Mitochondrial Therapeutics PDF

MODULE 5 11. MITOCHONDRIAL THERAPEUTICS 12. CELL SIGNALING I 13. CELL SIGNALING II - INSULIN 1  MODULE 5 11. MITOCHONDRIAL THERAPEUTICS 12. CELL SIGNALING I 13. CELL SIGNALING II - INSULIN 2 11 MITOCHONDRION-DRI...

MODULE 5 11. MITOCHONDRIAL THERAPEUTICS 12. CELL SIGNALING I 13. CELL SIGNALING II - INSULIN 1  MODULE 5 11. MITOCHONDRIAL THERAPEUTICS 12. CELL SIGNALING I 13. CELL SIGNALING II - INSULIN 2 11 MITOCHONDRION-DRIVEN THERAPEUTICS 3  MITOCHONDRION-DRIVEN THERAPEUTICS — CONTENTS — I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Transporters and bioenergetics Metabolic differences: normal and cancer cells Electron leak: generation of oxidants a. cancer cells and hypoxia Generation of messengers of cell death b. cancer cells and uncoupling Mitochondrial DNA and biogenesis program Anticancer targets in glycolytic tumors Regulation of PGC1a a. hexokinase-2 inhibitors AMPK b. Leading therapeutic compounds Sirt1 Anticancer targets in glycolytic tumors GSK3b Leading therapeutics in glycolytic tumors Mitochondrial dynamics Mitochondrial fusion IV. DICHLOROACETATE AND MITOCHONDRIAL DISEASES Mitochondrial fission Dichloroacetate and metabolic pathways Therapeutic roles of DCA II. STIMULATING MITOCHONDRIAL BIOGENESIS 1. The sirtuin- PGC1a axis V. TARGETING ANTIOXIDANTS TO MITOCHONDRIA 2. The AMPK- PGC1a axis 4  11. MITOCHONDRIAL THERAPEUTICS – LEARNING OBJECTIVES – Describe the role of mtDNA and nDNA in mitochondrial biogenesis Discuss the enzymic pathways that lead to activation of PGC1a Apply the above knowledge to inputs and outcomes of the biogenesis program Discuss stimulation of mitochondrial biogenesis as a function of the sirtuin-PGC1a axis and the AMPK-PGC1a axis Discuss how expression of HIF-1a supports glycolysis and lactic acid release in cancer cells Explain the metabolic basis for the use of dichloroacetate in mitochondrial diseases 5  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS 6  SOME MITOCHONDRIAL FUNCTIONS Mitochondria as energy-transducing organelles, the site of ATP production in ENERGY the cell and, as such, maintain cellular energy homeostasis OXIDATIVE Mitochondria generate oxidants (H2O2) that can cause mitochondrial oxida- STRESS tive damage but are also sources of redox signals regulating transcription CELL Mitochondria generate messengers (e.g., cytochrome c) of cell death that DEATH activate and assemble the intrinsic pathways of programmed cell death Mitochondria biogenesis is regulated by transcription factors under the BIOGENESIS control of the co-activator PGC-1a, which senses diverse physiological signals Mitochondria are dynamic organelles that undergo fusion and fission accord- DYNAMICS ing to the cell’s energy needs AUTOPHAGY These are important processes responsible for the breakdown of cellular MITOPHAGY contents, preserving energy and safe-guarding against damaged biomolecules. 7  SOME MITOCHONDRIAL FUNCTIONS Mitochondria as energy-transducing organelles, the site of ATP production in ENERGY the cell and, as such, maintain cellular energy homeostasis OXIDATIVE Mitochondria generate oxidants (H2O2) that can cause mitochondrial oxida- STRESS tive damage but are also sources of redox signals regulating transcription CELL Mitochondria generate messengers (e.g., cytochrome c) of cell death that DEATH activate and assemble the intrinsic pathways of programmed cell death Mitochondria biogenesis is regulated by transcription factors under the BIOGENESIS control of the co-activator PGC-1a, which senses diverse physiological signals Mitochondria are dynamic organelles that undergo fusion and fission accord- DYNAMICS ing to the cell’s energy needs AUTOPHAGY These are important processes responsible for the breakdown of cellular MITOPHAGY contents, preserving energy and safe-guarding against damaged biomolecules. 8  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS OMM IMS IMM MATRIX MATRIX 1. Transporters and Bioenergetics Mitochondria consist of an outer mitochondrial membrane, inter-membrane space, inner membr- ane, and matrix. The outer mitochondrial membr- VDAC ANT ANT ane is freely permeable to most metabolites but Phosphate Phosphate Transporter Transporter contains following transporters: voltage-dependent anion channel (VDAC), monocarboxylate transport- MCT MPC ers (MCT) (for ketone bodies and lactate), and the Tricarboxylate Tricarboxylate transpor Transporters Translocator of the Outer Membrane (TOM). Also Dicarboxylate transport Dicarboxylate Transporters embedded in the outer mitochondrial membrane TOM and constitutively present in mitochondria are the TIM TIM small proteins Bcl-2 and Bcl-xL that have anti- Bcl-2 apoptotic properties, i.e., inhibit the programmed Bcl-xL cell death triggered by mitochondrial dysfunction. 9  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS 1. Transporters and Bioenergetics OMM IMS IMM MATRIX MATRIX In the inner mitochondrial membrane: The adenine nucleotide translocator (ANT) in close proximity to VDAC. VDAC ANT ANT The Mitochondrial Pyruvate Carrier (MPC); pyruvate is not transported by MCT but it has its Phosphate Phosphate Transporter Transporter own transporter in the inner membrane. MCT MPC The tricarboxylate and dicarboxylate transporters Tricarboxylate The Translocator of the Inner Membrane (TIM) Tricarboxylate transpo Transporters Dicarboxylate transpo Dicarboxylate and TOM, on the outer mitochondrial membrane, Transporters constitute complexes involved in protein import TOM TIM TIM machinery into mitochondria and understanding of their function constitute the basis of mitochon- Bcl-2 drial drug delivery systems and their therapeutic Bcl-xL applications. 