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1.
Learn Mem ; 31(5)2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38876485

RESUMO

The common fruit fly Drosophila melanogaster provides a powerful platform to investigate the genetic, molecular, cellular, and neural circuit mechanisms of behavior. Research in this model system has shed light on multiple aspects of brain physiology and behavior, from fundamental neuronal function to complex behaviors. A major anatomical region that modulates complex behaviors is the mushroom body (MB). The MB integrates multimodal sensory information and is involved in behaviors ranging from sensory processing/responses to learning and memory. Many genes that underlie brain disorders are conserved, from flies to humans, and studies in Drosophila have contributed significantly to our understanding of the mechanisms of brain disorders. Genetic mutations that mimic human diseases-such as Fragile X syndrome, neurofibromatosis type 1, Parkinson's disease, and Alzheimer's disease-affect MB structure and function, altering behavior. Studies dissecting the effects of disease-causing mutations in the MB have identified key pathological mechanisms, and the development of a complete connectome promises to add a comprehensive anatomical framework for disease modeling. Here, we review Drosophila models of human neurodevelopmental and neurodegenerative disorders via the effects of their underlying mutations on MB structure, function, and the resulting behavioral alterations.


Assuntos
Modelos Animais de Doenças , Corpos Pedunculados , Doenças Neurodegenerativas , Transtornos do Neurodesenvolvimento , Animais , Corpos Pedunculados/fisiologia , Doenças Neurodegenerativas/fisiopatologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/fisiopatologia , Drosophila melanogaster , Humanos , Drosophila
2.
Learn Mem ; 31(5)2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38862172

RESUMO

How does the brain translate sensory information into complex behaviors? With relatively small neuronal numbers, readable behavioral outputs, and an unparalleled genetic toolkit, the Drosophila mushroom body (MB) offers an excellent model to address this question in the context of associative learning and memory. Recent technological breakthroughs, such as the freshly completed full-brain connectome, multiomics approaches, CRISPR-mediated gene editing, and machine learning techniques, led to major advancements in our understanding of the MB circuit at the molecular, structural, physiological, and functional levels. Despite significant progress in individual MB areas, the field still faces the fundamental challenge of resolving how these different levels combine and interact to ultimately control the behavior of an individual fly. In this review, we discuss various aspects of MB research, with a focus on the current knowledge gaps, and an outlook on the future methodological developments required to reach an overall view of the neurobiological basis of learning and memory.


Assuntos
Drosophila , Corpos Pedunculados , Corpos Pedunculados/fisiologia , Animais , Drosophila/fisiologia , Memória/fisiologia , Aprendizagem por Associação/fisiologia
3.
BMC Neurosci ; 25(1): 12, 2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38438989

RESUMO

BACKGROUND: Mutations in the gene DISC1 are associated with increased risk for schizophrenia, bipolar disorder and major depression. The study of mutated DISC1 represents a well-known and comprehensively characterized approach to understand neuropsychiatric disease mechanisms. However, previous studies have mainly used animal models or rather heterogeneous populations of iPSC-derived neurons, generated by undirected differentiation, to study the effects of DISC1 disruption. Since major hypotheses to explain neurodevelopmental, psychiatric disorders rely on altered neuronal connectivity observed in patients, an ideal iPSC-based model requires accurate representation of the structure and complexity of neuronal circuitries. In this study, we made use of an isogenic cell line with a mutation in DISC1 to study neuronal synaptic phenotypes in a culture system comprising a defined ratio of NGN2 and ASCL1/DLX2 (AD2)-transduced neurons, enriched for glutamatergic and GABAergic neurons, respectively, to mimic properties of the cortical microcircuitry. RESULTS: In heterozygous DISC1 mutant neurons, we replicated the expected phenotypes including altered neural progenitor proliferation as well as neurite outgrowth, deregulated DISC1-associated signaling pathways, and reduced synaptic densities in cultures composed of glutamatergic neurons. Cultures comprising a defined ratio of NGN2 and AD2 neurons then revealed considerably increased GABAergic synapse densities, which have not been observed in any iPSC-derived model so far. Increased inhibitory synapse densities could be associated with an increased efficiency of GABAergic differentiation, which we observed in AD2-transduced cultures of mutant neurons. Additionally, we found increased neuronal activity in GABAergic neurons through calcium imaging while the activity pattern of glutamatergic neurons remained unchanged. CONCLUSIONS: In conclusion, our results demonstrate phenotypic differences in a co-culture comprising a defined ratio of DISC1 mutant NGN2 and AD2 neurons, as compared to culture models comprising only one neuronal cell type. Altered synapse numbers and neuronal activity imply that DISC1 impacts the excitatory/inhibitory balance in NGN2/AD2 co-cultures, mainly through increased GABAergic input.


