A loss-of-function NCSTN mutation associated with familial Dowling Degos disease and hidradenitis suppurativa.

Dowling Degos disease (DDD) is a rare autosomal dominant genodermatosis characterized by acquired, slowly progressive reticulated pigmented lesions primarily involving flexural skin areas. Mutations in KRT5, POGLUT-1 and POFUT-1 genes have been associated with DDD, and loss-of-function mutations in PSENEN, a subunit of the gamma-secretase complex, were found in patients presenting with DDD or DDD comorbid with hidradenitis suppurativa (HS). A nonsense mutation in NCSTN, another subunit of the gamma-secretase, was already described in a patient suffering from HS and DDD but whether NCSTN could be considered a novel gene for DDD is still debated. Here, we enrolled a four-generation family with HS and DDD. Through Whole Exome Sequencing (WES) we identified a novel nonsense mutation in the NCSTN gene in all the affected family members. To study the impact of this variant, we isolated outer root sheath cells from patients' hair follicles. We showed that this variant leads to a premature stop codon, activates a nonsense-mediated mRNA decay, and causes NCSTN haploinsufficiency in affected individuals. In fact, cells treated with gentamicin, a readthrough agent, had the NCSTN levels corrected. Moreover, we observed that this haploinsufficiency also affects other subunits of the gamma-secretase complex, possibly causing DDD. Our findings clearly support NCSTN as a novel DDD gene and suggest carefully investigating this co-occurrence in HS patients carrying a mutation in the NCSTN gene.

Clinical and Molecular Characterization of Hidradenitis Suppurativa: A Practical Framework for Novel Therapeutic Targets.

BACKGROUND: The pathophysiological picture underlying hidradenitis suppurativa (HS) and its syndromic forms is still patchy, thus presenting a great challenge for dermatologists and researchers since just by better understanding the pathogenesis of disease we could identify novel therapeutic targets. METHODS: We propose a practical framework to improve subcategorization of HS patients and support the genotype-phenotype correlation, useful for endotype-directed therapies development. RESULTS: This framework includes (i) clinical work-up that involves the collection of demographic, lifestyle, and clinical data as well as the collection of different biological samples; (ii) genetic-molecular work-up, based on multi-omics analysis in combination with bioinformatics pipelines to unravel the complex etiology of HS and its syndromic forms; (iii) functional studies, - represented by skin fibroblast cell cultures, reconstructed epidermal models (both 2D and 3D) and organoids -, of candidate biomarkers and genetic findings necessary to validate novel potential molecular mechanisms possibly involved and druggable in HS; (iv) genotype-phenotype correlation and clinical translation in tailored targeted therapies. CONCLUSION: Omic findings should be merged and integrated with clinical data; moreover, the skin-omic profiles from each HS patient should be matched and integrated with the ones already reported in public repositories, supporting the efforts of the researchers and clinicians to discover novel biomarkers and molecular pathways with the ultimate goal of providing faster development of novel patient-tailored therapeutic approaches.

Notch Signaling in Health and Disease.

The Notch signaling pathway, a vital and evolutionarily conserved regulator of cellular processes, intricately shapes both health and disease [...].

Whole-Exome Sequencing in 10 Unrelated Patients with Syndromic Hidradenitis Suppurativa: A Preliminary Step for a Genotype-Phenotype Correlation.

BACKGROUND: The genetics of syndromic hidradenitis suppurativa (HS), an immune-mediated condition associated with systemic comorbidities such as inflammatory bowel diseases and arthritis, has not been completely elucidated. OBJECTIVE: To describe clinical features and genetic signature of patients with the main syndromic HS forms, i.e., PASH, PAPASH, and PASH/SAPHO overlapping. METHODS: Whole-exome sequencing (WES) approach was performed in ten patients with syndromic HS. RESULTS: Three clinical settings have been identified based on presence/absence of gut and joint inflammation. Four PASH patients who had also gut inflammation showed three different variants in NOD2 gene, two variants in OTULIN, and a variant in GJB2, respectively. Three PAPASH and three PASH/SAPHO overlapping patients who had also joint inflammation showed two different variants in NCSTN, one in WDR1 and PSTPIP1, and two variants in NLRC4, one of whom was present in a patient with a mixed phenotype characterized by gut and joint inflammation. LIMITATIONS: Limited number of patients that can be counterbalanced by the rarity of syndromic HS. CONCLUSION: Syndromic HS can be considered as a polygenic autoinflammatory condition; currently WES is a diagnostic tool allowing more accurate genotype-phenotype correlation.

