A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride-induced neuronal death.

Autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis. Dysfunction of autophagy is associated with the progression of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Although one of the typical features of brain aging is an accumulation of redox-active metals that eventually lead to neurodegeneration, a plausible link between trace metal-induced neurodegeneration and dysregulated autophagy has not been clearly determined. Here, we used a cupric chloride-induced neurodegeneration model in MN9D dopaminergic neuronal cells along with ultrastructural and biochemical analyses to demonstrate impaired autophagic flux with accompanying lysosomal dysfunction. We found that a surge of cytosolic calcium was involved in cupric chloride-induced dysregulated autophagy. Consequently, buffering of cytosolic calcium by calbindin-D28K overexpression or co-treatment with the calcium chelator BAPTA attenuated the cupric chloride-induced impairment in autophagic flux by ameliorating dysregulation of lysosomal function. Thus, these events allowed the rescue of cells from cupric chloride-induced neuronal death. These phenomena were largely confirmed in cupric chloride-treated primary cultures of cortical neurons. Taken together, these results suggest that abnormal accumulation of trace metal elements and a resultant surge of cytosolic calcium leads to neuronal death by impairing autophagic flux at the lysosomal level.

Asymmetric dimethylation of AMPKα1 by PRMT6 contributes to the formation of phase-separated puncta.

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase comprising α, ß, and γ subunits. AMPK is involved in intracellular energy metabolism and functions as a switch that turns various biological pathways in eukaryotes on and off. Several post-translational modifications regulating AMPK function have been demonstrated, including phosphorylation, acetylation, and ubiquitination; however, arginine methylation has not been reported in AMPKα1. We investigated whether arginine methylation occurs in AMPKα1. Screening experiments revealed arginine methylation of AMPKα1 mediated by protein arginine methyltransferase 6 (PRMT6). In vitro methylation and co-immunoprecipitation assays indicated that PRMT6 can directly interact with and methylate AMPKα1 without involvement of other intracellular components. In vitro methylation assays with truncated and point mutants of AMPKα1 revealed that Arg403 is the residue methylated by PRMT6. Immunocytochemical studies showed that the number of AMPKα1 puncta was enhanced in saponin-permeabilized cells when AMPKα1 was co-expressed with PRMT6, suggesting that PRMT6-mediated methylation of AMPKα1 at Arg403 alters the physiological characteristics of AMPKα1 and may lead to liquid-liquid phase separation.

Small extracellular vesicles derived from patients with persistent atrial fibrillation exacerbate arrhythmogenesis via miR-30a-5p.

Small extracellular vesicles (sEVs) are nanometer-sized membranous vesicles that contribute to the pathogenesis of atrial fibrillation (AF). Here, we investigated the role of sEVs derived from patients with persistent AF in the pathophysiology of AF. First, we evaluated the pathological effects of sEVs derived from the peripheral blood of patients with persistent AF (AF-sEVs). AF-sEVs treatment reduced cell viability, caused abnormal Ca2+ handling, induced reactive oxygen species (ROS) production and led to increased CaMKII activation of non-paced and paced atrial cardiomyocytes. Next, we analyzed the miRNA profile of AF-sEVs to investigate which components of AF-sEVs promote arrhythmias, and we selected six miRNAs that correlated with CaMKII activation. qRT-PCR experiment identified that miR-30a-5p was significantly down-regulated in AF-sEVs, paced cardiomyocytes, and atrial tissues of patients with persistent AF. CaMKII was predicted by bioinformatics analysis as a miR-30a-5p target gene and validated by a dual luciferase reporter; hence, we evaluated the effects of miR-30a-5p on paced cardiomyocytes and validated miR-30a-5p as a pro-arrhythmic signature of AF-sEVs. Consequently, AF-sEVs-loaded with miR-30a-5p attenuated pacing-induced Ca2+-handling abnormalities, whereas AF-sEVs-loaded with anti-miR-30a-5p reversed the change in paced cardiomyocytes. Taken together, the regulation of CaMKII by miR-30a-5p revealed that miR-30a-5p is a major mediator for AF-sEVs-mediated AF pathogenesis. Accordingly, these findings suggest that sEVs derived from patients with persistent AF exacerbate arrhythmogenesis via miR-30a-5p.

