RESUMO
Transposable elements (TEs) are active in neuronal cells raising the question whether TE insertions contribute to risk of neuropsychiatric disease. While genome-wide association studies (GWAS) serve as a tool to discover genetic loci associated with neuropsychiatric diseases, unfortunately GWAS do not directly detect structural variants such as TEs. To examine the role of TEs in psychiatric and neurologic disease, we evaluated 17,000 polymorphic TEs and find 76 are in linkage disequilibrium with disease haplotypes (P < 10-6 ) defined by GWAS. From these 76 polymorphic TEs, we identify potentially causal candidates based on having insertions in genomic regions of regulatory chromatin and on having associations with altered gene expression in brain tissues. We show that lead candidate insertions have regulatory effects on gene expression in human neural stem cells altering the activity of a minimal promoter. Taken together, we identify 10 polymorphic TE insertions that are potential candidates on par with other variants for having a causal role in neurologic and psychiatric disorders.
Assuntos
Transtornos Mentais , Retroelementos , Humanos , Retroelementos/genética , Estudo de Associação Genômica Ampla , Genoma , Loci Gênicos , Transtornos Mentais/genética , Elementos de DNA Transponíveis/genética , Evolução MolecularRESUMO
OBJECTIVE: Human endogenous retroviruses have been implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Expression of human endogenous retrovirus K (HERV-K) subtype HML-2 envelope (Env) in human neuronal cultures and in transgenic mice results in neurotoxicity and neurodegeneration, and mice expressing HML-2 Env display behavioral and neuromuscular characteristics resembling ALS. This study aims to characterize the neurotoxic properties of HML-2 Env. METHODS: Env neurotoxicity was detected in ALS cerebrospinal fluid and confirmed using recombinant Env protein in a cell-based assay and a mouse model. The mechanism of neurotoxicity was assessed with immunoprecipitation followed by mass spectrometry and Western blot, and by screening a panel of inhibitors. RESULTS: We observed that recombinant HML-2 Env protein caused neurotoxicity resulting in neuronal cell death, retraction of neurites, and decreased neuronal electrical activity. Injection of the Env protein into the brains of mice also resulted in neuronal cell death. HML-2 Env protein was also found in the cerebrospinal fluid of patients with sporadic ALS. The neurotoxic properties of the Env and the cerebrospinal fluid could be rescued with the anti-Env antibody. The Env was found to bind to CD98HC complexed to ß1 integrin on the neuronal cell surface. Using a panel of compounds to screen for their ability to block Env-induced neurotoxicity, we found that several compounds were protective and are linked to the ß1 integrin pathway. INTERPRETATION: HERV-K Env is released extracellularly in ALS and causes neurotoxicity via a novel mechanism. Present results pave the way for new treatment strategies in sporadic ALS. ANN NEUROL 2022;92:545-561.
Assuntos
Esclerose Lateral Amiotrófica , Retrovirus Endógenos , Esclerose Lateral Amiotrófica/genética , Animais , Produtos do Gene env , Humanos , Integrina beta1 , Camundongos , Camundongos TransgênicosRESUMO
Stem cells are capable of unlimited proliferation but can be induced to form brain cells. Factors that specifically regulate human development are poorly understood. We found that human stem cells expressed high levels of the envelope protein of an endogenized human-specific retrovirus (HERV-K, HML-2) from loci in chromosomes 12 and 19. The envelope protein was expressed on the cell membrane of the stem cells and was critical in maintaining the stemness via interactions with CD98HC, leading to triggering of human-specific signaling pathways involving mammalian target of rapamycin (mTOR) and lysophosphatidylcholine acyltransferase (LPCAT1)-mediated epigenetic changes. Down-regulation or epigenetic silencing of HML-2 env resulted in dissociation of the stem cell colonies and enhanced differentiation along neuronal pathways. Thus HML-2 regulation is critical for human embryonic and neurodevelopment, while it's dysregulation may play a role in tumorigenesis and neurodegeneration.
