Download [10] P. Paul, J de Belleroche, The role of D-amino acids in

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Short review
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Experimental approaches for elucidating co-agonist regulation of NMDA
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receptor in motor neurons: therapeutic implications for amyotrophic
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lateral sclerosis (ALS)
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Praveen Paul1 and Jackie de Belleroche1*
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1Neurogenetics
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of Medicine, Imperial College London, Hammersmith Hospital campus, Du Cane
Group, Division of Brain Sciences, Department of Medicine, Faculty
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Road, London W12 0NN, UK
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*correspondence
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Email [email protected]
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#44 (0) 207594 6649
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Abstract
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Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterised by selective
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loss of motor neurons leading to fatal paralysis. Although most cases are sporadic,
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approximately 10% of cases are familial and the identification of mutations in these kindred
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has greatly accelerated our understanding of disease mechanisms. To date, the causal
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genes in over 70% of these families have been identified. Recently, we reported a mutation
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(R199W) in the enzyme that degrades D-serine, D-amino acid oxidase (DAO) and co-
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segregates with disease in familial ALS. Moreover, D-serine and DAO are abundant in
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human spinal cord and severely depleted in ALS. Using cell culture models, we have defined
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the effects of R199W- DAO, and shown that it activates autophagy, leads to the formation of
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ubiquitinated aggregates and promotes apoptosis, all of which processes are attenuated by
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a D-serine/ glycine site antagonist of the N-methyl D aspartate receptor (NMDAR). These
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findings suggest that the toxic effects of R199W-DAO are at least in part mediated via the
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NMDAR involving the D-serine/ glycine site and that an excitotoxic mechanism may
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contribute to disease pathogenesis.
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Key words: D-serine, D-amino acid oxidase (DAO), NMDA receptors, Amyotrophic
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Lateral sclerosis (ALS), motor neurons, glycine
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Contents
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1.
The role of co-agonists at the NMDA receptor in mammalian forebrain.
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2.
The potential importance of D-serine in spinal cord is indicated from the identification of a
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mutation in D-amino acid oxidase (DAO) in amyotrophic lateral sclerosis/ motor neuron disease
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(ALS).
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3.
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Functional effects of DAO deficient models
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3.1
In vivo studies of DAO deficient models: determination of D-Ser
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3.2
The role of D-serine in the spinal cord: studies in cell culture
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3.3
Mechanisms of R199W-DAO toxicity: interaction between neuronal
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and glial cells
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3.4
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4.
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vulnerability?
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5.
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R199W-DAO causes a substantial increase in autophagy.
What are the unique properties of human motor neurones that underlie their selective
References
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1.The role of co-agonists at the NMDA receptor in mammalian forebrain.
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The major excitatory transmitter in the central nervous system is glutamate, whose
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powerful actions in fast conduction and synaptic plasticity are principally mediated through α-
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Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/ kainate and N-methyl-D-
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aspartate (NMDA) receptors respectively. Whilst AMPA and kainate receptors are activated
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solely by glutamate, NMDA receptors are co-incidence detectors, that require the binding of
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both glutamate and a co-agonist (D-serine or glycine) to GluN2 and GluN1 subunits
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respectively, combined with depolarisation to release the magnesium block present under
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resting conditions. NMDA receptors are heterotetrameric complexes usually composed of
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two GluN1 subunits and two GluN2A-D subunits, with the GluN1-GluN2A-GluN2B complex
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being the predominant receptor at hippocampal synapses [1].
