RESUMO
Enduring patterns of epigenomic and transcriptional plasticity within the mesolimbic dopamine system contribute importantly to persistent behavioral adaptations that characterize substance use disorders (SUD). While drug addiction has long been thought of as a disorder of dopamine (DA) neurotransmission, therapeutic interventions targeting receptor mediated DA-signaling have not yet resulted in efficacious treatments. Our laboratory recently identified a non-canonical, neurotransmission-independent signaling moiety for DA in brain, termed dopaminylation, whereby DA itself acts as a donor source for the establishment of post-translational modifications (PTM) on substrate proteins (e.g., histone H3 at glutamine 5; H3Q5dop). In our previous studies, we demonstrated that H3Q5dop plays a critical role in the regulation of neuronal transcription and, when perturbed within monoaminergic neurons of the ventral tegmental area (VTA), critically contributes to pathological states, including relapse vulnerability to both psychostimulants (e.g., cocaine) and opiates (e.g., heroin). Importantly, H3Q5dop is also observed throughout the mesolimbic DA reward pathway (e.g., in nucleus accumbens/NAc and medial prefrontal cortex/mPFC, which receive DA input from VTA). As such, we investigated whether H3Q5dop may similarly be altered in its expression in response to drugs of abuse in these non-dopamine-producing regions. In rats undergoing extended abstinence from cocaine self-administration (SA), we observed both acute and prolonged accumulation of H3Q5dop in NAc, but not mPFC. Attenuation of H3Q5dop in NAc during drug abstinence reduced cocaine-seeking and affected cocaine-induced gene expression programs associated with altered dopamine signaling and neuronal function. These findings thus establish H3Q5dop in NAc, but not mPFC, as an important mediator of cocaine-induced behavioral and transcriptional plasticity during extended cocaine abstinence.
Assuntos
Cocaína , Ratos , Animais , Cocaína/farmacologia , Núcleo Accumbens/metabolismo , Histonas/metabolismo , Ratos Sprague-Dawley , Área Tegmentar Ventral/metabolismo , Dopamina/metabolismo , Córtex Pré-Frontal/metabolismoRESUMO
During lactation, changes in maternal calcium metabolism are necessary to provide adequate calcium for newborn skeletal development. The calcium in milk is derived from the maternal skeleton through a process thought to be mediated by the actions of parathyroid hormone-related protein (PTHrP) in combination with decreased circulating estrogen concentrations. After weaning, bone lost during lactation is rapidly regained. Most studies of bone metabolism in lactating women have been performed in Caucasian subjects. There are well-documented differences between African American (AA) and Caucasian (C) bone metabolism, including higher bone mineral density (BMD), lower risk of fracture, lower 25-hydroxyvitamin D (25(OH) D), and higher PTH in AA compared to C. In this prospective paired cohort study, BMD and markers of bone turnover were compared in self-identified AA and C mothers during lactation and after weaning. BMD decreased in both AA and C women during lactation, with similar decreases at the lumbar spine (LS) and greater bone loss in the C group at the femoral neck (FN) and total hip (TH), demonstrating that AA are not resistant to PTHrP during lactation. BMD recovery compared to the 2 week postpartum baseline was observed 6 months after weaning, though the C group did not have complete recovery at the FN. Increases in markers of bone formation and resorption during lactation were similar in AA and C. C-terminal telopeptide (CTX) decreased to 30% below post-pregnancy baseline in both groups 6 months after weaning, while procollagen type 1 N-terminal (P1NP) returned to baseline in the AA group and fell to below baseline in the C group. Further investigation is required to determine impacts on long term bone health for women who do not fully recover BMD before a subsequent pregnancy.
