A century of exercise physiology: key concepts in neural control of the circulation.

Early in the twentieth century, Walter B. Cannon (1871-1945) introduced his overarching hypothesis of "homeostasis" (Cannon 1932)-the ability to sustain physiological values within a narrow range necessary for life during periods of stress. Physical exercise represents a stress in which motor, respiratory and cardiovascular systems must be integrated across a range of metabolic stress to match oxygen delivery to oxygen need at the cellular level, together with appropriate thermoregulatory control, blood pressure adjustments and energy provision. Of these, blood pressure regulation is a complex but controlled variable, being the function of cardiac output and vascular resistance (or conductance). Key in understanding blood pressure control during exercise is the coordinating role of the autonomic nervous system. A long history outlines the development of these concepts and how they are integrated within the exercise context. This review focuses on the renaissance observations and thinking generated in the first three decades of the twentieth century that opened the doorway to new concepts of inquiry in cardiovascular regulation during exercise. The concepts addressed here include the following: (1) exercise and blood pressure, (2) central command, (3) neurovascular transduction with emphasis on the sympathetic nerve activity and the vascular end organ response, and (4) tonic neurovascular integration.

Sex-dependent cholinergic effects on amyloid pathology: A translational study.

INTRODUCTION: About two-thirds of Alzheimer's Disease (AD) patients are women, who exhibit more severe pathology and cognitive decline than men. Whether biological sex causally modulates the relationship between cholinergic signaling and amyloid pathology remains unknown. METHODS: We quantified amyloid beta (Aß) in male and female App-mutant mice with either decreased or increased cholinergic tone and examined the impact of ovariectomy and estradiol replacement in this relationship. We also investigated longitudinal changes in basal forebrain (cholinergic function) and Aß in elderly individuals. RESULTS: We show a causal relationship between cholinergic tone and amyloid pathology in males and ovariectomized female mice, which is decoupled in ovary-intact and ovariectomized females receiving estradiol. In elderly humans, cholinergic loss exacerbates Aß. DISCUSSION: Our findings emphasize the importance of reflecting human menopause in mouse models. They also support a role for therapies targeting estradiol and cholinergic signaling to reduce Aß. HIGHLIGHTS: Cholinergic tone regulates amyloid beta (Aß) pathology in males and ovariectomized female mice. Estradiol uncouples the relationship between cholinergic tone and Aß. In elderly humans, cholinergic loss correlates with increased Aß in both sexes.

Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner.

BACKGROUND: Pannexin 3 (PANX3) is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggest that it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high-fat diet (HFD). METHODS: C57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 to 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression. RESULTS: In females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. In addition, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, multiple inflammatory genes were significantly different in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype in KO mice compared to WT. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs. CONCLUSION: PANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA and ß-Arrestin.

Proteinase-activated receptors (PARs) are a four-member family of G-protein-coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα q/11- and Gα i-signaling pathways and is dependent on signaling via the ß-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and ß-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.

Modulation of hippocampal neuronal resilience during aging by the Hsp70/Hsp90 co-chaperone STI1.

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.

Selective decrease of cholinergic signaling from pedunculopontine and laterodorsal tegmental nuclei has little impact on cognition but markedly increases susceptibility to stress.

