Calcium-mediated regulation of autophagy and programmed cell death in adult hippocampal neural stem cells: A story of calpains, ryanodine receptors, and presenilins

Importance of proper cell death regulation has received great recognitions in biomedical field as the greater understanding of cell death can provide better solutions to treatment of various human diseases, most notably neurodegenerative diseases. However, the underlying mechanisms of programmed cell death (PCD) are largely unknown, especially in neural stem cells (NSCs). Utilizing our well-established insulin withdrawal model of autophagic cell death (ACD) in adult hippocampal neural stem (HCN) cells, we explored the functional relevance of autophagic death of NSCs to pathogenesis of Alzheimer’s disease (AD). Aberrant neuronal Ca2+ levels in brains of AD patients have given a rise to Ca2+ hypothesis of AD which states that the amyloidogenic pathway leads to ultimate cognitive impairment in affected individuals through dysregulation of neuronal Ca2+ signaling. The aberrant Ca2+ homeostasis consequently mediates the abnormal activation of calpains, the Ca2+-dependent cysteine proteases which also play an essential role in diverse cellular events including cell development, differentiation and proliferation, and cell death. ⓒ 2016 DGISTCHAPTER 1: General Introduction 15-- 1.1 Adult Neurogenesis and Neural Stem Cells 15-- 1.1.1 Adult Neurogenesis: The Discovery 15-- 1.1.2 Adult Neural Stem Cells 17-- 1.2 Programmed Cell Death 19-- 1.2.1 PCD: Classifications and Features 19-- 1.2.2 PCD in the Brain 22-- 1.3 Autophagy 24-- 1.3.1 Autophagic Cell Death: Insulin Withdrawal Model in Hippocapal Neural Stem Cells 26-- 1.3.2 Autophagy in Neurodegeneration 29-- -- CHAPTER 2: General Materials and Common Techniques 32-- 2.1 Cell Culture 32-- 2.2 Western Blot Analysis 32-- 2.3 Immunohistochemistry 33-- 2.4 Immunocytochemistry 33-- 2.5 Real-Time Quantitative PCR 34-- 2.6 Cell Death Assay 34-- 2.7 Transfection for Delivery of DNAs or siRNAs 34-- 2.8 Flow Cytometry 34-- 2.9 Statistical Analysis 35-- -- CHAPTER 3: Calpain Determines the Propensity of Adult HCN Cells to ACD Following Insulin Withdrawal 36-- 3.1 Introduction 36-- 3.2 Materials and Methods 38-- 3.2.1 Antibodies and Reagents 38-- 3.2.2 Plasmids and siRNAs 38-- 3.2.3 Immunocytochemistry 38-- 3.2.4 Intracellular Calcium Imaging 38-- 3.2.5 Calpain Activity Assay 39-- 3.3 Results 40-- 3.3.1 Calpain 1 and 2 are Differentially Expressed in HCN Cells 40-- 3.3.2 Calpain 2 Inhibition Potentiates Autophagic Death of I(-) HCN Cells 40-- 3.3.3 Ectopic Expression of Calpain 1 Induces Apoptosis in HCN Cells Following Insulin Withdrawal 42-- 3.3.4 Degradation of Calpain 2 Is Achieved via UPS, Not Autophagy 45-- 3.3.5 Proteasome Inhibition Elevates the Concentration of Intracellular Calcium and Activates Calpain in I(-) HCN Cells 48-- 3.3.6 Lactacystin Switches the Default Autophagic Death of I(-) HCN Cells to Apoptosis 51-- 3.4 Discussion 56-- -- CHAPTER 4: Mediation of ACD by RyR3 in Adult HCN Cells 60-- 4.1 Introduction 60-- 4.2 Materials and Methods 65-- 4.2.1 Pharmacological Reagents 65-- 4.2.2 Immunofluorescnce-based Ca2+ Imaging 65-- 4.2.3 Autophagic Flux Assay 65-- 4.2.4 Generation of CRISPR/Cas9-mediated RYR3 Knockout HCN Cells 66-- 4.3 Results 67-- 4.3.1 ER-to-Cytosol Ca2+ efflux is Increased Following Insulin Withdrawal in HCN Cells 67-- 4.3.2 RyR3 is the Major RyR Isoform Expressed in HCN Cells and its Expression is Elevated Following Insulin Withdrawal 67-- 4.3.3 A RyR Agonist Caffeine Further Promotes ACD in I(-) HCN Cells 69-- 4.3.4 ACD Induction by Caffeine is Precluded in Autophagy-Defective HCN Cells Depleted of Atg7 69-- 4.3.5 Autophagy is Diminished by Pharmacological or Genetic RyR Inhibition in I(-) HCN Cells 73-- 4.3.6 Knockout of RyR3 Gene Occludes ER Ca2+ Release and Thereby Prevents ACD in I(-) HCN Cells 76-- 4.4 Discussion 81-- -- CHAPTER 5: A Novel Function of Presenilin-2 in Regulation of Autophagic Death of HCN Cells 86-- 5.1 Introduction 86-- 5.2 Materials and Methods 89-- 5.2.1 Antibodies and Reagents 89-- 5.2.2 Plasmids and siRNAs 89-- 5.2.3 Organotypic Hippocampal Slice Culture 89-- 5.2.4 Generation of CRISPR/Cas9-mediated Presenilin Knockout HCN Cell Lines 90-- 5.3 Results 91-- 5.3.1 Presenilin-2 Exhibits Distinct Expression Pattern in HCN Cells 91-- 5.3.2 Expression of Presenilin-2 in HCN Cells is Significantly Upregulated Upon Insulin Withdrawal 91-- 5.3.3 Genetic Depletion of PS2 Prevents Induction of ACD by Insulin Withdrawal in HCN Cells 91-- 5.3.4 PS2 Expression Potentiates ACD in I(-) HCN Cells, but Not in Atg7-Deficient I(-) HCN Cells 94-- -- CHAPTER 6: General Discussion 97DoctordCollectio

