KD Mice Research Articles

Aberrant cardiolipin metabolism is associated with cognitive deficiency and hippocampal alteration in tafazzin knockdown mice

Cardiolipin (CL) is a key mitochondrial phospholipid essential for mitochondrial energy production. CL is remodeled from monolysocardiolipin (MLCL) by the enzyme tafazzin (TAZ). Loss-of-function mutations in the gene which encodes TAZ results in a rare X-linked disorder called Barth Syndrome (BTHS). The mutated TAZ is unable to maintain the physiological CL:MLCL ratio, thus reducing CL levels and affecting mitochondrial function. BTHS is best known as a cardiac disease, but has been acknowledged as a multi-syndrome disorder, including cognitive deficits. Since reduced CL levels has also been reported in numerous neurodegenerative disorders, we examined how TAZ-deficiency impacts cognitive abilities, brain mitochondrial respiration and the function of hippocampal neurons and glia in TAZ knockdown (TAZ kd) mice. We have identified for the first time the profile of changes that occur in brain phospholipid content and composition of TAZ kd mice. The brain of TAZ kd mice exhibited reduced TAZ protein expression, reduced total CL levels and a 19-fold accumulation of MLCL compared to wild-type littermate controls. TAZ kd brain exhibited a markedly distinct profile of CL and MLCL molecular species. In mitochondria, the activity of complex I was significantly elevated in the monomeric and supercomplex forms with TAZ-deficiency. This corresponded with elevated mitochondrial state I respiration and attenuated spare capacity. Furthermore, the production of reactive oxygen species was significantly elevated in TAZ kd brain mitochondria. While motor function remained normal in TAZ kd mice, they showed significant memory deficiency based on novel object recognition test. These results correlated with reduced synaptophysin protein levels and derangement of the neuronal CA1 layer in hippocampus. Finally, TAZ kd mice had elevated activation of brain immune cells, microglia compared to littermate controls. Collectively, our findings demonstrate that TAZ-mediated remodeling of CL contributes significantly to the expansive distribution of CL molecular species in the brain, plays a key role in mitochondria respiratory activity, maintains normal cognitive function, and identifies the hippocampus as a potential therapeutic target for BTHS.

Fine‐tuning of hepatocyte calcium signaling and liver regeneration by the mitochondrial calcium uniporter

Calcium (Ca2+) signaling regulates a variety of cellular processes such as metabolism, cell proliferation, division and differentiation, gene transcription, stimulus‐secretion coupling and cell death. Ca2+ is able to regulate these diverse cellular processes due to the versatility of the Ca2+ signaling mechanism in terms of speed, amplitude and spatiotemporal patterning. Mitochondria form an integral part of this machinery, both as regulators and targets of Ca2+ signaling. In recent years, leading‐edge research in the field of mCa2+ (mitochondrial Ca2+) handling and its implications for cellular Ca2+ homeostasis opened up the possibility to investigate the functional implications of mCa2+ uptake in shaping the spatiotemporal dynamics of Ca2+ signaling, and regulating cellular metabolism (through activation of Ca2+ sensitive TCA cycle enzymes) and mitochondria‐mediated cell death (through mitochondrial permeability transition). mCa2+ is mediated by the mitochondrial uniporter complex, consisting of the pore forming MCU (Mitochondrial Calcium Uniporter) and regulators such as MICU1 (mitochondrial calcium uptake 1), MICU2 and EMRE (essential MCU regulator). MCU deletion abolishes mCa2+ uptake in mouse hepatocytes driven by vasopressin‐evoked cCa2+ (cytosolic Ca2+) increases. This lack of mCa2+ uptake resulted in alterations in cCa2+ dynamics, including an elevated resting [cCa2+] and a reduced cCa2+ peak and the total Ca2+ mobilization in response to vasopressin stimulation. These changes in cCa2+ dynamics could lead to alterations in downstream Ca2+ responsive signaling events. Indeed, we see suppression of AMPK activation through CAMKK2, in response to hormone‐evoked Ca2+ signals in MCU KD hepatocytes. Activation of Creb, CAMKK2, AMPKα and ERK1/2 are also more transient in the MCU KD hepatocytes compared to controls. Our previous studies have highlighted the importance of regulating MCU (through MICU1) under stress conditions involving enhanced mCa2+ entry, especially during liver regeneration following partial hepatectomy (PHx). Enhanced mCa2+ entry during liver regeneration maybe vital to cellular signaling events and increased mitochondrial ATP production. The lack of mCa2+ uptake in hepatocyte‐specific MCU KD mice resulted in decelerated kinetics of cell cycle progression after PHx reflected by the inhibition in BrdU incorporation and Ki67 upregulation at 30h post‐PHx, which recovers by 36h. Interestingly, apart from an inhibition in Cyclin D1 expression, many cell cycle regulatory proteins remained unchanged in the KD compared to WT controls. Cell signaling events such as AMPK, CAMKK2 and mTOR activation post‐PHx are altered. Gene expression analysis at 30h post‐PHx revealed massive changes in gene expression profiles between KD and control livers, especially in genes involving metabolic processes and protein synthesis in response to PHx. Overall, the lack of mCa2+ uptake affects multiple signaling and metabolic pathways in hepatocytes and impacts the kinetics of the liver regeneration response.Support or Funding InformationAA018873 and U01EB023224 (JBH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suppression of metastasis through inhibition of chitinase 3-like 1 expression by miR-125a-3p-mediated up-regulation of USF1.

