bluuebunny/crossref_metadata_2025 huggingface dataset:
Desmoglein 2 regulates PI3‐kinase/AKT‐dependent expression of Claudin 2 in intestinal epithelial cells
Authors: Burkard Natalie , Meir Michael , Flemming Sven , Germer Christoph‐Thomas , Waschke Jens , Schlegel Nicolas
Published: November 2023

Desmoglein 2 (Dsg2) is a desmosomal junctional protein expressed in all epithelia including the intestinal mucosa, and has been recently proposed to contribute in the pathogenesis of inflammatory bowel disease. In patients suffering from Crohn's disease (CD) it has been reported that reduced expression of intestinal Dsg2 is associated with impaired intestinal epithelial barrier function and accompanied with changes of tight junction (TJ) integrity. A previous study has provided that the application of a Dsg‐specific tandem peptide (TP) linking extracellular domains of adjacent desmogleins increases Dsg2‐mediated intercellular adhesion and strongly reduced TNFα‐induced expression of claudin2 (Cld2), which is referred to as “leaky” TJ protein. However the signaling pathways involved in the regulation of Cld2 by Dsg2 under inflammatory conditions are not elucidated yet.

The incubation of differentiated CaCo2 cells with TNFα led to an impaired epithelial barrier function as revealed by decreased trans‐epithelial electric resistance (TER) and loss of intercellular adhesion in dispase‐based enterocyte dissociation assay. Simultaneously, Dsg2 was reduced at the cell borders and claudin2 expression was significantly augmented as confirmed in Western blot and RT‐PCR analyses. Interestingly, the application of TP restored all TNFα‐induced effects.

Further analyses on signaling pathways demonstrated that both activation of the p38 mitogen‐activated protein kinase (p38MAPK) and phosphatidylinositol 3‐kinase (PI3K) with consecutive AKT phosphorylation are required for TNFα‐induced up‐regulation of Cld2 since the PI3‐kinase inhibitor LY2914002 and the p38MAPK inhibitor SB2021290 blocked up‐regulation of Cld2. Sequential experiments using these inhibitors determined that p38MAPK appears to be upstream of PI3K/AKT signaling. Interestingly, TP blocked both TNF‐induced activation of p38MAPK and PI3K/AKT signaling. By immunoprecipitation we found a direct interaction between Dsg2 and PI3‐kinase which was abrogated after TNFα incubation conforming to the observation in immunostaining that PI3‐kinase co‐localized with Dsg2 under steady state but dissociated from the cell borders to the cytoplasm under inflammatory conditions. These results were strengthened by knock‐down experiments using Dsg2‐siRNA where neither LY294002 nor TP were able to inhibit the PI3‐kinase signaling pathway and consecutive up‐regulation of Cld2.

Our findings provide new insights into the contribution of Dsg2 in regulating TJ composition under inflammatory conditions. Based on our in vitro observations Dsg2 regulates Cld2 expression in a p38MAPK/PI3K/AKT‐dependent manner.
Bacterial metabolism of Ursodeoxycholic Acid is necessary for its protective actions in a mouse model of intestinal inflammation
Authors: Lajczak Natalia Katarzyna , Ward Joseph B , Goggins Bridie J , Keely Simon , Keely Stephen J
Published: November 2023

Background

Although inflammatory bowel disease (IBD) represents a significant health and economic burden, current therapies are often ineffective. Altered intestinal epithelial barrier function has been well‐documented to contribute to IBD pathogenesis, with dysregulation of cytokine and antimicrobial protein (AMP) production, along with disrupted mucosal restitution being apparent. Although, the secondary bile acid, ursodeoxycholic acid (UDCA), has been well‐documented to have cytoprotective and anti‐inflammatory actions, its potential for treating IBD has not been well‐investigated.

Methods

C57BL/6 mice were given dextran sodium sulphate (DSS, 2.5%) in drinking water for 5 days to induce intestinal inflammation, with severity being recorded as disease activity index (DAI). Mice received Na+‐UDCA, a non‐metabolisable analogue of UDCA, 6‐methyl‐UDCA (6‐MUDCA), or lithocholic acid (LCA) via IP injection each day. Cytokine and HβD release from cultured colonic epithelial cells was analysed by Meso Scale Discovery Assay or ELISA. Wound healing was assessed by scratch assay with wound closure being measured after 48 hrs.

Results

In DSS‐treated mice, UDCA (30 mg/kg) significantly reduced DAI from 10.0 ± 0.3 in mice treated with DSS alone to 7.2 ± 0.7 in those co‐treated with UDCA (n = 6 – 12, p < 0.001). In cultured monolayers of T84 colonic epithelial cells, UDCA (50 – 150 μM) attenuated epithelial secretion of the proinflammatory AMP, HβD‐2, (n = 3, p < 0.001), while levels of TNF‐α, IL‐8 and Il‐1β were also significantly reduced (n = 4, p < 0.05). In wound healing studies in vitro, UDCA enhanced colonic epithelial migration and restitution, findings which were mirrored in an in vivo model of colonic mucosal healing in endoscopically wounded mice. Finally, further in vivo studies revealed that the metabolically stable UDCA analogue, 6‐MUDCA, was ineffective in preventing inflammation in the DSS model, while LCA (30 mg/kg), the primary bacterial metabolite of UDCA, was protective, reducing DAI to 5.2 ± 0.6 (n=5, p ≤ 0.001).

