Watching at the "D" side: D-amino acids and their significance in neurobiology June 05 -June 09, 2016 – Lake Como School of Advanced Studies

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Lake Como School of Advanced Studies
Watching at the “D” side: D-amino acids and ther
significance in neurobiology
6/9/16 1
Joseph T. Coyle, MD
Eben S. Draper Professor of Psychiatry and
Neuroscience
Harvard Medical School
D-Serine and Schizophrenia

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3. Clinical Features of the Schizophrenia
Syndrome
3
• Positive Symptoms: delusions,
hallucinations, thought
disorder (responsive to antipsychotic
drugs)
• Negative Symptoms: asociality, apathy,
poverty of thought, anhedonia
• Cognitive Deficits: memory, executive
functions, problem solving
• Cognitive and negative symptoms but not
positive symptoms predict disability

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Impact of Schizophrenia: HUGE
unmet needs
Affects 1% of the population.
Onset in early adulthood with lifetime disability.
Seventh most costly medical illness to Society
($66 Billion/year in USA).
High rates of substance abuse, homelessness and
imprisonment and early death.

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Anti-Psychotics Drugs Have Limited Efficacy in the
Treatment of Schizophrenia
1. In meta-analysis, second-generation (SG)
antipsychotics (clozapine, olanzapine, risperidone) are
marginally more effective than first-generation (FG)
antipsychotics (Leucht et al., Lancet 373: 31, 2009).
2. 74% of patients discontinue both FG and SG
antipsychotics within 18 months of treatment because
of side effects or lack of efficacy (Lieberman et al.,
NEJM 353: 1209, 2005).
3. Antipsychotics (except clozapine) have negligible
effects on cognition and negative symptoms (Essali et
al., Cochrane Database Syst Rev , ePub 2009).

7. Schizophrenia has a characteristic
neuropathology

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Specific Brain Structures are
Shrunken in Schizophrenia
1. Atrophy of the frontal cortex, temporal
cortex, hippocampus and thalamus (-3-5%)
2. Reductions in grey matter, loss of neuropil
and reduction in white matter.
1. WHY? No neuronal loss in cortex. Rather,
neuronal atrophy, skimpy dendrites and
reduced spines, resulting in ~35% reduction
in glutamatergic synapses.
ControlControl
Schizophrenia
Glantz LA, Lewis DA. Arch Gen Psychiatry. 57:65-73, 2000.
Konopaske et al., JAMA Psychiatry 71(12):1323-31, 2014.

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Cortical Atrophy and Schizophrenia: the
(Insurmountable?) Challenge
• Present at first episode.
• Pervasive but affects frontal cortex and temporal cortex
more severely.
• Progresses for ~10 years after onset of psychosis.
• Exacerbated by anti-psychotic treatment.
• Correlates with negative symptoms and with
cognitive impairment BUT NOT with positive symptoms

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GAD-67, parvalbumin-expressing, fast-firing
recurrent inhibitory inter-neurons are down-regulated
in schizophrenia
Tamminga et al., Am J Psychiatry 161: 1764, 2004

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Is There Evidence of NMDA
Receptor Dysfunction in
Schizophrenia?

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Tripartite Glutamatergic Synapse
JT Coyle: in Basic Neurochemistry, 2014

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The NMDA Receptor: molecular coincidence
detector
• Depolarization
Mg2+
GLU
DS
Ca2+
+
Na+
• Agonist binding at
glutamate site
• Agonist binding at glycine
modulatory site (GMS)
Expression of synaptic genes:
BDNF pathway, mTor pathway,
mir132, MeCP2
Ketamine

14. Blakely RD, Robinson MB, Thompson RC, Coyle JT.Hydrolysis of the brain
dipeptide N-acetyl-L-aspartyl-L-glutamate: subcellular and regional
distribution, ontogeny, and the effect of lesions on N-acetylated-alpha-linked
acidic dipeptidase activity. J Neurochem. 1988 Apr;50(4):1200-9.
Rhesus cortex
NAAG-IC
a, b, GCPII in situ; c, double label;
d, GFAP; e, control

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Arch Gen Psychiatry 52: 829-36, 1995.
Abnormal excitatory neurotransmitter metabolism in
schizophrenic brains.
Tsai G, Passani LA, Slusher BS, Carter R, Baer L,
Kleinman JE, Coyle JT.
“RESULTS: Our study demonstrates alterations in brain
levels of aspartate, glutamate, and NAAG and in
Glutamate Carboxypeptidase2 (GCP2) activity. Levels of
NAAG (an NMDA receptor antagonist) were increased
and GCP2 activity and glutamate levels were decreased in
the schizophrenic brains….
CONCLUSIONS: These findings support the hypothesis
that schizophrenia results from a hypofunction of certain
glutamatergic neuronal systems….”

