Podcasts about shrna

BUFFALO, NY- March 19, 2024 – A new #research paper was #published in Aging (listed by MEDLINE/PubMed as "Aging (Albany NY)" and "Aging-US" by Web of Science) Volume 16, Issue 5, entitled, “PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation.” Cellular senescence is a permanent cell cycle arrest that can be triggered by both internal and external genotoxic stressors, such as telomere dysfunction and DNA damage. The execution of senescence is mainly by two pathways, p16/RB and p53/p21, which lead to CDK4/6 inhibition and RB activation to block cell cycle progression. While the regulation of p53/p21 signaling in response to DNA damage and other insults is well-defined, the regulation of the p16/RB pathway in response to various stressors remains poorly understood. In this new study, researchers Chitra Palanivel, Lepakshe S. V. Madduri, Ashley L. Hein, Christopher B. Jenkins, Brendan T. Graff, Alison L. Camero, Sumin Zhou, Charles A. Enke, Michel M. Ouellette, and Ying Yan from the University of Nebraska Medical Center report a novel function of PR55α, a regulatory subunit of PP2A Ser/Thr phosphatase, as a potent inhibitor of p16 expression and senescence induction by ionizing radiation (IR), such as γ-rays. “During natural aging, there is a gradual accumulation of p16-expressing senescent cells in tissues [76]. To investigate the significance of PR55α in this up-regulation of p16, we compared levels of the p16 and PR55α proteins in a panel of normal tissue specimens derived from young (≤43 y/o) and old (≥68 y/o) donors.” The results show that ectopic PR55α expression in normal pancreatic cells inhibits p16 transcription, increases RB phosphorylation, and blocks IR-induced senescence. Conversely, PR55α-knockdown by shRNA in pancreatic cancer cells elevates p16 transcription, reduces RB phosphorylation, and triggers senescence induction after IR. Furthermore, this PR55α function in the regulation of p16 and senescence is p53-independent because it was unaffected by the mutational status of p53. Moreover, PR55α only affects p16 expression but not p14 (ARF) expression, which is also transcribed from the same CDKN2A locus but from an alternative promoter. In normal human tissues, levels of p16 and PR55α proteins were inversely correlated and mutually exclusive. “Collectively, these results describe a novel function of PR55α/PP2A in blocking p16/RB signaling and IR-induced cellular senescence.” DOI - https://doi.org/10.18632/aging.205619 Corresponding authors - Michel M. Ouellette - mouellet@unmc.edu, and Ying Yan - yyan@unmc.edu About Aging-US: Aging publishes research papers in all fields of aging research including but not limited, aging from yeast to mammals, cellular senescence, age-related diseases such as cancer and Alzheimer's diseases and their prevention and treatment, anti-aging strategies and drug development and especially the role of signal transduction pathways such as mTOR in aging and potential approaches to modulate these signaling pathways to extend lifespan. The journal aims to promote treatment of age-related diseases by slowing down aging, validation of anti-aging drugs by treating age-related diseases, prevention of cancer by inhibiting aging. Cancer and COVID-19 are age-related diseases. Aging is indexed by PubMed/Medline (abbreviated as “Aging (Albany NY)”), PubMed Central, Web of Science: Science Citation Index Expanded (abbreviated as “Aging‐US” and listed in the Cell Biology and Geriatrics & Gerontology categories), Scopus (abbreviated as “Aging” and listed in the Cell Biology and Aging categories), Biological Abstracts, BIOSIS Previews, EMBASE, META (Chan Zuckerberg Initiative) (2018-2022), and Dimensions (Digital Science). Please visit our website at https://www.Aging-US.com​​. MEDIA@IMPACTJOURNALS.COM

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537277v1?rss=1 Authors: Baidoe-Ansah, D., Mirzapourdelavar, H., Carceller, H., Perez, M., Strackeljan, L., Garcia, B., Seidenbecher, C. I., Kaushik, R., Nacher, J., Dityatev, A. Abstract: The condensed form of neural extracellular matrix (ECM), perineuronal nets (PNNs), is predominantly associated with parvalbumin-expressing (PV+) interneurons in the cortex and hippocampus. PNNs are enriched in several lecticans, including neurocan (Ncan). A polymorphism in the human Ncan gene has been associated with alterations in hippocampus-dependent memory function, variation of prefrontal cortex structure, and a higher risk for schizophrenia or bipolar disorder. Ncan knockout (KO) mice show related behavioral abnormalities, such as hyperactivity. Here we focused on studying how dysregulation of Ncan specifically in the mPFC may affect cognitive and synaptic functions. Intracortical adeno-associated virus (AAV) delivery was used to express shRNA against Ncan. Analysis of PNNs in Ncan shRNA-injected mice revealed a reduction in PNNs labelling by Wisteria floribunda agglutinin (WFA) around PV+ interneurons. Reduced Ncan expression resulted in a loss of the mPFC-dependent temporal order recognition and impairment of reversal spatial learning in a labyrinth (dry maze) task. As a potential synaptic substrate of these cognitive abnormalities, we report a robust reduction in the perisomatic GABAergic innervation of PV+ cells in Ncan KO and Ncan shRNA-injected mice. We also observed an increase in the density of vGLUT1-immunopositive synaptic puncta in the neuropil of Ncan shRNA-injected mice, which was, however, compensated in Ncan KO mice. Thus, our findings highlight a functional role of Ncan in supporting perisomatic GABAergic inhibition, temporal order recognition memory and cognitive flexibility, as one of the important cognitive resources depleted in neuropsychiatric disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.30.526323v1?rss=1 Authors: Hermanstyne, T. O., Yang, N.-D., Granados-Fuentes, D., Li, X., Mellor, R., Jegla, T., Herzog, E., Nerbonne, J. M. Abstract: Considerable evidence suggests that day-night rhythms in the functional expression of subthreshold potassium (K+) channels regulate daily oscillations in the rates of spontaneous action potential firing of neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The K+ conductance(s) driving these daily rhythms in repetitive firing rates, however, have not been identified. To test the hypothesis that subthreshold Kv12.1/Kv12.2-encoded K+ channels play a role, we obtained current-clamp recordings from SCN neurons in slices prepared from adult mice harboring targeted disruptions in the Kcnh8 (Kv12.1-/-) or Kcnh3 (Kv12.2-/-) locus. We found that mean nighttime repetitive firing rates were higher in Kv12.1-/- and Kv12.2-/-, than in wild type (WT), SCN neurons. In marked contrast, mean daytime repetitive firing rates were similar in Kv12.1-/-, Kv12.2-/- and WT SCN neurons, and the day-night difference in mean repetitive firing rates, a hallmark feature of WT SCN neurons, was eliminated in Kv12.1-/- and Kv12.2-/- SCN neurons. Similar results were obtained with in vivo shRNA-mediated acute knockdown of Kv12.1 or Kv12.2 in adult SCN neurons. Voltage-clamp experiments revealed that Kv12-encoded current densities in WT SCN neurons are higher at night than during the day. In addition, pharmacological block of Kv12-encoded currents increased the mean repetitive firing rate of nighttime, but not daytime, in WT SCN neurons. Dynamic clamp-mediated subtraction of modeled Kv12-encoded currents also selectively increased the mean repetitive firing rates of nighttime WT SCN neurons. Despite the elimination of nighttime decrease in the mean repetitive firing rates of SCN neurons, however, locomotor (wheel-running) activity remained rhythmic in Kv12.1-/-, Kv12.2-/-, Kv12.1-targeted shRNA-expressing, and Kv12.2-targeted shRNA-expressing animals. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.30.525978v1?rss=1 Authors: Reeb, T., Rhea, L., Adelizzi, E., Garnica, B., Dunnwald, E., Dunnwald, M. Abstract: BACKGROUND: RhoA GTPase plays critical roles in actin cytoskeletal remodeling required for controlling a diverse range of cellular functions including cell proliferation, cell adhesions, migration and changes in cell shape. RhoA cycles between an active GTP-bound and an inactive GDP-bound form, a process that is regulated by guanine nucleotide exchange factors (GEFs), and GTPase-activating proteins (GAPs). ARHGAP29 is a GAP expressed in keratinocytes of the skin and is decreased in the absence of Interferon Regulator Factor 6, a critical regulator of cell proliferation and migration. However, the role for ARHGAP29 in keratinocyte biology is unknown. RESULTS: Novel ARHGAP29 knockdown keratinocyte cell lines were generated using both CRISPR/Cas9 and shRNA technologies. Knockdown cells exhibited significant reduction of ARHGAP29 protein (50-80%) and displayed increased filamentous actin (stress fibers), phospho-myosin light chain (contractility), cell area and population doubling time. Furthermore, we found that ARHGAP29 knockdown keratinocytes displayed significant delays in scratch wound closure in both single cell and collective cell migration conditions. Particularly, our results show a reduction in path lengths, speed, directionality and persistence in keratinocytes with reduced ARHGAP29. The delay in scratch closure was rescued by both adding back ARHGAP29 or adding a ROCK inhibitor to ARHGAP29 knockdown cells. CONCLUSIONS: These data demonstrate that ARHGAP29 is required for keratinocyte morphology, proliferation and migration mediated through the RhoA pathway. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Everyone knows that we are not all the same, there is wonderful diversity in our bodies, our genetics, our lifestyles, and our preferences. And yet, when it comes to nutrition, the most successful public health messages are the broad guidelines, which suggests one size can fit all.   Think five-a-day, taking Vitamin D through the autumn and winter, and so on. At the same time, the science behind nutrition, the understanding of our metabolism and of our gut microbiome, has been increasing at a fantastic rate. The question is: how do you bring these two worlds together?   How do you bring the best of intricate nutritional science to a broader population? Could the answer lay in precision nutrition?   It is an emerging and exciting field which helps tailor dietary recommendations and nutritional guidelines, and there is some evidence it can have remarkable health impacts. It is an area which seems to offer huge potential, but exactly how much is yet to be discovered. Karen Vousden, Principal Group Leader, Francis Crick Institute Karen received her PhD from the University of London and following postdoctoral fellowships at the ICR and NCI, she returned to London to establish a research group at the Ludwig Institute.  Returning to the US, she was Chief of the Regulation of Cell Growth Laboratory at the NCI before coming back to the UK to take on the role of Director of the CRUK Beatson Institute in Glasgow.   In 2017, she moved her research group to the Francis Crick Institute in London and served as Chief Scientist for Cancer Research UK from 2016-2022. Karen's research has made contributions to our understanding of how the tumour suppressor protein p53 is regulated and the functions of p53 that contribute to its ability to control cancer progression.   During these studies, her group revealed an unexpected ability of p53 to help cells adapt and survive under transient periods of nutrient starvation.  This work led to a more general investigation of cancer cell metabolism, focused on exploring the role of oxidative stress and serine metabolism in cancer development and metastatic progression. Greg Hannon, Director of Cancer Research UK Cambridge Institute Greg Hannon FRS FMedSci is a professor of molecular cancer biology and director of the Cancer Research UK Cambridge Research Institute at the University of Cambridge.   Professor Hannon is internationally recognised for his contributions to small RNA biology, cancer biology, and mammalian genomics.  He has a long history in the discovery of cancer genes, beginning with work at CSHL that led to the identification of CDK inhibitors and their links to cancer.   More recently, his work has focused on small RNA biology, which led to an understanding of the biochemical mechanisms and biological functions of RNAi.   Building upon this foundation, he has developed widely-used tools and strategies for manipulation of gene expression in mammalian cells and animals and has generated genome-wide shRNA libraries that are available to the cancer community.   He was among the first to uncover roles for microRNAs in cancer, including the discovery of the miR-17-92 cluster as an oncogene, the placement of miR-34 within the p53 pathways, and the understanding that let-7 and miR-93 are critical regulators of both normal stem cells and tumour initiating cells in several tissues.   His laboratory also discovered the piRNA pathway and linked this to transposon repression and the protection of germ cell genomes. 