Rationale: Chitinase 3-like 1 (Chi3L1) protein is up-regulated in various diseases including solid cancers. According to Genome-Wide Association Study (GWAS)/Online Mendelian Inheritance in Man (OMIM)/Differentially Expressed Gene (DEG) analyses, Chi3L1 is associated with 38 cancers, and more highly associated with cancer compared to other oncogenes such as EGFR, TNFα, etc. However, the mechanisms and pathways by which Chi3L1 is associated with cancer are not clear. In current study, we investigated the role of Chi3L1 in lung metastasis.Methods: We performed the differentially expressed gene analysis to explore the genes which are associated with Chi3L1 using the web-based platform from Biomart. We investigated the metastases in lung tissues of C57BL/6 mice injected with B16F10 melanoma following treatment with Ad-shChi3L1. We also investigated the expression of USF1 and Chi3L1 in Chi3L1 KD mice lung tissues by Western blotting and IHC. We also analyzed lung cancer cells metastases induced by Chi3L1 using migration and cell proliferation assay in human lung cancer cell lines. The involvement of miR-125a-3p in Chi3L1 regulation was determined by miRNA qPCR and luciferase reporter assay.Results: We showed that melanoma metastasis in lung tissues was significantly reduced in Chi3L1 knock-down mice, accompanied by down-regulation of MMP-9, MMP-13, VEGF, and PCNA in Chi3L1 knock-down mice lung tissue, as well as in human lung cancer cell lines. We also found that USF1 was conversely expressed against Chi3L1. USF1 was increased by knock-down of Chi3L1 in mice lung tissues, as well as in human lung cancer cell lines. In addition, knock-down of USF1 increased Chi3L1 levels in addition to augmenting metastasis cell migration and proliferation in mice model, as well as in human cancer cell lines. Moreover, in human lung tumor tissues, the expression of Chi3L1 was increased but USF1 was decreased in a stage-dependent manner. Finally, Chi3L1 expression was strongly regulated by the indirect translational suppressing activity of USF1 through induction of miR-125a-3p, a target of Chi3L1.Conclusion: Metastases in mice lung tissues and human lung cancer cell lines were decreased by KD of Chi3L1. USF1 bound to the Chi3L1 promoter, however, Chi3L1 expression was decreased by USF1, despite USF1 enhancing the transcriptional activity of Chi3L1. We found that USF1 induced miR-125a-3p levels which suppressed Chi3L1 expression. Ultimately, our results suggest that lung metastasis is suppressed by knock-down of Chi3L1 through miR-125a-3p-mediated up-regulation of USF1.

CaMKIIα expression in a mouse model of NMDAR hypofunction schizophrenia: Putative roles for IGF-1R and TLR4

Schizophrenia (SCZ) is a neuropsychiatric disorder that is linked to social behavioral deficits and other negative symptoms associated with hippocampal synaptic dysfunction. Synaptic mechanism of schizophrenia is characterized by loss of hippocampal N-Methyl-d-Aspartate Receptor (NMDAR) activity (NMDAR hypofunction) and dendritic spines. Previous studies show that genetic deletion of hippocampal synaptic regulatory calcium-calmodulin dependent kinase II alpha (CaMKIIα) cause synaptic and behavioral defects associated with schizophrenia in mice. Although CaMKIIα is involved in modulation of NMDAR activity, it is equally linked to inflammatory and neurotropin signaling in neurons. Based on these propositions, we speculate that non-neurotransmitter upstream receptors associated with neurotropic and inflammatory signaling activities of CaMKIIα may alter its synaptic function. Besides, how these receptors (i.e. inflammatory and neurotropic receptors) alter CaMKIIα function (phosphorylation) relative to hippocampal NMDAR activity in schizophrenia is poorly understood. Here, we examined the relationship between toll-like receptor (TLR4; inflammatory), insulin-like growth factor receptor 1 (IGF-1R; neurotropic) and CaMKIIα expression in the hippocampus of behaviorally deficient schizophrenic mice after we induced schizophrenia through NMDAR inhibition.Schizophrenia was induced in WT (C57BL/6) mice through intraperitoneal administration of 30mg/Kg ketamine (NMDAR antagonist) for 5days (WT/SCZ). Five days after the last ketamine treatment, wild type schizophrenic mice show deficiencies in sociability and social novelty behavior. Furthermore, there was a significant decrease in hippocampal CaMKIIα (p<0.001) and IGF-1R (p<0.001) expression when assessed through immunoblotting and confocal immunofluorescence microscopy. Additionally, WT schizophrenic mice show an increased percentage of phosphorylated CaMKIIα in addition to upregulated TLR4 signaling (TLR4, NF-κB, and MAPK/ErK) in the hippocampus. To ascertain the functional link between TLR4, IGF-1R and CaMKIIα relative to NMDAR hypofunction in schizophrenia, we created hippocampal-specific TLR4 knockdown mouse using AAV-driven Cre-lox technique (TLR4 KD). Subsequently, we inhibited NMDAR function in TLR4 KD mice in an attempt to induce schizophrenia (TLR4 KD SCZ). Interestingly, IGF-1R and CaMKIIα expressions were preserved in the TLR4 KD hippocampus after attenuation of NMDAR function. Furthermore, TLR4 KD SCZ mice showed no prominent defects in sociability and social novelty behavior when compared with the control (WT).Our results show that a sustained IGF-1R expression may preserve the synaptic activity of CaMKIIα while TLR4 signaling ablates hippocampal CaMKIIα expression in NMDAR hypofunction schizophrenia. Together, we infer that IGF-1R depletion and increased TLR4 signaling are non-neurotransmitter pro-schizophrenic cues that can reduce synaptic CaMKIIα activity in a pharmacologic mouse model of schizophrenia.

