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1.  Safety evaluation and pharmacokinetics of A novel human tumor necrosis factor-alpha exhibited a higher anti-tumor activity and a lower systemic toxicity 
Anti-cancer drugs  2010;21(3):243-251.
We have previously prepared a prokaryotic expressed TNF-α mutant which exhibited a higher anti-tumor activity and a lower systemic toxicity compared with that of wild type TNF-α in both syngeneic murine tumor models and human tumor xenografts models. For its clinical use as an anti-tumor agent, we evaluate repeated dose toxicity, anaphylaxis, genetic toxicity, pharmacokinetic and metabolism in different animals according to the criteria for biological Investigational New Drug (IND) application. It was found to be safe at a dose of 4×106 IU/kg/day for 60 days after administration in rhesus monkeys but the TNF-α antibody level and liver toxicity needed to be monitored. No systemic anaphylaxis or genetic toxicity were found and the pharmacokinetic characteristics of the rmhTNF-α were suited for clinical use. Over 96.3% of rmhTNF-α could be reclaimed from the urine and feces in 24 hours after administration, which indicated the main excretion route. The results proved the characteristics of this rmhTNF-α satisfied clinical trial requirements. The related positive clinical trial results will be reported in future. This study of novel rmhTNF-α is of considerable importance, not only given the proven usefulness of TNF-α local application therapies under ILP (Isolated Limp Perfusion) and IHP (Isolated Hepatic Perfusion) conditions for selected indications, but also implicated for systemic application of TNF-α.
PMCID: PMC4266396  PMID: 20166241
Tumor necrosis factor-alpha; Mutation; Cancer therapy; Safety evaluation; Systemic toxicity; Pharmacokinetic
2.  Triptolide treatment reduces Alzheimer’s disease (AD)-like pathology through inhibition of BACE1 in a transgenic mouse model of AD 
Disease Models & Mechanisms  2014;7(12):1385-1395.
The complex pathogenesis of Alzheimer’s disease (AD) involves multiple contributing factors, including amyloid β (Aβ) peptide accumulation, inflammation and oxidative stress. Effective therapeutic strategies for AD are still urgently needed. Triptolide is the major active compound extracted from Tripterygium wilfordii Hook.f., a traditional Chinese medicinal herb that is commonly used to treat inflammatory diseases. The 5-month-old 5XFAD mice, which carry five familial AD mutations in the β-amyloid precursor protein (APP) and presenilin-1 (PS1) genes, were treated with triptolide for 8 weeks. We observed enhanced spatial learning performances, and attenuated Aβ production and deposition in the brain. Triptolide also inhibited the processing of amyloidogenic APP, as well as the expression of βAPP-cleaving enzyme-1 (BACE1) both in vivo and in vitro. In addition, triptolide exerted anti-inflammatory and anti-oxidative effects on the transgenic mouse brain. Triptolide therefore confers protection against the effects of AD in our mouse model and is emerging as a promising therapeutic candidate drug for AD.
PMCID: PMC4257007  PMID: 25481013
Alzheimer’s disease; Amyloid β; 5XFAD mice; BACE1; Inflammation; Triptolide
3.  The Stent-Assisted Coil-Jailing Technique Facilitates Efficient Embolization of Tiny Cerebral Aneurysms 
Korean Journal of Radiology  2014;15(6):850-857.
Tiny cerebral aneurysms are difficult to embolize because the aneurysm's sac is too small for a single small coil, and coils within the aneurysm may escape from the confinement of a stent. This study was performed to introduce the stent-assisted coil-jailing technique and to investigate its effect on the coil embolization of tiny intracranial aneurysms.
Materials and Methods
Sixteen patients with tiny intracranial aneurysms treated with the stent-assisted coil-jailing technique between January 2011 and December 2013 were retrospectively reviewed and followed-up.
All aneurysms were successfully treated with the coil-jailing technique, and at the end of embolization, complete occlusion of the aneurysm was achieved in 9 cases (56.3%), incomplete occlusion in 6 (37.5%), and partial occlusion in 1 (6.3%). Intraprocedural complications included acute thrombosis in one case (6.3%) and re-rupture in another (6.3%). Both complications were managed appropriately with no sequela. Follow-up was performed in all patients for 3-24 months (mean, 7.7 months) after embolization. Complete occlusion was sustained in the 9 aneurysms with initial complete occlusion, progressive thrombosis to complete occlusion occurred in the 6 aneurysms with initial near-complete occlusion, and one aneurysm resulted in progressive thrombosis to complete occlusion after initial partial occlusion. No migration of stents or coils occurred at follow-up as compared with their positions immediately after embolization. At follow-up, all patients had recovered with no sequela.
The stent-assisted coil-jailing technique can be an efficient approach for tiny intracranial aneurysms, even though no definite conclusion regarding its safety can be drawn from the current data.
