Subunit mRNA Expression Research Articles

Differential expression of components of the CGRP-receptor family in human coronary and human middle meningeal arteries: functional implications

BackgroundDifferent responses in human coronary arteries (HCA) and human middle meningeal arteries (HMMA) were observed for some of the novel CGRP receptor antagonists, the gepants, for inhibiting CGRP-induced relaxation. These differences could be explained by the presence of different receptor populations in the two vascular beds. Here, we aim to elucidate which receptors are involved in the relaxation to calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2) in HCA and HMMA.MethodsRNA was isolated from homogenized human arteries (23 HCAs; 12 F, 11 M, age 50 ± 3 years and 26 HMMAs; 14 F, 12 M, age 51 ± 3 years) and qPCR was performed for different receptor subunits. Additionally, relaxation responses to CGRP, AM or AM2 of the human arteries were quantified using a Mulvany myograph system, in the presence or absence of the adrenomedullin 1 receptor antagonist AM22-52 and/or olcegepant.ResultsCalcitonin-like receptor (CLR) mRNA was expressed equally in both vascular beds, while calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3) expression was low and could not be detected in all samples. RAMP1 expression was similar in HCA and HMMA, while RAMP2 expression was higher in HMMA. Moreover, receptor component protein (RCP) expression was higher in HMMA than in HCA. Functional experiments showed that olcegepant inhibits relaxation to all three agonists in both vascular beds. In HCA, antagonist AM22-52 did not inhibit relaxation to any of the agonists, while a trend for blocking relaxation to AM and AM2 could be observed in HMMA.ConclusionBased on the combined results from receptor subunit mRNA expression and the functional responses in both vascular tissues, relaxation of HCA is mainly mediated via the canonical CGRP receptor (CLR-RAMP1), while relaxation of HMMA can be mediated via both the canonical CGRP receptor and the adrenomedullin 1 receptor (CLR-RAMP2). Future research should investigate whether RAMP2 predominance over RAMP1 in the meningeal vasculature results in altered migraine susceptibility or in a different response to anti-migraine medication in these patients. Moreover, the exact role of RCP in CGRP receptor signalling should be elucidated in future research.

RIOK2 transcriptionally regulates TRiC and dyskerin complexes to prevent telomere shortening

Telomere shortening is a prominent hallmark of aging and is emerging as a characteristic feature of Myelodysplastic Syndromes (MDS) and Idiopathic Pulmonary Fibrosis (IPF). Optimal telomerase activity prevents progressive shortening of telomeres that triggers DNA damage responses. However, the upstream regulation of telomerase holoenzyme components remains poorly defined. Here, we identify RIOK2, a master regulator of human blood cell development, as a critical transcription factor for telomere maintenance. Mechanistically, loss of RIOK2 or its DNA-binding/transactivation properties downregulates mRNA expression of both TRiC and dyskerin complex subunits that impairs telomerase activity, thereby causing telomere shortening. We further show that RIOK2 expression is diminished in aged individuals and IPF patients, and it strongly correlates with shortened telomeres in MDS patient-derived bone marrow cells. Importantly, ectopic expression of RIOK2 alleviates telomere shortening in IPF patient-derived primary lung fibroblasts. Hence, increasing RIOK2 levels prevents telomere shortening, thus offering therapeutic strategies for telomere biology disorders.

Local synthesis of estradiol in the rostral ventromedial medulla protects against widespread muscle pain in male mice.

Animal studies consistently demonstrate that testosterone is protective against pain in multiple models, including an animal model of activity-induced muscle pain. In this model, females develop widespread muscle hyperalgesia, and reducing testosterone levels in males results in widespread muscle hyperalgesia. Widespread pain is believed to be mediated by changes in the central nervous system, including the rostral ventromedial medulla (RVM). The enzyme that converts testosterone to estradiol, aromatase, is highly expressed in the RVM. Therefore, we hypothesized that testosterone is converted by aromatase to estradiol locally in the RVM to prevent development of widespread muscle hyperalgesia in male mice. This was tested through pharmacological inhibition of estrogen receptors (ER), aromatase, or ER-α in the RVM which resulted in contralateral hyperalgesia in male mice (C57BL/6J). ER inhibition in the RVM had no effect on hyperalgesia in female mice. As prior studies show modulation of estradiol signaling alters GABA receptor and transporter expression, we examined if removal of testosterone in males would decrease mRNA expression of GABA receptor subunits and vesicular GABA transporter (VGAT). However, there were no differences in mRNA expression of GABA receptor subunits of VGAT between gonadectomized and sham control males. Lastly, we used RNAscope to determine expression of ER-α in the RVM and show expression in inhibitory (VGAT+), serotonergic (tryptophan hydroxylase 2+), and μ-opioid receptor expressing (MOR+) cells. In conclusion, testosterone protects males from development of widespread hyperalgesia through aromatization to estradiol and activation of ER-α which is widely expressed in multiple cell types in the RVM.Significance Statement This study's findings reveal, for the first time, that testosterone protects males from development of widespread muscle hyperalgesia through aromatization to estradiol and activation of ER-α within the RVM. The findings also show, for the first time, ER-α expression in the RVM in male mice, and the distribution patterns of this expression among multiple cell types. Thus, together our data suggest a unique endogenous mechanism in the RVM for protection against widespread pain in males only.

