Protein Tom40 Research Articles

Outer mitochondrial membrane E3Ub ligase MARCH5 controls de novo peroxisome biogenesis.

We report that the outer mitochondrial membrane (OMM)-associated E3Ub ligase MARCH5 is vital for generating mitochondria-derived pre-peroxisomes. In human immortalized cells, MARCH5 knockout leads to the accumulation of immature peroxisomes, reduced fatty-acid-induced peroxisomal biogenesis, and abnormal peroxisome biogenesis in MARCH5/Pex14 and MARCH5/Pex3 dko cells. Upon fatty-acid-induced peroxisomal biogenesis, MARCH5 redistributes to peroxisomes, and ubiquitination activity-deficient mutants of MARCH5 accumulate on peroxisomes containing high levels of the OMM protein Tom20 (mitochondria-derived pre-peroxisomes). Similarly, depletion of peroxisome biogenesis factor Pex14 leads to the accumulation of MARCH5- and Tom20-positive pre-peroxisomes, whereas no peroxisomes are detected in MARCH5/Pex14 dko cells. Inconsistent with MARCH5 merely acting as a quality factor, mitochondrial decline is not evident in tested models. Furthermore, reduced expression of peroxisomal proteins is detected in MARCH5-/- cells, whereas some of these proteins are stabilized in peroxisome biogenesis deficiency models lacking MARCH5 expression. Thus, MARCH5 is central for mitochondria-dependent peroxisome biogenesis.

Identification of a novel caspase cleavage motif AEAD

Infections of many viruses induce caspase activation to regulate multiple cellular pathways, including programmed cell death, immune signaling and etc. Characterizations of caspase cleavage sites and substrates are important for understanding the regulation mechanisms of caspase activation. Here, we identified and analyzed a novel caspase cleavage motif AEAD, and confirmed its caspase dependent cleavage activity in natural substrate, such as nitric oxide-associated protein 1 (NOA1). Fusing the enhanced green fluorescent protein (EGFP) with the mitochondrial marker protein Tom20 through the AEAD motif peptide localized EGFP to the mitochondria. Upon the activation of caspase triggered by Sendai virus (SeV) or herpes simplex virus type 1 (HSV-1) infection, EGFP diffusely localized to the cell due to the caspase-mediated cleavage, thus allowing visual detection of the virus-induced caspase activation. An AEAD peptide-derived inhibitor Z-AEAD-FMK were developed, which significantly inhibited the activities of caspases-1, -3, -6, -7, -8 and -9, exhibiting a broad caspase inhibition effect. The inhibitor further prevented caspases-mediated cleavage of downstream substrates, including BID, PARP1, LMNA, pro-IL-1β, pro-IL-18, GSDMD and GSDME, protecting cells from virus-induced apoptotic and pyroptotic cell death. Together, our findings provide a new perspective for the identification of novel caspase cleavage motifs and the development of new caspase inhibitors and anti-inflammatory drugs.

Role of FMRP in AKT/mTOR pathway-mediated hippocampal autophagy in fragile X syndrome

Fragile X syndrome (FXS) is caused by epigenetic silencing of the Fmr1 gene, leading to the deletion of the coding protein FMRP. FXS induces abnormal hippocampal autophagy and mTOR overactivation. However, it remains unclear whether FMRP regulates hippocampal autophagy through the AKT/mTOR pathway, which influences the neural behavior of FXS. Our study revealed that FMRP deficiency increased the protein levels of p-ULK-1 and p62 and decreased LC3II/LC3I level in Fmr1 knockout (KO) mice. The mouse hippocampal neuronal cell line HT22 with knockdown of Fmr1 by lentivirus showed that the protein levels of p-ULK-1 and p62 were increased, whereas LC3II/LC3I was unchanged. Further observations revealed that FMRP deficiency obstructed autophagic flow in HT22 cells. Therefore, FMRP deficiency inhibited autophagy in the mouse hippocampus and HT22 cells. Moreover, FMRP deficiency increased reactive oxygen species (ROS) level, decreased the co-localization between the mitochondrial outer membrane proteins TOM20 and LC3 in HT22 cells, and caused a decrease in the mitochondrial autophagy protein PINK1 in HT22 cells and Fmr1 KO mice, indicating that FMRP deficiency caused mitochondrial autophagy disorder in HT22 cells and Fmr1 KO mice. To explore the mechanism by which FMRP deficiency inhibits autophagy, we examined the AKT/mTOR signaling pathway in the hippocampus of Fmr1 KO mice, found that FMRP deficiency caused overactivation of the AKT/mTOR pathway. Rapamycin-mediated mTOR inhibition activated and enhanced mitochondrial autophagy. Finally, we examined whether rapamycin affected the neurobehavior of Fmr1 KO mice. The Fmr1 KO mice exhibited stereotypical behavior, impaired social ability, and learning and memory impairment, while rapamycin treatment improved behavioral disorders in Fmr1 KO mice. Thus, our study revealed the molecular mechanism by which FMRP regulates autophagy function, clarifying the role of hippocampal neuron mitochondrial autophagy in the pathogenesis of FXS, and providing novel insights into potential therapeutic targets of FXS.