PMCID: PMC4248643  PMID: 25469099
Tiny intracranial aneurysm; Stent-assisted coiling; Redundant coil tails; Coil migration
4.  SIRT1-mediated epigenetic downregulation of plasminogen activator inhibitor-1 prevents vascular endothelial replicative senescence 
Aging Cell  2014;13(5):890-899.
The inactivation of plasminogen activator inhibitor-1 (PAI-1) has been shown to exert beneficial effects in age-related vascular diseases. Limited information is available on the molecular mechanisms regarding the negatively regulated expression of PAI-1 in the vascular system. In this study, we observed an inverse correlation between SIRT1, a class III histone deacetylase, and PAI-1 expression in human atherosclerotic plaques and the aortas of old mice, suggesting that internal negative regulation exists between SIRT1 and PAI-1. SIRT1 overexpression reversed the increased PAI-1 expression in senescent human umbilical vein endothelial cells (HUVECs) and aortas of old mice, accompanied by decreased SA-β-gal activity in vitro and improved endothelial function and reduced arterial stiffness in vivo. Moreover, the SIRT1-mediated inhibition of PAI-1 expression exerted an antisenescence effect in HUVECs. Furthermore, we demonstrated that SIRT1 is able to bind to the PAI-1 promoter, resulting in a decrease in the acetylation of histone H4 lysine 16 (H4K16) on the PAI-1 promoter region. Thus, our findings suggest that the SIRT1-mediated epigenetic inhibition of PAI-1 expression exerts a protective effect in vascular endothelial senescence.
PMCID: PMC4331759  PMID: 25040736
atherosclerosis; endothelial replicative senescence; epigenetic; PAI-1; H4K16 acetylation; SIRT1
5.  Protein Phosphatase 2A Catalytic Subunit α (PP2Ac) Plays a MyD88-dependent, Central Role in the Gene-Specific Regulation of Endotoxin Tolerance (ET) 
Cell reports  2013;3(3):678-688.
MyD88, the intracellular adaptor of most TLRs, mediates either pro-inflammatory or immunosuppressive signaling that contributes to chronic inflammation-associated diseases. Although gene-specific chromatin modifications regulate inflammation, the role of MyD88 signaling in establishing such epigenetic landscapes under different inflammatory states remains elusive. Using quantitative proteomics to enumerate the inflammation-phenotypic constituents of MyD88 interactome, we found that in endotoxin-tolerant macrophages PP2Ac enhances its association with MyD88, and is constitutively activated. Knockdown of PP2Ac prevents suppression of pro-inflammatory genes and resistance to apoptosis. Through sitespecific dephosphorylation constitutively active PP2Ac disrupts the signal-promoting TLR4-MyD88 complex, and broadly suppresses the activities of multiple pro-inflammatory/proapoptotic pathways as well, shifting pro-inflammatory MyD88 signaling to a pro-survival mode. Constitutively active PP2Ac translocated with MyD88 into the nuclei of tolerant macrophages establishes the immunosuppressive pattern of chromatin modifications and represses chromatin remodeling to selectively silence pro-inflammatory genes, coordinating the MyD88-dependent inflammation control at both signaling and epigenetic levels under endotoxin-tolerant conditions.
PMCID: PMC4060247  PMID: 23434512
6.  The Involvement of NFAT Transcriptional Activity Suppression in SIRT1-Mediated Inhibition of COX-2 Expression Induced by PMA/Ionomycin 
PLoS ONE  2014;9(5):e97999.
SIRT1, a class III histone deacetylase, acts as a negative regulator for many transcription factors, and plays protective roles in inflammation and atherosclerosis. Transcription factor nuclear factor of activated T cells (NFAT) has been previously shown to play pro-inflammatory roles in endothelial cells. Inhibition of NFAT signaling may be an attractive target to regulate inflammation in atherosclerosis. However, whether NFAT transcriptional activity is suppressed by SIRT1 remains unknown. In this study, we found that SIRT1 suppressed NFAT-mediated transcriptional activity. SIRT1 interacted with NFAT, and the NHR and RHR domains of NFAT mediated the interaction with SIRT1. Moreover, we found that SIRT1 primarily deacetylated NFATc3. Adenoviral over-expression of SIRT1 suppressed PMA and calcium ionophore Ionomycin (PMA/Io)-induced COX-2 expression in human umbilical vein endothelial cells (HUVECs), while SIRT1 RNAi reversed the effects in HUVECs. Moreover, inhibition of COX-2 expression by SIRT1 in PMA/Io-treated HUVECs was largely abrogated by inhibiting NFAT activation. Furthermore, SIRT1 inhibited NFAT-induced COX-2 promoter activity, and reduced NFAT binding to the COX-2 promoter in PMA/Io-treated HUVECs. These results suggest that suppression of NFAT transcriptional activity is involved in SIRT1-mediated inhibition of COX-2 expression induced by PMA/Io, and that the negative regulatory mechanisms of NFAT by SIRT1 may contribute to its anti-inflammatory effects in atherosclerosis.
