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

NEK1 variants confer susceptibility to amyotrophic lateral sclerosis

To identify genetic factors contributing to amyotrophic lateral sclerosis (ALS), we conducted whole-exome analyses of 1,022 index familial ALS (FALS) cases and 7,315 controls. In a new screening strategy, we performed gene-burden analyses trained with established ALS genes and identified a significant association between loss-of-function (LOF) NEK1 variants and FALS risk. Independently, autozygosity mapping for an isolated community in the Netherlands identified a NEK1 p.Arg261His variant as a candidate risk factor. Replication analyses of sporadic ALS (SALS) cases and independent control cohorts confirmed significant disease association for both p.Arg261His (10,589 samples analyzed) and NEK1 LOF variants (3,362 samples analyzed). In total, we observed NEK1 risk variants in nearly 3% of ALS cases. NEK1 has been linked to several cellular functions, including cilia formation, DNA-damage response, microtubule stability, neuronal morphology and axonal polarity. Our results provide new and important insights into ALS etiopathogenesis and genetic etiology

Exome-wide Rare Variant Analysis Identifies TUBA4A Mutations Associated with Familial ALS

Exome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

Characterisation of the pathogenic effects of the <i>in vivo</i> expression of an ALS-linked mutation in D-amino acid oxidase: Phenotype and loss of spinal cord motor neurons - Fig 5

<p><b>Longitudinal assessment of neurological scores in female SOD1^G93A and double transgenic (SOD1^G93A/DAO^R199W) littermates (A) and for male mice (B).</b> Mean group combined neurological score plotted for SOD1^G93A and double transgenic (SOD1^G93A/DAO^R199W) mice over time from approximately 30 days of age until all the animals within a group died and the study was terminated (<i>n</i> ≥ 14 per experimental group). A combined neurological score was calculated for each mouse at each time point through summation of the neurological scores recorded for its left and right hind-limbs. Values are means with standard error of the mean (SEM) indicated by error bars. (A) SOD1^G93A females are presented in purple, and the double transgenic (SOD1^G93A/DAO^R199W) littermates are displayed in red. (B) SOD1^G93A males are presented in blue, and the double transgenic (SOD1^G93A/DAO^R199W) littermates are displayed in green.</p

Kaplan Meier time to event analyses for SOD1^G93A and double transgenic (SOD1^G93A/DAO^R199W) in both males and females.

<p>A. Median values obtained from Kaplan-Meier analysis for the time taken for mice to reach a score of 2 in both hindlimbs (onset of neurological scores), age at maximum weight (disease onset), disease progression and time taken for animals to reach the humane end point determined by its inability to right itself within 20 seconds of being placed on its side (survival) for both males and female littermates of either genotype groups. B. The Log-rank test assesses the null hypothesis that the Kaplan-Meier curves for all groups are identical (i.e. the presence of the DAO^R199W transgene did not change the time taken to reach the event being analysed). Thus, low P-values (shown below) indicate that differences between genotype groups did not occur due to chance. Log-rank analysis of the onset of neurological symptoms in females carrying the SOD1G93A transgene versus their double transgenic littermates revealed that there were significant differences between the groups for this particular measurement. A significant difference was also detected in survival between the double transgenic males and females via the log-rank test. Significant differences are indicated in bold. Threshold of significance was set at P < 0.05.</p

Longitudinal assessment of body weight in DAOR199W and wild-type littermates.

<p>Mean group body weight plotted over time from 30 days to 350 days of age. (A) females, n = 23 DAO^R199W (blue) and (n = 17) non-transgenic littermates (black). (B) males, n = 15 DAO^R199W (purple) and (n = 16) non-transgenic littermates (orange). Values are means± SEMs.</p