervous System (CNS) would be an attractive therapeutic target for the treatment of AD. Moreover, a large number of studies indicate that oxidative stress and mitochondrial dysfunction may play an important role in AD and their suppression or reduction via antioxidant use could be a promising preventive or therapeutic intervention for AD patients. Many antioxidant compounds have been demonstrated to protect the brain from Aβ neurotoxicity. Ferulic acid (FA) is an antioxidant naturally present in plant cell walls with anti-inflammatory activities and it is able to act as a free radical scavenger. Here we present the role of FA as inhibitor or disaggregating agent of amyloid structures as well as its effects on biological models.

Are oxidative stress and mitochondrial dysfunction the key players in the neurodegenerative diseases

Abstract Oxidative stress has long been linked to neuronal cell death that is associated with certain neurodegenerative conditions. Whether it is a primary cause or merely a downstream consequence of the neurodegenerative and aging process is still an open question. Mitochondria are deeply involved in the production of reactive oxygen species through the electron carriers of the respiratory chain and their role in neurodegenerative diseases is discussed here. Moreover, the input of new technological approaches in the study of oxidative stress response or in the evidence of an oxidative stress component in neurodegeneration is reviewed in this paper.

Systemic Immune Responses in Alzheimer's Disease: In Vitro Mononuclear Cell Activation and Cytokine Production

To investigate the systemic signs of immune-inflammatory responses in Alzheimers disease (AD), in the present study we have analyzed blood lymphocyte subsets and the expression of activation markers on peripheral blood mononuclear cells (PBMCs) from AD patients and age-matched healthy controls (HC) activated in vitro by recombinant amyloid-beta peptide (rAbeta42). Our study of AD lymphocyte subpopulations confirms the already described decrease of the absolute number and percentage of B cells when compared to HC lymphocytes, whereas the other subsets are not significantly different in patients and controls. We report the increased expression of the activation marker CD69 and of the chemokine receptors CCR2 and CCR5 on T cells but no changes of CD25 after activation. B cells are also activated by rAbeta42 as demonstrated by the enhanced expression of CCR5. Moreover, rAbeta42 induces an increased expression of the scavenger receptor CD36 on monocytes. Some activation markers and chemokine receptors are overexpressed in unstimulated AD cells when compared to controls. This is evidence of the pro-inflammatory status of AD. Stimulation by rAbeta42 also induces the production of the pro-inflammatory cytokines IL-1beta, IL-6, IFN-gamma, and TNF-alpha, and of the anti-inflammatory cytokines IL-10 and IL-1Ra. The chemokines RANTES, MIP-1beta, and eotaxin as well as some growth factors (GM-CSF, G-CSF) are also overproduced by AD-derived PBMC activated by rAbeta42. These results support the involvement of systemic immunity in AD patients. However, our study is an observational one so we cannot draw a conclusion about its contribution to the pathophysiology of the disease.

Insulin‐activated Akt rescues Aβ oxidative stress‐induced cell death by orchestrating molecular trafficking

Increasing evidence indicates that Alzheimer’s disease, one of the most diffused aging pathologies, and diabetes may be related. Here, we demonstrate that insulin signalling protects LAN5 cells by amyloid‐β42 (Aβ)‐induced toxicity. Aβ affects both activation of insulin receptors and the levels of phospho‐Akt, a critical signalling molecule in this pathway. In contrast, oxidative stress induced by Aβ can be antagonized by active Akt that, in turn, inhibits Foxo3a, a pro‐apoptotic transcription factor activated by reactive oxygen species generation. Insulin cascade protects against mitochondrial damage caused by Aβ treatment, restoring the mitochondrial membrane potential. Moreover, we show that the recovery of the organelle integrity recruits active Akt translocation to the mitochondrion. Here, it plays a role both by maintaining unimpaired the permeability transition pore through increase in HK‐II levels and by blocking apoptosis through phosphorylation of Bad, coming from cytoplasm after Aβ stimulus. Together, these results indicate that the Akt survival signal antagonizes the Aβ cell death process by balancing the presence and modifications of common molecules in specific cellular environments.

Metformin increases APP expression and processing via oxidative stress, mitochondrial dysfunction and NF-κB activation: Use of insulin to attenuate metformin's effect.