PMCID: PMC4032329  PMID: 24859347
7.  Shank3 Deficiency Induces NMDA Receptor Hypofunction via an Actin-Dependent Mechanism 
The Journal of Neuroscience  2013;33(40):15767-15778.
Shank3, which encodes a scaffolding protein at glutamatergic synapses, is a genetic risk factor for autism. In this study, we examined the impact of Shank3 deficiency on the NMDA-type glutamate receptor, a key player in cognition and mental illnesses. We found that knockdown of Shank3 with a small interfering RNA (siRNA) caused a significant reduction of NMDAR-mediated ionic or synaptic current, as well as the surface expression of NR1 subunits, in rat cortical cultures. The effect of Shank3 siRNA on NMDAR currents was blocked by an actin stabilizer, and was occluded by an actin destabilizer, suggesting the involvement of actin cytoskeleton. Since actin dynamics is regulated by the GTPase Rac1 and downstream effector p21-activated kinase (PAK), we further examined Shank3 regulation of NMDARs when Rac1 or PAK was manipulated. We found that the reducing effect of Shank3 siRNA on NMDAR currents was mimicked and occluded by specific inhibitors for Rac1 or PAK, and was blocked by constitutively active Rac1 or PAK. Immunocytochemical data showed a strong reduction of F-actin clusters after Shank3 knockdown, which was occluded by a PAK inhibitor. Inhibiting cofilin, the primary downstream target of PAK and a major actin depolymerizing factor, prevented Shank3 siRNA from reducing NMDAR currents and F-actin clusters. Together, these results suggest that Shank3 deficiency induces NMDAR hypofunction by interfering with the Rac1/PAK/cofilin/actin signaling, leading to the loss of NMDAR membrane delivery or stability. It provides a potential mechanism for the role of Shank3 in cognitive deficit in autism.
PMCID: PMC3787498  PMID: 24089484
8.  The Ability of BDNF to Modify Neurogenesis and Depressive-Like Behaviors Is Dependent upon Phosphorylation of Tyrosine Residues 365/367 in the GABAA-Receptor γ2 Subunit 
The Journal of Neuroscience  2013;33(39):15567-15577.
Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal activity, neurogenesis, and depressive-like behaviors; however, downstream effectors by which BDNF exerts these varying actions remain to be determined. Here we reveal that BDNF induces long-lasting enhancements in the efficacy of synaptic inhibition by stabilizing γ2 subunit-containing GABAA receptors (GABAARs) at the cell surface, leading to persistent reductions in neuronal excitability. This effect is dependent upon enhanced phosphorylation of tyrosines 365 and 367 (Y365/7) in the GABAAR γ2 subunit as revealed using mice in which these residues have been mutated to phenyalanines (Y365/7F). Heterozygotes for this mutation exhibit an antidepressant-like phenotype, as shown using behavioral-despair models of depression. In addition, heterozygous Y365/7F mice show increased levels of hippocampal neurogenesis, which has been strongly connected with antidepressant action. Both the antidepressant phenotype and the increased neurogenesis seen in these mice are insensitive to further modulation by BDNF, which produces robust antidepressant-like activity and neurogenesis in wild-type mice. Collectively, our results suggest a critical role for GABAAR γ2 subunit Y365/7 phosphorylation and function in regulating the effects of BDNF.
PMCID: PMC3782626  PMID: 24068823
9.  Adeno-associated virus serotype 9 efficiently targets ischemic skeletal muscle following systemic delivery 
Gene therapy  2013;20(9):930-938.