The pedunculopontine tegmental nucleus (PPT) and laterodorsal tegmental nucleus (LDT) are heterogeneous brainstem structures that contain cholinergic, glutamatergic, and GABAergic neurons. PPT/LDT neurons are suggested to modulate both cognitive and noncognitive functions, yet the extent to which acetylcholine (ACh) signaling from the PPT/LDT is necessary for normal behavior remains uncertain. We addressed this issue by using a mouse model in which PPT/LDT cholinergic signaling is highly decreased by selective deletion of the vesicular ACh transporter (VAChT) gene. This approach interferes exclusively with ACh signaling, leaving signaling by other neurotransmitters from PPT/LDT cholinergic neurons intact and sparing other cells. VAChT mutants were examined on different PPT/LDT-associated cognitive domains. Interestingly, VAChT mutants showed no attentional deficits and only minor cognitive flexibility impairments while presenting large deficiencies in both spatial and cued Morris water maze (MWM) tasks. Conversely, working spatial memory determined with the Y-maze and spatial memory measured with the Barnes maze were not affected, suggesting that deficits in MWM were unrelated to spatial memory abnormalities. Supporting this interpretation, VAChT mutants exhibited alterations in anxiety-like behavior and increased corticosterone levels after exposure to the MWM, suggesting altered stress response. Thus, PPT/LDT VAChT-mutant mice present little cognitive impairment per se, yet they exhibit increased susceptibility to stress, which may lead to performance deficits in more stressful conditions.-Janickova, H., Kljakic, O., Rosborough, K., Raulic, S., Matovic, S., Gros, R., Saksida, L. M., Bussey, T. J., Inoue, W., Prado, V. F., Prado, M. A. M. Selective decrease of cholinergic signaling from pedunculopontine and laterodorsal tegmental nuclei has little impact on cognition but markedly increases susceptibility to stress.

Nicotinamide Phosphoribosyltransferase in Smooth Muscle Cells Maintains Genome Integrity, Resists Aortic Medial Degeneration, and Is Suppressed in Human Thoracic Aortic Aneurysm Disease.

RATIONALE: The thoracic aortic wall can degenerate over time with catastrophic consequences. Vascular smooth muscle cells (SMCs) can resist and repair artery damage, but their capacities decline with age and stress. Recently, cellular production of nicotinamide adenine dinucleotide (NAD+) via nicotinamide phosphoribosyltransferase (Nampt) has emerged as a mediator of cell vitality. However, a role for Nampt in aortic SMCs in vivo is unknown. OBJECTIVES: To determine whether a Nampt-NAD+ control system exists within the aortic media and is required for aortic health. METHODS AND RESULTS: Ascending aortas from patients with dilated aortopathy were immunostained for NAMPT, revealing an inverse relationship between SMC NAMPT content and aortic diameter. To determine whether a Nampt-NAD+ control system in SMCs impacts aortic integrity, mice with Nampt-deficient SMCs were generated. SMC-Nampt knockout mice were viable but with mildly dilated aortas that had a 43% reduction in NAD+ in the media. Infusion of angiotensin II led to aortic medial hemorrhage and dissection. SMCs were not apoptotic but displayed senescence associated-ß-galactosidase activity and upregulated p16, indicating premature senescence. Furthermore, there was evidence for oxidized DNA lesions, double-strand DNA strand breaks, and pronounced susceptibility to single-strand breakage. This was linked to suppressed poly(ADP-ribose) polymerase-1 activity and was reversible on resupplying NAD+ with nicotinamide riboside. Remarkably, we discovered unrepaired DNA strand breaks in SMCs within the human ascending aorta, which were specifically enriched in SMCs with low NAMPT. NAMPT promoter analysis revealed CpG hypermethylation within the dilated human thoracic aorta and in SMCs cultured from these tissues, which inversely correlated with NAMPT expression. CONCLUSIONS: The aortic media depends on an intrinsic NAD+ fueling system to protect against DNA damage and premature SMC senescence, with relevance to human thoracic aortopathy.

Cardiac-specific inducible overexpression of human plasma membrane Ca2+ ATPase 4b is cardioprotective and improves survival in mice following ischemic injury.

Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca2+-ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo, and HF following experimental myocardial infarction (MI) in vivoMethods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca2+-regulatory genes, and induced hypertrophy without significant differences in Ca2+ transients or diastolic Ca2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF.Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF.

Deletion of the vesicular acetylcholine transporter from pedunculopontine/laterodorsal tegmental neurons modifies gait.

Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinson's disease (PD), have been suggested to be related to dysfunctional cholinergic signaling in the brainstem. We investigated how long-term loss of cholinergic signaling from mesopontine nuclei influence motor behaviors. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChTEn1-Cre-flox/flox ). VAChTEn1-Cre-flox/flox mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire-hang and grip-force tests. Young VAChTEn1-Cre-flox/flox mice (2-5 months-old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChTEn1-Creflox/flox mice (13-16 months-old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT-deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients. Read the Editorial Highlight for this article on page 688.

Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER).

Although aldosterone is a known regulator of renal and cardiovascular function, its role as a regulator of cancer growth and spread has not been widely considered. This study tested the hypothesis that aldosterone regulates cancer cell growth/spread via G protein-coupled estrogen receptor (GPER) activation. In vitro in murine renal cortical adenocarcinoma (RENCA) cells, a widely used murine in vitro model for the study of renal cell adenocarcinoma, aldosterone increased RENCA cell proliferation to a maximum of 125 ± 3% of control at a concentration of 10 nM, an effect blocked by the GPER antagonist G15 or by GPER knockdown using short interfering (sh) RNA techniques. Further, aldosterone increased RENCA cell migration to a maximum of 170 ± 20% of control at a concentration of 100 nM, an effect also blocked by G15 or by GPER down-regulation. In vivo, after orthotopic RENCA cell renal transplantation, pulmonary tumor spread was inhibited by pharmacologic blockade of aldosterone effects with spironolactone (percentage of lung occupied by metastasis: control = 68 ± 13, spironolactone = 26 ± 8, P < 0.05) or inhibition of aldosterone synthesis with a high dietary salt diet (percentage of lung: control = 44 ± 6, high salt = 12 ± 3, P < 0.05), without reducing primary tumor size. Additionally, adrenalectomy significantly reduced the extent of pulmonary tumor spread, whereas aldosterone infusion recovered pulmonary metastatic spread toward baseline levels. Finally, inhibition of GPER either with the GPER antagonist G15 or by GPER knockdown comparably inhibited RENCA cell pulmonary metastatic cancer spread. Taken together, these findings provide strong evidence for aldosterone serving a causal role in renal cell cancer regulation via its GPER receptor; thus, antagonism of GPER represents a potential new target for treatment to reduce metastatic spread.-Feldman, R. D., Ding, Q., Hussain, Y., Limbird, L. E., Pickering, J. G., Gros, R. Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER).

Cardiac acetylcholine inhibits ventricular remodeling and dysfunction under pathologic conditions.

Autonomic dysfunction is a characteristic of cardiac disease and decreased vagal activity is observed in heart failure. Rodent cardiomyocytes produce de novo ACh, which is critical in maintaining cardiac homeostasis. We report that this nonneuronal cholinergic system is also found in human cardiomyocytes, which expressed choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). Furthermore, VAChT expression was increased 3- and 1.5-fold at the mRNA and protein level, respectively, in ventricular tissue from patients with heart failure, suggesting increased ACh secretion in disease. We used mice with genetic deletion of cardiomyocyte-specific VAChT or ChAT and mice overexpressing VAChT to test the functional significance of cholinergic signaling. Mice deficient for VAChT displayed an 8% decrease in fractional shortening and 13% decrease in ejection fraction compared with angiotensin II (Ang II)-treated control animals, suggesting enhanced ventricular dysfunction and pathologic remodeling in response to Ang II. Similar results were observed in ChAT-deficient mice. Conversely, no decline in ventricular function was observed in Ang II-treated VAChT overexpressors. Furthermore, the fibrotic area was significantly greater (P < 0.05) in Ang II-treated VAChT-deficient mice (3.61 ± 0.64%) compared with wild-type animals (2.24 ± 0.11%). In contrast, VAChT overexpressing mice did not display an increase in collagen deposition. Our results provide new insight into cholinergic regulation of cardiac function, suggesting that a compensatory increase in cardiomyocyte VAChT levels may help offset cardiac remodeling in heart failure.

GRK2 targeted knock-down results in spontaneous hypertension, and altered vascular GPCR signaling.

