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Alessandro Usiello, PhD
D-Aspartate	exerts	an	opposing	role	upon	age-dependent	
NMDAR-related	synap7c	plas7city	and	memory...
Number of research articles on Pubmed (1990-2016):
comparison between D-serine and D-aspartate
0
500
1000
1500
2000
a.d-se...
Pharmacological features of free D-aspartate: what we already know
1. Can be stored in secretory granules and released by ...
(Hashimoto et al, J Neurochem, 1993)
D-aspartate levels do not display gross variations during
postnatal life in the human hippocampus and frontal cortex
(Unpu...
Free D-aspartate in the rat brain strongly decreases during
adulthood
.
Wolosker et al., Neuroscience (2000)
D-aspartate
Temporal decrease in D-aspartate concentration in mouse brain
Punzo, Errico et al 2016
The only enzyme known to catabolize D-aspartate is D-aspartate oxidase (DDO)
D-Asp + O2 + H2O α-oxalacetate + H2O2 + NH3
F...
D-aspartate causes NMDAR-dependent and independent inward currents in
striatum and in the hippocampus of mouse brain
(Erri...
DL-TBOA = glutamate/aspartate transporter inhibitor
CNQX = AMPAR antagonist
MCPG = metabotropic (mGluR) antagonist
CdCl2 =...
Example of HPLC chromatogram
1.  D-Asp
2.  L-Asp
3.  L-Glu
Semipermeable
Ultra-thin
Precision pump
In vivo microdialysis o...
(unpublished data)
D-aspartate occurs in the brain also at extracellular level and is released
in a calcium-dependent mann...
Pharmacological features of free D-aspartate
1. Can be stored in secretory granules and released by depolarizing stimuli
v...
Learning
Synaptic Plasticity
Memory
Does free D-aspartate modulate in vivo
NMDAR-dependent processes?
G
Sensory-gating int...
Higher extracellular D-aspartate concentrations in the brain of
freely moving DDO mutant mice
D-Aspartate + O2 + H2O α-Oxa...
(Errico et al, J Neurosci, 2008)
Higher levels of D-aspartate in Ddo-/- mice enhance hippocampal
NMDAR-dependent synaptic ...
Lack of Ddo is associated with
increased dendritic length and spine density in mouse brain
(Errico	et	al,	Transl	Psychiatr...
Mutant	mice	some;mes	show	molecular	compensa;ons	and/or	aspecific	effects	due	to	the	
gene;c	background……to	avoid	these	draw...
(unpublished data)
One-month oral administration of D-Aspartate in tap water increases its
extracellular concentration in ...
Acute and chronic D-aspartate trigger in vivo L-glutamate release
in the mouse prefrontal cortex by stimulating presynapti...
Four-week D-aspartate oral administration increases cerebral
blood volume in the prefrontal cortex and hippocampus of mice...
One month chronic D-aspartate oral administration enhances
dendritic length and increases spine density in mouse brain
(Er...
Oral D-aspartate supplementation enhances hippocampal NMDAR-
dependent synaptic plasticity and spatial memory of adult mic...
A genetic variant of DDO is associated with functional
prefrontal phenotypes relevant to schizophrenia in healthy humans
C...
“As the brain ages, the NMDA receptor system becomes progressively hypo-
functional, contributing to decreases in memory a...
***	
Water	
20	mM		
D-Asp	
	
Over 1-year old
***	
(Errico et al., Neurobiol Aging, 2011a)
Four-week oral treatment with D-...
F. Errico et al. / Neurobiology of Aging 32 (2011) 2229–2243 2235
Fig. 3. Effect of prolonged 12-month d-Asp exposure on h...
Prepulse inhibition of the startle reflex: a behavioural paradigm to
detect sensorimotor gating processes, from mice to hu...
Young adult Ddo-/- mice display attenuated sensorimotor gating
deficits induced by the hallucinogenic drug PCP
(Errico et ...
1.  D-aspartate mainly acts as an endogenous NMDA receptor agonist;
2.  D-aspartate is also present in the extracellular s...
…why Nature created an enzyme that during
post-natal life removes D-aspartate, if
increased content of this molecule impro...
Ddo gene ablation induces precocious spatial memory decay
Non	physiological	D-aspartate	level	modulate	the	cogni7ve	perfor...
Age-dependent LTP deterioration in the hippocampus of Ddo-/- mice
(Cristino et al, Neurobiol Aging, 2015)
Fig. 5. Elevatio...
Ddo mutant mice display age-dependent changes in hippocampal
ERK1/2 activation.
young
old
F. Errico et al. / Neurobiology ...
ACCEPTED MANUSCRIPT
Old Ddo-/- mice display increased sensorimotor gating deficits induced
by the hallucinogenic drug PCP
...
Non-physiological increase of D-aspartate in DDO mutant mice
is associated with age-related activation of caspase-3 in the...
D-aspartate oxidase prevents the age-related
cell death in dopaminergic neurons of the substantia nigra
(Punzo et al, J Ne...
D-aspartate oxidase, through the control of D-aspartate levels, prevents
accelerated age-related deterioration of brain pr...
What is the molecular mechanism
regulating the time-dependent increase
of D-aspartate oxidase activity the
mammalian brain?
Postnatal decrease in D-aspartate brain concentration is associated with
reciprocal increase in D-aspartate oxidase mRNA i...
Postnatal decrease of D-aspartate oxidase gene promoter methylation is
associated with the progressive increase in D-aspar...
The methylation state of the putative D-aspartate oxidase promoter
controls the expression of the Ddo mRNA in mice: Azacyt...
Neurobiology of Disease
Age-Related Changes in D-Aspartate Oxidase Promoter
Methylation Control Extracellular D-Aspartate ...
The methylation state of the Ddo gene promoter predicts its mRNA
expression in the adult mouse brain
RelativeDdomRNAexpres...
The methylation state of the region surrounding the start codon
of the DDO gene is higher in the frontal cortex than in th...
DDO mRNA expression levels in the adult hippocampus and frontal
cortex are complementary to DDO methylation profile
***
(U...
D-aspartate levels are higher in the frontal cortex
than in the hippocampus
**HPLC
(Unpublished data)
The regional differences in D-aspartate levels, DDO gene expression
and methylation profile are maintained in the brain of...
Age-related changes in D-aspartate concentration mirror
Ddo mRNA expression in the brain of Nothobranchius furzeri
Wang et...
Results
As found in rats and humans, also in mouse brain we documented a significant age-related decrease
of D-aspartate c...