Assuntos
Diferenciação Celular , Retrovirus Endógenos/fisiologia , Neurônios/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Biomarcadores , Diferenciação Celular/genética , Autorrenovação Celular/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Ligação Proteica , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen â¼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.
Assuntos
Antivirais/análise , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Inibidores de Proteases/análise , Inibidores de Proteases/farmacologia , Zika virus/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Inteligência Artificial , Chlorocebus aethiops , Modelos Animais de Doenças , Imunocompetência , Concentração Inibidora 50 , Metaciclina/farmacologia , Camundongos Endogâmicos C57BL , Inibidores de Proteases/uso terapêutico , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas Pequenas , Células Vero , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/virologiaRESUMO
TAR DNA-binding protein 43 (TDP-43) proteinopathy in brain cells is the hallmark of amyotrophic lateral sclerosis (ALS) but its cause remains elusive. Asparaginase-like-1 protein (ASRGL1) cleaves isoaspartates, which alter protein folding and susceptibility to proteolysis. ASRGL1 gene harbors a copy of the human endogenous retrovirus HML-2, whose overexpression contributes to ALS pathogenesis. Here we show that ASRGL1 expression was diminished in ALS brain samples by RNA sequencing, immunohistochemistry, and western blotting. TDP-43 and ASRGL1 colocalized in neurons but, in the absence of ASRGL1, TDP-43 aggregated in the cytoplasm. TDP-43 was found to be prone to isoaspartate formation and a substrate for ASRGL1. ASRGL1 silencing triggered accumulation of misfolded, fragmented, phosphorylated and mislocalized TDP-43 in cultured neurons and motor cortex of female mice. Overexpression of ASRGL1 restored neuronal viability. Overexpression of HML-2 led to ASRGL1 silencing. Loss of ASRGL1 leading to TDP-43 aggregation may be a critical mechanism in ALS pathophysiology.
Assuntos
Esclerose Lateral Amiotrófica , Asparaginase , Proteínas de Ligação a DNA , Neurônios , Proteinopatias TDP-43 , Animais , Feminino , Humanos , Masculino , Camundongos , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Asparaginase/genética , Asparaginase/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Córtex Motor/metabolismo , Córtex Motor/patologia , Neurônios/metabolismo , Neurônios/patologia , Proteinopatias TDP-43/metabolismo , Proteinopatias TDP-43/patologia , Proteinopatias TDP-43/genética , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismoRESUMO
Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is poorly defined, the pathophysiology is unknown, and no disease-modifying treatments are available. We used rigorous criteria to recruit PI-ME/CFS participants with matched controls to conduct deep phenotyping. Among the many physical and cognitive complaints, one defining feature of PI-ME/CFS was an alteration of effort preference, rather than physical or central fatigue, due to dysfunction of integrative brain regions potentially associated with central catechol pathway dysregulation, with consequences on autonomic functioning and physical conditioning. Immune profiling suggested chronic antigenic stimulation with increase in naïve and decrease in switched memory B-cells. Alterations in gene expression profiles of peripheral blood mononuclear cells and metabolic pathways were consistent with cellular phenotypic studies and demonstrated differences according to sex. Together these clinical abnormalities and biomarker differences provide unique insight into the underlying pathophysiology of PI-ME/CFS, which may guide future intervention.
