Kontinkangas, L101A Biochemistry of cellular organelles Lectures: 1. Membrane channels; 2. Membrane transporters; 3. Soluble lipid/metabolite-transfer proteins; 4. Mitochondria as cellular organelles; Seminar: Isolation of subcellular organelles; 5. Mitochondrial inheritance; 6. Mitochondria in health and disease; 7. Endoplasmic Reticulum (ER) and lipids; 8. Structure and function of peroxisomes; Seminar: Mitochondria in cellular life. Dr. Vasily Antonenkov, Visiting professor Dept. Biochemistry, Oulu University Oulu, Finland Web site: 1
Lecture 6: Mitochondria in health and disease Lecture content: Mitochondrial versus nuclear genome role in pathology; Maternal inheritance and mitochondrial diseases; MitochondrialDNA depletion syndrome; Mitophagy; Mitochondrial quality control; Mpv17 protein as potential mitochondrial patology sensor and mitokiller 2
Role of mitochondria in the cell The main function is an energy fixation in the form of ATP (OXPHOS); Pyruvate decarboxylation and citric acid cycle; Oxidation of long-chain fatty acids; Synthesis of some fatty acids (lipoic acid); Synthesis of cardiolipin; Ketogenesis; Metabolism of some amino acids; Storage of calcium ions; Heme synthesis, iron metabolism; Fixation of ammonium (urea cycle liver, small intestine); Initial steps of gluconeogenesis (liver, kidney); Steroidogenesis (adrenal glands); Heat production (brown fat tissue); Mitochondrial apoptosis; Cellular signaling. 3
Contribution of mitochondrial versus nuclear genome to mitochondrial diseases In total, the mitochondrion hosts about 1500 different types of proteins, but only 13 of them are coded on the mtdna; Only 3% of Mt proteins are involved in ATP production, others are responsible for another Mt functions; Therefore most of the genetic information for the Mt proteins is in nuclear DNA and is involved in processes other than ATP synthesis. This genetic information is inherited in a Mendelian pattern; However, large proportion of Mt diseases (about 15% of the total) is due to mutations in mtdna which are more friquent than in nuclear DNA thanks to high Mt ROS production and poor mtdna repair mechanisms. Mitochondrial diseases is a group of disorders caused by dysfunctional Mt. 4
Heteroplasmy versus homoplasmy Heteroplasmy cell contains both - Mt with mutated DNA and with healthy Mt. The proportion of mutant mtdna determines both the penetrance and severity of manifistations of Mt diseases; Penetrance a probability of expressing a phenotype caused by mtdna mutation; Homoplasmy cell contains a uniform collection of mtdna: either completely normal or completely mutant mtdna; At cell division, the mtdna replicates and sorts randomly among Mt. In turn, Mt sort randomly among daughter cells; In cells, where heteroplasmy is present, each daughter cell may receive different proportions of Mt carrying normal and mutant mtdna 5
Only egg cells contribute Mt to offspring, mutant mtdna is not inherited from a male. Each egg cell contains up to 100000 copies of Mt. If a mother is homoplasmic for an mtdna, then all of the Mt she passes to her children will also be homoplastic for the mutation; Therefore, all of an affected female s children will carry mutation; but none of the affected male s children inherited the condition. 6
Bottleneck effect Mother is heteroplasmic for an mtdna mutation she passes on small amounts of mutated and normal mtdna (bottleneck effect) randomly to egg cells; Due to low amount of transferred Mt it may be a significant shift in heteroplasmy in mature egg cells, that in turn will eventually determines the severity of manifestations of Mt disease; Moreover, deseased mother likely passes on disease to progeny, but not inevitably, because of bottleneck effect; The clinical manifestations of Mt diseases are quite variable due to bottleneck effect. 7
Mitochondrial and nuclear mutations in mitochondrial diseases Mt DNA mutations maternal inheritance; Mutations in nuclear DNA coding for Mt proteins dominant, recessive, X-linked inheritance. Lead to defects in: 1. Maintenance of mtdna, transcription and translation; 2. Mutations in Mt enzymes of intermediary metabolism or in components of respiratory chain; 3. Defects in biogenesis, mitophagy or fission/fusion machinery. Disorders caused by mutations in mtdna MtDNA mutations occur frequently due to high Mt ROS production and poor error checking and repair mechanisms relative to nuclear DNA. This means that mtdna disorders may occur spontaneously and relatively often. 8
Manifestations of mitochondrial disorders Disorders caused by mutations of nuclear DNA codes for the Mt proteins exhibit symptoms similar to the disorders caused by mtdna mutations; Many of the Mt disorders affect tissues that have a high energy demand, such as central nervous system, the heart, and muscle; Examples: 1. CNS: encephalopathy, psychosis and depression, ataxia; 2. Heart: cardiomyopathy, heart block; 3. Renal: renal tubular defects; 4. Endocrine: hypothyroidism, gonadal failure; 5. Eye: cataract, optic atrophy; 6. Periferal nervous system: myopathy, neuropathy. Mt disease may become clinically apparent once the number of affected Mt reaches a certain level so-called threshold expression. 9