Future studies
Francesco Napolitano and Francesco Errico
Anna	Di	Maio	
Tommaso	Nuzzo	
Daniela	Punzo	
Arianna	De	Rosa
Acknowledgements
	
Loredano	Pollegioni,	Silvia	Sacchi,	Gianluca	Molla	
Università	degli	Studi	dell’Insubria,	Varese,	Ital...
In the Hyppocampus of patients with schizophrenia, the state of DDO
methylation follows a peculiar profile of reduction wi...
Intermittent oral administration of D-aspartate exerts reversible influence
on hippocampal NMDAR-dependent synaptic plasti...
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D-Aspartate exerts an opposing role upon age-dependent NMDAR-related synaptic plasticity and memory decay - Alessandro Usiello

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

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D-Aspartate exerts an opposing role upon age-dependent NMDAR-related synaptic plasticity and memory decay - Alessandro Usiello

  1. 1. Alessandro Usiello, PhD D-Aspartate exerts an opposing role upon age-dependent NMDAR-related synap7c plas7city and memory decay Como 7th June 2016
  2. 2. Number of research articles on Pubmed (1990-2016): comparison between D-serine and D-aspartate 0 500 1000 1500 2000 a.d-serine d-asp
  3. 3. Pharmacological features of free D-aspartate: what we already know 1. Can be stored in secretory granules and released by depolarizing stimuli via Ca2+-dependent mechanism; 2. Binds L-Glu site of NMDARs in vitro with the same high affinity of NMDA and L-Asp). 3. Shows high and selective affinity for the L-Glu/L-Asp uptake system (EAATs). 1)  Does D-aspartate act selectively on NMDAR? 2) Does D-aspartate occur at extracellular level? 3) Does it across the blood brain barrier? 4) Does D-aspartate can be released also in vivo in a Ca2+-dependent manner ? Presynaptic glutamatergic terminal Postsynaptic neuron - dendritic spine Astrocyte Capillary
  4. 4. (Hashimoto et al, J Neurochem, 1993)
  5. 5. D-aspartate levels do not display gross variations during postnatal life in the human hippocampus and frontal cortex (Unpublished data) FRONTAL CORTEX HIPPOCAMPUS
  6. 6. Free D-aspartate in the rat brain strongly decreases during adulthood . Wolosker et al., Neuroscience (2000) D-aspartate
  7. 7. Temporal decrease in D-aspartate concentration in mouse brain Punzo, Errico et al 2016
  8. 8. The only enzyme known to catabolize D-aspartate is D-aspartate oxidase (DDO) D-Asp + O2 + H2O α-oxalacetate + H2O2 + NH3 FAD DDO D-aspartate oxidase activity in the brain increases during post-natal life (Van Veldhoven et al, Biochim Biophys Acta, 1991)
  9. 9. D-aspartate causes NMDAR-dependent and independent inward currents in striatum and in the hippocampus of mouse brain (Errico et al, Mol Cell Neurosci, 2008) fected by D-aspartate chronic treatment ( p Ͼ 0.1, per each protein) (Fig. 5e,f, respectively). Improved hippocampus-dependent memory in Ddo؊/؊ mice It is established that NMDAR signaling plays a pivotal role in hippocampus- related learning and memory (Lynch, 2004). Thus, based on the ability of D-aspartate to act as NMDAR agonist, we studied DdoϪ/Ϫ and D-aspartate chroni- cally treated animals, displaying exacer- bated hippocampal levels of this D-amino acid (Ddoϩ/ϩ vs DdoϪ/Ϫ , 33.8 Ϯ 4.0 vs 408.0 Ϯ 26.7 nmol/g tissue, p Ͻ 0.0001; H2O vs D-aspartate, 35.2 Ϯ 3.2 vs 133.6 Ϯ 9.5 nmol/g tissue, p Ͻ 0.0001; Student’s t test), in a hidden platform version of the Morris water maze and in a context- dependent fear conditioning paradigm. In the Morris maze test, we found unal- teredspatiallearningabilitiesinDdoϪ/Ϫ and D-aspartate chronically treated mice (Fig. 6a,d). Indeed, statistical analysis did not re- veal significant differences in spatial learn- ing, as confirmed by comparable escape la- tencies between genotypes (ANOVA, genotype effect and genotype ϫ sessions in- teraction, p Ͼ 0.1) and D-aspartate treat- 10410 • J. Neurosci., October 8, 2008 • 28(41):10404–10414 Errico et al. • D-Aspartate Has Antipsychotic-Like Properties NAcVTA Rhes Rhes DARP32 TH Merge Merge (Errico et al, J. Neurosci, 2008) fected by D-aspartate chronic treatment ( p Ͼ 0.1, per each protein) (Fig. 5e,f, respectively). Improved hippocampus-dependent memory in Ddo؊/؊ mice It is established that NMDAR signaling plays a pivotal role in hippocampus- related learning and memory (Lynch, 2004). Thus, based on the ability of D-aspartate to act as NMDAR agonist, we studied DdoϪ/Ϫ and D-aspartate chroni- cally treated animals, displaying exacer- bated hippocampal levels of this D-amino acid (Ddoϩ/ϩ vs DdoϪ/Ϫ , 33.8 Ϯ 4.0 vs 408.0 Ϯ 26.7 nmol/g tissue, p Ͻ 0.0001; H2O vs D-aspartate, 35.2 Ϯ 3.2 vs 133.6 Ϯ 9.5 nmol/g tissue, p Ͻ 0.0001; Student’s t test), in a hidden platform version of the Morris water maze and in a context- dependent fear conditioning paradigm. In the Morris maze test, we found unal- teredspatiallearningabilitiesinDdoϪ/Ϫ and D-aspartate chronically treated mice (Fig. 6a,d). Indeed, statistical analysis did not re- veal significant differences in spatial learn- ing, as confirmed by comparable escape la- tencies between genotypes (ANOVA, genotype effect and genotype ϫ sessions in- teraction, p Ͼ 0.1) and D-aspartate treat- ments (ANOVA, treatment effect and treat- ment ϫ sessions interaction, p Ͼ 0.1), 10410 • J. Neurosci., October 8, 2008 • 28(41):10404–10414 Errico et al. • D-Aspartate Has Antipsychotic-Like Properties
  10. 10. DL-TBOA = glutamate/aspartate transporter inhibitor CNQX = AMPAR antagonist MCPG = metabotropic (mGluR) antagonist CdCl2 = Voltage Gated Calcium Channel blocker The NMDAR-independent currents triggered by D-aspartate in pyramidal neurons of hippocampus are Ca2+-dependent Errico et al, Neurobiol Aging, 2011b
  11. 11. Example of HPLC chromatogram 1.  D-Asp 2.  L-Asp 3.  L-Glu Semipermeable Ultra-thin Precision pump In vivo microdialysis on freely moving mice dialysate samples collected at regular intervals at the tip of the probe a 1 mm long membraneUltra-thin concentric microdialysis probe perfusion with artificial CSF (flow rate 1-1.2 µl/min)