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Rowland et al., Schizophr Bull 39: 1096-1104, 2013
The levels of N-acetyl aspartyl glutamate in
forebrain significantly correllate with negative
symptoms in schizophrenia in vivo shown by MRS

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NMDA Receptor Hypofunction and
Schizophrenia
• Ketamine infusion in normal volunteers:
Replicates positive, negative and cognitive symptoms
Replicates physiologic signs: hypofrontality, elevated
subcortical dopamine release, eye tracking, disrupted
gamma rhythms.
• Endogenous NMDA GMS antagonists are elevated in
brain and CSF: kynurenic acid, N-acetylaspartyl-
glutamate (act at the glycine modulatory site).
• D-serine levels are reduced in CSF and serum
• Schizophrenia-like symptoms in early stages of NMDA
receptor auto-immune disorder: Brain On Fire

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SR-/- Mice as a Genetically Valid
Model of Cortical Pathology in
Schizophrenia

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Basu et al., Mol Psychiatry 14: 19-27, 2009
Serine Racemase (SR) Null Mutation
Prevents SR Expression in Brain
Cortical D-Serine <15% of WT
NMDA receptor function is decreased by >70%

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Acute Hippocampal Slice Preparation Shows Impaired
Long-Term Potentiation in SR-/- Mice
Basu et al., Mol Psychiatry 14:719-27, 2009
DeVito et al., Genes Brain Behav. 10:210-22, 2011
Balu et al., PNAS USA 110:E2400-9, 2013

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SR-/- Mice Exhibit Cognitive Impairments in Several
Domains
Cognitive Impairments: Probe
task in Morris water maze,
sequential memory, contextual
fear conditioning
Basu et al., Mol Psychiatry 14:719-27, 2009
DeVito et al., Genes Brain Behav. 10:210-22,
2011
Balu et al., PNAS USA 110:E2400-9, 2013
MWM Probe
Trial

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SR-/- mice primary sensory pyramidal neurons have
less complex dendritic structure and fewer spines
D Balu, A Basu and JT Coyle, Neurobiol Dis 45: 671-82, 2012
Cortical volume: -3.5%, P<0.05
~35% Reduction in glutamatergic synapses

23. Balu DT, Li Y, Puhl MD, Benneyworth MA, Basu AC, Takagi S,
Bolshakov VY, Coyle JT. Proc Natl Acad Sci U S A. 2013 Jun
25;110(26):E2400-9. 23
Schizophrenia and SR-/- mice have
reduced BDNF/TrkB signaling

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Akt/mTOR signaling are reduced in
schizophrenia and SR-/- mice
Balu DT, Li Y, Puhl MD, Benneyworth MA, Basu AC, Takagi S,
Bolshakov VY, Coyle JT. Proc Natl Acad Sci U S A. 2013 Jun
25;110(26):E2400-9.

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miRNA 132 is reduced in
schizophrenia and in SR-/-

26. Balu DT and Coyle JT. Neurosci
Letts 517: 77-81, 2012.
Silencing SR expression in cortical pyramidal neurons in
young adulthood (CamKIICre2834) partially impairs
dendrite and spine formation on S1 pyramidal neurons

27. SR-/- results in increased oxidative stress and decreased
parvalbumin staining GABAergic neurons in the anterior
cingulate cortex
Do et al., in
preparation
WPA is a marker for the
perineural net

28. SR-/- Mice Exhibit Anhedonia as Demonstrated
in Intracranial Self Stimulation
Carlezon WA Jr, Chartoff EH. Nat Protoc. 2007;2(11):2987-95;
Puhl M, in preparation,

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SR-­/-­ NMDA Receptor Hypofunction
Schizophrenia Endophenotype Replicates
Forebrain MRS Glutamate and GABA Findings
Puhl et al., Neurobiology of Disease, 73C: 269-74, 2014.
Schizophrenia: elevated MRS GABA: Kegeles et al., 2012; dela Fuente-Sndoval;
elevated MRS Glutamate: Kegeles et al., 2012; Ongur et al., 2010.

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Similarities between SR-/- mice and
schizophrenia cortical pathology
1. Cortical atrophy ✔ ✔
1. Reduced dendritic complexity ✔ ✔
2. Reduced spines ✔ ✔
3. Reduced neuroplastic pathway function
BDNF, Akt, mTor ✔ ✔
5. Oxidative stress, PV/GABA ✔ ✔
6. Cognitive impairments ✔ ✔
7. Anhedonia (Negative symptoms) ✔ ✔
Schiz SR-/-
DeVito et al., Genes Brain Behav. 10:210-22, 2011; Balu et al. ,
Neurobiology of Disease, 45: 671-82, 2012; Basu et al., Mol Psychiatry.
14:719-27, 2009; Balu et al., Proc Natl Acad Sci USA, 2013.