Hypertension, elevated arterial pressure, occurs as the consequence of increased peripheral resistance. G protein-coupled receptors (GPCRs) contribute to the regulation of vasodilator and vasoconstrictor responses, and their activity is regulated by a family of GPCR kinases (GRKs). GRK2 expression is increased in hypertension and this facilitates the development of the hypertensive state by increasing the desensitization of GPCRs important for vasodilation. We demonstrate here, that genetic knockdown of GRK2 using a small hairpin (sh) RNA results in altered vascular reactivity and the development of hypertension between 8-12 weeks of age in shGRK2 mice due to enhanced Gαq/11 signaling. Vascular smooth muscle cells (VSMCs) cultured from shGRK2 knockdown mice show increases in GPCR-mediated Gαs and Gαq/11 signaling, as the consequence of reduced GRK2-mediated desensitization. In addition, agonists and biased agonists exhibited age-dependent alterations in ERK1/2 and Akt signaling, as well as cell proliferation and migration responses in shGRK2 knockdown VSMCs when cultured from mice that are either 3 months or 6 months of age. Changes in angiotensin II-stimulated ERK1/2 phosphorylation are observed in VSMCs derived from 6-week-old shGRK2 mice prior to the development of the hypertensive phenotype. Thus, our findings indicate that the balance between mechanisms regulating vascular tone are shifted to favor vasoconstriction in the absence of GRK2 expression and that this leads to the age-dependent development of hypertension, as a consequence of global alterations in GPCR signaling. Consequently, therapeutic strategies that target GRK2 activity, not expression, may be more effective for the treatment of hypertension.

Fibroblast Growth Factor 9 Imparts Hierarchy and Vasoreactivity to the Microcirculation of Renal Tumors and Suppresses Metastases.

Tumor vessel normalization has been proposed as a therapeutic paradigm. However, normal microvessels are hierarchical and vasoreactive with single file transit of red blood cells through capillaries. Such a network has not been identified in malignant tumors. We tested whether the chaotic tumor microcirculation could be reconfigured by the mesenchyme-selective growth factor, FGF9. Delivery of FGF9 to renal tumors in mice yielded microvessels that were covered by pericytes, smooth muscle cells, and a collagen-fortified basement membrane. This was associated with reduced pulmonary metastases. Intravital microvascular imaging revealed a haphazard web of channels in control tumors but a network of arterioles, bona fide capillaries, and venules in FGF9-expressing tumors. Moreover, whereas vasoreactivity was absent in control tumors, arterioles in FGF9-expressing tumors could constrict and dilate in response to adrenergic and nitric oxide releasing agents, respectively. These changes were accompanied by reduced hypoxia in the tumor core and reduced expression of the angiogenic factor VEGF-A. FGF9 was found to selectively amplify a population of PDGFRß-positive stromal cells in the tumor and blocking PDGFRß prevented microvascular differentiation by FGF9 and also worsened metastases. We conclude that harnessing local mesenchymal stromal cells with FGF9 can differentiate the tumor microvasculature to an extent not observed previously.

G-protein estrogen receptor as a regulator of low-density lipoprotein cholesterol metabolism: cellular and population genetic studies.