Berberine Attenuates Acetamiprid Exposure-Induced Mitochondrial Dysfunction and Apoptosis in Rats via Regulating the Antioxidant Defense System.

Acetamiprid (ACMP) is a neonicotinoid insecticide that poses a significant threat to the environment and mankind. Oxidative stress and mitochondrial dysfunction are considered prime contributors to ACMP-induced toxic effects. Meanwhile, berberine (BBR) a natural plant alkaloid, is a topic of interest because of its therapeutic and prophylactic actions. Therefore, this study evaluated the effects of BBR on ACMP-mediated alterations in mitochondrial functions and apoptosis in rat liver tissue. Male Wistar rats were divided into four groups: (I) control, (II) BBR-treated, (III) ACMP-exposed, and (IV) BBR+ACMP co-treated groups. The doses of BBR (150 mg/kg b.wt) and ACMP (1/10 of LD50, i.e., 21.7 mg/kg b.wt) were given intragastrically for 21 consecutive days. The results showed that the administration of ACMP diminished mitochondrial complex activity, downregulated complex I (ND1 and ND2) and complex IV (COX1 and COX4) subunit mRNA expression, depleted the antioxidant defense system, and induced apoptosis in rat liver. BBR pre-treatment significantly attenuated ACMP-induced mitochondrial dysfunction by maintaining mitochondrial complex activity and upregulating ND1, ND2, COX1, and COX4 mRNA expression. BBR reversed ACMP-mediated apoptosis by diminishing Bax and caspase-3 and increasing the Bcl-2 protein level. BBR also improved the mitochondrial antioxidant defense system by upregulating mRNA expression of PGC-1α, MnSOD, and UCP-2 in rat liver tissue. This study is the first to evaluate the protective potential of BBR against pesticide-induced mitochondrial dysfunction in liver tissue. In conclusion, BBR offers protection against ACMP-induced impairment in mitochondrial functions by maintaining the antioxidant level and modulating the apoptotic cascade.

Renal upregulation of NCC counteracts empagliflozin-mediated NHE3 inhibition in normotensive but not in hypertensive rats

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in hypertensive patients. However, the underlying molecular mechanisms remain largely unknown. Proximal tubule (PT) NHE3-mediated sodium reabsorption is inhibited by SGLT2i in normotensive rodents, yet no BP-lowering effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. Administration of EMPA for 14 days reduced BP in 12-week-old SHRs but not in age-matched Wistar rats. PT NHE3 was inhibited by EMPA treatment in both Wistar and SHRs. EMPA increased NCC expression and phosphorylation levels in Wistar but not SHRs. SHRs exhibited higher cortical and medullary NKCC2 and β-ENaC subunit mRNA and higher medullary α-ENaC expression levels than Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP was reduced in response to EMPA and HCTZ combined but not by EMPA or HCTZ alone. Altogether, these data suggest that upregulation of NCC counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, thereby maintaining their baseline BP. Moreover, reducing NHE3 activity without further upregulation of the main apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension. FAPESP 2016/22140-7 and 2021/14534-3. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effect of morphine administration on GluN3B NMDA receptor subunit mRNA expression in rat brain.

Opioid addiction is critically dependent on the activation of N‑methyl‑D‑aspartate (NMDA) receptors, which are widely found in the mesocorticolimbic system. Meanwhile, opioid addiction may affect the expression level of NMDA receptor subunits. The existence of GluN3 subunits in the NMDA receptor's tetramer structure reduces the excitatory current of the receptor channel. We evaluated the changes in the mRNA expression pattern of the GluN3B subunit of the NMDA receptor in rat brains following acute and chronic exposure to morphine. Chronic, escalating intraperitoneal doses of morphine or saline were administered twice daily to male Wistar rats for six days. Two other groups were injected with a single acute dose of morphine or saline. The mRNA level of the GluN3B subunit of the NMDA receptor in the striatum, hippocampus, and nucleus accumbens (NAc) was measured by real‑time PCR. mRNA expression of the GluN3B subunit was considerably augmented (3.15 fold) in the NAc of animals chronically treated with morphine compared to the control group. The difference between rats that were chronically administered morphine and control rats was not statistically significant for other evaluated brain areas. In rats acutely treated with morphine, no significant differences were found for GluN3B subunit expression in the examined brain regions compared to the control group. It was concluded that chronic exposure to morphine notably increased the GluN3B subunit of the NMDA receptor in NAc. The extent of the impact of this finding on opioid addiction and its features requires further evaluation in future studies.

