Mitochondrial Debris Research Articles

When Our Best Friend Becomes Our Worst Enemy: The Mitochondrion in Trauma, Surgery, and Critical Illness.

Common for major surgery, multitrauma, sepsis, and critical illness, is a whole-body inflammation. Tissue injury is able to trigger a generalized inflammatory reaction. Cell death causes release of endogenous structures termed damage associated molecular patterns (DAMPs) that initiate a sterile inflammation. Mitochondria are evolutionary endosymbionts originating from bacteria, containing molecular patterns similar to bacteria. These molecular patterns are termed mitochondrial DAMPs (mDAMPs). Mitochondrial debris released into the extracellular space or into the circulation is immunogenic and damaging secondary to activation of the innate immune system. In the circulation, released mDAMPS are either free or exist in extracellular vesicles, being able to act on every organ and cell in the body. However, the role of mDAMPs in trauma and critical care is not fully clarified. There is a complete lack of knowledge how they may be counteracted in patients. Among mDAMPs are mitochondrial DNA, cardiolipin, N-formyl peptides, cytochrome C, adenosine triphosphate, reactive oxygen species, succinate, and mitochondrial transcription factor A. In this overview, we present the different mDAMPs, their function, release, targets, and inflammatory potential. In light of present knowledge, the role of mDAMPs in the pathophysiology of major surgery and trauma as well as sepsis, and critical care is discussed.

Mitochondria Directed Oxidative Stress and Metal Abnormalities at the Center of Alzheimer’s Disease

For nearly four decades, our research has focused on dissecting the cytopathology of Alzheimer’s disease (AD) with the goal of developing a cure. We have used oxidative stress as a window to view and understand AD. Oxidative damage to sugars, proteins, lipids, and nucleic acids increases in neuronal cytoplasm. The same neuronal compartment has increased redoxactive iron and copper, which can catalyze oxidative damage, and likely derive from mitochondrial debris (in and outside lysosomes) including cytochromes, mitochondria-specific prosthetic groups, and mtDNA. Mitochondria show altered axonal transport, size distribution, energetics, fusion/fission, and degradation in AD that correlate with the extent of oxidative damage suggesting they are the origin. Synaptic mitochondria abnormalities correlate with synaptic vesicular changes. Surprisingly, amyloidβ and tau are quantitatively associated with reduced neuronal oxidative damage. Copper sequestration by amyloidβ blocks copper-mediated oxidation of lipids and vitamin C indicating amyloidβ can be a protective response rather than the initiator of AD. Instead of being bound to amyloidβ, iron is present as 10nm magnetite crystals with super-paramagnetic properties as well as abundant metallic iron, along with metallic copper. This is the first report of metallic iron and copper in humans. Not just amyloidβ, but also tau, may be protective responses induced in AD to maintain neurons with altered balance for decades. While these studies put oxidative stress at the center of AD, they also highlight a complexity of multifaceted alterations that is homeostatic and requires a deeper level of understanding before an effective cure.

Mitochondria-derived damage-associated molecular patterns and inflammation in the ischemic-reperfused heart.

Cardiac cell death after myocardial infarction release endogenous structures termed damage-associated molecular patterns (DAMPs) that trigger the innate immune system and initiate a sterile inflammation in the myocardium. Cardiomyocytes are energy demanding cells and 30% of their volume are mitochondria. Mitochondria are evolutionary endosymbionts originating from bacteria containing molecular patterns similar to bacteria, termed mitochondrial DAMPs (mDAMPs). Consequently, mitochondrial debris may be particularly immunogenic and damaging. However, the role of mDAMPs in myocardial infarction is not clarified. Identifying the most harmful mDAMPs and inhibiting their early inflammatory signaling may reduce infarct size and the risk of developing post-infarct heart failure. The focus of this review is the role of mDAMPs in the immediate pro-inflammatory phase after myocardial infarction before arrival of immune cells in the myocardium. We discuss different mDAMPs, their role in physiology and present knowledge regarding their role in the inflammatory response of acute myocardial infarction.

Early Protective Role of Inflammation in Cardiac Remodeling and Heart Failure: Focus on TNFα and Resident Macrophages.