  12. 12. (unpublished data) D-aspartate occurs in the brain also at extracellular level and is released in a calcium-dependent manner in freely moving mice One-month chronic oral administration of 20 mM D-Asp in tap water Acute injection with 500 mg/kg D-Asp
  13. 13. Pharmacological features of free D-aspartate 1. Can be stored in secretory granules and released by depolarizing stimuli via Ca2+-dependent mechanism; 2. Binds L-Glu site of NMDARs in vitro with the same high affinity of NMDA and L-Asp. 3. Shows high and selective affinity for the L-Glu/L-Asp uptake system (EAATs). 4. D-aspartate mainly acts on NMDAR, although it also activates NMDA-independent currents 5. D-aspartate also occurs at extracellular level in the mouse brain 6. D-aspartate across the blood brain barrier 7. D-aspartate can be released in vivo in a Ca2+-dependent manner Presynaptic glutamatergic terminal Postsynaptic neuron - dendritic spine Astrocyte Capillary
  14. 14. Learning Synaptic Plasticity Memory Does free D-aspartate modulate in vivo NMDAR-dependent processes? G Sensory-gating integration processes
  15. 15. Higher extracellular D-aspartate concentrations in the brain of freely moving DDO mutant mice D-Aspartate + O2 + H2O α-Oxalacetate + H2O2 + NH3 D-Aspartate Oxidase (DDO) Errico et al, Gene, 2006 Francesco Errico Brain homogenates Cortex homogenates Cortex extracellular dialysate (Punzo, Errico et al, J Neurosci, 2016)
  16. 16. (Errico et al, J Neurosci, 2008) Higher levels of D-aspartate in Ddo-/- mice enhance hippocampal NMDAR-dependent synaptic plasticity and spatial memory (Errico et al, Mol Cell Neurosci, 2008)
  17. 17. Lack of Ddo is associated with increased dendritic length and spine density in mouse brain (Errico et al, Transl Psychiatry, 2014)
  18. 18. Mutant mice some;mes show molecular compensa;ons and/or aspecific effects due to the gene;c background……to avoid these drawbacks, we used another strategy for up-regula;ng D-aspartate levels in the adult brain Water 20 mM D-Asp D-Asp DDO Wild type mice (C57BL/6J) were administered with D-aspartate dissolved in tap water at the concentration of 20 mM An alternative (easier) strategy to increase D-aspartate brain levels without gene-targeting…
  19. 19. (unpublished data) One-month oral administration of D-Aspartate in tap water increases its extracellular concentration in the cortex of freely moving mice 20 mM D-Asp
  20. 20. Acute and chronic D-aspartate trigger in vivo L-glutamate release in the mouse prefrontal cortex by stimulating presynaptic NMDA and non-NMDA ionotropic and metabotropic receptors D-aspartate perfusion ± glutamatergic antagonists: MK801: NMDAR antagonist CNQX: AMPAR antagonist MTEP: mGluR5 negative allosteric modulator (unpublished data)
  21. 21. Four-week D-aspartate oral administration increases cerebral blood volume in the prefrontal cortex and hippocampus of mice (Errico et al, Transl Psychiat, 2014)
  22. 22. One month chronic D-aspartate oral administration enhances dendritic length and increases spine density in mouse brain (Errico et al, Transl Psychiatry, 2014)
  23. 23. Oral D-aspartate supplementation enhances hippocampal NMDAR- dependent synaptic plasticity and spatial memory of adult mice (Errico et al, Neurobiol Aging, 2011b) the last acquisition trial (data not shown). Oral administration of D-aspartate increases hippocampal D-aspartate levels and strongly enhances LTP at CA1 synapses In order to examine the consequences of deregulated levels of D-aspartate on hippocampus-dependent functions, avoiding Ddo gene targeting, we administered for 1 month a solution of 20 mM D-aspartate to 5 weeks old C57BL/6 mice. We used animals from C57BL/6 inbred strain because it represents the genetic background transmission and presynaptic transmission properties. To determine if this was confirmed in D-aspartate treated C57BL/6 mice, we tested the synaptic input–output relationship and the paired pulse facilitation in the CA1 region. As for Ddo−/− animals, also in this case no significant differences were observed (data not shown). In order to test whether alterations of synaptic plasticity might occur also in D-aspartate C57BL/6 treated mice, we conditioned the Schaffer commissural pathway with either TBS or depotentiation protocol, as previously shown in Figs. 1C and D. Interestingly, both PTP and LTP following TBS differed significantly between Fig. 5. Oral D-aspartate treatment enhances hippocampal LTP at CA1 synapses. (A) D-aspartate levels were measured by HPLC in the hippocampus of C57BL/6 untreated (n=7) and C57BL/6 D-aspartate treated (n=6) male mice. Treated animals drank 20 mM D-aspartate for 1 month prior to HPLC detection. ⁎⁎⁎ indicates pb0.0001 (Student's t test). Values are expressed as mean±S.E.M. Treatments are as indicated. (B) Superimposed pooled data showing the normalized changes in field potential slope (±S.E.M.) in H2O vs D-aspartate treated mice induced by TBS. The magnitude of potentiation measured throughout the experiment was significantly higher in D-aspartate (n=6) vs H2O (n=6) treated mice. The fEPSP traces were taken before and 60 min after TBS. Calibration bars: 0.5 mV, 10 ms. (C) Superimposed pooled data showing the normalized changes in field potential slope (±S.E.M.) in H2O vs D-aspartate treated mice induced by depotentiation protocol. Sixty minutes after a high-frequency tetanus (HFS, arrow) to produce LTP, slices were subjected to a LFS train (bar). The degree of depotentiation measured 60 min after LFS was significantly higher in Ddo+/+ (n=4) compared to Ddo−/− (n=4) mice. (D) Summary bar chart showing the effects of TBS and depotentiation on the fEPSP slopes (% of baseline) quantified 50–60 min after each protocol in the 4 groups under observation. ARTICLE IN PRESSNBA-7478; No.of Pages15 6 F. Errico et al. / Neurobiology of Aging xxx (2010) xxx–xxx Fig. 2. Effect of oral 3-month d-Asp administration on the modulation of hippocampal synaptic functions at CA1 synapses. (A) d-As by HPLC in the hippocampus of C57BL/6J mice treated for 3-months with a 20 mM d-Asp solution (H2O, n = 5; d-Asp, n = 4). Tre 45-day-old mice, significantly increased the levels of the d-amino acid, compared to respective controls. A time-course of hippocampa (Errico et al, Neurobiol Aging, 2011b) 20 mM D-Asp
