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1.
CNS Neurosci Ther ; 30(6): e14764, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38828629

ABSTRACT

AIMS: Neuropathic pain is a common chronic pain disorder, which is largely attributed to spinal central sensitization. Calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) activation in the spinal dorsal horn (SDH) is a major contributor to spinal sensitization. However, the exact way that CaMKIIα-positive (CaMKIIα+) neurons in the SDH induce neuropathic pain is still unclear. This study aimed to explore the role of spinal CaMKIIα+ neurons in neuropathic pain caused by chronic constriction injury (CCI) and investigate the potential epigenetic mechanisms involved in CaMKIIα+ neuron activation. METHODS: CCI-induced neuropathic pain mice model, Sirt1loxP/loxP mice, and chemogenetic virus were used to investigate whether the activation of spinal CaMKIIα+ neurons is involved in neuropathic pain and its involved mechanism. Transcriptome sequence, western blotting, qRT-PCR, and immunofluorescence analysis were performed to assay the expression of related molecules and activation of neurons. Co-immunoprecipitation was used to observe the binding relationship of protein. Chromatin immunoprecipitation (ChIP)-PCR was applied to analyze the acetylation of histone H3 in the Scn3a promoter region. RESULTS: The expression of sodium channel Nav1.3 was increased and the expression of SIRT1 was decreased in the spinal CaMKIIα+ neurons of CCI mice. CaMKIIα neurons became overactive after CCI, and inhibiting their activation relieved CCI-induced pain. Overexpression of SIRT1 reversed the increase of Nav1.3 and alleviated pain, while knockdown of SIRT1 or overexpression of Nav1.3 promoted CaMKIIα+ neuron activation and induced pain. By knocking down spinal SIRT1, the acetylation of histone H3 in the Scn3a (encoding Nav1.3) promoter region was increased, leading to an increased expression of Nav1.3. CONCLUSION: The findings suggest that an aberrant reduction of spinal SIRT1 after nerve injury epigenetically increases Nav1.3, subsequently activating CaMKIIα+ neurons and causing neuropathic pain.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Neuralgia , Sirtuin 1 , Animals , Neuralgia/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Sirtuin 1/metabolism , Sirtuin 1/genetics , Mice , Male , Neurons/metabolism , Spinal Cord/metabolism , Mice, Inbred C57BL
2.
Zhen Ci Yan Jiu ; 49(5): 480-486, 2024 May 25.
Article in English, Chinese | MEDLINE | ID: mdl-38764119

ABSTRACT

OBJECTIVES: To observe the activation state and neuronal types of somatosensory cortex and the primary motor cortex induced by electroacupuncture (EA) stimulation of "Sibai" (ST2) and "Quanliao" (SI18) acupoints in mice. METHODS: Male C57BL/6J mice were randomly divided into blank control and EA groups, with 6 mice in each group. Rats of the EA group received EA stimulation (2 Hz, 0.6 mA) at ST2 and SI18 for 30 minutes. Samples were collected after EA intervention, and immunofluorescence staining was performed to quantify the expression of the c-Fos gene (proportion of c-Fos positive cells) in the somatosensory cortex and primary motor cortex. The co-labelled cells of calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) and gamma-aminobutyric acid (GABA) in the somatosensory cortex and primary motor cortex were observed and counted by using microscope after immunofluorescence staining. Another 10 mice were used to detect the calcium activity of excitatory neurons in the somatosensory cortex and primary motor cortex by fiber photometry. RESULTS: In comparison with the blank control group, the number of c-Fos positive cells, and the proportion of c-Fos and CaMKⅡ co-labelled cells in both the somatosensory cortex and primary motor cortex were significantly increased after EA stimulation (P<0.05). No significant changes were found in the proportion of c-Fos and GABA co-labeled cells in both the somatosensory cortex and primary motor cortex after EA. Results of fiber optic calcium imaging technology showed that the spontaneous calcium activity of excitatory neurons in both somatosensory cortex and primary motor cortex were obviously increased during EA compared with that before EA (P<0.01), and strikingly reduced after cessation of EA compared with that during EA (P<0.05). CONCLUSIONS: Under physiological conditions, EA of ST2 and SI18 can effectively activate excitatory neurons in the somatosensory cortex and primary motor cortex.