RNF166 plays a dual role for Lys63-linked ubiquitination and sumoylation of its target proteins.

Ubiquitination and sumoylation are two important posttranslational modifications in cells. RING (Really Interesting New Gene)-type E3 ligases play essential roles in regulating a plethora of biological processes such as cell survival and death. In our previous study, we performed a microarray using inputs from MN9D dopaminergic neuronal cells treated with 6-hydroxydopamine and identified a novel RING-type E3 ligase, RNF166. We showed that RNF166 exerts proapoptotic effects via ubiquitin-dependent degradation of X-linked inhibitor of apoptosis and subsequent overactivation of caspase-dependent neuronal death following 6-hydroxydopamine treatment. In the present study, we further expanded the list of RNF166's binding substrates using mass spectral analyses of immunoprecipitates obtained from RNF166-overexpressing HEK293 cells. Poly (ADP-ribose) polymerase 1, ATPase WRNIP1, X-ray repair cross-complementing protein 5 (Ku80), and replication protein A 70 were identified as potential binding partners of RNF166. Additionally, we confirmed that RNF166 interacts with and forms lysine 63-linked polyubiquitin chains in Ku80. Consequently, these events promoted the increased stability of Ku80. Intriguingly, we found that RNF166 also contains distinct consensus sequences termed SUMO-interacting motifs and interacts with apoptosis signal-regulating kinase 1 (ASK1). We determined that RNF166 induces the sumoylation of ASK1. Overall, our data provide novel evidence that RNF166 has a dual function of Lys63-linked ubiquitination and sumoylation of its cellular targets.

Dysregulated autophagy is linked to BAX oligomerization and subsequent cytochrome c release in 6-hydroxydopmaine-treated neuronal cells.

Autophagy and apoptosis are essential physiological pathways that are required to maintain cellular homeostasis. Therefore, it is suggested that dysregulation in both pathways is linked to several disease states. Moreover, the crosstalk between autophagy and apoptosis plays an important role in pathophysiological processes associated with several neurodegenerative disorders. We have previously reported that 6-hydroxydopamine (6-OHDA)-triggered reactive oxygen species (ROS) induces dysregulated autophagy, and that a dysregulated autophagic flux contributes to caspase-dependent neuronal apoptosis. Based on our previous findings, we specifically aimed to elucidate the molecular mechanisms underlying the potential role of dysregulated autophagy in apoptotic neurodegeneration. The disuccinimidyl suberate (DSS) cross-linking assay and immunological analyses indicated that exposure of several types of cells to 6-OHDA resulted in BAX activation and subsequent oligomerization. Pharmacological inhibition and genetic perturbation of autophagy prevented 6-OHDA-induced BAX oligomerization and subsequent release of mitochondrial cytochrome c into the cytosol and caspase activation. These events were independent of expression levels of XIAP. Taken together, our results suggest that BAX oligomerization comprises a critical step by which 6-OHDA-induced dysregulated autophagy mediates neuronal apoptosis.

Therapeutic potential of miR-21 regulation by human peripheral blood derived-small extracellular vesicles in myocardial infarction.

Small extracellular vesicles (sEVs) as natural membranous vesicles are on the frontiers of nanomedical research, due to their ability to deliver therapeutic molecules such as microRNAs (miRNAs). The miRNA-21 (miR-21) is thought to be involved in the initiation and development of myocardial infarction (MI). Here, we examined whether miR-21 regulation using human peripheral blood-derived sEVs (PB-sEVs) could serve as a potential therapeutic strategy for MI. First, we examined miR-21 levels in hypoxic conditions and validated the ability of PB-sEVs to serve as a potential delivery system for miRNAs. Further, bioinformatics analysis and luciferase assay were performed to identify target genes of miR-21 mechanistically. Among numerous target pathways, we focused on nitrogen metabolism, which remains relatively unexplored compared with other possible miR-21-mediated pathways; hence, we aimed to determine novel target genes of miR-21 related to nitrogen metabolism. In hypoxic conditions, the expression of miR-21 was significantly up-regulated and correlated with nitric oxide synthase 3 (NOS3) levels, which in turn influences cardiac function. The down-regulation of miR-21 expression by PB-sEVs loaded with anti-miR-21 significantly improved survival rates, consistent with the augmentation of cardiac function. However, the up-regulation of miR-21 expression by PB-sEVs loaded with miR-21 reversed these effects. Mechanistically, miR-21 targeted and down-regulated the mRNA and protein expression of striatin (STRN), which could regulate NOS3 expression. In conclusion, we identified a novel therapeutic strategy to improve cardiac function by regulating the expression of miR-21 with PB-sEVs as an miR-21 or anti-miR-21 delivery vehicle and confirmed the miR-21-associated nitrogen metabolic disorders in MI.

