Table 1.
Na+,K+-ATPase α3 mutations selected for structural modelling.
Figure 1.
Structural modelling of Na+,K+-ATPase α3 mutations.
(A) Na+,K+-ATPase α3 wild-type (left), the I810S mutant (AHC; centre) and the I810N mutant (Myshkin; right). (B) Na+,K+-ATPase α3 wild-type (left), the I274N mutant (AHC; centre) and the I274T mutant (RDP; right). Side chain contact between Δ272 and Δ274 at the cytoplasmic end of the K+ pore is shown in yellow for the wild-type protein and the I274T mutant, but this contact is lost in the I274N mutant. (C) Na+,K+-ATPase α3 wild-type (left), the D801N mutant (AHC; centre) and D801Y mutant (RDP; right). In the D801N mutant, the electrostatic interaction at Δ801 with both K+ ions is lost due to replacement of terminal oxygen with nitrogen, resulting in the obstruction of the K+ pore, likely to markedly affect conductance rates through the pore, while the interaction of K+2 with E776 is maintained. In the D801Y mutant, there is a predicted loss of interaction of K+2 with E776. (D) Na+,K+-ATPase α3 wild-type (left), the D923Y mutant (AHC; centre) and the D923N mutant (RDP; right).
Figure 2.
Motor dysfunction in Myk/+ mice.
(A) Gait analysis. Left panel: Mean fore stride and hind stride distance (± SEM) per cm trunk of Myk/+ (n = 14) and +/+ (n = 17) female mice. There were significant main effects of genotype on fore stride length (F1,30 = 5.59, P = 0.025), hind stride length (F1,30 = 8.09, P = 0.008) and hind stride width (F1,30 = 24.44, P = 0.0001) (left panel). Middle panel: Typical examples of forepaw (red) and hindpaw (blue) placement of Myk/+ and +/+ mice are shown. Scale bar = 2 cm. Right panel: Myk/+ mouse showing splayed hindlimbs. (B) Balance beam. Mean number of foot slips (left panel) and traversal time (right panel) (± SEM) of Myk/+ (n = 26) and +/+ (n = 45) mice when traversing a narrow beam 24 hours after training. There were significant main effects of genotype on number of foot slips (F1,70 = 99.46, P = 0.0001) and traversal time (F1,70 = 43.38, P = 0.0001). (C) Tail suspension. Mean hindlimb retraction score (± SEM) of Myk/+ (n = 26) and +/+ (n = 26) mice suspended by the tail for 30 s. There was a significant main effect of genotype (F1,51 = 29.00, P = 0.0001). Hindlimb retraction is defined as the movement of one of both hindlimbs into the central body axis (photograph). (D) Accelerating rotarod. Mean latency (± SEM) of Myk/+ (n = 18) and +/+ (n = 21) mice to fall from a rotating rod over three training trials. There were significant main effects of sex (F1,38 = 9.94, P = 0.003) and genotype (F1,38 = 6.09, P = 0.019), but not genotype x sex interaction (F1,38 = 0.91, P = 0.346), females performing better than males regardless of genotype. (E) Tremor. Mean amplitude of displacement (± SEM) of Myk/+ (n = 36) and +/+ (n = 52) mice across a spectrum of frequencies. There was a significant main effect of genotype on frequency at the maximal amplitude (F1,87 = 57.1, P = 0.0001). *P<0.05; **P<0.01; ****P<0.0001 versus +/+ mice.
Figure 3.
Cognitive impairment in Myk/+ mice.
(A) Fear conditioning with 1.0-mA footshock. Mean freezing levels (± SEM) of Myk/+ (n = 24) and +/+ (n = 25) mice at baseline, during training, and in the contextual and cued conditioning tests. There were significant main effects of genotype on freezing in the context test (F1,48 = 8.52, P = 0.005) and in the cue test during presentation of the auditory tone (CS; F1,48 = 6.20, P = 0.016). (B) Conditioned taste aversion. Mean (± SEM) CS consumption scores (intake of saccharin/total fluid) 24 h following pairing with LiCl or saline treatment in Myk/+ and +/+ mice. There were significant main effects of genotype (F1,47 = 6.51, P = 0.014), treatment (F1,47 = 48.12, P = 0.0001), and genotype x treatment interaction (F1,47 = 4.09, P = 0.049). *P<0.05; ***P<0.001; ****P<0.0001 versus +/+ mice.
Figure 4.
Significant alterations in overt local cerebral glucose utilization in Myk/+ mice.
Data shown as mean ± SEM of the 14C-2-DG uptake ratio. *denotes P<0.05, **denotes P<0.01 and ***denotes P<0.001 significant genotype effect (Welch's t-test).
Figure 5.
Thalamocortical, thalamostriatal and intrathalamic functional connectivity in Myk/+ mice.
Summary diagrams showing altered functional connectivity of (A) frontal cortex (FCTX), (B) ventral anterior thalamic nucleus (VAthal), (C) ventromedial thalamic nucleus (VMthal), and (D) ventral posteromedial nucleus (VPMthal) in Myk/+ mice. Only regions where the 95% CI of the VIP exceeded 0.80, in either Myk/+ or +/+ mice, were considered to be functionally connected to the defined “seed” region of interest. The I810N Myshkin mutation-induced alterations in functional connectivity were analysed by permutation test (1000 random permutations of the real data) with significance set at P<0.05. Red denotes a significant increase, whereas blue denotes a significant decrease, in regional functional connectivity in Myk/+ mice relative to +/+.
Figure 6.
Altered periaqueductal grey and caudal motor cortex functional connectivity in Myk/+ mice.
Summary diagrams showing altered functional connectivity of (A) dorsomedial (DMPAG) and (B) rostral (RPAG) periaqueductal grey, and (C) caudal motor cortex (CMCTX) in Myk/+ mice. Only regions where the 95% CI of the VIP exceeded 0.80, in either Myk/+ or +/+ mice, were considered to be functionally connected to the defined “seed” region of interest. The I810N Myshkin mutation-induced alterations in functional connectivity were analysed by permutation test (1000 random permutations of the real data) with significance set at P<0.05. Red denotes a significant increase, whereas blue denotes a significant decrease, in regional functional connectivity in Myk/+ mice relative to +/+.
Figure 7.
Summary diagram of alterations in brain system functional connectivity and overt alterations in regional cerebral glucose metabolism seen in Myk/+ mice.
Blue shading of neural systems indicates a significant decrease in overt cerebral metabolism while red denotes a significant increase in overt cerebral metabolism (Figure 4). Blue/broken arrows indicate a decrease in functional connectivity between and within (periaqueductal grey subfields) neural systems in Myk/+ mice relative to +/+ littermates. Red/solid arrows indicate increased functional connectivity between and within (thalamic nuclei) neural systems in Myk/+ mice relative to +/+ littermates.