OBJECTIVE: Estrogen deficiency is linked with increased low-density lipoprotein (LDL) cholesterol. The hormone receptor mediating this effect is unknown. G-protein estrogen receptor (GPER) is a recently recognized G-protein-coupled receptor that is activated by estrogens. We recently identified a common hypofunctional missense variant of GPER, namely P16L. However, the role of GPER in LDL metabolism is unknown. Therefore, we examined the association of the P16L genotype with plasma LDL cholesterol level. Furthermore, we studied the role of GPER in regulating expression of the LDL receptor and proprotein convertase subtilisin kexin type 9. APPROACH AND RESULTS: Our discovery cohort was a genetically isolated population of Northern European descent, and our validation cohort consisted of normal, healthy women aged 18 to 56 years from London, Ontario. In addition, we examined the effect of GPER on the regulation of proprotein convertase subtilisin kexin type 9 and LDL receptor expression by the treatment with the GPER agonist, G1. In the discovery cohort, GPER P16L genotype was associated with a significant increase in LDL cholesterol (mean±SEM): 3.18±0.05, 3.25±0.08, and 4.25±0.33 mmol/L, respectively, in subjects with CC (homozygous for P16), CT (heterozygotes), and TT (homozygous for L16) genotypes (P<0.05). In the validation cohort (n=339), the GPER P16L genotype was associated with a similar increase in LDL cholesterol: 2.17±0.05, 2.34±0.06, and 2.42±0.16 mmol/L, respectively, in subjects with CC, CT, and TT genotypes (P<0.05). In the human hepatic carcinoma cell line, the GPER agonist, G1, mediated a concentration-dependent increase in LDL receptor expression, blocked by either pretreatment with the GPER antagonist G15 or by shRNA-mediated GPER downregulation. G1 also mediated a GPER- and concentration-dependent decrease in proprotein convertase subtilisin kexin type 9 expression. CONCLUSIONS: GPER activation upregulates LDL receptor expression, probably at least, in part, via proprotein convertase subtilisin kexin type 9 downregulation. Furthermore, humans carrying the hypofunctional P16L genetic variant of GPER have increased plasma LDL cholesterol. In aggregate, these data suggest an important role of GPER in the regulation of LDL receptor expression and consequently LDL metabolism.

Cholinergic activity as a new target in diseases of the heart.

The autonomic nervous system is an important modulator of cardiac signaling in both health and disease. In fact, the significance of altered parasympathetic tone in cardiac disease has recently come to the forefront. Both neuronal and nonneuronal cholinergic signaling likely play a physiological role, since modulating acetylcholine (ACh) signaling from neurons or cardiomyocytes appears to have significant consequences in both health and disease. Notably, many of these effects are solely due to changes in cholinergic signaling, without altered sympathetic drive, which is known to have significant adverse effects in disease states. As such, it is likely that enhanced ACh-mediated signaling not only has direct positive effects on cardiomyocytes, but it also offsets the negative effects of hyperadrenergic tone. In this review, we discuss recent studies that implicate ACh as a major regulator of cardiac remodeling and provide support for the notion that enhancing cholinergic signaling in human patients with cardiac disease can reduce morbidity and mortality. These recent results support the idea of developing large clinical trials of strategies to increase cholinergic tone, either by stimulating the vagus or by increased availability of Ach, in heart failure.

ChAT-ChR2-EYFP mice have enhanced motor endurance but show deficits in attention and several additional cognitive domains.

Acetylcholine (ACh) is an important neuromodulator in the nervous system implicated in many forms of cognitive and motor processing. Recent studies have used bacterial artificial chromosome (BAC) transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (ChAT) promoter (ChAT-ChR2-EYFP) to dissect cholinergic circuit connectivity and function using optogenetic approaches. We report that a mouse line used for this purpose also carries several copies of the vesicular acetylcholine transporter gene (VAChT), which leads to overexpression of functional VAChT and consequently increased cholinergic tone. We demonstrate that these mice have marked improvement in motor endurance. However, they also present severe cognitive deficits, including attention deficits and dysfunction in working memory and spatial memory. These results suggest that increased VAChT expression may disrupt critical steps in information processing. Our studies demonstrate that ChAT-ChR2-EYFP mice show altered cholinergic tone that fundamentally differentiates them from wild-type mice.

PPARδ activation attenuates hepatic steatosis in Ldlr-/- mice by enhanced fat oxidation, reduced lipogenesis, and improved insulin sensitivity.

PPARδ regulates systemic lipid homeostasis and inflammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPARδ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr(-/-) mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA ß-oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPKß1(-/-) hepatocytes. However, FA oxidation was only partially reduced in AMPKß1(-/-) hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPARδ-mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPKß1(-/-) hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinflammatory M1 state, which was reversed by GW1516 intervention. Thus, PPARδ agonist treatment inhibits the progression of preestablished hepatic steatosis.