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Serine Racemase is expressed in pyramidal and in
GABAergic inter-neurons but not in astrocytes in
human cortex
Balu et al., Cell Mol Neurobiol 34: 419, 2014

32. D-Serine does not co-localize with
astrocytic markers in cortex
Balu DT, Takagi S, Puhl MD, Benneyworth MA and Coyle, JT. Cell
Mol Neurobiol 34: 419-435, 2014.

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Does enhancing NMDA Receptor
Function Affect the Symptoms of
Schizophrenia?

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Goff et al., Am J Psych 152: 1213, 1995
Dose Finding Study for D-Cycloserine in
Schizophrenia

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GlyT1 inhibitor, NFPS, demonstrates that the glycine
modulatory site on synaptic NMDA receptors is not fully
occupied by glycine/D-serine
Bergeron et al., Proc Natl Acad Sci U S A. 95:15730-4, 1998.

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M
D-Cycloserine Treatment for 8 Weeks versus
Placebo: fMRI of a Memory Task
Yergulin-Todd et al., Psychiat Res (Neuroimaging) 138 (2005): 23-31.

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M
D-Cycloserine Treatment for 8 Weeks versus Placebo
Improves Memory: fMRI of a Memory Task
Yergulin-Todd et al., Psychiat Res (Neuroimaging) 138 (2005): 23-31.

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M
D-Cycloserine Treatment for 8 Weeks improves
memory, increases cortical blood flow and reduces
negative symptoms in schizophrenia
Right Superior Temporal Gyrus 0.82 +/- 0.58
Left Superior Temporal Gyrus 1.88 +/- 0.34*
Temporal Lobe Activation and
Negative Symptoms (PANSS) r= -0.77*
+/- S.D. * p< 0.05
Yergulin-Todd et al., Psychiat Res (Neuroimaging) 138 (2005) :23-31.

39. Tsai et al., 1998
(N=29)
Heresco-Leveyet al., 2005
(N=38)
BPRS/CGI -2.2** -5.7**
SANS -10.4** -7.9**
PANSS-Cog -1.7* -1.3**
PANSS-Pos -3.4** -1.4*
6/9/16 *p < 0.05; **p < 0.01
Addition of D-Serine (2g/day) to Anti-Psychotic
Drugs Reduces All Symptoms in Patients with
Chronic Schizophrenia

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Meta-analysis of Placebo-Controlled Glycine
Modulatory Site Agonist Clinical Trials in
Schizophrenia
“About eight hundred subjects from 26 studies were
included in current meta-analysis. Overall, the
NMDA-enhancing molecules are effective in most
schizophrenic symptom domains with the effect size
(ES) of total psychopathology of 0.40 (p<1x10-4).
The ES of clinical efficacy of the symptom domains
were in the order of depressive (0.40, p=3x10-4),
negative (0.38, p<1x10-4), cognitive (0.28, p=2x10-3),
positive symptom (0.26, p=0.0006), and general
psychopathology (0.26, p=0.006).”
Proof of concept but limitations as treatments…..
Tsai GE, Lin PY. Current Pharmaceutical Design 16: 522-37, 2010

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Is It Possible to Reverse the
Neuroplasticity Deficits in SR-/- Mice?

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3 Weeks of D-Serine Treatment Restores D-Serine
Levels in Brain in SR-/- Mice
Balu et al., PNAS USA 2013 Jun 25;110(26):E2400-9.

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Chronic D-Serine Reverses LTP, Spine Deficits and
Trophic Pathway Reductions in SR-/- Mice
Balu et al., PNAS USA 2013 Jun
25;110(26):E2400-9.
Balu and Coyle. Neurochem Intern.
75: 76-78, 2014.
P-TrkB, P-Akt, P-mTOR levels are
restored by D-serine.

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Chronic Systemic D-Serine Treatment Reverses
Memory Deficits (Hippocampal) in SR-/- Mice
Balu et al., Proc Natl Acad Sci USA, 2013.

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Genetic Studies Independently Confirm
Glutamatergic Hypothesis of
Schizophrenia

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Copy Number Variants (CNVs) and the Risk
for Schizophrenia
In an analysis of 18,492 subjects (7907 cases and
10585 controls), case CNVs were enriched for members
of the NMDAR complex (P=0.0015)…Our data indicate
that defects in NMDAR postsynaptic signaling…which
are known to be important in synaptic plasticity and
cognition, play a significant role in the pathogenesis of
schizophrenia.
Kirov et al., Mol Psychiatry 17: 142-­53, 2012.