Cardiac hypertrophy, initiated by a variety of physiological or pathological stimuli (hemodynamic or hormonal stimulation or infarction), is a critical early adaptive compensatory response of the heart. The structural basis of the progression from compensated hypertrophy to pathological hypertrophy and heart failure is still largely unknown. In most cases, early activation of an inflammatory program reflects a reparative or protective response to other primary injurious processes. Later on, regardless of the underlying etiology, heart failure is always associated with both local and systemic activation of inflammatory signaling cascades. Cardiac macrophages are nodal regulators of inflammation. Resident macrophages mostly attenuate cardiac injury by secreting cytoprotective factors (cytokines, chemokines, and growth factors), scavenging damaged cells or mitochondrial debris, and regulating cardiac conduction, angiogenesis, lymphangiogenesis, and fibrosis. In contrast, excessive recruitment of monocyte-derived inflammatory macrophages largely contributes to the transition to heart failure. The current review examines the ambivalent role of inflammation (mainly TNFα-related) and cardiac macrophages (Mφ) in pathophysiologies from non-infarction origin, focusing on the protective signaling processes. Our objective is to illustrate how harnessing this knowledge could pave the way for innovative therapeutics in patients with heart failure.

Skeletal Muscle Phenotype in Patients Undergoing Long-Term Hemodialysis Awaiting Kidney Transplantation.

Age and comorbidity-related sarcopenia represent a main cause of muscle dysfunction in patients on long-term hemodialysis. However, recent findings suggest muscle abnormalities that are not associated with sarcopenia. The aim of this study was to isolate functional and cellular muscle abnormalities independently of other major confounding factors, including malnutrition, age, comorbidity, or sedentary lifestyle, which are common in patients on maintenance hemodialysis. To overcome these confounding factors, alterations in skeletal muscle were analyzed in highly selected patients on long-term hemodialysis undergoing kidney transplantation. In total, 22 patients on long-term hemodialysis scheduled for kidney transplantation with few comorbidities, but with a long-term uremic milieu exposure, and 22 age, sex, and physical activity level frequency-matched control participants were recruited. We compared biochemical, functional, and molecular characteristics of the skeletal muscle using maximal voluntary force and endurance of the quadriceps, 6-minute walking test, and muscle biopsy of vastus lateralis. For statistical analysis, mean comparison and multiple regression tests were used. In patients on long-term hemodialysis, muscle endurance was lower, whereas maximal voluntary force was not significantly different. We observed a transition from type I (oxidative) to type II (glycolytic) muscle fibers, and an alteration of mitochondrial structure (swelling) without changes in DNA content, genome replication (peroxisome proliferator activator receptor γ coactivator-1α and mitochondrial transcription factor A), regulation of fusion (mitofusin and optic atrophy 1), or fission (dynamin-related protein 1). Notably, there were autophagosome structures containing glycogen along with mitochondrial debris, with a higher expression of light chain 3 (LC3) protein, indicating phagophore formation. This was associated with a greater conversion of LC3-I to LC3-II and the expression of Gabaralp1 and Bnip3l genes involved in mitophagy. In this highly selected long-term hemodialysis population, a low oxidative phenotype could be defined by a poor endurance, a fiber-type switch, and an alteration of mitochondria structure, without evidence of sarcopenia. This phenotype could be related to uremia through the activation of autophagy/mitophagy. NCT02794142 and NCT02040363.

Mitochondrial Quality Control in Cardiac-Conditioning Strategies against Ischemia-Reperfusion Injury.

Mitochondria are the central target of ischemic preconditioning and postconditioning cardioprotective strategies, which consist of either the application of brief intermittent ischemia/reperfusion (I/R) cycles or the administration of pharmacological agents. Such strategies reduce cardiac I/R injury by activating protective signaling pathways that prevent the exacerbated production of reactive oxygen/nitrogen species, inhibit opening of mitochondrial permeability transition pore and reduce apoptosis, maintaining normal mitochondrial function. Cardioprotection also involves the activation of mitochondrial quality control (MQC) processes, which replace defective mitochondria or eliminate mitochondrial debris, preserving the structure and function of the network of these organelles, and consequently ensuring homeostasis and survival of cardiomyocytes. Such processes include mitochondrial biogenesis, fission, fusion, mitophagy and mitochondrial-controlled cell death. This review updates recent advances in MQC mechanisms that are activated in the protection conferred by different cardiac conditioning interventions. Furthermore, the role of extracellular vesicles in mitochondrial protection and turnover of these organelles will be discussed. It is concluded that modulation of MQC mechanisms and recognition of mitochondrial targets could provide a potential and selective therapeutic approach for I/R-induced mitochondrial dysfunction.

