RESUMO
Friedreich ataxia (FRDA) is a life-threatening hereditary ataxia; its incidence is 1:50,000 individuals in the Caucasian population. A unique therapeutic drug for FRDA, the antioxidant Omaveloxolone, has been recently approved by the US Food and Drug Administration (FDA). FRDA is a multi-systemic neurodegenerative disease; in addition to a progressive neurodegeneration, FRDA is characterized by hypertrophic cardiomyopathy, diabetes mellitus and musculoskeletal deformities. Cardiomyopathy is the predominant cause of premature death. The onset of FRDA typically occurs between the ages of 5 and 15. Given the complexity and heterogeneity of clinical features and the variability of their onset, the identification of biomarkers capable of assessing disease progression and monitoring the efficacy of treatments is essential to facilitate decision making in clinical practice. We conducted an RNA-seq analysis in peripheral blood mononuclear cells from FRDA patients and healthy donors, identifying a signature of small non-coding RNAs (sncRNAs) capable of distinguishing healthy individuals from the majority of FRDA patients. Among the differentially expressed sncRNAs, microRNAs are a class of small non-coding endogenous RNAs that regulate posttranscriptional silencing of target genes. In FRDA plasma samples, hsa-miR-148a-3p resulted significantly upregulated. The analysis of the Receiver Operating Characteristic (ROC) curve, combining the circulating expression levels of hsa-miR-148a-3p and hsa-miR-223-3p (previously identified by our group), revealed an Area Under the Curve (AUC) of 0.86 (95%, Confidence Interval 0.77-0.95; p-value < 0.0001). An in silico prediction analysis indicated that the IL6ST gene, an interesting marker of neuroinflammation in FRDA, is a common target gene of both miRNAs. Our findings support the evaluation of combined expression levels of different circulating miRNAs as potent epi-biomarkers in FRDA. Moreover, we found hsa-miR-148a-3p significantly over-expressed in Intermediate and Late-Onset Friedreich Ataxia patients' group (IOG and LOG, respectively) compared to healthy individuals, indicating it as a putative prognostic biomarker in this pathology.
Assuntos
Biomarcadores , Ataxia de Friedreich , MicroRNAs , Humanos , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Ataxia de Friedreich/sangue , MicroRNAs/genética , MicroRNAs/sangue , Masculino , Biomarcadores/sangue , Prognóstico , Feminino , Adulto , RNA-Seq , Adolescente , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Criança , Adulto Jovem , Pessoa de Meia-Idade , Pré-Escolar , Curva ROC , Estudos de Casos e ControlesRESUMO
Frataxin (FXN) deficiency is responsible for Friedreich's ataxia (FRDA) in which, besides the characteristic features of spinocerebellar ataxia, two thirds of patients develop hypertrophic cardiomyopathy that often progresses to heart failure and premature death. Different mechanisms might underlie FRDA pathogenesis. Among them, the role of miRNAs deserves investigations. We carried out an miRNA PCR-array analysis of plasma samples of early-, intermediate- and late-onset FRDA groups, defining a set of 30 differentially expressed miRNAs. Hsa-miR223-3p is the only miRNA shared between the three patient groups and appears upregulated in all of them. The up-regulation of hsa-miR223-3p was further validated in all enrolled patients (n = 37, Fc = +2.3; P < 0.0001). Using a receiver operating characteristic curve analysis, we quantified the predictive value of circulating hsa-miR223-3p for FRDA, obtaining an area under the ROC curve value of 0.835 (P < 0.0001) for all patients. Interestingly, we found a significant positive correlation between hsa-miR223-3p expression and cardiac parameters in typical FRDA patients (onset < 25 years). Moreover, a significant negative correlation between hsa-miR223-3p expression and HAX-1 (HCLS1-associated protein X-1) at mRNA and protein level was observed in all FRDA patients. In silico analyses suggested HAX-1 as a target gene of hsa-miR223-3p. Accordingly, we report that HAX-1 is negatively regulated by hsa-miR223-3p in cardiomyocytes (AC16) and neurons (SH-SY5Y), which are critically affected cell types in FRDA. This study describes for the first time the association between hsa-miR223-3p and HAX-1 expression in FRDA, thus supporting a potential role of this microRNA as non-invasive epigenetic biomarker for FRDA.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ataxia de Friedreich , MicroRNAs , Neuroblastoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Ataxia de Friedreich/patologia , Humanos , MicroRNAs/sangue , Miócitos Cardíacos/metabolismo , Neuroblastoma/metabolismo , RNA Mensageiro/genéticaRESUMO