Abstract 2676: Role of the RON kinase in the regulation of the antitumor immune response in pancreatic cancer

Abstract RON is a cell-surface receptor for the macrophage-stimulating protein expressed in tissue-resident macrophages and overexpressed in multiple epithelial neoplasms including pancreatic cancer. Our laboratory recently found that RON serves to mediate pancreatic duct carcinogenesis initiated by KRAS. We demonstrated that RON overexpression in the pancreas results in more rapid local and metastatic progression ultimately associated with decreased survival. We also discovered profound changes in the immunophenotype within the tumor microenvironment associated with RON expression. We notably established that RON expression drives the polarization of tumor-associated macrophages into an alternative, pro-tumorigenic state. We hypothesize that these unique activities help to shape the immunosuppressive milieu of the pancreatic cancer microenvironment and that RON directed therapy has the potential to serve as a novel immunomodulatory strategy in this disease. The aim of this work is to define how RON signaling contributes to shaping the pancreatic cancer microenvironment. In order to further interrogate the role of RON during pancreatic carcinogenesis, we bred kinase-dead RON mice (KD) to Pdx1-cre/LSL-KRASG12D mice. We observed a significant delay in the onset of pancreatic neoplasia and a significant decrease in tumor weight. The absence of functional RON kinase markedly reduced trafficking of CD11b+ cells to the pancreas and immunophenotyping in tumors showed a clear reduction of alternatively polarized macrophages when compared to RON overexpression or KRAS mutation alone. Because RON is expressed in both epithelial and stromal cell populations, we further sought to dissect the relevance of RON function to tumor growth within these cellular compartments. We performed orthotopic injections of organoïd-3D cells derived from tumors of KPC mice (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre). These cells were injected in wild type (WT) or RON KD mice. Preliminary results show that tumor growth is impaired in KD mice, suggesting that RON activity in the host microenvironment influence tumor growth. In order to identify all immune populations regulated by RON, we will conduct an exhaustive immunophenotyping of murine pancreatic tumors. A transcriptomic analysis will then be conducted on selected cell populations to better define the molecular mechanisms associated with the RON regulation. Finally, given the effect of RON signaling on macrophage polarization, we will also dissect the effects of RON signaling from bone marrow derived myeloid cells versus resident tissue macrophages. Preliminary results and future experiments will significantly narrow the gap in our understanding of how RON signaling contributes to shaping the pancreatic cancer chimera and therefore how RON-directed therapies could be most successfully utilized to the benefit of those afflicted with this most deadly disease Citation Format: Alex Cazes, Michele L. Babicky, Jeffery Chakedis, Divya Sood, Dawn Jaquish, Andrew M. Lowy. Role of the RON kinase in the regulation of the antitumor immune response in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2676. doi:10.1158/1538-7445.AM2017-2676

Reduced Vesicular Acetylcholine Transporter favors antidepressant behaviors and modulates serotonin and dopamine in female mouse brain

Depression is extremely harmful to modern society. Despite its complex spectrum of symptoms, previous studies have mostly focused on the monaminergic system in search of pharmacological targets. However, other neurotransmitter systems have also been linked to the pathophysiology of depression. In this study, we provide evidence for a role of the cholinergic system in depressive-like behavior of female mice. We evaluated mice knockdown for the vesicular acetylcholine transporter (VAChT KD mice), which have been previously shown to exhibit reduced cholinergic transmission. Animals were subjected to the tail suspension and marble burying tests, classical paradigms to assess depressive-like behaviors and to screen for novel antidepressant drugs. In addition, brain levels of serotonin and dopamine were measured by high performance liquid chromatography. We found that female homozygous VAChT KD mice spent less time immobile during tail suspension and buried less marbles, indicating a less depressive phenotype. These differences in behavior were reverted by central, but not peripheral, acetylcholinesterase inhibition. Moreover, female homozygous VAChT KD mice exhibited higher levels of dopamine and serotonin in the striatum, and increased dopamine in the hippocampus. Our study thus shows a connection between depressive-like behaviors and the cholinergic system, and that the latter interacts with the monoaminergic system.

