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The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure-activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.
Atelopus is a species-rich group of Neotropical bufonids. Present knowledge on bioacoustics in this genus is relatively poor, as vocalisations have been described in only about one fifth of the ca. 100 species known. All studied members of the genus produce vocalisations although, with a few exceptions, most species lack a middle ear. Nonetheless, hearing has been demonstrated even in earless Atelopus making bioacoustics in these toads an inspiring research field. So far, three structural call types have been identified in the genus. As sympatry is uncommon in Atelopus, calls of the same type often vary little between species. Based on recordings from the 1980s, we describe vocalisations of three Venezuelan species (A. carbonerensis, A. mucubajiensis, A. tamaense) from the Cordillera de Mérida, commonly known as the Andes of Venezuela and the Tamá Massif, a Venezuelan spur of the Colombian Cordillera Oriental. Vocalisations correspond, in part, to the previously identified call types in Atelopus. Evaluation of the vocalisations of the three species presented in this study leads us to recognise a fourth structural call type for the genus. With this new addition, the Atelopus acoustic repertoire now includes (1) pulsed calls, (2) pure tone calls, (3) pulsed short calls and (4) pure tone short calls. The call descriptions provided here are valuable contributions to the bioacoustics of these Venezuelan Atelopus species, since all of them have experienced dramatic population declines that limit possibilities of further studies.
High tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. TIFP may hamper adequate uptake of macromolecular therapeutics in tumor tissue. In addition, TIFP generates mechanical forces affecting the tumor cortex, which might influence the growth parameters of tumor cells. This seems likely as, in other tissues (namely, blood vessels or the skin), mechanical stretch is known to trigger proliferation. Therefore, we hypothesize that TIFP-induced stretch modulates proliferation-associated parameters. Solid epithelial tumors (A431 and A549) were grown in Naval Medical Research Institute nude mice, generating a TIFP of about 10 mm Hg (A431) or 5 mm Hg (A549). Tumor drainage of the central cystic area led to a rapid decline of TIFP, together with visible relaxation of the tumor cortex. It was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis that TIFP lowering yields a decreased phosphorylation of proliferation-associated p44/42 mitogen-activated protein kinase and tumor relaxation. In confirmation, immunohistochemical staining showed a decrease of tumor-associated proliferation marker Ki-67 after TIFP lowering. These data suggest that the mechanical stretch induced by TIFP is a positive modulator of tumor proliferation.
Here, we describe a new immersion-based clearing method suitable for optical clearing of thick adult human brain samples while preserving its lipids and lipophilic labels such as 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). This clearing procedure is simple, easy to implement, and allowed for clearing of 5 mm thick human brain tissue samples within 12 days. Furthermore, we show for the first time the advantageous effect of the Periodate-Lysine-Paraformaldehyde (PLP) fixation as compared to the more commonly used 4% paraformaldehyde (PFA) on clearing performance.
High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease
(2019)
Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.
Elevated tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. Clinically, TIFP may hamper the uptake of chemotherapeutic drugs into the tumor tissue reducing their therapeutic efficacy. In this study, a means of modulating TIFP to increase the flux of macromolecules into tumor tissue is presented, which is based on the rationale that elevated plasma colloid osmotic pressure (COP) pulls water from tumor interstitium lowering the TIFP. Concentrated human serum albumin: (20% HSA), used as an agent to enhance COP, reduced the TIFP time-dependently from 8 to 2 mm Hg in human tumor xenograft models bearing A431 epidermoid vulva carcinomas. To evaluate whether this reduction facilitates the uptake of macromolecules, the intratumoral distribution of fluorescently conjugated dextrans (2.5 mg/ml) and cetuximab (2.0 mg/ml) was probed using novel time domain nearinfrared fluorescence imaging. This method permitted discrimination and semiquantification of tumor-accumulated conjugate from background and unspecific probe fluorescence. The coadministration of 20% HSA together with either dextrans or cetuximab was found to lower the TIFP significantly and increase the concentration of the substances within the tumor tissue in comparison to control tumors. Furthermore, combined administration of 20%HSA plus cetuximab reduced the tumor growth significantly in comparison to standard cetuximab treatment. These data demonstrate that increased COP lowers the TIFP within hours and increases the uptake of therapeutic macromolecules into the tumor interstitium leading to reduced tumor growth. This model represents a novel approach to facilitate the delivery of therapeutics into tumor tissue, particularly monoclonal antibodies.
Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.
Ataxin-2 (human gene symbol ATXN2) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide–sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters.
