Genetic Disruption of KLF1 K74 SUMOylation in Hematopoietic System Promotes Healthy Longevity in Mice.

The quest for rejuvenation and prolonged lifespan through transfusion of young blood has been studied for decades with the hope of unlocking the mystery of the key substance(s) that exists in the circulating blood of juvenile organisms. However, a pivotal mediator has yet been identified. Here, atypical findings are presented that are observed in a knockin mouse model carrying a lysine to arginine substitution at residue 74 of Krüppel-like factor 1 (KLF1/EKLF), the SUMOylation-deficient Klf1K74R/K74R mouse, that displayed significant improvement in geriatric disorders and lifespan extension. Klf1K74R/K74R mice exhibit a marked delay in age-related physical performance decline and disease progression as evidenced by physiological and pathological examinations. Furthermore, the KLF1(K74R) knockin affects a subset of lymphoid lineage cells; the abundance of tumor infiltrating effector CD8+ T cells and NKT cells is increased resulting in antitumor immune enhancement in response to tumor cell administration. Significantly, infusion of hematopoietic stem cells (HSCs) from Klf1K74R/K74R mice extends the lifespan of the wild-type mice. The Klf1K74R/K74R mice appear to be an ideal animal model system for further understanding of the molecular/cellular basis of aging and development of new strategies for antiaging and prevention/treatment of age-related diseases thus extending the healthspan as well as lifespan.

Secreted Neutrophil Gelatinase-Associated Lipocalin Shows Stronger Ability to Inhibit Cyst Enlargement of ADPKD Cells Compared with Nonsecreted Form.

Polycystic kidney disease (PKD) is one of the most common inherited diseases and is characterized by the development of fluid-filled cysts along multiple segments of the nephron. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of PKD, which is caused by mutations in either PKD1 or PKD2 genes that encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. As ADPKD progresses, cysts enlarge and disrupt normal kidney architecture, eventually leading to kidney failure. Our previous study showed that overexpression of exogenous kidney-specific neutrophil gelatinase-associated lipocalin (NGAL) reduced cyst progression and prolonged the lifespan of ADPKD mice (Pkd1L3/L3, 2L3 for short). In this study, we attempted to explore the underlying mechanism of reduced cyst progression in the presence of NGAL using immortalized 2L3 cells. The results of MTT and BrdU incorporation assays showed that recombinant mouse NGAL (mNGAL) protein significantly decreased the viability and proliferation of 2L3 cells. Flow cytometry and western blot analyses showed that mNGAL inhibited activation of the ERK and AKT pathways and induced apoptosis and autophagy in 2L3 cells. In addition, a 3D cell culture platform was established to identify cyst progression in 2L3 cells and showed that mNGAL significantly inhibited cyst enlargement in 2L3 cells. Overexpression of secreted mNGAL (pN + LS) and nonsecreted mNGAL (pN - LS) repressed cell proliferation and cyst enlargement in 2L3 cells and had effects on markers involved in proliferation, apoptosis, and autophagy. However, secreted mNGAL had a more pronounced and consistent effect than that of nonsecreted form. These results reveal that secreted mNGAL has stronger ability to inhibit cyst enlargement of ADPKD cells than that of nonsecreted form. These findings could help to identify strategies for the future clinical treatment of ADPKD.

Large deletion of Wdr19 in developing renal tubules disrupts primary ciliogenesis, leading to polycystic kidney disease in mice.

