Different States Of Activation Research Articles

Erratum: Figge et al., "Differential Activation States of Direct Pathway Striatal Output Neurons during l-DOPA-Induced Dyskinesia Development".

Erratum: Figge et al., "Differential Activation States of Direct Pathway Striatal Output Neurons during l-DOPA-Induced Dyskinesia Development".

Differential Activation States of Direct Pathway Striatal Output Neurons during l-DOPA-Induced Dyskinesia Development.

l-DOPA-induced dyskinesia (LID) is a debilitating motor side effect arising from chronic dopamine (DA) replacement therapy with l-DOPA for the treatment of Parkinson's disease. LID is associated with supersensitivity of striatal dopaminergic signaling and fluctuations in synaptic DA following each l-DOPA dose, shrinking the therapeutic window. The heterogeneous composition of the striatum, including subpopulations of medium spiny output neurons (MSNs), interneurons, and supporting cells, complicates the identification of cell(s) underlying LID. We used single-nucleus RNA sequencing (snRNA-seq) to establish a comprehensive striatal transcriptional profile during LID development. Male hemiparkinsonian mice were treated with vehicle or l-DOPA for 1, 5, or 10 d, and striatal nuclei were processed for snRNA-seq. Analyses indicated a limited population of DA D1 receptor-expressing MSNs (D1-MSNs) formed three subclusters in response to l-DOPA treatment and expressed cellular markers of activation. These activated D1-MSNs display similar transcriptional changes previously associated with LID; however, their prevalence and transcriptional behavior were differentially influenced by l-DOPA experience. Differentially expressed genes indicated acute upregulation of plasticity-related transcription factors and mitogen-activated protein kinase signaling, while repeated l-DOPA-induced synaptic remodeling, learning and memory, and transforming growth factor-β (TGF-β) signaling genes. Notably, repeated l-DOPA sensitized Inhba, an activin subunit of the TGF-β superfamily, in activated D1-MSNs, and its pharmacological inhibition impaired LID development, suggesting that activin signaling may play an essential role in LID. These data suggest distinct subsets of D1-MSNs become differentially l-DOPA-responsive due to aberrant induction of molecular mechanisms necessary for neuronal entrainment, similar to processes underlying hippocampal learning and memory.

Probing infrared dark clouds with class I methanol masers and thermal molecular emission using the Onsala 20 meter telescope

ABSTRACT Infrared dark clouds (IRDCs) represent the earliest stage of high-mass star formation and host molecular cores at different states of activity from quiet state without any IR-signature of star formation to active state with IR-signatures and pronounced maser activity. Many IRDCs show indications of shocked gas associated with protostar outflows and can contain class I methanol masers (cIMMs). The aim of this study is to probe different types of cores in IRDCs with cIMMs and thermal molecular emission and to investigate the relationship between presence of cIMMs and physical conditions in IRDCs. For a sample of 37 molecular cores, using the 20-m Onsala radio telescope, we collected molecular line data at 44, 85, and 97 GHz for more than 15 species including CH3OH, CH3CCH, and CS. Kinetic temperature of the gas and molecular column densities were obtained. Methanol emission at 44 GHz was detected in 29 sources, with 4 sources are being new discoveries. None of the cores in quiescent state show emission at 44 GHz. Our results testify that cIMM emission is a reliable marker of advanced state of molecular cores. The higher detection rate for intermediate and IR-quiet sources suggests that cIMMs most readily trace the early stages of star formation characterized by moderate IR-signatures. We found that masers with higher flux densities tend to be associated with emission in the CH3CCH lines with higher integrated intensities. Sources undetected at 44 GHz have 4 times lower integrated intensities of CH3CCH and exhibit poorer molecular spectra than the most sources with cIMMs.