Communication with the hematopoietic system is a vital component of regulating brain function in health and disease. Traditionally, the major routes considered for this neuroimmune communication are by individual molecules such as cytokines carried by blood, by neural transmission, or, in more severe pathologies, by the entry of peripheral immune cells into the brain. In addition, functional mRNA from peripheral blood can be directly transferred to neurons via extracellular vesicles (EVs), but the parameters that determine their uptake are unknown. Using varied animal models that stimulate neuronal activity by peripheral inflammation, optogenetics, and selective proteasome inhibition of dopaminergic (DA) neurons, we show that the transfer of EVs from blood is triggered by neuronal activity in vivo. Importantly, this transfer occurs not only in pathological stimulation but also by neuronal activation caused by the physiological stimulus of novel object placement. This discovery suggests a continuous role of EVs under pathological conditions as well as during routine cognitive tasks in the healthy brain.
Brain metastases are the most common intracranial tumor in adults and are associated with poor patient prognosis and median survival of only a few months. Treatment options for brain metastasis patients remain limited and largely depend on surgical resection, radio- and/or chemotherapy. The development and pre-clinical testing of novel therapeutic strategies require reliable experimental models and diagnostic tools that closely mimic technologies that are used in the clinic and reflect histopathological and biochemical changes that distinguish tumor progression from therapeutic response. In this study, we sought to test the applicability of magnetic resonance (MR) spectroscopy in combination with MR imaging to closely monitor therapeutic efficacy in a breast-to-brain metastasis model. Given the importance of radiotherapy as the standard of care for the majority of brain metastases patients, we chose to monitor the post-irradiation response by magnetic resonance spectroscopy (MRS) in combination with MR imaging (MRI) using a 7 Tesla small animal scanner. Radiation was applied as whole brain radiotherapy (WBRT) using the image-guided Small Animal Radiation Research Platform (SARRP). Here we describe alterations in different metabolites, including creatine and N-acetylaspartate, that are characteristic for brain metastases progression and lactate, which indicates hypoxia, while choline levels remained stable. Radiotherapy resulted in normalization of metabolite levels indicating tumor stasis or regression in response to treatment. Our data indicate that the use of MR spectroscopy in addition to MRI represents a valuable tool to closely monitor not only volumetrical but also metabolic changes during tumor progression and to evaluate therapeutic efficacy of intervention strategies. Adapting the analytical technology in brain metastasis models to those used in clinical settings will increase the translational significance of experimental evaluation and thus contribute to the advancement of pre-clinical assessment of novel therapeutic strategies to improve treatment options for brain metastases patients.
Aims: The examination of histological sections is still the gold standard in diagnostic pathology. Important histopathological diagnostic criteria are nuclear shapes and chromatin distribution as well as nucleus-cytoplasm relation and immunohistochemical properties of surface and intracellular proteins. The aim of this investigation was to evaluate the benefits and drawbacks of three-dimensional imaging of CD30+ cells in classical Hodgkin Lymphoma (cHL) in comparison to CD30+ lymphoid cells in reactive lymphoid tissues.
Materials and results: Using immunoflourescence confocal microscopy and computer-based analysis, we compared CD30+ neoplastic cells in Nodular Sclerosis cHL (NScCHL), Mixed Cellularity cHL (MCcHL), with reactive CD30+ cells in Adenoids (AD) and Lymphadenitis (LAD). We confirmed that the percentage of CD30+ cell volume can be calculated. The amount in lymphadenitis was approx. 1.5%, in adenoids around 2%, in MCcHL up to 4,5% whereas the values for NScHL rose to more than 8% of the total cell cytoplasm. In addition, CD30+ tumour cells (HRS-cells) in cHL had larger volumes, and more protrusions compared to CD30+ reactive cells. Furthermore, the formation of large cell networks turned out to be a typical characteristic of NScHL.
Conclusion: In contrast to 2D histology, 3D laser scanning offers a visualisation of complete cells, their network interaction and spatial distribution in the tissue. The possibility to differentiate cells in regards to volume, surface, shape, and cluster formation enables a new view on further diagnostic and biological questions. 3D includes an increased amount of information as a basis of bioinformatical calculations.
Cell–matrix adhesion and cell migration are physiologically important processes that also play a major role in cancer spreading. In cultured cells, matrix adhesion depends on integrin-containing contacts such as focal adhesions. Flotillin-1 and flotillin-2 are frequently overexpressed in cancers and are associated with poor survival. Our previous studies have revealed a role for flotillin-2 in cell–matrix adhesion and in the regulation of the actin cytoskeleton. We here show that flotillins are important for cell migration in a wound healing assay and influence the morphology and dynamics of focal adhesions. Furthermore, anchorage-independent growth in soft agar is enhanced by flotillins. In the absence of flotillins, especially flotillin-2, phosphorylation of focal adhesion kinase and extracellularly regulated kinase is diminished. Flotillins interact with α-actinin, a major regulator of focal adhesion dynamics. These findings are important for understanding the molecular mechanisms of how flotillin overexpression in cancers may affect cell migration and, especially, enhance metastasis formation.
