bluuebunny/crossref_metadata_2025 huggingface dataset:
Halocline Behavior and Salinity Preference in the Estuarine Teleost Fish, the Mummichog (Fundulus heteroclitus)
Authors: Marshall William Smithson , Tait Janet C , Mercer Evan W
Published: November 2023

Mummichogs prefer seawater but have wide ability to acclimate to extreme temperatures and salinities and spawn in dilute brackish water. Haloclines are common features of estuaries, but how fish react to them is not known. The objective was to reveal how mummichogs select microhabitats in the estuary and whether haloclines were a major feature affecting behavior. In the field, minnow trapping revealed that mummichogs move progressively into low salinity warmer water during early spring after ice melt and show significant aversion to colder temperatures and high salinity. First appearance in estuarine shallows occurred above 10 ºC and catch increased to 21 ºC over four weeks. Threespine stickleback (Gasterosteus aculeatus) also preferred warmer low salinity locations, but preferred slow moving streams, whereas mummichogs preferred tidal ponds. In the laboratory, artificial haloclines were made to test isothermal salinity preference, between 28 ‰ full strength seawater (SW, below) and 10 % SW (3.0 ‰, above). Mummichogs of both sexes acclimated to 5 ºC in SW strongly preferred SW. Surprisingly, freshwater (0 % SW) acclimated mummichogs at 21 ºC also preferred SW. In sexually mature fish acclimated to 21 ºC SW, only the males preferred SW; the females showed no significant preference for SW, meaning they freely entered low salinity. SW preference was manifested by a stereotypic passive aversion to the dilute upper layer at the halocline. We conclude that the overall movement of mummichogs into summer breeding grounds of low salinity is driven by maturation of females and their preference for warmer water (regardless of salinity) that overcomes the general tendency of the immature and male fish to avoid dilute waters.

Support or Funding Information

Supported by NSERC Discovery Grant RGPIN3698‐2009 to WSM and UCR scholarship to JCT.
Chemosensory Prey Preference in Neotropical Gastropod‐eating Snakes (Dipsadinae: Dipsadini)
Authors: Sheehy Coleman M. , Tovar Ruben U.
Published: November 2023

Neotropical snakes in the tribe Dipsadini are gastropod specialists, and many possess a suite of morphological and behavioral characteristics generally accepted as adaptations for extracting land snails from their shells. To test the assumption that these snakes prefer gastropod prey, we collected three Dipsas gaigeae, six Tropidodipsas philippii, two T. annuliferus and three Sibon nebulatus and offered to them the scents of four prey items (i.e., snail, earthworm, cricket and frog) plus two controls. Scents were offered via a cotton‐tipped swab, and the response variable was the number of tongue flicks directed towards the swab in 60s. Mean maximum tongue‐flick responses for each species were analyzed using ANOVA and rmANOVA. Dipsas gaigeae, S. nebulatus and T. annuliferus preferred gastropod prey to other potentially available prey, whereas T. philippii showed a strong preference for both gastropods and earthworms. Unlike the other species, T. phillippi consumed live earthworms, suggesting that this species likely eats earthworms in nature in addition to gastropods. These results suggest that earthworms may be the ancestral diet of the Dipsadini and highlight the importance of testing dietary assumptions in order to better understand the evolution of dietary specialization.

Support or Funding Information

We thank J. Reyes‐Velasco, C. Cox, J. Streicher, G. Rivas, T. Barros and M. Ingrasci for assistance with fieldwork and acquiring live snakes. A. Pires da Silva kindly provided space for conducting this study in the animal room facility at the University of Texas at Arlington. We thank O. Flores‐Villela, C. Franklin, A. (Beto) Mendoza and A. Monzón for their assistance with obtaining permits. This study was supported by NSF grant DEB 0613802 to J. Campbell and O. Flores‐Villela. The use of live snakes for this study fully complied with approved UTA Institutional Animal Care and Use Committee (IACUC) protocol A07.032.
Mutations in O‐GlcNAc Transferase Linked to X‐linked Intellectual Disability
Authors: Wells Lance
Published: November 2023

O‐GlcNAc is a regulatory post‐translational modification of serine and threonine residues of nuclear and cytoplasmic proteins that has been implicated in multiple biological processes including epigenetics. In humans, single genes encode enzymes for its attachment (O‐GlcNAc transferase (OGT)) and removal [O‐GlcNAcase (OGA)]. An X‐chromosome exome screen identified a missense mutation in the tetratricopeptide (TPR) domain of OGT (762G>T (p.L254F)) that segregates with X‐linked intellectual disability (XLID) in an affected family. A decrease in steady‐state OGT protein levels, in part due to a shorter half‐life of L254‐OGT, was observed, as predicted by in‐silico modeling, in isolated lymphoblastoid cell lines from affected individuals compared to controls. Recombinant expression of L254‐OGT demonstrated that the enzyme is active against in vivo protein substrates and in vitro peptide substrates. Surprisingly, steady‐state global O‐GlcNAc levels and the O‐GlcNAcome of the lymphoblasts remain grossly unaltered. Affected lymphoblastoids display a marked decrease in steady‐state OGA protein and mRNA levels, implying that a compensation mechanism exists, albeit imperfect given the phenotype of the patients, for maintaining global O‐GlcNAc levels. L254F‐OGT samples also display decreased luciferase reporter expression driven from the proximal promoter of OGA, suggesting that OGT regulates the gene expression of OGA. We observed an enrichment of the OGT containing transcriptional repressor complex mSin3A‐HDAC1 at the proximal promoter region of OGA in affected individuals, which would explain the decrease in OGA gene expression with reduced OGT protein levels. Global transcriptome analysis of L254F‐OGT lymphoblastoids compared to controls revealed a small subset of genes, many involved in chromatin remodeling, that are differentially expressed in affected patients. Further investigation has revealed another XLID family with the SNP E338G in OGT segregating with XLID and thus we have uncovered the first human disorder associated with mutations in OGT. Given that both mutations are in the TPR domains on OGT, we are currently exploring defining the OGT interactome.