Discussion/Conclusion

Together, our data demonstrate anti‐inflammatory and pro‐healing properties of UDCA in the colon, suggesting a potential role for the bile acid in treatment of IBD. However, in vivo, microbial metabolism to LCA appears to be important for the therapeutic actions of UDCA to be observed.
Thyroid hormone deficiency potentiates GABAergic inhibition of the brainstem respiratory network in newborn rat
Authors: Rousseau Jean‐Philippe , Tenorio Luana , Kinkead Richard
Published: November 2023

Because thyroid hormones (THs) are present in the brainstem and are essential for both the differentiation of the presynaptic GABAergic neurons and the maturation of the postsynaptic components of the GABAergic system, we proposed that TH deficiency during gestation is sufficient to disrupt GABAergic modulation of the brainstem's respiratory network in newborn rats. Thyroid hormone deficiency in the foetus was recreated by administrating an antithyroid susbstance (Methimazole;MMI) in drinking water of the pregnant dam at concentration of 0,02% (weight per volume; w/v) from the first day of pregnancy (gestational day 1; GD1) to the day of experiment with the newborn. In‐vitro measurements were performed on Sprague Dawley rats aged of 4 days. Fictive breathing (in‐vitro) was measured by extracting the brainstem with a part of the spinal cord and placing it in a chamber where it was superfused with an artificial cerebrospinal fluid (aCSF). The output signal from the respiratory network was recorded by a suction electrode placed on the fourth ventral root representing the inspiratory signal sent to the diaphragm via the phrenic nerve. Compared to controls, pups from MMI treated dams showed 69% decreased phrenic burst frequency under baseline conditions (5.5 ± 0.4 vs 8.0 ± 0.4 bursts/min; treatment effect: P=0.0003). Moreover, they presented an increased dose‐dependent depression of fictive breathing following Muscimol application (GABAA receptor agonist) (dose 0.1μM: −27 ± 7.9 vs −7.8 ± 3.4 % change from baseline; treatment effect: P=0.0233) and increased elevation of phrenic burst frequency following Bicuculline exposure (GABAA receptor antagonist) (dose 8μM: 25.4 ± 5.3 vs 5.3 ± 3.9 % change from baseline; treatment effect P=0.0085). This treatment‐dependent augmentation of GABAergic tone did not affect burst amplitude, thus indicating that the role of TH on GABAergic modulation is selective. We conclude that TH deficiency results in an increased GABAergic inhibition in the core elements of the respiratory control circuit which could explain the reduced phrenic bursts frequency observed in the treated pups. These results provide new hypotheses regarding the origins of respiratory disorders of the newborn involving reduced respiratory drive such as apnea of prematurity.

Support or Funding Information

Supported by NSERC.
Differential Effects of Systemic and Lung/Airway‐targeted Lipopolysaccharide (LPS) Administration on Inspiratory Motor Output in Anesthetized Spontaneously Breathing Female Versus Male Rats
Authors: Wadolowski Steven B , Wadolowski Robert M , Ahmad Mohammed S , Solomon Irene C
Published: November 2023

Both systemic (ip) and intratracheal (IT) administration of the bacterial endotoxin lipopolysaccharide (LPS) have been used independently to elicit neuroinflammation. While both methods have been reported to induce time‐dependent changes in expression levels of pro‐inflammatory cytokines in the CNS and/or alter some aspects of neural control of breathing, these studies have been performed in predominantly male rats. Thus, little is known regarding the impact of LPS adminiatration on ventilatory control in female rodents. Here, we characterize the effects of LPS delivered either systemically (3 mg/kg, ip) or IT (0.5 mg/kg) on basal inspiratory motor output (recorded as EMG activities from diaphragm (EMGDia) and genioglossus (EMGGG) muscles) and the acute hypoxic ventilatory response (HVR, 10% O2 for 90 s) at 2–5 hr post administration in urethane‐anesthetized spontaneously breathing female Sprague‐Dawley rats. Administration of saline served as a control, and data obtained here were also compared to data obtained from similar experiments in male rats. Compared to saline‐treated control rats, we found that both basal inspiratory motor activity and the HVR were altered by LPS administration albeit the effects were not identical between the administration methods. While both ip and IT LPS administration increased breathing frequency, the increase was more pronounced in rats receiving IT LPS (~18% vs 11%; P<0.05). For both administration methods, this increase was mediated predominantly by a decrease in the time between bursts (TE); however, a slight decrease in burst duration (TI) was noted in IT LPS‐treated rats. Both LPS treatments also blunted the HVR; however, the hypoxia‐induced increase in breathing frequency was greater for ip LPS than for IT LPS. Compared to the effects of LPS on similar measures in male rats, we found that the changes noted in female rats were less severe resulting in LPS‐treated female rats exhibiting lower basal breathing frequencies, longer TI, markedly longer TE, and a slightly more robust HVR. These findings indicate that while both methods of administration alter ventilatory activity (in both female and male rats), the resulting ventilatory patterns appear to be markedly different based on both the administration method and the sex of the rat. Whether these differences are due to administration method and/or sex differences in LPS‐induced changes in neuroinflammatory cytokines in respiratory‐related brainstem and spinal cord regions remains to be determined.
5HT Depletion Blunts Arousal and Chemoreflex Responses Coupled with Breathing Instability and Cardiac Dysfunction in Mice
Authors: Komnenov Dragana , Pop Alexandru , Kuhn Donald , Mateika Jason
Published: November 2023

Purpose

The present investigation was designed to examine if genetic depletion of serotonin (5HT) blunts the arousal response (AR) and chemoreflex sensitivity (CS) to hypercapnia and hypoxia leading to an increase in apnea frequency and duration. The study was also designed to determine if these modifications were coupled to coincident modulation of left ventricular (LV) function.

Methods

Telemetry transmitters were surgically implanted in wild type mice (Tph2+/+) (n = 12) and tryptophan hydroxylase 2 knockout mice (Tph2−/−) (n = 12) to measure electroencephalography, diaphragmatic muscle activity, core body temperature and gross motor activity. Following recovery, a subset of Tph2+/+ (n = 4) and Tph2−/− (n = 5) mice were placed in whole body plethysmographs and exposed to episodes of 7 % carbon dioxide or 10 % oxygen to determine the AR and CS to hypercapnia and hypoxia during non‐rapid eye movement (NREM) sleep. Cardiac parameters were also measured using echocardiography (n = 4).