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.11.519794v1?rss=1 Authors: Lopez-Erauskin, J., Bravo-Hernandez, M., Presa, M., Baughn, M. W., Melamed, Z., Beccari, M. S., Agra de Almeida Quadros, A. R., Zuberi, A., Ling, K., Platoshyn, O., Nino-Jara, E., Ndayambaje, I. S., Arnold-Garcia, O., McAlonis-Downes, M., Cabrera, L., Artates, J. W., Ryan, J., Bennett, F., Jafar-nejad, P., Rigo, F., Marsala, M., Lutz, C. M., Cleveland, D. W., Lagier-Tourenne, C. Abstract: The human mRNA most affected by TDP-43 loss-of-function is transcribed from the STMN2 gene and encodes stathmin-2 (also known as SCG10), whose loss is a neurodegenerative disease hallmark. Here using multiple in vivo approaches, including transient antisense oligonucleotide (ASO)-mediated suppression, chronic shRNA-mediated depletion in aging mice, and germline deletion, we establish stathmin-2 to be essential for acquisition and maintenance of neurofilament-dependent structuring of axoplasm critical for maintaining diameter and conduction velocity of large-myelinated axons. Sustained stathmin-2 loss from an otherwise mature adult nervous system is demonstrated over a time course of eight months to initiate and drive motor neuron disease that includes 1) shrinkage in inter-neurofilament spacing that is required to produce a three-dimensional space filling array that defines axonal caliber, 2) collapse of mature axonal caliber with tearing of outer myelin layers, 3) reduced conduction velocity, 4) progressive motor and sensory deficits (including reduction of the pain transducing neuropeptide CGRP), and 5) muscle denervation. Demonstration that chronic stathmin-2 reduction is itself sufficient to trigger motor neuron disease reinforces restoration of stathmin-2 as an attractive therapeutic approach for TDP-43-dependent neurodegeneration, including the fatal adult motor neuron disease ALS. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.09.515863v1?rss=1 Authors: Shouib, R., Eitzen, G. Abstract: Airway epithelial cells can respond to incoming pathogens, allergens and stimulants through the secretion of cytokines and chemokines. These pro-inflammatory mediators activate inflammatory signaling cascades that allow a robust immune response to be mounted. However, uncontrolled production and release of cytokines and chemokines can result in chronic inflammation and appears to be an underlying mechanism for the pathogenesis of pulmonary disorders such as asthma and COPD. The Rho GTPase, Cdc42, is an important signaling molecule that we hypothesize can regulate cytokine production and release from epithelial cells. We treated BEAS-2B lung epithelial cells with a set of stimulants to activate inflammatory pathways and cytokine release. The production, trafficking and secretion of cytokines were assessed when Cdc42 was pharmacologically inhibited with ML141 drug or silenced with lentiviral-mediated shRNA knockdown. We found that Cdc42 inhibition with ML141 differentially affected gene expression of a subset of cytokines; transcription of IL-6 and IL-8 were increased while MCP-1 was decreased. However, Cdc42 inhibition or depletion disrupted IL-8 trafficking and reduced its secretion even though transcription was increased. Cytokines transiting through the Golgi were particularly affected by Cdc42 disruption. Our results define a role for Cdc42 in the regulation of cytokine production and release in airway epithelial cells. This underscores the role of Cdc42 in coupling receptor activation to downstream gene expression and also as a regulator of cytokine secretory pathways. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.04.515170v1?rss=1 Authors: Datta, S., Sugatha, J., Priya, A., Raj, P., Jaimon, E., Jose, A. Abstract: Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Even with the vast amount of information unveiled systematically, the underlying mechanism of sorting remains elusive. Here, we established that SNX32, a SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member, is associated with SNX4 via its BAR domain. We identified A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 at the interface of these two SNX proteins that are important for maintaining the association. Via its PX domain, SNX32 interacts with the Transferrin receptor (TfR) and Cation Independent Mannose-6-Phosphate Receptor (CIMPR). We showed that the conserved F131 in its PX domain is important in stabilising the above interactions. Silencing of SNX32 led to a defect in intracellular trafficking of TfR and CIMPR, which could be rescued by overexpressing shRNA-resistant snx32. We also showed that both individual domains play an essential role in trafficking. Our results indicate that SNX4, SNX32 and Rab11 may participate in a common pathway regulating transferrin trafficking; however, the existence of an independent pathway for Rab11 and SNX32 could not be completely ruled out. Further, we established that the PX domain of SNX32 could bind to PI(3)P and PI(4)P, suggesting a possible explanation for its sub-cellular localization. Taken together, our study showed that SNX32 mediate the trafficking of specific cargo molecules along distinct pathway via its PX domain-directed binding to phosphoinositides and its BAR domain-mediated association with other SNX family members. Further, using SILAC-based differential proteomics of the wild type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin super family member, as a potential interactor of SNX32 in SH-SY-5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In Neuro-Glial cell lines, the silencing of SNX32 led to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32 depleted cells led us to propose that the SNX may contribute to maintaining the neuro-glial coordination via its role in BSG trafficking and the associated Monocarboxylate transporter activity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512949v1?rss=1 Authors: Merchant, J. P., Zhu, K., Henrion, M. Y. R., Zaidi, S. S. A., Branden, L., Moein, S., Alamprese, M. L., Pearse, R. V., Bennett, D. A., Ertekin-Taner, N., Young-Pearse, T. L., Chang, R. Abstract: Despite decades of genetic studies on late onset Alzheimer disease (LOAD), the molecular mechanisms of Alzheimer disease (AD) remain unclear. Furthermore, different cell types in the central nervous system (CNS) play distinct roles in the onset and progression of AD pathology. To better comprehend the complex etiology of AD, we used an integrative approach to build robust predictive (causal) network models which were cross-validated over multiple large human multi-omics datasets in AD. We employed a published method to delineate bulk-tissue gene expression into single cell-type gene expression and integrated clinical and pathologic traits of AD, single nucleotide variation, and deconvoluted gene expression for the construction of predictive network models for each cell type in AD. With these predictive causal models, we are able to identify and prioritize robust key drivers of the AD-associated network state. In this study, we focused on neuron-specific network models and prioritized 19 predicted key drivers modulating AD pathology. These targets were validated via shRNA knockdown in human induced pluripotent stem cell (iPSC) derived neurons (iNs), in which 10 out of the 19 neuron-related targets (JMJD6, NSF, NUDT2, YWHAZ, RBM4, DCAF12, NDRG4, STXBP1, ATP1B1, and FIBP) significantly modulated levels of amyloid-beta and/or phosphorylated tau peptides in the postmitotic iNs. Most notably, knockdown of JMJD6 significantly altered the neurotoxic ratios of A{beta} to 40 and p231-tau to total tau, indicating its potential therapeutic relevance to both amyloid and tau pathology in AD. Molecular validation by RNA sequencing (RNAseq) in iNs further confirmed the network structure, showing significant enrichment in differentially expressed genes after knockdown of the validated targets. Interestingly, our network model predicts that these 10 key drivers are upstream regulators of REST and VGF, two recently identified key regulators of AD pathogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.21.513252v1?rss=1 Authors: Yang, Q., Perfitt, T. L., Quay, J., Hu, L., Colbran, R. J. Abstract: Clustering of neuronal L-type voltage-gated Ca2+ channels (LTCC) in the plasma membrane is increasingly implicated in creating highly localized Ca2+ signaling nanodomains. For example, LTCC activation can increase phosphorylation of the nuclear CREB transcription factor by increasing Ca2+ concentrations within a nanodomain close to the channel, without requiring bulk Ca2+ increases in the cytosol or nucleus. However, the molecular basis for LTCC clustering is poorly understood. The postsynaptic scaffolding protein Shank3 specifically associates with one of the major neuronal LTCCs, the CaV1.3 calcium channel, and is required for optimal LTCC-dependent excitation-transcription coupling. Here, we co-expressed CaV1.3 1 subunits with two distinct epitope-tags with or without Shank3 in HEK cells. Co-immunoprecipitation studies using the cell lysates revealed that Shank3 can assemble multiple CaV1.3 1 subunits in a complex under basal conditions. Moreover, CaV1.3 LTCC complex formation was facilitated by CaV{beta} subunits ({beta}3 and {beta}2a), which also interact with Shank3. Shank3 interactions with CaV1.3 LTCCs and multimeric CaV1.3 LTCC complex assembly were disrupted following addition of Ca2+ and calmodulin (Ca2+/CaM) to cell lysates, perhaps simulating conditions within an activated CaV1.3 LTCC nanodomain. In intact HEK293T cells, co-expression of Shank3 enhanced the intensity of membrane-localized CaV1.3 LTCC clusters under basal conditions, but not after Ca2+ channel activation. Live cell imaging studies also revealed that Ca2+ influx through LTCCs disassociated Shank3 from CaV1.3 LTCCs clusters and reduced the CaV1.3 cluster intensity. Deletion of the PDZ domain from Shank3 prevented both binding to CaV1.3 and the changes in multimeric CaV1.3 LTCC complex assembly in vitro and in HEK293 cells. Finally, we found that shRNA knock-down of Shank3 expression in cultured rat primary hippocampal neurons reduced the intensity of surface-localized CaV1.3 LTCC clusters in dendrites. Taken together, our findings reveal a novel molecular mechanism contributing to neuronal LTCC clustering under basal conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.512990v1?rss=1 Authors: Kang, S., Choi, L. S., Im, S., Lee, K. W., Kim, D. H., Park, J. H., Park, M.-H., Lee, J., Park, S. K., Kim, K. P., Lee, H. M., Jeon, H. J., Park, H. S., Yoo, S.-K., Kim Pak, Y. Abstract: Parkinsons disease (PD), characterized by degeneration of dopaminergic neurons, share pathogenic features with obesity, including mitochondrial dysfunction and oxidative stress. Paraoxonase 2 (PON2) is an inner mitochondrial membrane protein that is highly expressed in dopaminergic neurons and is involved in the regulation of mitochondrial oxidative stress. However, no drug targeting PON2 has ever been developed for the treatment of PD. Here, we show that vutiglabridin, a clinical phase 2-stage drug for the treatment of obesity, has therapeutic effects in PD models, targeting mitochondrial PON2. Vutiglabridin penetrates into the brain, binds to PON2, and restores 1-methyl-4-phenylpyridinium (MPP+)-induced mitochondrial dysfunction in SH-SY5Y neuroblastoma cells. Knockdown of PON2 by lentiviral shRNA infection abolished the effects of vutiglabridin on mitochondria. In mice, vutiglabridin significantly alleviated motor impairments and damage to dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model, and these effects were also abolished in PON2-knockdown mice, suggesting that vutiglabridin is neuroprotective via PON2. Extensive in vitro and in vivo assessment of potential neurotoxicity showed vutiglabridin to be safe. Overall, these findings provide support for the clinical development of vutiglabridin as a novel PON2 modulator for the treatment of PD. One Sentence SummaryTargeting paraoxonase-2 by a clinical-stage compound vutiglabridin provides neuroprotective effects in preclinical models of Parkinsons disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512953v1?rss=1 Authors: Zhu, K., He, Q., Tsai, S.-F., Mudalige, D. M., Tang, A., Henrion, M. Y. R., Liu, Y., Vijayan, R., Zaidi, S. S. A., Branden, L., Cadiz, M. P., Hodos-Nkhereanye, R., Moein, S., Alamprese, M. L., Bennett, D. A., De Jager, P., Kuo, Y.-M., Ronaldson, P. T., Chang, R. Abstract: Microglia, the innate immune cells of the brain, are essential determinants of late-onset Alzheimer Disease (LOAD) neuropathology. Here, we developed an integrative computational systems biology approach to construct causal network models of genetic regulatory programs for microglia in Alzheimer Disease (AD). This model enabled us to identify novel key driver (KDs) genes for microglial functions that can be targeted for AD pharmacotherapy. We prioritized FCER1G, HCK, LAPTM5, ITGB2, SLC1A2, PAPLN, GSAP, NTRK2, and CIRBP as KDs of microglial phagocytosis promoting neuroprotection and/or neural repair. In vitro, shRNA knockdown of each KD significantly reduced microglial phagocytosis. We repurposed riluzole, an FDA-approved ALS drug that upregulates SLC1A2 activity, and discovered that it stimulated phagocytosis of A{beta}1-42 in human primary microglia and decreased hippocampal amyloid plaque burden/phosphorylated tau levels in the brain of aged 3xTg-AD mice. Taken together, these data emphasize the utlility of our integrative approach for repurposing drugs for AD therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.05.506638v1?rss=1 Authors: Uzay, B., Hokelekli, F. O., Yilmaz, M., Esen, E. C., Basar, K., Bahadir-Varol, A., Ayhan, Y., Dalkara, T., Eren-Kocak, E. Abstract: Fibroblast growth factor-2 (FGF2) is involved in the regulation of affective behavior and shows antidepressant effects through Akt and ERK1/2 pathways. NUDT6 is a protein encoded from the antisense strand of the FGF2 gene and its role in the regulation of affective behavior is unclear. Here, we show that increasing NUDT6 expression in the hippocampus results in depression-like behavior in rats without changing FGF2 levels or activating its downstream effectors, Akt and ERK1/2. Instead, NUDT6 acts by inducing inflammatory signaling, specifically by increasing S100A9 levels, activating NF-kB, and rising microglia number along with a reduction in neurogenesis. Conversely, inhibition of hippocampal NUDT6 expression by shRNA results in antidepressant effects and increases neurogenesis without altering FGF2 levels. Together these findings suggest that NUDT6 may play a role in major depression by inducing a proinflammatory state and serve as a novel therapeutic target for antidepressant development. The opposite effects of NUDT6 and FGF2 on depression-like behavior may serve as a mechanism to fine-tune affective behavior. Our findings open up new venues for studying the differential regulation and functional interactions of sense and antisense proteins in neural function and behavior as well as in neuropsychiatric disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Celyad Oncology's CMO, Dr. Charlie Morris, shares the benefits and challenges of the allogeneic approach versus the autologous approach and why a non-gene edited shRNA approach may result in better efficacy and safety for CAR-T therapies.