Support or Funding Information

This work was supported in part by a medical research grant from the W.M. Keck Foundation and by the National Center for Biomedical Glycomics (P41GM103490).
Synergistic role of hemin in endothelial cell activation in the lungs of sickle cell mice
Authors: Hu Xingang , Zheng Zhen , Alexeyev Mikhail F , Dickerson Carol , Meiler Steffen E , Wu Songwei
Published: November 2023

Although sickle cell disease (SCD) has long been characterized by typical clinical picture of sickle hemoglobin, chronic hemolytic anemia, and recurring episodic vaso‐occlusive crisis, mechanistic cascades linking hemolysis to vaso‐occlusion still remain largely unclear. Notably, the vascular endothelium in patients with SCD exhibits a consecutively activated, pro‐inflammatory phenotype even under steady state conditions, and a further enhanced inflammatory response occurs during vaso‐occlusive crisis. Recent studies have suggested that the hemolytic products, including heme and its oxidized form hemin, may directly target the endothelium, promote endothelial cell pro‐inflammatory activation, and consequently trigger vaso‐occlusion. Thus, the present study examined a potential mechanism of hemin in promoting pro‐inflammatory lung endothelial cell activation in SCD. Fluorescence microscopy was first performed in cultured rat pulmonary microvascular endothelial cells (PMVECs) that were stably transduced with a GFP‐tagged P‐selectin to examine the dynamics of P‐selectin translocation following stimulation with hemin (400 nmol/L). Indeed, hemin markedly stimulated P‐selectin translocation from cytosol to the plasma membrane leading to P‐selectin surface expression. Such hemin‐induced P‐selectin surface expression was nearly abolished by the membrane‐permeable radical scavenger tempol (20 μmol/L), indicating that the stimulative effect of hemin is mediated by cell oxidative stress. Next, an ex vivo polymorphonuclear neutrophil (PMN)‐perfused lung model was employed to determine whether hemin induces potentiated intracapillary neutrophil retention in SCD lungs. Lungs isolated from 12‐week‐old transgenic humanized homozygous SCD (SCD−/−) and heterozygous (SCD+/−) mice were assessed for intravascular PMN sequestration using immunohistochemistry for leukocyte myeloperoxidase following hemin perfusion (10 μmol/L, 15 min) followed by perfusion of non‐stimulated rat PMNs (1.0 × 105/mL, 20 min). Hemin‐stimulated intracapillary PMN sequestration was observed to be more pronounced in SCD−/− lungs as compared to SCD+/− lungs. The potentiated hemin‐induced PMN sequestration in SCD−/− lungs was dramatically attenuated when the lungs were perfused with a P‐selectin antibody (0.5 mg per lung set, 5 min), tempol (150 μmol/L, 5min), or, to a lesser degree, endothelin receptor A (ETA) antagonist BQ‐123 (1 μmol/L, 5 min), but not endothelin receptor B (ETB) antagonist BQ‐788 (1 μmol/L, 5 min), prior to hemin perfusion. Taken together, these observations suggest that hemin, by activating P‐selectin surface expression via intracellular reactive oxygen species generation, acts additively or synergistically to promote alveolar capillary endothelial pro‐inflammatory phenotype in SCD lungs.

Support or Funding Information

HL‐117684 (to S. Meiler) and HL‐066299 (to S. Wu)
NON‐MUSCLE MYOSIN LIGHT CHAIN KINASE INTERACTION WITH TRPC1 MEDIATES STORE‐OPERATED CALCIUM ENTRY (SOCE) INDEPENDENT OF STIM1
Authors: Tauseef Mohammad , Kini Vidisha , Knezevic Nebojsa , Schmidt Tracy Thennes , Amin Ruhul , Li Wennan , Obukhov Alexander , Malik Asrar , Mehta Dolly
Published: November 2023

Myosine light chain kinase (MYLK) is a calcium/calmodulin‐dependent enzyme that phosphorylates myosin II regulatory light chain (MLC), the only known substrate for MYLK. MYLK has two isoforms, the smooth muscle type, also known as short isoform, mainly present in smooth muscle cells; and a long isoform or non‐muscle type, is expressed in non‐muscle cells such endothelial cells and neutrophils. Non‐muscle MYLK (MYLK‐210) has an additional stretch of 922 amino acids at its N‐terminus, which contains sites that can be phosphorylated my multiple kinases such as PKA, PKC, PAK, Src, etc and regulate its kinase activity. Herein, we show that MYLK‐210, independent of its kinase function regulate store‐operated (SOC) Ca2+ entry. MYLK‐210 null endothelial cells (ECs) displayed diminished Ca2+ entry in response to SOC‐activator thapsigargin and thrombin. Additionally, activation of SOC failed to increase lung vascular hyper‐permeability in MYLK‐210 null mice. We generated MYLK‐210 constructs lacking C‐terminus (N‐MYLK) or N‐terminus (C‐MYLK) to investigate the role of specific domains of MYLK‐210 in inducing SOC activity. We found that N‐MYLK but not C‐MYLK rescued SOC activity in MYLK‐210 null ECs. Stromal interaction molecule‐1 (STIM1) and transient receptor potential canonical channel‐1 (TRPC1) form the key constituents of SOC. Thus, we isolated ECs from mice lacking STIM1 or TRPC1 to address the basis of MYLK‐N‐terminus regulation of SOC activity. We found that N‐MYLK rescued Ca2+ entry in STIM1 deleted ECs but failed to do so in TRPC1 null ECs. We identified “DFRDLL” motif in N‐MYLK interacted with TRPC1 and induces SOC activity. Transduction of N‐MYLK or N‐MYLK lacking DFRDLL domain in the vasculature of MYLK‐210 null mice showed that N‐MYLK not N‐MYLK lacking DFRDLL induced permeability in MYLK‐210 deleted mice. In summary, we discovered the non‐kinase mediated role of MYLK‐210 in regulating SOC activity and pulmonary vascular permeability.
Reduction in Edema Cuff Size is not the Mechanism of Lung Protection Afforded by Airway Pressure Release Ventilation (APRV)
Authors: Jain Sumeet Vidya , Kollisch‐Singule Michaela , Roy Shreyas , Pasquale Gabriella , Sweeney Tara , Satalin Josh , Andrews Penny , Habashi Nader , Nieman Gary , Gatto Louis A
Published: November 2023

