Sara Anna Bonini

Impaired Adult Neurogenesis Associated with Short-Term Memory Defects in NF-κB p50-Deficient Mice

Neurogenesis proceeds throughout adulthood in the brain of most mammalian species, but the molecular mechanisms underlying the regulation of stem/progenitor cell proliferation, survival, maturation, and differentiation have not been completely unraveled. We have studied hippocampal neurogenesis in NF-κB p50-deficient mice. Here we demonstrate that in absence of p50, the net rate of neural precursor proliferation does not change, but some of the steps leading to the final neuron differentiation status are hampered, resulting in ∼50% reduction in the number of newly born neurons in the adult mutant hippocampus. Additionally, in p50−/− mice, we observed a selective defect in short-term spatial memory performance without impairment of hippocampal-dependent spatial long-term memory and learning. Our results highlight the role of NF-κB p50 in hippocampal neurogenesis and in short-term spatial memory.

Nuclear Factor κB-Dependent Neurite Remodeling Is Mediated by Notch Pathway

In this study, we evaluated whether a cross talk between nuclear factor κB (NF-κB) and Notch may take place and contribute to regulate cell morphology and/or neuronal network in primary cortical neurons. We found that lack of p50, either induced acutely by inhibiting p50 nuclear translocation or genetically in p50−/− mice, results in cortical neurons characterized by reduced neurite branching, loss of varicosities, and Notch1 signaling hyperactivation. The neuronal morphological effects found in p50−/− cortical cells were reversed after treatment with the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-1-alanyl 1]-S-phenylglycine t-butyl ester) or Notch RNA interference. Together, these data suggested that morphological abnormalities in p50−/− cortical neurons were dependent on Notch pathway hyperactivation, with Notch ligand Jagged1 being a major player in mediating such effect. In this line, we demonstrated that the p50 subunit acts as transcriptional repressor of Jagged1. We also found altered distribution of Notch1 and Jagged1 immunoreactivity in the cortex of p50−/− mice compared with wild-type littermates at postnatal day 1. These data suggest the relevance of future studies on the role of Notch/NF-κB cross talk in regulating cortex structural plasticity in physiological and pathological conditions.

GPNMB/OA protein increases the invasiveness of human metastatic prostate cancer cell lines DU145 and PC3 through MMP-2 and MMP-9 activity

Non-metastatic glycoprotein melanoma protein B (GPNMB), also known as osteoactivin (OA) is expressed in a wide array of tumors and represents an emerging target for drug development. In this study, we investigated the role of GPNMB/OA in the progression of human metastatic DU145 and PC3 prostate cancer cells. GPNMB/OA contribution in PCa malignant phenotype has been analyzed by small interfering RNA-induced GPNMB/OA silencing. We found that following GPNMB/OA silencing the migration capability of both DU145 and PC3 cells, evaluated by using in vitro invasivity assay, as well as the metalloproteinases MMP-2 and MMP-9 activity were equally strongly inhibited. By contrast knocking down GPNMB/OA weakly attenuated cell proliferation rate of DU145, an effect that paralleled with an increase number of apoptotic cells. However, PC3 cell growth seems to be not affected by GPNMB/OA. Together, these data reveal that GPNMB/OA acts as a critical molecular mediator promoting the acquisition of the more aggressive, pro-metastatic phenotype distinctive of human DU145 and PC3 cell lines.

Microtubule stabilizing effect of notch activation in primary cortical neurons.

The appropriate level of microtubule stability is fundamental in neurons to assure correct polarity, migration, vesicles transport and to prevent axonal degeneration. In the present study, we have identified Notch pathway as an endogenous microtubule stabilizer. Stimulation of Notch receptors by exposure of mouse cortical neurons to the Notch ligand Jagged1 resulted in increased microtubule stability, as measured by using antibodies against post-translationally modified alpha tubulin, and changes in axonal morphology and branching, with varicosity loss, thicker neurites and enlarged growth cones. Similar effects were found after exposure of the cells to different doses of Taxol. However, contrary to Taxol, Jagged1 induced downregulation of the microtubule severing protein Spastin. We suggest that a fine-tuned manipulation of Notch signaling may represent a novel approach to modulate neuronal cytoskeleton plasticity.