Results

The AR and CS to hypercapnia and hypoxia were blunted in Tph2−/− compared to Tph2+/+ mice (AR hypercapnia − 31.3 ± 5.0 vs. 8.3 ± 4.4 s; hypoxia − 35.0 ± 1.6 vs. 18.6 ± 2.6 s; P < 0.05) (CS hypercapnia − 4.4 ± 0.6 vs. 6.9 ± 0.5; hypoxia − 1.8 ± 0.2 vs. 2.2 ± 0.2 ml/min/% CO2 or O2; P < 0.05). The frequency of apneic events was greater in Tph2−/− compared to Tph2+/+ mice during the light (8.6 ± 1.3 vs. 3.2 ± 1.0 events/hr; P < 0.05) and dark cycle (7.0 ± 1.5 events/hr vs. 1.8 ± 0.6 events/hr; P < 0.05). The duration index of apneic events was greater in Tph2−/− compared to Tph2+/+ mice during the light cycle (3.9 ± 0.3 vs. 2.8 ± 0.2; P < 0.05). The number of apneas terminated by arousal was reduced in the Tph2−/− compared to the Tph2+/+ mice (12.0 ± 1.9 vs.45.6 ± 8.5%; P < 0.05). LV diameter was smaller in Tph2−/− compared to Tph2+/+ mice (1.7 ± 0.1 vs. 2.4 ± 0.2 mm; P < 0.001), consistent with a higher ejection fraction (80.3 ± 1.5 vs. 63.3 ± 3.2 %; P < 0.005) and LV mass (143.4 ± 15.0 vs. 112.6 ± 8.2 mg; P = 0.07).

Conclusions

Genetic depletion of 5HT in a spontaneously breathing non‐anesthetized sleeping mouse model results in blunting of the AR and CS to hypercapnia and hypoxia. These blunted responses were coupled to exacerbation of breathing events during NREM sleep. The modifications in LV function indicates that 5HT depletion may be responsible for coincident respiratory and cardiac dysfunction in individuals with central sleep apnea. Our results provide a viable mechanism to explain the increased incidence of coincident respiratory and cardiac dysfunction in individuals with spinal cord injury.
Contribution Of The Pontine Medial Parabrachial Nucleus (mPBN) To The Respiratory Depression From The Benzodiazepine Midazolam (MDZ)
Authors: Zuperku Edward J. , Stucke Astrid G. , Hopp Francis A. , Tomlinson Jack , Krolikowski John G. , Stuth Eckehard A.
Published: November 2023

The risk of hypoventilation and apnea induced by systemic opioids appears to be greatly increased in the presence of sedatives such as MDZ. A subregion within the mPBN plays a key role in the control of expiratory duration (TE) and in addition, systemic opioid‐induced bradypnea can be reversed by naloxone microinjections in this subregion. We hypothesized that the enhanced depression of breathing may be due an enhanced GABAergic inhibition by MDZ in this subregion. The responses of phrenic nerve activity (PNA) to microinjections of MDZ (100 μM; 360 nl) and the mu‐opioid agonist [D‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO, 100 μM, 360 nl) into the mPBN subregion were compared in decerebrate, ventilated, vagotomized dogs. Responses to microinjected AMPA were used to identify the tachypneic subregion. Microinjections of MDZ had no effect on inspiratory duration (TI), TE, peak PNA, whereas DAMGO microinjections increased TE (by 85±48%; n=10), slightly increased TI and had no effect on peak PNA. Intravenous administration of MDZ produced a dose‐dependent reduction in TI (−18.5±4.5%) and peak PNA (−29.0±4.1%) with a small non‐significant increase in TE (n=13; peak dose: 238±10 μg/kg). Thus, the respiratory depressant effects of systemic MDZ on breathing do not appear to be mediated by the mPBN subregion where opioids induce bradypnea.

Support or Funding Information

Supported by VA grant 2 I01BX000721‐06.
Glutamatergic excitation in the parabrachial nucleus contributes significantly to respiratory rate control in adult rabbits
Authors: Stucke Astrid G , Stuth Eckehard AE , Hopp Francis A , Zuperku Edward J
Published: November 2023

We have previously identified a subarea of the parabrachial nucleus (PBN) in the adult, in vivo, decerebrate rabbit model where clinical doses of opioids significantly depressed respiratory rate. This study investigated the nature of glutamatergic excitatory drive to this area. The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3–4 kg) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Nerve activity was recorded from the c5 rootlet of the phrenic nerve, time averaged and used to calculate inspiratory and expiratory durations. The PBN was functionally identified by stereotaxic coordinates and a tachypneic response to localized pressure injection of α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA, 50 μM, 70 nl) utilizing a multibarrel glass pipette with a tip diameter of 20μm. The AMPA receptor antagonist 2,3‐ dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo[f]quinoxaline‐2,3‐dione (NBQX, 1mM, 350nl) and the NMDA receptor antagonist (2R)‐amino‐5‐ phosphonovaleric acid (AP5, 5mM, 350 nl) were injected into the bilateral “tachypneic area”. NBQX depressed respiratory rate by 39±7% (mean±SE, n=16, p<0.001). AP5 depressed respiratory rate by an additional 38±6% (p<0.001). The magnitude of respiratory rate depression was not different when antagonist order was reversed. Local injection of pharmacological concentrations of the clinically used NMDA receptor antagonist ketamine (4mM, 350nl, n=7) depressed respiratory rate by 22±2% (p<0.001). All rate changes were due to significant increases in inspiratory and expiratory duration with each drug (p<0.001). Peak phrenic activity, a measure of tidal volume, was minimally depressed (−8±2%, p<0.001). We conclude that glutamatergic excitation in the PBN that is mediated by AMPA and NMDA receptors contributes significantly to respiratory rate control.