BSBM-07 HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Rapidly growing solid tumors such as glioblastoma (GBM) are characteristically hypoxic, displaying large areas of necrosis surrounded by hyperproliferative pseudopalisading cells. Intra-tumoral hypoxia develops over time in the three-dimensional space and the degree of tissue oxygenation is a dynamic process that varies continuously. Combined with the extensive inter- and intra-tumoral heterogeneity associated with GBM at the bulk and single cell level, hypoxia contributes to a gradient of molecular alterations that are specific to the different cell populations that make up the bulk of the tumor and reside in specific niches. To date, high dimensional histopathologic analyses of the hypoxic regions within GBM tissue have not been performed. Here, we took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study where the exogenous hypoxia marker pimonidazole (PIMO) is administered to patients with GBM prior to surgery. Tissue specimens were subjected to imaging mass cytometry and serial immunohistochemistry using a panel of 27 markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. We took high-resolution imaging and statistical approaches to explore the interplay of the different markers within hypoxic regions of primary and recurrent GBMs, in addition to IDH-mutant gliomas. Our findings elucidate the expression pattern of key biological markers relative to one another, altered composition of different cell types, along with differential proliferative, transcriptional, and translational activation states associated with each cell type within the hypoxic regions of GBM.

Motor neurons within a network use cell-type specific feedback mechanisms to constrain relationships among ion channel mRNAs.

Recently, activity has been proposed as a primary feedback mechanism used by continuously bursting neurons to coordinate ion channel mRNA relationships that underlie stable output. However, some neuron types only have intermittent periods of activity and so may require alternative mechanisms that induce and constrain the appropriate ion channel profile in different states of activity. To address this, we used the pyloric dilator (PD; constitutively active) and the lateral gastric (LG; periodically active) neurons of the stomatogastric ganglion (STG) of the crustacean Cancer borealis. We experimentally stimulated descending inputs to the STG to cause release of neuromodulators known to elicit the active state of LG neurons and quantified the mRNA abundances and pairwise relationships of 11 voltage-gated ion channels in active and silent LG neurons. The same stimulus does not significantly alter PD activity. Activation of LG upregulated ion channel mRNAs and lead to a greater number of positively correlated pairwise channel mRNA relationships. Conversely, this stimulus did not induce major changes in ion channel mRNA abundances and relationships of PD cells, suggesting their ongoing activity is sufficient to maintain channel mRNA relationships even under changing modulatory conditions. In addition, we found that ion channel mRNA correlations induced by the active state of LG are influenced by a combination of activity- and neuromodulator-dependent feedback mechanisms. Interestingly, some of these same correlations are maintained by distinct mechanisms in PD, suggesting that these motor networks use distinct feedback mechanisms to coordinate the same mRNA relationships across neuron types.NEW & NOTEWORTHY Neurons use various feedback mechanisms to adjust and maintain their output. Here, we demonstrate that different neurons within the same network can use distinct signaling mechanisms to regulate the same ion channel mRNA relationships.

HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Rapidly growing solid tumors such as glioblastoma (GBM) are characteristically hypoxic, displaying large areas of necrosis surrounded by hyperproliferative pseudopalisading cells. Intra-tumoral hypoxia develops over time in the three-dimensional space and the degree of tissue oxygenation is a dynamic process that varies continuously. Combined with the extensive inter- and intra-tumoral heterogeneity associated with GBM at the bulk and single cell level, hypoxia contributes to a gradient of molecular alterations that are specific to the different cell populations that make up the bulk of the tumor and reside in specific niches. To date, high dimensional histopathologic analyses of the hypoxic regions within GBM tissue have not been performed. Here, we took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study where the exogenous hypoxia marker pimonidazole (PIMO) is administered to patients with GBM prior to surgery. Tissue specimens were subjected to imaging mass cytometry and serial immunohistochemistry using a panel of 27 markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. We took high-resolution imaging and statistical approaches to explore the interplay of the different markers within hypoxic regions of primary and recurrent GBMs, in addition to IDH-mutant gliomas. Our findings elucidate the expression pattern of key biological markers relative to one another, altered composition of different cell types, along with differential proliferative, transcriptional, and translational activation states associated with each cell type within the hypoxic regions of GBM.