Subject(s)
Acupuncture Points , Electroacupuncture , Mice, Inbred C57BL , Neurons , Animals , Male , Mice , Neurons/metabolism , Sensorimotor Cortex/metabolism , Humans , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-fos/genetics , Motor Cortex/metabolism , Somatosensory Cortex/metabolism
3.
Cardiovasc Diabetol ; 23(1): 150, 2024 May 03.
Article in English | MEDLINE | ID: mdl-38702777

ABSTRACT

BACKGROUND: Vasculopathy is the most common complication of diabetes. Endothelial cells located in the innermost layer of blood vessels are constantly affected by blood flow or vascular components; thus, their mechanosensitivity plays an important role in mediating vascular regulation. Endothelial damage, one of the main causes of hyperglycemic vascular complications, has been extensively studied. However, the role of mechanosensitive signaling in hyperglycemic endothelial damage remains unclear. METHODS: Vascular endothelial-specific Piezo1 knockout mice were generated to investigate the effects of Piezo1 on Streptozotocin-induced hyperglycemia and vascular endothelial injury. In vitro activation or knockdown of Piezo1 was performed to evaluate the effects on the proliferation, migration, and tubular function of human umbilical vein endothelial cells in high glucose. Reactive oxygen species production, mitochondrial membrane potential alternations, and oxidative stress-related products were used to assess the extent of oxidative stress damage caused by Piezo1 activation. RESULTS: Our study found that in VECreERT2;Piezo1flox/flox mice with Piezo1 conditional knockout in vascular endothelial cells, Piezo1 deficiency alleviated streptozotocin-induced hyperglycemia with reduced apoptosis and abscission of thoracic aortic endothelial cells, and decreased the inflammatory response of aortic tissue caused by high glucose. Moreover, the knockout of Piezo1 showed a thinner thoracic aortic wall, reduced tunica media damage, and increased endothelial nitric oxide synthase expression in transgenic mice, indicating the relief of endothelial damage caused by hyperglycemia. We also showed that Piezo1 activation aggravated oxidative stress injury and resulted in severe dysfunction through the Ca2+-induced CaMKII-Nrf2 axis in human umbilical vein endothelial cells. In Piezo1 conditional knockout mice, Piezo1 deficiency partially restored superoxide dismutase activity and reduced malondialdehyde content in the thoracic aorta. Mechanistically, Piezo1 deficiency decreased CaMKII phosphorylation and restored the expression of Nrf2 and its downstream molecules HO-1 and NQO1. CONCLUSION: In summary, our study revealed that Piezo1 is involved in high glucose-induced oxidative stress injury and aggravated endothelial dysfunction, which have great significance for alleviating endothelial damage caused by hyperglycemia.


Subject(s)
Blood Glucose , Diabetes Mellitus, Experimental , Human Umbilical Vein Endothelial Cells , Ion Channels , Mice, Knockout , Nitric Oxide Synthase Type III , Oxidative Stress , Animals , Humans , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Diabetes Mellitus, Experimental/metabolism , Ion Channels/metabolism , Ion Channels/genetics , Blood Glucose/metabolism , Nitric Oxide Synthase Type III/metabolism , Mechanotransduction, Cellular , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/deficiency , Cells, Cultured , Cell Proliferation , Apoptosis , Male , Diabetic Angiopathies/metabolism , Diabetic Angiopathies/physiopathology , Diabetic Angiopathies/pathology , Diabetic Angiopathies/genetics , Diabetic Angiopathies/etiology , Cell Movement , Mice, Inbred C57BL , Reactive Oxygen Species/metabolism , Aorta, Thoracic/metabolism , Aorta, Thoracic/pathology , Aorta, Thoracic/physiopathology , Mice , Streptozocin , Endothelium, Vascular/metabolism , Endothelium, Vascular/physiopathology , Endothelium, Vascular/pathology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics
4.
Skelet Muscle ; 14(1): 11, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38769542

ABSTRACT

BACKGROUND: Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. METHODS: We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1ΔE3/ΔE3 mice, two established mouse models of DM1. RESULTS: Muscle from DM1 mouse models showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models exhibited increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, the expression of synaptic genes was up-regulated in mutant innervated muscle, together with an abnormal accumulation of histone deacetylase 4 (HDAC4), a known target of CaMKII. Interestingly, denervation-induced increase in synaptic gene expression and AChR turnover was hampered in DM1 muscle. Importantly, CaMKIIß/ßM overexpression normalized endplate fragmentation and synaptic gene expression in innervated Mbnl1ΔE3/ΔE3 muscle, but it did not restore denervation-induced synaptic gene up-regulation. CONCLUSIONS: Our results indicate that CaMKIIß-dependent and -independent mechanisms perturb synaptic gene regulation and muscle response to denervation in DM1 mouse models. Changes in these signalling pathways may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Disease Models, Animal , Muscle, Skeletal , Myotonic Dystrophy , Neuromuscular Junction , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , Myotonic Dystrophy/physiopathology , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Neuromuscular Junction/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/innervation , Muscle, Skeletal/pathology , Mice , Humans , Histone Deacetylases/metabolism , Histone Deacetylases/genetics , Receptors, Cholinergic/metabolism , Receptors, Cholinergic/genetics , Male , Mice, Inbred C57BL
6.
Int J Mol Sci ; 25(8)2024 Apr 17.
Article in English | MEDLINE | ID: mdl-38674016

ABSTRACT

Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.


