The anti-leprosy drug clofazimine reduces polyQ toxicity through activation of PPARγ.

BACKGROUND: PolyQ diseases are autosomal dominant neurodegenerative disorders caused by the expansion of CAG repeats. While of slow progression, these diseases are ultimately fatal and lack effective therapies. METHODS: A high-throughput chemical screen was conducted to identify drugs that lower the toxicity of a protein containing the first exon of Huntington's disease (HD) protein huntingtin (HTT) harbouring 94 glutamines (Htt-Q94). Candidate drugs were tested in a wide range of in vitro and in vivo models of polyQ toxicity. FINDINGS: The chemical screen identified the anti-leprosy drug clofazimine as a hit, which was subsequently validated in several in vitro models. Computational analyses of transcriptional signatures revealed that the effect of clofazimine was due to the stimulation of mitochondrial biogenesis by peroxisome proliferator-activated receptor gamma (PPARγ). In agreement with this, clofazimine rescued mitochondrial dysfunction triggered by Htt-Q94 expression. Importantly, clofazimine also limited polyQ toxicity in developing zebrafish and neuron-specific worm models of polyQ disease. INTERPRETATION: Our results support the potential of repurposing the antimicrobial drug clofazimine for the treatment of polyQ diseases. FUNDING: A full list of funding sources can be found in the acknowledgments section.

Unraveling Transcriptome Profile, Epigenetic Dynamics, and Morphological Changes in Psoriasis-like Keratinocytes: "Insights into Similarity with Psoriatic Lesional Epidermis".

Keratinocytes are one of the primary cells affected by psoriasis inflammation. Our study aimed to delve deeper into their morphology, transcriptome, and epigenome changes in response to psoriasis-like inflammation. We created a novel cytokine mixture to mimic mild and severe psoriasis-like inflammatory conditions in cultured keratinocytes. Upon induction of inflammation, we observed that the keratinocytes exhibited a mesenchymal-like phenotype, further confirmed by increased VIM mRNA expression and results obtained from confocal microscopy. We performed RNA sequencing to achieve a more global view, revealing 858 and 6987 DEGs in mildly and severely inflamed keratinocytes, respectively. Surprisingly, we found that the transcriptome of mildly inflamed keratinocytes more closely mimicked that of the psoriatic epidermis transcriptome than the severely inflamed keratinocytes. Genes involved in the IL-17 pathway were a major contributor to the similarities of the transcriptomes between mildly inflamed KCs and psoriatic epidermis. Mild and severe inflammation led to the gene regulation of epigenetic modifiers such as HATs, HDACs, DNMTs, and TETs. Immunofluorescence staining revealed distinct 5-hmC patterns in inflamed versus control keratinocytes, and consistently low 5-mC intensity in both groups. However, the global DNA methylation assay detected a tendency of decreased 5-mC levels in inflamed keratinocytes versus controls. This study emphasizes how inflammation severity affects the transcriptomic similarity of keratinocytes to psoriatic epidermis and proves dynamic epigenetic regulation and adaptive morphological changes in inflamed keratinocytes.

Psoriasis-Associated Inflammatory Conditions Induce IL-23 mRNA Expression in Normal Human Epidermal Keratinocytes.

Psoriasis is a multifactorial, chronic inflammatory skin disease, the development of which is affected by both genetic and environmental factors. Cytosolic nucleic acid fragments, recognized as pathogen- and danger-associated molecular patterns, are highly abundant in psoriatic skin. It is known that psoriatic skin exhibits increased levels of IL-23 compared to healthy skin. However, the relationship between free nucleic acid levels and IL-23 expression has not been clarified yet. To examine a molecular mechanism by which nucleic acids potentially modulate IL-23 levels, an in vitro system was developed to investigate the IL-23 mRNA expression of normal human epidermal keratinocytes under psoriasis-like circumstances. This system was established using synthetic nucleic acid analogues (poly(dA:dT) and poly(I:C)). Signaling pathways, receptor involvement and the effect of PRINS, a long non-coding RNA previously identified and characterized by our research group, were analyzed to better understand the regulation of IL-23 in keratinocytes. Our results indicate that free nucleic acids regulate epithelial IL-23 mRNA expression through the TLR3 receptor and specific signaling pathways, thereby, contributing to the development of an inflammatory milieu favorable for the appearance of psoriatic symptoms. A moderate negative correlation was confirmed between the nucleic-acid-induced IL-23 mRNA level and the rate of its decrease upon PRINS overexpression.

