Cynthia Andreatta

Multiple antioxidants in the prevention and treatment of neurodegenerative disease: analysis of biologic rationale.

Parkinsons disease and Alzheimers disease are major progressive neurologic disorders, the risk of which increases with advancing age (65 years and over). In familial cases, however, early onset of disease (35-65 years) is observed. In spite of extensive basic and chemical research on Parkinsons disease and Alzheimers disease, no preventive or long-term effective treatment strategies are available. The analysis of existing literature suggests that oxidative stress is a major intermediary risk factor for the action of diverse groups of neurotoxins that are involved in these neurodegenerative diseases. In this review, it is proposed that the epigenetic components (mitochondria, other organelles, membranes, protein modification) rather than nuclear genes of neurons are the primary targets for the action of neurotoxins, including free radicals. In addition, a scientific rationale for using multiple antioxidants in clinical trials for the prevention of Parkinsons disease and Alzheimers disease among high-risk populations, and as an adjunct to standard therapy in the treatment of these diseases is presented.

Effects of altered cyclophilin A expression on growth and differentiation of human and mouse neuronal cells.

Abstract1. Cyclophilin A (CyP-A), a soluble cytoplasmic immunophilin, is known for its involvement in T cell differentiation and proliferation. Although CyP-A has a pivotal role in the immune response, it is most highly concentratedin brain, where its functions are largely unknown.2. We reported previously that a murine neuroblastoma (NB-P2) cellline can partially differentiate into neurons when treated with cyclosporin A (CyS-A), implicating a role for CyP-A in neuronal differentiation (Hovland et al. [1999]. Neurochem. Int. 3:229–235).3. The role of CyP-A in regulating neuronal growth and differentiation is not well defined. To investigate this, we first tested the utility of retroviral-mediated gene transfer and expression in human embryonic brain (HEB) and NB-P2 cells. Second, we examined the effects of retroviral-mediated overexpression or antisense-mediated reduction of CyP-A in HEB and NB-P2 cells.4. Our data show that retroviral vectors are efficient for stable gene transfer and expression in both cell lines. Moreover, neither overexpression nor reduction of CyP-A expression in NB-P2 cells altered the growth rate or induced differentiation. More importantly, the up- or down-regulation of CyP-A expressiondid not affect the magnitude of cAMP-induced NB-P2 differentiation. However, overexpression of CyP-A increased the growth rate of HEB cells.5. In summary, the utility of retroviral vectors for stable gene expression in human embryonic brain and murine neuroblastoma cells was shown. Furthermore,a novel role for CyP-A in augmenting the proliferation of human embryonic braincells was demonstrated in vitro.

Identifying genes involved in regulating differentiation of neuroblastoma cells

The genes regulating the induction of differentiation in neurons are not definitively known. Some neuronal tumors retain the ability to differentiate into mature, functional neurons in response to pharmacological agents, despite the presence of genetic anomalies. We hypothesized that some of the genes whose expression is altered between undifferentiated and differentiated states may be those responsible for inducing differentiation. To investigate this, we used a mouse neuroblastoma (NB) cell line, NBP2, in which ≥90% of the cells in the culture terminally differentiate upon elevation of intracellular adenosine 3′,5′‐cyclic monophosphate (cAMP) levels. Gene expression was analyzed using cDNA array blots containing 588 known genes. mRNA from cultures of undifferentiated and differentiated NB cells was used to make cDNA probes for blot hybridization. We identified several genes that are predominantly expressed in either undifferentiated or differentiated NB cells. In addition, numerous genes are moderately up‐ or down‐regulated during differentiation of NB cells. We identified the N‐myc protooncogene, cyclin B1, and protease nexin 1 as genes that are expressed in undifferentiated NB cells and whose levels are significantly down‐regulated upon differentiation. In contrast, the c‐fes and c‐fos protooncogenes and the RAG‐1 gene activator are genes whose expression is significantly up‐regulated during differentiation of NB cells. These findings were confirmed by RT‐PCR analysis. The transcript size and expression level of N‐myc, cyclin B1, protease nexin 1, c‐fes, and c‐fos were verified by Northern blotting. These genes may represent key mediators involved in the regulation of NB cell differentiation. J. Neurosci. Res. 64:302–310, 2001.

