Isabel Sastre

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.

Effect of Apolipoprotein E on the Cerebral Load of Latent Herpes Simplex Virus Type 1 DNA

ABSTRACT Herpes simplex virus type 1 (HSV-1) is neurotropic and enters a latent state lasting the lifetime of the host. This pathogen has recently been proposed as a risk factor for Alzheimers disease (AD) in conjunction with apolipoprotein E4 (ApoE4). In a murine acute infection model, we showed that viral neuroinvasiveness depends directly on the overall ApoE dosage and especially on the presence of isoform ApoE4. If an interaction between ApoE and HSV-1 is involved in AD, it may occur during latency rather than during acute infection. Certainly, ApoE plays an important role in late-onset AD, i.e., at a time in life when the majority of people harbor HSV-1 in their nervous system. In the present work, wild-type, APOE knockout, APOE3, and APOE4 transgenic mice were used to analyze the influence of the ApoE profile on the levels of latent virus DNA. The knockout mice had significantly lower concentrations of the virus in the nervous system than the wild-type mice, while the APOE4 mice had very high levels in the brain compared to the APOE3 animals. ApoE4 seems to facilitate HSV-1 latency in the brain much more so than ApoE3. The APOE dosage correlated directly with the HSV-1 DNA concentration in the brain, strengthening the hypothesis that HSV-1, together with ApoE, might be involved in AD.

Assessing the role of the TREM2 p.R47H variant as a risk factor for Alzheimer's disease and frontotemporal dementia

A non-synonymous genetic rare variant, rs75932628-T (p.R47H), in the TREM2 gene has recently been reported to be a strong genetic risk factor for Alzheimers disease (AD). Also, rare recessive mutations have been associated with frontotemporal dementia (FTD). We aimed to investigate the role of p.R47H variant in AD and FTD through a multi-center study comprising 3172 AD and 682 FTD patients and 2169 healthy controls from Spain. We found that 0.6% of AD patients carried this variant compared to 0.1% of controls (odds ratio [OR] = 4.12, 95% confidence interval [CI] = 1.21-14.00, p = 0.014). A meta-analysis comprising 32,598 subjects from 4 previous studies demonstrated the large effect of the p.R47H variant in AD risk (OR = 4.11, 95% CI = 2.99-5.68, p = 5.27×10(-18)). We did not find an association between p.R47H and age of onset of AD or family history of dementia. Finally, none of the FTD patients harbored this genetic variant. These data strongly support the important role of p.R47H in AD risk, and suggest that this rare genetic variant is not related to FTD.

Alzheimer's risk associated with human apolipoprotein E, alpha-2 macroglobulin and lipoprotein receptor related protein polymorphisms: absence of genetic interactions, and modulation by gender

Apolipoprotein E (apoE), the lipoprotein receptor related protein (LRP) and alpha-2 macroglobulin (alpha2M) have been proposed as a functional complex involved in amyloid clearance, a crucial event for Alzheimers disease development. In this work, we present an epidemiological approach aimed to study the interactions among these genes, age and gender. This approach did not reveal significant associations between the genes; however, the present study indicated that the risk associated with APOE promoter and LRP gene polymorphisms is modulated by gender.

Zebrafish as a new model for herpes simplex virus type 1 infection.

The zebrafish (Danio rerio) is rapidly gaining ground as a disease model. However, until now, the use of this species with human pathogens has been restricted to just three bacteria; no studies involving viruses that infect humans are recorded. In this study, the zebrafish was used as a model of herpes simplex virus type 1 (HSV-1) infection of the nervous system. Fish infected using viral culture supernatants showed detectable HSV-1 DNA concentrations 1-4 days after inoculation, indicating that this virus can experimentally infect and persist in this host. The kinetics of infection was dose dependent, especially in the head. Histological immunodetection of HSV-1 glycoproteins confirmed the presence of HSV-1 in the organs studied; infection led to histopathological changes. Moreover, the suppression of the immune system by cyclophosphamide and the antiviral effect of acyclovir were demonstrated. The infection of the encephalon was studied in detail, and the time course of viral colonization recorded. Immunofluorescence studies provided immunoreactive evidence of viral antigens in the encephalon and spinal cord. Viruses cleared from infected brains showed the ability to infect human neuroblastoma cells. This study is the first to demonstrate HSV-1 infection in the zebrafish and manifests the potential use of this species in herpesvirus studies.