Introduction

We have shown that preemptive application of APRV significantly reduces the incidence of acute respiratory distress syndrome (ARDS). The exact protective mechanism is currently not known.

Objective

To determine if the mechanism of this protection was a reduction in the size of pulmonary vascular edema cuffing.

Methods

14 swine were randomized into three groups: naïve (n=5), non‐protective ventilation (NPV; n=5), and airway pressure release ventilation (APRV; n=4). Naïve pigs did not receive lung injury. A fecal clot and gut ischemia/reperfusion‐induced ARDS model was used in both the NPV and APRV groups. Following injury, both groups were managed on their respective ventilator settings and sacrificed after 48 hours. Lung tissue was fixed and histology examined at 10× and 40× magnification. Lumen, tunica media, tunica adventitia, and lymphatics were identified, outlined, colored, and then quantified (Figure). The following parameters were further evaluated for each vessel: lumen area, lumen roundness, tunica media area, tunica adventitia area, and lymphatic area. Clinical parameters and histologic lung injury scoring were analyzed.

Results

Vessel lumen roundness and tunica media area did not vary between groups (p>0.05). Lumen area was smaller in the injury groups (NPV/APRV) than in the naïve animals (p<0.05). The perivascular cuff, i.e. tunica adventitia and lymphatic vessels, was larger in the injured animals compared with naïve (p<0.05), without difference between ventilatory strategy. The tunica adventitia occupied a larger percent of total vessel area in the injured animals; however, lymphatics did not occupy a larger percent of the cuff compared with naïve, correlating instead with vessel size. Naïve pigs had lower wet/dry ratio compared with NPV pigs (5.47 vs. 8.82; p<0.05) without difference between Naïve and APRV groups (5.47 vs. 7.11, p>0.05) or APRV and NPV groups (7.11 vs 8.82; p>0.05). PaO2/FiO2 ratio was higher in the APRV vs. NPV group (p<0.05), and histologic scoring showed decreased lung injury.

Conclusion

Despite increased lung protection using APRV, there was no difference in edema cuff size. Therefore, changes in edema cuff size do not contribute to APRV‐induced lung protection.
The Effect of Obese Levels of Leptin on Peripheral Chemoreception
Authors: Pye Richard L. , Roy Arijit , Wilson Richard J.A. , Wyatt Christopher N.
Published: November 2023

Obesity rates are increasing rapidly worldwide. Breathing and cardiovascular systems’ function are directly impacted by obesity and contribute to obesity related pathology. Leptin receptors are present in the oxygen sensing type I cells of the carotid body and in carotid sinus nerve afferent fibers. The adipokine leptin is a respiratory stimulant with known central effects. We have previously reported that acute administration of obese levels of leptin causes a measurable and long lasting change in Ca2+ signal in isolated carotid body type I cells as well as augmenting BKCa channel activity. Here we report that 100nM wortmannin, a PI3K inhibitor, blocks leptin induced Ca2+ signaling in these cells. Furthermore we show that in an ex‐vivo arterially perfused carotid body preparation that leptin causes a dose dependent increase in baseline firing rates under normoxia and an increase in the size of the sensory response to hypoxic challenge that does not wash out. These data show that leptin alters carotid body function acutely and suggest that studying carotid body function in chronic models of obesity is warranted.

Support or Funding Information

Supported by NIH R01‐HL091836
Activation of a Non‐Selective Cation Channel by Store‐Operated and Receptor‐Mediated Elevation of [Ca2+]i in Rat Carotid Body Glomus Cells
Authors: Kim Donghee , Wang Jiaju , Hogan James O
Published: November 2023

Our recent studies show that a non‐selective cation channel (NSC) is highly expressed in rat glomus cells. An increase in Ca2+ influx via the voltage‐dependent Ca2+ channel (VDCC) caused by depolarizing stimuli such as hypoxia, low pHo and high [KCl]o activated NSC. The effect of [Ca2+]i rise by mechanisms other than via VDCC on NSC activity is not yet known. Here we studied the role of store‐operated Ca2+ entry (SOCE) and receptor‐mediated [Ca2+]i rise on NSC activity. Patch‐clamp single channel recording and fura‐2 fluorescence signal were used. SOCE was produced by incubating cells in Ca2+‐free solution containing 1 uM thapsigargin and then adding Ca2+ back to the solution. The rapid rise in [Ca2+]i associated with SOCE caused a strong activation of NSC. Receptor‐mediated [Ca2+]i rise was produced using Angiotensin II (100 nM) and muscarine (10 uM), which stimulate the Gq‐PLC pathway and elevates [Ca2+]i in glomus cells. Both Ang II and muscarine produced a significant activation of NSC. Caffeine that causes release of Ca2+ from the endoplasimc reticulum also elevated [Ca2+]i and activated NSC. GABA, dopamine, endothelin‐1, 5‐HT did not elevate [Ca2+]i and did not activate NSC. Adenosine produced a weak elevation of [Ca2+]i but did not activate NSC. Our results indicate that a stimulus that produces an increase in [Ca2+]i, regardless of which signaling pathway (VDCC, SOCE and receptor‐mediated) is used, can activate NSC to help depolarize glomus cells.

