Aristolochic Acid I-Induced Hepatotoxicity in Tianfu Broilers Is Associated with Oxidative-Stress-Mediated Apoptosis and Mitochondrial Damage

Aristolochic acid (AA) is a component of traditional Chinese herbs and commonly used for farm animals in China. Over-exposure of AA has been proven to be associated with hepatotoxicity; however, the mechanism of action of AA-I-induced hepatotoxicity remains unknown. In the current study, a subchronic toxicity test was conducted to evaluate the mechanism of AA-induced hepatotoxicity in Tianfu broilers. According to the results, AA-I-induced hepatotoxicity in Tianfu broilers was evidenced by the elevation of liver weight, levels of serum glutamic oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). Furthermore, hepatocyte swelling, vesicular degeneration and steatosis were observed. Additionally, AA-I elevated the production of reactive oxygen species (ROS) and induced oxidative stress, which further led to excessive apoptosis, characterized by mitochondrial depolarization, upregulation of Bax, and down-regulation of Bcl-2 expression. In conclusion, the mechanism of AA-I-induced hepatotoxicity was associated with oxidative-stress-mediated apoptosis and mitochondrial damage.

Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy

The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1-PRKN pathway leading to mitophagy. We provide a detailed cell biological characterization of a benzosulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition after mitochondrial depolarization. This includes enhancement of TOMM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinson disease patients carrying loss of function PRKN mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1-independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U2OS cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover.

Mitochondrial Membrane Potential Influences Amyloid-β Protein Precursor Localization and Amyloid-β Secretion

Background: Amyloid-β (Aβ), which derives from the amyloid-β protein precursor (AβPP), forms plaques and serves as a fluid biomarker in Alzheimer’s disease (AD). How Aβ forms from AβPP is known, but questions relating to AβPP and Aβ biology remain unanswered. AD patients show mitochondrial dysfunction, and an Aβ/AβPP mitochondria relationship exists. Objective: We considered how mitochondrial biology may impact AβPP and Aβ biology. Methods: SH-SY5Y cells were transfected AβPP constructs. After treatment with FCCP (uncoupler), Oligomycin (ATP synthase inhibitor), or starvation Aβ levels were measured. β-secretase (BACE1) expression was measured. Mitochondrial localized full-length AβPP was also measured. All parameters listed were measured in ρ0 cells on an SH-SY5Y background. iPSC derived neurons were also used to verify key results. Results: We showed that mitochondrial depolarization routes AβPP to, while hyperpolarization routes AβPP away from, the organelle. Mitochondrial AβPP and cell Aβ secretion inversely correlate, as cells with more mitochondrial AβPP secrete less Aβ, and cells with less mitochondrial AβPP secrete more Aβ. An inverse relationship between secreted/extracellular Aβ and intracellular Aβ was observed. Conclusion: Our findings indicate mitochondrial function alters AβPP localization and suggest enhanced mitochondrial activity promote Aβ secretion while depressed mitochondrial activity minimize Aβ secretion. Our data complement other studies that indicate a mitochondrial, AβPP, and Aβ nexus, and could help explain why cerebrospinal fluid Aβ is lower in those with AD. Our data further suggest Aβ secretion could serve as a biomarker of cell or tissue mitochondrial function.

Mitochondrial Contributions in the Genesis of Delayed Afterdepolarizations in Ventricular Myocytes

Mitochondria fulfill the cell’s energy demand and affect the intracellular calcium (Ca2+) dynamics via direct Ca2+ exchange, the redox effect of reactive oxygen species (ROS) on Ca2+ handling proteins, and other signaling pathways. Recent experimental evidence indicates that mitochondrial depolarization promotes arrhythmogenic delayed afterdepolarizations (DADs) in cardiac myocytes. However, the nonlinear interactions among the Ca2+ signaling pathways, ROS, and oxidized Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways make it difficult to reveal the mechanisms. Here, we use a recently developed spatiotemporal ventricular myocyte computer model, which consists of a 3-dimensional network of Ca2+ release units (CRUs) intertwined with mitochondria and integrates mitochondrial Ca2+ signaling and other complex signaling pathways, to study the mitochondrial regulation of DADs. With a systematic investigation of the synergistic or competing factors that affect the occurrence of Ca2+ waves and DADs during mitochondrial depolarization, we find that the direct redox effect of ROS on ryanodine receptors (RyRs) plays a critical role in promoting Ca2+ waves and DADs under the acute effect of mitochondrial depolarization. Furthermore, the upregulation of mitochondrial Ca2+ uniporter can promote DADs through Ca2+-dependent opening of mitochondrial permeability transition pores (mPTPs). Also, due to much slower dynamics than Ca2+ cycling and ROS, oxidized CaMKII activation and the cytosolic ATP do not appear to significantly impact the genesis of DADs during the acute phase of mitochondrial depolarization. However, under chronic conditions, ATP depletion suppresses and enhanced CaMKII activation promotes Ca2+ waves and DADs.