Subject(s)
Dynamins , Graft Rejection , Heart Transplantation , Necroptosis , Receptor-Interacting Protein Serine-Threonine Kinases , Animals , Mice , Graft Rejection/metabolism , Graft Rejection/pathology , Heart Transplantation/adverse effects , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Dynamins/metabolism , Dynamins/genetics , Mitochondria/metabolism , Endothelial Cells/metabolism , Male , Mice, Inbred C57BL , Phosphoprotein Phosphatases/metabolism , Phosphoprotein Phosphatases/genetics , Phosphorylation , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Signal Transduction
7.
Nat Commun ; 15(1): 2694, 2024 Mar 27.
Article in English | MEDLINE | ID: mdl-38538603

ABSTRACT

Long noncoding RNAs (lncRNAs) play crucial roles in maintaining cell homeostasis and function. However, it remains largely unknown whether and how neuronal activity impacts the transcriptional regulation of lncRNAs, or if this leads to synapse-related changes and contributes to the formation of long-term memories. Here, we report the identification of a lncRNA, SLAMR, which becomes enriched in CA1-hippocampal neurons upon contextual fear conditioning but not in CA3 neurons. SLAMR is transported along dendrites via the molecular motor KIF5C and is recruited to the synapse upon stimulation. Loss of function of SLAMR reduces dendritic complexity and impairs activity-dependent changes in spine structural plasticity and translation. Gain of function of SLAMR, in contrast, enhances dendritic complexity, spine density, and translation. Analyses of the SLAMR interactome reveal its association with CaMKIIα protein through a 220-nucleotide element also involved in SLAMR transport. A CaMKII reporter reveals a basal reduction in CaMKII activity with SLAMR loss-of-function. Furthermore, the selective loss of SLAMR function in CA1 disrupts the consolidation of fear memory in male mice, without affecting their acquisition, recall, or extinction, or spatial memory. Together, these results provide new molecular and functional insight into activity-dependent changes at the synapse and consolidation of contextual fear.


Subject(s)
RNA, Long Noncoding , Mice , Male , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Neurons/metabolism , Hippocampus/physiology , Mental Recall/physiology , Neuronal Plasticity/genetics , Mice, Inbred C57BL
8.
Protein Sci ; 33(4): e4960, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38501502

ABSTRACT

Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a multidomain serine/threonine kinase that plays important roles in the brain, heart, muscle tissue, and eggs/sperm. The N-terminal kinase and regulatory domain is connected by a flexible linker to the C-terminal hub domain. The hub domain drives the oligomeric organization of CaMKII, assembling the kinase domains into high local concentration. Previous structural studies have shown multiple stoichiometries of the holoenzyme as well as the hub domain alone. Here, we report a comprehensive study of the hub domain stoichiometry and stability in solution. We solved two crystal structures of the CaMKIIß hub domain that show 14-mer (3.1 Å) and 16-mer (3.4 Å) assemblies. Both crystal structures were determined from crystals grown in the same drop, which suggests that CaMKII oligomers with different stoichiometries likely coexist. To further interrogate hub stability, we employed mass photometry and temperature denaturation studies of CaMKIIß and CaMKIIα hubs, which highlight major differences between these highly similar domains. We created a dimeric CaMKIIß hub unit using rational mutagenesis, which is significantly less stable than the oligomer. Both hub domains populate an intermediate during unfolding. We found that multiple CaMKIIß hub stoichiometries are present in solution and that larger oligomers are more stable. CaMKIIα had a narrower distribution of molecular weight and was distinctly more stable than CaMKIIß.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium , Male , Humans , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Semen/metabolism
9.
Ann Clin Lab Sci ; 54(1): 17-25, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38514055

