SOCS3 deregulation contributes to aberrant activation of the JAK/STAT pathway in precursor T-cell neoplasms.

Despite the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway being frequently altered in T-ALL/LBL, no specific therapy has been approved for T-ALL/LBL patients with constitutive signalling by JAK/STAT, so there is an urgent need to identify pathway members that may be potential therapeutic targets. In the present study, we searched for JAK/STAT pathway members potentially modulated through aberrant methylation and identified SOCS3 hypermethylation as a recurrent event in T-ALL/LBL. Additionally, we explored the implications of SOCS3 deregulation in T-ALL/LBL and demonstrated that SOCS3 counteracts the constitutive activation of the JAK/STAT pathway through different molecular mechanisms. Therefore, SOCS3 emerges as a potential therapeutic target in T-ALL/LBL.

Genetics of Wilson disease and Wilson-like phenotype in a clinical series from eastern Spain.

Wilson's disease (WD) is an autosomal recessive disorder caused by ATP7B mutations. Subjects with only one mutation may show clinical signs and individuals with biallelic changes may remain asymptomatic. We aimed to achieve a conclusive genetic diagnosis for 34 patients clinically diagnosed of WD. Genetic analysis comprised from analysis of exons to WES (whole exome sequencing), including promoter, introns, UTRs (untranslated regions), besides of study of large deletions/duplications by MLPA (multiplex ligation-dependent probe amplification). Biallelic ATP7B mutations were identified in 30 patients, so that four patients were analyzed using WES. Two affected siblings resulted to be compound heterozygous for mutations in CCDC115, which is involved in a form of congenital disorder of glycosylation. In sum, the majority of patients with a WD phenotype carry ATP7B mutations. However, if genetic diagnosis is not achieved, additional genes should be considered because other disorders may mimic WD.

Tetraspanin CD81 regulates HSV-1 infection.

Different members of the tetraspanin superfamily have been described to regulate different virus infectious cycles at several stages: viral entry, viral replication or virion exit or infectivity. In addition, tetraspanin CD81 regulates HIV reverse transcription through its association with the dNTP hydrolase SAMHD1. Here we aimed at analysing the role of CD81 in Herpes simplex virus 1 infectivity using a neuroblastoma cell model. For this purpose, we generated a CD81 KO cell line using the CRISPR/Cas9 technology. Despite being CD81 a plasma membrane protein, CD81 KO cells showed no defects in viral entry nor in the expression of early protein markers. In contrast, glycoprotein B and C, which require viral DNA replication for their expression, were significantly reduced in CD81 KO infected cells. Indeed, HSV-1 DNA replication and the formation of new infectious particles were severely compromised in CD81 KO cells. We could not detect significant changes in SAMHD1 total expression levels, but a relocalization into endosomal structures was observed in CD81 KO cells. In summary, CD81 KO cells showed impaired viral DNA replication and produced greatly diminished viral titers.

RNA-Seq reveals the existence of a CDKN1C-E2F1-TP53 axis that is altered in human T-cell lymphoblastic lymphomas.

BACKGROUND: Precursor T-cell lymphoblastic lymphomas (T-LBL) are rare aggressive hematological malignancies that mainly develop in children. As in other cancers, the loss of cell cycle control plays a prominent role in the pathogenesis in these malignancies that is primarily attributed to loss of CDKN2A (encoding protein p16INK4A). However, the impact of the deregulation of other genes such as CDKN1C, E2F1, and TP53 remains to be clarified. Interestingly, experiments in mouse models have proven that conditional T-cell specific deletion of Cdkn1c gene may induce a differentiation block at the DN3 to DN4 transition, and that the loss of this gene in the absence of Tp53 led to aggressive thymic lymphomas. RESULTS: In this manuscript, we demonstrated that the simultaneous deregulation of CDKN1C, E2F1, and TP53 genes by epigenetic mechanisms and/or the deregulation of specific microRNAs, together with additional impairing of TP53 function by the expression of dominant-negative isoforms are common features in primary human T-LBLs. CONCLUSIONS: Previous experimental work in mice revealed that T-cell specific deletion of Cdkn1c accelerates lymphomagenesis in the absence of Tp53. If, as expected, the consequences of the deregulation of the CDKN1C-E2F1-TP53 axis were the same as those experimentally demonstrated in mouse models, the disruption of this axis might be useful to predict tumor aggressiveness, and to provide the basis towards the development of potential therapeutic strategiesin human T-LBL.

Human in vivo evidence of associations between herpes simplex virus and cerebral amyloid-beta load in normal aging.