Genetic Investigation of Inverse Psoriasis.

Inverse psoriasis is considered to be a rare variant of plaque-type psoriasis and is associated with significantly impaired quality of life. Clinical manifestations and treatment options are somewhat different for each subtype. Identifying genetic variants that contribute to the susceptibility of different types of psoriasis might improve understanding of the etiology of the disease. Since we have no current knowledge about the genetic background of inverse psoriasis, whole exome sequencing was used to comprehensively assess genetic variations in five patients with exclusively inverse lesions. We detected six potentially pathogenic rare (MAF < 0.01) sequence variants that occurred in all investigated patients. The corresponding mutated genes were FN1, FBLN1, MYH7B, MST1R, RHOD, and SCN10A. Several mutations identified in this study are known to cause disease, but roles in psoriasis or other papulosquamous diseases have not previously been reported. Interestingly, potentially causative variants of established psoriasis-susceptibility genes were not identified. These outcomes are in agreement with our hypothesis that the inverse subtype is a different entity from plaque-type psoriasis.

Exosomal long non-coding RNAs as biomarkers in human diseases.

The intensive study of extracellular vesicles was started about a decade ago revealing alterations of their amount and content to several cellular stimuli, highly depending on the releasing cell type. Exosomes, a type of extracellular vesicles, are released by every cell type and are present in most body fluids, what makes them attractive targets of biomarker research. Several studies have indicated that their content - including proteins and coding, as well as non-coding nucleic acids - could represent the disease state and serves as specific disease biomarkers. Out of these molecules, a special interest was gained by long non-coding RNAs (lncRNAs). Just as exosomes, lncRNAs are specific to their cell of origin and often specific to diseases, also found extracellularly, mainly contained in extracellular vesicles. Thus, recent efforts in biomarker research has turned to circulating exosomal lncRNAs, which might lead to the development of highly specific disease markers. Here we summarize the current knowledge on disease-associated exosomal long non-coding RNAs. The intensive studies in this area have revealed numerous potential targets for biomarkers, and highlighted the potential of their combination with other exosomal markers to represent a highly sensitive and specific diagnostic tool. However, we believe that additional functional data on both exosomes and lncRNAs are necessary for understanding their deregulation in diseases and developing their use as diagnostic approaches.

Nuclear Factor κB Activation in a Type V Pityriasis Rubra Pilaris Patient Harboring Multiple CARD14 Variants.

Pityriasis rubra pilaris (PRP) is a rare papulosquamous skin disorder, which is phenotypically related to psoriasis. Some familial PRP cases show autosomal dominant inheritance due to CARD14 mutations leading to increased nuclear factor κB (NFκB) activation. Moreover, CARD14 polymorphisms have also been implicated in sporadic PRP. A Hungarian PRP patient with childhood onset disease showing worsening of the symptoms in adulthood with poor therapeutic response underwent genetic screening for the CARD14 gene, revealing four genetic variants (rs117918077, rs2066964, rs28674001, and rs11652075). To confirm that the identified genetic variants would result in altered NFκB activity in the patient, functional studies were carried out. Immunofluorescent staining of the NFκB p65 subunit and NFκB-luciferase reporter assay demonstrated significantly increased NFκB activity in skin samples and keratinocytes from the PRP patient compared to healthy samples. Characterization of the cytokine profile of the keratinocytes and peripheral blood mononuclear cells demonstrated that the higher NFκB activation in PRP cells induces enhanced responses to inflammatory stimuli. These higher inflammatory reactions could not be explained solely by the observed CARD14 or other inflammation-related gene variants (determined by whole exome sequencing). Thus our study indicates the importance of investigations on other genetic factors related to PRP and their further functional characterization to bring us closer to the understanding of cellular and molecular background of disease pathogenesis.