Assuntos
Transtorno Bipolar , Transtorno Depressivo Maior , Animais , Humanos , Técnicas de Cocultura , Neurônios GABAérgicos , Mutação , Proteínas do Tecido Nervoso/genética
4.
Arch Toxicol ; 98(9): 2919-2935, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38832940

RESUMO

Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Ácido Okadáico , Proteômica , Ácido Okadáico/toxicidade , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Linhagem Celular Tumoral , Carcinoma Hepatocelular/metabolismo , Fosforilação , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Transcriptoma/efeitos dos fármacos , Toxinas Marinhas , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Multiômica
5.
BMC Genomics ; 24(1): 702, 2023 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-37993800

RESUMO

Animals typically have either compound eyes, or camera-type eyes, both of which have evolved repeatedly in the animal kingdom. Both eye types include two important kinds of cells: photoreceptor cells, which can be excited by light, and non-neuronal support cells (SupCs), which provide essential support to photoreceptors. At the molecular level deeply conserved genes that relate to the differentiation of photoreceptor cells have fueled a discussion on whether or not a shared evolutionary origin might be considered for this cell type. In contrast, only a handful of studies, primarily on the compound eyes of Drosophila melanogaster, have demonstrated molecular similarities in SupCs. D. melanogaster SupCs (Semper cells and primary pigment cells) are specialized eye glia that share several molecular similarities with certain vertebrate eye glia, including Müller glia. This led us to question if there could be conserved molecular signatures of SupCs, even in functionally different eyes such as the image-forming larval camera eyes of the sunburst diving beetle Thermonectus marmoratus. To investigate this possibility, we used an in-depth comparative whole-tissue transcriptomics approach. Specifically, we dissected the larval principal camera eyes into SupC- and retina-containing regions and generated the respective transcriptomes. Our analysis revealed several common features of SupCs including enrichment of genes that are important for glial function (e.g. gap junction proteins such as innexin 3), glycogen production (glycogenin), and energy metabolism (glutamine synthetase 1 and 2). To evaluate similarities, we compared our transcriptomes with those of fly (Semper cells) and vertebrate (Müller glia) eye glia as well as respective retinas. T. marmoratus SupCs were found to have distinct genetic overlap with both fly and vertebrate eye glia. These results suggest that T. marmoratus SupCs are a form of glia, and like photoreceptors, may be deeply conserved.


Assuntos
Besouros , Drosophila melanogaster , Animais , Drosophila melanogaster/genética , Besouros/genética , Larva/genética , Retina , Neuroglia/metabolismo
6.
Proc Natl Acad Sci U S A ; 115(3): E448-E457, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29284750

