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Impaired metabolism of D-serine in a motor neuro disease, ALS - Jumpei Sasabe

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Watching at the "D" side: D-amino acids and their significance in neurobiology
June 05 -June

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Impaired metabolism of D-serine in a motor neuro disease, ALS - Jumpei Sasabe

  1. 1. Impaired metabolism of D-serine in a motor neuron disease, ALS Jumpei Sasabe, M.D., Ph.D. Department of Anatomy and Neuroscience Keio University School of Medicine Lake Como School on June 8th, 2016
  2. 2. D-Serine metabolism and chronic neurological disorders Schizophrenia Psychosis Prevalence: 1 in 250 Positive and negative symptoms Motor neuron disease Prevalence: 1 in 20,000 Progressive muscle weakness Amyotrophic lateral sclerosis(ALS)
  3. 3. Amyotrophic Lateral Sclerosis (ALS)
  4. 4. Amyotrophic Lateral Sclerosis (ALS) A motor neuron disease that involves both upper and lower motor neurons. Prevalence:5-6/0.1 million Annual incidence:1-2/0.1 million Life-long risk:1/1000 Dr Jean-Martin Charcot first described in 1869. No-muscle-nourishment involving Lateral corticospinal tracts
  5. 5. Amyotrophic Lateral Sclerosis (ALS)
  6. 6. Clinical Findings Symptoms: Upper motor Spasticity Hyperreflexia Lower motor Muscle atrophy Fasciculations Bulbar signs Dysphagia Slurred speech Negative symptoms: External ophtalmoplegia Bladder rectal disturbance Lethal event: Respiratory failure
  7. 7. Pathological feature Non-ALSALS Selective motor neuron death Inclusion body Gliosis Non-ALS ALS
  8. 8. Etiology Sporadic ALS (90%) Unknown Environmental risks: Neurotoxin, Pesticides, Military veterans, Heavy metals, Athletes, Residence in Guam or Kii district (Japan) Fuit bats eat cycad Beta-Methylamino-L-alanine: BMAA Cyanobacteria
  9. 9. Etiology Familial ALS (10%) More than 20 genes have been identified to associate with ALS DAO Time course of discovery of ALS genes Distribution of ALS genes in FALS and SALS (Gonzalez-Perez, Brown Jr, Motor Disorders, Chap 48)
  10. 10. Etiological hypotheses (Kaus and Sareen, Front Cell Neurosci 2015)
  11. 11. Glutamate toxicity in ALS 11 2 1 2 3 1 2 34 1 2 34 2. Reduced Ca-buffering protein 1. Ca-permeable AMPAR 3. Reduced Glu-uptake 4. Increased Glu-release 5. Riluzole is clinically effective 5 Inhibit NMDAR, Kainate R Improve Glu-uptake by astrocytes Reduce Glu-release by presynapse
  12. 12. ALS causative genes and cellular functions (Kaus and Sareen, Front Cell Neurosci 2015)
  13. 13. D-Serine hypothesis in ALS etiology ~associated with “glutamate toxicity”~ D-Ser is required for neuronal excitation Glu/D-Ser is accumulated
  14. 14. Model mice for ALS G93A SOD1-transgenic mouse Spontaneous motor neuron degeneration Wobbler Mendelian fALS SOD1 FUS TDP-43 Atypical and rare mendelian ALS Alsin VAP-B Dynactin P 150glued Genetic abnormality in fALS and sALS C9ORF72 Some features of ALS Some features of ALS Some features of ALS Subtle phenotype No phenotype More like ALS/FTD Strong ALS-like phenotype ALS-like phenotype
