Ultrastructural Changes In Mitochondria Research Articles

Berberine Alleviates Right Heart Dysfunction in MCT-Induced PAH Rats by Targeting Myocardial Mitochondrial Oxidative Phosphorylation

Background: Right ventricular failure (RVF) stands as a leading cause of mortality among pulmonary hypertension (PAH) patients. The underlying mechanisms contributing to Right Ventricular (RV) dysfunction remain enigmatic, with a conspicuous absence of effective treatments. RV functional deterioration is influenced by mitochondria oxidative phosphorylation and fatty acid oxidation. Berberine (BBR), a natural active compound extracted from Berberis vulgaris rhizomes, has shown promise for treating cardiovascular diseases due to its interactions with multiple intracellular targets. This study aimed to investigate the applicability and therapeutic effcacy of BBR in RVF and explore the mechanisms involved in its beneficial effects in monocrotaline (MCT)-induced PAH rats. Methods: Right ventricular hypertrophy (RVH) and RVF were induced via a single injection of MCT (60 mg/kg, ip) in Sprague-Dawley rats. BBR (4 mg/kg body weight per day) was administered through oral gavage. RVH and RVF were confirmed through transthoracic echocardiography (TTE). Transmission electron microscopy (TEM) was utilized to observe changes in cardiomyocyte and mitochondrial ultrastructure within the right ventricle tissue. Comparisons were made among Normal control (NC), RVH, RVH+BBR, RVF, and RVF+ BBR groups. RNA sequencing (RNA-seq) of right ventricle tissue was performed for both the NC (treated with normal saline, ip) and right ventricle dysfunction (RVH and RVF) groups. Gene Ontology and KEGG enrichment analyses were conducted using the R-package “Clusterprofiler.” RV transcriptome results were validated through qPCR and Western blot analysis. Results: Echocardiography and elevated ANP and BNP levels confirmed the presence of RVF following the development of MCT-induced PAH. TEM imaging revealed significant disruptions in mitochondrial ultrastructure, particularly in a subset of interfibrillar mitochondria, which exhibited swelling, disorganization, and reduced cristae density in RVH rats. These disruptions were even more pronounced in RVF rats. Transcriptomic profiles of RV tissue from RVH and RVF identified a total of 175 differentially expressed genes (DEGs). Gene Ontology and KEGG enrichment analyses pinpointed 38 genes associated with oxidative phosphorylation. qPCR demonstrated downregulation of mitochondrial oxidative phosphorylation-related genes (NDUFA2, NDUFA13, COX7B, COX6C, GSTK1, ECH1) in RVF. BBR ameliorated right heart function, leading to decreased RV internal diameter at end-diastole, a reduced RV hypertrophy index, and an increased RV fractional area change. This was accompanied by the recovery of ANP and BNP levels. Furthermore, after BBR application, the mitochondrial structure within RV tissue exhibited marked improvement. Lastly, Western blot analysis revealed that fatty acid oxidation-related proteins (Decr1, Acadm, Acacb, Hadh, Hadhb, Prkag3, Acaa2) were downregulated in right heart dysfunction, and BBR effectively mitigated the decline in oxidation-related protein levels in both RVH and RVF. Conclusion: In summary, our results underscore the critical role of BBR in preserving cardiac function from RVF in MCT-induced PAH rats. This preservation appears to occur through the upregulation of mitochondrial oxidative phosphorylation levels, ultimately enhancing energy metabolism within the mitochondria of RV cardiomyocytes in the right heart of PAH. This work is supported by Natural Science Foundation of Shandong Province(ZR2022MH052), China Postdoctoral Fund (NO.2021M691944) to XMY. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Therapeutic potential of dihydroartemisinin in mitigating radiation-induced lung injury: Inhibition of ferroptosis through Nrf2/HO-1 pathways in mice.

