Deficiency of nuclear receptor interaction protein leads to cardiomyopathy by disrupting sarcomere structure and mitochondrial respiration.

BACKGROUND: Cardiomyopathy is a common and lethal complication in patients with limb-girdle muscular dystrophy (LGMD), one of the most prevalent forms of muscular dystrophy. The pathogenesis underlying LGMD-related cardiomyopathy remains unclear. NRIP (gene name DCAF6), a Ca2+-dependent calmodulin binding protein, was reduced in dystrophic muscles from LGMD patients. Mice lacking NRIP exhibit a myopathic phenotype resembling that in LGMD patients, making NRIP deficiency a potential culprit leading to cardiomyopathy. This study aimed to determine if NRIP deficiency leads to cardiomyopathy and to explore the underlying molecular mechanisms. METHODS AND RESULTS: NRIP expression was reduced in both human and mouse failing hearts. Muscle-specific NRIP knockout (MCK-Cre::Dcaf6flox/flox) mouse heart and isolated cardiomyocytes exhibited markedly reduced contractility. Transmission electron microscopy revealed abnormal sarcomere structures and mitochondrial morphology in MCK-Cre::Dcaf6flox/flox hearts. Protein co-immunoprecipitation and confocal imaging revealed that NRIP interacts with α-actinin 2 (ACTN2) at the Z-disc. We found that NRIP facilitated ACTN2-mediated F-actin bundling, and that NRIP deficiency resulted in reduced binding between Z-disc proteins ACTN2 and Cap-Z. In addition, NRIP-deficiency led to increased mitochondrial ROS and impaired mitochondrial respiration/ATP production owing to elevated cellular NADH/NAD+ ratios. Treatment with mitochondria-directed antioxidant mitoTEMPO or NAD+ precursor nicotinic acid restored mitochondrial function and cardiac contractility in MCK-Cre::Dcaf6flox/flox mice. CONCLUSIONS: NRIP is essential to maintain sarcomere structure and mitochondrial/contractile function in cardiomyocytes. Our results revealed a novel role for NRIP deficiency in the pathogenesis of LGMD and heart failure. Targeting NRIP, therefore, could be a powerful new approach to treat myocardial dysfunction in LGMD and heart failure patients.

NRIP is newly identified as a Z-disc protein, activating calmodulin signaling for skeletal muscle contraction and regeneration.

Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) is a Ca(2+)-dependent calmodulin-binding protein. In this study, we newly identify NRIP as a Z-disc protein in skeletal muscle. NRIP-knockout mice were generated and found to have reduced muscle strength, susceptibility to fatigue and impaired adaptive exercise performance. The mechanisms of NRIP-regulated muscle contraction depend on NRIP being downstream of Ca(2+) signaling, where it stimulates activation of both 'calcineurin-nuclear factor of activated T-cells, cytoplasmic 1' (CaN-NFATc1; also known as NFATC1) and calmodulin-dependent protein kinase II (CaMKII) through interaction with calmodulin (CaM), resulting in the induction of mitochondrial activity and the expression of genes encoding the slow class of myosin, and in the regulation of Ca(2+) homeostasis through the internal Ca(2+) stores of the sarcoplasmic reticulum. Moreover, NRIP-knockout mice have a delayed regenerative capacity. The amount of NRIP can be enhanced after muscle injury and is responsible for muscle regeneration, which is associated with the increased expression of myogenin, desmin and embryonic myosin heavy chain during myogenesis, as well as for myotube formation. In conclusion, NRIP is a novel Z-disc protein that is important for skeletal muscle strength and regenerative capacity.

AAV-NRIP gene therapy ameliorates motor neuron degeneration and muscle atrophy in ALS model mice.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS. METHODS: We used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice. RESULTS: SOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice. CONCLUSIONS: AAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.

Breast carcinoma-amplified sequence 2 regulates adult neurogenesis via ß-catenin.

BACKGROUND: Breast carcinoma-amplified sequence 2 (BCAS2) regulates ß-catenin gene splicing. The conditional knockout of BCAS2 expression in the forebrain (BCAS2 cKO) of mice confers impaired learning and memory along with decreased ß-catenin expression. Because ß-catenin reportedly regulates adult neurogenesis, we wondered whether BCAS2 could regulate adult neurogenesis via ß-catenin. METHODS: BCAS2-regulating neurogenesis was investigated by characterizing BCAS2 cKO mice. Also, lentivirus-shBCAS2 was intracranially injected into the hippocampus of wild-type mice to knock down BCAS2 expression. We evaluated the rescue effects of BCAS2 cKO by intracranial injection of adeno-associated virus encoding BCAS2 (AAV-DJ8-BCAS2) and AAV-ß-catenin gene therapy. RESULTS: To show that BCAS2-regulating adult neurogenesis via ß-catenin, first, BCAS2 cKO mice showed low SRY-box 2-positive (Sox2+) neural stem cell proliferation and doublecortin-positive (DCX+) immature neurons. Second, stereotaxic intracranial injection of lentivirus-shBCAS2 knocked down BCAS2 in the hippocampus of wild-type mice, and we confirmed the BCAS2 regulation of adult neurogenesis via ß-catenin. Third, AAV-DJ8-BCAS2 gene therapy in BCAS2 cKO mice reversed the low proliferation of Sox2+ neural stem cells and the decreased number of DCX+ immature neurons with increased ß-catenin expression. Moreover, AAV-ß-catenin gene therapy restored neuron stem cell proliferation and immature neuron differentiation, which further supports BCAS2-regulating adult neurogenesis via ß-catenin. In addition, cells targeted by AAV-DJ8 injection into the hippocampus included Sox2 and DCX immature neurons, interneurons, and astrocytes. BCAS2 may regulate adult neurogenesis by targeting Sox2+ and DCX+ immature neurons for autocrine effects and interneurons or astrocytes for paracrine effects. CONCLUSIONS: BCAS2 can regulate adult neurogenesis in mice via ß-catenin.