Targeting therapeutic gene expression to skeletal muscle following intravenous administration is an attractive strategy for treating peripheral arterial disease (PAD), except that vector access to the ischemic limb could be a limiting factor. Since AAV serotype 9 transduces skeletal muscle at high efficiency following systemic delivery, we employed AAV-9 vectors bearing luciferase or enhanced green fluorescent protein (eGFP) reporter genes to test the hypothesis that increased desialylation of cell surface glycans secondary to hindlimb ischemia (HLI) might help offset the reduction in tissue perfusion that occurs in mouse models of PAD. The utility of the creatine kinase-based (CK6) promoter for restricting gene expression to skeletal muscle was also examined by comparing it to the cytomegalovirus (CMV) promoter after systemic administration following surgically-induced HLI. Despite reduced blood flow to ischemic limbs, CK6 promoter-driven luciferase activities in ischemic gastrocnemius (GA) muscles were ~34-, ~28-, and ~150-fold higher than in fully-perfused contralateral GA, heart, and liver, respectively, 10 days after intravenous administration. Furthermore, luciferase activity from the CK6 promoter in ischemic GA muscles was ~2-fold higher than with CMV, while in the liver CK6-driven activity was ~42-fold lower than with CMV, demonstrating that the specificity of ischemic skeletal muscle transduction can be further improved with muscle-specific promoters. Studies with Evans blue dye and fluorescently-labeled lectins revealed that vascular permeability and desialylation of cell surface glycans were increased in ischemic hindlimbs. Furthermore, AAV9/CK6/Luc vector genome copy numbers were ~6-fold higher in ischemic muscle compared to non-ischemic muscle in the HLI model, whereas this trend was reversed when the same genome was packaged in the AAV-1 capsid (which binds sialylated, as opposed to desialylated glycans), further underscoring the importance of desialylation in the ischemic enhancement of transduction displayed by AAV-9. Taken together, these findings suggest two complementary mechanisms contributing to the preferential transduction of ischemic muscle by AAV-9: increased vascular permeability and desialylation. In conclusion, ischemic muscle is preferentially targeted following systemic administration of AAV-9 in a mouse model of HLI. Unmasking of the primary AAV-9 receptor as a result of ischemia may contribute importantly to this effect.
PMCID: PMC3758463  PMID: 23535898
Adeno-associated virus; gene therapy; hindlimb ischemia; muscle-specific promoter
10.  A PGC-1α isoform induced by resistance training regulates skeletal muscle hypertrophy 
Cell  2012;151(6):1319-1331.
PGC-1α is a transcriptional coactivator induced by exercise that gives muscle many of the best known adaptations to endurance-type exercise, but has no effects on muscle strength or hypertrophy. We have identified a novel form of PGC-1α (PGC-1α4) that results from alternative promoter usage and splicing of the primary transcript. PGC-1α4 is highly expressed in exercised muscle but does not regulate most known PGC-1α targets such as the mitochondrial OXPHOS genes. Rather, it specifically induces IGF1 and represses myostatin, and expression of PGC-1α4 in vitro and in vivo induces robust skeletal muscle hypertrophy. Importantly, mice with skeletal muscle-specific transgenic expression of PGC-1α4 show increased muscle mass and strength, and dramatic resistance to the muscle wasting of cancer cachexia. Expression of PGC-1α4 is preferentially induced in mouse and human muscle during resistance exercise. These studies identify a novel PGC-1α protein that regulates and coordinates factors involved in skeletal muscle hypertrophy.
PMCID: PMC3520615  PMID: 23217713
11.  Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane☆ 
Molecular Metabolism  2013;3(2):114-123.
Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose-dependently protects mice from acute renal ischemic-reperfusion injury. From a technical standpoint, BAM15 represents an effective new tool that allows the study of mitochondrial function in the absence of off-target effects that can confound data interpretation. From a therapeutic perspective, BAM15-mediated protection from ischemia-reperfusion injury and its reduced toxicity will hopefully reignite interest in pharmacological uncoupling for the treatment of the myriad of diseases that are associated with altered mitochondrial function.
PMCID: PMC3953706  PMID: 24634817
ANT, adenine nucleotide translocase; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; ROS, reactive oxygen species; TCA cycle, tricarboxylic acid cycle; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride; TMRM, tetramethylrhodamine; Mitochondria; Bioenergetics; FCCP; CCCP; DNP; Ischemia
12.  Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion 
Nature  2013;497(7448):263-267.
Skeletal muscle arises from the fusion of precursor myoblasts into multinucleated myofibers1,2. While conserved transcription factors and signaling proteins involved in myogenesis have been identified, upstream regulators are less well understood. Here, we report an unexpected discovery that the membrane protein BAI1, previously linked to recognition of apoptotic cells by phagocytes3, promotes myoblast fusion. Endogenous BAI1 expression increased during myoblast fusion, and BAI1 overexpression enhanced myoblast fusion via signaling through ELMO/Dock180/Rac1 proteins4. During myoblast fusion, a fraction of myoblasts underwent apoptosis and exposed phosphatidylserine (PtdSer), an established ligand for BAI13. Blocking apoptosis potently impaired myoblast fusion, and adding back apoptotic myoblasts restored fusion. Furthermore, primary human myoblasts could be induced to form myotubes by adding apoptotic myoblasts, even under normal growth conditions. In vivo, myofibers from Bai1−/− mice are smaller than wild-type littermates. Muscle regeneration after injury was also impaired in Bai1−/− mice, highlighting a role for BAI1 in mammalian myogenesis. Collectively, these data identify signaling via the phosphatidylserine receptor BAI1 and apoptotic cells as novel promoters of myoblast fusion, with significant implications for muscle development and repair.