Single-cell quantitative expression of nicotinic acetylcholine receptor mRNA in rat hippocampal interneurons.

Hippocampal interneurons are a very diverse population of cells. Using single-cell quantitative PCR to analyze rat CA1 hippocampal interneurons, we quantified neuronal nicotinic acetylcholine receptor (nAChR) mRNA subunit expression and detailed possible nAChR subtype combinations for the α2, α3, α4, α5, α7, β2, β3, and β4 subunits. We also compared the expression detected in the stratum oriens and the stratum radiatum hippocampal layers. We show that the majority of interneurons in the CA1 of the rat hippocampus contain detectable levels of nAChR subunit mRNA. Our results highlight the complexity of the CA1 nAChR population. Interestingly, the α3 nAChR subunit is one of the highest expressed subunit mRNAs in this population, while the α4 is one of the least likely subunits to be detected in CA1 interneurons. The β2 nAChR subunit is the highest expressed beta subunit mRNA in these cells. In addition, Pearson's correlation coefficient values are calculated to identify significant differences between the nAChR subunit combinations expressed in the CA1 stratum oriens and the stratum radiatum. Statistical analysis also indicates that there are likely over 100 different nAChR subunit mRNA combinations expressed in rat CA1 interneurons. These results provide a valid avenue for identifying nAChR subtype targets that may be effective hippocampus-specific pharmacological targets.

Differential effects of interleukin-17A and 17F on cell interactions between immune cells and stromal cells from synovium or skin

We compared the contribution of IL-17A and IL-17F in co-culture systems mimicking cell interactions as found in inflamed synovium and skin. Synoviocytes or skin fibroblasts were co-cultured with activated PBMC, with IL-17A, IL-17 A/F, IL-17F, IL-23, anti-IL-17A, anti-IL-17A/F or anti-IL-17F antibodies. IL-17A, IL-17F, IL-6 and IL-10 production was measured at 48 h. mRNA expression of receptor subunits for IL-23, IL-12 and IL-17 was assessed at 24 h. Both cell activation and interactions were needed for a high IL-17A secretion while IL-17F was stimulated by PHA activation alone and further increased in co-cultures. IL-17F levels were higher than IL-17A in both co-cultures (p < 0.05). IL-17F addition decreased IL-17A secretion (p < 0.05) but IL-17A addition had no effect on IL-17F secretion. Interestingly, IL-17A and IL-17F upregulated IL-17RA and IL-17RC mRNA expression in PBMC/skin fibroblast co-cultures (p < 0.05) while only IL-17F exerted this effect in synoviocytes (p < 0.05). Monocyte exclusion in both co-cultures increased IL-17A and IL-17F (twofold, p < 0.05) while decreasing IL-10 and IL-6 secretion (twofold, p < 0.05). IL-17A and F had differential effects on their receptor expression with a higher sensitivity for skin fibroblasts highlighting the differential contribution of IL-17A and F in joint vs. skin diseases.

Potential of Quercetin to Protect Cadmium Induced Cognitive Deficits in Rats by Modulating NMDA-R Mediated Downstream Signaling and PI3K/AKT-Nrf2/ARE Signaling Pathways in Hippocampus.

