Nontransgenic Littermate Controls Research Articles

Early Dysbiosis and Dampened Gut Microbe Oscillation Precede Motor Dysfunction and Neuropathology in Animal Models of Parkinson's Disease.

Studies have shown different gut microbiomes in patients with Parkinson's disease (PD) compared to unaffected controls. However, when the gut microbiota shift toward dysbiosis in the PD process remains unclear. We aim to investigate the changes in gut microbiota, locomotor function, and neuropathology longitudinally in PD rodent models. Fecal microbiota were longitudinally assessed by sequencing the V4-V5 region of the 16S ribosomal RNA gene in a human mutant α-synuclein over-expressing mouse model of PD, SNCA p.A53T mice, and the non-transgenic littermate controls. The locomotor function, neuronal integrity, and α-synuclein expression in the different brain regions were compared between groups. Human fecal microbiota communities from 58 patients with PD and 46 unaffected controls were also analyzed using metagenomic sequencing for comparison. Compared to non-transgenic littermate controls, the altered gut microbiota of the SNCA p.A53T mice can be detected as early as 2 months old, and the diurnal oscillation of the gut microbiome was dampened throughout PD progression starting from 4 months old. However, neuropathology changes and motor deficits were observed starting at 6 months old. Similar changes in altered gut microbiota were also observed in another PD genetic mouse model carrying the LRRK2 p.G2019S mutation at 2 months old. Among the commonly enriched gut microbiota in both PD genetic mouse models, the abundance of Parabateroides Merdae and Ruminococcus torques were also increased in human PD patients compared to controls. These findings revealed the altered gut microbiota communities and oscillations preceding the occurrence of neuropathy and motor dysfunction in the PD process.

Fkbp38 deletion induces premature ovarian insufficiency in mice by activating mTOR signaling and inducing granulosa cell apoptosis

To investigate the role of tacrolimus-binding protein 38 (FKBP38) in follicle development and the mechanism by which Fkbp38 gene deletion causes premature ovarian insufficiency (POI). The Cre-loxp system was used to construct oocyte-specific Fkbp38 knockout transgenic mice. The genotype of the transgenic mice was identified using PCR, and the expression of FKBP38 in the oocytes was verified. The numbers of primordial follicles, primary follicles, secondary follicles and antral follicles in Fkbp38 knockout mice and non-transgenic littermate control mice were counted with HE staining under a microscope for analyzing the effect of Fkbp38 deletion on follicular development. The fertility and serum sex hormone levels of the mice were determined by reproduction experiments and ELISA to assess ovarian function. Ovarian granulosa cell apoptosis of the mice was assessed using TUNEL assay. The activity of the downstream target protein of phosphorylated ribosomal S6 (PS6) of mTOR signaling pathway was detected, and the expressions of BCL-2 and BAX proteins were determined using immunofluorescence assay for assessing oocyte development in the mice. The oocyte-specific Fkbp38 knockout transgenic mouse model was successfully constructed, which showed decreased fertility, disordered sex hormone levels, and significantly reduced primordial follicles, primary follicles and secondary follicles in the ovary (P < 0.05), demonstrating POI-like changes. Compared with the control mice, oocyte-specific Fkbp38 knockout caused activation of the mTOR signaling pathway, significantly increased apoptosis of the granulosa cells, and obviously increased the BAX/BCL- 2 ratio by increasing BAX expression and reducing BCL-2 expression in the oocytes (P < 0.05). FKBP38 plays an important role in follicle development, and Fkbp38 gene deletion in mice causes POI possibly by activating the mTOR signaling pathway and inducing granulosa cell apoptosis.

Age-Dependent Degradation of Locomotion Encoding in Huntington's Disease R6/2 Model Mice.

Background:Huntington’s disease (HD) is an inherited fatal neurodegenerative disease, leading to neocortical and striatal atrophy. The commonly studied R6/2 HD transgenic mouse model displays progressive motor and cognitive deficits in parallel to major pathological changes in corticostriatal circuitry.Objective:To study how disease progression influences striatal encoding of movement.Methods:We chronically recorded neuronal activity in the dorsal striatum of R6/2 transgenic (Tg) mice and their age-matched nontransgenic littermate controls (WTs) during novel environment exposure, a paradigm which engages locomotion to explore the novel environment.Results:Exploratory locomotion degraded with age in Tg mice as compared to WTs. We encountered fewer putative medium spiny neurons (MSNs)—striatal projection neurons, and more inhibitory interneurons—putative fast spiking interneurons (FSIs) in Tg mice as compared to WTs. MSNs from Tg mice fired less spikes in bursts without changing their firing rate, while FSIs from these mice had a lower firing rate and more of them were task-responsive as compared to WTs. Additionally, MSNs from Tg mice displayed a reduced ability to encode locomotion across age groups, likely associated with their low prevalence in Tg mice, whereas the encoding of locomotion by FSIs from Tg mice was substantially reduced solely in old Tg mice as compared to WTs.Conclusion:Our findings reveal an age-dependent decay in striatal information processing in transgenic mice. We propose that the ability of FSIs to compensate for the loss of MSNs by processes of recruitment and enhanced task-responsiveness diminishes with disease progression, possibly manifested in the displayed age-dependent degradation of exploratory locomotion.