  15. 15. Our strategies Motoneuronal death Neurotrophic factors Trigger of ALS Sporadic Familial ??? ACGATCGGCA ACGATTGGCA Sasabe J et al., Front Synaptic Neurosci 2014 Sasabe J et al., PNAS 2012 Sasabe J et al., EMBO J 2007 etc Mechanism of motoneuronal death Nawa M et al., Cell Death Differ 2012 Kanekura K et al., Mol Neurobiol 2009 Suzuki H et al., J Neurochem 2009 Nawa M et al., Cell Signal 2008 Obata Y et al., J Neurosci Res 2005 Kanekura K et al., J Biol Chem 2004 etc Matsuoka M et al., CNS Drug Rev 2006 Chiba T et al., BBRC 2006 Chiba T et al., J Neurosci Res 2004 etc
  16. 16. Accumulation of D-serine in ALS
  17. 17. D-Serine in ventral horn of ALS mice Survival ratio Phenotype of G93A-SOD1 transgenic mice Disease progression Presymptomatic Middle stage End stage D-serine Wild-typenon-TgG93A-SOD1Tg Presymptomatic Middle stage End stage 9 weeks 16 weeks 21 weeks (Sasabe et al., EMBO J 2007) D-serine is increased progressively in ALS mice D-Ser-Ab positive signals were diminished by preabsorption with D-serine
  18. 18. Quantitative D-serine in ALS mice Kenji Hamase Fluorescence intensity1D: Reversed-phase column 2D: Enantioselective column 3015 Ser 450 (min) 200 D-Ser L-Ser Fluorescence intensity Presymptomatic Middle stage End stage 9 w 16 w 21 w D-serine is accumulated in spinal cord with disease progression (Sasabe et al., PNAS 2012)
  19. 19. D-Serine in ALS patients (Sasabe et al., EMBO J, 2007) Non-ALS S4V-SOD1 ALS Sporadic N = 4 N = 1 N = 3 D-Ser enlarged (unpublished data) (unpublished data) There is a trend of D-serine increase in the spinal cord, but not in CSF in sporadic ALS patients.
  20. 20. Metabolism of D-serine in ALS
  21. 21. L-serine ⇆ D-serine Pyruvate D-Serine metabolism Levo- Dextro- serine racemase (SRR) PLP (Miya, J Comp Neurol. 2008) SRR D-amino acid oxidase (DAO) Hydroxypyruvate→ (Sasabe, PNAS 2012) DAO D-serine (Schell, J Neurosci. 1997)
  22. 22. What determines D-serine level in the hindbrain and spinal cord? D-Serine is produced, but actively degraded by DAO (Miyoshi et al, J Chromatogr B, 2010) (nmol/g) DAO+/+ DAO+/– DAO–/– D-Ser in DAO-null miceD-Ser in SR-ko mice SR+/+ SR+/– SR–/– (nmol/g) (Miyoshi, Sasabe, et al, Amino Acids, 2012) D-Ser in SR-ko DAO-null mice (Miyoshi, Sasabe, et al., Amino Acids 2012) SR+/+, DAO+/+ SR+/+, DAO–/– SR–/–, DAO+/+ SR–/–, DAO–/–
  23. 23. (Schell, J Neurosci. 1997) D-serine Local regulation of D-serine in forebrain (Modified from ‘serine shuttle’ model by H. Wolosker)
  24. 24. Local regulation of D-serine in hindbrain and spinal cord (Schell, J Neurosci. 1997) D-serine (Modified from ‘serine shuttle’ model by H. Wolosker)
  25. 25. SMI32, motoneuron marker; NeuN, neuron marker (Sasabe et al., EMBO J, 2007) Cellular accumulation of D-serine in ALS Presymptomatic 9 w End stage 21 w IB4, microglia marker; GFAP, astrocyte marker D-Serine accumulation involves glial D-serine metabolism
  26. 26. Involvement of serine racemase ~overproduction~ (Sasabe et al, EMBO J 2007; PNAS, 2012) Non-TgG93A-SOD1Tg A monoclonal antibody to mouse SR from BD Iba1, microglia marker; GFAP, astrocyte marker Increased expression of SR in microglia
  27. 27. Induction of serine racemase in microglia (Sasabe et al., EMBO J, 2007) Microglial cell line:MG5 JNK: c-Jun N-terminal Kinase LPS: lipopolysaccharide 72 h treatment 96 h treatment SP600125: JNK inhibitor 72 h after infection hSOD1