ABSTRACT

OBJECTIVE: Diabetic cardiomyopathy (DCM) is the most common cardiovascular complication of type 2 diabetes mellitus (T2DM). Patients affected with DCM face a notably higher risk of progressing to congestive heart failure compared to other populations. Myocardial hypertrophy, a clearly confirmed pathological change in DCM, plays an important role in the development of DCM, with abnormal Ca2+ homeostasis serving as the key signal to induce myocardial hypertrophy. Therefore, investigating the mechanism of Ca2+ transport is of great significance for the prevention and treatment of myocardial hypertrophy in T2DM. METHODS: The rats included in the experiment were divided into wild type (WT) group and T2DM group. The T2DM rat model was established by feeding the rats with high-fat and high-sugar diets for three months combined with low dose of streptozotocin (100mg/kg). Afterwards, primary rat cardiomyocytes were isolated and cultured, and cardiomyocyte hypertrophy was induced through high-glucose treatment. Subsequently, mechanistic investigations were carried out through transfection with si-STIM1 and oe-STIM1. Western blot (WB) was used to detect the expression of the STIM1, Orai1 and p-CaMKII. qRT-PCR was used to detect mRNA levels of myocardial hypertrophy marker proteins. Cell surface area was detected using TRITC-Phalloidin staining, and intracellular Ca2+ concentration in cardiomyocytes was measured using Fluo-4 fluorescence staining. RESULTS: Through animal experiments, an upregulation of Orai1 and STIM1 was revealed in the rat model of myocardial hypertrophy induced by T2DM. Meanwhile, through cell experiments, it was found that in high glucose (HG)-induced hypertrophic cardiomyocytes, the expression of STIM1, Orai1, and p-CaMKII was upregulated, along with increased levels of store-operated Ca2+ entry (SOCE) and abnormal Ca2+ homeostasis. However, when STIM1 was downregulated in HG-induced cardiomyocytes, SOCE levels decreased and p-CaMKII was downregulated, resulting in an improvement in myocardial hypertrophy. To further elucidate the mechanism of action involving SOCE and CaMKII in T2DM-induced myocardial hypertrophy, high-glucose cardiomyocytes were respectively treated with BTP2 (SOCE blocker) and KN-93 (CaMKII inhibitor), and the results showed that STIM1 can mediate SOCE, thereby affecting the phosphorylation level of CaMKII and improving cardiomyocyte hypertrophy. CONCLUSION: STIM1/Orai1-mediated SOCE regulates p-CaMKII levels, thereby inducing myocardial hypertrophy in T2DM.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium , Cardiomegaly , Diabetes Mellitus, Type 2 , Diabetic Cardiomyopathies , Glucose , ORAI1 Protein , Stromal Interaction Molecule 1 , Animals , Rats , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cardiomegaly/etiology , Cardiomegaly/metabolism , Diabetes Mellitus, Type 2/complications , Glucose/metabolism , Glucose/pharmacology , ORAI1 Protein/genetics , ORAI1 Protein/metabolism , Stromal Interaction Molecule 1/genetics , Stromal Interaction Molecule 1/metabolism , Up-Regulation , Diabetic Cardiomyopathies/complications , Rats, Sprague-Dawley , Male
10.
Signal Transduct Target Ther ; 9(1): 63, 2024 Mar 08.
Article in English | MEDLINE | ID: mdl-38453934

ABSTRACT

Neurotransmitter-initiated signaling pathway were reported to play an important role in regulating the malignant phenotype of tumor cells. Cancer cells could exhibit a "neural addiction" property and build up local nerve networks to achieve an enhanced neurotransmitter-initiated signaling through nerve growth factor-mediated axonogenesis. Targeting the dysregulated nervous systems might represent a novel strategy for cancer treatment. However, whether intrahepatic cholangiocarcinoma (ICC) could build its own nerve networks and the role of neurotransmitters in the progression ICC remains largely unknown. Immunofluorescence staining and Enzyme-linked immunosorbent assay suggested that ICC cells and the infiltrated nerves could generate a tumor microenvironment rich in acetylcholine that promotes ICC metastasis by inducing epithelial-mesenchymal transition (EMT). Acetylcholine promoted ICC metastasis through interacting with its receptor, alpha 5 nicotine acetylcholine receptor subunits (CHRNA5). Furthermore, acetylcholine/CHRNA5 axis activated GSK3ß/ß-catenin signaling pathway partially through the influx of Ca2+-mediated activation of Ca/calmodulin-dependent protein kinases (CAMKII). In addition, acetylcholine signaling activation also expanded nerve infiltration through increasing the expression of Brain-Derived Neurotrophic Factor (BDNF), which formed a feedforward acetylcholine-BDNF axis to promote ICC progression. KN93, a small-molecule inhibitor of CAMKII, significantly inhibited the migration and enhanced the sensitivity to gemcitabine of ICC cells. Above all, Acetylcholine/CHRNA5 axis increased the expression of ß-catenin to promote the metastasis and resistance to gemcitabine of ICC via CAMKII/GSK3ß signaling, and the CAMKII inhibitor KN93 may be an effective therapeutic strategy for combating ICC metastasis.