Cadmium exposure activates mitophagy through downregulating thyroid hormone receptor/PGC1α signal in preeclampsia

Gestational cadmium exposure increases the risk of preeclampsia. Placenta mitophagy was activated in preeclampsia. The aim of present study was to explore the mechanism of cadmium-induced mitophagy activation and its association with preeclampsia. Mitophagy markers expression levels were detected by quantitative real-time PCR, Western blot, immunofluorescence and immunochemistry in preeclampsia placenta. JEG3 cells were treated with CdCl2, iopanoic acid (IOP), 3-methyladenine and PGC1α SiRNA to verify mechanism of cadmium-induced mitophagy. Mitophagy marker LC3BII/I and P62 expression were increased and mitochondrial membrane receptor protein TOM20 and FUNDC1 expression were decreased in preeclampsia placenta as compared with that in normotension control. Mitophagy marker LC3BII/I and P62 expression were increased and TOM20 and FUNDC1 expression was decreased in CdCl2-treated JEG3 cells. Meanwhile, mitochondrial biogenesis regulator, PGC1α expression was decreased in preeclampsia and CdCl2-treated JEG3 cells. The expressions of LC3B and P62 were increased and the expressions of TOM20, FUNDC1 and PGC1α were decreased in IOP-treated cell. PGC1α SiRNA transfection led to increased expression of LC3BII/I and P62 and decreased expression of TOM20 and FUNDC1. The expression of sFlt1 was increased in preeclampsia placenta, CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. 3-methyladenine treatment protected the increased expression of sFlt1 in CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. Meanwhile, co-treatment of cadmium and IOP or PGC1αSiRNA led to a reduce expressions of OPA1, MFN1, MFN2 and FUNDC1 as compared to cadmium-treated, IOP-treated and PGC1α SiRNA-treated cells. These results elucidated that maternal cadmium exposure activated placenta mitophagy through downregulation of thyroid hormone receptor signal mediated decreased expression of PGC1α and was associated with the occurrence of preeclampsia.

The Role of Chorein Deficiency in Late Spermatogenesis.

VPS13A, also known as chorein, whose loss of function causes chorea-acanthocytosis (ChAc), is characterized by Huntington's-disease-like neurodegeneration and neuropsychiatric symptoms in addition to acanthocytosis in red blood cells. We previously reported that ChAc-model mice with a loss of chorein function exhibited male infertility, with asthenozoospermia and mitochondrial dysmorphology in the spermatozoa. Here, we report a novel aspect of chorein dysfunction in male fertility, particularly its role in spermatogenesis and mitochondrial integrity. An increase in anti-malondialdehyde antibody immunoreaction within the testes, predominantly observed at the advanced stages of sperm formation in chorein-deficient mice, suggests oxidative stress as a contributing factor to mitochondrial dysfunction and impaired sperm maturation. The chorein immunoreactivity in spermatids of wild-type mice accentuates its significance in sperm development. ChAc-model mice exhibit mitochondrial ultrastructural abnormalities, specifically during the late stages of sperm maturation, suggesting a critical timeframe for chorein's action in spermiogenesis. We observed an increase in TOM20 protein levels, indicative of disrupted mitochondrial import mechanisms. The concurrent decrease in metabolic enzymes such as IDH3A, LDHC, PGK2, and ACAT1 suggests a complex chorein-mediated metabolic network that is essential for sperm vitality. Additionally, heightened separation of cytoplasmic droplets from sperm highlights the potential membrane instability in chorein-deficient spermatozoa. Metabolomic profiling further suggests a compensatory metabolic shift, with elevated glycolytic and TCA-cycle substrates. Our findings suggest that chorein is involved in anti-ferroptosis and the maturation of mitochondrial morphology in the late stages of spermatogenesis, and its deficiency leads to asthenozoospermia characterized by membrane instability, abnormal cytosolic glycolysis, abnormal mitochondrial function, and a disrupted TCA cycle. Further analyses are required to unravel the molecular mechanisms that directly link these findings and to elucidate the role of chorein in spermatogenesis as well as its broader implications.