Exposure to cadmium, a heavy metal distributed in the environment is a cause of concern due to associated health effects in population around the world. Continuing with the leads demonstrating alterations in brain cholinergic signalling in cadmium induced cognitive deficits by us; the study is focussed to understand involvement of N-Methyl-D-aspartate receptor (NMDA-R) and its postsynaptic signalling and Nrf2-ARE pathways in hippocampus. Also, the protective potential of quercetin, a polyphenolic bioflavonoid, was assessed in cadmium induced alterations. Cadmium treatment (5mg/kg, body weight, p.o., 28days) decreased mRNA expression and protein levels of NMDA receptor subunits (NR1, NR2A) in rat hippocampus, compared to controls. Cadmium treated rats also exhibited decrease in levels of NMDA-R associated downstream signalling proteins (CaMKIIα, PSD-95, TrkB, BDNF, PI3K, AKT, Erk1/2, GSK3β, and CREB) and increase in levels of SynGap in hippocampus. Further, decrease in protein levels of Nrf2 and HO1 associated with increase in levels of Keap1 exhibits alterations in Nrf2/ARE signalling in hippocampus of cadmium treated rats. Degeneration of pyramidal neurons in hippocampus was also evident on cadmium treatment. Simultaneous treatment with quercetin (25mg/kg body weight p.o., 28days) was found to attenuate cadmium induced changes in hippocampus. The results provide novel evidence that cadmium exposure may disrupt integrity of NMDA receptors and its downstream signaling targets by affecting the Nrf2/ARE signaling pathway in hippocampus and these couldcontribute in cognitive deficits. It is further interesting that quercetin has the potential to protect cadmium induced changes by modulating Nrf2/ARE signaling which was effective to control NMDA-R and PI3K/AKT cell signaling pathways.

Interplay between proteasome function and inflammatory responses in e-cig vapor condensate-challenged lung epithelial cells.

Exposure to cigarettes and other nicotine-based products results in persistent inflammation in the lung. In recent years, electronic cigarettes (e-cigs) have become extremely popular among adults and youth alike. E-cigarette vapor-induced oxidative stress promotes protein breakdown, DNA damage and cell death, culminating in a variety of respiratory diseases. The proteasome, a multi-catalytic protease, superintends protein degradation within the cell. When cells are stimulated with inflammatory cytokines such as IFN-γ and TNF-α, the constitutive catalytic proteasome subunits are replaced by the inducible subunits-low-molecular mass polypeptide (LMP)2 (β1i), multi-catalytic endopeptidase complex-like (MECL)1 (β2i), and LMP7 (β5i), which are required for the production of certain MHC class I-restricted T-cell epitopes. In this study, we used human alveolar epithelial cells (A549) and exposed them to filtered air or (1%) tobacco-flavored (TF) electronic cigarette vapor condensate(ECVC) ± nicotine (6mg/ml) (TF-ECVC ± N) for 24h. We observed an increase in the levels of IFN-γ, TNF-α, and inducible proteasome subunits (LMP7/PSMB8, LMP2/PSMB9, MECL1/PSMB10), and a reduced expression of constitutive proteasome subunits (β1/PSMB6 and β2/PSMB7) in challenged A549 cells. Interestingly, knockdown of the inducible proteasome subunit LMP7 reversed ECVC-induced expression of NADPH oxidase and immunoproteasome subunits in A549 cells. In addition, pre-exposure to an LMP7 inhibitor (ONX-0914) abrogated the mRNA expression of several NOX subunits and rescued the excessive production/release of inflammatory cytokines/chemokines (IL-6, IL-8, CCL2, and CCL5) in ECVC-challenged cells. Our findings suggest an important role of LMP7 in regulating the expression of inflammatory mediators during ECVC exposure. Overall, our results provide evidence for proteasome-dependent ROS-mediated inflammation in ECVC-challenged cells.

242-LB: A Novel FoxO Target, LRRC2, Preserves Mitochondrial Function in Muscle from Insulin-Deficient Mice

Uncontrolled diabetes impairs muscle strength and mitochondrial metabolism, leading to increased mortality. FoxOs are key mediators of insulin signaling and we demonstrated that FoxOs mediate muscle atrophy and mitochondrial dysfunction in mice with streptozotocin (STZ) diabetes. Unfortunately, FoxOs are also tumor suppressors and knockout of FoxOs in proliferative tissues causes cancer. We aimed to investigate FoxO targets restricted to skeletal muscle in hopes of improving strength in diabetic conditions. RNA-seq from STZ mice with/without FoxO1/3/4 triple knockout showed several diabetes-induced FoxO targets, with the most robust being LRRC2 (Leucine rich repeating containing 2). The function of LRRC2 is unknown, but gene expression databases show LRRC2 is restricted to muscle and heart, and LRRC2 is enriched in human heart with dilated cardiomyopathy. Adenoviral overexpression of LRRC2 in C2C12 cells localized to mitochondria and showed increase mRNA levels of biogenesis marker PGC1α, and antioxidant genes SOD2 and Gpx3. However, LRRC2 overexpression did not change basal respiration measured in Seahorse. To determine LRRC2's effect on exercise tolerance and mitochondrial bioenergetics in vivo, we created muscle specific LRRC2 knockout (LKO) mice and rendered them diabetic with STZ. Diabetic LKO mice did worse than their littermate controls on treadmill exercise to exhaustion. Soleus permeabilized fibers showed significantly decreased ATP production that was uncoupled from respiration with glutamate/malate. Interestingly, soleus muscles from LKO mice were significantly larger than their littermate controls, whereas extensor digitorum longus and quadriceps were smaller in the diabetic LKO mice. Lastly, mRNA expression of mitochondrial subunits was not changed and thus did not explain the mitochondrial defects. In conclusion, LRRC2 is a novel FoxO target that helps preserve mitochondrial function and exercise tolerance in a Type 1 diabetes mouse model. Disclosure C. M. Penniman: None. G. Bhardwaj: None. C. Nowers: None. B. T. O'neill: Stock/Shareholder; Bristol-Myers Squibb Company, Pfizer Inc., Johnson &amp; Johnson. Funding U.S. Department of Veterans Affairs (lO1BXOO4468)

