Formation Of New Mitochondria Research Articles

Mitochondrial Quality Control and Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy.

Diabetic retinopathy continues to progress even when hyperglycemia is terminated, suggesting a 'metabolic memory' phenomenon. Mitochondrial dysfunction is closely associated with the development of diabetic retinopathy, and mitochondria remain dysfunctional. Quality control of mitochondria requires a fine balance between mitochondrial fission-fusion, removal of the damaged mitochondria (mitophagy) and formation of new mitochondria (biogenesis). In diabetes, while mitochondrial fusion protein (Mfn2) is decreased, fission protein (Drp1) is increased, resulting in fragmented mitochondria. Re-institution of normal glycemia fails to reverse mitochondrial fragmentation, and dysfunctional mitochondria continue to accumulate. Our aim was to investigate the direct effect of regulation of the mitochondrial fusion process during normal glycemia that follows a high glucose insult on mitochondrial quality control in the 'metabolic memory' phenomenon. Human retinal endothelial cells, incubated in 20 mM glucose for four days, followed by 5 mM glucose for four additional days, with or without the Mfn2 activator leflunomide, were analyzed for mitochondrial fission (live cell imaging), mitophagy (flow cytometry and immunofluorescence microscopy), and mitochondrial mass (mitochondrial copy numbers and MitoTracker labeling). Mitochondrial health was determined by quantifying mitochondrial reactive oxygen species (ROS), respiration rate (Seahorse XF96) and mitochondrial DNA (mtDNA) damage. Addition of leflunomide during normal glucose exposure that followed high glucose prevented mitochondrial fission, facilitated mitophagy and increased mitochondrial mass. Glucose-induced decrease in mitochondrial respiration and increase in ROS and mtDNA damage were also prevented. Thus, direct regulation of mitochondrial dynamics can help maintain mitochondrial quality control and interfere with the metabolic memory phenomenon, preventing further progression of diabetic retinopathy.

Mitochondrial network remodeling: an important feature of myogenesis and skeletal muscle regeneration.

The remodeling of the mitochondrial network is a critical process in maintaining cellular homeostasis and is intimately related to mitochondrial function. The interplay between the formation of new mitochondria (biogenesis) and the removal of damaged mitochondria (mitophagy) provide a means for the repopulation of the mitochondrial network. Additionally, mitochondrial fission and fusion serve as a bridge between biogenesis and mitophagy. In recent years, the importance of these processes has been characterised in multiple tissue- and cell-types, and under various conditions. In skeletal muscle, the robust remodeling of the mitochondrial network is observed, particularly after injury where large portions of the tissue/cell structures are damaged. The significance of mitochondrial remodeling in regulating skeletal muscle regeneration has been widely studied, with alterations in mitochondrial remodeling processes leading to incomplete regeneration and impaired skeletal muscle function. Needless to say, important questions related to mitochondrial remodeling and skeletal muscle regeneration still remain unanswered and require further investigation. Therefore, this review will discuss the known molecular mechanisms of mitochondrial network remodeling, as well as integrate these mechanisms and discuss their relevance in myogenesis and regenerating skeletal muscle.

Mitochondrial transformations in the aging human placenta.

Mitochondria play a key role in homeostasis and are central to one of the leading hypotheses of aging, the free radical theory. Mitochondria function as a reticulated network, constantly adapting to the cellular environment through fusion (joining), biogenesis (formation of new mitochondria), and fission (separation). This adaptive response is particularly important in response to oxidative stress, cellular damage, and aging, when mitochondria are selectively removed through mitophagy, a mitochondrial equivalent of autophagy. During this complex process, mitochondria influence surrounding cell biology and organelles through the release of signaling molecules. Given that the human placenta is a unique organ having a transient and somewhat defined life span of ∼280 days, any adaption or dysfunction associated with mitochondrial physiology as a result of aging will have a dramatic impact on the health and function of both the placenta and the fetus. Additionally, a defective placenta during gestation, resulting in reduced fetal growth, has been shown to influence the development of chronic disease in later life. In this review we focus on the mitochondrial adaptions and transformations that accompany gestational length and share similarities with age-related diseases. In addition, we discuss the role of such changes in regulating placental function throughout gestation, the etiology of gestational complications, and the development of chronic diseases later in life.

