bluuebunny/crossref_metadata_2025 huggingface dataset:
Discovery and Characterization of Small Molecule Potentiators of KCC2 Activity
Authors: Prael Francis J. , Delpire Eric , Weaver C. David
Published: April 2020

Deficits in inhibitory neurotransmission, and the resulting excitatory/inhibitory (E/I) imbalance, are a feature of a host of neurological disorders, including: epilepsy, chronic pain, schizophrenia, and autism. Increased intracellular Cl− ([Cl−]i) has been implicated as an underlying cause of the E/I imbalance via disruption of fast inhibitory neurotransmission. The K+‐Cl− cotransporter 2 (KCC2) is a principal exporter of neuronal Cl−. Owing to the effect of KCC2 on [Cl−]i, pharmacological potentiation of KCC2 activity has become a promising therapeutic approach for these neurological disorders. While there has been substantial progress in discovering pharmacological potentiators of KCC2 activity in recent years, current pharmacological tools targeting KCC2 either act by indirect mechanisms or have some controversy surrounding their activity at KCC2. Although these compounds represent major improvements to KCC2 potentiator pharmacology, the discovery of direct‐acting KCC2 potentiators would still be a substantial contribution to the field because: (a) direct‐acting KCC2 potentiators would be better chemical tools to assess the contribution of KCC2 to a given phenotype, since they would lack the off‐target effects associated with indirect KCC2 potentiators, and (b) de‐risk KCC2 itself as a therapeutic target for drug discovery. To discover direct‐acting KCC2 potentiators, we have developed and validated a suite of high throughput‐compatible assays to measure KCC2 activity. Using a primary assay measuring Cl− export via a genetically‐encoded Cl− sensor, we have iteratively screened over 30,000 compounds for their effect on KCC2 activity. Our iterative screening approach maximized our ability to explore chemical scaffolds that were active across primary and orthogonal assays – allowing us to discover a series of putative KCC2 potentiators, despite screening a relatively small chemical library. From these efforts, we have discovered compounds capable of decreasing [Cl−]i in a KCC2 inhibitor‐sensitive manner, potentiating transport of KCC2 substrate congeners in a KCC2‐dependent fashion, and blocking epileptiform calcium oscillations in cultured neurons. We are currently investigating the mechanism by which these compounds affect KCC2 activity. The compound class we have discovered may be useful as a pharmacological tool for evaluating the therapeutic potential of KCC2 in a host of neurological disorders which feature E/I imbalance.

Support or Funding Information

Vanderbilt Institute for Clinical and Translational Research Award (VR52556)

Chemistry‐Biology Interface T32 (5T32GM065086‐15)
How Do O‐Fucose Glycans Stabilize Thrombospondin Type 1 Repeats?
Authors: Berardinelli Steven J. , Manjunath Rajashri , Eletsky Alexander , Ito Atsuko , Haltiwanger Robert S.
Published: April 2020

Thrombospondin Type 1 Repeats (TSRs) are small cysteine‐rich domains found in multiple cell‐surface and secreted proteins. TSRs containing the consensus sequence Cxx(S/T)C are typically modified on the serine or threonine with an O‐linked Glcβ1‐3Fuc disaccharide. The O‐fucose is added by Protein O‐fucosyltransferase 2 (POFUT2), which is then elongated by β1‐3glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in early embryonic lethality, and human mutations in B3GLCT cause Peters Plus Syndrome (PPS), a Congenital Disorder of Glycosylation. POFUT2 and B3GLCT are localized in the endoplasmic reticulum (ER) ‐ the folding compartment for proteins in the secretory pathway, and POFUT2 only adds fucose to properly folded TSRs. Thus, POFUT2 appears to function as a folding sensor for TSRs, and both POFUT2 and B3GLCT have been proposed to assist in the folding of TSR‐containing proteins. Interestingly, loss of POFUT2 causes secretion defects for most TSR‐containing proteins, while loss of B3GLCT only effects secretion of a subset of these proteins. The mechanisms behind why POFUT2 is required for section of most TSR‐containing proteins but B3GLCT is required for only a few is not understood. We hypothesize that this unique disaccharide on TSRs is interacting with amino acids in close proximity to the sugars thereby stabilizing the folded state of TSRs and ultimately the protein as a whole, and that the fucose has the major stabilizing effect, while the glucose is only required for some TSRs. To test this hypothesis, we designed a Reductive Unfolding Assay to monitor the effects of the sugars on the stability of TSRs. We have identified amino acids affected by the presence of the fucose or glucose using NMR and molecular dynamics simulations. We have mutated several of these amino acids to determine if they reduce the stabilizing effects of the sugars using the reductive unfolding assay. One such mutant, P547A in TSR3 from human thrombospondin‐1, decreases the sugar‐mediated stabilization of TSR3, providing evidence that the disaccharide stabilizes the folded state by interacting with certain amino acids in close proximity. We are examining the stabilizing effects of the fucose and glucose on several other TSRs as well. These results provide further evidence that POFUT2 and B3GLCT act as quality control enzymes inside the ER.