Dynamikuntersuchungen des Wachstumshormon‐Sekreteagogum‐Rezeptors geben Einblicke in seine Energielandschaft

AbstractG‐Protein‐gekoppelte Rezeptoren initiieren die Signaltransduktion nach Ligandenbindung. Gegenstand der Studie ist der Wachstumshormon‐Sekreteagogum‐Rezeptor (GHSR), der durch das Peptid Ghrelin aktiviert wird. Obwohl es Strukturen des GHSR in verschiedenen Stadien der Aktivierung gibt, wurde die molekulare Dynamik dieser Zustände bisher nicht beschrieben. Wir analysierten die Trajektorien von Molekulardynamiksimulationen mittels Detektoranalyse, um die Dynamik des Apo‐ und des Liganden‐gebundenen Zustands zu vergleichen. Diese Analyse liefert zeitskalenspezifische Bewegungsamplituden. Wir identifizierten Unterschiede in der Dynamik des Apo‐ und Ghrelin‐gebundenen Zustands, insbesondere in der extrazellulären Schleife 2 und den Transmembranhelices 5–7. Kernresonanz‐Spektren der Histidinreste im GHSR zeigten Unterschiede in den chemischen Verschiebungen dieser Regionen. Wir evaluierten zeitskalenspezifische Korrelationen der Bewegungen zwischen Ligand und Rezeptor und fanden Korrelationen zwischen den N‐terminalen Ghrelin‐Resten und dem Rezeptor, nicht aber für den C‐Terminus von Ghrelin. Eine Hauptkomponentenanalyse lieferte Rückschlüsse auf die Energielandschaft des GHSR.

Analysis of the Dynamics of the Human Growth Hormone Secretagogue Receptor Reveals Insights into the Energy Landscape of the Molecule.

G protein-coupled receptors initiate signal transduction in response to ligand binding. Growth hormone secretagogue receptor (GHSR), the focus of this study, binds the 28 residue peptide ghrelin. While structures of GHSR in different states of activation are available, dynamics within each state have not been investigated in depth. We analyze long molecular dynamics simulation trajectories using "detec-tors" to compare dynamics of the apo and ghrelin-bound states yield-ing timescale-specific amplitudes of motion. We identify differ-ences in dynamics between apo and ghrelin-bound GHSR in the extracellular loop 2 and transmembrane helices 5-7. NMR of the GHSR histidine residues reveals chemical shift differences in these regions. We eva-luate timescale specific correlation of motions between residues of ghre-lin and GHSR, where binding yields a high degree of correlation for the first 8 ghrelin residues, but less correlation for the helical end. Finally, we investigate the traverse of GHSR over a rugged energy land-scape via principal component analysis.Insert abstract text here.

Assessing the stability conditions of a slope movement in Northern Italy interacting with the provincial road network

Landslides can have devastating consequences on communities and the environment, including loss of life, destruction of homes and infrastructure, and loss of valuable natural resources. In particular, slope movements involving transport infrastructures such as roads and railways can have a significant impact both in terms of network efficiency and safety for vehicles to use. Therefore, it is extremely important to be aware of the potential risks and take the necessary precautions to mitigate the effect of these phenomena. The case study described in this paper deals with a landslide interacting with a provincial road, which represents the main connection between the city of Berceto (Italy) and the Cisa Motorway. Due to the importance of this road, and after the occurrence of several instability events over the years, a series of investigations were carried out in order to gather information regarding the geological features of the site and to provide a preliminary assessment of the stability of the area. The geological and geomorphological evidences collected during the survey point to a condition of general slope instability, characterized by different states of activity. This is confirmed by the results obtained from the execution of a series of stability analyses based on the Limit Equilibrium Method (LEM), which also underlined the influence of the water level variation on the general stability of the area.

TMIC-73. HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Rapidly growing solid tumors such as glioblastoma (GBM) are characteristically hypoxic, displaying large areas of necrosis surrounded by hyperproliferative pseudopalisading cells. Intra-tumoral hypoxia develops over time in the three-dimensional space and the degree of tissue oxygenation is a dynamic process that varies continuously. Combined with the extensive inter- and intra-tumoral heterogeneity associated with GBM at the bulk and single cell level, hypoxia contributes to a gradient of molecular alterations that are specific to the different cell populations that make up the bulk of the tumor and reside in specific niches. To date, high dimensional histopathologic analyses of the hypoxic regions within GBM tissue have not been performed. Here, we took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study where the exogenous hypoxia marker pimonidazole (PIMO) is administered to patients with GBM prior to surgery. Tissue specimens were subjected to imaging mass cytometry and serial immunohistochemistry using a panel of 27 markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. We took high-resolution imaging and statistical approaches to explore the interplay of the different markers within hypoxic regions of primary and recurrent GBMs, in addition to IDH-mutant gliomas. Our findings elucidate the expression pattern of key biological markers relative to one another, altered composition of different cell types, along with differential proliferative, transcriptional, and translational activation states associated with each cell type within the hypoxic regions of GBM.

