Glutamate receptor, ionotropic, NMDA2B (GRIN2B), encoding the GluN2B/NR2B subunit of N-methyl-D-aspartate receptors (NMDARs), with various neurodevelopmental disorders, including autism spectrum disorders (ASDs), but the underlying mechanisms remain unclear. In addition, it remains unknown whether mutations in GluN2B, which starts to be expressed early in development, induces early pathophysiology that can be corrected by early treatments for long-lasting effects. We generated and characterized Grin2b-mutant mice that carry a heterozygous, ASD-risk C456Y mutation (Grin2b+/C456Y). In Grin2b+/C456Y mice, GluN2B protein levels were strongly reduced in association with decreased hippocampal NMDAR currents and NMDAR-dependent long-term depression (LTD) but unaltered long-term potentiation, indicative of mutation-induced protein degradation and LTD sensitivity. Behaviorally, Grin2b+/C456Y mice showed normal social interaction but exhibited abnormal anxiolytic-like behavior. Importantly, early, but not late, treatment of young Grin2b+/C456Y mice with the NMDAR agonist D-cycloserine rescued NMDAR currents and LTD in juvenile mice and improved anxiolytic-like behavior in adult mice. Therefore, GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, NMDAR-dependent LTD, and anxiety-like behavior, and early activation of NMDAR function has long-lasting effects on adult mouse behavior.Extensive evidence links  Mice that carry a heterozygous, autism spectrum disorder-risk C456Y mutation in the NMDA receptor (NMDAR) subunit GluN2B show decreased protein levels, hippocampal NMDAR currents, and NMDAR-dependent long-term depression and have abnormal anxiolytic-like behavior. Early, but not late, treatment of the young mice with the NMDAR agonist D-cycloserine rescues these phenotypes. Glutamate receptor, ionotropic, NMDA1 (GRIN1); GRIN2A; or GRIN2B genes encoding the main NMDAR subunits GluN1/NR1, GluN2A/NR2A, and GluN2B/NR2B, respectively.Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by social deficits and repetitive behaviors. Although a large number of ASD-risk mutations have been reported , the mecGRIN2B is one of the most frequently mutated ASD-risk genes, belonging to category 1 in the Simons Foundation Autism Research Initiative (SFARI) gene list, and shows stronger impacts on ASD than mutations in GRIN1 or GRIN2A [GRIN2B has been extensively associated with various neurodevelopmental disorders, including developmental delay, intellectual disability, attention-deficit/hyperactivity disorder, epilepsy, schizophrenia, obsessive-compulsive disorder, and encephalopathy [Among known NMDAR subunit genes, r GRIN2A –16. In aalopathy ,15,17,18GRIN2B in diverse brain disorders, mice carrying a conventional homozygous deletion of Grin2b display impaired suckling, neonatal death during postnatal day (P) 1–3, and impaired hippocampal long-term depression (LTD) in neonates [Grin2b deletion to specific cell types and developmental stages to circumvent the strong developmental impacts of Grin2b deletion, which revealed the important roles of GluN2B in the regulation of long-term potentiation (LTP), LTD, and cognitive behaviors [Grin2b deletion and reported impaired LTP at the mutant hippocampal fimbrial-CA3 synapses [Grin2b overexpression has been shown to enhance LTP and learning and memory in mice [In line with the strong involvement of neonates . Similarneonates . These eehaviors –23. Notasynapses , althoug in mice . These rGrin2b mice largely analyzing the synaptic and behavioral impacts of a homozygous Grin2b deletion, GRIN2B mutations identified in human brain disorders are preponderantly heterozygous mutations, and synaptic and behavioral phenotypes of heterozygous Grin2b-mutant mice remain largely unexplored. In addition, human GRIN2B mutations are often missense mutations that induce a single amino acid change in the encoded protein, again distinct from the null or truncation mutations previously studied in mice. Although many of the missense mutations of NMDAR subunits have been characterized in vitro [However, dissimilar to the previous studies on in vitro , their iGrin2b gene, a de novo mutation identified in a male individual with ASD and intellectual disability [Grin2b+/C456Y) showed substantial decreases in GluN2B protein levels, suggestive of mutation-induced protein degradation in vivo. Currents of GluN2B-containing NMDARs and NMDAR-dependent LTD (but not LTP) were also decreased, revealing sensitivity of LTD to GluN2B haploinsufficiency. Behaviorally, these mice showed normal social interaction but enhanced anxiety-like behavior in pups and contrasting anxiolytic-like behavior in juveniles and adults. These synaptic and behavioral effects were largely mimicked by an independent mouse line carrying a conventional heterozygous Grin2b deletion (Grin2b+/–). Importantly, early, but not late, treatment of young mice (P7–16) with the NMDAR agonist D-cycloserine normalized NMDAR currents and LTD in juvenile Grin2b+/C456Y mice and improved anxiolytic-like behavior in adult Grin2b+/C456Y mice, supporting the emerging concept in the field of neurodevelopmental and neuropsychiatric disorders that early and timely correction of key pathophysiological deficits is important for efficient and long-lasting beneficial effects.In the present study, we generated and characterized a knock-in mouse line carrying an ASD-risk mutation (GluN2B-C456Y) in the sability . These hGRIN2B gene leading to a C456Y mutation in the GluN2B subunit of NMDARs disrupts a disulfide bond within a loop residing at the interface between the amino-terminal domain (ATD) and ligand-binding domain (LBD) [Xenopus oocytes and human embryonic kidney 293 (HEK-293) cells have shown that the GluN2B-C456Y mutation induces multiple changes in the GluN2B protein, including protein degradation, limited surface trafficking, and gating alterations of GluN2B-containing NMDARs [Structural analysis has suggested that a missense mutation in the in (LBD) . In addig NMDARs .Xenopus oocytes yielded overall similar results. Specifically, structural modeling, based on the known structure of GluN1/GluN2B NMDARs [Our own structural investigation and functional characterization of the GluN2B-C456Y protein using B NMDARs –30, reveB NMDARs –30. It iDiheteromers produced by coexpressing GluN2B-C456Y and wild-type (WT) GluN1 yielded NMDAR currents that were <1% of the currents produced by WT GluN1/GluN2B diheteromers , thus reGrin2b gene (Grin2bC456Y/C456Y) mice showed neonatal death at P7 , similar to the case for mice with a conventional homozygous Grin2b deletion [Grin2b+/C456Y) mice were produced with the expected mendelian ratios and showed normal survival and growth.To explore the impacts of the C456Y mutation on the stability or function of GluN2B in mice, we generated a knock-in mouse line carrying the C456Y mutation in the n2b gene . Homozygdeletion ,20. In cGrin2b+/C456Y mice at embryonic day 20 (E20) and several postnatal stages  indicated approximately 30%–50% reductions in the levels of GluN2B protein (Grin2b or Grin1 (encoding GluN1)  . These rGrin2b+/C456Y mice. This mutation caused significant decreases in the ratio of NMDAR/AMPA receptor (AMPAR)-mediated evoked excitatory postsynaptic currents (EPSCs), tau of NMDAR current decay, and the amount of ifenprodil-sensitive current of GluN2B-containing NMDARs at Schaffer collateral-CA1 pyramidal (SC-CA1) synapses  and triheteromeric (1/2A/2B) NMDAR complexes [However, these observations are not congruent with human cases of revalent ,11–17. Tomplexes ,34,35.Grin2b+/C456Y hippocampus at juvenile stages (P16–33).To address this question, we measured several forms of synaptic plasticity in addition to low-frequency stimulation (LFS)-LTD, including LTP induced by high-frequency stimulation (HFS-LTP), theta burst stimulation–induced LTP (TBS-LTP), and metabotropic glutamate receptor (mGluR)-dependent LTD (mGluR-LTD) in the CA1 region of the Grin2b+/C456Y SC-CA1 synapses compared with WT mice  at SC-CA1 synapses of 56Y mice . It alsosynapses . These rGrin2b+/C456Y mice display altered density or morphology of excitatory synapses. However, there were no genotype differences in the density and morphology  of postsynaptic density (PSD) structures in the CA1 region of the WT and Grin2b+/C456Y hippocampus (P21)  , electroGrin2b+/C456Y synapses.A previous study employed single-neuron gene deletion to show that GluN2A and GluN2B distinctly regulate the number and strength of functional excitatory synapses . In addiGrin2b+/C456Y mice compared with those of WT animals  and miniature inhibitory postsynaptic currents (mIPSCs) did not differ in CA1 pyramidal neurons in the hippocampus of  animals , suggest animals , suggestfference .Grin2b+/C456Y CA1 pyramidal neurons, as shown by current-firing curves  in home cages with bedding but showed no repetitive self-grooming in a novel chamber without bedding , indicatGrin2b deletion (Grin2b+/–) to see whether the behavioral phenotypes observed in Grin2b+/C456Y mice could be reproduced. This mouse line has been used previously to demonstrate that a homozygous null Grin2b mutation entirely eliminates GluN2B protein and causes severe phenotypes, including impaired suckling and neonatal death [We next employed an independent mouse line carrying a conventional heterozygous al death .Grin2b+/–mice showed decreased (approximately 50%) whole-brain levels of the GluN2B, but not GluN1 or GluN2A, subunit of NMDARs at P14 and P21 (Grin2b+/C456Y mice in which both GluN2B and GluN1 levels were decreased (P14 and P21). Behaviorally, adult male Grin2b+/–mice showed phenotypes that were largely similar to those observed in adult male Grin2b+/C456Y mice, including hypoactivity and moderately anxiolytic-like behavior  of GluN2B function. The small differences in the behaviors of the two mouse lines may reflect minor effects attributable to the specific mutation/deletion in the Grin2b gene.These results indicate that the heterozygous C456Y mutation and conventional Grin2b+/C456Y pups (P4–12) were tested for the emission of USVs upon mother separation, a measure of anxiety in rodents responsive to anxiolytic medications [Grin2b+/C456Y pups display anxiety-like behaviors, similar to the anxiety symptoms comorbid with human ASD [ASD is characterized by early onset of core and comorbid symptoms. When uman ASD .Grin2b+/C456Y juveniles (P18–26) were subjected to a battery of behavioral tests, they displayed