Hereditary Parkinson’s disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCAA53T in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As an exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension, and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was also detected for other microtubule and microfilament factors, namely MAP2S1801, MARK1S394, MAP1AT1794, KIF1AS1537, 4.1NS541, 4.1GS86, and ADD2S528. While the MAP1B heavy chain supports regeneration and growth cones, its light chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2S299, VPS13DS2429, and VPS13CS2480 in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in Drosophila melanogaster that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly, ANK2 gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.
Microenvironmental regulation of tumor progression and therapeutic response in brain metastasis
(2019)
Cellular and non-cellular components of the tumor microenvironment (TME) are emerging as key regulators of primary tumor progression, organ-specific metastasis, and therapeutic response. In the era of TME-targeted- and immunotherapies, cancer-associated inflammation has gained increasing attention. In this regard, the brain represents a unique and highly specialized organ. It has long been regarded as an immunological sanctuary site where the presence of the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCB) restricts the entry of immune cells from the periphery. Consequently, tumor cells that metastasize to the brain were thought to be shielded from systemic immune surveillance and destruction. However, the detailed characterization of the immune landscape within border-associated areas of the central nervous system (CNS), such as the meninges and the choroid plexus, as well as the discovery of lymphatics and channels that connect the CNS with the periphery, have recently challenged the dogma of the immune privileged status of the brain. Moreover, the presence of brain metastases (BrM) disrupts the integrity of the BBB and BCB. Indeed, BrM induce the recruitment of different immune cells from the myeloid and lymphoid lineage to the CNS. Blood-borne immune cells together with brain-resident cell-types, such as astrocytes, microglia, and neurons, form a highly complex and dynamic TME that affects tumor cell survival and modulates the mode of immune responses that are elicited by brain metastatic tumor cells. In this review, we will summarize recent findings on heterotypic interactions within the brain metastatic TME and highlight specific functions of brain-resident and recruited cells at different rate-limiting steps of the metastatic cascade. Based on the insight from recent studies, we will discuss new opportunities and challenges for TME-targeted and immunotherapies for BrM.
Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.
The mechanistic target of rapamycin (mTOR) is elevated in prostate cancer, making this protein attractive for tumor treatment. Unfortunately, resistance towards mTOR inhibitors develops and the tumor becomes reactivated. We determined whether epigenetic modulation by the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), may counteract non-responsiveness to the mTOR inhibitor, temsirolimus, in prostate cancer (PCa) cells. Prostate cancer cells, sensitive (parental) and resistant to temsirolimus, were exposed to VPA, and tumor cell growth behavior compared. Temsirolimus resistance enhanced the number of tumor cells in the G2/M-phase, correlating with elevated cell proliferation and clonal growth. The cell cycling proteins cdk1 and cyclin B, along with Akt-mTOR signaling increased, whereas p19, p21 and p27 decreased, compared to the parental cells. VPA significantly reduced cell growth and up-regulated the acetylated histones H3 and H4. Cdk1 and cyclin B decreased, as did phosphorylated mTOR and the mTOR sub-complex Raptor. The mTOR sub-member Rictor and phosphorylated Akt increased under VPA. Knockdown of cdk1, cyclin B, or Raptor led to significant cell growth reduction. HDAC inhibition through VPA counteracts temsirolimus resistance, probably by down-regulating cdk1, cyclin B and Raptor. Enhanced Rictor and Akt, however, may represent an undesired feedback loop, which should be considered when designing future therapeutic regimens.
Synthesis and SAR of the antistaphylococcal natural product nematophin from Xenorhabdus nematophila
(2019)
The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead structures are needed. Here, the synthesis and an expanded structure–activity relationship of the simple and antistaphylococcal amide nematophin from Xenorhabdus nematophila and synthetic derivatives are described. Moreover, the synthesis of intrinsic fluorescent derivatives, incorporating azaindole moieties was achieved for the first time.
Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
Numerous cell–cell and cell–matrix interactions within the bone marrow microenvironment enable the controlled lifelong self-renewal and progeny of hematopoietic stem and progenitor cells (HSPCs). On the cellular level, this highly mutual interaction is granted by cell adhesion molecules (CAMs) integrating differentiation, proliferation, and pro-survival signals from the surrounding microenvironment to the inner cell. However, cell–cell and cell–matrix interactions are also critically involved during malignant transformation of hematopoietic stem/progenitor cells. It has become increasingly apparent that leukemia-associated gene products, such as activated tyrosine kinases and fusion proteins resulting from chromosomal translocations, directly regulate the activation status of adhesion molecules, thereby directing the leukemic phenotype. These observations imply that interference with adhesion molecule function represents a promising treatment strategy to target pre-leukemic and leukemic lesions within the bone marrow niche. Focusing on myeloid leukemia, we provide a current overview of the mechanisms by which leukemogenic gene products hijack control of cellular adhesion to subsequently disturb normal hematopoiesis and promote leukemia development.
Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.
During erythropoiesis, haematopoietic stem cells (HSCs) differentiate in successive steps of commitment and specification to mature erythrocytes. This differentiation process is controlled by transcription factors that establish stage- and cell type-specific gene expression. In this study, we demonstrate that FUSE binding protein 1 (FUBP1), a transcriptional regulator important for HSC self-renewal and survival, is regulated by T-cell acute lymphocytic leukaemia 1 (TAL1) in erythroid progenitor cells. TAL1 directly activates the FUBP1 promoter, leading to increased FUBP1 expression during erythroid differentiation. The binding of TAL1 to the FUBP1 promoter is highly dependent on an intact GATA sequence in a combined E-box/GATA motif. We found that FUBP1 expression is required for efficient erythropoiesis, as FUBP1-deficient progenitor cells were limited in their potential of erythroid differentiation. Thus, the finding of an interconnection between GATA1/TAL1 and FUBP1 reveals a molecular mechanism that is part of the switch from progenitor- to erythrocyte-specific gene expression. In summary, we identified a TAL1/FUBP1 transcriptional relationship, whose physiological function in haematopoiesis is connected to proper erythropoiesis.
Background: Altered neuronal development is discussed as the underlying pathogenic mechanism of autism spectrum disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region, quinolinate phosphoribosyltransferase (QPRT) showed the strongest regulation during neuronal differentiation of SH-SY5Y neuroblastoma cells. We hypothesized a causal relation between this tryptophan metabolism-related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on the differentiation of SH-SY5Y and specifically focused on neuronal morphology, metabolites of the tryptophan pathway, and the neurodevelopmental transcriptome.
Methods: The gene dosage-dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical mimicking of loss of QPRT, and (iii) complete CRISPR/Cas9-mediated knock out (KO). QPRT-KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain.
Results: QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with the neuritic complexity of SH-SY5Y. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT-associated tryptophan pathway was not affected by KO. At the transcriptome level, genes linked to neurodevelopmental processes and synaptic structures were affected. Differentially regulated genes were enriched for ASD candidates, and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala.
Conclusions: In this study, QPRT was causally related to in vitro neuronal differentiation of SH-SY5Y cells and affected the regulation of genes and gene networks previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.
Neurogenesis of hippocampal granule cells (GCs) persists throughout mammalian life and is important for learning and memory. How newborn GCs differentiate and mature into an existing circuit during this time period is not yet fully understood. We established a method to visualize postnatally generated GCs in organotypic entorhino-hippocampal slice cultures (OTCs) using retroviral (RV) GFP-labeling and performed time-lapse imaging to study their morphological development in vitro. Using anterograde tracing we could, furthermore, demonstrate that the postnatally generated GCs in OTCs, similar to adult born GCs, grow into an existing entorhino-dentate circuitry. RV-labeled GCs were identified and individual cells were followed for up to four weeks post injection. Postnatally born GCs exhibited highly dynamic structural changes, including dendritic growth spurts but also retraction of dendrites and phases of dendritic stabilization. In contrast, older, presumably prenatally born GCs labeled with an adeno-associated virus (AAV), were far less dynamic. We propose that the high degree of structural flexibility seen in our preparations is necessary for the integration of newborn granule cells into an already existing neuronal circuit of the dentate gyrus in which they have to compete for entorhinal input with cells generated and integrated earlier.
Low-frequency spike-field coherence is a fingerprint of periodicity coding in the auditory cortex
(2018)
The extraction of temporal information from sensory input streams is of paramount importance in the auditory system. In this study, amplitude-modulated sounds were used as stimuli to drive auditory cortex (AC) neurons of the bat species Carollia perspicillata, to assess the interactions between cortical spikes and local-field potentials (LFPs) for the processing of temporal acoustic cues. We observed that neurons in the AC capable of eliciting synchronized spiking to periodic acoustic envelopes were significantly more coherent to theta- and alpha-band LFPs than their non-synchronized counterparts. These differences occurred independently of the modulation rate tested and could not be explained by power or phase modulations of the field potentials. We argue that the coupling between neuronal spiking and the phase of low-frequency LFPs might be important for orchestrating the coding of temporal acoustic structures in the AC.
The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells.
The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent ApcMin/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.
Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear.
Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA.
Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors.
Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.