Oncotarget published this trending research paper on June 4, 2019, entitled, “Dissecting the role of RNA modification regulatory proteins in melanoma,” by researchers from the Department of Pathology, Yale University School of Medicine, and the Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham. “Since RNA is a key molecule that drives every cellular process, their deregulation is present in nearly all human disease and play a causative role.” The researchers explain that alterations among RNAs may arise due to altered activity or expression of the enzymes/proteins which are involved in the modification process. In this study, the team used multiple publicly available bioinformatics platforms to, first, analyze RNA alterations in melanoma samples, and then, to comprehensively analyze RNA modification regulatory proteins among melanoma samples. The publicly available datasets included: The Cancer Genome Atlas, The Human Protein Atlas, Oncomine, and the UALCAN database. “Our study started with the analysis of various genetic alterations (amplifications, mutations/deletion) as well as RNA overexpression of these RNA modification regulatory proteins in The Cancer Genome Atlas melanoma database.” Based on their analyses of these databases, reverse transcription quantitative PCR, soft-agar assays, validation by shRNA-mediated knockdown, and statistical analysis, the team identified what they believe are the most relevant RNA modifying proteins that play a crucial role in the development of melanoma. They found that METTL4 and DNMT3A RNA-modifying enzymes/proteins are both necessary for melanoma growth and overexpressed in melanoma. “Based on this we infer that the upregulated expression of RNA modification regulatory proteins METTL4 and DNMT3A play a key role in melanoma initiation or progression.” Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.26959 DOI - https://doi.org/10.18632/oncotarget.26959 Full text - https://www.oncotarget.com/article/26959/text/ Correspondence to - Romi Gupta - romigup@uab.edu Keywords - RNA modifications, epitranscriptome, melanoma, MAPK, BRAF mutant melanoma, skin cancer About Oncotarget Oncotarget is a bi-weekly, peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com or connect with: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget YouTube - https://www.youtube.com/c/OncotargetYouTube/ LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Oncotarget is published by Impact Journals, LLC please visit https://www.ImpactJournals.com or connect with @ImpactJrnls Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

This month on Episode 19 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the December 4 issue of Circulation Research. This episode features an in-depth conversation with Drs Mete Civelek and Redouane Aherrahrou, from the University of Virginia regarding their study titled Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells.   Article highlights:   Zahreddine, et al. Tamoxifen and E2 Effects On Reendothelialization   Zheng, et al. Arterial Stiffness Preceding Diabetes   Galang, et al. ATAC-seq Identifies Novel Isl1 SAN Enhancer   Cindy St. Hilaire:              Hi, welcome to Discover CircRes, the podcast to The American Heart Association’s journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from The Vascular Medicine Institute at the University of Pittsburgh, and today I will be highlighting three articles selected from the December 4’th issue of Circ Res. Drs Mete Civelek and Redouane Aherrahrou, from the University of Virginia, are here to discuss their study, Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells. Cindy St. Hilaire:              The first article I want to share is titled, Tamoxifen Accelerates Endothelial Healing by Targeting Estrogen Receptor-alpha in Smooth Muscle Cells. The first author is Rana Zahreddine, and the corresponding author is John Francois Arnal and they're from INSERM and the University of Toulouse, France. For breast cancers that contain high levels of estrogen receptor, a standard treatment is to give drugs that block either estrogen production or the receptor itself, such as tamoxifen. However, estrogen can elicit beneficial vascular protective effects, so treatment with tamoxifen might increase the risk of cardiovascular disease. Depending on the tissue, tamoxifen can both antagonize or activate estrogen receptor, so it's role in cardiovascular disease is unclear. Cindy St. Hilaire:              Some evidence even suggest tamoxifen might have protective effects, such as promoting vascular endothelial healing. Zahreddine and colleagues now show that while might suffering damage to the endothelial lining of a blood vessel have improved healing when treated with tamoxifen, or with estrogen, those suffering perivascular injury, that is to say, the injury that affects both the endothelial layer as well as the surrounding smooth muscle cell layer, heal only in response to estrogen. This suggests tamoxifen's healing effects might require smooth muscle cells. In mice, lacking the estrogen receptor and smooth muscle cells, they found estrogen, but not tamoxifen, healed endovascular injuries. While in mice lacking estrogen receptor and endothelial cells alone, they found the opposite. This work reveals nuances in the molecular actions of tamoxifen that should inform further assessment of its risk and benefits for use in patients. Cindy St. Hilaire:              The second article I want to share is titled, Arterial Stiffness Proceeding Diabetes, A Longitudinal Study. The first authors are Mengyi Zheng and Xinyuan Zhang and the corresponding authors are Xiang Gao and Shouling Wu, from Pennsylvania State University and North China University of Science and Technology. As a person ages, their risk of developing diabetes and cardiovascular disease increases. Aging is also linked to increase in arterial stiffness and high blood pressure, but how all these individual conditions affect and influence each other is not entirely clear. For example, while arterial stiffness and diabetes tend to correlate, whether one increases the risk, or the other, or the risk relationship or whether the risk relationship is bi-directional, is unknown. Cindy St. Hilaire:              To assess the interplay between these disease states, Zheng and colleagues studied diabetes and arterial stiffness in a cohort of 8,956 Chinese people between 2010 and 2015, none of whom had had diabetes or cardiovascular disease at the outset of the study. With repeated measures of fasting glucose levels, which is an indicator of diabetes, and pulse wave velocity, which is a measure of arterial stiffness, the team found that participants with a higher baseline arterial stiffness were more likely to develop diabetes during the five-year period than those with lower stiffness levels. Out of the original cohort of just over 8,900 individuals, a total of 979 individuals developed diabetes during the study. Higher baseline glucose levels did not predict future arterial stiffness; this suggests a risk relationship that is a one-way street. While the results require confirmation in additional cohorts, this finding is the first to identify the pathological mechanisms linking arterial stiffness to diabetes.  Cindy St. Hilaire:             The third article I want to share is titled, ATAC-Seq Reveals an ISL1 Enhancer That Regulates Sinoatrial Node Development and Function. The first author is Giselle Galang, Ravi Mandla, and Hongmei Ruan. And the corresponding author is Vasanth Vedantham, from the University of California, San Francisco. Pacemaker cells, of the sinoatrial node, establish and control the rhythmic contractions of the heart. These cells differ from regular cardiomyocytes in their transcription profiles, but how this transcriptional profile is established and maintained is not fully understood. To investigate the epigenetic landscape defining pacemaker cell fate, Galang and colleagues have employed a technique called ATAC-Seq, which identifies areas of the genome with accessible open chromatin structures, which is an indication of transcriptional activity. The team compared the genomes of pacemaker cells with atrial cardiomyocytes, and found a number of pacemaker cell-specific accessible loci that had both large numbers of transcription factor binding sequences and enhancer activity, when assayed in mice. Cindy St. Hilaire:              The team went on to specifically characterize one novel enhancer upstream of the gene, encoding ISL1, which is a key transcription factor for pacemaker cell identity. They showed that deleting the enhancer caused under development of the Sinoatrial node and arrhythmias in mice. They also noted that single cell nucleotide polymorphisms at the equivalent loci in humans, have been linked to variations in resting heart rate. The report verifies ATAC-Seq as an effective tool for identifying pacemaker enhancers and will launch future studies into how such enhancers function in heart development and disease. Cindy St. Hilaire:              Okay, so today with me is Dr Mete Civelek and Dr Redouane Aherrahrou, from the University of Virginia, and they're here to discuss their paper titled, Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells. And this article is featured in our December 4th issue. So thank you both so much for being here with me today. Mete Civelek:                   Great to talk to you Cindy. Thank you for choosing our paper. Cindy St. Hilaire:              Yeah, and seeing you over Zoom, I wish these were in person, but... Redouane Aherrahrou:  Thank you for having us. Cindy St. Hilaire:              Yeah. Great. So, before we dig into the details of this paper, which I think is a really nice paper, one of the things I like about it is that it couples GWAS with some functional things, which is obviously super important for figuring out what is important in that GWAS data. So, before we dig into the nitty-gritty of the paper, could you maybe explain what a GWAS study is, and what the strengths and weaknesses are, in terms of using that as an approach to figure out disease related pathophysiology? Mete Civelek:                   So, we know that coronary artery disease, or these cardio-metabolic diseases, have a genetic component, and in the past we used to do linkage studies, studying families, but in the last 15 years or so, because of the developments in technology, we can do these genome-wide association studies. And essentially what they do is they look at a population and some of the people in the population will have coronary artery disease and some people will not have coronary artery disease, will be otherwise healthy. And then you study the genetic variance across the entire genome and look for frequency differences in the people with the healthy phenotype and people who have the disease. And of course you do some statistical tests to find if this frequency differences is indeed statistic, the different between these two groups and you identify essentially loci that are associated with the disease. Mete Civelek:                   But I see GWAS as almost a detective work, you say something like, okay, let's say there was a murder in the United States, and then now you do GWAS of course to find the murder, right? But what that tells you is, okay, the murder occurred in, let's say Pittsburgh or Charlottesville or Washington DC, sure, it narrows down the scope of where you're going to look at, but it doesn't tell you exactly what happened and where it happened and things like that. And so after GWAS there many more questions to answer looking at the molecular mechanism of the locus, the tissue or cell type of action, the gene, which is being affected by the locus to affect the phenotype. So, it's very good at narrowing down possibilities and coming up with hypotheses, but then the real work begins. Cindy St. Hilaire:              I was wondering actually, as you were saying that, have there ever been... I guess like false discoveries, where people have really focused in, on a loci, because it came up maybe in one or multiple studies, but then maybe it didn't prove to be causative or they still can't figure it out. Are there examples of that? Mete Civelek:                   The most obvious example is actually the 9p21 locus. Cindy St. Hilaire:              Interesting. That's the one I was thinking of actually. Mete Civelek:                   Which has been associated with coronary artery disease susceptibility in all kinds of studies and in kinds of populations, this signal itself is real, what it's doing is been a lot of debate. Some people think that it's affecting the CDKN2A and 2B genes nearby. Cindy St. Hilaire:              Is that p21 or p16? Mete Civelek:                   One is p21 and one is p16, but I can't remember which one. Cindy St. Hilaire:              Yeah, I can't either. Mete Civelek:                   Right. And then there's a non-coding RNA in that region called lnRNA. Some people think it's affecting and lncRNA expression. Some people think it's affecting isoform abundance, so that's just probably the most famous locus in our field, in terms of figuring out what it's doing. Yeah. Cindy St. Hilaire:              Well, at least it's probably causing a lot of people to think of a lot of good questions to ask, so that's exciting. In your study, you state that you want to focus on the impact of coronary artery disease associated variants in atherosclerosis-relevant smooth muscle cell phenotypes, and the phenotypes you wanted to focus on were calcification, which is my personal favorite. So calcification, proliferation, and migration. So I was wondering why you wanted to focus on these phenotypes and then what kind of functional assays did you do? Redouane Aherrahrou:  So, the reason we choose those phenotypes because they are playing important role in the disease. So, for example, during the advanced stage of the disease, smooth muscles cells, they proliferate and migrate to make the fibrous cap. So the fibrous cap is actually stabilize the plaque against the rupture, and also during the advanced stage of the disease, the calcification also happening, a lot of people believe that the calcification also contribute to instability of the fibrous cap. So that's why we focus on those three phenotype, migration, proliferation, and calcification. Cindy St. Hilaire:              Interesting, and so I think you'd said you had about 150 patients in your study. Does that mean you did these functional assays in 150 different cell lines? Or how did you do that? Redouane Aherrahrou:  That's a good question. So we conducted actually, our assays from 150 healthy and multi-ethnic donors, so those people actually did die from motorcycle and car accidents, and the doctors actually use the chunk of the aorta where we'll actually isolate these cells from, and then they are actually healthy enough to use for the heart transplantation. Cindy St. Hilaire:              Wow. And so were you introducing known SNPs or SNPs that are pulled out of GWAS into the cells, or did the cells already have the SNPs available? How was the correlation done between functionality and SNPs? Redouane Aherrahrou:  That's a great question. So we actually use the natural SNPs that already exist in those donors. And we ask the question how the genetic variants of those donor affects migration, proliferation, and calcification phenotypes. Mete Civelek:                   So we essentially perform a GWAS in a dish- Cindy St. Hilaire:              Yeah, that's kind of what I was thinking- Mete Civelek:                   That's the bottom line, you just culture these cells and do these phenotypic characterizations, which you cannot do in healthy living human beings of course, and then just the naturally occurring genetic variation in these individuals, in these donors, to essentially calculate the association between the genetic variants and then these phenotypes. Cindy St. Hilaire:              And you were using aortic smooth muscle cells, right? Mete Civelek:                   Yes. Cindy St. Hilaire:              Do you think... this is one thing I always think about, especially because kind of harping back to Mark Majesky's early work with the chick embryo and developmental origins. Do you think if you had coronary arteries from the same individuals that the smooth muscle cells would respond similarly? Mete Civelek:                   This is a really good question, partially, yes and partially, no. I'll give you one specific example, for example, one of the loci that is associated with coronary artery disease is over a transcription factor called TCF21. And TCF21 is actually playing an important role in smooth muscle cell phenotypes, and that transcription factor is expressed only in coronary artery smooth muscle cells, but not in aortic smooth muscle cells. Cindy St. Hilaire:              Interesting. Mete Civelek:                   This was something that Dr Tom Quertermous from Stanford showed. So presumably we are capturing some of the genetic variation that's important in coronary artery disease as some of it was probably missing because we're using aortic smooth muscle cells. Cindy St. Hilaire:              Yeah. That is so neat. I really like that heat map you had, I think it was figure 4 because you really lined up along the SNPs that were identified in these patients you looked at the effect of that SNPs on a specific function test, you did, and you did, was it 11 functional tests? Mete Civelek:                   12 different functional tests. Cindy St. Hilaire:              12? Mete Civelek:                   Yes. Cindy St. Hilaire:              It's an amazing amount of work really. Well, how long have you been working on this project? Mete Civelek:                   Redouane, why don't you answer this question? Cindy St. Hilaire:              Or do you not want to talk about that? Redouane Aherrahrou:  Of course, it's not easy actually to culture and characterize 151 smooth muscle because you expect sometimes, you capture them, some of them, they will not grow, some of them they get contaminated, and you have to perform it again. And also, you cannot do the same experiment for all of them at the same time. So what we did actually, before we started the experiments, we decided to take a smooth muscle cell from one donor, and expanded many times and then we use the same donor to run each time for all the experiments, just to count for the batch and environment effects. Cindy St. Hilaire:              Yep. Redouane Aherrahrou:  So it took me actually almost one and a half year, to finish the characterization for all 151 smooth muscle cell. At that time I was also using also two incubators and then you can imagine, when you put the incubator- Cindy St. Hilaire:              It's full. Redouane Aherrahrou:  ...and I try to finish that, and then I again, start the experiment again to finish the other batch. Cindy St. Hilaire:              Oh God. Yeah, my lab also... we work only in primary human tissues from vessels, but also from valves. So my staff will