Assuntos
Doenças Transmissíveis , Síndrome de Fadiga Crônica , Humanos , Síndrome de Fadiga Crônica/metabolismo , Leucócitos Mononucleares/metabolismo , Doenças Transmissíveis/metabolismo , Biomarcadores/metabolismo , FenótipoRESUMO
Clinical trials in Parkinson's disease have shown that transplants of embryonic mesencephalic dopamine neurons form new functional connections within the host striatum, but the therapeutic benefits have been highly variable. One obstacle has been poor survival and integration of grafted dopamine neurons. Activation of Akt, a serine/threonine kinase that promotes cell survival and growth, increases the ability of neurons to survive after injury and to regenerate lost neuronal connections. Because the lipid phosphatase, phosphatase and tensin homolog (PTEN) inhibits Akt, we generated a mouse with conditional knock-out of PTEN in dopamine neurons, leading to constitutive expression of Akt in these neurons. Ventral mesencephalic tissue from dopamine phosphatase and tensin homologue knock-out or control animals was then transplanted bilaterally into the dopamine depleted striata of MitoPark mice that express a parkinsonian phenotype because of severe respiratory chain dysfunction in dopamine neurons. After transplantation into MitoPark mice, PTEN-deficient dopamine neurons were less susceptible to cell death, and exhibited a more extensive pattern of fibre outgrowth compared to control grafts. Voltammetric measurements demonstrated that dopamine release and reuptake were significantly increased in the striata of animals receiving dopamine PTEN knock-out transplants. These animals also displayed enhanced spontaneous and drug-induced locomotor activity, relative to control transplanted MitoPark mice. Our results suggest that disinhibition of the Akt-signalling pathway may provide a valuable strategy to enhance survival, function and integration of grafted dopamine neurons within the host striatum and, more generally, to improve survival and integration of different forms of neural grafts.
Assuntos
Sobrevivência Celular/genética , Neurônios Dopaminérgicos/transplante , Sobrevivência de Enxerto/genética , Mesencéfalo/transplante , Neuritos/metabolismo , PTEN Fosfo-Hidrolase/genética , Transtornos Parkinsonianos/cirurgia , Animais , Contagem de Células , Modelos Animais de Doenças , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Knockout , Atividade Motora/genética , PTEN Fosfo-Hidrolase/metabolismo , Transtornos Parkinsonianos/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
In the present study, we analyzed mice with a targeted deletion of ß-catenin in DA neurons (DA-ßcat KO mice) to address the functional significance of this molecule in the shaping of synaptic responses associated with motor learning and following exposure to drugs of abuse. Relative to controls, DA-ßcat KO mice showed significant deficits in their ability to form long-term memories and displayed reduced expression of methamphetamine-induced behavioral sensitization after subsequent challenge doses with this drug, suggesting that motor learning and drug-induced learning plasticity are altered in these mice. Morphological analyses showed no changes in the number or distribution of tyrosine hydroxylase-labeled neurons in the ventral midbrain. While electrochemical measurements in the striatum determined no changes in acute DA release and uptake, a small but significant decrease in DA release was detected in mutant animals after prolonged repetitive stimulation, suggesting a possible deficit in the DA neurotransmitter vesicle reserve pool. However, electron microscopy analyses did not reveal significant differences in the content of synaptic vesicles per terminal, and striatal DA levels were unchanged in DA-ßcat KO animals. In contrast, striatal mRNA levels for several markers known to regulate synaptic plasticity and DA neurotransmission were altered in DA-ßcat KO mice. This study demonstrates that ablation of ß-catenin in DA neurons leads to alterations of motor and reward-associated memories and to adaptations of the DA neurotransmitter system and suggests that ß-catenin signaling in DA neurons is required to facilitate the synaptic remodeling underlying the consolidation of long-term memories.
Assuntos
Inibidores da Captação de Dopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Deficiências da Aprendizagem/genética , Metanfetamina/farmacologia , Atividade Motora/efeitos dos fármacos , beta Catenina/deficiência , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Biofísica , Modelos Animais de Doenças , Estimulação Elétrica , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Força da Mão/fisiologia , Técnicas In Vitro , Locomoção/efeitos dos fármacos , Locomoção/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microdissecção , Atividade Motora/genética , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Teste de Desempenho do Rota-Rod , Substância Negra/citologia , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/citologia , beta Catenina/genéticaRESUMO
Prenatal Zika virus (ZIKV) infection is a serious global concern as it can lead to brain injury and many serious birth defects, collectively known as congenital Zika syndrome. Brain injury likely results from viral mediated toxicity in neural progenitor cells. Additionally, postnatal ZIKV infections have been linked to neurological complications, yet the mechanisms driving these manifestations are not well understood. Existing data suggest that the ZIKV envelope protein can persist in the central nervous system for extended periods of time, but it is unknown if this protein can independently contribute to neuronal toxicity. Here we find that the ZIKV envelope protein is neurotoxic, leading to overexpression of poly adenosine diphosphate -ribose polymerase 1, which can induce parthanatos. Together, these data suggest that neuronal toxicity resulting from the envelope protein may contribute to the pathogenesis of post-natal ZIKV-related neurologic complications.