hypoactivity, similar to adult mice, and, notably, strong anxiolytic-like behavior in the center region of the open-field arena  observed in adult (2–4-month-old) Grin2b+/C456Y mice. This hypothesis could be tested by normalizing the reduced NMDAR function and NMDAR-dependent LTD in early stages and examining whether these corrections are associated with behavioral rescues at late stages. To this end, we used D-cycloserine, a partial agonist at the glycine-binding site of NMDARs with increasing potential for the treatment of neurological and neuropsychiatric disorders [The reduced NMDAR function and LTD observed in young (2–3-week-old) isorders .Grin2b+/C456Y mice. In hippocampal slices from young Grin2b+/C456Y mice, application of D-cycloserine (10 μM), which can still activate mutant GluN2B-C456Y receptors , administered orally twice daily for 10 days (P7–16), followed by measurements of NMDA/AMPA ratio, paired-pulse facilitation, and LFS-LTD in juvenile mice (P17–21) and behavioral experiments in adult mice (>P56) (Grin2b+/C456Y SC-CA1 synapses in juvenile mice (P16–21), without affecting paired-pulse facilitation  . Early Dlitation . WT synaGrin2b+/C456Y mice in the elevated plus-maze test, without affecting WT mice (Grin2b+/C456Y mice (Grin2b+/C456Y mice because the time window for the treatment fell after the early time window for pup USV (P4–12).In addition, early D-cycloserine treatment improved anxiolytic-like behavior in  WT mice . In cont56Y mice . We coulGrin2b+/–mice carrying conventional heterozygous Grin2b deletion were tested for D-cycloserine-dependent rescue of synaptic and behavioral deficits, early chronic D-cycloserine treatment  normalized the decreased LFS-LTD at Grin2b+/C456Y SC-CA1 synapses, without affecting WT synapses  by the chronic drug treatment procedures .When synapses . Early Db+/–mice , similarb+/–mice  because Grin2b+/C456Y mice display decreased GluN2B levels at an adult stage (P56), suggesting that the continuing decrease in GluN2B levels in adult mutant mice, in addition to the reduced LFS-LTD in young mutant mice, might be associated with anxiety-like behavior or hypoactivity. To test this, we attempted to enhance NMDAR function in adult Grin2b+/C456Y mice by treating with D-cycloserine. In these experiments, we first used an acute treatment paradigm because acute D-cycloserine treatment has been previously shown to rescue ASD-like behaviors in many mouse models of ASD [s of ASD ,10,48–50Grin2b+/C456Y mice in open-field or elevated plus-maze tests  in the adult stage had no effect on anxiolytic-like behavior or hypoactivity in ze tests ; it also56Y mice . HoweverIn this study, we demonstrated that mice carrying a heterozygous C456Y mutation in the GluN2B subunit of NMDARs, an ASD-risk mutation in humans, exhibit decreased GluN2B and GluN1 protein levels, diminished currents of GluN2B-containing NMDARs, and reduced LFS-LTD. This mutation also induced anxiolytic-like behavior that can be corrected by early, but not late, D-cycloserine treatment that restores NMDAR function and NMDAR-dependent LTD.Grin2b+/C456Y brain at various developmental stages. A previous study on multiple GluN2A/B mutations using structural analysis and oocyte/HEK cell experiments reported similar findings on the impact of the GluN2B-C456Y mutation [A key finding in our study is that the GluN2B-C456Y mutation induces substantial degradation of the GluN2B protein in mice. This conclusion is supported by the measurement of GluN2B protein levels in the mutation . Interesmutation . Our stuGrin1 mRNA levels. It may occur because the reduction in GluN2B protein levels may lead to a situation in which some GluN1 proteins that can no longer associate with GluN2B to form heteromeric NMDAR complexes become destabilized and degraded. It is possible that some GluN1 proteins may fail to associate with mutant GluN2B proteins beginning in the endoplasmic reticulum [Our results further reveal an impact of reduced GluN2B protein levels on GluN1 protein levels, although the magnitude of this latter decrease was less than that of GluN2B. This further supports the previously reported importance of GluN2B in the maintenance of normal levels of GluN1 ,23. Thiseticulum  and are eticulum –54. Alteeticulum ,56.Complicating the situation is the fact that GluN2B can form a triheteromeric complex with GluN1 and GluN2A ,35,57,58Grin2b+/C456Y mice would affect both diheteromic and triheteromeric NMDAR complexes differentially in different brain regions, cell types, and developmental stages.In addition, given that diheteromic and triheteromeric NMDAR complexes display distinct biophysical and pharmacological properties in different spatiotemporal contexts ,61–63, tGrin2b+/C456Y hippocampal SC-CA1 synapses. Previous studies on genetic Grin2b deletion and its impacts on LTD found near-complete impairments of LTD in neonate mice (P1–3) carrying a conventional homozygous Grin2b deletion [Grin2b deletion restricted to Ca2+/calmodulin dependent protein kinase II (CaMKII)-positive principal neurons in the cortex and hippocampal CA1 region [Grin2b-mutant mouse study differs from those of the previous studies in the following respects: (1) use of a patient-derived knock-in mutation rather than a conventional, or conditional, gene deletion; (2) use of heterozygous instead of homozygous mutant mice  measured during a late juvenile stage (P27–33) could be because the postnatal switch from GluN2B to GluN2A by neuronal activity may be largely complete at this stage [Grin2b suppresses LTD and extends it by demonstrating that the patient-derived heterozygous GluN2B-C456Y mutation induces a selective reduction in LTD by approximately 50% in juvenile mice.Another key finding of our study is the reduced NMDAR-dependent LFS-LTD by about 50% at deletion  and in a1 region . The des not LTD ); and . This ds on LTD , differes on LTD ,62, and s on LTD ,72,73.Grin2b+/C456Y mice may contribute to the reduced LTD at SC-CA1 synapses. However, the previous single-neuron KO study employed AAV-dependent gene KO at the mouse age of P0–1, leaving GluN2B expression and function at embryonic stages unaffected. In addition, the single-neuron KO study would lead to homozygous (not heterozygous) Grin2b deletion, which might also affect the results.Notably, a recent study employing single-neuron gene knockout (KO) has reported that GluN2A or GluN2B is not critically required for ionotropic or non-ionotropic (not involving NMDAR-dependent ion flow –76) NMDAGrin2b+/C456Y mice showed moderately enhanced self-grooming, a core ASD-like behavior, in home cages with bedding, but normal self-grooming in a novel chamber without bedding, suggesting that these mice display moderately enhanced self-grooming that is suppressed by a novel environment. Moreover, enhanced self-grooming in home cages with bedding was observed in adult but not juvenile Grin2b+/C456Y mice, suggesting that repetitive self-grooming develops late in life in Grin2b+/C456Y mice and thus is unlikely to be ameliorated by early D-cycloserine treatment.Grin2b+/C456Y mice showed normal social approach, social novelty recognition, and social interaction in three-chamber and direct social-interaction tests. In addition, these animals showed normal social communication (USVs) during courtship. Juvenile Grin2b+/C456Y mice also displayed normal social interaction in the juvenile play test and spent normal amounts of time with reunited mothers. It is possible, however, that the social tests and variables that we employed in the present study may not be sensitive enough to detect certain social deficits.Contrary to our expectations, Grin2b+/C456Y mice showed hypoactivity in the open-field test, suggesting that this phenotype is established early (in juvenile or earlier stages) and persist into adulthood. Adult Grin2b+/C456Y mice show anxiolytic-like behavior in the elevated plus-maze test but normal anxiety-like behaviors in open-field and light-dark tests. Juvenile Grin2b+/C456Y mice show anxiolytic-like behavior in the open-field test, suggesting that adult and juvenile Grin2b+/C456Y mice show normal or anxiolytic-like behaviors. In contrast, Grin2b+/C456Y pups display strongly increased USV calls upon mother separation, suggestive of anxiety-like behavior. Therefore, the anxiety-like behavior of Grin2b+/C456Y pups seems to be rapidly weakened as these mice grow up, whereas self-grooming slowly develops at an adult stage, pointing to the contrasting trajectories of two important ASD-related phenotypes (anxiety-like behavior and self-grooming). How the early anxiety-like behavior in Grin2b+/C456Y pups is weakened or reversed as the pups grow into juveniles and adults remain unclear. This age-dependent reversal might reflect compensatory changes trying to overcome the over-activation of anxiety-related neural circuits. Although further details remain to be determined, our results are in line with the fact that anxiety is one of the key comorbidities of ASD [Both adult and juvenile s of ASD ,78 and ts of ASD ,79,80. Ns of ASD , implicas of ASD .Grin2b+/–mice  mice showed largely similar behaviors compared with those observed in Grin2b+/C456Y mice. Similar behaviors include hypoactivity in the open-field test and normal anxiety-like behavior in open-field and light-dark tests, but anxiolytic-like behavior of Grin2b+/–mice in the elevated plus-maze was much weaker than that in Grin2b+/C456Y mice. Biochemically, Grin2b+/–mice showed decreased levels of GluN2B but not GluN1 at P14 and P21, unlike the concomitant decreases in GluN2B and GluN1 in Grin2b+/C456Y mice, which may lead to subtle differences in synaptic and behavioral dysfunctions in these two mouse lines.Grin2b+/C456Y mice could not be compared with the symptoms of the human individual carrying GluN2B-C456Y mutation as they were minimally described in the previous study other than the fact that the mutation is a de novo mutation from a male individual with autism and intellectual disability [The behavioral phenotypes of sability . HoweverGrin2b+/C456Y mice normalizes NMDAR function and LTD in juvenile Grin2b+/C456Y mice and anxiolytic-like behavior in adult Grin2b+/C456Y mice. In addition, late acute treatment of adult Grin2b+/C456Y mice with D-cycloserine has no effect on abnormal behaviors probably because GluN2B expression is decreased at adult stages, and NMDAR-dependent LTD is difficult to induce at adult stages likely due to the switch of GluN2B to