Expression of miRNAs in circulating exosomes derived from patients with persistent atrial fibrillation.

Atrial fibrillation (AF), the most common type of cardiac arrhythmia, is thought to be regulated by changes in microRNA (miRNA) expression. However, the evidence for this is inconsistent. The high stability and expression of circulating exosomal miRNAs may allow their use as candidate biomarkers. For the discovery phase, exosomes were isolated from the serum of patients with supraventricular tachycardia (SVT) as the controls (n = 5) and with paroxysmal AF (n = 4) and persistent AF (n = 5) for microarray analysis of miRNAs. Forty-five miRNAs were expressed significantly higher (>1.5-fold) in patients with persistent AF, but not in patients with paroxysmal AF, relative to the levels in patients with SVT control. Notably, expression of 5 miRNAs (miRNA-103a, -107, -320d, -486, and let-7b) was elevated by more than 4.5-fold in patients with persistent AF. For the validation phase, miRNAs were analyzed using quantitative RT-PCR analysis in exosomes from the serum of patients with SVT control (n = 20) and patients with persistent AF (n = 40). These miRNAs and their target genes were involved in atrial function and structure, oxidative stress, and fibrosis pathways. These findings suggest that serum exosomal miRNAs might be used as novel biomarkers to reflect the progression of AF.-Mun, D., Kim, H., Kang, J.-Y., Park, H., Park, H., Lee, S.-H., Yun, N., Joung, B. Expression of miRNAs in circulating exosomes derived from patients with persistent atrial fibrillation.

Anamorsin attenuates cupric chloride-induced dopaminergic neuronal cell death.

Neurodegenerative diseases are associated with elevated levels of metal elements, which are well-known inducers of reactive oxygen species (ROS) in cells. Because dopaminergic neurons in the substantia nigra are vulnerable to ROS, dysregulation of metals and the resulting accumulation of ROS could be a cause of dopaminergic neurodegeneration. In this study, we showed that overexpression of anamorsin protected MN9D dopaminergic neuronal cells from cupric chloride-induced death. This cytoprotection was achieved by specifically decreasing ROS levels. As determined by mini two-dimensional electrophoretic assay, an acidic shift of anamorsin occurred during drug-induced death, which seemed to be mediated by oxidative modification of three of its CXXC motifs. Consequently, drug-induced dissociation of ASK1 from Trx1 and subsequent phosphorylation of JNK and p38 MAPK were inhibited in MN9D cells overexpressing anamorsin. Taken together, our results indicate that anamorsin exerts a neuroprotective effect by reducing intracellular ROS levels and subsequently attenuating activated stress-activated MAP kinases pathways.

Cardiac-specific delivery by cardiac tissue-targeting peptide-expressing exosomes.

Naturally occurring RNA carriers such as exosomes might be an untapped source of effective delivery vehicles. However, if exosomes are to be exploited for therapeutic applications, they must target specific tissues or cell types to avoid off-target effects. This study evaluated whether genetic modification of exosomes could enhance exosome delivery to heart cells and heart tissue without toxicity. Exosomes expressing cardiac-targeting peptide (CTP)-Lamp2b on the exosomal membrane (CTP-Exo) were generated by introducing vectors encoding CTP-Lamp2b into HEK 293 cells. The expression of CTP-Lamp2b peptide on exosomes was stabilized by attaching glycosylation sequences. Exosomes expressing only Lamp2b on exosomal membranes (CTL-Exo) were generated as a control. The in vitro and in vivo uptake of CTL-Exo and CTP-Exo was evaluated in cell lines and mice. Both exosomes were delivered to HEK 293 and H9C2 cells. The delivery of the exosome was not different between CTP-Exo and CTL-Exo in HEK 293 cells, whereas the delivery of CTP-Exo was 16% greater than that of CTL-Exo in H9C2 cells (P = 0.047). Cell viability was maintained at almost 100% with different dosages of both CTL-Exo and CTP-Exo. Moreover, compared with CTL-Exo, the in vivo delivery of exosomes to the hearts of mice was increased by 15% with CTP-Exo (P = 0.035). The delivery to livers and spleens was not different between the two exosomes. Genetic modification of exosomes by expressing CTP-Lamp2b on the exosomal membrane enhanced exosome delivery to heart cells and the heart tissue. These results suggested that CTP-Exo might be used as a therapeutic tool for heart disease.