certainly appreciate all your efforts for this paper. Mete Civelek:                   And you can also imagine there was this group of undergraduates, trailing- Cindy St. Hilaire:              An army yeah. Mete Civelek:                   Redouane wherever he goes….they were helping him out with many aspects. Cindy St. Hilaire:              Oh, sure that's amazing- Mete Civelek:                   He mentored, I think maybe five, six different undergraduate students throughout this project and they're all part of the paper. Yeah. Cindy St. Hilaire:              That's excellent. So towards the end of the study, you guys really focused on a gene, MIA3. So can we talk a little bit about that? What is this gene? What its normal function? Is it known? And then what did you find out in relation to smooth muscle cells? Mete Civelek:                   Well, I'll start and Redouane you can continue. So let me just walk back just a little bit to tell you how we kind of decided to focus on that. So we identified the 79 loci that are associated with smooth muscle cell phenotypes and coronary artery disease. So we wanted to show at least some kind of a validation and so we looked for loci that are not associated with lipids because we thought they will maybe not be important in smooth muscle cells. And then we then looked for loci out of those, who affect a nearby gene expression in aorta, in smooth muscle cells, but not in endothelial cells and in monocyte so we thought that will give us confidence- Cindy St. Hilaire:              So kind of enriching for this smooth muscle cell? Mete Civelek:                   And this MIA3 popped up and there was only one study actually that showed that, in codes for protein that localizes to the ER exit site and affects these COPII carriers, which secrete collagen into extracellular matrix. Well, collagen as you know, is important in cell stability and what smooth cell muscle cells produced. So, that's how we decided to focus on that gene. And I will let Redouane describe I guess, what we did with that gene. Redouane Aherrahrou:  Yeah. So after the function and mutation, we come up with this gene. So the first thing we did, because we found that the genetic variety in this locus affects actually proliferation in smooth muscle cell. So to what it did at least, this is actually SNP that's affecting the proliferation of smooth muscle cell by this gene, we down-regulate this gene actually in smooth muscle cell using two different shRNA, and we found actually the downregulation of this gene lead to affect the proliferation in sense that dominant regulation of this gene will affect the proliferation. Redouane Aherrahrou:  And also we found that the same genetic variance also in this gene lead to lower periphery, migration, sorry, the expression of this gene in small as I said, and then also in the aortic tissue. And interestingly, we did not see in the monocytes macrophages or other cell type, actually in the aortic tissue suggesting that this genetic variance affecting the coronary artery disease via smooth muscle cell. Mete Civelek:                   And we also collaborated with Renu Virmani's group at CVPath Institute, stained lesions, coronary artery lesions, which have these thick caps and thin caps as you very well know that is really relevant to plaque stability, and show that the thin caps have fewer smooth muscle cells which were positive for this protein MIA3, which was all in line with our genetic findings, because our genetic findings basically show that lower expression of this gene was associated with increased susceptibility to coronary artery disease. Cindy St. Hilaire:              Interesting. I wonder if it could be correlated with plaque rupture, right. If there's less smooth muscle cell, obviously, then a thin fibrous cap is nothing that anybody wants. So is the proliferation of the smooth muscle cell almost protective in a sense, that's one of the things people are starting to think about in calcification, [Alina has these amazing imaging studies, where she looks at matrix vesical accumulation and calcification mitosis. And really the field has noticed, with work by Linda Demer also, the field  has noticed that a large, huge chunk of calcification seems to be much less risky, I guess, compared to the microcalcifications, and I wonder if MIA3 might be like that too, if you can have just enough smooth muscle cell proliferation to kind of keep that cap thicker, is that more protective? Mete Civelek:                   I think that's a really good point that you are raising, because some of the answer is, in that figure 4 in the paper that you mentioned. What we find is that these loci, when people have the risk allele of these loci, so obviously people are at higher risk for coronary artery disease, some of them are associated with higher proliferation, but some of them associated with lower proliferation, same with calcification, same with migration. So it's really difficult to say, at least just looking at the genetic loci, yes, higher proliferation is always better. Cindy St. Hilaire:              Yeah. Mete Civelek:                   There's probably this really delicate balance that allows for plaque stability. Cindy St. Hilaire:              Yeah, it's reminisent, I guess, of the IO1-beta story, right? Mete Civelek:                   Exactly. Cindy St. Hilaire:              Gary Owens and colleagues have really shown that well. The role of it in early versus late plaque is different and it's complicated. That's what we learned. Mete Civelek:                   I agree. And it's specific to MIA3, that locus is also associated with myocardial infarction. Cindy St. Hilaire:              Oh, interesting. Mete Civelek:                   So indeed there is a possibility that really affects plaque stability. Cindy St. Hilaire:              Yeah. So Mete, say you and I are in the same faculty class in that we both started- Mete Civelek:                   Yes we are. We are classmates. Cindy St. Hilaire:              2015 we started our labs, and this is obviously a huge undertaking and really starting a project like this... when you're new, it's really a risk. You're proposing to collect 150 smooth muscle cell lines and characterize them all functionally and it turned out amazingly, but can you maybe talk about the early days in this project's development, and was there ever a moment where you're like, "What the heck am I doing? Is this going to work or..." Just kind of maybe talk to us a bit about that. Mete Civelek:                   That's a really excellent question. To be totally honest with you, I was really lucky to have recruited Redouane to the lab, but he and I worked together when I was a postdoc and when he was a PhD student, as part of this little consortium. So I knew he was going to work hard on his part and he was very driven- Cindy St. Hilaire:              He had magic hands. Mete Civelek:                   And he really want to do this project. So, I knew that it was going to work, but of course he and I both had these moments of, "Are we sure of what we're doing, and why are we doing this? Are we going to get something out of it?" And it was both of us kind of pumping each other, if you will say like, "Yep, this is going to work, we know it's going to work, we have faith in this," but I should also say that my lab also works on adipose tissue biology, and I already had another kind of a safe project going on in that realm. Cindy St. Hilaire:              So, that's funny, I started my lab that way too. I kind of had the project that was the direct continuation of my K grant, and then this kind of high risk, high reward project on valves and you know what, I think that's something that’s smart, it's kind of you have two tracks of research and hopefully one works and then the other one will work. And if they don't work at the same time, hopefully the other one can fill in the gaps, so... Mete Civelek:                   I totally agree with you, but truly, Redouane made a big difference in this project, imagine a postdoc, who's doing nothing but cell culture for two years, hoping that something is going to come out, that's a big risk for him too, it certainly paid off and it's paying off because he has two other papers in the pipeline from this project. Cindy St. Hilaire:              Wonderful. That's excellent. So what's the future for this project? What's kind of the next question you're going to ask if you don't mind sharing. Mete Civelek:                   Oh no, not at all. The most obvious one is looking at gene expression. So we have cultured these cells under two distinct conditions, one is the more contractile phenotype, one is the more productive phenotype. And we did RNA-Seq from, again 150 of these individuals in both conditions and we did what is known as eQTL mapping, so looking at the effect of the genetic loci on gene expression. In a separate project, we also actually collected media from these cells and looked at secreted proteins in the media and we're also finding the genetic loci that are affecting secreted protein, because as you very well know, smooth muscle cells secrete proteins to stabilize plaque stability. So those two papers are Redouane's next projects. And he's almost finished with one and has finished the analysis of the other ones, so hopefully- Cindy St. Hilaire:              That's exciting. Mete Civelek:                   ...more papers coming out in the next six months or so. Oh, I should have said the paper was chosen for the Genomic and Precision Medicine Counci; Young Investigator Award, so Redouane is competing- Cindy St. Hilaire:              Wonderful, also you are in that, excellent. Thank you both so much for joining me today. This was a lovely paper, it was actually inspiring. It made me think about some way to think about my calcification studies. Mete Civelek:                   Thank you so much, Cindy. This was really wonderful. Cindy St. Hilaire:              Absolutely. Thank you. That's it for the highlights from the December 4th issue of Circulation Research. Thank you very much for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Drs Mete Civelek and Redouane Aherrahrou. This podcast is produced by Rebecca McTavish and Ishara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire. And this Discover CircRes, your on the go source, for the most up-to-date and exciting discoveries in basic cardiovascular research.  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.385971v1?rss=1 Authors: Hernandez, L. M., Montersino, A., Niu, J., Guo, S., Thomas, G. Abstract: Janus Kinase-1 (JAK1) plays key roles in pro-survival signaling during neurodevelopment and in responses to neuronal injury. JAK1 was identified as a potential palmitoyl-protein in high-throughput studies, but the importance of palmitoylation for roles of JAK1 in neurons has not been addressed. Here, we report that JAK1 is endogenously palmitoylated in cultured Dorsal Root Ganglion (DRG) neurons and, using an shRNA knockdown/rescue approach, reveal that JAK1 palmitoylation is important for neuropoietic cytokine-dependent signaling and neuronal survival. We further identify the related palmitoyl acyltransferases (PATs) ZDHHC3 and ZDHHC7 as dominant regulators of JAK1 palmitoylation in transfected non-neuronal cells and endogenously in neurons. At the molecular level, palmitoylation is critical for JAK1 kinase activity in transfected cells and even in vitro, likely because palmitoylation facilitates transphosphorylation of key sites in the JAK1 activation loop. These findings provide new insights into palmitoylation-dependent control of neuronal development and survival. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378240v1?rss=1 Authors: Sanders, S. S., Hernandez, L. M., Soh, H., Karnam, S., Walikonis, R. S., Tzingounis, A., Thomas, G. M. Abstract: The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type I PDZ domain-containing proteins. However, ZDHHC14's neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the Axon Initial Segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14's importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.24.311985v1?rss=1 Authors: Snyder, N. W., Singh, B., Buchan, G., O'Brien, J., Arroyo, A. D., Liu, X., Sobol, R. W., Blair, I. A., Mesaros, C. A., Wendell, S. G. Abstract: Metabolism of PUFA results in the formation of hydroxylated fatty acids that can be further oxidized by dehydrogenases, often resulting in the formation of electrophilic, ,{beta}-unsaturated ketone containing fatty acids. As electrophiles are associated with redox signaling, we sought to investigate the metabolism of the oxo-fatty acid products in relation to their double bond architecture. Using an untargeted liquid chromatography mass spectrometry approach, we identified mono- and di-saturated products of the arachidonic acid-derived 11-oxoeicosatetraenoic acid (11-oxoETEs) and mono-saturated metabolites of 15-oxoETE and 17-oxoDHA in both human A549 lung carcinoma and umbilical vein endothelial cells. Notably, mono-saturated oxo-fatty acids maintained their electrophilicity as determined by nucleophilic conjugation to glutathione while a second saturation of 11-oxoETE resulted in a loss of electrophilicity. These results would suggest that prostaglandin reductase, known for its reduction of the ,{beta}-unsaturated double bond, was not responsible for saturation of oxo-fatty acids. Surprisingly, the knock down of prostaglandin reductase by shRNA confirmed its participation in the formation of 15-oxoETE and 17-oxoDHA mono-saturated metabolites. These findings will further facilitate the study of electrophilic fatty acid metabolism and signaling in the context of inflammatory diseases and cancer where they have been shown to have anti-inflammatory and anti-proliferative signaling properties. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.23.310284v1?rss=1 Authors: Hwa, L. S., Neira, S., Pati, D., Pina, M., Bowling, M. E., Calloway, R., kash, t. Abstract: The dynorphin/kappa opioid receptor (KOR) system in the brain regulates both stressful experiences and negative, aversive states during withdrawal from drugs of abuse. We explored the role of this system during acute withdrawal from long-term alcohol drinking, focusing on the bed nucleus of the stria terminalis (BNST), a region strongly implicated in alcohol-withdrawal induced alterations of behavior. Male C57BL/6J mice were subjected to repeated forced swim tests, home cage exposure to a predator odor, and a visual threat after eight weeks intermittent access to alcohol or water. Systemic injection of KOR antagonist norBNI reversed alcohol-related differences in immobility time during the second swim test and reduced burying behavior in response to predator odor, but did not affect behavioral response to visual threat. In dynorphin-GFP reporter mice, c-Fos immunoreactivity was increased in dynorphin-containing neurons after repeated swim stress and alcohol drinking. Using dynorphin-GFP mice, there was enhanced spontaneous excitatory synaptic drive onto dynorphin neurons in the BNST after alcohol-drinking mice underwent forced swim stress. Finally, knockdown of dynorphin in the BNST using a viral shRNA affected swim stress behavior and responses to TMT in alcohol drinkers and controls, but did not affect alcohol drinking. These studies confirm BNST dynorphin recruitment during acute withdrawal as playing a key role in altered behavioral responses to stressful stimuli. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.11.292722v1?rss=1 Authors: Mironov, A., Denisov, S., Gress, A., Kalinina, O. V., Pervouchine, D. Abstract: Tandem alternative splice sites (TASS) is a special class of alternative splicing events that are characterized by a close tandem arrangement of splice sites. Most TASS lack functional characterization and are believed to arise from splicing noise. Based on the RNA-seq data from the Genotype Tissue Expression project, we present an extended catalogue of TASS in healthy human tissues and analyze their tissue-specific expression. The expression of TASS is usually dominated by one major splice site (maSS), while the expression of minor splice sites (miSS) is at least an order of magnitude lower. Among 73k miSS with sufficient read support, 12k (17%) are significantly expressed above the expected noise level, and among them 2k are expressed tissue-specifically. We found significant correlations between tissue-specific expression of RNA-binding proteins (RBP) and tissue-specific expression of miSS that is consistent with miSS response to RBP inactivation by shRNA. In combination with RBP profiling by eCLIP, this allowed prediction of novel cases of tissue-specific splicing regulation including a miSS in QKI mRNA that is likely regulated by PTBP1. According to the structural annotation of the human proteome, tissue-specific miSS are enriched within disordered regions, and indels induced by miSS are enriched with short linear motifs and post-translational modification sites. Nonetheless, more than 15% of tissue-specific miSS affect structured protein regions and may adjust protein-protein interactions or modify the stability of the protein core. The significantly expressed miSS evolve under the same selection pressure as maSS, while other miSS lack signatures of evolutionary selection and conservation. Using mixture models, we estimated that not more than 10% of maSS and not more than 50% of significantly expressed miSS are noisy, while the proportion of noisy splice sites among not significantly expressed miSS is above 70%. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.249334v1?rss=1 Authors: Havrylov, S., Chrystal, P., van Baarle, S., French, C. R., MacDonald, I. M., Avasarala, J. R., Rogers, R. C., Berry, F. B., Kume, T., Waskiewicz, A. J., Lehmann, O. J. Abstract: Alterations to cilia are responsible for a wide range of severe disease; however, understanding of the transcriptional control of ciliogenesis remains incomplete. We evaluated whether ciliary dysfunction contributed to the pleiotropic phenotypes caused by the Forkhead transcription factor FOXC1. Here, we show that patients with FOXC1-attributable Axenfeld-Rieger Syndrome (ARS) have a prevalence of ciliopathy-associated phenotypes comparable to syndromic ciliopathies. We demonstrate that altering the level of Foxc1, via shRNA mediated inhibition and mRNA overexpression, modifies cilia length in vitro. These structural changes were associated with substantially perturbed cilia-dependent signaling [Hedgehog (Hh) and PDGFRalpha] and the altered ciliary compartmentalization of a major Hh pathway transcription factor, Gli2. Analyses of two Foxc1 murine mutant strains demonstrated altered axonemal length in the choroid plexus with the increased expression of an essential regulator of multi-ciliation, Foxj1. The novel complexity revealed in ciliation of the choroid plexus indicates a partitioning of function between these Forkhead transcription factors. Collectively, these results support a contribution from ciliary dysfunction to some FOXC1-induced phenotypes. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.30.229443v1?rss=1 Authors: Kohen, R., Baldwin, K. T., Garay, P. M., Tsukahara, T., Chen, A., Flynn, C. G., Johnson, C., Zhao, X., Sutton, M. A., Iwase, S., Giger, R. J. Abstract: Small hairpin RNAs (shRNAs) allow highly efficient gene knockdown. Here we employed different shRNAs to knock down the reticulon RTN4A/NogoA in primary neurons. Depletion of NogoA correlates with altered synaptic protein composition and spontaneous neurotransmission. However, similar phenotypes are not observed upon genetic deletion of Nogo or its receptors. Step-wise introduction of mismatches in the seed region of shNogoA provides further evidence that synaptic phenotypes are NogoA-independent. RNA sequencing revealed global changes in the neuronal transcriptome of cultures transduced with the original shNogoA or closely related variants. Transcriptomic changes are shRNA seed sequence dependent, but not target-specific. Parallel sequencing of small non-coding RNAs revealed dysregulation of microRNAs. Computational analysis shows that the altered miRNA composition correlates with changes in mRNA expression and preferentially affects protein-protein networks that function at synapses. Thus, off-target effects associated with shRNAs are an inherent property, and in particular, altered miRNA composition needs careful consideration. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.01.072926v1?rss=1 Authors: Zipperly, M. E., Sultan, F. A., Graham, G.-E., Brane, A. C., Simpkins, N. A., Ianov, L., Day, J. J. Abstract: Exposure to drugs of abuse produces robust transcriptional and epigenetic reorganization within brain reward circuits that outlives the direct effects of the drug and may contribute to addiction. DNA methylation is a covalent epigenetic modification that is altered following stimulant exposure and is critical for behavioral and physiological adaptations to drugs of abuse. Although activity-related loss of DNA methylation requires the Gadd45 (Growth arrest and DNA-damage-inducible) gene family, very little is known about how this family regulates the activity of brain reward circuits or behavioral responses to drugs of abuse. Here, we combined genome-wide transcriptional profiling, pharmacological manipulations, electrophysiological measurements, and CRISPR tools with traditional knockout and behavioral approaches in rodent model systems to dissect the role of Gadd45b in dopamine-dependent epigenetic regulation and cocaine reward. We show that acute cocaine administration induces rapid upregulation of Gadd45b mRNA in the rat nucleus accumbens, and that knockout or site-specific CRISPR/Cas9 gene knockdown of Gadd45b blocks cocaine conditioned place preference. In vitro, dopamine treatment in primary striatal neurons increases Gadd45b mRNA expression through a dopamine receptor type 1 (DRD1)-dependent mechanism. Moreover, shRNA-induced Gadd45b knockdown decreases expression of genes involved in psychostimulant addiction, blocks induction of immediate early genes by DRD1 stimulation, and prevents DRD1-mediated changes in DNA methylation. Finally, we demonstrate that Gadd45b knockdown decreases striatal neuron action potential burst duration in vitro, without altering other electrophysiological characteristics. These results suggest that striatal Gadd45b functions as a dopamine-induced gene that is necessary for cocaine reward memory and DRD1-mediated transcriptional activity. Copy rights belong to original authors. Visit the link for more info

This month on the Discover CircRes podcast, host Cindy St. Hilaire highlights five featured articles from recent issues of Circulation Research and talks with Matthew Stratton, Rushita Bagchi, and Tim McKinsey about their article on Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation. Article highlights: Vincentz, et al. HAND1 Enhancer Variation Impacts Heart Conduction   Zhuang, et al. EC-Klf2-Foxp1-Nlrp3 Regulates Atherogenesis   Quintanilla, et al. Robust Targets for Persistent AF Ablation   Lambert et al. Characterization of Kcnk3-Mutated Rats   Myagmar et al. Gq Mediates Cardioprotection Transcript Cindy St. H:       Hi, welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St Hilaire, and I'm an assistant professor at the University of Pittsburgh. My goal as host of this podcast is to share with you highlights from recent articles published in the August 30th and September 13th issues of Circulation Research. We'll also have an in-depth conversation with Drs. Matthew Stratton, Rushita Bagchi, and Tim McKinsey, who are the lead authors of one of the exciting discoveries presented in the September 13th issue. Cindy St. H:         The first article I want to share with you is titled, "Variation in a Left Ventricle–Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function." The first author is Joshua Vincentz and the corresponding author is Anthony Firulli, and this work was conducted in the Departments of Pediatrics, Anatomy, and Medical and Molecular Genetics at Indiana Medical School in Indianapolis, Indiana. Cindy St. H:         The heart's ventricular conduction system, or VCS, is composed of specialized muscle cells that propagate electrical signals through the working myocardium of the ventricles to coordinate the rhythmic contractions of the heart chambers. Disorders of the VCS can lead to certain types of arrhythmia. Genome-wide association studies have identified a number of single nucleotide polymorphisms, or SNPs, that appear to increase the risk of VCS-mediated arrhythmias. Two such SNPs are located in the upstream region of a gene encoding for Hand1. And Hand1 is a transcription factor that is involved in left ventricle development. Conditional cardiac Hand1 ablation during embryogenesis leads to ventricular septal defects and hyperplastic arterial ventricular valves, and a reduction in Hand1 expression could lead to morphological, and therefore functional defects. Vincentz and colleagues hypothesized that these SNPs might reside in an enhancer element, and that's a region of DNA and a promoter that allows for the increased expression of a gene. The region containing the SNPs is highly conserved from mammals to reptiles and includes two sequences that allow for the binding of GATA transcription factors. And GATA transcription factors are well known to drive cardiac development. So this team used CRISPR-Cas9 technology to show that the deletion of the enhancer impaired normal VCS morphology and therefore function. And they did this in a mouse model and in the in vitro electromobility shift assay (which frankly was one of my favorite love-to-hate experiments of my PhD). So this group did their own electromobility shift essay and showed that GATA-4 binds to these enhancer sites. And together, these results support a role for Hand1 in the formation and function of the VCS and offer insights to possible arrhythmia etiologies. And what I really love about this paper is that they could actually go from a SNP in a GWAS to a functional role of a protein, which is great. A lot of times with GWAS studies, you have no clue what the heck is going on. So this was a beautiful study where they actually could link a single nucleotide polymorphism to differential expression of a gene. Cindy St. H:         The next article I'd like to highlight is titled, "Endothelial Foxp1 Suppresses Atherosclerosis via Modulation of Nlrp3 Inflammasome Activation." The first authors are Tao Zhuang and Jie Liu, and the corresponding authors (there's three of them) are Zhongmin Liu, Muredach Reilly, and Yuzhen Zhang. The Liu and Zhang teams are from the Key Laboratory of Arrhythmias of the Ministry of Education of China, and the Research Center for Translational Medicine at Shanghai East Hospital, which is part of Tongji University School of Medicine in Shanghai. And the Reilly team is from the Cardiology Division in the Department of Medicine and the Irving Institute for Clinical and Translational Research at Columbia University in New York, New York. And I have to say my good friend Rob Bauer is also a coauthor on this article. So Rob, I hope you're listening. Cindy St. H:         Chronic inflammation contributes to atherosclerotic disease and is a major pathological mechanism contributing to the dysfunction of the vascular endothelium. So leukocytes, which are inflammatory cells that float around in your blood, leukocytes can adhere to the endothelial layer, and then they can migrate through the endothelial wall into the wall of the vasculature. And it's this activity, along with the uptake of oxidized LDL and the formation of a little fatty streak, that is the start of atherosclerosis. And now Zhuang and colleagues have identified that the transcription factor Foxp1 is a potential regulator of vascular endothelial health. So first they showed that while healthy arteries express Foxp1 robustly, atherosclerotic endothelium from both mice and humans exhibits reduced expression of this transcription factor. The team then generated atheroprone mice that either lacked Foxp1 or overexpressed Foxp1 specifically in the endothelium. The mice lacking Foxp1 were shown to have exacerbated athero with much larger plaque sizes and increased macrophage infiltration into the vessels, while overexpression of Foxp1 had largely the opposite effect. It actually curtailed progression of atherosclerotic disease. The team went on to examine the atherosclerosis-suppressing mechanism of Foxp1, showing that the factor suppressed expression of the inflammasome components in the endothelial cells. Cindy St. H:         So all together, these results highlight that Foxp1-mediated regulation of the inflammasome is a potential targetable pathway for atherosclerotic treatments, and having a new targetable pathway is important, as the CANTOS trial, which provides proof of concept of the inflammation hypothesis of atherosclerosis in humans, showed robust effects in only a small subset of the population tested. Thus, there is a need to identify other means, a plan B if you will, by which we can control the inflammation that contributes to atherosclerosis. Cindy St. H:         The next paper I want to highlight is titled, "Instantaneous Amplitude and Frequency Modulations Detect the Footprint of Rotational Activity and Reveal Stable Driver Regions as Targets for Persistent Atrial Fibrillation Ablation." The first author is Jorge Quintanilla, who is also a corresponding author alongside David Filgueiras-Rama, and they are from the National Center for Cardiovascular Research and the Center for Biomedical Research in Cardiovascular Diseases Network in Madrid, Spain. Uncoordinated contractions of the atria to the ventricles of the heart is called atrial fibrillation, or AFib, and AFib causes symptoms such as heart palpitations, dizziness, and taken to the extreme, AFib can actually cause death. To correct such rhythm problems, doctors can ablate certain regions of the heart suspected to be driving this misfiring. In an ablation procedure, a catheter is inserted through the blood vessels and into the heart. An electrophysiologist then identifies the locations of the heart that are sending abnormal electrical impulses, and with either delivery of tiny pulses of painless, low-level energy or using a catheter that has a cold tip to freeze the misfiring areas, the electrophysiologist can ablate and hopefully stop  AFib. The problem is that this approach often fails, and AFib still occurs or can reoccur after a length of time. So Quintanilla and colleagues wanted to develop a more personalized medicine approach to treating AFib. So to do this, they wanted to make it something simple, something affordable, and something that hospitals currently have access to. So they used the standard electroanatomical mapping system to track the amplitude and also the frequency modulations of the electrical signals from the hearts with AFib. And they found that regions with high and stable instantaneous frequency signals were the drivers of fibrillation in the hearts. When these regions were ablated in pigs with persistent AFib, the misfiring stopped in almost all cases and was sustained. The team went on to test the system in three patients with Afib, and two of the three remained arrhythmia-free without drugs for at least 16 months. So with further development and testing, this frequency mapping could potentially replace systems that are currently in use, and more importantly, this could provide a more accurate and patient-tailored way to find and ablate the drivers of AFib. Cindy St. H:       The next paper I want to highlight is titled, "Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension." Oh, now that was a nice title. That was nice and short. The first author is Mélanie Lambert, and the corresponding author is Fabrice Antigny, and they are from the INSERM Hôpital Marie Lannelongue in Le Plessis Robinson, France. Cindy St. H:       Pulmonary hypertension is a rare but life-threatening condition where the adverse remodeling of the pulmonary arteries causes an increase in the blood pressure that's needed to push the blood through the lungs, and this high blood pressure causes the heart to work harder, and it leads ultimately to right ventricular hypertrophy and heart failure. So genome-wide association studies have identified a number of mutations that have been linked to pulmonary hypertension and these include several loss-of-function mutations in the gene encoding for a potassium channel, and that's a protein that can release potassium from a cell to the extracellular environment. And the particular one that has been found to be mutated in pulmonary hypertension patients is Kcnk3. And this channel regulates the resting membrane potential of pulmonary artery smooth muscle cells. To date, it is not known how the loss of Kcnk3 contributes to pulmonary hypertension. To start to unravel this mystery, Lambert and colleagues created a full-body knockout of Kcnk3 in rats, and they used rats because that's a much more robust model for studying pulmonary hypertension than some of the murine models available. These knockout animals exhibited an increased pulmonary artery pressure. They also had faster heart rates and they were more susceptible than their wild-type counterparts to both pharmacological or hypoxia-induced pulmonary hypertension. These Kcnk3 knockout rats also had evidence of remodeled pulmonary vasculature, and this vasculature showed signs of endothelial dysfunction, altered vaso transcription, and altered neomuscularization. In in vitro studies, they used pulmonary artery smooth muscle cells that they isolated from these knockout rats, and these cells showed increased activation of proliferation markers, which is another signature of pulmonary hypertension. And this was also mirrored in human pulmonary artery smooth muscle cells that were treated with a Kcnk3 inhibitor. So together, this work starts to uncover the role of Kcnk3 in pulmonary hypertension pathogenesis. And it also provides the field with a novel model system from which people can learn more about the role of membrane potential of pulmonary artery smooth muscle cells in pulmonary hypertension. Cindy St. H:       The last paper I want to highlight before our interview is titled, "Coupling to Gq Signaling Is Required for Cardioprotection by an Alpha-1A-Adrenergic Receptor Agonist." The first author is Bat-Erdene Myagmar, and the corresponding author is Paul Simpson from the VA Medical Center in San Francisco, California. So like their name says, G-protein coupled receptors interact with G-protein subunits to propagate the signal when a ligand binds. The protein G alpha q has long been considered a key mediator of cardiac hypertrophy. And that's because in mice, when this Gq protein was overexpressed, it induced hypertrophy, myocardial apoptosis, and contractile failure. However, this sub unit Gq can interact with a multitude of G-protein coupled receptors that themselves bind a variety of ligands. So which receptor or which signaling pathway specifically is responsible for the hypertrophic phenotype? Recent studies by others had shown that stimulation of the alpha-1A adrenergic receptor prevents cardiotoxicity and heart failure. So Myagmar and colleagues asked whether this cardio-protective alpha-1A stimulation is dependent on the alpha q subunit. So using mice with a mutant version of alpha-1A that allows the binding of the ligand but does not couple with the Gq subunit, the team found that alpha-1A induced cardioprotection was absent. The mutant animals were more likely to die than their wild-type counterparts when hypertrophy was induced pharmacologically or surgically. And furthermore, in the mutant myocytes themselves, the group observed that alpha-1A induced ERK signaling, which is essential for the receptors cardioprotective activity, was impaired. So together these results showed that alpha-1A-induced cardioprotection is dependent on alpha q, and actually it showed that alpha q signaling is not always maladaptive. Cindy St. H:       Now we're going to move to our interview with Drs. Matthew Stratton, Rushita Bagchi and Tim McKinsey and we're going to talk about their great paper titled "Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation." Cindy St. H:       Okay, so now we're going to have our interview with Drs. Stratton, Bagchi, and McKinsey on their paper titled, "Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation." So welcome, everyone. Dr Tim M:          Thank you. Dr Rushita B:    Thank you. Dr Matt S:          Thank you. Cindy St. H:       I was wondering if you could just all maybe go around and introduce yourselves. Dr Tim M:          Sure. I'm Tim McKinsey. I'm a professor in the Division of Cardiology at the University of Colorado Anschutz Medical Campus. I also direct a newly formed fibrosis center on campus. It's called the CFReT, the Consortium for Fibrosis Research and Translation, and our goal is to understand new mechanisms that regulate fibrosis and develop new therapies to treat scarring, or fibrosis, in organs. Dr Rushita B:    I'm Rushita Bagchi. I'm currently a postdoctoral fellow in Dr McKinsey's lab. I grew up in India, and that's where I did my undergrad and master's degrees. Then I moved to Canada to do my PhD focusing on transcriptional regulation of cardiac fibrosis under the supervision of Dr Michael Czubryt. After that, I transitioned to Dr McKinsey's lab here in Denver to enhance or add to my expertise of transcription by studying epigenetics, and especially trying to find the underlying mechanisms that cause cardiovascular disease. The nice thing about this position for me has been that I have been able to constantly build up on my experience studying tissue fibrosis, but at the same time, Tim has been very generous and has let me develop projects of my own as well. Cindy St. H:       You're lucky. That's awesome. Thank you for joining us. And Dr Stratton. Dr Matt S:          I'm Matt Stratton. I'm an assistant professor in the Department of Physiology and Cell Biology at