Short review
Experimental approaches for elucidating co-agonist regulation of NMDA receptor in motor neurons: therapeutic implications for amyotrophic lateral sclerosis (ALS)
Praveen Paul1 and Jackie de Belleroche1*
1Neurogenetics Group, Division of Brain Sciences, Department of Medicine, Faculty of Medicine, Imperial College London, Hammersmith Hospital campus, Du Cane Road, London W12 0NN, UK
*correspondence
#44(0) 207594 6649
Abstract
Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterised by selective loss of motor neurons leading to fatal paralysis. Although most cases are sporadic, approximately 10% of cases are familial and the identification of mutations in these kindred has greatly accelerated our understanding of disease mechanisms. To date, the causal genes in over 70% of these families have been identified. Recently, we reported a mutation (R199W) in the enzyme that degrades D-serine, D-amino acid oxidase (DAO) and co-segregates with disease in familial ALS. Moreover, D-serine and DAO are abundant in human spinal cord and severely depleted in ALS. Using cell culture models, we have defined the effects of R199W- DAO, and shown that it activates autophagy, leads to the formation of ubiquitinated aggregates and promotes apoptosis, all of which processes are attenuated by a D-serine/glycine site antagonist of the N-methyl D aspartate receptor (NMDAR). These findings suggest that the toxic effects of R199W-DAO are at least in part mediated via the NMDARinvolving the D-serine/glycinesite and that anexcitotoxic mechanism may contribute to disease pathogenesis.
Key words: D-serine, D-amino acid oxidase (DAO), NMDA receptors, Amyotrophic Lateral sclerosis (ALS), motor neurons, glycine
Contents
1. The role of co-agonists at the NMDA receptor in mammalian forebrain.
2. The potential importance of D-serine in spinal cord is indicated from the identification of a mutation in D-amino acid oxidase (DAO) in amyotrophic lateral sclerosis/ motor neuron disease (ALS).
3. Functional effects of DAO deficient models
3.1In vivo studies of DAO deficient models:determination of D-Ser
3.2The role of D-serine in the spinal cord: studies in cell culture
3.3Mechanisms of R199W-DAO toxicity: interaction between neuronal and glial cells
3.4R199W-DAO causes a substantial increase in autophagy.
4.What are the unique properties of human motor neurones that underlie their selective vulnerability?
5. References
1.The role of co-agonists at the NMDA receptor in mammalian forebrain.
The major excitatory transmitter in the central nervous system is glutamate, whose powerful actions in fast conduction and synaptic plasticity are principally mediated through α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA)/ kainate and N-methyl-D-aspartate (NMDA) receptors respectively. Whilst AMPA and kainate receptors are activated solely by glutamate, NMDA receptors are co-incidence detectors,that require the binding of both glutamate and a co-agonist (D-serine or glycine) to GluN2 and GluN1 subunits respectively, combined with depolarisation to release the magnesium block present under resting conditions. NMDA receptors are heterotetrameric complexes usually composed of two GluN1 subunits and two GluN2A-D subunits, with the GluN1-GluN2A-GluN2B complex being the predominant receptor at hippocampal synapses [1].
There have been substantial advances in the characterisation of the diverse properties of different NMDA receptor subunits and the elucidation of their pivotal involvement in synaptic plasticity. One aspect that has only recently been fully recognised is the important role of the two co-agonists that function at the NMDA receptor, which are essential for operation of the NMDA receptor and differentially regulate receptor function. It is particularly in brain regions such as hippocampus, cerebral cortex and amygdala,that models of synaptic plasticity such as long term potentiation (LTP) have helped to establish the different effects of D-serine andglycine at NMDA receptors [2, 3, 4, 5]. One example is selective affinity shown byheterotetrameric NMDA receptors containing GluN1 and GluN2Asubunits,which have a greater affinity for D-serine compared to the NMDA receptor containing GluN1 and GluN2B subunits[6].On the other hand NMDA receptors containing GluN1 and GluN2B subunits have a much greater affinity for glycine compared to GluN2A containing receptors[6]. Both GluN2A and GluN2B containing receptors are found at the synapse and elegant work has been carried to show the association between GluN2B containing receptors and activated calcium and calmodulin-dependent kinase II which is translocated to the synaptic membrane during LTP [7, 8]. Current evidence from studies in the forebrain indicate that D-serine is the major co-agonist involved both in NMDA receptor mediated LTP and excitotoxicity[2, 3, 4, 5]. NMDAR–mediated currents (EPSCs) are diminished by D-amino acid oxidase (DAO) which metabolises D-serine, whereas NMDAR –mediated currents induced by afferent stimulation are diminished by glycine oxidase (GO) and not by DAO.