Hematopoietic differentiation is driven by transcription factors, which orchestrate a finely tuned transcriptional network. At bipotential branching points lineage decisions are made, where key transcription factors initiate cell type-specific gene expression programs. These programs are stabilized by the epigenetic activity of recruited chromatin-modifying cofactors. An example is the association of the transcription factor RUNX1 with protein arginine methyltransferase 6 (PRMT6) at the megakaryocytic/erythroid bifurcation. However, little is known about the specific influence of PRMT6 on this important branching point. Here, we show that PRMT6 inhibits erythroid gene expression during megakaryopoiesis of primary human CD34+ progenitor cells. PRMT6 is recruited to erythroid genes, such as glycophorin A. Consequently, a repressive histone modification pattern with high H3R2me2a and low H3K4me3 is established. Importantly, inhibition of PRMT6 by shRNA or small molecule inhibitors leads to upregulation of erythroid genes and promotes erythropoiesis. Our data reveal that PRMT6 plays a role in the control of erythroid/megakaryocytic differentiation and open up the possibility that manipulation of PRMT6 activity could facilitate enhanced erythropoiesis for therapeutic use.
Cytokine regulation of high-output nitric oxide (NO) derived from inducible NO synthase (iNOS) is critically involved in inflammation biology and host defense. Herein, we set out to characterize the role of type I interferon (IFN) as potential regulator of hepatic iNOS in vitro and in vivo. In this regard, we identified in murine Hepa1-6 hepatoma cells a potent synergism between pro-inflammatory interleukin-β/tumor necrosis factor-α and immunoregulatory IFNβ as detected by analysis of iNOS expression and nitrite release. Upregulation of iNOS by IFNβ coincided with enhanced binding of signal transducer and activator of transcription-1 to a regulatory region at the murine iNOS promoter known to support target gene expression in response to this signaling pathway. Synergistic iNOS induction under the influence of IFNβ was confirmed in alternate murine Hepa56.1D hepatoma cells and primary hepatocytes. To assess iNOS regulation by type I IFN in vivo, murine acetaminophen (APAP)-induced sterile liver inflammation was investigated. In this model of acute liver injury, excessive necroinflammation drives iNOS expression in diverse liver cell types, among others hepatocytes. Herein, we demonstrate impaired iNOS expression in type I IFN receptor-deficient mice which associated with diminished APAP-induced liver damage. Data presented indicate a vital role of type I IFN within the inflamed liver for fine-tuning pathological processes such as overt iNOS expression.
We have recently shown that caspase-8 is a new substrate of Polo-like kinase 3 (Plk3) that phosphorylates the protein on residue T273 thereby promoting its pro-apoptotic function. In the present study we aimed to investigate the clinical relevance of Plk3 expression and phosphorylation of caspase-8 at T273 in patients with anal squamous cell carcinoma (SSC) treated with 5-fluorouracil and mitomycin C-based chemoradiotherapy (CRT). Immunohistochemical detection of the markers was performed in pretreatment biopsy specimens of 95 patients and was correlated with clinical/histopathologic characteristics including HPV-16 virus load/p16INK4a expression and cumulative incidence of local and distant failure, cancer specific survival (CSS), and overall survival (OS). We observed significant positive correlations between Plk3 expression, pT273 caspase-8 signal, and levels of HPV-16 virus DNA load/p16INK4a detection. Patients with high scores of Plk3 and pT273 caspase-8 showed increased local control (p = 0.011; p = 0.001), increased CSS (p = 0.011; p = 0.013) and OS (p = 0.024; p = 0.001), while the levels of pT273 caspase-8 were significantly associated (p = 0.033) with distant metastases. In multivariate analyses Plk3 expression remained significant for local failure (p = 0.018), CSS (p = 0.016) and OS (p = 0.023). Moreover, a combined HPV16 DNA load and Plk3 or pT273 caspase-8 variable revealed a significant correlation to decreased local failure (p = 0.001; p = 0.009), increased CSS (p = 0.016; p = 0.023) and OS (p = 0.003; p = 0.003). In conclusion these data indicate that elevated levels of Plk3 and pT273 caspase-8 are correlated with favorable clinical outcome in patients with anal SCC treated with concomitant CRT.
SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.
Protein disulfide isomerases (PDIs) support endoplasmic reticulum redox protein folding and cell-surface thiol-redox control of thrombosis and vascular remodeling. The family prototype PDIA1 regulates NADPH oxidase signaling and cytoskeleton organization, however the related underlying mechanisms are unclear. Here we show that genes encoding human PDIA1 and its two paralogs PDIA8 and PDIA2 are each flanked by genes encoding Rho guanine-dissociation inhibitors (GDI), known regulators of RhoGTPases/cytoskeleton. Evolutionary histories of these three microsyntenic regions reveal their emergence by two successive duplication events of a primordial gene pair in the last common vertebrate ancestor. The arrangement, however, is substantially older, detectable in echinoderms, nematodes, and cnidarians. Thus, PDI/RhoGDI pairing in the same transcription orientation emerged early in animal evolution and has been largely maintained. PDI/RhoGDI pairs are embedded into conserved genomic regions displaying common cis-regulatory elements. Analysis of gene expression datasets supports evidence for PDI/RhoGDI coexpression in developmental/inflammatory contexts. PDIA1/RhoGDIα were co-induced in endothelial cells upon CRISP-R-promoted transcription activation of each pair component, and also in mouse arterial intima during flow-induced remodeling. We provide evidence for physical interaction between both proteins. These data support strong functional links between PDI and RhoGDI families, which likely maintained PDI/RhoGDI microsynteny along > 800-million years of evolution.
