Herpes simplex virus type 1 induces nuclear accumulation of hyperphosphorylated tau in neuronal cells.

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that remains latent in host neurons. Viral DNA replication is a highly structured process in which the redistribution of nuclear proteins plays an important role. Although tau is most widely known as a microtubule-associated protein found in a hyperphosphorylated state in the brains of patients with Alzheimer's disease (AD), this protein has also been detected at other sites such as the nucleolus. Here, we establish that HSV-1 infection gives rise to an increase in tau phosphorylation and that hyperphosphorylated tau accumulates in the nucleus, forming defined structures in HSV-1-infected neuronal cells reminiscent of the common sites of viral DNA replication. When tau expression in human neuroblastoma cells was specifically inhibited using an adenoviral vector expressing a short hairpin RNA to tau, viral DNA replication was not affected, indicating that tau is not required for HSV-1 growth in neuronal cells. Given that HSV-1 is considered a risk factor for AD, our results suggest a new way in which to understand the relationships between HSV-1 infection and the pathogenic mechanisms leading to AD.

Caspase-1 genetic variation is not associated with Alzheimer's disease risk.

BACKGROUND: Interleukin (IL)-1beta is a potent proinflammatory cytokine markedly overexpressed in the brains of patients with Alzheimer's disease (AD), and also involved in development of atherosclerosis and coronary artery disease. Caspase-1 (CASP1), formerly called IL-1beta converting enzyme (ICE), mediates the cleavage of the inactive precursor of IL-1beta into the biologically active form. CASP1 genetic variation (G+7/in6A, rs501192) has been associated with susceptibility to myocardial infarction and cardiovascular death risk. We examined the contribution of this gene to the susceptibility for AD. METHODS: We examined genetic variations of CASP1 by genotyping haplotype tagging SNPs (htSNPs) (rs501192, rs556205 and rs530537) in a group of 628 Spanish AD cases and 722 controls. RESULTS: There were no differences in the genotypic, allelic or haplotypic distributions between cases and controls in the overall analysis or after stratification by age, gender or APOE epsilon4 allele. CONCLUSION: Our negative findings in the Spanish population argue against the hypothesis that CASP1 genetic variations are causally related to AD risk.

A megalin polymorphism associated with promoter activity and Alzheimer's disease risk.

Elevated cerebral levels of amyloid beta-protein (Abeta) occur in Alzheimer's disease (AD), yet only a few patients show evidence of increased Abeta production. This observation suggests that many, perhaps most, cases of AD are caused by faulty clearance of Abeta. Megalin, which plays an important role in mediating Abeta clearance, is an attractive candidate gene for genetic association with AD. To investigate this hypothesis, we analyzed the megalin gene in a population of 2,183 subjects. Genetic analysis indicated that the rs3755166 (G/A) polymorphism located in the megalin promoter associated with risk for AD, dependently of apolipoprotein E genotype. The rs3755166 AA genotype frequency was significantly greater in AD patients than in control subjects. Furthermore, the luciferase reporter assay indicated that the rs3755166 A variant has 20% less transcriptional activity than the rs3755166 G variant. This study provides strong evidence that this megalin polymorphism confers a greater risk for AD, and supports a biological role for megalin in the neurodegenerative processes involved in AD.

DYRK1A genetic variants are not linked to Alzheimer's disease in a Spanish case-control cohort.

BACKGROUND: As dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) has been implicated in the abnormal hyperphosphorylation of tau in Alzheimer's disease (AD) brain, and the development of neurofibrillary tangles, we examined the contribution of this gene to the susceptibility for AD. METHODS: We examined genetic variations of DYRK1A by genotyping haplotype tagging SNPs (htSNPs) (rs11701483, rs2835740, rs1137600, rs2835761, rs2835762, rs2154545 and rs8132976) in a group of 634 Spanish AD cases and 733 controls. RESULTS: There were no differences in the genotypic, allelic or haplotypic distributions between cases and controls in the overall analysis or after stratification by APOE epsilon4 allele. CONCLUSION: Our negative findings in the Spanish population argue against the hypothesis that DYRK1A genetic variations are causally related to AD risk. Still, additional studies using different sets of patients and control subjects deserve further attention, since supporting evidence for association between DYRK1A gene and AD risk in the Japanese population exists.

Presenilin 1 polymorphism associated with Alzheimer's disease in apolipoprotein E4 carriers.