Co-agonist specificityatNMDA receptors in other CNS regions such spinal cord are less well characterised.
2.Thepotential importance of D-serine in spinal cord is indicated from the identification of a mutation in D-amino acid oxidase (DAO) in amyotrophic lateral sclerosis/ motor neuron disease (ALS).
The significance of DAO in spinal cord was only recently highlighted when our group identified a pathogenic mutation in theDAOgene that was associated with ALS [9].
Levels of DAO are highly enriched in brain stem, spinal cord and cerebellum in contrast to cerebral cortex [9, 10, 11, 12, 13], whereas serine racemase is most abundant in forebraincompared to brain stem [14, 15]. These high concentrations of DAO in spinal cord suggest that this region may have a selective vulnerability that requires a tight regulation of D-serine levels carried out in part by DAO though oxidative deamination of D-serine.
ALS is a devastating condition, causing muscle atrophy, paralysis, impaired speech and swallowing which rapidly progresses to death from respiratory failure in 3-5 years. The characteristic pathological features of the disease are loss of motor neurons in spinal cord, brain stem and motor cortex and sclerosis of the descending cortico-spinal tract from motor cortex (lateral crossed and ventral uncrossed). At the cellular level, the hall mark of disease is the presence of ubiquitinated inclusions positive for TDP-43 [16].
The most important and momentous advances in ALS research have come from the identification of mutations in genes that are responsible for the familial form of the disease which accounts for 5 to 10% of all cases. To date 18 ALS genes have been identified, the most prevalent FALS gene isC9orf72 [17, 18] followed by SOD1, TARDBP and FUS [19, 20, 21] andthese account for ~70% of all cases in our Imperial College cohort of 208 families, which is consistent with other UK, US and European cohorts. Outstanding FALS genes are currently emerging from exomiccapture/resequencingapproaches. The functional effects of these genes provide valuable clues about disease mechanisms which fit into 3 main categories, RNA binding and processing, protein quality control and excitotoxicity. The DNA/RNA binding proteins are TDP-43 and FUS encoded by TARDBP and FUS, respectively. These are nuclear proteins but they mislocalise to the cytoplasm in disease and accumulate in protein inclusions. C9orf72 is a gene containing an intronichexanucleotiderepeat of less than 30 units in controls which expands substantially to 500-2400 repeat units in ALS cases.Hexanucleotide expansionsin C9orf72account for 38% of FALS cases in UK, Europe and USA, but are more abundant in Scandinavia and rare in Asia. These expansions are also causal in ALS cases with fronto-temporal lobar degeneration (FTLD), familial FTLD and sporadic FTLD. Despite the different sites of pathology and phenotype, common cellular features are present. Most surprising, is the relatively high prevalence of hexanucleotide expansions in C9orf72 found in sporadic ALS cases (8%) indicating low penetrance of disease.
The second mechanism affected in ALS is proteostasis, mutations being found in genes functional in the unfolded protein response, ER stress, protein degradation pathways, carried out by the proteasome and autophagy, VAPB, p62, optineurin, ubiquilin2 [22]. Interestingly, VAPB is also significantly reduced in sporadic cases [23]. In cell culture, VAPB mutations cause endoplasmic reticulum (ER)fragmentation, protein aggregates and apoptotic cell death [24].
Now we come to D-amino acids and the third mechanism, excitotoxicty. This finding arose from linkage analysis carried out in an extended FALS kindred which showed significant association with disease for markers on chromosome 12. Subsequent sequencing of genes in this locusidentified a pathogenic mutation in D-amino acid oxidase(DAO) that segregated with disease. The mutation occurred in codon 199 and caused a non-synonymous change from arginine to tryptophan (R199W DAO) [9]. Furthermore, this arginine residue is highly conserved across species from Man to Fungi and Bacteria and the presence of this mutation severely impairs the kinetic characteristics of this enzyme. As DAO is known to catalyse the oxidative deamination of D-serine, an essential co-agonist at the NMDA subtype of glutamate receptor, enhanced levels of D-serine could potentiate NMDA responses and could implicate excitotoxity in disease pathogenesis.