GluN2A [Grin2b+/C456Y mice could not be tested because the chronic treatment procedure seemed to increase anxiety levels in these mice, blunting the baseline difference between WT and mutant mice. In addition, we could not test whether the increased USV calls in the mutant pups are associated with the reduced NMDAR function because the time window for early D-cycloserine treatment (P7–16) fell behind that for pup USV testing (P4–12). Together, these results suggest that early correction of NMDAR function and NMDAR-dependent LTD in young mice leads to long-lasting improvement of anxiolytic-like behavior in adult mice. Early treatment seems to be particularly important, not only because it has long-lasting effects, eliminating the necessity of repeated drug administration, but also because the small time window during which treatment is efficient appears to occur only during early developmental stages. Indeed, LTD is known to be most prominent during an early period (approximately 2–3 weeks) of postnatal brain development in mice and becomes weaker as the brain progressively matures and the ratio of GluN2B/GluN2A expression decreases [Importantly, our study suggests synaptic mechanisms that may be associated with the anxiolytic-like behavior, namely suppressed NMDAR functions and LFS-LTD at an early stage. In support of this hypothesis, early chronic D-cycloserine treatment of young o GluN2A ,84. The ecreases ,85–87.Shank2-mutant mice show increased NMDAR function (in contrast to decreased NMDAR function in later stages) [Shank2-mutant mice [Our findings are in line with the emerging concept that early and timely correction of key pathophysiological deficits in young mice is critical for the long-lasting and efficient rescue of synaptic and behavioral phenotypes in adult mice. For instance, early chronic fluoxetine treatment to restore reduced serotonin levels in young mice carrying a 15q11-13 duplication, a human ASD-risk mutation, has been shown to induce long-lasting normalization of serotonin levels and abnormal behaviors in adult mice . Similar stages) , and earant mice .Grin2b+/C456Y mice. In line with this idea, Grin2b is widely expressed in the brain [Our data indicate that LFS-LTD are similarly decreased in the hippocampus and mPFC. These results suggest that the decreased NMDAR function and LFS-LTD in the hippocampus may represent a proxy for changes occurring in other brain regions and that decreased NMDAR function and LFS-LTD in many brain areas, additional to the hippocampus, could contribute to the behavioral changes  observed in he brain –97.Grin2b+/C456Y mice be related to ASD pathophysiology? GluN2B-containing NMDARs that mediate large calcium influx are strongly expressed during early brain developmental stages to promote synapse and neuronal maturation through mechanisms, including posttranslational modification and gene expression [Grin2b+/C456Y mice would suppress these critical molecular and cellular early events. In addition, NMDAR-dependent LTD during early brain development is well known to sharpen neuronal circuits by promoting weakening of less active synapses and strengthening of more active synapses through redistribution of synaptic protein resources between these synapses [Grin2b+/C456Y mice would suppress LTD-dependent synapse-pruning and circuit-sharpening processes, leading to brain malfunctions and abnormal behaviors. This prediction, based on in vivo results, might apply not only to GluN2B-C456Y-related cases of ASD [Grin2b+/C456Y mice.Lastly, GluN2B-C456Y is a strong ASD-risk mutation . How migsynapses ,84. Thers of ASD ,11–16 bus of ASD ,17. How s of ASD . HoweverIn conclusion, the heterozygous ASD-risk mutation, GluN2B-C456Y, leads to decreased GluN2B protein levels, diminished currents of GluN2B-containing NMDARs, and reduced NMDAR-dependent LTD in young mice, as well as abnormal, anxiolytic-like behavior in adult mice. In addition, early D-cycloserine treatment of young mutant mice correcting NMDAR function and NMDAR-dependent LTD leads to long-lasting improvement of anxiolytic-like behaviors in adult mice.All animals were bred and maintained according to the Requirements of Animal Research at KAIST and all procedures were approved by the Committees of Animal Research at KAIST (KA2016-31).Grin2b+/cassette, Grin2b+/cassette mice were crossed with Protamine-Flp mice (C57BL/6J), which yielded floxed heterozygote mice (Grin2b+/C456Y).Grin2b knock-in mice under the genetic background of C57BL/6J carrying C456Y mutation in exon 6 with Frt sites and cassette were designed and generated by Biocytogen  Molecular modeling of the GluN2B-C456Y protein in complex with the GluN1 subunit of NMDARs. Note that the two cysteine residues in patch 1 of the LBD and patch 2 of the ATD in the WT GluN2B protein form a disulfide bond that strengthens the interaction between LBD and ATD, a bond that is disrupted by the GluN2B-C456Y mutation in patch 1 of the LBD. ATD, amino-terminal domain; LBD, ligand-binding domain; NMDAR, N-methyl-D-aspartate receptor; WT, wild type.(TIF)Click here for additional data file.S2 FigXenopus oocytes. Note that the amount of the mutant currents is <1% of the WT currents, despite the fact that mutant currents were recorded 1 day later than WT . n = 73 oocytes for WT (5.70 ± 0.61 μA) and 59 oocytes for C456Y (0.039 ± 0.004 μA), ***P < 0.001, Mann-Whitney. (B) The GluN2B-C456Y mutation increases maximal open probability, as assessed by measuring MK-801 inhibition kinetics. n = 22 oocytes for WT  and 19 oocytes for C456Y , ***P < 0.001, Mann-Whitney. (C) The GluN2B-C456Y mutation reduces the sensitivity to extracellular protons. n = 4 oocytes for WT (pH IC50 = 7.49 ± 0.016) and 5 oocytes for C456Y (pH IC50 = 7.11 ± 0.0075), *P = 0.016, Mann-Whitney. (D) The GluN2B-C456Y mutation decreases the spermine-dependent potentiation. n = 5 oocytes for WT  and 4 oocytes for C456Y ,*P = 0.016, Mann-Whitney. (E) The GluN2B-C456Y mutation does not affect the sensitivity to glutamate. n = 4 oocytes for WT (EC50 = 1.75 ± 0.04 μM) and 3 oocytes for C456Y (EC50 = 1.86 ± 0.02 μM), P = 0.23, Mann-Whitney. (F) The GluN2B-C456Y mutation decreases the sensitivity to glycine. n = 4 oocytes for WT (EC50 = 0.38 ± 0.017 μM) and 9 oocytes for C456Y (EC50 = 1.13 ± 0.049 μM), **P = 0.007, Mann-Whitney. (G) The GluN2B-C456Y mutation has minimal effect on the sensitivity to extracellular zinc. n = 11 oocytes for WT (IC50 = 0.68 ± 0.07 μM) and 11 oocytes for C456Y (IC50 = 0.97 ± 0.1 μM), ***P < 0.001, Mann-Whitney. (H) D-cycloserine is a partial agonist at GluN2B-C456Y mutant receptors. Currents recorded in 100 μM glutamate plus 100 μM D-cycloserine were normalized to currents recorded in 100 μM glutamate + 100 μM glycine (no D-cycloserine). n = 9 oocytes for WT (relative current: 0.57 ± 0.005) and 9 oocytes for C456Y (relative current: 0.40 ± 0.006), ***P < 0.001, Mann-Whitney. The numerical data underlying this figure can be found in 50, half maximal effective concentration; IC50, half maximal inhibitory concentration; NMDAR, N-methyl-D-aspartate receptor; ns, not significant; WT, wild type.(A) The GluN2B-C456Y mutation strongly decreases diheteromeric GluN1/GluN2B NMDAR currents in (TIF)Click here for additional data file.S3 Fig(A) Knock-in strategy for the GluN2B-C456Y mutation in mice. WT exon 6 was replaced with a mutant exon 6 containing the C456Y mutation. (B) PCR genotyping of homozygous (“Homo”) and HT KI mice. Ex, exon; Frt, flippase target site; Homo, homozygous; HT, heterozygous; KI, knock-in; Neo, neomycin gene; WT, wild type.(TIF)Click here for additional data file.S4 FigGrin2b+/C456Y brain at multiple developmental stages  were immunoblotted with the indicated antibodies. For quantification (bar graphs), average levels of GluN1, Glu2A, and Glu2B proteins from Grin2b+/C456Y mice were normalized to those from WT mice. n = 4 mice for WT and HT, *P < 0.05, **P < 0.01, ***P < 0.001, Student t test. (B) Normal levels of Grin2b and Grin1 (encoding GluN1) mRNAs in WT, HT, and homozygous (“Homo”) KI embryos (E20), as indicated by the results of RT-qPCR reactions targeting Grin2b mRNA exons 3, 4, 11, or 14, and Grin1 mRNA exons 3, 7, or 12. n = 4 mice for WT, 4 for HT, and 3 for Homo, one-way ANOVA with Tukey’s test. The numerical data underlying this figure can be found in (A) Crude synaptosomal fractions from the (TIF)Click here for additional data file.S5 FigGrin2b+/C456Y mice (P18–20). n = 15 neurons from 3 mice for WT and 15 (3) for HT, Mann-Whitney test (frequency) and Student t test (amplitude). (B) Normal mIPSCs in CA1 neurons of Grin2b+/C456Y mice (P21–23). n = 15 (3) for WT and HT, Student t test. (C) Normal sEPSCs in CA1 neurons of Grin2b+/C456Y mice (P22–24). n = 15 (3) for WT and 14 (4) for HT, Mann-Whitney test. (D) Normal sIPSCs in CA1 neurons of Grin2b+/C456Y mice (P22–24). n = 13 (3) for WT and 18 (4) for HT, Mann-Whitney test (frequency) and Student t test (amplitude). (E) Normal ratio of evoked IPSCs over evoked EPSCs in the CA1 region of Grin2b+/C456Y mice (P20–22). n = 8 (4) for WT and 8 (3) for HT, Mann-Whitney test. (F) Normal neuronal excitability in CA1 neurons of Grin2b+/C456Y mice (P21–23), as indicated by the current-firing relationship. n = 13 (3) for WT and 13 (4) for HT, two-way ANOVA. The numerical data underlying this figure can be found in (A) Normal mEPSCs in CA1 neurons of (TIF)Click here for additional data file.S6 FigGrin2b+/C456Y mice (P90–114) in the learning (A) and probe (B–E) phases of the initial (A–C) and reversal  sessions of the Morris water maze, as shown by time taken to escape to the platform, percent of time spent in target quadrant, and number of crossing over the former platform location. n = 12 mice for WT and HT, two-way ANOVA with Sidak’s test and Student t test. (F) Normal novel object–recognition memory in Grin2b+/C456Y mice (P70–80), as shown by the percent of time spent exploring a novel object relative to a familiar object. n = 12 mice for WT and HT, Student t test. The numerical data underlying this figure can be found in (A–E) Normal spatial learning and memory in (TIF)Click here for additional data file.S7 FigGrin2b+/C456Y mice (P79–80) in the three-chamber test under both bright-light and dark conditions, as shown by time spent sniffing the target and time spent in the chamber with the target. n = 8 mice for WT and HT , **P < 0.01, ***P < 0.001, two-way ANOVA with Sidak’s test. (I–K) Normal social interaction in freely