Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an extremely abundant protein in the brain where, remarkably, it is estimated to make up 1–5% of total neuronal protein. Although it comprises only 223 amino acids it has one of the most complicated 3D knotted structures yet discovered. Beyond its expression in neurons UCH-L1 has only very limited expression in other healthy tissues but it is highly expressed in several forms of cancer. Although UCH-L1 is classed as a deubiquitinating enzyme (DUB) the direct functions of UCH-L1 remain enigmatic and a wide array of alternative functions has been proposed. UCH-L1 is not essential for neuronal development but it is absolutely required for the maintenance of axonal integrity and UCH-L1 dysfunction is implicated in neurodegenerative disease. Here we review the properties of UCH-L1, and how understanding its complex structure can provide new insights into its roles in neuronal function and pathology. The identification and safe destruction of unwanted, misfolded or aggregated proteins is essential for cell viability. The complexity and sophistication of neuronal architecture and signalling pathways make them especially vulnerable to protein aggregation and failure to adequately destroy proteins underlies multiple neuropathologies .48-linked polyubiquitin chains enter the ubiquitin-proteasome system (UPS), which mediates their degradation via the 26S proteasome . Cr. Crapo) in vitro assays have shown that UCH-L1 can bind and efficiently hydrolyse ubiquitin-AMC–a ubiquitin molecule conjugated to a small organic fluorescent probe containing two benzene rings [The UCH class of DUBs all contain an unstructured loop that restricts access to the active site. UCH-L1 contains the shortest loop in the UCH class, which prevents access to the active site for all proteins except for very short disordered peptides (∼10 amino acids) conjugated to ubiquitin . In crysne rings –but it cne rings . In contne rings ,47, suggin vitro assays have also shown that the efficiency of UCH-L5 (UCH37) at cleaving ubiquitinated substrates can vary enormously depending on the reaction conditions used, suggesting that the simplified assays used so far may not accurately reflect the in vivo conditions necessary for UCH-L1 substrate hydrolysis [It should be noted, however, that drolysis ,49. For drolysis , althougin vitro assays [UCH-L1 has a high affinity for ubiquitin, which it can efficiently hydrolyse from small C-terminal extensions in o assays . It alsoo assays . Indeed,o assays  and its 48-polyubiquitin chains are recognized by proteasomal subunits and cofactors allowing the targeting of ubiquitinated substrates for degradation. The ubiquitin molecule itself, however, is recycled. The 19S proteasomal lid contains subunits that can recognize and deubiquitinate substrate proteins, releasing mono- and poly-ubiquitin chains, however it has been proposed that recycled ubiquitin molecules can still be linked to small peptide fragments, which need to be processed for re-use [in vitro substrate hydrolysis assays [Lysr re-use ,53. Conss assays . Furthers assays –56. Thess assays . This als assays .Although UCH-L1 has been proposed as crucial for maintaining proper proteasomal function ,58, protD and L [UCH-L1 deficient nm3419 mice show increased mRNA levels of the lysosomal enzymes cathepsins D and L . Down-reD and L . HoweverMonomeric ubiquitin is processed from proubiquitin precursor proteins. Four different genes encode mammalian proubiquitin precursors, two of which are synthesized as ribosomal subunit-fusion proteins and two as polyubiquitin precursors . In bactIndeed, although there is clearly a great deal to discover about the mechanisms underlying ubiquitin processing, work using rabbit reticulocytes, mouse liver and HeLa cells has provided evidence that polyubiquitin gene products can be co-translationally processed . UCH-L3 in vitro experiments using UCH DUBs and ubiquitinated substrates show that UCH-L1 is far less efficient than the homologous UCH-L3 [UCH-L1 has been proposed to deubiquitinate several exogenously expressed proteins in clonal cell lines, including NOXA and NOX4 ,64. Nones UCH-L3 . Overalls UCH-L3 .DUBs act as part of larger protein complexes and the identification of component proteins provides information about the pathways and functions regulated. As discussed below, proteins reported as UCH-L1 interactors from co-immunoprecipitation studies include amyloid precursor protein (APP)  and tubuIn addition to its monomeric DUB function, a dimeric form of UCH-L1 has been proposed to have ubiquitin E4 ligase function, acting to extend polyubiquitin chains on substrate proteins such as α-synuclein and tubulin ,66. Inte63 polyubiquitin chains on α-synuclein thereby preventing its proteasomal degradation [It has also been reported that UCH-L1 acts as a ligase to extend Lysradation . Howeverradation .Uchl1 mutant mice lines and an Uchl1 knockout mouse have been characterized [Two naturally spontaneously occurring cterized ,56,68. Tgad) phenotype developed spontaneously in a strain of lab mice, leading to sensory ataxia at approximately 3 months, and motor ataxia at 4 months, manifesting first as a hind limb paralysis and followed by death at approximately 6 months [Uchl1 gene, corresponding to the loss of 42 residues from 154 aa to 196 aa, including the catalytic His161 [gad mouse are due to UCH-L1 ablation [gad mice revealed inclusion bodies in axon nerve terminals in the gracile tract of the spinal cord. Axons from dorsal root ganglion cells that pass through the gracile tract possess the longest axons in the mammalian CNS [The recessive gracile axonal dystrophy , induced following ischaemic injury, and are implicated in the pathogenesis of neurological diseases [152, a residue that is not present in UCH-L3 [152 is situated in the short unstructured active site loop  and Parkinson's disease (PD). UCH-L1 solubility is decreased by the oxidation of cysteine and methionine residues and carbonyl formation –92 and tactivity . Mechaniactivity ,36,90.The loss of dopaminergic neurons in PD is preceded by the formation of Lewy Bodies, insoluble proteinaceous inclusions enriched with ubiquitinated aggregates, as well as displaying extensive protein oxidative modification . Most caPARK5 [The I93M point mutation in UCH-L1 has been the focus of considerable research because it occurred in four out of seven family members who developed PD. As a result UCH-L1 has been designated a Parkinson's susceptibility gene and given the alternative name of PARK5 . It shouPARK5 ,98.gad mice are healthy and UCH-L1-deficient mice do not develop Parkinsonian symptoms. Rather, I93M likely gives rise to a dominant toxic gain-of-function, so studies have focused on the physical properties of the mutant protein. The Ile93 site is located in an intramolecular α-helix near the active site and contacts the hydrophobic core of UCH-L1 -1,2,3,4-tetrahydroisoquinoline , suggesting a possible role in the pathogenesis of idiopathic PD . Moreovell death . Althoug152 residue (see above) acts as a redox buffer in neurons and reacts with, and chelates, free radicals to maintain short-term cellular function [The complex morphology of neurons dictates a high membrane-to-cytoplasm ratio and synapses require a high proportion of unsaturated fatty acids that regulate membrane fluidity . Howeverfunction .gad mice show increased vulnerability to lipid peroxidation, and damage is further increased in neurons cultured in media deficient in Vitamin-E (α-tocopherol), which is an antioxidant that protects cells from ROS (reactive oxygen species) damage. This is particularly relevant because chronic Vitamin-E deficiency causes gracile tract degeneration, similar to UCH-L1 deficient mouse models [Consistent with this, N2a cells treated with antisense UCH-L1 cDNA to down-regulate UCH-L1 expression were more susceptible to oxygen/glucose deprivation (OGD) induced cell death . Moreovee models –109.Overall, this hypothesis suggests that the abundance and diffuse cytoplasmic distribution of UCH-L1 allows for the chelation of excess ROS during acute damage, enabling the cell to continue to function in the short term at the expense of ubiquitin homeostasis.Despite intense research efforts the precise functions of UCH-L1 remain enigmatic. However, recent progress in defining the folding and tertiary structure has provided new insights. UCH-L1 has high affinity for monomeric ubiquitin, but is a poor hydrolase of ubiquitinated proteins due to restricted access to the active site . Thus, e90 mutation causes the same axonal degeneration and ataxic phenotype as the Glu7Ala mutation in vivo will help resolve these outstanding questions.UCH-L1 is necessary for the maintenance of axonal health and stability and its loss results in axonal degeneration and neuronal death. However, despite this clear and reproducible phenotype, as set about above, the mechanisms underlying this degeneration are unclear. We expect that future experiments exploring whether the CysMotor neurons may be particularly susceptible to UCH-L1 loss because they contain a specific ubiquitin pool or pathway that requires UCH-L1 regulation. Also, the high energy and protein turnover burdens required to maintain extensive axonal projections mean that they operate very close to their maximum capacity and that they are more vulnerable to defects that other neuronal types can withstand for longer. It is also possible that UCH-L1 may regulate axon maintenance via microtubule-associating proteins that are crucial for both axonal transport and stability ,85.152, are readily modified by oxidation and that this can lead to destabilization of the protein and exposure of the hydrophobic core, which results in cytotoxic gain-of-function of insoluble UCH-L1. Intriguingly, oxidative damaged induced instability and aggregation is prevented by Cys152Ala mutation, which presents an exciting possibility for therapeutic intervention.Beyond rare diseases caused by mutations in UCH-L1 that result in axonal degeneration, UCH-L1 is also implicated in other forms of neurodegenerative disease, most notably Parkinson's disease. Recent work highlights how residues in UCH-L1, particularly CysOverall, although UCH-L1 retains some mystique there has been significant progress towards defining its roles in healthy and diseased neurons. We anticipate that in the next few years a more complete understanding will lead to new strategies to exploit its potential as a therapeutic target.


