María Recuero

Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene

In this work, we explored the existence of genetic variants within the apolipoprotein E gene transcriptional regulatory region, using a denaturing gradient gel electrophoresis screening of a region comprising nucleotides −1017 to +406. Upon a population study, three new polymorphic sites (−491, −427 and −219) and two mutations were found. Functional effects of the polymorphisms, assayed by transient transfection and electrophoretic mobility shift assays in a human hepatoma cell line, showed that polymorphisms at sites −491 and −219 of the APOE promoter produce variations in the transcriptional activity of the gene, most probably through differential binding of nuclear proteins.

Herpes simplex virus type I induces the accumulation of intracellular β-amyloid in autophagic compartments and the inhibition of the non-amyloidogenic pathway in human neuroblastoma cells

Mounting evidence suggests that herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of Alzheimers disease (AD). Epidemiological analyses have shown that HSV-1 is a risk factor for AD in people with at least 1 type 4 allele of the apolipoprotein E gene. Recent studies have also suggested that HSV-1 contributes to the appearance of the biochemical anomalies characteristic of AD brains. In addition, autophagic activity appears to be reduced with aging, and the final stages of autophagy in neurodegenerative process appear to be impaired. The present work reports that HSV-1 provokes the strong intracellular accumulation of both the main species of β-amyloid (Aβ) in the autophagic compartments and that it is associated with a marked inhibition of Aβ secretion. Autophagosomes containing Aβ failed to fuse with lysosomes in HSV-1-infected cells, indicating the impaired degradation of Aβ localized in the autophagic vesicles. In addition, HSV-1 infection was associated with the inhibition of the nonamyloidogenic pathway of amyloid precursor protein (APP) processing without significantly affecting the activity of the secretases involved in the amyloidogenic pathway. Taken together, these data suggest that HSV-1 infection modulates autophagy and amyloid precursor protein processing, contributing to the accumulation of Aβ characteristic of AD.

Aβ production as consequence of cellular death of a human neuroblastoma overexpressing APP

In human brain the Aβ peptide is produced mainly by neurons and the overexpression of amyloid precursor protein (APP) that involves an increase in Aβ secretion, has been observed in some areas of the Alzheimers disease patients brain. We have generated two stably transfected human neuroblastoma lines which overexpress APP; both of them secreted Aβ and showed morphological changes and cell death with apoptotic program characteristics. Interestingly, coculture experiments with the untransfected human neuroblastoma cell line showed that the Aβ peptide was not responsible for the death in those cell lines; additionally, we indicate that upon cell death, Aβ peptide is secreted into cell medium.

A free radical-generating system induces the cholesterol biosynthesis pathway: a role in Alzheimer's disease

Oxidative stress, which plays a critical role in the pathogenesis of neurodegenerative diseases such as Alzheimers disease (AD), is intimately linked to aging – the best established risk factor for AD. Studies in neuronal cells subjected to oxidative stress, mimicking the situation in AD brains, are therefore of great interest. This paper reports that, in human neuronal cells, oxidative stress induced by the free radical‐generating xanthine/xanthine oxidase (X‐XOD) system leads to apoptotic cell death. Microarray analyses showed a potent activation of the cholesterol biosynthesis pathway following reductions in the cell cholesterol synthesis caused by the X‐XOD treatment; furthermore, the apoptosis was reduced by inhibiting 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGCR) expression with an interfering RNA. The potential importance of this mechanism in AD was investigated by genetic association, and it was found that HMGCR, a key gene in cholesterol metabolism and among those most strongly upregulated, was associated with AD risk. In summary, this work presents a human cell model prepared to mimic the effect of oxidative stress in neurons that might be useful in clarifying the mechanism involved in free radical‐induced neurodegeneration. Gene expression analysis followed by genetic association studies indicates a possible link among oxidative stress, cholesterol metabolism and AD.

Herpes Simplex Virus Type I Induces an Incomplete Autophagic Response in Human Neuroblastoma Cells

Autophagy is a homeostatic process involved in the turnover or elimination of cytoplasmic components, damaged organelles, and protein aggregates via a lysosomal degradation mechanism. Autophagy also provides a mechanism of innate immunity, known as xenophagy, designed to protect cells from intracellular pathogens, but it may unfortunately be subverted to act as a pro-viral pathway facilitating the replication of certain viruses. Herpes simplex virus type I (HSV-1) is a neurotropic virus that remains latent in host neurons; it is the most common cause of sporadic viral encephalitis. Moreover, HSV-1 has been related to the pathogenesis of Alzheimers disease. HSV-1 can modulate the autophagic process through a mechanism mediated by the viral protein ICP34.5. Here we report that HSV-1 induces a strong increase in GFP-LC3 and endogenous LC3 lipidation, and triggers the accumulation of intracellular autophagic compartments (mainly autophagosomes) without enhancing autophagic long-lived protein degradation in the late stages of infection. Autophagy inhibition mediated by ATG5 gene silencing had no effect on viral growth. The present results suggest that HSV-1 infection activates the host autophagic machinery and strongly controls the autophagic process, blocking the fusion of autophagosomes with lysosomes. These events might be important in the neurodegenerative process associated with HSV-1 infection.