Radiation-induced lung injury (RILI) is a common consequence of thoracic radiation therapy that lacks effective preventative and treatment strategies. Dihydroartemisinin (DHA), a derivative of artemisinin, affects oxidative stress, immunomodulation, and inflammation. It is uncertain whether DHA reduces RILI. In this work, we investigated the specific mechanisms of action of DHA in RILI. Twenty-four C57BL/6J mice were randomly divided into four groups of six mice each: Control group, irradiation (IR) group, IR + DHA group, and IR + DHA + Brusatol group. The IR group received no interventions along with radiation treatment. Mice were killed 30 days after the irradiation. Morphologic and pathologic changes in lung tissue were observed with hematoxylin and eosinstaining. Detection of hydroxyproline levels for assessing the extent of pulmonary fibrosis. Tumor necrosis factor α (TNF-α), transforming growth factor-β (TGF-β), glutathione peroxidase (GPX4), Nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) expression in lung tissues were detected. In addition, mitochondrial ultrastructural changes in lung tissues were also observed, and the glutathione (GSH) content in lung tissues was assessed. DHA attenuated radiation-induced pathological lung injury and hydroxyproline levels. Additionally, it decreased TNF-α and TGF-β after irradiation. DHA may additionally stimulate the Nrf2/HO-1 pathway. DHA upregulated GPX4 and GSH levels and inhibited cellular ferroptosis. Brusatol reversed the inhibitory effect of DHA on ferroptosis and its protective effect on RILI. DHA modulated the Nrf2/HO-1 pathway to prevent cellular ferroptosis, which reduced RILI. Therefore, DHA could be a potential drug for the treatment of RILI.

Alleviating CB2-Dependent ER Stress and Mitochondrial Dysfunction Improves Chronic Cerebral Hypoperfusion-Induced Cognitive Impairment

Augmentation of endoplasmic reticulum (ER) stress may trigger excessive oxidative stress, which induces mitochondrial dysfunction. The fatty acid amide hydrolase inhibitor, URB597, shows anti-oxidation characteristics in multiple neurological disorders. The present study aimed to determine whether inhibition of ER stress was involved in the protective effects of URB597 against chronic cerebral hypoperfusion (CCH)-induced cognitive impairment. Hippocampal HT-22 cells were exposed to oxygen-glucose deprivation. The cell viability, apoptosis, ER stress, mitochondrial ATP, and oxidative stress levels were assessed following treatment with URB597, benzenebutyric acid (4-PBA), and thapsigargin (TG). Furthermore, the effects of URB597 on ER stress and related pathways were investigated in the CCH animal model, including Morris water maze testing of cognition, western blotting analysis of ER stress signaling, and transmission electron microscopy of mitochondrial and ER ultrastructure changes. The results suggested that cerebral ischemia caused ER stress with upregulation of ER stress signaling-related proteins, mitochondrial dysfunction, neuronal apoptosis, ultrastructural injuries of mitochondria-associated ER membranes, and cognitive decline. Co-immunoprecipitation experiments confirmed the interaction between CB2 and β-Arrestin1. Inhibiting ER stress by URB597 improved these changes by activating CB2/β-Arrestin1 signaling, which was reversed by the CB2 antagonist, AM630. Together, the results identified a novel mechanism of URB597, involving CCH-induced cognitive impairment alleviation of CB2-dependent ER stress and mitochondrial dysfunction. Furthermore, this study identified CB2 as a potential target for therapy of ischemic cerebrovascular diseases.Graphical

Role of mitochondria in the regulation of ferroptosis and disease.

Ferroptosis is a distinctive form of iron-dependent cell death characterized by significant ultrastructural changes in mitochondria. Given the crucial involvement of mitochondria in various cellular processes such as reactive oxygen species production, energy metabolism, redox status, and iron metabolism, mounting evidence suggests a vital role of mitochondria in the regulation and execution of ferroptosis. Furthermore, there exists a strong correlation between ferroptosis and various diseases. In this review, we aim to summarize the mechanisms underlying the induction and defense of ferroptosis, emphasizing the influence of mitochondria on this intricate process. Additionally, we provide an overview of the role of ferroptosis in disease, particularly cancer, and elucidate the mechanisms by which drugs targeting mitochondria impact ferroptosis. By presenting a theoretical foundation and reference point, this review aims to contribute to both basic cell biology research and the investigation of clinically relevant diseases.

Ataxia Syndrome With Hearing Loss and Nephronophthisis Associated With a Novel Homozygous Variant in XPNPEP3.