WD repeat domain 19 (Wdr19) is a major component of the intraflagellar transport (IFT) machinery, which is involved in the function of primary cilia. However, the effects of Wdr19 on primary cilia formation, cystogenesis, and polycystic kidney disease (PKD) progression remain unclear. To study these effects, we generated three lines of kidney-specific conditional knockout mice: Wdr19-knockout (Wdr19-KO, Wdr19f/- ::Cdh16-CreTg/0 ), Pkd1-knockout (Pkd1-KO, Pkd1f/- ::Cdh16-CreTg/0 ), and Wdr19/Pkd1-double knockout (Wdr19&Pkd1-dKO, Wdr19f/- ;Pkd1f/- ::Cdh16-CreTg/0 ) mice. Ultrastructural analysis using transmission electron microscopy (TEM) indicated that the primary cilia were almost absent at postnatal day 10 in Wdr19-KO mice compared with Pkd1-KO and wild-type (WT) mice. However, the primary cilia appeared structurally normal even if malfunctional in Pkd1-deficient cysts. The Pkd1-KO mice had the most severe PKD progression, including the shortest lifespan (14 days) and the largest renal cysts, among the three knockout lines. Thus, the molecular mechanism of renal cystogenesis in Wdr19-KO mice (primary cilia abrogation) was different from that in Pkd1-KO mice (primary cilia malfunction). In summary, Wdr19 deficiency leads to primary cilia abrogation and renal cyst formation. Wdr19 is primarily proposed to participate in retrograde IFT and to be crucial for the construction of primary cilia, which are critical organelles for tubulogenesis in the developing kidneys. © 2022 The Pathological Society of Great Britain and Ireland.

Pdia4 regulates ß-cell pathogenesis in diabetes: molecular mechanism and targeted therapy.

Loss of ß-cell number and function is a hallmark of diabetes. ß-cell preservation is emerging as a promising strategy to treat and reverse diabetes. Here, we first found that Pdia4 was primarily expressed in ß-cells. This expression was up-regulated in ß-cells and blood of mice in response to excess nutrients. Ablation of Pdia4 alleviated diabetes as shown by reduced islet destruction, blood glucose and HbA1c, reactive oxygen species (ROS), and increased insulin secretion in diabetic mice. Strikingly, this ablation alone or in combination with food reduction could fully reverse diabetes. Conversely, overexpression of Pdia4 had the opposite pathophysiological outcomes in the mice. In addition, Pdia4 positively regulated ß-cell death, dysfunction, and ROS production. Mechanistic studies demonstrated that Pdia4 increased ROS content in ß-cells via its action on the pathway of Ndufs3 and p22phox . Finally, we found that 2-ß-D-glucopyranosyloxy1-hydroxytrideca 5,7,9,11-tetrayne (GHTT), a Pdia4 inhibitor, suppressed diabetic development in diabetic mice. These findings characterize Pdia4 as a crucial regulator of ß-cell pathogenesis and diabetes, suggesting Pdia4 is a novel therapeutic and diagnostic target of diabetes.

Endothelial-specific insulin receptor substrate-1 overexpression worsens neonatal hypoxic-ischemic brain injury via mTOR-mediated tight junction disassembly.

Hypoxic-ischemic (HI) encephalopathy is the major cause of mortality and disability in newborns. The neurovascular unit is a major target of acute and chronic brain injury, and therapies that protect simultaneously both neurons and vascular endothelial cells from neonatal HI injury are in demand. Insulin receptors and its key downstream molecule-insulin receptor substrate -1 (IRS-1) are potential neuroprotective targets and expressed both in neuron and endothelial cells. To investigate whether IRS-1 can act similarly in neurons and vascular endothelial cells in protecting neurovascular units and brain form HI injury, we found that neuron-specific IRS-1 transgenic rats showed reduced neurovascular injury and infarct volumes, whereas endothelial-specific IRS-1 transgenic rats showed increased blood-brain barrier (BBB) disruption and exaggerated neurovascular injury after neonatal HI brain injury. Endothelial-specific IRS-1 overexpression increased vascular permeability and disassembled the tight junction protein (zonula occludens-1) complex. Inhibition of mammalian target of rapamycin (mTOR) by rapamycin preserved tight junction proteins and attenuated BBB leakage and neuronal apoptosis after HI in the endothelial-specific IRS-1 transgenic pups. Together, our findings suggested that neuronal and endothelial IRS-1 had opposite effects on the neurovascular integrity and damage after neonatal HI brain injury and that endothelial IRS-1 worsens neurovascular integrity after HI via mTOR-mediated tight junction protein disassembly.