Identifying Sex‐dependent Markers of Heart Failure and Atrial Myopathy

Heart failure (HF) with preserved ejection fraction (HFpEF) and HF with reduced ejection fraction (HFrEF) each represent approximately ½ of current HF cases. HFrEF is a cardiac-centric syndrome, driven by cell loss and dysfunction leading to dilated cardiomyopathy. HFpEF, alternatively, is a systemic syndrome characterized by accumulated risk factors, comorbidities, and a stiff heart with diastolic dysfunction, sometimes clinically presenting as restrictive cardiomyopathy. Recent studies have established that women constitute the majority of patients with HFpEF and that female sex is one of the strongest distinguishing features of HFpEF versus HFrEF. Comorbidities in females include obesity, hypertension, and diabetes. HFrEF or systolic HF is more common in males, and risk factors include smoking and heart attack. While numerous devices and pharmacological agents reduce mortality in HFrEF, no current medications are similarly effective for HFpEF; thus, the un-responsive HFpEF proportion is rising. Atrial fibrillation (AF) is the most common sustained arrhythmia in patients with HF and also demonstrates sex differences in incidence. Both AF and HFpEF have shared risk factors and confer increased risks for adverse cardiovascular outcomes. Since sex-specific variables that contribute to HF development and progression are largely unknown, increasing our knowledge about molecular mechanisms involved in metabolic and structural changes in the male versus female failing heart will allow us to pinpoint possible targets for safer and more effective sex-specific therapeutic strategies. We haves recently developed a mouse model that recapitulates this sexual dimorphism in HF and atrial susceptibility. In this model, female mice only exhibit continuous left ventricular hypertrophy, diastolic dysfunction, preserved ejection fraction, pulmonary congestion, arrhythmia, and reduced exercise tolerance, mimicking the clinical symptoms of restrictive cardiomyopathy. Thus, this mouse line represents a novel tool to interrogate sex differences in cardiac remodeling at the functional, structural, and biochemical level at baseline or with coordinate cardiac or metabolic stress. These mice were generated by crossing the βARKnt line (residues 50-145) with the βARKct line, peptide fragments of the prototypical G protein-coupled receptor (GPCR) kinase (GRK) GRK2 (βARK1). Breeding of βARKnt (NT) with βARKct (M27) single-transgenic mice results in a combination of all four possible genotypes, non-transgenic littermate controls (NLC)/NLC, NT/NLC (TG/NLC), NLC/M27 (NLC/TG), and NT/M27 (TG/TG), born as littermate controls. Heart weight normalized to tibia length (HW/TL) was significantly increased in 9-12 week old female and male TG/NLC and TG/TG mice, but left atrial weight was enhanced only in the TG/TG mice and to a greater extent in females that males. Despite no difference in ventricular myocyte area, ventricular fibrosis, atrial myocyte area, and atrial fibrosis were significantly increased in female NT/M27 mice only. Further, this was accompanied by a significant increase in mitral valve E/A (ventricular relaxation/atrial contraction) ratio in these mice.

G protein-coupled receptor kinase 5 (GRK5) contributes to impaired cardiac function and immune cell recruitment in post-ischemic heart failure.

