bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pharmacological or genetic depletion of senescent astrocytes prevents whole brain irradiation‐induced impairment of neurovascular coupling responses protecting cognitive function in mice
Authors: Yabluchanskiy Andriy , Tarantini Stefano , Fulop Gabor , Kiss Tamas , Balasubramanian Priya , DelFavero Jordan , Ahire Chetan , Csipo Tamas , Nyúl-Tóth Ádam , Lipecz Agnes , Sonntag William , Schwartzman Michal , Campisi Judith , Csiszar Anna , Ungvari Zoltan 
Published: April 2020

Whole brain irradiation (WBI, also known as whole brain radiation therapy or WBRT) is a mainstream therapy for patients with identifiable brain metastases and as a prophylaxis for microscopic malignancies. WBI accelerates brain aging, causing progressive cognitive dysfunction in ~50% of surviving patients, thus compromising quality of life. The mechanisms responsible for this WBI side effect remain obscure, and there are no effective treatments or prevention strategies. Here, we test the hypothesis that WBI induces astrocyte senescence, which contributes to impaired astrocytic neurovascular coupling (NVC) responses and the genesis of cognitive decline. To achieve this goal, we used transgenic p16‐3MR mice, which allows the detection and selective elimination of senescent cells. We subjected these mice to a clinically relevant protocol of fractionated WBI (5 Gy twice weekly for 4 weeks). WBI‐treated and control mice were tested for spatial memory performance (radial arm water maze), astrocyte‐dependent NVC responses (whisker‐stimulation‐induced increases in cerebral blood flow, assessed by laser speckle contrast imaging), NVC‐related gene expression, astrocytic release of eicosanoid gliotransmitters and the presence of senescent astrocytes (by flow cytometry, immunohistochemistry and gene expression profiling) at 6 months post‐irradiation. WBI induced senescence in astrocytes, which associated with NVC dysfunction and impaired performance on cognitive tasks. To establish a causal relationship between WBI‐induced senescence and NVC dysfunction, se‐nescent cells were depleted from WBI‐treated animals (at 3 months post‐WBI) by genetic (ganciclovir treatment) or pharmacological (treatment with the BCL‐2/BCL‐xL inhibitor ABT263/Navitoclax, a known senolytic drug) means. In WBI treated mice, both treatments effectively eliminated senescent astrocytes, rescued NVC responses and improved cognitive performance. Our findings suggest that the use of senolytic drugs can be a promising strategy for preventing the cognitive impairment associated with WBI.Support or Funding InformationThis work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE).
Extracellular vesicles from tumor microenvironment transforms endothelial cell phenotype via downregulation of TRPV4 channels
Authors: Guarino Brianna Denise , Adapala Ravi Kumar , Kanugula Anantha K. , Dougherty Julie A. , Paruchuri Sailaja , Mahmood Khan A. , Thodeti Charles Kumar 
Published: April 2020

Angiogenesis, the formation of new blood vessels from existing ones, is a normal physiological process. However, deregulation of angiogenesis can lead to pathological states such as cancer, that is characterized by hyper‐permeable and tortuous vessels. We have recently shown a significant decrease in functional expression of the mechanosensitive ion channel, transient potential receptor vanilloid 4 (TRPV4), in tumor endothelial cells (TEC). Further, pharmacological activation of TRPV4 induced normalization of tumor vasculature and improved cancer therapy. However, the molecular mechanisms by which TRPV4 is downregulated in TEC is not yet known. To determine this mechanism, we focused on extracellular vesicles (EVs) derived from tumor cells. We first collected conditioned media (TCM) from tumor cells with and without pre‐treatment of an exosome inhibitor, GW4869. We found that treatment of human normal endothelial cells (hNEC) with TCM transformed them into tumor‐endothelial like (hTEC) phenotype as revealed by expression of TEM8, VEGFR2 membrane translocation, and abnormal tube formation. However, TCM from exosome inhibitor‐treated cells, failed to induce endothelial transformation. Further, we found that EVs isolated from TCM induced hNEC transformation to hTEC. Mechanistically, we found that tumor derived EVs induced functional downregulation of TRPV4 in hNEC as assessed by calcium imaging. Taken together, our results suggest that tumor derived EVs transforms normal endothelial cells via downregulation of TRPV4 channels.Support or Funding InformationNational Institutes of Health R15CA202847 and R01HL119705
Functional and Structural Characterization of Acrodysostosis Mutations of PKA RIα Reveals Mechanism Causing resistant to cAMP activation
Authors: Del-rio Jason , Wu Leon , Lu Tsanwen , Aoto Phillip , Taylor Susan S. 
Published: April 2020