Efr3a Insufficiency Attenuates the Degeneration of Spiral Ganglion Neurons after Hair Cell Loss

Sensorineural hearing loss (SNHL) is caused by an irreversible impairment of cochlear hair cells and subsequent progressive degeneration of spiral ganglion neurons (SGNs). Eighty-five requiring 3 (Efr3) is a plasma membrane protein conserved from yeast to human, and knockout of Efr3a was reported to facilitate the survival of hippocampal newborn neurons in adult mice. Previously, we found Efr3a expression in the auditory neural pathway is upregulated soon after the destruction of hair cells. Here we conducted a time-course analysis of drug-caused damage to hearing ability, hair cells and SGNs in Efr3a knocking down mice (Efr3a−/+, Efr3a KD) and their wild type littermates. Functional examination showed that both groups of mice suffered from serious hearing loss with a higher level of severity in wild type (WT) mice. Morphologic observation following drugs administration showed that both WT and Efr3a KD mice went through progressive loss of hair cells and SGNs, in association with degenerative changes in the perikarya, intracellular organelles, cell body conformation in SGNs, and the changes of SGNs in WT mice were more severe than in Efr3a KD mice. These beneficial effects of Efr3a KD could be ascribed to an increase in the expression of some neurotrophic factors and their receptors in Efr3a KD mice. Our results indicate that Efr3a insufficiency suppresses drug-caused SNHL neurodegeneration in association with an increase in the expression of some neurotrophic factors and their receptors, which may be targeted in the treatment of neurodegeneration.

116.2 Discovery and Development of Novel M1 and M4 PAMs for the Treatment of Schizophrenia.

Abstract Background: Multiple lines of evidence indicate that development of selective positive allosteric modulators (PAMs) of the M1 and M4 muscarinic acetylcholine receptor subtypes (mAChR) represent important novel therapeutic approaches for the complex symptoms associated schizophrenia. Over the last decade, Dr. Jones and colleagues at the Vanderbilt Center for Neuroscience Drug Discovery (VCNDD) have disclosed the discovery of several, selective mAChR PAMs, as represented by the M1 PAM VU6004256 and M4 PAM VU0467154. This presentation will focus on recent findings demonstrating the ability of M1 and M4 PAMs to reverse abnormalities in prefrontal cortical-mediated physiology, behaviors, and/or cognitive functions observed across preclinical models of N-methyl-D-aspartate subtype of the glutamate receptor (NMDAR) hypofunction, which are thought to underlie many of the symptoms observed in schizophrenia. Methods: Medicinal chemistry and high throughput screening methods were utilized to identify two different series of selective M1 and M4 PAMs, represented by VU6004256 and VU0467154. Disposition was determined in mouse and rat pharmacokinetic studies using LC/MS/MS quantitation. In vivo efficacy was determined across several mouse and rat models predictive of antipsychotic-like activity and enhancement of cognitive function, in particular using the genetic mouse model of global reduction in the NR1 subunit of the NMDAR (NR1 knockdown [KD]). Results: In vitro both VU6004256 and VU0467154 exhibit low nanomolar potency at M1 or M4, respectively, with no activity at the other mAChR subtypes up to 30 μM or significant off target activity at other GPRCs at 10 µM. These ligands are also highly brain penetrant with suitable pharmacokinetic properties for in vivo dosing. Both VU6004256 and VU0467154 produced dose-dependent reductions in preclinical models predictive of antipsychotic-like activity, including reversal of hyperlocomotion in the NR1 KD mice. In addition, the M1 PAM VU6004256 decreased excessive pyramidal neuron firing in layer V of the prefrontal cortex (PFC) in awake, freely moving NR1 KD mice, as well as reversed impairments in two PFC-mediated learning tasks observed in these mice. Interestingly, the M4 PAM VU0467154 also enhanced performance in preclinical models of associative learning and memory functions after acute and chronic dosing and induced state-dependent alterations in sleep architecture and arousal. Conclusion: Collectively, these findings indicate that VU6004256 and VU0467154 represent highly optimized M1 and M4 PAM tool compounds for the evaluation of the respective roles of these mAChR subtypes in vivo and provide further validation for the development of selective M1 and M4 PAMs for the treatment of the psychotic symptoms and cognitive impairments observed in schizophrenia.