Aims Myocardial infarction (MI) is the most common cause of heart failure (HF) worldwide. G protein-coupled receptor kinase 5 (GRK5) is upregulated in failing human myocardium and promotes maladaptive cardiac hypertrophy in animal models. However, the role of GRK5 in ischemic heart disease is still unknown. In this study, we evaluated whether myocardial GRK5 plays a critical role post-MI in mice and included the examination of specific cardiac immune and inflammatory responses.Methods and results Cardiomyocyte-specific GRK5 overexpressing transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice as well as cardiomyocyte-specific GRK5 knockout mice (GRK5cKO) and wild type (WT) were subjected to MI and, functional as well as structural changes together with outcomes were studied. TgGRK5 post-MI mice showed decreased cardiac function, augmented left ventricular dimension and decreased survival rate compared to NLC post-MI mice. Cardiac hypertrophy and fibrosis as well as fetal gene expression were increased post-MI in TgGRK5 compared to NLC mice. In TgGRK5 mice, GRK5 elevation produced immuno-regulators that contributed to the elevated and long-lasting leukocyte recruitment into the injured heart and ultimately to chronic cardiac inflammation. We found an increased presence of pro-inflammatory neutrophils and macrophages as well as neutrophils, macrophages and T-lymphocytes at 4-days and 8-weeks respectively post-MI in TgGRK5 hearts. Conversely, GRK5cKO mice were protected from ischemic injury and showed reduced early immune cell recruitment (predominantly monocytes) to the heart, improved contractility and reduced mortality compared to WT post-MI mice. Interestingly, cardiomyocyte-specific GRK2 transgenic mice did not share the same phenotype of TgGRK5 mice and did not have increased cardiac leukocyte migration and cytokine or chemokine production post-MI.Conclusions Our study shows that myocyte GRK5 has a crucial and GRK-selective role on the regulation of leucocyte infiltration into the heart, cardiac function and survival in a murine model of post-ischemic HF, supporting GRK5 inhibition as a therapeutic target for HF.

GRK5 is a regulator of fibroblast activation and cardiac fibrosis

Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.

2043-P: Hyaluronan Produced by Inflamed Islet Endothelial Cells Contributes to HIAPP Aggregation

In type 2 diabetes (T2D), aggregation of human islet amyloid polypeptide (hIAPP) occurs within the islet extracellular matrix (ECM) and is toxic to β cells. We recently showed that hIAPP aggregation also induces a pro-inflammatory response in islet endothelial cells (ECs). In most tissues, this inflammatory response in ECs is mediated by upregulating production of ECM components, such as hyaluronan (HA). Indeed, we find that HA accumulates in the islet ECM of hIAPP transgenic mice, where it was colocalized with amyloid deposits, while no HA accumulation occurred in non-transgenic littermate controls (which do not develop islet amyloid). Therefore, we hypothesized that hIAPP aggregation results in HA production from islet ECs, and that this HA production exacerbates hIAPP deposition/toxicity. To test this hypothesis, we utilized immortalized murine islet ECs (MS-1 cells), which under normal conditions express low levels of HA synthase (Has isoforms 2 and 3). Treatment of these cells for 48 h with hIAPP (20 µM) increased Has2 and Has3 mRNA levels (23±4 and 8±1 fold over vehicle; n=4, p&amp;lt;0.05 for both), which was abrogated upon co-incubation with Congo red, an inhibitor of hIAPP aggregation (1.4±0.3 and 1.4±0.2 for Has2 and Has3, respectively; n=4). Next, we used a thioflavin T assay, wherein fluorescence is proportional to fibril formation from amyloidogenic peptides, to determine the kinetics of hIAPP aggregation ± HA. We found co-incubation of HA with synthetic hIAPP peptide resulted in a marked acceleration (log time of 1 vs. 40 hours) and increase in maximal (greater than 2-fold) hIAPP accretion. In sum, exposure of islet ECs to aggregated hIAPP increases HA synthesis by these cells, which in turn further exacerbates aggregation of hIAPP to form amyloid. Thus, interventions targeting endothelial cell inflammation and/or HA production may be useful in blocking amyloid formation and its toxic effects in T2D. Disclosure J.J. Castillo: None. M.F. Hogan: None. A. Aplin: None. N. Esser: None. A.T. Templin: None. R.L. Hull: None. Funding National Institutes of Health (T32HL007028)

1884-P: Cardiomyocyte-Specific PKA Inhibition Ameliorates Cardiac Dysfunction Induced by Obesity

Obesity and type 2 diabetic (T2D) cause cardiac dysfunction, which could be related to chronically activated sympathoadrenergic system (SAS). SAS system activation leads to the activation of PKA in cardiomyocytes. In this study, we aimed to test the hypothesis that chronically activated PKA plays an important role in obese induced cardiac dysfunction. Methods and Results: 1. Cardiomyocyte specific PKA inhibition (cPKAi) transgenic (TG) mouse model (n=18) and nontransgenic littermate control (NLC, n=20) mice (3-month old) were fed with high fat food (D12451, Research Diet) or normal chow for 6 months.2. In 3-month old male TG mice, intraperitoneal glucose injection did not increase blood glucose as high as in 3-month old NLC mice. The area under the curve of cPKAi TG mice was significantly less than that of control mice. cPKAi transgenic mice had decreased cardiac function after overnight fasting while control mice did not have cardiac function decrement even though both types of mice had the same extend of glucose reduction after overnight fasting. 3. The BW increase was more in TG mice. 4. After HFD feeding, cPKAi TG group had better cardiac function: (1). The maximal systolic pressure (mmHg) of TG (107.8±4.49) was significantly higher than that of NLC (83.0±8.1) mice. (2). The end diastolic pressure (EDP, mmHg) of TG (2.7 ± 2.2) was significantly lower in TG mice (11.5 ± 3.3); (3). Maximum dp/dt was significantly greater in TG than in NLC mice; (4). The relaxation rate of the heart was significantly better in TG than in NLC mice; 5. The HW/BW (mg/g) was significantly less in TG mice (4.6 ± 0.2) than in control mice (5.2 ± 0.2). 6. HFD reduced myocytes contractions, Ca transients, and Ca currents in both groups but the reduction was less in TG myocytes. 7. TG hearts had less inflammatory cell infiltration and fibrosis than control hearts. Conclusion: cPKAi enhances cardiac glucose uptake and ameliorates cardiac remodeling and preserves cardiac function in HFD-induced obese. Disclosure X. Chen: None. Funding American Diabetes Association (1-19-IBS-210)