Biallelic variants in XPNPEP3 are associated with a rare mitochondrial syndrome characterized by nephronophthisis leading to kidney failure, essential tremor, hearing loss, seizures, and intellectual disability. Only 2 publications on this condition are available. We report a man with a complex ataxia syndrome, hearing loss, and kidney failure associated with a new biallelic variant in XPNPEP3. Clinical evaluation, neuroimaging studies, a kidney biopsy, and whole genome sequencing (WGS) were applied. Since the phenotype was compatible with a mitochondrial disease, a muscle biopsy with morphological and mitochondrial biochemical investigations was performed. Axial ataxia, cerebellar atrophy, hearing loss, myopathy, ptosis, supranuclear palsy, and kidney failure because of nephronophthisis were the prominent features in this case. WGS revealed the novel biallelic variant c.766C>T (p.Gln256*) in XPNPEP3. A muscle biopsy revealed COX negative fibers, a few ragged red fibers, and ultrastructural mitochondrial changes. Enzyme activity in respiratory chain complex IV was reduced in muscle and fibroblasts. This is the first report of a slowly progressive cerebellar ataxia associated with a novel biallelic variant in XPNPEP3. Abnormalities typical for mitochondrial disease and the slow progression of kidney disease are also striking. Our report expands the spectrum of XPNPEP3-related diseases.

Microglia/macrophages are ultrastructurally altered by their proximity to spinal cord injury in adult female mice

Traumatic spinal cord injury can cause immediate physical damage to the spinal cord and result in severe neurological deficits. The primary, mechanical tissue damage triggers a variety of secondary damage mechanisms at the injury site which significantly contribute to a larger lesion size and increased functional damage. Inflammatory mechanisms which directly involve both microglia (MG) and monocyte-derived macrophages (MDM) play important roles in the post-injury processes, including inflammation and debris clearing. In the current study, we investigated changes in the structure and function of MG/MDM in the injured spinal cord of adult female mice, 7 days after a thoracic contusion SCI. With the use of chip mapping scanning electron microscopy, which allows to image large samples at the nanoscale, we performed an ultrastructural comparison of MG/MDM located near the lesion vs adjacent regions to provide novel insights into the mechanisms at play post-injury. We found that MG/MDM located near the lesion had more mitochondria overall, including mitochondria with and without morphological alterations, and had a higher proportion of altered mitochondria. MG/MDM near the lesion also showed an increased number of phagosomes, including phagosomes containing myelin and partiallydigested materials. MG/MDM near the injury interacted differently with the spinal cord parenchyma, as shown by their reduced number of direct contacts with synaptic elements, axon terminals and dendritic spines. In this study, we characterized the ultrastructural changes of MG/MDM in response to spinal cord tissue damage in mice, uncovering changes in phagocytic activity, mitochondrial ultrastructure, and inter-cellular interactions within the spinal cord parenchyma.

Mitochondrial Dysfunction in Dopaminergic Neurons Derived from Patients with LRRK2- and SNCA-Associated Genetic Forms of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Some cases of PD may be caused by genetic factors, among which mutations in the LRRK2 and SNCA genes play an important role. To develop effective neuroprotective strategies for PD, it is important to diagnose the disease at the earliest stages of the neurodegenerative process. Therefore, the detection of diagnostic and prognostic markers of Parkinson's disease (PD) is an urgent medical need. Advances in induced pluripotent stem cell (iPSC) culture technology provide new opportunities for the search for new biomarkers of PD and its modeling in vitro. In our work, we used a new technology for multiplex profiling of gene expression using barcoding on the Nanostring platform to assess the activity of mitochondrial genes on iPSC-derived cultures of dopaminergic neurons obtained from patients with LRRK2- and SNCA-associated genetic forms PD and a healthy donor. Electron microscopy revealed ultrastructural changes in mitochondria in both LRRK2 and SNCA mutant cells, whereas mitochondria in cells from a healthy donor were normal. In a culture with the SNCA gene mutation, the ratio of the area occupied by mitochondria to the total area of the cytoplasm was significantly lower than in the control and in the line with the LRRK2 gene mutation. Transcriptome analysis of 105 mitochondria proteome genes using the Nanostring platform revealed differences between the diseased and normal cells in the activity of genes involved in respiratory complex function, the tricarboxylic acid cycle, ATP production, mitochondria-endoplasmic reticulum interaction, mitophagy, regulation of calcium concentration, and mitochondrial DNA replication.

