IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca²⁺ homeostasis. We have previously shown that the IP₃ receptor (IP₃R) plays a role in elevating basal cytoplasmic Ca²⁺ and that pharmacological blockade of IP₃R restores muscle function. Moreover, we have shown that the IP₃R pathway negatively regulates autophagy by controlling mitochondrial Ca²⁺ levels. Nevertheless, it remains unclear whether IP₃R is involved in abnormal mitochondrial Ca²⁺ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP₃R was downregulated in mdx fibers. Pharmacological blockade of IP₃R in mdx fibers restored both increased mitochondrial Ca²⁺ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP₃R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP₃R1 activity with changes in autophagy, mitochondrial Ca²⁺ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP₃R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP₃R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.     

Intracellular cholesterol accumulation and coenzyme Q10 deficiency in Familial Hypercholesterolemia

Familial Hypercholesterolemia (FH) is an autosomal co-dominant genetic disorder characterized by elevated low-density lipoprotein (LDL) cholesterol levels and increased risk for premature cardiovascular disease. Here, we examined FH pathophysiology in skin fibroblasts derived from FH patients harboring heterozygous mutations in the LDL-receptor.Fibroblasts from FH patients showed a reduced LDL-uptake associated with increased intracellular cholesterol levels and coenzyme Q₁₀ (CoQ₁₀) deficiency, suggesting dysregulation of the mevalonate pathway.Secondary CoQ₁₀ deficiency was associated with mitochondrial depolarization and mitophagy activation in FH fibroblasts. Persistent mitophagy altered autophagy flux and induced inflammasome activation accompanied by increased production of cytokines by mutant cells. All the pathological alterations in FH fibroblasts were also reproduced in a human endothelial cell line by LDL-receptor gene silencing.Both increased intracellular cholesterol and mitochondrial dysfunction in FH fibroblasts were partially restored by CoQ₁₀ supplementation. Dysregulated mevalonate pathway in FH, including increased expression of cholesterogenic enzymes and decreased expression of CoQ₁₀ biosynthetic enzymes, was also corrected by CoQ₁₀ treatment.Reduced CoQ₁₀ content and mitochondrial dysfunction may play an important role in the pathophysiology of early atherosclerosis in FH. The diagnosis of CoQ₁₀ deficiency and mitochondrial impairment in FH patients may also be important to establish early treatment with CoQ₁₀.     

Mitochondrial redox system,dynamics, and dysfunction in lung inflammaging and COPD

Myriad forms of endogenous and environmental stress disrupt mitochondrial function by impacting critical processes in mitochondrial homeostasis, such as mitochondrial redox system, oxidative phosphorylation, biogenesis, and mitophagy. External stressors that interfere with the steady state activity of mitochondrial functions are generally associated with an increase in reactive oxygen species, inflammatory response, and induction of cellular senescence (inflammaging) potentially via mitochondrial damage associated molecular patterns (DAMPS). Many of these are the key events in the pathogenesis of chronic obstructive pulmonary disease (COPD) and its exacerbations. In this review, we highlight the primary mitochondrial quality control mechanisms that are influenced by oxidative stress/redox system, including role of mitochondria during inflammation and cellular senescence, and how mitochondrial dysfunction contributes to the pathogenesis of COPD and its exacerbations via pathogenic stimuli.     

Atg8-family interacting motif crucial for selective autophagy

Autophagy is a bulk degradation system conserved among most eukaryotes. Recently, autophagy has been shown to mediate selective degradation of various targets such as aggregated proteins and damaged or superfluous organelles. Structural studies have uncovered the conserved specific interactions between autophagic receptors and Atg8-family proteins through WXXL-like sequences, which we term the Atg8-family interacting motif (AIM). AIM functions in various autophagic receptors such as Atg19 in the cytoplasm-to-vacuole targeting pathway, p62 and neighbor of BRCA1 gene 1 (NBR1) in autophagic degradation of protein aggregates, and Atg32 and Nix in mitophagy, and may link the target-receptor complex to autophagic membranes and/or their forming machineries.     

Role of autophagy in liver physiology and pathophysiology

Autophagy is a highly conserved intracellular degradation pathway by which bulk cytoplasm and superfluous or damaged organelles are enveloped by double membrane structures termed autophagosomes. The autophagosomes then fuse with lysosomes for degradation of their contents, and the resulting amino acids can then recycle back to the cytosol. Autophagy is normally activated in response to nutrient deprivation and other stressors and occurs in all eukaryotes. In addition to maintaining energy and nutrient balance in the liver, it is now clear that autophagy plays a role in liver protein aggregates related diseases, hepatocyte cell death, steatohepatitis, hepatitis virus infection and hepatocellular carcinoma. In this review, I discuss the recent findings of autophagy with a focus on its role in liver pathophysiology.     