Ezrin Expression is Increased During Disease Progression in a Tauopathy Mouse Model and Alzheimer's Disease.

The lack of diagnostic tools and disease-modifying treatments against Alzheimer's disease (AD) and related disorders, collectively known as tauopathies, has led to a socioeconomic burden of epidemic proportion. Proteomics approaches can be used to identify novel proteome changes that could help us understand the pathogenesis of tau-related pathological hallmarks and/or cellular stress responses associated with tauopathy. These studies, however, need to be conducted taking into consideration brain region specificity and stage of neurodegeneration in order to provide insights about the pathological role of the identified proteins. We used a tauopathy mouse model (JNPL3) that expresses human tau bearing a P301L mutation and develops motor impairment, the severity of which correlates with the increased accumulation of pathological tau. Tissue was dissected from asymptomatic and severely motor impaired JNPL3 mice as well as non-transgenic littermate controls and subjected to two-dimensional gel electrophoresis. Differentially abundant protein spots were identified by tandem mass spectrometry. Postmortem mild cognitive impairment (MCI), AD and normal aging controls were used to validate the pathological significance of the identified protein. Ezrin was identified as a protein that is upregulated in tau-mediated neurodegeneration. We demonstrate that Ezrin protein abundance increased in JNPL3 mice preceded motor impairment and was sustained in severely motor impaired mice. Ezrin expression was also increased in the temporal cortex of MCI and AD patients. The results demonstrate that increased Ezrin protein abundance changes are associated with the early stages of neurodegeneration in tauopathy models and human disease. Understanding the role of Ezrin in tauopathies such as AD may provide new insights for targeting tau-mediated neurodegeneration.

Abstract 574: An Amino-Terminal Peptide of GRK5 Prevents Pathological Cardiac Hypertrophy in Response to Pharmacological and Surgical Stress

Introduction: The pathogenesis and progression of heart failure (HF) encompasses maladaptive cardiac hypertrophy, ventricular remodeling, and contractile dysfunction. A gene expression maladaptation in HF is an increase in expression of G protein-coupled receptor kinase (GRK5). Recently, non-canonical roles for GRK5 have been uncovered that are crucial for the development of maladaptive hypertrophy and these are due to its nuclear translocation. We have shown that nuclear targeting of GRK5 is mediated by an amino-terminal (NT) domain that can bind calmodulin. Hypothesis: We hypothesize that expression of a peptide encoding the NT of GRK5 that includes this calmodulin binding domain will act as a calmodulin inhibitor preventing the nuclear accumulation of GRK5 and inhibit its non-canonical pathological activities. Methods: To further study this, neonatal rat ventricular cardiomyocytes overexpressing GRK5-NT or a negative control virus were treated with 50μM phenylephrine (PE) or vehicle for 48 hours to induce a hypertrophic response. Cell areas and transcript levels of fetal genes were measured after treatment. Novel transgenic GRK5-NT and non-transgenic littermate control (NLC) mice were characterized before and 4 weeks after transverse aortic constriction (TAC) or sham surgery. Echocardiography, myocyte cross-sectional area, and heart weight to tibia length metrics were used to evaluate TAC response. Results: In vitro, expression of the GRK5-NT peptide abrogated the hypertrophic effects of PE, both in terms of fetal gene re-activation and myocyte size. In vivo, transgenic GRK5-NT mice did not demonstrate any hypertrophy after 4 weeks of TAC and cardiac dysfunction seen in NLC mice at this time point was absent in GRK5-NT mice. Further, GRK5-NT TAC mice showed no increase in cell surface area compared to their respective sham controls post-TAC. Conclusion: In this study, we show that the GRK5-NT peptide prevents the pathological response to pharmacological and surgical models of cardiac hypertrophy. Further studies in vivo will reveal whether GRK5-NT can preserve function in the long-term, and further in vitro characterization will reveal whether the mechanism is GRK5-dependent.