Background: LRRK2 mutations are causative for Parkinson disease, but regulation of LRRK2 remains elusive.Results: Arsenite induces loss of LRRK2 Ser910/Ser935 phosphorylation and 14-3-3 binding, increased self-association, attenuated kinase activity and GTP binding, and translocation to centrosomes.Conclusion: LRRK2 is regulated by arsenite-induced signaling and oxidative stress.Significance: Understanding LRRK2 regulation will provide novel approaches toward developing therapeutic tools targeting LRRK2 activity. 910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function.Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser Ras of complex proteins) that is thought to operate in tandem with the adjacent COR  domain and a serine/threonine protein kinase domain (in vitro (in vivo (Parkinson disease (PD)1–e domain , 8. The e domain . The mosin vitro , 11 and   , 21 or re (MPTP) , 23. LRRe (MPTP) , wherease (MPTP) , 26.2O2-induced stress promoted the loss of LRRK2 phosphorylation at sites Ser910/Ser935 in stable inducible expression cell lines as well as of endogenous LRRK2 in a lymphoblastoid cell line, whereas this was rescued by inhibition of protein phosphatases. WT LRRK2 and the variants R1441C, D2017A (kinase-dead), and G2019S responded to oxidative stress in a similar manner with loss of constitutive phosphorylation and loss of 14-3-3 binding. Arsenite stress induced LRRK2 self-association as well as accumulation of very high molecular mass forms of LRRK2. Treatment with arsenite resulted in attenuation of kinase activity in LRRKtide phosphorylation and autophosphorylation assays and a decrease in LRRK2 binding to GTP in vitro. Arsenite stress also induced LRRK2 translocation to ubiquitin proteasomal centers localizing at centrosomes. Our data collectively support a role of oxidative stress in modulating LRRK2 activity and suggest a sequence of signaling events induced by arsenite stress.Based on these data, we hypothesized that LRRK2 is involved in the oxidative stress response and explored this using the oxidative stressor arsenite. We assessed changes in phosphorylation, self-association, kinase activity, GTP binding, and cellular localization of LRRK2 under arsenite stress. We found that both arsenite and H2O2 for 45 min. The LRRK2 inhibitor LRRK2-IN1 was used at 1 μm for 2 h. MG132, lactacystin, and nocodazole were used at 50 μm for 6 h. In the combination treatment experiments, cells were primed with IN-1 for 75 min or with MG132 or nocodazole for 5 h before the addition of arsenite for the last 45 min of the treatment. In the calyculin A experiments, cells were pretreated with 10 nm calyculin A for 15 min before the addition of arsenite, IN-1, or H2O2 and incubation for an additional 30 min.Inducible HEK-293T cell lines expressing different GFP LRRK2 variants were grown and cultured as described previously . Human lm Tris/HCl (pH 7.4), 137 mm NaCl, 3 mm KCl, 10% (v/v) glycerol, 1 mm EDTA, and 0.3% Triton X-100 supplemented with protease inhibitors (Roche Applied Science) and phosphatase inhibitors (Pierce). Lysates were centrifuged at 21,000 × g, 4 °C for 10 min, and the supernatants were analyzed for protein concentration (Pierce). 20 μg of total protein from each supernatant was analyzed by SDS-PAGE for expression of the proteins in question. 400 μg of each sample was precleared with protein G beads (Sigma-Aldrich) for 1 h at 4 °C, and subsequently, GFP-LRRK2 was immunoprecipitated with Chromotek-GFP-Trap-agarose resin (Allele Biotech) for 2 h at 4 °C. The GFP-agarose was gently washed six times with buffer containing 20 mm Tris/HCl (pH 7.4), 137 mm NaCl, 3 mm KCl , and 0.1% Triton X-100. The washed beads were boiled for 10 min in 4× NuPAGE loading buffer (Invitrogen) supplemented with 1.4 m β-mercaptoethanol and analyzed by SDS-PAGE. Each co-immunoprecipitation was repeated in three independent experiments, and quantification was performed by estimating the ratio of immunoprecipitated binding partner to the amount of LRRK2 construct pulled down on the beads.HEK-293T cells stably expressing GFP WT LRRK2 were transfected with 3×FLAG-LRRK2 variants using Lipofectamine 2000 (Invitrogen) as per the manufacturer's instructions. After 24 h, cells were lysed in buffer containing 20 mg, 4 °C for 10 min, and the supernatants were then passed through 0.45-μm filters  by centrifugation at 14,000 × g for 3 min to remove any insoluble material. Size exclusion chromatography was performed using a BioAssist G4SWXL column  with PBS as the mobile phase as described before  and lysed using five freeze-thaw cycles in liquid nitrogen. Lysates were centrifuged at 21,000 × d before . The col910 LRRK2 (UDD1 15(3)), anti-phospho-Ser935 LRRK2 (UDD2 10(12)), anti-phospho-Thr1410 LRRK2 (MJFR4-25-5), and anti-LRRK2 (C41-2) from Abcam; anti-phospho-Ser51 eIF2α from Epitomics; anti-eIF2α and anti-ubiquitin from Santa Cruz Biotechnology, Inc.; and anti-β-actin from Sigma-Aldrich.Standard Western blot protocols were used with the following antibodies: anti-phospho-Serm sodium arsenite for 45 min and lysed in buffer containing 20 mm Tris/HCl (pH 7.4), 137 mm NaCl, 3 mm KCl, 10% (v/v) glycerol, 1 mm EDTA, and 1% Triton X-100 supplemented with protease inhibitors (Roche Applied Science) and phosphatase inhibitors (Pierce), by 1 h of rotation at 4 °C. Lysates were centrifuged at 21,000 × g, 4 °C for 10 min, and supernatants were precleared with agarose beads for 1 h, rotating at 4 °C, followed by centrifugation at 3,000 × g, 4 °C for 5 min to separate the lysate from beads. Subsequently, equal amounts of protein were incubated with 30 μl of GTP-agarose  with rotation for 2 h at 4 °C with some samples supplemented with 10 mm GTP as a competitive inhibitor of binding to the beads. GTP-agarose beads were washed three times in lysis buffer, eluted with 4× NuPAGE loading buffer, and analyzed by SDS-PAGE. In a separate experiment, a titration of increasing concentrations of GTP was used to elute LRRK2 from GTP-agarose beads before analysis by Western blot.The GTP binding properties of LRRK2 were assessed as described previously . BrieflyIn vitro kinase assays were performed as described previously (m Tris/HCl (pH 7.4), 300 mm NaCl, 3 mm KCl, 10% (v/v) glycerol, 1 mm EDTA, and 1% Triton X-100 supplemented with protease inhibitors (Roche Applied Science) and phosphatase inhibitors (Pierce) with FLAG M2-agarose beads (Sigma). In vitro autophosphorylation kinase assays in the presence of different concentrations of sodium arsenite were performed with 10 nm recombinant GST-LRRK2(970–2527) (Life Technologies) or recombinant GST-CK1α  in 1× kinase buffer , 6 μCi of [33P]ATP , and 10 μm ATP for 30 min at 30 °C. Reactions were terminated by adding 4× NuPAGE loading buffer and analyzed by SDS-PAGE, whereas incorporated 33P was detected by autoradiography. In the LRRKtide phosphorylation assay, incorporation of 33P was detected by liquid scintillation counting.eviously , 29. For4 cells/well on 12-mm coverslips precoated with laminin/poly-d-lysine  and cultured as described before . Mean values ± S.E. are indicated.Experiments examining phosphorylation levels of LRRK2 under oxidative stress, LRRK2 self-association experiments, and centrosomal localization experiments were analyzed by one-way ANOVA with Tukey's post hoc test. A two-tailed Student's 2O2. We observed a concentration-dependent reduction in Ser935 phosphorylation with arsenite (>100 μm), and with H2O2 (>200 μm) (A). Arsenite stress promotes translational arrest by mediating phosphorylation of the mammalian translation initiation factor eIF2α. Activation of this cellular stress response was therefore verified by monitoring the concentration-dependent increase in eIF2α phosphorylation under the same conditions (A).To investigate how oxidative stress alters phosphorylation of endogenous LRRK2, we treated human lymphoblastoid cells with arsenite or H>200 μm) A. Arseninditions A.2O2 (B) or arsenite (C) induced loss of Ser910/Ser935 phosphorylation of WT as well as R1441C, D2017A, and G2019S mutants in a concentration-dependent manner. LRRK2 Y1699C displayed greatly reduced constitutive Ser910/Ser935 phosphorylation, in agreement with previous studies .To further evaluate the requirement of kinase activity, LRRK2-expressing cells were treated with arsenite in the presence or absence of the LRRK2 kinase inhibitor LRRK2IN-1. As with endogenous LRRK2, treatment with 0.5 mA mutant , C and D1410 autophosphorylation of immunoaffinity-purified GFP-LRRK2 (C and E) 1410 (E).We also looked at ThrFP-LRRK2 , C and EC and E) . We obser(P)1410 E.910/Ser935 phosphorylation should decrease LRRK2 binding to 14-3-3 proteins. To test this, we immunoprecipitated LRRK2 from cells expressing WT LRRK2 or mutants after treatment with arsenite and/or LRRK2IN-1. In all cases, loss of Ser910/Ser935 phosphorylation was accompanied by decreased co-immunoprecipitation of 14-3-3 (C).Loss of Serf 14-3-3 C.910/Ser935 of kinase-inactive D2017A suggest that the underlying mechanism does not rely on LRRK2 kinase activity. Because a recent study suggested that LRRK2 phosphorylation at Ser910/Ser935 is modulated by protein phosphatase 1 (PP1) . Arsenite promoted the association of LRRK2 with PP1α in a co-immunoprecipitation assay in cells transiently overexpressing FLAG LRRK2, whereas this effect was not observed for LRRK2IN-1 or H2O2 (C and D). These results suggest that oxidative stress can modulate LRRK2 constitutive phosphorylation