A first-in-class dimethyl 2-acetamido terephthalate inhibitor targeting Conyza canadensis SHMT1 with a novel herbicidal mode-of-action

IntroductionHerbicide application is a highly efficiency method of weed control that boots agricultural output and assures food security. The development of novel herbicides focuses on improved bioactivity and new modes of action. The amino acid biosynthesis was validated as a promising novel mode of action for herbicidal compounds. However, the amino acid biosynthesis enzyme remains largely unexplored for herbicidal targets. ObjectivesSerine hydroxymethyl transferase (SHMT) is an essentialenzyme in the photorespiratory cycle. The study aims to explore Conyza canadensis SHMT1 (CcSHMT1) as a promising target for herbicide discovery. MethodsStructure determination of CcSHMT1 was resolved by X-ray crystallography. Virtual screening docking experiments were performed with Glide version 5.5. Novel derivatives of dimethyl 2-acetamido terephthalate were further designed, synthesized, and bioassay. The druggability of the inhibitor was evidenced by ultrastructural changes in mitochondria, in vivo and vitro enzyme activity assays, and genetics analysis. ResultsCcSHMT1 has a typical PLP-dependent enzyme 3D structure. The dimethyl 2-acetamido terephthalate-containing compounds had herbicidal activity. Dimethyl 2-(2-(4-(2-(4-bromo-2-chlorophenoxy) acetyl)piperazin-1-yl)acetamido) terephthalate (Compound 9ay, EC50 = 193.8 g a.i./ ha) exhibited the highest herbicidal activity on tested weed among the synthesized compounds. Compound 9ay had no obvious adverse effect on the growth of maize and honeybees. Compound 9ay was verified to target CcSHMT1 as an herbicide candidate. ConclusionA first-in-class CcSHMT1 inhibitor that could be developed as a potent herbicide with a new mode of action and provide an avenue for discovering novel inhibitors of pyridoxal-5-phosphate-dependent enzymes.

Ultrastructural Changes in Hippocampal Region CA1 Neurons After Exposure to Permissive Hypercapnia and/or Normobaric Hypoxia.

Isolated exposure to intermittent hypoxia and permissive hypercapnia activates signaling mechanisms that induce ultrastructural changes in mitochondria and endoplasmic reticulum, accompanied by the development of maximal ischemic tolerance in neurons under the combined influence of these factors. However, there are a lack of data on the combined impact of these factors on the ultrastructure of neuronal organelles. The present study aims to comparatively assess the ultrastructural changes in neurons following isolated and combined exposure to hypoxia and hypercapnia, as well as to correlate these changes with the neuroprotective potential previously observed for these factors. Following a 15-session course of 30-min exposures to permissive hypercapnia (PCO2≈50mmHg) and/or normobaric hypoxia (PO2≈150mmHg), morphometric assessment was conducted to evaluate the extent of ultrastructural changes in hippocampal neurons (mitochondria, perinuclear space, and granular endoplasmic reticulum). It was found that in hippocampal neurons from the CA1 region, permissive hypercapnia resulted in increased mitochondrial size, expansion of membranous compartments of the granular endoplasmic reticulum, and perinuclear space. Normobaric hypoxia affected only mitochondrial size, while hypercapnic hypoxia specifically widened the perinuclear space. These ultrastructural changes objectively reflect varying degrees of the influence of hypoxia and hypercapnia on organelles responsible for energy metabolism, anti-apoptotic, and synthetic functions of neurons. This confirms the effect of potentiation of their neuroprotective effects under combined exposure and highlights the dominant role of the hypercapnic component in this mechanism.

Ultrastructure of Myofascial Trigger Points of Skeletal Muscles of Rats after Photobiostimulation with Low-Intensity Red Light.

The effectiveness of low-intensity red light on myofascial trigger points in skeletal muscle of mature rats was evaluated by electron microscopy and high-resolution respirometry. The revealed changes in mitochondrial ultrastructure and activity of the respiratory chain enzymes indicate the development of hypoxia in the simulation area. Under the influence of low-intensity red light on myofascial trigger points, a decrease in the number of destructively altered muscle fibers and stimulation of mitochondrial respiration were found. These findings indicate intracellular regeneration and the stimulating effect of low-intensity red light on plastic processes.