Altered brain activity during withdrawal from chronic alcohol is associated with changes in IL-6 signal transduction and GABAergic mechanisms in transgenic mice with increased astrocyte expression of IL-6

Interleukin-6 (IL-6) is an important neuroimmune factor that is increased in the brain by alcohol exposure/withdrawal and is thought to play a role in the actions of alcohol on the brain. To gain insight into IL-6/alcohol/withdrawal interactions and how these interactions affect the brain, we are studying the effects of chronic binge alcohol exposure on transgenic mice that express elevated levels of IL-6 in the brain due to increased astrocyte expression (IL-6 tg) and their non-transgenic (non-tg) littermate controls. IL-6/alcohol/withdrawal interactions were identified by genotypic differences in spontaneous brain activity in electroencephalogram (EEG) recordings from the mice, and by Western blot analysis of protein activation or expression in hippocampus obtained from the mice after the final alcohol withdrawal period. Results from EEG studies showed frequency dependent genotypic differences in brain activity during withdrawal. For EEG frequencies that were affected by alcohol exposure/withdrawal in both genotypes, the nature of the effect was similar, but differed across withdrawal cycles. Differences between IL-6 tg and non-tg mice were also observed in Western blot studies of the activated form of STAT3 (phosphoSTAT3), a signal transduction partner of IL-6, and subunits of GABAA receptors (GABAAR). Regression analysis revealed that pSTAT3 played a more prominent role during withdrawal in the IL-6 tg mice than in the non-tg mice, and that the role of GABAAR alpha-5 and GABAAR alpha-1 in brain activity varied across genotype and withdrawal. Taken together, our results suggest that IL-6 can significantly impact mechanisms involved in alcohol withdrawal.

Smooth Muscle PPARγ Mutation Causes Impaired Renal Blood Flow and Salt‐Sensitive Hypertension

Failure to vasodilate in systemic and renal vasculature in response to salt loading is a hallmark of salt sensitivity in hypertensive humans and experimental models. Human subjects carrying dominant negative mutations in PPARγ exhibit hypertension while global PPARγ activation consistently lowers blood pressure (BP). To determine whether tissue‐specific actions of PPARγ regulate vascular function, salt sensitivity and BP, we studied mice specifically expressing a human hypertension‐causing mutation in PPARγ (P467L) selectively in smooth muscle (S‐P467L). S‐P467L transgenic mice and non‐transgenic littermate controls (NT) were fed regular diet (0.4% salt) or high salt (HS) diet containing 4% salt for 4 weeks. Salt induced marked elevations in systolic BP in S‐P467L mice (136±4 mmHg salt n=6 vs 121±3 mmHg regular diet n=7, p&lt;0.01, two‐way ANOVA), but not in NT mice (121±4 mmHg salt n=4 vs 116±4.0 mmHg regular diet n=5). Because salt induced similar increases in cardiac output in S‐P467L and NT animals, the pressor effect of salt intake in S‐P467L mice was not due to a larger fluid volume, but likely higher peripheral vascular resistance. In keeping with this, HS‐fed S‐P467L mice, but not NT controls, exhibited severe impairment in acetylcholine (ACh)‐ and sodium nitroprusside (SNP)‐induced vasorelaxation in carotid artery (Maximal relaxation at 30 μM ACh: S‐P467L 31±4.9% n=4 vs. NT 90±1.8% n=6, p&lt;0.01; Maximal relaxation to 30 μM SNP: S‐P467L 38±2.8% n=4 vs. NT 89±2.6% n=6, p&lt;0.01, two‐way ANOVA Repeated Measurements) and in cerebral basilar artery (Maximal relaxation at 100 μM ACh: S‐P467L ‐3.2±9.3% n=7 vs. NT 57±5.9% n=5, p&lt;0.01). Importantly, salt‐induced vascular dysfunction rapidly developed in S‐P467L mice after receiving HS diet for only 3 days, preceding BP elevations. The impaired vasorelaxation in HS‐fed S‐P467L mice was associated with smaller renal artery diameter (S‐P467L 322±21 μm n=6 vs NT 389±22 μm n=5, p&lt;0.05 two‐way ANOVA) and blunted renal blood flow (S‐P467L 36±3.6 μL/min/g n=6 vs. NT 50±6.4 μL/min/g n=5, p&lt;0.05). These changes were not caused by differences in circulating renin‐angiotensin levels or renal sympathetic nerve activity between the two strains. Preliminary data indicated that during the 3rd week of HS diet, S‐P467L mice produced 36% less nitrate/nitrite in 24 hour urine compared to NT controls, which suggested blunted renal bioavailability of nitric oxide, a potent inhibitor of Na‐K‐2Cl cotransporter (NKCC2). This was associated with a declined capacity of HS‐fed S‐P467L mice to excrete an acute volume challenge, which was rescued by an NKCC2 inhibitor furosemide, but not by the Na‐Cl‐cotransporter inhibitor hydrochlorothiazide. Our data support the novel concept that smooth muscle PPARγ regulates systemic vascular resistance, renal perfusion and tubular sodium transport, and loss of these protective actions of PPARγ predisposes to salt sensitivity and hypertension.Support or Funding InformationDr. Jing Wu is supported by an American Heart Association Postdoctoral Fellowship (17POST33660685). This project is funded by R01 HL125603 and R01 HL131689 to Dr. Curt Sigmund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Altered hippocampal synaptic function in transgenic mice with increased astrocyte expression of CCL2 after withdrawal from chronic alcohol