PMCID: PMC3773542  PMID: 23615608
13.  Ebola Virus Does Not Block Apoptotic Signaling Pathways 
Journal of Virology  2013;87(10):5384-5396.
Since viruses rely on functional cellular machinery for efficient propagation, apoptosis is an important mechanism to fight viral infections. In this study, we sought to determine the mechanism of cell death caused by Ebola virus (EBOV) infection by assaying for multiple stages of apoptosis and hallmarks of necrosis. Our data indicate that EBOV does not induce apoptosis in infected cells but rather leads to a nonapoptotic form of cell death. Ultrastructural analysis confirmed necrotic cell death of EBOV-infected cells. To investigate if EBOV blocks the induction of apoptosis, infected cells were treated with different apoptosis-inducing agents. Surprisingly, EBOV-infected cells remained sensitive to apoptosis induced by external stimuli. Neither receptor- nor mitochondrion-mediated apoptosis signaling was inhibited in EBOV infection. Although double-stranded RNA (dsRNA)-induced activation of protein kinase R (PKR) was blocked in EBOV-infected cells, induction of apoptosis mediated by dsRNA was not suppressed. When EBOV-infected cells were treated with dsRNA-dependent caspase recruiter (dsCARE), an antiviral protein that selectively induces apoptosis in cells containing dsRNA, virus titers were strongly reduced. These data show that the inability of EBOV to block apoptotic pathways may open up new strategies toward the development of antiviral therapeutics.
PMCID: PMC3648168  PMID: 23468487
14.  BAF60c drives glycolytic muscle formation and improves glucose homeostasis through Deptor-mediated AKT activation 
Nature medicine  2013;19(5):640-645.
A shift from oxidative to glycolytic metabolism has been associated with skeletal muscle insulin resistance in type 2 diabetes1–5. However, whether this metabolic switch is deleterious or adaptive remains controversial6–8, in part due to limited understanding of the regulatory network that directs the metabolic and contractile specification of fast-twitch glycolytic muscle. Here we show that BAF60c, a transcriptional cofactor enriched in fast-twitch muscle, promotes a switch from oxidative to glycolytic myofiber type through Deptor-mediated AKT activation. Muscle-specific transgenic expression of BAF60c activates a program of molecular, metabolic, and contractile changes characteristic of glycolytic muscle. In addition, BAF60c is required for maintaining glycolytic capacity in adult skeletal muscle in vivo. BAF60c expression is significantly decreased in skeletal muscle from obese mice. Unexpectedly, transgenic activation of the glycolytic muscle program by BAF60c protects mice from diet-induced insulin resistance and glucose intolerance. Further mechanistic studies revealed that Deptor is induced by the BAF60c/Six4 transcriptional complex and mediates activation of AKT and glycolytic metabolism by BAF60c in a cell-autonomous manner. This work defines a fundamental mechanism underlying the specification of fast glycolytic muscle and illustrates that the oxidative to glycolytic metabolic shift in skeletal muscle is potentially adaptive and beneficial in the diabetic state.
PMCID: PMC3650110  PMID: 23563706
15.  Exercise Training Improves Plantarflexor Muscle Function in mdx Mice 
We tested the hypothesis that low intensity exercise in mdx mice improves plantarflexor muscle contractile function, resistance to fatigue, and mitochondrial adaptations without exacerbating muscular dystrophy.
We subjected mdx mice to 12 wk of voluntary, low-resistance wheel running (Run, n=17) or normal cage activities (sedentary; Sed, n=16) followed by in vivo analyses for plantarflexor torque generation and fatigue resistance, or running capacity on a treadmill. Gastrocnemius muscles were further evaluated for exercise-induced mitochondrial adaptations and fiber type distribution and central nuclei. T-tests were used to determine differences between the Sed and Run groups.
Plantarflexor submaximal isometric torques and maximal isometric torque at multiple ankle joint angles, and resistance to fatigue were greater in Run compared to Sed mdx mice (P<0.05). Citrate synthase and β-HAD enzyme activities and COX IV protein expression in gastrocnemius muscles were greater in Run than Sed mdx mice (P≤0.04), along with a trend of fiber type transformation from type IIb to type 2x fibers. Exercise training in mdx mice did not elevate serum creatine kinase levels, but led to a significant reduction of centrally-nucleated myofibers.
Voluntary, low-resistance wheel running in mdx mice can result in skeletal muscle adaptation, leading to improved contractile function and reduced fatigability, with no indication that exercise was detrimental. This study supports the need for further investigation of low intensity exercise as an early therapeutic intervention in ambulatory boys with DMD.
PMCID: PMC3470762  PMID: 22460476
Duchenne muscular dystrophy; fatigue; mitochondria; physical activity; skeletal muscle; strength
16.  Remodeling of Hyperpolarization-Activated Current, Ih, in Ah-Type Visceral Ganglion Neurons Following Ovariectomy in Adult Rats 
PLoS ONE  2013;8(8):e71184.
