developing cerebellum:
Neuronal migration is one of the key events that ensure proper layering and connectivity of the developing cerebellum (Ghashghaei et al., 2007; Gleeson and Walsh, 2000). The majority of the cerebellar development in rodents takes place postnatally, wherein the CGN precursors form a second germinal zone at the outer layer of the cerebellar anlage thereby forming the EGL. This event is followed by extensive proliferation and increase in CGN precursor numbers in the EGL and the proper migration of the newly differentiated CGNs
from the EGL, across the ML into the IGL (Alder et al., 1996; Altman, 1972b; Marzban et al., 2014; Sillitoe and Joyner, 2007; Sotelo, 2004). Various extrinsic guidance and cell-intrinsic mechanisms that regulate the cytoskeletal dynamics also govern successful CGN migration in the cerebellum (da Silva and Dotti, 2002; Kuijpers and Hoogenraad, 2011;
Yacubova and Komuro, 2003). A previous study from the lab identified the F-box protein FBXO41 as a cell-intrinsic regulator of neuronal migration in the developing cerebellum, showing a brain-dominant and neuronal expression that localizes to the centrosome as well as the cytoplasm (Dr. A. Holubowska PhD thesis). My results establish FBXO41 as a CNS-specific neuronal F-box protein with high levels of expression throughout the brain, including the cerebellum and some limited expression in the spinal cord. Using the FBXO41 knockout mouse line, I further investigated the role of FBXO41 in vivo.
Due to their high mortality in the first two postnatal weeks, FBXO41-/- mice were analyzed at P16. While the FBXO41+/- mice were indistinguishable from their wild type littermates, the FBXO41-/- mice displayed a severely ataxic gait characterized by uncoordinated movements, poor performance in the ledge test and rotarod and presence of severe hind limb clasping.
Upon histological analyses of the FBXO41-/- mouse brains, the most dramatic phenotype was observed in the cerebellum, which showed impaired neuronal migration. I analyzed the FBXO41-/- mice at three different ages, P12, P16 and P30 and found that all three ages showed more cells in the ML, most of which were CGNs. Interestingly, I also discovered that P12 FBXO41-/- cerebella displayed a thicker EGL as compared to wild type. Strikingly the P16 FBXO41-/- cerebella still possessed a residual EGL, while in FBXO41+/+ cerebella neuronal migration was almost completed and the EGL had vanished. These findings are indicative of impaired neuronal migration. Intriguingly, the residual EGL of P16 FBXO41-/- cerebella harbored, in addition to postmitotic CGNs, mitotic precursor cells, suggesting that there could be a delay in progenitor differentiation that may also contribute to the delayed neuronal migration phenotype.
Further analysis revealed that impaired neuronal migration was CGN-specific and did not affect the Purkinje cell layer. This observation was consistent with the finding that FBXO41 is abundantly expressed in the IGL, but is completely absent from the Purkinje cells and ML.
However, effects of impaired CGN migration on the synaptic connections between the Purkinje cell dendrites and parallel fibers (CGN axons) cannot be ruled out. By P30, the
residual EGL of FBXO41-/- mice had disappeared, but it still harbored more migrating CGNs when compared to the wild type. Collectively, these findings indicate that loss of FBXO41 decelerates migration of CGNs in the cerebellum, where they fail to migrate to the IGL within a developmentally appropriate time window. A similar developmental delay in CGN migration has also been reported in mouse models for the actin-binding protein profilin1 as well as the microtubule-binding protein adenomatous polyposis coli 2 (APC2) (Kullmann et al., 2012; Shintani et al., 2012). It is hence conceivable that FBXO41 might function as a cell-intrinsic regulator of the cytoskeleton either by directly influencing cytoskeletal dynamics or by intercepting extracellular, pro-migratory cues at the leading edge.
Previous results from the lab identified a dual localization of FBXO41 at the centrosome and the cytoplasm (Dr. A. Holubowska PhD thesis). The centrosome is known to serve as the major site of microtubule nucleation resulting in the formation of a microtubule cage thereby aiding in nucelokinesis and proper radial migration during neurodevelopment (Higginbotham and Gleeson, 2007; Tsai and Gleeson, 2005). Several centrosome-associated proteins including DISC1, NDEL1, Lis1, and the Par/aPKC complex have been previously reported to influence neuronal migration (Kamiya et al., 2005; Sasaki et al., 2005; Solecki et al., 2004;
Tanaka et al., 2004). Interestingly, a previous study from the lab also reported that FBXO41 interacts with DISC1 and possibly NDEL1 to regulate neuronal migration (Dr. A.