Assuntos
Densidade Óssea , Lactação , Feminino , Humanos , Recém-Nascido , Gravidez , Negro ou Afro-Americano , Cálcio , Cálcio da Dieta , Estudos de Coortes , Proteína Relacionada ao Hormônio Paratireóideo , Estudos Prospectivos , BrancosRESUMO
Relapse vulnerability in substance use disorder is attributed to persistent cue-induced drug seeking that intensifies (or "incubates") during drug abstinence. Incubated cocaine seeking has been observed in both humans with cocaine use disorder and in preclinical relapse models. This persistent relapse vulnerability is mediated by neuroadaptations in brain regions involved in reward and motivation. The dorsal hippocampus (DH) is involved in context-induced reinstatement of cocaine seeking but the role of the DH in cocaine seeking during prolonged abstinence has not been investigated. Here we found that transforming growth factor-ß (TGF-ß) superfamily member activin A is increased in the DH on abstinence day (AD) 30 but not AD1 following extended-access cocaine self-administration compared to saline controls. Moreover, activin A does not affect cocaine seeking on AD1 but regulates cocaine seeking on AD30 in a bidirectional manner. Next, we found that activin A regulates phosphorylation of NMDA receptor (NMDAR) subunit GluN2B and that GluN2B-containing NMDARs also regulate expression of cocaine seeking on AD30. Activin A and GluN2B-containing NMDARs have both previously been implicated in hippocampal synaptic plasticity. Therefore, we examined synaptic strength in the DH during prolonged abstinence and observed an increase in moderate long-term potentiation (LTP) in cocaine-treated rats compared to saline controls. Lastly, we examined the role of DH projections to the lateral septum (LS), a brain region implicated in cocaine seeking and found that DH projections to the LS govern cocaine seeking on AD30. Taken together, this study demonstrates a role for the DH in relapse behavior following prolonged abstinence from cocaine self-administration.
Assuntos
Comportamento de Procura de Droga/fisiologia , Hipocampo/metabolismo , Subunidades beta de Inibinas/metabolismo , Ativinas/metabolismo , Animais , Cocaína/farmacologia , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Extinção Psicológica/efeitos dos fármacos , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Fosforilação , Ratos , Ratos Sprague-Dawley , Recidiva , Autoadministração , Fator de Crescimento Transformador beta/metabolismoRESUMO
The numbers of insulin-secreting pancreatic beta cells are reduced in people with type 1 and type 2 diabetes. Driving beta cell regeneration in the pancreases of people with diabetes would be an attractive approach to reversing diabetes. While adult human beta cells have long been believed to be terminally differentiated and, therefore, irreversibly quiescent, it has become clear over recent years that this is not true. More specifically, both candidate and unbiased high-throughput screen approaches have revealed several classes of molecules that are clearly able to induce human beta cell proliferation. Here, we review recent approaches and accomplishments in human beta cell regenerative drug discovery. We also list the challenges that this rapidly moving field must confront to translate beta cell regenerative therapy from the laboratory to the clinic.
Assuntos
Descoberta de Drogas/métodos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Pâncreas/citologia , Pâncreas/metabolismo , Regeneração/fisiologiaRESUMO
Numerous compounds stimulate rodent ß-cell proliferation; however, translating these findings to human ß-cells remains a challenge. To examine human ß-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human ß-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled ß-cells with high specificity using both nuclear and cytoplasmic markers. ß-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1ß signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ ß-cells, whereas treatment with other compounds had limited to no effect on human ß-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human ß-cell proliferation, thus allowing for increased testing of candidate human ß-cell mitogens.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ativinas/farmacologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Adulto , Automação , Técnicas de Cultura de Células , Avaliação Pré-Clínica de Medicamentos , Eritropoetina/farmacologia , Exenatida , Feminino , GABAérgicos/farmacologia , Harmina/farmacologia , Humanos , Incretinas/farmacologia , Masculino , Pessoa de Meia-Idade , Inibidores da Monoaminoxidase/farmacologia , Miostatina/farmacologia , Nucleosídeos/farmacologia , Peptídeos/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Prolactina/farmacologia , Regeneração/efeitos dos fármacos , Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Vasodilatadores/farmacologia , Peçonhas/farmacologia , Adulto Jovem , Ácido gama-Aminobutírico/farmacologiaRESUMO
OBJECTIVE: All forms of diabetes result from insufficient functional ß-cell mass. Thus, achieving the therapeutic goal of expanding ß-cell mass requires a better mechanistic understanding of how ß-cells proliferate. Glucose is a natural ß-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood. METHODS: Here, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE. RESULTS: We found that ChREBP is essential for glucose-mediated transcriptional induction of Myc, and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1, 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc-Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc, as well as decreased glucose-stimulated ß-cell proliferation. CONCLUSIONS: The mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter.