Proteasome Activity Is Critical for the cAMP-Induced Differentiation of Neuroblastoma Cells

Abstract1. The ubiquitin–proteasome pathway is involved in a variety of cellular functions in mammalian cells. The role of proteasome, however, in the course of cell differentiation is not well characterized. We hypothesized that proteasome activity might be essential during neuronal cell differentiation.2. To investigate the role of proteasome during neuronal differentiation, we made use of a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons upon elevation of the intracellular level of adenosine 3′,5′-cyclic monophosphate (cAMP). To monitor proteasome activity in NBP2 cells, we integrated an expression cassette~for a short-lived green fluorescent protein (d2EGFP) into these cells, which were designated as NBP2-PN25. When NBP2-PN25 cells were treated with a proteasome inhibitor, lactacystin or MG132, a dose-dependent increase in the constitutive levels of d2EGFP expression was detected.3. We also found that proteasome inhibition by lactacystin during the cAMP-induced differentiation of NBP2-PN25 cells triggered cell death. Both lactacystin and cAMP induction reduced the expression of mRNA for the differentiation-associated genes, such as N-mycand cyclin B1. While cAMP-inducing agents decreased the level of N-myc and cyclin B1 proteins, lactacystin increased the level of these proteins.4. Our data suggest that a reduced level of N-myc and cyclin B1 proteins is critical to commence differentiation, and this can be blocked by a proteasome inhibitor, leading to cell death. Concomitant induction of differentiation and proteasome inhibition, may, therefore, be potentially useful for the treatment of human neuroblastomas.

Overexpression of α-synuclein decreased viability and enhanced sensitivity to prostaglandin E2, hydrogen peroxide, and a nitric oxide donor in differentiated neuroblastoma cells

Increased accumulation of α‐synuclein is associated with certain neurodegenerative diseases including Parkinsons disease (PD) and Alzheimers disease (AD). One mechanism of α‐synuclein‐induced toxicity involves increased oxidative stress. It was unknown whether neurons overexpressing α‐synuclein would exhibit increased sensitivity to hydrogen peroxide (H2O2) or 3‐morpholinosydnonimine (SIN‐1; a nitrous oxide donor). To study this, we developed a murine neuroblastoma (NB) cell line that overexpresses wild‐type human α‐synuclein (NBP2‐PN54) under the control of the cytomegalovirus (CMV) promoter using a retroviral vector. Human α‐synuclein mRNA and protein were readily detectable in NBP2‐PN54 cells. Results showed that differentiated NBP2‐PN54 cells exhibited decreased viability in comparison to differentiated vector (NBP2‐PN1) and parent (NBP2) control cells. These cells also exhibited increased sensitivity to PGE2, H2O2 and SIN‐1. Because of involvement of proteasome inhibition in neurodegeneration, we also investigated whether treatment of differentiated NBP2‐PN54 cells with PGE2, H2O2 or SIN‐1 inhibits proteasome activity. Results showed that H2O2 and SIN‐1 inhibited proteasome activity, but PGE2 did not. These results suggest that overexpression of α‐synuclein not only participates directly in degeneration of neurons, but it also increases the vulnerability of neurons to other potential neurotoxins.

Overexpression of amyloid precursor protein is associated with degeneration, decreased viability, and increased damage caused by neurotoxins (prostaglandins A1 and E2, hydrogen peroxide, and nitric oxide) in differentiated neuroblastoma cells.

Inflammatory reactions are considered one of the important etiologic factors in the pathogenesis of Alzheimers disease (AD). Prostaglandins such as PGE2 and PGA1 and free radicals are some of the agents released during inflammatory reactions, and they are neurotoxic. The mechanisms of their action are not well understood. Increased levels of β‐amyloid fragments (Aβ40 and Aβ42), generated through cleavage of amyloid precursor protein (APP), oxidative stress, and proteasome inhibition, are also associated with neurodegeneration in AD brains. Therefore, we investigated the effect of PGs and oxidative stress on the degeneration and viability of cyclic AMP‐induced differentiated NB cells overexpressing wild‐type APP (NBP2‐PN46) under the control of the CMV promotor in comparison with differentiated vector (NBP2‐PN1) or parent (NBP2) control cells. Results showed that differentiated NBP2‐PN46 cells exhibited enhanced spontaneous degeneration and decreased viability in comparison with differentiated control cells, without changing the level of Aβ40 and Aβ42. PGA1 or PGE2 treatment of differentiated cells caused increased degeneration and reduced viability in all three cell lines. These effects of PGs are not due to alterations in the levels of vector‐derived APP mRNA or human APP holoprotein, secreted levels of Aβ40 and Aβ42, or proteasome activity. H2O2 or SIN‐1 (an NO donor) treatment did not change vector‐derived APP mRNA levels, but H2O2 reduced the level of human APP protein more than SIN‐1. Furthermore, SIN‐1 increased the secreted level of Aβ40, but not of Aβ42, whereas H2O2 had no effect on the level of secreted Aβ fragments. Both H2O2 and SIN‐1 inhibited proteasome activity in the intact cells. The failure of neurotoxins to alter APP mRNA levels could be due to the fact that they do not affect CMV promoter activity. These results suggest that the mechanisms of action of PGs on neurodegeneration are different from those of H2O2 and SIN‐1 and that the mechanisms of neurotoxicity of H2O2 and SIN‐1 are, at least in part, different from each other.

