We have investigated a pathogenic mutation in D-amino acid oxidase (DAO), DAOR199W, associated with familial Amyotrophic Lateral Sclerosis (ALS) that impairs D-serine metabolism and causes protein aggregation, autophagy and cell death in motor neuron cell lines. These features are consistent with the pathogenic processes occurring in ALS but most importantly, we have demonstrated that activation of the formation of ubiquitinated protein inclusions, increased autophagosome production and apoptotic cell death caused by the mutation in cell lines are attenuated by 5,7-dichlorokynurenic acid (DCKA), a selective inhibitor of the glycine/D-serine binding site of the NMDA receptor. D-serine is an essential co-agonist at this glutamate receptor. This data provides insight into potential upstream mechanisms that involve the action of D-serine at the NMDA receptor and might contribute to neurodegeneration. This is highly relevant to sporadic ALS (SALS), familial ALS, as well as ALS models, where elevated levels of D-serine have been reported and hence has broader clinical therapeutic implications. In order to investigate this further, we have generated a transgenic line expressing the pathogenic mutation, in order to determine whether mice expressing DAOR199W develop a motor phenotype and whether crossing the SOD1G93A model of ALS with mice expressing DAOR199W affects disease progression. We found that heterozygous expression of DAOR199W led to a significant loss of spinal cord motor neurons at 14 months, which is similar to that found in homozygous mice expressing DAOG181R. We hypothesize that DAO has potential for development as a therapeutic agent in ALS.

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.