Warburg effect and its role in tumourigenesis.

Glucose is a crucial molecule in energy production and produces different end products in non-tumourigenic- and tumourigenic tissue metabolism. Tumourigenic cells oxidise glucose by fermentation and generate lactate and adenosine triphosphate even in the presence of oxygen (Warburg effect). The Na+/H+-antiporter is upregulated in tumourigenic cells resulting in release of lactate- and H+ ions into the extracellular space. Accumulation of lactate- and proton ions in the extracellular space results in an acidic environment that promotes invasion and metastasis. Otto Warburg reported that tumourigenic cells have defective mitochondria that produce less energy. However, decades later it became evident that these mitochondria have adapted with alterations in mitochondrial content, structure, function and activity. Mitochondrial biogenesis and mitophagy regulate the formation of new mitochondria and degradation of defective mitochondria in order to combat accumulation of mutagenic mitochondrial deoxyribonucleic acid. Tumourigenic cells also produce increase reactive oxygen species (ROS) resulting from upregulated glycolysis leading to pathogenesis including cancer. Moderate ROS levels exert proliferative- and prosurvival signaling, while high ROS quantities induce cell death. Understanding the crosstalk between aberrant metabolism, redox regulation, mitochondrial adaptions and pH regulation provides scientific- and medical communities with new opportunities to explore cancer therapies.

358. AAV-MitoTimer to Asses Mitochondrial Turnover

Mitochondrial turnover is a key marker in the process of cellular senescence. In order to track mitochondrial turnover rates in various cell lines, an indicator is necessary to identify the formation of new mitochondria along with the degradation of old mitochondria. This measure is critical for the study of disease processes in which there is an up or down regulation of mitochondrial turnover rates. One dynamic tool to measure this balance is the pTRE-Tight-MitoTimer plasmid. The MitoTimer transgene combines the red fluorescent protein, dsRed, and a human cytochrome c oxidase subunit VIII (Cox 8) mitochondrial localization sequence to traffic the protein to the mitochondria where the color shifts from green to red over time (approximately 24 hours). Our goal is to use this as a tool to assess the increase or decrease in mitochondrial turnover as a result of mutations that cause mitochondrial dysfunction. We successfully developed two plasmids through the ligation of the MitoTimer transgene into a double stranded AAV-ITR containing plasmid with a 1) Desmin promoter pds-AAV-Des-MitoTimer to restrict expression to striated muscle and 2) CMV promoter pds-AAV-CMV-MitoTimer to enable ubiquitous expression among many cell types. Once cloned, these novel plasmids were then transfected into an immortalized C2C12 mouse muscle cell line to confirm successful generation of functional plasmids. An immediate application for these novel plasmids in our laboratory is the characterization of mitochondrial turnover rates in Barth Syndrome (BTHS) patient derived cells. BTHS is an X-linked mitochondrial disease that results from mutations of the gene TAZ, which encodes tafazzin. This gene serves to orchestrate the production of mature cardiolipin (CL) within the mitochondrial membrane. Mutations in the TAZ gene result in altered CL maturation, with implications on mitochondrial function. Thus far, mitochondrial turnover rates have not been carefully assessed in this disorder. As a preliminary experiment using human patient derived BTHS fibroblasts (harboring an exon 10: 748G &gt T nonsense mutation), pds-AAV-CMV-MitoTimer was successfully transfected and displayed variation in green and red fluorescence ratios as compared to a healthy control cell line. This demonstration of the impact of BTHS on mitochondrial turnover dynamics supports future use of these plasmids in BTHS induced pluripotent stem cells (iPSCs) that have been differentiated into cardiomyocytes or skeletal myotubes - the two cell-types most affected in BTHS. This will enable future comparisons of mitochondrial turnover in clinically relevant BTHS cells harboring a variety of different mutations and provide another metric by which to measure the success of pre-clinical therapeutics for BTHS. We are preparing an AAV Quad-mutant serotype for in vitro testing, and other serotypes will be used for in vivo models of mitochondrial dysfunction. AAV-MitoTimer will continue to be used as a measure of mitochondrial turnover, and provide an effective characterization method for metabolic diseases.