Formyl Peptide Receptor-1 Blockade Prevents Receptor Regulation by Mitochondrial Danger-Associated Molecular Patterns and Preserves Neutrophil Function After Trauma.

Trauma predisposes to systemic sterile inflammation (systemic inflammatory response syndrome) as well as infection, but the mechanisms linking injury to infection are poorly understood. Mitochondrial debris contains formyl peptides. These bind formyl peptide receptor-1, trafficking neutrophils to wounds, initiating systemic inflammatory response syndrome, and wound healing. Bacterial formyl peptides, however, also attract neutrophils via formyl peptide receptor-1. Thus, mitochondrial formyl peptides might suppress neutrophils antimicrobial function. Also, formyl peptide receptor-1 blockade used to mitigate systemic inflammatory response syndrome might predispose to sepsis. We examined how mitochondrial formyl peptides impact neutrophils functions contributing to antimicrobial responses and how formyl peptide receptor-1 antagonists affect those functions. Prospective study of human and murine neutrophils and clinical cohort analysis. University research laboratory and level 1 trauma center. Trauma patients, volunteer controls. C57Bl/6, formyl peptide receptor-1, and formyl peptide receptor-2 knockout mice. Human and murine neutrophils functions were activated with autologous mitochondrial debris, mitochondrial formyl peptides, or bacterial formyl peptides followed by chemokines or leukotrienes. The experiments were repeated using formyl peptide receptor-1 antagonist cyclosporin H, "designer" human formyl peptide receptor-1 antagonists (POL7178 and POL7200), or anti-formyl peptide receptor-1 antibodies. Mouse injury/lung infection model was used to evaluate effect of formyl peptide receptor-1 inhibition. Human neutrophils cytosolic calcium, chemotaxis, reactive oxygen species production, and phagocytosis were studied before and after exposure to mitochondrial debris, mitochondrial formyl peptides, and bacterial formyl peptides. Mitochondrial formyl peptide and bacterial formyl peptides had similar effects on neutrophils. Responses to chemokines and leukotrienes were suppressed by prior exposure to formyl peptides. POL7200 and POL7178 were specific antagonists of human formyl peptide receptor-1 and more effective than cyclosporin H or anti-formyl peptide receptor-1 antibodies. Formyl peptides inhibited mouse neutrophils responses to chemokines only if formyl peptide receptor-1 was present. Formyl peptide receptor-1 blockade did not inhibit neutrophils bacterial phagocytosis or reactive oxygen species production. Cyclosporin H increased bacterial clearance in lungs after injury. Formyl peptides both activate and desensitize neutrophils. Formyl peptide receptor-1 blockade prevents desensitization, potentially both diminishing systemic inflammatory response syndrome and protecting the host against secondary infection after tissue trauma or primary infection.

MiR-125a induces apoptosis, metabolism disorder and migrationimpairment in pancreatic cancer cells by targeting Mfn2-related mitochondrial fission.

Mitochondrial fission is important for the development and progression of pancreatic cancer (PC). However, little is known regarding its role in pancreatic cancer apoptosis, metabolism and migration. In the current study, the mechanism by which mitochondrial fission modifies the biological characteristics of PC was explored. MicroRNA-125a (miR-125a) had the ability to inhibit mitochondrial fission and contributed to cellular survival. Suppressed mitochondrial fission led to a reduction in mitochondrial debris, preserved the mitochondrial membrane potential, inhibited mitochondrial permeability transition pore opening, ablated cytochrome c leakage into the cytoplasm and reduced the pro-apoptotic protein contents, finally blocking mitochondria related apoptosis pathways. Furthermore, defective mitochondrial fission induced by miR-125a enhanced mitochondria-dependent energy metabolism by promoting activity of electron transport chain complexes. Furthermore, suppressed mitochondrial fission also contributed to PANC-1 cell migration by preserving the F-actin balance. Furthermore, mitofusin 2 (Mfn2), the key defender of mitochondrial fission, is involved in inhibition of miR125a-mediated mitochondrial fission. Low contents of miR-125a upregulated Mfn2 transcription and expression, leading to inactivation of mitochondrial fission. Ultimately, the current study determined that miR-125a and Mfn2 are regulated by hypoxia-inducible factor 1 (HIF1). Knockdown of HIF1 reversed miR-125a expression, and therefore, inhibited Mfn2 expression, leading to activation of mitochondrial fission. Collectively, the present study demonstrated mitochondrial fission as a tumor suppression process that is regulated by the HIF/miR-125a/Mfn2 pathways, acting to restrict PANC-1 cell survival, energy metabolism and migration, with potential implications for novel approaches for PC therapy.