Neutrophils Facilitate Prolonged Inflammasome Response in the DAMP-Rich Inflammatory Milieu

Aberrant inflammasome activation contributes to various chronic inflammatory diseases; however, pyroptosis of inflammasome-active cells promptly terminates local inflammasome response. Molecular mechanisms underlying prolonged inflammasome signaling thus require further elucidation. Here, we report that neutrophil-specific resistance to pyroptosis and NLRP3 desensitization can facilitate sustained inflammasome response and interleukin-1β secretion. Unlike macrophages, inflammasome-activated neutrophils did not undergo pyroptosis, indicated by using in vitro cell-based assay and in vivo mouse model. Intriguingly, danger-associated molecular patterns (DAMP)-rich milieu in the inflammatory region significantly abrogated NLRP3-activating potential of macrophages, but not of neutrophils. This macrophage-specific NLRP3 desensitization was associated with DAMP-induced mitochondrial depolarization that was not observed in neutrophils due to a lack of SARM1 expression. Indeed, valinomycin-induced compulsory mitochondrial depolarization in neutrophils restored inflammasome-dependent cell death and ATP-induced NLRP3 desensitization in neutrophils. Alongside prolonged inflammasome-activating potential, neutrophils predominantly secreted interleukin-1β rather than other proinflammatory cytokines upon NLRP3 stimulation. Furthermore, inflammasome-activated neutrophils did not trigger efferocytosis-mediated M2 macrophage polarization essential for the initiation of inflammation resolution. Taken together, our results indicate that neutrophils can prolong inflammasome response via mitochondria-dependent resistance to NLRP3 desensitization and function as major interleukin-1β-secreting cells in DAMP-rich inflammatory region.

Selective localization of Mfn2 near PINK1 enable its preferential ubiquitination by Parkin on mitochondria

Mutations in Parkin and PINK1 cause an early-onset familial Parkinson's disease. Parkin is a RING-In-Between-RING (RBR) E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: 1) thioester intermediate formation on Parkin, and 2) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyzes ubiquitin transfer to substrates. We found that His433 in the RING2 domain catalyzes acyl transfer. In cells, mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via phospho-ubiquitin at these sites.

Miro1 Impairment in a Parkinson’s At-Risk Cohort

There is a lack of reliable molecular markers for Parkinson’s disease (PD) patients and at-risk individuals. The detection of the pre-symptomatic population of PD will empower more effective clinical intervention to delay or prevent disease onset. We have previously found that the mitochondrial protein Miro1 is resistant to mitochondrial depolarization-induced degradation in fibroblasts from a large number of PD patients and several at-risk individuals. Therefore, Miro1 has the potential to molecularly label PD populations. In order to determine whether Miro1 could serve as a molecular marker for the risk of PD, here we examine the Miro1 response to mitochondrial depolarization by biochemical approaches in induced pluripotent stem cells from a cohort of at-risk individuals. Our results show that the Miro1 phenotype is significantly associated with PD risk. We propose that Miro1 is a promising molecular marker for detecting both PD and at-risk populations. Tracking this Miro1 marker could aid in diagnosis and Miro1-based drug discoveries.

NAD(H) Regulates the Permeability Transition Pore in Mitochondria through an External Site

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD+ on: (1) the Ca2+-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca2+-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca2+-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca2+-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD+ increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca2+-uptake and the free Ca2+ concentration in the medium. NAD(H) suppressed the Ca2+-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca2+-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.