Support or Funding Information

This work was supported by NIH grant HD090156.
Saturated fatty acid‐enriched diet exacerbate hepatic mitochondrial dysfunction in rats subjected to maternal protein restriction
Authors: Simões-Alves Aiany C. , Costa-Silva João H. , Barros-Junior Idelfonso B. , da Silva-Filho Reginaldo Correia , Vasconcelos Diogo A. A. , Morio Béatrice , Fernandes Mariana P.
Published: April 2020

The nutritional transition that the western population has undergone is increasingly associated with chronic metabolic diseases. In this context, dietary factors has been linked to oxidative stress, making the body vulnerable to mitochondrial dysfunction and reactive oxygen species. In this work, we evaluated the impact of a saturated fatty acid‐enriched diet in the post‐weaning offspring subjected to maternal protein restriction on the liver mitochondrial bioenergetics. Wistar rats were mated and during gestation and lactation, mothers received control diets (NP, normal protein content 17%) or low protein (LP, 8% protein). After weaning, rats received either NL (normolipidic) or HL (+59% SFA) diets up to 90 days of life. It was verified that all respiratory states of hepatic mitochondria showed a reduction in the LP group subjected to the post‐weaning HL diet (LP‐HL), basal state (−43.7%), state 3 (−37.3%), state 4 (−32.1%) and uncoupled state (−60.4%). Similarly, respiratory control was significantly lower (p<0.01). This group also presented greater mitochondrial swelling compared to controls (p<0.01), potentiated after Ca2+ addition (p <0,01) and prevented in the presence of EGTA (calcium chelator) (p<0.001); and cyclosporin A (mitochondrial permeability transition pore inhibitor) (p<0.0001). These effects were associated to increased liver protein and lipid peroxidation and reduced anti‐oxidant enzymes activities in the LP group fed HL diet after weaning. Additionaly, it was observed increase in PGC‐1α and VDAC and decrease in Tfam gene expressions compared to LP‐NL (p<0.05). Our data suggest that SFA‐enriched HFD induces mitochondrial dysfunction associated with oxidative stress, greater expression of VDAC that may potentiate the opening of the mitochondrial permeability transition pore, and lower mitochondrial biogenesis in the liver of adults rats, with major effects in animals previously submitted to maternal protein restriction.

Support or Funding Information

This study was supported by FACEPE (Brazil), CNPq (Brazil) and CAPES/COFECUB (Brazil/France).
Does Acute Exercise Increase Insulin‐Stimulated Skeletal Muscle Glucose Uptake, Blood Flow And Insulin Signalling In Response To A Meal?
Authors: Mcconell Glenn , Sjoberg Kim , Wojtaszewski Jorgen , Kiens Bente , Richter Erik
Published: April 2020

A single bout of exercise increases skeletal muscle insulin sensitivity measured by euglycemic, hyperinsulinemic clamp (EHC) through coordinated increases in skeletal muscle insulin‐stimulated microvascular perfusion and molecular signalling at the level of TBC1D4
,
. Surpringly, however, skeletal muscle interstitial glucose concentration decreases substantially during a EHC, especially after one‐legged exercise, reaching 2–3 mM
. This result may, however, be a artifact of the EHC because, unlike after a meal, blood glucose does not rise during a EHC but is “clamped” at around 5 mM while plasma insulin is elevated. Therefore, the aim of this project was to determine the effect on leg blood flow and skeletal muscle glucose metabolism of meals begun 4 hours after one‐legged exercise. We hypothesized that hyperglycemia after the meal would prevent a drop in muscle interstitial glucose concentrations and result in large increases in leg glucose uptake. Four healthy young males performed 60 min of 1‐legged knee extensor exercise involving at 80% of 1‐legged peak work load (PWL) with three 5 min intervals at 100% 1‐legged PWL. Participants then rested for 4 hours and catheters were inserted into the femoral artery of one leg and the femoral vein of both legs and two microdialysis catheters for measurement of interstitial glucose concentration were inserted into the vastus lateral muscle of both legs. A solid meal was ingested (40 kJ.kg BW−1) 4 hrs after the 1‐legged exercise with smaller liquid meals (20 kJ.kg BW−1) ingested 4.5 and 5 hrs after the exercise. Four hours after exercise there was no significant difference between the exercised and rested leg for leg blood flow, leg arteriovenous glucose difference, leg glucose uptake, vastus lateralis interstitial glucose concentration or apparent muscle membrane glucose permeability. Meal ingestion increased arterial blood glucose concentration and increased leg blood flow, leg arteriovenous glucose difference and leg glucose uptake more in the exercised leg than the rested leg. Meals increased vastus lateralis interstitial glucose concentration, especially in the rested leg. Vastus lateralis apparent muscle membrane glucose permeability increased with the meals in both legs and moreso in the previously exercised leg. Ingestion of meals several hours after 1‐legged exercise results in hyperglycemia, increased skeletal muscle interstitial glucose concentration and increased muscle membrane permeability to glucose. This results in larger increases in muscle glucose uptake in the previously exercised than the rested leg. Real life feeding after exercise confirms and extends findings from clamp studies.
Spinal cord injury leads to increases in apnea frequency and duration in spontaneously breathing mice with and without central nervous system 5HT
Authors: Qiu Qingchao , Kariharan Thiruchelvam , Kuhn Donald , Mateika Jason
Published: April 2020

Purpose

The present investigation was designed to explore the impact of serotonin (5HT) on apnea severity and duration, arousal frequency and respiratory stability following spinal cord injury (SCI) in mice.

Methods

Telemetry transmitters were surgically implanted in wild type mice (Tph2+/+) (n = 4) and tryptophan hydroxylase 2 knockout mice (Tph2−/−) (n = 3) to measure electroencephalography, core body temperature and gross motor activity. Following recovery, the Tph2+/+ and Tph2−/− mice were placed in whole body plethysmographs and measures of apnea severity, coefficient of variation of the duration of each breath, duration index and arousal frequency during non‐rapid eye movement sleep were determined for 6 hours. Following these measures, a C2 hemisection of the spinal cord was completed and the physiological measurements were repeated 4 and 21 days following hemisection.