The role of Efr3a in age-related hearing loss

Efr3a has been found to be involved in the functional maintenance and structural degeneration of sensory and motor nervous tissues. Our previous data have suggested that Efr3a may be associated with the initiation of the degeneration of spiral ganglion neurons (SGNs). In this study, we used Efr3a knockdown (Efr3a KD) and Efr3a overexpression (Efr3a OE) mice to determine the role of Efr3a in age-related hearing loss. Measurements of hearing thresholds showed that Efr3a had little or no influence on the hearing threshold at all frequencies in adult mice, whereas in an early stage of senescence, Efr3a reduction resulted in better hearing function, especially at 10 and 12months of age. No significant differences were observed in hair cell loss among the three groups until 14months. The number of surviving hair cells in the OE mice was lower than that in the KD mice. As indicated by the density of SGNs in the upper basal turn, the Efr3a OE mice displayed earlier and more severe degeneration than the KD mice. In addition, the p-Akt levels in the cochlear spiral ganglions were higher in adult Efr3a KD mice than in WT and OE mice, although there was no difference in Akt expression among the three groups. Our study suggests that down-regulation of Efr3a might improve hearing function and alleviate the degeneration of SGNs in an early stage of senescence, probably via enhancing the basal expression of activated Akt.

Prefrontal Cortex-Mediated Impairments in a Genetic Model of NMDA Receptor Hypofunction Are Reversed by the Novel M1 PAM VU6004256.

Abnormalities in the signaling of the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) within cortical and limbic brain regions are thought to underlie many of the complex cognitive and affective symptoms observed in individuals with schizophrenia. The M1 muscarinic acetylcholine receptor (mAChR) subtype is a closely coupled signaling partner of the NMDAR. Accumulating evidence suggests that development of selective positive allosteric modulators (PAMs) of the M1 receptor represent an important treatment strategy for the potential normalization of disruptions in NMDAR signaling in patients with schizophrenia. In the present studies, we evaluated the effects of the novel and highly potent M1 PAM, VU6004256, in ameliorating selective prefrontal cortical (PFC)-mediated physiologic and cognitive abnormalities in a genetic mouse model of global reduction in the NR1 subunit of the NMDAR (NR1 knockdown [KD]). Using slice-based extracellular field potential recordings, deficits in muscarinic agonist-induced long-term depression (LTD) in layer V of the PFC in the NR1 KD mice were normalized with bath application of VU6004256. Systemic administration of VU6004256 also reduced excessive pyramidal neuron firing in layer V PFC neurons in awake, freely moving NR1 KD mice. Moreover, selective potentiation of M1 by VU6004256 reversed the performance impairments of NR1 KD mice observed in two preclinical models of PFC-mediated learning, specifically the novel object recognition and cue-mediated fear conditioning tasks. VU6004256 also produced a robust, dose-dependent reduction in the hyperlocomotor activity of NR1 KD mice. Taken together, the current findings provide further support for M1 PAMs as a novel therapeutic approach for the PFC-mediated impairments in schizophrenia.

Impaired Cardiolipin Biosynthesis Prevents Hepatic Steatosis and Diet-Induced Obesity.

Mitochondria are the nexus of energy metabolism, and consequently their dysfunction has been implicated in the development of metabolic complications and progression to insulin resistance and type 2 diabetes. The unique tetra-acyl phospholipid cardiolipin (CL) is located in the inner mitochondrial membrane, where it maintains mitochondrial integrity. Here we show that knockdown of Tafazzin (TAZ kd), a CL transacylase, in mice results in protection against the development of obesity, insulin resistance, and hepatic steatosis. We determined that hypermetabolism protected TAZ kd mice from weight gain. Unexpectedly, the large reduction of CL in the heart and skeletal muscle of TAZ kd mice was not mirrored in the liver. As a result, TAZ kd mice exhibited normal hepatic mitochondrial supercomplex formation and elevated hepatic fatty acid oxidation. Collectively, these studies identify a key role for hepatic CL remodeling in regulating susceptibility to insulin resistance and as a novel therapeutic target for diet-induced obesity.

Decreased spontaneous activity in AMPK α2 muscle specific kinase dead mice is not caused by changes in brain dopamine metabolism