10  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS 1. Transporters and Bioenergetics The mitochondrial matrix houses metabolic pathways, such as the tricarboxylic acid cycle (TCA), the PDH complex, b-oxidation, and ketolysis. Reducing IMM equivalents M Electron Transfer A (NADH and FPH2) from matrix metabolic pathways flow through the respiratory Chain chain Tricarbo Oxidative Phosphorylation creating a chemiosmotic gradient that results in ATP formation. IMM Electron Transfer Generation ofM A OMM (NA Pyruvate Fatty acyl-Coa Ketone Chain NADH Tricarbo bodies IMS Oxidative Phosphorylation Generation of Pyruvate pyruvate DH βb-oxidation -oxidation OMM (NA succinyl-CoA succinyl-CoA H+ I NADH NADH NADH transferase transferase IMS FADH2 pyruvate DH NAD FPH2 Pyruvate NADH acetyl-CoA Q acet H+ I FADH2 pyruvate DH citrate H+ III NADcitra oxaloacetate citrate citratesynthase synthase ! aconitase Q acet oxaloacetate c NADH isocitrate H++ III malate DH citra NADH H IV oxaloacetate isocitrate DH c NADH malate malate DH malate DH tricarboxylic NADH H+ IV acid malate cycle !-ketoglutarate malate Pi ADP fumarate suc ATPase H+ a !-ketoglu- tarate DH tarate DH Pi ATP FADH2 ADP fumarate NADH suc ATP ATP ATPase H+ fumarate succinyl-CoA succinate DH DH ATP FADH2 VDAC ANT succinate ADP ADP ATP ATP FADH2 11 VDAC ANT  MITOCHONDRIA-DEPENDENT PARACRINE SIGNALING Mitochondrial stress induces cells to release soluble molecules, such as metabolites (e.g., succinate), proteins (e.g., GDF15), and peptides that act on other cells or tissues in a paracrine fashion to elicit a systemic response. These signaling molecules are referred to as mitokines.  MITOCHONDRIAL SIGNALING REGULATES PHYSIOLOGY AND PATHOLOGY CELL 1 CELL 2 Succinate downstream REGULATION OF effectors IMMUNE RESPONSE TCA SUCNR1 ISR ATF4 TCA TCA REGULATION OF GDF15 downstream BODY WEIGHT, effectors GLUCOSE CONTROL, FOOD FATE Mitochondrial stress can induce a cell to release signaling molecules (mitokines): e.g., succinate and GDF15 (Growth Differentiation Factor 15). Succinate binds to the SUCNR1 and forward signaling is involved in the regulation of immune responses. GDF15, translated in the nucleus after integrated stress response from mitochondria releases ATF4 (Activating Transcription Factor 4). GDF15 is involved in regulation of body weight, glucose control, and food fate.  SOME MITOCHONDRIAL FUNCTIONS Mitochondria as energy-transducing organelles, the site of ATP production in ENERGY the cell and, as such, maintain cellular energy homeostasis OXIDATIVE Mitochondria generate oxidants (H2O2) that can cause mitochondrial oxida- STRESS tive damage but are also sources of redox signals regulating transcription CELL Mitochondria generate messengers (e.g., cytochrome c) of cell death that DEATH activate and assemble the intrinsic pathways of programmed cell death Mitochondria biogenesis is regulated by transcription factors under the BIOGENESIS control of the co-activator PGC-1a, which senses diverse physiological signals Mitochondria are dynamic organelles that undergo fusion and fission accord- DYNAMICS ing to the cell’s energy needs AUTOPHAGY These are important processes responsible for the breakdown of cellular MITOPHAGY contents, preserving energy and safe-guarding against damaged biomolecules. 14  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS 2. Electron Leak: Generation of Oxidants Mitochondria consume 98-99% of the cellular O2, I which is reduced at the cytochrome oxidase electron leak electron leak – O2 2-3% (complex IV) site of the respiratory chain to H2O. Q e O2.–, H2O2 oxi However, 1-2% of the electrons do not reach the III cytochrome oxidase site and leak from the respira- oxidants respiration c tory chain at the complex I / coenzyme Q site. O2 95-98% IV + 4e– Electron leak is the basis for the non-enzymatic H2O reduction of O2, reduced via one-electron transfer to a free radical species, superoxide anion radical (O2.–) oxidative damage and hydrogen peroxide (H2O2). 15  SOME MITOCHONDRIAL FUNCTIONS Mitochondria as energy-transducing organelles, the site of ATP production in ENERGY the cell and, as such, maintain cellular energy homeostasis OXIDATIVE Mitochondria generate oxidants (H2O2) that can cause mitochondrial oxida- STRESS tive damage but are also sources of redox signals regulating transcription CELL Mitochondria generate messengers (e.g., cytochrome c) of cell death that DEATH activate and assemble the intrinsic pathways of programmed cell death Mitochondria biogenesis is regulated by transcription factors under the BIOGENESIS control of the co-activator PGC-1a, which senses diverse physiological signals Mitochondria are dynamic organelles that undergo fusion and fission accord- DYNAMICS ing to the cell’s energy needs AUTOPHAGY These are important processes responsible for the breakdown of cellular MITOPHAGY contents, preserving energy and safe-guarding against damaged biomolecules. 16  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS 3 Generation of Messengers of Cell Death Mitochondria are also harbingers of cell death, for they release factors that activate a cell death program known as apoptosis. Cytochrome c is one of those factors that –when released from mitochondria– activates the assembly of the apoptotic machinery that leads to cell death. Cytochrome c is released upon disruption of the mitochondrial outer membrane or opening of the permeability transition pore. Several mitochondrion-based therapeutics are aimed at inhibiting the formation of the permeability transition pore, thus preventing the release of cytochrome c and activation of the cell death machinery. OMM IMS IMM activation of the intrinsic apoptotic activation of the activationpathway of the intrinsic apoptotic VDAC VD intrinsic apoptotic I pathway pathway OMM electron leakcyt c VDAC VDAC VDAC VDAC pe VDAC electron leak OMM OMM OMM t O2 2-3% permeability Q cyt c e– cyt c IMM permeability transition perme VDAC transition tran.