Mitofusins 1 and 2 Are Essential for Postnatal Metabolic Remodeling in Heart

At birth, there is a switch from placental to pulmonary circulation and the heart commences its aerobic metabolism. In cardiac myocytes, this transition is marked by increased mitochondrial biogenesis and remodeling of the intracellular architecture. The mechanisms governing the formation of new mitochondria and their expansion within myocytes remain largely unknown. Mitofusins (Mfn-1 and Mfn-2) are known regulators of mitochondrial networks, but their role during perinatal maturation of the heart has yet to be examined. The objective of this study was to determine the significance of mitofusins during early postnatal cardiac development. We genetically inactivated Mfn-1 and Mfn-2 in midgestational and postnatal cardiac myocytes using a loxP/Myh6-cre approach. At birth, cardiac morphology and function of double-knockout (DKO) mice are normal. At that time, DKO mitochondria increase in numbers, appear to be spherical and heterogeneous in size, but exhibit normal electron density. By postnatal day 7, the mitochondrial numbers in DKO myocytes remain abnormally expanded and many lose matrix components and membrane organization. At this time point, DKO mice have developed cardiomyopathy. This leads to a rapid decline in survival and all DKO mice die before 16 days of age. Gene expression analysis of DKO hearts shows that mitochondria biogenesis genes are downregulated, the mitochondrial DNA is reduced, and mitochondrially encoded transcripts and proteins are also reduced. Furthermore, mitochondrial turnover pathways are dysregulated. Our findings establish that Mfn-1 and Mfn-2 are essential in mediating mitochondrial remodeling during postnatal cardiac development, a time of dramatic transitions in the bioenergetics and growth of the heart.

Toxic effects of mitochondrial DNA inhibitors on insect growth and development

The formation of new mitochondria in insect tissues occurs during discrete periods of cell growth associated with the molting cycles or metamorphosis. Acriflavine and ethidium bromide are known to disrupt replication, transcription and translation of circular, mitochondrial DNA during mitochondriogenesis. Injection of acriflavine into Heliothis zea during metamorphosis resulted in adult deformities and the loss of flight ability in normal appearing individuals. A 45% mortality rate was found for doses of 20 μg acriflavine/individual. Both acriflavine and ethidium bromide were toxic to neonate Aedes aegypti larvae. Acriflavine was most toxic with an LD 50 of 0.48 ppm, and larvae required only 12 hr of exposure for a maximal mortality response. Surviving larvae took up to three times longer to reach the pupal stage.

ELEKTRONENMIKROSKOPISCHE UND ELEKTRONENMIKROSKOPISCH‐HISTOCHEMISCHE UNTERSUCHUNGEN UBER NEBENNIERENRINDE DER RATTE MIT AMINOGLUTÄTHIMID (ELIPTEN, CIBA) ÜND UBER IHREN REPARATURPROZESS.

Amlnoglutethimide blocks the conversion of free cholesterln to D́5‐pregnenolone. It's effects on the fasciculata cells were studied electron microscopically in Wistar rats fed with this chemical for a maximum of seven days. The animals surviving after this treatment were also examined to follow the process of restoration from the damage. Four to seven days after the initiation of the feeding, the mitochondria were enlarged, variously deformed and contained intramitochondrial vacuoles. The lipid droplets were increased in size. Three to four days after withdrawal of amlnoglutethimide, so‐called “Intramitochondrial mitochondria” appeared. The picture suggesting division of the mitochondrial matrx was also occasionally encountered. These features suggested formation of new mitochondria from preexisting mitochondria. The mltohchondrla resumed their normal shape and appearance starting about ten days after withdrawal of amlnoglutethimide. The electron microscopic histochemlcal method demonstrated tubular crystals of digitonin‐cholesterid in the intramitochondrial vacuoles as well as in the lipid droplets 4 days after the initiation of the feeding. The withdrawal of amlnoglutethimide was followed with disappearance of these crystals from the sites of deposition.