Targeting Notch pathway induces growth inhibition and differentiation of neuroblastoma cells

High-risk neuroblastoma is a severe pediatric tumor characterized by poor prognosis. Understanding the molecular mechanisms involved in tumor development and progression is strategic for the improvement of pharmacological therapies. Notch was recently proposed as a pharmacological target for the therapy of several cancers and is emerging as a new neuroblastoma-related molecular pathway. However, the precise role played by Notch in this cancer remains to be studied extensively. Here, we show that Notch activation by the Jagged1 ligand enhances the proliferation of neuroblastoma cells, and we propose the possible use of Notch-blocking γ-secretase inhibitors (GSIs) in neuroblastoma therapy. Two different GSIs, Compound E and DAPT, were tested alone or in combination with 13-cis retinoic acid (RA) on neuroblastoma cell lines. SH-SY5Y and IMR-32 cells were chosen as paradigms of lower and higher malignancy, respectively. Used alone, GSIs induced complete cell growth arrest, promoted neuronal differentiation, and significantly reduced cell motility. The combination of GSIs and 13-cis RA resulted in the enhanced growth inhibition, differentiation, and migration of neuroblastoma cells. In summary, our data suggest that a combination of GSIs with 13-cis RA offers a therapeutic advantage over a single agent, indicating a potential novel therapy for neuroblastoma.

Generation of reactive oxygen species by beta amyloid fibrils and oligomers involves different intra/extracellular pathways

A neuropathological characteristic of Alzheimer’s disease is the extracellular accumulation of amyloid beta peptide (Aβ) in neuritic plaques. Recent evidences suggested that soluble Aβ oligomers are the predominant neurotoxic species for neurons. Thus, considerable attention has been paid to discriminate the cytotoxic pathways of Aβ pre-fibrillar aggregates and mature fibrils. We showed that the mechanisms by which Aβ oligomers and fibrils generated reactive oxygen species differ in terms of site of production and kinetics, suggesting the involvement of different intra/extracellular pathways.

Blockade of the tumor necrosis factor-related apoptosis inducing ligand death receptor DR5 prevents beta-amyloid neurotoxicity.

We originally suggested that inhibition of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) death pathway could be taken into consideration as a potential therapeutic strategy for Alzheimers disease (AD). However, because the critical role of TRAIL in immune surveillance, the neutralization of TRAIL protein by an antibody to prevent its binding to death receptors is definitely a risky approach. Here, we demonstrated that the blockade of the TRAIL death receptor DR5 with a specific antibody completely prevented amyloid β peptide (Aβ) neurotoxicity in both neuronal cell line and primary cortical neurons. DR5 was demonstrated to be a key factor in TRAIL death pathway. In fact, whereas TRAIL expression was enhanced dose-dependently by concentrations of β amyloid ranging from 10 nM to 1 μM, only the highest toxic dose of Aβ (25 μM) induced the increased expression of DR5 and neuronal cell death. In addition, the increased expression of DR5 receptor after β amyloid treatment was sustained by p53 transcriptional activity, as demonstrated by the data showing that the p53 inhibitor Pifithrin α prevented both β amyloid-induced DR5 induction and cell death. These data suggest a sequential activation of p53 and DR5 upon β amyloid exposure. Further insight into the key role of DR5 in AD was suggested by data showing a significant increase of DR5 receptor in cortical slices of AD brain. Thus, these findings may give intracellular TRAIL pathway a role in AD pathophysiology, making DR5 receptor a possible candidate as a pharmacological target.

