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Assessing motor and gait phenotypes in Nedd4 and Nedd4-2-heterozygous mice

D. Bongiorno,1,2 N. Boase,3 S. Kumar3 and P. Poronnik,1,2 1School of Medical Science, RMIT University, Bundoora VIC 3083, Australia, 2Health Innovations Research Institute (HIRi), RMIT University, Bundoora, VIC 3083, Australia and 3Centre for Cancer Biology, SA Pathology, Frome road, Adelaide, SA 5000, Australia.

Introduction: Nedd4 and Nedd4-2 (Neural precursor cell Expressed Developmentally Down-regulated 4 and 4-2) are E3 ubiquitin ligases that share high degree of homology but distinct substrates within the central nervous system (CNS)(Yang & Kumar, 2010). Although the number of substrates has grown substantially, their in vivo roles are not well characterized. Previous studies in Nedd4 knockout embryos identified abnormalities in neuromuscular junctions and a reduction in motor neurons in the spinal cord (Liu et al., 2009). In addition, the gene for a novel interacting protein for Nedd4, Brain expressed associated with Nedd4 (BEAN), is disrupted in patients with spinocerebellar ataxia type 31 (SCN31) (Sato et al., 2009). These findings point to a potential role in motor function and gait. Nedd4-2 on the other hand, has been shown to regulate dopamine transporter (DAT) (Sorkina et al., 2006) and therefore may also contribute to motor function. Nedd4 and Nedd4-2 knockout mice are not viable and die shortly after birth due to growth (Cao et al., 2008) and lung defects (Boase et al., 2011), therefore in this study we wanted to determine whether a single copy deletion of either Nedd4 or Nedd4-2 (by using heterozygous mice) was sufficient to alter motor function and gait behaviours. Expression of Nedd4 and Nedd4-2 in the brain with particular localization to regions associated with motor function and control was also investigated.

Methods: Gait and motor function were assessed in young (2 month old) and old (6 month old) Nedd4 and Nedd4-2 heterozygous mice and age matched wild-type littermates (n = 8-14). RotaRod measures the latency to fall from rotating drum over an accelerated speed for a maximum of 5 minutes. Gait analysis was examined using the DigiGait system. Mice were placed on a transparent motorized treadmill and required to take 4-6 complete strides. The analysis software was then used to calculate a number of gait parameters. Standard immunohistochemical methods were used to identify the expression of Nedd4 and Nedd4-2 in the brain.

Results: Nedd4 and Nedd4-2 levels in whole brain lysates from heterozygous animals were reduced by at least 50%. Nedd4 and Nedd4-2 were strongly expressed in Purkinje neurons of the cerebellum. Nedd4-2 however, was also found to be strongly expressed in dopaminergic neurons of the substantia nigra. Gait analysis in Nedd4 heterozygous mice revealed abnormalities that became more pronounced with age. At 6 months, Nedd4 heterozygous mice have significant alterations in stride, swing and brake duration with a concomitant decrease in stride length frequency. Furthermore, stride length and stride length variability were also significantly increased. Paw angle dynamics showed an open splayed phenotype compared to wild-type littermates. In contrast Nedd4-2 heterozygous mice showed no changes in gait at 2 months, and only minor differences at 6 months. No change in motor function was found in Nedd4 heterozygous mice at both time points as assessed by RotaRod. Motor function was normal in 2 month old Nedd4-2 heterozygous mice, however by 6 months there was a significant reduction in motor capacity with a reduction in latency to fall from the RotaRod.

Conclusion: Motor function is impaired in older Nedd4-2 heterozygous mice, with no change evident in the gait. This impairment may be attributed to its role in regulating dopamine re-uptake. Nedd4 heterozygous mice conversely show significant and profound changes in gait with normal motor function capacity. The expression of Nedd4 and Nedd4-2 in the cerebellum was not surprising as this region is largely responsible for motor function and control. Furthermore, identifying strong expression of Nedd4-2 in dopaminergic neurons of the substantia nigra suggests that these two ligases affect motor function.

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