Ohio State University. I did my graduate training at Colorado State University in neurodevelopment and neuroendocrinology and then moved to Tim's lab for a postdoc and assumed my current position this past December. Cindy St. H:       Wow. How's it going? Dr Matt S:          It's going well. Starting a lab is a lot of fun and a lot of stuff going on. Cindy St. H:       Yeah, I'm four years in now and at the same time you feel brand new and excited and then, oh my God, what am I doing? So that's great. Well thank you all for joining me. So I really like this paper, mostly because I'm also a vascular biologist. I kind of focus more on the heart valves, but I have a real interest in cell phenotype transitioning and cell shifting, and so when you started to talk about chromatin remodeling and bromodomain protein, I was really interested and wanted to hear more. So maybe we can start by telling everyone what is the clinical need that your paper at base is trying to address? Dr Tim M:          Well before we get into that, could I start by saying that we're honored to have our work published in Circulation Research. We're really grateful for that. I also want to point out that this is the result of a very detailed collaborative effort involving at least six other labs, including the labs of Charles Lan at Baylor College of Medicine, Jun Qi at the Dana-Farber Cancer Institute, Kunhua Song and Maggie Lam here in Colorado, as well as Sap Haldar and Deepak Srivastava at the Gladstone Institutes in San Francisco. Without this collaborative effort, none of this would have been possible. Cindy St. H:       That's great to hear and I'm really happy you mentioned that. Team science is so important, and I feel like we almost can't get these big, groundbreaking papers unless we really work as a good team, so thank you for highlighting that. Dr Tim M:          So we're really interested in fibrosis, which is a hallmark of heart failure. Fibrosis can actually be a good thing for the heart. If you have a myocardial infarction, you need a strong scar to form to prevent the ventricle from rupturing. But in response to chronic stress like hypertension and other things, you can get this longstanding fibrosis that results in cardiac dysfunction. That's because fibrosis is essentially a scarring process and one of the things that that does is to create a stiff in the left ventricle that can't relax effectively. Unfortunately, despite the well-known roles of fibrosis in cardiac disease, there are no targeted anti-fibrotic therapies for the heart, and that's really our focus in the lab. We've had a long-standing interest in epigenetic regulation of heart failure and cardiac fibrosis, and we've known for some time that inhibitors have a family of epigenetic reader proteins called the bromodomain and extraterminal proteins, the BET proteins. Inhibitors of those BET proteins can block cardiac fibrosis in rodent models and improve cardiac function. What we knew going into this work is that systemic delivery of those compounds was efficacious. But as you know, the heart is made up of many different cell types. So we really wanted to understand if the efficacy of these compounds was related to effects in resident cardiac fibroblasts. Cindy St. H:       Excellent. So what is the role of a cardiac fibroblast in a healthy cell, and where does that go awry? Dr Matt S:            So in a undiseased heart, fibroblasts are necessary to provide structure, right? They lay down the extracellular matrix that really holds the heart together. Without them, you would not have a good pump function. Where they go awry, I mean, that's one of the things that we're trying to study, right? They become proliferative, they become contractile, and they secrete, or we call them super-secretors, of extracellular matrix. So TGF-beta is really a known signaling molecule that kicks the fibroblasts into this activated or myofibroblast state. We use that in the paper as a agonist for our cultured cells. Cindy St. H:         Great, thank you. So what was the hypothesis you were testing in this paper? Dr Matt S:            So what we wanted to know, if BRD4 and BET proteins are important for this activation of cardiac fibroblasts? So going from a quiescent fibroblast to a proliferative and super-secretor of extracellular matrix fibroblast in the heart. And those experiments hit right away. I mean, we did those experiments, and it was quite dramatic that if you use JQ1 to inhibit these BET proteins, you completely blocked this myofibroblast differentiation. We went in and did some siRNA and shRNA work to show that really BRD4 appears to be the main culprit of the BET protein families. Cindy St. H:         Rushita, could you tell us a little bit about what a bromodomain protein is and what maybe specifically BRD4 is in relation to the other bromodomain proteins? Dr Rushita B:      Sure. So when we talk about the chromatin, there are various players in there that are known as, in general, chromatin modifiers. So you have enzymes that add acetylation mark on lysine residues on histone tails, which is basically DNA is wound around these histones and those histones have lysine tails, but you have the big acetyl group sitting. Now when you have this acetyl group sitting, this makes it more accessible for the transcriptional machinery and allowing gene transcription to happen. Those enzymes are known as histone acetyltransferase, the ones that add the acetyl mark there. The ones that take it away, which is what our lab has been studying for a long time, and Tim is a known world expert in the field, those are known as histone deacetylases, or HDACs, which basically remove those acetyl marks and compact the chromatin, thereby suppressing gene expression. This BET proteins or bromodomains are transcriptional coactivators. So this bromodomain is actually in charge or takes up the duty of identifying these acetyl marks on the lysine residues and therefore, tells the transcription machinery to come in and allow gene transcription to happen. There are a few BET proteins. Of them, BRD4 has been studied extensively in cancer as well as in the heart. But as Tim mentioned, the role of BRD4 has been studied vastly in the heart in terms of the cardiac myocytes, but not so much in the non-myocyte population, which is where our work stands out really well and starts highlighting the role of this specific chromatin modifier protein in activation or control of profibrotic gene expression. Cindy St. H:         Yeah. So correct me if I'm wrong, but my understanding is, you're going to need a little bit of the cardiac fibroblasts remodeling in the early phase. But where it is really detrimental is when that overcompensates and overproliferates and throws down too much matrix and then is bad. So do you see your study as a way to kind of target that window of where a potential treatment might be applicable? Dr Tim M:            Yeah, we think that BRD4 is a nodal regulator of cardiac fibrosis and therefore, an excellent therapeutic target. The challenge will be developing selective BRD4 inhibitors that are safe, as well as effective. We know that BRD4 is not only expressed in cardiac fibroblasts-it's all over the body. But we think our work provides an entry point to the development of highly selective BRD4 inhibitors for fibrotic indications, including heart failure. Cindy St. H:         So that's one of the things I was wondering, how specific your drug is to BRD4 versus the other ones, but also you mentioned the myofibroblast versus the immune cells infiltrating the heart. Do we know what BRD4 is doing in those cells in this system? Dr Tim M:            BRD4 is definitely pro-inflammatory, and BET protein inhibitors like JQ1 are anti-inflammatory, that's for sure. Interestingly, there's a BET family inhibitor called Apabetalone, RVX-208, that's in Phase III clinical testing for people with atherosclerosis. So if that's successful, it will provide proof of concept that you can target this family of epigenetic readers to treat cardiovascular disease. I also wanted to point out that JQ1 was initially discovered by Jay Bradner's lab, in particular Jun Qi, who is a coauthor and collaborator on this paper. Cindy St. H:         Oh, very nice. Okay, good conflict of interest too, I guess. So maybe you guys can talk a little bit about how you managed to get this huge team of scientists together efficiently, and what were any hang-ups? Matt is laughing a bit, but you two are the lead authors, Matthew and Rushita. How did you two kind of lead the way on this and divvy up this huge project? Dr Matt S:            So it is definitely a project management-style approach I think you have to take. I mean, there's a lot of communication, really a lot of communication with bioinformatics, analysts, and getting the right sequencing done, and that was fun, but it took a lot of effort. And once you get this big data, how do you present it in an intelligible story and how do you pick things out that may lead to new discoveries, right? So we highlight Sertad4 in here as a gene that's very much BRD4-dependent. And I think this is a proof of concept for using this genomics, and particularly BRD4, as kind of a molecular string to pull on to unwind this puzzle. So that was a lot of fun. And you know, Rushita was super awesome in helping with this project. Dr Rushita B:      Yeah, I think having stared at cardiac fibroblasts for six years during my PhD definitely gave me the confidence that I could step up to the plate and deliver what was necessary. And like Matt said, there was a lot of omics-based stuff that we did in the paper. And that is actually one of the key highlights, because we see papers or manuscripts that are published that have RNA-Seq, ChIP-Seq and proteomics, but I believe the strength in our article is the combination of all three. So we were actually able to do overlapping ChIP-Seq and RNA-Seq experiments, and then there was proteomics involved. So we are looking at it at the genomic transcriptome and protium-wide changes that are happening all together, put in one manuscript. And the beauty of this work is it has now created data sets that people can mine and get more information out of. And this is something that will definitely continue to drive our future studies in the lab as well. Cindy St. H:         Can you maybe expand on that? Could you maybe describe briefly for the audience what ChIP-Seq is and what RNA-Seq is, and really the power that is created when you can couple those techniques with the same samples? Dr Matt S:            Sure. So BRD4 was the center point of the paper, right? So we did BRD4 ChIP-Seq and RNA Pol II ChIP-Seq in fibroblasts treated with TGF-beta or not. So in ChIP-Seq, you basically immunoprecipitated your target protein, and that brings with it, if it's bound to chromatin, that brings with it the DNA that it's bound to. And then you can sequence the DNA that comes out of your immunoprecipitation and map that to the genome, and you get a very nice picture of where is BRD4 enriched, and where does it go after stimulation like TGF-beta, when the fibroblast becomes a myofibroblast. So you can line all these up and you can pick out what gene changes we think are directly dependent on BRD4. That's something that we like, because we now know that BRD4 is a good target, right, so that kind of pulls it together. Cindy St. H:         Great. Thank you. What else do you want to bring up? Dr Matt S:            I think understanding how signals get translated to changes in gene expression is obviously something that the field is very much interested in. And because BRD4 is basically a step away from RNA polymerase II, it gives you a little bit more specificity in knowing that that's a disease-activated pathway, right? So trying to figure out what directs BRD4 to new locations in the chromatin and cause it to be removed from previous locations in the chromatin is really an interesting area of research. So we did a pathway screen basically using inhibitors, and we use Sertad4 as the readout, right. And we found that a p38 inhibitor was able to block the ability of TGF-beta to induce Sertad4. And we were able then to show that p38 had a role in targeting BRD4 specifically to the Sertad4 locus. Dr Tim M:            I wanted to say, you know, one of the challenges with this project is that fibroblasts are difficult to work with. You would think that they would be easier to work with than a myocyte. But when a fibroblast hits a plastic cell culture dish, it rapidly transforms into an activated cell, because that plastic has a very high tensile strength. So it took a lot of optimization to figure out methods to culture these cells to maintain them in a quiescent state. Cindy St. H:         What did you do? What was that trick? Dr Tim M:            I mean, it involves changing cell density, changing the constituents of the medium, and doing other things. Cindy St. H:         Science magic. Dr Tim M:            Yeah. Dr Rushita B:      And I'll just add to that. The nice thing about being able to contribute to a study like this is also that, like Tim said, fibroblasts, they change phenotype rapidly. You take them out of a biological system, whether it's a heart or any other tissue, you plate them out in cell culture, they start changing. The nice thing about the in vivo study, the RNA-Seq that was done using the in vivo study with JQ1, was that we used a very simple pressure overload model known as the TAC model, which is a very well-established and accepted model worldwide in the field of cardiovascular disease, treating animals with JQ1. So we isolated fibroblasts, but the time from the isolation of cells to the time an RNA was prepared was an hour or two. So we made sure that we minimally exposed them to culture conditions in the lab, so we retained their biology. So what we did on plastic dishes before, although they were plated on plastic, and we had RNA-Seq done on those cells, like Tim said, we did optimize the conditions. And then being able to similarly treat or use the cells that come from an animal directly and both of them contributing to a similar cohort of genes or pathways that we can look at, that has definitely given immense strength to this manuscript. Cindy St. H:         And that's why it's in Circ Research, so it's a beautiful paper. Very well done. So I can't imagine all these hearts that you had to isolate and get single cells of and culture. What kind of days were you pulling? What was the actual boots on the ground of getting this done? How did that work? Dr Tim M:            It wasn't uncommon for me to get emails from Matt and Rushita at very odd hours of the night or early in the morning. Dr Rushita B:      Yeah, it was like we had the animals being sacrificed, hearts taken, and running to the cell culture room to do everything under sterile conditions. Most important thing- I think what worked out really well is we made sure we had all the reagents prepared ahead of time, so that once the heart is out, it's weighed, because we were also looking at hypertrophy because of the TAC model. We weighed the heart and it goes into your BST right away. Cindy St. H:         I try to teach that to my lab. It's like the cooking idea of mise en place. I make them lay out everything in the cell culture hood ahead of time, and it's all in the order and you just boom, boom, boom, boom. Dr Rushita B:      And a lot of our experiments were done later in the evening, so the nice thing was we had access to multiple centrifuges, which is usually a huge plus. And I still remember Matt being on one side, I'd be on the other. And then we had help from members of the lab as well. They were running between the cell culture room and the centrifuge. So it was actually quite fun. It turned out really well. Cindy St. H:         I'm picturing like those old water brigades to put out a fire where like a bucket is just passed. Is that what this was? Dr Rushita B:      That was very similar to the situation you just talked about. Cindy St. H:         That's great. It sounds kind of painful, but also kind of fun. I guess lastly, maybe one of you can end with telling us what are the bigger picture results of this, and what are the next steps in terms of maybe possibly translating this to the clinic? Dr Tim M:            Well as I mentioned, one of the things we're trying to do is to selectively inhibit BRD4. We're also trying to inhibit it only in cardiac fibroblasts with the hope that we'll be able to improve the therapeutic index of BRD4 inhibition. So create a situation where patients can tolerate this anti-fibrotic therapy better than if it was delivered systemically. We're also looking at other regions of BRD4. BRD4 contains the bromodomains, and those are the targets of JQ1, but there are other interesting domains on BRD4 that we're actively pursuing. Cindy St. H:         Thank you. And Matthew, what are you doing in your new lab, or is it just set up right now? Dr Matt S:            Well I have a K Award from the National Institute of Aging. Cindy St. H:         Congratulations. Dr Matt S:            Thank you. To look at BRD4's role in the heart and cardiac aging. And I also have a couple projects based on some of the mining that we've done from these datasets. So hopefully those lead to good publications and follow-on grants. Cindy St. H:         Well, if this is a good start, I'm sure they will. And Rushita, what are your next plans? How long have you been with Tim? Dr Rushita B:      So I've been here with Tim for almost four years now, so I'm pretty much in the final leg of my postdoctoral training. So I'm still continuing to work on tissue fibrosis projects, including the heart. But I have been able to develop a new field of interest and something that Tim has entrusted me to carry on in the lab in the field of cardiometabolic disease, but definitely with an epigenetic focus. So hopefully in a year's time I see myself having an independent academic scientist position. My dream job will be to be at an academic institute where I can lead a research team which focuses on deciphering or trying to even find the most basic molecules that define the underlying mechanisms of tissue fibrosis and cardiometabolic disease. Cindy St. H:         That sounds like a great plan. Very best luck to you. Dr Rushita B:      Thank you. Cindy St. H:         Do you guys want to add anything else? Dr Tim M:            The field of cardiovascular epigenetics is in its infancy and we still have a lot to learn. Cindy St. H:         And I'm sure all of you will do your parts in moving that field forward. So with that, we're going to end our interview with Drs. Stratton, Bagchi, and McKinsey. Thank you all for joining me and thank you to the listeners for listening. Have a great day. Dr Tim M:            Thank you. Dr Rushita B:      Thank you. Dr Matt S:            Thank you. Cindy St. H:         That's it for highlights from the August 30th and September 13th issues of Circulation Research. Thank you for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.