Results

Following SCI the number of apnea events increased in the Tph2+/+ (baseline vs. 21 days: 52.1 ± 17.4 vs. 106.4 ± 21.2 events/hr, p = 0.02) and the Tph2−/− mice (30.7 ± 9.6 vs. 77.5 ± 34.1, p = 0.02). The coefficient of variance of the duration of each breath also increased after SCI (Tph2+/+ 0.25 ± 0.02 vs. 0.30 ± 0.06, Tph2−/− 0.25 ± 0.02 vs. 0.35 ± 0.06, p = 0.07). Likewise, the apnea duration index increased following SCI in the Tph2+/+ (baseline vs. 21 days: 2.4 ± 0.05 vs. 3.2 ± 0.6 events/hr, p = 0.047) and Tph2−/− mice (baseline vs. 21 days: 2.6 ± 0.5 vs. 3.8 ± 0.5 events/hr, p = 0.047). Lastly, the number of arousals increased after SCI in both groups (Tph2+/+ 19.0 ± 4.6 vs. 39.1 ± 13.8, Tph2−/− 5.1 ± 0.9 vs. 35.6 ± 19.4 arousal/hr, p = 0.03).

Conclusions

SCI leads to an increase in the frequency and duration of apneic events, which is coupled to an increase in the number of arousals. Likewise, alterations in the coefficient of variation indicate that breathing is more variable when present. However, the role of 5HT in mediating arousals and stabilizing breathing is less certain in this small sample size, since modifications in breathing stability and arousal frequency was not statistically different in the Tph2+/+ and Tph2−/− mice following SCI.

Support or Funding Information

Department of Veterans Affairs (I01BX003946 & 15SRCS003 JHM)
A fine‐tuned interaction between the ER and mitochondria regulates Ca2+‐dependent after hyperpolarizations in vasopressin supraoptic neurons
Authors: Kirchner Matthew Karl , Stern Javier Eduardo
Published: April 2020

It is known that vasopressin magnocellular neurons become hyperexcitable in cardiovascular diseases such as heart failure. This hyperexcitability is mediated by shifts in both synaptic and intrinsic mechanisms. One key intrinsic mechanism is the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium‐dependent K+ current (IsAHP) most notable for its role in spike frequency adaptation. While features of the sAHP and its effects on firing are well established, a comprehensive understanding of the underlying mechanisms are still unknown. Previous work demonstrated that sAHP activation requires Ca2+ entry via voltage‐gated Ca2+ channels (VGCCs). However, whether in VP neurons Ca2+ entry via these channels directly activates the sAHP, or alternatively, whether it stimulates Ca2+‐induced Ca2+ release from an internal source such as endoplasmic reticulum (ER) or mitochondria remains unknown. Combining patch clamp electrophysiology with simultaneous Ca2+ imaging in transgenic eGFP‐VP rats, we investigated the role of ER and mitochondrial in shaping the sAHP. We measured sAHPs in voltage clamp by evoking a train of action potentials and measuring the resulting outward current. Pre‐incubation of slices with thapsigargin (3μM), an inhibitor of the sarco/ER Ca2+ ATPase inhibited IsAHPs significantly (96%, p< 0.001) while leaving Ca2+ transients unaffected (p > 0.05). This demonstrates that ER Ca2+ release is necessary for sAHP activation. Conversely, blockade of mitochondrial Ca2+ release via blocking mitochondrial Ca2+ exchangers with TPP (20μM) failed to inhibit the IsAHP (p > 0.05). We next evaluated the role of mitochondrial Ca2+ uptake with bath application of the mitochondrial uncoupler CCCP (1μM) or the mitochondrial Ca2+ uniporter (MCU) blocker Ru360 (20μM) in the patch pipette. These manipulations significantly prolonged the decay time constant of both the IsAHP (p< 0.01) and the underlying Ca2+ transient (p< 0.05), demonstrating that the MCU regulates spatiotemporal dynamics of the sAHP. Finally, combining thapsigargin with Ru360 in the pipette showed an initial block of the sAHP, followed by an enhancement over 15 minutes as Ru360 dialyzed into the cell. These results support the ER (but not mitochondria) as a major source of Ca2+ activating the sAHP, as well as a role for the MCU in regulating the magnitude and time course of the sAHP. Taken together, our studies support a fine‐tuned interaction between VGCCs, ER, and mitochondria in modulating the magnitude and time course of the sAHP in VP neurons. Future studies warrant investigation into whether changes in these interactions contribute to altered VP neuronal activity in cardiovascular disease states.

Support or Funding Information

R01HL090948
Effects of Prostacyclin Signaling on Alzheimer’s Disease Associated Pathologies
Authors: Womack Tasha , Eriksen Jason L.
Published: April 2020

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder that is the most common cause of dementia in aged populations. A substantial amount of data demonstrates that chronic neuroinflammation can accelerate neurodegenerative pathologies, while epidemiological and experimental evidence suggests that use of anti‐inflammatory agents may be neuroprotective. In AD, chronic neuroinflammation results in the upregulation of cyclooxygenase and increased production of prostaglandin H2, a precursor for many vasoactive prostanoids. While is well‐established that many prostaglandins can modulate the progression of neurodegenerative disorders, little is known about the role of prostacyclin (PGI2) in the brain. We have conducted studies to assess the effect of elevated prostacyclin biosynthesis in a mouse model of AD. Upregulated prostacyclin expression significantly worsened cognitive abnormalities, accelerated amyloid pathology, and damaged the neurovasculature. PGI2 overexpression selectively increased soluble amyloid‐β 42 production, total amyloid accumulation, and burden. PGI2 altered microvessel length and branching, and PGI2 expression in combination with amyloid was more detrimental than amyloid expression alone. In vitro studies demonstrated that increased prostacyclin signaling inhibited microvessel formation and selectively altered gamma secretase subunit expression. Our findings demonstrate that chronic prostacyclin expression plays a novel and unexpected role that hastens the development of the AD phenotype.
Blood Pressure is Positively Associated with Glycosylated Hemoglobin but not the Hypoxic Ventilatory Response in Obstructive Sleep Apnea Patients
Authors: Puri Shipra , Panza Gino , Rimar Caroline , Lin Ho-Sheng , Mateika Jason
Published: April 2020