Regulated expression of VP16CREB in neuroblastoma cells: analysis of differentiation and apoptosis.

Highly malignant neuroblastoma tumors generally have defects in differentiation and apoptotic pathways. For a better understanding of these events, we use a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons in response to elevated levels of cAMP. Because one of the main downstream effectors of the cAMP signaling pathway is cAMP‐response element binding (CREB), we reasoned that it might affect the expression of genes associated with differentiation and apoptotic events in NBP2 cells. To investigate this, we established tetracycline‐regulated expression (TetOff) of VP16CREB, which constitutively transactivates promoters containing the CRE sequence motif. Using this system, we found that inducible expression of VP16CREB in NBP2 cells results in 1) morphological differentiation that is characterized by the formation of neurites and growth cones, 2) reversible cell differentiation unlike cAMP‐induced terminal differentiation, 3) cell cycle arrest at G1, 4) no apoptosis in the presence of partial inhibition of proteasome unlike an increase in cAMP levels, and 5) changes in the expression of many genes, including down‐regulation of N‐myc, cyclin B1, Dickkopf‐1, and Mad‐2 and up‐regulation of tyrosine hydroxylase, c‐fos, N10, and ICER genes. Although VP16CREB expression and activation of the cAMP pathway impart many similar effects in NBP2 cells, they also bear some distinct genetic and morphological differences. Our data suggest that increased levels of cAMP function through not only CREB but also other signaling pathways that account for the additional cAMP‐induced effects, including irreversible differentiation and onset of apoptosis during partial inhibition of proteasome in NBP2 cells.

Altered expression of genes regulating cell growth, proliferation, and apoptosis during adenosine 3',5'-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture.

SummaryAn elevation of the intracellular levels of adenosine 3′,5′-cyclic monophosphate (cAMP) induces terminal differentiation in neuroblastoma (NB) cells in culture; however, genetic alterations during differentiation have not been fully identified. To investigate this, we used Mouse Genome U74A microarray containing ∼6000 functionally characterized genes to measure changes in gene expression in murine NB cells 30 min and 4,24, and 72 h after treatment with cAMP-stimulating agents. Based on the time of increase in differentiated functions and their status (reversible versus irreversible) after treatment with cAMP-stimulating agents, the induction of differentiation in NB cells was divided into three distinct phases: initiation (about 4 h after treatment when no increase in differentiated functions is detectable), promotion (about 24 h after treatment when an increase in differentiated functions occurs, but they are reversible upon the removal of cAMP), and maintenance (about 72 h after treatment when differentiated functions are maximally expressed, but they are irreversible upon the removal of cAMP). Results showed that alterations in expression of genes regulating cell growth, proliferation, apoptosis, and necrosis occurred during cAMP-induced differentiation of NB cells. Genes that were upregulated during the initiation, promotion, or maintenance phase were called initiators, promoters, or maintainers of differentiation. Genes that were downregulated during the initiation, promotion, or maintenance phase were called suppresors of initiation, promotion, or maintenance phase. Genes regulating growth may act as initiators, promoters, maintainers, or suppressors of these phases. Genes regulating cell proliferation may primarily act as suppressors of promotion. Genes regulating cell cycle may behave as suppressors of initiation or promotion, whereas those regulating apoptosis and necrosis may act as initiators or suppressors of initiation or promotion. The fact that genetic signals for differentiation occurred 30 min after treatment with cAMP, whereas cell-cycle genes were downregulated at a later time, suggests that decision for NB cells to differentiate is made earlier and not at the cell-cycle stage, as commonly believed.

