Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive neuronal cell loss. Recently, dysregulation of intracellular Ca2+ homeostasis has been suggested as a common proximal cause of neural dysfunction in AD. Here, we investigated (1) the pathogenic role of destabilization of ryanodine receptor (RyR2) in endoplasmic reticulum (ER) upon development of AD phenotypes in AppNL-G-F mice, which harbor three familial AD mutations (Swedish, Beyreuther/Iberian, and Arctic), and (2) the therapeutic effect of enhanced calmodulin (CaM) binding to RyR2. In the neuronal cells from AppNL-G-F mice, CaM dissociation from RyR2 was associated with AD-related phenotypes, i.e. Aβ accumulation, TAU phosphorylation, ER stress, neuronal cell loss, and cognitive dysfunction. Surprisingly, either genetic (by V3599K substitution in RyR2) or pharmacological (by dantrolene) enhancement of CaM binding to RyR2 reversed almost completely the aforementioned AD-related phenotypes, except for Aβ accumulation. Thus, destabilization of RyR2 due to CaM dissociation is most likely an early and fundamental pathogenic mechanism involved in the development of AD. The discovery that neuronal cell loss can be fully prevented simply by stabilizing RyR2 sheds new light on the treatment of AD.
Introduction
Our previous studies demonstrated that dantrolene, a ryanodine receptor stabilizer, prevents endoplasmic reticulum (ER) stress in the heart. ER stress is a strong mediator of impaired lipid metabolism in the liver, thereby contributing to fatty liver disease. In this study, we investigated the effects of dantrolene on fatty liver disease in mice and ER stress in hepatocytes.
Methods and results
Eight weeks old C57BL/6 mice were fed high-fat diet (HFD) for 8 weeks with or without the oral administration of dantrolene (100 mg/kg/day). The livers of mice without dantrolene (HFD group) showed severe fatty liver, whereas the livers of the mice treated with dantrolene (HFD + DAN group) only showed slightly fatty liver. To address the preventive effects of dantrolene, primary hepatocytes were cultured with palmitate in the presence or absence of dantrolene. Dantrolene reduced lipid load and prevents palmitate-induced increase in cytoplasmic Ca
2+
and ER stress. Based on these findings, we propose that dantrolene is a potential new therapeutic agent against fatty liver disease.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in the cardiac type 2 ryanodine receptor (RyR2). Using knock-in mouse (KI) model (R2474S/+), we previously reported that a single point mutation within the RyR2 sensitizes the channel to agonists, primarily mediated by defective inter-domain interaction within the RyR2 molecule and subsequent dissociation of calmodulin (CaM) from the RyR2. Here, we examined whether CPVT can be genetically rescued by enhancing the binding affinity of CaM to the RyR2. We first determined whether there is a possible amino-acid substitution at the CaM-binding domain in the RyR2 (3584-3603) that can enhancethe binding affinity of CaM, and found that V3599K substitution showed the highest binding affinity of CaM to CaM-binding domain. Hence, we generated a heterozygous KI mouse model (V3599K/+) with a single amino acid substitution in the CaM-binding domain of the RyR2, and crossbred it with the heterozygous CPVT -associated R2474S/+ KI mouse to obtain a double heterozygous R2474S/V3599K KI mouse. The CPVT phenotypes—bidirectional or polymorphic ventricular tachycardia, spontaneous Ca2+ transients, and Ca2+ sparks—were all inhibited in the R2474S/V3599K mice. Thus, enhancement of the CaM binding affinity of the RyR2 is essential to prevent CPVT-associated arrhythmogenesis.
significantly associated with increased composite events (adjusted hazard ratio (HR), 1.38; 95% confidence interval (CI), 1.15-1.67, P50.001). Moreover, higher furosemide dose (O 40mg) was associated with incidence of composite events, especially in patients with reduced ejection fraction (!50%) (HR, 1.93; 95% CI, 1.37-2.71, P! 0.001) and in patients with lower glomerular filtration rate (!60%) (HR, 1.68; 95% CI, 1.262.24, P!0.001). Conclusions: Use of higher dose of furosemide is associated with worsened prognosis in CHF patients.
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