Formation of new mitochondria in plant cells.

Formation of new mitochondria in plant cells.

Fine structure of spermatogonia and primary spermatocytes in rabbits

The fine structure of rabbit Spermatogonia and primary spermatocytes in meiotic prophase has been studied with different methods of preparation, including a technique for acid phosphatase activity. The spermatogonial cytoplasm is rich in free ribosomes and containes moderate amounts of vesicular, smooth-surfaced endoplasmic reticulum and mitochondria, a simple Golgi-apparatus, some micropinocytotic vesicles, and occasional multivesicular bodies, vacuoles and dense bodies with acid phosphatase activity. The large type A Spermatogonia have a prominent nucleolus and their mitochondria sometimes form clusters with a dense intermitochondrial substance, similar to that in spermatocytes. The nucleus and cytoplasm of primary spermatocytes increase markedly in volume and density during meiotic prophase. The Golgi apparatus enlarges and becomes more differentiated and finally forms small proacrosome granules. The endoplasmic reticulum produces numerous small, mainly smooth vesicles and might also be the source of a new organelle: numerous piles of narrow cisternae with opaque contents. These piles disintegrate late in prophase. The mitochondria become aggregated in clusters with dense intermitochondrial substance and their internal structure is characterized by highly dilated cristae and small particles, interpreted as mitochondrial ribosomes, in the matrix. The role of these structures in the formation of new mitochondria is discussed. The clusters of mitochondria finally disperse and their cores of dense intermitochondrial substance, possibly containing ribonucleoprotein, coalesce into a large chromatoid body similar to that in spermatids. Micropinocytosis and a few lysosomes occur in most spermatocytes. The pachytene nuclei show prominent nucleoli and a distinct sex vesicle without any synaptinemal complex. The importance for spermatid differentiation of some events taking place in the cytoplasm of primary spermatocytes is emphasized.

Structures possibly related to the formation of new mitochondria in spinal ganglion neuroblasts

Spinal ganglion neuroblasts of chick embryos show a marked increase in the chondriome from the 84th to the 120th hour of incubation. These cells contain membranous whorls associated either with the nuclear envelope or with some mitochondria. These findings are discussed and some interpretations of them are put forward. The most likely hypothesis seems to be that the membranous whorls may be related to the formation of new mitochondria.

Die menschliche Leber im Elektronenmikroskop: Untersuchungen an Leberpunktaten

During the past few years, numerous publications appeared describing the ultrastructure of animal and human livers. Also in the past two or three years, a number of excellent monographs were published dealing with the same subject. The publication of Cossel's monograph at this time only supplements the information already available; however, his extensive use of human material, normal and pathologic, places this volume in a class by itself. The electron micrographs are of good quality and the three dimensional drawings are excellent. The volume contains 599 references which include almost all of the major publications in the field of hepatic ultrastructure. It is only fair to point out that this book is not without certain imaginative concepts which have insufficient scientific basis. Cossel's description of mitochondrial changes and their relation to hepatic pathology and formation of new mitochondria by division of preexisting mitochondria lacks adequate experimental proof. It also should

Processi di Neoformazione di Mitocondri Nella Cellula Vegetale

Abstract Processes of formation of new mitochondria in plant cells. — While studying with the electron microscope the quiescent centre in the onion (Allium cepa) root tips, the writer was able to observe the endoplasmic reticulum arranged into peculiar concentric ring systems, which are here described. In some of the ring structures, between a ring and the contigous one, mitochondria-like bodies are formed, which are then detached by a kind of budding process. A short review of the current literature on formation of the mitochondria is also reported.