BACKGROUND: Mounting data suggests that herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of AD, possibly instigating amyloid-beta (Aß) accumulation decades before the onset of clinical symptoms. However, human in vivo evidence linking HSV-1 infection to AD pathology is lacking in normal aging, which may contribute to the elucidation of the role of HSV-1 infection as a potential AD risk factor. METHODS: To shed light into this question, serum anti-HSV IgG levels were correlated with 18F-Florbetaben-PET binding to Aß deposits and blood markers of neurodegeneration (pTau181 and neurofilament light chain) in cognitively normal older adults. Additionally, we investigated whether associations between anti-HSV IgG and AD markers were more evident in APOE4 carriers. RESULTS: We showed that increased anti-HSV IgG levels are associated with higher Aß load in fronto-temporal regions of cognitively normal older adults. Remarkably, these cortical regions exhibited abnormal patterns of resting state-functional connectivity (rs-FC) only in those individuals showing the highest levels of anti-HSV IgG. We further found that positive relationships between anti-HSV IgG levels and Aß load, particularly in the anterior cingulate cortex, are moderated by the APOE4 genotype, the strongest genetic risk factor for AD. Importantly, anti-HSV IgG levels were unrelated to either subclinical cognitive deficits or to blood markers of neurodegeneration. CONCLUSIONS: All together, these results suggest that HSV infection is selectively related to cortical Aß deposition in normal aging, supporting the inclusion of cognitively normal older adults in prospective trials of antimicrobial therapy aimed at decreasing the AD risk in the aging population.

Cholesterol Modulation Attenuates the AD-like Phenotype Induced by Herpes Simplex Virus Type 1 Infection.

Cholesterol, a crucial component of cell membranes, influences various biological processes, including membrane trafficking, signal transduction, and host-pathogen interactions. Disruptions in cholesterol homeostasis have been linked to congenital and acquired conditions, including neurodegenerative disorders such as Alzheimer's disease (AD). Previous research from our group has demonstrated that herpes simplex virus type I (HSV-1) induces an AD-like phenotype in several cell models of infection. This study explores the interplay between cholesterol and HSV-1-induced neurodegeneration. The impact of cholesterol was determined by modulating its levels with methyl-beta-cyclodextrin (MßCD) using the neuroblastoma cell lines SK-N-MC and N2a. We have found that HSV-1 infection triggers the intracellular accumulation of cholesterol in structures resembling endolysosomal/autophagic compartments, a process reversible upon MßCD treatment. Moreover, MßCD exhibits inhibitory effects at various stages of HSV-1 infection, underscoring the importance of cellular cholesterol levels, not only in the viral entry process but also in subsequent post-entry stages. MßCD also alleviated several features of AD-like neurodegeneration induced by viral infection, including lysosomal impairment and intracellular accumulation of amyloid-beta peptide (Aß) and phosphorylated tau. In conclusion, these findings highlight the connection between cholesterol, neurodegeneration, and HSV-1 infection, providing valuable insights into the underlying mechanisms of AD.

Herpes Simplex Virus Type 1 Induces AD-like Neurodegeneration Markers in Human Progenitor and Differentiated ReNcell VM Cells.

An increasing body of evidence strongly suggests that infections or reactivations of herpes simplex virus type 1 (HSV-1) may be closely linked to Alzheimer's disease (AD). Promising results have been obtained using cell and animal models of HSV-1 infection, contributing to the understanding of the molecular mechanisms linking HSV-1 infection and AD neurodegeneration. ReNcell VM is a human neural stem cell line that has been used as a model system to study the impact of various infectious agents on the central nervous system. In this study, we demonstrate the suitability of the ReNcell VM cell line for developing a new in vitro model of HSV-1 infection. By following standard differentiation protocols, we were able to derive various nervous cell types, including neurons, astrocytes, and oligodendrocytes, from neural precursors. Additionally, we demonstrated the susceptibility of ReNcell VM cells, including precursor and differentiated cells, to HSV-1 infection and subsequent viral-induced AD-like neurodegeneration. Our findings support the use of this cell line to generate a new research platform for investigating AD neuropathology and its most significant risk factors, which may lead to important discoveries in the context of this highly impactful disease.

Health-Related Quality of Life in Patients Living with Wilson Disease in Spain: A Cross-Sectional Observational Study.

Wilson disease (WD) is a rare copper metabolism disorder caused by mutations in the ATP7B gene. It usually affects young individuals and can produce hepatic and/or neurological involvement, potentially affecting health-related quality of life (HRQoL). We assessed HRQoL in a cohort of Spanish patients with WD and evaluated disease impact on several domains of patients' lives, treatment adherence, drug preference and satisfaction, and healthcare resource utilisation in a cross-sectional, retrospective, multicentric, observational study. A total of 102 patients were included: 81.4% presented isolated liver involvement (group H) and 18.6% presented neurological or mixed involvement (group EH). Up to 30% of patients reported a deteriorated emotional status with anxiety and depression, which was greater in the EH subgroup; the use of neuropsychiatric drugs was high. Over 70% of the patients were satisfied with their current treatment but complained about taking too many pills, stating they would consider switching to another more patient-friendly treatment if available. The Simplified Medication Adherence Questionnaire revealed only 22.5% of patients were fully adherent to therapy, suggesting that alternative therapies are needed. This real-world study, even though is highly enriched with hepatic patients and mild disease, shows that WD impacts patients' HRQoL, especially in the emotional domain.