  24. 24. A genetic variant of DDO is associated with functional prefrontal phenotypes relevant to schizophrenia in healthy humans CC genotype predicts reduced DDO mRNA expression (In silico analysis, Braincloud database) (Fisher’s post hoc vs CT P=0.06; vs TT P=0.00003) CC genotype is associated with greater prefrontal gray matter volume (assayed by voxel-based morphometry) (Fisher’s post hoc, all P<0.005) CC and CT genotypes predict greater prefrontal activity during working memory tasks (n-back task) (assayed by BOLD fMRI) (Fisher’s post hoc P=0.02 and P=0.05, respectively). he influence of deregulated high levels of d functional synaptic plasticity, we also used o gene, that show a 10–20-fold increase in of D-Asp.41 In the PFC, total dendritic length was significantly increased in Ddo− / − mice, trols (Ddo+/+ vs Ddo− / − (mean ± s.e.m.): s 1537.47 ± 138.10 μm, Po0.05; Figure 3a). d no difference in the complexity of basal es between Ddo+/+ and Ddo− / − mice y, dendrites from Ddo− / − pyramidal neurons 2219.51 ± 43.86 μm, Po0.01; Figure 3d). Sholl analysis revealed a significantly different morphological organization of basal den- drites between genotypes (genotype: F(1,40) = 8.131, P = 0.0214; distance from soma × genotype: F(5,40) = 4.152, P = 0.0039; Figure 3e), as revealed by a greater number of intersections in Ddo− / − mice between 50 and 125 μm, compared with controls (Po0.01 at 50 μm; Po0.05 at 75, 100 and 125 μm). Apical intersections are not different between genotypes (Figure 3e). Also, spine density is significantly increased in Ddo− / − mice, compared with controls [Ddo+/+ vs Ddo− / − (mean ± s.e.m.): f DDO rs3757351 with prefrontal phenotypes in humans. (a) Association of DDO rs3757351 with DDO mRNA expression PFC (n = 268). Graph depicts normalized log2 ratios (sample/reference). Data from http://braincloud.jhmi.edu/; (b) 3757351 with prefrontal gray matter volume in Caucasian healthy subjects (n = 159). Left panel: three-dimensional ontal cluster associated with a main effect of rs3757351. Image thresholded at Po0.005, nonstationary cluster extend : graph showing mean ± 0.95 CIs of gray matter content extracted from the cluster depicted in the left panel. (c) 3757351 with prefrontal BOLD response during working memory in Caucasian healthy subjects (n = 143). Left panel: ndering of the prefrontal cluster associated with a main effect of rs3757351. Image thresholded at Po0.05, FWE : graph showing mean ± 0.95 CIs of parameter estimated extracted from the cluster depicted in the left panel. CI, LPFC, dorsolateral PFC; PFC, prefrontal cortex. To further evaluate the influence of deregulated high levels of D-Asp on structural and functional synaptic plasticity, we also used knockout mice for Ddo gene, that show a 10–20-fold increase in the cerebral content of D-Asp.41 In the PFC, total dendritic length of pyramidal neurons was significantly increased in Ddo− / − mice, compared with controls (Ddo+/+ vs Ddo− / − (mean ± s.e.m.): 1189.37 ± 28.63 μm vs 1537.47 ± 138.10 μm, Po0.05; Figure 3a). Sholl analysis revealed no difference in the complexity of basal and apical dendrites between Ddo+/+ and Ddo− / − mice (Figure 3b). Conversely, dendrites from Ddo− / − pyramidal neurons (Ddo vs Ddo (mean ± s.e.m.): 1765.25 ± 117.58 μm vs 2219.51 ± 43.86 μm, Po0.01; Figure 3d). Sholl analysis revealed a significantly different morphological organization of basal den- drites between genotypes (genotype: F(1,40) = 8.131, P = 0.0214; distance from soma × genotype: F(5,40) = 4.152, P = 0.0039; Figure 3e), as revealed by a greater number of intersections in Ddo− / − mice between 50 and 125 μm, compared with controls (Po0.01 at 50 μm; Po0.05 at 75, 100 and 125 μm). Apical intersections are not different between genotypes (Figure 3e). Also, spine density is significantly increased in Ddo− / − mice, compared with controls [Ddo+/+ vs Ddo− / − (mean ± s.e.m.): Figure 4. Association of DDO rs3757351 with prefrontal phenotypes in humans. (a) Association of DDO rs3757351 with DDO mRNA expression levels in postmortem PFC (n = 268). Graph depicts normalized log2 ratios (sample/reference). Data from http://braincloud.jhmi.edu/; (b) Association of DDO rs3757351 with prefrontal gray matter volume in Caucasian healthy subjects (n = 159). Left panel: three-dimensional rendering of the prefrontal cluster associated with a main effect of rs3757351. Image thresholded at Po0.005, nonstationary cluster extend corrected. Right panel: graph showing mean ± 0.95 CIs of gray matter content extracted from the cluster depicted in the left panel. (c) Association of DDO rs3757351 with prefrontal BOLD response during working memory in Caucasian healthy subjects (n = 143). Left panel: Three-dimensional rendering of the prefrontal cluster associated with a main effect of rs3757351. Image thresholded at Po0.05, FWE (n=159) dendritic length and converts E-LTP into L-LTP in the adult hippocampus To further evaluate the influence of deregulated high levels of D-Asp on structural and functional synaptic plasticity, we also used knockout mice for Ddo gene, that show a 10–20-fold increase in the cerebral content of D-Asp.41 In the PFC, total dendritic length of pyramidal neurons was significantly increased in Ddo− / − mice, compared with controls (Ddo+/+ vs Ddo− / − (mean ± s.e.m.): 1189.37 ± 28.63 μm vs 1537.47 ± 138.10 μm, Po0.05; Figure 3a). Sholl analysis revealed no difference in the complexity of basal and apical dendrites between Ddo+/+ and Ddo− / − mice (Figure 3b). Conversely, dendrites from Ddo− / − pyramidal neurons CA1 area of the hippocampus. Dendritic length was significantly increased in Ddo− / − mice, compared with Ddo+/+ animals (Ddo+/+ vs Ddo−/ − (mean ± s.e.m.): 1765.25 ± 117.58 μm vs 2219.51 ± 43.86 μm, Po0.01; Figure 3d). Sholl analysis revealed a significantly different morphological organization of basal den- drites between genotypes (genotype: F(1,40) = 8.131, P = 0.0214; distance from soma × genotype: F(5,40) = 4.152, P = 0.0039; Figure 3e), as revealed by a greater number of intersections in Ddo− / − mice between 50 and 125 μm, compared with controls (Po0.01 at 50 μm; Po0.05 at 75, 100 and 125 μm). Apical intersections are not different between genotypes (Figure 3e). Also, spine density is significantly increased in Ddo− / − mice, compared with controls [Ddo+/+ vs Ddo− / − (mean ± s.e.m.): Figure 4. Association of DDO rs3757351 with prefrontal phenotypes in humans. (a) Association of DDO rs3757351 with DDO mRNA expression levels in postmortem PFC (n = 268). Graph depicts normalized log2 ratios (sample/reference). Data from http://braincloud.jhmi.edu/; (b) Association of DDO rs3757351 with prefrontal gray matter volume in Caucasian healthy subjects (n = 159). Left panel: three-dimensional rendering of the prefrontal cluster associated with a main effect of rs3757351. Image thresholded at Po0.005, nonstationary cluster extend (n = 143) Intronic DDO variant rs3757351 (n=268) (Errico et al, Transl Psychiatry, 2014)
  25. 25. “As the brain ages, the NMDA receptor system becomes progressively hypo- functional, contributing to decreases in memory and learning performance” An increasing level of N-methyl-D-aspartate (NMDA) receptor hypofunction within the brain is associated with memory and learning impairments, with psychosis, and ultimately with excitotoxic brain injury. As the brain ages, the NMDA receptor system becomes progressively hypo- functional, contributing to decreases in memory and learning performance. In those individuals destined to develop Alzheimer’s disease, other abnormalities (eg, amyloidopathy and oxidative stress) interact to increase the NMDA receptor hypofunction (NRHypo) burden. In these vulnerable individuals, the brain then enters into a severe and persistent NRHypo state, which can lead to widespread neurodegeneration with accompanying men- tal symptoms and further cognitive deterioration. If the hypotheses described herein prove correct, treatment implications may be considerable. Pharmacological meth- ods for preventing the overstimulation of vulnerable cor- ticolimbic pyramidal neurons developed in an animal model may be applicable to the prevention and treat- he amino acid glutamate (Glu) plays a central role in both the normal and abnormal functioning of the central nervous system (CNS). Glu is recognized to be the main excitatory neurotransmitter in the CNS, estimated to be released at up to half of the synapses in the brain. In addition, Glu is also an excitotoxin that can destroy CNS neurons by excessive activation of excita- tory receptors on dendritic and somal surfaces. Two major classes of Glu receptors, ionotropic and metabotropic, have been identified. Glu exerts excito- toxic activity through three receptor subtypes, which belong to the ionotropic family. These three receptors are named after agonists to which they are differentially sensitive, N-methyl-D-aspartate (NMDA), amino-3- hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainic acid (KA). Of these three, the NMDA recep- tor has been the most extensively studied and the most frequently implicated in CNS diseases.1 Excessive activation of NMDA receptors (NMDA receptor hyperfunction [NRHyper]) plays an important role in the pathophysiology of acute CNS injury syn- dromes such as hypoxia-ischemia, trauma, and status epilepticus.1,2 Recently, hyperstimulation of AMPA/KA receptors and consequent excitotoxicity has been pro- posed to underlie neurodegeneration in amyotrophic lateral sclerosis (ALS, Lou Gerhig’s Disease3,4 ).The role of Glu excitotoxicity in the pathology of several other neuropsychiatric disorders has been extensively reviewed elsewhere1,5 and will not be the focus of this NMDA receptor function, memory, and brain aging John W. Newcomer, MD; Nuri B. Farber, MD; John W. Olney, MD T B a s i c r e s e a r c h An increasing level of N-methyl-D-aspartate (NMDA) receptor hypofunction within the brain is associated with memory and learning impairments, with psychosis, and ultimately with excitotoxic brain injury. As the brain ages, the NMDA receptor system becomes progressively hypo- functional, contributing to decreases in memory and learning performance. In those individuals destined to develop Alzheimer’s disease, other abnormalities (eg, amyloidopathy and oxidative stress) interact to increase the NMDA receptor hypofunction (NRHypo) burden. In these vulnerable individuals, the brain then enters into a severe and persistent NRHypo state, which can lead to widespread neurodegeneration with accompanying men- tal symptoms and further cognitive deterioration. If the hypotheses described herein prove correct, treatment implications may be considerable. Pharmacological meth- ods for preventing the overstimulation of vulnerable cor- ticolimbic pyramidal neurons developed in an animal model may be applicable to the prevention and treat- ment of Alzheimer’s disease. he role in both the central be the main estimated to the brain. In destroy CN tory recept major clas metabotrop toxic activi belong to th are named a sensitive, N hydroxy-5-m and kainic a tor has been frequently i Excessive receptor hy role in the dromes suc epilepticus.1 receptors an posed to un lateral scler of Glu exci neuropsych reviewed el paper. Inste underexcita hypofunctio Keywords: Alzheimer’s disease; brain aging; memory; NMDA receptor; NMDA receptor hypofunction Address for cor chiatry, Washing T
  26. 26. *** Water 20 mM D-Asp Over 1-year old *** (Errico et al., Neurobiol Aging, 2011a) Four-week oral treatment with D-aspartate in one-year aged female mice fully restores NMDAR-dependent synaptic plasticity deficits