Differential Activation and Desensitization States Promoted by Noncanonical α7 Nicotinic Acetylcholine Receptor Agonists.

A series of dipicolyl amine pyrimidines (DPPs) were previously identified as potential α7 agonists by means of a calcium influx assay in the presence of the positive allosteric modulator (PAM) 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596). The compounds lack the quaternary or strongly basic nitrogens of typical nicotinic agonists. Although differing in structure from typical nicotinic agonists, based on crystallographic data with the acetylcholine binding protein, they appeared to engage the site shared by such typical orthosteric agonists. Using oocytes expressing human α7 receptors, we found that the DPPs were efficacious activators of the receptor, with currents showing rapid desensitization characteristic of α7 receptors. However, we note that the rate of recovery from this desensitization depends strongly on structural features within the DPP family. Although the activation of receptors by DPP was blocked by the competitive antagonist methyllycaconitine (MLA), MLA had no effect on the DPP-induced desensitization, suggesting multiple modes of DPP binding. As expected, the desensitized conformational states could be reactivated by PAMs. Mutants made insensitive to acetylcholine by the C190A mutation in the agonist binding site were weakly activated by DPPs. The observation that activation of C190A mutants by the DPP compounds was resistant to the allosteric antagonist (-)cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide supports the hypothesis that the activity of these noncanonical agonists in the orthosteric binding sites was not entirely dependent on the classic epitopes controlling activation by typical agonists and that perhaps they may access alternative modes for promoting the conformational changes associated with activation and desensitization. SIGNIFICANCE STATEMENT: This study reports a family of nicotinic acetylcholine receptor agonists that break the rules about what the structure of a nicotinic acetylcholine receptor agonist should be. It shows that the activity of these noncanonical agonists in the orthosteric binding sites is not dependent on the classical epitopes controlling activation by typical agonists and that through different binding poses, they promote unique conformational changes associated with receptor activation and desensitization.

High-Resolution Imaging of Human Cancer Proteins Using Microprocessor Materials.

Mutations in tumor suppressor genes, such as Tumor Protein 53 (TP53), are heavily implicated in aggressive cancers giving rise to gain- and loss-of-function phenotypes. While individual domains of the p53 protein have been studied extensively, structural information for full-length p53 remains incomplete. Functionalized microprocessor chips (microchips) with properties amenable to electron microscopy permitted us to visualize complete p53 assemblies for the first time. The new structures revealed p53 in an inactive dimeric state independent of DNA binding. Residues located at the protein-protein interface corresponded with modification sites in cancer-related hot spots. Changes in these regions may amplify the toxic effects of clinical mutations. Taken together, these results contribute advances in technology and imaging approaches to decode native protein models in different states of activation.

Impact of Adolescent Intermittent Ethanol Exposure on Social Investigation, Social Preference, and Neuronal Activation in cFos-LacZ Male and Female Rats.