Anamorsin, a novel caspase-3 substrate in neurodegeneration.

Activated caspases play a central role in the execution of apoptosis by cleaving endogenous substrates. Here, we developed a high throughput screening method to identify novel substrates for caspase-3 in a neuronal cell line. Critical steps in our strategy consist of two-dimensional electrophoresis-based protein separation and in vitro caspase-3 incubation of immobilized proteins to sort out direct substrates. Among 46 putative substrates identified in MN9D neuronal cells, we further evaluated whether caspase-3-mediated cleavage of anamorsin, a recently recognized cell death-defying factor in hematopoiesis, is a general feature of apoptosis. In vitro and cell-based cleavage assays indicated that anamorsin was specifically cleaved by caspase-3 but not by other caspases, generating 25- and 10-kDa fragments. Thus, in apoptosis of neuronal and non-neuronal cells induced by various stimuli including staurosporine, etoposide, or 6-hydroxydopamine, the cleavage of anamorsin was found to be blocked in the presence of caspase inhibitor. Among four tetrapeptide consensus DXXD motifs existing in anamorsin, we mapped a specific cleavage site for caspase-3 at DSVD(209)↓L. Intriguingly, the 25-kDa cleaved fragment of anamorsin was also detected in post-mortem brains of Alzheimer and Parkinson disease patients. Although the RNA interference-mediated knockdown of anamorsin rendered neuronal cells more vulnerable to staurosporine treatment, reintroduction of full-length anamorsin into an anamorsin knock-out stromal cell line made cells resistant to staurosporine-induced caspase activation, indicating the antiapoptotic function of anamorsin. Taken together, our approach seems to be effective to identify novel substrates for caspases and has the potential to provide meaningful insights into newly identified substrates involved in neurodegenerative processes.

Gel-based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell.

Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.

Acetylation of cyclin-dependent kinase 5 is mediated by GCN5.

Cyclin-dependent kinase 5 (CDK5), a member of atypical serine/threonine cyclin-dependent kinase family, plays a crucial role in pathophysiology of neurodegenerative disorders. Its kinase activity and substrate specificity are regulated by several independent pathways including binding with its activator, phosphorylation and S-nitrosylation. In the present study, we report that acetylation of CDK5 comprises an additional posttranslational modification within the cells. Among many candidates, we confirmed that its acetylation is enhanced by GCN5, a member of the GCN5-related N-acetyl-transferase family of histone acetyltransferase. Co-immunoprecipitation assay and fluorescent localization study indicated that GCN5 physically interacts with CDK5 and they are co-localized at the specific nuclear foci. Furthermore, liquid chromatography in conjunction with a mass spectrometry indicated that CDK5 is acetylated at Lys33 residue of ATP binding domain. Considering this lysine site is conserved among a wide range of species and other related cyclin-dependent kinases, therefore, we speculate that acetylation may alter the kinase activity of CDK5 via affecting efficacy of ATP coordination.

Small extracellular vesicle-mediated CRISPR-Cas9 RNP delivery for cardiac-specific genome editing.

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Although clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing holds immense potential for genetic manipulation, its clinical application is hindered by the absence of an efficient heart-targeted drug delivery system. Herein, we developed CRISPR-Cas9 ribonucleoprotein (RNP)-loaded extracellular vesicles (EVs) conjugated with cardiac-targeting peptide (T) for precise cardiac-specific genome editing. RNP complexes containing Cas9 and single guide RNA targeting miR-34a, an MI-associated molecular target, were loaded into EVs (EV@RNP). Gene editing by EV@RNP attenuated hydrogen peroxide-induced apoptosis in cardiomyocytes via miR-34a inhibition, evidenced by increased B-cell lymphoma 2 levels, decreased Bcl-2-associated X protein levels, and the cleavage of caspase-3. Additionally, to improve cardiac targeting in vivo, we used click chemistry to form functional T-EV@RNP by conjugating T peptides to EV@RNP. Consequently, T-EV@RNP-mediated miR-34a genome editing might exert a protective effect against MI, reducing apoptosis, ameliorating MI injury, and facilitating the recovery of cardiac function. In conclusion, the genome editing delivery system established by loading CRISPR/Cas9 RNP with cardiac-targeting EVs is a powerful approach for precise and tissue-specific gene therapy for cardiovascular disease.