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Study
Genome-­Wide Association Study (GWAS)
of 113,075 controls and 36,989
schizophrenic subjects reveal 108 risk loci.
Ripke et al., Nature 511: 421, 2014.
Serine Racemase
17q13

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48Coyle JT and Balu DT. Current Opinion Pharmacol 20: 109-115, 2015.
Multiple Schizophrenia Risk Genes Converge
on NMDA Receptor Signaling
mGluR3'
Glu'
Astrocyte
GRIN2A
Risk'Genes'
GRM3
GRIA1
DLG1
DLG2
CLCN3
SRR
AKT3
CO2
0'
H'
NH3
+'
OH'
L"serine(
CO2
0'
NH3
+'
H'
OH'
D"serine(
SR'
Pre"synap.c(
MEF(
Nucleus(
PSD
Akt3(
SAP97'
DS'Glu'
NR2A'
mGluR3'
Glu'
PSD93' Cl-
ClC-3
Glu'
SAP97'
PSD93'
GluR1'
D-serine
Glutamate
Transcription
factor/repressor
MEF2

49. Genetic models are useful for
identifying treatments that address the
pathophysiology of schizophrenia.

50. 5 Day Treatment with a mGluR5 Positive Allosteric
Modulator (VU-551) Reverses Neuroplasticity Deficits
in SR-/- Mice
Balu DT, Li Y, Takagi S, Presti KT, Ramikie TS, Rook JM, Jones CK, Lindsley CW, Conn
PJ, Bolshakov VY, Coyle JT. Neuropsychopharmacology. 2016 Jan 7. doi:
10.1038/npp.2016.2.

51. Balu DT, Li Y, Takagi S, Presti KT, Ramikie TS, Rook JM, Jones CK, Lindsley
CW, Conn PJ, Bolshakov VY, Coyle JT. Neuropsychopharmacology. 2016 Jan
7. doi: 10.1038/npp.2016.2.
VU-551, a mGluR5 positive allosteric modulator,
restores CA3-CA1 LTP and contextual fear
conditioning

52. Complement C4 Gene
Major Histocompatability Complex Risk Locus for
Schizophrenia (HSA 6) Encodes Complement C4
“Each common CA4 allele associating with schizophrenia (was) in proportion
to its tendency to generate greater expression of C4A. In mice, C4 mediated
synapse elimination during post-natal development. The results…may help
explain the reduced number of synapses in the brains of individuals with
schizophrenia.”
Sekar A et al.,Schizophrenia risk from complex variation of complement
component 4. Nature. 2016 Feb 11;530(7589):177-83.

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Schizophrenia is a circuit
disorder offering many targets
for potential pharmacologic
interventions

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DA
Glu
Glu
GABA
Ventral
Striatum
Psychosis
Disinhibition
Cognitive Impairment NMDA Receptor
Hypofunction
+
+
VTA
NBM
↓Alpha 7 nAChR
+
Dendritic
dysplasia
Ketamine
NAAG/Kynurenate
mGluR3, Arc, NRG1,
GRIK3, GRIN2A, SR

55. Summary
• Many risk genes for schizophrenia including serine racemase and C4A
regulate synaptic plasticity and are associated with reduced cortical
glutamatergic synapses (~-30%).
• Knocking out SR replicates many of the pathologic features of
schizophrenia including reduced synaptic connectivity, cortical
atrophy, reduced PV+GABAergic staining, negative symptoms and
cognitive impairments (genetic model with construct validity)
• SR and D-serine are localized to forebrain glutamatergic and
PV+GABAergic neurons where D-serine serves as the synaptic
co-agonist at NMDA receptors
• Much of the SR-/- phenotype can be reversed by treating adult SR-/-
mice with D-serine or an mGluR5 PAM, holding out hope for
pharmacologic reversal of the glutamatergic disconnection in
schizophrenia and effective treatment of cognitive and negative
symptoms

56. Conclusions
1. Impairments in pathways mediating synaptic plasticity are a
common pathophysiologic feature of serious mental disorders
2. Glutamatergic neurotransmission is a critical mediator
3. Regions of impairments dictate symptoms: widespread in cortex
in schizophrenia; hippocampus, prefrontal cortex in mood
disorder; nucleus accumbens and prefrontal cortex in addictions;
striatum in obsessive compulsive disorder
4. Restorative treatments likely require enhancement of pro-
neuroplastic pathways AND targeted neurocognitive
interventions