– O2 , H2O2 oxidants oxidative ANT pore pore A po III VDAC cyt c IMM VDAC ANT IMM IMMdamage ANT poreANTopening oxidants ANT ANT cyt c respiration cyt c ANT ANT c pore opening pore pore opening O2 95-98% opening IV H2O 17 oxidative  SOME MITOCHONDRIAL FUNCTIONS Mitochondria as energy-transducing organelles, the site of ATP production in ENERGY the cell and, as such, maintain cellular energy homeostasis OXIDATIVE Mitochondria generate oxidants (H2O2) that can cause mitochondrial oxida- STRESS tive damage but are also sources of redox signals regulating transcription CELL Mitochondria generate messengers (e.g., cytochrome c) of cell death that DEATH activate and assemble the intrinsic pathways of programmed cell death Mitochondria biogenesis is regulated by transcription factors under the BIOGENESIS control of the co-activator PGC-1a, which senses diverse physiological signals Mitochondria are dynamic organelles that undergo fusion and fission accord- DYNAMICS ing to the cell’s energy needs AUTOPHAGY These are important processes responsible for the breakdown of cellular MITOPHAGY contents, preserving energy and safe-guarding against damaged biomolecules. 18  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial DNA and the biogenesis program Mitochondria contain their own DNA (mtDNA) that encodes 37 genes required for the oxidative phosphorylation machinery: 2 rRNAs, 22 tRNAs, and 13 protein-coding genes for the respiratory complexes. Each complex contains several subunits. mtDNA encodes 7 of the 45 subunits of complex I. No subunit in complex II is encoded by mtDNA. Of the 11 subunits of complex III, 1 is OMM encoded by mtDNA. Complex IV consists of 13 subunits and 3 are encoded by mtDNA. IMM Complex V, ATPase, consists of 16 subunits of which 2 are encoded by mtDNA. IMS MATRIX I 7 II Q SDH III 1 c IV 3 V 2 19 !  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial DNA and the biogenesis program There are about 1500 mitochondrial proteins and most of them are encoded by nuclear genes (nDNA), translated in the cytosol, and imported into mitochondria by a process known as mitochondrial biogenesis. Mitochondrial proteins have a dual origin that requires the coordination of mtDNA and nDNA. Disruption of mitochondrial assembly leads to several pathologies. Mitochondrial primary dysfunctions are the result of a mutation to a gene encoded by mtDNA or nDNA. Mutations in mtDNA can lead to defective assembly of oxidative phosphorylation; e.g., MELAS (Mitochondrial Encephalopathy Lactic Acidosis and Stroke-like episodes) is due to a mutation in the mitochondrial tRNA gene leading to a defective assembly of the oxidati- ve phosphorylation complexes and mtDNA nDNA impairment of energy metabolism. 37 genes mitochondrial biogenesis Mutation of a nuclear gene that encodes for an assembly factor for mutations in 1,500 PROTEINS mitochondrial complex I, NDUFAF3, results in a genes neonatal defect in energy metabol- neonatal defect; ism. MELAS lack of complex I assembly 20  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial DNA and the biogenesis program Many pathogenic mtDNA mutations are heteroplasmic, i.e., they coexist with a variable percentage of wild-type mtDNA. Clinical symptoms typically ensue when the amount of mutant mtDNA offsets a critical threshold, usually 50-60% of the total mtDNA, below which the mutation has virtually no clinical impact. The deleterious effects of either mtDNA- or nDNA mutations could be overcome by increasing the total amount of mitochondria and/or of functional active mitochondrial respiratory complex units, via activation of the mitochondrial biogenesis program. mtDNA nDNA mutations in mitochondrial mitochondrial genes 37 genes biogenesis 1,500 PROTEINS neonatal defect; lack MELAS of complex I assembly 21  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial DNA and the biogenesis program Many pathogenic mtDNA mutations are heteroplasmic, i.e., they coexist with a variable percentage of wild-type mtDNA. Clinical symptoms typically ensue when the amount of mutant mtDNA offsets a critical threshold, usually 50-60% of the total mtDNA, below which the mutation has virtually no clinical impact. The deleterious effects of either mtDNA- or nDNA mutations could be overcome by increasing the total amount of mitochondria and/or of functional active mitochondrial respiratory complex units, via activation of the mitochondrial biogenesis program. Pathogenicity Normal mtDNA Mutant mtDNA Threshold Mitochondria with normal Mitochondria with abnormal OXPHOS function OXPHOS function 50-60% Mutti et al., 2022, Fixing the powerhouse. The Biochemist 9-13 22  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS The Biogenesis Program The number of functional mitochondria is determined by transcriptional processes that regulate mitochondrial biogenesis: new mitochondria are formed within the cell. Mitochondrial biogenesis enables cellular adaptation to energetic and metabolic demands and it requires the synthesis, import, and incorporation of proteins and lipids to the existing mitochondrial reticulum, as well as replication of the mtDNA. The peroxisome