and 14-3-3 binding through a pathway that is largely independent of the kinase activity of LRRK2 but depends on PP1/PP2A phosphatase activity and that arsenite can act to stabilize the interaction of LRRK2 with PP1α.The differential effects of LRRK2IN-1 and arsenite stress on phosphorylation at Ser 1 (PP1) , we test protein , A and B or H2O2 , C and Din vitro enzymatic activity (33–A and B). This was not dependent on LRRK2 kinase activity because co-treatment with LRRK2IN-1 did not impede self-association.Although LRRK2 is believed to be largely monomeric in cells, it can also form dimers that may have enhanced activity , 33–35. ivity 33–, A and BC and D). Arsenite enhanced the association of LRRK2 K1906M and G2019S with WT, indicating that this is not dependent on kinase activity. Strikingly, stimulation of self-association by arsenite was attenuated in the case of ROC-COR domain mutants, compared with the other variants. Based on previous reports on the involvement of the ROC-COR domain in dimerization, our data support the formation of a functional LRRK2 complex induced by arsenite treatment.LRRK2 dimerization is thought to be mediated by its ROC domain , 36, 37 E and F). Interestingly, kinase inhibition by LRRK2IN-1 caused only a subtle alteration in the elution of WT LRRK2 in the presence or absence of arsenite. Dephosphorylation induced by LRRK2IN-1 is insufficient for LRRK2 self-assembly (A) and the formation of large native protein complexes (E), suggesting that additional mechanisms are involved in the formation of arsenite-induced LRRK2 molecular complexes. We also investigated whether PP1α is recruited to high molecular weight species and observed that its distribution in size exclusion chromatography is not altered with arsenite treatment . Although this suggests that the general pool of PP1α in cells is not shifted to apparent high molecular weight fractions, it does not argue against an involvement in arsenite-induced LRRK2 dephosphorylation because PP1α is broadly distributed throughout cells, and substoichiometric amounts of this enzyme should be able to dephosphorylate LRRK2.We next examined the effect of arsenite-induced stress on the apparent molecular mass of the LRRK2 complex under native conditions by size exclusion chromatography. WT LRRK2 eluted over a range of apparent molecular masses with the highest signal for immunoreactivity observed between 440 and 660 kDa, as described previously . Arsenitassembly A and theomplexes E, suggesin vitro assays using GTP-agarose to precipitate endogenous LRRK2 from lysates of lymphoblastoid cells or GFP LRRK2 from stably expressing HEK-293T cells. Arsenite treatment induced a significant decrease in the amount of LRRK2 bound to GTP-agarose at endogenous and overexpressed levels (A–D). We performed a specific elution of LRRK2 from the GTP resin with increasing concentrations of GTP (E and F). Our results are consistent with a decrease in in vitro GTP binding affinity for LRRK2 from arsenite-treated cells, the functional significance of which merits further investigation.Our data suggest that arsenite can induce LRRK2 self-association . Becaused levels , A–D. Wes of GTP , E and F935 phosphorylation is dependent on GTP binding, we examined the K1347A GTP binding-deficient mutant (G and H). When expressed in cells, K1347A LRRK2 is constitutively phosphorylated at Ser935 at lower levels than WT protein, suggesting that GTP binding supports this phosphorylation. However, arsenite stress was capable of diminishing Ser935 phosphorylation of K1347A LRRK2, suggesting that GTP binding is not required for arsenite-induced dephosphorylation.To investigate whether the effect of arsenite on Sert mutant , G and H910/Ser935 and affecting the autophosphorylation site Thr1410. We therefore tested the hypothesis that arsenite can affect the kinase activity of LRRK2, using an in vitro assay system. HEK-293T cells expressing FLAG LRRK2 were treated with LRRK2IN-1 or arsenite, and LRRK2 was purified and assayed for kinase activity, measuring the incorporation of 33P with the model substrate LRRKtide (m) attenuated the kinase activity of LRRK2 (A). We subsequently tested whether arsenite can directly inhibit LRRK2 when added to the kinase reaction. FLAG LRRK2 WT was purified from untreated cells and used in kinase assays with LRRK2IN-1 or arsenite (B). Whereas LRRK2IN-1 (10 μm) abolished LRRK2 kinase activity, arsenite (500 μm) decreased the incorporation of 33P on LRRKtide by ∼50% compared with untreated controls at 60 min of incubation. To address the specificity of the effect on LRRK2, an in vitro autophosphorylation kinase assay was performed with recombinant LRRK2 and an unrelated kinase casein kinase 1α (CK1α) in the presence of increasing concentrations of arsenite (C and D). Arsenite (500 μm) inhibited LRRK2 autophosphorylation, whereas CK1α autophosphorylation was largely unaffected (D). These results suggest that arsenite may have a direct effect on LRRK2 activity.LRRK2 possesses an active kinase domain, the activity of which is reportedly influenced by some genetic mutations, dimerization, and GTP binding. Our data indicate that arsenite can alter LRRK2 self-association and GTP binding while also modulating constitutive phosphorylation at Serof LRRK2 A. We subarsenite B. Whereaarsenite , C and Daffected D. These 910/Ser935 phosphorylation, triggered by kinase inhibition, alters the distribution of LRRK2 and promotes the formation of skein-like structures (A). Because there was only a single area of LRRK2 staining in each cell, we addressed whether these structures were related to centrosomes. Co-staining with γ-tubulin revealed strong co-localization of LRRK2 with large perinuclear centrosomes under arsenite treatment.LRRK2 is largely cytoplasmic but can also be associated with membranous structures , 41. Losructures , 15, a pructures . To examructures A. BecausA and C). Double staining with an anti-ubiquitin antibody revealed that LRRK2 is sequestered to ubiquitin-positive perinuclear centrosomes after acute arsenite treatment or 6-h treatment with MG132 or lactacystin (B). We further investigated whether LRRK2 is itself polyubiquitinated, by immunoprecipitation followed by Western blotting, which revealed that acute arsenite treatment induces ubiquitylation of GFP LRRK2 WT in cells (D). Similar effects were seen with proteasomal inhibition by either MG132 or lactacystin. Previous studies have reported that arsenite can induce accumulation of ubiquitylated proteins (935 phosphorylation of LRRK2 (D), suggesting that arsenite-induced dephosphorylation is not required for translocation of LRRK2 to centrosomes.The centrosome represents the cellular microtubule-organizing center, but it is also believed to be a center for proteolysis and protein folding, where factors involved in the ubiquitin proteasome pathway (UPS) are sequestered and associate with γ-tubulin , 43. LRRtacystin B. We furin cells D. Similaproteins , 47. MG1of LRRK2 D, sugges2O2. This did not alter the localization of LRRK2, indicating that increased cytosolic reactive oxygen species is not sufficient for centrosomal association (E). This also supports the proposition that loss of Ser910/Ser935 phosphorylation is not sufficient to drive translocation to centrosomes. We next examined whether kinase activity is required for centrosomal association after arsenite treatment. Kinase inhibition by LRRK2IN-1 produced skeinlike structures, as reported previously (F).To test whether oxidative stress is sufficient to drive LRRK2 intracellular translocation, we treated WT LRRK2-expressing cells with increasing concentrations of Hociation E. This aeviously ; howevereviously F.910/Ser935 phosphorylation is neither sufficient nor required for translocation of LRRK2 into centrosomes under arsenite stress (F).We further addressed whether loss of phosphorylation is required for WT LRRK2 translocation to centrosomes by treating with calyculin A prior to arsenite stress. Arsenite stress induced LRRK2 translocation to centrosomes even under calyculin A co-treatment, suggesting that loss of Sere stress F.F). Depolymerization of microtubules also did not alter arsenite-induced WT LRRK2 Ser935 dephosphorylation (data not shown).Microtubules that originate at the microtubule-organizing center mediate retrograde transport of vesicles and organelles. LRRK2 is believed to interact with microtubules through its ROC domain , 49. DepFig. 