Respiratory and mental health effects of wildfires: an ecological study in Galician municipalities (north-west Spain).

BACKGROUND: During the summer of 2006, a wave of wildfires struck Galicia (north-west Spain), giving rise to a disaster situation in which a great deal of the territory was destroyed. Unlike other occasions, the wildfires in this case also threatened farms, houses and even human lives, with the result that the perception of disaster and helplessness was the most acute experienced in recent years. This study sought to analyse the respiratory and mental health effects of the August-2006 fires, using consumption of anxiolytics-hypnotics and drugs for obstructive airway diseases as indicators. METHODS: We conducted an analytical, ecological geographical- and temporal-cluster study, using municipality-month as the study unit. The independent variable was exposure to wildfires in August 2006, with municipalities thus being classified into the following three categories: no exposure; medium exposure; and high exposure. Dependent variables were: (1) anxiolytics-hypnotics; and (2) drugs for obstructive airway diseases consumption. These variables were calculated for the two 12-month periods before and after August 2006. Additive models for time series were used for statistical analysis purposes. RESULTS: The results revealed a higher consumption of drugs for obstructive airway diseases among pensioners during the months following the wildfires, in municipalities affected versus those unaffected by fire. In terms of consumption of anxiolytics-hypnotics, the results showed a significant increase among men among men overall -pensioners and non-pensioners- in fire-affected municipalities. CONCLUSIONS: Our study indicates that wildfires have a significant effect on population health. The coherence of these results suggests that drug utilisation research is a useful tool for studying morbidity associated with environmental incidents.

Matrix metalloproteinase 14 regulates HSV-1 infection in neuroblastoma cells.

Growing evidence supports that chronic or latent infection of the central nervous system might be implicated in Alzheimer's disease (AD). Among them, Herpes simplex virus type 1 (HSV-1) has emerged as a major factor in the etiology of the disease. Our group is devoted to the study of the relationship among HSV-1, oxidative stress (OS) and neurodegeneration. We have found that HSV-1 induces the main neuropathological hallmarks of AD, including the accumulation of intracellular amyloid beta (Aß), hyperphosphorylated tau protein and autophagic vesicles, that OS exacerbates these effects, and that matrix metalloproteinase 14 (MMP-14) participates in the alterations induced by OS. In this work, we focused on the role of MMP-14 in the degenerative markers raised by HSV-1 infection. Interestingly, we found that MMP-14 blockage is a potent inhibitor of HSV-1 infection efficiency, that also reduces the degeneration markers, accumulation of Aß and hyperphosphorylated tau, induced by the virus. Our results point to MMP-14 as a potent antiviral target to control HSV-1 infection and its associated neurodegenerative effects.

LAMP2 deficiency attenuates the neurodegeneration markers induced by HSV-1 infection.

Mounting evidence suggests a major role of infectious agents in the pathogenesis of sporadic Alzheimer's disease (AD). Among them, herpes simplex virus type 1 (HSV-1) infection has emerged as a major factor in the etiology of AD. HSV-1 is able to induce some of the main alterations of the disease such as hyperphosphorylation of tau protein and accumulation of amyloid-ß peptide. Functional genomic analysis of a cell model of HSV-1 infection and oxidative stress developed in our laboratory revealed lysosomal system to be the main pathway altered, and the lysosome-associated membrane protein 2 (LAMP2) gene one of the most strongly modulated genes. The aim of this work is to study LAMP2 as an AD candidate gene and to investigate its role in the neurodegeneration induced by HSV-1 using a LAMP2 knockdown cell model. LAMP2 deficiency led to a significant reduction of viral DNA replication and formation of infectious particles. In addition, tau hyperphosphorylation and inhibition of Aß secretion induced by the virus were attenuated by the absence of LAMP2. Finally, genetic association studies revealed LAMP2 genetic variants to be associated with AD risk. In summary, our data indicate that LAMP2 could be a suitable candidate to mediate the AD-like phenotype caused by HSV-1.

Matrix Metalloproteinase 14 Mediates APP Proteolysis and Lysosomal Alterations Induced by Oxidative Stress in Human Neuronal Cells.