DAO is known to be localised to specific regions of the CNS, showing a strong enrichment in motor nuclei of the brain stem, such as the facial nerve nucleus. We carried out an extensive study of the distribution of DAO, D-serine and serine racemase (SR), the enzyme responsible for D-serine synthesis from L-serine, in human spinal cord from control cases compared to ALS cases [25]. In spinal cord, there is a prominent expression of DAO, D-serine and SR in large motor neurons present in the anterior horn cell region of spinal cord in control cases (Figure 1). In addition, DAO immunoreactivity is widely present in neuronal fibres and small glial-like cells fibres present in the grey matter.In ALS cases, there is a substantial depletion of the motor neuron pool as shown by loss of motor neuron markers such as choline acetyl transferase (ChAT) and vesicle associated membrane protein associated protein B (VAPB) which is accompanied by ~ 90% loss of DAO, SR and D-serine staining [25]. This further substantiates the localisation of D-serine in motor neurons together with enzymes involved in their synthesis and metabolism and their depletion in ALS.
3.Functional effects of DAO
3.1In vivo studies of DAO deficient models: determination of D-Serine
Extensive work carried out by Dr Konno’s group has characterised a naturally occurring mutation in DAO (G181R) found in mouse that reduces DAO activity and has proved to be extremely valuable in characterising behavioural effects of this mutation [26]. Using ddY/DAO-mice backcrosed with C57BL/6J, a homozygous mouse line (DAO-/-) was obtained which exhibited marked effects on motor phenotype. At 8 months, abnormal reflexes characterised by retraction of hind limbs, similar to that found in the G93ASODmouse model of ALS, were seen accompanied by a significant reduction of 24% in motor neuron number [27]. By 15 months, increased axonal degeneration with muscle atrophy was detected [27].
Furthermore, this group has also explored the role of D-serine and DAO in the G93ASOD1 mouse model of ALS and shown that DAO activity in lumbar spinal cord is decreasedby 42%, which is accompanied by reduced DAO protein expression. The magnitude of this decrease was comparable to that found in DAO(+/-)heterozygotes. The effect of reduced DAO enzyme activity on D-serine levels was assayed using a highly selective and sensitive 2D-HPLC method and showed an elevation in D-serine levels which increased with disease progression [27]. This confirmed earlier findings from this group, where D-serine was measured using a chemiluminescence assay, in which hydrogen peroxide generated in the presence of DAO and peroxidise was detected using luminol [28]. In the latter study, Sasabe et al [28] also presented preliminary results from immunohistochemical analysis, that D-serine was elevated in sporadic (two out three studied) and one familial ALS case (A4V SOD1).
We further examined the cellular effects of R199W-DAO on viability, the interaction between neurons and glial cells and developed a generic model with implications relevant to all forms of ALS.
3.2The role of D-serinein the spinal cord: studies in cell culture
When expressed in primary motor neuron cultures,R199W-DAO increases apoptosis, as indicated from TUNEL labelling, compared to wild-type DAO [9]. When expressed in motor neurone-like cell lines, NSC-34,R199W-DAO stimulates the generation of ubiquitinated protein aggregates, which are increased relative to the effects of transfectionwith wild type DAO and further enhanced by tunicamycin[9]. Our previous studies in primary cell cultures showed that R199W-DAO was not only toxic when expressed inmotor neuronsbut also when glial cells expressing R199W-DAOwere grown over a layer of motor neurons.This prompted us tolook at the cross talk between neuronal and glial cells using a co-culture approach.
3.3Mechanisms of R199W-DAO toxicity: interaction between neuronal and glial cells
In order to do this we made permanent C6 glial cell lines expressing either, mutant or wild-type DAO or vector and suspended these cells in a trans-well above NSC-34 cells. We found that C6 cells expressing R199W-DAO promoted apoptosis in motor neurons (not expressing the mutation) indicating that a glial factor was contributing to the cell death (Figure 2). The most likely candidate was D-serine as this would be predicted to be elevated by DAO inhibition and has been shown to be increased in the transgenic mouse model of ALS which overexpresses G93ASOD1 and also in a preliminary study of ALS cases[28]. In order to confirm this, we used a selective antagonist at the glycine/ D-serine binding site of the NMDA receptor, 5,7-Dichloro-4-hydroxyquinoline-2-carboxylic acid (DCKA). DCKA effectively prevents cell death due to NMDA or simulated ischaemia in brain slices [29]. Indeed, we found that DCKA reduced apoptosis in motor neurons co-cultured with C6 cells expressing R199W-DAO (Figure 2C). This observation that dysfunction of D-serine metabolism caused by a mutant allele demonstrates how glial cells can affect motor neuron survival and suggests that other perturbations of glial function that increase D-serine production through SR induction, such as amyloid beta, inflammatory mediators and lipopolysaccharide[30],may also contribute to motor neuron degeneration in ALS.