It is well known that physical activity has several health benefits, yet many people do not exercise. Dopamine levels in the striatum of the brain are thought to be important for the motivation to exercise. Conversely, we hypothesized that muscle quality can affect the motivation to exercise through alterations of the brain dopamine levels specifically in the striatal region. To test this hypothesis, transgenic mice overexpressing an inactivatable dominant negative α2 AMPK construct (AMPK α2 KD) in muscles and littermate wildtype (WT) mice were tested. AMPK α2 KD mice have impaired running capacity and display reduced voluntary wheel running activity. Striatal content of dopamine and its metabolites were measured under basal physiological conditions and after cocaine-induced dopamine efflux from the ventral striatum by in vivo microdialysis. Moreover, cocaine-induced locomotor activity was tested in an open field test. Furthermore, we investigated maximal running capacity and voluntary running over a period of 19days.AMPK α2 KD mice ran 30% less in daily distance compared to WT. Furthermore, AMPK α2 KD mice showed significantly decreased locomotor activity in the open field test compared to WT when treated with saline or cocaine, respectively, but the increase induced by cocaine was similar in AMPK α2 KD and WT mice. The efflux of dopamine in ventral striatum after cocaine treatment increased similarly by 2.5-fold in the two genotypes, and basal levels of dopamine and its metabolites DOPAC and HVA were also similar between genotypes. These findings show that decreased AMPK activity in muscle leads to decreased voluntary activity which is not due to secondary abnormalities in dopamine levels in the ventral striatum or sensitivity to cocaine. Thus, decreased voluntary activity in AMPK muscle deficient mice is most likely unrelated to regulation of brain dopamine content and metabolism.

NRF2 Intensifies Host Defense Systems to Prevent Lung Carcinogenesis, but After Tumor Initiation Accelerates Malignant Cell Growth.

Nrf2 activation promotes resistance to chemical carcinogenesis in animal models, but activating mutations in Nrf2 also confer malignant characters to human cells by activating antioxidative/detoxifying enzymes and metabolic reprogramming. In this study, we examined how these contradictory activities of Nrf2, cancer chemoprevention and cancer cell growth enhancement, can be reconciled in an established mouse model of urethane-induced lung carcinogenesis. Using Keap1-knockdown (kd) mice, which express high levels of Nrf2, we found that urethane was rapidly excreted into the urine, consistent with an upregulation in the expression of urethane detoxification genes. Consequently, urethane-induced tumors were significantly smaller and less frequent in Keap1-kd mice than in wild-type mice. In contrast, tumor cells derived from Keap1-kd mice and transplanted into nude mice exhibited higher tumorigenicity compared with cells derived from wild-type mice. To identify the factors contributing to the tumor growth phenotype in the transplantation model, we performed a microarray analysis and found that many antioxidative stress genes were upregulated in the Keap1-kd-derived tumors. Therefore, we suggest that Nrf2 activation in cancer cells enhances their tumorigenicity, but global Nrf2 activation, as in Keap1-kd mice, simultaneously enhances anticancer immunity, thereby suppressing the growth potential of Keap1-kd tumors. Our findings provide relevant insight into the dual role of Nrf2 in cancer and warrant further studies of Nrf2 function during different stages of carcinogenesis. Cancer Res; 76(10); 3088-96. ©2016 AACR.

Lifespan and reproduction in brain-specific miR-29-knockdown mouse

The microRNA miR-29 is widely distributed and highly expressed in adult mouse brain during the mouse's lifetime. We recently created conditional mutant mice whose miR-29 was brain-specifically knocked down through overexpression of an antisense RNA transgene against miR-29. To explore a role for brain miR-29 in maximizing organismal fitness, we assessed somatic growth, reproduction, and lifespan in the miR-29-knockdown (KD) mice and their wild-type (WT) littermates. The KD mice were developmentally indistinguishable from WT mice with respect to gross morphology and physical activity. Fertility testing revealed that KD males were subfertile, whereas KD females were hyperfertile, only in terms of reproductive success, when compared to their gender-matched WT correspondents. Another phenotypic difference between KD and WT animals appeared in their lifespan data; KD males displayed an overall increasing tendency in post-reproductive survival relative to WT males. In contrast, KD females were prone to shorter lifespans than WT females. These results clarify that brain-targeted miR-29 knockdown affects both lifespan and reproduction in a gender-dependent manner, and moreover that the reciprocal responsiveness to the miR-29 knockdown between these two phenotypes in both genders closely follow life-course models based on the classical trade-off prediction wherein elaborate early-life energetic investment in reproduction entails accelerated late-life declines in survival, and vice versa. Thus, this study identified miR-29 as the first mammalian miRNA that is directly implicated in the lifetime trade-off between the two major fitness components, lifespan and reproduction.

Abstract 16267: NADPH Oxidase Nox4 Up-regulation Contributes to the Worsening of Pressure Overload-induced Cardiac Dysfunction in Angiopoeitin-like 2 Knockdown Mice

Background: Angiopoietin-like 2 (angptl2) is a circulating protein promoting vascular inflammation and endothelial dysfunction in mice, but little is known on its impact on cardiac function. Our previous results suggest that knocking down angptl2 in mice (KD) worsens pressure overload-induced cardiac dysfunction while preserving cerebral artery structure and endothelial function. As NADPH oxidase NOX4 is known to produce H2O2, a deleterious hypertrophic stimulus in cardiomyocytes but also a vasodilatory factor, we hypothesized that increased expression of NOX4 contributes to the aggravated cardiac dysfunction observed in KD mice. Methods/Results: Cardiac function was measured in vivo by Millar catheter in KD mice versus their wild-type (WT) littermates in response to a 6-week pressure overload induced by transverse aortic constriction (TAC). Concomitantly to a worsened cardiac remodeling and an aggravated cardiac dysfunction compared to WT-TAC mice, only KD-TAC mice displayed an increase in cardiac mRNA and protein expression of NOX4 (p&lt;0.05 vs. WT, n=8). To specifically decrease cardiac NOX4 expression, we performed, two weeks after TAC surgery, a single i.v. injection of cardiac specific associated adenovirus AAV9 expressing a NOX4-targeted shRNA or a scrambled shRNA. In KD-TAC mice, the AAV9-shNOX4 limited cardiac hypertrophy as evidenced by a smaller heart/tibia ratio (Table, * p&lt;0.05). Moreover, cardiac function in KD-TAC-AAV9-shNOX4 mice was partly prevented by maintaining contraction and relaxation maximal velocities and by limiting the abnormal rise of minimal pressure and end diastolic pressure caused by TAC (Table, * p&lt;0.05). Conclusion: Angptl2 knockdown worsens cardiac hypertrophy and contractile dysfunction induced by pressure overload: cardiac up-regulation of NOX4 could contribute to these deleterious effects in angptl2 KD mice.

Autonomic cardiocirculatory control in mice with reduced expression of the vesicular acetylcholine transporter.

In cardiovascular diseases, sympathetic tone has been comprehensively studied, whereas parasympathetic tone has received minor attention. The vesicular ACh transporter (VAChT) knockdown homozygous (VAChT KD(HOM)) mouse is a useful model for examining the cardiocirculatory sympathovagal balance. Therefore, we investigated whether cholinergic dysfunction caused by reduced VAChT expression could adversely impact hemodynamic parameter [arterial pressure (AP) and heart rate (HR)] daily oscillation, baroreflex sensitivity, hemodynamic variability, sympathovagal balance, and cardiovascular reactivity to restraint stress. Wild-type and VAChT KD(HOM) mice were anesthetized for telemetry transmitter implantation, and APs and HRs were recorded 10 days after surgical recovery. Changes in HR elicited by methylatropine and propranolol provided the indexes of sympathovagal tone. Cardiovascular reactivity in response to a restraint test was examined 24 h after continuous recordings of AP and HR. VAChT KD(HOM) mice exhibited reduced parasympathetic and elevated sympathetic tone. Daily oscillations of AP and HR as well as AP variability were similar between groups. Nevertheless, HR variability, patterns with two dissimilar variations from symbolic analysis, and baroreflex sensitivity were reduced in VAChT KD(HOM) mice. The change in mean AP due to restraint stress was greater in VAChT KD(HOM) mice, whereas the tachycardic response was not. These findings demonstrate that the cholinergic dysfunction present in the VAChT KD(HOM) mouse did not adversely impact basal hemodynamic parameters but promoted autonomic imbalance, an attenuation of baroreflex sensitivity, and a greater pressure response to restraint stress. These results provide a framework for understanding how autonomic imbalance impacts cardiovascular function.

0159 : Increased NOX4 expression in mice knockdown of angiopoietin-like 2 worsens pressure overload-induced cardiac dysfunction but preserves vascular endothelial integrity

Angiopoietin-like 2 (angptl2) is a pro-inflammatory and pro-oxidative protein that induces endothelial dysfunction in mice. The impact of angptl2 on cardiac function is still unknown. We hypothesized that angptl2 could contribute to cardiac dysfunction and that knocking-down angptl2 would be protective against pressure overload. We investigated both cardiac and vascular endothelial functions in angptl2 knockdown mice (KD) versus wild-type (WT) littermates, in response to a 6-week pressure overload induced by transverse aortic constriction (TAC). TAC increased systolic pressure in the right carotid artery by 60% in WT, but only 28% in KD mice. In TAC-WT, but not in -KD mice, carotid and posterior cerebral arteries isolated from the highpressure right side displayed increased wall thickness, a remodeling associated with endothelial dysfunction. In contrast, and contrary to our hypothesis, TAC induced a more severe cardiac remodeling in KD than in WT mice: TAC-KD mice displayed a greater heart weight / tibia length ratio, as well as a higher gene expression of hypertrophic remodeling molecular markers, (ANP, BNP, MyoHβ/MyoHα) when compared to TAC-WT mice. Cardiac function measured by Millar catheter also showed greater alteration of the left ventricular relaxation in TAC-KD mice (increased minimal and end diastolic pressures, 25% reduced relaxation rate), suggesting cardiac contractile dysfunction. Finally, we observed, only in hearts from TAC-KD mice, an increase in mRNA and protein expression of NOX4 which is known to produce H 2 O 2 , a deleterious hypertrophic stimulus in cardiomyocytes, but is a vasodilatory factor. This is the first demonstration that angptl2 knockdown paradoxically worsens cardiac hypertrophy and contractile dysfunction induced by pressure overload, contrasting with the preserved arterial wall structure and endothelial integrity. Up-regulation of NOX4 could, at least partly, contribute to these opposite effects in angptl2 KD mice.

AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD.

The mitochondrial protein deacetylase sirtuin (SIRT) 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS) handling. We determined the requirement of AMP-activated protein kinase (AMPK) for exercise training-induced increases in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p < 0.01) and superoxide dismutase 2 (MnSOD; p < 0.05) protein abundance in quadriceps muscle of wild-type (WT; n = 13–15), but not AMPK α2 kinase dead (KD; n = 12–13) mice. We also observed a strong trend for increased MnSOD abundance in exercise-trained skeletal muscle of healthy humans (p = 0.051; n = 6). To further elucidate a role for AMPK in mediating these effects, we treated WT (n = 7–8) and AMPK α2 KD (n = 7–9) mice with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). Four weeks of daily AICAR injections (500 mg/kg) resulted in AMPK-dependent increases in SIRT3 (p < 0.05) and MnSOD (p < 0.01) in WT, but not AMPK α2 KD mice. We also tested the effect of repeated AICAR treatment on mitochondrial protein levels in mice lacking the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α KO; n = 9–10). Skeletal muscle SIRT3 and MnSOD protein abundance was reduced in sedentary PGC-1α KO mice (p < 0.01) and AICAR-induced increases in SIRT3 and MnSOD protein abundance was only observed in WT mice (p < 0.05). Finally, the acetylation status of SIRT3 target lysine residues on MnSOD (K122) or oligomycin-sensitivity conferring protein (OSCP; K139) was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling in skeletal muscle in response to exercise training.

Loss of PI3K-Gamma Scaffold Function causes Severe Electrical Remodeling in Mice Ventricular Myocytes

In the heart the kinase/scaffold protein phosphoinositide-3-kinase-γ (PI3Kγ) has been associated with maladaptive remodeling. However little is known about the relative contribution of kinase-scaffold roles on the normal electrical activity in cardiac cells.Here, we compared the action potentials and ionic currents in ventricular myocytes isolated from conventional KO mice or mice expressing a catalytically inactive PI3K (“kinase-dead”, KD). Action potentials were evoked at 1Hz (I-clamp). K+ and L-type Ca2+ (ICaL) currents were measured by step depolarizations (V-clamp).Membrane capacitances (Cm) were not different between genotypes. KO myocytes showed marked prolongation of the action potential duration (APD) at 90% of repolarization (APD90: WT 64.36±4.1 ms, KO 149.6±18 ms). APD90 beat-to-beat variability (BVR) was larger in KO and, consistently, the incidence of early afterdepolarizations was higher in KO than WT (respectively, 66% and 18%). In contrast, KD cells showed a slight prolongation of APD90 (96±7.9 ms), but BVR and EADs incidence were comparable to WT. While voltage-gated K+ currents (Ito, IKur and Iss) were strongly downregulated in KO, KD myocytes showed only a small reduction of Ito. ICaL density was significantly lower in both KO and KD mice at negative potentials only. Consistently, both KD and KO mice showed a positive shift of both steady-state activation (ΔV1/2 vs WT: KO 4.2 mV, KD 5.5 mV) and inactivation curves (ΔV1/2 vs WT: KO 3.2 mV, KD 3.4 mV). Addition of isoproterenol (100 nM) resulted in ICaL enhancement, whose magnitude was larger in KO compared to KD and WT myocytes (% increase,WT: 58±6%,KD:45±7%,KO:95.5±12%). These data suggest distinct action related to either kinase and scaffold functions: while the former has an impact on ICaL biophysical properties, the latter prevents arrhythmias in murine ventricular myocytes by preserving K+ currents.

Identification of miR-126 knockdown mouse and the change of blood glucose

To detect the expression of miR-126 in different tissues and organs and the change of peripheral blood glucose in microRNA-126 knock down (miR-126 KD) mouse, and to explore the pathological significance. Total RNAs were isolated from twelve kinds of tissues and organs in wild-type mouse (WT) and miR-126 KD mouse respectively. Th en, the expression level of miR-126 was detected by real-time PCR assay. Th e levels of peripheral blood glucose and body weight of miR-126 KD mice were measured. Th e pathologic changes of pancreas and lung tissue were observed by HE staining. Compared with the WT mice, the relative expression of miR-126 in spleen, liver, muscle and lung from the miR-126 KD mice were dramatically decreased respectively (P<0.05). The level of peripheral blood glucose in the miR-126 KD mouse increased significantly at seven week and sixteen week after the birth (P<0.05). HE staining showed that the pathological structure of pancreas and liver were abnormal. The body weight of miR-126 KD mice was increased obviously from thirteen week after birth (P<0.05). Peripheral blood glucose levels in the miR-126 KD mouse were dramatically elevated, which might be related to the pathological changes in the structure of pancreas and liver. These results suggest that miR-126 may play an important role in the metabolism of blood glucose and the development of type 2 diabetes mellitus.