Support or Funding Information

Supported by NIH grant
Acute Intermittent Hypoxia‐Induced Plasticity: What About Micturition?
Authors: Collins William F , Catege Marianna , Solomon Irene C
Published: November 2023

It is well documented that acute intermittent hypoxia (AIH) induces sustained respiratory motor plasticity (long‐term facilitation) lasting more than 60 minutes. The goal of the present study is to characterize possible similar effects of AIH on lower urinary tract function. To this end, bladder intravesical pressure and external urethral sphincter (EUS) EMG activity were recorded in urethane‐anesthetized, spontaneously breathing adult female Sprague Dawley rats during continuous infusion of saline into the bladder to elicit reflex micturition events. The rate of saline infusion was adjusted to achieve a baseline bladder inter‐contraction interval (ICI) of approximately 4 minutes. Following 30–40 minutes of baseline recording, rats were exposed to a single bout of AIH consisting of three five‐minute episodes of hypoxia (10% O2; 90% N2) each separated by five‐minute exposures to room air. Data acquisition continued for at least 90 minutes following the AIH exposure. In addition to the classic induction of respiratory motor plasticity, changes in reflex micturition were observed during the AIH recovery period and were characterized by sustained increases in bladder ICI (i.e., decreased voiding frequency), bladder pressure threshold (i.e., elevated intravesical pressure at the onset of an active bladder contraction) and EUS activity during bladder filling (i.e., increased EUS guarding reflex) lasting up to 60 minutes post AIH. In control experiments, sustained changes in bladder ICI, bladder pressure threshold and EUS activation were not observed following a single fifteen‐minute episode of sustained hypoxia (10% O2; 90% N2). These data suggest that AIH‐induced neuroplasticity extends beyond respiratory motor control and includes both somatic and autonomic motor function.

Support or Funding Information

Support for this project was provided by The Thomas Hartman Center for Parkinson's Research at Stony Brook University and The SUNY Brain Network of Excellence.
Role of PTEN in Hypoxic Pulmonary Vasoconstriction
Authors: Krauszman Adrienn , Mak Tak , Sheppard Dean , Szaszi Katalin , Kuebler Wolfgang
Published: November 2023

Rationale

Hypoxic pulmonary vasoconstricition (HPV) optimizes ventilation/perfusion matching in the lung, and critically depends on transient receptor potential canonical (TRPC) 6 activation and trafficking to caveolae. In endothelial cells, phosphatase and tensin homolog (PTEN) has been shown to serve as a scaffold for TRPC6, enabling cell surface expression of the channel and subsequent Ca2+ entry. Moreover, Rho kinase (ROCK) is known to phosphorylate PTEN and play a role in its localization in chemotaxing neutrophils. Hence, we hypothesized that the interaction between PTEN and TRPC6 in PASMC may be required for TRPC6 translocation to caveolae and an intact HPV response in a ROCK‐dependent manner.

Methods

Interaction between PTEN and TRPC6 was tested by co‐immunoprecipitation in whole lung lysates, and by proximity ligation assay in pulmonary artery smooth muscle cells (PASMC). Caveolar recruitment of proteins was assessed in PASMC by sucrose density gradient ultracentrifugation. HPV was analyzed in isolated perfused lungs as hypoxia‐induced increase in perfusion pressure. Calcium entry to PASMC was measured by Fura2 imaging.

Results

SMC specific PTEN KO mice had a lower HPV response compared to WT mice. Similarly, PTEN knock down in PASMC blunted Ca2+ influx in response to hypoxia. Hypoxia induced protein‐protein interaction between PTEN and TRPC6 in both whole lungs and PASMC, and translocated PTEN to caveolae (caveolin 1‐rich membrane fraction). Inhibition of ROCK (Y27632; 10 μM) prevented both effects and attenuated the HPV response in intact lungs.

Conclusion

Hypoxia induces PTEN interaction with TRPC6 in PASMC, and PTEN translocation to caveolae in a ROCK‐dependent manner. Our findings identify an important role for PTEN in TRPC6 recruitment and activation downstream of ROCK and thus, in triggering HPV in a manner that is independent of its phosphatase activity.

Support or Funding Information

Funded by a CIHR operational grant to WMK
Postinspiratory complex: a third excitatory respiratory oscillator
Authors: Anderson Tatiana M. , Garcia Alfredo J. , Bloom Jacob C. , Baertsch Nathan A. , Pollak Julia , Ramirez Jan‐Marino
Published: November 2023

Mammalian respiration consists of three phases: (1) inspiration, (2) an early phase of expiration, postinspiration, and (3) a late phase of expiration. Two excitatory oscillators have been proposed for the generation of inspiration and active expiration, a conditional expiration that occurs during the late expiratory phase. However, the cellular and circuit mechanisms underlying the generation of postinspiration are largely unknown. Here we describe a third, previously undiscovered excitatory rhythm generator, the Postinspiratory Complex, or PIC, located immediately caudal to the facial nucleus and just medial and dorsal to the rostral‐most portion of the nucleus ambiguus. In this study a variety of in vitro and in vivo approaches are employed to demonstrate that this rhythmogenic kernel generates activity in the postinspiratory phase. Immunohistochemical and optogenetic techniques are utilized to reveal that PIC neurons co‐express acetylcholine and glutamate. Differential responses to neuromodulators and a distinct anatomical location distinguish the PIC from other described respiratory oscillators. Inhibitory interactions between this rhythmogenic network and the preBötzinger Complex, the network responsible for generating inspiration, establish the relative timing, but are not responsible for the generation of the burst underlying inspiratory and postinspiratory activity. Light stimulation of cholinergic PIC neurons in Chat‐cre:Ai27 mice evokes postinspiratory vagal activity in vivo that can reset the inspiratory rhythm. We therefore propose that the three phases of mammalian breathing are generated by three excitatory rhythmogenic medullary networks that are coordinated by inhibitory mechanisms.