Hyperpolarization-activated currents (Ih) mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels modulate excitability of myelinated A− and Ah-type visceral ganglion neurons (VGN). Whether alterations in Ih underlie the previously reported reduction of excitability of myelinated Ah-type VGNs following ovariectomy (OVX) has remained unclear. Here we used the intact nodose ganglion preparation in conjunction with electrophysiological approaches to examine the role of Ih remodeling in altering Ah-type neuron excitability following ovariectomy in adult rats. Ah-type neurons were identified based on their afferent conduction velocity. Ah-type neurons in nodose ganglia from non-OVX rats exhibited a voltage ‘sag’ as well as ‘rebound’ action potentials immediately following hyperpolarizing current injections, which both were suppressed by the Ih blocker ZD7288. Repetitive spike activity induced afterhyperpolarizations lasting several hundreds of milliseconds (termed post-excitatory membrane hyperpolarizations, PEMHs), which were significantly reduced by ZD7288, suggesting that they resulted from transient deactivation of Ih during the preceding spike trains. Ovariectomy reduced whole-cell Ih density, caused a hyperpolarizing shift of the voltage-dependence of Ih activation, and slowed Ih activation. OVX-induced Ih remodeling was accompanied by a flattening of the stimulus frequency/response curve and loss of PEMHs. Also, HCN1 mRNA levels were reduced by ∼30% in nodose ganglia from OVX rats compared with their non-OVX counterparts. Acute exposure of nodose ganglia to 17beta-estradiol partly restored Ih density and accelerated Ih activation in Ah-type cells. In conclusion, Ih plays a significant role in modulating the excitability of myelinated Ah-type VGNs in adult female rats.
PMCID: PMC3741359  PMID: 23951107
17.  Exercise training-induced Regulation of Mitochondrial Quality 
Mitochondria are dynamic organelles in skeletal muscle critical in physical performance and disease. The mitochondrial life cycle spans biogenesis, maintenance, and clearance. Exercise training may promote each of these processes, conferring positive impacts on skeletal muscle contractile and metabolic functions. This review focuses on the regulation of these processes by endurance exercise and discusses potential benefits in health and disease.
PMCID: PMC3384482  PMID: 22732425
Skeletal muscle; exercise-induced adaptation; mitochondrial biogenesis; mitochondrial fission; mitochondrial fusion; autophagy; mitophagy
18.  Disconnecting mitochondrial content from respiratory chain capacity in PGC-1 deficient skeletal muscle 
Cell reports  2013;3(5):1449-1456.
The transcriptional coactivators PGC-1α and PGC-1β are widely thought to be required for mitochondrial biogenesis and fiber typing in skeletal muscle. We show here that mice lacking both PGC-1s in myocytes do indeed have profoundly deficient mitochondrial respiration, but surprisingly have preserved mitochondrial content, isolated muscle contraction capacity, fiber type composition, in-cage ambulation, and voluntary running capacity. Most of these findings are recapitulated in cell culture, and thus cell-autonomous. Functional electron microscopy reveals normal cristae density with decreased cytochrome oxidase activity. These data lead to the following surprising conclusions: that PGC-1s are in fact dispensable for baseline muscle function, mitochondrial content, and fiber typing; that endurance fatigue at low workloads is not limited by muscle mitochondrial capacity; and that mitochondrial content and cristae density can be dissociated from respiratory capacity.
PMCID: PMC3688451  PMID: 23707060
19.  Disrupted GABAAR Trafficking and Synaptic Inhibition in a Mouse Model of Huntington’s Disease 
Neurobiology of Disease  2012;46(2):497-502.
Growing evidence suggests that Huntington’s disease (HD), a neurodegenerative movement disorder caused by the mutant huntingtin (htt) with an expanded polyglutamine (polyQ) repeat, is associated with the altered intracellular trafficking and synaptic function. GABAA receptors, the key determinant of the strength of synaptic inhibition, have been found to bind to the huntingtin associated protein 1 (HAP1). HAP1 serves as an adaptor linking GABAA receptors to the kinesin family motor protein 5 (KIF5), controlling the transport of GABAA receptors along microtubules in dendrites. In this study, we found that GABAAR-mediated synaptic transmission is significantly impaired in a transgenic mouse model of HD expressing polyQ-htt, which is accompanied by the diminished surface expression of GABAA receptors. Moreover, the GABAAR/HAP1/KIF5 complex is disrupted and dissociated from microtubules in the HD mouse model. These results suggest that GABAAR trafficking and function is impaired in HD, presumably due to the interference of KIF5-mediated microtubule-based transport of GABAA receptors. The diminished inhibitory synaptic efficacy could contribute to the loss of the excitatory/inhibitory balance, leading to increased neuronal excitotoxicity in HD.