6G and H). These data suggest that recruitment of LRRK2 to the centrosome is not dependent on microtubule-associated transport mechanisms but may be influenced by association with γ-tubulin.We further investigated the recruitment of LRRK2 to centrosomes by examining a possible association with γ-tubulin. In co-immunoprecipitation experiments, arsenite treatment enhanced the association of GFP LRRK2 WT with endogenous γ-tubulin , G and HA and B). Perinuclear localization of the Y1699C and G2019S variants was evident in ∼20% of centrosomes in stable expression cell lines, whereas D2017A exhibited only minimal centrosomal localization. The S910A/S935A mutant that cannot be phosphorylated did not co-localize with centrosomes in the absence of treatment (A). Arsenite treatment induced accumulation of LRRK2 WT and variants into centrosomes stained for γ-tubulin with >95% of centrosomes positive for LRRK2 in WT as well as the R1441C, Y1699C, D2017A, and G2019S (A and C). However, the S910A/S935A mutant produced an intermediate phenotype following arsenite treatment, localizing into centrosomes as well as uncharacterized cytoplasmic foci. This suggests that whereas dephosphorylation may dynamically mobilize LRRK2, arsenite-induced stress invokes additional cell signaling events driving LRRK2 translocation to centrosomes.We also investigated the effect of LRRK2 mutations on the sequestration of LRRK2 to centrosomes in cells stably expressing mutant forms of LRRK2 . In contreatment A. Arsenid G2019S , A and CA, bottom panels). LRRK2 K1347A accumulated in centrosomes under arsenite treatment, suggesting that GTP binding is not required for arsenite-induced association of LRRK2 with centrosomes. The kinase-dead D2017A mutant was also recruited to centrosomes ; thus, centrosomal association is independent of the kinase activity of LRRK2. Collectively, these data suggest that the translocation of LRRK2 to centrosomes under arsenite is probably related to degradation of the protein by the UPS, and the pathway is independent of the activity of LRRK2 itself.To examine the requirement of GTP binding for the shift in localization following arsenite treatment, we transiently expressed LRRK2 K1347A in HEK-293T cells . This implies that arsenite-induced self-interaction may be dependent on GTP binding capacity. This is in accordance with a recent study suggesting that LRRK2 mutations that impair guanine nucleotide binding (K1347A and T1348N) attenuated the formation of dimers   , 58, whein vitro. It is possible that the loss of in vitro GTP binding following arsenite treatment is the result of the formation of a tight LRRK2 dimer or stable protein complex rendering the ROC domain inaccessible in vitro to GTP-agarose. Another possibility is that LRRK2 is GTPase-active, consistent with arsenite inducing formation of a dimer  can substitute phosphate in certain biological reactions (2), arsenate contaminants may potentially compete with phosphate in our in vitro kinase reactions. The fact that (a) autophosphorylation of the unrelated kinase CK1α is not affected in vitro and (b) arsenite treatment does not globally inhibit kinases . Interestingly, the ROC domain mutant R1441C shows some co-localization with centrosomes in untreated conditions. The fact that arsenite treatment produces a phenotype in all variants that is similar to the effect of this ROC domain mutation may suggest changes in LRRK2 conformation and autoinhibition. LRRK2 has been reported to localize in perinuclear bodies described as aggresomes (We show that arsenite treatment also causes LRRK2 to relocalize to the centrosome, co-localizing with γ-tubulin. Although pharmacological kinase inhibition by LRRK2IN-1 also induces dephosphorylation, this can result in translocation of LRRK2 to microtubule-like skein structures. Furthermore, S910A/S935A LRRK2, which is incapable of phosphorylation, exhibits a further distinct phenotype . Thus, t factors , 43. In  factors C. Interegresomes  that cangresomes  (data no2O2 levels (–Our data suggest that signaling of the cellular response to arsenite stress directly regulates LRRK2. Arsenite disrupts ATP production by inhibiting pyruvate dehydrogenase and induces generation of nitric oxide and reactive oxygen species in cells partly by mediating the increase in intracellular H2 levels –65. It a levels –. The var910/Ser935 is controlled by upstream kinases as well as a feedback loop pathway dependent on its kinase activity. Arsenite and H2O2 induce dephosphorylation of LRRK2 by stimulating phosphatases, such as PP1, and/or by modulating upstream kinase activity, such as IκB, causing dissociation from 14-3-3. This mobilizes and translocates LRRK2 to membranes, where it assembles into LRRK2 complexes (We propose a model describing arsenite-induced oxidative stress regulation of LRRK2 . Monomeromplexes . Modulat2O2 and arsenite, affect LRRK2 in several different ways that would be consistent with protein activation, namely dephosphorylation, formation of oligomeric species, and altered binding of GTP. However, arsenite, which can inhibit LRRK2 kinase directly, causes accumulation of LRRK2 at the centrosome, probably for protein degradation. We speculate that the difference between H2O2 and arsenite suggests that cells will remove an inactive LRRK2 molecule, which may be relevant for termination of cellular signaling by LRRK2 protein complexes. We therefore propose that the balance between activation of LRRK2 by oxidative stress and its degradation by the UPS is a critical mediator of protein function.In summary, our data show that two oxidative stressors, H


Since Majorana fermions are expected to reside at the core of the vortices, a close examination of the vortex dynamics in superconducting interface is of paramount importance. Here, we report the robustness of the interfacial superconductivity and 2D vortex dynamics in four as-grown and aged Bi2Te3/FeTe heterostructure with different Bi2Te3 epilayer thickness . After two years’ air exposure, superconductivity remains robust even when the thickness of Bi2Te3 epilayer is down to 3 nm. Meanwhile, a new feature at ~13 K is induced in the aged samples, and the high field studies reveal its relevance to superconductivity. The resistance of all as-grown and aged heterostructures, just below the superconducting transition temperature follows the Arrhenius relation, indicating the thermally activated flux flow behavior at the interface of Bi2Te3 and FeTe. Moreover, the activation energy exhibits a logarithmic dependence on the magnetic field, providing a compelling evidence for the 2D vortex dynamics in this novel system. The weak disorder associated with aging-induced Te vacancies is possibly responsible for these observed phenomena.The discovery of two-dimensional superconductivity in Bi Unlike conventional insulators, three-dimensional (3D) topological insulators, e.g. Bi2Se3 and Bi2Te3, are protected by the time reversal symmetry and have insulating bulk surrounded by metallic surface states with helical Dirac fermions. At the interface between the topological surface states and an s-wave superconductor, a two-dimensional (2D) spinless px + ipy topological superconductor was predicted to be induced by the proximity effect, which hosts the Majorana fermions2324252628293031i.e., Bi2Te3/FeTe heterostructure, where neither Bi2Te3 nor FeTe thin films was superconducting2Te3, their interplay is expected to provide a new platform for the search of Majorana fermions, which obey the non-Abelian statistics and may play an important role in the development of fault-tolerant quantum computerIn condensed matter physics, exotic physical phenomena usually emerge at the heterostructure interface of two materials with different topological characters. Recently, new topological materials including topological insulators12345678910111212141516181920212Te3/FeTe heterostructure using the electrical transport measurements. After two years’ air exposure, the aged heterostructures remain superconducting but with a broader transition region. A new feature appears at ~13 K after two years, and its relevance to superconductivity is further revealed by high field studies. The resistance of the Bi2Te3/FeTe heterostructure just below the transition temperature follows the Arrhenius relation, which we attribute to thermally activated flux flow (TAFF) behavior. The activation energy exhibits a logarithmic dependence on the applied magnetic field, indicating the existence of a 2D vortex system.Here, we study the robustness of the interfacial superconductivity and 2D vortex dynamics in the BiR(T) of Bi2Te3(7 nm)/FeTe in both as-grown and after-two-years measurements. Before the normalization, the normal state resistance of all after-two-years samples is generally higher: using R(18 K) as a benchmark, it is 12–64% larger compared with the as-grown samples. Since the formation of Te vacancies is always observed in the Bi2Te3 and FeTe after long-term exposure to air3536Tzero drops. Therefore, the weak disorder from the Te vacancies is not completely detrimental to superconductivity; instead it provides an interesting avenue for investigating the vortex dynamics, which will be discussed later. Compared with the as-grown result, in Bi2Te3(7 nm)/FeTe the temperature of the maximum resistance, Tmax, shifts from 12.4 K to 13 K and the resistance exhibits a two-step drop below Tmax in the after-two-years case. To provide a clearer view on these results, dR/dT curves are plotted in R(T) curve. As temperature further decreases, a sharp transition starts. We define the starting point of the sharp resistance drop as Tmid in after-two-years R(T) result, as shown in R(T) of after-two-years heterostructures with different Bi2Te3 layer thicknesses. Compared with the as-grown results in 2Te3 layer down to 3 nm as shown in dR/dT curves in the inset of Tmax shows an increase after two years and locates at ~13 K for all samples, manifesting that the new feature is indeed induced by the aging effect. Meanwhile, relative to the full superconducting transition, the shoulder is weakened as the Bi2Te3 thickness decreases as shown in the inset of To learn more about the observation of the new feature, we further measure 2Te3/FeTe heterostructure, the temperature dependent resistances in different magnetic fields are studied. R(T) results of Bi2Te3(14 nm)/FeTe in different magnetic fields applied perpendicular to the ab plane, respectively. With an increasing