Study Objective

The hypoxic ventilatory response (HVR) is considered an index of peripheral chemoreflex sensitivity. Previous work has suggested that an increase in the HVR is responsible for acute nocturnal blood pressure surges that ultimately lead to chronic elevations in blood pressure. Previous results have also indicated that glycosylation of hemoglobin (HbA1C) impairs nitric oxide (NO) relaxation of vascular smooth muscle leading to chronic elevations in blood pressure. Given these findings, we hypothesized that the HVR and HbA1C would be positively associated with blood pressure during sleep before and 3 weeks after treatment with mild intermittent hypoxia (MIH) in humans with obstructive sleep apnea (OSA).

Methods

Eight hypertensive participants with OSA were exposed to twelve 2‐minute episodes of hypoxia (PETO2 ≈ 50 mmHg) separated by 2‐minute normoxic intervals during wakefulness for 15 days over 3 weeks. PETCO2 was sustained at 2 mmHg above baseline during MIH administration. HbA1C was measured prior to completion of the protocol on Day 1 and 15. Moreover, blood pressure was measured over a period of 24 hours, before and after completion of the MIH protocol.

Results

On Day 1, the HVR was negatively associated with HbA1C (−0.74, p < 0.04) and systolic (SBP) (R = −0.73, p < 0.05), diastolic (DBP) (R = −0.69, p < 0.05) and mean arterial blood pressure (MAP) (R = −0.71, p < 0.05) recorded during the sleep period of a 24 hour cycle. In contrast, HbA1C was positively associated with measures of SBP (R = 0.82, p < 0.01), DBP (R = 0.81, p < 0.01) and MAP (R = 0.86, p < 0.01) during sleep. Following treatment with MIH, blood pressure during sleep decreased (Before vs. After MIH: SBP 131.9 ± 4.1 vs. 122.9 ± 3.1, DBP 77.8 ± 3.9 vs. 72.8 ± 3.9, MAP 95.8 ± 3.8 vs. 89.5 ± 3.4, P < 0.02). The change (Δ) in SBP during sleep following MIH was correlated to the Δ HbA1C (R = 0.85, p ≤ 0.01). Moreover, the degree to which SBP dipped before vs. after MIH was also correlated to Δ HbA1C (R = 0.87, p ≤ 0.01). In contrast, Δ HVR was not correlated to the Δ in blood pressure parameters.

Conclusion

Although peripheral chemoreflex sensitivity may be responsible for acute surges in blood pressure, this factor does not appear to be a strong predictor of chronic changes in blood pressure. In contrast, modifications in local signaling molecules may ultimately have the greatest impact on chronic modifications in blood pressure. Indeed, our findings lend support to previous results which suggest that increases in blood pressure may be coupled to the impairment of nitric oxide in response to glycosylated hemoglobin. This relationship may exist in the presence of a blunted HVR.

Support or Funding Information

Department of Veterans Affairs (I01CX000125 & 15SRCS003 JHM, 1IK1RX002945 GSP), and NIH (R56HL142757 & R01HL142757 JHM).
Identification of the Hsp90 Isoforms and Co‐Chaperones that Repress Opioid Anti‐Nociception in the Spinal Cord
Authors: Bejarano Paul , Duron David , Mishra Sanket , Blagg Brian S. J. , Streicher John M.
Published: April 2020

The various isoforms of the chaperone protein heat shock protein 90 (Hsp90) in conjunction with specific co‐chaperones are key regulators of signal transduction. We have recently identified a novel role for Hsp90 within downstream mu opioid receptor (MOR) signaling both within the brain and spinal cord. Within the brain, we found that Hsp90α and the co‐chaperones Cdc37 and p23 promote MOR‐induced ERK phosphorylation and subsequent anti‐nociception. However, in the spinal cord Hsp90 represses MOR‐induced ERK phosphorylation and subsequent anti‐nociception, so that Hsp90 inhibitor treatment in the spinal cord results in enhanced opioid pain relief. This study aimed to identify the Hsp90 isoforms and co‐chaperones necessary for this mechanism within the spinal cord, and whether they differ from the brain. We used novel isoform and co‐chaperone selective small molecule inhibitors and in vivo gene knockdown using CRISPR/Cas9 to selectively block each of the Hsp90 isoforms and co‐chaperones in the spinal cords of male and female CD‐1 mice. We then used opioid‐induced anti‐nociception in the tail flick assay as a readout, combined with immunohistochemistry to validate CRISPR/Cas9 knockdown in the spinal cord. In contrast to our earlier brain studies, we found that the isoforms Hsp90α, Hsp90β, and Grp94 all acted to repress opioid anti‐nociception in the spinal cord, in that inhibiting these isoforms resulted in increased opioid anti‐nociception. We similarly found that the co‐chaperones Cdc37, p23, and Aha1 all similarly repressed opioid anti‐nociception in the spinal cord. These findings suggest that Hsp90β, Grp94, and Aha1 all act within the spinal cord but not the brain to regulate opioid anti‐nociception. These findings also suggest, in line with our earlier studies, that blocking these isoforms with systemic selective inhibitors could represent a novel therapeutic approach to enhance opioid therapy, increasing analgesic efficacy while reducing side effects like tolerance and reward/addiction.