Herpes Simplex Virus Type 1 Infection via the Bloodstream with Apolipoprotein E Dependence in the Gonads Is Influenced by Gender

ABSTRACT Herpes simplex virus type 1 (HSV-1) causes disease in humans and animals. Infection usually occurs via the neural route and possibly occurs via the hematogenous route. The latter, however, is the main route by which immunosuppressed individuals and neonates are infected. Gender-dependent differences in the incidence and severity of some viral infections have been reported. To detect differences between the sexes with respect to HSV-1 colonization and disease, the characteristics of both acute and latent infections in hematogenously infected male and female mice were compared. In acute infection, the female mice had a poorer outcome: HSV-1 colonization was more effective, especially in the gonads and brain. In the encephalon, the midbrain had the highest viral load. In latent infection, brain viral loads were not significantly different with respect to sex. Significant differences were seen, however, in the blood and trigeminal ganglia: HSV-1 seroprevalence was observed in females, with no virus detected in males. In brain dissections, only the cerebral cortex of the females had viral loads statistically higher than those observed in the males. The spread of the virus to several organs of interest during acute infection was examined immunohistochemically. Female mice showed greater viral immunostaining, especially in the adrenal cortex, gonads, and midbrain. In male mice, HSV-1 was detected predominantly in the adrenal cortex. It was also found that apolipoprotein E promotes virus colonization of the ovaries, the APOE gene dose being directly related to viral invasiveness.

Polymorphism in genes involved in adrenergic signaling associated with Alzheimer's.

To investigate the potential involvement of adrenergic signaling in Alzheimers disease (AD) pathogenesis, we performed genetic and functional studies of genes initiating the cascade. We chose two functional single-nucleotide polymorphisms (SNPs) in the beta1-adrenergic receptor (ADRB1) and the G protein beta3 subunit (GNB3) genes, respectively, and analyzed their allelic frequencies in a case-control sample of AD. We found that the GNB3 T allele produces a significant risk for AD in individuals homozygous for the ADRB1 C allele, suggesting that the combined effect of both polymorphisms influences AD susceptibility. Interestingly, the co-expression of GNB3 T and ADRB1 C alleles, compared with GNB3 C and ADRB1 G, produced increased cAMP levels and MAPK activation following adrenergic stimulation of transfected human cell lines. Furthermore, the co-expression of these alleles also produced increases in APP expression. These data strongly indicate that the combination of GNB3 and ADRB1 polymorphisms produces AD susceptibility by changing the cell responsiveness to adrenergic stimulation, pointing to the modulation of brain adrenergic receptors as a potential target for novel AD therapeutic strategies.

Double stranded RNA activated EIF2 α kinase (EIF2AK2; PKR) is associated with Alzheimer's disease

Sporadic Alzheimers disease (AD) appears to be the consequence of the interaction between combinations of genes and environmental factors (for example virus infections). To test this hypothesis, we are examining human genes relevant to herpes simplex virus type 1 (HSV-1) infection via genetic association studies in AD case–control samples. Recently, we found that a variant in TAP2, a major target used by HSV-1 to evade immune surveillance, is associated with AD. The present work analyses another gene involved in the host cell response to HSV-1, EIF2AK2 (eukaryotic translation initiation factor 2-alpha kinase 2; coding for PKR); PKR mediates the virus-induced shut-off of translation, and levels of activated PKR are high in the brains of AD patients. An EIF2AK2 SNP (rs2254958) located in the 5′-UTR region within an exonic splicing enhancer was found to be associated with AD. More specifically: the C allele was more commonly found in the patients and, compared to non-CC genotypes, the CC homozygotes showed earlier (around 3.3 years) onset of AD, especially in the absence of the APOE4 allele. These results further support the hypothesis that variants of human genes participating in HSV-1 infection modulate the susceptibility and/or clinical manifestations of AD.

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.

A TAP2 genotype associated with Alzheimer's disease in APOE4 carriers

Sporadic Alzheimers disease (AD) appears to be the consequence of the interaction between combinations of genes and environmental factors. Binding with the transporter associated with antigen processing (TAP) is thought to be the main way in which herpes simplex virus type 1 (HSV-1) evades immune surveillance. Several TAP gene polymorphisms were examined and a TAP2 SNP (rs241448) associated with AD found in two independent case-control samples, especially in carriers of the APOE4 allele. These findings are consistent with the hypothesis that human genetic variants facilitating the access of HSV-1 to the brain might result in susceptibility to AD.