RESUMO

Learning and memory rely on dopamine and downstream cAMP-dependent plasticity across diverse organisms. Despite the central role of cAMP signaling, it is not known how cAMP-dependent plasticity drives coherent changes in neuronal physiology that encode the memory trace, or engram. In Drosophila, the mushroom body (MB) is critically involved in olfactory classical conditioning, and cAMP signaling molecules are necessary and sufficient for normal memory in intrinsic MB neurons. To evaluate the role of cAMP-dependent plasticity in learning, we examined how cAMP manipulations and olfactory classical conditioning modulate olfactory responses in the MB with in vivo imaging. Elevating cAMP pharmacologically or optogenetically produced plasticity in MB neurons, altering their responses to odorants. Odor-evoked Ca2+ responses showed net facilitation across anatomical regions. At the single-cell level, neurons exhibited heterogeneous responses to cAMP elevation, suggesting that cAMP drives plasticity to discrete subsets of MB neurons. Olfactory appetitive conditioning enhanced MB odor responses, mimicking the cAMP-dependent plasticity in directionality and magnitude. Elevating cAMP to equivalent levels as appetitive conditioning also produced plasticity, suggesting that the cAMP generated during conditioning affects odor-evoked responses in the MB. Finally, we found that this plasticity was dependent on the Rutabaga type I adenylyl cyclase, linking cAMP-dependent plasticity to behavioral modification. Overall, these data demonstrate that learning produces robust cAMP-dependent plasticity in intrinsic MB neurons, which is biased toward naturalistic reward learning. This suggests that cAMP signaling may serve to modulate intrinsic MB responses toward salient stimuli.


Assuntos
Condicionamento Clássico/fisiologia , AMP Cíclico/fisiologia , Corpos Pedunculados/fisiologia , Plasticidade Neuronal/fisiologia , Olfato/fisiologia , Animais , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Aprendizagem/fisiologia , Odorantes
7.
J Neurogenet ; 34(1): 36-46, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32043414

RESUMO

Recent years have witnessed significant progress in understanding how memories are encoded, from the molecular to the cellular and the circuit/systems levels. With a good compromise between brain complexity and behavioral sophistication, the fruit fly Drosophila melanogaster is one of the preeminent animal models of learning and memory. Here we review how memories are encoded in Drosophila, with a focus on short-term memory and an eye toward future directions. Forward genetic screens have revealed a large number of genes and transcripts necessary for learning and memory, some acting cell-autonomously. Further, the relative numerical simplicity of the fly brain has enabled the reverse engineering of learning circuits with remarkable precision, in some cases ascribing behavioral phenotypes to single neurons. Functional imaging and physiological studies have localized and parsed the plasticity that occurs during learning at some of the major loci. Connectomics projects are significantly expanding anatomical knowledge of the nervous system, filling out the roadmap for ongoing functional/physiological and behavioral studies, which are being accelerated by simultaneous tool development. These developments have provided unprecedented insight into the fundamental neural principles of learning, and lay the groundwork for deep understanding in the near future.


Assuntos
Comportamento Animal/fisiologia , Drosophila melanogaster/fisiologia , Aprendizagem/fisiologia , Memória/fisiologia , Corpos Pedunculados/fisiologia , Animais , Condicionamento Clássico/fisiologia , Vias Neurais/fisiologia , Percepção Olfatória/fisiologia
8.
J Exp Biol ; 222(Pt 15)2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31262786

RESUMO

Among animals with visual processing mechanisms, the leech Hirudo verbana is a rare example in which all neurons can be identified. However, little is known about its visual system, which is composed of several pigmented head eyes and photosensitive non-pigmented sensilla that are distributed across its entire body. Although several interneurons are known to respond to visual stimuli, their response properties are poorly understood. Among these, the S-cell system is especially intriguing: it is multimodal, spans the entire body of the leech and is thought to be involved in sensory integration. To improve our understanding of the role of this system, we tested its spectral sensitivity, spatial integration and adaptation properties. The response of the S-cell system to visual stimuli was found to be strongly dependent on the size of the area stimulated, and adaptation was local. Furthermore, an adaptation experiment demonstrated that at least two color channels contributed to the response, and that their contribution was dependent on the adaptation to the background. The existence of at least two color channels was further supported by transcriptomic evidence, which indicated the existence of at least two distinct groups of putative opsins for leeches. Taken together, our results show that the S-cell system has response properties that could be involved in the processing of spatial and color information of visual stimuli. We propose the leech as a novel system to understand visual processing mechanisms with many practical advantages.