Support or Funding Information

Supported by NIH R01GM112960‐01A1 (Dr. Stucke) and VA merit grant I01BX000721 (Dr. Zuperku).
Promoting Respiratory Motor Plasticity After Spinal Cord Injury Through High Frequency Stimulation (HF‐SCS)
Authors: Alilain Warren J , Stoltz Daimen R , Ritter Kyle J , Hager Lydia E , Ghoweri Adam O , Dick Thomas E , DiMarco Anthony F , Kowalski Krzysztof E
Published: November 2023

More than 50% of the spinal cord injury (SCI) population have injuries at the cervical level. Located at this level are the respiratory bulbospinal pathways which mediate breathing. Therefore, injuries at the cervical level can leave many of these patients unable to breath. One promising intervention to restore breathing is HF‐SCS (300Hz). In previous investigations, we demonstrated that HF‐SCS applied at the T2 level after an experimental complete C2 spinal cord section leads to evoked inspiratory diaphragmatic EMG activity that can enable survival of the animal. However, the extent to which HF‐SCS can promote spinal respiratory motor plasticity in order to promote restoration of breathing function is still unknown. To address this question, we performed a HF‐SCS at the T2 level in six anesthetized rats while recording bilateral diaphragmatic EMG activity one week after C2 hemisection. As expected, C2 hemisection abolished activity in the hemidiaphragm ipsilateral to the hemisection, while spontaneous activity contralateral to the C2 hemisection still remained. HF‐SCS resulted in robust inspiratory activity bilaterally in the diaphragm. Following discontinuation of HF‐SCS bilateral spontaneous activity persisted. Moreover, EMG activity on the hemisected side was synchronized with the contralateral side. Taken together, these results suggest that HF‐SCS after a C2 hemisection can enhance activity and promote plasticity of the crossed phrenic pathways which still remain. Further studies are planned to explore the mechanisms of these observations with the ultimate goal to restore function in patients following SCI.

Support or Funding Information

Funded by University of Kentucky College of Medicine Funds & The MetroHeath Foundation.

Disclosure: Dr. DiMarco has a significant financial interest in Synapse BioMedical, Inc, a manufacturer of diaphragm pacing systems.
Developmental nicotine exposure increases the response to muscimol in hypoglossal motoneurons.
Authors: Wollman Lila , Levine Richard B , Fregosi Ralph F
Published: November 2023

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) is known to cause complex changes to the brainstem respiratory circuit including increased sensitivity to inhibitory neurotransmitters and increased staining of GABAAR‐1a subunit in the hypoglossal nucleus. Here we test the hypothesis that DNE alters the response of hypoglossal motoneurons (XII MNs) to the GABAA receptor agonist Muscimol. We obtainedwhole cell patch clamp recordings from XII MNs in medullary slices (700 micron thick) from DNE pups and control pups. Cells were voltage clamped at −75mV. Using a cesium based intracellular solution, and in the presence of the AMPA receptor antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), the NMDA receptor antagonist D(‐)‐2‐Amino‐5‐phosphonopentanoic acid (AP‐5), the glycine receptor antagonist Strychnine, and the voltage gated sodium channel blocker Tetrodotoxin, bath application of 0.5 mM Muscimol produces an inward current in these cells. We measured peak inward current from 10 control cells and 10 DNE cells. Our results show that DNE causes in an increased GABAA receptor mediated inward current (Control {n=10} −239.3±30.75 pA, DNE {n=10} −364.4±38.7 pA, P=0.02). This result is consistent with DNE causing increased GABAA receptor expression in XII MNs.

Support or Funding Information

NIH RO1H071302 AHA 12GRXIT120S0354
Prenatal Nicotinic Exposure (PNE) Prolongs Superior Laryngeal C‐Fibers (SLCFs)‐mediated Apnea through Upregulating TRPV1 Receptor
Authors: Gao Xiuping , Zhao Lei , Zhuang Jianguo , Xu Fadi
Published: November 2023

Maternal cigarette smoke is a critical factor in the pathogenesis of sudden infant death syndrome (SIDS). Simulation of SLCFs evokes an immediate apnea and cardiovascular instability, which are potentially life‐threatening and implicated in pathogenesis of SIDS. To date, the impact of PNE on SLCFs has not been explored. We tested hypothesis whether PNE could prolong the SLCF‐mediated apnea, and if so, whether this prolongation is associated with an upregulated expression of TRPV1 on nodose/jugular (N/J) ganglionic neurons retrogradely traced from the superior laryngeal nerve (SLN). To this end, we compared following outcomes between Ctrl and PNE rat pups (postnatal days 11–14): 1) the apneic response to intralaryngeal application of capsaicin (10 μg/kg, 50 μl) to stimulate local TRPV1 in anesthetized preparation; 2) immunoreactivity of TRPV1 in laryngeal sensory neurons of N/J ganglia retrogradely traced by DiI; and 3) TRPV1 currents recorded by whole‐cell patch clamp on cultured sensory laryngeal C‐neurons from N/J ganglia retrogradely traced by DiI. Our results showed that PNE: 1) markedly prolonged the SLCF‐mediated apnea; 2) significantly increased the expression TRPV1 receptors in N/J ganglionic C‐neurons; and 3) augmented the amplitudes of TRPV1 currents on these neurons. The results suggest that PNE is able to prolong the SLCF‐mediated apnea likely though upregulating TRPV1 receptor expression, which may contribute to SIDS.