Subject(s)
Benzenesulfonamides , Benzylamines , Bile Duct Neoplasms , Cholangiocarcinoma , Humans , beta Catenin/metabolism , Brain-Derived Neurotrophic Factor/genetics , Nicotine , Acetylcholine , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Gemcitabine , Glycogen Synthase Kinase 3 beta , Cell Line, Tumor , Cholangiocarcinoma/drug therapy , Cholangiocarcinoma/genetics , Bile Ducts, Intrahepatic/metabolism , Bile Ducts, Intrahepatic/pathology , Bile Duct Neoplasms/drug therapy , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/metabolism , Neurotransmitter Agents , Receptors, Cholinergic , Tumor Microenvironment
11.
Food Chem Toxicol ; 186: 114577, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38458532

ABSTRACT

Silver nanoparticles (AgNPs) have been widely used in biomedicine and cosmetics, increasing their potential risks in neurotoxicity. But the involved molecular mechanism remains unclear. This study aims to explore molecular events related to AgNPs-induced neuronal damage by RNA-seq, and elucidate the role of Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22 cells synaptic degeneration induced by AgNPs. This study found that cell viabilities were decreased by AgNPs in a dose/time-dependent manner. AgNPs also increased protein expression of PINK1, Parkin, synaptophysin, and inhibited PGC-1α, MAP2 and APP protein expression, indicating AgNPs-induced synaptic degeneration involved in disturbance of mitophagy and mitochondrial biogenesis in HT22 cells. Moreover, inhibition of AgNPs-induced Ca2+/CaMKII activation and Drp1/ROS rescued mitophagy disturbance and synaptic degeneration in HT22 cells by reserving aforementioned protein express changes except for PGC-1α and APP protein. Thus, AgNPs-induced synaptic degeneration was mediated by Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22 cells, and mitophagy is the sensitive to the mechanism. Our study will provide in-depth molecular mechanism data for neurotoxic evaluation and biomedical application of AgNPs.


Subject(s)
Metal Nanoparticles , Mitochondrial Diseases , Humans , Silver/toxicity , Silver/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Mitochondria/metabolism , Metal Nanoparticles/toxicity
12.
J Neurosci ; 44(11)2024 Mar 13.
Article in English | MEDLINE | ID: mdl-38331582

ABSTRACT

Cerebellum has been implicated in drug addiction; however, its underlying cellular populations and neuronal circuitry remain largely unknown. In the current study, we identified a neural pathway from tyrosine hydroxylase (TH)-positive Purkinje cells (PCTH+) in cerebellar lobule VI to calcium/calmodulin-dependent protein kinase II (CaMKII)-positive glutamatergic neurons in the medial cerebellar nucleus (MedCaMKII), forming the lobule VI PCTH+-MedCaMKII pathway in male mice. In naive male mice, inhibition of PCTH+ neurons activated Med neurons. During conditioned place preference (CPP) training, exposure to methamphetamine (METH) inhibited lobule VI PCTH+ neurons while excited MedCaMKII neurons in mice. Silencing MedCaMKII using a tetanus toxin light chain (tettox) suppressed the acquisition of METH CPP in mice but resulted in motor coordination deficits in naive mice. In contrast, activating lobule VI PCTH+ terminals within Med inhibited the activity of Med neurons and subsequently blocked the acquisition of METH CPP in mice without affecting motor coordination, locomotor activity, and sucrose reinforcements in naive mice. Our findings identified a novel lobule VI PCTH+-MedCaMKII pathway within the cerebellum and explored its role in mediating the acquisition of METH-preferred behaviors.


Subject(s)
Central Nervous System Stimulants , Methamphetamine , Animals , Male , Mice , Methamphetamine/pharmacology , Tyrosine 3-Monooxygenase/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Reinforcement, Psychology , Cerebellum/metabolism , Central Nervous System Stimulants/pharmacology
13.
J Affect Disord ; 349: 286-296, 2024 Mar 15.
Article in English | MEDLINE | ID: mdl-38199412

ABSTRACT

BACKGROUND: Early life stress is a major risk factor for later development of psychiatric disorders, including post-traumatic stress disorder (PTSD). An intricate relationship exists between various neurotransmitters (such as glutamate, norepinephrine or serotonin), calcium/calmodulin-dependent protein kinase II (CaMKII), as an important regulator of glutamatergic synaptic function, and PTSD. Here, we developed a double-hit model to investigate the interaction of maternal deprivation (MD) as an early life stress model and single prolonged stress (SPS) as a PTSD model at the behavioral and molecular levels. METHODS: Male Wistar rats exposed to these stress paradigms were subjected to a comprehensive behavioral analysis. In hippocampal synaptosomes we investigated neurotransmitter release and glutamate concentration. The expression of CaMKII and the content of monoamines were determined in selected brain regions. Brain-derived neurotrophic factor (BDNF) mRNA was quantified by radioactive in situ hybridization. RESULTS: We report a distinct behavioral phenotype in the double-hit group. Double-hit and SPS groups had decreased hippocampal presynaptic glutamatergic function. In hippocampus, double-hit stress caused a decrease in autophosphorylation of CaMKII. In prefrontal cortex, both SPS and double-hit stress had a similar effect on CaMKII autophosphorylation. Double-hit stress, rather than SPS, affected the norepinephrine and serotonin levels in prefrontal cortex, and suppressed BDNF gene expression in prefrontal cortex and hippocampus. LIMITATIONS: The study was conducted in male rats only. The affected brain regions cannot be restricted to hippocampus, prefrontal cortex and amygdala. CONCLUSION: Double-hit stress caused more pronounced and distinct behavioral, molecular and functional changes, compared to MD or SPS alone.