Conformational altered p53 affects neuronal function: relevance for the response to toxic insult and growth-associated protein 43 expression

The role of p53 in neurodegenerative diseases is essentially associated with neuronal death. Recently an alternative point of view is emerging, as altered p53 conformation and impaired protein function have been found in fibroblasts and blood cells derived from Alzheimer’s disease patients. Here, using stable transfected SH-SY5Y cells overexpressing APP751wt (SY5Y-APP) we demonstrated that the expression of an unfolded p53 conformation compromised neuronal functionality. In particular, these cells showed (i) augmented expression of amyloid precursor protein (APP) and its metabolites, including the C-terminal fragments C99 and C83 and β-amyloid peptide (ii) high levels of oxidative markers, such as 4-hydroxy-2-nonenal Michael-adducts and 3-nitro-tyrosine and (iii) altered p53 conformation, mainly due to nitration of its tyrosine residues. The consequences of high-unfolded p53 expression resulted in loss of p53 pro-apoptotic activity, and reduction of growth-associated protein 43 (GAP-43) mRNA and protein levels. The role of unfolded p53 in cell death resistance and lack of GAP-43 transcription was demonstrated by ZnCl2 treatment. Zinc supplementation reverted p53 wild-type tertiary structure, increased cells sensitivity to acute cytotoxic injury and GAP-43 levels in SY5Y-APP clone.

Increased CD44 gene expression in lymphocytes derived from Alzheimer disease patients.

In this study, we demonstrated for the first time an increased CD44 gene expression in lymphocytes derived from Alzheimer’s disease (AD) patients in comparison with healthy subjects. CD44 is a surface antigen expressed by cells of the immune and central nervous system as well as in a variety of other tissues. Functioning as adhesion molecule, CD44 is furthermore involved in driving immune response into infected tissues, including the CNS. We also found that lymphocytes of the same patients expressed significant levels of unfolded p53 isoform, confirming what we already demonstrated in fibroblasts and lymphocytes derived from other cohorts of AD patients. A correlation between p53 and CD44 expression has been well demonstrated in cancer cells, suggesting that CD44 could be a target gene of mutant p53, or either mutant p53 could lack its ability to negatively regulate CD44 expression. The contemporaneous increased expression of unfolded p53 and CD44 in AD lymphocytes may suggest that these two molecules cross-talk together participating in peripheral immune response during the development of the disease.

Dietary zeolite supplementation reduces oxidative damage and plaque generation in the brain of an Alzheimer's disease mouse model

AIM Oxidative stress is considered one of the main events that lead to aging and neurodegeneration. Antioxidant treatments used to counteract oxidative damage have been associated with a wide variety of side effects or at the utmost to be ineffective. The aim of the present study was to investigate the antioxidant property of a natural mineral, the tribomechanically micronized zeolite (MZ). MAIN METHODS Cell death and oxidative stress were assessed in retinoic acid differentiated SH-SY5Y cells, a neuronal-like cell line, after a pro-oxidant stimulus. In vivo evaluation of antioxidant activity and amyloidogenic processing of beta amyloid have been evaluated in a transgenic model of aging related neurodegeneration, the APPswePS1dE9 transgenic mice (tg mice) after a five-month long period of water supplementation with MZ. KEY FINDINGS The study showed that 24h of cell pretreatment with MZ (1) protected the cells by radical oxygen species (ROS)-induced cell death and moreover (2) induced a reduction of the mitochondrial ROS production following a pro-oxidant stimulation. Looking for an antioxidant effect of MZ in vivo, we found (3) an increased activity of the endogenous antioxidant enzyme superoxide dismutase (SOD) in the hippocampus of tg mice and (4) a reduction in amyloid levels and plaque load in MZ treated tg mice compared to control tg mice. SIGNIFICANCE Our results suggest MZ as a novel potential adjuvant in counteracting oxidative stress and plaque accumulation in the field of neurodegenerative diseases.