Durch Fehler entstandene tetraploide Zellen sind chromosomal instabil und können zu Zelltransformation führen. Die Beweise verdichten sich, dass die Propagation von tetraploiden Säugetierzellen durch einen p53-vermittelten Arrest eingeschränkt wird; jedoch ist weiterhin unklar, was die Ursache dieses p53-vermittelten Arrests ist. Um die Ursache des p53-vermittelten Arrests zu identifizieren, wurden individuelle Zellen mittels zeitraffender Mikroskopie in Echtzeit verfolgt. Neu entstandene tetraploide Zellen können einen Zellzyklus vollenden, aber die Mehrzahl der Zellen starb oder verharrte in einem Arrest in der folgenden G1-Phase, abhängig davon ob die vorangegangene Mitose fehlerfrei verlief oder nicht. Tochterzellen, denen eine fehlerhafte Mitose voranging, akkumulierten p53 im Zellkern, was zum Zelltod oder einem irreversiblen Zellzyklusarrest führte. Es zeigte sich durch den Anstieg von 8-OHdG, einem Indikator für oxidative DNA Schädigung, dass tetraploide Zellen durch die vermehrten fehlerhaften Mitosen höheren Konzentrationen von reaktiven oxidativen Spezien (ROS) ausgesetzt sind. Der Anstieg von 8-OHdG korrelierte mit der p53-Akkumulation im Zellkern. Da keine vermehrte Phosphorylierung des Histons H2AX (γ-H2AX), ein Marker für DNA-Strangbrüche, detektiert wurde, lässt sich schlussfolgern, dass ROS entscheidend für den p53 vermittelten Arrest verantwortlich sind. Mehrere p53-aktivierende Kinasen wurden mittels RNA Interferenz (RNAi) und chemischer Genetik untersucht, ob sie einen Einfluss auf den Zellzyklusarrest von tetraploiden Zellen haben. Von den getesteten Kinasen hatte nur ATM einen Einfluss auf die Aktivierung von p53 nach fehlerhaften tetraploiden Mitosen. Zwar wird ATM in der Regel durch DNA-Schäden aktiviert, jedoch wurde bereits zuvor gezeigt, dass ATM auch durch erhöhte ROS Konzentrationen aktiviert werden kann. Um die Zusammenhänge des Zellzyklusarrests weiter aufzuklären, wurde ein genomübergreifender esiRNA Screen etabliert, der die Zellproliferation nach induzierter Tetraploidisierung analysiert. Durch Kombination der Zellzyklusanalyse an Hand des DNA-Gehalts zusammen mit den FUCCI-Zellzyklusindikatoren, konnten tetraploide und diploide Zellen nebeneinander mikroskopisch analysiert werden, ohne zuvor tetraploide und diploide Zellen isolieren zu müssen. Dieser neue experimentelle Ansatz ermöglichte die Identifikation von Genen, die spezifisch die Proliferation von tetraploiden Zellen verstärken oder einschränken Im Primärscreen wurden 1159 Gene identifiziert, deren Inhibition die Proliferation einschränken. Weiter wurden 431 Gene identifiziert, deren Inhibition die Proliferation der tetraploiden Zellen verstärken. Von den 431 Genen, deren Inhibition die Proliferation verstärken, wurden 371 Gene einem Konfirmationsscreen unterzogen, in dem 158 der identifizierten 371 Gene bestätigt wurden. Die bioinformatische Analyse der 158 Gene zeigte eine signifikante Anhäufung von Genen, die mit DNA-Replikation, dem kanonischen Wnt-Signalweg oder mit Tumorsignalwegen assoziiert sind. Unter letzteren ist CCDC6 sehr interessant, da dessen Genprodukt durch ATM phosphoryliert wird und nachgeschaltet den Tumorsuppressor 14-3-3σ reguliert. Des weiteren wurden mittels einer Meta Analyse der Ergebnisse des Primärscreens, zusammen mit den Daten aus dem “Project Achilles”, welches genomweit den Effekt von shRNA-vermittelter Geninhibition auf die Proliferation von 108 Krebszelllinien untersuchte, 18 Gene identifiziert, deren Inhibition sowohl die Proliferation von tetraploiden Zellen einschränkt, als auch die Proliferation von Zelllinien hemmt, welche von Krebsarten stammen, die zu meist chromosomale Instabilitäten (CIN) aufweisen. Damit bilden die präsentierten Daten nicht nur eine gute Basis zur Aufklärung des Zellzyklusarrests tetraploider Zellen, sondern auch für die Identifikation neuer potentieller Zielmoleküle, welche benutzt werden können um Tumorerkrankungen mit chromosomaler Instabilität zu behandeln, welche häufig resistent gegen die bislang verfügbaren Behandlungen sind.

Frontotemporal dementia is the second most common neurodegenerative disease in people younger than 65 years. Patients suffer from behavioral changes, language deficits and speech impairment. Unfortunately, there is no effective treatment available at the moment. Cytoplasmic inclusions of the DNA/RNA-binding protein TDP-43 are the pathological hallmark in the majority of FTLD cases, which are accordingly classified as FTLD-TDP. Mutations in GRN, the gene coding for the trophic factor progranulin, are responsible for the majority of familiar FTLD-TDP cases. The first genome-wide association study performed for FTLD-TDP led to the identification of risk variants in the so far uncharacterized gene TMEM106B. Initial cell culture studies revealed intracellular localization of TMEM106B protein in lysosomes but its neuronal function remained elusive. Based on these initial findings, I investigated the physiological function of TMEM106B in primary rat neurons during this thesis. I demonstrated that endogenous TMEM106B is localized to late endosomes and lysosomes in primary neurons, too. Notably, knockdown of the protein does neither impair general neuronal viability nor the protein level of FTLD associated proteins, such as GRN or TDP-43. However, shRNA-mediated knockdown of TMEM106B led to a pronounced withering of the dendritic arbor in developing and mature neurons. Moreover, the strong impairment of dendrite outgrowth and maintenance was accompanied by morphological changes and loss of dendritic spines. To gain mechanistic insight into the loss-of-function phenotypes, I searched for coimmunoprecipitating proteins by LC-MS/MS. I specifically identified the microtubule-binding protein MAP6 as interaction partner and was able to validate binding. Strikingly, overexpression of MAP6 in primary neurons phenocopied the TMEM106B knockdown effect on dendrites and loss of MAP6 restored dendritic branching in TMEM106B knockdown neurons, indicating functional interaction of the two proteins. The link between a lysosomal and a microtubule-binding protein made me study the microtubule dependent transport of dendritic lysosomes. Remarkably, live cell imaging studies revealed enhanced movement of dendritic lysosomes towards the soma in neurons devoid of TMEM106B. Again, MAP6 overexpression phenocopied and MAP6 knockdown rescued this effect, strengthening the functional link. The MAP6-independent rescue of dendrite outgrowth by enhancing anterograde lysosomal movement provided additional evidence that dendritic arborization is directly controlled by lysosomal trafficking. From these findings I suggest the following model: TMEM106B and MAP6 together act as a molecular brake for the retrograde transport of dendritic lysosomes. Knockdown of TMEM106B and (the presumably dominant negative) overexpression of MAP6 release this brake and enhance the retrograde movement of lysosomes. Subsequently, the higher protein turnover and the net loss of membranes in distal dendrites may cause the defect in dendrite outgrowth. The findings of this study suggest that lysosomal misrouting in TMEM106B risk allele carrier might further aggravate lysosomal dysfunction seen in patients harboring GRN mutations and thereby contribute to disease progression. Taken together, I discovered the first neuronal function for the FTLD-TDP risk factor TMEM106B: This lysosomal protein acts together with its novel, microtubule-associated binding partner MAP6 as molecular brake for the dendritic transport of lysosomes and thereby controls dendrite growth and maintenance.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic options. Activating β-catenin somatic mutations are found in ACC and have been associated with a poor clinical outcome. In fact, activation of the Wnt/β-catenin signaling pathway seems to play a major role in ACC aggressiveness, and might, thus, represent a promising therapeutic target. Similar to patient tumor specimen the H295 cell line derived from an ACC harbors a natural activating β-catenin mutation. We herein assess the in vitro and in vivo effect of β-catenin inactivation using a doxycyclin (dox) inducible shRNA plasmid in H295R adrenocortical cancer cells line (clone named shβ). Following dox treatment a profound reduction in β-catenin expression was detectable in shβ clones in comparison to control clones (Ctr). Accordingly, we observed a decrease in Wnt/βcatenin-dependent luciferase reporter activity as well as a decreased expression of AXIN2 representing an endogenous β-catenin target gene. Concomitantly, β-catenin silencing resulted in a decreased cell proliferation, cell cycle alterations with cell accumulation in the G1 phase and increased apoptosis in vitro. In vivo, on established tumor xenografts in athymic nude mice, 9 days of β-catenin silencing resulted in a significant reduction of CTNNB1 and AXIN2 expression. Moreover, continous β-catenin silencing, starting 3 days after tumor cell inoculation, was associated with a complete absence of tumor growth in the shβ group while tumors were present in all animals of the control group. In summary, these experiments provide evidences that Wnt/β-catenin pathway inhibition in ACC is a promising therapeutic target.

Stress is part of everyday life. And while acute and short periods of stress can help to overcome challenges, exposure to chronic - and especially uncontrollable - stress can lead to maladaption of the organism, which can ultimately increase the risk of disease. However, vulnerability to stress and vulnerability to risk increases are strongly dependent on the individual. The molecular underpinnings of this vulnerability and resilience are still largely unknown. Therefore, the present thesis aims at identifying novel molecules involved in modulating individual stress vulnerability in the brain of male mice. In a first step, we investigated long-term gene expression changes in the hippocampus of male mice that underwent chronic social stress. Adolescent male CD1 mice were subjected to 7 weeks of chronic social stress and were investigated after 5 weeks of recovery via an unbiased whole-genome approach utilising microarray technology. Here, we did not find strong differences caused by the stress exposure, possibly because not all animals were affected by the stress exposure. Nevertheless, we identified Iffo1 as a gene that seems to be affected by at least acute stress and might also be involved in the long-term effect of chronic stress exposure. In the next step, we classified the animals from the same paradigm into stress-vulnerable and stress-resilient individuals based on their corticosterone levels after recovery. Animals which still showed elevated levels of corticosterone 5 weeks after stress were defined as vulnerable, while animals in which levels returned to baseline comparable to controls were termed stress resilient. With an additional whole-genome experiment, we were able to show distinct patterns of gene expression between the groups, including genes like Arc, Gria1 and Gria2. In addition, we also investigated differences in peripheral lymphocytes, which showed regulation in genes like Hsp90b1 or SLA. When we compared the expression profiles between brain and peripheral blood, we showed that stress-vulnerable and stress-resilient animals show different patterns of correlations. In the final part of the thesis, we decreased the expression of Arc, one of the genes we found overrepresented in vulnerable individuals, in the hippocampal formation of male mice via AAV-mediated shRNA knockdown. As we performed the modulation of Arc before the stress exposure, we were able to investigate the causal influence of Arc expression on stress exposure. Animals that were subjected to 3 weeks of chronic social defeat, showed an increase in anxiety-related behaviour, impairment in spatial memory, an increase in social behaviour and did not differ in depression-like behaviour. Concomitant with the behavioural alterations, stressed animals showed alterations in multiple physiological parameters, like increased adrenal glands or corticosterone response. Intriguingly, we were able to prevent most of the behavioural, but not the physiological, changes with the Arc knockdown. This strongly suggests that Arc is at least partly causally involved in the molecular machinery that underlies stress vulnerability. As Arc is a downstream molecule in multiple pathways already connected to stress vulnerability or stress in general, it might be that Arc actually is one of the major molecular factors that translate the effects of these pathways.