Selenomethionine Prevents Degeneration Induced by Overexpression of Wild-Type Human α-Synuclein during Differentiation of Neuroblastoma Cells

Objective: High levels of wild-type α-synuclein are found in autopsied brain samples of idiopathic Parkinson’s disease (PD), some familial PD, some Alzheimer’s disease (AD) and Down’s syndrome with dementia. Therefore, we have investigated whether overexpression of wild-type α-synuclein causes degeneration during adenosine, 3′,5′-cyclic monophosphate (cAMP)-induced differentiation of murine neuroblastoma (NB) cells in culture. We have also studied whether selenomethionine can modify the effect of overexpression of α-synuclein during differentiation of NB cells. Methods: To study these issues, we established a murine neuroblastoma (NB) clone (NBP2-PN54-C20) that expressed high levels of wild-type human α-synuclein as determined by real time PCR and Western blot. We have utilized RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, and prostaglandin A1 (PGA1), a stimulator of adenylate cyclase, or RO20-1724 and dibutyryl cAMP to induce terminal differentiation in over 95% of the cell population by elevating the intracellular levels of cAMP in NB cells. The viability of cells was determined by MTT assay and LDH leakage assay, and the degeneration was documented by photomicrographs. Results: The results showed that overexpression of human wild-type α-synuclein decreased viability and increased degenerative changes in comparison to those observed in vector control cells, when differentiation was induced by treatment with RO20-1724 and PGA1, but not with RO20-1724 and dibutyryl cAMP. When selenomethionine was added to NB cells overexpressing α-synuclein immediately after the addition of RO20-1724 and PGA1, the viability and degenerative changes were markedly reduced, suggesting the involvement of increased oxidative stress in the mechanism of action of α-synuclein. This protective effect was not observed after treatment with sodium selenite or methionine. Conclusions: Data suggested that Overexpression of wild-type human α-synuclein-decreased viability and increased the levels of degenerative changes during differentiation of NB cells were reduced by selenomethionine treatment. This suggest that one of mechanisms of action α-synuclein may involve increased oxidative stress.

Role of the adenosine 3',5'-cyclic monophosphate (cAMP) in enhancing the efficacy of siRNA-mediated gene silencing in neuroblastoma cells.

Gene-silencing activity mediated by siRNA has been demonstrated in mammalian cells; however, the mechanism of its regulation is not well understood. Since downregulation of a number of genes occurs during adenosine 3′,5′-cyclic monophosphate (cAMP)-induced differentiation of neuroblastoma (NB) cells, it is possible that cAMP may play a role in regulating siRNA activity during differentiation. To study this, we utilized an NB cell line (NBP2-PN25) that expresses a short-lived green fluorescent protein (d2EGFP) under the CMV promoter. These cells were transfected with a retroviral plasmid that expresses U6 promoter-driven expression of siRNA targeted to d2EGFP and then were treated with cAMP-elevating agents (200 μg/ml RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, and 1 μg/ml prostaglandin A1, a stimulator of adenylate cyclase) for 2 or 24 h. The siRNA activity was measured by determining the level of intensity of d2EGFP protein by flow cytometry, and the level of d2EGFP mRNA by real-time PCR. The results showed that cAMP-elevating agents enhanced U6-driven siRNA activity directed towards d2EGFP in NB cells 24 h after treatment. One of the mechanisms of action of cAMP is mediated via phosphatidylinositol 3-kinase (PI3K) inhibition; therefore, we have investigated the effect of a PI3K inhibitor on siRNA activity. This study showed that inhibition of PI3K also enhanced U6-driven siRNA activity towards d2EGFP. cAMP-stimulating agents increased U6 transcript levels, perhaps suggesting that increased siRNA activity may in part be due to an increase in transcriptional activity. When NB cells were transfected with a synthetic siRNA directed to d2EGFP, both cAMP elevation and PI3K inhibition similarly enhanced siRNA activity. Sodium butyrate, which inhibits the growth of NB cells similar to the effect produced by cAMP, did not affect U6-driven siRNA activity towards d2EGFP. Protein kinase C (PKC) activation or inhibition also failed to affect siRNA activity in NB cells. This study also showed that cAMP elevation and PI3K inhibition increases U6-driven siRNA activity directed towards an endogenous gene, p53. Our data suggest a role for the cAMP pathway in affecting the efficacy of siRNA system during differentiation of NB cells.