Role of Histamine H3 Receptor Antagonist Pitolisant in Early Neural Differentiation of Mouse Embryonic Stem Cells.

The histamine H3 receptor, prominently expressed in neurons with a minor presence in glial cells, acts as both an autoreceptor and an alloreceptor, controlling the release of histamine and other neurotransmitters. The receptor impacts various essential physiological processes. Our team's initial investigations had demonstrated that the histamine H3 receptor antagonists could facilitate nerve regeneration by promoting the histamine H1 receptors on primary neural stem cells (NSCs) in the traumatic brain injury mouse, which suggested the potential of histamine H3 receptor as a promising target for treating neurological disorders and promoting nerve regeneration. Pitolisant (PITO) is the only histamine H3 receptor antagonist approved by the Food and Drug Administration (FDA) for treating narcolepsy. However, there is no report on Pitolisant in neural development or regeneration, and it is urgent to be further studied in strong biological activity models in vitro. The embryonic stem (ES) cells were differentiated into neural cells in vitro, which replicated the neurodevelopmental processes that occur in vivo. It also provided an alternative model for studying neurodevelopmental processes and testing drugs for neurological conditions. Therefore, we aimed to elucidate the regulatory role of Pitolisant in the early differentiation of ES cells into neural cells. Our results demonstrated that Pitolisant could promote the differentiation of ES cells toward NSCs and stimulated the formation of growth cones. Furthermore, Pitolisant was capable of inducing the polarization of NSCs through the cAMP-LKB1-SAD/MARK2 pathway, but had no significant effect on later neuronal maturation. Pitolisant altered mitochondrial morphology and upregulated the levels of mitochondrion-related proteins TOM20, Drp1, and p-Drp1, and reversed the inhibitory effect of Mdivi-1 on mitochondrial fission during the early neural differentiation of ES cells. In addition, Pitolisant induced the increase in cytosolic Ca2+. Our study provided an experimental foundation for the potential application of histamine H3 receptor-targeted modulators in the field of neuroregeneration.

Investigating proliferation and differentiation capacities of Hanwoo steer myosatellite cells at different passages for developing cell-cultured meat

The aim of study was to investigate proliferation and differentiation capacities of Hanwoo myosatellite cells for the development of Hanwoo cell cultures. From P1 to P19, the number of live cells decreased and the cell size increased. It was confirmed that the PAX7 mRNA was higher in P3 than P6 and P9 (p < 0.05). The maximum differentiation score was measured from P1 to P12. The maximum differentiation score maintained high from P1 to P10. Immunostaining was performed for both P1 and P10 cells to investigate differentiation characteristics. And there were no significant differences in differentiation characteristics between P1 and P10 cells. MYOG mRNA was low, whereas C-FOS mRNA was high (p < 0.05) in the late passage. Myosin and Tom20 protein also showed low values in the late passage (p < 0.05). In conclusion, our results suggest that it is appropriate to use P1 to P10 for the production of cultured meat using Hanwoo muscle cells. If cell culture meat production is performed without differentiation, the passage range may increase further. These results provide basic essential data required for further development of Hanwoo cell cultures, which could provide a valuable source of protein for human populations in the future.

The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.