Mutations of presenilin 1 (PSEN1) are associated with monogenic Alzheimer's disease (AD); polymorphisms at this gene may therefore be associated with the sporadic form of the disease. In fact, recent meta-analyses and whole-genome association studies indicate PSEN1 as one of the few genes significantly associated with AD risk. Several polymorphisms have been analyzed in PSEN1. The present work examined the possible modulation of the risk of AD by a PSEN1 polymorphism (dbSNP rs3025786) located in intron 7, which we found during a denaturing gradient gel electrophoresis mutation screening of the gene, and which was previously reported as 'suspected' in the public databases. The study of a Spanish case-control sample of 1,183 individuals showed this polymorphism to be associated with AD in an apolipoprotein E (APOE)-specific manner: more specifically, to carry the PSEN1 C allele was associated with a decreased AD risk among carriers of the APOE4 allele. Thus, the present results reinforce the possible involvement of PSEN1 in sporadic AD.

Non-invasive bioluminescence imaging for monitoring herpes simplex virus type 1 hematogenous infection.

Traditional studies on viral neuroinvasiveness and pathogenesis have generally relied on murine models that require the sacrifice of infected animals to determine viral distributions and titers. The present paper reports the use of in vivo bioluminescence imaging to monitor the replication and tropism of KOS strain HSV-1 viruses expressing the firefly luciferase reporter protein in hematogenously infected mice. Following intraperitoneal injection, a comparison was made between real-time PCR determinations of HSV-1 DNA concentrations (requiring the sacrifice of the experimental animals) and in vivo bioluminescence emissions in living animals. For further comparison, in vitro light emission was also measured in the ovaries and adrenal glands of sacrificed mice. After infection, HSV-1 spread preferentially to the ovaries and adrenal glands (these organs showed the highest virus levels). Both the PCR and bioluminescence methods detected low viral loads in the nervous system, where the virus was restricted to the spinal cord. The concentrations of viral DNA measured correlated with the magnitude of bioluminescence in vivo, and with the photon flux determined by the in vitro luciferase enzyme assay. The results show that bioluminescence imaging can be used for non-invasive, real-time monitoring of HSV-1 hematogenous infection in living mice, but that coupling this methodology with conventional techniques aids in the characterization of the infection.

Association of DSC1, a gene modulated by adrenergic stimulation, with Alzheimer's disease.

Alzheimer's disease (AD) is a complex multifactorial disorder involving a number of genetic and environmental factors, with severe head injury consistently reported as a major non-genetic risk factor. The adrenergic activation that occurs during major trauma increases cAMP levels, therefore the cAMP signaling pathway might be involved in AD pathogenesis. Time course of candidate gene expression following adrenergic stimulation with isoproterenol was assayed in neuroblastoma cells by quantitative reverse transcription (RT)-PCR. Then, genetic association studies of polymorphisms in several of these candidate genes were performed. Association studies in two independent case-control samples showed a polymorphism in DSC1, encoding desmocollin 1--a member of the desmosomal cadherins--which modulated AD susceptibility in a gender-specific manner. These results are in accordance with the potential involvement of the adrenergic signaling pathway in AD pathogenesis.

Transcriptional activation by AP-2alpha is modulated by the oncogene DEK.

Cell differentiation and development are highly regulated processes at the transcriptional level. One of the main transcription factors that regulate these processes is AP-2alpha, a cell-type specific protein required for vertebrate development and embryogenesis. AP-2alpha also regulates apoptosis and cell-cycle specific events by interacting with the oncogene c-Myc. In searching for novel AP-2alpha- interacting factors, using an affinity chromatography approach, we have observed that oncoprotein DEK interacts with AP-2alpha in vitro. The existence of an interaction between AP-2alpha and DEK in cellular cultures was demonstrated by expression of a tagged AP-2alpha form followed by immunodetection. By transient co-expression experiments using a reporter for APOE promoter activity we have found that DEK stimulates the transactivation activity of AP-2alpha over APOE promoter. Finally, electrophoretic mobility shift assays suggested that DEK enhances the DNA-binding activity of AP-2alpha. Our data suggest a novel cellular function of DEK as a transcriptional co-activator.

Specific interaction of heterogeneous nuclear ribonucleoprotein A1 with the -219T allelic form modulates APOE promoter activity.