The glycine receptor alpha 3 subunit mRNA expression shows sex-dependent differences in the adult mouse brain

BackgroundThe glycinergic system plays an important inhibitory role in the mouse central nervous system, where glycine controls the excitability of spinal itch- and pain-mediating neurons. Impairments of the glycine receptors can cause motor and sensory deficits. Glycine exerts inhibition through interaction with ligand-gated ion channels composed of alpha and beta subunits. We have investigated the mRNA expression of the glycine receptor alpha 3 (Glra3) subunit in the nervous system as well as in several peripheral organs of female and male mice.ResultsSingle-cell RNA sequencing (scRNA-seq) data analysis on the Zeisel et al. (2018) dataset indicated widespread but low expression of Glra3 in vesicular glutamate transporter 2 (Vglut2, Slc17a6) positive and vesicular inhibitory amino acid transporter (Viaat, Slc32a1)positive neurons of the mouse central nervous system. Highest occurrence of Glra3 expression was identified in the cortex, amygdala, and striatal regions, as well as in the hypothalamus, brainstem and spinal cord. Bulk quantitative real-time-PCR (qRT-PCR) analysis demonstrated Glra3 expression in cortex, amygdala, striatum, hypothalamus, thalamus, pituitary gland, hippocampus, cerebellum, brainstem, and spinal cord. Additionally, male mice expressed higher levels of Glra3 in all investigated brain areas compared with female mice. Lastly, RNAscope spatially validated Glra3 expression in the areas indicated by the single-cell and bulk analyses. Moreover, RNAscope analysis confirmed co-localization of Glra3 with Slc17a6 or Slc32a1 in the central nervous system areas suggested from the single-cell data.ConclusionsGlra3 expression is low but widespread in the mouse central nervous system. Clear sex-dependent differences have been identified, indicating higher levels of Glra3 in several telencephalic and diencephalic areas, as well as in cerebellum and brainstem, in male mice compared with female mice.

α-Synuclein Attenuates Maneb Neurotoxicity through the Modulation of Redox-Sensitive Transcription Factors.

The accumulation and aggregation of α-synuclein is a pathognomonic sign of Parkinson's disease (PD). Maneb (MB) exposure has also been reported as one environmental triggering factor of this multifactorial neurodegenerative disease. In our laboratory, we have previously reported that mild overexpression of α-synuclein (200% increase with respect to endogenous neuronal levels) can confer neuroprotection against several insults. Here, we tested the hypothesis that α-synuclein can modulate the neuronal response against MB-induced neurotoxicity. When exposed to MB, cells with endogenous α-synuclein expression displayed increased reactive oxygen species (ROS) associated with diminished glutamate-cysteine ligase catalytic subunit (GCLc) and hemeoxygenase-1 (HO-1) mRNA expressions and upregulation of the nuclear factor erythroid 2-related factor 2 (NRF2) repressor, BTB domain and CNC homolog 1 (BACH1). We found that α-synuclein overexpression (wt α-syn cells) attenuated MB-induced neuronal damage by reducing oxidative stress. Decreased ROS found in MB-treated wt α-syn cells was associated with unaltered GCLc and HO-1 mRNA expressions and decreased BACH1 expression. In addition, the increased SOD2 expression and catalase activity were associated with forkhead box O 3a (FOXO3a) nuclear compartmentalization. Cytoprotective effects observed in wt α-syn cells were also associated with the upregulation of silent information regulator 1 (SIRT1). In control cells, MB-treatment downregulated glutathione peroxidase 4 mRNA levels, which was coincident with increased ROS content, lipid peroxidation, and mitochondrial alterations. These deleterious effects were prevented by ferrostatin-1, an inhibitor of ferroptosis, under conditions of endogenous α-synuclein expression. The overexpression of α-synuclein attenuated MB toxicity by the activation of the same mechanisms as ferrostatin-1. Overall, our findings suggest that mild overexpression of α-synuclein attenuates MB-induced neurotoxicity through the modulation of NRF2 and FOXO3a transcription factors and prevents cell death probably by intervening in mechanisms associated with ferroptosis. Thus, we postulate that early stages of α-synuclein overexpression could be potentially neuroprotective against MB neurotoxicity.