Assuntos
Sanguessugas/fisiologia , Células Fotorreceptoras/classificação , Transcriptoma , Animais , Visão de Cores , Fenômenos Eletrofisiológicos , Interneurônios/fisiologia , Sanguessugas/genética , Sanguessugas/metabolismo , Opsinas/isolamento & purificação , Estimulação Luminosa , Células Fotorreceptoras/química , Células Fotorreceptoras/citologia
9.
Dev Genes Evol ; 227(4): 271-278, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28477155

RESUMO

The dioptric visual system relies on precisely focusing lenses that project light onto a neural retina. While the proteins that constitute the lenses of many vertebrates are relatively well characterized, less is known about the proteins that constitute invertebrate lenses, especially the lens facets in insect compound eyes. To address this question, we used mass spectrophotometry to define the major proteins that comprise the corneal lenses from the adult Drosophila melanogaster compound eye. This led to the identification of four cuticular proteins: two previously identified lens proteins, drosocrystallin and retinin, and two newly identified proteins, Cpr66D and Cpr72Ec. To determine which ommatidial cells contribute each of these proteins to the lens, we conducted in situ hybridization at 50% pupal development, a key age for lens secretion. Our results confirm previous reports that drosocrystallin and retinin are expressed in the two primary corneagenous cells-cone cells and primary pigment cells. Cpr72Ec and Cpr66D, on the other hand, are more highly expressed in higher order interommatidial pigment cells. These data suggest that the complementary expression of cuticular proteins give rise to the center vs periphery of the corneal lens facet, possibly facilitating a refractive gradient that is known to reduce spherical aberration. Moreover, these studies provide a framework for future studies aimed at understanding the cuticular basis of corneal lens function in holometabolous insect eyes.


Assuntos
Cristalinas/análise , Proteínas de Drosophila/análise , Drosophila melanogaster/química , Drosophila melanogaster/genética , Animais , Olho Composto de Artrópodes/química , Córnea/química , Cristalinas/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Evolução Molecular , Proteínas do Olho/genética , Regulação da Expressão Gênica , Hibridização In Situ , Cristalino/química , Espectrometria de Massas , Pupa/química , Pupa/citologia , Pupa/crescimento & desenvolvimento
10.
Evol Dev ; 18(4): 216-28, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27402568

RESUMO

Stemmata, the larval eyes of holometabolous insects are extremely diverse, ranging from full compound eyes, to a few ommatidial units as are typical in compound eyes, to sophisticated and functionally specialized image-forming camera-type eyes. Stemmata evolved from a compound eye ommatidial ancestor, an eye type that is morphologically well conserved in regards to cellular composition, and well studied in regards to development. However, despite this evolutionary origin it remains largely unknown how stemmata develop. In addition, it is completely unclear how development is altered to give rise to some of the functionally most complex stemmata, such as those of the sunburst diving beetle, Thermonectus marmoratus. In this study, we used histological methods to investigate the embryonic development of the functionally complex principal stemmata Eye 1 and Eye 2 of the larval visual system of T. marmoratus. To gain insights into how cellular components of their sophisticated camera-type eyes might have evolved from the cellular components of ommatidial ancestors, we contrast our findings against known features of ommatidia development, which are particularly well understood in Drosophila. We find many similarities, such as the early presence of a pseudostratified epithelium, and the order in which specific cell types are recruited. However, in Thermonectus each cell type is represented by a large number of cells from early on and major tissue re-orientation occurs as eye development progresses. This study provides insights into the timing of morphological features and represents the basis for future molecular studies.