Support or Funding Information

NIH NS087828‐01

NIH HL107084‐01
The pontine respiratory group partially mediates clinical opioid‐induced respiratory depression in young rabbits
Authors: Miller Justin Robert , Stuth Eckehard , Hopp Francis , Zuperku Edward , Stucke Astrid
Published: November 2023

We have recently shown that the parabrachial nucleus (PBN), a part of the pontine respiratory group, plays an important role in the respiratory depression by clinical concentrations of intravenous (IV) opioids in adult rabbits as well as adult dogs (Prkic et al 2012). This study investigated whether the area plays a similar role in young rabbits. Each rabbit (P13–25) was anesthetized with 1–1.5 MAC sevoflurane, tracheotomized, ventilated and decerebrated. The PBN was functionally identified by microinjecting 70 nl of 50 μM AMPA (α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid) in a grid‐wise fashion with a glass multi‐barrel electrode from 1 mm rostral to 1 mm caudal of the inferior colliculi and 1–3 mm lateral of midline into the dorso‐ventral center of neuronal activity. Bilateral microinjection of the mu‐opioid agonist [D‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO, 100 μM, 140–350nl) into this region led to a significant decrease in breathing frequency (−16±8 bpm; P < 0.001; n=15) via increased inspiratory (−0.5s, range −1.3‐(−0.3s); P<0.01) and expiratory duration (TE= −6.4s, range −11.7‐(−1.7)s; P < 0.001). This was reversed by local microinjection of the opioid‐antagonist naloxone (1mM, 420 nl). In a separate set of animals, bilateral local injection of naloxone into the same area partially reversed the respiratory rate depression caused by systemic opiate administration (0.5 mcg/kg/min IV remifentanil) by attenuating the opiate induced increase in expiration duration (+1.8±1.4s; P < 0.001; n=12). These studies suggest that in young rabbits mu‐opioid receptors in the PBN contribute to bradypnea caused by clinically relevant doses of systemically administered opioids. The magnitude of these effects is similar to the effects observed in adult rabbits.

Support or Funding Information

Supported by NIH R01GM112960‐01A1 and VA merit grant I01BX000721
Alterations in Doublecortin Expression in Human Neuronal Stem Cells in Response to Angiotensinergic Stimulation in Proliferation and Differentiation Conditions
Authors: Blanco Brigitte , Couling Leena E. , Siddiqi Nadia , Munoz James R , Speth Robert C
Published: November 2023

Activation or inhibition of the renin‐angiotensin system (RAS) affects neuronal function and viability. We showed previously that AT2 selective stimulation of human neuronal stem cells (H‐9 derived, Life Technologies) increased cell numbers in both differentiation and proliferation medium compared to control and AT1 stimulated human neuronal stem cells. We now show that doublecortin expression in human neuronal stem cells which were seeded in chamber slides and cultured under proliferation (with EGF and FGF‐2) or differentiation (no added growth factors) conditions is altered with chronic exposure to AT1 and AT2 selective agonists. Conditions included 14 days treatment with once daily addition of the AT2 selective agonist CGP42112 (final concentration 100 nM); once daily treatment with the non‐selective Ang II receptor agonist Sar1 angiotensin II (100 nM) plus the AT2 receptor selective antagonist PD123319 (10 μM) for selective AT1 receptor stimulation; once daily treatment with PD123319 (10 μM) alone; or once daily treatment with vehicle. Following treatment, cells were immunostained for the neuroblast marker doublecortin. Doublecortin expression was reduced by AT1 selective stimulation in differentiation conditions relative to control (Control 70.9 ± 17% and AT1 14.0 ± 6.2%, p<0.01), AT2 stimulated (AT2 86.7 ± 0.6%, p<0.01) and AT2 antagonized with PD123319 (PD123319 64.4 ± 9.3%, p<0.05). In proliferation conditions, doublecortin expression was reduced by AT2 selective stimulation and AT2 antagonism compared to control and AT1 selectively stimulated cells (p<0.01: Control 96.5 ± 1.1% ; AT1 88.7 ± 1.6% ; AT2 9.1 ± 6.1% ; PD123319 2.6 ± 1.0%). These results suggest that angiotensinergic inhibition of doublecortin expression is dependent upon the presence of the growth factors EGF and FGF‐2.

Support or Funding Information

R.C. Speth: Research Grant; Significant; NIH HL‐113905; Cardiovascular Neuroscience Research Fund, Nova Southeastern University. J. Munoz: President's Faculty Research Development Grant, Nova Southeastern University.
Reduced Glutamate Channel Phosphorylation in Pain‐Related Brain Areas in a Transgenic Model of Sickle Cell Disease
Authors: Towne Brooke V. , LeBlanc Dana M. , Paul Jinny A. , Gupta Kalpna , Edwards Scott
Published: November 2023