Genesis of mitochondria in neurospora crassa.

Observations with the light and electron microscopes have suggested three possible mechanisms for the formation of new mitochondria:' 2 (1) growth and subdivision of existing mitochondria, (2) denovo synthesis, and (3) derivation from other nonmitochondrial membranous structures. In an attempt to distinguish between these basic possibilities, we have carried out the following experiment: Cells fully labeled with a radioactive constituent of mitochondria were transferred to nonradioactive medium. During the subsequent logarithmic growth period purified mitochondrial fractions were isolated, and the distribution of label among their individual mitochondria was determined using a quantitative radioautographic technique.3, 4a In this type of experiment, the distribution of autographic grains among mitochondria could be expected to fit one of three patterns: (1) A dispersive pattern in which label would be distributed among all mitochondria, the average grain count per mitochondrion decreasing as a function of cell growth. This pattern is consistent with mitochondrial multiplication by division. (2) A conservative distribution in which the proportion of labeled mitochondria would decrease during growth in unlabeled medium as a logarithmic function of time, the average grain count per mitochondrion in the labeled population remaining the same. Such a pattern is consistent with de-novo synthesis. (3) An intermediate pattern, in which the label would not be randomly distributed, and the proportion of labeled individuals would decrease less rapidly than in the truly conservative case, the total grain count of the labeled population decreasing, remaining the same, or increasing slightly. This would be consistent with formation of mitochondria from labeled (possibly structural) precursors. In these studies radioactive choline was used as a label, because lecithin into which it is incorporated is a major constituent of mitochondrial phospholipids.5 6 Efficient and chemically specific labeling was obtained by working with a cholinerequiring strain of Neurospora crassa (chol-1, #34486).7 This cholineless strain appears to be blocked at the earliest step in the synthesis of choline from ethanolamine; it can make neither mononor dimethyl-ethanolamine, though it can use these compounds for the synthesis of choline.8 The experiment which has been outlined can yield the information needed, provided several prerequisites are fulfiled: (a) The cells under study must constitute a homogeneous population with a reproducible and prolonged logarithmic growth phase. (b) The mitochondrial fractions used for analysis must be free of contamination by other cellular structures. (c) The radioactive choline. which is incorporated must behave as a stable mitochondrial label. Much of the experimental evidence is directed toward demonstrating that these prerequisities are satisfied. Materials and Methods.-Materiais: Cytochrome c, Type III (horse heart) was purchased from Sigma Chemical Company; C14and H3-methyl-labeled choline chloride were purchased

A quantitative electron microscopic study of mitochondria in motor neurones following axonal section

An electron microscopic study was made of the motor neurones of the hypoglossal nuclei of a series of 10 rabbits, 10–11 days after unilateral hypoglossal neurotomy. Quantitative data obtained from the micrographs showed that on the operated side there was a significantly greater number of mitochondrial profiles per unit area of cytoplasm than on the control side. There was also a 70 per cent increase in the volume ratio of mitochondria to cytoplasm, approximately three-fifths of this being attributable to mitochondrial swelling and the remaining two-fifths to the increase in mitochondrial numbers. The findings may he interpreted as indicating that the increase in number of mitochondrial profiles on the operated side, was brought about by the formation of new mitochondria resulting in an increase in total functioning mitochondrial mass. There was a highly significant increase in the number of dense bodies (“microbodies”) per unit area of cytoplasm on the operated side, and a significant correlation was found between the average increase in the number of dense bodies and the average increase in mitochondrial numbers in the individual experiments. It has already been noted that the appearances of dense bodies in motor neurones of the rabbit were consistent with their bearing a developmental relationship to mitochondria and the present results would seem to favour the possibility that dense bodies may be concerned in mitochondrial formation. In the rat, other forms of dense body, containing areas of even greater density, were also noted.