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There have been substantial advances in the characterisation of the diverse
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properties of different NMDA receptor subunits and the elucidation of their pivotal
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involvement in synaptic plasticity. One aspect that has only recently been fully recognised is
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the important role of the two co-agonists that function at the NMDA receptor, which are
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essential for operation of the NMDA receptor and differentially regulate receptor function. It
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is particularly in brain regions such as hippocampus, cerebral cortex and amygdala, that
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models of synaptic plasticity such as long term potentiation (LTP) have helped to establish
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the different effects of D-serine and glycine at NMDA receptors [2, 3, 4, 5]. One example is
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selective affinity shown by heterotetrameric NMDA receptors containing GluN1 and GluN2A
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subunits, which have a greater affinity for D-serine compared to the NMDA receptor
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containing GluN1 and GluN2B subunits [6]. On the other hand NMDA receptors containing
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GluN1 and GluN2B subunits have a much greater affinity for glycine compared to GluN2A
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containing receptors [6]. Both GluN2A and GluN2B containing receptors are found at the
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synapse and elegant work has been carried to show the association between GluN2B
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containing receptors and activated calcium and calmodulin-dependent kinase II which is
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translocated to the synaptic membrane during LTP [7, 8]. Current evidence from studies in
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the forebrain indicate that D-serine is the major co-agonist involved both in NMDA receptor
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mediated LTP and excitotoxicity [2, 3, 4, 5]. NMDAR–mediated currents (EPSCs) are
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diminished by D-amino acid oxidase (DAO) which metabolises D-serine, whereas NMDAR –
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mediated currents induced by afferent stimulation are diminished by glycine oxidase (GO)
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and not by DAO.
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Co-agonist specificity at NMDA receptors in other CNS regions such spinal cord are less
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well characterised.
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2.The potential importance of D-serine in spinal cord is indicated from the
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identification of a mutation in D-amino acid oxidase (DAO) in amyotrophic lateral
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sclerosis/ motor neuron disease (ALS).
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The significance of DAO in spinal cord was only recently highlighted when our group
identified a pathogenic mutation in the DAO gene that was associated with ALS [9].
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Levels of DAO are highly enriched in brain stem, spinal cord and cerebellum in
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contrast to cerebral cortex [9, 10, 11, 12, 13], whereas serine racemase is most abundant in
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forebrain compared to brain stem [14, 15]. These high concentrations of DAO in spinal cord
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suggest that this region may have a selective vulnerability that requires a tight regulation of
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D-serine levels carried out in part by DAO though oxidative deamination of D-serine.
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ALS is a devastating condition, causing muscle atrophy, paralysis, impaired speech
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and swallowing which rapidly progresses to death from respiratory failure in 3-5 years. The
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characteristic pathological features of the disease are loss of motor neurons in spinal cord,
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brain stem and motor cortex and sclerosis of the descending cortico-spinal tract from motor
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cortex (lateral crossed and ventral uncrossed). At the cellular level, the hall mark of disease
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is the presence of ubiquitinated inclusions positive for TDP-43 [16].
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The most important and momentous advances in ALS research have come from the
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identification of mutations in genes that are responsible for the familial form of the disease
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which accounts for 5 to 10% of all cases. To date 18 ALS genes have been identified, the
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most prevalent FALS gene is C9orf72 [17, 18] followed by SOD1, TARDBP and FUS [19, 20,
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21] and these account for ~70% of all cases in our Imperial College cohort of 208 families,
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which is consistent with other UK, US and European cohorts. Outstanding FALS genes are
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currently emerging from exomic capture/ resequencing approaches. The functional effects of
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these genes provide valuable clues about disease mechanisms which fit into 3 main
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categories, RNA binding and processing, protein quality control and excitotoxicity. The
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DNA/RNA binding proteins are TDP-43 and FUS encoded by TARDBP and FUS,
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respectively. These are nuclear proteins but they mislocalise to the cytoplasm in disease and
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accumulate in protein inclusions. C9orf72 is a gene containing an intronic hexanucleotide
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repeat of less than 30 units in controls which expands substantially to 500-2400 repeat units
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in ALS cases. Hexanucleotide expansions in C9orf72 account for 38% of FALS cases in UK,
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Europe and USA, but are more abundant in Scandinavia and rare in Asia. These expansions
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are also causal in ALS cases with fronto-temporal lobar degeneration (FTLD), familial FTLD
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and sporadic FTLD. Despite the different sites of pathology and phenotype, common cellular
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features are present. Most surprising, is the relatively high prevalence of hexanucleotide
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expansions in C9orf72 found in sporadic ALS cases (8%) indicating low penetrance of
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disease.