Sickle cell disease (SCD) is associated with chronic, debilitating pain beginning in childhood and persisting throughout life. SCD patients also tend to exhibit elevated rates of depression and anxiety disorders, indicating a possible connection between recurring pain episodes and later affective or emotional dysregulation in this population. How SCD physiologically manifests within brain circuitry to mediate this link is currently unknown, although we hypothesize that alterations in glutamate plasticity in central pain centers is implicated. In the current study, we utilized a transgenic mouse model of SCD to measure protein kinase A (PKA)‐mediated phosphorylation of the excitatory AMPA glutamate receptor channel subunit GluR1 in specific brain regions associated with central nociception and the emotional dimension of pain, including the periaqueductal gray (PAG), prefrontal cortex (PFC), and hippocampus. Transgenic sickle cell mice (HbSS‐BERK) expressing human sickle hemoglobin displayed significant mechanical and thermal hyperalgesia across multiple tests of nociceptive sensitivity compared to healthy controls (HbAA‐BERK, expressing normal human adult hemoglobin). In addition, HbSS mice also exhibited reductions in GluR1 subunit phosphorylation in the PAG, PFC, and hippocampus relative to HbAA mice. This neuroadaptation is indicative of diminished excitatory transmission potential in these pain‐ and emotion‐associated brain areas. Moreover, our data are consistent with other preclinical pain models that have linked chronic, peripheral pain conditions with compromised excitatory signaling in central brain regions. These results make a strong case for greater appreciation and investigation into mechanisms linking pain sensitivity with possible affective disorder in individuals suffering from SCD. Our future plans include survey and functional magnetic resonance imaging (fMRI) studies of adolescent and young adult SCD patients to compare how our preclinical findings in HbSS mice associate with pain symptoms and brain activity in this patient population.

Support or Funding Information

R01HL103733

R00AA020839
Exploring neuroplasticity in acute mild traumatic brain injury
Authors: Arakaki Xianghong , Shoga Michael , Till Dale , Strickland David , Tran Thao , Gomez Megan , Li Lianyang , Zouridakis George , Dawlaty Jessica , Fonteh Alfred , Harrington Michael
Published: November 2023

Objectives

To explore neuroplasticity in a longitudinal study of acute mild traumatic brain injury (mTBI).

Methods

We are using quantitative electroencephalography (qEEG) and magnetoencephalography (MEG) during the resting state and during cognitive brain stress to explore neuroplasticity in an ongoing acute mild traumatic brain injury research. Acute mTBI patients are recruited from the emergency department of Huntington Memorial Hospital in Pasadena, CA, and controls are non‐head‐trauma patients. Brain stress includes the N‐back (0‐back and 2‐back) working memory test and Color‐Word Interference Test (CWIT), administered using E‐prime software. Data were collected at three time points: within 1 week of injury, 14 days, and 30 days after injury. Behavioral as well as MEG and qEEG analysis are performed to compare the two groups.

Results

Resting MEG detected low frequency activity in the mTBI group, consistent with previous publications. N‐back, in particular during 2‐back trials, and CWIT, in particular during incongruent trials, both show initial executive function impairment that improved on later visits. Time frequency analysis suggested corresponding compromised brain activity.

Conclusions

The EEG/MEG recordings during rest and brain stress are objective and sensitive to neuroplasticity in acute mTBI, and could be potential objective mTBI markers.

Support or Funding Information

Research supported by D.O.D. W81XWH‐13‐1‐0005
THE VOLTAGE‐GATED POTASSIUM CHANNEL KV1.3 AS A TARGET FOR INHIBITING DETRIMENTAL M1 MICROGLIA FUNCTIONS
Authors: Chen Yi‐Je (Jay) , Nguyen Hai M , Maezawa Izumi , Jin Lee‐Way , Wulff Heike
Published: November 2023

Kv1.3 was first discovered in human T cells in 1984 and has since then been pursued as a potential target for immunosuppression. It was later shown to be overexpressed in effector memory T cells, suggesting that Kv1.3 blockers would be particularly useful for the treatment of T cell mediated autoimmune diseases like multiple sclerosis, psoriasis and rheumatoid arthritis. In fact, the Kv1.3 blocking peptide ShK‐186 has recently been found to be effective in Phase‐1b study for psoriatic arthritis. We hypothesized that Kv1.3 blockers might also be useful for reducing microglia activation in ischemic stroke and other neurological diseases accompanied by neuroinflammation. Starting with cultured microglia, we observed that Kv1.3 expression is up‐regulated in pro‐inflammatory M1‐like microglia and that Kv1.3 blockers preferentially reduce the production of IL‐1beta and TNF‐alpha without affecting IL‐10 production. In organotypic hippocampal slices exposed to hypoxia/aglycemia Kv1.3 blockers significantly reduced microglia activation and increased neuronal survival. We further observed strong Kv1.3 expression on iNOS expressing inflammatory microglia in human stroke biopsies validating Kv1.3 as potential target. In both mouse and rat models of ischemic stroke the small molecule Kv1.3 blocker PAP‐1 significantly reduced infarct area and improved neurological deficit 7 days after reperfusion when administered 12 hours after reperfusion. In the mouse model, PAP‐1 selectively reduced brain levels of the inflammatory cytokines IL1‐beta and IFN‐gamma without affecting IL‐10 and BDNF. Based on these findings we propose Kv1.3 inhibitors as potential therapeutic agents for preferentially inhibiting pro‐inflammatory M1 microglia functions in ischemic stroke and other neurological diseases.

Support or Funding Information

Supported by GM076063 and AG043788.
Translating TNFR signalling into neuroprotective strategies: Proof of concept in acute in vivo models of neurodegeneration using humanized TNFR mice
Authors: Eisel Ulrich L. M. , Dong Yun , Fischer Roman , Naudé Pieter , Maier Olaf , Nyakas Csaba , van der Zee Eddy , Guenzi Eric , Herrmann Andreas , Kontermann Roland , Pfizenmaier Klaus
Published: November 2023

TNF is involved in several degenerative brain diseases including stroke, Multiple Sclerosis, Parkinson's disease and Alzheimer's disease (AD). It became apparent in recent years that TNFR1 and TNFR2 signalling diverges with respect to its involvement in neurodegeneration and neuroprotection, respectively (Fontaine et al., 2002; Marchetti et al., 2004; Dolga et al., 2008; 2009; Fischer et al., 2011; Naudé et al., 2012; Maier et al., 2013). Under chronic stimulation TNFR2 mediated signalling counteracts TNFR1 mediated pro‐apoptotic pathways by the activation of a PI3K, PKB/Akt dependent NF‐kB signalling pathway. This allows neuronal survival despite exposure to lethal concentrations of neurotoxins such as glutamate (NMDA) or oligomeric forms of Amyloid beta. Both agents are known to be involved in neuronal cell loss in the course of Alzheimer's disease.

