1. A pain physician’s tour of mitochondrial diseases

2. Outline Why Mitochondria? Who are the Mitochondria? Biochemistry Genetics Clinical syndromes Management Strategies Conclusion Mitochondria in Pain?

5. Why Mitochondria? Mitochondria involved in basic pain mechanisms Nociception Centralisation Analgesic tolerance Mitochondrial dysfunction underlies some clinically unexplained syndromes Fibromyalgia, Chronic Fatigue Syndrome Mitochondrial dysfunction in common disorders Nerve and Muscle disease eg Diabetic Neuropathy Depression Neurodegenerative Disorders

6. Who are the Mitochondria ? Mitochondria are the power supply for all cells Mitochondrial disease can impact on many systems Brain, Nerve and Muscle most energy dependent Mitochondrial and Nuclear genetic control

7. The humble mitochondrion Originally a symbiotic bacteria Each mitochondria has multiple copies of its own DNA Co dependence on nuclear derived genes and proteins Key function is oxidative phosphorlyation: Utilise O2 to produce ATP from ADP Respiratory Chain: 5 complexes working sequentially

10. Mitochondrial Biochemistry Complexes I to IV essentially strip electrons from hydrogen atoms (NADH/H2O), pumping protons into the INTERMEMBRANE SPACE This creates a large pH gradient, passage of protons through complex V is coupled to generation of ATP Process relies on ELECTRON TRANSFER CHAIN including various co-enzymes (Co Q10) Generates reactive oxygen species which are mopped up by other pathways (ubiquinone, lysosomes)

14. Mitochondrial Genetics 16,569 base pairs, closed loop of DNA Multiple copies in the matrix DNA encodes for 13 of 90 respiratory chain proteins the rest are coded from nuclear DNA Almost no noncoding areas, no introns – unstable Mitochondrial DNA maternally inherited Nuclear mutations follow Mendelian inheritance Mutations accumulate over time, heteroplasmic Tissue mosaicism

15. Mitochondrial DNA

16. Almost no introns, no histones: unstable No DNA repair enzymes Mitochondria dependent upon DNA replication to continue functioning Exposed to free radicals

17. Mitochondrial heteroplasmy

18. Mitochondrial genetic disorders Wide range of symptoms and syndromes Fatal encephalopathy or acidosis in first weeks of life Indolent muscle disorder such as ophthalmoplegia BRAIN: seizures, myoclonus, infarcts, ataxia, dystonia NERVE: deafness, blindness, peripheral neuropathy MUSCLE: eye muscles, proximal muscles, weakness, exercise intolerance, cardiomyopathy OTHER: Diabetes, Deafness, short stature, lactic acidosis, liver, pancreas, lipodystrophy

20. Mitochondrial genetic syndromes Mitochondrial Mutations Leber’s Hereditary Optic Neuropathy. Sequential, severe and irreversible visual loss in young males. LHON gene causes blindness in 50% m, 10% f pts CPEO: slowly progressive opthalmoplegia. Onset>50 usually a mtDNA deletion, sometimes point mutation CPEO plus: multiple DNA deletions can result in neuropathy, Parkinsonism, ataxia, retinopathy, cataract, deafness, hypogonadism, depression Kearns Sayre Syndrome (KSS): single large mt DNA deletion. Retinopathy, CPEO, cardiac conduction block. Limb myopathy, ataxia. Onset < 20. Limited lifespan.

22. Mitochondrial Syndromes Mitochondrial Mutations MELAS: Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke like events. Metabolic infarcts cross normal vascular territories. Headache, nausea, vomiting, seizures, hemiparesis, hemianopia or cortical blindness. Defect in mt transfer RNA 3243 A>G MERRF: Myoclonic Epilepsy with ragged red fibres. Stimulus sensitive Myoclonus, seizures, ataxia, myopathy, cardiomyopathy. Onset in childhood. mtDNA mutation

23. MELAS infarction

24. Ragged Red Fibre

25. Mitochondrial Syndromes Nuclear Mutations MNGIE: mitochondrial neurogastrointestinal encephalomyopathy. CPEO, myopathy, neuropathy, intestinal pseudo obstruction. Laparotomies, malnutrition. May have a leucoencephalopathy. Nuclear DNA mut. thymidine phosphorylase Leigh Syndrome: childhood onset symmetrical necrosis of brainstem and basal ganglia. Nystagmus, ataxia, dystonia, respiratory disease, pneumonia. Severe, progressive, fatal. Final common pathway of severe defects, many nuc DNA, some mtDNA

27. Mitochondrial Genetic Syndromes NARP: Neuropathy, ataxia, retinitis pigmentosa. Dementia, seizures, developmental delay. Neurogenic limb weakness. mtDNA point mutation affecting complex V. High mutation load causes Leigh Syn. Maternally inherited Deafness, Diabetes. Aminoglycoside Deafness. Mt DNA 1555A>G mut confers extreme sensitivity to AG. 1:500 population.