Assuntos
Lesões Encefálicas , Doenças do Sistema Nervoso , Síndromes Neurotóxicas , Infecção por Zika virus , Zika virus , Gravidez , Feminino , Humanos , Zika virus/metabolismo , Infecção por Zika virus/complicações , Infecção por Zika virus/patologia , Proteínas do Envelope Viral/metabolismo , Neurônios/patologiaRESUMO
Genome-wide (GWAS) and copy number variant (CNV) association studies have reproducibly identified numerous risk alleles associated with bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SCZ), but biological characterization of these alleles lags gene discovery, owing to the inaccessibility of live human brain cells and inadequate animal models for human psychiatric conditions. Human-derived induced pluripotent stem cells (iPSCs) provide a renewable cellular reagent that can be differentiated into living, disease-relevant cells and 3D brain organoids carrying the full complement of genetic variants present in the donor germline. Experimental studies of iPSC-derived cells allow functional characterization of risk alleles, establishment of causal relationships between genes and neurobiology, and screening for novel therapeutics. Here we report the creation and availability of an iPSC resource comprising clinical, genomic, and cellular data obtained from genetically isolated families with BD and related conditions. Results from the first 324 study participants, 61 of whom have validated pluripotent clones, show enrichment of rare single nucleotide variants and CNVs overlapping many known risk genes and pathogenic CNVs. This growing iPSC resource is available to scientists pursuing functional genomic studies of BD and related conditions.
Assuntos
Transtorno Depressivo Maior , Células-Tronco Pluripotentes Induzidas , Transtornos Psicóticos , Esquizofrenia , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/metabolismo , Transtornos Psicóticos/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Genômica , Estudo de Associação Genômica AmplaRESUMO
The discovery of the ability to induce somatic cells to a pluripotent state through the overexpression of specific transcription factors has the potential to transform the ways in which pharmaceutical agents and cellular transplantation therapies are developed. Proper utilization of the technology to generate induced pluripotent stem cells (iPSCs) requires that researchers select the appropriate reprogramming method for generating iPSCs so that the resulting iPSCs can be transitioned towards clinical applications effectively. This article reviews all of the currently available reprogramming techniques with a focus on critiquing them on the basis of their utility in translational medicine.
Assuntos
Diferenciação Celular , Engenharia Celular/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Pesquisa Translacional Biomédica/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Medicina Regenerativa , Reprodutibilidade dos Testes , Transfecção , TransgenesRESUMO
There is a continuing unmet medical need to develop neuroprotective strategies to treat neurodegenerative disorders. To address this need, we screened over 2000 compounds for potential neuroprotective activity in a model of oxidative stress and found that numerous antifungal agents were neuroprotective. Of the identified compounds, fluconazole was further characterized. Fluconazole was able to prevent neurite retraction and cell death in in vitro and in vivo models of toxicity. Fluconazole protected neurons in a concentration-dependent manner and exhibited efficacy against several toxic agents, including 3-nitropropionic acid, N-methyl D-aspartate, 6-hydroxydopamine, and the HIV proteins Tat and gp120. In vivo studies indicated that systemically administered fluconazole was neuroprotective in animals treated with 3-nitropropionic acid and prevented gp120-mediated neuronal loss. In addition to neuroprotection, fluconazole also induced proliferation of neural progenitor cells in vitro and in vivo. Fluconazole mediates these effects through upregulation and signaling via the insulin growth factor-1 receptor which results in decreased cAMP production and increased phosphorylation of Akt. Blockade of the insulin growth factor-1 receptor signaling with the selective inhibitor AG1024 abrogated the effects of fluconazole. Our studies suggest that fluconazole may be an attractive candidate for treatment of neurodegenerative diseases due to its protective properties against several categories of neuronal insults and its ability to spur neural progenitor cell proliferation.
