Currently, the incidence of Parkinson's disease (PD) is on the rise. More and more evidences suggest that mitochondrial dysfunction plays a crucial role in the etiology of PD, and dysfunction of mitochondrial complex I (MCI) is one of the most critical factors leading to mitochondrial dysfunction. On one hand, MCI dysfunction stimulates dopaminergic neurons to produce reactive oxygen species (ROS). On the other hand, MCI dysfunction decreases dopaminergic neuron viability and reduces ATP production. All these outcomes promote the pathological progression of PD. This review summarizes research progress on the role of MCI in the pathogenesis of PD, as well as PD treatment strategies based on MCI.
Parkinson Disease/metabolism* ; Humans ; Electron Transport Complex I/metabolism* ; Mitochondria/physiology* ; Reactive Oxygen Species/metabolism* ; Dopaminergic Neurons/metabolism* ; Animals ; Adenosine Triphosphate/metabolism*

BACKGROUND: Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) is a key protein that maintains myocardial Ca 2+ homeostasis. The present study aimed to investigate the mechanism underlying the SERCA2a-SUMOylation (small ubiquitin-like modifier) process after ischemia/reperfusion injury (I/RI) in vitro and in vivo . METHODS: Calcium transient and systolic/diastolic function of cardiomyocytes isolated from Serca2a knockout (KO) and wild-type mice with I/RI were compared. SUMO-relevant protein expression and localization were detected by quantitative real-time PCR (RT-qPCR), Western blotting, and immunofluorescence in vitro and in vivo . Serca2a-SUMOylation, infarct size, and cardiac function of Senp1 or Senp2 overexpressed/suppressed adenovirus infected cardiomyocytes, were detected by immunoprecipitation, triphenyltetrazolium chloride (TTC)-Evans blue staining, and echocardiography respectively. RESULTS: The results showed that the changes of Fura-2 fluorescence intensity and contraction amplitude of cardiomyocytes decreased in the I/RI groups and were further reduced in the Serca2a KO + I/RI groups. Senp1 and Senp2 messenger ribose nucleic acid (mRNA) and protein expression levels in vivo and in cardiomyocytes were highest at 6 h and declined at 12 h after I/RI. However, the highest levels in HL-1 cells were recorded at 12 h. Senp2 expression increased in the cytoplasm, unlike that of Senp1. Inhibition of Senp2 protein reversed the I/RI-induced Serca2a-SUMOylation decline, reduced the infarction area, and improved cardiac function, while inhibition of Senp1 protein could not restore the above indicators. CONCLUSION I/RI activated Senp1 and Senp2 protein expression, which promoted Serca2a-deSUMOylation, while inhibition of Senp2 expression reversed Serca2a-SUMOylation and improved cardiac function.
Animals ; Mice ; Calcium/metabolism* ; Cysteine Endopeptidases/metabolism* ; Myocardial Reperfusion Injury/metabolism* ; Myocardium/metabolism* ; Myocytes, Cardiac/metabolism* ; Proteins/metabolism* ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics*

OBJECTIVES: Application of ultrashort wave (USW) to rats with cerebral ischemia and reperfusion injury could inhibit the decrease of expression of secretory pathway Ca²⁺-ATPase 1 (SPCA1), an important participant in Golgi stress, reduce the damage of Golgi apparatus and the apoptosis of neuronal cells, thereby alleviating cerebral ischemia-reperfusion injury. This study aims to investigate the effect of USW on oxygen-glucose deprivation/reperfusion (OGD/R) injury and the expression of SPCA1 at the cellular level. METHODS: N2a cells were randomly divided into a control (Con) group, an OGD/R group, and an USW group. The cells in the Con group were cultured without exposure to OGD. The cells in the OGD/R group were treated with OGD/R. The cells in the USW group were treated with USW after OGD/R. Cell morphology was observed under the inverted phase-contrast optical microscope, cell activity was detected by cell counting kit-8 (CCK-8), apoptosis was detected by flow cytometry, and SPCA1 expression was detected by Western blotting. RESULTS: Most of the cells in the Con group showed spindle shape with a clear outline and good adhesion. In the OGD/R group, cells were wrinkled, with blurred outline, poor adhesion, and lots of suspended dead cells appeared; compared with the OGD/R group, the cell morphology and adherence were improved, with clearer outlines and fewer dead cells in the USW group. Compared with the Con group, the OGD/R group showed decreased cell activity, increased apoptotic rate, and down-regulating SPCA1 expression with significant differences (all <i>Pi><0.001); compared with the OGD/R group, the USW group showed increased cell activity, decreased apoptotic rate, and up-regulating SPCA1 expression with significant differences (<i>Pi><0.01 or <i>Pi><0.001). CONCLUSIONS USW alleviates the injury of cellular OGD/R, and its protective effect may be related to its up-regulation of SPCA1 expression.
Animals ; Rats ; Apoptosis ; Brain Ischemia ; Glucose/metabolism* ; Oxygen/metabolism* ; Reperfusion Injury/metabolism* ; Transcriptional Activation ; Up-Regulation ; Calcium-Transporting ATPases/metabolism*