28. Mitochondrial Dysfunction Genetic syndromes are striking but rare Increasing recognition of involvement of mitochondria in more common diseases, especially nerve and muscle disease

29. Mitochondrial Dysfunction Neuropathy HAART neuropathy Chemotherapy neuropathy: paclitaxel, Cis-Plat, Vinca Diabetic distal symmetric polyneuropathy Myopathy Statin myopathy Fibromyalgia CNS disorders Parkinson’s, Huntington’s, Friedreich’s, Migraine, Depression Cardiovascular Disorders Role in Endothelial function. CoQ10 levels predict mortality

30. HAART toxicity Reverse transcriptase inhibitors are nucleoside analogues. Inhibit mitochondrial as well as viral DNA synthesis Also inhibit adenylate kinase, ADP/ATP translocator R-T side effects include myopathy, neuropathy, lactic acidosis, fatty liver, pancreatitis, nephrotoxicity. Fatalities reported with Ribavirin (Hep C) plus didanosine HIV lipodystrophy closely resembles a mitochonrial disorder

31. HAART Neuropathy Occurs in up to two thirds of patients on RTIs zalcatibine>didanosine>stavudine>lamivudine>zidovudine>abacavir Neuropathic pain, ascending numbness Poorly responsive to conventional agents Forces a change in therapy, compromising efficacy Inhibition of mitochondrial DNA synthesis results in energy deficiency in peripheral nerve tissues Reduced epidermal nerve fibre density, sweat gland innervation.

32. Chemotherapy Neuropathy Paclitaxel, Doclitaxel, Platinum, Vinca Alkaloids Inhibit mitochondrial DNA and RNA synthesis Neuropathy frequent, limits dose and effectiveness Potential for treatment with metabolic support

33. Diabetic Neuropathy Mitochondrial dysfunction a key player in Type 2 DM Glucose binds to Hexokinase, located on outer mitochondrial membrane Glucose phosphorylation voltage dependent Glucose -6-P, ADP production stimulate oxidative phos G6P regulates mitochondrial function at several points NADH donation to complex I FADH to complex II ADP to Complex V In muscle, ATP turnover stimulates glycolysis, G6P Excess glucose levels PLUS reduced O2 tension lead to an increase in free radical production0.

34. Acetyl L Carnitine Maintains supply of acetyl-Co A Contributes to phospholipid synthesis Activates growth factor receptors Promotes regeneration, neuroprotective Analgesic properties Acute pain (cholinergic) Chronic pain (glutaminergic) Increases ACTH and beta endorphin levels

35. Acetyl L Carnitine Improves pain scores, NCS in diabetic neuropathy: 333 pts, ACL 2g/d. Pain reduced by 40%, NCV better 1364 pts Vibration sense up, Pain down, histological + Neuroprotective in Laboratory models of antiretroviral and chemotherapy induced neuropathy Clinically effective in HAART neuropathy 21 HIV patients on RTIs: ALC 1.5 grams bd improved neuropathic pain in 76% of pts skin and sweat gland nerve fibre density inc by 100%

36. ALC for Diabetic Neuropathy

37. Muscle Disease Statin Myopathy Statins inhibit CoQ10 synthesis Mitochondrial dysfunction. Ragged red fibres. Myalgia Co Q10 supplements reduce pain AND improve function by 40% (no improvement with Vit E) Am J Cardiol 2007;99(10):1409 Fibromyalgia Several lines of evidence indicating CoQ10 deficiency in fibromyalgia, chronic fatigue/ME