Pathogenesis of cardiac ischemia reperfusion injury is associated with CK2\u03b1-disturbed mitochondrial homeostasis via suppression of FUNDC1-related mitophagy

Disturbed mitochondrial homeostasis contributes to the pathogenesis of cardiac ischemia reperfusion (IR) injury, although the underlying mechanism remains elusive. Here, we demonstrated that casein kinase 2α (CK2α) was upregulated following acute cardiac IR injury. Increased CK2α was shown to be instrumental to mitochondrial damage, cardiomyocyte death, infarction area expansion and cardiac dysfunction, whereas cardiac-specific CK2α knockout (CK2αCKO) mice were protected against IR injury and mitochondrial damage. Functional assay indicated that CK2α enhanced the phosphorylation (inactivation) of FUN14 domain containing 1 (FUNDC1) via post-transcriptional modification at Ser13, thus effectively inhibiting mitophagy. Defective mitophagy failed to remove damaged mitochondria induced by IR injury, resulting in mitochondrial genome collapse, electron transport chain complex (ETC) inhibition, mitochondrial biogenesis arrest, cardiolipin oxidation, oxidative stress, mPTP opening, mitochondrial debris accumulation and eventually mitochondrial apoptosis. In contrast, loss of CK2α reversed the FUNDC1-mediated mitophagy, providing a survival advantage to myocardial tissue following IR stress. Interestingly, mice deficient in both CK2α and FUNDC1 failed to show protection against IR injury and mitochondrial damage through a mechanism possible attributed to lack of mitophagy. Taken together, our results confirmed that CK2α serves as a negative regulator of mitochondrial homeostasis via suppression of FUNDC1-required mitophagy, favoring the development of cardiac IR injury.

Intratracheal instillation of neutrophils rescues bacterial overgrowth initiated by trauma damage-associated molecular patterns.

Nosocomial pneumonias are common in trauma patients and so interventions to prevent and treat nosocomial pneumonia may improve outcomes. Our prior work strongly suggests that tissue injury predisposes to infections like nosocomial pneumonia because mitochondrial debris originating from injured cells contains damage-associated molecular patterns that can reduce neutrophil (PMN) migration into the airway and diminish PMN function in response to bacterial inoculation of the airway. This suggested that putting exogenous "normal" PMN into the airway might be beneficial. Postinjury pneumonia (PNA) commonly arises in two groups, early, community-acquired PNA (CAP) and later hospital-acquired PNA (HAP). Posttraumatic early-onset CAP and late-onset HAP were modeled in CD-1 mice using Staphylococcus aureus or Pseudomonas aeruginosa instilled intratracheal (i.t.) at clinically relevant times with or without extrapulmonary injuries mimicked by an intraperitoneal application of mitochondrial damage-associated molecular patterns. We applied bone marrow-derived PMN (BM-PMN) intratracheally to assess their effect on bacterial clearance in the lung. BM-PMN instillation i.t. had no untoward clinical effects on recipient animals. In both the early/CAP and late/HAP models, clearance of the bacterial inoculum from the lung was suppressed by mitochondrial debris and restored to uninjured levels by i.t. instillation of exogenous BM-PMN. Furthermore, PMN instillation cleared the inoculum of P. aeruginosa that could not be cleared by uninjured mice. Instillation of PMN into the lung, even across strains (CD-1 vs. C57BL/6) had no injurious effect. These initial studies suggest PMN instillation (i.t.) is worthy of further study as a potential adjunctive therapy aimed at decreasing the morbidity of lung infections in trauma patients. Moreover, PMN instillation (i.t.) may represent a unique means of preventing or treating pneumonia after serious injury that is completely independent of the need for antibiotic use.