Support or Funding Information

Supported by HL‐107462 and HL‐119683
Lethal Avian Influenza A (H5N1) Virus Induces Ataxic Breathing with Reduced Neurokinin 1 Receptor Expression in the Pre‐Botzinger Complex in Mice
Authors: Zhuang Jianguo , Zang Na , Ye Chunyan , Xu Fadi
Published: November 2023

The most common symptoms prior to death in patients infected by lethal influenza A (H5N1) are shortness of breath (tachypnea) and respiratory failure. Although the death has been observed in mice at 7 day postinfection, dynamic process of the ventilatory disorders leading to respiratory failure over the infection period and the underlying mechanisms has not been investigated. To this end, the mice were intranasally inoculated with vehicle (Ctrl), 100 PFU HK483 (lethal strain) or HK486 virus (nonlethal strain). We first measured ventilation and arterial blood pH and gases at 0 (before), 2, 4, and 6 days postinfection (dpi). Ctrl and HK486 mice showed no change over the infection period, while HK483 mice presented: 1) gradual body weight loss and hypothermia; 2) a remarkable tachypnea at 2–4 dpi and an ataxic breathing with appearance of long‐lasting apneas at 6 dpi; and 3) hypoxemia and hypercapnemia after 4 dpi that were worse at 6 dpi. Because lethal H5N1 virus is neurotropic and ataxic breathing (hypoxemia) has been reported in rats with an insufficient expression of neural neurokinin 1 receptor (NK1R) in the Pre‐Botzinger complex (PBC), we second asked if HK483 virus replicated in the PBC to induce apoptosis and reduce the population of neurons expressing NK1R. After euthanasia and fixation at 6 dpi, the medullary sections were processed to detect the nucleoprotein of H5N1 virus (NP) alone or coupled with immunoreactivity (IR) of NK1R or CASP3 (a marker for apoptosis). The results showed that HK483 but not HK486 replicated in PBC neurons, particularly local NK1R neurons and reduced the population of PBC NK1R neurons by 75%. CASP3‐IR was detectable in the PBC and NK1R neurons solely in HK483 mice. Our results suggest that lethal H5N1 virus replicates in the PBC to induce apoptosis and thereby reduce population of NK1R neurons, contributing to ataxic breathing and respiratory failure.

Support or Funding Information

Supported by HL‐107462 and HL‐119683
Progeria: How much information do we know?
Authors: Glymph Kelly , Agazuma Keshia , Raynes Edilberto , Waller Lauren , Coker Karen , Michaels Natalie , Banting Imelda
Published: November 2023

Hutchinson‐Gilford Progeria syndrome, also known as Progeria, is a rare genetic disorder, caused by LMNA gene mutation resulting in segmental aging syndrome. It affects one in 20 million people worldwide and has an estimated prevalence of one in four million. It has no sex predilection and affects all races. The syndrome does not manifest signs and symptoms until 9 to 12 months of life and the life expectancy is often only until age 12 to 16 years old (Mayo Clinic, 2016). The purpose of the study was to peruse the available research and to disseminate information to the public in order to increase awareness of the syndrome, and to rectify any misconceptions about the disorder. Using a systematic review of literature, the findings suggested that a multi‐disciplinary approach is needed to help the affected children increase their quality of life.

Edilberto A. Raynes, MD, PhD; Kelly Glymph, SPT; Keshia Agazuma, SPT; Lauren Waller, SPT; Karen Coker, PT, DPT, CWS, C/NDT, FACCWS; Natalie Michaels, PT, EdD, GCS Emeritus, and Imelda Banting, PT, MD
NF‐kB, but not HIF‐1a, Inhibition Gene‐specifically Blocks Hypoxia‐induced Microglial Priming
Authors: Ewald Andrea C , Kiernan Elizabeth A , Janke Anna C , Watters Jyoti J
Published: November 2023

The innate immune system has the capacity for “memory,” the ability to change future cellular responses based on prior experiences. Previous studies in our lab have shown that chronic intermittent hypoxia (IH), a hallmark of sleep‐disordered breathing, can induce cellular memory in both adult and neonatal microglia, CNS resident immune cells. When the immune system is challenged with lipopolysaccharide (LPS) the day following the last IH exposure, LPS‐induced microglial gene expression is augmented, indicating that IH pre‐exposure primes the microglial immune response. Hypoxia‐induced priming of microglial inflammatory gene responses are associated with upregulation of histone 3 lysine 4 trimethylation (H3K4me3) at gene promoters/enhancers, supporting an epigenetic role in microglial memory following hypoxia exposure. To begin to address the signaling mechanisms underlying IH‐induced priming, and the subsequent changes in H3K4me3, we developed an in vitro model of hypoxia which mirrors the microglial responses to IH observed in vivo. Using this model, we tested the hypothesis that hypoxia‐induced potentiation of inflammatory gene expression in microglia requires the activation of the hypoxia‐responsive transcription factors HIF‐1a and NF‐kB, key modulators of inflammatory gene expression. Immortalized N9 microglia were treated overnight with echinomycin (HIF‐1a inhibitor, 1 nM) or BOT‐64 (NF‐kB inhibitor, 3 μM) during the hypoxia exposure period (1.5% O2 for 16 hrs), after which time the cells were washed and stimulated with LPS (100 ng/mL; 3 hrs). We used qRT‐PCR to assess the gene expression of two inflammatory molecules that are potentiated by hypoxia pre‐exposure: inducible nitrogen oxide synthase (iNOS) and interleukin‐1 beta (IL‐1B). We found that while HIF‐1a inhibition did not attenuate the hypoxia‐induced potentiation of iNOS nor IL‐1B, NF‐kB inhibition had gene‐specific effects. Specifically, BOT‐64 blocked the potentiation of iNOS, but not IL‐1B. These results suggest that hypoxia‐induced priming of microglia involves the activation of NF‐kB, as well as at least one other unidentified pathway, to influence microglial gene expression. Ongoing studies are examining if NF‐kB inhibition also attenuates upregulation of H3K4me3 in inflammatory gene regulatory regions to provide insights into the mechanism whereby NF‐kB may regulate hypoxia‐induced microglial priming. Because this form of cellular memory augments the microglial inflammatory response, it may be associated with exacerbated CNS damage during sleep disordered breathing.