Autoantibody of NRIP, a novel AChR-interacting protein, plays a detrimental role in myasthenia gravis.

BACKGROUND: Nuclear receptor interaction protein (NRIP) co-localizes with acetylcholine receptor (AChR) at the neuromuscular junction (NMJ), and NRIP deficiency causes aberrant NMJ architecture. However, the normal physiological and pathophysiological roles of NRIP in NMJ are still unclear. METHODS: We investigated the co-localization and interaction of NRIP with AChR-associated proteins using immunofluorescence and immunoprecipitation assay, respectively. The binding affinity of AChR-associated proteins was analysed in muscle-restricted NRIP knockout mice and NRIP knockout muscle cells (C2C12). We further collected the sera from 43 patients with myasthenia gravis (MG), an NMJ disorder. The existence and features of anti-NRIP autoantibody in sera were studied using Western blot and epitope mapping. RESULTS: NRIP co-localized with AChR, rapsyn and α-actinin 2 (ACTN2) in gastrocnemius muscles of mice; and α-bungarotoxin (BTX) pull-down assay revealed NRIP with rapsyn and ACTN2 in complexes from muscle tissues and cells. NRIP directly binds with α subunit of AChR (AChRα) in vitro and in vivo to affect the binding affinity of AChR with rapsyn and rapsyn with ACTN2. In 43 patients with MG (age, 58.4 ± 14.5 years; female, 55.8%), we detected six of them (14.0%) having anti-NRIP autoantibody. The presence of anti-NRIP autoantibody correlated with a more severe type of MG when AChR autoantibody existed (P = 0.011). The higher the titre of anti-NRIP autoantibody, the more severe MG severity (P = 0.032). The main immunogenic region is likely on the IQ motif of NRIP. We also showed the IgG subclass of anti-NRIP autoantibody mainly to be IgG1. CONCLUSIONS: NRIP is a novel AChRα binding protein and involves structural NMJ formation, which acts as a scaffold to stabilize AChR-rapsyn-ACTN2 complexes. Anti-NRIP autoantibody is a novel autoantibody in MG and plays a detrimental role in MG with the coexistence of anti-AChR autoantibody.

Muscle-restricted nuclear receptor interaction protein knockout causes motor neuron degeneration through down-regulation of myogenin at the neuromuscular junction.

BACKGROUND: Nuclear receptor interaction protein (NRIP) is a calcium/calmodulin (CaM) binding protein. Nuclear receptor interaction protein interacts with CaM to activate calcineurin and CaMKII signalling. The conventional NRIP knockout mice (global knockout) showed muscular abnormality with reduction of muscle oxidative functions and motor function defects. METHODS: To investigate the role of NRIP on neuromuscular system, we generated muscle-restricted NRIP knockout mice [conditional knockout (cKO)]. The muscle functions (including oxidative muscle markers and muscle strength) and lumbar motor neuron functions [motor neuron number, axon denervation, neuromuscular junction (NMJ)] were tested. The laser-captured microdissection at NMJ of skeletal muscles and adenovirus gene therapy for rescued effects were performed. RESULTS: The cKO mice showed muscular abnormality with reduction of muscle oxidative functions and impaired motor performances as global knockout mice. To our surprise, cKO mice also displayed motor neuron degeneration with abnormal architecture of NMJ. Specifically, the cKO mice revealed reduced motor neuron number with small neuronal size in lumbar spinal cord as well as denervating change, small motor endplates, and decreased myonuclei number at NMJ in skeletal muscles. To explore the mechanisms, we screened various muscle-derived factors and found that myogenin is a potential candidate that myogenin expression was lower in skeletal muscles of cKO mice than wild-type mice. Because NRIP and myogenin were colocalized around acetylcholine receptors at NMJ, we extracted RNA from synaptic and extrasynaptic regions of muscles using laser capture microdissection and showed that myogenin expression was especially lower at synaptic region in cKO than wild-type mice. Notably, overexpression of myogenin using intramuscular adenovirus encoding myogenin treatment rescued abnormal NMJ architecture and preserved motor neuron death in cKO mice. CONCLUSIONS: In summary, we demonstrated that deprivation of NRIP decreases myogenin expression at NMJ, possibly leading to abnormal NMJ formation, denervation of acetylcholine receptor, and subsequent loss of spinal motor neuron. Overexpression of myogenin in cKO mice can partially rescue abnormal NMJ architecture and motor neuron death. Therefore, muscular NRIP is a novel trophic factor supporting spinal motor neuron via stabilization of NMJ by myogenin expression.