3.4R199W-DAO causes a substantial increase in autophagy.
These results clearly indicated that the D-serine/ glycine agonist binding site on the NMDA receptor could contribute to apoptotic cell death in motor neurons. In order to determine whether accumulation of ubiquitinated protein aggregates seen in NSC34 cells expressing R199W-DAO [9]was linked or triggered by effects of D-serine at the NMDA receptor, we characterised the effects of R199W-DAO on two major protein degradation process, the ubiquitin-proteasome system (UPS) and autophagy. Proteasomal activity was measured using GFP-CL1, a UPS reporter which accumulates in cells with impaired UPS [31] but activity was unaffected in NSC-34 cells expressing R199W-DAO compared to wild-type-DAO.However, marked effects of R199W-DAO were seen on autophagy. The effect of R199W-DAO on autophagy was measured by monitoring the conversion of microtubule associated protein light chain 3 (LC3) from LC3-I to its lipidated form, LC3-II, which occurs during the generation of autophagosomes [32]. GFP-LC3 was co-transfected with RFP-tagged DAO into NSC-34 cells and GFP-LC3 puncta were quantified. Cells expressing R199W DAO showed a five-fold increase in punctate GFP-LC3 staining compared to WT DAO expressing cells [25]. A significant increase in LC3-II and LC3-I protein was found with both DAO mutations compared to WT DAO substantiating the observation thatthe mutation caused an increase in autophagosomegeneration [25]. A similar increase in LC3-II levels is seen in spinal cord motor neurons of the SOD1 (G93A) mouse model of ALS [33] and increased autophagosomes are observed in motor neurons of ALS cases [34].
In view of the link between the D-serine/glycine binding site of the NMDAR and apoptosis, we investigated whether DCKA affected autophagy and levels of LC3-II protein in NSC-34 cells co-transfected with GFP-LC3 and RFP-tagged DAO. DCKA significantly reduced LC3-II levels in cells expressing R199W DAO but not in WT DAO strongly suggesting that the increased autophagy caused by R199W DAO was mediated via the NMDA receptor [25].
4.What are the unique properties of human motor neurones that underlie their selective vulnerability?
Studies on the functional properties of a mutation in DAO associated with ALS help to elucidate the potential reasons for the selective vulnerability of motor neurons in ALS. A key factor lies in the selective distribution of distribution of DAO in motor neurons and motor nuclei of the spinal cord and the consequences for impaired D-serine metabolism. Furthermore, the major transporter for D-serine, Asc1 has a high affinity for D-serine and is predominantly distributed in brain stem and spinal cord [35]. Other transporters for D-serine e.g. ASCT2(ASCT1) have a lower affinity for D-serine and do not show a differential distribution in spinal cord. ASCT2 is found both in glial and neuronal cells.Interestingly, the other co-agonist at the D-serine/glycine binding site of the NMDA receptor, glycine, is more highly concentrated in the spinal cord than brain, where it activates strychnine-sensitive glycine receptors as well as functioning as a co-agonist at NMDA receptors. Theglycine transporters, GlyT1 and GLT2 are also enriched in spinal cord compared to brain together with Asc1 which has a high affinity not only for D-serine but also for glycine (Km~ 8uM). Indeed, early studies have indicated that CSF levels are elevated in ALS and glycine challenge in ALS subjects is accompanied by a reduced clearance of glycine from plasma and CSF [36, 37].
High levels of DAO in motor neurons and motor nuclei indicate the importance of DAO in regulating D-serine levels and potential neurotoxic effects of D-serine.This is further supported by the enrichment of the main D-serine and glycine transporters, Asc-1[35]and GlyT2, in brain stem and spinal cord compared to brain that contribute to the regulation of the levels of NMDAR co-agonists [38].
Understanding the relative importance of glycine and D-serine at NMDARs in spinal cord compared to other brain regions is clearly fundamental. Each co-agonist shows differential selectivity for NMDA receptors containing different NR2subunits, D-serine showing slightly greater affinity for NMDA receptors GluN2Asubunitswhereasglycineshows a much greater affinity for NMDA receptors containing GluN2Bthan D-serine[6]. Future studiesare needed to characterise the properties and composition of synaptic and extrasynaptic NMDA receptors and their distribution on spinal cord motor neurons.
Acknowlegements
We are grateful to the motor Neurone Disease Association for funding this work.