proliferator-activated receptor g co-activator 1a (PGC1a) is the master regulator of mitochondrial biogenesis; mitochondrial biogenesis is driven by nuclear transcription factors (NRF-1 and NRF-2 (Nuclear Respiratory Factor-1 and -2) and ERRa (Estrogen-Related Receptor a)) that require PGC1a as co-activator. TRANSCRIPTION COACTIVATOR FACTORS NRF-1 NRF-2 MITOCHONDRIAL PGC1α BIOGENESIS ERRα 23  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial DNA and the Biogenesis Program Following activation by PGC1a, NRF-1 and NRF-2 contribute to the expression of many genes required for the maintenance and function of the mitochondrial respiratory apparatus. NRF-1 and NRF-2 act on genes encoding for different mitochondrial functions. Tfam, mitochondrial Protein importtranscription assembly factor a, facilitates the transcription of the (TOM, TIM) mitochondrial genome. Protein import assembly (TOM, TIM) Heme Biosynthesis TOM TOM PGC-1α Heme Biosynthesis TIM TIM V MITOCHONDRIA PGC1α Respiratory subunits Respiratory subunits I I 5ALS 5ALS mtDNA -1 (complexes I–V, cytochrome (complexes I-V, cyt c) c) mtDNA NRF-1 II II -2 NRF-2 α ERRα nuclear III III MITOCHONDRIA Nuclear genes IV IV Genes c V c NUCLEUS Mitochondrial translation Mitochondrial translation (ribosomal proteins) (ribosomal proteins) NUCLEUS mDNA transcription/replication (Tfam) mtDNA transcription and replication (Tfam, TFB1M) 24  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS The Biogenesis Program: Regulation of PGC1a Physical Oxidative Cytokines Exercise Stress PGC-1a plays a central role in the transcriptional Physical Exercise Hypoxia Oxidative Hypoxia Stress Nutrients Nutrients Cytokines control of mitochondrial biogenesis; PGC-1a is modulated by extracellular signals that effect PGC-1! metabolism, differentiation, and cell growth. The activation of PGC-1a entails post-translational TF Metabolic Genes modifications by energy sensors, such as AMPK (AMP-activated protein kinase) and the sirtuin Sirt1 (deacetylases). a. AMPK activates PGC-1a upon phosphorylation at threonine 177 and serine 538, thus leading to an increased mitochondrial biogenesis as well as an increased activity of mitochondrial enzymes Ser-P AMPK in skeletal muscle in response to exercise. PGC-1α PGC-1α inactive active Thr-P ATP ADP 25  IV. ENERGY- AND NUTRIENT SENSORS 1 AMPK Some of the metabolic effects that ensue following AMPK activation are particularly relevant to treatment of type 2 diabetes: Agonists → Ca2+ → CaMKK ANABOLISM Catabolic pathways are activated by AMPK: glucose uptake via GLUT4 and LKB1 AMPK GLUT1, glycolysis, fatty acid uptake, Metabolic → AMP CATABOLISM stress fatty acid oxidation, mitochondrial bio- inhibition of anabolism activation of catabolism genesis, and autophagy. fatty acid synthesis glucose uptake Agonists Anabolic Capathways 2+ CaMKKare inhibited by mTOR activation anabolism protein synthesis glycolysis AMPK: fatty acid, triglyceride, LKB1 AMPK chole- sterol, glycogen, protein, and rRNA. AMPK catabolism Metabolic synthesis; stress transcription AMP of lipogenic cholesterol synthesis fatty acid oxidation enzymes, transcription of gluconeogenic gluconeogenesis mitochondrial enzymes. biogenesis 26  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Sirt1 Biogenesis Program - Regulation of PGC1a: Sirt1 protein protein | | b. Sirt1 is a member of the Sirtuin family, deace- lys–Acetyl lys tylases that remove the acetyl group on lysine in NAD+ a reaction that is dependent on the co-substrate 2'-O-acetyl-ADP-ribose + NAD+. The reaction products include the nicotinamide deacetylated protein (now active), 2’-O-acetyl- caloric ADP-ribose and nicotinamide. restriction hydrophobic residue inactive Sirt1 acetylated active NRF-1 Nuclear PGC-1α PGC-1α NRF-2 deacetylated Genes target ERRα | | target lys–Acetyl protein acetyl-Lys lys protein target NAD+ NUCLEUS Sirt Sirt 2'-O-acetyl-ADP-ribose Sirt Sirt + D+ D+ nicotinamide NA NA inactive Sirt nicotinamide D+ NA 2-O-acetyl- ADP-ribose 27  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Biogenesis Program - Regulation of PGC1a: Sirt1 Sirt1 b. Sirt1 deacetylates PGC-1a and, thereby activatesprotein protein PGC-1a, which is required to | | sequester it to the nucleus and activate the transcription lys–Acetyl lys factors (NRF-1, NRF-2, NAD + ERRa) and the subsequent mitochondria biogenesis. Sirt1 is inhibited by its product, nicotinamide. Sirtuins 2'-O-acetyl-ADP-ribose are implicated in the mechanism of lifespan + nicotinamide extension observed under caloric restriction. caloric restriction Sirt1 NRF-1 NRF-1 Nuclear Nuclear mitochondrial PGC-1α PGC-1α NRF-2 NRF-2 Genes ERRα ERRα Genes biogenesis | | lys–Acetyl lys inactive NAD+ active NUCLEUS NUCLEUS 2'-O-acetyl-ADP-ribose + nicotinamide 28  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS The Biogenesis Program - Regulation of PGC1a: AMPK and Sirt1 The activities of AMPK and Sirt1 are connected: low energy status is sensed by AMPK and this results in an increase of the cellular NAD+ pool; NAD+ is a co-substrate for Sirt1: low energy status AMP/ATP NAD+/NADH AMPK P Sirt1 P PGC-1α PGC-1α PGC-1α Ac ATP Ac NAD+ active ADP nicotinamide 29  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS The Biogenesis Program - Regulation of PGC1a: AMPK and Sirt1 caloric high fat, sedentary INPUTS restriction exercise high sugar life lipids EFFECTORS ↑NAD+ ↑AMP ↓ amino ↓glucose insulin acids Sirtuins AMPK mTOR IIS SENSORS DNA repair Chromatin modifications Mitochondrial biogenesis and function Reduced inflammation PHYSIOLOGICAL Stress resistance Translation