Assessment of the neurotoxic effect of iron (III) oxide nanoparticles at the subcellular level

Introduction. Both naturally occurring and artificially produced nanoparticles are ubiquitous; their high concentrations can be detected in the environment, thus posing risks of toxic effects in humans. Penetrating the blood-brain barrier by metal nanoparticles has been already proven and is currently of interest from the point of view of toxicology and hygiene.
 Materials and methods. Female rats were exposed to ferric oxide nanoparticles administered intranasally with a 25 mg/ml suspension at a dose of 50 µl three times a week during six weeks. The experimental and control groups contained seven animals each. Tissue samples for testing were taken from the olfactory bulbs of the rat’s brain. Iron (III) oxide nanoparticles were identified by electron microscopy and energy-dispersive X-ray spectroscopy. The cytotoxic effect of ferric oxide nanoparticles was assessed by ranking mitochondria by mitochondrial membrane morphotypes and comparing their distribution in the experimental and control groups.
 Results. We confirmed the presence of nanoparticles in tissues of the olfactory bulbs of the exposed rodents. The morphotype pattern of mitochondria showed significant changes following the exposure to ferric oxide nanoparticles: the proportion of mitochondria with normal and vesicular swollen morphotypes decreased by 36.4 and 4.9%, respectively, compared with the control group of animals, the proportion of mitochondria with normal vesicular and vesicular morphotypes increased by 19.8 and 21.8%, while the proportion of vesicular swollen mitochondria decreased from 9.5% to 4.6%.
 Limitations. The study was limited to examining ultrastructural changes in mitochondria and identifying ferric oxide nanoparticles in tissues.
 Conclusions. Further studies of the impact of iron-containing nanoparticles on the structure and functions of the mitochondrial apparatus can help to identify their potential harm at the subcellular level and provide information for the development of appropriate health protective measures and new strategies for prevention and treatment of metal toxicity-induced diseases in humans.

Acid ground nano-realgar processed product inhibits breast cancer by inducing mitophagy via the p53/BNIP3/NIX pathway.

Breast cancer is a highly prevalent malignancy with the first morbidity and the primary reason for female cancer-related deaths worldwide. Acid ground nano-realgar processed product (NRPP) could inhibit breast cancer cell proliferation and induce autophagy in our previous research; however, the underlying mechanisms are still unclear. Therefore, this research aimed to verify whether NRPP induces breast cancer mitophagy and explore the mitophagy-mediated mechanism. Primarily, rhodamine-123 assay and transmission electron microscopy were applied to detect mitochondrial membrane potential (MMP) and ultrastructural changes in the MDA-MB-435S cells, respectively. Mito-Tracker Green/Lyso-Tracker Red staining, western blot, immunofluorescence and RT-PCR were used to explore molecular mechanisms of NRPP-induced mitophagy in vitro. MDA-MB-435S breast cancer xenograft models were established to assess the activity and mechanisms of NRPP in vivo. Our results showed that NRPP decreased MMP and increased autophagosome numbers in MDA-MB-435S cells and activated mitophagy. Furthermore, mitophagy was consolidated because mitochondria and lysosomes colocalized phenomenology were observed, and the expression of LC3II/I and COXIV was upregulated. Additionally, we found the p53/BNIP3/NIX pathway was activated. Finally, NRPP inhibited tumour growth and downregulated the levels of TNF-α, IL-1β and IL-6. Necrosis, damaged mitochondria and autophagosomes were observed in xenograft tumour cells, and proteins and mRNA levels of LC3, p53, BNIP3 and NIX were increased. Overall, NRPP inhibited MDA-MB-435S cell proliferation and tumour growth by inducing mitophagy via the p53/BNIP3/NIX pathway. Thus, NRPP is a promising candidate for breast cancer treatment.