40. More Disorders Depression CoQ10 levels lower in depressed pts p<0.0002 Lower again in patients with CFS or treatment resistant depression Link to cardiovascular mortality Cardiovascular Disease Efficacy of statins predicted by CoQ10 levels CoQ10 levels an independent predictor of survival CoQ10 improves CHF: increased LVEF, Cardiac OP

41. Coenzyme Q10

42. Q is for Quinone, 10 is the number of isoprene units Complex synthesis, Quinone group from tyrosine, isoprene side chains from acetyl CoA (Cholesterol) Statins reduce CoQ10 levels by 40% Oil soluble, poor oral absorption Found in heart, liver, nuts, soy or grapeseed oil. Average intake 3-6mg/d, supplement at 150-300mg/d Can lower BP by 17mmHg. Reduces insulin resistance Studies in aging, dementia, PAIN….

44. Mitochondrial Mechanisms in Pain Capsaicin induced hyperalgesia Reactive Oxygen species (ROS) from mitochondria Superoxide Dismutase SOD2 Mechanical Hyperalgesia ROS scavengers powerful antinociceptives Mitochondrial Complex I or complex III inhibitors induce long lasting hyperalgesia Morphine Antinociceptive tolerance Inactivation of mitoSOD leads to excess peroxynitrite Nitrative and Oxidative stress results in sensitisation Effect reversed by peroxynitrite scavengers

45. Mitochondria and hyperalgesia

46. Making the Diagnosis Well developed syndromes present no difficulty Pointers to possible mitochondrial disorder: Myopathy, Neuropathy, Deafness, Diabetes, Cardiomyopathy or conduction defect. Stroke, migraine, seizure, dementia Serum Lactate, lactate/ pyruvate ratio. CK rise. Changes on ncs/EMG. CNS imaging. Muscle Biopsy Gomori trichrome stain demonstrates accumulation of abnormal mitochondria – Ragged Red Fibre

47. Ragged Red Fibre

48. Diagnosis Muscle Biopsy: other techniques Cytochrome C (COX) and Succinate DH staining Specific enzymatic analysis. Tricky: sample handling, differing lab evaluations. Age related changes. Looking for reduced biochemical activity of various complexes. Genetic Analysis Specific tests for specific syndromes: MELAS, MERRF, LHON, adult Leigh syndrome. Deletion analysis for CPEO, KSS, myopathies

49. Management Supportive Diabetes – due to energy failure not insulin resistance. OHAs, minimal insulin. NO metformin!! Seizures – AVOID valproate as it inhibits function. Levetiracetam, Lamotrigene, Clonazepam Respiratory and Bulbar weakness. Noninvasive ventilation, PEG tubes etc. Cardiac – KSS and others may require Pacemaker. Be alert for WPW and SVT.

50. Management Pharmacology Coenzyme Q10 Acetyl Carnitine Limited efficacy: Riboflavin, alpha lipoic acid, Vitamin A,D,E,K Trials: benzafibrate, reservatrol, PCC1 alpha. L- arginine for MELAS Metabolic agents: bicarbonate, dichloroacetate MNGIE- stem cell transplantation

51. Management Resistance Training Mitochondrial genetic material is heteroplasmic, and dynamic. Mitochondria have a social life! Proportions of mutant and wild type DNA are not stable. Resistance training can apply selection pressure in favour of more functional DNA. Population of “satellite cells” in muscle with high proportions of wild type DNA. Exercise physiologist supervised protocols available.

52. Resistance Training

53. Management Gene Therapy In vitro studies. All promising, none proven. Insertion of nuclear ATP6 gene to replace defective mt gene in NARP Similar approach to correct ND4 mutation in rat model of LHON Zinc finger nucleases to bind and compromise mutant mtDNA Introduction of cytosolic tRNA to replace defective mt tRNA

54. Conclusion Complex group of diseases Genetic disorders have CPEO, Myopathy, Neuropathy, Retinopathy, Cardiomyopathy, myoclonus, seizures, funny stroke syndromes, migraine, ataxia, dystonia, acidosis, diabetes, deafness, short stature Metabolic and Neurological Disease: Diabetes, Heart Disease, Neurodegenerative disease. Pain and related disorders: Depression, Fibromyalgia, CFS Important role in basic pain mechanisms

55. Conclusion Treatment is largely supportive Coenzyme Q10 supplements generally worthwhile Resistance training useful for muscle disease Increasingly wide spectrum of disorders affecting or affected by mitochondrial function.