fidelity PROCESSES Stem cell maintenance Telomere maintenance Autophagy Disease Health Frailty Homeostasis Pathophysiologies OUTCOMES Disease-free Compressed morbidity Bonkowski & Sinclair (2016) Nat Rev Mol Cell Biol 17, 679 30  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS The Biogenesis Program – Regulation of PGC1a: GSK3b Glycogen synthase kinase 3b (GSK3b) is a negative regulator of PGC-1a since its phosphorylation (at Threonine295) targets PGC-1a for nuclear proteasomal degradation. GSK3b has been linked to the pathogenesis of Alzheimer's disease, although its role in PGC-1a regulation has not been explored in detail. proteasome Thr295–P Thr295–P GSK3β PGC-1α PGC-1α PGC-1α ATP ADP nucleus 31  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS The Biogenesis Program – Regulation of PGC1a: AMPK, SIRT, and GSK3b AMPK PGC1α SIRT GSK3β 32  SOME MITOCHONDRIAL FUNCTIONS Mitochondria as energy-transducing organelles, the site of ATP production in ENERGY the cell and, as such, maintain cellular energy homeostasis OXIDATIVE Mitochondria generate oxidants (H2O2) that can cause mitochondrial oxida- STRESS tive damage but are also sources of redox signals regulating transcription CELL Mitochondria generate messengers (e.g., cytochrome c) of cell death that DEATH activate and assemble the intrinsic pathways of programmed cell death Mitochondria biogenesis is regulated by transcription factors under the BIOGENESIS control of the co-activator PGC-1a, which senses diverse physiological signals Mitochondria are dynamic organelles that undergo fusion and fission accord- DYNAMICS ing to the cell’s energy needs AUTOPHAGY These are important processes responsible for the breakdown of cellular MITOPHAGY contents, preserving energy and safe-guarding against damaged biomolecules. 33  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial Dynamics Mitochondria are highly dynamic organelles whose morphology and activity can be regulated by two processes: fusion and fission. Unbalanced fission leads to mitochondrial fragmentation and unbalanced fusion leads to mitochondrial elongation. Mitochondrial dynamics allows mitochondria to interact with each other: without such dynamics mitochondria population would consist of autonomous organelles that have impaired function. Disruption of the fusion machinery leads to neurodegenerative diseases. fusion + fission 34  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial Dynamics a. Mitochondrial fusion – Mitochondrial fusion results in mixing of the outer-, inner mitochondrial membranes, and matrix contents. The fusion machinery consists of mitofusins (Mfn1 and Mfn2) and OPA1 (optic atrophy protein 1). Mitofusins are located on the outer membrane and OPA1 on the inner membrane. Interactions between Mfn molecules spanning adjacent mitochondria result in fusion of the outer membranes whereas OPA1 is involved in inner mitochondrial membrane fusion. Outer Intermembrane Membrane Space Mfn1 OPA1 Inner Matrix Membrane 35  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial Dynamics a. Mitochondrial fusion – The fusion machinery consists of mitofusins (Mfn1 and Mfn2) and OPA1 (optic atrophy protein 1). Mitofusins are located on the outer membrane and OPA1 on the inner membrane. Outer Membrane Mfn1 1 A1 OPA OP A1 OP Inner Membrane Mfn2 Matrix 36  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial Dynamics b. Mitochondrial fission – The fission machinery consists largely of Drp1 (dynamin-related protein 1), which is in the cytosol. Another key component of the mitochondrial fission machinery is Fis1 (fission 1), which is a small protein located on the outer mitochondrial membrane. Drp1 Fis1 fission 37 David C. Chan  MITOCHONDRIAL DYNAMICS FISSION AND FUSION Caloric restriction Sirtuin 1 / 3 Drp1 Mild Stress OPA1 Mitofusin PKA - cAMP Fis1 MITOCHONDRIAL FISSION MITOCHONDRIAL FUSION ROS OXPHOS Excess nutrients mTOR1 Oxidative stress Increased Ca2+ 38  I. BRIEF DESCRIPTION OF MITOCHONDRIAL FUNCTIONS Mitochondrial Dynamics in Neurodegenerative Diseases Mitochondrial dynamics has an impact on a wide variety of human diseases through interactions with other cellular processes. These neurodegenerative diseases affect distinct regions of the brain as well as the peripheral nervous system. Mitochondrial dynamics is essential to maintain mito- chondrial function in healthy neurons. Drp1 defects are always associated with abnor- mal brain development. 39  SOME MITOCHONDRIAL FUNCTIONS Mitochondria as energy-transducing organelles, the site of ATP production in ENERGY the cell and, as such, maintain cellular energy homeostasis OXIDATIVE Mitochondria generate oxidants (H2O2) that can cause mitochondrial oxida- STRESS tive damage but are also sources of redox signals regulating transcription CELL Mitochondria generate messengers (e.g., cytochrome c) of cell death that DEATH activate and assemble the intrinsic pathways of programmed cell death Mitochondria biogenesis is regulated by transcription factors under the BIOGENESIS control of the co-activator PGC-1a, which senses diverse physiological signals Mitochondria are dynamic organelles that undergo fusion and fission accord- DYNAMICS ing to the cell’s energy needs AUTOPHAGY These are important processes responsible for the breakdown of cellular MITOPHAGY contents, preserving energy and safe-guarding against damaged biomolecules. 