Iron induces B cell pyroptosis through Tom20–Bax–caspase–gasdermin E signaling to promote inflammation post-spinal cord injury

BackgroundImmune inflammatory responses play an important role in spinal cord injury (SCI); however, the beneficial and detrimental effects remain controversial. Many studies have described the role of neutrophils, macrophages, and T lymphocytes in immune inflammatory responses after SCI, although little is known about the role of B lymphocytes, and immunosuppression can easily occur after SCI.MethodsA mouse model of SCI was established, and HE staining and Nissl staining were performed to observe the pathological changes. The size and morphology of the spleen were examined, and the effects of SCI on spleen function and B cell levels were detected by flow cytometry and ELISA. To explore the specific mechanism of immunosuppression after SCI, B cells from the spleens of SCI model mice were isolated using magnetic beads and analyzed by 4D label-free quantitative proteomics. The level of inflammatory cytokines and iron ions were measured, and the expression of proteins related to the Tom20 pathway was quantified by western blotting. To clarify the relationship between iron ions and B cell pyroptosis after SCI, we used FeSO4 and CCCP, which induce oxidative stress to stimulate SCI, to interfere with B cell processes. siRNA transfection to knock down Tom20 (Tom20-KD) in B cells and human B lymphocytoma cell was used to verify the key role of Tom20. To further explore the effect of iron ions on SCI, we used deferoxamine (DFO) and iron dextran (ID) to interfere with SCI processes in mice. The level of iron ions in splenic B cells and the expression of proteins related to the Tom20–Bax–caspase–gasdermin E (GSDME) pathway were analyzed.ResultsSCI could damage spleen function and lead to a decrease in B cell levels; SCI upregulated the expression of Tom20 protein in the mitochondria of B cells; SCI could regulate the concentration of iron ions and activate the Tom20–Bax–caspase–GSDME pathway to induce B cell pyroptosis. Iron ions aggravated CCCP-induced B cell pyroptosis and human B lymphocytoma pyroptosis by activating the Tom20–Bax–caspase–GSDME pathway. DFO could reduce inflammation and promote repair after SCI by inhibiting Tom20–Bax–caspase–GSDME-induced B cell pyroptosis.ConclusionsIron overload activates the Tom20–Bax–caspase–GSDME pathway after SCI, induces B cell pyroptosis, promotes inflammation, and aggravates the changes caused by SCI. This may represent a novel mechanism through which the immune inflammatory response is induced after SCI and may provide a new key target for the treatment of SCI.

The Translocase of the Outer Mitochondrial Membrane (TOM40) is required for mitochondrial dynamics and neuronal integrity in Dorsal Root Ganglion Neurons

Polymorphisms and altered expression of the Translocase of the Outer Mitochondrial Membrane – 40 kD (Tom40) are observed in neurodegenerative disease subjects. We utilized in vitro cultured dorsal root ganglion (DRG) neurons to investigate the association of TOM40 depletion to neurodegeneration, and to unravel the mechanism of neurodegeneration induced by decreased levels of TOM40 protein. We provide evidence that severity of neurodegeneration induced in the TOM40 depleted neurons increases with the increase in the depletion of TOM40 and is exacerbated by an increase in the duration of TOM40 depletion. We also demonstrate that TOM40 depletion causes a surge in neuronal calcium levels, decreases mitochondrial motility, increases mitochondrial fission, and decreases neuronal ATP levels. We observed that alterations in the neuronal calcium homeostasis and mitochondrial dynamics precede BCL-xl and NMNAT1 dependent neurodegenerative pathways in the TOM40 depleted neurons. This data also suggests that manipulation of BCL-xl and NMNAT1 may be of therapeutic value in TOM40 associated neurodegenerative disorders.

Reduced OPA1, Mitochondrial Fragmentation and Increased Susceptibility to Apoptosis in Granular Corneal Dystrophy Type 2 Corneal Fibroblasts.