Establishing F1A-CreERT2 Mice to Trace Fgf1 Expression in Adult Mouse Cardiomyocytes.

Fibroblast growth factor 1 (FGF1) regulates many biological and physiological processes. In mice, Fgf1 gene contains at least three upstream promoters and are alternatively spliced to the first protein coding exon, giving rise to different Fgf1 mRNA variants (1A, 1B and 1G). Among them, the Fgf1A transcript is predominantly expressed in the heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line using the Fgf1A promoter (F1A) driving the expression of the inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A-CreERT2 mice, similar to that of Fgf1A mRNA. The F1A-CreERT2 mice were crossed with ROSA26 mice, and the F1 mice were analyzed. The LacZ-positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2-mediated recombination in the tissues is monitored through LacZ-positive signals, indicating the in situ localization of F1A-positive cells. Consistently, these F1A-positive cells with RFP-positive signals or LacZ-positive blue signals were co-localized with cardiomyocytes expressing cardiac troponin T, suggesting cardiomyocyte-specific activation of Fgf1A promoter. Our data suggested that the F1A-CreERT2 mouse line could be used for time-dependent and lineage tracing of Fgf1A-expressing cells in vivo.

Negative Regulation of the Differentiation of Flk2- CD34- LSK Hematopoietic Stem Cells by EKLF/KLF1.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2- CD34- Lin- Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor ß subunit (CSF2RB). As a result, Flk2- CD34- LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2- CD34- LSK-HSCs.

Kidney-based in vivo model for drug-induced nephrotoxicity testing.

The need is critical and urgent for a real-time, highly specific, and sensitive acute kidney injury biomarker. This study sought to establish a sensitive and specific Miox-NanoLuc transgenic mouse for early detection of drug-induced nephrotoxicity. We generated Miox-NanoLuc transgenic mice with kidney-specific NanoLuc overexpression. Our data showed that Miox-NanoLuc-produced luminescence was kidney-specific and had good stability at room temperature, 4 °C, - 20 °C, and repeated freeze-thaw cycles. Serum levels of BUN and creatinine were significantly increased at day 2 or 3 in cisplatin-treated mice and at day 5 in aristolochic acid (AAI)-treated mice. Particularly, the serum and urine Miox-NanoLuc luminescence levels were significantly increased at day 1 in cisplatin-treated mice and at day 3 in AAI-treated mice. Renal pathological analysis showed that the kidney sections of cisplatin-treated mice at day 5 and AAI-treated mice at day 13 showed cytolysis and marked vacuolization of tubular cells. In conclusion, we developed a new platform to early quantify drug-induced nephrotoxicity before serum BUN and creatinine levels increased and pathological tubular cell injury occurred. This model may serve as an early detection for drug- and food-induced nephrotoxicity and as an animal model to investigate tubular cell injury.

A reporter mouse for non-invasive detection of toll-like receptor ligands induced acute phase responses.

The acute phase response (APR) is a systemic first-line defense against challenges including infection, trauma, stress, and neoplasia. Alteration of acute phase protein (APP) levels in plasma is the most important change during acute phase response. C-reactive protein (CRP), which increases dramatically during inflammation onset, is an indicator of inflammation. To monitor the process of APR, we generated human CRP promoter-driven luciferase transgenic (hCRP-Luc) mice to quantify the hCRP promoter activation in vivo. The naïve female hCRP-Luc mice express low basal levels of liver bioluminescence, but the naïve male hCRP-Luc mice do not. Thus, female hCRP-Luc mice are suitable for monitoring the process of APR. The liver bioluminescence of female hCRP-Luc mice can be induced by several toll-like receptor (TLR) ligands. The expression of liver bioluminescence was highly sensitive to endotoxin stimulation in a dose-dependent manner. On-off-on bioluminescence response was noted in female hCRP-Luc mice upon two endotoxin stimulations one month apart. The LPS-induced bioluminescence of the female hCRP-Luc mice was IL-6-mediated and associated with APP alpha-1-acid glycoprotein expression. In conclusion, the female hCRP-Luc mouse is a non-invasive, sensitive and reusable reporter tool for APR.

Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Polycystic kidney disease (PKD) is characterized by the expansion of fluid-filled cysts in the kidney, which impair the function of kidney and eventually leads to end-stage renal failure. It has been previously demonstrated that transgenic overexpression of prothymosin α (ProT) induces the development of PKD; however, the underlying mechanisms remain unclear. In this study, we used a mouse PKD model that sustains kidney-specific low-expression of Pkd1 to illustrate that aberrant up-regulation of ProT occurs in cyst-lining epithelial cells, and we further developed an in vitro cystogenesis model to demonstrate that the suppression of ProT is sufficient to reduce cyst formation. Next, we found that the expression of ProT was accompanied with prominent augmentation of protein acetylation in PKD, which results in the activation of downstream signal transducer and activator of transcription (STAT) 3. The pathologic role of STAT3 in PKD has been previously reported. We determined that this molecular mechanism of protein acetylation is involved with the interaction between ProT and STAT3; consequently, it causes the deprivation of histone deacetylase 3 from the indicated protein. Conclusively, these results elucidate the significant role of ProT, including protein acetylation and STAT3 activation in PKD, which represent potential for ameliorating the disease progression of PKD.-Chen, Y.-C., Su, Y.-C., Shieh, G.-S., Su, B.-H., Su, W.-C., Huang, P.-H., Jiang, S.-T., Shiau, A.-L., Wu, C.-L. Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Targeted Delivery of Curcumin Rescues Endoplasmic Reticulum-Retained Mutant NOX2 Protein and Avoids Leukocyte Apoptosis.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by defects in the leukocyte NADP oxidase. We previously reported that sarcoplasmic/endoplasmic reticulum calcium pump (SERCA) inhibitors could be used to rescue mutant H338Y-gp91phox protein of a particular type of CGD with a CybbC1024T mutation, leading to endoplasmic reticulum (ER) retention of the mutant protein. In this study, we developed a novel mouse model with the CybbC1024T mutation on a Cybb knockout background and investigated the therapeutic effects of ER-targeted delivery of the SERCA inhibitor, curcumin, with poly(lactic-coglycolic acid) (PLGA) nanoparticles (NPs). We found that PLGA encapsulation improved the efficacy of curcumin as a SERCA inhibitor to induce ER calcium release. ER-targeting curcumin-loaded PLGA NPs reduced and delayed extracellular calcium entry and protected the cells from mitochondrial damage and apoptosis. In vivo studies showed that ER-targeting curcumin-loaded PLGA NPs treatment enhanced neutrophil gp91phox expression, ROS production and peritoneal bacterial clearance ability of the CybbC1024T transgenic Cybb -/- mice. Our findings indicate that ER-targeted delivery of curcumin not only rescues ER-retained H338Y-gp91phox protein, and hence leukocyte function, but also enhances the bioavailability and reduces cytotoxicity. Modulation of ER function by using organelle-targeted NPs may be a promising strategy to improve the therapeutic potential of curcumin as a treatment for CGD.

MST3 is involved in ENaC-mediated hypertension.

Liddle syndrome is an inherited form of human hypertension caused by increasing epithelial Na+ channel (ENaC) expression. Increased Na+ retention through ENaC with subsequent volume expansion causes hypertension. In addition to ENaC, the Na+-K+-Cl- cotransporter (NKCC) and Na+-Cl- symporter (NCC) are responsible for Na+ reabsorption in the kidneys. Several Na+ transporters are evolutionarily regulated by the Ste20 kinase family. Ste20-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 phosphorylate downstream NKCC2 and NCC to maintain Na+ and blood pressure (BP) homeostasis. Mammalian Ste20 kinase 3 (MST3) is another member of the Ste20 family. We previously reported that reduced MST3 levels were found in the kidneys in spontaneously hypertensive rats and that MST3 was involved in Na+ regulation. To determine whether MST3 is involved in BP stability through Na+ regulation, we generated a MST3 hypomorphic mutation and designated MST3+/- and MST3-/- mice to examine BP and serum Na+ and K+ concentrations. MST3-/- mice exhibited hypernatremia, hypokalemia, and hypertension. The increased ENaC in the kidney played roles in hypernatremia. The reabsorption of more Na+ promoted more K+ secretion in the kidney and caused hypokalemia. The hypernatremia and hypokalemia in MST3-/- mice were significantly reversed by the ENaC inhibitor amiloride, indicating that MST3-/- mice reabsorbed more Na+ through ENaC. Furthermore, Madin-Darby canine kidney cells stably expressing kinase-dead MST3 displayed elevated ENaC currents. Both the in vivo and in vitro results indicated that MST3 maintained Na+ homeostasis through ENaC regulation. We are the first to report that MST3 maintains BP stability through ENaC regulation.