CNS actions of the chemokine CCL2 are thought to play a role in a variety of conditions that can have detrimental consequences to CNS function, including alcohol use disorders. We used transgenic mice that express elevated levels of CCL2 in the CNS (CCL2-tg) and their non-transgenic (non-tg) littermate control mice to investigate long-term consequences of CCL2/alcohol/withdrawal interactions on hippocampal synaptic function, including excitatory synaptic transmission, somatic excitability, and synaptic plasticity. Two alcohol exposure paradigms were tested, a two-bottle choice alcohol (ethanol) drinking protocol (2BC drinking) and a chronic intermittent alcohol (ethanol) (CIE/2BC) protocol. Electrophysiological measurements of hippocampal function were made ex vivo, starting ∼0.6 months after termination of alcohol exposure. Both alcohol exposure/withdrawal paradigms resulted in CCL2-dependent interactions that altered the effects of alcohol on synaptic function. The synaptic alterations differed for the two alcohol exposure paradigms. The 2BC drinking/withdrawal treatment had no apparent long-term consequences on synaptic responses and long-term potentiation (LTP) in hippocampal slices from non-tg mice, whereas synaptic transmission was reduced but LTP was enhanced in hippocampal slices from CCL2-tg mice. In contrast, the CIE/2BC/withdrawal treatment enhanced synaptic transmission but reduced LTP in the non-tg hippocampus, whereas there were no apparent long-term consequences to synaptic transmission and LTP in hippocampus from CCL2-tg mice, although somatic excitability was enhanced. These results support the idea that alcohol-induced CCL2 production can modulate the effects of alcohol exposure/withdrawal on synaptic function and indicate that CCL2/alcohol interactions can vary depending on the alcohol exposure/withdrawal protocol used.

Acute Down-regulation of BDNF Signaling Does Not Replicate Exacerbated Amyloid-β Levels and Cognitive Impairment Induced by Cholinergic Basal Forebrain Lesion.

Degeneration of basal forebrain cholinergic neurons (BFCNs) precedes hippocampal degeneration and pathological amyloid-beta (Aβ) accumulation, and underpins the development of cognitive dysfunction in sporadic Alzheimer’s disease (AD). We hypothesized that degeneration of BFCNs causes a decrease in neurotrophin levels in innervated brain areas, which in turn promotes the development of Aβ pathology and cognitive impairment. Here we show that lesion of septo-hippocampal BFCNs in a pre-symptomatic transgenic amyloid AD mouse model (APP/PS1 mice) increases soluble Aβ levels in the hippocampus, and induces cognitive deficits in a spatial memory task that are not seen in either unlesioned APP/PS1 or non-transgenic littermate control mice. Furthermore, the BFCN lesion results in decreased levels of brain-derived neurotrophic factor (BDNF). However, viral knockdown of neuronal BDNF in the hippocampus of APP/PS1 mice (in the absence of BFCN loss) neither increased the level of Aβ nor caused cognitive deficits. These results suggest that the cognitive decline and Aβ pathology induced by BFCN loss occur independent of dysfunctional neuronal BDNF signaling, and may therefore be directly underpinned by reduced cholinergic neurotransmission.

Abstract 24071: A Peptide Of The Amino-terminus Of Grk2 Induces Hypertrophy And Yet Elicits Cardioprotection After Pressure Overload.