  28. 28. L-ProD-Pro D-Pro DAO G181R DAO WT DAO WT Involvement of D-amino acid oxidase ~less degradation~ Activity based staining of DAO in tissue Non-Tg G93A-SOD1 Tg (Sasabe et al., PNAS 2012) DAO Non-Tg G93A-SOD1 Tg Reticulospinal tract
  29. 29. Reduction of DAO Region-specific reduction of DAO activity (Sasabe et al., PNAS 2012) Downregulation of spinal DAO
  30. 30. What is the main contributor? Over-synthesis Loss of degradationvs Loss of DAO Elevated SR Reduction of DAO activity is mainly responsible to D-serine accumulation in ALS mice (Sasabe et al., PNAS 2012)
  31. 31. Summary-1
  32. 32. Relevance of reticulospinal pathway to motor function
  33. 33. What’s reticulospinal pathway? Corticospinal pathway Corticospinal pathway: Connects cortex and spinal cord directly Fine movements by hands Reticulospinal pathway: (i) one of descending motor pathways (i) it arises from the pontine and medullary reticular formation and terminates in ventromedial part of anterior horn (iii) Responsible for gross movements primarily of proximal muscles Brainstem pathways (Lemon, Ann Rev Neurosci, 2008)
  34. 34. Reticulospinal pathway is dominant in non-primates In non-primates, there are few functional corticospinal connection and the reticulospinal control seems to be more important. (Alstermark & Ogawa, J Neurophysiol, 2004) Development of corticospinal pathway and the emergence of fine motor control abilities (Lemon, Ann Rev Neurosci, 2008)
  35. 35. DAO distribution in human vs mouse Human Mouse (Sasabe et al., Front Synap Neurosci 2014) MedullaOblongataSpinalcord
  36. 36. (Mitchel, PNAS 2010) Classical Adult Onset ALS with autosomal dominant (AD) inheritance Autosomal dominant inheritanceR199W
  37. 37. R199W mutation abolishes DAO activity Dimerized human DAO (Cappelletti et al., BBA 2015)
  38. 38. R199W-DAO is a dominat-negative mutant Cotransfection of WT and R199W-DAO into COS cells results in inhibition of DAO activity. (Mitchell, PNAS 2010)
  39. 39. DAO activity in the spinal cord and DAO substrates D-alanine is not affected when DAO is not fully inactivated (Sasabe et al., PNAS 2012) Spinalcord
  40. 40. Is D-serine toxic to motor neurons?
  41. 41. NMDA toxicity on primary cultured spinal cord cells (Sasabe et al., EMBO J, 2007)
  42. 42. NMDA/D-serine toxicity on primary cultured spinal neurons Neurons from ALS mice are sensitive to NMDA/D-serine-toxicity in part through NR1 subunit of NMDAR (Sasabe et al., EMBO J, 2007)
  43. 43. Toxicity of R199W-DAO to motor neurons (Mitchell, PNAS 2010) MN: Primary cultured motor neurons MN: NSC-34 cells Glia: C6 cells R199W-DAO neurotoxicity mediates NR1 subunit of NMDAR
  44. 44. Summary-2 Cell death
  45. 45. Disease progression and D-serine metabolism
  46. 46. Role of D-serine metabolism in survival of ALS mice SR–/– mSOD1 SR+/+ mSOD1 SR+/– mSOD1 (Thompson et al., J Neurochem 2012) (unpublished data) D-serine level negatively affects survival of ALS mice
  47. 47. Role of D-serine metabolism in onset of ALS mice (Thompson et al., J Neurochem 2012) SR–/– mSOD1 SR+/– mSOD1 SR+/+ mSOD1 SR–/– mSOD1 SR+/+ mSOD1 SR+/– mSOD1 Forelimb muscle strengthHindlimb muscle weakness (gait abnormality) D-Serine level has a positive correlation with onset, but a negative correlation with progression
  48. 48. Oral D-serine intake and disease progression
  49. 49. Therapeutic strategy targeting D-serine in ALS