Caspase-3-mediated cleavage of PICOT in apoptosis.

Mammalian protein kinase C-interacting cousin of thioredoxin (PICOT) is a multi-domain mono-thiol glutaredoxin that is involved in several signal transduction pathways and is necessary for cell growth and metastasis. Here, we demonstrate that PICOT is a cleavage substrate of the apoptosis-related protein caspase-3. In vitro cleavage assays indicated that PICOT was specifically cleaved by caspase-3. Similarly, endogenous PICOT was cleaved in cell death responses induced by staurosporine and etoposide. These phenomena were blocked in the presence of a pan-caspase inhibitor. Using site-directed mutagenesis, we identified two putative caspase-3 cleavage sequences in PICOT, DRLD(101)/G and EELD(226)/T. Interestingly, overexpression of either PICOT wild type or the D101A/D226A double point mutant accelerated etoposide-induced activation of caspase-3 whereas siRNA-mediated knockdown of PICOT blocked this phenomenon. Our data raise the possibility that the pro-apoptotic role of PICOT is actively regulated via caspase-3-mediated cleavage.

Engineered small extracellular vesicle-mediated NOX4 siRNA delivery for targeted therapy of cardiac hypertrophy.

Small-interfering RNA (siRNA) therapy is considered a powerful therapeutic strategy for treating cardiac hypertrophy, an important risk factor for subsequent cardiac morbidity and mortality. However, the lack of safe and efficient in vivo delivery of siRNAs is a major challenge for broadening its clinical applications. Small extracellular vesicles (sEVs) are a promising delivery system for siRNAs but have limited cell/tissue-specific targeting ability. In this study, a new generation of heart-targeting sEVs (CEVs) has been developed by conjugating cardiac-targeting peptide (CTP) to human peripheral blood-derived sEVs (PB-EVs), using a simple, rapid and scalable method based on bio-orthogonal copper-free click chemistry. The experimental results show that CEVs have typical sEVs properties and excellent heart-targeting ability. Furthermore, to treat cardiac hypertrophy, CEVs are loaded with NADPH Oxidase 4 (NOX4) siRNA (siNOX4). Consequently, CEVs@siNOX4 treatment enhances the in vitro anti-hypertrophic effects by CEVs with siRNA protection and heart-targeting ability. In addition, the intravenous injection of CEVs@siNOX4 into angiotensin II (Ang II)-treated mice significantly improves cardiac function and reduces fibrosis and cardiomyocyte cross-sectional area, with limited side effects. In conclusion, the utilization of CEVs represents an efficient strategy for heart-targeted delivery of therapeutic siRNAs and holds great promise for the treatment of cardiac hypertrophy.

Serum exosomal long noncoding RNAs as a diagnostic biomarker for atrial fibrillation.

BACKGROUND: Exosomal long noncoding RNAs (lncRNAs) are known as ideal diagnostic biomarkers of various diseases. However, there are no reports on the use of serum exosomal lncRNAs as diagnostic biomarkers for atrial fibrillation (AF). OBJECTIVE: The purpose of this study was to explore serum exosomal lncRNAs as a useful tool for diagnosing AF. METHODS: Serum exosomes from patients with persistent AF and controls were isolated using a polymer-based exosome precipitation kit. We conducted a multiphase process including screening and 2 independent validation phases. In the screening phase, serum exosomal lncRNA expression profiles were examined using RNA sequencing analysis. In 2 validation phases, we evaluated the expression levels of candidate exosomal lncRNAs using quantitative reverse transcription polymerase chain reaction. Finally, we performed different statistical and functional analyses. RESULTS: After the screening phase, we identified 26 differentially expressed lncRNAs (ie, 15 upregulated and 11 downregulated lncRNAs with a |fold change| ≥2 and P <.05) in serum exosomes from patients with persistent AF compared with controls. We then screened out 6 exosomal lncRNAs as biomarker candidates following parameters: read length ≥200 nucleotides; exon number ≥2; and coding potential score <0.1. In 2 validation phases, exosomal lncRNAs LOC105377989 and LOC107986997 were consistently upregulated in the serum of patients with persistent AF compared with controls (P <.0001). Moreover, both exosomal lncRNAs exhibited significant diagnostic validity for AF. Notably, exosomal lncRNA LOC107986997 was involved in AF-related pathophysiological mechanisms. CONCLUSION: Serum-derived exosomal lncRNA LOC107986997 could serve as a potential biomarker for AF diagnosis.