Heart failure (HF) is a leading cause of death worldwide and a growing burden on public health, and the underlying mechanisms of cardiac remodeling and decompensation to HF remain a focus of research efforts towards therapeutic development. Signaling via G protein-coupled receptors (GPCRs) is critical for normal heart function and is tightly controlled by GPCR kinases (GRKs) with GRK2 (originally βARK1), being intimately involved in HF progression. In addition to its well-characterized role in regulating GPCRs, ongoing research has demonstrated great diversity in the functional roles of GRK2. I have recently investigated GRK2 amino terminal binding interactions through the generation of transgenic (Tg) mice with cardiac-targeted expression of the amino-terminal peptide βARKnt (residues 50-145). In a murine model of trans-aortic constriction (TAC)-induced pressure overload, echocardiography revealed increased left ventricular (LV) posterior wall thickness (1.57 versus 1.37 ± 0.02; n = 16) and LV mass in TgβARKnt compared to non-transgenic littermate controls (NLC) 4 weeks after TAC or Sham surgery. Interestingly, despite enhanced hypertrophy at baseline and after acute pressure overload, the progression to HF was paradoxically inhibited in TgβARKnt mice during chronic pressure overload with preserved cardiac function (% Ejection Fraction 57.3 versus 37.3 ± 2.0; n = 11, 10). Further, βARKnt expression limited adverse left ventricular remodeling, with reduced interstitial fibrosis (% area fibrosis 4.1 versus 9.2 ± 0.8; n = 11, 9 hearts) and preserved β-adrenergic receptor density 4 weeks after surgery. The effect of cardiac βARKnt expression was not consistent with alterations in GRK2 activity at GPCRs as neither GRK2 peptide inhibition (TgβARKct) nor GRK2 overexpression alter hypertrophy, and overexpression hastens HF development. Further, TgβARKnt mice exhibit reduced epididymal white adipose content and altered mitochondrial respiration, suggesting altered cardiac metabolism. These data support the idea that the βARKnt peptide embodies a distinct functional interaction and that βARKnt-mediated regulation of β-adrenergic receptor density may provide a novel means of cardioprotection during pressure-overload induced HF.

Abstract 396: GRK5-mediated Exacerbation of Ischemic Heart Failure Involves Cardiac Immune-Inflammatory Responses

Rationale: Coronary artery disease and subsequent myocardial ischemia (MI) are the most common cause of heart failure (HF) in the US. G protein-coupled receptor (GPCR) kinase 5 (GRK5) has been shown to be upregulated in failing human myocardium. While the canonical role of GRKs is to desensitize receptors via phosphorylation, it has been shown that GRK5 can also locate to the nucleus of cardiomyocytes where it can exert GPCR-independent effects that promote maladaptive cardiac hypertrophy after hypertrophic stress. Despite numerous data indicating the importance of GRK5 in hypertrophy, it is still unknown if GRK5 has a role in ischemic heart disease. Objective: In this study, we investigated the critical role that GRK5 plays after myocardial ischemic injury with a novel aspect being the regulation of immune and inflammatory responses including recruitment of immune cells to the injured heart. Methods and Results: Cardiac-specific GRK5 transgenic mice (Tg-GRK5) and non-transgenic littermate (NLC) control mice were subjected to MI. Tg-GRK5-HF mice showed decreased cardiac function (both global and segmental contractility) as well as augmented left ventricular diameters and volumes compared to NLC-HF mice. Heart weight (HW) to body weight or tibia length ratios as well as mRNA expression of all major adverse remodeling-associated biomarkers (ANF, BNP, b-MHC) were increased in the TgGRK5-HF compared to NLC-HF. Cardiac fibrosis in the border zone (BZ) area as well as mRNA levels of Collagen-1 (Col-1), Col-3, MMP2 and CTGF were higher in TgGRK5-HF compared to NLC-HF, strongly suggesting increased adverse remodeling. Inflammatory Cytokines (IL-6 and IL-1beta) were augmented in TgGRK5-HF compared to NLC-HF and contribute at least in part to increased immune cell recruitment in the heart. In fact, we found both Neutrophils and T-lymphocytes (T- cells) augmented in the BZ and infarct zone. Conclusions: Our study shows that cardiac GRK5 has a detrimental effect during ischemic HF. GRK5 overexpression causes reduced cardiac function and increased immune cell recruitment/inflammation. Further, these results suggest GRK5 as a potential therapeutic target to limit HF development after ischemic injury.

Protective effect and mechanism of estrogen receptor β on myocardial infarction in mice.

The protective effect and the mechanism of estrogen receptor β (ERβ) on myocardial infarction (MI) in mice were explored. A total of 12 female Tg-ERβ transgenic mice and 12 non-transgenic littermate control (NLC) wild-type C57 mice were used for the present study. Both transgenic and wild-type mice had similar baseline data such as age, sex, and weight. The mouse model of MI was established by coronary artery ligation method, and the cardiac structure and function changes of the mouse were observed by ultrasonic echocardiography on days 1, 3 and 7 after the operation. RT-PCR method was used to detect the expression of collagen I, α-SMA, TGF-β mRNA in the mouse heart, and Masson staining was used to detect cardiac fibrosis. At 3 days after operation, echocardiographic posterior wall thickness at end diastole (PWTD) and end systolic PWTS of Tg-ERβ mice were significantly reduced, and left ventricular systolic diameter and left ventricular diastolic diameter significantly increased (P<0.05) compared with NLC mice. The levels of expression of Tg-ERβ cardiac tissue collagen I, α-SMA, TGF-β mRNA were significantly lower than those in the NLC mice (P<0.05). In conclusion, Tg-ERβ exerts a protective effect on MI.