Acrodysostosis is a type of disease related to bone development which usually happened on gene PRKAR1A(PKA RIα) or PDE4D(PDE4D). Mutations in the PRKAR1A gene are linked to Carney complex disease (CNC) and Acrodysostosis‐1 (ACRDYS1) which show contrasting phenotypes. CNC mutations show increased protein kinase A (PKA) activity, whereas ACRDYS1 mutations result in decreased PKA activity and cAMP resistant holoenzymes. Here we characterize three ACRDYS1 mutants: Y371C, R366X and G198R biochemically and structurally to understand the molecular basis of Acrodysostosis disease mutants of PKA. The activation assay indicated that these three mutations show resistant to the cAMP activation, and almost complete loss of cooperativity for cAMP activation with a shift in the Hill coefficient from 1.7 in wild‐type to 1.2 in the mutants. The crystal structure of Y371C mutant at 3.36 Å resolution shows how PKA utilizes cyclic nucleotide‐binding (CNB) domains as building blocks to create a unique isoform‐specific homodimer interface, which agrees well with previously published wild‐type RIα structure. This structure also demonstrates the important role of Y371 which is essential for cAMP binding. The residues after Y371 are disordered in the mutant, and only one cyclic nucleotide ligand can be seen at its two CNB‐B sites. The molecular dynamics simulations show increased dynamics in Y371C mutant compared to wild type protein which agrees to the functional and structural results. These data allow us to better understand the molecular mechanism of Acrodysostosis and it might help the therapeutical and drugs development.Support or Funding InformationThis work was supported by NIH grant GM34921 (to S.S.T.).
Uncovering the Regulation of Fibrotic Signaling in Scleroderma by the Ubiquitin E3 Ligase KLHL42
Authors: Lear Travis , Lockwood Karina C. , Tuncer Ferhan , Larsen Mads , Evankovich John , Lafyatis Robert , Liu Yuan , Chen Bill B. 
Published: April 2020

Systemic scleroderma (SSc) is an autoimmune disease of unknown cause that affects over 2.5 million people globally. SSc results in dysfunctional connective tissues with excessive pro‐fibrotic signaling, affecting skin, cardiovascular, and particularly lung tissue. Over three‐quarters of SSc patients develop pulmonary fibrosis within five years; excessive scarring in SSc lung fibrosis is the main driver of mortality. There are no approved medicines to treat lung SSc. Recent research suggests that TGFb signaling is directly tied to SSc disease, through its pro‐fibrotic transcription factor signaling. Previous studies have shown ubiquitin e3 ligases potently control TGFb signaling through the targeted degradation of key regulator proteins, however their role in SSc‐TGFb signaling remains unclear. We utilized primary SSc patient lung cells for High Throughput Screening of modulators of TGFb signaling through High Content Imaging of the fibrotic transcription factor SMAD2/3 nuclear translocation. We screened an RNAi library targeting Ubiquitin E3 ligases and observed that the knockdown of the E3 ligase KLHL42 impaired TGFb‐dependent fibrotic signaling. KLHL42 knockdown reduced the production of fibrotic material and decreased TGFb‐mediated SMAD activation. We utilized unbiased ubiquitin proteomics to identify the phosphatase PP2A regulator PPP2R5e to be a KLHL42 substrate. Mechanistic studies validated the ubiquitin‐mediated control of PPP2R5e stability through KLHL42. Knockdown of PPP2R5e exacerbated TGFb‐mediated fibrotic signaling, demonstrating a role for PPP2R5e in the regulation of fibrotic signaling in SSc. Finally, we observed KLHL42 affected overall PP2A phosphatase activity through its targeted degradation of the subunit PPP2R5e. Here we report the characterization of the KLHL42‐PPP2R5e axis regulating fibrotic signaling in SSc lung fibroblasts. Future studies will investigate the potential for chemical inhibition of KLHL42 to ameliorate fibrotic signaling in SSc.Support or Funding Information5F31HL143843, 1R01DK119627, 5P50AR060780‐08, 1K08HL144820, 5R01HL142777, 5R35HL139860, and 5R01HL133184.
Assembly of Hypothalamic Perineuronal Nets Contributes to Sustained Blood Glucose Lowering by FGF1 Action in the Brain
Authors: Alonge Kimberly M. , Mirzadeh Zaman , Scarlett Jarrad M. , Brown Jenny M. , Bentsen Marie A. , Logsdon Aric F. , Banks William A. , Guttman Miklos , Wight Thomas N. , Morton Gregory J. , Schwartz Michael W. 
Published: April 2020