Subject(s)
Serotonin , Stress Disorders, Post-Traumatic , Humans , Rats , Male , Animals , Serotonin/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/pharmacology , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , Rats, Wistar , Glutamic Acid/metabolism , Norepinephrine , Maternal Deprivation , Down-Regulation , Brain/metabolism , Hippocampus/metabolism , Stress Disorders, Post-Traumatic/genetics , Disease Models, Animal
14.
Am J Hum Genet ; 111(2): 364-382, 2024 Feb 01.
Article in English | MEDLINE | ID: mdl-38272033

ABSTRACT

The calcium/calmodulin-dependent protein kinase type 2 (CAMK2) family consists of four different isozymes, encoded by four different genes-CAMK2A, CAMK2B, CAMK2G, and CAMK2D-of which the first three have been associated recently with neurodevelopmental disorders. CAMK2D is one of the major CAMK2 proteins expressed in the heart and has been associated with cardiac anomalies. Although this CAMK2 isoform is also known to be one of the major CAMK2 subtypes expressed during early brain development, it has never been linked with neurodevelopmental disorders until now. Here we show that CAMK2D plays an important role in neurodevelopment not only in mice but also in humans. We identified eight individuals harboring heterozygous variants in CAMK2D who display symptoms of intellectual disability, delayed speech, behavioral problems, and dilated cardiomyopathy. The majority of the variants tested lead to a gain of function (GoF), which appears to cause both neurological problems and dilated cardiomyopathy. In contrast, loss-of-function (LoF) variants appear to induce only neurological symptoms. Together, we describe a cohort of individuals with neurodevelopmental disorders and cardiac anomalies, harboring pathogenic variants in CAMK2D, confirming an important role for the CAMK2D isozyme in both heart and brain function.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Cardiomyopathy, Dilated , Intellectual Disability , Neurodevelopmental Disorders , Animals , Humans , Mice , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Heart , Neurodevelopmental Disorders/genetics
15.
PLoS One ; 19(1): e0285651, 2024.
Article in English | MEDLINE | ID: mdl-38180986

ABSTRACT

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium , Humans , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Baculoviridae/genetics , Biophysics , Calcium Signaling , Escherichia coli/genetics
16.
Rev Endocr Metab Disord ; 25(2): 369-382, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38064002

ABSTRACT

Diabetes mellitus is a metabolic disorder denoted by chronic hyperglycemia that drives maladaptive structural changes and functional damage to the vasculature. Attenuation of this pathological remodeling of blood vessels remains an unmet target owing to paucity of information on the metabolic signatures of this process. Ca2+/calmodulin-dependent kinase II (CaMKII) is expressed in the vasculature and is implicated in the control of blood vessels homeostasis. Recently, CaMKII has attracted a special attention in view of its chronic upregulated activity in diabetic tissues, yet its role in the diabetic vasculature remains under investigation.This review highlights the physiological and pathological actions of CaMKII in the diabetic vasculature, with focus on the control of the dialogue between endothelial (EC) and vascular smooth muscle cells (VSMC). Activation of CaMKII enhances EC and VSMC proliferation and migration, and increases the production of extracellular matrix which leads to maladaptive remodeling of vessels. This is manifested by activation of genes/proteins implicated in the control of the cell cycle, cytoskeleton organization, proliferation, migration, and inflammation. Endothelial dysfunction is paralleled by impaired nitric oxide signaling, which is also influenced by CaMKII signaling (activation/oxidation). The efficiency of CaMKII inhibitors is currently being tested in animal models, with a focus on the genetic pathways involved in the regulation of CaMKII expression (microRNAs and single nucleotide polymorphisms). Interestingly, studies highlight an interaction between the anti-diabetic drugs and CaMKII expression/activity which requires further investigation. Together, the studies reviewed herein may guide pharmacological approaches to improve health-related outcomes in patients with diabetes.