Clinical appearance of disorders caused by mtdna mutations Mt disease caused by mtdna mutations may become clinically apparent once the number of affected Mt reaches a certain level so-called threshold expression ; The effects of disease caused by mtdna mutations can be quite varied. Since the distribution of the defective mtdna may vary from organ to organ within the body, the mutation that in one individual may cause liver disease might in another person cause a brain disorder; The severity of the specific defect may also be great or small. Some minor defects cause only exercise intolerance, with no serious illness or disability; Defects often affect the function of the Mt in multiple tissues leading to multisystem diseases. 10
Example of mtdna-related Mt disease MELAS syndrome MELAS mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Symptoms: brain dysfunction (encephalopathy) with seizures and headaches, muscle disease with a build-up of lactic acid in the blood (lactic acidosis), temporary local paralysis (stroke-like episodes), and abnormal thinking (dementia). Onset: MELAS can affect people from age 4 to age 40 or more. Diagnosis: Muscle biopsy shows characteristic ragged red fibers and abnormal mitochondria. Genetics: MELAS is caused by mutations in the genes of mtdna coding for subunits of respiratory complex I (NADH dehydrogenase) and trnas. Maternal inheritance. Other important mt DNA-related Mt diseases: (1) eye disease Leber hereditary optic atrophy, (2) a type of epilesy MERRF Myoclonus Epilepsy with Ragged Red Fibers, (3) a neuromuscular disease Kearns-Sayre syndrome. 11
Example of nuclear DNA-related Mt disease Leigh s syndrome Leigh s syndrome is a rare genetic neurometabolic disorder that affects mainly the central nervous system. Symptoms: infants with poor sucking ability, the loss of head control and motor skills, tremor, rigidity, and hypokinesia. Onset: affect infants between the age of three months and two years. Diagnosis: Clinical observations. Genetics: The vast majority of Leigh disease cases are due to mutations in nuclear encoded genes, especially coding for pyruvate dehydrogenase and cytochrome oxidase complexes. Hence, the mutations affect oxidative phosphorylation (OXPHOS). Some Leigh disease cases are due to mutations in mtdna. Inherited in Mendelian patterns. 12
Mitochondrial DNA depletion syndromes (MDSs) MDSs form a group of autosomal, mainly recessive disorders characterized by decreased mtdna copy numbers in affected tissues; Three main clinical presentations: (1) myopathic, (2) encephalomyopathic, and (3) hepatocerebral; The affected genes are all of nuclear origin; Disease leads to depletion 30-80% of all mtdna; Age of onset: neonatal, infancy, early childhood; Clinical symptoms: CNS pathology ataxia, epilepsy, sensory neuropathy; Liver disease hepatic failure, hypoglycaemia; Muscular disorders myopathy, hypotonia, lactic acidosis. The vicious cycle leading to progressive alterations of the mtdna. 13
Mutations in genes shown on the table lead to decrease in mtdna copy numbers in different tissues that manifests in the clinical symptoms of mtdna depletion syndrome. 14
Nucleotide metabolism for mtdna synthesis and replication Genes in bold have been associated with diseases characterized by multiple mdna deletions and/or mtdna depletion. da deoxyadenine, dg deoxyguanine, dc deoxycytidine, dt deoxythymidine, rndp ribonucleotides (RNA constituents). 15
Mt fusion allows for tras-complementation or mitophagy of dysfunctional Mt (A) One Mt with defective genome (red) will fully fuse with a functional Mt (green) to restore the healthy mtdna; (B) One Mt with defective genome and low membrane potential (red) will transiently fuse with healthy Mt (green) to assess its ability to be rescued. If too deficient, it will be directed to mitophagy one of the mechanisms of Mt quality control. 16
Mitochondria and aging Mt hypothesis of aging predicts that the aging of a particular cell is triggered by accumulation of unhealthy mitochondria. This process may not kill the cell but should make it old, i.e. with low functional capasity; Proof: Mutator mice animals with mutated mtdna polymerase (point mutation D257A). This mutation compromises proof-reading activity of the enzyme and leads to accumulation of mutations in the mtdna; The mutator mice appear normal until the age of appr. 25 weeks, and thereafter they develop a progressive premature aging sindrome with weight loss, reduced subcutaneous fat, alopecia (loss of hair from the head), kyphosis (hump), osteoporosis, anemia, reduced fertility, heart enlargement, graying of the hair, hearing loss. Mutator mice has been created in 2004. Currently there are several models of premature aging where different mitochondrial proteins are deleted. One of them is Parkin protein. 17