Assuntos
Insulinas , Doenças Neurodegenerativas , Fármacos Neuroprotetores , Animais , Receptor IGF Tipo 1/metabolismo , Neuroproteção , Fluconazol/farmacologia , Fármacos Neuroprotetores/farmacologia , Proteínas Proto-Oncogênicas c-akt , Oxidopamina , Antifúngicos , Ácido D-AspárticoRESUMO
IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the intraflagellar transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5 and 13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling.
Assuntos
Padronização Corporal , Encéfalo/embriologia , Cílios/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mamíferos/embriologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Biomarcadores/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Morte Celular , Cílios/ultraestrutura , Proteínas do Citoesqueleto , Embrião de Mamíferos/anormalidades , Endoderma/embriologia , Endoderma/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Proteínas Hedgehog/metabolismo , Holoprosencefalia/embriologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteína Nodal/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. Here we describe the generation of a recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain was developed using a simplified gene delivery system integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter into a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter, cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5 kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain showed that Cre DNA-mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows. Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages and in adult animals. Background recombination levels were low during development and most prominent in nervous tissue. Cre recombinase expression could not be effectively induced in adult animals. While rtTA mRNA levels were high in developmental and adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. The mouse strain described here provides a valuable tool to further analyze the function of genes during specific developmental windows, by allowing the effective inactivation of their function throughout defined stages of embryonic development.
Assuntos
Antibacterianos/farmacologia , Doxiciclina/farmacologia , Expressão Gênica/efeitos dos fármacos , Integrases/metabolismo , Proteínas/metabolismo , Recombinação Genética/efeitos dos fármacos , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Expressão Gênica/fisiologia , Integrases/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas/genética , RNA Mensageiro/metabolismo , RNA não Traduzido , Recombinação Genética/fisiologiaRESUMO
The Lass gene family contains a group of highly conserved genes that are found in eukaryotic species. The founding member, lag1, was discovered in a screen for yeast longevity genes. Subsequently, lag1 homologs were discovered in other organisms including six mammalian paralogs. All Lass genes encode a highly conserved Lag1 domain and many also have an additional Hox domain. Lass proteins are ceramide synthases and therefore are critical for ceramide biosynthesis. Ceramide synthase is also a critical enzyme in the sphingolipid biosynthetic pathway. As ceramide and sphingolipids are key intermediates in diverse cellular processes such as cell growth, apoptosis, and stress response and may also play a role in cancer development, the function of Lass proteins is of great interest. In this review, we summarize the state of knowledge regarding Lass protein structure, biological function, and their emerging role in cancer development.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Ceramidas/biossíntese , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias/metabolismo , Oxirredutases/metabolismo , Esfingosina N-Aciltransferase , Relação Estrutura-AtividadeRESUMO
Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.
Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Animais , Contagem de Células , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dimetil Sulfóxido/toxicidade , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacosRESUMO
The LIM-HD proteins interact with different cofactors, including Ssdp1 to regulate development in a diverse range of species. The single stranded DNA binding protein (Ssdp1) is a member of an evolutionarily conserved family of proteins that regulate critical transcriptional processes during embryonic development. Ssdp1 is localized predominantly in the cytoplasm of 293T cells but is translocated to the nucleus when co-transfected with Lck, a member of the Src family of non-receptor tyrosine kinases. The Src tyrosine kinase inhibitor PP2 blocked the nuclear translocation of Ssdp1. Western blot analysis showed that co-expression of Ssdp1 and Lck in 293T cells induces Ssdp1 phosphorylation. Mutation of the Ssdp1 N terminal tyrosine residues 23 and 25 markedly reduced both the phosphorylation and the nuclear localization of Ssdp1. Lck enhanced the transcriptional activity of Ssdp1 in the context of known components of a LIM-homeodomain (LIM-HD)/cofactor complex. We propose that phosphorylation involving N-terminal tyrosine residues of Ssdp1 is a means of regulating its nuclear localization and subsequent transcriptional activation of LIM-HD complexes.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Tirosina/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Mutação , Fosforilação , Ligação Proteica , Pirimidinas/farmacologia , Transdução de Sinais , Ativação TranscricionalRESUMO
Olive leaf extract (OLE) has been used traditionally as a herbal supplement since it contains polyphenolic compounds with beneficial properties ranging from increasing energy levels, lowering blood pressure, and supporting the cardiovascular and immune systems. In addition to the beneficial effects on human health, OLE also has antimicrobial properties. The aim of this work was to investigate the antimicrobial effect of OLE against major foodborne pathogens, including Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Enteritidis. Our results demonstrated that at a concentration of 62.5 mg/ml, OLE almost completely inhibited the growth of these three pathogens. In addition, OLE also reduced cell motility in L. monocytogenes, which correlated with the absence of flagella as shown by scanning electron microscopy. Moreover, OLE inhibited biofilm formation in L. monocytogenes and S. Enteritidis. Taken together, OLE, as a natural product, has the potential to be used as an antimicrobial to control foodborne pathogens.
