Focus on the Role of D-serine and D-amino Acid Oxidase in Amyotrophic Lateral Sclerosis/Motor Neuron Disease (ALS)

Highly pathogenic avian influenza (HPAI) H5N1 virus has been circulating in Vietnam since 2003, while outbreaks of HPAI H5N6 virus are more recent, having only been reported since 2014. Although the spatial distribution of H5N1 outbreaks and risk factors for virus occurrence have been extensively studied, there have been no comparative studies for H5N6. Data collected through active surveillance of Vietnamese live-bird markets (LBMs) between 2011 and 2015 were used to explore and compare the spatio-temporal distributions of H5N1- and H5N6-positive LBMs. Conditional autoregressive models were developed to quantify spatio-temporal associations between agro-ecological factors and the two HPAI strains using the same set of predictor variables. Unlike H5N1, which exhibited a strong north-south divide, with repeated occurrence in the extreme south of a cluster of high-risk provinces, H5N6 was homogeneously distributed throughout Vietnam. Similarly, different agro-ecological factors were associated with each strain. Sample collection in the months of January and February and higher average maximum temperature were associated with higher likelihood of H5N1 positive market-day status. The likelihood of market-days being positive for H5N6 increased with decreased river density, and with successive Rounds of data collection. This study highlights marked differences in spatial patterns and risk factors for H5N1 and H5N6 in Vietnam, suggesting the need for tailored surveillance and control approaches

pLG72 modulates intracellular D-serine levels through its interaction with D-amino acid oxidase - Effect on schizophrenia susceptibility

Human genes coding for pLG72 and d-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein d-amino acid oxidase (hDAAO). In the brain this oxidizes d-serine, a potent activator of N-methyl-d-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of d-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of d-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility

PLG72 Modulates Intracellular D-Serine Levels through Its Interaction with D-Amino Acid Oxidase : EFFECT ON SCHIZOPHRENIA SUSCEPTIBILITY

Human genes coding for pLG72 and d-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein d-amino acid oxidase (hDAAO). In the brain this oxidizes d-serine, a potent activator of N-methyl-d-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of d-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of d-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility

Synaptic and extrasynaptic NMDA receptors are gated by different endogenous coagonists.

N-methyl-D-aspartate receptors (NMDARs) are located in neuronal cell membranes at synaptic and extrasynaptic locations, where they are believed to mediate distinct physiological and pathological processes. Activation of NMDARs requires glutamate and a coagonist whose nature and impact on NMDAR physiology remain elusive. We report that synaptic and extrasynaptic NMDARs are gated by different endogenous coagonists, D-serine and glycine, respectively. The regionalized availability of the coagonists matches the preferential affinity of synaptic NMDARs for D-serine and extrasynaptic NMDARs for glycine. Furthermore, glycine and D-serine inhibit NMDAR surface trafficking in a subunit-dependent manner, which is likely to influence NMDARs subcellular location. Taking advantage of this coagonist segregation, we demonstrate that long-term potentiation and NMDA-induced neurotoxicity rely on synaptic NMDARs only. Conversely, long-term depression requires both synaptic and extrasynaptic receptors. Our observations provide key insights into the operating mode of NMDARs, emphasizing functional distinctions between synaptic and extrasynaptic NMDARs in brain physiology

Contribution of the d-Serine-Dependent Pathway to the Cellular Mechanisms Underlying Cognitive Aging

An association between age-related memory impairments and changes in functional plasticity in the aging brain has been under intense study within the last decade. In this article, we show that an impaired activation of the strychnine-insensitive glycine site of N-methyl-d-aspartate receptors (NMDA-R) by its agonist d-serine contributes to deficits of synaptic plasticity in the hippocampus of memory-impaired aged rats. Supplementation with exogenous d-serine prevents the age-related deficits of isolated NMDA-R-dependent synaptic potentials as well as those of theta-burst-induced long-term potentiation and synaptic depotentiation. Endogenous levels of d-serine are reduced in the hippocampus with aging, that correlates with a weaker expression of serine racemase synthesizing the amino acid. On the contrary, the affinity of d-serine binding to NMDA-R is not affected by aging. These results point to a critical role for the d-serine-dependent pathway in the functional alterations of the brain underlying memory impairment and provide key information in the search for new therapeutic strategies for the treatment of memory deficits in the elderly

Dysfunctional d-aspartate metabolism in BTBR mouse model of idiopathic autism.