Integrative Analysis of the AMPK subunits in Colorectal Adeno Carcinoma.

The 5-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an emerging cancer treatment and therapeutic target. Due to the enzyme's complexity and dual nature, it is a confounding target in the treatment strategy. The study aimed to conduct an integrative analysis of the seven subunits and twelve isoforms of AMPK, which is not reported so far in colorectal adenocarcinoma patients. The web-based tools UALCAN, Timer 2.0, KM Plotter, cBioPortal, COSMIC, and STRING were used to investigate the differential expression of AMPK subunits, protein-level Expression, promoter methylation status, survival analyses, Enrichment analysis, and protein-protein interaction. The mRNA expression of AMPK subunits are upregulated in Colorectal Adeno Carcinoma (COAD), while the protein expression is comparatively reduced in colon tumors. The protein-level expression of α2 and β2 is decreased significantly in COAD patients. The γ3 subunit in colon tumor is hypermethylated. The study also reports that Liver Kinase B1 mutation in 7% of CRC patients, which might be the reason for downregulation of the gene and the protein expression of AMPK subunits in COAD. The Overall analysis of the subunits affirms that AMPK expression is beneficial in cancer.

Impact of Ovariectomy on the Anterior Pituitary Gland in Female Rats.

Ovariectomy (OVX) causes a depletion of circulating estradiol (E2) and influences hypothalamic kisspeptin neurons, which govern gonadotropin-releasing hormone (GnRH) release and ultimately gonadotropin secretion. In this study, we examined the changes induced by OVX on the anterior pituitary gland in female rats. OVX significantly increased the mRNA expression of gonadotropin α, luteinizing hormone (LH) β, and follicle-stimulating hormone (FSH) β subunits within the pituitary gland compared with control (sham-operated) rats, and this was completely suppressed by E2 supplementation. High-dose dihydrotestosterone supplementation also prevented the OVX-induced increase in the expression of the three gonadotropin subunits. GnRH receptor mRNA expression within the pituitary was significantly increased in OVX rats, and this increase was completely inhibited by E2 supplementation. The mRNA expression of the receptors for adenylate cyclase-activating polypeptide and kisspeptin was unchanged by OVX. Although the mRNA levels of inhibin α, βA, and βB subunits within the pituitary gland were not modulated by OVX, follistatin gene expression within the pituitary gland was increased by OVX, and this increase was completely inhibited by E2 supplementation after OVX. In experiments using a pituitary gonadotroph cell model (LβT2 cells), follistatin itself did not modulate the mRNA expression of gonadotropin LHβ and FSHβ subunits, and the GnRH-induced increase in the expression of these genes was slightly inhibited in the presence of follistatin. Our current observations suggest that OVX induces several characteristic changes in the pituitary gland of rats.

Effects of simultaneous versus post exposure epigallocatechin-3-gallate treatment on aluminum induced neurotoxicity in rat hippocampus: A multi-approach study

Chronic aluminium(Al) exposure can affect the antioxidant and glutaminergic systems through N-methyl-D-aspartate receptors (NMDAR). This study was aimed to investigate the neurotoxic effect of Al through different mechanisms in rat hippocampus and to evaluate the protective role of epigallocatechin gallate (EGCG), a well-known antioxidant, with simultaneous administration of Al,as well as post-treatment after Al exposure.For this purpose, aluminum chloride(AlCl3) was administered simultaneously with two different EGCG doses for 8 weeks as the first part of the study.In the second part of the study, after 4 weeks of AlCl3 pre-administration, two different EGCG doses were also administered during four additional weeks as post-treatment.Al administration led to oxidative stress and increased acetylcholinesterase levels.NMDAR subunit mRNA expressions were down-regulated by Al, which was apparent in NMDAR1/2B subunits.Simultaneous EGCG treatment has shown a better neuroprotective effect in terms of these mechanisms and represents novel approach for the prevention of neurodegenerative diseases likely to be induced by Al.

