El lunes y martes 20 y 21 de noviembre coordinamos un simposio internacional en la Fundación Ramón Areces, sobre los defectos del transporte de aminoácidos.

1. The glycine transporter GlyT1, NMDARs and schizophrenia
Francisco Zafra,
Centro de Biología Molecular “Severo Ochoa”, CSIC-UAM,
28049, Madrid, Spain
Simposio Internacional: Defectos del transporte de aminoácidos
Fundación Areces, Noviembre 2017

2. GLY
Astrocytes
Glu
NMDA-R
AMPA-R
Postsynaptic
neuron
Presynaptic
neuron
Na+
Glu
Gln
Glu Gln2Na, Cl -+
GlyT1
GLY
Glu
Gln
?
SNAT5?
SNAT3
SNAT5?
SNAT1?
SNAT2?
GLT1
EAAT3
GLT1
Na, Ca+ ++
GS
Gln-asa
GLY
GlyT1
Glutamate-
Glutamine Cycle
GLUTAMATERGIC SYNAPSE
Na+
H+
Na+
SNAT2?
NFPS NFPS
CLOZAPINA
+
ORG23798 ORG23798
GlyT1
NMDAR Functions: Development, Plasticity, Memory,
Learning…
Pathology:
Overstimulation- Cell death (Ischemia, TBI, ALS…)
Hypofunction- Schizophrenia

4. Harvey & Yee, Nat Rev Drug Discov. 2013;12(11):866-85
Glutamatergic and glycinergic neurons control dopaminergic reward circuits

5. Harvey & Yee, Nat Rev Drug Discov. 2013;12(11):866-85
Glycinergic interneurons control dopamine release

6. LOCALIZATION OF GlyT1 IN BRAIN

7. GlyT1: mRNA and protein display partially
discordant distributions
GlyT1mRNA GlyT1

8. MAVAHGPVATSSPEQ-
MVGKGAKGML-GlyT1a
GlyT1b
Ab 176
Ab 224
Development of a new anti-GlyT1 antibody

9. LOCALIZATION OF GlyT1 IN HIPPOCAMPUS AND NEOCORTEX

10. LOCALIZATION OF GlyT1 IN GLUTAMATERGIC TERMINALS

11. ULTRASTRUCTURAL LOCALIZATION OF GlyT1 IN HIPPOCAMPUS AND NEOCORTEX

12. Dentate Gyrus
Cerebellum
Pre Post
Pre Post
ULTRASTRUCTURAL LOCALIZATION OF GlyT1 IN DENTATE GYRUS AND CEREBELLUM.
COLOCALIZATION WITH NMDAR

13. GlyT1 CO-IMMUNOPRECIPITATES WITH NMDAR AND PSD95

14. LOCALIZATION OF GlyT1 IN SYNAPTIC VESICLES

15. Rat brain homogenate
P1 S1
S2P2
S3P3
Lysis
S4P4
gradient
SV

16. + antiSynaptophisin
+ protein A sepharose
AntiSynaptophysin Ab
Protein A - sepharose
GlyT1
Synaptophysin
LOCALIZATION OF GlyT1 IN SYNAPTIC VESICLES
Immunoisolation of synaptic vesicles

17. Sinaptophysin (10nm)
GlyT1(5nm)
VGLUT1 (10nm)
GlyT1(5nm)
LOCALIZATION OF GlyT1 IN SYNAPTIC VESICLES

18. ZINC INHIBITS GlyT1 MEDIATED UPTAKE AND STIMULATES EFFLUX
m
m
m
Gly
H+
Glu
VGLUT
Na+/K+ATPase
K+
ATP
Na+
SSV
ATP
H+ ATPase
GluGlu Gly
Glutamatergic neuron
Zn2+
GlyT1
GlyT1
GlyT1
Gly?
Gly
Gly?
?
Efflux Uptake
Zn2+Zn2+

19. This is the first report on mutant mice lacking forebrain neuronal GlyT1,
clearly showing the presence of schizophrenia-resilient phenotypes