A Three-Dimensional Technique for The Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

#4181 THE AUTOSOMAL RECESSIVE POLYCYSTIC KIDNEY DISEASE PROTEIN, FPC, RELEASES MITOCHONDRIA LOCALISING FRAGMENTS INVOLVED IN PREVENTING CYSTOGENESIS

Abstract Background and Aims Autosomal recessive polycystic kidney disease is caused by mutations in PKHD1 encoding FPC, and is characterized by severe renal cystogenesis in neonates, yet mouse models do not fully recapitulate the human phenotype. Indeed, even the Pkhd1 null allele does not cause renal cystogenesis in the mouse. Several cleavage products of FPC are reported yet their function remains unknown. The aim of this study was to determine the function of the FPC cleavage products and their effects on cyst development in ARPKD. Method Three Pkhd1 mutant mouse lines and the cystic Pkd1V mouse were crossed to produce digenic mice with which to study renal cystogenesis. Biochemical analysis was used to investigate FPC cleavage patterns using a panel of new antibodies. Cell models and electron microscopy revealed underlying mitochondrial defects in Pkhd1 Knockout mice. Results Pkhd1 mutation modifies a Pkd1 uncleavable mutant (Pkd1V), enhancing the cystic phenotype in both the kidney and pancreas. The hypomorphic Pkhd1 mutant and Pkhd1 KO both enhance the Pkd1V kidney phenotype, making distal tubule cysts more severe and initiating cystogenesis in the proximal tubules. FPC displays differential cleavage to produce fragments of unknown function. New antibodies were generated to interrogate FPC cleavage products. Three small C terminal cleavage fragments were identified which contain a mitochondrial targeting sequence and are recruited to mitochondria. Mitochondrial ultrastructural changes were evident after deletion of Pkhd1 including mitochondrial fragmentation and dilated cristae, suggesting disrupted mitochondrial function. Deletion of just the C-terminal fragment of FPC (ΔCT), the portion that directly corresponds to the portion that cleaves and localises to the mitochondria, is sufficient to enhance the renal cystic phenotype of the PC1 cleavage mutant. Unlike the other Pkhd1 mutants however, FPC (ΔCT) does not result in the pancreatic cystogenesis when combined with Pkd1V, suggesting that the FPC C terminus in not required to prevent pancreatic cyst development. Conclusion Our results suggest that the C-terminus of FPC plays an important role in preventing renal cystogenesis via a newly discovered mitochondria specific function. Our work reveal some aspects of FPC's function, in particular a previously unrecognised mitochondria function that is mediated through FPC cleavage products and is essential in the kidney to prevent the enhancement of cystogenesis in the digenic model.

Characterization of Mitochondria Degeneration in Spinal Motor Neurons Triggered by Chronic Over-activation of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Receptors in the Rat Spinal Cord in Vivo

Mitochondrial damage is a central mechanism involved in neurological disorders as Alzheimer’s, and Parkinson’s diseases and amyotrophic lateral sclerosis. Energy production is the most studied mitochondrial function; however, mitochondria are also involved in processes like calcium buffering homeostasis, and cell death control during apoptosis and necrosis. Using transmission electron microscopy, in this in vivo study in male rats, we describe ultrastructural mitochondrial alterations of spinal motor neurons along chronic AMPA-induced excitotoxicity, which has been described as one of the most relevant mechanisms in ALS disease. Mitochondrial alterations begin with a crest swelling, which progresses to a full mitochondrial swelling and crest disruption. Changes on the mitochondrial morphology from elongated to a circular shape also occur along the AMPA-excitotoxicity process. In addition, by combining the TUNEL assay and immunohistochemistry for mitochondrial enzymes, we show evidence of mitochondrial DNA damage. Evidence of mitochondrial alterations during an AMPA-excitotoxic event is relevant because resembles the mitochondrial alterations previously reported in ALS patients and in transgenic familial ALS models, suggesting that a chronic excitotoxic model can be related to sporadic ALS (as has been shown in recent papers), which represent more than the 90% of the ALS cases. Understanding the mechanisms involved in motor neuron degenerative process, such as the ultrastructural mitochondrial changes permits to design strategies for MN-degeneration treatments in ALS.

Astrocytes derived from neural progenitor cells are susceptible to Zika virus infection.