BACKGROUND: The COVID-19 pandemic-related health crisis has imposed measures aimed at reducing the overcrowding of health facilities, by developing telemedicine and by forcing many sexually transmitted infection (STI) clinics to book appointments by telephone. In this work, we evaluate the performance of the nursing telephone triage system, introduced in the major STI center in Northwest Italy, for the adequacy of clinical pathways for of symptomatic STI patients. METHODS: From January to March 2021, all symptomatic patients wishing to access the CeMuSS center first underwent nurse-led telephone triage. Symptoms suggestive of STIs were further classified into four syndromic presentations: cutaneous neoformations, genital and oral ulcers, anogenital discharge, and finally other dermatological manifestations. All other clinical pictures were properly managed and eventually referred to other centers and not considered in the analysis. During the following medical examinations, the concordance between presumptive syndromic diagnosis and confirmed clinical diagnosis were recorded. Cohen k test was used to assess concordance. RESULTS: According to the Cohen k test, a good concordance between telephone presumptive diagnoses and medical clinical assessment was found (73.79% with a k = 0.611), whereas only a scarcely acceptable concordance between expected and real waiting time was established (75.51%, k = 0.34). CONCLUSIONS: Concordance between nursing syndromic diagnosis and syndromic medically confirmed diagnosis is good from a clinical point of view but there is a limitation when considering a public health perspective. An optimal training of nurses may improve the method of telephone triage. For future ongoing emergencies, the implementation of telemedicine with accurate patient management systems is mandatory.
Assuntos
COVID-19 , Saúde Sexual , Infecções Sexualmente Transmissíveis , Humanos , COVID-19/diagnóstico , COVID-19/epidemiologia , Triagem/métodos , Projetos Piloto , Pandemias , Infecções Sexualmente Transmissíveis/diagnóstico , Infecções Sexualmente Transmissíveis/epidemiologia , Telefone , HospitaisRESUMO
Friedreich's ataxia (FRDA) is a rare monogenic disease characterized by multisystem, slowly progressive degeneration. Because of the genetic defect in a non-coding region of FXN gene, FRDA cells exhibit severe deficit of frataxin protein levels. Hence, FRDA pathophysiology is characterized by a plethora of metabolic disruptions related to iron metabolism, mitochondrial homeostasis and oxidative stress. Importantly, an impairment of the antioxidant defences exacerbates the oxidative damage. This appears closely associated with the disablement of key antioxidant proteins, such as the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and the mitochondrial superoxide dismutase (MnSOD). The cytokine interferon gamma (IFN-γ) has been shown to increase frataxin expression in FRDA cells and to improve functional deficits in FRDA mice. Currently, IFN-γ represents a potential therapy under clinical evaluation in FRDA patients. Here, we show that IFN-γ induces a rapid expression of Nrf2 and MnSOD in different cell types, including FRDA patient-derived fibroblasts. Our data indicate that IFN-γ signals two separate pathways to enhance Nrf2 and MnSOD levels in FRDA fibroblasts. MnSOD expression increased through an early transcriptional regulation, whereas the levels of Nrf2 are induced by a post-transcriptional mechanism. We demonstrate that the treatment of FRDA fibroblasts with IFN-γ stimulates a non-canonical Nrf2 activation pathway through p21 and potentiates antioxidant responses under exposure to hydrogen peroxide. Moreover, IFN-γ significantly reduced the sensitivity to hydrogen peroxide-induced cell death in FRDA fibroblasts. Collectively, these results indicate the presence of multiple pathways triggered by IFN-γ with therapeutic relevance to FRDA.