Transgenic mice with increased astrocyte expression of CCL2 show altered behavioral effects of alcohol

Emerging research provides strong evidence that activation of CNS glial cells occurs in neurological diseases and brain injury and results in elevated production of neuroimmune factors. These factors can contribute to pathophysiological processes that lead to altered CNS function. Recently, studies have also shown that both acute and chronic alcohol consumption can produce activation of CNS glial cells and the production of neuroimmune factors, particularly the chemokine ligand 2 (CCL2). The consequences of alcohol-induced increases in CCL2 levels in the CNS have yet to be fully elucidated. Our studies focus on the hypothesis that increased levels of CCL2 in the CNS produce neuroadaptive changes that modify the actions of alcohol on the CNS. We utilized behavioral testing in transgenic mice that express elevated levels of CCL2 to test this hypothesis. The increased level of CCL2 in the transgenic mice involves increased astrocyte expression. Transgenic mice and their non-transgenic littermate controls were subjected to one of two alcohol exposure paradigms, a two-bottle choice alcohol drinking procedure that does not produce alcohol dependence or a chronic intermittent alcohol procedure that produces alcohol dependence. Several behavioral tests were carried out including the Barnes maze, Y-maze, cued and contextual conditioned fear test, light–dark transfer, and forced swim test. Comparisons between alcohol naïve, non-dependent, and alcohol-dependent CCL2 transgenic and non-transgenic mice show that elevated levels of CCL2 in the CNS interact with alcohol in tests for alcohol drinking, spatial learning, and associative learning.

NLRP3 Inflammasome Inhibitor Ameliorates Amyloid Pathology in a Mouse Model of Alzheimer's Disease.

The activation of the NLRP3 inflammasome signaling pathway plays an important role in the neuroinflammation in Alzheimer's disease (AD). In this study, we investigated the effects of JC-124, a rationally designed NLRP3 inflammasome inhibitor, on AD-related deficits in CRND8 APP transgenic mice (TgCRND8). We first demonstrated increased formation and activation of NLRP3 inflammasome in TgCRND8 mice compared to non-transgenic littermate controls, which was inhibited by the treatment with JC-124. Importantly, JC-124 treatment led to decreased levels of Aβ deposition and decreased levels of soluble and insoluble Aβ1-42 in the brain of CRND8 mice which was accompanied by reduced β-cleavage of APP, reduced activation of microglia but enhanced astrocytosis. Oxidative stress was decreased and synaptophysin was increased in the CRND8 mice after JC-124 treatment, demonstrating a neuroprotective effect. Overall, these data demonstrated beneficial effects of JC-124 as a specific NLRP3 inflammasome inhibitor in AD mouse model and supported the further development of NLRP3 inflammasome inhibitors as a viable option for AD therapeutics.

Inhibition of farnesyl pyrophosphate synthase improves pressure overload induced chronic cardiac remodeling.

Farnesyl pyrophosphate synthase (FPPS) is a key enzyme in the mevalonate pathway. In our previous studies, we find that inhibition of FPPS attenuates angiotensin II-induced cardiac hypertrophy and fibrosis by suppressing RhoA while FPPS and Ras are up-regulated in pressure overload rats. In this study, we evaluate the effects and mechanisms of FPPS inhibition in pressure overload mice. Male FPPS-small interfering RNA (SiRNA) transgenic (Tg) mice and non-transgenic littermate control (NLC) were randomly divided into suprarenal abdominal aortic constriction (AAC) group and sham operation group. 12 weeks following AAC, mice were sacrificed by cervical dislocation. Histological and echocardiographic assessments showed that inhibition of FPPS improved chronic cardiac remodeling which was induced by AAC. The reductions of Ras farnesylation and GTP-Ras, as well as their downstream extracellular signal-related kinases 1/2 (ERK1/2) expression were observed in the heart of Tg-AAC mice compared with NLC-AAC mice, along with the reduction of fetal gene expression. We provide here important experimental evidence that inhibition of FPPS improves AAC induced chronic cardiac remodeling and fibrosis by the reduction of farnesylated Ras and the downregulation of Ras-ERK1/2 pathway.