Background: Autism spectrum disorders (ASD) comprise a heterogeneous group of neurodevelopmental conditions characterized by impairment in social interaction, deviance in communication, and repetitive behaviors. Dysfunctional ionotropic NMDA and AMPA receptors, and metabotropic glutamate receptor 5 activity at excitatory synapses has been recently linked to multiple forms of ASD. Despite emerging evidence showing that d-aspartate and d-serine are important neuromodulators of glutamatergic transmission, no systematic investigation on the occurrence of these D-amino acids in preclinical ASD models has been carried out. Methods: Through HPLC and qPCR analyses we investigated d-aspartate and d-serine metabolism in the brain and serum of four ASD mouse models. These include BTBR mice, an idiopathic model of ASD, and Cntnap2-/-, Shank3-/-, and 16p11.2+/- mice, three established genetic mouse lines recapitulating high confidence ASD-associated mutations. Results: Biochemical and gene expression mapping in Cntnap2-/-, Shank3-/-, and 16p11.2+/- failed to find gross cerebral and serum alterations in d-aspartate and d-serine metabolism. Conversely, we found a striking and stereoselective increased d-aspartate content in the prefrontal cortex, hippocampus and serum of inbred BTBR mice. Consistent with biochemical assessments, in the same brain areas we also found a robust reduction in mRNA levels of d-aspartate oxidase, encoding the enzyme responsible for d-aspartate catabolism. Conclusions: Our results demonstrated the presence of disrupted d-aspartate metabolism in a widely used animal model of idiopathic ASD. General significance: Overall, this work calls for a deeper investigation of D-amino acids in the etiopathology of ASD and related developmental disorders

Glial D-Serine Gates NMDA Receptors at Excitatory Synapses in Prefrontal Cortex.

N-methyl-D-aspartate receptors (NMDARs) subserve numerous neurophysiological and neuropathological processes in the cerebral cortex. Their activation requires the binding of glutamate and also of a coagonist. Whereas glycine and D-serine (D-ser) are candidates for such a role at central synapses, the nature of the coagonist in cerebral cortex remains unknown. We first show that the glycine-binding site of NMDARs is not saturated in acute slices preparations of medial prefrontal cortex (mPFC). Using enzymes that selectively degrade either D-ser or glycine, we demonstrate that under the present conditions, D-ser is the principle endogenous coagonist of synaptic NMDARs at mature excitatory synapses in layers V/VI of mPFC where it is essential for long-term potentiation (LTP) induction. Furthermore, blocking the activity of glia with the metabolic inhibitor, fluoroacetate, impairs NMDAR-mediated synaptic transmission and prevents LTP induction by reducing the extracellular levels of D-serine. Such deficits can be restored by exogenous D-ser, indicating that the D-amino acid mainly originates from glia in the mPFC, as further confirmed by double-immunostaining studies for D-ser and anti-glial fibrillary acidic protein. Our findings suggest that D-ser modulates neuronal networks in the cerebral cortex by gating the activity of NMDARs and that altering its levels is relevant to the induction and potentially treatment of psychiatric and neurological disorders

Characterisation of the pathogenic effects of the in vivo expression of an ALS-linked mutation in D-amino acid oxidase: Phenotype and loss of spinal cord motor neurons

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neuromuscular disorder characterised by selective loss of motor neurons leading to fatal paralysis. Current therapeutic approaches are limited in their effectiveness. Substantial advances in understanding ALS disease mechanisms has come from the identification of pathogenic mutations in dominantly inherited familial ALS (FALS). We previously reported a coding mutation in D-amino acid oxidase (DAOR199W) associated with FALS. DAO metabolises D-serine, an essential co-agonist at the N-Methyl-D-aspartic acid glutamate receptor subtype (NMDAR). Using primary motor neuron cultures or motor neuron cell lines we demonstrated that expression of DAOR199W, promoted the formation of ubiquitinated protein aggregates, activated autophagy and increased apoptosis. The aim of this study was to characterise the effects of DAOR199W in vivo, using transgenic mice overexpressing DAOR199W. Marked abnormal motor features, e.g. kyphosis, were evident in mice expressing DAOR199W, which were associated with a significant loss (19%) of lumbar spinal cord motor neurons, analysed at 14 months. When separated by gender, this effect was greater in females (26%; p< 0.0132). In addition, we crossed the DAOR199W transgenic mouse line with the SOD1G93A mouse model of ALS to determine whether the effects of SOD1G93A were potentiated in the double transgenic line (DAOR199W/SOD1G93A). Although overall survival was not affected, onset of neurological signs was significantly earlier in female double transgenic animals than their female SOD1G93A littermates (125 days vs 131 days, P = 0.0239). In summary, some significant in vivo effects of DAOR199W on motor neuron function (i.e. kyphosis and loss of motor neurons) were detected which were most marked in females and could contribute to the earlier onset of neurological signs in double transgenic females compared to SOD1G93A littermates, highlighting the importance of recognizing gender effects present in animal models of ALS