Aquaporins Are One of the Critical Factors in the Disruption of the Skin Barrier in Inflammatory Skin Diseases.

The skin is the largest organ of the human body, serving as an effective mechanical barrier between the internal milieu and the external environment. The skin is widely considered the first-line defence of the body, with an essential function in rejecting pathogens and preventing mechanical, chemical, and physical damages. Keratinocytes are the predominant cells of the outer skin layer, the epidermis, which acts as a mechanical and water-permeability barrier. The epidermis is a permanently renewed tissue where undifferentiated keratinocytes located at the basal layer proliferate and migrate to the overlying layers. During this migration process, keratinocytes undertake a differentiation program known as keratinization process. Dysregulation of this differentiation process can result in a series of skin disorders. In this context, aquaporins (AQPs), a family of membrane channel proteins allowing the movement of water and small neutral solutes, are emerging as important players in skin physiology and skin diseases. Here, we review the role of AQPs in skin keratinization, hydration, keratinocytes proliferation, water retention, barrier repair, wound healing, and immune response activation. We also discuss the dysregulated involvement of AQPs in some common inflammatory dermatological diseases characterised by skin barrier disruption.

Variant Enrichment Analysis to Explore Pathways Functionality in Complex Autoinflammatory Skin Disorders through Whole Exome Sequencing Analysis.

The challenge of unravelling the molecular basis of multifactorial disorders nowadays cannot rely just on association studies searching for potential causative variants shared by groups of patients and not present in healthy individuals; indeed, association studies have as a main limitation the lack of information on the interactions between the disease-causing variants. Thus, new genomic analysis tools focusing on disrupted pathways rather than associated gene variants are required to better understand the complexity of a disease. Therefore, we developed the Variant Enrichment Analysis (VEA) workflow, a tool applicable for whole exome sequencing data, able to find differences between the numbers of genetic variants in a given pathway in comparison with a reference dataset. In this study, we applied VEA to discover novel pathways altered in patients with complex autoinflammatory skin disorders, namely PASH (n = 9), 3 of whom are overlapping with SAPHO) and PAPASH (n = 3). With this approach we have been able to identify pathways related to neutrophil and endothelial cells homeostasis/activations, as disrupted in our patients. We hypothesized that unregulated neutrophil transendothelial migration could elicit increased neutrophil infiltration and tissue damage. Based on our findings, VEA, in our experimental dataset, allowed us to predict novel pathways impaired in subjects with autoinflammatory skin disorders.

Multiomics Integration in Skin Diseases with Alterations in Notch Signaling Pathway: PlatOMICs Phase 1 Deployment.

The high volume of information produced in the age of omics was and still is an important step to understanding several pathological processes, providing the enlightenment of complex molecular networks and the identification of molecular targets associated with many diseases. Despite these remarkable scientific advances, the majority of the results are disconnected and divergent, making their use limited. Skin diseases with alterations in the Notch signaling pathway were extensively studied during the omics era. In the GWAS Catalog, considering only studies on genomics association (GWAS), several works were deposited, some of which with divergent results. In addition, there are thousands of scientific articles available about these skin diseases. In our study, we focused our attention on skin diseases characterized by the impairment of Notch signaling, this pathway being of pivotal importance in the context of epithelial disorders. We considered the pathologies of five human skin diseases, Hidradenitis Suppurativa, Dowling Degos Disease, Adams-Oliver Syndrome, Psoriasis, and Atopic Dermatitis, in which the molecular alterations in the Notch signaling pathway have been reported. To this end, we started developing a new multiomics platform, PlatOMICs, to integrate and re-analyze omics information, searching for the molecular interactions involved in the pathogenesis of skin diseases with alterations in the Notch signaling pathway.

Notch Signaling Regulation in Autoinflammatory Diseases.

Notch pathway is a highly conserved intracellular signaling route that modulates a vast variety of cellular processes including proliferation, differentiation, migration, cell fate and death. Recently, the presence of a strict crosstalk between Notch signaling and inflammation has been described, although the precise molecular mechanisms underlying this interplay have not yet been fully unravelled. Disruptions in Notch cascade, due both to direct mutations and/or to an altered regulation in the core components of Notch signaling, might lead to hypo- or hyperactivation of Notch target genes and signaling molecules, ultimately contributing to the onset of autoinflammatory diseases. To date, alterations in Notch signaling have been reported as associated with three autoinflammatory disorders, therefore, suggesting a possible role of Notch in the pathogenesis of the following diseases: hidradenitis suppurativa (HS), Behçet disease (BD), and giant cell arteritis (GCA). In this review, we aim at better characterizing the interplay between Notch and autoinflammatory diseases, trying to identify the role of this signaling route in the context of these disorders.