Assuntos
Besouros/anatomia & histologia , Besouros/crescimento & desenvolvimento , Animais , Diferenciação Celular , Besouros/classificação , Embrião não Mamífero/anatomia & histologia , Olho/embriologia , Larva/anatomia & histologia , Cristalino/embriologia
11.
J Exp Biol ; 219(Pt 24): 3866-3874, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27974533

RESUMO

The highly specialized evolution of Strepsiptera has produced one of the most unusual eyes among mature insects, perhaps in line with their extremely complex and challenging life cycle. This relatively rare insect order is one of the few for which it has been unclear what spectral classes of photoreceptors any of its members may possess, an even more apt question given the nocturnal evolution of the group. To address this question, we performed electroretinograms on adult male Xenos peckii: we measured spectral responses to equi-quantal monochromatic light flashes of different wavelengths, and established VlogI relationships to calculate spectral sensitivities. Based on opsin template fits, we found maximal spectral sensitivity (λmax) in the green domain at 539 nm. Application of a green light to 'bleach' green receptors revealed that a UV peak was contributed to by an independent UV opsin with a λmax of 346 nm. Transcriptomics and a phylogenetic analysis including 50 other opsin sequences further confirmed the presence of these two opsin classes. While these findings do not necessarily indicate that these unorthodox insects have color vision, they raise the possibility that UV vision plays an important role in the ability of X. peckii males to find the very cryptic strepsipteran females that are situated within their wasp hosts.


Assuntos
Células Fotorreceptoras de Invertebrados/fisiologia , Raios Ultravioleta , Vespas/citologia , Vespas/fisiologia , Animais , Eletrorretinografia , Feminino , Masculino , Opsinas/genética , Opsinas/metabolismo , Parasitos/genética , Parasitos/ultraestrutura , Células Fotorreceptoras de Invertebrados/ultraestrutura , Filogenia , Análise Espectral , Transcriptoma/genética , Vespas/genética , Vespas/ultraestrutura
12.
Nat Commun ; 15(1): 6873, 2024 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-39127721

RESUMO

Ribosomes are regulated by evolutionarily conserved ubiquitination/deubiquitination events. We uncover the role of the deubiquitinase OTUD6 in regulating global protein translation through deubiquitination of the RPS7/eS7 subunit on the free 40 S ribosome in vivo in Drosophila. Coimmunoprecipitation and enrichment of monoubiquitinated proteins from catalytically inactive OTUD6 flies reveal RPS7 as the ribosomal substrate. The 40 S protein RACK1 and E3 ligases CNOT4 and RNF10 function upstream of OTUD6 to regulate alkylation stress. OTUD6 interacts with RPS7 specifically on the free 40 S, and not on 43 S/48 S initiation complexes or the translating ribosome. Global protein translation levels are bidirectionally regulated by OTUD6 protein abundance. OTUD6 protein abundance is physiologically regulated in aging and in response to translational and alkylation stress. Thus, OTUD6 may promote translation initiation, the rate limiting step in protein translation, by titering the amount of 40 S ribosome that recycles.


Assuntos
Proteínas de Drosophila , Biossíntese de Proteínas , Proteínas Ribossômicas , Ubiquitinação , Animais , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Ribossomos/metabolismo , Estresse Fisiológico , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética
13.
bioRxiv ; 2024 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-39149363