Consequently in recent years new compounds have been developed that – instead of targeting TNF itself – selectively target TNFR1 or TNFR2 as potential therapeutics in human neurodegenerative diseases. Here we show the results of blocking either human TNFR1 or activating human TNFR2 in a mouse model with humanized TNFRs. For generating humanized TNFR mice we used gene targeting to replace the extracellular domains of the mouse TNFR genes by their human counterpart.

Loss of cholinergic innervation is one of the earliest hallmarks in AD, which leads to severe cognitive impairment. Cholinergic neurons project their fibres from the Nucleus Basalis of Meynert (NBM) into the hippocampus and prefrontal cortex. Therefore loss of neurons in the NBM will reduce fibre density in the hippocampus and prefrontal cortex and lead to memory and learning deficits. We have established the NBM lesion model in humanized TNFR mice to investigate in vivo the potential therapeutic role of either TNFR1 antagonists or TNFR2 agonists. By using optical density measurements of stained cholinergic fibres and microglial activation in brain sections we were able to quantify the amount of neuronal cell loss eight days upon delivery of a toxic dose of NMDA into the Nucleus basalis via stereotactic injection. Controlateral NBM damage was used as internal standard. Behavioural testing was performed to assess cognitive function. Our results suggest that both, blocking TNFR1 or activating TNFR2, results in neuroprotection in this acute lesioning model, which serves as a paradigm for neuronal loss in chronic neurodegenerative diseases such as AD.

Support or Funding Information

Parts of this work was funded by EU FP6 NeuroproMiSe, ZonMW Deltaplan Dementie Memorabel Program, Internationale Stichting Alzheimer Onderzoek, Netherlands, Carl‐Zeiss‐Stiftung, Hertie Stiftung, BMBF, Germany.
Winter‐Like Conditions Do Not Enhance Neuroprotection from OGD in Syrian Hamsters
Authors: Tangog Ekaterina Aldonza , Pérez Iris , Hamilton Jock S , Horowitz John M , Horwitz Barbara A
Published: November 2023

Neurons in rodent species capable of hibernation are more tolerant to oxygen glucose deprivation (OGD), a model simulating stroke, than those not capable of hibernation. We previously demonstrated that OGD in hippocampal slices from hibernating Syrian hamsters (HH; core temperature <10°C) exhibited enhanced neuroprotection compared to hamsters exposed to summer‐like conditions (SH; 14:10 h light: dark, 22 ± 2°C). To examine the role that preparation for hibernation might play in OGD tolerance, we compared hamsters acclimated to winter‐like conditions (i.e., more than three weeks in 8:16 h light:dark at 6 ± 2°C), but without entry into torpor (WH) to HH and SH. To assess acclimation to winter‐like conditions, gonadal weights were measured. We tested the hypothesis that neuroprotection from OGD would be greater in WH than in SH. Hippocampal slices were placed into a recording chamber perfused with artificial cerebrospinal fluid, and electrodes were positioned to stimulate the Schaffer collateral nerve bundle innervating CA1 pyramidal neurons. Population spike amplitudes (PSA) were recorded from the CA1 cell layer before, during, and after exposure to 10 min of OGD. Even though gonadal weights of WH decreased (as would be expected in preparation for hibernation), the PSA response to OGD did not differ between WH and SH groups. In contrast, the HH group exhibited enhanced tolerance to OGD. These results negate our hypothesis, but support an alternative hypothesis – namely that entry into hibernation is required for the increased OGD tolerance of hibernating hamsters.
Role of endothelial nitric oxide in control of peripheral vascular conductance during muscle metaboreflex activation
Authors: Senador Danielle , Kaur Jasdeep , Alvarez Alberto , Hanna Hanna W. , Krishnan Abhinav C. , Altamimi Yasir H. , O'Leary Donal S.
Published: November 2023

The muscle metaboreflex is a powerful pressor reflex induced by the activation of chemically sensitive muscle afferents as a result of metabolite accumulation. During submaximal dynamic exercise, the rise in arterial pressure is primarily due to increases in cardiac output as there is little systemic vasoconstriction. Indeed, in normal animals we have shown a small, but significant, peripheral vasodilation during metaboreflex activation which is mediated at least in part by release of epinephrine and activation of vascular β2 receptors (Kaur et al., Am. J. Physiol. Heart Circ., 2015). We tested whether this vasodilation is in part due to increased release of nitric oxide caused by the rise in cardiac output eliciting endothelium‐dependent flow‐mediated vasodilation. The muscle metaboreflex was activated via graded reductions in hindlimb blood flow during mild exercise with and without nitric oxide synthesis blockade (L‐nitroarginine methyl ester (L‐NAME); 5mg/kg). We accessed the role of increased cardiac output in mediating peripheral vasodilation via the slope of the relationship between the rise in non‐ischemic vascular conductance (conductance of all vascular beds excluding hindlimbs) vs. the rise in cardiac output. L‐NAME increased mean arterial pressure at rest and during exercise. The metaboreflex‐induced increases in mean arterial pressure were unaltered by L‐NAME; whereas, the increases in cardiac output and non‐ischemic vascular conductance were attenuated. However, the slope of the relationship between non‐ischemic vascular conductance and cardiac output was not affected by L‐NAME indicating that the rise in cardiac output did not elicit vasodilation via increased release of nitric oxide. Thus, although nitric oxide is intrinsic to the vascular tonus, endothelial‐dependent flow‐mediated vasodilation plays little role in the small peripheral vasodilation observed during muscle metaboreflex activation.