The progressive degeneration of granular corneal dystrophy type 2 (GCD2) corneal fibroblasts is associated with altered mitochondrial function, but the underlying mechanisms are incompletely understood. We investigated whether an imbalance of mitochondrial dynamics contributes to mitochondrial dysfunction of GCD2 corneal fibroblasts. Transmission electron microscopy revealed several small, structurally abnormal mitochondria with altered cristae morphology in GCD2 corneal fibroblasts. Confocal microscopy showed enhanced mitochondrial fission and fragmented mitochondrial tubular networks. Western blotting revealed higher levels of MFN1, MFN2, and pDRP1 and decreased levels of OPA1 and FIS1 in GCD2. OPA1 reduction by short hairpin RNA (shRNA) resulted in fragmented mitochondrial tubular networks and increased susceptibility to mitochondrial stress-induced apoptosis. A decrease in the mitochondrial biogenesis-related transcription factors NRF1 and PGC1α was observed, while there was an increase in the mitochondrial membrane proteins TOM20 and TIM23. Additionally, reduced levels of mitochondrial DNA (mtDNA) were exhibited in GCD2 corneal fibroblasts. These observations suggest that altered mitochondrial fission/fusion and biogenesis are the critical molecular mechanisms that cause mitochondrial dysfunction contributing to the degeneration of GCD2 corneal fibroblasts.

Loss of parkin causes endoplasmic reticulum calcium dyshomeostasis by upregulation of reticulocalbin 1.

Increasing evidence suggests that astrocytes play an important role in the progression of Parkinson's disease (PD). Previous studies on our parkin knockout mouse demonstrated a higher accumulation of damaged mitochondria in astrocytes than in surrounding dopaminergic (DA) neurons, suggesting that Parkin plays a crucial role regarding their interaction during PD pathogenesis. In the current study, we examined primary mesencephalic astrocytes and neurons in a direct co-culture system and discovered that the parkin deletion causes an impaired differentiation of mesencephalic neurons. This effect required the parkin mutation in astrocytes as well as in neurons. In Valinomycin-treated parkin-deficient astrocytes, ubiquitination of Mitofusin 2 was abolished, whereas there was no significant degradation of the outer mitochondrial membrane protein Tom70. This result may explain the accumulation of damaged mitochondria in parkin-deficient astrocytes. We examined differential gene expression in the substantia nigra region of our parkin-KO mouse by RNA sequencing and identified an upregulation of the endoplasmic reticulum (ER) Ca2+ -binding protein reticulocalbin 1 (RCN1) expression, which was validated using qPCR. Immunostaining of the SN brain region revealed RCN1 expression mainly in astrocytes. Our subcellular fractionation of brain extract has shown that RCN1 is located in the ER and in mitochondria-associated membranes (MAM). Moreover, a loss of Parkin function reduced ATP-stimulated calcium-release in ER mesencephalic astrocytes that could be attenuated by siRNA-mediated RCN1 knockdown. Our results indicate that RCN1 plays an important role in ER-associated calcium dyshomeostasis caused by the loss of Parkin function in mesencephalic astrocytes, thereby highlighting the relevance of astrocyte function in PD pathomechanisms.

Abstract 15560: Mitophagy Dysfunction and Activation of the Inflammasome Cause Aortic Stiffness in Low Aerobic Capacity Aged Rats

Aging and low aerobic capacity are associated with an increased risk of cardiovascular events and mortality. We reported that a polygenic rat model selectively bred for low aerobic capacity (LCR) showed aortic stiffness and dysfunction with age compared to rats bred for a high aerobic capacity (HCR). We observed increased aortic mitochondrial ROS and extracellular mtDNA DAMPs levels, activation of AIM2 inflammasome and its downstream effectors and increased collagen content in old LCR rat aortas. However, the molecular mechanisms linking low aerobic capacity to inflammation in aging have not been elucidated. In order to decipher the changes in genetic networks in low aerobic capacity and aging-associated aortic stiffness, a global transcriptome analysis was performed by RNA-Seq and gene ontology analysis of aortas from young (4 month) and old (27 month) LCR and HCR rats. Down-regulated pathways in old LCR aortas include mitochondrial biogenesis and mitophagy and up-regulated pathways include pro-inflammatory pathways, apoptosis/necroptosis/ferroptosis pathways, in contrast to HCR aortas which have higher levels of pathways regulating longevity, oxidative phosphorylation, and mitophagy and autophagy. In line with that, old LCR rats displayed a 54% reduction in mitophagy (colocalization of mitochondrial marker protein TOM20 with lysosomal marker protein LAMP1) in aortic media and a 76% reduction in cultured VSMCs in comparison to older HCR rats. Mitophagy levels did not differ significantly between young LCR and HCR rats. The autophagic vacuole formation rate was a 12-fold lower in old LCR than in old HCR aortic VSMCs, suggesting that impaired mitophagy may be responsible for accumulation of mtDNA-derived damage associated molecular patterns (mtDNA DAMPs). Treatment with various inducers of mitophagy (rapamycin, deferiprone, and MitoTEMPO) significantly increased mitophagy and decreased mtDNA DAMPs levels in old LCR VSMCs. This was also associated with a reduction in inflammasome activation, mitochondrial dysfunction, and ROS levels. We conclude that impaired mitophagy causes accumulation of DAMPs and inflammasome activation, inducing aortic stiffness and dysfunction in old age LCR rats.