NRIP/DCAF6 stabilizes the androgen receptor protein by displacing DDB2 from the CUL4A-DDB1 E3 ligase complex in prostate cancer.

Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer.

A light-field-based method to adjust on-axis rounded leaf end MLC position to predict off-axis MLC penumbra region dosimetric performance in a radiation therapy planning system.

PURPOSE: An analytical and experimental study of split shape dose calculation correction by adjusting the position of the on-axis round leaf end position is presented. We use on-axis corrected results to predict off-axis penumbra region dosimetric performance in an intensity-modulated radiation therapy treatment planning system. MATERIALS AND METHODS: The precise light-field edge position (X(tang.p)) was derived from the on-axis 50% dose position created by using the nominal light field for geometric and mathematical manipulation. Leaf position (X(mlc.p)) could be derived from X(tang.p) by defining in the treatment planning system for monitor unit calculation. On-axis offset (correction) could be obtained from the position corresponding to 50% of the central axis dose minus the X(mlc.p) position. The off-axis 50% dose position can then be derived from the on-axis 50% dose position. RESULTS: The monitor unit calculation of the split shape using the on-axis rounded leaf end MLC penumbra region could provide an under-or overdose of 7.5% per millimeter without an offset correction. When using the on-axis rounded leaf end offset correction to predict the off-axis dose, the difference between the off- and on-axis 50% dose position is within ±1.5 mm. CONCLUSIONS: It is possible to achieve a dose calculation within 0.5% error for an adjusted MLC leaf edge location in the treatment planning system with careful measurement and an accurate on-axis offset correction. Dose calculations located at an off-axis spilt shape region should be used carefully due to noncorrectable errors which were found to be up to 10%.

Optimizing the TLD-100 readout system for various radiotherapy beam doses using the Taguchi methodology.

The TLD-100 readout system was optimized for various radiotherapy beam doses using the Taguchi method. The radiotherapy beam was produced by a Varian 21EX linear accelerator (LINAC) at 6MV. The beam doses were 50, 100 and 150cGy, and the measured data in each group were averaged from three TLD-100 chips. A total of nine combinations of four parameters were arranged, in the manner suggested by Taguchi. The four parameters were defined as initial temperature, heating rate, preheat time and maximum set temperature of the readout system during TLD reading. The loss function eta adopted herein was specifically defined to satisfy the requirements of both sharp linearity and good reproducibility of the TLD reading at various radiotherapy beam doses. The optimized values were: (1) 50( composite function)C for initial temperature, (2) 3 (degrees C)/s for heating rate, (3) 5 min for the TLD preheat time and (4) 250 degrees C for the maximum temperature for TLD reading. Additionally, the parameters that dominated the TLD readout were: (1) initial temperature, (2) heating rate and (4) maximum temperature setting for TLD reading; and the minor parameter was (3) TLD preheat time before reading. The interactions among the dominant parameters were also studied: no significant cross interaction occurred between initial temperature and heating rate or between initial temperature and maximum temperature. However, a complex cross-interaction existed between optimal heating rate and maximum temperature.

Impact of flood damage on pollutant removal efficiencies of a subtropical urban constructed wetland.

Typhoons and hurricanes in subtropical/tropical regions can induce significant environmental changes (e.g., mass flooding and inundations). However, the damage to the pollutant removal efficiencies of constructed wetlands brought about by these natural disturbances has been neglected in major studies conducted in temperate climates. Therefore, this study compares the pollutant removal performance of a constructed wetland in the Danshui River Basin, before and after the system was inundated with flooding from Typhoon Krosa in 2007. The pollutant removal performance of the free water surface (FWS) constructed wetland was investigated monthly from September 2006 to April 2008. Results of the study demonstrated that this FWS wetland effectively removed 64.3% BOD, 98.9% NH(4)-N, and 39.5% Total-P before Typhoon Krosa. However, the extensive flooding caused by Typhoon Krosa swept over most of the aboveground plant community and deposited the sediment onto the bottom of each compartment. Subsequently, reduced pollutant removal efficiencies were observed. Only 37.7% BOD, 35.1% NH(4)-N, and 31.8% Total-P were removed after this event, although the flow regime was immediately restored. Comparing the water quality data for the FWS wetland before and after Typhoon Krosa revealed the immediate, quantitative damage to the pollutant removal performance caused by the typhoon's inundation. Consequently, a high-flow bypass and additional preventive measures would protect any constructed wetland in areas subject to typhoons.