Pleiotropic Role of Notch Signaling in Human Skin Diseases.

Notch signaling orchestrates the regulation of cell proliferation, differentiation, migration and apoptosis of epidermal cells by strictly interacting with other cellular pathways. Any disruption of Notch signaling, either due to direct mutations or to an aberrant regulation of genes involved in the signaling route, might lead to both hyper- or hypo-activation of Notch signaling molecules and of target genes, ultimately inducing the onset of skin diseases. The mechanisms through which Notch contributes to the pathogenesis of skin diseases are multiple and still not fully understood. So far, Notch signaling alterations have been reported for five human skin diseases, suggesting the involvement of Notch in their pathogenesis: Hidradenitis Suppurativa, Dowling Degos Disease, Adams-Oliver Syndrome, Psoriasis and Atopic Dermatitis. In this review, we aim at describing the role of Notch signaling in the skin, particularly focusing on the principal consequences associated with its alterations in these five human skin diseases, in order to reorganize the current knowledge and to identify potential cellular mechanisms in common between these pathologies.

Autophagy in Zika Virus Infection: A Possible Therapeutic Target to Counteract Viral Replication.

Zika virus (ZIKV) still constitutes a public health concern, however, no vaccines or therapies are currently approved for treatment. A fundamental process involved in ZIKV infection is autophagy, a cellular catabolic pathway delivering cytoplasmic cargo to the lysosome for degradation-considered as a primordial form of innate immunity against invading microorganisms. ZIKV is thought to inhibit the Akt-mTOR signaling pathway, which causes aberrant activation of autophagy promoting viral replication and propagation. It is therefore appealing to study the role of autophagic molecular effectors during viral infection to identify potential targets for anti-ZIKV therapeutic intervention.

25-hydroxycholesterol reduces inflammation, viral load and cell death in ZIKV-infected U-87 MG glial cell line.

Zika virus (ZIKV) infection is a global health issue due to its worldwide diffusion and to the related effects on neural progenitor cells with severe consequences on developing brain as well as on the central nervous system of adults. Previous studies showed that ZIKV infection induces an increment of IL1B expression in the central nervous system and also in the blood. IL-1ß is a pro-inflammatory cytokine essential for cellular defence, tissue repair and neuroinflammation, a mechanism seen to be associated with several neuroinflammatory diseases. 25-hydroxycholesterol (25-HC) is a natural oxysterol, derived from hydroxylation of cholesterol, possessing important antiviral activity possibly correlated to its ability to alter host membrane structures. Furthermore, 25-HC is involved in the modulation of IL1B gene expression, being able to suppress IL-1ß driven inflammation probably by blocking the activation of the SREB proteins. In our study, we analysed the antiviral action of 25-HC in ZIKV-infected U-87 MG cells, also evaluating its impact on inflammation and cell death. We demonstrated that 25-HC is able to reduce inflammation and cell death caused by ZIKV infection and also to diminish intracellular ZIKV load in U-87 MG glial cell line. Considering its antiviral activity and its ability to penetrate blood-brain barrier, 25-HC could be proposed, based on our results and literature findings, as a potential anti-ZIKV agent.

Antiviral properties of blue laser in an in vitro model of HSV-1 infection.

Herpes simplex virus type-1 (HSV-1) is known to cause lifelong infections in humans. First infection is characterized by gingiva-stomatitis and pharyngitis, while virus reactivation causes recurrent herpes labialis with ulcerations on intraoral mucosa, mouth or external facial skin [1]. Laser therapy (LT), set at red and infrared wavelengths, has been reported as able to reduce HSV-1 recurrence and duration of herpetic sores [2]. Despite the blue wavelength already showed its efficacy in killing different strains of bacteria, it has never been tested on viruses [3].

MMAB, a novel candidate gene to be screened in the molecular diagnosis of Mevalonate Kinase Deficiency.