  27. 27. F. Errico et al. / Neurobiology of Aging 32 (2011) 2229–2243 2235 Fig. 3. Effect of prolonged 12-month d-Asp exposure on hippocampal NMDAR-related synaptic plasticity. (A) d-Asp levels were detected by HPLC in the hippocampus of C57BL/6J mice treated for 12 months with a 20 mM d-Asp solution (H2O, n = 3; d-Asp, n = 3). Long-term treatment, that started on 45-day-old mice, significantly increased the levels of this d-amino acid, compared to respective controls. (B) Basal synaptic transmission was normal in 12-month d-Asp- treated synapses. (C) PPF was comparable at every tested interval between both treated and untreated groups. Data represent the mean ± SEM for at least 8 separate experiments/group. (D) Superimposed pooled data showing the effects of 1-year d-Asp administration on LTP (n = 8) vs. control (n = 7) (left panel) and following 1-month withdrawal (n = 6) vs. control (n = 6) (right panel). A different control group was used for each condition. Chronic long-term administration of d-Asp reduced NMDAR-dependent synaptic plasticity at CA1 synapses while successive 1-month removal of d-Asp from drinking solution was able to reinstate LTP at control levels. *p < 0.05, compared to control H2O-treated group (Student’s t test). Values are expressed as mean ± SEM. Treatments are as indicated. fully restore long-term potentiation (48.2 ± 8.1% above base- line) at levels recorded in untreated controls (49.1 ± 6.4% above baseline) (p > 0.05; Fig. 3D, right panel). 3.4. Effects of increased hippocampal d-Asp content on the expression of NMDAR and AMPAR subunits levels To investigate whether the changes of hippocampal LTP in d-Asp-treated mice may depend on an abnormal expression of glutamate receptors, we examined the levels of NMDAR and AMPAR subunits in the hippocampus of mice treated 3.5. d-Asp oral administration influence NMDAR-dependent memory In light with the pharmacological feature of d-Asp to acti- vate NMDARs (Errico et al., 2008a,b), here we investigated the NMDAR-dependent behavioral correlate of an abnormal hippocampal d-Asp content in treated animals. Firstly, to exclude potential sensori/motor/motivational alterations in 3- and 12-month treated mice, we performed a visual version of the Morris water maze. Overall, our behavioral data indi- cated no statistical difference between treatments over test sessions, at each regimen of treatment analysed (two-way F. Errico et al. / Neurobiology of Aging 32 (2011) 2229–2243 2237 Fig. 5. Consequences of chronic 3-month d-Asp treatment on the modulation of spatial memory in C57BL/6J mice. (A) Mice treated for 3 months with d-Asp One year D-Aspartate supplementation in C57B6 mice improves emotional learning without perturbing spatial memory sets of animals. First, we analysed motor activity of animals in the novelty-induced exploration paradigm over a 60-min session (Fig. 6A). Our data indicated no main influence of d-Asp treatment on motor response. Statistical analysis revealed a comparable locomotor profile of habituation between d-Asp- and H2O-treated mice after both 3-month [two-wayANOVA:timeeffect,F(5, 120) =52.085,p<0.0001; time×treatment interaction, F(5, 120) =2.215, p>0.05] and 12-month administration [two-way ANOVA: time effect, F(5, 110) =32.017, p<0.0001; time×treatment interaction, F(5, 120) =1.702, p>0.1]. arms, after both 3-month [F(1, 40) =1.581, p>0.1] and 12- month chronic administration [F(1, 23) =2.689, p>0.1]. Then, we assessed the effect of abnormal higher d-Asp brain levels in other cognitive tasks, such as object recogni- tion test (Winters et al., 2008) and fear conditioning (Phillips and LeDoux, 1992). In the object recognition test, our behav- ioral analysis failed to reveal differences between groups. Indeed, in the retention session performed 24h after the first exposure to the objects, d-Asp- and H2O-treated mice showed similar preference towards the novel object [one- way ANOVA: 3-month treatment: F(1, 37) =3.353, p>0.05; Fig. 6. Behavioral analysis of locomotor, anxiety-like and cognitive abilities in 3- and 12-month d-Asp-treated mice. (A) d-Asp treatment does not induce motor effects. Mice treated for 3 (n=13, per treatment) or 12 months with d-Asp (n=12, per treatment) were submitted to a novelty-induced exploration task. 20 mM D- Asp Morris water maze Fear Conditioning
  28. 28. Prepulse inhibition of the startle reflex: a behavioural paradigm to detect sensorimotor gating processes, from mice to humans Prepulse Inhibition (PPI) is a neurological phenomenon in which a weak anticipatory stimulus (prepulse) inhibits the startle reflex produced by a subsequent strong startling stimulus (pulse). The inhibition of the startle mirrors the ability of the nervous system to give the adequate saliency to sensory information. PPI deficits manifest in the inability to filter out the unnecessary information, a condition producing sensory hallucinations and cognitive fragmentation in patients with schizophrenia.
  29. 29. Young adult Ddo-/- mice display attenuated sensorimotor gating deficits induced by the hallucinogenic drug PCP (Errico et al, Transl Psychiatry, 2015)
  30. 30. 1.  D-aspartate mainly acts as an endogenous NMDA receptor agonist; 2.  D-aspartate is also present in the extracellular space and is released in vivo in a Ca2+-dependent manner; 3.  Extracellular D-aspartate triggers the release of L-glutamate. This effect is induced through the stimulation of pre-synaptic NMDA and non-NMDA (AMPA and mGlu5) receptors; 4.  D-aspartate can efficiently cross the blood brain barrier and reaches the brain parenchyma; 5.  Increased D-aspartate levels enhance dendritic length, spiny density, synaptic plasticity and cognition in young adult mice. 6.  Short-term administration of D-aspartate in aged mice rescues deterioration of NMDAR-dependent synaptic plasticity. Results
  31. 31. …why Nature created an enzyme that during post-natal life removes D-aspartate, if increased content of this molecule improves synaptic plasticity and cognitive abilities?