Adolescence is a sensitive developmental period during which alcohol use is often initiated and consumed in high quantities, often at binge or even high-intensity drinking levels. Our lab has repeatedly found that adolescent intermittent ethanol (AIE) exposure in rats results in long-lasting social impairments, specifically in males, however our knowledge of the neuronal underpinnings to this sex-specific effect of AIE is limited. The present study was designed to test whether social anxiety-like alterations in AIE-exposed males would be accompanied by alterations of neuronal activation across brain regions associated with social behavior, with AIE females demonstrating no social impairments and alterations in neuronal activation. Adolescent male and female cFos-LacZ transgenic rats on a Sprague-Dawley background were exposed to ethanol (4 g/kg, 25% v/v) or water via intragastric gavage every other day during postnatal days (P) 25–45 for a total of 11 exposures (n = 13 per group). Social behavior of adult rats was assessed on P70 using a modified social interaction test, and neuronal activation in brain regions implicated in social responding was assessed via β-galactosidase (β-gal) expression. We found that AIE exposure in males resulted in a significantly lower social preference coefficient relative to water-exposed controls, with no effect evident in females. Exposure-specific relationships between social behavior and neuronal activation were identified, with AIE eliminating correlations found in water controls related to social interaction, and eliciting negative correlations mainly in limbic regions in a sex-specific manner. AIE exposure in the absence of social testing was also found to differentially affect neural activity in the orbitofrontal cortex and central amygdala in males and females. These data suggest that AIE produces sex-specific social impairments that are potentially driven by differential neuronal activation states in regions important for social behavior, including the medial prefrontal and orbitofrontal cortices, nucleus accumbens, lateral septum, and central amygdala. Future studies should be focused on identification of specific neuronal phenotypes activated by interaction with a social partner in AIE-exposed subjects and their control counterparts.

Metastable dynamics of neural circuits and networks.

Cortical neurons emit seemingly erratic trains of action potentials or “spikes,” and neural network dynamics emerge from the coordinated spiking activity within neural circuits. These rich dynamics manifest themselves in a variety of patterns, which emerge spontaneously or in response to incoming activity produced by sensory inputs. In this Review, we focus on neural dynamics that is best understood as a sequence of repeated activations of a number of discrete hidden states. These transiently occupied states are termed “metastable” and have been linked to important sensory and cognitive functions. In the rodent gustatory cortex, for instance, metastable dynamics have been associated with stimulus coding, with states of expectation, and with decision making. In frontal, parietal, and motor areas of macaques, metastable activity has been related to behavioral performance, choice behavior, task difficulty, and attention. In this article, we review the experimental evidence for neural metastable dynamics together with theoretical approaches to the study of metastable activity in neural circuits. These approaches include (i) a theoretical framework based on non-equilibrium statistical physics for network dynamics; (ii) statistical approaches to extract information about metastable states from a variety of neural signals; and (iii) recent neural network approaches, informed by experimental results, to model the emergence of metastable dynamics. By discussing these topics, we aim to provide a cohesive view of how transitions between different states of activity may provide the neural underpinnings for essential functions such as perception, memory, expectation, or decision making, and more generally, how the study of metastable neural activity may advance our understanding of neural circuit function in health and disease.

Monitoring the Levels of Cellular NF-κB Activation States.

Simple SummaryIn stress and disease situations, cells must rapidly and in a coordinated manner change their gene expression patterns to respond adequately. The NF-κB system comprises five transcription factors that are released from the cytosol to enter the nucleus in response to a wide range of extracellular stimuli via a complex cytosolic signaling system. In the nucleus, activated NF-κB dimers bind to specific chromatin loci across the entire genome and induce the expression of a broad repertoire of genes that regulate immune and inflammatory responses. Consistent with its biological importance, the extent of NF-κB activity is regulated and controlled at multiple levels. The aim of this review is to comprehensively present and discuss the currently available conceptual and methodological approaches to monitor the molecular activation status of the NF-κB system, including multi-level single cell analysis.The NF-κB signaling system plays an important regulatory role in the control of many biological processes. The activities of NF-κB signaling networks and the expression of their target genes are frequently elevated in pathophysiological situations including inflammation, infection, and cancer. In these conditions, the outcome of NF-κB activity can vary according to (i) differential activation states, (ii) the pattern of genomic recruitment of the NF-κB subunits, and (iii) cellular heterogeneity. Additionally, the cytosolic NF-κB activation steps leading to the liberation of DNA-binding dimers need to be distinguished from the less understood nuclear pathways that are ultimately responsible for NF-κB target gene specificity. This raises the need to more precisely determine the NF-κB activation status not only for the purpose of basic research, but also in (future) clinical applications. Here we review a compendium of different methods that have been developed to assess the NF-κB activation status in vitro and in vivo. We also discuss recent advances that allow the assessment of several NF-κB features simultaneously at the single cell level.