Mevalonate kinase deficiency (MKD) is an autosomal recessive inflammatory disease. Mutations in MVK gene are associated with MKD with modest genotype-phenotype correlation. In spite of recent guidelines indicating specific MVK mutations for the more severe form or the milder one, little is known about MVK variability within and between populations. The aim of this work is to provide supplementary information about MVK variability useful in the molecular diagnosis of MKD, as well as to unravel the presence of novel genes potentially involved as involved in the clinical heterogeneity of MKD phenotype. We used a population-based approach, coupled with Combined Annotation-Dependent Depletion (CADD) score, to analyze the level of genetic variability for common and putatively deleterious MVK variants. We also performed Exome screening with the Illumina Human Exome Bead Chip on 21 MKD patients to double-check our in silico findings. Haplotype block detection in different populations revealed the existence of two blocks in MVK; interestingly, the first haploblock comprises the promoter region shared with MMAB gene. Analyses of MMAB and MVK genetic variants in 21 MKD patients strengthen our observations showing a novel scenario in which the same mutations commonly associated with MKD are found coupled with different combination of MMAB rs7134594 SNP was already described as associated with HDL cholesterol level and present in the haploblock promoter region. The rs7134594 SNP is reported as an eQTL for MVK and MMAB. Hypothesizing the presence of genetic variants modulating the complex phenotypic spectrum of MKD, we suggest that future directions in screening for MKD pathogenic variants should focus both MMAB and MVK genes.

Alendronate treatment induces IL-1B expression and apoptosis in glioblastoma cell line.

Alendronate (ALD), one among the nitrogen-containing bisphosphonates (NBPs), is currently used for the treatment of many pathological conditions. Unfortunately, although generally tolerated, NBPs treatment has been associated with central nervous system (CNS) adverse outcomes, such as amnesia, hallucinations and visual disturbances. So, we analyzed the effect of ALD treatment in glial cells, the main sources of cholesterol for neurons and principal cells involved in the immunological defense of the brain. We treated a glial cell line (U87-MG) with increasing doses of ALD (0.1, 1, 10, 25, 50 µM) for 48 h, aimed at evaluating the influence of this drug treatment on IL-1B expression, NLRP3 and CASP1 expression, mitochondrial activity and apoptotic cell death. We observed that ALD treatment, at the higher concentrations, induced a significant increase of IL-1B, NLRP3, CASP1 expression, provoked apoptosis and also mitochondrial damage in U87-MG. Considering the reported CNS adverse outcomes of NBPs treatment, our results confirm ALD side-effects on glial cell model.

Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency.

BACKGROUND/AIMS: Mevalonate Kinase Deficiency (MKD), is a hereditary disease due to mutations in mevalonate kinase gene (MVK). MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA), the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. METHODS: SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. RESULTS: MVK mutants' over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor) further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. CONCLUSIONS: We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation.

Zika virus induces inflammasome activation in the glial cell line U87-MG.

In the last years, neurological complications related to Zika virus (ZIKV) infection have emerged as an important threat to public health worldwide. ZIKV infection has been associated to neurological disorders such as congenital microcephaly in newborns and Guillain-Barré syndrome, myelopathy and encephalitis in adults. ZIKV is characterized by neurotropism and neurovirulence. Several studies have identified microglial nodules, gliosis, neuronal and glial cells degeneration and necrosis in the brain of ZIKV infected infants, suggesting that ZIKV could play a role in these neurological disorders through neuroinflammation and microglial activation. Little information is available about neuroinflammation and ZIKV-related neurological disorders. Therefore, we investigated if ZIKV is able to infect a glial cell line (U87-MG) and how the glial cell line responds to this infection in terms of inflammation (IL-1ß, NLRP-3 and CASP-1), oxidative stress (SOD2 and HemeOX) and cell death. We observed a significant increase of ZIKV load in both cells and supernatants after 72 h, compared to 48 h of infection. We found that ZIKV infection induces an increase of IL-1ß, NLRP-3 and CASP-1 genes expression. Significant increase of IL-1ß and unchanged pro-IL-1ß protein levels have also been detected. Moreover, we observed SOD2 and HemeOX increased gene expression mainly after 72 h post ZIKV infection. Subsequently, we found a decrease of U87-MG cell viability, after both 48 h and 72 h of ZIKV infection. Our results show that U87-MG cells are susceptible to ZIKV infection. ZIKV is able to successfully replicate in infected cells causing oxidative stress, NLRP3 inflammasome activation and subsequent release of mature IL-1ß; this process culminates in cell death. Thus, considering the central role of neuroinflammation in neurological disorders, it is important to comprehend every aspect of this mechanism in order to better understand the pathogenesis of ZIKV infection and to identify possible strategies to fight the virus by rescuing cell death.

Antiretroviral Treatment in HIV-1-Positive Mothers: Neurological Implications in Virus-Free Children.