Assuntos
Genes myc , Glucose , Animais , Ratos , Cromatina/genética , DNA , Glucose/metabolismo , RNA Guia de Sistemas CRISPR-Cas , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Proteínas Proto-Oncogênicas c-mycRESUMO
Five hundred thirty-seven million people globally suffer from diabetes. Insulin-producing ß cells are reduced in number in most people with diabetes, but most individuals still have some residual ß cells. However, none of the many diabetes drugs in common use increases human ß cell numbers. Recently, small molecules that inhibit dual tyrosine-regulated kinase 1A (DYRK1A) have been shown to induce immunohistochemical markers of human ß cell replication, and this is enhanced by drugs that stimulate the glucagon-like peptide 1 (GLP1) receptor (GLP1R) on ß cells. However, it remains to be demonstrated whether these immunohistochemical findings translate into an actual increase in human ß cell numbers in vivo. It is also unknown whether DYRK1A inhibitors together with GLP1R agonists (GLP1RAs) affect human ß cell survival. Here, using an optimized immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+) protocol in mouse kidneys bearing human islet grafts, we demonstrate that combination of a DYRK1A inhibitor with exendin-4 increases actual human ß cell mass in vivo by a mean of four- to sevenfold in diabetic and nondiabetic mice over 3 months and reverses diabetes, without alteration in human α cell mass. The augmentation in human ß cell mass occurred through mechanisms that included enhanced human ß cell proliferation, function, and survival. The increase in human ß cell survival was mediated, in part, by the islet prohormone VGF. Together, these findings demonstrate the therapeutic potential and favorable preclinical safety profile of the DYRK1A inhibitor-GLP1RA combination for diabetes treatment.
Assuntos
Quinases Dyrk , Exenatida , Harmina , Células Secretoras de Insulina , Peptídeos , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Animais , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Exenatida/farmacologia , Exenatida/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Harmina/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Camundongos , Peptídeos/farmacologia , Peptídeos/metabolismo , Peçonhas/farmacologia , Peçonhas/uso terapêutico , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Quimioterapia Combinada , Proliferação de Células/efeitos dos fármacos , XenoenxertosRESUMO
The small molecule DYRK1A inhibitor, harmine, induces human beta cell proliferation, expands beta cell mass, enhances expression of beta cell phenotypic genes, and improves human beta cell function i n vitro and in vivo . It is unknown whether the "pro-differentiation effect" is a DYRK1A inhibitor class-wide effect. Here we compare multiple commonly studied DYRK1A inhibitors. Harmine, 2-2c and 5-IT increase expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC2A2, SLC30A8, ENTPD3 in normal and T2D human islets. Unexpectedly, GNF4877, CC-401, INDY, CC-401 and Leucettine fail to induce expression of these essential beta cell molecules. Remarkably, the pro-differentiation effect is independent of DYRK1A inhibition: although silencing DYRK1A induces human beta cell proliferation, it has no effect on differentiation; conversely, harmine treatment enhances beta cell differentiation in DYRK1A-silenced islets. A careful screen of multiple DYRK1A inhibitor kinase candidate targets was unable to identify pro-differentiation pathways. Overall, harmine, 2-2c and 5-IT are unique among DYRK1A inhibitors in their ability to enhance both beta cell proliferation and differentiation. While beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor pharmaceutical development.