The mediobasal hypothalamus (MBH) plays a key role in the homeostatic control of blood glucose levels, and defective activity of glucoregulatory neurocircuits in this brain area is implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). We have identified irregularities in the abundance of perineuronal nets (PNNs), specialized extracellular matrix (ECM) lattices that regulate neurocircuit network interactions, surrounding MBH neurons in rodent models of T2DM. Specifically, diabetic Zucker Diabetic Fatty (ZDF) rats exhibit a 56% decrease in aggrecan+ PNN structures in the MBH compared to age‐matched normoglycemic WT controls, suggestive of destabilized glucose‐sensing neurocircuit interactions. To determine whether this effect is associated with changes in the relative abundance of chondroitin sulfate (CS)‐glycosaminoglycan (GAG) isomers (Δ0S‐, Δ4S‐, Δ6S‐, Δ4S6S, Δ2S6S‐CS), which constitute the glycan component of PNNs and influence the function of these matrices, we characterized CS‐GAG sulfation patterns using state‐of‐the‐art LC‐MS/MS + MRM analysis. Here, we report that diabetic ZDF rats exhibit a significant increase in Δ4S‐CS and corresponding decrease in Δ6S‐ and Δ2S6S‐CS isomers within the MBH that are suggestive of altered protein‐GAG interactions, axonal repulsion and ECM stiffening. We next sought to determine if these PNNs are targets for the effect of fibroblast growth factor 1 (FGF1) to induce sustained blood glucose lowering in ZDF rats following intracerebroventricular (icv) administration. We found that in ZDF rats, icv FGF1 injection normalized CS‐GAG composition while inducing a 2.2‐fold increase in aggrecan+ PNN structures. To determine whether this response is required for blood glucose lowering induced by central FGF1 treatment, ZDF rats received an icv injection of FGF1 followed immediately by bilateral microinjection of either vehicle or the CS‐GAG digesting enzyme, Chondroitinase ABC (ChABC), directed to the MBH to disassemble PNNs. Our finding that the duration of blood glucose lowering induced by icv FGF1 injection was markedly shortened by digestion of hypothalamic PNNs identifies PNNs as key targets for the action of FGF1 to induce sustained remission of diabetes. These data identify a key role for MBH PNNs in glucoregulatory neural network interactions involved in the homeostatic control of blood glucose.Support or Funding InformationThis project was supported by the National Institutes of Health Awards DK12266201 (to K.M.A) and DK083042 (to M.W.S). Research support also provided by Novo Nordisk, Inc (CMS‐431104).
Mild Cognitive Impairment in the Presence of Depressive Symptoms Related to Impaired Cerebrovascular Function in the Obese Zucker Rat
Authors: Halvorson Brayden , Whitehead Shawn , Frisbee Jefferson 
Published: April 2020

