TGR5 Activation Ameliorates Mitochondrial Homeostasis via Regulating the PKCδ/Drp1-HK2 Signaling in Diabetic Retinopathy

Background: Diabetic retinopathy (DR) is one of the most important microvascular diseases of diabetes. Our previous research demonstrated that bile acid G-protein-coupled membrane receptor (TGR5), a novel cell membrane receptor of bile acid, ameliorates the vascular endothelial cell dysfunction in DR. However, the precise mechanism leading to this alteration remains unknown. Thus, the mechanism of TGR5 in the progress of DR should be urgently explored.Methods: In this study, we established high glucose (HG)-induced human retinal vascular endothelial cells (RMECs) and streptozotocin-induced DR rat in vitro and in vivo. The expression of TGR5 was interfered through the specific agonist or siRNA to study the effect of TGR5 on the function of endothelial cell in vitro. Western blot, immunofluorescence and fluorescent probes were used to explore how TGR5 regulated mitochondrial homeostasis and related molecular mechanism. The adeno-associated virus serotype 8-shTGR5 (AAV8-shTGR5) was performed to evaluate retinal dysfunction in vivo and further confirm the role of TGR5 in DR by HE staining, TUNEL staining, PAS staining and Evans Blue dye.Results: We found that TGR5 activation alleviated HG-induced endothelial cell apoptosis by improving mitochondrial homeostasis. Additionally, TGR5 signaling reduced mitochondrial fission by suppressing the Ca2+-PKCδ/Drp1 signaling and enhanced mitophagy through the upregulation of the PINK1/Parkin signaling pathway. Furthermore, our result indicated that Drp1 inhibited mitophagy by facilitating the hexokinase (HK) 2 separation from the mitochondria and HK2-PINK1/Parkin signaling. In vivo, intraretinal microvascular abnormalities, including retinal vascular leakage, acellular capillaries and apoptosis, were poor in AAV8-shTGR5-treated group under DR, but this effect was reversed by pretreatment with the mitochondrial fission inhibitor Mdivi-1 or autophagy agonist Rapamycin.Conclusion: Overall, our findings indicated that TGR5 inhibited mitochondrial fission and enhanced mitophagy in RMECs by regulating the PKCδ/Drp1-HK2 signaling pathway. These results revealed the molecular mechanisms underlying the protective effects of TGR5 and suggested that activation of TGR5 might be a potential therapeutic strategy for DR.

Mitochondria as a target and central hub of energy division during cold stress in insects

Temperature stress is one of the crucial factors determining geographical distribution of insect species. Most of them are active in moderate temperatures, however some are capable of surviving in extremely high as well as low temperatures, including freezing. The tolerance of cold stress is a result of various adaptation strategies, among others the mitochondria are an important player. They supply cells with the most prominent energy carrier—ATP, needed for their life processes, but also take part in many other processes like growth, aging, protection against stress injuries or cell death. Under cold stress, the mitochondria activity changes in various manner, partially to minimize the damages caused by the cold stress, partially because of the decline in mitochondrial homeostasis by chill injuries. In the response to low temperature, modifications in mitochondrial gene expression, mtDNA amount or phosphorylation efficiency can be observed. So far study also showed an increase or decrease in mitochondria number, their shape and mitochondrial membrane permeability. Some of the changes are a trigger for apoptosis induced via mitochondrial pathway, that protects the whole organism against chill injuries occurring on the cellular level. In many cases, the observed modifications are not unequivocal and depend strongly on many factors including cold acclimation, duration and severity of cold stress or environmental conditions. In the presented article, we summarize the current knowledge about insect response to cold stress focusing on the role of mitochondria in that process considering differences in results obtained in different experimental conditions, as well as depending on insect species. These differentiated observations clearly indicate that it is still much to explore. Graphical Abstract

P. Edulis Extract Protects Against Amyloid-β Toxicity in Alzheimer’s Disease Models Through Maintenance of Mitochondrial Homeostasis via the FOXO3/DAF-16 Pathway

Alzheimer’s disease (AD) is a common and devastating disease characterized by pathological aggregations of beta-amyloid (Aβ) plaques extracellularly, and Tau tangles intracellularly. While our understandings of the aetiologies of AD have greatly expanded over the decades, there is no drug available to stop disease progression. Here, we demonstrate the potential of P. edulis pericarp extract in protecting against Aβ-mediated neurotoxicity in mammalian cells and Caenorhabditis elegans models of AD. We show P. edulis pericarp protects against memory deficit, neuronal loss, and promotes longevity in the Aβ model of AD via stimulation of mitophagy, a selective cellular clearance of damaged and dysfunctional mitochondria. P. edulis pericarp also restores memory and increases neuronal resilience in a C. elegans Tau model of AD. While defective mitophagy-induced accumulation of damaged mitochondria contributes to AD progression, P. edulis pericarp improves mitochondrial homeostasis through NIX/DCT1-dependent mitophagy and SOD3-dependent mitochondrial resilience, both via increased nuclear translocation of the upstream transcriptional regulator FOXO3/DAF-16. Further studies to identify active molecules in P. edulis pericarp that could maintain neuronal mitochondrial homeostasis may enable the development of potential drug candidates for AD.

