Florence Molinari - INMED-INSERM U901. Marseille.
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GC1 mutations can cause early epileptic encephalopathies. The document discusses: 1) GC1 mutations have been found in children with early epileptic encephalopathies and suppression bursts. GC1 is the mitochondrial glutamate carrier that transports glutamate into mitochondria. 2) Inactivating GC1 in astrocyte cultures leads to decreased NADH production, impaired mitochondrial membrane potential activation by glutamate, and decreased ATP levels. 3) This suggests GC1 is crucial for astrocyte glutamate metabolism and mitochondrial function, and its deficiency may cause excitotoxicity through impaired glutamate clearance from the extracellular space.
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Florence Molinari - INMED-INSERM U901. Marseille.
- 1. Early Epileptic Encephalopathies related to the deficit of the mitochondrial glutamate carrier SLC25A22 Florence Molinari INMED, INSERM U901 Parc Scientifique de Luminy, Marseille, France florence.molinari@inserm.fr
- 2. Early Epileptic Encephalopathies Severe brain disorders in which the epileptic electrical discharges may result in a progressive psychomotor dysfunction Early onset < 6 months of life Severe EEG abnormalities Multi-form of seizures (Myoclonic seizures, spasms…), focal or multi-focal Seizures are intractable Cognitive, sensitive, motor impairments and behavioral disturbances Very poor prognostic
- 3. Suppression Bursts (SB) « Bursts of polyspikes which alternate with lack of electric activity » Early Infantile Epileptic Encephalopathy Description in 1976 by Ohtahara Early Myoclonic Encephalopathy Description in 1978 by Aicardi & Goutières Ohtahara S and Yamatogi Y, J Clin Neurophysiol, 2003 Early Epileptic Encephalopathies with « suppression bursts »
- 4. Ethiological factors: Brain lesions or malformations Metabolic disorders : Non-ketotic hyperglycinaemia; propionic aciduria, methylmalonic acidaemia, D-glyceric acidaemia… Mitochondrial complexes disorders Early Epileptic Encephalopathies
- 5. Gene Chromosome Function Reference ARX * Aristaless Related homeobox Xp21 Transcription factor Regulation of differentiation, proliferation and tangential migration (neurons precursors and interneurons) Kato et al., AmJ HumGenet 2007 CDKL5 Cyclin-Dependent Kinase-Like 5 Xp22 Serine-Threonine kinase Rosas-Vargas et al., J Med Genet 2008 ErbB4 erythroblastic leukemia viral oncogene homolog 4 2q34 Epidermal growth factor receptor (EGFR) family Backxet al, Eur J HumGenet, 2009 GLUL or GS Glutamine synthétase 1q31 Astroglial glutamate degradation Haberle et al., N Engl J Med, 2005 GRIN2A Glutamate Receptor, Ionotropic, N-methyl D-aspartate 2A 16p13 NMDA-R2A Endele et al., Nat Genet, 2010 KCNQ2 Potassium voltage-gated channel, KQT-like subfamily, member 2 20q13.3 IM Potassium Channel, neuronal excitability break Weckhuysen et al, Ann Neurol, 2012 Kato et al, Epilepsia, 2013 Milh et al, Orphanet J Rare Dis, 2013 KCNT1 Potassium channel, subfamily T, member 1 9q34.3 Sodium-activated potassium channel neuronal excitability break Barcia et al, Nat Genet, 2012 MAGI2 Membrane-associated Guanylate Cyclase Inverted 2 7q11.3-q21.1 Scaffolding enzyme (interaction with NMDA-R) Marshall et al., AmJ HumGenet, 2008 PCDH19 Protocadherin 19 Xq22 Control of the calcium-dependent cell-cell adhesion Dibbens et al., Nat Genet, 2008 PLC b 1 * Phospholipase Cb1 20p12.3 Cellular signaling