RESUMO
UNLABELLED: Astrocytes are the predominant cell type in the nervous system and play a significant role in maintaining neuronal health and homeostasis. Recently, astrocyte dysfunction has been implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Astrocytes are thus an attractive new target for drug discovery for neurological disorders. Using astrocytes differentiated from human embryonic stem cells, we have developed an assay to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. This phenotypic oxidative stress assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, we identified a set of 22 that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress. Nine of these compounds were also found to be protective of induced pluripotent stem cell-differentiated astrocytes in a related assay. These compounds are thought to confer protection through hormesis, activating stress-response pathways and preconditioning astrocytes to handle subsequent exposure to hydrogen peroxide. In fact, four of these compounds were found to activate the antioxidant response element/nuclear factor-E2-related factor 2 pathway, a protective pathway induced by toxic insults. Our results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development. SIGNIFICANCE: Astrocytes play a key role in neurological diseases. Drug discovery efforts that target astrocytes can identify novel therapeutics. Human astrocytes are difficult to obtain and thus are challenging to use for high-throughput screening, which requires large numbers of cells. Using human embryonic stem cell-derived astrocytes and an optimized astrocyte differentiation protocol, it was possible to screen approximately 4,100 compounds in titration to identify 22 that are cytoprotective of astrocytes. This study is the largest-scale high-throughput screen conducted using human astrocytes, with a total of 17,536 data points collected in the primary screen. The results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development.
Assuntos
Antioxidantes/farmacologia , Astrócitos/efeitos dos fármacos , Descoberta de Drogas/métodos , Células-Tronco Embrionárias/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Neurogênese , Estresse Oxidativo/efeitos dos fármacos , Elementos de Resposta Antioxidante/efeitos dos fármacos , Astrócitos/metabolismo , Citoproteção , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Hep G2 , Humanos , Peróxido de Hidrogênio/toxicidade , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxidantes/farmacologia , Fenótipo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas PequenasRESUMO
Smith-Lemli-Opitz syndrome (SLOS) is a malformation disorder caused by mutations in DHCR7, which impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. SLOS results in cognitive impairment, behavioral abnormalities and nervous system defects, though neither affected cell types nor impaired signaling pathways are fully understood. Whether 7DHC accumulation or cholesterol loss is primarily responsible for disease pathogenesis is also unclear. Using induced pluripotent stem cells (iPSCs) from subjects with SLOS, we identified cellular defects that lead to precocious neuronal specification within SLOS derived neural progenitors. We also demonstrated that 7DHC accumulation, not cholesterol deficiency, is critical for SLOS-associated defects. We further identified downregulation of Wnt/ß-catenin signaling as a key initiator of aberrant SLOS iPSC differentiation through the direct inhibitory effects of 7DHC on the formation of an active Wnt receptor complex. Activation of canonical Wnt signaling prevented the neural phenotypes observed in SLOS iPSCs, suggesting that Wnt signaling may be a promising therapeutic target for SLOS.