Support or Funding Information

This work was supported by NIH R01 NS085226.
Discriminative Stimulus Effects of Naltrexone and Haloperidol in Rats with Chronic, Intermittent Access to Sucrose
Authors: Marek Morgan , Moline Adam , Altendorf Luke , Vogelsang Jenna , Cyra Matthew , James Kimberly , Herrmann Jody , Levine Allen , Jewett David
Published: November 2023

Previous research demonstrated chronic, intermittent sucrose access increased endorphin and dopamine function, and naltrexone elicited some withdrawal‐like behaviors in rats. We determined the naltrexone serves as a discriminative stimulus (3.2 mg/kg, s.c.) in subjects with 12 hr access to sucrose (10–32%) followed by 12 hr access to water, but not in subjects with 24 hr access to water. We wondered if smaller naltrexone doses could be established as discriminative stimuli. Groups of rats were injected with naltrexone (0.1–1.0 mg/kg) or saline 1 hr after sucrose access and 15 min prior to discrimination training. Left lever presses were reinforced under a FR 15 schedule following drug administration, and right lever presses were reinforced following saline administration. Daily training session continued until rats emitted 80% or greater condition‐appropriate responses both prior to the first consequence and for the total session. Most subjects trained with either 0.32 or 1.0 mg/kg acquired the discrimination. Naltrexone (0.1 mg/kg) served as a discriminative stimulus in approximately one‐third of the subjects. We also attempted to establish the dopamine antagonist haloperidol as a discriminative stimulus. Haloperidol (0.056 mg/kg, 30 min PT) was established as a discriminative stimulus in 4 of 4 subjects with chronic, intermittent sucrose access (M=49 sessions) and 5 of 6 subjects with 24 hr water access (M=82 sessions). Two of 5 subjects with chronic, intermittent sucrose access discriminated haloperidol (0.018 mg/kg) from saline (M=84 sessions). Other subjects failed to acquire the discrimination in 120 sessions under these conditions. Haloperidol (0.056 mg/kg) significantly reduced rates of lever pressing. This effect of haloperidol was independent of liquid consumption. In sum, chronic sucrose consumption alters opioid function, naltrexone's ability to serve as a discriminative stimulus depends on the training dose, and sucrose consumption does not consistently alter the ability of haloperidol to serve as a discriminative stimulus.

Support or Funding Information

The University of Wisconsin‐Eau Claire Office of Research and Sponsored Programs and the Minnesota Obesity Center
CRISPR/Cas9‐Mediated Genome Editing of the Murine Pulmonary Circulation
Authors: Marsboom Glenn , Yuan Yang , Rehman Jalees , Malik Asrar B.
Published: November 2023

CRISPR/Cas9 is increasingly being tested for in vivo genome editing and we describe here the optimization, pharmacokinetic properties, and efficiency of this system to target the pulmonary vasculature in mice. We used cationic liposomes to deliver a PX458 vector expressing Cas9, eGFP, and gRNA to the pulmonary circulation and studied both its bio‐distribution and time needed to knock down proteins.

Different amounts of liposomes were injected containing 25, 50, 75, and 100μg of plasmid DNA. After 4 days, mice were sacrificed and several organs were studied for eGFP expression. We found a dose‐dependent increase in pulmonary expression. The only other organ with detectable expression was the liver, but expression was at least 7‐fold higher in the lungs compared to the liver even at the highest dose tested. We found no eGFP expression in kidney, heart, brain, or skeletal muscle (n=4 animals/dose). Within the lung, we used immunohistochemistry to show that endothelial cells and to a lesser degree pulmonary artery smooth muscle cells were selectively targeted. We did not detect any expression in airway epithelial cells or airway macrophages. Lung expression was very temporary, with expression peaking at 4 days and returning to baseline after 28 days (n=4 mice/group), minimizing the chances of side effects, more specifically non‐specific alterations in the DNA that could result from continued expression of the endonuclease Cas9. Using mT mice that ubiquitously express tandem dimer Tomato fluorescent protein, were able to show a downregulation of fluorescence specifically in the majority of the pulmonary blood vessels after just 7 days (n=3 mice/group). Genomic alterations were confirmed by the T7 endonuclease assay and Next‐Generation Sequencing. Importantly, we did not observe any thrombosis or other adverse side‐effects that might be associated with expression of Cas9 in the pulmonary endothelium.

In conclusion, we used a non‐viral delivery method to specifically deliver CRISPR/Cas9 to the pulmonary vasculature. The temporary expression of CRISPR/Cas9 led to a pronounced downregulation of the targeted fluorescent protein within 7 days, but could easily be adapted to target almost any gene. Currently available animal models do not allow to knock out a gene specifically in the pulmonary endothelial cells and effects on the pulmonary circulation might be compounded by effects in the systemic circulation. Therefore, our work provides a novel tool to specifically target and study genes in the pulmonary circulation.

Support or Funding Information

This work was supported in part by a Parker B. Francis fellowship and American Heart Scientist Development grant 15SDG23250002 (GM), NIH‐R01‐GM094220 (JR) and NIH‐R01‐HL045638, R01‐HL090152, R01‐HL118068, P01‐HL060678, P01‐HL077806 (ABM).
Histone deacetylase 9 (HDAC9) is associated with increased lysine acetylation levels in diabetic mice kidneys: a potential mechanism underlying renal impairment in type 2 diabetes
Authors: Polce Simran A , Carrillo‐Sepulveda Maria Alicia
Published: November 2023