The rs13388259 Intergenic Polymorphism in the Genomic Context of the BCYRN1 Gene Is Associated with Parkinson's Disease in the Hungarian Population.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by bradykinesia, resting tremor, and muscle rigidity. To date, approximately 50 genes have been implicated in PD pathogenesis, including both Mendelian genes with rare mutations and low-penetrance genes with common polymorphisms. Previous studies of low-penetrance genes focused on protein-coding genes, and less attention was given to long noncoding RNAs (lncRNAs). In this study, we aimed to investigate the susceptibility roles of lncRNA gene polymorphisms in the development of PD. Therefore, polymorphisms (n=15) of the PINK1-AS, UCHL1-AS, BCYRN1, SOX2-OT, ANRIL and HAR1A lncRNAs genes were genotyped in Hungarian PD patients (n=160) and age- and sex-matched controls (n=167). The rare allele of the rs13388259 intergenic polymorphism, located downstream of the BCYRN1 gene, was significantly more frequent among PD patients than control individuals (OR = 2.31; p=0.0015). In silico prediction suggested that this polymorphism is located in a noncoding region close to the binding site of the transcription factor HNF4A, which is a central regulatory hub gene that has been shown to be upregulated in the peripheral blood of PD patients. The rs13388259 polymorphism may interfere with the binding affinity of transcription factor HNF4A, potentially resulting in abnormal expression of target genes, such as BCYRN1.

Differential Inflammatory-Response Kinetics of Human Keratinocytes upon Cytosolic RNA- and DNA-Fragment Induction.

Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types-normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line-upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor α in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor κB (NF-κB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-κB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT).

PRINS Non-Coding RNA Regulates Nucleic Acid-Induced Innate Immune Responses of Human Keratinocytes.

Cytosolic DNA fragments are recognized as pathogen- and danger-associated molecular patterns that induce a cascade of innate immune responses. Moreover, excessive cytosolic DNA can enhance chronic inflammation, predominantly by activating inflammasomes, and thereby contributing to the pathogenesis of chronic diseases, such as psoriasis. Psoriasis associated non-protein coding RNA induced by stress (PRINS) is a long non-coding RNA, which has been shown to be associated with psoriasis susceptibility and cellular stress responses; however, the precise mechanism of its action has not been determined. Here, we provide evidence that, in addition to inflammasome activation, cytosolic DNA induces intracellular inflammatory reactions while decreasing PRINS gene expression. Furthermore, PRINS overexpression can ameliorate the inflammatory-mediator production of keratinocytes induced by cytosolic DNA. Overexpression of PRINS resulted in decreased interleukin-6 (IL-6) and chemokine (C-C motif) ligand 5 (CCL-5) expression and secretion. In silico analysis predicted direct binding sites between PRINS and the mRNAs, which was confirmed by an in vitro binding assay and on cellular level. Our results indicated that PRINS binds directly to the mRNAs of IL-6 and CCL-5 at specific binding sites and eventually destabilizes these mRNAs, leading to a decrease in their expression and secretion of the corresponding proteins. These results may indicate a restrictive role for PRINS in inflammatory processes.

Keratinocytes express functional CARD18, a negative regulator of inflammasome activation, and its altered expression in psoriasis may contribute to disease pathogenesis.