moving Grin2b+/C456Y mice (P62–89) in the direct social-interaction test, as shown by time spent in nose-to-nose sniffing or following and total time spent in social interaction . n = 11 mice for WT and 13 for HT, Student t test (except for Mann-Whitney test for following). (L–N) Normal courtship USVs in male Grin2b+/C456Y mice (P74–101) upon encountering a novel female mouse, as shown by the number of calls, duration of each call, and latency to the first call. n = 21 mice for WT and 17 for HT, Student t test . (O–P) Enhanced repetitive self-grooming  by Grin2b+/C456Y mice (P68–88) in home cages with bedding, but normal self-grooming in a novel chamber without bedding, as shown by time spent self-grooming (or digging). n = 21 mice for WT and 17 for HT for self-grooming in home cages with bedding, n = 16 mice for WT and HT for self-grooming in a novel chamber without bedding, *P < 0.05, Student t test. The numerical data underlying this figure can be found in (A–H) Normal social approach and social novelty recognition in (TIF)Click here for additional data file.S8 FigGrin2b+/–mice brain. Whole-brain total lysates from Grin2b+/–mice at P14 and P21 were immunoblotted with anti-GluN1/2A/2B antibodies. For quantification, average levels of GluN1/2A/2B proteins from Grin2b+/–mice were normalized to those from WT mice. n = 4 mice for WT and HT, *P < 0.05, **P < 0.01, Student t test. (C–F) Hypoactivity and normal anxiety-like behavior in Grin2b+/–mice (P61–71) in the open-field test, as shown by distance moved and time spent in the center region of the open-field arena. n = 16 mice for WT and 14 for HT, *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Sidak’s test and Student t test. (G–J) Anxiolytic-like behavior in Grin2b+/–mice (P65–75) in the elevated plus-maze, as shown by the number of entries into and time spent in open/closed arms. n = 16 mice (WT) and 14 (HT), ***P < 0.001, Student t test. (K) Normal anxiety-like behavior in Grin2b+/–mice (P68–78) in the light-dark test, as shown by time spent in the light chamber. n = 16 mice (WT) and 14 (HT), Mann-Whitney test. The numerical data underlying this figure can be found in (A and B) Decreased levels of the GluN2B, but not GluN1 or GluN2A, protein in the (TIF)Click here for additional data file.S9 FigGrin2b+/–mice (P75–85) in the three-chamber test under both bright-light and dark conditions, as shown by time spent sniffing the target and time spent in the chamber with the target. n = 8 mice for WT and 7 for HT, *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Sidak’s test. (I–K) Normal courtship USVs in Grin2b+/–mice (P77–87) upon encountering a novel female mouse, as shown by the number of calls, duration of each call, and latency to the first call. n = 16 for WT and 14 for HT, Student t test except Mann-Whitney test for latency to first all. (L and M) Normal repetitive self-grooming and digging in Grin2b+/–mice (P77–87) in home cages with bedding, as shown by time spent self-grooming or digging. n = 16 (WT) and 14 (HT), Mann-Whitney test. (N) Normal novel object–recognition memory in Grin2b+/–mice (P63–73), as shown by the percent of time spent exploring a novel object relative to a familiar object. n = 16 (WT) and 14 (HT), Student t test. The numerical data underlying this figure can be found in (A–H) Normal social approach and social novelty recognition in (TIF)Click here for additional data file.S10 FigGrin2b+/C456Y juveniles (P22–24) show normal behaviors in the maternal-homing test, as shown by the time spent with the reunited mother. Note that these mice showed normal exploration of the bedding materials from previous home cages (“Home”) suggestive of normal olfactory function; it is unclear why these mice prefer to explore the new corner (“New”). n = 24 mice for WT and 20 for HT, *P < 0.05, ***P < 0.001, two-way ANOVA with Sidak’s test. (C) Grin2b+/C456Y juveniles (P28–20) show normal social interaction in the juvenile play test, as shown by the total time spent in social interaction . n = 9 pairs for WT and 5 pairs for HT, Student t test. (D and E) Grin2b+/C456Y juveniles (P24–26) show normal repetitive self-grooming and digging in home cages with bedding, as shown by time spent self-grooming or digging. n = 24 mice for WT and 20 for HT, Mann-Whitney test. The numerical data underlying this figure can be found in (A and B) (TIF)Click here for additional data file.S11 FigGrin2b+/–mice followed by LTD measurements in juvenile mice (P17–21) and behavioral tests (OFT and EPM) in adult mice (>P56). (B) Early chronic DCS treatment normalizes LFS-LTD at SC-CA1 synapses in juvenile Grin2b+/–mice (P17–20). n = 14 mice (6) for WT_V (78.0% ± 4.1%), 15 (5) for WT_D (85.2% ± 4.2%), 17 (6) for HT_V (96.9% ± 4.5%), 14 (5) for HT_D (79.6% ± 3.7%), *P < 0.05, two-way ANOVA with Tukey’s test. (C) Early chronic DCS treatment has no effect on the hypoactivity in adult Grin2b+/–mice (P56–62). n = 11 mice for WT_D, 12 for WT_D, 9 for HT_V, 11 for HT_D, ***P < 0.001, two-way ANOVA with Tukey’s test. (D) Early chronic DCS treatment has no effect on the anxiety-like behavior in adult Grin2b+/–mice (P58–76), as shown by closed-arm time and open-arm entry/time. Note, however, that the early chronic drug treatment procedure  seems to modestly increase anxiety levels in the mutant mice, blunting the baseline difference in the closed-arm entry between WT-V and HT-V  Experimental strategy for chronic, oral DCS treatment (40 mg/kg), twice daily for 10 days (P7–16), in young (TIF)Click here for additional data file.S12 FigGrin2b+/C456Y mice (P57–66) does not affect anxiolytic-like behavior in the EPM. Note, however, that the chronic drug treatment procedure  seems to substantially increase anxiety levels in the mutant mice, blunting the baseline difference in EPM variables between WT-V and HT-V  Late chronic oral DCS treatment  in adult Grin2b+/C456Y mice (P57–66) does not affect hypoactivity in the OFT. n = 10 mice for WT_D, 11 for WT_D, 11 for HT_V, 12 for HT_D, **P < 0.01, ***P < 0.001, two-way ANOVA with Tukey’s test. The numerical data underlying this figure can be found in (A–E) Late chronic oral DCS treatment  in adult see also , making (TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(DOCX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(PDF)Click here for additional data file. 24 Feb 2020AttachmentPoint by point responses to referee comments.docxSubmitted filename: Click here for additional data file. 27 Feb 2020Dear Eunjoon, Thank you for submitting your revised manuscript entitled "Early correction of synaptic long-term depression improves abnormal anxiety-like behavior in adult GluN2B-C456Y-mutant mice" for consideration as a Research Article by PLOS Biology.Your revision has now been evaluated by the PLOS Biology editorial staff, as well as by the original Academic Editor, and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.Please re-submit your manuscript within two working days, i.e. by Mar 02 2020 11:59PM.https://www.editorialmanager.com/pbiologyLogin to Editorial Manager here: http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit. During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review. plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,Gabriel Gasque, Ph.D.,Senior EditorPLOS Biology 28 Feb 2020AttachmentPoint by point responses to referee comments.docxSubmitted filename: Click here for additional data file. 26 Mar 2020Dear Eunjoon,Thank you for submitting your revised Research Article entitled "Early correction of synaptic long-term depression improves abnormal anxiety-like behavior in adult GluN2B-C456Y-mutant mice" for publication in PLOS Biology. I have now obtained advice from the original reviewers and have discussed their comments with the Academic Editor. Based on the reviews, we will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by reviewer 2. 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To submit your revision, please go to Please do not hesitate to contact me should you have any questions.Sincerely,Gabriel Gasque, Ph.D., Senior EditorPLOS Biology------------------------------------------------------------------------DATA POLICY:--Please update your S3 Data file to include Figure S2C.-- Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend. ------------------------------------------------------------------------BLOT AND GEL REPORTING REQUIREMENTS:https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirementsFor manuscripts submitted on or after 1st July 2019, we require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: ------------------------------------------------------------------------Reviewer remarks:Reviewer #1: The authors have adequately addressed the majority of this reviewer's concerns. With the significant changes made to the manuscript during the revision, the paper is suitable for publication.Reviewer #2, Marc Fuccillo: The authors have attempted to address many of my requests. In doing so, they have strengthened the idea that the C456Y point mutant functions like the NR2B heterozygous LOF mutation, both at the synaptic, protein and behavioral level. While perhaps expected, these data provide clarity to the role of ASD-associated NMDAR mutations. Another strong plus is the addition of data showing a similar plasticity effect in the mPFC. these data suggest that the data in hippo are a proxy for other areas (something that should be mentioned in the text). Overall, I feel like this is a strong contribution to the asd pathophysiology literature. while not incredibly surprising, the data is clear and makes a strong point supporting this specific disease-associated mutation as GRIN2B haploinsufficiency. the reproducibility of DCS rescue across assays is also a strength. A few short textual things should be added:1. discuss the relationship between anxiogenic and anxiolytic behaviors as they relate to ASD. these are not part of the core behavioral phenotype but are the most clearly altered in this work. the literature on ASD and anxiety-related behaviors should be discussed.2. please discuss that in light of the mPFC data, it is unclear which brain regions are contributing to the mutation-associated behavioral (and rescue-related) changes.3. "suppressed recognition of the fact that a dark and closed place is generally safe" - this isn't any better than before - perhaps just describe the phenotype.Reviewer #3: The authors performed extra experiments and gave explanations to address all the questions raised by the reviewers. The manuscript became even more data heavy and the conclusions are now better discussed to support the authors ideas. Although there are some technical issues, the authors do demonstrate GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, LTD, and anxiety-like behavior. The overall behavioral and physiological experiments provide an insight for the importance of early correction of pathophysiological deficits.The findings are important in the field for the treatment of neurodevelopmental or psychiatric diseases caused by NMDAR mutations.Reviewer #4: Satisfied with the manuscript as edited. Again, it is an important contribution to the literature as I stated in my initial review, though there are not major new insights here. Addition of the PFC data at least provides some additional evidence for more general deficits. 8 Apr 2020AttachmentResponse to reviewers Shin et al.docxSubmitted filename: Click here for additional data file. 15 Apr 2020Dear Dr Kim,On behalf of my colleagues and the Academic Editor, Thomas C Südhof, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology. The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. 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Please do not hesitate to contact me if I can provide any assistance during the production process.Kind regards,Vita UsovaPublication Editor, PLOS Biologyon behalf ofGabriel Gasque,Senior EditorPLOS Biology