Zika virus (ZIKV) was first isolated in 1947. From its isolation until 2007, symptoms of ZIKV-caused disease were limited (e.g., fever, hives, and headache); however, during the epidemic in Brazil in 2014, ZIKV infection caused Guillain-Barré syndrome in adults and microcephaly in fetuses and infants of women infected during pregnancy. The neurovirulence of ZIKV has been studied using neural progenitor cells (NPCs), brain organoids, neurons, and astrocytes. NPCs and astrocytes appear to be the most susceptible cells of the Central Nervous System to ZIKV infection. In this work, we aimed to develop a culture of astrocytes derived from a human NPC cell line. We analyze how ZIKV affects human astrocytes and demonstrate that 1) ZIKV infection reduces cell viability, increases the production of Reactive Oxygen Species (ROS), and results in high viral titers; 2) there are changes in the expression of genes that facilitate the entry of the virus into the cells; 3) there are changes in the expression of genes involved in the homeostasis of the glutamatergic system; and 4) there are ultrastructural changes in mitochondria and lipid droplets associated with production of virions. Our findings reveal new evidence of how ZIKV compromises astrocytic functionality, which may help understand the pathophysiology of ZIKV-associated congenital disease.

The Role of Pyruvate Metabolism in Mitochondrial Quality Control and Inflammation.

Pyruvate metabolism, a key pathway in glycolysis and oxidative phosphorylation, is crucial for energy homeostasis and mitochondrial quality control (MQC), including fusion/fission dynamics and mitophagy. Alterations in pyruvate flux and MQC are associated with reactive oxygen species accumulation and Ca2+ flux into the mitochondria, which can induce mitochondrial ultrastructural changes, mitochondrial dysfunction and metabolic dysregulation. Perturbations in MQC are emerging as a central mechanism for the pathogenesis of various metabolic diseases, such as neurodegenerative diseases, diabetes and insulin resistance-related diseases. Mitochondrial Ca2+ regulates the pyruvate dehydrogenase complex (PDC), which is central to pyruvate metabolism, by promoting its dephosphorylation. Increase of pyruvate dehydrogenase kinase (PDK) is associated with perturbation of mitochondria-associated membranes (MAMs) function and Ca2+ flux. Pyruvate metabolism also plays an important role in immune cell activation and function, dysregulation of which also leads to insulin resistance and inflammatory disease. Pyruvate metabolism affects macrophage polarization, mitochondrial dynamics and MAM formation, which are critical in determining macrophage function and immune response. MAMs and MQCs have also been intensively studied in macrophage and T cell immunity. Metabolic reprogramming connected with pyruvate metabolism, mitochondrial dynamics and MAM formation are important to macrophages polarization (M1/M2) and function. T cell differentiation is also directly linked to pyruvate metabolism, with inhibition of pyruvate oxidation by PDKs promoting proinflammatory T cell polarization. This article provides a brief review on the emerging role of pyruvate metabolism in MQC and MAM function, and how dysfunction in these processes leads to metabolic and inflammatory diseases.

Flavonoids from Hippophae rhamnoides Linn. Revert Doxorubicin-Induced Cardiotoxicity through Inhibition of Mitochondrial Dysfunction in H9c2 Cardiomyoblasts In Vitro

Doxorubicin (Dox) is one of the most frequently prescribed anti-cancer drugs. However, treatment with Dox is limited due to cumulative cardiotoxicity. 3-O-β-d-Sophorosylkaempferol-7-O-{3-O-[2(E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoyl]}-α-L-rhamnoside (F-A), kaempferol 3-sophoroside 7-rhamnoside (F-B), and hippophanone (F-C) were successfully obtained by purification and separation of seabuckthorn seed residue in our previous research. This study was undertaken to investigate the protective effect of three flavonoids against Dox-induced H9c2 cell apoptosis. Cell proliferation was detected by MTT assay. 2',7'-Dichlorofluorescein diacetate (DCFH-DA) was used to determine the production of intracellular reactive oxygen species (ROS). ATP content was measured using an assay kit. Transmission electron microscopy (TEM) was used to observe changes in mitochondrial ultrastructure. The expression levels of proteins (p-JNK, JNK, p-Akt, Akt, p-P38, P38, p-ERK, ERK, p-Src, Src, Sab, IRE1α, Mfn1, Mfn2, and cleaved caspase-3) were evaluated by Western blot. Molecular docking was performed using AutoDock Vina. The three flavonoids could significantly relieve Dox-induced cardiac injury and inhibit cardiomyocyte apoptosis. The mechanisms were mainly related to the stability of mitochondrial structure and function maintained by suppressing the production of intracellular ROS, p-JNK and cleaved caspase-3, and increasing ATP contents and protein expression of mitochondrial mitofusin (Mfn1, Mfn2), Sab and p-Src. Pretreatment with flavonoids from Hippophae rhamnoides Linn. can reduce Dox-induced H9c2 cell apoptosis based on the 'JNK-Sab-Ros' signal pathway.

