III

1. Georgia Neurological SocietyAnnual Meeting 2011Neurometabolic Disease in Adult Neurologic Practice H+ H2O O2 NADH ATP ADP NAD+ II V I III IV CoQ10 C H+ H+ H+ H+

2. Research Grants 2009-2010 Department of Defense, Grant: Mitochondrial Defects in Autism AR080046 2009-2012 Department of Defense, Grant: Mechanisms of mitochondrial defects in Gulf War Syndrome GW080138 2010-2013 Department of Defense, Grant: Mechanisms of mitochondrial dysfunction in autism. Combined laboratory analysis and functional MRI assessments. AR093329 2010-2011 PND Association Research Grant Program titled "Keratinocytes and / or melanocytes as a cell source for the enzyme assay of tyrosine hydroxylase" 2005-2010 Subcontract for NIH HPA grant (UO1 AI057565): Hyperphenylalaninemia in cerebral malaria

3. Cerebral Folate Defects Mitochondrial Diseases Treatment

4. Cerebral Folate Defects: Definition What are cerebral folate defects and how are they diagnosed? Any neurological syndrome associated with a low CSF 5-methyltetrahydrofolate (5-MTHF) (<50 mmol/L) in the presence of NORMAL peripheral folate status. (Dev Med Child Neurol 46:843;2004) If diagnosed early enough, these defects are treatable.

5. Cerebral Folate Defects: Causes What causes cerebral folate defects? Genetic Disease Drugs Autoimmune Disease Cerebral folate defects affect ALL AGE GROUPS. For more detailed information about various conditions causing CFD, email me at: jshoffner@mnglab.com Hyland K, Shoffner J, Heales SJ. J Inherit Metab Dis. 2010 Oct;33(5):563-70

6. Cerebral Folate Defects: Symptoms What are the symptoms of CFD? Demyelination Epilepsy (essentially all types) Movement disorders Neuropathies Neurobehavioral (autism) and psychiatric disorders Cognitive impairment and mental retardation Spasticity NOT ANEMIA IN MOST CASES !!! Symptoms evolve over time

7. Cerebral Folate Defects: Mechanisms Plasma Choroid Plexus CSF 5-MTHF ATP 5-MTHF RFC FR1 3-4x Higher than plasma FolatePolyglutamate Pool 5-MTHF = 5-methyltetrahydrofolate; FR1 = Folate receptor 1; RFC = Reduced Folate Carrier

8. Cerebral Folate Defects: Causes

9. Mitochondrial Disease Not just a defect in ATP production ATP production Free radical generation Apoptosis Mitochondrial/Endoplasmic Reticulum Calcium regulation Effects on other metabolic pathways Toxic interactions

10. Glucose + Fatty Acids = ATP! GLYCOGEN Medium Chain (C8-C12) Long Chain (C14-C20) Short Chain (C4-C6) GLYCOLYSIS Fatty Acids Alanine Pyruvate Lactate L-Carnitine CPT I CPT II VLCAD Long Chain (C14-C20) Pyruvate PDHC Acetyl -CoA Medium Chain Acetyl -CoA Short Chain TCA -oxidation H+ H2O O2 NADH ATP ATP ADP ADP NAD+ II A N T V I III IV CoQ10 C H+ H+ H+ H+ P O R ADP ATP

11. Complex III Complex I Complex IV HOW IT REALLY WORKS: SUPERCOMPLEXES Stability and AssemblyJ BiolChem 279;36349,2004

12. Supercomplexes + Monomeric OXPHOS Enzymes Blue Native Electrophoresis The higher molecular weight bands may be oligomeric forms of Complex V. I1III2IVn + CVn * * I1III2IV1 CI1CIII2 Complex III Complex I Dimeric CV Monomeric CV Dimeric CIII Monomeric CIV Complex IV NormalPatient 1Patient 3 Pediatric Adult SURF1 SURF1

13. Complex IV: CLEAR NATIVE GEL Monomeric OXPHOS Enzyme Assembly Monomeric Complex IV Monomeric Complex IV * * * * NormalPatient 2 Patient 3 Normal Pediatric Adult NormalPatient 1 Patient 2 Normal Pediatric Pediatric