Prevention of mitochondrial sickness versus removal of sick mitochondria How to cope with the problem of unhealthy mitochondria? 1. One way is to prevent their sickness: removal of brocken (denaturated) proteins from Mt by specific proteases, antioxidant defence, repearing of mtdna, repearing of Mt membranes, fusion with healthy Mt, etc. 2. Another way is to remove unhealthy mitochondria from the cell the question is: how to recognize that the mitochondrion is sick? It should be mechanisms of mitochondrial quality control. 18
How to eliminate mitochondria? 19
Different variants of mitophagy Mitophagy requires specific labelling of mitochondria and their recruiment into isolation membranes which form autophagosomes; Mitophagy during development of erythrocytes is activated due to increased expression of the NIX protein (outer Mt membrane) which interacts with the LC3 protein (isolation membrane) using WXXL motif; It seems that in yeast and erythrocytes the quality control of Mt is not crucial; PINK/parkin - dependent mitophagy selects the only unhealthy Mt. 20
PINK1/Parkin-dependent mitophagy PINK1 is a protein kinase that is constantly synthesized in the cytosol and is delivered to mitochondria. If the mitochondrion is healthy, the PINK1 protein is degraded; If the mitochondrion is sick the PINK1 protein is not degraded but instead is accumulated on the surface of outer Mt membrane; The PINK1 protein attarcts the Parkin protein (ubiquitin ligase); The Parkin protein add ubiquitin (small protein) to several outer memdrane proteins including VDAC. This signals that the mitochondrion should be degraded in the proteasome. 21
Mitophagy depends on membrane potential In Mt with high membrane potential the PINK1 protein containing the N-terminal Mt targeting sequence is delivered into the inner Mt membrane by TOM and TIM protein import complexes. Thei import is potential-dependent. In the inner membrane the PINK1 is constantly degraded by the corresponding proteases. In Mt with low membrane potential the PINK1 protein is not delivered into the inner membrane but instead is accumulated in the outer membrane where it interacts with Parkin. Defects in PINK1/Parkin-dependent mitophagy lead to mtdna instability. 22
How to find out unhealthy mitochondria? Possible symptoms of Mt sickness poteintial markers to detect unhealthy Mt: 1. Membrane potential; 2. Level of ROS production; 3. ATP/AMP raitio; 4. Matrix ph; 5.Piconcentration. Mt membrane potential is the difference in concentration of charged molecules on the opposite sides of the inner Mt membrane. It is high (140-180 mv) at normal conditions; Relatively low membrane potential not always indicate Mt sickness, but instead is a result of certain physiological conditions high ATP demand at low supply of substrates to the electron chain, e.g., stress conditions, hunger, etc. Therefore, to find out unhealthy Mt the cell should use detection of membrane potential in combination with some other markers. 23
Mpv17 protein Mpv17 is a small (18 kda) inner Mt membrane protein homologous to peroxisomal membrane non-selective channel Pxmp2; Knock-out of Mpv17p leads to premature aging: gray coat early in adulthood, agedependent high blood pressure, kidney desease, deafness, and cataract; Mutations in human Mpv17 protein lead to severe mtdna depletion syndrome. Hump 24
Mpv17p is a redox-sensitive channel Voltage-dependent closing of the Mpv17 channel depends on several parameters: redox state, protein phosphorylation (may reflect ATP/AMP raitio), and ph; At oxidative conditions (i.e., high production of ROS) the channel is resistant to closing. At reductive conditions the channel is prone to gating; Because the channel should be closed at normal conditions, its opening may signal extreme conditions or sickness of the particle. 25
Sensing the signal Opening the channel What is next? Leakage of ions and small solutes in and out of the Mt matrix. It seems that Mpv17 channel is a sensor of Mt pathology and a mitokiller. Consequences: 1. Drop in membrane potential; 2. Swelling/shrinking of mitochondria; 3. Decrease in the content of compounds that can be used as a fuel for respiratory chain (NAD redusers: pyruvate, malate; succinate, fatty acids, ketone bodies, etc). 26
Suggested questions Role of mitochondrial and nuclear genomes in mitochondrial disorders, heteroplasmy and homoplasmy; Maternal inheritance what is it? Clinical manifestations of mitochondrial disorders; Melas and Leigh s syndromes describe shortly, what is the mechanistic difference between these disorders? Mitochondrial DNA depletion syndrome; Mitochondrial hypothesis of aging, mutator mice; Mitophagy what is it? Different variants of mitophagy; PINK1/Parkin-dependent mitophagy; Mpv17 protein and its apparent role in mitochondrial quality control and mitophagy. 27