Objective: To investigate the expression of cation chloride cotransporter (NKCC1/KCC2) in the neurons from cerebral lesions of children with focal cortical dysplasia (FCD) type Ⅱ, to provide a morphological basis for revealing the possible mechanism of epilepsy. Methods: Eight cases of FCD type Ⅱ diagnosed at Beijing Haidian Hospital, Beijing, China and 12 cases diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from February 2017 to December 2019 were included. The expression of NKCC1 and KCC2 in FCD type Ⅱa and FCD type Ⅱb was detected using immunohistochemistry and double immunohistochemical stains. The average optical density of NKCC1 in dysmorphic neurons and normal neurons was also determined using immunohistochemical staining in FCD type Ⅱa (10 cases). Results: The patients were all younger than 14 years of age. Ten cases were classified as FCD type IIa, and 10 cases as FCD type Ⅱb. NKCC1 was expressed in the cytoplasm of normal cerebral cortex neurons and KCC2 expressed on cell membranes. In dysmorphic neurons of FCD type Ⅱa, expression of NKCC1 increased, which was statistically higher than that of normal neurons (<i>P<i>0.01). Aberrant expression of KCC2 in dysmorphic neurons was also noted in the cytoplasm. In the FCD Ⅱb type, the expression pattern of NKCC1/KCC2 in dysmorphic neurons was the same as that of FCD type Ⅱa. The aberrant expression of NKCC1 in balloon cells was negative or weakly positive on the cell membrane, while the aberrant expression of KCC2 was absent. Conclusions: The expression pattern of NKCC1/KCC2 in dysmorphic neurons and balloon cells is completely different from that of normal neurons. The NKCC1/KCC2 protein-expression changes may affect the transmembrane chloride flow of neurons, modify the effect of inhibitory neurotransmitters γ-aminobutyric acid and increase neuronal excitability. These effects may be related to the occurrence of clinical epileptic symptoms.
Child ; Humans ; Brain/pathology* ; Cations/metabolism* ; Chlorides/metabolism* ; Epilepsy/metabolism* ; Malformations of Cortical Development, Group I/metabolism* ; Solute Carrier Family 12, Member 2/metabolism* ; Symporters/metabolism*

Cardiovascular Diseases ; Heart Failure ; Humans ; Protein Processing, Post-Translational ; Sarcoplasmic Reticulum Calcium-Transporting ATPases