Finally, 112 patients were eligible for analysis. To identify the factors influencing LDL-C levels, univariate and multivariate linear regression analyses were performed after 52 weeks of ezetimibe treatment. Overall, advanced age, T2DM, and high baseline LDL-C were significantly associated with a greater decrease in LDL-C levels. In the non-T2DM group, advanced age and high baseline LDL-C were associated with greater decrease in LDL-C levels. In the T2DM group, baseline LDL-C was the only factor that influenced the change in LDL-C levels. Advanced age was significantly associated with higher LDL-C reduction in non-T2DM patients, but not in T2DM patients. Ezetimibe use might be beneficial in older patients without T2DM. The lack of association between age and the LDL-C lowering effect by ezetimibe in patients with T2DM may be due to yet unknown mechanism except low statistical power.Ezetimibe reduces cardiovascular risk by lowering the levels of low-density lipoprotein cholesterol (LDL-C). However, there is limited information regarding the factors associated with ezetimibe-mediated LDL-C reduction. We investigated the factors associated with LDL-C reduction after ezetimibe administration in Japanese patients with or without type 2 diabetes mellitus (T2DM). This single-center retrospective observational study enrolled a total of 266 consecutive ezetimibe-naïve patients, of which 154 were excluded because of either switching from statin or fenofibrate to ezetimibe ( Hyperlipidemia accelerates the progression of atherosclerosis and raises the risk of cardiovascular diseases . Statin,In Improved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE-IT) and other clinical trials ,13,14,15The present study aimed to examine the clinical factors that influence the change in LDL-C level after one year of ezetimibe treatment in patients with or without T2DM.n = 52) and discontinuation of ezetimibe within 52 weeks or missing from follow up during 52 weeks (n = 102). Finally, 112 patients were eligible for analysis (n = 33) and without T2DM .We conducted a single-center retrospective observational study. A total of 266 ezetimibe-naïve patients, who were started on ezetimibe treatment between August 2007 and December 2012, were included. Out of these patients, we excluded 154 patients because of the following reasons: switching from statin or fenofibrate to ezetimibe . Medical technicians reanalyzed the data to check for out of reference values.The primary outcome was the % change in LDL-C from baseline to follow up (52 weeks after the treatment). LDL-C levels were calculated using the Friedewald equation when the triglyceride (TG) level was equal to or less than 400 mg/dL, and were directly measured when the TG level exceeded 400 mg/dL.2), antihyperlipidemic pretreatment (>4 weeks prior to start of ezetimibe administration), and laboratory data.We reviewed the medical chart to collect data on clinical characteristics, comorbidities (including hypertension and T2DM), coronary artery disease , chronic kidney disease , and TG after 52 weeks of ezetimibe treatment, the Wilcoxon signed-rank test was used.For the two-group comparison, the Student’s p < 0.10) were assessed using a multivariate linear regression model in the overall population and stratified under T2DM or non-T2DM.We examined age, gender, T2DM, hypertension, coronary artery disease, statin pretreatment, and baseline LDL-C of the patients using univariate linear regression analysis, to identify the factors associated with the change in LDL-C after ezetimibe treatment in the overall population and stratified them under T2DM or non-T2DM. The factors suggestive to be associated with LDL-C reduction in the univariate analysis  and Prism 8.0.0 . All statistical tests were two-tailed and In overall, the median age was 60 years. There were 64/112 (56%) male patients, of which 33/112 (29%) patients had T2DM. There were no significant differences in age, gender, body weight, and body mass index between the patients with and without T2DM. Hypertension, coronary artery disease, chronic kidney disease, and hemodialysis were more frequently seen in the T2DM group than in the non-T2DM group .p = 0.004; There were no significant differences in baseline LDL-C and HDL-C between the non-T2DM and T2DM groups . Baselinp = 0.003, p < 0.001, p = 0.056). In overall, multivariate linear regression analysis showed that age and baseline LDL-C were significantly associated with a reduction in LDL-C (age, coefficient = −0.75, p = 0.016; baseline LDL-C, coefficient = −0.48, p < 0.001; In overall, univariate linear regression analysis revealed age and baseline LDL-C to be significant factors associated with the % change in LDL-C (age, coefficient = −0.99, p = 0.006, p < 0.001, p = 0.024, p < 0.001, In the non-T2DM group, age and baseline LDL-C showed significant correlation with % change in LDL-C (age, coefficient = −1.22, p = 0.61; p < 0.001; In the T2DM group, there was no significant correlation between age and the % change in LDL-C (The estimated statistical power for the two-group comparison of the % change in LDL-C between the non-T2DM and T2DM groups was 0.96. The calculated statistical power of a univariate linear regression model to estimate % change in LDL-C using age in overall, non-T2DM and T2DM were 0.84, 0.81, and 0.09. The multivariate linear regression model to determine the factors that influence the LDL-C lowering effect of ezetimibe in the overall population and the non-T2DM group had 0.98 and 0.94 of statistical power.The present study compared the factors associated with ezetimibe-mediated LDL-C reduction in Japanese patients with or without T2DM. There were two major findings. First, the T2DM group exhibited a larger LDL-C reduction than the non-T2DM group . Second,T2DM and high baseline LDL-C have previously been reported to be associated with the LDL-C lowering effects of ezetimibe ,15. MeanThe IMPROVE-IT trial ,14, a laThe present study had several limitations. The patients’ compliance with the ezetimibe regimen was not assured, although a registered nurses confirmed their compliance. We could not address the clinical information that could modify the LDL-C lowering effect of ezetimibe, such as severity of T2DM, diet, and daily exercise. We could not follow up clinical events entirely such as cardiovascular events or new-onset malignancy. Despite the limitations of this study, we demonstrated the importance of age in patients without T2DM while considering cost-effectiveness of the ezetimibe treatment for hyperlipidemia. The lack of association between age and LDL-C lowering effect by ezetimibe in patients with T2DM might be due to low statistical power. Well-designed studies to further elucidate the factors that are associated with the response to ezetimibe are warranted.A greater decrease in LDL-C levels was observed in the T2DM group than in the non-T2DM group. There was a significant negative correlation between age and change in LDL-C levels after 52 weeks of ezetimibe treatment in the overall population and in the non-T2DM patients. Advanced age is a significant factor for the LDL-C lowering effect of ezetimibe in non-T2DM patients, but not in T2DM patients. Ezetimibe should be recommended to patients with T2DM, although it might be most beneficial for older patients without T2DM.


Mycobacterium tuberculosis and one of the top 10 causes of death worldwide. Treating TB is challenging; successful treatment requires a long course of multiple antibiotics. Rifampicin (RIF) is a first-line drug for treating TB, and the development of RIF-resistant M. tuberculosis makes treatment even more difficult. To determine the mechanism of RIF resistance in these strains, we searched for novel mutations by sequencing. Four isolates, CDC-1, CDC-2, CDC-3, and CDC-4, had high-level RIF resistance and unique mutations encoding RpoB G158R, RpoB V168A, RpoB S188P, and RpoB Q432insQ, respectively. To evaluate their correlation with RIF resistance, plasmids carrying rpoB genes encoding these mutant proteins were transfected into the H37Rv reference strain. The plasmid complementation of RpoB indicated that G158R, V168A, and S188P did not affect the MIC of RIF. However, the MIC of RIF was increased in H37Rv carrying RpoB Q432insQ. To confirm the correlation between RIF resistance and Q432insQ, we cloned an rpoB fragment carrying the insertion (encoding RpoB Q432insQ) into H37Rv by homologous recombination using a suicide vector. All replacement mutants expressing RpoB Q432insQ were resistant to RIF (MIC > 1 mg/L). These results indicate that RpoB Q432insQ causes RIF resistance in M. tuberculosis.Tuberculosis (TB) is an infectious respiratory disease caused by  Mycobacterium tuberculosis is an important human pathogen that causes tuberculosis (TB). It was first isolated by Robert Koch in 1882, and numerous TB drugs have been developed since the 1930s, although the first effective anti-TB drug was not discovered until 1944  of rpoB that is called the RIF resistance-determining region (RRDR) (E. coli)  . AccordiE. coli) . Drug reM. tuberculosis H37Rv was used as the reference strain. M. tuberculosis H37Rv and clinical isolates were cultured in DifcoTM Middlebrook 7H9 medium  supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% tween-80 at 37°C . All experiments involving M. tuberculosis strains were conducted at the Biosafety level 3 lab in the National Taiwan University College of Medicine and followed institutional biosafety procedures.Clinical isolates were obtained from the Reference Laboratory of Mycobacteriology of the Taiwan Centers for Disease Control.  