A previous study from the lab reported that FBXO41 is developmentally upregulated but is maintained at steady state levels in the postnatal brain including the cerebellum, suggesting post-developmental functions of FBXO41 (Holubowska, 2013). These might include maintenance of synapses, axonal transport and neuronal health. It is interesting to note that while FBXO41-/- mice display a striking motor phenotype characterized by an ataxic gait and uncoordinated movement at P16 as well as P30, the FBXO41+/- mice were indistinguishable from their wild type littermates. Since further analysis of the P30 FBXO41-/- mouse brains and in vitro cell culture experiments highlighted the role of FBXO41 in neuronal survival, I
speculated if loss of one of the FBXO41+/- gene alleles would result in an ageing-related onset behavioral phenotype, possibly as a consequence of late-onset neurodegenerative events. Hence, I subjected a 10 month-old cohort of male mice to a battery of behavioral tests to assess their general anxiety, locomotor activity, motor coordination and olfaction. At 10 months of age, I neither observed any differences in body weights nor did the genotypes show abnormal changes in anxiety when subjected to the elevated plus maze. Both genotypes had no differences in the amount of locomotor activity represented by track lengths.
Interestingly, while FBXO41+/+ mice preferred to spend time in the periphery of the open field close to the walls, FBXO41+/- mice showed a slight increase in the time spent in the intermediate quadrant compared to the periphery. This difference was very minute, however significant, which could be attributed to fewer numbers of wild types or be non-specific. On the other hand, it could suggest a mild decrease in anxiety levels in the FBXO41+/- mice.
Some neurodegenerative disease patients as well as mouse models, for example those being affected by Parkinson’s disease, display disinhibition in behavior as one of the early symptoms (Paumier et al., 2013). Interestingly, a recent study analyzing a Chinese Han population implicated specific SNPs in the Fbxo41 gene as risk factors for Parkinson’s Disease in females (Liang et al., 2013). Additionally, I analyzed the mice for signs of neurological defects by subjecting them to the hind limb clasping test. Both FBXO41+/+ and FBXO41+/- mice were indistinguishable with no pronounced signs of hind limb clasping. I further subjected the animals to the balance beam test as well as rotarod to gauge their motor coordination. There was no difference between the genotypes in the balance beam test. In the rotarod test however, the FBXO41+/- mice seemed to outdo their wild type littermates, showing a steeper learning curve. This, however, should be considered as false-positive effect due to the fact that several wild type FBXO41+/+ animals were unmotivated and gave up easily. Additional tests on olfaction as well as the pole test showed no difference within the two genotypes. The results of behavioral testing revealed that FBXO41+/- mice show neither signs of motor impairment nor olfactory and neurological defects, suggesting that half the dosage of Fbxo41 is not enough to elicit obvious behavioral differences.
Even though there were no significant differences observed in motor coordination and gait between FBXO41+/+ and FBXO41+/- mice, deficits in learning and memory, due to the high expression of FBXO41 in other areas of the brain, cannot be ruled out. On the other hand, the mice were only 10 months in age at the time of analysis, while most age dependent
Further analyses of the FBXO41+/- mice with a larger cohort should be hence performed at older ages, followed up by thorough histological analyses, in order to rule out any age dependent phenotypes.
4.4 Open questions and scope for further research:
Apart from the high expression in the cerebellum, FBXO41 is also abundantly expressed in other parts of the brain, for example the cortex, hippocampus and striatum. Hence, it is conceivable that the FBXO41-/- mice display impaired neuronal migration and deferred wiring in other regions of the brain. Analysis of the cortical and hippocampal regions at P12, P16 and P30 did not yield any obvious defects in these regions. These results suggest that either FBXO41 related impaired neuronal migration phenotype is cerebellum-specific, or that the migrational defects in the cortex or hippocampus have caught up over the course of development, which might be likely. Preliminary data from the lab for instance suggests that FBXO41-/- mice also show improper neuronal migration in the embryonic and neonatal cortex (A.Gellerer, Master’s thesis; Dr. M Vadhvani, unpublished data). Thorough histological marker analysis of the developing cortex as well as the hippocampus would however be necessary in order to gain more clarity. Since FBXO41 is also very highly expressed in the postnatal adult brain, including the cortex, hippocampus and cerebellum, it could also serve in the formation and maintenance of synapses, which requires further investigation.
Owing to the increased apoptosis in the hippocampus it is also possible that along with the severe motor deficits the FBXO41-/- mice also suffer from impaired cognition. Interestingly, several studies have also associated the cerebellum to proper learning, memory, cognition and speech (Boyden et al., 2004; Ferrucci et al., 2012; Marien et al., 2001; Stoodley, 2012; cerebellum. Usage of cultured CGNs as a model system identified that the novel E3 ligase FBXO41-Cul7 regulates axon growth together with NFM via its non-proteolytic ubiquitnation, leading to NFM stabilization. Since FBXO41 as well as NFM are both
neuronal proteins expressed abundantly in the brain, it is likely their function in axon growth regulation is also conserved in other neuronal types. However, further experiments for example in cortical or hippocampal neurons will be important as a proof of principal. Since FBXO41 forms a cullin7 based E3 ligaseit is very likely that, similar to axon growth, it also regulates neuronal migration and cell death in a ligase activity-dependent manner. Further experiments using the ligase-dead FBXO41 mutants would be necessary to confirm the same.
Apart from impaired cerebellar development, defects in neuronal migration, deferred wiring of the other regions of the mouse brain could also greatly contribute to the motor symptoms observed in the FBXO41-/- mice. Further identification of FBXO41-Cul7 targets would greatly shed light on other FBXO41- mediated pathways and their implications.