Subject(s)
Diabetes Mellitus , Vascular System Injuries , Animals , Humans , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Signal Transduction
17.
Dev Dyn ; 253(4): 390-403, 2024 Apr.
Article in English | MEDLINE | ID: mdl-37860955

ABSTRACT

BACKGROUND: Noncanonical Wnts are morphogens that can elevate intracellular Ca2+, activate the Ca2+/calmodulin-dependent protein kinase, CaMKII, and promote cell movements during vertebrate gastrulation. RESULTS: Zebrafish express seven CaMKII genes during embryogenesis; two of these, camk2b1 and camk2g1, are necessary for convergent extension (CE) cell movements. CaMKII morphant phenotypes were observed as early as epiboly. At the 1-3 somite stage, neuroectoderm and paraxial cells remained unconverged in both morphants. Later, somites lacked their stereotypical shape and were wider, more closely spaced, and body gap angles increased. At 24hpf, somite compression and notochord undulation coincided with a shorter and broader body axis. A camk2b1 crispant was generated which phenocopied the camk2b1 morphant. The levels of cell proliferation, apoptosis and paraxial and neuroectodermal markers were unchanged in morphants. Hyperactivation of CaMKII during gastrulation by transient pharmacological intervention (thapsigargin) also caused CE defects. Mosaically expressed dominant-negative CaMKII recapitulated these phenotypes and showed significant midline bifurcation. Finally, the introduction of CaMKII partially rescued Wnt11 morphant phenotypes. CONCLUSIONS: Overall, these data support a model whereby cyclically activated CaMKII encoded from two genes enables cell migration during the process of CE.


Subject(s)
Zebrafish Proteins , Zebrafish , Animals , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Gastrulation/physiology , Cell Movement/physiology
18.
Biochim Biophys Acta Mol Cell Res ; 1871(2): 119610, 2024 02.
Article in English | MEDLINE | ID: mdl-37913845

ABSTRACT

BACKGROUND: We tested whether enhancing the capacity for calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling would delay fatigue of excitation-induced calcium release and improve contractile characteristics of skeletal muscle during fatiguing exercise. METHODS: Fast and slow type muscle, gastrocnemius medialis (GM) and soleus (SOL), of rats and mouse interosseus (IO) muscle fibers, were transfected with pcDNA3-based plasmids for rat α and ß CaMKII or empty controls. Levels of CaMKII, its T287-phosphorylation (pT287-CaMKII), and phosphorylation of components of calcium release and re-uptake, ryanodine receptor 1 (pS2843-RyR1) and phospholamban (pT17-PLN), were quantified biochemically. Sarcoplasmic calcium in transfected muscle fibers was monitored microscopically during trains of electrical excitation based on Fluo-4 FF fluorescence (n = 5-7). Effects of low- (n = 6) and high- (n = 8) intensity exercise on pT287-CaMKII and contractile characteristics were studied in situ. RESULTS: Co-transfection with αCaMKII-pcDNA3/ßCaMKII-pcDNA3 increased α and ßCaMKII levels in SOL (+45.8 %, +250.5 %) and GM (+40.4 %, +89.9 %) muscle fibers compared to control transfection. High-intensity exercise increased pT287-ßCaMKII and pS2843-RyR1 levels in SOL (+269 %, +151 %) and GM (+354 %, +119 %), but decreased pT287-αCaMKII and p17-PLN levels in GM compared to SOL (-76 % vs. +166 %; 0 % vs. +128 %). α/ß CaMKII overexpression attenuated the decline of calcium release in muscle fibers with repeated excitation, and mitigated exercise-induced deterioration of rates in force production, and passive force, in a muscle-dependent manner, in correlation with pS2843-RyR1 and pT17-PLN levels (|r| > 0.7). CONCLUSION: Enhanced capacity for α/ß CaMKII signaling improves fatigue-resistance of active and passive contractile muscle properties in association with RyR1- and PLN-related improvements in sarcoplasmic calcium release.


Subject(s)
Calcium , Ryanodine Receptor Calcium Release Channel , Rats , Mice , Animals , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolism , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium Signaling , Muscle Contraction
19.
Environ Toxicol ; 39(3): 1494-1504, 2024 Mar.
Article in English | MEDLINE | ID: mdl-37994244