Support or Funding Information

(NIH HL55473)
Centrally Acting Perindopril Attenuates the Exercise Induced Increase in Muscle Sympathetic Nerve Activity during Heavy Dynamic Exercise
Authors: Moralez Gilbert , Jouett Noah P , Tian Jun , Zimmerman Matthew C , Raven Peter B
Published: November 2023

Central angiotensin II (Ang II) linked free radical (FR) production scavenges nitric oxide (NO) enabling an increased central sympathetic neural outflow (SNA). The pathophysiological increase in Ang II linked FR production is recognized as a major mechanism involved in neurogenic hypertension. During exercise, there is a physiological increase in Ang II and muscle sympathetic nerve activity (MSNA) in direct relation to increasing exercise intensity. We tested the hypothesis that the exercise induced increase in Ang II linked FR production and MSNA activity during exercise is located within the brain. Six healthy subjects performed three randomly ordered trials of 70 ° upright back‐supported dynamic leg cycling after ingestion of two different lipid soluble Angiotensin converting enzyme inhibitors ((ACEi) Perindopril (PER) ‐ highly lipid soluble; Captopril (CAP) non‐lipid soluble)) and/or placebo (PL). Repeated measurements of whole venous blood, MSNA, and mean arterial pressures (MAP) were obtained at rest and during steady‐state heavy intensity exercise at heart rates (HR) of 120 bpm (e120). Peripheral venous superoxide concentrations as measured by electron paramagnetic resonance (EPR) spectroscopy were not significantly altered at rest (P≥0.4) and during E120 by the ACE inhibitors (P≥0.07). Likewise, baseline MSNA (PL, 25 ± 1.5 bust/min; CAP, 21 ± 0.7 bust/min; PER, 25 ± 0.7 bust/min) and MAP (PL, 86 ± 2.8 mmHg vs. CAP, 84 ± 2.6 mmHg; PER, 84 ± 0.7 mmHg) were unchanged at rest (P≥0.1; P≥0.8 respectively). However, during E120 central acting PER attenuated the increases in MSNA and MAP. Increasing only 15±6% for MAP and 24±8% for MSNA when compared to PL (26 ± 6% MAP; 57±16% MSNA; P<0.05) and CAP (26±4%MAP; 69±13%MSNA P<0.05). From these data we conclude that centrally acting PER attenuated the central increase in the exercise induced Ang II linked free radical production resulting in an increased central/peripheral NO activity induced reduction in MSNA during heavy intensity exercise.
Structural Characterization of the Emergence and Evolution of Catalysis in the Chalcone Isomerase Family Fold
Authors: Burke Jason , Kaltenbach Miriam , Tawfik Dan , Noel Joe
Published: November 2023

Chalcone Isomerase (CHI) evolved from a non‐enzymatic Fatty Acid‐binding protein (FAP) into a highly‐efficient catalyst for the stereospecific conversion of naringenin chalcone into (2S)‐naringenin, an early committed step of the plant flavanoid pathway. To understand the structural basis for the emergence and evolution of enzyme activity in CHI, we reconstructed a probable ancestor which represents a form of the protein that has yet to acquire canonical CHI activity. Directed evolution of this “ancestral” protein uncovered three substitutions, each of which are capable of independently licensing CHI activity. High‐resolution protein crystal structures of the inactive CHI ancestor and an active mutant reveal that a single activating substitution stabilizes an alternative structural network, which includes several known catalytic residues within the active site. Comparison of backbone spin‐relaxation rates by NMR suggests that the conformational changes observed between the crystal structures of the inactive and active variants are consistent with changes to the dynamic state of the enzyme in solution. Specifically, we observe changes in fast time‐scale backbone motions of secondary structure motifs bordering the active site. Additional protein crystal structures along the directed evolutionary trajectory reveal that progressive reorientation of ground state conformations are coupled with changes to the active site architecture, and these changes underlie the increases in catalytic activity.
Crystal structure and mutagenesis studies of mammalian fucosyltransferase‐9 (FUT9) reveals the catalytic core and substrate specificity
Authors: Boruah Bhargavi M , Kadirvalraj Renu , Wang Shuo , Ramiah Annapoorani , Wood Zachary A , Moremen Kelley W
Published: November 2023

Mammalian fucosyltransferases (FUTs) fall into 5 CAZy families, all of which employ GDP‐Fucose as a sugar donor, but each has a unique acceptor specificity. FUT9 is one of the several α1,3‐fucosyltransferases in CAZy GT10, which catalyzes the last step in the biosynthesis of Lewis X antigen by addition of a fucose residue to precursor glycan structures. Recombinant expression and purification of soluble glycosyltransferases for biochemical and structural characterization has been a challenge, since these enzymes require efficient glycosylation and disulphide bonding for correct folding and function. In our study, we employ a mammalian cell expression platform for generation of soluble, secreted forms of glycosylation enzyme catalytic domains in large scale for structural and functional studies of these enzymes. Based on this approach, we report the 1.2 Å crystal structure of mammalian FUT9 catalytic domain that highlights elements of substrate specificity and catalysis. FUT9 has been co‐crystallized with GDP and lacto‐N‐neotetraose (LNnT) to reveal details of the binding sites for the donor and the acceptor, respectively. Enzymatic characterization of wild type and mutant forms of FUT9 using a collection of acceptor substrates have correlated the positions of active site residues with their contributions to catalysis and acceptor specificity. Together, these results provide insights into the enzyme structure and details of substrate interactions and specificity.

Support or Funding Information

This study is supported as an ARRA and administrative supplement to NIH NCRR grant RR005351 and P01GM107012