Ligand stimulation of CD95 induces activation of Plk3 followed by phosphorylation of caspase-8
(2016)
Upon interaction of the CD95 receptor with its ligand, sequential association of the adaptor molecule FADD (MORT1), pro-forms of caspases-8/10, and the caspase-8/10 regulator c-FLIP leads to the formation of a death-inducing signaling complex. Here, we identify polo-like kinase (Plk) 3 as a new interaction partner of the death receptor CD95. The enzymatic activity of Plk3 increases following interaction of the CD95 receptor with its ligand. Knockout (KO) or knockdown of caspase-8, CD95 or FADD prevents activation of Plk3 upon CD95 stimulation, suggesting a requirement of a functional DISC for Plk3 activation. Furthermore, we identify caspase-8 as a new substrate for Plk3. Phosphorylation occurs on T273 and results in stimulation of caspase-8 proapoptotic function. Stimulation of CD95 in cells expressing a non-phosphorylatable caspase-8-T273A mutant in a rescue experiment or in Plk3-KO cells generated by CRISPR/Cas9 reduces the processing of caspase-8 prominently. Low T273 phosphorylation correlates significantly with low Plk3 expression in a cohort of 95 anal tumor patients. Our data suggest a novel mechanism of kinase activation within the Plk family and propose a new model for the stimulation of the extrinsic death pathway in tumors with high Plk3 expression.
Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with β-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.
Lymphocyte function-associated antigen 1 (LFA-1) affinity and avidity changes have been assumed to mediate adhesion to intercellular adhesion molecule-1 for T-cell conjugation to dendritic cells (DC). Although the T-cell receptor (TCR) and LFA-1 can generate intracellular signals, the immune cell adaptor protein linker for the activation of T cells (LAT) couples the TCR to downstream events. Here, we show that LFA-1 can mediate both adhesion and de-adhesion, dependent on receptor clustering. Although increased affinity mediates adhesion, LFA-1 cross-linking induced the association and activation of the protein-tyrosine kinases FAK1/PYK1 that phosphorylated LAT selectively on a single Y-171 site for the binding to adaptor complex GRB-2-SKAP1. LAT-GRB2-SKAP1 complexes were distinct from canonical LAT-GADs-SLP-76 complexes. LFA-1 cross-linking increased the presence of LAT-GRB2-SKAP1 complexes relative to LAT-GADs-SLP-76 complexes. LFA-1-FAK1 decreased T-cell-dendritic cell (DC) dwell times dependent on LAT-Y171, leading to reduced DO11.10 T cell binding to DCs and proliferation to OVA peptide. Overall, our findings outline a new model for LFA-1 in which the integrin can mediate both adhesion and de-adhesion events dependent on receptor cross-linking.
Dendrites form predominantly binary trees that are exquisitely embedded in the networks of the brain. While neuronal computation is known to depend on the morphology of dendrites, their underlying topological blueprint remains unknown. Here, we used a centripetal branch ordering scheme originally developed to describe river networks—the Horton-Strahler order (SO)–to examine hierarchical relationships of branching statistics in reconstructed and model dendritic trees. We report on a number of universal topological relationships with SO that are true for all binary trees and distinguish those from SO-sorted metric measures that appear to be cell type-specific. The latter are therefore potential new candidates for categorising dendritic tree structures. Interestingly, we find a faithful correlation of branch diameters with centripetal branch orders, indicating a possible functional importance of SO for dendritic morphology and growth. Also, simulated local voltage responses to synaptic inputs are strongly correlated with SO. In summary, our study identifies important SO-dependent measures in dendritic morphology that are relevant for neural function while at the same time it describes other relationships that are universal for all dendrites.
Objective: Loss of function mutations in PINK1 typically lead to early onset Parkinson disease (PD). Zebrafish (Danio rerio) are emerging as a powerful new vertebrate model to study neurodegenerative diseases. We used a pink1 mutant (pink−/−) zebrafish line with a premature stop mutation (Y431*) in the PINK1 kinase domain to identify molecular mechanisms leading to mitochondrial dysfunction and loss of dopaminergic neurons in PINK1 deficiency.
Methods: The effect of PINK1 deficiency on the number of dopaminergic neurons, mitochondrial function, and morphology was assessed in both zebrafish embryos and adults. Genome-wide gene expression studies were undertaken to identify novel pathogenic mechanisms. Functional experiments were carried out to further investigate the effect of PINK1 deficiency on early neurodevelopmental mechanisms and microglial activation.