Extracellular Mitochondria and Mitochondrial Components Act as Damage-Associated Molecular Pattern Molecules in the Mouse Brain

Mitochondria and mitochondrial debris are found in the brain's extracellular space, and extracellular mitochondrial components can act as damage associated molecular pattern (DAMP) molecules. To characterize the effects of potential mitochondrial DAMP molecules on neuroinflammation, we injected either isolated mitochondria or mitochondrial DNA (mtDNA) into hippocampi of C57BL/6 mice and seven days later measured markers of inflammation. Brains injected with whole mitochondria showed increased Tnfα and decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation. Some of these effects were also observed in brains injected with mtDNA (decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation), and mtDNA injection also caused several unique changes including increased CSF1R protein and AKT phosphorylation. To further establish the potential relevance of this response to Alzheimer's disease (AD), a brain disorder characterized by neurodegeneration, mitochondrial dysfunction, and neuroinflammation we also measured App mRNA, APP protein, and Aβ1-42 levels. We found mitochondria (but not mtDNA) injections increased these parameters. Our data show that in the mouse brain extracellular mitochondria and its components can induce neuroinflammation, extracellular mtDNA or mtDNA-associated proteins can contribute to this effect, and mitochondria derived-DAMP molecules can influence AD-associated biomarkers.

Circulating Mitochondrial DAMPs Are Not Effective Inducers of Proteinuria and Kidney Injury in Rodents.

Mitochondria in eukaryotic cells are derived from bacteria in evolution. Like bacteria, mitochondria contain DNA with unmethylated CpG motifs and formyl peptides, both of which have recently been shown to be damage associated molecular patterns (DAMPs) and induce immune response and cell injury. Based on the facts that circulating mitochondrial DAMPs (mtDAMPs) are increased in the patients of trauma or burn injury who also have proteinuria, that mtDAMPs can activate immune cells which in turn secrete glomerular permeability factors, that renal intrinsic cells express a variety of DAMP receptors, and that mtDAMPs can directly increase endothelial cell permeability in vitro, we hypothesized that mtDAMPs may be novel circulating factors inducing proteinuria and kidney injury. We tested this hypothesis by directly injecting mtDAMPs into rodents and examining urinary protein and kidney histology. We prepared mtDAMP samples, including mitochondrial DNA (mtDNA) and mitochondrial debris (MTD), from rodent liver. In mice, injection of mtDNA for 20 μg/ml initial concentration in circulation (much higher than the clinical range), did not cause any renal manifestations. However, an increased dose leading to 45 μg/ml initial concentration in circulation resulted in a transient, slight increase in urinary albumin. In rats, MTD injection resulting in 450 μg/ml initial concentration of MTD protein in circulation, which was much higher than the clinical range, caused mild, transient proteinuria and lung lesions. Multiple injections of such large amount of either mtDNA or MTD into rodents on 3 consecutive days also failed in inducing proteinuria and kidney injury. In summary, clinical levels of circulating mtDAMPs do not induce proteinuria and clinically irrelevant high levels of mtDAMPs cause only a transient and slight increase in urinary protein in rodents, suggesting that circulating mtDAMPs may not be responsible for the proteinuria and kidney injury in patients with trauma, burn injury, and other diseases.