14. Mitochondrial Disease: Treatment Irreversible Reversible Normal

15. Mitochondrial Dysfunction: Threshold Expression Normal OXPHOS Function Abnormal Normal Phenotype

16. Cerebral Folate Deficiency: Adolescent Mitochondrial disease: Adolescent • Kearnes-Sayre Syndrome • Sporadic mtDNA Deletion • Abnormal choroid plexus Progressive loss of balance and motor function. Became confined to bed over several months. CSF 5-MTHF 8 (NL 50-120) CFD observed in over 80% of Kearnes-Sayre cases

17. Genetic Diagnosis Rapidly Evolving: mtDNA ----16,569 nucleotides Nuclear DNA----~1400 genes Next Generation Sequencing

18. Cerebral Folate Deficiency: Adolescent Mitochondrial disease: Adolescent Kearnes-Sayre Syndrome

19. Cerebral Folate Deficiency: Adolescent Mitochondrial disease: Adolescent Treatment with Leucovorin (Folinic acid) 25mg po bid. Resolution of symptoms and return to baseline: Walking independently, going to school, complete self care

20. Fatigue and Myalgias Cerebral Folate Deficiency: Adult Onset Mitochondrial disease: Adult Onset 41 year old woman 20-25 years: Less endurance than peers 26 years – present: Progressive fatigue, severely limiting activity 36 years: Distal pain, paresthesias in hands and feet; Cognitive complaints (calculations, word retrieval, memory) Brain and spinal cord imaging: Negative EMG/NCV: Negative Rheumatological Testing: Negative Two sons: One healthy (9 years old). One with muscle weakness, leg cramps, myopathic EMG, normal CPK.

21. Exercise Testing: Essential for diagnosing and following fatigue Anaerobic Threshold (AT) Patient: 10 ml/kg/min Predicted: >11 ml/kg/min Peak VO2 Max Patient: 1.44 L/min Predicted: ≥ 1.83 L/Min Predicted normal ranges: Based on age, gender, weight, height, BMI) Normal Pulmonary Function Testing Exercise Testing: Met-Test

22. Fatigue and Myalgias Cerebral Folate Deficiency: Adult Onset Mitochondrial disease: Adult Onset Muscle Biopsy: Sarcoplasmic Masses Epidermal Nerve Fiber Density: Decreased consistent with a small fiber neuropathy CSF 5-methyltetrahydrofolate: 25 (NL >50 mmol/L) CSF protein: 45 (NL 15-45) Mitochondrial Disease CSF Alanine: 44.9 µmol/L (NL 20.8-35.4 µmol/L) Muslce Biochemistry: Complex I and Complex III Defect

23. Fatigue and Myalgias Cerebral Folate Deficiency: Adult Onset Mitochondrial disease: Adult Onset Genetics: Mitochondrial DNA (nuclear and mtDNA) Pending FOLR1 (Folate Receptor) Single mutation in Exon 3: c.352C>T P.Q118X Stop codon producing a truncated protein May increase susceptibility to developing a cerebral folate defect

24. Fatigue and Myalgias Cerebral Folate Deficiency: Adult Onset Mitochondrial disease: Adult Onset Folinic acid Treatment: 25mg PO every 8 hours Improved neuropathic symptoms: Pain and dysaesthesias Improved episodes with difficulty thinking (calculations, word retrieval, memory)

25. Adult Onset Fatigue and MyalgiasSyndromes A retrospective analysis of evaluations performed on 62 patients was performed. 36 adults (>19 years) (11 male; 25 female) Mean age ± SD = 39.2 ± 9.2 years; Median = 39.5 years; Range 20-60 years

26. Fatty acid oxidation diseases, glycogen storage diseases, autoimmune disease, congenital myopathies and muscular dystrophies were excluded.

27. Mitochondrial Disease Treatment Coenzyme Q10: Mildly effective Exception: Primary (genetic) CoQ10 deficiency. Response can be dramatic. Clinical investigations: EPI-743 (Edison Pharmaceutical): Effect not clear in Leigh Disease (2 patients). Drug currently under study Other companies: Collaboration moving forward on agents showing improvements in cells with mitochondrial dysfunction

28. Mitochondrial ATP Generation: Medical Neurogenetics ResearchMitochondrial and Cytoplasmic Luciferase

29. Georgia Neurological SocietyAnnual Meeting 2011Neurometabolic Disease in Adult Neurologic Practice H+ H2O O2 NADH ATP ADP NAD+ II Thank You! V I III IV CoQ10 C H+ H+ H+ H+