20. GlyT1 IN GLUTAMATERGIC SYNAPSES/SCHYZOPHRENIA
ALLELIX: WO01187855 (2001)
GLAXO: WO0200413280 (2004)
WO2005058885 (2005)
WO2005058317 (2005)
WO2005058882 (2005)
MERCK: WO2005046601 (2005)
ROCHE: WO2004407034 (2004)
WO 200504166 (2005)
WO2005014563 (2005)
WO20055023261 (2005)
WO2005023260 (2005)
YAMANOUCHI:DE10315570
SANOFI: WO2004013100 (2004)
WO2004013101 (2004)
WO2005037782 (2005)
WO2005037783 (2005)
WO200537785 (2005)
WO2005037785 (2005)
GlyT1 INHIBITORS

21. SOME GlyT1 INHIBITORS

22. Bitopertin/RG1678/RO4917838

23. Might other transporters contribute to regulation of [Gly] in
glutamatergic synapses?
Might trafficking of GlyT1 counterbalance the effect of
treatments?

24. Might other transporters contribute to regulation of [Gly] in
glutamatergic synapses? For instance SNAT5/SN2, or Asc1
Pochini et al Front. Chem., 11 August 2014
Nakanishi et al. Am J Physiol
Cell Physiol
2001;281:C1757-C1768

25. DISTRIBUTION OF SNAT5 AND VGLUT1 IN BRAIN
SNAT5 VGLUT1

26. LOCALIZATION OF SNAT5 IN NEOCORTEX

27. SNAT5 DOESN’T COLOCALIZES WITH NEURONAL MARKERS

28. ULTRASTRUCTURAL LOCALIZATION OF SNAT5 IN NEOCORTEX

29. Might trafficking of GlyT1 counterbalance
the effect of treatments?

30. INTRACELLULAR TRAFFIC OF GlyT1
Dep Ub
DUBs

31. Lysosomes
PKC
CONSTITUTIVE AND REGULATED TURNOVER OF GlyT1
Early endosome

32. Clathrin
Early endosome PKC
Dynamin 2
Lysosomes
REGULATED ENDOCYTOSIS OF GLTY1 DEPENDS ON DYNAMIN 2 AND CLATHRIN
Monensin

33. CONSTITUTIVE AND PKC REGULATED ENDOCYTOSIS
OF GlyT1

34. CONSTITUTIVE AND PKC REGULATED ENDOCYTOSIS
THROUGH CLATHTIN VESICLES

35. K25
K26
K619
K577
K581
CONSTITUTIVE ENDOCYTOSIS OF GlyT1 DEPENDS ON LYSINE K619

36. PKC REGULATED ENDOCYTOSIS OF GlyT1 DEPENDS ON LYSINE K619

37. ACTIVATION OF PKC PROMOTES UBIQUITINATION OF GlyT1

38. GLT1 TURNOVER REGULATION BY UBIQUITINATION/DEUBIQUITINATION

39. THE UBIQUITIN LIGASE NEDD4-2 PROMOTES UBIQUITINATION AND
DOWN-REGULATION OF GLT-1 IN EUKARYOTIC CELLS
García-Tardón et al., J. Biol Chem. 2012, 287:19177-87.

40. NEDD4-2 MEDIATES THE PKC-DEPENDENT ENDOCYTOSIS OF GLT-1
García-Tardón et al., J. Biol Chem. 2012, 287:19177-87.

41. Glutamate induced internalization of GLT-1 in mixed
primary cultures from rat cortex
L-trans-2,4-PDC
WAY 213613
Ibañez et al., Neuropharmacology 2016
Adapted from Danbolt
http://www.cmbn.no/group-danbolt.html
B
180 -
100 -
200µM PDC
100µM WAY
1mM glutamate
CNX
WB:
GLT-1
CNX
GLT-1
180 -
100 -
SurfaceTotal
-
-
-
+
-
+
- +
-
+
- +
-
+
- +
-
-
200µM PDC
100µM WAY
1mM glutamate
SurfaceGLT-1(%ofcontrols)
-
-
-
+
-
+
- +
-
+
- +
-
+
- +
-
-
***
100
80
60
40
20
0
120
L-trans-2,4-PDC
WAY 213613