BACKGROUND: Chronic hyperglycemia has deleterious effects on pancreatic β-cell function and turnover. Recent studies support the view that cyclin-dependent kinase 5 (CDK5) plays a role in β-cell failure under hyperglycemic conditions. However, little is known about how CDK5 impair β-cell function. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. In this study, we examined the effect of myricetin on high glucose (HG)-induced β-cell apoptosis and explored the relationship between myricetin and CDK5. METHODS: To address this question, we subjected INS-1 cells and isolated rat islets to HG conditions (30 mM) in the presence or absence of myricetin. Docking studies were conducted to validate the interaction between myricetin and CDK5. Gene expression and protein levels of endoplasmic reticulum (ER) stress markers were measured by real-time reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: Activation of CDK5 in response to HG coupled with the induction of ER stress via the down regulation of sarcoendoplasmic reticulum calcium ATPase 2b (SERCA2b) gene expression and reduced the nuclear accumulation of pancreatic duodenal homeobox 1 (PDX1) leads to β-cell apoptosis. Docking study predicts that myricetin inhibit CDK5 activation by direct binding in the ATP-binding pocket. Myricetin counteracted the decrease in the levels of PDX1 and SERCA2b by HG. Moreover, myricetin attenuated HG-induced apoptosis in INS-1 cells and rat islets and reduce the mitochondrial dysfunction by decreasing reactive oxygen species production and mitochondrial membrane potential (Δψm) loss. CONCLUSION: Myricetin protects the β-cells against HG-induced apoptosis by inhibiting ER stress, possibly through inactivation of CDK5 and consequent upregulation of PDX1 and SERCA2b.
Animals ; Apoptosis ; Blotting, Western ; Calcium-Transporting ATPases ; Cyclin-Dependent Kinase 5 ; Diabetes Mellitus, Type 2 ; Down-Regulation ; Endoplasmic Reticulum Stress ; Endoplasmic Reticulum ; Gene Expression ; Genes, Homeobox ; Glucose ; Hyperglycemia ; Insulin-Secreting Cells ; Membrane Potential, Mitochondrial ; Polymerase Chain Reaction ; Rats ; Reactive Oxygen Species ; Reticulum ; Reverse Transcription ; Up-Regulation

BACKGROUND/AIMS: The Gastroesophageal Reflux Disease Questionnaire (GerdQ) has been developed and validated as a tool for the diagnosis of gastroesophageal reflux disease (GERD) in patients with gastrointestinal symptoms. However, the GerdQ and the cutoff value for determining GERD has not been validated in Korea. METHODS: Patients with symptoms suggestive of GERD were consecutively recruited. The Korean version of GerdQ was developed through a forward-backward translation process according to the cross-cultural adaptation method. Endoscopically documented esophagitis, abnormal results on 24-hour ambulatory pH recording with symptom association monitoring, or response to proton pump inhibitor treatment were used as diagnostic references for GERD. The reproducibility and test characteristics of the Korean version of GerdQ were assessed. RESULTS: A total of 149 patients with a median age of 55 years were analyzed. The intra-class correlation coefficient of 2 subsequently measured GerdQ scores was 0.651 (95% CI, 0.518–0.748). The cutoff value of 8 was found to have the highest sensitivity (64.9%; 95% CI, 56.2–73.7) and specificity (71.4%; 95% CI, 56.5–86.4) for the diagnosis of GERD. The questionnaire had a high positive predictive value (88.1%; 95% CI, 81.2–95.0), but a low negative predictive value (38.5%; 95% CI, 26.2–50.3) for GERD. Any symptom improvement on proton pump inhibitor treatment showed a sensitivity of 93.0% (95% CI, 88.3–97.7) and a specificity of 48.6% (95% CI, 32.0–65.1) for GERD. CONCLUSION: The Korean version of GerdQ is a useful complementary tool in the diagnosis of GERD.
Diagnosis ; Esophagitis ; Gastroesophageal Reflux ; Humans ; Hydrogen-Ion Concentration ; Korea ; Methods ; Proton Pumps ; Sensitivity and Specificity ; Surveys and Questionnaires ; Symptom Assessment

Gastroesophageal reflux disease (GERD) is diagnosed according to the medical history or in response to proton pump inhibitor therapy. However, the need for further testing is always appropriate. The decisive evidence for the current diagnosis of GERD is severe erosive esophagitis of Los Angeles grade C or D, long-segment Barrett's mucosa or peptic strictures seen on endoscopy or an acid exposure time >6% on ambulatory pH or pH impedance monitoring. If ambulatory reflux monitoring correlates between reflux and the symptoms, then the diagnosis and treatment are certain. If it is difficult to clearly diagnose this malady as seen upon endoscopy and ph/pH impedance monitoring, then this review recommends the biopsy findings, motor evaluation and novel impedance metrics. Novel impedance metrics include the baseline impedance and the post reflux swallow-induced peristaltic wave index. Therefore, making a future GERD diagnosis should focus on defining the patient's phenotype. The phenotype is determined by the level of reflux exposure, clearance efficacy, anatomy of the esophageal gastric junction, and the psychological state of the patient. The purpose of this review is to clarify the diagnostic guideline for GERD according to several test methods.
Biopsy ; Consensus ; Constriction, Pathologic ; Diagnosis ; Electric Impedance ; Endoscopy ; Esophageal pH Monitoring ; Esophagitis ; Gastroesophageal Reflux ; Humans ; Hydrogen-Ion Concentration ; Manometry ; Mucous Membrane ; Phenotype ; Proton Pumps