MITOCHONDRIAL DNA IS RELEASED BY SHOCK AND ACTIVATES NEUTROPHILS VIA P38 MAP KINASE

Bacterial DNA (bDNA) can activate an innate-immune stimulatory "danger" response via toll-like receptor 9 (TLR9). Mitochondrial DNA (mtDNA) is unique among endogenous molecules in that mitochondria evolved from prokaryotic ancestors. Thus, mtDNA retains molecular motifs similar to bDNA. It is unknown, however, whether mtDNA is released by shock or is capable of eliciting immune responses like bDNA. We hypothesized shock-injured tissues might release mtDNA and that mtDNA might act as a danger-associated molecular pattern (or "alarmin") that can activate neutrophils (PMNs) and contribute to systemic inflammatory response syndrome. Standardized trauma/hemorrhagic shock caused circulation of mtDNA as well as nuclear DNA. Human PMNs were incubated in vitro with purified mtDNA or nuclear DNA, with or without pretreatment by chloroquine (an inhibitor of endosomal receptors like TLR9). Neutrophil activation was assessed as matrix metalloproteinase (MMP) 8 and MMP-9 release as well as p38 and p44/42 mitogen-activated protein kinase (MAPK) phosphorylation. Mitochondrial DNA induced PMN MMP-8/MMP-9 release and p38 phosphorylation but did not activate p44/42. Responses were inhibited by chloroquine. Nuclear DNA did not induce PMN activation. Intravenous injection of disrupted mitochondria (mitochondrial debris) into rats induced p38 MAPK activation and IL-6 and TNF-alpha accumulation in the liver. In summary, mtDNA is released into the circulation by shock. Mitochondrial DNA activates PMN p38 MAPK, probably via TLR9, inducing an inflammatory phenotype. Mitochondrial DNA may act as a danger-associated molecular pattern or alarmin after shock, contributing to the initiation of systemic inflammatory response syndrome.

Mitochondrial Peptides Are Potent Immune Activators That Activate Human Neutrophils Via FPR-1

Tissue injury from mechanical trauma modulates innate immunity. The resultant systemic inflammatory response syndrome (SIRS) closely mimics clinical sepsis, and bacterial n-formyl peptides are septic mediators. Similar formyl peptides exist in mitochondria but little is known about their actions on human neutrophils (PMN). Mitochondria were isolated from rat heptocytes and disrupted. Soluble mitochondrial degradation products (MDP) were used to stimulate human PMN. Cytosolic calcium ([Ca]i) responses to MDP were assayed with and without antibody blockade of formyl peptide receptor 1 (FPR-1) or formyl peptide receptor like-1 (FPRL-1). Chemotaxis toward MDP was assessed in trans-wells. Oxidative burst to MDP was assessed using carboxycarboxy-2', 7'-dichlorodihydrofluorescein diacetate. MDP caused [Ca]i responses similar to those caused by formyl-Met-Leu-Phe. [Ca]i responses were completely blocked by anti-FPR-1 antibodies, showing activation occurred via the high-affinity, G-protein-coupled FPR-1 receptor. MDP acted on FPR-1 to give chemotactic responses similar to 10 nM formyl-Met-Leu-Phe. MDP also caused dose-dependent oxidative burst. Formylated mitochondrial proteins are potent immune activators. Acting through the FPR-1 receptor on professional phagocytes, MDP elicits [Ca]i release responses and Ca entry via G-protein-coupled pathways. MDP activates chemotaxis and respiratory burst. Our findings suggest a novel paradigm wherein one root cause of SIRS after trauma may be the release of mitochondrial fragments from mechanically damaged tissues. In this paradigm, mitochondrial debris "alarmins" alter host PMN phenotype, activating or suppressing immunity, predisposing to SIRS, sepsis or organ failure.

Accumulation of DNA, Nuclear and Mitochondrial Debris, and ROS at Sites of Age-Related Cortical Cataract in Mice

Lenses from young and old mice were analyzed by laser scanning confocal microscopy (LSCM) with vital dyes, to determine whether age-related subcapsular and cortical cataracts were linked to the failure of lens fiber cells to degrade nuclei, DNA, and mitochondria properly and whether they result in the overproduction of reactive oxygen species (ROS) at the same sites. As opposed to the clear DNA-free subcapsular and cortical areas of young adult mouse lenses, these areas in cataractous old mouse lenses were found to contain accumulations of nuclei, nuclear fragments, aggregated mitochondria, and amorphous DNA as cortical inclusions (P < 0.001 between young and old lenses). These inclusions correlated spatially with age-related cataracts and with the presence of ROS. The source of such undegraded material was a large expansion of transition nuclei in the bow region and also direct involution of surface lens epithelial cells (LECs) into the underlying cortex, frequently leaving bare patches devoid of nuclei on the surface of the anterior epithelium. Live lenses were stained vitally for DNA with Hoechst 33342. ROS and mitochondria were stained and quantified with dihydrorhodamine 123 (DHR). In fixed lenses, DNA was stained with propidium iodide (PI) or 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI). The intensity and position of each probe's fluorescence was determined by LSCM. Cataract localization was ascertained by digitalized microscopy of reflected light. In aged mice, most subcapsular and cortical cataracts colocalize with accumulations of nuclei, mitochondria, and DNA, These effects are accompanied at the same sites by the production of ROS. The condition is due to the failure of lens fiber cells in the bow region to differentiate properly into the clear fiber state and to the improper involution of cells from the anterior epithelium directly into the underlying cortex, resulting in cataractous opacities.