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The second mechanism affected in ALS is proteostasis, mutations being found in
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genes functional in the unfolded protein response, ER stress, protein degradation pathways,
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carried out by the proteasome and autophagy, VAPB, p62, optineurin, ubiquilin2 [22].
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Interestingly, VAPB is also significantly reduced in sporadic cases [23]. In cell culture, VAPB
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mutations cause endoplasmic reticulum (ER) fragmentation, protein aggregates and
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apoptotic cell death [24].
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Now we come to D-amino acids and the third mechanism, excitotoxicty. This finding
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arose from linkage analysis carried out in an extended FALS kindred which showed
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significant association with disease for markers on chromosome 12. Subsequent sequencing
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of genes in this locus identified a pathogenic mutation in D-amino acid oxidase (DAO) that
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segregated with disease.
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synonymous change from arginine to tryptophan (R199W DAO) [9]. Furthermore, this
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arginine residue is highly conserved across species from Man to Fungi and Bacteria and the
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presence of this mutation severely impairs the kinetic characteristics of this enzyme. As DAO
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is known to catalyse the oxidative deamination of D-serine, an essential co-agonist at the
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NMDA subtype of glutamate receptor, enhanced levels of D-serine could potentiate NMDA
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responses and could implicate excitotoxity in disease pathogenesis.
The mutation occurred in codon 199 and caused a non-
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DAO is known to be localised to specific regions of the CNS, showing a strong
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enrichment in motor nuclei of the brain stem, such as the facial nerve nucleus. We carried
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out an extensive study of the distribution of DAO, D-serine and serine racemase (SR), the
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enzyme responsible for D-serine synthesis from L-serine, in human spinal cord from control
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cases compared to ALS cases [25]. In spinal cord, there is a prominent expression of DAO,
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D-serine and SR in large motor neurons present in the anterior horn cell region of spinal cord
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in control cases (Figure 1). In addition, DAO immunoreactivity is widely present in neuronal
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fibres and small glial-like cells fibres present in the grey matter. In ALS cases, there is a
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substantial depletion of the motor neuron pool as shown by loss of motor neuron markers
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such as choline acetyl transferase (ChAT) and vesicle associated membrane protein
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associated protein B (VAPB) which is accompanied by ~ 90% loss of DAO, SR and D-serine
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staining [25]. This further substantiates the localisation of D-serine in motor neurons together
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with enzymes involved in their synthesis and metabolism and their depletion in ALS.
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3.Functional effects of DAO
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3.1
In vivo studies of DAO deficient models: determination of D-Serine
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Extensive work carried out by Dr Konno’s group has characterised a naturally
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occurring mutation in DAO (G181R) found in mouse that reduces DAO activity and has
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proved to be extremely valuable in characterising behavioural effects of this mutation [26].
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Using ddY/DAO- mice backcrosed with C57BL/6J, a homozygous mouse line (DAO-/-) was
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obtained which exhibited marked effects on motor phenotype. At 8 months, abnormal
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reflexes characterised by retraction of hind limbs, similar to that found in the
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mouse model of ALS, were seen accompanied by a significant reduction of 24% in motor
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neuron number [27]. By 15 months, increased axonal degeneration with muscle atrophy was
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detected [27].
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G93A
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Furthermore, this group has also explored the role of D-serine and DAO in the
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G93A
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decreased by 42%, which is accompanied by reduced DAO protein expression. The
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magnitude of this decrease was comparable to that found in DAO(+/-) heterozygotes. The
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effect of reduced DAO enzyme activity on D-serine levels was assayed using a highly
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selective and sensitive 2D-HPLC method and showed an elevation in D-serine levels which
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increased with disease progression [27]. This confirmed earlier findings from this group,
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where D-serine was measured using a chemiluminescence assay, in which hydrogen
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peroxide generated in the presence of DAO and peroxidise was detected using luminol [28].
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In the latter study, Sasabe et al [28] also presented preliminary results from
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immunohistochemical analysis, that D-serine was elevated in sporadic (two out three
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studied) and one familial ALS case (A4V SOD1).
SOD1 mouse model of ALS and shown that DAO activity in lumbar spinal cord is
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We further examined the cellular effects of R199W-DAO on viability, the interaction
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between neurons and glial cells and developed a generic model with implications relevant to
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all forms of ALS.