Support or Funding Information

These studies were supported by an Arizona Biomedical Research Commission New Investigator Award (#ADHS18‐198875) and institutional funds from the University of Arizona to JMS, and NIH R01CA213566 to BB. BB is a founder with an equity stake in Grannus Therapeutics, a virtual startup for developing novel Hsp90 inhibitors. No other author has any relevant conflicts to declare.
Peri‐renal Adipose Tissue Inflammation Possibly Underlies Mild Renal Dysfunction in Early Metabolic Challenge
Authors: Hammoud Safaa H. , Koubar Sahar , Mougharbil Nahed Hisham , Eid Assaad Hisham , El-Yazbi Ahmed F.
Published: April 2020

Cardiovascular disease remains a primary cause of morbidity and mortality in patients with diabetes. Cardiovascular complications in those patients are independent of glycemic control; intensive antihyperglycemic therapy was found to be ineffective in improving the prognosis of these complications. Evidence of vascular function deterioration in prediabetic patients implicates the involvement of mechanisms other than hyperglycemia in the development of cardiovascular dysfunction. Adipose inflammation constitutes a possible mechanism that is involved in the pathogenesis of diabetes and metabolic syndrome. We developed a rat model of mild hypercaloric (HC) stress with a delayed development of hyperglycemia to investigate the mechanism of cadiovascular dysfunction in early stages of metabolic challenge. We previously reported that prior to the development of hyperglycemia, perivascular adipose tissue inflammation was associated with cardiovascular structural and functional anomalies. No changes in blood glucose, body weight or blood pressure levels were observed in this rat model. Systemic inflammation was not detected in this rat model, however following 12 weeks of HC feeding, peri‐renal adipose tissue showed increased oxidative stress and inflammatory cytokine expression (IL‐1b). Moreover, proteinuria and hyperfiltration indicated an early stage of renovascular dysfunction. This renovascular dysfunction manifested as (1) loss of proper endothelial feedback, (2) loss of prostaglandin and nitric oxide activity, and (3) an uncontrolled vasodilation induced by upregulation of epoxyeicosatrienoic acid effect. Moreover, the renovascular dysfunction was associated with significant increases in renal oxidative stress, inflammatory mediators (IL‐1b and TGF‐b1) and culminated in renal structural damage. Glomerulosclerosis was increased following HC feeding as well as significant increases in the expression of a‐smooth muscle actin in proximal tubular cells suggesting possible epithelial to mesenchymal transformation. Ameliorating adipose inflammation by a two‐week treatment with non‐hypoglycemic doses of metformin (100 mg/Kg/day) or pioglitazone (2.5 mg/Kg/day), improved renovascular function and structural nephrotoxicity. These results provide evidence of an early progressive renal damage induced by localized perirenal adipose tissue inflammation in prediabetes.

Support or Funding Information

Research support: AUB‐FM grant #320186
Acute Fatiguing Exercise Alters Sarco‐endoplasmic Reticulum Stress‐related Signaling in Equine Skeletal Muscle
Authors: Klein Dylan Joseph , McKeever Kenneth , Anthony Tracy
Published: April 2020

Sarco‐endoplasmic reticulum (S‐ER) stress results from physiological and pathological disturbances that alter S‐ER function. In turn, the unfolded protein response (UPR) and ubiquitin proteasome pathway (UPP) are activated in order to re‐establish S‐ER homeostasis and promote cellular resilience in the face of future cellular stressors. Unaccustomed, vigorous exercise (e.g. aerobic or resistance exercise) has been shown in the comparative mammalian literature to activate the UPR in skeletal muscle. As such, the S‐ER may play a crucial role in the adaptations of skeletal muscle to exercise. The horse represents a highly athletic and muscular mammal, yet no such research has been conducted in the horse. Eight (n=4 males, n=4 females; 3–8 yrs) unconditioned Standardbred horses completed a bout of high‐intensity fatiguing exercise in the form of a graded exercise test to determine the effects of exercise on UPR and UPP‐related markers in skeletal muscle. Biopsies were taken from the M. gluteus medius before, and at 3 h and 24 h post‐exercise, alongside time‐matched standing controls. Results showed that acute fatiguing exercise significantly increased phosphorylated eukaryotic initiation factor 2‐alpha (p<0.05) and tended to increase activating transcription factor 4 gene expression (p=0.096), but had no effect on additional UPR (e.g. Xbp‐1, Chop, Bip) or UPP (e.g. Fbxo32, Trim63)‐related mRNA transcripts. These results suggest that high‐intensity fatiguing exercise can alter UPR‐related signaling but does not induce a full transcriptional UPR or UPP in equine skeletal muscle at the time points measured.

Support or Funding Information

USDA NIFA NC1184, NIH DK109714
Kir4.1 is Critical for Regulation of ENaC and ROMK Activity in the Late Distal Nephron
Authors: Mutig Kerim , Wang Ming-Xiao , Zhang Dan-Dan , Lin Dao-Hong , Ellison David H. , Thomson Martin N. , Bachmann Sebastian , Wang Wen-Hui
Published: April 2020