Since the worldwide introduction of antiretroviral therapy (ART) in human immunodeficiency virus type 1, HIV-1-positive mothers, together with HIV-1 testing prior to pregnancy, caesarian birth and breastfeeding cessation with replacement feeding, a reduction of HIV-1 mother-to-child transmission (MTCT) has been observed in the last few years. As such, an increasing number of children are being exposed in utero to ART. Several questions have arisen concerning the neurological effects of ART exposure in utero, considering the potential effect of antiretroviral drugs on the central nervous system, a structure which is in continuous development in the fetus and characterized by great plasticity. This review aims at discussing the possible neurological impairment of children exposed to ART in utero, focusing attention on the drugs commonly used for HIV-1 MTCT prevention, clinical reports of ART neurotoxicity in children born to HIV-1-positive mothers, and neurologic effects of protease inhibitors (PIs), especially ritonavir-"boosted" lopinavir (LPV/r) in cell and animal central nervous system models evaluating the potential neurotoxic effect of ART. Finally, we present the findings of a meta-analysis to assess the effects on the neurodevelopment of children exposed to ART in utero.

HIV Protease Inhibitors Apoptotic Effect in SH-SY5Y Neuronal Cell Line.

BACKGROUND: Prophylactic treatment regimens to prevent mother-to-child HIV transmission include protease inhibitors Lopinavir and Ritonavir. Lopinavir and Ritonavir have been reported to be able to induce intracellular oxidative stress in diverse cellular models, however scarce informations are available about protease inhibitor effects of in the central nervous system (CNS). In our study we evaluated the impact of protease inhibitors on a cell neuronal model. METHODS: We treated a neuroblastoma cell line (SH-SY5Y) with increasing doses of Lopinavir and Ritonavir (0.1-1-10-25-50 µM), used alone or in combination, evaluating the impact of these drugs in terms of mitochondrial activity, with MTT cell proliferation assay; mRNA expression of heme oxygenase (HemeOH) and reactive oxygen species (ROS) levels with 2',7'-dichlorofluorescin diacetate (H2DCFDA) in order to assess oxidative stress; apoptotic cell death with flow cytometry. RESULTS: We observed that Lopinavir and Ritonavir treatment, at 25 and/or 50 µM concentrations, induced mitochondrial damage, increase of heme oxygenase RNA expression levels and ROS generation, followed by apoptosis in SH-SY5Y. CONCLUSIONS: Our in vitro model demonstrates a damaging effect of HIV protease inhibitors on the neuroblastoma cell line, thus partially mimicking the impact of these drugs on the CNS of children born to HIV positive mothers undergone to antiretroviral treatment.

Microglia activation and interaction with neuronal cells in a biochemical model of mevalonate kinase deficiency.

Mevalonate kinase deficiency is a rare disease whose worst manifestation, characterised by severe neurologic impairment, is called mevalonic aciduria. The progressive neuronal loss associated to cell death can be studied in vitro with a simplified model based on a biochemical block of the mevalonate pathway and a subsequent inflammatory trigger. The aim of this study was to evaluate the effect of the mevalonate blocking on glial cells (BV-2) and the following effects on neuronal cells (SH-SY5Y) when the two populations were cultured together. To better understand the cross-talk between glial and neuronal cells, as it happens in vivo, BV-2 and SH-SY5Y were co-cultured in different experimental settings (alone, transwell, direct contact); the effect of mevalonate pathway biochemical block by Lovastatin, followed by LPS inflammatory trigger, were evaluated by analysing programmed cell death and mitochondrial membrane potential, cytokines' release and cells' morphology modifications. In this experimental condition, glial cells underwent an evident activation, confirmed by elevated pro-inflammatory cytokines release, typical of these disorders, and a modification in morphology. Moreover, the activation induced an increase in apoptosis. When glial cells were co-cultured with neurons, their activation caused an increase of programmed cell death also in neuronal cells, but only if the two populations were cultured in direct contact. Our findings, being aware of the limitations related to the cell models used, represent a preliminary step towards understanding the pathological and neuroinflammatory mechanisms occurring in mevalonate kinase diseases. Contact co-culture between neuronal and microglial cells seems to be a good model to study mevalonic aciduria in vitro, and to contribute to the identification of potential drugs able to block microglial activation for this orphan disease. In fact, in such a pathological condition, we demonstrated that microglial cells are activated and contribute to neuronal cell death. We can thus hypothesise that the use of microglial activation blockers could prevent this additional neuronal death.