Kurian et al., Brain, 2010. PNKP * Polynucleotide Kinase 3’-Phosphate 19q13.33 DNA repair network Shen et al., Nat Genet, 2010. PNPO * Pyridoxamine 5'-phosphate oxidase 17q21 Vitamin B6 metabolism. Mills et al., HumMol Genet, 2005 SCN1A, SCN2A *, SCN8A Sodium Channel Neuronal Type 1, 2, 8 a-Subunit 2q24.3 12q13.13 NaV1.1, NaV1.2, NaV1.6 Generation and propagation of action potentials Heron et al., J Med Genet, 2009 Ogiwara et al., Neurology, 2009 Veeramah et al., Am. J. Hum. Genet, 2012 SLC25A22 or GC1 * Mitochondrial Glutamate Carrier 1 11p15 Mitochondrial glutamate transport Molinari et al, AmJ HumGenet, 2005; Molinari et al, Clin Genet, 2009; Poduri et al., Ann Neurol, 2013; Cohen et al., Eur j Paediatr Neurol, 2014 SPTAN1 Nonerythrocytic a-Spectrin-1 9q33-q34 Regulation of the axonal structure stability Saitsu et al., AmJ HumGenet, 2010 SRGAP2 Slit-Robo GTPase activating Protein 2 1q32.1 Regulation of neuronal migration and neurite outgrowth and branching Saitsu et al., AmJ Med Genet, 2011 STXBP1 * Syntaxin Binding Protein 1 9q34 Synaptic vesicular release Saitsu et al., Nat Genet, 2008 TBC1D24 TBC1 domain family, member 24 16p13.3 Maturation of neuronal circuits Milh et al, HumMut, 2013 Early Epileptic Encephalopathies * with suppression burst
- 6. The SLC25 family 53 members that transport metabolites, nucleotides and cofactors across the IMM 4 are involved in the mitochondrial glutamate transport The mitochondrial aspartate-glutamate carriers SLC25A12 or AGC1 SLC25A13 orAGC2 The mitochondrial glutamate carriers SLC25A22 or GC1 SLC25A18 or GC2 Glu- H+ GC Glu- H+ Glu- H+ AGC Glu- H+ Asp- Asp- cytosol mitochondria Mainly expressed in neurons Expressed in neurons and astrocytes
- 7. 3 different GC1 mutations in 4 families SLC25A22 mutations are involved in EEE 7 children with EEE 2 children with MPSI (Migrating Partial Seizures in Infancy) EEEwithSB
- 8. SLC25A22 mutations are involved in EEE EEEwithSBMPSI Neonatal Onset Intractable Epileptic Encephalopathy Corpus calosum hypoplasia 3 different GC1 mutations in 4 families 7 children with EEE 2 children with MPSI (Migrating Partial Seizures in Infancy)
- 9. GC1 mutations lead to non functional carrier Wild-type protein + glutamate Mutated protein + glutamate p.Pro206Leu p.Gly236Trp In collaboration with Ferdinando Palmieri’s Lab Molinari et al, Am J Hum Genet, 2005 Molinari et al, Clin Genet, 2009
- 10. Physiopathology Hypothesis EEE related to GC1 mutations Network hyperexcitability Disturbance of enzymes or transporters ATP-dependent (GS, EAAT, Na/K ATPase, …) Glutamate clearance dysregulation Mitochondrial disorder with ATP synthesis deficiency Astroglial disorder with glutamate catabolism impairment AND/OR
- 11. GC1 inactivation in astrocyte primary culture Cellular Model : Astrocyte culture and inhibition by shRNA Biochemical parameters analysis : ● NADH ● Mitochondrial Respiratory Chain activation ● Cellular ATP level ● Amino-acids concentration Generation of a cellular model to study the absence of GC1
- 12. C D 5’ 3’ E SLC25A22 or GC1 (Rattus norvegicus) 2,3 kb, 9 coding exons Relative expression of GC1 48h after transfection in C6 shRNA generation and validation Goubert et al, Front Cell Neurosci, 2017 -74% -68% -53%
- 13. Clone generation and validation Francesco M. Lasorsa (Italy) Protein expression/function of GC1 in C6-clones Protein level Western blot in C6 mitochondria extract AGC1 shR NA -GC 1.C clone2.21 GC1 C ontrol shRN A -G C1.C clone 2.9 m m RN A clone 1.5 b-ATPase 68 kDa 55 kDa 30 kDa Goubert et al, Front Cell Neurosci, 2017 * * Our shRNA-GC1 successfully knock-downed GC1 expression