The extent of NaCl reabsorption in the early distal convoluted tubule (DCT1) via the apical Na+‐Cl− cotransporter (NCC) defines the sodium load of the ensuing late DCT (DCT2) and connecting tubule (CNT). DCT2 and CNT express the epithelial sodium channel (ENaC) and potassium Kir1.1 channel (ROMK) to perform electrogenic sodium‐dependent potassium secretion into the urine. Kidney‐specific deletion of Kir4.1 (Ks‐Kir4.1−/−) causes profound hypokalemia in mice, which may result from reduced DCT function and increased sodium load of DCT2/CNT, because potassium sensing via Kir4.1 is a critical step for NCC activation. Since Kir4.1 expression extends throughout DCT2 and CNT, we hypothesized that the deletion of Kir4.1 may also affect the expression and activity of ENaC and ROMK. To address this hypothesis we examined ENaC and ROMK functions by measuring the amiloride‐sensitive Na+ currents and TPNQ‐sensitive K+ currents in the DCT2/CNT of wild‐type (WT) and Ks‐Kir4.1−/− mice fed regular or K+‐deficient diets for three days. Cellular distribution of ENaC and ROMK was studied by immunofluorescence. K+‐deficient diet decreased amiloride‐sensitive Na currents from 205 pA to 120 pA at −60 mV and decreased TPNQ‐sensitive K currents from 1100 pA to 600 pA at −40 mV in WT animals. Ks‐Kir4.1−/− mice showed increased baseline Na+ (520 pA at −60 mV) and K+ currents (1600 pA at −40 mV). Feeding Ks‐Kir4.1−/− mice with K+‐deficient diet failed to inhibit ENaC and ROMK. Localization experiments revealed substantially increased ROMK signal intensities along DCT2 and CNT, which were identified by double immunostaining for parvalbumin, calbindin, or aquaporin 2. Moreover, DCT2 exhibited significant elongation in Ks‐Kir4.1−/− mice indicating nephron remodeling. We conclude that Kir4.1 regulates ENaC and ROMK functions in the DCT2 and CNT. Their upregulation in Ks‐Kir4.1−/− mice likely contributes to renal K wasting.

Support or Funding Information

Deutsche Forschungsgemeinschaft grants MU2924/2‐2, BA700/22‐2, SFB 1365; National Institute of Health grants DK 54983 and DK 115366; Fondation LeDucq and NIDDK R0151496; Russian Academic Excellence project “5–100”.
Endothelial specific ERG gene is required for preventing pulmonary vascular complications by regulating sprouting angiogenesis and inflammation
Authors: Vellingiri Vigneshwaran , Avin BR Vijay , Joshi Jagdish Chandra , Kang Hojin , Chen Jiwang , Mehta Dolly
Published: April 2020

ERG, a member of ETS family of transcription factors, acts as a key modulator of endothelial cell differentiation and haematopoiesis. Despite the high degree expression of ERG in endothelial cells, its role in maintaining adult vascular architecture remain elusive. We show that ERG is expressed at the highest level in the adult lung than other vascularized organs such as kidney and brain. Intriguingly, we found that conditional deletion of ERG in endothelial cells of ERGCDh5‐Cre‐ERT mice (ERGiΔEC) spontaneously compromised lung vessel density and these lungs showed severe hemorrhage with vascular injury. The defective angiogenesis was further confirmed by regressed invasive and migratory efficiency of ERG silenced human pulmonary endothelial cells as evaluated by collagen invasion and scratch assay. Further experiments reveled that loss of endothelial ERG markedly reduced several arterial specific genes including DLL4 expression in the lungs and pulmonary ECs. We also found that loss of ERG induced the expression of IL8 and its receptor CXCR2, a critical inducer of inflammation. Immunocytochemistry demonstrated downregulated Dll4 in the sprouting front of the ERG silenced endothelial cells. Dll4 is the only Notch ligand which is expressed predominantly in the vascular endothelium that regulate vascular patterning and vessel density in mature tissues. Ongoing studies are underway to address the linkage between DLL4 with IL8‐mediated inflammatory signalling on vascular pruning and thereby impaired lung functions.
Adrenergic receptors signal to β‐arrestins to control the membrane trafficking of Monocarboxylate transporter 1 (MCT1) in glioblastoma
Authors: John Kristen Mary , Arain Hirra , Dores Michael Robert
Published: April 2020

Monocarboxylate transporters (MCTs) are a family of 12‐pass transmembrane domain proteins that mediate the rapid symport of protons and monocarboxylates, including lactate, across the plasma membrane. MCT‐1, the primary MCT in glioblastoma cells, helps regulate mammalian metabolism and is thought to act as a scaffold for and facilitate the activation of pro‐migratory and pro‐invasive signaling pathways. Uncovering the cellular mechanisms governing MCT‐1 trafficking is crucial for the development of therapeutic treatments for MCT‐1 as an anticancer target. Here, we describe the role of signaling by β2‐adrenergic (β2AR) and α2‐adrenergic (α2AR) receptors in mediating MCT‐1 internalization into caveolae and sorting endosomes. Through MCT‐1 and Caveolin‐1 colocalization analysis, we show an increase in MCT‐1 internalization upon β2AR agonist stimulation and even greater increase in internalization upon simultaneous β2AR and α2AR agonist stimulation, which suggest that both receptors mediate this response in a synergistic manner. Furthermore, we demonstrate that siRNA depletion of endogenous β‐arrestin inhibits MCT‐1 internalization in cells when stimulated with a β2AR agonist, indicating that β2AR likely signals through β‐arrestin to mediate MCT‐1 internalization. We are currently investigating whether these results are recapitulated in single‐cell derived β‐arrestin knockout clones. Our current research also focuses on identifying which β‐arrestin‐dependent signaling pathways promote synergistic MCT‐1 internalization in response to adrenergic receptor activation.
ET‐1 Receptor Function in Women with Advancing Reproductive Age
Authors: McGinty Shane J. , Welti Laura M. , Kuczmarski Andrew V. , Shoemaker Leena N. , Moreau Kerrie L. , Wenner Megan M.
Published: April 2020

Endothelin‐1 (ET‐1) contributes to endothelial dysfunction, a primary driver of hypertension and cardiovascular disease. Our lab has previously shown that the ETB receptor mediates vasodilation in premenopausal women (PRE), but this effect is lost in postmenopausal women (POST). Noticeable declines in endothelial function are evident early in the menopausal transition, but the function of the ETB receptor in perimenopausal women (PERI) is unclear.