Clinical and experimental biomedical studies have shown Type 2 diabetes mellitus (T2DM) to be a risk factor for the development of Alzheimers disease (AD). This study demonstrates the effect of metformin, a therapeutic biguanide administered for T2DM therapy, on β-amyloid precursor protein (APP) metabolism in in vitro, ex vivo and in vivo models. Furthermore, the protective role of insulin against metformin is also demonstrated. In LAN5 neuroblastoma cells, metformin increases APP and presenilin levels, proteins involved in AD. Overexpression of APP and presenilin 1 (Pres 1) increases APP cleavage and intracellular accumulation of β-amyloid peptide (Aβ), which, in turn, promotes aggregation of Aβ. In the experimental conditions utilized the drug causes oxidative stress, mitochondrial damage, decrease of Hexokinase-II levels and cytochrome C release, all of which lead to cell death. Several changes in oxidative stress-related genes following metformin treatment were detected by PCR arrays specific for the oxidative stress pathway. These effects of metformin were found to be antagonized by the addition of insulin, which reduced Aβ levels, oxidative stress, mitochondrial dysfunction and cell death. Similarly, antioxidant molecules, such as ferulic acid and curcumin, are able to revert metformins effect. Comparable results were obtained using peripheral blood mononuclear cells. Finally, the involvement of NF-κB transcription factor in regulating APP and Pres 1 expression was investigated. Upon metformin treatment, NF-κB is activated and translocates from the cytoplasm to the nucleus, where it induces increased APP and Pres 1 transcription. The use of Bay11-7085 inhibitor suppressed the effect of metformin on APP and Pres 1 expression.

Ferulic Acid: a Natural Antioxidant Against Oxidative Stress Induced by Oligomeric A-beta on Sea Urchin Embryo

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder, characterized by loss of memory and impairment of multiple cognitive functions. Amyloid beta peptide (Aβ) is the main component of amyloid plaques observed in the brain of individuals affected by AD. Oxidative stress and mitochondrial dysfunction, induced by Aβ, are among the earliest events in AD, triggering neuronal degeneration and cell death. Use of natural molecules with antioxidant properties could be a suitable strategy for inhibiting the cell death cascade. Here, by employing the sea urchin Paracentrotus lividus as a model system, and Aβ oligomers, we tested the effectiveness of ferulic acid (FA), a natural antioxidant, as a putative AD neuroprotective compound. By microscopic inspection we observed that FA is able to reverse morphological defects induced by Aβ oligomers in P. lividus embryos. In addition, FA is able to neutralize reactive oxygen species (ROS), recover mitochondrial membrane potential, and block apoptotic pathways. Moreover, this model system has allowed us to obtain information about down- or up-regulation of some key molecules—Foxo3a, ERK, and p53—involved in the antioxidant mechanism.

Mediterranean Diet and Healthy Ageing: A Sicilian Perspective

Traditional Mediterranean diet (MedDiet) is a common dietary pattern characterizing a lifestyle and culture proven to contribute to better health and quality of life in Mediterranean countries. By analyzing the diet of centenarians from the Sicani Mountains and eating habits of inhabitants of Palermo, it is reported that a close adherence to MedDiet is observed in the countryside, whereas in big towns this adherence is not so close. This has an effect on the rates of mortality at old age (and reciprocally longevity) that are lower in the countryside than in big towns. Concerning the health effects of the diet, the low content of animal protein and the low glycaemic index of the Sicilian MedDiet might directly modulate the insulin/IGF-1 and the mTOR pathways, known to be involved in ageing and longevity. In particular, the reduction of animal protein intake may significantly reduce serum IGF-1 concentrations and inhibit mTOR activity with a down-regulation of the signal that leads to the activation of FOXO3A and, consequently, to the transcription of homeostatic genes that favour longevity. The down-regulation of both IGF-1 and mTORC1 also induces an anti-inflammatory effect. In addition to the effects on sensing pathways, many single components of MedDiet are known to have positive effects on health, reducing inflammation, optimizing cholesterol and other important risk factors of age-related diseases. However, a key role is played by polyphenols represented in high amount in the Sicilian MedDiet (in particular in extra virgin olive oil) that can work as hormetins that provide an environmental chemical signature regulating stress resistance pathways such as nuclear factor erythroid 2-related factor 2.