at 37°C . E. coliM. tuberculosis were tested in the Reference Laboratory of Mycobacteriology of the Taiwan Centers for Disease Control. Each isolate was initially obtained from a patient and was inoculated in solid and liquid culture. The minimum inhibitory concentration (MIC) of RIF was determined using a SensititreTM Mycobacterium tuberculosis MIC Plate  according to the manufacturer’s instructions. The bacterial culture was adjusted to a 0.5 McFarland standard and then added to the SensititreTM plate, which was covered with an adhesive plastic seal. After incubation at 37°C, the results were recorded with a SensititreTM VizionTM Digital MIC Viewing System. The critical concentration of RIF was 1 mg/L.Clinical isolates of M. tuberculosis strains were cultured in Middlebrook 7H9 medium, and then the OD600nm was adjusted to 0.1. A 200-μL aliquot of each prepared bacterial suspension was placed in a 96-well sterile plate  and incubated at 37°C for 14 days with and without RIF (0.03–512 mg/L). After 14 days of incubation, 50 μL of a freshly prepared 1:1 mixture of Alamar blue  and 10% tween 80 was added to each well. Then, the plate was incubated at 37°C for 48 h, and the color was recorded. Blue indicates no growth, and pink indicates growth. During incubation and staining, the plate was sealed with an adhesive plate seal.Microplate alamar blue assays were performed as previously described with minor modifications . BrieflyM. tuberculosis . The primers used are shown in M. tuberculosis was transformed by electroporation at 2,500 V, 1,000 Ω, and 25 μF and recovered in 10 mL of Middlebrook 7H9 medium supplemented with 10% OADC and 0.5% glycerol for 24 h  containing 50 mg/L hygromycin and incubated at 37°C for 4 weeks.The plasmid pMN437 was used to express RpoB in rculosis . Derivatfor 24 h . RecoverrpoB mutations were amplified by PCR from the genomic DNA of the resistant strains as a template and then cloned into the ScaI site of the suicide plasmid pGOAL19   . The priescribed .Mycobacterium tuberculosis isolates were lysed with InstantGetTM DNA Extraction Solution  and heat-killed (100°C for 15 min). Then, the supernatants of heat-killed M. tuberculosis isolates were added to the PCR reagent containing KOD XtremeTM Hot Start DNA Polymerase  and gene-specific primers, rpoB out F1 and rpoB-out-R  and were verified by capillary electrophoresis . The rpoB sequences of clinical strains CDC-1, CDC-2, and CDC-4 were submitted to NCBI .oB-out-R , to amplrpoB gene. The RIF resistance levels of clinical strains were determined using a SensititreTM Mycobacterium tuberculosis MIC Plate. The MIC of RIF for CDC-1 was 8 mg/L, and the MICs of RIF for CDC-2, CDC-3, and CDC-4 were >16 mg/L  and the V at codon 170 was replaced with F (V170F). In CDC-2, the V at codon 168 was replaced with A (V168A) and the V at codon 170 was replaced with F (V170F). In CDC-3, the V at codon 170 of RpoB was substituted with F (V170F), and the S at codon 188 was replaced with P (S188P). In CDC-4, the S at codon 431 was replaced with G (S431G) and a Q was inserted at codon 432 (Q432insQ). However, G158R, V168A, S188P, and Q432insQ had not previously been confirmed to be related to drug resistance.Four clinical isolates, CDC-1, CDC-2, CDC-3, and CDC-4, had high-level RIF resistance and different novel mutations within the >16 mg/L . Sequenc158R, V168A, S188P, and Q432insQ, respectively). To evaluate the association between these unique amino acid changes and the RIF resistance of the clinical isolates, plasmids (pMN437-derived) expressing these RpoB proteins were transformed into H37Rv . The MIC of RIF for H37Rv:pMN437-CDC-4 was the same as that of clinical strain CDC-4, and the MIC of RIF for H37Rv:pMN437-RpoB Q432insQ was two-fold lower than that for H37Rv:pMN437-CDC-4 and clinical strain CDC-4. This suggests that RpoB G158R, RpoB V168A, and RpoB S188P likely do not contribute to RIF resistance, whereas RpoB Q432insQ contributes to high-level RIF resistance in M. tuberculosis.Clinical isolates CDC-1, CDC-2, CDC-3, and CDC-4 had high-level RIF resistance and harbored unique RpoB amino acid changes  was selected after two rounds of homologous recombination. DNA sequencing confirmed that seven strains  had the CAA insertion in codon 432 of rpoB, which leads to a Q insertion   . AccordioB V170F , suggestrculosis . Drug rerculosis .rpoB are located in the RRDR  were previously reported to be associated with RIF resistance  can be found in the article/J-TW designed the study. L-YL, P-FH, T-LL, and J-TW discussed the results and revised the manuscript. W-TL, H-YT, W-HL, and RJ provided and analyzed the clinical strains. L-YL, S-HW, S-EC, L-YH, and H-EK prepared materials and performed experiments. L-YL and P-FH analyzed the data. L-YL wrote the manuscript. All authors reviewed and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ischemic heart disease is the main cardiovascular complication of diabetes patients which is mainly caused by oxidative stress. DJ-1 is the key regulator for myocardial protection through inhibiting phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and activating Akt . This research is to investigate whether the antioxidant N-acetylcysteine (NAC) could alleviate diabetic myocardial ischemia/reperfusion (I/R) injury by the protective molecule DJ-1. DJ-1 in rat myocardial H9c2 cells and cardiac tissue was respectively knocked down by siRNA and adeno-associated virus (AAV). From the present study, it could be found that compared with high glucose (HG)-normal (N)/DM group, hypoxia/reoxygenation (H/R) or I/R injury can aggravate oxidative stress injury and apoptosis rate of myocardial cells, inhibit the expression of Bcl-2, activate the BAX and cleaved caspase-3 (c-caspase-3) protein and PTEN/Akt pathway. However, in the groups of HG-N, DM, HG-N+I/R and DM+I/R, NAC can significantly reduce oxidative stress injury and apoptosis rate of myocytes, promote the Bcl-2 and DJ-1 molecules, inhibit BAX and c-caspase-3 protein and PTEN/Akt pathway. Compared with HG-N+I/R+NAC and DM+I/R+NAC groups, the oxidative stress injury, apoptosis rate of myocardial cells and heart tissues increased after the knockdown of DJ-1, the expression of Bcl-2 and DJ-1 were inhibited, the BAX and c-caspase-3 expression was increased, and PTEN/Akt pathway was activated. Taken together, the findings suggest that NAC can reduce I/R injury in diabetic myocardium by up-regulating the PTEN/Akt pathway through the level of DJ-1. Diabetes is an independent risk factor for cardiovascular disease. The vulnerability of myocardial ischemia/reperfusion (I/R) injury in diabetic patients is increased ,2. The IDJ-1 gene is an oncogene firstly discovered in NIH3T3 cells in 1997, and its encoded protein is widely expressed in various tissues [The  tissues . It part tissues , molecul tissues , inhibit tissues . Mitocho tissues . Under t tissues . When ge tissues . As an i tissues .N-acetylcysteine (NAC) is a thiol-containing free radical scavenger and precursor to the antioxidant glutathione (GSH), and is therefore widely used to remove ROS from oxidative stress . AvailabNormal myocardial H9c2 cell line was purchased from China Center for Type Culture Collection (Wuhan University). Dulbecco’s modified Eagle’s medium (DMEM) low-glucose medium (sugar concentration 5.5 mmol/l) and 100 μ/ml penicillin + 0.1 g/l streptomycin double antibiotic were purchased from Gibco . Fetal bovine serum was purchased from Sijiqing (China). Trypsin was purchased from GSEE-TECH (China). DJ-1, cleaved caspase-3  PTEN, Akt, p-Akt, Bcl-2, BAX and GAPDH primary antibodies were purchased from CST (U.S.A.). The Prime-Script RT reagent kit, SYBR Premix Ex Taq kit and TRIzol were purchased from TAKARA (China). Fluorescent secondary antibody IRDye800CW and Odyssey Infrared Imaging System were purchased from LI-COR (U.S.A.). Victor X-type microplate reader was purchased from PerkinElmer (U.S.A.). Flow kit was purchased from Nanjing built (China). Flow cytometry was purchased from BD (U.S.A.).2). The cells in HG-H/R + NAC + siRNA-DJ-1 group were given siRNA (Santa Cruz Biotechnology) knockdown cells before modeling and intervention. The DJ-1 molecule was then pretreated with siRNA in the HG-I/R+NAC group cells for 24 h before NAC modelling, and the HG model was prepared by adding HG medium for 24 h after 24 h. The HG-H/R cell model was placed in a three-gas incubator (volume fraction 94% N2 + volume fraction 5% CO2 + volume fraction 1% O2) for hypoxia for 4 h, and the H/R model was prepared by reoxygenation for 2 h in a common incubator.The normal growth logarithmic H9c2 cardiomyocytes were randomly divided into five groups: high glucose (HG) and normoxia group (HG-N), HG hypoxia/reoxygenation group (HG-H/R), HG normoxia + NAC group (HG-N+NAC), HG hypoxia/reoxygenation + NAC group (HG-H/R + NAC), HG hypoxia/reoxygenation + NAC + siRNA-DJ-1 group (HG-H/R + NAC + siRNA-DJ -1). The cells in each group were cultured in a common incubator  rats weighing 210–240 g were purchased from Beijing Huakang Biotechnology Co., Ltd. The animal experiment was conducted in the animal experiment center of Renmin Hospital of Wuhan University at temperature of 25 ± 2°C, relative humidity of 50 ± 15% and normal circadian rhythm (12-h dark/12-h light). All study protocols were in accordance with internationally accepted principles and the Guidelines for the Care and Use of Laboratory Animals of Wuhan University , approved by the Ethics Committee of Renmin Hospital of Wuhan University and consistent with the Ethical Guidelines of the International Association for Pain Research. The animal experiment number was SYXK (HUBEI) 2014-0080. Animal quality certificate number was NO.11400700265563. All the rats were randomly divided into five groups: diabetic mellitus group (DM), diabetic myocardial