magnetic field, Tmax, Tmid and Tzero gradually shift to lower temperature together as superconductivity is suppressed. Similar behavior is also observed in the parallel fields. From the R(T) curves in different magnetic fields, the corresponding upper critical fields Hmax, Hmid and Hzero in both as-grown and after-two-years cases can be obtained, and the H - T phase diagrams of Bi2Te3(7 nm)/FeTe and Bi2Te3(14 nm)/FeTe are plotted in Hzero, comparing with the as-grown one. For both field directions, the as-grown Hmax locates between the after-two-years Hmax and Hmid, and all three curves show the same variation trend as magnetic field changes. It further manifests that the new feature at 13 K, i.e., the after-two-years Hmax, are relevant to the interfacial superconductivity of the heterostructure. In the study of BiS2-based superconductor LaO0.5F0.5BiS2, two-step drop of R(T) in different magnetic fields was also observed0.5F0.5BiS2 sample was polycrystalline, the origin of two anisotropic upper critical fields was attributed to the anisotropy of the grains in different directions2 atmosphere, at the expense of lowering the superconductivity transition temperature (see To further investigate the aging effect on the superconducting transition of Biture see .2Te3/FeTe heterostructure samples show a relatively broad superconducting transition regime (>3 K) in magnetic fields even for the as-grown samples. This means the mixed states exist in the transition regime and vortex dynamics studies will be important and can provide useful information about the interfacial superconducting behavior. Thermally activated flux flow (TAFF) describes the motion of vortices due to the activation over some energy barriers, e.g. pinning centers3940β-FeSe single crystal1.03Te0.55Se0.451.14Te0.91S0.090.7F0.32Sr2CaCu2O8+δ2Cu3O7-δ2ρ in the TAFF region follows the Arrhenius relation394041Comparing with type-I superconductors, our Biρ0 is a temperature independent constant, U is the thermal activation energy of the flux flow. Therefore, ρ = ln ρ0(H) − U(H)/T. At a fixed magnetic field, ln ρ(T) − 1/T plot is expected to have a linear relation in the TAFF regime. Two samples, Bi2Te3(7 nm)/FeTe and Bi2Te3(14 nm)/FeTe, are carefully studied by applying the TAFF theory, as shown in ρ(T) − 1/T axes in Tm should be equal to the Tc of the system. For the as-grown case, Tm of Bi2Te3(7 nm)/FeTe and Bi2Te3(14 nm)/FeTe are obtained as 11.6 K and 10.2 K, which are close to their Tmax = 12.4 K and 11.4 K, respectively. However, for after-two-years results (c.f.Tm of Bi2Te3(7 nm)/FeTe and Bi2Te3(14 nm)/FeTe are obtained as 9 K and 8.7 K, which are closer to their Tmid of ~ 11 K than Tmax of ~ 13 K. This indicates that as-grown samples fall into the TAFF region much faster than the after-two-years samples when the superconducting transition commences. This slower approach to the TAFF region in the after-two-years samples, to a large extent, is affected by the new feature around 13 K, although the origin of this new feature remains unclear.where lts c.f., Tm of Bρ − 1/T curves, the activation energy U(H) can be obtained from the slope value. U at different magnetic fields for Bi2Te3(7 nm)/FeTe and Bi2Te3(14 nm)/FeTe, respectively. For the as-grown samples , with H0 ≈ Hc2, as observed in other 2D systems48492Te3/FeTe interface332Te3(7 nm)/FeTe and Bi2Te3(14 nm)/FeTe samples, the fitted value of H0 decreases after two years’ air exposure as displayed in Hzero in d is the superconducting layer thickness, λ is the penetration depth and U shows an overall decrease, implying that the aging weakens the vortex pinning behavior or enhances the flux flow of the system. Interestingly, the logarithmic dependence of U on H remains valid, albeit with a lower U0  ≈ 1.05×10−3(ρ0/Tc)1/2, where ρ0 is the residual resistivity of the normal state and λ(0) is the zero temperature limit of the penetration depthλ(T) and ρ(T) do not vary strongly with age, we can estimate 2Te3(7 nm)/FeTe, take 2Te3(14 nm)/FeTe, From the linear fitting of ln U is the nucleation and the subsequent motion of the dislocation pairs associated with the vortex lattice. In this model, U is primarily the energy cost of nucleating the pair49U can be associated with the relative ease of nucleating the dislocation pairs. Since exposing Bi2Te3 and FeTe to air inevitably promotes the formation of Te vacancies35362Te3/FeTe heterostructures.One possible scenario responsible for the logarithmic magnetic field dependence of 2Te3/FeTe heterostructures. Superconductivity is robust after two years’ air exposure, even when the thickness of the Bi2Te3 layer is down to 3 nm. Comparing with the upper critical fields of the as-grown measurements, a new feature around 13 K induced by the aging effect is demonstrated to be relevant to the interfacial superconductivity. The resistance of the Bi2Te3/FeTe heterostructures below the superconducting transition temperature obeys the Arrhenius relation, which demonstrates the TAFF behaviour. The activation energy U(H) follows a logarithmic dependence on the applied magnetic field in the as-grown samples, indicating that the vortex system is two-dimensional. The logarithmic dependence remains valid in the aged samples, although U(H) becomes lower at all magnetic fields studied, leading to the conclusion that the 2D vortex system in Bi2Te3/FeTe heterostructures is robust.In conclusion, we study the superconducting properties of as-grown and aged Bi2Te3/FeTe heterostructure samples used in the experiment were grown by molecular beam epitaxy on a (111) semi-insulating GaAs substrate with an undoped 50 nm-thick ZnSe buffer layer. A 140 nm-thick FeTe layer was first deposited onto the ZnSe buffer, followed by a growth of the Bi2Te3 layer on the FeTe layer via van der Waals expitaxy. The thicknesses of the Bi2Te3 epilayer were 3 nm, 5 nm, 7 nm and 14 nm for four different wafers, respectively. Detailed structural characterizations can be found in the early worki.e. Bi2Te3(3 nm)/FeTe, Bi2Te3(5 nm)/FeTe, Bi2Te3(7 nm)/FeTe, and Bi2Te3(14 nm)/FeTe, which were measured in the second round after two years, are exactly the same strips as the ones used in the first round. All transport measurements were conducted in a Quantum Design physical property measurement system with a 14-Tesla superconducting magnet and a base temperature of 2 K.The BiHow to cite this article: Liu, H.-C. et al. Robust two-dimensional superconductivity and vortex system in Bi2Te3/FeTe heterostructures. Sci. Rep.6, 26168; doi: 10.1038/srep26168 (2016).


Here we investigate the expression and the possible physiologic role of β3-adrenergic receptor (β3-AR) in mouse kidney. The β3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that β3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the β3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of β3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of β3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of β3-AR stimulation in the kidney. Hence, β3-AR agonism might be useful to bypass AVPR2-inactivating mutations.To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β In the kidney, the antidiuretic hormone arginine vasopressin (AVP) is a critical regulator of water and electrolyte homeostasis. AVP is released from the pituitary gland into the bloodstream and binds to the type 2 vasopressin receptor (AVPR2),In the thick ascending limb of Henle, AVP stimulates NaCl reabsorption across the Na-K-Cl cotransporter (NKCC2), increasing its phosphorylation,AVPR2 gene cause X-linked nephrogenic diabetes insipidus (XNDI), characterized by constant diuresis and the risk of severe dehydration.In the CD, AVP stimulates the exocytosis of the water channel aquaporin 2 (AQP2)1-2-AR)et al.The β-adrenergic system controls several renal functions. In particular, types 1 and 2 β-adrenoreceptors  the thick ascending limb of Henle, expressing the apical NKCC2 cotransporter3-AR completely disappeared when the anti–β3-AR antibody used for immunofluorescence was preadsorbed on its immunizing peptide  or the AVP analog 1-deamino-8-d-arginine-vasopressin (dDAVP), 10−7 M, used as positive control for cAMP production (P < 0.0001).Our finding that βoduction a. Result3-AR in the inner medullary CD, BRL37344 failed to induce AQP2 apical accumulation in this portion of the CD (not shown). Importantly, the effect of BRL37344 was prevented by preincubation with either the β3-AR-selective antagonist L748,337Accordingly, we used 10 μM BRL37344 for all the following experiments performed in freshly isolated live mouse kidney slices, untreated (resting) or incubated with either dDAVP or BRL37344 b. ConfocNext, we evaluated the level of NKCC2 phosphorylation under the same experimental conditions using an antibody against the regulatory phosphothreonine residues in the N-terminus of NKCC2.3-AR stimulation, we repeated these experiments on live kidney slices from β3-AR–null mice (β3-AR−/−).3-AR functional expression, BRL37344 promoted neither AQP2 apical accumulation .To confirm that these effects of BRL37344 were ascribable to βmulation a nor NKCmulation b and c. 3-AR agonism on AQP2 and NKCC2, the major players involved in antidiuresis, prompted us to investigate whether β3-AR inactivation may affect water and electrolyte handling in the kidney in vivo. To this end, we evaluated these parameters in β3-AR−/− mice3-AR functional expression and β1-2-AR−/− knockout mice,3-AR is the only expressed β-AR. Age-matched wild-type (wt) mice of each strain were used as controls (β3-AR+/+ and β1-2-AR+/+). Strikingly, in β3-AR−/−, diuresis was higher (by 77%), urine osmolality was lower (by 30%), and water intake was increased (by 40%) compared with β3-AR+/+ , confirming that β3-AR−/− mice have less basal NKCC2 cotransporter activity to inhibit.To further support this evidence, we analyzed the effects of bumetanide injection on natriuresis in both β+/+ mice c and d. 3-AR−/− mice on a water deprivation test was comparable to that of β3-AR+/+ mice  or phosphate-buffered saline (PBS) alone (vehicle). Urine samples were collected for 4 hours after injections, the first time point at which all BRL37344-treated animals began to urinate. Diuresis, urine osmolality, and urine electrolyte excretion were analyzed and are shown in 3-AR−/− and β3-AR+/+ mice, we expressed our results as a percentage of the values measured in vehicle-treated animals of each genotype. Strikingly, BRL37344 greatly reduced the diuresis in β3-AR+/+ mice but not in β3-AR−/− mice  can explain the polyuria of β3-AR−/− mice because these mice show normal urine-concentrating abilities under a water deprivation challenge.The fact that food consumption in β+/+ mice  seems to3-AR−/− mice have normal plasma levels of Na+, K+, Cl−, and Ca++ indicating that their polyuric phenotype is neither induced by hypercalciuria/hypercalcemia nor by hypokalemia.3-AR−/− mice is not a consequence of an increased GFR, which is comparable to that in β3-AR+/+ mice. On the other hand, β1-2-AR−/− do not show alterations of urine output and osmolality, suggesting that β3-AR, rather β1-AR and β2-AR, regulate these urine parameters. However, the question whether β3-AR is more important than β1-AR and β2-AR in baseline renal function cannot be solved by the current study as we cannot compare the urine-concentrating ability of β3-AR−/− and β1-2-AR−/− mice. The 2 strains result from a different genetic background, and early studies showed that renal parameters significantly differ in mice of different strains.We also show that β3-AR activation on AQP2 subcellular localization and NKCC2 phosphorylation, BRL37344 exerts a potent antidiuretic effect in β3-AR+/+ mice but not in β3AR−/− mice, thus confirming our ex vivo data on its specific action at the β3-AR. The additional finding that in β3-AR+/+ mice, BRL37344 reduces urinary excretion of Na+, K+, and Cl− but not Ca++ and induces a 70% reduction of the urine output, whereas urine osmolality is increased by ∼40% may be explained by assuming that β3-AR stimulation promotes not only water but also salt reabsorption in the kidney. In line with this possibility, the strong reduction of urine output observed in BRL37344-treated mice is independent of the decrease in the GFR.In line with the stimulatory effect of β3-ARs are also expressed in the hypothalamusin vivo, β3-AR agonism triggers AVP-independent antidiuresis. In addition, results in live kidney slices, demonstrating that BRL37344 induces cAMP production, AQP2 plasma membrane accumulation, and NKCC2 phosphorylation/activation in the thick ascending limb of Henle, provide additional, although indirect, evidence that BRL37344 triggers its effect independently of central β3-AR activation.βThe current results cannot exclude that the effects of BRL37344 on urine output may be related to the systemic effects of the drug on arterial pressure. However, it has been shown in rats that BRL37344 reduces arterial pressure by ∼14%3-ARs are expressed in most of the AVP-sensitive nephron segments; (ii) β3-AR stimulation promotes AQP2 plasma membrane accumulation and NKCC2 activation, thus increasing water and salt reabsorption in the kidney tubule; (iii) this effect is likely mediated by an increase of intracellular cAMP; and (iv) β3-AR agonism induces antidiuresis in mice lacking AVPR2.In conclusion, our experimental data indicate that (i) in mice, β3-AR in promoting antidiuresis under physiologic conditions. Some evidence indicates that there is a synaptic contact between renal sympathetic varicosities and renal tubular epithelial cell basolateral membranes.3-ARs, upregulating NKCC2 and AQP2 activity, can enhance solutes and water reabsorption in the nephron, thus eliciting an antidiuretic effect. Although we restricted our investigation to the regulatory role of β3-ARs on AQP2 and NKCC2, the possible effect of β3-AR stimulation on other Na/Cl transporters or additional AQPs, participating in the countercurrent multiplier system, is worth further investigation.Taken together, these data suggest an unexplored role of sympathetic stimulation via the β3-AR−/− mice are polyuric but show normal urine-concentrating ability during water deprivation suggest that, under physiologic conditions, β3-AR activation by sympathetic nerves does not provide an additional mechanism, corroborating the kidney antidiuretic response to AVP. Although much work remains to be done to fully understand the role of β3-ARs in water and salt reabsorption during sympathetic activation, the current results are potentially relevant for the development of novel pharmacologic approaches to the treatment of diseases caused by AVPR2-altered signaling, including XNDI, polycystic kidney diseases, and the syndrome of inappropriate secretion of AVP. For instance, in XNDI patients, β3-AR agonists may bypass the lack of AVPR2 function, restore NKCC2 and AQP2 activity, and improve the unpaired urine concentration mechanism. It must be emphasized that patients with autosomal forms of NDIAQP2 gene would not benefit from this potential treatment.The observation that β3-AR, such as mirabegron,Further studies are needed to verify this proof-of-concept, but the ameliorative effect of BRL37344 on renal concentrating abilities of AVPR2-null mice strongly encourages studies in this direction. In particular, we suggest that agonists of the human β3-AR (cat. nos. sc-50436 and sc-1473) were obtained from Santa Cruz Biotechnology  and were previously validated for Western blotting and immunofluorescence analysis.Polyclonal antibodies against β3-AR agonist3-AR > β2-AR > β1-AR, with an approximately 20-fold and 100-fold higher selectivity for β3-AR versus β2-AR and β1-AR, respectively.BRL37344 is a well-known β3-AR.3-AR antagonist currently available.L748,337 has been reported as one of the very few antagonists with a high selectivity for βTotal RNA was extracted from mouse brown adipose tissue, the bladder, and the kidney by the TRIzol reagent and reverse-transcribed into cDNA using SuperScript VILO cDNA Synthesis Kit .3-AR intron-spanning primers were previously reported.The mouse βBrown adipose tissue, bladder, and kidney cortex/total medulla were isolated from male C57BL/6J mice and homogenized in radioimmunoprecipitation assay buffer.3-AR antibody  and anti–p-NKCC2 antibodies (1:1000) followed by horseradish peroxidase–conjugated secondary antibody.Fifteen micrograms of each lysate were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting. After blocking with 3% bovine serum albumin, blots were incubated with anti–βBlots were revealed by enhanced chemiluminescence, with Chemidoc XRS, equipped with Image Lab Software  and quantified with ImageJ software.3-AR (sc-1473), AQP2, AQP1, CLC-K, NKCC2, CD31, NCC, and phosphorylated NKCC2.Mouse kidneys were fixed with 4% paraformaldehyde in PBS at 4°C, dehydrated in graded ethanol, and embedded in paraffin wax. Serial sections, 5 μm thick, were deparaffinized, rehydrated, and subjected to immunofluorescence analysis. Antigen retrieval was performed by boiling sections in citrate buffer . After blocking with 1% bovine serum albumin in PBS for 30 minutes, sections were incubated with the primary antibodies βSections were incubated with AlexaFluor-conjugated secondary antibodies (Life Technologies). Confocal images were obtained with a confocal microscope .Kidneys from FVB/C57/129/DBA mice  were minced and enzymatically digested as previously reported.2, then stimulated for 40 minutes with dDAVP (10−7 M) or BRL37344 (10−5 M), the latter alone or after 30 minutes of preincubation with either L748,337 (10−7 M) or H89 (10−5 M). Slices were either processed for immunoblotting analysis or fixed in 4% paraformaldehyde and processed for immunofluorescence as described previously.C57BL/6J male mice were anesthetized with tribromoethanol (250 mg/kg) and killed by cervical dislocation. Kidneys were excised, and thin transversal slices (250 μm) were cut using a McIlwain Tissue Chopper . Slices were left at 37°C for 15 minutes in Dulbecco’s Modified Eagle Medium/F12 medium pre-equilibrated with 5% COAll animal experiments were approved by the Institutional Committee on Research Animal Care, in accordance with the Italian Institute of Health Guide for the Care and Use of Laboratory Animals. Mice were maintained on a 12-hour light/12-hour dark cycle, with free access to water and food.3-AR−/− and β3-AR+/+ mice1-2-AR−/− and β1-2-AR+/+ miceβfl/fl and V2Rfl/y Esr1-Cre mice) were previously described.The GFR of conscious mice was measured as previously reported.fl/y Esr1-Cre mice received a single i.p. injection of BRL37344 (0.6 mg/kg) or PBS alone and urine output and osmolality were monitored every hour for 3 hours. Urine osmolality was measured using a vapor pressure osmometer.V2RFor statistical analysis, GraphPad Prism software  was used. The statistical analysis performed is indicated in the figure legends.All the authors declared no competing interests.