TOMM40 genetic variants associated with healthy aging and longevity: a systematic review

IntroductionHealthy aging relies on mitochondrial functioning because this organelle provides energy and diminishes oxidative stress. Single nucleotide polymorphisms (SNPs) in TOMM40, a critical gene that produces the outer membrane protein TOM40 of mitochondria, have been associated with mitochondrial dysfunction and neurodegenerative processes. Yet it is not clear whether or how the mitochondria may impact human longevity. We conducted this review to ascertain which SNPs have been associated with markers of healthy aging.MethodsUsing the PRISMA methodology, we conducted a systematic review on PubMed and Embase databases to identify associations between TOMM40 SNPs and measures of longevity and healthy aging.ResultsTwenty-four articles were selected. The TOMM40 SNPs rs2075650 and rs10524523 were the two most commonly identified and studied SNPs associated with longevity. The outcomes associated with the TOMM40 SNPs were changes in BMI, brain integrity, cognitive functions, altered inflammatory network, vulnerability to vascular risk factors, and longevity.DiscussionsOur systematic review identified multiple TOMM40 SNPs potentially associated with healthy aging. Additional research can help to understand mechanisms in aging, including resilience, prevention of disease, and adaptation to the environment.

Exosome mediated Tom40 delivery protects against hydrogen peroxide-induced oxidative stress by regulating mitochondrial function.

Mitochondrial dysfunction is a hallmark of neurodegeneration. The expression level of Tom40, a crucial mitochondrial membrane protein, is significantly reduced in neurodegenerative disease subjects. Tom40 overexpression studies have shown to protect the neurons against oxidative stress by improving mitochondrial function. Thus, successful delivery of Tom40 protein to the brain could lead to a novel therapy for neurodegenerative diseases. However, delivering protein to the cell may be difficult. Especially the blood-brain barrier (BBB) is a big hurdle to clear in order to deliver the protein to the brain. In the current study, we engineered exosomes, which are the extracellular vesicles of endosomal origin, and able to cross BBB as delivery vehicles packing human Tom40. We found Tom40 protein delivery by the exosome successfully protected the cells against hydrogen peroxide-induced oxidative stress. This result suggests that exosome-mediated delivery of Tom40 may potentially be useful in restoring mitochondrial functions and alleviating oxidative stress in neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases.

Steady-State Levels of Miro1 Linked to Phosphorylation at Serine 156 and Mitochondrial Respiration in Dopaminergic Neurons.