A neuronal population model based on cellular automata to simulate the electrical waves of the brain

Neural oscillations as synchronized activity of large numbers of neurons in the neural ensembles have always been a hot topic of research for experimental and theoretical studies because of their high importance in human behaviors and functions. Since there is not yet a comprehensive mathematical model for simulating brainwaves, we proposed a cellular automaton (CA) model of a neuronal population by considering the different states of an action potential at the cellular level and simple connectivity patterns . Some important characteristics of a neural network are included in the model, such as different states of activation of a neuron, and excitatory and inhibitory synapses. Our computational model can display different dynamics from fixed-point and limit-cycle to chaotic behaviors similar to different dynamics of a real neuronal population in the brain. Qualitative and quantitative comparisons of the real electroencephalogram data and the CA simulations in the linear and nonlinear domains demonstrated the efficiency of our CA network to simulate the electrical brain activity. Time series from the proposed model display a high-dimensional stochastic behavior that corresponds to the behavior of a healthy brain. Therefore, this model can be used to study biological neuronal populations and provide more insight into their different mechanisms.

Electroencephalographic Microstates in Schizophrenia and Bipolar Disorder.

Schizophrenia (SCH) and bipolar disorder (BD) are characterized by many types of symptoms, damaged cognitive function, and abnormal brain connections. The microstates are considered to be the cornerstones of the mental states shown in EEG data. In our study, we investigated the use of microstates as biomarkers to distinguish patients with bipolar disorder from those with schizophrenia by analyzing EEG data measured in an eyes-closed resting state. The purpose of this article is to provide an electron directional physiological explanation for the observed brain dysfunction of schizophrenia and bipolar disorder patients.Methods: We used microstate resting EEG data to explore group differences in the duration, coverage, occurrence, and transition probability of 4 microstate maps among 20 SCH patients, 26 BD patients, and 35 healthy controls (HCs).Results: Microstate analysis revealed 4 microstates (A–D) in global clustering across SCH patients, BD patients, and HCs. The samples were chosen to be matched. We found the greater presence of microstate B in BD patients, and the less presence of microstate class A and B, the greater presence of microstate class C, and less presence of D in SCH patients. Besides, a greater frequent switching between microstates A and B and between microstates B and A in BD patients than in SCH patients and HCs and less frequent switching between microstates C and D and between microstates D and C in BD patients compared with SCH patients.Conclusion: We found abnormal features of microstate A, B in BD patients and abnormal features of microstate A, B, C, and D in SCH patients. These features may indicate the potential abnormalities of SCH patients and BD patients in distributing neural resources and influencing opportune transitions between different states of activity.

Transcriptomic Analysis of Rat Macrophages.

The laboratory rat is widely used as a model for human diseases. Many of these diseases involve monocytes and tissue macrophages in different states of activation. Whilst methods for in vitro differentiation of mouse macrophages from embryonic stem cells (ESC) and bone marrow (BM) are well established, these are lacking for the rat. The gene expression profiles of rat macrophages have also not been characterised to the same extent as mouse. We have established the methodology for production of rat ESC-derived macrophages and compared their gene expression profiles to macrophages obtained from the lung and peritoneal cavity and those differentiated from BM and blood monocytes. We determined the gene signature of Kupffer cells in the liver using rats deficient in macrophage colony stimulating factor receptor (CSF1R). We also examined the response of BM-derived macrophages to lipopolysaccharide (LPS). The results indicate that many, but not all, tissue-specific adaptations observed in mice are conserved in the rat. Importantly, we show that unlike mice, rat macrophages express the CSF1R ligand, colony stimulating factor 1 (CSF1).