Alzheimer's amyloid precursor protein is expressed on the surface of hematopoietic cells upon activation

A4-amyloid is the major component of senile plaques and neurofibrillary tangles found in the brain of patients suffering Alzheimers disease. This 39-42 amino acid peptide is derived from a larger precursor protein (APP). Since APP gene encodes for a putative membrane protein, the study of APP expression at the cell surface may provide useful data for understanding its physiological function. In this report, we present data on APP expression, that was detected by APP specific mAbs in cells of the hematopoietic system. APP was weakly expressed on the cell surface of resting human lymphocytes and monocytes, but it could be induced to the surface of those cells upon stimulation. The cell activators capable of inducing APP membrane expression comprehended mitogenic lectins, calcium ionophores, phosphatase inhibitors, and anti mu-chain or anti-CD3 antibodies in B and T cells, respectively. Interestingly, phorbol esters were able to induce APP membrane expression in monocytic, but not in lymphoid cells. In contrast to lymphocytes and monocytes, granulocytes never expressed cell surface or cytoplasmic APP, even after the activation. The induction of membrane APP in response to lymphocyte activation signals, including antibodies to the antigen receptor of B and T cells, raises the possibility that APP might play the role of a cell surface receptor in the immune system.

PLA2G3, a Gene Involved in Oxidative Stress Induced Death, is Associated with Alzheimer's Disease

Oxidative stress, which plays a critical role in the pathogenesis of neurodegenerative diseases such as Alzheimers disease (AD), is intimately linked to aging, the best established risk factor for AD. Studies in neuronal cells subjected to oxidative stress, mimicking such stress in AD brains, are therefore of great interest. PLA2G3 is the most overexpressed gene in a human neuronal model of oxidative stress induced by the free radical-generating xanthine/xanthine oxidase (X-XOD) system, which provokes apoptotic cell death. In this work, we describe that PLA2G3 gene silencing produced a marked inhibition of X-XOD induced cell death, and that PLA2G3 polymorphisms are associated with AD in a Spanish case-control sample. The capacity to respond to oxidative stress may therefore modulate the risk of AD, and PLA2G3 is a potential target to regulate neuronal damage induced by free radicals.

Oxidative Stress Enhances Neurodegeneration Markers Induced by Herpes Simplex Virus Type 1 Infection in Human Neuroblastoma Cells

Mounting evidence suggests that Herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of Alzheimer’s disease (AD). Previous work from our laboratory has shown HSV-1 infection to induce the most important pathological hallmarks of AD brains. Oxidative damage is one of the earliest events of AD and is thought to play a crucial role in the onset and development of the disease. Indeed, many studies show the biomarkers of oxidative stress to be elevated in AD brains. In the present work the combined effects of HSV-1 infection and oxidative stress on Aβ levels and autophagy (neurodegeneration markers characteristic of AD) were investigated. Oxidative stress significantly potentiated the accumulation of intracellular Aβ mediated by HSV-1 infection, and further inhibited its secretion to the extracellular medium. It also triggered the accumulation of autophagic compartments without increasing the degradation of long-lived proteins, and enhanced the inhibition of the autophagic flux induced by HSV-1. These effects of oxidative stress were not due to enhanced virus replication. Together, these results suggest that HSV-1 infection and oxidative damage interact to promote the neurodegeneration events seen in AD.

A free radical-generating system regulates APP metabolism/processing.

Oxidative stress, a risk factor in the pathophysiology of Alzheimers disease, is intimately associated with aging. We previously reported that the X‐XOD free radical generating system acts as a modulator of lipid metabolism and a mild inducer of apoptotic death. Using the same cell model, the present study examines the metabolism/processing of the amyloid precursor protein (APP). Prior to inducing cell death, X‐XOD promoted the secretion of α‐secretase‐cleaved soluble APP (sAPPα) and increased the level of APP carboxy‐terminal fragments produced by α and γ secretase (αCTF and γCTF/AICD). In contrast, it reduced the activity of β‐secretase and the level of secreted Aβ. The present results indicate that mild oxidative stress maintained throughout culturing regulates APP metabolism/processing in SK‐N‐MC human neuroblastoma cells.

Herpes simplex virus type 2 infection induces AD-like neurodegeneration markers in human neuroblastoma cells

Herpes simplex virus (HSV) types 1 and 2 are neurotropic viruses that establish lifelong latent infections in neurons. Mounting evidence suggests that HSV-1 infection is involved in the pathogenesis of Alzheimers disease (AD). The relationships between other herpesvirus infections and events associated with neurodegeneration have not, however, been extensively studied. The present work reports that HSV-2 infection leads to the strong accumulation of hyperphosphorylated tau and the amyloid-β peptides Aβ40 and Aβ42 (all major pathological hallmarks of AD) in human SK-N-MC neuroblastoma cells. Infection is also associated with a marked reduction in the amount of Aβ40 secreted and in the proteolytic fragments of the amyloid-β precursor protein (APP) (secreted APPα and the α-C-terminal fragment). These results indicate that HSV-2 infection inhibits the nonamyloidogenic pathway of APP processing and impairs Aβ secretion in these cells. In addition, HSV-2 induces the accumulation of intracellular autophagic compartments containing Aβ due to a failure in the late stages of autophagy. To our knowledge, this is the first report to show that HSV-2 infection strongly alters the tau phosphorylation state, APP processing, and autophagic process in human neuroblastoma cells, leading to the appearance of AD-like neurodegeneration markers.