Holubowska PhD thesis). In order to establish if the centrosomal localization of FBXO41 was crucial for its neuronal migration regulating effect, we monitored the migration behavior of CGNs using in vivo experiments in the developing rat cerebellum. First and foremost the results showed that loss of FBXO41 is reproducible and confirms that the impaired neuronal migration phenotype observed in the FBXO41-/- mice is specifically due to loss of FBXO41.The results also suggest that the cytoplasmic FBXO41 is necessary and sufficient to stimulate neuronal migration, while the centrosomal FBXO41 has little or no influence highlighting FBXO41’s centrosome-independent control of neuronal migration. This finding is both surprising and interesting. Since DISC1 for instance is also expressed extensively in the cytoplasm, nucleus and mitochondria (James et al., 2004), it is possible that FBXO41 interacts with DISC1 in the cytoplasm to exert its function on neuronal migration.
Interestingly, FBXO41 was also previously reported to promote axon growth independent of its centrosomal localization (Dr. A. Houlubowska, PhD Thesis). These findings suggest that FBXO41 may have other important functions at the centrosome, e.g. regulation of progenitor
Apart from impaired CGN migration, P30 FBXO41-/- mice also displayed abnormal foliation of the cerebellum, with reduced cerebellar size and intralobular outfoldings in folia IV and V.
Since the most obvious histological phenotype observed in FBXO41-/- mice was in the cerebellum, the severely ataxic phenotype of the mice can be majorly attributed to the cerebellar defects. The absence inflammation and neurodegenration in the P16 brain suggests that the motor phenotypes observed at P16 might be due to developmental abnormalities including impaired neuronal migration. Several studies have previously attributed ataxia-like phenotypes to dysfunction in cerebellar development, for instance a recent study reporting that deletion of the CNS-specific TGF-ß signaling protein Smad2 results in aberrant cerebellar morphology, impaired CGN migration, impaired Purkinje cell synaptic connections and early postnatal ataxia (Wang et al., 2011). Moreover, several human cerebellar ataxias have also been successfully modeled in mice including the Ahi1 knockout mouse, which displays abnormal cerebellar development and is viewed as a mouse model for Joubert’s syndrome congenital ataxia (Lancaster et al., 2011a).
Interestingly, several forms of ataxias are also associated with neurodegenerative events in the cerebellum (Assadi et al., 2008; Cummings et al., 1999), for example the SCA1 transgenic mice modeling spinocerebellar ataxia type 1 displaying neurodegeneration of Purkinje cells as a result of nuclear localization and toxic accumiliation of ataxin1 (Klement et al., 1998). Although the P30 FBXO41-/- cerebellum appears to catch up in neuronal migration, FBXO41-/- mice still show a persistent ataxic gait, uncoordinated movement and severe hind limb clasping. Moreover, P30 FBXO41-/- mice display a significantly smaller cerebellum together with pronounced foliation defects in folia IV, V and IV, which turned out to be a result of ongoing neurodegeneration as FBXO41-/- brains revealed a significant increase in apoptosis in the cerebella. Interestingly, the majority of the apoptotic cells appeared to be clustered in the abnormal folias. These results suggest that while the motor deficits observed at P16 are largely due to neurodevelopmental defects, the persistent motor phenotype in P30 FBXO41-/- mice as well as the reduced cerebellar size are a result of faulty wiring and compromised cerebellar architecture. Additionally, in vitro survival assays on cultured CGNs confirm that the observation of cell death in the FBXO41-/- brain can be attributed to loss of FBXO41. This further highlights the prosurvial nature of FBXO41, and its necessity in maintaining health and long-term integrity of neurons.
The delay in cerebellar development is accompanied by neurodegeneration and motor defects in the FBXO41-/- mice and thus accentuates the crucial function of FBXO41 in cerebellar development, cell health and proper motor coordination. The FBXO41-/- mice could hence be viewed as a model of cerebellar ataxia. Interestingly, several genes that are nowadays implicated in human cerebellar ataxias were first identified several decades ago in knockout mice models displaying cerebellar abnormalities and ataxia-like phenotypes. The staggerer mouse model, which is a knockout for the retinoic acid-related orphan nuclear receptor-α (ROR-α), has been generated more than 50 years ago, but its interaction with ATAXIN1 and the link to spinocerebellar ataxia 1 (SCA1) has only recently been made (Gold et al., 2003;
Hamilton et al., 1996; Serra et al., 2004; Serra et al., 2006). Similarly, human mutations in GIRD2 gene associated with cerebellar ataxia were identified 60 years after the GIRD2 gain of function mouse (lurcher) was generated (Hills et al., 2013; Utine et al., 2013). Most recently, mutations in the TRCP3 gene as observed in the Moonwalker mouse, have been implicated in human cerebellar ataxia (Becker, 2014). Since ~40% of cerebellar ataxias originate from unknown causes (Sailer and Houlden, 2012), it is conceivable that unidentified SNPs or mutations in the Fbxo41 gene could be the underlying cause for some of them and hence genetic screens for mutations or variations in the Fbxo41 gene are essential to establish the link from mouse to human cerebellar ataxias.