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3.2 The role of D-serine in the spinal cord: studies in cell culture
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When expressed in primary motor neuron cultures, R199W-DAO increases
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apoptosis, as indicated from TUNEL labelling, compared to wild-type DAO [9]. When
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expressed in motor neurone-like cell lines, NSC-34, R199W-DAO stimulates the generation
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of ubiquitinated protein aggregates, which are increased relative to the effects of transfection
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with wild type DAO and further enhanced by tunicamycin [9].
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primary cell cultures showed that R199W-DAO was not only toxic when expressed in motor
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neurons but also when glial cells expressing R199W-DAO were grown over a layer of motor
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neurons. This prompted us to look at the cross talk between neuronal and glial cells using a
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co-culture approach.
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3.3 Mechanisms of R199W-DAO toxicity: interaction between neuronal and glial cells
Our previous studies in
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In order to do this we made permanent C6 glial cell lines expressing either, mutant or
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wild-type DAO or vector and suspended these cells in a trans-well above NSC-34 cells. We
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found that C6 cells expressing R199W-DAO promoted apoptosis in motor neurons (not
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expressing the mutation) indicating that a glial factor was contributing to the cell death
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(Figure 2). The most likely candidate was D-serine as this would be predicted to be elevated
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by DAO inhibition and has been shown to be increased in the transgenic mouse model of
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ALS which overexpresses
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order to confirm this, we used a selective antagonist at the glycine/ D-serine binding site of
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the NMDA receptor, 5,7-Dichloro-4-hydroxyquinoline-2-carboxylic acid (DCKA). DCKA
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effectively prevents cell death due to NMDA or simulated ischaemia in brain slices [29].
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Indeed, we found that DCKA reduced apoptosis in motor neurons co-cultured with C6 cells
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expressing R199W-DAO (Figure 2C). This observation that dysfunction of D-serine
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metabolism caused by a mutant allele demonstrates how glial cells can affect motor neuron
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survival and suggests that other perturbations of glial function that increase D-serine
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production through SR induction, such as amyloid beta, inflammatory mediators and
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lipopolysaccharide [30], may also contribute to motor neuron degeneration in ALS.
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3.4 R199W-DAO causes a substantial increase in autophagy.
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SOD1 and also in a preliminary study of ALS cases [28]. In
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These results clearly indicated that the D-serine/ glycine agonist binding site on the
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NMDA receptor could contribute to apoptotic cell death in motor neurons. In order to
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determine whether accumulation of ubiquitinated protein aggregates seen in NSC34 cells
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expressing R199W-DAO [9] was linked or triggered by effects of D-serine at the NMDA
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receptor, we characterised the effects of R199W-DAO on two major protein degradation
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process, the ubiquitin-proteasome system (UPS) and autophagy. Proteasomal activity was
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measured using GFP-CL1, a UPS reporter which accumulates in cells with impaired UPS
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[31] but activity was unaffected in NSC-34 cells expressing R199W-DAO compared to wild-
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type-DAO. However, marked effects of R199W-DAO were seen on autophagy. The effect of
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R199W-DAO on autophagy was measured by monitoring the conversion of microtubule
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associated protein light chain 3 (LC3) from LC3-I to its lipidated form, LC3-II, which occurs
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during the generation of autophagosomes [32]. GFP-LC3 was co-transfected with RFP-
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tagged DAO into NSC-34 cells and GFP-LC3 puncta were quantified. Cells expressing
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R199W DAO showed a five-fold increase in punctate GFP-LC3 staining compared to WT
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DAO expressing cells [25]. A significant increase in LC3-II and LC3-I protein was found with
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both DAO mutations compared to WT DAO substantiating the observation that the mutation
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caused an increase in autophagosome generation [25]. A similar increase in LC3-II levels is
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seen in spinal cord motor neurons of the SOD1 (G93A) mouse model of ALS [33] and
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increased autophagosomes are observed in motor neurons of ALS cases [34].