Assuntos
Ataxia de Friedreich , Interferon gama , Animais , Camundongos , Interferon gama/farmacologia , Fator 2 Relacionado a NF-E2/genética , Antioxidantes/farmacologia , Ataxia de Friedreich/genética , Peróxido de Hidrogênio , Superóxido DismutaseRESUMO
Frataxin deficiency, responsible for Friedreich's ataxia (FRDA), is crucial for cell survival since it critically affects viability of neurons, pancreatic beta cells and cardiomyocytes. In FRDA, the heart is frequently affected with typical manifestation of hypertrophic cardiomyopathy, which can progress to heart failure and cause premature death. A microarray analysis performed on FRDA patient's lymphoblastoid cells stably reconstituted with frataxin, indicated HS-1-associated protein X-1 (HAX-1) as the most significantly upregulated transcript (FC = +2, P < 0.0006). quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and western blot analysis performed on (I) HEK293 stably transfected with empty vector compared to wild-type frataxin and (II) lymphoblasts from FRDA patients show that low frataxin mRNA and protein expression correspond to reduced levels of HAX-1. Frataxin overexpression and silencing were also performed in the AC16 human cardiomyocyte cell line. HAX-1 protein levels are indeed regulated through frataxin modulation. Moreover, correlation between frataxin and HAX-1 was further evaluated in peripheral blood mononuclear cells (PBMCs) from FRDA patients and from non-related healthy controls. A regression model for frataxin which included HAX-1, group membership and group* HAX-1 interaction revealed that frataxin and HAX-1 are associated both at mRNA and protein levels. Additionally, a linked expression of FXN, HAX-1 and antioxidant defence proteins MnSOD and Nrf2 was observed both in PBMCs and AC16 cardiomyocytes. Our results suggest that HAX-1 could be considered as a potential biomarker of cardiac disease in FRDA and the evaluation of its expression might provide insights into its pathogenesis as well as improving risk stratification strategies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cardiomiopatia Hipertrófica/patologia , Ataxia de Friedreich/complicações , Regulação da Expressão Gênica , Insuficiência Cardíaca/patologia , Proteínas de Ligação ao Ferro/metabolismo , Miócitos Cardíacos/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Cardiomiopatia Hipertrófica/etiologia , Cardiomiopatia Hipertrófica/metabolismo , Feminino , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Humanos , Proteínas de Ligação ao Ferro/genética , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Adulto Jovem , FrataxinaRESUMO
Four pre-exposure prophylaxis (PrEP) users with gastro-intestinal disorders (sleeve gastrectomy, terminal ileitis, celiac disease or chronic diarrhea) and receiving oral tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) were included. Despite a self-reported high adherence, trough plasma tenofovir concentrations (after a supervised intake) were significantly lower than those observed in PrEP recipients without gastrointestinal disorders [21 (±9.1) vs. 138 (±85) ng/mL]. PrEP users with gastrointestinal disorders may need increased TDF doses or alternative prophylactic measures.
RESUMO
Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases.
Assuntos
Ataxia de Friedreich/genética , Regulação da Expressão Gênica , Proteínas de Ligação ao Ferro/genética , Modelos Biológicos , RNA não Traduzido/metabolismo , Aconitato Hidratase/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Humanos , Linfócitos/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , FrataxinaRESUMO
BACKGROUND: Friedreich's ataxia is an autosomal-recessive cerebellar ataxia caused by mutation of the frataxin gene, resulting in decreased frataxin expression, mitochondrial dysfunction, and oxidative stress. Currently, no treatment is available for Friedreich's ataxia patients. Given that levels of residual frataxin critically affect disease severity, the main goal of a specific therapy for Friedreich's ataxia is to increase frataxin levels. OBJECTIVES: With the aim to accelerate the development of a new therapy for Friedreich's ataxia, we took a drug repositioning approach to identify market-available drugs able to increase frataxin levels. METHODS: Using a cell-based reporter assay to monitor variation in frataxin amount, we performed a high-throughput screening of a library containing 853 U.S. Food and Drug Administration-approved drugs. RESULTS: Among the potentially interesting candidates isolated from the screening, we focused our attention on etravirine, an antiviral drug currently in use as an anti-human immunodeficiency virus therapy. Here, we show that etravirine can promote a significant increase in frataxin levels in cells derived from Friedreich's ataxia patients, by enhancing frataxin messenger RNA translation. Importantly, frataxin accumulation in treated patient cell lines is comparable to frataxin levels in unaffected carrier cells, suggesting that etravirine could be therapeutically relevant. Indeed, etravirine treatment restores the activity of the iron-sulphur cluster containing enzyme aconitase and confers resistance to oxidative stress in cells derived from Friedreich's ataxia patients. CONCLUSIONS: Considering its excellent safety profile along with its ability to increase frataxin levels and correct some of the disease-related defects, etravirine represents a promising candidate as a therapeutic for Friedreich's ataxia. © 2019 International Parkinson and Movement Disorder Society.