OBJECTIVE: To establish a congenital chloride diarrhea (CCD)-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model, and to investigate its biological function. METHODS: The sequence of the SLC26A3 gene in GenBank was used to design the upstream and downstream single-guide RNA (sgRNA) that could specifically recognize the 392 locus of the SLC26A3 gene, and the sgRNA was mixed with the pSpCas9-puro vector after enzyme digestion to construct an eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3). Caco-2 cells were transfected with the recombinant plasmid and synthesized single-stranded DNA oligonucleotides (ssODNs), and Taqman genotyping assay and Sanger sequencing were used to identify the expression of SLC26A3 c.392C>G (p.P131R) in Caco-2 cells. Wild-type Caco-2 cells were selected as normal control group and the Caco-2 cells with successful expression of SLC26A3 c.392C>G (p.P131R) was selected as P131R group. Both groups were treated with 100 ng/mL tumor necrosis factor-α (TNF-α), and then the normal control group was named as TNF-α group, and the P131R group was named as TNF-α+P131R group. Electric cell-substrate impedance sensing (ECIS) assay was used to evaluate the change in the monolayer barrier function of intestinal epithelial cells in the above four groups, and Western blot was used to measure the change in the expression of SLC26A3 protein in the normal control group and the P131R group. RESULTS: The eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3) was successfully constructed. Both Taqman genotyping assay and Sanger sequencing confirmed the successful establishment of the Caco-2 cell model of SLC26A3 c.392C>G (p.P131R) expression. ECIS assay showed that compared with the normal control group, the P131R group had a significant increase in the monolayer permeability of intestinal epithelial cells (P<0.05), and at the same time, the P131R group had a significantly greater increase in cell membrane permeability after the induction with 100 ng/mL TNF-α (P<0.05). Western blot showed that compared with the normal control group, the P131R group had a significant reduction in the expression of SLC26A3 protein (P=0.001). CONCLUSIONS SLC26A3 c.392C>G (p.P131R) can reduce the expression of SLC26A3 protein, increase the monolayer permeability of intestinal epithelial cells, and thus lead to diarrhea.
Caco-2 Cells ; Chloride-Bicarbonate Antiporters ; genetics ; Diarrhea ; congenital ; genetics ; Humans ; Intestinal Mucosa ; Metabolism, Inborn Errors ; genetics ; Polymorphism, Single Nucleotide ; Sulfate Transporters ; genetics ; Tight Junctions ; Tumor Necrosis Factor-alpha

OBJECTIVE: To explore the molecular basis for a pedigree affected with Darier-White disease. METHODS: Genomic DNA was isolated from 3 patients and 1 unaffected member from the pedigree, as well as 80 healthy controls. Targeted sequence capture and next-generation sequencing were used to screen mutations of skin disease-related genes. Candidate mutations were verified by Sanger sequencing, and co-segregation analysis was carried out to confirm the pathogenicity of mutation. Conservation analysis and protein structure and function were also predicted with Bioinformatic tools. RESULTS: A heterozygous mutation c.2246G>T (p.G749V) was identified in exon 15 of ATP2A2 gene in all 3 patients from the pedigree, but not in the unaffected member or 80 healthy controls. The corresponding amino acid was highly conserved, and mutation of which can lead to structural and functional changes of the protein. CONCLUSION The c.2246G>T missense mutation of the ATP2A2 gene probably underlies the Darier-White disease in this pedigree by causing damages to the structure and function of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2).
Darier Disease ; genetics ; Heterozygote ; Humans ; Mutation, Missense ; Pedigree ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics