Mitochondrial Network Formation Research Articles

Developmental dynamics of mitochondrial fission and fusion proteins in functionally divergent skeletal muscles of goat.

During skeletal muscle development, the intricate mitochondrial network formation relies on continuous fission and fusion. This process in larger mammals differs from rodents, the most used animal models. However, the expression pattern of proteins regulating mitochondrial dynamics in developing skeletal muscle remains unexplored in larger mammals. Therefore, we characterized the cellular expression and tissue-level distribution of these proteins during development taking goat as a model. We have performed histological and immunohistochemical analyses to study metabolic features in various muscles. Neonatal muscles display uniform distribution of mitochondrial activity. In contrast, adult muscles exhibit clear distinctions based on their function, whether dedicated for posture maintenance or facilitating locomotion. Mitochondrial fission proteins like DRP-1, MFF, and fusion proteins like MFN-1 and 2 are abundantly expressed in neonatal muscles. Fission proteins exhibit drastic downregulation with limited peripheral expression, whereas fusion proteins continue to express in a fiber-specific manner during adulthood. Locomotory muscles exhibit different fibers based on mitochondrial activity and peripheralization with high SDH activity. The proximity ligation assay between MFN1 and MFN2 demonstrates that their interaction is restricted to subsarcolemmal mitochondria in adult fibers while distributed evenly in neonatal fibers. These differences between postural and locomotory muscles suggest their physiological and metabolic properties are different.

Analysis of the Mouse Hepatic Peroxisome Proteome-Identification of Novel Protein Constituents Using a Semi-Quantitative SWATH-MS Approach.

Ongoing technical and bioinformatics improvements in mass spectrometry (MS) allow for the identifying and quantifying of the enrichment of increasingly less-abundant proteins in individual fractions. Accordingly, this study reassessed the proteome of mouse liver peroxisomes by the parallel isolation of peroxisomes from a mitochondria- and a microsome-enriched prefraction, combining density-gradient centrifugation with a semi-quantitative SWATH-MS proteomics approach to unveil novel peroxisomal or peroxisome-associated proteins. In total, 1071 proteins were identified using MS and assessed in terms of their distribution in either high-density peroxisomal or low-density gradient fractions, containing the bulk of organelle material. Combining the data from both fractionation approaches allowed for the identification of specific protein profiles characteristic of mitochondria, the ER and peroxisomes. Among the proteins significantly enriched in the peroxisomal cluster were several novel peroxisomal candidates. Five of those were validated by colocalization in peroxisomes, using confocal microscopy. The peroxisomal import of HTATIP2 and PAFAH2, which contain a peroxisome-targeting sequence 1 (PTS1), could be confirmed by overexpression in HepG2 cells. The candidates SAR1B and PDCD6, which are known ER-exit-site proteins, did not directly colocalize with peroxisomes, but resided at ER sites, which frequently surrounded peroxisomes. Hence, both proteins might concentrate at presumably co-purified peroxisome-ER membrane contacts. Intriguingly, the fifth candidate, OCIA domain-containing protein 1, was previously described as decreasing mitochondrial network formation. In this work, we confirmed its peroxisomal localization and further observed a reduction in peroxisome numbers in response to OCIAD1 overexpression. Hence, OCIAD1 appears to be a novel protein, which has an impact on both mitochondrial and peroxisomal maintenance.

In vitro and in vivo studies on the effect of a mitochondrial fusion promoter on Leydig cell integrity and function.

Background: The interstitial testicular Leydig cells are responsible for the production of testosterone, which functionally deteriorate with normal aging. Decreased expression of mitochondrial steroidogenic interactome proteins and diminished mitochondrial function in aging Leydig cells suggest that mitochondrial dynamics play a role in maintaining adequate levels of testosterone. Optic atrophy 1 (OPA1) protein regulates mitochondrial dynamics and cristae formation in many cell types. Previous studies showed that increasing OPA1 expression in dysfunctional Leydig cells restored mitochondrial function and recovered androgen production to levels found in healthy Leydig cells. These findings suggested that mitochondrial dynamics may be a promising target to ameliorate diminished testosterone levels in aging males. Methods: We used twelve-month-old rats to explore the relationship between mitochondrial dynamics and Leydig cell function. Isolated Leydig cells from aged rats were treated ex vivo with the cell-permeable mitochondrial fusion promoter 4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl)hydrazono)ethyl) phenol (mitochondrial fusion promoter M1), which enhances mitochondrial tubular network formation. In parallel, rats were treated with 2mg/kg/day M1 for 6weeks before Leydig cells were isolated. Results: Ex vivo M1-treated cells showed enhanced mitochondrial tubular network formation by transmission electron microscopy, enhanced Leydig cell mitochondrial integrity, improved mitochondrial function, and higher testosterone biosynthesis compared to controls. However, in vivo treatment of aged rats with M1 not only failed to re-establish testosterone levels to that of young rats, it also led to further reduction of testosterone levels and increased apoptosis, suggesting M1 toxicity in the testis. The in vivo M1 toxicity seemed to be tissue-specific, however. Conclusion: Promoting mitochondrial fusion may be one approach to enhancing cell health and wellbeing with aging, but more investigations are warranted. Our findings suggest that fusion promoters could potentially enhance the productivity of aged Leydig cells when carefully regulated.

M-type pyruvate kinase 2 (PKM2) tetramerization alleviates the progression of right ventricle failure by regulating oxidative stress and mitochondrial dynamics

BackgroundRight ventricle failure (RVF) is a progressive heart disease that has yet to be fully understood at the molecular level. Elevated M-type pyruvate kinase 2 (PKM2) tetramerization alleviates heart failure, but detailed molecular mechanisms remain unclear.ObjectiveWe observed changes in PKM2 tetramerization levels during the progression of right heart failure and in vitro cardiomyocyte hypertrophy and explored the causal relationship between altered PKM2 tetramerization and the imbalance of redox homeostasis in cardiomyocytes, as well as its underlying mechanisms. Ultimately, our goal was to propose rational intervention strategies for the treatment of RVF.MethodWe established RVF in Sprague Dawley (SD) rats by intraperitoneal injection of monocrotaline (MCT). The pulmonary artery pressure and right heart function of rats were assessed using transthoracic echocardiography combined with right heart catheterization. TEPP-46 was used both in vivo and in vitro to promote PKM2 tetramerization.ResultsWe observed that oxidative stress and mitochondrial disorganization were associated with increased apoptosis in the right ventricular tissue of RVF rats. Quantitative proteomics revealed that PKM2 was upregulated during RVF and negatively correlated with the cardiac function. Facilitating PKM2 tetramerization promoted mitochondrial network formation and alleviated oxidative stress and apoptosis during cardiomyocyte hypertrophy. Moreover, enhancing PKM2 tetramer formation improved cardiac mitochondrial morphology, mitigated oxidative stress and alleviated heart failure.ConclusionDisruption of PKM2 tetramerization contributed to RVF by inducing mitochondrial fragmentation, accumulating ROS, and finally promoted the progression of cardiomyocyte apoptosis. Facilitating PKM2 tetramerization holds potential as a promising therapeutic approach for RVF.

SIRT1 inhibits mitochondrial hyperfusion associated mito-bulb formation to sensitize oral cancer cells for apoptosis in a mtROS-dependent signalling pathway

SIRT1 (NAD-dependent protein deacetylase sirtuin-1), a class III histone deacetylase acting as a tumor suppressor gene, is downregulated in oral cancer cells. Non-apoptotic doses of cisplatin (CDDP) downregulate SIRT1 expression advocating the mechanism of drug resistance. SIRT1 downregulation orchestrates inhibition of DNM1L-mediated mitochondrial fission, subsequently leading to the formation of hyperfused mitochondrial networks. The hyperfused mitochondrial networks preserve the release of cytochrome C (CYCS) by stabilizing the mitochondrial inner membrane cristae (formation of mitochondrial nucleoid clustering mimicking mito-bulb like structures) and reducing the generation of mitochondrial superoxide to inhibit apoptosis. Overexpression of SIRT1 reverses the mitochondrial hyperfusion by initiating DNM1L-regulated mitochondrial fission. In the overexpressed cells, inhibition of mitochondrial hyperfusion and nucleoid clustering (mito-bulbs) facilitates the cytoplasmic release of CYCS along with an enhanced generation of mitochondrial superoxide for the subsequent induction of apoptosis. Further, low-dose priming with gallic acid (GA), a bio-active SIRT1 activator, nullifies CDDP-mediated apoptosis inhibition by suppressing mitochondrial hyperfusion. In this setting, SIRT1 knockdown hinders apoptosis activation in GA-primed oral cancer cells. Similarly, SIRT1 overexpression in the CDDP resistance oral cancer-derived polyploid giant cancer cells (PGCCs) re-sensitizes the cells to apoptosis. Interestingly, synergistically treated with CDDP, GA induces apoptosis in the PGCCs by inhibiting mitochondrial hyperfusion.

PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics

Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.

Exercise and mitochondrial mechanisms in patients with sarcopenia.

Sarcopenia is a severe loss of muscle mass and functional decline during aging that can lead to reduced quality of life, limited patient independence, and increased risk of falls. The causes of sarcopenia include inactivity, oxidant production, reduction of antioxidant defense, disruption of mitochondrial activity, disruption of mitophagy, and change in mitochondrial biogenesis. There is evidence that mitochondrial dysfunction is an important cause of sarcopenia. Oxidative stress and reduction of antioxidant defenses in mitochondria form a vicious cycle that leads to the intensification of mitochondrial separation, suppression of mitochondrial fusion/fission, inhibition of electron transport chain, reduction of ATP production, an increase of mitochondrial DNA damage, and mitochondrial biogenesis disorder. On the other hand, exercise adds to the healthy mitochondrial network by increasing markers of mitochondrial fusion and fission, and transforms defective mitochondria into efficient mitochondria. Sarcopenia also leads to a decrease in mitochondrial dynamics, mitophagy markers, and mitochondrial network efficiency by increasing the level of ROS and apoptosis. In contrast, exercise increases mitochondrial biogenesis by activating genes affected by PGC1-ɑ (such as CaMK, AMPK, MAPKs) and altering cellular calcium, ATP-AMP ratio, and cellular stress. Activation of PGC1-ɑ also regulates transcription factors (such as TFAM, MEFs, and NRFs) and leads to the formation of new mitochondrial networks. Hence, moderate-intensity exercise can be used as a non-invasive treatment for sarcopenia by activating pathways that regulate the mitochondrial network in skeletal muscle.

Evidence of a role for cAMP in mitochondrial regulation in ovarian granulosa cells.

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes.

Cultivation of Cells in a Physiological Plasmax Medium Increases Mitochondrial Respiratory Capacity and Reduces Replication Levels of RNA Viruses.

Changes in metabolic pathways are often associated with the development of various pathologies including cancer, inflammatory diseases, obesity and metabolic syndrome. Identification of the particular metabolic events that are dysregulated may yield strategies for pharmacologic intervention. However, such studies are hampered by the use of classic cell media that do not reflect the metabolite composition that exists in blood plasma and which cause non-physiological adaptations in cultured cells. In recent years two groups presented media that aim to reflect the composition of human plasma, namely human plasma-like medium (HPLM) and Plasmax. Here we describe that, in four different mammalian cell lines, Plasmax enhances mitochondrial respiration. This is associated with the formation of vast mitochondrial networks and enhanced production of reactive oxygen species (ROS). Interestingly, cells cultivated in Plasmax displayed significantly less lysosomes than when any standard media were used. Finally, cells cultivated in Plasmax support replication of various RNA viruses, such as hepatitis C virus (HCV) influenza A virus (IAV), severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and several others, albeit at lower levels and with delayed kinetics. In conclusion, studies of metabolism in the context of viral infections, especially those concerning mitochondria, lysosomes, or redox systems, should be performed in Plasmax medium.

A mixture of persistent organic pollutants detected in human follicular fluid increases progesterone secretion and mitochondrial activity in human granulosa HGrC1 cells

Disruption of granulosa cells (GCs), the main functional cells in the ovary, is associated with impaired female fertility. Epidemiological studies demonstrated that women have detectable levels of organic pollutants (e.g., perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, polychlorinated biphenyl 153, and hexachlorobenzene) in their follicular fluid (FF), and thus these compounds may directly affect the function of GCs in the ovary. Considering that humans are exposed to multiple pollutants simultaneously, we elucidated the effects of a mixture of endocrine-disrupting chemicals (EDCs) on human granulosa HGrC1 cells. The EDC mixture directly increased progesterone secretion by upregulating 3β-hydroxysteroid dehydrogenase (3βHSD) expression. Furthermore, the EDC mixture increased activity of mitochondria, which are the central sites for steroid hormone biosynthesis, and the ATP content. Unexpectedly, the EDC mixture reduced glucose transporter 4 (GLUT4) expression and perturbed glucose uptake; however, this did not affect the glycolytic rate. Moreover, inhibition of GLUT1 by STF-31 did not alter the effects of the EDC mixture on steroid secretion but decreased basal estradiol secretion. Taken together, our results demonstrate that the mixture of EDCs present in FF can alter the functions of human GCs by disrupting steroidogenesis and may thus adversely affect female reproductive health. This study highlights that the EDC mixture elicits its effects by targeting mitochondria and increases mitochondrial network formation, mitochondrial activity, and expression of 3βHSD, which is associated with the inner mitochondrial membrane.

Flutamide treatment reveals a relationship between steroidogenic activity of Leydig cells and ultrastructure of their mitochondria

Our present knowledge on interrelation between morphology/ultrastructure of mitochondria of the Leydig cell and its steroidogenic function is far from satisfactory and needs additional studies. Here, we analyzed the effects of blockade of androgen receptor, triggered by exposure to flutamide, on the expression of steroidogenic proteins (1) and ultrastructure of Leydig cells’ constituents (2). We demonstrated that increase in the expression level of steroidogenic (StAR, CYP11A1, 3β-HSD, and CYP19A1) proteins (and respective mRNAs) in rat testicular tissue as well as elevation of intratesticular sex steroid hormone (testosterone and estradiol) levels observed in treated animals correspond well to morphological alterations of the Leydig cell ultrastructure. Most importantly, up-regulation of steroidogenic proteins’ expression apparently correlates with considerable multiplication of Leydig cell mitochondria and subsequent formation of local mitochondrial networks. Interestingly, we showed also that the above-mentioned processes were associated with elevated transcription of Drp1 and Mfn2 genes, encoding proteins implicated in mitochondrial dynamics. Collectively, our studies emphasize the importance of mitochondrial homeostasis to the steroidogenic function of Leydig cells.

BNIP3L-mediated mitophagy is required for mitochondrial remodeling during the differentiation of optic nerve oligodendrocytes

ABSTRACT Retinal ganglion cell axons are heavily myelinated (98%) and myelin damage in the optic nerve (ON) severely affects vision. Understanding the molecular mechanism of oligodendrocyte progenitor cell (OPC) differentiation into mature oligodendrocytes will be essential for developing new therapeutic approaches for ON demyelinating diseases. To this end, we developed a new method for isolation and culture of ON-derived oligodendrocyte lineage cells and used it to study OPC differentiation. A critical aspect of cellular differentiation is macroautophagy/autophagy, a catabolic process that allows for cell remodeling by degradation of excess or damaged cellular molecules and organelles. Knockdown of ATG9A and BECN1 (pro-autophagic proteins involved in the early stages of autophagosome formation) led to a significant reduction in proliferation and survival of OPCs. We also found that autophagy flux (a measure of autophagic degradation activity) is significantly increased during progression of oligodendrocyte differentiation. Additionally, we demonstrate a significant change in mitochondrial dynamics during oligodendrocyte differentiation, which is associated with a significant increase in programmed mitophagy (selective autophagic clearance of mitochondria). This process is mediated by the mitophagy receptor BNIP3L (BCL2/adenovirus E1B interacting protein 3-like). BNIP3L-mediated mitophagy plays a crucial role in the regulation of mitochondrial network formation, mitochondrial function and the viability of newly differentiated oligodendrocytes. Our studies provide novel evidence that proper mitochondrial dynamics is required for establishment of functional mitochondria in mature oligodendrocytes. These findings are significant because targeting BNIP3L-mediated programmed mitophagy may provide a novel therapeutic approach for stimulating myelin repair in ON demyelinating diseases. Abbreviations: A2B5: a surface antigen of oligodendrocytes precursor cells, A2B5 clone 105; ACTB: actin, beta; APC: an antibody to label mature oligodendrocytes, anti-adenomatous polyposis coli clone CC1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9A: autophagy related 9A; AU: arbitrary units; BafA1: bafilomycin A1; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CNP: 2′,3′-cyclic nucleotide 3′-phosphodiesterase; Ctl: control; COX8: cytochrome c oxidase subunit; CSPG4/NG2: chondroitin sulfate proteoglycan 4; DAPI: 4′6-diamino-2-phenylindole; DNM1L: dynamin 1-like; EGFP: enhanced green fluorescent protein; FACS: fluorescence-activated cell sorting; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; GFP: green fluorescent protein; HsESC: human embryonic stem cell; IEM: immunoelectron microscopy; LAMP1: lysosomal-associated membrane protein 1; LC3B: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MFN2: mitofusin 2; Mito-Keima: mitochondria-targeted monomeric keima-red; Mito-GFP: mitochondria-green fluorescent protein; Mito-RFP: mitochondria-red fluorescent protein; MitoSOX: red mitochondrial superoxide probe; MKI67: antigen identified by monoclonal antibody Ki 67; MMP: mitochondrial membrane potential; O4: oligodendrocyte marker O4; OLIG2: oligodendrocyte transcription factor 2; ON: optic nerve; OPA1: OPA1, mitochondrial dynamin like GTPase; OPC: oligodendrocyte progenitor cell; PDL: poly-D-lysine; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; RGC: retinal ganglion cell; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SEM: standard error of the mean; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; TUBB3: tubulin, beta 3 class III.

Studying Mitochondrial Network Formation by In Vivo and In Vitro Reconstitution Assay.

Mitochondria change their morphologies from small isolated vesicles to large continuous networks across the cell cycles. The mitochondrial network formation (MNF) plays an important role in maintaining mitochondrial DNA integrity and interchanging mitochondrial materials. The disruption of the mitochondrial network affects mitochondrial functions, such as ATP production, integration of metabolism, calcium homeostasis, and regulation of apoptosis, leading to the abnormal development and several human diseases including neurodegenerative disease. In this unit, we describe the method of studying MNF, which is driven by microtubule-dependent motor protein, by in vivo imaging and single-molecule in vitro reconstitution assays.

Role of autophagy in Dynamic Remodeling of Cultured Mouse Hepatocytes

Primary culture of hepatocytes is a valuable tool to study mechanisms involved in liver metabolic, endocrine and secretory functions. However, freshly isolated hepatocytes dedifferentiate and lose many features of hepatocyte including the transporters and metabolic enzymes in the course of primary culture. Here, we investigated the role and mechanisms of autophagy in the dynamic remolding of long‐term cultured mouse hepatocytes. Mouse hepatocytes were freshly isolated from wild type mice and liver‐specific Atg5 as well as Drp1 knockout mice and cultured for different time points from 2 hours to 7 days. Autophagy and mitophagy flux were determined using the tandem GFP‐mCherry‐LC3 and Cox8‐GFP‐mCherry. We found that hepatocytes gradually lost their typical cuboidal morphology and displayed fibroblast like morphology, which was accompanied by increased stress fibers and highly fused mitochondrial network formation in the course of culture. These mitochondrial dynamic changes were associated with decreased major mitochondrial fusion proteins Mfn1/2 and fission protein Drp1. The levels of hepatic cytochrome P450 enzymes (such as 2E1 and 2B10) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) markedly decreased, suggesting loss of hepatic metabolic features of drug and glucose metabolism in cultured hepatocytes. GFP‐mCherry‐LC3 puncta analysis and Cox8‐GFP‐mCherry analysis revealed that basal autophagic flux and mitophagy increased as early as day 2 and continue to increase in dedifferentiated hepatocytes during the culture. This mitophagy was independent of Parkin, as the levels of Parkin were dramatically decreased after 12 hours culture in hepatocytes. Mitophagy was markedly blunted in cultured Drp1 knockout hepatocytes, suggesting that elongated mitochondrial may be protective against autophagic degradation. Importantly, the levels of P450 enzymes, Parkin and GADPH remained higher in Atg5 knockout hepatocytes compared with wild type hepatocytes during the culture, suggesting the autophagy may be involved in the degradation of these metabolic enzymes. In conclusion, these findings indicate that autophagy is important for the remodeling of cultured hepatocytes and manipulation of autophagy process may be helpful to maintain some of the hepatocyte features during the culture.

Experimental studies of mitochondrial and lysosomal function in in vitro and in vivo models relevant to Parkinson's disease genetic risk.

Several studies have identified the involvement of mitochondrial and lysosomal dysfunction in Parkinson's disease (PD) pathology. In this review we discuss recent work that has identified deficits in mitophagy, mitochondrial network formation, increased sensitivity to mitochondrial stressors and alterations in proteins regulating mitochondrial fission and fusion associated with patient-derived fibroblasts harboring mutations in LRRK2 gene and from sporadic PD patient cells. We further focus on alterations of lysosomal enzymes, in particular glucocerebrosidase activity, and resultant lipid dyshomeostasis in PD and aging, in human tissue and in vivo rodent models. Future studies aimed at understanding the convergence of mitochondrial and lysosomal pathways will be of essence for the identification of unique cellular defects in PD and for the development of new treatments.

55Identification of celastramycin as a novel therapeutic agent for pulmonary arterial hypertension - high-throughput screening of 5,562 compounds

Abstract Background Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) accompanying increased production of inflammatory factors and adaptation of mitochondrial metabolism to a hyperproliferative state. However, at present, since all the drugs in clinical use target pulmonary vascular dilatation, they may not be so effective for patients with advanced PAH. Purposes We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. Methods In the first screening, we examined 5,562 compounds from our original library using high-throughput screening system to discover a compound that inhibits proliferation of PASMCs from PAH patients (PAH-PASMCs). In the second screening, we performed concentration-dependent assays and counter assays with PAH-PASMCs and PASMCs from healthy donors. We also performed apoptosis assays and mechanistic analysis for inflammation, reactive oxygen species (ROS), and mitochondrial function. Results We found that celastramycin, a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PAH-PASMCs in a dose-dependent manner with minimal effects on PASMCs from healthy donors. Moreover, celastramycin inhibited proliferation with minimal increase in apoptosis and low rate of cell death. Then, we synthesized 25 analogues of celastramycin, and finally selected the lead compound that significantly inhibited proliferation of PAH-PASMCs and reduced cytosolic ROS levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of hypoxia-inducible factor-1α, which was abnormally activated in PAH-PASMCs and impaired aerobic metabolism, and nuclear factor-κB, which induces pro-inflammatory signals, in PAH-PASMCs compared with vehicle controls, leading to reduced secretion of inflammatory cytokines. Importantly, celastramycin treatment reduced the ROS levels in PAH-PASMCs with increased protein levels of NF-E2-related factor 2 (Nrf2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. We also discovered that celastramycin-mediated effects on these transcriptional modulators could be regulated by zinc finger C3H1 domain-containing protein, which is a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in three experimental animal models of PH in mice and rats, accompanied by reduced inflammatory changes in the lungs. Conclusions These results indicate that celastramycin ameliorates pulmonary hypertension through inhibition of excessive proliferation of PAH-PASMCs, for which its anti-inflammatory and beneficial effects on mitochondrial energy metabolism may be involved. Thus, celastramycin could be a novel drug for PAH as it exerts anti-proliferative effects on PAH-PASMCs.