Abstract 810: Beyond genomics - A precision oncology approach utilizing multiplex protein profiling for tumor and tumor organoid samples

Abstract Precision medicine's goal of achieving a better response rate by avoiding ineffective therapies has sparked new approaches, including the testing of patient-derived 3d (PD3Ds) tumor cultures for modeling individual patient response, and the use of various molecular pathology techniques for advanced tumor profiling. An intrinsic limitation of well-established genomics methodologies is, however, that they cannot directly assess cell-signaling activity within the tumor cells defined by the phosphorylation status of cellular signaling pathway networks. Here we present the development of a robust protein profiling strategy utilizing the DigiWest immuno-assay platform, to obtain data on the activation status of key cellular signaling networks implicated in cancer, and on proteins targeted by FDA-approved drugs. This includes targeted cancer therapies for e.g. EGFR, HER2, PI3K, mTOR, ALK and AKT. Accordingly, we compiled a list of relevant pathway nodes and their phosphorylation sites that yield activity information on RAS/RAF/ERK, PI3K/AKT and mTOR signaling pathways. Based on this, we validated 242 total and phospho antibodies in a pre-set oncoproteomic DigiWest panel that yields information on the activity of all major signaling pathways from the receptor level down to transcription factors, apoptosis and proliferation. This oncoproteomic panel can be utilized for elucidating drug response in pre-clinical cell models, in PD3D organoid models or in clinical tumor samples. Exemplary, we show differential activation states in cancer cell lines representing the most prevalent cancer types and the impact of pathway activation in different models. Finally, we tested this panel in PD3D organoids that were subjected to screening against common targeted therapies. While the initial results are promising, further work on evaluating how such an oncoproteomic panel profiling might contribute to the rationale for personalized therapy decisions is required. Disclosure Statement: NMI TT Pharmaservices offers DigiWest protein profiling as a service. CELLphenomics offers PD3D cell cultures and drug testing as a service. Citation Format: Gerrit Erdmann, Przemylaw Dudys, Anja Arndt, Christian Regenbrecht, Markus Templin, Christoph Sachse. Beyond genomics - A precision oncology approach utilizing multiplex protein profiling for tumor and tumor organoid samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 810.

MP07-19 ACYLOXYACYL HYDROLASE REGULATES MICROGLIAL PHENOTYPE IN A MOUSE MODEL OF INTERSTITIAL CYSTITIS

INTRODUCTION AND OBJECTIVE: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a devastating condition of chronic pelvic pain. We previously linked a locus encoding acyloxyacyl hydrolase (Aoah) with pain severity in a murine model of IC. AOAH-deficient mice develop pelvic allodynia and exhibit symptoms co-morbid with IC/BPS, such as altered voiding and a depressive phenotype. Microglia are the resident immune cells of the central nervous system (CNS), and studies have linked altered microglial morphology and activation to neuropathic pain and depression. It is also well established that microglial activation can be modulated by the gut microbiota, and AOAH dysbiosis has been reported in IC/BPS patients. Therefore, we examined the microglial phenotype in AOAH-deficient mice and the potential role of microglia in modulating pelvic pain. METHODS: To analyze changes in microglial morphology, brains from wild type (WT) C57BL/6 and AOAH-deficient mice were sectioned and stained for the microglial marker P2RY12. Following immunohistochemistry, z-stacks were acquired and skeletal analyses were performed using Image J. Activation of microglia was measured through Western blotting and a proteome profiler array of mouse cytokines using brain lysates. To address the functional role of microglia in pelvic pain, microglia were eliminated from the CNS of AOAH-deficient and WT mice by oral administration of chow containing PLX5622. Mice were exposed to mechanical stimulation of the pelvic area with von Frey filaments prior to and following microglial elimination. RESULTS: Our preliminary data revealed altered microglial morphology in AOAH-deficient mice compared to WT mice. AOAH-deficient mice exhibited hyper-ramified microglia, suggesting a surveillance phenotype, in the cortex and Barrington’s nucleus, an essential region for micturition. We also observed hyper-ramified microglia in the paraventricular nucleus, a regulator of stress and depression. Microglia were less ramified in the hippocampus, consistent with an activated phenotype. Due to the morphological changes, we expect to observe differences in cytokine release between WT and AOAH-deficient mice, reflecting these differential activation states. In addition, we expect that microglia elimination will alleviate pelvic allodynia and depression in AOAH-deficient mice. CONCLUSIONS: Overall, we observed that microglial phenotype is dependent on AOAH, suggesting microglia-specific receptors or cytokines may be considered as future therapeutic targets for treating IC/BPS. Source of Funding: NIDDK awards U01 DK082342 and R01 DK066112-06S1