  32. 32. Ddo gene ablation induces precocious spatial memory decay Non physiological D-aspartate level modulate the cogni7ve performances in age-dependent manner : ü  Enhances spa;al memory at adulthood ü  Worsens cogni;ve abili;es in elderly phases (Errico et al, Neurobiol Aging, 2011a) young old
  33. 33. Age-dependent LTP deterioration in the hippocampus of Ddo-/- mice (Cristino et al, Neurobiol Aging, 2015) Fig. 5. Elevation of d-Asp levels produces opposite, age-related modulatory effects on hippocampal LTP. Superimposed pooled data showing the normalized changes in field potential slope (±SEM) in 4/5-, 9/10- and 13/14-month-old Ddo+/+ and Ddo−/− mice and, induced by HFS protocol (100 Hz for 1 s). fEPSP slopes were recorded and expressed as the percentage of the pre-tetanus baseline. A stimulation intensity that evoked 50% of maximal fEPSP response was used. Superimposed representative EPSPs shown were recorded before and 1 hr after LTP induction, as indicated by the corresponding numbers. Calibration bars, 1 mV and 10 ms. young old (Errico et al, Neurobiol Aging, 2011)
  34. 34. Ddo mutant mice display age-dependent changes in hippocampal ERK1/2 activation. young old F. Errico et al. / Neurobiology of Aging 32 (2011) 2061–2074 2071 (Errico et al, Neurobiol Aging, 2011a)
  35. 35. ACCEPTED MANUSCRIPT Old Ddo-/- mice display increased sensorimotor gating deficits induced by the hallucinogenic drug PCP (Cristino et al, Neurobiol Aging, 2015)
  36. 36. Non-physiological increase of D-aspartate in DDO mutant mice is associated with age-related activation of caspase-3 in the prefrontal cortex of Ddo-/- mice D aspartate oxidase, through the control of D-aspartate levels, counteracts brain deterioration caused by excessive NMDAR stimulation
  37. 37. D-aspartate oxidase prevents the age-related cell death in dopaminergic neurons of the substantia nigra (Punzo et al, J Neurosci, 2016) DARP32 TH Merge Merge
  38. 38. D-aspartate oxidase, through the control of D-aspartate levels, prevents accelerated age-related deterioration of brain processes caused by excessive NMDAR stimulation induction and maintenance of LTP. These effects are likely the cause of the cognitive decay experienced by aged mammals (Rosenzweig and Barnes 2003). Therefore, in the light of the ability of D-Asp to increase hippocampal NMDAR-dependent LTP and, in turn, spatial memory of adult Ddo-/- mice, one could question whether the lifelong excess of this D-amino acid may modulate the natural decay of cognitive processes at aging. In response to this ques- tion, results indicate that while increased D-Asp content enhances NMDAR-dependent LTP in 4/5-month-old Ddo-/- mice, persistent deregulation of this D-amino acid dramatically accelerates the age-dependent decay of syn- aptic plasticity in 13-/14-month-old knockout mice (Errico et al. 2011a). Consistently, spatial memory improvement found in 4-/5-month-old Ddo-/- mice turns into a drastic worsening of learning and memory abilities in mutants of 13–14 months of age (Fig. 1) (Errico et al. 2011a). Simi- larly to aged Ddo-/- mice, persistent administration of D-Asp for 12 months to C57BL/6 mice produces a signif- Fig. 1 Schematic representation of the opposite, age-related effects The role of free D-asp in the mammalian brain
  39. 39. What is the molecular mechanism regulating the time-dependent increase of D-aspartate oxidase activity the mammalian brain?
  40. 40. Postnatal decrease in D-aspartate brain concentration is associated with reciprocal increase in D-aspartate oxidase mRNA in mouse brain Punzo, Errico et al 2016
  41. 41. Postnatal decrease of D-aspartate oxidase gene promoter methylation is associated with the progressive increase in D-aspartate oxidase mRNA Punzo, Errico et al 2016 Ddo mRNA Ddo methylation
  42. 42. The methylation state of the putative D-aspartate oxidase promoter controls the expression of the Ddo mRNA in mice: Azacytidine treatment of primary cortical neurons Punzo, Errico et al 2016 Azacytidine:DNA demethylating agent
  43. 43. Neurobiology of Disease Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging Daniela Punzo,1,2* XFrancesco Errico,3,4* Luigia Cristino,5* XSilvia Sacchi,6,7 Simona Keller,1,4 Carmela Belardo,8 Livio Luongo,8 XTommaso Nuzzo,2,3 Roberta Imperatore,5 Ermanno Florio,1,4 Vito De Novellis,8 XOrnella Affinito,1,4 XSara Migliarini,9 XGiacomo Maddaloni,9 Maria Jose` Sisalli,1 XMassimo Pasqualetti,9,10 XLoredano Pollegioni,6,7 Sabatino Maione,8 Lorenzo Chiariotti,1,4 and Alessandro Usiello2,3 1Istituto di Endocrinologia ed Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (CNR), 80131 Naples, Italy, 2Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, 81100 Caserta, Italy, 3Laboratory of Behavioural Neuroscience, Ceinge Biotecnologie Avanzate, 80145 Naples, Italy, 4Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, 80131 Naples, Italy, 5Institute of Biomolecular Chemistry, CNR, 80078 Pozzuoli, Italy, 6Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy, 7Protein Factory Research Center, Politecnico di Milano and University of Insubria, 20133 Milan, Italy, 8Department of Experimental Medicine, Section of Pharmacology, Second University of Naples, 80138 Naples, Italy, 9Department of Biology, Unit of Cell and Developmental Biology, University of Pisa, 56126 Pisa, Italy, and 10Center for Neuroscience and Cognitive Systems, Istituto Italiano di Tecnologia, 38068 Rovereto, Italy TheendogenousNMDAreceptor(NMDAR)agonistD-aspartateoccurstransientlyinthemammalianbrainbecauseitisabundantduring embryonicandperinatalphasesbeforedrasticallydecreasingduringadulthood.Itiswellestablishedthatpostnatalreductionofcerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicar- boxylicD-aminoacids.Inthepresentwork,weshowthatD-aspartatecontentinthemousebraindrasticallydecreasesafterbirth,whereas DdomRNAlevelsconcomitantlyincrease.Interestingly,postnatalDdogeneexpressionisparalleledbyprogressivedemethylationwithin its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (DdoϪ/Ϫ ), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of D-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in DdoϪ/Ϫ brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in DdoϪ/Ϫ brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate. Key