Diabetic nephropathy is one of the major complications faced by patients with type 2 diabetes (T2DM). It is characterized by fibrosis, mesangial matrix expansion, loss of proper glomerular filtration, and marked renal vascular dysfunction. The mechanism by which diabetic milieu promotes renal dysfunction is not entirely understood. Recent studies have demonstrated that nephropathy in type 1 diabetes is correlated with high levels of renal lysine acetylation. Importantly, histone deacetylase 9 (HDAC9), a class II subclass IIa mammalian HDAC that catalyze the removal of acetyl groups from ɛ‐amino groups of lysine's, has been linked to T2DM complications. Thus, we hypothesize that T2DM increases levels of renal lysine acetylation which is associated with downregulation of HDAC9. To address our hypothesis, male fourteen‐weeks‐old db/db mice, an experimental model of T2DM, and same age‐matched heterozygous nondiabetic (ND) mice (db/m+) were utilized in this current study. As expected, db/db mice exhibited robust increase in body weight (39.8g vs. 19.5g ND, p<0.05, n=7), kidney weight (238.8mg vs. 163.9mg ND, p<0.05, n=6) and blood glucose levels (265 mg/dl vs. 104.5mg/dl ND, p<0.05, n=7). Together these results confirm the diabetic state in the model utilized. Further, to determine whether HDAC9 and lysine acetylation are altered in kidneys from diabetic mice, western blot analysis was performed. Kidneys from diabetic mice exhibited markedly increases in lysine acetylation levels (80% increase vs. ND, p<0.001, n=5), which was correlated with significant reduction in HDAC9 expression (60% reduction vs. ND, p<0.05, n=5), suggesting that decreased HDAC9 may contribute to the elevated levels of lysine acetylation. Of note, no changes in others HDAC class II subclass IIa, including HDAC4, HDAC5 and HDAC7 were found in kidneys from db/db mice. Our results demonstrate that diabetic milieu increases lysine acetylation in kidney and downregulated HDAC9 epigenetic enzyme expression. Thus, targeting HDAC9 may offer novel therapeutic strategies for treating diabetic nephropathy. Studies using overexpression of HDAC9 has been initiated to confirm the role of HDCA9 in the diabetic renal dysfunction.

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NIH
Regulator of G‐protein Signaling 10 (RGS10) Expression is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity
Authors: Alqinyah Mohammed , Maganti Nagini , Ali Mourad , Yadav Ruchi , Weng Han‐Rong , Hooks Shelley
Published: November 2023

Regulator of G‐protein Signaling 10 (RGS10) has emerged as a key regulator of pro‐inflammatory cytokine production in microglia, functioning as an important neuroprotective factor. While RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor‐activation. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 expression in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is silenced in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation‐dependent silencing of RGS10 expression in microglia. We demonstrate that RGS10 is endogenously expressed in spinal cord microglia, and expression is suppressed in the spinal cord in a nerve injury induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor Trichostatin A blocks the ability of Lipopolysaccharide (LPS) to suppress RGS10 transcription in BV‐2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of RGS10. Further, we used chromatin immunoprecipitation to demonstrate that H3 histones at the RGS10 proximal promoter are deacetylated in BV‐2 microglia following LPS activation, and HDAC1 association at the RGS10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1‐phosphate, an endogenous microglial signaling mediator which inhibits HDAC activity, enhances basal RGS10 expression in BV‐2 microglia, suggesting that RGS10 expression is dynamically regulated in microglia in response to multiple signals.

Support or Funding Information

This work was supported by grants from the National Multiple Sclerosis Society (SBH) and the National Institutes of Neurological Disorders and Stroke [RO1NS064289] (HRW).
Regulation of the Expression and Function of Exchange Protein Directly Activated by cAMP (EPAC) by Hypoxia
Authors: Olah Mark , Justus Lillian , Kiefer Erika , Shelley Jessica
Published: November 2023

Exchange protein directly activated by cAMP (EPAC) is a signaling protein implicated in several physiologic and pathophysiologic processes. Following binding of cAMP, EPAC activates small GTPases such as Rap1 to induce downstream effects. In vascular endothelial cells, EPAC may have a role in the regulation of permeability, vascular tone and angiogenesis. As low oxygen tension initiates many vascular events including those modulated by EPAC, we hypothesized that hypoxia regulates EPAC expression and function in human microvascular endothelial cells (HMVEC). HMVEC maintained in 2.0% oxygen for 48 hours demonstrated a significant decrease in expression of EPAC protein relative to normoxic cells or HMVEC exposed to hypoxia for shorter periods. At 48 hours of hypoxia, EPAC expression was 53.9 ± 7.4% of that in normoxic cells. To begin to delineate the mechanism underlying this downregulation of EPAC, we examined the role of the transcription factor hypoxia‐inducible factor‐1α (HIF‐1α). Deferoxamine, an iron chelator, and IOX‐2, an inhibitor of prolyl hydroxylase, both increased expression of HIF‐1α at short time periods as did exposure to 2.0% oxygen. At 48 hours of treatment, deferoxamine and IOX‐2 mimicked the effects of hypoxia by decreasing EPAC expression to levels 40.5 ± 12.1% and 43.0 ± 12.7% of control, respectively. To assess the functional significance of hypoxia‐induced downregulation of EPAC, we examined activation of the downstream effector Akt by determining levels of phospho‐Akt (Ser473) by Western blotting. The A2B adenosine receptor agonist 5′‐N‐ethylcarboxamidoadenosine (NECA) and the adenylyl cyclase activator forskolin, both of which increase intracellular cAMP levels, as well as the EPAC specific activator 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine 3′,5′‐cyclic monophosphate (8‐CPT) induced a decreased Akt stimulation in hypoxic HMVEC relative to normoxic cells. In conclusion, hypoxia downregulates protein expression of EPAC in HMVEC and this may be mediated by HIF‐1α. This decrease may result in reduced EPAC‐mediated Akt activation. Studies are currently further exploring the mechanism of the hypoxia‐induced downregulation of EPAC and examining effects on Akt‐mediated functional responses such as cell survival.