ABSTRACT

As a synthetic androgen, 17α-methyltestosterone (MT) is widely used in aquaculture to induce sex reversal and may pose a potential risk to aquatic organisms. This ecological risk has attracted the attention of many scholars, but it is not comprehensive enough. Thus, the adverse effects of MT on zebrafish (Danio rerio) were comprehensively evaluated from gonadal histology, as well as the mRNA expression levels of 47 genes related to hypothalamic-pituitary-gonadal (HPG) axis, germ cell differentiation, sex determination, and hypothalamus-pituitary-thyroid (HPT) axis. Adult zebrafish with a female/male ratio of 5:7 were exposed to a solvent control (0.001% dimethyl sulfoxide) and three measured concentrations of MT (5, 51 and 583 ng/L) for 50 days. The results showed that MT had no significant histological effects on the ovaries of females, but the frequency of late-mature oocytes (LMO) showed a downward trend, indicating that MT could induce ovarian suppression to a certain extent. The transcriptional expression of activating transcription factor 4b1 (atf4b1), activating transcription factor 4b2 (atf4b2), calcium/calmodulin-dependent protein kinase II delta 1 (camk2d1), calcium/calmodulin-dependent protein kinase II delta 2 (camk2d2) and calcium/calmodulin-dependent protein kinase II inhibitor 2 (camk2n2) genes in the brain of females increased significantly at all treatment groups of MT, and the mRNA expression of forkhead box L2a (foxl2) and ovarian cytochrome P450 aromatase (cyp19a1a) genes in the ovaries were down-regulated by 5 and 583 ng/L group, which would translate into inhibition of oocyte development. As compared to females, MT had relatively little effects on the reproductive system of males, and only the transcriptional alterations of synaptonemal complex protein 3 (sycp3) and 17-alpha-hydroxylase/17,20-lyase (cyp17) genes were observed in the testes, not enough to affect testicular histology. In addition, MT at all treatments strongly increased corticotropin-releasing hormone (crh) transcript in the brain of females, as well as deiodinase 2 (dio2) transcript in the brain of males. The paired box protein 8 (pax8) gene was significantly decreased at 51 or 583 ng/L of MT in both female and male brains. The above results suggest that MT can pose potential adverse effects on the reproductive and thyroid endocrine system of fish.


Subject(s)
Water Pollutants, Chemical , Zebrafish , Animals , Male , Female , Zebrafish/metabolism , Methyltestosterone/metabolism , Methyltestosterone/pharmacology , Hypothalamic-Pituitary-Gonadal Axis , Thyroid Gland/metabolism , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/pharmacology , Gonads , Gene Expression , Germ Cells , RNA, Messenger/metabolism , Activating Transcription Factors/genetics , Activating Transcription Factors/metabolism , Activating Transcription Factors/pharmacology , Water Pollutants, Chemical/metabolism
20.
Acupunct Med ; 42(1): 23-31, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38126262

ABSTRACT

BACKGROUND: This study aimed to investigate the effects of electroacupuncture (EA) on cognitive recovery and synaptic remodeling in a rat model of middle cerebral artery occlusion (MCAO) followed by reperfusion and explore the possible mechanism. METHOD: Focal cerebral ischemia was modeled in healthy adult Sprague-Dawley rats by MCAO. The MCAO rats were classified into four groups: sham, MCAO, MCAO + GB20 (receiving EA at GB20) and MCAO + NA (receiving EA at a "non-acupoint" location not corresponding to any traditional acupuncture point location about 10 mm above the iliac crest). Neurological deficit scores and behavior were assessed before and during the treatment. After intervention for 7 days, the hippocampus was dissected to analyze growth-associated protein (GAP)-43, synaptophysin (SYN) and postsynaptic density protein (PSD)-95 expression levels by Western blotting. Bioinformatic analysis and primary hippocampal neurons with calcium-voltage gated channel subunit alpha 1B (CACNA1B) gene overexpression were used to screen the target genes for EA against MCAO. RESULTS: Significant amelioration of neurological deficits and learning/memory were found in MCAO + GB20 rats compared with MCAO or MCAO + NA rats. Protein levels of GAP-43, SYN and PSD-95 were significantly improved in MCAO + GB20-treated rats together with an increase in the number of synapses in the hippocampal CA1 region. CACNA1B appeared to be a target gene of EA in MCAO. There were increased mRNA levels of CACNA1B, calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and cyclic adenosine monophosphate response element binding (CREB) and increased phosphorylation of CaM, CaMKII and CREB in the hippocampal region in MCAO + GB20 versus MCAO and MCAO + NA groups. CACNA1B overexpression modulated expression of the CaM-CaMKII-CREB axis. CONCLUSION: EA treatment at GB20 may ameliorate the negative effects of MCAO on cognitive function in rats by enhancing synaptic plasticity. EA treatment at GB20 may exert this neuroprotective effect by regulating the CACNA1B-CaM-CaMKII-CREB axis.


Subject(s)
Brain Ischemia , Electroacupuncture , Reperfusion Injury , Rats , Animals , Rats, Sprague-Dawley , Calmodulin/metabolism , Calmodulin/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/pharmacology , Cognition , Signal Transduction , Brain Ischemia/genetics , Brain Ischemia/therapy , Brain Ischemia/metabolism , Infarction, Middle Cerebral Artery/metabolism , Reperfusion Injury/genetics , Reperfusion Injury/therapy , Neuronal Plasticity
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