RESUMO
BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo. On the other hand, single-nucleus RNA sequencing (snRNA-seq) has compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and affords enhanced information from intronic sequences that can be leveraged to identify pre-mRNA transcripts. METHODS: We obtained nuclear preparations from fresh human islet cells and generated snRNA-seq datasets. We compared these datasets to scRNA-seq output obtained from human islet cells from the same donor. We employed snRNA-seq to obtain the transcriptomic profile of human islets engrafted in immunodeficient mice. In both analyses, we included the intronic reads in the snRNA-seq data with the GRCh38-2020-A library. RESULTS: First, snRNA-seq analysis shows that the top four differentially and selectively expressed genes in human islet endocrine cells in vitro and in vivo are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT (ß-cells); PTPRT, FAP, PDK4, LOXL4 (α-cells); LRFN5, ADARB2, ERBB4, KCNT2 (δ-cells); and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 (γ-cells). Second, by integrating information from scRNA-seq and snRNA-seq of human islet cells, we distinguish three ß-cell sub-clusters: an INS pre-mRNA cluster (ß3), an intermediate INS mRNA cluster (ß2), and an INS mRNA-rich cluster (ß1). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA-rich cluster (ß1) becomes the predominant sub-cluster in vivo. CONCLUSIONS: In summary, snRNA-seq and pre-mRNA analysis of human islet cells can accurately identify human islet cell populations, subpopulations, and their dynamic transcriptome profile in vivo.
Assuntos
Ilhotas Pancreáticas , Transcriptoma , Humanos , Camundongos , Animais , Perfilação da Expressão Gênica , Precursores de RNA/metabolismo , Ilhotas Pancreáticas/metabolismo , Análise de Sequência de RNA , RNA Nuclear Pequeno/metabolismo , RNA Mensageiro/metabolismo , Análise de Célula Única , Canais de Potássio Ativados por Sódio/genética , Canais de Potássio Ativados por Sódio/metabolismo , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
Mutations in CDKN1C, encoding p57KIP2, a canonical cell cycle inhibitor, underlie multiple pediatric endocrine syndromes. Despite this central role in disease, little is known about the structure and function of p57KIP2 in the human pancreatic beta cell. Since p57KIP2 is predominantly nuclear in human beta cells, we hypothesized that disease-causing mutations in its nuclear localization sequence (NLS) may correlate with abnormal phenotypes. We prepared RIP1 insulin promoter-driven adenoviruses encoding deletions of multiple disease-associated but unexplored regions of p57KIP2 and performed a comprehensive structure-function analysis of CDKN1C/p57KIP2. Real-time polymerase chain reaction and immunoblot analyses confirmed p57KIP2 overexpression, construct size, and beta cell specificity. By immunocytochemistry, wild-type (WT) p57KIP2 displayed nuclear localization. In contrast, deletion of a putative NLS at amino acids 278-281 failed to access the nucleus. Unexpectedly, we identified a second downstream NLS at amino acids 312-316. Further analysis showed that each individual NLS is required for nuclear localization, but neither alone is sufficient. In summary, p57KIP2 contains a classical bipartite NLS characterized by 2 clusters of positively charged amino acids separated by a proline-rich linker region. Variants in the sequences encoding these 2 NLS sequences account for functional p57KIP2 loss and beta cell expansion seen in human disease.