Support or Funding Information

Supported by funds from The Raabe College of Pharmacy, Ohio Northern University
UCH‐L1 as a Susceptibility Factor for Nigrostriatal and Mesolimbic Dopamine Neurons: Neurotoxicant Exposure and Aging
Authors: Winner Brittany Michele , Zhang Harue , Lookingland Keith , Goudreau John
Published: November 2023

Parkinson disease (PD) is a debilitating neurodegenerative disorder in which nigrostriatal (NSDA) dopamine neurons die, affecting signaling within the basal ganglia pathway. Loss of dopamine input to the striatum (ST) results in the motor symptoms required to diagnose PD. Although the majority of cases are idiopathic, genes mutated in familial PD can provide clues to highlight important pathways important in pathogenesis and progression of PD. One such pathway is the ubiquitin proteasome system (UPS) which degrades proteins tagged with ubiquitin (Ub) chains. Since accumulation and aggregation of misfolded proteins such as alpha synuclein is a hallmark of PD, enhancing degradation of key substrates is a lucrative target for therapy. One UPS‐related protein mutated in familial PD is UCH‐L1, a deubiquitinating enzyme responsible for maintaining available pools of free monomeric Ub. UCH‐L1 is an abundant neuron‐specific protein that is critical for normal synaptic function and to maintain the structure of the neuromuscular junction. In a mouse model of oxidative damage specific to dopamine neurons, systemic administration of neurotoxicant MPTP resulted in loss of UCH‐L1 expression and function in the substantia nigra after 24 h. Since loss of UCH‐L1 and loss of phenotypic markers both occur concurrently in NSDA neurons, we hypothesized that loss of UCH‐L1 contributes to susceptibility to neurotoxicant administration. Not all dopamine neurons are equally susceptible in PD: mesolimbic dopamine neurons (MLDA) are spared while NSDA neurons degenerate. We compared NSDA to MLDA neurons and found that MLDA neurons are less susceptible to MPTP‐induced loss of ST dopamine and tyrosine hydroxylase (TH), which coincides with maintenance of UCH‐L1 expression in the ventral tegmental area. In addition, we performed an aging study to determine if UCH‐L1 is lost in aged mice versus young mice and if aged mice are more sensitive to loss of UCH‐L1 after MPTP. Finally, we hypothesized that overexpression of UCH‐L1 in NSDA neurons via rAAV 2/5 will be protective against proteasome impairment and loss of dopamine phenotypic markers after MPTP. Preliminary results indicate that aged mice are more sensitive to MPTP‐induced loss of dopamine in the ST compared to young mice, but not more sensitive to loss of TH and dopamine transporter expression. However, aged mice show decreased metabolism of dopamine as evidenced by lower levels of one of its metabolites, DOPAC. Aged mice also show similar levels of UPS impairment compared to young mice. Further study will determine if UCH‐L1 expression is influenced by aging in NSDA neurons, and if UCH‐L1 overexpression rescues UPS impairment in young mice.

Support or Funding Information

Institute for Integrative Toxicology T32 Predoctoral Fellowship National Institutes of Health NINDS
Behavioral and Biochemical Consequences of Simulated Vehicle Exhaust Exposure (SVEE) in Rats
Authors: Salvi Ankita , Patki Gaurav , Liu Hesong , Salim Samina
Published: November 2023

Air pollution from vehicle exhaust is a serious environmental threat, known to contribute to cardiovascular, respiratory and neurological complications. While the effect of vehicle exhaust exposure on heart and lungs is well known, adverse effects of these emissions on the brain and its psychological impact has been ignored. Here, we examined exhaust‐induced psychological and neurobiological alterations in rats using a simulated vehicle exhaust exposure (SVEE) model. Prolonged exposures to the gaseous constituents of vehicle exhaust, carbon dioxide (CO2), carbon monoxide (CO) and nitrogen dioxide (NO2) are known to increase oxidative stress. And, our previous studies have established a causal link between oxidative stress and behavioral as well as cognitive deficits. Therefore, we hypothesized that prolonged exposures to pro‐oxidants from vehicle exhaust elevate oxidative stress leading to behavioral and cognitive deficits. And, interventions that limit/prevent oxidative stress such as a mitochondria‐permeable antioxidant, MitoQ, can protect SVEE‐induced impairments. Four groups of Sprague Dawley rats were included: Control+Vehicle (C+V), Control+MitoQ (C+M), Exposure+Vehicle (E+V) and Exposure+MitoQ (E+M). The MitoQ groups received 250μM of MitoQ for 4 weeks via drinking water. Vehicle groups received normal drinking water during this period. Following the treatment, exposure rats were exposed to a simulated mixture of vehicle exhaust containing 0.04% CO2, 0.9 ppm NO2 and 3 ppm CO. Exposures were performed in whole body exposure chambers 5h daily for 2 weeks. Duration of exposures was comparable to daily exposure of exhaust levels in areas of high traffic. Control rats were exposed to normal air for the same duration. Following SVEE, anxiety‐ and depression‐like behavior, as well as cognitive function and intelligence quotient (IQ) levels were assessed. An increase in anxiety‐like and depression‐like behavior as well as impaired memory and low IQ was observed in E+V rats as compared to C+V and C+M rats. However these deficits were not seen in E+M rats. At the biochemical level, effect of SVEE on mitochondrial function (one of the targets of increased oxidative stress) was assessed. Mitochondrial oxygen consumption in three brain regions, namely, prefrontal cortex, hippocampus and amygdala was determined using high resolution respirometry. SVEE led to decreased mitochondrial oxygen consumption in rats suggesting SVEE‐induced mitochondrial dysfunction. Oxygen consumption was unaltered in rats that received MitoQ.

Thus our study has identified behavioral and biochemical impairments associated with exposure to vehicle exhaust in rats and identified a potential role of the antioxidant MitoQ in preventing these impairments.

Support or Funding Information

Grant Support: 2R15MH093918‐02