Identification of Celastramycin as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) accompanying increased production of inflammatory factors and adaptation of the mitochondrial metabolism to a hyperproliferative state. However, all the drugs in clinical use target pulmonary vascular dilatation, which may not be effective for patients with advanced PAH. We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. We screened 5562 compounds from original library using high-throughput screening system to discover compounds which inhibit proliferation of PASMCs from patients with PAH (PAH-PASMCs). We found that celastramycin, a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PAH-PASMCs in a dose-dependent manner with relatively small effects on PASMCs from healthy donors. Then, we made 25 analogs of celastramycin and selected the lead compound, which significantly inhibited cell proliferation of PAH-PASMCs and reduced cytosolic reactive oxygen species levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of HIF-1α (hypoxia-inducible factor 1α), which impairs aerobic metabolism, and κB (nuclear factor-κB), which induces proinflammatory signals, in PAH-PASMCs, leading to reduced secretion of inflammatory cytokine. Importantly, celastramycin treatment reduced reactive oxygen species levels in PAH-PASMCs with increased protein levels of Nrf2 (nuclear factor erythroid 2-related factor 2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. Moreover, these celastramycin-mediated effects were regulated by ZFC3H1 (zinc finger C3H1 domain-containing protein), a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in 3 experimental animal models, accompanied by reduced inflammatory changes in the lungs. These results indicate that celastramycin ameliorates pulmonary hypertension, reducing excessive proliferation of PAH-PASMCs with less inflammation and reactive oxygen species levels, and recovered mitochondrial energy metabolism. Thus, celastramycin is a novel drug for PAH that targets antiproliferative effects on PAH-PASMCs.

Protein composition of the muscle mitochondrial reticulum during postnatal development.

Muscle mitochondrial networks changed from a longitudinal, fibre parallel orientation to a perpendicular configuration during postnatal development. Mitochondrial dynamics, mitophagy and calcium uptake proteins were abundant during early postnatal development. Mitochondrial biogenesis and oxidative phosphorylation proteins were upregulated throughout muscle development. Postnatal muscle mitochondrial network formation is accompanied by a change in protein expression profile from mitochondria designed for co-ordinated cellular assembly to mitochondria highly specialized for cellular energy metabolism. Striated muscle mitochondria form connected networks capable of rapid cellular energy distribution. However, the mitochondrial reticulum is not formed at birth and the mechanisms driving network development remain unclear. In the present study, we aimed to establish the network formation timecourse and protein expression profile during postnatal development of the murine muscle mitochondrial reticulum. Two-photon microscopy was used to observe mitochondrial network orientation in tibialis anterior (TA) muscles of live mice at postnatal days (P) 1, 7, 14, 21 and 42, respectively. All muscle fibres maintained a longitudinal, fibre parallel mitochondrial network orientation early in development (P1-7). Mixed networks were most common at P14 but, by P21, almost all fibres had developed the perpendicular mitochondrial orientation observed in mature, glycolytic fibres. Tandem mass tag proteomics were then applied to examine changes in 6869 protein abundances in developing TA muscles. Mitochondrial proteins increased by 32% from P1 to P42. In addition, both nuclear- and mitochondrial-DNA encoded oxidative phosphorylation (OxPhos) components were increased during development, whereas OxPhos assembly factors decreased. Although mitochondrial dynamics and mitophagy were induced at P1-7, mitochondrial biogenesis was enhanced after P14. Moreover, calcium signalling proteins and the mitochondrial calcium uniporter had the highest expression early in postnatal development. In conclusion, mitochondrial networks transform from a fibre parallel to perpendicular orientation during the second and third weeks after birth in murine glycolytic skeletal muscle. This structural transition is accompanied by a change in protein expression profile from mitochondria designed for co-ordinated cellular assembly to mitochondria highly specialized for cellular energy metabolism.