I/R group (DM-I/R), diabetes + NAC intervention group (DM + NAC), diabetic myocardial I/R + NAC intervention group (DM-I/R + NAC), diabetic myocardial I/R +NAC intervention +DJ-1 adeno-associated virus (AAV) knockdown group (DM-I/R + NAC + AAV-DJ-1). DM-I/R + NAC + AAV-DJ-1 rats were targeted to knock down the expression of DJ-1 in rat hearts using tail vein injection prior to intervention.To prepare diabetic rat model, the rats were injected with 1% of STZ (60 mg/kg) once in the tail vein after fasting for 12 h, and fasted for 6 h after 3 days. The fasting blood glucose level was greater than 16.7 mmol/l prompted that the diabetes model had been successfully modelled. Thereafter, the amount of water drunk and food eaten in the rats were recorded daily, and blood glucose and body weight were monitored weekly. Establishment of a DJ-1 silent rat model using serotype AAV9  tail vein injection. One week after successful induction of the diabetes model, rats in the DM + NAC, DM-I/R + NAC and DM-I/R + NAC + AAV-DJ-1 groups were treated with a dose of 1.5 g/kg.d NAC for 4 weeks.To prepare myocardial I/R rat model, rats were fasted for 12 h before operation, anesthetized by intraperitoneal injection of 1% pentobarbital sodium 60 mg/kg and fixed on the homeothermic table to keep 37°C core body temperature, connected to electrocardiograph (ECG) monitoring, connected after tracheal intubation. The ventilator mechanically controls breathing. The venous access was established by separating the right internal jugular vein. The right femoral artery was inserted to record the invasive arterial pressure. The needle electrode was inserted under the skin and chest of the rat, and the small animal ECG was connected. A 75% ethanol solution was used to disinfect the chest skin, and a 1-cm incision was made in the left side of the sternum. The layers were separated and exposed, and the third and fourth ribs were cut. The hooks were opened along the intercostal space, and the capsule was cut to fully expose the heart. A ligature was placed ∼2 mm below the branch of the left anterior descending coronary artery (LAD) of the coronary artery with a 6-0 lesion-free line for ligation. At the time of ligation, a thin plastic tube having a diameter of 0.2 cm was used to pass through the ligature wire and clamped with a microscopic hemostat. After 10 min of stabilization, LAD was ligated to cause myocardial ischemia. After 30 min, the ligature was released and re-perfused for 2 h. All rats were killed by cervical dislocation after 2 h of reperfusion. The success criteria for LAD ligation were: whitening of the anterior region, ST-segment elevation in the ECG II lead . The criteria for reperfusion success are: ST segment fall, apex redness.5 cells were added to each well, and cultured until cell density was 50%, then the cell transfection was performed. The siRNA was diluted with the optimized medium and mixed; the liposomes were diluted and diluted for 10 min at room temperature; the diluted siRNA was thoroughly mixed with the liposomes and incubated for 25 min at room temperature. One milliliter of the fully mixed liposome was added to the culture dish and placed in a 37°C incubator for 6 h. After siRNA transfection for 6 h, the medium was discarded, and the appropriate amount of the medium without the anti-antibiotic was added and placed in a 37°C cell culture incubator. Continue to culture for 24 h. The corresponding drug treatment and modeling intervention were given. The siRNA was provided by Gena Pharma, and the sequence (5′–3′) was CGAGCUGGGAUUAAAGUCATTUGACUUUAAUCCCAGCUCGTT.H9c2 cells were seeded in six-well plates. A 1 × 10H9c2 cardiomyocytes were collected by 0.25% trypsin digestion, and seeded in 96-well culture plates at 5000 cells per well. After incubation at 37°C in a 5% carbon dioxide incubator for 24 h, the cells in each group were given the corresponding intervention. The 96-well plate aspirated the drug-containing medium and 10 μl of cell counting kit-8 (CCK-8) solution were added per well and 90 μl of serum-free medium, and incubated it at 37°C for 4 h, then measured the absorbance at 450 nm.The supernatant, lysate and serum samples of each group were collected, centrifuged at 4°C, 3000 rpm for 15 min, and the supernatant or serum was taken and stored in a refrigerator at −20°C. Specific procedures were based on the kit instructions, the levels of LDH, GSH in the cell supernatant, 15-F2t-Isoprostane (15-F2t-isop) in the cell supernatant and lysate, serum CK-MB and 15-F2t-isop were measured under a microplate reader.GAPDH forward and reverse primer sequences were 5′-AAC GGCACA GTCAAGGCTGA-3′ and 5′-AACGGCACAGTCAAGGCTGA-3′. Total RNA for myocardial tissue and H9c2 cells was extracted by TRIzol. According to the Prime-Script RT reagent kit and the SYBR Premix Ex Taq kit instructions, the RNA was reverse-transcribed into cDNA. Then the polymerase chain reaction initiated at 95°C for 30 s, followed 40 cycles of amplification of denaturation at 95°C for 5 s, annealing at 60°C for 34 s by using a StepOne Plus device (Applied Biosystems). The 2CT−ΔΔ method was used to analyze the data.The primers were designed and synthesized by Wuhan Qingke Biotechnology Co., Ltd . The primer sequences for DJ-1 and GAPDH were listed as follows. DJ-1 forward and reverse primer sequences were 5′-ACCGCGCAGGAAAAACACGC-3′ and 5′-CTGCCAGACGGCTCTGCAC-3′. The housekeeping gene The treated cells and the heart tissue were fully lysed with pre-cooled RIPA lysate, the supernatant was taken and the protein concentration was determined by BCA kit, and one-fourth volume of protein loading buffer was added, heated at 100°C for 5 min. After denaturation, it was separated by SDS/PAGE. The protein was transferred using a PVDF membrane, and 5% skim milk was prepared to block the PVDF membrane for 1 h, and then DJ-1, PTEN, Akt, p-Akt, Bcl-2, BAX, c-caspase-3 were respectively diluted at a ratio of 1:1000 primary antibody for overnight incubation at 4°C. Then, the fluorescent secondary antibody IRDye800CW  was diluted at a ratio of 1:10000, and the PVDF membrane was incubated at room temperature for 1 h, and TBST was washed three times for 10 min each time. The corresponding gray value was measured by Odyssey Infrared Imaging Technique, and the internal reference of each protein was GAPDH.g for 10 min. The supernatant was discarded, washed twice with PBS, centrifuged to remove supernatant. A total of 100 μl of 1× Annexin-binding buffer was added. The cells were suspended and added with Annexin V (5 μl) and propidium iodide (PI) 5 μl at room temperature for 20 min in the dark. Then 400 μl Annexin-binding buffer was added and mixed, stored in an ice box and detected by flow cytometry.Each group of treated H9c2 cardiomyocytes were digested with trypsin, centrifuged at 300×5 per well, and cultured overnight at 37°C in a 5% CO2 incubator. The cells were treated according to different groups, and the corresponding treatments were given respectively. The cells were incubated at 37°C for 30 min in the dark. After the incubation, the cells were washed with fresh medium; the cells were fixed and stored, and microscopic photographs were taken. Three sheets were selected for each slice. Photographs were taken for optical density analysis of fluorescent photographs using IPP 6.0 software.The cells were given the corresponding intervention, and then washed with PBS three times for 3 min each time, fixed with 4% paraformaldehyde for 30 min at room temperature, and washed three times with PBS for 3 min each time. Goat serum was added dropwise to the slide, blocked at room temperature for 30 min, and the primary antibody of DJ-1 (1:200) was added to the blocking solution by suction paper and incubated overnight at 4°C. The excess primary antibody on the slide was washed, and the fluorescent secondary antibody with cy3 label was incubated for 1 h at room temperature, incubated with DAPI in the dark for 5 min, the cover was washed, and the image was observed with a fluorescence microscope. The ROS fluorescence assay was performed on H9c2 cells in a logarithmic growth phase with good growth conditions, seeded on 20 mm-slides at 2 ×10At the end of reperfusion, six rats were randomly selected from each group, LAD was ligated again, and 3% Evans Blue 1 ml was injected into the left ventricular cavity. The residual blood was washed with pre-cooled saline, and the dye was distributed in the myocardium through the coronary artery. The heart was quickly removed and stored at −20°C for 2 h. The sections were cut perpendicular to the long axis of the myocardium, and the thickness was 2 mm. The cells were incubated in 1% triphenyltetrazolium chloride (TTC) buffer at 37°C for 15 min in the dark, and then placed in 4% paraformaldehyde for 30 min. The blue staining part of Evans Blue is a viable myocardial tissue called normal myocardial tissue (non-ischemic myocardium), the TTC staining area is brick red (ischemic myocardium), and the non-TTC staining area is gray (infarcted myocardium). Each layer of the slice was photographed on both sides, and the photos were analyzed by ImagePro Plus 6.0 software. The area of myocardial ischemic area and percentage of infarct area were measured as representative of the percentage of ischemic myocardium area to the left ventricular area (AAR/LV%) and the percentage of infarcted myocardium area to the area of ischemic myocardium (IA/AAR%).After reperfusion for 2 h, the apical tissue was excised, fixed in 4% paraformaldehyde solutionand sliced by paraffin embedding. Myocardial pathological morphology was observed under microscope by HE staining; myocardial cell apoptosis rate was detected by Tunel staining. DJ-1 protein expression and distribution in myocardial tissue of each group were detected by immunohistochemistry.t test, and the differences between groups were evaluated using the one-way analysis of variance (ANOVA). The post hoc test was performed using the LSD test. P<0.05 was considered statistically significant.Statistical analysis was performed using SPSS 17.0 software. The measurement data were expressed as means ± standard deviation. After the homogeneity test for variance, the differences between two groups were analyzed using a Student’s P<0.05). As shown in P<0.05). Compared with HG-N group, the DJ-1 protein level in HG-N+NAC group was increased (P<0.05). When compared with HG-H/R group, DJ-1 protein level was increased in HG-H/R+NAC group (P<0.05). Compared with HG-H/R+NAC group, the DJ-1 protein level in HG-H/R+NAC+siRNA-DJ-1 group was decreased (P<0.05).SiRNA was first used to down-regulate DJ-1 in H9c2 cells. The DJ-1 protein level was detected to verify the transfection effects A–C, compP<0.05). Compared with HG-N group, the cell viability increased and the LDH level decreased in HG-N+NAC group (P<0.05). Compared with HG-H/R group, the cell viability was increased and LDH level was decreased in HG-H/R+NAC group (P<0.05). Compared with HG-H/R+NAC group, the cell viability was decreased and LDH level was increased in the HG-H/R+NAC+siRNA-DJ-1 group (P<0.05).As shown in P<0.05). Compared with HG-N group, GSH level was increased, ROS and 15-F2t-isop levels were decreased in HG-N+NAC group (P<0.05). Compared with HG-H/R group, GSH level was increased, ROS and 15-F2t-isop were decreased in HG-H/R+NAC group (P<0.05). Compared with HG-H/R+NAC group, GSH level was decreased, ROS and 15-F2t-isop levels were increased in HG-H/R+NAC+siRNA-DJ-1 group (P<0.05).GSH is a special substance that relieves toxins. It is a small molecular peptide composed of three amino acids and acts as an important antioxidant and free radical scavenger in the body . 