40  AUTOPHAGY Autophagy is triggered by ULK1 complex and includes several other factors, such as PI3K class III, Beclin 1, and importantly, LC3-II, which anchors to the membrane and interacts with the cargo (orga- nelles, cytosolic proteins, etc) through receptors LC3-II LC3-II that recognize the cargo. receptors receptors Once the autophagosome is formed, it fuses with lysosomes, whose proteolytic enzymes degrade ➁ ➁ ➂ ➂ phagophore phagophore nucleation expansion the cargo. nucleation expansion AMPK ULK1 ULK1 PI3K PI3K class class III III Beclin Beclin 11 ① ① initiation autophagosome autophagosome initiation mTOR ➃ ➃ fusion fusion ➅ ➅ release release autolysosome lysosome lysosome clearance autolysosome clearance ➄➄ degradation degradation 41  AUTOPHAGY AND MITOPHAGY MITOPHAGY IS A QUALITY CONTROL MECHANISM Non-selective autophagy degrades Mitophagy selectively cytosolic proteins and organelles degrades mitochondria Pathology Parkinson’s disease, Isolation Alzheimer’s disease, and/or ALS membrane Retinopathy Pulmonary hypertension Pink1 Cardiomyocyte senescence Fatty liver disease HEALTHY Cytosolic Damaged Parkin Mitochondrial damage proteins and Mitochondrion organelles Removal of damaged mitochondria PARKINSON’S DISEASE Autophagosome Mitochondrial damage Removal of damaged Ubiquitin mitochondria Lysosome Lysosome 42 Damaged Mitochondrion MITOPHAGY is an autophagic process for removal of damaged mitochondria. Initially, translocation and stabilization of PINK1 on damaged mitochondria facilitate recruitment of Parkin to the mitochondrial membrane from the cytosol. Once Parkin translocated, PINK1 phosphorylates Parkin (E3 ubiquitin PINK1 P ligase) as well as ubiquitin to enhance ubiquitination of the LC3 mitochondrial outer membrane proteins. Mitochondria, Adaptor Protein Ub thus marked, are subsequently engulfed by AUTOPHAGO- SOMES (LC3, Ubiquitin) for lysosomal degrada- Engulfment into tion. (See slides in The Starve feed Cycle) autophagosome Autolysosome Lysosomal degradation of damaged mitochondria II. STIMULATING MITOCHONDRIAL BIOGENESIS 44  SOME MITOCHONDRIAL FUNCTIONS Mitochondria as energy-transducing organelles, the site of ATP production in ENERGY the cell and, as such, maintain cellular energy homeostasis OXIDATIVE Mitochondria generate oxidants (H2O2) that can cause mitochondrial oxida- STRESS tive damage but are also sources of redox signals regulating transcription CELL Mitochondria generate messengers (e.g., cytochrome c) of cell death that DEATH activate and assemble the intrinsic pathways of programmed cell death Mitochondria biogenesis is regulated by transcription factors under the BIOGENESIS control of the co-activator PGC-1a, which senses diverse physiological signals Mitochondria are dynamic organelles that undergo fusion and fission accord- DYNAMICS ing to the cell’s energy needs AUTOPHAGY These are important processes responsible for the breakdown of cellular MITOPHAGY contents, preserving energy and safe-guarding against damaged biomolecules. 45  II. STIMULATING MITOCHONDRIAL BIOGENESIS 1 The sirtuin-PGC1a axis The nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase activity of sirtuins is the basis for modulation of at least 34 distinct targets in mammalian cells. The human sirtuins (Sirt1-7) have been identified to regulate a variety of biological processes, such as: Sirt1 protein protein glucose homeostasis | | gluconeogenesis lys–Acetyl lys mitochondrial biogenesis (PGC-1a) NAD+ insulin secretion 2'-O-acetyl-ADP-ribose adipogenesis and adipolysis + nicotinamide apoptosis senescence metabolism Sirt1 is one of the most versatile and dynamic regulators of cell biology. This invites consideration of sirtuins (with emphasis on Sirt1) as potential drug targets, especially because Sirt1 activators are entering clinical trials in humans. 46  II. STIMULATING MITOCHONDRIAL BIOGENESIS 1 The sirtuin-PGC1a axis Two compounds that activate the PGC1a axis are currently in clinical trials: resveratrol SRT-2104 and SRT-1720. Screening libraries of compounds for their ability to increase the enzymic activity of Sirt1 identified a number of polyphenols –among them resveratrol– that increase the catalytic activity of Sirt1. SRT-1720 and SRT-2104 has a well behaved pharmacokinetic profile and OH is more HO potent than resveratrol. Data are accumulating on the safety and tolerability of resveratrol resveratrol in humans, while the clinical pharmacokinetic and metabolism profiles OH are becoming reasonably well defined. OH H3CO OCH3 HO H3CO resveratrol OH O SRT-1720 N N H3CO OCH3 S N S N H3CO N N N N HN O N O S O SRT-1720 SRT-2104 N N S N 47  II. STIMULATING MITOCHONDRIAL BIOGENESIS 1 The sirtuin-PGC1a axis __________ CLINICAL TRIALS OF SIRTUIN-ACTIVATING COMPOUNDS (2000-2016) _________ Sirtuin-activating Phase I (safety) Phase II (safety and Phase III and IV (pivotal compound efficacy) and post-marketing) Resveratrol Cardiovascular Cardiovascular Cardiovascular Cancer Diabetes Pulmonary Diabetes Neuropathy Diabetes Neuropathy Inflammation Neuropathy SRT-2104 Cardiovascular Inflammation None Diabetes Diabetes Inflammation Bonkowski & Sinclair (2016) Nat Rev Mol Cell Biol 17, 679 48  II. STIMULATING MITOCHONDRIAL BIOGENESIS 2 The AMPK-PGC1a axis NH2 AICAR (5-AminoImidazole-4-CArboxamide Ribonucleoside) O N activates PGC1a by generating inosine monophosphate (IMP), H2N which acts as an AMPK agonist by mimicking AMP. AICAR is transported across the plasma membrane as inosine mono- HO phosphate. Treatment with AICAR in animal models of HO OH cytochrome oxidase (COX) deficiency led to an activation of AICAR 5-AminoImidazole-4- CArboxamide Ribonucleoside the AMPK-PGC1a axis and correction of COX deficiency. β α γ AMPK AMPK IMP IMP active AMPK allosteric site binding of AMP 49  II. STIMULATING MITOCHONDRIAL