The alteration of amyloid precursor protein (APP) proteolysis is a hallmark of Alzheimer's disease (AD). Recent studies have described noncanonical pathways of APP processing that seem partly executed by lysosomal enzymes. Our laboratory's in vitro human SK-N-MC model has shown that oxidative stress (OS) alters the lysosomal degradation pathway and the processing/metabolism of APP. The present study identifies the lysosomal protein matrix metalloproteinase 14 (MMP14) as a protease involved in the APP noncanonical processing. Previous expression analyses of the above cells showed MMP14 to be overexpressed under OS. In the present work, its role in changes in OS-induced APP proteolysis and lysosomal load was examined. The results show that MMP14 mediates the accumulation of an ≈85 kDa N-terminal APP fragment and increases the lysosome load induced by OS. These results were validated in neurons and neural progenitor cells generated from the induced pluripotent stem cells of patients with sporadic AD, reinforcing the idea that MMP14 may offer a therapeutic target in this disease.

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.

Downregulation of specific FBXW7 isoforms with differential effects in T-cell lymphoblastic lymphoma.

FBXW7 is a driver gene in T-cell lymphoblastic neoplasia acting through proteasome degradation of key proto-oncogenes. FBXW7 encodes three isoforms, α, ß and γ, which differ only in the N-terminus. In this work, massive sequencing revealed significant downregulation of FBXW7 in a panel of primary T-cell lymphoblastic lymphomas characterised by the absence of mutations in its sequence. We observed that decreased expression mainly affected the FBXW7ß isoform and to a lesser extent FBXW7α and may be attributed to the combined effect of epigenetic changes, alteration of upstream factors and upregulation of miRNAs. Transient transfections with miRNA mimics in selected cell lines resulted in a significant decrease of total FBXW7 expression and its different isoforms separately, with the consequent increment of critical substrates and the stimulation of cell proliferation. Transient inhibition of endogenous miRNAs in a T-cell lymphoblastic-derived cell line (SUP-T1) was capable of reversing these proliferative effects. Finally, we show how FBXW7 isoforms display different roles within the cell. Simultaneous downregulation of the α and γ isoforms modulates the amount of CCNE1, whilst the ß-isoform alone was found to have a prominent role in modulating the amount of c-MYC. Our data also revealed that downregulation of all isoforms is a sine qua non condition to induce a proliferative pattern in our cell model system. Taking these data into account, potential new treatments to reverse downregulation of all or a specific FBXW7 isoform may be an effective strategy to counteract the proliferative capacity of these tumour cells.

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.

A Free Radical-Generating System Regulates Amyloid Oligomers: Involvement of Cathepsin B.

Amyloid-ß (Aß), a major component of senile plaques, is generated via the proteolysis of amyloid-ß protein precursor (AßPP). This cleavage also produces AßPP fragment-derived oligomers which can be highly neurotoxic. AßPP metabolism/processing is affected by many factors, one of which is oxidative stress (OS). Associated with aging, OS is an important risk factor for Alzheimer's disease. In addition, the protein degradation systems, especially those involving cathepsins, are impaired in aging brains. Moreover, cathepsin B (CTSB) is a cysteine protease with potentially specific roles in AßPP proteolysis (ß-secretase activity) and Aß clearance (Aß degradative activity). The present work examines the effect of OS and the involvement of CTSB in amyloid oligomer formation. The xanthine/xanthine oxidase (X-XOD) free radical generating system induced the partial inhibition of CTSB activity, which was accompanied by an increase in large amyloid oligomers. These were located throughout the cytosol and in endo-lysosomal vesicles. Cells treated with the CTSB inhibitor CA-074Me also showed increased amyloid oligomer levels, whereas those subjected to OS in the presence of the inhibitor showed no such increase. However, CTSB inhibition clearly modulated the AßPP metabolism/processing induced by X-XOD, as revealed by the increase in intracellular AßPP and secreted α-secretase-cleaved soluble AßPP. The present results suggest that CTSB participates in the changes of amyloid oligomer induced by mild OS.

The lysosome system is severely impaired in a cellular model of neurodegeneration induced by HSV-1 and oxidative stress.

The causal agent(s) and molecular mechanisms of Alzheimer's disease (AD) remain unclear. Mounting evidence suggests that herpes simplex virus type 1 (HSV-1) infection is involved in the AD pathogenesis. Oxidative stress (OS) may also be crucial in the AD development. Our group previously reported that both HSV-1 and OS trigger the appearance of AD-type neurodegeneration markers. The main aim of the present study was to identify the mechanisms involved in this triggering. Expression studies revealed the involvement of a set of OS-regulated genes in HSV-1-infected cells and in cells harboring the Swedish mutation of the amyloid beta precursor protein gene. Functional annotation of these genes revealed the lysosome system to be impaired, suggesting that the interaction of OS with both HSV-1 and amyloid beta precursor protein mutations affects lysosomal function. Functional studies revealed HSV-1 infection and OS to increase the lysosome load, reduce the activity of lysosomal hydrolases, affect cathepsin maturation, and inhibit the endocytosis-mediated degradation of the epidermal growth factor receptor. These findings suggest alterations in the lysosome system to be involved in different forms of 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.