Early deficits in GABA inhibition parallels an increase in L-type Ca2+ currents in the jaw motor neurons of SOD1G93A mouse model for ALS

Amyotrophic Lateral Sclerosis (ALS) involves protracted pre-symptomatic periods of abnormal motor neuron (MN) excitability occurring in parallel with central and peripheral synaptic perturbations. Focusing on inhibitory control of MNs, we first compared longitudinal changes in pre-synaptic terminal proteins for GABA and glycine neurotransmitters around the soma of retrogradely identified trigeminal jaw closer (JC) MNs and ChAT-labeled midbrain extraocular (EO) MNs in the SOD1G93A mouse model for ALS. Fluorescence immunocytochemistry and confocal imaging were used to quantify GAD67 and GlyT2 synaptic bouton density (SBD) around MN soma at pre-symptomatic ages ∼P12 (postnatal), ∼P50 (adult) and near disease end-stage (∼P135) in SOD1G93A mice and age-matched wild-type (WT) controls. We noted reduced GAD67 innervation in the SOD1G93A trigeminal jaw closer MNs around P12, relative to age-matched WT and no significant difference around P50 and P135. In contrast, both GAD67 and GlyT2 innervation were elevated in the SOD1G93A EO MNs at the pre-symptomatic time points. Considering trigeminal MNs are vulnerable in ALS while EO MNs are spared, we suggest that upregulation of inhibition in the latter might be compensatory. Notable contrast also existed in the innate co-expression patterns of GAD67 and GlyT2 with higher mutual information (co-dependency) in EO MNs compared to JC in both SOD1G93A and WT mice, especially at adult stages (P50 and P135). Around P12 when GAD67 terminals expression was low in the mutant, we further tested for persistent GABA inhibition in those MNs using in vitro patch-clamp electrophysiology. Our results show that SOD1G93A JC MNs have reduced persistent GABA inhibition, relative to WT. Pharmacological blocking of an underlying tonically active GABA conductance using the GABA-α5 subunit inverse agonist, L-655-708, disinhibited WT JC MNs and lowered their recruitment threshold, suggesting its role in the control of intrinsic MN excitability. Quantitative RT-PCR in laser dissected JC MNs further supported a reduction in GABA-α5 subunit mRNA expression in the mutant. In light of our previous report that JC MNs forming putative fast motor units have lower input threshold in the SOD1G93A mice, we suggest that our present result on reduced GABA-α5 tonic inhibition provides for a mechanism contributing to such imbalance. In parallel with reduced GABA inhibition, we noted an increase in voltage-gated L-type Ca2+ currents in the mutant JC MNs around P12. Together these results support that, early modifications in intrinsic properties of vulnerable MNs could be an adaptive response to counter synaptic deficits.

OR09-3 Integrins as Potential Molecular Targets in Thyroid Cancer Imaging and Therapy

Abstract Background Integrins are cell adhesion receptors consisting of 24 transmembrane heterodimers generated from a combination of 18α integrin and 8β integrin subunits. A subset of integrins consists of receptors recognizing Arg-Gly-Asp (RGD) peptide motifs. One of the RGD-recognizing receptors is integrin αvβ3 that has been recently shown to play a role in neovascularization and progression of several cancers. Radiolabeled RGD analogs have emerged as potential imaging and therapeutic options in cancers characterized by a high expression of integrin αvβ3. Therefore, the aim of this study was to establish the expression of integrin αvβ3 in thyroid cancer (TC). Methods We analyzed the mRNA expression of integrin αvβ3 in 496 BRAF-like and RAS-like human TC tissue samples, including 65 paired samples of tumor vs normal tissue based on The Cancer Genome Atlas. We assessed the protein expression of integrin αvβ3 in 70 TC tissue samples and 10 normal thyroid tissues, as well as in 14 TC cell lines. BRAF-like or RAS-like tumor status was determined by BRS score based upon standard expression profiles ranging from -1 to 0 for BRAF-like cancer and 0 to 1 for RAS-like tumors. The association between BRS and αvβ3 expression was tested using the Spearman correlation coefficient (r). T-tests and paired T-tests were used to compare the continuous variables between the two groups as appropriate, and Kruskal-Wallis test was used for multiple group comparisons with an adjusted p-value of ≤ 0.05 as statistically significant. Results αv integrin subunit mRNA expression was significantly higher in TC than normal thyroid (log fold change 0.3, p=0.001), while the expression of the β3 subunit was similar between paired normal and malignant samples (log fold change -0.2, p=0.30). BRAF-like tumors were characterized by a higher mRNA expression of αvβ3 integrins as documented by a moderate negative correlation between BRS and αv (r=-0.5, p&amp;lt;0.001) as well as β3 (r=-0.27, p&amp;lt;0.001). Consistently, the BRAF-like TC cell lines OCUT2 (BRS=-1), TPC1 (BRS=-0.4), K1 (BRS=-0.29), as well as Hurthle cell TC cell line XTC1 (BRS=-0.46), were characterized by the highest αvβ3 mRNA and/or protein expression. Immunostaining revealed αv expression in all malignant samples, with classic papillary TC characterized by the highest expression as compared with follicular TC (p&amp;lt;0.001), poorly-differentiated TC (p=0.006) and normal thyroid (p&amp;lt;0.001). β3 protein expression had lower intensity than αv integrin and was present in 31.8% of papillary TC, 15% of follicular TC and was not detected in poorly-differentiated TC nor normal thyroid. Conclusions TC is characterized by a differential expression of αvβ3 integrin, which is particularly high in the most common type of TC - BRAF-like papillary TC. The αvβ3 integrin could potentially serve as a molecular target for imaging and therapy with radiolabeled RGD analogs in TC. Presentation: Saturday, June 11, 2022 12:00 p.m. - 12:15 p.m.