BIOGENESIS The Biogenesis Program - Regulation of PGC1a: AMPK and Sirt1 caloric high fat, sedentary INPUTS restriction exercise high sugar life lipids EFFECTORS ↑NAD+ ↑AMP ↓ amino ↓glucose insulin acids AICAR Sirtuins AMPK mTOR IIS SENSORS Resveratrol DNA repair Chromatin modifications Mitochondrial biogenesis and function Reduced inflammation PHYSIOLOGICAL Stress resistance Translation fidelity PROCESSES Stem cell maintenance Telomere maintenance Autophagy Disease Health Frailty Homeostasis Pathophysiologies OUTCOMES Disease-free Compressed morbidity 50  ENERGY- AND NUTRIENT SENSORS 1 AMPK Some of the metabolic effects that ensue following AMPK activation are particularly relevant to treatment of type 2 diabetes: Agonists → Ca2+ → CaMKK ANABOLISM Catabolic pathways are activated by AMPK: glucose uptake via GLUT4 and LKB1 AMPK GLUT1, glycolysis, fatty acid uptake, Metabolic → AMP CATABOLISM stress fatty acid oxidation, mitochondrial bio- inhibition of anabolism activation of catabolism genesis, and autophagy. fatty acid synthesis glucose uptake Agonists Anabolic Capathways 2+ CaMKKare inhibited by mTOR activation anabolism protein synthesis glycolysis AMPK: fatty acid, triglyceride, LKB1 AMPK chole- sterol, glycogen, protein, and rRNA. AMPK catabolism Metabolic synthesis; stress transcription AMP of lipogenic cholesterol synthesis fatty acid oxidation enzymes, transcription of gluconeogenic gluconeogenesis mitochondrial enzymes. biogenesis 51  AMPK STIMULATES GLUCOSE UPTAKE (GLUT4) AICAR (an activator of AMPK) facilitates glucose uptake in muscle (GLUT4) by two mechanisms: Similar to the effect of insulin, AMPK activates the translocation of GLUT4 from the intracellular vesicles to the plasma membrane upon activation of a monomeric GTPase AMPK phosphorylates HDAC5, thereby releasing MEF2 (Myocyte Enhancer Factor- 2) from the complex. MEF2 translocates to the nucleus and activates the expression of GLUT4 AICAR GLUT4 Rab-GTP AS160 AMPK GAP Rab-GDP GLUT4 P GL UT HDAC5 4 HDAC5 T4 GLU MEF2 GLUT4 MEF2 HDAC5: Histone DeACetylase 5 MEF2: Myocyte Enhancer Factor-2 52 II. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 53  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 gluc glucose NORMAL Metabolic differences between normal- and cancer cells CELL GLUT Normal cells primarily metabolize glucose to pyruvate for glucos glucose-6P growth and survival, followed by complete mitochondrial oxidation of pyruvate to CO2 through the TCA cycle and the pyruvate pyru oxidative phosphorylation, generating 36 ATP/glucose. O2 is TCA OXPHOS essential for it is required as the final electron acceptor. O2 When O2 is limited, pyruvate is metabolized to lactate. CO2 ATP Aerobic glycolysis Ana 36 ATP / Glucose 2A glucose glucose NORMAL CANCER CELL Cancer cells convert most glucose to lactate regardless of GLUT GLUT CELL the availability of O2 (the Warburg effect), diverting glucose-6P glucose glucose-6P metabolites from energy production to anabolic processes to accelerate cell proliferation, at the expense pyruvate of generating pyruvate lactate MCT4 lactate only 2 ATP/glucose. Increases in glucose consumption TCA OXPHOS and lactic acid release are characteristics of the cancer cell; O2 CO2 ATP lactate levels positively correlate with the aggressiveness of several types of human cancers. Aerobic glycolysis 36 ATP / Glucose Anaerobic glycolysis 2 ATP / Glucose 54  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Metabolic differences between normal- and cancer cells a. Cancer cells produce enough energy to survive when supplies and waste disposal are limited At a remote location from perfused blood vessels, hypoxic tumor cells rely on glycolysis for survival and proliferation. High ATP production depends on glucose availability and is associated with lactate release (facilitated by MCT4). Oxygenated tumor cells have a metabolic preference for lactate versus glucose. In the presence of O2, lactate is oxidized to pyruvate by lactate dehydrogenase 1 (LDH1) and pyruvate fuels the tricarboxylic acid cycle to produce ATP. The metabolic preference of oxidative tumor cells for lactate allows hypoxic tumor cells to get access to high levels of glucose. OXYGENATED OXIDATIVE HYPOXIA GLYCOLYTIC CANCER CELL CANCER CELL MCT4 MCT4 lactate lactate lactate pyruvate BLOOD VESSEL LDH1 TCA OXPHOS glucose glucose GLUT CO2 ATP glucose glucose O2 55  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Metabolic differences between normal- and cancer cells glucose a. Cancer cells produce enough energy to GLUT survive when supplies and waste disposal glucose are limited. HIF-1a is major key player in ↓ HK2 glucose-6P ↓ isomerase cancer cells; it is activated under hypoxic fructose-6P ↓ PFK (low pO2) conditions. HIF-1a increases the fructose-1,6-P ↓ aldolase expression of GLUT1 and GLUT3 as well ↓ glyceraldehyde 3P as of the monocarboxylate transporter HIF-1 ↓ GAPDH HIF-1 1,3-bisP glycerate MCT4, which releases lactate and H+, ↓ P-glycerate kinase 3P-glycerate ↓ P-glycerate mutase resulting in extracellular acidification. HIF- 2P-glycerate ↓ enolase PDH kinase-1 1a increases the expression of enzymes of P-enolpyruvate ↓ pyruvate kinase anaerobic glycolysis: hexokinase-2 (HK2), Pyruvate PDH pyruvate → acetyl-CoA LDH5 phosphofructokinase-2 (PFK), pyruvate lactate kinase (PK), and lactate dehydrogenase 5 MCT4 (LDH5). lactate 56  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Metabolic differences between normal- and cancer cells b. Cancer cells uncouple the TCA from the respiratory chain to enable biosynthesis of cell constituents even in the presence