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In view of the link between the D-serine/glycine binding site of the NMDAR and
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apoptosis, we investigated whether DCKA affected autophagy and levels of LC3-II protein in
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NSC-34 cells co-transfected with GFP-LC3 and RFP-tagged DAO. DCKA significantly
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reduced LC3-II levels in cells expressing R199W DAO but not in WT DAO strongly
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suggesting that the increased autophagy caused by R199W DAO was mediated via the
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NMDA receptor [25].
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4.What are the unique properties of human motor neurones that underlie their
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selective vulnerability?
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Studies on the functional properties of a mutation in DAO associated with ALS help
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to elucidate the potential reasons for the selective vulnerability of motor neurons in ALS. A
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key factor lies in the selective distribution of distribution of DAO in motor neurons and motor
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nuclei of the spinal cord and the consequences for impaired D-serine metabolism.
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Furthermore, the major transporter for D-serine, Asc1 has a high affinity for D-serine and is
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predominantly distributed in brain stem and spinal cord [35]. Other transporters for D-serine
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e.g. ASCT2 (ASCT1) have a lower affinity for D-serine and do not show a differential
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distribution in spinal cord. ASCT2 is found both in glial and neuronal cells. Interestingly, the
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other co-agonist at the D-serine/glycine binding site of the NMDA receptor, glycine, is more
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highly concentrated in the spinal cord than brain, where it activates strychnine-sensitive
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glycine receptors as well as functioning as a co-agonist at NMDA receptors. The glycine
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transporters, GlyT1 and GLT2 are also enriched in spinal cord compared to brain together
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with Asc1 which has a high affinity not only for D-serine but also for glycine (Km~ 8uM).
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Indeed, early studies have indicated that CSF levels are elevated in ALS and glycine
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challenge in ALS subjects is accompanied by a reduced clearance of glycine from plasma
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and CSF [36, 37].
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High levels of DAO in motor neurons and motor nuclei indicate the importance of
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DAO in regulating D-serine levels and potential neurotoxic effects of D-serine. This is further
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supported by the enrichment of the main D-serine and glycine transporters, Asc-1 [35] and
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GlyT2, in brain stem and spinal cord compared to brain that contribute to the regulation of
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the levels of NMDAR co-agonists [38].
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Understanding the relative importance of glycine and D-serine at NMDARs in spinal
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cord compared to other brain regions is clearly fundamental. Each co-agonist shows
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differential selectivity for NMDA receptors containing different NR2 subunits, D-serine
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showing slightly greater affinity for NMDA receptors GluN2A subunits whereas glycine
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shows a much greater affinity for NMDA receptors containing GluN2B than D-serine [6].
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Future studies are needed to characterise the properties and composition of synaptic and
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extrasynaptic NMDA receptors and their distribution on spinal cord motor neurons.
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Acknowlegements
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We are grateful to the motor Neurone Disease Association for funding this work.
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Conflicts of interest
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The authors have no conflicts of interest.
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Figure legends
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Figure 1.
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Distribution of DAO, SR and D-Serine in lumbar spinal cord. Lumbar sections (L5) from
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control cases were stained for (A) DAO distribution in control cases. (B) SR distribution in
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control cases, (C) D-serine distribution in control cases, a polarised distribution is indicated
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(arrow). Data is taken from Paul et al [25].
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Figure 2
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Annexin V levels show that R199W DAO promotes apoptosis in glial cells and
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neighbouring neuronal cells. Annexin V levels (A) C6 glial cells permanently expressing
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WT or R199W DAO, (B) NSC-34 neuronal cells co-cultured with C6 glial cells, (C) NSC-34
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cells treated with DCKA and co-cultured with C6 cells. Significant one-way ANOVA subject
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to post-hoc testing with Bonferroni correction (A, B). Paired t-test used in (C). Values are
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means ± SEM, for P values shown, *P < 0.05, ** P < 0.01. n = 5, except (C) where n=3. The
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control in Figure 1B, ‘No cells medium alone’ corresponds to the naive medium. It should be
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noted that levels of Annexin V in adherent cells are higher than those obtained in cell
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suspensions due to some activation caused by mechanical cell detachment. Data is taken
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from Paul et al [25]
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