- 14. Primary culture of Astrocytes From cortex of E18 rats embryos 2-3 weeks of culture minimum to obtain confluent flasks (T75) 100µm 50µm Cultures are highly enriched in Astrocytes : 86,1% ( 1,2 %) DAPI GFAP (Glial fibrillary acid protein) Goubert et al, Front Cell Neurosci, 2017
- 15. Transfection of primary Astrocytes Nucleofection using the Neon System (Invitrogen) GFAP mRFP1 100 µm Goubert et al, Front Cell Neurosci, 2017
- 16. GC1 inactivation in astrocyte primary culture Cellular Model : Astrocyte culture and inhibition by shRNA Biochemical parameters analysis : ● NADH ● Mitochondrial Respiratory Chain activation ● Cellular ATP level ● Amino-acids concentration
- 17. Biochemical Parameters Analysis Cytosol Intermembrane Space OMM IMM Mitochondrial Matrix Glu- H+ Glu- H+ a-KG NH4+GDH NAD+ NADH C IVC III C II ATP CoQ c H+ H+ H+ e- e- e- ADP C VC I Krebs Cycle GC1
- 18. Biochemical Parameters Analysis Cytosol Intermembrane Space OMM IMM Mitochondrial Matrix Glu- H+ Glu- H+ a-KG NH4+GDH NAD+ NADH C IVC III C II ATP CoQ c H+ H+ H+ e- e- e- ADP C VC I Krebs Cycle GC1
- 19. Biochemical Parameters Analysis Cytosol Intermembrane Space OMM IMM Mitochondrial Matrix Glu- H+ NADH C IVC III C II ATP CoQ c H+ H+ H+ e- e- e- ADP C VC I Krebs Cycle GC1 H+ Gradient ( Dym) ? ATP ?
- 20. NAD(P)H measurement in primary Astrocytes, 48h post-transfection NAD(P)H Measurement NADH is an autofluorescent molecule (lexc = 360 nm et lemi = 450-490 nm) Goubert et al, Front Cell Neurosci, 2017
- 21. NAD(P)H measurement in primary Astrocytes, 48h post-transfection NAD(P)H Measurement Absence of GC1 results in a lower NADH production NADH is an autofluorescent molecule (lexc = 360 nm et lemi = 450-490 nm) Goubert et al, Front Cell Neurosci, 2017
- 22. GC1 inactivation in astrocyte primary culture Cellular Model : Astrocyte culture and inhibition by shRNA Biochemical parameters analysis : ● NADH ● Mitochondrial Respiratory Chain activation ● Cellular ATP level ● Amino-acids concentration
- 23. The mitochondrial membrane potential Dym The Dym reflects the pumping of H+ across the IMM by the mitochondrial respiratory chain, the driving force behind ATP production The Dym was monitored with the R123, in real time (1 image/30sec) R123: cell-permeant fluorescent dye sequestred by active mitochondria. This probe is used in quenching mode: H+ R123 Fluorescence = Hyperpolarization H+ R123 Fluorescence = Depolarization Goubert et al, Front Cell Neurosci, 2017
- 24. The mitochondrial membrane potential Dym MRC is functional but not activated by glutamate alone Goubert et al, Front Cell Neurosci, 2017 (n=28) (n=23) (n=12) (n=29)
- 25. The mitochondrial membrane potential Dym 0,0 0,5 1,0 1,5 2,0 Succinate Oxidation C+ Patient Fibroblasts p.Pro206Leu Glutamate Oxidation From Molinari F, JBB, 2010 Molinari et al, Am J Hum Genet, 2005
- 26. GC1 inactivation in astrocyte primary culture Cellular Model : Astrocyte culture and inhibition by shRNA Biochemical parameters analysis : ● NADH ● Mitochondrial Respiratory Chain activation ● Cellular ATP level ● Amino-acids concentration
- 27. Global ATP level measurement ATP synthesis was measured in real time (1 image/30sec) by bioluminescence using the luciferase-luciferin reaction lemi = 562 nm The light emission is directly proportional to ATP quantity