PMCID: PMC3323696  PMID: 22402331
huntingtin; GABAA receptor; IPSC; KIF5; microtubule; trafficking
Free radical biology & medicine  2012;52(9):1708-1715.
Activation of the transcription factor NF-E2 related factor 2 (Nrf2) by oxidative stress induces the expression of a variety of antioxidant and anti-inflammatory genes. Yet, genetic ablation of Nrf2 was shown to protect mice from high-fat diet (HFD)-induced obesity and insulin resistance. The mechanisms that underlay this seemingly paradoxical finding remain largely unexplored.
Here we examined whether Nrf2 deficiency in myeloid cells contributes to protection against HFD-induced metabolic changes by decreasing adipose tissue inflammation. In vitro, induction of IL-1β by inflammatory stimuli was significantly reduced in Nrf2-deficient macrophages. While, inflammatory gene expression in the stromal vascular fraction was reduced in both global and chimeric Nrf2 KO mice, only global Nrf2-deficient but not bone marrow-transplanted Nrf2 chimeric mice were protected against HFD-induced adipose tissue inflammation. While global Nrf2 deficiency resulted in significantly decreased expression of inflammatory genes and PPARγ2, there was no difference when Nrf2 was absent only in myeloid cells. In vitro co-culture with adipocytes demonstrated that macrophage Nrf2 regulated inflammatory gene expression in macrophages, however, was not required to induce inflammatory gene expression in adipocytes. Finally, in contrast to global Nrf2 knock-out, Nrf2 deficiency in myeloid cells did not protect against HFD-induced insulin resistance.
Together, our data demonstrate a dominant role of nonmyeloid Nrf2 in controlling HFD-induced adipose tissue inflammation and the development of insulin resistance.
PMCID: PMC3383807  PMID: 22370093
Nrf2; inflammation; insulin resistance; bone marrow transplantation; stromal vascular fraction and adipocyte/macrophage co-culture
21.  Repeated Stress Causes Cognitive Impairment by Suppressing Glutamate Receptor Expression and Function in Prefrontal Cortex 
Neuron  2012;73(5):962-977.
Chronic stress could trigger maladaptive changes associated with stress-related mental disorders, however, the underlying mechanisms remain elusive. In this study, we found that exposing juvenile male rats to repeated stress significantly impaired the temporal order recognition memory, a cognitive process controlled by prefrontal cortex (PFC). Concomitantly, significantly reduced AMPAR- and NMDAR-mediated synaptic transmission and glutamate receptor expression were found in PFC pyramidal neurons from repeatedly stressed animals. All these effects relied on activation of glucocorticoid receptors and the subsequent enhancement of ubiquitin/proteasome-mediated degradation of GluR1 and NR1 subunits, which was controlled by the E3 ubiquitin ligase Nedd4-1 and Fbx2, respectively. Inhibition of proteasomes or knockdown of Nedd4-1 and Fbx2 in PFC prevented the loss of glutamatergic responses and recognition memory in stressed animals. Our results suggest that repeated stress dampens PFC glutamatergic transmission by facilitating glutamate receptor turnover, which causes the detrimental effect on PFC-dependent cognitive processes.
PMCID: PMC3302010  PMID: 22405206
stress; corticosterone; glucocorticoid receptor; NMDA receptor; AMPA receptor; ubiquitination; degradation; recognition memory
22.  Construction, Expression, and Characterization of Thymosin Alpha 1 Tandem Repeats in Escherichia coli 
BioMed Research International  2013;2013:720285.
Thymosin alpha 1 (Tα1), which is composed of 28 amino acids, has been commercialized worldwide for its immune-modulatory and antitumor effects. Tα1 can stimulate T cell proliferation and differentiation from bone marrow stem cells, augment cell-mediated immune responses, and regulate homeostasis of immune system. In this study, we developed a novel strategy to produce Tα1 concatemer (Tα1③) in Escherichia coli and compared its activity with chemically synthesized Tα1. Results showed that Tα1③ can more effectively stimulate T cell proliferation and significantly upregulate IL-2 receptor expression. We concluded that the expression system for Tα1 concatemer was constructed successfully, which could serve as an efficient tool for the production of large quantities of the active protein.
PMCID: PMC3600210  PMID: 23555093
23.  Induction of Osteoarthritis and Metabolic Inflammation by a Very High Fat Diet in Mice: Effects of Short-term Exercise 
Arthritis and Rheumatism  2012;64(2):443-453.
To test the hypotheses that obesity due to a very high fat diet induces knee osteoarthritis, and that short-term wheel running exercise protects against obesity-induced knee osteoarthritis by reducing systemic inflammation and metabolic dysregulation.