Mitophagy in Yeast: Molecular Mechanism and Regulation

Mitophagy is a type of autophagy that selectively degrades mitochondria. Mitochondria, known as the “powerhouse of the cell”, supply the majority of the energy required by cells. During energy production, mitochondria produce reactive oxygen species (ROS) as byproducts. The ROS damages mitochondria, and the damaged mitochondria further produce mitochondrial ROS. The increased mitochondrial ROS damages cellular components, including mitochondria themselves, and leads to diverse pathologies. Accordingly, it is crucial to eliminate excessive or damaged mitochondria to maintain mitochondrial homeostasis, in which mitophagy is believed to play a major role. Recently, the molecular mechanism and physiological role of mitophagy have been vigorously studied in yeast and mammalian cells. In yeast, Atg32 and Atg43, mitochondrial outer membrane proteins, were identified as mitophagy receptors in budding yeast and fission yeast, respectively. Here we summarize the molecular mechanisms of mitophagy in yeast, as revealed by the analysis of Atg32 and Atg43, and review recent progress in our understanding of mitophagy induction and regulation in yeast.

PGC1α protects against cisplatin-induced skeletal muscle dysfunction

Background and Hypothesis: We and others have shown that chemotherapy promotes skeletal muscle wasting and weakness (i.e., cachexia) by disrupting mitochondrial homeostasis and causing oxidative stress. Peroxisome proliferative-activated receptor gamma coactivator 1-alpha (PGC1α) is a pivotal regulator of mitochondrial biogenesis and is involved in reducing oxidative damage in skeletal muscle. Hence, in the present study we investigated whether overexpression of skeletal muscle PGC1α (mPGC1α) was sufficient to preserve skeletal muscle mass and function in young and old mice treated with cisplatin. Experimental Design or Project Methods: Young (2-month; n = 5) and old (18-month; n = 5-8) male wild type (WT) or mPGC1α transgenic mice were treated with cisplatin (2.5mg/kg), while age-matched WT mice received vehicle for 2 weeks. Animals were assessed for muscle force and motor unit number estimation (MUNE). Skeletal muscles were weighed and processed for molecular analyses, including assessment of mitochondrial protein content. Results: Young WT mice exposed to cisplatin showed evidence of cachexia, as indicated by reduced gastrocnemius size (-16%), plantarflexion force (-8%) and MUNE (-56%), whereas mPGC1α mice were only partially protected. Interestingly, despite exacerbated cachexia in aged WT mice treated with chemotherapy, as demonstrated by markedly decreased gastrocnemius size (-22%), plantarflexion force (-18%) and MUNE (-80%) compared to untreated WT, muscle mass, strength and innervation were fully preserved in age-matched mPGC1α mice. Follow-up molecular analyses revealed that WT animals exposed to chemotherapy present loss of muscle mitochondrial proteins PGC1α, OPA1 and CytochromeC, whereas their levels in mPGC1α mice were robustly increased. Conclusion and Potential Impact: Altogether, our data suggest that PGC1α plays a pivotal role in preserving skeletal muscle mass and function, usually impaired by anticancer treatments. These findings enforce developing mitochondria-targeting therapeutics to combat the negative consequences that chemotherapy has on skeletal muscle.

Mitochondrial Dysfunction and Diabetic Nephropathy: Nontraditional Therapeutic Opportunities

Diabetic nephropathy (DN) is a progressive microvascular diabetic complication. Growing evidence shows that persistent mitochondrial dysfunction contributes to the progression of renal diseases, including DN, as it alters mitochondrial homeostasis and, in turn, affects normal kidney function. Pharmacological regulation of mitochondrial networking is a promising therapeutic strategy for preventing and restoring renal function in DN. In this review, we have surveyed recent advances in elucidating the mitochondrial networking and signaling pathways in physiological and pathological contexts. Additionally, we have considered the contributions of nontraditional therapy that ameliorate mitochondrial dysfunction and discussed their molecular mechanism, highlighting the potential value of nontraditional therapies, such as herbal medicine and lifestyle interventions, in therapeutic interventions for DN. The generation of new insights using mitochondrial networking will facilitate further investigations on nontraditional therapies for DN.

Mitochondrial calcium uniporter affects neutrophil bactericidal activity during Staphylococcus aureus infection

Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus ; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1 -/- ) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1 -/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus . Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1 -/- neutrophils alone are not more antibacterial towards S. aureus , but rather enhanced suicidal NETosis by MICU1 -/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1 -/- neutrophils in the heart produced higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.

PINK1 deficiency impairs osteoblast differentiation through aberrant mitochondrial homeostasis

Background PTEN-induced kinase 1 (PINK1) is a serine/threonine-protein kinase in mitochondria that is critical for mitochondrial quality control. PINK1 triggers mitophagy, a selective autophagy of mitochondria, and is involved in mitochondrial regeneration. Although increments of mitochondrial biogenesis and activity are known to be crucial during differentiation, data regarding the specific role of PINK1 in osteogenic maturation and bone remodeling are limited. Methods We adopted an ovariectomy model in female wildtype and Pink1−/− mice. Ovariectomized mice were analyzed using micro-CT, H&E staining, Masson’s trichrome staining. RT-PCR, western blot, immunofluorescence, alkaline phosphatase, and alizarin red staining were performed to assess the expression of PINK1 and osteogenic markers in silencing of PINK1 MC3T3-E1 cells. Clinical relevance of PINK1 expression levels was determined via qRT-PCR analysis in normal and osteoporosis patients. Results A significant decrease in bone mass and collagen deposition was observed in the femurs of Pink1−/− mice after ovariectomy. Ex vivo, differentiation of osteoblasts was inhibited upon Pink1 downregulation, accompanied by impaired mitochondrial homeostasis, increased mitochondrial reactive oxygen species production, and defects in mitochondrial calcium handling. Furthermore, PINK1 expression was reduced in bones from patients with osteoporosis, which supports the practical role of PINK1 in human bone disease. Conclusions In this study, we demonstrated that activation of PINK1 is a requisite in osteoblasts during differentiation, which is related to mitochondrial quality control and low reactive oxygen species production. Enhancing PINK1 activity might be a possible treatment target in bone diseases as it can promote a healthy pool of functional mitochondria in osteoblasts.