Assuntos
Inibidor de Quinase Dependente de Ciclina p57 , Células Secretoras de Insulina , Sinais de Localização Nuclear , Humanos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Núcleo Celular/metabolismo , Células Secretoras de Insulina/metabolismo , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genéticaRESUMO
Prior studies have shown that pancreatic α-cells can transdifferentiate into ß-cells, and that ß-cells de-differentiate and are prone to acquire an α-cell phenotype in type 2 diabetes (T2D). However, the specific human α-cell and ß-cell subtypes that are involved in α-to-ß-cell and ß-to-α-cell transitions are unknown. Here, we have integrated single cell RNA sequencing (scRNA-seq) and single nucleus RNA-seq (snRNA-seq) of isolated human islets and human islet grafts and provide additional insight into α-ß cell fate switching. Using this approach, we make seven novel observations. 1) There are five different GCG -expressing human α-cell subclusters [α1, α2, α-ß-transition 1 (AB-Tr1), α-ß-transition 2 (AB-Tr2), and α-ß (AB) cluster] with different transcriptome profiles in human islets from non-diabetic donors. 2) The AB subcluster displays multihormonal gene expression, inferred mostly from snRNA-seq data suggesting identification by pre-mRNA expression. 3) The α1, α2, AB-Tr1, and AB-Tr2 subclusters are enriched in genes specific for α-cell function while AB cells are enriched in genes related to pancreatic progenitor and ß-cell pathways; 4) Trajectory inference analysis of extracted α- and ß-cell clusters and RNA velocity/PAGA analysis suggests a bifurcate transition potential for AB towards both α- and ß-cells. 5) Gene commonality analysis identifies ZNF385D, TRPM3, CASR, MEG3 and HDAC9 as signature for trajectories moving towards ß-cells and SMOC1, PLCE1, PAPPA2, ZNF331, ALDH1A1, SLC30A8, BTG2, TM4SF4, NR4A1 and PSCK2 as signature for trajectories moving towards α-cells. 6) Remarkably, in contrast to the events in vitro , the AB subcluster is not identified in vivo in human islet grafts and trajectory inference analysis suggests only unidirectional transition from α-to-ß-cells in vivo . 7) Analysis of scRNA-seq datasets from adult human T2D donor islets reveals a clear unidirectional transition from ß-to-α-cells compatible with dedifferentiation or conversion into α-cells. Collectively, these studies show that snRNA-seq and scRNA-seq can be leveraged to identify transitions in the transcriptional status among human islet endocrine cell subpopulations in vitro , in vivo , in non-diabetes and in T2D. They reveal the potential gene signatures for common trajectories involved in interconversion between α- and ß-cells and highlight the utility and power of studying single nuclear transcriptomes of human islets in vivo . Most importantly, they illustrate the importance of studying human islets in their natural in vivo setting.
RESUMO
Senescent cells are a major contributor to age-dependent cardiovascular tissue dysfunction, but knowledge of their in vivo cell markers and tissue context is lacking. To reveal tissue-relevant senescence biology, we integrate the transcriptomes of 10 experimental senescence cell models with a 224 multi-tissue gene co-expression network based on RNA-seq data of seven tissues biopsies from â¼600 coronary artery disease (CAD) patients. We identify 56 senescence-associated modules, many enriched in CAD GWAS genes and correlated with cardiometabolic traits-which supports universality of senescence gene programs across tissues and in CAD. Cross-tissue network analyses reveal 86 candidate senescence-associated secretory phenotype (SASP) factors, including COL6A3. Experimental knockdown of COL6A3 induces transcriptional changes that overlap the majority of the experimental senescence models, with cell-cycle arrest linked to modulation of DREAM complex-targeted genes. We provide a transcriptomic resource for cellular senescence and identify candidate biomarkers, SASP factors, and potential drivers of senescence in human tissues.
Assuntos
Senescência Celular , Transcriptoma , Humanos , Transcriptoma/genética , Senescência Celular/genética , Fenótipo , Biomarcadores , Colágeno , Colágeno Tipo VI/genéticaRESUMO
Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.
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BACKGROUND: Thyroid papillary microcarcinoma (TPMC) is an incidentally discovered papillary carcinoma that measures ≤1.0 cm in size. Most TPMCs are indolent, whereas some behave aggressively. The objective of the study was to evaluate whether the combination of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and specific histopathologic features allows risk stratification of TPMC. METHODS: A group aggressive TPMCs was selected based on the presence of lymph node metastasis or tumor recurrence. Another group of nonaggressive tumors included TPMCs matched with the first group for age, sex, and tumor size, but with no extrathyroid spread. A molecular analysis was performed, and histologic slides were scored for multiple histopathologic criteria. A separate validation cohort of 40 TPMCs was evaluated. RESULTS: BRAF mutations were detected in 77% of aggressive TPMCs and in 32% of nonaggressive tumors (P = .001). Several histopathologic features differed significantly between the groups. By using multivariate regression analysis, a molecular-pathologic (MP) score was developed that included BRAF status and 3 histopathologic features: superficial tumor location, intraglandular tumor spread/multifocality, and tumor fibrosis. By adding the histologic criteria to BRAF status, sensitivity was increased from 77% to 96%, and specificity was increased from 68% to 80%. In the independent validation cohort, the MP score stratified tumors into low-risk, moderate-risk, and high-risk groups with the probability of lymph node metastases or tumor recurrence in 0%, 20%, and 60% of patients, respectively. CONCLUSIONS: BRAF status together with several histopathologic features allowed clinical risk stratification of TPMCs. The combined MP risk stratification model was a better predictor of extrathyroid tumor spread than either mutation or histopathologic findings alone.