  50. 50. What is a good therapeutic target to control D-serine level? 1. Inhibition of SR 2. Blockade of NR1 3. Inhibit D-Ser transport 4. Activation of DAO
  51. 51. How is DAO down-regulated? MEK/ERK activation appears to be involved in DAO downregulation (Sasabe et al., PNAS 2012)
  52. 52. Searching for DAO-inducing small molecules DAO
  53. 53. (unpublished data) Searching for DAO-inducing small molecules Antagonists to a certain molecule5 2 PD0325901U0126 MEK/ERK inhibitor Distribution of molecule X in CNS Non-Tg G93A-SOD1 Tg MoleculeXDAO
  54. 54. What remains elusive? 1. Cellular regulation of D-serine in hindbrain and spinal cord regions 2. Mechanism of how glial activation triggers DAO downregulation 3. Frequency of abnormal D-serine metabolism in ALS 4. Therapeutic effectiveness of D-serine removal in ALS
  55. 55. D-Amino acid metabolism modulates microbial community and innate defense
  56. 56. D-Amino acids in life Levo- Dextro- L-amino acids D-amino acids Protein synthesis + | D-Ala & D-Glu: Component of peptidoglycan Bacteria Peptidoglycan Mammals Bacteria specific D-amino acids? D-Ser & D-Asp: Neurotransmitter D-Ser & D-Asp D-Val D-Phe D-Tyr D-Thr D-Ile D-Met D-Leu D-Met & D-Leu: Trigger of remodeling cell wall in the stationary phase (Lam, 2009)
  57. 57. (Kenji Hamase) Chiral separation of proteinogenic amino acids “Two-dimensional HPLC system” His Asn Ser GlnArg Asp Gly allo-Thr Glu Thr Ala Pro Met Val allo-Ile Ile Leu Phe Trp Lys Cys Tyr 1D: reversed-phase separation 2D: chiral separation
  58. 58. Microbiota produce free D-amino acids in the gut L-Amino acidsD-Amino acids D-Ala, D-Asp, D-Glu, and D-Pro are predominantly found in cecal content of SPF-mice.
  59. 59. Intestinal regulation of D-amino acids Intestinal D-amino acids are regulated by host-microbe interaction L-amino acids ⇆ D-amino acids D-amino acid oxidase (DAO) α-keto acids + H2O2 + NH3→ DAO activity is restricted in the small intestine DAO is localized to enterocytes and goblet cellsDAO affects luminal D-AA level Microbiota regulate DAO expressionDAO limits proliferation of V. cholerae DAOG181R DAOWT
  60. 60. D-amino-acid metabolite, H2O2, is toxic to V.cholerae oxyR: a transcriptional regulator activated by H2O2 H2O2
  61. 61. DAO modulates microbiota composition (LEfSe, Huttenhower lab)
  62. 62. Lactobacilli are increased in DAO-null mice Fluorescence in situ hybridization (FISH)
  63. 63. Alanine synthetic pathway (Kaaij et al, AEM 2004) What affects abundance of Lactobacilli? L. johnsonii L.johnsonii completelylacks genes encoding biosynthetic pathways for amino acids, purine nucleotides, and most cofactors. (Pridmore, et al., PNAS 2004)
  64. 64. Gut-Brain cross talk (Mol Psychiatry review 2016) Indication of microbiota involvement Nervous system development Cognition and emotion Autism・Depression・Schizophrenia Short-chain fatty acids Neurotransmitters (Dopamine、GABA、Histamine、Ach) Tryptophan metabolism etc Microbial metabolites
  65. 65. Keio University Sadakazu Aiso Masataka Suzuki Kyushu University Kenji Hamase Yurika Miyoshi Shiseido Co., Ltd. Masashi Mita IUHW Ryuichi Konno BWH/ Harvard Med School Matthew K Waldor Brigid Davis Seth Rakoff-nahoum Ting Zhang

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