Generation of a heterozygous TPM1-E192K knock-in human induced pluripotent stem cell line using CRISPR/Cas9 system.

E192K missense mutation of TPM1 has been found in different types of cardiomyopathies (e.g., hypertrophic cardiomyopathy, dilated cardiomyopathy, and left ventricular non-compaction), leading to systolic dysfunction, diastolic dysfunction, and/or tachyarrhythmias. Here, we generated a heterozygous TPM1-E192K knock-in human induced pluripotent stem cell (iPSC) line using CRISPR/Cas9-based genome editing system. The cells exhibit normal karyotype, typical stem cell morphology, expression of pluripotency markers and differentiation ability into three germ layers. Accordingly, this cell line could provide a useful cell resource for exploring the pathogenic role of TPM1-E192K mutation in different types of cardiomyopathies.

Generation of two PITX2 knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system.

PITX2 is a homeobox gene located in the human 4q25 locus and is commonly associated with atrial fibrillation (AF). Here, we generated two PITX2 knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 genome editing. The edited iPSCs maintained fullpluripotency, normal karyotype and spontaneousdifferentiation capability. This cell line provides a suitable model for investigating the physiopathologyof PITX2 mutation in atrial fibrillation.

Generation of three TTN knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system.

TTN mutations are the common genetic cause for various types of cardiomyopathies (e.g., dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy) and skeletal myopathies. Here, we generated three TTN knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 system. These cell lines, which exhibit normal karyotype, typical morphology and pluripotency, could provide useful platform for investigating the role of TTN in associated disorders.

Nuclear translocation of anamorsin during drug-induced dopaminergic neurodegeneration in culture and in rat brain.

Anamorsin, also called cytokine-induced apoptosis inhibitor 1 (CIAPIN1), was recently identified to confer resistance to apoptosis induced by growth factor deprivation and to be indispensible for hematopoiesis. Recently, it was demonstrated that anamorsin is also widely distributed in both fetal and adult tissues. In this study, we evaluated the tissue distribution of anamorsin in the central nervous system (CNS) during development. In situ hybridization and immunoblot analyses revealed that anamorsin mRNA and protein were both highly and widely expressed in various regions of the CNS, including the cerebral cortex, hippocampus, midbrain, cerebellum, medulla, and spinal cord. Based on these findings, we examined its cellular localization during drug-induced neurodegeneration in MN9D dopaminergic cells. Both immunocytochemical localization and immunoblot analyses indicated that cytosolic anamorsin was translocated into the nucleus in a time-dependent manner following treatment with a reactive oxygen species (ROS)-inducing drug, 6-hydroxydopamine (6-OHDA). Treatment of cells with the apoptosis-inducing reagent, staurosporine, did not appear to cause translocation of anamorsin into the nucleus. When cells were treated with the nuclear export inhibitor, Leptomycin B, alone or with 6-OHDA, nuclear anamorsin levels increased, indicating that nuclear influx and efflux of anamorsin are regulated by 6-OHDA treatment. In rat brain injected with 6-OHDA, nuclear translocation of anamorsin was identified in certain tyrosine hydroxylase (TH)-positive neurons as well as TH-negative cells. Furthermore, treatment of MN9D cells with hydrogen peroxide or ROS-inducing trace metals caused nuclear translocation of anamorsin. Taken together, our data indicate that nuclear translocation of anamorsin is a ROS-dependent event and may participate in the regulation of transcription of critical molecules during dopaminergic neurodegeneration.