Assuntos
Ataxia de Friedreich/tratamento farmacológico , Proteínas de Ligação ao Ferro/metabolismo , Piridazinas/uso terapêutico , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Humanos , Proteínas de Ligação ao Ferro/genética , Nitrilas , Pirimidinas , FrataxinaRESUMO
Defective expression of frataxin is responsible for the inherited, progressive degenerative disease Friedreich's Ataxia (FRDA). There is currently no effective approved treatment for FRDA and patients die prematurely. Defective frataxin expression causes critical metabolic changes, including redox imbalance and ATP deficiency. As these alterations are known to regulate the tyrosine kinase Src, we investigated whether Src might in turn affect frataxin expression. We found that frataxin can be phosphorylated by Src. Phosphorylation occurs primarily on Y118 and promotes frataxin ubiquitination, a signal for degradation. Accordingly, Src inhibitors induce accumulation of frataxin but are ineffective on a non-phosphorylatable frataxin-Y118F mutant. Importantly, all the Src inhibitors tested, some of them already in the clinic, increase frataxin expression and rescue the aconitase defect in frataxin-deficient cells derived from FRDA patients. Thus, Src inhibitors emerge as a new class of drugs able to promote frataxin accumulation, suggesting their possible use as therapeutics in FRDA.
Assuntos
Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/biossíntese , Quinases da Família src/genética , Trifosfato de Adenosina/deficiência , Trifosfato de Adenosina/genética , Inibidores Enzimáticos/farmacologia , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Ligação ao Ferro/genética , Oxirredução , Ubiquitinação/genética , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo , FrataxinaRESUMO
Friedreich's ataxia is an autosomal recessive progressive degenerative disorder caused by deficiency of the protein frataxin. The most common genetic cause is a homozygotic expansion of GAA triplets within intron 1 of the frataxin gene leading to impaired transcription. Preclinical in vivo and in vitro studies have shown that interferon gamma (IFNγ) is able to up-regulate the expression of frataxin gene in multiple cell types. We designed a phase IIa clinical trial, the first in Italy, aimed at assessing both safety and tolerability of IFNγ in Friedreich's patients and ability to increase frataxin levels in peripheral blood mononuclear cells. Nine patients (6 female and 3 males aged 21-38 years) with genetically confirmed disease were given 3 subcutaneous escalating doses (100, 150 and 200 µg) of IFNγ (human recombinant interferon 1 b gamma, trade name IMUKIN(®)), over 4 weeks. The primary end-point was the assessment of the safety and tolerability of IFNγ by means of standard clinical and hematological criteria. The secondary end-point was the detection of changes of frataxin levels in peripheral blood mononuclear cells after each single escalating dose of the drug. IFNγ was generally well tolerated, the main adverse event was hyperthermia/fever. Although, increases in frataxin levels could be detected in a minority of patients, these changes were not significant. A large phase III multicenter, randomized clinical trial with IFNγ in Friedreich's ataxia patients is currently ongoing. This study is expected to conclusively address the clinical efficacy of IFNγ therapy in patients with Friedreich's ataxia.