The polymorphic -219T/G variant in the APOE promoter has been associated with variations in basal transcriptional activity as well as with the risk of developing Alzheimer's disease, myocardial infarction and early-onset coronary heart disease. The molecular mechanisms underlying these effects are presently unknown. In this report, we show that nuclear extracts from Jurkat cells form a T-specific complex with a motif including the -219 site within the APOE promoter. By DNA-affinity chromatography and mass spectrometry, the human heterogeneous nuclear ribonucleoprotein hnRNPA1(A1) was identified as one component of the complex. In vitro binding analysis indicated that a fragment of A1 had a marked binding specificity for the T form. Interaction of A1 with this region is driven by an adjacent telomeric-like sequence; however, the presence of G, but not T, at -219 position inhibited this interaction. The differences in transcriptional activity between the -219T and -219G promoter allelic forms correlated with the expression levels of A1 in several cell lines; also, over-expression of A1 increased the activity of the T form relative to that of the G form. These results indicate that A1 transactivates APOE promoter activity by direct and specific interaction with the -219T site.

Neuronal specific regulatory elements in apolipoprotein E gene proximal promoter.

Systemic and local changes in apolipoprotein E (ApoE) quantity have been related with Alzheimer's and other neurodegenerative diseases, showing the relevance of maintaining physiological ApoE levels. However, APOE transcription has not been extensively studied in neural cells. In this report, we study the transcriptional activity of different APOE proximal promoter regions and their binding to nuclear proteins from human neural (astrocytoma and neuroblastoma) and peripheral (hepatoma and lymphoma) cell lines. We define several regions with a negative regulatory effect in all the cells and a region with a strong positive activity in neuroblastoma cells. Additionally, we show that the -219T/G polymorphism produces variations in APOE transcriptional activity, with the G allele showing higher activity.

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

To investigate the potential involvement of adrenergic signaling in Alzheimer's 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.

Abeta production as consequence of cellular death of a human neuroblastoma overexpressing APP.

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

ApoE4 is more efficient than E3 in brain access by herpes simplex virus type 1.

Apolipoprotein E (ApoE) plays a relevant role in herpes simplex type 1 (HSV-1) infection of the CNS; after infection by the hematogenous route, the viral neuroinvasiveness directly depends on the APOE gene dose. To analyze the effect of ApoE isoforms on the HSV-1 infectivity to the brain, we have used a model of hematogenous infection of mice humanized for the ApoE3 or the ApoE4 alleles, and we have analyzed the presence of viral DNA in several organs by real time quantitative PCR. We have found that animals expressing human ApoE4 present very high levels of virus in the brain when compared to those expressing the ApoE3 allele; in contrast, there were no significant differences in the viral levels found in peripheral organs. Apolipoprotein E4 seems to facilitate the entry and/or spread of HSV-1 in the brain much more efficiently than E3, pointing to a novel potential mechanism underlying the susceptibility to neurodegenerative processes associated with the ApoE4 allele.

A free radical-generating system regulates AßPP metabolism/processing: involvement of the ubiquitin/proteasome and autophagy/lysosome pathways.

Oxidative stress is an early event in the pathogenesis of Alzheimer's disease (AD). We previously reported that, in SK-N-MC cells, the xanthine/xanthine oxidase (X-XOD) free radical generating system regulates the metabolism/processing of the amyloid-ß protein precursor (AßPP). Oxidative stress alters the two main cellular proteolytic machineries, the ubiquitin/proteasome (UPS) and the autophagy/lysosome systems, and recent studies have established connections between the malfunctioning of these and the pathogenesis of AD. The aim of the present work was to examine the involvement of these proteolytic systems in the regulation of AßPP metabolism by X-XOD. The proteasome inhibitor MG132 was found to accelerate the metabolism/processing of AßPP promoted by X-XOD because it significantly enhances the secretion of α-secretase-cleaved soluble AßPP and also the levels of both carboxy-terminal fragments (CTFs) produced by α- and ß-secretase. Further, MG132 modulated the intracellular accumulation of holo-AßPP and/or AßPP CTFs. This indicates that the X-XOD modulation of AßPP metabolism/processing involves the UPS pathway. With respect to the autophagy/lysosome pathway, the AßPP processing and intracellular location patterns induced by X-XOD treatment closely resembled those produced by the lysosome inhibitor ammonium chloride. The present results suggest that the regulation of AßPP metabolism/processing by mild oxidative stress requires UPS activity with a simultaneous reduction in that of the autophagy/lysosome system.

Aging-related neurostructural, neuropathological, and behavioral changes associated with herpes simplex virus type 1 brain infection in mice.