of O2. This leads to the leakage of citrate, isocitrate and other intermediates of the TCA cycle to be used for biosynthetic purposes. Glycolysis remains the main provider of ATP and serves as a hub for biosynthetic pathways. NORMAL CELL CANCER CELL CANCER CELL glucose glucose glucose glycolysis glycolysis glycolysis ATP ATP ATP pyruvate pyruvate lactate pyruvate lactate cytosol citrate lipids pyruvate mitochondria pyruvate pyruvate acetyl-CoA acetyl-CoA acetyl-CoA TCA TCA TCA O2 O2 O2 NADH I Q III c IV NADH X I Q III c IV NADH X I Q III c IV H 2O 57  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Metabolic differences between normal- and cancer cells: Glutaminolysis b. Cancer cells uncouple the TCA from the respiratory chain to enable biosynthesis of cell constituents. Glutaminolysis is a major cancer cell mechanism to uncouple the tricarboxylic acid cycle from oxidative phosphorylation that –even in the presence of pyruvate nucleotides fatty acids O2. Glutamine activates mTOR; Manipula- NADPH tion of glutaminolysis and mTOR is an acetyl-CoA NADP+ ME important drug target for cancer treatment. nucleotides glutamine malate aspartate citrate GT OAA citrate glutamine mTOR malate isocitrate NH4+ NH4+ H2O TCA GLS GDH fumarate α-ketoglutarate glutamate AT succinate succinyl-CoA amino keto acid GSH acid 58  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Oncogenesis ­ Tumor suppressors ¯ Hypoxia (e.g., PI3K/Akt) (e.g., p53, PTEN) HIF1a ­ Glycolysis ­ Glutaminolysis ­ OXPHOS ¯ TUMORIGENESIS ­ 59  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 ADP Anticancer targets in glycolytic tumors ATP GLUCOSE GLUCOSE-6P Mammalian tissues have 4 hexokinase (HK) HK isoforms that catalyze the first irreversible step HK1, HK2 HK4 (glucokinase) associated with the liver and pancreas of glycolysis: glucose + ATP ® glucose-6-P + outer mitochondrial low affinity for glucose membrane ADP. HK-1-3 have a high affinity for glucose high affinity for glucose HK3 perinuclear compartment relative to HK-4 (also termed glucokinase). HK- high affinity for glucose 1 and HK-2 are associated with the outer mito- glucose-6P chondrial membrane; HK-4 occurs in cytosol of ADP ADP pancreas. VDAC ANT ATPase ATP ATP Hexokinase-2 Inhibitors VDAC in the outer glucose membrane is associated with ANT in the inner membrane that, in turn, is associated with complex V (ATPase). This complex is termed glucose-6P ATP synthasome. The association of HK-2 with ADP ADP VDAC provides preferential access to mito- HK VDAC ANT ATPase ATP ATP chondrion-generated ATP, a requirement for glucose phosphorylation of glucose to glucose-6P. 60  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Anticancer targets in glycolytic tumors ! Hexokinase-2 Inhibitors The association of HK-2 with the ATP synthasome supports ATP — Hexokinase OMM activation of glycolysis by HK2 having — VDAC immediate access to mitochondrion- IMM generated ATP. This makes HK2 a target for cancer therapy, with strategies aimed ATP ATP Synthase (Complex V) either at dis-lodging HK2 from VDAC or inhibiting HK2 or inhibiting mitochondrial ATP HK2 VDAC pyruvate dehydrogenase kinase. ! ! 61  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Anticancer Targets in the Glycolytic Metabolism of Tumors HK-2 is overexpressed in tumors and this overexpression disrupts the bioenergetic flux as observed in cancer cells. In normal cells –with a low expression of HK-2– glycolysis (glucose ® pyruvate flux) coordinates with oxidative phosphorylation (pyruvate ® ATP flux), whereas in cancer cells, the overexpression of HK-2 drives the conversion of glucose to lactate, thus disrupting this metabolic coupling. Glycolysis Oxidative phosphorylation glucose pyruvate metabolic flux pyruvate ATP flux metabolic coupling normal cells Glycolysis Oxidative phosphorylation glucose lactate metabolic flux pyruvate ATP flux dysregulation of metabolic coupling by overexpression of HK-2 62  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Anticancer Targets in the Glycolytic Metabolism of Tumors Targeting tumors for destruction via small molecule-mediated inhibition of the HK-2/VDAC glucose interaction GLUT4 Plasma " membrane 6P-gluconolactone pentose phosphate pathway glucose glucose-6P clotrimazole glycolysis bifanazole Dislodge HK-2 methyl jasmonate from VDAC 3-Br-Pyruvate Analogs of HK-2 N-terminal ATP ATP ADP cinnamic acid chlotrimazole disloge VDAC !HK-2 HK2 pyruvate derivatives cinnamic acid derivatives bifanazole dislodge Inhibition (#-cyano-4-OH-cinnamic acid) methyl jasmonate from3-Br-Pyruvate HK2 ! —VDAC VDAC LDH of MCT DCA lactate lactate ATP dichloroacetate " pyruvate MCT PDHK PDH–P PDH acetyl-CoA 63  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Anticancer Targets in the Glycolytic Tumors Hexokinase-2 Inhibitors: 3-Br-pyruvate has been very effective in eradicating tumors in immuno-competent animals, exerting a rapid loss of cellular ATP in those tumors that show a robust Warburg effect, and eradicate- ing a large rat hepatocellular carcinoma in 3 weeks without recurrence (Ko et al, Biochem. Biophys. Res. Commun 324). 64  III. CANCER, THE WARBURG EFFECT, AND HEXOKINASE-2 Anticancer Targets in the Glycolytic Metabolism of Tumors Leading therapeutic compounds targeting glycolytic metabolism of tumors ___________________________________________________________________________________________________________________ Target Compound Mode of Action Current Clinical Status ___________________________________________________________________________________________________________________ GLUTs 2DG Compet