Assuntos
Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Medição de Risco/métodos , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Carcinoma , Carcinoma Papilar , Humanos , Metástase Linfática , Recidiva , Câncer Papilífero da TireoideRESUMO
Persistent transcriptional events in ventral tegmental area (VTA) and other reward relevant brain regions contribute to enduring behavioral adaptations that characterize substance use disorder. Recent data from our laboratory indicate that aberrant accumulation of the newly discovered histone post-translational modification (PTM), H3 dopaminylation at glutamine 5 (H3Q5dop), contributes significantly to cocaine-seeking behavior following prolonged periods of abstinence. It remained unclear, however, whether this modification is important for relapse vulnerability in the context of other drugs of abuse, such as opioids. Here, we showed that H3Q5dop plays a critical role in heroin-mediated transcriptional plasticity in midbrain regions, particularly the VTA. In rats undergoing abstinence from heroin self-administration (SA), we found acute and persistent accumulation of H3Q5dop in VTA. Attenuation of H3Q5dop during abstinence induced persistent changes in gene expression programs associated with neuronal signaling and dopaminergic function in heroin abstinence and led to reduced heroin-seeking behavior. Interestingly, the observed changes in molecular pathways after heroin SA showed significant yet reversed overlap with the same genes altered in cocaine SA. These findings establish an essential role for H3Q5dop, and its downstream transcriptional consequences, in heroin-induced functional plasticity in VTA.
Assuntos
Transtornos Relacionados ao Uso de Cocaína , Cocaína , Animais , Cocaína/farmacologia , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Heroína/farmacologia , Histonas/metabolismo , Masculino , Ratos , Autoadministração , Área Tegmentar Ventral/metabolismoRESUMO
Resistance to regeneration of insulin-producing pancreatic ß cells is a fundamental challenge for type 1 and type 2 diabetes. Recently, small molecule inhibitors of the kinase DYRK1A have proven effective in inducing adult human ß cells to proliferate, but their detailed mechanism of action is incompletely understood. We interrogated our human insulinoma and ß cell transcriptomic databases seeking to understand why ß cells in insulinomas proliferate, while normal ß cells do not. This search reveals the DREAM complex as a central regulator of quiescence in human ß cells. The DREAM complex consists of a module of transcriptionally repressive proteins that assemble in response to DYRK1A kinase activity, thereby inducing and maintaining cellular quiescence. In the absence of DYRK1A, DREAM subunits reassemble into the pro-proliferative MMB complex. Here, we demonstrate that small molecule DYRK1A inhibitors induce human ß cells to replicate by converting the repressive DREAM complex to its pro-proliferative MMB conformation.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Insulinoma , Neoplasias Pancreáticas , Adulto , Proliferação de Células , Humanos , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/metabolismoRESUMO
Preservation and expansion of ß-cell mass is a therapeutic goal for diabetes. Here we show that the hyperactive isoform of carbohydrate response-element binding protein (ChREBPß) is a nuclear effector of hyperglycemic stress occurring in ß-cells in response to prolonged glucose exposure, high-fat diet, and diabetes. We show that transient positive feedback induction of ChREBPß is necessary for adaptive ß-cell expansion in response to metabolic challenges. Conversely, chronic excessive ß-cell-specific overexpression of ChREBPß results in loss of ß-cell identity, apoptosis, loss of ß-cell mass, and diabetes. Furthermore, ß-cell "glucolipotoxicity" can be prevented by deletion of ChREBPß. Moreover, ChREBPß-mediated cell death is mitigated by overexpression of the alternate CHREBP gene product, ChREBPα, or by activation of the antioxidant Nrf2 pathway in rodent and human ß-cells. We conclude that ChREBPß, whether adaptive or maladaptive, is an important determinant of ß-cell fate and a potential target for the preservation of ß-cell mass in diabetes.