Abstract P343: Adaptive Immune Mechanisms Exacerbate Cholinergic Activation-mediated Hypertension In Dahl Salt-sensitive Rats

Hypertension affects over one billion people worldwide and significantly contributes to the pathogenesis of cardiovascular and renal disease. Activation of nicotinic acetylcholine receptors (nAChR) in immune cells regulates immune responses. We have shown earlier that the development of hypertension in a genetic model of human essential hypertension, the Spontaneously Hypertensive Rats (SHR), is dependent on nAChR-mediated renal inflammation. Broadening the application of this mechanism, in the present study we hypothesized that nAChR activation plays a role in inflammation and the development of hypertension in a salt-sensitive model of hypertension, the Dahl salt-sensitive (DSS) rat. To test the hypothesis, we infused 4 weeks old DSS (n=12) and Dahl salt resistant (DSR, n=12) rats with either saline (n=6) or nicotine (15mg/kg/day, n=6) for 4 weeks. Animals were fed a 4% NaCl diet for the entire duration and moved into metabolic cages 3 days prior to euthanasia. Systolic blood pressure (SBP) measured by tail cuff method remained unaltered in DSR. However, SBP was significantly elevated in DSS rats that received nicotine as compared to saline (196.33±7.8 vs. 168±5.2 mmHg, p&lt;0.05). SBP changes in nicotine infused DSS were not accompanied by differences in albuminuria or urinary sodium excretion compared to saline controls. RT-PCR revealed significantly increased CD3e mRNA expression (a marker of T-cell, P=0.04) in the immune cells derived from nicotine infused DSS as compared to saline controls. Increased CD3e mRNA expression in the kidney of nicotine infused DSS compared to saline was observed. Nicotine infusion did not change mRNA expression of nAChR subunits in DSR immune cells. In contrast, there was a ≥50% decrease in mRNA expression of α1-α7, α9, α10, β2, and β3 nAChR in immune cells derived from nicotine infused DSS compared to saline controls. There was a small yet significant increase in the mRNA expression of the β1-nAChR subunit in the immune cells of nicotine infused DSS compared to saline controls. We conclude that nAChR activation potentiates hypertension in the DSS independent of sodium retention or renal function. Our preliminary data suggests a possible role for nAChR mediated adaptive immune mechanisms in salt-sensitive hypertension.

Nucleosome remodeling and deacetylation complex and MBD3 influence mouse embryonic stem cell naïve pluripotency under inhibition of protein kinase C

The pluripotency of naïve mouse embryonic stem cells (mES) is regulated by multiple signaling pathways, with inhibition of protein kinase C (PKCi) playing a particularly important role in maintaining naïve mES. However, the regulatory function of nucleosome remodeling and deacetylase (NuRD) complex in mES cultured in a PKCi system is unknown. We found that, compared with 2iL-derived mES, PKCi-derived mES showed low mRNA expression of NuRD complex subunits, including MBD3, HDAC1/HDAC2, MTA1, and RbAP46/RbAP48. Western blot showed that PKCi-derived mES expressed lower protein levels of MBD3 and HDAC2 at passage 3, as well as MBD3, HDAC2, and MTA1 at passage 10, indicating that PKCi suppressed NuRD complex expression. Knockdown of MBD3 increased PKCi-derived mES pluripotency by increasing NANOG and OCT4 expression and colony formation. By contrast, overexpression of MBD3 or removal of PKC inhibitor-induced differentiation of mES, results in reduced NANOG, OCT4, and REX1 expression and colony formation, increased differentiation-related gene expression, and differentiation into flat cells. Knockdown of MBD3 in mES upon PKC inhibitor removal partially reversed cell differentiation. Our results show that the regulatory NuRD complex and its MBD3 subunit influence the naïve pluripotency of mES cultured in a PKCi system.