42. Ibañez et al., Neuropharmacology 2016
GLT-1 internalization depends on ubiquitination
Adapted from Gabriel et al., J. Neuroscience 33 (45) 17836-46, 2013
Adapted from Yernool et al., Nature 431: 811–818, 2004
0 15 30 60 (min)
250-
100-
250-
100-
1mM Glu
0 20 40 60
0
50
100
150
HAGLT-1 HAGLT-1-7KR
(mM Glu) 0 1.0 10.0 0 1.0 10.0
CNX
CNX
SurfaceGLT-1(%ofcontrols)
0 1.0 10.0 (mM Glu)
HA-GLT-1
HA-GLT-1-7KR
A B
IP:HA
WB:
UB
C
HA
HA
HA
D
Banddensity(%controls)
180 -
100 -
- + - + +
- - + + + 1µM PMA
1mM Glu
180 -
100 -
SurfaceTotal
CNX
WB:
HA
CNX
HA
CTR Glu PMA
GluPMA
Glu PMA
CTR Glu PMA GluPMA
Glu PMA
SurfaceGLT-1(%ofcontrols)
CNX
HA
180 -
100 -
Surface
- + - +
Vehicle PYR41
180 -
100 - CNX
HA
Total
Vehicle
PYR41
SurfaceGLT-1(%ofcontrols)
Glu (1mM)
120
100
80
60
40
20
0
Controls Glu (1mM)
120
100
80
60
40
20
0
*
WB:
WB:
180 -
100 -
180 -
100 -
* *
*
0
50
100
*
*
*
SurfaceTotal
Time

43. Arrestins as adaptors between GLT-1 and Nedd4-2
B
D
GFP
+ + +
- + +
100 -
75 -
180 -
100 -
75 -
180 -
GFP
- - +
Nedd4-2
Nedd4-2
IP:HALysates
180 -
100 -
50 -
Surface
- + - +
Scr
siRNA
b-ARR1
siRNA
180 -
100 -
Total
C
HA
HA
- + +HAGLT1
180 -
100 -
180 -
100 -
50 - b-ARR1
HA
Nedd4-2
HA
Nedd4-2
IP:Nedd4-2Lysates
Scr siRNA
b-ARR1 siRNA
- +
- +
-
-
180 -
100 -
50 -
180 -
100 -
50 -
IP:Nedd4-2Lysates
HA
Nedd4-2
HA
Nedd4-2
Flag
Flag
ScrsiRNA
b-ARR1siRNA
HAGLT1/Nedd4-2binding(%ofcontrols)
Scr siRNA
β-ARR1 siRNA
Glu (1 mM)Vehicle
Vehicle
Glu (1 mM)
Controls
Banddensity(%ofcontrols)
Nedd4-2
GFP-b-ARR1
HA
HA
CNX
CNX
b-ARR1
HAGLT1
GFP-b-ARR1 Glu (1 mM)
Glu (1 mM)
HAGLT1
Flag-b-ARR1
- + +
- - +
A
100
50
0
SurfaceHAGLT1(%ofcontrols)
100
50
0
0
100
200
300
400
500
WB:
WB:
WB:
WB:
*
*
*
*
Ibañez et al., Neuropharmacology 2016

44. Arrestins as adaptors between GLT-1 and Nedd4-2 mediated
ubiquitination
PKC
Glutamate
Lysosomes

45. Some pending challenges for a better understanding of
the role of glycine in the glutamatergic (and
glycerinergic) synapses
- The role of other Gly transporters located in these
synapses
- Identification of regulatory pathways and interacting
partners of GlyT1
- Response of these regulatory mechanisms to
treatments with bitopertin and other ligands of GlyT1
- Adaptive responses of D-ser or other ligands of the
glycine-B site on NMDAR.

46. Former lab members
Beatriz Cubelos
Enrique Fernández
Noemí García Tardón