Assuntos
Ataxia de Friedreich/tratamento farmacológico , Interferon gama/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Adulto , Análise Química do Sangue , Esquema de Medicação , Feminino , Ataxia de Friedreich/sangue , Humanos , Interferon gama/efeitos adversos , Proteínas de Ligação ao Ferro/sangue , Itália , Masculino , Fármacos Neuroprotetores/efeitos adversos , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Resultado do Tratamento , Adulto Jovem , FrataxinaRESUMO
Friedreich ataxia is an inherited neurodegenerative disease that leads to progressive disability. There is currently no effective treatment and patients die prematurely. The underlying genetic defect leads to reduced expression of the mitochondrial protein frataxin. Frataxin insufficiency causes mitochondrial dysfunction and ultimately cell death, particularly in peripheral sensory ganglia. There is an inverse correlation between the amount of residual frataxin and the severity of disease progression; therefore, therapeutic approaches aiming at increasing frataxin levels are expected to improve patients' conditions. We previously discovered that a significant amount of frataxin precursor is degraded by the ubiquitin/proteasome system before its functional mitochondrial maturation. We also provided evidence for the therapeutic potential of small molecules that increase frataxin levels by docking on the frataxin ubiquitination site, thus preventing frataxin ubiquitination and degradation. We called these compounds ubiquitin-competing molecules (UCM). By extending our search for effective UCM, we identified a set of new and more potent compounds that more efficiently promote frataxin accumulation. Here we show that these compounds directly interact with frataxin and prevent its ubiquitination. Interestingly, these UCM are not effective on the ubiquitin-resistant frataxin mutant, indicating their specific action on preventing frataxin ubiquitination. Most importantly, these compounds are able to promote frataxin accumulation and aconitase rescue in cells derived from patients, strongly supporting their therapeutic potential.
Assuntos
Aconitato Hidratase/metabolismo , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Fármacos Neuroprotetores/farmacologia , Sítios de Ligação , Linhagem Celular , Desenho de Fármacos , Fluorescência , Células HEK293 , Humanos , Immunoblotting , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/genética , Simulação de Acoplamento Molecular , Mutação , Fármacos Neuroprotetores/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitinação/efeitos dos fármacos , FrataxinaRESUMO
The analyses of the insect species found on decomposing remains may provide useful information for the estimation of the minimum time elapsed since death and other parameters, such as causes and circumstances of death. The majority of research has focused on the early colonizing species, typically blowflies, while research concerning late colonizing insects is currently sparse. Dermestid beetles of the genus Dermestes L. (Coleoptera: Dermestidae) are one of the predominant insect species associated with decomposing remains during dry decay and skeletal stages of decomposition. In some dry environments, Dermestes species are likely to be the only necrophagous insects feeding on the decomposing remains. Furthermore, Dermestes species (immature and adults), their remains (cast skins and fecal material), and their artifacts (pupal chambers) are frequently found associated with ancient remains (e.g., mummies, fossils). Dermestes species have a worldwide distribution and are considered important in decomposition processes, forensic investigations, and economically as a known pest of stored products. Despite their recognized forensic importance, there is limited data documenting the ecology, biology, and the growth rates of the forensically relevant species. The aim of this review is to provide a comprehensive synopsis on the available literature concerning Dermestes species associated with forensic cases. In particular, aspects of colonization behavior, growth rates for forensic taxa and potential best practice guidelines for forensic casework encompassing late colonizing Dermestes species are discussed.
Assuntos
Besouros/fisiologia , Entomologia/métodos , Ciências Forenses/métodos , Animais , Cadáver , Besouros/crescimento & desenvolvimento , Ciências Forenses/normas , Humanos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Guias de Prática Clínica como Assunto/normas , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , ReproduçãoAssuntos
Autopsia , Betacoronavirus , Líquidos Corporais , Infecções por Coronavirus , Necrotério , Equipamento de Proteção Individual , Pneumonia Viral , Humanos , Autopsia/métodos , Autopsia/normas , Líquidos Corporais/virologia , Cadáver , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , COVID-19 , Pessoal de Saúde/normas , Itália/epidemiologia , Necrotério/métodos , Necrotério/normas , Pandemias , Equipamento de Proteção Individual/normas , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , SARS-CoV-2RESUMO
Friedreich's ataxia (FRDA) is the most common hereditary ataxia, affecting â¼3 in 100 000 individuals in Caucasian populations. It is caused by intronic GAA repeat expansions that hinder the expression of the FXN gene, resulting in defective levels of the mitochondrial protein frataxin. Sensory neurons in dorsal root ganglia (DRG) are particularly damaged by frataxin deficiency. There is no specific therapy for FRDA. Here, we show that frataxin levels can be upregulated by interferon gamma (IFNγ) in a variety of cell types, including primary cells derived from FRDA patients. IFNγ appears to act largely through a transcriptional mechanism on the FXN gene. Importantly, in vivo treatment with IFNγ increases frataxin expression in DRG neurons, prevents their pathological changes and ameliorates the sensorimotor performance in FRDA mice. These results disclose new roles for IFNγ in cellular metabolism and have direct implications for the treatment of FRDA.