- 28. Global ATP level measurement Goubert et al, Front Cell Neurosci, 2017
- 29. ATP H+ 3Na+ K+ NH3, ATP ADP, Pi 2K+ ATP ADP GS Na/K ATPase Glu entry [ATP] but weak synthesis Ouabain Na/K ATPase Inhibition (ouabain) + Glu Ø consumption ATP + Glc [ATP] In absence of GC1 [ATP] but Ø synthesis [ATP] ?? Global ATP level measurement Glc O2
- 30. Global ATP level measurement Goubert et al, Front Cell Neurosci, 2017
- 31. The equilibrium ATP synthesis/consumption is disturbed in absence of GC1 Global ATP level measurement Goubert et al, Front Cell Neurosci, 2017
- 32. GC1 inactivation in astrocyte primary culture Cellular Model : Astrocyte culture and inhibition by shRNA Biochemical parameters analysis : ● NADH ● Mitochondrial Respiratory Chain activation ● Cellular ATP level ● Amino-acids concentration
- 33. Glutamate catabolism in astrocytes GS: Glutamine synthetase GDH: Glutamate dehydrogenase ALAT: Alanine amino-transferase AAT: Aspartate amino-transferase g-GCS: Glu GDH ALAT AAT: :g-glutamyl-cysteine synthetase Glu In the mitochondria Glutamate + NAD+ a-KG+NADH + NH4 + Glutamate + oxaloacetate Aspartate + GDH AAT Cytosol Mitochondria a-KG 4 5 ~30% EAAT Astrocytes are considered to be responsible for the absorption and metabolism of the major part of glutamate in the brain via EAAT 1 2 3 H+ TCA 4 5 Glutamate + NH4 ++ ATP Glutamine + ADP + Pi Glutamate + Pyruvate Alanine +a-KG GS ALAT In the cytosol Glutamate + Cysteine + ATP g-glutamylcysteine g-GCS - GS ALAT 1 2 3 ~40%
- 34. Analysis of Amino-Acids concentration Control shGC1.C shGC1.D The absence of GC1 in astrocytes leads to an intracellular glutamate accumulation Goubert et al, Front Cell Neurosci, 2017
- 35. Conclusion First study of GC1 inactivation in cerebral cells In the absence of GC1, the MRC is functional but not activated by glutamate Lack of glutamate oxidation results in a lower global ATP level Absence of mitochondrial glutamate transport results in intracellular glutamate accumulation
- 36. Pathophysiological Mechanism hypothesis Seizures and Early Epileptic Encephalopathy Development Glutamate Accumulation Low level of global ATP EAAT dysfunction Slow down of the glutamate clearance Glutamate Spillover and neuronal hyperexcitability
- 37. Astrocyte PRE-SYNAPTIC POST-SYNAPTIC SYNAPTIC CLEFT Signal Transmission NMDA Receptors Glutamate AP [Ca²+] Neuron Excitatory Amino-Acid Transporters (EAAT) AMPA Receptors
- 38. Neuron Astrocyte PRE-SYNAPTIC POST-SYNAPTIC SYNAPTIC CLEFT Signal Transmission Glutamate Glutamine 1 Glutamine synthase 2 NH3, ATP ADP, Pi 1 H+ SLC25A22 H+ 3Na+ K+ 2 Glutaminase NMDA Receptors AMPA Receptors Excitatory Amino-Acid Transporters (EAAT)
- 39. Neuron Astrocyte PRE-SYNAPTIC POST-SYNAPTIC SYNAPTIC CLEFT Signal Transmission Glutamate Glutamine 1 Glutamine synthase 2 NH3, ATP ADP, Pi 1 2 Glutaminase NMDA Receptors AMPA Receptors Excitatory Amino-Acid Transporters (EAAT) SLC25A22 H+ 3Na+ K+
- 40. Neuron Astrocyte PRE-SYNAPTIC POST-SYNAPTIC SYNAPTIC CLEFT Signal Transmission 1 2 NH3, ATP ADP, Pi Glutamate Glutamine 1 Glutamine synthase 2 Glutaminase NMDA Receptors AMPA Receptors Excitatory Amino-Acid Transporters (EAAT) SLC25A22 H+ 3Na+ K+
- 41. Pathophysiological Mechanism hypothesis Trabelsi et al, Glia, 2017
- 42. Pathophysiological Mechanism hypothesis Trabelsi et al, Glia, 2017 GS inhibition and intracellular glutamate increase in astrocytes slow down the time course of STC Peri/extrasynaptic NMDAR-mediated EPSC evoked by HFS is amplified after GS inhibition.