Results: PINK1 deficiency results in loss of dopaminergic neurons as well as early impairment of mitochondrial function and morphology in Danio rerio. Expression of TigarB, the zebrafish orthologue of the human, TP53-induced glycolysis and apoptosis regulator TIGAR, was markedly increased in pink−/− larvae. Antisense-mediated inactivation of TigarB gave rise to complete normalization of mitochondrial function, with resulting rescue of dopaminergic neurons in pink−/− larvae. There was also marked microglial activation in pink−/− larvae, but depletion of microglia failed to rescue the dopaminergic neuron loss, arguing against microglial activation being a key factor in the pathogenesis.
Interpretation: Pink1−/− zebrafish are the first vertebrate model of PINK1 deficiency with loss of dopaminergic neurons. Our study also identifies TIGAR as a promising novel target for disease-modifying therapy in PINK1-related PD. Ann Neurol 2013;74:837–847
Hematopoietic differentiation is controlled by key transcription factors, which regulate stem cell functions and differentiation. TAL1 is a central transcription factor for hematopoietic stem cell development in the embryo and for gene regulation during erythroid/megakaryocytic differentiation. Knowledge of the target genes controlled by a given transcription factor is important to understand its contribution to normal development and disease. To uncover direct target genes of TAL1 we used high affinity streptavidin/biotin-based chromatin precipitation (Strep-CP) followed by Strep-CP on ChIP analysis using ChIP promoter arrays. We identified 451 TAL1 target genes in K562 cells. Furthermore, we analysed the regulation of one of these genes, the catalytic subunit beta of protein kinase A (PRKACB), during megakaryopoiesis of K562 and primary human CD34+ stem cell/progenitor cells. We found that TAL1 together with hematopoietic transcription factors RUNX1 and GATA1 binds to the promoter of the isoform 3 of PRKACB (Cβ3). During megakaryocytic differentiation a coactivator complex on the Cβ3 promoter, which includes WDR5 and p300, is replaced with a corepressor complex. In this manner, activating chromatin modifications are removed and expression of the PRKACB-Cβ3 isoform during megakaryocytic differentiation is reduced. Our data uncover a role of the TAL1 complex in controlling differential isoform expression of PRKACB. These results reveal a novel function of TAL1, RUNX1 and GATA1 in the transcriptional control of protein kinase A activity, with implications for cellular signalling control during differentiation and disease.
Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair.
Objective: To determine the role of the AMPKα2 subunit in vascular repair.
Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice.
Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.
Parkinson's disease (PD) is a frequent neurodegenerative process in old age. Accumulation and aggregation of the lipid-binding SNARE complex component α-synuclein (SNCA) underlies this vulnerability and defines stages of disease progression. Determinants of SNCA levels and mechanisms of SNCA neurotoxicity have been intensely investigated. In view of the physiological roles of SNCA in blood to modulate vesicle release, we studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation) to identify effects of SNCA gain of function as potential disease biomarkers. Downregulation of complexin 1 (CPLX1) mRNA was correlated with genotype, but the expression of other Parkinson's disease genes was not. In global RNA-seq profiling of blood from presymptomatic PARK4 indviduals, bioinformatics detected significant upregulations for platelet activation, hemostasis, lipoproteins, endocytosis, lysosome, cytokine, Toll-like receptor signaling and extracellular pathways. In PARK4 platelets, stimulus-triggered degranulation was impaired. Strong SPP1, GZMH and PLTP mRNA upregulations were validated in PARK4. When analysing individuals with rapid eye movement sleep behavior disorder, the most specific known prodromal stage of general PD, only blood CPLX1 levels were altered. Validation experiments confirmed an inverse mutual regulation of SNCA and CPLX1 mRNA levels. In the 3′-UTR of the CPLX1 gene we identified a single nucleotide polymorphism that is significantly associated with PD risk. In summary, our data define CPLX1 as a PD risk factor and provide functional insights into the role and regulation of blood SNCA levels. The new blood biomarkers of PARK4 in this Turkish family might become useful for PD prediction.
The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.
Autophagy can act either as a tumor suppressor or as a survival mechanism for established tumors. To understand how autophagy plays this dual role in cancer, in vivo models are required. By using a highly heterogeneous C. elegans germline tumor, we show that autophagy-related proteins are expressed in a specific subset of tumor cells, neurons. Inhibition of autophagy impairs neuronal differentiation and increases tumor cell number, resulting in a shorter life span of animals with tumors, while induction of autophagy extends their life span by impairing tumor proliferation. Fasting of animals with fully developed tumors leads to a doubling of their life span, which depends on modular changes in transcription including switches in transcription factor networks and mitochondrial metabolism. Hence, our results suggest that metabolic restructuring, cell-type specific regulation of autophagy and neuronal differentiation constitute central pathways preventing growth of heterogeneous tumors.