A Novel Murine Model Expressing a Chimeric mSCARB2/hSCARB2 Receptor Is Highly Susceptible to Oral Infection with Clinical Isolates of Enterovirus 71.

Enterovirus 71 (EV71) infection is generally associated with hand-foot-and-mouth disease (HFMD) and may cause severe neurological disorders and even death. An effective murine oral infection model for studying the pathogenesis of various clinical EV71 isolates is lacking. We developed a transgenic (Tg) mouse that expresses an EV71 receptor, that is, human scavenger receptor class B member 2 (hSCARB2), in a pattern highly similar to that of endogenous murine SCARB2 (mSCARB2) protein. A FLAG-tagged SCARB2 cDNA fragment composed of exons 3 to 12 was inserted into a murine Scarb2 gene-containing bacterial artificial chromosome (BAC) clone, and the resulting transgene was used for establishment of chimeric receptor-expressing Tg mice. Tg mice intragastrically (i.g.) infected with clinical isolates of EV71 showed neurological symptoms, such as ataxia and paralysis, and fatality. There was an age-dependent decrease in susceptibility to viral infection. Pathological characteristics of the infected Tg mice resembled those of encephalomyelitis in human patients. Viral infection was accompanied by microglial activation. Clodronate treatment of the brain slices from Tg mice enhanced viral replication, while lipopolysaccharide treatment significantly inhibited it, suggesting an antiviral role for microglia during EV71 infection. Taken together, this Tg mouse provides a model that closely mimics natural infection for studying EV71 pathogenesis and for evaluating the efficacy of vaccines or other antiviral drugs.IMPORTANCE The availability of a murine model of EV71 infection is beneficial for the understanding of pathogenic mechanisms and the development and assessment of vaccines and antiviral drugs. However, the lack of a murine oral infection model thwarted the study of pathogenesis induced by clinically relevant EV71 strains that are transmitted via the oral-oral or oral-fecal route. Our Tg mice could be intragastrically infected with clinically relevant EV71 strains in an efficient way and developed neurological symptoms and pathological changes strikingly resembling those of human infection. Moreover, these mice showed an age-dependent change in susceptibility that is similar to the human case. This Tg mouse, when combined with the use of other genetically modified mice, potentially contributes to studying the relationship between developmental changes in immunity and susceptibility to virus.

Safe Nanocomposite-Mediated Efficient Delivery of MicroRNA Plasmids for Autosomal Dominant Polycystic Kidney Disease (ADPKD) Therapy.

There is currently no cure for gene mutation-caused autosomal dominant polycystic kidney disease (ADPKD). Over half of patients with ADPKD eventually develop kidney failure, requiring dialysis or kidney transplantation. Current treatment modalities for ADPKD focus on reducing morbidity and mortality from renal and extrarenal complications of the disease. MicroRNA has been shown to be useful in treating ADPKD. This study combines anti-miRNA plasmids and iron oxide/alginate nanoparticles for conjugation with antikidney antibodies. These nanocomposites can specifically target renal tubular cells, providing a potential treatment for ADPKD. Magnetic resonance imaging and in vivo imaging system results show effective targeting of renal cells. Anti-miRNA plasmids released from the nanocomposites inhibit cell proliferation and cyst formation in the PKD cellular and animal models. The results suggest the novel combination of the anti-miRNA plasmids and nanomaterials provides potential clinical implications for ADPKD treatment.

CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation.

Casein kinase 1α (CK1α), a component of the ß-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded by Csnk1a1) in skin physiology, we crossed mice harboring floxed Csnk1a1 with mice expressing K14-Cre-ERT2 to generate mice in which tamoxifen induces the deletion of Csnk1a1 exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by ß-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo after Csnk1a1 ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14-Cre-ERT2 CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte-stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn.

The type VI adenylyl cyclase protects cardiomyocytes from ß-adrenergic stress by a PKA/STAT3-dependent pathway.

BACKGROUND: The type VI adenylyl cyclase (AC6) is a main contributor of cAMP production in the heart. The amino acid (aa) sequence of AC6 is highly homologous to that of another major cardiac adenylyl cyclase, AC5, except for its N-terminus (AC6-N, aa 1-86). Activation of AC6, rather than AC5, produces cardioprotective effects against heart failure, while the underlying mechanism remains to be unveiled. Using an AC6-null (AC6-/-) mouse and a knockin mouse with AC6-N deletion (AC6 ΔN/ΔN), we aimed to investigate the cardioprotective mechanism of AC6 in the heart. METHODS: Western blot analysis and immunofluorescence staining were performed to determine the intracellular distribution of AC6, AC6-ΔN (a truncated AC6 lacking the first 86 amino acids), and STAT3 activation. Activities of AC6 and AC6-ΔN in the heart were assessed by cAMP assay. Apoptosis of cardiomyocytes were evaluated by the TUNEL assay and a propidium iodine-based survival assay. Fibrosis was examined by collagen staining. RESULTS: Immunofluorescence staining revealed that cardiac AC6 was mainly anchored on the sarcolemmal membranes, while AC6-ΔN was redistributed to the sarcoplasmic reticulum. AC6ΔN/ΔN and AC6-/- mice had more apoptotic myocytes and cardiac remodeling than WT mice in experimental models of isoproterenol (ISO)-induced myocardial injury. Adult cardiomyocytes isolated from AC6ΔN/ΔN or AC6-/- mice survived poorly after exposure to ISO, which produced no effect on WT cardiomyocytes under the condition tested. Importantly, ISO treatment induced cardiac STAT3 phosphorylation/activation in WT mice, but not in AC6ΔN/ΔN and AC6-/- mice. Pharmacological blockage of PKA-, Src-, or STAT3- pathway markedly reduced the survival of WT myocytes in the presence of ISO, but did not affect those of AC6ΔN/ΔN and AC6-/- myocytes, suggesting an important role of AC6 in mediating cardioprotective action through the activation of PKA-Src-STAT3-signaling. CONCLUSIONS: Collectively, AC6-N controls the anchorage of cardiac AC6 on the sarcolemmal membrane, which enables the coupling of AC6 with the pro-survival PKA-STAT3 pathway. Our findings may facilitate the development of novel therapies for heart failure.

Overexpression of exogenous kidney-specific Ngal attenuates progressive cyst development and prolongs lifespan in a murine model of polycystic kidney disease.

Neutrophil gelatinase-associated lipocalin (Ngal) is a biomarker for acute and chronic renal injuries, including polycystic kidney disease (PKD). However, the effect of Ngal on PKD progression remains unexplored. To study this, we generated 3 strains of mice with different expression levels of Ngal within an established PKD model (Pkd1L3/L3): Pkd1L3/L3 (with endogenous Ngal), Pkd1L3/L3; NgalTg/Tg (with endogenous and overexpression of exogenous kidney-specific Ngal) and Pkd1L3/L3; Ngal-/- mice (with Ngal deficiency). Knockout of endogenous Ngal had no effect on phenotypes, cystic progression, or survival of the PKD mice. However, the transgenic mice had a significantly longer lifespan, smaller (but not fewer) renal cysts, and less interstitial fibrosis than the mice without or with endogenous Ngal. Western-blot analyses showed significant increases in Ngal and cleaved caspase-3 and decreases in α-smooth muscle actin, hypoxia-inducible factor 1-α, pro-caspase 3, proliferating cell nuclear antigen, Akt, mammalian target of rapamycin, and S6 Kinase in the transgenic mice as compared with the other 2 strains of PKD mice. Thus, overexpression of exogenous kidney-specific Ngal reduced cystic progression and prolonged the lifespan in PKD mice, was associated with reductions in interstitial fibrosis and proliferation, and augmented apoptosis.