Krüppel-like factor 8 (KLF8) has only recently been identified to be involved in tumor cell proliferation and invasion of several different tumor entities like renal cell carcinoma, hepatocellular carcinoma and breast cancer. In the present study, we show for the first time the expression of KLF8 in gliomas of different WHO grades and its functional impact on glioma cell proliferation. In order to get information about KLF8-mRNA regulation qPCR was performed and did not reveal any significant difference in samples (n = 10 each) of non-neoplastic brain (NNB), low-grade gliomas (LGG, WHO°II) and glioblastomas (GBM, WHO°IV). Immunohistochemistry of tissue samples (n = 7 LGG, 11 AA and 12 GBM) did not show any significant difference in the fraction of KLF8-immunopositive cells of all analyzed cells in LGG (87%), AA (80%) or GBM (89%). Tissue samples from cerebral breast cancer metastasis, meningiomas but also non-neoplastic brain demonstrated comparable relative cell counts as well. Moreover, there was no correlation between KLF8 expression and the expression pattern of the assumed proliferation marker Ki67, which showed high variability between different tumor grade (9% (LGG), 6% (AA) and 15% (GBM) of Ki67-immunopositive cells). Densitometric analysis of Western blotting revealed that the relative amount of KLF8-protein did also not differ between the highly aggressive and proliferative GBM (1.05) compared to LGG (0.93; p

Introduction: Human mesenchymal stem cells (hMSC) are easily obtainable from bone mar-row and possess the ability to differentiate into osteoblasts. Therefore, they have been sug-gested as a suitable source for bone regeneration. HMSC are equipped with a variety of in-tegrins that mediate essential cell-matrix interactions. Collagen I represent approximately 90% of the bone protein content. Cell attachment to collagen I is mediated by three members of the integrin receptor family named a1b1, a2b1 and a11b1 integrins. The main aim of this doctoral thesis was to investigate the basal expression of those integrins in hMSC and to func-tionally analyze the knockdown effect of a single collagen I-binding integrin on hMSC behav-ior in vitro. Materials and methods: HMSC were cultured on collagen I-coated surface. A lentiviral trans-fer of a1-, a2- and a11-specific shRNA was applied for downregulation of the corresponding integrin mRNA. Quantitative PCR and western blot analysis were used to assess the basal ex-pression, knockdown efficiency and integrin compensation. Colorimetric adhesion assay was used for estimation of the extent of cells attachment. HMSC spreading and migration was ob-served by time lapse experiments. JC-1 staining was used for investigation of the initiation of apoptosis. Results: Quantitative PCR were used to assess the basal expression of collagen I-binding integrins in three hMSC donors. We found that these integrins are differently expressed as integrin a11 had the highest and integrin a2 the lowest expression. Next, we applied lentiviral delivery of target-specific short hairpin RNA (shRNA) in order to knockdown each of the collagen I-binding integrins and compared them to the hMSC transduced with a sequence against a non-human gene abbreviated as shRNA control. We achieved significant downregulation (> 80%) of the collagen I-binding integrin mRNA and protein. Subsequently to the transduction, we did not noticed pronounce morphological cell changes, however, a clear decrease of a2- and a11-knockdown hMSC numbers was observed during cultivation. Using a quantitative adhesion assay, we estimated that 120 min after plating only 30% of integrin a11-deficent cells were able to attach to collagen I. In contrast, at the same time point, 70% of integrin a2-knockdown hMSC were attached while integrin a1- and shRNA control hMSC have already reached 100% cell adhesion. Furthermore, a time lapse-based investigation showed that integrin a1- and shRNA control hMSC need approximately 35 min to fully spread on collagen I. In contrast, integrin a2- and a11-knockdown hMSC took approximately double more time for spreading in comparison to shRNA control hMSC. Additionally, we analyzed the migration capability of the four different hMSC lines. The average path which integrin a1- and shRNA control hMSC passed was approximately 170 µm with mean speed of 11.5 µm/h. In parallel integrin a2 and a11-deficient hMSC migrated to a distance of approximately 70 µm with a velocity of 5 µm/h. Since it was observed a lost of a2- and a11-deficient hMSC, next we performed JC-1 staining that visualizes mitochondrial leakage, a hallmark of apoptosis. The majority of integrin a2- and a11-knockdown hMSC exhibited mitochondrial leakage whereas integrin a1- and shRNA control hMSC showed intact mitochondria. Finally, we used quantitative PCR to investigate whether there were compensatory effects between the three integrin receptors. We detect that knockdown of integrin a1 led to upregulation of a2 and a11. Similarly, when integrin a2 was downregulated, integrin a1 and a11 expression increased. Interestingly, knockdown of integrin a11 caused only a slight increase in integrin a1 but not in a2 expression. We also observed that upon osteogenic stimulation, integrin a2 and a11-deficient hMSC further reduced in number and did not mineralize the matrix even on a single cell level. Moreover, our preliminary investigation in hMSC-derived from osteoporosis suffering patients showed a tremendous downregulation of integrin a2. Conclusions: Our results strongly suggested that integrins a2b1 and a11b1 mediate an indis-pensible signaling for hMSC. Once these receptors were ablated from cell surface, hMSC re-duced their spreading, adhesion, migration and survival rates. Our integrin knockdown mod-els can be used for further investigations and understanding of the integrins a2b1 and a11b1 importance and signaling in hMSC and hOB since we observed a strong downregulation of integrin a2 expression in osteoporosis.

Podosomen sind aktinreiche Adhäsionsstrukturen, die vor allem in monozytären Zellen, aber auch in dendritischen Zellen, Osteoklasten, Endothelzellen oder glatten Muskelzel-len vorkommen. In primären humanen Makrophagen gibt es zwei Subpopulationen von Podosomen: größere, hochdynamische Precursor in der Peripherie sowie kleinere, sta-bilere Podosomen im Zellzentrum. Die Regulation der Podosomendynamik in der Zell-peripherie erfolgt durch das Mikrotubuli-basierte Motorprotein KIF1C, wahrscheinlich durch den Transport von Regulationsfaktoren. Ein Schwerpunkt der vorliegenden Ar-beit lag daher in der Identifizierung dieser Regulatoren. • Die Aufreinigung KIF1C-GFP positiver Vesikel mittels FACS ist grundsätzlich funk-tionell. Die Analyse zahlreicher Vesikel-assoziierter Proteine spricht weiter für die Hypothese, dass es sich bei der von KIF1C transportierten Fracht um vesikuläre Struk-turen handelt. Die Detektion zahlreicher unspezifischer Proteine zeigt jedoch auch, dass die Methode der Aufreinigung zukünftig noch verbessert werden muss. • RabGTPasen, die auch am Transport von Vesikeln beteiligt sind, haben oftmals eine ähnliche subzelluläre Lokalisation wie KIF1C. Vor allem zwischen Rab6a und KIF1C war ein häufiger und länger dauernder Kontakt in der Zellperipherie zu beobachten. Mittels GFP-Immunpräzipitation konnte eine Interaktion bestätigt werden. • Auf der Suche nach weiteren potentiellen Interaktionspartnern von KIF1C wurde das Protein HAX1 identifiziert. Sowohl in fixierten als auch in lebenden primären humanen Makrophagen konnte eine eindeutige Kolokalisation der Proteine in der Zellperipherie beobachtet werden. Bei Einsatz der Rigormutante von KIF1C (KIF1C-K103A) akku-mulierten beide Proteine am MTOC. Diese Ergebnisse lassen auf eine Interaktion zwi-schen KIF1C und HAX1 schließen. Das Motorprotein KIF9 lokalisiert vor allem an den stabileren Podosomen im Zentrum der Zelle. Bei der Ermittlung der Rolle von KIF9 hinsichtlich der Regulation dieser Po-dosomensubpopulation wurden folgende Erkenntnisse gewonnen: • Knock-down von KIF9 reduziert die Anzahl der Podosomen und inhibiert bei noch bestehenden Podosomen den Abbau extrazellulärer Matrix. Für KIF9 konnte demnach nicht nur eine Beteiligung an der Podosomenregulation sondern auch eine Rolle im Matrixabbau zugewiesen werden. • KIF9-GFP positive Vesikel assoziieren mit Mikrotubuli und kontaktieren mehrere Podosomen nacheinander. Dies spricht für eine direkte Verbindung von KIF9-vermitteltem, mikrotubuli-basiertem Transport mit Podosomen, die durch KIF9 regu-liert werden. • Durch Immunpräzipitationsversuche wurden Hinweise gefunden, dass KIF9 mögli-cherweise in unterschiedlichen Spleißvarianten oder verschieden phosphorylierten Zu-ständen existiert. • Als Interaktionspartner für KIF9 konnte Reggie-1 identifiziert werden. Durch knock-down von Reggie-1 und auch Reggie-2 konnte diesen Proteinen eine Beteiligung am Abbau extrazellulärer Matrix zugeschrieben werden. Die Teilung der Podosomen-Precursor sowie Auflösung der regulären Podosomen sind grundlegende Vorgänge. Unterschiede in der molekularen Zusammensetzung der Podo-somen-Subpopulationen waren bisher allerdings unbekannt. • Supervillin konnte als erstes Protein identifiziert werden, das differentiell an die unter-schiedlichen Subpopulationen lokalisiert. Dies zeigt zum ersten Mal eine unterschiedli-che molekulare Zusammensetzung der Podosomen-Subpopulationen. • Podosomen reichern Supervillin an, bevor diese sich auflösen. Überexpression von GFP-Supervillin führte außerdem zu einem Verlust von Podosomen, wohingegen shRNA-basierter knock-down die Lebensdauer verlängerte. Supervillin scheint somit eine Rolle in der Regulation von Podosomen zu spielen. • Die Myosin IIA-Bindedomäne ist sowohl für die Anzahl der Podosomen als auch für die differentielle Rekrutierung an die unterschiedlichen Subpopulationen essentiell. • Supervillin steht mit Myosin IIA und der phosphorylierten leichten Kette von Myosin in Verbindung und koppelt kontraktiles Myosin an Podosomen, was deren Auflösung auslöst. • Durch siRNA-basierten knock-down konnte gezeigt werden, dass Supervillin erst zu-sammen mit Myosin IIA und/oder Gelsolin die Effektivität der Podosomen hinsichtlich Matrixabbau beeinflusst. Die Podosomenanzahl hingegen war nicht verändert.

Der akute Myokardinfarkt stellt in den Industrienationen immer noch eine der häufigsten Todesursachen dar. Auch nach Wiedereröffnen des Gefäßes führt eine prolongierte myokardiale Ischämie zur Ausbildung eines Infarktareals. Neben der irreversiblen Schädigung der Myozyten während der Ischämie kommt es auch zu dem so genannten Reperfusionsschaden, dieser kann aber, zumindest tierexperimentell, durch eine entsprechende Therapie verringert werden. Wir konnten bereits zeigen, dass die retrograde Applikation von embryonalen endothelialen Vorläuferzellen, von murinen Embryonen Tag 7,5 (Tie-2+, c-Kit+, Sca-1+, flk-1 low, MHC-1-) eine Kardioprotektion über lösliche Faktoren vermittelt. Diese Reduktion der Infarktgöße war über einen PI3K-AKT Signaltransduktionsweg vermittet. In der hier vorliegenden Studie haben wir uns mit dem Einfluss von Thymosin β4 auf die eEPC vermittelte Kardioprotektion beschäftigt. Methoden: In vitro wurden neonatale ventrikuläre Myozyten der Ratte einer Hypoxie (4 h) und Reoxygenation (1 h) ausgesetzt. Die überlebenden Zellen wurden mittels Trypan-Blau-Exklusion identifiziert. Des Weiteren wurden neonatale ventrikuläre Endothelzellen der Ratte auch einer Hypoxie (18 h) und Reoxygenation (4 h) ausgesetzt und die Apoptoserate mittles TUNEL-Färbung analysiert. Embryonale EPCs mit/ohne Thymosin β4 shRNA Transfektion wurden während Hypoxie kokultiviert oder Thymosin β4 Protein wurde dem Medium zugesetzt. In Schweinen (n= 9 pro Gruppe) wurde am Tag 1 mittels LAD-Verschluß (1 h) ein Infarkt induziert. 5x106 eEPCs mit/ohne Thymosin β4 shRNA Transfektion oder Thymosin β4 Protein wurden nach 55 min Ischämie in die anteriore interventrikulare Herzvene retroinfundiert. Nach 24 h Reperfusion wurden die globale und regionale Myokardfunktion (Sonomikrometrie) sowie die Infarktgröße bestimmt. Darüber hinaus wurde die Inflammation mittels Myeloperoxidase Analyse im Gewebe untersucht. Ergebnisse: Die „short hairpin“ Ribonukleinsäure (shRNA) Transfektion führte zu einer verringerten Thymosin „messanger“ RNA Expression in „real time“ Polimerase Kettenreaktions-Untersuchungen (rt-PCR). In Zellkultur war der Anteil überlebender neonataler Kardiomyozyten in Anwesenheit von eEPCs signifikant erhöht, wenn diese Zellen Thymosin β4 exprimierten. Die Analyse der TUNEL-Färbung zeigte eine deutlich geringere Apoptoserate der neonatalen Endothelzellen, die mit eEPCs kokultiviert wurden, es sei denn die Thymosin β4 Expression wurde durch Transfektion der shRNA reduziert. Die Applikation von Thymosin β4 Protein zeigte bei beiden Zellarten ein ähnliches Ergebnis wie die Kokultivierung mit den eEPCs. In vivo waren nach 24 h zahlreiche Zellen im ischämischen Areal nachweisbar. Die Anzahl der Zellen war durch die Reduktion der Thymosin β4 Expression nicht beeinträchtigt. Die regionale Applikation der eEPCs reduzierte die Infarktgröße signifikant gegenüber der Kontrollgruppe, wohingegen die Thymosin β4 shRNA Transfektion der eEPCs diesen Effekt inhibierte. Auch hier zeigte die retrograde Applikation des Thymosin β4 Proteins eine kardioprotektive Wirkung, die ähnlich ausgeprägt war wie die der eEPCs. Die Analyse der TUNEL-positiven Zellen zeigte eine deutliche Reduktion der Apoptoserate nach Retroinfusion der eEPCs oder des Thymosin β4 Protein, auch hier verloren die eEPCs ihre protektiven Eigenschaften nach der Transfektion mit Thymosin β4 shRNA. Die Inflammation im Ischämieareal, ein wichtiges Kennzeichen für die Ausprägung des Ischämie/Reperfusionsschadens, konnte durch die Verabreichung von eEPCs und auch Thymosin β4 Protein signifikant reduziert werden. Die Reduktion der Thymosin β4 Expression verhinderte wiederum diesen kardioprotektiven Effekt. Diese Untersuchungen zeigen, dass embryonale endotheliale Vorläuferzellen den Ischämie/Reperfusionsschaden zu einem frühen postischämischen Zeitpunkt verringern. Der kardioprotektive Effekt dieser Zellen ist zumindest teilweise Thymosin β4 abhängig, da eine analoge Protektion durch die lokale Applikation von Thymosin β4 Protein erreicht werden kann. Generell zeigt diese Arbeit, dass neben dem direkten Einsatz von Vorläuferzellen und Stammzellen zur Behandlung des Reperfusionsschadens diese Zellen auch genutzt werden können, um mögliche Kandidatenproteine zur Kardioprotektion nach akutem Myokardinfarkt zu identifizieren und somit eine effektive Therapie des Reperfusions-schadens beim Menschen zu ermöglichen.

VI - Maturazione dell'mRNA e RNA catalitici VI-C) Applicazioni pratiche dei