Low birth weight followed by postnatal over-nutrition in the guinea pig exposes a predominant player in the development of vascular dysfunction.

The association between intrauterine growth restriction (IUGR) and hypertension is well established, yet the interaction between IUGR and other pathogenic contributors remains ill-defined. This study examined the independent and interactive effects of fetal growth reduction resulting in low birth weight (LBW), and postnatal Western diet (WD) on vascular function. Growth reduction was induced in pregnant guinea pigs by uterine artery ablation. LBW and normal birth weight (NBW) offspring were randomly assigned to a control diet (CD) or a WD. In young adulthood, length-tension curves were generated in aortic rings and responses to methacholine (MCh) were evaluated in the carotid and aorta using wire myography. Relative to NBW/CD, aortae of NBW/WD offspring were stiffer, as determined by a leftward shift in the length-tension curve, yet the shift in the LBW/CD curve was considerably greater. Aortic stiffening was most severe in LBW/WD (slope: NBW/CD, 1.97 ± 0.04; NBW/WD, 2.16 ± 0.04; LBW/CD, 2.28 ± 0.05; LBW/WD, 2.34 ± 0.07). Maximal responses (Emax) to MCh were significantly blunted in the aorta of LBW/CD vs. NBW/CD (P < 0.05) and in LBW/WD vs. NBW/WD offspring (P < 0.05); but WD alone had no influence on MCh responses. Emax values for carotid responses to MCh were reduced in LBW/CD vs. NBW/CD (P < 0.05). Thus, aortic stiffening was influenced more by LBW than by a postnatal WD and the most severe stiffening was observed in LBW/WD offspring. In contrast, blunted endothelial responses in LBW/CD offspring were not exacerbated by WD. IUGR may have a greater independent impact on vascular function than a postnatal WD.

Nuclear 82-kDa choline acetyltransferase decreases amyloidogenic APP metabolism in neurons from APP/PS1 transgenic mice.

Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to ß-amyloid peptides (Aß), cholinergic neuron loss with decreased choline acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aß production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aß1-42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aß1-42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aß production. This decreased formation of Aß could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to increased levels of Aß. Decreasing levels of 82-kDa ChAT due to increasing age or neurodegeneration could alter the balance towards increasing Aß production, with this potentiating the decline in function of cholinergic neurons.

Cardiomyocyte-secreted acetylcholine is required for maintenance of homeostasis in the heart.

Heart activity and long-term function are regulated by the sympathetic and parasympathetic branches of the nervous system. Parasympathetic neurons have received increased attention recently because acetylcholine (ACh) has been shown to play protective roles in heart disease. However, parasympathetic innervation is sparse in the heart, raising the question of how cholinergic signaling regulates cardiomyocytes. We hypothesized that non-neuronal secretion of ACh from cardiomyocytes plays a role in cholinergic regulation of cardiac activity. To test this possibility, we eliminated secretion of ACh exclusively from cardiomyocytes by targeting the vesicular acetylcholine transporter (VAChT). We find that lack of cardiomyocyte-secreted ACh disturbs the regulation of cardiac activity and causes cardiomyocyte remodeling. Mutant mice present normal hemodynamic parameters under nonstressful conditions; however, following exercise, their heart rate response is increased. Moreover, hearts from mutant mice present increased oxidative stress, altered calcium signaling, remodeling, and hypertrophy. Hence, without cardiomyocyte-derived ACh secretion, hearts from mutant mice show signs of imbalanced autonomic activity consistent with decreased cholinergic drive. These unexpected results suggest that cardiomyocyte-derived ACh is required for maintenance of cardiac homeostasis and regulates critical signaling pathways necessary to maintain normal heart activity. We propose that this non-neuronal source of ACh boosts parasympathetic cholinergic signaling to counterbalance sympathetic activity regulating multiple aspects of heart physiology.