Miro1 has emerged as an interesting target to study Parkinson’s disease-relevant pathways since it is a target of PINK1 and Parkin. Miro1 is a mitochondrial GTPase with the primary function of facilitating mitochondrial movement, and its knockout in mice is postnatally lethal. Here, we investigated the effect of the artificial RHOT1/Miro1 S156A mutation since it is a putative PINK1 phosphorylation site shown to be involved in Miro1 degradation and mitochondrial arrest during mitophagy. We gene-edited a homozygous phospho-null Miro1 S156A mutation in induced pluripotent stem cells to study the mutation in human dopaminergic neurons. This mutation causes a significant depletion of Miro1 steady-state protein levels and impairs further Miro1 degradation upon CCCP-induced mitophagy. However, mitochondrial mass measured by Tom20 protein levels, as well as mitochondrial area, are not affected in Miro1 S156A neurons. The mitochondria are slightly lengthened, which is in line with their increased turnover. Under basal conditions, we found no discernable effect of the mutation on mitochondrial movement in neurites. Interestingly, the S156A mutation leads to a significant reduction of mitochondrial oxygen consumption, which is accompanied by a depletion of OXPHOS complexes III and V. These effects are not mirrored by Miro1 knockdown in neuroblastoma cells, but they are observed upon differentiation. Undifferentiated Miro1 S156A neural precursor cells do not have decreased Miro1 levels nor OXPHOS complexes, suggesting that the effect of the mutation is tied to development. In mature dopaminergic neurons, the inhibition of Miro1 Ser156 phosphorylation elicits a mild loss of mitochondrial quality involving reduced mitochondrial membrane potential, which is sufficient to induce compensatory events involving OXPHOS. We suggest that the mechanism governing Miro1 steady-state levels depends on differentiation state and metabolic demand, thus underscoring the importance of this pathway in the pathobiology of Parkinson’s disease.

Tourniquet-induced lower limb ischemia/reperfusion reduces mitochondrial function by decreasing mitochondrial biogenesis in acute kidney injury in mice.

The mechanisms by which lower limb ischemia/reperfusion induces acute kidney injury (AKI) remain largely uncharacterized. We hypothesized that tourniquet‐induced lower limb ischemia/reperfusion (TILLIR) would inhibit mitochondrial function in the renal cortex. We used a murine model to show that TILLIR of the high thigh regions inflicted time‐dependent AKI as determined by renal function and histology. This effect was associated with decreased activities of mitochondrial complexes I, II, V and citrate synthase in the kidney cortex. Moreover, TILLIR reduced mRNA levels of a master regulator of mitochondrial biogenesis PGC‐1α, and its downstream genes NDUFS1 and ATP5o in the renal cortex. TILLIR also increased serum corticosterone concentrations. TILLIR did not significantly affect protein levels of the critical regulators of mitophagy PINK1 and PARK2, mitochondrial transport proteins Tom20 and Tom70, or heat‐shock protein 27. TILLIR had no significant effect on mitochondrial oxidative stress as determined by mitochondrial ability to generate reactive oxygen species, protein carbonylation, or protein levels of MnSOD and peroxiredoxin1. However, TILLIR inhibited classic autophagic flux by increasing p62 protein abundance and preventing the conversion of LC3‐I to LC3‐II. TILLIR increased phosphorylation of cytosolic and mitochondrial ERK1/2 and mitochondrial AKT1, as well as mitochondrial SGK1 activity. In conclusion, lower limb ischemia/reperfusion induces distal AKI by inhibiting mitochondrial function through reducing mitochondrial biogenesis. This AKI occurs without significantly affecting PINK1‐PARK2‐mediated mitophagy or mitochondrial oxidative stress in the kidney cortex.

Dengzhan Xixin injection derived from a traditional Chinese herb Erigeron breviscapus ameliorates cerebral ischemia/reperfusion injury in rats via modulation of mitophagy and mitochondrial apoptosis