RESUMO

Genetic disorders such as neurofibromatosis type 1 increase vulnerability to cognitive and behavioral disorders, such as autism spectrum disorder and attention-deficit/hyperactivity disorder. Neurofibromatosis type 1 results from loss-of-function mutations in the neurofibromin gene and subsequent reduction in the neurofibromin protein (Nf1). While the mechanisms have yet to be fully elucidated, loss of Nf1 may alter neuronal circuit activity leading to changes in behavior and susceptibility to cognitive and behavioral comorbidities. Here we show that mutations decreasing Nf1 expression alter motor behaviors, impacting the patterning, prioritization, and behavioral state dependence in a Drosophila model of neurofibromatosis type 1. Loss of Nf1 increases spontaneous grooming in a nonlinear spatial and temporal pattern, differentially increasing grooming of certain body parts, including the abdomen, head, and wings. This increase in grooming could be overridden by hunger in food-deprived foraging animals, demonstrating that the Nf1 effect is plastic and internal state-dependent. Stimulus-evoked grooming patterns were altered as well, with nf1 mutants exhibiting reductions in wing grooming when coated with dust, suggesting that hierarchical recruitment of grooming command circuits was altered. Yet loss of Nf1 in sensory neurons and/or grooming command neurons did not alter grooming frequency, suggesting that Nf1 affects grooming via higher-order circuit alterations. Changes in grooming coincided with alterations in walking. Flies lacking Nf1 walked with increased forward velocity on a spherical treadmill, yet there was no detectable change in leg kinematics or gait. Thus, loss of Nf1 alters motor function without affecting overall motor coordination, in contrast to other genetic disorders that impair coordination. Overall, these results demonstrate that loss of Nf1 alters the patterning and prioritization of repetitive behaviors, in a state-dependent manner, without affecting motor coordination.

14.
Nat Commun ; 15(1): 1287, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38346946

RESUMO

Fibroblast growth factor receptor (FGFR)-2 can be inhibited by FGFR-selective or non-selective tyrosine kinase inhibitors (TKIs). Selective TKIs are approved for cholangiocarcinoma (CCA) with FGFR2 fusions; however, their application is limited by a characteristic pattern of adverse events or evocation of kinase domain mutations. A comprehensive characterization of a patient cohort treated with the non-selective TKI lenvatinib reveals promising efficacy in FGFR2-driven CCA. In a bed-to-bench approach, we investigate FGFR2 fusion proteins bearing critical tumor-relevant point mutations. These mutations confer growth advantage of tumor cells and increased resistance to selective TKIs but remain intriguingly sensitive to lenvatinib. In line with clinical observations, in-silico analyses reveal a more favorable interaction pattern of lenvatinib with FGFR2, including an increased flexibility and ligand efficacy, compared to FGFR-selective TKIs. Finally, the treatment of a patient with progressive disease and a newly developed kinase mutation during therapy with a selective inhibitor results in a striking response to lenvatinib. Our in vitro, in silico, and clinical data suggest that lenvatinib is a promising treatment option for FGFR2-driven CCA, especially when insurmountable adverse reactions of selective TKIs or acquired kinase mutations occur.


Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Compostos de Fenilureia , Quinolinas , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Ductos Biliares Intra-Hepáticos , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia
15.
J Exp Clin Cancer Res ; 43(1): 77, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38475864

RESUMO

BACKGROUND: The DNA damage response (DDR) is a physiological network preventing malignant transformation, e.g. by halting cell cycle progression upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary tumors of the nervous system and DDR dysregulation contributes to acquired treatment resistance. Therefore, DDR targeting is a promising therapeutic anti-glioma strategy. Here, we investigated Ataxia telangiectasia and Rad3 related (ATR) inhibition (ATRi) and functionally-instructed combination therapies involving ATRi in experimental glioma. METHODS: We used acute cytotoxicity to identify treatment efficacy as well as RNAseq and DigiWest protein profiling to characterize ATRi-induced modulations within the molecular network in glioma cells. Genome-wide CRISPR/Cas9 functional genomic screens and subsequent validation with functionally-instructed compounds and selected shRNA-based silencing were employed to discover and investigate molecular targets modifying response to ATRi in glioma cell lines in vitro, in primary cultures ex vivo and in zebrafish and murine models in vivo. RESULTS: ATRi monotherapy displays anti-glioma efficacy in vitro and ex vivo and modulates the molecular network. We discovered molecular targets by genome-wide CRISPR/Cas9 loss-of-function and activation screens that enhance therapeutic ATRi effects. We validated selected druggable targets by a customized drug library and functional assays in vitro, ex vivo and in vivo. CONCLUSION: In conclusion, our study leads to the identification of novel combination therapies involving ATRi that could inform future preclinical studies and early phase clinical trials.