3D mitochondrial network formation during postnatal skeletal muscle development of mice

Mitochondrial connectivity plays an important role for muscle energy distribution. However, it is obscure how their structural formation is developed during postnatal muscle growth. Here, using a high‐resolution electron microscopy (i.e., FIB‐SEM), we have sought to examine three dimensional (3D) mitochondrial network formations in oxidative (soleus) and glycolytic (gastrocnemius) muscles of C57BL/6N mice at postnatal (P) 1, 7, 14, 21, and 42 day, respectively. After semi‐automated machine learning process, 3D mitochondrial reticulum formations were rendered and quantitatively analyzed by ImageJ software. Both types of skeletal muscles showed similarly fiber parallel mitochondrial network formations during early days (P1–P7). After P14, oxidative muscles developed grid‐like mitochondrial reticulum, while glycolytic muscle mitochondria were found as perpendicular formations along the Z‐lines. Unlikely to glycolytic muscles where individual mitochondrial volumes were similarly maintained across the development, oxidative muscles significantly increased those volumes by P14 and then largely decreased to the level comparable to glycolytic muscles. After P14, oxidative muscles maintained notably higher total mitochondrial volumes as compared to glycolytic muscles. Furthermore, we observed that at P14, glycolytic muscles showed remarkably higher ratio of mitochondrial surface area to volume, as well as sphericity, than oxidative muscles. Another interesting point was that during early muscle growth (P1–P7), mitochondrial elongation, aspect ratio, and length were all shown to be higher in glycolytic muscles than in oxidative muscles. In conclusion, this preliminary study provides how 3D mitochondrial networks are changed over the course of postnatal muscle development, and also suggests that P14 may be a critical time point for the mitochondrial transformation. However, further studies are needed to better understand 3D muscle mitochondrial development along with contractile components and other subcellular elements such as lipid droplets and sarcoplasmic reticulum.Support or Funding InformationThis work is supported by the Division of Intramural Research of the National Heart, Lung and Blood Institute and the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial Dysfunction in Astrocytes Impairs the Generation of Reactive Astrocytes and Enhances Neuronal Cell Death in the Cortex Upon Photothrombotic Lesion.

Mitochondria are key organelles in regulating the metabolic state of a cell. In the brain, mitochondrial oxidative metabolism is the prevailing mechanism for neurons to generate ATP. While it is firmly established that neuronal function is highly dependent on mitochondrial metabolism, it is less well-understood how astrocytes function rely on mitochondria. In this study, we investigate if astrocytes require a functional mitochondrial electron transport chain (ETC) and oxidative phosphorylation (oxPhos) under physiological and injury conditions. By immunohistochemistry we show that astrocytes expressed components of the ETC and oxPhos complexes in vivo. Genetic inhibition of mitochondrial transcription by conditional deletion of mitochondrial transcription factor A (Tfam) led to dysfunctional ETC and oxPhos activity, as indicated by aberrant mitochondrial swelling in astrocytes. Mitochondrial dysfunction did not impair survival of astrocytes, but caused a reactive gliosis in the cortex under physiological conditions. Photochemically initiated thrombosis induced ischemic stroke led to formation of hyperfused mitochondrial networks in reactive astrocytes of the perilesional area. Importantly, mitochondrial dysfunction significantly reduced the generation of new astrocytes and increased neuronal cell death in the perilesional area. These results indicate that astrocytes require a functional ETC and oxPhos machinery for proliferation and neuroprotection under injury conditions.