Caspase recruitment domain family member 18 (CARD18, Iceberg) is known as a negative regulatory molecule that inhibits inflammatory events by terminating inflammasome activation due to a direct interaction with pro-caspase-1. During the investigation of molecular mechanisms in keratinocytes that contribute to the pathogenesis of psoriasis, we found that CARD18 expression differs in healthy and psoriatic skin; moreover, CARD18 demonstrated altered response under inflammatory conditions in healthy and psoriatic skin. In healthy skin, low basal CARD18 expression was detected, which showed significant elevation in response to inflammatory stimuli (lymphokine treatment or mechanical injury). In contrast, higher basal expression was observed in psoriatic non-involved skin, but no further induction could be detected. We demonstrated that keratinocytes express CARD18 both at mRNA and protein levels and the expression increased in parallel with differentiation. The investigation of cellular inflammatory processes revealed that psoriasis-associated danger signals triggered the expression of inflammasome components (AIM2, Caspase-1) and CARD18 as well as IL-1ß production of keratinocytes. Furthermore, gene-specific silencing of CARD18 in cells treated with cytosolic DNA (poly(dA:dT)) resulted in increased IL-1ß secretion, suggesting a negative regulatory role for CARD18 in keratinocyte inflammatory signaling. The differential regulation of CARD18 in healthy and psoriatic uninvolved epidermis may contribute to the susceptibility of psoriasis. Furthermore, our in vitro results indicate that CARD18 may contribute to the fine tuning of keratinocyte innate immune processes.

Switching on RNA silencing suppressor activity by restoring argonaute binding to a viral protein.

We found that Sweet potato feathery mottle virus (SPFMV) P1, a close homologue of Sweet potato mild mottle virus P1, did not have any silencing suppressor activity. Remodeling the Argonaute (AGO) binding domain of SPFMV P1 by the introduction of two additional WG/GW motifs converted it to a silencing suppressor with AGO binding capacity. To our knowledge, this is the first instance of the transformation of a viral protein of unknown function to a functional silencing suppressor.

Expression and functional studies on the noncoding RNA, PRINS.

PRINS, a noncoding RNA identified earlier by our research group, contributes to psoriasis susceptibility and cellular stress response. We have now studied the cellular and histological distribution of PRINS by using in situ hybridization and demonstrated variable expressions in different human tissues and a consistent staining pattern in epidermal keratinocytes and in vitro cultured keratinocytes. To identify the cellular function(s) of PRINS, we searched for a direct interacting partner(s) of this stress-induced molecule. In HaCaT and NHEK cell lysates, the protein proved to be nucleophosmin (NPM) protein as a potential physical interactor with PRINS. Immunohistochemical experiments revealed an elevated expression of NPM in the dividing cells of the basal layers of psoriatic involved skin samples as compared with healthy and psoriatic uninvolved samples. Others have previously shown that NPM is a ubiquitously expressed nucleolar phosphoprotein which shuttles to the nucleoplasm after UV-B irradiation in fibroblasts and cancer cells. We detected a similar translocation of NPM in UV-B-irradiated cultured keratinocytes. The gene-specific silencing of PRINS resulted in the retention of NPM in the nucleolus of UV-B-irradiated keratinocytes; suggesting that PRINS may play a role in the NPM-mediated cellular stress response in the skin.

Neuroprotection with a new kynurenic acid analog in the four-vessel occlusion model of ischemia.

Global forebrain ischemia results in damage to the pyramids in the CA1 hippocampal subfield, which is particularly vulnerable to excitotoxic processes. Morphological and functional disintegration of this area leads to a cognitive dysfunction and neuropsychiatric disorders. Treatment with N-methyl-d-aspartate receptor antagonists is a widely accepted method with which to stop the advance of excitotoxic processes and concomitant neuronal death. From a clinical aspect, competitive glycine- and polyamine-site antagonists with relatively low affinity and moderate side-effects are taken into account. Endogenous kynurenic acid acts as an antagonist on the obligatory co-agonist glycine site, and has long been at the focus of neuroprotective trials. In the present study, we estimated the neuroprotective capability of a novel kynurenic acid analog in transient global forebrain ischemia, measuring the rate of hippocampal CA1 pyramidal cell loss and the preservation of long-term potentiation at Schaffer collateral-CA1 synapses. The neuroprotective potential was reflected by a significantly diminished hippocampal CA1 cell loss and preserved long-term potentiation expression. The neuroprotective effect was robust in the event of pretreatment, and also when the drug was administered at the time of reperfusion. This result is beneficial since a putative neuroprotectant proven to be effective as post-treatment is of much greater benefit.