Male C57BL/6J mice were fed either a control (13.5% kcal fat) or very high fat diet (60% kcal fat) from 12–24 wks of age. From 20–24 wks, half of the animals were housed with running wheels. Knee osteoarthritis severity was determined via histopathology, and serum cytokines were measured using a multiplex bead immunoassay and ELISAs. Body composition was quantified by dual-energy X-ray absorptiometry, and insulin resistance was assessed by glucose tolerance testing.
A very high fat diet increased osteoarthritis scores and serum leptin, adiponectin, KC (mouse analog of IL-8), MIG (monokine induced by interferon-gamma, or CXCL9), and interleukin 1 receptor antagonist (IL-1Ra) levels in proportion to percent body fat, which increased 3-fold compared to controls. Wheel running reduced osteoarthritis progression in the medial femur of obese mice. Exercise disrupted the clustering of cytokine expression and improved glucose tolerance without reducing body fat or cytokine levels.
Obesity induced by a very high-fat diet causes osteoarthritis and systemic inflammation in proportion to body fat. Increased joint loading is not sufficient to explain the increased incidence of knee osteoarthritis with obesity as wheel running is protective rather than damaging. Exercise improves glucose tolerance and disrupts the co-expression of pro-inflammatory cytokines, suggesting that increased aerobic exercise may act independent of weight loss in promoting joint health.
PMCID: PMC3268860  PMID: 21953366
obesity; exercise; knee joint; inflammation; body fat; cartilage; proteoglycan
24.  Aβ impairs nicotinic regulation of inhibitory synaptic transmission and interneuron excitability in prefrontal cortex 
Accumulation of β-amyloid (Aβ) and cholinergic deficiency are two prominent features of Alzheimer’s disease (AD). To understand how Aβ-induced dysfunction of the nicotinic system may contribute to cognitive impairment in AD, we examined the effect of Aβ on nicotinic regulation of synaptic transmission and neuronal excitability in prefrontal cortex (PFC), a brain region critical for cognitive processes.
We found that activation of nicotinic acetylcholine receptors (nAChRs) with nicotine increased the inhibitory postsynaptic currents recorded in PFC pyramidal neurons, which was associated with the nicotine-induced increase in the excitability of PFC layer I GABAergic interneurons. Both effects of nicotine were disrupted by Aβ. However, Aβ did not impair nicotinic regulation of excitatory neurotransmission in PFC interneurons. The nicotinic effect on synaptic inhibition was also lost in transgenic mice with five familial Alzheimer’s disease mutations. Inhibiting PKC attenuated nicotinic regulation of inhibitory, but not excitatory, neurotransmission.
Our study suggests that Aβ selectively impairs nicotinic regulation of inhibitory inputs to PFC pyramidal neurons, which might be due to its interference with PKC activation. Thus, in the PFC circuits of AD, the balance between inhibition and excitation under the control of nAChRs may be disturbed by Aβ.
PMCID: PMC3610117  PMID: 23327202
Alzheimer’s disease; β-amyloid; Nicotinic acetylcholine receptor; Prefrontal cortex; Interneuron; Pyramidal neuron; Inhibitory postsynaptic current; Firing; Protein kinase C
25.  Subtype Identification in Acutely Dissociated Rat Nodose Ganglion Neurons Based on Morphologic Parameters 
Nodose ganglia are composed of A-, Ah- and C-type neurons. Despite their important roles in regulating visceral afferent function, including cardiovascular, pulmonary, and gastrointestinal homeostasis, information about subtype-specific expression, molecular identity, and function of individual ion transporting proteins is scarce. Although experiments utilizing the sliced ganglion preparation have provided valuable insights into the electrophysiological properties of nodose ganglion neuron subtypes, detailed characterization of their electrical phenotypes will require measurements in isolated cells. One major unresolved problem, however, is the difficulty to unambiguously identify the subtype of isolated nodose ganglion neurons without current-clamp recording, because the magnitude of conduction velocity in the corresponding afferent fiber, a reliable marker to discriminate subtypes in situ, can no longer be determined. Here, we present data supporting the notion that application of an algorithm regarding to microscopic structural characteristics, such as neuron shape evaluated by the ratio between shortest and longest axis, neuron surface characteristics, like membrane roughness, and axon attachment, enables specific and sensitive subtype identification of acutely dissociated rat nodose ganglion neurons, by which the accuracy of identification is further validated by electrophysiological markers and overall positive predictive rates is 89.26% (90.04%, 76.47%, and 98.21% for A-, Ah, and C-type, respectively). This approach should aid in gaining insight into the molecular correlates underlying phenotypic heterogeneity of nodose ganglia. Additionally, several critical points that help for neuron identification and afferent conduction calibration are also discussed.
PMCID: PMC3729014  PMID: 23904796
visualization; visceral sensory neuron; conduction velocity; whole-cell patch; action potential

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