Ethnopharmacological relevanceDengzhan Xixin injection (DX), a preparation of extracts from traditional Chinese medicine Erigeron breviscapus (Vaniot) Hand.-Mazz., has been widely used in clinical treatment of cerebral ischemia sequelae in China for a long history. However, its underlying mechanisms remain unclear. Aim of the studyThe objective of this present study aimed to investigate the therapeutic effects of DX on cerebral ischemia/reperfusion (I/R) injury in a rat model. Meanwhile, its underlying molecular mechanisms on mitochondrial protection were further interpreted. Materials and methodsThe major components of DX were detected by high-performance liquid chromatography analysis. The model of cerebral I/R injury was established by middle cerebral artery occlusion (MCAO) in SD rats. We firstly performed neurobehavioral score, the regional cerebral blood flow (rCBF) assay, and TTC, HE and Nissl staining for evaluating the effects of DX on I/R injury. And then, the cortical levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) were determined by commercial kits. Whereafter, real time-PCR and transmission electron microscopy were employed to investigate the relative copy number of mitochondrial DNA (mtDNA) and neuronal ultrastructure changes, respectively. Further, the potential interactions of major components in DX with mitophagy/apoptosis-related proteins were predicted by Schrodinger molecular docking. The expression of mitophagy-related proteins LC3, p62, TOM20, PINK1 and Parkin was estimated by western blot and immunofluorescence analyses. Furthermore, TUNEL staining and western blot were used to detect the apoptotic phenomenon and the protein expression of Bax, Bcl-2, Cytochrome c (Cyto-c) and cleaved Caspase-3. ResultsDX mainly contains scutellarin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, caffeic acid and 5-O-caffeoylquinic acid. Compared with the model group, DX could remarkably relieve ischemia-provoked neurological deficit, rCBF deficiency and cerebral infarction. Pathological changes and neuronal loss in a MCAO model of rats were memorably ameliorated by DX administration. Meanwhile, DX reduced the surged ROS and MDA, while increased the level of SOD. Notably, DX treatment conversed the collapse of ATP and MMP, along with decreased in the relative copy number of mtDNA, contributing to the maintaining of mitochondrial ultrastructure via the increased number of autophagy lysosomes. The representative ingredients in DX had a potential bind with the active sites of mitophagy/apoptosis-related proteins. DX stimulated the protein expression of LC3, PINK1 and Parkin, while reduced the levels of p62 and TOM20. In addition, DX confined TUNEL-positive cell rate with the decreased expressions of Bax, Cyto-c and cleaved Caspase-3 as well as the increased Bcl-2 level. ConclusionsWe demonstrated that the protection of DX against brain ischemia could attribute to alleviating mitochondrial damage by upregulating mitophagy and inhibiting mitochondria-mediated apoptosis.

Determine the Function of the Exocyst in Vesicle Tethering by Ectopic Targeting.

We describe an assay, in which ectopically targeting the exocyst subunit Sec3 to mitochondria is used to determine its role in tethering of post-Golgi vesicles to the plasma membrane. In the assay, we use a plasmid that encodes a fusion protein of the mitochondria protein Tom20 and Sec3 N-terminally tagged with the florescence protein mCherry, and coexpress the plasmid in yeast cells with CIT1-GFP, a marker protein of mitochondria. We then detect the colocalization between Sec3 and CIT1 and other exocyst subunits such as Sec5 on mitochondria using fluorescence microscopy. We further detect the colocalization between Sec3 and Sec4, a Rab protein and a marker of post-Golgi vesicles. Through this assay, we propose that the exocyst subunit Sec3 recruits the other exocyst subunits and secretory vesicles to a target membrane, suggesting that it plays a pivotal role in vesicle tethering. This approach is likely appropriate for studying other tethering complexes at their specific stages of trafficking and may also be used in other eukaryotic cells such as the cultured mammalian cells.

Apoptin induces pyroptosis of colorectal cancer cells via the GSDME-dependent pathway.

Apoptin is a small molecular weight protein encoded by the VP3 gene of chicken anemia virus (CAV). It can induce apoptosis of tumor cells and play anti-tumorigenic functions. In this study, we identified a time-dependent inhibitory role of apoptin on the viability of HCT116 cells. We also demonstrated that apoptin induces pyroptosis through cleaved caspase 3, and with a concomitant cleavage of gasdermin E (GSDME) rather than GSDMD. GSDME knockdown switched the apoptin-induced cell death from pyroptosis to apoptosis in vitro. Furthermore, we demonstrated that the effect of apoptin on GSDME-dependent pyroptosis could be mitigated by caspase-3 and caspase-9 siRNA knockdown. Additionally, apoptin enhanced the intracellular reactive oxygen species (ROS), causing aggregation of the mitochondrial membrane protein Tom20. Moreover, bax and cytochrome c were released to the activating caspase-9, eventually triggering pyroptosis. Therefore, GSDME mediates the apoptin-induced pyroptosis through the mitochondrial apoptotic pathway. Finally, using nude mice xenografted with HCT116 cells, we found that apoptin induces pyroptosis and significantly inhibits tumor growth. Based on this mechanism, apoptin may provide a new strategy for colorectal cancer therapy.