Friedreich ataxia mouse models with progressive cerebellar and sensory ataxia reveal autophagic neurodegeneration in dorsal root ganglia.

Friedreich ataxia (FRDA), the most common recessive ataxia, is characterized by degeneration of the large sensory neurons of the spinal cord and cardiomyopathy. It is caused by severely reduced levels of frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biosynthesis. Through a spatiotemporally controlled conditional gene-targeting approach, we have generated two mouse models for FRDA that specifically develop progressive mixed cerebellar and sensory ataxia, the most prominent neurological features of FRDA. Histological studies showed both spinal cord and dorsal root ganglia (DRG) anomalies with absence of motor neuropathy, a hallmark of the human disease. In addition, one line revealed a cerebellar granule cell loss, whereas both lines had Purkinje cell arborization defects. These lines represent the first FRDA models with a slowly progressive neurological degeneration. We identified an autophagic process as the causative pathological mechanism in the DRG, leading to removal of mitochondrial debris and apparition of lipofuscin deposits. These mice therefore represent excellent models for FRDA to unravel the pathological cascade and to test compounds that interfere with the degenerative process.

FRACTIONATION AND PROPERTIES OF AN EXTRA-MITOCHONDRIAL ENZYME SYSTEM FROM PEANUTS CATALYZING THE BETA-OXIDATION OF PALMITIC ACID.

An extra-mitochondrial system prepared from germinating peanut cotyledons by differential centrifugation and ammonium sulfate precipitation is able to catalyze the conversion of C14-labeled palmitic acid to C14-labeled acetyl-CoA, in the presence of CoA, ATP, DPN and Mg (9). This system is found in the supernatant fraction left after centrifugation of the mitochondria at 10,000 X g for 30 minutes. Of course this fraction is heterogeneous containing some mitochondrial debris, microsomes, as well as soluble proteins. The question of intracellular localization of the enzymes catalyzing this ,6-oxidation activity was raised (9) and 3 possibilities were advanced: A) The /3-oxidation activity of this in vitro extra-mitochondrial fraction is derived from ruptured mitochondria; B) The /8-oxidation activity in the 10,COO X g supernatant is indeed of extra-mitochondrial origin, i.e., microsomal, hyaloplasmic or both. C) In vivo ,6-oxidation activity is present both inside and outside the mitochondria. During the course of this study we were able to show that in vitro, the activation of long chain fatty acids is particulate, while the conversion of the activated acids into acetyl-CoA is catalyzed by very soluble enzymes.

Incorporation of labeled amino acids into interior sites of protein by a cell-free system from rat skeletal muscle

Amino acid incorporation into protein by microsomal systems from bacteria, plants, and various mammalian tissues has been studied extensively, but thus far incorporation by similar systems from skeletal muscle has not been reported. ( McLean et al. (1958) obtained low levels of incorporation into mitochondria from rat skeletal muscle; they did not report incorporation into microsomes in vitro.) However, when muscle microsomes ∗ ∗ For purposes of this paper, microsomes are defined as those cellular constituents which are not sedimented at 10,000 × g (15 min.) but are sedimented at 105,000 × g (60 min.). There is reason to believe that such preparations contain mitochondrial debris as well as fragments of the endoplasmic reticulum ( Siekevitz, 1962). from young, rapidly growing rats were incubated with amino acids of extremely high specific activity ∗∗ ∗∗ L-leucine-U-C 14, 143.4 mc/mmole, was purchased from the New England Nuclear Corporation. L-Phenylalanine-U-C 14, 140 mc/mmole, was purchased from Schwarz BioResearch. , it was possible to demonstrate amino acid incorporation into protein.