15-F2t P<0.05). BAX, c-caspase-3 and cell apoptosis rate levels were increased (P<0.05). Compared with HG-N group, Bcl-2 protein level was increased in HG-N+NAC group (P<0.05). BAX, c-caspase-3 and cell apoptosis rate levels were decreased (P<0.05). Compared with HG-H/R group, the level of Bcl-2 protein in HG -H/R+NAC group was increased (P<0.05). BAX, c-caspase-3 and cell apoptosis rate levels were decreased (P<0.05). Compared with HG-H/R+NAC group, the level of Bcl-2 protein in HG-H/R+NAC+siRNA-DJ-1 group was decreased (P<0.05). BAX, c-caspase-3 and cell apoptosis rate levels were elevated (P<0.05).As shown in P<0.05) and the level of p-Akt protein was slightly up-regulated in HG-H/R group, but the difference was not statistically significant (P>0.05). PTEN protein level was decreased in HG-N+NAC and HG-H/R+NAC group respectively compared with HG-N and HG-H/R (P<0.05) and p-Akt protein level was increased in HG-N+NAC and HG-H/R+NAC group respectively compared with HG-N and HG-H/R (P<0.05). Compared with HG-H/R+NAC, the level of PTEN protein in HG-H/R+NAC+siRNA-DJ-1 group was increased (P<0.05) and the level of p-Akt protein in HG-H/R+NAC+siRNA-DJ-1 group was decreased (P<0.05). There was no significant difference of Akt molecule in each group. There was no significant difference in the expression of caspase3 between the groups.As shown in P<0.05). As shown in P>0.05). Compared with DM group, the level of DJ-1 protein in DM+NAC group was increased (P<0.05). Compared with DM-I/R group, the expression of DJ-1 protein was increased in DM-I/R+NAC group (P<0.05). However, the expression of DJ-1 protein in the DM-I/R+NAC+AAV-DJ-1 group was lower than that in the DM-I/R+NAC group (P<0.05).The above experimental results verified that NAC can reduce H/R injury in HG-H/R-induced cardiomyocytes by regulating the PTEN/Akt pathway through up-regulating the level of DJ-1. Similarly, to further verify the efficacy of NAC in diabetic I/R rats, we first used the AAV to target knockdown of DJ-1 molecules in myocardial tissue, and then modeled and intervened. The general situation of each group of rats is shown in As shown in P<0.05). Compared with DM group, CK-MB and 15-F2t-isop for plasma and cardiac levels in DM+NAC group were decreased (P<0.05). Compared with DM-I/R group, CK-MB and 15-F2t-isop for plasma and cardiac levels in DM-I/R+NAC group were decreased (P<0.05). Compared with DM-I/R+NAC group, CK-MB and 15-F2t-isop for plasma and cardiac levels in DM-I/R+NAC+AAV-DJ-1 group were increased (P<0.05).As shown in P<0.05). Compared with DM group, the cell apoptosis rate in DM+NAC were decreased (P<0.05). Compared with DM-I/R group, the cell apoptosis rate in DM-I/R+NAC group was decreased (P<0.05). Compared with DM-I/R+NAC, the cell apoptosis rate DM-I/R+NAC+AAV-DJ-1 group was elevated (P<0.05).As shown in P<0.05). BAX and c-caspase-3 were increased (P<0.05). Compared with DM group, Bcl-2 protein level was increased in DM+NAC group (P<0.05). BAX and c-caspase-3 were decreased (P<0.05). Compared with DM-I/R group, the level of Bcl-2 protein in DM-I/R+NAC were increased. BAX, c-caspase-3 and myocardial infarction area were decreased (P<0.05). Compared with DM-I/R+NAC, the level of Bcl-2 protein in DM-I/R+NAC+AAV-DJ-1 group was decreased (P<0.05). BAX, c-caspase-3 and myocardial infarction area were elevated (P<0.05). There was no significant difference in the expression of caspase 3 between the groups.As shown in P<0.05), DJ-1 and p-Akt protein levels were slightly up-regulated but the difference was not statistically significant (P>0.05). Compared with DM group, PTEN protein level in DM+NAC group was decreased (P<0.05), p-Akt protein level was increased (P<0.05). Compared with DM-I/R group, PTEN protein level was decreased in DM-I/R+NAC group (P<0.05), p-Akt protein level was increased (P<0.05). Compared with DM-I/R+NAC group, the protein level of PTEN in DM-I/R+NAC+AAV-DJ-1 group was increased (P<0.05), p-Akt protein level was decreased (P<0.05). There was no significant difference for Akt molecule in each group.As shown in The 2015 International Diabetes Conference focused on the high incidence of diabetes and its prevention and treatment of cardiovascular complications, two important critical medical conferences. One of the common concerns is the prevention and molecular mechanism of perioperative myocardial I/R injury. Recent epidemiological investigations still show that ischemic heart disease is the main cardiovascular complication of diabetes patients and the leading cause of death , again i2O2 and 6-hydroxydopamine (6-OHDA)-induced cells apoptosis [2O2 and apoptosis were increased in the zebrafish DJ-1 knockout model [Oxidative stress is a key link in myocardial I/R injury , which ipoptosis . Baulac ut model . Studiesut model ,38. DJ-1ut model . Jain etut model . Xue et ut model . Shimizuut model . Billia ut model . Similarut model , and itsut model . Therefoin vivo. In the HG state, the level of DJ-1 protein is elevated, which is a negative regulator for PTEN [An important pathophysiological mechanism of damage to diabetic myocardial I/R injury is the inactivation of the PI-3K/Akt pathway . Siddallfor PTEN . Meanwhifor PTEN . PTEN cafor PTEN . For furNAC is a product of acetylation of L-cysteine, it acts as an antioxidant and a thiol-based donor to increase the antioxidant enzymes such as intracellular reduced GSH. The mechanism of action of NAC may be that NAC contains a sulfhydryl group, which induces aggregation of intracellular GSH by deacetylation, promotes antioxidation, enhances intracellular detoxification and reduces release of oxygen free radicals, thereby blocking the second messenger effect of ROS. Unlike other antioxidants, NAC regulates the activity of activator protein-1 (AP-1) and the secretion of cytokines, exerting antioxidant and immunomodulatory activities. At the same time, NAC can also enter into white blood cells to convert into physiological antioxidants, increase the level of reduction in cells, and inactivate reactive oxygen in white blood cells and medium . The conin vitro experiments, DJ-1 in rat myocardial H9c2 cells was first knocked down by siRNA. Then the cells and rats were built as HG and hypoxia reoxygenation/ischemia reperfusion models. NAC drug intervention was used to intervene the cells. In vitro experiments, compared with HG-N group, H/R injury can aggravate oxidative stress injury and apoptosis rate of myocardial cells, down-regulate the expression of DJ-1 molecule, and activate the PTEN/Akt pathway. However, for the groups of HG-N and HG-H/R, NAC can significantly reduce oxidative stress injury and apoptosis rate of myocytes, promote the expression of DJ-1 molecule, and inhibit PTEN/Akt pathway. Compared with the groups of HG-N+H/R+NAC and DM+I/R+NAC, the oxidative stress injury and apoptosis rate of myocardial cells tissues increased after the targeted knockdown of DJ-1. The expression of DJ-1 molecule was inhibited, and the PTEN/Akt pathway was subsequently activated. Similarity, in the in vivo experiments, DJ-1 in rat cardiac tissue was knocked down by siRNA adeno-associated viral vector. Then the rats were built as diabetes mellitus and I/R models. In the DM and DM+I/R groups, NAC could significantly reduce oxidative stress injury and apoptosis rate of myocytes, promote the expression of DJ-1 molecule, and inhibit PTEN/Akt pathway. Compared with DM+I/R+NAC group, the oxidative stress injury and apoptosis rate of myocardial cells in heart tissues were increased after the targeted knockdown of DJ-1. The expression of DJ-1 molecule was inhibited, and the PTEN/Akt pathway was activated. It's worth noting that in vivo experiments, compared with DM group the expression of DJ-1 in DM-I/R group had a slight increase . The above result was different with the in vitro experiments. In vitro experiments, compared with normal cells the level of DJ-1 increased after HG stimulation, because HG stimulation could reduce the compensatory protective stress response [In vivo experiments, the diabetic model was a long-term pathological process. Therefore, compared with normal animals, the level of DJ-1 in diabetic rats was decreased. And the level of DJ-1 protein was slightly increased after diabetic myocardial I/R, but there was no statistical significance, which was coincidence with the previous study [in vitro and in vivo results, since the NAC-treated group showed less damage characteristics than the untreated group, and the DJ-1 protein showed more damage after inhibition, we concluded that the protective effect after NAC treatment may be mediated by up-regulation of DJ-1. However, as we have previously reported, NAC as an antioxidant can also protect diabetic myocardium by regulating the caveolin-3/eNOS signaling pathway [For the results, in the response . After Hresponse . In vivous study . Moreoveus study . Therefous study ,40. In g pathway , so the In summary, NAC can attenuate myocardial I/R injury by up-regulating DJ-1 level regulation of PTEN/Akt pathway, which provides new treatment and clinical basis for clinical treatment of diabetic myocardial I/R injury. However, its specific regulation of DJ-1 by NAC needs further study.Click here for additional data file.