Assuntos
Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Interferon gama/farmacologia , Interferon gama/fisiologia , Proteínas de Ligação ao Ferro/biossíntese , Animais , Células Cultivadas , Modelos Animais de Doenças , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/patologia , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Células HeLa , Humanos , Interferon gama/uso terapêutico , Proteínas de Ligação ao Ferro/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Transcrição Gênica , Ativação Transcricional , FrataxinaRESUMO
Friedreich's ataxia (FRDA) is a devastating orphan disease, with no specific treatment. The disease is caused by reduced expression of the protein frataxin, which results in mitochondrial defects and oxidative damage. Levels of residual frataxin critically affect onset and progression of the disease. Understanding the molecular mechanisms that regulate frataxin stability and degradation may, therefore, be exploited for the design of effective therapeutics. Here we show that frataxin is degraded by the ubiquitin-proteasome system and that K(147) is the critical residue responsible for frataxin ubiquitination and degradation. Accordingly, a K(147)R substitution generates a more stable frataxin. We then disclose a set of lead compounds, computationally selected to target the molecular cleft harboring K(147), that can prevent frataxin ubiquitination and degradation, and increase frataxin levels in cells derived from FRDA patients. Moreover, treatment with these compounds induces substantial recovery of aconitase activity and adenosine-5'-triphosphate levels in FRDA cells. Thus, we provide evidence for the therapeutic potential of directly interfering with the frataxin degradation pathway.
Assuntos
Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Aconitato Hidratase/genética , Aconitato Hidratase/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/genética , Células HEK293 , Humanos , Proteínas de Ligação ao Ferro/genética , Mutação de Sentido Incorreto , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitina/genética , FrataxinaRESUMO
Prion disease is a fatal neurodegenerative disorder characterized by accumulation of an abnormal prion protein (PrPSc) in the central nervous system. To identify PrPSc aggregates for diagnostic purposes, pathologists use immunohistochemical staining of prion protein antibodies on tissue samples. With digital pathology, artificial intelligence can now analyze stained slides. In this study, we developed an automated pipeline for the identification of PrPSc aggregates in tissue samples from the cerebellar and occipital cortex. To the best of our knowledge, this is the first framework to evaluate PrPSc deposition in digital images. We used two strategies: a deep learning segmentation approach using a vision transformer, and a machine learning classification approach with traditional classifiers. Our method was developed and tested on 64 whole slide images from 41 patients definitively diagnosed with prion disease. The results of our study demonstrated that our proposed framework can accurately classify WSIs from a blind test set. Moreover, it can quantify PrPSc distribution and localization throughout the brain. This could potentially be extended to evaluate protein expression in other neurodegenerative diseases like Alzheimer's and Parkinson's. Overall, our pipeline highlights the potential of AI-assisted pathology to provide valuable insights, leading to improved diagnostic accuracy and efficiency.
Assuntos
Doenças Priônicas , Proteínas Priônicas , Humanos , Proteínas Priônicas/metabolismo , Inteligência Artificial , Doenças Priônicas/diagnóstico , Doenças Priônicas/patologia , Encéfalo/metabolismo , Aprendizado de MáquinaRESUMO
The inability to produce normal levels of the mitochondrial protein frataxin causes the hereditary degenerative disorder Friedreich's Ataxia (FRDA), a syndrome characterized by progressive gait instability, cardiomyopathy and high incidence of diabetes. Frataxin is an iron-binding protein involved in the biogenesis of iron-sulfur clusters (ISC), prosthetic groups allowing essential cellular functions such as oxidative phosphorylation, enzyme catalysis and gene regulation. Although several evidence suggest that frataxin acts as an iron-chaperone within the mitochondrial compartment, we have recently demonstrated the existence of a functional extramitochondrial pool of mature frataxin in various human cell types. Here, we show that a similar proteolytic process generates both mature mitochondrial and extramitochondrial frataxin. To address the physiological function of human extramitochondrial frataxin, we searched for ISC-dependent interaction partners. We demonstrate that the extramitochondrial form of frataxin directly interacts with cytosolic aconitase/iron regulatory protein-1 (IRP1), a bifunctional protein alternating between an enzymatic and a RNA-binding function through the 'iron-sulfur switch' mechanism. Importantly, we found that the cytosolic aconitase defect and consequent IRP1 activation occurring in FRDA cells are reversed by the action of extramitochondrial frataxin. These results provide new insight into the control of cytosolic aconitase/IRP1 switch and expand current knowledge about the molecular pathogenesis of FRDA.