- 43. Conclusion ASTROGLIAL GLUTAMATE CATABOLISM IMPAIRMENT ROLE IN EARLY EPILEPTIC ENCEPHALOPATHIES
- 44. Dr. Laurent Aniksztejn Dr. Hélène Becq Emmanuelle Goubert Yanina Mircheva Julie Sutera-Sardo Prs. Luigi and Ferdinando Palmieri Dr. F. Massimo Lasorsa Acknowledgements Christophe Melon
- 45. Acknowledgments
- 46. GC1 mutations lead to non functional carrier Wild-type protein + glutamate ▼ Wild-type protein + NaCl Mutated protein + glutamate Mutated protein + NaCl p.Pro206Leu p.Gly236Trp Molinari et al, Am J Hum Genet, 2005 Molinari et al, Clin Genet, 2009 In collaboration with Ferdinando Palmieri’s Lab
- 47. GC1 model Generation of cellular /animal models to study GC1 absence Barely enter into the cells and risks of side effects shRNA In Vivo Studies No Animal Model for GC1 Drugs injection : Mersalyl, Tanic acid, Bromocresol purple, Pyridoxal-5’- Phosphate to inhibit GC1 and GC2 (Fiermonte et al, 2002) In Vitro Studies Inactivation of GC1 in INS-1E cells with shRNA (Casimir et al., 2009) Role of GC1 in glucose-stimulated insulin secretion No study of GC1 in cerebral cells
- 48. shRNA generation and validation -74% -68% -53% Relative expression of GC1 48h after transfection in C6 C D 5’ 3’ E SLC25A22 or GC1 (Rattus norvegicus) 2,3 kb, 9 coding exons Goubert et al, Front Cell Neurosci, 2017
- 49. Clone generation and validation Reconstitution of integral mitochondrial protein into liposome [14C]-Glu (1mM) Glu (20mM) Our shRNA-GC1 successfully knock-downed GC1 expression * * Francesco M. Lasorsa (Italy) Goubert et al, Front Cell Neurosci, 2017
- 50. NAD(P)H measurement in primary Astrocytes, 48h post-transfection NAD(P)H Measurement Absence of GC1 results in a lower NADH production NADH is an autofluorescent molecule (lexc = 360 nm et lemi = 450-490 nm)
- 51. « Quenching mode » : R123 Fluo λexc = 488 – 500 nm λemm = 520 nm R + R + R + -------+++++ R + R + R + R + R + R + R + R + R + R + R + CR ∆Ψm GC1 R + R + R + ------- + ++++++ CR ∆Ψm GC1 Glutamate ou Glucose H + R + R + R + R + R + R + R + R + The mitochondrial membrane potential Dym
- 52. Global ATP level measurement
- 53. Glutamate catabolism in astrocytes H+ 3Na+ K+ GS Glu entry [Glu]i [Gln]i Ratio Glu/Gln unchanged ?
- 54. Analysis of Amino-Acids concentration
- 55. Analysis of Amino-Acids concentration Table 1: Aspartate and Alanine contents determined by high-performance liquid chromatography (HPLC) in astrocytes transfected with mRFP1 alone (Control, n=8), with shRNA GC1.C (n=8) or with shRNA GC1.D (n=10). Data are normalized on the resting condition and expressed as mean ± sem
- 56. SLC25A22 mutations are involved in EOEE Poduri et al, Annals Neurology, 2013
Hinweis der Redaktion
- Pour mesurer le gradient de prtones et ainsi mettre en évidence les conséquences de l’invalidation de GC1 sur la force protonomotrice, nous avons incubées les astrocytes en présence de la Rhodamine 123, marqueur fluorescent des mitochondries actives des cellules vivantes. R123 est une molécule qui fonctionne en quenching mode cad l’accumulation des molécules résulte en la répartition de l’énergie propre de chaque molécule parmis toutes les molécules, nous observons donc une diminution de la fluorescence. Séquestrée dans la matrice des mitochondries , la R123 chargé positivement s’alligne de façon homogène en suivant le gradient de proton, du côté interne de la membrane chargée négativement. L’entrée de glutamate va provoquer la diminution du potentiel mitochondriale et accumulation de plus de molécules de rhodamine sur la membrane. L’entrée de glutamate este associé donc à la diminution de la baisse de l’intensité de la fluorecsence.