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Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Células Secretoras de Insulina , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Retroalimentação , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
Substance use disorders (SUDs) are chronic brain diseases characterized by transitions from recreational to compulsive drug use and aberrant drug craving that persists for months to years after abstinence is achieved. The transition to compulsive drug use implies that plasticity is occurring, altering the physiology of the brain to precipitate addicted states. Epigenetic phenomena represent a varied orchestra of transcriptional tuning mechanisms that, in response to environmental stimuli, create and maintain gene expression-mediated physiological outcomes. Therefore, epigenetic mechanisms represent a convergent regulatory framework through which the plasticity required to achieve an addicted state can arise and then persist long after drug use has ended. In the first section, we will introduce basic concepts in epigenetics, such as chromatin architecture, histones and their posttranslational modifications, DNA methylation, noncoding RNAs, and transcription factors, along with methods for their investigation. We will then examine the implications of these mechanisms in SUDs, with a particular focus on cocaine-mediated neuroepigenetic plasticity across multiple behavioral models of addiction.
Assuntos
Epigênese Genética , Transtornos Relacionados ao Uso de Substâncias/genética , HumanosRESUMO
According to the World Health Organization (WHO), 422 million people are suffering from diabetes worldwide. Current diabetes therapies are focused on optimizing blood glucose control to prevent long-term diabetes complications. Unfortunately, current therapies have failed to achieve glycemic targets in the majority of people with diabetes. In this context, regeneration of functional insulin-producing human ß-cells in people with diabetes through the use of DYRK1A inhibitor drugs has recently received special attention. Several small molecule DYRK1A inhibitors have been identified that induce human ß-cell proliferation in vitro and in vivo. Furthermore, DYRK1A inhibitors have also been shown to synergize ß-cell proliferation with other classes of drugs, such as TGFß inhibitors and GLP-1 receptor agonists. In this perspective, we review the status of DYRK1A as a therapeutic target for ß-cell proliferation and provide perspectives on technical and scientific challenges for future translational development.
Assuntos
Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus/tratamento farmacológico , Células Secretoras de Insulina/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Diabetes Mellitus/metabolismo , Descoberta de Drogas , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinases DyrkRESUMO
A quantitative deficiency of normally functioning insulin-producing pancreatic beta cells is a major contributor to all common forms of diabetes. This is the underlying premise for attempts to replace beta cells in people with diabetes by pancreas transplantation, pancreatic islet transplantation, and transplantation of beta cells or pancreatic islets derived from human stem cells. While progress is rapid and impressive in the beta cell replacement field, these approaches are expensive, and for transplant approaches, limited by donor organ availability. For these reasons, beta cell replacement will not likely become available to the hundreds of millions of people around the world with diabetes. Since the large majority of people with diabetes have some residual beta cells in their pancreata, an alternate approach to reversing diabetes would be developing pharmacologic approaches to induce these residual beta cells to regenerate and expand in a way that also permits normal function. Unfortunately, despite the broad availability of multiple classes of diabetes drugs in the current diabetes armamentarium, none has the ability to induce regeneration or expansion of human beta cells. Development of such drugs would be transformative for diabetes care around the world. This picture has begun to change. Over the past half-decade, a novel class of beta cell regenerative small molecules has emerged: the DYRK1A inhibitors. Their emergence has tremendous potential, but many areas of uncertainty and challenge remain. In this review, we summarize the accomplishments in the world of beta cell regenerative drug development and summarize areas in which most experts would agree. We also outline and summarize areas of disagreement or lack of unanimity, of controversy in the field, of obstacles to beta cell regeneration, and of challenges that will need to be overcome in order to establish human beta cell regenerative drug therapeutics as a clinically viable class of diabetes drugs.