Commitments have been shown to promote cooperation if, on the one hand, they can be sufficiently enforced, and on the other hand, the cost of arranging them is justified with respect to the benefits of cooperation. When either of these constraints is not met it leads to the prevalence of commitment free-riders, such as those who commit only when someone else pays to arrange the commitments. Here, we show how intention recognition may circumvent such weakness of costly commitments. We describe an evolutionary model, in the context of the one-shot Prisoner's Dilemma, showing that if players first predict the intentions of their co-player and propose a commitment only when they are not confident enough about their prediction, the chances of reaching mutual cooperation are largely enhanced. We find that an advantageous synergy between intention recognition and costly commitments depends strongly on the confidence and accuracy of intention recognition. In general, we observe an intermediate level of confidence threshold leading to the highest evolutionary advantage, showing that neither unconditional use of commitment nor intention recognition can perform optimally. Rather, our results show that arranging commitments is not always desirable, but that they may be also unavoidable depending on the strength of the dilemma. Since Darwin, the problem of explaining the evolution of cooperative behavior has been actively investigated in many fields, from Evolutionary Biology, Ecology, to Economics and Social Science. Several mechanisms responsible for the evolution of cooperation have been proposed, from kin and group selection to direct and indirect reciprocity, to structured population, and to punishment123467891011121311781513Additionally, human beings are experts in mind reading, particularly at discerning what others are perceiving and intending198162122212425c) for its co-player to receive a benefit (b > c), while a defective act (D) is to spend nothing and thus provides its co-player with no benefit. In a one-shot pairwise interaction, for each player it is better to play D, leading to a zero payoff for both, while both can obtain a higher payoff (b–c) if they simultaneously choose C. Here, we consider a strategy, which, at each interaction, attempts first to assess the co-player's intention (whether to cooperate or to defect). Only when it is not confident about what the co-player intends to do in the current interaction, does it propose to the co-player a commitment deal. A commitment proposer pays a cost of arrangement (δ)111214Thus motivated, here we investigate whether a conditional use of commitment through intention recognition can promote the emergence of cooperation in the one-shot Prisoner's Dilemma. In its simple form, a cooperative act (C) is to pay a cost (confidence threshold (θ), which is utilized to decide when intention recognition can be relied on (to choose a move), or a commitment deal needs to be arranged to clarify the co-player's intention. The questions we would like to ask here are whether such a conditional use of commitment can resolve the commitment free-riding issues, particularly when a strong commitment cannot be arranged. Furthermore, what is the appropriate confidence threshold, inasmuch the benefit and the cost of commitments and the accuracy of the intention recognition vary?We show that a conditional use of commitments, by means of first assessing intentions of the co-player, can facilitate the commitment free-riding issue, ameliorating the performance of commitment and leading to improved cooperation. The key parameter in our model is a x ∈ , is assigned to the recognition result. It defines the degree of confidence, in terms of a probability, that IRCOM predicts the co-player's intention correctly. Then, if it is confident enough about the prediction, that is if x is greater than a given, so-called, confidence threshold, θ ∈ , then in the current interaction it cooperates if the recognized intention of the co-player is to cooperate, and defects otherwise.We consider here, next to the traditional pure cooperator (C) and defector (D) strategies, a new strategy which combines intention recognition and commitment arrangement, denoted by IRCOM. In an interaction, IRCOM recognizes the intention (to cooperate or to defect) of its co-player. A confidence level, x < θ, it proposes a commitment to others and subsequently cooperates if the opponent accepts the deal. If the deal is not accepted, then this IRCOM refuses to play the game. We consider two additional commitment free-riding strategies14When IRCOM is not sufficiently confident about its co-player's intention, i.e. y = r × x, where r > 0 is dubbed the accuracy-to-confidence ratio. Assuming that the confidence, x, is uniformly distributed in , the payoff matrix for IRCOM readsM1 and M2 are the payoff matrices when IRCOM plays without proposing a commitment (i.e. when x > θ) and when it does so (i.e. when x ≤ θ), respectively. For details of the computation of the two matrices see Methods and However, the prediction being made can be wrong. We assume that prediction accuracy and confidence are positively correlated2829x ≤ θ, i.e. IRCOM is not confident enough about its intention prediction, it behaves the same as a pure commitment proposer (COMP)θ is, the more cautious IRCOM is about its intention recognition result, thereby tending to use commitments more frequently. In an interaction between IRCOM and COMP, we consider that COMP always proposes first and pays the arrangement cost Note that if θ, IRCOM is dominant, whereas the population spends most of the time in the homogenous state of IRCOM, regardless of the initial composition of the population  commitment deal.We first study the stationary distribution in a population of the six above described strategies, namely IRCOM, COMP, C, D, FAKE and FREE (see Methods). The results show that, for a large range of the confidence threshold pulation . Howeverδ, as long as an appropriate θ is adopted. Interestingly, in contrast to COMPr, as shown in r is too small. That is, whenever intention recognition can be performed with a sufficiently high accuracy, as are the case for instance in repeated games1621see also  in SI. Iδ, see δ). It is exactly when COMP does not perform well, as it is dominated by the commitment free-riders FREE and FAKE in either condition (i.e. high δ), respectivelyr, as can be seen in r. Furthermore, the improvement is substantial for large We now analyze whether and when the conditional use of commitment can actually facilitate the evolution of cooperation. To that end, we make a direct comparison in terms of the level of cooperation obtained through commitment strategies in our model, i.e. from IRCOM and COMP, and such a level in the unconditional commitment model where IRCOM is not included, see δ, as well as varying the accuracy over confidence ratio r. In general, the higher r, the lower confidence level needs to be attained to rely on intention recognition predictions. That is, as the PD becomes more beneficial and the intention recognition prediction can be carried out more accurately, a smaller confidence is exacted to rely on intention recognition, thereby avoiding the cost of arranging commitment. We also observe that this confidence level does not significantly depend on δ, see We now ask, when should one take more risk, avoiding to arrange costly commitment? In We have shown, within the context of the one-shot Prisoner's Dilemma (PD), that a conditional use of commitment based on a subjective confidence in assessing a co-player's intention can lead to improved levels of commitment and cooperation. In general, by avoiding the payment of the cost of arranging commitments whenever gaining a sufficient confidence about the co-player's intention, an evolutionary advantage can be achieved. Waiting for a too large confidence may lead to unnecessarily paying the cost, though it can be avoided. However, doing so when confidence is low allows defectors and commitment free-riders to exploit, leading to the destruction of cooperation. Our results show that the gained improvement via the intention recognition capability is more significant when the PD is less harsh, and as more accurate predictions can be achieved. Interestingly, such an improvement is most significant when the cost of arranging commitments is high, thereby overcoming the weaker cases of using the pure commitment strategy91819Our results are in line with the work in Ref. The key role of intention recognition in the current model is to allow choosing cooperative partners and avoid reliance on arranging a costly explicit commitment. In environments where partner selection is possible—that is, when people can choose with whom they associate for mutualistic endeavors—then implicit commitments are evolved, by which people behave as if they had bargained with others in order to reach an agreement, in accordance with contractualist moral psychology32Several behavioral experiments on intention based strategies exist that are closely related to our model. The experiment in Ref. θ = 0) performs quite well, complying with the results obtained in Refs. Several extensions to the present model can be described. In our model we have considered a general one-shot interaction scenario, but we envisage that as more prior experience is incorporated, for instance by observing direct or indirect past actions of the co-player, intention recognition can be performed better, thereby leading to better performance of IRCOM. Indeed, in Refs. 35Overall, our work indicates that, on the one hand, it is evolutionarily advantageous to be able to avoid arranging costly commitments whenever the co-player's intention can be assessed with sufficient confidence and accuracy. On the other hand, arranging prior commitments may be also unavoidable, depending on the strength of the dilemma, in order to reach a high level of cooperation.fitness or social success. The dynamics of strategy change in a population is governed by social learning, that is, the most successful agents will tend to be imitated by the others. There are many ways to model social learning539A with fitness Af adopts the strategy of another agent B with fitness Bf with probability given byβ controls the ‘imitation strength', i.e., how strongly the agents are basing the decision to imitate on fitness comparisons. For β = 0, we obtain the limit of neutral drift – the imitation decision is random. For large β, imitation becomes increasingly deterministic.Our analysis is based on evolutionary game theory methods for finite populations37μ > 0  and (N − k) agents using strategy B. Thus, the (average) payoff of the agent that uses A or uses B can be written as follows, respectively,XπY, stands for the payoff an agent using strategy X obtained in an interaction with another agent using strategy Y, given by the payoff matrix (9).More precisely, let k of agents using strategy A at each time step can be written asN − 1) agents using B is given by3841434648β = 0), T−(j) = T+(j) µj. Thus, BρA, = 1/N. Considering a set {1, …, q} of different strategies, these fixation probabilities determine a transition matrix ijTj≠i, = jiρ/(q − 1) and M provides the stationary distribution described above384346Now, the probability to change, by ±1, the number R  for mutual cooperation . Unilateral cooperation provides the sucker's payoff S for the cooperative player and the temptation to defect T for the defecting one. The payoff matrix corresponds to the preferences associated with the Prisoner's Dilemma when the parameters satisfy the ordering, T > R > P > S5T = b; R = b − c; P = 0; S = −c, where b and c are the benefit and cost of cooperation, respectively.The one-shot Prisoner's Dilemma can be described with the following payoff matrix:θ + cp.When proposing commitment, the average payoff of IRCOM, as the row player, reads27The payoff matrix for IRCOM when relying on intention recognition readsT.A.H., F.C.S., T.L. and L.M.P. designed the research. The models were implemented by T.A.H. Results were analyzed and improved by T.A.H., F.C.S., T.L. and L.M.P. T.A.H., F.C.S., T.L. and L.M.P. wrote the paper together.Supporting Information