A Passivity-based Nonlinear Admittance Control with Application to Powered Upper-limb Control under Unknown Environmental Interactions

This paper presents an admittance controller based on the passivity theory for a powered upper-limb exoskeleton robot which is governed by the nonlinear equation of motion. Passivity allows us to include a human operator and environmental interaction in the control loop. The robot interacts with the human operator via F/T sensor and interacts with the environment mainly via end-effectors. Although the environmental interaction cannot be detected by any sensors (hence unknown), passivity allows us to have natural interaction. An analysis shows that the behavior of the actual system mimics that of a nominal model as the control gain goes to infinity, which implies that the proposed approach is an admittance controller. However, because the control gain cannot grow infinitely in practice, the performance limitation according to the achievable control gain is also analyzed. The result of this analysis indicates that the performance in the sense of infinite norm increases linearly with the control gain. In the experiments, the proposed properties were verified using 1 degree-of-freedom testbench, and an actual powered upper-limb exoskeleton was used to lift and maneuver the unknown payload.Comment: Accepted in IEEE/ASME Transactions on Mechatronics (T-MECH

Short term outcomes of topiramate monotherapy as a first-line treatment in newly diagnosed West syndrome

PurposeTo investigate the efficacy of topiramate monotherapy in West syndrome prospectively.MethodsThe study population included 28 patients (15 male and 13 female children aged 2 to 18 months) diagnosed with West syndrome. After a 2-week baseline period for documentation of the frequency of spasms, topiramate was initiated at 2 mg/kg/day. The dose was increased by 2 mg/kg every week to a maximum of 12 mg/kg/day. Clinical assessment was based on the parents' report and a neurological examination every 2 weeks for the first 2 months of treatment. The baseline electroencephalograms (EEGs) were compared with the post-treatment EEGs at 2 weeks and 1 month.ResultsWest syndrome was considered to be cryptogenic in 7 of the 28 patients and symptomatic in 21 patients. After treatment, 11 patients (39%) became spasm-free, 6 (21%) had more than 50% spasmsreduction, 3 (11%) showed less than 50% reduction, and 8 (29%) did not respond. The effective daily dose for achieving more than 50% reduction in spasm frequency, including becoming spasm-free, was found to be 5.8±1.1 mg/kg/day. Nine patients (32%) showed complete disappearance of spasms and hypsarrhythmia, and 11 (39%) showed improved EEG results. Despite adverse events (4 instances of irritability, 3 of drowsiness, and 1 of decreased feeding), no patients discontinued the medication.ConclusionTopiramate monotherapy seems to be effective and well tolerated as a first line therapy for West syndrome and is not associated with serious adverse effects

Peripheral arterial endothelial dysfunction predicts future cardiovascular events in diabetic patients with albuminuria: a prospective cohort study

Background Reactive hyperemia-peripheral arterial tonometry (RH-PAT) is a noninvasive and simple test for evaluating the endothelial function. There has been sparse evidence on the usefulness of the RH-PAT index (RHI) in predicting future cardiovascular diseases among diabetic patients. Methods Asymptomatic diabetic patients with albuminuria were selected; their medical history and laboratory findings were evaluated every 3 to 4 months, respectively. The primary outcome was a composite of three-point major adverse cardiovascular events (3-point MACE): death from cardiovascular causes, acute coronary events, or nonfatal stroke. On the contrary, secondary outcomes included a composite of 3-point MACE, hospitalization for heart failure, or chronic kidney disease (CKD) progression. RHI was measured using the Endo-PAT2000 at the baseline. RHI < 1.67 was considered to indicate peripheral endothelial dysfunction (PED). Results In total, 149 subjects were included (mean age, 61.8 ± 9.2 years; duration of diabetes was 12 years). During the follow-up period (median, 49.7 months), of the 149 subjects, primary outcomes were detected in 12 (1 [2.3%] and 11 [10.5%] of those without and with PED, respectively). The presence of PED in baseline measurements significantly increased both primary and secondary outcomes, following adjustment for age, sex, hypertension, glycated hemoglobin, low-density lipoprotein cholesterol, triglyceride, systolic blood pressure, baseline estimated glomerular filtration rate, overt proteinuria, duration of diabetes, premedical history of ischemic events, anti-platelet agents, and smoking history (hazard ratio [HR]: 10.95; 95% confidence interval CI 1.00–119.91 for the primary outcome; HR, 4.12; 95% CI 1.37–12.41 for secondary outcome). In addition, PED could predict secondary outcomes independent of the risk score according to the American College of Cardiology/American Heart Association (HR: 3.24; 95% CI 1.14–9.17). Conclusions PED can independently predict future cardiovascular events among diabetic patients with albuminuria.This study was supported by Health Connect Research Fund (No. 16-2013-87)