Assuntos
Glioma , Peixe-Zebra , Camundongos , Animais , Linhagem Celular Tumoral , Reparo do DNA , Dano ao DNA , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
16.
bioRxiv ; 2023 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-37503285

RESUMO

Animals generally have either compound eyes, which have evolved repeatedly in different invertebrates, or camera eyes, which have evolved many times across the animal kingdom. Both eye types include two important kinds of cells: photoreceptor cells, which can be excited by light, and non-neuronal support cells (SupCs), which provide essential support to photoreceptors. Despite many examples of convergence in eye evolution, similarities in the gross developmental plan and molecular signatures have been discovered, even between phylogenetically distant and functionally different eye types. For this reason, a shared evolutionary origin has been considered for photoreceptors. In contrast, only a handful of studies, primarily on the compound eyes of Drosophila melanogaster , have demonstrated molecular similarities in SupCs. D. melanogaster SupCs (Semper cells and primary pigment cells) are specialized eye glia that share several molecular similarities with certain vertebrate eye glia, including Müller glia. This led us to speculate whether there are conserved molecular signatures of SupCs, even in functionally different eyes such as the image-forming larval camera eyes of the sunburst diving beetle Thermonectus marmoratus . To investigate this possibility, we used an in-depth comparative whole-tissue transcriptomics approach. Specifically, we dissected the larval principal camera eyes into SupC- and retina-containing regions and generated the respective transcriptomes. Our analysis revealed several conserved features of SupCs including enrichment of genes that are important for glial function (e.g. gap junction proteins such as innexin 3), glycogen production (glycogenin), and energy metabolism (glutamine synthetase 1 and 2). To evaluate the extent of conservation, we compared our transcriptomes with those of fly (Semper cells) and vertebrate (Müller glia) eye glia as well as respective retinas. T. marmoratus SupCs were found to have distinct genetic overlap with both fly and vertebrate eye glia. These results provide molecular evidence for the deep conservation of SupCs in addition to photoreceptor cells, raising essential questions about the evolutionary origin of eye-specific glia in animals.

17.
Biochem Pharmacol ; 197: 114905, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34971590

RESUMO

The constitutive androstane receptor (CAR) controls xenobiotic clearance, regulates liver glucose, lipid metabolism, and energy homeostasis. These functions have been mainly discovered using the prototypical mouse-specific CAR ligand TCPOBOP in wild-type or CAR null mice. However, TCPOBOP is reported to result in some off-target metabolic effects in CAR null mice. In this study, we compared the metabolic effects of TCPOBOP using lipidomic, transcriptomic, and proteomic analyses in wild-type and humanized CAR-PXR-CYP3A4/3A7 mice. In the model, human CAR retains its constitutive activity in metabolism regulation; however, it is not activated by TCPOBOB. Notably, we observed that TCPOBOP affected lipid homeostasis by elevating serum and liver triglyceride levels and promoted hepatocyte hypertrophy in humanized CAR mice. Hepatic lipidomic analysis revealed a significant accumulation of triglycerides and decrease of its metabolites in humanized CAR mice. RNA-seq analysis has shown divergent gene expression levels in wild-type and humanized CAR mice. Gene expression regulation in humanized mice is mainly involved in lipid metabolic processes and in the PPAR, leptin, thyroid, and circadian clock pathways. In contrast, CAR activation by TCPOBOP in wild-type mice reduced liver and plasma triglyceride levels and induced a typical transcriptomic proliferative response in the liver. In summary, we identified TCPOBOP as a disruptor of lipid metabolism in humanized CAR mice. The divergent effects of TCPOBOP in humanized mice in comparison with the prototypical CAR-mediated response in WT mice warrant the use of appropriate model ligands and humanized animal models during the testing of endocrine disruption and the characterization of adverse outcome pathways.