PURPOSE

The purpose of this study was to test the hypothesis that ETB‐mediated vasodilation progressively declines with advancing reproductive age in women.

METHODS

We used the STRAW+10 staging criteria to classify women as PRE (n=12, 26±2 yrs, 23±1 kg/m2, 83±2 mmHg), PERI (n=9, 50±1 yrs, 24±1 kg/m2, 84±3 mmHg), or POST (n=11, 56±1 yrs, 24±1 kg/m2, 87±2 mmHg). Cutaneous vasodilatory responses to local heating were measured using laser doppler flowmetry during microdialysis perfusions of lactated Ringer’s (Control), ETB receptor blockade (BQ‐788, 300nM), and ETA receptor blockade (BQ‐123, 500nM). Cutaneous vascular conductance (CVC) was calculated during the plateau phase of local heating (42°C), and normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C.

RESULTS

PRE showed the greatest cutaneous vasodilation in response to local heating (PRE: 93±2; PERI: 84±2; POST: 88±2 %CVC max, P<0.05 vs PERI and POST). There was a trend for increased vasodilatory responses to ETB receptor blockade with advancing reproductive age (PRE: 85±3; PERI: 91±2; POST: 92±3 %CVC max, P=0.13). However, there were no differences in vasodilatory responses during ETA receptor blockade (PRE: 90±2; PERI: 91±3; POST: 92±3 %CVC max, P=0.84). Finally, brachial artery flow‐mediated dilation tended to decline (PRE: 7±1; PERI: 6±1; POST: 5±1 %Δ, P=0.09) while pulse wave velocity increased with advancing reproductive age (PRE: 6±0; PERI: 7±0; POST: 7±0 m/s, P<0.05 vs PERI and POST).

CONCLUSIONS

These preliminary data suggest that adverse changes in vascular function, and potentially ETB receptor function, are evident early in PERI. Therefore, future research examining the role of the ET‐1 pathway in progressive vascular dysfunction throughout the menopausal transition is warranted.

Support or Funding Information

NIH R01 HL 146558, P20 GM 113125
Defining Cell Cycle‐Specific Interactions of the pRB Tumor Suppressor
Authors: Stadnicki Emily Jane , Manning Amity
Published: April 2020

The retinoblastoma protein, pRB, functions as a tumor suppressor that is inactivated in most types of cancer cells. This tumor suppressive function is achieved through binding to E2F and regulating transcription. Other important cellular functions of pRB include DNA damage response and repair and mitotic chromosome segregation. Recently, Mass Spec approaches have identified over 300 distinct proteins that interact with pRB. Many of these proteins have known functions outside of transcriptional regulation, suggesting that they may represent the functional link between pRB and its non‐transcriptional roles in the cell. However, this approach was completed with an asynchronous population of cells, where cells undergoing mitosis are likely to be underrepresented. Nevertheless, some key interactors that were identified through the mass spec including several proteins with known roles in mitotic chromosome organization and mitotic fidelity. We hypothesize that pRB interactions with and regulation of a subset of proteins are important for pRB’s role in mitosis, and that loss of these interactions when pRB function is compromised in cancer may explain the mitotic defects seen in these cancers. To test this hypothesis, we have confirmed that pRB interacts with specific proteins during mitosis by performing reciprocal immunoprecipitation experiments in nocodazole‐synchronized cells. Using silver stain analysis of pRB immunoprecipitation samples, we find that many distinct proteins appear to interact with pRB during mitosis. We will use western blot approaches to attempt to identify specific proteins that are potential pRB mitotic interactors. We will then use Mass Spec to identify these additional interactors. By defining cell cycle specific complexes formed by pRB we hope to elucidate the non‐canonical role of pRB in regulating mitotic chromosome segregation and to understand how this function of pRB may be altered in cancer contexts.
Signal Processing and Machine‐learning Algorithm to classify anesthesia depth
Authors: Reyes-Cruz Dario , Mosquera Oscar Leonardo , Botero-Rosas Daniel Alfonso , Gallego-Correa John Jairo , Leon-Ariza Henry H. , Duarte-Tamara Santiago , Botero-Machado Juan David , Rozo-Cespedes Laura , Garcia-Ramos Andres Felipe , Padron-Ayala Antonietta
Published: April 2020

Background

An algorithm with complexity measures, nonlinear dynamics, and neural networks were developed to detect and classify the anesthetic deep (AD). It has been shown that extreme anesthetic depth has been correlated with an increased risk of mortality. Likewise, intraoperative awareness has been reported in the anesthetic drugs under dosage.

Methods

Our artificial intelligence classifier algorithm was developed using a Complexity Brainwave Index (CBI) and a heart rate variability parameter (HRVP). Data from 60 patients (25 men and 35 women), adults between 18 and 65 years of age undergoing surgical procedures (ASA I–II) were collected. The classifier was designed using multi‐layer feedforward neural networks with a hyperbolic activation function using the patient data set and their anesthesia records.

Results

The CBI by itself showed a better prediction probability with Pk of 0.935 when compared with Datex‐Ohmeda index in which State Entropy and Response Entropy had a Pk of 0.884 and 0.899 respectively; also, our CBI showed a performance of 99% of the awake state and 93.3% for deep anesthesia state. Additionally, when combining the CBI and the HRVP, the C‐Statistic was 99% for the awake state, 87.41% for light anesthesia, 82,46% for general anesthesia, and 93.33% for deep anesthesia.

Conclusions

Our results demonstrate that special biological signal filtering and a machine learning algorithm may be used to classify AD during a surgical procedure on ASA I–II patients, assisting anesthesiologists and clinicians in decision making.