words: aging; D-amino acids; DNA methylation; neurodegeneration; NMDA receptor 3064 • The Journal of Neuroscience, March 9, 2016 • 36(10):3064–3078 “…..interes;ngly, the evidence that azaci;dine induces 4-fold increase in Ddo mRNA levels, compared with vehicle-treated embryonic primary neurons, indicates the existence of a finely tuned demethyla;on process at the basis of the observed postnatal increase in Ddo gene expression. ….we argue that the epigene;c changes within the Ddo regulatory region herein inves;gated are part of a developmental program to regulate the ;med expression of the Ddo gene during prenatal and postnatal brain development. …....ISH clearly indicated that postnatal transcrip;onal ac;va;on of Ddo gene differs from one brain area to another, sugges7ng that Ddo gene demethyla7on occurs with a peculiar spa7o- temporal paCern to properly regulate the regional postnatal occurrence of DDO mRNA, protein and, ul7mately, free D-Asp concentra7ons…”
  44. 44. The methylation state of the Ddo gene promoter predicts its mRNA expression in the adult mouse brain RelativeDdomRNAexpressionlevel H p C x 0 .0 0 .5 1 .0 1 .5 ** C p G -3 6 3 C p G -3 3 0 C p G -3 1 8 C p G -2 4 6 C p G -1 7 5 C p G -1 2 5 0 1 0 2 0 3 0 4 0 H p C x H p C x 0 1 0 2 0 3 0 Average methylation(%) Methylation(%) ** ** ** ** ** * studies would be advisable to elucidate how extracellular concentrations of D-Asp relate to the affinity of this D-amino acid for NMDARs, under both physiological conditions and in mice with enriched content of endoge- (Morris et al. 2003). Interestingly, in the hidden-platform version of the Morris water maze, Ddo-/- mice exhibit an improved spatial memory compared to controls (Errico et al. 2008a, 2011a). Likewise, in the contextual fear Table 1 Free D-aspartate levels in different brain regions of Ddo-/- mice Brain area Age of mice (month) D-Asp content (nmol/g tissue) References Ddo?/? mice Ddo-/- mice Hippocampus 4–5 119 ± 12 1847 ± 180 Errico et al. (2011b) 9–10 115 ± 7 1810 ± 129 13–14 140 ± 13 2048 ± 276 Striatum 4 31 ± 6 325 ± 23 Errico et al. (2008a, 2011c) Cortex 43 ± 6 817 ± 40 Cerebellum 34 ± 8 447 ± 22 Olfactory bulbs 33 ± 4 319 ± 34 Table 2 Free D-aspartate levels in different brain regions of D-aspartate-treated mice Brain area 20 mM D-asp oral administration delivery time (month) Age of mice (month) D-Asp content (nmol/g tissue) References H2O-treated mice D-Asp-treated mice Hippocampus 1 2 22 ± 3 102 ± 11 Errico et al. (2008b) 3 4.5 41 ± 2 99 ± 5 Errico et al. (2011a) 12 13.5 71 ± 11 158 ± 29 Striatum 2 3.5 17 ± 4 91 ± 14 Errico et al. (2008a) Cortex 58 ± 4 153 ± 11 Cerebellum 18 ± 1 81 ± 12 F. Errico et al. (Unpublished data)
  45. 45. The methylation state of the region surrounding the start codon of the DDO gene is higher in the frontal cortex than in the hippocampus of human brain ** ** ** ** ** * ** * ** (Unpublished data)
  46. 46. DDO mRNA expression levels in the adult hippocampus and frontal cortex are complementary to DDO methylation profile *** (Unpublished data)
  47. 47. D-aspartate levels are higher in the frontal cortex than in the hippocampus **HPLC (Unpublished data)
  48. 48. The regional differences in D-aspartate levels, DDO gene expression and methylation profile are maintained in the brain of patients with schizophrenia ** ****** * * * * (Unpublished data)
  49. 49. Age-related changes in D-aspartate concentration mirror Ddo mRNA expression in the brain of Nothobranchius furzeri Wang et al., 2015 Cell Age-related decrease of D-aspartate brain levels Age-related increase of Ddo mRNA brain levels DDO DAPI DDO DAPIDDO DAPI DDO DAPI 3 w 27 w 3 w 27 w 3 w 27 w DdomRNAlevels *L-Asp nmol/mgproteins D-Asp/(D-Asp+L-Asp) D-Asp/tot AspD-Asp nmol/mgproteins 0 2 4 6 8 10 12 14 a3 weeks b31 weeks 0 ,05 ,1 ,15 ,2 ,25 ,3 a3 weeks b31 weeks 0 ,25 ,5 ,75 1 1,25 1,5 1,75 2 2,25 a3 weeks b31 weeks 3w 31w 31w3w 31w3w *** *** ...in progress Ddo methylation analysis(Unpublished data)
  50. 50. Results As found in rats and humans, also in mouse brain we documented a significant age-related decrease of D-aspartate concentration. The age-related decrease in D-aspartate level is associated with reciprocal increase in D-aspartate oxidase mRNA in mouse brain Age-related decrease of D-aspartate oxidase gene methylation is associated with the progressive temporal increase in D-aspartate oxidase mRNA The hyper-methylation state of D-aspartate oxidase gene promoter during embryonic life prevents the expression of the Ddo mRNA in mice The methylation state of the Ddo gene regulates its mRNA expression and predict in the adult mouse and human brain the concentration of D-aspartate
  51. 51. Future studies
  52. 52. Francesco Napolitano and Francesco Errico Anna Di Maio Tommaso Nuzzo Daniela Punzo Arianna De Rosa
  53. 53. Acknowledgements Loredano Pollegioni, Silvia Sacchi, Gianluca Molla Università degli Studi dell’Insubria, Varese, Italy. Robert Nis7cò Sapienza University of Rome, Rome, Italy. Gina Cris7no Ins>tute of Biomolecular Chemistry, CNR, Pozzuoli, Italy Alessandro Gozzi IIT, Center for Neuroscience and Cogni>ve Systems, Rovereto, Italy. Alessandro Bertolino University of Bari “Aldo Moro”, Bari, Italy Saba7no Maione Second University of Naples (SUN), Caserta, Italy Livia D’Angelo and Paolo De Girolamo Federico II, University of Naples , Italy Lorenzo Chiario6 Federico II, University of Naples , Italy Michele Morari University of Ferrara Fabrizio Gardoni and Monica Di Luca University of Milan Diego Centonze, Nicola Mercuri and Antonio Pisani University of Roma Tor Vergata Paolo Calabresi University of Perugia Micaela Morelli University of Cagliari Massimo Pasqualetti University of Pisa Marco Feligioni Casa di Cura del Policlinico Dezza, Milano Nadia Canu University Tor Vergata Rome
  54. 54. In the Hyppocampus of patients with schizophrenia, the state of DDO methylation follows a peculiar profile of reduction with age
  55. 55. Intermittent oral administration of D-aspartate exerts reversible influence on hippocampal NMDAR-dependent synaptic plasticity (Errico et al, Neurobiol Aging, 2011) 20mM D-Asp (3 months) Water (3 weeks) 20mM D-Asp (1 month)D-Asp DDO

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