Assuntos
Receptor Constitutivo de Androstano/agonistas , Receptor Constitutivo de Androstano/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Piridinas/administração & dosagem , Animais , Humanos , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
18.
Elife ; 112022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35285796

RESUMO

Anatomical and physiological compartmentalization of neurons is a mechanism to increase the computational capacity of a circuit, and a major question is what role axonal compartmentalization plays. Axonal compartmentalization may enable localized, presynaptic plasticity to alter neuronal output in a flexible, experience-dependent manner. Here, we show that olfactory learning generates compartmentalized, bidirectional plasticity of acetylcholine release that varies across the longitudinal compartments of Drosophila mushroom body (MB) axons. The directionality of the learning-induced plasticity depends on the valence of the learning event (aversive vs. appetitive), varies linearly across proximal to distal compartments following appetitive conditioning, and correlates with learning-induced changes in downstream mushroom body output neurons (MBONs) that modulate behavioral action selection. Potentiation of acetylcholine release was dependent on the CaV2.1 calcium channel subunit cacophony. In addition, contrast between the positive conditioned stimulus and other odors required the inositol triphosphate receptor, which maintained responsivity to odors upon repeated presentations, preventing adaptation. Downstream from the MB, a set of MBONs that receive their input from the γ3 MB compartment were required for normal appetitive learning, suggesting that they represent a key node through which reward learning influences decision-making. These data demonstrate that learning drives valence-correlated, compartmentalized, bidirectional potentiation, and depression of synaptic neurotransmitter release, which rely on distinct mechanisms and are distributed across axonal compartments in a learning circuit.


Assuntos
Acetilcolina , Olfato , Animais , Axônios , Drosophila/fisiologia , Drosophila melanogaster , Corpos Pedunculados/fisiologia , Plasticidade Neuronal/fisiologia , Neurotransmissores , Olfato/fisiologia
19.
Cancers (Basel) ; 14(18)2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36139700

RESUMO

In cancer, the complex interplay between tumor cells and the tumor microenvironment results in the modulation of signaling processes. By assessing the expression of a multitude of proteins and protein variants in cancer tissue, wide-ranging information on signaling pathway activation and the status of the immunological landscape is obtainable and may provide viable information on the treatment response. Archived breast cancer tissues from a cohort of 84 patients (no adjuvant therapy) were analyzed by high-throughput Western blotting, and the expression of 150 proteins covering central cancer pathways and immune cell markers was examined. By assessing CD8α, CD11c, CD16 and CD68 expression, immune cell infiltration was determined and revealed a strong correlation between event-free patient survival and the infiltration of immune cells. The presence of tumor-infiltrating lymphocytes was linked to the pronounced activation of the Jak/Stat signaling pathway and apoptotic processes. The elevated phosphorylation of PPARγ (pS112) in non-immune-infiltrated tumors suggests a novel immune evasion mechanism in breast cancer characterized by increased PPARγ phosphorylation. Multiplexed immune cell marker assessment and the protein profiling of tumor tissue provide functional signaling data facilitating breast cancer patient stratification.

20.
ACS Infect Dis ; 7(6): 1596-1606, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33724771

RESUMO

The presence of antibodies against endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We developed a serum screening platform using a bead-based Western blot system called DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. Characterization of the immunoassay for detection of SARS-CoV-2 specific antibodies revealed a sensitivity of 90.3% and a diagnostic specificity of 98.1%. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates comparable assay performances (Cohen's κ ranging from 0.8874 to 0.9508). Analogous assays for OC43, 229E, and NL63 were established and combined into one multiplex with the SARS-CoV-2 assay. Seroreactivity for different coronaviruses was detected with high incidence, and the multiplex assay was adapted for serum screening.


Assuntos
COVID-19 , Coronaviridae , Teste para COVID-19 , Humanos , Extratos Vegetais , SARS-CoV-2
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