Support or Funding Information

University of La Sabana
Effect of Streptozotocin (STZ) Concentration on Chemoreflex Function and Morphology of the Nucleus Tractus Solitarii in STZ‐Induced Alzheimer’s Disease
Authors: Humphrey Chuma M. , Hooker John W. , Thapa Mahima , Wilcox Mason J. , Ostrowski Daniela , Ostrowski Tim D.
Published: April 2020

Alzheimer’s disease (AD) is associated with deterioration of memory and cognition due to changes in brain areas important for these functions. The majority of AD patients also suffer multiple episodes of breathing cessations (apneas) during sleep. AD‐related sleep apnea may be associated with the decline of brainstem centers that control breathing and chemoreflex function. The nucleus tractus solitarii (nTS) is a key region for respiratory control and any AD‐related deterioration of this nucleus critically impacts its function.

In this study, we used the streptozotocin (STZ) ‐induced rat model for sporadic AD (6‐week old rats, N = 6–8 rats/group) and compared chemoreflex dysfunction to indices of morphological alterations in the nTS. Particularly, we compared data obtained two weeks following injections of either 2 mg/kg or 2× 1.5 mg/kg STZ. Peripheral chemoreflex function was tested with 10% O2 in a plethysmography chamber. While increased respiratory rate thirty minutes into hypoxia was significantly blunted with both STZ dosages, animals receiving 2 mg/kg STZ partially compensated with larger tidal volumes. Hence, minute ventilation (rate x volume) was only significantly decreased in animals that received 2× 1.5 mg/kg STZ. Consistent with previously published data analyzing memory dysfunction in this model, there was a trend for reduced overall hippocampal size for both STZ concentrations. Immunohistochemical analysis of neuronal density (Anti‐NeuN antibody) in the CA1 region showed no difference between both STZ concentrations and control. However, synaptic density (Anti‐Synaptophysin antibody) was significantly reduced in both STZ concentrations. The overall size of the caudal nTS (4 sections each 180 μm apart) in the brainstem exhibited a greater reduction in 2× 1.5 mg/kg STZ. NeuN density was not altered between STZ concentrations and control. Interestingly, despite the stronger decline of chemoreflex function and nTS size with 2× 1.5 mg/kg STZ, synaptic density in the nTS (calamus scriptorius area) yielded a significant reduction for 2 mg/kg STZ only.

Our study shows that reduced nTS size and synaptic density may be underlying the early changes of chemoreflex dysfunction in this model of sporadic AD. 2× 1.5 mg/kg STZ seemed to have a greater impact on respiratory function and nTS size. However, the varying change in synaptic density of each STZ concentration may be due to regional differences, since nTS size at calamus scriptorius did not differ between groups. Future studies will incorporate analysis of more caudal and rostral sections.

Support or Funding Information

TruScholar and MoLSAMP grant from Truman State University (TSU) to CMH, GIASR from TSU to MW, seed money from A.T. Still University to TDO
Exosomal Signaling by Skeletal Muscle: Role in Neuromuscular Ageing
Authors: Hemmings Kay , Pollock Natalie , Owen Euan , Mora Silvia , Brooks Susan , McArdle Anne
Published: April 2020

Muscle mass and function are reduced with age in mammals and this is associated with disrupted neuromuscular interactions. Skeletal muscle mounts a robust stress response to contractions by an increased expression of Heat Shock Proteins (HSPs) that facilitate muscle remodeling, promote protein folding and clearance of damaged proteins. In contrast, other cell types including neuronal cells, are unable to mount a stress response. It has been proposed that HSPs may be transferred from one cell type to another in exosomes to maintain proteostasis in the recipient cells. Thus, speculation that the robust HSP response by muscle provides support to peripheral neuronal cells via exosomal transfer of HSPs is tempting. However, HSP generation by muscle following contractions is attenuated in old mice and humans. We hypothesise that inability of muscles of old mice to produce HSPs in response to contraction results in altered exosomal HSP transfer, and therefore a failure to maintain proteostasis in motor neurons, resulting in neuronal and muscle degeneration
(1).

Muscle fibres were isolated from flexor digitorum bravis (FDB) muscles of adult (6–8 month) and old (24–26 month) mice, and initiated to contract using a non‐damaging electrical stimulation protocol
(2). Media was collected from quiescent fibres, and fibres immediately following contraction, and Extracellular Vesicles (EVs) purified using a Total Exosome Isolation kit (Thermo). NanoSight analysis was used to characterise the size and number of the EVs. Total protein content was determined and HSP levels were analysed by western blotting.

Data demonstrated no significant difference in the total number of EVs released from quiescent fibres from adult and old mice, along with a similar increase in EVs released by muscle fibres of adult (2.7 fold) and old mice (3.1 fold) immediately following contraction compared with quiescent fibres. In contrast, the size distribution of the EVs from fibres of old mice was altered compared with EVs from adult fibres, and the total protein content of EVs was significantly less (40% decrease) in those released by quiescent old fibres compared with EVs of quiescent adult fibres. The lower protein content for EVs from old compared with adult fibres was maintained following stimulation.

A 2.5 fold increase in HSP25 and a substantial increase in HSP60 content (from undetectable levels) was seen in EVs from fibres of adult mice immediately following contraction. In contrast, preliminary data suggested that the HSP60 content of EVs produced by quiescent muscle fibres from old mice is already elevated, with little further increase following contraction in a similar manner to muscles in vivo
(3). Additional data will examine the uptake of EVs by neuronal cells and the effect of age of donor on their uptake ability.

Support or Funding Information

With thanks to National Institute of Ageing (AG051442) and the University of Liverpool for their generous support.