Ryanodine receptor hyperactivity promotes mitochondrial dysfunction via calpain-dependent OPA1 proteolysis.

Hereditary RyR2 gain-of function mutations are linked to the highly malignant arrhythmia syndrome, catecholaminergic polymorphic tachycardia (CPVT). A mutation-mediated increase in RyR2 activity not only results in unstable sarcoplasmic reticulum Ca2+ release, but also alters mitochondrial structure and function, leading to excessive emission of damaging reactive oxygen species (ROS) by the organelle. However, the exact mechanisms underlying RyR2 hyperactivity-dependent mitochondrial damage remain elusive. We hypothesized that RyR2 hyperactivity activates the intermembrane space (IMS)-residing mitochondrial Ca2+-dependent protease calpain, driving proteolysis of OPA1, a protein responsible for tight cristae arrangement. Concomitant changes in cristae architecture promotes mito-ROS production, thereby aggravating the CPVT phenotype. To test this, we generated a unique CPVT RyR2-S2222L(+/-) rat model. Electron microscopy demonstrated significantly increased mitochondrial cristae diameter in tissue slices from CPVT rat hearts. Biochemical analysis showed reduced OPA1 expression in isolated CPVT myocytes, suggesting that mitochondrial ultrastructural changes in CPVT could be driven by Ca2+-dependent proteolysis of OPA1 by calpain. Importantly, specific inhibition of calpain via adeno-associated virus mediated expression of IMS-targeted calpastatin reduced the incidence of ventricular tachycardia in CPVT rats. At the cellular level, adenoviral expression of IMS-CAST restored OPA1 expression levels and significantly improved intracellular Ca2+ homeostasis in CPVT, indicative of normalized RyR2 function. Furthermore, IMS-calpain inhibition attenuated the reduction in mitochondrial matrix Ca2+ and increase in mito-ROS emission observed in CPVT myocytes using genetic probes mtRCamp1h and MLS-HyPer7. All protective effects were lost in myocytes with additional shRNA-mediated knockdown of OPA1. We conclude that RyR2 hyperactivity promotes mitochondrial structural damage via IMS-calpain-mediated degradation of OPA1 and contributes to proarrhythmic remodeling in CPVT.

Heme oxygenase-1 prevents non-alcoholic steatohepatitis through modulating mitochondrial quality control.

Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease (NAFLD) and lacks effective treatment options. Heme oxygenase-1 (HO-1) is a critical defense against oxidative stress and inflammation in the liver injury. This study aims to investigate the protective role and underlying mechanisms of HO-1 in NASH pathogenesis. The hepatocyte-specific HO-1 knockout (HO-1HEPKO ) mice on a C57BL/6J background (HO-1fl/fl /Alb-Cre) were generated and fed a high-fat/western-style diet (HFD) or methionine-choline-deficient diet (MCD). Changes in mitochondrial ultrastructure were observed by transmission electron microscopy and confocal microscopy. A mitochondrial PCR array was used to identify the crucial genes associated with mitochondrial dysfunction. Hepatocyte-specific HO-1HEPKO mice developed steatohepatitis with severe steatosis, ballooning, and necroinflammation. Dysregulated hepatic expression of mitochondria-related proteins, including DRP1, Tomm20, MFN1 and MFN2 were detected in NASH animals. Ultrastructural mitochondrial damage was observed in HO-1HEPKO mice. Mitochondrial dysfunction was recapitulated in HO-1-knockdown cells in vitro, as evidenced by decreased membrane potential, reduced ATP content, and mtDNA damage. Conversely, HO-1 overexpression restored these changes in vitro. Mechanistically, HO-1 deficiency reduced the inhibitory effect on Tomm20, leading to mitochondrial dysfunction, and thereby causing steatohepatitis. HO-1 attenuates diet-induced steatohepatitis by preventing mitochondrial dysfunction, indicating that HO-1 may constitute a potential therapeutic target for NASH.