Deficiency of CPEB2-Confined Choline Acetyltransferase Expression in the Dorsal Motor Nucleus of Vagus Causes Hyperactivated Parasympathetic Signaling-Associated Bronchoconstriction.

Cytoplasmic polyadenylation element binding protein 2 (CPEB2) is an RNA-binding protein and translational regulator. To understand the physiological function of CPEB2, we generated CPEB2 knock-out (KO) mice and found that most died within 3 d after birth. CPEB2 is highly expressed in the brainstem, which controls vital functions, such as breathing. Whole-body plethysmography revealed that KO neonates had aberrant respiration with frequent apnea. Nevertheless, the morphology and function of the respiratory rhythm generator and diaphragm neuromuscular junctions appeared normal. We found that upregulated translation of choline acetyltransferase in the CPEB2 KO dorsal motor nucleus of vagus resulted in hyperactivation of parasympathetic signaling-induced bronchoconstriction, as evidenced by increased pulmonary acetylcholine and phosphorylated myosin light chain 2 in bronchial smooth muscles. Specific deletion of CPEB2 in cholinergic neurons sufficiently caused increased apnea in neonatal pups and airway hyper-reactivity in adult mice. Moreover, inhalation of an anticholinergic bronchodilator reduced apnea episodes in global and cholinergic CPEB2-KO mice. Together, the elevated airway constriction induced by cholinergic transmission in KO neonates may account for the respiratory defect and mortality. SIGNIFICANCE STATEMENT: This study first generated and characterized cpeb2 gene-deficient mice. CPEB2-knock-out (KO) mice are born alive but most die within 3 d after birth showing no overt defects in anatomy. We found that the KO neonates showed severe apnea and altered respiratory pattern. Such respiratory defects could be recapitulated in mice with pan-neuron-specific or cholinergic neuron-specific ablation of the cpeb2 gene. Further investigation revealed that cholinergic transmission in the KO dorsal motor nucleus of vagus was overactivated because KO mice lack CPEB2-suppressed translation of the rate-limiting enzyme in the production of acetylcholine (i.e., choline acetyltransferase). Consequently, increased parasympathetic signaling leads to hyperactivated bronchoconstriction and abnormal respiration in the KO neonates.

Early Detection of T cell Transfer-induced Autoimmune Colitis by In Vivo Imaging System.

Inflammatory bowel disease is a chronic and progressive inflammatory intestinal disease that includes two major types, namely ulcerative colitis and Crohn's disease (CD). CD is characterized by intestinal epithelial hyperplasia and inflammatory cell infiltration. Transfer of CD25-CD45RBhiCD4+ (naïve) T cells into immunodeficiency mice induces autoimmune colitis with pathological lesions similar to CD and loss of body weight 4 weeks after cell transfer. However, weight loss neither has sufficient sensitivity nor totally matches the pathological findings of CD. To establish an early and sensitive indicator of autoimmune colitis model, the transferred T cell-induced colitis mouse model was modified by transferring luciferase-expressing donor T cells and determining the colitis by in vivo imaging system (IVIS). Colitis was detected with IVIS 7-10 days before the onset of body weight loss and diarrhea. IVIS was also applied in the dexamethasone treatment trial, and was a more sensitive indicator than body weight changes. All IVIS signals were parallel to the pathological abnormalities of the gut and immunological analysis results. In summary, IVIS provides both sensitive and objective means to monitor the disease course of transferred T cell-induced CD and fulfills the 3Rs principle of humane care of laboratory animals.