Assuntos
Aconitato Hidratase/metabolismo , Citosol/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteínas de Ligação ao Ferro/farmacologia , Aconitato Hidratase/genética , Células Cultivadas , Ataxia de Friedreich/genética , Regulação da Expressão Gênica , Humanos , Proteína 1 Reguladora do Ferro/genética , FrataxinaRESUMO
Friedreich ataxia is a rare neurodegenerative disorder caused by insufficient levels of the essential mitochondrial protein frataxin. It is a severely debilitating disease that significantly impacts the quality of life of affected patients and reduces their life expectancy, however, an adequate cure is not yet available for patients. Frataxin function, although not thoroughly elucidated, is associated with assembly of iron-sulfur cluster and iron metabolism, therefore insufficient frataxin levels lead to reduced activity of many mitochondrial enzymes involved in the electron transport chain, impaired mitochondrial metabolism, reduced ATP production and inefficient anti-oxidant response. As a consequence, neurons progressively die and patients progressively lose their ability to coordinate movement and perform daily activities. Therapeutic strategies aim at restoring sufficient frataxin levels or at correcting some of the downstream consequences of frataxin deficiency. However, the classical pathways of drug discovery are challenging, require a significant amount of resources and time to reach the final approval, and present a high failure rate. Drug repositioning represents a viable alternative to boost the identification of a therapy, particularly for rare diseases where resources are often limited. In this review we will describe recent efforts aimed at the identification of a therapy for Friedreich ataxia through drug repositioning, and discuss the limitation of such strategies.
RESUMO
Introduction and Aims: The increase in stress levels, social confinement, and addiction's physical consequences play an essential role in the proliferation of drug abuse. In this context, the Covid-19 pandemic produced remarkable effects on those individuals prone to addictions, especially to alcohol. Alcohol is linked to multiple dangerous conditions such as social issues, severe medical conditions, and road accidents. The determination of ethylglucuronide (EtG) in hair is frequently performed to test and monitor chronic excessive alcohol intake conditions, as it allows differentiation among low-risk/moderate drinkers, and excessive/chronic drinkers. Our study aimed to explore hair EtG levels in a controlled population to assess the impact of Covid-19 lockdown on alcohol intake along March-May 2020. Materials and Methods: EtG levels were measured in all hair samples collected in the months following April 2020 to evaluate the behaviors related to alcohol intake along with the time frame from March to May 2020. The measured concentration distributions for each month were compared with those reported in the same month during the previous 4 years (2016-2019). The dataset was built to highlight possible differences between genders, and the different categories of alcohol consumption, separately. Results: The samples collected from April to August 2020 (500 < N <1,100 per month) showed an increase in the percentage of subjects classified as abstinent/low-risk drinkers (from 60 up to 79%) and a decrease of subjects classified as moderate and chronic drinkers (-12 and -7%, respectively) when compared to the previous 4 years. A decrease in the overall mean value of EtG in the period April-June 2020 was observed, while the EtG levels of both June and July 2020 provided an increasing trend for chronic/excessive consumers (+27 and +19% for June and July 2020, respectively). A peculiar rise in the EtG levels of moderate and chronic/excessive female consumers was observed along April-June 2020, too. Discussion and Conclusions: Behavioral and social studies generally report a decrease in alcohol consumption during the Covid-19 lockdown. However, people already suffering from drug or alcohol addictions before Covid-19 pandemic seemingly enhance their harmful behavior. Our data from April to August 2020 are consistent with both suppositions. Our observations confirm once again the utility of EtG to investigate the patterns of alcohol consumption in the population.