Metabolic syndrome is a highly prevalent constellation of pathologies that frequently combines overweight/obesity, moderate hypertension, atherogenic dyslipidemia, and type II diabetes mellitus, and is accompanied by a systemic vascular pro‐inflammatory, pro‐oxidant condition. The evolution of metabolic syndrome is associated with elevated cerebrovascular disease risk including elevations in both the occurrence and the severity of stroke and transient ischemic attacks within the cerebral circulation. Frequently, these impairments to cerebral vascular function also result in cognitive and behavioral impairments to the afflicted individual even in the absence of an acute pathological event.Given the established elevation in depressive symptoms in OZR that develop in parallel to cerebrovascular dysfunction, we sought to determine the extent to which cognition was impaired.In this study 17–18‐wk‐old male lean and (LZR) obese Zucker rats (OZR) were tested for learning, memory, and behavioral flexibility using an operant conditioning chamber (OCC) and the Morris Water Maze (MWM). Learning was assessed by number of trials to reach 8 correct consecutive responses in OCC or a 6 trial MWM protocol. Performance in a retrieval or probe session 24 h after learning was used to measure memory in OCC and MWM respectively. Behavioral flexibility was measured by analyzing error types in OCC and search strategy in the MWM probe test. Depressive symptoms were measured using coat score, novelty suppressed feeding response, and latency to start grooming and frequency of subsequent grooming after sucrose spray. Ex vivo pressurized middle cerebral arteries (MCA) were used to determine the extent to which metabolic syndrome in OZR alters key indices of vascular reactivity of cerebral resistance vessels and the mechanics of the vascular wall.Endothelium dependent vasodilator reactivity was attenuated in OZR vs LZR in response to acetylcholine. Further impairments were demonstrated in OZR with an increase in depressive symptoms as measured by an increase in coat score and latency to start grooming after sucrose spray as well as mild cognitive impairment measured by increased time to reach the platform in MWM learning trials. In contrast, no signs of cognitive impairment were demonstrated in OCC. These results suggest that the changes in cerebrovascular function in OZR are associated with the development of depressive symptoms and mild cognitive impairment in MWM learning with no changes in learning, memory, or behavior flexibility in OCC at this age.Support or Funding InformationCIHR (#130138 and #389769); NSERC (RGPIN‐2018‐05450)
Estrogen modulation of fate choice during kidney development
Authors: Wesselman Hannah , Pfaff Mairead , Wingert Rebecca 
Published: April 2020

Vertebrate kidney formation is a nuanced process that directs renal progenitors to form the functional units of the kidney, known as nephrons, which are comprised of specialized cell types organized into discrete functional segments. The zebrafish embryo forms two nephrons by 24 hours post fertilization (hpf) and offers a simple but tractable genetic model to study nephron patterning. A chemical screen carried out by our lab coupled with whole mount in situ hybridization (WISH) identified 17ß‐estradiol (E2), the dominant form of estrogen in vertebrates, as a potential regulator of segmentation. Further investigation has revealed that E2 results in an expansion of the distal early (DE) and decrease of the distal late (DL) nephron segments due to alterations in cell number that were quantified with fluorescent ISH. Furthermore, E2 treated animals had expanded expression of the essential DE lineage transcription factor irx3b and its target, irx1a, suggesting E2 affects specification of DE precursors. To delineate the possible mechanism of action of E2’s renal effects, we conducted a targeted chemical screen with selective estrogen receptor modulators (SERMs) that exhibit preferential binding for known E2 receptors. The esr2 antagonist PHTPP similarly reduced the DE segment, suggesting that Esr2 may be involved in nephrogenesis. Consistent with this hypothesis, esr2a and esr2b transcripts are expressed during renal progenitor specification. To investigate which of these receptors (or both) are a major player in distal cell fate choice, we have created CRISPR/Cas9 mutants using guides designed for esr2a and esr2b, respectively. With additional genetic studies, we hope to further our understanding of the role of estrogen signaling in kidney development. Combined, these preliminary findings have relevance for understanding kidney disease models, as well as efforts to recapitulate development in vivo for drug discovery and regenerative therapies.Support or Funding InformationR01DK100237
A link between the inflammatory mediator interleukin‐13, the transcriptional repressor REST, the KCNQ5 gene, and Kv7.5 potassium channel function in vascular smooth muscle cells
Authors: Byron Kenneth L. , Brueggemann Lyubov I. , Sandhu Gurpreet , Cabe Maleen , Cribbs Leanne L. 
Published: April 2020