It is long known that Kasugamycin inhibits translation of canonical transcripts containing a 5’-UTR with a Shine Dalgarno (SD) motif, but not that of leaderless transcripts. To gain a global overview of the influence of Kasugamycin on translation efficiencies, the changes of the translatome of Escherichia coli induced by a 10 minutes Kasugamycin treatment were quantified. The effect of Kasugamycin differed widely, 102 transcripts were at least twofold more sensitive to Kasugamycin than average, and 137 transcripts were at least twofold more resistant, and there was a more than 100-fold difference between the most resistant and the most sensitive transcript. The 5’-ends of 19 transcripts were determined from treated and untreated cultures, but Kasugamycin resistance did neither correlate with the presence or absence of a SD motif, nor with differences in 5’-UTR lengths or GC content. RNA Structure Logos were generated for the 102 Kasugamycin-sensitive and for the 137 resistant transcripts. For both groups a short Shine Dalgarno (SD) motif was retrieved, but no specific motifs associated with resistance or sensitivity could be found. Notably, this was also true for the region -3 to -1 upstream of the start codon and the presence of an extended SD motif, which had been proposed to result in Kasugamycin resistance. Comparison of the translatome results with the database RegulonDB showed that the transcript with the highest resistance was leaderless, but no further leaderless transcripts were among the resistant transcripts. Unexpectedly, it was found that translational coupling might be a novel feature that is associated with Kasugamycin resistance. Taken together, Kasugamycin has a profound effect on translational efficiencies of E. coli transcripts, but the mechanism of action is different than previously described.
Elevated tumor interstitial fluid pressure (TIFP) is a prominent feature of solid tumors and hampers the transmigration of therapeutic macromolecules, for example, large monoclonal antibodies, from tumor-supplying vessels into the tumor interstitium. TIFP values of up to 40 mm Hg have been measured in experimental solid tumors using two conventional invasive techniques: the wick-in-needle and the micropuncture technique. We propose a novel noninvasive method of determining TIFP via ultrasonic investigation with scanning acoustic microscopy at 30-MHz frequency. In our experimental setup, we observed for the impedance fluctuations in the outer tumor hull of A431-vulva carcinoma–derived tumor xenograft mice. The gain dependence of signal strength was quantified, and the relaxation of tissue was calibrated with simultaneous hydrostatic pressure measurements. Signal patterns from the acoustical images were translated into TIFP curves, and a putative saturation effect was found for tumor pressures larger than 3 mm Hg. This is the first noninvasive approach to determine TIFP values in tumors. This technique can provide a potentially promising noninvasive assessment of TIFP and, therefore, can be used to determine the TIFP before treatment approach as well to measure therapeutic efficacy highlighted by lowered TFP values.
Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.
Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends.
Pathogenicity of many microbes relies on their capacity to resist innate immunity, and to survive and persist in an immunocompetent human host microbes have developed highly efficient and sophisticated complement evasion strategies. Here we show that different human pathogens including Gram-negative and Gram-positive bacteria, as well as the fungal pathogen Candida albicans, acquire the human terminal complement regulator vitronectin to their surface. By using truncated vitronectin fragments we found that all analyzed microbial pathogens (n = 13) bound human vitronectin via the same C-terminal heparin-binding domain (amino acids 352–374). This specific interaction leaves the terminal complement complex (TCC) regulatory region of vitronectin accessible, allowing inhibition of C5b-7 membrane insertion and C9 polymerization. Vitronectin complexed with the various microbes and corresponding proteins was thus functionally active and inhibited complement-mediated C5b-9 deposition. Taken together, diverse microbial pathogens expressing different structurally unrelated vitronectin-binding molecules interact with host vitronectin via the same conserved region to allow versatile control of the host innate immune response.
Background: Sphingolipids constitute bioactive molecules with functional implications in liver homeostasis. Particularly, ablation of very long chain ceramides in a knockout mouse model has been shown to cause a severe hepatopathy.
Methods: We aimed to evaluate the serum sphingolipid profile of 244 patients with cirrhosis prospectively followed for a median period of 228±217 days via mass spectrometry.
Results: We thereby observed a significant decrease of long and very long chain ceramides, particularly of C24ceramide, in patients with increasing severity of cirrhosis (p<0.001). Additionally, hydropic decompensation, defined by clinical presentation of ascites formation, was significantly correlated to low C24ceramide levels (p<0.001) while a significant association to hepatic decompensation and poor overall survival was observed for low serum concentrations of C24ceramide (p<0.001) as well. Multivariate analysis further identified low serum C24ceramide to be independently associated to overall survival (standard beta = -0.001, p = 0.022).
Conclusions: In our current analysis serum levels of very long chain ceramides show a significant reciprocal correlation to disease severity and hepatic decompensation and are independently associated with overall survival in patients with cirrhosis. Serum sphingolipid metabolites and particularly C24ceramide may constitute novel molecular targets of disease severity, hepatic decompensation and overall prognosis in cirrhosis and should be further evaluated in basic research studies.