To date, the main advances in understanding Alzheimer's disease (AD) have revolved around the genetic variants associated with the familial form of the disease, yet the majority of cases are sporadic. The main risk factor for AD is aging, followed by production of the E4 isoform of apolipoprotein E (APOE). Female gender also increases the risk of developing AD. Herpes simplex virus type 1 (HSV-1) has been epidemiologically and experimentally associated with AD, although no studies on its effects over aging have been undertaken. To assess the potential aging-related consequences of HSV-1 brain infection, 2 month-old wild-type and apoE-deficient mice were infected with the virus, and over the next 16 months analyses made of cerebral viral load, neuropathological, morphological, and metabolic changes in the brain, and cognitive performance. Viral load in the central nervous system (CNS) increased with age. The viral load in the brains of aged apoE+/+ female mice was 43 times that seen in apoE-/- male mice. No MRI-detectable morphological differences nor any clear neuropathological differences were seen between 18 month-old infected and mock-infected mice, although differences were seen in younger animals. Neuroinfection was associated with memory deficit and a reduction in metabolic indicators of CNS health.

Herpes simplex virus type I induces the accumulation of intracellular ß-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 Alzheimer's 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 ß-amyloid (Aß) in the autophagic compartments and that it is associated with a marked inhibition of Aß secretion. Autophagosomes containing Aß failed to fuse with lysosomes in HSV-1-infected cells, indicating the impaired degradation of Aß 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 Aß characteristic of 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 Alzheimer's 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.

How statins could be evaluated successfully in clinical trials for Alzheimer's disease?

Over the last decade, a large number of experimental observations have suggested a relationship between alterations in cholesterol homeostasis and Alzheimer's disease (AD). Moreover, epidemiological studies have pointed an association between statin treatment and a decrease in the risk of having AD. For these reasons, a large number of clinical trials have been carried out to determine whether the statins can prevent the progression of AD. However, these studies did not provide clear evidence for the therapeutic efficacy in AD. We consider that there are a number of explanations for this failure that may provide guidance for selecting and clinically developing statins with therapeutic efficacy in AD.

Genetic variations in tau-tubulin kinase-1 are linked to Alzheimer's disease in a Spanish case-control cohort.

Neurofibrillary tangles, one of the characteristic neuropathological lesions found in Alzheimer's disease (AD) brains, are composed of abnormally hyperphosphorylated tau protein. Tau-tubulin kinase-1 (TTBK1) is a brain-specific protein kinase involved in tau phosphorylation at AD-related sites. We examined genetic variations of TTBK1 by genotyping nine haplotype tagging SNPs (htSNPs) (rs2104142, rs2651206, rs10807287, rs7764257, rs3800294, rs1995300, rs2756173, rs6936397, and rs6458330) in a group of 645 Spanish late-onset AD patients and 738 healthy controls. Using a recessive genetic model, minor allele homozygotes for rs2651206 in intron 1 (OR=0.50, p=0.0003), rs10807287 in intron 5 (OR=0.49, p=0.0002), and rs7764257 in intron 9 (OR=0.57, p=0.023), which are in strong linkage disequilibrium, had a lower risk of developing AD than subjects homozygotes and heterozygotes for the major allele. TTBK1 is a promising new candidate tau phosphorylation-related gene for AD risk.

Genetic cross-interaction between APOE and PRNP in sporadic Alzheimer's and Creutzfeldt-Jakob diseases.

Alzheimer's disease (AD) and Creutzfeldt-Jakob disease (CJD) represent two distinct clinical entities belonging to a wider group, generically named as conformational disorders that share common pathophysiologic mechanisms. It is well-established that the APOE ε4 allele and homozygosity at polymorphic codon 129 in the PRNP gene are the major genetic risk factors for AD and human prion diseases, respectively. However, the roles of PRNP in AD, and APOE in CJD are controversial. In this work, we investigated for the first time, APOE and PRNP genotypes simultaneously in 474 AD and 175 sporadic CJD (sCJD) patients compared to a common control population of 335 subjects. Differences in genotype distribution between patients and control subjects were studied by logistic regression analysis using age and gender as covariates. The effect size of risk association and synergy factors were calculated using the logistic odds ratio estimates. Our data confirmed that the presence of APOE ε4 allele is associated with a higher risk of developing AD, while homozygosity at PRNP gene constitutes a risk for sCJD. Opposite, we found no association for PRNP with AD, nor for APOE with sCJD. Interestingly, when AD and sCJD patients were stratified according to their respective main risk genes (APOE for AD, and PRNP for sCJD), we found statistically significant associations for the other gene in those strata at higher previous risk. Synergy factor analysis showed a synergistic age-dependent interaction between APOE and PRNP in both AD (SF = 3.59, p = 0.027), and sCJD (SF = 7.26, p = 0.005). We propose that this statistical epistasis can partially explain divergent data from different association studies. Moreover, these results suggest that the genetic interaction between APOE and PRNP may have a biological correlate that is indicative of shared neurodegenerative pathways involved in AD and sCJD.