The rat model of cardiac remodeling was established by subcutaneous injection of isoproterenol(ISO) at the dose of 0.25 mg·kg−1·d−1 for 7days. We found that the expression of β1- and β2-ARs decreased in the remodeling heart. The mechanisms may include the inhibition of DNA transcription and the increase of mRNA degradation. cAMP-response element binding protein(CREB) is a well-known transcription factor of β-AR. However, the expression and activation of CREB was not changed in the remodeling heart. Further, human Antigen-R (HuR), a RNA binding protein, which binds to the 3'-untranslated region of the β-AR mRNA and promotes RNA degradation, was increased in the remodeling model. And in vitro, HuR deficiency reversed the reduction of β-AR mRNA induced by ISO. Therefore, the present findings indicate that HuR, but not CREB, is responsible for the reduction of β-AR expression in ISO induced cardiac remodeling.β-adrenergic receptors (β-ARs) play an important role in cardiac remodeling, which is the key pathological process in various heart diseases and leads to heart failure. However, the regulation of β-AR expression in remodeling hearts is still unclear. This study aims to clarify the possible mechanisms underlying the regulation of β Cardiac remodeling referrers mainly to cardiac myocyte hypertrophy and interstitial fibrosis, which results in the pathologic and functional alterations of heart and promotes the progression of heart failure, ventric1 induced hypertrophy [2-AR expression and a decrease of β1-AR was showed at the early stage of Doxorubicin-induced cardiomyopathy[The change in β-AR expression is important to signaling alteration in the pertrophy . In addiomyopathy. However1- and β2-AR through binding to their UTRs [Gene expression is regulated at both transcriptional and post-transcriptional levels. cAMP-response element binding protein(CREB), a 43-kD basic leucine zipper transcription factor, regulates the transcription of many genes through binding to the CRE, an 8-bp palindromic consensus element (TGACGTCA), in their promoters.CREB alseir UTRs –18.1-and β2-ARs in ISO-induced cardiac remodeling and the regulation mechanisms at transcriptional and post-transcriptional levels.In the present study, we investigated the expression of β−1·d−1, dissolved in saline, Sigma–Aldrich, St. Louis, MO, USA) once daily for seven consecutive days. In vehicle group, rats were infused with saline as the control. All animals care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center. Rats were housed in a temperature-and humidity-controlled room on a 12 h light/dark cycle and given free access to water and food. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals[In the present experiments, 14 SD rats of 6–8 weeks old, weighing 280–320g, were randomly divided into two groups, ISO group and vehicle group. In ISO group, rats were treated with subcutaneous injection of isoproterenol equipped with a 30 MHz sectorial probe. Rats were anaesthetized with gas-mixture of 3% isoflurane.And rats were positioned on a heating platform with a small dose of isoflurane to stabilize physiological values especially heart rate.The parasternal long-axis image was viewed with B-mode, and the parasternal short-axis(SAX) image at the level of papillary muscles was acquired when the scanhead was rotated clockwise 90°. Then, the M-mode image was obtained using the M-mode cursor to measure wall thickness in the end-diastole and systole of left ventricular. We took ejection fraction(EF%), fractional shortening(FS%), LV anterior wall thickness at diastole and systole  and LV posterior wall thickness at diastole and systole as recommended by the American Society of Echocardiography. Measurements were made on three continuous cardiac cycles per loop and averaged for each data.Heart tissues fixed with 10% formalin(PH 7.4) were embedded in paraffin, sectioned into 5-μm slices, and stained with wheat germ agglutinin(WGA).The myocytes cross-sectional areas were analyzed by Image-Pro Plus  and at least 50 cardiomyocytes were measured in one mid-ventricular cross-section. The sections were stained with picrosirius red. The ratio of sirius red-stained area to total ventricular area, termed as the collagen volume fraction (CVF), was calculated by Image-Pro Plus. And we randomly selected 10 fields from each section.1-AR and HuRat 4°C overnight. Afterward, sections were incubated with the secondary antibody at 37°C for 1h, visualized with the DAB substrate system(Zhongshan Golden Bridge Biotechnology Laboratories) and counterstained with hematoxylin. The sections were then photographed with the Leica Q500 IW light microscope.Heart tissues were fixed with 4% formaldeyde, embeded in paraffin, and made in 5-μm slices. Antigen retrieval were measured by heating sections at 92–93°C for 10minutes in natrium citricum buffer(PH = 6.0). The sections were blocked with 5% goat serum and incubated with the primary antibody of β2-AR at 4°C overnight. The second antibody, donkey anti-rabbit Alexa Fluor 568,was incubated at room temperature for 1h and nuclei were counterstained with Hoechst 33258. Images were acquired using Zeiss LSM 780 confocal microscope .Heart tissues were fixed with 4% formaldeyde, embeded in paraffin, and made in 5-μm slices. After antigen retrieval, the sections were blocked with 5%BSA  and incubated with the primary antibody ofβ1-AR, β2-AR and CREB were determined by real time PCR . The reaction conditions involved denaturation at 95°C for15s, annealing at 60°C for 30s,and extension at 72°C for 30s. The PCR products were sequenced. The gene expression level was calculated by 2-ΔCT. Primers were shown in the Total RNA was isolated from the left ventricle using TRIzol reagent  and quantified by measuring the absorbance at 260nm. One microgram of total RNA was used for reverse transcription. And rat type I collagen, rat type III collagen, ANP, β1-AR(Santa Cruz Biotechnology), β2-AR(Santa Cruz Biotechnology), phospho-CREB, CREB and HuR(Santa Cruz Biotechnology) were examined by western-blot. All cell samples were lysed in lysis buffer. The protein concentration was assessed by BCA protein assay kit (Life Technologies). Proteins were subjected to electrophoresis with 10% SDS polyacrylamide gel and transferred to NC membranes. The membranes were analyzed with antibodies according to the supplier’s protocol, and immunolabelled bands were visualized by use of the Pierce ECL Western Blotting Substrate .The expressions of βPrimary neonatal rat cardiac myocytes (NRCMs) were harvested from ventricles of 1–3 day-old rats as previously described . VentricTMA-siRNA complexes were prepared in dilution buffer according to manufacturer’s protocol. Cells were transfected with either 100nMHuR siRNAs or scrambled siRNA complexes for 24h in opti-MEM I . HuR expression was assessed by western blot.The specific siRNA targeting rat HuR were synthesized with the sequence 5’-AAG AGG CAA UUA CCA GUU UCA -3’. The scrambled sequences were synthesized with the sequence 5’-UUC UCC GAA CGU GUC ACG UTT-3’.The day before transfection, cells were seeded in 6 well plate in 10%FBS/DMEM without antibiotics and grew overnight. At the day of transfection, ScreenFectResults were expressed as mean±SEM and all data were analyzed with Graph Pad Prism 5. t-test and two-way ANOVA were used to compare differences, P <0.05 was considered to be statistically significant between groups.Myocardial hypertrophy is the major structural change of cardiac remodeling. In the present study, we evaluated cardiac hypertrophy by using echocardiography. The results of echocardiography showed that LVPWd and LVAWd were increased by ISO, as wellIn addition, the size of myocytes and reactivation of fetal genes were detected to evaluate cardiac hypertrophy.ISO infusion increased the myocytes cross-sectional area. The celThese data suggested that myocardial hypertrophy was induced by subcutaneous administration of ISO(0.25mg/kg/d) up to seven days.As previously mentioned, cardiac fibrosis is another structural change of cardiac remodeling. We quantitatively evaluated cardiac fibrosis with two methods, including robust morphological and biochemical assay. With picrosirius red staining, the collagen volume fraction of left ventricular was increased by ISO infusion . The bio1-AR was significantly reduced, 3 α2-ARs , and 3 β-ARs [1- and β2-AR, the major subtypes in heart, is a main mechanism in the progression of cardiac remodeling[Cardiac remodeling appears in many pathologic conditions including myocardial infarction, hypertension, hypertrophic cardiomyopathy, dilated cardiomyopathy and diabetic cardiomyopathy. The excβ2and β3). Activatemodeling. Thus, w1-AR and β2-AR mRNA expression was down-regulated in cardiac remodeling model. Consistent with our result, β1-AR and β2-AR mRNA decreased by 30% and 42% respectively with isoproterenol treatment in H9C2 cell[2-AR was constant in the failing heart[Besides effectors, gene expressions of β-ARs were regulated by stress stimulation. In the H9C2 cell. In patiH9C2 cell.Cardiac 2-AR agonist[1- and β2-AR mRNA expression and CREB was involved in this phenomenon in rat lung[CREB, binding to the cAMP response element, mediateR agonist. In respR agonist.Further, rat lung.The phos rat lung. However rat lung, 37. How rat lung, 39.1-AR[2-AR[1-AR mRNA induced by ISO treatment. Meanwhile, β2-AR expression was regulated partially through HuR. It is possible that β2-AR expression was regulated by other mechanisms, like receptor degradation [The post-transcriptional regulation was another major way to control the expression of β-ARs. HuR is an important protein to regulate gene expression at the post-transcriptional level, which could regulate the mRNA stability of β-AR. HuR, consisting of four members, HuA(HuR),HuB, HuC and HuD, binds to AUUUA pentamers or derivative sequences.HuR reco1-AR,and was 1-AR[2-AR. In our radation .1- and β2- AR is decreased, which is regulated by HuR at post-transcriptional level rather than by CREB at transcriptional level  LVPWs: LV posterior wall thickness at systole. (B) LVAWs: LV anterior wall thickness at systole.(TIF)Click here for additional data file.S2 Fig(A)The myocytes size was evaluated. The myocytes was stimulated with ISO for 12h, 24h and 48h. (B)The myocytes size was quantified for S2A Fig. n = 4, * P<0.05, *** P<0.001 vs CON.(TIF)Click here for additional data file.