Genetic Insights into Sporadic Parkinson's Disease Pathogenesis

Intensive research over the last 15 years has led to the identification of several autosomal recessive and dominant genes that cause familial Parkinson’s disease (PD). Importantly, the functional characterization of these genes has shed considerable insights into the molecular mechanisms underlying the etiology and pathogenesis of PD. Collectively; these studies implicate aberrant protein and mitochondrial homeostasis as key contributors to the development of PD, with oxidative stress likely acting as an important nexus between the two pathogenic events. Interestingly, recent genome-wide association studies (GWAS) have revealed variations in at least two of the identified familial PD genes (i.e. α-synuclein and LRRK2) as significant risk factors for the development of sporadic PD. At the same time, the studies also uncovered variability in novel alleles that is associated with increased risk for the disease. Additionally, in-silico meta-analyses of GWAS data have allowed major steps into the investigation of the roles of gene-gene and gene-environment interactions in sporadic PD. The emergent picture from the progress made thus far is that the etiology of sporadic PD is multi-factorial and presumably involves a complex interplay between a multitude of gene networks and the environment. Nonetheless, the biochemical pathways underlying familial and sporadic forms of PD are likely to be shared.     

SIRT3抑制线粒体自噬并减轻高糖加重的神经元缺氧再灌注损伤*

目的:探讨SIRT3调控的线粒体自噬对高糖加重神经元缺氧再灌注损伤的影响及机制。方法:高糖(50 mmol/L)干预HT22细胞后,构建细胞缺氧/复氧模型,利用SIRT3抑制剂3-TYP抑制SIRT3表达。倒置显微镜观察细胞形态改变,CCK8法检测细胞存活率,流式细胞术检测细胞凋亡率,TMRE荧光试剂盒检测细胞线粒体膜电位,RT-qPCR、Western blot检测相关分子的基因和蛋白质表达。结果:高糖使神经元缺氧再灌注后的细胞碎片进一步增加,细胞存活率降低,细胞凋亡率升高(P<0.05)。此外,高糖降低了神经元缺氧再灌注后的线粒体膜电位(P<0.05)。进一步研究发现,高糖上调神经元缺氧再灌注后线粒体分裂相关蛋白DRP1的表达水平,降低了线粒体融合相关蛋白OPA1和线粒体外膜蛋白TOM20的表达;并且增加了自噬相关蛋白LC3Ⅱ、Beclin-1和线粒体自噬相关蛋白PINK1、Parkin的表达;同时,高糖升高了SIRT3的基因和蛋白质表达(P<0.05)。而SIRT3抑制剂3-TYP使神经元高糖缺氧再灌注损伤加重,同时进一步上调DRP1、LC3Ⅱ和PINK1的蛋白质表达(P<0.05)。结论:高糖可显著加重神经元缺氧再灌注损伤,破坏细胞线粒体功能,激活细胞线粒体自噬;SIRT3可抑制PINK1-Parkin通路介导的线粒体自噬并减轻神经元高糖缺氧再灌注损伤。     

Chronic Deletion and Acute Knockdown of Parkin Have Differential Responses to Acetaminophen-induced Mitophagy and Liver Injury in Mice

We previously demonstrated that pharmacological induction of autophagy protected against acetaminophen (APAP)-induced liver injury in mice by clearing damaged mitochondria. However, the mechanism for removal of mitochondria by autophagy is unknown. Parkin, an E3 ubiquitin ligase, has been shown to be required for mitophagy induction in cultured mammalian cells following mitochondrial depolarization, but its role in vivo is not clear. The purpose of this study was to investigate the role of Parkin-mediated mitophagy in protection against APAP-induced liver injury. We found that Parkin translocated to mitochondria in mouse livers after APAP treatment followed by mitochondrial protein ubiquitination and mitophagy induction. To our surprise, we found that mitophagy still occurred in Parkin knock-out (KO) mice after APAP treatment based on electron microscopy analysis and Western blot analysis for some mitochondrial proteins, and Parkin KO mice were protected against APAP-induced liver injury compared with wild type mice. Mechanistically, we found that Parkin KO mice had decreased activated c-Jun N-terminal kinase (JNK), increased induction of myeloid leukemia cell differentiation protein (Mcl-1) expression, and increased hepatocyte proliferation after APAP treatment in their livers compared with WT mice. In contrast to chronic deletion of Parkin, acute knockdown of Parkin in mouse livers using adenovirus shRNA reduced mitophagy and Mcl-1 expression but increased JNK activation after APAP administration, which exacerbated APAP-induced liver injury. Therefore, chronic deletion (KO) and acute knockdown of Parkin have differential responses to APAP-induced mitophagy and liver injury in mice.