Inflammatory cytokines may contribute to development of allergic hyperresponsiveness in excitable tissues. In peripheral neurons, development of neuropathic pain was found to be associated with overexpression of the repressor element 1‐silencing transcription factor (REST) and subsequently linked to suppression of KCNQ gene transcription, which was associated with a reduction of M‐current and increased neuronal excitability. We previously found that Kv7.5 channels encoded by the KCNQ5 gene are important regulators of smooth muscle cell relaxation in both the vasculature and airways. We also found previously that both vascular and airway smooth muscle cells express REST, which translocated to the nucleus upon short term treatment with interleukin‐13 (IL‐13, 100 ng/ml for 24–48 hr). Chronic treatment of A7r5 vascular smooth muscle cells with IL‐13 for 5–6 days resulted in a 37±12% reduction of KCNQ5 mRNA level and a 34% reduction of endogenous whole cell Kv7.5 current amplitude. We performed a search for genomic sequences that match the REST consensus sequence and using chromatin immunoprecipitation techniques, we have now identified a REST binding region on the rat KCNQ5 gene. The link between IL‐13‐induced REST translocation and suppression of Kv7.5 functional expression was investigated using a REST knock‐out strategy in the A7r5 cell line, using CRISPR‐Cas9 technology. REST KO A7r5 cells had functional endogenous Kv7.5 currents that were insensitive to chronic treatment with IL‐13 and which trended to have higher current densities than those measured in A7r5 cells expressing non‐targeting guide RNA. In summary, REST appears to function as a link between the inflammatory cytokine IL‐13 and suppression of KCNQ5 gene expression/Kv7.5 current in vascular smooth muscle cells.Support or Funding InformationThe project described was supported by Award Number 01BX002344 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development.
Vascular ATP‐sensitive K+ (KATP) Channels: Sex and Fiber‐type Differences in the Support of Contracting Muscle Blood Flow and Interstitial PO2
Authors: Colburn Trenton D. , Weber Ramona E. , Schulze Kiana M. , Hageman K. Sue , Behnke Bradley J. , Musch Timothy I. , Poole David C. 
Published: April 2020

Glibenclamide (GLI), prescribed to Type II diabetes patients, enhances insulin release by inhibiting pancreatic KATP channels. KATP channels support maximal aerobic capacity (V̇O2max; determined by cardiac output (Q̇) and O2 utilization within contracting skeletal muscle (arterial‐venous O2 difference)) and blood flow during treadmill running in male rats. High‐intensity exercise tolerance (critical speed, CS) is impaired by GLI but is not correlated with reductions in resting cardiac function (Q̇) which highlights the role for peripheral vascular KATP channel function to protect against fatigue. Whether muscle O2 delivery‐utilization matching (interstitial PO2, PO2is) is impaired by GLI in a fiber‐type specific manner, and whether sex differences exist in KATP function, are unknown.PURPOSEWe hypothesized that local inhibition of KATP channels via GLI would decrease PO2is and blood flow within contracting fast‐twitch oxidative (mixed gastrocnemius (MG)) and slow‐twitch oxidative (soleus (SOL)) muscles with females (F) exhibiting the greatest reduction.METHODSMale (n=8), female (n=9, proestrus) and ovariectomized female (F+OVX; n=11) Sprague‐Dawley rats were utilized in these experiments. PO2is was determined, before and after GLI superfusion (5 mg kg−1), via phosphorescence quenching (G4) in the exposed MG and SOL muscles during electrically‐induced twitch contractions (180 s, 7V, 1Hz). Blood flow was assessed via infusion of fluorescent‐labeled microspheres (15 μm) at 180 s of contractions.RESULTSGLI reduced MG blood flow (female: 49 ± 9 vs 34 ± 5; male: 50 ± 5 vs 35 ± 4 ml min−1 100 g−1), nadir PO2is (female: 5.9 ± 0.3 vs 4.7 ± 0.3; male: 6.8 ± 0.7 vs 5.5 ± 0.4) and end‐contraction PO2is (female: 7.3 ± 0.5 vs 6.1 ± 0.5; male: 8.9 ± 1.1 vs 7.2 ± 0.5 mmHg), but did not impact SOL, of female and male rats (p<0.05). Conversely, in F+OVX, PO2is was reduced in the SOL (nadir: 12.5 ± 1.5 vs 8.8 ± 0.9; end‐contraction: 14.5 ± 1.5 vs 10.2 ± 1.1 mmHg; p<0.05), but not MG.CONCLUSIONThese data support the role of vascular KATP channels in supporting muscle O2 delivery and high‐intensity exercise tolerance by matching O2 delivery‐utilization with ovariectomy shifting KATP channel effects from fast‐ to slow‐twitch muscles. Thus GLI treatment in diabetic and other cardiovascular disease patients may potentiate exercise intolerance, especially for post‐menopausal females where compromised slow‐twitch fiber function impairs the ability or willingness to engage in low intensity exercise.Support or Funding InformationSupported by NIH Grants: HL108328 (D.C.P and T.I.M) and F31HL145981 (T.D.C)